AtPHT4;4 is a chloroplast-localized ascorbate transporter in Arabidopsis.

Ascorbate is an antioxidant and coenzyme for various metabolic reactions in vivo. In plant chloroplasts, high ascorbate levels are required to overcome photoinhibition caused by strong light. However, ascorbate is synthesized in the mitochondria and the molecular mechanisms underlying ascorbate transport into chloroplasts are unknown. Here we show that AtPHT4;4, a member of the phosphate transporter 4 family of Arabidopsis thaliana, functions as an ascorbate transporter. In vitro analysis shows that proteoliposomes containing the purified AtPHT4;4 protein exhibit membrane potential- and Cl(-)-dependent ascorbate uptake. The AtPHT4;4 protein is abundantly expressed in the chloroplast envelope membrane. Knockout of AtPHT4;4 results in decreased levels of the reduced form of ascorbate in the leaves and the heat dissipation process of excessive energy during photosynthesis is compromised. Taken together, these observations indicate that the AtPHT4;4 protein is an ascorbate transporter at the chloroplast envelope membrane, which may be required for tolerance to strong light stress.

Arabidopsis mutant of AtABCG26, an ABC transporter gene, is defective in pollen maturation.

In plants, pollen is the male gametophyte that is generated from microspores, which are haploid cells produced after meiosis of diploid pollen mother cells in floral anthers. In normal maturation, microspores interact with the tapetum, which consists of one layer of metabolically active cells enclosing the locule in anthers. The tapetum plays several important roles in the maturation of microspores. ATP-binding cassette (ABC) transporters are a highly conserved protein super-family that uses the energy released in ATP hydrolysis to transport substrates. The ABC transporter gene family is more diverse in plants than in animals. Previously, we reported that an Arabidopsis half-size type ABC transporter gene, COF1/AtWBC11/AtABCG11, is involved in lipid transport for the construction of cuticle layers and pollen coats in normal organ formation, as compared to CER5/AtWBC12/AtABCG12. However, physiological functions of most other ABCG members are unknown. Here, we identified another family gene, AtABCG26, which is required for pollen development in Arabidopsis. An AtABCG26 mutant developed very few pollen grains, resulting in a male-sterile phenotype. By investigating microspore and pollen development in this mutant, we observed that there was a slight abnormality in tetrad morphology prior to the formation of haploid microspores. At a later stage, we could not detect exine deposition on the microspore surface. During pollen maturation, many grains in the mutant anthers got aborted, and surviving grains were found to be defective in mitosis. Transmission of the mutant allele through male gametophytes appeared to be normal in genetic transmission analysis, supporting the view that the pollen function was disturbed by sporophytic defects in the AtABCG26 mutant. AtABCG26 can be expected to be involved in the transport of substrates such as sporopollenin monomers from tapetum to microspores, which both are plant-specific structures critical to pollen development.

Arabidopsis mutants of AtABCG22, an ABC transporter gene, increase water transpiration and drought susceptibility.

In plants, water vapour is released into the atmosphere through stomata in a process called transpiration. Abscisic acid (ABA) is a key phytohormone that facilitates stomatal closure through its action on guard cells. Recently, ATP-binding cassette (ABC) transporter genes, AtABCG25 and AtABCG40, were shown to be involved in ABA transport and responses. However, the functions of many other AtABCG family genes are still unknown. Here, we identified another ABCG gene (AtABCG22) that is required for stomatal regulation in Arabidopsis. The atabcg22 mutant plants had lower leaf temperatures and increased water loss, implying elevated transpiration through an influence on stomatal regulation. We also found that atabcg22 plants were more suspectible to drought stress than wild-type plants. AtABCG22 was expressed in aerial organs, mainly guard cells, in which the gene expression pattern was consistent with the mutant phenotypes. Using double mutants, we investigated the genetic relationships between the mutations. The atabcg22 mutation further increased the water loss of srk2e/ost1 mutants, which were defective in ABA signalling in guard cells. Also, the atabcg22 mutation enhanced the phenotype of nced3 mutants, which were defective in ABA biosynthesis. Accordingly, the additive roles of AtABCG22 functions in ABA signalling and ABA biosynthesis are discussed.

