Floral Meristem Research Articles

Evaluation of the Possible Contribution of Various Regulatory Genes to Determination of Carpel Number as a Potential Mechanism for Optimal Agricultural Yield

Various regulatory genes encoding transcription factors and miRNAs regulate carpel number. Multicarpelly is normally associated with increased size of the floral meristem, and several genetic factors have been discovered that influence this characteristic. A fundamental understanding of the regulatory genes affecting carpel number can facilitate strategies for agricultural yield improvement, which is crucial, given that the global population is growing rapidly. A multicarpellate plant may provide a significantly higher yield than a plant bearing fewer carpels. Higher yields can be achieved via various means; in this review, we provide an overview of the current knowledge of the various regulatory factors that contribute to multicarpelly and the potential of increasing carpel number to achieve an increased yield.

De novo stem cell establishment in meristems requires repression of organ boundary cell fate.

Stem cells play important roles in animal and plant biology, as they sustain morphogenesis and tissue replenishment following aging or injury. In plants, stem cells are embedded in multicellular structures called meristems. The formation of new meristems is essential for the plastic expansion of the highly branched shoot and root systems. In particular, axillary meristems (AMs) that produce lateral shoots arise from the division of boundary domain cells at the leaf base. The CUP-SHAPED COTYLEDON (CUC) genes are major determinants of the boundary domain and are required for AM initiation. However, how AMs get structured and how stem cells become established de novo remain elusive. Here, we show that two NGATHA-LIKE (NGAL) transcription factors, DEVELOPMENT-RELATED PcG TARGET IN THE APEX4 (DPA4)/NGAL3 and SUPPRESSOR OF DA1-1 7 (SOD7)/NGAL2, redundantly repress CUC expression in initiating AMs of Arabidopsis thaliana. Ectopic boundary fate leads to abnormal growth and organization of the AM and prevents de novo stem cell establishment. Floral meristems of the dpa4 sod7 double mutant show a similar delay in de novo stem cell establishment. Altogether, while boundary fate is required for the initiation of AMs, our work reveals how it is later repressed to allow proper meristem establishment and de novo stem cell niche formation.

Disruption of transcription factor RhMYB123 causes the transformation of stamen to malformed petal in rose (Rosa hybrida)

We find that the R2R3 MYB transcription factor RhMYB123 has a novel function to regulate stamen-petal organ specification in rose. Rose is one of the ornamental plants with economic importance worldwide. Malformed flower seriously affects the ornamental value and fertility of rose. However, the regulatory mechanism is largely unknown. In this work, we identified a R2R3 MYB transcription factor RhMYB123 from rose, the expression of which significantly decreased from flower differentiation stage to floral organ development stage. Phylogenetic analysis indicated that it belongs to the same subgroup as MYB123 of Arabidopsis and located in nucleus. In addition, RhMYB123 was confirmed to have transcriptional activation function by dual luciferase assay. Silencing RhMYB123 using Virus-Induced Gene Silencing (VIGS) in rose could increase the number of malformed petaloid stamen. Furthermore, we identified 549 differential expressed genes (DEGs) in TRV-RhMYB123 lines compared to TRV controls by RNA-seq of floral buds (flower differentiation stage). Among of those genes, expression of 3 MADS box family genes related to floral organ development reduced in TRV-RhMYB123 lines, including AGAMOUS (RhAG), AGAMOUS LIKE 15 (RhAGL15), and SHATTERPROOF 1 (RhSHP1). Given, previous studies have shown that auxin plays a crucial role in floral meristem initiation and stamen-petal organ specification. We also found 6 DEGs were involved in auxin signal transduction, of which five were reduced expression in TRV-RhMYB123. Taken together, our findings suggested that RhMYB123 may govern the development of malformed petaloid stamen by regulating the expressions of some MADS box family members and auxin signaling pathway elements.

Cell- and noncell-autonomous AUXIN RESPONSE FACTOR3 controls meristem proliferation and phyllotactic patterns.

