Salt-sensitive Rice Cultivar Research Articles

Long term exogenous putrescine application improves grain yield of a salt-sensitive rice cultivar exposed to NaCl

In order to determine the impact of polyamines on the yield-related parameters of rice (Oryza sativa L.) exposed to NaCl, the plants belonging to a salt-sensitive cultivar I Kong Pao were maintained from the young seedling stage until harvest on nutrient solutions containing 0 or 30 mM NaCl in the presence or absence of 10 μM putrescine (Put), 10 μM spermidine (Spd) or 10 μM spermine (Spm). Exogenous Put and to a lesser extent exogenous Spd improved growth and yield of salt-treated plants in relation to an increase in K+/Na+ ratio of shoots and roots as compared to plants exposed to NaCl in the absence of exogenous polyamines. Exogenous Put also improved the net CO2 assimilation, at least partly as a consequence of an increase in the stomatal conductance. Yield increase of salt-treated plants exposed to Put was related to an improvement of floral morphogenesis leading to a higher number of fertile tillers per plant and a higher number of spikelets per panicle. Putrescine also improved the pollen viability in salt-treated plants, allowing a higher seed set and thus a higher grain yield per plant. Although polyamines accumulated in the shoots to some extent in response to exogenous application, neither Put nor Spd accumulated in the seeds. In contrast, Spm did not afford any protection of salt-treated plants but was translocated to the seeds during maturation. Seeds with a high internal Spm concentration exhibited delayed germination in the presence of NaCl. These data are discussed in relation to the implication of polyamine in the metabolism and physiology of salt-treated plants.

An ABRE-binding factor, OSBZ8, is highly expressed in salt tolerant cultivars than in salt sensitive cultivars of indica rice

BackgroundThe bZIP class Abscisic acid Responsive Element (ABRE)-binding factor, OSBZ8 (38.5 kD) has been considered to regulate ABA-mediated transcription in the suspension cultured cells of japonica rice. Still, nothing is known about the expression of OSBZ8 at protein level in vegetative tissue of salt sensitive and salt tolerant rice plants. In our previous study, Electrophoretic Mobility Shift Assay (EMSA) of [32P]ABRE-DNA and nuclear extracts prepared from the lamina of Pokkali rice plants has detected the presence of an ABRE-binding factor. Northern analysis has also detected salinity stress induced accumulation of transcripts for bZIP class of factor. Therefore, OSBZ8 was considered to play an important role in the regulation of transcription in the vegetative tissue of rice. The aim of this study is to find out whether OSBZ8 has any role in regulating the NaCl-stress induced gene expression in vegetative tissue and whether the expression of OSBZ8 factor directly correlates with the stress tolerance of different varieties of indica type rice.ResultsNorthern analysis of total RNA from roots and lamina of salt-sensitive M-I-48 and salt-tolerant Nonabokra, when probed with the N-terminal unique region of OSBZ8 (OSBZ8p, without the highly conserved basic region), a transcript of 1.3 kb hybridized and its level was much higher in tolerant cultivar. EMSA with Em1a, the strongest ABA Responsive Element till reported from the upstream of EmBP1, and the nuclear extracts from laminar tissue of untreated and salt-treated seedlings of three salt sensitive, one moderately sensitive and two salt tolerant indica rice cultivars showed specific binding of nuclear factor to ABRE element. Intensity of binding was low and inducible in salt sensitive rice cultivars while high and constitutive in salt tolerant cultivars. EMSA with 300 bp 5'upstream region of Rab16A gene, a well known salt stress and ABA-inducible gene of rice, showed formation of two complexes, again very weak in salt sensitive and strong in salt tolerant rice cultivar.ConclusionThe bZIP factor OSBZ8 was found to be present in the ABRE-DNA: protein complex as shown by the supershift of the complex by the purified antiserum raised against OSBZ8p. Treatment of the seedlings with NaCl was found to enhance the complex formation, suggesting the regulation of OSBZ8 gene at both transcriptional and post-translational steps. Comparative EMSA with different varieties of rice suggests a positive correlation with the expression pattern of OSBZ8 and salt tolerance in rice cultivars.

