78. Thomas, R; Kermode, AR. (2019) Enzyme enhancement therapeutics for lysosomal storage diseases: Current status and perspective.Mol. Genet. Metab. 126 Enzyme enhancement therapeutics for lysosomal storage diseases: Current status and perspective
Pharmacological chaperones; Proteostasis regulators; High-throughput screening; Lysosomal storage diseases; Missense mutations; ER-associated degradation; Enzyme modulation; Proteostasis network; ER homeostasis; Blood-brain-barrier; Adjunctive therapies
Small-molecule- enzyme enhancement therapeutics (EETs) have emerged as attractive agents for the treatment of lysosomal storage diseases (LSDs), a broad group of genetic diseases caused by mutations in genes encoding lysosomal enzymes, or proteins required for lysosomal function. The underlying enzyme deficiencies characterizing LSDs cause a block in the stepwise degradation of complex macromolecules (e.g. glycosaminoglycans, glycolipids and others), such that undegraded or partially degraded substrates progressively accumulate in lysosomal and non-lysosomal compartments, a process leading to multisystem pathology via primary and secondary mechanisms. Missense mutations underlie many of the LSDs; the resultant mutant variant enzyme hydrolase is often impaired in its folding and maturation making it subject to rapid disposal by endoplasmic reticulum (ER)-associated degradation (ERAD). Enzyme deficiency in the lysosome is the result, even though the mutant enzyme may retain significant catalytic functioning. Small molecule modulators pharmacological chaperones (PCs), or proteostasis regulators (PRs) are being identified through library screens and computational tools, as they may offer a less costly approach than enzyme replacement therapy (ERT) for LSDs, and potentially treat neuronal forms of the diseases. PCs, capable of directly stabilizing the mutant protein, and PRs, which act on other cellular elements to enhance protein maturation, both allow a proportion of the synthesized variant protein to reach the lysosome and function. Proof-of-principle for PCs and PRs as therapeutic agents has been demonstrated for several LSDs, yet definitive data of their efficacy in disease models and/or in downstream clinical studies in many cases has yet to be achieved. Basic research to understand the cellular consequences of protein misfolding such as perturbed organellar crosstalk, redox status, and calcium balance is needed. Likewise, an elucidation of the early in cellulo pathogenic events underlying LSDs is vital and may lead to the discovery of new small molecule modulators and/or to other therapeutic approaches for driving proteostasis toward protein rescue. DOI PubMed
77. Zeng, Y; He, X; Danyukova, T; Pohl, S; Kermode, AR. (2019) Toward Engineering the Mannose 6-Phosphate Elaboration Pathway in Plants for Enzyme Replacement Therapy of Lysosomal Storage Disorders.J. Clin. Med. 8 Toward Engineering the Mannose 6-Phosphate Elaboration Pathway in Plants for Enzyme Replacement Therapy of Lysosomal Storage Disorders
alpha-L-iduronidase (IDUA); mannose 6-phosphate (M6P); M6P elaboration machinery; plant-based platform; enzyme replacement therapy (ERT); transgenic plant; lysosomal enzyme
Mucopolysaccharidosis (MPS) I is a severe lysosomal storage disease caused by alpha-L-iduronidase (IDUA) deficiency, which results in accumulation of non-degraded glycosaminoglycans in lysosomes. Costly enzyme replacement therapy (ERT) is the conventional treatment for MPS I. Toward producing a more cost-effective and safe alternative to the commercial mammalian cell-based production systems, we have produced recombinant human IDUA in seeds of an Arabidopsis mutant to generate the enzyme in a biologically active and non-immunogenic form containing predominantly high mannose N-linked glycans. Recombinant enzyme in ERT is generally thought to require a mannose 6-phosphate (M6P) targeting signal for endocytosis into patient cells and for intracellular delivery to the lysosome. Toward effecting in planta phosphorylation, the human M6P elaboration machinery was successfully co-expressed along with the recombinant human IDUA using a single multi-gene construct. Uptake studies using purified putative M6P-IDUA generated in planta on cultured MPS I primary fibroblasts indicated that the endocytosed recombinant lysosomal enzyme led to substantial reduction of glycosaminoglycans. However, the efficiency of the putative M6P-IDUA in reducing glycosaminoglycan storage was comparable with the efficiency of the purified plant mannose-terminated IDUA, suggesting a poor in planta M6P-elaboration by the expressed machinery. Although the in planta M6P-tagging process efficiency would need to be improved, an exciting outcome of our work was that the plant-derived mannose-terminated IDUA yielded results comparable to those obtained with the commercial IDUA (Aldurazyme (R) (Sanofi, Paris, France)), and a significant amount of the plant-IDUA is trafficked by a M6P receptor-independent pathway. Thus, a plant-based platform for generating lysosomal hydrolases may represent an alternative and cost-effective strategy to the conventional ERT, without the requirement for additional processing to create the M6P motif. DOI PubMed
76. Guenin, S; Hardouin, J; Paynel, F; Muller, K; Mongelard, G; Driouich, A; Lerouge, P; Kermode, AR; Lehner, A; Mollet, JC; Pelloux, J; Gutierrez, L; Mareck, A. (2017) AtPME3, a ubiquitous cell wall pectin methylesterase of Arabidopsis thaliana, alters the metabolism of cruciferin seed storage proteins during post-germinative growth of seedlings.Journal of Experimental Botany 68: 1083-1095 AtPME3, a ubiquitous cell wall pectin methylesterase of Arabidopsis thaliana, alters the metabolism of cruciferin seed storage proteins during post-germinative growth of seedlings
Arabidopsis; AtPME3; CRUCIFERIN; etiolated hypocotyl; pectin methylesterase; seed germination; transcriptomic analyses
AtPME3 (At3g14310) is a ubiquitous cell wall pectin methylesterase. Atpme3-1 loss-of-function mutants exhibited distinct phenotypes from the wild type (WT), and were characterized by earlier germination and reduction of root hair production. These phenotypical traits were correlated with the accumulation of a 21.5-kDa protein in the different organs of 4-day-old Atpme3-1 seedlings grown in the dark, as well as in 6-week-old mutant plants. Microarray analysis showed significant down-regulation of the genes encoding several pectin-degrading enzymes and enzymes involved in lipid and protein metabolism in the hypocotyl of 4-day-old dark grown mutant seedlings. Accordingly, there was a decrease in proteolytic activity of the mutant as compared with the WT. Among the genes specifying seed storage proteins, two encoding CRUCIFERINS were up-regulated. Additional analysis by RT-qPCR showed an overexpression of four CRUCIFERIN genes in the mutant Atpme3-1, in which precursors of the alpha- and beta-subunits of CRUCIFERIN accumulated. Together, these results provide evidence for a link between AtPME3, present in the cell wall, and CRUCIFERIN metabolism that occurs in vacuoles. DOI
75. Pierce, OM; McNair, GR; He, X; Kajiura, H; Fujiyama, K; Kermode, AR. (2017) N-glycan structures and downstream mannose-phosphorylation of plant recombinant human alpha-l-iduronidase: toward development of enzyme replacement therapy for mucopolysaccharidosis I.Plant Mol.Biol. 95: 593-606 N-glycan structures and downstream mannose-phosphorylation of plant recombinant human alpha-l-iduronidase: toward development of enzyme replacement therapy for mucopolysaccharidosis I
Lysosomal storage diseases; Mucopolysaccharidosis I; Enzyme replacement therapy; Mannose-6-phosphate tag; Lysosomal targeting; Alpha-L-iduronidase
Arabidopsis N-glycan processing mutants provide the basis for tailoring recombinant enzymes for use as replacement therapeutics to treat lysosomal storage diseases, including N-glycan mannose phosphorylation to ensure lysosomal trafficking and efficacy. Functional recombinant human alpha-l-iduronidase (IDUA; EC 3.2.1.76) enzymes were generated in seeds of the Arabidopsis thaliana complex-glycan-deficient (cgl) C5 background, which is deficient in the activity of N-acetylglucosaminyl transferase I, and in seeds of the Arabidopsis gm1 mutant, which lacks Golgi alpha-mannosidase I (GM1) activity. Both strategies effectively prevented N-glycan maturation and the resultant N-glycan structures on the consensus sites for N-glycosylation of the human enzyme revealed high-mannose N-glycans of predominantly Man(5) (cgl-IDUA) or Man(6-8) (gm1-IDUA) structures. Both forms of IDUA were equivalent with respect to their kinetic parameters characterized by cleavage of the artificial substrate 4-methylumbelliferyl-iduronide. Because recombinant lysosomal enzymes produced in plants require the addition of mannose-6-phosphate (M6P) in order to be suitable for lysosomal delivery in human cells, we characterized the two IDUA proteins for their amenability to downstream in vitro mannose phosphorylation mediated by a soluble form of the human phosphotransferase (UDP-GlcNAc: lysosomal enzyme N-acetylglucosamine [GlcNAc]-1-phosphotransferase). Gm1-IDUA exhibited a slight advantage over the cgl-IDUA in the in vitro M6P-tagging process, with respect to having a better affinity (i.e. lower K (m)) for the soluble phosphotransferase. This may be due to the greater number of mannose residues comprising the high-mannose N-glycans of gm1-IDUA. Our elite cgl- line produces IDUA at > 5.7% TSP (total soluble protein); screening of the gm1 lines showed a maximum yield of 1.5% TSP. Overall our findings demonstrate the relative advantages and disadvantages associated with the two platforms to create enzyme replacement therapeutics for lysosomal storage diseases. DOI
74. Cliff, MA; Law, JR; Lucker, J; Scaman, CH; Kermode, AR. (2016) Descriptive and hedonic analyses of low-Phe food formulations containing corn (Zea mays) seedling roots: toward development of a dietary supplement for individuals with phenylketonuria.Journal of the Science of Food and Agriculture 96: 140-149 Descriptive and hedonic analyses of low-Phe food formulations containing corn (Zea mays) seedling roots: toward development of a dietary supplement for individuals with phenylketonuria
corn (Zeamays) roots; descriptive analysis; hedonic evaluations; phenylketonuria; phenylalanine ammonia lyase
BACKGROUND: Seedling roots of anthocyanin-rich corn (Zea mays) cultivars contain high levels of phenylalanine ammonia lyase (PAL) activity. The development of a natural dietary supplement containing corn roots could provide the means to improve the restrictive diet of phenylketonuria (PKU) patients by increasing their tolerance to dietary phenylalanine (Phe). Therefore this research was undertaken to explore the sensory characteristics of roots of four corn cultivars as well as to develop and evaluate food products (cereal bar, beverage, jam-like spread) to which roots had been added. RESULTS: Sensory profiles of corn roots were investigated using ten trained judges. Roots of Japanese Striped corn seedlings were more bitter, pungent and astringent than those of white and yellow cultivars, while roots from the Blue Jade cultivar had a more pronounced earthy/mushroom aroma. Consumer research using 24 untrained panelists provided hedonic (degree-of-liking) assessments for products with and without roots (controls). The former had lower mean scores than the controls; however, the cereal bar had scores above 5 on the nine-point scale for all hedonic assessments compared with the other treated products. CONCLUSION: By evaluating low-Phe food products containing corn roots, this research ascertained that the root-containing low-Phe cereal bar was an acceptable 'natural' dietary supplement for PKU-affected individuals. (C) 2015 Her Majesty the Queen in Right of Canada Journal of the Science of Food and Agriculture (C) 2015 Society of Chemical Industry DOI
73. Liu, Y; Muller, K; El-Kassaby, YA; Kermode, AR. (2015) Changes in hormone flux and signaling in white spruce (Picea glauca) seeds during the transition from dormancy to germination in response to temperature cues.BMC Plant Biology 15 Changes in hormone flux and signaling in white spruce (Picea glauca) seeds during the transition from dormancy to germination in response to temperature cues
Seed dormancy; Auxin; ABA; GAs; Moist-chilling; Seed germination; White spruce
Background: Seeds use environmental cues such as temperature to coordinate the timing of their germination, allowing plants to synchronize their life history with the seasons. Winter chilling is of central importance to alleviate seed dormancy, but very little is known of how chilling responses are regulated in conifer seeds. White spruce (Picea glauca) is an important conifer species of boreal forests in the North American taiga. The recent sequencing and assembly of the white spruce genome allows for comparative gene expression studies toward elucidating the molecular mechanisms governing dormancy alleviation by moist chilling. Here we focused on hormone metabolite profiling and analyses of genes encoding components of hormone signal transduction pathways, to elucidate changes during dormancy alleviation and to help address how germination cues such as temperature and light trigger radicle emergence. Results: ABA, GA, and auxin underwent considerable changes as seeds underwent moist chilling and during subsequent germination; likewise, transcripts encoding hormone-signaling components (e.g. ABI3, ARF4 and Aux/IAA) were differentially regulated during these critical stages. During moist chilling, active IAA was maintained at constant levels, but IAA conjugates (IAA-Asp and IAA-Glu) were substantially accumulated. ABA concentrations decreased during germination of previously moist-chilled seeds, while the precursor of bioactive GA1 (GA53) accumulated. We contend that seed dormancy and germination may be partly mediated through the changing hormone concentrations and a modulation of interactions between central auxin-signaling pathway components (TIR1/AFB, Aux/IAA and ARF4). In response to germination cues, namely exposure to light and to increased temperature: the transfer of seeds from moist-chilling to 30 degrees C, significant changes in gene transcripts and protein expression occurred during the first six hours, substantiating a very swift reaction to germination-promoting conditions after seeds had received sufficient exposure to the chilling stimulus. Conclusions: The dormancy to germination transition in white spruce seeds was correlated with changes in auxin conjugation, auxin signaling components, and potential interactions between auxin-ABA signaling cascades (e.g. the transcription factor ARF4 and ABI3). Auxin flux adds a new dimension to the ABA: GA balance mechanism that underlies both dormancy alleviation by chilling, and subsequent radicle emergence to complete germination by warm temperature and light stimuli. DOI
72. Yadav, AK; Shen, DL; Shan, X; He, X; Kermode, AR; Vocadlo, DJ. (2015) Fluorescence-Quenched Substrates for Live Cell Imaging of Human Glucocerebrosidase Activity.Journal of the American Chemical Society 137: 1181-1189 Fluorescence-Quenched Substrates for Live Cell Imaging of Human Glucocerebrosidase Activity
Deficiency of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) leads to abnormal accumulation of glucosyl ceramide in lysosomes and the development of the lysosomal storage disease known as Gauchers disease. More recently, mutations in the GBA1 gene that encodes GCase have been uncovered as a major genetic risk factor for Parkinsons disease (PD). Current therapeutic strategies to increase GCase activity in lysosomes involve enzyme replacement therapy (ERT) and molecular chaperone therapy. One challenge associated with developing and optimizing these therapies is the difficulty in determining levels of GCase activity present within the lysosomes of live cells. Indeed, visualizing the activity of endogenous levels of any glycoside hydrolases, including GCase, has proven problematic within live mammalian cells. Here we describe the successful modular design and synthesis of fluorescence-quenched substrates for GCase. The selection of a suitable fluorophore and quencher pair permits the generation of substrates that allow convenient time-dependent monitoring of endogenous GCase activity within cells as well as localization of activity within lysosomes. These efficiently quenched (similar to 99.9%) fluorescent substrates also permit assessment of GCase inhibition in live cells by either confocal microscopy or high content imaging. Such substrates should enable improved understanding of GCase in situ as well the optimization of small-molecule chaperones for this enzyme. These findings also suggest routes to generate fluorescence-quenched substrates for other mammalian glycoside hydrolases for use in live cell imaging. DOI PubMed
71. He, X; Galpin, JD; Miao, YS; Jiang, LW; Grabowski, GA; Kermode, AR. (2014) Membrane anchors effectively traffic recombinant human glucocerebrosidase to the protein storage vacuole of Arabidopsis seeds but do not adequately control N-glycan maturation.Plant Cell Reports 33: 2023-2032 Membrane anchors effectively traffic recombinant human glucocerebrosidase to the protein storage vacuole of Arabidopsis seeds but do not adequately control N-glycan maturation
Arabidopsis thaliana; Gaucher disease; Human glucocerebrosidase; Protein storage vacuole; N-glycosylation
Key message Human glucocerebrosidase with vacuolar anchoring domains was targeted to protein storage vacuoles (PSVs) of Arabidopsis seeds, but unexpectedly via the Golgi complex. PSV-targeting to effectively avoid problematic N-glycans is protein dependent. Plant-specific N-glycosylation patterns elaborated within the Golgi complex are a major limitation of using plants to produce biopharmaceuticals as the presence of beta 1,2 xylose and/or alpha 1,3 fucose residues on the recombinant glycoprotein can render the product immunogenic if administrated parenterally. A reporter protein fused to a vacuolar membrane targeting motif comprised of the BP-80 transmembrane domain (TMD), and the cytoplasmic tail (CT) of alpha-tonoplast intrinsic protein (alpha-TIP) is delivered to protein storage vacuoles (PSVs) of tobacco seeds by ER-derived transport vesicles that bypass the Golgi complex. This prompted us to investigate whether a pharmaceutical glycoprotein is targeted to PSVs using the same targeting sequences, thus avoiding the unwanted plant-Golgi-specific complex N-glycan modifications. The human lysosomal acid beta-glucosidase (glucocerebrosidase; GCase) (EC 3.2.1.45) fused to the BP-80 TMD and alpha-TIP CT was produced in Arabidopsis thaliana wild-type (Col-0) seeds. The chimeric GCase became localized in PSVs but transited through the Golgi complex, as indicated by biochemical analyses of the recombinant protein's N-glycans. Our findings suggest that use of this PSV-targeting strategy to avoid problematic N-glycan maturation on recombinant therapeutic proteins is not consistently effective, as it is likely protein- and/or species-specific. DOI PubMed
70. Lopez-Villalobos, A; Lucker, J; Lopez-Quiroz, AA; Yeung, EC; Palma, K; Kermode, AR. (2014) Preservation of high phenylalanine ammonia lyase activities in roots of Japanese Striped corn: A potential oral therapeutic to treat phenylketonuria.Cryobiology 68: 436-445 Preservation of high phenylalanine ammonia lyase activities in roots of Japanese Striped corn: A potential oral therapeutic to treat phenylketonuria
