BacA: a possible regulator that contributes to the biofilm formation of Pseudomonas aeruginosa.

Previously, we pointed out in P. aeruginosa PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes (pa3732 to pa3729) that we named bac for "Biofilm Associated Cluster." The present study focuses on the PA14_16140 protein, i.e., the PA3732 (BacA) homolog in the PA14 strain. The role of BacA in rhamnolipid secretion, biofilm formation and virulence, was confirmed by phenotypic experiments with a bacA mutant. Additional investigations allow to advance that the bac system involves in fact 6 genes organized in operon, i.e., bacA to bacF. At a molecular level, quantitative proteomic studies revealed an accumulation of the BAC cognate partners by the bacA sessile mutant, suggesting a negative control of BacA toward the bac operon. Finally, a first crystallographic structure of BacA was obtained revealing a structure homologous to chaperones or/and regulatory proteins.

Additive and Specific Effects of Elicitor Treatments on the Metabolic Profile of Arabidopsis thaliana.

Several elicitors of plant defense have been identified and numerous efforts to use them in the field have been made. Exogenous elicitor treatments mimic the in planta activation of pattern-triggered immunity (PTI), which relies on the perception of pathogen-associated molecular patterns (PAMPs) such as bacterial flg22 or fungal chitins. Early transcriptional responses to distinct PAMPs are mostly overlapping, regardless of the elicitor being used. However, it remains poorly known if the same patterns are observed for metabolites and proteins produced later during PTI. In addition, little is known about the impact of a combination of elicitors on PTI and the level of induced resistance to pathogens. Here, we monitored Arabidopsis thaliana resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) following application of flg22 and chitosan elicitors, used individually or in combination. A slight, but not statistically significant increase in induced resistance was observed when the elicitors were applied together when compared with individual treatments. We investigated the effect of these treatments on the metabolome by using an untargeted analysis. We found that the combination of flg22 and chitosan impacted a higher number of metabolites and deregulated specific metabolic pathways compared with the elicitors individually. These results contribute to a better understanding of plant responses to elicitors, which might help better rationalize their use in the field. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Towards understanding the extensive diversity of protein N-glycan structures in eukaryotes.

N-glycosylation is an important post-translational modification of proteins that has been highly conserved during evolution and is found in Eukaryota, Bacteria and Archaea. In eukaryotes, N-glycan processing is sequential, involving multiple specific steps within the secretory pathway as proteins travel through the endoplasmic reticulum and the Golgi apparatus. In this review, we first summarize the different steps of the N-glycan processing and further describe recent findings regarding the diversity of N-glycan structures in eukaryotic clades. This comparison allows us to explore the different regulation mechanisms of N-glycan processing among eukaryotic clades. Recent findings regarding the regulation of protein N-glycosylation are highlighted, especially the regulation of the biosynthesis of complex-type N-glycans through manganese and calcium homeostasis and the specific role of transmembrane protein 165 (TMEM165) for which homologous sequences have been identified in several eukaryotic clades. Further research will be required to characterize the function of TMEM165 homologous sequences in different eukaryotic clades.

Two Carbohydrate-Based Natural Extracts Stimulate in vitro Pollen Germination and Pollen Tube Growth of Tomato Under Cold Temperatures.

To date, it is widely accepted by the scientific community that many agricultural regions will experience more extreme temperature fluctuations. These stresses will undoubtedly impact crop production, particularly fruit and seed yields. In fact, pollination is considered as one of the most temperature-sensitive phases of plant development and until now, except for the time-consuming and costly processes of genetic breeding, there is no immediate alternative to address this issue. In this work, we used a multidisciplinary approach using physiological, biochemical, and molecular techniques for studying the effects of two carbohydrate-based natural activators on in vitro tomato pollen germination and pollen tube growth cultured in vitro under cold conditions. Under mild and strong cold temperatures, these two carbohydrate-based compounds significantly enhanced pollen germination and pollen tube growth. The two biostimulants did not induce significant changes in the classical molecular markers implicated in pollen tube growth. Neither the number of callose plugs nor the CALLOSE SYNTHASE genes expression were significantly different between the control and the biostimulated pollen tubes when pollens were cultivated under cold conditions. PECTIN METHYLESTERASE (PME) activities were also similar but a basic PME isoform was not produced or inactive in pollen grown at 8°C. Nevertheless, NADPH oxidase (RBOH) gene expression was correlated with a higher number of viable pollen tubes in biostimulated pollen tubes compared to the control. Our results showed that the two carbohydrate-based products were able to reduce in vitro the effect of cold temperatures on tomato pollen tube growth and at least for one of them to modulate reactive oxygen species production.

