Xyloglucan Endotransglycosylase Gene Research Articles

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

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

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

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

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

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

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

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

Variability in CitXET expression and XET activity in Citrus cultivar Huangguogan seedlings with differed degrees of etiolation.

Considering the known effects of xyloglucan endotransglycosylase (XET) on plant growth and development, we aimed to determine whether XETs help to regulate the growth and elongation of Huangguogan shoots and roots. We confirmed a possible role for XET during seedling etiolation. Our results revealed that the roots of etiolated seedlings (H-E) were longer than those of green seedlings (H-G). However, shoot length exhibited the opposite pattern. We also observed positive and negative effects on the xyloglucan-degrading activity of XET in the root sub-apical region and shoots of etiolated Huangguogan seedling, respectively. There was a significant down-regulation in CitXET expression in the etiolated shoots at 15 days after seed germination. On the contrary, it was significantly increased in the root sub-apical region of etiolated and multicolored seedlings at 15 days after seed germination. The XET coding sequence (i.e., CitXET) was cloned from Huangguogan seedlings using gene-specific primers. The encoded amino acid sequence was predicted by using bioinformatics-based methods. The 990-bp CitXET gene was highly homologous to other XET genes. The CitXET protein was predicted to contain 319 amino acids, with a molecular mass of 37.45 kDa and an isoelectric point of 9.05. The predicted molecular formula was C1724H2548N448O466S14, and the resulting protein included only one transmembrane structure. The CitXET secondary structure consisted of four main structures (i.e., 21% α-helix, 30.72% extended strand, 9.09% β-turn, and 39.18% random coil). Analyses involving the NCBI Conserved Domains Database (NCBI-CDD), InterPro, and ScanProsite revealed that CitXET was a member of the glycosyl hydrolase family 16 (GH16), and included the DEIDFEFLG motif. Our results indicate that the differed degrees of etiolation influenced the CitXET expression pattern and XET activity in Huangguogan seedlings. The differential changes in XET activity and CitXET expression levels in Huangguogan seedlings may influence the regulation of root and shoot development, and may be important for seedling etiolation.

Gibberellin-Regulation and Genetic Variations in Leaf Elongation for Tall Fescue in Association with Differential Gene Expression Controlling Cell Expansion.

Leaf elongation rate (LER) is an important factor controlling plant growth and productivity. The objective of this study was to determine whether genetic variation in LER for a fast-growing (‘K-31’), and a dwarf cultivar (‘Bonsai’) of tall fescue (Festuca arundinacea) and gibberellic acid (GA) regulation of LER were associated with differential expression of cell-expansion genes. Plants were treated with GA3, trinexapac-ethyl (TE) (GA inhibitor), or water (untreated control) in a hydroponic system. LER of ‘K-31’ was 63% greater than that of ‘Bonsai’, which corresponded with 32% higher endogenous GA4 content in leaf and greater cell elongation and production rates under the untreated control condition. Exogenous application of GA3 significantly enhanced LER while TE treatment inhibited leaf elongation due to GA3-stimulation or TE-inhibition of cell elongation and production rate in leaves for both cultivars. Real-time quantitative polymerase chain reaction analysis revealed that three α-expansins, one β-expansin, and three xyloglucan endotransglycosylase (XET) genes were associated with GA-stimulation of leaf elongation, of which, the differential expression of EXPA4 and EXPA7 was related to the genotypic variation in LER of two cultivars. Those differentially-expressed expansin and XET genes could play major roles in genetic variation and GA-regulated leaf elongation in tall fescue.

Co-expression of MtDREB1C and RcXET Enhances Stress Tolerance of Transgenic China Rose (Rosa chinensis Jacq.)

