Metabolism Of Cell Wall Polysaccharides Research Articles

The roles of cell wall polysaccharides in response to waterlogging stress in Brassica napus L. root

BackgroundBrassica napus L. (B. napus) is susceptible to waterlogging stress during different cultivation periods. Therefore, it is crucial to enhance the resistance to waterlogging stress to achieve a high and stable yield of B. napus.ResultsHere we observed significant differences in the responses of two B. napus varieties in root under waterlogging stress. The sensitive variety (23651) exhibited a more pronounced and rapid reduction in cell wall thickness and root integrity compared with the tolerant variety (Santana) under waterlogging stress. By module clustering analysis based on transcriptome data, we identified that cell wall polysaccharide metabolism responded to waterlogging stress in root. It was found that pectin content was significantly reduced in the sensitive variety compared with the tolerant variety. Furthermore, transcriptome analysis revealed that the expression of two homologous genes encoding polygalacturonase-inhibiting protein 2 (PGIP2), involved in polysaccharide metabolic pathways, was highly upregulated in root of the tolerant variety under waterlogging stress. BnaPGIP2s probably confer waterlogging resistance by inhibiting the activity of polygalacturonases (PGs), which in turn reduces the degradation of the pectin backbone polygalacturonic acid.ConclusionsOur findings demonstrate that cell wall polysaccharides in root plays a vital role in response to the waterlogging stress and provide a theoretical foundation for breeding waterlogging resistance in B. napus varieties.

Melatonin-Induced Chromium Tolerance Requires Hydrogen Sulfide Signaling in Maize.

Both melatonin and hydrogen sulfide (H2S) mitigate chromium (Cr) toxicity in plants, but the specific interaction between melatonin and H2S in Cr detoxification remains unclear. In this study, the interaction between melatonin and H2S in Cr detoxification was elucidated by measuring cell wall polysaccharide metabolism and antioxidant enzyme activity in maize. The findings revealed that exposure to Cr stress (100 μM K2Cr2O7) resulted in the upregulation of L-/D-cysteine desulfhydrase (LCD/DCD) gene expression, leading to a 77.8% and 27.3% increase in endogenous H2S levels in maize leaves and roots, respectively. Similarly, the endogenous melatonin system is activated in response to Cr stress. We found that melatonin had a significant impact on the relative expression of LCD/DCD, leading to a 103.3% and 116.7% increase in endogenous H2S levels in maize leaves and roots, respectively. In contrast, NaHS had minimal effects on the relative mRNA expression of serotonin-Nacetyltransferase (SNAT) and endogenous melatonin levels. The production of H2S induced by melatonin is accompanied by an increase in Cr tolerance, as evidenced by elevated gene expression, elevated cell wall polysaccharide content, increased pectin methylesterase activity, and improved antioxidant enzyme activity. The scavenging of H2S decreases the melatonin-induced Cr tolerance, while the inhibitor of melatonin synthesis, p-chlorophenylalanine (p-CPA), has minimal impact on H2S-induced Cr tolerance. In conclusion, our findings suggest that H2S serves as a downstream signaling molecule involved in melatonin-induced Cr tolerance in maize.

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

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

Transcriptome analysis reveals genes related to the synthesis and metabolism of cell wall polysaccharides in goji berry (Lycium barbarum L.) from various regions.

