Antisense-mediated suppression of tomato zeaxanthin epoxidase alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance

A tomato (Lycopersicon esculentum Mill.) zeaxanthin epoxidase gene (LeZE) was isolated and antisense transgenic tomato plants were produced. Northern, southern, and western blot analyses demonstrated that antisense LeZE was transferred into the tomato genome and the expression of LeZE was inhibited. The ratio of (A+Z)/(V+A+Z) in antisense transgenic plants was maintained at a higher level than in the wild type (WT) plants under high light and chilling stress with low irradiance. The value of non-photochemical quenching (NPQ) in WT and transgenic plants was not affected during the stresses. The oxidizable P700 and the maximal photochemical efficiency of PSII (F_v/F_m) in transgenic plants decreased more slowly at chilling temperature under low irradiance. These results suggested that suppression of LeZE caused zeaxanthin accumulation, which was helpful in alleviating photoinhibition of PSI and PSII in tomato plants under chilling stress.     

Transgenic tobacco with suppressed zeaxanthin formation is susceptible to stress-induced photoinhibition

Tobacco (Nicotiana tabacum cv. Xanthi) transformed with the antisense construct of tobacco violaxanthin de-epoxidase was analyzed for responses in growth chambers to both short and long-term stress treatments. Following a short-term (2 or 3 h) high-light treatment, antisense plants had a greater reduction in F_v/F_m relative to wild-type, indicating a greater susceptibility to photoinhibition. The responses of antisense plants to long-term stress were examined in two separate experiments, one with high light alone and the other wherein high light and water stress were combined. In the light-stress experiment, plants were grown at 1300 mol photons m^–2 s^–1 under a 12 h photoperiod. In the light and water-stress experiment, plants were grown under moderately high light of 900 mol photons m^–2 s^–1, under a 16 h photoperiod, in combination with water stress. Both conditions caused formation of high antheraxanthin and zeaxanthin levels in wild-type plants but not in antisense plants. In both cases, antisense plants showed significant reductions in F_v/F_m and total leaf-pigment content relative to wild-type. The data demonstrate a critical photoprotective function of the xanthophyll cycle-dependent energy dissipation in tobacco exposed suddenly to high amounts of excess light over extended times.This revised version was published online in October 2005 with corrections to the Cover Date.     

Grafting onto Cucurbita moschata rootstock alleviates salt stress in cucumber plants by delaying photoinhibition

To determine how the use of a given rootstock can influence the functioning of the photosynthetic apparatus of the scion under salt stress, the growth, gas exchange, photosystem II (PSII) efficiency, xanthophyll cycle, and chloroplast ultrastructure of nongrafted, self-grafted, and pumpkin-grafted (hereafter referred to as rootstock-grafted) cucumber (Cucumis sativus L.) plants were investigated at day 15 after being treated with 90 mM NaCl. The reductions in plant growth of the rootstock-grafted plants were lower than those of the nongrafted and self-grafted plants under 90 mM NaCl. The net photosynthetic rate, stomatal conductance, maximal and effective quantum yield of PSII photochemistry, photochemical quenching coefficient, and effective quantum-use efficiency of PSII in the light-adapted state of the nongrafted and self-grafted plants were significantly decreased under 90 mM NaCl. However, these reductions were alleviated when the cucumber plants were grafted onto the pumpkin (Cucurbita moschata Duch.) rootstock. The intercellular CO₂ concentrations were significantly increased in the nongrafted and self-grafted plants under 90 mM NaCl, whereas it was decreased in the rootstock-grafted plants. Nonphotochemical quenching (NPQ) and the deepoxidation state of the xanthophyll cycle were significantly increased under 90 mM NaCl, particularly in the rootstockgrafted plants, suggesting the rootstock-grafted plants had higher potential to dissipate excess excitation energy and reduce the probability of photodamage to PSII. Under 90 mM NaCl, the number of grana was reduced, the thylakoids were swollen, and starch granules accumulated in all plants. However, the damage of chloroplast ultrastructure was alleviated in the rootstock-grafted plants. Taken together, the use of C. moschata rootstock alleviated salt stress in cucumber plants by delaying photoinhibition, probably due to a lower incidence of both stomatal and nonstomatal factors limiting photosynthesis.     

