The Biosynthesis of Heterophyllin B in Pseudostellaria heterophylla From prePhHB-Encoded Precursor

Abstract

Plant cyclic peptides (CPs) are a large group of small molecule metabolites found in a wide variety of plants, including traditional Chinese medicinal plants. However, the majority of plant CPs have not been studied for their biosynthetic mechanisms, including heterophyllin B (HB), which is one of the characteristic chemical components of Pseudostellaria heterophylla. Here, we screened the precursor gene (prePhHB) of HB in P. heterophylla and functionally identified its correctness in vivo and in vitro. First, we developed a new method to screen the precursors of HB from 16 candidate linear peptides. According to transcriptome sequencing data, we cloned the genes that encoded the HB precursor peptides and confirmed that the prePhHB-encoded precursor peptide could enzymatically synthesize HB. Next, we generated the transgenic tobacco that expressed prePhHB, and the results showed that HB was detected in transgenic tobacco. Moreover, we revealed that prePhHB gene expression is positively correlated with HB accumulation in P. heterophylla. Mutations in the prePhHB gene may influence the accumulation of HB in P. heterophylla. These results suggest that HB is ribosomally synthesized and posttranslationally modified peptide (RiPP) derived from the precursor gene prePhHB-encoded precursor peptide, and the core peptide sequence of HB is IFGGLPPP in P. heterophylla. This study developed a new idea for the rapid identification of Caryophyllaceae-type CP precursor peptides via RNA-sequencing data mining.

Keywords: Pseudostellaria heterophylla; cyclic peptide; gene; heterophyllin B; prePhHB.

Figure 1

There is a difference in the content of HB of P. heterophylla with respect to the areas of major cultivation in China. (A) Morphological pictures of the flowers, leaves, and tuberous roots of cultivated P. heterophylla in five areas, including Jiangsu Province, Fujian Province, Anhui Province, Guizhou Province, and Shandong Province, China. (B) Analysis of fresh weight per tuberous roots in five provinces of China. Small circles of different colors were shown as the means of 3 biological replicates each containing 20 tuberous roots, and bars represent means ± SEM of 4 independent regions for “Shandong,” 3 independent regions for “Jiangsu”, 8 independent regions for “Anhui”, 16 independent regions for “Guizhou”, and 13 independent regions for “Fujian”. (C) The content of HB in the tuberous root of cultivated P. heterophylla in five provinces. Small circles of different colors also represent means of three biological replicates, and bars represent means ± SEM of n independent regions (n = 4 for “Shandong”, n = 3 for “Jiangsu”, n = 8 for “Anhui”, n = 16 for “Guizhou”, n = 13 for “Fujian”). Data between provinces (Shandong Province vs. Fujian Province, Jiangsu Province vs. Fujian Province, Anhui Province vs. Fujian Province, Guizhou Province vs. Fujian Province) were analyzed by one-way ANOVA, and **** denotes statistical significance at p < 0.0001. (D) Structure of HB (a cyclic octapeptide). (E) HPLC analysis of HB in tuberous roots of cultivated P. heterophylla from five provinces. The structure of HB, corresponding to the peaks, is marked by the red arrow.

Figure 2

Screening and identification of the preheterophyllin B gene from the P. heterophylla RNA-seq database. (A) Schematic diagram of the preheterophyllin B gene acquisition. (B) Manual alignment of the predicted amino acid sequences of cDNAs encoding putative CPs of (i) P. heterophylla, (ii) Saponaria vaccaria, and (iii) Dianthus caryophyllus. Core peptide motifs are shown in gray. (C) Nucleotide and deduced amino acid sequences of the prePhHB gene. The start codon (ATG) and the stop codon (TAA) are shaded with gray. The nucleotide sequences (yellow) and deduced amino acid sequences (dark blue) of the core peptide motif are shown. The AATAAAA frame is shaded in pink. Blue indicates the poly (A) tail. (D) Phylogenetic analysis and motif analysis of the amino acid sequences that encode the Caryophyllaceae-like CP precursors. Class I are precursors of segetalin A, segetalin B, segetalin D, segetalin G, segetalin H, segetalin K, and segetalin L of S. vaccaria. Class II is precursors of segetalin F and segetalin J of S. vaccaria. Schematic diagram for the motifs is indicated on the right of each precursors. (Yellow: leader peptide motifs, red: core peptide motifs, blue: recognition sequence motifs).

Figure 3

The relative expression level of prePhHB gene was positively correlated with the content of HB in the tissues of P. heterophylla. (A) Heat-map of the differentially expressed unigenes associated with biosynthesis of HB in phloem of tuberous roots, flower, stems, leaves, and xylem of tuberous roots of P. heterophylla. prePhHB is highlighted with a red box. Three biological replicates were plant numbers 1, 3, and 4, respectively (Li et al., 2016). (B) The content of HB in phloem of tuberous roots, stems, leaves, and xylem of tuberous roots of P. heterophylla. Bars represent the means values ± SEM of three biologically independent replicates. (C) Correlation between the relative expression level of prePhHB and the content of HB (i.e., Fig 3A vs. 3B), each independent point represents means of three technical replicates and the independent points, with same color, which indicate three biological replicates (r² = 0.8768, p < 0.0001).

Figure 4

Enzymatic verification of the conversion of preheterophyllin B to HB in vitro. (A) Schematic diagram in which crude enzymes are combined with polypeptide in an aqueous environment to convert the substrate to HB through a series of reactions. (B and C) Quantitative analysis of the HB is formed by polypeptides under the action of crude enzymes in vitro, bars represent means ± SEM of four independent experiments, and asterisks represent significant difference between control and polypeptide, with a one-way ANOVA test (***p < 0.001).

Figure 5

Recombinant expression of prePhHB in tobacco leaves enables HB production. (A) Quantification of the RT-PCR analysis results of the Figure (S4B) using ImageJ software. All analyses were performed with three biological replicates, and bars represent means ± SEM. (B) Total ion chromatograms (TIC) of the extracts of wild-type and transgenic tobacco are shown. (i) HB standard test via UPLC-MS/MS; (ii) no HB for WT tobacco was detected; (iii) expression of prePhHB leads to accumulation of HB (RT = 4.12 min). (C) Production of HB in transformed tobacco generated using Agrobacterium tumefaciens strain harboring pLGNL-prePhHB or WT tobacco. HB was determined by UHPLC-MS/MS. N.D. is not detected, and data are the means ± SEM of three biological replicates.

Figure 6

Mutations of prePhHB may result in accumulation of HB in “FJZR”. (A) HB was identified and quantified using HPLC-DAD, and N.D. is not detected. Bars represent the mean values ± SEM of three biological replicates. (B) The expression pattern of prePhHB in tuberous roots of P. heterophylla that were originated in different regions: “JSJR”, “FJZR”, “AHXZ”, and “GZSB”, respectively, using RT-qPCR. The values are the means ± SEM of three biological replicates. (C) The sequence alignment of prePhHB among P. heterophylla from the “JSJR”, “FJZR”, “AHXZ”, and “GZSB”. Mutation sites were highlighted by striking color. Red areas represent insertion mutation, and purple areas represent deletion mutation.