Inactivation of genes,encoding tocopherol biosynthetic pathway enzymes,results in oxidative stress in outdoor grown Arabidopsis thaliana

Tocopherols (α-, β-, γ- and δ-tocopherols) represent a group of lipophilic antioxidants which are synthesized only by photosynthetic organisms. It is widely believed that protection of pigments and proteins of photosynthetic system and polyunsaturated fatty acids from oxidative damage caused by reactive oxygen species (ROS) is the main function of tocopherols. The wild type Columbia and two mutants of Arabidopsis thaliana with T-DNA insertions in tocopherol biosynthesis genes – tocopherol cyclase (vte1) and γ-tocopherol methyltransferase (vte4) – were analyzed after long-term outdoor growth. The concentration of total tocopherol was up to 12-fold higher in outdoor growing wild type and vte4 plant lines than in plants grown under laboratory conditions. The vte4 mutant plants had a lower concentration of chlorophylls and carotenoids, whereas the mutant plants had a higher level of total glutathione than of wild type. The activities of antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate oxidase (AO, EC 1.10.3.3) were lower in both mutants, whereas activities of catalase (EC 1.11.1.6) and ascorbate peroxidase (APx, EC 1.11.1.11) were lower only in vte1 mutant plants in comparison to wild type plants. However, the activity of guaiacol peroxidase (GuPx, EC 1.11.1.7) was higher in vte1 and vte4 mutants than that in wild type. Additionally, both mutant plant lines had higher concentration of protein carbonyl groups and oxidized glutathione compared to the wild type, indicating the development of oxidative stress. These results demonstrate in plants that tocopherols play a crucial role for growth of plants under outdoor conditions by preventing oxidation of cellular components.     

RNAi-mediated tocopherol deficiency impairs photoassimilate export in transgenic potato plants

Tocopherols (vitamin E) are lipophilic antioxidants presumed to play a key role in protecting chloroplast membranes and the photosynthetic apparatus from photooxidative damage. Additional nonantioxidant functions of tocopherols have been proposed after the recent finding that the Suc export defective1 maize (Zea mays) mutant (sxd1) carries a defect in tocopherol cyclase (TC) and thus is devoid of tocopherols. However, the corresponding vitamin E deficient1 Arabidopsis mutant (vte1) lacks a phenotype analogous to sxd1, suggesting differences in tocopherol function between C4 and C3 plants. Therefore, in this study, the potato (Solanum tuberosum) ortholog of SXD1 was isolated and functionally characterized. StSXD1 encoded a protein with high TC activity in vitro, and chloroplastic localization was demonstrated by transient expression of green fluorescent protein-tagged fusion constructs. RNAi-mediated silencing of StSXD1 in transgenic potato plants resulted in the disruption of TC activity and severe tocopherol deficiency similar to the orthologous sxd1 and vte1 mutants. The nearly complete absence of tocopherols caused a characteristic photoassimilate export-defective phenotype comparable to sxd1, which appeared to be a consequence of vascular-specific callose deposition observed in source leaves. CO2 assimilation rates and photosynthetic gene expression were decreased in source leaves in close correlation with excess sugar accumulation, suggesting a carbohydrate-mediated feedback inhibition rather than a direct impact of tocopherol deficiency on photosynthetic capacity. This conclusion is further supported by an increased photosynthetic capacity of young leaves regardless of decreased tocopherol levels. Our data provide evidence that tocopherol deficiency leads to impaired photoassimilate export from source leaves in both monocot and dicot plant species and suggest significant differences among C3 plants in response to tocopherol reduction.     

Characterization of tocopherol cyclases from higher plants and cyanobacteria. Evolutionary implications for tocopherol synthesis and function

