Abstract
Cyclin-dependent kinase inhibitors known as KRPs (kip-related proteins) control the progression of plant cell cycles and modulate various plant developmental processes. However, the function of KRPs in rice remains largely unknown. In this study, two rice KRPs members, KRP1 and KRP2, were found to be predominantly expressed in developing seeds and were significantly induced by exogenous abscisic acid (ABA) and Brassinosteroid (BR) applications. Sub-cellular localization experiments showed that KRP1 was mainly localized in the nucleus of rice protoplasts. KRP1 overexpression transgenic lines (OxKRP1), krp2 single mutant (crkrp2), and krp1/krp2 double mutant (crkrp1/krp2) all exhibited significantly smaller seed width, seed length, and reduced grain weight, with impaired seed germination and retarded early seedling growth, suggesting that disturbing the normal steady state of KRP1 or KRP2 blocks seed development partly through inhibiting cell proliferation and enlargement during grain filling and seed germination. Furthermore, two cyclin-dependent protein kinases, CDKC;2 and CDKF;3, could interact with KRP1 in a yeast-two-hybrid system, indicating that KRP1 might regulate the mitosis cell cycle and endoreduplication through the two targets. In a word, this study shed novel insights into the regulatory roles of KRPs in rice seed maturation and germination.
Highlights
In eukaryotes, the cell cycle is strictly regulated by cyclin-dependent kinases (CDKs), a type of Ser-Thr protein kinases, together with specific cyclin (CYC) partners during plant growth and development [1,2]
Given that some genes with minor, but statistically significant, differences may be undetected by RNA-seq, we further examined the transcription level of numerous key grain filling regulators in OxKRP1 6 DAP developing seeds, including ADP-glucose pyrophosphorylase (AGPase), soluble starch synthase (SS), starch branching enzyme (SBE), starch debranching enzyme (DBE), and starch phosphorylase L (PHOL) directly involved in amylose and amylopectin biosynthesis, and two transcription factors regulating rice starch biosynthesis such as RSR1 (Rice Starch Regulator1, a rice AP2/EREBP family transcription factor) and bZIP58 (RISBZ1, a basic leucine zipper transcription factor) during seed development [23,27]
The biological functions of these KRP inhibitors are mainly by the inhibiting the activities of CDK and endoreduplication during endosperm development [33]
Summary
The cell cycle is strictly regulated by cyclin-dependent kinases (CDKs), a type of Ser-Thr protein kinases, together with specific cyclin (CYC) partners during plant growth and development [1,2]. As the central regulators of cell proliferation, the activity of CDKs is finely modulated by numerous molecular mechanisms, including post-translational modification (PTM) such as phosphorylation, proteolysis, and other regulatory proteins [3,4,5,6]. Among these regulatory proteins, CDK inhibitors (CKIs) directly bind to CDK/CYC complexes and inhibit their activities, thereby arresting the cell cycle in response to internal and external cues [7,8,9]. LeKRP1, another KRP inhibitor isolated in tomato (Lycopersicon esculentum), mainly accumulates in the jelly-like locular (gel) tissue and inhibits CDK/CYC kinase activities in endoreduplicating cells, thereby arresting the mitotic activities to determine the gel tissue development [15]
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