CsCIPK11-Regulated Metalloprotease CsFtsH5 Mediates the Cold Response of Tea Plants

Abstract

Photosystem II repair in chloroplasts is a critical process involved in maintaining a plant's photosynthetic activity under cold stress. FtsH (filamentation temperature-sensitive H) is an essential metalloprotease that is required for chloroplast photosystem II repair. However, the role of FtsH in tea plants and its regulatory mechanism under cold stress remains elusive. In this study, we cloned a FtsH homolog gene in tea plants, named CsFtsH5, and found that CsFtsH5 was located in the chloroplast and cytomembrane. RT-qPCR showed that the expression of CsFtsH5 was increased with leaf maturity and was significantly induced by light and cold stress. Transient knockdown CsFtsH5 expression in tea leaves using antisense oligonucleotides resulted in hypersensitivity to cold stress, along with higher relative electrolyte leakage and lower Fv/Fm values. To investigate the molecular mechanism underlying CsFtsH5 involvement in the cold stress, we focused on the calcineurin B-like-interacting protein kinase 11 (CsCIPK11), which had a tissue expression pattern similar to that of CsFtsH5 and was also upregulated by light and cold stress. Yeast two-hybrid and dual luciferase (Luc) complementation assays revealed that CsFtsH5 interacted with CsCIPK11. Furthermore, the Dual-Luc assay showed that CsCIPK11-CsFtsH5 interaction might enhance CsFtsH5 stability. Altogether, our study demonstrates that CsFtsH5 is associated with CsCIPK11 and plays a positive role in maintaining the photosynthetic activity of tea plants in response to low temperatures.

Keywords: CsCIPK11; CsFtsH5; cold; photosynthetic activity; tea plant.

Figure 1

Phylogenetic analyses and protein sequence alignment of CsFtsH5. (a) Neighbor-joining phylogenetic tree of CsFtsH5 and AtFtsHs. The red dot indicates CsFtsH5. (b) Bioinformatic features of CsFtsH5 domains and multiple sequences alignment of CsFtsH5. At: Arabidopsis thaliana; Pt: Populus trichocarpa; Nt: Nicotiana tabacum; Zm: Zea mays L.; Vv: Vitis vinifera; Os: Oryza sativa; Ac: Actinidia Chinensis.

Figure 2

Expression profiles and sub-cellular localization of CsFtsH5. (a) Sub-cellular localization of CsFtsH5 protein in H2B-RFP transgenic Nicotiana benthamiana leaves. The empty 35S-GFP was a positive control. Nuclei are indicated by the H2B-RFP (red), and the chloroplasts are shown in blue. Scale bar = 20 µm. (b)The expression patterns of CsFtsH5 in tea plant tissues, including bud, 1st L, 2nd L, 3rd L, mature L, stem, flower, seed, and root. Samples were collected on April, 2020, and October, 2020. (c) The CsFtsH5 expression in tea plants under 22 °C and 4 °C treatments for 1, 3, 6, and 12 h, as well as 1, 2, and 3 d. Data are shown as the mean ± SEM (n = 3). Asterisks indicate significant differences according to t-tests (* p < 0.05, ** p < 0.01). (d) The phenotype of 2 leaves and 1 bud of ZC604 and ZH1 under 100% and 10% sunlight conditions. Scale bar = 1 cm. (e) The expression patterns of CsFtsH5 in leaves of the green-leaf cultivar of ZC604 cultivar and the yellow-leaf cultivar of ZH1 under 100% and 10% sunlight conditions. Means of three replicates and standard errors are presented; different letters above the column indicate a significant difference at p < 0.05 using LSD’s test. Values in (b,e) are expressed relative to the expression levels of the reference gene using the formula 2^−ΔCt. Values in (c) are expressed relative to the expression levels of the 22 °C group using the formula 2^−ΔΔCt. CsPTB was used as a reference gene in expression analysis.

Figure 3

CsFtsH5-AsODN tea plants showed hypersensitivity to cold stress. (a) Phenotypes of CsFtsH5-AsODN and CsFtsH5-sODN plants treated with cold stress (4 °C for 5 d followed by 22 °C recovery for 2 d) and freezing stress (22 °C for 4.5 d followed by −16 °C for 12 h, then 22 °C recovery for 2 d). Control group was grown at 22 °C for 7 d. Scale bar = 2 cm. (b–d). Relative electrolyte leakage, chlorophyll fluorescence images, and Fv/Fm value of CsFtsH5-AsODN and CsFtsH5-sODN plants treated with low temperature. (e) The relative expression of CsCBF and CsCOR in CsFtsH5-AsODN and CsFtsH5-sODN plants under 4 °C low temperature. Mean and standard deviation values were obtained from at least three independent experiments. The asterisks indicated significantly different values between CsFtsH5-AsODN and CsFtsH5-sODN plants according to t-tests (* p < 0.05, ** p < 0.01).

Figure 4

CsFtsH5’s interaction with CsCIPK11. (a) The expression patterns of CsCIPK11 in tea plant tissues, including bud, 1st L, 2nd L, 3rd L, mature L, stem, flower, seed, and root. Samples were collected on April 2020 and October 2020. (b) The CsCIPK11 expression in tea plants under 22 °C and 4 °C treatments for 1, 3, 6, and 12 h, as well as 1, 2, and 3 d. Data are shown as the mean ± SEM (n = 3). Asterisks indicate significant differences according to t-tests (** p < 0.01). (c) The expression patterns of CsCIPK11 in leaves of ZC604 cultivar and ZH1 cultivar under 100% and 10% sunlight conditions. Means of three replicates and standard errors are presented; different letters above the column indicate significant difference at p < 0.05 using LSD’s test. Values in (a,c) are expressed relative to the expression levels of reference gene using formula 2^−ΔCt. Values in (b) are expressed relative to the expression levels of 22 °C group using formula 2^−ΔΔCt. CsPTB was used as a reference gene in expression analysis. (d) Y2H assay of pGBKT7-CsCIPK11 and pGADT7-CsFtsH5 in AH109 strain. DDO, SD/−Leu/−Trp; QDO, SD/−Leu/−Trp/−His/−Ade; X, X-α-gal. pGBKT7-53 and pGADT7-T acted as the positive control, while pGBKT7-lam and pGADT7-T served as the negative control. (e) Luciferase complementation imaging analysis of CsCIPK11-nLuc and CsFtsH5-cLuc in Nicotiana benthamiana leaves. CsCIPK11-nLuc with cLuc, nLuc with CsFtsH5-cLuc, and nLuc with cLuc served as controls.

Figure 5

CsCIPK11 may stabilize the CsFtsH5 protein. (a) Fluorescence observations in Nicotiana benthamiana leaves that expressed 35S-CsCIPK11/35S-CsFtsH5-Luc and 35S-empty vector/35S-CsFtsH5-Luc. (b) Relative LUC/REN activity measurements in dual-Luciferase assays. The relative LUC/REN value is the average of six biological replicates. Error bars indicate the SEM of six biological replicates. Asterisks indicate significant differences according to t-tests (** p < 0.01).