Isolation And Functional Analysis Of Tomato Glutaredoxin SlGRX1

Reactive oxygen species (ROS) are redox signals essential to many physiological processes in plants. But high concentration of ROS can cause damage to macromoleces thus disrupt normal signaling in plant. Glutaredoxins (Grxs) are ubiquitous small heat-stable disulfide oxidoreductases that play a crucial role in plant response to oxidative stress. Recently, studies have extended our knowledge on the physiological and molecular functions of Grxs in plant. In the present study, a novel CGFS type Grx gene was isolated from tomato and its functions were validated using loss-of-function and gain-of-function approaches by virus induced gene silencing (VIGS) of the gene in tomato and over-expression of the gene in Arabidopsis.Based on the sequence information of CGFS type Grxs from Arabidopsis and rice, a cDNA encoding a protein containing the consensus Grx family domain with a CGFS active site was isolated using homologue cloning and in silio cloning methods. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed that SlGRX1 is expressed ubiquitously in tomato including leaf, root, stem and flower and can be induced by oxidative, drought and salt stresses.To investigate the subcellular localization of SlGRX1 protein in plant cells, the SlGRX1 protein was fused to both C-terminus and N-terminus of GFP and the construct was transiently expressed in Nicotiana benthamiana leaf epidermal cells then monitored by capturing GFP fluorescence. Our results suggested that SlGRX1 protein was localized in both nucleus and cytoplasm.SlGRX1 gene silencing vector DNAmβ-SlGRX1 was constructed based on the geminivirus satellite DNAmβinduced silencing system and then introduced into Agrobacterium tumefaciens strain EHA105. Agrobecterium harboring recombinant silencing vector DNAmβ-SlGRX1 and TYLCCNV (Tomato yellow leaf curl China virus) infectious clone were co-agroinoculated into tomato plants. Meanwhile, tomato plants co-agroinoculated with TYLCCNV and empty silencing vector DNAmP was used as the control. VIGS mediated silencing of SlGRX1 in tomato led to increased sensitivity to oxidative and salt stresses with decreased relative chlorophyll content, and reduced tolerance to drought stress with decreased relative water content. These results suggested that SlGRX1 was required for plant resistance to oxidative, drought and salt stresses.The coding sequence of SlGRX1 was then cloned into the binary vector pBin438 to produce pBin438-SlGRX1 in which the SlGRX1 gene was expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The plasmid pBin438-SlGRX1 was then introduced into A. tumefaciens strain EHA105. Transformation of Arabidopsis was performed using the floral dip method. The empty vector pBin438 was also transformed into Arabidopsis as the control. Two independent homozygous lines (Y1, Y2) with a single copy of SlGRX1 were allowed to grow for 3 generations for further studies. We found over-expression of SlGRX1 in Arabidopsis plants enhanced lines Yl and Y2 the tolerance to oxidative, drought and salt stresses in both in vitro and in soil-grown conditions.Expression levels of some oxidative related ascorbate peroxidase (Apx) genes Apx2, Apx3, Apx4, Apx5, Apx6, and well-known drought/salt stress marker genes RD29A, RD22 and RAB18 in Y1,Y2 and the control plants were analyzed by qRT-PCR to explore the possible mechanism of the enhanced abiotic stress tolerance in SlGRX1 over-expression plants. We found that over-expression of the SlGRX1 gene in Arabidopsis can activate the expression of the Arabidopsis Apx2 and Apx6, indicating that the SlGRX1 gene may positively regulate the Apx2 and Apx6 genes to improve plants resistance to oxidative stress.