Analysis of ambient temperature-responsive transcriptome in shoot apical meristem of heat-tolerant and heat-sensitive broccoli inbred lines during floral head formation

Abstract

Background: Head formation of broccoli (Brassica oleracea var. italica) is greatly reduced under high temperature (22 °C and 27 °C). Broccoli inbred lines that are capable of producing heads at high temperatures in summer are varieties that are unique to Taiwan. However, knowledge of the early-activated pathways of broccoli head formation under high temperature is limited.

Results: We compared heat-tolerant (HT) and heat-sensitive (HS) transcriptome of broccoli under different temperatures. Weighted gene correlation network analysis (WGCNA) revealed that genes involved in calcium signaling pathways, mitogen-activated protein kinase (MAPK) cascades, leucine-rich repeat receptor-like kinases (LRR-RLKs), and genes coding for heat-shock proteins and reactive oxygen species homeostasis shared a similar expression pattern to BoFLC1, which was highly expressed at high temperature (27 °C). Of note, these genes were less expressed in HT than HS broccoli at 22 °C. Co-expression analysis identified a model for LRR-RLKs in survival-reproduction tradeoffs by modulating MAPK- versus phytohormones-signaling during head formation. The difference in head-forming ability in response to heat stress between HT and HS broccoli may result from their differential transcriptome profiles of LRR-RLK genes. High temperature induced JA- as well as suppressed auxin- and cytokinin-related pathways may facilitate a balancing act to ensure fitness at 27 °C. BoFLC1 was less expressed in HT than HS at 22 °C, whereas other FLC homologues were not. Promoter analysis of BoFLC1 showed fewer AT dinucleotide repeats in HT broccoli. These results provide insight into the early activation of stress- or development-related pathways during head formation in broccoli. The identification of the BoFLC1 DNA biomarker may facilitate breeding of HT broccoli.

Conclusions: In this study, HT and HS broccoli genotypes were used to determine the effect of temperature on head formation by transcriptome profiling. On the basis of the expression pattern of high temperature-associated signaling genes, the HS transcriptome may be involved in stress defense instead of transition to the reproductive phase in response to heat stress. Transcriptome profiling of HT and HS broccoli helps in understanding the molecular mechanisms underlying head-forming capacity and in promoting functional marker-assisted breeding.

Keywords: Biomarker; Broccoli; Genomic analysis; Head formation; High temperatures.

Fig. 1

Weighted gene co-expression network analysis (WGCNA) of temperature-associated genes in the heat-tolerant and heat-sensitive broccoli genotypes. a Clustering dendrogram of genes showing module membership in colours. The y axis represents network distance as determined by 1 - topological overlap (TO), where values closer to 0 indicate greater similarity of probe expression profiles across samples (experimental treatments). Bottom: the first band shows module membership in colours. Additional bands indicate positive (red) and negative (blue) correlation to 15 °C, 22 °C, and 27 °C (see scale bar in B). b Colours to the left represent the 14 modules in the gene co-expression network. For each module, the heatmap shows module eigengen (ME) correlations to traits (4 groups of experimental treatment). Numbers in each cell report the correlation coefficients and Student asymptotic P value (parentheses) for significant ME-trait relationship. Scale bar, right, indicates the range of possible correlations from positive (red, 1) to negative (blue, − 1). c Relationship between the WGCNA modules (brown and magenta) and the three different temperatures (upper panel) and expression of the corresponding eigengene across the samples in the modules (lower panel). The heatmap (upper panel) and barplot of eigengene expression (lower panel) have the same samples (x axis). Rows of the heatmap correspond to genes, columns to samples; red in the color key denotes overexpression, green underexpression

