Integrated transcriptomic and metabolomic analyses elucidate the mechanism by which grafting impacts potassium utilization efficiency in tobacco

Abstract

Background: Potassium plays a crucial role in determining the quality of flue-cured tobacco leaves. Our prior investigations have demonstrated that using potassium-efficient rootstocks through grafting offers a viable solution to the prevalent issue of low potassium levels in Chinese flue-cured tobacco leaves. Nevertheless, the specific molecular mechanisms responsible for the increase in potassium content following grafting in tobacco leaves have yet to be elucidated. This study revealing for the first time how grafting improves potassium utilization efficiency through combined transcriptome and metabolome analysis.

Results: This study selected Wufeng NO. 2, a potassium-efficient variety, and Yunyan 87, a main cultivar, as the research subjects to investigate the underlying reasons for differential potassium utilization efficiency among different tobacco rootstocks through transcriptome and metabolic data analysis of grafted tobacco. The results showed a considerable increment of 90.1% in the potassium content of the grafted tobacco leaves. Overall, 2044 differentially expressed genes were identified through transcriptome analysis, with the majority being enriched in plant hormone signal transduction and the MAPK pathway. Metabolome analysis revealed 175 metabolites with significant differences, primarily involving primary metabolites such as amino acids and carbohydrates. Among these, there was an increase in the metabolites levels related to glycolysis, amino acid metabolism, and the TCA cycle pathway in grafted tobacco leaves. The key metabolites and genes in the above pathways were selected for Mantel-Pearson correlation analysis, leading to the identification of 2 genes and 3 metabolites, including IAA, CIP1, D-fructose, Fumaric acid and Oxoglutaric acid, that were significantly associated with the increased potassium content in grafted tobacco.

Conclusions: This study uncovers the intricate molecular mechanism behind grafting tobacco to enhance potassium utilization efficiency, thereby offering theoretical support for enhancing crop nutrient utilization efficiency through grafting technology.

Keywords: Grafting; Metabolome; Potassium; Tobacco; Transcriptome.

Fig. 1

The effects of grafting on the chemical composition of FCTLs. Y, Yunyan 87; Y/W, grafted tobacco treatment (Y grafted onto Wufeng No.2). * and ** stand for P < 0.05 and P < 0.01, respectively

Fig. 2

Circle and (A) and bubble (B) charts of KEGG enrichment. (A) The pathway of the first 20 enrichments are shown by the outer circle (first circle). Different A classes are distinguishable by their different colors. The Q values and the number of pathways in the differential background of the studied genes are shown in the second circle. The more length of the bars and their higher red color intensity indicate smaller Q values and the larger number of genes in the background, respectively. The third circle is a bar chart showing the proportion of up- (dark purple) and down-adjusted (light purple) differential genes that have been adjusted up and down; (B) Various colors represent different functional categories. The orange line indicates a threshold with a P_value=0.05. The larger size of a bubble in this chart means more enriched genes in that pathway

Fig. 3

KEGG gene - pathway network. Stars represent pathways, dots represent genes, and straight lines represent the connections between pathways and genes

Fig. 4

Circle (A) and bubble (B) charts of GO enrichment. (A) The outer (first) circle indicates the GO term of the first 20 enrichments. Each color represents a specific A class. The second circle shows Q values and the number of GO terms in the background of differential gene. The more length of a bar and its higher red color intensity illustrate the smaller Q value and more background genes. The third circle shows a bar chart of the proportion of up- (dark purple) and down-adjusted (light purple) differential genes; (B) Each color displays a specific functional classification. The orange line shows a threshold at P_value=0.05. The larger size of the bubbles means a higher number of genes enriched in the current GO term

Fig. 5

Differentially expressed metabolites(DEMs) analysis between Y and Y/W. (A) Primary classification of DEMs and the numbers of up- and down-regulated metabolites. (B) The Y/W upregulated metabolites include 11 amino acid metabolites. (C) The Y/W upregulated metabolites include 14 carbohydrate metabolites. (D) The Y/W upregulated certain metabolites include 11 lipid metabolites. (E) A column chart depicting the impact and P_value of various KEGG pathways of differentially expressed metabolites (DEMs). Y, Yunyan 87; Y/W, grafted tobacco treatment (Y grafted onto Wufeng No.2)

Fig. 6

DEGs and DAMs of the key pathway (alanine, aspartate and glutamate metabolism) in tobacco leaves in response to grafting treatment. The pastel yellow background represents the metabolism pathway of aspartate, alanine, and glutamate. The mint green box illustrates the pathway of citrate cycle (TCA cycle). The red font represents metabolites with upregulated abundance in Y/W. The white font denotes the genes influenced by the grafting treatment in Y/W (Genes with upregulated expression were highlighted in white font on a red background, whereas genes with downregulated expression were highlighted in white font on a green background). The color change from deep blue to deep red in the color block represents differences in the abundance or expression levels of metabolites or genes between Y and Y/W. Deep blue indicates a lower level of gene expression or metabolite abundance, whereas deep red signifies the opposite

Fig. 7

The Mantel test revealed the key drivers of the chemical composition changes in tobacco leaves caused by grafting. Edge width of the lines recourses to “r” of Mantel for statistic of relevant distance correlations. The statistical importance is highlighted by line colors. Gradient color bar on the right side displays Spearman’s correlation coefficients to compare key metabolites and genes pairwise. TN, Total nitrogen; K, Potassium; RS, Reducing sugar

Fig. 8

Genes and metabolites closely related to the effect of grafting on K content in tobacco leaves