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. 2011 May;107(7):1087–1101. doi: 10.1093/aob/mcr057

Analyses of two rice (Oryza sativa) cyclin-dependent kinase inhibitors and effects of transgenic expression of OsiICK6 on plant growth and development

Ruifang Yang 1, Qicai Tang 1, Huimei Wang 1, Xiaobo Zhang 1, Gang Pan 1, Hong Wang 2, Jumin Tu 1,*
PMCID: PMC3091807  PMID: 21558459

Abstract

Background and Aims

Plants have a family of proteins referred to as ICKs (inhibitors of cyclin-dependent kinase, CDK) or KRPs (Kip-related proteins) that function to regulate the activities of CDK. Knowledge of these plant CDK inhibitors has been gained mostly from studies of selected members in dicotyledonous plants, particularly Arabidopsis. Much remains to be learned regarding the differences among various members of the ICK/KRP family, and regarding the function and regulation of these proteins in monocotyledonous plants.

Methods

We analysed ICK-related sequences in the rice (Orysa sativa L. subsp. indica) genome and determined that there are six members with the conserved C-terminal signature region for ICK/KRP proteins. They are referred to as OsiICKs and further analyses were performed. The interactions with CDKs and cyclins were determined by a yeast two-hybrid assay, and cellular localization by fusion with the enhanced green fluorescence protein (EGFP). The expression of OsiICK6 in different tissues and in response to several treatments was analysed by reverse transcriptase-mediated polymerase chain reaction (RT-PCR) and real-time PCR. Furthermore, OsiICK6 was over-expressed in transgenic rice plants and significant phenotypes were observed.

Key Results and Conclusions

Based on putative protein sequences, the six OsiICKs are grouped into two classes, with OsiICK1 and OsiICK6 in each of the two classes, respectively. Results showed that OsiICK1 and OsiICK6 interacted with OsCYCD, but differed in their interactions with CDKA. Both EGFP:OsiICK1 and EGFP:OsiICK6 were localized in the nucleus. Whereas EGFP:OsiICK6 showed a punctuate subnuclear distribution, OsiICK1 had a homogeneous pattern. Over-expression of OsiICK6 resulted in multiple phenotypic effects on plant growth, morphology, pollen viability and seed setting. In OsiICK6-over-expressing plants, leaves rolled toward the abaxial side, suggesting that cell proliferation is critical in maintaining an even growth along the dorsal–ventral plane of leaf blades.

Keywords: Cell cycle, cyclin-dependent kinase inhibitor, leaf curvature, ICK, nuclear localization, Oryza sativa, plant growth, protein–protein interaction, rice, seed yield, stress-induced expression

INTRODUCTION

It is now well known that the cell cycle in plants as in other eukaryotic organisms is regulated by the cyclin-dependent kinases (CDKs). To control the cell cycle and cell proliferation, endogenous and exogenous factors need to modulate the activity of CDK during plant development and in the response to environmental changes. The activity of CDKs is regulated by several biochemical mechanisms, including protein phosphorylation, proteolysis and binding by CDK inhibitors.

In plants, there exists a family of CDK inhibitors referred to as ICKs (inhibitors of cyclin-dependent kinase; Wang et al., 1997; Lui et al., 2000; Zhou et al., 2002a) or KRPs (Kip-related proteins; De Veylder et al., 2001). ICK/KRP genes have been identified from different plant species including Arabidopsis (Wang et al., 1997; Lui et al., 2000; De Veylder et al., 2001; Zhou et al., 2002a), tobacco (Jasinski et al., 2002a, 2003), maize (Coelho et al., 2005), alfalfa (Pettko-Szandtner et al., 2006), tomato (Bisbis et al., 2006) and rice (Barrôco et al., 2006). Plant ICK/KRP proteins have a characteristic conserved short C-terminal region, which is similar to an N-terminal region in the mammalian Kip/Cip family of CDK inhibitors (Wang et al., 1997; Lui et al., 2000; De Veylder et al., 2001). Domain-mapping studies have shown that ICK/KRP proteins interact with A-type CDKs and D-type cyclins through the conserved C-terminal region (Wang et al., 1998; Lui et al., 2000; Jasinski et al., 2002b). Apart from this region, there is wide sequence divergence among different members of Arabidopsis ICK/KRP proteins, implying possible functional differentiation among the members. In addition to the ICK/KRP family, a second family of CDK inhibitors has been identified with two members in Arabidopsis, named SIAMESES (SIM) and EL2, which share a conserved EIEDFF sequence with the ICK/KRP proteins (Churchman et al., 2006; Peres et al., 2007).

The expression of Arabidopsis ICK/KRP genes varies in different tissues (Wang et al., 1998; Lui et al., 2000; De Veylder et al., 2001; Ormenese et al., 2004) and during the cell cycle (Menges et al., 2005). Several ICK/KRP genes are also regulated by environmental signals. The expression of Arabidopsis ICK1 and alfalfa KRPMt is induced by abscisic acid (ABA) (Wang et al., 1998; Pettko-Szandtner et al., 2006). ICK1 expression is also induced by salt stress (NaCl treatment) and this induction may be mediated by ABA (Ruggiero et al., 2004). These observations support the suggestion that ABA may inhibit the cell cycle under stressful conditions through CDK inhibitors (Wang et al., 1998). On the other hand, the expression of Arabidopsis ICK2 is suppressed by auxin (Himanen et al., 2002; Richard et al., 2002).

Several studies have shown that over-expression of an ICK/KRP gene driven by a constitutive promoter dramatically affects plant growth and development with the common phenotypes being reduced plant size, serrated leaves, reduced cell number and enlarged cells (Wang et al., 2000; De Veylder et al., 2001; Jasinski et al., 2002a, 2003; Zhou et al., 2002a; Barrôco et al., 2006). Targeted expression of an ICK/KRP gene also has specific effects on plant morphology and cellular differentiation. Expression of Arath;ICK1 under the control of a petal-specific promoter resulted in petals with dramatically altered shapes while Arath;ICK1 expression under the control of a pollen promoter reduced fertility (Zhou et al., 2002b). When Arath;ICK1 was expressed under the control of a trichome-specific GL2 promoter, the trichomes of the transgenic Arabidopsis plants had a reduced size and number of branches, and these trichomes collapsed and died earlier compared with the wild-type plants (Schnittger et al., 2003). Arath;ICK1 has also been expressed in the L1 layer using the L1 layer-specific AtML1 promoter (Bemis and Torii, 2007). In those transgenic Arabidopsis plants, cell division in the epidermal layer was inhibited with an enlarged cell size whereas the underlying mesophyll/cortex had a normal cell number and size, indicating autonomy of cell proliferation in the three cell layers. Furthermore, the phenotypic effects from ICK/KRP over-expression could be attenuated by co-expressing a D-type cyclin, which interacts with ICK/KRPs (Jasinski et al., 2002a; Schnittger et al., 2003; Zhou et al., 2003b).

