Morphological and stage-specific transcriptome analyses reveal distinct regulatory programs underlying yam (Dioscorea alata L.) bulbil growth

Abstract

In yam (Dioscorea spp) species, bulbils at leaf axils are the most striking species-specific axillary structure and exhibit important ecological niches. Genetic regulation underlying bulbil growth remains largely unclear so far. Here, we characterize yam (Dioscorea alata L.) bulbil development using histological analysis, and perform full transcriptional profiling on key developmental stages together with phytohormone analyses. Using the stage-specific scoring algorithm, we have identified 3451 stage-specifically expressed genes that exhibit a tight link between major transcriptional changes and stages. Co-expressed gene clusters revealed an obvious over-representation of genes associated with cell division and expansion at the initiation stage of bulbils (T1). Transcriptional changes of hormone-related genes highly coincided with hormone levels, indicating that bulbil initiation and growth are coordinately controlled by multiple phytohormones. In particular, localized auxin is transiently required to trigger bulbil initiation, and be further depleted or exported from bulbils to promote growth by up-regulation of genes involved in auxinconjugation and efflux. The sharp increase in supply of sucrose and an enhanced trehalose-6-phophate pathway at T1 were observed, suggesting that sucrose probably functions as a key signal and promotes bulbil initiation. Analysis of the expression of transcription factors (TFs) predicated 149 TFs as stage-specifically expressed; several T1-specific TFs (from Aux/IAA, E2F, MYB, and bHLH families) have been shown to play key roles in triggering bulbil formation. Together, our work provides a crucial angle for in-depth understanding of the molecular programs underlying yam's unique bulbil development processes. Stage-specific gene sets can be queried to obtain key candidates regulating bulbil growth, serving as valuable resources for further functional research.

Keywords: Bulbil; genetic regulation; growth; phytohormone; transcriptome; yam (Dioscorea alata L).

Fig. 1.

Morphology of the bulbil during key developmental stages. (A) Bulbil phenotype. (B) Photographs of the bulbil at the initiation (T1), early (T2), middle (T3), and mature stages (T4). (C–F) Paraffin sections of bulbils at T1 (C), T2 (D), T3 (E), and T4 (F) stages. The images show the zone of the junction region between the bulbil and axil. (G) Showing root primordia (RP). (H) Showing the meristematic zone (MZ). The arrows in T1 and T2 show the BP and root primordia, respectively. Scale bars (A and B), 1 cm; (C), 500 µm; (D–H), 200 µm.

Fig. 2.

Gene expression profiles during bulbil growth. (A) Principal component analysis for 12 bulbil types showing the four stage-specific groups based on all gene expression profiles. (B) Venn diagram showing the numbers of unique and overlapping expressed genes in bulbils among different developmental stages (T1–T4). (C) Comparison of the number of up- and down-regulated genes between different stages.

Fig. 3.

Stage-specific gene clusters. (A) Heat map of scaled FPKM values of 3451 stage-specific genes (with SS score >0.3) during bulbil growth. (B) Average expression profiles of each gene cluster and its corresponding enriched GO functional category. The sets of stage-specific genes are available in Supplementary Table S7.

Fig. 4.

Heat maps of gene sets involved in the cell cycle and proliferation (A), and cell wall expansion (B). The gene expression levels are standardized into Z-scores and colored in red and blue for high and low expression, respectively. Gene names (shown on the right) are described in detail in Supplementary Dataset S1.

Fig. 5.

Heat maps of gene sets associated with hormone (auxin, CK, and ABA) biosynthsis and catabolism, transport, and signaling. The expression levels are standardized per gene into Z-scores and colored in red and blue for high and low expression, respectively. Gene names (shown on the right) are described in detail in Supplementary Dataset S1.

Fig. 6.

Gene and metabolite regulation involved in starch and sucrose metabolic processes, and sucrose signaling. (A) Starch biosynthesis process. (B) Sucrose metabolism. (C) Sucrose signaling involved in the trehalose metabolism pathway. Heat maps next to the arrows represent changes in expression of genes encoding corresponding enzymes for reactions. The expression levels are standardized per gene into Z-scores and colored in red and green for high and low expression, respectively. (D) Verification of expression levels of genes encoding SUS1, VIF1, and TPS1 determined by qRT–PCR. (E) Sucrose accumulation in bulbils, demonstrating that sucrose is sharply increased at the early stage of bulbil formation (T1). Data show the mean ±SD (n=5). ADG, glucose-1-phosphate adenylyltransferase; CIN, cell wall invertase; FRK, fructokinase; HXK, hexokinase; SBE, glucan-branching enzyme; SUS, sucrose synthase; TPP, trehalose-phosphate phosphatase; TPS, trehalose-phosphate synthase; VIF1, vacuolar invertase1.

Fig. 7.

Expression profiles of transcription factors (TFs) and hypothetical regulatory modules. (A) Heat map of scaled FPKM values of 149 stage-specific TFs (with SS score >0.3) identified during bulbil growth. The TF families listed on the right show predominantly expression in corresponding stage-specific clusters. The numbers in parentheses represent the number of members from TF families in this cluster. (B) Cumulative expression profiles of AUX/IAA (six members) and E2F-DP (four members) TFs in the T1-specific cluster. (C) Hypothetical models of AUX/IAA- and E2F-DP- mediated gene regulation in the T1-specific cluster. The heat map shows the expression of genes involved in GO functional categories by AUX/IAA and E2F-DP induction, suggesting that the two TFs and their targets are co-over-represented in the T1-specific cluster.

Fig. 8.

Schematic model of gene regulation by auxin, CK, ABA, and sucrose during bulbil growth. The localized auxin in the bulbil is transiently produced by stimulation of auxin synthesis (TAR3 and YUCCA1), and activates bulbil initiation. Further, the excess auxin is depleted and transported to maintain bulbil outgrowth by up-regulation of auxin-conjugated genes (GH3.5 and GH3.6) and auxin efflux proteins (PIN1B, LAX2, and the ABC transporter B family). CKs promote its growth through rapid activation of the CK transporter (ENT3) gene, together with suppression of CK degradative genes (CKX1/3/9/11). ABA contributes to bulbil growth through repression of ABA synthesis genes (NECDs and ZEPs) at the early stage of bulbil formation. Sucrose probably acts as a signal, and promotes bulbil growth through up-regulation of genes involved in the T6P pathway (TPS1 and TPS5). The integration of these hormones and sucrose provides a forward signaling to activate genes associated with cell division, proliferation, and expansion, which contributes key roles in maintaining cell enlargement during bulbil growth.