Exogenous Putrescine Reduces Sodium and Chloride Accumulation in NaCl-Treated Calli of the Salt-Sensitive Rice Cultivar I Kong Pao

In order to gain information on the putative involvement of polyamines (PAs) in the response of rice cells to salinity, mature embryo-derived calli issued from the salt-sensitive cultivar I Kong Pao were exposed for 3 months to the simultaneous presence of NaCl (0, 150 and 300 mM) and exogenous polyamines (putrescine (Put): 1 and 10 mM; spermidine (Spd): 1 and 10 mM; spermine (Spm): 1 mM). Callus growth, endogenous PAs, Na+, K+ and Cl− concentrations were quantified and analysed in relation to cell viability based on 2,3,5-triphenytetrazolium chloride (TTC) reduction. All exogenous PAs were efficiently absorbed from the external medium. Exogenous Put 1 mM clearly stimulated growth of salt-stressed calli in relation to a decrease in both Na+ and Cl− accumulation. In contrast, Spd 10 mM and Spm 1 mM exacerbated the deleterious impact of NaCl on callus growth and induced a decrease in K+ concentration. While Put helped in the maintenance of cell viability, Spd 10 mM and Spm 1 mM decreased cell viability, mainly in relation to an inhibition of the alternative respiratory pathway. It is proposed that Put may assume positive functions in salt stress resistance in rice.

Do exogenous polyamines have an impact on the response of a salt-sensitive rice cultivar to NaCl?

In order to analyze the putative impact of polyamines (PAs) on the plant response to salt, seedlings from the salt-sensitive rice cultivar I Kong Pao (IKP) were exposed for 5, 12 and 19 days to 0, 50 or 100 mM NaCl in the absence, or in the presence of exogenous PAs (putrescine (Put), spermidine (Spd) or spermine (Spm) 1mM) or inhibitors of PA synthesis (methylglyoxalbis-guanyl hydrazone (MGBG) 1mM, cyclohexylammonium (CHA) 5mM and D-arginine (D-Arg) 5mM). The addition of PAs in nutritive solution reduced plant growth in the absence of NaCl and did not afford protection in the presence of salt. PA-treated plants exhibited a higher K+/Na+ ratio in the shoots, suggesting an improved discrimination among monovalent cations at the root level, especially at the sites of xylem loading. The diamine Put induced a decrease in the shoot water content in the presence of NaCl, while Spd and Spm had no effects on the plant water status. In contrast to Spd, Spm was efficiently translocated to the shoots. Both PAs (Spd and Spm) induced a decrease in cell membrane stability as suggested by a strong increase in malondialdehyde content of PA-treated plants exposed to NaCl. These results are discussed in relation to the putative functions of PAs in stressed plant metabolism.

Regulation of Ammonium Accumulation during Salt Stress in Rice (Oryza sativa L.) Seedlings

Metabolic processes related to ammonium release and assimilation were investigated in a salt-sensitive rice (Oryza sativa L.) cultivar Anapurna. Ammonium content of the 3rd leaves increased 3-4 times when seedlings were treated with 100 mM NaCl for 6 days under both growth chamber light condition and in darkness (nonphotorespiration). An in vitro experiment revealed strong inhibition of protein synthesis as an effect of NaCl on the incorporation of 14C-leucine into protein. Exposure to salt stress slightly increased leaf proteolytic activity. The increase of proteolytic activity and decrease of protein synthesis, which directly causes accumulation of free amino acids, might lower the need for ammonium incorporation to form amino acids and indirectly cause the excessive accumulation of ammonium. No significant changes in the assimilatory activities of glutamine synthetase (GS; EC 6.3.1.2) and ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) were found under salt stress. Salt treatment changed the balance of the direction of glutamate dehydrogenase (NAD(H); EC 1.4.1.2); the aminating (NADH-GDH) activity increased while deaminating (NAD-GDH) activity decreased. We conclude that the accumulation of ammonium under salt stress was not due to inhibition of assimilatory activity of GS/GOGAT cycle or aminating GDH. Since these enzymes require supply of C-skeleton in the form of 2-oxoglutarate, reductant and energy to function, the reduction of photosynthetic capacity and the decrease of 2-oxoglutarate might be responsible for the excess accumulation of ammonium in salt-stressed seedlings.

