Transcriptome Analysis Reveals Key Pathways and Hormone Activities Involved in Early Microtuber Formation of Dioscorea opposita

Abstract

Chinese yam (Dioscorea opposita) is an important tuberous crop used for both food and medicine. Despite a long history of cultivation, the understanding of D. opposita genetics and molecular biology remains scant, which has limited its genetic improvement. This work presents a de novo transcriptome sequencing analysis of microtuber formation in D. opposita. We assembled cDNA libraries from different stages during the process of microtuber formation, designated as initial explants (EXP), axillary bud proliferation after three weeks (BUD), and microtuber visible after four weeks (MTV). More differentially expressed genes (DEGs) and pathways were identified between BUD vs. EXP than in MTV vs. BUD, indicating that proliferation of the axillary bud is the key stage of microtuber induction. Gene classification and pathway enrichment analysis showed that microtuber formation is tightly coordinated with primary metabolism, such as amino acid biosynthesis, ribosomal component biosynthesis, and starch and sucrose metabolism. The formation of the microtuber is regulated by a variety of plant hormones, including ABA. Combined with analysis of physiological data, we suggest that ABA positively regulates tuberization in D. opposita. This study will serve as an empirical foundation for future molecular studies and for the propagation of D. opposita germplasm in field crops.

Figure 1

Microtuber induction and formation stages in D. opposita. Pictures of D. opposita: at the stages of initial explant (EXP), axillary bud proliferation after three weeks (BUD), and microtuber visible after four weeks (MTV). The arrow indicates a microtuber. Bar = 5 mm.

Figure 2

Gene Ontology (GO) assignments for the microtuber induction and formation transcriptome of D. opposita. Results are summarized under three main GO categories: biological process, cellular component, and molecular function. The left y-axis indicates the percentage of a specific subcategory of genes in each main category. The right y-axis indicates the number (count) of genes.

Figure 3

KOG functional classification for the microtuber induction and formation transcriptome of D. opposita. From a total of 181,047 de novo assembled transcripts, 9,404 transcripts with significant homologies in the KOG database (E value ≤ 1e − 3) were classified into 26 KOG categories.

Figure 4

KEGG functional classification for the microtuber induction and formation transcriptome of D. opposita. From a total of 181,047 de novo assembled transcripts, 7,278 transcripts with significant homologies in the KEGG database were classified into 5 KEGG categories. A: cellular processes; B: environmental information processing; C: genetic information processing; D: metabolism; and E: organismal systems.

Figure 5

Differentially expressed genes (DEGs) in the microtuber formation-stage comparisons. (a) DEG Venn diagram. The sum of the numbers in each large circle represents the total number of DEGs of the comparative combination, and the circle overlapping part represents what the DEG combination has in common. (b) Whole-study overview of log-fold changes in gene expression in comparisons BUD vs. EXP (upper left), MTV vs. EXP (upper right), and MTV vs. BUD (down left). The x-axis indicates the log-fold changes between the two samples. The y-axis indicates the absolute expression levels (–log₁₀ (Padj)). The number of up- or downregulated genes in BUD vs. EXP, MTV vs. EXP, and MTV vs. BUD are shown in the down-right panel.

Figure 6

The influence of exogenous ABA on the rates of microtuber formation. ST: sodium tungstate. Data are presented as means ± SD. Asterisks indicate significance (^∗P < 0.01 versus control, Student's t-test).