Antagonistic regulation of the gibberellic acid response during stem growth in rice.

The size of plants is largely determined by growth of the stem. Stem elongation is stimulated by gibberellic acid1-3. Here we show that internode stem elongation in rice is regulated antagonistically by an 'accelerator' and a 'decelerator' in concert with gibberellic acid. Expression of a gene we name ACCELERATOR OF INTERNODE ELONGATION 1 (ACE1), which encodes a protein of unknown function, confers cells of the intercalary meristematic region with the competence for cell division, leading to internode elongation in the presence of gibberellic acid. By contrast, upregulation of DECELERATOR OF INTERNODE ELONGATION 1 (DEC1), which encodes a zinc-finger transcription factor, suppresses internode elongation, whereas downregulation of DEC1 allows internode elongation. We also show that the mechanism of internode elongation that is mediated by ACE1 and DEC1 is conserved in the Gramineae family. Furthermore, an analysis of genetic diversity suggests that mutations in ACE1 and DEC1 have historically contributed to the selection of shorter plants in domesticated populations of rice to increase their resistance to lodging, and of taller plants in wild species of rice for adaptation to growth in deep water. Our identification of these antagonistic regulatory factors enhances our understanding of the gibberellic acid response as an additional mechanism that regulates internode elongation and environmental fitness, beyond biosynthesis and gibberellic acid signal transduction.

Structural Cerebral Network Differences in Prodromal Alzheimer's Disease and Prodromal Dementia with Lewy Bodies.

INTRODUCTION: Alzheimer's disease (AD) and dementia with Lewy bodies (DLB) have long prodromal phases without dementia. However, the patterns of cerebral network alteration in this early stage of the disease remain to be clarified. METHOD: Participants were 48 patients with mild cognitive impairment (MCI) due to AD (MCI-AD), 18 patients with MCI with DLB (MCI with Lewy bodies: MCI-LB), and 23 healthy controls who underwent a 1.5-Tesla magnetic resonance imaging scan. Cerebral networks were extracted from individual T1-weighted images based on the intracortical similarity, and we estimated the differences of network metrics among the three diagnostic groups. RESULTS: Whole-brain analyses for degree, betweenness centrality, and clustering coefficient images were performed using SPM8 software. The patients with MCI-LB showed significant reduction of degree in right putamen, compared with healthy subjects. The MCI-AD patients showed significant lower degree in left insula and bilateral posterior cingulate cortices compared with healthy subjects. There were no significant differences in small-world properties and in regional gray matter volume among the three groups. CONCLUSIONS: We found the change of degree in the patients with MCI-AD and with MCI-LB, compared with healthy controls. These findings were consistent with the past single-photon emission computed tomography studies focusing on AD and DLB. The disease-related difference in the cerebral neural network might provide an adjunct biomarker for the early detection of AD and DLB.

High affinity promoter binding of STOP1 is essential for early expression of novel aluminum-induced resistance genes GDH1 and GDH2 in Arabidopsis.

Malate efflux from roots, which is regulated by the transcription factor STOP1 (SENSITIVE-TO-PROTON-RHIZOTOXICITY1) and mediates aluminum-induced expression of ALUMINUM-ACTIVATED-MALATE-TRANSPORTER1 (AtALMT1), is critical for aluminum resistance in Arabidopsis thaliana. Several studies showed that AtALMT1 expression in roots is rapidly observed in response to aluminum; this early induction is an important mechanism to immediately protect roots from aluminum toxicity. Identifying the molecular mechanisms that underlie rapid aluminum resistance responses should lead to a better understanding of plant aluminum sensing and signal transduction mechanisms. In this study, we observed that GFP-tagged STOP1 proteins accumulated in the nucleus soon after aluminum treatment. The rapid aluminum-induced STOP1-nuclear localization and AtALMT1 induction were detected in the presence of a protein synthesis inhibitor, suggesting that post-translational regulation is involved in these events. STOP1 also regulated rapid aluminum-induced expression for other genes that carry a functional/high-affinity STOP1-binding site in their promoter, including STOP2, GLUTAMATE-DEHYDROGENASE1 and 2 (GDH1 and 2). However STOP1 did not regulate Al resistance genes which have no functional STOP1-binding site such as ALUMINUM-SENSITIVE3, suggesting that the binding of STOP1 in the promoter is essential for early induction. Finally, we report that GDH1 and 2 which are targets of STOP1, are novel aluminum-resistance genes in Arabidopsis.

