SlMDH3 Interacts with Autophagy Receptor Protein SlATI1 and Positively Regulates Tomato Heat Tolerance

Abstract

Autophagy, a highly conserved protein degradation system, plays an important role in protecting cells from adverse environmental conditions. ATG8-INTERACTING PROTEIN1 (ATI1) acts as an autophagy receptor, but its functional mechanisms in plants' heat stress tolerance remain unclear. In this study, using LC-MS/MS, we identified malate dehydrogenase (SlMDH3) as a SlATI1-interacting protein. Further studies showed that heat stress induced the expression of SlMDH3 and SlMDH3 co-localized with SlATI1 under both 22 °C and 42 °C heat treatment conditions. Moreover, silencing of SlMDH3 increased the sensitivity of tomato to heat stress, as evidenced by exacerbated degradation of chlorophyll; accumulation of MDA, H₂O₂, and dead cells; increased relative conductivity; and inhibition of stress-related gene expression. Conversely, overexpression of SlMDH3 improved tomato's heat tolerance, leading to opposite effects on physiological indicators and gene expression compared to SlMDH3 silencing. Taken together, our study suggests that SlMDH3 interacts with SlATI1 and positively regulates tomato heat tolerance.

Keywords: ATI1; autophagy; heat stress; malate dehydrogenase; tomato.

Figure 1

SlMDH3 interacts with SlATI1. (A) Interaction of SlATI1 and SlMDH3 in Y2H assay. Nub1, positive control; pPR3-N, negative control. (B) Co-IP assay of in vivo interaction between SlMDH3 and SlATI1. Constructs of SlMDH3-GFP and SlATI1-Myc were co-transfected in N. benthamiana leaves. Protein samples of inputs and outputs were immunoprecipitated with anti-GFP or anti-Myc antibodies.* represents unknown protein. (C) Interactions of SlMDH3 and SlATI1 in bimolecular fluorescence complementation (BiFC) assays. SlATI1 and SlMDH3 were fused to N-terminal or C-terminal portions of yellow fluorescent protein (NE or CE) and then transiently co-expressed after agroinfiltration in N. benthamiana and incubated at 22 °C for 3 d under light conditions. Construct pair CYFP-SlMDH3 + nYFP was co-transfected as negative control. Microscope’s magnification: 10 × 40. (D) Split luciferase (Luc) assay of SlATI1-SlMDH3 interaction. SlMDH3 and SlATI1 were fused to CLuc and NLuc, respectively, and were transiently co-expressed in N. benthamiana leaves. Construct pairs SlATI1-CLuc + NLuc, NLuc-SlMDH3+ CLuc, and NLuc + CLuc were co-transfected as negative controls.

Figure 2

Analysis of SlMDH3 expression during heat stress in tomato (cv. Micro-Tom). Plants were subjected to heat treatment at 42 °C with three biological replications, each consisting of three plants at 5–6 leaf stage. **, p < 0.01; ***, p < 0.001.

Figure 3

SlMDH3 co-localizes with SlATI1. (A) Subcellular localization of SlMDH3 in cytoplasm. (B) SlATI1 co-localizes with SlMDH3 under normal and high temperatures. All constructs were infiltrated into N. benthamiana leaves using Agrobacterium strain GV3101. Green, red, and yellow colors show signals of GFP-SlMDH3, mCherry-SlATI1, and their merged protein, respectively. Microscope’s magnification: 10 × 40.

Figure 4

SlMDH3 silencing increases tomato’s sensitivity to heat stress. (A) MDH activity in TRV2:00 and TRV2:SlMDH3 plants. (B) Phenotypes of tomato seedlings. (C) Relative conductivity, total chlorophyll content, and MDA content in TRV2:00 and TRV2:SlMDH3 plants. (D) Accumulation of dead cells (stained using Trypan blue) and H₂O₂ (stained using DAB) after heat stress in tomato leaves. (E) Changes in relative expression levels of heat tolerance marker SlHsp70 and SlHsfA1 after heat stress in tomato leaves. TRV2:SlMDH3, SlMDH3-silenced tomato plants; TRV2:00, negative control plants. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance.

Figure 5

SlMDH3 overexpression enhances tomato’s tolerance against heat stress. (A) Phenotypes of tomato seedlings after exposure to heat stress (42 °C for 10 h). (B) MDH activity, relative conductivity, and MDA content. (C) Accumulation of dead cells (stained using Trypan blue) and H₂O₂ (stained using DAB) in tomato leaves. (D) Changes in relative expression levels of heat tolerance marker SlHsp70 and SlHsfA1 after heat stress in tomato leaves. WT, wild type; OE3 and OE6, SlMDH3 overexpression lines. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance.