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Background and Aims Pit pairs and their filter-like partition, i.e. pit membranes, play important roles as water pathways, barriers and regulators in the water-conducting system of angiosperms. In Fraxinus species, the intervessel and vessel–parenchyma pit membranes in sapwood are normally encrusted during winter. Although these encrustations inevitably influence the performance of pits, their properties and functions remain unclear. This study aimed to reveal the morphological and chemical characteristics of encrustations in F. mandshurica in order to deepen understanding of the seasonal encrustation of pit membranes. Methods Seasonal and positional variations in the presence and morphology of encrustations were examined by field-emission scanning electron microscopy (FE-SEM). Cryo-FE-SEM for freeze-fixed greenwood samples was conducted to clarify whether encrustations were present in living trees. Chemical components were examined by histochemical staining using light and electron microscopy, immunofluorescence labelling and ultraviolet microspectroscopy. Key Results Encrustations began to deposit in autumn before leaf senescence and disappeared in spring before bud flushing. They infiltrated within the pit membranes, which suggested that they severely limit the permeation of pits. The encrustations differed in morphology among positions: they entirely filled the pit chambers in latewood, while they covered the pit membranes in earlywood. The encrustations were similarly observed in the samples that were freeze-fixed immediately after collection, indicating that they are present in living trees. The encrustations contained polysaccharides, including xyloglucan and homogalacturonan, and phenolic compounds, possibly including flavonoids and coumarins. These chemical components were also detected in droplets found in the latewood vessels with the encrustations, suggesting that the materials constituting encrustations were supplied through the vessel lumens. Conclusions Encrustations undoubtedly cover the pit membranes in living F. mandshurica trees in winter and their morphology and chemical composition indicate that they are impermeable, have positional differences in function and are characterized by elaborate deposition/removal processes. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 system has been successfully implemented in animals and plants is a second-generation genome editing tool. We are able to optimize a Cas9 system to edited Ntab06050 and Ntab0857410 genes in HD and K326 tobacco cultivars respectively. The gene Ntab06050 is related to lignin synthesis while the gene Ntab0857410 belongs to pectin synthesis by utilizing Agrobacterium-mediated leaf disc method. We have constructed total eight different constructs for the lignin related gene family CCoAMT, out of which three constructs have been selected from Ntab0184090, two constructs from Ntab0392460 while one construct from each Ntab0540120, Ntab0857410 and Ntab0135940 gene. To study the Cas9 system in pectin related genes, total five constructs have been utilized under Cas9 system and multiple target sites were selected by identifying PAM sequences. Out of which three constructs were targeted from NtabGAE1and NtabGAE6 homologous while two were targeted from NtabGAUT4 homologous. Where as, UDP-D-glucuronate 4-epimerase gene family is a Golgi localized, might have a role in the interconvertion of UDP-D-GlcA and UDP-D-GalA in pectin synthesis. We have succeeded in the mutation of pectin related NtabGAUT4 and lignin related NtabCCoAMT genes with 6.2% and 9.4% mutation frequency. [ABSTRACT FROM AUTHOR]

Australian saltbush (Atriplex spp.) survive in exceptionally saline environments and are often used for pasture in semi-arid areas. To investigate the impact of salinity on saltbush root morphology and root exudates, three Australian native saltbush species (Atriplex nummularia , Atriplex amnicola , and Atriplex vesicaria) were grown in vitro in optimised sterile, semi-hydroponic systems in media supplemented with different concentrations of salt (NaCl). Histological stains and chromatographic techniques were used to characterise the root apical meristem (RAM) type and root exudate composition of the saltbush seedlings. We report that saltbush species have closed-type RAMs, which release border-like cells (BLCs). Monosaccharide content, including glucose and fructose, in the root mucilage of saltbush was found to be uniquely low, suggesting that saltbush may minimise carbon release in polysaccharides of root exudates. Root mucilage also contained notable levels of salt, plus increasing levels of unidentified compounds at peak salinity. Un-esterified homogalacturonan, xyloglucan, and arabinogalactan proteins between and on the surface of BLCs may aid intercellular adhesion. At the highest salinity levels, root cap morphology was altered but root:shoot ratio remained consistent. While questions remain about the identity of some components in saltbush root mucilage other than the key monosaccharides, this new information about root cap morphology and cell surface polysaccharides provides avenues for future research. Australian saltbush (Atriplex spp.), used for pasture in semi-arid areas, survive in exceptionally salty environments, but little is known about how salinity affects the root morphology and root exudates. We discovered that saltbush have closed-type root apical meristems and release limited sugars as root exudates, maintaining root:shoot ratio even at extreme salinity. These findings offer new insights into saltbush resilience and encourage further research into their unique root properties. [ABSTRACT FROM AUTHOR]

Main conclusion: The ultrastructural design and biochemical organization of the significantly thickened outer tissues of the gametophytic stem of Hypnodendron menziesii optimizes load bearing of the stem. Hypnodendron menziesii is a bryoid umbrella moss growing in high humid conditions on the forest floors of New Zealand. The erect gametophyte bears up to eight whorls of branches in succession, spreading across the stem that bears the heavy weight of branches with highly hydrated leaves. Our investigation using a combination of light microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and TEM-immunolabeling techniques provided novel information on the structural design and biochemical organization of greatly thickened cell walls of epidermal, hypodermal, and outermost cortical tissues, comparing underlying thin-walled cortical tissues in the gametophytic stem. Probing into the ultrastructure of the cell wall architecture of these target tissues by TEM and SEM revealed the cell walls to display a multilamellar organization, in addition to demonstrating the presence of an electron-dense substance in the cell wall, presumably flavonoids. The pattern of distribution and concentration of rhamnogalacturonan, homogalacturonan, and heteromannan, as determined by immunogold labeling, suggests that it is the combination of structural and molecular design of the cell wall that may optimize the mechanical function of the epidermal, hypodermal, and outer cortical tissues. Statistical relationships between the overall thickness of epidermal, hypodermal, and outer cortical cell walls, the lumen area of cells and the percentage area of cell wall occupied in these tissues at different heights of the stem, and thickness of secondary cell wall layers (L1-L4/5) were explored. The results of these analyses unequivocally support the contribution of outer tissues to the mechanical strength of the resilient stem. [ABSTRACT FROM AUTHOR]

BACKGROUND: Lime peel, obtained from juice factory waste, is considered as a source of pectin. Lime peel pectin samples, extracted by three extraction procedures of 90 °C heating for 120 min, 90 °C heating for 90 min and then 32 min of ultrasound, and 80 °C heating for 60 min and then 22 min of ultrasound, and purified by two methods using ethanol and sodium caseinate (SC), were prepared and characterized. RESULTS: The results showed that the purification method significantly affected the lime pectin samples properties. Pectin samples purified with SC had a transparent and film‐like appearance, whereas the ethanol‐purified pectin samples showed an amorphous and opaque appearance. Pectin samples purified with ethanol showed higher extraction recovery (approximately 20%), whereas the lowest pectin yield was observed for the purification with SC (approximately 10%). Although SC purification did not enhance the pectin yield, it diminished the level of 'non‐pectin' components and resulted in purer pectin. The bands at 1045–1076 cm−1, relating to neutral sugars, had higher intensities in ethanol‐purified pectin samples, indicating their higher sugar contents. Also the samples purified with SC exhibited more thermal stability, probably as a result of the presence of protein in their structure. CONCLUSION: In the present study, under the same or varying extraction conditions, the most important element distinguishing pectins in terms of appearance, physicochemical, thermal and Fourier transform infrared characteristics was the type of purification method. Purification with SC may result in pectins with more non‐esterified galacturonic acids from the homogalacturonan fraction. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

SUMMARY: Plant cell wall polysaccharides, including xylan, mannan, xyloglucan, and pectins, are often acetylated and members of the domain of unknown function 231 (DUF231)/trichome birefringence‐like (TBL) family have been shown to be O‐acetyltransferases mediating the acetylation of xylan, mannan, and xyloglucan. However, little is known about the O‐acetyltransferases responsible for pectin acetylation. In this report, we biochemically characterized a suite of Arabidopsis DUF231/TBL proteins for their roles in pectin acetylation. We generated 24 TBL recombinant proteins in mammalian cells and demonstrated that 10 of them were able to transfer acetyl groups from acetyl‐CoA onto the pectins homogalacturonan (HG) or rhamnogalacturonan‐I (RG‐I), and thus were named pectin O‐acetyltransferase 1 to 10 (POAT1 to 10). It was found that POAT2,4,9,10 specifically acetylated HG and POAT5,6 acetylated RG‐I, whereas POAT1,3,7,8 could act on both HG and RG‐I. The acetylation of HG and RG‐I by POATs was further corroborated by hydrolysis with pectin acetylesterases and by nuclear magnetic resonance spectroscopy. In addition, mutations of the conserved GDS and DXXH motifs in POAT3 and POAT8 were shown to lead to a loss of their ability to acetylate HG and RG‐I. Furthermore, simultaneous RNA interference downregulation of POAT1,3,6,7,8 resulted in reduced cell expansion, impaired plant growth, and decreased pectin acetylation. Together, our findings indicate that these POATs are pectin O‐acetyltransferases involved in acetylation of the pectin polysaccharides HG and RG‐I. [ABSTRACT FROM AUTHOR]