In cell-cell communication, noncell-autonomous transcription factors play vital roles in controlling plant stem cell fate. We previously reported that AUXIN RESPONSE FACTOR3 (ARF3), a member of the ARF family with critical roles in floral meristem maintenance and determinacy, has a distinct accumulation pattern that differs from the expression domain of its encoding gene in the shoot apical meristem (SAM). However, the biological meaning of this difference is obscure. Here, we demonstrate that ARF3 expression in Arabidopsis (Arabidopsis thaliana) is mainly activated at the periphery of the SAM by auxin where ARF3 cell autonomously regulates the expression of meristem-organ boundary-specific genes, such as CUP-SHAPED COTYLEDON1-3 (CUC1-3), BLADE ON PETIOLE1-2 (BOP1-2), and TARGETS UNDER ETTIN CONTROL3 (TEC3) to regulate the arrangement of organs in regular pattern, a phenomenon referred to as phyllotaxis. We also show that ARF3 is translocated into the organizing center where it represses cytokinin activity and WUSCHEL expression to regulate meristem activity noncell-autonomously. Therefore, ARF3 acts as a molecular link that mediates the interaction of auxin and cytokinin signaling in the SAM while coordinating the balance between meristem maintenance and organogenesis. Our findings reveal an ARF3-mediated coordination mechanism through cell-cell communication in dynamic SAM maintenance.

An optimized protocol using Steedman's wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber

In situ mRNA hybridization (ISH) is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber. The most common ISH protocol uses paraffin wax; however, embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased signals. Here, we developed an optimized protocol to simplify the process and improve RNA sensitivity. We combined embedding tissue in low melting-point Steedman's wax with processing tissue sections in solution, as in the whole-mount ISH method in the optimized protocol. Using the optimized protocol, we examined the expression patterns of the CLAVATA3 (CLV3) and WUSCHEL (WUS) genes in shoot apical meristems and floral meristems of Cucumis sativus (cucumber) and Arabidopsis thaliana (Arabidopsis). The optimized protocol saved 4–5 days of experimental period compared with the standard ISH protocol using paraffin wax. Moreover, the optimized protocol achieved high signal sensitivity. The optimized protocol was successful for both cucumber and Arabidopsis, which indicates it might have general applicability to most plants.

Role of grapevine SEPALLATA-related MADS-box gene VvMADS39 in flower and ovule development.

Seedlessness is one of the most important breeding goals for table grapes; thus, understanding the molecular genetic regulation of seed development and abortion is critical for the development of seedless cultivars. In the present study, we characterized VvMADS39, a class E MADS-box gene of grapevine (Vitis vinifera) orthologous to Arabidopsis SEP2. Heterologous overexpression of VvMADS39 in tomato reduced the fruit and seed size and seed number. Targeted mutagenesis of the homologous SlMADS39 in tomato induced various floral and fruit defects. It could reasonable to suppose that active VvMADS39 expression in "Thompson Seedless" may restrict cellular expansion, resulting in the development of smaller fruits and seeds, VvMADS39 may play a role in the regulation of ovule development in grapevine and contributes to seedless fruit formation. In contrast, VvMADS39 suppression in "Red Globe" was associated with enhanced histone H3 lysine 27 trimethylation in the promoter region of VvMADS39, allowing normal ovule and fruit development; Meanwhile, VvMADS39 interacts with VvAGAMOUS, and the activity of the VvMADS39-VvAGAMOUS dimer to induce integument development requires the activation and maintenance of VvINO expression. The synergistic cooperation between VvMADS39 and related proteins plays an important role in maintaining floral meristem characteristics, and fruit and ovule development.

Genetic architecture underlying variation in floral meristem termination in Aquilegia.

Floral organs are produced by floral meristems (FMs), which harbor stem cells in their centers. Since each flower only has a finite number of organs, the stem cell activity of an FM will always terminate at a specific time point, a process termed floral meristem termination (FMT). Variation in the timing of FMT can give rise to floral morphological diversity, but how this process is fine-tuned at a developmental and evolutionary level is poorly understood. Flowers from the genus Aquilegia share identical floral organ arrangement except for stamen whorl number (SWN), making Aquilegia a well-suited system for investigation of this process: differences in SWN between species represent differences in the timing of FMT. By crossing A. canadensis and A. brevistyla, quantitative trait locus (QTL) mapping has revealed a complex genetic architecture with seven QTL. We explored potential candidate genes under each QTL and characterized novel expression patterns of select loci of interest using in situ hybridization. To our knowledge, this is the first attempt to dissect the genetic basis of how natural variation in the timing of FMT is regulated, and our results provide insight into how floral morphological diversity can be generated at the meristematic level.

