The role of ABA in freezing tolerance and cold acclimation in barley

The role of ABA in freezing resistance in nonacclimated and cold‐acclimated barley ( Hordeum vulgare L.) was studied. Eleven nonacclimated cultivars differed in their LT₅₀, ranging from −10.8 to −4.8°C. Sugars, free proline, soluble proteins and ABA were analyzed in nonacclimated cultivars and during cold acclimation of one cultivar. There was an inverse correlation between LT₅₀ and both ABA and sucrose contents. Exogenous ABA caused a decrease in the freezing point of leaf tissue in the cultivar with the lowest level of endogenous ABA, but not in the cultivar with the highest level, suggesting that ABA in the latter may be near the optimum endogenous level to induce freezing tolerance. Plants of cv. Aramir treated with ABA or allowed to acclimate to cold temperature increased their soluble sugar content to a similar level. The LT₅₀ of leaves of cold‐acclimated cv. Aramir decreased from −5.8 to −11.4°C, with biphasic kinetics, accumulating proline and soluble sugars with similar kinetics. The biphasic profile observed during cold acclimation could be a direct consequence of cryoprotectant accumulation kinetics. ABA and soluble protein accumulation showed a single step profile, associated mainly with the second phase of the LT₅₀ decrease. Thus, a significant increase in endogenous ABA is part of the response of barley to low temperature and may be required as a signal for the second phase of cold acclimation. Endogenous ABA contents in the nonacclimated state may determine constitutive freezing tolerance.     

Involvement of abscisic acid and ethylene in the responses of citrus seedlings to salt shock

The responses of salt‐sensitive citrus rootstocks to 200 m M NaCl were periodically determined on seedlings of citrange Carrizo ( Citrus sinensis [L.] Osbeck × Poncirus trifoliata [L.] Raf) during 30 days. The stressed seedlings adjusted osmotically, reduced stomatal conductance, increased proline content and ethylene production, and showed massive leaf abscission (92%). The salt shock also increased abscisic acid (ABA) and aminocyclopropane‐1‐carboxylic acid (ACC) in roots, xylem fluid and leaves, and in addition promoted Cl⁻ accumulation. The pattern of change of ABA, ACC and proline followed a two‐phase response: an initial transient increase (10‐12 days) overlapping with a gradual and continuous accumulation. This biphasic response appears to be compatible with the proposal that the transitory hormonal rises are induced by the osmotic component of salinity, whereas the Cl⁻ increase determines the subsequent accumulations. During the second phase, Cl⁻ levels correlated with abscission in leaves. Production of leaf ethylene was also concomitant with the increase in the abscission rate. Salt‐induced abscission was either reduced with CoCl₂ (52%) or inhibited with silver thiosulphate (14%). The results suggest that in salt‐stressed citrus, leaf abscission is induced by the chloride build‐up through a mechanism that stimulates leaf ACC synthesis and further conversion to ethylene.     

K+ status and ABA affect both exudation rate and hydraulic conductivity in sunflower roots

Twenty‐day‐old sunflower plants ( Helianthus annuus L. cv. Sun‐Gro 380) grown in nutrient solutions with different KCl levels were used to study the effects of K⁺ status of the root and of abcisic acid (ABA) on the exudation rate (J_v), the hydraulic conductivity of the root (L_p), the fluxes of exuded K⁺ and Na⁺ (J_K and J_Na), and the gradient of osmotic pressure between the xylem and the external medium. J_v and L_p increased in direct proportion to the K⁺ starvation of the root. Also addition of ABA (4 µ M ) at the onset of exudation in the external medium made J_v and L_p rise, and this effect also increased with the degree of K⁺ starvation. Similarly, K⁺ starvation and ABA promoted both the flux of exuded Na⁺ and the accumulation of Na⁺ in the root. We suggest that ABA acts as a regulating signal for the radial transport of water across the root, and that potassium may be an effector of this mechanism.     

