Jatropha curcas, whose seed content is approximately 30–40% oil, is an ideal feedstock for producing biodiesel and bio-jet fuels. However, Jatropha plants have a low number of female flowers, which results in low seed yield that cannot meet the needs of the biofuel industry. Thus, increasing the number of female flowers is critical for the improvement of Jatropha seed yield. Our previous findings showed that cytokinin treatment can increase the flower number and female to male ratio and also induce bisexual flowers in Jatropha. The mechanisms underlying the influence of cytokinin on Jatropha flower development and sex determination, however, have not been clarified. This study examined the transcriptional levels of genes involved in the response to cytokinin in Jatropha inflorescence meristems at different time points after cytokinin treatment by 454 sequencing, which gave rise to a total of 294.6 Mb of transcript sequences. Up-regulated and down-regulated annotated and novel genes were identified, and the expression levels of the genes of interest were confirmed by qRT-PCR. The identified transcripts include those encoding genes involved in the biosynthesis, metabolism, and signaling of cytokinin and other plant hormones, flower development and cell division, which may be related to phenotypic changes of Jatropha in response to cytokinin treatment. Our analysis indicated that Jatropha orthologs of the floral organ identity genes known as ABCE model genes, JcAP1,2, JcPI, JcAG, and JcSEP1,2,3, were all significantly repressed, with an exception of one B-function gene JcAP3 that was shown to be up-regulated by BA treatment, indicating different mechanisms to be involved in the floral organ development of unisexual flowers of Jatropha and bisexual flowers of Arabidopsis. Several cell division-related genes, including JcCycA3;2, JcCycD3;1, JcCycD3;2 and JcTSO1, were up-regulated, which may contribute to the increased flower number after cytokinin treatment. This study presents the first report of global expression patterns of cytokinin-regulated transcripts in Jatropha inflorescence meristems. This report laid the foundation for further mechanistic studies on Jatropha and other non-model plants responding to cytokinin. Moreover, the identification of functional candidate genes will be useful for generating superior varieties of high-yielding transgenic Jatropha.

Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔC_t), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.

Jatropha curcas L. is a potential biofuel plant. Application of exogenous cytokinin (6-benzyladenine, BA) on its inflorescence buds can significantly increase the number of female flowers, thereby improving seed yield. To investigate which genes and signal pathways are involved in the response to cytokinin in J. curcas inflorescence buds, we monitored transcriptional activity in inflorescences at 0, 3, 12, 24, and 48 h after BA treatment using a microarray. We detected 5,555 differentially expressed transcripts over the course of the experiment, which could be grouped into 12 distinct temporal expression patterns. We also identified 31 and 131 transcripts in J. curcas whose homologs in model plants function in flowering and phytohormonal signaling pathways, respectively. According to the transcriptional analysis of genes involved in flower development, we hypothesized that BA treatment delays floral organ formation by inhibiting the transcription of the A, B and E classes of floral organ-identity genes, which would allow more time to generate more floral primordia in inflorescence meristems, thereby enhancing inflorescence branching and significantly increasing flower number per inflorescence. BA treatment might also play an important role in maintaining the flowering signals by activating the transcription of GIGANTEA (GI) and inactivating the transcription of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and TERMINAL FLOWER 1b (TFL1b). In addition, exogenous cytokinin treatment could regulate the expression of genes involved in the metabolism and signaling of other phytohormones, indicating that cytokinin and other phytohormones jointly regulate flower development in J. curcas inflorescence buds. Our study provides a framework to better understand the molecular mechanisms underlying changes in flowering traits in response to cytokinin treatment in J. curcas inflorescence buds. The results provide valuable information related to the mechanisms of cross-talk among multiple phytohormone signaling pathways in woody plants.

