Shi-Qing Peng

Identification and expression profiles of the WRKY transcription factor family in Ricinus communis

In plants, WRKY proteins constitute a large family of transcription factors. They are involved in many biological processes, such as plant development, metabolism, and responses to biotic and abiotic stresses. A large number of WRKY transcription factors have been reported from Arabidopsis, rice, and other higher plants. The recent publication of the draft genome sequence of castor bean (Ricinus communis) has allowed a genome-wide search for R. communis WRKY (RcWRKY) transcription factors and the comparison of these positively identified proteins with their homologs in model plants. A total of 47 WRKY genes were identified in the castor bean genome. According to the structural features of the WRKY domain, the RcWRKY are classified into seven main phylogenetic groups. Furthermore, putative orthologs of RcWRKY proteins in Arabidopsis and rice could now be assigned. An analysis of expression profiles of RcWRKY genes indicates that 47 WRKY genes display differential expressions either in their transcript abundance or expression patterns under normal growth conditions.

Genome-wide identification and characterization of WRKY gene family in Hevea brasiliensis

WRKY proteins constitute a large family of transcription factors. In this study, we identified 81 WRKY genes (named HbWRKY1 to HbWRKY81) in the latest rubber tree genome. Tissue-specific expression profiles showed that 74 HbWRKYs were expressed in at least one of the tissues and the other 7 genes showed very low expression in all tissues tested, which suggested that HbWRKYs took part in many cellular processes. The responses of 20 selected HbWRKYs to jasmonic acid (JA) and ethylene (ET) were analyzed in the latex. 17 HbWRKYs responded to at least one treatment, which included 15 HbWRKYs responding to JA treatment, 15 HbWRKYs to ET, which suggested that these HbWRKYs were regulated by JA and ET. We also observed that HbWRKY3, 14, and 55 bind HbSRPP promoter and activate the transcription in yeast. This study suggests that HbWRKY proteins maybe involved in the transcriptional regulation of nature rubber biosynthesis.

Complete Chloroplast Genome Sequence of Aquilaria sinensis (Lour.) Gilg and Evolution Analysis within the Malvales Order

Aquilaria sinensis (Lour.) Gilg is an important medicinal woody plant producing agarwood, which is widely used in traditional Chinese medicine. High-throughput sequencing of chloroplast (cp) genomes enhanced the understanding about evolutionary relationships within plant families. In this study, we determined the complete cp genome sequences for A. sinensis. The size of the A. sinensis cp genome was 159,565 bp. This genome included a large single-copy region of 87,482 bp, a small single-copy region of 19,857 bp, and a pair of inverted repeats (IRa and IRb) of 26,113 bp each. The GC content of the genome was 37.11%. The A. sinensis cp genome encoded 113 functional genes, including 82 protein-coding genes, 27 tRNA genes, and 4 rRNA genes. Seven genes were duplicated in the protein-coding genes, whereas 11 genes were duplicated in the RNA genes. A total of 45 polymorphic simple-sequence repeat loci and 60 pairs of large repeats were identified. Most simple-sequence repeats were located in the noncoding sections of the large single-copy/small single-copy region and exhibited high A/T content. Moreover, 33 pairs of large repeat sequences were located in the protein-coding genes, whereas 27 pairs were located in the intergenic regions. Aquilaria sinensis cp genome bias ended with A/T on the basis of codon usage. The distribution of codon usage in A. sinensis cp genome was most similar to that in the Gonystylus bancanus cp genome. Comparative results of 82 protein-coding genes from 29 species of cp genomes demonstrated that A. sinensis was a sister species to G. bancanus within the Malvales order. Aquilaria sinensis cp genome presented the highest sequence similarity of >90% with the G. bancanus cp genome by using CGView Comparison Tool. This finding strongly supports the placement of A. sinensis as a sister to G. bancanus within the Malvales order. The complete A. sinensis cp genome information will be highly beneficial for further studies on this traditional medicinal plant. Moreover, the results will enhance our understanding about the evolution of cp genomes of the Malvales order, particularly with regard to the role of A. sinensis in plant systematics and evolution.

