Genome-wide analysis and environmental response profiling of dirigent family genes in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>)

Dirigent (DIR) and DIR-like family genes were involved in lignification or in the response to pathogen infection and abiotic stress in plants. Little is known to us about how rice DIR genes respond to adverse conditions. In this study, we reported genome-wide analysis of 49 DIR or DIR-likes genes in rice. The 49 OsDIRs or OsDIR-likes were tandem arranged into ten clusters. The phylogenetic analysis indicated that the 49 rice DIR and DIR-like genes cluster into five distinct subfamilies, DIR-a and four DIR-like subfamilies (DIR-b/d, and DIR-g, DIR-c, DIR-e). Meta-analysis of microarray gene expression datas indicated that all the OsDIRs or OsDIR-likes were expressed almost at the same level but with different patterns: most OsDIRs or OsDIR-likes were expressed exclusively in stigma and ovary and were induced by IAA and BAP; several genes were induced by trans-zeatin (tZ) and DMSO; 23 OsDIRs or OsDIR-likes were responded to abiotic stress. Our analysis also showed that most of these genes could respond to abiotic stresses, which contained cis-regulatory elements. The present study will provide a useful reference for further functional analysis of the DIR genes in rice.     

Effect of 1-aminocyclopropane-1-carboxylic acid (ACC)-induced ethylene on <Emphasis Type="Italic">cellulose synthase A</Emphasis> (<Emphasis Type="Italic">CesA</Emphasis>) genes in flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L. ‘Nike’) seedlings

Introduction

Cellulose microfibril is a major cell wall polymer that plays an important role in the growth and development of plants. The gene cellulose synthase A (CesA), encoding cellulose synthases, is involved in the synthesis of cellulose microfibrils. However, the regulatory mechanism of CesA gene expression is not well understood, especially during the early developmental stages.

Objective

To identify factor(s) that regulate the expression of CesA genes and ultimately control seedling growth and development.

Methods

The presence of cis-elements in the promoter region of the eight CesA genes identified in flax (Linum usitatissimum L. ‘Nike’) seedlings was verified, and three kinds of ethylene-responsive cis-elements were identified in the promoters. Therefore, the effect of ethylene on the expression of four selected CesA genes classified into Clades 1 and 6 after treatment with 10^?4 and 10^?3 M 1-aminocyclopropane-1-carboxylic acid (ACC) was examined in the hypocotyl of 4–6-day-old flax seedlings.

Results

ACC-induced ethylene either up- or down-regulated the expression of the CesA genes depending on the clade to which these genes belonged, age of seedlings, part of the hypocotyl, and concentration of ACC.

Conclusion

Ethylene might be one of the factors regulating the expression of CesA genes in flax seedlings.

     

Constitutive expression of <Emphasis Type="Italic">SlTrxF</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant saccharide metabolism. In this study, a gene encoding the TrxF protein, named SlTrxF, was isolated from tomato. The coding region of SlTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants exhibited increased starch accumulation compared to the wild-type (WT). Real-time quantitative PCR analysis showed that constitutive expression of SlTrxF up-regulated the expression of ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthase (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that SlTrxF may improve starch content of Arabidopsis by regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis.     

The lignin synthesis related genes and lodging resistance of <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis>

Lignin is closely related to the lodging resistance of common buckwheat (Fagopyrum esculentum Moench.). However, the characteristics of lignin synthesis related genes have not yet been reported. We investigated the lignin biosynthesis gene expression, activities of related enzymes, and accumulation of lignin monomers during branching stage, bloom stage, and milky ripe stage by real-time quantitative PCR, UVspectrophotometry, and gas chromatography-mass spectrometry in the 2^nd internode of three common buckwheat cultivars with different lodging resistance. The results showed that lignin content and the activity of phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) were closely related to the lodging resistance of common buckwheat. Further, we studied gene expression of cinnamate 4-hydroxylase (C4H), caffeoyl-CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamoyl-CoA reductase (CCR), and caffeic acid O-methyltransferase (COMT). The lignin biosynthesis genes were divided into three classes according to their expression pattern: 1) expression firstly increasing and then descending (PAL, 4CL, CAD, C4H, CCoAOMT, F5H, and CCR), 2) expression remaining constant during maturation (C3H), and 3) expression decreasing with maturation (COMT). The present study provides preliminary insights into the expression of lignin biosynthesis genes in common buckwheat, laying a foundation for further understanding the lignin biosynthesis.     

