Effects of ALA on Photosynthesis,Antioxidant Enzyme Activity,and Gene Expression,and Regulation of Proline Accumulation in Tomato Seedlings Under NaCl Stress

This study examined the effects of 5-aminolevulinic acid (ALA) application on photosynthesis, activity and gene expression of key antioxidant enzymes, and on proline accumulation in tomato (Lycopersicon esculentum Mill. ‘Hezuo 903’) seedlings under NaCl stress. NaCl stress significantly decreased the net photosynthetic rates and inhibited the activity of photosystem II, whereas exogenous ALA application significantly restored the net photosynthetic rates, quantum yield of electron transport, and energy conversion efficiency of photosystem II of tomato under NaCl stress. Production of superoxide, hydrogen peroxide, and malondialdehyde strongly increased in response to NaCl stress, and these increases were significantly counteracted by ALA. ALA increased the activity of reactive oxygen species (ROS) scavenging antioxidant enzymes, including superoxide dismutase, catalase, ascorbate peroxidase, and peroxidase, and upregulated the expression of SOD, APX, and POD, genes that encode these enzymes in NaCl-treated plants. ALA simultaneously increased proline accumulation in tomato seedlings under NaCl stress by regulating the expression of genes that encode ALA biosynthetic enzymes and that control proline biosynthesis and metabolism, for example, expression of GluRS and GluTR was downregulated, accompanied by a significant increase in the expression of P5CS and decline in the expression of ProDH. ALA provided protection against NaCl stress by increasing photosynthetic capacity, regulating antioxidant enzyme gene expression and proline accumulation, and decreasing ROS accumulation and lipid peroxidation in tomato.     

Reactive oxygen species, ascorbate–glutathione pool, and enzymes of their metabolism in drought-sensitive and tolerant indica rice (Oryza sativa L.) seedlings subjected to progressing levels of water deficit

Water deficit for rice is a worldwide concern, and to produce drought-tolerant varieties, it is essential to elucidate molecular mechanisms associated with water deficit tolerance. In the present study, we investigated the differential responses of nonenzymatic antioxidants ascorbate (AsA), glutathione (GSH), and their redox pool as well as activity levels of enzymes of ascorbate–glutathione cycle in seedlings of drought-sensitive rice (Oryza sativa L.) cv. Malviya-36 and drought-tolerant cv. Brown Gora subjected to water deficit treatment of ?1.0 and ?2.1 MPa for 24–72 h using PEG-6000 in sand cultures. Water deficit caused increased production of reactive oxygen species such as O₂?^?, H₂O₂, and HO? in the tissues, and the level of production was higher in the sensitive than the tolerant cultivar. Water deficit caused reduction in AsA and GSH and decline in their redox ratios (AsA/DHA and GSH/GSSG) with lesser decline in tolerant than the sensitive seedlings. With progressive level of water deficit, the activities of monodehydroascorbate reductase, dehydroascorbate reductase, ascorbate peroxidase (APX), and glutathione transferase increased in the seedlings of both rice cultivars, but the increased activity levels were higher in the seedlings of drought-tolerant cv. Brown Gora compared to the sensitive cv. Malviya-36. Greater accumulation of proline was observed in stressed seedlings of tolerant than the sensitive cultivar. In-gel activity staining of APX revealed varying numbers of their isoforms and their differential expression in sensitive and tolerant seedlings under water deficit. Results suggest that an enhanced oxidative stress tolerance by a well-coordinated cellular redox state of ascorbate and glutathione in reduced forms and induction of antioxidant defense system by elevated activity levels of enzymes of ascorbate–glutathione cycle is associated with water deficit tolerance in rice.     

Improving drought stress tolerance in fenugreek (Trigonella foenum-graecum) by exogenous melatonin

Abstract

The present study was conducted to determine the changes which occurred in the water-stressed fenugreek (Trigonella foenum-gracum L.) in response to melatonin treatment. Fenugreek is a valuable medicinal plant which contains trigonelline that plays an osmoregulatory role in abiotic stress. To this end, the effects of three levels of water deficit in the first experiment and four concentrations of melatonin in the second experiment were investigated on the physiological and biochemical responses. The findings of the first experiment demonstrated that water deficit decreased the weight and length of fenugreek shoots as well as pigments contents, and catalase activity. In addition, such a deficit led to an increase in proline, trigonelline, melatonin and enhanced the activity of other antioxidant enzymes. Finally, the hydrogen peroxide and malondialdehyde concentrations increased under severe stress. The results of the second experiment indicated that melatonin treatment significantly reduced chlorophyll degradation. Furthermore, the activities of the ROS scavenging enzymes were increased by melatonin. Moreover, exogenous melatonin led to an increase in endogenous melatonin and trigonelline whereas it led to a decrease in proline content. These results revealed that fenugreek tolerated water deficit by increasing the endogenous melatonin and trigonelline, as well as the general physiological responses.     

