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Pectin is an important component of cell wall polysaccharides and is important for normal plant growth and development. As a major component of pectin in the primary cell wall, homogalacturonan (HG) is a long-chain macromolecular polysaccharide composed of repeated α-1,4-D-GalA sugar units. At the same time, HG is synthesized in the Golgi apparatus in the form of methyl esterification and acetylation. It is then secreted into the plasmodesmata, where it is usually demethylated by pectin methyl esterase (PME) and deacetylated by pectin acetylase (PAE). The synthesis and modification of HG are involved in polysaccharide metabolism in the cell wall, which affects the structure and function of the cell wall and plays an important role in plant growth and development. This paper mainly summarizes the recent research on the biosynthesis, modification and the roles of HG in plant cell wall.

Perception of pathogen-associated molecular patterns (PAMPs) triggers mitogen-activated protein (MAP) kinase 4 (MPK4)-mediated phosphorylation and induces downstream transcriptional reprogramming, but the mechanisms of the MPK4 defense pathway are poorly understood. Here, we showed that phosphorylation-mediated inactivation of the CCCH protein C3H14 by MPK4 positively regulates the immune response in Arabidopsis (Arabidopsis thaliana). Compared with wild-type plants, loss-of-function mutations in C3H14 and its paralog C3H15 resulted in enhanced defense against Pst DC3000 in infected leaves and the development of systemic acquired resistance (SAR), whereas C3H14 or C3H15 overexpression enhanced susceptibility to this pathogen and failed to induce SAR. The functions of C3H14 in PAMP-triggered immunity (PTI) and SAR were dependent on MPK4-mediated phosphorylation. Challenge with Pst DC3000 or the flagellin peptide flg22 enhanced the phosphorylation of C3H14 by MPK4 in the cytoplasm, relieving C3H14-inhibited expression of PTI-related genes and attenuating C3H14-activated expression of its targets NIM1-INTERACTING1 (NIMIN1) and NIMIN2, two negative regulators of SAR. Salicylic acid (SA) affected the MPK4–C3H14–NIMIN1/2 cascades in immunity, but SA signaling mediated by the C3H14-NIMIN1/2 cascades was independent of MPK4 phosphorylation. Our study suggests that C3H14 might be a negative component of the MPK4 defense signaling pathway.

Plant CCCH proteins participate in the control of multiple developmental and adaptive processes, but the regulatory mechanisms underlying these processes are not well known. In this study, we showed that the Arabidopsis (Arabidopsis thaliana) CCCH protein C3H15 negatively regulates cell elongation by inhibiting brassinosteroid (BR) signaling. Genetic and biochemical evidence showed that C3H15 functions downstream of the receptor BR INSENSITIVE 1 (BRI1) as a negative regulator in the BR pathway. C3H15 is phosphorylated by the GLYCOGEN SYNTHASE KINASE 3 -like kinase BR-INSENSITIVE 2 (BIN2) at Ser111 in the cytoplasm in the absence of BRs. Upon BR perception, C3H15 transcription is enhanced, and the phosphorylation of C3H15 by BIN2 is reduced. The dephosphorylated C3H15 protein accumulates in the nucleus, where C3H15 regulates transcription via G-rich elements (typically GGGAGA). C3H15 and BRASSINAZOLE RESISTANT 1 (BZR1)/BRI1-EMS-SUPPRESSOR 1 (BES1), two central transcriptional regulators of BR signaling, directly suppress each other and share a number of BR-responsive target genes. Moreover, C3H15 antagonizes BZR1 and BES1 to regulate the expression of their shared cell elongation-associated target gene, SMALL AUXIN-UP RNA 15 (SAUR15). This study demonstrates that C3H15-mediated BR signaling may be parallel to, or even attenuate, the dominant BZR1 and BES1 signaling pathways to control cell elongation. This finding expands our understanding of the regulatory mechanisms underlying BR-induced cell elongation in plants.

Activity of the vascular cambium gives rise to secondary xylem for wood formation in trees. The transcription factor WUSCHEL-related HOMEOBOX4 (WOX4) is a central regulator downstream of the hormone and peptide signaling pathways that maintain cambial activity. However, the genetic regulatory network underlying WOX4-mediated wood formation at the post-transcriptional level remains to be elucidated. In this study, we identified the ubiquitin receptor PagDA1 in hybrid poplar (Populus alba × Populus glandulosa clone 84K) as a negative regulator of wood formation, which restricts cambial activity during secondary growth. Overexpression of PagDA1 in poplar resulted in a relatively reduced xylem due to decreased cambial cell division. By contrast, mutation of PagDA1 by CRISPR/Cas9 resulted in an increased cambial cell activity and promoted xylem formation. Genetic analysis demonstrated that PagDA1 functions antagonistically in a common pathway as PagWOX4 to regulate cambial activity. We propose that PagDA1 physically associates with PagWOX4 and modulates the degradation of PagWOX4 by the 26S proteasome. Moreover, genetic analysis revealed that PagDA1 exerts its negative effect on cambial development by modulating the stability of PagWOX4 in a ubiquitin-dependent manner mediated by the E3 ubiquitin ligase PagDA2. In sum, we have identified a cambial regulatory protein complex, PagDA1–PagWOX4, as a potential target for wood biomass improvement.

