Degree Of Pectin Methylesterification Research Articles

Phosphorus deficiency-induced cell wall pectin demethylesterification enhances cadmium accumulation in roots of Salix caprea

Regions affected by heavy metal contamination frequently encounter phosphorus (P) deficiency. Numerous studies highlight crucial role of P in facilitating cadmium (Cd) accumulation in woody plants. However, the regulatory mechanism by which P affects Cd accumulation in roots remains ambiguous. This study aims to investigate the effects of phosphorus (P) deficiency on Cd accumulation, Cd subcellular distribution, and cell wall components in the roots of Salix caprea under Cd stress. The results revealed that under P deficiency conditions, there was a 35.4% elevation in Cd content in roots, coupled with a 60.1% reduction in Cd content in shoots, compared to the P sufficiency conditions. Under deficient P conditions, the predominant response of roots to Cd exposure was the increased sequestration of Cd in root cell walls. The sequestration of Cd in root cell walls increased from 37.1% under sufficient P conditions to 66.7% under P deficiency, with pectin identified as the primary Cd binding site under both P conditions. Among cell wall components, P deficiency led to a significant 31.7% increase in Cd content within pectin compared to P sufficiency conditions, but did not change the pectin content. Notably, P deficiency significantly increased pectin methylesterase (PME) activity by regulating the expression of PME and PMEI genes, leading to a 10.4% reduction in the degree of pectin methylesterification. This may elucidate the absence of significant changes in pectin content under P deficiency conditions and the concurrent increase in Cd accumulation in pectin. Fourier transform infrared spectroscopy (FTIR) results indicated an increase in carboxyl groups in the root cell walls under P deficiency compared to sufficient P treatment. The results provide deep insights into the mechanisms of higher Cd accumulation in root mediated by P deficiency.

Texture classification and in situ cation measurements by SEM-EDX provide detailed quantitative insights into cell wall cation interaction changes during ageing, soaking, and cooking of red kidney beans

Hard-to-cook (HTC) is a textural defect that delays the softening of common bean seeds during cooking. While this defect is commonly associated with conventionally stored beans, soaking/cooking of beans in CaCl2 solutions or sodium acetate buffer can also prolong the cooking time of beans due to formation of Ca2+ crosslinked pectin retarding bean softening during cooking. In this study, the role of the cell wall-bound Mg2+/Ca2+ content and the degree of pectin methyl esterification (DM) was quantified, as important factors for bean texture-related changes stipulated in the pectin-cation-phytate hypothesis, the most plausible hypothesis of HTC development. Evaluation of texture changes during cooking of conventionally aged beans (35 °C and 83% RH for up to 20 weeks), beans soaked/cooked in CaCl2 solutions (0.01 to 0.1 M) or soaked in 0.1 M sodium acetate buffer (pH 4.4) revealed large bean-to-bean variations. Therefore a texture-based classification approach was used to better capture the relation between texture characteristics and cell wall polymer, in particular pectin, related changes. While cell wall-bound Ca2+ and pectin DM did not change/were not related to the texture variation during cooking of fresh beans, increased cell wall-bound Ca2+ and decreased pectin DM were associated with prolonged conventional storage of beans and their texture changes during subsequent cooking (due to pectin cross linking, retarding its solubilization during cooking). Exogenously added Ca2+ from pre-treating beans in CaCl2 solutions promoted to a great extent the cell wall-bound Ca2+ during soaking but even more so during cooking, complementing the harder texture associated with these beans during cooking (compared to conventionally stored and fresh beans). Similarly, free Ca2+ endogenously generated by phytase-catalysed phytate hydrolysis (beans treated by acetate buffer) promoted crosslinking of pectin by Ca2+ (cell wall-bound Ca2+), delaying softening of beans during cooking.

Maintenance of Methyl-Esterified Pectin Level in Pollen Mother-Cell Stages Is Required for Microspore Development.

