Ryoichi Yano

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.

Temporal Expression Patterns of Hormone Metabolism Genes during Imbibition of Arabidopsis thaliana Seeds: A Comparative Study on Dormant and Non-Dormant Accessions

Seed imbibition is a prerequisite for subsequent dormancy and germination control. Here, we investigated imbibition responses of Arabidopsis seeds by transcriptomic and hormone profile analyses using dormant [Cape Verde Islands (Cvi)] and non-dormant [Columbia (Col)] accessions. Once imbibed, seeds of both accessions swelled most up to 3 h, reflecting water uptake. Microarray analysis showed that in both accessions, seeds imbibed for 15 min, 30 min and 1 h were less active in gene expression than at 3 h. More than 2,000 genes were either up-regulated or down-regulated in seeds imbibed for 3 h. Some genes up-regulated at 3 h were already induced in seeds imbibed for 1 h, suggestive of genome reprogramming early after the onset of imbibition. Imbibition-induced genes in seeds imbibed for 3 h included those up-regulated in both Col and Cvi (common) or unique to either accession (accession specific). Up-regulated genes that were both common and Cvi-specific were over-represented for sugar metabolism and the pentose phosphate pathway, whereas Col-specific genes were over-represented for ribosomal protein genes. Quantification of plant hormones showed that ABA and salicylic acid (SA) contents were higher, but gibberellin A(4) (GA(4)), N(6)-(Delta(2)-isopentenyl)adenine (iP), jasmonic acid (JA), JA-isoleucine (JA-Ile) and IAA were lower in imbibed seeds of Cvi compared with Col. In addition, changes in IAA and JA were initiated before 1 h, whereas ABA and JA-Ile declined 3 h after the onset of imbibition. An increase in GA(4) and iP appeared to be correlated temporally with the initiation of secondary water uptake, which marks the completion of germination.

The Time Required for Dormancy Release in Arabidopsis Is Determined by DELAY OF GERMINATION1 Protein Levels in Freshly Harvested Seeds

Seed dormancy prevents germination of freshly harvested seeds and is slowly released during seed storage by an unknown mechanism. This work shows that posttranslational modifications of the Arabidopsis thaliana protein DELAY OF GERMINATION1 occurring during seed storage can explain dormancy release. Seed dormancy controls the start of a plant’s life cycle by preventing germination of a viable seed in an unfavorable season. Freshly harvested seeds usually show a high level of dormancy, which is gradually released during dry storage (after-ripening). Abscisic acid (ABA) has been identified as an essential factor for the induction of dormancy, whereas gibberellins (GAs) are required for germination. The molecular mechanisms controlling seed dormancy are not well understood. DELAY OF GERMINATION1 (DOG1) was recently identified as a major regulator of dormancy in Arabidopsis thaliana. Here, we show that the DOG1 protein accumulates during seed maturation and remains stable throughout seed storage and imbibition. The levels of DOG1 protein in freshly harvested seeds highly correlate with dormancy. The DOG1 protein becomes modified during after-ripening, and its levels in stored seeds do not correlate with germination potential. Although ABA levels in dog1 mutants are reduced and GA levels enhanced, we show that DOG1 does not regulate dormancy primarily via changes in hormone levels. We propose that DOG1 protein abundance in freshly harvested seeds acts as a timer for seed dormancy release, which functions largely independent from ABA.

Starch-Related α-Glucan/Water Dikinase Is Involved in the Cold-Induced Development of Freezing Tolerance in Arabidopsis

Cold-induced soluble sugar accumulation enhances the degree of freezing tolerance in various cold-hardy plants including Arabidopsis (Arabidopsis thaliana), where soluble sugars accumulate in only a few hours at 2°C. Hence, along with photosynthesis, starch degradation might play a significant role in cold-induced sugar accumulation and enhanced freezing tolerance. Starch-related α-glucan/water dikinase (EC 2.7.9.4), encoded by Arabidopsis STARCH EXCESS 1 (SEX1), is hypothesized to regulate starch degradation in plastids by phosphorylating starch, thereby ensuring better accessibility by starch-degrading enzymes. Here, we show that Arabidopsis sex1 mutants, when incubated at 2°C for 1 d, were unable to accumulate maltooligosaccharides or normal glucose and fructose levels. In addition, they displayed impaired freezing tolerance. After 7 d at 2°C, sex1 mutants did not show any of the above abnormal phenotypes but displayed slightly higher leaf starch contents. The impaired freezing tolerance of sex1 mutants was restored by overexpression of wild-type SEX1 cDNA using the cauliflower mosaic virus 35S promoter. The results demonstrate a genetic link between the SEX1 locus and plant freezing tolerance, and show that starch degradation is important for enhanced freezing tolerance during an early phase of cold acclimation. However, induction of starch degradation was not accompanied by significant changes in α-glucan/water dikinase activity in leaf extracts and preceded cold-induced augmentation of SEX1 transcripts. Therefore, we conclude that augmentation of SEX1 transcripts might be a homeostatic response to low temperature, and that starch degradation during an early phase of cold acclimation could be regulated by a component(s) of a starch degradation pathway(s) downstream of SEX1.

