A CsWRKY48 Gene from Tea Plants Intercropped with Chinese Chestnut Plays an Important Role in Resistance to Biotic and Abiotic Stresses

Abstract

Tea plant (Camellia sinensis) is an important horticultural crop. The quality and productivity of tea plants is always threatened by various adverse environmental factors. Numerous studies have shown that intercropping tea plants with other plants can greatly improve the quality of their products. The intercropping system of Chinese chestnut (Castanea mollissima) and tea plants is an agricultural planting model in which the two species are grown on the same piece of land following a specific spacing and cultivation method. Based on a comparative transcriptome analysis between Chinese chestnut tea intercropped plantations and a pure tea plantation, it was found that the expression levels of the WRKY genes were significantly upregulated under the intercropping pattern. In this study, we cloned a candidate gene, CsWRKY48, and verified its functions in tobacco (Nicotiana tabacum) via heterologous transformation. The contents of protective enzyme activities and osmoregulatory substances were significantly increased, and the trichomes length and density were improved in the transgenic tobacco lines. This phenotype offered an enhanced resistance to both low temperatures and aphids for transgenic lines overexpressing CsWRKY48. Further analysis indicated that the CsWRKY48 transcription factor might interact with other regulators, such as CBF, ERF, MYC, and MYB, to enhance the resistance of tea plants to biotic and abiotic stresses. These findings not only confirm the elevated resistance of tea plants under intercropping, but also indicate a potential regulatory network mediated by the WRKY transcription factor.

Keywords: CsWRKY48; cold resistance; insect resistance; tea plants intercropped with Chinese chestnut; trichomes.

Figure 1

Expression analysis of WRKY48. (a) WRKY48 expression under low temperature; (b) WRKY48 expression under H₂O (as a control), MJ (Methyl Jasmonate), SA (Salicylic acid), GA3 (Gibberellin A3), ETH (Ethylene), ABA (Abscisic acid), PEG (Polyethylene glycol), and NaCl. Note: The bar chart represents the mean of three biological replicates, with error bars showing standard deviations. Asterisks indicate statistical significance based on one-way analysis of variance (** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 2

Analysis results of amino acid sequences for CsWRKY48. (a) Phylogenetic tree and (b) sequence analysis. Note: The target protein was marked with a red, ClWRKY48 (Camellia lanceoleosa, KAI8010938.1), NsWRKY48 (Nyssa sinensis, KAA8531200.1), CfWRKY48 (Cornus florida, XP059632820.1), VvWRKY48 (Vitis vinifera, RVW69378.1), and VrWRKY48 (Vitis rotundifolia, KAJ9699830.1).

Figure 3

Genetic transformation, positive seedling screening, and phenotypic observation of transgenic tobacco. (a) Tobacco leaf discs infected with Agrobacterium tumefaciens; (b) induction of resistant buds; (c) expansion of resistant buds; (d) isolation of resistant buds; (e) formation of independent lines; (f) expression analysis of transgenic lines; (g) transgenic tobacco after transplanting; (h) seeds harvested from mature transgenic tobacco for subsequent experiments; (i,m) control tobacco plants; (j,n) CsWRKY48-OE4; (k,o) CsWRKY48-OE16; and (l,p) CsWRKY48-OE40. Note: The bar chart represents the mean of three biological replicates, with error bars showing standard deviations. Asterisks indicate statistical significance based on one-way analysis of variance (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 4

Phenotype analysis of transgenic tobacco after low-temperature treatment and aphid feeding. (a,e,i–k) WT (wild-type); (b,f) CsWRKY48-OE4; (c,g) CsWRKY48-OE16; (d,h) CsWRKY48-OE40; (a–d) after low-temperature treatment for 12h; (e–h) after low-temperature treatment for 24 h; (i–n) after aphid feeding for 12 h.

Figure 5

Physiological and biochemical indices and relative expression of two cold resistance genes in transgenic tobacco at 4 °C. (a) Relative conductivity; (b) MDA content; (c) soluble protein content; (d) soluble sugar content; (e) proline content; (f) POD activity; (g) SOD activity; (h) CAT activity; (i) relative expression of NtCBF1; and (j) relative expression of NtDREB2B. Note: The bar chart represents the mean of three biological replicates, with error bars showing standard deviations. Asterisks indicate statistical significance based on one-way analysis of variance (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 6

Antioxidant enzyme activities and the expression of two insect resistance genes in the control tobacco plant and the CsWRKY48 transgenic plant after aphid feeding. (a) Relative conductivity; (b) MDA content; (c) soluble protein content; (d) soluble sugar content; (e) proline content; (f) POD activity; (g) SOD activity; (h) CAT activity; (i) relative expression of NtTD; and (j) relative expression of NtChiA. Note: The bar chart represents the mean of three biological replicates, with error bars indicating standard deviations. Asterisks denote statistical significance based on one-way analysis of variance (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 7

Prediction of cis-elements in the promoter of CsWRKY48, and analysis of the expression of relevant transcription factors and resistance genes in the transcriptomic data of the tea plants intercropped with Chinese chestnut. Note: (a) Prediction of cis-element in the promoter of CsWRKY48; analysis of the expression of relevant transcription factors and resistance genes in the transcriptomic data of the tea plants intercropped with Chinese chestnut. (b–i) Analysis of the expression of relevant transcription factors and resistance genes in the transcriptomic data of the tea plants intercropped with Chinese chestnut (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 8

Resistance mechanism of Chinese chestnut tea plantation. Note: Grey box indicates that tea plants in pure tea plantations were susceptible to low temperatures and aphid stresses; pink box indicates that tea plants in Chinese chestnut tea intercropped tea plantations were more resistant to low temperatures and aphids. POD: Peroxidase; SOD: Superoxide dismutase; CAT: Catalase; ERF: Ethylene responsive factor; bHLH: Basic helix–loop–helix; bZIP: Basic leucine zippers; MYB: Myeloblastosis viral; CBF1: C-repeat binding factor; DREB2B: Dehydration responsive element binding; Chia: Chitinase; TD: Threonine deaminase.