Pectin modification and degradation are vital for plant development, although the underlying mechanisms are still not well understood. Furthermore, reports on the function of pectin in early pollen development are limited. We generated OsPME-FOX rice lines with little methyl-esterified pectin even in the early-pollen mother-cell stage due to overexpression of the gene encoding pectin-methylesterase. Overexpression of OsPME1 in rice increased the activity of PME, which decreased the degree of pectin methyl esterification in the cell wall. OsPME1-FOX grew normally and showed abnormal phenotypes in anther and pollen development, especially in terms of the pollen mother-cell stage. In addition, we examined modifications of cell-wall polysaccharides at the cellular level using antibodies against polysaccharides. Immunohistochemical staining using LM19 and LM20 showed that methyl-esterified pectin distribution and the pectin contents in pollen mother-cell wall decreased in OsPME1-FOX compared with the wild type. Thus, the maintenance of methyl-esterified pectin plays a role in degrading and maintaining the pollen mother-cell wall during microspore development.

硼添加对铝胁迫栝楼生长和生理的影响

PDF HTML阅读 XML下载 导出引用 引用提醒 硼添加对铝胁迫栝楼生长和生理的影响 DOI: 10.5846/stxb202202220419 作者: 作者单位: 作者简介: 通讯作者: 中图分类号: 基金项目: 国家自然科学基金（32001224，41702181）；2021年国家级大学生创新创业训练计划（202110345017） Effects of exogenous boron on growth and physiology of Trichosanthes kirilowii Maxim. under aluminum stress Author: Affiliation: Fund Project: The National Natural Science Foundation of China (General Program, Key Program, Major Research Plan)，the 2021 National Undergraduate Training Program for Innovation and Entrepreneurship 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为揭示硼对缓解栝楼（Trichosanthes kirilowii Maxim.）铝（Aluminum）毒害的生理机制，以耐铝性强的安国栝楼和耐铝性弱的浦江栝楼为材料，研究了50 μmol/L硼酸（H3BO3）对300 μmol/L Al3+胁迫下栝楼幼苗生长、铝积累、抗氧化能力和细胞壁组分的影响。结果表明：铝胁迫下，植株的根长、株高、鲜重和干重降低，超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）活性受到显著抑制，安国栝楼、浦江栝楼根尖丙二醛（MDA）含量分别增加256.1%、278.2%，细胞壁多糖含量、果胶甲酯酶（PME）活性、根尖铝积累量显著提高，果胶甲基酯化度（DM）和3-脱氧-D-甘露-辛酮糖酸（KDO）含量降低。外源硼可以缓解铝胁迫对栝楼幼苗生长的抑制作用，安国与浦江栝楼的抗氧化酶活性显著提升，MDA含量、细胞壁多糖含量、PME酶活性均降低，安国栝楼果胶DM值恢复至正常水平的91.5%，浦江栝楼KDO含量较单铝处理组上升52.0%，活性铝结合位点减少，有效降低了根尖铝含量并维持其形态结构。因此，硼信号能通过调节细胞壁多糖组分且稳定其网格结构，减少根尖对铝的吸收，同时增强抗氧化酶活性，缓解铝胁迫导致栝楼幼苗生长中的次生氧化胁迫，提高栝楼幼苗对铝毒环境的耐受性，且对耐铝性强的安国栝楼作用效果较好。研究结果可为改良酸铝胁迫下作物的生长环境提供理论指导。 Abstract:Aluminum toxicity, a major abiotic stress that inhibits plant growth and development, has become one of the main factors reducing agricultural yield. To reveal the physiological mechanism of boron on alleviating aluminum toxicity of Trichosanthes kirilowii Maxim., seedlings from Anguo (with strong Al-tolerance) and Pujiang (with weak Al-tolerance) varieties of T. kirilowii were selected for water culture experiments and the effects of boron treatment (50 μmol/L) on varieties' growth, aluminum accumulation, antioxidant capacity, and cell wall components under aluminum stress (300 μmol/L) were studied. The results showed that 300 μmol/L Al3+ stress inhibited the root length, plant height, fresh weight, and dry weight of the plant. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were significantly decreased, and the content of malondialdehyde (MDA) in root tip of T. kirilowii in Anguo and Pujiang varieties increased by 256.1% and 278.2%, respectively. Aluminum stress also significantly enhanced the content of cell wall polysaccharide, pectin methylesterase (PME) activity, and root tip aluminum accumulation. However, the degree of pectin methyl-esterification (DM) and the content of 3-deoxy-d-mannose-octanoic acid (KDO) were strongly inhibited. Exogenous boron could alleviate the inhibitory effect of aluminum stress on the growth of T. kirilowii seedlings. The antioxidant enzyme activities of T. kirilowii in Anguo and Pujiang varieties were increased significantly. The MDA content, cell wall polysaccharide content, and PME enzyme activity were decreased. In addition, the pectin DM value of Trichosanthes in Anguo variety returned to 91.5% of the normal level, and the KDO content of T. kirilowii in Pujiang variety increased by 52.0% compared with that in the single aluminum treatment group. As a result, the active aluminum binding sites decreased, effectively reducing the aluminum content in the root tip and maintaining its morphological structure. Therefore, boron improved the tolerance of T. kirilowii seedlings to aluminum toxic environment by regulating the polysaccharide components of cell wall and stabilizing its grid structure to reduce the absorption of aluminum by root tips, and enhancing the activity of antioxidant enzymes to alleviate the secondary oxidative stress in the growth of T. kirilowii seedlings, which has a better effect on Anguo variety with stronger tolerance to aluminum toxicity. The research in this paper can provide theoretical guidance for improving the environment for crop growth under acid-aluminum stress. 参考文献 相似文献 引证文献