Spermidine treatment to rice seedlings recovers salinity stress-induced damage of plasma membrane and PM-bound H +-ATPase in salt-tolerant and salt-sensitive rice cultivars

Due to their polycationic nature, Spermidine (Spd 3+) and Spermine (Spm 4+) are known to interact with polyanionic compounds, e.g. negatively charged head group of phospholipid membrane components, thereby stabilizing salinity stress-induced damage of plasma membrane (PM). But to what extent polyamine-mediated restoration of activities of PM-bound enzymes occurs and differs within salt-sensitive and salt-tolerant rice cultivars is totally unknown. Therefore, PM was isolated from the roots of 3-day-old rice seedlings from two salt-tolerant (Nonabokra and Pokkali) and two salt-sensitive (M-1-48 and IR8) cultivars treated with none (control) or with NaCl (150 mM, 16 h) alone or with Spd (1 mM, 16 h). Vanadium sensitive but K + stimulated H +-ATPase activity from equal amount of PM was measured by estimating released Pi. Results showed that nine-fold higher level of H +-ATPase (100% vanadium sensitive) was detected from PM of Nonabokra roots in comparison to M-1-48 roots. Salinity stress alone to the seedlings significantly reduces the activity of PM-bound H +-ATPase. The activity of H +-ATPase was restored to some extent in the roots treated with NaCl stress in presence of 1 mM Spd. Analysis of PM-bound polyamine from untreated control roots showed only Putrescine from M-1-48 and IR8 cultivars, whereas roots of salt-tolerant plants, Nonabokra and Pokkali, have only Spermidine and Spermine. PM-bound H +-ATPase activity of control and treated plants, when measured by NADH oxidation (coupled reaction), 2.5–3.0-fold higher activity was detected from salt-tolerant cultivars. Salinity stress to the plants severely inhibits H +-ATPase activity and Spermidine co-treatment significantly recovers its activity in all four cultivars. Western Blot with equal amount of 5% SDS extracted protein from roots when analyzed by the polyclonal antibody raised against H +-ATPase (PM-bound) of Arabidopsis thaliana showed NaCl stress-induced decrease and Spermidine-induced recovery of 100 kDa polypeptide (known MW of 100 kDa H +-ATPase from rice). These results clearly demonstrate for the first time that the deficit of salt-sensitive rice cultivars, e.g. high accumulation of Na +, loss of K + ion, salinity stress-induced sharp inhibition of PM-bound H +-ATPase activity, could be overcome by supplying Spermidine exogenously.

Effects of 24-epibrassinolide on seed germination, seedling growth, lipid peroxidation, proline content and antioxidative system of rice (Oryza sativa L.) under salinity stress

The effects of 24-epibrassinolide (24-epiBL) on seedling growth, antioxidative system, lipid peroxidation, proline and soluble protein content were investigated in seedlings of the salt-sensitive rice cultivar IR-28. Seedling growth of rice plants was improved by 24-epiBL treatment under salt stress conditions. When seedlings treated with 24-epiBL were subjected to 120 mM NaCl stress, the activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and glutathione reductase (EC 1.6.4.2) did not show significant difference, whereas the activity of ascorbate peroxidase (EC 1.11.1.11) significantly increased. Increased activity of peroxidase (EC 1.11.1.7) under NaCl stress showed remarkable decrease in the 24-epiBL+NaCl-applied group. Lipid peroxidation level significantly increased under salt stress but decreased with 24-epiBL application revealing that less oxidative damage occurred in this group (24-epiBL+NaCl). In addition, increased proline content in the NaCl-applied group was decreased by 24-epiBL application in the 24-epiBL+NaCl-applied group. Soluble protein content was increased by 24-epiBL application even under NaCl stress, being also higher than control conditions (no 24-epiBL or NaCl treatment). 24-epiBL treatment considerably alleviated oxidative damage that occurred under NaCl-stressed conditions and improved seedling growth in part under salt stress in sensitive IR-28 seedlings.