Miscanthus is a promising lignocellulosic bioenergy crop for the production of cellulosic ethanol. The recalcitrance of lignocellulose hampers the efficient conversion of Miscanthus biomass to fermentable sugars. Therefore, screening of Miscanthus germplasms with enhanced lignocellulose saccharification efficiency is of important significance to the cellulosic ethanol production from Miscanthus biomass. In this study, we utilize heavy‐ion irradiation mutagenesis to establish a mutant library of Miscanthus lutarioriparius, and screened a reddish stem (rs) mutant with improved lignocellulose saccharification efficiency. The rs mutant was characterized by a noticeable reddish coloration in various lignified cell walls in phloem and vascular bundle sheath, whereas the biomass yield was not compromised. The reddish coloration was attributed to the reduced incorporation of several phenolic compounds including 3‐caffeoylquinic acid, dihydroflavonol and proanthocyanin A, which shared common precursors with lignin monolignol biosynthesis via the phenylpropanoid pathway. Correspondingly, the lignin content was decreased by 10.2% and the cellulose content was concomitantly increased by 9.5% in rs mutant compared with the control plant. Meanwhile, the saccharification efficiency was substantially improved by 22.4%–25.6% in rs mutant compared to the control, when the lignocellulosic biomass was pretreated with or without 1.5% H2SO4. The rs mutant identified here holds a potential utilization as an ideal candidate feedstock in cellulosic bioethanol production.

Duckweed is one kind of promising bioenergy plant with prominent advantages such as fast growth rate and high starch content. However, almost all previous studies focused on the natural duckweed germplasms. In this study, heavy‐ion irradiation was used to establish a mutant library of Lemna aequinoctialis 6002, and one mutant named submarine‐1 (sub‐1) was screened, which could accumulate more starch but with smaller granules. Unexpectedly, under proper external growth conditions such as poor nutritional status and insufficient growth space, sub‐1 mutant would sink underwater due to formation of dense tissue structure and large amount of fine starch particles with the extension of cultivation time. The starch content in the sinking sub‐1 increased to over 45% (dry weight) and was 12% higher than the floating sub‐1, highlighting that submergence can be considered as a spontaneous and efficient indicator for screening of high‐starch duckweed. Additionally, the saccharification efficiency of starch and ethanol yield had increased in sub‐1 mutant compared to the wild type. Based on the unique characteristics of sub‐1 mutant, a cultivation model of submerged duckweed in a simulated aquaculture pond was designed to get more starch‐rich biomass, enabling effective production of renewable bioenergy.

Development of strategies to deconstruct lignocellulosic biomass in tree species is essential for biofuels and biomaterials production. We applied a wood forming tissue-specific system in a hybrid poplar to express both PdSuSy (a sucrose synthase gene from Populus deltoides × P. euramericana that has not been functionally characterized) and HCHL (the hydroxycinnamoyl-CoA hydratase-lyase gene from Pseudomonas fluorescens, which inhibits lignin polymerization in Arabidopsis). The PdSuSy-HCHL overexpression poplars correspondingly driven by the promoters of Arabidopsis AtCesA7 and AtC4H resulted in a significant increase in cellulose (> 8%), xylan (> 12%) and glucose (> 29%) content, accompanying a reduction in galacturonic acid (> 36%) content, compared to control plants. The saccharification efficiency of these overexpression poplars was dramatically increased by up to 27%, but total lignin content was unaffected. These transgenic poplars showed inhibited growth characteristics, including > 16% reduced plant height, > 10% reduced number of internodes, and > 18% reduced fresh weight after growth of 4 months, possibly due to relatively low expression of HCHL in secondary xylem. Our results demonstrate the structural complexity and interaction of the cell wall polymers in wood tissue and outline a potential method to increase biomass saccharification in woody species.

The mucilage surrounding hydrated Arabidopsis thaliana seeds is a specialized extracellular matrix composed mainly of the pectic polysaccharide rhamnogalacturonan I (RG-I). Although, several genes responsible for RG-I biosynthesis have been identified, the transcriptional regulatory mechanisms controlling RG-I production remain largely unknown. Here we report that the trihelix transcription factor DE1 BINDING FACTOR 1 (DF1) is a key regulator of mucilage RG-I biosynthesis. RG-I biosynthesis is significantly reduced in loss-of-function mutants of DF1. DF1 physically interacts with GLABRA2 (GL2) and both proteins transcriptionally regulate the expression of the RG-I biosynthesis genes MUCILAGE MODIFIED 4 (MUM4) and GALACTURONOSYLTRANSFERASE-LIKE5 (GATL5). Through chromatin immunoprecipitation-quantitative PCR and transcriptional activation assays, we uncover a cooperative mechanism of the DF1–GL2 module in activating MUM4 and GATL5 expression, in which DF1 binds to the promoters of MUM4 and GATL5 through interacting with GL2 and facilitates the transcriptional activity of GL2. The expression of DF1 and GL2 is directly regulated by TRANSPARENT TESTA GLABRA2 (TTG2) and, in turn, DF1 directly represses the expression of TTG2. Taken together, our data reveal that the transcriptional regulation of mucilage RG-I biosynthesis involves a regulatory module, comprising DF1, GL2, and TTG2.

