Extended mining of the oil biosynthesis pathway in biofuel plant Jatropha curcas by combined analysis of transcriptome and gene interactome data.

BACKGROUND: Jatropha curcas L. is an important non-edible oilseed crop with a promising future in biodiesel production. However, little is known about the molecular biology of oil biosynthesis in this plant when compared with other established oilseed crops, resulting in the absence of agronomically improved varieties of Jatropha. To extensively discover the potentially novel genes and pathways associated with the oil biosynthesis in J. curcas, new strategy other than homology alignment is on the demand. RESULTS: In this study, we proposed a multi-step computational framework that integrates transcriptome and gene interactome data to predict functional pathways in non-model organisms in an extended process, and applied it to study oil biosynthesis pathway in J. curcas. Using homologous mapping against Arabidopsis and transcriptome profile analysis, we first constructed protein-protein interaction (PPI) and co-expression networks in J. curcas. Then, using the homologs of Arabidopsis oil-biosynthesis-related genes as seeds, we respectively applied two algorithm models, random walk with restart (RWR) in PPI network and negative binomial distribution (NBD) in co-expression network, to further extend oil-biosynthesis-related pathways and genes in J. curcas. At last, using k-nearest neighbors (KNN) algorithm, the predicted genes were further classified into different sub-pathways according to their possible functional roles. CONCLUSIONS: Our method exhibited a highly efficient way of mining the extended oil biosynthesis pathway of J. curcas. Overall, 27 novel oil-biosynthesis-related gene candidates were predicted and further assigned to 5 sub-pathways. These findings can help better understanding of the oil biosynthesis pathway of J. curcas, as well as paving the way for the following J. curcas breeding application.

De novo genome assembly and Hi-C analysis reveal an association between chromatin architecture alterations and sex differentiation in the woody plant Jatropha curcas.

BACKGROUND: Chromatin architecture is an essential factor regulating gene transcription in different cell types and developmental phases. However, studies on chromatin architecture in perennial woody plants and on the function of chromatin organization in sex determination have not been reported. RESULTS: Here, we produced a chromosome-scale de novo genome assembly of the woody plant Jatropha curcas with a total length of 379.5 Mb and a scaffold N50 of 30.7 Mb using Pacific Biosciences long reads combined with genome-wide chromosome conformation capture (Hi-C) technology. Based on this high-quality reference genome, we detected chromatin architecture differences between monoecious and gynoecious inflorescence buds of Jatropha. Differentially expressed genes were significantly enriched in the changed A/B compartments and topologically associated domain regions and occurred preferentially in differential contact regions between monoecious and gynoecious inflorescence buds. Twelve differentially expressed genes related to flower development or hormone synthesis displayed significantly different genomic interaction patterns in monoecious and gynoecious inflorescence buds. These results demonstrate that chromatin organization participates in the regulation of gene transcription during the process of sex differentiation in Jatropha. CONCLUSIONS: We have revealed the features of chromatin architecture in perennial woody plants and investigated the possible function of chromatin organization in Jatropha sex differentiation. These findings will facilitate understanding of the regulatory mechanisms of sex determination in higher plants.

Flower-Specific Overproduction of Cytokinins Altered Flower Development and Sex Expression in the Perennial Woody Plant Jatropha curcas L.

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.

Developmental basis for flower sex determination and effects of cytokinin on sex determination in Plukenetia volubilis (Euphorbiaceae).

KEY MESSAGE: Cytokinin might be an important factor to regulate floral sex at the very early stage of flower development in sacha inchi. Sacha inchi (Plukenetia volubilis, Euphorbiaceae) is characterized by having female and male flowers in a thyrse with particular differences. The mechanisms involved in the development of unisexual flowers are very poorly understood. In this study, the inflorescence and flower development of P. volubilis were investigated using light microscopy and scanning electron microscopy. We also investigated the effects of cytokinin on flower sex determination by exogenous application of 6-benzyladenine (BA) in P. volubilis. The floral development of P. volubilis was divided into eight stages, and the first morphological divergence between the male and female flowers was found to occur at stage 3. Both female and male flowers can be structurally distinguished by differences in the shape and size of the flower apex after sepal primordia initiation. There are no traces of gynoecia in male flowers or of androecia in female flowers. Exogenous application of BA effectively induced gynoecium primordia initiation and female flower development, especially at the early flower developmental stages. We propose that flower sex is determined earlier and probably occurs before flower initiation, either prior to or at inflorescence development due to the difference in the position of the female and male primordia in the inflorescence and in the time of the female and male primordia being initiated. The influence of cytokinin on female primordia during flower development in P. volubilis strongly suggests a feminization role for cytokinin in sex determination. These results indicate that cytokinin could modify the fate of the apical meristem of male flower and promote the formation of carpel primordia in P. volubilis.

