Showing papers in "Plant and Cell Physiology in 2013"

Rice Annotation Project Database (RAP-DB): an integrative and interactive database for rice genomics.

Abstract: The Rice Annotation Project Database (RAP-DB, http://rapdb.dna.affrc.go.jp/) has been providing a comprehensive set of gene annotations for the genome sequence of rice, Oryza sativa (japonica group) cv. Nipponbare. Since the first release in 2005, RAP-DB has been updated several times along with the genome assembly updates. Here, we present our newest RAP-DB based on the latest genome assembly, Os-Nipponbare-Reference-IRGSP-1.0 (IRGSP-1.0), which was released in 2011. We detected 37,869 loci by mapping transcript and protein sequences of 150 monocot species. To provide plant researchers with highly reliable and up to date rice gene annotations, we have been incorporating literature-based manually curated data, and 1,626 loci currently incorporate literature-based annotation data, including commonly used gene names or gene symbols. Transcriptional activities are shown at the nucleotide level by mapping RNA-Seq reads derived from 27 samples. We also mapped the Illumina reads of a Japanese leading japonica cultivar, Koshihikari, and a Chinese indica cultivar, Guangluai-4, to the genome and show alignments together with the single nucleotide polymorphisms (SNPs) and gene functional annotations through a newly developed browser, Short-Read Assembly Browser (S-RAB). We have developed two satellite databases, Plant Gene Family Database (PGFD) and Integrative Database of Cereal Gene Phylogeny (IDCGP), which display gene family and homologous gene relationships among diverse plant species. RAP-DB and the satellite databases offer simple and user-friendly web interfaces, enabling plant and genome researchers to access the data easily and facilitating a broad range of plant research topics.

Benzylisoquinoline Alkaloid Metabolism: A Century of Discovery and a Brave New World

Abstract: Benzylisoquinoline alkaloids (BIAs) are a structurally diverse group of plant specialized metabolites with a long history of investigation. Although the ecophysiological functions of most BIAs are unknown, the medicinal properties of many compounds have been exploited for centuries. These include the narcotic analgesics codeine and morphine, the antimicrobial agents sanguinarine and berberine, and the antitussive and anticancer drug noscapine. BIA biosynthesis involves a restricted number of enzyme types that catalyze landmark coupling reactions and subsequent functional group modifications. A pathogenesis-related (PR)10/Bet v1 'Pictet-Spenglerase', several O-methyl-, N-methyl- and O-acetyltransferases, cytochromes P450, FAD-dependent oxidases, non-heme dioxygenases and NADPH-dependent reductases have been implicated in the multistep pathways leading to structurally diverse alkaloids. A small number of plant species, including opium poppy (Papaver somniferum) and other members of the Ranunculales, have emerged as model systems to study BIA metabolism. The expansion of resources to include a wider range of plant species is creating an opportunity to investigate previously uncharacterized BIA pathways. Contemporary knowledge of BIA metabolism reflects over a century of research coupled with the development of key innovations such as radioactive tracing, enzyme isolation and molecular cloning, and functional genomics approaches such as virus-induced gene silencing. Recently, the emergence of transcriptomics, proteomics and metabolomics has expedited the discovery of new BIA biosynthetic genes. The growing repository of BIA biosynthetic genes is providing the parts required to apply emerging synthetic biology platforms to the development of production systems in microbes as an alternative to plants as a commecial source of valuable BIAs.

Stress Enhances the Synthesis of Secondary Plant Products: The Impact of Stress-Related Over-Reduction on the Accumulation of Natural Products

Abstract: Spice and medicinal plants grown under water deficiency conditions reveal much higher concentrations of relevant natural products compared with identical plants of the same species cultivated with an ample water supply. For the first time, experimental data related to this well-known phenomenon have been collected and a putative mechanistic concept considering general plant physiological and biochemical aspects is presented. Water shortage induces drought stress-related metabolic responses and, due to stomatal closure, the uptake of CO2 decreases significantly. As a result, the consumption of reduction equivalents (NADPH + H(+)) for CO2 fixation via the Calvin cycle declines considerably, generating a large oxidative stress and an oversupply of reduction equivalents. As a consequence, metabolic processes are shifted towards biosynthetic activities that consume reduction equivalents. Accordingly, the synthesis of reduced compounds, such as isoprenoids, phenols or alkaloids, is enhanced.

ZmLEA3, a multifunctional group 3 LEA protein from maize (Zea mays L.), is involved in biotic and abiotic stresses

Abstract: Late embryogenesis abundant (LEA) proteins accumulate to high levels during the late stage of seed maturation and in response to water deficit, and are involved in protecting higher plants from damage caused by environmental stresses, especially drought. In the present study, a novel maize (Zea mays L.) group 3 LEA gene, ZmLEA3, was identified and later characterized using transgenic tobacco plants to investigate its functions in abiotic and biotic stresses. Transcript accumulation demonstrated that ZmLEA3 was induced in leaves by high salinity, low temperature, osmotic and oxidative stress as well as by signaling molecules such as ABA, salicylic acid (SA) and methyl jasmonate (MeJA). The transcript of ZmLEA3 could also be induced by pathogens [Pseudomonas syringae pv. tomato DC3000 (pst dc3000)]. ZmLEA3 is located in the cytosol and the nucles. Further study indicated that the ZmLEA3 protein could bind Mn(2+), Fe(3+), Cu(2+) and Zn(2+). Overexpression of ZmLEA3 in transgenic tobacco (Nicotiana tabacum) and yeast (GS115) conferred tolerance to osmotic and oxidative stresses. Interestingly, we also found that overexpression of ZmLEA3 in transgenic tobacco increased the hypersensitive cell death triggered by pst dc3000 and enhanced the expression of PR1a, PR2 and PR4 when compared with the wild type. Thus, we proposed that the ZmLEA3 protein plays a role in protecting plants from damage by protecting protein structure and binding metals under osmotic and oxidative stresses. In addition, ZmLEA3 may also enhance transgenic plant tolerance to biotic stress.

