Interactions between abscisic acid, ethylene and gibberellin in internodal elongation in floating rice: the promotive effect of abscisic acid at low humidity

The enhancement of internodal elongation in floating or deepwater rice (Oryza sativa L. cv. Habiganj Aman II) by treatment with ethylene or gibberellic acid (GA₃) at high relative humidity (RH) is inhibited by abscisic acid (ABA). Here, we examined the interactive effects of ethylene, gibberellin (GA) and ABA at low RH on internodal elongation of deepwater rice stem segments. Although ethylene alone hardly promoted internodal elongation of stem sections at 30% RH, it enhanced the internodal elongation induced by GA₃. Application of ABA alone to stem segments had no effect on internodal elongation. However, in the presence of ethylene and GA₃ at 30% RH, ABA further promoted internodal elongation. This promotive effect of ABA was not found in the internodes of stem segments treated either with ethylene or with GA₃ at 30% RH or in the internodes of stem segments treated with ethylene and/or GA₃ at 100% RH.     

Molecular Events Underlying Coordinated Hormone Action in Submergence Escape Response of Deepwater Rice

Recent studies revealed that some rice varieties adopt opposite strategies to overcome flooding stress. While certain varieties hold metabolism and stay stunted until floodwater recedes, deepwater rice varieties undergo rapid stem elongation and do not suffer drowning problems. Both varieties use the same signaling agents, the ethylene response factors, as key factors even though they display opposite submergence responses. In deepwater rice, ethylene response factor genes SNORKEL1 and SNORKEL2 are believed to play a major role in submergence escape by mediating ethylene signaling, which leads to rapid stem elongation. These genes connect hormone signaling cascades from ethylene to ABA and gibberellins (GAs). Submergence increases ethylene levels in the internodal space, ethylene upregulates an ABA inactivating enzyme gene, OsCYP707A5 or OsABA8ox1, and some GA metabolism genes such as OsGA20ox genes and OsGA3ox genes. As a result of gene regulation by ethylene, internodal ABA levels decrease while GA levels increase, finally upregulating growth-related genes like expansin genes (OsEXPs). Along with the ethylene signaling in submergence, it is necessary to consider an alternative signaling pathway induced by hypoxia. Taken together, study on the submergence responses of rice plants will lead to improvement of crop production and contribution to basic research on plant growth.     

Expression of novel rice gibberellin 2-oxidase gene is under homeostatic regulation by biologically active gibberellins

We have cloned two genes for gibberellin (GA) 2-oxidase from rice (Oryza sativa L.). Expression of OsGA2ox2 was not observed. The other gene, OsGA2ox3, was expressed in every tissue examined and was enhanced by the application of biologically active GA. Recombinant OsGA2ox3 protein catalyzed the metabolism of GA₁ to GA₈ and GA₂₀ to GA₂₉-catabolite. These results indicate that OsGA2ox3 is involved in the homeostatic regulation of the endogenous level of biologically active GA in rice. Electronic Publication     

Comparison of the role of gibberellins and ethylene in response to submergence of two lowland rice cultivars, Senia and Bomba

We examined the gibberellin (GA) and ethylene regulation of submergence-induced elongation in seedlings of the submergence-tolerant lowland rice (Oryza sativa L.) cvs Senia and Bomba. Elongation was enhanced after germination to facilitate water escape and reach air. We found that submergence-induced elongation depends on GA because it was counteracted by paclobutrazol (an inhibitor of GA biosynthesis), an effect that was negated by GA₃. Moreover, in the cv Senia, submergence increased the content of active GA₁ and its immediate precursors (GA₅₃, GA₁₉ and GA₂₀) by enhancing expression of several GA biosynthesis genes (OsGA20ox1 and -2, and OsGA3ox2), but not by decreasing expression of several OsGA2ox (GA inactivating genes). Senia seedlings, in contrast to Bomba seedlings, did not elongate in response to ethylene or 1-aminocyclopropane-1-carboxylic-acid (ACC; an ethylene precursor) application, and submergence-induced elongation was not reduced in the presence of 1-methylcyclopropene (1-MCP; an ethylene perception inhibitor). Ethylene emanation was similar in Senia seedlings grown in air and in submerged-grown seedlings following de-submergence, while it increased in Bomba. The expression of ethylene biosynthesis genes (OsACS1, -2 and -3, and OsACO1) was not affected in Senia, but expression of OsACS5 was rapidly enhanced in Bomba upon submergence. Our results support the conclusion that submergence elongation enhancement of lowland rice is due to alteration of GA metabolism leading to an increase in active GA (GA₁) content. Interestingly, in the cv Senia, in contrast to cv Bomba, this was triggered through an ethylene-independent mechanism.     