Arabidopsis tiling array analysis to identify the stress-responsive genes.

Plants respond and adapt to drought, cold, and high-salinity stresses. Stress-inducible gene products function in the stress response and tolerance in plants. Using cDNA microarrays and oligonucleotide microarrays, stress-inducible genes have been identified in various plant species so far. Recently, tiling array technology has become a powerful tool for the whole-genome transcriptome analysis. We applied the Arabidopsis Affymetrix tiling arrays to study the whole-genome transcriptome under drought, cold, and high-salinity stresses and identified a large number of drought, cold, and high-salinity stress-inducible genes and transcriptional units (TUs).

Genome sequence of the palaeopolyploid soybean.

Soybean (Glycine max) is one of the most important crop plants for seed protein and oil content, and for its capacity to fix atmospheric nitrogen through symbioses with soil-borne microorganisms. We sequenced the 1.1-gigabase genome by a whole-genome shotgun approach and integrated it with physical and high-density genetic maps to create a chromosome-scale draft sequence assembly. We predict 46,430 protein-coding genes, 70% more than Arabidopsis and similar to the poplar genome which, like soybean, is an ancient polyploid (palaeopolyploid). About 78% of the predicted genes occur in chromosome ends, which comprise less than one-half of the genome but account for nearly all of the genetic recombination. Genome duplications occurred at approximately 59 and 13 million years ago, resulting in a highly duplicated genome with nearly 75% of the genes present in multiple copies. The two duplication events were followed by gene diversification and loss, and numerous chromosome rearrangements. An accurate soybean genome sequence will facilitate the identification of the genetic basis of many soybean traits, and accelerate the creation of improved soybean varieties.

CNI1/ATL31, a RING-type ubiquitin ligase that functions in the carbon/nitrogen response for growth phase transition in Arabidopsis seedlings.

Plants are able to sense and respond to changes in the balance between carbon (C) and nitrogen (N) metabolite availability, known as the C/N response. During the transition to photoautotrophic growth following germination, growth of seedlings is arrested if a high external C/N ratio is detected. To clarify the mechanisms for C/N sensing and signaling during this transition period, we screened a large collection of FOX transgenic plants, overexpressing full-length cDNAs, for individuals able to continue post-germinative growth under severe C/N stress. One line, cni1-D (carbon/nitrogen insensitive 1-dominant), was shown to have a suppressed sensitivity to C/N conditions at both the physiological and molecular level. The CNI1 cDNA encoded a predicted RING-type ubiquitin ligase previously annotated as ATL31. Overexpression of ATL31 was confirmed to be responsible for the cni1-D phenotype, and a knock-out of this gene resulted in hypersensitivity to C/N conditions during post-germinative growth. The ATL31 protein was confirmed to contain ubiquitin ligase activity using an in vitro assay system. Moreover, removal of this ubiquitin ligase activity from the overexpressed protein resulted in the loss of the mutant phenotype. Taken together, these data demonstrated that CNI1/ATL31 activity is required for the plant C/N response during seedling growth transition.

The glycerophosphoryl diester phosphodiesterase-like proteins SHV3 and its homologs play important roles in cell wall organization.

Despite the importance of extracellular events in cell wall organization and biogenesis, the mechanisms and related factors are largely unknown. We isolated an allele of the shaven3 (shv3) mutant of Arabidopsis thaliana, which exhibits ruptured root hair cells during tip growth. SHV3 encodes a novel protein with two tandemly repeated glycerophosphoryl diester phosphodiesterase-like domains and a glycosylphosphatidylinositol anchor, and several of its paralogs are found in Arabidopsis. Here, we report the detailed characterization of mutants of SHV3 and one of its paralogs, SVL1. The shv3 and svl1 double mutant exhibited additional defects, including swollen guard cells, aberrant expansion of the hypocotyl epidermis and ectopic lignin deposits, suggesting decreased rigidity of the cell wall. Fourier-transform infrared spectroscopy and measurement of the cell wall components indicated an altered cellulose content and pectin modification with cross-linking in the double mutant. Furthermore, we found that the ruptured root hair phenotype of shv3 was suppressed by increasing the amount of borate, which is supposed to be involved in pectic polysaccharide cross-linking, in the medium. These findings indicate that SHV3 and its paralogs are novel important factors involved in primary cell wall organization.

Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili.

BACKGROUND: Cryptomeria japonica D. Don is one of the most commercially important conifers in Japan. However, the allergic disease caused by its pollen is a severe public health problem in Japan. Since large-scale analysis of expressed sequence tags (ESTs) in the male strobili of C. japonica should help us to clarify the overall expression of genes during the process of pollen development, we constructed a full-length enriched cDNA library that was derived from male strobili at various developmental stages. RESULTS: We obtained 36,011 expressed sequence tags (ESTs) from either one or both ends of 19,437 clones derived from the cDNA library of C. japonica male strobili at various developmental stages. The 19,437 cDNA clones corresponded to 10,463 transcripts. Approximately 80% of the transcripts resembled ESTs from Pinus and Picea, while approximately 75% had homologs in Arabidopsis. An analysis of homologies between ESTs from C. japonica male strobili and known pollen allergens in the Allergome Database revealed that products of 180 transcripts exhibited significant homology. Approximately 2% of the transcripts appeared to encode transcription factors. We identified twelve genes for MADS-box proteins among these transcription factors. The twelve MADS-box genes were classified as DEF/GLO/GGM13-, AG-, AGL6-, TM3- and TM8-like MIKCC genes and type I MADS-box genes. CONCLUSION: Our full-length enriched cDNA library derived from C. japonica male strobili provides information on expression of genes during the development of male reproductive organs. We provided potential allergens in C. japonica. We also provided new information about transcription factors including MADS-box genes expressed in male strobili of C. japonica. Large-scale gene discovery using full-length cDNAs is a valuable tool for studies of gymnosperm species.

Arabidopsis transcriptome analysis under drought, cold, high-salinity and ABA treatment conditions using a tiling array.

Plants respond and adapt to drought, cold and high-salinity stresses in order to survive. In this study, we applied Arabidopsis Affymetrix tiling arrays to study the whole genome transcriptome under drought, cold, high-salinity and ABA treatment conditions. The bioinformatic analysis using the tiling array data showed that 7,719 non-AGI transcriptional units (TUs) exist in the unannotated "intergenic" regions of Arabidopsis genome. These include 1,275 and 181 TUs that are induced and downregulated, respectively, by the stress or ABA treatments. Most of the non-AGI TUs are hypothetical non-protein-coding RNAs. About 80% of the non-AGI TUs belong to pairs of the fully overlapping sense-antisense transcripts (fSATs). Significant linear correlation between the expression ratios (treated/untreated) of the sense TUs and the ratios of the antisense TUs was observed in the SATs of AGI code/non-AGI TU. We studied the biogenesis mechanisms of the stress- or ABA-inducible antisense RNAs and found that the expression of sense TUs is necessary for the stress- or ABA-inducible expression of the antisense TUs in the fSATs (AGI code/non-AGI TU).

The AtGenExpress hormone and chemical treatment data set: experimental design, data evaluation, model data analysis and data access.