Phenylalanine ammonia lyase (PAL); Oral enzyme therapeutic; Phenylketonuria (PKU); Freezing preservation; Seedling roots; Japanese Striped corn; Oral therapeutic; Enzyme substitution therapy; Simulated digestion
Phenylketonuria (PKU) is an inherited metabolic disorder caused by deficient phenylalanine hydroxylase (PAH) activity, the enzyme responsible for the disposal of excess amounts of the essential amino acid phenylalanine (Phe). Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) has potential to serve as an enzyme substitution therapy for this human genetic disease. Using 7-day-old Japanese Striped corn seedlings (Japonica Striped maize, Zea mays L. cv. japonica) that contain high activities of PAL, we investigated a number of methods to preserve the roots as an intact food and for long-term storage. The cryoprotectant effects of maple syrup and other edible sugars (mono- and oligosaccharides) were evaluated. Following thawing, the preserved roots were then examined to determine whether the rigid plant cell walls could protect the PAL enzyme from proteolysis during simulated (in vitro) digestion comprised of gastric and intestinal phases. While several treatments led to retention of PAL activity during freezing, upon thawing and in vitro digestion, root tissues that had been previously frozen in the presence of maple syrup exhibited the highest residual PAL activities (similar to 50% of the initial enzyme activity), in marked contrast to all of the treatments using other edible sugars. The structural integrity of the root cells, and the stability of the functional PAL tetramer were also preserved with the maple syrup protocol. These results have significance for the formulation of oral enzyme/protein therapeutics. When plant tissues are adequately preserved, the rigid cell walls constitute a protective barrier even under harsh (e.g. gastrointestinal-like) conditions. (C) 2014 Elsevier Inc. All rights reserved. DOI
69. Bie, HY; Yin, J; He, X; Kermode, AR; Goddard-Borger, ED; Withers, SG; James, MNG. (2013) Insights into mucopolysaccharidosis I from the structure and action of alpha-L-iduronidase.Nature Chemical Biology 9: 739-+ Insights into mucopolysaccharidosis I from the structure and action of alpha-L-iduronidase
GLYCOSYL-ENZYME INTERMEDIATE; THERMOANAEROBACTERIUM-SACCHAROLYTICUM; PHARMACOLOGICAL CHAPERONES; BETA-GLUCOCEREBROSIDASE; GAUCHER-DISEASE; IDENTIFICATION; MUTATIONS; XYLOSIDASE; MODEL; NUCLEOPHILE
Mucopolysaccharidosis type I (MPS I), caused by mutations in the gene encoding alpha-L-iduronidase (IDUA), is one of approximately 70 genetic disorders collectively known as the lysosomal storage diseases. To gain insight into the basis for MPS I, we crystallized human IDUA produced in an Arabidopsis thaliana cgl mutant. IDUA consists of a TIM barrel domain containing the catalytic site, a beta-sandwich domain and a fibronectin-like domain. Structures of IDUA bound to iduronate analogs illustrate the Michaelis complex and reveal a B-2,B-5 conformation in the glycosyl-enzyme intermediate, which suggest a retaining double displacement reaction involving the nucleophilic Glu299 and the general acid/base Glu182. Unexpectedly, the N-glycan attached to Asn372 interacts with iduronate analogs in the active site and is required for enzymatic activity. Finally, these IDUA structures and biochemical analysis of the disease-relevant P533R mutation have enabled us to correlate the effects of mutations in IDUA to clinical phenotypes. DOI
68. He, X; Pierce, O; Haselhorst, T; von Itzstein, M; Kolarich, D; Packer, NH; Gloster, TM; Vocadlo, DJ; Qian, Y; Brooks, D; Kermode, AR. (2013) Characterization and downstream mannose phosphorylation of human recombinant alpha-L-iduronidase produced in Arabidopsis complex glycan-deficient (cgl) seeds.Plant Biotechnol. J. 11: 1034-1043 Characterization and downstream mannose phosphorylation of human recombinant alpha-L-iduronidase produced in Arabidopsis complex glycan-deficient (cgl) seeds
Arabidopsis cgl mutant; mucopolysaccharidosis I; human -L-iduronidase; mannose-6-phosphate recognition marker; N-glycosylation
Mucopolysaccharidosis (MPS) I is a lysosomal storage disease caused by a deficiency of -L-iduronidase (IDUA) (EC 3.2.1.76); enzyme replacement therapy is the conventional treatment for this genetic disease. Arabidopsis cgl mutants are characterized by a deficiency of the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of hybrid and complex N-glycan biosynthesis. To develop a seed-based platform for the production of recombinant IDUA for potential treatment of MPS I, cgl mutant seeds were generated to express human IDUA at high yields and to avoid maturation of the N-linked glycans on the recombinant human enzyme. Enzyme kinetic data showed that cgl-IDUA has similar enzymatic properties to the commercial recombinant IDUA derived from cultured Chinese hamster ovary (CHO) cells (Aldurazyme(TM)). The N-glycan profile showed that cgl-derived IDUA contained predominantly high-mannose-type N-glycans (94.5%), and the residual complex/hybrid N-glycan-containing enzyme was efficiently removed by an additional affinity chromatography step. Furthermore, purified cgl-IDUA was amenable to sequential in vitro processing by soluble recombinant forms of the two enzymes that mediate the addition of the mannose-6-phosphate (M6P) tag in mammalian cellsUDP-GlcNAc:lysosomal enzyme N-acetylglucosamine (GlcNAc)-1-phosphotransferaseand GlcNAc-1-phosphodiester -N-acetylglucosaminidase (the uncovering enzyme'). Arabidopsis seeds provide an alternative system for producing recombinant lysosomal enzymes for enzyme replacement therapy; the purified enzymes can be subjected to downstream processing to create the M6P, a recognition marker essential for efficient receptor-mediated uptake into lysosomes of human cells. DOI PubMed
67. Muller, K; Levesque-Tremblay, G; Bartels, S; Weitbrecht, K; Wormit, A; Usadel, B; Haughn, G; Kermode, AR. (2013) Demethylesterification of Cell Wall Pectins in Arabidopsis Plays a Role in Seed Germination.Plant Physiology 161: 305-316 Demethylesterification of Cell Wall Pectins in Arabidopsis Plays a Role in Seed Germination
ENDO-BETA-MANNANASE; ABSCISIC-ACID; XYLOGLUCAN ENDOTRANSGLYCOSYLASE; METHYLESTERASE INHIBITORS; ENDOSPERM CAP; DORMANCY; GROWTH; THALIANA; ESTERIFICATION; EXPRESSION
The methylesterification status of cell wall homogalacturonans, mediated through the action of pectin methylesterases (PMEs), influences the biophysical properties of plant cell walls such as elasticity and porosity, important parameters for cell elongation and water uptake. The completion of seed germination requires cell wall extensibility changes in both the radicle itself and in the micropylar tissues surrounding the radicle. In wild-type seeds of Arabidopsis (Arabidopsis thaliana), PME activities peaked around the time of testa rupture but declined just before the completion of germination (endosperm weakening and rupture). We overexpressed an Arabidopsis PME inhibitor to investigate PME involvement in seed germination. Seeds of the resultant lines showed a denser methylesterification status of their cell wall homogalacturonans, but there were no changes in the neutral sugar and uronic acid composition of the cell walls. As compared with wild-type seeds, the PME activities of the overexpressing lines were greatly reduced throughout germination, and the low steady-state levels neither increased nor decreased. The most striking phenotype was a significantly faster rate of germination, which was not connected to altered testa rupture morphology but to alterations of the micropylar endosperm cells, evident by environmental scanning electron microscopy. The transgenic seeds also exhibited an apparent reduced sensitivity to abscisic acid with respect to its inhibitory effects on germination. We speculate that PME activity contributes to the temporal regulation of radicle emergence in endospermic seeds by altering the mechanical properties of the cell walls and thereby the balance between the two opposing forces of radicle elongation and mechanical resistance of the endosperm. DOI
66. Zeng, Y; Zhao, TH; Kermode, AR. (2013) A Conifer ABI3-Interacting Protein Plays Important Roles during Key Transitions of the Plant Life Cycle.Plant Physiology 161: 179-195 A Conifer ABI3-Interacting Protein Plays Important Roles during Key Transitions of the Plant Life Cycle
LATERAL ROOT DEVELOPMENT; OF-FUNCTION MUTATION; ABSCISIC-ACID; ARABIDOPSIS-THALIANA; GENE-EXPRESSION; STORAGE PROTEIN; FLOWERING TIME; ABI3 GENE; CELL ELONGATION; SEED DORMANCY
ABI3 (for ABSCISIC ACID INSENSITIVE3), a transcription factor of the abscisic acid signal transduction pathway, plays a major role during seed development, dormancy inception, and dormancy maintenance. This protein appears to also function in meristematic and vegetative plant tissues and under certain stress conditions. We have isolated the ABI3 gene ortholog (CnABI3) from yellow cedar (Callitropsis nootkatensis) and found that it was functionally similar to other ABI3 genes of angiosperms. Here, we report that using a yeast (Saccharomyces cerevisiae) two-hybrid approach, we have identified another protein of yellow cedar (CnAIP2; for CnABI3 INTERACTING PROTEIN2) that physically interacts with CnABI3. Functional analyses revealed that CnAIP2 plays important roles during key transitions in the plant life cycle: (1) CnAIP2 impaired seed development and reduced seed dormancy; (2) CnAIP2 promoted root development, particularly the initiation of lateral roots, and the CnAIP2 gene promoter was exquisitely auxin sensitive; and (3) CnAIP2 promoted the transition from vegetative growth to reproductive initiation (i.e. flowering). The nature of the effects of CnAIP2 on these processes and other evidence place CnAIP2 in the category of a "global" regulator, whose actions are antagonistic to those of ABI3. DOI
65. Babajani, G; Tropak, MB; Mahuran, DJ; Kermode, AR. (2012) Pharmacological chaperones facilitate the post-ER transport of recombinant N370S mutant beta-glucocerebrosidase in plant cells: Evidence that N370S is a folding mutant.Molecular Genetics and Metabolism 106: 323-329 Pharmacological chaperones facilitate the post-ER transport of recombinant N370S mutant beta-glucocerebrosidase in plant cells: Evidence that N370S is a folding mutant
beta-glucocerebrosidase; N370S mutation; Pharmacological chaperones; Tobacco BY2 cells; Protoplasts; Secretion
Gaucher disease is a prevalent lysosomal storage disease in which affected individuals inherit mutations in the gene (GBA1) encoding lysosomal acid beta-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45). One of the most prevalent disease-causing mutations in humans is a N370S missense mutation in the GCase protein. As part of a larger endeavor to study the fate of mutant human proteins expressed in plant cells, the N370S mutant protein along with the wild-type- (WT)-GCase, both equipped with a signal peptide, were synthesized in transgenic tobacco BY2 cells, which do not possess lysosomes. The enzymatic activity of plant-recombinant N370S GCase lines was significantly lower (by 81-95%) than that of the WT-GCase lines. In contrast to the WT-GCase protein, which was efficiently secreted from tobacco BY2 cells, and detected in large amounts in the culture medium, only a small proportion of the N370S GCase was secreted. Pharmacological chaperones such as N-(n-nonyl) deoxynojirimycin and ambroxol increased the. steady-state mutant protein levels both inside the plant cells and in the culture medium. These findings contradict the assertion that small molecule chaperones increase N370S GCase activity (as assayed in treated patient cell lysates) by stabilizing the enzyme in the lysosome, and suggest that the mutant protein is impaired in its ability to obtain its functional folded conformation, which is a requirement for exiting the lumen of the ER. (C) 2012 Elsevier Inc. All rights reserved. DOI
64. He, X; Galpin, JD; Tropak, MB; Mahuran, D; Haselhorst, T; von Itzstein, M; Kolarich, D; Packer, NH; Miao, YS; Jiang, LW; Grabowski, GA; Clarke, LA; Kermode, AR. (2012) Production of active human glucocerebrosidase in seeds of Arabidopsis thaliana complex-glycan-deficient (cgl) plants.Glycobiology 22: 492-503 Production of active human glucocerebrosidase in seeds of Arabidopsis thaliana complex-glycan-deficient (cgl) plants
Arabidopsis cgl mutant; Gaucher disease; human glucocerebrosidase; mannose-terminated N-glycans; N-glycosylation
There is a clear need for efficient methods to produce protein therapeutics requiring mannose-termination for therapeutic efficacy. Here we report on a unique system for production of active human lysosomal acid beta-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45) using seeds of the Arabidopsis thaliana complex-glycan-deficient (cgl) mutant, which are deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101). Gaucher disease is a prevalent lysosomal storage disease in which affected individuals inherit mutations in the gene (GBA1) encoding GCase. A gene cassette optimized for seed expression was used to generate the human enzyme in seeds of the cgl (C5) mutant, and the recombinant GCase was mainly accumulated in the apoplast. Importantly, the enzymatic properties including kinetic parameters, half-maximal inhibitory concentration of isofagomine and thermal stability of the cgl-derived GCase were comparable with those of imiglucerase, a commercially available recombinant human GCase used for enzyme replacement therapy in Gaucher patients. N-glycan structural analyses of recombinant cgl-GCase showed that the majority of the N-glycans (97%) were mannose terminated. Additional purification was required to remove similar to 15% of the plant-derived recombinant GCase that possessed potentially immunogenic (xylose- and/or fucose-containing) N-glycans. Uptake of cgl-derived GCase by mouse macrophages was similar to that of imiglucerase. The cgl seed system requires no addition of foreign (non-native) amino acids to the mature recombinant GCase protein, and the dry transgenic seeds represent a stable repository of the therapeutic protein. Other strategies that may completely prevent plant-like complex N-glycans are discussed, including the use of a null cgl mutant. DOI
63. He, X; Haselhorst, T; von Itzstein, M; Kolarich, D; Packer, NH; Gloster TM;Vocadlo, DJ; Clarke, LA; Qian, Y; Kermode, AR. (2012) Production of α-L-iduronidase in maize for the potential treatment of a human lysosomal storage disease.Nature Communications 3: Article number: 1062 Production of α-L-iduronidase in maize for the potential treatment of a human lysosomal storage disease
Lysosomal storage diseases are a class of over 70 rare genetic diseases that are amenable to enzyme replacement therapy. Towards developing a plant-based enzyme replacement therapeutic for the lysosomal storage disease mucopolysaccharidosis I, here we expressed α-L-iduronidase in the endosperm of maize seeds by a previously uncharacterized mRNA-targeting-based mechanism. Immunolocalization, cellular fractionation and in situ RT-PCR demonstrate that the α-L-iduronidase protein and mRNA are targeted to endoplasmic reticulum (ER)-derived protein bodies and to protein body-ER regions, respectively, using regulatory (5′- and 3′-UTR) and signal-peptide coding sequences from the γ-zein gene. The maize α-L-iduronidase exhibits high activity, contains high-mannose N-glycans and is amenable to in vitro phosphorylation. This mRNA-based strategy is of widespread importance as plant N-glycan maturation is controlled and the therapeutic protein is generated in a native form. For our target enzyme, the N-glycan structures are appropriate for downstream processing, a prerequisite for its potential as a therapeutic protein. DOI PubMed
62. He, X; Haselhorst, T; von Itzstein, M; Kolarich, D; Packer, NH; Kermode, AR. (2012) Influence of an ER-retention signal on the N-glycosylation of recombinant human alpha-l-iduronidase generated in seeds of Arabidopsis.Plant Molecular Biology 79: 157-169 Influence of an ER-retention signal on the N-glycosylation of recombinant human alpha-l-iduronidase generated in seeds of Arabidopsis
Arabidopsis cgl mutant; ER-retention; Mucopolysaccharidosis (MPS) I; Human alpha-L-iduronidase; N-glycosylation; Complex N-glycans
Processes associated with late events of N-glycosylation within the plant Golgi complex are a major limitation to the use of plant-based systems to produce recombinant pharmaceutical proteins for parenteral administration. Specifically, sugars added to the N-glycans of a recombinant protein during glycan maturation to complex forms (e.g. beta 1,2 xylose and alpha 1,3 fucose) can render the product immunogenic. In order to avoid these sugars, the human enzyme alpha-l-iduronidase (IDUA, EC 3.2.1.76), with a C-terminal ER-retention sequence SEKDEL, was expressed in seeds of complex-glycan-deficient (cgl) mutant and wild-type (Col-0) Arabidopsis thaliana, under the control of regulatory (5'-, signal-peptide-encoding-, and 3'-) sequences from the arcelin 5-I gene of Phaseolus vulgaris (cgl-IDUA-SEKDEL and Col-IDUA-SEKDEL, respectively). The SEKDEL motif had no adverse effect on the specific activity of the purified enzyme. Surprisingly, the majority of the N-glycans of Col-IDUA-SEKDEL were complex N-glycans (i.e. contained xylose and/or fucose) (88 %), whereas complex N-glycans comprised a much lower proportion of the N-glycans of cgl-IDUA-SEKDEL (26 %), in which high-mannose forms were predominant. In contrast to the non-chimeric IDUA of cgl seeds, which is mainly secreted into the extracellular spaces, the addition of the SEKDEL sequence to human recombinant IDUA expressed in the same background led to retention of the protein in ER-derived vesicles/compartments and its partial localization in protein storage vacuoles. Our data support the contention that the use of a C-terminal ER retention motif as an effective strategy to prevent or reduce complex N-glycan formation, is protein specific. DOI
61. Müller K, Bouyer D, Schnittger A, Kermode AR. (2012) Evolutionarily Conserved Histone Methylation Dynamics during Seed Life-Cycle Transitions.PLOS One 7(12): e51532 Evolutionarily Conserved Histone Methylation Dynamics during Seed Life-Cycle Transitions
Plants have a remarkable ability to react to seasonal changes by synchronizing life-cycle transitions with environmental conditions. We addressed the question of how transcriptional re-programming occurs in response to an environmental cue that triggers the major life cycle transition from seed dormancy to germination and seedling growth. We elucidated an important mechanistic aspect of this process by following the chromatin dynamics of key regulatory genes with a focus on the two antagonistic marks, H3K4me3 and H3K27me3. Histone methylation patterns of major dormancy regulators changed during the transition to germination and seedling growth. We observed a switch from H3K4me3 and high transcription levels to silencing by the repressive H3K27me3 mark when dormancy was broken through exposure to moist chilling, underscoring that a functional PRC2 complex is necessary for this transition. Moreover, this reciprocal regulation by H3K4me3 and H3K27me3 is evolutionarily conserved from gymnosperms to angiosperms.
