Identification and functional analysis of the ICK gene family in maize

Inhibitors of cyclin-dependent kinases (ICKs) are key regulators of cyclin-dependent kinase activities and cell division. Herein, we identified eight ICKs in maize, which we named Zeama;ICKs (ZmICKs). Primary sequencing and phylogenetic analyses were used to divide the ZmICK family into two classes: group B and group C. Subcellular localization analysis of ZmICK:enhanced green fluorescent protein (eGFP) fusion constructs in tobacco leaf cells indicated that ZmICKs are principally nuclear. Co-localization analysis of the ZmICKs and maize A-type cyclin-dependent kinase (ZmCDKA) was also performed using enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) fusion constructs. The ZmICKs and ZmCDKA co-localized in the nucleus. Semi-quantitative RT-PCR analysis of the ZmICKs showed that they were expressed at different levels in all tissues examined and shared similar expression patterns with cell cycle-related genes. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that ZmICK1, ZmICK2, ZmICK3, and ZmICK4 interact with ZmCDKA1 and ZmCDKA3. Interestingly, ZmICK7 interacts with D-type cyclins. Transformed and expressed ZmCDKA1 and ZmICKs together in fission yeast revealed that ZmICK1, ZmICK3, and ZmICK4 can affect ZmCDKA1 function. Moreover, the C-group of ZmICKs could interact with ZmCDKA1 directly and affect ZmCDKA1 function, suggesting that C-group ZmICKs are important for cell division regulation.


List of Supplementary Information
. The basic information about the identified ICK/KRP candidate genes in Maize.
Length of bp* means the length of cDNA; Length of aa* shows the number of amino acid; In/ex means the number of Intron-exon; Mw is the molecular weight of the protein; Chr represents the chromosome. Table S2. The basic information about the ICK/KRP gene family of Arabidopsis and Orysa sativa. Length of bp* means the length of cDNA; Length of aa* shows the number of amino acid; In/ex means the number of Intron-exon; Mw is the molecular weight of the protein; Chr represents the chromosome; Location shows the detail location on the chromosome. Table S3. Primers for the main research. PCR annealing temperature is 57°C to 61 °C for all genes based on the primers information. "Category" indicates the carriers constructed and expression analysis in research, "Gene Name" represents the name of gene in the article, "Primer name" and "Primer Sequence" represents the primer name and sequence of related gene in this article. Table S4. The conserved motifs sequence information of ICK in maize.       SD/-Leu/-Trp indicates media lacking tryptophan (Trp) and leucine (Leu). SD/-Ade/-His/-Leu/-Trp represents the synthetic dextrose (SD) media lacking tryptophan (Trp), leucine (Leu), adenine (Ade) and histidine (His).
SD/-Ade/-His/-Leu/-Trp/30mM3-AT/10uM X-a-gal indicates the synthetic dextrose (SD) media contain 30 mM 3-AT and 10 uM X-a-gal. The gray box indicates the growth condition on the same medium and every yeast plaque indicates the growth of the transformants. "+" represents the positive control of the yeast two-hybrid, the combination of pGADT7 and pGBKT7-53. "-" shows the negative control of yeast two-hybrid, the pGADT7-T co-transformed with pGBKT7-lam. Each alphabet labelled with 1 to 3 represents the same combination. Bar=50 µm.

Fig. S8 Cytological analysis of fission yeast cells expressing C-group ZmICKs, ZmCDKA3
and a combination of the proteins. A1 represents wild-type fission yeast cells transformed with pREP42 and pESP-3. A2 shows yeast cells expressing ZmCDKA3 and pREP42. B1 to E1 presents the yeast cells expressing the C-group ZmICKs-pREP42 with pESP-3. B2 to E2 shows the yeast cells co-expressing C-group ZmICKs and ZmCDKA3. The ZmICKs-pREP42 and the corresponding ZmICKs-pREP42 with ZmCDKA3-pESP-3 images are shown side-by-side. Bars=10 µm.

Fig. S9
The averages with standard deviations of the actual cell size (um 2 ). Averages are the average of the transformation of the same combination between different views and standard deviations represent the degree of dispersion of them. The X-axis represents the actual cell area size (um 2 ). The Y-axis shows the different transformation of combination. "**" indicates significant differences in mean values between the different treatments and controls.

Figure S1
Physical locations information about maize ICK genes, the grey boxes on chromosomes indicate the approximate locations of the 9 maize ICK genes. CHR is the abbreviation of chromosomes and represents as black bars. The number corresponds to the number of chromosomes.    SD/-Ade/-His/-Leu/-Trp/30mM3-AT/10uM X-α-gal indicates the synthetic dextrose (SD) media contain 30 mM 3-AT and 10 uM X-α-gal. The gray box indicates the growth condition on the same medium and every yeast plaque indicates the growth of the transformants. "+" represents the positive control of the yeast two-hybrid, the combination of pGADT7 and pGBKT7-53. "-" shows the negative control of yeast two-hybrid, the pGADT7-T co-transformed with pGBKT7-lam. ZmCDKA3 and a combination of the proteins. A1 represents wild-type fission yeast cells transformed with pREP42 and pESP-3. A2 shows yeast cells expressing ZmCDKA3 and pREP42. B1 to E1 presents the yeast cells expressing the C-group ZmICKs-pREP42 with pESP-3. B2 to E2 shows the yeast cells co-expressing C-group ZmICKs and ZmCDKA3.

Figure S9
The averages with standard deviations of the actual cell size (um 2 ). Averages are the average of the transformation of the same combination between different views and standard deviations represent the degree of dispersion of them. The X-axis represents the actual cell area size (um 2 ). The Y-axis shows the different transformation of combination. "**" indicates significant differences in mean values between the different treatments and controls.