The Molecular Mass and Isoelectric Point of Cytokinin Riboside 5'-Monophosphate Phosphoribohydrolase Sequences for Wheat Genome

1. Introduction

As one of most important plant hormone group, cytokinin is play strongly role and implicated in plant breeding of wheat in terms of flowering and yield [1,2] and including other role such as environmental stress (biotic and abiotic stresses) [3]. The metabolic cycling of cytokinin involves initial by isopentenyl transferase enzymes in order to formation of cytokinin nucleotides and thus activation into active free-base forms by lonely guy enzymes (LOG) [4]. The pathway of signaling cytokinins have multi-step systems of phosphorylation by involving histidine kinases, histidine phosphotransfer proteins, and regulators response [5]. In wheat, cytokinins occur during grain development especially in rapid endosperm nuclear divisions and can help to delaying senescence by stimulating sucrose by stay-green phonotypes in late stage of development. In addition, cytokinin can manipulate amino acid metabolism by distribution of nutrient inside plan and enhance the expression of transported and mobilization [6].

A common wheat (Triticum aestivum) is allohexaploid that has three genomes, which has been driven from ancestral species (AABBDD, A genome from Triticum Urartu, B genome from Aegilops speltoides, and D genome: from Aegilops tauschii). This genome reflects complex instruction of genetic wheat, which has six sets of chromosomes as hexa-ploid [7]. The benefit of this complexity of wheat genome leads to genetic diversity and adaptability to grown and survive in a variety of environmental conditions. However, wheat under drought stress has been classified based on its ability by different mechanisms such as mange water or reduced transpiration, or improved root depth [6]. Cytokine influence stomatal closure to reduce water loss and can develop and elongation of root growth to access water deeper from soil. Under water limitation, wheat species were significantly noted by reduction in traits such as photosynthesis rate (14% and 10% compared to 24% and 12%), stomatal conduction, total sugar, and total starch content [1,2,3,4,5,6,7,8]. The metabolism of cytokinin in wheat regulates by active cytokinin level by dephoshorylation and deribosylation during development processes. This processes included shoot apical meristem, flower, and vascular developments, and retaining sink activity to inhibit nitrogen remobilization, and maintaining proteins synthesis [9]. The objective of study wheat’s cytokinin expression through amine acid is crucial for development wheat stage. The aims here was to explain the wheat genomes (mRNA and proteins) for its properties and function from two different databases through the cytokinin riboside 5'-monophosphate phosphoribohydrolase sequences using relative synonymous codon usage (RSCU), molecular weight (g/mol), theoretical isoelectric point, instability index, aliphatic index, and hydrophobicity. The future objective from these results would be benefit for in nutrition and industrial applications such as digestibility and nutritional supplements that can aid in breeding programs [10].

2. Results

2.2. The Number of Chromosomes for Cytokinin Riboside 5'-Monophosphate Phosphoribohydrolase Genes Through NCBI and Ensemble Databases

NCBI had 35 genes in different chromosomes while ensemble had 38 genes and both databases had able to show these genes in different chromosomes. Figure 1 shows that A, B, and D genomes with different numbers of chromosomes in each one. 2A, 2B, and 2D was zero number from NCBI databases comparing with ensemble databases that shows 1 genes in both genomes 2A and 2D and 2 genes in 2B genome. Some genomes had more genes than other genes. For example, genome 4A and 5B had five genes from NCBI databases while 4 genes shown in genomes 1B, 1D, 4D, and 5D from ensemble databases. This gene differences came from key factors such as accumulation over times of genetic differences, repeated sequences due to transposable elements (TEs) that could insert into different locations, gene loss as shown in several reports by 10 to 16 thousand genes losses, and hybridization of wheat from breeding programs. In addition, the Cytokinin riboside 5'-monophosphate phosphoribohydrolase has multiple members from 1 to 11 reflect the functional redundancy for cytokinin metabolism roles across developmental stages. Gene expression of cytokinin riboside 5'-monophosphate phosphoribohydrolase has different elements of cis-regulatory.