Influence of exogenously supplied potassium and sodium salts on the abscisic acid-induced proline accumulation in barley leaf segments

A stimulation of the abscisic acid (ABA)-induced increase in proline was observed in leaf segments of barley ( Hordeum vulgare L. cv. Georgie) if K⁺ or Na⁺ were supplied in the external medium as salts of monovalent anions such as NO₃, Br, Cr and I, but not when sulphate or phosphate were used. To a lesser extent, the effect was evident also with RbCl, but it did not occur when chlorides of Li⁺. Cs⁺, NH₄⁺, Mg:⁺ and Ca²⁺ were used. Both KC1 and NaCl in the concentration range 2–100 m M influence the ABA-dependent proline accumulation to the same extent; the increase induced was about 100% at 10 m M , and reached a maximum between 60 and 100 m M. The effect is not due to the osmotic activity of the salts and does not seem to depend on changes in K⁺ and Na⁺ levels within the leaf tissue, but it is somehow linked to their external concentration. The existence of a specific interaction between ABA and K⁺ or Na⁺, possibly at the cell membrane level, is proposed.     

一氧化氮参与调节盐胁迫下脱落酸诱导的小麦幼苗叶片脯氨酸的累积

不同浓度(0.01～5.00mmol/L)的外源一氧化氮(NO)供体硝普钠(SNP)以浓度依赖性的性式诱导150mmol/LNaCl胁迫下小麦(Triticum aestivum L.cv.Yangmai 158)幼苗叶片脯氨酸的累积.其中0.1 mmol/L的SNP效果最明显,而结合采用NO清除剂c-PTIO和血红蛋白的处理均分别逆转了该效应.研究结果还发现:0.1 mmol/L SNP诱导的脯氨酸累积还可能有利于盐胁迫下小麦幼苗的保水性;0.1 mmol/L的SNP显著激活了内源ABA的合成,而结合血红蛋白的处理则证实,在外源ABA诱导脯氨酸累积的过程中NO可能作用于ABA信号分子的下游,但NO和ABA信号分子在此诱导反应中不存在累积效应.进一步研究脯氨酸合成和降解的酶促反应途径,发现外源NO处理前4天内可能主要是通过提高△'-吡咯啉-5-羧酸合成酶(P5CS)的活性来促进脯氨酸的合成,以后直至第8天主要是通过抑制脯氨酸脱氢酶(ProDH)的活性来抑制脯氨酸的降解;ABA对于P5CS和ProDH活性的调节能力弱于NO.此外,Ca2 在NO诱导的盐胁迫下小麦叶片脯氨酸累积的信号分子途径中起重要的介导作用.     

Effects of abscisic acid and proline on adaptation of tobacco callus cultures to salinity and osmotic shock

Tobacco callus ( Nicotina tabacum cv. Badischer Geudertheimer) took up sorbitol rapidly and without a lag period from media with up to 0.7 M of the polyol. Accumulation of proline was greatly enhanced under these conditions and was proportional to the absorbed sorbitol, while the viability of the callus cultures was quite low after a few hours of incubation. Under moderate conditions (0.1 M sorbitol) as well as under severe osmotic shock (0.7 M sorbitol), the cells adapted by adjusting the sorbitol/proline ratio to ca 3. NaCl (0.1 M ) had the same effect as sorbitol (0.7 M ) on the survival rate, but only slightly affected proline synthesis in the first hours of incubation. Addition of 10⁷ or 10 ⁵ M abscisic acid (ABA) did not increase the proline content, but 10 ⁷ M ABA delayed the deleterious effect of NaCl and improved the state of the cells. No influence of abscisic acid during the incubation with sorbitol could be detected. Two different strategies for the adjustment of tobacco callus to salinity and sorbitol are suggested: Non-ionic stress is controlled by the accumulation of proline, whereas ABA could be involved in the adaptation to ionic stress.     

