Shuangchen Chen

Overexpression of mitochondrial uncoupling protein conferred resistance to heat stress and Botrytis cinerea infection in tomato

The mitochondrial uncoupling protein genes improve plant stress tolerance by minimizing oxidative damage. However, the underlying mechanism of redox homeostasis and antioxidant signaling associated with reactive oxygen species (ROS) accumulation remained poorly understood. We introduced LeUCP gene into tomato line Ailsa Craig via Agrobacterium-mediated method. Transgenic lines were confirmed for integration into the tomato genome using PCR and Southern blot hybridization. One to three copies of the transgene were integrated into the tomato nuclear genome. Transcription of LeUCP in various transgenic lines was determined using real-time PCR. Transgenic tomato overexpressing LeUCP showed higher growth rate, chlorophyll content, maximum photochemical efficiency of PSII (Fv/Fm), photochemical quenching coefficient (qP) and electron transport rate (ETR), increased contents of AsA and proline, higher AsA/DHA ratio and GalLDH activity, reduced ROS accumulation, and enhanced heat stress tolerance compared with the control plants. The transgenic tomato plants also exhibited significant increases in tolerance against the necrotrophic fungus Botrytis cinerea. Taken together, our results suggest that LeUCP may play a pivotal role in controlling a broad range of abiotic and biotic stresses in plants by increasing redox level and antioxidant capacity, elevating electron transport rate, lowering H2O2 and lipid peroxidation accumulation.

Overexpression of E3 Ubiquitin Ligase Gene AdBiL Contributes to Resistance against Chilling Stress and Leaf Mold Disease in Tomato

Ubiquitination is a common regulatory mechanism, playing a critical role in diverse cellular and developmental processes in eukaryotes. However, a few reports on the functional correlation between E3 ubiquitin ligases and reactive oxygen species (ROS) or reactive nitrogen species (RNS) metabolism in response to stress are currently available in plants. In the present study, the E3 ubiquitin ligase gene AdBiL (Adi3 Binding E3 Ligase) was introduced into tomato line Ailsa Craig via Agrobacterium-mediated method. Transgenic lines were confirmed for integration into the tomato genome using PCR. Transcription of AdBiL in various transgenic lines was determined using real-time PCR. Evaluation of stress tolerance showed that T1 generation of transgenic tomato lines showed only mild symptoms of chilling injury as evident by higher biomass accumulation and chlorophyll content than those of non-transformed plants. Compared with wild-type plants, the contents of AsA, AsA/DHA, GSH and the activity of GaILDH, γ-GCS and GSNOR were increased, while H2O2, O2.−, MDA, NO, SNOs, and GSNO accumulations were significantly decreased in AdBiL overexpressing plants in response to chilling stress. Furthermore, transgenic tomato plants overexpressing AdBiL showed higher activities of enzymes such as G6PDH, 6PGDH, NADP-ICDH, and NADP-ME involved in pentose phosphate pathway (PPP). The transgenic tomato plants also exhibited an enhanced tolerance against the necrotrophic fungus Cladosporium fulvum. Tyrosine nitration protein was activated in the plants infected with leaf mold disease, while the inhibition could be recovered in AdBiL gene overexpressing lines. Taken together, our results revealed a possible physiological role of AdBiL in the activation of the key enzymes of AsA–GSH cycle, PPP and down-regulation of GSNO reductase, thereby reducing oxidative and nitrosative stress in plants. This study demonstrates an optimized transgenic strategy using AdBiL gene for crop improvement against biotic and abiotic stress factors.

Combined Inoculation with Multiple Arbuscular Mycorrhizal Fungi Improves Growth, Nutrient Uptake and Photosynthesis in Cucumber Seedlings

Mycorrhizal inoculation stimulates growth, photosynthesis and nutrient uptake in a wide range of host plants. However, the ultimate effects of arbuscular mycorrhyzal (AM) symbiosis vary with the plants and fungal species involved in the association. Therefore, identification of the appropriate combinations of AM fungi (AMF) that interact synergistically to improve their benefits is of high significance. Here, three AM fungal compositions namely VT (Claroideoglomus sp., Funneliformis sp., Diversispora sp., Glomus sp., and Rhizophagus sp.) and BF (Glomus intraradices, G. microageregatum BEG and G. Claroideum BEG 210), and Funneliformis mosseae (Fm) were investigated with respect to the growth, gas exchange parameters, enzymes activities in Calvin cycles and related gene expression in cucumber seedlings. The results showed that VT, BF and Fm could successfully colonize cucumber root to a different degree with the colonization rates 82.38, 74.65, and 70.32% at 46 days post inoculation, respectively. The plant height, stem diameter, dry weight, root to shoot ratio of cucumber seedlings inoculated with AMF increased significantly compared with the non-inoculated control. Moreover, AMF colonization greatly increased the root activity, chlorophyll content, net photosynthetic rate, light saturated rate of the CO2 assimilation (Asat), maximum carboxylation rate (Vcmax) and maximum ribulose-1,5-bis-phosphate (RuBP) regeneration rate (Jmax), which were increased by 52.81, 30.75, 58.76, 47.00, 69.15, and 65.53% when inoculated with VT, respectively. The activities of some key enzymes such RuBP carboxylase/oxygenase (RuBisCO), D-fructose-1,6-bisphosphatase (FBPase), D-fructose-6-phosphatase (F6P) and ribulose-5-phosphate kinase (Ru5PK), and related gene expression involved in the Calvin cycle including RCA, FBPase, FBPA, SBPase, rbcS and rbcL were upregulated by AMF colonization. AMF inoculation also improved macro- and micro nutrient contents such as N, P, K, S, Ca, Cu, Fe, Mn, Mg, and Zn in roots. Further analysis revealed that inoculation with VT had relatively better effect on growth of cucumber seedling followed by BF and Fm, indicating that AMF composition consisting of distant AMF species may have a better effect than a single or closely related AMF spp. This study advances the understanding of plant responses to different AM fungi toward development of strategies on AMF-promoted vegetable production.