The mechanisms underlying susceptibility to and defense against Pseudomonas syringae (Pph) of the common bean (Phaseolus vulgaris) have not yet been clarified. To investigate these, 15-day-old plants of the variety Riñón were infected with Pph and the transcriptomic changes at 2 h and 9 h post-infection were analysed. RNA-seq analysis showed an up-regulation of genes involved in defense/signaling at 2 h, most of them being down-regulated at 9 h, suggesting that Pph inhibits the transcriptomic reprogramming of the plant. This trend was also observed in the modulation of 101 cell wall-related genes. Cell wall composition changes at early stages of Pph infection were associated with homogalacturonan methylation and the formation of egg boxes. Among the cell wall genes modulated, a pectin methylesterase inhibitor 3 (PvPMEI3) gene, closely related to AtPMEI3 , was detected. PvPMEI3 protein was located in the apoplast and its pectin methylesterase inhibitory activity was demonstrated. PvPMEI3 seems to be a good candidate to play a key role in Pph infection, which was supported by analysis of an Arabidopsis pmei3 mutant, which showed susceptibility to Pph, in contrast to resistant Arabidopsis Col-0 plants. These results indicate a key role of the degree of pectin methylesterification in host resistance to Pph during the first steps of the attack. [ABSTRACT FROM AUTHOR]

Introduction: Postharvest textural changes in fruit are mainly divided into softening and lignification. Loquat fruit could have severe lignification with increased firmness during postharvest storage. Pectin is mainly associated with the postharvest softening of fruit, but some studies also found that pectin could be involved in strengthening the mechanical properties of the plant. Objectives: This study focused on characterizing the dynamics of pectin and its complexation in the cell wall of lignified loquat fruit during postharvest storage, and how these changes could influence fruit firmness. Methods: The homogalacturonan (HG) pectin in the cell wall of loquat fruit was identified using monoclonal antibodies. An oligogalacturonide (OG) probe was used to label the egg-box structure formed by Ca2+ cross-linking with low-methylesterified HG. An exogenous injection was used to verify the role of egg-box structures in the firmness increase in loquat fruit. Results: The JIM5 antibody revealed that low-methylesterified HG accumulated in the tricellular junctions and middle lamella of loquat fruit that had severe lignification symptoms. The pectin methylesterase (PME) activity increased during the early stages of storage at 0 °C, and the calcium-pectate content and flesh firmness constantly increased during storage. The OG probe demonstrated the accumulation of egg-box structures at the cellular level. The exogenous injection of PME and Ca2+ into the loquat flesh led to an increase in firmness with more low-methylesterified HG and egg-box structure signals. Conclusion: PME-mediated demethylesterification generated large amounts of low-methylesterified HG in the cell wall. This low-methylesterified HG further cross-linked with Ca2+ to form egg-box structures. The pectin-involved complexations then contributed to the increased firmness in loquat fruit. Overall, besides being involved in fruit softening, pectin could also be involved in strengthening the mechanical properties of postharvest fruit. This study provides new ideas for obtaining a better texture of postharvest loquat fruits based on pectin regulation.

The rapid ripening of soursop (Annona muricata L.) fruits is owing to its high respiration rate. Several enzymes affect the fruit cell wall in this process, resulting in the depolymerization of pectin primarily in the homogalacturonan. The main group of enzymes affecting the pectin content of soursop fruits include polygalacturonase (PG), pectate lyase (PL), pectin methylesterase (PME), and PME inhibitors (PMEis). In this study, pectin-degrading enzymes were analyzed using bioinformatic tools to uncover the gaps in our knowledge of this fruit. In this context, 67 genes encoding PG, 33 PL, 58 PME, and 39 PMEi isoenzymes were found. These genes were categorized into several families based on the results of phylogenetic analysis. Regarding the analysis of gene expression, a total of 25 were identified as differentially expressed genes (DEGs) in PG, while 3, 21, and 15 were found for PL, PME, and PMEis, respectively. Likewise, functional enrichment analysis demonstrated that the DEGs are involved in the modification of the cell wall, specifically in the degradation of pectin. On the other hand, gene co-expression networks revealed that the genes PG32 and PG35 affect the expression of PGs, as well as PL19 of the PL family, PME20, PME32, and PME35 of the PME family, and PMEi04 of the PMEi family. This suggests that they have a significant impact on the softening of soursop fruits. [ABSTRACT FROM AUTHOR]

[Display omitted] • Pectin metabolism patterns in lignified loquat fruit were different from other fruits. • Low-methylesterified HG accumulated in cell walls of loquat fruit during cold storage. • Low-methylesterified HG formed egg-box structure with Ca2+ in lignified loquat fruit. • LTC treatment inhibited the increase of low-methylesterified HG and egg-box structure. • Accumulation of pectin-involved egg-box structure was related to firmness increase. Postharvest textural changes in fruit are mainly divided into softening and lignification. Loquat fruit could have severe lignification with increased firmness during postharvest storage. Pectin is mainly associated with the postharvest softening of fruit, but some studies also found that pectin could be involved in strengthening the mechanical properties of the plant. This study focused on characterizing the dynamics of pectin and its complexation in the cell wall of lignified loquat fruit during postharvest storage, and how these changes could influence fruit firmness. The homogalacturonan (HG) pectin in the cell wall of loquat fruit was identified using monoclonal antibodies. An oligogalacturonide (OG) probe was used to label the egg-box structure formed by Ca2+ cross-linking with low-methylesterified HG. An exogenous injection was used to verify the role of egg-box structures in the firmness increase in loquat fruit. The JIM5 antibody revealed that low-methylesterified HG accumulated in the tricellular junctions and middle lamella of loquat fruit that had severe lignification symptoms. The pectin methylesterase (PME) activity increased during the early stages of storage at 0 °C, and the calcium-pectate content and flesh firmness constantly increased during storage. The OG probe demonstrated the accumulation of egg-box structures at the cellular level. The exogenous injection of PME and Ca2+ into the loquat flesh led to an increase in firmness with more low-methylesterified HG and egg-box structure signals. PME-mediated demethylesterification generated large amounts of low-methylesterified HG in the cell wall. This low-methylesterified HG further cross-linked with Ca2+ to form egg-box structures. The pectin-involved complexations then contributed to the increased firmness in loquat fruit. Overall, besides being involved in fruit softening, pectin could also be involved in strengthening the mechanical properties of postharvest fruit. This study provides new ideas for obtaining a better texture of postharvest loquat fruits based on pectin regulation. [ABSTRACT FROM AUTHOR]

Main conclusion: The phylogenetic relationship and evolutionary history of the GAUT gene family were identified in 8 Rosaseae species. PbrGAUT22 was involved in controlling pollen tube growth by regulating the content of pectins. In plants, galacturonosyltransferases (GAUTs) were involved in homogalacturonan biosynthesis and functioned in maintaining pollen tube cell wall integrity. However, the feature and evolutionary history of the GAUT gene family in Rosaceae species and candidates in pear pollen tube growth remain unclear. Here, we identified 190 GAUT genes in 8 Rosaceae species, including Chinese white pear (Pyrus bretschneideri), European pear (Pyrus communis), apple (Malus × domestica), peach (Prunus persica), Japanese apricot (Prunus mume), sweet cherry (Prunus avium), woodland strawberry (Fragaria vesca) and black raspberry (Rubus occidentalis). Members in GAUT gene family were divided into 4 subfamilies according to the phylogenetic and structural analysis. Whole-genome duplication events and dispersed duplicates drove the expansion of the GAUT gene family. Among 23 pollen-expressed PbrGAUT genes in pear, PbrGAUT22 showed increased expression level during 1–6 h post-cultured pollen tubes. PbrGAUT22 was localized to the cytoplasm and plasma membrane. Knockdown of PbrGAUT22 expression in pollen tubes caused the decrease of pectin content and inhibited pear pollen tubes growth. Taken together, we investigated the identification and evolution of the GAUT gene family in Rosaceae species, and found that PbrGAUT22 played an essential role in the synthesis of pectin and the growth of pear pollen tubes. [ABSTRACT FROM AUTHOR]