The Plant Ontology as a Tool for Comparative Plant Anatomy and Genomic Analyses

Abstract: The Plant Ontology (PO; http://www.plantontology.org/) is a publicly available, collaborative effort to develop and maintain a controlled, structured vocabulary (‘ontology’) of terms to describe plant anatomy, morphology and the stages of plant development. The goals of the PO are to link (annotate) gene expression and phenotype data to plant structures and stages of plant development, using the data model adopted by the Gene Ontology. From its original design covering only rice, maize and Arabidopsis, the scope of the PO has been expanded to include all green plants. The PO was the first multispecies anatomy ontology developed for the annotation of genes and phenotypes. Also, to our knowledge, it was one of the first biological ontologies that provides translations (via synonyms) in non-English languages such as Japanese and Spanish. As of Release #18 (July 2012), there are about 2.2 million annotations linking PO terms to >110,000 unique data objects representing genes or gene models, proteins, RNAs, germplasm and quantitative trait loci (QTLs) from 22 plant species. In this paper, we focus on the plant anatomical entity branch of the PO, describing the organizing principles, resources available to users and examples of how the PO is integrated into other plant genomics databases and web portals. We also provide two examples of comparative analyses, demonstrating how the ontology structure and PO-annotated data can be used to discover the patterns of expression of the LEAFY (LFY) and terpene synthase (TPS) gene homologs.

Copper Regulates Primary Root Elongation Through PIN1-Mediated Auxin Redistribution

Abstract: The heavy metal copper (Cu) is an essential microelement required for normal plant growth and development, but it inhibits primary root growth when in excess. The mechanism underlying how excess Cu functions in this process remains to be further elucidated. Here, we report that a higher concentration of CuSO ₄ inhibited primary root elongation of Arabidopsis seedlings by affecting both the elongation and meristem zones. In the meristem zone, meristematic cell division potential was reduced by excess Cu. Further experiments showed that Cu can modulate auxin distribution, resulting in higher auxin activities in both the elongation and meristem zones of Cu-treated roots based on DR5::GUS expression patterns. This Cu-mediated auxin redistribution was shown to be responsible for Cu-mediated inhibition of primary root elongation. Additional genetic and physiological data demonstrated that it was PINFORMED1 (PIN1), but not PIN2 or AUXIN1 (AUX1), that regulated this process. However, Cu-induced hydrogen peroxide accumulation did not contribute to Cu-induced auxin redistribution for inhibition of root elongation. When the possible role of ethylene in this process was analyzed, Cu had a similar impact on the root elongation of both the wild type and the ein2-1 mutant, implying that Cu-mediated inhibition of primary root elongation was not due to the ethylene signaling pathway.

Grass meristems II: inflorescence architecture, flower development and meristem fate.

Abstract: Plant development depends on the activity of various types of meristems that generate organs such as leaves and floral organs throughout the life cycle. Grass species produce complex inflorescences and unique flowers. The grass inflorescence is composed of different types of branches, including a specialized branch called a spikelet. The spikelet is a special unit of the inflorescence and forms one to several florets, depending on the species. In the floret, floral organs such as perianth organs, carpels and stamens are formed. In Arabidopsis, because the inflorescence meristem (IM) forms the floral meristems (FMs) directly on its flanks, the change of meristem fate is relatively simple. In contrast, in grasses, different types of meristem, such as the IM, the branch meristem (BM), the spikelet pair meristem (SPM) in some grasses, the spikelet meristem (SM) and the FM, are responsible for the elaboration of their complex inflorescences and flowers. Therefore, sequential changes of meristem fate are required, and a number of genes involved in the specification of the fate of each meristem have been identified. In this review, we focus on the following issues concerning the fate of the reproductive meristems in two grass species, maize (Zea mays) and rice (Oryza sativa): (i) meristem regulation during inflorescence development; (ii) specification and fate change of the BM and the SM; (iii) determinacy of the FM; and (iv) communication between the meristem and lateral organs.

Mutation of the Arabidopsis NAC016 Transcription Factor Delays Leaf Senescence

Abstract: The highly ordered process of senescence forms the final stage of leaf development; a large set of senescence-associated genes (SAGs) execute this orderly dismantling of the photosynthetic apparatus and remobilization of cellular components. A number of transcription factors (TFs) modulate SAG expression to promote or delay senescence. Here we show that NAC016, the previously uncharacterized senescence-associated NAM/ATAF1/2/CUC2 (senNAC) TF in Arabidopsis thaliana, promotes senescence. Leaves of nac016 mutants remained green under senescence-inducing conditions, and leaves of NAC016-overexpressing (NAC016-OX) plants senesced early. Under dark-induced senescence (DIS) conditions, nac016 mutants had low ion leakage, and retained the proper balance of photosystem proteins and normal grana thylakoid shape much longer than wild-type plants, suggesting that nac016 acts as a functional stay-green type senescence mutant. Under DIS conditions, SAGs (NYC1, PPH, SGR1/NYE1 and WRKY22), including senNACs (JUB1, NAP, ORE1, ORS1 and VNI2), were down-regulated in nac016 mutants and up-regulated in NAC016-OX plants. In addition to its role in senescence, NAC016 also affects abiotic stress. Under salt and oxidative stress conditions, NAC016 expression rapidly increased in developing leaves, possibly to promote senescence. Indeed, under the stress conditions, nac016 mutants stayed green and NAC016-OX plants senesced rapidly. To identify direct targets of the NAC016 TF in the regulation of leaf senescence, we conducted yeast one-hybrid assays, which strongly suggested that NAC016 binds to the promoters of NAP and ORS1. Based on these results, we propose that NAC016 regulatory mechanisms promoting leaf senescence exhibit cross-talk with the salt and oxidative stress-responsive signaling pathways.