Using Speech Data From Interactions With a Voice Assistant to Predict the Risk of Future Accidents for Older Drivers: Prospective Cohort Study.

BACKGROUND: With the rapid growth of the older adult population worldwide, car accidents involving this population group have become an increasingly serious problem. Cognitive impairment, which is assessed using neuropsychological tests, has been reported as a risk factor for being involved in car accidents; however, it remains unclear whether this risk can be predicted using daily behavior data. OBJECTIVE: The objective of this study was to investigate whether speech data that can be collected in everyday life can be used to predict the risk of an older driver being involved in a car accident. METHODS: At baseline, we collected (1) speech data during interactions with a voice assistant and (2) cognitive assessment data-neuropsychological tests (Mini-Mental State Examination, revised Wechsler immediate and delayed logical memory, Frontal Assessment Battery, trail making test-parts A and B, and Clock Drawing Test), Geriatric Depression Scale, magnetic resonance imaging, and demographics (age, sex, education)-from older adults. Approximately one-and-a-half years later, we followed up to collect information about their driving experiences (with respect to car accidents) using a questionnaire. We investigated the association between speech data and future accident risk using statistical analysis and machine learning models. RESULTS: We found that older drivers (n=60) with accident or near-accident experiences had statistically discernible differences in speech features that suggest cognitive impairment such as reduced speech rate (P=.048) and increased response time (P=.040). Moreover, the model that used speech features could predict future accident or near-accident experiences with 81.7% accuracy, which was 6.7% higher than that using cognitive assessment data, and could achieve up to 88.3% accuracy when the model used both types of data. CONCLUSIONS: Our study provides the first empirical results that suggest analysis of speech data recorded during interactions with voice assistants could help predict future accident risk for older drivers by capturing subtle impairments in cognitive function.

Sensitive to Proton Rhizotoxicity1 Regulates Salt and Drought Tolerance of Arabidopsis thaliana through Transcriptional Regulation of CIPK23.

The transcription factor sensitive to proton rhizotoxicity 1 (STOP1) regulates multiple stress tolerances. In this study, we confirmed its involvement in NaCl and drought tolerance. The root growth of the T-DNA insertion mutant of STOP1 (stop1) was sensitive to NaCl-containing solidified MS media. Transcriptome analysis of stop1 under NaCl stress revealed that STOP1 regulates several genes related to salt tolerance, including CIPK23. Among all available homozygous T-DNA insertion mutants of the genes suppressed in stop1, only cipk23 showed a NaCl-sensitive root growth phenotype comparable to stop1. The CIPK23 promoter had a functional STOP1-binding site, suggesting a strong CIPK23 suppression led to NaCl sensitivity of stop1. This possibility was supported by in planta complementation of CIPK23 in the stop1 background, which rescued the short root phenotype under NaCl. Both stop1 and cipk23 exhibited a drought tolerant phenotype and increased abscisic acid-regulated stomatal closure, while the complementation of CIPK23 in stop1 reversed these traits. Our findings uncover additional pleiotropic roles of STOP1 mediated by CIPK23, which regulates various ion transporters including those regulating K+-homeostasis, which may induce a trade-off between drought tolerance and other traits.

STOP1 regulates the expression of HsfA2 and GDHs that are critical for low-oxygen tolerance in Arabidopsis.

The SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) transcription factor regulates gene expression associated with multiple stress tolerances in plant roots. In this study, we investigated the mechanism responsible for the sensitivity of the stop1 mutant to low-oxygen stress in Arabidopsis. Transcriptomic analyses revealed that two genes involved in low-oxygen tolerance, namely GLUTAMATE DEHYDROGENASE 1 (GDH1) and GDH2, showed lower expression levels in the stop1 mutant than in the wild-type. Sensitivity of the gdh1gdh2 double-mutant to low-oxygen conditions was partly attributable to the low-oxygen sensitivity of the stop1 mutant. Two transcription factors, STOP2 and HEAT SHOCK FACTOR A2 (HsfA2), were expressed at lower levels in the stop1 mutant. An in planta complementation assay indicated that CaMV35S::STOP2 or CaMV35S::HsfA2 partially rescued the low-oxygen tolerance of the stop1 mutant, which was concomitant with recovered expression of genes regulating low-pH tolerance and genes encoding molecular chaperones. Prediction of cis-elements and in planta promoter assays revealed that STOP1 directly activated the expression of HsfA2. Similar STOP1-dependent low-oxygen sensitivity was detected in tobacco. Suppression of NtSTOP1 induced low-oxygen sensitivity, which was associated with lower expression levels of NtHsfA2 and NtGDHs compared with the wild-type. Our results indicated that STOP1 pleiotropically regulates low-oxygen tolerance by transcriptional regulation.

Involvement of phosphatidylinositol metabolism in aluminum-induced malate secretion in Arabidopsis.

To identify the upstream signaling of aluminum-induced malate secretion through aluminum-activated malate transporter 1 (AtALMT1), a pharmacological assay using inhibitors of human signal transduction pathways was performed. Early aluminum-induced transcription of AtALMT1 and other aluminum-responsive genes was significantly suppressed by phosphatidylinositol 4-kinase (PI4K) and phospholipase C (PLC) inhibitors, indicating that the PI4K-PLC metabolic pathway activates early aluminum signaling. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and PI4K reduced aluminum-activated malate transport by AtALMT1, suggesting that both the PI3K and PI4K metabolic pathways regulate this process. These results were validated using T-DNA insertion mutants of PI4K and PI3K-RNAi lines. A human protein kinase inhibitor, putatively inhibiting homologous calcineurin B-like protein-interacting protein kinase and/or Ca-dependent protein kinase in Arabidopsis, suppressed late-phase aluminum-induced expression of AtALMT1, which was concomitant with the induction of an AtALMT1 repressor, WRKY46, and suppression of an AtALMT1 activator, Calmodulin-binding transcription activator 2 (CAMTA2). In addition, a human deubiquitinase inhibitor suppressed aluminum-activated malate transport, suggesting that deubiquitinases can regulate this process. We also found a reduction of aluminum-induced citrate secretion in tobacco by applying inhibitors of PI3K and PI4K. Taken together, our results indicated that phosphatidylinositol metabolism regulates organic acid secretion in plants under aluminum stress.

FPX is a Novel Chemical Inducer that Promotes Callus Formation and Shoot Regeneration in Plants.

Auxin and cytokinin control callus formation from developed plant organs as well as shoot regeneration from callus. Dedifferentiation and regeneration of plant cells by auxin and cytokinin stimulation are considered to be caused by the reprogramming of callus cells, but this hypothesis is still argued to this day. Although an elucidation of the regulatory mechanisms of callus formation and shoot regeneration has helped advance plant biotechnology research, many plant species are intractable to transformation because of difficulties with callus formation. In this study, we identified fipexide (FPX) as a useful regulatory compound through a chemical biology-based screening. FPX was shown to act as a chemical inducer in callus formation, shoot regeneration and Agrobacterium infection. With regards to morphology, the cellular organization of FPX-induced calli differed from those produced under auxin/cytokinin conditions. Microarray analysis revealed that the expression of approximately 971 genes was up-regulated 2-fold after a 2 d FPX treatment compared with non-treated plants. Among these 971 genes, 598 genes were also induced by auxin/cytokinin, whereas 373 genes were specifically expressed upon FPX treatment only. FPX can promote callus formations in rice, poplar, soybean, tomato and cucumber, and thus can be considered a useful tool for revealing the mechanisms of plant development and for use in plant transformation technologies.

A plant U-box protein, PUB4, regulates asymmetric cell division and cell proliferation in the root meristem.