Introduction: GoSAMTs play a role in the methylation of polysaccharides synthesized by the Golgi. Pectin homogalacturonan (HG) methyl-esterification is essential for the proper function of this polysaccharide in cell walls. In order to better understand the role of GoSAMTs in HG biosynthesis, we analyzed mucilage methyl-esterification in gosamt mutants. Methods: To determine the function of GoSAMT1 and GoSAMT2 in HG methylesterification we utilized epidermal cells of seed coats, as these structures produce mucilage, which is a pectic matrix. We evaluated differences in seed surface morphology and quantified mucilage release. We measured methanol release, and used antibodies and confocal microscopy to analyze HG methyl-esterification in mucilage. Results: We observed morphological differences on the seed surface and delayed, uneven mucilage release in gosamt1-1gosamt2-1 double mutants. We also found changes in the distal wall length indicating abnormal cell wall breakage in this double mutant. Using methanol release and immunolabeling, we confirmed that GoSAMT1 and GoSAMT2 are involved in HG methyl-esterification in mucilage. However, we did not find evidence of decreasing HG in the gosamt mutants. Confocal microscopy analyses detected different patterns in the adherent mucilage and a greater number of low-methyl-esterified domains near the seed coat surface, which correlates with a greater number of “egg-box” structures in this region. We also detected a shift in the partitioning between the Rhamnogalacturonan-I soluble and adherent layers of the double mutant, which correlated with increased amounts of arabinose and arabinogalactan-protein in the adherent mucilage. Discussion: The results show that the HG synthesized in gosamt mutant plants is less methyl esterified, resulting in more egg-box structures, which stiffen the cell walls in epidermal cells and change the rheological properties of the seed surface. The increased amounts of arabinose and arabinogalactan-protein in adherent mucilage, also suggests that compensation mechanisms were triggered in the gosamt mutants. [ABSTRACT FROM AUTHOR]

A homogalacturonan pectin (HG, designated as WJP-F80) was extracted from the peel of winter jujube (Zizyphus jujuba Mill. Cv. Dongzao) and separated via ethanol-graded precipitation. The structural and conformational features were elucidated through HPAEC-PAD, GC-MS, 2D NMR, and HPSEC-MALLS studies. In vivo assessments were carried out to evaluate the hepatoprotective effects of WJP-F80 against CCl4-induced injury of mice. Results showed that WJP-F80 was a linear 1,4-α-galacturonan with partially methyl-esterified at O-6 of GalpA and occasionally acetylation. The Mw of WJP-F80 was determined as 45.3 kDa, the polydispersity was calculated as 1.56, and the Rg was measured as 22.7 nm in 0.1 M NaNO3. The conformational analysis revealed that WJP-F80 exhibited as rigid stiff chain in low Mw range, while aggregation by self-assembly of HG chains lead to high Mw and random coil conformation. In vivo studies indicated that WJP-F80 can protect the livers of mice from acute injury induced via CCl4 by decreasing the serum biochemical markers of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) to normal levels. This work provides a theoretical basis for the value-added deep processing of winter jujube. [ABSTRACT FROM AUTHOR]

Pectinolytic enzymes have diverse industrial applications. Among these, pectate lyases act on the internal α-1,4-linkage of the pectate backbone, playing a critical role in pectin degradation. While most pectate lyases characterized thus far are of bacterial origin, fungi can also be excellent sources of pectinolytic enzymes. In this study, we performed biochemical characterization of the pectate lyase AnPL9 belonging to the polysaccharide lyase family 9 (PL9) from the filamentous fungus Aspergillus nidulans. Recombinant AnPL9 was produced using a Pichia pastoris expression system and purified. AnPL9 exhibited high activity on homogalacturonan (HG), pectin from citrus peel, pectin from apple, and the HG region in rhamnogalacturonan-I. Although digalacturonic acid and trigalacturonic acid were not degraded by AnPL9, tetragalacturonic acid was converted to 4,5-unsaturated digalacturonic acid and digalacturonic acid. These results indicate that AnPL9 degrades HG oligosaccharides with a degree of polymerization > 4. Furthermore, AnPL9 was stable within a neutral-to-alkaline pH range (pH 6.0–11.0). Our findings suggest that AnPL9 is a candidate pectate lyase for biotechnological applications in the food, paper, and textile industries. This is the first report on a fungal pectate lyase belonging to the PL9 family. [ABSTRACT FROM AUTHOR]

A homogeneous polysaccharide named as LJW2F2 was extracted and purified from the flowers of Lonicera japonica Thunb. Structural characteristic indicated that LJW2F2 was a homogalacturonan composed of α-1,4-D-galacturonic acid with a molecular weight of 7.2 kDa. Previous investigation suggested that homogalacturonan might impede angiogenesis, however the mechanism is still vague. Here we reported that LJW2F2 significantly disrupted capillary-like tube formation of human microvascular endothelia cells (HMEC-1) on matrigel as well as the cells migration. Mechanism study revealed that LJW2F2 might inactivate phosphorylation of epidermal growth factor receptor (EGFR), subsequently suppress Raf, mitogen-activated protein kinase (MEK) and extracellular-related kinase (ERK) phosphorylation. Moreover, LJW2F2 markedly decreased the expression of Notch1 and Delta-like ligand 4 (Dll4). Therefore, our results suggested that LJW2F2 might be a potential angiogenesis inhibitor via disturbing multiple signaling pathways. [ABSTRACT FROM AUTHOR]

The rapid ripening of soursop (Annona muricata L.) fruits is owing to its high respiration rate. Several enzymes affect the fruit cell wall in this process, resulting in the depolymerization of pectin primarily in the homogalacturonan. The main group of enzymes affecting the pectin content of soursop fruits include polygalacturonase (PG), pectate lyase (PL), pectin methylesterase (PME), and PME inhibitors (PMEis). In this study, pectin-degrading enzymes were analyzed using bioinformatic tools to uncover the gaps in our knowledge of this fruit. In this context, 67 genes encoding PG, 33 PL, 58 PME, and 39 PMEi isoenzymes were found. These genes were categorized into several families based on the results of phylogenetic analysis. Regarding the analysis of gene expression, a total of 25 were identified as differentially expressed genes (DEGs) in PG, while 3, 21, and 15 were found for PL, PME, and PMEis, respectively. Likewise, functional enrichment analysis demonstrated that the DEGs are involved in the modification of the cell wall, specifically in the degradation of pectin. On the other hand, gene co-expression networks revealed that the genes PG32 and PG35 affect the expression of PGs, as well as PL19 of the PL family, PME20, PME32, and PME35 of the PME family, and PMEi04 of the PMEi family. This suggests that they have a significant impact on the softening of soursop fruits.

IntroductionGoSAMTs play a role in the methylation of polysaccharides synthesized by the Golgi. Pectin homogalacturonan (HG) methyl-esterification is essential for the proper function of this polysaccharide in cell walls. In order to better understand the role of GoSAMTs in HG biosynthesis, we analyzed mucilage methyl-esterification in gosamt mutants.MethodsTo determine the function of GoSAMT1 and GoSAMT2 in HG methyl-esterification we utilized epidermal cells of seed coats, as these structures produce mucilage, which is a pectic matrix. We evaluated differences in seed surface morphology and quantified mucilage release. We measured methanol release, and used antibodies and confocal microscopy to analyze HG methyl-esterification in mucilage.ResultsWe observed morphological differences on the seed surface and delayed, uneven mucilage release in gosamt1-1gosamt2-1 double mutants. We also found changes in the distal wall length indicating abnormal cell wall breakage in this double mutant. Using methanol release and immunolabeling, we confirmed that GoSAMT1 and GoSAMT2 are involved in HG methyl-esterification in mucilage. However, we did not find evidence of decreasing HG in the gosamt mutants. Confocal microscopy analyses detected different patterns in the adherent mucilage and a greater number of low-methyl-esterified domains near the seed coat surface, which correlates with a greater number of “egg-box” structures in this region. We also detected a shift in the partitioning between the Rhamnogalacturonan-I soluble and adherent layers of the double mutant, which correlated with increased amounts of arabinose and arabinogalactan-protein in the adherent mucilage.DiscussionThe results show that the HG synthesized in gosamt mutant plants is less methyl esterified, resulting in more egg-box structures, which stiffen the cell walls in epidermal cells and change the rheological properties of the seed surface. The increased amounts of arabinose and arabinogalactan-protein in adherent mucilage, also suggests that compensation mechanisms were triggered in the gosamt mutants.

