The C2H2 zinc‐finger protein SlZF3 regulates AsA synthesis and salt tolerance by interacting with CSN5B

Abiotic stresses are a major cause of crop loss. Ascorbic acid (AsA) promotes stress tolerance by scavenging reactive oxygen species (ROS), which accumulate when plants experience abiotic stress. Although the biosynthesis and metabolism of AsA are well established, the genes that regulate these pathways remain largely unexplored. Here, we report on a novel regulatory gene from tomato (Solanum lycopersicum) named SlZF3 that encodes a Cys2/His2‐type zinc‐finger protein with an EAR repression domain. The expression of SlZF3 was rapidly induced by NaCl treatments. The overexpression of SlZF3 significantly increased the levels of AsA in tomato and Arabidopsis. Consequently, the AsA‐mediated ROS‐scavenging capacity of the SlZF3‐overexpressing plants was increased, which enhanced the salt tolerance of these plants. Protein–protein interaction assays demonstrated that SlZF3 directly binds CSN5B, a key component of the COP9 signalosome. This interaction inhibited the binding of CSN5B to VTC1, a GDP‐mannose pyrophosphorylase that contributes to AsA biosynthesis. We found that the EAR domain promoted the stability of SlZF3 but was not required for the interaction between SlZF3 and CSN5B. Our findings indicate that SlZF3 simultaneously promotes the accumulation of AsA and enhances plant salt‐stress tolerance.     

Exploring the impact of wounding and jasmonates on ascorbate metabolism

Vitamin C (ascorbate, AsA) is the most abundant water-soluble antioxidant in plants. Ascorbate provides the first line of defense against damaging reactive oxygen species (ROS), and helps protect plant cells from many factors that induce oxidative stress, including wounding, ozone, high salinity, and pathogen attack. Plant defenses against these stresses are also dependent upon jasmonates (JAs), a class of plant hormones that promote ROS accumulation. Here, we review evidence showing that wounding and JAs influence AsA accumulation in various plant species, and we report new data from Arabidopsis and tomato testing the influence of JAs on AsA levels in wounded and unwounded plants. In both species, certain mutations that impair JA metabolism and signaling influence foliar AsA levels, suggesting that endogenous JAs may regulate steady-state AsA. However, the impact of wounding on AsA accumulation was similar in JA mutants and wild type controls, indicating that this wound response does not require JAs. Our findings also indicate that the effects of wounding and JAs on AsA accumulation differ between species; these factors both enhanced AsA accumulation in Arabidopsis, but depressed AsA levels in tomato. These results underscore the importance of obtaining data from more than one model species, and demonstrate the complexity of AsA regulation.     

Overexpression in tobacco of a tomato GMPase gene improves tolerance to both low and high temperature stress by enhancing antioxidation capacity

GDP-mannose pyrophosphorylase (GMPase: EC 2.7.7.22) plays a crucial role in the synthesis of l-ascorbate (AsA) and the consequent detoxification of reactive oxygen species (ROS). Herein, a GMPase (accession ID DQ449030) was identified and cloned from tomato. The full-length cDNA sequence of this gene contains 1,498 bp nucleotides encoding a putative protein with 361 amino acid residues of approximate molecular weight 43 kDa. Northern blot analysis revealed that the GMPase was expressed in all examined tomato tissues, but its expression level was up-regulated in tomato plants subjected to abnormal temperatures. We then overexpressed this tomato GMPase in tobacco plants and observed that the activity of GMPase and the content of AsA were significantly increased by two- to fourfold in the leaves of transgenic tobacco plants. The effect of this gene overexpression was superimposed by the treatments of high or low temperature in tobacco, since the activities of both chloroplastic SOD (superoxide dismutase EC 1.15.1.1), APX (ascorbate peroxidase EC 1.11.1.7) and the content of AsA in leaves were significantly higher in transgenic plants than those of WT, while the contents of H₂O₂ and O₂ ^−· were reduced. Meanwhile, relative electric conductivity increased less in transgenic plants than that in WT, and the net photosynthetic rate (P _n) and the maximal photochemical efficiency of PSII (F _v/F _m) of transgenic plants were notably higher than those of WT under temperature stresses. In conclusion, the overexpression of GMPase increased the content of AsA, thereby leading to the increase in tolerance to temperature stress in transgenic plants.     

