Cytokinins play key roles in plant growth and development, and hence their biosynthesis and degradation have been extensively studied. Cytokinin oxidase/dehydrogenases (CKXs) are a group of enzymes that regulate oxidative cleavage to maintain cytokinin homeostasis. In rice, 11 CKX genes have been identified to date; however, most of their functions remain unknown. In this study, we comprehensively examined the expression patterns and functions of the CKXs in rice by using CRISPR/Cas9 technology to construct mutants of all 11 genes. The results revealed that the ckx single-mutants and higher-order ckx4 ckx9 mutant lines showed functional overlaps and sub-functionalization. Notably, the ckx1 ckx2 and ckx4 ckx9 double-mutants displayed contrasting phenotypic changes in tiller number and panicle size compared to the wild-type. In addition, we identified several genes with significantly altered expression in both the ckx4 and ckx9 single-mutant and double-mutant plants. Many of the differentially expressed genes were found to be associated with auxin and cytokinin pathways, and cytokinins in the ckx4 ckx9 double-mutant were increased compared to the wild-type. Taken together, our findings provide new insights into the functions of CKX genes in rice growth and may provide the foundations for future studies aimed at improving rice yield.
細胞分裂素在植物的生長發(fā)育中起到重要作用，因此細胞分裂素的生物合成與生物降解過程被廣泛研究。細胞分裂素氧化/脫氫酶（CKX）參與細胞分裂素的氧化裂解，具有調(diào)控細胞分裂素穩(wěn)態(tài)的作用。水稻中一共鑒定出11個CKX基因，但是大部分CKX的功能依舊不明確。本研究首先分析了各個OsCKX基因的親緣關(guān)系和表達模式，并利用CRISPR/Cas9技術(shù)在粳稻品種中花11號和日本晴中創(chuàng)制了9個OsCKX基因的單突變體，以及osckx1 osckx2、osckx4 osckx9雙突變體和osckx6 osckx7 osckx10三突變體。細致觀察各突變體的表型，對各OsCKX基因的功能做了總結(jié)歸納。其中OsCKX1、OsCKX2與OsCKX4、OsCKX9在調(diào)控分蘗和穗型的功能相反，osckx1 osckx2雙突變體表現(xiàn)為分蘗數(shù)減少，穗增大，結(jié)實率下降及千粒重增加，而osckx4 osckx9雙突變體的分蘗數(shù)增加，根系和穗變小，結(jié)實率以及千粒重降低。
進一步檢測日本晴和osckx4 osckx9突變體根系和基部組織中的細胞分裂素含量，證實在根系中，順式玉米素（cZ）、反式玉米素（tZ）、異戊烯基腺苷（IPA）和反式玉米素核苷（tZR）的含量出現(xiàn)極顯著上升。熒光定量PCR實驗發(fā)現(xiàn)osckx4 osckx9突變體腋芽中大部分A型RR基因（細胞分裂素誘導響應因子）表達的水平顯著提高，間接說明突變體腋芽內(nèi)細胞分裂素含量較日本晴提高。接著，對日本晴和突變體的根尖和基部節(jié)進行轉(zhuǎn)錄組測序，推測生長素轉(zhuǎn)運和信號轉(zhuǎn)導相關(guān)基因表達量的變化可能是導致osckx4 osckx9突變體的分蘗數(shù)和根系出現(xiàn)顯著變化的原因，而根尖赤霉素合成相關(guān)基因OsKS1和OsKS2表達量的下調(diào)可能導致了osckx4 osckx9突變體株高的下降?？傮w而言，該研究全面分析了CKX基因在水稻生長發(fā)育中的功能，為進一步提高水稻產(chǎn)量提供了理論基礎(chǔ)。