We analyzed global gene expression in Arabidopsis in response to various hormones and in related experiments as part of the AtGenExpress project. The experimental agents included seven basic phytohormones (auxin, cytokinin, gibberellin, brassinosteroid, abscisic acid, jasmonate and ethylene) and their inhibitors. In addition, gene expression was investigated in hormone-related mutants and during seed germination and sulfate starvation. Hormone-inducible genes were identified from the hormone response data. The effects of each hormone and the relevance of the gene lists were verified by comparing expression profiles for the hormone treatments and related experiments using Pearson's correlation coefficient. This approach was also used to analyze the relationships among expression profiles for hormone responses and those included in the AtGenExpress stress-response data set. The expected correlations were observed, indicating that this approach is useful to monitor the hormonal status in the stress-related samples. Global interactions among hormones-inducible genes were analyzed in a pairwise fashion, and several known and novel hormone interactions were detected. Genome-wide transcriptional gene-to-gene correlations, analyzed by hierarchical cluster analysis (HCA), indicated that our data set is useful for identification of clusters of co-expressed genes, and to predict the functions of unknown genes, even if a gene's function is not directly related to the experiments included in AtGenExpress. Our data are available online from AtGenExpressJapan; the results of genome-wide HCA are available from PRIMe. The data set presented here will be a versatile resource for future hormone studies, and constitutes a reference for genome-wide gene expression in Arabidopsis.

The Arabidopsis SDG4 contributes to the regulation of pollen tube growth by methylation of histone H3 lysines 4 and 36 in mature pollen.

Plant SET domain proteins are known to be involved in the epigenetic control of gene expression during plant development. Here, we report that the Arabidopsis SET domain protein, SDG4, contributes to the epigenetic regulation of pollen tube growth, thus affecting fertilization. Using an SDG4-GFP fusion construct, the chromosomal localization of SDG4 was established in tobacco BY-2 cells. In Arabidopsis, sdg4 knockout showed reproductive defects. Tissue-specific expression analyses indicated that SDG4 is the major ASH1-related gene expressed in the pollen. Immunological analyses demonstrated that SDG4 was involved in the methylation of histone H3 in the inflorescence and pollen grains. The significant reduction in the amount of methylated histone H3 K4 and K36 in sdg4 pollen vegetative nuclei resulted in suppression of pollen tube growth. Our results indicate that SDG4 is capable of modulating the expression of genes that function in the growth of pollen tube by methylation of specific lysine residues of the histone H3 in the vegetative nuclei.

Arabidopsis MALE STERILITY1 encodes a PHD-type transcription factor and regulates pollen and tapetum development.

The Arabidopsis thaliana MALE STERILITY1 (MS1) gene encodes a nuclear protein with Leu zipper-like and PHD-finger motifs and is important for postmeiotic pollen development. Here, we examined MS1 function using both cell biological and molecular biological approaches. We introduced a fusion construct of MS1 and a transcriptional repression domain (MS1-SRDX) into wild-type Arabidopsis, and the transgenic plants showed a semisterile phenotype similar to that of ms1. Since the repression domain can convert various kinds of transcriptional activators to dominant repressors, this suggested that MS1 functioned as a transcriptional activator. The Leu zipper-like region and the PHD motif were required for the MS1 function. Phenotypic analysis of the ms1 mutant and the MS1-SRDX transgenic Arabidopsis indicated that MS1 was involved in formation of pollen exine and pollen cytosolic components as well as tapetum development. Next, we searched for MS1 downstream genes by analyzing publicly available microarray data and identified 95 genes affected by MS1. Using a transgenic ms1 plant showing dexamethasone-inducible recovery of fertility, we further examined whether these genes were immediately downstream of MS1. From these results, we discuss a role of MS1 in pollen and tapetum development and the conservation of MS1 function in flowering plants.

Cytological and biochemical analysis of COF1, an Arabidopsis mutant of an ABC transporter gene.

