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.     

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.     

Enhancement of transpiration by ethylene is responsible for absence of internodal elongation in floating rice at low humidity

Internodal elongation in floating rice (Oryza sativa) is known to be enhanced by treatment with ethylene or gibberellic acid (GA3) at high relative humidity (RH). However, ethylene-induced internodal elongation is inhibited at low RH, while GA3-induced internodal elongation is hardly affected by humidity. We examined the effects of ethylene and GA3 on the rate of transpiration in stem segments incubated at 30% or 100% RH. Ethylene promoted the transpiration of stem segments at 30% RH, but not at 100% RH, while GA3 had little effect on transpiration at either 30% or 100% RH. We propose that the absence of ethylene-induced internodal elongation at low RH is due, at least in part, to ethylene-induced transpiration.     

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.     

Gibberellin-induced elongation and osmoregulation in internodes of floating rice

Internodal elongation in floating rice ( Oryza sativa L. cv. Habiganj Aman II) is known to be enhanced by treatment with ethylene or gibberellic acid (GA₃) at high relative humidity (RH). However, ethylene-induced internodal elongation is inhibited at low RH. while GA₃-induced internodal elongation is hardly affected by humidity. We examined the possible involvement of osmoregulation in the stimulation by GA₃ of the elongation of internodes at low RH. Submergence and treatment with ethylene or GA3₃ at 100% RH increased the osmotic potential in internodes of excised stem segments, while GA₃ at 20% RH maintained the osmotic potential at a low level. In internodes of stem segments that had been treated with GA₃ at 20% RH, the activity of invertase and the level of soluble sugars were almost 2- and 1.5-fold higher, respectively, than those in internodes that had been treated with GA₃ at 100% RH. These results indicate that one of the possible mechanisms by which GA₃ promotes elongation of internodes at low RH involves the osmoregulation that is achieved by promotion of the synthesis of invertase.     

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.     

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.     

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     

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.     

Exogenous Jasmonic and Abscisic Acids Act Differentially in Elongating Tissues from Oat Stem Segments

Segments can be cut from the peducular-1 internode of oat (Avena sativa L.) shoots so as to contain the graviresponsive, auxin-sensitive leaf sheath pulvinus, and the gibberellin-sensitive internodal tissue. These two growth-capable tissues were used to study the effects and interactions of jasmonic acid (JA) and abscisic acid (ABA) in regulating cell elongation. When supplied alone at physiologic concentrations (10⁻⁵, 10⁻⁴ m), JA promoted growth and cell wall synthesis in the internodal tissue, whereas by itself, ABA inhibited internodal elongation and even inhibited JA-promoted growth. When gibberellic acid (GA₃) was used to stimulate internodal elongation, JA and ABA caused similar levels of inhibition and, at certain concentrations, were synergistic. Inhibition by ABA was initiated several hours earlier than inhibition by JA, and only the ABA effect could be partially overcome by 10⁻³ m aminoethoxyvinylglycine. Both JA and ABA inhibited elongation of pulvinar tissue that was induced to grow by gravistimulus or auxin, although here JA was more potent than ABA at equimolar concentrations. When 10⁻⁵ m fusicoccin was used as a general nonphysiologic growth stimulus, JA had no effect on the internode but inhibited the pulvinus, whereas ABA had no effect on the pulvinus but inhibited the internode. These results provide strong physiologic evidence that JA and ABA act by different mechanisms in the regulation of elongation, at least in this representative grass. Received May 28, 1996; accepted November 7, 1996     

Expansins and Internodal Growth of Deepwater Rice

The distribution and activity of the cell wall-loosening protein expansin is correlated with internodal growth in deepwater rice (Oryza sativa L.). Acid-induced extension of native cell walls and reconstituted extension of boiled cell walls were confined to the growing region of the internode, i.e. to the intercalary meristem (IM) and the elongation zone. Immunolocalization by tissue printing and immunoblot analysis, using antibody against cucumber expansin 29 as a probe, confirmed that rice expansin occurred primarily in the IM and elongation zone. Rice expansin was localized mainly around the vascular bundles at the base of the IM and along the inner epidermal cell layer surrounding the internodal cavity. Submergence greatly promoted the growth of rice internodes, and cell walls of submerged internodes extended much more in response to acidification than did the cell walls of air-grown internodes. Susceptibility of cell walls to added expansin was also increased in submerged internodes, and analysis by immunoblotting showed that cell walls of submerged internodes contained more expansin than did cell walls of air-grown internodes. Based on these data, we propose that expansin is involved in mediating rapid internodal elongation in submerged deepwater rice internodes.     

