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Background Xyloglucan endotransglucosylase/hydrolase (XTH) family plays an important role in cell wall reconstruction and stress resistance in plants. However, the detailed characteristics of XTH family genes and their expression pattern under salt stress have not been reported in poplar. Results In this study, a total of 43 PtrXTH genes were identified from Populus simonii × Populus nigra, and most of them contain two conserved structures (Glyco_hydro_16 and XET_C domain). The promoters of the PtrXTH genes contain mutiple cis-acting elements related to growth and development and stress responses. Collinearity analysis revealed that the XTH genes from poplar has an evolutionary relationship with other six species, including Eucalyptus robusta, Solanum lycopersicum, Glycine max, Arabidopsis, Zea mays and Oryza sativa. Based on RNA-Seq analysis, the PtrXTH genes have different expression patterns in the roots, stems and leaves, and many of them are highly expressed in the roots. In addition, there are11 differentially expressed PtrXTH genes in the roots, 9 in the stems, and 7 in the leaves under salt stress. In addition, the accuracy of RNA-Seq results was verified by RT-qPCR. Conclusion All the results indicated that XTH family genes may play an important role in tissue specificity and salt stress response. This study will lay a theoretical foundation for further study on molecular function of XTH genes in poplar.

All land plants encode large multigene families of xyloglucan endotransglucosylase/hydrolases (XTHs), plant-specific enzymes that cleave and reconnect plant cell-wall polysaccharides. Despite the ubiquity of these enzymes, considerable uncertainty remains regarding the evolutionary history of the XTH family. Phylogenomic and comparative analyses in this study traced the non-plant origins of the XTH family to Alphaproteobacteria ExoKs, bacterial enzymes involved in loosening biofilms, rather than Firmicutes licheninases, plant biomass digesting enzymes, as previously supposed. The relevant horizontal gene transfer (HGT) event was mapped to the divergence of non-swimming charophycean algae in the Cryogenian geological period. This HGT event was the likely origin of charophycean EG16-2s, which are putative intermediates between ExoKs and XTHs. Another HGT event in the Cryogenian may have led from EG16-2s or ExoKs to fungal Congo Red Hypersensitive proteins (CRHs) to fungal CRHs, enzymes that cleave and reconnect chitin and glucans in fungal cell walls. This successive transfer of enzyme-encoding genes may have supported the adaptation of plants and fungi to the ancient icy environment by facilitating their sessile lifestyles. Furthermore, several protein evolutionary steps, including coevolution of substrate-interacting residues and putative intra-family gene fusion, occurred in the land plant lineage and drove diversification of the XTH family. At least some of those events correlated with the evolutionary gain of broader substrate specificities, which may have underpinned the expansion of the XTH family by enhancing duplicated gene survival. Together, this study highlights the Precambrian evolution of life and the mode of multigene family expansion in the evolutionary history of the XTH family.

Flower opening is an important phenomenon in plant that indicates the readiness of the flower for pollination leading to petal expansion and pigmentation. This phenomenon has great impact on crop yield, which makes researches of its mechanism attractive for both plant physiology study and agriculture. Gene promoters directing the expression in petal during the petal cell wall modification and expansion when flower opens could be a convenient tool to analyze or monitor gene expression targeting this event. However, there are no reports of isolated gene promoters that can direct gene expression in petal or petal limb during the rapid cell wall dynamics when the flower opens. Xyloglucan endotransglucosylase/hydrolase 7 (XTH7), a cell wall modifying enzyme, was reported having up-regulated gene expression in the petal of Arabidopsis thaliana and Petunia hybrida. In this study, we fused a 1,904 bp length P. hybrida XTH7 promoter with a gene encoding a bright bioluminescent protein (Green enhanced Nano-lantern) to report gene expression and observed petal up-regulated bioluminescence activity by means of a consumer-grade camera. More importantly, this novel promoter demonstrated up-regulated activity in the petal limb of P. hybrida matured flower during flower opening. P. hybrida XTH7 promoter would be a useful tool for flowering study, especially for petal expansion research during flower opening.

Summary The xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes involved in cell wall assembly and growth regulation, cleaving and re‐joining hemicellulose chains in the xyloglucan–cellulose network. Here, in a homologous system, we compare the secretion patterns of XTH11, XTH33 and XTH29, three members of the Arabidopsis thaliana XTH family, selected for the presence (XTH11 and XTH33) or absence (XTH29) of a signal peptide, and the presence of a transmembrane domain (XTH33). We show that XTH11 and XTH33 reached, respectively, the cell wall and plasma membrane through a conventional protein secretion (CPS) pathway, whereas XTH29 moves towards the apoplast following an unconventional protein secretion (UPS) mediated by exocyst‐positive organelles (EXPOs). All XTHs share a common C‐terminal functional domain (XET‐C) that, for XTH29 and a restricted number of other XTHs (27, 28 and 30), continues with an extraterminal region (ETR) of 45 amino acids. We suggest that this region is necessary for the correct cell wall targeting of XTH29, as the ETR‐truncated protein never reaches its final destination and is not recruited by EXPOs. Furthermore, quantitative real‐time polymerase chain reaction analyses performed on 4‐week‐old Arabidopsis seedlings exposed to drought and heat stress suggest a different involvement of the three XTHs in cell wall remodeling under abiotic stress, evidencing stress‐, organ‐ and time‐dependent variations in the expression levels. Significantly, XTH29, codifying the only XTH that follows a UPS pathway, is highly upregulated with respect to XTH11 and XTH33, which code for CPS‐secreted proteins., Significance Statement The cellular distribution of three xyloglucan endotransglucosylase/hydrolases (XTHs) was monitored comparatively while expressing respective fluorescent constructs in Arabidopsis cotyledons. We show that XTH11 and XTH33 follow conventional protein secretion towards cell wall and plasma membrane, respectively, whereas XTH29, the only XTH predicted to lack a signal peptide, is secreted into the wall via exocyst‐positive organelle‐mediated unconventional protein secretion (UPS). Significantly, XTH29, poorly expressed in physiological conditions, demonstrates upregulation under drought and high temperature conditions, suggesting a specific possible role for UPS‐secreted XTH29 in stress responses.

Background Fruit softening is a major determinant of commercial value and shelf life. A transcriptomic analysis of 'Golden Delicious' and 'Golden Del. Reinders' (a bud mutation of 'Golden Delicious' that readily softens) apple fruit was conducted during storage. Results A comparative analysis of the obtained expression profiles of fruit between two cultivars identified 1345 upregulated and 3475 downregulated differentially expressed genes (DEGs). The DEGs identified were associated with cellular processes and carbohydrate metabolism and were especially enriched in cell-wall-related genes. Among the cell-wall-related genes, the xyloglucan endotransglucosylase/hydrolases (XTH) gene MdXTHB was significantly upregulated and exhibited high expression levels in 'Golden Del. Reinders' fruit, which had a lower level of firmness relative to 'Golden Delicious'. Overexpression of MdXTHB in both 'Golden Delicious' and 'Fuji', which typically maintain high levels of firmness in storage, exhibited faster rates of softening and an earlier peak of ethylene production than empty-vector-infiltrated fruit did. Conclusion The results of this study indicate that MdXTHB potentially promotes apple fruit softening by degrading the fruit cell wall. This result is also useful to designing further experiments on the molecular regulation of fruit softening in apple. © 2020 Society of Chemical Industry.

Lead (Pb) is a heavy metal with high toxicity to plants. Root is the major organ to respond to Pb stress. However, little is known about how plant roots perceive Pb stress signaling. Here, we describe the transcriptome of Arabidopsis root tips under Pb stress using the RNA-seq assay. A total of 703 differentially expressed genes (DEGs) were identified and expressed at every time points. Some early-responsive DEGs (1 h) were predicted to be negatively involved in cell elongation and cell expansion, while some late-responsive DEGs (24 h) positively participated in defense of oxidative stress. Hydrogen peroxide (H2O2) and superoxide (O2−) were increased significantly in root tips under Pb stress. Cell wall extension related gene XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE 18 (XTH18) was induced in root tips, and xth18 showed reduced root growth inhibition by Pb stress. Our results revealed the potential mechanism of root growth inhibition by Pb stress and shed light for the further study.

