Xyloglucan Endotransglycosylase Research Articles

Dicyclohexylcarbodiimide and disodium succinate regulate the pulp softening and breakdown in fresh longan by modulating the metabolism of cell wall polysaccharides

Cell wall polysaccharides (CWP) are an important component of cell structure, and its content variation is a primary factor affecting the texture of fresh longan. The influences of dicyclohexylcarbodiimide (DCC) and disodium succinate (DS) treatments on the pulp softening and pulp breakdown, and their relation to CWP metabolism in pulp of longan fruit were explored. Compared with the control longan, DCC-treated fruit showed lower value of pulp firmness, higher index of pulp breakdown, higher expression levels of cell wall disassembly enzymes (CWDEs)-related genes (DlPME1, DlPME3, DlPG1, Dlβ-Gal1, Dlβ-Gal16, DlCx7, DlXET3, DlXET27), and higher activities of CWDEs including pectin methyl esterase (PME), polygalacturonase (PG), β-galactosidase (β-Gal), cellulase (Cx), xyloglucan endotransglycosylase (XET), lower values of cell wall materials, and lower levels of CWP including ionic-soluble pectin, covalent-soluble pectin, cellulose, and hemicellulose. Whereas, the opposite effects were exhibited in DS-treated fruit. The above data indicate that DCC-hastened longan pulp softening and pulp breakdown was due to the up-regulating expressions of CWDEs-related genes, the activating activities of CWDEs, and the accelerating CWP disassembly by DCC treatment. Whereas, DS repressed longan pulp softening and pulp breakdown through down-regulating the expressions of CWDEs-related genes, inhibiting CWDEs activities, and reducing CWP degradation.

Transcriptomic and sugar metabolic analysis reveals molecular mechanisms of peach gummosis in response to Neofusicoccum parvum infection.

Peach gummosis, a devastating disease caused by Neofusicoccum parvum, significantly shortens peach tree lifespan and reduces the yield of peach trees. Despite its impact, the molecular mechanism underlying this disease remains largely unexplored. In this study, we used RNA-seq, sugar metabolism measurements, and an integrated transcriptional and metabolomic analysis to uncover the molecular events driving peach gummosis. Our results revealed that N. parvum infection drastically altered the transcripts of cell wall degradation-related genes, the log2Fold change in the transcript level of Prupe.1G088900 encoding xyloglucan endotransglycosylase decreased 2.6-fold, while Prupe.6G075100 encoding expansin increased by 2.58-fold at 12 hpi under N. parvum stress. Additionally, sugar content analysis revealed an increase in maltose, sucrose, L-rhamnose, and inositol levels in the early stages of infection, while D-galactose, D-glucose, D-fructose consistently declined as gummosis progressed. Key genes related to cell wall degradation and starch degradation, as well as UDP-sugar biosynthesis, were significantly upregulated in response to N. parvum. These findings suggest that N. parvum manipulates cell wall degradation and UDP-sugar-related genes to invade peach shoot cells, ultimately triggering gum secretion. Furthermore, weighted gene co-expression network analysis (WGCNA) identified two transcription factors, ERF027 and bZIP9, as central regulators in the downregulated and upregulated modules, respectively. Overall, this study enhances our understanding of the physiological and molecular responses of peach trees to N. parvum infection and provide valuable insights into the mechanisms of peach defense against biotic stresses.