Levels of {beta}-Glucan and Lignin in Elongating Internodes of Deepwater Rice

Partial submergence or treatment with either ethylene or gibberellicacid (GA₃ induces rapid growth in deepwater rice (Oryza sativaL.). We correlated the synthesis of two cell wall componentswith two phases of internodal elongation, namely (13,14)-ß-glucanformation with cell elongation and lignification with differentiationof the secondary cell wall and cessation of growth. The contentof ß-glucan was highest in the zone of cell elongationin internodes of air-grown plants and plants that were inducedto grow rapidly by submergence. In the intercalary meristemand in the differentiation zone of the internode, ß-glucanlevels were ca. 70% lower than in the zone of cell elongation.The outer cell layers, enriched in epidermis, contained moreß-glucan in submerged, rapidly growing internodesthan in air-grown, control internodes. The ß-glucancontent of the inner, parenchymal tissue was unaffected or slightlylowered by submergence. The epidermis appears to be the growth-limitingstructure of rapidly growing rice internodes. We hypothesizethat elevated levels of ß-glucan contribute to elongationgrowth by increasing the extensibility of the cell wall. Lignificationwas monitored by measuring the content of lignin and the activitiesof two enzymes of the lignin biosynthetic pathway, coniferylalcohol dehydrogenase (CAD) and phenylalanine ammonia-lyase(PAL), in growing and non-growing regions of the internode.Using submerged whole plants and GA₃-treated excised stem segments,we showed that lignin content and CAD activity were up to sixfoldlower in newly formed internodal tissue of rapidly growing ricethan in slowly growing tissue. No differences were observedin parts of the internode that had been formed prior to inductionof growth. PAL activity was reduced throughout the internodeof submerged plants. We conclude that lignification is one ofthe processes that is suppressed to permit rapid growth. ¹ This work was supported by the National Science Foundationthrough grants No. DCB-8718873 and DCB-9103747 and by the Departmentof Energy through grant No. DE-FGO2-90ER20021. M.S. was therecipient of a fellowship from the Max Kade Foundation.     

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     

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     

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。     

Response of Amaranthus retroflexus L. seeds to gibberellic acid, ethylene and abscisic acid depending on duration of stratification and burial

Freshly harvested, dormant seeds of Amaranthus retroflexus were unable to germinate at 25 and 35 °C. To release their dormancy at the above temperatures, the seeds were stratified at a constant temperature (4 °C) under laboratory conditions or at fluctuating temperatures in soil or by outdoor burial in soil. Fully dormant, or seeds stratified or buried (2006/2007 and 2007/2008) for various periods were treated with exogenous gibberellic acid (GA₃), ethephon and abscisic acid (ABA). Likewise, the effects of these regulators, applied during stratification, on seed germination were determined. The results indicate that A. retroflexus seed dormancy can be released either by stratification or by autumn–winter burial. The effect of GA₃ and ethylene, liberated from ethephon, applied after various periods of stratification or during stratification, depends on dormancy level. GA₃ did not affect or only slightly stimulated the germination of non-stratified, fully dormant seeds at 25 and 35 °C respectively. Ethylene increased germination at both temperatures. Seed response to GA₃ and ethylene at 25 °C was increased when dormancy was partially removed by stratification at constant or fluctuating temperatures or autumn–winter burial. The response to GA₃ and ethylene increased with increasing time of stratification. The presence of GA₃ and ethephon during stratification may stimulate germination at 35 °C. Thus, both GA₃ and ethylene can partially substitute the requirement for stratification or autumn–winter burial. Both hormones may also stimulate germination of secondary dormant seeds, exhumed in September. The response to ABA decreased in parallel with an increasing time of stratification and burial up to May 2007 or March 2008. Endogenous GA_n, ethylene and ABA may be involved in the control of dormancy state and germination of A. retroflexus. It is possible that releasing dormancy by stratification or partial burial is associated with changes in ABA/GA and ethylene balance and/or sensitivity to these hormones.     