Introduction. The functional basis of protopectin complex can be represented as a network of regions that consist of homogalacturonan sequences and a base of rhamnogalacturonans-I, i.e. rhamnosyl-containing branching sites. Enzymatic isolation of these regions is possible only at a certain minimal native degree of polymerization. The research objective was to develop a system of criteria for assessing the potential applicability of the enzymatic transformation of plant protopectin complex. Study objects and methods. The research featured the polymerization degree of the homogalacturonan regions of the protopectin complex and produced a system of assessment criteria for the enzymatic fragmentation potential of the protopectin complex. The theoretical calculations were based on the values of the mass fractions of rhamnosyl and galacturonide residues in plant cell walls. The result was a new polymerization degree analytical function. Results and discussion. The ratio of the mass fractions of rhamnosyl and galacturonide residues in the water-insoluble plant tissue served as a dimensionless criterion of applicability. The rational condition for the dimensionless criterion of applicability was based on the fundamental constraint for homogalacturonan regions in the protopectin complex. It was expressed by a fundamental inequation. The rational area for determining the numerical values of the applicability criterion was presented as . The functional dependence was reduced to a two-dimensional criteria space as “width of rhamnosyl branches vs. the criterion of applicability”, where each pectin-containing raw material was given a single uniquely defined position. The boundary conditions for the criteria space were determined analytically. Conclusion. The new approach offers an assessment of the enzymatic fragmentation potential of the plant protopectin complex by homoenzyme preparations. The approach is in fact the second stage of the decision tree in the science-based technology for pectin and its products.

De-methyl esterification of homogalacturonan and subsequent cross-linking with Ca2+ is hypothesized to enhance the freezing survival of cold acclimated plants by reducing the porosity of primary cell walls. To test this theory, we collected leaf epidermal peels from non- (23/18 °C) and cold acclimated (2 weeks at 12/4 °C) Japanese bunching onion (Allium fistulosum L.). Cold acclimation enhanced the temperature at which half the cells survived freezing injury by 8 °C (LT50 =–20 °C), and reduced tissue permeability by 70-fold compared with non-acclimated epidermal cells. These effects were associated with greater activity of pectin methylesterase (PME) and a reduction in the methyl esterification of homogalacturonan. Non-acclimated plants treated with 50 mM CaCl2 accumulated higher concentrations of galacturonic acid, Ca2+ in the cell wall, and a lower number of visible cell wall pores compared with that observed in cold acclimated plants. Using cryo-microscopy, we observed that 50 mM CaCl2 treatment did not lower the LT50 of non-acclimated cells, but reduced the lethal intracellular ice nucleation to temperatures observed in cold acclimated epidermal cells. We postulate that the PME-homogalacturonan-mediated reduction in cell wall porosity is integral to intracellular freezing avoidance strategies in cold acclimated herbaceous cells. [ABSTRACT FROM AUTHOR]

The impact of pectin structure on carotenoid bioaccessibility is still uncertain. This study aims to investigate how the different pectic polymers affected the bioaccessibility of carotenoids in a simulated juice model during static in vitro digestion. This study includes homogalacturonan (HG), which is a linear pectic polymer, rhamnogalacturonan-I (RG-I), which is a branched pectic polymer, and rhamnogalacturonan (RG), which is a diverse pectic polymer rich in RG-I, rhamnogalacturonan-II (RG-II), and xylogalacturonan domains. Juice models without pectin had the highest carotenoid bioaccessibility, suggesting pectin has negative effects on carotenoid bioaccessibility. During the intestinal phase, systems with HG showed the highest viscosity, followed by systems with RG and systems with RG-I. Systems with RG-I had lower carotenoid bioaccessibility than systems with HG and RG-II. Both the percentage of RG-I and the average side chain length of RG-I had negative correlations with carotenoid bioaccessibility. RG-I side chains with more arabinose and/or galactose might cause lower carotenoid bioaccessibility in this juice model system. This study offers valuable insights into the relationship between pectin structure and carotenoid bioaccessibility in a simulated juice model, highlighting the importance of considering pectin composition for maximizing carotenoid bioaccessibility and potential health benefits in fruit-based beverages. • Simulated models reveal links between pectin and carotenoid bioaccessibility. • Carotenoid bioaccessibility was lower in rhamnogalacturonan-I system. • Rhamnogalacturonan-I ratio is negatively linked with carotenoid bioaccessibility. • Longer side chains of rhamnogalacturonan-I reduce carotenoid bioaccessibility. [ABSTRACT FROM AUTHOR]

Discarded carrots were repurposed to produced bio-based films through a pilot-scale hydrothermal treatment of the pulp. The process, conducted at 140 °C and 180 °C using single and multiple flow-through reactors, successfully extracted high molecular weight pectin-containing arabinogalactan (P-AG). Separation and purification through ultrafiltration and diafiltration yielded fractions with molecular weights ranging from 3.48 to 102.75 kDa. The solid residue from the hydrothermal process was subjected to mechanical treatments to obtain lignin-containing cellulose nanofibers (L-CNFs). Comparative analysis showed the P-AG film possessed superior oxygen barrier properties (67.73 vs. 239.83 cm3·µm/m2/kPa/day), while the L-CNFs film displayed higher tensile strength (7.74 vs. 3.14 MPa). Combining both fractions revealed that L-CNFs could be incorporated at percentages exceeding 15% (w/w) without compromising the P-AG-based film's integrity, resulting in a 57.5% increase in water contact angle (125.8°). In examining P-AG samples, a higher molecular weight reduced oxygen permeability by 14.6% (41.14 cm3·µm/m2/kPa/day) but increased the water vapor permeability by 11.4% (2.78·10−10 g·m/m2/s/Pa). Tensile strength showed a 150.9% increase at lower molecular weights and higher pectin content (2.84 MPa), attributed to arabinogalactan's pronounced branching. Elongation increased by 157.9% at higher arabinogalactan content (15.28%). This study achieved a groundbreaking milestone by obtaining purified fractions of P-AG and L-CNFs from carrot waste, demonstrating promising film properties suitable for food packaging applications. [Display omitted] • Films were produced from pectin-containing arabinogalactan of discarded carrots. • Films were produced from lignin-containing cellulose nanofibers of discarded carrots. • The combination of both fractions increased the hydrophobicity of the surface (126°). • Higher arabinogalactan content improved elongation but not tensile strength. • Acceptable films were obtained 100% from new fractions of discarded carrots. [ABSTRACT FROM AUTHOR]

Significance Statement: In Arabidopsis, mutations in xyloglucan xylosyltransferases result in the complete absence of this hemicellulose in the cell wall. Remarkably, the plant undergoes normal development and flowering without structural abnormalities other than a smaller stature compared to wild type and aberrant swelling of root hairs. We show, using quantitative linkage analysis, that the xxt1/xxt2 double mutant exhibits an enhanced accumulation of homogalacturonan and glucomannan, possibly to maintain structural integrity of the cell wall during growth in the absence of xyloglucan. [ABSTRACT FROM AUTHOR]

Tea is a popular beverage with a long history of safe and healthy use. Tea polysaccharide is a bioactive component extracted from tea, which has attracted more and more attention in recent decades. In this article, an acidic polysaccharide Gougunao tea polysaccharide (GPS) was isolated from Gougunao green tea by hot water extraction and ethanol precipitation. After purification by a diethylaminoethyl (DEAE) Sepharose Fast Flow column and a Sephacryl S-400 column, several homogalacturonan (HG) and rhamnogalacturonan-I (RG-I) fractions were obtained. Fraction GPS2b with the highest yield was selected for structural characterization by methylation and nuclear magnetic resonance (NMR) analysis. GPS2b was found to be an HG-type pectic polysaccharide (degree of methyl esterification [DE], 51.6%) with low molecular weight (Mw, 36.8 kDa). It was mainly composed of →4)-α-GalpA- (1→ and →4)-α-GalpA-6-OMe-(1→. In addition, a minor highly branched RG-I domain was identified in this fraction. The investigation of structural features of tea polysaccharides can provide insights to understand their structure-bioactivity relationship.

Antibiotic-associated diarrhea (AAD) is a common side-effect of antibiotic treatment resulting from an imbalance in the colonic bacteria. The hypothesis of this study is to ask whether polysaccharide from the rhizome of Dioscorea opposita which is recorded as conventional herbs and food for diarrhea treatment in Southeast Asia, may be an active compound against diarrhea induced by antibiotics. To address, firstly, a homogenous polysaccharide, DOP0.2-S-3 was characterized as a homogalacturonan containing linear repeating units of → 4)-α-D-GalAp(1 → 4)-α-D-GalAp(1 → with the average molecular weight of 14 kDa. DOP0.2-S-3 significantly reduced the water content and defecation times caused by AAD in mice, while it also remarkably attenuated the cytokines of IL-1β and IL-6 expression in mice colon tissues. DOP0.2-S-3 decreased potential pathogen and increased Bacteroidetes in the mice gut. These results suggested DOP0.2-S-3 might be a new leading compound for the functional foods or drug candidate development against AAD partially through regulating gut flora. [ABSTRACT FROM AUTHOR]

A homogalacturonan pectin (HG, designated as WJP-F80) was extracted from the peel of winter jujube (Zizyphus jujuba Mill. Cv. Dongzao) and separated via ethanol-graded precipitation. The structural and conformational features were elucidated through HPAEC-PAD, GC-MS, 2D NMR, and HPSEC-MALLS studies. In vivo assessments were carried out to evaluate the hepatoprotective effects of WJP-F80 against CCl4-induced injury of mice. Results showed that WJP-F80 was a linear 1,4-α-galacturonan with partially methyl-esterified at O-6 of GalpA and occasionally acetylation. The Mw of WJP-F80 was determined as 45.3 kDa, the polydispersity was calculated as 1.56, and the Rg was measured as 22.7 nm in 0.1 M NaNO3. The conformational analysis revealed that WJP-F80 exhibited as rigid stiff chain in low Mw range, while aggregation by self-assembly of HG chains lead to high Mw and random coil conformation. In vivo studies indicated that WJP-F80 can protect the livers of mice from acute injury induced via CCl4 by decreasing the serum biochemical markers of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) to normal levels. This work provides a theoretical basis for the value-added deep processing of winter jujube.