The xyloglucan endotransglycosylase gene RcXET of China Rose (Rosa chinensis Jacq.) and the MtDREB1C gene of Medicago truncatula Gaertn. were pyramided into the plant expression vector pBin438 and transformed into China Rose. Southern blot and Northern blot analyses showed that the heterologous gene MtDREB1C was integrated into the genome of surviving transgenic rose plants and expressed at different levels. Real-time PCR analysis demonstrated that robust expression of the congenetic gene RcXET was activated in the five surviving transgenic rose plants. The performance of the five transgenic lines under freezing and drought stress was superior to that of non-transformed controls. Thus, pyramiding of the genes MtDREB1C and RcXET in China Rose was more effective to enhance freezing and drought tolerance than untransformed controls. A positive correlation was observed between the expression of RcXET and the growth rate in contrast to the non-transgenic plants. The physiological assay showed that co-expression had greater effects on EC%, contents of proline, soluble sugar, photosynthesis rate, negative water potential, and turgor loss point than activities of POD and SOD under stress. The study also highlights the utility of a simple and rapid approach to express two or even more genes in one expression vector.

Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing.

BackgroundIn the era of DNA throughput sequencing, assembling and understanding gymnosperm mega-genomes remains a challenge. Although drafts of three conifer genomes have recently been published, this number is too low to understand the full complexity of conifer genomes. Using techniques focused on specific genes, gene models can be established that can aid in the assembly of gene-rich regions, and this information can be used to compare genomes and understand functional evolution.ResultsIn this study, gene capture technology combined with BAC isolation and sequencing was used as an experimental approach to establish de novo gene structures without a reference genome. Probes were designed for 866 maritime pine transcripts to sequence genes captured from genomic DNA. The gene models were constructed using GeneAssembler, a new bioinformatic pipeline, which reconstructed over 82 % of the gene structures, and a high proportion (85 %) of the captured gene models contained sequences from the promoter regulatory region. In a parallel experiment, the P. pinaster BAC library was screened to isolate clones containing genes whose cDNA sequence were already available. BAC clones containing the asparagine synthetase, sucrose synthase and xyloglucan endotransglycosylase gene sequences were isolated and used in this study. The gene models derived from the gene capture approach were compared with the genomic sequences derived from the BAC clones. This combined approach is a particularly efficient way to capture the genomic structures of gene families with a small number of members.ConclusionsThe experimental approach used in this study is a valuable combined technique to study genomic gene structures in species for which a reference genome is unavailable. It can be used to establish exon/intron boundaries in unknown gene structures, to reconstruct incomplete genes and to obtain promoter sequences that can be used for transcriptional studies. A bioinformatics algorithm (GeneAssembler) is also provided as a Ruby gem for this class of analyses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2490-z) contains supplementary material, which is available to authorized users.

Kiwifruit EIL and ERF Genes Involved in Regulating Fruit Ripening

Kiwifruit (Actinidia deliciosa) is a climacteric fruit sensitive to low concentrations of ethylene. To investigate the transcriptional mechanisms underlying kiwifruit ethylene response, transcription factors encoding four EIN3-Like (EILs) and 14 Ethylene Response Factors (ERFs) were cloned from kiwifruit. Expression of these transcription factors was examined during fruit development. The expression of transcripts of most AdERFs was higher during early fruit development, with the exception of AdERF3, which increased with maturity. Several AdERFs were apparently down-regulated by ethylene, as they were affected by the ethylene inhibitor 1-methylcyclopropene and by antisense suppression of ACO (for 1-aminocyclopropane-1-carboxylic acid oxidase) in the fruit. In contrast, AdEILs were constitutively expressed during fruit development and ripening. The transcription factors AdEIL2 and AdEIL3 activated transcription of the ripening-related genes AdACO1 and AdXET5 (xyloglucan endotransglycosylase gene) and, when overexpressed in Arabidopsis (Arabidopsis thaliana), stimulated ethylene production. The potential repressor AdERF9 suppressed this promoter activity. These results support a role for kiwifruit EILs and ERFs in transcriptional regulation of ripening-related genes and in the regulation of kiwifruit fruit-ripening processes.

Cloning of XET gene from Anthocephalus chinensis and its plant expression vector construction

A full-length cDNA sequence of xyloglucan endotransglycosylase gene (XET), abundantly expressed in the cambium of Anthocephalus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromosome walking. Analytical results of the DNA sequence show that a 912 bp complete open reading frame (ORF) encoded a 303-amino acid protein was in the 1205 bp full cDNA sequence. The deduced amino acid sequence of AcXET, which contained the conserved specific EIDFE catalytic site sequence to XETs was homologous to the other known XET proteins. In order to study the gene function of AcXET and obtain transgenic plants, a plant expression vector pBIAcXET was constructed by recombinating the AcXET fragment from the cloning vector pMD19AcXET and the binary vector pBI121 between the XbaI and SmaI sites. The fragment of AcXET gene was inserted between the CaMV 35S promotor and the coding region of the GUS gene in pBI121. The identification results show that the plant expression binary vector pBIAcXET was constructed successfully. These results lay the foundation for studying the molecular mechanism of AcXET gene during wood formation.

