Genome-wide identification and expression analysis of WRKY transcription factors in pearl millet (Pennisetum glaucum) under dehydration and salinity stress

Abstract

Background: Plants have developed various sophisticated mechanisms to cope up with climate extremes and different stress conditions, especially by involving specific transcription factors (TFs). The members of the WRKY TF family are well known for their role in plant development, phytohormone signaling and developing resistance against biotic or abiotic stresses. In this study, we performed a genome-wide screening to identify and analyze the WRKY TFs in pearl millet (Pennisetum glaucum; PgWRKY), which is one of the most widely grown cereal crops in the semi-arid regions.

Results: A total number of 97 putative PgWRKY proteins were identified and classified into three major Groups (I-III) based on the presence of WRKY DNA binding domain and zinc-finger motif structures. Members of Group II have been further subdivided into five subgroups (IIa-IIe) based on the phylogenetic analysis. In-silico analysis of PgWRKYs revealed the presence of various cis-regulatory elements in their promoter region like ABRE, DRE, ERE, EIRE, Dof, AUXRR, G-box, etc., suggesting their probable involvement in growth, development and stress responses of pearl millet. Chromosomal mapping evidenced uneven distribution of identified 97 PgWRKY genes across all the seven chromosomes of pearl millet. Synteny analysis of PgWRKYs established their orthologous and paralogous relationship among the WRKY gene family of Arabidopsis thaliana, Oryza sativa and Setaria italica. Gene ontology (GO) annotation functionally categorized these PgWRKYs under cellular components, molecular functions and biological processes. Further, the differential expression pattern of PgWRKYs was noticed in different tissues (leaf, stem, root) and under both drought and salt stress conditions. The expression pattern of PgWRKY33, PgWRKY62 and PgWRKY65 indicates their probable involvement in both dehydration and salinity stress responses in pearl millet.

Conclusion: Functional characterization of identified PgWRKYs can be useful in delineating their role behind the natural stress tolerance of pearl millet against harsh environmental conditions. Further, these PgWRKYs can be employed in genome editing for millet crop improvement.

Keywords: Abiotic stress; Cis-regulatory elements; Pearl millet; Synteny; WRKY transcription factors.

Fig. 1

Multiple sequence alignment of identified PgWRKY proteins. The amino acid conservation is shown in shaded colours, while domain conservation is shown through underline colours. The shaded colours indicate low to high residue conservation i.e., blue to red. The domain conservation for WRKY, C-C and H-H/C domains are shown through underline red, blue and green colour respectively

Fig. 2

The circular phylogenetic representation of P. glaucum WRKY proteins with A. thaliana, O. sativa & S. italica: A total of 379 WRKY proteins were aligned by MUSCLE, and a phylogenetic tree was constructed by MEGA v7.0 using maximum likelihood method with 1000 bootstrap replication. Each colour indicates an individual group (I-III) of ancestral relationship

Fig. 3

The chromosomal distribution and positioning of PgWRKYs across all seven chromosomes of P. glaucum. Seven chromosomes with varying lengths are shown in Mb (million base pair) scale in the left, where individual chromosomes (bars) are labelled with respective PgWRKY genes

Fig. 4

Structural elucidation of identified 97 PgWRKY genes: The structural features of PgWRKYs are represented in different colours, where yellow indicates an exonic region, blue indicates upstream/downstream region, black indicates intronic region and pink indicates no sequence information

Fig. 5

The schematic representation of motif analysis: The upper panel indicates predicted motifs in PgWRKYs, represented in different colour using MEME suite v5.1.0. Whereas, the lower panel shows the signature of each motif with conserved amino acid residues

Fig. 6

The Circos plot representation for synteny relationship between P. glaucum, A. thaliana, O. sativa and S. italica WRKY genes: The synteny analysis of PgWRKYs were mapped in a circular form using CIRCOS v0.52. Each coloured curve indicates the orthologous positions of PgWRKYs across the chromosomes

Fig. 7

Gene ontology analysis of identified PgWRKYs: The enrichment analysis of PgWRKY shows significantly enriched GO terms involved in cellular, molecular and biological processes

Fig. 8

In-silico analysis of Cis-regulatory elements: The frequency of identified putative cis-acting elements in the 1.5 kb upstream region of PgWRKY genes. The letter on each bar indicates the class (shown in legend) it belongs

Fig. 9

Tissue specific expression analysis. Heat map showing the differential expression level of selected PgWRKYs in leaves, stem and root tissues of pearl millet. The transcript abundance level has been normalized and hierarchically clustered. Block with colours indicate decreased (green) or increased (red) transcript accumulation among the analyzed tissues. The heat map was generated using TBtools v0.66831 [37]

Fig. 10

Expression analysis of PgWRKYs under different abiotic stress conditions determined by qRT-PCR. The Y-axis indicates relative expression level and the X-axis represents different time points of stress treatment taken for expression analysis. a). Differential expression level of PgWRKYs in response to drought stress at 0th day, 4th day, 6th day and 8th day time points. b). Expression pattern of PgWRKYs under salt stress at 0-h, 3-h and 24-h time points. Each data point represents mean ± standard deviation (SD) (n = 3). Significant difference in mean between the samples for a given set is indicated by *P < 0.05, **P < 0.01, as obtained by Student’s t-test