Biochemical,physiological and growth changes in response to salinity in callus cultures of <Emphasis Type="Italic">Sesuvium portulacastrum</Emphasis> L.

In vitro-grown cells of Sesuvium portulacastrum L., an important ‘salt accumulator’ mangrove associate, were incubated on a medium containing different levels of salt, including 0, 100, 200, or 400 mM NaCl, in order to evaluate biochemical, physiological, and growth responses. A significant decrease in callus growth, water status, and cell membrane damage was observed under salt stress. Osmotic adjustment was revealed by the accumulation of inorganic ions, such as sodium (Na⁺), and organic osmolytes (proline, glycine betaine, and total soluble sugars) in NaCl-treated calli compared to control. However, accretion of osmolytes and inorganic ions did not support growth of calli under NaCl stress conditions. The observed reduced growth rate in calli subjected to stress, up to 200 mM NaCl, was coupled with lower catalase and ascorbate peroxidase activities and with a significantly higher superoxide dismutase activity. These findings suggested that S. portulacastrum cell cultures exhibited higher osmotic adjustment to salt stress.     

Compatible solute accumulation and stress-mitigating effects in barley genotypes contrasting in their salt tolerance

The accumulation of compatible solutes is often regarded as a basic strategy for the protection and survival of plants under abiotic stress conditions, including both salinity and oxidative stress. In this work, a possible causal link between the ability of contrasting barley genotypes to accumulate/synthesize compatible solutes and their salinity stress tolerance was investigated. The impact of H(2)O(2) (one of the components of salt stress) on K(+) flux (a measure of stress 'severity') and the mitigating effects of glycine betaine and proline on NaCl-induced K(+) efflux were found to be significantly higher in salt-sensitive barley genotypes. At the same time, a 2-fold higher accumulation of leaf and root proline and leaf glycine betaine was found in salt-sensitive cultivars. The total amino acid content was also less affected by salinity in salt-tolerant cultivars. In these, potassium was found to be the main contributor to cytoplasmic osmolality, while in salt-sensitive genotypes, glycine betaine and proline contributed substantially to cell osmolality, compensating for reduced cytosolic K(+). Significant negative correlations (r= -0.89 and -0.94) were observed between Na(+)-induced K(+) efflux (an indicator of salt tolerance) and leaf glycine betaine and proline. These results indicate that hyperaccumulation of known major compatible solutes in barley does not appear to play a major role in salt-tolerance, but rather, may be a symptom of salt-susceptibility.     

Effects of salt stress in relation to osmotic adjustment on sugarcane (<Emphasis Type="Italic">Saccharum officinarum</Emphasis> L.) callus cultures

In vitro responses of embryogenic sugarcane (Saccharum officinarum L.; cv. CoC-671) calli stressed with different levels of NaCl (0.0, 42.8, 85.6, 128.3, 171.1, 213.9 or 256.7 mM) were studied. The results showed that a significant decrease in callus growth and cell viability occurred with ≥85.6 mM NaCl. Higher amounts of free proline and glycine betaine were accumulated in NaCl-stressed calli. Although the leached and retained Na⁺ contents increased, the retained K⁺ content decreased with increasing levels of NaCl. Such a mechanism implies that sugarcane can be considered as a Na⁺-excluder. The accumulation of salt ions and osmolytes could play an important role in osmotic adjustment in sugarcane cells under salt stress.     

Protection of Bacillus subtilis against cold stress via compatible-solute acquisition

Accumulation of compatible solutes is a strategy widely employed by bacteria to achieve cellular protection against high osmolarity. These compounds are also used in some microorganisms as thermostress protectants. We found that Bacillus subtilis uses the compatible solute glycine betaine as an effective cold stress protectant. Glycine betaine strongly stimulated growth at 15°C and permitted cell proliferation at the growth-inhibiting temperature of 13°C. Initial uptake of glycine betaine at 15°C was low but led eventually to the buildup of an intracellular pool whose size was double that found in cells grown at 35°C. Each of the three glycine betaine transporters (OpuA, OpuC, and OpuD) contributed to glycine betaine accumulation in the cold. Protection against cold stress was also accomplished when glycine betaine was synthesized from its precursor choline. Growth of a mutant defective in the osmoadaptive biosynthesis for the compatible solute proline was not impaired at low temperature (15°C). In addition to glycine betaine, the compatible solutes and osmoprotectants l-carnitine, crotonobetaine, butyrobetaine, homobetaine, dimethylsulfonioactetate, and proline betaine all served as cold stress protectants as well and were accumulated via known Opu transport systems. In contrast, the compatible solutes and osmoprotectants choline-O-sulfate, ectoine, proline, and glutamate were not cold protective. Our data highlight an underappreciated facet of the acclimatization of B. subtilis to cold environments and allow a comparison of the characteristics of compatible solutes with respect to their osmotic, heat, and cold stress-protective properties for B. subtilis cells.     