DOI
60. Muller, K; Linkies, A; Leubner-Metzger, G; Kermode, AR. (2012) Role of a respiratory burst oxidase of Lepidium sativum (cress) seedlings in root development and auxin signalling.Journal of Experimental Botany 63: 6325-6334 Role of a respiratory burst oxidase of Lepidium sativum (cress) seedlings in root development and auxin signalling
AtrbohB; auxin; Lepidium sativum; Rboh; reactive oxygen species; RNAi; root development; superoxide
Reactive oxygen species are increasingly perceived as players in plant development and plant hormone signalling pathways. One of these species, superoxide, is produced in the apoplast by respiratory burst oxidase homologues (rbohs), a family of proteins that is conserved throughout the plant kingdom. Because of the availability of mutants, the focus of research into plant rbohs has been on Arabidopsis thaliana, mainly on AtrbohD and AtrbohF. This study investigates: (i) a different member of the Atrboh family, AtrbohB, and (ii) several rbohs from the close relative of A. thaliana, Lepidium sativum (ocress'). Five cress rbohs (Lesarbohs) were sequenced and it was found that their expression patterns were similar to their Arabidopsis orthologues throughout the life cycle. Cress plants in which LesarbohB expression was knocked down showed a strong seedling root phenotype that resembles phenotypes associated with defective auxin-related genes. These transgenic plants further displayed altered expression of auxin marker genes including those encoding the auxin responsive proteins 14 and 5 (IAA14 and IAA5), and LBD16 (LATERAL ORGAN BOUNDARIES DOMAIN16), an auxin-responsive protein implicated in lateral root initiation. It is speculated that ROS produced by rbohs play a role in root development via auxin signalling. DOI
59. Zhao, TH; Zeng, Y; Kermode, AR. (2012) A plant cell-based system that predicts alpha beta 42 misfolding: Potential as a drug discovery tool for Alzheimer's disease.Molecular Genetics and Metabolism 107: 571-579 A plant cell-based system that predicts alpha beta 42 misfolding: Potential as a drug discovery tool for Alzheimer's disease
Alzheimer's disease; Amyloid beta (A beta); Protein misfolding/aggregation; GFP; Hptll; Plant cell
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid beta (A beta) peptides and the failure of mechanisms to clear toxic aggregates. The A beta 42 peptide is considered to be a causative factor that underlies the pathophysiology of AD, in part due to its propensity for misfolding and aggregation; the small oligomers that result represent toxic species. Thus agents that prevent A beta 42 misfolding/ aggregation or, alternatively improve A beta 42 oligomer clearance, may have significant therapeutic value. We have developed the basis for a drug screening system based on transgenic plant cells that express A beta 42 fusion proteins to serve as the reliable indicators of the general conformational status of A beta 42. Within cells of transgenic tobacco and Nicotiana benthamiana, misfolding of A beta 42 causes the misfolding of a GFP fusion partner, and consequently there is a loss of fluorescence associated with the native GFP protein. In a similar fusion consisting of A beta 42 linked to hygromycin phosphotransferase II (Hpt II), a hygromycin-resistance marker, misfolding of A beta 42 leads to a misfolded Hpt II, and consequently the transgenic cells are unable to grow on media containing hygromycin. Importantly, substitution of the 'aggregation-prone' A beta 42 with a missense mutant of A beta 42 (F19S/L34F) that is not prone to misfolding/aggregation, 'rescues' both fusion partners. Several 'positive control' chemicals that represent inhibitors of A beta 42 aggregation, including curcumin, epigallocatechin-3-gallate (EGCG), and resveratrol show efficacy in preventing the A beta 42-fusion proteins from misfolding/aggregating in the transgenic plant cells. We discuss the potential of the two fusion protein systems to serve as the basis for an inexpensive, selective, and efficient screening system in which a plant cell can fluoresce or survive only in the presence of drug candidates that are able to prevent A beta 42 misfolding/aggregation. (C) 2012 Elsevier Inc. All rights reserved. DOI
58. Galpin, JD; Clemens, S; Kermode, AR. (2010) The carboxy-terminal ER-retention motif, SEKDEL, influences the N-linked glycosylation of recombinant human alpha-L-iduronidase but has little effect on enzyme activity in seeds of Brassica napus and Nicotiana tabacum.Plant Science 178: 440-447 The carboxy-terminal ER-retention motif, SEKDEL, influences the N-linked glycosylation of recombinant human alpha-L-iduronidase but has little effect on enzyme activity in seeds of Brassica napus and Nicotiana tabacum
Recombinant proteins; Human alpha-L-iduronidase; Mucopolysaccharidosis I; ER-retention; N-linked glycosylation; Enzyme replacement therapy
One of the potential drawbacks of the use of plant-based systems for production of human glycoproteins is the presence of immunogenic sugars within the protein's glycans, especially xylose. This can occur as a consequence of the protein undergoing transit through the plant Golgi complex, in which modifying enzymes may convert the high-mannose N-glycans of the recombinant protein to complex forms. In an effort to minimize this problem, the human enzyme alpha-L-iduronidase (IDUA, EC 3.2.1.76), with and without a C-terminal ER-retention sequence SEKDEL, was expressed in seeds of Brassica napus and Nicotiana tabacum under the control of regulatory (5'-, signal-peptide-encoding- and 3'-) sequences from the arcelin 5-I gene of Phaseolus vulgaris. The specific activities of the purified IDUA and IDUA-SEKDEL enzymes from seeds of both plant species were consistently between 32,000 and 40,000 nmol 4-methylumbelliferone/min/mg protein. Mass analysis of the hydrolyzed monosaccharides from N-glycans demonstrated that the presence of the SEKDEL sequence significantly diminished the amount of xylose and fucose present. This indicates that the proportion of synthesized recombinant proteins in which their constituent N-linked glycans underwent maturation (and hence, the addition of potentially immunogenic sugars) had been reduced, but not eliminated. However, importantly, the C-terminal modification of human IDUA expressed in seeds had little effect on the stability or activity of the enzyme. (C) 2010 Elsevier Ireland Ltd. All rights reserved. DOI
57. He, X; Kermode, AR. (2010) Programmed Cell Death of the Megagametophyte during Post-germinative Growth of White Spruce (Picea glauca) Seeds is Regulated by Reactive Oxygen Species and the Ubiquitin-mediated Proteolytic System.Plant and Cell Physiology 51: 1707-1720 Programmed Cell Death of the Megagametophyte during Post-germinative Growth of White Spruce (Picea glauca) Seeds is Regulated by Reactive Oxygen Species and the Ubiquitin-mediated Proteolytic System
Programmed cell death; Picea glauca; Megagametophyte; Reactive oxygen species; Ubiquitin-proteasome; Catalase; Protein degradation
The megagametophyte of white spruce (Picea glauca) seeds undergoes programmed cell death following seed germination. This process is characterized by distinct morphological and biochemical features, such as DNA fragmentation and the induction of proteases. Biphasic production of hydrogen peroxide was detected in the megagametophyte following seed germination. ROS scavengers or inhibitors of ROS production decreased caspase-like protease activity and slowed the progression of cell death. One catalase (CAT) of white spruce reacted with antibodies directed against cotton-seed CAT. The corresponding CAT gene was cloned and compared with the catalase genes of other plant species. The activity of the white spruce CAT enzyme was stimulated by tyrosine phosphorylation. The phosphorylated CAT was subjected to ubiquitination and degraded by the proteasome. Furthermore, the proteasome inhibitor MG132 inhibited the degradation of CAT and delayed cell death. These results suggest that the interplay of CAT and the ubiquitin-mediated proteolytic system is critical in the control of ROS production and subsequent cell death. DOI
56. Fu, LH; Miao, YS; Lo, SW; Seto, TC; Sun, SSM; Xu, ZF; Clemens, S; Clarke, LA; Kermode, AR; Jiang, LW. (2009) Production and characterization of soluble human lysosomal enzyme alpha-iduronidase with high activity from culture media of transgenic tobacco BY-2 cells.Plant Science 177: 668-675 Production and characterization of soluble human lysosomal enzyme alpha-iduronidase with high activity from culture media of transgenic tobacco BY-2 cells
GLYCAN-DEFICIENT SEEDS; HAMSTER OVARY CELLS; PLANT-CELLS; PREVACUOLAR COMPARTMENTS; PHARMACEUTICAL PROTEINS; REPLACEMENT THERAPY; EXPRESSION; ANTIBODIES; DEGLYCOSYLATION; GLYCOPROTEINS
Lysosomal storage diseases (LSDs), that collectively represent over 50 disorders, are amenable to enzyme replacement therapies. However, the current methods used to commercially produce recombinant lysosomal enzymes for this purpose, most commonly Chinese Hamster Ovary cells and human fibroblasts, are prohibitively costly. Plant bioreactors; hold great promise for economic production of functional human alpha-L-iduronidase (hIDUA; glycosaminoglycan alpha-L-iduronohydrolase: EC 3.2.1.76), the enzyme deficient in the human LSD, Mucopolysaccharidosis I. We have developed and tested an expression system using transgenic tobacco BY-2 cells to produce high amounts of active hIDUA. A plant signal peptide was essential for proper expression and secretion of the 78 kDa glycosylated hIDUA into the cultured media of transgenic BY-2 cells. The yield and activity of the secreted hIDUA from long-term cultures of transgenic BY-2 cell lines were as high as 10 mu g/mL media and 53,000 pmol/min/mg proteins, respectively. Thus, this transgenic BY-2 cell line presents an attractive platform for economic production and easy downstream purification of hIDUA for enzyme replacement therapy. Furthermore, this system can be used for the production and purification of other human lysosomal enzymes or pharmaceuticals. (C) 2009 Elsevier Ireland Ltd. All rights reserved. DOI
55. McInnis, S; Clemens, S; Kermode, AR. (2009) The ornamental variety, Japanese striped corn, contains high anthocyanin levels and PAL specific activity: establishing the potential for development of an oral therapeutic.Plant Cell Reports 28: 503-515 The ornamental variety, Japanese striped corn, contains high anthocyanin levels and PAL specific activity: establishing the potential for development of an oral therapeutic
PHENYLALANINE AMMONIA-LYASE; PHENYLPROPANOID METABOLISM; PETROSELINUM-CRISPUM; GENE-EXPRESSION; BIOSYNTHESIS; MAIZE; PHENYLKETONURIA; ARABIDOPSIS; IDENTIFICATION; INDUCTION
Phenylalanine ammonia-lyase [PAL, EC 4.3.1.24 (formerly EC 4.3.1.5)], functions in the plant phenylpropanoid biosynthetic pathway to deaminate the amino acid l-phenylalanine forming trans-cinnamic acid and ammonia. The human inherited metabolic disorder phenylketonuria (PKU) is characterized by an inability of individuals to metabolize phenylalanine. Toward the development of a plant-PAL based therapeutic for the treatment of this disorder, a comparative analysis of PAL activities within various members of the Poaceae was undertaken. This led to the identification of a Zea mays cultivar, Japanese Striped corn with very high levels of PAL specific activity in seedling tissues. The root tissues of this corn variety contain greater levels of PAL gene transcripts and PAL activities, compared to those of the shoot tissues, and are intensely colored due to the accumulation of anthocyanin pigments. PAL activities in the root tissues of young seedlings of another corn variety that lacked root anthocyanins (Indian Blue corn) were generally 30-50% lower than those of Japanese Striped corn seedlings at equivalent growth stages. In general, various stress or hormonal treatments led to minimal changes in PAL specific activity of maize tissues, as compared to controls. The PAL enzymes of Japanese Striped corn root tissues are robust; roots retained 90% of their PAL activity after freeze-drying and > 50% activity after freeze-drying and a subsequent 15-week storage at 4A degrees C. This work serves as a prelude to the formulation of a dietary supplement for treatment of PKU based on preserved edible cereal root tissues with high levels of intrinsic PAL activity. DOI
54. Feurtado, JA; Ren, C; Ambrose, SJ; Cutler, AJ; Ross, ARS; Abrams, SR; Kermode, AR. (2008) The coat-enhanced dormancy mechanism of western white pine (Pinus monticola Dougl. ex D. Don) seeds is mediated by abscisic acid homeostasis and mechanical restraint.Seed Science and Technology 36: 283-300 The coat-enhanced dormancy mechanism of western white pine (Pinus monticola Dougl. ex D. Don) seeds is mediated by abscisic acid homeostasis and mechanical restraint
The dormancy mechanism of western white Pine (Pinus monticola Dougl. ex D. Don) seeds was determined to be "coat-enhanced"; sequential removal of tissues surrounding the embryo - the seed coat, nucellar membrane/cap and megagametophyte - showed that all of these contribute to dormancy maintenance. Transfer of seeds to germination conditions following moist chilling of the seed decreased the mechanical restraint of the enclosing seed tissues. Coincidentally, the embryo growth potential increased, as demonstrated by an increased mechanical strength of the embryo during moist chilling and germination. and its reduced sensitivity to highly negative osmotic potential. The seed coat and underlying structures also influence the ability of embryos to metabolize abscisic acid (ABA). Unlike the intact dormant-imbibed seed, which maintained high levels of ABA in both the embryo and megagametophyte. removal of structures surrounding tire embryo decreased ABA levels and promoted ABA metabolism and/or transport. ABA levels in the embryo and megagametophyte were inversely correlated with increases in the germination capacity of seed populations resulting from treatments to remove tissues surrounding the embryo (e.g. the seed coat and other tissues). Changes in ABA metabolites showed no clear relationship to the germination capacity of seed Populations. which may have been due to the further flux of phaseic acid, dihydrophaseic acid, and 7 '-hydroxy ABA to downstream metabolites and/or transport of ABA and metabolites. This Study provides insight into the dormancy mechanism of western white pine seeds, and underscores tire importance of the surrounding seed tissues in maintaining ABA homeostasis in the embryo and megagametophyte.
53. Goldson, A., Lam, M., Scaman, C.H., Clemens, S., Kermode, A. (2008) Screening of phenylalanine ammonia lyase in plant tissues, and retention of activity during dehydration.Journal of the Science of Food and Agriculture 88: 619 - 625 Screening of phenylalanine ammonia lyase in plant tissues, and retention of activity during dehydration.
phenylalanine ammonia lyase; wheat seedlings; hyper phenylalaninemia; dehydration CELL-SUSPENSION CULTURES; MEDICAGO-SATIVA L; PHENYLPROPANOID METABOLISM; STRESS RESPONSES; WHEAT SEEDLINGS; PHENYLKETONURIA; INDUCTION; ACID; PURIFICATION; GERMINATION
BACKGROUND: Oral therapy with phenylalanine ammonia lyase (PAL), naturally encapsulated in plant cells, may provide a potential alternative treatment for hyperphenylalaninemic patients, including those with phenylketonuria. Therefore different sources of plant tissue were investigated for PAL activity.
RESULTS: Enzyme activity was highest in grain seedlings, with maximal enzyme activity in 7-day-old red spring wheat (Triticum aestivum L.) seedlings. The PAL activities of leaves and roots/endosperm of wheat seedlings were 11.90 +/- 2.64 and 6.48 +/- 1.59 mu mol h(-1) g(-1) dry weight respectively. Three PAL-related polypeptides with molecular weights of 74, 83 and 103 kDa were identified in wheat seedling leaf tissues, while only the 74 kDa polypeptide was detected in root/endosperm tissues. Dehydration was investigated as a method of concentrating PAL in wheat seedlings. Freeze-drying was found to retain the most PAL activity (> 90% recovery on a dry weight basis) compared with air drying and vacuum microwave drying for both leaf and root/endosperm samples.