Effects of exogenous spermidine on antioxidant system of tomato seedlings exposed to high temperature stress

The effects of foliar spraying with spermidine (Spd) on antioxidant system in tomato (Lycopersicon esculentum Mill.) seedlings were investigated under high temperature stress. The high temperature stress significantly inhibited plant growth and reduced chlorophyll (Chl) content. Application of exogenous 1 mM Spd alleviated the inhibition of growth induced by the high temperature stress. Malondialdehyde (MDA), hydrogen peroxide (H₂O₂) content and superoxide anion (O₂) generation rate were significantly increased by the high temperature stress, but Spd significantly reduced the accumulation of reactive oxygen species (ROS) and MDA content under the stress. The high temperature stress significantly decreased glutathione (GSH) content and activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR), but increased contents of dehydroascorbic acid (DHA), ascorbic acid (AsA), and oxidized glutathione (GSSG) in tomato leaves. However, Spd significantly increased the activities of antioxidant enzymes, levels of antioxidants and endogenous polyamines in tomato leaves under the high temperature stress. In addition, to varying degrees, Spd regulated expression of MnSOD, POD, APX2, APX6, GR, MDHAR, DHAR1, and DHAR2 genes in tomato leaves exposed to the high temperature stress. These results suggest that Spd could change endogenous polyamine levels and alleviate the damage by oxidative stress enhancing the non-enzymatic and enzymatic antioxidant system and the related gene expression.     

Exogenous 5-aminolevulinic acid pretreatment ameliorates oxidative stress triggered by low-temperature stress of <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Low temperature is an important limiting factor in tomato production in early spring and winter. 5-Aminolevulinic acid (ALA) protects crops against varied abiotic stresses. However, the methodology to precisely use ALA to increase the cold tolerance in tomatoes is still not fully known. We therefore explored the effects of ALA concentration, application period, and dose on membrane lipid peroxidation, antioxidation, photosynthesis, and plant growth in different tomato cultivars (Zhongza No. 9, ZZ and Jinpeng No. 1, JP) at low-temperature stress. Results revealed that low temperature caused plants oxidative damage and growth inhibition in both ZZ and JP plants. The ROS (hydrogen peroxide and superoxide anion) accumulation and membrane lipid peroxidation (malondialdehyde content and the relative electrical conductivity) were more remarkable in JP plants than ZZ plants under low temperature. The catalase (CAT) and ascorbate–glutathione cycle (AsA–GSH) induced by ALA reliably eliminated excessive ROS to maintain the redox balance in both tomato cultivars under low-temperature stress. In AsA–GSH cycle, AsA regeneration was mainly catalyzed by dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR), from dehydroascorbate (DHA) to AsA and monodehydroascorbate (MDA) to AsA in ZZ plants, while AsA regeneration in JP plants was mostly catalyzed by DHAR, from DHA to AsA. The ALA optimum concentration was 25 mg L^?1. The tomato plants with five true leaves pretreated with 6 mL ALA were more effective than spraying after cold occurred. In conclusion, the two tomato varieties illustrated different capacities to bear low-temperature stress. And ZZ plants were more tolerant to low temperature than JP plants. Precise ALA pretreatment observably alleviated low temperature induced-damage via CAT and AsA–GSH cycle in both cultivars. The regeneration of AsA in AsA–GSH cycle may be more comprehensive in ZZ plants than JP plants, to better tolerate low-temperature stress.     

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.

