Downregulation of multiple CDK inhibitor ICK/KRP genes upregulates the E2F pathway and increases cell proliferation, and organ and seed sizes in Arabidopsis

Abstract

SUMMARYThe ICK/KRP cyclin-dependent kinase (CDK) inhibitors are important plant cell cycle factors sharing only limited similarity with the metazoan CIP/KIP family of CDK inhibitors. Little is known about the specific functions of different ICK/KRP genes in planta. In this study, we created double and multiple mutants from five single Arabidopsis ICK/KRP T-DNA mutants, and used a set of 20 lines for the functional investigation of the important gene family. There were gradual increases in CDK activity from single to multiple mutants, indicating that ICK/KRPs act as CDK inhibitors under normal physiological conditions in plants. Whereas lowerorder mutants showed no morphological phenotypes, the ick1 ick2 ick6 ick7 and ick1 ick2 ick5 ick6 ick7 mutants had a slightly altered leaf shape. The quintuple mutant had larger cotyledons, leaves, petals and seeds than the wild-type control. At the cellular level, the ICK/KRP mutants had more but smaller cells in all the organs examined. These phenotypic effects became more apparent as more ICK/KRPs were downregulated, suggesting that to a large extent ICK/KRPs function in plants redundantly in a dosage-dependent manner. Analyses also revealed increased expression of E2F-dependent genes, and elevated RBR1 as well as an increased level of phospho-RBB1 protein in the quintuple mutant. Thus, downregulation of multiple ICK/ KRP genes increases CDK activity, upregulates the E2F pathway and stimulates cell proliferation, resulting in increased cell numbers, and larger organs and seeds.

Ethylene Inhibits Cell Proliferation of the Arabidopsis Root Meristem

“…Auxindependent mechanisms mediate these effects of ethylene on endoreduplication at the RAM based on the auxin-repressor SHY2 facilitating the ethylene response and auxin being a modulator of the mitotic-to-endocycle transition in roots (Ishida et al, 2010). We also find that ethylene induces expression of the cyclin-dependent kinase inhibitor ICK1/KRP1 (Cheng et al, 2013;Wen et al, 2013), suggesting that ethylene may have more direct effects on the cell cycle. Some mechanisms used by ethylene to negatively regulate cell proliferation in the RAM may also function in the shoot, because unlike cytokinin, which positively regulates cell proliferation in the shoot, ethylene seems to negatively regulate cell proliferation in both the shoot and the root (Skirycz et al, 2011).…”

“…SHY2 encodes the auxin repressor INDOLE-3-ACETIC ACID INDUCIBLE3 (IAA3), and its induction by cytokinin has been implicated in negatively regulating cell proliferation at the RAM (Dello Ioio et al, 2008). ICK1/KRP1 encodes a cyclin-dependent kinase inhibitor, with the ICK/KRP genes serving to negatively regulate cell proliferation in plants based on genetic analysis using LOF mutants (Cheng et al, 2013;Wen et al, 2013); increased levels of ICK1/KRP1 correlate with a developmental decrease in cell cycle progression in both roots and shoots (Beemster et al, 2005). Other genes examined were largely unresponsive to ethylene.…”

Why do plants need so many cyclin-dependent kinase inhibitors?

“…While individual krp knockouts have minimal affects on plant morphology, knocking out multiple KRP genes results in larger organ size due to increased cell proliferation via increased expression of E2F target genes, 20 consistent with the proposed G1 checkpoint role of KRPs. However, unique roles for some individual KRPs have emerged.…”

“…redundancy in both CKI gene families, suggesting that divergence in biochemical function plays a minor role in maintaining the number of genes. 18,20,21 Of particular note, Kumar and coworkers found that 10 different SMRs, including an SMR from the bryophyte Physcomitrella patens, can complement the trichome cell division phenotype of Arabidopsis sim mutants. 13 Additionally, individual KRP and the SMR genes show distinct patterns of tissue-specific and stress-regulated expression, consistent with the individual genes playing roles in integrating the cell cycle with a wide variety of signals.…”

Enhanced levels of plant cell cycle inhibitors hamper root-knot nematode-induced feeding site development

“…This accelerated mitotic activity in galls is consistent with recent data, showing that loss-of-function of multiple KRP mutants promote the accumulation of CDK/CYC complexes, facilitating and triggering plant cell proliferation. [18][19][20] Increased CDK levels above the normal endogenous cell threshold most likely triggers a shift toward a faster and longer mitotic phase in galls. Despite the accelerated mitotic activity in gall tissues, development of the associated nematodes seems unchanged and these physiological alterations display no synergetic effect to RKN reproduction.…”