Functional Characterization of JcSWEET12 and JcSWEET17a from Physic Nut.

Physic nut (Jatropha curcas L.) has attracted extensive attention because of its fast growth, easy reproduction, tolerance to barren conditions, and high oil content of seeds. SWEET (Sugar Will Eventually be Exported Transporter) family genes contribute to regulating the distribution of carbohydrates in plants and have great potential in improving yield and stress tolerance. In this study, we performed a functional analysis of the homology of these genes from physic nut, JcSWEET12 and JcSWEET17a. Subcellular localization indicated that the JcSWEET12 protein is localized on the plasma membrane and the JcSWEET17a protein on the vacuolar membrane. The overexpression of JcSWEET12 (OE12) and JcSWEET17a (OE17a) in Arabidopsis leads to late and early flowering, respectively, compared to the wild-type plants. The transgenic OE12 seedlings, but not OE17a, exhibit increased salt tolerance. In addition, OE12 plants attain greater plant height and greater shoot dry weight than the wild-type plants at maturity. Together, our results indicate that JcSWEET12 and JcSWEET17a play different roles in the regulation of flowering time and salt stress response, providing a novel genetic resource for future improvement in physic nut and other plants.

RNA-Seq Reveals That Multiple Pathways Are Involved in Tuber Expansion in Tiger Nuts (Cyperus esculentus L.).

The tiger nut (Cyperus esculentus L.) is a usable tuber and edible oil plant. The size of the tubers is a key trait that determines the yield and the mechanical harvesting of tiger nut tubers. However, little is known about the anatomical and molecular mechanisms of tuber expansion in tiger nut plants. This study conducted anatomical and comprehensive transcriptomics analyses of tiger nut tubers at the following days after sowing: 40 d (S1); 50 d (S2); 60 d (S3); 70 d (S4); 90 d (S5); and 110 d (S6). The results showed that, at the initiation stage of a tiger nut tuber (S1), the primary thickening meristem (PTM) surrounded the periphery of the stele and was initially responsible for the proliferation of parenchyma cells of the cortex (before S1) and then the stele (S2-S3). The increase in cell size of the parenchyma cells occurred mainly from S1 to S3 in the cortex and from S3 to S4 in the stele. A total of 12,472 differentially expressed genes (DEGs) were expressed to a greater extent in the S1-S3 phase than in S4-S6 phase. DEGs related to tuber expansion were involved in cell wall modification, vesicle transport, cell membrane components, cell division, the regulation of plant hormone levels, signal transduction, and metabolism. DEGs involved in the biosynthesis and the signaling of indole-3-acetic acid (IAA) and jasmonic acid (JA) were expressed highly in S1-S3. The endogenous changes in IAA and JAs during tuber development showed that the highest concentrations were found at S1 and S1-S3, respectively. In addition, several DEGs were related to brassinosteroid (BR) signaling and the G-protein, MAPK, and ubiquitin-proteasome pathways, suggesting that these signaling pathways have roles in the tuber expansion of tiger nut. Finally, we come to the conclusion that the cortex development preceding stele development in tiger nut tubers. The auxin signaling pathway promotes the division of cortical cells, while the jasmonic acid pathway, brassinosteroid signaling, G-protein pathway, MAPK pathway, and ubiquitin protein pathway regulate cell division and the expansion of the tuber cortex and stele. This finding will facilitate searches for genes that influence tuber expansion and the regulatory networks in developing tubers.

Functional characterization of polyol/monosaccharide transporter 1 in Lotus japonicus.

Polyol/Monosaccharide Transporters (PLTs/PMTs) localized in the plasma membrane have previously been identified in plants. The physiological role and the functional properties of these proteins in legume plants are, however, unclear. Here we describe the functional analysis of LjPLT1, a plasma membrane-localized PLT protein from Lotus japonicus. The LjPLT1 gene was strongly expressed in the vascular tissue of roots, stems and leaves. Expression of the LjPLT1 cDNAs in yeast revealed that the protein functions as a broad-spectrum H+ -symporter for both linear polyols of sorbitol and mannitol, and cyclic polyol myo-inositol. It also catalyzes the transport of different hexoses, including fructose, glucose, galactose and mannose. Overexpression of LjPLT1 (OELjPLT1) results in inhibition of plant growth and a decrease in nodule nitrogenase activity in L. japonicus. The soluble sugars were increased in newly expanded leaves, roots and nodules but decreased in mature leaves in OELjPLT1 plants. In addition, the OELjPLT1 seedlings displayed an increased sensitivity to high content mannitol and boron toxicity, but neither drought nor salinity stresses. Taken together, the present study indicates that the LjPLT1 protein may participate in the translocation of hexoses/polyols to regulate multiple physiological and growth processes in L. japonicus.