In goji berries (Lycium barbarum L.), the cell wall properties and ripening environment affect fruit quality and their economic benefits. However, the mechanism underlying the cell wall remains to be fully elucidated. The results showed that total sugar content was higher in Qinghai berries (13.87%, P < 0.01), whereas cellulose content peaked in Zhongning berries (28%, P < 0.05). Arabinose, galactose, and galacturonic acid were the principal components of the cell wall polysaccharides in goji berries. Among them, the content of galactose in Zhongning was significantly the highest (P < 0.05). Interestingly, we found that highly expressed β-glucosidase and lowly expressed endoglucanase led to cellulose accumulation by RNA-sequencing analysis. The expression analysis results suggested that pectate lyase and pectinesterase enzymes could be major factors related to higher galactose and galacturonic acid contents in Zhongning compared to in Qinghai and Gansu. The starch and sucrose metabolism pathway, pentose and glucuronate interconversions pathway, and galactose metabolism pathway played a significant role in cell wall polysaccharide synthesis and metabolism. In the present study, we aimed to provide some insights into the cell wall on polysaccharide composition, structural features, and gene analysis in goji berries from Zhongning, Qinghai, and Gansu in China. These results might help to clarify the molecular function of the major genes in the cell wall polysaccharides of goji berries and provide a solid foundation for further study. © 2023 Society of Chemical Industry.

Zizania latifolia Cell Wall Polysaccharide Metabolism and Changes of Related Enzyme Activities during Postharvest Storage.

The metabolism of polysaccharides in the Zizania latifolia cell wall helps maintain the postharvest quality during storage. Fresh Z. latifolia was stored at 4 °C and 25 °C to evaluate the hardness, cell wall polysaccharide composition, cell wall structure, active ingredients, and cell wall metabolism-related enzyme activities. The results showed that hardness declined concomitantly with an increase in water-soluble pectin content during storage, as well as with a decrease in propectin and cellulose contents. Correlation analysis showed that lower activities of cell wall-degrading enzymes, such as polygalacturonase, cellulase, and β-galactosidase in Z. latifolia stored at 4 °C, were associated with lighter fiberization and greater hardness, compared with those stored at 25 °C. Additionally, the results of infrared spectroscopy showed that texture softening may be attributed to a decrease in the degree of esterification of water-soluble polysaccharides at 25 °C compared to that at 4 °C.

Effects of hydrogen peroxide treatment on pulp breakdown, softening, and cell wall polysaccharide metabolism in fresh longan fruit

Longan (Dimocarpus longan Lour.) is prone to pulp softening and pulp breakdown, leading to a loss of its nutrients including polysaccharides. ROS is one main factor affecting fruit quality. This work intended to explicate the influences of hydrogen peroxide, acting as a ROS, on pulp softening, pulp breakdown, and cell wall polysaccharides metabolism in longan fruit during storage. Contrasted to the control group, hydrogen peroxide-treated samples exhibited lower firmness, lower amounts of CWM, ISP, CSP, hemicellulose and cellulose, but higher breakdown index, WSP amount, expression levels of DlPG, DlPE, Dlβ-Gal, DlCx and DlXET and activities of their corresponding enzymes (PG, PE, β-Gal, Cx, XET). These results suggested that hydrogen peroxide reduced longan pulp firmness due to the increased gene expression levels and enzymes activities related to cell wall polysaccharide degradation to boost their decomposition, thereby led to the accelerated pulp softening and the expedited pulp breakdown of harvested longans.

Transcriptome analysis of atemoya pericarp elucidates the role of polysaccharide metabolism in fruit ripening and cracking after harvest

