A transcriptomic evaluation of the mechanism of programmed cell death of the replaceable bud in Chinese chestnut

Abstract

Previous studies suggest that the senescence and death of the replaceable bud of the Chinese chestnut cultivar (cv.) "Tima Zhenzhu" involves programmed cell death (PCD). However, the molecular network regulating replaceable bud PCD is poorly characterized. Here, we performed transcriptomic profiling on the chestnut cv. "Tima Zhenzhu" replaceable bud before (S20), during (S25), and after (S30) PCD to unravel the molecular mechanism underlying the PCD process. A total of 5,779, 9,867, and 2,674 differentially expressed genes (DEGs) were discovered upon comparison of S20 vs S25, S20 vs S30, and S25 vs S30, respectively. Approximately 6,137 DEGs common to at least two comparisons were selected for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to interrogate the main corresponding biological functions and pathways. GO analysis showed that these common DEGs could be divided into three functional categories, including 15 cellular components, 14 molecular functions, and 19 biological processes. KEGG analysis found that "plant hormone signal transduction" included 93 DEGs. Overall, 441 DEGs were identified as related to the process of PCD. Most of these were found to be genes associated with ethylene signaling, as well as the initiation and execution of various PCD processes.

Keywords: Chinese chestnut; programmed cell death; replaceable bud; transcriptomics.

Figure 1

The process of replaceable bud senescence in Chinese chestnut cv. “Tima Zhenzhu.” S20: 20 days after flowering, before PCD, green buds. S25: 25 days after flowering, during PCD, yellow-green buds but no abscission layer formed. S30: 30 days after flowering, after PCD, brown-yellow buds with partial abscission layer formation. Bars = 6 mm.

Figure 2

Assessment of the relationships among different replaceable bud samples of chestnut cv. “Tima Zhenzhu” using heat mapping (a) and PCA (b), with each sample (S20, S25, and S30) containing three biological replicates (−1, −2, and −3). The triangle represents the coordinates (center point) of the mean of the three biological replicates for each group of samples.

Figure 3

(a–c) Expression profiles of DEGs in three comparison groups. Green and red dots represent down- and upregulated DEGs with FDR ≤ 0.01 and |log₂(FC)| > 1, respectively. Black dots indicate genes without expression changes. FC (fold change) was calculated based on the FPKM values in S20 vs S25, S20 vs S30, and S25 vs S30. (d) Venn diagram of DEGs in three comparison groups.

Figure 4

GO classification of the 6,137 common DEGs. GO terms were divided into three categories: molecular functions, BPs, and CCs.

Figure 5

Top 20 enriched KEGG pathways of the 6,137 common DEGs. Coloring indicates lower (red) or higher (blue) significance (P-value). The dot size is proportional to the DEG number.

Figure 6

Heat maps showing phytohormone-related DEG expression patterns, including ethylene (a), auxin (b), cytokinin (c), ABA (d), GA (e), SA (f), and BR (g).

Figure 7

The network of protein–protein interaction displayed by common DEGs involved in replaceable bud PCD of chestnut cv. “Tima Zhenzhu.” Target proteins are identified by their gene names and represented by nodes. I-V represent five network modules.

Figure 8

qRT-PCR validation of DEG expression levels obtained from RNA-seq.

Figure 9

Tentative model of PCD during replaceable bud senescence in chestnut cv. “Tima Zhenzhu.”