Chemical constituents from leaves of Jatropha curcas.

Objective: To investigate the chemical constituents from the leaves of Jatropha curcas and evaluate their inhibition on lipopolysaccharide (LPS)-activated BV-2 microglia cells. Methods: The n-BuOH extract of the leaves of J. curcas was isolated by macroporous adsorption resin, silica gel, ODS, column chromatography and semi-preparative HPLC. The structures of the compounds were identified by MS, NMR, ECD, and other spectroscopic methods. In addition, anti-neuroinflammatory effects of isolated compounds were evaluated by measuring the production of nitric oxide (NO) in over-activated BV-2 cells. Results: Seventeen compounds, including (7R,8S)-crataegifin A-4-O-ß-D-glucopyranoside (1), (8R,8'R)-arctigenin (2), arctigenin-4'-O-ß-D-glucopyranoside (3), (-)-syringaresinol (4), syringaresinol-4'-O-ß-D-glucopyranoside (5), (-)-pinoresinol (6), pinoresinol-4'-O-ß-D-glucopyranoside (7), buddlenol D (8), (2R,3R)-dihydroquercetin (9), (2S,3S)-epicatechin (10), (2R,3S)-catechin (11), isovitexin (12), naringenin-7-O-ß-D-glucopyranoside (13), chamaejasmin (14), neochamaejasmin B (15), isoneochamaejasmin A (16), and tomentin-5-O-ß-D-glucopyranoside (17) were isolated and identified. Compounds 2, 4 and 8 significantly inhibited the release of NO in BV-2 microglia activated by LPS, with IC50 values of 18.34, 29.33 and 26.30 µmol/L, respectively. Conclusion: Compound 1 is a novel compound, and compounds 2, 3, 8, 14-17 are isolated from Jatropha genus for the first time. In addition, the lignans significantly inhibited NO release and the inhibitory activity was decreased after glycosylation.

Overexpression of LjPLT3 Enhances Salt Tolerance in Lotus japonicus.

Intracellular polyols are used as osmoprotectants by many plants under environmental stress. However, few studies have shown the role of polyol transporters in the tolerance of plants to abiotic stresses. Here, we describe the expression characteristics and potential functions of Lotus japonicus polyol transporter LjPLT3 under salt stress. Using LjPLT3 promoter-reporter gene plants showed that LjPLT3 was expressed in the vascular tissue of L. japonicus leaf, stem, root, and nodule. The expression was also induced by NaCl treatment. Overexpression of LjPLT3 in L. japonicus modified the growth rate and saline tolerance of the transgenic plants. The OELjPLT3 seedlings displayed reduced plant height under both nitrogen-sufficient and symbiotic nitrogen fixation conditions when 4 weeks old. The nodule number of OELjPLT3 plants was reduced by 6.7-27.4% when 4 weeks old. After exposure to a NaCl treatment in Petri dishes for 10 days, OELjPLT3 seedlings had a higher chlorophyll concentration, fresh weight, and survival rate than those in the wild type. For symbiotic nitrogen fixation conditions, the decrease in nitrogenase activity of OELjPLT3 plants was slower than that of the wild type after salt treatment. Compared to the wild type, both the accumulation of small organic molecules and the activity of antioxidant enzymes were higher under salt stress. Considering the concentration of lower reactive oxygen species (ROS) in transgenic lines, we speculate that overexpression of LjPLT3 in L. japonicus might improve the ROS scavenging system to alleviate the oxidative damage caused by salt stress, thereby increasing plant salinity tolerance. Our results will direct the breeding of forage legumes in saline land and also provide an opportunity for the improvement of poor and saline soils.

Ectopic expression of WRINKLED1 in rice improves lipid biosynthesis but retards plant growth and development.

