BLISTER promotes seed maturation and fatty acid biosynthesis by interacting with WRINKLED1 to regulate chromatin dynamics in Arabidopsis.

WRINKLED1 (WRI1) is an important transcription factor that regulates seed oil biosynthesis. However, how WRI1 regulates gene expression during this process remains poorly understood. Here, we found that BLISTER (BLI) is expressed in maturing Arabidopsis thaliana seeds and acts as an interacting partner of WRI1. bli mutant seeds showed delayed maturation, a wrinkled seed phenotype, and reduced oil content, similar to the phenotypes of wri1. In contrast, BLI overexpression resulted in enlarged seeds and increased oil content. Gene expression and genetic analyses revealed that BLI plays a role in promoting the expression of WRI1 targets involved in fatty acid biosynthesis and regulates seed maturation together with WRI1. BLI is recruited by WRI1 to the AW boxes in the promoters of fatty acid biosynthesis genes. BLI shows a mutually exclusive interaction with the Polycomb-group protein CURLY LEAF (CLF) or the chromatin remodeling factor SWITCH/SUCROSE NONFERMENTING 3B (SWI3B), which facilitates gene expression by modifying nucleosomal occupancy and histone modifications. Together, these data suggest that BLI promotes the expression of fatty acid biosynthesis genes by interacting with WRI1 to regulate chromatin dynamics, leading to increased fatty acid production. These findings provide insights into the roles of the WRI1-BLI-CLF-SWI3B module in mediating seed maturation and gene expression.

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.

Arsenic inhibits citric acid accumulation via downregulating vacuolar proton pump gene expression in citrus fruits.

Citric acid content is a critical quality determinant in citrus (Citrus spp.) fruits. Although arsenic (As) can effectively reduce citric acid content to improve citrus fruit quality, it can have adverse environmental effects. The discovery of nontoxic substitutes is hampered by the incomplete elucidation of the underlying mechanisms of As action in citrus fruits. Metabolic, transcriptomic, and physiological analyses were employed to investigate As action on citric acid accumulation to discover the mechanisms of As action in citrus. The enzyme activity related to citrate biosynthesis was not inhibited and the content of the involved metabolites was not reduced in As-treated fruits. However, the proton pump genes CitPH5 and CitPH1 control the vacuolar citric acid accumulation and transcription factor genes CitTT8 and CitMYB5, which regulate CitPH5 and CitPH1, were downregulated. The oxidative stress-response genes were upregulated in As-treated fruits. The reactive oxygen species (ROS) treatment also downregulated CitTT8 and CitMYB5 in juice cells. The mitochondrial ROS production rate increased in As-treated fruits. AsIII was more potent in stimulating isolated mitochondria to overproduce ROS compared to AsV. Our results indicate that the As inhibition of citric acid accumulation may be primarily due to the transcriptional downregulation of CitPH5, CitPH1, CitTT8, and CitMYB5. As-induced oxidative stress signaling may operate upstream to downregulate these acid regulator genes. Mitochondrial thiol proteins may be the principal targets of As action in citrus fruits.

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.

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.

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.

The Pumilio RNA-binding protein APUM24 regulates seed maturation by fine-tuning the BPM-WRI1 module in Arabidopsis.

Pumilio RNA-binding proteins participate in messenger RNA (mRNA) degradation and translational repression, but their roles in plant development are largely unclear. Here, we show that Arabidopsis PUMILIO PROTEIN24 (APUM24), an atypical Pumilio-homology domain-containing protein, plays an important part in regulating seed maturation, a major stage of plant development. APUM24 is strongly expressed in maturing seeds. Reducing APUM24 expression resulted in abnormal seed maturation, wrinkled seeds, and lower seed oil contents, and APUM24 knockdown resulted in lower levels of WRINKLED 1 (WRI1), a key transcription factor controlling seed oil accumulation, and lower expression of WRI1 target genes. APUM24 reduces the mRNA stability of BTB/POZMATH (BPM) family genes, thus decreasing BPM protein levels. BPM is responsible for the 26S proteasome-mediated degradation of WRI1 and has important functions in plant growth and development. The 3' untranslated regions of BPM family genes contain putative Pumilio response elements (PREs), which are bound by APUM24. Reduced BPM or increased WRI1 expression rescued the deficient seed maturation of apum24-2 knockdown mutants, and APUM24 overexpression resulted in increased seed size and weight. Therefore, APUM24 is crucial to seed maturation through its action as a positive regulator fine-tuning the BPM-WRI1 module, making APUM24 a promising target for breeding strategies to increase crop yields.