BackgroundMature fruit cracking during the normal season in African Pride (AP) atemoya is a major problem in postharvest storage. Our current understanding of the molecular mechanism underlying fruit cracking is limited. The aim of this study was to unravel the role starch degradation and cell wall polysaccharide metabolism in fruit ripening and cracking after harvest through transcriptome analysis.ResultsTranscriptome analysis of AP atemoya pericarp from cracking fruits of ethylene treatments and controls was performed. KEGG pathway analysis revealed that the starch and sucrose metabolism pathway was significantly enriched, and approximately 39 DEGs could be functionally annotated, which included starch, cellulose, pectin, and other sugar metabolism-related genes. Starch, protopectin, and soluble pectin contents among the different cracking stages after ethylene treatment and the controls were monitored. The results revealed that ethylene accelerated starch degradation, inhibited protopectin synthesis, and enhanced the soluble pectin content, compared to the control, which coincides with the phenotype of ethylene-induced fruit cracking. Key genes implicated in the starch, pectin, and cellulose degradation were further investigated using RT-qPCR analysis. The results revealed that alpha-amylase 1 (AMY1), alpha-amylase 3 (AMY3), beta-amylase 1 (BAM1), beta-amylase 3 (BAM3), beta-amylase 9 (BAM9), pullulanase (PUL), and glycogen debranching enzyme (glgX), were the major genes involved in starch degradation. AMY1, BAM3, BAM9, PUL, and glgX all were upregulated and had higher expression levels with ethylene treatment compared to the controls, suggesting that ethylene treatment may be responsible for accelerating starch degradation. The expression profile of alpha-1,4-galacturonosyltransferase (GAUT) and granule-bound starch synthase (GBSS) coincided with protopectin content changes and could involve protopectin synthesis. Pectinesterase (PE), polygalacturonase (PG), and pectate lyase (PEL) all involved in pectin degradation; PE was significantly upregulated by ethylene and was the key enzyme implicated pectin degradation.ConclusionBoth KEGG pathway enrichment analysis of DEGs and material content analysis confirmed that starch decomposition into soluble sugars and cell wall polysaccharides metabolism are closely related to the ripening and cracking of AP atemoya. A link between gene up- or downregulation during different cracking stages of atemoya fruits and how their expression affects starch and pectin contents were established by RT-qPCR analysis.

Changes in cell wall polysaccharide composition, gene transcription and alternative splicing in germinating barley embryos

Barley (Hordeum vulgare L.) seed germination initiates many important biological processes such as DNA, membrane and mitochondrial repairs. However, little is known on cell wall modifications in germinating embryos. We have investigated cell wall polysaccharide composition change, gene transcription and alternative splicing events in four barley varieties at 24h and 48h germination. Cell wall components in germinating barley embryos changed rapidly, with increases in cellulose and (1,3)(1,4)-β-d-glucan (20–100%) within 24h, but decreases in heteroxylan and arabinan (3–50%). There were also significant changes in the levels of type I arabinogalactans and heteromannans. Alternative splicing played very important roles in cell wall modifications. At least 22 cell wall transcripts were detected to undergo either alternative 3′ splicing, alternative 5′ splicing or intron retention type of alternative splicing. These genes coded enzymes catalyzing synthesis and degradation of cellulose, heteroxylan, (1,3)(1,4)-β-d-glucan and other cell wall polymers. Furthermore, transcriptional regulation also played very important roles in cell wall modifications. Transcript levels of primary wall cellulase synthase, heteroxylan synthesizing and nucleotide sugar inter-conversion genes were very high in germinating embryos. At least 50 cell wall genes changed transcript levels significantly. Expression patterns of many cell wall genes coincided with changes in polysaccharide composition. Our data showed that cell wall polysaccharide metabolism was very active in germinating barley embryos, which was regulated at both transcriptional and post-transcriptional levels.

Identification of Hypoxia-Inducible Target Genes of Aspergillus fumigatus by Transcriptome Analysis Reveals Cellular Respiration as an Important Contributor to Hypoxic Survival

Aspergillus fumigatus is an opportunistic, airborne pathogen that causes invasive aspergillosis in immunocompromised patients. During the infection process, A. fumigatus is challenged by hypoxic microenvironments occurring in inflammatory, necrotic tissue. To gain further insights into the adaptation mechanism, A. fumigatus was cultivated in an oxygen-controlled chemostat under hypoxic and normoxic conditions. Transcriptome analysis revealed a significant increase in transcripts associated with cell wall polysaccharide metabolism, amino acid and metal ion transport, nitrogen metabolism, and glycolysis. A concomitant reduction in transcript levels was observed with cellular trafficking and G-protein-coupled signaling. To learn more about the functional roles of hypoxia-induced transcripts, we deleted A. fumigatus genes putatively involved in reactive nitrogen species detoxification (fhpA), NAD(+) regeneration (frdA and osmA), nitrogen metabolism (niaD and niiA), and respiration (rcfB). We show that the nitric oxygen (NO)-detoxifying flavohemoprotein gene fhpA is strongly induced by hypoxia independent of the nitrogen source but is dispensable for hypoxic survival. By deleting the nitrate reductase gene niaD, the nitrite reductase gene niiA, and the two fumarate reductase genes frdA and osmA, we found that alternative electron acceptors, such as nitrate and fumarate, do not have a significant impact on growth of A. fumigatus during hypoxia, but functional mitochondrial respiratory chain complexes are essential under these conditions. Inhibition studies indicated that primarily complexes III and IV play a crucial role in the hypoxic growth of A. fumigatus.