Tocopherols are lipophilic antioxidants synthesized exclusively by photosynthetic organisms and collectively constitute vitamin E, an essential nutrient for both humans and animals. Tocopherol cyclase (TC) catalyzes the conversion of various phytyl quinol pathway intermediates to their corresponding tocopherols through the formation of the chromanol ring. Herein, the molecular and biochemical characterization of TCs from Arabidopsis (VTE1 [VITAMIN E 1]), Zea mays (SXD1 [Sucrose Export Deficient 1]) and Synechocystis sp. PCC6803 (slr1737) are described. Mutations in the VTE1, SXD1, or slr1737 genes resulted in both tocopherol deficiency and the accumulation of 2,3-dimethyl-6-phytyl-1,4-benzoquinone (DMPBQ), a TC substrate. Recombinant SXD1 and VTE1 proteins are able to convert DMPBQ to gamma-tocopherol in vitro. In addition, expression of maize SXD1 in a Synechocystis sp. PCC6803 slr1737 knockout mutant restored tocopherol synthesis, indicating that TC activity is evolutionarily conserved between plants and cyanobacteria. Sequence analysis identified a highly conserved 30-amino acid C-terminal domain in plant TCs that is absent from cyanobacterial orthologs. vte1-2 causes a truncation within this C-terminal domain, and the resulting mutant phenotype suggests that this domain is necessary for TC activity in plants. The defective export of Suc in sxd1 suggests that in addition to presumed antioxidant activities, tocopherols or tocopherol breakdown products also function as signal transduction molecules, or, alternatively, the DMPBQ that accumulates in sxd1 disrupts signaling required for efficient Suc export in maize.     

Characterization of an Arabidopsis mutant deficient in γ-tocopherol methyltransferase

Alpha-tocopherol (vitamin E) is synthesized from gamma-tocopherol in chloroplasts by gamma-tocopherol methyltransferase (gamma-TMT; VTE4). Leaves of many plant species including Arabidopsis contain high levels of alpha-tocopherol, but are low in gamma-tocopherol. To unravel the function of different forms of tocopherol in plants, an Arabidopsis plant (vte4-1) carrying a functional null mutation in the gene gamma-TMT was isolated by screening a mutant population via thin-layer chromatography. A second mutant allele (vte4-2) carrying a T-DNA insertion in the coding sequence of gamma-TMT was identified in a T-DNA tagged mutant population. In vte4-1 and vte4-2 leaves, high levels of gamma-tocopherol accumulated, whereas alpha-tocopherol was absent indicating that, presumably, these two mutants represents null alleles. Over-expression of the gamma-TMT cDNA in vte4-1 restored wild-type tocopherol composition. Mutant plants were very similar to wild type. During oxidative stress (high light, high temperature, cold treatment) the amounts of alpha-tocopherol and gamma-tocopherol increased in wild type, and gamma-tocopherol in vte4-1. However, chlorophyll content and photosynthetic quantum yield were very similar in wild type and vte4-1, suggesting that alpha-tocopherol can be replaced by gamma-tocopherol in vte4-1 to protect the photosynthetic apparatus against oxidative stress. Fatty acid and lipid composition were very similar in WT, vte4-1 and vte1, an Arabidopsis mutant previously isolated which is completely devoid of tocopherol. Therefore, a shift in tocopherol composition or the absence of tocopherol has no major impact on the amounts of specific fatty acids or on lipid hydrolysis.     

Remobilization of Phytol from Chlorophyll Degradation Is Essential for Tocopherol Synthesis and Growth of Arabidopsis

Phytol from chlorophyll degradation can be phosphorylated to phytyl-phosphate and phytyl-diphosphate, the substrate for tocopherol (vitamin E) synthesis. A candidate for the phytyl-phosphate kinase from Arabidopsis thaliana (At1g78620) was identified via a phylogeny-based approach. This gene was designated VITAMIN E DEFICIENT6 (VTE6) because the leaves of the Arabidopsis vte6 mutants are tocopherol deficient. The vte6 mutant plants are incapable of photoautotrophic growth. Phytol and phytyl-phosphate accumulate, and the phytyl-diphosphate content is strongly decreased in vte6 leaves. Phytol feeding and enzyme assays with Arabidopsis and recombinant Escherichia coli cells demonstrated that VTE6 has phytyl-P kinase activity. Overexpression of VTE6 resulted in increased phytyl-diphosphate and tocopherol contents in seeds, indicating that VTE6 encodes phytyl-phosphate kinase. The severe growth retardation of vte6 mutants was partially rescued by introducing the phytol kinase mutation vte5. Double mutant plants (vte5 vte6) are tocopherol deficient and contain more chlorophyll, but reduced amounts of phytol and phytyl-phosphate compared with vte6 mutants, suggesting that phytol or phytyl-phosphate are detrimental to plant growth. Therefore, VTE6 represents the missing phytyl-phosphate kinase, linking phytol release from chlorophyll with tocopherol synthesis. Moreover, tocopherol synthesis in leaves depends on phytol derived from chlorophyll, not on de novo synthesis of phytyl-diphosphate from geranylgeranyl-diphosphate.     