The cell walls of forage chicory (Cichorium intybus) leaves are known to contain high proportions of pectic polysaccharides. However, little is known about the distribution of pectic polysaacharides among walls of different cell types/tissues and within walls. In this study, immunolabelling with four monoclonal antibodies was used to map the distribution of pectic polysaccharides in the cell walls of the laminae and midribs of these leaves. The antibodies JIM5 and JIM7 are specific for partially methyl-esterified homogalacturonans; LM5 and LM6 are specific for (1→4)-β-galactan and (1→5)-α-arabinan side chains, respectively, of rhamnogalacturonan I. All four antibodies labelled the walls of the epidermal cells with different intensities. JIM5 and JIM7, but not LM5 or LM6, labelled the middle lamella, tricellular junctions, and the corners of intercellular spaces of ground, xylem and phloem parenchyma. LM5, but not LM6, strongly labelled the walls of the few sclerenchyma fibres in the phloem of the midrib and lamina vascular bundles. The LM5 epitope was absent from some phloem parenchyma cells. LM6, but not LM5, strongly labelled the walls of the stomatal guard cells. The differential distribution of pectic epitopes among walls of different cell types and within walls may reflect the deposition and modification of these polysaccharides which are involved in cell wall properties and cell development. [ABSTRACT FROM AUTHOR]

Background and Aims The programmed softening occurring during fruit development requires scission of cell wall polysaccharides, especially pectin. Proposed mechanisms include the action of wall enzymes or hydroxyl radicals. Enzyme activities found in fruit extracts include pectate lyase (PL) and endo-polygalacturonase (EPG), which, in vitro , cleave de-esterified homogalacturonan in mid-chain by β-elimination and hydrolysis, respectively. However, the important biological question of whether PL exhibits action in vivo had not been tested. Methods We developed a method for specifically and sensitively detecting in-vivo PL products, based on Driselase digestion of cell wall polysaccharides and detection of the characteristic unsaturated product of PL action. Key Results In model in-vitro experiments, pectic homogalacturonan that had been partially cleaved by commercial PL was digested to completion with Driselase, releasing an unsaturated disaccharide ('ΔUA–GalA'), taken as diagnostic of PL action. ΔUA–GalA was separated from saturated oligogalacturonides (EPG products) by electrophoresis, then subjected to thin-layer chromatography (TLC), resolving ΔUA–GalA from higher homologues. The ΔUA–GalA was confirmed as 4-deoxy-β- l - threo -hex-4-enopyranuronosyl-(1→4)- d -galacturonic acid by NMR spectroscopy. Driselase digestion of cell walls from ripe fruits of date (Phoenix dactylifera), pear (Pyrus communis), rowan (Sorbus aucuparia) and apple (Malus pumila) yielded ΔUA–GalA, demonstrating that PL had been acting in vivo in these fruits prior to harvest. Date-derived ΔUA–GalA was verified by negative-mode mass spectrometry, including collision-induced dissociation (CID) fragmentation. The ΔUA–GalA:GalA ratio from ripe dates was roughly 1:20 (mol mol–1), indicating that approx. 5 % of the bonds in endogenous homogalacturonan had been cleaved by in-vivo PL action. Conclusions The results provide the first demonstration that PL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening. [ABSTRACT FROM AUTHOR]

The cell walls of forage chicory (Cichorium intybus) leaves are known to contain high proportions of pectic polysaccharides. However, little is known about the distribution of pectic polysaacharides among walls of different cell types/tissues and within walls. In this study, immunolabelling with four monoclonal antibodies was used to map the distribution of pectic polysaccharides in the cell walls of the laminae and midribs of these leaves. The antibodies JIM5 and JIM7 are specific for partially methyl-esterified homogalacturonans; LM5 and LM6 are specific for (1→4)-β-galactan and (1→5)-α-arabinan side chains, respectively, of rhamnogalacturonan I. All four antibodies labelled the walls of the epidermal cells with different intensities. JIM5 and JIM7, but not LM5 or LM6, labelled the middle lamella, tricellular junctions, and the corners of intercellular spaces of ground, xylem and phloem parenchyma. LM5, but not LM6, strongly labelled the walls of the few sclerenchyma fibres in the phloem of the midrib and lamina vascular bundles. The LM5 epitope was absent from some phloem parenchyma cells. LM6, but not LM5, strongly labelled the walls of the stomatal guard cells. The differential distribution of pectic epitopes among walls of different cell types and within walls may reflect the deposition and modification of these polysaccharides which are involved in cell wall properties and cell development.

In present study, two water-soluble polysaccharides designated as POL-1 and POL-2 were purified from purslane and their structural characteristics as well as immunomodulatory activity were investigated. The weight-average molecular weight (M w) of POL-1 and POL-2 were determined to be 64,100 Da and 21,000 Da, respectively. Comprehensive techniques including UV, IR, GC–MS, and NMR were applied to deduced that POL-1 was a pectin polysaccharide homogalacturonan (HG) consisting of →4)- α -Gal p A-(1→ with methyl ester degree of 9.71 % and acetylation degree of 0.34 %, while POL-2 was composed of a 1, 4-linked β -Gal p backbone substituted by short side chain →4)- α -Glc p -(1→ and →6)- α -Glc p -(1→. The →4)- α -Glc p -(1→ was attached at the O -6 position of →4)- β -Gal p -(1→. TEM further revealed that POL-1 was non-branched single chains, while POL-2 was entangled microstructure with side chains. Moreover, POL-2 significantly promoted macrophage phagocytosis as well as the secretion of NO and cytokines (TNF- α , IL-6) through activating NF-κB signaling pathway, thus demonstrating potential immunomodulatory activity. These findings suggested that purslane may be exploited as a potential adjuvant and dietary supplement with immunostimulatory purpose. [Display omitted] • An acidic polysaccharide POL-1 and a neutral polysaccharide POL-2 were purified from Portulaca oleracea L. • The structural characterization of POL-1 and POL-2were conducted. • Polysaccharide POL-2 displayed immunomodulatory effects on RAW264.7 cells in vitro. • The mechanism of immunomodulatory activity of POL-2 is involved in activating NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

In order to understand mechanisms of ontogenic resistance to apple scab, we analyzed various aspects of young and old leaves. We have introduced an apple plants cultivation system where in vitro propagated and rooting explants produce a genetically uniform population of apple (Malus domestica cv. Idared) plants. In this work, we demonstrate that apple plants produced in our cultivation system showed susceptibility to Venturia inaequalis, the cause of apple scab disease in young leaves and resistance in old leaves, which is similar to orchard situation. Our analysis shows that the cessation of epidermal cell expansion and shape formation coincided with the onset of ontogenic resistance in older leaves. Formation of specific cuticular lamellar structures did not coincide with ontogenic resistance onset. Further, chemical composition analysis of wax from young susceptible and old resistant leaves did not reveal specific compounds involved in ontogenic resistance. Differences in homogalacturonan content in cell walls in susceptible and resistant cells as well as decreased methylesterification of pectin in resistant leaves suggest that polysaccharide composition of the cell wall may play a role in mycelium growth and nutrition.

