Masanori Okamoto

CYP707A1 and CYP707A2, which encode abscisic acid 8'-hydroxylases, are indispensable for proper control of seed dormancy and germination in Arabidopsis

Endogenous abscisic acid (ABA) levels are regulated by both biosynthesis and catabolism of the hormone. ABA 8′-hydroxylase is considered to be the key catabolic enzyme in many physiological processes. We have previously identified that four members of the Arabidopsis (Arabidopsis thaliana) CYP707A gene family (CYP707A1 to CYP707A4) encode ABA 8′-hydroxylases, and that the cyp707a2 mutants showed an increase in ABA levels in dry and imbibed seeds. In this study, we showed that the cyp707a1 mutant accumulated ABA to higher levels in dry seeds than the cyp707a2 mutant. Expression analysis showed that the CYP707A1 was expressed predominantly during mid-maturation and was down-regulated during late-maturation. Concomitantly, the CYP707A2 transcript levels increased from late-maturation to mature dry seed. Phenotypic analysis of single and double cyp707a mutants indicates that the CYP707A1 is important for reducing ABA levels during mid-maturation. On the other hand, CYP707A2 is responsible for the regulation of ABA levels from late-maturation to germination. Moreover, CYP707A1 and CYP707A3 were also shown to be involved in postgermination growth. Spatial expression analysis suggests that CYP707A1 was expressed predominantly in embryo during mid-maturation, whereas CYP707A2 expression was detected in both embryo and endosperm from late-maturation to germination. Our results demonstrate that each CYP707A gene plays a distinct role during seed development and postgermination growth.

High Temperature-Induced Abscisic Acid Biosynthesis and Its Role in the Inhibition of Gibberellin Action in Arabidopsis Seeds

Suppression of seed germination at supraoptimal high temperature (thermoinhibiton) during summer is crucial for Arabidopsis (Arabidopsis thaliana) to establish vegetative and reproductive growth in appropriate seasons. Abscisic acid (ABA) and gibberellins (GAs) are well known to be involved in germination control, but it remains unknown how these hormone actions (metabolism and responsiveness) are altered at high temperature. Here, we show that ABA levels in imbibed seeds are elevated at high temperature and that this increase is correlated with up-regulation of the zeaxanthin epoxidase gene ABA1/ZEP and three 9-cis-epoxycarotenoid dioxygenase genes, NCED2, NCED5, and NCED9. Reverse-genetic studies show that NCED9 plays a major and NCED5 and NCED2 play relatively minor roles in high temperature-induced ABA synthesis and germination inhibition. We also show that bioactive GAs stay at low levels at high temperature, presumably through suppression of GA 20-oxidase genes, GA20ox1, GA20ox2, and GA20ox3, and GA 3-oxidase genes, GA3ox1 and GA3ox2. Thermoinhibition-tolerant germination of loss-of-function mutants of GA negative regulators, SPINDLY (SPY) and RGL2, suggests that repression of GA signaling is required for thermoinibition. Interestingly, ABA-deficient aba2-2 mutant seeds show significant expression of GA synthesis genes and repression of SPY expression even at high temperature. In addition, the thermoinhibition-resistant germination phenotype of aba2-1 seeds is suppressed by a GA biosynthesis inhibitor, paclobutrazol. We conclude that high temperature stimulates ABA synthesis and represses GA synthesis and signaling through the action of ABA in Arabidopsis seeds.

Drought Induction of Arabidopsis 9-cis-Epoxycarotenoid Dioxygenase Occurs in Vascular Parenchyma Cells

The regulation of abscisic acid (ABA) biosynthesis is essential for plant responses to drought stress. In this study, we examined the tissue-specific localization of ABA biosynthetic enzymes in turgid and dehydrated Arabidopsis (Arabidopsis thaliana) plants using specific antibodies against 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3), AtABA2, and Arabidopsis aldehyde oxidase 3 (AAO3). Immunohistochemical analysis revealed that in turgid plants, AtABA2 and AAO3 proteins were localized in vascular parenchyma cells most abundantly at the boundary between xylem and phloem bundles, but the AtNCED3 protein was undetectable in these tissues. In water-stressed plants, AtNCED3 was detected exclusively in the vascular parenchyma cells together with AtABA2 and AAO3. In situ hybridization using the antisense probe for AtNCED3 showed that the drought-induced expression of AtNCED3 was also restricted to the vascular tissues. Expression analysis of laser-microdissected cells revealed that, among nine drought-inducible genes examined, the early induction of most genes was spatially restricted to vascular cells at 1 h and then some spread to mesophyll cells at 3 h. The spatial constraint of AtNCED3 expression in vascular tissues provides a novel insight into plant systemic response to drought stresses.

