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)     

Transformation of Synechococcus with a gene for choline oxidase enhances tolerance to salt stress

Choline oxidase, isolated from the soil bacterium Arthrobacter globiformis, converts choline to glycinebetaine (N-trimethylglycine) without a requirement for any cofactors. The gene for this enzyme, designated codA, was cloned and introduced into the cyanobacterium Synechococcus sp. PCC 7942. The codA gene was experssed under the control of a strong constitutive promoter, and the transformed cells accumulated glycinebetaine at intracellular levels of 60–80 mM. Consequently the cells acquired tolerance to salt stress, as evaluated in terms of growth, accumulation of chlorophyll and photosynthetic activity.     

Accumulation of glycinebetaine and its synthesis from radioactive precursors under salt-stress in the cyanobacterium Aphanothece halophytica

Growth in salt-stressed (2.0 M NaCl) Aphanothece halophytica was initially delayed during the first two days of cultivation and eventually attained the same growth rate as the control (0.5 M NaCl) cells. Glycinebetaine accumulation increased slightly in control cells but a dramatic increase of glycinebetaine occurred in salt-stressed cells during a growth period of six days. There was no apparent increase in the synthesis of [¹⁴C] glycinebetaine in the control cells, in contrast to the marked increase in its synthesis in the salt-stressed cells. Increasing NaCl concentration in the growth medium induced both the accumulation and the synthesis of glycinebetaine. Time course experiments provided evidence that [¹⁴C] choline was first oxidized to [¹⁴C] betaine aldehyde which was further oxidized to [¹⁴C] glycinebetaine in A. halophytica. The supporting data for such a pathway were obtained from the presence of choline and betaine aldehyde dehydrogenase activities found in the membrane and cytoplasmic fractions, respectively. The activities of these two enzymes were also enhanced upon increasing NaCl concentration in the growth medium from 0.5 M to 2.0 M. Under this condition an increaseof approximately 1.5-fold was observed for choline dehydrogenase activity as compared to 2.5-fold for betaine aldehyde dehydrogenase activity, suggesting a preferable induction of the latter enzyme by salt stress. A. halophytica was able to utilize [¹⁴C] ethanolamine and [¹⁴C] glycine for the synthesis of [¹⁴C] glycinebetaine. This revised version was published online in August 2006 with corrections to the Cover Date.     

Degradation of Glycinebetaine by Betaine-Homocysteine Methyltransferase in Aphanothece halophytica: Effect of Salt Downshock and Starvation

We have investigated conditions leading to the degradation of glycinebetaine in Aphanothece halophytica and have shown the activity of betaine-homocysteine methyltransferase (BHMT). The intracellular glycinebetaine level was decreased approximately 50% after 36 h salt downshock from 2.0 m NaCl medium to 0.5 m NaCl medium. A slight additional decrease of glycinebetaine occurred when salt downshock was combined with dark treatment. The omission of carbon and nitrogen sources in the growth medium further decreased intracellular glycinebetaine. The activity of BHMT increased from 0 to 460 nmol h⁻¹mg⁻¹ after 3 h salt downshock. Higher strength of salt downshock resulted in higher activity of the enzyme. Small increase of the enzyme activity was also observed when A. halophytica was deprived of carbon and nitrogen sources in the growth medium. Received: 17 March 2000 / Accepted: 24 April 2000     

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.     

An inland and a coastal population of the Mediterranean xero-halophyte species Atriplex halimus L. differ in their ability to accumulate proline and glycinebetaine in response to salinity and water stress

