Maimoona Ayyaz Ansari
Nishtar Medical University Multan
Members of the Alfin-like (AL) transcription factor family, which includes a highly conserved DUF3594 domain and a PHD-finger motif, play critical roles in plant abiotic stress responses during a variety of metabolic and physiological processes. Plant homeo-domain (PHD) finger proteins have been linked to plant responses to salinity stress. There has been no systematic, comprehensive study of this family in Hordeum vulgare to date. This research has identified 8 putative Alfin like genes in H.vulgare. these genes are distributed among 5 chromosomes. Using Ensembl plant Database. The PHD family TFs’ amino acid sequence, phylogenetic tree, physical characterizations, and conserved motifs were predicted and analysed. The analysis of PHD family TFs in various species revealed that the number of PHD TFs in various species had a close relationship with plant evolution.
Throughout their lives, plants are subjected to stressful environmental conditions such as drought, high salinity, low temperature, and biotic stresses. These stresses wreak havoc on the environment, making it unfavorable for plant growth, development, and productivity (Urano, Kurihara, Seki, & Shinozaki, 2010). Plants modify or adjust a variety of metabolic processes related to genetics and physiology during the responses and adaptations that allow them to survive such stresses (Yamaguchi-Shinozaki & Shinozaki, 2006).
A variety of gene families act as transcription factors (TFs) and are related with functional regulation of plants from the metabolic and physiological characteristics to deal with abiotic stresses (Qu & Zhu, 2006). Transcription factors (TFs) perform their functions by binding directly to the promoters of target genes in a sequence-specific manner to either activate or repress the transcription of downstream target genes (Qu & Zhu, 2006). The activity of these transcription factors can maintain, enhance, or repress gene expression at the molecular level. The responsive control of these transcription factors aids higher plants in adapting to their environment, as well as other developmental and genetic responses (Carroll, 2005).
Members of the Alfin-like TF family are playing a significant character in root growth and expansion, root hair enlargement, meristem development, and salt stress resistance (Song et al., 2013). In Arabidopsis, Alfin-like 6 TFs regulate root hair elongation in phosphate-deficient conditions. (Chandrika, Sundaravelpandian, Yu, & Schmidt, 2013). The AL gene family was discovered in the alfalfa (Medicago sativa) plant as a 7S storage protein. The cDNA of the Alfin1 gene was isolated from a salt-tolerant alfalfa plant (J. E. Krochko, S. K. Pramanik, & J. D. Bewley, 1992). The AL TFs family contains genes that encode a DUF3594 domain with nearly 140 conserved amino acids in length and a PHD finger motif with nearly 50 conserved amino acids in length at the N and C termini (Kayum et al., 2016). The presence of the zinc-enriched PHD-finger motif in various plant species proposes that Alfin-like proteins may play a role in vital functional practices linked to plant system biology (Lee, Lee, Chung, & Kwon, 2009). The highly conserved DUF3594 domain is functionally unknown, and no proteins containing this domain have been identified in animals, fungi, or prokaryotes (Krochko & Bewley, 1988).
Hordeum vulgare is the world’s fourth most cultivated cereal crop, after rice, maize, and wheat, and its grains are primarily used for human, cattle, and beer brewing (Malcomber, Preston, Reinheimer, Kossuth, & Kellogg, 2006). The flowering process and spikelet formation significantly contribute to barley grain yield (Giraldo, Benavente, Manzano-Agugliaro, & Gimenez, 2019).
Several studies have been conducted on the functional studies of the Alfin like family, but not enough work has been done on in silico genome wide analysis of the Alfin like family in Hordeum vulgare. The published whole genome in barley allows us to understand the functions of Alfin like transcription factor proteins in this crop and study phylogenetic analysis using bioinformatics analysis. The recently released barley genome was used to identify 7 Alfin like genes in this study.
3. Materials and Methods:
3.1 Database search and sequence retrieval:
The accession number for Alfin like transcription factor family were retrieved from Arabidopsis information resource (TAIR) (https://www.arabidopsis.org/browse/genefamily/index.jsp) (Waziri, Singh, Sharma, Chatterjee, & Purty, 2020). The gene sequences of seven members of Alfin like transcription factor family were downloaded from NCBI using TAIR accession number. The protein sequences for Hordeum vulgare plant were obtained by performing protein blast of sequences of Arabidopsis thaliana sequences obtained from TAIR.
