Influence of NaCl on Growth, Proline, and Phosphoenolpyruvate Carboxylase Levels in Mesembryanthemum crystallinum Suspension Cultures

The facultative halophyte Mesembryanthemum crystallinum responds to salt stress by increasing the levels of phosphoenolpyruvate carboxylase (PEPCase) and other enzymes associated with Crassulacean acid metabolism. A more common response to salt stress in sensitive and tolerant species, including M. crystallinum, is the accumulation of proline. We have established M. crystallinum suspension cultures to investigate whether both these salt-induced responses occur at the cellular level. Leaf-and root-derived cultures maintain 5% of the total soluble amino acids as proline. Cell culture growth slows upon addition of 400 millimolar NaCl, and proline levels increase to 40% of the total soluble amino acids. These results suggest a functional salt-stress and response program in Mesembryanthemum cells. Suspension cultures grown with or without 400 millimolar NaCl have PEPCase levels that compare with those from roots and unstressed leaves. The predominant protein cross-reacting with an anti-PEPCase antibody corresponds to 105 kilodaltons (apparent molecular mass), whereas a second species of approximately 110 kilodaltons is present at low levels. In salt-stressed leaves, the 110 kilodalton protein is more prevalent. Levels of mRNA for both ppc1 (salt stress induced in leaves) and ppc2 (constitutive) genes in salt-treated suspensions cultures are equal to unstressed leaves, and only twice the levels found in untreated suspension cultures. Whereas cells accumulate proline in response to NaCl, PEPCase protein amounts remain similar in salt-treated and untreated cultures. The induction upon salt stress of the 110 kilodalton PEPCase protein and other Crassulacean acid metabolism enzymes in organized tissues is not observed in cell culture and may depend on tissue-dependent or photoautotrophy-dependent programs.     

Influence of proline and hydroxyproline on salt-stressed axillary bud cultures of two varieties of potato (Solanum tuberosum)

Summary The effects of exogenously supplied proline and hydroxyproline on the potato (Solanum tuberosum) varieties L.T.8 and Desiree were studied using axillary bud cultures both in the presence and absence of 0.6% salt stress. In both varieties, the effects of exogenously supplied proline and hydroxyproline at 1.0 mM and 2.5 mM were less severe than 0.6% salt alone. At the same time, the accumulation of proline, protein, carbohydrates, sodium, and potassium were similar. However, when both the salt and proline/hydroxyproline were supplied, proline and hydroxyproline provided some measure of protection against salt stress. It is believed that increased proline levels in L.T.8 and increased carbohydrates in Desiree due to the presence of exogenously supplied proline/hydroxyproline were responsible for the additional protection against salt stress in the axillary bud cultures of these varieties.     

Comparative physiological and metabolomic responses of four <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> varieties contrasting in drought stress resistance

Brachypodium distachyon (Brachypodium) is not only a monocot grass species, but also a promising model organism of crop research. In this study, the drought resistance of four Brachypodium varieties was identified including drought stress-tolerant Bd1-1 and Bd21, drought stress-susceptible Bd3-1 and Bd18-1. Physiological assay showed that drought-tolerant varieties (Bd1-1 and Bd21) were more effective in maintenance of leaf water content, activation of catalase and peroxidase activities and accumulation of reduced glutathione, resulting in alleviated cell damage and lower reactive oxygen species level than drought-susceptible varieties (Bd3-1 and Bd18-1) in response to drought stress. In addition, 54 primary metabolites were differentially regulated among Brachypodium varieties and after drought stress treatment, indicating the complexity of Brachypodium response to drought stress. We also identified several commonly regulated metabolites especially some compatible solutes including proline and soluble sugars, which exhibited higher concentrations in the drought-tolerant varieties. Taken together, this study suggested that natural variation of Brachypodium varieties in response to drought stress might be connected with higher leaf water, enhanced accumulation of osmolyte and more effective antioxidant system, as well as the modulation of metabolic profiles under drought stress conditions.     

Influence of salinity on axillary bud cultures of six lowland tropical varieties of potato (Solanum tuberosum)

Axillary bud cultures of six low-land tropical varieties of potato Solanum tuberosum (L. T. 2, 5, 6, 7, 8 and 9) were studied for their response to salinity stress using crude sea salt. Concentrations of 0.8% and above severely inhibited the growth, while decreased growth was observed between 0.4% and 0.6% salt in all the varieties, with occasional shoot tip necrosis and compound leaf formation. A lower concentration of salt (0.2%) was beneficial and increased shoot weight in most varieties. Protein content showed a positive correlation with proline levels, indicating that proline accumulation was due to new synthesis rather than breakdown of existing proteins. Soluble carbohydrates increased in the stem with increasing salt. Roots showed an increase in sodium content while shoots showed increased potassium levels, with increasing salt concentrations. There was a positive correlation between dry weight yields and K/Na ratio. It is apparent that the synthesis of proline, accumulation of soluble carbohydrates as well as increase in potassium and sodium content were all used to adjust the physiology in response to salt stress by the different varieties of potato, with varying degrees, in the present study.     