New Insights into Plant Extracellular DNA. A Study in Soybean Root Extracellular Trap.

exDNA is found in various organisms, including plants. However, plant exDNA has thus far received little attention related to its origin and role in the RET (root extracellular trap). In this study, we performed the first high-throughput genomic sequencing of plant exDNA from a Fabaceae with worldwide interest: soybean (Glycine max (L.) Merr.). The origin of this exDNA was first investigated in control condition, and the results show high-coverage on organelles (mitochondria/plastid) DNA relative to nuclear DNA, as well as a mix of coding and non-coding sequences. In the second part of this study, we investigated if exDNA release was modified during an elicitation with PEP-13 (a peptide elicitor from oomycete genus Phytophthora). Our results show that treatment of roots with PEP-13 does not affect the composition of exDNA.

Identification of two compounds able to improve flax resistance towards Fusarium oxysporum infection.

Plants are surrounded by a diverse range of microorganisms that causes serious crop losses and requires the use of pesticides. Flax is a major crop in Normandy used for its fibres and is regularly challenged by the pathogenic fungus Fusarium oxysporum (Fo) f. sp. lini. To protect themselves, plants use "innate immunity" as a first line of defense level against pathogens. Activation of plant defense with elicitors could be an alternative for crop plant protection. A previous work was conducted by screening a chemical library and led to the identification of compounds able to activate defense responses in Arabidopsis thaliana. Four compounds were tested for their abilities to improve resistance of two flax varieties against Fo. Two of them, one natural (holaphyllamine or HPA) and one synthetic (M4), neither affected flax nor Fo growth. HPA and M4 induced oxidative burst and callose deposition. Furthermore, HPA and M4 caused changes in the expression patterns of defense-related genes coding a glucanase and a chitinase-like. Finally, plants pre-treated with HPA or M4 exhibited a significant decrease in the disease symptoms. Together, these findings demonstrate that HPA and M4 are able to activate defense responses in flax and improve its resistance against Fo infection.

Characterization of a GDP-Fucose Transporter and a Fucosyltransferase Involved in the Fucosylation of Glycoproteins in the Diatom Phaeodactylum tricornutum.

Although Phaeodactylum tricornutum is gaining importance in plant molecular farming for the production of high-value molecules such as monoclonal antibodies, little is currently known about key cell metabolism occurring in this diatom such as protein glycosylation. For example, incorporation of fucose residues in the glycans N-linked to protein in P. tricornutum is questionable. Indeed, such epitope has previously been found on N-glycans of endogenous glycoproteins in P. tricornutum. Meanwhile, the potential immunogenicity of the α(1,3)-fucose epitope present on plant-derived biopharmaceuticals is still a matter of debate. In this paper, we have studied molecular actors potentially involved in the fucosylation of the glycoproteins in P. tricornutum. Based on sequence similarities, we have identified a putative P. tricornutum GDP-L-fucose transporter and three fucosyltransferase (FuT) candidates. The putative P. tricornutum GDP-L-fucose transporter coding sequence was expressed in the Chinese Hamster Ovary (CHO)-gmt5 mutant lacking its endogenous GDP-L-fucose transporter activity. We show that the P. tricornutum transporter is able to rescue the fucosylation of proteins in this CHO-gmt5 mutant cell line, thus demonstrating the functional activity of the diatom transporter and its appropriate Golgi localization. In addition, we overexpressed one of the three FuT candidates, namely the FuT54599, in P. tricornutum and investigated its localization within Golgi stacks of the diatom. Our findings show that overexpression of the FuT54599 leads to a significant increase of the α(1,3)-fucosylation of the diatom endogenous glycoproteins.