Transglycanases remodel cell-wall polymers, having critical impact on many physiological processes. Unlike xyloglucan endotransglucosylase (XET) activity, widely studied in land-plants, very little is known about charophyte wall-modifying enzymes - information that would promote our understanding of the 'primordial' wall, revealing how the wall matrix is re-modelled in the closest living algal relatives of land-plants, and what changed during terrestrialisation. We conducted various in-vitro assays for wall-remodelling transglycosylases, monitoring either (a) polysaccharide-to-[3 H]oligosaccharide transglycosylation or (b) non-radioactive oligosaccharide-to-oligosaccharide transglycosylation. We screened a wide collection of enzyme extracts from charophytes (and early-diverging land-plants for comparison) and discovered several homo- and hetero-transglycanase activities. In contrast to most land-plants, charophytes possess high trans-s-1,4-mannanase activity, suggesting that land-plants' algal ancestors prioritised mannan remodelling. Trans-s-1,4-xylanase activity was also found, most abundantly in Chara, Nitella and Klebsormidium. Exo-acting transglycosidase activities (trans-s-1,4-xylosidase and trans-s-1,4-mannosidase) were also detected. In addition, charophytes exhibited homo- and hetero-trans-s-glucanase activities [XET, mixed-linkage-glucan:xyloglucan endotransglucosylase, and cellulose:xyloglucan endotransglucosylase] despite the paucity or lack of land-plant-like xyloglucan and mixed-linkage-glucan (MLG) as potential donor substrates in their cell walls. However, trans-α-xylosidase activity (which remodels xyloglucan in angiosperms) was absent in charophytes and early-diverging land-plants. Transglycanase action was also found in situ, acting on endogenous algal polysaccharides as donor-substrates and fluorescent xyloglucan-oligosaccharides as acceptor-substrates. We conclude that trans-s-mannanase and trans-s-xylanase activities are present and thus may play key roles in charophyte walls (most of which possess little or no xyloglucan and MLG, but often contain abundant s-mannans and s-xylans), comparable to the roles of XET in xyloglucan-rich land-plants.

A comparative transcriptome analysis between unripe and ripe berries of Vitis flexuosa, a Korean native wild grape, was conducted to identify differentially expressed genes (DEGs). About 12 billion nucleotides derived from unripe and ripe berries were sequenced and 180,503 contigs were obtained from the assembly results. Expression profiles of transcriptome data revealed that 5249 DEGs showed significant differences at transcript levels between unripe and ripe berries. The top five up-regulated genes in ripe berries against unripe berries were cupin family protein, cytochrome p450 79a2, HSP20-like chaperones superfamily protein, chalcone and stilbene synthase family protein, and beta-tonoplast intrinsic protein. The five most down-regulated genes in ripe berries against unripe berries were cellulose synthase family protein, laccase 17, proline-rich protein 2, subtilase 1.3, and laccase/diphenol oxidase family protein. Among DEGs, top 10 up- and down-regulated carbohydrate, organic substance, and phenylpropanoid metabolic process-related genes were selected from transcriptome analysis and their expression profiles were validated by real-time PCR. Real-time PCR analysis revealed higher expression level of DEGs encoding xyloglucan endotransglucosylase/hydrolase 32 and pectin lyase-like superfamily protein, which are related to starch and sucrose metabolism, cruciferin 3 and AGAMOUS-like 104, which are involved in organic acid metabolism, and MYB domain protein 113 and chalcone and stilbene synthase family protein, which play a role in the phenylalanine ammonia-lyase pathway in ripe than in unripe berries. Overall, we present an overview of transcriptome changes and carbohydrate, organic substance, and phenylpropanoid metabolic process-related expression patterns in unripe and ripe berries of V. flexuosa during berry ripening.

Plants have evolved various strategies to sense and respond to saline environments, which severely reduce plant growth and limit agricultural productivity. Alteration to the cell wall is one strategy that helps plants adapt to salt stress. However, the physiological mechanism of how the cell wall components respond to salt stress is not fully understood. Here, we show that expression of XTH30, encoding xyloglucan endotransglucosylase-hydrolase30, is strongly up-regulated in response to salt stress in Arabidopsis. Loss-of-function of XTH30 leads to increased salt tolerance and overexpression of XTH30 results in salt hypersensitivity. XTH30 is located in the plasma membrane and is highly expressed in the root, flower, stem, and etiolated hypocotyl. The NaCl-induced increase in xyloglucan (XyG)-derived oligosaccharide (XLFG) of the wild type is partly blocked in xth30 mutants. Loss-of-function of XTH30 slows down the decrease of crystalline cellulose content and the depolymerization of microtubules caused by salt stress. Moreover, lower Na+ accumulation in shoot and lower H2O2 content are found in xth30 mutants in response to salt stress. Taken together, these results indicate that XTH30 modulates XyG side chains, altered abundance of XLFG, cellulose synthesis, and cortical microtubule stability, and negatively affecting salt tolerance., Xyloglucan endotransglucosylase-hydrolase30 modulates xyloglucan side chains by altering abundance of xyloglucan-derived oligosaccharide, negatively affecting salt tolerance.

Mangalore melon (Cucumis melo ssp. agrestis var. acidulus) is a non-dessert melon, extensively grown in the coastal districts of South India, but hardly known to the rest of the World. Immature or mature fruits of Mangalore melon are used in preparation of delicious dishes such as vegetable stew, chutneys and curries. They are appreciated for nutritional values, long shelf life and biotic stress resistance. Seventy-nine accessions of Mangalore melon were collected from five states of South India and their genetic diversity was assessed using inter simple sequence repeat (ISSR) markers. Putative candidate genes of extended shelf life in Mangalore melon were studied by quantitative reverse transcription polymerase chain reaction in comparison with cantaloupe (Cucumis melo L.). Shelf life varied from 65 days to 300 days at room temperature. Six ISSR primers amplified 142 fragments ranging from 80 bp to 2380 bp with an average of 23.66 bands per marker on a high-resolution capillary electrophoresis system. Neighbor joining phylogenetic tree construction from the ISSR allele similarity based genetic distance revealed two major clusters with 46 and 33 accessions in each cluster. Expression of fruit ripening related genes of ethylene biosynthesis (1-aminocyclopropane-1-carboxylate synthase, 1-aminocyclopropane-1-carboxylate oxidase) and cell wall metabolism (polygalacturonase, xyloglucan endotransglucosylase/hydrolase and expansin) in Mangalore melons was significantly lower than the cantaloupe melon at 180 days after harvest. Mangalore melon is a promising genetic resource for enhancing the shelf life of melons and the putative candidate genes are useful in enhancing shelf life of cantaloupe following validation and conformation.

Organ abscission in plants requires the hydrolysis of cell wall components, mainly including celluloses, pectins, and xyloglucans. However, how the genes that encode those hydrolytic enzymes are regulated and their function in abscission remains unclear. Previously we revealed that two cellulase genes LcCEL2/8 and two polygalacturonase genes LcPG1/2 were responsible for the degradation of celluloses and pectins, respectively, during fruitlet abscission in litchi. Here, we further identified three xyloglucan endotransglucosylase/hydrolase genes (LcXTH4, LcXTH7, LcXTH19) that are also involved in this process. Nineteen LcXTHs, named LcXTH1-19, were identified in the litchi genome. Transcriptome data and qRT-PCR confirmed that LcXTH4/7/19 were significantly induced at the abscission zone (AZ) during fruitlet abscission in litchi. The GUS reporter driven by each promoter of LcXTH4/7/19 was specifically expressed at the floral abscission zone of Arabidopsis, and importantly ectopic expression of LcXTH19 in Arabidopsis resulted in precocious floral organ abscission. Moreover, electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter analysis showed that the expression of LcXTH4/7/19 could be directly activated by two ETHYLENE INSENSITIVE 3-like (EIL) transcription factors LcEIL2/3. Collectively, we propose that LcXTH4/7/19 are involved in fruitlet abscission, and LcEIL2/3-mediated transcriptional regulation of diverse cell wall hydrolytic genes is responsible for this process in litchi.

Frankincense tree (Boswellia sacra Fluek) has been poorly known on how it responds to tapping and wound-recovery process at molecular levels. Here, we used RNA-sequencing analysis to profile transcriptome of B. sacra after 30 min, 3 h and 6 h of post-tapping. Results showed 5525 differentially expressed genes (DEGs) that were related to terpenoid biosynthesis, phytohormonal regulation, cellular transport, and cell-wall synthesis. Plant-growth-regulators were applied exogenously which showed regulation of endogenous jasmonates and resulted in rapid recovery of cell-wall integrity by significantly up-regulated gene expression of terpenoid biosynthesis (germacrene-D synthase, B-amyrin synthase, and squalene epioxidase-1) and cell-wall synthesis (xyloglucan endotransglucosylase, cellulose synthase-A, and cell-wall hydrolase) compared to control. These findings suggest that tapping immediately activated several cell-developmental and regeneration processes, alongwith defense-induced terpenoid metabolism, to improve the healing process in epidermis. Exogenous growth regulators, especially jasmonic acid, can drastically help tree recovery from tissue degeneration and might help in tree conservation purposes.

