Induction of a polyubiquitin gene promoter by dehydration stresses in transformed rice cells

The expression of the maize polyubiquitin gene promoter UBI1 in rice cells has been used to study the involvement of ubiquitin in cell protection responses to dehydration caused by osmotic, saline or freezing stress. The effect of these stresses on UBI1 activity was investigated by the use of stably transformed rice calli (UBI1:GUS), as well as by transient expression experiments performed with cell lines with high or low tolerance to each type of stress. The theoretical analysis of the UBI1 promoter shows several putative stress-regulated boxes that could account for the stress-related UBI1 induction pattern described in this work. We suggest that the study of the differential UBI1 promoter-driven expression in rice cell lines with different level of tolerance to stress might be useful to elucidate complex signal transduction pathways in response to dehydration stresses in monocots.     

Uptake of an endocytic marker by rice cells: variations related to osmotic and saline stress

Saline and osmotic stress are the main abiotic factors limiting the productivity of rice and other crop plants. Although both coincide in generating water deficit and affect many aspects of plant growth and development similarly, some effects of salinity are distinctively related to the ionic component of salt stress. At the cellular level, dessication tolerance is largely dependent on the cell's ability for osmotic adjustment. Here, we have studied the effects of saline and osmotic stress on endocytosis by rice cells, to investigate the common and distinctive effects of saline-generated stress and osmotically generated stress, and the possible involvement of endocytosis in tolerance mechanisms. For this purpose, we have used rice cell lines with different levels of tolerance and biotinylated bovine serum albumin (bBSA) as an endocytic marker, which in our previous experiments has been shown to enter rice cells by a process with the characteristics of receptor-mediated endocytosis. Our results indicate that the pattern of uptake is common to both types of stress. Thus, when rice cells were subjected to saline or osmotic stress there was an initial dose-dependent inhibition of uptake. However, after more extended stress periods, there was an activation of uptake in the stressed cells. This late activation appears mainly related to the inhibition of growth commonly caused by the different stress agents used in this study. When using cell lines with different degrees of tolerance, the level of uptake activation varied as a function of the type of stress. Thus, under osmotic stress, a higher stress tolerance was directly related to a higher bBSA uptake, while the opposite occurred under saline stress. The possible role of endocytosis in the cellular responses to osmotic and saline stress is discussed.     

Glycine betaine involvement in freezing tolerance and water stress in Arabidopsis thaliana

Levels of endogenous glycine betaine in the leaves were measured in response to cold acclimation, water stress and exogenous ABA application in Arabidopsis thaliana. The endogenous glycine betaine level in the leaves increased sharply during cold acclimation treatment as plants gained freezing tolerance. When glycine betaine (10 mM) was applied exogenously to the plants as a foliar spray, the freezing tolerance increased from -3.1 to -4.5 degrees C. In addition, when ABA (1 mM) was applied exogenously, the endogenous glycine betaine level and the freezing tolerance in the leaves increased. However, the increase in the leaf glycine betaine level induced by ABA was only about half of that by the cold acclimation treatment. Furthermore, when plants were subjected to water stress (leaf water potential of approximately -1.6 MPa), the endogenous leaf glycine betaine level increased by about 18-fold over that in the control plants. Water stress lead to significant increase in the freezing tolerance, which was slightly less than that induced by the cold acclimation treatment. The results suggest that glycine betaine is involved in the induction of freezing tolerance in response to cold acclimation, ABA, and water stress in Arabidopsis plants.     

