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

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.

Abstract SaPIN2a encodes a proteinase inhibitor in nightshade (Solanum americanum), which is specifically localized to the enucleate sieve elements.It has been proposed to play an important role in phloem development by regulating proteolysis in sieve elements. In this study, we purified and characterized native SaPIN2a from nightshade stems and recombinant SaPIN2a expressed in Escherichia coli. Purified native SaPIN2a was found as a charge isomer family of homodimers, and was weakly glycosylated. Native SaPIN2a significantly inhibited serine proteinases such as trypsin, chymotrypsin, and subtilisin, with the most potent inhibitory activity on subtilisin. It did not inhibit cysteine proteinase papain and aspartic proteinase cathepsin D. Recombinant SaPIN2a had a strong inhibitory effect on chymotrypsin, but its inhibitory activities toward trypsin and especially toward subtilisin were greatly reduced. In addition, native SaPIN2a can effectively inhibit midgut trypsin-like activities from Trichoplusia ni and Spodoptera litura larvae, suggesting a potential for the production of insect-resistant transgenic 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.