HIGHLY METHYL ESTERIFIED SEEDS is a pectin methyl esterase involved in embryo development.

Homogalacturonan pectin domains are synthesized in a highly methyl-esterified form that later can be differentially demethyl esterified by pectin methyl esterase (PME) to strengthen or loosen plant cell walls that contain pectin, including seed coat mucilage, a specialized secondary cell wall of seed coat epidermal cells. As a means to identify the active PMEs in seed coat mucilage, we identified seven PMEs expressed during seed coat development. One of these, HIGHLY METHYL ESTERIFIED SEEDS (HMS), is abundant during mucilage secretion, peaking at 7 d postanthesis in both the seed coat and the embryo. We have determined that this gene is required for normal levels of PME activity and homogalacturonan methyl esterification in the seed. The hms-1 mutant displays altered embryo morphology and mucilage extrusion, both of which are a consequence of defects in embryo development. A significant decrease in the size of cells in the embryo suggests that the changes in embryo morphology are a consequence of lack of cell expansion. Progeny from a cross between hms-1 and the previously characterized PME inhibitor5 overexpression line suggest that HMS acts independently from other cell wall-modifying enzymes in the embryo. We propose that HMS is required for cell wall loosening in the embryo to facilitate cell expansion during the accumulation of storage reserves and that its role in the seed coat is masked by redundancy.

Trans-Golgi network localized ECHIDNA/Ypt interacting protein complex is required for the secretion of cell wall polysaccharides in Arabidopsis.

The secretion of cell wall polysaccharides through the trans-Golgi network (TGN) is required for plant cell elongation. However, the components mediating the post-Golgi secretion of pectin and hemicellulose, the two major cell wall polysaccharides, are largely unknown. We identified evolutionarily conserved YPT/RAB GTPase Interacting Protein 4a (YIP4a) and YIP4b (formerly YIP2), which form a TGN-localized complex with ECHIDNA (ECH) in Arabidopsis thaliana. The localization of YIP4 and ECH proteins at the TGN is interdependent and influences the localization of VHA-a1 and SYP61, which are key components of the TGN. YIP4a and YIP4b act redundantly, and the yip4a yip4b double mutants have a cell elongation defect. Genetic, biochemical, and cell biological analyses demonstrate that the ECH/YIP4 complex plays a key role in TGN-mediated secretion of pectin and hemicellulose to the cell wall in dark-grown hypocotyls and in secretory cells of the seed coat. In keeping with these observations, Fourier transform infrared microspectroscopy analysis revealed that the ech and yip4a yip4b mutants exhibit changes in their cell wall composition. Overall, our results reveal a TGN subdomain defined by ECH/YIP4 that is required for the secretion of pectin and hemicellulose and distinguishes the role of the TGN in secretion from its roles in endocytic and vacuolar trafficking.

Tuning of pectin methylesterification: consequences for cell wall biomechanics and development.

MAIN CONCLUSION: Recent publications have increased our knowledge of how pectin composition and the degree of homogalacturonan methylesterification impact the biochemical and biomechanical properties of plant cell walls, plant development, and plants' interactions with their abiotic and biotic environments. Experimental observations have shown that the relationships between the DM, the pattern of de-methylesterificaton, its effect on cell wall elasticity, other biomechanical parameters, and growth are not straightforward. Working towards a detailed understanding of these relationships at single cell resolution is one of the big tasks of pectin research. Pectins are highly complex polysaccharides abundant in plant primary cell walls. New analytical and microscopy techniques are revealing the composition and mechanical properties of the cell wall and increasing our knowledge on the topic. Progress in plant physiological research supports a link between cell wall pectin modifications and plant development and interactions with the environment. Homogalacturonan pectins, which are major components of the primary cell wall, have a potential for modifications such as methylesterification, as well as an ability to form cross-linked structures with divalent cations. This contributes to changing the mechanical properties of the cell wall. This review aims to give a comprehensive overview of the pectin component homogalacturonan, including its synthesis, modification, regulation and role in the plant cell wall.