Comparative transcriptome analysis of Discosporangium mesarthrocarpum (Phaeophyceae), Schizocladia ischiensis (Schizocladiophyceae), and Phaeothamnion confervicola (Phaeothamniophyceae), with special reference to cell wall-related genes.

Within the stramenopile lineage, only brown algae (Phaeophyceae) have evolved complex multicellularity, although some other members of the lineage (e.g., Schizocladia in Schizocladiophyceae; Phaeothamnion in Phaeothamniophyceae) also develop simple multicellular thalli. The development of an adherent extracellular matrix (ECM) is considered to be one of the key steps in the evolution of multicellularity, because ECM is involved in adhesion of cells to each other and in cell-cell communication essential for developmental, reproductive, and sophisticated defense systems. Because there are no unicellular organisms within brown algae, we considered that comparison of other stramenopile taxa closely related to brown algae and having multicellular thalli could yield clues to elucidate the evolution of multicellularity in brown algae. In this study, we investigated transcriptomes involved in cell wall polysaccharide metabolism of three stramenopile species, Discosporangium mesarthrocarpum, which is suggested to be one of the most basal taxa within Phaeophyceae, S.ischiensis, and P.confervicola. We employed 454-FLX high-throughput pyrosequencing to generate expressed sequence tag (EST) databases for these species, and performed comparative analyses between these databases and the genome sequence of the brown alga Ectocarpus siliculosus. Results indicate that cell wall polysaccharide metabolism pathways of D.mesarthrocarpum are similar to E.siliculosus, whereas those of S.ischiensis and P.confervicola are significantly different from E.siliculosus, suggesting that the components of the cell wall in S.ischiensis and P.confervicola are likely to be different from those of E.siliculosus.

Changes in metabolism of cell wall polysaccharides in oat leaves during senescence: relevance to the senescence-promoting effect of methyl jasmonate

Changes in cell wall polysaccharides in oat (Avena sativa L.) leaf segments during senescence promoted by methyl jasmonate (JA-Me) were studied. During the incubation with water at 25 °C in the dark, the loss of chlorophyll of the segments excised from the primary leaves of 8-day-old green seedlings was found dramatically just after leaf excision, and leaf color completely turned to yellow after the 3- to 4-day incubation in the dark. Application of 10 µM JA-Me substantially promoted the loss of chlorophyll corresponding with the chloroplast degradation. Cell wall polysaccharides in oat leaf segments mainly consisted of hemicellulosic and cellulosic ones. During the process of leaf senescence, the amount of hemicellulosic I and II, and cellulosic polysaccharides decreased, but little in pectic polysaccharides. JA-Me significantly enhanced the decrease in cellulosic polysaccharides, but little in hemicellulosic ones. Arabinose, xylose and glucose were identified as main constituents of neutral sugars of hemicellulosic polysaccharides. The neutral sugar compositions of hemicellulosic polysaccharides changed little during leaf senescence both in the presence or absence of JA-Me. These facts suggest that JA-Me affects sugar metabolism relating to cellulosic polysaccharides during leaf senescence.

The cell wall polysaccharide metabolism of the brown alga Ectocarpus siliculosus. Insights into the evolution of extracellular matrix polysaccharides in Eukaryotes.

