Qinghua Shi

Ascorbic acid contents in transgenic potato plants overexpressing two dehydroascorbate reductase genes

Ascorbic acid (AsA, vitamin C) is one of the most important nutritional quality factors in many horticultural crops and has many biological activities in the human body. Dehydroascorbate reductase (EC 1.8.5.1; DHAR) plays an important role in maintaining the normal level of ascorbic acid (AsA) by recycling oxidized ascorbic acid. To increase AsA content of potato, we isolated and characterized the cDNAs encoding two isoform DHARs localized in cytosol and chloroplast from potato, and developed two types of transgenic potato plants overexpressing cytosolic DHAR gene and chloroplastic DHAR, respectively. Incorporation of the transgene in the genome of potato was confirmed by PCR and real time RT-PCR. The overexpression of cytosolic DHAR significantly increased DHAR activities and AsA contents in potato leaves and tubers, whereas chloroplastic DHAR overexpression only increased DHAR activities and AsA contents in leaves, and did not change them in tubers. These results indicated that AsA content of potato can be elevated by enhancing recycling ascorbate via DHAR overexpression, moreover, cytosolic DHAR might play main important roles in improving the AsA contents of potato tubers.

Overexpression of S‐adenosyl‐l‐methionine synthetase increased tomato tolerance to alkali stress through polyamine metabolism

S-adenosyl-L-methionine (SAM) synthetase is the key enzyme involved in the biosynthesis of SAM, which serves as a common precursor for polyamines (PAs) and ethylene. A SAM synthetase cDNA (SlSAMS1) was introduced into the tomato genome using the Agrobacterium tumefaciens transformation method. Transgenic plants overexpressing SlSAMS1 exhibited a significant increase in tolerance to alkali stress and maintained nutrient balance, higher photosynthetic capacity and lower oxidative stress compared with WT lines. Both in vivo and in vitro experiments indicated that the function of SlSAMS1 mainly depended on the accumulation of Spd and Spm in the transgenic lines. A grafting experiment showed that rootstocks from SlSAMS1-overexpressing plants provided a stronger root system, increased PAs accumulation, essential elements absorption, and decreased Na(+) absorption in the scions under alkali stress. As a result, fruit set and yield were significantly enhanced. To our knowledge, this is the first report to provide evidence that SlSAMS1 positively regulates tomato tolerance to alkali stress and plays a major role in modulating polyamine metabolism, resulting in maintainability of nutrient and ROS balance.

Effects of Exogenous Silicon on Photosynthetic Capacity and Antioxidant Enzyme Activities in Chloroplast of Cucumber Seedlings Under Excess Manganese

Effects of silicon on photosynthetic parameters and antioxidant enzymes of chloroplast in cucumber seedlings under excess Mn were studied. Compared with the control, excess Mn significantly inhibited net photosynthetic rate (Pn), stomatal conductance, as well as the maximum yield of the photosystem Ⅱ photochemical reactions (Fv/Fm) and the quantum yield of photosysytem Ⅱ electron transport (PSⅡ), application of Si reversed the negative effects of excess Mn. In the further investigation, it was obtained that application of Si significantly increased the activities of enzymes related with ascorbate-glutathione cycle including ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in cucumber chloroplast under excess Mn, this could be responsible for the lower accumulation of H2O2 and lower lipid peroxidation of chloroplast induced by Mn, and resulted in keeping higher photosynthesis.

Overexpression of CsNMAPK in tobacco enhanced seed germination under salt and osmotic stresses

In this research, biological function of CsNMAPK, encoding a mitogen-activated protein kinase of cucumber, was investigated under salt and osmotic stresses. Northern blot analysis showed that the expression of CsNMAPK was induced by salt and osmotic stresses in the cucumber root. In order to determine whether CsNMAPK was involved in plant tolerance to salt and osmotic stresses, transgenic tobacco plants constitutively overexpressing CsNMAPK were generated. Northern and Western blot analysis showed that strong signals were detected in the RNA and protein samples extracted from transgenic lines, whereas no signal was detected in the wild type tobacco, indicating that CsNMAPK was successfully transferred into tobacco genome and overexpressed. The results of seed germination showed that germination rates of transgenic lines were significantly higher than wild type under high salt and osmotic stresses. In addition, seed growth of transgenic lines was much better than wild type under salt and osmotic stresses. These results indicated that overexpression of CsNMAPK positively regulated plant tolerance to salt and osmotic stresses.