The root meristem (RM) is a fundamental structure that is responsible for postembryonic root growth. The RM contains the quiescent center (QC), stem cells and frequently dividing meristematic cells, in which the timing and the frequency of cell division are tightly regulated. In Arabidopsis thaliana, several gain-of-function analyses have demonstrated that peptide ligands of the Clavata3 (CLV3)/embryo surrounding region-related (CLE) family are important for maintaining RM size. Here, we demonstrate that a plant U-box E3 ubiquitin ligase, PUB4, is a novel downstream component of CLV3/CLE signaling in the RM. Mutations in PUB4 reduced the inhibitory effect of exogenous CLV3/CLE peptide on root cell proliferation and columella stem cell maintenance. Moreover, pub4 mutants grown without exogenous CLV3/CLE peptide exhibited characteristic phenotypes in the RM, such as enhanced root growth, increased number of cortex/endodermis stem cells and decreased number of columella layers. Our phenotypic and gene expression analyses indicated that PUB4 promotes expression of a cell cycle regulatory gene, CYCD6;1, and regulates formative periclinal asymmetric cell divisions in endodermis and cortex/endodermis initial daughters. These data suggest that PUB4 functions as a global regulator of cell proliferation and the timing of asymmetric cell division that are important for final root architecture.

Characterization of a helminthosporic acid analog that is a selective agonist of gibberellin receptor.

Helminthosporol, a natural growth regulator isolated from a fungus, stimulates hypocotyl growth and seed germination, similar to gibberellin (GA). We recently reported that helminthosporic acid (H-acid), a synthetic analog of helminthosporol, acts as an agonist of GA receptor. In this study, we showed that a H-acid analog, in which the hydroxymethyl group at the C-8 position of H-acid was converted to a keto group, acts as a selective GA receptor agonist. 1) This analog shows higher hypocotyl elongation activity in Arabidopsis than H-acid does, and induces the degradation of DELLA protein and 2) leads to the formation of the GID1-DELLA complex and 3) regulates the expression of GA-related genes. In addition, 4) its hypocotyl elongation activity was not observed in a atgid1a single mutant, and 5) this analog could promote only the interaction between specific GA receptors and DELLA proteins in vitro. Taken together, our results strongly suggest that the selectivity of the reported H-acid analog depends on the specificity of its GA receptor binding activity.

The chemical NJ15 affects hypocotyl elongation and shoot gravitropism via cutin polymerization.

We previously found a chemical, designated as NJ15, which inhibited both auxin and brassinosteroid responses in dark-grown Arabidopsis. To study its mode of action, we performed a phenotypic screening of NJ15-low-sensitive lines among mutant pools of Arabidopsis. One line (f127) showed clear NJ15-low-sensitivity in terms of hypocotyl elongation and shoot gravitropism. After further testing, it was determined that DCR, an enzyme involved in cutin polymerization, had lost its function in the mutant, which caused its low sensitivity to NJ15. Fatty acids are the base materials for polymers such as cutin and cuticular wax. We confirmed that NJ15 affects fatty acid biosynthesis, and that it does differently from cafenstrole, a known inhibitor of cuticular wax formation. Based on these results, we propose that the target of NJ15 is likely located within the cutin polymer formation pathway. ABBREVIATIONS: Caf: cafenstrole; DEG: differentially expressed gene; FDR: false discovery rate; FOX: full length cDNA-overexpressor; VLCFA: very-long-chain fatty acid.

Helminthosporic acid functions as an agonist for gibberellin receptor.

Helminthosporol was isolated from a fungus, Helminthosporium sativum, as a natural plant growth regulator in 1963. It showed gibberellin-like bioactivity that stimulated the growth of the second leaf sheath of rice. After studying the structure-activity relationship between the compound and some synthesized analogs, it was found that helminthosporic acid (H-acid) has higher gibberellin-like activity and chemical stability than helminthosporol. In this study, we showed that (1) H-acid displays gibberellin-like activities not only in rice but also in Arabidopsis, (2) it regulates the expression of gibberellin-related genes, (3) it induces DELLA degradation through binding with a gibberellin receptor (GID1), and (4) it forms the GID1-(H-acid)-DELLA complex to transduce the gibberellin signal in the same manner as gibberellin. This work shows that the H-acid mode of action acts as an agonist for gibberellin receptor.