Genome-wide characterization of the xyloglucan endotransglucosylase/hydrolase family genes and their response to plant hormone in sugar beet

Xyloglucan endotransglucosylase/hydrolases (XTHs) play a crucial role in plant growth and development. However, their functional response to phytohormone in sugar beet still remains obscure. In this study, we identified 30 putative BvXTH genes in the sugar beet genome. Phylogenetic and evolutionary relationship analysis revealed that they were clustered into three groups and have gone through eight tandem duplication events under purifying selection. Gene structure and motif composition analysis demonstrated that they were highly conserved and all contained one conserved glycoside hydrolase family 16 domain (Glyco_hydro_16) and one xyloglucan endotransglycosylase C-terminus (XET_C) domain. Transcriptional expression analysis exhibited that all BvXTHs were ubiquitously expressed in leaves, root hairs and tuberous roots, and most of them were up-regulated by brassinolide (BR), jasmonic acid (JA), abscisic acid (ABA) and gibberellic acid (GA3). Further mutant complementary experiment demonstrated that expression of BvXTH17 rescued the retarded growth phenotype of xth22, an Arabidopsis knock out mutant of AtXTH22. The findings in our work provide fundamental information on the structure and evolutionary relationship of the XTH family genes in sugar beet, and reveal the potential function of BvXTH17 in plant growth and hormone response.

Enzymes in 3D: Synthesis, remodelling, and hydrolysis of cell wall (1,3;1,4)-β-glucans.

Recent breakthroughs in structural biology have provided valuable new insights into enzymes involved in plant cell wall metabolism. More specifically, the molecular mechanism of synthesis of (1,3;1,4)-β-glucans, which are widespread in cell walls of commercially important cereals and grasses, has been the topic of debate and intense research activity for decades. However, an inability to purify these integral membrane enzymes or apply transgenic approaches without interpretative problems associated with pleiotropic effects has presented barriers to attempts to define their synthetic mechanisms. Following the demonstration that some members of the CslF sub-family of GT2 family enzymes mediate (1,3;1,4)-β-glucan synthesis, the expression of the corresponding genes in a heterologous system that is free of background complications has now been achieved. Biochemical analyses of the (1,3;1,4)-β-glucan synthesized in vitro, combined with 3-dimensional (3D) cryogenic-electron microscopy and AlphaFold protein structure predictions, have demonstrated how a single CslF6 enzyme, without exogenous primers, can incorporate both (1,3)- and (1,4)-β-linkages into the nascent polysaccharide chain. Similarly, 3D structures of xyloglucan endo-transglycosylases and (1,3;1,4)-β-glucan endo- and exohydrolases have allowed the mechanisms of (1,3;1,4)-β-glucan modification and degradation to be defined. X-ray crystallography and multi-scale modeling of a broad specificity GH3 β-glucan exohydrolase recently revealed a previously unknown and remarkable molecular mechanism with reactant trajectories through which a polysaccharide exohydrolase can act with a processive action pattern. The availability of high-quality protein 3D structural predictions should prove invaluable for defining structures, dynamics, and functions of other enzymes involved in plant cell wall metabolism in the immediate future.

Reduced degradation of the cell wall polysaccharides maintains higher tissue integrity of papaya (Carica papaya L.) during chilling storage

Papaya (Carica papaya L.) fruit are vulnerable to chilling injury (CI), and the symptoms of CI develop more intensively when the fruit are stored at 6 ℃ rather than 1 ℃. This is considered to be abnormal CI behavior of papaya fruit, a phenomenon that is unclear but thought to be related to cell wall metabolism. Thus, this study examined the relationship between the development of CI and alterations of the cell wall metabolism in papaya fruit stored at 1 ºC and 6 ℃. The 1 ℃-storage maintained the integrity of the papaya exocarp and alleviated the development of CI and the loss of free moisture. Ultrastructural observations demonstrated that the middle lamellae were denser in papaya fruit stored at 1 ℃ than those in 6 ℃-storage, which alleviated the disintegration of cell wall at 1 ℃-storage. Compared with 6 ℃-storage, 1 ℃-storage inhibited the degradation of Na2CO3-soluble pectin, CDTA-soluble pectin, and cellulose. The 1 ℃-storage also delayed the solubilization of water-soluble pectin and the transformation of 24% KOH-soluble fraction to the 4% KOH-soluble fraction. Lower activities of cell wall-modifying enzymes, including pectin methylesterase, polygalacturonase, pectate lyase, β-galactosidase, xyloglucan endotransglycosylase, and cellulase, were observed in papaya fruit stored at 1 ℃. These results suggest that alleviating the degradation of cell wall polysaccharides maintains higher tissue integrity in papaya fruit during chilling storage.

