Huawu Jiang

Global analysis of gene expression profiles in developing physic nut (Jatropha curcas L.) seeds.

Background Physic nut (Jatropha curcas L.) is an oilseed plant species with high potential utility as a biofuel. Furthermore, following recent sequencing of its genome and the availability of expressed sequence tag (EST) libraries, it is a valuable model plant for studying carbon assimilation in endosperms of oilseed plants. There have been several transcriptomic analyses of developing physic nut seeds using ESTs, but they have provided limited information on the accumulation of stored resources in the seeds. Methodology/Principal Findings We applied next-generation Illumina sequencing technology to analyze global gene expression profiles of developing physic nut seeds 14, 19, 25, 29, 35, 41, and 45 days after pollination (DAP). The acquired profiles reveal the key genes, and their expression timeframes, involved in major metabolic processes including: carbon flow, starch metabolism, and synthesis of storage lipids and proteins in the developing seeds. The main period of storage reserves synthesis in the seeds appears to be 29–41 DAP, and the fatty acid composition of the developing seeds is consistent with relative expression levels of different isoforms of acyl-ACP thioesterase and fatty acid desaturase genes. Several transcription factor genes whose expression coincides with storage reserve deposition correspond to those known to regulate the process in Arabidopsis. Conclusions/Significance The results will facilitate searches for genes that influence de novo lipid synthesis, accumulation and their regulatory networks in developing physic nut seeds, and other oil seeds. Thus, they will be helpful in attempts to modify these plants for efficient biofuel production.

Cloning and functional characterization of an acyl-acyl carrier protein thioesterase (JcFATB1) from Jatropha curcas

A full-length cDNA of an acyl-acyl carrier protein (ACP) thioesterase (TE) (EC 3.1.2.14), named JcFATB1, was isolated from the woody oil plant Jatropha curcas L. The deduced amino acid sequence of the cDNA shares about 78% identity with FATB TEs, but only about 33% identity with FATA TEs from other plants. The deduced sequence also contains two essential residues (H(317) and C(352)) for TE catalytic activity and a putative chloroplast transit peptide at the N-terminal. Southern blot analysis revealed that a single copy of JcFATB1 is present in the J. curcas genome, and semi-quantitative PCR analysis showed that JcFATB1 was expressed in all tissues that were examined, most strongly in seeds, in which its expression peaked in late developmental stages. Seed-specific overexpression of the JcFATB1 cDNA in Arabidopsis resulted in increased levels of saturated fatty acids, especially palmitate, and in reduced levels of unsaturated fatty acids. The findings suggest that JcFATB1 from this woody oil plant can function as a saturated acyl-ACP TE and could potentially modify the seed oil of J. curcas to increase its levels of palmitate.

Integrated genome sequence and linkage map of physic nut (Jatropha curcas L.), a biodiesel plant

The family Euphorbiaceae includes some of the most efficient biomass accumulators. Whole genome sequencing and the development of genetic maps of these species are important components in molecular breeding and genetic improvement. Here we report the draft genome of physic nut (Jatropha curcas L.), a biodiesel plant. The assembled genome has a total length of 320.5 Mbp and contains 27,172 putative protein-coding genes. We established a linkage map containing 1208 markers and anchored the genome assembly (81.7%) to this map to produce 11 pseudochromosomes. After gene family clustering, 15,268 families were identified, of which 13,887 existed in the castor bean genome. Analysis of the genome highlighted specific expansion and contraction of a number of gene families during the evolution of this species, including the ribosome-inactivating proteins and oil biosynthesis pathway enzymes. The genomic sequence and linkage map provide a valuable resource not only for fundamental and applied research on physic nut but also for evolutionary and comparative genomics analysis, particularly in the Euphorbiaceae.

Genome-wide analysis of the WRKY gene family in physic nut (Jatropha curcas L.)

The WRKY proteins, which contain highly conserved WRKYGQK amino acid sequences and zinc-finger-like motifs, constitute a large family of transcription factors in plants. They participate in diverse physiological and developmental processes. WRKY genes have been identified and characterized in a number of plant species. We identified a total of 58 WRKY genes (JcWRKY) in the genome of the physic nut (Jatropha curcas L.). On the basis of their conserved WRKY domain sequences, all of the JcWRKY proteins could be assigned to one of the previously defined groups, I-III. Phylogenetic analysis of JcWRKY genes with Arabidopsis and rice WRKY genes, and separately with castor bean WRKY genes, revealed no evidence of recent gene duplication in JcWRKY gene family. Analysis of transcript abundance of JcWRKY gene products were tested in different tissues under normal growth condition. In addition, 47 WRKY genes responded to at least one abiotic stress (drought, salinity, phosphate starvation and nitrogen starvation) in individual tissues (leaf, root and/or shoot cortex). Our study provides a useful reference data set as the basis for cloning and functional analysis of physic nut WRKY genes.