Differential effects of cold, osmotic stress and abscisic acid on polyamine accumulation in wheat

The effects of cold, osmotic stress and abscisic acid (ABA) on polyamine accumulation were compared in the moderately freezing-sensitive wheat (Triticum aestivum L.) variety Chinese Spring (CS) and in two derived chromosome 5A substitution lines, CS(T. spelta 5A) and CS(Cheyenne 5A), exhibiting lower and higher levels of freezing tolerance, respectively. When compared with the other treatments, putrescine (Put) and spermidine (Spd) levels were much greater after cold treatment, spermine (Spm) following polyethylene glycol-induced (PEG) osmotic stress and Spm and cadaverine (Cad) after ABA treatment. During 3-week cold stress, the Put concentration, first exhibited a transient increase and decrease, and then gradually increased. These alterations may be due to changes in the expression of genes encoding the enzymes of Put synthesis. The Put content was higher in the freezing-tolerant chromosome 5A substitution line than in the sensitive one after 3 weeks of cold. In contrast to cold, ABA and PEG induced a continuous decrease in the Spd level when applied for a period of 3 weeks. The Spm content, which increased after PEG and ABA addition, was twice as high as that of Put during ABA treatment at most sampling points, but this difference was lower in the case of PEG. The Cad level, induced to a great extent by ABA, was much lower when compared with that of the other polyamines. The present experiments indicate that cold, osmotic stress and ABA have different effects on polyamines, and that some of these changes are affected by chromosome 5A and correlate with the level of stress tolerance.     

Photoperiodic induction of dormancy and freezing tolerance in Betula pubescens. Involvement of ABA and dehydrins

In many woody plants photoperiod signals the initiation of dormancy and cold acclimation. The photoperiod-specific physiological and molecular mechanisms have remained uncharacterised. The role of abscisic acid (ABA) and dehydrins in photope-riod-induced dormancy and freezing tolerance was investigated in birch, Betula pubescens Ehrh. The experiments were designed to investigate if development of dormancy and freezing tolerance under long-day (LD) and short-day (SD) conditions could be affected by manipulation of the endogenous ABA content, and if accumulation of dehydrin-like proteins was correlated with SD and/or the water content of the buds. Experimentally, the internal ABA content was increased by ABA application and by water stress treatment under LD, and decreased by blocking the synthesis of ABA with fluridone under SD. Additionally, high humidity (95% RH) was applied to establish if accidental water stress was involved in SD. ABA content was monitored by gas chromatography-mass spectrometry with selective ion monitoring (SIM). Short days induced a transient increase in ABA content, which was absent in 95% RH, whereas fluridone treatment decreased ABA. Short days induced a typical pattern of bud desiccation and growth cessation regardless of the treatment, and improved freezing tolerance except in the fluridone treatment. ABA content of the buds was significantly increased after spraying ABA on leaves and after water stress, treatments that did not induce cessation of growth and dormancy, but improved freezing tolerance. In addition to several constitutively produced dehydrins, two SD-specific proteins of molecular masses 34 and 36 kDa were found. Photoperiod- and experimentally-induced alterations in ABA contents affected freezing tolerance but not cessation of growth and dormancy. Therefore, involvement of ABA in the photoperiodic control of cold acclimation is more direct than in growth cessation and dormancy. As the typical desiccation pattern of the buds was found in all SD plants, and was not directly related to ABA content or to freezing tolerance, this pattern characterises the onset of photo-period-induced growth cessation and dormancy. The results provide evidence for the existence of various constitutively and two photoperiod-induced dehydrins in buds of birch, and reveal characteristics of dormancy and freezing tolerance that may facilitate further investigations of photoperiodic control of growth in trees.     

Osmotic stress and water stress have opposite effects on putrescine and proline production in excised rice leaves

The effects of water stress and osmotic stress (sorbitol treatment) on the production of putrescine and proline in excised rice leaves were compared. Osmotic stress and water stress were found to affect differentially the levels of putrescine and proline in excised rice leaves. Putrescine accumulation is induced by osmotic stress, whereas proline accumulation is induced by water stress. The effects of ABA on the levels of proline and putrescine are similar to those of water stress, whereas the effects of jasmonic acid methyl ester (JA-Me) are similar to those of osmotic stress. Water stress results in an increase of endogenous ABA is excised rice leaves. However, neither osmotic stress nor JA-Me has effect on endogenous ABA levels in excised rice leaves. Of particular interest is the finding that proline levels increase when putrescine levels induced by osmotic stress or JA-Me are reduced by D-arginine and -methylornithine. L-arginine and L-ornithine applied exogenously also cause an increase in proline levels. It seems that L-arginine and L-ornithine are preferentially utilized as precursors for putrescine accumulation in excised rice leaves treated with osmotic stress and JA-Me, and for proline accumulation in excised rice leaves exposed to water stress and ABA.Abbreviations ABA abscisic acid - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - HPLC high performance chromatography - JA-Me jasmonic acid methyl ester - PVP poly-vinylpyrrolidone     

