Arabidopsis ICK/KRP cyclin-dependent kinase inhibitors function to ensure the formation of one megaspore mother cell and one functional megaspore per ovule.

Ling Cao,Sheng Wang,Raju Datla,Shengjian Ye,Hong Wang,Yan Cheng,Prakash Venglat,Yongming Zhou,Lihua Zhao,Yuan Qin

doi:10.1371/journal.pgen.1007230

Abstract

In most plants, the female germline starts with the differentiation of one megaspore mother cell (MMC) in each ovule that produces four megaspores through meiosis, one of which survives to become the functional megaspore (FM). The FM further develops into an embryo sac. Little is known regarding the control of MMC formation to one per ovule and the selective survival of the FM. The ICK/KRPs (interactor/inhibitor of cyclin-dependent kinase (CDK)/Kip-related proteins) are plant CDK inhibitors and cell cycle regulators. Here we report that in the ovules of Arabidopsis mutant with all seven ICK/KRP genes inactivated, supernumerary MMCs, FMs and embryo sacs were formed and the two embryo sacs could be fertilized to form two embryos with separate endosperm compartments. Twin seedlings were observed in about 2% seeds. Further, in the mutant ovules the number and position of surviving megaspores from one MMC were variable, indicating that the positional signal for determining the survival of megaspore was affected. Strikingly, ICK4 fusion protein with yellow fluorescence protein was strongly present in the degenerative megaspores but absent in the FM, suggesting an important role of ICKs in the degeneration of non-functional megaspores. The absence of or much weaker phenotypes in lower orders of mutants and complementation of the septuple mutant by ICK4 or ICK7 indicate that multiple ICK/KRPs function redundantly in restricting the formation of more than one MMC and in the selective survival of FM, which are critical to ensure the development of one embryo sac and one embryo per ovule.

Highlights

The cyclin-dependent kinase (CDK) inhibitors are proteins of usually small molecular masses able to inhibit CDKs through direct binding
The female germline starts with the differentiation of one megaspore mother cell (MMC) in each ovule that produces multiple megaspores through meiosis
In mutant ovules the number and position of surviving megaspores from one MMC were variable and ICK4-YFP fusion protein was strongly expressed in the degenerative megaspores but absent in the FM. Those findings together with other results in our study indicate that multiple ICK/KRPs function redundantly in controlling the formation of one MMC per ovule and in the degeneration of non-functional megaspores, which are critical for the subsequent development of one embryo sac per ovule and one embryo per seed

Summary

Introduction

The cyclin-dependent kinase (CDK) inhibitors are proteins of usually small molecular masses able to inhibit CDKs through direct binding. The ICK/KRP family of plant CDK inhibitors was initially discovered in Arabidopsis and share limited similarity in the Cterminal region with the mammalian Kip/Cip family of CDK inhibitors [1,2,3]. There are seven ICK/KRP genes in Arabidopsis [2]. Apart from the C-terminal conserved regions, plant ICK/KRP inhibitors differ at the protein sequence level greatly from the animal Kip/Cip CDK inhibitors and among themselves, implying possible functional differences. ICK/KRP genes are present in the genomes of all seed plants examined but absent from bryophytes and algae, and sequence analysis suggests that the plant ICK/KRP family and animal KIP/CIP family might have evolved independently [4]

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