CHOTTO1, a Putative Double APETALA2 Repeat Transcription Factor, Is Involved in Abscisic Acid-Mediated Repression of Gibberellin Biosynthesis during Seed Germination in Arabidopsis

The phytohormones abscisic acid (ABA) and gibberellins (GAs) are the primary signals that regulate seed dormancy and germination. In this study, we investigated the role of a double APETALA2 repeat transcription factor, CHOTTO1 (CHO1), in seed dormancy, germination, and phytohormone metabolism of Arabidopsis (Arabidopsis thaliana). Wild-type seeds were dormant when freshly harvested seeds were sown, and these seeds were released from dormancy after a particular period of dry storage (after-ripening). The cho1 mutant seeds germinated easily even in a shorter period of storage than wild-type seeds. The cho1 mutants showed reduced responsiveness to ABA, whereas transgenic plants constitutively expressing CHO1 (p35S∷CHO1) showed an opposite phenotype. Notably, after-ripening reduced the ABA responsiveness of the wild type, cho1 mutants, and p35S∷CHO1 lines. Hormone profiling demonstrated that after-ripening treatment decreased the levels of ABA and salicylic acid and increased GA4, jasmonic acid, and isopentenyl adenine when wild-type seeds were imbibed. Expression analysis showed that the transcript levels of genes for ABA and GA metabolism were altered in the wild type by after-ripening. Hormone profiling and expression analyses indicate that cho1 seeds, with a short period of storage, resembled fully after-ripened wild-type seeds. Genetic analysis showed that the cho1 mutation partially restored delayed seed germination and reduced GA biosynthesis activity in the ABA-overaccumulating cyp707a2-1 mutant background but did not restore seed germination in the GA-deficient ga1-3 mutant background. These results indicate that CHO1 acts downstream of ABA to repress GA biosynthesis during seed germination.

CHOTTO1, a Double AP2 Domain Protein of Arabidopsis thaliana, Regulates Germination and Seedling Growth Under Excess Supply of Glucose and Nitrate

Arabidopsis chotto1 (cho1) mutants show resistance to (-)-R-ABA, an ABA analog, during germination and seedling growth. Here, we report cloning and characterization of the CHO1 gene. cho1 mutants showed only subtle resistance to (+)-S-ABA during germination. The cho1 mutation acts as a strong enhancer of the abi5 mutant, whereas the cho1 abi4 double mutant showed ABA resistance similar to the abi4 single mutant. This suggests that CHO1 and ABI4, but not ABI5, act in the same genetic pathway. Map-based cloning revealed that the CHO1 gene encodes a putative transcription factor containing double AP2 domains. The CHO1 gene was expressed predominantly in seed, with the strongest expression in imbibed seed. Induction of CHO1 expression was observed 4 h after seed imbibition and reached a maximum level at 24 h. Induction of CHO1 expression did not occur in the abi4 mutants, indicating that this is an ABI4-dependent process. Microarray experiments showed that a large number of genes involved in primary metabolism and the stress response were up-regulated in the cho1 mutant. Growth of abi4 and cho1 mutant seedlings was resistant to high concentrations of glucose. In addition, growth of cho1 mutant seedlings was partially resistant to excess nitrate (50 mM), as evident from their expanded green cotyledons. However, their growth was normal under moderate nitrate concentrations (< 10 mM). This nitrate response was specific to the cho1 mutants and was not observed in the abi4 mutants. Taken together, our results indicate that CHO1 regulates nutritional responses downstream of ABI4 during germination and seedling growth.