Soil salinity and drought compromise water uptake and lead toosmotic adjustment in xero-halophyte plant species. These importantenvironmental constraints may also have specific effects onplant physiology. Stress-induced accumulation of osmocompatiblesolutes was analysed in two Tunisian populations of the Mediteraneanshrub Atriplex halimus L.—plants originating from a salt-affectedcoastal site (Monastir) or from a non-saline semi-arid area(Sbikha)—were exposed to nutrient solution containingeither low (40 mM) or high (160 mM) doses of NaCl or 15% polyethyleneglycol. The low NaCl dose stimulated plant growth in both populations.Plants from Monastir were more resistant to high salinity andexhibited a greater ability to produce glycinebetaine in responseto salt stress. Conversely, plants from Sbikha were more resistantto water stress and displayed a higher rate of proline accumulation.Proline accumulated as early as 24 h after stress impositionand such accumulation was reversible. By contrast, glycinebetaineconcentration culminated after 10 d of stress and did not decreaseafter the stress relief. The highest salt resistance of Monastirplants was not due to a lower rate of Na⁺ absorption; plantsfrom this population exhibited a higher stomatal conductanceand a prodigal water-use strategy leading to lower water-useefficiency than plants from Sbikha. Exogenous application ofproline (1 mM) improved the level of drought resistance in Monastirplants through a decrease in oxidative stress quantified bythe malondialdehyde concentration, while the exogenous applicationof glycinebetaine improved the salinity resistance of Sbikhaplants through a positive effect on photosystem II efficiency. Key words: Atriplex halimus, glycinebetaine, halophyte, NaCl, osmotic adjustment, proline, salinity, water stress     

Response of Melanthera biflora to Salinity and Water Stress

Melanthera biflora (Asteraceae) is a moderately salt-tolerantplant from the Indo-Pacific region. In laboratory studies itsgrowth was inhibited by salt above 50 mol m^–3, but itwas able to survive salinities approaching that of seawater,namely 400 mol m^–3. Shoot potassium concentrations weremaintained over a range of salinities up to 400 mol m^–3,while sodium and chloride accumulation followed closely theincrease in external osmotic pressure. In contrast, the increasein osmotic pressure of the leaf sap of Melanthera biflora, subjectedto water stress, was due mainly to a decrease in the ratio offresh weight/dry weight. 3-dimethylsulphoniopropionate (3-DMSP)and glycinebetaine were identified by fast atom bombardmentmass and ¹H -NMR spectroscopy, with 3-DMSP being the main oniumcompound and glycinebetaine absent in some accessions. Onium(quaternary ammonium and/or tertiary sulphonium) compounds andproline increased during salt and water stress due mainly toa decrease in the fresh weight/dry weight ratio of tissue, althoughpart of the increase in salt-stressed tissue was due to an increasein the accumulation of the onium compound. This salt-inducedincrease in 3-DMSP was inhibited in conditions of low sulphursupply and there was no compensatory increase in proline. Key words: Melanthera biflora, Asteraceae, salinity, glycinebetaine, 3-dimethylsulphonioproprionate     

Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress

Glycinebetaine is one of the compatible solutes that accumulate in the chloroplasts of certain halotolerant plants when these plants are exposed to salt or cold stress. The codA gene for choline oxidase, the enzyme that converts choline into glycinebetaine, has previously been cloned from a soil bacterium, Arthrobacter globiformis. Transformation of Arabidopsis thaliana with the cloned codA gene under the control of the 35S promoter of cauliflower mosaic virus enabled the plant to accumulate glycinebetaine and enhanced its tolerance to salt and cold stress. At 300 mM NaCl, considerable proportions of seeds of transformed plants germinated well, whereas seeds of wild-type plants failed to germinate. At 100 mM NaCl, transformed plants grew well whereas wild-type plants did not do so. The transformed plants tolerated 200 mM NaCl, which was lethal to wild-type plants. After plants had been incubated with 400 mM NaCl for two days, the photosystem II activity of wild-type plants had almost completely disappeared, whereas that of transformed plants remained at more than 50% of the original level. When exposed to a low temperature in the light, leaves of wild-type plants exhibited symptoms of chlorosis, whereas those of transformed plants did not. These observations demonstrate that the genetic modification of Arabidopsis thaliana that allowed it to accumulate glycinebetaine enhanced its ability to tolerate salt and cold stress.     