Genome wide identification of MADS box gene family in Musa balbisiana and their divergence during evolution

MADS box gene family is transcription factor gene family that is involved in growth and development of eukaryotes. In plants the MADS box gene family is mainly associated with floral meristem identity and flower development, apart from being involved in nearly all the phases of plant growth. The MADS box gene family has also been shown to be involved during fruit development and ripening. In this study the MADS box gene family from Musa balbisiana was identified and the divergence of this gene family between Musa balbisiana and Musa acuminata studied. A total of 97 MADS box genes were identified from the genome of Musa balbisiana. Phylogenetic analysis showed that the MbMADS box genes were categorised into type I (α and γ; the β group was not distinguishable) and type II groups (MIKCc and MIKC* and MIKCc was further divided into 13 subfamilies). The typeII group has the largest number of genes and also showed the most expansion which could be correlated with the whole genome duplications. There were significant differences in the MADS box genes from Musa acuminata and Musa balbisiana during evolution that can be correlated with different floral phenotype and fruit ripening pattern. The divergence of the MADS RIN genes in Musa balbisiana as compared to Musa acuminata might play an important role in the slow ripening of Musa balbisiana fruits.

A 3D gene expression atlas of the floral meristem based on spatial reconstruction of single nucleus RNA sequencing data

Cellular heterogeneity in growth and differentiation results in organ patterning. Single-cell transcriptomics allows characterization of gene expression heterogeneity in developing organs at unprecedented resolution. However, the original physical location of the cell is lost during this methodology. To recover the original location of cells in the developing organ is essential to link gene activity with cellular identity and function in plants. Here, we propose a method to reconstruct genome-wide gene expression patterns of individual cells in a 3D flower meristem by combining single-nuclei RNA-seq with microcopy-based 3D spatial reconstruction. By this, gene expression differences among meristematic domains giving rise to different tissue and organ types can be determined. As a proof of principle, the method is used to trace the initiation of vascular identity within the floral meristem. Our work demonstrates the power of spatially reconstructed single cell transcriptome atlases to understand plant morphogenesis. The floral meristem 3D gene expression atlas can be accessed at http://threed-flower-meristem.herokuapp.com.

Regulatory Networks of Flowering Genes in Angelica sinensis during Vernalization.

Angelica sinensis is a low-temperature and long-day perennial herb that has been widely used for cardio-cerebrovascular diseases in recent years. In commercial cultivation, up to 40% of flowering decreases the officinal yield of roots and accumulation of bioactive compounds. Although the regulatory mechanism of flowering genes during the photoperiod has been revealed, the networks during vernalization have not been mapped. Here, transcriptomics profiles of A. sinensis with uncompleted (T1), completed (T2) and avoided vernalization (T3) were performed using RNA-seq, and genes expression was validated with qRT-PCR. A total of 61,241 isoforms were annotated on KEGG, KOG, Nr and Swiss-Prot databases; 4212 and 5301 differentially expressed genes (DEGs) were observed; and 151 and 155 genes involved in flowering were dug out at T2 vs. T1 and T3 vs. T1, respectively. According to functional annotation, 104 co-expressed genes were classified into six categories: FLC expression (22; e.g., VILs, FCA and FLK), sucrose metabolism (12; e.g., TPSs, SUS3 and SPSs), hormone response (18; e.g., GID1B, RAP2s and IAAs), circadian clock (2; i.e., ELF3 and COR27), downstream floral integrators and meristem identity (15; e.g., SOC1, AGL65 and SPLs) and cold response (35; e.g., PYLs, ERFs and CORs). The expression levels of candidate genes were almost consistent with FPKM values and changes in sugar and hormone contents. Based on their functions, four pathways that regulate flowering during vernalization were mapped, including the vernalization pathway, the autonomic pathway, the age pathway and the GA (hormone) pathway. This transcriptomic analysis provides new insights into the gene-regulatory networks of flowering in A. sinensis.