CONCLUSION: This study has led to a better understanding of PAL activity and stability in plant tissues and provides the basis for developing a natural plant preparation as a dietary supplement for the treatment of hyperphenylalaninemia. (c) 2007 Society of Chemical Industry. DOI
52. Lam, A; Scaman, CH; Clemens, S; Kermode, A. (2008) Retention of Phenylalanine Ammonia-lyase Activity in Wheat Seedlings during Storage and in Vitro Digestion.Journal of Agricultural and Food Chemistry 56: 11407-11412 Retention of Phenylalanine Ammonia-lyase Activity in Wheat Seedlings during Storage and in Vitro Digestion
TRANS-CINNAMIC ACID; ENZYME REPLACEMENT; PHENYLKETONURIA; STRATEGY
The retention of phenylalanine ammonia-lyase (PAL) activity in Red Spring wheat seedlings during storage and in vitro protein digestion was evaluated toward assessing the efficacy of plant PAL as a dietary supplement for patients suffering from the metabolic disease, phenylketonuria. Retention of PAL activity in freeze-dried wheat seedling tissues following three months of storage at -20 degrees C ranged from 62% in the leaf to 89% in root/residual seed tissues. After a 3-h two-stage ("gastric-intestinal") in vitro digestion, 36% and 42% recovery of PAL activity was associated with chopped fresh leaf and root/residual seed tissues respectively; however, no activity was recovered from freeze-dried tissues. High performance liquid chromatographic analysis of the residual phenylalanine (Phe) after in vitro digestion confirmed that the fresh tissues effected a significantly higher conversion of exogenous Phe than freeze-dried tissues. These results demonstrate that the plant cell walls provide protection of PAL during in vitro digestion. In cases where exogenous Phe (100 mg; 24 mM) was supplied to the tissues, the product of the reaction, trans-cinnamic acid, may have exerted a significant inhibitory effect on PAL activity. DOI
51. Terskikh, VV; Zeng, Y; Feurtado, JA; Giblin, M; Abrams, SR; Kermode, AR. (2008) Deterioration of western redcedar (Thuja plicata Donn ex D. Don) seeds: protein oxidation and in vivo NMR monitoring of storage oils.Journal of Experimental Botany 59: 765-777 Deterioration of western redcedar (Thuja plicata Donn ex D. Don) seeds: protein oxidation and in vivo NMR monitoring of storage oils
conifer seeds; in vivo NMR spectroscopy; MRI; oil peroxidation; protein carbonylation; seed deterioration; seed storage; storage lipids; western redcedar
Deterioration of conifer seeds during prolonged storage has a negative impact on reforestation and gene conservation efforts. Western redcedar (Thuja plicata Donn ex D. Don) is a species of tremendous value to the forest industry. The seeds of this species are particularly prone to viability losses during long-term storage. Reliable tools to assess losses in seed viability during storage and their underlying causes, as well as the development of methods to prevent storage-related deterioration of seeds are needed by the forest industry. In this work, various imaging methods and biochemical analyses were applied to study deterioration of western redcedar seeds. Seedlots that exhibited poor germination performance, i.e. those that had experienced the greatest losses of viability during prolonged storage, exhibited greater abundance of oxidized proteins, detected by protein oxidation assays, and more pronounced changes in their in vivo C-13 NMR spectra, most likely due to storage oil oxidation. The proportion of oxidized proteins also increased when seeds were subjected to accelerated ageing treatments. Detection of oxidized oils and proteins may constitute a reliable and useful tool for the forest industry. DOI
50. Downing, WL; Hu, XK; Kermode, AR. (2007) Post-transcriptional factors are important for high-level expression of the human alpha-L-iduronidase gene in Arabidopsis cgl (complex-glycan-deficient) seeds.Plant Science 172: 327-334 Post-transcriptional factors are important for high-level expression of the human alpha-L-iduronidase gene in Arabidopsis cgl (complex-glycan-deficient) seeds
human alpha-L-iduronidase; arabidopsis; molecular farming; transgenic plants; signal peptide; post-transcriptional regulation
We are developing various plant-based systems to produce enzymes for the treatment of lysosomal storage disorders. One such system is seeds of the Arabidopsis thaliana cgl mutant, which are deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), and thus produce recombinant proteins containing glycans primarily in the high-mannose form. We sought to identify gene regulatory sequences that would enhance the expression of a human recombinant gene encoding alpha-L-iduronidase (IDUA; EC 3.2.1.76) in mature seeds of the cgl mutant. Three promoters derived from temporally-regulated seed genes, those of the napin, vicilin, and arcelin genes, led to similar human IDUA activities at seed maturity for the highest-expressing lines (0.6-1.1 units/mg total soluble protein; TSP). In constructs containing the arcelin (arc 5-I) gene promoter, exchange of the 5'-UTR and signal peptide sequences of the IDUA gene with those of the arcelin gene resulted in a considerable increase in IDUA enzyme activity level which was also reflected in an increase in the accumulation of protein and in steady-state mRNA levels. Replacement of the nopaline synthase gene 3' end with the arcelin gene 3' end also resulted in a significant increase in IDUA activity. However, the increase was highest in the presence of other gene regulatory sequences from the arcelin gene (5'-UTR and signal peptide), which appeared to act synergistically to enhance expression at the protein and activity level. Three lines expressing this 'optimized' construct exhibited extremely high IDUA protein levels and activities (e.g., 820 +/- 63 units/mg TSP), which may have been due to transcriptional and post-transcriptional factors. The human enzyme possessed moderate stability in seeds stored in the dry state following harvest. (c) 2006 Published by Elsevier Ireland Ltd. DOI
49. Feurtado, JA; Yang, J; Ambrose, SJ; Cutler, AJ; Abrams, SR; Kermode, AR. (2007) Disrupting abscisic acid homeostasis in western white pine (Pinus monticola Dougl. Ex D. Don) seeds induces dormancy termination and changes in abscisic acid catabolites.Journal of Plant Growth Regulation 26: 46-54 Disrupting abscisic acid homeostasis in western white pine (Pinus monticola Dougl. Ex D. Don) seeds induces dormancy termination and changes in abscisic acid catabolites
abscisic acid metabolism; fluridone; germination; seed dormancy; Pinus
To investigate the role of abscisic acid (ABA) biosynthesis and catabolism in dormant imbibed seeds of western white pine (Pinus monticola), ABA and selected catabolites were measured during a combined treatment of the ABA biosynthesis inhibitor fluridone, and gibberellic acid (GA). Fluridone in combination with GA effectively disrupted ABA homeostasis and replaced the approximately 90-day moist chilling period normally required to break dormancy in this species. Individually, both fluridone and GA treatments decreased ABA levels in the embryos and megagametophytes of white pine seeds compared to a water control; however, combined fluridone/GA treatment, the only treatment to terminate dormancy effectively, led to the greatest decline in ABA content. Fluridone treatments revealed that a high degree of ABA turnover/transport occurred in western white pine seeds during the initial stages of dormancy maintenance; at this time, ABA levels decreased by approximately two-thirds in both embryo and megagametophyte tissues. Gibberellic acid treatments, both alone and in combination with fluridone, suggested that GA acted transiently to disrupt ABA homeostasis by shifting the ratio between biosynthesis and catabolism to favor ABA catabolism or transport. Increases in phaseic acid (PA) and dihydrophaseic acid (DPA) were observed during fluridone/GA treatments; however, increases in ABA metabolites did not account for the reduction in ABA observed; additional catabolism and/or transport of ABA and selected metabolites in all probability accounts for this discrepancy. Finally, levels of 7' hydroxy-ABA (7'OH-ABA) were higher in dormant-imbibed seeds, suggesting that metabolism through this pathway is increased in seeds that maintain higher levels of ABA, perhaps as a means to further regulate ABA homeostasis. DOI
48.Kermode, AR; Zeng, Y; Hu, XK; Lauson, S; Abrams, SR; He, X. (2007) Ectopic expression of a conifer Abscisic Acid Insensitive3 transcription factor induces high-level synthesis of recombinant human alpha-L-iduronidase in transgenic tobacco leaves.Plant Molecular Biology 63: 763-776 Ectopic expression of a conifer Abscisic Acid Insensitive3 transcription factor induces high-level synthesis of recombinant human alpha-L-iduronidase in transgenic tobacco leaves
molecular pharming; recombinant human alpha-L-iduronidase; Abscisic Acid Insensitive3 gene; transactivation; transgenic tobacco
We are examining various plant-based systems to produce enzymes for the treatment of human lysosomal storage disorders. Constitutive expression of the gene encoding the human lysosomal enzyme, alpha-l-iduronidase (IDUA; EC 3.2.1.76) in leaves of transgenic tobacco plants resulted in low-enzyme activity, and the protein appeared to be subject to proteolysis. Toward enhancing production of this recombinant enzyme in vegetative tissues, transgenic tobacco plants were generated to co-express a CaMV35S:Chamaecyparis nootkatensis Abscisic Acid Insensitive3 (CnABI3) gene construct, along with the human gene construct. The latter contained regulatory sequences of the Phaseolus vulgaris arcelin 5-I gene (5'-flanking, signal-peptide-encoding, and 3'-flanking regions). Ectopic synthesis of the CnABI3 protein led to the transactivation of the arcelin promoter and accordingly high activity (e.g., 25,000 pmol/min/mg total soluble protein) and levels of recombinant IDUA mRNA and protein were induced in leaves of transgenic tobacco, particularly in the presence of 150-200 mu M S-(+)-ABA. Synthesis of human IDUA containing a carboxy-terminal ER retention (SEKDEL) sequence was also inducible by ABA in leaves co-transformed with the CnABI3 gene. As compared to the natural S-(+)-ABA, two persistent ABA analogues, (+)-8' acetylene ABA and (+)-8'methylene ABA, led to greater levels of beta-glucuronidase (GUS) reporter activities in leaves co-expressing the CnABI3 gene and a vicilin:GUS chimeric gene. In contrast, (+)-8' acetylene ABA and natural ABA appeared to be equally effective in stimulating the CnABI3-induced expression of an arcelin:GUS gene, and of the human IDUA gene, the latter also driven by arcelin-gene-regulatory sequences. Various stress-related treatments, particularly high concentrations of NaCl, had an even greater effect than ABA in promoting accumulation of human IDUA in co-transformed tobacco leaves. This strategy provides the means of enhancing the yields of recombinant proteins in transgenic plant vegetative tissues and potentially in cultured plant cells. The human recombinant protein can be readily induced in the presence of chemicals such as NaCl that can be added to cell cultures or even whole plants without a significant increase in production costs. DOI
47. Wan, LL; Ross, ARS; Yang, JY; Hegedus, DD; Kermode, AR. (2007) Phosphorylation of the 12 S globulin cruciferin in wild-type and abi1-1 mutant Arabidopsis thaliana (thale cress) seeds.Biochemical Journal 404: 247-256 Phosphorylation of the 12 S globulin cruciferin in wild-type and abi1-1 mutant Arabidopsis thaliana (thale cress) seeds
Arabidopsis thaliana (thale cress) abi1 mutant; cruciferin; mass spectrometry (MS); protein phosphorylation; seed storage proteins; two-dimensional electrophoresis
Cruciferin (a 12 S globulin) is the most abundant storage protein in the seeds of Arabidopsis thaliana (thale cress) and other crucifers, sharing structural similarity with the cupin superfamily of proteins. Cruciferin is synthesized as a precursor in the rough endoplasmic reticulum. Subunit assembly is accompanied by structural rearrangements involving proteolysis and disulfide-bond formation prior to deposition in protein storage vacuoles. The A. thaliana cv. Columbia genome contains four cruciferin loci, two of which, on the basis of cDNA analysis, give rise to three alternatively spliced variants. Using MS, we confirmed the presence of four variants encoded by genes At4g28520.1, At5g44120.3, At1g03880.1 and At1g3890.1 in A. thaliana seeds. Two-dimensional gel electrophoresis, along with immunological detection using anti-cruciferin antiserum and antibodies against phosphorylated amino acid residues, revealed that cruciferin was the major phosphorylated protein in Arabidopsis seeds and that polymorphism far exceeded that predicted on the basis of known isoforms. The latter may be attributed, at least in part, to phosphorylation site heterogeneity. A total of 20 phosphorylation sites, comprising nine serine, eight threonine and three tyrosine residues, were identified by MS. Most of these are located on the IE (interchain disulfide-containing) face of the globulin trimer, which is involved in hexamer formation. The implications of these findings for cruciferin processing, assembly and mobilization are discussed. In addition, the protein phosphatase 2C-impaired mutant, abil-1, was found to exhibit increased levels of cruciferin phosphorylation, suggesting either that cruciferin may be an in vivo target for this enzyme or that abil-1 regulates the protein kinase/phosphatase system required for cruciferin phosphorylation. DOI
46. Downing, WL; Galpin, JD; Clemens, S; Lauzon, SM; Samuels, AL; Pidkowich, MS; Clarke, LA; Kermode, AR. (2006) Synthesis of enzymatically active human alpha-L-iduronidase in Arabidopsis cgl (complex glycan-deficient) seeds.Plant Biotechnol J 4: 169-181 Synthesis of enzymatically active human alpha-L-iduronidase in Arabidopsis cgl (complex glycan-deficient) seeds
enzyme replacement therapy; human alpha-L-iduronidase; lysosomal storage diseases; molecular farming; N-linked glycosylation
As an initial step to develop plants as systems to produce enzymes for the treatment of lysosomal storage disorders, Arabidopsis thaliana wild-type (Col-0) plants were transformed with a construct to express human alpha-L-iduronidase (IDUA; EC 3.2.1.76) in seeds using the promoter and other regulatory sequences of the Phaseolus vulgaris arcelin 5-I gene. IDUA protein was easily detected on Western blots of extracts from the T-2 seeds, and extracts contained IDUA activity as high as 2.9 nmol 4-methylumbelliferone (4 MU)/min/mg total soluble protein (TSP), corresponding to approximately 0.06 mu g IDUA/mg TSP. The purified protein reacted with an antibody specific for xylose-containing plant complex glycans, indicating its transit through the Golgi complex. In an attempt to avoid maturation of the N-linked glycans of IDUA, the same IDUA transgene was introduced into the Arabidopsis cgl background, which is deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of complex glycan biosynthesis. IDUA activity and protein levels were significantly higher in transgenic cgl vs. wild-type seeds (e.g. maximum levels were 820 nmol 4 MU/min/mg TSP, or 18 mu g IDUA/mg TSP). Affinity-purified IDUA derived from cgl mutant seeds showed a markedly reduced reaction with the antibody specific for plant complex glycans, despite transit of the protein to the apoplast. Furthermore, gel mobility changes indicated that a greater proportion of its N-linked glycans were susceptible to digestion by Streptomyces endoglycosidase H, as compared to IDUA derived from seeds of wild-type Arabidopsis plants. The combined results indicate that IDUA produced in cgl mutant seeds contains glycans primarily in the high-mannose form. This work clearly supports the viability of using plants for the production of human therapeutics with high-mannose glycans.
45.Kermode, AR. (2006) Plants as factories for production of biopharmaceutical and bioindustrial proteins: lessons from cell biology.Can J Bot 84: 679-694 Plants as factories for production of biopharmaceutical and bioindustrial proteins: lessons from cell biology
human recombinant proteins; molecular pharming; transgenic plants; gene expression; protein stability; post-translational modification; N-glycosylation
Transgenic plants, seeds, and cultured plant cells are potentially one of the most economical systems for large-scale production of recombinant proteins for industrial and pharmaceutical uses. Biochemical, technical, and economic concerns with current production systems have generated enormous interest in developing plants as alternative production systems. However, various challenges must be met before plant systems can fully emerge as suitable, viable alternatives to Current animal-based systems for large-scale production of biopharmaceuticals and other products. Aside from regulatory issues and developing efficient methods for downstream processing of recombinant proteins, there are at least two areas of challenge: (1) Can we engineer plant cells to accumulate recombinant proteins to sufficient levels? (2) Can we engineer plant cells to post-translationally modify recombinant proteins so that they are structurally and functionally similar to the native proteins? Attempts to improve the accumulation of a recombinant protein in plant cells require an appreciation of the processes of gene transcription, mRNA stability, processing, and export, and translation initiation and efficiency. Likewise, many post-translational factors must be considered, including protein stability, protein function and activity, and protein targeting. Moreover, we need to understand how the various processes leading from the gene to the functional protein are interdependent and functionally linked. Manipulation of the post-translational processing machinery of plant cells, especially that for N-linked glycosylation and glycan processing, is a challenging and exciting area. The functions of N-glycan heterogeneity and microheterogeneity, especially with respect to protein function, stability, and transport, are poorly understood and this represents an important area of cell biology.
44. Chiwocha, SDS; Cutler, AJ; Abrams, SR; Ambrose, SJ; Yang, J; Ross, ARS; Kermode, AR. (2005) The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and germination.Plant Journal 42: 35-48 The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and germination
hormone metabolites; ethylene response mutant; auxins; cytokinins; abscisic acid; gibberellins; dormancy; germination
In Arabidopsis thaliana, the etr1-2 mutation confers dominant ethylene insensitivity and results in a greater proportion of mature seeds that exhibit dormancy compared with mature seeds of the wild-type. We investigated the impact of the etr1-2 mutation on other plant hormones by analyzing the profiles of four classes of plant hormones and their metabolites by HPLC-ESI/MS/MS in mature seeds of wild-type and etr1-2 plants. Hormone metabolites were analyzed in seeds imbibed immediately under germination conditions, in seeds subjected to a 7-day moist-chilling (stratification) period, and during germination/early post-germinative growth. Higher than wild-type levels of abscisic acid (ABA) appeared to contribute, at least in part, to the greater incidence of dormancy in mature seeds of etr1-2. The lower levels of abscisic acid glucose ester (ABA-GE) in etr1-2 seeds compared with wild-type seeds under germination conditions (with and without moist-chilling treatments) suggest that reduced metabolism of ABA to ABA-GE likely contributed to the accumulation of ABA during germination in the mutant. The mutant seeds exhibited generally higher auxin levels and a large build-up of indole-3-aspartate when placed in germination conditions following moist-chilling. The mutant manifested increased levels of cytokinin glucosides through zeatin-O-glucosylation (Z-O-Glu). The resulting increase in Z-O-Glu was the largest and most consistent change associated with the ETR1 gene mutation. There were more gibberellins (GA) and at higher concentrations in the mutant than in wild-type. Our results suggest that ethylene signaling modulates the metabolism of all the other plant hormone pathways in seeds. Additionally, the hormone profiles of etr1-2 seed during germination suggest a requirement for higher than wild-type levels of GA to promote germination in the absence of a functional ethylene signaling pathway.
43.Kermode, AR. (2005) Role of abscisic acid in seed dormancy.Journal of Plant Growth Regulation 24: 319-344 Role of abscisic acid in seed dormancy
seed development; dormancy; germination; seedling vigor; abscisic acid; ABA biosynthesis/catabolism; hormonal cross-talk; signal transduction
Seed dormancy is an adaptive trait that improves survival of the next generation by optimizing the distribution of germination over time. The agricultural and forest industries rely on seeds that exhibit high rates of germination and vigorous, synchronous growth after germination; hence dormancy is sometimes considered an undesirable trait. The forest industry encounters problems with the pronounced dormancy of some conifer seeds, a feature that can lead to non-uniform germination and poor seedling vigor. In cereal crops, an optimum balance is most sought after; some dormancy at harvest is favored because it prevents germination of the physiologically mature grain in the head prior to harvest (that is, preharvest sprouting), a phenomenon that leads to considerable damage to grain quality and is especially prominent in cool moist environments. The sesquiterpene abscisic acid (ABA) regulates key events during seed formation, such as the deposition of storage reserves, prevention of precocious germination, acquisition of desiccation tolerance, and induction of primary dormancy. Its regulatory role is achieved in part by cross-talk with other hormones and their associated signaling networks, via mechanisms that are largely unknown. Quantitative genetics and functional genomics approaches will contribute to the elucidation of genes and proteins that control seed dormancy and germination, including components of the ABA signal transduction pathway. Dynamic changes in ABA biosynthesis and catabolism elicit hormone-signaling changes that affect downstream gene expression and thereby regulate critical checkpoints at the transitions from dormancy to germination and from germination to growth. Some of the recent developments in these areas are discussed.
42. Terskikh, VV; Feurtado, JA; Borchardt, S; Giblin, M; Abrams, SR; Kermode, AR. (2005) In vivo C-13 NMR metabolite profiling: potential for understanding and assessing conifer seed quality.Journal of Experimental Botany 56: 2253-2265 In vivo C-13 NMR metabolite profiling: potential for understanding and assessing conifer seed quality
amino acids; conifer seeds; germination; imbibition; in vivo C-13 NMR spectroscopy; metabolite profiling; monoterpenes; storage lipids; sucrose
High-resolution C-13 MAS NMR spectroscopy was used to profile a range of primary and secondary metabolites in vivo in intact whole seeds of eight different conifer species native to North America, including six of the Pinaceae family and two of the Cupressaceae family. In vivo C-13 NMR provided information on the total seed oil content and fatty acid composition of the major storage lipids in a non-destructive manner. In addition, a number of monoterpenes were identified in the C-13 NMR spectra of conifer seeds containing oleoresin; these compounds showed marked variability in individual seeds of Pacific silver fir within the same seed lot. In imbibed conifer seeds, the C-13 NMR spectra showed the presence of considerable amounts of dissolved sucrose presumed to play a protective role in the desiccation-tolerance of seeds. The free amino acids arginine and asparagine, generated as a result of storage protein mobilization, were detected in vivo during seed germination and early seedling growth. The potential for NMR to profile metabolites in a non-destructive manner in single conifer seeds and seed populations is discussed. It is a powerful tool to evaluate seed quality because of its ability to assess reserve accumulation during seed development or at seed maturity; it can also be used to monitor reserve mobilization, which is critical for seedling emergence.
