Counteraction of exogenous L-proline with NaCl in salt-sensitive cultivar of rice

The counteraction of exogenous L-proline at different concentrations with salinity (100 mM NaCl at sublethal concentration) were observed on germinating rice. Supplemented 30 mM L-proline was shown to be the most effective; at higher concentration it reduced seedling growth and K⁺/Na⁺ ratio. Thus 30 mM L-proline can alleviate the salinity stress in rice seedlings.     

Effect of sodium chloride-induced stress on growth, proline, glycine betaine accumulation, antioxidative defence and bacoside A content in in vitro regenerated shoots of Bacopa monnieri (L.) Pennell

Growth, osmotic adjustment, antioxidant enzyme defense and the principle medicinal component bacoside A were studied in the in vitro raised shoot cultures of Bacopa monnieri, a known medicinal plant, under different concentrations of NaCl [0.0 (control), 50, 100, 150 or 200 mM]. A sharp increase in Na⁺ content was observed at 50 mM NaCl level and it was about 6.4-fold higher when compared with control. While Na⁺ content increased in the shoots with increasing levels of NaCl in the medium, both K⁺ and Ca²⁺ concentrations decreased. Significant reduction was observed in shoot number per culture; shoot length, fresh weight (FW), dry weight (DW) and tissue water content (TWC) when shoots were exposed to increasing NaCl concentrations (50–200 mM) as compared with the control. Decrease in TWC was not significant at higher NaCl level (150 and 200 mM). At 200 mM NaCl, growth of shoots was adversely affected and microshoots died under prolonged stress. Minimum damage to the membrane as assessed by malondialdehyde (MDA) content was noticed in the controls in contrast to sharp increase of it in NaCl-stressed shoots. Higher amounts of free proline, glycinebetaine and total soluble sugars (TSS) accumulated in NaCl-stressed shoots indicating that it is a glycinebetaine accumulator. About 2.11-fold higher H₂O₂ content was observed at 50 mM NaCl as compared with control and it reached up to 7.1-folds more at 200 mM NaCl. Antioxidant enzyme activities (superoxide dismutase, catalase, ascorbate peroxidase and guaiacol peroxidase) also increased with a rise in NaCl level. Increase in bacoside A, a triterpene saponin content was observed only up to 100 mM NaCl level. Higher salt concentrations inhibited the accumulation of bacoside A. It appears from the data that accumulation of osmolytes, ions and elevated activities of antioxidant enzymes play an important role in osmotic adjustment in shoot cultures of Bacopa under salt stress.     

Metabolic engineering of rice leading to biosynthesis of glycinebetaine and tolerance to salt and cold

Genetically engineered rice (Oryza sativa L.) with the ability to synthesize glycinebetaine was established by introducing the codA gene for choline oxidase from the soil bacterium Arthrobacter globiformis. Levels of glycinebetaine were as high as 1 and 5 mol per gram fresh weight of leaves in two types of transgenic plant in which choline oxidase was targeted to the chloroplasts (ChlCOD plants) and to the cytosol (CytCOD plants), respectively. Although treatment with 0.15 m NaCl inhibited the growth of both wild-type and transgenic plants, the transgenic plants began to grow again at the normal rate after a significantly less time than the wild-type plants after elimination of the salt stress. Inactivation of photosynthesis, used as a measure of cellular damage, indicated that ChlCOD plants were more tolerant than CytCOD plants to photoinhibition under salt stress and low-temperature stress. These results indicated that the subcellular compartmentalization of the biosynthesis of glycinebetaine was a critical element in the efficient enhancement of tolerance to stress in the engineered plants.     

Effect of nacl- salinity on metabolism of proline in salt- sensitive and salt- resistant cultivars of rice

The effect of NaCl at sublethal concentration was observed on germinating seeds of salt-sensitive and -resistant rice cultivars with respect to the level of proline regulatory enzymes and the growth of seedlings on different days of early germination period. The two enzymes of proline biosynthesis and catabolism, Δ-pyrroline-5-carboxylate reductase and L-proline dehydrogenase, were taken into consideration to observe the effects of 100 mM NaCl on their activities in both rice cultivars. The activity of Δ-pyrroline-5-carboxylate reductase in salt-resistant cultivar was increased twice after 5 d in 100 mM NaCl. Simultaneously, the activity of L-proline dehydrogenase was decreased significantly. High activities of Δ-pyrroline-5-carboxylate reductase may be regarded as a biological marker for screening the sensitive and resistant cultivars of rice seed under NaCl-salinity.     