Variation in Growth and Ion Uptake in Salt Tolerant and Sensitive Rice Cultivars under NaCl Salinity

Variation in Growth and Ion Uptake in Salt Tolerant and Sensitive Rice Cultivars under NaCl Salinity

Calcium and Salt‐Tolerance of Rice

The impact of calcium (Ca) supply on the growth and yield of rice was studied in nutrient and soil culture experiments. In solution culture experiment, the effect of external‐Ca supply (0.5, 1.0, 1.5, 2.0, 3.0, 4.0 mM Ca) on the growth and shoot composition of two rice cultivars, varying in salt‐tolerance, in the presence and absence of 100 mol m− 3 NaCl was measured in a four week trial. In soil culture experiment, Ca (as gypsum; CaSO4 · 2H2O) was applied at the rates of 0, 10, 25, 50 and 100% of gypsum requirement (GR) of the saline‐sodic soil (after determining its GR). A positive, improving and stimulative effect of Ca was noted in growth characteristics (shoot and root weight and tillering capacity) in both the cultivars at 1.0 to 1.5 mM Ca; above this concentration, Ca had inhibitory effect. Although increasing supply of Ca tended to decrease sodium (Na) and chloride (Cl) concentration in the shoots of both rice cultivars, shoot‐concentrations of Na and Cl were much lower in NIAB‐6 (salt tolerant) than IR 1561 (salt sensitive). An increase in potassium(K):Na ratio, an indication of salt tolerance, was observed at 1.0 mM Ca and above in both rice cultivars under saline environment. Result, confirmed in salt affected soils of two different types (saline and saline sodic), showed an improvement in the paddy yield of both salt tolerant and salt sensitive rice cultivars due to Ca application as gypsum at the rate of 25% of GR of soil. Excessive rates of Ca either in solution culture or soil were detrimental for plant growth and paddy yield. The ameliorative effect of Ca was because of increased Ca:Na ratio of the growth medium.

Isolation and expression analysis of salt stress-associated ESTs from contrasting rice cultivars using a PCR-based subtraction method.

Salt stress adversely affects the growth of rice plants. To understand the molecular basis of salt-stress response, four subtracted cDNA libraries were constructed employing specific NaCl-stressed tissues from salt-tolerant (CSR 27 and Pokkali) and salt-sensitive (Pusa basmati 1) rice cultivars. An efficient PCR-based cDNA subtraction method was employed for the isolation of the salt-stress responsive cDNA clones. In all, 1,266 cDNA clones were isolated in the course of this study, out of which 85 clones were end-sequenced. Database search of the sequenced clones showed that 22 clones were homologous to genes that have earlier been implicated in stress response, 34 clones were novel with respect to their function and six clones showed no homology to sequences in any of the public database. Northern analysis showed that the transcript expression pattern of selected clones was variable amongst the cultivars tested with respect to stress-regulation.

Induction of Oxidative Stress in Roots of Oryza sativa L. in Response to Salt Stress

With the imposition of salt stress (0.5 to 3 % NaCl or CaCl2) a decrease in germination rate and accumulation of proline was observed in the root tissue. Both NaCl and CaCl2 solutions induced an increase in the total peroxide content and lipid peroxidation and decrease in catalase, guaiacol peroxidase and superoxide dismutase activities in root tissues suggesting an oxidative stress in the salt sensitive rice cultivar.