On the role of abscisic Acid and gibberellin in the regulation of growth in rice

Submergence induces rapid elongation of rice coleoptiles (Oryza sativa L.) and of deepwater rice internodes. This adaptive feature helps rice to grow out of the water and to survive flooding. Earlier, we found that the growth response of submerged deepwater rice plants is mediated by ethylene and gibberellin (GA). Ethylene promotes growth, at least in part, by increasing the responsiveness of the internodal tissue to GA. In the present work, we examined the possibility that increased responsiveness to GA was based on a reduction in endogenous abscisic acid (ABA) levels. Submergence and treatment with ethylene led, within 3 hours, to a 75% reduction in the level of ABA in the intercalary meristem and the growing zone of deepwater rice internodes. The level of GA₁ increased fourfold during the same time period. An interaction between GA and ABA could also be shown by application of the hormones. ABA inhibited growth of submerged internodes, and GA counteracted this inhibition. Our results indicate that the growth rate of deepwater rice internodes is determined by the ratio of an endogenous growth promoter (GA) and a growth inhibitor (ABA). We also investigated whether ABA is involved in regulating the growth of rice coleoptiles. Rice seedlings were grown on solutions containing fluridone, an inhibitor of carotenoid and, indirectly, of ABA biosynthesis. Treatment with fluridone reduced the level of ABA in coleoptiles and first leaves by more than 75% and promoted coleoptile growth by more than 60%. Little or no enhancement of growth by fluridone was observed in barley, oat, or wheat. The involvement of ABA in determining the growth rate of rice coleoptiles and deepwater rice internodes may be related to the semiaquatic growth habit of this plant.     

Physiological characteristics and related gene expression of after‐ripening on seed dormancy release in rice

After‐ripening is a common method used for dormancy release in rice. In this study, the rice variety Jiucaiqing (Oryza sativa L. subsp. japonica) was used to determine dormancy release following different after‐ripening times (1, 2 and 3 months). Germination speed, germination percentage and seedling emergence increased with after‐ripening; more than 95% germination and 85% seedling emergence were observed following 1 month of after‐ripening within 10 days of imbibition, compared with <45% germination and 20% seedling emergence in freshly harvested seed. Hence, 3 months of after‐ripening could be considered a suitable treatment period for rice dormancy release. Dormancy release by after‐ripening is mainly correlated with a rapid decline in ABA content and increase in IAA content during imbibition. Subsequently, GA₁/ABA, GA₇/ABA, GA₁₂/ABA, GA₂₀/ABA and IAA/ABA ratios significantly increased, while GA₃/ABA, GA₄/ABA and GAs/IAA ratio significantly decreased in imbibed seeds following 3 months of after‐ripening, thereby altering α‐amylase activity during seed germination. Peak α‐amylase activity occurred at an earlier germination stage in after‐ripened seeds than in freshly harvested seeds. Expression of ABA, GA and IAA metabolism genes and dormancy‐related genes was regulated by after‐ripening time upon imbibition. Expression of OsCYP707A5, OsGA2ox1, OsGA2ox2, OsGA2ox3, OsILR1, OsGH3‐2, qLTG3‐1 and OsVP1 increased, while expression of Sdr4 decreased in imbibed seeds following 3 months of after‐ripening. Dormancy release through after‐ripening might be involved in weakening tissues covering the embryo via qLTG3‐1 and decreased ABA signalling and sensitivity via Sdr4 and OsVP1.     