Overexpression of zeaxanthin epoxidase gene enhances the sensitivity of tomato PSII photoinhibition to high light and chilling stress

A tomato ( Lycopersicon esculentum Mill.) zeaxanthin epoxidase gene ( LeZE ) was isolated. The deduced amino acid sequence of LeZE showed high identities with zeaxanthin epoxidase in other plant species. Northern blot analysis showed that the mRNA accumulation of LeZE in the wild-type (WT) was not induced by light and temperature but regulated by the diurnal rhythm. The sense transgenic plants were obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Northern and western blot analysis confirmed that sense LeZE was transferred into the tomato genome and overexpressed. The ratio of (A + Z)/(V + A + Z) and the values of non-photochemical quenching were lower in transgenic plants than in WT plants under high light and chilling stress with low irradiance. The O₂ evolution rate and the maximal photochemical efficiency of PSII (Fv/Fm) in transgenic plants decreased more quickly during both stresses and recovered slower than that in WT under optimal conditions. These results suggested that overexpression of LeZE impaired the function of the xanthophyll cycle and aggravated PSII photoinhibition in tomato under high light and chilling stress.     

Kinetic correlation of recovery from photoinhibition and zeaxanthin epoxidation

The generation of non-photochemical fluorescence quenching under photoinhibitory illumination and its relaxation under subsequent low light illumination in leaves from intermittent-light-grown pea (Pisum sativum L.) plants (IML-plants) has been investigated. In parallel, we studied (i) the activity of the xanthophyll cycle with emphasis on zeaxanthin formation and reconversion to violaxanthin and (ii) the degradation rate of D1 protein. In comparison to control plants grown in continuous light, IML-plants were much more susceptible to photoinhibition as determined from the increase of slowly (halftimes > 20 min) relaxing quenching (qI) of variable chlorophyll fluorescence. The relaxation (recovery) kinetics of qI (under weak light) in both types of plant depended on the photon flux density, temperature and duration of pre-illumination. The recovery time generally increased with an increasing degree of qI. In IML-plants, relaxation of qI was kinetically closely related to the epoxidation of zeaxanthin. At high degrees of photosystem II inhibition the kinetics resembled those of D1 degradation. The results are discussed in terms of the mechanisms of photosystem II inactivation in vivo.     

Exogenous putrescine alleviates photoinhibition caused by salt stress through cooperation with cyclic electron flow in cucumber

Photosynthesis Research - When plants suffer from abiotic stresses, cyclic electron flow (CEF) is induced for photo-protection. Putrescine (Put), a primary polyamine in chloroplasts, plays a...     

Garden-waste-vermicompost leachate alleviates salinity stress in tomato seedlings by mobilizing salt tolerance mechanisms

The incidence of salinity-induced plant stress as a result of natural and anthropogenic factors in arid and semi-arid agricultural lands is great. In South Africa alone, 9 % of irrigated agricultural land is salt-affected. Commercial fertilizers used for improving soil nutrient levels are costly and affect the quality, lifespan and sustainability of soil and water resources. Organic farming practices are based on cost-effective and environmentally-aware management systems. Vermicompost leachate (VCL) is a vermicompost-derived liquid product that has become recognised as a suitable soil amendment product. Commercial tomato (Lycopersicon esculentum Mill var. Heinz-1370) seedlings were subjected to sodium chloride (NaCl) concentrations of 0, 25, 50 and 100 mM and were treated with 1:10 (v/v) WizzardWorms VCL prepared in Hoagland’s nutrient solution under greenhouse conditions. Morphological characters of VCL-treated tomato seedlings showed improved root growth and stimulated overall aboveground growth with significantly higher numbers of leaves, greater stem thickness and increased leaf area, even at a high NaCl-tested concentration (100 mM). The accumulation of compatible solutes such as proline and total soluble sugars indicate an induced salt tolerance or adaptive mechanism in VCL-treated tomato seedlings. The current investigation demonstrates the potential of an organic liquid to maximise tomato productivity by improving seedling growth performance under salt stress conditions.     

