Systematic analysis and expression of Gossypium 2ODD superfamily highlight the roles of GhLDOXs responding to alkali and other abiotic stress in cotton

Abstract

Background: 2-oxoglutarate-dependent dioxygenase (2ODD) is the second largest family of oxidases involved in various oxygenation/hydroxylation reactions in plants. Many members in the family regulate gene transcription, nucleic acid modification/repair and secondary metabolic synthesis. The 2ODD family genes also function in the formation of abundant flavonoids during anthocyanin synthesis, thereby modulating plant development and response to diverse stresses.

Results: Totally, 379, 336, 205, and 204 2ODD genes were identified in G. barbadense (Gb), G. hirsutum (Gh), G. arboreum (Ga), and G. raimondii (Gb), respectively. The 336 2ODDs in G. hirsutum were divided into 15 subfamilies according to their putative functions. The structural features and functions of the 2ODD members in the same subfamily were similar and evolutionarily conserved. Tandem duplications and segmental duplications served essential roles in the large-scale expansion of the cotton 2ODD family. Ka/Ks values for most of the gene pairs were less than 1, indicating that 2ODD genes undergo strong purifying selection during evolution. Gh2ODDs might act in cotton responses to different abiotic stresses. GhLDOX3 and GhLDOX7, two members of the GhLDOX subfamily from Gh2ODDs, were significantly down-regulated in transcription under alkaline stress. Moreover, the expression of GhLDOX3 in leaves was significantly higher than that in other tissues. These results will provide valuable information for further understanding the evolution mechanisms and functions of the cotton 2ODD genes in the future.

Conclusions: Genome-wide identification, structure, and evolution and expression analysis of 2ODD genes in Gossypium were carried out. The 2ODDs were highly conserved during evolutionary. Most Gh2ODDs were involved in the regulation of cotton responses to multiple abiotic stresses including salt, drought, hot, cold and alkali.

Keywords: Cotton; Gene family; Leucocyanidin dioxygenase; Phylogenetic analysis; Structural analysis.

Fig. 1

Two rootless phylogenetic trees for 2ODDs. A A phylogenetic tree of 336 2ODDs in G. hirsutum by Neighbor-Joining (NJ) method. B A phylogenetic tree for the 1124 identified 2ODDs from four Gossypium species by Maximum Likelihood (ML) method. Boxes with varying color represent different clades of 2ODDs

Fig. 2

Chromosomal positions of 2ODDs from four Gossypium species. Gene IDs are shown on the right side. The vertical bar on the left side represents the position of the genes and length of chromosomes. Black lines indicate tandem duplication gene pairs

Fig. 3

Collinearity relationships of 2ODDs in Gossypium. A The collinearity relationship of repeated gene pairs of 2ODDs among G. hirsutum, G. barbadense, G. arboreum and G. raimondii. The chromosomal lines in different colors indicate the collinearity area around the 2ODDs. B Collinearity relationships of Gh2ODDs and Gb2ODDs with their homologs in the ancestor species through multiple synteny plots. Dense grey lines in the background reveal collinear blocks, while blue lines represent syntenic 2ODD gene pairs

Fig. 4

Selection pressure based on non-synonymous to synonymous ratio (Ka/Ks) for 2ODDs. A The density of duplicated gene pairs in various ranges of Ka/Ks. Different colors mean different combinations of gene pairs among four Gossypium species. B Prediction of a number of duplicated gene pairs of different combinations from four Gossypium species. The blue, orange and grey colors show the different selection pressure

Fig. 5

Conserved protein motifs of Gh2ODDs along the phylogenic tree and subfamily classification patterns. A Phylogenetic tree of Gh2ODDs. B Conserved motifs of Gh2ODDs. Boxes with varying color represent different motifs. C Gene structure of Gh2ODDs. Green boxes indicate exons, and black lines show introns

Fig. 6

Analysis of cis elements and expression profiles of Gh2ODDs. A Phylogenetic tree of Gh2ODD genes. B Cis regulatory elements present in promoter regions of Gh2ODDs. Bars in diverse colors indicate different cis-acting elements. C Differentially expressed Gh2ODDs under cold, hot, salt or drought stress, as well as tissues expression patterns of Gh2ODDs at various growth stages. The expression level from lower to high are represented by red bar to blue one

Fig. 7

Tissue specific expression profilings of different Gh2ODDs in the stem, root, stamen, leaf, pistil, petal, ovule and fiber. Error bars show standard deviations among three independent biological replications

Fig. 8

Expression patterns of Gh2ODD genes under different saline-alkali stress (100 mM NaCl, 50 mM Na₂CO₃and 0.125 mM NaOH). Statistical analyses were performed by Student’s t-test (*P < 0.05 and **P < 0.01)

Fig. 9

Expression analysis of Gh2ODDs in response to alkaline stress in leaves by qRT-PCR. Cotton seedlings were treated with Na₂CO₃ stress. The mean values were from three independent biological replicates. Statistical analyses were performed by Student’s t-test (*P < 0.05 and **P < 0.01)

Fig. 10

Protein–protein interaction analysis of 2ODD proteins. Protein–protein interaction network produced by the STRINGV9.1. Each node represents a protein and each edge represents an interaction. They are colored by evidence type

Fig. 11

Silencing GhLDOX3 via VIGS reduced sensitivity to Na₂CO₃ stress. A The phenotype of cotton after GhLDOX3 gene silencing under Na₂CO₃ stress. pYL156:PDS as a positive control, pYL156 was a negative control (containing empty vector), and pYL156:GhLDOX3 shows a VIGS plant silencing GhLDOX3. B The expression levels of GhLDOX3 by qRT-PCR under Na₂CO₃ stress. C Proline (Pro) contents. D Chlorophyll contents. VIGS plants containing vectors pYL156 and pYL156:GhLDOX3 were treated with 0 and 50 mM Na₂CO₃ for 24 h. Then, the parameters above were assayed in leaves. Statistical analyses were performed by Student’s t-test (*P < 0.05 and **P < 0.01)