The effects of NaCl on growth,water relations,osmolytes and ion content in <Emphasis Type="Italic">Kochia prostrata</Emphasis>

The effects of NaCl (0, 50, 100, 150 and 200 mM) on growth, water relations, glycinebetaine, free proline, ion contents, stomata number and size of Kochia prostrata (L.) Schard were determined. Shoot and root fresh and dry matter, root and shoot length, relative growth rate, net assimilation rate, relative water content, water use efficiency, soluble sugars and glycinebetaine contents were not changed at low NaCl concentrations, but they were significantly decreased at 200 mM NaCl. The K⁺, Mg²⁺ and Ca²⁺ contents, water potential, chlorophyll a+b and carotenoides contents, and stomata number and size were reduced already at low concentrations of NaCl. In contrast, the Na⁺, Cl^– and proline contents increased several times with increasing NaCl concentration. Kochia prostrata is a salt tolerant species, the optimal growth of this plant occurred up to 150 mM NaCl. The mechanisms of salt tolerance in the plant may be balance among ion accumulation and production of glycinebetaine, proline, soluble sugars for maintenance of pressure potential.     

Salt Tolerance of Glycinebetaine-Deficient and -Containing Maize Lines

Pairs of homozygous near-isogenic glycinebetaine-containing (Bet1/Bet1) and -deficient (bet1/bet1) F8 lines of Zea mays L. (maize) were tested for differences in salt (150 mM NaCl or 127.25 mM NaCl plus 22.5 mM CaCl2) tolerance. The Bet1/Bet1 lines exhibited less shoot growth inhibition (as measured by dry matter accumulation, leaf area expansion rate and/or, plant height extension rate) under salinized conditions in comparison to their nearisogenic bet1/bet1 sister lines. These growth differences were associated with maintenance of a significantly higher leaf relative water content, a higher rate of carbon assimilation, and a greater turgor in Bet1/Bet1 lines than in bet1/bet1 lines under salinized conditions. These results strongly suggest that a single gene conferring glycinebetaine accumulation (and/or a tightly linked locus) plays a key role in osmotic adjustment in maize.     

Over-expression of a barley aquaporin increased the shoot/root ratio and raised salt sensitivity in transgenic rice plants

Barley HvPIP2;1 is a plasma membrane aquaporin and its expression was down-regulated after salt stress in barley [Katsuhara et al. (2002) Plant Cell Physiol. 43: 885]. We produced and analyzed transgenic rice plants over-expressing barley HvPIP2;1 in the present study. Over-expression of HvPIP2;1 increased (1) radial hydraulic conductivity of roots (Lp(r)) to 140%, and (2) the mass ratio of shoot to root up to 150%. In these transgenic rice plants under salt stress of 100 mM NaCl, growth reduction was greater than in non-transgenic plants. A decrease in shoot water content (from 79% to 61%) and reduction of root mass or shoot mass (both less than 40% of non-stressed plants) were observed in transgenic plants under salt stress for 2 weeks. These results indicated that over-expression of HvPIP2;1 makes rice plants sensitive to 100 mM NaCl. The possible involvement of aquaporins in salt tolerance is discussed.     

Enhancement of NaCl tolerance in Arabidopsis thaliana by exogenous L-asparagine and D-asparagine

The tolerances of Columbia Arabidopsis thaliana (L.) Heynh. to NaCl, L-asparagine (L-Asn) and D-asparagine (D-Asn) during seedling establishment on sterile agar medium were determined. Germination and the establishment of upright seedlings with expanded green cotyledons were increasingly inhibited by NaCl concentrations from 20 to 180 m M and radicle growth was prevented at 225 m M NaCl. Tolerance of established seedlings to NaCl was similar at these concentrations. Seedling establishment was prevented at 20 m M L-Asn and 60 m M D-Asn, but L-Asn was not toxic to established seedlings. At lower concentrations, exogenous L- and D-Asn enhanced NaCl tolerance during germination and seedling establishment. Inhibition of seedling establishment by NaCl concentrations below 225 m M was reduced by the addition of L- and D-Asn to the medium. Maximal reduction of NaCl inhibition occurred between 2 and 4 m M for both L- and D-Asn. Higher concentrations of NaCl prevented establishment whether exogenous Asn was present or not. Reduction of NaCl inhibition occurred to the same extent whether L-Asn was presented simultaneously with the NaCl or preloaded for up to 24 h. The total seedling content of Na⁺ increased about 4-fold to 55 μg (mg dry weight)⁻¹ as the medium concentration of NaCl was increased from 9 μ M to 150 m M NaCl. Total K⁺ content declined about 80% from about 34 μg (mg dry weight)⁻¹ over the same range of NaCl concentrations. The Na⁺ uptake and K⁺ efflux by whole seedlings were similar whether or not NaCl tolerance was increased by exogenous Asn.     