WRINKLED1 (WRI1) is a transcription factor which is key to the regulation of seed oil biosynthesis in Arabidopsis. In the study, we identified two WRI1 genes in rice, named OsWRI1a and OsWRI1b, which share over 98% nucleotide similarity and are expressed only at very low levels in leaves and endosperms. The subcellular localization of Arabidopsis protoplasts showed that OsWRI1a encoded a nuclear localized protein. Overexpression of OsWRI1a under the control of the CaMV 35S promoter severely retarded plant growth and development in rice. Expressing the OsWRI1a gene under the control of the P1 promoter of Brittle2 (highly expressed in endosperm but low in leaves and roots) increased the oil content of both leaves and endosperms and upregulated the expression of several genes related to late glycolysis and fatty acid biosynthesis. However, the growth and development of the transgenic plants were also affected, with phenotypes including smaller plant size, later heading time, and fewer and lighter grains. The laminae (especially those of flag leaves) did not turn green and could not unroll normally. Thus, ectopic expression of OsWRI1a in rice enhances oil biosynthesis, but also leads to abnormal plant growth and development.

Roles of AGD2a in Plant Development and Microbial Interactions of Lotus japonicus.

Arabidopsis AGD2 (Aberrant Growth and Death2) and its close homolog ALD1 (AGD2-like defense response protein 1) have divergent roles in plant defense. We previously reported that modulation of salicylic acid (SA) contents by ALD1 affects numbers of nodules produced by Lotus japonicus, but AGD2's role in leguminous plants remains unclear. A combination of enzymatic analysis and biological characterization of genetic materials was used to study the function of AGD2 (LjAGD2a and LjAGD2b) in L. japonicus. Both LjAGD2a and LjAGD2b could complement dapD and dapE mutants of Escherichia coli and had aminotransferase activity in vitro. ljagd2 plants, with insertional mutations of LjAGD2, had delayed flowering times and reduced seed weights. In contrast, overexpression of LjAGD2a in L. japonicus induced early flowering, with increases in seed and flower sizes, but reductions in pollen fertility and seed setting rates. Additionally, ljagd2a mutation resulted in increased expression of nodulin genes and corresponding increases in infection threads and nodule numbers following inoculation with Rhizobium. Changes in expression of LjAGD2a in L. japonicus also affected endogenous SA contents and hence resistance to pathogens. Our results indicate that LjAGD2a functions as an LL-DAP aminotransferase and plays important roles in plant development. Moreover, LjAGD2a activates defense signaling via the Lys synthesis pathway, thereby participating in legume-microbe interaction.

Genome-Wide Identification, Expression Patterns and Sugar Transport of the Physic Nut SWEET Gene Family and a Functional Analysis of JcSWEET16 in Arabidopsis.

The Sugars Will Eventually be Exported Transporters (SWEET) family is a class of sugar transporters that play key roles in phloem loading, seed filling, pollen development and the stress response in plants. Here, a total of 18 JcSWEET genes were identified in physic nut (Jatropha curcas L.) and classified into four clades by phylogenetic analysis. These JcSWEET genes share similar gene structures, and alternative splicing of messenger RNAs was observed for five of the JcSWEET genes. Three (JcSWEET1/4/5) of the JcSWEETs were found to possess transport activity for hexose molecules in yeast. Real-time quantitative PCR analysis of JcSWEETs in different tissues under normal growth conditions and abiotic stresses revealed that most are tissue-specifically expressed, and 12 JcSWEETs responded to either drought or salinity. The JcSWEET16 gene responded to drought and salinity stress in leaves, and the protein it encodes is localized in both the plasma membrane and the vacuolar membrane. The overexpression of JcSWEET16 in Arabidopsis thaliana modified the flowering time and saline tolerance levels but not the drought tolerance of the transgenic plants. Together, these results provide insights into the characteristics of SWEET genes in physic nut and could serve as a basis for cloning and further functional analysis of these genes.

Anti-neuroinflammatory effects in vitro and in vivo, and chemical profile of Jatropha curcas L.

