Genome-Wide Identification and Characterization of Maize Long-Chain Acyl-CoA Synthetases and Their Expression Profiles in Different Tissues and in Response to Multiple Abiotic Stresses

Abstract

Long-chain acyl-CoA synthetases (LACSs) are essential enzymes that activate free fatty acids to fatty acyl-CoA thioesters, playing key roles in fatty acid (FA) catabolism, lipid synthesis and storage, epidermal wax synthesis, and stress tolerance. Despite their importance, comprehensive information about LACS genes in maize, a primary food crop, remains scarce. In the present work, eleven maize LACS genes were identified and mapped across five chromosomes. Three pairs of segmentally duplicated genes were detected in the maize LACS gene family, which underwent significant purifying selection (Ka/Ks < 1). Subsequently, phylogenetic analysis indicated that ZmLACS genes were divided into four subclasses, as supported by highly conserved motifs and gene structures. On the basis of the PlantCARE database, analysis of the ZmLACS promoter regions revealed various cis-regulatory elements related to tissue-specific expression, hormonal regulation, and abiotic stress response. RT-qPCR analysis showed that ZmLACS genes exhibit tissue-specific expression patterns and respond to diverse abiotic stresses including drought and salt, as well as phytohormone abscisic acid. Furthermore, using the STRING database, several proteins involved in fatty acid and complex lipid synthesis were identified to be the potential interaction partners of ZmLACS proteins, which was also confirmed by the yeast two-hybrid (Y2H) assay, enhancing our understanding of wax biosynthesis and regulatory mechanisms in response to abiotic stresses in maize. These findings provide a comprehensive understanding of ZmLACS genes and offer a theoretical foundation for future research on the biological functions of LACS genes in maize environmental adaptability.

Keywords: expression profiles; genome-wide investigation; long-chain acyl-CoA synthetase; maize (Zea mays L.).

Figure 1

Chromosomal locations of LACS genes in maize. Eleven ZmLACS genes were distributed across chromosomes 2, 3, 4, 9, and 10. The corresponding chromosome numbers are illustrated at the uppermost section of each chromosome.

Figure 2

Synteny relationships of the ZmLACS gene family. The red lines in the circle represent the duplicated LACS gene pairs in the maize. The gray lines illustrate gene collinearity regions in the maize genome. The outer circle segments represent individual maize chromosomes. Scale bars on chromosomes provide a visual reference for chromosomal lengths in megabases (Mb).

Figure 3

Phylogenetic analysis and classification of LACS proteins in maize, Arabidopsis, wheat, and rice. The phylogenetic tree, constructed with 1000 bootstrap replicates, was visually drawn by categorizing it into distinct subgroups. Each subgroup, denoted by different colors, was further labeled as I–IV, representing the four identified subgroups. Arrowheads represent LACS proteins in Arabidopsis, circles indicate LACS proteins in maize, square frames represent LACS proteins in wheat, and five-pointed stars indicate LACS proteins in rice.

Figure 4

Phylogenetic relationship, conserved motifs, and gene structure of maize LACSs: (A) The neighbor-joining (NJ) phylogenetic tree of maize LACS proteins. (B) Conserved motifs distributions of 11 ZmLACS proteins. Diverse colored boxes illustrate conserved motifs (1–10). (C) Gene structure of maize LACSs. The yellow box represents the coding region (CDS), the green box represents the untranslated region (UTR), and the black line represents the intron.

Figure 5

Cis-elements analysis in ZmLACS promoter regions. Distinct colored boxes at the bottom represent diverse cis-elements.

Figure 6

Relative expression analysis of ZmLACS genes in different tissues. The expression levels of ZmLACS1 (A), ZmLACS2 (B), ZmLACS 4.1 (C), ZmLACS4.2 (D), ZmLACS4.3 (E), ZmLACS6.1(F), ZmLACS6.2 (G), ZmLACS8.1 (H), ZmLACS8.2 (I), ZmLACS9.1 (J), and ZmLACS9.2 (K) in primary root (V1) (1), pooled leaves (V1) (2), stem and SAM (V1) (3), first internode (V5) (4), immature tassel (V13) (5), meiotic tassel (V18) (6), silks (R1) (7), anthers (R1) (8), pericarp 18 DAP (9), embryo 22 DAP (10), endosperm 22 DAP (11), seed 2 DAP (12), seed 4 DAP (13), seed 8 DAP (14), seed 16 DAP (15), and seed 20 DAP (16) using RT-qPCR analysis. Expression in primary root (V1) was set to 1.00. Data shown are means ± SD of three biological replicates.

Figure 7

Relative expression of ZmLACS genes under drought treatment. The expression levels of ZmLACS1 (A), ZmLACS2 (B), ZmLACS 4.1 (C), ZmLACS4.2 (D), ZmLACS4.3 (E), ZmLACS6.1 (F), ZmLACS6.2 (G), ZmLACS8.1 (H), ZmLACS8.2 (I), ZmLACS9.1 (J), and ZmLACS9.2 (K) using RT-qPCR analysis. Expression in control was set to 1.00. Data shown are means ± SD of three biological replicates. ns indicates no significant difference to the corresponding controls. *, **, ***, and **** represent p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 vs. control, respectively (Student’s t-test).

Figure 8

Relative expression of ZmLACS genes under NaCl treatment. The expression levels of ZmLACS1 (A), ZmLACS2 (B), ZmLACS 4.1 (C), ZmLACS4.2 (D), ZmLACS4.3 (E), ZmLACS6.1 (F), ZmLACS6.2 (G), ZmLACS8.1 (H), ZmLACS8.2 (I), ZmLACS9.1 (J), and ZmLACS9.2 (K) using RT-qPCR analysis. Expression in control was set to 1.00. Data shown are means ± SD of three biological replicates. ns indicates no significant difference to the corresponding controls. *, **, ***, and **** represent p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 vs. control, respectively (Student’s t-test).

Figure 9

Relative expression of ZmLACS genes under ABA treatment. The expression levels of ZmLACS1 (A), ZmLACS2 (B), ZmLACS 4.1 (C), ZmLACS4.2 (D), ZmLACS4.3 (E), ZmLACS6.1 (F), ZmLACS6.2 (G), ZmLACS8.1 (H), ZmLACS8.2 (I), ZmLACS9.1 (J), and ZmLACS9.2 (K) using RT-qPCR analysis. Expression in control was set to 1.00. Data shown are means ± SD of three biological replicates. ns indicates no significant difference to the corresponding controls. *, **, ***, and **** represent p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 vs. control, respectively (Student’s t-test).

Figure 10

Predicted protein–protein interaction networks (PPIs) using the STRING tool. Within the PPI network, every node encapsulates all proteins derived from the corresponding single gene. The size of each node indicates the degree of interaction, while the thickness of the edges signifies the strength of protein–protein interactions. Nodes representing ZmLACSs are drawn in yellow, while proteins interacting with ZmLACSs are highlighted in blue.

Figure 11

(A) Y2H assay showing the interaction between ZmPAT1/2/3 and ZmLACS1/4.1/8.1. (B) Y2H assay showing the interaction between ZmVLCAD1-4/1-5 and ZmLACS2/4.2/6.2/9.2. Yeast cells were grown on selection plates: SD-LW(-Leu-Trp) and SD-LWHA(-Leu-Trp-His-Ade). BD: pGBKT7, AD: pGADT7.