Modulation in growth,photosynthetic efficiency,activity of antioxidants and mineral ions by foliar application of glycinebetaine on pea (Pisum sativum L.) under salt stress

A pot experiment was carried out to explore the role of glycinebetaine (GB) as foliar spray foliar on two pea (Pisum sativum L.) varieties (Pea 09 and Meteor Fsd) under saline and non-saline conditions. Thirty-two-day-old plants were subjected to two levels 0 and 150 mM of NaCl stress. Salt treatment was applied in full strength Hoagland’s nutrient solution. Three levels 0, 5 and 10 mM of GB were applied as foliar treatment on 34-day-old pea plants. After 2 weeks of foliar treatment with GB data for various growth and physiochemical attributes were recorded. Rooting-medium applied salt (150 mM NaCl) stress decreased growth, photosynthesis, chlorophyll, chlorophyll fluorescence and soluble protein contents, while increasing the activities of enzymatic (POD and CAT) and non-enzymatic (ascorbic acid and total phenolics) antioxidant enzymes. Foliar application of GB decreased root and shoot Na⁺ under saline conditions, while increasing shoot dry matter, root length, root fresh weight, stomatal conductance (g _s), contents of seed ascorbic acid, leaf phenolics, and root and shoot Ca²⁺ contents. Of three GB (0, 5, 10 mM) levels, 10 mM proved to be more effective in mitigating the adverse effects of salinity stress. Overall, variety Pea 09 showed better performance in comparison to those of var. Meteor Fsd under both normal and salinity stress conditions. GB-induced modulation of seed ascorbic acid, leaf phenolics, g _s, and root Ca²⁺ values might have contributed to the increased plant biomass, reduction of oxidative stress, increased osmotic adjustment and better photosynthetic performance of pea plants under salt stress.     

Genetic and physiological relationships between L-asparaginase I and asparaginase II in Saccharomyces cerevisiae.

The cistron that codes for L-asparaginase I in Saccharomyces cerevisiae (aspl) is not genetically linked to either of the cistrons coding for expression of asparaginase II (asp2 and asp3). Cells containing different combinations of theses enzymes grow at different rates in media in which L-asparagine or D-asparagine is the only source of nitrogen for cell replication. Cells lacking L-asparaginase I but possessing asparaginase II grow more rapidly in medium containing D-asparagine as a nitrogen source than cells containing both enzymes, even though D-asparagine is not a substrate of L-asparaginase I. These results indicate that L-asparaginase I and asparaginase II interact in some way to regulate the utilization of asparagine as a nitrogen source for cell growth.     

Possible Involvement of Greening in Cell Growth of Suaeda japonica under Salt Stress

Suaeda japonica, a member of the family Chenopodiaceae, is ahalophyte that grows on the shores of the Ariake Sea in Japan.Using yellowish and selected green callus tissues of S. japonica,we examined the correlation between cell growth and glycinebetainecontent, as well as the activity of betaine aldehyde dehydrogenase(BADH), under salt stress. Although the growth of yellowishcallus tissues was markedly inhibited by 0.1 M NaCl, that ofgreen callus tissues was not. However, in 0.3 M NaCl, the freshweight of green callus tissues was decreased by 15%. Both theendogenous level of glycinebetaine and the activity of BADHin yellowish callus tissues were very low, but green callustissues contained very high levels of glycinebetaine and highBADH activity. The activity in green callus increased significantlywith increases in the concentration of NaCl. The increase inlevels of Chl was also observed, but the increase occurred earlierthan that in the level of glycinebetaine. Functional developmentof chloroplasts (an increase in the level of Chl) seems to beinvolved in the increase in the activity of BADH, and the resultantincrease in levels of glycinebetaine seems to facilitate callusgrowth under salt stress. (Received October 2, 1996; Accepted November 18, 1996)     

Compatibility of glycinebetaine in rice plants: evaluation using transgenic rice plants with a gene for peroxisomal betaine aldehyde dehydrogenase from barley

