Mutation of AtPME2, a pH-Dependent Pectin Methylesterase, Affects Cell Wall Structure and Hypocotyl Elongation.

Pectin methylesterases (PMEs) modify homogalacturonan's chemistry and play a key role in regulating primary cell wall mechanical properties. Here, we report on Arabidopsis AtPME2, which we found to be highly expressed during lateral root emergence and dark-grown hypocotyl elongation. We showed that dark-grown hypocotyl elongation was reduced in knock-out mutant lines as compared to the control. The latter was related to the decreased total PME activity as well as increased stiffness of the cell wall in the apical part of the hypocotyl. To relate phenotypic analyses to the biochemical specificity of the enzyme, we produced the mature active enzyme using heterologous expression in Pichia pastoris and characterized it through the use of a generic plant PME antiserum. AtPME2 is more active at neutral compared to acidic pH, on pectins with a degree of 55-70% methylesterification. We further showed that the mode of action of AtPME2 can vary according to pH, from high processivity (at pH8) to low processivity (at pH5), and relate these observations to the differences in electrostatic potential of the protein. Our study brings insights into how the pH-dependent regulation by PME activity could affect the pectin structure and associated cell wall mechanical properties.

The maize low-lignin brown midrib3 mutant shows pleiotropic effects on photosynthetic and cell wall metabolisms in response to chilling.

Maize (Zea mays L.) is one of the major cereal crops in the world and is highly sensitive to low temperature. Here, changes in photosynthetic and cell wall metabolisms were investigated during a long chilling exposure in inbred line F2 and a low-lignin near-isogenic brown midrib3 mutant (F2bm3), which has a mutation in the caffeic acid O-methyltransferase (COMT) gene. Results revealed that the plant biomass was reduced, and this was more pronounced in F2bm3. Photosynthesis was altered in both lines with distinct changes in photosynthetic pigment content between F2bm3 and F2, indicating an alternative photoprotection mechanism between lines under chilling. Starch remobilization was observed in F2bm3 while concentrations of sucrose, fructose and starch increased in F2, suggesting a reduced sugar partitioning in F2. The cell wall was altered upon chilling, resulting in changes in the composition of glucuronorabinoxylan and a reduced cellulose level in F2. Chilling shifted lignin subunit composition in F2bm3 mutant to a higher proportion of p-hydroxyphenyl (H) units, whereas it resulted in lignin with a higher proportion of syringyl (S) residues in F2. On average, the total cell wall ferulic acid (FA) content increased in both genotypes, with an increase in ether-linked FA in F2bm3, suggesting a greater degree of cross-linking to lignin. The reinforcement of the cell wall with lignin enriched in H-units and a higher concentration in cell-wall-bound FA observed in F2bm3 as a response to chilling, could be a strategy to protect the photosystems.

In situ ESEM using 3-D printed and adapted accessories to observe living plantlets and their interaction with enzyme and fungus.

This paper describes an innovative way of using environmental scanning electron microscopy (ESEM) and the development of a suitable accessory to perform in situ observation of living seedlings in the ESEM. We provide details on fabrication of an accessory that proved to be essential for such experiments but inexpensive and easy to build in the laboratory, and present our in situ observations of the tissue and cell surfaces. Sample-specific configurations and optimized tuning of the ESEM were defined to maintain Arabidopsis and flax seedlings viable throughout repetitive exposure to the imaging conditions in the microscope chamber. This method permitted us to identify cells and tissues of the live plantlets and characterize their surface morphology during their early stage of growth and development. We could extend the application of this technique, to visualize the response of living cells and tissues to exogenous enzymatic treatments with polygalacturonase in Arabidopsis, and their interaction with hyphae of the wilt fungus Verticillium dahliae during artificial infection in flax plantlets. Our results provide an incentive to the use of the ESEM for in situ studies in plant science and a guide for researchers to optimize their electron microscopy observation in the relevant fields.

Integument-Specific Transcriptional Regulation in the Mid-Stage of Flax Seed Development Influences the Release of Mucilage and the Seed Oil Content.

