Ethylene Responses in Rice Roots and Coleoptiles Are Differentially Regulated by a Carotenoid Isomerase-Mediated Abscisic Acid Pathway

Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice.     

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.     

Phytochrome Action in Oryza sativa L: IV. Red and Far Red Reversible Effect on the Production of Ethylene in Excised Coleoptiles

Excised apical segments of etiolated rice (Oryza sativa L.) coleoptiles produced ethylene. Increasing the number of cut sites per coleoptile increased the rate of ethylene formation. Ethylene produced by an etiolated-intact seedling in the dark was about a half of that by the excised coleoptile segment. Red light of low energy as well as of continuous irradiation inhibited the production of ethylene. The inhibition by a low energy dose of red light was partly relieved, if the red light was followed immediately by a small dose of far red light. The effect of red and far red light was repeatedly reversible, indicating that ethylene production was regulated by a phytochrome system. If the exposure to far red light was preceded by a period of darkness, this photoreversibility disappeared; 50% of the initial reversibility was lost within 5 hours. Applied ethylene (10 microliters per liter) significantly promoted the growth of intact coleoptiles of either totally etiolated or red light-treated seedlings, but had no effect on the excised apical segment of coleoptile.     

Ethylene-Enhanced Transport of Uranine, a Fluorescent Dye, in Rice-Seedling Explants in Relation to Ethylene-Stimulated Coleoptile Growth

Ethylene enhanced the transport of uranine, a fluorescent dye,through the coleoptiles of rice (Oryza sativa L. cv. Sasanishiki)seedlings explants after a 2-h lag period. The uranine transportincreased with increasing concentration of ethylene, reachinga maximum at 1 µ1.liter^–1 ethylene. It decreasedwhen some metabolic inhibitors, 2,4-dinitrophenol, carbonylcyanide 3-chlorophenylhydrazone and N-ethylmaleimide, were appliedto the scutella. Mechanical rupture or heating at the vascular-bundlesides of the coleoptiles also interfered with the ethylene-enhancedtransport of uranine. The wounding and removal of the scutellasimilarly blocked uranine transport. Moreover, strong fluorescenceof uranine was observed on the phloem regions of two vascularbundles of a coleoptile under a fluorescence microscope. Itis suggested that the ethylene-enhanced transport of uraninein the coleoptile of rice-seedling explants occurs through thephloem. On the other hand, the removal of the endosperm or therupture of the vascular bundles inhibited the ethylene-stimulatedelongation of the rice coleoptiles. Moreover, ethylene increaseddry weight of the shoots. These results suggest that the ethylene-stimulatedelongation of rice coleoptiles was supported partly by the ethylene-enhancedtranslocation of materials from the endosperms to the coleoptiles. (Received September 6, 1984; Accepted November 15, 1984)     

Ethylene and the growth of rice seedlings

Etiolated whole rice seedlings enclosed in sealed vials produced ethylene at a rate of 0.9 picomole per hour per seedling. When 2-centimeter-long shoots were subdivided into 5-millimeter-long sections, the sections containing the tip of the shoot evolved 37% of the total ethylene with the remaining 63% being produced along a gradient decreasing to the base of the shoot. The tip of the coleoptile also had the highest level of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid and of the ethylene-forming enzyme activity. Ethylene is one of the factors controlling coleoptile elongation. Decapitation of the seedling reduced ethylene evolution to one-third its original level and inhibited coleoptile growth. In short-term experiments, the growth rate of decapitated seedlings was restored to almost that of intact seedlings by application of ethylene at a concentration of 10 microliters per liter. Apart from ethylene, O₂ also participates in the control of coleoptile growth. When rice seedlings were grown in a gas mixture of N₂ and O₂, the length of the coleoptiles reached a maximum at a concentration of 2.5% O₂. Lower and higher concentrations of O₂ reduced coleoptile growth. The effect of exogenous ethylene on coleoptile growth was also O₂ dependent.     