Analysis of Codon Usage Bias in Xyloglucan Endotransglycosylase (XET) Genes

Xyloglucan endotransglycosylase (XET) genes are widely distributed in most plants, but the codon usage bias of XET genes has remained uncharacterized. Thus, we analyzed the codon usage bias using 4500 codons of 20 XET genes to elucidate the genetic and evolutionary patterns. Phylogenetic and hierarchical cluster analyses revealed that the 20 XET genes belonged to two groups. The closer the genetic distance, the more similar the codon usage preference. The codon usage bias of most XET genes was weak, but there was also some codon usage bias. AGA, AGG, AUC, and GUG were the top four codons (RSCU > 1.5) in the 20 XET genes. CitXET had a stronger codon usage bias, and there were eight optimal codons of CitXET (i.e., AGA, AUU, UCU, CUU, CCA, GCU, GUU, and AAA). The RSCU values underwent a correspondence analysis. The two main factors affecting codon usage bias (i.e., Axes 1 and 2) accounted for 54.8% and 17.6% of the total variation, respectively. Multiple correspondence analysis revealed that XET genes were widely distributed, with Group 1 genes being closer to Axis 1 than Group 2 genes, which were closer to Axis 2. Codons with A/U at the third codon position were distributed closer to Axis 1 than codons with G/C at the third codon position. PgXET, ZmXET, VlXET, VrXET, and PcXET were biased toward codons ending with G/C. In contrast, CitXET, DpXET, and BrpXET were strongly biased toward codons ending with A/U, indicating that these XET genes have a strong codon usage bias. Translational selection and base composition (especially A and U at the third codon position), followed by mutation pressure and natural selection, may be the most important factors affecting codon usage of 20 XET genes. These results may be useful in clarifying the codon usage bias of XET genes and the relevant evolutionary characteristics.

Identification and analysis of the xyloglucan endotransferase/hydrolase (XTH) family genes in apple

Fruit firmness directly determines the commercial value of apples (Malus × domestica Borkh.). The change in its variation is influenced by cell structure and cell wall hydrolases. The significant cell wall hydrolytic enzyme, xyloglucan endotransglycosylase/hydrolase (XTH), is essential for the process of ripening and softening of fruit. In this study, we identified 33 MdXTHs and named them MdXTH1–33 according to their position on the chromosome of apples. Phylogenetic analysis clustered MdXTHs into six groups (A-F), and those with similar gene structure were observed in the same group. Multiple sequence alignment results showed that all MdXTHs have the glycoside hydrolase family 16 domain (Glyco_hydro_16) and the xyloglucan endotransglycosylase C-terminus (XET_C), two highly conserved domains. The promoter of the MdXTHs contained multiple cis-acting elements associated with hormone response. Quantitative real-time PCR analysis suggested that MdXTH3, MdXTH25, and MdXTH26 were most probably involved in fruit softening. The expression of the three MdXTHs was changed and the firmness of fruit was decreased with hormone treaments. In summary, this study provides an extensive analysis of MdXTHs in apples and offers a reference for apple fruit texture breeding through the study of cell wall-related basis.

Changes in the cell walls on fruit skin of Beurré D´Anjou pears (Pyrus communis L.) associated with sunburn injury