Long-term effects of abscisic acid on K+ transport in young wheat plants of different K+ status

The effects of abscisic acid (ABA) on growth, uptake and translocation of potassium ions, K⁺,Mg²⁺-ATPase activity and transpiration were investigated in young wheat ( Triticum aestivum L. cv. Martonvásári-8) plants grown at different K⁺ supplies. Long-term treatment with ABA (10 μ M ) reduced growth in high-K⁺ plants, but had less effect under low-K⁺ conditions. K⁺(⁸⁶Rb) uptake was inhibited by about 70 and 40% in low- and high-K⁺ plants, respectively. The stimulation by K⁺ of the Mg²⁺-ATPase activity in the root microsomal fraction was lost with ABA treatment. It is suggested that the inhibitory effect of ABA on K⁺ uptake may be related to this effects on the K⁺,Mg²⁺-ATPase. Translocation of K⁺ to the shoot was inhibited in low-K⁺ plants only, and it was not affected in high-K⁺ plants. In parallel to this, ABA treatment reduced transpiration by about 50% in low-K⁺ plants, whereas a much smaller effect was seen in high-K⁺ plants. These observations suggest that the regulation by ABA of the stomatal movements is strongly counteracted by high-K⁺ status.     

Proline accumulation inside symbiosomes of faba bean nodules under salt stress

A two‐week salt treatment (NaCl, 100 m M ) induced a 50% inhibition of acetylene reduction activity (ARA) of faba bean ( Vicia faba L. var. minor cv. Soravi) nodules, associated with a large increase in the nodule pool of amino acids. The concentration of proline in the different nodule compartments was determined after calculating their respective volumes from their areas on electron micrographs. The proline concentration exhibited a large increase, especially in the cytosol where its amount was 8‐fold enhanced under salt stress, whereas the low proline content of bacteroids was less affected. Increase of proline concentration in faba bean nodules subjected to salt stress was correlated with an enhancement of the cytosolic Δ¹‐pyrroline‐5‐carboxylate synthetase (EC 2.7.2.11 + EC 1.2.1.41; P5CS) activity. Experiments with purified symbiosome preparations showed that the greatest proline content occurred in the peribacteroid space (PBS), where proline was the most abundant amino acid, with a concentration reaching 15.3 m M under salt stress. Proline accumulation in the PBS resulted both from a diffusive transport from the host cell to the symbiosomes through the peribacteroid membrane (PBM) and from the very low rate of uptake by faba bean bacteroids. This accumulation could be partly responsible for the 1.7‐fold enlargement of the symbiosome volume observed in salt‐stressed nodules. In incubations of bacteroids, isolated from salt‐stressed or unstressed plants and supplied with O₂ by purified oxyleghemoglobin, addition of proline stimulated neither O₂ consumption nor ARA. These results were consistent with proline playing a role as osmoticum, rather than energy source for bacteroid N₂ fixation in amide‐exporting legumes such as faba bean.     

Development and accumulation of ABA in fluridone-treated and drought-stressed Vicia faba plants under different light conditions

The effects of fluridone on guard cell morphology, chloroplast ultrastructure and accumulation of drought stress-induced abscisic acid (ABA) were studied in Vicia faba L. plants grown under different light conditions. Drought stress was induced by allowing the leaves to lose 12% of their fresh weight. The appearance of defective and undeveloped stomata, and chloroplasts with a destroyed thylakoid membrane system was found in fluridone-treated plants grown at a photosynthetic photon flux (PPF) of 600 μmol m^-2 s^-1. Plants grown at a PPF of 40 μmol m^-2 s^-1 had diminished levels of ABA after imposition of dehydration. Fluridone treatment reduced the level of ABA in both unstressed and dehydrated leaves. Accumulation of ABA in the control plants was considerably reduced when they were exposed to dark periods of 24, 48 and 72 h just before imposition of the stress. Twenty-four hours after the dark treatment dehydration of the leaves resulted in a 3-fold decrease in the level of stress-induced ABA, and 72 h after dark treatment the amount of stress-induced ABA approximated the prestressed values. Fluridone-treated plants failed to accumulate ABA under water stress. In addition to functionally active chloroplasts, well-developed and functional stomata are required for drought stress to elicit a rise in ABA.     