Glycinebetaine is synthesized in plants by the two‐step oxidation of choline, with betaine aldehyde as the intermediate. The reactions are catalyzed by choline mono‐oxygenase and betaine aldehyde dehydrogenase. Rice plants, which do not accumulate glycinebetaine, possess a gene encoding betaine aldehyde dehydrogenase, whose activity is detectable at low levels. To evaluate the compatibility in rice of glycinebetaine on growth and tolerance to salt, cold and heat, we produced transgenic rice plants by introduction of a cDNA for betaine aldehyde dehydrogenase of barley, which is localized in peroxisomes unlike the chloroplast‐specific localization of betaine aldehyde dehydrogenase in spinach and sugar beet. The transgenic rice plants converted high levels of exogenously applied betaine aldehyde (up to 10 mol m^–3) to glycinebetaine more efficiently than did wild‐type plants. The elevated level of glycinebetaine in transgenic plants conferred significant tolerance to salt, cold and heat stress. However, very high levels of glycinebetaine, resulting from conversion of applied betaine aldehyde to glycinebetaine or from exogenous application, inhibited increases in length of rice plants but not increases in dry weight. Our results suggested that the benefits of accumulation of glycinebetaine by rice plants might be considerable under high light conditions.     

Amelioration of the salt-stressed root growth of rice and normalization of the Na+ distribution between the shoot and root by (S)-alpha-methylbenzyl-2-fluoro-4-methylphenylurea

Optically active alpha-methylbenzyl phenyl ureas (MBPUs) show diverse plant physiological properties. Experiments were conducted to evaluate the salt-stress response of just-germinated rice seedlings supplemented with the S-enantiomer of MBPUs by assessing the growth and Na+ content. This study indicates that S-MBPUs served as a unique stress reliever for just-germinated young seedlings of rice injured by salinity. NaCl severely affected the root growth of rice seedlings. Concomitant treatment with S-MBPUs effectively ameliorated the growth inhibition of rice by NaCl. Glycine betaine (GB) did not act as a reliever of the NaCl stress. The addition of S-alpha-methylbenzyl 2-fluoro-4-methylphenyl urea (7, denoted as S-FM) to the saline medium ameliorated not only the root growth but also the protein content and dry weight of roots depending upon its concentration. The protein content, Na+ content and growth rate were correlated to each other with a positive relationship. The Na+ distribution ratio (S/R(Na+)) between the shoot and root increased with increasing concentration of NaCl when added alone, viz. with increasing growth reduction. A concomitant treatment with S-FM (7), however, resulted in the S/R(Na+) value becoming smaller with growth amelioration. This indicates that S-FM (7) controlled the translation of Na+ from the roots to shoots. S-FM (7) would have influenced some inherent functions connected with the Na+ behavior in the rice plant, although details of the mechanism for normalization of the S/R(Na+) ratio are still not clear.     

Utilization of D-asparagine by Saccharomyces cerevisiae.

Yeast strains sigma1278b and Harden and Young, which synthesize only an internal constitutive form of L-asparaginase, do not grow on D-asparagine, as a sole source of nitrogen, and whole cell suspensions of these strains do not hydrolyze D-asparagine. Strains X2180-A2 and D273-10B, which possess an externally active form of asparaginase, are able to grow slowly on D-asparagine, and nitrogen-starved suspensions of these strains exhibit high activity toward the D-isomer. Nitrogen starvation of strain X218O-A2 results in coordinate increase of D- and L-asparaginase activity; the specific activity observed for the D-isomer is approximately 20% greater than that observed for the L-isomer. It was observed, in studies with cell extracts, that hydrolysis of D-asparagine occurred only with extracts from nitrogen-starved cells of strains that synthesize the external form of asparaginase. Furthermore, the activity of the extracts toward the D-isomer was always higher than that observed with the L-isomer. A 400-fold purified preparation of external asparaginase from Saccharomyces cerevisiae X218U-A2 hydrolyzed D-asparagine with an apparent Km of 0.23 mM and a Vmax of 38.7 mumol/min per mg of protein. D-Asparagine was a competitive inhibitor of L-asparagine hydrolysis and the Ki determined for this inhibition was approximately equal to its Km. These data suggest that D-asparagine is a good substrate for the external yeast asparaginase but is a poor substrate for the internal enzyme.     

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.     