41. Terskikh, VV; Feurtado, JA; Ren, CW; Abrams, SR; Kermode, AR. (2005) Water uptake and oil distribution during imbibition of seeds of western white pine (Pinus monticola Dougl. ex D. Don) monitored in vivo using magnetic resonance imaging.Planta 221: 17-27 Water uptake and oil distribution during imbibition of seeds of western white pine (Pinus monticola Dougl. ex D. Don) monitored in vivo using magnetic resonance imaging
imbibition; magnetic resonance imaging; H-1 NMR; Pinus monticola; seed
Dry or fully imbibed seeds of western white pine (Pinus monticola Dougl. ex D. Don) were studied using high-resolution magnetic resonance imaging (MRI). Analyses of the dry seed revealed many of the gross anatomical features of seed structure. Furthermore, the non-invasive nature of MRI allowed for a study of the dynamics of water and oil distribution during in situ imbibition of a single seed with time-lapse chemical shift selective MRI. During soaking of the dry seed, water penetrated through the seed coat and megagametophyte. The cotyledons of the embryo (located in the chalazal end of the seed) were the first to show hydration followed by the hypocotyl and later the radicle. After penetrating the seed coat, water in the micropylar end of the seed likely also contributed to further hydration of the embryo; however, the micropyle itself did not appear to be a site for water entry into the seed. A model that describes the kinetics of the earlier stages of imbibition is proposed. Non-viable pine seeds captured with MRI displayed atypical imbibition kinetics and were distinguished by their rapid and uncontrolled water uptake. The potential of MR microimaging for detailed studies of water uptake and distribution during the soaking, moist chilling ("stratification"), and germination of conifer seeds is discussed.
40. Feurtado, JA; Ambrose, SJ; Cutler, AJ; Ross, ARS; Abrams, SR; Kermode, AR. (2004) Dormancy termination of western white pine (Pinus monticola Dougl. Ex D. Don) seeds is associated with changes in abscisic acid metabolism.Planta 218: 630-639 Dormancy termination of western white pine (Pinus monticola Dougl. Ex D. Don) seeds is associated with changes in abscisic acid metabolism
abscisic acid metabolism; germination; seed dormancy; Pinus
Western white pine (Pinus monticola) seeds exhibit deep dormancy at maturity and seed populations require several months of moist chilling to reach their uppermost germination capacities. Abscisic acid (ABA) and its metabolites, phaseic acid (PA), dihydrophaseic acid (DPA), 7'-hydroxy ABA (7'OH ABA) and ABA-glucose ester (ABA-GE), were quantified in western white pine seeds during dormancy breakage (moist chilling) and germination using an HPLC-tandem mass spectrometry method with multiple reaction monitoring and internal standards incorporating deuterium-labeled analogs. In the seed coat, ABA and metabolite levels were high in dry seeds, but declined precipitously during the pre-moist-chilling water soak to relatively low levels thereafter. In the embryo and megagametophyte, ABA levels decreased significantly during moist chilling, coincident with an increase in the germination capacity of seeds. ABA catabolism occurred via several routes, depending on the stage and the seed tissue. Moist chilling of seeds led to increases in PA and DPA levels in both the embryo and megagametophyte. Within the embryo, 7'OH ABA and ABA-GE also accumulated during moist chilling; however, 7'OH ABA peaked early in germination. Changes in ABA flux, i.e. shifts in the ratio between biosynthesis and catabolism, occurred at three distinct stages during the transition from dormant seed to seedling. During moist chilling, the relative rate of ABA catabolism exceeded ABA biosynthesis. This trend became even more pronounced during germination, and germination was also accompanied by a decrease in the ABA catabolites DPA and PA, presumably as a result of their further metabolism and/or leaching/transport. The transition from germination to post-germinative growth was accompanied by a shift toward ABA biosynthesis. Dormant imbibed seeds, kept in warm moist conditions for 30 days (after an initial 13 days of soaking), maintained high ABA levels, while the amounts of PA, 7'OH ABA, and DPA decreased or remained at steady-state levels. Thus, in the absence of conditions required to break dormancy there were no net changes in ABA biosynthesis and catabolism.
39. Gulden, RH; Chiwocha, S; Abrams, S; McGregor, I; Kermode, A; Shirtliffe, S. (2004) Response to abscisic acid application and hormone profiles in spring Brassica napus seed in relation to secondary dormancy.Canadian Journal of Botany-Revue Canadienne de Botanique 82: 1618-1624 Response to abscisic acid application and hormone profiles in spring Brassica napus seed in relation to secondary dormancy
abscisic acid (ABA); Brassica napus; fluridone; induced dormancy; osmotic stress; sensitivity
The plant hormone abscisic acid (ABA) has been implicated in the inception and maintenance of seed dormancy, while gibberellins promote dormancy breakage and germination in some species. We investigated whether osmotic stress induced secondary dormancy in Brassica napus L. is associated with changes in ABA sensitivity and metabolism, as well as changes in gibberellin levels. Seeds of two genotypes, one with low dormancy potential (LDP) and one with high dormancy potential (HDP) for secondary dormancy, were exposed to a dormancy-inducing osmotic treatment for up to 4 weeks and then germinated in the presence of increasing ABA concentrations. Even at relatively high concentrations of supplied ABA, germination of LDP seed was not inhibited, while relatively low ABA concentrations inhibited the germination of HDP seed after osmotic treatment. Fluridone was highly effective in suppressing secondary dormancy development in HDP seed, but had no effect on germinability in LDP seed. Despite the lack of differences in nonosmotically treated seed, ABA and ABA-glucose ester accumulated to higher levels, and gibberellin A, accumulated to lower levels, in HDP relative to LDP seed by the end of the osmotic treatment. Our findings indicate an association among ABA sensitivity, biosynthesis and accumulation, and secondary dormancy potential in B. napus seed.
38. Raimondi, N; Kermode, AR. (2004) Seedling growth and establishment in natural stands of yellow-cedar (Chamaecyparis nootkatensis) seedlings derived from the use of modified seed dormancy-breaking treatments.New Forests 27: 55-67 Seedling growth and establishment in natural stands of yellow-cedar (Chamaecyparis nootkatensis) seedlings derived from the use of modified seed dormancy-breaking treatments
Chamaecyparis nootkatensis; emergence; natural stands; seed dormancy; seedling growth; yellow-cedar
Following dispersal from the parent tree, seeds of yellow-cedar ( Chamaecyparis nootkatensis [ D. Don] Spach) exhibit low germination, primarily as a result of coat-imposed dormancy. Dormancy of the mature ( intact) seed is effectively terminated by traditional warm/cold treatments. A chemical treatment using the anaesthetic 1-propanol combined with a three day warm water soak ( 30 degreesC), a two day GA(3) treatment and 60 d of moist chilling not only promotes high germinability of yellow-cedar seeds, but also elicits vigorous post-germinative growth following seedling emergence under nursery greenhouse conditions. Here we compare the effectiveness of the more traditional warm/cold treatments with the chemical treatment in terms of their capacity to elicit vigorous growth and establishment in natural stands following transplant of seedlings from a nursery greenhouse environment. Two seed lots (42313 and 43697) and open-pollinated seed from parent trees 13-6 and 19-8 showed equivalent seedling growth in natural stands following the chemical treatment and two traditional warm/cold treatments typically used for dormancy breakage by the forest industry and by the Ministry of Forests in British Columbia. The chemical protocol offers the advantage of reducing the time required to break seed dormancy. We have now demonstrated that it yields seedlings that exhibit vigorous growth and are capable of withstanding the vagaries of the environment.
37. Ross, ARS; Ambrose, SJ; Cutler, AJ; Feurtado, JA; Kermode, AR; Nelson, K; Zhou, R; Abrams, SR. (2004) Determination of endogenous and supplied deuterated abscisic acid in plaint tissues by high-performance liquid chromatography-electro-spray ionization tandem mass spectrometry with multiple reaction monitoring.Analytical Biochemistry 329: 324-333 Determination of endogenous and supplied deuterated abscisic acid in plaint tissues by high-performance liquid chromatography-electro-spray ionization tandem mass spectrometry with multiple reaction monitoring
abscisic acids; deuterated analogs; electrospray ionization-tandem mass spectrometry; Arabidopsis thaliana; western white pine; Brassica naplis
A specific, sensitive, and accurate method for determination of abscisic acid (ABA) in plant tissues is described. The method employs reversed-phase high-performance liquid chromatography and electrospray ionization-tandem mass spectrometry for multiple reaction monitoring of underivatized ABA and deuterated ABA analogs. Specific analogs were used to study the mechanism of ABA fragmentation, to select appropriate standards, and to identify compounds suitable for metabolic studies involving the supply of differentially labeled ABA. Limits of detection and quantification of 1.9 and 4.7 pg, respectively, were obtained over a linear calibration range of 0-1.5 ng ABA (on-column injected) using 5,8', 8', 8'-d(4) ABA as the internal standard. Accuracy and precision were within 15% for routine quality control samples. The method of standard additions, as applied to Arabidopsis thaliana seed extracts, was also used to validate the method for analysis of plant tissue samples. The utility of the method was further demonstrated by determining levels of ABA in western white pine seeds and of ABA and supplied 8', 8', 8', 9' 9' 9'-d(6) ABA in Brassica napus tissues, using 5,8 8 8 -d(4) ABA or 8 8 8 -d(3) ABA as the internal standard. Limits of quantification as low as 0.89 ng/g were achieved by optimizing the extraction procedure for each type of plant tissue. Crown Copyright (C) 2004 Published by Elsevier Inc. All rights reserved.
36. Zeng, Y; Kermode, A. (2004) A gymnosperm ABI3 gene functions in a severe abscisic acid-insensitive mutant of Arabidopsis (abi3-6) to restore the wild-type phenotype and demonstrates a strong synergistic effect with sugar in the inhibition of post-germinative growth.Plant Molecular Biology 56: 731-746 A gymnosperm ABI3 gene functions in a severe abscisic acid-insensitive mutant of Arabidopsis (abi3-6) to restore the wild-type phenotype and demonstrates a strong synergistic effect with sugar in the inhibition of post-germinative growth
Abscisic Acid Insensitive3; Arabidopsis; Chamaecyparis nootkatensis; functional complementation; post-germinative growth; sugar hypersensitivity
The CnABI3 gene of yellow-cedar is an orthologue of the ABI3/VP1 gene of angiosperms; it shares many common characteristics with other ABI3/VP1 genes, yet has unique characteristics as well. We examined whether this gymnosperm transcription factor can functionally complement an angiosperm species with a defective ABI3 gene. A severe Arabidopsis abi3 null mutant abi3-6 was stably transformed with the CnABI3 gene coding-region driven by a modified CaMV 35S promoter. Several of the visible mutant phenotypes ( e. g., production of green seeds due to a lack of chlorophyll breakdown) were fully restored to those of the wild-type and the transformed seeds acquired desiccation tolerance. The functional complementation of the mutant also extended to the accumulation of several seed proteins ( including seed-storage-proteins, alpha-tonoplast intrinsic protein, dehydrin-related polypeptides and oleosin), which were restored to wild-type levels. However, not all phenotypes were fully restored; sensitivities of transgenic seeds to exogenous ABA ( as far as germination is concerned) were lower than that of the wild-type seeds, and flowering times were intermediate of those characteristic of wild-type and abi3-6 plants. A novel function for CnABI3, potentially related to a direct or indirect role in ER homeostasis was revealed. Two proteins with a molecular chaperone function in the ER (BiP and protein disulphide isomerase) were elevated in mutant seeds ( indicative of ER stress); expression of the CnABI3 gene decreased the accumulation of these proteins to levels characteristic of the wild-type. These studies reveal the degree of conservation of ABI3 functions between gymnosperms and angiosperms as well as some novel functions of ABI3-related genes.
35. Chiwocha, SDS; Abrams, SR; Ambrose, SJ; Cutler, AJ; Loewen, M; Ross, ARS; Kermode, AR. (2003) A method for profiling classes of plant hormones and their metabolites using liquid chromatography-electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds.Plant Journal 35: 405-417 A method for profiling classes of plant hormones and their metabolites using liquid chromatography-electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds
high-performance liquid chromatography (HPLC); electrospray ionization-tandem mass spectrometry (ESI-MS/MS); abscisic acid; gibberellins; cytokinins; auxins
A highly selective and sensitive method for the simultaneous analysis of several plant hormones and their metabolites is described. The method combines high-performance liquid chromatography (HPLC) with positive and negative electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to quantify a broad range of chemically and structurally diverse compounds. The addition of deuterium-labeled analogs for these compounds prior to sample extraction permits accurate quantification by multiple reaction monitoring (MRM). Endogenous levels of abscisic acid (ABA), abscisic acid glucose ester (ABA-GE), 7'-hydroxy-abscisic acid (7'-OH-ABA), phaseic acid (PA), dihydrophaseic acid (DPA), indole-3-acetic acid (IAA), indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine (2iP), isopentenyladenosine (IPA), and gibberellins (GA)(1), GA(3), GA(4), and GA(7) were determined simultaneously in a single run. Detection limits ranged from 0.682 fmol for Z to 1.53 pmol for ABA. The method was applied to the analysis of plant hormones and hormonal metabolites associated with seed dormancy and germination in lettuce (Lactuca sativa L. cv. Grand Rapids), using extracts from only 50 to 100 mg DW of seed. Thermodormancy was induced by incubating seeds at 33degreesC instead of 23degreesC. Germinating seeds transiently accumulated high levels of ABA-GE. In contrast, thermodormant seeds transiently accumulated high levels of DPA after 7 days at 33degreesC. GA(1) and GA(3) were detected during germination, and levels of GA(1) increased during early post-germinative growth. After several days of incubation, thermodormant seeds exhibited a striking transient accumulation of IAA, which did not occur in seeds germinating at 23degreesC. We conclude that hormone metabolism in thermodormant seeds is surprisingly active and is significantly different from that of germinating seeds.
34. Feurtado, JA; Xia, JH; Ma, Y; Kermode, AR. (2003) Increasing the temperature of the water soak preceding moist-chilling promotes dormancy-termination of seeds of western white pine (Pinus monticola Dougl.).Seed Science and Technology 31: 275-288 Increasing the temperature of the water soak preceding moist-chilling promotes dormancy-termination of seeds of western white pine (Pinus monticola Dougl.)
Seeds of western white pine (Pinus monticola Dougl.) exhibit a very pronounced and deep primary dormancy at maturity which often leads to erratic germination after the implementation of dormancy-breaking treatments. Toward increasing the efficiency of dormancy-breakage and improving the rate and synchronicity of germination, the effectiveness of various modified pre-chilling and chilling treatments were determined using five seedlots of western white pine. Treatments that combined chemicals (hormones or anaesthetics such as gibberellic acid and 1-propanol) with moist chilling were not effective in accelerating dormancy termination. Control of seed moisture content during chilling through the use of solid matrix priming was not effective in eliciting high germinability and proved more deleterious especially for longer chilling periods. The most effective treatment to terminate dormancy was an increased-temperature water soak prior to moist chilling. At an optimum temperature of 27degreesC, a 12-day water soak could effectively shorten the requirement for moist chilling by approximately 20-30 days (from 96 to 75 or 60 days).
33. He, X; Kermode, AR. (2003) Proteases associated with programmed cell death of megagametophyte cells after germination of white spruce (Picea glauca) seeds.Plant Molecular Biology 52: 729-744 Proteases associated with programmed cell death of megagametophyte cells after germination of white spruce (Picea glauca) seeds
caspase-like proteases; cysteine endoproteases; megagametophyte; Picea glauca; programmed cell death
During post-germinative seedling growth, the major storage organ of the white spruce (Picea glauca) seed, the megagametophyte, undergoes programmed cell death (PCD). Protease activities in megagametophyte cells that arise post-germinatively were investigated. The accumulation of protease activities can be divided into two phases: the first phase correlated with degradation of storage proteins while the second phase was temporally associated with cell death, although some of the early proteases were also active during the later phase. Proteases induced during PCD were mainly serine and cysteine proteases. One of the PCD-associated cysteine proteases had homology to Cys-EP, a PCD-related cysteine protease of the castor bean endosperm. Transcripts encoding a Cys-EP-related protein were not present in megagametophytes when seeds were imbibed, nor were they present during germination and early post-germinative growth (radicle length ca. 2-5 mm). At a later post-germinative stage (i.e when the seed's radicle was ca. 15 mm), the Cys-EP-related transcripts (ca. 1.3 kb) became abundant and, at this time, the 48 kDa proform of the enzyme first appeared. The mature form of the Cys-EP (ca. 38 kDa) was predominant at a very late stage of post-germinative growth. Immunocytochemistry showed that the Cys-EP-related protein was localized to spherical organelles (ca. 2 mum) that may be equivalent to the 'ricinosomes' of castor bean endosperm cells. Caspase-like protease (CLP) activities were first detected 3 days after germination with the caspase-specific substrate Ac-DEVD-AMC; maximum activities occurred when the seed's radicle was ca. 20-25 mm. When germinated seeds were treated with a caspase-3 inhibitor, both the peak of CLP activities and the death of megagametophyte cells were delayed. We propose that the Cys-EP-related protein and CLP activity are involved in PCD of white spruce megagametophyte cells.
32. He, X; Kermode, AR. (2003) Nuclease activities and DNA fragmentation during programmed cell death of megagametophyte cells of white spruce (Picea glauca) seeds.Plant Molecular Biology 51: 509-521 Nuclease activities and DNA fragmentation during programmed cell death of megagametophyte cells of white spruce (Picea glauca) seeds
DNA fragmentation; megagametophyte; nucleases; Picea glauca; programmed cell death
The haploid megagametophyte of white spruce (Picea glauca) seeds undergoes programmed cell death (PCD) during post-germinative seedling growth. Death of the megagametophyte storage parenchyma cells was preceded by reserve mobilization and vacuolation. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling)-positive nuclei indicated that the first megagametophyte cells to die were those closest to the radicle at the micropylar end of the seed as well as those that comprised the most peripheral and innermost layers at the chalazal end of the seed. The death process was accompanied by nuclear fragmentation and internucleosomal DNA cleavage and the sequential activation of several nucleases. The latter comprised at least two groups: those induced relatively early during post-germinative seedling growth, that had pH optima in the neutral range (33, 31, 17 and 15 kDa), and those induced later that had pH optima in the acidic range (73, 62, 48, 43 and 29 kDa). Activities of all of the nucleases were stimulated by Ca2+, Mg2+ and Mn2+; only the nucleases active at neutral pH were inhibited by Zn2+. The temporal pattern of induction of the neutral and acidic nucleases may suggest that the latter function after tonoplast rupture.