• Sunburn reduced cuticular microcrack area and skin thickness, and increased cell wall thickness. • Neutral sugars (NS) content increased in two pectin fractions (WSP and CSP) in sunburn skin. • Hemicellulose and lignin contents increased in sunburn skin. • The expression of Exp and XTH genes increased in response to sunburn. The purpose of this work was to evaluate the effects of sunburn on pear fruit skin at the structural, biochemical, and molecular levels. Pears ( Pyrus communis L.) cv. Beurré D'Anjou were collected at the official commercial harvest time and classified into three groups based on levels of sublethal sunburn: no sunburn (S0), mild sunburn (S1), and moderate sunburn (S2). Skin anatomy was characterized by SEM imaging, cell wall components were quantified by spectrophotometric techniques, and the expression of different genes related to cell wall remodeling was measured in S0, S1, and S2 tissues. Sun-damaged skin cells had reduced size and thickened walls. In the cuticular surface, a reduction of microcracking area, correlated with the intensity of the damage, was further noted. Cell wall fractionation revealed changes in the distribution of neutral sugars (NS) following sunburn, although no differences in uronic acid (UA) content were observed in the different pectin fractions. Hemicellulose and lignin contents were significantly ( P <0.05) increased in sun-damaged tissues. Expression levels of genes encoding polygalacturonase (PcPG2), β-galactosidase (PcGAL2 and PcGAL4), xyloglucan endotransglycosylase (PcXET), and expansin (PCExp1) were increased in the skin of sun-damaged (S1 and S2) fruit. These results indicate that sunburn alters the anatomy and cell wall structure and composition of pear fruit skin, and stimulates different mechanisms involved in the acquisition of stress tolerance.

Definition of the Acceptor Substrate Binding Specificity in Plant Xyloglucan Endotransglycosylases Using Computational Chemistry.

Xyloglucan endotransglycosylases (XETs) play key roles in the remodelling and reconstruction of plant cell walls. These enzymes catalyse homo-transglycosylation reactions with xyloglucan-derived donor and acceptor substrates and hetero-transglycosylation reactions with a variety of structurally diverse polysaccharides. In this work, we describe the basis of acceptor substrate binding specificity in non-specific Tropaeolum majus (TmXET6.3) and specific Populus tremula x tremuloides (PttXET16A) XETs, using molecular docking and molecular dynamics (MD) simulations combined with binding free energy calculations. The data indicate that the enzyme-donor (xyloglucan heptaoligosaccharide or XG-OS7)/acceptor complexes with the linear acceptors, where a backbone consisted of glucose (Glc) moieties linked via (1,4)- or (1,3)-β-glycosidic linkages, were bound stably in the active sites of TmXET6.3 and PttXET16A. Conversely, the acceptors with the (1,6)-β-linked Glc moieties were bound stably in TmXET6.3 but not in PttXET16A. When in the (1,4)-β-linked Glc containing acceptors, the saccharide moieties were replaced with mannose or xylose, they bound stably in TmXET6.3 but lacked stability in PttXET16A. MD simulations of the XET-donor/acceptor complexes with acceptors derived from (1,4;1,3)-β-glucans highlighted the importance of (1,3)-β-glycosidic linkages and side chain positions in the acceptor substrates. Our findings explain the differences in acceptor binding specificity between non-specific and specific XETs and associate theoretical to experimental data.

Root Growth of Transgenic Tobacco Plants with Overexpression of Expansin and Xyloglucan endotransglycosylase Genes under Cadmium Stress

Expansins and xyloglucan endotransglycosylases play an important role in the regulation of plant growth under optimal and stressful conditions. Transgenic tobacco plants overexpressing NtEXPA1 and NtEXPA5 expansin genes and NtEXGT xyloglucan endotransglycosylase of Nicotiana tabacum L. have been previously created by the authors. The aim of this work was the morphophysiological analysis of the roots of these transgenic tobacco plants under conditions of cadmium stress. Transgenic tobacco plants were characterized by increased root length compared to wild type plants, both under optimal conditions and when exposed to cadmium. The area of parenchyma cells of roots of transgenic tobacco plants overexpressing NtEXPA1 and NtEXPA5 expansin genes was greater than the wild type, while the cell sizes, on the contrary, were smaller in the case of the transgene NtEXGT. Overexpression of NtEXPA1,NtEXPA5, and NtEXGT genes contributed to an increase in the total antioxidant capacity and activity of ascorbate peroxidases and a decrease in the content of proline in the roots under the action of cadmium. In the shoots of plants transgenic for the expansin genes, a lower content of MDA was found both under optimal conditions and under the action of cadmium. Thus, it has been shown that NtEXPA1 and NtEXPA5 transgenes have a stimulating effect on the growth of tobacco roots under conditions of cadmium stress by enhancing cell expansion and a positive effect on the components of the antioxidant system. The NtEXGT gene is also involved in root growth under the action of cadmium, including through the effect on the antioxidant system.

