Overexpression of SlGGP-LIKE gene enhanced the resistance of tomato to salt stress

Ascorbic acid (AsA) plays an important role in scavenging reactive oxygen species (ROS) and reducing photoinhibition in plants, especially under stress. The function of SlGGP which encodes the key enzyme GDP-L-galactose phosphorylase in AsA synthetic pathway is relatively clear. However, there is another gene SlGGP-LIKE that encodes this enzyme in tomato, and there are few studies on it, especially under salt stress. In this study, we explored the function of this gene in tomato salt stress response using transgenic lines overexpressing SlGGP-LIKE (OE). Under normal conditions, overexpressing SlGGP-LIKE can increase the content of reduced AsA and the ratio of AsA/ DHA (dehydroascorbic acid), as well as the level of xanthophyll cycle. Under salt stress, compared with the wild-type plants (WT), the OE lines can maintain higher levels of reduced AsA. In addition, OE lines also have higher levels of reduced GSH (glutathione) and total GSH, higher ratios of AsA/DHA and GSH/oxidative GSH (GSSR), and higher level of xanthophyll cycle. Therefore, the OE lines are more tolerant to salt stress, with higher photosynthetic activity, higher antioxidative enzyme activities, higher content of D1 protein, lower production rate of ROS, and lighter membrane damage. These results indicate that overexpressing SlGGP-LIKE can enhance tomato resistance to salt stress through promoting the synthesis of AsA.

     

Overexpression of tomato GDP-l-galactose phosphorylase gene in tobacco improves tolerance to chilling stress

Key message

The overexpression of tomato GDP- l -galactose phosphorylase gene enhanced tolerance to chilling stress and reduced photoinhibition of photosystems I and II in transgenic tobacco.

Abstract

Chilling stress is a crucial factor that limits the geographical distribution and yield of chilling-sensitive plants. Ascorbate (AsA) protects plants by scavenging reactive oxygen species and reduces photoinhibition by promoting the conversion of violaxanthin to zeaxanthin in the xanthophyll cycle to dissipate excess excitation energy. Possible mechanisms of AsA for plant photoprotection under chilling stress were investigated by isolating the tomato GDP-l-galactose phosphorylase gene (SlGGP) and producing transgenic tobacco plants with overexpression of SlGGP. The transgenic plants subjected to chilling stress accumulated less H₂O₂, demonstrated lower levels of ion leakage and malondialdehyde, and acquired higher net photosynthetic rate, higher maximum photochemical efficiency of PSII, and higher D1 protein content compared with the wild-type (WT) plants. The transgenic plants subjected to chilling stress also showed higher GDP-l-galactose phosphorylase activity, increased AsA content as well as ascorbate peroxidase and oxidizable P700 activities than WT plants. Thus, SlGGP overexpression is crucial in promoting AsA synthesis and alleviating photoinhibition of two photosystems.     

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.     

Overexpression of thaumatin-like protein in transgenic tomato plants confers enhanced resistance to Alternaria solani

Abstract

A cDNA encoding thaumatin-like protein (TLP) from rice was cloned into the binary vector pMON410 under the control of the CaMV 35S promoter for Agrobacterium-mediated transformation of tomato. All putative transformants were tested for the integration and expression of the chimeric gene by polymerase chain reaction (PCR) for hygromycin resistance gene (hph) and enzyme-linked immunosorbent assay (ELISA) for TLP respectively. Constitutive, high-level expression of TLP was observed in transgenic plants. The transgenic lines exhibited increased resistance to Alternaria solani, the early blight pathogen compared to non-transgenic tomato plants.     

Overexpression of an Arabidopsis peroxisomal ascorbate peroxidase gene in tobacco increases protection against oxidative stress.