31. Lait, CG; Miller, DR; Bates, SL; Borden, JH; Kermode, AR. (2003) Biochemical assay detects feeding damage to loblolly pine seeds caused by the leaffooted pine seed bug (Hemiptera : Coreidae).Journal of Entomological Science 38: 644-653 Biochemical assay detects feeding damage to loblolly pine seeds caused by the leaffooted pine seed bug (Hemiptera : Coreidae)
seed bug; Pinus taeda; antibody; immunodetection; damage
A large number of proteins in salivary gland extracts of the leaffooted pine seed bug, Leptoglossus corculus Say, were strongly recognized by a polyclonal antibody-based assay developed for detecting saliva of the western conifer seed bug. Leptoglossus occidentalis Heide-mann, in lodgepole pine, Pinus contorta var. latifolia Engelmann, seeds. An average of approximately 85% of loblolly pine, Pinus taeda L., seeds exposed to feeding by L. corculus for 1 to 4 weeks in the laboratory contained detectable amounts of salivary proteins when the antibody assays were performed weekly on samples (n = 10) of seed. In comparison, radiography of exposed seed detected an average of approximately 63% damaged seed over the same 4-wk period, indicating that the antibody assay increased sensitivity of damage detection by approximately one-third. Depletion of insoluble polypeptides and proliferation of soluble polypeptides less than or equal to23.5 kDa was apparent after SDS-PAGE and quantitative assays were performed on proteins extracted from seeds that were damaged by exposure to L. corculus feeding. Our data suggest that the antibody-based test could be used to obtain accurate estimates of seed losses attributable to L. corculus feeding in southern pine seed orchards.
30. Ma, YL; Feurtado, JA; Kermode, AR. (2003) Effect of solid matrix priming during moist chilling on dormancy breakage and germination of seeds of four fir species.New Forests 25: 49-66 Effect of solid matrix priming during moist chilling on dormancy breakage and germination of seeds of four fir species
germination capacity/rate; post-germinative growth; solid matrices; true firs
The ability of solid matrix priming (SMP) to overcome seed dormancy of seeds of four fir species (Pacific silver fir, subalpine fir, grand fir and noble fir), was examined. Seeds were subjected to treatments that combined moist chilling and SMP (SMP-chilling) and their germination compared to that elicited by traditional moist chilling. For the SMP-chilling, six solid matrices with different water holding capacities were tested (sphagnum, cat litter, peat moss, Agro-Lig Greens Grade, Agro-Lig Micro Fine and sand). For each of the species tested, germination performance was significantly improved by SMP-chilling; the results were dependent on the type of solid matrix, the matrix water content and the treatment duration. SMP-chilling, using the best solid matrices for each of the species tested, resulted in germination percentages and rates that were 24-35% and 8-21% higher, respectively, as compared with that yielded from control moist-chilling. High germination percentages (79% or greater) of three of the four species (Pacific silver fir, subalpine fir, and grand fir) were promoted after just four weeks (grand fir in sand) or eight weeks of treatment (Pacific silver fir in peat moss and subalpine fir in Agro-Lig Greens Grade). The germination rate of grand fir seeds benefited from SMP-chilling periods longer than four weeks, but this resulted in germination during the dormancy-breaking treatment. The water contents of the solid matrices that were optimal in terms of eliciting the best germination performance were 320% (w/w) for peat moss, 40% for Agro-Lig Greens Grade and 5-15% for sand. Conducting SMP at chilling temperatures (4 degreesC) is a practical approach to shorten the time required for effective dormancy termination of Abies seeds; treatment times were shortened by 27 days for Pacific silver fir and subalpine fir and more vigorous seedling growth was exhibited by grand fir and noble fir.
29. Palma, K; Kermode, AR. (2003) Metabolism of hydrogen peroxide during reserve mobilization and programmed cell death of barley (Hordeum vulgare L.) aleurone layer cells.Free Radical Biology and Medicine 35: 1261-1270 Metabolism of hydrogen peroxide during reserve mobilization and programmed cell death of barley (Hordeum vulgare L.) aleurone layer cells
programmed cell death; reactive oxygen species; catalase; glyoxysome; aleurone layer cells; abscisic acid; gibberellic acid; barley; free radicals
During germination, aleurone layer cells of barley (Hordeum vulgare) grains synthesize and secrete hydrolytic enzymes (principally a-amylase) in response to gibberellic acid (GA); shortly thereafter, the aleurone layer cells undergo programmed death. Gluconeogenesis of lipid reserves within aleurone cells, which supports this hydrolytic enzyme synthesis, results in the generation of H2O2, which is catabolized by glyoxysomal catalase. Lowered amounts of catalase may contribute to aleurone cell death because of a compromised capacity to cope with reactive oxygen species generated by glyoxysomes and mitochondria. In the presence of GA, cells of intact aleurone layers underwent programmed death between 18 and 48 h; in the presence of ABA, no cell death was evident over 60 h. The capacity of GA-treated layers to metabolize exogenous H2O2 increased steadily over the first 24 h, during the stage of lipid mobilization and the major synthesis and secretion of a-amylase; thereafter, this capacity declined markedly. In contrast, cells of ABA-treated aleurone layers exhibited little change in their capacity for H2O2-metabolism. Glyoxysomal catalase increased in activity over the first 12-24 h of GA treatment, which was accompanied by an increase in catalase-1 transcripts between 12 and 18 h. Catalase protein and activity declined after 24 h in GA-treated layers, prior to the onset of rapid programmed death at 30 h. These data suggest that a decline in glyoxysomal catalase precedes death of aleurone cells and may indeed contribute to an increase in cellular oxidative stress. (C) 2003 Elsevier Inc.
28. Zeng, Y; Raimondi, N; Kermode, AR. (2003) Role of an ABI3 homologue in dormancy maintenance of yellow-cedar seeds and in the activation of storage protein and Em gene promoters.Plant Molecular Biology 51: 39-49 Role of an ABI3 homologue in dormancy maintenance of yellow-cedar seeds and in the activation of storage protein and Em gene promoters
abscisic acid insensitive 3 homologue; Chamaecyparis nootkatensis; Em gene; germination; seed dormancy; storage; protein genes; trans-activation
ABI3/VP1 proteins are members of a large group of transcription factors that act as intermediaries in regulating abscisic acid (ABA)-responsive genes during seed development, including those involved in reserve deposition, acquisition of desiccation tolerance and dormancy induction. CnABI3, an ABI3/VP1 gene homologue was recently cloned from yellow cedar, a conifer species that produces seeds that are deeply dormant at maturity. Here, we investigated whether the conifer ABI3/VP1 gene homologue shares characteristics with its angiosperm counterparts. CnABI3 was synthesized exclusively in seeds, with no detectable protein in leaves and roots. Stable expression of the CnABI3 gene in two transgenic tobacco lines previously transformed with chimeric constructs (vicilin and napin 5 regions linked to a beta-glucuronidase (GUS) gene-coding region) showed that the ectopic expression of the CnABI3 protein strongly activated both the vicilin and napin storage protein gene promoters in leaves and other vegetative tissues. GUS activities were up to more than 1000-fold of those in control plants. ABA had a synergistic effect, further enhancing GUS activity levels. When expressed transiently in yellow-cedar embryos, CnABI3 activated the expression of a chimeric Em-GUS gene in the presence of ABA. The role of CnABI3 in dormancy maintenance of yellow-cedar seeds was examined by monitoring the expression of the CnABI3 gene at the mRNA and protein levels before, during and after dormancy termination. CnABI3 protein was present in the megagametophyte and embryo of dormant mature and warm stratified seed, but declined during subsequent moist chilling, a treatment effective in breaking dormancy. In contrast, the protein was preserved (albeit in lower amounts) in seeds subjected to a control treatment (12 weeks in warm, moist conditions) that is ineffective in breaking dormancy. A decline in CnABI3 gene transcripts was also positively correlated with dormancy breakage, but did not occur during moist chilling itself, but rather during subsequent germination, indicating potential control at the post-transcriptional level.
27. Bates, SL; Lait, CG; Borden, JH; Kermode, AR. (2002) Measuring the impact of Leptoglossus occidentalis (Heteroptera : Coreidae) on seed production in lodgepole pine using an antibody-based assay.Journal of Economic Entomology 95: 770-777 Measuring the impact of Leptoglossus occidentalis (Heteroptera : Coreidae) on seed production in lodgepole pine using an antibody-based assay
Coreidae; Leptoglossus occidentalis; Pinus contorta variety latifolia; seed orchard; radiography; antibody
We measured the impact of Leptoglossus occidentalis on seed production in lodgepole pine, Pinus contorta variety latifolia Engelmann, using an antibody marker de eloped to detect residual saliva in fed-on seeds. Nymphs, adult females, and adult males Were caged on cones during early, mid-and late season cone development. Individual analysis of 12,887 seeds extracted from 365 cones revealed that 37.3% seeds tested positive for seed bug saliva. The antibody assay was 38 times more effective than radiography at detecting seed bug damage. Radiography can detect partially emptied seed but cannot discriminate between aborted seeds and those emptied by seed bugs. The antibody marker was least sensitive in detecting early season damage compared with mid-and late season damage. We hypothesize that residual saliva in seeds fed on early in the season was either absorbed by the damaged seed or degraded over time. Earls season feeding resulted in the greatest number of seeds fused to cone scales and the extraction efficiency for cones exposed to feeding during this time was reduced by 64% compared With control cones. Adding fused seeds to antibudy-positive seeds raised the proportion of damaged seeds to 48.3%. At all stages of cone development, adult females Were the most destructive life stage, damaging up to two seeds per clay late in the season, When seed losses Were adjusted to damage per degree-day. female damage was greatest early in the season, while males caused the same amount of damage regardless of cone development period. The results of the antibody assay provide baseline data for developing damage prediction formulae, and establish L. occidentalis as a potentially serious pest in lodgepole pine seed orchards.
26. Crowe, AU; Plant, AL; Kermode, AR. (2002) Effects of an industrial effluent on plant colonization and on the germination and post-germinative growth of seeds of terrestrial and aquatic plant species.Environmental Pollution 117: 179-189 Effects of an industrial effluent on plant colonization and on the germination and post-germinative growth of seeds of terrestrial and aquatic plant species
germination; seedling growth; oil sands effluent; osmotic potential; wetlands
Major oil sands industrial companies are located in the Athabasca Oil Sands Deposit in northeastern Alberta, Canada. During the process used to extract light crude oil (via hot water digestion and flotation), gypsum is usually added to produce consolidated tails (CT) and CT release water. The vast volumes of process-treated waters (effluent) are held within large dyked tailings ponds. Toward testing viable options for reclamation, various hummock-wetlands systems have been constructed; in addition, natural wetlands (inhabited by obligate wetland plant species) have become established as a result of seeping of the effluents held within the large dyked ponds. Vegetation surveys conducted on and around the industrial site revealed that the constructed wetlands associated with the dyke drainage (effluent treated with phosphorous) and consolidated tails (CT; effluent treated with gypsum) had low biodiversity and were not invaded by many aquatic plants. Although the natural wetland was also not invaded by many aquatic species, it was found to be as diverse as the reference wetlands (i.e. off-site wetlands not exposed to the effluents). Exposure to oil sands effluents had an inhibitory effect on the germination (percent and/or rate) of several plant species (tomato, clover, wheat, rye, pea, reed canary grass, loblolly pine) clover and tomato seed germination were most affected. Two treatments in particular (effluents from the natural on-site wetland and the CT constructed wetland), delayed germination, and also led to reduced fresh weight of seedlings of tomato, wheat, clover and loblolly pine. The osmolarities of the effluents associated with the natural on-site wetland and CT constructed wetland were 712 and 728 mOs/kg, respectively; substituting these effluents with solutions of polyethylene glycol of the same osmotic potentials had a greater inhibitory effect on germination rate. The negative effects of the effluents on seed germination may account for the paucity of aquatic species that invaded the oil sands impacted wetlands. This factor will also be critical in determining the long-term feasibility of hummock-wetland systems. (C) 2002 Elsevier Science Ltd. All rights reserved.
25. Lazarova, G; Zeng, Y; Kermode, AR. (2002) Cloning and expression of an ABSCISIC ACID-INSENSITIVE 3 (ABI3) gene homologue of yellow-cedar (Chamaecyparis nootkatensis).Journal of Experimental Botany 53: 1219-1221 Cloning and expression of an ABSCISIC ACID-INSENSITIVE 3 (ABI3) gene homologue of yellow-cedar (Chamaecyparis nootkatensis)
abscisic acid; ABI3; seed dormancy; yellow-cedar
A homologue of the ABI3 gene was isolated from the conifer species, Chamaecyparis nootkatensis. The deduced protein of 794 amino acids exhibited sequence similarity to other VP1/ABI3 proteins within four regions. Expression occurs exclusively in seeds, with no detectable mRNA in leaves and roots. Unlike the homologues of angiosperms, CnABI3 may be encoded by more than one gene.
24. Schmitz, N; Abrams, SR; Kermode, AR. (2002) Changes in ABA turnover and sensitivity that accompany dormancy termination of yellow-cedar (Chamaecyparis nootkatensis) seeds.Journal of Experimental Botany 53: 89-101 Changes in ABA turnover and sensitivity that accompany dormancy termination of yellow-cedar (Chamaecyparis nootkatensis) seeds
coat-imposed dormancy; embryo; abscisic acid metabolism; seed germination; yellow cedar
Yellow-cedar (Chamaecyparis nootkatensis [D. Don] Spach) seeds exhibit prolonged coat-imposed dormancy following their dispersal from the parent plant. Analyses were undertaken using S-(+)-[H-3] abscisic acid (ABA) to monitor the capacity of embryos to metabolize ABA following their isolation from seeds subjected to various dormancy-breaking and control treatments. Radiolabelled phaseic acid (PA) and dihydrophaseic acid (DPA) were detected in embryos and, to a greater extent in the surrounding media, by 48 h regardless of whether the embryos had been excised from seed previously subjected to only a 3 d soak or to a full dormancy-breaking treatment. Of the two enantiomers of ABA, only the natural S-(+)-ABA effectively inhibited germination of isolated embryos. A metabolism-resistant synthetic ABA analogue S-[8',8',8',9',9',9']-hexadeuteroabscisic acid, S-(+)-d6-ABA, consistently slowed the germination rate of excised embryos to a greater extent than that caused by natural S-(+)-ABA. The deuterium-labelled ring methyl groups of the analogue made it more resistant to oxidation by yellow-cedar embryos and thus rendered the analogue more persistent and possessing greater activity. With increasing time of exposure to moist chilling, yellow-cedar embryos became increasingly insensitive to both ABA and to the analogue. Subjecting seed to chemical treatments (GA(3) in combination with 1-propanol) prior to moist chilling strongly enhanced the germinability of whole seeds. This treatment also had a relatively greater impact on ABA metabolism than did moist chilling alone, as indicated by a greater capacity of S-(+)-d6-ABA to inhibit the germination of embryos as compared to S-(+)-ABA. Moist chilling was most critical for reduced ABA sensitivity of embryos. A change in the embryo's ability to metabolize ABA and reduced embryo sensitivity to ABA are two factors associated with dormancy termination of whole seeds of yellow cedar; a change in only one of these factors is insufficient to elicit high germinability.
23. Schmitz, N; Xia, JH; Kermode, AR. (2002) Emergence and growth of yellow-cedar (Chamaecyparis nootkatensis) seedlings following modified dormancy-breaking treatments.Seed Science and Technology 30: 249-262 Emergence and growth of yellow-cedar (Chamaecyparis nootkatensis) seedlings following modified dormancy-breaking treatments
Following dispersal from the parent tree, seeds of yellow-cedar (Chamaecyparis nootkatensis [D. Don] Spach) exhibit a low capacity for germination, primarily as a result of coat-imposed dormancy. Dormancy of the mature (intact) seed is effectively terminated by a three day soak (21degreesC) and a warm moist period (four weeks at 20degreesC) followed by at least eight weeks of moist chilling (2-4degreesC). This treatment is used by most nurseries and the B.C. Ministry of Forests, albeit with some variation. A chemical treatment using the anaesthetic I-propanol promotes high germinability of yellow-cedar seeds, when combined with a three day warm water soak (30degreesC), a two day GA(3) treatment and 60 days of moist chilling. Here we compare the effectiveness of the more traditional warm/cold treatments (performed on moistened Kimpak or in moist sand) with our chemical treatment in terms of their capacity to elicit vigorous post-germinative growth following seedling emergence under nursery greenhouse conditions. Two seed lots (42313 and 43697) and open-pollinated seed from parent trees 19-8 and 13-6 showed equivalent emergence and vigorous post-germinative growth following the chemical and more traditional dormancy-breaking treatments. The chemical protocol offers three advantages over more traditional procedures: (1) it reduces the time required to break seed dormancy; (2) it leads. to. synchronous and high germinability and rapid post-germinative growth and (3) it minimizes the unpredictability of traditional protocols that require more rigorous monitoring of moisture content.
22. Xia, JH; Stewart, D; Kermode, AR. (2002) Modified moist chilling treatments that promote germination and post-germinative reserve mobilization of different seed lots of yellow-cedar (Chamaecyparis nootkatensis [D. Don] Spach).Seed Science and Technology 30: 263-277 Modified moist chilling treatments that promote germination and post-germinative reserve mobilization of different seed lots of yellow-cedar (Chamaecyparis nootkatensis [D. Don] Spach)
Seeds of yellow-cedar (Chamaecyparis nootkatensis [D. Don] Spach) often exhibit sporadic and non-synchronous germination even after prolonged moist chilling. Treatments that combine chemicals (anaesthetics like I-propanol, gibberellic acid and nitrate) with moist chilling greatly accelerate dormancy termination. Here we report on the effectiveness of extended experiments using several treatment combinations on seven seed lots of yellow-cedar as well as open-pollinated seed from parent trees 19-8 and 13-6. The most effective combination overall, as far as the percentage and rate of germination are concerned, consisted of a three day soak at elevated temperature (30degreesC), a one day 1-propanol treatment, a two day GA(3) treatment and 60 days of moist chilling. More traditional warm/cold treatments were also effective, provided that high seed moisture content was strictly maintained, particularly during moist chilling (35-40% on a fresh weight basis). Protein reserve mobilization and induction of isocitrate lyase activities in the embryo and megagametophyte following germination indicated that early post-germinative events were equivalent when dormancy termination of seeds was achieved by the chemical versus the more traditional warm/cold treatment.
