Coordinate expression of AOS genes and JA accumulation: JA is not required for initiation of closing layer in wound healing tubers

Wounding induces a series of coordinated physiological responses essential for protection and healing of the damaged tissue. Wound-induced formation of jasmonic acid (JA) is important in defense responses in leaves, but comparatively little is known about the induction of JA biosynthesis and its role(s) in tuber wound-healing. In this study, the effects of wounding on JA content, expression of JA biosynthetic genes, and the involvement of JA in the initiation of closing layer formation in potato tubers were determined. In addition, the role of abscisic acid (ABA) in wound-induced JA accumulation was examined. The basal JA content in non-wounded tuber tissues was low (<3 ng g⁻¹ FW). Two hours after wounding, the JA content increased by >5-fold, reached a maximum between 4 and 6 h after wounding, and declined to near-basal levels thereafter. Tuber age (storage duration) had little effect on the pattern of JA accumulation. The expressions of the JA biosynthetic genes (StAOS2, StAOC, and StOPR3) were greatly increased by wounding reaching a maximum 2-4 h after wounding and declining thereafter. A 1-h aqueous wash of tuber discs immediately after wounding resulted in a 94% inhibition of wound-induced JA accumulation. Neither JA treatment nor inhibition of JA accumulation affected suberin polyphenolic accumulation during closing layer development indicating that JA was not essential for the initiation of primary suberization. ABA treatment did not restore JA accumulation in washed tuber tissues suggesting that leaching of endogenous ABA was either not involved or not solely involved in this loss of JA accumulation by washing. Collectively, these results indicate that JA is not required for the induction of processes essential to the initiation of suberization during closing layer development, but do not exclude the possibility that JA may be involved in other wound related responses.     

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.     

Effects of postharvest storage and dormancy status on ABA content, metabolism, and expression of genes involved in ABA biosynthesis and metabolism in potato tuber tissues

At harvest, and for an indeterminate period thereafter, potato tubers will not sprout and are physiologically dormant. Abscisic acid (ABA) has been shown to play a critical role in tuber dormancy control but the mechanisms controlling ABA content during dormancy as well as the sites of ABA synthesis and catabolism are unknown. As a first step in defining the sites of synthesis and cognate processes regulating ABA turnover during storage and dormancy progression, gene sequences encoding the ABA biosynthetic enzymes zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED) and three catabolism-related genes were used to quantify changes in their relative mRNA abundances in three specific tuber tissues (meristems, their surrounding periderm and underlying cortex) by qRT-PCR. During storage, StZEP expression was relatively constant in meristems, exhibited a biphasic pattern in periderm with transient increases during early and mid-to-late-storage, and peaked during mid-storage in cortex. Expression of two members of the potato NCED gene family was found to correlate with changes in ABA content in meristems (StNCED2) and cortex (StNCED1). Conversely, expression patterns of three putative ABA-8′-hydroxylase (CYP707A) genes during storage varied in a tissue-specific manner with expression of two of these genes rising in meristems and periderm and declining in cortex during storage. These results suggest that ABA synthesis and metabolism occur in all tuber tissues examined and that tuber ABA content during dormancy is the result of a balance of synthesis and metabolism that increasingly favors catabolism as dormancy ends and may be controlled at the level of StNCED and StCYP707A gene activities Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

内源ABA信号水平动态调控的分子机制

从逆境信号感知、ABA合成的触发到ABA水平的动态调控,是细胞内重要的逆境信号传导途径,相对于应答ABA的下游信号事件,该领域研究滞后。研究显示,根系中ZEP、限速酶NCED、AtRGS1等合成酶基因及ABA2基因响应胁迫反应上调ABA信号水平。而7′-,8′-,9′-hydroxylase和糖基转移酶基因受逆境诱导激活,负调节ABA的积累。同时,提高的内源ABA信号水平能激活合成酶基因和代谢酶基因的表达。此外,基因表达和源库动力学分析显示,叶片ABA动态库的维持依赖根源ABA的持续供应。值得一提的是,miRNA与ABA信号起源及动态水平维持有关。进一步的代谢动力学分析揭示,ABA信号水平受合成酶基因和代谢酶基因表达的协同控制,多因素共同参与内源ABA信号水平的动态调控。     

