Salt tolerance in crop plants monitored by chlorophyll fluorescence in vivo

The potential of measurements of chlorophyll fluorescence in vivo to detect cellular responses to salinity and degrees of salt stress in leaves was investigated for three crop plants. Sugar beet (Beta vulgaris L.) (salt tolerant), sunflower (Helianthus annuus L.) (moderately salt tolerant), and bean (Phaseolus Vulgaris L. cv Canadian Wonder) (salt intolerant) were grown in pots and watered with mineral nutrient solution containing 100 millimolar NaCl. The fast rise in variable chlorophyll fluorescence yield that is correlated with photoreduction of photosystem II acceptors increased in leaves of sugar beet plants treated with salt suggesting stimulation of photosystem II activity relative to photosystem I. In sunflower, this fast rise was depressed by approximately 25% and the subsequent slow rate of quenching of the chlorophyll fluorescence was stimulated. These differences were more marked in the older mature leaves indicating an increasing gradient of salt response down the plant. The salt effect in vivo was reversible since chloroplasts isolated from mature leaves of salt-treated and control sunflower plants gave similar photosystem II activities. Unlike in sugar beet and sunflower, leaves of salt-treated bean progressively lost chlorophyll. The rate of slow quenching of chlorophyll fluorescence decreased indicating development of a partial block after photosystem II and possible initial stimulation of photosystem II activity. With further loss of chlorophyll photosystem II activity declined. It was concluded that measurements of chlorophyll fluorescence in vivo can provide a rapid means of detecting salt stress in leaves, including instances where photosynthesis is reduced in the absence of visible symptoms. The possible application to screening for salt tolerance is discussed.     

Contrasting Physiological Responses of Jatropha curcas Plants to Single and Combined Stresses of Salinity and Heat

In this study we evaluated the contrasting major physiological responses of Jatropha curcas L. to salinity alone and in combination with high temperature. The plants were subjected to salinity (100 mM NaCl) before and after exposure to 43 °C (heat stress) for 6 h. The effects of salinity were more harmful than heat stress, and the effects of salt stress were increased when both stress factors were combined. The negative effects of the combined treatments included strong impairment of the CO₂ assimilation rate and stomata conductance and increased Na⁺ and Cl^? accumulation in the leaves associated with increased membrane damage and lipid peroxidation. Heat favorably stimulated the accumulation of glycine betaine and chlorophyll in the salt-stressed leaves. Treatments with salt, heat, and their combination stimulated the antioxidant enzymatic defense system, that is, the expression of ascorbate peroxidase (APX) and superoxide dismutase (SOD), whereas the expression of catalase (CAT) was stimulated through treatments with salt alone and in combination with heat; treatment with heat alone did not affect CAT expression. The ascorbate redox state was decreased under salinity stress alone and in combination with heat but remained unaffected when treated with heat alone. Overall, the leaf H₂O₂ concentration did not change in response to these stresses, but lipid peroxidation and membrane damage was increased. Moreover, high temperature increases the negative effects of salt stress on key physiological processes, but treatment with heat alone is favorable for several metabolic indicators of young J. curcas plants. In contrast with heat, these plants exhibit higher physiological disturbances under isolated salinity stress.     

Cloning of a cytosolic ascorbate peroxidase gene from Lycium chinense Mill. and enhanced salt tolerance by overexpressing in tobacco

To evaluate the physiological importance of cytosolic ascorbate peroxidase (APX) in the reactive oxygen species (ROS)-scavenging system, a full-length cDNA clone, named LmAPX, encoding a cytosolic ascorbate peroxidase was isolated from Lycium chinense Mill. using homologous cloning, then the expression of LmAPX under salt stress was investigated. After sequencing and related analysis, the LmAPX cDNA sequence was 965 bp in length and had an open reading frame (ORF) of 750 bp coding for 250 amino acids. Furthermore, the LmAPX sequence was sub-cloned into prokaryotic expression vector pET28a and the recombinant proteins had a high expression level in Escherichia coli. Results from a southern blot analysis indicated that three inserts of this gene existed in the tobacco genome encoding LmAPX. Compared with the control plants (wild-type and empty vector control), the transgenic plants expressing the LmAPX gene exhibited lower amount of hydrogen peroxide (H₂O₂) and relatively higher values of ascorbate peroxidase activity, proline content, and net photosynthetic rate (P_n) under the same salt stress. These results suggested that overexpression of the LmAPX gene could decrease ROS production caused by salt stress and protect plants from oxidative stress.     