Localization to a particular compartment or cellular region is important for the functions of cell cycle regulators (Pines, 1999). Results from green fluorescent protein fusion experiments showed that all seven Arabidopsis ICKs were exclusively localized in the nucleus (Bird et al., 2007; Wang et al., 2008). Similar localization results were also observed for two tobacco ICK/KRP proteins NtKIS1a and NtKIS2 (Jasinski et al., 2002a) and SIM, a member of the SIM/EL2 family of plant CDK inhibitors, from Arabidopsis (Churchman et al., 2006). These results indicate that nuclear localization is a functional property of the plant ICKs, and perhaps SIM-type inhibitors as well.

The understanding of plant ICK/KRP CDK inhibitors has come mostly from studies performed with dicotyledonous species, particularly Arabidopsis. Rice is a prominent model for monocotyledonous plants and one of the most important food crops. However, only limited information is available on ICK/KRP inhibitors in rice or monocot plants (Coelho et al., 2005; Barrôco et al., 2006; Guo et al., 2007; Mizutani et al., 2010). Seven KRP genes have been reported for rice (Barrôco et al., 2006; Guo et al., 2007), but one is probably a pseudogene (Guo et al., 2007). Orysa;KRP3 is highly expressed in multinucleate syncytial endosperm, suggesting that it is involved in cell cycle control of developing endosperm (Mizutani et al., 2010). In addition, transgenic over-expression of Orysa;KRP1 affected endosperm development and greatly reduced seed filling (Barrôco et al., 2006). Despite these observations, our understanding on monocot CDK inhibitors is still rudimentary. In this study, we characterized two rice ICK/KRP genes functionally in terms of protein–protein interaction, subcellular localization, gene expression and transgenic expression.

MATERIALS AND METHODS

Analysis of phylogeny and conserved sequences

For the initial identification of ICK-related sequences from indica rice, Arabidopsis ICK/KRP sequences were used to search the BGI Rice Database (http://rise2.genomics.org.cn/) by BLAST. We refer to ICK/KRP genes from Oryza sativa subspecies indica as Orysa;ICKs or in short OsiICKs. For phylogenetic analysis, sequences of japonica subspecies were from the NCBI database (http://www.ncbi.nlm.nih.gov/), and poplar (Populus trichocarpa) sequences from the poplar genome database (Poplar v 1·0: http://genome.jgi-psf.org/Poptr1/Poptr1.home.html). Phylogenetic analysis was conducted using MEGA4 by the neighbour-joining method (Tamura et al., 2007).

For analysis of conserved sequences, and subsequent molecular analysis, we isolated the actual cDNA sequences from an indica line MH63 (see below). We cloned the complete coding sequences of OsiICK1, OsiICK4, OsiICK5 and OsiICK6, but were unsuccessful in cloning the cDNAs of OsiICK2 and OsiICK3. Protein sequences derived from these cDNAs and BGI sequences for OsiICK2 and OsiICK3 were used for the analysis of conserved domains with MEME (http://meme.sdsc.edu/meme4_3_0/cgi-bin/meme.cgi). Amino acid sequences were aligned using UniProt (http://www.uniprot.org/). Putative nuclear localization signals were identified using Psort (http://psort.ims.u-tokyo.ac.jp/).

Cloning of rice cell cycle genes

Total RNAs were isolated from calli derived from the indica line MH63 using Trizol reagent (Invitrogen; www.invitrogen.com) according to the manufacturer's instructions. First-strand cDNA synthesis of rice cell cycle genes OsiICK1, OsiICK4, OsiICK5, OsiICK6, OsCYCA2;1, OsCYCB2;2, OsCYCD2;1, OsCDKA;1 and OsCDKB2;1 was carried out using a cDNA synthesis kit (TaKaRa; http://TaKaRa.com.cn). The complete coding sequences (CDSs) were amplified from these cDNAs using gene-specific primers with SalI and NotI restriction sites, respectively (Supplementary Data Table S1, available online). The amplified cDNA products were cloned into pMD-18T vector (TaKaRa). The resulting plasmids (named pMD-OsiICK1, pMD-OsiICK4, PMD-OsiICK5, pMD-OsiICK6, pMD-OsCDKA, pMD-OsCDKB, pMD-OsCYCA, pMD-OsCYCB and pMD-OsCYCD) were verified by sequencing.

Yeast two-hybrid analysis

Interactions of OsiICK1 and OsiICK6 with different rice CDKs or cyclins were analysed using the ProQuest yeast two-hybrid system (Invitrogen). The CDSs of rice OsiICK1, OsiICK6, OsCYCA2;1, OsCYCB2;2, OsCYCD2;1, OsCDKA;1 and OsCDKB2;1 were digested with SalI and NotI from pMD-OsiICK1, pMD-OsiICK6, pMD-OsCDKA, pMD-OsCDKB, pMD-OsCYCA, pMD-OsCYCB, and pMD-OsCYCD, respectively. The purified cDNA fragments were cloned into the yeast two-hybrid DNA binding-domain (BD) vector pDBLeu, as well as the yeast two-hybrid DNA activation-domain (AD) vector pPC86. All the resulting constructs were confirmed by DNA sequencing. Different combinations between BD and AD constructs were introduced into Saccharomyces cerevisiae strain Mav203 using a lithium acetate transformation method (Gietz and Woods, 1998). The transformants were then incubated on synthetic dextrose (SD) medium either lacking tryptophan (Trp) and leucine (Leu), or SD medium lacking Trp, Leu and histidine (His) but supplemented with 10 mm 3-aminotriazole (3-AT), for 3 d. The colonies growing on SD-Trp-Leu medium were assayed for β-galactosidase activity by the X-Gal filter assays. For all interactions tested, yeast double transformants were cultured in SD-Trp-Leu liquid medium overnight. After the cultures were diluted to an absorbance of 0·5 at OD600, 10 µL of cell suspension were placed onto solid (a) SD-Trp-Leu medium, (b) SD-Trp-Leu-His plus 10 mm 3-AT medium and (c) SD-Trp-Leu medium on which a filter was previously deposited. Growth was recorded and X-Gal filter assay was performed after 3 d of incubation at 30 °C. The procedures for X-Gal filter assay were as follows: the colonies were replicated onto a piece of nitrocellulose filter paper that had been saturated with Z-buffer (60 mm Na2HPO4, 40 mm NaH2PO4, 10 mm KCl, 1 mm MgSO4, pH 7·0) plus 0·1 % Triton X-100 and 1·5 mg mL−1 X-Gal. The filter was submerged in liquid nitrogen for 5–10 s to crack cells and then placed into a Petri dish with Z-buffer plus 0·1 % Triton X-100 and 1·5 mg mL−1 X-Gal. The filter was incubated at 30 °C, and the appearance of blue colour was monitored at 2 h.