Demethylesterification of cell wall pectins in Arabidopsis plays a role in seed germination.

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.

Cell wall polysaccharides are mislocalized to the Vacuole in echidna mutants.

During cell wall biosynthesis, the Golgi apparatus is the platform for cell wall matrix biosynthesis and the site of packaging, of both matrix polysaccharides and proteins, into secretory vesicles with the correct targeting information. The objective of this study was to dissect the post-Golgi trafficking of cell wall polysaccharides using echidna as a vesicle traffic mutant of Arabidopsis thaliana and the pectin-secreting cells of the seed coat as a model system. ECHIDNA encodes a trans-Golgi network (TGN)-localized protein, which was previously shown to be required for proper structure and function of the secretory pathway. In echidna mutants, some cell wall matrix polysaccharides accumulate inside cells, rather than being secreted to the apoplast. In this study, live cell imaging of fluorescent protein markers as well as transmission electron microscopy (TEM)/immunoTEM of cryofixed seed coat cells were used to examine the consequences of TGN disorganization in echidna mutants under conditions of high polysaccharide production and secretion. While in wild-type seed coat cells, pectin is secreted to the apical surface, in echidna, polysaccharides accumulate in post-Golgi vesicles, the central lytic vacuole and endoplasmic reticulum-derived bodies. In contrast, proteins were partially mistargeted to internal multilamellar membranes in echidna. These results suggest that while secretion of both cell wall polysaccharides and proteins at the TGN requires ECHIDNA, different vesicle trafficking components may mediate downstream events in their secretion from the TGN.

The chloroplastic lipocalin AtCHL prevents lipid peroxidation and protects Arabidopsis against oxidative stress.

Lipocalins are small ligand-binding proteins with a simple tertiary structure that gives them the ability to bind small, generally hydrophobic, molecules. Recent studies have shown that animal lipocalins play important roles in the regulation of developmental processes and are involved in tolerance to oxidative stress. Plants also possess various types of lipocalins, and bioinformatics analyses have predicted that some lipocalin members may be present in the chloroplast. Here we report the functional characterization of the Arabidopsis thaliana chloroplastic lipocalin AtCHL. Cellular fractionation showed that AtCHL is a thylakoid lumenal protein. Drought, high light, paraquat and abscisic acid treatments induce AtCHL transcript and protein accumulation. Under normal growth conditions, knockout (KO) and over-expressing (OEX) lines do not differ from wild-type plants in terms of phenotype and photosynthetic performance. However, KO plants, which do not accumulate AtCHL, show more damage upon photo-oxidative stress induced by drought, high light or paraquat. In contrast, a high level of AtCHL allows OEX plants to cope better with these stress conditions. When exposed to excess light, KO plants display a rapid accumulation of hydroxy fatty acids relative to the wild-type, whereas the lipid peroxidation level remains very low in OEX plants. The increased lipid peroxidation in KO plants is mediated by singlet oxygen and is not correlated with photo-inhibition of the photosystems. This work provides evidence suggesting that AtCHL is involved in the protection of thylakoidal membrane lipids against reactive oxygen species, especially singlet oxygen, produced in excess light.

Overexpression of a pectin methylesterase inhibitor in Arabidopsis thaliana leads to altered growth morphology of the stem and defective organ separation.

The methylesterification status of cell wall pectins, mediated through the interplay of pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs), influences the biophysical properties of plant cell walls. We found that the overexpression of a PMEI gene in Arabidopsis thaliana plants caused the stems to develop twists and loops, most strongly around points on the stem where leaves or inflorescences failed to separate from the main stem. Altered elasticity of the stem, underdevelopment of the leaf cuticle, and changes in the sugar composition of the cell walls of stems were evident in the PMEI overexpression lines. We discuss the mechanisms that potentially underlie the aberrant growth phenotypes.