Alterations in Water Status,Endogenous Abscisic Acid Content,and Expression of rab18 Gene during the Development of Freezing Tolerance in Arabidopsis thaliana

Treatments as diverse as exposure to low temperature (LT), exogenous abscisic acid (ABA), or drought resulted in a 4 to 5[deg]C increase in freezing tolerance of the annual herbaceous plant Arabidopsis thaliana. To correlate the increase in freezing tolerance with the physiological changes that occur in response to these treatments, we studied the alterations in water status, endogenous ABA levels, and accumulation of rab18 (V. Lang and E.T. Palva [1992] Plant Mol Biol 20: 951-962) mRNA. Exposure to LT and exogenous ABA caused only a minor decline in total water potential ([psi]w), in contrast to a dramatic decrease in [psi]w during drought stress. Similarly, the endogenous ABA levels were only slightly and transiently increased in LT-treated plants in contrast to a massive increase in ABA levels in drought-stressed plants. The expression of the ABA-responsive rab18 gene was low during the LT treatment but could be induced to high levels by exogenous ABA and drought stress. Taken together, these results suggest that the moderate increases in freezing tolerance of A. thaliana might be achieved by different mechanisms. However, ABA-deficient and ABA-insensitive mutants of A. thaliana have impaired freezing tolerance, suggesting that ABA is, at least indirectly, required for the development of full freezing tolerance.     

Tomato tos1 mutation identifies a gene essential for osmotic tolerance and abscisic acid sensitivity

Osmotic stress severely limits plant growth and agricultural productivity. We have used mutagenesis to identify plant genes that are required for osmotic stress tolerance in tomato. As a result, we have isolated a novel mutant in tomato (tos1) caused by a single recessive nuclear mutation that is hypersensitive to general osmotic stress. Growth measurements demonstrated that the tos1 mutant is less sensitive to intracellular abscisic acid (ABA) and this decreased ABA sensitivity of tos1 is a basic cellular trait expressed by the mutant at all developmental stages analysed. It is not caused by a deficiency in the synthesis of ABA because the tos1 seedlings accumulated more ABA than the wild type (WT) after osmotic stress. In contrast, the tss2 tomato mutant, which is also hypersensitive to osmotic stress, is hypersensitive to exogenous ABA. Comparative analysis of tos1 and tss2 indicates that appropriate ABA perception and signalling is essential for osmotic tolerance.     

Drought tolerance, abscisic acid and electrolyte leakage in fast-and slow-growing black spruce (Picea mariana) progenies

To investigate the role that drought tolerance plays in growth, abscisic acid (ABA) accumulation and electrolyte leakage during water stress were compared in fast- and slow-growing black spruce ( Picea mariana [Mill.] B. S. P.) progenies. Changes in the ABA content of the needles were quantified using an indirect enzyme-linked immuno-sorbent assay validated by gas chromatography electron capture detection. Needle electrolyte leakage was estimated using a conductivity bridge. Seedlings were stressed using (1) osmotic stress, induced by a stepwise increase in concentrations of polyethylene glycol 3 350 (PEG) for ABA study and (2) air drying for electrolyte leakage study. Progenies did not differ in ABA levels under unstressed conditions, but progeny differences were observed under osmotic stress. Needle ABA content increased up to 500% under osmotic stress. Slow-growing black spruce progenies (25 and 46) accumulated more ABA under moderate (18% PEG), but not severe (25% PEG), osmotic stress. The slow-growing progenies also leaked more electrolytes under moderate to severe water stress and lost 50% electrolytes at a higher xylem tension, suggesting they suffered more injury and were less dehydration tolerant. Our previously-published results showed that slow-growing progenies lost their photosynthesis and stomatal conductance more quickly during osmotic stress and recovered more slowly after rehydration. Therefore, tolerance of dehydration leading to a maintenance of physiological integrity during drought stress could explain the fast growth rates of more vigorous black spruce progenies.     

