Identification of histone acetyltransferase genes responsible for cannabinoid synthesis in hemp | Chinese Medicine

Identification and characterization of HAT genes in hemp

On the basis of the BLASTP, Pfam, and SMART results, 11 acetyltransferase genes were identified in the hemp genome (Table 1). We named the family members, CsHATs-1CsHATs-11, according to their chromosomal location. Eleven CsHAT genes were unevenly distributed on chr1, chr3, chr4, chr6, and chr8 (Fig. 1). Seven CsHAT genes (CsHATs-3, CsHATs-4, CsHATs-5, CsHATs-6, CsHATs-7, CsHATs-8, and CsHATs-9) were located on chr4, harboring the largest number of genes. CsHATs-1, CsHATs-2, CsHATs-10, and CsHATs-11 were located on chr1, chr3, chr6, and chr8, respectively.

Table 1 Detailed information of 11 predicted HATs in hemp.
Fig. 1
figure 1

Chromosomal location of 11 histone acetyltransferase genes (HATs) in hemp

Six CsHATs comprised 440–750 amino acids, and five CsHATs comprised 1700–1900 amino acids. The molecular weights (MWs) of CsHATs varied from 49115.2 to 214202.7 Da. CsHATs-1 encoded the shortest protein, whereas CsHATs-10 encoded the longest protein with the highest molecular weight of 214202.7 Da. The isoelectric points (PIs) varied from 5.11 to 8.72. Six CsHATs were detected in the cytoplasm.

Phylogenetic analysis of CsHATs

To understand the phylogenetic relationships among HATs from different species, we constructed a neighbor-joining (NJ) tree with 12 AtHATs, 8 OsHATs, and 11 CsHATs. As shown in Fig. 2, the HAT proteins of these species were divided into six categories, which shared a high homology. The HAC group was the largest, comprising 12 HAT proteins, including four CsHATs (CsHATs-3, CsHATs-4, CsHATs-5, and CsHATs-6), five AtHATs (AtHAC1, AtHAC2, AtHAC4, AtHAC5, and AtHAC12), and three OsHATs (Os06g49130, Os02g04490, and Os01g14370). The HAG1 group contained five HAT proteins, including three CsHATs (CsHATs-7, CsHATs-8, and CsHATs-9), one AtHAT (AtHAG1), and one OsHAT (Os10g28040). The HAG2 and HAG3 groups contained three HAT proteins each. The HAG2 group contained CsHATs-11, AtHAG2, and Os09g17850, whereas the HAG3 group contained CsHATs-2, AtHAG3, and Os04g40840. The HAF and HAM groups contained four HAT proteins. The HAF group contained CsHATs-10, AtHAF1, AtHAF2, and Os06g43790, whereas the HAM group contained CsHATs-1, AtHAM1, AtHAM2, and Os07g43360.

Fig. 2
figure 2

Phylogenetic tree of CsHATs from Arabidopsis, Oryza sativa, and hemp. A phylogenetic tree was constructed using the NJ (neighbour-joining) method. Each group is shown in different colours. Triangles represent A. thaliana, quadrangles represent O. sativa, and circles represent hemp

Gene structures and conserved motifs of CsHATs

The comparison of gene structures provides insight into the evolution of the gene family. Thus, we analyzed the structures of the CsHATs. The positions and numbers of exons were significantly different for CsHATs across different phylogenetic groups and were relatively conserved among those within a group. The introns also varied significantly among the different phylogenetic groups (Fig. 3A, B). All genes contained introns and exons, and the number of introns ranged from 8 to 20, whereas those of exons ranged from 9 to 21 (Fig. 3B). Genes clustered in the same group shared similar gene structures.

Fig. 3
figure 3

Phylogenetic relationships, gene structure, and conserved domains of histone acetyltransferase gene (HATs) in hemp. A Phylogenetic tree was constructed based on the sequences of CsHAT proteins using MEGA 7 software. The details of the clusters are shown in different colours. B Exon–intron structure of CsHATs. Yellow boxes indicate exons and black lines indicate introns. C Conserved domains of the HAT proteins in Cannabis sativa. Motifs numbered 1–10 are displayed in different coloured boxes

The predicted amino acid sequences of the 11 CsHAT proteins were queried on MEME to characterize the putative motifs in the hemp HAT family. Ten motifs were predicted for these proteins, namely, motifs 1–10 (Fig. 3C). Members of the same group contained similar motifs, suggesting similar functions. Three CsHAT proteins did not contain any of the ten motifs, and eight CsHAT proteins contained varying numbers of motifs. All 10 motifs were found in CsHATs-3, CsHATs-4, CsHATs-5, and CsHATs-6. Five motifs (motif1, motif2, motif6, motif7, and motif10) were found in CsHATs-7, CsHATs-8, and CsHATs-9. Motif2 was found in CsHATs-10. These results indicated conserved motif compositions and similar gene structures among HAT members in the same group. Together with the phylogenetic analysis results, the reliability of the group classifications was validated. The motif structure is shown in Additional file 3: Figure S1.