• Brown algal cell walls share some components with plants (cellulose) and animals (sulfated fucans), but they also contain some unique polysaccharides (alginates). Analysis of the Ectocarpus genome provides a unique opportunity to decipher the molecular bases of these crucial metabolisms. • An extensive bioinformatic census of the enzymes potentially involved in the biogenesis and remodeling of cellulose, alginate and fucans was performed, and completed by phylogenetic analyses of key enzymes. • The routes for the biosynthesis of cellulose, alginates and sulfated fucans were reconstructed. Surprisingly, known families of cellulases, expansins and alginate lyases are absent in Ectocarpus, suggesting the existence of novel mechanisms and/or proteins for cell wall expansion in brown algae. • Altogether, our data depict a complex evolutionary history for the main components of brown algal cell walls. Cellulose synthesis was inherited from the ancestral red algal endosymbiont, whereas the terminal steps for alginate biosynthesis were acquired by horizontal gene transfer from an Actinobacterium. This horizontal gene transfer event also contributed genes for hemicellulose biosynthesis. By contrast, the biosynthetic route for sulfated fucans is an ancestral pathway, conserved with animals. These findings shine a new light on the origin and evolution of cell wall polysaccharides in other Eukaryotes.

The rice dynamin-related protein DRP2B mediates membrane trafficking, and thereby plays a critical role in secondary cell wall cellulose biosynthesis

Membrane trafficking between the plasma membrane (PM) and intracellular compartments is an important process that regulates the deposition and metabolism of cell wall polysaccharides. Dynamin-related proteins (DRPs), which function in membrane tubulation and vesiculation are closely associated with cell wall biogenesis. However, the molecular mechanisms by which DRPs participate in cell wall formation are poorly understood. Here, we report the functional characterization of Brittle Culm3 (BC3), a gene encoding OsDRP2B. Consistent with the expression of BC3 in mechanical tissues, the bc3 mutation reduces mechanical strength, which results from decreased cellulose content and altered secondary wall structure. OsDRP2B, one of three members of the DRP2 subfamily in rice (Oryza sativa L.), was identified as an authentic membrane-associated dynamin via in vitro biochemical analyses. Subcellular localization of fluorescence-tagged OsDRP2B and several compartment markers in protoplast cells showed that this protein not only lies at the PM and the clathrin-mediated vesicles, but also is targeted to the trans-Golgi network (TGN). An FM4-64 uptake assay in transgenic plants that express green fluorescent protein-tagged OsDRP2B verified its involvement in an endocytic pathway. BC3 mutation and overexpression altered the abundance of cellulose synthase catalytic subunit 4 (OsCESA4) in the PM and in the endomembrane systems. All of these findings lead us to conclude that OsDRP2B participates in the endocytic pathway, probably as well as in post-Golgi membrane trafficking. Mutation of OsDRP2B disturbs the membrane trafficking that is essential for normal cellulose biosynthesis of the secondary cell wall, thereby leading to inferior mechanical properties in rice plants.

Transcriptome analysis of secondary-wall-enriched seed coat tissues of canola (Brassica napus L.)

The seed coat of Brassica napus (canola, oilseed rape) is derived from ovule integuments and contains a layer of palisade cells, which have thick secondary walls. Because cellulosic walls and other indigestible components of the seed coat contribute negatively to the value of oilseeds, efforts are underway to alter seed development. To facilitate these efforts, and to better understand the biology of seed coats, we used a 90,000 element microarray to identify genes whose transcripts were expressed in developing seed coats of B. napus. After dissecting seed coats into three layers, and comparing transcript expression in the middle fraction (which contained the palisade-enriched tissue and bulk of inner integument) to transcript expression in developing hypocotyls, we identified 674 genes whose transcripts were more abundant in the middle fraction of the seed coat. Among these were well-characterized markers of seed coat identity and many genes associated with metabolism of cell wall polysaccharides, flavonoids and various cell wall proteins and transcription factors. Conversely, we identified 1,203 genes whose transcripts were more abundant in the hypocotyl tissue as compared to seed coat, including xylem-specific markers, such as XCP1 and XCP2. We validated 21 of the differentially expressed transcripts using quantitative RT-PCR. The results define a set of transcripts that are highly enriched in the developing seed coat of B. napus.