OsGA2ox5, a Gibberellin Metabolism Enzyme,Is Involved in Plant Growth,the Root Gravity Response and Salt Stress

Gibberellin (GA) 2-oxidases play an important role in the GA catabolic pathway through 2β-hydroxylation. There are two classes of GA2oxs, i.e., a larger class of C₁₉-GA2oxs and a smaller class of C₂₀-GA2oxs. In this study, the gene encoding a GA 2-oxidase of rice, Oryza sativa GA 2-oxidase 5 (OsGA2ox5), was cloned and characterized. BLASTP analysis showed that OsGA2ox5 belongs to the C₂₀-GA2oxs subfamily, a subfamily of GA2oxs acting on C₂₀-GAs (GA₁₂, GA₅₃). Subcellular localization of OsGA2ox5-YFP in transiently transformed onion epidermal cells revealed the presence of this protein in both of the nucleus and cytoplasm. Real-time PCR analysis, along with GUS staining, revealed that OsGA2ox5 is expressed in the roots, culms, leaves, sheaths and panicles of rice. Rice plants overexpressing OsGA2ox5 exhibited dominant dwarf and GA-deficient phenotypes, with shorter stems and later development of reproductive organs than the wild type. The dwarfism phenotype was partially rescued by the application of exogenous GA₃ at a concentration of 10 µM. Ectopic expression of OsGA2ox5 cDNA in Arabidopsis resulted in a similar phenotype. Real-time PCR assays revealed that both GA synthesis-related genes and GA signaling genes were expressed at higher levels in transgenic rice plants than in wild-type rice; OsGA3ox1, which encodes a key enzyme in the last step of the bioactive GAs synthesis pathway, was highly expressed in transgenic rice. The roots of OsGA2ox5-ox plants exhibited increased starch granule accumulation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Furthermore, rice and Arabidopsis plants overexpressing OsGA2ox5 were more resistant to high-salinity stress than wild-type plants. These results suggest that OsGA2ox5 plays important roles in GAs homeostasis, development, gravity responses and stress tolerance in rice.     

A role of <Emphasis Type="Italic">OsGA20ox1</Emphasis>, encoding an isoform of gibberellin 20-oxidase,for regulation of plant stature in rice

Gibberellin (GA) 20-oxidase (GA20ox) is a key enzyme that normally catalyzes the penultimate steps in GA biosynthesis. One of the GA20ox genes in rice (Oryza sativaL.), OsGA20ox2 (SD1), is well known as the Green Revolution gene, and loss-of function mutation in this locus causes semi-dwarfism. Another GA20ox gene, OsGA20ox1, has also been identified, but its contribution to plant stature has remained unclear because no suitable mutants have been available. We isolated a mutant, B142, tagged with a T-DNA containing three CaMV 35S promoters, which showed a tall, GA-overproduction phenotype. The final stature of the B142 mutant reflects internode overgrowth and is approximately twice that of its wild-type parent. This mutant responds to application of both GA3 and a GA biosynthesis inhibitor, indicating that it is a novel tall mutant of rice distinct from GA signaling mutants such as slr1. The integrated T-DNAs, which contain three CaMV 35S promoters, are located upstream of the OsGA20ox1 open reading frame (ORF) in the B142 mutant genome. Analysis of mRNA and the endogenous GAs reveal that biologically active GA level is increased by up-regulation of the OsGA20ox1 gene in B142. Introduction of OsGA20ox1 cDNA driven by 35S promoter into the wild type phenocopies the morphological characteristics of B142. These results indicate that the elongated phenotype of the B142 mutant is caused by up-regulation of the OsGA20ox1 gene. Moreover, the final stature of rice was reduced by specific suppression of the OsGA20ox1 gene expression. This result indicates that not only OsGA20ox2 but also OsGA20ox1 affects plant stature.     