21. Bates, SL; Lait, CG; Borden, JH; Kermode, AR. (2001) Effect of feeding by the western conifer seed bug, Leptoglossus occidentalis, on the major storage reserves of developing seeds and on seedling vigor of Douglas-fir.Tree Physiology 21: 481-487 Effect of feeding by the western conifer seed bug, Leptoglossus occidentalis, on the major storage reserves of developing seeds and on seedling vigor of Douglas-fir
lipid; protein; Pseudotsuga menziesii; seed storage reserves; seedling vigor
The impact of feeding by the western conifer seed bug (Leptoglossus occidentalis Heidemann) on storage reserves of developing seeds of Douglas-fir (Pseudostuga menziesii (Mirb.) France) was studied by caging nymphal and adult seed bugs on cones during late-season development, and nymphs on cones during early, mid- and late-season development. Analysis of the major storage reserves of partially damaged seeds revealed that late-season feeding by each life stage and feeding by nymphs at all three stages of cone development significantly reduced the amounts of lipid and buffer-insoluble (crystalloid) protein in seeds at harvest by up to 78 and 97%, respectively. Seeds showing light to moderate damage on radiographs did not exhibit a reduction in the amount of buffer-soluble (matrix) protein. Seeds damaged by feeding during early development compensated in part by continuing to synthesize lipid and crystalloid protein. Light or moderate damage to mature Douglas-fir seeds exposed to L. occidentalis in the laboratory reduced seedling emergence by > 80%, but the seedlings that emerged successfully appeared to suffer no adverse effects when grown under standard nursery conditions.
20. Crowe, AU; Han, B; Kermode, AR; Bendell-Young, LI; Plant, AL. (2001) Effects of oil sands effluent on cattail and clover: photosynthesis and the level of stress proteins.Environmental Pollution 113: 311-322 Effects of oil sands effluent on cattail and clover: photosynthesis and the level of stress proteins
cattail; Typha latifolia L.; clover; Trifolium hybridum L.; apparent photosynthesis; RuBisCo; osmotic stress
The oil sands industry located in northeastern Alberta, Canada, generates large volumes of effluent characterized by a high level of dissolved ions and naphthenic acids. The dikes used to store the effluent seep, creating wetlands which are subsequently invaded by obligate wetland flora such as cattail (Typha latifolia L.). The appearance of these wetlands prompted the oil sands industry to consider wetlands as part of their reclamation strategy. However, to ensure long-term viability of such wetlands, the response of the flora to the industrial effluent needed to be determined. To this end, apparent photosynthesis (APS), the level of ribulose-1,5-bisphosphate carboxylase (RuBisCo) large subunit, dehydrin-related polypeptides, and protein disulphide isomerase (PDI) were evaluated in cattail and alsike clover plants (Trifolium hybridum L.) exposed to the oil sands effluent. APS measured in plants impacted by oil sands effluent was significantly higher than that of plants in the non-impacted off-site location. Among the on-site locations, plants growing in the natural wetlands site had higher APS compared to all other sites. The level of RuBisCo was not increased in cattail or clover growing in effluent-contaminated sites indicating that enhanced photosynthesis was not due to greater levels of this enzyme. Dehydrin-related polypeptides were detected only in the roots of cattail and were absent in clover. The polypeptide profile was altered in cattail exposed to oil sands effluent indicating that they were responding to an osmotic stress. The level of PDI was unaffected in the leaves of cattail regardless of the nature of the effluent to which they were exposed. Overall, the data indicate that cattail and clover are adapted to the oil sands effluent, although further studies are needed to assess their longterm ability to survive in the presence of this anthropogenic stress. (C) 2001 Elsevier Science Ltd. All rights reserved.
19. Lait, CG; Bates, SL; Borden, JH; Kermode, AR. (2001) Protein reserve hydrolysis in Douglas-fir seeds (Pseudotsuga menziesii [Mirb.] Franco) following feeding by the western conifer seed bug.Seed Science and Technology 29: 609-617 Protein reserve hydrolysis in Douglas-fir seeds (Pseudotsuga menziesii [Mirb.] Franco) following feeding by the western conifer seed bug
Nymphs and adults of the western conifer seed bug, Leptoglossus occidentalis Heidemann (Hemiptera: Coreidae) feed on maturing seeds of many economically important coniferous tree species in natural stands and seed orchards throughout western North America. Changes to the profiles of soluble (matrix) and insoluble (crystalloid) protein reserves of mature Douglas-fir seeds were examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to assess reserve hydrolysis caused by feeding of the seed bug. The major soluble and insoluble storage proteins of Douglas-fir seeds were rapidly hydrolyzed following feeding by L. occidentalis, in which female adults appeared to be more efficient in hydrolyzing seed reserves than adult males or nymphs. The higher molecular weight (34.5-39.5 kDa) acidic subunit polypeptides of the crystalloid protein complex (fractionated by SDS-PAGE under reducing conditions) were almost completely hydrolyzed in seeds sustaining as little as light feeding damage, in which > 66% of seed contents remained (as determined by radiographic analysis). Only traces of crystalloid protein remained in seeds that had sustained severe damage, in which < 33% of the seed contents remained. Similarly, the major matrix (soluble) storage proteins also underwent rapid hydrolysis. Hydrolysis of soluble storage proteins following germination of Douglas-fir seeds showed striking similarities to that induced by seed bug feeding and hydrolysis of insoluble crystalloid proteins, albeit slower in germinated seeds, led to similar soluble breakdown products as those produced in fed-on seeds.
18. Lait, CG; Bates, SL; Kermode, AR; Morrissette, KK; Borden, JH. (2001) Specific biochemical marker-based techniques for the identification of damage to Douglas-fir seed resulting from feeding by the western conifer seed bug, Leptoglossus occidentalis Heidemann (Hemiptera : Coreidae).Insect Biochemistry and Molecular Biology 31: 739-746 Specific biochemical marker-based techniques for the identification of damage to Douglas-fir seed resulting from feeding by the western conifer seed bug, Leptoglossus occidentalis Heidemann (Hemiptera : Coreidae)
hemiptera; coreidae; Leptoglossus occidentalis; Pseudotsuga menziesii; pinaceae; seed storage proteins; saliva; biochemical markers; polyclonal antibody; immunodetection; protein hydrolysis
Specific biochemical marker-based techniques were tested for their ability to distinguish between seeds of Douglas-fir, Pseudotsuga menziesii (Mirbel) France, that were filled or unfilled (aborted) at maturity and those that were damaged or emptied by the western conifer seed bug, Leptoglossus occidentalis Heidemann. A polyclonal antibody raised against salivary gland extracts from L. occidentalis successfully identified residual salivary proteins on Western blots containing proteins from Douglas-fir seeds that had sustained various degrees of seed bug feeding damage. In a single blind experiment. the polyclonal antibody correctly identified 100% of undamaged control, 97% of unfilled control (aborted), and 98% of seed bug damaged seeds. Polyclonal antibodies raised against insoluble alfalfa crystalloid storage protein (11S globulin) detected the depletion of 11S globulin and the subsequent appearance of its hydrolyzed fragments in the soluble protein fraction of Douglas-fir seeds that were fed-upon by the seed bug. Feeding by L. occidentalis nymphs caused ca. 98% depletion of insoluble protein, but only ca. 53% reduction in the amount of soluble protein in seeds that appeared empty on radiographs. By comparison, unfilled (aborted) seeds contained significantly less insoluble and soluble protein than empty seeds that were fed-upon by L. occidentalis: moreover, no crystalloid (11S globulin) breakdown products were generated. The biochemical markers described in this study are reliable tools that can be used to identify conifer seeds that have sustained light to severe damage from L. occidentalis feeding. (C) 2001 Elsevier Science Ltd. All rights reserved.
17. Lait, CG; Bates, SL; Morrissette, KK; Borden, JH; Kermode, AR. (2001) Biochemical assays for identifying seeds of lodgepole pine and other conifers fed on by Leptoglossus occidentalis Heidemann (Hemiptera : Coreidae).Canadian Journal of Botany-Revue Canadienne de Botanique 79: 1349-1357 Biochemical assays for identifying seeds of lodgepole pine and other conifers fed on by Leptoglossus occidentalis Heidemann (Hemiptera : Coreidae)
Leptoglossus occidentalis; saliva; biochemical markers; polyclonal antibody; immunodetection; Pinus contorta
Radiography is a valuable tool for assessing quality of conifer seeds, but it cannot differentiate between aborted seeds and seeds that have been emptied by western conifer seed bug (Leptoglossus occidentalis Heidemann) feeding. We tested three biochemical marker-based assays that were developed to identify L. occidentalis damage to seeds of Douglas-fir, Pseudostuga menziesii (Mirb.) Franco, for their use in lodgepole pine, Pinus contorta var. latifolia Engelmann. The three assays included measurement of storage protein reserve depletion, immunodetection of fragments of insoluble (crystalloid) storage proteins, and immunodetection of L. occidentalis salivary proteins. Aborted seeds contained significantly less soluble and insoluble protein than seeds that were fed on by L. occidentalis. Polyclonal antibodies raised against 11S globulin crystalloid proteins or L. occidentalis salivary gland extracts only immunoreacted with proteins in seeds exposed to L. occidentalis feeding. In a single-blind test, antibody raised against salivary-gland extracts correctly distinguished between undamaged full seeds, unfilled aborted seeds, and seeds fed on by L. occidentalis. Immunodetection of L. occidentalis salivary proteins was also performed on seeds of Abies amabilis Dougl. ex J. Forbes, Tsuga heterophylla (Raf.) Sarg., Picea sitchensis Bong (Carr.), Pinus ponderosa Lawson, and Pinus monticola Dougl. ex D. Don. For all species, immunoreactive polypeptides were only detected in seeds fed on by L. occidentalis. These biochemical marker-based techniques could help researchers and seed orchard managers estimate seed losses caused by L. occidentalis in commercial seed orchards and natural forest stands.
16. Schmitz, N; Xia, JH; Kermode, AR. (2001) Dormancy of yellow cedar seeds is terminated by gibberellic acid in combination with fluridone or with osmotic priming and moist chilling.Seed Science and Technology 29: 331-346 Dormancy of yellow cedar seeds is terminated by gibberellic acid in combination with fluridone or with osmotic priming and moist chilling
Following dispersal from the parent tree, seeds of yellow cedar (Chamaecyparis nootkatensis D. Don Spach) exhibit a low capacity for germination, primarily as a result of coat-imposed dormancy. Combined warm and cold stratification (generally 4 weeks and 8 weeks, respectively) is often used to break dormancy. Here we investigate the effectiveness of different chemical treatments in replacing warm stratification and in reducing the requirement for cold stratification (moist chilling). Treatments comprised of gibberellic acid (GA(3)) in combination with osmotic priming (using polyethylene glycol, PEG) or with other hormones (gibberellin (4+7) and benzylaminopurine) had a promotive effect on subsequent germination of whole seeds of yellow cedar after a 60-day moist chilling period. GA3 had virtually no effect on germination when used alone and even when used with 60 days of moist chilling. Whole seeds exposed to GA, and to fluridone (the latter to diminish endogenous abscisic acid, ABA), exhibited increased germination with an increasing time of exposure to fluridone, even in the complete absence of moist chilling, but seedlings were abnormal and unable to survive. Embryos excised from mature seeds which had been subjected to dormancy-breaking treatments (osmotic priming, GA3, and moist chilling), exhibited a lowered sensitivity to ABA compared to embryos excised from mature seeds that had received only a 3-day water soak. A decline in ABA within the seed and/or reduced embryo sensitivity to ABA may be two critical factors leading to dormancy breakage of yellow cedar seed.
15. Bates, SL; Borden, JH; Kermode, AR; Bennett, RG. (2000) Impact of Leptoglossus occidentalis (Hemiptera : Coreidae) on Douglas-fir seed production.Journal of Economic Entomology 93: 1444-1451 Impact of Leptoglossus occidentalis (Hemiptera : Coreidae) on Douglas-fir seed production
Coreidae; Leptoglossus occidentalis; Pseudostuga menzieisii; seed loss; radiography
We investigated the effect of feeding by the western conifer-seed bug, Leptoglossus occidentalis Heidemann, on seed production in developing cones of coastal Douglas-Br, Pseudostuga menziesii (Mirb.) France. with respect to seed bug life stage and sex (nymphs, adult females, and adult males) and timing of feeding (early, mid-, and late season cone development). Feeding by females on caged cones for a a-wk period during late season cone development reduced the proportion of full seeds in cones by approximate to 70% compared with caged control cones. There was no significant difference among nymphs, adult females, and adult males with respect to the proportion of empty or partially fed-upon seeds produced during the same feeding period. Feeding by nymphs for 2 wk early in the season resulted in a threefold increase in the number of unextractable seeds fused to cones compared with the control. Weight measurements of harvested seeds indicated that radiography is an accurate tool to distinguish among Douglas-fir seeds that have sustained light, moderate, or severe damage. Determining the full impact of L. occidentalis on conifer seed production will require the development of a reliable method to distinguish between naturally alerted seeds and seeds emptied through feeding by seed bugs.
14. Bates, SL; Borden, JH; Savoie, A; Blatt, SE; Lait, CG; Kermode, AR; Bennett, RG. (2000) Impact of feeding by Leptoglossus occidentalis (Hemiptera : Coreidae) on the major storage reserves of mature Douglas-fir (Pinaceae) seeds.Canadian Entomologist 132: 91-102 Impact of feeding by Leptoglossus occidentalis (Hemiptera : Coreidae) on the major storage reserves of mature Douglas-fir (Pinaceae) seeds
In laboratory experiments adults and nymphs of the western conifer seed bug, Leptoglossus occidentalis Heidemann, were allowed to feed on mature seeds of Douglas-fir, Pseudotsuga menziesii (Mirbel) France. Weight-loss measurements and scanning electron microscopy provided strong supporting evidence for the use of simple radiographic diagnosis as a method of classifying feeding damage to seeds into four categories: Light (greater than two thirds of seed contents remaining), moderate (one third to two thirds of seed contents remaining), severe (less than one third of seed contents remaining), and extreme (seed empty). Scanning electron micrographs showed the apparent depletion of lipid and protein storage reserves which was verified by quantitative analyses that showed significant loss of lipid and buffer-insoluble (crystalloid) storage protein from seeds in all damage categories. The amount of buffer-soluble (matrix) protein was reduced in seeds from the severe and extreme damage categories. The increase in buffer-soluble protein observed in lightly damaged seeds was likely due to the solubilization of crystalloid storage protein, as a result of its breakdown into smaller peptides. Our results suggest that through the action of both lipases and proteases, L. occidentalis can have a serious impact on the major storage reserves of conifer seeds. Moreover, our data suggest that L. occidentalis feeds in a different manner than the laceration and flushing method found in other seed-feeding Hemiptera.
13. Bendell-Young, LI; Bennett, KE; Crowe, A; Kennedy, CJ; Kermode, AR; Moore, MM; Plant, AL; Wood, A. (2000) Ecological characteristics of wetlands receiving an industrial effluent.Ecological Applications 10: 310-322 Ecological characteristics of wetlands receiving an industrial effluent
anthropogenic impacts; benthic community structure; ecosystem characteristics; fish acute lethality and stress; oil sands; wetlands
The primary objectives of this study were to evaluate the ecological characteristics of wetland ecosystems that had developed in response to oil sands effluent relative to reference wetland ecosystems and, from such an evaluation, to assess whether these wetlands were viable systems capable of integrating into the northern Canadian landscape. A secondary objective was to evaluate the use of several ecologically relevant endpoints as indicators of an ecosystem response to a known anthropogenic stress, in this case, wetlands receiving oil sands effluent. To achieve this, a suite of endpoints were compared between effluent-impacted wetlands and nonimpacted reference wetlands. Endpoints for comparison included: (1) benthic macroinvertebrate community structure, (2) chironomid density and biomass, (3) the incidence of chironomid mentum deformities, (4) the mutagenetic potential of sediment-dwelling chironomids, (5) growth and photosynthetic rare for the aquatic plant Typha latifolia (cattail), and (6) fish acute lethality and stress response as measured by changes in blood chemistry (percentage hematocrit [%hct], percentage leucocrit [%lct], and differential white blood cell count). Wetlands receiving oil sands effluent supported a low-diversity benthic community, dominated primarily by the Chironomidae and cattail. There was no evidence of mentum deformities or mutagenicity in chironomids sampled from the oil-impacted wetlands. Cattails grown in oil sands effluent and sediment demonstrated increased photosynthetic rates; however, these increased rates did not translate into increased plant growth. In contrast to the benthic community and the cattail, indigenous fish were unable to survive in wetlands containing oil sands effluent. Fish displayed altered blood chemistry (elevated %hct, depressed %lct) and ultimately death when held beyond 14 d in the oil-impacted wetlands. Of the various ecological endpoints measured, the macroinvertebrate community and changes in fish blood chemistry were the most sensitive indicators of an anthropogenic stress, demonstrating distinct differences in response between impacted and reference wetlands. To ensure that these wetlands can safely integrate into the northern Canadian landscape, future studies need to focus on their impacts at higher trophic levels indigenous to the wetland.
12. Ren, CW; Kermode, AR. (2000) An increase in pectin methyl esterase activity accompanies dormancy breakage and germination of yellow cedar seeds.Plant Physiology 124: 231-242 An increase in pectin methyl esterase activity accompanies dormancy breakage and germination of yellow cedar seeds
Pectin methyl esterase (PME) (EC 3.1.1.11) catalyzes the hydrolysis of methylester groups of cell wall pectins. We investigated the role of this enzyme in dormancy termination and germination of yellow cedar (Chamaecyparis nootkatensis [D. Don] Spach) seeds. PME activity was not detected in dormant seeds of yellow cedar but was induced and gradually increased during moist chilling; high activity coincided with dormancy breakage and germination. PME activity was positively correlated to the degree of dormancy breakage of yellow cedar seeds. The enzyme produced in different seed parts and in seeds at different times during moist chilling, germination, and early post-germinative growth consisted of two isoforms, both basic with isoelectric points of 8.7 and 8.9 and the same molecular mass of 62 kD. The pH optimum for the enzyme was between 7.4 and 8.4. In intact yellow cedar seeds, activities of the two basic isoforms of PME that were induced in embryos and in megagametophytes following dormancy breakage were significantly suppressed by abscisic acid. Gibberellic acid had a stimulatory effect on the activities of these isoforms in embryos and megagametophytes of intact seeds at the germinative stage. We hypothesize that PME plays a role in weakening of the megagametophyte, allowing radicle emergence and the completion of germination.
11. Schmitz, N; Abrams, SR; Kermode, AR. (2000) Changes in abscisic acid content and embryo sensitivity to (+)-abscisic acid during the termination of dormancy of yellow cedar seeds.Journal of Experimental Botany 51: 1159-1162 Changes in abscisic acid content and embryo sensitivity to (+)-abscisic acid during the termination of dormancy of yellow cedar seeds
coat-imposed dormancy; embryo; megagametophyte; abscisic acid; seed germination; yellow cedar
Yellow cedar seeds are dormant at maturity. The abscisic acid (ABA) content of the embryo (but not the megagametophyte) decreased approximately 2-fold following exposure of seeds to a dormancy-breaking treatment; this process was also accompanied by a 10-fold lowered sensitivity of the embryo to S-(+)-ABA. A decline in ABA within the seed is not sufficient for dormancy breakage; reduced embryo sensitivity to ABA is also required.