Transcriptome analysis of Japanese pear (Pyrus pyrifolia Nakai) flower buds transitioning through endodormancy.

Abstract: The transcriptomes of endodormant and ecodormant Japanese pear (Pyrus pyrifolia Nakai 'Kosui') flower buds were analyzed using RNA-seq technology and compared. Among de novo assembly of 114,191 unigenes, 76,995 unigenes were successfully annotated by BLAST searches against various databases. Gene Ontology (GO) enrichment analysis revealed that oxidoreductases were enriched in the molecular function category, a result consistent with previous observations of notable changes in hydrogen peroxide concentration during endodormancy release. In the GO categories related to biological process, the abundance of DNA methylation-related gene transcripts also significantly changed during endodormancy release, indicating the involvement of epigenetic regulation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also showed the changes in transcript abundance of genes involved in the metabolism of various phytohormones. Genes for both ABA and gibberellin biosynthesis were down-regulated, whereas the genes encoding their degradation enzymes were up-regulated during endodormancy release. In the ethylene pathway, 1-aminocyclopropane-1-carboxylate synthase (ACS), a gene encoding the rate-limiting enzyme for ethylene biosynthesis, was induced towards endodormancy release. All of these results indicated the involvement of phytohormones in endodormancy release. Furthermore, the expression of dormancy-associated MADS-box (DAM) genes was down-regulated concomitant with endodormancy release, although changes in the abundance of these gene transcripts were not as significant as those identified by transcriptome analysis. Consequently, characterization of the Japanese pear transcriptome during the transition from endormancy to ecodormancy will provide researchers with useful information for data mining and will facilitate further experiments on endodormancy especially in rosaceae fruit trees.

Characterization of AMT-mediated High-affinity Ammonium Uptake in Roots of Maize (Zea mays L.)

Abstract: High-affinity ammonium uptake in plant roots is mainly mediated by AMT1-type ammonium transporters, and their regulation varies depending on the plant species. In this study we aimed at characterizing AMT-mediated ammonium transport in maize, for which ammonium-based fertilizer is an important nitrogen (N) source. Two ammonium transporter genes, ZmAMT1;1a and ZmAMT1;3 , were isolated from a maize root-specific cDNA library by functional complementation of an ammonium uptake-defective yeast mutant. Ectopic expression of both genes in an ammonium uptake-defective Arabidopsis mutant conferred high-affinity ammonium uptake capacities in roots with substrate affinities of 48 and 33 μM for ZmAMT1;1a and ZmAMT1;3, respectively. In situ hybridization revealed co-localization of both ZmAMT genes on the rhizodermis, suggesting an involvement in capturing ammonium from the rhizosphere. In N-deficient maize roots, [Formula] influx increased significantly while ZmAMT expression did not. Ammonium resupply to N-deficient or nitrate-pre-cultured roots, however, rapidly enhanced both [Formula] influx and ZmAMT transcript levels, revealing a substrate-inducible regulation of ammonium uptake. In conclusion, the two rhizodermis-localized transporters ZmAMT1;1a and ZmAMT1;3 are most probably the major components in the high-affinity transport system in maize roots. A particular regulatory feature is their persistent induction by ammonium rather than an up-regulation under N deficiency.

Cytosolic Glutamine Synthetase1;2 is Responsible for the Primary Assimilation of Ammonium in Rice Roots

Abstract: Among three genes for cytosolic glutamine synthetase (OsGS1;1, OsGS1;2 and OsGS1;3) in rice (Oryza sativa L.) plants, the OsGS1;2 gene is known to be mainly expressed in surface cells of roots, but its function was not clearly understood. We characterized knock-out mutants caused by the insertion of an endogenous retrotransposon Tos17 into exon 2 of OsGS1;2. Homozygously inserted mutants showed severe reduction in active tiller number and hence panicle number at harvest. Other yield components, such as spikelet number per panicle, 1,000-spikelet weight and proportion of well ripened grains, were nearly identical between the mutants and wild-type plants. When the contents of free amino acids in roots were compared between the mutants and the wild type, there were marked reductions in contents of glutamine, glutamate, asparagine and aspartate, but a remarkable increase in free ammonium ions in the mutants. Concentrations of amino acids and ammonium ions in xylem sap behaved in a similar fashion. Re-introduction of OsGS1;2 cDNA under the control of its own promoter into the knock-out mutants successfully restored yield components to wild-type levels as well as ammonium concentration in xylem sap. The results indicate that GS1;2 is important in the primary assimilation of ammonium ions taken up by rice roots, with GS1;1 in the roots unable to compensate for GS1;2 functions.