Evolution of Cell Wall Polymers in Tip-Growing Land Plant Gametophytes: Composition, Distribution, Functional Aspects and Their Remodeling.

During evolution of land plants, the first colonizing species presented leafy-dominant gametophytes, found in non-vascular plants (bryophytes). Today, bryophytes include liverworts, mosses, and hornworts. In the first seedless vascular plants (lycophytes), the sporophytic stage of life started to be predominant. In the seed producing plants, gymnosperms and angiosperms , the gametophytic stage is restricted to reproduction. In mosses and ferns, the haploid spores germinate and form a protonema, which develops into a leafy gametophyte producing rhizoids for anchorage, water and nutrient uptakes. The basal gymnosperms (cycads and Ginkgo) reproduce by zooidogamy. Their pollen grains develop a multi-branched pollen tube that penetrates the nucellus and releases flagellated sperm cells that swim to the egg cell. The pollen grain of other gymnosperms (conifers and gnetophytes) as well as angiosperms germinates and produces a pollen tube that directly delivers the sperm cells to the ovule (siphonogamy). These different gametophytes, which are short or long-lived structures, share a common tip-growing mode of cell expansion. Tip-growth requires a massive cell wall deposition to promote cell elongation, but also a tight spatial and temporal control of the cell wall remodeling in order to modulate the mechanical properties of the cell wall. The growth rate of these cells is very variable depending on the structure and the species, ranging from very slow (protonemata, rhizoids, and some gymnosperm pollen tubes), to a slow to fast-growth in other gymnosperms and angiosperms. In addition, the structural diversity of the female counterparts in angiosperms (dry, semi-dry vs wet stigmas, short vs long, solid vs hollow styles) will impact the speed and efficiency of sperm delivery. As the evolution and diversity of the cell wall polysaccharides accompanied the diversification of cell wall structural proteins and remodeling enzymes, this review focuses on our current knowledge on the biochemistry, the distribution and remodeling of the main cell wall polymers (including cellulose, hemicelluloses, pectins, callose, arabinogalactan-proteins and extensins), during the tip-expansion of gametophytes from bryophytes, pteridophytes (lycophytes and monilophytes), gymnosperms and the monocot and eudicot angiosperms.

Comparative in depth RNA sequencing of P. tricornutum's morphotypes reveals specific features of the oval morphotype.

Phaeodactylum tricornutum is the most studied diatom encountered principally in coastal unstable environments. It has been hypothesized that the great adaptability of P. tricornutum is probably due to its pleomorphism. Indeed, P. tricornutum is an atypical diatom since it can display three morphotypes: fusiform, triradiate and oval. Currently, little information is available regarding the physiological significance of this morphogenesis. In this study, we adapted P. tricornutum Pt3 strain to obtain algal culture particularly enriched in one dominant morphotype: fusiform, triradiate or oval. These cultures were used to run high-throughput RNA-Sequencing. The whole mRNA transcriptome of each morphotype was determined. Pairwise comparisons highlighted biological processes and molecular functions which are up- and down-regulated. Finally, intersection analysis allowed us to identify the specific features from the oval morphotype which is of particular interest as it is often described to be more resistant to stresses. This study represent the first transcriptome wide characterization of the three morphotypes from P. tricornutum performed on cultures specifically enriched issued from the same Pt3 strain. This work represents an important step for the understanding of the morphogenesis in P. tricornutum and highlights the particular features of the oval morphotype.

Endoplasmic Reticulum Body-Related Gene Expression in Different Root Zones of Arabidopsis Isolated by Laser-Assisted Microdissection.