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.

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.

The Phenylalanine Ammonia Lyase Gene LjPAL1 Is Involved in Plant Defense Responses to Pathogens and Plays Diverse Roles in Lotus japonicus-Rhizobium Symbioses.

Phenylalanine ammonia lyase (PAL) is important in the biosynthesis of plant secondary metabolites that regulate growth responses. Although its function is well-established in various plants, the functional significance of PAL genes in nodulation is poorly understood. Here, we demonstrate that the Lotus japonicus PAL (LjPAL1) gene is induced by Mesorhizobium loti infection and methyl-jasmonate (Me-JA) treatment in roots. LjPAL1 altered PAL activity, leading to changes in lignin contents and thicknesses of cell walls in roots and nodules of transgenic plants and, hence, to structural changes in roots and nodules. LjPAL1-knockdown plants (LjPAL1i) exhibited increased infection thread and nodule numbers and the induced upregulation of nodulin gene expression after M. loti infection. Conversely, LjPAL1 overexpression delayed the infection process and reduced infection thread and nodule numbers after M. loti inoculation. LjPAL1i plants also exhibited reduced endogenous salicylic acid (SA) accumulation and expression of the SA-dependent marker gene. Their infection phenotype could be partially restored by exogenous SA or Me-JA application. Our data demonstrate that LjPAL1 plays diverse roles in L. japonicus-rhizobium symbiosis, affecting rhizobial infection progress and nodule structure, likely by inducing lignin modification, regulating endogenous SA biosynthesis, and modulating SA signaling.

Integrated genome sequence and linkage map of physic nut (Jatropha curcas L.), a biodiesel plant.

The family Euphorbiaceae includes some of the most efficient biomass accumulators. Whole genome sequencing and the development of genetic maps of these species are important components in molecular breeding and genetic improvement. Here we report the draft genome of physic nut (Jatropha curcas L.), a biodiesel plant. The assembled genome has a total length of 320.5 Mbp and contains 27,172 putative protein-coding genes. We established a linkage map containing 1208 markers and anchored the genome assembly (81.7%) to this map to produce 11 pseudochromosomes. After gene family clustering, 15,268 families were identified, of which 13,887 existed in the castor bean genome. Analysis of the genome highlighted specific expansion and contraction of a number of gene families during the evolution of this species, including the ribosome-inactivating proteins and oil biosynthesis pathway enzymes. The genomic sequence and linkage map provide a valuable resource not only for fundamental and applied research on physic nut but also for evolutionary and comparative genomics analysis, particularly in the Euphorbiaceae.

Global analysis of gene expression profiles in physic nut (Jatropha curcas L.) seedlings exposed to drought stress.

BACKGROUND: Physic nut (Jatropha curcas L.) is a small perennial tree or large shrub, which is well-adapted to semi-arid regions and is considered to have potential as a crop for biofuel production. It is now regarded as an excellent model for studying biofuel plants. However, our knowledge about the molecular responses of this species to drought stress is currently limited. RESULTS: In this study, genome-wide transcriptional profiles of roots and leaves of 8-week old physic nut seedlings were analyzed 1, 4 and 7 days after withholding irrigation. We observed a total of 1533 and 2900 differentially expressed genes (DEGs) in roots and leaves, respectively. Gene Ontology analysis showed that the biological processes enriched in droughted plants relative to unstressed plants were related to biosynthesis, transport, nucleobase-containing compounds, and cellular protein modification. The genes found to be up-regulated in roots were related to abscisic acid (ABA) synthesis and ABA signal transduction, and to the synthesis of raffinose. Genes related to ABA signal transduction, and to trehalose and raffinose synthesis, were up-regulated in leaves. Endoplasmic reticulum (ER) stress response genes were significantly up-regulated in leaves under drought stress, while a number of genes related to wax biosynthesis were also up-regulated in leaves. Genes related to unsaturated fatty acid biosynthesis were down-regulated and polyunsaturated fatty acids were significantly reduced in leaves 7 days after withholding irrigation. As drought stress increased, genes related to ethylene synthesis, ethylene signal transduction and chlorophyll degradation were up-regulated, and the chlorophyll content of leaves was significantly reduced by 7 days after withholding irrigation. CONCLUSIONS: This study provides us with new insights to increase our understanding of the response mechanisms deployed by physic nut seedlings under drought stress. The genes and pathways identified in this study also provide much information of potential value for germplasm improvement and breeding for drought resistance.

