Overexpression of endoplasmic reticulum omega-3 fatty acid desaturase gene improves chilling tolerance in tomato

An endoplasmic reticulum-localized tomato omega-3 fatty acid desaturase gene (LeFAD3) was isolated and characterized with regard to its sequence, response to various temperatures and function in transgenic tomato plants. Northern blot analysis showed that LeFAD3 was expressed in all organs tested and was markedly abundant in roots. Meanwhile, the expression of LeFAD3 was induced by chilling stress (4 °C), but inhibited by high temperature (40 °C). The transgenic plants were obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Northern and western blot analyses confirmed that sense LeFAD3 was transferred into tomato genome and overexpressed. Level of linolenic acids (18:3) increased and correspondingly level of linoleic acid (18:2) decreased in leaves and roots. After chilling stress, the fresh weight of the aerial parts of transgenic plants was higher than that of the wild type (WT) plants, and the membrane system ultrastructure of chloroplast in leaf cell and all the subcellular organelles in root tips of transgenic plants kept more intact than those of WT. Relative electric conductivity increased less in transgenic plants than that in WT, and the respiration rate of the transgenic plants was notably higher than that of WT. The maximal photochemical efficiency of PSII (F_v/F_m) and the O₂ evolution rate in WT decreased more than those in transgenic plants under chilling stress. Together with other data, results showed that the overexpression of LeFAD3 led to increased level of 18:3 and alleviated the injuries under chilling stress.     

Antisense expression of tomato chloroplast omega-3 fatty acid desaturase gene (<Emphasis Type="Italic">LeFAD7</Emphasis>) enhances the tomato high-temperature tolerance through reductions of trienoic fatty acids and alterations of physiological parameters

We studied how the reductions of trienoic fatty acids (TAs) and increases of dienoic fatty acids (DAs) enhanced high-temperature tolerance in antisense expression of tomato chloroplast omega-3 fatty acid desaturase gene (LeFAD7) transgenic tomato (Lycopersicon esculentum Mill.) plants. In transgenic plants, the content of linolenic acid (18:3) was markedly decreased, while linoleic acid (18:2) was increased correspondingly and the similar changes were observed under high-temperature stress as well. Under high-temperature stress, transgenic plants can maintain a relatively higher level of net photosynthetic rate (P _N) and chlorophyll (Chl) content than that of wild type (WT) plants. A decreased Chl/Carotenoids (xanthophylls and carotenes, Car) ratio and Chl a/b ratio were observed in transgenic plants. Transgenic plants exhibited visible decrease in the relative electrolyte conductivity, higher activities of antioxidative enzymes and lower reactive oxygen species correspondingly than WT. In addition, high-temperature stress for 24 h caused more extensive changes of chloroplast ultrastructure in WT than in transgenic plants. We therefore suggested that the enhancement of high-temperature tolerance in antisense expression of LeFAD7 transgenic plants might be raised from the reduction of TAs and increase of DAs subsequently leading to series of physiological alterations.     

Antisense-Mediated Depletion of Tomato Chloroplast Omega-3 Fatty Acid Desaturase Enhances Thermal Tolerance

A chloroplast-localized tomato （Lycopersicon esculentum Mill.） ω-3 fatty acid desaturase gene （LeFADT） was isolated and characterized with regard to its sequence, response to various temperatures, and function in antisense transgenic tomato plants. The deduced amino acid sequence had four histidine-rich regions, of which three regions were highly conserved throughout the whole ω-3 fatty acid desaturasegene family. Southern blotting analysis showed that LeFAD7was encoded by a single copy gene and had two homologous genes in the tomato genome. Northern blot showed that LeFAD7 was expressed in all organs and was especially abundant in leaf tissue. Meanwhile, expression of LeFAD7 was induced by chilling stress （4 ℃）, but was inhibited by high temperature （45 ℃）, in leaves. Transgenic tomato plants were produced by integration of the antisense LeFAD7DNA under the control of a CaMV35S promoter into the genome. Antisense transgenic plants with lower 18 ： 3 content could maintain a higher maximal photochemical efficiency （Fv/Fm） and O2 evolution rate than wild-type plants. These results suggested that silence of the LeFAD7 gene alleviated high-temperature stress. There was also a correlation between the low content of 18 ： 3 resulting from silence of the LeFAD7 gene and tolerance to high-temperature stress.     

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.     

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.     