2.3. The Relative Synonymous Codon Usage (RSCU)

The results of RSCU values (data not shown due to large table) above 1.6 reflect significant overrepresentation such as phenylalanine (TTC) located in 5B chromosomes, Leucine (TTA) located in 5A, and 1D chromosomes, leucine (TTG) located in 5B chromosome, leucine (CTC) located in 5D, 7D, and 7B. Leucine (CTA) located in 5D. Isoleucine (ATT) located in 5A, and 1D. Valine (GTC) located in 5D, and 4A. Serine (TCT) located in 5D, 1B, and 4A. Proline (CCT) located in 4B. Threonine (ACG) located in 4D, 5B, 5A, and 4B. Arginine (CGC) located in 5B, 5D, and 1B. Glycine (GGC) 5D, 4A, 5B, 3D, 7B, and 5D. However, RSCU values below 0.6 suggest underrepresentation such as leucine (CTA) located in 3A, 5D, 1B, 5B, and 7B. Valine (GTT) located in 4D, 5B, 4A, and 5B. Proline (CCC) located in 5B, 5D, 5A, 4B, 1B, and 4A. Threonine (ACC) located in 3A, 4D, 5B, 5A, and 4B. Glutamic Acid (GAA) located in 5B, 5D, 5A, 4A, 1D, 5B, and 7B. Arginine (CGT) located in 3A, 5B, 1B, 5B, and 7D. Serine (AGT) located in 4D, 5B, 5A, 4D, 4B, and 7B. Glycine (GGT) located in 3A, 5B, 5D, 7D, and 7B. Figure 2 is data of principal component analysis that shown amino acid patterns. From this Figure 2, amino acids that explained main of variation is assigned to PCA1 with total variation about 72.11% and these amino acids are Isoleucine, Leucine, Lysine, Aspartic Acid, and Serine and more shown in Figure 2. While the PCA2 and PCA3 had total variation, 17.04% and 10.85 respectively assigned with arginine, glutamic acid and threonine for PCA2 and assigned with aspartic Acid.

The physiochemical characterization:

Table 1 shows the results values of molecular weight (g/mol), theoretical isoelectric point, instability index, aliphatic index, and hydrophobicity. Theoretical isoelectric point (PI) is the pH that ranged from 4.81 to 6.6 in chromosomes 3A and 4D respectively (average 5.37 ± 0.297 Figure 3). However, the instability index showed that these two chromosomes (3A and 4D) were at 34.3 and 38.16 respectively. In addition, these two chromosomes (3A, 4D) had 88.69 and 89.59 of aliphatic index indicate increasing of their stability and their hydrophobicity were lower 0.04 and -0.04 respectively. The high instability was found at 1D and 5D with 54.16 and 50.36 respectively with decreasing in their stability as shown in aliphatic index at 92.98 and 88.91. Figure 4 is data of principal component analysis that shown amino acid patterns. From this Figure 4, amino acids that explained main of variation is assigned to PCA1 with total variation about 58.88 % and these chromosomes are 5D, 4D, 1A, 4B, 3D and more shown in Figure 4. While the PCA2 and PCA3 had total variation, 27.52% and 13.6% respectively assigned with 1D, 3B, 3A, 2D, 2A and 2B.

3. Discussion

These cytokinins are important phytohormones encoded by LOG family of genes in wheat and other crops. It is play roles in several stage of plant development such as regulating growth and affecting of yield component of wheat [11]. Its biosynthesis occurs in the plant cell and. It also showed about 11 to 14 genes family allocated in three chromosomes of wheat. In barely, report showed that the pattern of expression of these genes reflects role in growth and reproductive development. In addition, the results showed the possibility of selection natural variation in gene expression for incorporation into wheat genotypes breeding material. In Arabidopsis, this cytokinin acted at specific level with redundantly at vascular tissues of developing flowers and leaves [12,13].