The Arabidopsis gene APX3 that encodes a putative peroxisomal membrane-bound ascorbate peroxidase was expressed in transgenic tobacco plants. APX3-expressing lines had substantial levels of APX3 mRNA and protein. The H2O2 can be converted to more reactive toxic molecules, e.g. .OH, if it is not quickly removed from plant cells. The expression of APX3 in tobacco could protect leaves from oxidative stress damage caused by aminotriazole which inhibits catalase activity that is found mainly in glyoxysomes and peroxisomes and leads to accumulation of H2O2 in those organelles. However, these plants did not show increased protection from oxidative damage caused by paraquat which leads to the production of reactive oxygen species in chloroplasts. Therefore, protection provided by the expression of APX3 seems to be specific against oxidative stress originated from peroxisomes, not from chloroplasts, which is consistent with the hypothesis that APX3 is a peroxisomal membrane-bound antioxidant enzyme.     

Calcium is involved in the abscisic acid-induced ascorbate peroxidase, superoxide dismutase and chilling resistance in Stylosanthes guianensis

The objective of this work was to test whether Ca²⁺, a second messenger in stress response, is involved in ABA-induced antioxidant enzyme activities in Stylosanthes guianensis. Plants were sprayed with abscisic acid (ABA), calcium channel blocker, LaCl₃, calcium chelator, ethylene glycol-bis(β-amino ethyl ether)-N,N,N′, N′-tetraacetid acid (EGTA), and ABA in combination with LaCl₃ or EGTA. Their effects on superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities and chilling resistance were compared. The results showed that ABA decreased electrolyte leakage and lipid peroxidation but increased maximum photochemical efficiency measured as variable to maximum fluorescence ratio (F_v/F_m) under chilling stress. Treatment with LaCl₃ or EGTA alone and in combination with ABA increased electrolyte leakage and lipid peroxidation, decreased Fv/Fm, suggesting that the block in Ca²⁺ signalling decreased chilling resistance of S. guianensis and the ABA-enhanced chilling resistance. ABA-induced SOD and APX activities were suppressed by LaCl₃ or EGTA. The results suggested that Ca²⁺ is involved in the ABA-enhanced chilling resistance and the ABA-induced SOD and APX activities in S. guianensis.     

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.     

Overexpression of glycerol-3-phosphate acyltransferase gene improves chilling tolerance in tomato

A tomato (Lycopersicon esculentum Mill.) glycerol-3-phosphate acyltransferase gene (LeGPAT) was isolated. The deduced amino acid sequence revealed that LeGPAT contained four acyltransferase domains, showing high identities with GPAT in other plant species. A GFP fusion protein of LeGPAT was targeted to chloroplast in cowpea mesophyll protoplast. RNA gel blot showed that the mRNA accumulation of LeGPAT in the wild type (WT) was induced by chilling temperature. Higher expression levels were observed when tomato leaves were exposed to 4 degrees C for 4 h. RNA gel and western blot analysis confirmed that the sense gene LeGPAT was transferred into the tomato genome and overexpressed under the control of 35S-CaMV. Although tomato is classified as a chilling-sensitive plant, LeGPAT exhibited selectivity to 18:1 over 16:0. Overexpression of LeGPAT increased total activity of LeGPAT and cis-unsaturated fatty acids in PG in thylakoid membrane. Chilling treatment induced less ion leakage from the transgenic plants than from the WT. The photosynthetic rate and the maximal photochemical efficiency of PS II (Fv/Fm) in transgenic plants decreased more slowly during chilling stress and recovered faster than in WT under optimal conditions. The oxidizable P700 in both WT and transgenic plants decreased obviously at chilling temperature under low irradiance, but the oxidizable P700 recovered faster in transgenic plants than in the WT. These results indicate that overexpression of LeGPAT increased the levels of PG cis-unsaturated fatty acids in thylakoid membrane, which was beneficial for the recovery of chilling-induced PS I photoinhibition in tomato.     