Tissue-specific pectin methylesterification and pectin methylesterase activities play a role in lettuce seed germination

Cell wall mechanical strength is partially controlled by the degree of pectin methylesterification that is mediated through the action of pectin methylesterases (PMEs). Changes in cell wall mechanical strength are involved in the endosperm cap weakening and radicle elongation growth during the germination of some endospermic seeds. However, little is known about the roles of the status of pectin methylesterification and PME activities during seed germination, especially in lettuce, a traditional model plant for seed germination research. In the present study, we demonstrated that the rupture of lettuce endosperm cap during seed germination was caused by cell rupture, not by cell separation as formerly reported in other species. Next, we investigated the status of pectin methylesterification of the endosperm cap and radicle during germination, using immunolabeling by specific monoclonal antibodies against methyl-esterified pectins and calcium bridges. We also analyzed the expression patterns of PME activities and an encoding gene LsPME/PMEI51 in the endosperm cap and radicle. It was found that cell walls of the endosperm cap comprise more methyl-esterified pectins than radicle, while PME activities and the abundance of LsPME/PMEI51 transcripts in the endosperm cap are lower than those in the radicle. In addition, the status of pectin methylesterification and PME expression patterns changed during seed germination. Low PME activities and high pectin methylesterification in the endosperm cap is hypothesized to be the reason of why endosperm does not rupture by cell separation. It is concluded that tissue-specific pectin methylesterification and PME activities play a role in lettuce seed germination.

Mutation of CESA1 phosphorylation site influences pectin synthesis and methylesterification with a role in seed development

Cell wall biogenesis is required for the production of seeds of higher plants. However, little is known about regulatory mechanisms underlying cell wall biogenesis during seed formation. Here we show a role for the phosphorylation of Arabidopsis cellulose synthase 1 (AtCESA1) in modulating pectin synthesis and methylesterification in seed coat mucilage. A phosphor-null mutant of AtCESA1 on T166 (AtCESA1T166A) was constructed and introduced into a null mutant of AtCESA1 (Atcesa1-1). The resulting transgenic lines showed a slight but significant decrease in cellulose contents in mature seeds. Defects in cellulosic ray architecture along with reduced levels of non-adherent and adherent mucilage were observed on the seeds of the AtCESA1T166A mutant. Reduced mucilage pectin synthesis was also reflected by a decrease in the level of uronic acid. Meanwhile, an increase in the degree of pectin methylesterification was also observed in the seed coat mucilage of AtCESA1T166A mutant. Change in seed development was further reflected by a delayed germination and about 50% increase in the accumulation of proanthocyanidins, which is known to bind pectin and inhibit seed germination as revealed by previous studies. Taken together, the results suggest a role of AtCESA1 phosphorylation on T166 in modulating mucilage pectin synthesis and methylesterification as well as cellulose synthesis with a role in seed development.

