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.

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.

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.

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.

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.

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.

Effects of LAZY family genes on shoot gravitropism in Lotus japonicus.

Plant architecture is an important agronomic trait to determine the biomass and sward structure of forage grass. The IGT family plays a pivotal role in plant gravitropism, encompassing both the gravitropic response and the modulation of plant architecture. We have previously shown that LjLAZY3, one of the IGT genes, plays a distinct role in root gravitropism in L. japonicus. However, the function of LAZY proteins on shoot gravitropism in this species is poorly understood. In this study, we identified nine IGT genes in the L. japonicus genome, which have been categorized into four clades based on the phylogenetic relationships of IGT proteins from 18 legumes: LAZY1, NGR (NEGATIVE GRAVITROPIC RESPONSE OF ROOTS), IGT-LIKE, and TAC1. We found that LAZY genes in the first three clades have demonstrated distinct role for modulating plant gravitropism in L. japonicus with specific impacts as follows. Mutation of the LAZY1 gene, LjLAZY1, defected the gravitropic response of hypocotyl without impacting the main stem's branch angle. In contrast, the overexpression of the NGR gene, LjLAZY3, substantially modulated the shoot's gravitropism, leading to narrower lateral branch angles. Additionally, it enhanced the shoots' gravitropic response. The overexpression of another NGR gene, LjLAZY4, specifically reduced the main stem's branch angle and decreased plant stature without affecting the shoot gravitropic response. The phenotype of IGT-LIKE gene LjLAZY2 overexpression is identical to that of LjLAZY4. While overexpression of the IGT-LIKE gene LjLAZY5 did not induce any observable changes in branch angle, plant height, or gravitropic response. Furthermore, the LjLAZYs were selectively interacted with different BRXL and RLD proteins, which should the important factor to determine their different functions in controlling organ architecture in L. japonicus. Our results deepen understanding of the LjLAZY family and its potential for plant architecture improvement in L. japonicus.

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.

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.

Overexpression of SGR results in oxidative stress and lesion-mimic cell death in rice seedlings.

It is thought that the Stay Green Rice (SGR) gene is involved in the disaggregation of the light harvesting complex and in the subsequent breakdown of chlorophyll and apo-protein during senescence. In this study, we found that overexpression of SGR (Ov-SGR) resulted in the generation of singlet oxygen and other reactive oxygen species and produced a chlorophyll-dependent regional cell death phenotype on leaves of rice seedlings. Transcriptome analyses using Affymetrix Rice GeneChips revealed that Ov-SGR rice seedlings exhibited a number of signs of singlet oxygen response. The genes and their associated biochemical pathways identified provide an insight into how rice plants respond to singlet oxygen at the molecular and physiologic level.

Isoforms of GBSSI and SSII in four legumes and their phylogenetic relationship to their orthologs from other angiosperms.

Multiple isoforms of starch synthases (SSs) have been found in plants. In addition, at least two isoforms of granule-bound SS (GBSS) and SSII have further diverged in cereals into two or three subisoforms. Here, we report the occurrence, phylogeny, and expression patterns of two different forms of both GBSSI and SSII in four legumes: birdsfoot trefoil, cowpea, mung bean, and soybean. The phylogenetic data acquired indicate that the putative proteins of both SS duplicates have diverged into two different isoforms: GBSSIa and GBSSIb, and SSIIa and SSIIb. The SSIIb genes appear to have become non-functional in soybean as a result of two nonsense mutations in the putative coding region. Transcripts of the GBSSIa and SSIIa genes were found to be abundant in cotyledons, but had lower expression levels in the leaves of the two starchy seed legumes. However, these genes were expressed at moderate levels in the leaves of the two oilseed legumes. In contrast, the GBSSIb and SSIIb genes were mainly expressed in the leaves of the legumes we examined. In both the legume and cereal species we studied, the GBSS orthologs that were mainly expressed in sink tissues, were more hydrophilic and may have been subjected to more intense purifying selection than those that were mainly expressed in source tissues. These findings provide evidence that the GBSSI and SSII genes in the starchy seed legumes and cereals studied have undergone convergent evolution with respect to evolutionary constraints, amino acid sequences, and expression divergence after gene duplication.

Comparison of the starch synthesis genes between maize and rice: copies, chromosome location and expression divergence.

Gene duplication and divergence are important evolutionary processes. It has been suggested that a whole genome duplication (WGD) event occurred in the Gramineae, predating its divergence, and a second WGD occurred in maize during its evolution. In this study we compared the fate of the genes involved in the core pathway of starch biosynthesis following the ancient and second WGDs in maize and rice. In total, thirty starch synthesis genes were detected in the maize genome, which covered all the starch synthesis gene families encoded by 27 genes in rice. All of these genes, except ZmGBSSIIb and ZmBEIII, are anchored within large-scale synteny blocks of rice and maize chromosomes. Previous findings and our results indicate that two of the current copies of many starch synthesis genes (including AGPL, AGPS, GBSS, SSII, SSIII, and BEII) probably arose from the ancient WGD in the Gramineae and are still present in the maize and rice genome. Furthermore, two copies of at least six genes (AGPS1, SSIIb, SSIIIb, GBSSII, BEI, and ISA3) appear to have been retained in the maize genome after its second WGD, although complete coding regions were only detected among the duplicate sets of AGPS1, SSIIb, and SSIIIb. The expression patterns of the remaining duplicate sets of starch synthesis genes (AGPL1/2, AGPS1/2, SSIIa/b, SSIIIa/b, GBSSI/II, and BEIIa/b) differ in their expression and could be classified into two groups in maize. The first group is mainly expressed in the endosperm, whereas the second is expressed in other organs and the early endosperm development. The four duplicate sets of ZmGBSSII, ZmSSIIb, ZmSSIIIb and AGPS1, which arose from the second WGD diverged in gene structure and/or expression patterns in maize. These results indicated that some duplicated starch synthesis genes were remained, whereas others diverged in gene structure and/or expression pattern in maize. For most of the duplicated genes, one of the copies has disappeared in the maize genome after the WGD and the subsequent "diploidization".