RNA isolation from different tissues and treatments with ABA and stress conditions

As the main focus of this study was OsiICK6, we further analysed its expression in different tissues and in response to different conditions. For determination of OsiICK6 expression in normal tissues, total RNAs were isolated from leaves, stems, roots, young panicles and maturing florets using Trizol reagent (Invitrogen) according to the manufacturer's instructions. To analyse the expression in response to low temperature, ABA and high osmotic treatments, an embryonic cell culture line from the indica line MH63 was used. The culture was routinely maintained in Petri dishes containing 10 mL medium and incubated at 28 °C in a darkroom. The low-temperature treatment was conducted by directly transferring the embryogenic calli from the normal growth conditions to a 4 °C incubator. Cultures grown at 28 °C in the dark were used as a control. For ABA and osmolarity treatments, embryogenic calli were transferred from semi-solid MS medium (Murashige and Skoog, 1962) to liquid MS medium supplemented with 50 µm ABA, or 300 mm mannitol. The calli grown in the same liquid MS medium without the supplementary component were used as a control. All the cultures were cultured in a shaker at 250 r.p.m. at 28 °C. They were sampled at 0, 2, 4, 8, 16 and 24 h and total RNAs were isolated using Trizol reagent. Each treatment experiment was repeated three times.

Expression analysis by RT-PCR and real-time PCR

Two micrograms of freshly extracted RNAs was used as template for synthesis of first-strand cDNAs of OsiICK6, which were used for semi-quantitative RT-PCR and real-time PCR. In the semi-quantitative RT-PCR, the primers sqICK6F and sqICK6R (Supplementary Data Table S1), and Taq polymerase were used. The PCR was performed under the following conditions: cDNA was predenatured at 94 °C for 5 min, followed by 28 cycles of 30 s at 94 °C, 30 s at 56 °C and 30 s at 72 °C in 25 µL of reaction mixture containing 1× PCR buffer (10 mm Tris-HCl, 50 mm KCl and 1·5 mm MgCl2), 0·2 mm dNTPs, 0·2 µm of each primer, 0·625 units of Taq polymerase and 8 % dimethyl sulfoxide. To ensure linearity of response, the RT-PCR reactions were performed for no more than 28 cycles. Rice Actin1 cDNA amplified with primers ACTINF and ACTINR (Supplementary Data Table S1) was used as a reference. Also, to avoid DNA contamination in cDNA amplification, total RNAs were treated with DNase I prior to cDNA synthesis and primers were designed to cover a region containing at least one intron. All PCRs were repeated using at least three independent samples. PCR products were fractionated on 1 % agarose gel containing ethidium bromide and photographed under UV light.

For real-time PCR analysis of OsiICK6 expression, the SYBR Green I PCR master mix kit (TaKaRa) was used with the primer pair qICKF and qICKR (Supplementary Data Table S1). Relative expression levels of OsiICK6 were detected using the method (Livak and Schmittgen, 2001; Peres et al., 2007). Here, ΔΔCT = (CT, Target – CT, Actin) Time x – (CT, Target – CT, Actin) Time 0. The CT (cycle threshold) value for both target and internal control genes are the means of the triplicate independent PCRs. ‘Time x’ in the formula means a given time point among the time points when samples were taken, i.e. 0, 2, 4, 8, 16 and 24 h. As OsiICK6 showed the lowest expression level in maturing florets among all tissues sampled, the expression level in maturing florets was used as the base value for calculating the differences (changes in CT) among different tissues. Therefore, the corresponding formula was modified as follows: ΔΔCT = (CT, Target – CT, Actin)DST – (CT, Target – CT, Actin)MF, where DST refers to the developmental stage tissue.

Subcellular localization of OsiICK1 and OsiICK6

The subcellular localization of OsiICK1 and OsiICK6 was analysed by transient expression of a fusion construct containing enhanced green fluorescence protein (EGFP). To prepare EGFP–OsiICK fusion constructs, the complete coding sequence of OsiICK6 was removed with SalI and NotI digestion from pMD-OsiICK6 and cloned into pUC-35S::EGFP::NOS (modified from pEGFP vector of Clontech) as a fusion to the 3′-end of EGFP. The 35S::EGFP:OsiICK6::NOS fragment was removed from the plasmid by digestion with HindIII and EcoRI and cloned into pCAMBIA1300 (CAMBIA), resulting in the construct pYG6 (Supplementary Data Fig. S1A, available online). OsiICK1 cDNA was obtained with SalI and SacI digestion from pMD-OsiICK1 and cloned into pYG6 to replace OsiICK6, resulting in the construct pYG1 (Supplementary Data Fig. S1A). The constructs were introduced into Agrobacterium tumefaciens strain EHA105. Transient expression in tobacco cells was performed according to previously described procedures (Van den Ackerveken et al., 1996; Zhou et al., 2006). Agrobacterium cultures were infiltrated into tobacco leaves with a 1-mL syringe. Leaf sections with size around 1 cm2 were visualized under a laser scanning confocal microscope (Leica TCS SP5) 24–48 h after infiltration.

Generation of OsiICK6 over-expression lines

Rice transformation depends on plant regeneration from transformed callus and the over-expression of ICKs driven by a strong 35S promoter is expected to suppress callus regeneration and limit plant transformation events. Accordingly, we used a less strong promoter from a rice 33-kDa secretory protein (33SP) gene (GenBank accession no. AF311908). A 1·1-kb promoter region of the 33SP gene (nucleotides from –995 to 83 relative to the transcriptional start site) was amplified from genomic DNA of MH63 using 33SPF1 and 33SPR1 primers (Supplementary Data Table S1), with restriction sites HindIII and BamHI, respectively. The fragment was cloned into pMD-18T (TaKaRa) and sequence verified by DNA sequencing. The promoter in pMD-18T was then digested with HindIII and BamHI and inserted into pCAMBIA1300-35S::EGFP::NOS to replace the 35S promoter, resulting in p33G (Supplementary Data Fig. S1A). This construct was used to assess the expression pattern and efficiency of the 33SP promoter via green fluorescence visualization and semi-quantitative RT-PCR.

To prepare the EGFP-free over-expression construct, the 33SP promoter fragment was amplified from p33G using primers 33SPF1 and 33SPR1 (Supplementary Data Table S1) with restriction sites KpnI and SmaI, respectively, and cloned into pBluescript SK+ (pBSK) (Stratagene; http://www.stratagene.com) to produce pBSK-SP. The nitric oxide synthase (NOS) terminator was amplified from pUC-35S::EGFP::NOS using primers NOSF and NOSR (Supplementary Data Table S1). The NOS terminator fragment was ligated into pBSK to produce pBSK-NOS.