Mutations in both ArabidopsisXyloglucan Xylosyltransferase1 (XXT1) and Xyloglucan Xylosyltransferase2 (XXT2) result in the absence of the major hemicellulose, xyloglucan (XyG). The growth physiology, tensile strength, and cell wall compositions of etiolated hypocotyls of xxt1/xxt2 double and xxt1/xxt2/xxt5 triple mutants were characterized and compared with those of mur2 and mur3 mutations that alter the side group composition of XyG. Wild type and mur2 demonstrated normal elongation growth, whereas the xxt double and triple mutants and mur3 showed reduced elongation and cell swelling at the base of the hypocotyl at the later stages of elongation. Whereas mur3 hypocotyls had reduced tensile strength, the xxt double and triple mutants had similar tensile strengths as wild type, but reached breaking points at lower strains. Loss of XyG in hypocotyls and rosette leaf cell walls was associated with specific increases in the relative abundance of homogalacturonan and the hemicellulose glucomannan, but not arabinoxylan. The increased homogalacturonan and glucomannan abundance did not result from enhanced expression of their polymer synthases. An analysis of published transcriptomic data indicated that genes encoding some members of the expansin, xyloglucan endotransglucosylase/hydrolase, arabinogalactan-protein, and structural protein families were upregulated in the xxt mutants. We hypothesize that an as yet unknown mechanism exists by which pectin and hemicellulose abundance can be altered other than by increased rates of biosynthesis to maintain a structurally sound cell wall.

Wood (secondary xylem) formation in tree species is dependent on auxin-mediated vascular cambium activity in stems. However, the complex regulatory networks underlying xylem formation remain elusive. Xylem development in Populus was characterized based on microscopic observations of stem sections in transgenic plants. Transcriptomic, quantitative real-time PCR, chromatin immunoprecipitation PCR, and electrophoretic mobility shift assay analyses were conducted to identify target genes involved in xylem development. Yeast two-hybrid, pull-down, bimolecular fluorescence complementation, and co-immunoprecipitation assays were used to validate protein-protein interactions. PaC3H17 and its target PaMYB199 were found to be predominantly expressed in the vascular cambium and developing secondary xylem in Populus stems and play opposite roles in controlling cambial cell proliferation and secondary cell wall thickening through an overlapping pathway. Further, PaC3H17 interacts with PaMYB199 to form a complex, attenuating PaMYB199-driven suppression of its xylem targets. Exogenous auxin application enhances the dual control of the PaC3H17-PaMYB199 module during cambium division, thereby promoting secondary cell wall deposition. Dual regulation of xylem formation by an auxin-mediated PaC3H17-PaMYB199 module represents a novel regulatory mechanism in Populus, increasing our understanding of the regulatory networks involved in wood formation.

Homogalacturonan (HG), a component of pectin, is synthesized in the Golgi apparatus in its fully methylesterified form. It is then secreted into the apoplast where it is typically de-methylesterified by pectin methylesterases (PME). Secretion and de-esterification are critical for normal pectin function, yet the underlying transcriptional regulation mechanisms remain largely unknown. Here, we uncovered a mechanism that fine-tunes the degree of HG de-methylesterification (DM) in the mucilage that surrounds Arabidopsis thaliana seeds. We demonstrate that the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor (TF) ERF4 is a transcriptional repressor that positively regulates HG DM. ERF4 expression is confined to epidermal cells in the early stages of seed coat development. The adhesiveness of the erf4 mutant mucilage was decreased as a result of an increased DM caused by a decrease in PME activity. Molecular and genetic analyses revealed that ERF4 positively regulates HG DM by suppressing the expression of three PME INHIBITOR genes (PMEIs) and SUBTILISIN-LIKE SERINE PROTEASE 1.7 (SBT1.7). ERF4 shares common targets with the TF MYB52, which also regulates pectin DM. Nevertheless, the erf4-2 myb52 double mutant seeds have a wild-type mucilage phenotype. We provide evidence that ERF4 and MYB52 regulate downstream gene expression in an opposite manner by antagonizing each other’s DNA-binding ability through a physical interaction. Together, our findings reveal that pectin DM in the seed coat is fine-tuned by an ERF4–MYB52 transcriptional complex.

Numerous proteins involved in cellulose biosynthesis and assembly have been functionally characterized. Nevertheless, we have a limited understanding of the mechanisms underlying the transcriptional regulation of the genes that encode these proteins. Here, we report that HOMEODOMAIN GLABROUS2 (HDG2), a Homeobox-Leucine Zipper IV transcription factor, regulates cellulose biosynthesis in Arabidopsis (Arabidopsis thaliana) seed coat mucilage. HDG2 is a transcriptional activator with the transactivation domain located within its Leucine-Zipper domain. Transcripts of HDG2 were detected specifically in seed coat epidermal cells with peak expression at 10 d postanthesis. Disruptions of HDG2 led to seed coat mucilage with aberrant morphology due to a reduction in its crystalline cellulose content. Electrophoretic mobility shift and yeast one-hybrid assays, together with chromatin immunoprecipitation and quantitative PCR, provided evidence that HDG2 directly activates CELLULOSE SYNTHASE5 (CESA5) expression by binding to the L1-box cis-acting element in its promoter. Overexpression of CESA5 partially rescued the mucilage defects of hdg2-3. Together, our data suggest that HDG2 directly activates CESA5 expression and thus is a positive regulator of cellulose biosynthesis in seed coat mucilage.