Ectopic over-expression of peroxisomal ascorbate peroxidase (SbpAPX) gene confers salt stress tolerance in transgenic peanut (Arachis hypogaea)

Peroxisomal ascorbate peroxidase gene (SbpAPX) of an extreme halophyte Salicornia brachiata imparts abiotic stress endurance and plays a key role in the protection against oxidative stress. The cloned SbpAPX gene was transformed to local variety of peanut and about 100 transgenic plants were developed using optimized in vitro regeneration and Agrobacterium mediated genetic transformation method. The T₀ transgenic plants were confirmed for the gene integration; grown under controlled condition in containment green house facility; seeds were harvested and T₁ plants were raised. Transgenic plants (T₁) were further confirmed by PCR using gene specific primers and histochemical GUS assay. About 40 transgenic plants (T₁) were selected randomly and subjected for salt stress tolerance study. Transgenic plants remained green however non-transgenic plants showed bleaching and yellowish leaves under salt stress conditions. Under stress condition, transgenic plants continued normal growth and completed their life cycle. Transgenic peanut plants exhibited adequate tolerance under salt stress condition and thus could be explored for the cultivation in salt affected areas for the sustainable agriculture.     

Peroxisomal APX knockdown triggers antioxidant mechanisms favourable for coping with high photorespiratory H2O2 induced by CAT deficiency in rice

The physiological role of peroxisomal ascorbate peroxidases (pAPX) is unknown; therefore, we utilized pAPX4 knockdown rice and catalase (CAT) inhibition to assess its role in CAT compensation under high photorespiration. pAPX4 knockdown induced co‐suppression in the expression of pAPX3. The rice mutants exhibited metabolic changes such as lower CAT and glycolate oxidase (GO) activities and reduced glyoxylate content; however, APX activity was not altered. CAT inhibition triggered different changes in the expression of CAT, APX and glutathione peroxidase (GPX) isoforms between non‐transformed (NT) and silenced plants. These responses were associated with alterations in APX, GPX and GO activities, suggesting redox homeostasis differences. The glutathione oxidation‐reduction states were modulated differently in mutants, and the ascorbate redox state was greatly affected in both genotypes. The pAPX suffered less oxidative stress and photosystem II (PSII) damage and displayed higher photosynthesis than the NT plants. The improved acclimation exhibited by the pAPX plants was indicated by lower H₂O₂ accumulation, which was associated with lower GO activity and glyoxylate content. The suppression of both pAPXs and/or its downstream metabolic and molecular effects may trigger favourable antioxidant and compensatory mechanisms to cope with CAT deficiency. This physiological acclimation may involve signalling by peroxisomal H₂O₂, which minimized the photorespiration.     

Light and Excess Manganese : Implications for Oxidative Stress in Common Bean

The effect of light intensity on antioxidants, antioxidant enzymes, and chlorophyll content was studied in common bean (Phaseolus vulgaris L.) exposed to excess Mn. Leaves of bean genotypes contrasting in Mn tolerance were exposed to two different light intensities and to excess Mn; light was controlled by shading a leaflet with filter paper. After 5 d of Mn treatment ascorbate was depleted by 45% in leaves of the Mn-sensitive genotype ZPV-292 and by 20% in the Mn-tolerant genotype CALIMA. Nonprotein sulfhydryl groups and glutathione reductase were not affected by Mn or light treatment. Ten days of Mn-toxicity stress increased leaf ascorbate peroxidase activity of cv ZPV-292 by 78% in low light and by 235% in high light, and superoxide dismutase activity followed a similar trend. Increases of ascorbate peroxidase and superoxide dismutase activity observed in cv CALIMA were lower than those observed in the susceptible cv ZPV-292. The cv CALIMA had less ascorbate oxidation under excess Mn-toxicity stress. Depletion of ascorbate occurred before the onset of chlorosis in Mn-stressed plants, especially in cv ZPV-292. Lipid peroxidation was not detected in floating leaf discs of mature leaves exposed to excess Mn. Our results suggest that Mn toxicity may be mediated by oxidative stress, and that the tolerant genotype may maintain higher ascorbate levels under stress than the sensitive genotype.     