Salinity stress responses in the plant growth promoting rhizobacteria,Azospirillum spp

In order to adapt to the fluctuations in soil salinity/osmolarity the bacteria of the genusAzospirillum accumulate compatible solutes such as glutamate, proline, glycine betaine, trehalose, etc. Proline seems to play a major role in osmoadaptation. With increase in osmotic stress the dominant osmolyte inA. brasilense shifts from glutamate to proline. Accumulation of proline inA. brasilense occurs by both uptake and synthesis. At higher osmolarityA. brasilense Sp7 accumulates high intracellular concentration of glycine betaine which is taken up via a high affinity glycine betaine transport system. A salinity stress induced, periplasmically located, glycine betaine binding protein (GBBP) of ca. 32 kDa size is involved in glycine betaine uptake inA. brasilense Sp7. Although a similar protein is also present inA. brasilense Cd it does not help in osmoprotection. It is not known ifA. brasilense Cd can also accumulate glycine betaine under salinity stress and if the GBBP-like protein plays any role in glycine betaine uptake. This strain, under salt stress, seems to have inadequate levels of ATP to support growth and glycine betaine uptake simultaneously. ExceptA. halopraeferens, all other species ofAzospirillum lack the ability to convert choline into glycine betaine. Mobilization of thebet ABT genes ofE. coli intoA. brasilense enables it to use choline for osmoprotection. Recently, aproU-like locus fromA. lipoferum showing physical homology to theproU gene region ofE. coli has been cloned. Replacement of this locus, after inactivation by the insertion of kanamycin resistance gene cassette, inA. lipoferum genome results in the recovery of mutants which fail to use glycine betaine as osmoprotectant.     

Accumulation of proline and glycine betaine in Ipomoea pes-caprae induced by NaCl

The present study pertains to the effect of different concentration of NaCl on the contents of proteins, free amino acids, proline and glycine betaine in leaves, stems and roots of Ipomoea pes-caprae. The protein content of the tissues increased in response to salinity upto 200 mM NaCl; the free amino acids content showed a reversal trend. The proline and glycine betaine contents increased with increasing salinity upto 500 mM NaCl. The accumulation of proline and glycine betaine might play a role in the alleviation of salt stress. This revised version was published online in July 2006 with corrections to the Cover Date.     

In planta function of compatible solute transporters of the AtProT family

The three proline transporters of Arabidopsis thaliana (AtProTs) transport the compatible solutes proline and glycine betaine and the stress-induced compound γ-aminobutyric acid when expressed in heterologous systems. The aim of the present study was to show transport and physiological relevance of these three AtProTs in planta. Using single, double, and triple knockout mutants and AtProT-overexpressing lines, proline content, growth on proline, transport of radiolabelled betaine, and expression of AtProT genes and enzymes of proline metabolism were analysed. AtProT2 was shown to facilitate uptake of L- and D-proline as well as [(14)C]glycine betaine in planta, indicating a role in the import of compatible solutes into the root. Toxic concentrations of L- and D-proline resulted in a drastic growth retardation of AtProT-overexpressing plants, demonstrating the need for a precise regulation of proline uptake and/or distribution. Furthermore evidence is provided that AtProT genes are highly expressed in tissues with elevated proline content--that is, pollen and leaf epidermis.     

Effects of optimal and supra-optimal salinity stress on antioxidative defence,osmolytes and in vitro growth responses in <Emphasis Type="Italic">Sesuvium portulacastrum</Emphasis> L.