Xyloglucan (XyG) is the predominant hemicellulose in the primary cell walls of most dicotyledonous plants. Current models of these walls predict that XyG interacts with cellulose microfibrils to provide the wall with the rigidity and strength necessary to maintain cell integrity. Remodeling of this network is required to allow cell elongation and plant growth. In this study, homologs of Arabidopsis thaliana MURUS3 (MUR3), which encodes a XyG-specific galactosyltransferase, were obtained from Brassica rapa (BrMUR3) to Brassica oleracea (BoMUR3). Genetic complementation showed that BrMUR3 and BoMUR3 rescue the phenotypic defects of the mur3-3 mutant. Xyloglucan subunit composition analysis provided evidence that BrMUR3 and BoMUR3 encode a galactosyltransferase, which transfers a galactose residue onto XyG chains. The detection of XXFG and XLFG XyG subunits (restoration of fucosylated side chains) in mur3-3 mutants overexpressing BrMUR3 or BoMUR3 show that MUR3 from Brassica to Arabidopsis are comparable as they add Gal to the third xylosyl residue of the XXXG subunit. Our results provide additional information for functional dissection and evolutionary analysis of MUR3 genes derived from brassicaceous species.

Necrotrophic pathogens such as Botrytis cinerea cause significant crop yield losses. Plant CCCH proteins play important roles in pathogen resistance responses. However, the CCCH-mediated defense mechanisms against necrotrophic pathogens are unclear. Here, we report that the Arabidopsis CCCH protein C3H14 positively regulates basal defense against B. cinerea mainly by WRKY33 signaling. Simultaneous mutation of C3H14 and its paralog C3H15 resulted in enhanced susceptibility to B. cinerea, while C3H14 or C3H15 overexpression lines exhibited reduced susceptibility. A large number of differentially expressed genes (DEGs) were present in the c3h14c3h15 double mutant and C3H14 overexpression plants compared with wild-type plants at 24 hr post infection. These DEGs covered over one third of B. cinerea-responsive WRKY33 targets, including genes involved in jasmonic acid (JA)/ethylene (ET) signaling, and camalexin biosynthesis. Genetic analysis indicated that C3H14 mainly depended on WRKY33 to modulate defense against B. cinerea. Moreover, C3H14 activated the WRKY33-ORA59 and -PAD3 cascades to correspondingly control JA/ET- and camalexin-mediated defense responses. However, C3H14 was essential for B. cinerea-induced production of 12-oxo-phytodienoic acid and it also directly mediated ORA59-dependent JA/ET signaling after infection. Therefore, C3H14 may act as a novel transcriptional regulator of the WRKY33-mediated defense pathway.

The Arabidopsis (Arabidopsis thaliana) seed coat mucilage is a specialized cell wall with pectin as its major component. Pectin is synthesized in the Golgi apparatus with homogalacturonan fully methylesterified, but it must undergo de-methylesterification by pectin methylesterase (PME) after being secreted into the cell wall. This reaction is critical for pectin maturation, but the mechanisms of its transcriptional regulation remain largely unknown. Here, we show that the Arabidopsis ERF4 transcription factor positively regulates pectin de-methylesterification during seed development and directly suppresses the expression of PME INHIBITOR13 (PMEI13), 14, 15 and SUBTILISIN-LIKE SERINE PROTEASE 1.7 (SBT1.7). The erf4 mutant seeds showed repartitioning of mucilage between soluble and adherent layers as a result of decreased PME activity and increased degree of pectin methylesterification. ERF4 physically associates with and antagonizes MYB52 in activating PMEI6, 14 and SBT1.7 and MYB52 also antagonizes ERF4 activity in the regulation of downstream targets. Gene expression studies revealed that ERF4 and MYB52 have opposite effects on pectin de-methylesterification. Genetic analysis indicated that the erf4-2 myb52 double mutant seeds show mucilage phenotype similar to wild-type. Taken together, this study demonstrates that ERF4 and MYB52 antagonize each other’s activity to maintain the appropriate degree of pectin methylesterification, expanding our understanding of how pectin de-methylesterification is fine-tuned by the ERF4-MYB52 transcriptional complex in the seed mucilage.One-sentence summaryArabidopsis ERF4 and MYB52 transcription factors interact and play antagonistic roles in regulating homogalacturonan de-methylesterification related genes in the seed coat mucilage.

The polysaccharide pectin is a major component of the plant cell wall. The pectic glycan homogalacturonan (HG) is a proportionally small but important component of a specialized seed cell wall called mucilage. HG is synthesized in a highly methylesterified form, and, following secretion, is de-methylesterified by pectin methylesterases (PMEs). The degree of methylesterification of HG determines the structural and functional properties of pectin, but how methylesterification is regulated remains largely unknown. Here, we identified two BEL1-Like homeodomain (BLH) transcription factors, BLH2 and BLH4, as positive regulators of HG de-methylesterification in Arabidopsis (Arabidopsis thaliana) seed coat mucilage. BLH2 and BLH4 were significantly expressed in mucilage secretory cells during seed mucilage production. BLH2 and BLH4 single mutants exhibited no obvious mucilage phenotype, but the blh2 blh4 double mutant displayed significantly reduced mucilage adherence to the seed. Reduced mucilage adherence in blh2 blh4 was caused by decreased PME activity in the seed coat, which increased the degree of methylesterification of HG in mucilage. The expression of several PME metabolism-related genes, including PME58, PECTIN METHYLESTERASE INHIBITOR6, SEEDSTICK, and MYB52 was significantly altered in blh2 blh4 seeds. BLH2 and BLH4 directly activated PME58 expression by binding to its TGACAGGT cis-element. Moreover, pme58 mutants exhibited reduced mucilage adherence similar to that of blh2 blh4, and the blh2 blh4 pme58 triple mutant exhibited no additional mucilage adherence defects. Furthermore, overexpression of PME58 in blh2 blh4 rescued the mucilage adherence defect. Together, these results demonstrate that BLH2 and BLH4 redundantly regulate de-methylesterification of HG in seed mucilage by directly activating PME58.