Ethylene-induced putrescine accumulation modulates K+ partitioning between roots and shoots in barley seedlings

We investigated the cause and effect relationships among ethylene, polyamines, and K⁺ in barley ( Hordeum vulgare L. cv. Amagi) seedlings. Application of 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene, to the growth medium caused a decrease in K⁺ concentration in roots and an increase in shoots. Addition of ACC induced putrescine accumulation in roots, while spermidine and spermine levels remained unchanged. Exogenous supply of putrescine led to putrescine accumulation and reduced K⁺ concentration. Application of Co²⁺, an inhibitor of ethylene biosynthesis, together with ACC, inhibited putrescine accumulation with a decrease in K⁺ concentration in roots. ACC-treated roots showed K⁺ uptake capacity equivalent to that of control roots, implying that the majority of K⁺ is translocated to shoots. These results suggest that ethylene regulates K⁺ partitioning between roots and shoots through the level of accumulation of putrescine in barley seedlings.     

Mannitol metabolism in cultured plant cells

Non-structural storage carbohydrates were measured in 9-day-old barley ( Hordeum vulgare L. cv. Brant) primary leaves. Accumulation rates of starch, sucrose and total non-structural carbohydrates (TNC) were approximately linear when measured between 2- and 12-h of light. Progressively higher TNC accumulation rates were observed at higher irradiance levels (i.e., comparing 250, 550 and 1050 ·mol m⁻² s⁻¹). Synthesis of a low-molecular-weight fructan also was enhanced by high irradiances. Low irradiance treatments decreased leaf sucrose levels and there was a corresponding increase in the lag period preceding starch synthesis in the light. Increased starch accumulation rates were usually observed when sucrose concentrations were high. These and other results suggested that cytosolic sucrose concentrations affected starch metabolism in the chloroplast. However, sucrose accumulation rates increased and starch storage decreased when barley seedlings were transferred from 20 to 10°C during the light period. Lowering the night temperature from 20 to 10°C for a single dark period 8-days after planting increased the TNC content of barley primary leaves at the beginning of day nine. In this experiment, TNC accumulation rates of treated and untreated leaves were similar. Changes in the accumulation rate of TNC were usually observed within 2- to 4-h after barley seedlings were exposed to altered environmental conditions. Monitoring rapid changes in leaf carbohydrate levels is a sensitive method for assessing the effects of environmental treatments on photosynthetic metabolism.     

Salt stress in cultured rice cells: effects of proline and abscisic acid

Abstract. The presence of 1 and 10 mol m⁻³ proline in media containing 100 and 200 mol m⁻³ of NaCl, had little effect on the growth of salt-adapted callus of rice. However, in such callus proline accumulation was stimulated by 10 mol m⁻³ proline in the presence of 100 mol m⁻³ NaCl. On the other hand, with 100 mol m⁻³ NaCl, both 1 and 10 mol m⁻³ proline significantly increased both the growth and proline content of salt-unadapted callus. On replacing NaCl with KCl (100 and 200 mol m⁻³), growth of saltadapted as well as unadapted callus was inhibited, but the presence of 10 mol m⁻³ proline had an ameliorating effect. Abscisic acid (ABA) supressed the growth of both salt-adapted and unadapted callus of rice in the absence of salt stress. ABA inhibited the growth of callus adapted to and grown in 100 and 200 mol m⁻³ of NaCl or when it was replaced by equimolar concentrations of KCl. Growth of 100 mol m⁻³ NaCl adapted cells was inhibited when they were transferred to a medium containing 200 mol m⁻³ of NaCl, but in the presence of ABA it was stimulated. ABA increased the growth of unadapted cells when subjected to different salts. Also, ABA accelerated the adaptation of cells exposed to salt but not to water deficits imposed by nonionic solutes.     

