The classical cytokinin signal transduction in plants depends on the transfer of phosphate groups between histidine (H) and aspartate (d) in histidine receptor kinase HK, histidine phosphotransferase HP and cytokinin response factor RR. However, the molecular mechanism of the regulation of this phosphate relay process is still poorly understood. In rice, cytokinin can significantly regulate the number of grains per panicle, but its regulatory function on grain weight or grain size is not clear.

Through years of efforts, Dr. Liu Dapu and Dr. Tong Hongning were surprised to find that ppkl1, which controls grain size, is a new cytokinin signal component. The d364 site of ppkl1 reduces the phosphate relay efficiency from ahp2 to cytokinin response factor rr21 protein and inhibits rice grain development by inducing the phosphate group on cytokinin phosphate transfer protein ahp2.

Through large-scale mutagenesis, the researchers screened a large grain mutant S48 and cloned the mutant gene ppkl1. It was found that it mutated at the same site as the previously reported gene gl3.1 controlling rice grain size, but caused different changes in amino acids. Similar to RR protein, ppkl1 can interact directly with ahp2 protein, and the amino acid composition and sequence of d364 site are very similar to that of RR protein. However, d364 can not receive phosphate group like RR protein. When ppkl1-d364 was present, the phosphate relay efficiency from ahp2 to rr21 decreased significantly, while d364 mutation had no effect on phosphate relay, and the grain increased significantly accordingly. Ppkl1-d364 may be a key inhibitor of cytokinin signal to avoid excessive grain development in rice.

Ppkl family in rice contains three members, which can interfere with cytokinin phosphate relay signal and have redundant function. The mutation of ppkl1-d364 lost the inhibitory function of phosphate relay, but because the mutant protein still occupied the interaction position with ahp2, it constitutively activated cytokinin signal through dominant negative regulation mechanism, resulting in significant grain increase. Using rice variety kongyu131 as material, multiple non frameshift mutant genotypes were obtained by in-situ gene editing in the region where d364 is located, which can increase grain in varying degrees, and are semi dominant like d364 mutation, and some genotypes can significantly increase yield. In this study, researchers created rice materials with 1000 grain weight gradually distributed from 20g to 38g, suggesting that ppkl1 has great application potential in crop precision design and breeding.

Interestingly, ppkl family is an important component of brassinolide main signal pathway, and its carboxyl terminal phosphatase region is necessary for brassinolide signal transmission. D364 site is located at the amino end, and its inhibition of cytokinin signal does not depend on phosphatase activity, suggesting that ppkl1 may mediate the interaction between two plant hormones, which provides a clue for understanding the function of brassinolide in regulating cell division. The inhibitory effect of ppkl family proteins on cytokinin signal may be an ancient function. Its functional site is hidden in brassinolide signal component. It is of great application value to explore and use it to improve crop molecular design. The research results are published online at molecular plant under the title of a cryptic inhibitor of cytokinin phosphoray controls rice grain size.