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. 2024 Feb 26:15:1352040.
doi: 10.3389/fpls.2024.1352040. eCollection 2024.

"Genome-wide identification of bZIP gene family in Pearl millet and transcriptional profiling under abiotic stress, phytohormonal treatments; and functional characterization of PgbZIP9"

Deepak Kumar Jha #  1   2 , Jeky Chanwala #  1   2 , Preeti Barla  1 , Nrisingha Dey  1
Affiliations

"Genome-wide identification of bZIP gene family in Pearl millet and transcriptional profiling under abiotic stress, phytohormonal treatments; and functional characterization of PgbZIP9"

Deepak Kumar Jha et al. Front Plant Sci. .

Abstract

Abiotic stresses are major constraints in crop production, and are accountable for more than half of the total crop loss. Plants overcome these environmental stresses using coordinated activities of transcription factors and phytohormones. Pearl millet an important C4 cereal plant having high nutritional value and climate resilient features is grown in marginal lands of Africa and South-East Asia including India. Among several transcription factors, the basic leucine zipper (bZIP) is an important TF family associated with diverse biological functions in plants. In this study, we have identified 98 bZIP family members (PgbZIP) in pearl millet. Phylogenetic analysis divided these PgbZIP genes into twelve groups (A-I, S, U and X). Motif analysis has shown that all the PgbZIP proteins possess conserved bZIP domains and the exon-intron organization revealed conserved structural features among the identified genes. Cis-element analysis, RNA-seq data analysis, and real-time expression analysis of PgbZIP genes suggested the potential role of selected PgbZIP genes in growth/development and abiotic stress responses in pearl millet. Expression profiling of selected PgbZIPs under various phytohormones (ABA, SA and MeJA) treatment showed differential expression patterns of PgbZIP genes. Further, PgbZIP9, a homolog of AtABI5 was found to localize in the nucleus and modulate gene expression in pearl millet under stresses. Our present findings provide a better understanding of bZIP genes in pearl millet and lay a good foundation for the further functional characterization of multi-stress tolerant PgbZIP genes, which could become efficient tools for crop improvement.

Keywords: ABI5 (ABA insensitive 5); abiotic stress; bZIP transcription factors; pearl millet; plant genomics.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Location of bZIP family members on 7 chromosomes of pearl millet. The identified bZIP members were distributed on all the 7 chromosomes of pearl millet.
Figure 2
Figure 2
Phylogenetic tree of P. glaucum bZIP proteins with bZIP proteins of A. thaliana, O. sativa, and S. italica. A total of 356 proteins were aligned by MUSCLE, and a phylogenetic tree was constructed by MEGA v7.0 using the maximum likelihood method with 1000 bootstrap replication. The phylogeny classified the identified bZIP members into 12 groups.
Figure 3
Figure 3
(A) Schematic representation of conserved motifs in PgbZIP proteins. Motifs are indicated by different color boxes for motifs 1–20, (B) Gene structure of the 98 putative pearl millet bZIP genes. Yellow boxes indicate exons, black lines indicate introns and blue boxes indicate upstream/downstream. Motif distribution and exon-intron arrangement was conserved among the groups.
Figure 4
Figure 4
Synteny relationship between P. glaucum, A. thaliana, O. sativa, and S. italica bZIP genes. Each connected colored line shows duplicated gene pairs among the species. Closest relationship was observed between bZIP members of P. glaucum and S. italica.
Figure 5
Figure 5
Expression analysis of PgbZIP genes under Drought stress at day 0, 5, 7, 9 and 11 post-treatment. Nearly All the candidate PgbZIP genes were upregulated under drought stress. The X-axis represents different time points and the Y-axis indicates relative expression level. A significant difference in the mean is indicated by *P < 0.05, as obtained by Student’s t-test.
Figure 6
Figure 6
Expression analysis of PgbZIP genes under Heat stress at 0h, 3h and 24h post-treatment. Majority of PgbZIP genes were upregulated on heat application. The X-axis represents different time points and the Y-axis indicates relative expression level. The significant difference in the mean is indicated by *P < 0.05, as obtained by Student’s t-test.
Figure 7
Figure 7
Expression analysis of PgbZIP genes under Salt stress at day 0h, 12h and 24h post-treatment. Majority of the PgbZIP genes were upregulated. The X-axis represents different time points and the Y-axis indicates relative expression level. A significant difference in the mean is indicated by *P < 0.05, as obtained by Student’s t-test.
Figure 8
Figure 8
Heat map showing expression analysis of PgbZIP genes under SA, MeJA, and ABA hormonal stress at 0-hour, 3-hour, and 24-hour time points. The mRNA level has been normalized and the heat map was generated using TBtools v0.66831. The scale bar indicates lower (Red) to higher (Green) expressions level. The differential expression pattern was obtained for selected PgbZIP genes under all the phytohormonal treatments.
Figure 9
Figure 9
(A) Protein sequence alignment of PgbZIP9 protein along with bZIP proteins of other species confirmed the presence of bZIP domain, (B) Phylogenetic analysis of PgbZIP9 showed its closeness to AtABI5, (C) Exon-intron organization done with GSDS server, (D) bZIP domain confirmation with SMART server, (E) PgbZIP9 Structure prediction through Alpha fold server showed disordered N-terminal and C-terminal regions.
Figure 10
Figure 10
Subcellular localization and transactivation activity analysis of PgbZIP9. (A) Schematic representation of 35S:PgbZIP9-GFP construct. (B) Confocal image of subcellular location of PgbZIP9 gene along with vector control. GFP, Green fluorescence; DAPI, 4’,6-diamidino-2-phenylindole; Merged, Merged image of GFP; DAPI and Bright field (BF). PgbZIP9 was a nuclear protein. (C) Transcriptional activation assay of PgbZIP9 in a yeast expression system. No transcriptional activity was observed in yeast.
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