The contamination of soils with cadmium (Cd) has become a serious environmental issue that needs to be addressed. Elucidating the mechanisms underlying Cd accumulation may facilitate the development of plants that accumulate both high and low amounts of Cd. In this study, a combination of phenotypic, physiological, and comparative transcriptomic analyses was performed to investigate the effects of different Cd concentrations (0, 5, 10, 30, 50 mg/kg) on Brassica juncea L. Our results suggest that B. juncea L. seedlings had a degree of tolerance to the 5 mg/kg Cd treatment, whereas higher Cd stress (10–50 mg/kg) could suppress the growth of B. juncea L. seedlings. The contents of soluble protein, as well as MDA (malondialdehyde), were increased, but the activities of CAT (catalase) enzymes and the contents of soluble sugar and chlorophyll were decreased, when B. juncea L. was under 30 and 50 mg/kg Cd treatment. Comparative transcriptomic analysis indicated that XTH18 (xyloglucan endotransglucosylase/hydrolase enzymes), XTH22, and XTH23 were down-regulated, but PME17 (pectin methylesterases) and PME14 were up-regulated, which might contribute to cell wall integrity maintenance. Moreover, the down-regulation of HMA3 (heavy metal ATPase 3) and up-regulation of Nramp3 (natural resistance associated macrophage proteins 3), HMA2 (heavy metal ATPase 2), and Nramp1 (natural resistance associated macrophage proteins 1) might also play roles in reducing Cd toxicity in roots. Taken together, the results of our study may help to elucidate the mechanisms underlying the response of B. juncea L. to various concentrations of Cd.

Low-temperature atmospheric pressure plasma technology has been used in agriculture and plant science by direct and indirect treatment of bio-samples. However, the cellular and molecular mechanisms affected by plasma-activated water (PAW) are largely unexplored. In this study, PAW generated from a surface dielectric barrier discharge (SDBD) device was used for plant development. Physicochemical analysis was performed to confirm the PAW properties that correlated with the plasma treatment time. Arabidopsis thaliana L. was utilized to study the effect of the PAW treatment in the early developmental stage. The plasma-activated water samples are denoted as PAW5 time in minutes (min), PAW7 min, PAW12 min, PAW19 min and PAW40 min with the plasma treatment time. Seedlings grown in the PAW5, PAW7 and PAW12 had increased root lengths while the root lengths were decreased in the PAW19 and PAW40. In the cellular level observation, the PAW treatment specifically increased the root hair numbers per unit of the root but suppressed the root hair length in the PAW, indicating that PAW mainly modulates the root hair cell density in the root. Furthermore, we found that the root hair density and length at PAW5 in maximal observed conditions were positively regulated by root developmental-related genes including COBRA-LIKE9 (COBL9), XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE9 (XTH9), XTH17, AUXIN1 (AUX1) and LIKE-AUXIN (LAX3).

Fruit ripening and abscission are the results of the cell wall modification concerning different components of the signaling network. However, molecular-genetic information on the cross-talk between ripe fruit and their abscission zone (AZ) remains limited. In this study, we investigated transcriptional and hormonal changes in olive (Olea europaea L. cv Picual) pericarp and AZ tissues of fruit at the last stage of ripening, when fruit abscission occurs, to establish distinct tissue-specific expression patterns related to cell-wall modification, plant-hormone, and vesicle trafficking in combination with data on hormonal content. In this case, transcriptome profiling reveals that gene encoding members of the &alpha, galactosidase and &beta, hexosaminidase families associated with up-regulation of RabB, RabD, and RabH classes of Rab-GTPases were exclusively transcribed in ripe fruit enriched in ABA, whereas genes of the arabinogalactan protein, laccase, lyase, endo-&beta, mannanase, ramnose synthase, and xyloglucan endotransglucosylase/hydrolase families associated with up-regulation of RabC, RabE, and RabG classes of Rab-GTPases were exclusively transcribed in AZ-enriched mainly in JA, which provide the first insights into the functional divergences among these protein families. The enrichment of these protein families in different tissues in combination with data on transcript abundance offer a tenable set of key genes of the regulatory network between olive fruit tissues in late development.

Transglycanases are enzymes that remodel the primary cell wall in plants, potentially loosening and/or strengthening it. Xyloglucan endotransglucosylase (XET; EC 2.4.1.207), ubiquitous in land plants, is a homo-transglucanase activity (donor, xyloglucan; acceptor, xyloglucan) exhibited by XTH (xyloglucan endotransglucosylase/hydrolase) proteins. By contrast, hetero-trans-β-glucanase (HTG) is the only known enzyme that is preferentially a hetero-transglucanase. Its two main hetero-transglucanase activities are MLG : xyloglucan endotransglucosylase (MXE) and cellulose : xyloglucan endotransglucosylase (CXE). HTG is highly acidic and found only in the evolutionarily isolated genus of fern-allies, Equisetum. We now report genes for three new highly acidic HTG related XTHs in E. fluviatile (EfXTH-A, EfXTH-H and EfXTH-I). We expressed them heterologously in Pichia and tested the encoded proteins’ enzymic activities to determine whether their acidity and/or their Equisetum-specific sequences might confer high hetero-transglucanase activity. Untransformed Pichia was found to secrete MLG-degrading enzyme(s), which had to be removed for reliable MXE assays. All three acidic EfXTHs exhibited very predominantly XET activity, although low but measurable hetero-transglucanase activities (MXEand CXE) were also detected in EfXTH-H and EfXTH-I. We conclude that the extremely high hetero-transglucanase activities of Equisetum HTG are not emulated by similarly acidic Equisetum XTHs that share up to 55.5% sequence identity with HTG.

The notion that xyloglucans (XG) play a pivotal role in tethering cellulose microfibrils in the primary cell wall of plants can be traced back to the first molecular model of the cell wall proposed in 1973, which was reinforced in the 1990s by the identification of Xyloglucan Endotransglucosylase/Hydrolase (XTH) enzymes that cleave and reconnect xyloglucan crosslinks in the cell wall. However, this tethered network model has been seriously challenged since 2008 by the identification of the Arabidopsis thaliana xyloglucan-deficient mutant (xxt1 xxt2), which exhibits functional cell walls. Thus, the molecular mechanism underlying the physical integration of cellulose microfibrils into the cell wall remains controversial. To resolve this dilemma, we investigated the cell wall regeneration process using mesophyll protoplasts derived from xxt1 xxt2 mutant leaves. Imaging analysis revealed only a slight difference in the structure of cellulose microfibril network between xxt1 xxt2 and wild-type (WT) protoplasts. Additionally, exogenous xyloglucan application did not alter the cellulose deposition patterns or mechanical stability of xxt1 xxt2 mutant protoplasts. These results indicate that xyloglucan is not essential for the initial assembly of the cellulose network, and the cellulose network formed in the absence of xyloglucan provides sufficient tensile strength to the primary cell wall regenerated from protoplasts.

Buckwheat (Fagopyrum esculentum) exhibits high Al tolerance, but only a few genes have been functionally characterized. We previously characterized a half-type ABC transporter, Fagopyrum esculentum sensitive to Al rhizotoxicity1 (FeSTAR1), in buckwheat Al tolerance. This study aims to investigate whether and how another half-type ABC transporter (FeSTAR2) plays role in Al tolerance in buckwheat. The expression of FeSTAR2 and complementation test in Arabidopsis als3 mutant was examined. The interaction between FeSTAR1 and FeSTAR2 and subcellular location were analyzed by bimolecular fluorescence complementation (BiFC) and yeast two-hybrid (Y2H) assays. Al rapidly and specifically induced FeSTAR2 expression. Having transmembrane domains, FeSTAR2 localizes to membrane. BiFC and Y2H assays showed that FeSTAR2 could interact with FeSTAR1 which contains only nucleotide binding domain. Intriguingly, interaction between FeSTAR1 and FeSTAR2 altered their locations. Both heterologous expression of FeSTAR2 in als3 and exogenous UDP-glucose rescued its Al hypersensitivity of als3 mutant, suggesting that involvement of FeSTAR2 in Al tolerance requires UDP-glucose. Furthermore, Al-induced inhibition of xyloglucan endotransglucosylase (XET) activity in both atstar1 and als3 mutants could be restored by UDP-glucose. Our results indicate that FeSTAR2 interacts with FeSTAR1 to form an ABC transporter to regulate Al tolerance by vesicular transport of UDP-glucose which affects hemicellulose metabolism by regulating XET activity.