In transposon-tagged lines of Arabidopsis we found two new mutants, cof1-1 and cof1-2 (cuticular defect and organ fusion), that show the phenotype of wilting when grown in soil, organ fusion of rosette leaves and infertility. Toluidine blue testing and scanning electron microscopy observation revealed that these mutants had cuticular defects in the stems and adult leaves, but not in cotyledones. Transmission electron microscopy observation revealed thinner cuticle layers in the mutants, and cuticular materials interspersed between the two fused epidermal layers were observed in the mutant rosette leaves. These two mutants had a transposon insertion in the coding regions of WBC11, which was classified as a member of ABC transporter genes in Arabidopsis. WBC11 showed high sequence similarity to CER5 (also called WBC12), which was involved in cuticular lipid export. Gas chromatographic analysis revealed that C29 alkane extracted from the stem surface of cof1 mutants was reduced whereas C29 ketone was accumulated, which was different from the case of cer5 mutants. While cer5 mutants had fairly normal morphology, cof1 mutants had pleiotropic phenotypes so that COF1/WBC11 could have important roles different from those of CER5/WBC12. We also found that C29 alkane was accumulated in the intracellular extract of cof1 mutants, suggesting a function for WBC11 in the direct transport of lipid molecules. Pollen observation showed that mutant pollen grains were irregularly shaped. The function of COF1/WBC11 in lipid transport for the construction of cuticle layers and pollen coats for normal organ formation is discussed.

Co-expression of the stress-inducible zinc finger homeodomain ZFHD1 and NAC transcription factors enhances expression of the ERD1 gene in Arabidopsis.

The ZFHD recognition sequence (ZFHDRS) and NAC recognition sequence (NACRS) play an important role in the dehydration-inducible expression of the Arabidopsisthaliana EARLY RESPONSIVETO DEHYDRATION STRESS 1 (ERD1) gene. Using the yeast one-hybrid system, we isolated a cDNA encoding the ZFHD1 transcriptional activator that specifically binds to the 62 bp promoter region of ERD1, which contains the ZFHDRS. Both in vitro and in vivo analyses confirmed specific binding of the ZFHD1 to ZFHDRS, and the expression of ZFHD1 was induced by drought, high salinity and abscisic acid. The DNA-binding and activation domains of ZFHD1 were localized on the C-terminal homeodomain and N-terminal zinc finger domain, respectively. Microarray analysis of transgenic plants over-expressing ZFHD1 revealed that several stress-inducible genes were upregulated in the transgenic plants. Transgenic plants exhibited a smaller morphological phenotype and had a significant improvement of drought stress tolerance. Using the yeast two-hybrid system, we detected an interaction between ZFHD1 and NACRS-binding NAC proteins. Moreover, co-over-expression of the ZFHD1 and NAC genes restored the morphological phenotype of the transgenic plants to a near wild-type state and enhanced expression of ERD1 in both Arabidopsis T87 protoplasts and transgenic Arabidopsis plants.

Catalysis, subcellular localization, expression and evolution of the targeting peptides degrading protease, AtPreP2.

We have previously identified a zinc metalloprotease involved in the degradation of mitochondrial and chloroplast targeting peptides, the presequence protease (PreP). In the Arabidopsis thaliana genomic database, there are two genes that correspond to the protease, the zinc metalloprotease (AAL90904) and the putative zinc metalloprotease (AAG13049). We have named the corresponding proteins AtPreP1 and AtPreP2, respectively. AtPreP1 and AtPreP2 show significant differences in their targeting peptides and the proteins are predicted to be localized in different compartments. AtPreP1 was shown to degrade both mitochondrial and chloroplast targeting peptides and to be dual targeted to both organelles using an ambiguous targeting peptide. Here, we have overexpressed, purified and characterized proteolytic and targeting properties of AtPreP2. AtPreP2 exhibits different proteolytic subsite specificity from AtPreP1 when used for degradation of organellar targeting peptides and their mutants. Interestingly, AtPreP2 precursor protein was also found to be dual targeted to both mitochondria and chloroplasts in a single and dual in vitro import system. Furthermore, targeting peptide of the AtPreP2 dually targeted green fluorescent protein (GFP) to both mitochondria and chloroplasts in tobacco protoplasts and leaves using an in vivo transient expression system. The targeting of both AtPreP1 and AtPreP2 proteases to chloroplasts in A. thaliana in vivo was confirmed via a shotgun mass spectrometric analysis of highly purified chloroplasts. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that AtPreP1 and AtPreP2 are differentially expressed in mature A. thaliana plants. Phylogenetic evidence indicated that AtPreP1 and AtPreP2 are recent gene duplicates that may have diverged through subfunctionalization.