Characterization of genes encoding ABA 8'-hydroxylase in ethylene-induced stem growth of deepwater rice (Oryza sativa L.)

Ethylene and submergence enhance stem elongation of deepwater rice, at least in part, by reducing in the internode the endogenous abscisic acid (ABA) content and increasing the level of gibberellin A1 (GA1). We cloned and characterized the CYP707A5 and CYP707A6 genes, which encode putative ABA 8'-hydroxylase, the enzyme that catalyzes the oxidation of ABA. Expression of CYP707A5 was upregulated significantly by ethylene treatment, whereas that of CYP707A6 was not altered. Recombinant proteins from both genes expressed in yeast cells showed activity of ABA 8'-hydroxylase. This finding indicates that CYP707A5 may play a role in ABA catabolism during submergence- or ethylene-induced stem elongation in deepwater rice. Taken together, these results provide links between the molecular mechanisms and physiological phenomena of submergence- and ethylene-induced stem elongation in deepwater rice.     

Differences and similarities in the photoregulation of gibberellin metabolism between rice and dicots

In rice seedlings, elongation of leaf sheaths is suppressed by light stimuli. The response is mediated by two classes of photoreceptors, phytochromes and cryptochromes. However, it remains unclear how these photoreceptors interact in the process. Our recent study using phytochrome mutants and novel cryptochrome RNAi lines revealed that cryptochromes and phytochromes function cooperatively, but independently to reduce active GA contents in seedlings in visible light. Blue light captured by cryptochrome 1 (cry1a and cry1b) induces robust expression of GA 2-oxidase genes (OsGA2ox4-7). In parallel, phytochrome B with auxiliary action of phytochrome A mediates repression of GA 20-oxidase genes (OsGA20ox2 and OsGA20ox4). The independent effects cumulatively reduce active GA contents, leading to a suppression of leaf sheath elongation. These regulatory mechanisms are distinct from phytochrome B function in dicots. We discuss reasons why the distinct system appeared in rice, and advantages of the rice system in early photomorphogenesis.     

Amylase activity and growth in internodes of deepwater rice

Isoelectrofocusing, product analysis, thermal denaturation studies and affinity chromatography on cycloheptaamylose-Sephadex were used to identify the amylolytic enzymes in internodes of deepwater rice (Oryza sativa L.). Amylolytic activity in internodes of deepwater rice consists of -amylase (sometimes separated into two isoforms) and of -amylase. During submergence of whole plants, -amylase activity increases in young, growing internodes, but -amylase activity declines. Although non-growing, mature internodes contain higher levels of -amylase than do the elongating younger internodes, the effect of submergence on amylase activities in both tissues follows the same trend. Submergence, gibberellic acid (GA₃) and ethylene all promote -amylase activity in growing and non-growing internodes of excised deepwater-rice stem sections. Inhibitor studies showed that submergence and ethylene promote -amylase activity in the absence of endogenous gibberellin (GA), and GA₃ enhances -amylase activity when ethylene action is inhibited. Therefore, ethylene and GA appear to increase -amylase activity independently of each other. Enhanced -amylase activities are probably responsible for the mobilization of carbohydrates which are needed to support internode elongation during submergence of deepwater rice.Abbreviations CHA cycloheptaamylose - GA₃ gibberellic acid - NBD 2,5-norbornadiene - TCY tetcyclacis     

Expression of a gibberellin 2-oxidase gene around the shoot apex is related to phase transition in rice