The ethyl acetate extract of the stems of Jatropha curcas (ESJ) exerted prominent anti-neuroinflammatory effect through inhibiting microglial overactivation, and reducing mRNA expression of inflammatory factors, including nitric oxide (NO), inducible nitric oxide synthase, and interleukin-1ß in the cortex and the formation of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasomes in C57BL/6 mice. Phytochemical research afforded twenty-three major constituents, including five undescribed components (diterpenes 1-3, 7 and a triterpene 18) and a new natural product [a diterpene, (3S,5S,10R)-3-hydroxy-12-methoxy-13-methylpodopcarpa-8,11,13-trien-7-one (8)], by comprehensive analysis of spectroscopic data. Bioassay showed that ESJ (IC50: 6.49 µg/mL), diterpenes 1, 5, 12, 14, 15, 17, triterpenes 18, 19, preussomerin 22, and lactone 23 (IC50 values from 0.10 to 49.05 µM) inhibited NO production more strongly than the positive control in lipopolysaccharide-stimulated BV-2 cells. HPLC experiment further substantiated that 1, 5, 12, 14-15, 17-19, 22-23 are the characteristic constituents of ESJ, suggesting they might possess the potential for the treatment of neuroinflammation.

Ectopic Expression of JcCPL1, 2, and 4 Affects Epidermal Cell Differentiation, Anthocyanin Biosynthesis and Leaf Senescence in Arabidopsis thaliana.

The CAPRICE (CPC)-like (CPL) genes belong to a single-repeat R3 MYB family, whose roles in physic nut (Jatropha curcas L.), an important energy plant, remain unclear. In this study, we identified a total of six CPL genes (JcCPL1-6) in physic nut. The JcCPL3, 4, and 6 proteins were localized mainly in the nucleus, while proteins JcCPL1, 2, and 5 were localized in both the nucleus and the cytoplasm. Ectopic overexpression of JcCPL1, 2, and 4 in Arabidopsis thaliana resulted in an increase in root hair number and decrease in trichome number. Consistent with the phenotype of reduced anthocyanin in shoots, the expression levels of anthocyanin biosynthesis genes were down-regulated in the shoots of these three transgenic A. thaliana lines. Moreover, we observed that OeJcCPL1, 2, 4 plants attained earlier leaf senescence, especially at the late developmental stage. Consistent with this, the expression levels of several senescence-associated and photosynthesis-related genes were, respectively, up-regulated and down-regulated in leaves. Taken together, our results indicate functional divergence of the six CPL proteins in physic nut. These findings also provide insight into the underlying roles of CPL transcription factors in leaf senescence.

A d-pinitol transporter, LjPLT11, regulates plant growth and nodule development in Lotus japonicus.

Polyol transporters have been functionally characterized in yeast and Xenopus laevis oocytes as H+-symporters with broad substrate specificity, but little is known about their physiological roles in planta. To extend this knowledge, we investigated the role of LjPLT11 in Lotus japonicus-Mesorhizobium symbiosis. Functional analyses of LjPLT11 in yeast characterized it as an energy-independent transporter of xylitol, two O-methyl inositols, xylose, and galactose. We showed that LjPLT11 is located on peribacteroid membranes and functions as a facilitative transporter of d-pinitol within infected cells of L. japonicus nodules. Knock-down of LjPLT11 (LjPLT11i) in L. japonicus accelerated plant growth under nitrogen sufficiency, but resulted in abnormal bacteroids with corresponding reductions in nitrogenase activity in nodules and plant growth in the nitrogen-fixing symbiosis. LjPLT11i nodules had higher osmotic pressure in cytosol, and lower osmotic pressure in bacteroids, than wild-type nodules both 3 and 4 weeks after inoculation of Mesorhizobium loti. Levels and distributions of reactive oxygen species were also perturbed in infected cells of 4-week-old nodules in LjPLT11i plants. The results indicate that LjPLT11 plays a key role in adjustment of the levels of its substrate pinitol, and thus maintenance of osmotic balance in infected cells and peribacteroid membrane stability during nodule development.

Ectopic expression of an AGAMOUS homologue gene in Jatropha curcas causes early flowering and heterostylous phenotypes.