As a major hemicellulose component of plant cell walls, xylans play a determining role in maintaining the wall structure. However, the mechanisms of transcriptional regulation of xylan biosynthesis remain largely unknown. Arabidopsis seed mucilage represents an ideal system for studying polysaccharide biosynthesis and modifications of plant cell walls. Here, we identify KNOTTED ARABIDOPSIS THALIANA 7 (KNAT7) as a positive transcriptional regulator of xylan biosynthesis in seed mucilage. The xylan content was significantly reduced in the mucilage of the knat7-3 mutant and this was accompanied by significantly reduced expression of the xylan biosynthesis-related genes IRREGULAR XYLEM 14 (IRX14) and MUCILAGE MODIFIED 5/MUCILAGE-RELATED 21 (MUM5/MUCI21). Electrophoretic mobility shift assays, yeast one-hybrid assays, and chromatin immunoprecipitation with quantitative PCR verified the direct binding of KNAT7 to the KNOTTED1 (KN1) binding site [KBS,TGACAG(G/C)T] in the promoters of IRX7, IRX14, and MUM5/MUCI21 in vitro, in vivo, and in planta. Furthermore, KNAT7 directly activated the expression of IRX14 and MUM5/MUCI21 in transactivation assays in mesophyll protoplasts, and overexpression of IRX14 or MUM5/MUCI21 in knat7-3 partially rescued the defects in mucilage adherence. Taken together, our results indicate that KNAT7 positively regulates xylan biosynthesis in seed-coat mucilage via direct activation of the expression of IRX14 and MUM5/MUCI21.

Apart from providing the much-needed strength, plant cell walls define the shape, size and function of cells. As such, there is a constant change in the cell wall dynamics. These are facilitated by various enzymes and proteins. Expansins are a typical example of those cell wall proteins that are involved in cell wall modifications underlying many plant developmental and physiological processes. In this work, we investigated the role of NtEXPA11 gene in tobacco by generating transgenic plants ectopically expressing NtEXPA11 under the control of CaMV35S promoter. Gene expression analysis revealed that although this gene was present in all the studied tissues in WT plants, its transcript levels were highest in the stems, flowers and leaves and lowest in the roots. Following its overexpressing in tobacco, the NtEXPA11-OX plants exhibited an enhanced growth phenotype. Compared to WT plants, these plants demonstrated an increased growth rate which was characterized by a vigorous root system as well as an accelerated growth rate during their early developmental stages. NtEXPA11-OX plants also developed significantly bigger leaves and internode lengths. They exhibited a 57% increase (NtEXPA11-2) and 98% increase (NtEXPA11-19) in leaf area when grown on MS media. Most interestingly, NtEXPA11-OX plants had significantly bigger pith and parenchyma cells compared to their WT counterparts. Furthermore, we noted that NtEXPA11 plays an important role in plant adaptation to stresses as indicated by the improved tolerance to drought and salt stress of the NtEXPA11-OX plants compared to the WT plants.

Virus-induced gene silencing (VIGS) is a transient based reverse genetic tool used to elucidate the function of novel gene in N. benthamiana. In current study, 14 UDP-D-glucuronate 4-epimerase (GAE) family members were identified and their gene structure, phylogeny and expression pattern were analyzed. VIGS system was optimized for the functional characterization of NbGAE6 homologous genes in N. benthamiana. Whilst the GAE family is well-known for the interconversion of UDP-D-GlcA and UDP-D-GalA during pectin synthesis. Our results revealed that the downregulation of these genes significantly reduced the amount of GalA in the homogalacturunan which is the major component of pectin found in primary cell wall. Biphenyl assay and high performance liquid chromatography analysis (HPLC) depicted that the level of 'GalA' monosaccharide reduced to 40-51% in VIGS plants as compared to the wild type plants. Moreover, qRT-PCR also confirmed the downregulation of the NbGAE6 mRNA in VIGS plants. In all, this is the first comprehensive study of the optimization of VIGS system for the provision of rapid silencing of GAE family members in N. benthamiana, eliminating the need of stable transformants.