Abstract Background Grafting is a technique widely used in horticulture. The processes involved in grafting are diverse, and the technique is commonly employed in studies focusing on the mechanisms that regulate cell differentiation or response of plants to abiotic stress. Information on the changes in the composition of the cell wall that occur during the grafting process is scarce. Therefore, this study was carried out for analyzing the composition of the cell wall using Arabidopsis hypocotyls as an example. During the study, the formation of a layer that covers the surface of the graft union was observed. So, this study also aimed to describe the histological and cellular changes that accompany autografting of Arabidopsis hypocotyls and to perform preliminary chemical and structural analyses of extracellular material that seals the graft union. Results During grafting, polyphenolic and lipid compounds were detected, along with extracellular deposition of carbohydrate/protein material. The spatiotemporal changes observed in the structure of the extracellular material included the formation of a fibrillar network, polymerization of the fibrillar network into a membranous layer, and the presence of bead-like structures on the surface of cells in established graft union. These bead-like structures appeared either “closed” or “open”. Only three cell wall epitopes, namely: LM19 (un/low-methyl-esterified homogalacturonan), JIM11, and JIM20 (extensins), were detected abundantly on the cut surfaces that made the adhesion plane, as well as in the structure that covered the graft union and in the bead-like structures, during the subsequent stages of regeneration. Conclusions To the best of our knowledge, this is the first report on the composition and structure of the extracellular material that gets deposited on the surface of graft union during Arabidopsis grafting. The results showed that unmethyl-esterified homogalacturonan and extensins are together involved in the adhesion of scion and stock, as well as taking part in sealing the graft union. The extracellular material is of importance not only due to the potential pectin–extensin interaction but also due to its origin. The findings presented here implicate a need for studies with biochemical approach for a detailed analysis of the composition and structure of the extracellular material.

Comprehensive microarray polymer profiling, fruit softening, homogalacturonan, pectin, polygalacturonase, RNA-Seq. [Extracted from the article]

The water-soluble fractions of pectin extracted from the pulp of ripe papayas have already been found to exert positive effects on cancer cell cultures. However, the mechanisms that lead to these beneficial effects and the pectin characteristics that exert these effects are still not well understood. Characteristics such as molecular size, monosaccharide composition and structural conformation are known as polysaccharide factors that can cause alterations in cellular response. During fruit ripening, a major polysaccharide solubilization, depolymerization, and chemical modification occur. The aims of this work are to fractionate the pectin extracted from the pulp of papayas at two stages of ripening (fourth and ninth day after harvesting) into uronic and neutral fractions and to test them for the inhibition of human recombinant galectin-3 and the inhibition of colon cancer cell growth. The structures of the fractions were chemically characterized, and the uronic fraction extracted from the fourth day after harvesting presented the best biological effects across different concentrations in both galectin-3 inhibition and viability assays. The results obtained may help to establish a relationship between the chemical structures of papaya pectins and the positive in vitro biological effects, such as inhibiting cancer cell growth. [ABSTRACT FROM AUTHOR]

The major cell wall pectic glycan homogalacturonan (HG) is crucial for plant growth, development, and reproduction. HG synthesis occurs in the Golgi and is catalyzed by members of the galacturonosyltransferase (GAUT) family with GAUT1 being the archetypal and best studied family member. In Arabidopsis suspension culture cells and tobacco leaves, the Golgi localization of Arabidopsis GAUT1 has been shown to require protein-protein interactions with its homolog GAUT7. Here we show that in pollen tubes GAUT5 and GAUT6, homologs of GAUT7, also target GAUT1 to the Golgi apparatus. Pollen tube germination and elongation in double homozygous knock-out mutants (gaut5 gaut6, gaut5 gaut7, and gaut6 gaut7) are moderately impaired, whereas gaut5−/−gaut6−/−gaut7+/− triple mutant is severely impaired and male infertile. Amounts and distributions of methylesterified HG in the pollen tube tip were severely distorted in the double and heterozygous triple mutants. A chimeric protein comprising GAUT1 and a non-cleavable membrane anchor domain was able to partially restore pollen tube germination and elongation and to reverse male sterility in the triple mutant. These results indicate that GAUT5, GAUT6, and GAUT7 are required for synthesis of native HG in growing pollen tubes and have critical roles in pollen tube growth and male fertility in Arabidopsis.

Understanding plant adaptation mechanisms to prolonged water immersion provides options for genetic modification of existing crops to create cultivars more tolerant of periodic flooding. An important advancement in understanding flooding adaptation would be to elucidate mechanisms, such as aerenchyma air-space formation induced by hypoxic conditions, consistent with prolonged immersion. Lysigenous aerenchyma formation occurs through programmed cell death (PCD), which may entail the chemical modification of polysaccharides in root tissue cell walls. We investigated if a relationship exists between modification of pectic polysaccharides through de-methyl esterification (DME) and the formation of root aerenchyma in select Fabaceae species. To test this hypothesis, we first characterized the progression of aerenchyma formation within the vascular stele of three different legumes—Pisum sativum, Cicer arietinum, and Phaseolus coccineus—through traditional light microscopy histological staining and scanning electron microscopy. We assessed alterations in stele morphology, cavity dimensions, and cell wall chemistry. Then we conducted an immunolabeling protocol to detect specific degrees of DME among species during a 48-hour flooding time series. Additionally, we performed an enzymatic pretreatment to remove select cell wall polymers prior to immunolabeling for DME pectins. We were able to determine that all species possessed similar aerenchyma formation mechanisms that begin with degradation of root vascular stele metaxylem cells. Immunolabeling results demonstrated DME occurs prior to aerenchyma formation and prepares vascular tissues for the beginning of cavity formation in flooded roots. Furthermore, enzymatic pretreatment demonstrated that removal of cellulose and select hemicellulosic carbohydrates unmasks additional antigen binding sites for DME pectin antibodies. These results suggest that additional carbohydrate modification may be required to permit DME and subsequent enzyme activity to form aerenchyma. By providing a greater understanding of cell wall pectin remodeling among legume species, we encourage further investigation into the mechanism of carbohydrate modifications during aerenchyma formation and possible avenues for flood-tolerance improvement of legume crops.

Cell wall structural modifications through pectin cross-linkages between calcium ions and/or boric acid may be key to mitigating dehydration stress and fungal pathogens. Water loss was profiled in a pure pectin system and in vivo. While calcium and boron reduced water loss in pure pectin standards, the impact on Allium species was insignificant (p > 0.05). Nevertheless, synchrotron X-ray microscopy showed the localization of exogenously applied calcium to the apoplast in the epidermal cells of Allium fistulosum. Exogenous calcium application increased viscosity and resistance to shear force in Allium fistulosum, suggesting the formation of calcium cross-linkages (“egg-box” structures). Moreover, Allium fistulosum (freezing tolerant) was also more tolerant to dehydration stress compared to Allium cepa (freezing sensitive). Furthermore, the addition of boric acid (H3BO3) to pure pectin reduced water loss and increased viscosity, which indicates the formation of RG-II dimers. The Arabidopsis boron transport mutant, bor1, expressed greater water loss and, based on the lesion area of leaf tissue, a greater susceptibility to Colletotrichum higginsianum and Botrytis cinerea. While pectin modifications in the cell wall are likely not the sole solution to dehydration and biotic stress resistance, they appear to play an important role against multiple stresses.

An efficient plant regeneration protocol has been established for two commercial Populus hybrid clones, MC (Populus × euramericana) and UNAL (Populus × interamericana). The culture of internode segments on Murashige and Skoog (MS) medium with 0.5 μM α-naphthalene acetic acid (NAA) and 4 μM N6-benzyladenine for 7 weeks (2 weeks in absence of activated charcoal and 5 weeks in its presence) resulted in the highest frequency of shoot regeneration (100 % for MC and 82 % for UNAL). All regenerated shoots longer than 2 cm rooted on half-strength MS medium, independent of the addition of 0.1 μM NAA. Nevertheless, shoots developed better-formed roots in NAA-free medium, which had a positive effect on the acclimatization of plants. In order to know the cellular processes underlying in vitro shoot organogenesis, a histological study was made in UNAL internode-explants. Results revealed that in vitro culture caused swelling around the cut-off zones in all explants, but only those undergoing organogenesis formed proliferation centers under subepidermal cells, which led to formation of bud primordia. Moreover, in vivo tissues and explants with different in vitro response showed different immunolabelling patterns when they were treated with fluorescentmonoclonal antibodies directed to several pectin-polysaccharides of the cell wall. Results allow us to assign a predominant role of homogalacturonan with a low degree of methyl-esterification in the initiation of bud primordia, role of β-1,4-D-galactan side chains of rhamnogalacturonan-I in the cellular differentiation, role of α-1,5-L-arabinan side chains of rhamnogalacturonan-I and of homogalacturonan with a high degree of methyl-esterification in cell division and growth.

