Ethylene-induced stem growth of deepwater rice is correlated with expression of gibberellin- and abscisic acid-biosynthetic genes

Ethylene decreases the content of endogenous abscisic acid (ABA) and increases the level of bioactive gibberellin A₁ (GA₁) in the submerged internodes of deepwater rice. During partial submergence, internodes of deepwater rice undergo rapid elongation as a result of ethylene accumulation in the internodal lacunae. In anin vitro experiment using stem sections from deepwater rice, treatment with 5 μL L^-1 ethylene promoted stem growth by up to 3.2-foId times over air treatment. Expression patterns were analyzed for genes that encode GA- and ABA-biosynthesis enzymes to determine any possible molecular basis for the changes observed in GA₁ and ABA contents as a result of ethylene action. Expression of theOsGA20ox2 andOsGA20ox4 genes, which encode GA 20-oxidase, and of theOsGA3ox2 gene, which encodes the enzyme that converts GA₂₀ to CA₁, was up-regulated, whereas that of three ABA-biosynthetic genes —OsNCED1, OsNCED2, andOsNCEDS-was down-regulated in the presence of ethylene. These results indicate that GA and ABA contribute equally to the submergence-or ethylene-induced stem elongation of deepwater rice via the coordinated and opposite regulation of biosynthesis.     

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.     

Growth promotion and inhibition of the Amazonian wild rice species Oryza grandiglumis to survive flooding

In Asian cultivated rice (Oryza sativa), distinct mechanisms to survive flooding are activated in two groups of varieties. Submergence-tolerant rice varieties possessing the SUBMERGENCE1A (SUB1A) gene display reduced growth during flash floods at the seedling stage and resume growth after the flood recedes, whereas deepwater rice varieties possessing the SNORKEL1 (SK1) and SNORKEL2 (SK2) genes display enhanced growth based on internodal elongation during prolonged submergence at the mature stage. In this study, we investigated the occurrence of these growth responses to submergence in the wild rice species Oryza grandiglumis, which is native to the Amazon floodplains. When subjected to gradual submergence, adult plants of O. grandiglumis accessions showed enhanced internodal elongation with rising water level and their growth response closely resembled that of deepwater varieties of O. sativa with high floating capacity. On the other hand, when subjected to complete submergence, seedlings of O. grandiglumis accessions displayed reduced shoot growth and resumed normal growth after desubmergence, similar to the response of submergence-tolerant varieties of O. sativa. Neither SUB1A nor the SK genes were detected in the O. grandiglumis accessions. These results indicate that the O. grandiglumis accessions are capable of adapting successfully to flooding by activating two contrasting mechanisms as the situation demands and that each mechanism of adaptation to flooding is not mediated by SUB1A or the SK genes.     

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.     

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.     

A comparison of the submergence response of deepwater and non-deepwater rice

Twelve cultivars of rice (Oryza sativa L.), representing deepwater, short-statured, and semidwarf types, were tested for their response to submergence. The magnitude of the response varied between cultivars; however, all cultivars responded to submergence by rapid growth once internodal elongation had started. Three of these cultivars were tested for elongation capacity at four ages. The deepwater rice was capable of rapid internodal elongation in response to submergence at 4 weeks of age. Growth of the short-statured and semidwarf cultivars was not stimulated by submergence until about 10 weeks of age. In air, the internodes of deepwater rice grew slower than did those of the short-statured and semidwarf cultivars. We also investigated the elongation response of stem sections of all 12 cultivars to an atmosphere containing 3% O₂, 6% CO₂, 91% N₂ (all by volume), and 1 microliter per liter ethylene. We found that the response of each of the non-deepwater cultivars was qualitatively and quantitatively similar to that of the deepwater rice.     

深水稻节间伸长生长的机制

淹水可促进深水稻节间快速伸长生长,其主要受内源赤霉素、乙烯、脱落酸等激素信号分子的调控。淹水能促进深水稻植株体内乙烯、赤霉素的生物合成、抑制脱落酸的生物合成,外源乙烯、赤霉素会加速深水稻节间伸长,而外源脱落酸抑制淹水节间的伸长,其中赤霉素是直接作用因子,乙烯能降低内源脱落酸水平、增加节间对赤霉素的敏感性;还与渗透调节、细胞壁组份如膨胀素等有关,淹水及赤霉素都大大增加了膨胀素基因的表达。并就深水稻的进一步研究进行了展望。     

深水稻节间伸长生长的机制

淹水可促进深水稻节间快速伸长生长,其主要受内源赤霉素、乙烯、脱落酸等激素信号分子的调控。淹水能促进深水稻植物株体内乙烯、赤霉素的生物合成、抑制脱落酸的生物合成,外源乙烯、赤霉素会加速深水稻节间伸长,而外源脱落酸抑制淹水节间的伸长,其中赤霉素是直接作用因子,乙烯能降低内源脱落酸水平、增加节地赤霉素的敏感性;还与渗透调节、细胞壁组份如膨胀素等有关,淹水及赤霉素都大大增加了膨胀素基因的表达。并就深水稻的     

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.     