Flax (Linum usitatissimum L.) seed oil, which accumulates in the embryo, and mucilage, which is synthesized in the seed coat, are of great economic importance for food, pharmaceutical as well as chemical industries. Theories on the link between oil and mucilage production in seeds consist in the spatio-temporal competition of both compounds for photosynthates during the very early stages of seed development. In this study, we demonstrate a positive relationship between seed oil production and seed coat mucilage extrusion in the agronomic model, flax. Three recombinant inbred lines were selected for low, medium and high mucilage and seed oil contents. Metabolite and transcript profiling (1H NMR and DNA oligo-microarrays) was performed on the seeds during seed development. These analyses showed main changes in the seed coat transcriptome during the mid-phase of seed development (25 Days Post-Anthesis), once the mucilage biosynthesis and modification processes are thought to be finished. These transcriptome changes comprised genes that are putatively involved in mucilage chemical modification and oil synthesis, as well as gibberellic acid (GA) metabolism. The results of this integrative biology approach suggest that transcriptional regulations of seed oil and fatty acid (FA) metabolism could occur in the seed coat during the mid-stage of seed development, once the seed coat carbon supplies have been used for mucilage biosynthesis and mechanochemical properties of the mucilage secretory cells.

The exogenous application of AtPGLR, an endo-polygalacturonase, triggers pollen tube burst and repair.

Plant cell wall remodeling plays a key role in the control of cell elongation and differentiation. In particular, fine-tuning of the degree of methylesterification of pectins was previously reported to control developmental processes as diverse as pollen germination, pollen tube elongation, emergence of primordia or elongation of dark-grown hypocotyls. However, how pectin degradation can modulate plant development has remained elusive. Here we report the characterization of a polygalacturonase (PG), AtPGLR, the gene for which is highly expressed at the onset of lateral root emergence in Arabidopsis. Due to gene compensation mechanisms, mutant approaches failed to determine the involvement of AtPGLR in plant growth. To overcome this issue, AtPGLR has been expressed heterologously in the yeast Pichia pastoris and biochemically characterized. We showed that AtPGLR is an endo-PG that preferentially releases non-methylesterified oligogalacturonides with a short degree of polymerization (< 8) at acidic pH. The application of the purified recombinant protein on Amaryllis pollen tubes, an excellent model for studying cell wall remodeling at acidic pH, induced abnormal pollen tubes or cytoplasmic leakage in the subapical dome of the pollen tube tip, where non-methylesterified pectin epitopes are detected. Those leaks could either be repaired by new ß-glucan deposits (mostly callose) in the cell wall or promoted dramatic burst of the pollen tube. Our work presents the full biochemical characterization of an Arabidopsis PG and highlights the importance of pectin integrity in pollen tube elongation.

Cytological Approaches Combined With Chemical Analysis Reveals the Layered Nature of Flax Mucilage.

The external seed coat cell layer of certain species is specialized in the production and extrusion of a polysaccharide matrix called mucilage. Variations in the content of the released mucilage have been mainly associated with genetically regulated physiological modifications. Understanding the mucilage extrusion process in crop species is of importance to gain deeper insight into the complex cell wall biosynthesis and dynamics. In this study, we took advantage of the varying polysaccharide composition and the size of the flax mucilage secretory cells (MSCs) to study mucilage composition and extrusion in this species of agricultural interest. We demonstrate herein that flax MSCs are structured in four superimposed layers and that rhamnogalacturonans I (RG I) are firstly synthesized, in the upper face, preceding arabinoxylan and glucan synthesis in MSC lower layers. Our results also reveal that the flax mucilage release originates from inside MSC, between the upper and deeper layers, the latter collaborating to trigger polysaccharide expansion, radial cell wall breaking and mucilage extrusion in a peeling fashion. Here, we provide evidence that the layer organization and polysaccharide composition of the MSCs regulate the mucilage release efficiency like a peeling mechanism. Finally, we propose that flax MSCs may represent an excellent model for further investigations of mucilage biosynthesis and its release.

PME58 plays a role in pectin distribution during seed coat mucilage extrusion through homogalacturonan modification.

Pectins are major components of plant primary cell walls. They include homogalacturonans (HGs), which are the most abundant pectin and can be the target of apoplastic enzymes like pectin methylesterases (PMEs) that control their methylesterification level. Several PMEs are expressed in the seed coat of Arabidopsis thaliana, particularly in mucilage secretory cells (MSCs). On the basis of public transcriptomic data, seven PME genes were selected and checked for their seed-specific expression by quantitative reverse transcription PCR. Of these, PME58 presented the highest level of expression and was specifically expressed in MSCs at the early stages of seed development. pme58 mutants presented two discrete phenotypes: (i) their adherent mucilage was less stained by ruthenium red when compared to wild-type seeds, but only in the presence of EDTA, a Ca(2+)chelator; and (ii) the MSC surface area was decreased. These phenotypes are the consequence of an increase in the degree of HG methylesterification connected to a decrease in PME activity. Analysis of the sugar composition of soluble and adherent mucilage showed that, in the presence of EDTA, sugars of adherent mucilage were more readily extracted in pme58 mutants. Immunolabelling with LM19, an antibody that preferentially recognizes unesterified HGs, also showed that molecular interactions with HGs were modified in the adherent mucilage of pme58 mutants, suggesting a role of PME58 in mucilage structure and organization. In conclusion, PME58 is the first PME identified to play a direct role in seed mucilage structure.