Overexpression of tomato chloroplast omega-3 fatty acid desaturase gene alleviates the photoinhibition of photosystems 2 and 1 under chilling stress

In transgenic (TG) tomato (Lycopersicon esculentum Mill.) overexpressed ω-3 fatty acid desaturase gene (LeFAD7) was identified, which was controlled by the cauliflower mosaic virus 35S promoter and induced increased contents of unsaturated fatty acids in thylakoid membrane. Under chilling stress at low irradiance (4 °C, 100 μmol m⁻² s⁻¹) TG plants with higher linolenic acids (18: 3) content maintained a higher O₂ evolution rate, oxidizable P700 content, and maximal photochemical efficiency (F_v/F_m) than wild type (WT) plants. Low temperature treatment for 6 h resulted in extensive changes of chloroplast ultrastructure: in WT plants most chloroplasts became circular, the number of amyloids increased, appressed granum stacks were dissolved, grana disappeared, and the number of grana decreased, while only a few grana were found in leaves of TG plants. Hence the overexpression of LeFAD7 could increase the content of 18: 3 in thylakoid membrane, and this increase alleviated the photoinhibition of photosystem (PS) 1 and PS2 under chilling at low irradiance.     

Silicon alleviates the deleterious salt effect on tomato plant growth by improving plant water status

In order to investigate the role of Si in alleviating the deleterious effects of salinity on tomato plant growth, the tomato cultivar Moneymaker was grown with 0 or 80mM NaCl combined with 0 and 2.5mM Si. Plant growth parameters, salt accumulation in plant tissues and plant water relations were analysed. Si treatment did not alter salt input into the plant or salt distribution between plant organs. There were non-significant differences in plant water uptake, but plant water content in salinised plants supplied with Si was 40% higher than in salinised plants that were not supplied with Si. Plants treated with NaCl alone showed a reduction in plant dry weight and total plant leaf area of 55% and 58%, respectively, while the reduction in plants treated with NaCl plus Si was only 31% and 22%, respectively. Leaf turgor potential and net photosynthesis rates were 42% and 20% higher in salinised plants supplied with Si than in salinised plants that were not supplied with Si. Water use efficiency calculated from instantaneous gas exchange parameters and as the ratio between plant dry matter and plant water uptake were, respectively, 17% and 16% higher in salinised plants supplied with Si. It can be concluded that Si improves the water storage within plant tissues, which allows a higher growth rate that, in turn, contributes to salt dilution into the plant, mitigating salt toxicity effects.     

Salicylic acid increased aldose reductase activity and sorbitol accumulation in tomato plants under salt stress

Increased aldose reductase (ALR) activities were detected in the leaf tissues of tomato plants grown for 3 weeks in culture medium containing 10^?7 or 10^?4 M salicylic acid (SA), and in the roots after the 10^?4 M SA pretreatment. The ALR activity changed in parallel with the sorbitol content in the leaves of the SA-treated plants. Salt stress elicited by 100 mM NaCl enhanced the accumulation of sorbitol in the leaves of control plants and as compared with the untreated control the sorbitol content in the SA-pretreated leaves remained elevated under salt stress. DEAE cellulose anionexchange column purification of the protein precipitated with 80 % (NH₄)₂SO₄ revealed two enzyme fractions with ALR activity in both the leaf and the root tissues. The fraction of the leaf extract that was not bound to the column reacted with glucose and glucose-6-P as substrates, whereas glucose was not a substrate for the bound fraction or for root isoenzymes. The root enzyme was less sensitive to salt treatment: 50 mM NaCl caused 30 % inhibition in the leaf extract, whereas the enzyme activity of the root extract was not affected. It is suggested that increased ALR activity and sorbitol synthesis in the leaves of SA-treated tomato plants may result in an improved salt stress tolerance.     