Enhancement of starch content by constitutive expression of <Emphasis Type="Italic">GmTrxF</Emphasis> in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant carbohydrate metabolism biosynthesis. In this study, a gene encoding the TrxF protein, named GmTrxF, was isolated from soybean. The open reading frame (ORF) contained 540 nucleotides encoding 179 amino acids. The coding region of GmTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis. The starch content in GmTrxF expressing plants was increased by 57–109% compared to that in wild-type (WT). Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of GmTrxF up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthases (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that GmTrxF may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes invovled in starch biosynthesis. The manipulation of GmTrxF expression might be used for increasing starch accumulation of plants in the future.     

A soybean plastidic ATP/ADP transporter gene, <Emphasis Type="Italic">GmAATP</Emphasis>, is involved in carbohydrate metabolism in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic ATP/ADP transporter (AATP) imports adenosine triphosphate (ATP) from the cytosol into plastids, resulting in the increase of the ATP supply to facilitate anabolic synthesis in heterotrophic plastids of dicotyledonous plants. The regulatory role of GmAATP from soybean in increasing starch accumulation has not been investigated. In this study, a gene encoding the AATP protein, named GmAATP, was successfully isolated from soybean. Transient expression of GmAATP in Arabidopsis protoplasts and Nicotiana benthamiana leaf epidermal cells revealed the plastidic localization of GmAATP. Its expression was induced by exogenous sucrose treatment in soybean. The coding region of GmAATP was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis to obtain transgenic plants. Constitutive expression of GmAATP significantly increased the sucrose and starch accumulation in the transgenic plants. Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of GmAATP up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2), granule-bound starch synthase (AtGBSS I and AtGBSS II), soluble starch synthases (AtSSS I, AtSSS II, AtSSS III, and AtSSS IV), and starch branching enzyme (AtSBE I and AtSBE II) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses indicated that the major enzymes (AGPase, GBSS, SSS, and SBE) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to the wild type (WT). These findings suggest that GmAATP may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis. All these results suggest that GmAATP could be used as a candidate gene for developing high starch-accumulating plants as alternative energy crops.     

Identification and functional characterization of DzS3GT,a cytoplasmic glycosyltransferase catalyzing biosynthesis of diosgenin 3-<Emphasis Type="Italic">O</Emphasis>-glucoside in <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis>

Dioscorea plants produce pharmaceutical diosgenin, which usually exists in plants in the form of saponins and has been a starting material for the production of steroids over seven decades. The first step of steroidal saponin biosynthesis from the corresponding aglycone is glycosylation by 3-O-sterol glycosyltransferase (S3GT), transferring the glycosyl from a sugar donor to the 3-OH position of the aglycone. In this study, a DzS3GT gene from Dioscorea zingiberensis was cloned and expressed in Escherichia coli, and the recombinant DzS3GT protein showed 3-O-sterol glycosyltransferase activity in vitro. Subcellular localization analysis revealed that the DzS3GT protein is located in the cytoplasm in rice protoplasts. The tissue profiles of DzS3GT differ from those reported SGT genes. DzS3GT is expressed strongly in leaves and very weakly in stems. The diosgenin 3-O-glucoside (trillin) content is much higher in the leaves than in other organs. The specificity of gene expression and saponins accumulation suggest that the biosynthesis of trillin may occur mainly in the leaves of D. zingiberensis. This is the first report of the cloning and biochemical characterization of a glycosyltransferase gene involved in the biosynthesis of diosgenin 3-O-glucoside in Dioscorea plants. In addition, the study provides a potential relevance to the biosynthesis and transport mechanism of steroidal saponins in Dioscorea plants.     