Transgenic tomato plants overexpressing chloroplastic monodehydroascorbate reductase are resistant to salt- and PEG-induced osmotic stress

RNA gel hybridization showed that the expression of monodehydroascorbate reductase (MDHAR) in the wild type (WT) tomato was decreased firstly and then increased under salt- and polyethylene glycol (PEG)-induced osmotic stress, and the maximum level was observed after treatment for 12 h. WT, sense transgenic and antisense transgenic tomato plants were used to analyze the antioxidative ability to cope with osmotic stresses. After salt stress, the fresh mass (FM) and height of sense transgenic lines were greater than those of antisense lines and WT plants. Under salt and PEG treatments, sense transgenic plants showed a lower level of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), a higher net photosynthetic rate (P _N), and the maximal photochemical efficiency of PSII (F_v/F_m) compared with WT and antisense transgenic plants. Moreover, sense lines maintained higher ascorbate peroxidase (APX) activity than WT and antisense plants under salt- and PEG-induced osmotic stress. These results indicate that chloroplastic MDHAR plays an important role in alleviating photoinhibition of PSII by elevating ascorbate (AsA) level under salt- and PEG-induced osmotic stress.     

Enhancement of low-temperature tolerance in transgenic tomato plants overexpressing Lefad7 through regulation of trienoic fatty acids

We studied how tomato (Lycopersicon esculentum Mill.) chloroplast omega-3 fatty acid desaturase gene (Lefad7) overexpression enhanced low-temperature (LT) tolerance in transgenic tomato plants. In these plants, the content of linolenic acid (18:3) markedly increased and, correspondingly, the content of linoleic acid (18:2) decreased. Similar changes were found after 6 h under LT (4°C) treatment. Under LT stress, wild type (WT) tomato plants showed a much greater increase in relative electrolyte leakage and malondialdehyde (MDA) contents compared with transgenic plants. Transgenic plants exhibited higher activities of antioxidative enzymes and a lower content of reactive oxygen species (ROS). Transgenic plants maintained a relatively higher level of the net photosynthetic rate (P _N) and chlorophyll (Chl) content than WT plants under LT stress. Taken together, we suggested that overexpression of Lefad7 enhanced LT tolerance by changing the composition of membrane lipids in tomato plants, with the increased content of trienoic fatty acids and reduced content of dienoic fatty acids that led to series of physiological alterations.     

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.     

Constitutive accumulation of zeaxanthin in tomato alleviates salt stress-induced photoinhibition and photooxidation

Zeaxanthin (Z) has a role in the dissipation of excess excitation energy by participating in non‐photochemical quenching (NPQ) and is essential in protecting the chloroplast from photooxidative damage. To investigate the physiological effects and functional mechanism of constitutive accumulation of Z in the tomato at salt stress‐induced photoinhibition and photooxidation, antisense‐mediated suppression of zeaxanthin epoxidase transgenic plants and the wild‐type (WT) tomato were used. The ratio of Z/(V + A + Z) and (Z + 0.5A)/(V + A + Z) in antisense transgenic plants were maintained at a higher level than in WT plants under salt stress, but the value of NPQ in WT and transgenic plants was not significantly different under salt stress. However, the maximal photochemical efficiency of PSII (Fv/Fm) and the net photosynthetic rate (Pn) in transgenic plants decreased more slowly under salt stress. Furthermore, transgenic plants showed lower level of hydrogen peroxide (H₂O₂), superoxide anion radical (O₂^??) and ion leakage, lower malondialdehyde content. Compared with WT, the content of D1 protein decreased slightly in transgenic plants under salt stress. Our results suggested that the constitutive accumulation of Z in transgenic tomatoes can alleviate salt stress‐induced photoinhibition because of the antioxidant role of Z in the scavenging quenching of singlet oxygen and/or free radicals in the lipid phase of the membrane.     

The increase in unsaturation of fatty acids of phosphatidylglycerol in thylakoid membrane enhanced salt tolerance in tomato