The unsaturation of phosphatidylglycerol in thylakoid membrane alleviates PSII photoinhibition under chilling stress

Over-expression of chloroplast glycerol-3-phosphate acyltransferase gene (LeGPAT) in tomato increased cis-unsaturated fatty acid content in phosphatidylglycerol (PG) of the thylakoid membrane. Under chilling stress, the oxygen evolving activity, the maximal photochemical efficiency of PSII (F _v/F _m), and superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities decreased less in sense lines than in antisense lines compared to wild-type (WT) plants. Consistently, the relative electric conductivity, \textO₂ ^{. -} {\text{O}}_{2} ^{{. - }} and H₂O₂ contents in sense lines were lower than those of WT and antisense lines. The antisense lines with low level of unsaturated fatty acids in PG were extremely susceptible to photoinhibition of PSII and had a significant reduction in the D1 protein content of PSII reaction center under chilling stress. However, in the presence of streptomycin (SM), the degradation of D1 protein was faster in sense lines than in WT and antisense plants. These results suggested that, under chilling stress conditions, increasing cis-unsaturated fatty acids in PG through over-expression of LeGPAT can alleviate PSII photoinhibition by accelerating the repair of D1 protein and improving the activities of antioxidant enzymes in chloroplasts.     

Chemical characterization of stress-induced vascular coating in tomato

Indirect evidence suggests that vascular coatings formed by plants in response to stress consist of suberin-like substances containing lipid and phenolic compounds. To provide more direct chemical evidence that coatings are suberin, we used a natural pathogen, Verticillium albo-atrum, or a stress-responsive hormone, abscisic acid, to induce coating in two isolines of tomato (Lycopersicon esculentum L. cultivar Craigella) that are resistant or susceptible to the pathogen. Using treated petioles that had been monitored cytologically, chemical depolymerization followed by combined gas-liquid chromatography-mass spectrometry analysis of alkane-α,ω-diol levels confirmed the presence of suberin after induction of coating and showed quantitative differences between the isolines that correlated with cytological measurements of the coating response. Northern analysis of suberization-associated anionic peroxidase mRNA showed corresponding increases, and tissue blot analysis further indicated that induction of the mRNA was localized in the responding vascular bundles, as determined by suberin histochemistry. Taken together, these results provide chemical evidence that the coatings are mainly suberin.     

The effects of moderately high temperature on zeaxanthin accumulation and decay

Moderately high temperature reduces photosynthetic capacities of leaves with large effects on thylakoid reactions of photosynthesis, including xanthophyll conversion in the lipid phase of the thylakoid membrane. In previous studies, we have found that leaf temperature of 40°C increased zeaxanthin accumulation in dark-adapted, intact tobacco leaves following a brief illumination, but did not change the amount of zeaxanthin in light-adatped leaves. To investigate heat effects on zeaxanthin accumulation and decay, zeaxanthin level was monitored optically in dark-adapted, intact tobacco and Arabidopsis thaliana leaves at either 23 or 40°C under 45-min illumination. Heated leaves had more zeaxanthin following 3-min light but had less or comparable amounts of zeaxanthin by the end of 45?min of illumination. Zeaxanthin accumulated faster at light initiation and decayed faster upon darkening in leaves at 40°C than leaves at 23°C, indicating that heat increased the activities of both violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE). In addition, our optical measurement demonstrated in vivo that weak light enhances zeaxanthin decay relative to darkness in intact leaves of tobacco and Arabidopsis, confirming previous observations in isolated spinach chloroplasts. However, the maximum rate of decay is similar for weak light and darkness, and we used the maximum rate of decay following darkness as a measure of the rate of ZE during steady-state light. A simulation indicated that high temperature should cause a large shift in the pH dependence of the amount of zeaxanthin in leaves because of differential effects on VDE and ZE. This allows for the reduction in ΔpH caused by heat to be offset by increased VDE activity relative to ZE.     

Antioxidant metallothionein alleviates endoplasmic reticulum stress-induced myocardial apoptosis and contractile dysfunction