Proline and glycinebetaine enhance antioxidant defense and methylglyoxal detoxification systems and reduce NaCl-induced damage in cultured tobacco cells

Salt stress impairs reactive oxygen species (ROS) and methylglyoxal (MG) detoxification systems, and causes oxidative damage to plants. Up-regulation of the antioxidant and glyoxalase systems provides protection against NaCl-induced oxidative damage in plants. Thiol–disulfide contents, glutathione content and its associated enzyme activities involved in the antioxidant defense and glyoxalase systems, and protein carbonylation in tobacco Bright Yellow-2 cells grown in suspension culture were investigated to assess the protection offered by proline and glycinebetaine against salt stress. Salt stress increased protein carbonylation, contents of thiol, disulfide, reduced (GSH) and oxidized (GSSG) forms of glutathione, and the activity of glutathione-S-transferase and glyoxalase II enzymes, but decreased redox state of both thiol–disulfide and glutathione, and the activity of glutathione peroxidase and glyoxalase I enzymes involved in the ROS and MG detoxification systems. Exogenous application of proline or glycinebetaine resulted in a reduction of protein carbonylation, and in an increase in glutathione redox state and activity of glutathione peroxidase, glutathione-S-transferase and glyoxalase I under salt stress. Neither proline nor glycinebetaine, however, had any direct protective effect on NaCl-induced GSH-associated enzyme activities. The present study, therefore, suggests that both proline and glycinebetaine provide a protective action against NaCl-induced oxidative damage by reducing protein carbonylation, and enhancing antioxidant defense and MG detoxification systems.     

The Distribution of Na+, K+ and Glycinebetaine in Salicornia bigelovii

Stems of young actively growing Salicornia bigelovii were dissectedinto the three major tissue layers: vascular, spongy mesophylland palisade. Each layer was analysed for chlorophyll, ash (salt),protein and glycinebetaine content. When glycinebetaine contentwas based on protein content, the vascular and spongy mesophylllayers had nearly identical values. Correction for probableRuBP carboxylase content in the palisade layer gave a glycinebetaine/proteinratio similar to that of the other tissues. All three tissuelayers were found to contain significant amounts of salt. Key words: Salicornia bigelovii, Salt distribution     

Photosynthetic Adaptation to Salt Stress in Three-Color Leaves of a C4 Plant Amaranthus tricolor

We examined the photosynthetic adaptation mechanisms for saltstress in Amaranthus tricolor, which has leaves with green,yellow and red regions, in relation to the accumulation of glycinebetaineas osmoprotectants. The content of Chl, especially of Chl bin the red and yellow regions was 3{small tilde}4% of that inthe green region. The levels of Chl proteins such as LHCII,PSI and PSII were significantly lower than those in the greenregion. However, the contents of other photosynthetic proteinsin these regions seem to be relatively high. We observed thenet photosynthetic CO₂ fixation activity in the red and yellowregions which was about 40% of that in the green region. Uponsalt stress (0.3 M NaCl) for 5 d the levels of Chl, PSI, PSII,ribulose 1,5-bis phosphate carboxygenase and oxygenase, andthe CO₂ fixation rate in the green region decreased by about20{small tilde}35% whereas those in the non-green regions remainedalmost at the same levels. A. tricolor was found to accumulatesglycinebetaine, betainealdehyde dehydrogenase and choline monooxygenaseat similar levels in all three color regions and their contentsincreased upon salt stress. These results suggest that the lowcapacity of light harvesting in non-green regions would be favorof salt stress since the photosynthetic components in theseregions were retained at relatively high levels under high salinity. (Received February 9, 1999; Accepted April 16, 1999)     

Enhanced germination under high-salt conditions of seeds of transgenicArabidopsis with a bacterial gene (codA) for choline oxidase

Arabidopsis thaliana was transformed previously with thecodA gene from the soil bacteriumArthrobacter globiformis. This gene encodes choline oxidase, the enzyme that converts choline to glycinebetaine. Transformation with thecodA gene significantly enhanced the tolerance of transgenic plants to low temperature and high-salt stress. We report here that seeds of transgenic plants that expressed thecodA gene were also more tolerant to salt stress during germination than seeds of non-transformed wild-type plants. Seedlings of transgenic plants grew more rapidly than those of wild-type plants under salt-stress conditions. Furthermore, exogenously applied glycinebetaine was effective in alleviating the harmful effects of salt stress during germination of seeds and growth of young seedlings, a result that suggests that it was glycinebetaine that had enhanced the tolerance of the transgenic plants. These observations indicate that synthesis of glycinebetaine in transgenic plantsin vivo, as a result of the expression of thecodA gene, might be veryuseful in improving the ability of crop plants to tolerate salt stress. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

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.     