Knockdown of LjIPT3 influences nodule development in Lotus japonicus.

Cytokinins play important roles in legume-rhizobia symbiosis. Here we report isolation of six genes encoding isopentenyl transferase (IPT) from Lotus japonicus, which catalyze the rate-limiting step of cytokinin biosynthesis. The LjIPT3 gene was found to be up-regulated in infected roots and mature nodules. Histochemical analysis demonstrated expression of Pro(LjIPT3):GUS (ß-glucuronidase) in vegetative and reproductive organs, and was especially high in the vascular bundles of roots. When inoculated with Mesorhizobium loti MAFF303099, LjIPT3 was undetectable in the nodule primordia and developing nodules, and later it was expressed only in the vascular bundles of mature nodules. In addition, knockdown of LjIPT3 (LjIPT3i) by RNA interference reduced levels of endogenous cytokinins, affected plant development and accelerated Chl degradation during dark-induced leaf senescence. Compared with the wild type, LjIPT3i plants produced fewer infection threads and nodules. In addition, expression of downstream nodulation-related transcription factor genes LjNSP1, LjNSP2 and LjNIN decreased dramatically in LjIPT3i plants. These results suggest that LjIPT3 regulates the CRE1-dependent cytokinin pathway, affecting nodule initiation and thereby influencing the number of infection threads and nodules. Detection of nitrogenase activity and observation of nodule structure showed that endogenous cytokinins are required for full development of the infected cells in mature nodules by preventing early senescence. Therefore, our results indicate that the LjIPT3 gene product is required for nodule initiation and development, and does not appear to be involved in early infection events.

Knockdown of LjALD1, AGD2-like defense response protein 1, influences plant growth and nodulation in Lotus japonicus.

The discovery of the enzyme L,L-diaminopimelate aminotransferase (LL-DAP-AT, EC 2.6.1.83) uncovered a unique step in the L-lysine biosynthesis pathway in plants. In Arabidopsis thaliana, LL-DAP-AT has been shown to play a key role in plant-pathogen interactions by regulation of the salicylic acid (SA) signaling pathway. Here, a full-length cDNA of LL-DAP-AT named as LjALD1 from Lotus japonicus (Regel) Larsen was isolated. The deduced amino acid sequence shares 67% identity with the Arabidopsis aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (AtALD1) and is predicted to contain the same key elements: a conserved aminotransferase domain and a pyridoxal-5'-phosphate cofactor binding site. Quantitative real-time PCR analysis showed that LjALD1 was expressed in all L. japonicus tissues tested, being strongest in nodules. Expression was induced in roots that had been infected with the symbiotic rhizobium Mesorhizobium loti or treated with SA agonist benzo-(1, 2, 3)-thiadiazole-7-carbothioic acid. LjALD1 Knockdown exhibited a lower SA content, an increased number of infection threads and nodules, and a slight reduction in nodule size. In addition, compared with wild-type, root growth was increased and shoot growth was suppressed in LjALD1 RNAi plant lines. These results indicate that LjALD1 may play important roles in plant development and nodulation via SA signaling in L. japonicus.

Cloning and functional characterization of an acyl-acyl carrier protein thioesterase (JcFATB1) from Jatropha curcas.

