Full-length transcriptome analysis of shade-induced promotion of tuber production in Pinellia ternata

Abstract

Background: Pinellia ternata is native to China and has been used as a traditional herb due to its antiemetic, antitussive, analgesic, and anxiolytic effects. When exposed to strong light intensity and high temperature during the reproductive growth process, P. ternata withers in a phenomenon known as "sprout tumble", which largely limits tuber production. Shade was previously found to delay sprout tumble formation (STF); however, no information exists regarding this process at the molecular level. Hence, we determined the genes involved in tuber development and STF in P. ternata.

Results: Compared to that with natural sun-light (control), shade significantly induced chlorophyll accumulation, increased chlorophyll fluorescence parameters including initial fluorescence, maximal fluorescence, and qP, and dramatically repressed chlorophyll a:b and NPQ. Catalase (CAT) activity was largely induced by shade, and tuber products were largely increased in this environment. Transcriptome profiles of P. ternata grown in natural sun-light and shaded environments were analyzed by a combination of next generation sequencing (NGS) and third generation single-molecule real-time (SMRT) sequencing. Corrections of SMRT long reads based on NGS short reads yielded 136,163 non-redundant transcripts, with an average N50 length of 2578 bp. In total, 6738 deferentially-expressed genes (DEGs) were obtained from the comparisons, specifically D5S vs D5CK, D20S vs D20CK, D20S vs D5S, and D20CK vs D5CK, of which, 6384 DEGs (94.8%) were generated from the D20S vs D20CK comparison. Gene annotation and functional analyses revealed that these genes were related to auxin signal transduction, polysaccharide and sugar metabolism, phenylpropanoid biosynthesis, and photosynthesis. Moreover, the expression of genes enriched in photosynthesis appeared to be significantly altered by shade. The expression patterns of 16 candidate genes were consistent with changes in their transcript abundance as identified by RNA-Seq, and these might contribute to STF and tuber production.

Conclusion: The full-length transcripts identified in this study have provided a more accurate depiction of P. ternata gene transcription. Further, we identified potential genes involved in STF and tuber growth. Such data could serve as a genetic resource and a foundation for further research on this important traditional herb.

Keywords: Pinellia ternata; Shade; Single-molecule real-time sequencing; Sprout tumble; Transcriptome; Tuber production.

Fig. 1

Effect of shade on the growth of P. ternata. Effect of shade on the rate of STF (a) and tuber weight (b) of P. ternata grown in a control and a shaded environment. Tuber weight of P. ternata grown in control and shade environment. Tubers harvested in control (c) and shaded environment (d) after all P. ternata had STF. Data are presented as mean ± SD (n > 30). * indicates significant difference (P < 0.05)

Fig. 2

Effect of the 20-d-shade treatment on SOD, CAT, and POD enzymatic activity in P. ternata. Data are presented as mean ± SD (n > 30). * indicates significant difference (P < 0.05)

Fig. 3

Length distribution of the P. ternata transcripts

Fig. 4

Clustering analysis of the DEGs. a Hierarchical clustering graph of the 6738 DEGs based on average log₁₀(FPKM+ 1) values of all genes in each cluster. b statistics of upregulated- and downregulated-expression of DEGs in each cluster

Fig. 5

Enriched GO Terms of DEGs generated from the D20S vs D20CK group. BP and MF are abbreviations of biological process and molecular function, respectively

Fig. 6

KEGG enrichment analyses with the DEGs generated from the D20S vs D20CK group. The rich factor is the ratio of differentially expressed genes versus all annotated genes in corresponding pathways

Fig. 7

The 16 genes with differential expression when P. ternata is grown for 5 and 20 d in control and shaded environment, respectively. All the data represent the values relative to those of control at 5d. Data are presented as mean ± SD. * means differed significantly (P < 0.05). PIF3: phytochrome-interacting factor 3; HY5: elongated hypocotyls 5; CAB151: chlorophyll a/b-binding proteins 151; RUB: rubisco; SOD: superoxide dismutase; CAT: catalase; POD: polyamine oxidase; GR: glutathione reductase; ARF1/19: auxin response factor 1/19; PIN1/6: PIN FORMED1/6; XET: β-xyloglucan endotransglycosylase; RGP: reversible glycosylated polypeptides; SUS: sucrose synthase; and CHS: chalcone synthase

Fig. 8

Putative gene interaction model for tuber growth of P. ternata in a shaded environment. A shaded environment suppresses light signaling and increases the photosynthetic efficiency by repressing PtHY5 expression and increasing the transcripts of PtPIF3, PtCAB151, and PtRUB, which not only promote tuber growth directly, but also reduces ROS content via remitting photoinhibition, thereby delaying STF and facilitating tuber growth indirectly. The reduction in PtHY5 expression causes auxin signaling by up-regulating the expression of PtPIN1, PtPIN6, PtARF1, and PtARF19 which further increases the expression of PtXET, PtRGP, and PtSUS, accelerating tuber growth. Additionally, the induction of PtCAT and PtGR in a shaded environment promotes tuber growth by indirectly eliminating ROS accumulation while the up-regulation of PtCHS expedites tuber growth