Effects of weak acids on proline accumulation in barley leaves: a comparison between abscisic acid and isobutyric acid

Abstract. When isobutyric acid (IBA) or abscisic acid (ABA) are supplied to leaf sections a similar rapid and marked decrease in the intracellular pH is observed. This acidification is accompanied by an increase in proline level which is about the same for both 3 mol m⁻³ IBA and 1 mol m⁻³ ABA treatments.
Fusicoccin (FC), known to act at the proton pump level, almost completely suppresses the ABA-induced acidification of the cell sap, whereas it only partially counteracts the acidifying effect of IBA, in particular during short periods of treatment. This effect of FC is paralleled by a similar inhibition of the induced proline accumulation: in fact, FC completely suppresses the ABA-induced increase in proline during short treatment periods, whereas it is only effective in inhibiting the IBA-induced proline accumulation after long treatment periods.
These data seem to suggest that the ABA- and IBA-induced changes in proline level might be mediated by changes in the intracellular pH.     

Role of sodium in the ABA‐mediated long‐term growth response of bean to salt stress

Long‐term salt effects on plant growth have often been related to direct ion toxicity due to the accumulation of high ion concentrations in plant tissue. This work examines the relative importance of endogenous ABA, as well as Na⁺ and Cl⁻ toxicity, in the inhibition of leaf growth and photosynthesis, in bean plants grown at 1, 25, 50 and 75 m M NaCl until the fruit‐bearing stage. All salt‐treated plants showed very high leaf Cl⁻ concentrations, with little difference between plants exposed to 50 or 75 m M NaCl. The 25 and 50 mM salt‐treated plants were able to successfully exclude Na⁺ from their leaves, and only suffered an initial decline in the rate of leaf growth. Plants exposed to 75 m M NaCl showed an increase in Na⁺ leaf concentrations with an accompanying decrease in growth and photosynthesis as salt exposure progressed. A high correlation was found between leaf Na⁺ and leaf growth. Leaf ABA significantly increased with salt supply, and was highly correlated with both leaf Na⁺ and leaf growth. Our results suggest that in bean plants under long‐term salt stress, leaf ABA may participate in the regulation of leaf growth, and leaf Na⁺ would be at least partly responsible for increased ABA levels.     

CO2, light and temperature influence senescence and protein degradation in wheat leaf segments

Effects of environmental conditions influencing photosynthesis and photorespiration on senescence and net protein degradation were investigated in segments from the first leaf of young wheat ( Triticum aestivum L. cv. Arina) plants. The segments were floated on H₂O at 25, 30 or 35°C in continuous light (PAR: 50 or 150 µmol m⁻² s⁻¹) in ambient air and in CO₂‐depleted air. Stromal enzymes, including phosphoglycolate phosphatase, glutamine synthetase, ferredoxin‐dependent glutamate synthase, phosphoribulokinase, and the peroxisomal enzyme, glycolate oxidase, were detected by SDS‐PAGE followed by immunoblotting with specific antibodies. In general, the net degradation of proteins and chlorophylls was delayed in CO₂‐depleted air. However, little effect of CO₂ on protein degradation was observed at 25°C under the lower level of irradiance. The senescence retardation by the removal of CO₂ was most pronounced at 30°C and at the higher irradiance. The stromal enzymes declined in a coordinated manner. Immunoreactive fragments from the degraded polypeptides were in most cases not detectable. However, an insolubilized fragment of glycolate oxidase accumulated in vivo, especially at 25°C in the presence of CO₂. Detection of this fragment was minimal after incubation at 30°C and completely absent on blots from segments kept at 35°C. In CO₂‐depleted air, the fragment was only weakly detectable after incubation at 25°C. The results from these investigations indicate that environmental conditions that influence photosynthesis may interfere with senescence and protein catabolism in wheat leaves.     