Sacha Inchi (Plukenetia volubilis L.), which belongs to the Euphorbiaceae, has been considered a new potential oil crop because of its high content of polyunsaturated fatty acids in its seed oil. The seed oil especially contains high amounts of α-linolenic acid (ALA), which is useful for the prevention of various diseases. However, little is known about the genetic information and genome sequence of Sacha Inchi, which has largely hindered functional genomics and molecular breeding studies. In this study, a de novo transcriptome assembly based on transcripts sequenced in eight major organs, including roots, stems, shoot apexes, mature leaves, male flowers, female flowers, fruits, and seeds of Sacha Inchi was performed, resulting in a set of 124,750 non-redundant putative transcripts having an average length of 851 bp and an N50 value of 1909 bp. Organ-specific unigenes analysis revealed that the most organ-specific transcripts are found in female flowers (2244 unigenes), whereas a relatively small amount of unigenes are detected to be expressed specifically in other organs with the least in stems (24 unigenes). A total of 42,987 simple sequence repeats (SSRs) were detected, which will contribute to the marker assisted selection breeding of Sacha Inchi. We analyzed expression of genes related to the α-linolenic acid metabolism based on the de novo assembly and annotation transcriptome in Sacha Inchi. It appears that Sacha Inchi accumulates high level of ALA in seeds by strong expression of biosynthesis-related genes and weak expression of degradation-related genes. In particular, the up-regulation of FAD3 and FAD7 is consistent with high level of ALA in seeds of Sacha Inchi compared with in other organs. Meanwhile, several transcription factors (ABI3, LEC1 and FUS3) may regulate key genes involved in oil accumulation in seeds of Sacha Inchi. The transcriptome of major organs of Sacha Inchi has been sequenced and de novo assembled, which will expand the genetic information for functional genomic studies of Sacha Inchi. In addition, the identification of candidate genes involved in ALA metabolism will provide useful resources for the genetic improvement of Sacha Inchi and the metabolic engineering of ALA biosynthesis in other plants.

Gibberellin (GA) is an important phytohormone and has been reported to promote flowering in annual and biennial plants, but may play an opposite role in woody perennial plants. In this study, the promoting or inhibiting effect of GA on floral initiation was investigated in Jatropha curcas L., a perennial woody plant, which has great potential as renewable energy plant due to the high oil content in the seed. We found that exogenous application of gibberellic acid (GA₃) inhibited flowering, whereas GA biosynthesis inhibitors promoted flowering. The promotive effect of the GA biosynthesis inhibitor paclobutrazol on floral initiation could be diminished by GA₃ treatment. The expression levels of GA biosynthesis genes Jatropha GA 3-oxidase 3 (JcGA3ox3) and the GA receptor genes, Jatropha GA-INSENSITIVE DWARF 1C (JcGID1C) were decreased, whereas a GA catabolism gene, Jatropha GA 2-oxidase 8 (JcGA2ox8), was elevated during floral transition. These results suggest that GA inhibits floral initiation in J. curcas.

Trehalose-6-phosphate (T6P) phosphatase (TPP), a dephosphorylating enzyme, catalyzes the dephosphorylation of T6P, generating trehalose. In Jatropha, we found six members of the TPP family. Five of them JcTPPA, JcTPPC, JcTPPD, JcTPPG, and JcTPPJ are highly expressed in female flowers or male flowers, or both, suggesting that members of the JcTPP family may participate in flower development in Jatropha. The wide expression of JcTPPJ gene in various organs implied its versatile roles and thus was chosen for unraveling its biological functions during developmental process. We constructed an overexpression vector of JcTPPJ cDNA driven by the cauliflower mosaic virus (CaMV) 35S promoter for genetic transformation. Compared with control Arabidopsis plants, 35S:JcTPPJ transgenic Arabidopsis plants presented greater sucrose contents in their inflorescences and displayed late-flowering and heterostylous phenotypes. Exogenous application of sucrose to the inflorescence buds of wild-type Arabidopsis repressed the development of the perianth and filaments, with a phenocopy of the 35S:JcTPPJ transgenic Arabidopsis. These results suggested that the significantly increased sucrose level in the inflorescence caused (or induced) by JcTTPJ overexpression, was responsible for the formation of heterostylous flower phenotype. However, 35S:JcTPPJ transgenic Jatropha displayed no obvious phenotypic changes, implying that JcTPPJ alone may not be sufficient for regulating flower development in Jatropha. Our results are helpful for understanding the function of TPPs, which may regulate flower organ development by manipulating the sucrose status in plants.