The effect of optimal and supra-optimal concentrations (0, 200, 400 or 600 mM) of NaCl on the growth, osmotic adjustment and antioxidant enzyme defence was studied in the in vitro cultures of Sesuvium portulacastrum. A significant increase in growth, tissue water content (TWC) and fresh to dry weight ratio (FW/DW) was observed in the shoots exposed to 200 mM salt. Minimum damage to the membrane in terms of low relative electrolytic leakage (REL) and malondialdehyde (MDA) content and better osmotic adjustment at 200 mM salt stress was coupled with the higher accumulation of sodium ions and total soluble sugars as against low proline and glycine betaine contents. A fine tuning of antioxidant enzyme activities (superoxide dismutase, catalase and ascorbate peroxidase) was also found to be responsible for the optimum growth of shoots. In contrast, sub-optimal (0 mM) and supra-optimal concentrations (400–600 mM) of NaCl significantly affected the growth, water status and increased the REL as well as MDA content of the shoots due to the accumulation of toxic concentrations of saline ions. The highest accumulation of proline and glycine betaine in addition to antioxidant enzyme activities exhibited higher osmotic adjustment and survival of the shoots under sub- or supra-optimal concentrations of NaCl as a penalty to reduced growth.     

Role for glycine betaine transport in Vibrio cholerae osmoadaptation and biofilm formation within microbial communities

Vibrio cholerae is a halophilic facultative human pathogen found in marine and estuarine environments. Accumulation of compatible solutes is important for growth of V. cholerae at NaCl concentrations greater than 250 mM. We have identified and characterized two compatible solute transporters, OpuD and PutP, that are involved in uptake of glycine betaine and proline by V. cholerae. V. cholerae does not, however, possess the bet genes, suggesting that it is unable to synthesize glycine betaine. In contrast, many Vibrio species are able to synthesize glycine betaine from choline. It has been shown that many bacteria not only synthesize but also secrete glycine betaine. We hypothesized that sharing of compatible solutes might be a mechanism for cooperativity in microbial communities. In fact, we have demonstrated that, in high-osmolarity medium, V. cholerae growth and biofilm development are enhanced by supplementation with either glycine betaine or spent media from other bacterial species. Thus, we propose that compatible solutes provided by other microorganisms may contribute to survival of V. cholerae in the marine environment through facilitation of osmoadaptation and biofilm development.     

Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes.

The concentrations of intracellular solutes in Listeria monocytogenes were examined in cells grown at various concentrations of NaCl. At 5% NaCl, cells contained elevated concentrations of potassium and glycine betaine compared with concentrations in cells grown without NaCl. At 7.5% NaCl, cells contained increased concentrations of K+, glycine betaine, glycine, alanine, and proline. Only glycine betaine, choline, or glycine promoted growth on a solidified defined medium containing 4% NaCl; there was no growth at higher concentrations of NaCl in the defined medium.     

Biochemical changes induced by salt stress in halotolerant bacterial isolates are media dependent as well as species specific

Halophilic bacteria respond to salt stress by regulating the cytosolic pools of organic solutes to achieve osmotic equilibrium. In order to understand the metabolic regulation of these organic solutes, for the first time, we have investigated the effect of salt on growth and biochemical changes in four major moderately halophilic bacterial strains isolated from a saltern region of the Kumta coast, India. The strains under study were Halomonas hydrothermalis VITP9, Bacillus aquimaris VITP4, Planococcus maritimus VITP21, and Virgibacillus dokdonensis VITP14, which exhibited similar salt tolerance (0% to 10% w/v NaCl) with optimal growth at 5% w/v NaCl. Biochemical analysis showed that the total intracellular organic solutes increased significantly with increasing NaCl concentration in the growth medium, and the compositions of the solutes were dependent on the type of strain and also on the nutrient richness of the growth medium. Glutamic acid levels increased in all the strains under salt stress, indicating the significance of glutamic acid as the anionic counterpart of K⁺/Na⁺ ions and precursor for other synthesized nitrogenous osmolytes. Though initial studies were performed with thin-layer chromatography, mass spectrometry was used to identify the major solutes accumulated by the strains under salt stress, such as proline (VITP4), ectoine (VITP14 and VITP9), and sugars (VITP21) under minimal medium and glycine betaine (by all the strains under study) under complex growth medium conditions. Such comparative study on the stress-dependent metabolic differences of different microbes, under identical experimental condition, helps to identify possible bacterial sources for the production of industrially important solutes.     

Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes.

The concentrations of intracellular solutes in Listeria monocytogenes were examined in cells grown at various concentrations of NaCl. At 5% NaCl, cells contained elevated concentrations of potassium and glycine betaine compared with concentrations in cells grown without NaCl. At 7.5% NaCl, cells contained increased concentrations of K+, glycine betaine, glycine, alanine, and proline. Only glycine betaine, choline, or glycine promoted growth on a solidified defined medium containing 4% NaCl; there was no growth at higher concentrations of NaCl in the defined medium.     

Accumulation of free proline and glycine betaine in Aster tripolium subjected to a saline shock: A kinetic study related to light period

On transferring three-week-old plants of Aster tripolium L. growing in a half strength Hoagland's medium to the same medium containing 333 m M NaCl a very quick uptake of salt and, after a lag phase of 3 to 5 h, an increase in free proline level was observed. During the time course of imino acid storage, the accumulation rates were higher in the light than in the dark, thereby suggesting some kind of photocontrol on solute metabolism. At zero time, high levels of glycine betaine were present in young plants grown without salt. However, after the application of saline shock, the betaine level also increased significantly. The highest rate of betaine accumulation was detected during the third day of treatment when the rate of proline storage decreased. Glycine betaine storage could also be linked to light dependent processes; whatever its importance in response to salt shock was, the levels observed were lower than those of plants directly grown on 333 m M NaCl for three weeks. When saline stressed plants were transferred to a medium without NaCl, the proline level quickly decreaed while that of glycine betaine remained stable.     

Functional expression of Sinorhizobium meliloti BetS, a high-affinity betaine transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Osteras, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na(+)/H(+) antiporters in B. japonicum could explain its very high Na(+) sensitivity.     

LeProT1, a transporter for proline, glycine betaine, and gamma-amino butyric acid in tomato pollen

During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes. Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70% of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and gamma-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.     

耐盐药蒲公英(Taraxacum officinale Weber)愈伤组织筛选及生理生化特性分析

为获得耐1.5% NaCl的药蒲公英(Taraxacum officinale Weber)愈伤组织, 以药蒲公英叶片外植体为材料诱导愈伤组织。以NaCl为选择因子, 从愈伤组织直接筛选。在选择培养基上, 大部分愈伤组织褐化死亡, 个别褐化死亡的愈伤组织周围有少量新的细胞团长出, 将其转接到新鲜的选择培养基上, 每3周继代一次, 经3个月继代筛选获得了耐1.5% NaCl的药蒲公英细胞团。以普通愈伤组织为对照, 发现随着NaCl浓度升高, 耐盐愈伤组织的相对生长率下降但显著高于对照; 且随着盐胁迫处理时间延长持续升高, 而普通愈伤组织对照几乎停止生长, 说明耐盐愈伤组织具有相对稳定的耐盐性。在蛋白水平上, 耐盐愈伤组织与对照愈伤组织差异明显, SDS-PAGE分析显示: 耐盐愈伤组织比对照多出一条34 kD大小的蛋白带, 且30 kD、18 kD左右的蛋白带明显上调。相同处理条件下耐盐愈伤组织脯氨酸的增加幅度高于对照。盐胁迫条件下, 耐盐愈伤组织的超氧化物歧化酶(Super oxidase dimutase, SOD)、过氧化物酶(Peroxidase, POD)和过氧化氢酶(Catalase, CAT)活性明显高于对照,且随着处理时间的延长和盐浓度的增加呈现升高的趋势, 而对照则呈现先升高后下降的趋势。结果说明耐盐愈伤组织一方面通过小分子有机溶质如脯氨酸的方式调节其渗透平衡, 另一方面还可通过提高抗氧化能力降低盐分造成的次级伤害。积累蛋白也可能是耐盐愈伤组织调节渗透平衡的一种方式。通过生理生化分析确定我们获得的耐盐愈伤组织为耐盐变异体。