Light-Dark Changes in Proline Content of Barley Leaves under Salt Stress

Proline accumulation in leaves of barley (Hordeum vulgare L. cv. Alfa) seedlings treated with 150 mM NaCl was promoted in the light and suppressed in the dark. The light/dark changes of proline content was enhanced with each 12 h light/12 h dark cycle and the proline content increased steadily. Root and shoot concentrations of Na⁺ and Cl^– in salt treated plants increased about 10 to 25 times as compared to the control. The content of these ions and the content of malondialdehyde were higher in the shoot of seedlings exposed to salt stress for 4 d in the light in comparison with the seedlings exposed to NaCl for 4 d in darkness. Light stimulated both ions and proline accumulation in the leaves and has no effect in the roots. Oxygen uptake was higher in the seedlings kept 4 d in the light which have higher endogenous free proline content. Chlorophyll fluorescence measurements showed that the photochemical activity of PS 2 slightly decreased as a result of salt stress and was not influenced by light regimes during plant growth.     

Apoplastic and membrane‐associated Ca2+ in leaves and roots as affected by boron deficiency

A combination of fluorescein‐isothiocyanate (FITC), coumarin‐benzothiazol (BTC), and chlorotetracycline (CTC) fluorescence was used to simultaneously monitor apoplastic pH, apoplastic free Ca²⁺, and plasma membrane‐bound Ca²⁺. As early boron deficiency reactions supposedly include alterations of plasma membrane‐bound transport processes besides rapid effects on cell wall physical properties, the corresponding changes were followed in leaves and roots of Vicia faba L. cv. Troy.
Boron deficiency did not alter the apoplastic pH, but it reduced plasma membrane‐bound Ca²⁺ in roots at 4 h and leaves at 3 days after starting the deficiency treatment. The decrease in plasma membrane‐bound Ca²⁺ coincided with an increase in apoplastic free Ca²⁺ and K⁺, and occurred before the first visible symptoms were noticed.
It is proposed that less Ca²⁺ is bound to the plasma membrane due to a reduction of specific Ca²⁺‐binding sites (borate esters with vic ‐diols or polyhydroxy‐carboxylates) before plasma membrane integrity deteriorates.     

Membrane and ion transport properties in cereals under acidic and alkaline stress

The effects of pH on the growth and the K⁺ (⁸⁶Rb) uptake and K⁺ content of excised rice ( Oryza sativa L. cv. Dunghan Shali) and wheat ( Triticum aestivum L. cv. GK Szeged) roots were investigated. Rice roots responded to H⁺ stress with an increased K⁺(⁸⁶Rb) influx and a decreased K⁺ content, suggesting an increased exchange between the cytoplasmic K⁺ pool and the external medium. Under the same experimental conditions wheat did not show any anomalous K⁺(⁸⁶Rb) influx. Growth of both rice and wheat was relatively insensitive to pH between 4 to 10.     

Effect of excess Cu on the photosynthetic apparatus of runner bean leaves treated at two different growth stages

Variations in water binding strength, water relations and the accumulation of solutes during water stress of three durum wheat ( Triticum durum Desf.) cultivars are reported and discussed. Water binding strength was determined by constructing adsorption isotherms at 5 and 20°C and by calculating the differential enthalpy (ΔH) after van't Hoff.
Reducing sugars, proline, K⁺ and Cl⁻ were the major contributors to osmotic adjustment. Solutes, such as quaternary ammonium compounds, and non-reducing sugars contributed to osmotic potential at full turgor, but did not increase in proportion to water stress. Genotypic differences have been observed for K⁺ accumulation capability, the water-stressed leaves of cv. Capeiti 8 showing the largest increase. The same cultivar demonstrated the most negative ΔH values, indicative of strongly bound water, and the highest integrated enthalpy (ΔH_inf) values for leaf moisture below 0.1 g H₂O per g dry weight, i.e. in the isotherm region where water was presumably chemisorbed to the charged groups of macromolecules. The accumulation of ions (Cl⁻, K⁺) and proline was concurrent with an increase in the binding-strength of tightly and weakly bound water, respectively.