A major catabolic pathway for gibberellin (GA) is initiated by 2beta-hydroxylation, a reaction catalyzed by GA 2-oxidase. We have isolated and characterized a cDNA, designated Oryza sativa GA 2-oxidase 1 (OsGA2ox1) from rice (Oryza sativa L. cv Nipponbare) that encodes a GA 2-oxidase. The encoded protein, produced by heterologous expression in Escherichia coli, converted GA(1), GA(4), GA(9), GA(20), and GA(44) to the corresponding 2beta-hydroxylated products GA(8), GA(34), GA(51), GA(29), and GA(98), respectively. Ectopic expression of the OsGA2ox1 cDNA in transgenic rice inhibited stem elongation and the development of reproductive organs. These transgenic plants were deficient in endogenous GA(1). These results indicate that OsGA2ox1 encodes a GA 2-oxidase, which is functional not only in vitro but also in vivo. OsGA2ox1 was expressed in shoot apex and roots but not in leaves and stems. In situ hybridization analysis revealed that OsGA2ox1 mRNA was localized in a ring at the basal region of leaf primordia and young leaves. This ring-shaped expression around the shoot apex was drastically decreased after the phase transition from vegetative to reproductive growth. It was absent in the floral meristem, but it was still present in the lateral meristem that remained in the vegetative phase. These observations suggest that OsGA2ox1 controls the level of bioactive GAs in the shoot apical meristem; therefore, reduction in its expression may contribute to the early development of the inflorescence meristem.     

Development of Nitrogen Assimilation Enzymes during Photoautotrophic Growth of Chenopodium rubrum Suspension Cultures

We have shown previously that ethylene, which accumulates in the air spaces of submerged stem sections of rice (Oryza sativa L. cv “Habiganj Aman II”), is involved in regulating the growth response caused by submergence. The role of gibberellins in the submergence response was studied using tetcyclacis (TCY), a new plant growth retardant, which inhibits gibberellin biosynthesis. Stem sections excised from plants that had been watered with a solution of 1 micromolar TCY for 7 to 10 days did not elongate when submerged in the same solution or when exposed to 1 microliter per liter ethylene in air. Gibberellic acid (GA₃) at 0.3 micromolar overcame the effect of TCY and restored the rapid internodal elongation in submerged and ethylene-treated sections to the levels observed in control sections that had not been treated with TCY. The effect of 0.01 to 0.2 micromolar GA₃ on internodal elongation was enhanced two- to eight-fold when 1 microliter per liter ethylene was added to the air passing through the chamber in which the sections were incubated. GA₃ and ethylene caused a similar increase in cell division and cell elongation in rice internodes. Thus, ethylene may cause internodal elongation in rice by increasing the activity of endogenous GAs. In internodes from which the leaf sheath had been peeled off, growth in response to submergence, ethylene and GA₃ was severely inhibited by light.     

Function and Expression Analysis of Gibberellin Oxidases in Apple

Three cDNAs, encoding gibberellin (GA) 20-oxidase (MdGA20ox1, identical to AB037114), 3-oxidase (MdGA3ox1), and 2-oxidase (MdGA2ox1), were isolated from apple cv. Fuji (Malus x domestica). Southern blot analysis indicated that each of these genes belongs to a gene family. Standard enzyme assays show that the MdGA20ox1-MBP fusion protein can sequentially oxidize three times at C-20 position of GA₁₂ and GA₅₃ and generate GA₉ and GA₂₀; the MdGA3ox1-MBP fusion protein converts GA₂₀ and GA₉ to GA₄ and GA₁, and the MdGA2ox1-MBP fusion protein converts GA₄ and GA₁ to GA₃₄ and GA₈, respectively. In addition, we confirmed that MdGA20ox1 is strongly expressed in immature seeds and scarcely detected in other tissues, whereas MdGA3ox1 and MdGA2ox1 are mainly expressed in flowers. Therefore, all the three cDNAs are localized in reproductive tissues. Functional and expression analysis of the three GA oxidases would provide fundamental molecular information to analyze GA metabolic regulation in apple.     