Silicon deposition in the root reduces sodium uptake in rice (Oryza sativa L.) seedlings by reducing bypass flow

Sodium chloride reduces the growth of rice seedlings, which accumulate excessive concentrations of sodium and chloride ions in their leaves. In this paper, we describe how silicon decreases transpirational bypass flow and ion concentrations in the xylem sap in rice (Oryza sativa L.) seedlings growing under NaCl stress. Salt (50 mM NaCl) reduced the growth of shoots and roots: adding silicate (3 mM) to the saline culture solution improved the growth of the shoots, but not roots. The improvement of shoot growth in the presence of silicate was correlated with reduced sodium concentration in the shoot. The net transport rate of Na from the root to shoot (expressed per unit of root mass) was also decreased by added silicate. There was, however, no effect of silicate on the net transport of potassium. Furthermore, in salt-stressed plants, silicate did not decrease the transpiration, and even increased it in seedlings pre-treated with silicate for 7 d prior to salt treatment, indicating that the reduction of sodium uptake by silicate was not simply through a reduction in volume flow from root to shoot. Experiments using trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS), an apoplastic tracer, showed that silicate dramatically decreased transpirational bypass flow in rice (from about 4.2 to 0.8%), while the apparent sodium concentration in the xylem, which was estimated indirectly from the flux data, decreased from 6.2 to 2.8 mM. Direct measurements of the concentration of sodium in xylem sap sampled using Philaenus spumarius confirmed that the apparent reduction was not a consequence of sodium recycling. X-ray microanalysis showed that silicon was deposited in the outer part of the root and in the endodermis, being more obvious in the latter than in the former. The results suggest that silicon deposition in the exodermis and endodermis reduced sodium uptake in rice (Oryza sativa L.) seedlings under NaCl stress through a reduction in apoplastic transport across the root.     

Effect of aqueous leaf extract of common lambsquarters (Chenopodium album L.) and NaCl on germination and seedling growth of rice

Aqucous leaf extract of common lambsquarters (Chenopodium album L.) was evaluated alone or in combination with NaCl for its effect on germination and rice seedling growth. The treatments have no effect on germination. However, growth of both shoot and root significantly decreased under NaCl alone and in combination with leaf extract. Root growth was affected more than the shoot and the effect of NaCl was accentuated in the presence of leaf extract.     

Comparative effects of NaCl and polyethylene glycol on root distribution,growth, and stomatal conductance of sour orange seedlings

An experiment was conducted to study sour orange (Citrus aurantium L.) seedling root density, distribution, and morphological development under NaCl and polyethylene glycol (PEG) stresses in relation to shoot growth and stomatal conductance. Plants were treated with 2 stress levels (– 0.12 and – 0.24 MPa) of NaCl and PEG 4000 for 7 months. Root observation chambers were used to monitor root growth and distribution under stressed and non-stressed conditions. Seedlings receiving NaCl or PEG treatments produced fewer roots and shallower root systems with 46 to 65% of the roots occurring in the top portion of the soil. Fibrous root weight per unit length was increased by 24 to 30% by PEG but was not significantly increased by NaCl.Root growth rate usually alternated with shoot growth in a 2-month cycle. This alternating pattern was not shifted by NaCl and PEG stresses. In all NaCl and PEG treatments, growth was depressed and stomatal conductance was reduced. Compared to controls, plants that received NaCl or PEG had smaller shoot and root dry weights, fewer leaves, shorter height, and fewer roots. Sodium chloride usually caused less damage than PEG to sour orange seedlings suggesting that NaCl and PEG acted through different mechanisms.Florida Agricultural Experiment Station Journal Series No. 9941.     

Inducers of glycinebetaine synthesis in barley.

Glycinebetaine is an osmoprotectant accumulated by barley (Hordeum vulgare) plants in response to high levels of NaCl, drought, and cold stress. Using barley seedlings in hydroponic culture, we characterized additional inducers of glycinebetaine accumulation. These included other inorganic salts (KCl, MgCl(2), LiCl, and Na(2)SO(4)), oxidants (H(2)O(2) and cumene hydroperoxide), and organic compounds (abscisic acid, polymixin B, n-butanol, salicylic acid, and aspirin). Stress symptoms brought on by high NaCl and other inducers, and not necessarily correlated with glycinebetaine accumulation, include wilting, loss of chlorophyll, and increase in thiobarbituric acid reacting substances. For NaCl, Ca(2+) ions at 10 to 20 mM decrease these stress symptoms without diminishing, or even increasing, glycinebetaine induction. Abscisic acid induces glycinebetaine accumulation without causing any of the stress symptoms. NaCl, KCl, and H(2)O(2) (but not other inducers) induce glycinebetaine at concentrations below those needed for the other stress symptoms. Mg(2+) at 10 to 20 mM induces both stress symptoms and glycinebetaine, but only at low (0.2 mM) Ca(2+). Although illumination is needed for optimal induction, a significant increase in the leaf glycinebetaine level is found in complete darkness, also.