Chemically forced dormancy termination mimics natural dormancy progression in potato tuber meristems by reducing ABA content and modifying expression of genes involved in regulating ABA synthesis and metabolism

The length of potato tuber dormancy depends on both the genotype and the environmental conditions during growth and storage. Abscisic acid (ABA) has been shown to play a critical role in tuber dormancy control but the mechanisms regulating ABA content during dormancy, as well as the sites of ABA synthesis, and catabolism are unknown. Recently, a temporal correlation between changes in ABA content and certain ABA biosynthetic and catabolic genes has been reported in stored field tubers during physiological dormancy progression. However, the protracted length of natural dormancy progression complicated interpretation of these data. To address this issue, in this study the synthetic dormancy-terminating agent bromoethane (BE) was used to induce rapid and highly synchronous sprouting of dormant tubers. The endogenous ABA content of tuber meristems increased 2-fold 24 h after BE treatment and then declined dramatically. By 7 d post-treatment, meristem ABA content had declined by >80%. Exogenous [(3)H]ABA was readily metabolized by isolated meristems to phaseic and dihydrophaseic acids. BE treatment resulted in an almost 2-fold increase in the rate of ABA metabolism. A differential expression of both the StNCED and StCYP707A gene family members in meristems of BE-treated tubers is consistent with a regulatory role for StNCED2 and the StCYP707A1 and StCYP707A2 genes. The present results show that the changes in ABA content observed during tuber dormancy progression are the result of a dynamic equilibrium of ABA biosynthesis and degradation that increasingly favours catabolism as dormancy progresses.     

Chemical inhibition of potato ABA-8'-hydroxylase activity alters in vitro and in vivo ABA metabolism and endogenous ABA levels but does not affect potato microtuber dormancy duration

The effects of azole-type P450 inhibitors and two metabolism-resistant abscisic acid (ABA) analogues on in vitro ABA-8'-hydroxylase activity, in planta ABA metabolism, endogenous ABA content, and tuber meristem dormancy duration were examined in potato (Solanum tuberosum L. cv. Russet Burbank). When functionally expressed in yeast, three potato CYP707A genes were demonstrated to encode enzymatically active ABA-8'-hydroxylases with micromolar affinities for (+)-ABA. The in vitro activity of the three enzymes was inhibited by the P450 azole-type inhibitors ancymidol, paclobutrazol, diniconazole, and tetcyclasis, and by the 8'-acetylene- and 8'-methylene-ABA analogues, with diniconazole and tetcyclasis being the most potent inhibitors. The in planta metabolism of [(3)H](±)-ABA to phaseic acid and dihydrophaseic acid in tuber meristems was inhibited by diniconazole, tetcyclasis, and to a lesser extent by 8'-acetylene- and 8'-methylene-ABA. Continuous exposure of in vitro generated microtubers to diniconazole resulted in a 2-fold increase in endogenous ABA content and a decline in dihydrophaseic acid content after 9 weeks of development. Similar treatment with 8'-acetylene-ABA had no effects on the endogenous contents of ABA or phaseic acid but reduced the content of dihydrophaseic acid. Tuber meristem dormancy progression was determined ex vitro in control, diniconazole-, and 8'-acetylene-ABA-treated microtubers following harvest. Continuous exposure to diniconazole during microtuber development had no effects on subsequent sprouting at any time point. Continuous exposure to 8'-acetylene-ABA significantly increased the rate of microtuber sprouting. The results indicate that, although a decrease in ABA content is a hallmark of tuber dormancy progression, the decline in ABA levels is not a prerequisite for dormancy exit and the onset of tuber sprouting.     

AtPER1 enhances primary seed dormancy and reduces seed germination by suppressing the ABA catabolism and GA biosynthesis in Arabidopsis seeds