盐旱交叉胁迫对灰胡杨(Populus pruinosa)幼苗生长和生理生化特性的影响

以一年生灰胡杨幼苗为试验材料,利用田间控盐控水的方法,进行干旱和盐胁迫试验,通过测定生长和生理生化指标探讨幼苗在盐旱交叉胁迫下的生长发育及适应规律,旨在阐明干旱及盐交叉胁迫下植物抗旱抗盐机理。研究结果表明:在盐、旱及交叉胁迫下,灰胡杨幼苗抗氧化酶活性、MDA和脯氨酸含量与对照存在显著差异(P0.05)。(1)在8、11 g/L和15 g/L盐处理下,灰胡杨幼苗相对高生长、相对枝长和冠幅增量均受到抑制,且差异显著(P0.05),而干旱胁迫和盐旱交互胁迫下差异不显著。(2)在盐胁迫、盐旱交叉胁迫下,随着胁迫程度的加重,抗氧化酶SOD、POD、CAT活性表现出先增加后降低的趋势,三者协调一致;仅干旱胁迫时,抗氧化酶SOD、POD、CAT活性显著增加;(3)在盐、旱及其盐旱交叉胁迫下,脯氨酸含量呈上升趋势,MDA含量则表现出先降低后升高趋势,这与抗氧化酶活性先升高后降低的趋势相对应。因此,抗氧化酶活性对缓解脂膜过氧化的伤害具有一定限度,MDA含量与抗氧化酶活性呈负相关,灰叶胡杨幼苗在盐旱交叉胁迫下表现出一定的耐性。     

Ameliorative Effect by Calcium on NaCI Salinity Stress Related to Proline Metabolism in the Callus of Centella asiatica L

Soil salinity affects plant growth and development by way of osmotic stress. Compatible osmolytes are potent osmoprotectants that playa role in counteracting the effect of saline stress. Proline biosynthesis and catabolism were investigated in both the control and salt stressed calli. Proline content showed a steady increase in the calli of all NaCI treated media. Calli on CaCl₂ containing media did not show any increase in proline level compared to control calli. When the salinized media were supplemented with CaCl₂ the proline level drastically increased compared to the corresponding calli grown on salt alone. Similarly, the activity of proline biosynthetic enzyme, pyrroline-5-carboxylate synthetase (P5CS) under salt stress was higher in NaCl + CaCl₂ supplemented medium than the calli on the salinized medium alone. This suggested that the alleviation effect of calcium under saline condition was through modulation of the enzyme complexes that accelerate the rate of proline biosynthesis under salt stress. Similarly, the activity of proline degrading enzyme, proline oxidase was found to be lower in calli of all salt stressed media than control.     

Cytochemical localization of ATPase plasma membrane and acid phosphatase by cerium-based method in a salt-adapted cell line of Pisum sativum

In order to study a possible application of cerium-based techniquesin plant cells, ATPase and acid phosphatase activities havebeen compared in two cell lines of Pisum sativum calli, onesensitive to NaCI and the other selected to be grown under salinity(85 mM NaCI). ATPase activity was unchanged and localized inthe plasma membrane of both cell lines. Acid phosphatase activitywas significantly increased in the salt-selected line and localizedin the cell walls, Golgi complex, multivesicular bodies andvacuoles. These results indicated a possible involvement ofboth activities in the maintenance of cell growth in the selectedline under saline conditions. Key words: Acid phosphatase, Pisum sativum, plasma membrane ATPase, salt stress, ultrastructure     