Endoplasmic reticulum (ER) bodies are important organelles for root defense. However, little is known regarding the genetic control of their formation in root tissues. In the present study, (L.) Heynh. roots were dissected using laser-assisted microdissection (LAM) with minimal sample preparation (no fixation or embedding steps) and the expression of genes associated with ER body formation and function was assessed by real-time quantitative reverse-transcription polymerase chain reaction (RT-qRT-PCR) in the presence and absence of the defense phytohormone methyl jasmonate (MeJA). Zones of interest were identified in plants overexpressing a fluorescent construct; these being the root cap zone, meristematic zone, elongation zone, and differentiation zone. Given their role in ER body formation, the expression of the genes , , , , and was evaluated in the whole root and in the four dissected root zones using RT-qRT-PCR. Our data show that the expression level of all five genes differs in a root-zone-specific manner in untreated roots. They also reveal that all of them are overexpressed in response to MeJA with the two genes being the most highly overexpressed in the EZ. Finally, the gene, encoding for a transcription factor that regulates the expression of the four other genes, is the first to respond to MeJA, supporting its central role in ER body formation and function in root defense.

PECTIN METHYLESTERASE48 is involved in Arabidopsis pollen grain germination.

Germination of pollen grains is a crucial step in plant reproduction. However, the molecular mechanisms involved remain unclear. We investigated the role of PECTIN METHYLESTERASE48 (PME48), an enzyme implicated in the remodeling of pectins in Arabidopsis (Arabidopsis thaliana) pollen. A combination of functional genomics, gene expression, in vivo and in vitro pollen germination, immunolabeling, and biochemical analyses was used on wild-type and Atpme48 mutant plants. We showed that AtPME48 is specifically expressed in the male gametophyte and is the second most expressed PME in dry and imbibed pollen grains. Pollen grains from homozygous mutant lines displayed a significant delay in imbibition and germination in vitro and in vivo. Moreover, numerous pollen grains showed two tips emerging instead of one in the wild type. Immunolabeling and Fourier transform infrared analyses showed that the degree of methylesterification of the homogalacturonan was higher in pme48-/- pollen grains. In contrast, the PME activity was lower in pme48-/-, partly due to a reduction of PME48 activity revealed by zymogram. Interestingly, the wild-type phenotype was restored in pme48-/- with the optimum germination medium supplemented with 2.5 mm calcium chloride, suggesting that in the wild-type pollen, the weakly methylesterified homogalacturonan is a source of Ca(2+) necessary for pollen germination. Although pollen-specific PMEs are traditionally associated with pollen tube elongation, this study provides strong evidence that PME48 impacts the mechanical properties of the intine wall during maturation of the pollen grain, which, in turn, influences pollen grain germination.

Protein N-glycosylation in eukaryotic microalgae and its impact on the production of nuclear expressed biopharmaceuticals.

Microalgae are currently used for the production of food compounds. Recently, few microalgae species have been investigated as potential biofactories for the production of biopharmaceuticals. Indeed in this context, microalgae are cheap, classified as Generally Recognized As Safe (GRAS) organisms and can be grown easily. However, problems remain to be solved before any industrial production of microalgae-made biopharmaceuticals. Among them, post-translational modifications of the proteins need to be considered. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. Therefore, the evaluation of microalgae as alternative cell factory for biopharmaceutical productions thus requires to investigate their N-glycosylation capability in order to determine to what extend it differs from their human counterpart and to determine appropriate strategies for remodeling the microalgae glycosylation into human-compatible oligosaccharides. Here, we review the secreted recombinant proteins which have been successfully produced in microalgae. We also report on recent bioinformatics and biochemical data concerning the structure of glycans N-linked to proteins from various microalgae phyla and comment the consequences on the glycan engineering strategies that may be necessary to render those microalgae-made biopharmaceuticals compatible with human therapy.

The cell wall pectic polymer rhamnogalacturonan-II is required for proper pollen tube elongation: implications of a putative sialyltransferase-like protein.

