Shenghua Wang

Growth and antioxidant responses in Jatropha curcas seedling exposed to mercury toxicity.

Jatropha curcas seedlings were exposed to varying concentrations of mercury in order to investigate mercury accumulation, and the changes in growth and antioxidant enzyme activities using in vitro embryo germination and culture. Our results showed that mercury is readily accumulated by germinating embryos and growing seedlings, and its content was greater in the radicles than those of in the cotyledons and hypocotyls. This accumulation was directly correlated with an increase in tested mercury concentrations in the medium. Biomass in the cotyledons, hypocotyls and radicles increased gradually with increasing mercury concentrations, peaking in seedlings exposed to mercury concentration of 50 microM, and then decreased. Superoxide dismutase activities in the cotyledons, hypocotyls and radicles showed largest increment at mercury concentration of 100 microM. Peroxidase activities in the cotyledons and hypocotyls reached peaks at mercury concentration of 200 microM, and the highest activity in the radicles was observed at 100 microM. Catalase activities in the cotyledons and hypocotyls were significantly induced, and the highest activity in the radicles was observed at mercury concentration of 200 microM. Phenylalanine ammonia-lyase activities in the hypocotyls had a positive correlation to mercury concentrations, and the highest activities in the cotyledons and radicles were found at mercury concentrations of 200 and 100 microM, respectively. Analysis of superoxide dismutase, peroxidase and catalase isoenzymes suggested that different patterns depend on mercury concentrations and tissue types, and the staining intensities of these isoenzymes are consistent with the changes of these enzyme activities assayed in solutions.

In vitro flowering of bitter melon

Abstract. Flowers were formed from shoot tips of bitter melon (Momordica charantia L.) cultured on Murashige and Skoog medium supplemented with 90xa0mM sucrose, 0.05xa0mM Fe2+ and 4xa0µM N6-benzyladenine (BA). The addition of 0.05xa0mM Fe2+ to the medium prevented chlorosis of the explant and promoted normal flowering. Increasing the ratio of carbon to nitrogen promoted male flower formation but intensively inhibited vegetative growth. The influence of cytokinin on the morphogenesis of the explant was highly notable. Flowers could be formed after a 15- to 20-day exposure to kinetin (Kin) or BA. Kin and BA had opposite effects with regard to the development of the explant. Kin promoted flower formation, especially female, but inhibited branch bud formation. Conversely, BA promoted branch bud formation and also promoted male flower formation when present at a concentration of 1–2xa0µM, but completely inhibited flower formation at 4–8xa0µM. Fluorescein diacetate staining and in vitro germination showed that in vitro pollen were of a fairly high viability.

MicroRNA profiles and their control of male gametophyte development in rice

Plant microRNAs (miRNAs) act as negative regulators of gene expression by slicing target transcripts or inhibiting translation. A number of miRNAs play important roles in development. In order to investigate the potential function of miRNAs during male gametogenesis in rice, we obtained both gene and small RNA expression profiles by combining microarray and high-throughput sequencing technologies. From the microarray datasets, 2,925 male gametophyte-specific genes were identified, including 107 transcription factors and three significant Argonaute genes (AGO12, AGO13, and AGO17). From the sRNA-Seq datasets, 104 unique miRNAs (miRus) were identified, including 47 known miRus and 57 novel miRus; interestingly, most of the new miRus are pollen-specific and not conserved among species. Furthermore, an interactive network of miRNA-target was constructed based on the two datasets. By employing enrichment analysis, the miRNA-regulated targets were found to be involved in both the up and down pathways, but predominantly in the down pathways, including 37 GO biological processes and 32 KEGG pathways. These findings indicate that miRNAs play a broad regulatory role during male gametophyte development in rice.

Effects of low nitrogen and drought stresses on proline synthesis of Jatropha curcas seedling

Proline is one of the most important osmoregulatory solutes subjected to osmotic stresses. In this study, low nitrogen supply suppressed the dry biomass, leaf area, and proline biosynthesis of the seedlings of the energy plant Jatropha curcas, which could grow in poor, dry soil. Low-nitrogen stress induced JcP5CS mRNA expression and decreased the activity of P5CS enzyme and the content of free proline in leaves of J. curcas seedlings. When the seedlings grown in low-nitrogen conditions were suddenly exposed to PEG-6000 (−1.6xa0MPa) stress, the expression of JcP5CS gene was highly induced, and both the activity of P5CS and the content of free proline increased and maintained at high levels to mitigate the impact of drought stresses. This may be one of the reasons why J. curcas could adapt to poor and drought conditions.

Molecular cloning and characterization of phospholipase D from Jatropha curcas

Phospholipase D (PLD, EC 3.1.4.4) is a key enzyme involved in phospholipid catabolism, initiating a lipolytic cascade in membrane deterioration during senescence and stress, which was cloned from Jatropha curcas L., an important plant species as its seed is the raw material for biodiesels. The cDNA was 2,886xa0bp in length with a complete open reading frame of 2,427xa0bp which encoded a polypeptide of 808 amino acids including a putative signal peptide of 53 amino acid residues and a mature protein of 755 amino acids with a predicted molecular mass of 86xa0kD and a pI of 5.44, having two highly conserved ‘HKD’ motifs. Phylogenetic analysis indicated the J. curcas PLD alpha (JcPLDα) showed a high similarity to other PLD alpha from plants. Semi-quantitative RT-PCR analysis revealed that it was especially abundant in root, stem, leaf, endosperm and flower, weakly in seed. And the JcPLDα was increasedly expressed in leaf undergoing environmental stress such as salt (300xa0mM NaCl), drought (30% PEG), cold (4°C) and heat (50°C). The JcPLDα protein was successfully expressed in Escherichia coli and showed high enzymatic activities. Maximal activity was at pH 8 and 60°C.