Abscisic acid and the control of seed dormancy and germination.

Abscisic acid (ABA) is a plant hormone that regulates seed dormancy and germination. Seeds undergo changes in both ABA content and sensitivity during seed development and germination in response to internal and external cues. Recent advances in functional genomics have revealed the integral components involved in ABA metabolism (biosynthesis and catabolism) and perception, the core signalling pathway, as well as the factors that trigger ABA-mediated transcription. These allow for comparative studies to be conducted on seeds under different environmental conditions and from different genetic backgrounds. This review summarizes our understanding of the control of ABA content and the responsiveness of seeds to afterripening, light, high temperature and nitrate, with a focus on which tissues are involved in its metabolism and signalling. Also described are the regulators of ABA metabolism and signalling, which potentially act as the node for hormone crosstalk. Integration of such knowledge into the complex and diverse events occurring during seed germination will be the next challenge, which will allow for a clearer understanding of the role of ABA.

Genome-wide analysis of endogenous abscisic acid-mediated transcription in dry and imbibed seeds of Arabidopsis using tiling arrays.

The phytohormone abscisic acid (ABA) plays important roles in the induction and maintenance of seed dormancy. Although application of exogenous ABA inhibits germination, the effects of exogenous ABA on ABA-mediated gene transcription differ from those of endogenous ABA. To understand how endogenous ABA regulates the transcriptomes in seeds, we performed comprehensive expression analyses using whole-genome Affymetrix tiling arrays in two ABA metabolism mutants - an ABA-deficient mutant (aba2) and an ABA over-accumulation mutant (cyp707a1a2a3 triple mutant). Hierarchical clustering and principal components analyses showed that differences in endogenous ABA levels do not influence global expression of stored mRNA in dry seeds. However, the transcriptome after seed imbibition was related to endogenous ABA levels in both types of mutant. Endogenous ABA-regulated genes expressed in imbibed seeds included those encoding key ABA signaling factors and gibberellin-related components. In addition, cohorts of ABA-upregulated genes partially resembled those of dormant genes, whereas ABA-downregulated genes were partially overlapped with after-ripening-regulated genes. Bioinformatic analyses revealed that 6105 novel genes [non-Arabidopsis Genome Initiative (AGI) transcriptional units (TUs)] were expressed from unannotated regions. Interestingly, approximately 97% of non-AGI TUs possibly encoded hypothetical non-protein-coding RNAs, including a large number of antisense RNAs. In dry and imbibed seeds, global expression profiles of non-AGI TUs were similar to those of AGI genes. For both non-AGI TUs and AGI code genes, we identified those that were regulated differently in embryo and endosperm tissues. Our results suggest that transcription in Arabidopsis seeds is more complex and dynamic than previously thought.

Ectopic Expression of ABSCISIC ACID 2/GLUCOSE INSENSITIVE 1 in Arabidopsis Promotes Seed Dormancy and Stress Tolerance