Stress‐driven increase in proline levels,and not proline levels themselves,correlates with the ability to withstand excess salt in a group of 17 Italian rice genotypes

• In most plant species, a rapid increase in free proline content occurs following exposure to hyperosmotic stress conditions. However, inconsistent results were reported concerning the role of such an increase on the plant response to water shortage or excess salt. Therefore, the possibility that proline accumulation may help the cell to withstand stress conditions, or that it simply represents a stress marker, is still a matter of debate. A possible relationship between proline accumulation and salt tolerance was investigated in a set of 17 Italian rice varieties.
• Rice seedlings were exposed to increasing salt concentrations during germination and early growth. The resulting levels of free proline were measured separately in shoots and roots and compared to those in untreated controls. Results were related to the corresponding ability of a given genotype to tolerate stress conditions.
• Neither absolute proline levels in untreated or in salt‐stressed seedlings showed a straightforward relationship to the relative tolerance to salt, estimated as conductivity values able to reduce growth by 10 or 50%. Conversely, a highly significant correlation was found between the increase in proline levels in shoots and the ability to withstand stress.
• The results strengthen a recent hypothesis suggesting than an increase in proline metabolic rates, more than the resulting proline content, may help the cell to counteract the effects of abiotic stress conditions.

     

Effect of water stress on proline synthesis from radioactive precursors

Barley (Hordeum vulgare L. var. Prior) leaves converted more ¹⁴C-glutamic acid to free proline when water-stressed than when turgid; neither decreased protein synthesis nor isotope trapping by the enlarged free proline pools found in wilted tissue seemed to account for the result. This apparent stimulation of proline biosynthesis in wilted leaves was not observed when radioactive ornithine or P5C (Δ¹-pyrroline-5-carboxylate, an intermediate following glutamate in proline synthesis) were used as proline precursors unless proline levels were high as a result of previous water stress. We interpret this to mean that any stimulation of proline synthesis by water stress must act on P5C formation rather than its reduction to proline. Experiments showing greater apparent conversion of ¹⁴C-glutamate to proline do not unequivocally prove that proline synthesis is stimulated by water stress, as P5C feeding studies show that proline oxidation is inhibited under comparable conditions. This inhibition could account, at least in part, for increased proline labeling, and must be considered an alternate possibility.     

Antioxidant response to drought in red and white clover

Antioxidant response to drought in red (Trifolium pratense L., cv. ??Start??) and white clover (Trifolium repens L, cv. ??Haifa?? and cv. ??Debut??) grown as soil cultures was evaluated in water-deprived and recovered plants. Drought provoked oxidative stress in leaves confirmed by the considerable changes in electrolyte leakage, malondialdehyde, hydrogen peroxides and proline contents. Immunoblot of ??-1-pyrroline-5-carboxylate synthetase (P5CS), which catalyzes the first two steps in proline biosynthesis, revealed strong induction of the enzyme in red clover plants submitted to drought. Water-deprived white clover plants exhibited distinct P5CS profiles. This was related to different drought tolerance of the studied T. repens cultivars. Isoenzyme analyses of superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) demonstrated certain differences in antioxidant defence among the tested varieties. It was confirmed that MnSOD (in both T. repens and T pratense) and FeSOD (in T. repens) isoforms were the most affected by drought. The red clover cultivar ??Start?? exhibited the lowest FeSOD and POX activities which could contribute to its poor performance under water deprivation.     

Effect of ploidy levels on the activities of Δ1-pyrroline-5-carboxylate synthetase,superoxide dismutase and peroxidase in Cenchrus species grown under water stress

The effects of ploidy levels on the activities of Δ¹-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned), superoxide dismutase (SOD; EC 1.15.1.1) and guaiacol peroxidase (POX; EC 1.11.1.7), as well as malondialdehyde (MDA) and proline contents were studied in two months old plants of Cenchrus species. The Cenchrus species represent three ploidy levels: diploid, tetraploid, hexaploid and two life spans: annual and perennial. Plants were subjected to water stress for 2, 4, 6 and 8 d by withholding water under glasshouse conditions. Although the levels of proline increased with the magnitude of water stress, the P5CS activity did not show a corresponding increase in all species. Peroxidase and superoxide dismutase activities showed an increase or steady state in the early phase of drought and then declined with the further increase in the magnitude of water stress, indicating differing behaviors of species towards drought tolerance. Under drought, diploid Cenchrus species had a higher POX activity, MDA accumulation and lower proline content than tetraploid species. Lower POX and higher P5CS activities and proline contents, however, were observed in hexaploid and tetraploid species. Taken together, our findings suggest that diploid species have a less efficient antioxidant system to scavenge reactive oxygen species than tetra and hexaploid Cenchrus. This may result in a corresponding variability in growth and persistence under natural grasslands. The study also paves the way for investigations on the molecular events associated with drought in Cenchrus species differing in ploidy and life span.     