Two \u03b2-glucuronosyltransferases involved in the biosynthesis of type II arabinogalactans function in mucilage polysaccharide matrix organization in Arabidopsis thaliana

BackgroundArabinogalactan-proteins (AGPs) are heavily glycosylated with type II arabinogalactan (AG) polysaccharides attached to hydroxyproline residues in their protein backbone. Type II AGs are necessary for plant growth and critically important for the establishment of normal cellular functions. Despite the importance of type II AGs in plant development, our understanding of the underlying role of these glycans/sugar residues in mucilage formation and seed coat epidermal cell development is poorly understood and far from complete. One such sugar residue is the glucuronic acid residues of AGPs that are transferred onto AGP glycans by the action of β-glucuronosyltransferase genes/enzymes.ResultsHere, we have characterized two β-glucuronosyltransferase genes, GLCAT14A and GLCAT14C, that are involved in the transfer of β-glucuronic acid (GlcA) to type II AGs. Using a reverse genetics approach, we observed that glcat14a-1 mutants displayed subtle alterations in mucilage pectin homogalacturonan (HG) compared to wild type (WT), while glcat14a-1glcat14c-1 mutants displayed much more severe mucilage phenotypes, including loss of adherent mucilage and significant alterations in cellulose ray formation and seed coat morphology. Monosaccharide composition analysis showed significant alterations in the sugar amounts of glcat14a-1glcat14c-1 mutants relative to WT in the adherent and non-adherent seed mucilage. Also, a reduction in total mucilage content was observed in glcat14a-1glcat14c-1 mutants relative to WT. In addition, glcat14a-1glcat14c-1 mutants showed defects in pectin formation, calcium content and the degree of pectin methyl-esterification (DM) as well as reductions in crystalline cellulose content and seed size.ConclusionsThese results raise important questions regarding cell wall polymer interactions and organization during mucilage formation. We propose that the enzymatic activities of GLCAT14A and GLCAT14C play partially redundant roles and are required for the organization of the mucilage matrix and seed size in Arabidopsis thaliana. This work brings us a step closer towards identifying potential gene targets for engineering plant cell walls for industrial applications.

Anti-inflammatory activity of high and low methoxylated apple pectins, in vivo and in vitro

One of the possible mechanisms of the anti-inflammatory action of pectins is associated with the inhibition of excessive pro-inflammatory activity of macrophages - the cells that regulate inflammation intensity and reparative regeneration. It has been found that pectins with a low degree of methyl esterification of the carboxyl groups of the galacturonan core of the macromolecule exhibit this effect. In addition to leukocytes, intestinal epithelial cells are also involved in the pathogenesis of inflammatory bowel diseases. However, to date, there have been insufficient studies of the effect of pectin methyl esterification on the inflammatory response of enterocytes. The aim of the research was to evaluate the effect of the degree of pectin methyl esterification on inflammation of the colon in mice after oral administration and on the inflammatory response of human colon epithelium cells of the Caco-2 line in vitro. Material and methods. In a prospective study, 40 male BALB/c mice weighing 20-25 g were used, 10 animals in each group. Solutions of apple pectins (200 mg/0.2 ml) were orally administered to mice through a plastic catheter 24 h before the induction of colitis. The control mice received water, and prednisone administration at a dose of 5 mg/kg of body weight was used as a positive control. Colon inflammation in mice was induced by a single rectal administration of 5% acetic acid (0.1 ml). A day later, the degree and area of the lesion was assessed using a light microscope, the activity of myeloperoxidase in the wall of the colon was determined by spectrophotometry. The effect of pectins on metabolic activity, intercellular permeability, Tumor Necrosis Factor α generation and alkaline phosphatase (ALP) activity in Caco-2 cells was assessed. Results. It was found that low-methyl esterified pectin AU701, which contains more than 70% of free carboxyl groups, inhibited colon inflammation in mice. High methyletherified pectin AU201, in which more than 70% of the carboxyl groups are replaced by methyl ester, didn't affect inflammation. It was revealed that pretreatment of Caco-2 cells with AU701 and AU201 pectins prevented lipopolysaccharide-induced increase in intercellular permeability and reduced the pro-inflammatory response of Caco-2 cells to LPS. After incubation of Caco-2 cells with AU701 pectin, the rate of hydrolysis of p-nitrophenyl phosphate (an alkaline phosphate substrate) increased by 40%. Pectin AU201 had no effect on the alkaline phosphatase activity of enterocytes. Conclusion. Thus, it was found that low-methyl esterified pectin AU701 inhibits inflammation both in vivo and in vitro. High-methyl esterified pectin AU201 suppresses pro-inflammatory reactions only in vitro. The ability of pectins to inhibit intestinal inflammation has a multifactorial nature, and is due, inter alia, to their ability to stimulate the expression of alkaline phosphatase by enterocytes.