     

Oxidative stress and antioxidant responses of mulberry (Morus alba) plants subjected to deficiency and excess of manganese

The aim of the study was to relate the effects of deficiency and excess of Mn with the generation of reactive oxygen species (ROS) and altered cellular redox environment in mulberry (Morus alba L.) cv. Kanva-2 plants. Mn deficiency symptom appeared as mild interveinal chlorosis in middle leaves. Mn-excess did not produce any specific symptom. Leaf water potential (Ψ) was increased in Mn-deficient and Mn-excess mulberry plants. Mn-deficient leaves contained less Mn, less chloroplastic pigments and high tissue Fe, Zn and Cu concentrations. Starch content was increased with increasing Mn supply. While reducing sugar content increased in Mn-deficient and Mn-excess plants as well, non-reducing sugars remained unaffected in Mn-deficient plants and decreased in Mn-excess plants. Moreover, study of antioxidative responses, oxidative stress (H₂O₂ and lipid peroxidation) and cellular redox environment [dehydroascorbate (DHA)/ascorbic acid (AsA) ratio] in Mn-stressed mulberry plants was also undertaken. Both hydrogen peroxide and lipid peroxidation were enhanced in the leaves of Mn-deficient plants. Increased H₂O₂ concentration in Mn-excess leaves did not induce oxidative damage as indicated by no change in lipid peroxidation. The ratio of the redox couple (DHA/AsA) was increased both in Mn-deficient or Mn-excess plants. The activities of superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) increased in Mn-deficient plants. The activity of ascorbate peroxidase (EC 1.11.1.11) increased with increasing Mn supply. The results suggest that deficiency or excess of Mn induces oxidative stress through enhanced ROS generation and disturbed redox couple in mulberry plants.     

Combined action of antioxidant defense system and osmolytes in chilling shock-induced chilling tolerance in Jatropha curcas seedlings

Low non-freezing temperature is one of the major environmental factors affecting growth, development and geographical distribution of chilling-sensitive plants, Jatropha curcas is considered as a sustainable energy plants with great potential for biodiesel production. In this study, chilling shock at 5 °C followed by recovery at 26 °C for 4 h significantly improved survival percentage of J. curcas seedlings under chilling stress at 1 °C. In addition, chilling shock could obviously enhance the activities of antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR), and the levels of antioxidants ascorbic acid (AsA) and glutathione (GSH), as well as the contents of osmolytes proline and betaine in leaves of seedlings of J. curcas compared with the control without chilling shock. During the process of recovery, GR activity, AsA, GSH, proline and betaine contents sequentially increased, whereas SOD, APX and CAT activities gradually decreased, but they markedly maintained higher activities than those of control. Under chilling stress, activities of SOD, APX, CAT, GR and GPX, and contents of AsA, GSH, proline and betaine, as well as the ratio of the reduced antioxidants to total antioxidants [AsA/(AsA + DHA) and GSH/(GSH + GSSG)] in the shocked and non-shock seedlings all dropped, but shocked seedlings sustained significantly higher antioxidant enzyme activity, antioxidant and osmolyte contents, as well as ratio of reduced antioxidants to total antioxidants from beginning to end compared with control. These results indicated that the chilling shock followed by recovery could improve chilling tolerance of seedlings in J. curcas, and antioxidant enzymes and osmolytes play important role in the acquisition of chilling tolerance.     

Metal/metalloid stress tolerance in plants: role of ascorbate,its redox couple,and associated enzymes

The enhanced generation of reactive oxygen species (ROS) under metal/metalloid stress is most common in plants, and the elevated ROS must be successfully metabolized in order to maintain plant growth, development, and productivity. Ascorbate (AsA) is a highly abundant metabolite and a water-soluble antioxidant, which besides positively influencing various aspects in plants acts also as an enigmatic component of plant defense armory. As a significant component of the ascorbate-glutathione (AsA-GSH) pathway, it performs multiple vital functions in plants including growth and development by either directly or indirectly metabolizing ROS and its products. Enzymes such as monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) and dehydroascorbate reductase (DHAR, EC 1.8.5.1) maintain the reduced form of AsA pool besides metabolically controlling the ratio of AsA with its oxidized form (dehydroascorbate, DHA). Ascorbate peroxidase (APX, EC 1.11.1.11) utilizes the reduced AsA pool as the specific electron donor during ROS metabolism. Thus, AsA, its redox couple (AsA/DHA), and related enzymes (MDHAR, DHAR, and APX) cumulatively form an AsA redox system to efficiently protect plants particularly against potential anomalies caused by ROS and its products. Here we present a critical assessment of the recent research reports available on metal/metalloid-accrued modulation of reduced AsA pool, AsA/DHA redox couple and AsA-related major enzymes, and the cumulative significance of these antioxidant system components in plant metal/metalloid stress tolerance.     