Effect of nitric oxide and putrescine on antioxidative responses under NaCl stress in chickpea plants

Chickpea plants were subjected to salt stress for 48 h with 100 mM NaCl, after 50 days of growth. Other batches of plants were simultaneously treated with 0.2 mM sodium nitroprusside (NO donor) or 0.5 mM putrescine (polyamine) to examine their antioxidant effects. Sodium chloride stress adversely affected the relative water content (RWC), electrolyte leakage and lipid peroxidation in leaves. Sodium nitroprusside and putrescine could completely ameliorate the toxic effects of salt stress on electrolyte leakage and lipid peroxidation and partially on RWC. No significant decline in chlorophyll content under salt stress as well as with other treatments was observed. Sodium chloride stress activated the antioxidant defense system by increasing the activities of peroxidase (POX), catalase (CAT) superoxide dismutase (SOD) and ascorbate peroxidase (APX). However no significant effect was observed on glutathione reductase (GR) and dehydro ascorbate reductase (DHAR) activities. Both putrescine and NO had a positive effect on antioxidant enzymes under salt stress. Putrescine was more effective in scavenging superoxide radical as it increased the SOD activity under salt stress whereas nitric oxide was effective in hydrolyzing H₂O₂ by increasing the activities of CAT, POX and APX under salt stress.     

The inductive responses of the antioxidant enzymes by salt stress in the rice (Oryza sativa L.)

To access contributions of inductive responses of the antioxidant enzymes in the resistance to salt stress, activities of the enzymes were determined in the rice (Oryza sativaL. cv. Dongjin) plant. In the leaves of the rice plant, salt stress preferentially enhanced the content of H₂O₂ as well as the activities of the superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxidase specific to guaiacol, whereas it induced the decrease of catalase activity. On the other hand, salt stress had little effect on the activity levels of glutathione reductase (GR). In order to analyze the changes of antioxidant enzyme isoforms against salt stress, plant extracts were subjected to native PAGE. Leaves of the rice plant had two isoforms of Mn-SOD and five isoforms of Cu/Zn-SOD. Fe-SOD isoform was not observed in the activity gels. Expression of Cu/Zn-1, -2, and Mn-SOD-2 isoforms was preferentially enhanced by salt stress. Seven APX isoforms were presented in the leaves of the rice plants. The intensities of APX-4 to -7 were enhanced by salt stress, whereas those of APX-1 to -3 were minimally in changed response to salt stress. There were seven GR isoforms in the leaves of rice plants. Levels of activity for most GR isoforms did not change in the stressed plants compared to the control plants. On the other hand, the levels of activity for most antioxidant enzymes changed little in the roots of stressed plants compared to the control plants. These results collectively suggest that SOD leads to the overproduction of hydrogen peroxide in the leaves of rice plants subjected to salt stress: The overproduction of hydrogen peroxide functions as the signal of salt stress, which induces the induction of specific APX isoforms but not specific GR isoforms under catalase deactivation.     

Short-term UV-B radiation effects on morphology,physiological traits and accumulation of bioactive compounds in Prunella vulgaris L.