Jatropha curcasseeds are abundant in biodiesel, and low seed yields are linked to poor quality female flowers, which creates a bottleneck for Jatropha seed utilization. Therefore, identifying the genes associated with flowering is crucial for the genetic enrichment of seed yields. Here, we identified an AGAMOUS homologue gene (JcAG) from J. curcas. We found that reproductive organs had higher JcAG expression than vegetative organs, particularly the carpel. Rosette leaves were small and misshapen in 35S:JcAG transgenic lines in comparison with those in wild-type plants. JcAG overexpression caused an extremely early flowering, delayed perianth and stamen filament development, small flowers, and significantly shorter Arabidopsis plants with little fruit. In the JcAG-overexpressing line, the homeotic transformation of sepals into pistillate organs was observed, and floral meristem and organ identity genes were regulated. This study provides insights into the JcAG's function and benefits to our knowledge of the underlying the genetic mechanisms related to floral sex differentiation in Jatropha.

LAZY3 plays a pivotal role in positive root gravitropism in Lotus japonicus.

LAZY1 family genes play important roles in both shoot and root gravitropism in plants. Here we report a Lotus japonicus mutant that displays negative gravitropic response in primary and lateral roots. Map-based cloning identified the mutant gene LAZY3 as a functional ortholog of the LAZY1 gene. Mutation of the LAZY3 gene reduced rootward polar auxin transport (PAT) in the primary root, which was also insensitive to the PAT inhibitor N-1-naphthylphthalamic acid. Moreover, immunolocalization of enhanced green fluorescent protein-tagged LAZY3 in L. japonicus exhibited polar localization of LAZY3 on the plasma membrane in root stele cells. We therefore suggest that the polar localization of LAZY3 in stele cells might be required for PAT in L. japonicus root. LAZY3 transcripts displayed asymmetric distribution at the root tip within hours of gravistimulation, while overexpression of LAZY3 under a constitutive promoter in lazy3 plants rescued the gravitropic response in roots. These data indicate that root gravitropism depends on the presence of LAZY3 but not on its asymmetric expression in root tips. Expression of other LAZY genes in a lazy3 background did not rescue the growth direction of roots, suggesting that the LAZY3 gene plays a distinct role in root gravitropism in L. japonicus.

Correlation analysis of cold-related gene expression with physiological and biochemical indicators under cold stress in oil palm.

Oil palm (Elaeis guineensis Jacq.) is a representative tropical oil crop that is sensitive to low temperature. Oil palm can experience cold damage when exposed to low temperatures for a long period. During these unfavorable conditions, a series of gene induction/repression and physico-chemical changes occur in oil palm. To better understand the link between these events, we investigated the expression levels of various genes (including COR410, COR413, CBF1, CBF2, CBF3, ICE1-1, ICE1-2, ICE1-4, SIZ1-1, SIZ1-2, ZAT10, ZAT12) and the accumulation of osmolytes (proline, malondialdehyde and sucrose). Likewise, the activity of superoxide dismutase (SOD) in oil palm under cold stress (4°C, 8°C and 12°C) was examined. The results showed a clear link among the expression of CBFs (especially CBF1 and CBF3) and the all genes examined under cold stress (12°C). The expression of CBF1 and CBF2 also exhibited a positive link with the accumulation of sucrose and proline under cold stress in oil palm. At 4°C, the proline content exhibited a very significant correlation with electrolyte leakage in oil palm. The results of this study provide necessary information regarding the mechanism of the response and adaption of oil palm to cold stress. Additionally, they offer clues for the selection or development of cold-tolerant cultivars from the available germplasms of oil palm.

The role of endogenous thiamine produced via THIC in root nodule symbiosis in Lotus japonicus.

Thiamine is a pivotal primary metabolite which is indispensable to all organisms. Although its biosynthetic pathway has been well documented, the mechanism by which thiamine influences the legume-rhizobium symbiosis remains uncertain. Here, we used overexpressing transgenic plants, mutants and grafting experiments to investigate the roles played by thiamine in Lotus japonicus nodulation. ljthic mutants displayed lethal phenotypes and the defect could be overcome by supplementation of thiamine or by overexpression of LjTHIC. Reciprocal grafting between L. japonicus wild-type Gifu B-129 and ljthic showed that the photosynthetic products of the aerial part made a major contribution to overcoming the nodulation defect in ljthic. Overexpression of LjTHIC in Lotus japonicus (OE-LjTHIC) decreased shoot growth and increased the activity of the enzymes 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase. OE-LjTHIC plants exhibited an increase in the number of infection threads and also developed more nodules, which were of smaller size but unchanged nitrogenase activity compared to the wildtype. Taken together, our results suggest that endogenous thiamine produced via LjTHIC acts as an essential nutrient provided by the host plant for rhizobial infection and nodule growth in the Lotus japonicus - rhizobium interaction.