Mineral composition of and proline accumulation by zostera marina L. in response to environmental salinity

Plants of Zostera marina L. collected in the field were grown for several weeks at different salinities under controlled conditions. After this period plants were harvested and the plant tissue water was analysed for proline, Na, K and Cl, which increased in response to seawater concentration as did osmolality. Proline accumulated up to 22 mmoll⁻¹ tissue water at the highest seawater concentration. Proline synthesis or breakdwon was found to be slow in response to changes in environmental salinity. Nitrogen levels in Zostera were high (5% dry weight); a possible drain of proline on plant nitrogen is discussed. In vitro activity of malate dehydrogenase (MDH) was inhibited by NaCl; proline did not affect MDH activity, not even at 1 mol proline l⁻¹. The role of proline in osmotic adaptation of Zostera marina is discussed.     

Medicago truncatula stress associated protein 1 gene (MtSAP1) overexpression confers tolerance to abiotic stress and impacts proline accumulation in transgenic tobacco

Stress associated proteins (SAP) have been already reported to play a role in tolerance acquisition of some abiotic stresses. In the present study, the role of MtSAP1 (Medicago truncatula) in tolerance to temperature, osmotic and salt stresses has been studied in tobacco transgenic seedlings. Compared to wild type, MtSAP1 overexpressors were less affected in their growth and development under all tested stress conditions. These results confirm that MtSAP1 is involved in the response processes to various abiotic constraints. In parallel, we have performed studies on an eventual link between MtSAP1 overexpression and proline, a major player in stress response. In an interesting way, the results for the transgenic lines did not show any increase of proline content under osmotic and salt stress, contrary to the WT which usually accumulated proline in response to stress. These data strongly suggest that MtSAP1 is not involved in signaling pathway responsible for the proline accumulation in stress conditions. This could be due to the fact that the overexpression of MtSAP1 provides sufficient tolerance to seedlings to cope with stress without requiring the free proline action. Beyond that, the processes by which the MtSAP1 overexpression lead to the suppression of proline accumulation will be discussed in relation with data from our previous study involving nitric oxide.     

Jasmonic acid interacts with abscisic acid to regulate plant responses to water stress conditions

Phytohormones are key players in signaling environmental stress conditions. Hormone profiling together with proline accumulation were studied in leaves and roots of different mutant lines of Arabidopsis. Regulation of proline accumulation in this system seems complex and JA-deficient (jar1-1) and JA-insensitive (jai1) lines accumulating high levels of proline despite their very low ABA levels seems to discard an ABA-dependent response. However, the pattern of proline accumulation in jai1 seedlings parallels that of ABA. Under stress conditions, there is an opposite pattern of ABA accumulation in roots of jar1-1/coi1-16 (in which ABA only slightly increase) and jai1 (in which ABA increase is even higher than in WT plants). This also makes JA-ABA crosstalk complex and discards any lineal pathway that could explain this hormonal interaction.     

Analysis of Natural Variation in Bermudagrass (Cynodon dactylon) Reveals Physiological Responses Underlying Drought Tolerance

Bermudagrass (Cynodon dactylon) is a widely used warm-season turfgrass and one of the most drought tolerant species. Dissecting the natural variation in drought tolerance and physiological responses will bring us powerful basis and novel insight for plant breeding. In the present study, we evaluated the natural variation of drought tolerance among nine bermudagrass varieties by measuring physiological responses after drought stress treatment through withholding water. Three groups differing in drought tolerance were identified, including two tolerant, five moderately tolerant and two susceptible varieties. Under drought stress condition, drought sensitive variety (Yukon) showed relative higher water loss, more severe cell membrane damage (EL), and more accumulation of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), while drought tolerant variety (Tifgreen) exhibited significantly higher antioxidant enzymes activities. Further results indicated that drought induced cell injury in different varieties (Yukon, SR9554 and Tifgreen) exhibited liner correlation with leaf water content (LWC), H₂O₂ content, MDA content and antioxidant enzyme activities. Additionally, Tifgreen plants had significantly higher levels of osmolytes (proline level and soluble sugars) when compared with Yukon and SR9554 under drought stress condition. Taken together, our results indicated that natural variation of drought stress tolerance in bermudagrass varieties might be largely related to the induced changes of water status, osmolyte accumulation and antioxidant defense system.     