The fleshy taproot of radish is an important storage organ determining its yield and quality. Taproot thickening is a complex developmental process in radish. However, the molecular mechanisms governing this process remain unclear at the proteome level. In this study, a comparative proteomic analysis was performed to analyze the proteome changes at three developmental stages of taproot thickening using iTRAQ approach. In total, 1862 differentially expressed proteins (DEPs) were identified from 6342 high-confidence proteins, among which 256 up-regulated proteins displayed overlapped accumulation in S1 (pre-cortex splitting stage) vs. S2 (cortex splitting stage) and S1 vs. S3 (expanding stage) pairs, whereas 122 up-regulated proteins displayed overlapped accumulation in S1 vs. S3 and S2 vs. S3 pairs. Gene Ontology (GO) and pathway enrichment analysis showed that these DEPs were mainly involved in several processes such as “starch and sucrose metabolism”, “plant hormone signal transduction”, and “biosynthesis of secondary metabolites”. A high concordance existed between iTRAQ and RT-qPCR at the mRNA expression levels. Furthermore, association analysis showed that 187, 181, and 96 DEPs were matched with their corresponding differentially expressed genes (DEGs) in S1 vs. S2, S1 vs. S3, and S2 vs. S3 comparison, respectively. Notably, several functional proteins including cell division cycle 5-like protein (CDC5), expansin B1 (EXPB1), and xyloglucan endotransglucosylase/hydrolase protein 24 (XTH24) were responsible for cell division and expansion during radish taproot thickening process. These results could facilitate a better understanding of the molecular mechanism underlying taproot thickening, and provide valuable information for the identification of critical genes/proteins responsible for taproot thickening in root vegetable crops., Root vegetables: tapping into root thickening for improved yields An investigation into the proteins and genes responsible for taproot thickening in radish could help improve yields of root vegetables. The fleshy taproot of vegetables like radish are vital to food supplies worldwide, but the precise mechanisms behind healthy, high quality taproot formation, and thickening are unclear. Liwang Liu at Nanjing Agricultural University in Nanjing, China, and co-workers have identified several functional proteins and corresponding genes responsible for cell division in taproot growth and thickening. The team analyzed changes in protein expression at three different developmental stages of radish taproot. They identified 1862 differentially expressed proteins involved in several processes including sugar and starch metabolism, hormone signaling, and biosynthesis of metabolites. Their results provide fundamental insights into the regulation of taproot thickening in radish and could be expanded to other root vegetables.

Recently, two rice genes, OsAPETALA2 (OsAP2) and OsWRKY24 have been reported to be positive regulators involved in increased lamina inclination and grain size through cell elongation. Here, we found that the two genes have tightly linked expression patterns and functional convergence in rice, and are also likely to play an opposite role in Arabidopsis. Overexpression of the two rice transcription factors in Arabidopsis caused smaller plant size with reduced cell size, and the expression of a series of genes encoding expansins and xyloglucan endotransglucosylase/hydrolases (XTHs) involved in cell elongation was reduced. However, transgenic Arabidopsis expressing OsWRKY24-SRDX as a synthetic chimeric repressor displayed indistinguishable phenotypes from wild-type plants. Moreover, the subcellular localization pattern of OsWRKY24 in Arabidopsis was different from that in rice. Thus, we demonstrate an example of transcription factors from one species playing distinct roles in different plant species.

The role of xyloglucan endotransglycosylases in the regulation and promotion of plant growth in response to such widespread stress factors as drought, salinization, and hypothermia remains poorly understood. The tXET-B2 (SlXTH10) gene encodes one of the xyloglucan endotransglycosylases of tomato, which is most closely related in the nucleotide sequence to the AtXTH15 and AtXTH16 genes of Arabidopsis thaliana. At present, the specific functions of the tXET-B2 gene, as well as of its homologs, AtXTH15 and AtXTH16, remain obscure. To study the role of tXET-B2 in the regulation of growth and adaptation to abiotic stress factors, transgenic tobacco plants with estradiol-inducible expression of the tomato tXET-B2 gene were generated. Overexpression of this gene promoted tobacco root growth in a medium containing 50 mM NaCl. Under drought conditions, exogenous treatment with estradiol resulted in a considerable increase in fresh and dry weight in many of the studied transgenic lines. Under normal conditions, as well as under salinization and hypothermia stress, such positive effect was detected only for some transgenic lines. The obtained data point to the possibility of using genetically engineered constructs of the tXET-B2 gene to correct growth parameters of transgenic plants under the influence of stress factors.

Aluminum (Al) toxicity is one of the primary factors limiting crop production in acid soils worldwide. The cell wall is the major target of Al toxicity owing to the presence of many Al binding sites. Previous studies have found that XTH, encoding xyloglucan endohydrolase (XEH) and xyloglucan endotransglucosylase (XET), could participate in cell wall extension and affect the binding ability of the cell wall to Al by impeding the activities of these two enzymes. In this study, we found that ZmXTH, an XTH gene in maize, was involved in Al detoxification. The Al-induced up-regulation of ZmXTH occurred in the roots, prominently in the root tips. Additionally, the expression of ZmXTH was specifically induced by Al3+ but no other divalent or trivalent cations. Compared with the wild-type Arabidopsis, ZmXTH overexpressing plants grew more healthy and had decreased Al content in their root and root cell wall after Al stress. Overall, the results suggest that ZmXTH could confer the Al tolerance of transgenic Arabidopsis plants by reducing the Al accumulation in their roots and cell walls.

Brassinosteroids (BRs) play numerous important roles in plant growth and development. Previous studies reported that BRs could promote stem growth by regulating the expression of xyloglucan endotransglucosylase/hydrolases (XTHs). However, the mechanism of XTHs involved in stem growth remains unclear. In this study, PcBRU1, which belonged to the XTH family, was upregulated by exogenous BL treatment in Pyrus communis. The expression of PcBRU1 was highest in stems and lowest in leaves. Subcellular localization analysis indicated that PcBRU1 was located in the plasma membrane. Furthermore, overexpressing PcBRU1 in tobaccos promoted the plant height and internode length. Electron microscopy and anatomical structure analysis showed that the cell wall was significantly thinner and the cells were slenderer in transgenic tobacco lines overexpressing PcBRU1 than in wild-type tobaccos. PcBRU1 promoted stem growth as it loosened the cell wall, leading to the change in cell morphology. In addition, overexpressing PcBRU1 altered the root development and leaf shape of transgenic tobaccos. Taken together, the results could provide a theoretical basis for the XTH family in regulating cell-wall elongation and stem growth.

‘Royal Gala’ apple fruit (Malus domestica) is one of the popular apple fruit consumed world-wide. A great amount literature was accumulated for this classical apple variety about the molecular mechanisms of individual gene or gene family transcript changes during ripening, yet few of them on the large scale at transcriptomic levels. The present study was designed to use RNA-Seq to compare expression profiles between commercial ripening and physiological ripening stages of apple fruit in order to provide a deeper whole view for understanding the molecular control of ‘Royal Gala’ fruit ripening on transcript level. 13.78 G and 12.28 G clean reads were obtained for the commercial ripening and physiological ripening stages apple fruit, respectively acquired and were blasted against the apple genome. 78,553 genes were expressed in ripening apple fruit and among them 860 genes were differentially expressed between the two maturity stages, with 199 up-regulated and 661 down-regulated. The enrichment of differentially expressed genes (DEGs) in gene ontology (GO) pathways revealed that genes in 8 GO terms were more activated at physiological maturity stage. Meanwhile, KEGG enrichment showed that secondary metabolism contained more up-regulated DEGs at physiological maturity stage than at commercial maturity stage. Furthermore, genes working on ripening-related processes were examined. For transcription factors regulating ethylene biosynthesis such as ERFs and HBs, the expression levels were significantly different between the two samples. Regarding fruit firmness decline, expression variation happened to genes encoding enzymes involving in cell wall metabolism, such as polygalacturonase PG1, xyloglucan endotransglucosylase XTHs and EXP3, as well as cell wall structure protein gene FLA. In conclusion, the RNA-Seq data presents the global expression changes of ripening related transcripts in ‘Royal Gala’ apple and novel identified genes and/or transcription factors may provide new clues to illuminate this complicated event.

Xyloglucan endotransglucosylase / hydrolase family genes encode enzymatic proteins that potentially have two different catalytic activities either endotransglucosylase ( XET ) or endohydrolase ( XEH ) activity. Endotransglucosylase catalyze the breakdown and molecular grafting of the β-(1-4)-xyloglucan backbone and endohydrolase catalyze the irreversible shortening. We are interested in understanding the molecular mechanism of the upstream region of the XET gene. An expression unit consisting of the b-glucuronidase (gusA) reporter gene under the control of 1831 bp fragment of the Populus euphratica XET ( PeXET ) promoter was introduced into tobacco via Agrobacterium tumefaciens. The sequence was identified as PeXET promoter as it contain all the upstream sequences required for a typical promoter. Analysis of the promoter sequence indicated some basic cis-acting elements were related to various environmental stresses, developmental response and plant hormones responses. Functional properties of the promoter were examined by GUS staining and fluorescence quantitative analyses using at least three PCR-positive transgenic tobacco plants, treated with various plant hormones iso-osmotic and environmental stresses for different times. We demonstrated that the PeXET promoter was a multistress-inducible promoter. To explain the unknown features of the upstream regulatory mechanism of the PeXET gene in desert plants, we suggest that the function of the PeXET promoter, especially under environmental stress conditions, to be studied for possible value in molecular breeding as new promoters of stress-resistance genes for desert plants.