3.2 Identification of conserved motifs in protein domain:
To identify protein conserved motifs multiple sequence alignment is performed using Clustal W (https://www.ebi.ac.uk/Tools/msa/clustalo/) (Okonechnikov, Golosova, Fursov, & Bioinformatics, 2012) and MEGA7.0 software were used to show motifs and conserved regions among seven Alfin like proteins. Weblogo was used to represent the conserved motifs (https://weblogo.berkeley.edu/logo.cgi).
3.3 Phylogenetic analysis:
A Neighbor Joining method-based phylogenetic tree was constructed using the molecular evolution genetic analysis (MEGA7) to investigate the evolutionary relationships among the Alfin like genes. The Neighbor joining (NJ) strategy was used to generate the phylogenetic tree, which summarizes the evolutionary distances between seven members of the Alfin-like transcription factor family. The tree was depicted and presented in a circular format (Sukenik, Kaplan‐Levy, Viner‐Mozzini, Quesada, & Hadas, 2013).
3.4 Gene structure analysis:
TAIR (www.arabidopsis.org/) was used to extract and visualize exon, intron, and UTR organization for each Alfin-like gene. These gene features were then mapped using the GSDS gene structure display server (Waziri et al., 2020).
3.5 Promoter analysis:
Cisregulatory elements were searched from upstream region (1000-1100bp) of all characterized genes. The cisregulatory elements in the promoter region were analyzed by Plant Care website.
4.1 Identification of Alfin like transcription factor family members and their chromosomal location:
In this study, a total of 8 genes from Hordeum vulgare genome were identified as the members of Alfin like transcription factor family. The accession number, chromosomal location, genomic and peptide length and other related information of the identified genes is shown in Table 1. The chromosomal localization studies revealed the uneven distribution of these 8 candidates on all the 5 chromosomes of Hordeum vulgare. The first chromosome 1H contains 2 genes; 2H contains 2 genes while there is 1 gene on each 3H, 4H and 5H. Nucleotides length, exon count and chromosomal location is known for 7 genes coding different protein while for 1 protein KAE8821247 chromosomal location and its exon count is unknown.
4.2 Phylogenetic analysis and identification of conserved motifs:
The phylogenetic relationship of Alfin like transcriptional factor family was examined by multiple sequence alignment of their amino acid sequences using crustalW and a tree was generated using MEGA 7 software with Neighbour joining method (Tamura et al., 2013). The alignment showed that Alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, aspartic acid, histidine, lycine. Luciene, methionine are highly conserved Fig1. We identiﬁed 8 putative HvAL genes based on the presence of conserved DUF3594 domains and the PHD-ﬁnger
motif, which were designated HvAL1–18 according to their positions on chromosomes 1–5. The lengths of the HvAL proteins range from 125 to 271 amino acids (aa), except for KAE8821247, which is 692 aa in length. Multiple sequence alignments of these proteins clearly revealed the presence of a highly similar DUF3594 domain and PHD-ﬁnger motif at their N-termini and C-termini, respectively. The PHDﬁnger motif, like the C4HC3 zinc-ﬁnger motif, is approximately 50 aa in length. Three motifs were predicted as a part of PHD finger domain using MEME Suite (motif based sequence analysis tool) (http://meme.nbcr.net/meme/cgi-bin/meme.cgi). All of three were found to be highly conserved as shown in. Fig 1.2 showed 3 conserved motif among AL genes in H.vulgare.
4.3 Promoter Analysis:
Gene regulation and expression is controlled by regulatory elements present in the promoter sequence (Rushton et al., 2012). The regulatory elements are known as cis-acting regulatory DNA elements. We selected five elements and mapped them on 1 KB promoter sequence upstream to the start codon. These are CCAAT-Box, CGTCA-motif, LTR, ABRE, TGACG-motif.
4.4 Gene Structure analysis:
Gene structure analysis was done to obtain the ratio of intron exon structures in all Alfin like genes of Hordium Vulgare Based on these structures; it was observed that the average exon and intron number in the AL genes as shown in Fig 1.4.
