Genome-wide identification of rice CXE gene family and mining of alleles for potential application in rice improvement

Abstract

Carboxylesterases (CXE, EC 3.1.1.1), a class of hydrolases with an α/β folding domain, play important roles in plant growth and development and stress response. Here, we identified 32, 63, 41, and 45 CXE genes in Oryza sativa Japonica (Nipponbare), Oryza sativa Indica (93-11), Oryza sativa Indica (Xian-1B1 var.IR64), and Oryza sativa Japonica (Geng-sbtrp var.ChaoMeo), respectively. Then, we analyzed the chromosomal location, physical and chemical properties, subcellular localization, collinearity, and selection pressure of CXE genes in four rice varieties. We also analyzed the functional interaction network, cis-regulatory elements, evolutionary relationship, and protein tertiary structure, and performed gene expression profiling and qPCR verification under abiotic stress, as well as diversity analysis of 3010 gene-CDS-haplotype (gcHap) rice samples, aiming to understand the potential function of the 32 OsCXE genes. Our results indicated that fragment replication is the main reason for amplification of the CXE gene family in rice, and the gene family has undergone strong purification selection. OsCXE3.1, OsCXE3.2, OsCXE3.3, OsCXE5.1, and OsCXE7.3 may be used to improve the tolerance of rice to abiotic stress. OsCXE play important roles in rice population differentiation and improvement, and the major gcHaps at most OsCXE locus are significantly associated with yield traits. Therefore, natural variations of most OsCXE locus have great potential value for improvement of rice productivity.

Keywords: biological stress; carboxylesterase; gene-CDS-haplotype (gcHap) diversity; rice; yield traits.

Figure 1

Phylogenetic trees of CXE family members. (A) Phylogenetic tree of four rice species; (B) Phylogenetic tree of four rice and Arabidopsis Thaliana.

Figure 2

Collinearity of CXE genes in rice. (A) Collinearity between Os and 93-11. (B) Collinearity between Os and ChaoMeo. (C) Collinearity between Os and IR64. (D) Collinearity between ChaoMeo and IR64. (E) Collinearity between 93-11 and ChaoMeo. (F) Collinearity between 93-11 and IR64.

Figure 3

Selection pressure (Ka/Ks) analysis. (A) Selection pressure diagram. (B) Prediction of the number of genes in different combinations of four rice species,Where positive indicates Ka/Ks> 1 and pure selection indicates Ka/Ks<1.

Figure 4

From left to right, phylogenetic tree,conserved motif,conserved domainc and gene structure analysis of 32 OsCXE (Nipponbare).

Figure 5

Detection of cis-regulatory elements in 32 OsCXE (Nipponbare) genes. Cis-elements with similar functions are shown in the same color. Black lines indicate the promoter length of OsCXE genes, and boxes of different colors represent cis-regulatory elements with different functions.

Figure 6

3D structure and network interactions of 32 OsCXE (Nipponbare) proteins. (A) Prediction of OsCXE protein tertiary structure. According to the phylogenetic relationship, these proteins were divided into seven clades, and the blue and red color of the three-dimensional protein structure represent low activity and high activity, respectively. (B) OsCXE protein interaction network. Red denotes the central gene, and lines indicate interactions.

Figure 7

Analysis of 32 OsCXE (Nipponbare) gene expression. Color markers indicate changes in gene expression, with red indicating high expression and green indicating low expression. (A) Expression of OsCXE in the root, leaf, seedlings, shoot, stem, meristem, flower, seed, embryo, endosperm, panicle, female reproductive tissue, and male reproductive tissue under normal conditions. (B) Expression of OsCXE genes in shoots and roots at different developmental stages. (C) Expression levels of OsCXE genes in shoots and roots at different time after ABA treatment. (D) Expression levels of OsCXE genes in shoots and roots at different time after JA treatment. (E) Expression levels of OsCXE genes in stems and leaves under heat and salt stress at different time. (F) Expression levels of OsCXE genes at different time during flooding, cold, osmotic, and drought stress. (G) Expression of OsCXE genes at different time in roots under flooding, cold, osmotic and drought conditions.

Figure 8

Analysis of expression levels of five genes of the OsCXE (Nipponbare) family under different treatments. (A) ABA treatment. (B) MeJA treatment. (C) Drought stress. (D) NaCl stress. (E) High temperature stress. (F) Low temperature stress. Statistical analysis of the data was performed using WPS2023 software, and IBM SPSS Statistics 25 statistics analysis software was used to perform analysis of variance; the significance level was defined as **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 9

Population diversity of OsCXE genes in 3010 rice accessions. (A) Conserved genes (HK) (E_H = 0 and gcHapN = 1), low diversity genes (0< E_H < 0.05, $\overline{EH}$ = 0.020 ± 0.015, and gcHapN =12 ± 10), medium-low diversity genes (0.05 ≤ E_H < 0.3, $\overline{EH}$ = 0.171 ± 0.065, and gcHapN = 82 ± 67), medium-high diversity gene (0.3≤E_H <0.7, $\overline{EH}$ =0.444 ± 0.110,and gcHapN=498 ± 305), and high diversity gene (0.7≤E_H ≤ 1, $\overline{EH}$ =0.804 ± 0.075,and gcHapN =1767 ± 458). (B) Relationship between Shannon fairness (E_H ) of 32 OsCXE gene and gcHapN. (C) The E_H value of 32 OsCXE genes in different populations. (D) gcHap number (gcHapN) of OsCXE gene in different populations. (E) I_Nei values of OsCXE genes between all paired populations calculated from gcHap data.

Figure 10

Haplotype network analysis of OsCXE genes and their associations with four agronomic traits in 3KRG.P-values under the trait names indicate differences between haplotypes assessed by a two-factor ANOVA, where different letters on the boxplot indicate statistically significant differences at P< 0.05 based on Duncan’s multiple range test. The bar chart on the right shows the difference in frequency of dominant gcHaps between local varieties (LANs) and modern varieties (MVs) of Xian and Geng. Chi-square tests were used to determine significant differences in the proportion of the same gcHap between different populations ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05 and N.S., not significant.

Figure 11

Favorable gcHap frequencies of 32 OsCXE genes affecting TGW, GL, GW, PL and CN in Xian/Indica rice (XI), Geng/Japonica rice (GJ) and different rice subpopulations.The “favorable” gcHaps of a gene are defined as the gcHAPs associated with the highest trait values. “#accession” indicates the number of accessions with “favorable” gcHaps. Distribution frequency of five subpopulations of XI (XI-1A, XI-1B, XI-2, XI-3, and XI-ADM) and four subpopulations of GJ (temperate GJ [GJ-TMP], subtropical GJ [GJ-SBTRP], tropical GJ [GJ-TRP], and GJ-ADM).