Comparison of the starch synthesis genes between maize and rice: copies, chromosome location and expression divergence

Gene duplication and divergence are important evolutionary processes. It has been suggested that a whole genome duplication (WGD) event occurred in the Gramineae, predating its divergence, and a second WGD occurred in maize during its evolution. In this study we compared the fate of the genes involved in the core pathway of starch biosynthesis following the ancient and second WGDs in maize and rice. In total, thirty starch synthesis genes were detected in the maize genome, which covered all the starch synthesis gene families encoded by 27 genes in rice. All of these genes, except ZmGBSSIIb and ZmBEIII, are anchored within large-scale synteny blocks of rice and maize chromosomes. Previous findings and our results indicate that two of the current copies of many starch synthesis genes (including AGPL, AGPS, GBSS, SSII, SSIII, and BEII) probably arose from the ancient WGD in the Gramineae and are still present in the maize and rice genome. Furthermore, two copies of at least six genes (AGPS1, SSIIb, SSIIIb, GBSSII, BEI, and ISA3) appear to have been retained in the maize genome after its second WGD, although complete coding regions were only detected among the duplicate sets of AGPS1, SSIIb, and SSIIIb. The expression patterns of the remaining duplicate sets of starch synthesis genes (AGPL1/2, AGPS1/2, SSIIa/b, SSIIIa/b, GBSSI/II, and BEIIa/b) differ in their expression and could be classified into two groups in maize. The first group is mainly expressed in the endosperm, whereas the second is expressed in other organs and the early endosperm development. The four duplicate sets of ZmGBSSII, ZmSSIIb, ZmSSIIIb and AGPS1, which arose from the second WGD diverged in gene structure and/or expression patterns in maize. These results indicated that some duplicated starch synthesis genes were remained, whereas others diverged in gene structure and/or expression pattern in maize. For most of the duplicated genes, one of the copies has disappeared in the maize genome after the WGD and the subsequent “diploidization”.

The Stay-Green Rice like (SGRL) gene regulates chlorophyll degradation in rice.

The Stay-Green Rice (SGR) protein is encoded by the SGR gene and has been shown to affect chlorophyll (Chl) degradation during natural and dark-induced leaf senescence. An SGR homologue, SGR-like (SGRL), has been detected in many plant species. We show that SGRL is primarily expressed in green tissues, and is significantly downregulated in rice leaves undergoing natural and dark-induced senescence. As the light intensity increases during the natural photoperiod, the intensity of SGRL expression declines while that of SGR expression increases. Overexpression of SGRL reduces the levels of Chl and Chl-binding proteins in leaves, and accelerates their degradation in dark-induced senescence leaves in rice. Our results suggest that the SGRL protein is also involved in Chl degradation. The relationship between SGRL and SGR and their effects on the degradation of the light-harvesting Chl a/b-binding protein are also discussed.

Global analysis of gene expression profiles in physic nut (Jatropha curcas L.) seedlings exposed to salt stress.

Background Salt stress interferes with plant growth and production. Plants have evolved a series of molecular and morphological adaptations to cope with this abiotic stress, and overexpression of salt response genes reportedly enhances the productivity of various crops. However, little is known about the salt responsive genes in the energy plant physic nut (Jatropha curcas L.). Thus, excavate salt responsive genes in this plant are informative in uncovering the molecular mechanisms for the salt response in physic nut. Methodology/Principal Findings We applied next-generation Illumina sequencing technology to analyze global gene expression profiles of physic nut plants (roots and leaves) 2 hours, 2 days and 7 days after the onset of salt stress. A total of 1,504 and 1,115 genes were significantly up and down-regulated in roots and leaves, respectively, under salt stress condition. Gene ontology (GO) analysis of physiological process revealed that, in the physic nut, many “biological processes” were affected by salt stress, particular those categories belong to “metabolic process”, such as “primary metabolism process”, “cellular metabolism process” and “macromolecule metabolism process”. The gene expression profiles indicated that the associated genes were responsible for ABA and ethylene signaling, osmotic regulation, the reactive oxygen species scavenging system and the cell structure in physic nut. Conclusions/Significance The major regulated genes detected in this transcriptomic data were related to trehalose synthesis and cell wall structure modification in roots, while related to raffinose synthesis and reactive oxygen scavenger in leaves. The current study shows a comprehensive gene expression profile of physic nut under salt stress. The differential expression genes detected in this study allows the underling the salt responsive mechanism in physic nut with the aim of improving its salt resistance in the future.