Genomic and expression analysis of glycosyl hydrolase family 35 genes from rice (Oryza sativa L.)

BackgroundMany plant β-galactosidases (Bgals) have been well characterized and their deduced biological functions mainly involve degradation of structural pectins, xyloglucans or arabinogalactoproteins in plant cell walls. However, gene multiplicity in glycosyl hydrolase family 35 (GH35), to which these proteins belong, implies diverse functions. In this study, the gene multiplicity, apparent evolutionary relationships and transcript expression of rice Bgal genes were examined, in order to predict their biological functions.ResultsFifteen rice Bgal genes were identified in the plant genome, one of which encodes a protein similar to animal Bgals (OsBgal9), and the remaining 14 fall in a nearly plant-specific subfamily of Bgals. The presence of both classes of Bgals in bryophytes, as well as vascular plants, suggests both gene lineages were present early in plant evolution. All 15 proteins were predicted to contain secretory signal sequences, suggesting they have secretory pathway or external roles. RT-PCR and database analysis found two distinct lineages to be expressed nearly exclusively in reproductive tissues and to be closely related to Arabidopsis Bgals expressed most highly in flower and pollen. On the other hand, OsBgal6 is expressed primarily in young vegetative tissues, and alternative splicing in panicle prevents its production of full-length protein in this reproductive tissue. OsBgal11 also showed alternative splicing to produce different length proteins. OsBgal13 produced by recombinant expression in Escherichia coli hydrolyzed α-L-arabinoside in addition to β-D-galactoside and β-(1→3)-, β-(1→4)- and β-(1→6)- linked galacto-oligosaccharides.ConclusionRice GH35 contains fifteen genes with a diversity of protein sequences, predicted locations and expression and splicing patterns that suggest that OsBgals enzymes may play a variety of roles in metabolism of cell wall polysaccharides, glycoproteins and glycolipids.

Physiological roles of plant glycoside hydrolases

The functions of plant glycoside hydrolases and transglycosidases have been studied using different biochemical and molecular genetic approaches. These enzymes are involved in the metabolism of various carbohydrates containing compounds present in the plant tissues. The structural and functional diversity of the carbohydrates implies a vast spectrum of enzymes involved in their metabolism. Complete genome sequence of Arabidopsis and rice has allowed the classification of glycoside hydrolases in different families based on amino acid sequence data. The genomes of these plants contain 29 families of glycoside hydrolases. This review summarizes the current research on plant glycoside hydrolases concerning their principal functional roles, which were attributed to different families. The majority of these plant glycoside hydrolases are involved in cell wall polysaccharide metabolism. Other functions include their participation in the biosynthesis and remodulation of glycans, mobilization of energy, defence, symbiosis, signalling, secondary plant metabolism and metabolism of glycolipids.

Quantitative trait loci analysis of primary cell wall composition in Arabidopsis.

Quantitative trait loci (QTL) analysis was used to identify genes underlying natural variation in primary cell wall composition in Arabidopsis (Arabidopsis thaliana). The cell walls of dark-grown seedlings of a Bay-0 x Shahdara recombinant inbred line population were analyzed using three miniaturized global cell wall fingerprinting techniques: monosaccharide composition analysis by gas chromatography, xyloglucan oligosaccharide mass profiling, and whole-wall Fourier-transform infrared microspectroscopy. Heritable variation and transgression were observed for the arabinose-rhamnose ratio, xyloglucan side-chain composition (including O-acetylation levels), and absorbance for a subset of Fourier-transform infrared wavenumbers. In total, 33 QTL, corresponding to at least 11 different loci controlling dark-grown hypocotyl length, pectin composition, and levels of xyloglucan fucosylation and O-acetylation, were identified. One major QTL, accounting for 51% of the variation in the arabinose-rhamnose ratio, affected the number of arabinan side chains presumably attached to the pectic polysaccharide rhamnogalacturonan I, paving the way to positional cloning of the first gene underlying natural variation in pectin structure. Several QTL were found to be colocalized, which may have implications for the regulation of xyloglucan metabolism. These results demonstrate the feasibility of combining fingerprinting techniques, natural variation, and quantitative genetics to gain original insight into the molecular mechanisms underlying the structure and metabolism of cell wall polysaccharides.