The major cell wall pectic glycan homogalacturonan (HG) is crucial for plant growth, development, and reproduction. HG synthesis occurs in the Golgi and is catalyzed by members of the galacturonosyltransferase (GAUT) family with GAUT1 being the archetypal and best studied family member. In Arabidopsis suspension culture cells and tobacco leaves, the Golgi localization of Arabidopsis GAUT1 has been shown to require protein-protein interactions with its homolog GAUT7. Here we show that in pollen tubes GAUT5 and GAUT6, homologs of GAUT7, also target GAUT1 to the Golgi apparatus. Pollen tube germination and elongation in double homozygous knock-out mutants (gaut5 gaut6 , gaut5 gaut7 , and gaut6 gaut7) are moderately impaired, whereas gaut5 −/− gaut6 −/− gaut7 +/− triple mutant is severely impaired and male infertile. Amounts and distributions of methylesterified HG in the pollen tube tip were severely distorted in the double and heterozygous triple mutants. A chimeric protein comprising GAUT1 and a non-cleavable membrane anchor domain was able to partially restore pollen tube germination and elongation and to reverse male sterility in the triple mutant. These results indicate that GAUT5, GAUT6, and GAUT7 are required for synthesis of native HG in growing pollen tubes and have critical roles in pollen tube growth and male fertility in Arabidopsis. [ABSTRACT FROM AUTHOR]

Aphids are a major issue for commercial crops. These pests drain phloem nutrients and transmit ~50% of the known insect-borne viral diseases. During aphid feeding, trophic structures called stylets advance toward the phloem intercellularly, disrupting cell wall polymers. It is thought that cell wall-modifying enzymes (CWMEs) present in aphid saliva facilitate stylet penetration through this intercellular polymer network. Additionally, different studies have demonstrated that host settling preference, feeding behavior, and colony performance of aphids are influenced by modulating the CWME expression levels in host plants. CWMEs have been described as critical defensive elements for plants, but also as a key virulence factor for plant pathogens. However, whether CWMEs are elements of the plant defense mechanisms or the aphid infestation process remains unclear. Therefore, in order to better consider the function of CWMEs and cell wall-derived damage-associated molecular patterns (DAMPs) during plant–aphid interactions, the present review integrates different hypotheses, perspectives, and experimental evidence in the field of plant–aphid interactions and discusses similarities to other well-characterized models such as the fungi–plant pathosystems from the host and the attacker perspectives. [ABSTRACT FROM AUTHOR]

De-esterification of homogalacturonan (HG) is thought to stiffen pectin gels and primary cell walls by increasing calcium cross-linking between HG chains. Contrary to this idea, recent studies found that HG de-esterification correlated with reduced stiffness of living tissues, measured by surface indentation. The physical basis of such apparent wall softening is unclear, but possibly involves complex biological responses to HG modification. To assess the direct physical consequences of HG de-esterification on wall mechanics without such complications, we treated isolated onion (Allium cepa) epidermal walls with pectin methylesterase (PME) and assessed wall biomechanics with indentation and tensile tests. In nanoindentation assays, PME action softened the wall (reduced the indentation modulus). In tensile force/extension assays, PME increased plasticity, but not elasticity. These softening effects are attributed, at least in part, to increased electrostatic repulsion and swelling of the wall after PME treatment. Despite softening and swelling upon HG de-esterification, PME treatment alone failed to induce cell wall creep. Instead, acid-induced creep, mediated by endogenous α-expansin, was reduced. We conclude that HG de-esterification physically softens the onion wall, yet reduces expansin-mediated wall extensibility. [ABSTRACT FROM AUTHOR]

Understanding plant adaptation mechanisms to prolonged water immersion provides options for genetic modification of existing crops to create cultivars more tolerant of periodic flooding. An important advancement in understanding flooding adaptation would be to elucidate mechanisms, such as aerenchyma air-space formation induced by hypoxic conditions, consistent with prolonged immersion. Lysigenous aerenchyma formation occurs through programmed cell death (PCD), which may entail the chemical modification of polysaccharides in root tissue cell walls. We investigated if a relationship exists between modification of pectic polysaccharides through de-methyl esterification (DME) and the formation of root aerenchyma in select Fabaceae species. To test this hypothesis, we first characterized the progression of aerenchyma formation within the vascular stele of three different legumes— Pisum sativum , Cicer arietinum , and Phaseolus coccineus —through traditional light microscopy histological staining and scanning electron microscopy. We assessed alterations in stele morphology, cavity dimensions, and cell wall chemistry. Then we conducted an immunolabeling protocol to detect specific degrees of DME among species during a 48-hour flooding time series. Additionally, we performed an enzymatic pretreatment to remove select cell wall polymers prior to immunolabeling for DME pectins. We were able to determine that all species possessed similar aerenchyma formation mechanisms that begin with degradation of root vascular stele metaxylem cells. Immunolabeling results demonstrated DME occurs prior to aerenchyma formation and prepares vascular tissues for the beginning of cavity formation in flooded roots. Furthermore, enzymatic pretreatment demonstrated that removal of cellulose and select hemicellulosic carbohydrates unmasks additional antigen binding sites for DME pectin antibodies. These results suggest that additional carbohydrate modification may be required to permit DME and subsequent enzyme activity to form aerenchyma. By providing a greater understanding of cell wall pectin remodeling among legume species, we encourage further investigation into the mechanism of carbohydrate modifications during aerenchyma formation and possible avenues for flood-tolerance improvement of legume crops. [ABSTRACT FROM AUTHOR]

This article deals with the distribution of callose and of the homogalacturonan (HG) epitopes recognized by LM20, JIM5, and 2F4 antibodies in cell walls of differentiating and functioning stomatal complexes of the monocotyledon Zea mays and the dicotyledon Vigna sinensis. The findings revealed that, during stomatal development, in these plant species, callose appears in an accurately spatially and timely controlled manner in cell walls of the guard cells (GCs). In functioning stomata of both plants, callose constitutes a dominant cell wall matrix material of the polar ventral cell wall ends and of the local GC cell wall thickenings. In Zea mays, the LM20, JIM5, or 2F4 antibody-recognized HG epitopes were mainly located in the expanding cell wall regions of the stomatal complexes, while in Vigna sinensis, they were deposited in the local cell wall thickenings of the GCs as well as at the ledges of the stomatal pore. Consideration of the presented data favors the view that in the stomatal complexes of the monocotyledon Z. mays and the dicotyledon V. sinensis, the esterified HGs contribute to the cell wall expansion taking place during GC morphogenesis and the opening of the stomatal pore. Besides, callose and the highly de-esterified HGs allow to GC cell wall regions to withstand the mechanical stresses exerted during stomatal function. [ABSTRACT FROM AUTHOR]

Summary: How cell wall elasticity, plasticity, and time‐dependent extension (creep) relate to one another, to plant cell wall structure and to cell growth remain unsettled topics. To examine these issues without the complexities of living tissues, we treated cell‐free strips of onion epidermal walls with various enzymes and other agents to assess which polysaccharides bear mechanical forces in‐plane and out‐of‐plane of the cell wall. This information is critical for integrating concepts of wall structure, wall material properties, tissue mechanics and mechanisms of cell growth. With atomic force microscopy we also monitored real‐time changes in the wall surface during treatments. Driselase, a potent cocktail of wall‐degrading enzymes, removed cellulose microfibrils in superficial lamellae sequentially, layer‐by‐layer, and softened the wall (reduced its mechanical stiffness), yet did not induce wall loosening (creep). In contrast Cel12A, a bifunctional xyloglucanase/cellulase, induced creep with only subtle changes in wall appearance. Both Driselase and Cel12A increased the tensile compliance, but differently for elastic and plastic components. Homogalacturonan solubilization by pectate lyase and calcium chelation greatly increased the indentation compliance without changing tensile compliances. Acidic buffer induced rapid cell wall creep via endogenous α‐expansins, with negligible effects on wall compliances. We conclude that these various wall properties are not tightly coupled and therefore reflect distinctive aspects of wall structure. Cross‐lamellate networks of cellulose microfibrils influenced creep and tensile stiffness whereas homogalacturonan influenced indentation mechanics. This information is crucial for constructing realistic molecular models that define how wall mechanics and growth depend on primary cell wall structure. Significance Statement: To assess how cell wall elasticity, plasticity and slow time‐dependent extension during growth relate to one another and to wall structure, we measured the biomechanical actions of various enzymes by uniaxial tensile testing, nano‐indentation, creep assays, and atomic force microscopy (AFM) imaging. These responses were separable, unlike those expected of a simple fiber‐reinforced hydrogel. Our results identify distinctive roles for homogalacturonan and cross‐lamellate networks of cellulose microfibrils in epidermal wall mechanics and growth. [ABSTRACT FROM AUTHOR]