Tissue-Specific Expression of SMALL AUXIN UP RNA41 Differentially Regulates Cell Expansion and Root Meristem Patterning in Arabidopsis

Abstract: Among the three primary auxin-induced gene families, Auxin/Indole-3-Acetic Acid (Aux/IAA), Gretchen Hagen3 (GH3) and SMALL AUXIN UP RNA (SAUR), the function of SAUR genes remains unclear. Arabidopsis SAUR genes have been phylogenetically classified into three clades. Recent work has suggested that SAUR19 (clade II) and SAUR63 (clade I) promote cell expansion through the modulation of auxin transport. Herein, we present our work on SAUR41, a clade III SAUR gene with a distinctive expression pattern in root meristems. SAUR41 was normally expressed in the quiescent center and cortex/endodermis initials; upon auxin stimulation, the expression was provoked in the endodermal layer. During lateral root development, SAUR41 was expressed in prospective stem cell niches of lateral root primordia and in expanding endodermal cells surrounding the primordia. SAUR41-EGFP (enhanced green fluorescent protein) fusion proteins localized to the cytoplasm. Overexpression of SAUR41 from the Cauliflower mosaic virus 35S promoter led to pleiotropic auxin-related phenotypes, including long hypocotyls, increased vegetative biomass and lateral root development, expanded petals and twisted inflorescence stems. Ectopic SAUR41 proteins were able to promote auxin transport in hypocotyls. Tissue-specific expression of SAUR41 from the PIN1, WOX5, PLT2 and ACR4 promoters induced the formation of new auxin accumulation/signaling peaks above the quiescent centers, whereas tissue-specific expression of SAUR41 from the PIN2 and PLT2 promoters enhanced root gravitropic growth. Cells in the root stem cell niches of these transgenic seedlings were differentially enlarged. The distinctive expression pattern of the SAUR41 gene and the explicit function of SAUR41 proteins implied that further investigations on the loss-of-function phenotypes of this gene in root development and environmental responses are of great interest.

Analysis of the Alfalfa Root Transcriptome in Response to Salinity Stress

Abstract: Salinity is one of the major abiotic factors affecting alfalfa productivity. Identifying genes that control this complex trait will provide critical insights for alfalfa breeding programs. To date, no studies have been published on a deep sequencing-based profiling of the alfalfa transcriptome in response to salinity stress. Observations gathered through research on reference genomes may not always be applicable to alfalfa. In this work, Illumina RNA-sequencing was performed in two alfalfa genotypes contrasting in salt tolerance, in order to estimate a broad spectrum of genes affected by salt stress. A total of 367,619,586 short reads were generated from cDNA libraries originated from roots of both lines. More than 60,000 tentative consensus sequences (TCs) were obtained and, among them, 74.5% had a significant similarity to proteins in the NCBI database. Mining of simple sequence repeats (SSRs) from all TCs revealed 6,496 SSRs belonging to 3,183 annotated unigenes. Bioinformatics analysis showed that the expression of 1,165 genes, including 86 transcription factors (TFs), was significantly altered under salt stress. About 40% of differentially expressed genes were assigned to known gene ontology (GO) categories using Arabidopsis GO. A random check of differentially expressed genes by quantitative real-time PCR confirmed the bioinformatic analysis of the RNA-seq data. A number of salt-responsive genes in both tested genotypes were identified and assigned to functional classes, and gene candidates with roles in the adaptation to salinity were proposed. Alfalfa-specific data on salt-responsive genes obtained in this work will be useful in understanding the molecular mechanisms of salinity tolerance in alfalfa.

Combinatorial Biosynthesis of Legume Natural and Rare Triterpenoids in Engineered Yeast

Abstract: Triterpenoid saponins are a diverse group of specialized (secondary) metabolites with many biological properties. The model legume Medicago truncatula has an interesting profile of triterpenoid saponins from which sapogenins are differentiated into hemolytic and non-hemolytic types according to the position of their functional groups and hemolytic properties. Gene co-expression analysis confirmed the presence of candidate P450s whose gene expression correlated highly with that of b-amyrin synthase (bAS). Among these, we identified CYP716A12 and CYP93E2 as key enzymes in hemolytic and non-hemolytic sapogenin biosynthetic pathways. The other candidate P450s showed no b-amyrin oxidation activity. However, among the remaining candidate P450s, CYP72A61v2 expression highly correlated with that of CYP93E2, and CYP72A68v2 expression highly correlated with that of CYP716A12. These correlation values were higher than occurred with bAS expression. We generated yeast strains expressing bAS, CPR, CYP93E2 and CYP72A61v2, and bAS, CPR, CYP716A12 and CYP72A68v2. These transgenic yeast strains produced soyasapogenol B and gypsogenic acid, respectively. We were therefore able to identify two CYP72A subfamily enzymes: CYP72A61v2, which modifies 24-OH-b-amyrin, and CYP72A68v2, which modifies oleanolic acid. Additionally, P450s that seemed not to work together in planta were combinatorially expressed in transgenic yeast. The yeast strains (expressing bAS, CPR, CYP72A63 and CYP93E2 or CYP716A12) produced rare triterpenoids that do not occur in M. truncatula. These results show the potential for combinatorial synthesis of diverse triterpenoid structures and enable identification of the enzymes involved in their biosynthesis.

