Gibberellins (GAs) are plant hormones that play fundamental roles in plant growth and development. Gibberellin 2-oxidase (GA2ox) plays a direct role in determining the levels of bioactive GAs by catalyzing bioactive GAs or their immediate precursors to inactive forms. In this study, a GA2ox gene, designated JcGA2ox6, was isolated from Jatropha curcas. JcGA2ox6 is expressed in all tissues of adult Jatropha, with the highest expression level in male flowers and the lowest expression level in young leaves. Overexpression of JcGA2ox6 in Arabidopsis resulted in a typical dwarf phenotype, along with late flowering, smaller leaves and flowers, shorter siliques and smaller seeds. Similarly, when JcGA2ox6 was overexpressed in Jatropha, the transgenic plants exhibited a dwarf phenotype with dark-green leaves and smaller inflorescences, flowers, fruits and seeds. However, the flowering time of Jatropha was not affected by overexpression of JcGA2ox6, unlike that in the transgenic Arabidopsis. Moreover, the number of flowers per inflorescence, the weight of 10 seeds and the seed oil content were significantly decreased in transgenic Jatropha. The results indicated that overexpression of JcGA2ox6 had a great impact on the vegetative and reproductive growth of transgenic Jatropha. Furthermore, we found that the dwarf phenotype of transgenic Jatropha was caused by a decrease in endogenous bioactive GA₄, which was correlated with the degree of dwarfism.

Jatropha curcas is considered a potential biodiesel feedstock crop. Currently, the value of J. curcas is limited because its seed yield is generally low. Transgenic modification is a promising approach to improve the seed yield of J. curcas. Although Agrobacterium-mediated genetic transformation of J. curcas has been pursued for several years, the transformation efficiency remains unsatisfying. Therefore, a highly efficient and simple Agrobacterium-mediated genetic transformation method for J. curcas should be developed. We examined and optimized several key factors that affect genetic transformation of J. curcas in this study. The results showed that the EHA105 strain was superior to the other three Agrobacterium tumefaciens strains for infecting J. curcas cotyledons, and the supplementation of 100 mM acetosyringone slightly increased the transient transformation frequency. Use of the appropriate inoculation method, optimal kanamycin concentration and appropriate duration of delayed selection also improved the efficiency of stable genetic transformation of J. curcas. The percentage of β-glucuronidase positive J. curcas shoots reached as high as 56.0 %, and 1.70 transformants per explant were obtained with this protocol. Furthermore, we optimized the root-inducing medium to achieve a rooting rate of 84.9 %. Stable integration of the T-DNA into the genomes of putative transgenic lines was confirmed by PCR and Southern blot analysis. Using this improved protocol, a large number of transgenic J. curcas plantlets can be routinely obtained within approximately 4 months. The detailed information provided here for each step of J. curcas transformation should enable successful implementation of this transgenic technology in other laboratories.

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.

Diacylglycerol acyltransferase (DGAT) is the only enzyme that catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol (DAG) to form triacylglycerol (TAG). The two main types of DGAT enzymes in the woody perennial biofuel plant Jatropha curcas, JcDGAT1 and JcDGAT2, were previously characterized only in heterologous systems. In this study, we investigated the functions of JcDGAT1 and JcDGAT2 in J. curcas.JcDGAT1 and JcDGAT2 were found to be predominantly expressed during the late stages of J. curcas seed development, in which large amounts of oil accumulated. As expected, overexpression of JcDGAT1 or JcDGAT2 under the control of the CaMV35S promoter gave rise to an increase in seed kernel oil production, reaching a content of 53.7% and 55.7% of the seed kernel dry weight, respectively, which were respectively 25% and 29.6% higher than that of control plants. The increase in seed oil content was accompanied by decreases in the contents of protein and soluble sugars in the seeds. Simultaneously, there was a two- to four-fold higher leaf TAG content in transgenic plants than in control plants. Moreover, by analysis of the fatty acid (FA) profiles, we found that JcDGAT1 and JcDGAT2 had the same substrate specificity with preferences for C18:2 in seed TAGs, and C16:0, C18:0, and C18:1 in leaf TAGs. Therefore, our study confirms the important role of JcDGAT1 and JcDGAT2 in regulating oil production in J. curcas.

Jatropha curcas L. is monoecious with a low female-to-male ratio, which is one of the factors restricting its seed yield. Because the phytohormone cytokinins play an essential role in flower development, particularly pistil development, in this study, we elevated the cytokinin levels in J. curcas flowers through transgenic expression of a cytokinin biosynthetic gene (AtIPT4) from Arabidopsis under the control of a J. curcas orthologue of TOMATO MADS BOX GENE 6 (JcTM6) promoter that is predominantly active in flowers. As expected, the levels of six cytokinin species in the inflorescences were elevated, and flower development was modified without any alterations in vegetative growth. In the transgenic J. curcas plants, the flower number per inflorescence was significantly increased, and most flowers were pistil-predominantly bisexual, i.e., the flowers had a huge pistil surrounded with small stamens. Unfortunately, both the male and the bisexual flowers of transgenic J. curcas were infertile, which might have resulted from the continuously high expression of the transgene during flower development. However, the number and position of floral organs in the transgenic flowers were well defined, which suggested that the determinacy of the floral meristem was not affected. These results suggest that fine-tuning the endogenous cytokinins can increase the flower number and the female-to-male ratio in J. curcas.

Background: Jatropha curcasL., a perennial oilseed plant, is considered as a promising feedstock for biodiesel production. Genetic modification of flowering characteristics is critical forJatrophabreeding. However, analysis of floral-specific promoters inJatrophais limited.Methods: In this study, we isolated theJatrophaortholog ofTM6(JcTM6) gene fromJatrophaflower cDNA library and detected the expression pattern ofJcTM6gene by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). We isolated a 1.8-kb fragment from the 5’ region of theJcTM6gene and evaluated its spatiotemporal expression pattern inArabidopsisusing theβ-glucuronidase(GUS) reporter gene andArabidopsis ATP/ADP isopentenyltransferase 4(AtIPT4) gene, respectively.Results: JcTM6was identified as a flower-specific gene inJatropha. As expected,JcTM6promoter was only active in transgenicArabidopsisflowers with the strongest activity in stamens. Moreover,JcTM6:AtIPT4transgenicArabidopsisshowed a phenotype of large flowers without any alterations in other organs. Furthermore, deletion of the region from –1,717 to –876 bp resulted in the disappearance of promoter activity in stamens but an increase in promoter activity in young leaves, sepals, and petals. Deletion analysis suggests that the –1,717- to –876-bp promoter fragment contains regulatory elements that confer promoter activity in stamens and inhibit activity in young leaves, sepals, and petals.