Transcriptomic and Metabolomic Reprogramming from Roots to Haustoria in the Parasitic Plant, Thesium chinense

Abstract

Most plants show remarkable developmental plasticity in the generation of diverse types of new organs upon external stimuli, allowing them to adapt to their environment. Haustorial formation in parasitic plants is an example of such developmental reprogramming, but its molecular mechanism is largely unknown. In this study, we performed field-omics using transcriptomics and metabolomics to profile the molecular switch occurring in haustorial formation of the root parasitic plant, Thesium chinense, collected from its natural habitat. RNA-sequencing with de novo assembly revealed that the transcripts of very long chain fatty acid (VLCFA) biosynthesis genes, auxin biosynthesis/signaling-related genes and lateral root developmental genes are highly abundant in the haustoria. Gene co-expression network analysis identified a network module linking VLCFAs and the auxin-responsive lateral root development pathway. GC-TOF-MS analysis consistently revealed a unique metabolome profile with many types of fatty acids in the T. chinense root system, including the accumulation of a 25-carbon long chain saturated fatty acid in the haustoria. Our field-omics data provide evidence supporting the hypothesis that the molecular developmental machinery used for lateral root formation in non-parasitic plants has been co-opted into the developmental reprogramming of haustorial formation in the linage of parasitic plants.

Fig. 1

Haustorial formation in T. chinense. (A) T. chinense in their natural habitat (near the Kizu River, Kyoto, Japan). The blue dashed line traces a T. chinense individual; blue arrowheads indicate several T. chinense individuals. (B) Gross morphology of T. chinense haustorium and host root. (C–E) Safranin O-stained lateral haustoria at various stages: host invasion (C), xylem formation (D) and host connection (E). Arrowheads indicate haustoria. (F–H) Longitudinal sections of T. chinense haustorium and host root: whole haustorium (F), basal part of the haustorium, where de novo-formed xylem connects to the parasite root xylem (G), apical part of the haustorium, where intrusive cells invaded the host xylem (H). P, parasite; H, host; Xb, xylem bridge; Px, parasite xylem; Hx, host xylem. Scale bars = 1 mm for (A–E) and 200 μm for (F–H).

Fig. 2

Transcriptomic change in T. chinense haustorial formation. (A) Tissues from T. chinense haustorium and root regions were analyzed and are highlighted by pink and green, respectively. To remove host contamination using bioinformatics, the host root highlighted in purple was also analyzed. The detailed bioinformatics pipeline is shown in Supplementary Fig. S1. (B) Volcano plot showing the differentially expressed transcripts between T. chinense haustoria and roots. The logarithms of the fold change (log FC) of individual genes are plotted against the negative logarithm of their FDR. Negative log FC values represent up-regulation in haustoria, and positive values represent up-regulation in roots. Blue dots represent differentially expressed genes between haustoria and roots with an FDR <0.05. The most significantly up-regulated genes, as well as VLCFA biosynthesis genes, are shown. UP in haustoria, up-regulated in haustoria; UP in roots, up-regulated in roots.

Fig. 3

Gene regulatory network in developmental reprogramming of T. chinense haustorial formation. (A) Weighted gene co-expression network for the differentially expressed genes in Fig. 2B. Nodes and edges represent genes and co-expression relationships between genes, respectively. Edge width indicates the weight of gene co-expression. The seven modules determined by the Fast-Greedy modularity optimization algorithm are represented by different colored nodes (M1–M7). Selected GO terms enriched in each module (FDR <0.05) are shown, and the detailed results of the GO enrichment analysis are shown in Supplementary Dataset S7. (B) The detailed network structure of M1. The size and color coding of nodes indicate log strength and strength, respectively, whose value sums up the weights of the adjacent edges for each node. Genes related to VLCFA biosynthesis, auxin response and lateral root development are highlighted by the different gray background shading.

Fig. 4

Lipophilic metabolite profiling of the T. chinense root system. (A) Relative abundance of annotated fatty acids detected in the lipophilic metabolite profiles in T. chinense roots and haustoria. Data presented are the means of four independent biological replicates. (B) Scatter plot of standard compounds of retention index vs. chain length of alkane groups from C6 to C30 saturated fatty acids. R² is shown. The fitted line identified that the unknown fatty acid accumulated in haustoria was a 25-carbon long chain saturated fatty acid (pentacosanoic acid). (C) The chemical structure of pentacosanoic acid and its abundance (CRMN normalized values) between T. chinense roots and haustoria. (D) Schematic diagram representing recruitment of the lateral root developmental pathway associated with the accumulation of VLCFAs into developmental reprogramming of T. chinense haustorial formation. TF, transcription factor.