Monitoring the expression profiles of genes induced by hyperosmotic, high salinity, and oxidative stress and abscisic acid treatment in Arabidopsis cell culture using a full-length cDNA microarray.

Transcriptional regulation in response to hyperosmotic, high-salinity and oxidative stress, and abscisic acid (ABA) treatment in Arabidopsis suspension-cultured cell line T87 was investigated with a cDNA microarray containing 7000 independent full-length Arabidopsis cDNAs. The transcripts of 102, 11, 84 and 73 genes were increased more than 5-fold within 5h after treatment with 0.5M mannitol, 0.1M NaCl, 50 microM ABA and 10mM H2O2, respectively. On the other hand, the transcripts of 44, 57, 25 and 34 genes were down-regulated to less than one-third within 5h after treatment with 0.5M mannitol, 0.1M NaCl, 50 microM ABA and 10mM H2O2, respectively. Venn diagram analysis revealed 11 genes were induced significantly by mannitol, NaCl, and ABA, indicating crosstalk among these signaling pathways. Comparison of the genes induced by each stress revealed that 32%, 17% and 33% of mannitol-, NaCl- and ABA-inducible genes were also induced by H2O2, indicating the crosstalk between the signaling pathways for osmotic stress and oxidative stress. Although the expression profiles revealed that the T87 cells had most of the regulatory systems seen in Arabidopsis seedlings, the T87 cells did not have one of ABA-dependent signaling pathways.

Folate synthesis in plants: the last step of the p-aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate lyase.

In plants, the last step in the synthesis of p-aminobenzoate (PABA) moiety of folate remains to be elucidated. In Escherichia coli, this step is catalyzed by the PabC protein, a beta-lyase that converts 4-amino-4-deoxychorismate (ADC)--the reaction product of the PabA and PabB enzymes--to PABA and pyruvate. So far, the only known plant enzyme involved in PABA synthesis is ADC synthase, which has fused domains homologous to E. coli PabA and PabB and is located in plastids. ADC synthase has no lyase activity, implying that plants have a separate ADC lyase. No such lyase is known in any eukaryote. Genomic and phylogenetic approaches identified Arabidopsis and tomato cDNAs encoding PabC homologs with putative chloroplast-targeting peptides. These cDNAs were shown to encode functional enzymes by complementation of an E. coli pabC mutant, and by demonstrating that the partially purified recombinant proteins convert ADC to PABA. Plant ADC lyase is active as dimer and is not feedback inhibited by physiologic concentrations of PABA, its glucose ester, or folates. The full-length Arabidopsis ADC lyase polypeptide was translocated into isolated pea chloroplasts and, when fused to green fluorescent protein, directed the passenger protein to Arabidopsis chloroplasts in transient expression experiments. These data indicate that ADC lyase, like ADC synthase, is present in plastids. As shown previously for the ADC synthase transcript, the level of ADC lyase mRNA in the pericarp of tomato fruit falls sharply as ripening advances, suggesting that the expression of these two enzymes is coregulated.

The MALE STERILITY1 gene of Arabidopsis, encoding a nuclear protein with a PHD-finger motif, is expressed in tapetal cells and is required for pollen maturation.

We identified the Arabidopsis MALE STERILITY1 (MS1) gene by transposon-mediated mutagenesis. In the transposon-inserted allele ms1-8, normal immature microspores separated from tetrads, but their subsequent maturation was abnormal: the outer layer of the microspore was absent, and both the microspore and the tapetal layer gradually became vacuolated. Empty locules resulted. The MS1 gene was expressed only in the tapetal layer during a very short period when the microspores were packed as tetrads. By the time the microspores had separated, the gene was no longer expressed. MS1 was not expressed in microspores. MS1 encodes a protein with a PHD-finger motif characteristic of some transcriptional regulators. A fusion protein consisting of the N-terminus of MS1 and green fluorescent protein was localized in the nucleus. These results suggest that MS1 protein is a nuclear signal molecule indispensable for pollen maturation.