Molecular cloning,characterization, and functional analysis of a gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A synthase from <Emphasis Type="Italic">Matricaria chamomilla</Emphasis>

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA as the first committed enzyme in the mevalonate (MVA) pathway. HMGS plays an important role in the biosynthesis of the sesquiterpene, which is the main constituent of essential oil in Matricaria chamomilla. In this paper, a HMGS gene designated as McHMGS (GenBank Accession No. KU529970) was successfully cloned from M. chamomilla. The full-length cDNA of McHMGS was 1495-bp and contained a 1374-bp open reading frame. It encoded a 458-amino-acid protein with a calculated molecular weight of about 50.7 kDa and isoelectric point of 5.69. Sequence comparison revealed that McHMGS showed extensive homology with HMGSs from other plant species. Phylogenetic tree analysis indicated that McHMGS is clustered with the HMGS of Asteraceae in the dicotyledoneae clade. Further functional complementation of McHMGS in hmgs-deficient mutant yeast strain YSC6274 demonstrated that cloned McHMGS cDNA encodes a functional HMGS and mediates the MVA biosynthesis in yeasts. The tissue expression pattern analysis revealed that McHMGS expression level is highest in the flowers and lowest in the stems. Quantitative real-time PCR analysis showed that the expression of McHMGS was induced by MeJA, and the expression level is highest 24 h after induction. The characterization and expression of McHMGS can help in further studying the role of McHMGS gene in the biosynthesis of sesquiterpene in M. chamomilla.     

A tomato plastidic ATP/ADP transporter gene <Emphasis Type="Italic">SlAATP</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

A plastidic ATP/ADP transporter (AATP) is responsible for importing ATP from the cytosol into plastids. Increasing the ATP supply is a potential way to facilitate anabolic synthesis in heterotrophic plastids of plants. In this work, a gene encoding the AATP protein, named SlAATP, was successfully isolated from tomato. Expression of SlAATP was induced by exogenous sucrose treatment in tomato. The coding region of SlAATP was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis to obtain transgenic plants. Constitutive expression of SlAATP significantly increased the starch accumulation in the transgenic plants. Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of StAATP up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AtAGPase), granule-bound starch synthase (AtGBSS I and AtGBSS II), soluble starch synthases (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) and starch branching enzyme (AtSBE I and AtSBE II) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses indicated that the major enzymes (AGPase, GBSS, SSS and SBE) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to the wild-type (WT). These findings suggest that SlAATP may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes invovled in starch biosynthesis. The manipulation of SlAATP expression might be used for increasing starch accumulation of plants in the future.     

Putrescine biosynthesis and export genes are essential for normal growth of avian pathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background

Avian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T).

Results

Following whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (ΔspeB, ΔspeC, ΔspeF, ΔspeB/C and ΔspeD/E), and in polyamines transport systems (ΔpotE, ΔyeeF, ΔpotABCD and ΔpotFGHI). Contrary to what was observed for MG1655, the ΔpotE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ΔspeB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ΔspeB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed.

Conclusions

Deletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.

     

Identification and functional analysis of anthocyanin biosynthesis genes in <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrids

Phalaenopsis species are among the most popular potted flowers for their fascinating flowers. When their whole-genome sequencing was completed, they have become useful for studying the molecular mechanism of anthocyanin biosynthesis. Here, we identified 49 candidate anthocyanin synthetic genes in the Phalaenopsis genome. Our results showed that duplication events might contribute to the expansion of some gene families, such as the genes encoding chalcone synthase (PeCHS), flavonoid 3′-hydroxylase (PeF3′H), and myeloblastosis (PeMYB). To elucidate their functions in anthocyanin biosynthesis, we conducted a global expression analysis. We found that anthocyanin synthesis occurred during the very early flower development stage and that the flavanone 3-hydroxylase (F3H), F3′H, and dihydroflavonol 4-reductase (DFR) genes played key roles in this process. Over-expression of Phalaenopsis flavonoid 3′,5′-hydroxylase (F3′5′H) in petunia showed that it had no function in anthocyanin production. Furthermore, global analysis of sequences and expression patterns show that the regulatory genes are relatively conserved and might be important in regulating anthocyanin synthesis through different combined expression patterns. To determine the functions of MYB2, 11, and 12, we over-expressed them in petunia and performed yeast two-hybrid analysis with anthocyanin (AN)1 and AN11. The MYB2 protein had strong activity in regulating anthocyanin biosynthesis and induced significant pigment accumulation in transgenic plant petals, whereas MYB11 and MYB12 had lower activities. Our work provided important improvement in the understanding of anthocyanin biosynthesis and established a foundation for floral colour breeding in Phalaenopsis through genetic engineering.