Seed is vital to the conservation of germplasm and plant biodiversity. Seed dormancy is an adaptive trait in numerous seed‐plant species, enabling plants to survive under stressful conditions. Seed dormancy is mainly controlled by abscisic acid (ABA) and gibberellin (GA) and can be classified as primary and secondary seed dormancy. The primary seed dormancy is induced by maternal ABA. Here we found that AtPER1, a seed‐specific peroxiredoxin, is involved in enhancing primary seed dormancy. Two loss‐of‐function atper1 mutants, atper1‐1 and atper1‐2, displayed suppressed primary seed dormancy accompanied with reduced ABA and increased GA contents in seeds. Furthermore, atper1 mutant seeds were insensitive to abiotic stresses during seed germination. The expression of several ABA catabolism genes (CYP707A1, CYP707A2, and CYP707A3) and GA biosynthesis genes (GA20ox1, GA20ox3, and KAO3) in atper1 mutant seeds was increased compared to wild‐type seeds. The suppressed primary seed dormancy of atper1‐1 was completely reduced by deletion of CYP707A genes. Furthermore, loss‐of‐function of AtPER1 cannot enhance the seed germination ratio of aba2‐1 or ga1‐t, suggesting that AtPER1‐enhanced primary seed dormancy is dependent on ABA and GA. Additionally, the level of reactive oxygen species (ROS) in atper1 mutant seeds was significantly higher than that in wild‐type seeds. Taken together, our results demonstrate that AtPER1 eliminates ROS to suppress ABA catabolism and GA biosynthesis, and thus improves the primary seed dormancy and make the seeds less sensitive to adverse environmental conditions.     

Brassinolide Effect on Growth of Apical Meristems, Ethylene Production, and Abscisic Acid Content in Potato Tubers

Treatment of intact potato (Solanum tuberosum L., cv. Nevskii) tubers with 24-epibrassinolide (EB) resulted in prolonged deep dormancy, increased production of ethylene and higher contents of free and bound abscisic acid (ABA) in buds. EB at the most efficient concentration 0.021 mg dm^–3, applied immediately after tuber harvest, inhibited sprouting by 36 – 38 d, increased ethylene formation after 1 and 7 d of storage by almost 300 and 150%, respectively, and increased the content of both free and bound ABA during the whole period of storage (on average by about 80%). Electron microscopic and morphometric studies showed that EB brings about a decrease in cell volume in tunica and all types of meristems and an increase in the number of vacuoles, accompanied by a decrease in their volume.     

Wounding of potato tubers induces increases in ABA biosynthesis and catabolism and alters expression of ABA metabolic genes

The effects of physical wounding on ABA biosynthesis and catabolism and expression of genes encoding key ABA metabolic enzymes were determined in potato tubers. An increase in ABA and ABA metabolite content was observed 48 h after wounding and remained elevated through 96 h. Wounding induced dramatic increases in the expression of the ABA metabolic genes encoding zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), and ABA-8′-hydroxylase. Although the patterns of wound-induced expression of individual genes varied, increased gene expression was observed within 3 h of wounding and remained elevated through 96 h. An apparent correlation between expression of the gene encoding ZEP and the increase in ABA content suggested that the wound-induced increase in ABA biosynthesis was regulated by both substrate availability and increased NCED activity. Suppression of wound-induced jasmonic acid accumulation by rinsing the wounded tissue with water did not inhibit the subsequent increase in ABA content. Exogenous ethylene completely suppressed the wound-induced increase in ABA content and dramatically reduced wound-induced up-regulation of ABA metabolic genes. This study is the first to identify the molecular bases for increased ABA accumulation following physical trauma in potato tubers and highlights the complex physiological interactions between various wound-induced hormones.     

Seed dormancy and ABA metabolism in Arabidopsis and barley: the role of ABA 8'-hydroxylase

We have investigated the relationship between seed dormancy and abscisic acid (ABA) metabolism in the monocot barley and the dicot Arabidopsis. Whether dormant (D) or non-dormant (ND), dry seed of Arabidopsis and embryos of dry barley grains all had similarly high levels of ABA. ABA levels decreased rapidly upon imbibition, although they fell further in ND than in D. Gene expression profiles were determined in Arabidopsis for key ABA biosynthetic [the 9-cis epoxycarotenoid dioxygenasegene family] and ABA catabolic [the ABA 8'-hydroxylase gene family (CYP707A)] genes. Of these, only the AtCYP707A2 gene was differentially expressed between D and ND seeds, being expressed to a much higher level in ND seeds. Similarly, a barley CYP707 homologue, (HvABA8'OH-1) was expressed to a much higher level in embryos from ND grains than from D grains. Consistent with this, in situ hybridization studies showed HvABA8'OH-1 mRNA expression was stronger in embryos from ND grains. Surprisingly, the signal was confined in the coleorhiza, suggesting that this tissue plays a key role in dormancy release. Constitutive expression of a CYP707A gene in transgenic Arabidopsis resulted in decreased ABA content in mature dry seeds and a much shorter after-ripening period to overcome dormancy. Conversely, mutating the CYP707A2 gene resulted in seeds that required longer after-ripening to break dormancy. Our results point to a pivotal role for the ABA 8'-hydroxylase gene in controlling dormancy and that the action of this enzyme may be confined to a particular organ as in the coleorhiza of cereals.     