To generate the OsiICK6 over-expression construct, the SalI and NotI OsiICK6 cDNA fragment from pMD-OsiICK6 was cloned into pBSK-NOS, resulting in the construct pBSK-OsiICK6::NOS. Digestion of this construct with SalI and then treatment with Pyrobest (TaKaRa) created blunt ends, which was followed by further digestion with KpnI to create a sticky 5′-end. A KpnI–SmaI 33SP promoter fragment from pBSK-SP was cloned into the above vector, resulting in a pBSK-based construct containing the 33SP promoter::OsiICK6::NOS expression cassette. The expression cassette was removed and cloned into a plant expression vector pCAMBIA1301 to produce the construct p1301Y6, as shown (Supplementary Data Fig. S1B). p1301Y6 was introduced into A. tumefaciens strain EHA105, which was used to transform rice embryogenic callus cultures from MH63 to generate transformed calli and OsiICK6-over-expression (OsiICK6OE) plants. Transformants were screened by PCR using primers (Supplementary Data Table S1) specific for the hygromycin B phosphotransferase gene (hpt) present in pCAMBIA1301.

Phenotypic evaluations of OsiICK6-over-expressing lines

The evaluation of seed yield-related traits of three independent transformants OsiICK6OE-7, OsiICK6OE-16 and OsiICK6OE-34 were performed in the T2 generation. For each independent transformant, three progeny lines and ten plants in each line were evaluated. The wild-type plants were used as the control. Traits evaluated were the number of panicles per plants, number of grains per panicles, seed-setting rate and thousand-seed weight. The data were analysed statistically using SPSS software (SPSS Inc.).

Microscopy and analysis of cell size and number

Stems at certain developmental stages were sampled and prepared for paraffin embedding using a procedure described by Ogawa et al. (2003), with modifications. Briefly, the samples were fixed in 10 % formalin in phosphate buffer (10 % formalin in 8 mm sodium phosphate, pH 7·4), at room temperature for 8–24 h, and dehydrated through a graded ethanol series from low to high concentration. Then, ethanol was replaced with xylene. The treated samples were then embedded in paraffin. Sections (4 µm) were cut on a microtome and floated on 40 °C water bath. Thereafter, the sections were transferred onto plus slides and the slides were allowed to dry. Sections were cleared of paraffin with xylene and then re-hydrated through a graded ethanol series from high to low concentrations before being stained with 0·1 % toluidine blue (TBO) solution. The sections were examined under a Leica DMIRB microscope.

To analyse pollen development, pollen sampled from spikelet of control and OsiICK6OE plants just before flowering was stained with 1 % (w/v) iodine and potassium iodide (I2-KI) solution for viability or 1·0 µg mL−1 4′,6-diamidino-2-phenylindole (DAPI) in 1× phosphate-buffered saline solution for nuclear staining (Barrôco et al., 2006). The stained pollen grains were then visualized and images were recorded using a Leica DMIRB fluorescence microscope.

For cell size and number determinations, the longitudinal sections from both control and transgenic stems were used and the cell size was measured using ImageJ software (http://rsb.info.nih.gov/ij/docs/intro.html). The average cell size was calculated based on 20 cells randomly selected from each of the four cell layers per stem. The number of cells per unit was calculated using the average cell size to divide by 1 µm2.

For scanning electron microscopy, the fourth leaves of the control and OsiICK6-over-expressing plants were collected and dehydrated in a graded ethanol series. Once dehydrated, the samples were placed in a critical point dryer, mounted and coated with gold. The treated samples were examined and photographed using a scanning electron microscope (Philips XL-30-ESEM).

RESULTS

Identification of ICK-related genes from the indica rice genome

Seven ICK/KRP-related sequences were identified from the BGI Rice database of Oryza sativa subspecies indica ‘93-11’ (http://rise2.genomics.org.cn/), consistent with previous reports (Barrôco et al., 2006; Guo et al., 2007). One rice ICK/KRP gene lacks the conserved C-terminal CDK- and cyclin-binding region, while it shares similarities in the N-terminal part with other rice ICK/KRP sequences (Guo et al., 2007). As the conserved C-terminal region is required for CDK inhibition function (Zhou et al., 2003a), we consider this gene not to be a true ICK/KRP member. Thus, we consider that the ICK/KRP family in rice has six members. The six ICK/KRP proteins from the two rice subspecies match each other well.

The putative OsiICK protein sequences were aligned with Oryza sativa subspecies japonica, Arabidopsis and poplar ICK/KRP sequences. As shown in Fig. 1, the rice ICK/KRP sequences can be grouped into two classes, with OsiICK1–OsiICK4 in one class and OsiICK5 and OsiICK6 in the other. Interestingly, OsiICK5 (Orysa;KRP5) and OsiICK6 (Orysa;KRP4) are more closely related to three ICKs from Arabidopsis and poplar than to other rice ICK/KRP proteins, implying that there are probably functional differences between the two groups of rice ICK/KRP proteins.

Fig. 1.

Fig. 1.

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Phylogenetic analysis of plant ICKs/KRPs. Full-length ICK/KRP amino acid sequences from Arabidopsis thaliana, two subspecies of Oryza sativa and Populus trichocarpa were aligned and a phylogenetic tree was produced using MEGA4 software. The rice ICK/KRP sequences are clustered into two groups.

In addition to motifs 1 and 2 involved in the interactions with the CDK and cyclin, three other conserved motifs are present in rice ICKs (Fig. 2). Motif 4 of Arabidopsis ICK1 and ICK7 has been shown to confer nuclear localization in a punctate pattern (Zhou et al., 2006; Bird et al., 2007). More conserved motifs could be identified (Torres-Acosta et al., 2011). However, little is known about the functional roles for these motifs. In addition, nuclear localization signals (NLSs) are also identified in some of the rice ICKs (Fig. 2).

Fig. 2.

Fig. 2.

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Sequence analysis of OsiICK proteins. (A) Schematic overview of five conserved motifs (boxes 1–5) identified by MEME in different OsiICK proteins. The scale of amino acid length (light grey bar), nuclear localization signal (NLS, dark grey bar) and the conserved amino acid sequences is indicated. (B) Uniprot alignment of six OsiICKs. The conserved amino acid residues are indicated by dark grey or light grey shading, respectively. Putative NLSs are underlined. Five conserved motifs are marked on the top of sequences by numbers 1–5.

