MsHDZ23, a Novel Miscanthus HD-ZIP Transcription Factor, Participates in Tolerance to Multiple Abiotic Stresses.

The homeodomain-leucine zipper (HD-ZIP) transcription factors, representing one of the largest plant-specific superfamilies, play important roles in the response to various abiotic stresses. However, the functional roles of HD-ZIPs in abiotic stress tolerance and the underlying mechanisms remain relatively limited in Miscanthus sinensis. In this study, we isolated an HD-ZIP TF gene, MsHDZ23, from Miscanthus and ectopically expressed it in Arabidopsis. Transcriptome and promoter analyses revealed that MsHDZ23 responded to salt, alkali, and drought treatments. The overexpression (OE) of MsHDZ23 in Arabidopsis conferred higher tolerance to salt and alkali stresses compared to wild-type (WT) plants. Moreover, MsHDZ23 was able to restore the hb7 mutant, the ortholog of MsHDZ23 in Arabidopsis, to the WT phenotype. Furthermore, MsHDZ23-OE lines exhibited significantly enhanced drought stress tolerance, as evidenced by higher survival rates and lower water loss rates compared to WT. The improved drought tolerance may be attributed to the significantly smaller stomatal aperture in MsHDZ23-OE lines compared to WT. Furthermore, the accumulation of the malondialdehyde (MDA) under abiotic stresses was significantly decreased, accompanied by dramatically enhanced activities in several antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the transgenic plants. Collectively, these results demonstrate that MsHDZ23 functions as a multifunctional transcription factor in enhancing plant resistance to abiotic stresses.

Fine-tuning brassinosteroid biosynthesis via 3'UTR-dependent decay of CPD mRNA modulates wood formation in Populus.

Brassinosteroids (BRs) are plant hormones that regulate wood formation in trees. Currently, little is known about the post-transcriptional regulation of BR synthesis. Here, we show that during wood formation, fine-tuning BR synthesis requires 3'UTR-dependent decay of Populus CONSTITUTIVE PHOTOMORPHOGENIC DWARF 1 (PdCPD1). Overexpression of PdCPD1 or its 3' UTR fragment resulted in a significant increase of BR levels and inhibited secondary growth. In contrast, transgenic poplars repressing PdCPD1 3' UTR expression displayed moderate levels of BR and promoted wood formation. We show that the Populus GLYCINE-RICH RNA-BINDING PROTEIN 1 (PdGRP1) directly binds to a GU-rich element in 3' UTR of PdCPD1, leading to its mRNA decay. We thus provide a post-transcriptional mechanism underlying BRs synthesis during wood formation, which may be useful for genetic manipulation of wood biomass in trees.

Efficient Accumulation of Amylopectin and Its Molecular Mechanism in the Submerged Duckweed Mutant.

Large-scale use of fossil fuels has brought about increasingly serious problems of environmental pollution, development and utilization of renewable energy is one of the effective solutions. Duckweed has the advantages of fast growth, high starch content and no occupation of arable land, so it is a promising starchy energy plant. A new submerged duckweed mutant (sub-1) with abundant starch accumulation was obtained, whose content of amylopectin accounts for 84.04% of the starch granules. Compared with the wild type (Lemna aequinoctialis), the branching degree of starch in sub-1 mutant was significantly increased by 19.6%. Chain length DP 6-12, DP 25-36 and DP > 36 of amylopectin significantly decreased, while chain length DP 13-24 significantly increased. Average chain length of wild-type and sub-1 mutant starches were greater than DP 22. Moreover, the crystal structure and physical properties of starch have changed markedly in sub-1 mutant. For example, the starch crystallinity of sub-1 mutant was only 8.94%, while that of wild-type was 22.3%. Compared with wild type, water solubility of starch was significantly reduced by 29.42%, whereas swelling power significantly increased by 97.07% in sub-1 mutant. In order to further analyze the molecular mechanism of efficient accumulation of amylopectin in sub-1 mutant, metabolome and transcriptome were performed. The results showed that glucose accumulated in sub-1 mutant, then degradation of starch to glucose mainly depends on α-amylase. At night, the down-regulated ß-amylase gene resulted in the inhibition of starch degradation. The starch and sucrose metabolism pathways were significantly enriched. Up-regulated expression of SUS, AGPase2, AGPase3, PYG, GPI and GYS provide sufficient substrate for starch synthesis in sub-1 mutant. From the 0H to 16H light treatment, granule-bound starch synthase (GBSS1) gene was inhibited, on the contrary, the starch branching enzyme (SBE) gene was induced. Differential expression of GBSS1 and SBE may be an important reason for the decrease ratio of amylose/amylopectin in sub-1 mutant. Taken together, our results indicated that the sub-1 mutant can accumulate the amylopectin efficiently, potentially through altering the differential expression of AGPase, GBSS1, SBE, and BAM. This study also provides theoretical guidance for creating crop germplasm with high amylopectin by means of synthetic biology in the future.