内源ABA信号水平动态调控的分子机制

从逆境信号感知、ABA合成的触发到ABA水平的动态调控, 是细胞内重要的逆境信号传导途径, 相对于应答ABA的下游信号事件, 该领域研究滞后。研究显示, 根系中ZEP、限速酶NCED、AtRGS1等合成酶基因及ABA2基因响应胁迫反应上调ABA信号水平。而7′-, 8′-, 9′-hydroxylase和糖基转移酶基因受逆境诱导激活, 负调节ABA的积累。同时, 提高的内源ABA信号水平能激活合成酶基因和代谢酶基因的表达。此外, 基因表达和源库动力学分析显示, 叶片ABA动态库的维持依赖根源ABA的持续供应。值得一提的是, miRNA与ABA信号起源及动态水平维持有关。进一步的代谢动力学分析揭示, ABA信号水平受合成酶基因和代谢酶基因表达的协同控制, 多因素共同参与内源ABA信号水平的动态调控。     

高等植物脱落酸生物合成途径及其酶调控

脱落酸(ABA)生物合成一般有两条途径:C15直接途径和C40间接途径,前者经C15法呢焦磷酸(FPP)直接形成ABA;后者经由类胡萝卜素的氧化裂解间接形成ABA,是高等植物ABA生物合成的主要途径.9-顺式环氧类胡萝卜素氧化裂解为黄质醛是植物ABA生物合成的关键步骤,然后黄质醛被氧化形成一种酮,该过程需NAD为辅因子,酮再转变形成ABA-醛,ABA-醛氧化最终形成ABA.在该途径中,玉米黄质环氧化酶(ZEP)、9-顺式环氧类胡萝卜素双加氧酶(NCED)和醛氧化酶(AO)可能起重要作用.     

Kinetics and localization of wound-induced DNA biosynthesis in potato tuber

Tuber wounding induces a cascade of biological responses that are involved in processes required to heal and protect surviving plant tissues. Little is known about the coordination of these processes, including essential wound-induced DNA synthesis, yet they play critical roles in maintaining marketability of the harvested crop and tubers cut for seed. A sensitive “Click-iT EdU Assay” employing incorporation of the thymidine analog, 5-ethynyl-2′-deoxyuridine (EdU), in conjunction with 4′,6-diamindino-2-phenylindole (DAPI) counter labeling, was employed to objectively identify and determine the time course and spatial distribution of tuber nuclei that were wound-induced to enter S-phase of the cell cycle. Both labeling procedures are rapid and sensitive in situ. Following wounding, EdU incorporation (indicating DNA synthesis) was not detectable until after 12 h, rapidly reached a maximum at about 18 h and then declined to near zero at 48 h. About 28% of the nuclei were EdU labeled at 18 h reflecting the proportion of cells in S-phase of the cell cycle. During the ∼30 h in which induced cells were progressing through S-phase, de novo DNA synthesis extended 7–8 cell layers below the wound surface. Cessation of nuclear DNA synthesis occurred about 4 d prior to completion of wound closing layer formation. Initiation of wound periderm development followed at 7 d, i.e. about 5 d after cessation of nuclear DNA biosynthesis; at this time the phellogen developed and meristematic activity was detected via the production of new phellem cells. Collectively, these results provide new insight into the coordination of wound-induced nucleic acid synthesis with associated tuber wound-healing processes.     

Regulatory involvement of abscisic acid in potato tuber wound-healing

Rapid wound-healing is crucial in protecting potato tubers frominfection and dehydration. Wound-induced suberization and theaccumulation of hydrophobic barriers to reduce water vapourconductance/loss are principal protective wound-healing processes.However, little is known about the cognate mechanisms that effector regulate these processes. The objective of this researchwas to determine the involvement of abscisic acid (ABA) in theregulation of wound-induced suberization and tuber water vapourloss (dehydration). Analysis by liquid chromatography–massspectrometry showed that ABA concentrations varied little throughoutthe tuber, but were slightly higher near the periderm and lowestin the pith. ABA concentrations increase then decrease duringtuber storage. Tuber wounding induced changes in ABA content.ABA content in wound-healing tuber discs decreased after wounding,reached a minimum by 24 h, and then increased from the 3rd tothe 7th day after wounding. Wound-induced ABA accumulationswere reduced by fluridone (FLD); an inhibitor of de novo ABAbiosynthesis. Wound-induced phenylalanine ammonia lyase activitywas slightly reduced and the accumulation of suberin poly(phenolics)and poly(aliphatics) noticeably reduced in FLD-treated tissues.Addition of ABA to the FLD treatment restored phenylalanineammonia lyase activity and suberization, unequivocally indicatingthat endogenous ABA is involved in the regulation of these wound-healingprocesses. Similar experiments showed that endogenous ABA isinvolved in the regulation of water vapour loss, a process linkedto wax accumulation in wound-healing tubers. Rapid reductionof water vapour loss across the wound surface is essential inpreventing desiccation and death of cells at the wound site;live cells are required for suberization. These results unequivocallyshow that endogenous ABA is involved in the regulation of wound-inducedsuberization and the processes that protect surface cells fromwater vapour loss and death by dehydration. Key words: Abscisic acid, poly(aliphatic), poly(phenolic), potato, Solanum tuberosum L., suberin     

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.     

