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.     

Antisense-mediated Depletion of Tomato Endoplasmic Reticulum Omega-3 Fatty Acid Desaturase Enhances Thermal Tolerance

An endoplasmic reticulum-localized tomato ω-3 fatty acid desaturase gene(LeFAD3) was isolated.The antisense tomato plants were obtained under the control of the cauliflower mosaic virus 35S promoter(35S-CaMV).Northern blot analysis confirmed that the expression of LeFAD3 was inhibited in the tomato genome.Levels of 18:3 decreased and correspondingly levels of 18:2 increased in total lipids of leaves and roots.After heat stress,the fresh weight of the aerial parts of antisense transgenic plants was higher than that of the wild type(WT) plants.The membrane system ultrastructure of chloroplasts in leaf cells and all of the subcellular organelles in the root tips of transgenic plants remained more intact than those of WT.Relative electric conductivity increased less in transgenic plants than in WT.Under heat stress,the maximal photochemical efficiency of photosystem II(Fv/Fm) and the O2 evolution rate decreased more in WT than in transgenic plants.These results suggested that the depletion of LeFAD3 increased the saturation of fatty acids and alleviated high temperature stress.     

Fatty-acid-induced release of manganes from chloroplasts

The effect of both endogenous and exogenous unsaturated free fatty acids on manganese release from chloroplasts of chill-resistant (spinach) and chill-sensitive (tomato, bean) plants was studied. The level of endogenous free fatty acids increased 2–3-fold during cold and dark storage of leaves of chill-sensitive plants and was accompanied by depletion of about 60% of total chloroplast manganese content. Similar effects were observed when accumulation of free fatty acids in chloroplasts was achieved by storage of growing tomato plants for a few days in the dark at room temperature. In contrast, the cold and dark treatment of leaves of chill-resistant plant (spinach) affected neither free fatty acid, manganese levels nor Hill-reaction activity in chloroplasts. Incubation of chloroplasts of both chill-sensitive and chill-resistant plants with bean leaf galactolipase resulted in an accumulation of free fatty acids and a release of approx. 60% of total manganese content. The same amount of total manganese content was released following 3 h incubation of chloroplasts with linolenic acid at fatty acid/chlorophyll ratio (w/w, 2:1–10:1). The efficiency of C₁₈ unsaturated fatty acids/linolenic, linoleic, oleic on manganese release from chloroplasts was established in decreasing order C_18:3 > C_18:2 > C_18:1. The results indicate that the inhibitory effect of both endogenous and exogenous fatty acids on Hill reaction depends on the release from chloroplasts of functionally active, loosely bound manganese. Thus, similarly to both Tris and hydroxylamine treatments of chloroplasts, the incubation of chloroplast preparations with unsaturated fatty acids may be a useful tool for manganese depletion of chloroplasts.     

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.     

Physiological and Molecular Effects of 24-Epibrassinolide, a Brassinosteroid on Thermotolerance of Tomato

Brassinosteroids are naturally occurring plant growth regulators, which exhibit structural similarities to animal steroid hormones. Recent studies have indicated that besides an essential role in plant growth and development, brassinosteroids also exert anti-stress effects on plants. We show here that tomato plants treated with 24-epibrassinolide (EBR) are more tolerant to high temperature than untreated plants. An analysis of mitochondrial small heat shock proteins (MT-sHSP) in tomato leaves by western blotting revealed that the MT-sHSP did not preferentially accumulate in EBR treated plants at 25 °C. However, treatment of plants at 38 °C induced much more accumulation of MT-sHSP in EBR treated than in untreated plants. Results of this study provide the first direct evidence for EBR induced expression of MT-sHSP, which possibly induced thermotolerance in tomato plants. EBR treated tomato plants had better photosynthetic efficiency. We also observed significantly higher in vitro pollen germination, enhanced pollen tube growth and low pollen bursting in the presence of EBR at 35 °C, a temperature high enough to induce heat-stress symptoms in tomato, indicating a possible role of EBR during plant reproduction.     

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.     

Effects of mycorrhizal infection and soil phosphorus availability on in vitro and in vivo pollen performance in Lycopersicon esculentum (Solanaceae)

The effects of mycorrhizal infection and soil P availability on in vitro and in vivo pollen performance were studied in two cultivars of tomato (Lycopersicon esculentum). In the first study, plants were grown in a greenhouse under three treatment combinations: nonmycorrhizal, low P (NMPO); nonmycorrhizal, high P (NMP3); and mycorrhizal, low P (MPO). Mycorrhizal infection and high soil P conditions significantly increased in vitro pollen tube growth rates but not percentage of germination. In addition, pollen from NMP3 and MPO plants sired significantly more seeds than pollen from NMPO plants in pollen mixture studies. In the second study, plants were grown initially in a greenhouse under two treatment combinations: NMPO and MPO. After all plants began to flower, they were placed in experimental arrays in the field. Under open pollination, pollen from MPO plants sired significantly more seeds than pollen from NMPO plants. This result was primarily attributed to increased flower production (and thus pollen production) in MPO plants. Thus, mycorrhizal infection and high soil P conditions can increase pollen quality (in vitro and in vivo pollen performance) as well as pollen quantity, thereby enhancing fitness through the male function. Anthocyanin production (used to determine paternity) also affected pollen performance.     