Control of abscisic acid catabolism and abscisic acid homeostasis is important for reproductive stage stress tolerance in cereals

Drought stress at the reproductive stage causes pollen sterility and grain loss in wheat (Triticum aestivum). Drought stress induces abscisic acid (ABA) biosynthesis genes in anthers and ABA accumulation in spikes of drought-sensitive wheat varieties. In contrast, drought-tolerant wheat accumulates lower ABA levels, which correlates with lower ABA biosynthesis and higher ABA catabolic gene expression (ABA 8'-hydroxylase). Wheat TaABA8'OH1 deletion lines accumulate higher spike ABA levels and are more drought sensitive. ABA treatment of the spike mimics the effect of drought, causing high levels of sterility. ABA treatment represses the anther cell wall invertase gene TaIVR1, and drought-tolerant lines appeared to be more sensitive to the effect of ABA. Drought-induced sterility shows similarity to cold-induced sterility in rice (Oryza sativa). In cold-stressed rice, the rate of ABA accumulation was similar in cold-sensitive and cold-tolerant lines during the first 8 h of cold treatment, but in the tolerant line, ABA catabolism reduced ABA levels between 8 and 16 h of cold treatment. The ABA biosynthesis gene encoding 9-cis-epoxycarotenoid dioxygenase in anthers is mainly expressed in parenchyma cells surrounding the vascular bundle of the anther. Transgenic rice lines expressing the wheat TaABA8'OH1 gene under the control of the OsG6B tapetum-specific promoter resulted in reduced anther ABA levels under cold conditions. The transgenic lines showed that anther sink strength (OsINV4) was maintained under cold conditions and that this correlated with improved cold stress tolerance. Our data indicate that ABA and ABA 8'-hydroxylase play an important role in controlling anther ABA homeostasis and reproductive stage abiotic stress tolerance in cereals.     

水稻突变体M-20的耐盐生理特性

不同盐（NaCl）浓度的水培条件下,水稻耐盐突变体M－20幼苗生长及生存能力优于其原始品系77－170,膜透性测定表明盐胁迫对M－20质膜的损伤较小;同时,外界盐浓度小于100mmol／L时,M－20幼苗根、地上部的钠离子含量低于其原始品系。土培幼苗浇灌盐水（10mmol／L）时,M－20成熟及生长叶片的内源ABA水平增加时期晚于原始品系,增幅也小;而游离脯氨酸的增加时期虽晚,但增幅远高于原始品系。     

Levels of endogenous abscisic acid and indole-3-acetic acid influence shoot organogenesis in callus cultures of rice subjected to osmotic stress

Osmotic stress and endogenous hormone levels may have a role in shoot organogenesis, but a systematic study has not yet to investigate the links. We evaluated the changes of the endogenous indole-3-acetic acid (IAA) and abscisic acid (ABA) levels in rice (Oryza sativa L. cv. Tainan 5) callus during shoot organogenesis induced by exogenous plant growth regulator treatments or under osmotic stress. Non-regenerable callus showed low levels of endogenous ABA and IAA, with no fluctuation in level during the period evaluated. The addition of 100 μM ABA or 2 mM anthranilic acid (IAA precursor) into Murashige and Skoog basal induction medium containing 10 μM 2,4-D enhanced the regeneration frequency slightly, to 5 and 35%, respectively, and their total cellular ABA or IAA levels were increased significantly, correspondingly to the treatments. However, the regeneration frequency was greatly increased to 80% after treatment with 0.6 M sorbitol or 100 μM ABA and 2 mM anthranilic acid combined. Both treatments produced high levels of total cellular ABA and IAA at the callus stage, which was quickly decreased on the first day after transfer to regeneration medium. Thus, osmotic stress-induced simultaneous accumulation of endogenous ABA and IAA is involved in shoot regeneration in rice callus.