Regulation and manipulation of ABA biosynthesis in roots

Overexpression of 9-cis-epoxycarotenoid dioxygenase (NCED) is known to cause abscisic acid (ABA) accumulation in leaves, seeds and whole plants. Here we investigated the manipulation of ABA biosynthesis in roots. Roots from whole tomato plants that constitutively overexpress LeNCED1 had a higher ABA content than wild-type (WT) roots. This could be explained by enhanced in situ ABA biosynthesis, rather than import of ABA from the shoot, because root cultures also had higher ABA content, and because tetracycline (Tc)-induced LeNCED1 expression caused ABA accumulation in isolated tobacco roots. However, the Tc-induced expression led to greater accumulation of ABA in leaves than in roots. This demonstrates for the first time that NCED is rate-limiting in root tissues, but suggests that other steps were also restrictive to pathway flux, more so in roots than in leaves. Dehydration and NCED overexpression acted synergistically in enhancing ABA accumulation in tomato root cultures. One explanation is that xanthophyll synthesis was increased during root dehydration, and, in support of this, dehydration treatments increased beta-carotene hydroxylase mRNA levels. Whole plants overexpressing LeNCED1 exhibited greatly reduced stomatal conductance and grafting experiments from this study demonstrated that this was predominantly due to increased ABA biosynthesis in leaves rather than in roots. Genetic manipulation of both xanthophyll supply and epoxycarotenoid cleavage may be needed to enhance root ABA biosynthesis sufficiently to signal stomatal closure in the shoot.     

<Emphasis Type="Italic">Strboh A</Emphasis> homologue of NADPH oxidase regulates wound-induced oxidative burst and facilitates wound-healing in potato tubers

During 30-months of storage at 4°C, potato (Solanum tuberosum L.) tubers progressively lose the ability to produce superoxide in response to wounding, resist microbial infection, and develop a suberized wound periderm. Using differentially aged tubers, we demonstrate that Strboh A is responsible for the wound-induced oxidative burst in potato and aging attenuates its expression. In vivo superoxide production and NADPH oxidase (NOX) activity from 1-month-old tubers increased to a maximum 18–24 h after wounding and then decreased to barely detectable levels by 72 h. Wounding also induced a 68% increase in microsomal protein within 18 h. These wound-induced responses were lost over a 25- to 30-month storage period. Superoxide production and NOX activity were inhibited by diphenylene iodonium chloride, a specific inhibitor of NOX, which in turn effectively inhibited wound-healing and increased susceptibility to microbial infection and decay in 1-month-old tubers. Wound-induced superoxide production was also inhibited by EGTA-mediated destabilization of membranes. The ability to restore superoxide production to EGTA-treated tissue with Ca⁺² declined with advancing tuber age, likely a consequence of age-related changes in membrane architecture. Of the five homologues of NOX (Strboh A-D and F), wounding induced the expression of Strboh A in 6-month-old tubers but this response was absent in tubers stored for 25–30 months. Strboh A thus mediates the initial burst of superoxide in response to wounding of potato tubers; loss of its expression increases the susceptibility to microbial infection and contributes to the age-induced loss of wound-healing ability.     

高等植物脱落酸生物合成途径及其酶调控

脱落酸（ABA）生物合成一般有两条途径：C15直接途径和C40间接途径, 前者经C15法呢焦磷酸（FPP）直接形成ABA;后者经由类胡萝卜素的氧化裂解间接形成ABA, 是高等植物ABA生物合成的主要途径。9-顺式环氧类胡萝卜素氧化裂解为黄质醛是植物ABA生物合成的关键步骤, 然后黄质醛被氧化形成一种酮, 该过程需NAD为辅因子, 酮再转变形成ABA-醛, ABA-醛氧化最终形成ABA。在该途径中,玉米黄质环氧化酶（ZEP）、9-顺式环氧类胡萝卜素双加氧酶（NCED）和醛氧化酶（AO）可能起重要作用。     

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.     

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.