Introduction of a Na+/H+ antiporter gene from Atriplex gmelini confers salt tolerance to rice

We engineered a salt-sensitive rice cultivar (Oryza sativa cv. Kinuhikari) to express a vacuolar-type Na+/H+ antiporter gene from a halophytic plant, Atriplex gmelini (AgNHX1). The activity of the vacuolar-type Na+/H+ antiporter in the transgenic rice plants was eight-fold higher than that in wild-type rice plants. Salt tolerance assays followed by non-stress treatments showed that the transgenic plants overexpressing AgNHX1 could survive under conditions of 300 mM NaCl for 3 days while the wild-type rice plants could not. These results indicate that overexpression of the Na+/H+ antiporter gene in rice plants significantly improves their salt tolerance.

Crosses between cultivars and tissue culture-selected plants for salt resistance improvement in rice, Oryza sativa

Several R-2 somaclonal families were derived from plants regenerated from a salt-resistant callus of the salt-sensitive rice cultivar 'I Kong Pao' (IKP). The family R-2-1-23, in the presence of NaCl exhibited higher yield performances than the initial cultivar. This improvement in salinity resistance, however, was not transmitted to following generations, despite a higher number of spikelets per plant, family R-3-1-23 did not perform better than the initial cultivar because of a very low seed set. This somaclonal family, its initial being the cultivar IKP, the breeding line lR31785 (extremely salt-sensitive) and the cultivar 'Aiwu' (moderately salt-resistant), were used as parents for production of hybrids. Four crosses, IKP x R-3-1-23, IR31785 x R-3-1-23, IR31785 x IKP and IKP x 'Aiwu', were performed. Most of the F-1 hybrids cultivated in the absence of salt exhibited increased performances compared with the mid-parent. suggesting an heterosis effect for yield-related parameters. F-2 populations were screened for salinity resistance and a clear improvement for yield in stress conditions was recorded for populations derived from IKP x R-3-1-23, IR31785 x R-3-1-23 and IR31785 x IKP, although the mean level of increase over the mid-parent (RIMP) varied depending on the population, the presence or absence of stress, and the quantified parameters. The results are discussed in relation to the usefulness of in vitro selection for obtaining interesting somaclonal variants useful to be integrated in classical breeding programmes for salinity resistance in rice.

Salinity induced behavioural changes in malate dehydrogenase and glutamate dehydrogenase activities in rice seedlings of differing salt tolerance

The activities of malate dehydrogenase in whole tissue extract (NAD +-MDH) as well as in mitochondrial (NAD +-MDH) and chloroplastic (NADP +-MDH) preparations of aminating (NADH-GDH) and deaminating(NAD +-GDH) glutamate dehydrogenases were studied in two sets of rice cultivars differing in salt tolerance grown under moderate (7 dS m −1) and high (14 dS m −1) NaCl salinity levels. A contrasting response to salinity on enzyme activities was found between the sensitive and tolerant cultivars during a 5–20-day growth period of study. NaCl salinity in situ caused increase in all three MDH activities in salt tolerant cvs. CSR-1 and CSR-3 whereas in salt sensitive cvs. Ratna and Jaya 16–100% inhibition in activities was noted. Chloroplastic MDH was extremely sensitive to NaCl. In seedlings of salt tolerant cultivars concomitant increase in both aminating and deaminating GDH activities was observed with increase in salinity level, whereas in sensitive cultivars under higher salinity level decrease in GDH activity was noted. Under in vitro conditions NaCl concentration in the range 1–1000 mM caused gradual inhibition in MDH activity. With 400 mM NaCl in vitro, complete loss of mitochondrial and chloroplastic MDH activities was observed. GDH activity increased with increasing concentration of NaCl up to 200 mM NaCl and other salts in vitro and was inhibited thereafter. However 800 mM NaCl caused complete loss of deaminating GDH activity from sensitive cultivar but not from tolerant cultivar. Results suggest varying behaviour of MDH and GDH in two sets of rice cultivars differing in salt tolerance and that inhibition in the activities of dehydrogenases in salt sensitive rice cultivars due to salinity may be one of the possible reasons for decreased growth of rice plants under saline conditions.