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.     

The Critical Requirement for Linolenic Acid Is Pollen Development,Not Photosynthesis,in an Arabidopsis Mutant

The very high proportions of trienoic fatty acids found in chloroplast membranes of all higher plants suggest that these lipid structures might be essential for photosynthesis. We report here on the production of Arabidopsis triple mutants that contain negligible levels of trienoic fatty acids. Photosynthesis at 22[deg]C was barely affected, and vegetative growth of the mutants was identical with that of the wild type, demonstrating that any requirement for trienoic acyl groups in membrane structure and function is relatively subtle. Although vegetative growth and development were unaffected, the triple mutants are male sterlle and produce no seed under normal conditions. Comparisons of pollen development in wild-type and triple mutant flowers established that pollen grains in the mutant developed to the tricellular stage. Exogenous applications of [alpha]-llnolenate or jasmonate restored fertility. Taken together, the results demonstrate that the critical role of trienoic acids in the life cycle of plants is as the precursor of oxylipin, a signaling compound that regulates final maturation processes and the release of pollen.     

The effect of heat stress on tomato pollen characteristics is associated with changes in carbohydrate concentration in the developing anthers

Continuous exposure of tomato 'Trust' to high temperatures (day/night temperatures of 32/26 degrees C) markedly reduced the number of pollen grains per flower and decreased viability. The effect of heat stress on pollen viability was associated with alterations in carbohydrate metabolism in various parts of the anther during its development. Under control, favourable temperature conditions (28/22 degrees C), starch accumulated in the pollen grains, where it reached a maximum value 3 d before anthesis; it then diminished towards anthesis. During anther development, the concentration of total soluble sugars gradually increased in the anther walls and in the pollen grains (but not in the locular fluid), reaching a maximum at anthesis. Continuous exposure of the plants to high temperatures (32/26 degrees C) prevented the transient increase in starch concentration and led to decreases in the concentrations of soluble sugars in the anther walls and the pollen grains. In the locular fluid, however, a higher soluble sugar concentration was detected under the high-temperature regime throughout anther development. These results suggest that a major effect of heat stress on pollen development is a decrease in starch concentration 3 d before anthesis, which results in a decreased sugar concentration in the mature pollen grains. These events possibly contribute to the decreased pollen viability in tomato.     

Overexpression of Pto induces a salicylate-independent cell death but inhibits necrotic lesions caused by salicylate-deficiency in tomato plants

Tomato plants overexpressing the disease resistance gene Pto (35S::Pto) exhibit spontaneous cell death, accumulation of salicylic acid (SA), elevated expression of pathogenesis-related genes, and enhanced resistance to a broad range of pathogens. Because salicylate plays an important role in the cell death and defense activation in many lesion mimic mutants, we investigated the interaction of SA-mediated processes and the 35S::Pto-mediated defense pathway by introducing the nahG transgene that encodes salicylate hydroxylase. Here, we show that SA is not required for the 35S::Pto-activated microscopic cell death and plays a minor role in defense gene activation and general disease resistance in 35S::Pto plants. In contrast, temperature greatly affects the spontaneous cell death and general resistance in 35S::Pto plants, and high temperature inhibits the cell death. The NahG tomato plants develop spontaneous, unconstrained necrotic lesions on leaves. These lesions also are initiated by the inoculation of a virulent strain of Pseudomonas syringae pv. tomato. However, the NahG-dependent necrotic lesions are inhibited in the NahG/35S::Pto plants. This inhibition is most pronounced under conditions favoring the 35S::Pto-mediated spontaneous cell death development. These results indicate that the signaling pathways activated by Pto overexpression suppress the cellular damage that is caused by SA depletion. We also found that ethylene is dispensable for the 35S::Pto-mediated general defense.     

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.     