The Temperature-Dependent Retention of Introns in GPI8 Transcripts Contributes to a Drooping and Fragile Shoot Phenotype in Rice.

Attachment of glycosylphosphatidylinositols (GPIs) to the C-termini of proteins is one of the most common posttranslational modifications in eukaryotic cells. GPI8/PIG-K is the catalytic subunit of the GPI transamidase complex catalyzing the transfer en bloc GPI to proteins. In this study, a T-DNA insertional mutant of rice with temperature-dependent drooping and fragile (df) shoots phenotype was isolated. The insertion site of the T-DNA fragment was 879 bp downstream of the stop codon of the OsGPI8 gene, which caused introns retention in the gene transcripts, especially at higher temperatures. A complementation test confirmed that this change in the OsGPI8 transcripts was responsible for the mutant phenotype. Compared to control plants, internodes of the df mutant showed a thinner shell with a reduced cell number in the transverse direction, and an inhomogeneous secondary wall layer in bundle sheath cells, while many sclerenchyma cells at the tops of the main veins of df leaves were shrunken and their walls were thinner. The df plants also displayed a major reduction in cellulose and lignin content in both culms and leaves. Our data indicate that GPI anchor proteins play important roles in biosynthesis and accumulation of cell wall material, cell shape, and cell division in rice.

Overexpression of a Phosphate Starvation Response AP2/ERF Gene From Physic Nut in Arabidopsis Alters Root Morphological Traits and Phosphate Starvation-Induced Anthocyanin Accumulation.

Physic nut (Jatropha curcas L.) is highly tolerant of barren environments and a significant biofuel plant. To probe mechanisms of its tolerance mechanisms, we have analyzed genome-wide transcriptional profiles of 8-week-old physic nut seedlings subjected to Pi deficiency (P-) for 2 and 16 days, and Pi-sufficient conditions (P+) controls. We identified several phosphate transporters, purple acid phosphatases, and enzymes of membrane lipid metabolism among the 272 most differentially expressed genes. Genes of the miR399/PHO2 pathway (IPS, miR399, and members of the SPX family) showed alterations in expression. We also found that expression of several transcription factor genes was modulated by phosphate starvation stress in physic nut seedlings, including an AP2/ERF gene (JcERF035), which was down-regulated in both root and leaf tissues under Pi-deprivation. In JcERF035-overexpressing Arabidopsis lines both numbers and lengths of first-order lateral roots were dramatically reduced, but numbers of root hairs on the primary root tip were significantly elevated, under both P+ and P- conditions. Furthermore, the transgenic plants accumulated less anthocyanin but had similar Pi contents to wild-type plants under P-deficiency conditions. Expression levels of the tested genes related to anthocyanin biosynthesis and regulation, and genes induced by low phosphate, were significantly lower in shoots of transgenic lines than in wild-type plants under P-deficiency. Our data show that down-regulation of the JcERF035 gene might contribute to the regulation of root system architecture and both biosynthesis and accumulation of anthocyanins in aerial tissues of plants under low Pi conditions.

Biochemical characterization of microbial type terpene synthases in two closely related species of hornworts, Anthoceros punctatus and Anthoceros agrestis.