Prunella vulgaris plants (full-bloom stage) were subjected to short-term (15 days) UV-B radiation in a growth chamber. UV-B radiation was effective at enhancing morphological and biomass characteristics and decreasing chlorophyll contents of P. vulgaris. Treatment of P. vulgaris with artificial UV-B radiation significantly increased peroxidase (POD), superoxide dismutase (SOD) and glutathione (GSH) activities compared to the control treatment. UV-B radiation significantly increased the levels of hydrogen peroxide (H₂O₂), malondialdehyde (MDA) and proline in leaves of P. vulgaris compared to those of control plants. In addition, the contents of total flavonoids, rosmarinic acid, caffeic acid and hyperoside significantly increased under UV-B radiation. The total phenolic levels also increased under UV-B treatment. These results demonstrated that short-term UV-B radiation can enhance production of secondary metabolites in P. vulgaris, resulting in increased spica yield compared to that of control plants.     

Arbuscular mycorrhizal symbiosis modulates antioxidant response in salt-stressed Trigonella foenum-graecum plants

An experiment was conducted to evaluate the influence of Glomus intraradices colonization on the activity of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (PX), ascorbate peroxidase (APX), and glutathione reductase (GR)] and the accumulation of nonenzymatic antioxidants (ascorbic acid, α-tocopherol, glutathione, and carotenoids) in roots and leaves of fenugreek plants subjected to varying degrees of salinity (0, 50, 100, and 200 mM NaCl) at two time intervals (1 and 14 days after saline treatment, DAT). The antioxidative capacity was correlated with oxidative damage in the same tissue. Under salt stress, lipid peroxidation and H₂O₂ concentration increased with increasing severity and duration of salt stress (DoS). However, the extent of oxidative damage in mycorrhizal plants was less compared to nonmycorrhizal plants. The study reveals that mycorrhiza-mediated attenuation of oxidative stress in fenugreek plants is due to enhanced activity of antioxidant enzymes and higher concentrations of antioxidant molecules. However, the significant effect of G. intraradices colonization on individual antioxidant molecules and enzymes varied with plant tissue, salinity level, and DoS. The significant effect of G. intraradices colonization on antioxidative enzymes was more evident at 1DAT in both leaves and roots, while the concentrations of antioxidant molecules were significantly influenced at 14DAT. It is proposed that AM symbiosis can improve antioxidative defense systems of plants through higher SOD activity in M plants, facilitating rapid dismutation of O₂ ^- to H₂O₂, and subsequent prevention of H₂O₂ build-up by higher activities of CAT, APX, and PX. The potential of G. intraradices to ameliorate oxidative stress generated in fenugreek plants by salinity was more evident at higher intensities of salt stress.     

A salt-inducible chloroplastic monodehydroascorbate reductase from halophyte Avicennia marina confers salt stress tolerance on transgenic plants

Plant growth and productivity are adversely affected by various abiotic stress factors. In our previous study, we used Avicennia marina, a halophytic mangrove, as a model plant system for isolating genes functioning in salt stress tolerance. A large scale random EST sequencing from a salt stressed leaf tissue cDNA library of one month old A. marina plants resulted in identification of a clone showing maximum homology to Monodehydroascorbate reductase (Am-MDAR). MDAR plays a key role in regeneration of ascorbate from monodehydroascorbate for ROS scavenging. In this paper, we report the cellular localization and the ability to confer salt stress tolerance in transgenic tobacco of this salt inducible Am-MDAR. A transit peptide at the N-terminal region of Am-MDAR suggested that it encodes a chloroplastic isoform. The chloroplastic localization was confirmed by stable transformation and expression of the Am-MDAR-GFP fusion protein in tobacco. Transgenic tobacco plants overexpressing Am-MDAR survived better under conditions of salt stress compared to untransformed control plants. Assays of enzymes involved in ascorbate–glutathione cycle revealed an enhanced activity of MDAR and ascorbate peroxidase whereas the activity of dehyroascorbate reductase was reduced under salt stressed and unstressed conditions in Am-MDAR transgenic lines. The transgenic lines showed an enhanced redox state of ascorbate and reduced levels of malondialdehyde indicating its enhanced tolerance to oxidative stress. The results of our studies could be used as a starting point for genetic engineering of economically important plants tolerant to salt stress.     