Cell walls are basically complex with dynamic structures that are being involved in several growth and developmental processes, as well as responses to environmental stresses and the defense mechanism. Pectin is secreted into the cell wall in a highly methylesterified form. It is able to perform function after the de-methylesterification by pectin methylesterase (PME). Whereas, the pectin methylesterase inhibitor (PMEI) plays a key role in plant cell wall modification through inhibiting the PME activity. It provides pectin with different levels of degree of methylesterification to affect the cell wall structures and properties. The PME activity was analyzed in six tissues of Sorghum bicolor, and found a high level in the leaf and leaf sheath. PMEI families have been identified in many plant species. Here, a total of 55 pectin methylesterase inhibitor genes (PMEIs) were identified from S. bicolor whole genome, a more detailed annotation of this crop plant as compared to the previous study. Chromosomal localization, gene structures and sequence characterization of the PMEI family were analyzed. Moreover, cis-acting elements analysis revealed that each PMEI gene was regulated by both internal and environmental factors. The expression patterns of each PMEI gene were also clustered according to expression pattern analyzed in 47 tissues under different developmental stages. Furthermore, some SbPMEIs were induced when treated with hormonal and abiotic stress. Taken together, these results laid a strong foundation for further study of the functions of SbPMEIs and pectin modification during plant growth and stress responses of cereal.

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

Miscanthus is a promising lignocellulosic bioenergy crop for bioethanol production. To identify candidate genes and regulation networks involved in secondary cell wall (SCW) development in Miscanthus, we performed de novo transcriptome analysis of a developing internode. According to the histological and in-situ histochemical analysis, an elongating internode of M. lutarioriparius can be divided into three distinct segments, the upper internode (UI), middle internode (MI) and basal internode (BI), each representing a different stage of SCW development. The transcriptome analysis generated approximately 300 million clean reads, which were de novo assembled into 79,705 unigenes. Nearly 65% of unigenes was annotated in seven public databases. Comparative profiling among the UI, MI and BI revealed four distinct clusters. Moreover, detailed expression profiling was analyzed for gene families and transcription factors (TFs) involved in SCW biosynthesis, assembly and modification. Based on the co-expression patterns, putative regulatory networks between TFs and SCW-associated genes were constructed. The work provided the first transcriptome analysis of SCW development in M. lutarioriparius. The results obtained provide novel insights into the biosynthesis and regulation of SCW in Miscanthus. In addition, the genes identified represent good candidates for further functional studies to unravel their roles in SCW biosynthesis and modification.

The homeodomain-leucine zipper (HD-ZIP) transcription factors are important regulators in various developmental processes and responses to environmental stimuli. Currently, little information is available for HD-ZIP gene family in Medicago truncatula. Here we perform a genome-wide analysis of HD-ZIP gene family in M. truncatula. Totally 52 M. truncatula HD-ZIPs (MtHDZs) were identified and classified into four distinctive subfamilies (I to IV). Members clustered in the same subfamily shared similar gene structure and protein motifs. Fifty-one MtHDZs were non-evenly distributed onto eight chromosomes. Segmental duplication and purifying selection mainly contributed to the expansion and retention of M. truncatula HD-ZIP gene family. Expression profiling using the publicly available microarray data revealed that MtHDZ genes exhibited distinctive tissue-specific patterns and divergent responses to drought and salt stresses. In addition, the expression profile between each paralogous pair diverged differentially. Our results identified potential targets for the genetic improvement of abiotic stress tolerance in Medicago.

The Cys2His2 (C2H2)-type zinc-finger protein (ZFP) family, which includes 176 members in Arabidopsis thaliana, is one of the largest families of putative transcription factors in plants. Of the Arabidopsis ZFP members, only 33 members are conserved in other eukaryotes, with 143 considered to be plant specific. C2H2-type ZFPs have been extensively studied and have been shown to play important roles in plant development and environmental stress responses by transcriptional regulation. The ethylene-responsive element binding-factor-associated amphiphilic repression (EAR) domain (GCC box) has been found to have a critical role in the tolerance response to abiotic stress. Many of the plant ZFPs containing the EAR domain, such as AZF1/2/3, ZAT7, ZAT10, and ZAT12, have been shown to function as transcriptional repressors. In this review, we mainly focus on the C1-2i subclass of C2H2 ZFPs and summarize the latest research into their roles in various stress responses. The role of C2H2-type ZFPs in response to the abiotic and biotic stress signaling network is not well explained, and amongst them, C1-2i is one of the better-characterized classifications in response to environmental stresses. These studies of the C1-2i subclass ought to furnish the basis for future studies to discover the pathways and receptors concerned in stress defense. Research has implied possible protein-protein interactions between members of C1-2i under various stresses, for which we have proposed a hypothetical model.

Peanut seeds are ideal bioreactors for the production of foreign recombinant proteins and/or nutrient metabolites. Seed-Specific Promoters (SSPs) are important molecular tools for bioreactor research. However, few SSPs have been characterized in peanut seeds. The mining of Seed-Specific Candidate Genes (SSCGs) is a prerequisite for promoter cloning. Here, we described an approach for the genome-wide mining of SSCGs via comparative gene expression between seed and nonseed tissues. Three hundred thirty-seven SSCGs were ultimately identified, and the top 108 SSCGs were characterized. Gene Ontology (GO) analysis revealed that some SSCGs were involved in seed development, allergens, seed storage and fatty acid metabolism. RY REPEAT and GCN4 motifs, which are commonly found in SSPs, were dispersed throughout most of the promoters of SSCGs. Expression pattern analysis revealed that all 108 SSCGs were expressed specifically or preferentially in the seed. These results indicated that the promoters of the 108 SSCGs may perform functions in a seed-specific and/or seed-preferential manner. Moreover, a novel SSP was cloned and characterized from a paralogous gene of SSCG29 from cultivated peanut. Together with the previously characterized SSP of the SSCG5 paralogous gene in cultivated peanut, these results implied that the method for SSCG identification in this study was feasible and accurate. The SSCGs identified in this work could be widely applied to SSP cloning by other researchers. Additionally, this study identified a low-cost, high-throughput approach for exploring tissue-specific genes in other crop species.

