A Comprehensive Analysis In Silico of KCS Genes in Maize Revealed Their Potential Role in Response to Abiotic Stress

Abstract

β-ketoacyl-CoA synthase (KCS) enzymes play a pivotal role in plants by catalyzing the first step of very long-chain fatty acid (VLCFA) biosynthesis. This process is crucial for plant development and stress responses. However, the understanding of KCS genes in maize remains limited. In this study, we present a comprehensive analysis of ZmKCS genes, identifying 29 KCS genes that are unevenly distributed across nine maize chromosomes through bioinformatics approaches. These ZmKCS proteins varied in length and molecular weight, suggesting functional diversity. Phylogenetic analysis categorized 182 KCS proteins from seven species into six subgroups, with maize showing a closer evolutionary relationship to other monocots. Collinearity analysis revealed 102 gene pairs between maize and three other monocots, whereas only five gene pairs were identified between maize and three dicots, underscoring the evolutionary divergence of KCS genes between monocotyledonous and dicotyledonous plants. Structural analysis revealed that 20 out of 29 ZmKCS genes are intronless. Subcellular localization prediction and experimental validation suggest that most ZmKCS proteins are likely localized at the plasma membrane, with some also present in mitochondria and chloroplasts. Analysis of the cis-acting elements within the ZmKCS promoters suggested their potential involvement in abiotic stress responses. Notably, expression analysis under abiotic stresses highlighted ZmKCS17 as a potential key gene in the stress response of maize, which presented an over 10-fold decrease in expression under salt and drought stresses within 48 h. This study provides a fundamental understanding of ZmKCS genes, paving the way for further functional characterization and their potential application in maize breeding for enhanced stress tolerance.

Keywords: KCS genes; Zea mays; abiotic stress response; gene family.

Figure 1

Chromosomal distribution of maize KCS genes and their interchromosomal relationships. The innermost ring represents the syntenic blocks across the maize B73 genome, with grey lines indicating all such blocks. The colorful lines denote the collinear blocks of ZmKCS genes within the maize genome. The subsequent yellow and red rings correspond to gene density and the GC ratio, respectively, with each ring reflecting these genomic features at their respective locations. The outermost ring shows the physical positions of the ZmKCS genes within the maize genome.

Figure 2

Phylogenetic analysis of KCS gene family across different species. (A) This section presents a phylogenetic tree of KCS gene family members from seven species, including maize and three monocots (sorghum, rice, Brachypodium distachyon), as well as three dicots (Arabidopsis thaliana, tomato, soybean). The tree was constructed using MEGA 7.0 software employing the maximum likelihood (ML) method. The tree is organized into six subgroups, each identified by a distinct background color. Monocot branches are highlighted in red, with maize KCS genes marked by green stars. The abbreviations used are as follows: EER, EES for sorghum; KQJ, KQK, PNT for Brachypodium distachyon; At for Arabidopsis thaliana; Zm for maize; Os for rice; solyc for tomato; and KRH for soybean. (B) This panel shows the results of a collinearity analysis of KCS genes between maize and three dicots: Glycine max (Gm), Solanum lycopersicum (Sl), and Arabidopsis thaliana (At). (C) This panel shows the collinearity analysis of KCS genes between maize and three monocots: Sorghum bicolor (Sb), Oryza sativa (Os), and Brachypodium distachyon (Bd). The background gray lines represent genome-wide collinear blocks, while the blue lines specifically highlight the collinearity of KCS genes, illustrating evolutionary connections and genomic conservation across these species.

Figure 3

Gene structure and conserved motifs in ZmKCS genes. (A) Exon–intron structure of ZmKCS genes. (B) Motif analysis of ZmKCS proteins. Ten conserved motifs across 29 ZmKCS proteins, with each conserved motif represented by a unique color. Motif lengths are proportional to their representation in each protein.

Figure 4

Subcellular localization of four of the ZmKCS genes in tobacco epidermal cells. The KCS-GFP fusion proteins are predominantly localized to the cell membrane and chloroplasts, as indicated by their fluorescence, indicating specific localization patterns.

Figure 5

Salt stress response of ZmKCS genes. The expression levels of seven ZmKCS genes (A–G) were assessed via qRT-PCR. Maize seedlings were subjected to salt stress (150 mM NaCl), and leaf samples were collected at 0, 6, 12, 24, 36, and 48 h. The data are presented as the means ± standard errors (SEs) of three biological replicates. Statistically significant differences between the control (CK) and salt treatment groups are denoted by asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (determined by independent Student’s tests).

Figure 6

Drought stress response of ZmKCS genes. The expression levels of seven ZmKCS genes (A–G) were validated via qRT-PCR. Seedings were subjected to drought (20% PEG6000), and leaves were sampled at 0, 6, 12, 24, 36 and 48 h. Data represent the means ± standard errors (SEs) of three biological replicates. Statistically significant differences between the control (CK) and treatment groups (PEG) are indicated by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; independent Student’s t-test).