Theophylline-Dependent Riboswitch as a Novel Genetic Tool for Strict Regulation of Protein Expression in Cyanobacterium Synechococcus elongatus PCC 7942

Abstract: The cyanobacterium Synechococcus elongatus PCC 7942 is a major model species for studies of photosynthesis. It is are also a potential cell factory for the production of renewable biofuels and valuable chemicals. We employed engineered riboswitches to control translational initiation of target genes in this cyanobacterium. A firefly luciferase reporter assay revealed that three theophylline riboswitches performed as expected in the cyanobacterium. Riboswitch-E* exhibited very low leaky expression of luciferase and superior and dose-dependent on/off regulation of protein expression by theophylline. The maximum magnitude of the induction vs. basal level was ∼190-fold. Furthermore, the induction level was responsive to a wide range of theophylline concentrations in the medium, from 0 to 2 mM, facilitating the fine-tuning of luciferase expression. We adapted this riboswitch to another gene regulation system, in which expression of the circadian clock kaiC gene product is controlled by the theophylline concentration in the culture medium. The results demonstrated that the adequately adjusted expression level of KaiC restored complete circadian rhythm in the kaiC-deficient arrhythmic mutant. This theophylline-dependent riboswitch system has potential for various applications as a useful genetic tool in cyanobacteria.

ABCG15 encodes an ABC transporter protein, and is essential for post-meiotic anther and pollen exine development in rice.

Abstract: In flowering plants, anther and pollen development is critical for male reproductive success. The anther cuticle and pollen exine play an essential role, and in many cereals, such as rice, orbicules/ubisch bodies are also thought to be important for pollen development. The formation of the anther cuticle, exine and orbicules is associated with the biosynthesis and transport of wax, cutin and sporopollenin components. Recently, progress has been made in understanding the biosynthesis of sporopollenin and cutin components in Arabidopsis and rice, but less is known about the mechanisms by which they are transported to the sites of deposition. Here, we report that the rice ATP-binding cassette (ABC) transporter, ABCG15, is essential for post-meiotic anther and pollen development, and is proposed to play a role in the transport of rice anther cuticle and sporopollenin precursors. ABCG15 is highly expressed in the tapetum at the young microspore stage, and the abcg15 mutant exhibits small, white anthers lacking mature pollen, lipidic cuticle, orbicules and pollen exine. Gas chromatography-mass spectrometry (GC-MS) analysis of the abcg15 anther cuticle revealed significant reductions in a number of wax components and aliphatic cutin monomers. The expression level of genes involved in lipid metabolism in the abcg15 mutant was significantly different from their levels in the wild type, possibly due to perturbations in the homeostasis of anther lipid metabolism. Our study provides new insights for understanding the molecular mechanism of the formation of the anther cuticle, orbicules and pollen wall, as well as the machinery for lipid metabolism in rice anthers.

CathaCyc, a Metabolic Pathway Database Built from Catharanthus roseus RNA-Seq Data

Abstract: The medicinal plant Madagascar periwinkle (Catharanthus roseus) synthesizes numerous terpenoid indole alkaloids (TIAs), such as the anticancer drugs vinblastine and vincristine. The TIA pathway operates in a complex metabolic network that steers plant growth and survival. Pathway databases and metabolic networks reconstructed from 'omics' sequence data can help to discover missing enzymes, study metabolic pathway evolution and, ultimately, engineer metabolic pathways. To date, such databases have mainly been built for model plant species with sequenced genomes. Although genome sequence data are not available for most medicinal plant species, next-generation sequencing is now extensively employed to create comprehensive medicinal plant transcriptome sequence resources. Here we report on the construction of CathaCyc, a detailed metabolic pathway database, from C. roseus RNA-Seq data sets. CathaCyc (version 1.0) contains 390 pathways with 1,347 assigned enzymes and spans primary and secondary metabolism. Curation of the pathways linked with the synthesis of TIAs and triterpenoids, their primary metabolic precursors, and their elicitors, the jasmonate hormones, demonstrated that RNA-Seq resources are suitable for the construction of pathway databases. CathaCyc is accessible online (http://www.cathacyc.org) and offers a range of tools for the visualization and analysis of metabolic networks and 'omics' data. Overlay with expression data from publicly available RNA-Seq resources demonstrated that two well-characterized C. roseus terpenoid pathways, those of TIAs and triterpenoids, are subject to distinct regulation by both developmental and environmental cues. We anticipate that databases such as CathaCyc will become key to the study and exploitation of the metabolism of medicinal plants.

The Arabidopsis DUF231 Domain-Containing Protein ESK1 Mediates 2-O- and 3-O-Acetylation of Xylosyl Residues in Xylan

Abstract: Xylan, a major polysaccharide in plant lignocellulosic biomass, is acetylated at O-2 and/or O-3 and its acetylation impedes the use of biomass for biofuel production. Currently, it is not known what genes encode acetyltransferases that are responsible for xylan O-acetylation. In this report, we demonstrate an essential role for the Arabidopsis gene ESKIMO1 (ESK1) in xylan O-acetylation during secondary wall biosynthesis. ESK1 expression was found to be regulated by the secondary wall master regulator SND1 (secondary wall-associated NAC domain protein1) and specifically associated with secondary wall biosynthesis. Its encoded protein was localized in the Golgi, the site of xylan biosynthesis. The esk1 mutation caused reductions in secondary wall thickening and stem mechanical strength. Chemical analyses of cell walls revealed that although the esk1 mutation did not cause apparent alterations in the xylan chain length and the abundance of the reducing end sequence, it resulted in a significant reduction in the degree of xylan acetylation. The reduced acetylation of esk1 xylan rendered it more accessible and digestible by endoxylanase, leading to generation of shorter xylooligomers compared with the wild type. Further structural analysis of xylan showed that the esk1 mutation caused a specific reduction in 2-O- and 3-O-monoacetylation of xylosyl residues but not in 2,3-di-O-acetylation or 3-O-acetylation of xylosyl residues substituted at O-2 with glucuronic acid. Consistent with ESK1's involvement in xylan O-acetylation, an activity assay revealed that the esk1 mutation led to a significant decrease in xylan acetyltransferase activity. Together, these results demonstrate that ESK1 is a putative xylan acetyltransferase required for 2-O- and 3-O-monoacetylation of xylosyl residues and indicate the complexity of the biochemical mechanism underlying xylan O-acetylation.