Accurate analysis of gene expression requires selection of appropriate reference genes. In this study, we report analysis of eight candidate reference genes (ACTIN, UBQ, EF-1&alpha, UBC, IF-4&alpha, TUB, PP2A, and HIS), which were screened from the genome and transcriptome data in Brassica juncea. Four statistical analysis softwares geNorm, NormFinder, BestKeeper, and RefFinder were used to test the reliability and stability of gene expression of the reference genes. To further validate the stability of reference genes, the expression levels of two CYCD3 genes (BjuB045330 and BjuA003219) were studied. In addition, all genes in the xyloglucan endotransglucosylase/hydrolase (XTH) family were identified in B. juncea and their patterns at different periods of stem enlargement were analyzed. Results indicated that UBC and TUB genes showed stable levels of expression and are recommended for future research. In addition, XTH genes were involved in regulation of stem enlargement expression. These results provide new insights for future research aiming at exploring important functional genes, their expression patterns and regulatory mechanisms for mustard development.

Withering is an important biological process accompanied by dehydration and cell wall metabolism in postharvest plant organs during curing/processing and storage. However, dynamics involved in cell wall metabolism and resultant water loss during withering in postharvest tobacco leaves is not well-documented. Here, tandem mass tag (TMT)-based quantitative proteomic analysis in postharvest tobacco leaves (cultivar K326) under different withering conditions was performed. In total, 11,556 proteins were detected, among which 496 differentially abundant proteins (DAPs) were identified. To elucidate the withering mechanism of tobacco leaves, 27 DAPs associated with cell wall metabolism were screened. In particular, pectin acetylesterases, glucan endo-1,3-beta-glucosidases, xyloglucan endotransglucosylase/hydrolase, alpha-xylosidase 1-like, probable galactinol-sucrose galactosyltransferases, endochitinase A, chitotriosidase-1-like and expansin were the key proteins responsible for the withering of postharvest tobacco leaves. These DAPs were mainly involved in pectin metabolism, cellulose, hemicellulose and galactose metabolism, amino sugar and nucleotide sugar metabolism as well as cell wall expansion. Furthermore, relative water content and softness values were significantly and positively correlated. Thus, dehydration and cell wall metabolism were crucial for tobacco leaf withering under different conditions. Nine candidate DAPs were confirmed by parallel reaction monitoring (PRM) technique. These results provide new insights into the withering mechanism underlying postharvest physiological regulatory networks in plants/crops.

Lateral roots (LRs) are the main component of the root system architecture in Arabidopsis. The plasticity of LR development has an important role in improving plant survival in response to the external environment. Previous studies have revealed a number of genetic pathways that control plant growth in response to environmental stimuli. Here, we find that the xyloglucan endotransglucosylase 19 (XTH19) and XTH23 genes are involved in LR development under salt stress. The density of LRs was decreased in the xth23 single mutant, which was also more sensitive to salt than the wild type, and the xth19xth23 double mutant exhibited additive downregulated LR initiation and salt sensitivity compared with the single mutant. On the contrary, constitutive overexpression of XTH19 or XTH23 caused increased LR densities. Furthermore, XTH19 and XTH23 were induced by salt via the key brassinosteroid signaling pathway transcription factor BES1. In addition, we found that 35S::BES1 increased salt tolerance and the phenotype of xth19xth23 & 35S::BES1 was partially complementary to the wild-type level. In vivo and in vitro assays demonstrated that BES1 acts directly upstream of XTH19 and XTH23 to control their expression. Overall, our results revealed that XTH19 and XTH23 are involved in LR development via the BES1-dependent pathway, and contribute to LR adaptation to salt.

Summary Fruit softening in Fragaria (strawberry) is proposed to be associated with the modification of cell wall components such as xyloglucan by the action of cell wall‐modifying enzymes. This study focuses on the in vitro and in vivo characterization of two recombinant xyloglucan endotransglucosylase/hydrolases (XTHs) from Fragaria vesca, FvXTH9 and FvXTH6. Mining of the publicly available F. vesca genome sequence yielded 28 putative XTH genes. FvXTH9 showed the highest expression level of all FvXTHs in a fruit transcriptome data set and was selected with the closely related FvXTH6 for further analysis. To investigate their role in fruit ripening in more detail, the coding sequences of FvXTH9 and FvXTH6 were cloned into the vector pYES2 and expressed in Saccharomyces cerevisiae. FvXTH9 and FvXTH6 displayed xyloglucan endotransglucosylase (XET) activity towards various acceptor substrates using xyloglucan as the donor substrate. Interestingly, FvXTH9 showed activity of mixed‐linkage glucan:xyloglucan endotransglucosylase (MXE) and cellulose:xyloglucan endotransglucosylase (CXE). The optimum pH of both FvXTH9 and FvXTH6 was 6.5. The prediction of subcellular localization suggested localization to the secretory pathway, which was confirmed by localization studies in Nicotiana tabacum. Overexpression showed that Fragaria × ananassa fruits infiltrated with FvXTH9 and FvXTH6 ripened faster and showed decreased firmness compared with the empty vector control pBI121. Thus FvXTH9 and also FvXTH6 might promote strawberry fruit ripening by the modification of cell wall components., Significance Statement This study suggests that xyloglucan endotransglucosylase/hydrolases from Fragaria vesca, namely FvXTH9 and FvXTH6, promote strawberry fruit ripening through involvement in the modification of cell wall components. Overexpression of FvXTH9 and FvXTH6 in Fragaria × ananassa fruits resulted in accelerated fruit softening.

Background and aims: Xyloglucan endotransglucosylase/hydrolase (XTH) proteins that possess xyloglucan endotransglucosylase (XET) activity contribute to cell-wall assembly and remodelling, orchestrating plant growth and development. Little is known about in-vivo XET regulation, other than at the XTH transcriptional level. Plants contain ‘cold-water-extractable, heat-stable polymers’ (CHPs) which are XTH-activating factors (XAFs) that desorb and thereby activate wall-bound XTHs. Since XAFs may control cell-wall modification in vivo, we have further explored their nature. Methods: Material was cold-water-extracted from 25 plant species; proteins were precipitated by heat-denaturation, then CHP was ethanol-precipitated. For XAF assays, CHP (or sub-fractions thereof) was applied to washed Arabidopsis thaliana cell walls, and enzymes thus solubilised were assayed radiochemically for XET activity. In some experiments, the CHP was pre-treated with trifluoroacetic acid (TFA), alkali (NaOH) or glycanases. Key results: · CHP specifically desorbed wall-bound XTHs, but not β-glucosidases, phosphatases or peroxidases.· · CHP preparations from 25 angiosperms all possessed XAF activity but had no consistent monosaccharide composition. · Of eleven individual plant polymers tested, only gum arabic and tamarind xyloglucan were XAF-active, albeit less so than CHP. · On gel-permeation chromatography, XAF-active cauliflower CHP eluted with molecular weight ~7,000–140,000, though no specific sugar residue(s) co-eluted exactly with XAF activity. · Cauliflower XAF activity survived cold alkali and warm dilute TFA (which break ester and glycofuranosyl linkages respectively), but was inactivated by hot 2M TFA (which breaks glycopyranosyl linkages). · Cauliflower XAF activity was remarkably stable to diverse glycanases and glycosidases. Conclusions: XAFs are naturally occurring heat-stable polymers that specifically desorb (thereby activating) wall-bound XTHs. Their XAF activity considerably exceeds that of gum arabic and tamarind xyloglucan, and they were not identifiable as any major plant polysaccharide. We propose that XAF is a specific, minor, plant polymer that regulates xyloglucan transglycosylation in vivo, and thus wall assembly and restructuring

Adventitious roots (ARs) play an irreplaceable role in the uptake of water and nutrients due to under-developed principle root in plants. The process of ARs formation is affected by plant hormone. In this study, by employing High-Throughout Tag-sequencing Technique and ELISA method, we analyzed of the transcriptome and indole-3-acetic acid (IAA) content to monitor the changes of metabolism regulated by ethylene signaling in lotus. Exogenous application of ethephon (ethylene precursor) dramatically accelerated ARs development, and while restrained by 1-methylcyclopropene (1-MCP, the ethylene perception inhibitor), indicating the crucial role ethylene in ARs development. Transcriptomic analysis showed that both treatment of ethephon and 1-MCP dramatically altered the expression of numerous genes. In total, transcriptional expressions of 694 genes were induced and 554 genes were suppressed in ETH/CK0 stages compared with MCP/CK0 stages. Most of these up-regulated genes exhibited the one-three folds changes. In ETH/MCP libraries, we found nine and five genes involved in the metabolism or transcriptional responses to ethylene and IAA, and fourteen genes, which were considered to NAC, bHLH, AP2-EREBP, MYB, LOB, bHLH and bZIP families, respectively, exhibited an increase in transcriptional level. In addition, an enhanced mRNA levels of seven genes [1-aminocyclopropane-1-carboxylate oxidase (ACO), leucine-rich repeat receptor, pectinesterase, pyruvate decarboxylase, ethylene oxide synthase, respiratory burst oxidase homolog protein and xyloglucan endotransglucosylase] relevant to ARs formation were detected in was detected in ETH/MCP libraries. Furthermore, we found that IAA content was obviously decreased after applications were detected on ethephon and 1-MCP. However, the decreased IAA level in 1-MCP treatment was more pronounced than that in ethephon treatment, and kept a low level during the whole periods of ARs development. Taken together, our findings provided a comprehensive understanding of ethylene’s regulation during ARs formation in lotus seedlings.