Fig. 2

Gene Ontology (GO) enrichment analysis of WGCNA gene modules. GO circle plot displaying gene-annotation enrichment analysis. a The brown module. b The magenta module. Radar chart shows the distribution of individual terms in the annotation categories. The expression profiles were normalized with the RMA algorithm based on median baseline. The fold changes (FC) of gene expression values (log₂ FC) were derived from 3 biological replications corresponding to each sample. Within each selected GO term, blue dot shows a gene downregulated at 22 °C (HT/HS) and red dot indicates a gene upregulated at 22 °C (HT/HS). The outer to inner layers of gray circles indicate the relative fold-change of gene expression (from higher to lower). The height of the inner rectangle represents the P value of the GO term. The rectangle is coloured with the blue-red gradient according to the z score. (P < 0.05, FDR adjusted P < 0.05) Z-score = (upregulated – downregulated) /$\sqrt{\text{upregulated}+\text{downregulated}}$

Fig. 3

Network component analysis for the proteins within the WGCNA modules. Genes belonging to the brown (a) or magenta (b) modules were used as a query in the STRING database (http://string-db.org). After excluding proteins without interaction, the visualization network contained 114 nodes (genes) and 188 edges (connections). The hub proteins are marked by circles lines. Red indicates the top 5 connection proteins; Blue indicates the proteins involved in floral development

Fig. 4

Growth and the expression of flowering-associated genes in the heat-tolerant and heat-sensitive broccoli genotypes. a Growth of the two broccoli lines at 22 °C at different times post germination (18, 28, 38, 48, 58 and 68 days). d, day. b RT-PCR analysis of flowering-associated genes at different growth stages in two broccoli genotypes. The cDNA sequences of BoFLC1–5, BoFT, BoSOC1 and Bo18S with their corresponding accession numbers were obtained from GenBank of National Center for Biotechnology Information (Additional file 9: Table S7). The primers were designed to amplify these gene transcripts specifically. Bo18s was used as an internal control for RT-PCR analysis. White bar equals 4 cm

Fig. 5

Heatmap of stress-associated signal component expression in the brown module. a Heatmap of genes encoding stress-associated signal proteins, including calmodulin (CaM), calmodulin binding protein (CaMBP), CBL-interacting protein kinase (CIPK), mitogen-activated protein kinase kinase kinase (MEKK), and calmodulin binding protein (CBP). b Heatmap of genes encoding HSP70 and HSP20 gene family. c Heatmap of genes encoding ROS homeostasis-associated proteins, including peroxidase (POD), thioredoxin (TRx), ferredoxin (FDX), catalase (CAT), and glutathione peroxidase (GPx). d Heatmap of genes encoding phytohormone-associated proteins, including auxin, cytokinin, and jasmonate

Fig. 6

Distribution and functional classification of the differentially expressed genes. a Venn diagram showing the number of upregulated genes (numbers in black) in the HT genotype and the upregulated (numbers in blue) in the HS genotypes at 15 °C, 22 °C, and 27 °C. Results were based on the FDR < 0.05 and two-fold change in expression. b Gene Ontology analysis of the significantly temperature-associated genes (fold change ≥2) by microarray analysis. The agriGO database was used to perform enrichment analysis and the negative log of the P value is shown for the significantly enriched gene categories. c MapMan analysis of the secondary metabolism-associated genes. Each gene displayed as a square, red for upregulation and blue for downregulation. d The relative expression of probes assigned as epithiospecifier protein (ESP) and branched-chain aminotransferase4 (BCAT4) at 15 °C, 22 °C, and 27 °C in the HT and HS genotypes

Fig. 7

Broccoli FLC1 promoter architecture. The sequences spanning the promoter region upstream of the start codon (− 1500 bp) were obtained by PCR using gene-specific primers. The PCR products were cloned into a TA-vector and 10 individual clones per genotype were sequenced. a Comparison of the FLC1 promoter sequences of Arabidopsis and broccoli genotypes. The distinct conserved motifs in the promoter regions are marked with vertical black lines. The gray boxes indicate identical nucleotides. b The EcoRI restriction enzyme site is denoted with triangles. c Validation of EcoRI recognition site by restriction enzyme digestion of PCR amplicons and gel electrophoresis analysis. d The AT-rich region is denoted with a black line. e The ANAERO1CONSENSUS element is denoted with a black line