Highly divergent methyltransferases catalyze a conserved reaction in tocopherol and plastoquinone synthesis in cyanobacteria and photosynthetic eukaryotes

Tocopherols are lipid-soluble compounds synthesized only by photosynthetic eukaryotes and oxygenic cyanobacteria. The pathway and enzymes for tocopherol synthesis are homologous in cyanobacteria and plants except for 2-methyl-6-phytyl-1,4-benzoquinone/2-methyl-6-solanyl-1,4-benzoquinone methyltransferase (MPBQ/MSBQ MT), which catalyzes a key methylation step in both tocopherol and plastoquinone (PQ) synthesis. Using a combined genomic, genetic, and biochemical approach, we isolated and characterized the VTE3 (vitamin E defective) locus, which encodes MPBQ/MSBQ MT in Arabidopsis. The phenotypes of vte3 mutants are consistent with the disruption of MPBQ/MSBQ MT activity to varying extents. The ethyl methanesulfonate-derived vte3-1 allele alters tocopherol composition but has little impact on PQ levels, whereas the null vte3-2 allele is deficient in PQ and alpha- and gamma-tocopherols. In vitro enzyme assays confirmed that VTE3 is the plant functional equivalent of the previously characterized MPBQ/MSBQ MT (Sll0418) from Synechocystis sp PCC6803, although the two proteins are highly divergent in primary sequence. Sll0418 orthologs are present in all fully sequenced cyanobacterial genomes, Chlamydomonas reinhardtii, and the diatom Thalassiosira pseudonana but absent from vascular and nonvascular plant databases. VTE3 orthologs are present in all vascular and nonvascular plant databases and in C. reinhardtii but absent from cyanobacterial genomes. Intriguingly, the only prokaryotic genomes that contain VTE3-like sequences are those of two species of archea, suggesting that, in contrast to all other enzymes of the plant tocopherol pathway, the evolutionary origin of VTE3 may have been archeal rather than cyanobacterial. In vivo analyses of vte3 mutants and the corresponding homozygous Synechocystis sp PCC6803 sll0418::aphII mutant revealed important differences in enzyme redundancy, the regulation of tocopherol synthesis, and the integration of tocopherol and PQ biosynthesis in cyanobacteria and plants.     

The role of homogentisate phytyltransferase and other tocopherol pathway enzymes in the regulation of tocopherol synthesis during abiotic stress

Tocopherols are amphipathic antioxidants synthesized exclusively by photosynthetic organisms. Tocopherol levels change significantly during plant growth and development and in response to stress, likely as a consequence of the altered expression of pathway-related genes. Homogentisate phytyltransferase (HPT) is a key enzyme limiting tocopherol biosynthesis in unstressed Arabidopsis leaves (E. Collakova, D. DellaPenna [2003] Plant Physiol 131: 632-642). Wild-type and transgenic Arabidopsis plants constitutively overexpressing HPT (35S::HPT1) were subjected to a combination of abiotic stresses for up to 15 d and tocopherol levels, composition, and expression of several tocopherol pathway-related genes were determined. Abiotic stress resulted in an 18- and 8-fold increase in total tocopherol content in wild-type and 35S::HPT1 leaves, respectively, with tocopherol levels in 35S::HPT1 being 2- to 4-fold higher than wild type at all experimental time points. Increased total tocopherol levels correlated with elevated HPT mRNA levels and HPT specific activity in 35S::HPT1 and wild-type leaves, suggesting that HPT activity limits total tocopherol synthesis during abiotic stress. In addition, substrate availability and expression of pathway enzymes before HPT also contribute to increased tocopherol synthesis during stress. The accumulation of high levels of beta-, gamma-, and delta-tocopherols in stressed tissues suggested that the methylation of phytylquinol and tocopherol intermediates limit alpha-tocopherol synthesis. Overexpression of gamma-tocopherol methyltransferase in the 35S::HPT1 background resulted in nearly complete conversion of gamma- and delta-tocopherols to alpha- and beta-tocopherols, respectively, indicating that gamma-tocopherol methyltransferase activity limits alpha-tocopherol synthesis in stressed leaves.     

Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination

Tocopherols (vitamin E) are lipophilic antioxidants synthesized by all plants and are particularly abundant in seeds. Despite cloning of the complete suite of tocopherol biosynthetic enzymes and successful engineering of the tocopherol content and composition of Arabidopsis thaliana leaves and seeds, the functions of tocopherols in plants have remained elusive. To address this issue, we have isolated and characterized two VITAMIN E loci (VTE1 and VTE2) in Arabidopsis that when mutated result in tocopherol deficiency in all tissues. vte1 disrupts tocopherol cyclase activity and accumulates a redox-active biosynthetic intermediate, whereas vte2 disrupts homogentisate phytyl transferase activity and does not accumulate pathway intermediates. Mutations at either locus cause significantly reduced seed longevity compared with the wild type, indicating a critical role for tocopherols in maintaining viability during quiescence. However, only vte2 mutants exhibited severe seedling growth defects during germination and contained levels of lipid hydroperoxides and hydroxy fatty acids elevated up to 4- and 100-fold, respectively, relative to the wild type. These data demonstrate that a primary function of tocopherols in plants is to limit nonenzymatic lipid oxidation during seed storage, germination, and early seedling development. The vte mutant phenotypes also explain the strong selection for retention of tocopherol biosynthesis during the evolution of seed-bearing plants.     

Glutathione and transpiration as key factors conditioning oxidative stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> exposed to uranium

Although oxidative stress has been previously described in plants exposed to uranium (U), some uncertainty remains about the role of glutathione and tocopherol availability in the different responsiveness of plants to photo-oxidative damage. Moreover, in most cases, little consideration is given to the role of water transport in shoot heavy metal accumulation. Here, we investigated the effect of uranyl nitrate exposure (50 μM) on PSII and parameters involved in water transport (leaf transpiration and aquaporin gene expression) of Arabidopsis wild type (WT) and mutant plants that are deficient in tocopherol (vte1: null α/γ-tocopherol and vte4: null α-tocopherol) and glutathione biosynthesis (high content: cad1.3 and low content: cad2.1). We show how U exposure induced photosynthetic inhibition that entailed an electron sink/source imbalance that caused PSII photoinhibition in the mutants. The WT was the only line where U did not damage PSII. The increase in energy thermal dissipation observed in all the plants exposed to U did not avoid photo-oxidative damage of mutants. The maintenance of control of glutathione and malondialdehyde contents probed to be target points for the overcoming of photoinhibition in the WT. The relationship between leaf U content and leaf transpiration confirmed the relevance of water transport in heavy metals partitioning and accumulation in leaves, with the consequent implication of susceptibility to oxidative stress.     

Light,temperature and tocopherol status influence foliar vascular anatomy and leaf function in Arabidopsis thaliana

This study addressed whether the winter annual Arabidopsis thaliana can adjust foliar phloem and xylem anatomy both differentially and in parallel. In plants acclimated to hot vs cool temperature, foliar minor vein xylem‐to‐phloem ratio was greater, whereas xylem and phloem responded concomitantly to growth light intensity. Across all growth conditions, xylem anatomy correlated with transpiration rate, while phloem anatomy correlated with photosynthetic capacity for two plant lines (wild‐type Col‐0 and tocopherol‐deficient vte1 mutant) irrespective of tocopherol status. A high foliar vein density (VD) was associated with greater numbers and cross‐sectional areas of both xylem and phloem cells per vein as well as higher rates of both photosynthesis and transpiration under high vs low light intensities. Under hot vs cool temperature, high foliar VD was associated with a higher xylem‐to‐phloem ratio and greater relative rates of transpiration to photosynthesis. Tocopherol status affected development of foliar vasculature as dependent on growth environment. The most notable impact of tocopherol deficiency was seen under hot growth temperature, where the vte1 mutant exhibited greater numbers of tracheary elements (TEs) per vein, a greater ratio of TEs to sieve elements, with smaller individual sizes of TEs, and resulting similar total areas of TEs per vein and transpiration rates compared with Col‐0 wild‐type. These findings illustrate the plasticity of foliar vascular anatomy acclimation to growth environment resulting from independent adjustments of the vasculature's components.     

