Genome-Wide Identification, Classification, and Expression Analyses of the CsDGAT Gene Family in Cannabis sativa L. and Their Response to Cold Treatment

Abstract

Hempseed is a nutrient-rich natural resource, and high levels of hempseed oil accumulate within hemp seeds, consisting primarily of different triglycerides. Members of the diacylglycerol acyltransferase (DGAT) enzyme family play critical roles in catalyzing triacylglycerol biosynthesis in plants, often governing the rate-limiting step in this process. As such, this study was designed to characterize the Cannabis sativa DGAT (CsDGAT) gene family in detail. Genomic analyses of the C. sativa revealed 10 candidate DGAT genes that were classified into four families (DGAT1, DGAT2, DGAT3, WS/DGAT) based on the features of different isoforms. Members of the CsDGAT family were found to be associated with large numbers of cis-acting promoter elements, including plant response elements, plant hormone response elements, light response elements, and stress response elements, suggesting roles for these genes in key processes such as development, environmental adaptation, and abiotic stress responses. Profiling of these genes in various tissues and varieties revealed varying spatial patterns of CsDGAT expression dynamics and differences in expression among C. sativa varieties, suggesting that the members of this gene family likely play distinct functional regulatory functions CsDGAT genes were upregulated in response to cold stress, and significant differences in the mode of regulation were observed when comparing roots and leaves, indicating that CsDGAT genes may play positive roles as regulators of cold responses in hemp while also playing distinct roles in shaping the responses of different parts of hemp seedlings to cold exposure. These data provide a robust basis for further functional studies of this gene family, supporting future efforts to screen the significance of CsDGAT candidate genes to validate their functions to improve hempseed oil composition.

Keywords: diacylglycerol acyltransferase (DGAT); expression patterns; gene family; hemp.

Figure 1

Phylogenetic analysis of CsDGAT gene family. A phylogenetic tree of CsDGAT proteins from C. sativa and other plants was constructed by MEGA 10.0 using full-length protein sequences. Different branches were distinguished with different color shades. CsDGAT proteins were represented by red color. For the description of other species abbreviations involved in the figure, please see Additional Table S1.

Figure 2

Chromosomal location and synteny analysis of CsDGATs in C. sativa genome. (A) Chromosomal locations of CsDGATs. Tandem-duplicated genes are indicated with red boxes, and the chromosome number is indicated above each chromosome. The scale is in megabases (Mb). (B) Syntenic relationship of CsDGATs. The annotations on the fragments represent different chromosomes, and the numbers in the outermost circle represent the positions of the corresponding chromosomes. The CsDGATs involved in segmental duplications in the CsDGATs gene family are mapped to their respective locations of the C. sativa genome in the circular diagram. The different color lines represent the segmental duplication pairs between the CsDGATs and the DGAT gene of other plant genomes. Gm, Glycine max (yellow font), Ha, Helianthus annuus (orange font), Jr, Juglans regia (grey font), Rc, Ricinus communis (blue font), Si, Sesamum indicum (green font), Ah, Arachis hypogaea (brown font).

Figure 3

Structures for the CsDGAT gene family. (A) Phylogenetic relationships and gene structures for the CsDGAT gene family. Phylogenetic tree generated by the neighbor-joining tree method with bootstrapping analysis (1000 replicates) based on the protein sequences of CsDGAT. The exon-intron structures for CsDGAT gene family members were visualized with GSDS2.0. The horizontal black lines, red boxes, and gray boxes show introns, coding sequence, and untranslated region, and the scale displays the relative length and position of the introns and exons. (B) Model illustrating transmembrane regions and putative conserved domain structure of 10 CsDGAT protein identified in C. sativa. The CsDGAT family can be divided into three subgroups based on the different conserved domains: CsDGAT1, CsDGAT2, CsDGAT3, and CsWSD1. The grey bars represent the length of each protein sequence, and conserved domains are shown as colored boxes. The conserved domain architectures prediction relies on SMART, and IBS software was used for visualization with default parameters. MBOAT = membrane-bound O-acyl transferase domain, LPAT = lysophospholipid acyltransferase domain, TRX = thioredoxin-like, ferredoxin family domain, WES = wax ester synthase-like Acyl-CoA acyltransferase domain, DUF = domain of unknown function, AATase = alcohol acetyltransferase domain, T: transmembrane domains; The numbered bar indicates the position of amino acid.

Figure 4

The schematic model of Cis-acting elements distribution pattern in 10 CsDGATs gene promoter regions. (A) These cis-acting elements were classified into four groups: stress-responsive, light-responsive, phytohormone-responsive, and plant-responsive, as shown in the heatmap. (B) The total count of these four categories is displayed in the bar plot.

Figure 5

Subcellular localization analysis of CsDGATs proteins. GFP blank vector (control); Tobacco (Nicotiana benthamiana) leaves transiently expressed CsDGATs-GFP fusion proteins were observed through the laser scanning confocal microscope. The scale bar label in the lower right corner of each picture represents 20 μm.

Figure 6

Normalized expression profiles of CsDGAT family genes in C. sativa. (A) Normalized expression profiles of CsDGAT genes in female inflorescences of ten hemp varieties based on transcriptome expression data. (B) Normalized expression profiles of CsDGAT genes in different tissues of identically hemp varieties based on transcriptome expression data. Each column represents a different tissue of hemp. (C) Normalized expression profiles of CsDGAT genes in developing seeds at different growth stages after fertilization based on transcriptome expression data. Each column represents a different growth stage of hemp. The size of circles with different colors represents the value of expression quantity. The bottom bar indicates high to low normalized expression data (red to blue).

Figure 7

Expression patterns of the CsDGAT genes in different tissues detected by RT-qPCR. The characters on the X-axis show the different tissues of C. sativa (root, stem, leaf, flower, and seed). The Y-axis indicates the relative expression level of different genes. Three replicates were performed, and the vertical bar is the standard error. The actin7 gene was used as an internal reference. Significant differences between the different group are indicated by different letters a–d (p < 0.05).

Figure 8

Phenotype and CsDGAT expression pattern analysis under cold stress response in C. sativa. (A) Phenotype analysis under cold stress in C. sativa. a. Phenotypic changes of hemp plants under cold stress; b. Phenotypic changes of hemp leaves under cold stress.(B) Expression patterns of the CsDGAT genes in leaves during different cold treatment time points detected by RT-qPCR. (C) Expression patterns of the CsDGAT genes in roots during different cold treatment time points detected by RT-qPCR. The characters on the X-axis show the different time points of cold treatment (0, 12, 24, 48, and 72 h). The Y-axis indicates the relative expression level of different genes. Three replicates were performed, and the vertical bar is the standard error. The actin7 gene was used as an internal reference. Significant differences between the control group and cold treatment samples are indicated by * (p < 0.05) and ** (p < 0.01).