Modeling basal angiosperm floral phyllotaxis

ABSTRACT : We have developed mathematical models that accurately reproduce the floral meristems of seven angiosperm species from the ANA grade (Amborellales and Austrobaileyales). This allowed us to shed new light on the main problems posed by their phyllotaxis, such as whether it is whorled or spiral (in Nuphar), the origin of organ dédoublement or post-meristematic development.

Molecular mechanisms and evolutionary history of phytomelatonin in flowering

Flowering is a critical stage in plant life history, which is coordinated by environmental signals and endogenous cues. Phytomelatonin is a widely distributed indoleamine present in all living organisms and plays pleiotropic roles in plant growth and development. Recent evidence has established that phytomelatonin could modulate flowering in many species, probably in a concentration-dependent manner. Phytomelatonin seems to associate with floral meristem identification and floral organ formation, and the fluctuation of phytomelatonin might be important for flowering. Regarding the underlying mechanisms, phytomelatonin interacts with the central components of floral gene regulatory networks directly or indirectly, including the MADS-box gene family, phytohormones, and reactive oxygen species (ROS). From an evolutionary point of view, the actions of phytomelatonin in flowering probably evolved during the period of the diversification of flowering plants and could be regarded as a functional extension of its primary activities. The presumed evolutionary history of phytomelatonin-modulated flowering is proposed, presented in the chronological order of the appearance of phytomelatonin and core flowering regulators, namely DELLA proteins, ROS, and phytohormones. Further efforts are needed to address some intriguing aspects, such as the exploration of the association between phytomelatonin and photoperiodic flowering, phytomelatonin-related floral MADS-box genes, the crosstalk between phytomelatonin and phytohormones, as well as its potential applications in agriculture.

Stigma/Style Cell-Cycle Inhibitor 1, a Regulator of Cell Proliferation, Interacts With a Specific 14-3-3 Protein and Is Degraded During Cell Division.

The final shape and size of plant organs are determined by a network of genes that modulate cell proliferation and expansion. Among those, SCI1 (Stigma/style Cell-cycle Inhibitor 1) functions by inhibiting cell proliferation during pistil development. Alterations in SCI1 expression levels can lead to remarkable stigma/style size changes. Recently, we demonstrated that SCI1 starts to be expressed at the specification of the Nicotiana tabacum floral meristem and is expressed at all floral meristematic cells. To elucidate how SCI1 regulates cell proliferation, we screened a stigma/style cDNA library through the yeast two-hybrid (Y2H) system, using SCI1 as bait. Among the interaction partners, we identified the 14-3-3D protein of the Non-Epsilon group. The interaction between SCI1 and 14-3-3D was confirmed by pulldown and co-immunoprecipitation experiments. 14-3-3D forms homo- and heterodimers in the cytoplasm of plant cells and interacts with SCI1 in the nucleus, as demonstrated by Bimolecular Fluorescence Complementation (BiFC). Analyses of SCI1-GFP fluorescence through the cell-cycle progression revealed its presence in the nucleoli during interphase and prophase. At metaphase, SCI1-GFP fluorescence faded and was no longer detected at anaphase, reappearing at telophase. Upon treatment with the 26S proteasome inhibitor MG132, SCI1-GFP was stabilized during cell division. Site-directed mutagenesis of seven serines into alanines in the predicted 14-3-3 binding sites on the SCI1 sequence prevented its degradation during mitosis. Our results demonstrate that SCI1 degradation at the beginning of metaphase is dependent on the phosphorylation of serine residues and on the action of the 26S proteasome. We concluded that SCI1 stability/degradation is cell-cycle regulated, consistent with its role in fine-tuning cell proliferation.

Isolation and Functional Characterization of a LEAFY Gene in Mango (Mangifera indica L.).

