The Biosynthesis of Heterophyllin B in <em>Pseudostellaria heterophylla</em> From <em>prePhHB</em>-Encoded Precursor
Supplementary information for Figure 4. Identification of prePhHB-encoded linear peptide via HPLC and LC-MS. (A) Purity testing via HPLC. (B) Molecular weight detection via LC-MS.
Supplementary information for Figure 4. HPLC chromatograms showing products of reactions with the conversion of preheterophyllin B (linear polypeptide) to HB (cyclic peptide) in vitro. (i) HB standard; (ii) Control group, no polypeptide was added; (iii) Polypeptide group (mAU, milli-absorbance unit; the unit of retention time is min).
PCR amplification of the full-length coding sequence of prePhHB from genomic DNA extracted from transgenic tobacco. (A) PCR amplification using specific primers for the prePhHB gene. (B) PCR amplification using specific primers for the plant expression vector PLGNL. The left M is 5000 bp Marker and the right M is 500 bp Marker; CK1: Wild-type tobacco; CK2: PLGNL-prePhHB plasmid DNA; CK3: LBA4404-PLGNL-prePhHB bacteria liquid; Numbers 1 to 50: transgenic tobacco plants.
Supplementary information for Figure 5. (A) Kanamycin sensitivity tests that the above picture is kanamycin-resistant plants and the following picture is albino plants. (B) RT-PCR analysis of relative mRNA levels of prePhHB and ACTIN in stems, leaves, and roots of transgenic and wild-type plants. ACTIN gene was used as an internal control. Transgenic lines (L1, L9, L10, L22, L25, L42 and L47) were transformed by Agrobacterium-mediated transformation containing the pLGNL-prePhHB.
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.
We thank Jun Li, who provided us with the vectors pLGNL and wild-type tobacco seeds. We thank Ling Ding, Chuanzhi Kang, Dengkai Long, Haixia Shi, Jie Chen, Wangyin Feng, and Qing Liang for their help in experiments. We thank Guanghong Cui for her valuable comments in the revision of this article.