Three MADS-box genes of Hevea brasiliensis expressed during somatic embryogenesis and in the laticifer cells

Three MADS-box genes, designated HbMADS1, HbMADS2 and HbMADS3, were isolated from Hevea brasiliensis. HbMADS1, HbMADS2 and HbMADS3 encode polypetides consisting of 245, 217 and 239 amino acids, respectively, containing conserved MADS-box motifs at N-terminus. Transcription pattern analysis revealed that three MADS-box genes had highly transcription in the laticifer cells. The transcriptions of HbMADS1and HbMADS3 were induced in the laticifer cells by jamonic acid, while HbMADS2 was not induction by jamonic acid. Ethephone is not effective in inducing their expression. The three genes were differentially expressed during somatic embryogenesis of rubber tree. Characterization of HbMADSs will attribute to understand their possible function in rubber tree.

Molecular characterization of a thioredoxin h gene (HbTRX1) from Hevea brasiliensis showing differential expression in latex between self-rooting juvenile clones and donor clones

The cDNA code of thioredoxin h, designated as HbTRX1, was isolated from Hevea brasiliensis by rapid amplification of cDNA ends. HbTRX1 contained a 542-bp open reading frame encoding 123 amino acids. The deduced HbTRX1 protein showing high identity to thioredoxin h of other plant species was predicted to possess the conserved catalytic site WCXPC. Semiquantitative reverse transcription-polymerase chain reaction analysis revealed that HbTRX1 was constitutively expressed in all tested tissues. HbTRX1 transcripts accumulated at relatively low levels in the flower, somatic embryo, and leaves, while HbTRX1 transcripts accumulated at relatively high levels in the callus and latex. The HbTRX1 transcript was expressed at different levels, with higher levels in self-rooting juvenile clones than in their donor clones. HbTRX1 was expressed in Escherichiacoli, and its activity was demonstrated using the dithiothreitol-dependent insulin assay. This work provides a basis for studying the biological function of thioredoxin h in rubber tree.

Comparative Transcriptome Analysis of Latex Reveals Molecular Mechanisms Underlying Increased Rubber Yield in Hevea brasiliensis Self-Rooting Juvenile Clones

Rubber tree (Hevea brasiliensis) self-rooting juvenile clones (JCs) are promising planting materials for rubber production. In a comparative trial between self-rooting JCs and donor clones (DCs), self-rooting JCs exhibited better performance in rubber yield. To study the molecular mechanism associated with higher rubber yield in self-rooting JCs, we sequenced and comparatively analyzed the latex of rubber tree self-rooting JCs and DCs at the transcriptome level. Total raw reads of 34,632,012 and 35,913,020 bp were obtained from the library of self-rooting JCs and DCs, respectively, by using Illumina HiSeq 2000 sequencing technology. De novo assemblies yielded 54689 unigenes from the library of self-rooting JCs and DCs. Among 54689 genes, 1716 genes were identified as differentially expressed between self-rooting JCs and DCs via comparative transcript profiling. Functional analysis showed that the genes related to the mass of categories were differentially enriched between the two clones. Several genes involved in carbohydrate metabolism, hormone metabolism and reactive oxygen species scavenging were up-regulated in self-rooting JCs, suggesting that the self-rooting JCs provide sufficient molecular basis for the increased rubber yielding, especially in the aspects of improved latex metabolisms and latex flow. Some genes encoding epigenetic modification enzymes were also differentially expressed between self-rooting JCs and DCs. Epigenetic modifications may lead to gene differential expression between self-rooting JCs and DCs. These data will provide new cues to understand the molecular mechanism underlying the improved rubber yield of H. brasiliensis self-rooting clones.