ABA Inducible Rice Protein Phosphatase 2C Confers ABA Insensitivity and Abiotic Stress Tolerance in Arabidopsis

Arabidopsis PP2C belonging to group A have been extensively worked out and known to negatively regulate ABA signaling. However, rice (Oryza sativa) orthologs of Arabidopsis group A PP2C are scarcely characterized functionally. We have identified a group A PP2C from rice (OsPP108), which is highly inducible under ABA, salt and drought stresses and localized predominantly in the nucleus. Genetic analysis revealed that Arabidopsis plants overexpressing OsPP108 are highly insensitive to ABA and tolerant to high salt and mannitol stresses during seed germination, root growth and overall seedling growth. At adult stage, OsPP108 overexpression leads to high tolerance to salt, mannitol and drought stresses with far better physiological parameters such as water loss, fresh weight, chlorophyll content and photosynthetic potential (Fv/Fm) in transgenic Arabidopsis plants. Expression profile of various stress marker genes in OsPP108 overexpressing plants revealed interplay of ABA dependent and independent pathway for abiotic stress tolerance. Overall, this study has identified a potential rice group A PP2C, which regulates ABA signaling negatively and abiotic stress signaling positively. Transgenic rice plants overexpressing this gene might provide an answer to the problem of low crop yield and productivity during adverse environmental conditions.     

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.     

Preferential Induction of Alcohol Dehydrogenase in Coleoptiles of Rice Seedlings Germinated in Submergence Condition

Difference in the growth response to submergence between coleoptiles and roots of rice (Oryza sativa L.) was investigated in 9-d-old rice seedlings. The coleoptile length in the submergence condition was much greater than that in aerobic condition, whereas the root length in the submergence condition was less than that in the aerobic condition. Alcohol dehydrogenase (ADH) activity in the coleoptiles in the submergence condition was much greater than that in the aerobic condition, but ADH activity in the roots in the submergence condition increased slightly. These results suggest that the preferential ADH induction in rice seedlings may contribute to the difference in the growth response between the coleoptiles and roots under low oxygen conditions.     

Cooperation of Ethylene and Auxin in the Growth Regulation of Rice Coleoptile Segments

Elongation of coleoptile segments, having or not having a tip,excised from rice (Oryza sativa L. cv. Sasanishiki) seedlingswas promoted by exogenous ethylene above 0.3 µl l^–1as well as by IAA above 0.1 µM. Ethylene production ofdecapitated segments was stimulated by IAA above 1.0µM,and this was strongly inhibited by 1.0 µM AVG. AVG inhibitedthe IAA-stimulated elongation of the decapitated segment witha 4 h lag period, and this was completely recovered by ethyleneapplied at the concentration of 0.03 µl l^–1, whichhad no effect on elongation without exogenous IAA. The effectsof IAA and ethylene on elongation were additive. These factsshow that ethylene produced in response to IAA promotes ricecoleoptile elongation in concert with IAA, probably by prolongingthe possible duration of the IAA-stimulated elongation, butthat they act independently of each other. Moreover, AVG stronglyinhibited the endogenous growth of coleoptile segments withtips and this effect was nullified by the exogenous applicationof 0.03 µl l^–1 ethylene. These data imply that theelongation of intact rice coleoptiles may be regulated cooperativelyby endogenous ethylene and auxin in the same manner as foundin the IAA-stimulated elongation of the decapitated coleoptilesegments. Key words: oryza sativa, Ethylene, Auxin, Coleoptile growth     

Abscisic acid is a negative regulator of root gravitropism in Arabidopsis thaliana

The plant hormone abscisic acid (ABA) plays a role in root gravitropism and has led to an intense debate over whether ABA acts similar to auxin by translating the gravitational signal into directional root growth. While tremendous advances have been made in the past two decades in establishing the role of auxin in root gravitropism, little progress has been made in characterizing the role of ABA in this response. In fact, roots of plants that have undetectable levels of ABA and that display a normal gravitropic response have raised some serious doubts about whether ABA plays any role in root gravitropism. Here, we show strong evidence that ABA plays a role opposite to that of auxin and that it is a negative regulator of the gravitropic response of Arabidopsis roots.     

Indole-3-acetic acid and rice coleoptile elongation under anoxia

To investigate the presence of a possible synergistic effect of IAA and anaerobiosis on rice coleoptile elongation, excised coleoptiles grown in aerobic and anaerobic conditions were tested and compared with intact seedling aerial parts for response to exogenous IAA and for levels of endogenous IAA. Excised coleoptiles were fed with³H-IAA to study aerobic and anaerobic IAA metabolism. Our results can be summarized as follows. (1) IAA and anaerobiosis have no synergistic effect on rice coleoptile elongation. (2) This behavior is due not to an inhibition of IAA uptake but probably to a reduced and different IAA metabolism in coleoptile grown in the absence of oxygen. (3) In anaerobic rice coleoptiles, the conversion to inactive conjugate (IAA-Asp) could be adopted as means of detoxification in the case of abnormally high and unutilized IAA levels. (4) The increase in IAA level found in coleoptiles of intact seedlings during anaerobic treatment could be due, as in the roots, to a translocation from the endosperm, in which the hormone is contained in a great quantity.     