SENSITIVE TO PROTON RHIZOTOXICITY1, CALMODULIN BINDING TRANSCRIPTION ACTIVATOR2, and other transcription factors are involved in ALUMINUM-ACTIVATED MALATE TRANSPORTER1 expression.

In Arabidopsis (Arabidopsis thaliana) the root apex is protected from aluminum (Al) rhizotoxicity by excretion of malate, an Al chelator, by ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (AtALMT1). AtALMT1 expression is fundamentally regulated by the SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) zinc finger protein, but other transcription factors have roles that enable Al-inducible expression with a broad dynamic range. In this study, we characterized multiple cis-elements in the AtALMT1 promoter that interact with transcription factors. In planta complementation assays of AtALMT1 driven by 5' truncated promoters of different lengths showed that the promoter region between -540 and 0 (the first ATG) restored the Al-sensitive phenotype of atalm1 and thus contains cis-elements essential for AtALMT1 expression for Al tolerance. Computation of overrepresented octamers showed that eight regions in this promoter region contained potential cis-elements involved in Al induction and STOP1 regulation. Mutation in a position around -297 from the first ATG completely inactivated AtALMT1 expression and Al response. In vitro binding assays showed that this region contained the STOP1 binding site, which accounted for the recognition by four zinc finger domains of the protein. Other positions were characterized as cis-elements that regulated expression by repressors and activators and a transcription factor that determines root tip expression of AtALMT1. From the consensus of known cis-elements, we identified CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR2 to be an activator of AtALMT1 expression. Al-inducible expression of AtALMT1 changed transcription starting sites, which increased the abundance of transcripts with a shortened 5' untranslated region. The present analyses identified multiple mechanisms that regulate AtALMT1 expression.

Joint genetic and network analyses identify loci associated with root growth under NaCl stress in Arabidopsis thaliana.

Plants have evolved a series of tolerance mechanisms to saline stress, which perturbs physiological processes throughout the plant. To identify genetic mechanisms associated with salinity tolerance, we performed linkage analysis and genome-wide association study (GWAS) on maintenance of root growth of Arabidopsis thaliana in hydroponic culture with weak and severe NaCl toxicity. The top 200 single-nucleotide polymorphisms (SNPs) determined by GWAS could cumulatively explain approximately 70% of the variation observed at each stress level. The most significant SNPs were linked to the genes of ATP-binding cassette B10 and vacuolar proton ATPase A2. Several known salinity tolerance genes such as potassium channel KAT1 and calcium sensor SOS3 were also linked to SNPs in the top 200. In parallel, we constructed a gene co-expression network to independently verify that particular groups of genes work together to a common purpose. We identify molecular mechanisms to confer salt tolerance from both predictable and novel physiological sources and validate the utility of combined genetic and network analysis. Additionally, our study indicates that the genetic architecture of salt tolerance is responsive to the severity of stress. These gene datasets are a significant information resource for a following exploration of gene function.

Epoxycarotenoid-mediated synthesis of abscisic acid in Physcomitrella patens implicating conserved mechanisms for acclimation to hyperosmosis in embryophytes.

Plants acclimate to environmental stress signals such as cold, drought and hypersalinity, and provoke internal protective mechanisms. Abscisic acid (ABA), a carotenoid-derived phytohormone, which increases in response to the stress signals above, has been suggested to play a key role in the acclimation process in angiosperms, but the role of ABA in basal land plants such as mosses, including its biosynthetic pathways, has not been clarified. Targeted gene disruption of PpABA1, encoding zeaxanthin epoxidase in the moss Physcomitrella patens was conducted to determine the role of endogenous ABA in acclimation processes in mosses. The generated ppaba1 plants were found to accumulate only a small amount of endogenous ABA. The ppaba1 plants showed reduced osmotic acclimation capacity in correlation with reduced dehydration tolerance and accumulation of late embryogenesis abundant proteins. By contrast, cold-induced freezing tolerance was less affected in ppaba1, indicating that endogenous ABA does not play a major role in the regulation of cold acclimation in the moss. Our results suggest that the mechanisms for osmotic acclimation mediated by carotenoid-derived synthesis of ABA are conserved in embryophytes and that acquisition of the mechanisms played a crucial role in terrestrial adaptation and colonization by land plant ancestors.

