Effects Of Tomato Mitochondrial Uncoupling Protein (LeUCP) Gene Silencing On Photosynthesis, Respiration And Stress Resistance In The Leaves Of Tomato

Plant mitochondrion is the important place to provide energy for cell respiration. It plays an important role for plants in perceiving in stress signal, regulating the defense response. Not only plant mitochondrial alternative oxidase electron transport chain in the mechanism of oxidative stress, it also posses mitochondrial uncoupling proteins to decrease reactive oxygen species. Recent studies found that AtUCPl gene play an active role in abiotics stress tolerance to low temperature, drought and other oxidative stress and effective photosynthesis. However, the mechanism in the regulation function for the vegetable crops and its role in stress resistance mechanism are not clear. Therefore, it is great importance to investigate mechanism of plant UCP gene in the regulation of photosynthesis, respiration and the reaction in stress tolerance.In this study, tobacco rattle virus (TRV) and tomato plants (Lycopersicon esculentum Mill. cv. Zongza 9) were used to construct the gene silencing vector carrying the fragment of LePDS and LeUCP gene.VIGS transform system of LePDS and LeUCP genes were established. The silencing of the reference gene LePDS appeared in photo-bleached young leaves; RT-PCR analysis showed that LeUCP gene was degraded significantly.TRV-LeUCP gene silencing plants were used to study the effect mitochondria LeUCP genes of photosynthetic parameters and related enzymes activity, fluorescence, electron flow distribution. Mitochondria respiration, reactive oxygen species accumulation and alternative oxidase (AOX) were also analyzed. The effects on redox state of mitochondrial uncoupling protein were studied combination with aconitase citric acid content in the Krebs cycle activity and redox state changes. Under PQ and high temperature stress, the roles of mitochondrial uncoupling protein in oxidative stress were also investgate. The following are the major results:1. The effects of tomato LeUCP gene silencing on photosynthesis were examined in the present study. No phenotypic differences were detected in the growth of tomato plants infected with TRV-LeUCP viral vector. The photosynthesis rate in the leaves of LeUCP gene silencing was lower than the control plants under different light intensities. Net Photosynthetic rate, Asat, Vc,max values decrease, stomatal conductance increase with LeUCP gene silence. However, no significant effects were observed on intercellular CO2 concentralion between different treatments. Activity of Rubisco and FBPase of tomato plants with LeUCP gene silencing was inhibited, it decreased by 23.3% and 20.3% respectively.2. We have studied tomato LeUCP gene silencing on the chlorophyll fluorescence parameters. LeUCP gene expression reduce had no effect on Fv/Fm, but decreased photochemical quenching and electron transport rate. Compared with mock treatment plants qP values decrease by 54%. Meanwhile, non-photochemical quenching and Je (PSII), the distribution of Je (PCR) and Je (PCO) and the ratio of NAD+/NADH ratio were significantly reduced.The results demonstrated that LeUCP gene is necessary for effective photosynthesis. LeUCP gene may play a role through influencing Calvin cycle key enzymes activities, re-distributing of the proportion of PSII electron flow, reducing electron transfer and light respiration rate.3. Mitochondrial LeUCP gene silencing significantly decreased respiration and alternative respiratory and inhibited of AOX1 gene family expression, especially AOXla gene expression. LeUCP gene silencing reduce content of ubiquinone and pyruvic acid, which decreased by 54.3% and 29.1% respectively compared with Mock and. It indicated that mitochondrial alternative oxidase and uncoupling protein expression appear synergistic. Compared with mock, aconitase activity and citric acid content decreased as 34.6%,14.4% respectively.NADH was increased and NAD+ /NADH decreased, it showed mitochondrial uncoupling protein played important role in redox state maintenance.4. Compared with mock and TRV treatment, LeUCP gene silencing induced of H2O2 and O2·-accumulation on leaves. Meanwhile, ROS accumulation increased significantly under PQ and heat stress treatments, in which H2O2 and O2·- increase 44.67% and 23% after PQ treatment respectively compared with Mock. PQ treatment caused severe oxidative stress, Fv/Fm decreased significantly and MDA content increased. PQ treatment induced antioxidant genes such as CAT1, CAT2, SOD, cAPX, GR, POD, but the expression levels of TRV-LeUCP+PQ were still below Mock+PQ and TRV+PQ treatment. Compared with Mock, PQ treatment plants improved AsA, GSH content associated with AsA/DHA, GSH+GSSG increased. However, the AsA content and GSH content of TRV-LeUCP+PQ treatment leaves were lower than Mock+PQ and the TRV+PQ treatment. Therefore, it was further evidence that LeUCP gene had an important role in promoting stress resistance and redox signaling balance under oxidative stress.