Proteome analysis of sugar beet (Beta vulgaris L.) elucidates constitutive adaptation during the first phase of salt stress

Salinity is one of the major stress factors responsible for growth reduction of most of the higher plants. In this study, the effect of salt stress on protein pattern in shoots and roots of sugar beet (Beta vulgaris L.) was examined. Sugar beet plants were grown in hydroponics under control and 125 mM salt treatments. A significant growth reduction of shoots and roots was observed. The changes in protein expression, caused by salinity, were monitored using two-dimensional gel-electrophoresis. Most of the detected proteins in sugar beet showed stability under salt stress. The statistical analysis of detected proteins showed that the expression of only six proteins from shoots and three proteins from roots were significantly altered. At this stage, the significantly changed protein expressions we detected could not be attributed to sugar beet adaptation under salt stress. However, unchanged membrane bound proteins under salt stress did reveal the constitutive adaptation of sugar beet to salt stress at the plasma membrane level.     

Antioxidant responses of halophyte plant Aeluropus littoralis under long-term salinity stress

Salinity influences the agricultural production all over the world. This constrain, similar to others biotic and abiotic stresses generate the reactive oxygen species such as superoxide, hydrogen peroxide and hydroxyl radicals. In the evolution process of halophyte plants the mechanisms to detoxify ROS, such as antioxidant enzymes, have been developed. Aeluropus littoralis is a special halophyte that selected to our research, so the plants treated with NaCl at different salt concentration (0, 250, 450 and 650 mM) for a period 45 days. Leaves and roots (separately) collected and their proteins extracted for superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD) activity assay. Meanwhile the electrolyte leakage of leaves analyzed and increased at 450 and 650 mM of NaCl concentrations. Superoxide dismutase and catalase showed same pattern for changing in enzymatic activities (increasing activity by salt stress in roots and decreasing in shoot at 450 and 650 mM stress), also peroxidase and ascorbate peroxidase activity almost increased in all stress conditions.     

Salt and drought tolerance of sugarcane under iso-osmotic salt and water stress: growth,osmolytes accumulation,and antioxidant defense

In order to discriminate between the ionic and osmotic components of salt stress, sugarcane (Saccharum officinarum L. cv. Co 86032) plants were treated with salt-NaCl or polyethylene glycol-PEG 8000 solutions (?0.7 MPa) for 15 days. Both the salt and PEG treatments significantly reduced leaf width, number of green leaves, and chlorophyll stability index. Osmotic adjustment (OA) indicated that both the stresses led to significant accumulation of osmolytes and sugars. Salt stressed plants appeared to use salt as an osmoticum while the PEG stressed plants showed an accumulation of sugars. Oxidative damage to membranes was not severe in plants subjected to salt or PEG stress. The salt stressed plants showed an increase in the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX), while PEG stress led to an increase in SOD but not APX activity as compared to the control. Thus, results indicate that the iso-osmotic salt or PEG stress led to differential responses in plants especially with respect to growth, OA, and antioxidant enzyme activities.     

Cytosolic ascorbate peroxidase from Arabidopsis thaliana L. is encoded by a small multigene family

A second cytosolic ascorbate peroxidase (cAPX; EC 1.11.1.11) gene from Arabidopsis thaliana has been characterised. This second gene (designated APX1b) maps to linkage group 3 and potentially encodes a cAPX as closely related to that from other dicotyledonous species as to the other member of this gene family (Kubo et al, 1993, FEBS Lett 315: 313–317; here designated APX1a), which maps to linkage group 1. In contrast, the lack of sequence similarity in non-coding regions of the genes implies that they are differentially regulated. Under non-stressed conditions only APX1a is expressed. APX1b was identified during low-stringency probing using a cDNA coding for pea cAPX which, in turn, was recovered from a cDNA library by immunoscreening with an antiserum raised against tea plastidial APX (pAPX). No pAPX cDNAs were recovered, despite the antiserum displaying specificity for pAPX in Western blots.Abbreviations ATG methionine translation initiation codon - bp base pair - cAPX cytosolic ascorbate peroxidase - pAPX plastidial ascorbate peroxidase - RFLP restriction fragment length polymorphism Accession numbers: The APX1b sequence is in the EMBL database under accession number X80036M.S. gratefully acknowledges the support from the Junta Nacional de Investigaçâo Cientifica e Tecnológia, Portugal (grant number BD/394/90-IE). This work was supported by the Biotechnological and Biological Sciences Research Council through a grant-in-aid to the John Innes Centre.     

