CsWRKY31 gene from Camellia sinensis intercropped with chinese chestnut enhances plant resistance to low-temperature (Abiotic) and aphid (Biotic) stresses

In China, the Chinese chestnut-tea intercropping system as an eco-agricultural practice improves the growing environment of tea plants and significantly enhances their stress resistance. WRKY proteins are important transcription factors (TFs) in plants, involved in growth and development and responses to environmental changes. We have identified a WRKY transcription factor gene CsWRKY31 which was highly expressed in tea plants likely induced by the intercropping. The aim of this study was to predict the resistance mechanism of CsWRKY31 gene at the genome level using heterologous transformation and physiological biochemical experiments. To investigate its function, we cloned CsWRKY31 and heterologously overexpressed it in Nicotiana tabacum . After heterologously overexpression, we found that the number and length of trichomes in transgenic tobacco were significantly greater than those in wild-type plants. Increased trichome density correlated with more glandular structures on stems and leaves suggesting enhanced resistance. To further characterize CsWRKY31 mediated resistance, we subjected transgenic tobacco lines to cold stress (abiotic) and aphid stress (biotic). Under cold stress, transgenic tobacco recovered more rapidly from wilting and resumed normal growth compared to wild-type plants; under insect stress, no yellowing or leaf senescence was observed in transgenic tobacco leaves. Under both cold and aphid stresses, transgenic tobacco exhibited higher contents of soluble proteins, soluble sugars and proline, as well as lower malondialdehyde levels and relative conductivity than wild-type plants. Additionally, transgenic lines showed enhanced antioxidant enzyme activities and higher expression of stress-related genes. These results indicate that CsWRKY31 plays a key role in improving plant stress resistance, which may be associated with altered trichome development and the antioxidant enzyme network. Bioinformatics analysis indicated that CsWRKY31 may function coordinately with regulatory factors including CBF , ERF , MYC , and MYB to enhance comprehensive stress resistance in tea plants. This study advances our understanding of the mechanisms by which chestnut-tea intercropping systems improve tea plant resistance, highlighting the critical role of CsWRKY31 in enhancing plant stress resistance.