Isolation of the promoter of a cotton β-galactosidase gene (GhGal1) and its expression in transgenic tobacco plants

Beta-galactosidases (EC 3.2.1.23) constitute a widespread family of glycosyl hydrolases in plants and are thought to be involved in metabolism of cell wall polysaccharides. A cDNA of the cotton (Gossypium hirsutum) beta-galactosidase gene, designated GhGal1, has previously been identified and its transcripts are highly abundant at the elongation stage of the cotton fiber. To examine the temporal and spatial control of GhGal1 expression, a transcriptional fusion of the GhGal1 promoter region (1770 bp) with the beta-glucuronidase (GUS) reporter gene was introduced into tobacco plants by the Agrobacterium infection method. The resulting transgenic plants showed higher GUS activity of fruit in the transgenic plants than that in the negative and positive controls. Histochemical localization of GUS activity demonstrated that the expression of the GUS gene could be found in the meristem zones of roots, cotyledons, vascular tissues, fruit and trichomes in transgenic tobacco plants. Additionally, sequence analysis of the regulatory region also revealed several conserved motifs among which some were shared with previously reported fruit/seed-specific elements and the others were related with trichome expression. These results indicated the temporal and spatial expression characterization of the GhGal1 promoter in transgenic tobacco plants and provided an important insight into the roles of GhGal1 in cotton fiber development.

31.Jasmonate-induced dehiscence of coleoptiles in etiolated maize seedlings : Relevance to metabolism of cell wall polysaccharides

31.Jasmonate-induced dehiscence of coleoptiles in etiolated maize seedlings : Relevance to metabolism of cell wall polysaccharides

Cloning and characterization of cottonGhBGgene encoding β-glucosidase

Beta-1,4-glucosidase (BG, EC3.2.1.21), one of three cellulases, is a widespread family of enzymes involved in the metabolism of cell wall polysaccharides in both prokaryocytes and eukaryotes. Here, we report the isolation of a full-length cDNA encoding beta-1,4-glucosidase protein (designated as GhBG) and its putative function in the process of fiber development and in yeast. Through random sequencing of the cotton fiber cDNA library from 7235 germplasm line, with elite fiber quality in Gossypium hirsutum L. and utilizing the 5' rapid amplification of cDNA ends (RACE) technique, a 2133 bp cDNA clone encoding a cotton fiber specifically expressed protein (accession number: DQ103699) was isolated. GhBG was composed of a 1884 bp open reading frame (ORF) encoding 627 amino acid residues. This putative protein had an isoelectric point of 8.17, a calculated molecular weight of 68.78 KD and a signal peptide with 23 amino acid residues at the N-terminal. RT-PCR analysis indicated GhBG was specifically expressed in fiber cells and was highly abundant in 5-17 day post anthesis (DPA). It was not, however, expressed in root, hypocotyls or leaves. Southern blotting analysis showed there were two copies of GhBG in the upland cotton genome; most likely contained in sub-genome A and sub-genome D. GhBG was then integrated into a yeast expression vector, pREP-5N and electro-transformed into fission yeast Schizosaccharomyces pombe Q-01. The results demonstrated that GhBG led to a significant increase in cell length and width and a remarkable decrease of the length/width ratio. Compared to vector control transformants, cells were significantly larger and rounder and their growth velocity was also reduced.