Microbial terpene synthase-like (MTPSL) genes are a type of terpene synthase genes only recently identified in plants. In contrast to typical plant terpene synthase genes, which are ubiquitous in land plants, MTPSL genes appear to occur only in nonseed plants. Our knowledge of catalytic functions of MTPSLs is very limited. Here we report biochemical characterization of the enzymes encoded by MTPSL genes from two closely related species of hornworts, Anthoceros punctatus and Anthoceros agrestis. Seven full-length MTPSL genes were identified in A. punctatus (ApMTPSL1-7) based on the analysis of its genome sequence. Using homology-based cloning, the apparent orthologs for six of the ApMTPSL genes, except ApMTPSL2, were cloned from A. agrestis. They were designated AaMTPSL1, 3-7. The coding sequences for each of the 13 Anthoceros MTPSL genes were cloned into a protein expression vector. Escherichia coli-expressed recombinant MTPSLs from hornworts were assayed for terpene synthase activities. Six ApMTPSLs and five AaMTPSLs, except for ApMTPSL5 and AaMTPSL5, showed catalytic activities with one or more isoprenyl diphosphate substrates. All functional MTPSLs exhibited sesquiterpene synthase activities. In contrast, only ApMTPSL7 and AaMTPSL7 showed monoterpene synthase activity and only ApMTPSL2, ApMTPSL6 and AaMTPSL6 showed diterpene synthase activity. Most MTPSLs from Anthoceros contain uncanonical aspartate-rich motif in the form of either 'DDxxxD' or 'DDxxx'. Homology-based structural modeling analysis of ApMTPSL1 and ApMTPSL7, which contain 'DDxxxD' and 'DDxxx' motif, respectively, showed that 'DDxxxD' and 'DDxxx' motifs are localized in the similar positions as the canonical 'DDxxD' motif in known terpene synthases. To further understand the role of individual aspartate residues in the motifs, ApMTPSL1 and ApMTPSL7 were selected as two representatives for site-directed mutagenesis studies. No activities were detected when any of the conserved aspartic acid was mutated into alanine. This study provides new information about the catalytic functions of MTPSLs and the functionality of their uncanonical aspartate-rich motifs, and builds a knowledge base for studying the biological importance of MTPSL genes and their terpene products in nonseed plants.

Analysis of Transcriptional Responses of the Inflorescence Meristems in Jatropha curcas Following Gibberellin Treatment.

Jatropha curcas L. seeds an oilseed plant with great potential for biodiesel production. However, low seed yield, which was limited by its lower female flowers, was a major drawback for its utilization. Our previous study found that the flower number and female-to-male ratio were increased by gibberellin treatment. Here, we compared the transcriptomic profiles of inflorescence meristem at different time points after gibberellic acid A3 (GA3) treatment. The present study showed that 951 differentially expressed genes were obtained in response to gibberellin treatment, compared with control samples. The 6-h time point was an important phase in the response to exogenous gibberellin. Furthermore, the plant endogenous gibberellin, auxin, ethylene, abscisic acid, and brassinolide-signaling transduction pathways were repressed, whereas the genes associated with cytokinin and jasmonic acid signaling were upregulated for 24-h time point following GA3 treatment. In addition, the floral meristem determinacy genes (JcLFY, JcSOC1) and floral organ identity genes (JcAP3, JcPI, JcSEP1-3) were significantly upregulated, but their negative regulator (JcSVP) was downregulated after GA3 treatment. Moreover, the effects of phytohormone, which was induced by exogenous plant growth regulator, mainly acted on the female floral differentiation process. To the best of our knowledge, this data is the first comprehensive analysis of the underlying transcriptional response mechanism of floral differentiation following GA3 treatment in J. curcas, which helps in engineering high-yielding varieties of Jatropha.

Transcriptome profile analysis reveals the regulation mechanism of floral sex differentiation in Jatropha curcas L.

The seeds of Jatropha curcas contain a high percentage of biodiesel. However, low seed yield which was limited by its poor female flowers was a bottleneck for its utilization. Here, we compared the transcriptomic profiles of five different samples during floral sex differentiation stages using Illumina Hiseq 4000. Our results showed that hundreds of differentially expressed genes (DEGs) were detected in floral sex initiation period, but thousands of DEGs were involved in the stamens and ovules development process. Moreover, the DEGs were mainly shown up-regulation in male floral initiation, but mainly down-regulation in female floral initiation. Male floral initiation was associated with the flavonoid biosynthesis pathway while female floral initiation was related to the phytohormone signal transduction pathway. Cytokinin (CTK) signaling triggered the initiation of female floral primordium, thereafter other phytohormones co-promoted the female floral development. In addition, the floral organ identity genes played important roles in floral sex differentiation process and displayed a general conservation of the ABCDE model in J. curcas. To the best of our knowledge, this data is the first comprehensive analysis of the underlying regulatory mechanism and the related genes during floral sex differentiation in J. curcas, which help in engineering high-yielding varieties of J. curcas.