Jia-Hong Zhu

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.

De Novo transcriptome characterization of Dracaena cambodiana and analysis of genes involved in flavonoid accumulation during formation of dragon’s blood

Dragon’s blood is a red resin mainly extracted from Dracaena plants, and has been widely used as a traditional medicine in East and Southeast Asia. The major components of dragon’s blood are flavonoids. Owing to a lack of Dracaena plants genomic information, the flavonoids biosynthesis and regulation in Dracaena plants remain unknown. In this study, three cDNA libraries were constructed from the stems of D. cambodiana after injecting the inducer. Approximately 266.57 million raw sequencing reads were de novo assembled into 198,204 unigenes, of which 34,873 unique sequences were annotated in public protein databases. Many candidate genes involved in flavonoid accumulation were identified. Differential expression analysis identified 20 genes involved in flavonoid biosynthesis, 27 unigenes involved in flavonoid modification and 68 genes involved in flavonoid transport that were up-regulated in the stems of D. cambodiana after injecting the inducer, consistent with the accumulation of flavonoids. Furthermore, we have revealed the differential expression of transcripts encoding for transcription factors (MYB, bHLH and WD40) involved in flavonoid metabolism. These de novo transcriptome data sets provide insights on pathways and molecular regulation of flavonoid biosynthesis and transport, and improve our understanding of molecular mechanisms of dragon’s blood formation in D. cambodiana.

Identification and characterization of the abscisic acid (ABA) receptor gene family and its expression in response to hormones in the rubber tree

Abscisic acid (ABA) is an essential phytohormone involved in diverse physiological processes. Although genome-wide analyses of the ABA receptor PYR/PYL/RCAR (PYL) protein/gene family have been performed in certain plant species, little is known about the ABA receptor protein/gene family in the rubber tree (Hevea brasiliensis). In this study, we identified 14 ABA receptor PYL proteins/genes (designated HbPYL1 through HbPYL14) in the most recent rubber tree genome. A phylogenetic tree was constructed, which demonstrated that HbPYLs can be divided into three subfamilies that correlate well with the corresponding Arabidopsis subfamilies. Eight HbPYLs are highly expressed in laticifers. Five of the eight genes are simultaneously regulated by ABA, jasmonic acid (JA) and ethylene (ET). The identification and characterization of HbPYLs should enable us to further understand the role of ABA signal in the rubber tree.

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.

Identification and Functional Characterization of the DcF3’H Promoter from Dracaena cambodiana

Dragon’s blood is a traditional medicine that is produced mainly from Dracaenas plants and is widely used in the world. The main chemical compounds in dragon’s blood are flavonoids. Flavonoid 3′-hydroxylase (F3’H) is an enzyme that synthesizes 3′-hydroxylated flavonoids. DcF3’H plays a vital role in determining the flavonoid content during the production of dragon′s blood; however, the regulation of DcF3’H expression in Dracaenas plants is poorly understood. In this study, a 759-bp promoter region of DcF3’H was isolated from Dracaena cambodiana. The promoter sequence contained cis-acting elements related to various environmental stresses and hormones responses. Dual-luciferase assays showed that the DcF3’H promoter was up-regulated by 6-benzyl aminopurine (6-BA) and down-regulated by methyl jasmonate (MeJA), and these results were consistent with transcription levels of DcF3’H in stems that were verified by quantitative real-time PCR (qPCR). DcHLH1, a bHLH transcription factor identified in yeast one-hybrid screening, was capable of both binding to the promoter and activating the transcription of DcF3’H by ~10-fold. The results provide new insights into the regulatory mechanisms of DcF3’H in Dracaena cambodiana.