Abscisic acid (ABA) is an important phytohormone that plays a critical role in seed development, dormancy, and stress tolerance. 9-cis-Epoxycarotenoid dioxygenase is the key enzyme controlling ABA biosynthesis and stress tolerance. In this study, we investigated the effect of ectopic expression of another ABA biosynthesis gene, ABA2 (or GLUCOSE INSENSITIVE 1 [GIN1]) encoding a short-chain dehydrogenase/reductase in Arabidopsis (Arabidopsis thaliana). We show that ABA2-overexpressing transgenic plants with elevated ABA levels exhibited seed germination delay and more tolerance to salinity than wild type when grown on agar plates and/or in soil. However, the germination delay was abolished in transgenic plants showing ABA levels over 2-fold higher than that of wild type grown on 250 mm NaCl. The data suggest that there are distinct mechanisms underlying ABA-mediated inhibition of seed germination under diverse stress. The ABA-deficient mutant aba2, with a shorter primary root, can be restored to normal root growth by exogenous application of ABA, whereas transgenic plants overexpressing ABA2 showed normal root growth. The data reflect that the basal levels of ABA are essential for maintaining normal primary root elongation. Furthermore, analysis of ABA2 promoter activity with ABA2∷β-glucuronidase transgenic plants revealed that the promoter activity was enhanced by multiple prolonged stresses, such as drought, salinity, cold, and flooding, but not by short-term stress treatments. Coincidently, prolonged drought stress treatment led to the up-regulation of ABA biosynthetic and sugar-related genes. Thus, the data support ABA2 as a late expression gene that might have a fine-tuning function in mediating ABA biosynthesis through primary metabolic changes in response to stress.

NIN-like protein 8 is a master regulator of nitrate-promoted seed germination in Arabidopsis

Seeds respond to multiple different environmental stimuli that regulate germination. Nitrate stimulates germination in many plants but how it does so remains unclear. Here we show that the Arabidopsis NIN-like protein 8 (NLP8) is essential for nitrate-promoted seed germination. Seed germination in nlp8 loss-of-function mutants does not respond to nitrate. NLP8 functions even in a nitrate reductase-deficient mutant background, and the requirement for NLP8 is conserved among Arabidopsis accessions. NLP8 reduces abscisic acid levels in a nitrate-dependent manner and directly binds to the promoter of CYP707A2, encoding an abscisic acid catabolic enzyme. Genetic analysis shows that NLP8-mediated promotion of seed germination by nitrate requires CYP707A2. Finally, we show that NLP8 localizes to nuclei and unlike NLP7, does not appear to be activated by nitrate-dependent nuclear retention of NLP7, suggesting that seeds have a unique mechanism for nitrate signalling.

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).

Substantial expression of novel small open reading frames in Oryza sativa

In our previous integrated study combining informatics and molecular biology analyses, we revealed that Arabidopsis small open reading frames (sORFs) predicted by computational analysis have biological functions in morphogenesis. Here, we report that sequences homologous to Arabidopsis sORFs are abundant in intergenic regions of the rice genome. These sequences represent a subset of non-protein-coding DNA, and some are transcribed into mRNA. These results indicate that many sORFs associated with morphogenesis are hidden in the genomes of crop species.

Positional correlation analysis improves reconstruction of full-length transcripts and alternative isoforms from noisy array signals or short reads

Motivation: A reconstruction of full-length transcripts observed by next-generation sequencer or tiling arrays is an essential technique to know all phenomena of transcriptomes. Several techniques of the reconstruction have been developed. However, problems of high-level noises and biases still remain and interrupt the reconstruction. A method is required that is robust against noise and bias and correctly reconstructs transcripts regardless of equipment used. Results: We propose a completely new statistical method that reconstructs full-length transcripts and can be applied on both next-generation sequencers and tiling arrays. The method called ARTADE2 analyzes ‘positional correlation’, meaning correlations of expression values for every combination on genomic positions of multiple transcriptional data. ARTADE2 then reconstructs full-length transcripts using a logistic model based on the positional correlation and the Markov model. ARTADE2 elucidated 17 591 full-length transcripts from 55 transcriptome datasets and showed notable performance compared with other recent prediction methods. Moreover, 1489 novel transcripts were discovered. We experimentally tested 16 novel transcripts, among which 14 were confirmed by reverse transcription–polymerase chain reaction and sequence mapping. The method also showed notable performance for reconstructing of mRNA observed by a next-generation sequencer. Moreover, the positional correlation and factor analysis embedded in ARTADE2 successfully detected regions at which alternative isoforms may exist, and thus are expected to be applied for discovering transcript biomarkers for a wide range of disciplines including preemptive medicine. Availability: http://matome.base.riken.jp Contact: toyoda@base.riken.jp Supplementary information:Supplementary data are available at Bioinformatics online.