Sorghum (Sorghum bicolor) is an important bioenergy crop. Its biomass mainly consists of the cellulosic and non-cellulosic polysaccharides, both which can be converted to biofuels. The biosynthesis of non-cellulosic polysaccharides involves several glycosyltransferases (GT) families including GT47. However, there was no systemic study on GT47 family in sorghum to date. Here, we identified 39 sorghum GT47 family members and showed the functional divergences of MURUS3 (MUR3) homologs. Sorghum GT47 proteins were phylogenetically clustered into four distinct subfamilies. Within each subfamily, gene structure was relatively conserved between the members. Ten gene pairs were identified from the 39 GT47 genes, of which two pairs might be originated from tandem duplication. 25.6% (10/39) of sorghum GT47 genes were homologous to Arabidopsis MUR3, a xyloglucan biosynthesis gene in primary cell walls. SbGT47_2, SbGT47_7, and SbGT47_8, three most homologous genes of MUR3, exhibited different tissue expression patterns and were selected for complementation into Arabidopsis mur3-3. Physiological and cell wall analyses showed that SbGT47_2 and SbGT47_7 may be two functional xyloglucan galactosyltransferases in sorghum. Further studies found that MUR3-like genes are widely present in the seed plants but not in the chlorophytic alga Chlamydomonas reinhardtii. Our results provide novel information for evolutionary analysis and functional dissection of sorghum GT47 family members.

This review archives the achievements made in the last two decades and presents a brief outline of some significant factors influencing the Agrobacterium-mediated transformation of Sorghum bicolor. Recently, progress in successful transformation has been made for this particular monocot crop through direct DNA delivery method and indirect method via Agrobacterium. However, lower transformation rate still proved to be a bottleneck in genetic modification of sorghum. An efficient Agrobacterium transformation system could be attained by optimizing the preliminary assays, comprising of explant source, growth media, antibiotics, Agrobacterium strains and agro-infection response of callus. The selection of competent strains for genetic transformation is also one of the key factors of consideration. Successful transformation is highly dependent on genome configuration of selected cultivar, where non-tannin genotype proved the best suited. Immature embryos from the field source have higher inherent adaptation chances than that of the greenhouse source. A higher concentration of Agrobacterium may damage the explant source. Utilization of anti-necrotic treatments and optimized tissue culture timeframe are the adequate strategies to lower down the effect of phenolic compounds. Appropriate selection of culture media vessels at different stages of tissue culture may also assist in a constructive manner. In conclusion, some aspects such as culture environment with medium composition, explant sources, and genotypes play an indispensable role in successful Agrobacterium-mediated sorghum transformation system.

Biochar amendment has been proposed to be as a promising means to improve soil properties and enhance plant productivity in different types of soils. However, biochar application in the improvement of plant productivity in saline-alkali soils has received less attention. In the present study, we carried out a pot experiment in greenhouse to evaluate the effect of biochar amendment on the growth of Miscanthus lutarioriparius, a promising bioenergy crop, in a coastal saline-alkali soil during a 92-day growth period. The biochar was added at 0, 1.0%, 2.0%, 2.5%, 5.0%, and 10.0% (w/w) levels in a random block design with three replicates. The results showed that biochar applications at 2.0% and 2.5% levels significantly promoted Miscanthus growth by ameliorating the physicochemical and biological properties of the saline-alkali soil. The soil moisture, soil organic matter (SOM), nitrate nitrogen (NO3−-N), available phosphorus (Olsen-P), and concentrations of potassium (K+), calcium (Ca2+) and magnesium (Mg2+) ions were significantly increased, while soil pH, bulk density, ammonium nitrogen (NH4+-N), sodium (Na+) ion, and exchangeable sodium percentage (ESP) contents were substantially reduced. In addition, the soil bacterial community structure and diversity were substantially shifted after the biochar amendment, which in turn affected soil N and P cycling and improved soil physicochemical properties. Meanwhile, biochar application considerably mitigated the salinity stress to Miscanthus plants by activating anti-oxidative enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), therefore enhancing the scavenging capability of reactive oxygen species (ROS). Together, our results indicated that biochar amendment effectively promoted Miscanthus growth by ameliorating soil physical, chemical and biological properties, and alleviated salt stress injuries to plants in a coastal saline-alkali soil.

