Integrated transcriptomics and proteomics revealed that exogenous spermidine modulated signal transduction and carbohydrate metabolic pathways to enhance heat tolerance of lettuce

Abstract

Lettuce (Lactuca sativa L.) is sensitive to high temperatures, and the growth is inhibited under excessive temperature. Spermidine can improve the ability of lettuce to tolerate high temperatures, however, the molecular mechanism was poorly understood. The molecular mechanism of lettuce response to heat stress (2h) were investigated by physiology, transcriptome, and proteome. The results showed that 781 differentially expressed genes (DEGs) and 255 differentially expressed proteins (DEPs) were detected in lettuce treated with spermidine under heat stress. The DEGs and DEPs of lettuce were treated with 1 mM spermidine under high temperatures stress. There were 718/236 genes/proteins with the same expression trend. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed the genes were mainly enriched in intracellular signal transduction and carbohydrate metabolism pathways, which stimulated the expression of genes/proteins related to hormone and mitogen-activated protein kinase (MAPK) signal transduction pathways, starch and sucrose metabolism, pentose and glucose mutual transformation pathways. It also increased the contents of auxin and cytokinin, starch and soluble sugar. String network analysis showed that spermidine promoted material transport and antioxidant enzyme activity to improve lettuce resist high-temperature stress by removing superoxide radicals, binding and central transport of nuclear pores. In summary, signal transduction and gluconeogenic pathways may be the main ways in which spermidine improve lettuce to tolerate in heat stress. These results increase the understanding of the heat tolerance of lettuce at the transcriptional and protein levels, and provide a better understanding of the heat tolerance mechanism of lettuce.

Keywords: Exogenous spermidine; Heat stress; Lettuce; Proteome; Transcriptome.

Fig. 1

Proteome analysis of exogenous spermidine on lettuce seedlings under high-temperature stress. A DEPs in each treatment group. B Volcano plot of DEPs in HSvsH. The volcano plot features left red dots for downregulated DEPs, right red dots for upregulated DEPs, and blue-green dots for metabolites with nonsignificant differences. C GO analysis of significantly up-regulated proteins in HSvsH. D GO analysis of significantly down-regulated proteins in HSvsH

Fig. 2

KEGG analysis of DEPs of HSvsH. A KEGG-enriched histogram of up-regulated proteins. B KEGG-enriched histogram of down-regulated proteins. C KEGG-enriched bubble plot of up-regulated proteins. D KEGG-enriched bubble plot of down-regulated proteins. The size of the circle represents the number of DEPs

Fig. 3

Transcriptome group analysis of exogenous spermidine on lettuce seedlings under high-temperature stress. A DEGs in each treatment group. B GO analysis of DEGs in HSvsH. C KEGG pathway categorization of DEGs in HSvsH. D KEGG-enriched histogram of DEGs in HSvsH. E KEGG-enriched bubble diagram of DEGs in HSvsH. The size of the circle represents the number of DEGs

Fig. 4

Transcriptome and proteome integration analysis of exogenous spermidine on lettuce seedlings under high-temperature stress. A GO pathway categorization of DEGs in HSvsH. B GO analysis of DEGs in HSvsH. C KEGG-enriched histogram of DEGs in HSvsH. D KEGG pathway categorization of DEGs in HSvsH. E KEGG-enriched bubble diagram of DEGs in HSvsH. The size of the circle represents the number of DEGs

Fig. 5

Heatmap of gene/protein expression related to phytohormone signaling pathways. Red color indicates an increase; green and color indicate decreases. Double lines, cytomembrane; Grey wide dotted line, nuclear membrane; Grey wide line, endoplasmic reticulum membrane; +u, ubiquitination; +p, phosphorylation; -p, dephosphorylation , indirect effect; , activation; , inhibition; , expression; , indirect effect; , dissociation. In the heat map, red indicates high expression and blue indicates low expression

Fig. 6

Effect of exogenous spermidine on phytohormones in lettuce under high-temperature stress. A IAA content. B ABA content. C CTK content. D BR content. Values are the mean ± SD (n = 3). Different lowercase letters indicate a significant difference among treatments (P < 0.05)

Fig. 7

Heatmap of gene/protein expression related to MAPK signaling pathways. Red color indicates an increase; green and color indicate decreases. Double lines, cytomembrane; Grey wide dotted line, nuclear membrane; Grey wide line, endoplasmic reticulum membrane; +u, ubiquitination; +p, phosphorylation; -p, dephosphorylation ,indirect effect; ,activation; , inhibition; , expression; , indirect effect; ,dissociation. In the heat map, red indicates high expression and blue indicates low expression

Fig. 8

Effect of exogenous spermidine on the carbohydrate content of lettuce under high-temperature stress. A starch content. B soluble sugar content. C fructose con-tent. D sucrose content. Values are the mean ± SD (n = 3). Different lowercase letters indicate a significant difference among treatments (P < 0.05)

Fig. 9

Heatmap of annotated carbohydrate metabolism pathways of DEGs in associated genes/proteins. In the heat map, red indicates high expression and blue indicates low expression

Fig. 10

Heatmap of expression of relevant DEGs in associated genes/proteins in the starch and sucrose metabolic pathways. FRK, Fructokinase, beta-fructofuranosidase (INV), glucan endo-1,3-beta-D-glucosidase (EGLC), beta-glucosidase (BG), endoglucanase (EG). In the heat map, red indicates high expression and blue indicates low expression

Fig. 11

Heatmap of expression of relevant DEGs in associated genes/proteins in the pentose and glucuronide interconversion pathway. Pectinesterase (PE), polygalacturonase (PG), galacturan 1,4-alpha-galacturonidase (GH), pectate lyase (PLY). In the heat map, red indicates high expression and blue indicates low expression

Fig. 12

Interaction network of DEPs in HSvsH. In the network, each node represents a specific protein, and each connecting line represents a predicted or experimentally verified protein-protein interaction