The maize DWARF1 encodes a gibberellin 3-oxidase and is dual localized to the nucleus and cytosol.

The maize (Zea mays) gibberellin (GA)-deficient mutant dwarf1 (d1) displays dwarfism and andromonoecy (i.e. forming anthers in the female flower). Previous characterization indicated that the d1 mutation blocked three steps in GA biosynthesis; however, the locus has not been isolated and characterized. Here, we report that D1 encodes a GA 3-oxidase catalyzing the final step of bioactive GA synthesis. Recombinant D1 is capable of converting GA20 to GA1, GA20 to GA3, GA5 to GA3, and GA9 to GA4 in vitro. These reactions are widely believed to take place in the cytosol. However, both in vivo GFP fusion analysis and western-blot analysis of organelle fractions using a D1-specific antibody revealed that the D1 protein is dual localized in the nucleus and cytosol. Furthermore, the upstream gibberellin 20-oxidase1 (ZmGA20ox1) protein was found dual localized in the nucleus and cytosol as well. These results indicate that bioactive GA can be synthesized in the cytosol and the nucleus, two compartments where GA receptor Gibberellin-insensitive dwarf protein1 exists. Furthermore, the D1 protein was found to be specifically expressed in the stamen primordia in the female floret, suggesting that the suppression of stamen development is mediated by locally synthesized GAs.

Overexpression of LOV KELCH protein 2 confers dehydration tolerance and is associated with enhanced expression of dehydration-inducible genes in Arabidopsis thaliana.

KEY MESSAGE: The overexpression of LKP2 confers dehydration tolerance in Arabidopsis thaliana ; this is likely due to enhanced expression of dehydration-inducible genes and reduced stomatal opening. LOV KELCH protein 2 (LKP2) modulates the circadian rhythm and flowering time in plants. In this study, we observed that LKP2 overexpression enhanced dehydration tolerance in Arabidopsis. Microarray analysis demonstrated that expression of water deprivation-responsive genes was higher in the absence of dehydration stress in transgenic Arabidopsis plants expressing green fluorescent protein-tagged LKP2 (GFP-LKP2) than in control transgenic plants expressing GFP. After dehydration followed by rehydration, GFP-LKP2 plants developed more leaves and roots and exhibited higher survival rates than control plants. In the absence of dehydration stress, four dehydration-inducible genes, namely DREB1A, DREB1B, DREB1C, and RD29A, were expressed in GFP-LKP2 plants, whereas they were not expressed or were expressed at low levels in control plants. Under dehydration stress, the expression of DREB2B and RD29A peaked faster in the GFP-LKP2 plants than in control plants. The stomatal aperture of GFP-LKP2 plants was smaller than that of control plants. These results suggest that the dehydration tolerance of GFP-LKP2 plants is caused by upregulation of DREB1A-C/CBF1-3 and their downstream targets; restricted stomatal opening in the absence of dehydration stress also appears to contribute to the phenotype. The rapid and high expression of DREB2B and its downstream target genes also likely accounts for some features of the GFP-LKP2 phenotype. Our results suggest that LKP2 can be used for biotechnological applications not only to adjust the flowering time control but also to enhance dehydration tolerance.

Natural variation in a polyamine transporter determines paraquat tolerance in Arabidopsis.