JCDB: a comprehensive knowledge base for Jatropha curcas, an emerging model for woody energy plants.

BACKGROUND: Jatropha curcas is an oil-bearing plant, and has seeds with high oil content (~ 40%). Several advantages, such as easy genetic transformation and short generation duration, have led to the emergence of J. curcas as a model for woody energy plants. With the development of high-throughput sequencing, the genome of Jatropha curcas has been sequenced by different groups and a mass of transcriptome data was released. How to integrate and analyze these omics data is crucial for functional genomics research on J. curcas. RESULTS: By establishing pipelines for processing novel gene identification, gene function annotation, and gene network construction, we systematically integrated and analyzed a series of J. curcas transcriptome data. Based on these data, we constructed a J. curcas database (JCDB), which not only includes general gene information, gene functional annotation, gene interaction networks, and gene expression matrices but also provides tools for browsing, searching, and downloading data, as well as online BLAST, the JBrowse genome browser, ID conversion, heatmaps, and gene network analysis tools. CONCLUSIONS: JCDB is the most comprehensive and well annotated knowledge base for J. curcas. We believe it will make a valuable contribution to the functional genomics study of J. curcas. The database is accessible at http://jcdb.xtbg.ac.cn.

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.

De novo transcriptome assembly of the eight major organs of Sacha Inchi (Plukenetia volubilis) and the identification of genes involved in α-linolenic acid metabolism.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

The complete chloroplast genome sequence of the biofuel plant Sacha Inchi, Plukenetia volubilis.

Sacha Inchi (Plukenetia volubilis) is a potential woody oil seed plant for producing healthy vegetable oil due to high content of α-linolenic acid in its seeds. In this study, we report the structure of the complete chloroplast genome of P. volubilis using high-throughput next-generation sequencing technology. The circular chloroplast genome is 161,733 bp in size, containing a pair of inverted repeat regions (IR) of 27,382 bp each, which were separated by a large single copy region (LSC) of 88,843 bp and a small single copy region (SSC) of 18,126 bp. The chloroplast genome harbors 135 genes, including 92 protein-coding genes, 35 tRNA genes and 8 rRNA genes. Based on the phylogenetic relationships between the chloroplast genome of P. volubilis and those of the other species, P. volubilis is most closely related to castor bean (Ricinus communis).

De novo transcriptome assembly and comparative analysis between male and benzyladenine-induced female inflorescence buds of Plukenetia volubilis.

Plukenetia volubilis is a promising oilseed crop due to its seeds being rich in unsaturated fatty acids, especially alpha-linolenic acid. P. volubilis is monoecious, with separate male and female flowers on the same inflorescence. We previously reported that male flowers were converted to female flowers by exogenous cytokinin (6-benzyladenine, 6-BA) treatment in P. volubilis. To identify candidate genes associated with floral sex differentiation of P. volubilis, we performed de novo transcriptome assembly and comparative analysis on control male inflorescence buds (MIB) and female inflorescence buds (FIB) induced by 6-BA using Illumina sequencing technology. A total of 57,664 unigenes with an average length of 979 bp were assembled from 104.1 million clean reads, and 45,235 (78.45%) unigenes were successfully annotated in the public databases. Notably, Gene Ontology analyses revealed that 4193 and 3880 unigenes were enriched in the categories of reproduction and reproductive processes, respectively. Differential expression analysis identified 1385 differentially expressed unigenes between MIB and FIB, of which six unigenes related to cytokinin and auxin signaling pathways and 16 important transcription factor (TF) genes including MADS-box family members were identified. In particular, several unigenes encoding important TFs, such as homologs of CRABS CLAW, RADIALIS-like 1, RADIALIS-like 2, HECATE 2, WUSCHEL-related homeobox 9, and SUPERMAN, were expressed at higher levels in FIB than in MIB. The expression patterns of the 36 selected unigenes revealed by transcriptome analysis were successfully validated by quantitative real-time PCR. This study not only provides comprehensive gene expression profiles of P. volubilis inflorescence buds, but also lays the foundation for research on the molecular mechanism of floral sex determination in P. volubilis and other monoecious plants.

