Gene expression profiling reveals large regulatory switches between succeeding stipe stages in Volvariella volvacea

Abstract

The edible mushroom Volvariella volvacea is an important crop in Southeast Asia and is predominantly harvested in the egg stage. One of the main factors that negatively affect its yield and value is the rapid transition from the egg to the elongation stage, which has a decreased commodity value and shelf life. To improve our understanding of the changes during stipe development and the transition from egg to elongation stage in particular, we analyzed gene transcription in stipe tissue of V. volvacea using 3'-tag based digital expression profiling. Stipe development turned out to be fairly complex with high numbers of expressed genes, and regulation of stage differences is mediated mainly by changes in expression levels of genes, rather than on/off modulation. Most explicit is the strong up-regulation of cell division from button to egg, and the very strong down-regulation hereof from egg to elongation, that continues in the maturation stage. Button and egg share cell division as means of growth, followed by a major developmental shift towards rapid stipe elongation based on cell extension as demonstrated by inactivation of cell division throughout elongation and maturation. Examination of regulatory genes up-regulated from egg to elongation identified three potential high upstream regulators for this switch. The new insights in stipe dynamics, together with a series of new target genes, will provide a sound base for further studies on the developmental mechanisms of mushroom stipes and the switch from egg to elongation in V. volvacea in particular.

Figure 1. Four developmental stages of V. volvacea and corresponding gene expression.

(A) Showing the four developmental stages of V. volvacea as used in this study; BU, EG, EL and MA, and the three intermediate transitions; T1, T2 and T3. Size of the fruiting bodies is indicated in centimeters by the ruler included in the four pictures. The button stage (BU) shows a closed pileus, a ∼0.2 cm long stipe and universal veil. The egg stage (EG) is larger, the universal veil still enveloping the ∼0.5–1 cm long stipe and pileus. The elongation stage (EL) shows a still closed pileus and largely elongated stipe (∼5–7 cm), the ruptured veil will form the volva attached to the bottom of the stipe (better visible in MA stage). The maturation stage (MA) ends with a completely unfolded pileus (picture shows pileus in process of being unfolded) and maximal stipe size of ∼5–10 cm in length and ∼0.5–1.5 cm in diameter. (B) Venn diagram showing numbers of expressed genes in stipe tissue of the four depicted stages BU, EG, EL and MA. Each stage has one corresponding circle in the Venn diagram. Overlap between circles and gene numbers herein indicate co-expression of that number of genes between respective stages. For example, the BU stage has 182 uniquely expressed genes, 327 genes that are expressed in the BU stage and the EG stage but not in EL and MA, 371 genes expressed in BU, EG and EL but not in MA, and so on. 5,439 genes are expressed in all four stages, and co-expression between BU and EG (327) is considerably higher than between EG and EL, or EL and MA.

Figure 2. Changes in gene expression between succeeding stages; transitions T1, T2 and T3.

Differentially expressed genes (DEGs) are divided over three groups of absolute log2 fold changes in expression; 2 to 4 fold (orange), 4 to 8 fold (purple) or 8 or more fold (green). Genes with expression in only one of two compared stages (thus uniquely expressed in that particular comparison) predominantly show >8 fold changes and are additionally indicated between brackets within this group. Considerably more genes are up-regulated from BU to EG, whereas more genes are down-regulated from EG to EL. Numbers of up- and down-regulated genes from EL to MA are more balanced, although more genes are down- than up-regulated. In each transition, the largest number of DEGs is represented by the 2–4 fold group (orange). Relative high numbers of genes are completely turned off from EG to EL (135) and turned on from EL to MA (97).

Figure 3. COG and KEGG based functional annotation of differentially expressed genes.

Clear changes in regulation of biological functions are shown. The most important changes are: almost exclusive up-regulation of DNA replication and cell cycle related processes from BU to EG, down-regulation of those processes from EG to EL, and up-regulation of transport related processes from EL to MA (even though more genes are down- than up-regulated during transition from EL to MA).

Figure 4. Differentially expressed genes associated to the cell cycle pathway in transition T1 and T2.

(A) DEGs between BU and EG, associated to the KEGG cell cycle pathway in the KEGG database. All except one gene are up-regulated. Up-regulated genes are boxed red, down-regulated genes are boxed green. Asterisks indicate genes for which DGE data was verified by relative Q-PCR (Table S7B). (B) DEGs between EG and EL, associated to the KEGG cell cycle pathway in the KEGG database. All except one gene are down-regulated. Up-regulated genes are boxed red, down-regulated genes are boxed green. Asterisks indicate genes for which DGE data was verified by relative Q-PCR (Table S7B).