Based on their conserved domains, TFs have been classified into numerous families; approximately 84 families have been discovered to date. The Alfin-like (AL) TF family, first discovered in alfalfa as a 7S storage protein, is involved in a number of biological processes, including root growth and development, root elongation, and meristem development. Members of the AL TF family are distinguished by the presence of two highly conserved domains, the DUF3594 domain and the PHD-finger motif, at their N-termini and C-termini, respectively (J. E. Krochko, S. K. Pramanik, & J. D. J. P. P. Bewley, 1992). Plant homeo-domain (PHD) finger proteins have been linked to plant responses to a variety of abiotic stresses. The PHD finger is a chromatin-interacting domain found in the nucleus of eukaryotic organisms that regulates transcription. A typical PHD is made up of 50–80 stabilized amino acid residues (Wang et al., 2015). A typical PHD is composed of 50–80 amino acid residues that are stabilised by two zinc ions as metal binding RING domains in a “cross-brace” topology and have a distinct Cys4-His-Cys3 pattern (Sanchez & Zhou, 2011). It is conserved and has been discovered in over 400 proteins from eukaryotes, including yeast. The PHD finger protein has a variety of roles in plants, including development and growth (Wilson, Morroll, Dawson, Swarup, & Tighe, 2001).
The PHD-finger motif not only promotes protein–protein interactions, but also cis-acting (C/A)CAC elements in target gene promoter regions, which are found in both the plant and animal kingdoms. The discovery that AL proteins in plants contain both conserved domains suggests that they play important biological roles in plants (Bastola, Pethe, & Winicov, 1998). Gene expansion, including tandem and segmental duplication, may be the source of gene duplication events from an evolutionary standpoint. One of the major driving forces in the evolution of gene families and genetic systems is thought to be gene duplication. The expansion of various maize gene families was primarily due to segmental duplication (Bancroft, 2001).
Furthermore, the majority of conserved motifs were found in all subfamilies, while some distinct motifs were found in individual subfamilies. The functions of these distinct motifs are unknown at this time, but they may have assisted plants in adapting their various regulatory processes to environmental changes during the evolution of the maize genome (Cannon, Mitra, Baumgarten, Young, & May, 2004). According to the prompter cis-element analysis, all HvAL, TFs have stress responsive element and abscisic acid responsive element (Table 1.2). However, the other genes are not clearly responsive to this stress, which could be due to a lack of a stress-related cis-element in the promoter region (Benfey & Weigel, 2001).
In conclusion, the AL family is involved in a variety of developmental and physiological processes in plants. Few plants AL families have been identified at the genome-wide level to date.
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7. Tables and Figures:
Table1.1 Alfin like transcription factor genes of Hordeum vulgare, details e.g. chromosome number, number of exons in each gene, protein length, start and ending position of genes are indicated in the table.
|Accession number||Gene symbol||Protein name||Chromosome #||Exon count||Protein length||Start & End Position of Each Gene on genome|
Fig1.1 Multiple sequence alignments of all Alﬁn-like proteins of Hordeum Vulgare . The conserved DUF3594 and PHD domains are labeled. The C4HC3 zinc ﬁnger motifs are indicated by asterisks.
PHD Zinc finger Domain
Fig1.2 Phylogenetic tree of full length AL genes from Hordium vulgare.
Fig1.3 Conserved motif designed using Web logo server
Table 1.2 Regulatory elements and their functions in Hordeum vulgare
|Regulatory element||Core sequence||HvAL1||HvAL2.1||HvAL2.2||HvAL4||HvAL6||HvAL7.1||HvAL7.2||Functions|
|CCAAT-box||CAACGG||1||1||1||MYBHv1 binding site.|
|CGTCA-motif||CGTCA||4||3||3||3||3||2||2||cis-acting regulatory element involved in the MeJA-responsiveness.|
|LTR||CCGAAA||3||2||2||1||2||cis-acting element involved in low-temperature responsiveness.|
|TGACG-motif||TGACG-motif||4||3||3||3||3||2||2||cis-acting regulatory element involved in the MeJA-responsiveness.|
|ABRE||CGCACGTGTC||1||1||cis-acting element involved in the abscisic acid responsiveness.|
Fig 1.4 Conserved motif in AL genes of Hordium vulgare generated using MEME suite software. Different Colorful blocks shows 3 conserved motifs.