7. 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
6. 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
5. 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
4. 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
3. 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
2. 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
1. 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