BACKGROUND AND AIMS: Rhamnogalacturonan-II (RG-II) is one of the pectin motifs found in the cell wall of all land plants. It contains sugars such as 2-keto-3-deoxy-d-lyxo-heptulosaric acid (Dha) and 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo), and within the wall RG-II is mostly found as a dimer via a borate diester cross-link. To date, little is known regarding the biosynthesis of this motif. Here, after a brief review of our current knowledge on RG-II structure, biosynthesis and function in plants, this study explores the implications of the presence of a Golgi-localized sialyltransferase-like 2 (SIA2) protein that is possibly involved in the transfer of Dha or Kdo in the RG-II of Arabidopsis thaliana pollen tubes, a fast-growing cell type used as a model for the study of cell elongation. METHODS: Two heterozygous mutant lines of arabidopsis (sia2-1+/- and qrt1 × sia2-2+/-) were investigated. sia2-2+/- was in a quartet1 background and the inserted T-DNA contained the reporter gene ß-glucuronidase (GUS) under the pollen-specific promoter LAT52. Pollen germination and pollen tube phenotype and growth were analysed both in vitro and in vivo by microscopy. KEY RESULTS: Self-pollination of heterozygous lines produced no homozygous plants in the progeny, which may suggest that the mutation could be lethal. Heterozygous mutants displayed a much lower germination rate overall and exhibited a substantial delay in germination (20 h of delay to reach 30 % of pollen grain germination compared with the wild type). In both lines, mutant pollen grains that were able to produce a tube had tubes that were either bursting, abnormal (swollen or dichotomous branching tip) or much shorter compared with wild-type pollen tubes. In vivo, mutant pollen tubes were restricted to the style, whereas the wild-type pollen tubes were detected at the base of the ovary. CONCLUSIONS: This study highlights that the mutation in arabidopsis SIA2 encoding a sialyltransferase-like protein that may transfer Dha or Kdo on the RG-II motif has a dramatic effect on the stability of the pollen tube cell wall.

Deciphering the responses of root border-like cells of Arabidopsis and flax to pathogen-derived elicitors.

Plant pathogens including fungi and bacteria cause many of the most serious crop diseases. The plant innate immune response is triggered upon recognition of microbe-associated molecular patterns (MAMPs) such as flagellin22 and peptidoglycan. To date, very little is known of MAMP-mediated responses in roots. Root border cells are cells that originate from root caps and are released individually into the rhizosphere. Root tips of Arabidopsis (Arabidopsis thaliana) and flax (Linum usitatissimum) release cells known as "border-like cells." Whereas root border cells of pea (Pisum sativum) are clearly involved in defense against fungal pathogens, the function of border-like cells remains to be established. In this study, we have investigated the responses of root border-like cells of Arabidopsis and flax to flagellin22 and peptidoglycan. We found that both MAMPs triggered a rapid oxidative burst in root border-like cells of both species. The production of reactive oxygen species was accompanied by modifications in the cell wall distribution of extensin epitopes. Extensins are hydroxyproline-rich glycoproteins that can be cross linked by hydrogen peroxide to enhance the mechanical strength of the cell wall. In addition, both MAMPs also caused deposition of callose, a well-known marker of MAMP-elicited defense. Furthermore, flagellin22 induced the overexpression of genes involved in the plant immune response in root border-like cells of Arabidopsis. Our findings demonstrate that root border-like cells of flax and Arabidopsis are able to perceive an elicitation and activate defense responses. We also show that cell wall extensin is involved in the innate immunity response of root border-like cells.

Exploring the N-glycosylation pathway in Chlamydomonas reinhardtii unravels novel complex structures.

Chlamydomonas reinhardtii is a green unicellular eukaryotic model organism for studying relevant biological and biotechnological questions. The availability of genomic resources and the growing interest in C. reinhardtii as an emerging cell factory for the industrial production of biopharmaceuticals require an in-depth analysis of protein N-glycosylation in this organism. Accordingly, we used a comprehensive approach including genomic, glycomic, and glycoproteomic techniques to unravel the N-glycosylation pathway of C. reinhardtii. Using mass-spectrometry-based approaches, we found that both endogenous soluble and membrane-bound proteins carry predominantly oligomannosides ranging from Man-2 to Man-5. In addition, minor complex N-linked glycans were identified as being composed of partially 6-O-methylated Man-3 to Man-5 carrying one or two xylose residues. These findings were supported by results from a glycoproteomic approach that led to the identification of 86 glycoproteins. Here, a combination of in-source collision-induced dissodiation (CID) for glycan fragmentation followed by mass tag-triggered CID for peptide sequencing and PNGase F treatment of glycopeptides in the presence of (18)O-labeled water in conjunction with CID mass spectrometric analyses were employed. In conclusion, our data support the notion that the biosynthesis and maturation of N-linked glycans in the endoplasmic reticulum and Golgi apparatus occur via a GnT I-independent pathway yielding novel complex N-linked glycans that maturate differently from their counterparts in land plants.