XET activity determination in powdered wood samples as an indicator of tension wood, tested on juvenile Populus x euramericana exposed to severe long-term static bending

Abstract Leaning stems of woody plants form reaction wood, in hardwood trees termed tension wood (TW). Typical TW fibers, gelatinous fibers (G-fibers), are characterized by an inner gelatinous cell wall layer (G-layer). Xyloglucan endotransglycosylases (XETs) was proposed as the essential enzyme in cell wall modifications in TW, by making xyloglucan (XG) cross-links between G- and S2-layers in G-fibers, and thus maintaining their contact. The determination of TW presence in a sample is of great importance for the forest products industry, biofuel production, and tree physiology studies. However, TW is not easy to detect visually. The colorimetric assay for XET activity determination as an indicator of TW presence in a sample was tested on powdered stem segments of juvenile Populus x euramericana trees exposed to severe long-term static bending. In parallel, histochemical and ultrastructural characterization of stem samples of bent and control trees was performed. The tested colorimetric assay for XET activity determination could be suggested as a useful and easily applicable tool for fast screening of powdered wood samples for the presence of TW.

The Sequence and Integrated Analysis of Competing Endogenous RNAs Originating from Tea Leaves Infected by the Pathogen of Tea Leaf Spot, Didymella segeticola

Tea leaf spot, caused by Didymella segeticola, is an important disease which negatively affects the productivity and the quality of tea leaves. During infection by the pathogen, competing endogenous RNAs (ceRNAs) from tea leaves could contribute to achieving pathogenicity. In this study, circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs), constituting ceRNAs, which share binding sites on microRNAs (miRNAs), and messenger RNAs (mRNAs) from infected and uninfected leaves of tea (Camellia sinensis 'Fuding-dabaicha') were sequenced and analyzed, and the identity and expression levels of the target genes of miRNA-mRNA and miRNA-lncRNA/circRNA were predicted. Analysis indicated that 10 mRNAs were bound by 20 miRNAs, 66 lncRNAs were bound by 40 miRNAs, and 17 circRNAs were bound by 29 miRNAs, respectively. For the regulation modes of ceRNAs, five ceRNA pairs were identified by the correlation analysis of lncRNA-miRNA-mRNA. For instance, expression of the xyloglucan endotransglycosylase gene in infected leaves was downregulated at the level of mRNA through miRNA PC-5p-3511474_3 binding with lncRNA TEA024202.1:MSTRG.37074.1. Gene annotation indicated that expression of this gene was significantly enriched in cell wall biogenesis and in the pathway of plant hormone signal transduction. The functional analysis of ceRNAs isolated from infected tea leaves will provide a valuable resource for future research on D. segeticola pathogenicity.

Broad Specific Xyloglucan:Xyloglucosyl Transferases Are Formidable Players in the Re-Modelling of Plant Cell Wall Structures.