ABA 9'-hydroxylation is catalyzed by CYP707A in Arabidopsis

Abscisic acid (ABA) catabolism is important for regulating endogenous ABA levels. To date, most effort has focused on catabolism of ABA to phaseic acid (PA), which is generated spontaneously after 8′-hydroxylation of ABA by cytochrome P450s in the CYP707A subfamily. Neophaseic acid (neoPA) is another well-documented ABA catabolite that is produced via ABA 9′-hydroxylation, but the 9′-hydroxylase has not yet been defined. Here, we show that endogenous neoPA levels are reduced in loss-of-function mutants defective in CYP707A genes. In addition, in planta levels of both neoPA and PA are reduced after treatment of plants with uniconazole-P, a P450 inhibitor. These lines of evidence suggest that CYP707A genes also encode the 9′-hydroxylase required for neoPA synthesis. To test this, in vitro enzyme assays using microsomal fractions from CYP707A-expressing yeast strains were conducted and these showed that all four Arabidopsis CYP707As are 9′-hydroxylases, although this activity is minor. Collectively, our results demonstrate that ABA 9′-hydroxylation is catalyzed by CYP707As as a side reaction.     

The Arabidopsis cytochrome P450 CYP707A encodes ABA 8'-hydroxylases: key enzymes in ABA catabolism

The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism. In plants, ABA 8'-hydroxylation is thought to play a predominant role in ABA catabolism. ABA 8'-hydroxylase was shown to be a cytochrome P450 (P450); however, its corresponding gene had not been identified. Through phylogenetic and DNA microarray analyses during seed imbibition, the candidate genes for this enzyme were narrowed down from 272 Arabidopsis P450 genes. These candidate genes were functionally expressed in yeast to reveal that members of the CYP707A family, CYP707A1-CYP707A4, encode ABA 8'-hydroxylases. Expression analyses revealed that CYP707A2 is responsible for the rapid decrease in ABA level during seed imbibition. During drought stress conditions, all CYP707A genes were upregulated, and upon rehydration a significant increase in mRNA level was observed. Consistent with the expression analyses, cyp707a2 mutants exhibited hyperdormancy in seeds and accumulated six-fold greater ABA content than wild type. These results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.     

Abscisic acid catabolism enhances dormancy release of grapevine buds

The molecular mechanism regulating dormancy release in grapevine buds is as yet unclear. It was formerly proposed that dormancy is maintained by abscisic acid (ABA)‐mediated repression of bud–meristem activity and that removal of this repression triggers dormancy release. It was also proposed that such removal of repression may be achieved via natural or artificial up‐regulation of VvA8H‐CYP707A4, which encodes ABA 8′‐hydroxylase, and is the most highly expressed paralog in grapevine buds. The current study further examines these assumptions, and its experiments reveal that (a) hypoxia and ethylene, stimuli of bud dormancy release, enhance expression of VvA8H‐CYP707A4 within grape buds, (b) the VvA8H‐CYP707A4 protein accumulates during the natural transition to the dormancy release stage, and (c) transgenic vines overexpressing VvA8H‐CYP707A4 exhibit increased ABA catabolism and significant enhancement of bud break in controlled and natural environments and longer basal summer laterals. The results suggest that VvA8H‐CYP707A4 functions as an ABA degrading enzyme, and are consistent with a model in which the VvA8H‐CYP707A4 level in the bud is up‐regulated by natural and artificial bud break stimuli, which leads to increased ABA degradation capacity, removal of endogenous ABA‐mediated repression, and enhanced regrowth. Interestingly, it also hints at sharing of regulatory steps between latent and lateral bud outgrowth.     