Determination of the interactions of OsiICK1 and OsiICK6 with cyclins and CDKs

OsiICK1 and OsiICK6, each from the two groups of rice ICK/KRP proteins (Fig. 1), were selected to investigate their interactions with CDKs and cyclins by the yeast two-hybrid system. It was observed that both OsiICK1 and OsiICK6 were able to interact with OsCYCD2;1, but not with OsCYCA2;1 or OsCYCB2;1 (Table 1). In terms of interactions with OsCDKs, neither rice ICK protein interacted with OsCDKB2;1, but OsiICK6 interacted with OsCDKA;1 (Supplementary Data Fig. S2A, available online). These results were further confirmed by a β-galactosidase assay (Supplementary Data Fig. S2B). These observations suggest that OsiICK6 interacts with both OsCDKA;1 and OsCYCD2;1, while OsiICK1 interacts only with OsCYCD2;1. Neither of them interacts with OsCDKB2;1, OsCYCA2;1 or OsCYCB2;2.

Table 1.

Interactions of OsiICK6 and OsiICK1 with different OsCDKs and OsCYCs

OsiICK6
 OsiICK1
GAL-AD GAL-BD GAL-AD GAL-BD
OsCDKA – ++ – –
OsCDKB – – – –
OsCYCA – – – –
OsCYCB – – – –
OsCYCD ++ ++ ++ ++
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The OsCDK and OsCYC constructs were prepared in the yeast two-hybrid DNA binding-domain (BD) vector pDBLeu, as well as the yeast two two-hybrid DNA activation-domain (AD) vector pPC86. They were tested against OsCDK and OsCYC constructs in pPC66 and pDBleu, respectively. Yeast double transformants could all grow normally on SD-Trp-Leu medium. To determine the interactions, they were grown on solid medium of SD-Trp-Leu-His plus 10 mm 3-amino-1,2,4-triazole (3-AT) at 30 °C for 3 d. Key: –, no interaction; +, a positive interaction. The images of yeast cell growth for the positive interactions are presented in Supplementary Fig. S2A, available online.

Expression of OsiICK6 was induced by stress conditions

The expression pattern of OsiICK6, which belongs to a class of ICK/KRP sequences from both dicot and monocot plant species, and more closely related to Arath;KRP4/ICK6 and Arath;KRP5/ICK7 than Arath;ICK1 and Arath;ICK2 (Fig. 1; Torres-Acosta et al., 2011), was investigated by quantifying the relative abundance of the mRNA in different tissues, including leaves, stems, roots, young panicles and maturing florets using real-time PCR. The results revealed that OsiICK6 was expressed in all the tissues analysed, and was highly expressed in leaves (Fig. 3A).

Fig. 3.

Fig. 3.

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Real-time PCR analysis of OsiICK6 expression in various tissues and in response low temperature, abscisic acid (ABA) and mannitol treatments. (A) OsiICK6 expression in various tissues of the wild-type indica rice of MH63. MF, maturing florets; YP, young panicles; L, leaf; S, stem; R, root. (B-D) Expression of OsiICK6 in rice embryogenic calli in response to low temperature (4 °C), ABA (50 mm) and mannitol (300 mm) treatments. Samples were taken at the time points indicated and RNA samples isolated. cDNAs were synthesized and used in real-time PCR. The RT-PCR was performed three times and the data are presented as means ± s.d.; treated and untreated controls as indicated.

To assess the expression of OsiICK6 in response to environmental conditions, its transcript level was analysed by real-time PCR after treatments with low temperature (4 °C), ABA (50 µm) and osmotic pressure (300 mm mannitol). Freshly cultured embryogenic cell cultures were used because of their uniform genetic background and growth status. The data indicate that expression of OsiICK6 was induced by all three stress conditions (Fig. 3B–D). For low temperature and mannitol treatments, after 8 h or more, there were about two-fold or more transcripts in the treated samples than in controls (Fig. 3B, D). For the ABA treatment, there was a higher level of induction, but the level appeared to decrease after 8 h presumably due to the depletion and degradation of ABA in the cell culture (Fig. 3C).

OsiICK1 and OsiICK6 were localized in the nucleus of tobacco leaf cells

Previous studies indicated that all the Arabidopsis ICKs are localized in the nucleus (Bird et al., 2007). It is thus interesting to determine the cellular localization of rice ICKs, particularly OsiICK1 and OsiICK6 in this study. Putative nuclear localization signals could be found in OsiICK1 and OsiICK6 sequences (Fig. 2). The control EGFP was localized in both the nucleus and cytoplasm (Fig. 4A–C). In contrast, both EGFP:OsiICK1 and EGFP:OsiICK6 were localized in the nucleus with little fluorescence present in the cytoplasm (Fig. 4D–K). It was interesting to observe that while EGFP:OsiICK6 showed a strong punctuate pattern of subnuclear distribution in the nucleus (Fig. 4G), the subnuclear distribution of EGFP:OsiICK1 was much more homogeneous (Fig. 4K). Based on the observation that the nuclear location patterns for the Arabidopsis ICK/KRPs are consistent in the tobacco leaves and in Arabidopsis plants (Bird et al., 2007), and also the similarities in nuclear localization of the two rice ICKs to Arabidopsis ICK/KRPs, it is very likely that the localization patterns observed in this study reflect the localization of the two rice ICK proteins in rice.

Fig. 4.

Fig. 4.

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Subcellular localization of OsiICK1 and OsiICK6 in transiently transfected tobacco cells. (A–C) Localization of control EGFP. (D–G) Localization of EGFP–OsiICK6 fusion protein, showing a non-homogeneous punctuate subnuclear distribution in the nucleus (G). (H–K) Localization of EGFP–OsiICK1 fusion protein which shows more homogeneous subnuclear distribution in the nucleus (K). Images were taken in dark field for green fluorescence (A, D, G, H, K) and bright field (B, E, I) for the cell outline. Images (C), (F) and (J) are superimposed images between (A) and (B), (D) and (E), and (H) and (I), respectively. Scale bars: (A–F, H–J) = 25 µm, (G, K) = 10 µm.

Plant growth and development were significantly influenced by OsiICK6 over-expression

OsiICK6 was over-expressed in rice plants to determine its effect on plant growth and development. First, the expression of EGFP driven by the 33SP-promoter was evaluated. Analysis of gene expression by both semi-quantitative and real-time RT-PCR methods showed that the EGFP was expressed in all tissues analysed, including roots, stems and leaves, but that levels were lower than those of 35S-promoter-drived EGFP (Fig. 5). Thus, the 33SP promoter was used subsequently to drive OsiICK6 over-expression.

Fig. 5.

Fig. 5.

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Comparative analysis of the EGFP (GFP) reporter gene driven by the 33SP promoter and 35S promoter in root (R), stem (S) and leaf (L) tissues. For the labels above the figure, 33 stands for 33SP promoter, 35 for 35S promoter and CK for the negative control. The bottom row shows RT-PCR amplification of the rice Actin1 cDNA as a reference.