S-Nitrosoglutathione Reductase-Modulated Redox Signaling Controls Sodic Alkaline Stress Responses in Solanum lycopersicum L.

S-Nitrosoglutathione reductase (GSNOR) plays an important role in regulating nitric oxide (NO) and S-nitrosothiol (SNO) homeostasis, and is therefore involved in the modulation of processes mediated by reactive nitrogen species (RNS). Although RNS have emerged as a key component in plant response to abiotic stress, knowledge of their regulation by GSNOR under alkaline stress was lacking. In this study, metabolic regulation of NO and SNOs was investigated in tomato plants of the wild type (WT), GSNOR overexpression lines (OE-1/2) and GSNOR suppression lines (AS-1/2) grown under either control conditions or sodic alkaline stress. Phenotype, photosynthesis, reactive oxygen species (ROS) metabolism, Na(+)-K(+) homeostasis and expression of genes encoding ROS scavenging, Na(+) detoxification and programmed cell death (PCD) were also analyzed. Compared with WT lines, OE-1/2 lines were alkaline tolerant, while AS-1/2 lines were alkaline sensitive. In AS-1/2 lines, although genetic expression of Na(+) detoxification was activated by GSNOR-regulated NO and ROS signaling, excess RNS and ROS accumulation also led to serious oxidative stress and induced PCD. In contrast, overexpression of GSNOR significantly increased ROS scavenging efficiency. Thus, it seemed that increasing alkaline tolerance via GSNOR overexpression in tomato was attributed to the regulation of redox signaling including RNS and ROS.

Sodic alkaline stress mitigation by interaction of nitric oxide and polyamines involves antioxidants and physiological strategies in Solanum lycopersicum

Nitric oxide (NO) and polyamines (PAs) are two kinds of important signal in mediating plant tolerance to abiotic stress. In this study, we observed that both NO and PAs decreased alkaline stress in tomato plants, which may be a result of their role in regulating nutrient balance and reactive oxygen species (ROS), thereby protecting the photosynthetic system from damage. Further investigation indicated that NO and PAs induced accumulation of each other. Furthermore, the function of PAs could be removed by a NO scavenger, cPTIO. On the other hand, application of MGBG, a PA synthesis inhibitor, did little to abolish the function of NO. To further elucidate the mechanism by which NO and PAs alleviate alkaline stress, the expression of several genes associated with abiotic stress was analyzed by qRT-PCR. NO and PAs significantly upregulated ion transporters such as the plasma membrane Na(+)/H(+) antiporter (SlSOS1), vacuolar Na(+)/H(+) exchanger (SlNHX1 and SlNHX2), and Na(+) transporter and signal components including ROS, MAPK, and Ca(2+) signal pathways, as well as several transcription factors. All of these play important roles in plant adaptation to stress conditions.

Identification of NaCl and NaHCO3 stress responsive proteins in tomato roots using iTRAQ-based analysis

Soil salinity and alkalinity are common constraints to crop productivity in low rainfall regions of the world. However, the physiological difference of plant response to these two stresses was short of deep investigation. This study has identified a set of differentially expressed proteins of tomato root exploring to NaCl and NaHCO3 stress by iTRAQ (isobaric tags for relative and absolute quantitation) assay. A total of 313 proteins responsive to NaCl and NaHCO3 were observed. Among these proteins, 70 and 114 proteins were up-regulated by salt and alkali stress, respectively. While down-regulated proteins were 80 in salt treatment and 83 in alkali treatment. Only 39 up-regulated proteins and 30 down-regulated proteins were shared by salt and alkali stresses. The majority of the down-regulated proteins accounted for metabolism and energy conversion, and the up-regulated proteins were involved in signaling or transport. Compared with salt stress, alkali stress down-regulated proteins related with the respiratory metabolism, fatty acid oxidative metabolism and nitrogenous metabolism of tomato roots, and up-regulated protein with the reactive oxygen species (ROS) scavenging and ion transport. This study provides a novel insight into tomato roots response to salt and alkali stress at a large translation level.