Exogenous glycinebetaine reduces sodium accumulation in salt-stressed rice plants

Glycinebetaine (GB) is a compatible organic solute produced by several plant species to cope with salt or water stress. This compound is involved in osmotic adjustment as well as in protecting several cellular structures. It is well established that rice is unable to synthesize this compound because it lacks choline monooxygenase(CMO), which is involved in the synthesis of betaine aldehyde, the immediate precursor of GB. It has also been reported that exogenous GB protects rice from salt stress by maintaining photosystem II integrity and relative water content (Harinasut et al 1996). Its effect on sodium nutrition in salt-stressed rice, however, was never reported. In a phytotron experiment, a salt-sensitive rice cultivar--I Kong Pao--maintained on nutrient solution (Yoshida et al 1976; renewed each week) was subjected to various NaCl doses (0, 30, 50, and 100 mM), with or without simultaneous exposure to 1 mM GB (Sigma Chemical). Plants were exposed to salt stress at the seedling stage (20 d old; 100 plants per treatment) on 25-L tanks in a complete randomized block. Ten surviving plants were collected each week during a subsequent 1-mo experiment. Relative growth rate was quantified on a dry-weight basis and ion concentration was determined after digestion of organic matter with nitric acid by an inductively coupled argon plasma emission spectrophotometer.

Expression of arginine decarboxylase in seedlings of indica rice (Oryza sativa L.) cultivars as affected by salinity stress.

The effect of salinity stress on the activity of arginine decarboxylase (ADC, EC 4.1.1.19), the first enzyme in biosynthesis of polyamines (PA) from arginine, as well as its transcript level has been compared in salt-sensitive (M-1-48) and salt-tolerant (Pokkali) rice cultivars. Treatment of 72 h grown seedlings either with increasing concentrations of NaCl or with 150 mM NaCl for different time periods, showed a gradual increase of activity in Pokkali. In M-1-48 an immediate increase followed by sharp decrease was observed on prolonged treatment beyond 6 h or above 150 mM NaCl. To generate a DNA probe for ADC, the polymerase chain reaction was used with oat genomic DNA and sequence-specific primers. A region of oat genomic DNA containing a coding sequence for 166 amino acids of the C-terminal part of the ADC enzyme was amplified and called OAD1. Southern analysis of EcoRI- or BamHI-cut genomic DNAs from different cultivars of rice with OAD1 as the probe revealed strong hybridization with one DNA fragment of rice and restriction fragment length polymorphism (RFLP) was noticed. Northern analysis of total RNA of rice with OAD1 as the probe revealed hybridization with a transcript of similar size to the ADC transcript in oat. While in Pokkali, at least a 20-fold accumulation of OAD1 homologous transcript was detected after treatment with 200 mM NaCl, only a seven-fold increase in transcript level was found in M-1-48 after 150 mM NaCl treatment. Results suggest that in the salt-tolerant rice cultivar Pokkali, ADC enzyme activity increases and its transcript also accumulates during the prolonged salinity stress, this mechanism is absent in the salt-sensitive rice cultivar M-1-48 where a prolonged period of salinity stress down-regulates both ADC activity and its transcript level.

Characterization of a rice gene showing organ-specific expression in response to salt stress and drought.

Protein changes induced by salinity stress were investigated in the roots of the salt-sensitive rice cultivar Taichung native 1. We found eight proteins to be induced and obtained partial sequences of one with a molecular mass of 15 kilodaltons and an isoelectric point of 5.5. Using an oligonucleotide probe based on this information, a cDNA clone, salT, was selected and found to contain an open reading frame coding for a protein of 145 amino acid residues. salT mRNA accumulates very rapidly in sheaths and roots from mature plants and seedlings upon treatment with Murashige and Skoog salts (1%), air drying, abscisic acid (20 microM), polyethylene glycol (5%), sodium chloride (1%), and potassium chloride (1%). Generally, no induction was seen in the leaf lamina even when the stress should affect all parts of the plant uniformly. The organ-specific response of salT is correlatable with the pattern of Na+ accumulation during salt stress.