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.

Structural alteration of cell wall pectins accompanies pea development in response to cold.

Pea (Pisum sativum) cell wall metabolism in response to chilling was investigated in a frost-sensitive genotype 'Terese' and a frost-tolerant genotype 'Champagne'. Cell walls isolated from stipules of cold acclimated and non-acclimated plants showed that cold temperatures induce changes in polymers containing xylose, arabinose, galactose and galacturonic acid residues. In the tolerant cultivar Champagne, acclimation is accompanied by increases in homogalacturonan, xylogalacturonan and highly branched Rhamnogalacturonan I with branched and unbranched (1→5)-α-arabinans and (1→4)-ß-galactans. In contrast, the sensitive cultivar Terese accumulates substantial amounts of (1→4)-ß-xylans and glucuronoxylan, but not the pectins. Greater JIM7 labeling was observed in Champagne compared to Terese, indicating that cold acclimation also induces an increase in the degree of methylesterification of pectins. Significant decrease in polygalacturonase activities in both genotypes were observed at the end of cold acclimation. These data indicate a role for esterified pectins in cold tolerance. The possible functions for pectins and their associated arabinans and galactans in cold acclimation are discussed.

The transcription factor BELLRINGER modulates phyllotaxis by regulating the expression of a pectin methylesterase in Arabidopsis.

Plant leaves and flowers are positioned along the stem in a regular pattern. This pattern, which is referred to as phyllotaxis, is generated through the precise emergence of lateral organs and is controlled by gradients of the plant hormone auxin. This pattern is actively maintained during stem growth through controlled cell proliferation and elongation. The formation of new organs is known to depend on changes in cell wall chemistry, in particular the demethylesterification of homogalacturonans, one of the main pectic components. Here we report a dual function for the homeodomain transcription factor BELLRINGER (BLR) in the establishment and maintenance of the phyllotactic pattern in Arabidopsis. BLR is required for the establishment of normal phyllotaxis through the exclusion of pectin methylesterase PME5 expression from the meristem dome and for the maintenance of phyllotaxis through the activation of PME5 in the elongating stem. These results provide new insights into the role of pectin demethylesterification in organ initiation and cell elongation and identify an important component of the regulation mechanism involved.

Identification of pectin methylesterase 3 as a basic pectin methylesterase isoform involved in adventitious rooting in Arabidopsis thaliana.

• Here, we focused on the biochemical characterization of the Arabidopsis thaliana pectin methylesterase 3 gene (AtPME3; At3g14310) and its role in plant development. • A combination of biochemical, gene expression, Fourier transform-infrared (FT-IR) microspectroscopy and reverse genetics approaches were used. • We showed that AtPME3 is ubiquitously expressed in A. thaliana, particularly in vascular tissues. In cell wall-enriched fractions, only the mature part of the protein was identified, suggesting that it is processed before targeting the cell wall. In all the organs tested, PME activity was reduced in the atpme3-1 mutant compared with the wild type. This was related to the disappearance of an activity band corresponding to a pI of 9.6 revealed by a zymogram. Analysis of the cell wall composition showed that the degree of methylesterification (DM) of galacturonic acids was affected in the atpme3-1 mutant. A change in the number of adventitious roots was found in the mutant, which correlated with the expression of the gene in adventitious root primordia. • Our results enable the characterization of AtPME3 as a major basic PME isoform in A. thaliana and highlight its role in adventitious rooting.

Major changes in the cell wall during silique development in Arabidopsis thaliana.

Fruit development is a highly complex process, which involves major changes in plant metabolism leading to cell growth and differentiation. Changes in cell wall composition and structure play a major role in modulating cell growth. We investigated the changes in cell wall composition and the activities of associated enzymes during the dry fruit development of the model plant Arabidopsis thaliana. Silique development is characterized by several specific phases leading to fruit dehiscence and seed dispersal. We showed that early phases of silique growth were characterized by specific changes in non-cellulosic sugar content (rhamnose, arabinose, xylose, galactose and galacturonic acid). Xyloglucan oligosaccharide mass profiling further showed a strong increase in O-acetylated xyloglucans over the course of silique development, which could suggest a decreased capacity of xyloglucans to be associated with each other or to cellulose. The degree of methylesterification, mediated by the activity of pectin methylesterases (PMEs), decreased over the course of silique growth and dehiscence. The major changes in cell wall composition revealed by our analysis suggest that it could be major determinants in modulating cell wall rheology leading to growth or growth arrest.