Overexpression of chloroplastic glycerol-3-phosphate acyltransferase gene (LeGPAT) in tomato increased cis-unsaturated fatty acid content in phosphatidylglycerol (PG) of thylakoid membrane. By contrast, suppressing the expression of LeGPAT decreased the content of cis-unsaturated fatty acid in PG. Under salt stress, sense transgenic plants exhibited higher activities of chloroplastic antioxidant enzymes, lower content of reactive oxygen species (ROS) and less ion leakage compared with the wild type (WT) plants. The net photosynthetic rate (P _N) and the maximal photochemical efficiency (F_v/F_m) of photosystem II (PSII) decreased more slightly in sense lines but more markedly in the antisense ones, compared to WT. D1 protein, located in the reactive center of the PSII, is the primary target of photodamage and has the highest turnover rate in the chloroplast. Under salt stress, compared with WT, the content of D1 protein decreased slightly in sense lines and significantly in the antisense ones. In the presence of streptomycin (SM), the net degradation of the damaged D1 protein was faster in sense lines than in other plants. These results suggested that, under salt-stress conditions, increasing cis-unsaturated fatty acids in PG by overexpression of LeGPAT can alleviate PSII photoinhibition by accelerating the repair of D1 protein and improving the activity of antioxidant enzymes in chloroplasts.     

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.     

Antisense-mediated depletion of tomato chloroplast glycerol-3-phosphate acyltransferase affects male fertility and increases thermal tolerance

A chloroplast-localized tomato ( Lycopersicon esculentum Mill.) glycerol-3-phosphate acyltransferase (LeGPAT) gene was isolated. The antisense tomato plants were got under the control of the caulifower mosaic virus 35S promoter (35S-CaMV). RNA gel blot analysis confirmed that the expression of LeGPAT was inhibited in the tomato genome. The depletion of LeGPAT caused a massive arrest in pollen development. It also increased the size of tapetal cells, delayed tapetum degeneration, reduced ER membrane biogenesis and altered oil body size. Results therefore suggested that LeGPAT played a crucial role in pollen development. There was, however, no effect on the ovule. The depletion of LeGPAT also increased the saturation of phosphatidylglycerol (PG) fatty acids in thylakoid membranes. Increase of PG-saturated fatty acids was helpful in alleviating photoinhibition of PSII in tomato plants under heat stress.     

Genetic Engineering of Glycine Betaine Biosynthesis Reduces Heat-Enhanced Photoinhibition by Enhancing Antioxidative Defense and Alleviating Lipid Peroxidation in Tomato

Glycine betaine (GB) is a compatible solute that accumulates rapidly to enhance heat tolerance in many plants grown under heat stress. In this study, a BADH gene (betaine aldehyde dehydrogenase) from spinach was introduced into tomato (Lycopersicon esculentum cv. ‘Moneymaker’) via Agrobacterium-mediated transformation. Transgenic tomato lines expressing BADH exhibited higher capabilities for GB accumulation. Chlorophyll fluorescence analysis of wild type (WT) and transgenic plants exposed to heat treatment (42 °C) showed that transgenic plants exhibited higher photosynthetic capacities than WT plants. This finding suggests that GB accumulation increases tolerance to heat-enhanced photoinhibition. This increased tolerance was associated with an improvement in D1 protein content, which accelerated the repair of photosystem II (PSII) following heat-enhanced photoinhibition. Significant accumulations of hydrogen peroxide (H₂O₂) and superoxide radical (O₂ ^?) were observed in WT plants under heat stress. However, these accumulations were much less for the transgenic plants. An important finding reported herein is that exogenous GB cannot directly reduce the content of reactive oxygen species (ROS). In accordance with a lower relative electrolyte conductivity and malondialdehyde content, the activities of antioxidant enzymes were higher in transgenic lines than in WT plants, indicating that the degree of membrane injury in the transgenic plants was lower compared to the WT plants. These results suggest that GB accumulation in vivo cannot directly eliminate ROS. Rather, higher antioxidant enzyme activities must be maintained to lessen the accumulation of ROS in transgenic plants and to decrease the degree of membrane injury.     

Overexpression of chloroplastic monodehydroascorbate reductase enhanced tolerance to temperature and methyl viologen‐mediated oxidative stresses

A tomato (Lycopersicon esculentum Mill.) monodehydroascorbate reductase gene (LeMDAR) was isolated. The LeMDAR–green fluorescence protein (GFP) fusion protein was targeted to chloroplast in Arabidopsis mesophyll protoplast. RNA and protein gel blot analyses confirmed that the sense‐ and antisense‐ LeMDAR were integrated into the tomato genome. The MDAR activities and the levels of reduced ascorbate (AsA) were markedly increased in sense transgenic lines and decreased in antisense transgenic lines compared with wild‐type (WT) plants. Under low and high temperature stresses, the sense transgenic plants showed lower level of hydrogen peroxide (H₂O₂), lower thiobarbituric acid reactive substance (TBARS) content, higher net photosynthetic rate (P_n), higher maximal photochemical efficiency of PSII (F_v/F_m) and fresh weight compared with WT plants. The oxidizable P700 decreased more obviously in WT and antisense plants than that in sense plants at chilling temperature under low irradiance. Furthermore, the sense transgenic plants exhibited significantly lower H₂O₂ level, higher ascorbate peroxidase (APX) activity, greater P_n and F_v/F_m under methyl viologen (MV)‐mediated oxidative stresses. These results indicated that overexpression of chloroplastic MDAR played an important role in alleviating photoinhibition of PSI and PSII and enhancing the tolerance to various abiotic stresses by elevating AsA level.     