Banana is a climacteric fruit whose ripening once initiated is irreversible and proceeds very fast making the fruits highly perishable. Application of various post-harvest technologies has been shown to slow down the ripening process in climacteric fruits such as banana. One of these technologies is hexanal, a naturally occurring compound that delays the ripening process in banana fruits without compromising the quality. As the molecular mechanisms underlying the mode of action of hexanal in delaying ripening are yet to be elucidated, we undertook a comparative transcriptomic analysis using banana fruits treated with either hexanal, ethylene or untreated controls. Results of our study show that hexanal significantly delayed the rate of pulp softening throughout the storage period. Sequencing results showed that 776 genes were up-regulated and 2146 were down-regulated upon hexanal treatment while 4 genes were up-regulated and 76 were down-regulated upon ethylene treatment at day one of storage. Additionally, 2423 genes were up-regulated and 2862 were down-regulated upon hexanal treatment while a total of 4820 genes were up-regulated and 5395 were down-regulated upon ethylene treatment at day four. We found that hexanal treatment transiently suppressed the expression of genes involved in ethylene biosynthesis, cell membrane deterioration and cell wall degradation by day four of storage contrary to the observed induction of the same genes in ethylene-treated fruits. The particular genes are; Aminocyclopropane-1-Carboxylic Acid Oxidase, 1-Aminocyclopropane-1-Carboxylic Acid, Phospholipase D, Polygalacturonase, Expansin and Xyloglucan Endotransglucosylase. Later on at day 18 of storage, genes involved in ethylene biosynthesis, cell wall and cell membrane degradation and aroma synthesis were induced in the hexanal-treated fruits. These findings reveal that hexanal works by temporarily suppressing the expression of genes involved in ethylene biosynthesis, cell wall degradation and cell membrane deterioration.

Plant height is determined by the processes of cell proliferation and elongation. Plant hormones play key roles in a species-dependent manner in these processes. We used paclobutrazol (PAC) at 400 mg·L-1 in this study to spray Agapanthus praecox ssp. orientalis plants in order to induce dwarf scape (inflorescence stem). Morphological examination showed that PAC reduced scape height by inhibiting the cell elongation by 54.56% and reducing cell proliferation by 10.45% compared to the control. Quantification and immunolocalization of endogenous gibberellins (GAs) and indole-3-acetic acid (IAA) showed that the GA1, GA3, and GA4 levels and the IAA gradient were reduced. Among these hormones, GA4 was the key component of GAs, which decreased 59.51-92.01% compared to the control in scape. The expression of cell wall synthesis related genes cellulose synthase (CESA) and UDP-glucuronic acid decarboxylase (UXS) were upregulated significantly, whereas cell wall loosening gene xyloglucan endotransglucosylase 2 (XET2) was downregulated by 99.99% surprisingly. Correlation analysis suggested GA regulated cell elongation and auxin modulated cell proliferation in Agapanthus scape. Additionally, the accumulation of sugars played roles in cell wall synthesis and cell expansion. These results indicated GA and IAA signals triggered a downstream signaling cascade, controlled cell expansion and proliferation during scape elongation.

Background and aims Aluminium (Al) toxicity and drought are two major limiting factors for common bean (Phaseolus vulgaris) production on tropical acid soils. Polyethylene glycol (PEG 6000)-induced osmotic stress (OS) simulating drought stress reduces Al accumulation in the entire root tips of common bean by alteration of cell-wall (CW) porosity, which might be regulated by two genes encoding xyloglucan endotransglucosylase/hydrolase, PvXTH9 and PvXTHb The aim of this research was to understand the spatial and temporal regulation of both XTH genes in PEG-mediated Al accumulation in the root tips. Methods In this study the spatial and temporal expression patterns of Al-inhibited root elongation, Al accumulation, XTH gene expression and xyloglucan endotransglucosylase (XET) enzyme action in the root tips were analysed under PEG-induced OS by a combination of physiological and molecular approaches such as quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ fluorescence detection of XET in root tips. Key results The results showed that Al accumulation, expression of XTH genes and XET action were distinctly reduced in the apical 0-2, 2-7 and 7-12 mm zones under OS, implying a potential regulatory role of XTH genes and XET enzyme in CW Al accumulation in these zones. Conclusions The results provide novel insights into the physiological and molecular mechanisms of CW structure modification as a response of plant roots to OS, which will contribute to mitigate Al and drought stresses, severely limiting crop yields on acid soils.

The xyloglucan endotransglucosylase/hydrolase (XTH) genes in Arabidopsis thaliana (L.) Heynh. form part of a group of mechano-stimulated genes and play an important role in abiotic stress tolerance. Mining the RNAseq transcriptomic database of 40,430 potato (Solanum tuberosum L.) genes based on functional annotation and homology search, our objective was to discover potentially homologous XTH genes. A Gene Ontology-based XTH homology search and functional annotation discovered, from among the 33 A. thaliana (AtXTH) and 25 tomato (Solanum lycopersicum L.) (SlXTH) XTH genes, 35 gene sequences corresponding to 20 AtXTH genes and 40 gene sequences corresponding to 21 SlXTH genes, respectively. Thirteen sequences corresponding to 11 putative XTH genes in potato, named as StXTH after SlXTH genes, were significantly up- or down-regulated in response to ultrasound. These putative StXTH genes in potato can serve for future functional genetic analyses.

The field application of safe chemical inducers plays a vital role in the stimulation of systematic acquired resistance (SAR) of plants. In this study, the efficacy use of three and six field applications with chitosan, lithovit, and K-thiosulfate at 4 gL&minus, 1 and salicylic acid at 1.5 gL&minus, 1 in improving tomato productivity, quality, and modifying the defense signaling pathways to the Alternaria alternata infection was investigated. Salicylic acid was the most effective in vitro where it completely inhibited the growth of Alternaria alternata. The highest yield quantity was recorded with six applications with Chitosan followed by Salicylic acid, also, they were the most effective treatments in controlling the Alternaria alternata infection in tomato fruits. The maximum increase in chitinase and catalase activity of tomato fruits was observed at five days after inoculation, following treatment with six sprays of salicylic acid followed by chitosan. The transcript levels of seven defense-related genes: ethylene-responsive transcription factor 3 (RAP), xyloglucan endotransglucosylase 2 (XET-2), catalytic hydrolase -2 (ACS-2), proteinase inhibitor II (PINII), phenylalanine ammonia-lyase 5 (PAL5), lipoxygenase D (LOXD), and pathogenesis-related protein 1 (PR1) were upregulated in response to all treatments. The highest expression levels of the seven studied genes were recorded in response to six foliar applications with chitosan. Chitosan followed by salicylic acid was the most effective among the tested elicitors in controlling the black mold rot in tomato fruits. In conclusion, pre-harvest chitosan and salicylic acid in vivo application with six sprays could be recommended as effective safe alternatives to fungicides against black mold disease in tomato fruits.

Plants are continuously infected by various pathogens throughout their lifecycle. Previous studies have reported that the expression of Class III acyl-CoA-binding proteins (ACBPs) such as the Arabidopsis ACBP3 and rice ACBP5 were induced by pathogen infection. Transgenic Arabidopsis AtACBP3-overexpressors (AtACBP3-OEs) displayed enhanced protection against the bacterial biotroph, Pseudomonas syringae, although they became susceptible to the fungal necrotroph Botrytis cinerea. A Class III ACBP from a monocot, rice (Oryza sativa) OsACBP5 was overexpressed in the dicot Arabidopsis. The resultant transgenic Arabidopsis lines conferred resistance not only to the bacterial biotroph P. syringae but to fungal necrotrophs (Rhizoctonia solani, B. cinerea, Alternaria brassicicola) and a hemibiotroph (Colletotrichum siamense). Changes in protein expression in R. solani-infected Arabidopsis OsACBP5-overexpressors (OsACBP5-OEs) were demonstrated using proteomic analysis. Biotic stress-related proteins including cell wall-related proteins such as FASCILIN-LIKE ARABINOGALACTAN-PROTEIN10, LEUCINE-RICH REPEAT EXTENSIN-LIKE PROTEINS, XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE PROTEIN4, and PECTINESTERASE INHIBITOR18; proteins associated with glucosinolate degradation including GDSL-LIKE LIPASE23, EPITHIOSPECIFIER MODIFIER1, MYROSINASE1, MYROSINASE2, and NITRILASE1; as well as a protein involved in jasmonate biosynthesis, ALLENE OXIDE CYCLASE2, were induced in OsACBP5-OEs upon R. solani infection. These results indicated that upregulation of these proteins in OsACBP5-OEs conferred protection against various plant pathogens.