Polyamines (PAs) are ubiquitous, polycationic compounds that are essential for the growth and survival of all organisms. Although the PA-uptake system plays a key role in mammalian cancer and in plant survival, the underlying molecular mechanisms are not well understood. Here, we identified an Arabidopsis L-type amino acid transporter (LAT) family transporter, named RMV1 (resistant to methyl viologen 1), responsible for uptake of PA and its analog paraquat (PQ). The natural variation in PQ tolerance was determined in 22 Arabidopsis thaliana accessions based on the polymorphic variation of RMV1. An RMV1-GFP fusion protein localized to the plasma membrane in transformed cells. The Arabidopsis rmv1 mutant was highly resistant to PQ because of the reduction of PQ uptake activity. Uptake studies indicated that RMV1 mediates proton gradient-driven PQ transport. RMV1 overexpressing plants were hypersensitive to PA and PQ and showed elevated PA/PQ uptake activity, supporting the notion that PQ enters plant cells via a carrier system that inherently functions in PA transport. Furthermore, we demonstrated that polymorphic variation in RMV1 controls PA/PQ uptake activity. Our identification of a molecular entity for PA/PQ uptake and sensitivity provides an important clue for our understanding of the mechanism and biological significance of PA uptake.

Integrated analysis of transcriptome and metabolome of Arabidopsis albino or pale green mutants with disrupted nuclear-encoded chloroplast proteins.

We used four mutants having albino or pale green phenotypes with disrupted nuclear-encoded chloroplast proteins to analyze the regulatory system of metabolites in chloroplast. We performed an integrated analyses of transcriptomes and metabolomes of the four mutants. Transcriptome analysis was carried out using the Agilent Arabidopsis 2 Oligo Microarray, and metabolome analysis with two mass spectrometers; a direct-infusion Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR/MS) and a gas chromatograph-time of flight mass spectrometer. Among approximately 200 known metabolites detected by the FT-ICR/MS, 71 metabolites showed significant changes in the mutants when compared with controls (Ds donor plants). Significant accumulation of several amino acids (glutamine, glutamate and asparagine) was observed in the albino and pale green mutants. Transcriptome analysis revealed altered expressions of genes in several metabolic pathways. For example, genes involved in the tricarboxylic acid cycle, the oxidative pentose phosphate pathway, and the de novo purine nucleotide biosynthetic pathway were up-regulated. These results suggest that nitrogen assimilation is constitutively promoted in the albino and pale green mutants. The accumulation of ammonium ions in the albino and pale green mutants was consistently higher than in Ds donor lines. Furthermore, genes related to pyridoxin accumulation and the de novo purine nucleotide biosynthetic pathway were up-regulated, which may have occurred as a result of the accumulation of glutamine in the albino and pale green mutants. The difference in metabolic profiles seems to be correlated with the disruption of chloroplast internal membrane structures in the mutants. In albino mutants, the alteration of metabolites accumulation and genes expression is stronger than pale green mutants.

SABRE2: a database connecting plant EST/full-length cDNA clones with Arabidopsis information.

The SABRE (Systematic consolidation of Arabidopsis and other Botanical REsources) database cross-searches plant genetic resources through publicly available Arabidopsis information. In SABRE, plant expressed sequence tag (EST)/cDNA clones are related to TAIR (The Arabidoposis Information Resource) gene models and their annotations through sequence similarity. By entering a keyword, SABRE searches and retrieves TAIR gene models and annotations, together with homologous gene clones from various plant species. SABRE thus facilitates using TAIR annotations of Arabidopsis genes for research on homologous genes from other model plants. To expand the application range of SABRE to crop breeding, we have recently upgraded SABRE to SABRE2 (http://sabre.epd.brc.riken.jp/SABRE2.html), by newly adding six model plants (including the major crops barley, soybean, tomato and wheat), and by improving the retrieval interface. The present version has integrated information on >1.5 million plant EST/cDNA clones from the National BioResource Project (NBRP) of Japan. All clones are actual experimental resources from 14 plant species (Arabidoposis, barley, cassava, Chinese cabbage, lotus, morning glory, poplar, Physcomitrella patens, Striga hermonthica, soybean, Thellungiella halophila, tobacco, tomato and wheat), and are available from the core facilities of the NBRP. SABRE2 is thus a useful tool that can contribute towards the improvement of important crop breeds by connecting basic research and crop breeding.