The 7B-1 mutant in tomato shows blue-light-specific resistance to osmotic stress and abscisic acid

Germination of wild-type (WT) tomato ( Lycopersicon esculentum Mill.) seed is inhibited by mannitol (100-140 mM) in light, but not in darkness, suggesting that light amplifies the responsiveness of the seed to osmotic stress (M. Fellner, V.K. Sawhney (2001) Theor Appl Genet 102:215-221). Here we report that white light (W) and especially blue light (B) strongly enhance the mannitol-induced inhibition of seed germination, and that the effect of red light (R) is weak or nil. The inhibitory effect of mannitol could be completely overcome by fluridone, an inhibitor of abscisic acid (ABA) biosynthesis, indicating that mannitol inhibits seed germination via ABA accumulation in seeds. The inhibition of WT seed germination by exogenous ABA was also amplified by W or B, but not by R. In a recessive, ABA-overproducing, 7B-1 mutant of tomato, seed germination and hypocotyl growth were resistant to inhibition by mannitol or exogenous ABA, both in W or B. Experiments with fluridone suggested that inhibition of hypocotyl growth by W or B is also partially via ABA accumulation. De-etiolation in the mutant was especially less in B compared to the WT, and there was no difference in hypocotyl growth between the two genotypes in R. Our data suggest that B amplifies the responsiveness of tomato seeds and hypocotyls to mannitol and ABA, and that W- or B-specific resistance of the 7B-1 mutant to osmotic stress or ABA is a consequence of a defect in B perception or signal transduction.     

Rapid inactivation of chloroplastic ascorbate peroxidase is responsible for oxidative modification to Rubisco in tomato (Lycopersicon esculentum) under cadmium stress

To investigate the sensitive site of antioxidant systems in chloroplast under cadmium stress and its consequence on reactive oxygen species production and action, the sub-organellar localization of chloroplast superoxide dismutases (SOD,EC 1.15.1.1) and ascorbic peroxidase (APX, EC 1.11.1.11) isoenzymes and changes of enzymes activities under cadmium stress were investigated in tomato seedlings. Two APX isoforms, one thylakoid-bound and one stromal, were detected. Cd at 50 μM induced a moderate increase of SOD activities but a rapid inactivation of both APX isoenzymes. APX inactivation was mainly related to the decrease of ascorbate concentration, as supported by in vitro treatment of exogenous ascorbate and APX kinetic properties under Cd stress. H2O2 accumulation in chloroplast, as a consequence of APX inactivation,was associated with a 60% loss of Rubisco (EC 4.1.1.39) activity, which could be partially accounted for by a 10% loss of Rubisco content. Protein oxidation assay found that the Rubisco large subunit was the most prominent carbonylated protein; the level of carbonylated Rubisco large subunit increased fivefold after Cd exposure. Thiol groups in the Rubisco large subunit were oxidized, as indicated by non-reducing electrophoresis. Treating crude extract with H2O2 resulted in a similar pattern of protein oxidation and thiols oxidation with that observed in Cd-treated plants. Our study indicates that APXs in the chloroplast is a highly sensitive site of antioxidant systems under Cd stress, and the inactivation of APX could be mainly responsible for oxidative modification to Rubisco and subsequent decrease in its activity.     

The protein kinase CPK28 phosphorylates ascorbate peroxidase and enhances thermotolerance in tomato

High temperatures are a major threat to plant growth and development, leading to yield losses in crops. Calcium-dependent protein kinases (CPKs) act as critical components of Ca²⁺ sensing in plants that transduce rapid stress-induced responses to multiple environmental stimuli. However, the role of CPKs in plant thermotolerance and their mechanisms of action remain poorly understood. To address this issue, tomato (Solanum lycopersicum) cpk28 mutants were generated using a CRISPR-Cas9 gene-editing approach. The responses of mutant and wild-type plants to normal (25°C) and high temperatures (45°C) were documented. Thermotolerance was significantly decreased in the cpk28 mutants, which showed increased heat stress-induced accumulation of reactive oxygen species (ROS) and levels of protein oxidation, together with decreased activities of ascorbate peroxidase (APX) and other antioxidant enzymes. The redox status of ascorbate and glutathione were also modified. Using a yeast two-hybrid library screen and protein interaction assays, we provide evidence that CPK28 directly interacts with cytosolic APX2. Mutations in APX2 rendered plants more sensitive to high temperatures, whereas the addition of exogenous reduced ascorbate (AsA) rescued the thermotolerance phenotype of the cpk28 mutants. Moreover, protein phosphorylation analysis demonstrated that CPK28 phosphorylates the APX2 protein at Thr-59 and Thr-164. This process is suggested to be responsive to Ca²⁺ stimuli and may be required for CPK28-mediated thermotolerance. Taken together, these results demonstrate that CPK28 targets APX2, thus improving thermotolerance. This study suggests that CPK28 is an attractive target for the development of improved crop cultivars that are better adapted to heat stress in a changing climate.