Low levels of oxygen promote internodal elongation in floating rice independently of enhanced ethylene production

Submergence induces rapid elongation of internodes in floating rice(Oryza sativa L. cv. Habiganj Aman II). The initial signalfor such internodal elongation has been considered to be the reduced partialpressure of oxygen in submerged internodal cavities, which promotes theelongation of internodes through the enhancement of ethylene synthesis. Weexamined the relationship between low oxygen pressure and ethylene production inthe rapid elongation of floating rice internodes using ethylene biosynthesisinhibitors, aminooxyacetic acid (AOA) and CoCl₂. When floating ricestem segments were incubated in an atmosphere of low O₂, internodalelongation accelerated and ethylene production increased. However, in stemsegments treated with AOA or CoCl₂, low O₂ stillstimulated the elongation of internodes although the ethylene production by theinternodes was less than by those in control stem segments where internodalelongation was not promoted. These results indicate that low O₂ iscapable of causing rapid elongation of internodes of floating rice independentlyof enhanced production of ethylene. In addition to low O₂,submergence, ethylene and gibberellic acid each enhanced the production ofethylene by internodal tissues, suggesting that enhanced ethylene production isa common phenomenon accompanied by the acceleration of internodal elongation infloating rice.     

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.     

Interactions between ethylene, gibberellin and abscisic acid regulate emergence and growth rate of adventitious roots in deepwater rice

Growth of adventitious roots is induced in deepwater rice (Oryza sativa L.) when plants become submerged. Ethylene which accumulates in flooded plant parts is responsible for root growth induction. Gibberellin (GA) is ineffective on its own but acts in a synergistic manner together with ethylene to promote the number of penetrating roots and the growth rate of emerged roots. Studies with the GA biosynthesis inhibitor paclobutrazol revealed that root emergence was dependent on GA activity. Abscisic acid (ABA) acted as a competitive inhibitor of GA activity. Root growth rate on the other hand was dependent on GA concentration and ABA acted as a potent inhibitor possibly of GA but also of ethylene signaling. The results indicated that root emergence and elongation are distinct phases of adventitious root growth that are regulated through different networking between ethylene, GA and ABA signaling pathways. Adventitious root emergence must be coordinated with programmed death of epidermal cells which cover root primordia. Epidermal cell death is also controlled by ethylene, GA and ABA albeit with cell-type specific cross-talk. Different interactions between the same hormones may be a means to ensure proper timing of cell death and root emergence and to adjust the growth rate of emerged adventitious roots.     

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.     

Epidermal cell death in rice is regulated by ethylene, gibberellin, and abscisic acid

Programmed cell death (PCD) of epidermal cells that cover adventitious root primordia in deepwater rice (Oryza sativa) is induced by submergence. Early suicide of epidermal cells may prevent injury to the growing root that emerges under flooding conditions. Induction of PCD is dependent on ethylene signaling and is further promoted by gibberellin (GA). Ethylene and GA act in a synergistic manner, indicating converging signaling pathways. Treatment of plants with GA alone did not promote PCD. Treatment with the GA biosynthesis inhibitor paclobutrazol resulted in increased PCD in response to ethylene and GA presumably due to an increased sensitivity of epidermal cells to GA. Abscisic acid (ABA) was shown to efficiently delay ethylene-induced as well as GA-promoted cell death. The results point to ethylene signaling as a target of ABA inhibition of PCD. Accumulation of ethylene and GA and a decreased ABA level in the rice internode thus favor induction of epidermal cell death and ensure that PCD is initiated as an early response that precedes adventitious root growth.     

In vivo 1-aminocyclopropane-1-carboxylate synthase activity in internodes of deepwater rice : enhancement by submergence and low oxygen levels

Inasmuch as the activity of 1-aminocyclopropane-1-carboxylate (ACC) synthase cannot be measured in homogenates of deepwater rice internodes (Oryza sativa L.), we have employed an in vivo assay to determine the activity of this enzyme. This assay is based on the accumulation of ACC in tissue kept under N₂. Submergence of whole plants or stem sections containing the uppermost, developing internode enhances the in vivo activity of ACC synthase in the stem. This stimulation of in vivo ACC-synthase activity is especially pronounced in the region of the internode containing the intercalary meristem and the elongation zone above it. Enhancement of in vivo ACC-synthase activity is evident after 2 hours of submergence and shows a peak after 4 hours. Reduced levels of atmospheric O₂, which promote ethylene synthesis and growth in internodes of deepwater rice, also enhance the in vivo activity of ACC synthase. Our results are consistent with the hypothesis that induction of ACC-synthase activity at low partial O₂ pressures is among the first biochemical events leading to internodal growth in deepwater rice.     