STIFDB2: an updated version of plant stress-responsive transcription factor database with additional stress signals, stress-responsive transcription factor binding sites and stress-responsive genes in Arabidopsis and rice.

Abstract: Understanding the principles of abiotic and biotic stress responses, tolerance and adaptation remains important in plant physiology research to develop better varieties of crop plants. Better understanding of plant stress response mechanisms and application of knowledge derived from integrated experimental and bioinformatics approaches are gaining importance. Earlier, we showed that compiling a database of stress-responsive transcription factors and their corresponding target binding sites in the form of Hidden Markov models at promoter, untranslated and upstream regions of stress-up-regulated genes from expression analysis can help in elucidating various aspects of the stress response in Arabidopsis. In addition to the extensive content in the first version, STIFDB2 is now updated with 15 stress signals, 31 transcription factors and 5,984 stress-responsive genes from three species (Arabidopsis thaliana, Oryza sativa subsp. japonica and Oryza sativa subsp. indica). We have employed an integrated biocuration and genomic data mining approach to characterize the data set of transcription factors and consensus binding sites from literature mining and stress-responsive genes from the Gene Expression Omnibus. STIFDB2 currently has 38,798 associations of stress signals, stress-responsive genes and transcription factor binding sites predicted using the Stress-responsive Transcription Factor (STIF) algorithm, along with various functional annotation data. As a unique plant stress regulatory genomics data platform, STIFDB2 can be utilized for targeted as well as high-throughput experimental and computational studies to unravel principles of the stress regulome in dicots and gramineae. STIFDB2 is available from the URL: http://caps.ncbs.res.in/stifdb2.

Thermotolerance Responses in Ripening Berries of Vitis vinifera L. cv Muscat Hamburg

Abstract: This study was supported by Genoma Espana [within a collaborative agreement with Genome Canada (Grapegen Project)]; the Spanish Ministry of Science and Innovation [grant Nos. BIO2008-03892, BIO2011-26229]; a bilateral collaborative grant with the University of Cuyo (Mendoza, Argentina) [grant No. AR2009-0021].

LITTLE NUCLEI 1 and 4 Regulate Nuclear Morphology in Arabidopsis thaliana

Abstract: The morphology of plant nuclei varies among different species, organs, tissues and cell types. However, mechanisms and factors involved in the maintenance of nuclear morphology are poorly understood. Because nuclei retain their shapes even after cytoskeletal inhibitor treatments both in vivo and in vitro, we assumed involvement of the nuclear lamina, which plays a critical role in the regulation of nuclear morphology in animals. The crude nuclear lamina fraction isolated from Arabidopsis thaliana leaves was analyzed by mass spectrometry, and putative nuclear lamina proteins were identified. Among their T-DNA insertion lines, nuclei of little nuclei1 (linc1) and linc4 disruptants were more spherical than those of wild-type plants. Because A. thaliana harbors four LINC genes, we prepared all single and linc1/4 and linc2/3 double disruptants. In leaf epidermal cells, the circularity index of the nucleus in all linc disruptants except linc3 was significantly higher than that in the wild-type plants. The extent of the effects of LINC1 and/or LINC4 disruption was significantly higher than that of the effects of LINC2 disruption. The nuclear area was significantly smaller in the linc1, linc4 and linc1/4 disruptants than in the wild-type plants. Regardless of the defects in nuclear morphology, all linc disruptants exhibited a normal ploidy level. In interphase cells, LINC1 and LINC4 were mainly localized to the nuclear periphery, whereas LINC2 was in the nucleoplasm and LINC3 was detected in both regions. From prometaphase to anaphase in mitotic root tip cells, LINC1 was co-localized with chromosomes, whereas other LINCs were dispersed in the cytoplasm.

Ionomic responses and correlations between elements and metabolites under salt stress in wild and cultivated barley.

Abstract: A thorough understanding of ionic detoxification and homeostasis is imperative for improvement of salt tolerance in crops. However, the homeostasis of elements and their relationship to metabolites under salt stress have not been fully elucidated in plants. In this study, Tibetan wild barley accessions, XZ16 and XZ169, differing in salt tolerance, and a salt-tolerant cultivar CM72 were used to investigate ionomic profile changes in tissues in response to 150 and 300 mM NaCl at the germination and seedling stages. At the germination stage, the contents of Ca and Fe significantly decreased in roots, while K and S contents increased, and Ca and Mg contents decreased in shoots, after 10 d of treatment. At the seedling stage, the contents of K, Mg, P and Mn in roots and of K, Ca, Mg and S in shoots decreased significantly after 21 d of treatment. Moreover, Na had a significant negative correlation with metabolites involved in glycolysis, α-ketoglutaric acid, maleic acid and alanine in roots, and metabolites associated with the tricarboxylic acid (TCA) cycle, sucrose, polyols and aspartate in leaves. The salt-tolerant genotypes XZ16 and CM72 showed a lower Na content in tissues, and less reduction in Zn and Cu in roots, of Ca, Mg and S in leaves, and shoot DW than the sensitive genotype XZ169, when exposed to a higher salt level. The results indicated that restriction of Na accumulation and rearrangement of nutrient elements and metabolites in barley tissues are possibly attributable to development of salt tolerance.