OsDOG, a gibberellin-induced A20/AN1 zinc-finger protein, negatively regulates gibberellin-mediated cell elongation in rice

The A20/AN1 zinc-finger proteins (ZFPs) play pivotal roles in animal immune responses and plant stress responses. From previous gibberellin (GA) microarray data and A20/AN1 ZFP family member association, we chose Oryza sativa dwarf rice with overexpression of gibberellin-induced gene (OsDOG) to examine its function in the GA pathway. OsDOG was induced by gibberellic acid (GA₃) and repressed by the GA-synthesis inhibitor paclobutrazol. Different transgenic lines with constitutive expression of OsDOG showed dwarf phenotypes due to deficiency of cell elongation. Additional GA₁ and real-time PCR quantitative assay analyses confirmed that the decrease of GA₁ in the overexpression lines resulted from reduced expression of GA3ox2 and enhanced expression of GA2ox1 and GA2ox3. Adding exogenous GA rescued the constitutive expression phenotypes of the transgenic lines. OsDOG has a novel function in regulating GA homeostasis and in negative maintenance of plant cell elongation in rice.     

The Response to Gibberellin in Pisum sativum Grown under Alternating Day and Night Temperature

The application of gibberellins (GA) reduces the difference in stem elongation observed under a low day (DT) and high night temperature (NT) combination (negative DIF) compared with the opposite regime, a high DT/low NT (positive DIF). The aim of this work was to investigate possible thermoperiodic effects on GA metabolism and tissue sensitivity to GA by comparing the response to exogenously applied GA (in particular, GA₁ and GA₃) in pea plants (Pisum sativum cv. Torsdag) grown under contrasting DIF. Control plants not treated with growth inhibitors or additional GA were 38% shorter under negative (DT/NT 13/21°C) than positive DIF (DT/NT 21/13°C) because of shorter internodes. Additional GA₁ or GA₃ decreased the difference between positive and negative DIF. In pea plants dwarfed with paclobutrazol, which inhibits GA biosynthesis at an early step, the response to GA₁ was reduced more strongly by negative compared with positive DIF than the response to GA₃. The induced stem elongation by GA₁₉ and GA₂₀ did not deviate significantly from the response to GA₁. Plants treated with prohexadione-calcium, an inhibitor of both the production and the inactivation of GA₁, grew equally tall under the two temperature regimes in response to both GA₁ and GA₃. We hypothesize that the reduced response to GA₁ compared with GA₃ in paclobutrazol-treated plants grown under negative DIF is caused by a higher rate of 2β-hydroxylation of GA₁ into GA₈ under negative than positive DIF. This contributes to lower levels of GA₁ and consequently shorter stems and internodes in pea plants grown under negative than positive DIF. Differences in tissue sensitivity to GA alone cannot account for this specific thermoperiodic effect on stem elongation. Received May 28, 1998; accepted May 29, 1998     

Gibberellin Metabolism and Transport During Germination and Young Seedling Growth of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

The role of gibberellins (GAs) during germination and early seedling growth is examined by following the metabolism and transport of radiolabeled GAs in cotyledon, shoot, and root tissues of pea (Pisum sativum L.) using an aseptic culture system. Mature pea seeds have significant endogenous GA₂₀ levels that fall during germination and early seedling growth, a period when the seedling develops the capacity to transport GA₂₀ from the cotyledon to the shoot and root of the seedling. Even though cotyledons at 0–2 days after imbibition have appreciable amounts of GA₂₀, the cotyledons retain the ability to metabolize labeled GA₁₉ to GA₂₀ and express significant levels of PsGA20ox2 message (which encodes a GA biosynthesis enzyme, GA 20-oxidase). The large pool of cotyledonary GA₂₀ likely provides substrate for GA₁ synthesis in the cotyledons during germination, as well as for shoots and roots during early seedling growth. The shoots and roots express GA metabolism genes (PsGA3ox genes which encode GA 3-oxidases for synthesis of bioactive GA₁, and PsGA2ox genes which encode GA 2-oxidases for deactivation of GAs to GA₂₉ and GA₈), and they develop the capacity to metabolize GAs as necessary for seedling establishment. Auxins also show an interesting pattern during early seedling growth, with higher levels of 4-chloro-indole-3-acetic acid (4-Cl-IAA) in mature seeds and higher levels of indole-3-acetic acid (IAA) in young root and shoot tissues. This suggests a changing role for auxins during early seedling development.