Dong Guo

Coordinated regulation of anthocyanin biosynthesis through photorespiration and temperature in peach (Prunus persica f. atropurpurea)

The usual red color of young leaves of peach (Prunus persica f. atropurpurea) is due to the accumulation of anthocyanin. Real-time PCR analysis revealed a strong correlation between the expression levels of anthocyanin biosynthetic genes and anthocyanin content in leaves at different developmental stages. The expression profiles of both anthocyanin biosynthetic genes and photorespiratory genes showed significant changes in leaves held in the dark or exposed to heat stress, compared with controls. The expression of anthocyanin biosynthetic genes dramatically decreased in peach red leaves following dark or heat treatments, resulting in a significant decrease of anthocyanin accumulation. However, the photorespiration-related genes GDCH and GOX exhibited increased expression in peach leaves after dark or heat treatment. Moreover, the expression levels of GDCH and GOX in the Arabidopsis chi/f3′h mutant that does not accumulate anthocyanins were higher than in the wild type. Overall, these results support the hypothesis that photorespiration-related genes might be involved in the regulation of anthocyanin biosynthesis. This finding provides a new insight into our understanding of the mechanism underlying the control of anthocyanin biosynthesis in plants.

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.

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 EIN3/EIL gene family in Hevea brasiliensis

Ethylene-insensitive3/ethylene-insensitive3-like (EIN3/EIL) transcription factor is a key transcription factor involved in the ethylene signal transduction pathway in plants. Although the genome-wide analysis of this family has been carried out in certain plant species, little is known about EIN3/EIL genes in rubber tree (Hevea brasiliensis). In this study, we identified four EIN3/EIL genes (designated as HbEIN3-1 to HbEIN3-4) in the rubber tree genome annotation. Tissue-specific expression profiles showed that four HbEIN3s were expressed in at least one of the tested tissues. The four HbEIN3s responded to ethylene (ET) and jasmonic acid (JA) treatment, which suggested that HbEIN3s were involved in response to JA and ET signaling. HbEIN3s showed transactivation activity in yeast. HbEIN3-1 bound to the promoter of the rubber transferase gene, suggesting that HbEIN3-1 may be involved in the regulation of natural rubber biosynthesis. The N-terminal basic domain II was required for the HbEIN3-1 binding with the promoter of the rubber transferase gene.

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.

Transcriptome-wide identification and expression analysis of glutathione S-transferase genes involved in flavonoids accumulation in Dracaena cambodiana.

Dragons blood is a traditional medicine widely used in the world, and the main components of which are flavonoids. However, little is known about its formation mechanism. Previous studies indicate that plant glutathione S-transferase (GST) genes are involved in transportation of flavonoids from cytosolic synthesis to vacuolar accumulation. In this study, 20 Dracaena cambodiana GST genes (DcGSTs) were identified based on transcriptome database. Phylogenetic analysis revealed that 20 DcGSTs belonged to seven different classes. Tissue-specific expression analysis suggested that DcGSTs displayed differential expressions either in their transcript abundance or expression patterns under normal growth conditions. The transcript profiles of three DcGSTs in response to the inducer of dragons blood were strongly correlated with flavonoids biosynthetic genes, consistent with dragons blood accumulation. Our survey provides useful information for future studies on GST genes involved in dragons blood formation in D.xa0cambodiana.

Molecular characterization of HbCZF1, a Hevea brasiliensis CCCH-type zinc finger protein that regulates hmg1

Key messageThe HbCZF1 protein binds to thehmg1promoter in yeast and this interaction was confirmed in vitro. Thehmg1promoter was activated in transgenic plants by HbCZF1.AbstractBiosynthesis of natural rubber is known to be based on the mevalonate pathway in Hevea brasiliensis. The final step in the mevalonate production is catalyzed by the branch point enzyme, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR), which shunts HMG-CoA into the isoprenoid pathway, leading to the synthesis of natural rubber. However, molecular regulation of HMGR expression is not known. To study the transcriptional regulation of HMGR, the yeast one-hybrid experiment was performed to screen the latex cDNA library using the hmg1 (one of the three HMGR in H. brasiliensis) promoter as bait. One cDNA that encodes the CCCH-type zinc finger protein, designated as HbCZF1, was isolated from H. brasiliensis. HbCZF1 interacted with the hmg1 promoter in yeast one-hybrid system and in vitro. HbCZF1 contains a 1110xa0bp open reading frame that encodes 369 amino acids. The deduced HbCZF1 protein was predicted to possess a typical C-X7-C-X5-C3-H CCCH motif and RNA recognition motif. HbCZF1 was predominant in the latex, but little expression was detected in the leaves, barks, and roots. Furthermore, in transgenic tobacco plants, over-expression of HbCZF1 highly activated the hmg1 promoter. These results suggested that HbCZF1 may participate in the regulation of natural rubber biosynthesis in H. brasiliensis.

The 14-3-3 protein HbGF14a interacts with a RING zinc finger protein to regulate expression of the rubber transferase gene in Hevea brasiliensis

Hevea brasiliensis is a key commercial source of natural rubber (cis 1,4-polyisoprene). In H. brasiliensis, rubber transferase is responsible for cis-1,4-polymerization of isoprene units from isopentenyl diphosphate and thus affects the yield of rubber. Little is known about the regulatory mechanisms of the rubber transferase gene at a molecular level. In this study we show that the 5UTR intron of the promoter of the rubber transferase gene (HRT2) suppresses the expression of HRT2. A H. brasiliensis RING zinc finger protein (designated as HbRZFP1) was able to interact specifically with the HRT2 promoter to down-regulate its transcription in vivo. A 14-3-3 protein (named as HbGF14a) was identified as interacting with HbRZFP1, both in yeast and in planta. Transient co-expression of HbGF14a and HbRZFP1-encoding cDNAs resulted in HbRZFP1-mediated HRT2 transcription inhibition being relieved. HbGF14a repressed the protein-DNA binding of HbRZFP1 with the HRT2 promoter in yeast. We propose a regulatory mechanism by which the binding of HbGF14a to HbRZFP1 interferes with the interaction of HbRZFP1 with the HRT2 promoter, thereby repressing the protein-DNA binding between them. This study provides new insights into the role of HbGF14a in mediating expression of the rubber transferase gene in Hevea brasiliensis.

Identification and characterization of MAGO and Y14 genes in Hevea brasiliensis

Abstract Mago nashi (MAGO) and Y14 proteins are highly conserved among eukaryotes. In this study, we identified two MAGO (designated as HbMAGO1 andHbMAGO2) and two Y14 (designated as HbY14aand HbY14b) genes in the rubber tree (Hevea brasiliensis) genome annotation. Multiple amino acid sequence alignments predicted that HbMAGO and HbY14 proteins are structurally similar to homologous proteins from other species. Tissue-specific expression profiles showed that HbMAGO and HbY14 genes were expressed in at least one of the tissues (bark, flower, latex, leaf and root) examined. HbMAGOs and HbY14s were predominately located in the nucleus and were found to interact in yeast two-hybrid analysis (YTH) and bimolecular fluorescence complementation (BiFC) assays. HbMAGOs and HbY14s showed the highest transcription in latex and were regulated by ethylene and jasmonate. Interaction between HbMAGO2 and gp91phox (a large subunit of nicotinamide adenine dinucleotide phosphate) was identified using YTH and BiFC assays. These findings suggested that HbMAGO may be involved in the aggregation of rubber particles in H. brasiliensis.