Overexpression of a pectin methylesterase gene PtoPME35 from Populus tomentosa influences stomatal function and drought tolerance in Arabidopsis thaliana

Poplar is a superior forestation species with high adaptability. The woody tissue of poplar is mainly derived from cell wall. Cell wall formation determines cell shape and woody growth. Pectin is rich in primary cell wall, but it is also involved in the regulation of wood formation. In our study, we cloned a gene from poplar (Populus tomentos), designed as PtoPME35, which encodes a putative pectin methylesterase. PtoPME35 has higher sequence similarity with Arabidopsis AtPME35. Gene expression analysis shows that PtoPME35 has a constitutive expression pattern in multiple tissues, with the highest expression in stem. Subcellular localization result indicates that PtoPME35 is localized to the cell wall. To elucidate the biological function of PtoPME35 in vivo, we generated overexpression plants in poplar and Arabidopsis. The degree of pectin methylesterification is decreased in PtoPME35-overexpressing transgenic poplar, although no obvious phenotypes were displayed. In PtoPME35-overexpressing Arabidopsis plants, stomatal opening is inhibited and water loss rate is decreased under the drought condition. Moreover, the expression levels of drought-stress responsive genes were higher with mannitol treatment in PtoPME35-overexpressing Arabidopsis plants than in wild type controls. Accordingly, these results suggest that PtoPME35 may regulate osmotic stress responses by modulating stomatal functions.

Arabidopsis FLYING SAUCER 2 Functions Redundantly with FLY1 to Establish Normal Seed Coat Mucilage.

Following exposure to water, mature Arabidopsis seeds are surrounded by a gelatinous capsule, termed mucilage. The mucilage consists of pectin-rich polysaccharides, which are produced in epidermal cells of the seed coat. Although pectin is a major component of plant cell walls, its biosynthesis and biological functions are not fully understood. Previously, we reported that a transmembrane RING E3 ubiquitin ligase, FLYING SAUCER 1 (FLY1) regulates the degree of pectin methyl esterification for mucilage capsule formation. The Arabidopsis thaliana genome has a single FLY1 homolog, FLY2. In this study, we show that the FLY2 protein functions in mucilage modification together with FLY1. FLY2 was expressed in seed coat epidermal cells during mucilage synthesis, but its expression level was much lower than that of FLY1. While fly2 showed no obvious difference in mucilage capsule formation from wild type, the fly1 fly2 double mutants showed more severe defects in mucilage than fly1 alone. FLY2-EYFP that was expressed under the control of the FLY1 promoter rescued fly1 mucilage, showing that FLY2 has the same molecular function as FLY1. FLY2-EYFP colocalized with marker proteins of Golgi apparatus (sialyltransferase-mRFP) and late endosome (mRFP-ARA7), indicating that as FLY1, FLY2 controls pectin modification by functioning in these endomembrane organelles. Furthermore, phylogenetic analysis suggests that FLY1 and FLY2 originated from a common ancestral gene by gene duplication prior to the emergence of Brassicaceae. Taken together, our findings suggest that FLY2 functions in the Golgi apparatus and/or the late endosome of seed coat epidermal cells in a manner similar to FLY1.

PECTIN ACETYLESTERASE9 Affects the Transcriptome and Metabolome and Delays Aphid Feeding.

The plant cell wall plays an important role in damage-associated molecular pattern-induced resistance to pathogens and herbivorous insects. Our current understanding of cell wall-mediated resistance is largely based on the degree of pectin methylesterification. However, little is known about the role of pectin acetylesterification in plant immunity. This study describes how one pectin-modifying enzyme, PECTIN ACETYLESTERASE 9 (PAE9), affects the Arabidopsis (Arabidopsis thaliana) transcriptome, secondary metabolome, and aphid performance. Electro-penetration graphs showed that Myzus persicae aphids established phloem feeding earlier on pae9 mutants. Whole-genome transcriptome analysis revealed a set of 56 differentially expressed genes (DEGs) between uninfested pae9-2 mutants and wild-type plants. The majority of the DEGs were enriched for biotic stress responses and down-regulated in the pae9-2 mutant, including PAD3 and IGMT2, involved in camalexin and indole glucosinolate biosynthesis, respectively. Relative quantification of more than 100 secondary metabolites revealed decreased levels of several compounds, including camalexin and oxylipins, in two independent pae9 mutants. In addition, absolute quantification of phytohormones showed that jasmonic acid (JA), jasmonoyl-Ile, salicylic acid, abscisic acid, and indole-3-acetic acid were compromised due to PAE9 loss of function. After aphid infestation, however, pae9 mutants increased their levels of camalexin, glucosinolates, and JA, and no long-term effects were observed on aphid fitness. Overall, these data show that PAE9 is required for constitutive up-regulation of defense-related compounds, but that it is not required for aphid-induced defenses. The signatures of phenolic antioxidants, phytoprostanes, and oxidative stress-related transcripts indicate that the processes underlying PAE9 activity involve oxidation-reduction reactions.