Rapid Construction of a Plant RNA Interference Expression Vector for Hairpin RNA–Mediated Targeting Using a PCR-Based Method

Here we describe a rapid and efficient PCR-mediated ligation protocol for constructing a plant RNA interference vector to express long hairpin RNA (hpRNA). In the protocol, four oligonucleotide primers were used and three rounds of PCRs performed. The product of the first PCR was used as a megaprimer for the second PCR to generate a chimeric molecule with a gene-specific sequence and a spacer spliced together. The chimeric product could be used as another megaprimer for the third PCR to ligate another gene-specific sequence to the other end of the spacer, but in the reverse orientation. Thus, within a few days, two gene-specific sequences could be ligated to a spacer in the antisense and sense orientations using the PCR-mediated ligation method, without reliance on restriction cleavage and DNA ligation. The ligated product could be inserted into the plant expression vector for plant transformation. The transcribed RNA formed hpRNA constructs containing sense/antisense arms for specific gene targeting. Overexpression of hpRNA constructed by a Medicago truncatula xyloglucan endotransglycosylase gene retarded the growth of transgenic M. truncatula roots.

Differential expression and regulation of longan XET genes in relation to fruit growth

Xyloglucan endotransglycosylase (XET) catalyses the transglycosylation of xyloglucan, the major hemicellulose polymer, which has been thought to mediate the cross-linking of cellulose microfibrils in cellular walls and proposed to be involved in the control of cell wall relaxation. To understand the relationship between longan fruit growth and XET gene expression patterns, three XET genes from fruit were identified and then their expression profiles in pericarp and aril tissues of fruit at different development stages were investigated. Three full-length cDNAs of 1077, 1093 and 1181 bp encoding XETs, named DlXET1, DlXET2 and DlXET3, respectively, were isolated from expanding fruit using RT-PCR and RACE-PCR (rapid amplification of cDNA ends) methods. Northern blotting analysis showed that three DlXET mRNAs exhibited different patterns during fruit growth and development. Accumulation of DlXET1 kept changeable during the fruit growth and development, Dl-XET2 mRNA accumulations in the pericarp and in the aril increased gradually following fruit growth during the whole fruit developmental stage. In addition, DlXET3 did not accumulate in the pericarp during the whole fruit development stage, but it could be detected in the aril of fruit during the rapid aril growth stage (63–74 DAA). These results indicated that DlXET2 was associated with growth of pericarp and aril in longan fruit, while DlXET1 was related to pericarp growth and DlXET3 was primarily responsible for aril growth. To further characterize the expressions of three XETs regulated by plant growth substances, α-naphthalene acetic acid (NAA) and thidiazuron (TDZ) were used to treat longan fruit at 21 DAA and 56 DAA when pericarp and aril grew rapidly respectively, it was found that treatment at 21 DAA, only TDZ increased the accumulation of DlXET2 mRNA at 12 h after treatment. However, both NAA and TDZ increased the accumulation of DlXET3 after 36 h treatment, although DlXET3 did not accumulate in the whole fruit. While, treatment at 56 DAA, both NAA and TDZ increased the accumulations of DlXETs in aril except DlXET3 in aril by NAA treatment. In addition, the expression patterns of the three XETs showed different tissue specificity. These results suggested that XET genes played a different role in longan fruit growth and showed different response to plant growth substances which existed difference at different growth stage.

