Genome wide identification of LcC2DPs gene family in Lotus corniculatus provides insights into regulatory network in response to abiotic stresses

Abstract

Low temperatures and drought reduce forage yield and quality, with protein kinases crucial for plant stress response. This study examines the LcC2DPs protein kinase family in Lotus corniculatus, identifying 90 members, with some tandemly distributed on chromosomes 2-6, and grouped into 5 subfamilies (I-V). 34 homologous gene pairs were found in Arabidopsis thaliana. LcC2DP genes promoters contain hormone and stress response elements. GO analysis highlights enrichment in hormone response and kinase activity. Transcriptomic analysis links 78 genes to environmental response and stress growth, with 10 validated by qRT-PCR after treatment with 100 μM ABA and IAA, 20% PEG6000, and 4 °C. Protein interaction analysis identifies 5 core proteins (LcC2DP5, 11, 15, 38, and 58) activated by drought and cold stress. Gene analysis revealed that only LcC2DP5 and LcC2DP15 share co-expression transcription factors, with bZIP, bHLH, WRKY, NAC, MYB-related, MYB, C3H, and C2H2 being prominent. These proteins are expressed under drought and cold conditions, highlighting LcC2DP5 and LcC2DP15 activity. NAC and C2H2 are vital for drought response, while bZIP and MYB-related are important for cold response. This suggests that various LcC2DPs in Lotus corniculatus respond to hormones and stress via a TF regulatory network.

Keywords: Lotus corniculatus; C2 domain; Calcium-dependent protein kinase; Cold stress; Drought response; Signal transduction.

Fig. 1

Phylogenetic relationship, gene structure, and motif composition of LcC2DPs. A Rootless phylogenetic tree from MEGA7.0. B Motifs 1–10 shown in different colors. C Illustration of the C2 domain. D Exon–intron structure: green for UTR, yellow for CDS, black for intron.

Fig. 2

Chromosomal localization, phylogenetic, and collinearity analysis of LcC2DPs family. A Chromosomal positions with length scale. The left axis indicates the length of each chromosome, measured in megabases (Mb). B Phylogenetic tree with colored groups using Neighbor-joining (NJ) method and 1000 Bootstrap replications. The color-coded groups represent distinct subfamilies (I-V), with each color indicating a different subfamily. C Intra-specific collinearity with red lines for duplicated genes; colored rectangles for chromosomes; blue circle for gene density. D Inter-specific collinearity analysis of C2DPs between Lotus corniculatus and Arabidopsis thaliana: Chr1-5 for Arabidopsis, chr1-6 for Lotus, red lines for duplicated LcC2DPs and AtC2DPs.

Fig. 3

Cis-regulatory element analysis of LcC2DPs promoter regions. The promoter region is situated upstream of the gene, spanning from -2000 to -1 base pairs, different colors represent distinct promoter elements.

Fig. 4

Functional annotation and expression analysis of the LcC2DPs gene family. A GO enrichment analysis, where the size and color of dots correspond to the number of genes and the q-value, respectively. B KEGG pathway enrichment analysis, with dot size and color indicating gene count and statistical significance (q-value). C Heatmap depicting the temporal expression patterns of LcC2DPs under submergence stress.

Fig. 5

LcC2DPs gene expression profiles. A Expression in roots, stems, and leaves. B Lotus phenotype after 100 uM ABA and IAA treatment. C qRT-PCR expression levels post hormone treatment. Data are mean ± SD (n = 3). Significance assessed by t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 6

Plant phenotypes and LcC2DPs expression with 20% PEG and 4 °C treatments. A Phenotype comparison. B Expression under drought. C Expression in low temperatures. Data are mean ± SD (n = 3). Significance assessed by t-test: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 7

Analysis of protein–protein interactions, core gene expression, and co-expression. A Protein interactions. Expression of LcC2DP5, 11, 15, 38, and 58 under drought (B) and low temperature (C). D Co-expression of LcC2DP5 and LcC2DP15, with solid lines for positive and dashed for negative correlations.

Fig. 8

TF expression via qRT-PCR under drought (A) and low temperature (B), with significance assessed by T- test. Data are mean ± SD (n = 3). Significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 9

Transcriptional regulatory network of LcC2DP5 and LcC2DP15 under drought and low temperature. ¹When drought and low-temperature signals are detected by membrane receptors, calcium ions are released into the cytoplasm, activating protein kinases LcC2DP5 and LcC2DP15. This triggers phosphorylation cascades that activate transcription factors like bZIP, bHLH, WRKY, NAC, MYB-related, MYB, C3H, and C2H2, leading to the transcription of stress-related genes and enhancing plant resilience. ²NAC, C2H2, bHLH, MYB, bZIP, and MYB-related TFs move to the nucleus after activation, while WRKY and C3H are activated in the nucleus. ³NAC and C2H2 are key in LcC2DP5 and LcC2DP15 pathways during drought, whereas bHLH and MYB-related are crucial in low temperature.