Identification and functional analysis of novel precursor genes in cyclic peptide biosynthesis in Pseudostellaria heterophylla

Abstract

Background: Pseudostellaria heterophylla, a member of the Caryophyllaceae family, is widely used in traditional Chinese medicine due to its bioactive cyclic peptides (CPs) with immunomodulatory functions. Caryophyllaceae- like CPs, one of the largest types plant-derived CPs, typically consist of 5–12 amino acids and are derived from ribosomally synthesized peptide precursors. The diversity of CPs arises from variations in their core peptide sequences. However, the precursor genes responsible for Caryophyllaceae-like CPs biosynthesis in P. heterophylla remain largely uncharacterized.

Results: In this study, barcoding PCR combined with high-throughput sequencing was used to efficiently genotype precursor genes encoding CPs in P. heterophylla. This approach enabled the identification of known and novel precursor genes, including prePhHB_1, prePhHB_2, prePhPE and prePhPN. The core peptide regions showed high variability, while the leader and follower regions were relatively conserved, with a few nucleotide mutations. Tissue-specific expression analysis revealed that prePhHB was predominantly expressed in the phloem and fibrous roots, while prePhPE was specifically expressed in the xylem. prePhPN exhibited low expression level and was mainly detected in the phloem and stem. Moreover, the expression of these precursor genes was responsive to abscisic acid and nitrogen stress. RNA in situ hybridization revealed that prePhPE transcripts were primarily localized in the xylem and phellem of the roots. Transient co-expression in Nicotiana benthamiana indicated that prePhPE is involved in the biosynthesis of Pseudostellarin E (PE).

Conclusions: Barcoding PCR combined with high-throughput sequencing provides an effective strategy for investigating CP precursor genes, including those with low expression. The results reveal conserved features in CP precursor genes and highlight a previously unrecognized mechanism contributing to CP diversity. The prePhPE gene was identified as the precursor gene of PE, which accumulates mainly in the xylem of P. heterophylla roots. prePhPN may be a precursor gene for a novel CP.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12870-025-06972-2.

Keywords: PrePhPE; Pseudostellaria heterophylla; High-throughput sequencing; Precursor gene; Pseudostellarin E biosynthesis.

Fig. 1

Nucleotide sequence alignment of putative cyclic peptide precursor genes identified from barcode sequence of Pseudostellaria heterophylla. These precursor genes encoded the precursor peptides of 3 different CPs: PE, HB, and PN. Red frame represents the variation of the nucleotide sequence. Blue frame indicates a nonsense mutation. The conserved sequences are visualized

Fig. 2

Amino acid sequence analysis of precursor genes from P. heterophylla. A Conserved and variation regions of precursor peptides, with red frame indicating amino acid variation. B Phylogenetic analyses of precursor peptides detected from P. heterophylla. Black lines represent precursor peptides of PE, red line represents precursor peptides of HB, and the purple lines represent precursor peptides of PN

Fig. 3

Visualization of three clade precursor peptides from P. heterophylla. Red letters indicate conserved C-terminal residues. Purple letters indicate mutated amino acids in the follower region of HB-type peptides. And 3 genes encode precursors peptide of HB, including PrePhHB, PrePhHB_1 and PrePhHB_2. One precursor gene was detected for encoding precursors peptide of PE and HN, respectively. Blue letters indicate amino acids variations in the leader peptide region

Fig. 4

Expression analysis of three precursor genes in different tissues of Pseudostellaria heterophylla. The relative expression levels of prePhHB (A), prePhPE (B), and prePhHN (C) in roots, phloem, xylem, leaf, fibrous roots, stem, and leaf. All data are mean ± SD (n = 3). Different lowercase letters denote significant differences by multiple comparisons using SPSS Statistics

Fig. 5

In situ hybridization localization of prePhPE in P. heterophylla. A In root, the expression of prePhPE was detected in the xylem and phellem, while weak signals were observed in the cambium, phloem, and rays. (ii) showd hybridization in root using the prePhPE sense probe. Scale bars are 1000 μm (i), 200 μm (ii, iii), and 100 μm (iv). B In stem, the expression of prePhPE was localized to the phellem, cortex and xylem. Scale bars are 100 μm (i), 100 μm (ii). ii, the hybridization in stem using the prePhPE sense probe

Fig. 6

Transient expression of prePhPE in tobacco cells leads to PE biosynthesis. A Extracted ion chromatogram trace of prePhPE overexpression in tobacco with a mass spectra peak at a [M + H]⁺ m/z value of 878.516. B Extracted ion chromatogram trace of prePhPE overexpression in tobacco and P. heterophylla tuberous roots. (C) MS data extracted at retention times 28.82- and 28.83-min. Cyclic peptides were detected by UPLC-QTOF-MS/MS in both P. heterophylla and tobacco, with peaks marked by retention time (RT) and measured molecular weight (MW)

Fig. 7

Proposed biosynthetic pathway of cyclic peptides in P. heterophylla. The precursor peptide of CPs was encoded by specific precursor genes in P. heterophylla, which were cleaved to form linear intermediates. Subsequent removal of the C-terminal region obtained the core peptide, followed by cyclization to generate mature CPs. The expression of precursor genes were in response to ABA and nitrogen