Reproduction of sugar beet mosaic and tobacco mosaic viruses in anthocyanized beet plants

During our studies on the interaction of anthocyanins and plant virus diseases, reproduction of sugar beet mosaic (SBMV) and tobacco mosaic viruses (TMV) was investigated. Experiments were carried out in leaves of sugar beet,Beta vulgaris cv. Dobrovicka N and its spontaneous anthocyanized mutant. SBMV induces a systemic infection while TMV is responsible for primary local symptoms in sugar beet leaves only. Our quantitative analyses onAmaranthus caudatus L. andChenopodium quinoa Wilid. showed a significant decrease in concentration of SBMV in juice extracted from anthocyanized beet plants as compared with extracts from normal green infected plants. Significant differences were also obtained when SBMV — containing juice was tested in mixtures with healthy extracts from anthocyanized and normal green plants. Also the intensity of TMV symptoms in beet leaves was considerably decreased in leaves of antho-eyanized plants.     

Does Water Deficit Stress Promote Ethylene Synthesis by Intact Plants?

The effect of plant water deficit on ethylene production by intact plants was tested in three species, beans (Phaseolus vulgaris L.), cotton (Gossypium hirsutum L.) and miniature rose (Rosa hybrida L., cv Bluesette). Compressed air was passed through glass, plant-containing cuvettes, ethylene collected on chilled columns, and subsequently assayed by gas chromatography. The usual result was that low water potential did not promote ethylene production. When plants were subjected to cessation of irrigation, ethylene production decreased on a per plant or dry weight basis of calculation. No significant promotion of ethylene production above control levels was detected when water deficit-treated bean or cotton plants were rewatered. The one exception to this was for cotton subjected to a range of water deficits, plants subjected to deficits of −1.4 to −1.6 MPa exhibited a transient increase of ethylene production of 40 to 50% above control levels at 24 or 48 hours. Ethylene was collected from intact leaves while plants developed a water deficit stress of −2.9 megapascals after rewatering, and no significant promotion of ethylene production was detected. The shoots of fruited, flowering cotton plants produced less ethylene when subjected to cessation of irrigation. In contrast, the ability of bench drying of detached leaves to increase ethylene production several-fold was verified for both beans and cotton. The data indicate that detached leaves react differently to rapid drying than intact plants react to drying of the soil with regard to ethylene production. This result suggests the need for additional attention to ethylene as a complicating factor in experiments employing excised plant parts and the need to verify the relevance of shock stresses in model systems.     

Changes in antioxidant enzyme activities in detached leaves of cucumber exposed to chilling

We studied changes in biochemical and physiological status, level of oxidative damage, and antioxidant enzyme activities in detached leaves of cucumber plants (Cucumis sativus L. cv. Pyunggangnaebyungsamchuk) that were exposed to a low temperature (4°C). Chlorophyll fluorescence (F_v/F_m) declined during the chilling treatment, but was slowly restored after the tissues were returned to 25°C. Likewise, the fluorescence quenching coefficient and relative water content decreased during the stress period, but then increased during recovery. In contrast, we detected a significant rise in protein and hydrogen peroxide contents in the chilled leaves, as well as higher activities for superoxide dismutase, ascorbate peroxidase, peroxidase, and glutathione reductase. However, the level of catalase decreased not only during chilling but also after 24 h of recovery. These results indicate that exposure to low temperatures acts as an oxidative stress. Moreover, we propose that a regulating mechanism exists in the detached cucumber leaves and contains an antioxidant defense system that induces active oxygen species, thereby alleviating the effects of chilling stress within 12 h.