10. Xia, JH; Kermode, AR. (2000) Dormancy of yellow cedar (Chamaecyparis nootkatensis [D. Don] Spach) seeds is effectively terminated by treatment with 1-propanol or nitrate in combination with a warm water soak, gibberellin and moist chilling.Seed Science and Technology 28: 227-240 Dormancy of yellow cedar (Chamaecyparis nootkatensis [D. Don] Spach) seeds is effectively terminated by treatment with 1-propanol or nitrate in combination with a warm water soak, gibberellin and moist chilling
seed dormancy; germination; yellow cedar; gibberellin; nitrate; propanol; moist chilling
Following dispersal from the parent tree, seeds of yellow cedar (Chamaecyparis nootkatensis [D. Don] Spach) exhibit a low capacity for germination as a result of coat-imposed dormancy. Germination of the mature (intact) seed requires several months of moist chilling; however, even after prolonged moist chilling, germination is typically sporadic and nonsynchronous. Here we investigate the effectiveness of chemical treatments in reducing the requirement for moist chilling. The most effective agent in this regard was the anaesthetic 1-propanol, which promoted 75-85% germination of yellow cedar seeds, when combined with a two day GA, treatment and 30 days of moist chilling. The promotive effect of 1-propanol was specific; 2-propanol and 1-pentanol were ineffective. In the absence of propanol, nitrate in the form of KNO3, along with GA(3), was effective for dormancy alleviation when appropriate conditions were used. Soaking of seeds in warm running water (30 or 40 degrees C instead of 21 degrees C) before chemically treating them resulted in a further improvement in germination percentage. Moist chilling of seeds after chemical treatment was essential for improving overall germination performance and post-germination performance (i.e. seedling emergence). The duration of the traditional dormancy-breaking treatment for yellow cedar seed (87 days), could be reduced to 36 days without a compromise in percentage or rate of germination, by combining a three day soak in running water at 30 degrees C, a one day 1-propanol treatment, Two days of GA(3) and 30 days of moist chilling.
9. Ren, CW; Kermode, AR. (1999) Analyses to determine the role of the megagametophyte and other seed tissues in dormancy maintenance of yellow cedar (Chamaecyparis nootkatensis) seeds: morphological, cellular and physiological changes following moist chilling and during germination.Journal of Experimental Botany 50: 1403-1419 Analyses to determine the role of the megagametophyte and other seed tissues in dormancy maintenance of yellow cedar (Chamaecyparis nootkatensis) seeds: morphological, cellular and physiological changes following moist chilling and during germination
coat-imposed dormancy; embryo; megagametophyte; nucellar cap; seed germination; yellow cedar
Yellow cedar (Chamaecyparis nootkatensis) seeds exhibit prolonged dormancy following their dispersal from the parent plant. Embryos excised fully from their enclosing seed tissues exhibit 100% germination, indicating that the seed tissues enclosing the embryo (the testa, remnants of the nucellus and the megagametophyte) play an inhibitory role and prevent radicle emergence. As part of an assessment of the role of seed tissues in the dormancy mechanism of yellow cedar seeds, light microscopy was used to examine changes within the major structures of the seed following a 90 d warm (26 degrees C)/cold (4 degrees C) moist treatment ('stratification') and during germination. In the micropylar tip of the seed, the nucellus forms a hard nucellar cap covering the radicle. The nucellar cap is composed primarily of degenerated cells; histological staining with ruthenium red revealed a predominance of pectins. There were no obvious cellular or morphological differences (detected by light microscopy) between mature seeds subjected to a 3 d soak and seeds subjected to a 3 d soak and the 90 d dormancy-breaking treatment. However, just prior to germination there was an outward projection of the nucellar cap through the micropyle, which appeared to be caused by the extension of highly folded proteinaceous strands lying immediately in front of the radicle. When the testa was removed, the embryo enclosed within the intact megagametophyte was incapable of germination. If, however, the megagametophyte surrounding the embryo was slit or the embryo surrounded by an intact megagametophyte was subjected to a 3 d rinse in water, some germination occurred, perhaps as a result of an enhanced release of inhibitors from the megagametophyte. After stratification, dormancy of yellow cedar seeds is broken; concurrent with dormancy breakage, there was; a mechanical weakening of the megagametophyte. The embryo also underwent changes that included an increase in turgor and a reduced sensitivity to highly negative osmotic potential. It is concluded that coat-imposed dormancy of yellow cedar seeds is enforced by mechanical restraint of the megagametophyte as well as a leachable chemical inhibitor (most probably ABA).
8. Xia, JH; Kermode, AR. (1999) Analyses to determine the role of embryo immaturity in dormancy maintenance of yellow cedar (Chamaecyparis nootkatensis) seeds: synthesis and accumulation of storage proteins and proteins implicated in desiccation tolerance.Journal of Experimental Botany 50: 107-118 Analyses to determine the role of embryo immaturity in dormancy maintenance of yellow cedar (Chamaecyparis nootkatensis) seeds: synthesis and accumulation of storage proteins and proteins implicated in desiccation tolerance
embryo; megagametophyte; seed development; yellow cedar; storage proteins; dehydrins; alpha-TiP
Development of yellow cedar seeds is completed by about 17-21 months after pollination. Following dispersal from the parent plant, the seeds exhibit a low capacity for germination and typically require an additional year to meet their moist chilling requirements and break dormancy, Biochemical analyses were undertaken in order to address whether seed dormancy is imposed and maintained because the embryo or megagametophyte is immature at the time of seed shedding and hence requires time to complete developmental events before dormancy can be terminated. Major protein reserves of the embryo and megagametophyte are the buffer-insoluble crystalloid (legumin) storage proteins and the water-soluble albumin proteins. SDS-PAGE, fluorography of in vivo synthesized proteins and Western blot analyses showed that the greatest increase in protein reserve synthesis and accumulation occurred between the first and second years of development; deposition of soluble and insoluble storage protein was largely completed in seeds of second-year cones by August, 2-3 months prior to seed dispersal, The period associated with greatest accumulation of storage proteins was accompanied by an increased accumulation of two ER-resident proteins associated with post-translational maturation of storage proteins (binding protein and protein disulphide isomerase), Accumulation of proteins implicated in the acquisition of desiccation tolerance (dehydrins and the tonoplast intrinsic protein, a-Tip) occurred between the first and second years of development. Several heat-stable proteins and some of the proteins associated with late development continued to be synthesized after seed shedding and in 13 d moist-chilled mature seeds. However, this did not include the major dehydrin-like protein of yellow cedar seeds. Further, the continued synthesis of heat-stable proteins does not appear to be a factor preventing the germination of yellow cedar seeds following dispersal from the parent plant; rather, the mechanism of dormancy is primarily coat-imposed.
7. Han, B; Berjak, P; Pammenter, N; Farrant, J; Kermode, AR. (1997) The recalcitrant plant species, Castanospermum australe and Trichilia dregeana, differ in their ability to produce dehydrin-related polypeptides during seed maturation and in response to ABA or water-deficit-related stresses.Journal of Experimental Botany 48: 1717-1726 The recalcitrant plant species, Castanospermum australe and Trichilia dregeana, differ in their ability to produce dehydrin-related polypeptides during seed maturation and in response to ABA or water-deficit-related stresses
dehydrin; ABA; desiccation; recalcitrant; seed
In constrast to seeds of orthodox species, those of recalcitrant species do not acquire desiccation tolerance during their development and are shed from the parent plant at high water contents, Dehydrin production in seeds of recalcitrant species was examined during development and germination, in response to abscisic acid (ABA), and following the imposition of various water-deficit-related stresses, including desiccation, water stress, high salt, high osmolarity, and low temperature. Two tropical species exhibited a differential capacity to produce dehydrin-related proteins during seed maturation. Dehydrins were present in axes and cotyledons of Castanospermum australe seeds during mid-maturation and at maturity. In Trichilia dregeana, no dehydrin-related polypeptides were detected in the mature seed. During the development of C. australe seeds, the nature of the dehydrin-related polypeptides accumulated in the cotyledons and axis changed and new polypeptides were detected in the mature seeds that were not present during mid-maturation. The dehydrins present in cotyledons of mature seeds (31, 37 and 40 kDa) were still detectable after germination (i.e. in untreated seedlings). These dehydrins became less abundant in the cotyledons of C. australe seedlings following ABA and all stress treatments except cold, although most of the dehydrins were still detectable, An exception was the desiccation-treated seedlings, in which no dehydrins were detected. In the roots of C. australe seedlings, no dehydrins were found after germination nor were they induced in the root by ABA or any of the stress treatments imposed on seedlings. Seedlings of Trichilia dregeana did not produce dehydrins in the roots or cotyledons when exposed to ABA or water-deficit-related stresses.
6. Han, B; Hughes, DW; Galau, GA; Bewley, JD; Kermode, AR. (1997) Changes in late-embryogenesis-abundant (LEA) messenger RNAs and dehydrins during maturation and premature drying of Ricinus communis L seeds.Planta 201: 27-35 Changes in late-embryogenesis-abundant (LEA) messenger RNAs and dehydrins during maturation and premature drying of Ricinus communis L seeds
desiccation; endosperm; late embryogenesis abundant proteins; Ricinus
In Ricinus communis L. (castor bean) endosperms, two classes of Late Embryogenesis Abundant (Lea) transcripts were first detected during mid-development (at 30-35 days after pollination, DAP) and peaked at 50 DAP,just prior to the onset of desiccation. Most of the Class I mRNAs declined substantially during desiccation itself; Class II mRNAs remained abundant in the mature dry (60 DAP) seed. Following imbibition, all Lea mRNAs abundant in the mature dry seed declined rapidly (within 5-24 h). Premature drying of developing 35-DAP seeds resulted in the loss of storage-protein mRNAs (Leg B Mat I); following rehydration, mRNAs encoding postgerminative proteins (Germ D91, D30 and D38) increased in the endosperm. The Lea mRNAs present in the developing fresh seed at 35 DAP were preserved, but did not increase in response to premature desiccation; upon rehydration these Lea mRNAs declined within 5 h. During seed development, substantial changes occurred in the synthesis of a subset of LEA proteins referred to as 'dehydrins'; in particular, new dehydrin polypeptides were induced between 40 and 60 DAP. Such proteins were not as evident in prematurely dried endosperms. In contrast to the rapid loss of Lea mRNAs during germination, many of the dehydrin proteins abundant in the dried seed persisted following imbibition or rehydration.
5.Kermode, AR. (1997) Approaches to elucidate the basis of desiccation-tolerance in seeds.Seed Science Research 7: 75-95 Approaches to elucidate the basis of desiccation-tolerance in seeds
dehydrin; LEA; ABA; water stress; sugars; desiccation; recalcitrant; orthodox; seed
Plants undergo a series of physiological, biochemical and molecular changes in response to adverse environmental conditions or stresses such as drought, low temperature or high salt. Several genes and their corresponding proteins have been described that may play a role in withstanding water-deficit-related stresses or full desiccation. In particular, sugars and late-embryogenesis-abundant (LEA) proteins have received the most attention. Plant responses to water-deficit and desiccation have been well-characterized at the molecular level; however, pinpointing the precise roles of the gene products in protecting cells under conditions of water deficit remains a challenging task. While few plants are capable of withstanding full desiccation, most seeds undergo this event as a pre-programmed and final stage in their development. These are the so-called 'orthodox' seeds. In contrast to seeds of orthodox species, those of recalcitrant species do not acquire desiccation tolerance during their development and are shed from the parent plant at relatively high water contents. The essential components of desiccation tolerance of seeds are likely to involve the ability to effect repair upon subsequent rehydration as well as the ability to accumulate protective substances that limit the amount of damage which otherwise would be caused by water loss. Studies have begun to examine whether the desiccation sensitivity of recalcitrant seeds is at least partially the result of an insufficient accumulation of LEA-type proteins, or whether other factors (including a lack of protective sugars) are more important. This review assesses some of these studies as well as recent research to understand gene and protein function using transgenic host plant systems.
4. Han, B; Kermode, AR. (1996) Dehydrin-like proteins in castor bean seeds and seedlings are differentially produced in response to ABA and water-deficit-related stresses.Journal of Experimental Botany 47: 933-939 Dehydrin-like proteins in castor bean seeds and seedlings are differentially produced in response to ABA and water-deficit-related stresses
dehydrin; castor bean; ABA; desiccation
The stress inducibility of dehydrin protein production in seedlings of castor bean was analysed by subjecting them to ABA and various water-deficit-related treatments including desiccation, water stress, high salt, high osmolarity, and low temperature. A further goal was to determine whether the immature seed (at stages prior to major dehydrin synthesis) would respond in a similar manner to these stresses. A number of dehydrin-like proteins increased in seedlings subjected to the various stress treatments. In the endosperm, these appear to be different from the dehydrin-related polypeptides that are induced during late seed development and which persist following germination/growth of mature seeds. In the endosperm of seedlings, ABA, water stress and desiccation induced the same dehydrin polypeptides, while high osmolarity, high salt and low temperature induced a different set. Stress-specific differences in dehydrin synthesis were also found in the cotyledons and radicle of castor bean seedlings; however, dehydrins inducible by exogenous ABA were consistently produced. Immature seeds treated with ABA or subjected to stress responded by producing dehydrin-like proteins associated with late development; however, the same proteins were induced following detachment of immature seeds from the parent plant and maintenance on water. When seedlings were exposed simultaneously to GA and either ABA, high salt, or low temperature, dehydrin production was suppressed. It is concluded that dehydrin production in castor bean is tissue-specific and is dependent upon the physiological stage of the seed. In the endosperm, the response to different stresses may rely upon more than one signal transduction pathway.
3. Jiang, LW; Abrams, SR; Kermode, AR. (1996) Vicilin and napin storage-protein gene promoters are responsive to abscisic acid in developing transgenic tobacco seed but lose sensitivity following premature desiccation.Plant Physiology 110: 1135-1144 Vicilin and napin storage-protein gene promoters are responsive to abscisic acid in developing transgenic tobacco seed but lose sensitivity following premature desiccation
In transgenic tobacco (Nicotiana tabacum L.) seed, expression of chimeric beta-glucuronidase (GUS) genes containing the vicilin or napin storage-protein gene promoters is responsive to premature drying and declines upon rehydration (L. Jiang, W.L. Downing, C.L. Baszczynski, A.R. Kermode [1995] Plant Physiol 107: 1439-1449). Desiccation may cause changes in the content of or sensitivity to abscisic acid (ABA), partially or wholly removing the effects of this ''modulator'' of developmental gene expression. We studied:the enhancement of GUS reporter enzyme activities in transgenic tobacco by exogenous ABA before and after drying. A racemic mixture of ABA at 10 mu M caused a 2- to 3-fold increase in GUS activity in developing transgenic seed expressing chimeric genes containing the vicilin or napin gene promoters. However, when these seeds were prematurely dried prior to their culture on ABA medium, enhancement of GUS activity was virtually abolished. Use of optically pure ABAs revealed that the enhancement in GUS activity in developing fresh seed was due largely to the natural (+) form of ABA. Chimeric constructs containing a viral 35S promoter did not respond to ABA whether or not premature drying was applied. Thus, vicilin and napin chimeric genes, initially sensitive to ABA, become relatively insensitive to the hormone following drying. A decline in ABA sensitivity may be an important factor in the cessation of storage-protein gene expression.
2. Jiang, LW; Kermode, AR; Jones, RL. (1996) Premature drying increases the GA-responsiveness of developing aleurone layers of barley (Hordeum vulgare L) grain.Plant and Cell Physiology 37: 1116-1125 Premature drying increases the GA-responsiveness of developing aleurone layers of barley (Hordeum vulgare L) grain
alpha-amylase; barley; desiccation; development; GA sensitivity
The effect of premature drying on the sensitivity of aleurone layer cells of developing barley (Hordeum vulgare L.) grain to gibberellic acid (GA(3)) was investigated. The capacity of barley aleurone layer cells to respond to GA(3), as indicated by alpha-amylase synthesis and secretion by de-embryonated grain, increased during the later stages of development. Aleurone layers of immature grain (younger than 30 d after anthesis; DAA) were not capable of producing alpha-amylase in response to GA(3); however, premature drying at this stage promoted GA-responsiveness resulting in the induction of mRNA for alpha-amylase and increased alpha-amylase synthesis and secretion. Preincubation of the immature grain or its maintenance at 100% relative humidity prior to exposure of the de-embryonated grain to GA(3) also led to an enhanced capacity of the aleurone layer to produce alpha-amylase and its mRNA as compared to the fresh, untreated grain. However, the amount of mRNA and enzyme produced was much lower than that induced by premature drying. Moreover, following these nondrying treatments, the aleurone layer cells remained unresponsive to exogenous GA(3); the same amount of enzyme was produced in the absence of applied GA(3). Transient expression of chimeric gene constructs in aleurone layer cells of de-embryonated grain suggest that drying up-regulates the alpha-amylase gene promoter in response to GA(3). We conclude that desiccation is required for barley aleurone layer cells to become responsive to GA(3) and hence express their full potential for alpha-amylase synthesis and secretion.
1.Kermode, AR. (1996) Mechanisms of intracellular protein transport and targeting in plant cells.Critical Reviews in Plant Sciences 15: 285-423 Mechanisms of intracellular protein transport and targeting in plant cells
protein targeting; protein folding; protein assembly; protein stability; secretory pathway; molecular chaperone
The specificity of protein targeting processes is the basis of maintaining structural and functional integrity of the cell, enabling the various subcellular compartments to carry out their unique metabolic roles. Studies in plants have progressed markedly in the last 5 years, and many of the specific signals involved in the transport and targeting of proteins to the nucleus, chloroplast, mitochondrion and microbody, and to organelles along the secretory pathway (endoplasmic reticulum [ER], Golgi complex, and vacuole) have been characterized. Exciting prospects include the identification of receptors involved in the recognition of protein targeting signals, mechanisms of vesicle targeting, and the role of mRNA targeting. Although important exceptions exist, a striking feature of the mechanisms and cellular machinery of protein targeting is their universality - among plants, animals, and eukaryotic microorganisms - and even between prokaryotes and eukaryotes. More information is required about the structural features of proteins that allow for their stable accumulation in a particular subcellular compartment, of particular interest to the plant genetic engineer. Our understanding of the rules that govern protein folding and oligomer assembly and how these processes relate to a protein's ultimate stability in the cell is limited.