Large quantities of mucilage are synthesized in seed coat epidermis cells during seed coat differentiation. This process is an ideal model system for the study of plant cell wall biosynthesis and modifications. In this study, we show that mutation in Irregular Xylem 7 (IRX7) results in a defect in mucilage adherence due to reduced xylan biosynthesis. IRX7 was expressed in the seeds from 4 days post-anthesis (DPA) to 13 DPA, with the peak of expression at 13 DPA. The seed coat epidermis cells of irx7 displayed no aberrant morphology during differentiation, and these cells synthesized and deposited the same amount of mucilage as did wild type (WT) cells. However, the distribution of the water-soluble vs. adherent mucilage layers was significantly altered in irx7 compared to the WT. Both the amount of xylose and the extent of glycosyl linkages of xylan was dramatically decreased in irx7 water-soluble and adherent mucilage compared to the WT. The polymeric structure of water-soluble mucilage was altered in irx7, with a total loss of the higher molecular weight polymer components present in the WT. Correspondingly, whole-seed immunolabeling assays and dot-immunoassays of extracted mucilage indicated dramatic changes in rhamnogalacturonan I (RG I) and xylan epitopes in irx7 mucilage. Furthermore, the crystalline cellulose content was significantly reduced in irx7 mucilage. Taken together, these results indicate that xylan synthesized by IRX7 plays an essential role in maintaining the adhesive property of seed coat mucilage, and its structural role is potentially implemented through its interaction with cellulose.

Expansins are pH-dependent cell wall loosening proteins which form a large family in plants. They have been shown to be involved in various developmental processes and been implicated in enabling plants' ability to absorb nutrients from the soil as well as conferring biotic and abiotic stress resistances. It is therefore clear that they can be potential targets in genetic engineering for crop improvement. Tobacco (Nicotiana tabacum) is a major crop species as well as a model organism. Considering that only a few tobacco expansins have been studied, a genome-wide analysis of the tobacco expansin gene family is necessary. In this study, we identified 52 expansins in tobacco, which were classified into four subfamilies: 36 NtEXPAs, 6 NtEXPBs, 3 NtEXLAs and 7 NtEXLBs. Compared to other species, the NtEXLB subfamily size was relatively larger. Phylogenetic analysis showed that the 52 tobacco expansins were divided into 13 subgroups. Gene structure analysis revealed that genes within subfamilies/subgroups exhibited similar characteristics such as gene structure and protein motif arrangement. Whole-genome duplication and tandem duplication events may have played important roles in the expanding of tobacco expansins. Cis-Acting element analysis revealed that each expansin gene was regulated or several expansin genes were co-regulated by both internal and environmental factors. 35 of these genes were identified as being expressed according to a microarray analysis. In contrast to most NtEXPAs which had higher expression levels in young organs, NtEXLAs and NtEXLBs were preferentially expressed in mature or senescent tissues, suggesting that they might play different roles in different organs or at different developmental stages. As the first step towards genome-wide analysis of the tobacco expansin gene family, our work provides solid background information related to structure, evolution and expression as well as regulatory cis-acting elements of the tobacco expansins. This information will provide a strong foundation for cloning and functional exploration of expansin genes in tobacco.

Highlight Xylan synthesized by IRX14 maintains seed coat mucilage structure potentially through altering the crystallization and organization of cellulose., During differentiation, the Arabidopsis seed coat epidermal cells synthesize and secrete large quantities of pectinaceous mucilage into the apoplast, which is then released to encapsulate the seed upon imbibition. In this study, we showed that mutation in Irregular Xylem 14 (IRX14) led to a mucilage cohesiveness defect due to a reduced xylan content. Expression of IRX14 was detected specifically in the seed coat epidermal cells, reaching peak expression at 13 days post-anthesis (DPA) when the accumulation of mucilage polysaccharides has ceased. Sectioning of the irx14-1 seed coat revealed no visible structural change in mucilage secretory cell morphology. Although the total amount of mucilage was comparable with the wild type (WT), the partition between water-soluble and adherent layers was significantly altered in irx14-1, with redistribution from the adherent layer to the water-soluble layer. The monosaccharide composition analysis revealed that xylose content was significantly reduced in irx14-1 water-soluble and adherent mucilage compared with the WT. The macromolecular characteristics of the water-soluble mucilage were modified in irx14-1 with a loss of the larger polymeric components. In accordance, glycome profiling and dot immunoblotting of seed mucilage using antibodies specific for rhamnogalacturonan I (RG I) and xylan confirmed the ultra-structural alterations in the irx14-1 mucilage. Meanwhile, the crystalline cellulose content was reduced in the irx14-1 mucilage. These results demonstrated that IRX14 was required for the biosynthesis of seed mucilage xylan, which plays an essential role in maintaining mucilage architecture potentially through altering the crystallization and organization of cellulose.

Key message Results from various expansin related studies have demonstrated that expansins present an opportunity to improve various crops in many different aspects ranging from yield and fruit ripening to improved stress tolerance. Abstract The recent advances in expansin studies were reviewed. Besides producing the strength that is needed by the plants, cell walls define cell shape, cell size and cell function. Expansins are cell wall proteins which consist of four sub families; α-expansin, β-expansin, expansin-like A and expansin-like B. These proteins mediate cell wall loosening and they are present in all plants and in some microbial organisms and other organisms like snails. Decades after their initial discovery in cucumber, it is now clear that these small proteins have diverse biological roles in plants. Through their ability to enable the local sliding of wall polymers by reducing adhesion between adjacent wall polysaccharides and the part they play in cell wall remodeling after cytokinesis, it is now clear that expansins are required in almost all plant physiological development aspects from germination to fruiting. This is shown by the various reports from different studies using various molecular biology approaches such as gene achieve these many roles through their non-enzymatic wall loosening ability. This paper reviews and summarizes some of the reported functions of expansins and outlines the potential uses of expansins in crop improvement programs.