The expression driven by the 33SP promoter was independently verified using plants transformed by the 33SP promoter–OsiICK6 construct (Supplementary Data Fig. S1B). T0 transformants regenerated from independent transformed calli were selected for expression and phenotypic analyses, with plants regenerated from non-transformed calli as controls. OsiICK6 transcript levels in stems and leaves of OsiICK6OE and control plants were examined by both semi-quantitative RT-PCR and real-time PCR. The results revealed that levels of OsiICK6 in the stems and leaves of the OsiICK6OE plant were 5–10 times higher than those in the control plant (Fig. 6A).

Fig. 6.

Fig. 6.

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Expression and phenotypic analyses of rice plants over-expressing OsiICK6. (A) Real-time PCR (top panel) and semi-quantitative PCR (bottom panel) analysis of OsiICK6 in stems (S1 and S2) and leaves (L1 and L2) of OsiICK6-over-expressing (OsiICK6OE) (S2 and L2) and control (S1 and L1) plants. (B) Comparison of control (left) and OsiICK6OE (right) plants at maximum tillering stage. (C) Tillers of control (left) and OsiICK6OE (right) plants showing the positions of internodes indicated by arrows. (D, E) Transverse cross-sections of stems sampled from control (D) and OsiICK6OE (E) plants. Scale bars = 100 µm. (F, G) Enlarged images of the areas indicated in (D) and (E), respectively. Scale bars = 50 µm. (H, I) Longitudinal sections of the main stems from the control (left) and OsiICK6OE (right) plants. Outer to inner orientation is shown from left to right. Scale bars represent 50 µm. (J) The leaf blades of control and homozygous OsiICK6OE plants. Top: the adaxial (Ad) and abaxial (Ab) epidermis of control (1) and OsiICK6OE (2) leaves. Bottom: cross sections of control (left) and OsiICK6OE (right) leaves. (K, L) Scanning electron micrographs of adaxial epidermis from control (K) and OsiICK6 °E (L) plants. Stomata (st), lateral vein (lv) and dumbbell-shaped silicon cell (dsc) are indicated by arrow lines. Scale bars = 40 µm. (M, N) Comparisons of stomatal size on the adaxial (Ad1 and Ad2) and abaxial (Ab1 and Ab2) sides of leaves from control (Ad1 and Ab1) and OsiICK6OE (Ad2 and Ab2) plants. Scale bars = 3 µm.

OsiICK6-over-expressing T0 plants derived from different transgenic events (independent transformed calli) were clearly dwarfed (Fig. 6B). The average plant height of these OsiICK6OE T0 plants was 35–45 % shorter than that of control plants (Table 2). Further investigation on two OsiICK6OE T2 lines revealed that the shorter stem was mainly due to a reduced length of the first and second internodes counted from the top (Fig. 6C). Measurements of the internode lengths showed that the reduced length of those two internodes accounted for over 60 % of the total reduction in the height of the transformants (Table 3).

Table 2.

The height of the OsiICK6-over-expressing plants regenerated from independently transformed calli compared with control plants

Transformed calli Plants regenerated Plant height (cm) Percentage of the height of controls
OsiICK6OE-8 3 46·7 ± 12·6 54
OsiICK6OE-19 6 50·3 ± 8·2 50
OsiICK6OE-25 8 53·8 ± 5·7 62
Wild-type control 3 86·3 ± 2·1 100
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Plants were regenerated from independent calli transformed using OsiICK6 or control constructs. Transformants were transferred into soil and grown to maturity. Plant height (defined as the length of the highest tiller above ground) was measured for each plant just before harvest.

Table 3.

Reduction of internode lengths in OsiICK6-over-expressing plants compared with wild-type plants

Lines Length of internodes from upmost (cm)
 Total
5th 4th 3rd 2nd 1st
Wild-type control (cm) 2·8 ± 0·6 6·2 ± 1·2 11·3 ± 1·4 18·9 ± 1·2 34·4 ± 2·1 73·6 ± 3·6
OsiICK6OE-7-134 (cm) 1·8 ± 0·7 4·3 ± 1·1 8·0 ± 1·8 12·7 ± 2·8 30·3 ± 2·3 57·1 ± 2·9
Reduction in length (cm) 1·0 1·9 3·3 6·2 4·1 16·5
RCH* ( %) 6·1 11·5 20·0 37·6 24·8 100·0
OsiICK6OE-7-129 (cm) 2·1 ± 0·8 4·5 ± 1·6 8·8 ± 1·9 15·9 ± 2·1 28·2 ± 2·6 59·5 ± 3·7
Reduction in length (cm) 0·7 1·7 2·5 3·0 6·2 14·1
RCH (%) 5·0 12·1 17·7 21·3 43·9 100·0
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The OsiICK6-over-expressing and control plants were grown under normal conditions in an isolated experimental plot. The lengths of internodes were measured at maturity and 20 tillers were measured for each line.

* RCH, relative contribution to the reduction in height. RCH (%) = reduction in length of individual internode/total reduction in all internodes measured × 100.

To gain further insight into the phenotypic effects of OsiICK6 over-expression, leaf and stem tissues from OsiICK6OE and control plants were examined by microscopy. Comparisons of transverse stem sections showed that all tissue layers including the vascular bundle sheath from the OsiICK6OE plant had much larger but fewer cells than the corresponding tissues from the control plant (Fig. 6D–G). This observation was confirmed with longitudinal sections (Fig. 6H, I), in which the four cell layers from the epidermis were analysed. The average cell size for these cells increased almost three-fold from 2060 ± 730 µm2 in the control plants to 6980 ± 1890 µm2 in the transgenic plants. These results collectively demonstrate that OsiICK6 over-expression resulted in a reduced cell number, but larger cells in various tissues examined, with the overall result being dwarfed plants.

Morphological changes were also evident in the OsiICK6OE plants. In control plants, the leaves normally rolled slightly toward the adaxial side (Fig. 6J, Ad1 and Ab1). In contrast, leaves of the OsiICK6OE plants were rolled severely toward the abaxial side (Fig. 6J, Ad2 and Ab2). This phenomenon has been consistently observed in different OsiICK6OE plants. There were also other structural changes displayed by the OsiICK6OE plants, including decreased spacing between lateral veins, a reduced diameter of lateral veins with an increased size of the dumbbell-shaped silicon cells, reduced number of stomatal lines, increased distance longitudinally between stomatas on both the adaxial and abaxial sides of leaves, and increased size of stomata on the adaxial side of leaves (Fig. 6K–M). The average length of stomata on the adaxial leaves of OsiICK6OE plants was 18·21 ± 1·43 µm, compared with 13·56 ± 0·92 µm in the leaves of control plants (Fig. 6M). However, the stomatal size on the abaxial side of leaves on OsiICK6OE plants appeared not to be affected (Fig. 6N), although the stomata were reduced from two to one line (data not shown).