Sequestration of Mn into the cell wall contributes to Mn tolerance in sugarcane (Saccharum officinarum L.)

Sugarcane (Saccharum officinarum L.) grown on acid soils suffers serious chlorosis resulting from excess manganese (Mn). We explored the mechanisms underlying sugarcane tolerance to excess Mn. Seedlings of genotypes YC96–66 (66) and YC58–21 (21) were cultured hydroponically with MnCl2, then Mn effects on leaf chlorophyll concentration, Mn subcellular distribution, cell wall polysaccharides were determined. A Mn adsorption kinetics assay was also conducted to examine the association of Mn-induced pectin modification with Mn-binding to cell walls. Excess Mn caused significant dose- and time-dependent decrease in chlorophyll, with serious chlorosis in genotype 21 compared to 66. Genotype 66 absorbed more Mn and maintained higher leaf Mn, with greater proportions of Mn retained in cell walls. Up to 93.2% and 90.0% of cell wall-bound Mn was found in the pectin fraction in genotype 66 and 21, respectively. Mn induced pectin accumulation and pectin methylesterase activation, along with a decrease in the degree of pectin methylesterification, which thereby enhanced cell wall Mn-binding capacity in genotype 66. In contrast, no remarkable change was observed in genotype 21. Mn-induced pectin accumulation, pectin demethylesterification and subsequent sequestration of Mn into the cell wall contribute to Mn tolerance in the sugarcane.

Cell wall features transferred from common into durum wheat to improve Fusarium Head Blight resistance

Durum wheat is naturally more susceptible to Fusarium graminerum infection in comparison to common wheat. The improvement of durum wheat resistance against F. graminearum is a challenge due to the lack of resistance sources in its gene pool. FHB-resistance factors were introduced in durum wheat by generating recombinant inbred lines (RILs), obtained by crossing the hexaploid resistant accession 02-5B-318 with the susceptible durum wheat cv. Saragolla. In this work we explored the possible contribution of cell wall (CW) in RILs with improved FHB resistance. We thoroughly studied CW components, mycotoxins content and the expression of related genes in different RILs selected for their extremely high and low resistance to FHB. Differences were found in resistant and susceptible lines in the degree of pectin methylesterification and in deoxynivalenol (DON) accumulation after fungal infection. Genes involved in biochemical modification of CW structure (WheatPme-1, Glu-1) and mycotoxins accumulation (ns-LTP-1) were analyzed as putative candidates for FHB resistance. Our results indicate that durum wheat plants with cell wall structure and gene response acquired from common wheat displayed an increased resistance to FHB.

MYB52 Negatively Regulates Pectin Demethylesterification in Seed Coat Mucilage.

Pectin, which is a major component of the plant primary cell walls, is synthesized and methyl-esterified in the Golgi apparatus and then demethylesterified by pectin methylesterases (PMEs) located in the cell wall. The degree of methylesterification affects the functional properties of pectin, and thereby influences plant growth, development and defense. However, little is known about the mechanisms that regulate pectin demethylesterification. Here, we show that in Arabidopsis (Arabidopsis thaliana) seed coat mucilage, the absence of the MYB52 transcription factor is correlated with an increase in PME activity and a decrease in the degree of pectin methylesterification. Decreased methylesterification in the myb52 mutant is also correlated with an increase in the calcium content of the seed mucilage. Chromatin immunoprecipitation analysis and molecular genetic studies suggest that MYB52 transcriptionally activates PECTIN METHYLESTERASE INHIBITOR6 (PMEI6), PMEI14, and SUBTILISIN-LIKE SER PROTEASE1.7 (SBT1.7) by binding to their promoters. PMEI6 and SBT1.7 have previously been shown to be involved in seed coat mucilage demethylesterification. Our characterization of two PMEI14 mutants suggests that PMEI14 has a role in seed coat mucilage demethylesterification, although its activity may be confined to the seed coat in contrast to PMEI6, which functions in the whole seed. Our demonstration that MYB52 negatively regulates pectin demethylesterification in seed coat mucilage, and the identification of components of the molecular network involved, provides new insight into the regulatory mechanism controlling pectin demethylesterification and increases our understanding of the transcriptional regulation network involved in seed coat mucilage formation.