A comparative molecular-physiological study of submergence response in lowland and deepwater rice

Survival of rice (Oryza sativa) upon an extreme rise of the water level depends on rapid stem elongation, which is mediated by ethylene. A genomic clone (OS-ACS5) encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, which catalyzes a regulatory step in ethylene biosynthesis, has been isolated from cv IR36, a lowland rice variety. Expression was induced upon short- and long-term submergence in cv IR36 and in cv Plai Ngam, a Thai deepwater rice variety. Under hypoxic conditions, abscisic acid and gibberellin had a reciprocal opposite effect on the activity of OS-ACS5. Gibberellin up-regulated and abscisic acid down-regulated OS-ACS5 mRNA accumulation. Growth experiments indicated that lowland rice responded to submergence with a burst of growth early on, but lacked the ability to sustain elongation growth. Sustained growth, characteristic for deepwater rice, was correlated with a prolonged induction of OS-ACS5. In addition, a more pronounced capacity to convert ACC to ethylene, a limited ACC conjugation, and a high level of endogenous gibberellin(20) were characteristic for the deepwater variety. An elevated level of OS-ACS5 messenger was found in cv IR36 plants treated with exogenous ACC. This observation was concomitant with an increase in the capacity of converting ACC to ethylene and in elongation growth, and resulted in prolonged survival. In conclusion, OS-ACS5 is involved in the rapid elongation growth of deepwater rice by contributing to the initial and long-term increase in ethylene levels. Our data also suggest that ACC limits survival of submerged lowland rice seedlings.     

Anatomical analysis of growth and developmental patterns in the internode of deepwater rice

Submergence of the stem induces rapid internodal elongation in deepwater rice (Oryza sativa L. cv. Habiganj Aman II). A comparative anatomical study of internodes isolated from airgrown and partially submerged rice plants was undertaken to localize and characterize regions of growth and differentiation in rice stems. Longitudinal sections were examined by light and scanning-electron microscopy. Based on cell-size analysis, three zones of internodal development were recognized: a zone of cell division and elongation at the base of the internode, designated the intercalary meristem (IM); a zone of cell elongation without concomitant cell division; and a zone of cell differentiation where neither cell division nor elongation occur. The primary effects of submergence on internodal development were a threefold increase in the number of cells per cell file resulting from a decrease in the cell-cycle time from 24 to 7 h within the IM; an expansion of the cell-elongation zone from 5 to 15 mm leading to a threefold greater final cell length; and a suppression of tissue differentiation as indicated by reduced chlorophyll content and a lack of secondary wall formation in xylem and cortical sclerenchyma. These data indicate that growth of deepwater-rice internoes involves a balance between elongation and differentiation of the stem. Submergence shifts this balance in favor of growth.Abbreviations GA gibberellin - IM intercalary meristem     

Effect of submergence on translocation,starch content and amylolytic activity in deep-water rice

Submergence induces rapid internodal elongation in deep-water rice (Oryza sativa L. cv. Habiganj Aman II). We investigated the metabolic activities which help to support such fast growth. Three days of submergence in water under continuous light led to the mobilization of 65% of the starch from those regions of rice internodes which had been formed prior to submergence. Disappearance of starch was accompanied by a 70-fold enhancement of amylolytic activity. Similar increases in amylolytic activity were detected in response to ethylene and gibberellic acid. Submergence also caused a 26-fold increase in the translocation of newly synthesized photosynthetic assimilates from the leaves to the internodes and younger regions of the culms. These physiological processes are likely to provide the metabolic energy required for internodal elongation in response to submergence.Abbreviation GA₃ gibberellic acid     

Submergence Enhances Expression of a Gene Encoding 1-Aminocyclopropane-1-Carboxylate Oxidase in Deepwater Rice

Partial submergence greatly stimulates internodal growth indeepwater rice (Oryza sativa L.). Previous work has shown thatthe effect of submergence is, at least in part, mediated byethylene, which accumulates in the air spaces of submerged internodes.To investigate the expression of the genes encoding ethylenebiosynthetic enzymes during accelerated growth of deepwaterrice, we cloned a 1-aminocyclopropane- 1-carboxylate (ACC) oxidasecDNA (OSACO1) from internodes of submerged plants and measuredthe activity of the enzyme in tissue extracts with an improvedassay. We found an increase in ACC oxidase mRNA levels and enzymeactivity after 4 to 24 h of submergence. Thus, it is likelythat ethylene biosynthesis in internodes of deepwater rice iscontrolled, at least in part, at the level of ACC oxidase. (Received January 6, 1996; Accepted April 6, 1996)