Plant xyloglucan:xyloglucosyl transferases, known as xyloglucan endo-transglycosylases (XETs) are the key players that underlie plant cell wall dynamics and mechanics. These fundamental roles are central for the assembly and modifications of cell walls during embryogenesis, vegetative and reproductive growth, and adaptations to living environments under biotic and abiotic (environmental) stresses. XET enzymes (EC 2.4.1.207) have the β-sandwich architecture and the β-jelly-roll topology, and are classified in the glycoside hydrolase family 16 based on their evolutionary history. XET enzymes catalyse transglycosylation reactions with xyloglucan (XG)-derived and other than XG-derived donors and acceptors, and this poly-specificity originates from the structural plasticity and evolutionary diversification that has evolved through expansion and duplication. In phyletic groups, XETs form the gene families that are differentially expressed in organs and tissues in time- and space-dependent manners, and in response to environmental conditions. Here, we examine higher plant XET enzymes and dissect how their exclusively carbohydrate-linked transglycosylation catalytic function inter-connects complex plant cell wall components. Further, we discuss progress in technologies that advance the knowledge of plant cell walls and how this knowledge defines the roles of XETs. We construe that the broad specificity of the plant XETs underscores their roles in continuous cell wall restructuring and re-modelling.

Phomopsis longanae Chi-induced longan pulp breakdown and softening in relation to cell wall polysaccharides disassembly

Cell wall polysaccharides are crucial contributors to the structural properties of plants and perform as a barrier against fungal invasion. Phomopsis longanae Chi, a principal devastating pathogenic fungus, can induce a severe pulp breakdown and softening of postharvest longan. P. longanae-induced longan pulp breakdown and softening in relation to the disassembly of cell wall polysaccharides were explicated. Results suggested that a higher pulp breakdown index and a lower pulp firmness were obtained in P. longanae-infected fruit. More importantly, P. longanae infection promoted gene expression levels and activities of cell wall-disassembling enzymes (pectin methylesterase, polygalacturonase, β-galactosidase, cellulase, and xyloglucan endotransglycosylase), and facilitated the disassembly of polysaccharides like ionic-soluble pectin, covalent-soluble pectin, cellulose, and hemicellulose, which were closely relevant to pulp breakdown and softening. Given these results, the aggravated pulp breakdown and softening of longan after P. longanae inoculation were attributed to cell wall polysaccharides modification regulated by enzyme and related genes.

Exogenously Applied Chitosan and Chitosan Nanoparticles Improved Apple Fruit Resistance to Blue Mold, Upregulated Defense-Related Genes Expression, and Maintained Fruit Quality

Blue rot disease caused by Penicillium expansum is one of the most widespread fungal diseases that affects apples worldwide. This work was to verify the effect of chitosan (2 and 4 g/L) and its nano-form (0.2 and 0.4 g/L) against blue rot disease on apples and their effect on the expression of six defense-related genes as well as fruit quality parameters. Regarding disease incidence, in most cases, chitosan NPs performed better as compared to their raw materials for both artificial and natural infections. The highest efficacy was obtained for chitosan NPs at 0.4 g/L for artificial and natural infection in both 2019 and 2020 seasons. All treatments kept fruit quality parameters regarding firmness, total soluble solids, and titratable acidity for artificial and natural infection in both seasons. As expected, the exogenous application of chitosan NPs and bulk form triggered an increase in the expression levels of six defense-related genes including chitinase, peroxidase, β-1,3-glucanase, Xyloglucan endotransglycosylase (XET), pathogenesis-related protein (PR8), and phenylalanine ammonia lyase-1 (PAL1). Moreover, the highest mRNA quantity of all the studied genes was detected in leaves treated with chitosan NPs at both concentrations compared to other treatments. Chitosan NPs can be considered an eco-friendly and effective approach against blue mold of apples and can be integrated into management programs to maintain postharvest quality and extend the shelf life of fruits.

Changes in secondary metabolites and fiber quality of cotton (Gossypium hirsutum) seed under consecutive water stress and in silico analysis of cellulose synthase and xyloglucan endotransglucosylase.

