Overexpression of watermelon ClWRKY20 in transgenic Arabidopsis improves salt and low-temperature tolerance

The WRKY transcription factor plays an important regulatory role in plant responses to biotic and abiotic stressors. In this study, ClWRKY20, whose expression was strongly induced by chilling in watermelon, was isolated and characterized. ClWRKY20 belongs to group III of the WRKY family. The subcellular localization analysis found that ClWRKY20 was localized only in the nucleus. Expression characteristic analysis found that it was upregulated in response to drought, salt, and exogenous phytohormones, including salicylic acid (SA), ethylene (ET), and abscisic acid (ABA). The overexpression of ClWRKY20 enhanced the salt and low-temperature tolerance of transgenic Arabidopsis determined through apparent symptoms and physiological analyses of the root growth, the lethal rate, the relative electrical leakage, MDA and proline contents, the seed germination rate, and the Fv/Fm value. Furthermore, the ClWRKY20 overexpression lines showed increased sensitivity to ABA during seed germination, while did not at the seedling stage. The RNA sequencing and quantitative real-time PCR analyses revealed that several stress-related genes induced by chilling stress in wild-type plants were also upregulated by the overexpression of ClWRKY20 in transgenic plants, relative to their expressions in the WT Arabidopsis. Additionally, transcript levels of several genes involved in the ABA signaling pathway, in the ethylene biosynthetic and ethylene signaling pathway, in the jasmonic acid biosynthesis and jasmonate signaling, or related to auxin transport and auxin-responsive, were significantly increased in the transgenic Arabidopsis compared with that in the WT plants. Taken together, ClWRKY20 positively modulated plant tolerance to salt and low temperature stresses, and the enhanced chilling tolerance in transgenic Arabidopsis was accompanied by the increased expressions of stress-related genes. Moreover, ClWRKY20 not only modulated chilling responses through ABA signaling, but also might function as an integrator among several signaling pathways.