Identification of a BAHD Acyltransferase Gene Involved in Plant Growth and Secondary Metabolism in Tea Plants

Abstract

Plant acyl-CoA dominated acyltransferases (named BAHD) comprise a large appointed protein superfamily and play varied roles in plant secondary metabolism like synthesis of modified anthocyanins, flavonoids, volatile esters, etc. Tea (Camellia sinensis) is an important non-alcoholic medicinal and fragrancy plant synthesizing different secondary metabolites, including flavonoids. In the tea (C.A sinensis) cultivar Longjing 43 (LJ43), eight samples were performed into three groups for transcriptome analysis under three biological replications. Among the BAHD acyltransferase genes in tea cultivars, the expression of TEA031065 was highest in buds and young leaves following the RNA sequencing data, which was coincident with the tissue rich in catechins and other flavonoids. We then transformed this gene into wild-type Arabidopsis as an over-expression (OX) line 1 and line 2 in ½ MS media to verify its function. In the wild types (WT), the primary root length, number of secondary roots, and total root weight were significantly higher at 24%, 15%, and 53.92%, respectively, compared to the transgenic lines (OX1 and OX2). By contrast, the leaves displayed larger rosettes (21.58%), with higher total leaf weight (32.64%) in the transgenic lines than in the wild type (WT). This result is consistent with DCR mutant At5g23940 gene in Arabidopsis thaliana. Here, anthocyanin content in transgenic lines was also increased (21.65%) as compared to WT. According to the RNA sequencing data, a total of 22 growth regulatory genes and 31 structural genes with TFs (transcription factors) that are correlative with plant growth and anthocyanin accumulation were identified to be differentially expressed in the transgenic lines. It was found that some key genes involved in IAA (Auxin) and GA (Gibberellin) biosynthesis were downregulated in the transgenic lines, which might be correlated with the phenotype changes in roots. Moreover, the upregulation of plant growth regulation genes, such as UGT73C4 (zeatin), ARR15, GH3.5, ETR2, ERS2, APH4, and SAG113 might be responsible for massive leaf growth. In addition, transgenic lines shown high anthocyanin accumulation due to the upregulation of the (1) 3AT1 and (3) GSTF, particularly, GSTF12 genes in the flavonoid biosynthesis pathway. However, the TFs such as, CCoAMT, bHLH, WRKY, CYP, and other MYBs were also significantly upregulated in transgenic lines, which increased the content of anthocyanins in A. thaliana seedlings. In conclusion, a BAHD acyltransferase (TEA031065) was identified, which might play a vital role in tea growth and secondary metabolites regulation. This study increases our knowledge concerning the combined functionality of the tea BAHD acyltransferase gene (TEA031065).

Keywords: Arabidopsis thaliana; BAHD acyltransferase (TEA031065) gene; Camellia sinensis; gene expression; secondary metabolites.

Figure 1

Expression of 21 BAHD Acyltransferase genes in tea (C. sinensis). Expression level of tea plant 21 BAHD acyltransferase genes at eight representative tissues (apical bud, flower, fruit, young leaf, mature leaf, old leaf, root, stem). The means of RNA-seq. analysis data are the ± SD (n = 3) p ˂ 0.05.

Figure 2

Expression level of BAHD acyltransferase (TEA031065) gene in three different tissues (root, stem, and leaf) in tea plant (C. sinensis). The eight samples were divided into three groups: 1-R, newly growing root; 1-S, newly growing shoot; 2-L, second-stage leaf; 2-R, second-stage root; 2-S, second-stage stem; 3-L, third-stage leaf; 3-R, third-stage root; 3-S, third-stage stem. The means of RNA-seq. analysis data are the ± SD (n = 1) p ˂ 0.05.

Figure 3

Light-microscopy images of different TEA031065-over-expression allele phenotypes for roots and their statistical growth rates in transgenic plants (A. thaliana). (A) Phenotypes of 21-day-old OX-1 and OX-2 versus wild type; (B) primary root length; (C) root weight; and (D) secondary root number/plant of control (CK)/wild-type and transgenic (lines 1 and 2 gene overexpression) plants for 14 days. The LSD analysis shown the significant differences among the different treatment by the lowercase letter a_>b_>c. The values imply means of three biological replicates ± SD. p ˂ 0.05.

Figure 4

Light-microscopy images of different TEA031065-over-expression line phenotypes for leaves and their statistical growth-rate capacity in A. thaliana. (A) Phenotypes of 21-days old OX-1 and OX-2 versus wild-type leaves; (B) leaf fresh weight; (C) leaf length; and (D) leaf number/plant of control (CK)/wild-type and transgenic (overexpression) lines 1 and 2 gene plants for 21 days. The LSD analysis shown the significant differences among the different treatment by the lowercase letter a_>b_>c. The values imply means of three biological replicates ± SD. p ˂ 0.05.

Figure 5

Total Anthocyanin analysis in A. thaliana under ½ MS media. Anthocyanin accumulations of newly grown Arabidopsis transgenic leaves in OX (line 1 and line 2) and WT (wild type) by 760CRT dual-wavelength, double-beam Prove UV-2500 spectrophotometer (mean ± standard deviation, n = 3). Means showing effective difference (p < 0.05) are labeled with different letters based on one-way ANOVA.

Figure 6

DEGs (differentially expressed genes) in OX1 vs. WT, OX2 vs. WT, and OX vs. WT groups in A. thaliana. The means of RNA-seq. analysis data are the ± SD (n = 3) and means showing effective difference (p < 0.05).

Figure 7

Expression levels of potential DEGs for plant-growth regulation in transgenic plant (A. thaliana). The heat map was originated from the log2-fold change (log2FC) and mean value calculated from three replicates of RNA-Seq. data. The color shows the fold change of DEGs under OX1, OX2, and WT, displayed on the right side. The means of RNA-seq. analysis data are the ± SD (n = 3) and means showing effective difference (p < 0.05).

Figure 8

Expression levels of potential DEGs for flavonoid production in transgenic plant (A. thaliana). The heat map was created through the log2-fold change (log2FC) and mean value calculated from three replicates of RNA-Seq. data. The color shows the fold change of DEGs under OX1, OX2, and WT, displayed on the right side. The means of RNA-seq. analysis data are the ± SD (n = 3) and means showing effective difference (p < 0.05).