Exogenous Nitric Oxide Alleviated the Inhibition of Photosynthesis and Antioxidant Enzyme Activities in Iron-Deficient Chinese Cabbage (Brassica chinensis L.)

Abstract The effects of exogenous nitric oxide (NO) on plant growth, chlorophyll contents, photosynthetic and chlorophyll fluorescence parameters as well as lipid peroxidation and activities of antioxidant enzymes were investigated in Chinese cabbage plants exposed to iron (Fe) deficiency. Iron deficiency led to serious chlorosis in Chinese cabbage leaves, and resulted in significant decrease in plant growth, photosynthetic pigments, net photosynthetic rate, Fv/Fm, Φ PS II and activities of antioxidant enzymes, and increase in lipid peroxidation. While treatment with SNP, a NO donor, it could revert the iron deficiency symptoms, increased photosynthetic rate as well as activities of antioxidant enzymes, and protected membrane from lipid peroxidation, as a result, the growth inhibition of Chinese cabbage by Fe deficiency was alleviated.

Promoting Roles of Melatonin in Adventitious Root Development of Solanum lycopersicum L. by Regulating Auxin and Nitric Oxide Signaling

Melatonin (MT) plays integral roles in regulating several biological processes including plant growth, seed germination, flowering, senescence, and stress responses. This study investigated the effects of MT on adventitious root formation (ARF) of de-rooted tomato seedlings. Exogenous MT positively or negatively influenced ARF, which was dependent on the concentration of MT application. In the present experiment, 50 μM MT showed the best effect on inducing ARF. Interestingly, exogenous MT promoted the accumulation of endogenous nitric oxide (NO) by down-regulating the expression of S-nitrosoglutathione reductase (GSNOR). To determine the interaction of MT and NO in ARF, MT synthesis inhibitor p-chlorophenylalanine, NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt as well as GSNOR-overexpression plants with low NO levels were used. The function of MT was removed by NO scavenger or GSNOR-overexpression plants. However, application of MT synthesis inhibitor did little to abolish the function of NO. These results indicate that NO, as a downstream signal, was involved in the MT-induced ARF. Concentrations of indole-3-acetic acid and indole-3-butyric acid, as well as the expression of several genes related to the auxin signaling pathway (PIN1, PIN3, PIN7, IAA19, and IAA24), showed that MT influenced auxin transport and signal transduction as well as auxin accumulation through the NO signaling pathway. Collectively, these strongly suggest that elevated NO levels resulting from inhibited GSNOR activity and auxin signaling were involved in the MT-induced ARF in tomato plants. This can be applied in basic research and breeding.

Overexpression of S-adenosylmethionine synthetase 1 enhances tomato callus tolerance to alkali stress through polyamine and hydrogen peroxide cross-linked networks

S-adenosylmethionine synthetase is a member of the stress-induced family genes. Our previous research indicated that overexpression of SlSAMS1 confers alkali stress tolerance to tomato seedlings. However, information regarding the alkali stress tolerance mechanism of SlSAMS1 and the cross-linked network between SlSAMS1 and downstream signal has been limited. To study how SlSAMS1 improves alkali stress tolerance, we manipulated the SlSAMS1 transgenic calluses through a pharmacological approach and found that overexpression of SlSAMS1 was positively correlated with polyamine (PA) and hydrogen peroxide (H2O2) accumulation leading to improve alkali stress tolerance. Additionally, the accumulation of H2O2 in SlSAMS1 overexpression calluses depended on polyamine oxidase activity. The activities of antioxidant system, accumulation of organic acid, Na+ detoxification as well as alkali stress tolerance of the SlSAMS1 transgenic calluses were reversed by PA biosynthesis inhibitors, but not significantly influenced by ethylene biosynthesis inhibitors. These results suggest that overexpression of SlSAMS1 enhances alkali stress tolerance through PA and H2O2 cross-linked networks, which provide new insight into how SlSAMS1 functions as a stress mediatory element in regulating plants tolerance to alkali stress.