Analysis of Cis-acting elements in promoters of CsHATs

To further study the potential regulatory mechanisms of CsHATs during stress responses, 2000 bp upstream sequences from the translation start sites of CsHATs were retrieved from TBtools and submitted to PlantCARE to detect the cis-elements. Eight stress response elements, namely, salicylic acid, abscisic acid, jasmonic acid, auxin, gibberellin, flavonoid biosynthesis gene regulatory elements, defense and stress response elements, and low-temperature response elements were analyzed (Fig. 4). Some stress-response-related cis-elements were detected in the promoters of CsHATs. These hormone regulatory elements widely exist in CsHAT promoters, and the auxin-response element unique to CsHAT-10. Defense and stress response elements existed in the promoters of all CsHATs, except for CsHATs-2, CsHATs-10, and CsHATs-11. The flavonoid biosynthesis gene regulation elements were present in four CsHATs (CsHATs-7, CsHATs-8, CsHATs-9, and CsHATs-10); low-temperature response elements were only found in CsHATs-2. The cis-element analysis illustrated that CsHATs could respond to various stresses.

Fig. 4
figure 4

Prediction of cis-acting elements in the CsHAT promoters. The phylogenetic tree was constructed based on the sequences of CsHAT proteins using MEGA 7 software. The details of the clusters are shown in different colours. The type, quantity, and position of the elements in the CsHAT promoters are shown

Collinearity analysis for CsHATs

We constructed three comparative syntenic maps of hemp associated with three representative species, two dicots (Arabidopsis and grape), and one monocot (maize) to further infer the phylogenetic mechanisms of CsHATs (Fig. 5). Three genes in Arabidopsis were collinear with CsHATs; likewise, two in grapes and one in maize were observed. The number of orthologous pairs between hemp and the three species (Arabidopsis, grape, and maize) was 3, 2, and 1. This finding indicated that these orthologous pairs may have already existed prior to the divergence of dicotyledonous and monocotyledonous plants.

Fig. 5
figure 5

Synteny analysis of HATs between hemp and three representative plant species (Arabidopsis, grape, and maize). Grey lines in the background indicate the collinear blocks within hemp and other plant genomes, whereas the red lines highlight the syntenic HAT pairs

Expression pattern analysis for CsHATs

Previously, we obtained RNA-seq data containing different organs and inflorescence developmental stages of hemp (S1: apical meristem, female flowers absent; S2: the female flowers appear and stigma is white; S3: the stigma is orange when pollination is complete; S4: when the seeds are green and not yet ripe, and S5: when the seeds are mature and brown) (unpublished). Based on the RNA-seq data, we examined the expression patterns of CsHATs, some of which showed similar expression patterns across organs (Fig. 6A). CsHATs-6 and CsHATs-9 were highly expressed in roots and stems but were rarely detected in seeds, female flowers, and male flowers. CsHATs-2 was highly expressed only in the seeds. The expression of some genes showed significant trends at different developmental stages (Fig. 6B). For example, the levels of CsHATs-2 and CsHATs-6 expression gradually increased with inflorescence development, whereas those of CsHATs-1, CsHATs-9, and CsHATs-11 declined towards the subsequent developmental stages of inflorescence, involving seed development and maturation.

Fig. 6
figure 6

Expression patterns of HATs and histone deacetylase genes (HDACs) in hemp. A Expression of HATs and HDACs across different organs. B Expression of HATs and HDACs at different inflorescence developmental stages. Heat maps reflect the fragments per kilobase of transcript per million mapped fragments (FPKM) values of HATs and HDACs. Colours from red to blue indicate high to low expression

The levels of deacetylation gene expression were analyzed in hemp. According to the members of the CsHDAC gene family screened in a previous study [37], 10 CsHDACs were identified based on our transcriptomic data. These CsHDACs were highly expressed in different hemp organs, especially in almost all female flowers, male flowers, and stems (Fig. 6A). The expression of CsHDA4, CsHDA6, CsHDA7, CsHDA10, and CsHDT2 gradually increased from S3–S5 (Fig. 6B). These results indicated that CsHATs and CsHDACs were widely and differently expressed in different organs and developmental stages of hemp, and they may synergistically affect protein acetylation in these organs and across developmental stages.