Biochemical,physiological and molecular evaluation of rice cultivars differing in salt tolerance at the seedling stage

Changes in the antioxidant enzymes, lipid peroxidation, sodium and potassium, chlorophyll, H₂O₂ and proline content were monitored in the leaves of 42 rice varieties which were not yet well-documented for the salinity tolerance under different salinity levels. The tolerant varieties (FL478, Hassani, Shahpasand, Gharib and Nemat) showed signs of tolerance (lower Na⁺/K⁺ ratio, high proline accumulation, less membrane damage, lower H₂O₂ production, and higher superoxide dismutase and catalase activity) very well. The positive relationship between the level of salt tolerance and the amount of proline accumulation in the rice varieties support the important role of proline under the salt stress. The varieties were genotyped for 12 microsatellite markers that were closely linked to SalTol QTL. The results of association analysis indicated that RM1287, RM8094, RM3412 and AP3206 markers had the high value of R² for the regression models of the studied traits. It shows the important role of SalTol in controlling physio-biochemical traits. The results can be used in the future marker assisted selection (MAS) directly, if the results are confirmed.     

Effect of sodium and calcium chlorides,abscisic acid and proline on callus cultures ofArachis hypogaea L.

Callus cultures ofArachis hypogaea L. cv. JL-24 adapted to 200 mM NaCl (otherwise lethal to cells) were used for the study. Calli grew slowly when transferred to 250 mM NaCl, but the growth was enhanced when ABA was included in the medium. ABA induced increase in growth of callus was not accompanied by corresponding increase in internal free proline levels. 0.5 mM of CaCl₂ ameliorated the negative effect of NaCl indicating that cells require a specific Ca²⁺/Na⁺ ratio for their growth. Proline content also increased at this ratio thereby suggesting that increase in growth at 0.5 mM Ca²⁺ may be due to an increase in proline content. However, exogenous proline did not increase the growth of callus (adapted to 200 mM), and higher concentrations even inhibited the growth. This shows that proline is not required for growth or adaptation of cells to salt stress, but is produced as a consequence of stress.     

Changes in abscisic acid and proline levels in maize varieties of different drought resistance

Four varieties of maize differing in drought resistance and geographical origin (Swabi White and Shaheen from Pakistan, Garbo and Goldprinz from Germany) were analyzed for their proline and abscisic acid (ABA) accumulation during a prolonged water stress period. Proline levels increased continuously during the stress period in all the four varieties, but to different amounts. The drought-susceptible varieties Shaheen and Goldprinz produced higher levels of proline than the drought-resistant varieties Swabi White and Garbo. A negative correlation was also found between maximal ABA contents and degree of drought resistance during prolonged stress of younger plants. ABA levels did not increase steadily, but reached a maximum long before the end of the stress phase, and then declined. The results are discussed in relation to the possibility of using proline and ABA levels as biochemical indicators of resistance against drought.     

Variability in Proline-Accumulating Ability of Barley (Hordeum vulgare L.) Cultivars Induced by Vapor Pressure Deficit

This work was undertaken in an effort to reconcile the conflicting proline-accumulating responses of the barley (Hordeum vulgare L.) cultivars, Excelsior and Proctor, reported by Singh et al. (1972) and Hanson et al. (1976). It deals with the effects of different vapor pressure deficits (VPD) during growth and subsequent drought stress on several barley cultivars. A higher VPD (1.2 kilopascals) during Clipper seedling growth resulted in higher solute-accumulating ability, seemingly independently of leaf water potential, than a lower VPD (0.12 kilopascals). The higher VPD during stress also resulted in higher solute contents, and this response may be more closely related to leaf water potential. When the responses of Excelsior and Proctor were examined in detail, it was found that the relative proline-accumulating ability of the two cultivars was dependent upon the VPD under which they were grown. At low VPD, Proctor accumulated significantly more proline than did Excelsior; whereas at higher VPD, Excelsior accumulated more proline than did Proctor. The crossover occurred at a VPD of about 0.72 kilopascals. This reversal of cultivar response was enhanced by multiplying seed under the two VPD extremes. Glycinebetaine accumulation did not demonstrate the crossover effect, although the concentration of this compound in all cultivars also depended on the VPD prevailing during growth and/or stress. Solute levels, in general, were more closely related to the decrease in relative water content than to a decrease in leaf water potential. It is concluded that the conflicting proline-accumulating responses of Excelsior and Proctor could be explained by these findings.