Systematic Analysis of the 4-Coumarate:Coenzyme A Ligase (4CL) Related Genes and Expression Profiling during Fruit Development in the Chinese Pear

Abstract

In plants, 4-coumarate:coenzyme A ligases (4CLs), comprising some of the adenylate-forming enzymes, are key enzymes involved in regulating lignin metabolism and the biosynthesis of flavonoids and other secondary metabolites. Although several 4CL-related proteins were shown to play roles in secondary metabolism, no comprehensive study on 4CL-related genes in the pear and other Rosaceae species has been reported. In this study, we identified 4CL-related genes in the apple, peach, yangmei, and pear genomes using DNATOOLS software and inferred their evolutionary relationships using phylogenetic analysis, collinearity analysis, conserved motif analysis, and structure analysis. A total of 149 4CL-related genes in four Rosaceous species (pear, apple, peach, and yangmei) were identified, with 30 members in the pear. We explored the functions of several 4CL and acyl-coenzyme A synthetase (ACS) genes during the development of pear fruit by quantitative real-time PCR (qRT-PCR). We found that duplication events had occurred in the 30 4CL-related genes in the pear. These duplicated 4CL-related genes are distributed unevenly across all pear chromosomes except chromosomes 4, 8, 11, and 12. The results of this study provide a basis for further investigation of both the functions and evolutionary history of 4CL-related genes.

Keywords: 4-coumarate:coenzyme A ligases (4CL); acyl-coenzyme A synthetase (ACS); gene family; pear; qRT-PCR.

Figure 1

Phylogenetic relationships of 4-coumarate:coenzyme A ligase (4CL)-related proteins in Malus x domestica, Prunus persica, Prunus mume, Oryza sativa, Arabidopsis, and Pyrus bretschneideri. Asterisks represent bootstrap values of at least 70%. The large dashed arc indicates a large clade of acyl-activating enzymes (AAEs) from four Rosaceous plants (Malus x domestica, Prunus persica, Prunus mume, and Pyrus bretschneideri) as well as Oryza sativa and Arabidopsis. The red dashed ellipse indicates the clade of 4CL and acyl-coenzyme A synthetases (ACS) genes. The gene name species prefixes: Pb, Pyrus bretschneideri; Pp, Prunus persica; Pm, Prunus mume; Md, Malus x domestica; At, Arabidopsis thaliana; and Os, Oryza sativa. Gene names and models are listed in Table 1 and Table S1. The scale represents 0.2 amino acid changes.

Figure 2

Phylogenetic and exon–intron structure analyses of the 30 predicted pear 4CL-related genes. The neighbor joining (NJ) tree is shown on the left. The phylogenetic tree was constructed with MEGA 6.0 [52] using the full-length amino acid sequences of the 30 pear 4CL-related proteins. Bootstrap values are represented by black numbers. The gene structures are shown on the right. Exons, introns and untranslated regions (UTRs) are indicated by green boxes, gray lines, and blue boxes, respectively. The length of each 4CL-related gene can be estimated based on the scale at the bottom.

Figure 3

Collinearity analysis of 4CL-related regions among pear, rice, Arabidopsis, apple, peach, and yangmei. To identify the species of origin for each chromosome, a species identifier is included before the chromosome name: At, Arabidopsis thaliana; Os, Oryza sativa; Pb, Pyrus bretschneideri; Pp, Prunus persica; and Pm, Prunus mume. Different colored bars connect collinear regions between Pyrus bretschneideri and the other plant species.

Figure 4

Chromosomal locations and gene duplication of 4CL-related genes in pear. Chromosome numbers are indicated at the top of each vertical black bar, the names on the left side of each chromosome correspond to the approximate locations of each 4CL-related gene. Dashed lines represent segmental duplicated genes, and the tandem duplicated gene pairs are joined by a red line. The scale is in megabases (Mb).

Figure 5

Distribution of conserved motifs in pear 4CL and ACS numbers. Motifs of 4CL and ACS proteins were identified with the Multiple Expectation Maximization for Motif Elicitation (MEME) online tool. Note that the length of each box in the proteins does not represent the actual motif size, and different motifs are indicated by different colored boxes based on results of the MEME analysis. The conserved amino acid sequences and length of each motif are shown in Table S2. Clades are defined in Figure 1.

Figure 6

Expression levels of Pb4CL/PbACS genes during the eight stages of pear fruit development at 15 days after flowering (DAF), 39 DAF, 47 DAF, 55 DAF, 63 DAF, 79 DAF, 102 DAF and 145 DAF. These expression patterns were obtained using qRT-PCR, and the relative expression was log2 transformed. B: clade B genes; C: clade C genes; D: clade D genes; E: clade E genes; F: clade F genes; and 4CLs: 4CL genes. Clades are defined in Figure 1.

Figure 7

Expression patterns of ACS/4CL genes in Arabidopsis in different organs. R, root; St, stem; L, leaf; S3, siliques with seeds, stage 3 (mid-globular to early heart embryos); S4, siliques with seeds, stage 4 (early to late heart embryos); S5, siliques with seeds, stage 5 (late heart to mid torpedo embryos); S6, seeds, stage 6, without siliques; mid to late torpedo embryos; S7, seeds, stage 7, without siliques (late torpedo to early walking-stick embryos); S8, seeds, stage 8, without siliques (walking-stick to early curled cotyledons embryos); S9 seeds, stage 9, without siliques (curled cotyledons to early green cotyledons embryos); and S10, seeds, stage 10, without siliques (green cotyledons embryos). Clades are defined in Figure 1.

Figure 8

Expression patterns of ACS/4CL genes in rice in different organs. Se5, seeds at 5 DAP; Se10, seeds at 10 DAP; Sh, shoots; Le20, leaves at 20 days; Le, seedlings at the four-leaf stage; Em25, embryos at 25 DAP; En25, endosperm at 25 DAP; An, anthers; and Pi, pistils. Clades are defined in Figure 1.