Regulation of growth in rice seedlings

Etiolated rice seedlings (Oryza sativa L.) exhibited marked morphological differences when grown in sealed containers or in containers through which air was passed continuously. Enhancement of coleoptile and mesocotyl growth and inhibition of leaf and root growth in the sealed containers (“enclosure syndrome”) were accompanied by accumulation of CO₂ and C₂H₄ in and depletion of O₂ from the atmosphere. Ethylene (1 μl 1^?1), high levels of CO₂, and reduced levels of O₂ contributed equally to the increase in coleoptile and mesocotyl growth. The effect of enclosure could be mimicked by passing a gas mixture of 3% O₂, 82% N₂, 15% CO₂ (all v/v), and 1 μl l^?1) C₂H₄ through the vials containing the etiolated seedlings. The effects of high CO₂ and low O₂ concentrations were not mediated through increased C₂H₄ production. The enclosure syndrome was also observed in rice seedlings grown under water either in darkness or in light. The length of the rice coleoptile was positively correlated with the depth of planting in water-saturated vermiculite. The length of coleoptiles of wheat, barley, and oats was not affected by the depth of planting. In rice, the length of coleoptile was determined by the levels of O₂, CO₂, and ethylene, rather than by light. This regulatory mechanism allows rice seedlings to grow out of shallow water in which the concentration of O₂ is limiting.     

Gradients of polyamines and their biosynthetic enzymes in coleoptiles and roots of corn

The distribution of diamines, polyamines, and their biosynthetic enzymes arginine decarboxylase and ornithine decarboxylase in roots and coleoptiles of corn (Zea mays var Golden Cross Bantam) seedlings have been determined. Putrescine content, expressed on either a fresh weight or protein basis, increases from the tip to the base in both roots and coleoptiles. In roots, this gradient is paralleled by an activity gradient of arginine and ornithine decarboxylases. Spermidine is distributed equally along the length of coleoptiles; in roots, this is true only on a protein basis. Free spermine is detectable only in the root tip, but a bound form is present throughout the root and coleoptile. The results are compared with gradients in protein and DNA content and discussed in relation to the possible cellular roles of polyamines.     

Polyamines in Rice Seedlings under Oxygen-Deficit Stress

Incubation of 3-d-old seedlings of Oryza sativa L. cv Arborio under anaerobic conditions, leads to a large increase in the titer of free putrescine while aerobic incubation causes a slight decrease. After 2 days, the putrescine level is about 2.5 times greater without oxygen than in air. The rice coleoptile also accumulates a large amount of bound putrescine and, to a lesser extent, spermidine and spermine (mainly as acid-soluble conjugates). Accumulation of conjugates in the roots is severely inhibited by the anaerobic treatment. Feeding experiments with labeled amino acids showed that anoxia stimulates the release of ¹⁴CO₂ from tissues fed with [¹⁴C]arginine and that arginine is the precursor in putrescine biosynthesis. After 2 d of anoxia, the activity of arginine decarboxylase was 42% and 89% greater in coleoptile and root, respectively, than in the aerobic condition. The causes of the differences in polyamine metabolism in anoxic coleoptiles and roots are discussed.     

Root Growth of Arabidopsis thaliana Is Regulated by Ethylene and Abscisic Acid Signaling Interaction in Response to HrpNEa, a Bacterial Protein of Harpin Group