Degree of pectin methyl esterification in endosperm cell walls is involved in embryo bending in Arabidopsis thaliana

The endosperm is a transitory structure involved in proper embryo elongation. The cell walls of mature seed endosperm are generally composed of a uniform distribution of cellulose, unesterified homogalacturonans, and arabinans. Recent studies suggest that changes in cell wall properties during endosperm development could be related to embryo growth. The degree of methyl esterification of homogalacturonans may be involved in this endosperm tissue remodelling. The relevance of the degree of homogalacturonan methyl esterification during seed development was determined by immunohistochemical analyses using a panel of probes with specificity for homogalaturonans with different degrees of methyl esterification. Low-esterified and un-esterified homogalacturonans were abundant in endosperm cells during embryo bending and were also detected in mature embryos. BIDXII (BDX) could be involved in seed development, because bdx-1 mutants had misshapen embryos. The methyl esterification pattern described for WT seeds was different during bdx-1 seed development; un-esterified homogalacturonans were scarcely present in the cell walls of endosperm in bending embryos and mature seeds. Our results suggested that the degree of methyl esterification of homogalacturonans in the endosperm cell wall may be involved in proper embryo development.

Pectic enzymes as potential enhancers of ascorbic acid production through the D-galacturonate pathway in Solanaceae

The increase of L-Ascorbic Acid (AsA) content in tomato (Solanum lycopersicum) is a common goal in breeding programs due to its beneficial effect on human health. To shed light into the regulation of fruit AsA content, we exploited a Solanum pennellii introgression line (IL12-4-SL) harbouring one quantitative trait locus that increases the content of total AsA in the fruit. Biochemical and transcriptomic analyses were carried out in fruits of IL12-4-SL in comparison with the cultivated line M82 at different stages of ripening. AsA content was studied in relation with pectin methylesterase (PME) activity and the degree of pectin methylesterification (DME). Our results indicated that the increase of AsA content in IL12-4-SL fruits was related with pectin de-methylesterification/degradation. Specific PME, polygalacturonase (PG) and UDP-D-glucuronic-acid-4-epimerase (UGlcAE) isoforms were proposed as components of the D-galacturonate pathway leading to AsA biosynthesis. The relationship between AsA content and PME activity was also exploited in PMEI tobacco plants expressing a specific PME inhibitor (PMEI). Here we report that tobacco PMEI plants, altered in PME activity and degree of pectin methylesterification, showed a reduction in low methylesterified pectic domains and exhibited a reduced AsA content. Overall, our results provide novel biochemical and genetic traits for increasing antioxidant content by marker-assisted selection in the Solanaceae family.

Pectin methylesterases contribute the pathogenic differences between races 1 and 4 of Fusarium oxysporum f. sp. cubense

Plant cell walls, which are mainly composed of pectin, play important roles in plant defence responses to pathogens. Pectin is synthesised in a highly esterified form and then de-esterified by pectin methylesterases (PMEs). Because of this, PMEs are directly involved in plant defence. However, the molecular mechanisms of their interactions with pectins remain unclear. In this study, we compared the expression level and enzyme activities of PMEs in a banana Cavendish cultivar (Musa AAA ‘Brazilian’) inoculated with Fusarium oxysporum f. sp. cubense pathogenic races 1 (Foc1) and 4 (Foc4). We further examined the spatial distribution of PMEs and five individual homogalacturonans (HGs) with different degree of pectin methylesterification (DM). Results suggested that the banana roots infected with Foc1 showed lower PME activity than those infected with Foc4, which was consisted with observed higher level of pectin DM. The level of HGs crosslinked with Ca2+ was significantly higher in roots infected with Foc1 compared with those infected with Foc4. Therefore, banana exhibited significantly different responses to Foc1 and Foc4 infection, and these results suggest differences in PME activities, DM of pectin and Ca2+-bridged HG production. These differences could have resulted in observed differences in virulence between Foc1 and Foc4.

The subtilisin-like protease SBT3 contributes to insect resistance in tomato.