LEAFY (LFY) plays an important role in the flowering process of plants, controlling flowering time and mediating floral meristem differentiation. Owing to its considerable importance, the mango LFY gene (MiLFY; GenBank accession no. HQ585988) was isolated, and its expression pattern and function were characterized in the present study. The cDNA sequence of MiLFY was 1152 bp, and it encoded a 383 amino acid protein. MiLFY was expressed in all tested tissues and was highly expressed in flowers and buds. Temporal expression analysis showed that MiLFY expression was correlated with floral development stage, and two relative expression peaks were detected in the early stages of floral transition and floral organ differentiation. Moreover, 35S::GFP-MiLFY fusion protein was shown to be localized to the nucleus of cells. Overexpression of MiLFY in Arabidopsis promoted early flowering and the conversion of lateral meristems into terminal flowers. In addition, transgenic plants exhibited obvious morphological changes, such as differences in cauline leaf shape, and the number of lateral branches. When driven by the MiLFY promoter, GFP was highly expressed in leaves, floral organs, stems, and roots, during the flowering period. Exogenous gibberellin (GA3) treatment downregulated MiLFY promoter expression, but paclobutrazol (PPP333) upregulated it. Bimolecular fluorescence complementation (BiFC) assays showed that the MiLFY protein can interact with zinc-finger protein 4 (ZFP4) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (MiSOC1D). Taken together, these results indicate that MiLFY plays a pivotal role in controlling mango flowering, and that it is regulated by gibberellin and paclobutrazol.

The auxin-responsive transcription factor SlDOF9 regulates inflorescence and flower development in tomato.

Understanding the mechanisms underlying differentiation of inflorescence and flower meristems is essential towards enlarging our knowledge of reproductive organ formation and to open new prospects for improving yield traits. Here, we show that SlDOF9 is a new modulator of floral differentiation in tomato. CRISPR/Cas9 knockout strategy uncovered the role of SlDOF9 in controlling inflorescence meristem and floral meristem differentiation via the regulation of cell division genes and inflorescence architecture regulator LIN. Tomato dof9-KO lines have more flowers in both determinate and indeterminate cultivars and produce more fruit upon vibration-assisted fertilization. SlDOF9 regulates inflorescence development through an auxin-dependent ARF5-DOF9 module that seems to operate, at least in part, differently in Arabidopsis and tomato. Our findings add a new actor to the complex mechanisms underlying reproductive organ differentiation in flowering plants and provide leads towards addressing the diversity of factors controlling the transition to reproductive organs.

Tomato CRABS CLAW paralogues interact with chromatin remodelling factors to mediate carpel development and floral determinacy.

Summary CRABS CLAW (CRC) orthologues play a crucial role in floral meristem (FM) determinacy and gynoecium formation across angiosperms, the key developmental processes for ensuring successful plant reproduction and crop production. However, the mechanisms behind CRC mediated FM termination are far from fully understood.Here, we addressed the functional characterization of tomato (Solanum lycopersicum) paralogous CRC genes. Using mapping‐by‐sequencing, RNA interference and CRISPR/Cas9 techniques, expression analyses, protein–protein interaction assays and Arabidopsis complementation experiments, we examined their potential roles in FM determinacy and carpel formation.We revealed that the incomplete penetrance and variable expressivity of the indeterminate carpel‐inside‐carpel phenotype observed in fruit iterative growth (fig) mutant plants are due to the lack of function of the S. lycopersicum CRC homologue SlCRCa. Furthermore, a detailed functional analysis of tomato CRC paralogues, SlCRCa and SlCRCb, allowed us to propose that they operate as positive regulators of FM determinacy by acting in a compensatory and partially redundant manner to safeguard the proper formation of flowers and fruits.Our results uncover for the first time the physical interaction of putative CRC orthologues with members of the chromatin remodelling complex that epigenetically represses WUSCHEL expression through histone deacetylation to ensure the proper termination of floral stem cell activity.

The SINGLE FLOWER (SFL) gene encodes a MYB transcription factor that regulates the number of flowers produced by the inflorescence of chickpea.

Summary Legumes usually have compound inflorescences, where flowers/pods develop from secondary inflorescences (I2), formed laterally at the primary inflorescence (I1). Number of flowers per I2, characteristic of each legume species, has important ecological and evolutionary relevance as it determines diversity in inflorescence architecture; moreover, it is also agronomically important for its potential impact on yield. Nevertheless, the genetic network controlling the number of flowers per I2 is virtually unknown.Chickpea (Cicer arietinum) typically produces one flower per I2 but single flower (sfl) mutants produce two (double‐pod phenotype). We isolated the SFL gene by mapping the sfl‐d mutation and identifying and characterising a second mutant allele. We analysed the effect of sfl on chickpea inflorescence ontogeny with scanning electron microscopy and studied the expression of SFL and meristem identity genes by RNA in situ hybridisation.We show that SFL corresponds to CaRAX1/2a, which codes a MYB transcription factor specifically expressed in the I2 meristem.Our findings reveal SFL as a central factor controlling chickpea inflorescence architecture, acting in the I2 meristem to regulate the length of the period for which it remains active, and therefore determining the number of floral meristems that it can produce.