N-glycans of Phaeodactylum tricornutum diatom and functional characterization of its N-acetylglucosaminyltransferase I enzyme.

N-glycosylation, a major co- and post-translational event in the synthesis of proteins in eukaryotes, is unknown in aquatic photosynthetic microalgae. In this paper, we describe the N-glycosylation pathway in the diatom Phaeodactylum tricornutum. Bio-informatic analysis of its genome revealed the presence of a complete set of sequences potentially encoding for proteins involved in the synthesis of the lipid-linked Glc(3)Man(9)GlcNAc(2)-PP-dolichol N-glycan, some subunits of the oligosaccharyltransferase complex, as well as endoplasmic reticulum glucosidases and chaperones required for protein quality control and, finally, the α-mannosidase I involved in the trimming of the N-glycan precursor into Man-5 N-glycan. Moreover, one N-acetylglucosaminyltransferase I, a Golgi glycosyltransferase that initiates the synthesis of complex type N-glycans, was predicted in the P. tricornutum genome. We demonstrated that this gene encodes for an active N-acetylglucosaminyltransferase I, which is able to restore complex type N-glycans maturation in the Chinese hamster ovary Lec1 mutant, defective in its endogeneous N-acetylglucosaminyltransferase I. Consistent with these data, the structural analyses of N-linked glycans demonstrated that P. tricornutum proteins carry mainly high mannose type N-glycans ranging from Man-5 to Man-9. Although representing a minor glycan population, paucimannose N-glycans were also detected, suggesting the occurrence of an N-acetylglucosaminyltransferase I-dependent maturation of N-glycans in this diatom.

Cytosolic N-terminal arginine-based signals together with a luminal signal target a type II membrane protein to the plant ER.

BACKGROUND: In eukaryotic cells, the membrane compartments that constitute the exocytic pathway are traversed by a constant flow of lipids and proteins. This is particularly true for the endoplasmic reticulum (ER), the main "gateway of the secretory pathway", where biosynthesis of sterols, lipids, membrane-bound and soluble proteins, and glycoproteins occurs. Maintenance of the resident proteins in this compartment implies they have to be distinguished from the secretory cargo. To this end, they must possess specific ER localization determinants to prevent their exit from the ER, and/or to interact with receptors responsible for their retrieval from the Golgi apparatus. Very few information is available about the signal(s) involved in the retention of membrane type II protein in the ER but it is generally accepted that sorting of ER type II cargo membrane proteins depends on motifs mainly located in their cytosolic tails. RESULTS: Here, using Arabidopsis glucosidase I as a model, we have identified two types of signals sufficient for the location of a type II membrane protein in the ER. A first signal is located in the luminal domain, while a second signal corresponds to a short amino acid sequence located in the cytosolic tail of the membrane protein. The cytosolic tail contains at its N-terminal end four arginine residues constitutive of three di-arginine motifs (RR, RXR or RXXR) independently sufficient to confer ER localization. Interestingly, when only one di-arginine motif is present, fusion proteins are located both in the ER and in mobile punctate structures, distinct but close to Golgi bodies. Soluble and membrane ER protein markers are excluded from these punctate structures, which also do not colocalize with an ER-exit-site marker. It is hypothesized they correspond to sites involved in Golgi to ER retrotransport. CONCLUSION: Altogether, these results clearly show that cytosolic and luminal signals responsible for ER retention could coexist in a same type II membrane protein. These data also suggest that both retrieval and retention mechanisms govern protein residency in the ER membrane. We hypothesized that mobile punctate structures not yet described at the ER/Golgi interface and tentatively named GERES, could be involved in retrieval mechanisms from the Golgi to the ER.

