Expression pattern analysis of heat shock transcription factors (HSFs) gene family in Aeluropus littoralis under salinity stress

Heat shock proteins (HSPs) are one of the main stress-responsive genes in plants that highly express in response to variable environmental stresses. Expression of HSP genes is primarily regulated by attaching heat shock transcription factors (HSFs) to binding sites at their promoter region. HSFs and their coding genes have been widely characterized in several plant species, but so far no report is available on the structure, organization, phylogenetic relationships and expression profile of these genes in halophyte species. Identification and characterization of gene family members properties in stress tolerant plant can be useful to understanding their molecular function and biological processes. The present study aimed to identify the HSF transcription factor gene families in halophyte plant Aeluropus littoralis.

The protein sequences of Arabidopsis thaliana HSF gene families were blasted against Aeluropus littoralis genomic sequences by local TblastN tools. After removing repetitive sequences, all sequences were verified by BlastP. In order to identification, annotation and analysis of domain architectures, the identified proteins were analyzed in different protein domain databases including Pfam 32.0, PROSITE and InterProScan. Similarity clustering based on motifs patterns were done by SALAD tool in all AlHSFs. The structure of exon and intron was generated by comparing the predicted CDS against genomic sequences of AlHSFs in gene structure display server (GSDS). The expression pattern analysis of AlHSFs genes was carried in leaf and root tissues under salinity stress and recovery conditions by transcriptome analysis.

In total, 11 non-redundant HSF genes encoding HSF domain-containing proteins were identified in A. littoralis genomes. Aeluropus HSF genes were named based on their identity to Arabidopsis AtHSF homologous proteins. All 11 AlHSFs were divided into three classes (A, B, and C), based on homology with Arabidopsis. Seven genes belonging to the HSFA class and three genes belonging to the HSFB class, and finally HSFC class like Arabidopsis had one gene. The gene structure analysis showed that AlHSF gene family member had distinct gene features, such as the composition and position of exons, introns, and conserved elements. The most AlHSF genes had two exons and one intron, while two genes (AlHSFA6B.1 and AlHSFA1B) contained three exons and two introns. AlHSFA6B.3 had six exons and five introns while AlHSFA6B.2 with one exon was intronless. Based on SALAd tool, 13 conserved motifs were identified from 11 AlHSFs. Motifs of 1, 2, 3, 4 and 5 were presented in most AlHSF proteins, except for AlHSFA6B.2 which lacked the motif 4 and 5. Motif 2 was also absent only in AlHSFA6B.2 and AlHSFB4. Removal of these motifs may have occurred during gene duplication in the evolutionary process of this family, resulting in shorter coding regions. Also, the results revealed that some motifs were present in specific AlHSF proteins. For example, motif 10 only existed in AlHSFA6B.1 and AlHSFB1. Motif 12 was only present in AlHSFA6B.1 and AlHSFA1B. The phylogenetic tree divided the proteins into three groups based on the existence and distribution of different motifs. The expression pattern of AlHSFs genes in leaf and root tissues under salinity and recovery conditions showed that AlHSFA6B.3 gene was not expressed, indicating that this gene was silent in these tissues under corresponding stress. AlHSFB2A gene had the highest expression level (1.11) in leaf tissue under salinity stress. After that, AlHSFA6B.1 gene with expression levels of 0.96 and 0.87 was expressed more in leaf tissue under salinity stress and recovery, respectively. The least expression level was observed in AlHSFC1 and AlHSFA3 genes, respectively, which was four times less than the control. AlHSFC1 gene showed a significant expression decrease in the recovery conditions after a little expression increase in leaf tissue under salinity stress. Significant expression decrease of AlHSFA3 gene was observed in root tissue under salinity stress which indicates its role as a negative regulator in response to salt stress.

Different expression patterns of AlHSF family member suggest distinct modes of positive and negative gene expression regulation. These may also be related to their different molecular functions as well as diverse regulatory mechanisms involved in controlling the activation of these genes. The findings of this study reveal the functional characteristics of the AlHSF genes and provide a foundation for future functional research regarding their biological roles in plant tolerance to stress.