Combining association mapping and transcriptomics identify HD2B histone deacetylase as a genetic factor associated with seed dormancy in Arabidopsis thaliana

Seed dormancy is an important adaptive trait that enables germination at the proper time, thereby ensuring plant survival after germination. In Arabidopsis, considerable variation exists in the degree of seed dormancy among wild-type accessions (ecotypes). In this paper, we identify a plant-specific HD2 histone deacetylase gene, HD2B (At5g22650), as a genetic factor associated with seed dormancy. First, genome-wide association mapping of 113 accessions was used to identify single nucleotide polymorphisms that possibly explain natural variation for seed dormancy. Integration of genome-wide association mapping and transcriptome analysis during cold-induced dormancy cycling identified HD2B as the most plausible candidate gene, and quantitative RT-PCR analysis demonstrated that HD2B expression was up-regulated by cold and after-ripening (dry storage of mature seed), treatments that are known to break seed dormancy. Interestingly, quantitative RT-PCR analysis in 106 accessions revealed that the expression of HD2B in imbibed seeds was significantly suppressed in most of the dormant accessions compared with less-dormant accessions, suggesting that suppression of HD2B expression may be important to maintain seed dormancy in dormant accessions. In addition, transgenic seeds of a dormant Cvi-0 accession that carried a 2.5 kb genomic DNA fragment of HD2B cloned from a less-dormant Col-0 accession ((Col)HD2B/Cvi-0) exhibited reduced seed dormancy accompanied by enhanced expression of HD2B when after-ripened or cold-imbibed. Endogenous levels of gibberellin were found to be increased in the imbibed seeds of after-ripened (Col)HD2B/Cvi-0 compared with wild-type Cvi-0. These results suggest that HD2B plays a role in seed dormancy and/or germinability in Arabidopsis thaliana.

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.

The Glycine max cv. Enrei Genome for Improvement of Japanese Soybean Cultivars

We elucidated the genome sequence of Glycine max cv. Enrei to provide a reference for characterization of Japanese domestic soybean cultivars. The whole genome sequence obtained using a next-generation sequencer was used for reference mapping into the current genome assembly of G. max cv. Williams 82 obtained by the Soybean Genome Sequencing Consortium in the USA. After sequencing and assembling the whole genome shotgun reads, we obtained a data set with about 928 Mbs total bases and 60,838 gene models. Phylogenetic analysis provided glimpses into the ancestral relationships of both cultivars and their divergence from the complex that include the wild relatives of soybean. The gene models were analyzed in relation to traits associated with anthocyanin and flavonoid biosynthesis and an overall profile of the proteome. The sequence data are made available in DAIZUbase in order to provide a comprehensive informatics resource for comparative genomics of a wide range of soybean cultivars in Japan and a reference tool for improvement of soybean cultivars worldwide.

Metabolic switching of astringent and beneficial triterpenoid saponins in soybean is achieved by a loss-of-function mutation in Cytochrome P450 72A69.

&NA; Triterpenoid saponins are major components of secondary metabolites in soybean seeds and are divided into two groups: group A saponins, and 2,3‐dihydro‐2,5‐dihydroxy‐6‐methyl‐4H‐pyran‐4‐one (DDMP) saponins. The aglycone moiety of group A saponins consists of soyasapogenol A (SA), which is an oxidized &bgr;‐amyrin product, and the aglycone moiety of the DDMP saponins consists of soyasapogenol B (SB). Group A saponins produce a bitter and astringent aftertaste in soy products, whereas DDMP saponins have known health benefits for humans. We completed map‐based cloning and characterization of the gene Sg‐5, which is responsible for SA biosynthesis. The naturally occurring sg‐5 mutant lacks group A saponins and has a loss‐of‐function mutation (L164*) in Glyma15g39090, which encodes the cytochrome P450 enzyme, CYP72A69. An enzyme assay indicated the hydroxylase activity of recombinant CYP72A69 against SB, which also suggested the production of SA. Additionally, induced Glyma15g39090 mutants (R44* or S348P) lacked group A saponins similar to the sg‐5 mutant, indicating that Glyma15g39090 corresponds to Sg‐5. Endogenous levels of DDMP saponins were higher in the sg‐5 mutant than in the wild‐type lines due to the loss of the enzyme activity that converts SB to SA. Interestingly, the genomes of palaeopolyploid soybean and the closely related common bean carry multiple Sg‐5 paralogs in a genomic region syntenic to the soybean Sg‐5 region. However, SA did not accumulate in common bean samples, suggesting that Sg‐5 activity evolved after gene duplication event(s). Our results demonstrate that metabolic switching of undesirable saponins with beneficial saponins can be achieved in soybean by disabling Sg‐5. Significance Statement Triterpenoid saponins in soybeans are divided into two groups: bitter and astringent group A saponins, and health‐promoting DDMP saponins. Disabling a single cytochrome P450 gene (Sg‐5) can switch the metabolism from the undesirable group A saponins to the beneficial DDMP saponins. This finding will help soybean breeders improve the quality and consumer acceptance of soy food products.