Subtilisin-like proteases (SBTs) constitute a large family of extracellular plant proteases, the function of which is still largely unknown. In tomato plants, the expression of SBT3 was found to be induced in response to wounding and insect attack in injured leaves but not in healthy systemic tissues. The time course of SBT3 induction resembled that of proteinase inhibitor II and other late wound response genes suggesting a role for SBT3 in herbivore defense. Consistent with such a role, larvae of the specialist herbivore Manduca sexta performed better on transgenic plants silenced for SBT3 expression (SBT3-SI). Supporting a contribution of SBT3 to systemic wound signaling, systemic induction of late wound response genes was attenuated in SBT3-SI plants. The partial loss of insect resistance may thus be explained by a reduction in systemic defense gene expression. Alternatively, SBT3 may play a post-ingestive role in plant defense. Similar to other anti-nutritive proteins, SBT3 was found to be stable and active in the insect's digestive system, where it may act on unidentified proteins of insect or plant origin. Finally, a reduction in the level of pectin methylesterification that was observed in transgenic plants with altered levels of SBT3 expression suggested an involvement of SBT3 in the regulation of pectin methylesterases (PMEs). While such a role has been described in other systems, PME activity and the degree of pectin methylesterification did not correlate with the level of insect resistance in SBT3-SI and SBT3 overexpressing plants and are thus unrelated to the observed resistance phenotype.

Molecular and biochemical characterization of rice pectin methylesterase inhibitors (OsPMEIs)

Cell wall modifications such as partial degradation and depolymerization by cell wall hydrolases are normal cellular processes and are required for the functionalities of different cell types. Pectin, one of the major cell wall polysaccharides, is predominantly found in primary cell walls and middle lamellae and is subjected to in muro modification, primarily by cell wall-localized pectin methylesterases (PMEs). Molecular biochemical studies have demonstrated that enzymatic activities of PMEs are governed by multiple pectin methylesterase inhibitors (PMEIs), which consequently control the pectin methylesterification status. Although a few studies in Arabidopsis have shown the importance of this PMEI-mediated regulation in the biophysical properties of cell walls, little is known about the molecular physiological functions of rice PMEIs. We found 49 members of the PMEI family in the rice genome. Analysis of their transcript levels by quantitative real-time PCR and meta expression analysis showed that they are regulated spatially and temporally, as well as in response to diverse stresses. Quantification of cell wall-bound methylesters indicated that the degree of pectin methylesterification is developmentally regulated; in particular, higher PMEI activities were detected in cell wall proteins prepared from young leaves. Furthermore, an activity assay demonstrated that two recombinant OsPMEI proteins (OsPMEI8 and 12) were able to inhibit the enzymatic activity of a commercial PME protein. Subcellular localization indicated that OsPMEI8 is targeted to the middle lamella and OsPMEI12 is localized in the plasma membrane and nucleus. Taken together, our findings provide the first molecular and biochemical evidence for functional characterization of PMEIs in rice growth and development.

Novel roles of hydrogen peroxide (H₂O₂) in regulating pectin synthesis and demethylesterification in the cell wall of rice (Oryza sativa) root tips.

Hydrogen peroxide (H₂O₂) has been reported to increase lignin formation, enhance cell wall rigidification, restrict cell expansion and inhibit root elongation. However, our results showed that it not only inhibited rice (Oryza sativa) root elongation, but also increased root diameter. No study has reported how and why H₂O₂ increases cell expansion and root diameter. Exogenous H₂O₂ and its scavenger 4-hydroxy-Tempo were applied to confirm the roles of H₂O₂. Immunofluorescence, fluorescence probe, ruthenium red staining, histological section and spectrophotometry were used to monitor changes in the degree of pectin methylesterification, pectin content, pectin methylesterase (PME) activity and H₂O₂ content. Exogenous H₂O₂ inhibited root elongation, but increased cell expansion and root diameter significantly. H₂O₂ not only increased the region of pectin synthesis and pectin content in root tips, but also increased PME activity and pectin demethylesterification. The scavenger 4-hydroxy-Tempo reduced root H₂O₂ content and recovered H₂O₂-induced increases in cell expansion and root diameter by inhibiting pectin synthesis, PME activity and pectin demethylesterification. H₂O₂ plays a novel role in the regulation of pectin synthesis, PME activity and pectin demethylesterification. H₂O₂ increases cell expansion and root diameter by increasing pectin content and demethylesterification.