Enhanced tolerance to drought stress in transgenic tobacco plants overexpressing VTE1 for increased tocopherol production from Arabidopsis thaliana

Tocopherol cyclase (VTE1, encoded by VTE1 gene) catalyzes the penultimate step of tocopherol synthesis. Transgenic tobacco plants overexpressing VTE1 from Arabidopsis were exposed to drought conditions during which transgenic lines had decreased lipid peroxidation, electrolyte leakage and H(2)O(2) content, but had increased chlorophyll compared with the wild type. Thus VTE1 can be used to increase vitamin E content of plants and also to enhance tolerance to environmental stresses.     

The Arabidopsis vitamin E pathway gene5-1 mutant reveals a critical role for phytol kinase in seed tocopherol biosynthesis

We report the identification and characterization of a low tocopherol Arabidopsis thaliana mutant, vitamin E pathway gene5-1 (vte5-1), with seed tocopherol levels reduced to 20% of the wild type. Map-based identification of the responsible mutation identified a G-->A transition, resulting in the introduction of a stop codon in At5g04490, a previously unannotated gene, which we named VTE5. Complementation of the mutation with the wild-type transgene largely restored the wild-type tocopherol phenotype. A knockout mutation of the Synechocystis sp PCC 6803 VTE5 homolog slr1652 reduced Synechocystis tocopherol levels by 50% or more. Bioinformatic analysis of VTE5 and slr1652 indicated modest similarity to dolichol kinase. Analysis of extracts from Arabidopsis and Synechocystis mutants revealed increased accumulation of free phytol. Heterologous expression of these genes in Escherichia coli supplemented with free phytol and in vitro assays of recombinant protein produced phytylmonophosphate, suggesting that VTE5 and slr1652 encode phytol kinases. The phenotype of the vte5-1 mutant is consistent with the hypothesis that chlorophyll degradation-derived phytol serves as an important intermediate in seed tocopherol synthesis and forces reevaluation of the role of geranylgeranyl diphosphate reductase in tocopherol biosynthesis.     

Ultraviolet-B-induced oxidative stress and antioxidant defense system responses in ascorbate-deficient vtc1 mutants of Arabidopsis thaliana

Ultraviolet-B (UV-B) radiation has a negative impact on plant cells, and results in the generation of reactive oxygen species (ROS). In order to increase our understanding of the effects of UV-B on antioxidant processes, we investigated the response of an ascorbate-deficient Arabidopsis thaliana mutant vtc1 to short-term increased UV-B exposure. After UV-B supplementation, vtc1 mutants exhibited oxidative damage. Evidence for damage included an increase in H(2)O(2) content and the production of thiobarbituric acid reactive substances (TBARS); a decrease in chlorophyll content and chlorophyll fluorescence parameters were also reported. The vtc1 mutants had higher total glutathione than the wild type (WT) during the first day of UV-B treatment. We found reduced ratio of glutathione/total glutathione and increased ratio of dehydroascorbate/total ascorbate in the vtc1 mutants, compared to the WT plants. In addition, the enzymes responsible for ROS scavenging, including superoxide dismutase, catalase, and ascorbate peroxidase, had insufficient activity in the vtc1 mutants, compared to the WT plants. The same reduced activity in the vtc1 mutants was reported for the enzymes responsible for the regeneration of ascorbate and glutathione (including monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase). These results suggest that the ascorbate-deficient mutant vtc1 is more sensitive to supplementary UV-B treatment than WT plants and ascorbate can be considered an important antioxidant for UV-B radiation.     