The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development

Jasmonic acid (JA) is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores and some microbial pathogens. Molecular genetic studies in Arabidopsis have established that JA also performs a critical role in anther and pollen development but is not essential for other developmental aspects of the plant's life cycle. Here, we describe the phenotypic and molecular characterization of a sterile mutant of tomato (jasmonic acid-insensitive1 [jai1]) that is defective in JA signaling. Although the mutant exhibited reduced pollen viability, sterility was caused by a defect in the maternal control of seed maturation, which was associated with the loss of accumulation of JA-regulated proteinase inhibitor proteins in reproductive tissues. jai1 plants exhibited several defense-related phenotypes, including the inability to express JA-responsive genes, severely compromised resistance to two-spotted spider mites, and abnormal development of glandular trichomes. We demonstrate that these defects are caused by the loss of function of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. These findings indicate that the JA/COI1 signaling pathway regulates distinct developmental processes in different plants and suggest a role for JA in the promotion of glandular trichome-based defenses.     

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.     

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.     

Ultrastructure and lipid alterations induced by cadmium in tomato (Lycopersicon esculentum) chloroplast membranes

The effects of cadmium (Cd) uptake on ultrastructure and lipid composition of chloroplasts were investigated in 28-day-old tomato plants (Lycopersicon esculentum var. Ibiza F1) grown for 10 days in the presence of various concentrations of CdCl2. Different growth parameters, lipid and fatty acid composition, lipid peroxidation, and lipoxygenase activity were measured in the leaves in order to assess the involvement of this metal in the generation of oxidative stress. We first observed that the accumulation of Cd increased with external metal concentration, and was considerably higher in roots than in leaves. Cadmium induced a significant inhibition of growth in both plant organs, as well as a reduction in the chlorophyll and carotenoid contents in the leaves. Ultrastructural investigations revealed that cadmium induced disorganization in leaf structure, essentially marked by a lowered mesophyll cell size, reduced intercellular spaces, as well as severe alterations in chloroplast fine structure, which exhibits disturbed shape and dilation of thylakoid membranes. High cadmium concentrations also affect the main lipid classes, leading to strong changes in their composition and fatty acid content. Thus, the exposure of tomato plants to cadmium caused a concentration-related decrease in the fatty acid content and a shift in the composition of fatty acids, resulting in a lower degree of fatty acid unsaturation in chloroplast membranes. The level of lipid peroxides and the activity of lipoxygenase were also significantly enhanced at high Cd concentrations. These biochemical and ultrastructural changes suggest that cadmium, through its effects on membrane structure and composition, induces premature senescence of leaves.     

Nutritional content of fresh, bee-collected and stored pollen of Aloe greatheadii var. davyana (Asphodelaceae)

Aloe greatheadii var. davyana is the most important indigenous South African bee plant. Fresh, bee-collected and stored pollen of this aloe was collected and analysed for its nutritional content, including amino acid and fatty acid composition. Highly significant differences were found between the three types of pollen. Collection and storage by the bees resulted in increased water (13-21% wet weight) and carbohydrate content (35-61% dry weight), with a resultant decrease in crude protein (51-28% dry weight) and lipid content (10-8% dry weight). Essential amino acids were present in equal or higher amounts than the required minimum levels for honeybee development, with the exception of tryptophan. Fatty acids comprised a higher proportion of total lipid in fresh pollen than in bee-collected and stored pollen. This study is the first to compare the changes that occur in pollen of a single species after collection by honeybees.     

CYP703 is an ancient cytochrome P450 in land plants catalyzing in-chain hydroxylation of lauric acid to provide building blocks for sporopollenin synthesis in pollen

CYP703 is a cytochrome P450 family specific to land plants. Typically, each plant species contains a single CYP703. Arabidopsis thaliana CYP703A2 is expressed in the anthers of developing flowers. Expression is initiated at the tetrad stage and restricted to microspores and to the tapetum cell layer. Arabidopsis CYP703A2 knockout lines showed impaired pollen development and a partial male-sterile phenotype. Scanning electron and transmission electron microscopy of pollen from the knockout plants showed impaired pollen wall development with absence of exine. The fluorescent layer around the pollen grains ascribed to the presence of phenylpropanoid units in sporopollenin was absent in the CYP703A2 knockout lines. Heterologous expression of CYP703A2 in yeast cells demonstrated that CYP703 catalyzes the conversion of medium-chain saturated fatty acids to the corresponding monohydroxylated fatty acids, with a preferential hydroxylation of lauric acid at the C-7 position. Incubation of recombinant CYP703 with methanol extracts from developing flowers confirmed that lauric acid and in-chain hydroxy lauric acids are the in planta substrate and product, respectively. These data demonstrate that in-chain hydroxy lauric acids are essential building blocks in sporopollenin synthesis and enable the formation of ester and ether linkages with phenylpropanoid units. This study identifies CYP703 as a P450 family specifically involved in pollen development.