Changes in antioxidant enzyme activities and gene expression in two muskmelon genotypes under progressive water stress

Responses of two muskmelon (Cucumis melo L.) genotypes (drought tolerant SC-15 and drought susceptible EC-564755) were analyzed at 0, 7, 14, and 21 d of progressive water stress. Although water deficit caused a significant decline in relative water content, the magnitude of reduction was lower in SC-15. Electrolyte leakage, hydrogen peroxide, and malonydialdehyde generation were higher in EC-564755, whereas accumulation of proline was higher in SC-15. Higher activities of antioxidant enzymes, such as catalase, superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, and glutathione reductase, and higher expression of the respective genes were recorded in SC-15 than in EC-564755. Expressions of DREB2C and DREB3 in SC-15 revealed a fluctuating pattern with down-regulation on days 7 and 21 of water stress, whereas up-regulation was observed on day 14. Concurrently, both genes in EC-564755 showed continuous down-regulation on days 7, 14, and 21 of water stress. Expressions of RD22 and dehydrin recorded on days 7, 14, and 21 were lower in SC-15. The cluster analysis showed that, these two genotypes had a clear distinction in physiological and biochemical properties and gene expressions under water stress and the genotype SC-15 had more efficient osmoprotectant mechanism than genotype EC-564755 under water deficit conditions.     

Brassinosteroid-mediated evaluation of antioxidant system and nitrogen metabolism in two contrasting cultivars of Vigna radiata under different levels of nickel

The role of 28-homobrassinolide (HBL) in countering nickel-induced oxidative damage through overexpression of antioxidant enzymes and proline in Vigna radiata has been investigated. Two varieties of V. radiata, one sensitive to Ni (PDM-139) and the other tolerant to Ni (T-44), were sown in the soil fed with different levels (0, 50, 100 or 150 mg kg⁻¹) of Ni, and at 29-day stage, foliage of plants was applied with deionized water (control), 10⁻⁸ or 10⁻⁶ M of HBL. The plants were sampled at 45-day stage of growth to assess various physiological as well as biochemical characteristics. The remaining plants were allowed to grow up to maturity to study the yield characteristics. The growth traits, leghemoglobin, nitrogen and carbohydrate content in the nodules, leaf chlorophyll content, photosynthesis efficiency, leaf water potential, activities of nitrate reductase, carbonic anhydrase and nitrogenase decreased proportionately with the increasing concentrations of nickel, whereas electrolyte leakage, various antioxidant enzymes viz. catalase, peroxidase and superoxide dismutase and accumulation of proline increased at 45-day stage. However, the exogenously applied HBL to the nickel-stressed or non-stressed plants improved growth, nodulation and photosynthesis and further enhanced the various antioxidant enzymes viz. catalase, peroxidase and superoxide dismutase and accumulation of proline. The deleterious impact of Ni on the plants was concentration dependent where HBL applied to the foliage induced overexpression of antioxidant enzyme and accumulation of proline (osmolyte) which could have conferred tolerance to Ni up to 100 mg kg⁻¹, resulting in improved growth, nodulation, photosynthesis and yield attributes.     

Cold acclimation induces freezing tolerance via antioxidative enzymes, proline metabolism and gene expression changes in two chrysanthemum species

Cold acclimation is necessary for chrysanthemum to achieve its genetically determined maximum freezing tolerance, but the underlying physiological and molecular mechanisms are unclear. The aim of this study was to discover whether changes in antioxidative enzymes, proline metabolism and frost-related gene expression induced by cold acclimation are related to freezing tolerance. Our results showed that the semi-lethal temperature (LT₅₀) decreased from ?7.3 to ?23.5 °C in Chrysanthemum dichrum and ?2.1 to ?7.1 °C in Chrysanthemum makinoi, respectively, after cold acclimation for 21 days. The activities of SOD, CAT and APX showed a rapid and transient increase in the two chrysanthemum species after 1 day of cold acclimation, followed by a gradual increase during the subsequent days and then stabilization. qRT-PCR analysis showed that the expression levels of some isozyme genes (Mn SOD, CAT and APX) were upregulated, which was consistent with the SOD, CAT and APX activities, while others remained relatively constant (Fe SOD and Cu/Zn SOD). P5CS and PDH expression were increased under cold acclimation and the level of P5CS presented similar trends as proline content, indicating proline accumulation was via P5CS and PDH cooperation. Cold acclimation also promoted DREB, COR413 and CSD gene expression. The activities of three enzymes and gene expression were higher in C. dichrum than in C. makinoi after cold acclimation. Our data suggested that cold-inducible freezing-tolerance could be attributed to higher activity of antioxidant enzymes, and increased proline content and frost-related gene expression during different periods.     