Ascorbate plays a key role in alleviating low temperature-induced oxidative stress in Arabidopsis

Low temperature has a negative impact on plant cells and results in the generation of reactive oxygen species (ROS). In order to study the role of ascorbate under chilling stress, the response of an ascorbate-deficient Arabidopsis thaliana mutant vtc2-1 to low temperature (2°C) was investigated. After chilling stress, vtc2-1 mutants exhibited oxidative damage. An increase in the H₂O₂ generation and the production of thiobarbituric acid reactive substances (TBARS), and a decrease in chlorophyll content, the maximal photochemical efficiency of PSII (F_v/F_m) and oxidizable P₇₀₀ were also noted. The ratio of ascorbate/dehydroascorbate and reduced glutathione/oxidzed glutathione in the vtc2-1 mutants were reduced, compared with the wild type (WT) plants. The activities of antioxidant enzymes, such as catalase (CAT) and ascorbate peroxidase (APX), and soluble antioxidants were lower in the vtc2-1 mutants than those in WT plants. These results suggested that the ascorbate-deficient mutant vtc2-1 was more sensitive to chilling treatment than WT plants. The low temperature-induced oxidative stress was the major cause of the decrease of PSII and PSI function in the vtc2-1 mutants. Ascorbate plays a critical role of defense without which the rest of the ROS defense network is unable to react effectively.     

Singlet oxygen scavenging activity of tocopherol and plastochromanol in Arabidopsis thaliana: relevance to photooxidative stress

In the present study, singlet oxygen (¹O₂) scavenging activity of tocopherol and plastochromanol was examined in tocopherol cyclase‐deficient mutant (vte1) of Arabidopsis thaliana lacking both tocopherol and plastochromanol. It is demonstrated here that suppression of tocopherol and plastochromanol synthesis in chloroplasts isolated from vte1 Arabidopsis plants enhanced ¹O₂ formation under high light illumination as monitored by electron paramagnetic resonance spin‐trapping spectroscopy. The exposure of vte1 Arabidopsis plants to high light resulted in the formation of secondary lipid peroxidation product malondialdehyde as determined by high‐pressure liquid chromatography. Furthermore, it is shown here that the imaging of ultra‐weak photon emission known to reflect oxidation of lipids was unambiguously higher in vte1 Arabidopsis plants. Our results indicate that tocopherol and plastochromanol act as efficient ¹O₂ scavengers and protect effectively lipids against photooxidative damage in Arabidopsis plants.     

Overexpression of Arabidopsis homogentisate phytyltransferase or tocopherol cyclase elevates vitamin E content by increasing gamma-tocopherol level in lettuce (Lactuca sativa L.)

Tocopherols, essential components of the human diet, are synthesized exclusively by photosynthetic organisms. To increase tocopherol content by increasing total flux to the tocopherol biosynthetic pathway, genes encoding Arabidopsis homogentisate phytyltransferase (HPT/V-TE2) and tocopherol cyclase (TC/VTE1) were constitutively overexpressed in lettuce (Lactuca sativa L.). Total tocopherol content of the transgenic plants overexpressing either of the genes was increased by more than 2-fold mainly due to an increase in gamma-tocopherol. However, chlorophyll content in the HPT/VTE2 and TC/VTE1 transgenic lines decreased by up to 20% and increased by up to 35%, respectively (P < 0.01). These results demonstrate that manipulation of the tocopherol biosynthetic pathway can increase or decrease chlorophyll content depending on the gene introduced.     

Drought stress induces oxidative stress and the antioxidant defense system in ascorbate-deficient vtc1 mutants of Arabidopsis thaliana

Drought stress has a negative impact on plant cells and results in the generation of reactive oxygen species (ROS). To increase our understanding of the effects of drought stress on antioxidant processes, we investigated the response of the ascorbate-deficient Arabidopsis thaliana vtc1 mutant to drought stress. After drought stress, vtc1 mutants exhibited increases in several oxidative parameters, including H₂O₂ content and the production of thiobarbituric acid reactive substances. Decreases in chlorophyll content and chlorophyll fluorescence parameters were also observed. The vtc1 mutants had higher total glutathione than did wild-type (WT) plants after 48 h of drought stress. A reduced ratio of glutathione/total glutathione and an increased ratio of dehydroascorbate/total ascorbate were observed in the vtc1 mutants compared with the WT plants. In addition, the activities of enzymes that are responsible for ROS scavenging, including superoxide dismutase, catalase, and ascorbate peroxidase, were decreased in the vtc1 mutants compared with the WT plants. Similar reductions in activity in the vtc1 mutant were observed for the enzymes that are responsible for the regeneration of ascorbate and glutathione, including monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase. These results suggest that low intrinsic ascorbate and impaired ascorbate–glutathione cycling in the vtc1 mutant induced a decrease in the reduced form of ascorbate, which enhanced sensitivity to drought stress.