Comparative transcriptome analysis of axillary buds in response to the shoot branching regulators gibberellin A3 and 6-benzyladenine in Jatropha curcas.

Cytokinin (CK) is the primary hormone that positively regulates axillary bud outgrowth. However, in many woody plants, such as Jatropha curcas, gibberellin (GA) also promotes shoot branching. The molecular mechanisms underlying GA and CK interaction in the regulation of bud outgrowth in Jatropha remain unclear. To determine how young axillary buds respond to GA3 and 6-benzyladenine (BA), we performed a comparative transcriptome analysis of the young axillary buds of Jatropha seedlings treated with GA3 or BA. Two hundred and fifty genes were identified to be co-regulated in response to GA3 or BA. Seven NAC family members were down-regulated after treatment with both GA3 and BA, whereas these genes were up-regulated after treatment with the shoot branching inhibitor strigolactone. The expressions of the cell cycle genes CDC6, CDC45 and GRF5 were up-regulated after treatment with both GA3 and BA, suggesting they may promote bud outgrowth via regulation of the cell cycle machinery. In the axillary buds, BA significantly increased the expression of GA biosynthesis genes JcGA20oxs and JcGA3ox1, and down-regulated the expression of GA degradation genes JcGA2oxs. Overall, the comprehensive transcriptome data set provides novel insight into the responses of young axillary buds to GA and CK.

Comparative Transcriptome Analysis between Gynoecious and Monoecious Plants Identifies Regulatory Networks Controlling Sex Determination in Jatropha curcas.

Most germplasms of the biofuel plant Jatropha curcas are monoecious. A gynoecious genotype of J. curcas was found, whose male flowers are aborted at early stage of inflorescence development. To investigate the regulatory mechanism of transition from monoecious to gynoecious plants, a comparative transcriptome analysis between gynoecious and monoecious inflorescences were performed. A total of 3,749 genes differentially expressed in two developmental stages of inflorescences were identified. Among them, 32 genes were involved in floral development, and 70 in phytohormone biosynthesis and signaling pathways. Six genes homologous to KNOTTED1-LIKE HOMEOBOX GENE 6 (KNAT6), MYC2, SHI-RELATED SEQUENCE 5 (SRS5), SHORT VEGETATIVE PHASE (SVP), TERMINAL FLOWER 1 (TFL1), and TASSELSEED2 (TS2), which control floral development, were considered as candidate regulators that may be involved in sex differentiation in J. curcas. Abscisic acid, auxin, gibberellin, and jasmonate biosynthesis were lower, whereas cytokinin biosynthesis was higher in gynoecious than that in monoecious inflorescences. Moreover, the exogenous application of gibberellic acid (GA3) promoted perianth development in male flowers and partly prevented pistil development in female flowers to generate neutral flowers in gynoecious inflorescences. The arrest of stamen primordium at early development stage probably causes the abortion of male flowers to generate gynoecious individuals. These results suggest that some floral development genes and phytohormone signaling pathways orchestrate the process of sex determination in J. curcas. Our study provides a basic framework for the regulation networks of sex determination in J. curcas and will be helpful for elucidating the evolution of the plant reproductive system.

An efficient protocol for Agrobacterium-mediated transformation of the biofuel plant Jatropha curcas by optimizing kanamycin concentration and duration of delayed selection.

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 ß-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.

Gibberellin Promotes Shoot Branching in the Perennial Woody Plant Jatropha curcas.

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.

Analysis of the transcriptional responses in inflorescence buds of Jatropha curcas exposed to cytokinin treatment.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Transcriptome of the inflorescence meristems of the biofuel plant Jatropha curcas treated with cytokinin.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.