Effects of abscisic acid on the orientation and cold stability of cortical microtubules in epicotyl cells of the dwarf pea

Summary The effects of abscisic acid (ABA) on the orientation and cold stability of cortical microtubules (MTs) in epidermal cells of epicotyls of the dwarf pea,Pisum sativum L. cv. Little Marvel, were examined by immunofluorescence microscopy. The effect of ABA on the elongation of epicotyls and on the orientation of cortical MTs was opposite to that of gibberellin A₃ (GA₃). Treatment with ABA, which reduced the promotion of epicotyl elongation by GA₃, eliminated the GA₃-induced predominance of transverse MTs and resulted in a predominance of longitudinal MTs. The effect of ABA on the cold stability of cortical MTs was also opposite to that of GA₃. ABA increased the cold stability of MTs, while GA₃ decreased it. The predominance of longitudinal MTs brought about by ABA may have some relationship to ABA-induced inhibition of the elongation of the epicotyl. ABA may alter membrane proteins to stabilize cortical MTs and induce cold hardiness of plants.Abbreviations ABA abscisic acid - DMSO dimethylsulfoxide - FITC fluorescein isothiocyanate - GA₃ gibberellin A₃ - MT microtubule - PBS phosphate-buffered saline Dedicated to the memory of Professor Oswald Kiermayer     

Floral initiation in sorghum hastened by gibberellic acid and far-red light

Combinations of far-red light (FR) (4 min) and gibberellic acid (GA₃), given at the beginning of a daily 12-h dark period in a growth room, were used to study floral induction in four maturity genotypes of the milo group of sorghum (Sorghum bicolor (L.) Moench). The 12-h dark period without GA₃ application or FR induced flowering in only the early genotype; FR hastened initiation in the early genotype, while GA₃ hastened floral initiation in the two intermidiate-flowering genotypes. GA₃ and FR together had a strong synergistic effect, hastening floral initiation by 30 to more than 80 d in the early and intermediate genotypes. Red light (R) did not hasten flowering; FR preceded by R gave the same effect as FR alone. GA₃ promoted stem elongation equally whether floral initiation occurred or not; thus, its effect on stem elongation was independent of floral initiation. The capacity of GA₃ to induce flowering in sorghum, a short-day plant, seems to be enhanced by phytochrome being in the P_R form at the beginning of the night when GA₃ was applied.Abbreviations FR far-red light - GA(s) gibberellin(s) - GA₃ gibberellic acid - R red light     

Changes in expansin activity and cell wall susceptibility to expansin action during cessation of internodal elongation in floating rice

We investigated the involvement of expansin action in determining the growth rate of internodes of floating rice (Oryza sativa L.). Floating rice stem segments in which rapid internodal elongation had been induced by submergence for 2 days were exposed to air or kept in submergence for 2 more days. Both treatments reduced the elongation rate of the internodes, and the degree of reduction was much greater in air-exposed stem segments than in continually submerged segments. These rates of internodal elongation were correlated with acid-induced cell wall extensibility and cell wall susceptibility to expansins in the cell elongation zone of the internodes, but not with extractable expansin activity. These results suggest that the reduced growth rate of internodes must be due, at least in part, to the decrease in acid-induced cell wall extensibility, which can be modulated through changes in the cell wall susceptibility to expansins rather than through expansin activity. Analysis of the cell wall composition of the internodes showed that the cellulosic and noncellulosic polysaccharide contents increased in response to exposure to air, but they remained almost constant under continued submergence although the cell wall susceptibility to expansins gradually declined even under continued submergence. The content of xylose in noncellulosic neutral sugars in the cell walls of internodes was closely and negatively correlated with changes in the susceptibility of the walls to expansins. These results suggest that the deposition of xylose-rich polysaccharides into the cell walls may be related to a decrease in acid-induced cell wall extensibility in floating rice internodes through the modulation of cell wall susceptibility to expansins.