Mycorrhizal-Mediated Lower Proline Accumulation in Poncirus trifoliata under Water Deficit Derives from the Integration of Inhibition of Proline Synthesis with Increase of Proline Degradation

Proline accumulation was often correlated with drought tolerance of plants infected by arbuscular mycorrhizal fungi (AMF), whereas lower proline in some AM plants including citrus was also found under drought stress and the relevant mechanisms have not been fully elaborated. In this study proline accumulation and activity of key enzymes relative to proline biosynthesis (▵¹-pyrroline-5-carboxylate synthetase, P5CS; ornithine-δ-aminotransferase, OAT) and degradation (proline dehydrogenase, ProDH) were determined in trifoliate orange (Poncirus trifoliata, a widely used citrus rootstock) inoculated with or without Funneliformis mosseae and under well-watered (WW) or water deficit (WD). AMF colonization significantly increased plant height, stem diameter, leaf number, root volume, biomass production of both leaves and roots and leaf relative water content, irrespectively of water status. Water deficit induced more tissue proline accumulation, in company with an increase of P5CS activity, but a decrease of OAT and ProDH activity, no matter whether under AM or no-AM. Compared with no-AM treatment, AM treatment resulted in lower proline concentration and content in leaf, root, and total plant under both WW and WD. The AMF colonization significantly decreased the activity of both P5CS and OAT in leaf, root, and total plant under WW and WD, except for an insignificant difference of root OAT under WD. The AMF inoculation also generally increased tissue ProDH activity under WW and WD. Plant proline content significantly positively correlated with plant P5CS activity, negatively with plant ProDH activity, but not with plant OAT activity. These results suggest that AM plants may suffer less from WD, thereby inducing lower proline accumulation, which derives from the integration of an inhibition of proline synthesis with an enhancement of proline degradation.     

Effect of spermine treatment on the functioning of Thellungiella salsuginea antioxidant system

The effect of spermine (Spm) treatment on the content of polyamines (PAs) and activities of antioxidant enzymes in the roots and leaves of Thellungiella salsuginea (Pall.) O.E. Schulz plants grown under optimal conditions were studied. The genes encoding three forms of ascorbate peroxidase (APX; APX1, APX2, and APX4) and genes of key enzymes of proline metabolism (Pro, P5CS1, 1P5CD) were identified, and their expression intensity was measured. Six-day-old plants were treated with Spm (1 and 2 mM) and with the inhibitor of polyamine oxidase (PAO) activity, N,N-(2-hydroxyethyl)hydrazine (HEH, 1 and 2 mM) separately or in combination by adding these compounds to nutrient medium. Roots and leaves responded differently to Spm treatment. In the leaves, the content of PAs reduced due to a decreased in the spermidine (Spd) content, whereas in the roots the total pool of PAs increased due to putrescine (Put) and Spd accumulation. Treatment with Spm activated PAO in the roots but not in the leaves; HEH removed this increase, but the intercellular Spm concentration was not substantially changed. It was suggested that treatment with Spm suppressed the biosynthesis of intracellular Spm and, on the other hand, stimulated the reverse conversion of Spm into Spd and further into Put due to the activation of one of the PAO isoforms. Plant treatment with Spm was not accompanied by a noticeable activation enzymes degrading hydrogen peroxide in the roots: APX, (except of peroxidase II), and catalase. However, the activity of Cu/Zn-SOD doubled and the activity of Mn-SOD reduced. In the leaves, slight activation of peroxidases I and III, the inhibition of Cu/Zn- and Mn-SOD, differential changes in the time-coursed of gene expression of three APX isoforms, and activated gene expression of key enzymes of Pro metabolism were observed. At the same time, the level of MDA did not increase either in the leaves or in the roots. This indicates that treatment of Th. salsuginea plants with Spm under optimal growing conditions did not enhance ROS generation and did not manifest prooxidant properties.     