The protein kinase CPK28 regulates thermotolerance in plants by targeting APX2, thus regulating cellular redox homeostasis.     

Double antisense plants lacking ascorbate peroxidase and catalase are less sensitive to oxidative stress than single antisense plants lacking ascorbate peroxidase or catalase

The plant genome is a highly redundant and dynamic genome. Here, we show that double antisense plants lacking the two major hydrogen peroxide-detoxifying enzymes, ascorbate peroxidase (APX) and catalase (CAT), activate an alternative/redundant defense mechanism that compensates for the lack of APX and CAT. A similar mechanism was not activated in single antisense plants that lacked APX or CAT, paradoxically rendering these plants more sensitive to oxidative stress compared to double antisense plants. The reduced susceptibility of double antisense plants to oxidative stress correlated with suppressed photosynthetic activity, the induction of metabolic genes belonging to the pentose phosphate pathway, the induction of monodehydroascorbate reductase, and the induction of IMMUTANS, a chloroplastic homologue of mitochondrial alternative oxidase. Our results suggest that a co-ordinated induction of metabolic and defense genes, coupled with the suppression of photosynthetic activity, can compensate for the lack of APX and CAT. In addition, our findings demonstrate that the plant genome has a high degree of plasticity and will respond differently to different stressful conditions, namely, lack of APX, lack of CAT, or lack of both APX and CAT.     

The polycotyledon mutant of tomato shows enhanced polar auxin transport

The polycotyledon mutant of tomato (Lycopersicon esculentum L. cv Ailsa Craig) showed altered development during embryogenesis and during vegetative and reproductive phases. The phenotype was pleiotropic and included the formation of extra cotyledons, changes in leaf shape, increased number of flowers (indeterminacy) with abnormal floral organs, the formation of epiphyllous structures, and altered gravitropism. The earliest defects were observed at the transition from the globular to the heart stage of embryogenesis with the formation of multiple cotyledons. Epidermal cells in the mutant embryo were smaller and less expanded compared with wild type. Examination of polar auxin transport (PAT) showed a striking enhancement in the case of the mutant. Increase in PAT did not appear to be caused by a decrease in flavonoids because the mutant had normal flavonoid levels. Application of 2,3,5-triiodobenzoic acid, an inhibitor of polar transport of auxin, rescued postgermination phenotypes of young seedlings. Our analysis reveals a level of control that negatively regulates PAT in tomato and its contribution to plant development and organogenesis.     

Brassinosteroid signaling positively regulates abscisic acid biosynthesis in response to chilling stress in tomato

Brassinosteroids (BRs) and abscisic acid (ABA) are essential regulators of plant growth and stress tolerance. Although the antagonistic interaction of BRs and ABA is proposed to ensure the balance between growth and defense in model plants, the crosstalk between BRs and ABA in response to chilling in tomato (Solanum lycopersicum), a warm-climate horticultural crop, is unclear. Here, we determined that overexpression of the BR biosynthesis gene DWARF (DWF) or the key BR signaling gene BRASSINAZOLE-RESISTANT1 (BZR1) increases ABA levels in response to chilling stress via positively regulating the expression of the ABA biosynthesis gene 9-CIS-EPOXYCAROTENOID DIOXYGENASE1 (NCED1). BR-induced chilling tolerance was mostly dependent on ABA biosynthesis. Chilling stress or high BR levels decreased the abundance of BRASSINOSTEROID-INSENSITIVE2 (BIN2), a negative regulator of BR signaling. Moreover, we observed that chilling stress increases BR levels and results in the accumulation of BZR1. BIN2 negatively regulated both the accumulation of BZR1 protein and chilling tolerance by suppressing ABA biosynthesis. Our results demonstrate that BR signaling positively regulates chilling tolerance via ABA biosynthesis in tomato. The study has implications in production of warm-climate crops in horticulture.