Polyploidy is an important evolutionary factor in most land plant lineages which possess more than two complete sets of chromosomes. Radish (Raphanus sativus L.) is an economically annual/biennial root vegetable crop worldwide. However, the expression patterns of duplicated homologs involved in the autopolyploidization remains unclear. In present study, the autotetraploid radish plants (2n = 4x = 36) were produced with colchicine and exhibited an increase in the size of flowers, leaves, stomata and pollen grains. The differential gene expression (DGE) profiling was performed to investigate the differences in gene expression patterns between diploid and its corresponding autotetraploid by RNA-Sequencing (RNA-Seq). Totally, 483 up-regulated differentially expressed genes (DEGs) and 408 down-regulated DEGs were detected in diploid and autotetraploid radishes, which majorly involved in the pathways of hormones, photosynthesis and stress response. Moreover, the xyloglucan endotransglucosylase/hydrolase (XTH) and pectin methylesterases (PME) family members related to cell enlargement and cell wall construction were found to be enriched in GO enrichment analysis, of which XTH family members enriched in “apoplast” and “cell wall” terms, while PME family members enriched in “cell wall” term. Reverse-transcription quantitative PCR (RT-qPCR) analysis indicated that the expression profile of DEGs were consistent with results from the RNA-Seq analysis. The DEGs involved in cell wall construction and auxin metabolism were predicted to be associated with organs size increase of autotetraploid radishes in the present study. These results could provide valuable information for elucidating the molecular mechanism underlying polyploidization and facilitating further genetic improvements of important traits in radish breeding programs.

The expression of two genes encoding xyloglucan endotransglucosylase/hydrolases (XTHs), Cs-XTH1 and Cs-XTH3, was upregulated during the onset of cucumber somatic embryogenesis. As a means of characterising the developmental regulation of these genes, the activity of the respective upstream regulatory regions was investigated in seedlings and somatic embryos of Arabidopsis thaliana and Cucumis sativus. GUS assays revealed that both genes are under developmental control. In addition, elevated promoter activity was found in the tension-bearing regions of the plant and in response to touch and wounding, which is consistent with the existence of numerous stress-related cis elements in the 5′-regulatory regions. In vivo xyloglucan endotransglucosylase (XET) action assays were performed to gain an overview on the role of XTHs during somatic embryogenesis. The highest XET action was observed in the external cell layers of somatic embryos in the cotyledonary region and in the presumptive region of peg formation. Based on the results, we propose a dual mechanism (one developmental and the second adaptive) for the regulation of Cs-XTH1 and Cs-XTH3 activity wherein the developmental pattern can be modified by mechanical stimuli.

Xyloglucan endotransglucosylase/hydrolase genes (XTHs) encode enzymes required for the reconstruction and modification of xyloglucan backbones, which will result in changes of cell wall extensibility during growth. A total of 56 NtXTH genes were identified from common tobacco, and 50 cDNA fragments were verified by PCR amplification. The 56 NtXTH genes could be classified into two subfamilies: Group I/II and Group III according to their phylogenetic relationships. The gene structure, chromosomal localization, conserved protein domains prediction, sub-cellular localization of NtXTH proteins and evolutionary relationships among Nicotiana tabacum, Nicotiana sylvestrisis, Nicotiana tomentosiformis, Arabidopsis, and rice were also analyzed. The NtXTHs expression profiles analyzed by the TobEA database and qRT-PCR revealed that NtXTHs display different expression patterns in different tissues. Notably, the expression patterns of 12 NtXTHs responding to environment stresses, including salinity, alkali, heat, chilling, and plant hormones, including IAA and brassinolide, were characterized. All the results would be useful for the function study of NtXTHs during different growth cycles and stresses.

Plant cell walls are affected by many biotic and abiotic stress conditions. The aim of this study is to determine the effects of 24-Epibrassinolide (EBL) on some cell wall-related genes in root tissue of five- and ten-week-old Arabidopsis thaliana plants exposed to boron (B) deficiency (0 µM) or toxicity (3000 µM) at the transcriptional level. Expressions of the genes that encode cellulose synthase (CESA1, CESA4, CESA6 and CESA8), cellulose synthase-like (CSLB5), expansin (EXPA5, EXPA8 and EXPA14) and cell wall protein (SEB1) decreased under conditions of B deficiency and toxicity. EBL treatments, in general, led the expressions of these genes to reduce significantly. Expressions of xyloglucan endotransglucosylase/hydrolase genes (XTH21 and XTH23) changed only under conditions of B toxicity. Boron stress and/or EBL treatments caused different responses in expression of pectin methylesterase (PME2 and PME41) genes. As a result of B stress, the expression levels of investigated genes changed more in roots of five-week-old plants than in roots of ten-week-old plants. Results of the present study include new findings that support the ability of BRs to increase molecular aspects of tolerance to stress in plants.

Highlight Expression of cell wall-related genes marks the onset of haustorium development in the parasitic plant Cuscuta. Action assays suggest a central role for xyloglucan endotransglucosylases/hydrolases in host plant infection., Changes in cell walls have been previously observed in the mature infection organ, or haustorium, of the parasitic angiosperm Cuscuta, but are not equally well charted in young haustoria. In this study, we focused on the molecular processes in the early stages of developing haustoria; that is, before the parasite engages in a physiological contact with its host. We describe first the identification of differentially expressed genes in young haustoria whose development was induced by far-red light and tactile stimuli in the absence of a host plant by suppression subtractive hybridization. To improve sequence information and to aid in the identification of the obtained candidates, reference transcriptomes derived from two species of Cuscuta, C. gronovii and C. reflexa, were generated. Subsequent quantitative gene expression analysis with different tissues of C. reflexa revealed that among the genes that were up-regulated in young haustoria, two xyloglucan endotransglucosylase/hydrolase (XTH) genes were highly expressed almost exclusively at the onset of haustorium development. The same expression pattern was also found for the closest XTH homologues from C. gronovii. In situ assays for XTH-specific action suggested that xyloglucan endotransglucosylation was most pronounced in the cell walls of the swelling area of the haustorium facing the host plant, but was also detectable in later stages of haustoriogenesis. We propose that xyloglucan remodelling by Cuscuta XTHs prepares the parasite for host infection and possibly aids the invasive growth of the haustorium.

The LONGIFOLIA1 (LNG1) gene of Arabidopsis regulates leaf shape by polar cell elongation independent of ROTUNDAFOLIA3 (ROT3). To expand our knowledge on the function of this gens in plant systems, Arabidopsis LNG1 (AtLNG1) was introduced both sense and antisense orientation under the control of 35S CaMV promoter into tobacco plants that lack AtLNG1 homolog. Resulting transgenic tobacco plants were analyzed by their phenotype, anatomy and transcript levels. AtLNG1-overexpressing tobacco lines showed increase in the leaf petiole and leaf blade compared with wild type tobacco line. The overexpressors also showed elongated palisade cells as well as epidermal cells in the leaf length direction, but no increase in cell number. Ectopic expression of AtLNG1 in tobacco plants also increased the expression of cell wall modification-related genes, such as NT_XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE9 (NT_XTH9), NT_XTH15 and NT_XTH33, indicating that these genes appear to be target of AtLNG1. As results of molecular and cellular examination, AtLNG1 seemed to have a conserved functional role in shaping leaf morphology in both Arabidopsis and tobacco.

Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the most destructive soil-borne diseases. In this study, young tissue-cultured plantlets of banana (Musa spp. AAA) cultivars differing in Foc susceptibility were used to reveal their differential responses to this pathogen using digital gene expression (DGE). Data were evaluated by various bioinformatic tools (Venn diagrams, gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses) and immunofluorescence labelling method to support the identification of gene candidates determining the resistance of banana against Foc. Interestingly, we have identified MaWRKY50 as an important gene involved in both constitutive and induced resistance. We also identified new genes involved in the resistance of banana to Foc, including several other transcription factors (TFs), pathogenesis-related (PR) genes and some genes related to the plant cell wall biosynthesis or degradation (e.g., pectinesterases, β-glucosidases, xyloglucan endotransglucosylase/hydrolase and endoglucanase). The resistant banana cultivar shows activation of PR-3 and PR-4 genes as well as formation of different constitutive cell barriers to restrict spreading of the pathogen. These data suggest new mechanisms of banana resistance to Foc.

