School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, 230009, China.
School of Horticulture and State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, 230036, China.
Planta. 2023 Jan 16;257(2):38. doi: 10.1007/s00425-023-04070-x.
This study unravels a novel regulatory module (CRL4-CK2α-CDK2) involving fruit size control by mediating cell division homeostasis (SlCK2α and SlCDK2) in tomato. Fruit size is one of the crucial agronomical traits for crop production. UV-damaged DNA binding protein 1 (DDB1), a core component of Cullin4-RING E3 ubiquitin ligase complex (CRL4), has been identified as a negative regulator of fruit size in tomato (Solanum lycopersicum). However, the underlying molecular mechanism remains largely unclear. Here, we report the identification and characterization of a SlDDB1-interacting protein putatively involving fruit size control through regulating cell proliferation in tomato. It is a tomato homolog SlCK2α, the catalytic subunit of the casein kinase 2 (CK2), identified by yeast two-hybrid (Y2H) assays. The interaction between SlDDB1 and SlCK2α was demonstrated by bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP). RNA interference (RNAi) and CRISPR/Cas9-based mutant analyses showed that lack of SlCK2α resulted in reduction of fruit size with reduced cell number, suggesting it is a positive regulator on fruit size by promoting cell proliferation. We also showed SlDDB1 is required to ubiquitinate SlCK2α and negatively regulate its stability through 26S proteasome-mediated degradation. Furthermore, we found that a tomato homolog of cell division protein kinase 2 (SlCDK2) could interact with and specifically be phosphorylated by SlCK2α, resulting in an increase of SlCDK2 protein stability. CRISPR/Cas9-based genetic evidence showed that SlCDK2 is also a positive regulator of fruit size by influencing cell division in tomato. Taken together, our findings, thus, unravel a novel regulatory module CRL4-CK2α-CDK2 in finely modulating cell division homeostasis and the consequences on fruit size.
本研究揭示了一个新的调控模块（CRL4-CK2α-CDK2），通过调节番茄细胞分裂平衡（SlCK2α 和 SlCDK2）来控制果实大小。果实大小是作物生产中关键的农艺性状之一。紫外线损伤 DNA 结合蛋白 1（DDB1）是 Cullin4-RING E3 泛素连接酶复合物（CRL4）的核心组成部分，已被鉴定为番茄果实大小的负调控因子。然而，其潜在的分子机制仍不清楚。在这里，我们通过调节番茄细胞增殖，鉴定并表征了一个 SlDDB1 相互作用蛋白，该蛋白可能参与果实大小的控制。它是番茄同源物 SlCK2α，即酪蛋白激酶 2（CK2）的催化亚基，通过酵母双杂交（Y2H）实验鉴定。SlDDB1 和 SlCK2α 之间的相互作用通过双分子荧光互补（BiFC）和免疫共沉淀（Co-IP）实验得到证实。RNA 干扰（RNAi）和基于 CRISPR/Cas9 的突变分析表明，SlCK2α 的缺失导致果实变小，细胞数量减少，表明它通过促进细胞增殖正向调控果实大小。我们还表明，SlDDB1 需要泛素化 SlCK2α，并通过 26S 蛋白酶体介导的降解来负调控其稳定性。此外，我们发现细胞分裂蛋白激酶 2（SlCDK2）的番茄同源物可以与 SlCK2α 相互作用，并特异性地被其磷酸化，导致 SlCDK2 蛋白稳定性增加。基于 CRISPR/Cas9 的遗传证据表明，SlCDK2 通过影响番茄细胞分裂也是果实大小的正向调控因子。综上所述，我们的研究结果揭示了一个新的调控模块 CRL4-CK2α-CDK2，它可以精细地调节细胞分裂平衡及其对果实大小的影响。