Identification, characterization and expression analysis of genes involved in steroidal saponin biosynthesis in Dracaena cambodiana

Dracaena cambodiana is a traditional medicinal plant used for producing dragon’s blood. The plants and dragon’s blood of D. cambodiana contain a rich variety of steroidal saponins. However, little is known about steroidal saponin biosynthesis and its regulation in D. cambodiana. Here, 122 genes encoding enzymes involved in steroidal saponin biosynthesis were identified based on transcriptome data, with 29 of them containing complete open reading frames (ORF). Transcript expression analysis revealed that several genes related to steroidal saponin biosynthesis showed distinct tissue-specific expression patterns; the expression levels of genes encoding the key enzymes involved in the biosynthesis and early modification of steroidal saponins were significantly down-regulated in the stems in response to the inducer of dragon’s blood, exhibiting positive correlations with the content of steroidal saponins. These results provide insights on the steroidal saponins biosynthetic pathway and mechanisms underlying induced formation of dragon’s blood in D. cambodiana.

Genome-wide identification, characterization, and expression analysis of SnRK2 family in Hevea brasiliensis

The sucrose non-fermenting 1-related protein kinase 2 (SnRK2) gene family belongs to a group of plant-specific serine/threonine kinase family involved in abscisic acid (ABA) signaling and biotic and abiotic stress response. Although genome-wide analyses of the SnRK2 gene family have been conducted in some species, little is known about the SnRK2 gene family in rubber tree (Hevea brasiliensis). In this study, we identified 10 SnRK2s designated as HbSnRK2.1 to HbSnRK2.10 in the rubber tree genome. The subsequently constructed phylogenetic tree demonstrated that HbSnRK2s have three subfamilies that correlate well with those of Arabidopsis sp. and rice subfamilies. All SnRK2 genes contained nine exons and eight introns. Although the C-terminus was divergent, eight conserved motifs were found. Motifs 1–6 were common to all HbSnRK2s. Expression analysis results showed that 7 of the 10 HbSnRK2s were highly expressed in latex. HbSnRK2.7 was predominantly expressed and simultaneously regulated by abscisic acid, jasmonic acid, and ethylene treatment in laticifers. HbSnRK identification and characterization provided further understanding on the role of ABA signal in the rubber tree.

Identification and expression analysis of genes involved in histone acetylation in Hevea brasiliensis

Histone post-translational modifications play a crucial role in epigenetic regulation of plant growth and development. Histone acetylation is a major and important post-translational modification of core histones. Based on the recently released draft of the rubber tree (Hevea brasiliensis) genome sequences, 23 genes encoding histone acetyltransferases (HATs) and 14 genes encoding histone deacetylases (HDACs) were identified in silico and were compared them with those detected in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) orthologs. Phylogenetic relationship and domain architecture were comprehensively analyzed. Additionally, expression profiles of HbHATs and HbHDACs were analyzed in different tissues and four periods during somatic embryogenesis of rubber tree. The transcription profiles of HbHATs and HbHDACs within rubber tree organs revealed a broad functional role for HbHATs and HbHDACs, suggesting key HbHATs and HbHDACs regulators in rubber tree development. Such a comprehensive analysis of these HbHATs and HbHDACs will provide fundamental understanding of their diverse roles in rubber tree development and be useful for future functional genomic studies on regulations of histone acetylation modifications in rubber tree.

Transcriptome-Wide Identification and Characterization of MYB Transcription Factor Genes in the Laticifer Cells of Hevea brasiliensis

MYB transcription factors hold vital roles in the regulation of plant secondary metabolic pathways. Laticifers in rubber trees (Hevea brasiliensis) are of primary importance in natural rubber production because natural rubber is formed and stored within these structures. To understand the role of MYB transcription factors in the specialized cells, we identified 44 MYB genes (named HblMYB1 to HblMYB44) by using our previously obtained transcriptome database of rubber tree laticifer cells and the public rubber tree genome database. Expression profiles showed that five MYB genes were highly expressed in the laticifers. HblMYB19 and HblMYB44 were selected for further study. HblMYB19 and HblMYB44 bound the promoters of HbFDPS1, HbSRPP, and HRT1 in yeast. Furthermore, the transient overexpression of HblMYB19 and HblMYB44 in tobacco plants significantly increased the activity of the promoters of HbFDPS1, HbSRPP, and HRT1. Basing on this information, we proposed that HblMYB19 and HblMYB44 are the regulators of HbFDPS1, HbSRPP, and HRT1, which are involved in the biosynthesis pathway of natural rubber.