Grass Meristems I : Shoot Apical Meristem Maintenance, Axillary Meristem Determinacy and the Floral Transition

Abstract: The vegetative and reproductive shoot architectures displayed by members of the grass family are critical to reproductive success, and thus agronomic yield. Variation in shoot architecture is explained by the maintenance, activity and determinacy of meristems, pools of pluripotent stem cells responsible for post-embryonic plant growth. This review summarizes recent progress in understanding the major properties of grass shoot meristems, focusing on vegetative phase meristems and the floral transition, primarily in rice and maize. Major areas of interest include: the control of meristem homeostasis by the CLAVATA-WUSCHEL pathway and by hormones such as cytokinin; the initiation of axillary meristems and the control of axillary meristem dormancy; and the environmental and endogenous cues that regulate flowering time. In an accompanying paper, Tanaka et al. review subsequent stages of shoot development, including current knowledge of reproductive meristem determinacy and the fate transitions associated with these meristems. © 2013 The Author 2013.

The Involvement of β-Amyrin 28-Oxidase (CYP716A52v2) in Oleanane-Type Ginsenoside Biosynthesis in Panax ginseng

Abstract: Panax species are the most popular medicinal herbs. The root of these plants contains pharmacologically active triterpene saponins, also known as ginsenosides, compounds that are divided into dammarane- and oleanane-type triterpenes. Two CYP716A subfamily genes (CYP716A47 and CYP716A53v2) were recently characterized, encoding an enzyme catalyzing the hydroxylation of dammarane-type triterpenes in Panax ginseng. Herein, we report that one CYP716A subfamily gene (CYP716A52v2) isolated from P. ginseng encodes a β-amyrin 28-oxidase, which is suggested to modify β-amyrin into oleanolic acid, a precursor of an oleanane-type saponin (mainly ginsenoside Ro) in P. ginseng. The ectopic expression of both PNY1 and CYP716A52v2 in recombinant yeast resulted in erythrodiol and oleanolic acid production, respectively. In vitro enzymatic activity assays biochemically confirmed that CYP716A52v2 catalyzed the oxidation of β-amyrin to produce oleanolic acid, and the chemical structure of the oleanolic acid product was confirmed using gas chromatography-mass spectrometry (GC/MS). Transgenic P. ginseng plants were generated via Agrobacterium tumefaciens-mediated transformation: the overexpression of CYP716A52v2 greatly increased the content of oleanane-type ginsenoside (ginsenoside Ro), whereas RNA interference against CYP716A52v2 markedly reduced it. Furthermore, the levels of other dammarene-type ginsenosides were not affected in these transgenic lines. These results indicate that CYP716A52v2 is a β-amyrin 28-oxidase that plays a key role in the biosynthesis of oleanane-type triterpenes in P. ginseng.

Systematic exploration of thioredoxin target proteins in plant mitochondria.

Abstract: The thioredoxin (Trx) system is known to play a pivotal role in cellular redox regulation, but its target proteins in plant mitochondria remain largely uncharacterized. In this study, we systemically screened Trx target candidates in plant mitochondria. Mitochondrial protein extracts were prepared from Arabidopsis shoots, spinach leaves and potato tubers, and then subfractionated into soluble matrix and insoluble membrane fractions. Protein extracts were loaded onto an affinity column immobilizing Arabidopsis mitochondria-localized o-type Trx mutant protein, in which one of two internal cysteines at the active site was substituted by serine. Proteins forming mixed-disulfide intermediates with the mutated Trx were identified by proteomic approaches. This procedure allowed the determination of 101 Trx target candidate proteins involved in a broad spectrum of mitochondrial processes. Furthermore, biochemical assay revealed that one of the potential Trx target proteins, alternative oxidase, is actually redox regulated by Trx. This study provides insights into the regulatory mechanism of diverse functions in mitochondrial biology that are mediated through the Trx system.

Evolution of three LOV blue light receptor families in green plants and photosynthetic stramenopiles: Phototropin, ZTL/FKF1/LKP2, and Aureochrome

Abstract: Many organisms, including bacteria, fungi, animal, plants and algae, utilize blue light to adapt to a fluctuating light environment. Plants and algae, and photosynthetic stramenopiles in particular, require light energy for photosynthesis and have thus evolved a range of sophisticated light-sensing systems to utilize light information efficiently for growth, development and physiological responses. LOV (light, oxygen or voltage) domain photoreceptors are widely distributed among prokaryotic and eukaryotic organisms, and the number of specific LOV photoreceptors are increased in certain taxa. In this review, we summarize the molecular basis and physiological functions of three different families of LOV blue light receptors specific to green plants and photosynthetic stramenopiles: phototropin, ZEITLUPE/FLAVIN-BINDING, KELCH REPEAT, F-BOX 1/LOV KELCH PROTEIN 2 (ZTL/FKF1/LKP2) and aureochrome.