Abstract

Aims. Endoplasmic reticulum (ER) stress exerts myocardial oxidative stress, apoptosis, and contractile anomalies, although the precise interplay between ER stress and apoptosis remains elusive. This study was designed to examine the impact of the cysteine-rich free radical scavenger metallothionein on ER stress-induced myocardial contractile defect and underlying mechanisms. Methods and results. Wild-type friendly virus B and transgenic mice with cardiac-specific overexpression of metallothionein were challenged with the ER stress inducer tunicamycin (1 mg/kg, intraperitoneal, 48 h) prior to the assessment of myocardial function, oxidative stress, and apoptosis. Our results revealed that tunicamycin promoted cardiac remodeling (enlarged left ventricular end systolic/diastolic diameters with little changes in left ventricular wall thickness), suppressed fractional shortening and cardiomyocyte contractile function, elevated resting Ca²⁺, decreased stimulated Ca²⁺ release, prolonged intracellular Ca²⁺ clearance, and downregulated sarco(endo)plasmic reticulum Ca²⁺-ATPase levels, the effects of which were negated by metallothionein. Treatment with tunicamycin caused cardiomyocyte mitochondrial injury, as evidenced by decreased mitochondrial membrane potential (??m, assessed by JC-1 staining), the effect of which was negated by the antioxidant. Moreover, tunicamycin challenge dramatically facilitated myocardial apoptosis as manifested by increased Bax, caspase 9, and caspase 12 protein levels, as well as elevated caspase 3 activity. Interestingly, metallothionein transgene significantly alleviated tunicamycin-induced myocardial apoptosis. Conclusion. Taken together, our data favor a beneficial effect of metallothionein against ER stress-induced cardiac dysfunction possibly associated with attenuation of myocardial apoptosis.     

Increasing salt tolerance in the tomato

In this paper, a number of strategies to overcome the deleterious effects of salinity on plants will be reviewed; these strategies include using molecular markers and genetic transformation as tools to develop salinity-tolerant genotypes, and some cultural techniques. For more than 12 years, QTL analysis has been attempted in order to understand the genetics of salt tolerance and to deal with component traits in breeding programmes. Despite innovations like better marker systems and improved genetic mapping strategies, the success of marker-assisted selection has been very limited because, in part, of inadequate experimental design. Since salinity is variable in time and space, experimental design must allow the study of genotype x environment interaction. Genetic transformation could become a powerful tool in plant breeding, but the growing knowledge from plant physiology must be integrated with molecular breeding techniques. It has been shown that the expression of several transgenes promotes a higher level of salt tolerance in some species. Despite this promising result, the development of a salt-tolerant cultivar by way of transgenesis has still not been achieved. Future directions in order to overcome the present limitations are proposed. Three cultural techniques have proved useful in tomato to overcome, in part, the effects of salinity: treatment of seedlings with drought or NaCl ameliorates the adaptation of adult plants to salinity; mist applied to tomato plants grown in Mediterranean conditions improves vegetative growth and yield in saline conditions; and grafting tomato cultivars onto appropriate rootstocks could reduce the effects of salinity.     

Metabolic implications of stress-induced proline accumulation in plants

In many plants, free proline accumulates in response to the imposition of a wide range of biotic and abiotic stresses. Controversy has surrounded the extent to which this shift in nitrogen metabolism benefits plants under adverse environmental conditions. Most attempts to account for the phenomenon have focused on the ability of proline to mediate osmotic adjustment, stabilise subcellular structures and scavenge free radicals. However, often the cytoplasmic pool of free proline even after the imposition of stress is insufficient size to account for pronounced biophysical effects.Alternatively, selective preservation of this stress-induced response may relate to endpoints other than simply augmenting the cellular pool of free proline. Proline accumulation may reduce stress-induced cellular acidification or prime oxidative respiration to provide energy needed for recovery. High levels of proline synthesis during stress may maintain NAD(P)+/NAD(P)H ratios at values compatible with metabolism under normal conditions. Consideration of the cofactor preference of plant 1-pyrroline-5-carboxylate (P5C) reductase as well as the in vivo concentrations of the two pyridine nucleotide cofactors and their respective redox ratios suggests that even a small increase in proline biosynthesis might have a large impact on the level of reduction of the cellular NADP pool. The increased NADP+/NADPH ratio mediated by proline biosynthesis is likely to enhance activity of the oxidative pentose phosphate pathway. This would provide precursors to support the demand for increased secondary metabolite production during stress as well as nucleotide synthesis accompanying the accelerated rate of cell division upon relief from stress, when oxidation of proline is likely to provide an important energy source for ADP phosphorylation. Thus, the extreme sensitivity of the metabolic processes of proline synthesis and degradation themselves may be of benefit by regulating metabolic processes adversely affected by stress. This viewpoint is supported by consideration of other physiological phenomena not directly related to stress responses, but in which proline metabolism may also play a regulatory role.A mechanism is proposed whereby the interconversions of proline and P5C in different cell types and the associated transfer of redox potential between tissues may constitute a form of metabolic signalling within higher plants. Stress-related alterations in proline metabolism may impinge on systems of redox control of plant gene expression.     

