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. 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

Strigolactone (SL), auxin and cytokinin (CK) interact to regulate shoot branching. CK has long been considered to be the only key phytohormone to promote lateral bud outgrowth. Here we report that gibberellin also acts as a positive regulator in the control of shoot branching in the woody plant Jatropha curcas. We show that gibberellin and CK synergistically promote lateral bud outgrowth, and that both hormones influence the expression of putative branching regulators, J. curcas BRANCHED1 and BRANCHED2, which are key transcription factors maintaining bud dormancy. Moreover, treatment with paclobutrazol, an inhibitor of de novo gibberellin biosynthesis, significantly reduced the promotion of bud outgrowth by CK, suggesting that gibberellin is required for CK-mediated axillary bud outgrowth. In addition, SL, a plant hormone involved in the repression of shoot branching, acted antagonistically to both gibberellin and CK in the control of lateral bud outgrowth. Consistent with this, the expression of JcMAX2, a J. curcas homolog of Arabidopsis MORE AXILLARY GROWTH 2 encoding an F-box protein in the SL signaling pathway, was repressed by gibberellin and CK treatment. We also provide physiological evidence that gibberellin also induces shoot branching in many other trees, such as papaya, indicating that a more complicated regulatory network occurs in the control of shoot branching in some perennial woody plants.

Jatropha curcas, a monoecious perennial biofuel shrub belonging to the family Euphorbiaceae, has few female flowers, which is one of the most important reasons for its poor seed yield. This study was undertaken to determine the effects of the plant growth regulator 6-benzyladenine (BA) on floral development and floral sex determination of J. curcas. Exogenous application of BA significantly increased the total number of flowers per inflorescence, reaching a 3.6-fold increase (from 215 to 784) at 160 mg/l of BA. Furthermore, BA treatments induced bisexual flowers, which were not found in control inflorescences, and a substantial increase in the femaleto- male flower ratio. Consequently, a 4.5-fold increase in fruit number and a 3.3-fold increase in final seed yield were observed in inflorescences treated with 160 mg/L of BA, which resulted from the greater number of female flowers and the newly induced bisexual flowers in BA-treated inflorescences. This study indicates that the seed yield of J. curcas can be increased by manipulation of floral development and floral sex expression.

The most commonly used protocol of the RNA isolation, the guanidine thiocyanate method, was unsuitable for recalcitrant plant tissues containing a large amount of storage proteins and secondary metabolites. We demonstrated that RNA could bind to the silica particles, which have been used successfully in DNA isolation from various sources, under a high concentration of NaCl in the presence of ethanol and sodium acetate. Based on this observation, an efficient, inexpensive, and highly reproducible technique, the acid phenol silica method, was developed to isolate high-quality RNAs from various plant tissues recalcitrant to extraction in guanidine thiocyanate.

SaPIN2a, a plant proteinase inhibitor from nightshade(Solanum americanum), was located to the enucleate sieve elements (SEs) of phloem. The expressed SaPIN2a in transgenic lettuce showed inhibition of plant endogenous trypsin- and chymotrypsin-like activities,suggesting that SaPIN2a can regulate proteolysis in plant cells. To further investigate the physiological role of SaPIN2a, we produced transgenic nightshade and lettuce plants overexpressing SaPIN2a from the cauliflower mosaic virus (CaMV) 35S promoter using Agrobacterium- mediated transformation. Overexpression of SaPIN2a in transgenic plants was demonstrated by northern blot and western blot analysis. SaPIN2aoverexpressing transgenic nightshade plants showed significantly lower height than wild-type plants. Transmission electron microscopy analysis showed that chloroplast- like organelles with thylakoids, which are not present in enucleate SEs of wild-type plants, were present in the enucleate SEs of SaPIN2a-overexpressing transgenic plants. This finding is discussed in terms of the possible role played by SaPIN2a in the regulation of proteolysis in SEs.