Deoxyuridine triphosphatase expression defines the transition from dormant to sprouting potato tuber buds

Identification of molecular markers defining the end of tuber dormancy prior to visible sprouting is of agronomic interest for potato growers and the potato processing industry. In potato tubers, breakage of dormancy is associated with the reactivation of meristem function. In dormant meristems, cells are arrested in the G₁/G₀ phase of the cell cycle and re-entry into the G₁ phase followed by DNA replication during the S phase enables bud outgrowth. Deoxyuridine triphosphatase (dUTPase) is essential for DNA replication and was therefore tested as a potential marker for meristem reactivation in tuber buds. The corresponding cDNA clone was isolated from potato by PCR. The deduced amino acid sequence showed 94% similarity to the tomato homologue. By employing different potato cultivars, a positive correlation between dUTPase expression and onset of tuber sprouting could be confirmed. Moreover, gene expression analysis of tuber buds during storage time revealed an up-regulation of the dUTPase 1 week before visible sprouting occurred. Further analysis using an in vitro sprout assay supported the assumption that dUTPase is a good molecular marker to define the transition from dormant to active potato tuber meristems.     

Abscisic acid biosynthesis in tomato: regulation of zeaxanthin epoxidase and 9-cis-epoxycarotenoid dioxygenase mRNAs by light/dark cycles,water stress and abscisic acid

Two genes encoding enzymes in the abscisic acid (ABA) biosynthesis pathway, zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED), have previously been cloned by transposon tagging in Nicotiana plumbaginifolia and maize respectively. We demonstrate that antisense down-regulation of the tomato gene LeZEP1 causes accumulation of zeaxanthin in leaves, suggesting that this gene also encodes ZEP. LeNCED1 is known to encode NCED from characterization of a null mutation (notabilis) in tomato. We have used LeZEP1 and LeNCED1 as probes to study gene expression in leaves and roots of whole plants given drought treatments, during light/dark cycles, and during dehydration of detached leaves. During drought stress, NCED mRNA increased in both leaves and roots, whereas ZEP mRNA increased in roots but not leaves. When detached leaves were dehydrated, NCED mRNA responded rapidly to small reductions in water content. Using a detached leaf system with ABA-deficient mutants and ABA feeding, we investigated the possibility that NCED mRNA is regulated by the end product of the pathway, ABA, but found no evidence that this is the case. We also describe strong diurnal expression patterns for both ZEP and NCED, with the two genes displaying distinctly different patterns. ZEP mRNA oscillated with a phase very similar to light-harvesting complex II (LHCII) mRNA, and oscillations continued in a 48 h dark period. NCED mRNA oscillated with a different phase and remained low during a 48 h dark period. Implications for regulation of water stress-induced ABA biosynthesis are discussed.     

Epoxycarotenoid cleavage by NCED5 fine-tunes ABA accumulation and affects seed dormancy and drought tolerance with other NCED family members

Carotenoid cleavage, catalyzed by the 9-cis-epoxycarotenoid dioxygenase (NCED) constitutes a key step in the regulation of ABA biosynthesis. In Arabidopsis, this enzyme is encoded by five genes. NCED3 has been shown to play a major role in the regulation of ABA synthesis in response to water deficit, whereas NCED6 and NCED9 have been shown to be essential for the ABA production in the embryo and endosperm that imposes dormancy. Reporter gene analysis was carried out to determine the spatiotemporal pattern of NCED5 and NCED9 gene expression. GUS activity from the NCED5 promoter was detected in both the embryo and endosperm of developing seeds with maximal staining after mid-development. NCED9 expression was found at early stages in the testa outer integument layer 1, and after mid-development in epidermal cells of the embryo, but not in the endosperm. In accordance with its temporal- and tissue-specific expression, the phenotypic analysis of nced5 nced6 nced9 triple mutant showed the involvement of the NCED5 gene, together with NCED6 and NCED9, in the induction of seed dormancy. In contrast to nced6 and nced9, however, nced5 mutation did not affect the gibberellin required for germination. In vegetative tissues, combining nced5 and nced3 mutations reduced vegetative growth, increased water loss upon dehydration, and decreased ABA levels under both normal and stressed conditions, as compared with nced3. NCED5 thus contributes, together with NCED3, to ABA production affecting plant growth and water stress tolerance.