Over-expression of OsiICK6 reduced rice seed production

The effects of over-expressing OsiICK6 on several traits related to seed yield were evaluated in T2 populations derived from three independent transgenic lines (OsiICK6OE-7, OsiICK6OE-16, OsiICK6OE-34). Quantitative measurements indicated that the number of panicles per plant, number of grains per panicle, seed-setting rate and thousand-seed weight were significantly affected by over-expression of OsiICK6. These four agronomically important traits were reduced from control levels of 16·5 ± 6·0, 156·4 ± 22·7, 80·9 ± 3·4 % and 26·9 ± 1·2 g to 5·7 ± 0·8, 87·4 ± 8·3, 27·7 ± 4·4 % and 21·3 ± 1·5 g in the transgenic OsiICK6OE T2 lines, respectively (Table 4).

Table 4.

Analysis of traits related to seed yield in OsiICK6-over-expressing and wild-type plants

Transformant Panicles per plant Grains per panicle Seed-setting rate (%) Thousand-seed weight (g)
OsiICK6OE-7 6·1 ± 0·7* 99·8 ± 7·8** 33·6 ± 5·3** 21·2 ± 1·9**
OsiICK6OE-16 7·0 ± 0·8* 79·7 ± 6·2** 29·4 ± 2·4** 22·4 ± 1·1**
OsiICK6OE-34 4·0 ± 0·9** 82·7 ± 10·8** 20·1 ± 5·5** 20·4 ± 1·5**
Mean 5·7 ± 0·8** 87·4 ± 8·3** 27·7 ± 4·4** 21·3 ± 1·5**
Wild-type 16·5 ± 6·0 156·4 ± 22·7 80·9 ± 3·4 26·9 ± 1·2
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Ten plants from each of the OsiICK6OE (T2) and wild-type lines were evaluated and the measurements were taken when plants were mature.

Asterisks indicate statistically significant differences from wild-type control at *P < 0·05 and **P < 0·01.

To further determine the reasons for the drastic reduction of seed-setting rate, pollen viability was examined under a microscope following I2-KI (Fig. 7A, B) and PI (Fig. 7C, D) staining. In the control plants, the majority of pollen grains appeared normal and had three nuclei, whereas the majority of pollen grains in the transgenic plants appeared abnormal (Fig. 7B, D). Quantitative analysis showed that in the control plants, about 78 % of pollen were normal as compared with an average of 22 % normal pollen grains in OsiICK6OE plants (Table 5). Analysis of 33SP promoter–EGFP expression showed that EGFP was clearly expressed in both stamen and anther tissues (Fig. 7F), supporting the phenotypic observations. Together, these data suggest that the poorly developed pollen was at least partially responsible for the reduction of seed-setting rate.

Fig. 7.

Fig. 7.

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(A–D) Analysis of pollen from control (A, C) and OsiICK6OE (B, D) plants. Pollen grains were sampled from spikelets just before flowering and stained with iodine and potassium iodide (I2-KI) solution (A, B). Pollen grains stained with DAPI (C, D). (E, F) Images (fluorescence and bright field merged) to show that GFP fluorescence was absent in the stamen and anther of the wild-type plant (E), but was present in the stamen and anther of 33SP promoter-EGFP plant (F). Scale bars: (A, B) = 50 µm; (C, D) = 25 µm; (E, F) = 600 µm.

Table 5.

Viability of pollen from T2 transgenic lines of OsiICK6OE-7, OsiICK6OE-16 and OsiICK6OE-34 and control plants

Lines Percentage of sterile pollen
I II III Mean
OsiICK6OE-7 76·6 (174/227) 76·0 (146/192) 72·4 (126/174) 75·0 ± 2·3
OsiICK6OE-16 80·9 (178/220) 82·5 (156/189) 78·5 (139/177) 80·1 ± 2·0
OsiICK6OE-34 78·6 (165/210) 74·4 (134/180) 82·2 (111/135) 78·4 ± 3·9
Wild-type 21·8 (39/179) 22·8 (42/184) 21·7 (39/190) 21·7 ± 1·2
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Pollen grains were collected from flowers just before flowering. They were stained with 1 % iodine and potassium iodide (I2-KI) solution for viability or 1·0 µg mL−1 DAPI in phosphate-buffered saline solution for nuclear number. Three anthers from each floret, three florets from each plant and three plants from each line were used for determination of pollen viability.

DISCUSSION

In this study, rice ICK/KRP putative protein sequences were compared with Arabidopsis ICK/KRPs. Based on the conserved C-terminal region shared by Arabidopsis ICK/KRPs (Wang et al., 1997; De Veylder et al., 2001; Zhou et al., 2002a), it is determined that rice has a family of six authentic members in the ICK/KRP family and the genes in the indica subspecies investigated in this study are referred to as OsiICKs (Figs 1 and 2). Based on the analysis of a large number of ICK/KRP sequences from various plant species, it has been found that plant ICK/KRP sequences can be classified into three classes (Torres-Acosta et al., 2011). The rice ICK/KRPs belong to two classes, with OsiICK1–OsiICK4 in one class and OsiICK5 and OsiICK6 in another (Fig. 1). Interestingly, OsiICK5 and OsiICK6 are more closely related to Arabidopsis ICK1 than other OsiICKs, suggesting that they share a common evolutionary ancestor (Torres-Acosta et al., 2011). To gain understanding of rice OsiICKs and the differences between the two classes, we investigated OsiICK1 and OsiICK6 as representatives from the B and C classes, respectively. We focused more on OsiICK6 as little is known about it.

The ICK/KRP family of proteins function by directly binding CDK complexes. Thus, it is important to understand whether different ICK/KRPs differ in their interactions with various CDKs and cyclins. Unfortunately, little was known regarding the interactions of rice ICK/KRP proteins with CDKs. Mizutani et al. (2010) showed that in a yeast two-hybrid system, Orysa;KRP3 (corresponding to OsiICK3) interacted with two rice CDKAs, OsCYCA1;1 and OsCYCD2;2, whereas it did not interact with OsCDKB2;1 or OsCYCB2;2. In the present study, two additional rice ICK/KRPs were shown to interact with OsCYCD2;1. Also, it was observed that OsiICK6 interacted with OsCDKA, while OsiICK1 showed little interaction with OsCDKA (Table 1; Supplementary Data Fig. S2). The difference between OsiICK1 and OsiICK6 in the same yeast two-hybrid system suggests that there may be a difference between the two proteins in terms of the interaction with OsCDKA. Furthermore, none of the three rice ICK/KRPs studied here and earlier (Mizutani et al., 2010) interacted with OsCDKB in the yeast two-hybrid system. These observations are similar to what was observed with Arabidopsis ICK/KRPs (Zhou et al., 2002a). Based on the yeast two-hybrid data, it has been suggested that CYCD and CDKA are the main targets of plant ICK/KRPs (Wang et al., 2006), which gained further support from a recent proteomics study (Van Leene et al., 2010).