The important of the relative synonymous codon usage (RSCU) in plant breeding is to help breeders for understanding the mechanisms and functional wheat genomes and thus wheat genomes can be build for environmental adaptations. Selection optimal codon helps for transcription termination of mRNA expression especially in binding sites by either generating or depleting this site of gene expression. RSCU values above 1.6 is strong positive codon in several codons such as TTC, TTA, TTG, CTC, CTA, ATT, and GTC these codons are hold T codon. In addition, codons TCT, CCT, ACG have C codon while codons CGC and GGC have G codon. Thus, these codons can be used for optimizing gene expressions in order to modifying endogenous of some genes to increase protein productions [14]. The important of principal component analysis here is to identify important amino acid for the Cytokinin riboside 5'-monophosphate phosphoribohydrolase to make best decision about selection in plant breeding programs. From PCA1 that explain total variation as largest loading has highlights most important amino acids that are associated to variability of this enzyme and thus more attention in breeding program to these amino acids [15,16]. Thus, the beneficial of these results would be developed wheat in nutrition and industrial applications such as increasing essential amino acids that related to improve proteins quality for valuable nutrition and to address starch for better wheat digestible and thus enhance human consumption [17].

4. Materials and Methods

The DNA and proteins sequences of cytokinin riboside 5'-monophosphate phosphori-bohydrolase have been retrieved from two different databases names the National Center for Biotechnology Information (NCBI) and ensemble plants websites as shown in Table 2. All of the studied sequences were annotated such as genes, regulatory elements and location of genes. From NCBI, the relative synonymous codon usage (RSCU) were calculated individually as the ration between the observed frequency and expected frequency for the particular codon by using website such as https://jamiemcgowan.ie/bioinf/index.html#. Some of codons were excluded from the calculation such as methionine (ATG), and tryptophas (TGG), and (TAA, TAG, TGA). The formula of calculation RSCU for codon j of amino acid I is by using formula as below: RSCU= ( n_(i x_(i,j ) ))/(∑_(j=1)^(n_i)▒〖xi,j〗) where n_i is the number of codon that code amino acid i, and the x_(i,j )is the number of occurrences of codon j [18]. The interpretation for RSCU values equals to 1 indicated no codon preferences (no bias) while values less than 1 indicated notable negative bias in codon usage. Values above 1.6 reflects significant overrepresentation while values below 0.6 suggest underrepresentation. Once, the RSCU values were determined, all values were analysis through SAS for more analysis by using principal component.

The physiochemical characterization

From ensemble plants were used to analysis some of chromosomes characterization such as molecular weight (g/mol), theoretical isoelectric point, instability index, aliphatic index, and hydrophobicity. These characterizations were contacted through web.expasy.org. The theoretical isoelectric point (PI) was the pH value that can be calculated through the formula pI = (pKa1 + pKa2) / 2 where pKa1 refers to dissociation constant for group of carboxylic acid and pKa2 refers to amino group. Both molecular weight and isoelectric point help to understand of biochemical and functional chromosomes of wheat breeding programs. The theoretical isoelectric point helps to know the stability of protein charge. The instability index refers to stability of protein if the values less than 40 reflects stable proteins while above 40 showed instability protein. This instability index can be calculated through the sequences length and the weighted sum of dipeptides. The aliphatic index refers to relative volume through using mole percent of amino acid (alanine, valine, isoleucine, and leucine). Lastly, the hydrophobicity refers to tendency of amino acid [19].

Principal component analysis

Principal component analysis of the RSCU and physiochemical characterization values and the physiochemical characterization were used through SAS software version 9.4 by using excel file format. The PCA plot can help to analyze values for identify patterns in amino acid or codons that were favored or disfavored in specific or major trends with reducing complexity of datasets. In general, PCA were analytical method for enhances our understanding of protein characteristics and for gene expression and evolutionary biology.

Contribution statement: Meshal M. Almutairi: Conceptualization, Writing-original draft, Data curation, Formal analysis, methodology. ???Conceptualization, writing editing, Data curation.