A full-length cDNA of an acyl-acyl carrier protein (ACP) thioesterase (TE) (EC 3.1.2.14), named JcFATB1, was isolated from the woody oil plant Jatropha curcas L. The deduced amino acid sequence of the cDNA shares about 78% identity with FATB TEs, but only about 33% identity with FATA TEs from other plants. The deduced sequence also contains two essential residues (H(317) and C(352)) for TE catalytic activity and a putative chloroplast transit peptide at the N-terminal. Southern blot analysis revealed that a single copy of JcFATB1 is present in the J. curcas genome, and semi-quantitative PCR analysis showed that JcFATB1 was expressed in all tissues that were examined, most strongly in seeds, in which its expression peaked in late developmental stages. Seed-specific overexpression of the JcFATB1 cDNA in Arabidopsis resulted in increased levels of saturated fatty acids, especially palmitate, and in reduced levels of unsaturated fatty acids. The findings suggest that JcFATB1 from this woody oil plant can function as a saturated acyl-ACP TE and could potentially modify the seed oil of J. curcas to increase its levels of palmitate.

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.

Molecular cloning and expression analysis of a gene encoding a putative beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III) from Jatropha curcas.

A cDNA clone encoding a putative beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III) was isolated from Jatropha curcas L., a woody oil plant. The cDNA clone (named JcKAS III) contained a 1203-bp open reading frame coding for 400 amino acids with a predicted molecular mass of about 42 kDa. The deduced amino acid sequence of the cDNA clone shares about 80% identity to KAS III from other plants, and contains a conserved Cys(176) in the active site and the amino acid motif G(355)NTSAAS(361) which is responsible for binding regulatory acyl-ACPs. Southern blotting analysis indicated that JcKAS III is a single copy gene in the J. curcas genome. Quantitative real-time PCR analysis showed that JcKAS III was expressed in all tissues examined with highest expression in roots, and that expression of JcKAS III increased as seeds developed.

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.

Global gene expression analysis of the response of physic nut (Jatropha curcas L.) to medium- and long-term nitrogen deficiency.

Jatropha curcas L. is an important biofuel plant with excellent tolerance of barren environments. However, studies on the regulatory mechanisms that operate in this plant in response to nitrogen (N) shortage are scarce. In this study, genome-wide transcriptional profiles of the roots and leaves of 8-week old physic nut seedlings were analyzed after 2 and 16 days of N starvation. Enrichment results showed that genes associated with N metabolism, processing and regulation of RNA, and transport predominated among those showing alterations in expression. Genes encoding transporter families underwent major changes in expression in both roots and leaves; in particular, those with roles in ammonia, amino acid and peptide transport were generally up-regulated after long-term starvation, while AQUAPORIN genes, whose products function in osmoregulation, were down-regulated. We also found that ASPARA-GINASE B1 and SARCOSINE OXIDASE genes were up-regulated in roots and leaves after 2 and 16 d N starvation. Genes associated with ubiquitination-mediated protein degradation were significantly up-regulated. In addition, genes in the JA biosynthesis pathway were strongly activated while expression of those in GA signaling was inhibited in leaves. We showed that four major classes of genes, those with roles in N uptake, N reutilization, C/N ratio balance, and cell structure and synthesis, were particularly influenced by long-term N limitation. Our discoveries may offer clues to the molecular mechanisms that regulate N reallocation and reutilization so as to maintain or increase plant performance even under adverse environmental conditions.

Molecular cloning and characterization of two ß-ketoacyl-acyl carrier protein synthase I genes from Jatropha curcas L.

The ß-ketoacyl-acyl carrier protein synthase I (KASI) is involved in de novo fatty acid biosynthesis in many organisms. Two putative KASI genes, JcKASI-1 and JcKASI-2, were isolated from Jatropha curcas. The deduced amino acid sequences of JcKASI-1 and JcKASI-2 exhibit around 83.8% and 72.5% sequence identities with AtKASI, respectively, and both contain conserved Cys-His-Lys-His-Phe catalytic active sites. Phylogenetic analysis indicated that JcKASI-2 belongs to a clade with several KASI proteins from dicotyledonous plants. Both JcKASI genes were expressed in multiple tissues, most strongly in filling stage seeds of J. curcas. Additionally, the JcKASI-1 and JcKASI-2 proteins were both localized to the plastids. Expressing JcKASI-1 in the Arabidopsis kasI mutant rescued the mutant's phenotype and restored the fatty acid composition and oil content in seeds to wild-type, but expressing JcKASI-2 in the Arabidopsis kasI mutant resulted in only partial rescue. This implies that JcKASI-1 and JcKASI-2 exhibit partial functional redundancy and KASI genes play a universal role in regulating fatty acid biosynthesis, growth, and development in plants.