Transcriptome analysis of two inflorescence branching mutants reveals cytokinin is an important regulator in controlling inflorescence architecture in the woody plant Jatropha curcas.

BACKGROUND: In higher plants, inflorescence architecture is an important agronomic trait directly determining seed yield. However, little information is available on the regulatory mechanism of inflorescence development in perennial woody plants. Based on two inflorescence branching mutants, we investigated the transcriptome differences in inflorescence buds between two mutants and wild-type (WT) plants by RNA-Seq to identify the genes and regulatory networks controlling inflorescence architecture in Jatropha curcas L., a perennial woody plant belonging to Euphorbiaceae. RESULTS: Two inflorescence branching mutants were identified in germplasm collection of Jatropha. The duo xiao hua (dxh) mutant has a seven-order branch inflorescence, and the gynoecy (g) mutant has a three-order branch inflorescence, while WT Jatropha has predominantly four-order branch inflorescence, occasionally the three- or five-order branch inflorescences in fields. Using weighted gene correlation network analysis (WGCNA), we identified several hub genes involved in the cytokinin metabolic pathway from modules highly associated with inflorescence phenotypes. Among them, Jatropha ADENOSINE KINASE 2 (JcADK2), ADENINE PHOSPHORIBOSYL TRANSFERASE 1 (JcAPT1), CYTOKININ OXIDASE 3 (JcCKX3), ISOPENTENYLTRANSFERASE 5 (JcIPT5), LONELY GUY 3 (JcLOG3) and JcLOG5 may participate in cytokinin metabolic pathway in Jatropha. Consistently, exogenous application of cytokinin (6-benzyladenine, 6-BA) on inflorescence buds induced high-branch inflorescence phenotype in both low-branch inflorescence mutant (g) and WT plants. These results suggested that cytokinin is an important regulator in controlling inflorescence branching in Jatropha. In addition, comparative transcriptome analysis showed that Arabidopsis homologous genes Jatropha AGAMOUS-LIKE 6 (JcAGL6), JcAGL24, FRUITFUL (JcFUL), LEAFY (JcLFY), SEPALLATAs (JcSEPs), TERMINAL FLOWER 1 (JcTFL1), and WUSCHEL-RELATED HOMEOBOX 3 (JcWOX3), were differentially expressed in inflorescence buds between dxh and g mutants and WT plants, indicating that they may participate in inflorescence development in Jatropha. The expression of JcTFL1 was downregulated, while the expression of JcLFY and JcAP1 were upregulated in inflorescences in low-branch g mutant. CONCLUSIONS: Cytokinin is an important regulator in controlling inflorescence branching in Jatropha. The regulation of inflorescence architecture by the genes involved in floral development, including TFL1, LFY and AP1, may be conservative in Jatropha and Arabidopsis. Our results provide helpful information for elucidating the regulatory mechanism of inflorescence architecture in Jatropha.

Selection of reliable reference genes for gene expression studies in the biofuel plant Jatropha curcas using real-time quantitative PCR.

Jatropha curcas is a promising renewable feedstock for biodiesel and bio-jet fuel production. To study gene expression in Jatropha in different tissues throughout development and under stress conditions, we examined a total of 11 typical candidate reference genes using real-time quantitative polymerase chain reaction (RT-qPCR) analysis, which is widely used for validating transcript levels in gene expression studies. The expression stability of these candidate reference genes was assessed across a total of 20 samples, including various tissues at vegetative and reproductive stages and under desiccation and cold stress treatments. The results obtained using software qBasePLUS showed that the top-ranked reference genes differed across the sample subsets. The combination of actin, GAPDH, and EF1α would be appropriate as a reference panel for normalizing gene expression data across samples at different developmental stages; the combination of actin, GAPDH, and TUB5 should be used as a reference panel for normalizing gene expression data across samples under various abiotic stress treatments. With regard to different developmental stages, we recommend the use of actin and TUB8 for normalization at the vegetative stage and GAPDH and EF1α for normalization at the reproductive stage. For abiotic stress treatments, we recommend the use of TUB5 and TUB8 for normalization under desiccation stress and GAPDH and actin for normalization under cold stress. These results are valuable for future research on gene expression during development or under abiotic stress in Jatropha. To our knowledge, this is the first report on the stability of reference genes in Jatropha.

Identification and expression analysis of two small heat shock protein cDNAs from developing seeds of biodiesel feedstock plant Jatropha curcas.

Plant small heat shock proteins (sHSPs) are known to be important for environmental stress tolerance and involved in various developmental processes. In this study, two full-length cDNAs encoding sHSPs, designated JcHSP-1 and JcHSP-2, were identified and characterized from developing seeds of a promising biodiesel feedstock plant Jatropha curcas by expressed sequence tag (EST) sequencing of embryo cDNA libraries and rapid amplification of cDNA ends (RACE). JcHSP-1 and JcHSP-2 contained open-reading frames encoding sHSPs of 219 and 157 amino acids, with predicted molecular weights of 24.42kDa and 18.02kDa, respectively. Sequence alignment indicated that both JcHSP-1 and JcHSP-2 shared high similarity with other plant sHSPs. Real-time quantitative RT-PCR analysis showed that the transcriptional level of both JcHSP-1 and JcHSP-2 increased along with natural dehydration process during seed development. A sharp increase of JcHSP-2 transcripts occurred in response to water content dropping from 42% in mature seeds to 12% in dry seeds. Western blot analysis revealed that the accumulation profile of two cross-reacting proteins, whose molecular weight corresponding to the calculated size of JcHSP-1 and JcHSP-2, respectively, was well consistent with the mRNA expression pattern of JcHSP-1 and JcHSP-2 in jatropha seeds during maturation and natural dehydration. These results indicated that both JcHSPs might play an important role in cell protection and seed development during maturation of J. curcas seeds.

[Expression profiles of AtWRKY25, AtWRKY26 and AtWRKY33 under abiotic stresses.].

The transcription factor WRKY family is one type of key regulatory components of plant development and defense against stress factors. The expression profiles of three AtWRKY genes under abiotic stresses were analyzed by Northern blotting analysis. The expression of AtWRKY25, AtWRKY26, and AtWRKY33 changed during stress treatments including thermal factors, NaCl, abscisic acid (ABA) and osmotic stress, and significantly under NaCl and cold treatments, suggesting a specific role of the three AtWRKYs in adaptation to environmental stresses in plants. We also found that the three AtWRKY genes showed distinct expression patterns under thermal stresses. AtWRKY25 and AtWRKY26 were gradually induced during heat and cold treatments, whereas AtWRKY33 was suppressed by heat treatment and induced rapidly during cold stress, indicating that the three AtWRKYs may play different roles in response to temperature factors. In addition, we analyzed the sequence of the promoters with bioinformatics approach, and some cis-elements involved in abiotic stresses and hormonal responses were revealed. The results provided important information for studying biological functions of three AtWRKY genes.