The evolving views of the simplest pectic polysaccharides: homogalacturonan.

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.

Phosphorylation-mediated inactivation of C3H14 by MPK4 enhances bacterial-triggered immunity in Arabidopsis.

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.

Ubiquitinated DA1 negatively regulates vascular cambium activity through modulating the stability of WOX4 in Populus.

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.

Using CRISPR-Cas9 Technology to Eliminate Xyloglucan in Tobacco Cell Walls and Change the Uptake and Translocation of Inorganic Arsenic.

Xyloglucan is a quantitatively major polysaccharide in the primary cell walls of flowering plants and has been reported to affect plants' ability to tolerate toxic elements. However, it is not known if altering the amounts of xyloglucan in the wall influences the uptake and translocation of inorganic arsenic (As). Here, we identified two Nicotiana tabacum genes that encode xyloglucan-specific xylosyltransferases (XXT), which we named NtXXT1 and NtXXT2. We used CRISPR-Cas9 technology to generate ntxxt1, ntxxt2, and ntxxt1/2 mutant tobacco plants to determine if preventing xyloglucan synthesis affects plant growth and their ability to accumulate As. We show that NtXXT1 and NtXXT2 are required for xyloglucan biosynthesis because no discernible amounts of xyloglucan were present in the cell walls of the ntxxt1/2 double mutant. The tobacco double mutant (ntxxt1/2) and the corresponding Arabidopsis mutant (atxxt1/2) do not have severe growth defects but do have a short root hair phenotype and a slow growth rate. This phenotype is rescued by overexpressing NtXXT1 or NtXXT2 in atxxt1/2. Growing ntxxt mutants in the presence of AsIII or AsV showed that the absence of cell wall xyloglucan affects the accumulation and translocation of As. Most notably, root retention of As increased substantially and the amounts of As translocated to the shoots decreased in ntxxt1/2. Our results suggest that xyloglucan-deficient plants provide a strategy for the phytoremediation of As contaminated soils.

The CCCH zinc finger protein C3H15 negatively regulates cell elongation by inhibiting brassinosteroid signaling.

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.

A DE1 BINDING FACTOR 1-GLABRA2 module regulates rhamnogalacturonan I biosynthesis in Arabidopsis seed coat mucilage.

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.

MUD1, a RING-v E3 ubiquitin ligase, has an important role in the regulation of pectin methylesterification in Arabidopsis seed coat mucilage.

Pectin is one of the major components of plant primary cell wall polysaccharides. The degree of pectin methylesterification (DM) plays an important role in the process of plant growth. However, little is known about the underlying regulatory mechanisms during the process of pectin demethylesterification. Here, we characterized mucilage defect 1 (mud1), a novel Arabidopsis thaliana mutant, which displays increased mucilage adherence resulting from increased activities of pectin methylesterases (PMEs) and decreased degree of pectin methylesterification (DM). MUD1 encodes a nuclear protein with a Really Interesting New Gene (RING)-v domain and is highly expressed in developing seed coat when seed coat mucilage starts to accumulate. We have demonstrated that MUD1 has E3 ubiquitin ligase activity in vitro. The expression of PME-related genes, including MYB52, LUH, SBT1.7, PMEI6, and PMEI14 decreased considerably in mud1. We propose that MUD1 acts as an ubiquitin ligase potentially regulating the DM of pectin by post-transcriptionally removing proteins that normally negatively regulate the level or activity of PMEs in the seed coat mucilage.

ERF4 and MYB52 transcription factors play antagonistic roles in regulating homogalacturonan de-methylesterification in Arabidopsis seed coat mucilage.

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.

HOMEODOMAIN GLABROUS2 regulates cellulose biosynthesis in seed coat mucilage by activating CELLULOSE SYNTHASE5.

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.

Identification of two functional xyloglucan galactosyltransferase homologs BrMUR3 and BoMUR3 in brassicaceous vegetables.

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.

Overexpression of NtEXPA11 modulates plant growth and development and enhances stress tolerance in tobacco.

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.

The Arabidopsis CCCH protein C3H14 contributes to basal defense against Botrytis cinerea mainly through the WRKY33-dependent pathway.

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.

KNAT7 regulates xylan biosynthesis in Arabidopsis seed-coat mucilage.

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.

Genome-wide identification and expression profiling of HD-ZIP gene family in Medicago truncatula.

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 on 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.

Identification and characterization of pectin related gene NbGAE6 through virus-induced gene silencing in Nicotiana benthamiana.

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.