Overexpression of SGR Results in Oxidative Stress and Lesion-mimic Cell Death in Rice Seedlings

It is thought that the Stay Green Rice (SGR) gene is involved in the disaggregation of the light harvesting complex and in the subsequent breakdown of chlorophyll and apo-protein during senescence. In this study, we found that overexpression of SGR (Ov-SGR) resulted in the generation of singlet oxygen and other reactive oxygen species and produced a chlorophyll-dependent regional cell death phenotype on leaves of rice seedlings. Transcriptome analyses using Affymetrix Rice GeneChips revealed that Ov-SGR rice seedlings exhibited a number of signs of singlet oxygen response. The genes and their associated biochemical pathways identified provide an insight into how rice plants respond to singlet oxygen at the molecular and physiologic level.

Knockdown of LjIPT3 influences nodule development in Lotus japonicus

Cytokinins play important roles in legume-rhizobia symbiosis. Here we report isolation of six genes encoding isopentenyl transferase (IPT) from Lotus japonicus, which catalyze the rate-limiting step of cytokinin biosynthesis. The LjIPT3 gene was found to be up-regulated in infected roots and mature nodules. Histochemical analysis demonstrated expression of Pro(LjIPT3):GUS (β-glucuronidase) in vegetative and reproductive organs, and was especially high in the vascular bundles of roots. When inoculated with Mesorhizobium loti MAFF303099, LjIPT3 was undetectable in the nodule primordia and developing nodules, and later it was expressed only in the vascular bundles of mature nodules. In addition, knockdown of LjIPT3 (LjIPT3i) by RNA interference reduced levels of endogenous cytokinins, affected plant development and accelerated Chl degradation during dark-induced leaf senescence. Compared with the wild type, LjIPT3i plants produced fewer infection threads and nodules. In addition, expression of downstream nodulation-related transcription factor genes LjNSP1, LjNSP2 and LjNIN decreased dramatically in LjIPT3i plants. These results suggest that LjIPT3 regulates the CRE1-dependent cytokinin pathway, affecting nodule initiation and thereby influencing the number of infection threads and nodules. Detection of nitrogenase activity and observation of nodule structure showed that endogenous cytokinins are required for full development of the infected cells in mature nodules by preventing early senescence. Therefore, our results indicate that the LjIPT3 gene product is required for nodule initiation and development, and does not appear to be involved in early infection events.

Genome-Wide Analysis of the AP2/ERF Gene Family in Physic Nut and Overexpression of the JcERF011 Gene in Rice Increased Its Sensitivity to Salinity Stress

The AP2/ERF transcription factors play crucial roles in plant growth, development and responses to biotic and abiotic stresses. A total of 119 AP2/ERF genes (JcAP2/ERFs) have been identified in the physic nut genome; they include 16 AP2, 4 RAV, 1 Soloist, and 98 ERF genes. Phylogenetic analysis indicated that physic nut AP2 genes could be divided into 3 subgroups, while ERF genes could be classed into 11 groups or 43 subgroups. The AP2/ERF genes are non-randomly distributed across the 11 linkage groups of the physic nut genome and retain many duplicates which arose from ancient duplication events. The expression patterns of several JcAP2/ERF duplicates in the physic nut showed differences among four tissues (root, stem, leaf, and seed), and 38 JcAP2/ERF genes responded to at least one abiotic stressor (drought, salinity, phosphate starvation, and nitrogen starvation) in leaves and/or roots according to analysis of digital gene expression tag data. The expression of JcERF011 was downregulated by salinity stress in physic nut roots. Overexpression of the JcERF011 gene in rice plants increased its sensitivity to salinity stress. The increased expression levels of several salt tolerance-related genes were impaired in the JcERF011-overexpressing plants under salinity stress.