Wheat yellow mosaic virus NIb targets TaVTC2 to elicit broad-spectrum pathogen resistance in wheat

GDP-L-galactose phosphorylase (VTC2) catalyses the conversion of GDP-L-galactose to L-galactose-1-P, a vital step of ascorbic acid (AsA) biosynthesis in plants. AsA is well known for its function in the amelioration of oxidative stress caused by most pathogen infection, but its function against viral infection remains unclear. Here, we have identified a VTC2 gene in wheat named as TaVTC2 and investigated its function in association with the wheat yellow mosaic virus (WYMV) infection. Our results showed that overexpression of TaVTC2 significantly increased viral accumulation, whereas knocking down TaVTC2 inhibited the viral infection in wheat, suggesting a positive regulation on viral infection by TaVTC2. Moreover, less AsA was produced in TaVTC2 knocking down plants (TaVTC2-RNAi) which due to the reduction in TaVTC2 expression and subsequently in TaVTC2 activity, resulting in a reactive oxygen species (ROS) burst in leaves. Furthermore, the enhanced WYMV resistance in TaVTC2-RNAi plants was diminished by exogenously applied AsA. We further demonstrated that WYMV NIb directly bound to TaVTC2 and inhibited TaVTC2 enzymatic activity in vitro. The effect of TaVTC2 on ROS scavenge was suppressed by NIb in a dosage-dependent manner, indicating the ROS scavenging was highly regulated by the interaction of TaVTC2 with NIb. Furthermore, TaVTC2 RNAi plants conferred broad-spectrum disease resistance. Therefore, the data indicate that TaVTC2 recruits WYMV NIb to down-regulate its own enzymatic activity, reducing AsA accumulation to elicit a burst of ROS which confers the resistance to WYMV infection. Thus, a new mechanism of the formation of plant innate immunity was proposed.     

南方根结线虫对不同砧木嫁接番茄苗活性氧清除系统的影响

采用盆栽人工接种方法,对番茄嫁接苗进行了抗性评价,研究了番茄嫁接苗叶片中抗氧化酶活性和活性氧代谢的动态变化。结果表明,接种南方根结线虫(J2)后,砧木嫁接苗表现为高抗,自根嫁接苗为高感。通过嫁接换根,与自根嫁接苗相比,砧木嫁接苗明显提高了接穗叶片的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性,降低了超氧阴离子(O.2-)产生速率以及过氧化氢(H2O2)和丙二醛(MDA)含量。表明番茄植株体内的活性氧水平和抗氧化酶活性的高低与其抗根结线虫的能力密切相关,较低的活性氧水平和较高的抗氧化酶活性有利于减轻对膜系统的伤害,提高番茄植株的抗根结线虫能力。     

Suppression of tomato <Emphasis Type="Italic">SlGGP</Emphasis> aggravates methyl viologen-mediated oxidative stress

Ascorbate (AsA) is an important antioxidant that can scavenge reactive oxygen species to protect plant cells against oxidative stress. Guanosine 5'-diphosphate (GDP)-L-galactose phosphorylase (GGP) is a key enzyme in the AsA biosynthetic pathway. To investigate the functions of GGP in AsA synthesis and oxidative stress tolerance in tomato, antisense lines with a reduced expression of SlGGP were obtained. Photobleaching after treatment of leaf disks with methyl viologen was more severe in transgenic lines compared to wild type (WT) plants. Moreover, compared with the WT plants, the transgenic plants showed a higher content of hydrogen peroxide, superoxide anion, malondialdehyde, as well as ion leakage, but a lower content of AsA and chlorophylls, ascorbate peroxidase activity, net photosynthetic rate, and maximal photochemical efficiency of photosystem II. Results of real-time quantitative polymerase chain reaction show that suppression of the SlGGP gene in the transgenic plants reduced their oxidative stress tolerance.