In Silico Characterization of a Cyclin Dependent Kinase -A (CDKA) and its Coding Gene in some Oryza Species

Rice (Oryza sativa) is a fundamental food for the majority of world population. Cyclin Dependent Kinase -A (CDKA) accelerates transition through different stages of cell cycle and contributes in gametes formation. In the present investigation, a CDKA encoding gene along with the corresponding protein were characterized in O. sativa Indica Group, O. glaberrima, O. barthii, O. brachyantha, O. glumipatula, O. longistaminata, O. meridionalis, O. nivara, O. punctata and O. rufipogon using in silico analyses. The results reflected little variation in most species except O. longistaminata and O. brachyantha. Compared with the remaining species, O. longistaminata lacked a negative regulatory binding site and had a modified cyclin binding site (PSTAICE instead of PSTAIRE) that may lead to future characterization of a new distinct subclass of CDKAs. O. brachyantha had a modified SUC/CKS (suppressor of CDC2/cyclin dependentkinase regulatory subunit)-binding motif. The observed variations can be exploited through traditional breeding or molecular approaches to manipulate cell division and growth of cultivated Oryza species.


Introduction:
Rice is an important strategic world crop stands second among the cultivated cereal crops with annual yield of 770 million ton from about 2.38 million acre (1). Possessing balanced contents of carbohydrates, lipids and proteins put rice as the predominant food for more than two thirds of the world population (2,3). The global climate change in addition to the growing world population necessitates breeding for new cultivars with better quantitative and qualitative traits (3,4).
Rice breeding suffers from the limited genetic variations of cultivated Oryza species (O. glaberrima and O. sativa) that do not exceed 20% of those recorded in wild Oryza species (5,6). Thus efforts should be continued to demonstrate interand intraspecific genetic variations in wild Oryza species to compensate shortage in variations observed in cultivated rice (5). Fortunately, the full sequence genomes of many wild Oryza species are now available and ready to be used for mining of valuable genetic information.
The in silico analyses provide precious genetic information for rice breeding including identification of important sequences in wild Oryza species such as regulatory elements for pathogenesis-related proteins (7), cyclin dependent kinase-B coding gene (5) and a gene encoding a Pathogenesis-Related Protein-10 (6).
Growth and development are regulated through strict control of cell cycle (8,9). All eukaryotic cells possess a group of Ser/Thr protein kinases known as cyclin dependent protein kinases (CDKs); that form complexes with cyclin then phosphorylate proteins crucial for cell cycle progression (10). All eukaryotes have a class of Ser/Thr protein kinases, known as CDKs that control progression through cell cycle checkpoints (7). CDKs contain a cyclin-binding domain, one or more phospho-regulatory sites in addition to an ATP binding site (11). Based on cyclin-binding domains, CDKs are clustered into seven classes (CDKA to CDKG) in addition to CDK-like kinases (CKLs) (8).
CDKAs constitute the biggest group of plant CDKs, distinguished with conserved PSTAIRE motif devoted for binding to cyclins (12). Through the constitutive expression of their coding genes (13), CDKAs are produced in plant cells to control plant growth through accelerating transition through different stages of cell cycle (14), phosphorylation of phosphatidic acid phosphohydrolase1 (15) and development of cytoskeleton and cell walls (16). Also, CDKAs contribute in gametes formation through a meiotic role recognized through preventing premature meiotic exit (17) and controlling chromosome axis assembly (18).
CDKA and its gene were characterized in rice (O. sativa Japonica group) for the first time by Hashimoto et al. (19). Thus, the present study aims at in silico characterizing of CDKA and its coding gene in cultivated and some wild Oryza species. Exon-intron structures of the retrieved genes were built up using coding and genomic sequences with the aid of Gene Structure Display Server website (http://gsds.cbi.pku.edu.cn/). The mined CDKA genes and the corresponding ones in the closest Gramineae species available in Gene Bank were aligned using Clustal W. The aligned sequences were employed to establish a phylogenetic tree based on Maximum Likelihood (ML) method in MEGA v. 6 (20) according to Hasegawa-Kishino-Yano model (21) with gamma distribution. 1000 replica-Bootstrap was utilized to judge significance of grouping patterns support (22).