Global warming has led to severe drought conditions. The selection of plant varieties that can withstand drought and produce increased yields are of utmost importance. In the current study, secondary metabolites, seed trait and fiber characteristic of cottonseeds (Gossypium hirsutum) exposed to double and third water stress exposure was investigated. Total phenol and tannin content in W1S33 increased significantly after third water stress exposure. Accumulation of wax was enhanced in seeds of W3S33 and W3S34 that were subjected to third water stress. Fiber quality parameters decreased when cottonseeds were rainfed. High irrigation resulted in fragile and delicate fiber. Seeds grown under 66% FC irrigation saved water and produced seeds that had the potential of producing high quality fibers. In silico analysis was performed on cellulose synthase A (CesA) and xyloglucan endotransglycosylase (XET) enzymes present in Gossypium hirsutum. The intracellular locations of the CesA and XET1 enzymes are the plasma membrane and cell wall, respectively. Proline is conserved in the C-terminal of the CesA enzyme and plays an important role in enzyme functionality. This study provides a better understanding as to the mechanisms by which the plant can tolerate and combat water stress conditions as well as reduce water consumption. In order to grow cotton seeds with desirable morphometric characteristics and optimal fibers under water stress exposure and in dry areas, it is better to use seeds that are irrigated under optimal irrigation conditions, ie 66% FC.

Plant Xyloglucan Xyloglucosyl Transferases and the Cell Wall Structure: Subtle but Significant.

Plant xyloglucan xyloglucosyl transferases or xyloglucan endo-transglycosylases (XET; EC 2.4.1.207) catalogued in the glycoside hydrolase family 16 constitute cell wall-modifying enzymes that play a fundamental role in the cell wall expansion and re-modelling. Over the past thirty years, it has been established that XET enzymes catalyse homo-transglycosylation reactions with xyloglucan (XG)-derived substrates and hetero-transglycosylation reactions with neutral and charged donor and acceptor substrates other than XG-derived. This broad specificity in XET isoforms is credited to a high degree of structural and catalytic plasticity that has evolved ubiquitously in algal, moss, fern, basic Angiosperm, monocot, and eudicot enzymes. These XET isoforms constitute gene families that are differentially expressed in tissues in time- and space-dependent manners during plant growth and development, and in response to biotic and abiotic stresses. Here, we discuss the current state of knowledge of broad specific plant XET enzymes and how their inherently carbohydrate-based transglycosylation reactions tightly link with structural diversity that underlies the complexity of plant cell walls and their mechanics. Based on this knowledge, we conclude that multi- or poly-specific XET enzymes are widespread in plants to allow for modifications of the cell wall structure in muro, a feature that implements the multifaceted roles in plant cells.

Structural characterization of the Pet c 1.0201 PR-10 protein isolated from roots of Petroselinum crispum (Mill.) Fuss

The native dimeric Petroselinum crispum (Mill.) Fuss protein Pet c 1.0201 and a monomeric xyloglucan endotransglycosylase enzyme (Garajova et al., 2008) isolated from the root cells co-purify and share similar molecular masses and acidic isoelectric points. In this work, we determined the complete primary structure of the parsley Pet c 1.0201 protein, based on tryptic and chymotryptic peptides followed by the manual micro-gradient chromatographic separation coupled with offline MALDI-TOF/TOF mass spectrometry. The bioinformatics approach enabled us to include the parsley protein into the PR-10 family, as it exhibited the highest protein sequence identity with the Apium graveolens Api g 1.0201 allergen and the major Daucus carota allergen Dau c 1.0201. Hence, we designated the Petroselinum crispum protein as Pet c 1.0201 and deposited it in the UniProt Knowledgebase under the accession C0HKF5. 3D protein homology modelling and molecular dynamics simulations of the Pet c 1.0201 dimer confirmed the typical structure of the Bet v 1 family allergens, and the potential of the Pet c 1.0201 protein to dimerize in water. However, the behavioural properties of Pet c 1.0201 and the celery allergen Api g 1.0101 differed in the presence of salts due to transiently and stably formed dimeric forms of Pet c 1.0201 and Api g 1.0101, respectively.