Short-term salt tolerance mechanisms in differentially salt tolerant tomato species

The physiological changes induced by a daily increase of NaCl level, over a period of 4 d, were studied in leaves of the salt-sensitive cultivated tomato species Lycopersicon esculentum and its wild salt-tolerant relative Lycopersicon pennellii. A higher solute contribution to the osmotic adjustment was observed in NaCl-treated leaves of L. pennellii than in those of L. esculentum. This response together with the higher accumulation of inorganic solutes in the wild species and of organic solutes in the cultivated species verified the different salt tolerance mechanisms operating in the two species in the short-term. With regard to the changes induced by salt stress on the free polyamine levels, the putrescine and spermine levels increased with salinity, whereas the spermine levels decreased in both tomato species; nevertheless, the main difference between the two species lays in an earlier and greater accumulation of putrescine induced by salinity in L. pennellii than in L. esculentum. The changes in putrescine levels were associated to changes in amino acids related to its synthesis, and the changes were different in both species. In L. esculentum, the high concentrations of some intermediate compounds (glutamate and arginine) were related to the low accumulation rate of both proline and putrescine. In contrast, in L. pennellii, important reductions in glutamate and arginine levels were found at the end of the salinization period. Moreover, in this last situation, a decline in the putrescine level ran parallel to a high proline accumulation, which suggests that the higher the stress level, the higher the deviation of glutamate to proline occurring in the salt tolerant species. It could be concluded that an early accumulation of the diamine putrescine seems to be associated with salt tolerance in the short-term.     

Drought-stress-induced polypeptide accumulation in tomato leaves

Abstract. Tomato ( Lycopersicon esculentum Mill ev. Ailsa Craig) leaf polypeptides, radiolabelled with L-[³⁶S] methionine during drought stress, were separated by two-dimensional polyacrylamide gel electrophoresis. Three sets of polypeptides were distinguished: those that decreased, increased or transiently increased in response to drought stress. Three sets of polypeptides also could be distinguished during recovery from drought stress: those that increased, decreased rapidly or decreased slowly and could still be detected after 6 h of recovery. The set of polypeptides that decreased during drought stress was the same set that increased during recovery and those that increased during stress, decreased during recovery. The leaf ABA concentration increased rapidly in response to drought stress and decreased rapidly during recovery from drought stress, returning to the nonstress level after 6 h of rehydration. There was a correlation between the accumulation of some of the drought-induced polypeptides and the pattern of ABA accumulation during stress and recovery from stress.     

Overexpression of SlGMEs leads to ascorbate accumulation with enhanced oxidative stress, cold, and salt tolerance in tomato

GDP-Mannose 3′,5′-epimerase (GME; EC 5.1.3.18) catalyses the conversion of GDP-d-mannose to GDP-l-galactose, an important step in the ascorbic acid (AsA) biosynthesis pathway in higher plants. In this study, two members of the GME gene family were isolated from tomato (Solanum lycopersicum). Both SlGME genes encode 376 amino acids and share a 92% similarity with each other. Semi-quantitative RT-PCR indicated that SlGME1 was constantly expressed in various tissues, whereas SlGME2 was differentially expressed in different tissues. Transient expression of fused SlGME1-GFP (green fluorescent protein) and SlGME2-GFP in onion cells revealed the cytoplasmic localisation of the two proteins. Transgenic plants over-expressing SlGME1 and SlGME2 exhibited a significant increase in total ascorbic acid in leaves and red fruits compared with wild-type plants. They also showed enhanced stress tolerance based on less chlorophyll content loss and membrane-lipid peroxidation under methyl viologen (paraquat) stress, higher survival rate under cold stress, and significantly higher seed germination rate, fresh weight, and root length under salt stress. The present study demonstrates that the overexpression of two members of the GME gene family resulted in increased ascorbate accumulation in tomato and improved tolerance to abiotic stresses.