Plants possess exuberant plasticity that facilitates its ability to adapt and survive under challenging environmental conditions. The developmental plasticity largely depends upon cellular elongation which is governed by a complex network of environmental and phytohormonal signals. Here, we report role of glucose (Glc) and Glc-regulated factors in controlling elongation growth and shade response in Arabidopsis. Glc controls shade induced hypocotyl elongation in a dose dependent manner. We have identified a Glc repressed factor REGULATED BY SUGAR AND SHADE1 (RSS1) encoding for an atypical basic helix-loop-helix (bHLH) protein of unknown biological function that is required for normal Glc actions. Phenotype analysis of mutant and overexpression lines suggested RSS1 to be a negative regulator of elongation growth. RSS1 affects overall auxin homeostasis. RSS1 interacts with the elongation growth-promoting proteins HOMOLOG OF BEE2 INTERACTING WITH IBH 1 (HBI1) and BR ENHANCED EXPRESSION2 (BEE2) and negatively affects the transcription of their downstream targets such as YUCs, INDOLE-3-ACETIC ACID INDUCIBLE (IAAs), LONG HYPOCOTYL IN FAR-RED1 (HFR1), HOMEOBOX PROTEIN 2 (ATHB2), XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASES (XTHs) and EXPANSINS. We propose, Glc signals might maintain optimal hypocotyl elongation under multiple signals such as light, shade and phytohormones through the central growth regulatory bHLH/HLH module.

Populus euphratica Olivier, which has been considered as a tree model for the study of higher plant response to abiotic stresses, survive in the desert ecosystem characterized by extreme drought stress. To survive in the harsh environmental condition the plant species have developed some plasticity such as the development of heteromorphic leaves and well-developed roots system. We investigated the physiological and molecular mechanisms enabling this species to cope with severe stress caused by drought. The heterophylly, evolved from linear to toothed-ovate shape, showed the significant difference in cuticle thickness, stomata densities, and sizes. Physiological parameters, SOD, POD, PPO, CAT activity, free proline, soluble protein and MDA contents fluctuated in response to soil drying. Gene expression profile of roots monitored at control and 4 moisture gradients regimes showed the up-regulation of 124, 130, 126 and 162 and down-regulation of 138, 251, 314, 168 DEGs, respectively. Xyloglucan endotransglucosylase/ hydrolase gene (XET) up-regulated at different moisture gradients, was cloned and expressed in tobacco. The XET promoter sequence harbors the drought signaling responsive cis-elements. The promoter expression activity varies in different organs. Over-expression and knocked down transgenic tobacco plant analysis confirmed the role of XET gene in roots growth and drought resistance.

In this work, we genetically characterized the function of Arabidopsis thaliana, LONGIFOLIA (LNG1), LNG2, LNG3, LNG4, their contribution to regulate vegetative architecture in plant. We used molecular and biophysical approaches to elucidate a gene function that regulates vegetative architecture, as revealed by the leaf phenotype and later effects on flowering patterns in Arabidopsis loss-of-function mutants. As a result, LNG genes play an important role in polar cell elongation by turgor pressure controlling the activation of XTH17 and XTH24. Plant vegetative architecture is related to important traits that later influence the floral architecture involved in seed production. Leaf morphology is the primary key trait to compose plant vegetative architecture. However, molecular mechanism on leaf shape determination is not fully understood even in the model plant A. thaliana. We previously showed that LONGIFOLIA (LNG1) and LONGIFOLIA2 (LNG2) genes regulate leaf morphology by promoting longitudinal cell elongation in Arabidopsis. In this study, we further characterized two homologs of LNG1, LNG3, and LNG4, using genetic, biophysical, and molecular approaches. Single loss-of-function mutants, lng3 and lng4, do not show any phenotypic difference, but mutants of lng quadruple (lngq), and lng1/2/3 and lng1/2/4 triples, display reduced leaf length, compared to wild type. Using the paradermal analysis, we conclude that the reduced leaf size of lngq is due to decreased cell elongation in the direction of longitudinal leaf growth, and not decreased cell proliferation. This data indicate that LNG1/2/3/4 are functionally redundant, and are involved in polar cell elongation in Arabidopsis leaf. Using a biophysical approach, we show that the LNGs contribute to maintain high turgor pressure, thus regulating turgor pressure-dependent polar cell elongation. In addition, gene expression analysis showed that LNGs positively regulate the expression of the cell wall modifying enzyme encoded by a multi-gene family, xyloglucan endotransglucosylase/hydrolase (XTH). Taking all of these together, we propose that LNG related genes play an important role in polar cell elongation by changing turgor pressure and controlling the activation of XTH17 and XTH24.

The exosome complex plays a central and essential role in RNA metabolism. However, current research on functions of exosome subunit in plants is limited. Here, we used an egg cell-specific promoter-controlled CRISPR/Cas9 system to knock out RRP42 which encodes a core subunit of the Arabidopsis exosome and presented evidence that RRP42 is essential for the development of female gametophytes. Next, we designed three different amiRNAs targeting RRP42. The rrp42 knock-down mutants mainly displayed variegated and serrated leaves, especially in cauline leaves. The internal anatomy of cauline leaves displayed irregularly shaped palisade cells and a reduced density of mesophyll cells. Interestingly, we detected highly accumulated mRNAs that encode xyloglucan endotransglucosylase/hydrolases (XTHs) and expansins (EXPAs) during later growth stages in rrp42 knock-down mutants. The mRNA decay kinetics analysis for XTH19, EXPA10, and EXPA11 revealed that RRP42 had a role in the decay of these mRNAs in the cytoplasm. RRP42 is localized to both the nucleus and cytoplasm, and RRP42 is preferentially expressed in cauline leaves during later growth stages. Altogether, our results demonstrate that RRP42 is essential for the development of female gametophytes and plays an important role in mesophyll cell morphogenesis.

Previously, we reported that although the Arabidopsis (Arabidopsis thaliana) Xyloglucan Endotransglucosylase-Hydrolase31 (XTH31) has predominately xyloglucan endohydrolase activity in vitro, loss of XTH31 results in remarkably reduced in vivo xyloglucan endotransglucosylase (XET) action and enhanced Al resistance. Here, we report that XTH17, predicted to have XET activity, binds XTH31 in yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitations assays and that this interaction may be required for XTH17 XET activity in planta. XTH17 and XTH31 may be colocalized in plant cells because tagged XTH17 fusion proteins, like XTH31 fusion proteins, appear to target to the plasma membrane. XTH17 expression, like that of XTH31, was substantially reduced in the presence of aluminum (Al), even at concentrations as low as 10 µm for 24 h or 25 µm for just 30 min. Agrobacterium tumefaciens-mediated transfer DNA insertion mutant of XTH17, xth17, showed low XET action and had moderately shorter roots than the wild type but was more Al resistant than the wild type. Similar to xth31, xth17 had low hemicellulose content and retained less Al in the cell wall. These data suggest a model whereby XTH17 and XTH31 may exist as a dimer at the plasma membrane to confer in vivo XET action, which modulates cell wall Al-binding capacity and thereby affects Al sensitivity in Arabidopsis.

Summary EDT1/HGD11 coordinately upregulates gene families of cell-wall-loosening proteins to alter cell-wall extensibility and promote primary root elongation., The gain-of-function mutant edt1 shows significantly enhanced drought tolerance and a well-developed root system including deeper primary roots and more lateral roots. To explore the molecular mechanisms underlying the improved root system of edt1, we performed transcriptome comparison between the wild-type and edt1 roots. One of the interesting findings from the analysis was that several gene families of cell-wall-loosening proteins were upregulated in the mutant roots, including expansins, extensins, xyloglucan endotransglucosylase/hydrolases (XTHs), pectin-related enzymes, and cellulases. Most of these genes contain HD-binding cis-elements in their promoters predominantly with the TTTAATTT sequence, which can be bound by HDG11 in vitro and in vivo. The coordinated expression of these gene families overlaps fast root elongation. Furthermore, overexpression of AtEXPA5, which was dramatically upregulated in edt1, resulted in longer primary roots because cells were more extended longitudinally. When combined by crossing the AtEXPA5-overexpression lines with one pectin methylesterase inhibitor family protein (PMEI) gene (At5g62360)- or one cellulase (CEL) gene (At2g32990)-overexpression lines, the primary roots of the progeny even exceeded both parents in length. Our results demonstrate that HDG11 directly upregulates cell-wall-loosening protein genes, which is correlated with altered root system architecture, and confirm that cell-wall-loosening proteins play important roles in coordinating cell-wall extensibility with root development. The results of transgene experiments showed that expansin works together with PMEI and CEL to generate synergistic effects on primary root elongation, suggesting that different cell-wall-loosening protein families may function in combination to generate optimal effects on root extensibility.