Tissue samples of P. polyphylla var. yunnanensis during the four dominant developmental stages were collected and investigated using high-performance liquid chromatography and RNA sequencing. Polyphyllin concentrations in the different tissues were found to be highly dynamic across developmental stages. Specifically, decreasing trends in polyphyllin concentration were observed in the aerial vegetative tissues, whereas an increasing trend was observed in the rhizomes. Consistent with the aforementioned polyphyllin concentration trends, different patterns of spatiotemporal gene expression in the vegetative tissues were found to be closely related with polyphyllin biosynthesis. Additionally, molecular dissection of the pathway components revealed 137 candidate genes involved in the upstream pathway of polyphyllin backbone biosynthesis. Furthermore, gene co-expression network analysis revealed 74 transcription factor genes and one transporter gene associated with polyphyllin biosynthesis and allocation.
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A schematic diagram was drawn to understand polyphyllin biosynthesis and the underlying regulatory mechanism (Fig. 7). Polyphyllin concentration in different tissues of P. polyphylla var. yunnanensis constantly varied with plant development. Polyphyllin concentration in the aerial parts, specifically the leaves, gradually declined, while that in the rhizomes elevated and remained high even at the senescence stage. The cubic heat map showed that 31 unigenes encoded for 16 key enzymes in the upstream of polyphyllin biosynthesis pathway, which were observed in both the spatiotemporal gene expression patterns and the modules associated with polyphyllin. In particular, the PBGs in the rhizomes were highly expressed at the pollination stage, while those in the leaves were highly expressed at the vegetative stage and gradually decreased with development. Based on gene co-expression network of the TFs and PBGs, six typical TF gene types previously reported to be involved in triterpene saponin biosynthesis exhibited putative regulation. Additionally, the key candidate transporter for polyphyllin accumulation, ABCB1, was also observed in the schematic diagram.
In addition, the genes responsible for synthesizing metabolites may be highly expressed in the tissues where metabolites are mainly stored, while translocation of natural compounds among plant organs also often occurs [31]. Through GCNA, we screened 13 transporter-coding genes belonging to GST, SLC, TMED, and ABC transmembrane transporter families that highly correlated with the identified PBGs. Notably, we discovered that the predicted ABCB1 was closely associated with polyphyllin. ABCB1 was highly expressed in the rhizomes during pollination and fruiting stages and in the leaves at the pollination stage. Plant ABC proteins are commonly classified into 13 subfamilies based on protein size (full or half), orientation (forward or reverse), idiotypic transmembrane or linker domains (presence or absence), and overall sequence similarity [47]. Interestingly, plants harboring mutations in ABC transporters exhibit deformed phenotypes, many of which are related to developmental processes and environmental adaptations [48]. For instance, the CjABCB1 localized at the plasma membrane of Coptis japonica is preferentially expressed in the xylem tissue of the rhizomes and catalyzes berberine translocation from the root to the rhizome to protect against pathogens [49]. ABC transporters are also involved in root exudation processes: one ABC transporter can transport structurally different compounds [50]. A pleiotropic drug resistance (PDR)-type ABC transporter in Arabidopsis and Spirodela can recognize sclareol or other natural compounds with similar structures and export them [51]. Thus, we speculate that ABCB1 is a crucial polyphyllin transporter for P. polyphylla. Taken together, GCNA utilization can facilitate the identification of transcription regulators and transporters that play a combinatorial role in regulating polyphyllin biosynthesis and accumulation. Additionally, we found that de novo transcriptome assembly of species with huge genome through next generation sequencing has some difficulties. The genome size of P.polyphylla was estimated over 50 Gb and it probably contains enormous homologs according to plant species with giant genome. In absence of the genome, billions of short reads generated from next generation sequencing bring the difficulties in transcriptome assembly. The data redundancy and fragments of genes inevitably occur and stand out in case of giant genome, absence of genome, and increasing sequence depth, though transcriptome sequencing quality was strictly controlled and the data cluttering and filtration analyses were conducted. These factors can influence the related ratios like proportion of protein-coding gene and noncoding gene. We will consider the Single-molecule, Real-time (SMRT) sequencing to reduce the impacts of technology and method on transcriptome assembly in the further research. 2ff7e9595c
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