The gall structure is built from redifferentiation of host plant tissues, changing the cell wall proprieties and associated functions. Because of the oxidative stress imposed by the galling insect on the plant tissues, some phenotypic traits associated with cell wall metabolism may be suppressed during gall development. Using an immunocytochemical approach we investigated the pectin and glycoprotein composition of the cell walls during non-galled leaf development and galls induced on Aspidosperma tomentosum (Apocynaceae). Embedding in historesin was used for anatomical characterization, as well as for immunocytochemical analyses. The samples were probed with the primary monoclonal antibodies JIM5, JIM7, LM1, LM2, LM5 and LM6. The gall develops especially from cell hypertrophy and hyperplasia of the spongy and palisade parenchyma, which determine its shape. Epitopes of β-D-galactan and α-L-arabinan in the galls were totally suppressed or less expressed. The increased labeling of homogalacturonan epitopes, especially in senescent galls, is a clear indicator that pectin methylesterase activity is maintained despite oxidative stresses imposed by the galling insect. Arabinogalactan proteins were not detected in any non-galled leaf or gall stages. The galling insect deeply changed the structure and deposition pattern of the cell wall, promoting new functionalities in the gall tissue compartments. • The galling insect is a phenotype manipulator of cell wall traits of the host organ. • Pectin dynamics is operational during A. tomentosum gall formation and suggest a linear process de-methyl esterification. • The gall cell walls have reduced abundance of galactan and arabinan epitopes. [ABSTRACT FROM AUTHOR]

In this work, the effects of four different extraction methods, acid (HCl), alkali (NaOH), enzymes (cellulase/pectinase), and buffer (pH 7.0) on the physicochemical properties and functionalities of burdock pectin were systematically investigated and compared. Buffer extraction gave a low yield (2.8 %) and is therefore limited in its application. The acid treatment hydrolyzed the neutral sidechains and gave a homogalacturonan content of 72.6 %. By contrast, alkali and enzymes preserved the sidechains while degrading the polygalacturonan backbone, creating a rhamnogalacturonan-I dominant structure. The branched structure, low molecular weight, and high degree of methylation (42.3 %) contributed to the interfacial adsorption, emulsifying capacity, and cellular antioxidant activity of the enzyme-extracted product. For the acid-extracted product, the strong intramolecular electrostatic repulsion restricted the formation of a contact interface to prevent coalescence of the emulsion. In addition, they did not have sufficient reducing ends to scavenge free radicals. Although a high branching size (5.0) was adopted, the low degree of methylation (19.5 %) affected the emulsifying capacity of the alkali-extracted products. These results provide useful information for pectic polysaccharides production with tailored properties. [Display omitted] • Acid, alkali, enzymes and buffer were used to extract pectic polysaccharides from burdock. • Acid treatment yielded homogalacturonan-rich pectic polysaccharides • Alkaline or enzyme extraction provided the rhamnogalacturonan-I dominant structure. • The emulsifying and antioxidant capacity of acid-extracted samples were constrained. • The small molecular size and branched structure favored the enzyme-extracted samples. [ABSTRACT FROM AUTHOR]

Land plants inherited several traits from their green algal ancestors (Zygnematophyceae), including a polysaccharide-rich cell wall, which is a prerequisite for terrestrial survival. A major component of both land plant and Zygnematophyceaen cell walls is the pectin homogalacturonan (HG), and its high water holding capacity may have helped algae to colonize terrestrial habitats, characterized by water scarcity. To test this, HG was removed from the cell walls of Zygnema filaments by pectate lyase (PL), and their effective quantum yield of photosystem II (YII) as a proxy for photosynthetic performance was measured in response to desiccation stress by pulse amplitude modulation (PAM). Old filaments were found to contain more HG and are more resistant against desiccation stress but relatively lose more desiccation resistance after HG removal than young filaments. After rehydration, the photosynthetic performance recovered less efficiently in filaments with a HG content reduced by PL, independently of filament age. Immunolabeling showed that partial or un-methylesterified HG occurs throughout the longitudinal cell walls of both young and old filaments, while no labeling signal occurred when filaments were treated with PL prior labeling. This confirmed that most HG can be removed from the cell walls by PL. The initial labeling pattern was restored after ~3 days. A different form of methylesterified HG was restricted to cell poles and cross cell walls. In conclusion, it was shown that the accumulation of HG in Zygnema filaments increases their resistance against desiccation stress. This trait might have played an important role during the colonization of land by Zygnematophyceae, which founded the evolution of all land plants.

The mechanisms underlying susceptibility to and defense against Pseudomonas syringae (Pph) of the common bean (Phaseolus vulgaris) have not yet been clarified. To investigate these, 15-day-old plants of the variety Riñón were infected with Pph and the transcriptomic changes at 2 h and 9 h post-infection were analysed. RNA-seq analysis showed an up-regulation of genes involved in defense/signaling at 2 h, most of them being down-regulated at 9 h, suggesting that Pph inhibits the transcriptomic reprogramming of the plant. This trend was also observed in the modulation of 101 cell wall-related genes. Cell wall composition changes at early stages of Pph infection were associated with homogalacturonan methylation and the formation of egg boxes. Among the cell wall genes modulated, a pectin methylesterase inhibitor 3 (PvPMEI3) gene, closely related to AtPMEI3, was detected. PvPMEI3 protein was located in the apoplast and its pectin methylesterase inhibitory activity was demonstrated. PvPMEI3 seems to be a good candidate to play a key role in Pph infection, which was supported by analysis of an Arabidopsis pmei3 mutant, which showed susceptibility to Pph, in contrast to resistant Arabidopsis Col-0 plants. These results indicate a key role of the degree of pectin methylesterification in host resistance to Pph during the first steps of the attack., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Plants have evolved different strategies to utilize various forms of nitrogen (N) from the environment. While regulation of plant growth and development in response to application of inorganic N forms has been characterized, our knowledge about the effect on cell wall structure and composition is quite limited. In this study, we analysed cell walls of Brachypodium distachyon supplied with three types of inorganic N (NH4NO3, NO3−, or NH4+). Cell wall profiles showed distinct alterations in both the quantity and structures of individual polymers. Nitrate stimulated cellulose, but inhibited lignin deposition at the heading growth stage. On the other hand, ammonium supply resulted in higher concentration of mixed linkage glucans. In addition, the chemical structure of pectins and hemicelluloses was strongly influenced by the form of N. Supply of only NO3− led to alteration in xylan substitution and to lower esterification of homogalacturonan. We conclude that the physiological response to absorption of different inorganic N forms includes pleotropic remodelling of type II cell walls. [ABSTRACT FROM AUTHOR]

The rheological properties of the optimized high-methoxyl pectin (PPOP), obtained from ponkan peel using nitric acid extraction (pH 1.6, 100 min, 36 ml/g liquid:solid ratio) were characterized. The flow behavior of aqueous and 0.1 M NaCl solutions at concentrations of 1%, 3% and 5% (w/w) was evaluated. The apparent viscosity of the solutions was directly related to the concentration. At the same concentrations and low shear rates the apparent viscosity values were close to those of a commercial citrus pectin. However, at higher shear rates, PPOP had a more shear thinning behavior. For both pectins, the presence of 0.1 M NaCl did not affect the apparent viscosity. The influence of pH, pectin and sucrose concentration on the gelling behavior of PPOP was investigated. Gelation occurred at pH < 3.5 and the strongest gel was obtained with 2.5% (w/w) pectin and 60% (w/w) sucrose at pH 2.5. The gel prepared under these conditions had viscoelastic behavior comparable to the gel prepared with a high-methoxyl commercial citrus pectin under the same conditions and displayed high thermal stability. Image 1 • The rheological properties of a pectin from ponkan peel (PPOP) were evaluated. • The strongest gel was obtained using 2.5% (w/w) PPOP, 60% (w/w) sucrose at pH 2.5. • The gel formed under these conditions was thermally stable. • The rheological properties of PPOP were comparable to a commercial citrus pectin. [ABSTRACT FROM AUTHOR]

Highlights • Polysaccharides from the chamomile tea were purified and characterized. • Inulin, HM homogalacturonan and a type II arabinogalactan were obtained. • Fructooligosaccharides ranging from GF2 (m / z 543) to GF10 (m / z 1839) were also present. • Chamomile is a source of structurally diverse dietary fibers. • Chamomile tea polysaccharides could have potential prebiotic function. Abstract Chamomile is one of most known species of medicinal plants. It has valuable pharmacological properties that produce positive effects in many therapeutical uses. Some of these properties are attributed to the presence of secondary metabolites but is already known that primary metabolites can also produce positive effects. In this study we elucidate the fine chemical structure of polysaccharides present in the infusion of chamomile flower chapters. After ethanolic precipitation, polysaccharides were obtained from the tea (fraction MRW, 3.2% yield), purified and characterized as an inulin type fructan, a highly methyl esterified and acetylated homogalacturonan (DE = 87% and DA = 19%), and a type II arabinogalactan. From ethanolic supernatant (20.2% yield), fructooligosaccharides (FOS) ranging from GF2 (m / z 543) to GF10 (m / z 1839) were detected. Inulin and FOS are well-established prebiotics, as well as the pectic polysaccharides. Thus, chamomile could be a source of structurally diverse dietary fibers with potential prebiotic, gastrointestinal and immunological functions. [ABSTRACT FROM AUTHOR]