COMT1 Silencing Aggravates Heat Stress-Induced Reduction in Photosynthesis by Decreasing Chlorophyll Content, Photosystem II Activity, and Electron Transport Efficiency in Tomato

Despite a range of initiatives to reduce global carbon emission, the mean global temperature is increasing due to climate change. Since rising temperatures pose a serious threat of food insecurity, it is important to further explore important biological molecules that can confer thermotolerance to plants. Recently, melatonin has emerged as a universal abiotic stress regulator that can enhance plant tolerance to high temperature. Nonetheless, such regulatory roles of melatonin were unraveled mainly by assessing the effect of exogenous melatonin on plant tolerance to abiotic stress. Here, we generated melatonin deficient tomato plants by silencing of a melatonin biosynthetic gene, CAFFEIC ACID O-METHYLTRANSFERASE 1 (COMT1), to unveil the role of endogenous melatonin in photosynthesis under heat stress. We examined photosynthetic pigment content, leaf gas exchange, and a range of chlorophyll fluorescence parameters. The results showed that silencing of COMT1 aggravated heat stress by inhibiting both the light reactions and the carbon fixation reactions of photosynthesis. The photosynthetic pigment content, light absorption flux, trapped energy flux, energy dissipation, density of active reaction center per photosystem II (PSII) cross-section, the photosynthetic electron transport rate, the maximum photochemical efficiency of PSII photochemistry, and the rate of CO2 assimilation all decreased in COMT1-silenced plants compared with that of non-silenced plants particularly under heat stress. However, exogenous melatonin alleviated heat-induced photosynthetic inhibition in both genotypes, indicating that melatonin is essential for maintaining photosynthetic capacity under stressful conditions. These findings provide genetic evidence on the vital role of melatonin in photosynthesis and thus may have useful implication in horticultural crop management in the face of climate change.

FIRST REPORT OF LEAF SPOT CAUSED BY BARTALINIA ROBILLARDOIDES ON PARTHENOCISSUS QUINQUEFOLIA IN CHINA

In late August 2013, leaf spot symptoms were first noticed on Virginia creeper [Parthenocissus quinquefolia (L.) Planch], an ornamental cum medicinal plant of vitaceae family, in the Forest Park, Luoyang City of Henan Province, China. Symptoms appeared as small circular brown dots on the leaves, gradually expanding into circular or irregularly-shaped yellow-brown to dark brown spots with raised margins and black dots in the center of the lesions. A fungus was carefully isolated from the leaf spot and cultured on potato dextrose agar, which produced dark brown colonies. Pycnidia were brown, ovoid, 13.3 μm in diameter. Conidiophores were cylindric and colorless, 8.1-10.l × 3.8-4.0 μm. Conidia (n = 50) were 18.1-20.8 × 2.5-4.2 μm, 2 to 4 septate, with apical cell hyaline, other cells pale brown. One eccentric pedicel, hyaline, 8-10.6 × 1.2 μm. Apical appendages 1 to 4, 13.3-16.0 × 1.2 μm, unbranched. Based on the isolate characteristics (Sutton, 1980; Barnett and Hunter, 1998), the fungus was subsequently confirmed as Bartalinia robillardoides by DNA sequence analysis of internal transcribed spacer, which was 100% identical to other known B. robillardoides isolates (GenBank Accession No. KF656706.1). To confirm pathogenicity of the fungus, ten surface disinfected healthy leaves of Virginia creeper were inoculated with a fresh conidial suspension (40 μl, 5 × 106 conidia/ml) of B. robillardoides at 26°C. Leaf necrosis symptoms appeared on the inoculated leaves within 7 days. The same fungus was successfully reisolated from the lesions, confirming Koch’s postulates, whereas, control plants showed no symptoms. To our knowledge, this is the first report of B. robillardoides infecting P. quinquefolia in China.

Combined effects of hypoxia and excess Mn2+ on oxidative stress and antioxidant enzymes in tomato seedlings

Effects of high level of Mn2+ on the changes in ROS generation, root cell viability, antioxidant enzyme activities, and related gene expression in tomato (Solanum lycopersicum L., cv. Zhongza 9) seedlings were studied under normoxic and hypoxia conditions. Mn2+ concentrations, ranged between 10 and 200 μM, led to significantly higher activities of superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APOD), glutathione reductase (GR), and also ascorbic acid (AsA) content in leaves and roots, improved root cell viability, and decreased O2·− accumulation compared with the higher Mn2+ level under hypoxia stress, which indicated that low Mn2+ could eliminate the active oxygen and protect the membrane lipid from the hypoxia hurt. When the concentration of Mn2+ reached 400–600 μM under hypoxia stress, the activities of SOD, POD, APOD, and GR and AsA content were decreased remarkably. In contrast, the MDA content was increased at the higher Mn2+ concentration. A number of antioxidant-related genes showed high expression at the lower level of Mn2+. The expression levels of SOD, POD, CAT, APOD, and GR genes were 7.95, 5.27, 3.18, 5.54, and 8.81 times compared to control, respectively. These results illustrated that the appropriate amount of Mn2+ could alleviate the detrimental effects of hypoxia stress, but reversely, the high level of Mn2+ just aggravated the existing damage to the tomato seedlings.