Functional Analysis Of ICK Gene Family In Arabidopsis

The ICK/KRP cyclin-dependent kinase (CDK) inhibitors play pivotal roles in cell cycle regulation. Present studies revealed that ICK/KRP proteins can inhibit CDK activity through binding to the CDK complex, leading to the effects of inhibiting the cell division and altering the plant growth and development. There are seven ICK/KRP genes in Arabidopsis genome. Current understanding of ICK/KRP functions has mostly come from overexpression studies. We know little regarding the functions of ICK/KRPs in planta under normal physiological conditions. In this study, we took reverse genetic techniques including T-DNA inserted mutation and RNAi interference to systematically analyze the functions of ICK gene family. The main results are as follows:1) Based on the identification of ick1, ick2, and ick7single mutants, we obtained two double mutants ickl/2and ick2/7. Gene expression analysis showed that the corresponding genes had no expressions in these mutants. We thus analyzed rosette leaf number, branch number, plant height and fresh weight on ick1-1, ick1-2, ick2, ick7, ickl/2, ick2/7, and two wild types (Col-0wild-type and segregated wild-type), and did not observe any significant differences, whereas, the surveys on plant growth showed a trend of growth enhancement in the double mutants.2) Two RNAi vectors pFGC5941-ICK1and pFGC5941-ICK△were constructed using full length CDS of ICK1and the sequence of ICK1conserved region as interference fragments, respectively, and were used for transformation of Arabidopsis. Under100mmol/L NaCl,10umol/L ABA,10umol/L IAA and cold (4℃) treatment conditions, we conducted fresh weight analyses on ickl-2, pFGC5941-ICK1, pFGC5941-ICK△and wild-type. The results showed that ickl-2line was more insensitive to NaCl and ABA than the wild-type, whereas, the sensitivities of two RNAi transgenic lines to NaCl and ABA treatments were moderate in comparison with these of wild-type and ickl-2. Gene expression analyses by RT-PCR revealed that this observation may relate to the inducible expression of ICK genes in response to stress. We also prepared a series of RNAi constructs using pHGRV vector system, and obtained corresponding transgenic lines. No significant phenotypic changes have been observed in these transgenic lines.3) Using ick1, ick2, ick7, ick5and ick6single mutants, we generated18T-DNA lines including five single mutants, five double mutants, five triple mutants, two quadruple mutants, and one quintuple mutant through crosses. Gene expression analysis showed that the corresponding genes were knocked out successfully in these mutants.4) Phenotypically, while lower-order mutants showed no morphological changes compared to wild-type, the ick1/2/6/7quadruple mutant and the ick1/2/5/6/7quintuple mutant had narrower and longer leaves than wild-type, suggesting that down-regulation of multiple ICKs affected leaf shape. Fresh/dry weight analysis of a series lines showed significant growth enhancement in both ick1/2/5/6quadruple mutant and ick1/2/5/6/7quintuple mutant. Moreover, ick1/2/5/6/7quintuple mutant had larger leaves, petals, and seeds than wild-type control. Investigations on the cellular level revealed that the increase in cell proliferation was responsible for organ size increases. As feedback of increased cell proliferation, the cell sizes of all the organs surveyed in ick1/2/5/6/7quintuple mutant were decreased.5) In complementation assay, re-introducing of ICK7gene to ick1/2/5/6/7quintuple can at least partially rescue the fresh weight increase and leaf shape alteration phenotypes observed in ick1/2/5/6/7quintuple mutant.6) In CDK activity assay on10lines (ick1, ick2, ick5, ick6, ick7, ick1/2, ick6/7, ick1/2/6/7, ick1/2/5/6/7, Wild-type, and ICK1OE line), ick1/2/5/6/7quintuple mutant had the highest CDK activity, followed by ick1/2/5/6/7quadruple mutant, double mutant, single mutant, wild-type and ICK1E, suggesting that the knocking-out of ICK genes accumulatively increased CDK activity in planta.7) In ick1/2/5/6/7quintuple mutant, real-time PCR results showed that most of the E2F-inducible genes investigated was up-regulated, which is accompanied by the protein level increases of both RB and p-RB. Therefore, knocking-out of ICK genes increases CDK activity.Taken above data together, in the ick1/2/5/6/7quintuple mutant, down-regulation of ICK genes increases CDK activity, results in more phosphorylated RB protein, up-regulates the E2F pathway, stimulates transactivation activity of E2F, tunes up expressions of E2F-inducible genes and stimulates cell proliferation, which resulted in increased cell numbers, and larger organs and seeds. For the first time, we provided the evidence for the functions of multiple ICK genes in planta under normal physiological conditions.