Molecular cloning and characterization of a jasmonate biosynthetic pathway gene for allene oxide cyclase from Jatropha curcas.

Herein, we cloned a full-length cDNA encoding allene oxide cyclase (AOC, EC 5.3.99.6) that is a key enzyme in jasmonates (JAs) biosynthetic pathway from Jatropha curcas L., an important plant species as its seed is the raw material for biodiesels, named as JcAOC (GenBank accession no. FJ874630). The cDNA was 924xa0bp in length with a complete open reading frame of 750xa0bp, which encoded a polypeptide of 250 amino acids including a putative signal peptide of 65 amino acid residues and a mature protein of 185 amino acids with a predicted molecular mass of 20.7xa0kDa and a isoelectric point of 6.24. Phylogenetic analysis indicated that JcAOC belonged to the AOC superfamily. Semi-quantitative RT-PCR analysis revealed that JcAOC mRNA was expressed in roots, stems, leaves, young seeds, endosperms, and flowers, but that the expression level was highest in leaves and lowest in seeds, and mRNA expression of JcAOC could be induced by salt stress (300xa0mM NaCl) and low temperature (4°C). Furthermore, the full-length coding region of JcAOC excluding signal peptide sequence was inserted into pET-30a and was successfully expressed in Escherichia coli. Overexpression of JcAOC in E. coli conferred its resistance to salt stress and low temperature.

Cloning and characterization of a novel Δ12-fatty acid desaturase gene from the tree Sapium sebiferum

A new full-length cDNA (Ssd12) encoding a Δ12-fatty acid desaturase (Δ12-FAD) was cloned from Sapium sebiferum using RT-PCR and RACE methods. Ssd12 contained a 1146xa0bp open reading frame encoding a protein of 381 amino acids. The amino acid sequence showed a much higher match with microsomal Δ12-FAD amino acid sequences than chloroplast Δ12-FAD amino acid sequences. Genomic Southern blot analysis suggested that Ssd12 had at least two copies. Ssd12 transcripts were detected in roots, leaves, stems, and seeds by real time PCR.

Genome-Wide Analysis Reveals Diversity of Rice Intronic miRNAs in Sequence Structure, Biogenesis and Function

Intronic microRNAs (in-miRNAs) as a class of miRNA family that regulates gene expression are still poorly understood in plants. In this study, we systematically identified rice in-miRNAs by re-mining eight published small RNA-sequencing datasets of rice. Furthermore, based on the collected expression, annotation, and putative target data, we investigated the structures, potential functions, and expression features of these in-miRNAs and the expression patterns of their host genes. A total of 153 in-miRNAs, which account for over 1/4 of the total rice miRNAs, were identified. In silico expression analysis showed that most of them (∼63%) are tissue or stage-specific. However, a majority of their host genes, especially those containing clustered in-miRNAs, exhibit stable high-level expressions among 513 microarray datasets. Although in-miRNAs show diversity in function and mechanism, the DNA methylation directed by 24 nt in-miRNAs may be the main pathway that controls the expressions of target genes, host genes, and even themselves. These findings may enhance our understanding on special functions of in-miRNAs, especially in mediating DNA methylation that was concluded to affect the stability of expression and structure of host and target genes.

Cloning and characterization of a cDNA encoding type 1 diacylglycerol acyltransferase from sunflower (Helianthus annuus L.).

A full-length cDNA encoding a putative diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) was obtained from sunflower (Helianthus annuus L.) seeds. The 1524-bp open reading frame of this cDNA, designated as HaDGAT1, encodes a protein of 507 amino acids with a molecular mass of 58.5 kDa showing high homology to DGAT1 enzymes of other plants. The protein characters, such as a predicted structure with a long N-terminal hydrophilic domain followed by 9 transmembrane domains, acyl-CoA-binding signature, diacylglycerol (DAG)-binding and putative endoplasmic reticulum retrieval motifs (ER-DIR), also indicated that HaDGAT belongs to the DGAT1 family. HaDGAT1 is expressed in all plant tissues especially in developing seeds. Expression of recombinant HaDGAT1 in yeast showed an 1.76-fold increase of total fatty acids, especially unsaturated fatty acids such as palmitoleic acid (enhanced by 86.6%) and oleic acid (enhanced by 81.6%).

Molecular cloning, characterization, and expression of an omega-3 fatty acid desaturase gene from Sapium sebiferum

A full-length cDNA (SsFAD3) for an omega-3 fatty acid desaturase (omega-3 FAD) was cloned from Sapium sebiferum (L.) Roxb. using rapid amplification of cDNA ends and reverse transcription polymerase chain reaction methods. SsFAD3 contained a 1119-bp open reading frame encoding a 372-amino acid polypeptide. The genomic sequence region of the SsFAD3 ORF was composed of 8 exons and 7 introns, similar to other omega-3 FADs found in most plants. The amino acid sequence showed a higher identity with microsomal omega-3 FADs than plastidial omega-3 FADs. Southern blot analysis of SsFAD3 suggested the existence of a small gene family composed of several copies or closely linked genes. SsFAD3 transcripts were detected in shoots, roots, leaves, stems, and seeds, but were most abundant in shoots. The function of SsFAD3 was confirmed by the accumulation of alpha-linolenic acid (alpha-18:3) in Saccharomyces cerevisiae transformants.