Down-regulated expression of plant-specific glycoepitopes in alfalfa.

SUMMARY: Compared with other plant expression systems used for pharmaceutical protein production, alfalfa offers the advantage of very homogeneous N-glycosylation. Therefore, this plant was selected for further attempts at glycoengineering. Two main approaches were developed in order to humanize N-glycosylation in alfalfa. The first was a knock-down of two plant-specific N-glycan maturation enzymes, beta1,2-xylosyltransferase and alpha1,3-fucosyltransferases, using sense, antisense and RNA interference strategies. In a second approach, with the ultimate goal of rebuilding the whole human sialylation pathway, human beta1,4-galactosyltransferase was expressed in alfalfa in a native form or in fusion with a targeting domain from N-acetylglucosaminyltransferase I, a glycosyltransferase located in the early Golgi apparatus in Nicotiana tabacum. Both knock-down and knock-in strategies strongly, but not completely, inhibited the biosynthesis of alpha1,3-fucose- and beta1,2-xylose-containing glycoepitopes in transgenic alfalfa. However, recombinant human beta1,4-galactosyltransferase activity in transgenic alfalfa completely prevented the accumulation of the Lewis a glycoepitope on complex N-glycans.

Water transport by aquaporins in the extant plant Physcomitrella patens.

Although aquaporins (AQPs) have been shown to increase membrane water permeability in many cell types, the physiological role of this increase was not always obvious. In this report, we provide evidence that in the leafy stage of development (gametophore) of the moss Physcomitrella patens, AQPs help to replenish more rapidly the cell water that is lost by transpiration, at least if some water is in the direct vicinity of the moss plant. Three AQP genes were cloned in P. patens: PIP2;1, PIP2;2, and PIP2;3. The water permeability of the membrane was measured in protoplasts from leaves and protonema. A significant decrease was measured in protoplasts from leaves and protonema of PIP2;1 or PIP2;2 knockouts but not the PIP2;3 knockout. No phenotype was observed when knockout plants were grown in closed petri dishes with ample water supply. Gametophores isolated from the wild type and the pip2;3 mutant were not sensitive to moderate water stress, but pip2;1 or pip2;2 gametophores expressed a water stress phenotype. The knockout mutant leaves were more bent and twisted, apparently suffering from an important loss of cellular water. We propose a model to explain how the AQPs PIP2;1 and PIP2;2 delay leaf dessication in a drying atmosphere. We suggest that in ancestral land plants, some 400 million years ago, APQs were already used to facilitate the absorption of water.

Targeting of proConA to the plant vacuole depends on its nine amino-acid C-terminal propeptide.

Concanavalin A (ConA) is a well characterized and extensively used lectin accumulated in the protein bodies of jack bean cotyledons. ConA is synthesized as an inactive precursor proConA. The maturation of inactive proConA into biologically active ConA is a complex process including the removal of an internal glycopeptide and a C-terminal propeptide (CTPP), followed by a head-to-tail ligation of the two largest polypeptides. The cDNA encoding proConA was cloned and expressed in tobacco BY-2 cells. ProConA was slowly transported to the vacuole where its maturation into ConA was similar to that in jack bean cotyledons, apart from an incomplete final ligation. To investigate the role of the nine amino acid CTPP, a truncated form lacking the propeptide (proConADelta9) was expressed in BY-2 cells. In contrast to proConA, proConADelta9 was rapidly chased out of the endoplasmic reticulum (ER) and secreted into the culture medium. The CTPP was then fused to the C-terminal end of a secreted form of green fluorescent protein (secGFP). When expressed in tobacco BY-2 cells and leaf protoplasts, the chimaeric protein was located in the vacuole whereas secGFP was located in the culture medium and in the vacuole. Altogether, our results show we have isolated a new C-terminal vacuolar sorting determinant.