Genome-wide identification and characterization of the KCS gene family in sorghum (Sorghum bicolor (L.) Moench)

Abstract

The aboveground parts of plants are covered with cuticle, a hydrophobic layer composed of cutin polyester and cuticular wax that can protect plants from various environmental stresses. β-Ketoacyl-CoA synthase (KCS) is the key rate-limiting enzyme in plant wax synthesis. Although the properties of KCS family genes have been investigated in many plant species, the understanding of this gene family in sorghum is still limited. Here, a total of 25 SbKCS genes were identified in the sorghum genome, which were named from SbKCS1 to SbKCS25. Evolutionary analysis among different species divided the KCS family into five subfamilies and the SbKCSs were more closely related to maize, implying a closer evolutionary relationship between sorghum and maize. All SbKCS genes were located on chromosomes 1, 2, 3, 4, 5, 6, 9 and 10, respectively, while Chr 1 and Chr 10 contained more KCS genes than other chromosomes. The prediction results of subcellular localization showed that SbKCSs were mainly expressed in the plasma membrane and mitochondria. Gene structure analysis revealed that there was 0-1 intron in the sorghum KCS family and SbKCSs within the same subgroup were similar. Multiple cis-acting elements related to abiotic stress, light and hormone response were enriched in the promoters of SbKCS genes, which indicated the functional diversity among these genes. The three-dimensional structure analysis showed that a compact spherical space structure was formed by various secondary bonds to maintain the stability of SbKCS proteins, which was necessary for their biological activity. qRT-PCR results revealed that nine randomly selected SbKCS genes expressed differently under drought and salt treatments, among which SbKCS8 showed the greatest fold of expression difference at 12 h after drought and salt stresses, which suggested that the SbKCS genes played a potential role in abiotic stress responses. Taken together, these results provided an insight into investigating the functions of KCS family in sorghum and in response to abiotic stress.

Keywords: Abiotic stress; KCS gene family; Sorghum bicolor.

Figure 1. Chromosomal localization of the SbKCS genes.

25 SbKCS genes were mapped on eight sorghum chromosomes. The chromosome numbers were indicated at the top of each vertical green bar. The gene names on each chromosome corresponded to the approximate locations of each SbKCS genes. The number shown to the left of each vertical green bar was the specific physical position of each SbKCS gene on the chromosome.

Figure 2. The prediction of subcellular localization for SbKCS proteins.

The color and the size of the circles indicated the reliability of the prediction results. The name of each protein was shown on the left. The site name for the predicted subcellular localization of each SbKCS protein was shown at the bottom.

Figure 3. Phylogenetic tree of KCS gene family members in five species.

The full-length protein sequences from Arabidopsis, sorghum, maize, rice and Brachypodium distachyon were used to construct the phylogenetic tree by MEGA 7.0 software based on a maximum likelihood (ML) method with bootstrap replicates of 1,000. Five groups were highlighted in different colors. The members of KCS family in sorghum were marked in red.

Figure 4. The motif and gene structure analysis of the KCS family members in sorghum.

(A) Motif analysis of the 25 SbKCS proteins. The conserved motifs in the SbKCS proteins were identified with MEME software. The grey lines denoted the non-conserved sequences, and each motif was showed by a different color. The length of motifs in each protein was presented in proportion. (B) The exon-intron structure analysis of the 25 SbKCS genes. Exons were represented by yellow rectangles, introns were represented by gray lines, and green rectangles represented UTR regions.

Figure 5. Three-dimensional (3D) models of the SbKCS proteins.

The 3D model of SbKCS protein was constructed by using the method of homologous protein modeling on SWISS-MODEL online website. Proteins of the different subfamily were shown in different colors at the bottom of each 3D model. Orange fonts represented the Group 1 subfamily; blue fonts represented the Group 2 subfamily; black fonts represent the Group 3 subfamily; purple fonts represented the Group 4 subfamily and red fonts represented the Group 5 subfamily.

Figure 6. Cis-acting elements in the SbKCS gene promoters.

The different cis-acting elements were shown in different colors. The name of each gene was shown on the left. Promoter sequence length was displayed in proportion.

Figure 7. Expression profile of nine SbKCS genes under drought stress treatment.

The expression level of the nine SbKCS genes (A-I) were validated by qRT-PCR. Seedings were treated with drought (20% PEG6000) and leaves were sampled at 0, 6, 12 and 24 h. Data represent mean ± standard error (SE) of three biological replicates. Statistically significant differences between the control (CK) and treatment groups (PEG) are indicated using asterisks (* p < 0.05, ** p < 0.01, *** P < 0.001, **** P < 0.0001; independent Student’s t-test).

Figure 8. Expression profile of nine SbKCS genes under salt stress treatment.

The expression level of the nine SbKCS genes (A–I) were validated by qRT-PCR. Seedings were treated with salt (150 mM NaCl) and leaves were sampled at 0, 6, 12 and 24 h. Data represent mean ± standard error (SE) of three biological replicates. Statistically significant differences between the control (CK) and treatment groups (Salt) are indicated using asterisks (* p < 0.05, ** p < 0.01, *** P < 0.001, **** P < 0.0001; independent Student’s t-test).