Results and Discussion:
The  (Fig. 1). The same exon-intron structure for CDKA coding gene was observed in walnut hybrid (30) and Physcomitrella patens (31). On the other hand, Gao et al. (32) recorded 8 exons spaced with 7 introns studying the same gene in Arabidopsis thaliana and Gossypium hirsutum. Such species-dependent exon-intron arrangement for CDKA coding gene was also recorded upon studying a gene encoding class B of these kinases (5).  Oryza species upon employing different sequences including a supermatrix of more than 4600 nuclear gene (33), sequences of centromeres in addition to centromere-linked genes (34), CDKB1 coding gene (5) and PR-10 coding gene (6).
Supported with instability index value lower than 40 (Table 1), all the retrieved CDKAs exhibited in vitro stability (35). Subcellular location analysis reflected cytoplasmic and nuclear distribution of the predicted CDKA protein (Table  1) which is suitable for roles recorded to be played by such protein. In nucleus, CDKA is involved in DNA replication (36) and formation of synaptonemal complex at the beginning of meiosis (18). In cytoplasm, CDKA is associated with spindle (37, 38) and phragmoplast formation (39   Analyses of the amino acid sequences of CDKA mined from the studied Oryza genomes showed 291-292 amino acid length in all species except O. longistaminata that appeared as a shorter amino acid chain of 273 residues (Fig. 3). Working

threonine (T) and tyrosine (Y) residues, 2. PSTAIRE motif, 3. T-loop preceded with asparagine (D) and 4. SUC/CKS -binding motif
Regarding the functionally important binding sites, the retrieved CDKAs showed the same sites appeared in CDKA characterized in O. sativa Japonica Group by Hashimoto et al. (19) with few but important exceptions (Fig. 3). Except O. longistaminata, all CDKAs showed threonine (T) and tyrosine (Y) residues whose phosphorylation blocks enzymatic activity. Absence of this binding site in O. longistaminata indicates a new mechanism for negative regulation that may be beneficial for breeding of cultivated Oryza.
A second interesting variation in functionally important sites was also demonstrated in O. longistaminata where PSTAIRE motif, specialized for cyclin binding, was modified to PSTAICE. Docking with cyclin D reflected absolute energy score of 3276 to 3479 kcal/mol in species having PSTAIRE motif ( Table 2). Within this range, O. longistaminata having PSTAICE motif showed 3342 kcal/mol absolute energy score for the same docking process strongly highlighting insignificant effect for difference between the two motifs on binding to cyclin.
Though PSTAIRE was known to be evolutionarily conserved signature for CDKA, it was modified to PSTALRE in diatoms (41) and sea lettuce (42) that adds to the importance of our finding in O. longistaminata and necessitates wet lab-based future investigations to characterize this CDKA that may lead to a distinct subclass of these important kinases.
The third functionally important area was identical in all Oryza species of the present study; it consists of asparagine (D) and adjacent T-loop (Fig.  3). Asparagine is required for positioning of the bound ATP essential for kinase activity. The T-loop consists of 27 residue centered around threonine (T) whose phosphorylation stabilizes the cyclin-binding (43).
With one exception observed in O. brachyantha, SUC/CKS (suppressor of CDC2/cyclin dependent-kinase regulatory subunit)binding motif showed complete matching in all studied species. Three substitutions were recorded in where serine, isoleucine and threonine in the consensus sequence where replaced with arginine,  Lysine and alanine. The same substitutions were also recorded in Jerusalem artichoke (44), coconut palm (45), Dendrobium candidum (46) and Lolium temulentum (47).
Secondary structures (Fig. 4 and Table 2) of the retrieved CDKAs showed PSTAIRE motif in the first α-helix as described by Sorrell et al. (50). 3-D models (Fig. 5 and Table 4) supported with negative Z-score also showed the pattern described for such kinases consisting of joined couple of αhelices and β-strands (46).  In conclusion, in silico analyses provided a time and cost effective tool to highlight valuable genetic variations in wild relatives of rice. The unique CDKA predicted in O. longistaminata lacking the negative regulatory binding site observed in other species may be exploited to accelerate growth in cultivated species through traditional breeding or molecular approaches.
Similarly, polymorphism in SUC/CKS -binding motif recorded in O. brachyantha, can be employed in cultivated species to make benefit of such variation in manipulating cell division.