Growth,Na, K,osmolyte accumulation and lipid membrane peroxidation of two provenances of Cakile maritima during water deficit stress and subsequent recovery

The effects of water deficit stress on growth, Na⁺, K⁺ and osmolyte accumulation in the halophyte species Cakile maritima were investigated. Two Tunisian provenances, Tabarka and Chaffar, belonging to different bioclimatic stages, humid and arid, respectively, were compared. After germination, thirty-day-old seedlings were cultivated for 4 weeks under optimal or limiting water supply, at 100% and 25% of field capacity (FC), respectively. A subset of stressed plants was thereafter rehydrated. The final harvest was carried out after 60 days of treatment. Upon water deficit stress, Chaffar provenance showed significantly lower reduction in biomass production, net CO₂ assimilation and stomatal conductance as well as of leaf water content. Leaf malondialdehyde (MDA) content was significantly increased in the two provenances but this effect was more pronounced in Tabarka plants than in Chaffar ones. Several criteria seem to be associated with the relative tolerance of Chaffar to water deficit: a slow growth rate, a greater ability to control photosynthetic gas exchange, a high ability to preferentially allocate photoassimilates to its roots, and a greater capacity for osmotic adjustment ensured by K⁺ and some compatible solutes such as proline and glycine betaine, but not soluble sugars. The superiority of Chaffar provenance also appeared at the level of its ability to recover after a severe water deficit stress (irrigation at 25% FC only during one month). The data suggest that compatible osmolytes (proline and glycine betaine) accumulated upon water deficit stress play important roles in this halophyte, being involved not only in osmotic adjustment but probably serving also in preservation of the structural and functional integrity at the cellular level during water deficit.     

Changes in the pattern of antioxidant enzymes in wheat exposed to water deficit and rewatering

Antioxidant defenses in two wheat cultivars differing in sensitivity to dehydration (YouJian (YJ-24) more sensitive than LongChun (LC-20) were analyzed during water deficit and rewatering. Resistant cultivar (LC-20) showed a higher relative water content than the sensitive cultivar (YJ-24) during the whole period of water withholding. In order to analyze the changes of antioxidant enzymes, native PAGE analysis of protein extract were performed. Wheat leaves had two isoforms of Mn-superoxide dismutase (SOD), two isoforms of Cu/Zn-SOD and one of Fe-SOD. Three catalase (CAT) isoforms were identified in the leaves of wheat. The activities of SOD and CAT isoforms were increased in two cultivars under water deficit. The intensities of SOD and CAT isoforms were slightly lower in LC-20 and increased continuously in YJ-24 after rewatering. Peroxidase (POD) isoforms were significantly increased during the whole dehydration-rehydration period. Three ascorbate peroxidase (APX) isoforms were present in gel. APX-1 and APX-3 were enhanced during water deficit and decreased during rewatering in LC-20. In YJ-24 only the activities of APX-2 were increased under water deficit. Seven isoforms of glutathione reductase (GR) were detected in the native gel. Activities of most of GR isoforms were higher in tolerant (LC-20) than in sensitive cultivar (YJ-24). Different isoforms of GR in two wheat cultivars behaved differently under water deficit and rewatering. These results collectively suggest that water deficit activates the SOD, CAT and ascorbate-glutathione cycle in wheat leaves. The response of enzyme isoforms to drought is not the same for all isoforms of antioxidant enzymes in two wheat cultivars.     

Effect of drought on pigments, osmotic adjustment and antioxidant enzymes in six woody plant species in karst habitats of southwestern China

Drought stress is one of the most important factors limiting the survival and growth of plants in the harsh karst habitats of southwestern China. Detailed knowledge about the ecophysiological responses of native plants with different growth forms to drought stress could contribute to the success of re-vegetation programs. Two shrubs, Pyracantha fortuneana and Rosa cymosa, and four trees, Broussonetia papyrifera, Cinnamomum bodinieri, Platycarya longipes and Pteroceltis tatarinowii, were randomly assigned to four drought treatments, i.e. well-watered, mild drought stress, moderate drought stress, and severe drought stress. Midday water potential, the maximum quantum efficiency of PSII photochemistry (F_v/F_m), pigments, osmotic solutes (soluble sugars and proline), cellular damages, and antioxidant enzymes (superoxide dismutase, catalase and peroxidase) were investigated. Drought stress significantly decreased pigments content, but increased the ratio of carotenoids to total chlorophylls in the studied species. After prolonged severe drought stress, the two shrubs exhibited higher F_v/F_m, less reductions of midday water potential, and lower increases of malondialdehyde content and ion leakage than the four trees. Prolonged severe drought stress largely decreased accumulations of osmotic solutes and activities of antioxidant enzymes in the four trees, but significantly increased proline content and superoxide dismutase activity in the two shrubs and peroxidase activity in P. fortuneana. The positive relationships were observed among activities of antioxidant enzymes, and between contents of osmotic solutes and activities of antioxidant enzymes. These findings suggested that the two shrubs had higher tolerance to severe drought stress than the four trees due to higher capacities of osmotic adjustment and antioxidant protection.