Conferring high‐temperature tolerance to nontransgenic tomato scions using graft transmission of RNA silencing of the fatty acid desaturase gene

We investigated graft transmission of high‐temperature tolerance in tomato scions to nontransgenic scions from transgenic rootstocks, where the fatty acid desaturase gene (LeFAD7) was RNA‐silenced. Tomato was transformed with a plasmid carrying an inverted repeat of LeFAD7 by Agrobacterium. Several transgenic lines showed the lower amounts of LeFAD7 RNA and unsaturated fatty acids, while nontransgenic control did not, and siRNA was detected in the transgenic lines, but not in control. These lines grew under conditions of high temperature, while nontransgenic control did not. Further, the nontransgenic plants were grafted onto the silenced transgenic plants. The scions showed less of the target gene RNA, and siRNA was detected. Under high‐temperature conditions, these grafted plants grew, while control grafted plants did not. Thus, it was shown that high‐temperature tolerance was conferred in the nontransgenic scions after grafting onto the silenced rootstocks.     

Response of xanthophyll cycle and chloroplastic antioxidant enzymes to chilling stress in tomato over-expressing glycerol-3-phosphate acyltransferase gene

Over-expression of chloroplastic glycerol-3-phosphate acyltransferase gene (LeGPAT) increased unsaturated fatty acid contents in phosphatidylglycerol (PG) of thylakoid membrane in tomato. The effect of this increase on the xanthophyll cycle and chloroplast antioxidant enzymes was examined by comparing wild type (WT) tomato with the transgenic (TG) lines at chilling temperature (4 °C) under low irradiance (100 μmol m⁻² s⁻¹). Net photosynthetic rate and the maximal photochemical efficiency of photosystem (PS) 2 (F_v/F_m) in TG plants decreased more slowly during chilling stress and F_v/F_m recovered faster than that in WT plants under optimal conditions. The oxidizable P700 in both WT and TG plants decreased during chilling stress under low irradiance, but recovered faster in TG plants than in the WT ones. During chilling stress, non-photochemical quenching (NPQ) and the de-epoxidized ratio of xanthophyll cycle in WT plants were lower than those of TG tomatoes. The higher activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in TG plants resulted in the reduction of O₂ ^−· and H₂O₂ contents during chilling stress. Hence the increase in content of unsaturated fatty acids in PG by the over-expression of LeGPAT could alleviate photoinhibition of PS2 and PS1 by improving the de-epoxidized ratio of xanthophyll cycle and activities of SOD and APX in chloroplast.     

Tomato plants ectopically expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress

A DNA cassette containing an Arabidopsis C repeat/dehydration-responsive element binding factor 1 (CBF1) cDNA and a nos terminator, driven by a cauliflower mosaic virus 35S promoter, was transformed into the tomato (Lycopersicon esculentum) genome. These transgenic tomato plants were more resistant to water deficit stress than the wild-type plants. The transgenic plants exhibited growth retardation by showing dwarf phenotype, and the fruit and seed numbers and fresh weight of the transgenic tomato plants were apparently less than those of the wild-type plants. Exogenous gibberellic acid treatment reversed the growth retardation and enhanced growth of transgenic tomato plants, but did not affect the level of water deficit resistance. The stomata of the transgenic CBF1 tomato plants closed more rapidly than the wild type after water deficit treatment with or without gibberellic acid pretreatment. The transgenic tomato plants contained higher levels of Pro than those of the wild-type plants under normal or water deficit conditions. Subtractive hybridization was used to isolate the responsive genes to heterologous CBF1 in transgenic tomato plants and the CAT1 (CATALASE1) was characterized. Catalase activity increased, and hydrogen peroxide concentration decreased in transgenic tomato plants compared with the wild-type plants with or without water deficit stress. These results indicated that the heterologous Arabidopsis CBF1 can confer water deficit resistance in transgenic tomato plants.     

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.