Functional Analysis And RNA-Sequencing Of LeWhy1 In Tomato Under Chilling Stress

Whirly, a dual targeted protein in plant, is a transcription factor, which can respond to environmental stimulation by regulating gene expression. Temperature stress is one of the major factors limiting tomato growth. So it is important to investigate the roles of whirly protein in tomato under chilling stress.In this study, a dual targeted whirly protein gene LeWhy1 was cloned from tomato leaves.The roles of LeWhy1 in chilling stress tolerance were investigated here using sense and antisense transgenic tomatoes. The major results are as follows:(1) The full length cDNA of LeWhy1 was isolated by RT-PCR using a pair of primers designed based on the LeWhy1 sequence(GenBank accession number: JQ670889). The full length of LeWhy1 is 807 bp encoding 268 amino acids residues.(2) p35S-LeWhy1-GFP fusion protein was constructed and transiently expressed in Arabidopsis protoplasts derived from leaf tissue. It was observed with confocal microscopy that the green fluorescence was clearly associated with chloroplasts and colocalized with the red autofluorescence of chloroplasts, demonstrating the LeWhy1 subcellular localization in chloroplast. And we observeded that Le Why1 located in both chloroplast and nucleus of the transgene tobacco cells with p35S-Le Why1-GFP.(3) The transcript of LeWhy1 was higher in the tissues with abundant of chlorophyll by qRT-PCR analysis. The LeWhy1 expression was induced by 4℃, 45℃, NaCl, PEG, SA, GA and ABA.(4) The full-length ORFs of LeWhy1 was connected with the expression vector pBI121 under the control of the 35S-CaMV promoter to form sense and antisense constructs. The Agrobacterium tumefaciens-mediated leaf disk method was used to generate transgenic tomato plants. Kanamycin-resistant plants were detected by PCR and qRT-PCR. The results suggested that sense lines and antisense lines were obtained.(5) Under chilling stress, the sense plants showed less membrane damage, lowersuperoxide radical(O2?-) and hydrogen peroxide(H2O2) levels and maintained a higher level of APX, CAT and POD enzyme activity than wide type. These results showed that overexpression of LeWhy1 enhanced the tolerance to chilling stress.(6) Transcription sequencing analysis results showed that the genes regulated by Le Why1 are most participate in photosynthesis and plant-pathogen interaction pathway.Genes for stress response, exonuclease, energy production and conversion and transport protein were obviously regulated by Le Why1. Some of them located in the nucleus, and others located in chloroplast, which is in line with the dual targeted of LeWhy1.(7) Under chilling stress, the genes correlated with seed maturation protein LEA4, IST1 homologues, zinc finger proteins, cytochrome P450, heat shock protein, ethylene response factors, glutathione transferases et al. were regulated significantly by Le Why1. Transcription factors regulated by low temperature included three helix transcription factor, heat stress transcription factor, ethylene-responsive transcription factors and WRKY transcription factors.(8) Two genes regulated by LeWhy1 significantly were selected from 443 new genes.They located in the 9th and 11 th chromosomes, respectively. The protein coded by the gene which was positively regulated by LeWhy1 showed cyclic compound binding activity. The gene which was negatively regulated by Le Why1 located in both chloroplast and mitochondria, and it acted as a transcription factor, regulating gene expression. These mechanisms need further study.