Differential expression of litchi XET genes in relation to fruit growth

Xyloglucan endotransglycosylase (XET) catalyses the transglycosylation of xyloglucan, the major hemicellulose polymer, which has been thought to mediate the cross-linking of cellulose microfibrils in cellular walls and proposed to be involved in the control of cell wall relaxation. To understand the relationship between litchi fruit cracking and gene expression patterns, three XET genes from litchi fruit were identified and then examined for their expression profiles in pericarp and aril tissues at different development stages, using a cracking-resistant cultivar, ‘Huaizhi’, and a cracking-susceptible cultivar, ‘Nuomici’. Three full-length cDNAs of 1267, 1095 and 1156 bp encoding XETs, named LcXET1, LcXET2 and LcXET3, respectively, were isolated from expanding fruit using RT-PCR and RACE-PCR (rapid amplification of cDNA ends) methods. Northern blotting analysis showed that LcXET1 mRNA accumulation occurred much earlier in aril tissues at 59 days after anthesis (DAA) than in pericarp tissues at 73 DAA in ‘Nuomici’. However, it appeared at almost the same time (66 DAA) in pericarp and aril tissues in ‘Huaizhi’, which suggested that differential accumulation of LcXET1 in pericarp and aril tissues in ‘Nuomici’ and ‘Huaizhi’ was closely associated with fruit cracking. LcXET2 mRNA accumulation could be detected in pericarp and aril tissues throughout fruit development but exhibited a differential accumulation pattern between pericarp and aril tissues. In the aril of ‘Nuomici’, intensive signal bands were detectable at 59−73 DAA in rapidly expanding fruits of ‘Nuomici’ but only weak bands could be found in the pericarp tissues. In contrast, moderate signal bands were detectable both in pericarp and aril tissues of ‘Huaizhi’ fruits. Furthermore, LcXET3 showed constitutive expression in both pericarp and aril tissues of developing ‘Nuomici’ and ‘Huaizhi’ litchi fruit. In addition, differential expression patterns of three XETs genes were observed in different tissues of litchi, with only LcXET1 being fruit-specific. To further address the role of LcXET in fruit cracking, α-naphthalene acetic acid (NAA) was used to treat ‘Nuomoci’ to reduce fruit cracking. Enhanced LcXET1 mRNA accumulation appeared in pericarp while LcXET2 and LcXET3 mRNA accumulation enhanced in aril tissues in the NAA-treated fruits. Thus, LcXET1 is more likely to play a role in reducing litchi fruit cracking than LcXET2 and LcXET3.

A gibberellin-regulated xyloglucan endotransglycosylase gene is expressed in the endosperm cap during tomato seed germination.

Xyloglucan endotransglycosylases (XETs) modify xyloglucans, major components of primary cell walls in dicots. A cDNA encoding an XET (LeXET4) was isolated from a germinating tomato (Lycopersicon esculentum Mill.) seed cDNA library. DNA gel blot analysis showed that LeXET4 is a single-copy gene in the tomato genome. LeXET4 mRNA was strongly expressed in germinating seeds, was much less abundant in stems, and was not detected in roots, leaves or flower tissues. During germination, LeXET4 mRNA was detected in seeds within 12 h of imbibition with maximum mRNA abundance at 24 h. Tissue prints showed that LeXET4 mRNA was localized exclusively to the endosperm cap region. Expression of LeXET4 was dependent on exogenous gibberellin (GA) in GA-deficient (gib-1 mutant) tomato seeds, while abscisic acid, a seed germination inhibitor, had no effect on LeXET4 mRNA expression in wild-type seeds. LeXET4 mRNA disappeared after radicle emergence, even though degradation of the lateral endosperm cell walls continued. The temporal, spatial and hormonal regulation pattern of LeXET4 gene expression suggests that XET has a role in endosperm cap weakening, a key process regulating tomato seed germination.

Expression of a xyloglucan endo-transglycosylase gene is closely related to grape berry softening

Six partial cDNAs for cell wall degradation-related enzymes, xyloglucan endo-transglycosylase (XET), polygalacturonase (PG), pectin methylesterase (PME), pectate lyase (PL), cellulase (Cel), and β- d-galactosidase (GAL), were isolated from a véraison-specific subtractive library made from Kyoho grape ( Vitis labruscana) berries. During the development of Kyoho grape berries, the expression of these genes was analyzed. Among the genes analyzed, XET gene expression was closely related to berry softening; slight XET gene expression was detected before véraison and was markedly increased at véraison (the stage of the onset of berry softening). In addition, the expression of the gene was berry specific. In the other genes, except for PL, however, no expression was detected during the berry development; PL was detected only after the coloring stage began. These observations suggest that XET plays an important role in grape berry softening. To obtain further information about this gene, a full-length cDNA clone ( VXET 1) encoding XET was isolated from a cDNA library of Kyoho grape berries and characterized. The VXET 1 was 1326 bp in length and contained a 5′-untranslated region of 67 bp, an open reading frame of 873 bp, and a 3′-untranslated region of 386 bp. The deduced amino acid sequence of the VXET 1 showed 73.5% identity with the corresponding XET (NXG 1) from nasturtium ( Tropaeolum majus) that has been shown to have endo-glucanase activity. These findings suggest that the VXET 1 product cleaves a cellulose–xyloglucan network of cell wall and induces the softening of Kyoho grape berries at véraison.

Characterization of a tomato xyloglucan endotransglycosylase gene that is down-regulated by auxin in etiolated hypocotyls.

The reorganization of the cellulose-xyloglucan matrix is proposed to serve as an important mechanism in the control of strength and extensibility of the plant primary cell wall. One of the key enzymes associated with xyloglucan metabolism is xyloglucan endotransglycosylase (XET), which catalyzes the endocleavage and religation of xyloglucan molecules. As with other plant species, XETs are encoded by a gene family in tomato (Lycopersicon esculentum cv T5). In a previous study, we demonstrated that the tomato XET gene LeEXT was abundantly expressed in the rapidly expanding region of the etiolated hypocotyl and was induced to higher levels by auxin. Here, we report the identification of a new tomato XET gene, LeXET2, that shows a different spatial expression and diametrically opposite pattern of auxin regulation from LeEXT. LeXET2 was expressed more abundantly in the mature nonelongating regions of the hypocotyl, and its mRNA abundance decreased dramatically following auxin treatment of etiolated hypocotyl segments. Analysis of the effect of several plant hormones on LeXET2 expression revealed that the inhibition of LeXET2 mRNA accumulation also occurred with cytokinin treatment. LeXET2 mRNA levels increased significantly in hypocotyl segments treated with gibberellin, but this increase could be prevented by adding auxin or cytokinin to the incubation media. Recombinant LeXET2 protein obtained by heterologous expression in Pichia pastoris exhibited greater XET activity against xyloglucan from tomato than that from three other species. The opposite patterns of expression and differential auxin regulation of LeXET2 and LeEXT suggest that they encode XETs with distinct roles during plant growth and development.

Characterization of a Tomato Xyloglucan Endotransglycosylase Gene That Is Down-Regulated by Auxin in Etiolated Hypocotyls

Characterization of a Tomato Xyloglucan Endotransglycosylase Gene That Is Down-Regulated by Auxin in Etiolated Hypocotyls

A new cDNA encoding a xyloglucan endo-transglycosylase-related polypeptide ( AtXTR8) preferentially expressed in seedling, root and stem of Arabidopsis thaliana

A new cDNA clone ( AtXTR8) encoding a xyloglucan endo-transglycosylase (XET)-related protein was isolated from an Arabidopsis thaliana germinating seed cDNA library, using a differential screen. The predicted amino acid sequence displays only 38 to 42% amino acid identity with other members of the Arabidopsis XTR (XET-related) protein family but shows 64% identity to a xyloglucan endo-transglycosylase enzyme (TmNXG1) expressed in nasturtium ( Tropaeolum majus) germinating seeds. The AtXTR8 transcipts are expressed in germinating seeds as early as 24 h after imbibition with highest level of accumulation reached at about 72 h. In adult plants, transcripts are mainly detected in roots and to a lower extent in inflorescence stems. During imbibition of the severely gibberellin-deficient gal-3 mutant seeds, transcripts are detected only if exogenous gibberellins are supplied in the medium. suggesting that gibberellins are directly or indirectly required for AtXTR8 gene expression. Unlike other XET genes such as TCH4, EXT and XTR3 which are rapidly activated by auxins in wild type Arabidopsis, the steady state level of AtXTR8 transcripts is not markedly increased by auxin treatment of 6-day-old seedlings. Phylogenetic analysis confirms that the XET and XTR protein sequences so far identified in various plant species split in three groups and Arabidopsis AtXTR8, which enzymatic activity has not been assessed, belongs to the least related group III.