Expression patterns of cannabinoid biosynthesis genes and PTM of histones in hemp

Cannabinoid content shows significant changes during inflorescence development [38]. We detected the expression of cannabinoid biosynthesis pathway genes based on RNA-seq data. As shown in the heatmap (Fig. 7A), these genes were differentially expressed across different inflorescence developmental stages. For example, LOX4, LOX8, and LOX9 were expressed only at S4. Some genes were expressed at most stages, such as AACT2, AAE4, HDS1 and MVK in S1, S3, S4, and S5. Some genes, such as PT6, GPP4, AAE5, OAC1, and OAC2, were expressed only in S1 and S4.

Fig. 7
figure 7

The expression patterns of the cannabinoid synthesis pathway genes and acetylation patterns of different histone lysines in hemp. A Expression of cannabinoid synthesis pathway genes at different inflorescence developmental stages. Heat maps reflect the fragments per kilobase of transcript per million mapped fragments (FPKM) values of cannabinoid synthesis pathway genes. Colours from red to blue indicate high to low expression. B Acetylation pattern of different histone lysines across inflorescence developmental stages of hemp

HATs and HDACs can catalyze histone acetylation to regulate gene expression [39]. Thus, we examined the acetylation patterns of histones during the five inflorescence developmental stages (Fig. 7B) (laboratory self-test, unpublished). Ten histones were acetylated during the inflorescence development. As shown in the heat map, XP_030484463.1, XP_030496658.1, XP_030506295.1, and XP_030488338.1 were highly acetylated in S1 and S2, whereas XP_030503049.1 was highly acetylated in S4 and S5. XP_030499096.1, XP_030504381.1, XP_030485570.1, and XP_030492994.1 were highly acetylated throughout all stages. These results indicated that histone acetylation occurs widely during inflorescence development and may affect the expression of cannabinoid biosynthesis pathway genes. In addition, we found that the expression of CsHATs and CsHDACs genes were significantly associated with cannabinoid synthesis genes. As shown in Additional file 4: Figure S2, CsHATs (CsHATs-1, CsHATs-2, CsHATs-6, CsHATs-9, and CsHATs-11) and CsHDACs (CsHDA2, CsHDA3, CsHDA4, CsHDA6, CsHDA7, CsHDA8, CsHDA9, CsHDA10, CsHDT1, and CsHDT2) were positively correlated with some cannabinoid synthesis genes. It indicated that CsHATs and CsHDACs may regulate the expression of cannabinoid synthesis genes.

Effect of histone acetylation inhibitor treatment on cannabinoid biosynthesis gene expression and contents of CBD and CBG

We treated hemp inflorescences with a histone acetylation inhibitor (PU139) to further investigate whether histone acetylation could affect cannabinoid biosynthesis. PU139 effectively inhibits the expression of the CsHATs. The levels of six CsHATs (CsHATs-1, -2, -7, -8, -9, and -11) were downregulated at different time points after inhibitor treatment (Fig. 8A). The expression of cannabinoid biosynthesis pathway genes, AAE18, GPP2, PT7, CBDAS4, and CBDAS6 was generally downregulated upon exposure to PU139 for different times (Fig. 8B). We also determined the CBD and CBG contents before and after treatment. The CBD content declined only 3 h after inhibitor administration (Fig. 8C). The CBG content decreased at 3 h and 24 h after inhibitor treatment and increased at 72 h (Fig. 8D). Therefore, inhibition of histone acetylation resulted in the inhibition of cannabinoid biosynthesis pathway gene expression and further decreased CBD and CBG contents in hemp inflorescence.

Fig. 8
figure 8

Relative expression of genes and the contents of CBD and CBG after PU139 treatment. A Levels of CsHAT expression after inhibitor treatment (measured by qRT-PCR). B Gene expression associated with cannabinoid synthesis after inhibitor treatment (measured by qRT-PCR). Data were normalised with the levels of EF1α, and vertical bars indicate the standard deviation. CD Contents of CBD and CBG after PU139 treatment. Vertical bars indicate the standard deviation. (**P < 0.01; *P < 0.05)

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