Genetic Engineering of Novel Bluer-Colored Chrysanthemums Produced by Accumulation of Delphinidin-Based Anthocyanins

Abstract: Chrysanthemums (Chrysanthemum morifolium Ramat.) have no purple-, violet- or blue-flowered cultivars because they lack delphinidin-based anthocyanins. This deficiency is due to the absence of the flavonoid 3′,5′-hydroxylase gene (F3′5′H), which encodes the key enzyme for delphinidin biosynthesis. In F3′5′H -transformed chrysanthemums, unpredictable and unstable expression levels have hampered successful production of delphinidin and reduced desired changes in flower color. With the aim of achieving delphinidin production in chrysanthemum petals, we found that anthocyanin biosynthetic gene promoters combined with a translational enhancer increased expression of some F3′5′H genes and accompanying delphinidin-based anthocyanin accumulation in transgenic chrysanthemums. Dramatic accumulation of delphinidin (up to 95%) was achieved by simple overexpression of Campanula F3′5′H controlled by a petal-specific flavanone 3-hydroxylase promoter from chrysanthemum combined with the 5′-untranslated region of the alcohol dehydrogenase gene as a translational enhancer. The flower colors of transgenic lines producing delphinidin-based anthocyanins changed from a red–purple to a purple–violet hue in the Royal Horticultural Society Colour Charts. This result represents a promising step toward molecular breeding of blue chrysanthemums.

TOO MUCH LOVE, a Novel Kelch Repeat-Containing F-box Protein, Functions in the Long-Distance Regulation of the Legume–Rhizobium Symbiosis

Abstract: The interaction of legumes with N2-fixing bacteria collectively called rhizobia results in root nodule development. The number of nodules formed is tightly restricted through the systemic negative feedback control by the host called autoregulation of nodulation (AON). Here, we report the characterization and gene identification of TOO MUCH LOVE (TML), a root factor that acts during AON in a model legume Lotus japonicus. In our genetic analyses using another root-regulated hypernodulation mutant, plenty, the tml-1 plenty double mutant showed additive effects on the nodule number, whereas the tml-1 har1-7 double mutant did not, suggesting that TML and PLENTY act in different genetic pathways and that TML and HAR1 act in the same genetic pathway. The systemic suppression of nodule formation by CLE-RS1/RS2 overexpression was not observed in the tml mutant background, indicating that TML acts downstream of CLE-RS1/RS2. The tml-1 Snf2 double mutant developed an excessive number of spontaneous nodules, indicating that TML inhibits nodule organogenesis. Together with the determination of the deleted regions in tml-1/-2/-3, the fine mapping of tml-4 and the next-generation sequencing analysis, we identified a nonsense mutation in the Kelch repeat-containing F-box protein. As the gene knockdown of the candidate drastically increased the number of nodules, we concluded that it should be the causative gene. An expression analysis revealed that TML is a root-specific gene. In addition, the activity of ProTML-GUS was constitutively detected in the root tip and in the nodules/nodule primordia upon rhizobial infection. In conclusion, TML is a root factor acting at the final stage of AON.

Chemical Synthesis of Arabidopsis CLV3 Glycopeptide Reveals the Impact of Hydroxyproline Arabinosylation on Peptide Conformation and Activity

Abstract: Arabinosylation of hydroxyproline (Hyp) is a post-translational modification often found in secreted peptide signals in plants. The physiological importance of this modification was highlighted by the finding that CLAVATA3 (CLV3), a key peptide signal for regulating the fate of stem cells in the shoot apical meristem in Arabidopsis, contains three l-arabinose residues linked via linear β-1,2-linkages. However, understanding the functions and properties of arabinosylated peptides has been hindered by difficulties in synthesizing the complex arabinose chain. Here we report the stereoselective total synthesis of β-1,2-linked triarabinosylated CLV3 peptide ([Ara3]CLV3). Chemically synthesized [Ara3]CLV3 restricted stem cell activity more effectively than did unmodified CLV3 peptide. Comparison of mono-, di- and triarabinosylated CLV3 glycopeptides revealed that the biological activity increased progressively as the arabinose chain length increased. Thus, the arabinose chain length of CLV3 is important for its biological activity. Nuclear magnetic resonance spectroscopy and nuclear Overhauser effect-based structure calculations further revealed the structural impact of the arabinose chain on peptide conformation. The arabinose chain of [Ara3]CLV3 extends toward the C-terminal end of the peptide, and its non-reducing end is positioned proximal to the peptide backbone. Consequently, the arabinose chain causes distinct distortion in the C-terminal half of the peptide in a highly directional manner. The established synthetic route of [Ara3]CLV3 will greatly contribute to our understanding of the biology and biochemistry of arabinosylated peptide signals in plants.

Nitrogen-dependent regulation of de novo cytokinin biosynthesis in rice: the role of glutamine metabolism as an additional signal.

Abstract: Cytokinin activity in plants is closely related to nitrogen availability, and an Arabidopsis gene for adenosine phosphate-isopentenyltransferase (IPT), IPT3, is regulated by inorganic nitrogen sources in a nitrate-specific manner. In this study, we have identified another regulatory system of cytokinin de novo biosynthesis in response to nitrogen status. In rice, OsIPT4, OsIPT5, OsIPT7 and OsIPT8 were up-regulated in response to exogenously applied nitrate and ammonium, with accompanying accumulation of cytokinins. Pre-treatment of roots with l-methionine sulfoximine, a potent inhibitor of glutamine synthetase, abolished the nitrate- and ammonium-dependent induction of OsIPT4 and OsIPT5, while glutamine application induced their expression. Thus, neither nitrate nor ammonium, but glutamine or a related metabolite, is essential for the induction of these IPT genes in rice. On the other hand, glutamine-dependent induction of IPT3 occurs in Arabidopsis, at least to some extent. In transgenic lines repressing the expression of OsIPT4, which is the dominant IPT in rice roots, the nitrogen-dependent increase of cytokinin in the xylem sap was significantly reduced, and seedling shoot growth was retarded despite sufficient nitrogen. We conclude that plants possess multiple regulation systems for nitrogen-dependent cytokinin biosynthesis to modulate growth in response to nitrogen availability.