Cloning and characterization of a novel cysteine protease gene (HbCP1) from Hevea brasiliensis

The full-length cDNA encoding a cysteine protease, designated HbCP1, was isolated for the first time from Hevea brasiliensis by the rapid amplification of cDNA ends (RACE) method. HbCP1 contained a 1371 bp open reading frame encoding 457 amino acids. The deduced HbCP1 protein, which showed high identity to cysteine proteases of other plant species, was predicted to possess a putative repeat in toxin (RTX) domain at the N-terminal and a granulin (GRAN) domain at the C-terminal. Southern blot analysis indicated that the HbCP1 gene is present as a single copy in the rubber tree. Transcription pattern analysis revealed that HbCP1 had high transcription in laticifer, and low transcription in bark and leaf. The transcription of HbCP1 in latex was induced by ethylene and tapping. Cloning of the HbCP1 gene will enable us to further understand the molecular characterization of cysteine protease and its possible function in the rubber tree.

Protein differential expression in the latex from Hevea brasiliensis between self-rooting juvenile clones and donor clones

To better understand molecular mechanism underlying the difference between self-rooting juvenile clones and donor clones, a proteomic approach was used to profile protein changes in the latex between self-rooting juvenile clones and donor clones. Total soluble proteins were extracted from latex in self-rooting juvenile clones and donor clones. Two-dimensional gel electrophoresis (2-DE) was used to identify proteins that were differentially expressed in self-rooting juvenile clones and donor clones and image analysis was used to determine which proteins were up- or down-regulated. Twenty-four differentially expressed protein spots were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Among the 24 proteins identified, 13 proteins were up-regulated, 11 proteins were decreased in self-rooting juvenile clones. These proteins were classified as carbohydrate and energy metabolism, secondary metabolism, signal translocation, transcriptional regulation-related, protein synthesis and degradation, transport, nucleoside acid process, lipid metabolism. Perhaps, the present study contributes towards an understanding of the molecular mechanism underlying the difference between self-rooting juvenile clones and donor clones.

HbMADS4, a MADS-box Transcription Factor from Hevea brasiliensis, Negatively Regulates HbSRPP

In plants MADS-box transcription factors (TFs) play important roles in growth and development. However, no plant MADS-box gene has been identified to have a function related to secondary metabolites regulation. Here, a MADS-box TF gene, designated as HbMADS4, was isolated from Hevea brasiliensis by the yeast one-hybrid experiment to screen the latex cDNA library using the promoter of the gene encoding H. brasiliensis small rubber particle protein (HbSRPP) as bait. HbMADS4 was 984-bp containing 633-bp open reading frame encoding a deduced protein of 230 amino acid residues with a typical conserved MADS-box motif at the N terminus. HbMADS4 was preferentially expressed in the latex, but little expression was detected in the leaves, flowers, and roots. Its expression was inducible by methyl jasmonate and ethylene. Furthermore, transient over-expression and over-expression of HbMADS4 in transgenic tobacco plants significantly suppressed the activity of the HbSRP promoter. Altogether, it is proposed that HbMADS4 is a negative regulator of HbSRPP which participates in the biosynthesis of natural rubber.

Molecular characterization of the Jatropha curcas JcR1MYB1 gene encoding a putative R1-MYB transcription factor

The cDNA encoding the R1-MYB transcription factor, designated as JcR1MYB1, was isolated from Jatropha curcas using rapid amplification of cDNA ends. JcR1MYB1 contains a 951 bp open reading frame that encodes 316 amino acids. The deduced JcR1MYB1 protein was predicted to possess the conserved, 56-amino acid-long DNA-binding domain, which consists of a single helix-turn-helix module and usually occurs in R1-MYBs. JcR1MYB1 is a member of the R1-MYB transcription factor subfamily. A subcellular localization study confirmed the nuclear localization of JcR1MYB1. Expression analysis showed that JcR1MYB1 transcripts accumulated in various examined tissues, with high expression levels in the root and low levels in the stem. JcR1MYB1 transcription was up-regulated by polyethylene glycol, NaCl, and cold treatments, as well as by abscisic acid, jasmonic acid, and ethylene treatment. Analysis of transgenic tobacco plants over-expressing JcR1MYB1 indicates an inportant function for this gene in salt stress.