HrpN_Ea is a harpin protein from Erwinia amylovora, a bacterial pathogen that causes fire blight in rosaceous plants. Treating plants with HrpN_Ea stimulates ethylene and abscisic acid (ABA) to induce plant growth and drought tolerance, respectively. Herein, we report that both growth hormones cooperate to mediate the role of HrpN_Ea in promoting root growth of Arabidopsis thaliana seedlings. Root growth is promoted coordinately with elevation in levels of ABA and ethylene subsequent to soaking of germinating seeds of wild-type (WT) Arabidopsis in a solution of HrpN_Ea. However, these responses are arrested by inhibiting WT roots from synthesizing ethylene as well as sensing of ABA and ethylene. The effects of HrpN_Ea on roots are also nullified in ethylene-insensitive etr1-1 and ein5-1 mutants and in the ABA-insensitive mutant abi2-1 of Arabidopsis. These results provide evidence for presence of a relationship between root growth enhancement and signaling by ABA and ethylene in response to HrpN_Ea. Nevertheless, when HrpN_Ea is applied to leaves, ethylene signaling is active in the absence of ABA signaling to promote plant growth. This suggests the presence of a different signaling mechanism in leaves from that in roots. X. Ren and F. Liu contributed equally to this study and are regarded as joint first authors     

The influence of plant growth regulators and storage on root induction and growth in <Emphasis Type="Italic">Pelargonium zonale</Emphasis> cuttings

The effects of post harvest application of ethylene, abscisic acid (ABA), indole-3-butyric acid (IBA) treatments or dark storage on root induction and continued growth of regenerated roots in Pelargonium cuttings were investigated using hydroponics in the greenhouse. Ethylene markedly increased rooting percentage in ‘Greco’ and ‘Surfing’, reduced the number of roots per cutting in ‘Surfing’ and had no effect on the total root lengths in the two cultivars. Ethylene treatment reduced fresh root mass in ‘Surfing’, increased dry root mass and reduced root water content in both cultivars. ABA (50 μM) enhanced rooting percentage in ‘Greco’, reduced the number of roots per cutting, reduced total root lengths and fresh root mass in both cultivars. ABA increased dry root mass and reduced root water content in ‘Surfing’ but this effect was not apparent in ‘Greco’. Storing cuttings in the dark for 4 days had no effect on rooting percentage and number of roots per cutting in ‘Greco’ and ‘Surfing’. However, dark storage reduced total root lengths in ‘Surfing’ and reduced fresh root mass in ‘Greco’. Dark storage had no effect on dry root mass and water content in both cultivars. Applying 4 μl l⁻¹ IBA in the rooting solution induced maximum (100%) root induction in ‘Surfing’. However, IBA reduced the number of roots per cutting in ‘Greco’, reduced total root lengths and fresh root mass in the two cultivars. IBA treatment profoundly increased and reduced dry root mass and root water content, respectively, in ‘Greco’ and ‘Surfing’. The enhanced root induction observed after IBA and ABA applications could be ascribed to their influence on ethylene biosynthesis, since ethylene treatment increased rooting percentage in both cultivars. However, high ABA (100 μM) and IBA (12 μl l⁻¹) levels or dark storage reduced the ability of induced roots to continue growth. We attribute our results to plant stress-response mechanism and ethylene appears to play an important role in the process of root initiation and root growth in Pelargonium cuttings.     

Effects of o(2) concentration on rice seedlings

The ability of rice, wheat, and oat seedlings to germinate and grow as the O₂ concentration was lowered to zero was compared. The germination of rice was completely unaffected by O₂ supply, whereas that of oats and wheat was strongly retarded at levels below 5% O₂. In contrast to the coleoptiles of oats and wheat and to roots of all three species where growth was progressively diminished as the O₂ concentration was lowered, that of the rice coleoptile was progressively increased. However, the dry weight and content of protein, sugars, and cellulose were all depressed in the rice coleoptile in anoxia, and the levels of several respiratory enzymes, particularly those of mitochondria, were also much lower than those of the coleoptiles grown in air. In 1% O₂, the growth of the rice coleoptile was similar to that in air. The effect of ethanol concentration on germination and growth of rice was measured. Coleoptile growth was reduced when the ethanol concentration exceeded 40 millimolarity, and root growth was somewhat more sensitive. Coleoptiles of all three species grown in air were transferred to N₂, and ethanol accumulation was measured over 24 hours. The rate of ethanol accumulation in oats was close to that in rice, and in all three species the amounts of ethanol lost to the surrounding medium were those expected from simple diffusion from the tissue. The ability of the rice coleoptile to grow in anoxia is apparently not due to a particularly low rate of ethanol formation or to unusual ethanol tolerance. Any explanation of the success of rice in anoxia must encompass the much lower rate of ATP synthesis than that in air and account for the biochemical deficiencies of the coleoptile.