All seven Arabidopsis ICK/KRPs are localized in the nucleus (Bird et al., 2007). The observation that two rice ICK proteins representing two different classes are both localized in the nucleus strongly indicates that it is likely that rice ICKs as the Arabidopsis counterparts are localized in the nucleus. While EGFP:OsiICK6 shows a prominent punctate pattern of subnuclear distribution, EGFP:OsiICK1 has a more homogeneous subnuclear distribution (Fig. 4). It has been shown that the punctuate pattern of subnuclear distribution was conferred by a motif in four Arabidopsis ICKs with the consensus sequence ‘YLQLRSRRL’ (Zhou et al., 2006; Bird et al., 2007). In addition to this motif, it was reported recently based on the study of the tomato ICK/KRP SlKRP1 that another motif could also confer a punctate pattern of subnuclear distribution (Nafati et al., 2010). The ‘YLQLRSRRL’ motif corresponds to motif 4 found in four of the OsiICKs (Fig. 2). Among the OsiICKs, the third residue in this motif is variable (Fig. 2). For the eighth residue, three OsiICKs have the conserved ‘R’ while OsiICK1 has a variant ‘M’. It is possible that the change of R to M in the YLQLRSRML of OsiICK1 has rendered the subnuclear distribution of EGFP:OsiICK1 more homogeneous (Fig. 4).

One critical issue in studying cell cycle regulators is to determine the relationship between cell proliferation and plant growth and development. Prior to the present study, only one rice ICK/KRP gene, Orysa;KRP1(OsiICK1), had been over-expressed in plants. Over-expression of Orysa;KRP1 was found to reduce cell production and seed filling, but did not significantly affect plant vegetative growth (Barrôco et al., 2006). In contrast, over-expression of OsiICK6 (corresponding to Orysa;KRP4 in japonica rice) in this study dramatically reduced plant vegetative growth and seed production. A series of characteristics related to plant growth and seed production such as plant height, leaf area, number of panicles per plant, number of grains per panicle, seed-setting rate and thousand-seed weight were affected. The transgenic plants became dwarf and semi-sterile with altered leaf morphology (Fig. 6 and Table 4). The reduced height of the OsiICK6OE plants was for the most part due to the reduced lengths of the first two internodes, suggesting that OsiICK6 over-expression had a major impact on cell proliferation in these two internodes. The observation that OsiICK6 over-expression resulted in more significant phenotypes than the over-expression of Orysa;KRP1 reported previously (Barrôco et al., 2006) may be due to the functional differences between the two genes. Alternatively, the difference is more probably due to the two different promoters used. In OsiICK6OE plants, pollen viability and seed-setting rate were also reduced. These results are consistent with the previous observation that Brassica plants expressing Arabidopsis ICK1 with a pollen-specific promoter showed drastically reduced pollen viability and seed setting (Zhou et al., 2002b).

Another interesting effect is on leaf development. Several factors have been reported to be involved in the regulation of leaf morphology, including axis-determining leaf genes (Eshed et al., 2001; Bowman et al., 2002), miRNAs and phytohormones (Juarez et al., 2004), as well as factors controlling the establishment of leaf polarity (Zhang et al., 2009). The leaves of OsiICK6OE plants were severely rolled upward. No such effect was reported with the over-expression of Orysa;KRP1 (Barrôco et al., 2006). This difference may be due either to the functional difference between OsiICK6 and Orysa;KRP1 or to the two different promoters used in the present study and the study by Barrôco et al. (2006). It is conceivable that upward rolling is due to differential growth between the adaxial and abaxial sides as a result of the difference in either cell proliferation or cell size increase, as the primary effect of OsiICK6 over-expression is the inhibition of cell proliferation. It could be further inferred that cell proliferation on the adaxial side was more affected than that on the abaxial side. This suggestion is also supported by the observation that cell size increased more on the adaxial side, an indication of a greater reduction in cell number. Thus, cell cycle regulation is a critical factor regulating the flatness or curvature of leaves in plants. Strong evidence for this suggestion has been obtained by targeted expression of the Arabdopsis ICK1 and KRP4/ICK7 with the L1-specific promoter, which resulted in leaves with strong negative curvature (severely rolled-up blades) (Bemis and Torii, 2007). Further investigation found that the stomata differentiating from the L1 layer during leaf development are larger than that from control (Bemis and Torii, 2007).

Orysa;KRP1, Orysa;KRP4 and Orysa;KRP5 are shown to be expressed in most tissues examined (Guo et al., 2007), while Orysa;KRP3 is highly expressed in endosperm (Mizutani et al., 2010). In addition to the possible roles in plant growth and development, cell cycle regulators may also function to integrate environmental cues. Thus, CDK inhibitors may link certain conditions that inhibit plant growth to the cell cycle machinery. In this regard, Arabidopsis ICK/KRPs have been shown to be induced by ABA, low temperature and salt (Wang et al., 1998; Ruggiero et al., 2004; Pettko-Szandtner et al., 2006). The increased expression of OsiICK6 following ABA, low temperature and mannitol treatments observed in this study is consistent with this view.

In this study, OsiICK1 and OsiICK6 representing two rice ICK groups were investigated in terms of their interactions with CDKs and cyclins, and their cellular localization. Differences between the two CDK inhibitors were observed. These results provide useful information regarding the basic characteristics of rice ICK/KRPs. Furthermore, the expression of OsiICK6 was shown to be induced by several conditions. The significant changes displayed by the transgenic rice plants over-expressing OsiICK6 demonstrate that alterations in the expression of a CDK inhibitor can significantly affect plant growth and development in rice.

SUPPLEMENTARY DATA

Supplementary data are available online at www.aob.oxfordjournals.org and consist of the following. Table S1: List of primers used in this study. Figure S1: Plasmid constructs used in this study. Figure S2: Analysis of physical interactions of OsiICK1 and OsiICK6 with OsCDKA;1 (OsCDKA), OsCDKB2;1 (OsCDKB) and OsCYCD2;1 (OsCYCD) in a yeast two-hybrid assay.

Supplementary Data
supp_107_7_1087__index.html (854B, html)

ACKNOWLEDGMENTS

This work was supported by the Talent Admission Scheme (grant number 317000-361303), Zhejiang University. H.W. gratefully acknowledges financial support from the Natural Sciences and Engineering Research Council of Canada.

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