SEPALLATA--like genes of Isatis indigotica can affect the architecture of the inflorescences and the development of the floral organs.

BackgroundThe architecture of inflorescence and the development of floral organs can influence the yield of seeds and have a significant impact on plant propagation. E-class floral homeotic MADS-box genes exhibit important roles in regulation of floral transition and differentiation of floral organs. Woad (Isatis indigotica) possesses unique inflorescence, floral organs and fruit. However, very little research has been carried out to determine the function of MADS-box genes in this medicinal cruciferous plant species.ResultsSEPALLATA orthologs in I. indigotica were cloned by degenerate PCR. The sequence possessing the highest identity with SEP2 and SEP4 of Arabidopsis were named as IiSEP2 and IiSEP4, respectively. Constitutive expression of IiSEP2 in Columbia (Col-0) ecotype of Arabidopsis led to early flowering, and the number of the flowers and the lateral branches was reduced, indicating an alteration in architecture of the inflorescences. Moreover, the number of the floral organs was declined, the sepals were turned into carpelloid tissues bearing stigmatic papillae and ovules, and secondary flower could be produced in apetalous terminal flowers. In 35S::IiSEP4-GFP transgenic Arabidopsis plants in Landsberg erecta (Ler) genetic background, the number of the floral organs was decreased, sepals were converted into curly carpelloid structures, accompanied by generation of ovules. Simultaneously, the size of petals, stamens and siliques was diminished. In 35S::IiSEP4-GFP transgenic plants of apetalous ap1 cal double mutant in Ler genetic background, the cauliflower phenotype was attenuated significantly, and the petal formation could be rescued. Occasionally, chimeric organs composed of petaloid and sepaloid tissues, or petaloid and stamineous tissues, were produced in IiSEP4 transgenic plants of apl cal double mutant. It suggested that overexpression of IiSEP4 could restore the capacity in petal differentiation. Silencing of IiSEP4 by Virus-Induced Gene Silencing (VIGS) can delay the flowering time, and reduce the number and size of the floral organs in woad flowers.ConclusionAll the results showed that SEPALLATA-like genes could influence the architecture of the inflorescence and the determinacy of the floral meristems, and was also related to development of the floral organs.

Gynoecium and fruit development in Arabidopsis.

Flowering plants produce flowers and one of the most complex floral structures is the pistil or the gynoecium. All the floral organs differentiate from the floral meristem. Various reviews exist on molecular mechanisms controlling reproductive development, but most focus on a short time window and there has been no recent review on the complete developmental time frame of gynoecium and fruit formation. Here, we highlight recent discoveries, including the players, interactions and mechanisms that govern gynoecium and fruit development in Arabidopsis. We also present the currently known gene regulatory networks from gynoecium initiation until fruit maturation.

Promotion of natural flowers by JcFT depends on JcLFY in the perennial woody species Jatropha curcas

Production of normal gametes is necessary for flowering plant reproduction, which involves the transition from vegetative to reproductive stage and floral organ development. Such transitions and floral development are modulated by various environmental and endogenous stimuli and controlled by sophisticated regulatory networks. FLOWERING LOCUS T (FT) and LEAFY (LFY) are two key genes that integrate signals from multiple genetic pathways in Arabidopsis. However, the comprehensive functions and relationship between these two genes in trees are poorly understood. In this study, we found that JcFT played a vital role in regulating the flowering transition in the perennial woody species Jatropha curcas. JcLFY also involved in regulating this transition and controlled floral organ development. The non–flowering phenotype of JcFT-RNAi was rescued successfully by overexpression of JcLFY, while the abnormal flowers produced by JcLFY silencing were not recovered by JcFT overexpression via hybridization. These results indicate that JcFT, in which a mutation leads to a nonflowering phenotype, is the central gene of the floral meristem transition and that JcLFY, in which a mutation leads to striking changes in flowering and often sterility, is the central floral and inflorescence development gene. Moreover, our hybridization results suggest that JcLFY acts downstream of JcFT in Jatropha.