High post-anthesis temperature effects on bread wheat (Triticum aestivum L.) grain transcriptome during early grain-filling

BackgroundHigh post-anthesis (p.a) temperatures reduce mature grain weights in wheat and other cereals. However, the causes of this reduction are not entirely known. Control of grain expansion by the maternally derived pericarp of the grain has previously been suggested, although this interaction has not been investigated under high p.a. temperatures. Down-regulation of pericarp localised genes that regulate cell wall expansion under high p.a. temperatures may limit expansion of the encapsulated endosperm due to a loss of plasticity in the pericarp, reducing mature grain weight. Here the effect of high p.a. temperatures on the transcriptome of the pericarp and endosperm of the wheat grain during early grain-filling was investigated via RNA-Seq and is discussed alongside grain moisture dynamics during early grain development and mature grain weight.ResultsHigh p.a. temperatures applied from 6-days after anthesis (daa) and until 18daa reduced the grain’s ability to accumulate water, with total grain moisture and percentage grain moisture content being significantly reduced from 14daa onwards. Mature grain weight was also significantly reduced by the same high p.a. temperatures applied from 6daa for 4-days or more, in a separate experiment. Comparison of our RNA-Seq data from whole grains, with existing data sets from isolated pericarp and endosperm tissues enabled the identification of subsets of genes whose expression was significantly affected by high p.a. temperature and predominantly expressed in either tissue. Hierarchical clustering and gene ontology analysis resulted in the identification of a number of genes implicated in the regulation of cell wall expansion, predominantly expressed in the pericarp and significantly down-regulated under high p.a. temperatures, including endoglucanase, xyloglucan endotransglycosylases and a β-expansin. An over-representation of genes involved in the ‘cuticle development’ functional pathway that were expressed in the pericarp and affected by high p.a. temperatures was also observed.ConclusionsHigh p.a. temperature induced down-regulation of genes involved in regulating pericarp cell wall expansion. This concomitant down-regulation with a reduction in total grain moisture content and grain weight following the same treatment period, adds support to the theory that high p.a. temperatures may cause a reduction in mature grain weight as result of decreased pericarp cell wall expansion.

Transcriptional Differences in Peanut (Arachis hypogaea L.) Seeds at the Freshly Harvested, After-ripening and Newly Germinated Seed Stages: Insights into the Regulatory Networks of Seed Dormancy Release and Germination.

Seed dormancy and germination are the two important traits related to plant survival, reproduction and crop yield. To understand the regulatory mechanisms of these traits, it is crucial to clarify which genes or pathways participate in the regulation of these processes. However, little information is available on seed dormancy and germination in peanut. In this study, seeds of the variety Luhua No.14, which undergoes nondeep dormancy, were selected, and their transcriptional changes at three different developmental stages, the freshly harvested seed (FS), the after-ripening seed (DS) and the newly germinated seed (GS) stages, were investigated by comparative transcriptomic analysis. The results showed that genes with increased transcription in the DS vs FS comparison were overrepresented for oxidative phosphorylation, the glycolysis pathway and the tricarboxylic acid (TCA) cycle, suggesting that after a period of dry storage, the intermediates stored in the dry seeds were rapidly mobilized by glycolysis, the TCA cycle, the glyoxylate cycle, etc.; the electron transport chain accompanied by respiration was reactivated to provide ATP for the mobilization of other reserves and for seed germination. In the GS vs DS pairwise comparison, dozens of the upregulated genes were related to plant hormone biosynthesis and signal transduction, including the majority of components involved in the auxin signal pathway, brassinosteroid biosynthesis and signal transduction as well as some GA and ABA signal transduction genes. During seed germination, the expression of some EXPANSIN and XYLOGLUCAN ENDOTRANSGLYCOSYLASE genes was also significantly enhanced. To investigate the effects of different hormones during seed germination, the contents and differential distribution of ABA, GAs, BRs and IAA in the cotyledons, hypocotyls and radicles, and plumules of three seed sections at different developmental stages were also investigated. Combined with previous data in other species, it was suggested that the coordination of multiple hormone signal transduction nets plays a key role in radicle protrusion and seed germination.