Regulation of Ethylene Biosynthesis Under Salt Stress in Red Pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) by 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Deaminase-producing Halotolerant Bacteria

The present study was carried out to understand the mechanism of salt stress amelioration in red pepper plants by inoculation of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing halotolerant bacteria. In general, ethylene production, ACC concentration, ACC synthase (ACS), and ACC oxidase (ACO) enzyme activities increased with increasing levels of salt stress. Treatment with halotolerant bacteria reduced ethylene production by 47–64%, ACC concentration by 47–55% and ACO activity by 18–19% in salt-stressed (150 mmol NaCl) red pepper seedlings compared to uninoculated controls. ACS activity was lower in red pepper seedlings treated with Bacillus aryabhattai RS341 but higher in seedlings treated with Brevibacterium epidermidis RS15 (44%) and Micrococcus yunnanensis RS222 (23%) under salt-stressed conditions as compared to uninoculated controls. A significant increase was recorded in red pepper plant growth under salt stress when treated with ACC deaminase-producing halotolerant bacteria as compared to uninoculated controls. The results of this study collectively suggest that salt stress enhanced ethylene production by increasing enzyme activities of the ethylene biosynthetic pathway. Inoculation with ACC deaminase-producing halotolerant bacteria plays an important role in ethylene metabolism, particularly by reducing the ACC concentration, although a direct effect on reducing ACO activity was also observed. It is suggested that growth promotion in inoculated red pepper plants under inhibitory levels of salt stress is due to ACC deaminase activity present in the halotolerant bacteria.     

Enhanced tolerance to light stress of transgenic Arabidopsis plants that express the codA gene for a bacterial choline oxidase

Arabidopsis thaliana was transformed with the codA gene from Arthrobacter globiformis. This gene encodes choline oxidase, an enzyme that converts choline to glycinebetaine. The photosynthetic activity, monitored in terms of chlorophyll fluorescence, of transformed plants was more tolerant to light stress than that of wild-type plants. This enhanced tolerance to light stress was caused by acceleration of the recovery of the photosystem II (PS II) complex from the photo-inactivated state. The transformed plants synthesized glycinebetaine, but no changes were detected in the relative levels of membrane lipids or in the relative levels of fatty acids in the various membrane lipids. Transformation with the codA gene increased levels of H₂O₂, a by-product of the reaction catalyzed by choline oxidase, by only 50% to 100% under stress or non-stress conditions. The activity of ascorbate peroxidase and, to a lesser extent, that of catalase in transformed plants were significantly higher than in the wild-type plants. These observations suggest that H₂O₂ produced by choline oxidase in the transformed plants might have stimulated the expression of H₂O₂ scavenging enzymes, with resultant maintenance of the level of H₂O₂ within a certain limited range. It appears that glycinebetaine produced in vivo, but not changes in membrane lipids or in the level of H₂O₂, protected the PS II complex in transformed plants from damage due to light stress.     

Transformation of Brassica juncea (L.) Czern with bacterial codA gene enhances its tolerance to salt stress

The codA gene for biosynthesis of glycinebetaine from Arthrobacter globiformis was used for transforming Brassica juncea cv. Pusa Jaikisan (which lack any means to synthesize glycinebetaine) through Agrobacterium mediated transformation. The stable insertion of the codA gene in the shoots obtained on medium with kanamycin and hygromycin was confirmed by PCR analysis of the nptII gene. Southern hybridization with a codA probe further demonstrated its successful integration. Immunoblot analysis revealed the presence of choline oxidase demonstrating that the bacterial codA gene had been successfully transcribed and translated. The seeds of transgenic lines showed enhanced capacity to germinate under salt stress as compared to that of the wild type. Further, the seedlings of transgenic plants that expressed codA gene showed significantly higher growth than that of the wild type under salt stress conditions. These results demonstrated that the introduction of a biosynthetic pathway for glycinebetaine into Brassica juncea significantly enhanced their salt tolerance. Hence, homozygous genotypes of selected transformed lines can be exploited for improving the salt tolerance of the desirable cultivars of Brassica juncea through breeding programmes.