Integrated transcriptomic and metabolomic analyses elucidate the mechanism by which grafting impacts potassium utilization efficiency in tobacco
Potassium (K) is a necessary nutrient that significantly impacts the quality of flue-cured tobacco leaves. It is well-documented that K influences several critical attributes, including processability, aromatic taste, and flammability of tobacco leaves, which are vital for high-quality tobacco production . Statistical analyses reveal that the optimal K content in flue-cured tobacco leaves ranges between 2.5% and 3.5%, aligning with the standards of internationally recognized high-quality tobacco . However, a substantial disparity exists in the content of this element in Chinese flue-cured tobacco leaves, which often falls short of these benchmarks, thus affecting their global competitiveness . Efforts to enhance the K content of flue-cured tobacco leaves through fertilizer or variety have faced numerous challenges. Conventional methods, such as soil fertilization and foliar application of K are not only costly but also come up with limited success rate due to factors like soil K deficiency and environmental conditions . This has motivated researchers to explore alternative approaches, including the technique of grafting, which has demonstrated great potential in addressing these limitations. Grafting has been widely recognized for its application and advantages in crop cultivation. It offers several benefits, such as improved disease resistance, enhanced stress tolerance, and better nutrient uptake efficiency . The success of grafting in improving K utilization efficiency in various crops have been reported in recent studies. For instance, grafting has been shown to enhance K uptake in tomatoes and cucumbers , leading to better growth and yield. Several studies have demonstrated that rootstock grafting can alter the expression of regulatory metabolic pathways or specific genes in scions . Additionally, epigenetic mechanisms also contribute to the plant’s response to grafting . However, there is a lack of data in the literature regarding the influence of grafting on K content in tobacco leaves. In our previous studies , it was observed that the grafting of Maryland tobacco with flue-cured tobacco enhanced K + utilization efficiency and improved certain tobacco quality indicators such as combustion. However, further exploration is needed to elucidate the underlying response signal pathway and metabolic pathway mechanisms. In tobacco, the process of K uptake and transport is controlled by various gene families with K + channels, high-affinity transporters, co-transporters, reverse transporters, and proton pumps, which are essential for energy supply . Besides the genes directly involved in K transport, certain genes associated with K signal transduction, such as cyclin-dependent kinase inhibitor 1 A ( CIP1 ) and calcineurin B-like 1 ( CBL1 ), also exhibited elevated expression levels in grafted tobacco. These genes greatly influence plant response to K stress by modulating the activity of downstream K transporters . In this research, rootstocks with high K efficiency were grafted with primary tobacco varieties. By conducting integrated analysis of transcriptomic and metabolomic data, molecular and biochemical pathways related to K utilization efficiency of grafted tobacco were revealed. The utilization of these advanced omics technologies lets us comprehensively understand the complex interactions between genetic expression and metabolic processes in grafted tobacco plants. The significance of this research lies in filling the gap regarding the impact of grafting on K utilization in tobacco. Moreover, the achievements of this work offer a scientific basis for developing more efficient and sustainable K management strategies in tobacco cultivation, which could lead to the production of flue-cured tobacco leaves with higher quality. 

Experimental design Experiments were performed at the Western Chongqing Vegetable Research and Development Center, Chongqing Academy of Agricultural Sciences, China (106 ◦ 18′ E, 29 ◦ 47′ N). K-efficient genotype tobacco ‘Wufeng No.2’ ( N.tabacum , Yichang Tobacco Company of Hubei Province, Yichang, China) and the main cultivar ‘Yunyan 87’ ( Nicotiana tabacum , Yunnan Tobacco Research Institute, Yuxi, China) were employed. The soil was a Dystric Regosol (FAO classification with a pH of 6.4) . The experiment consisted of two treatments, grafted seedling Y/W (Y grafted to Wufeng No.2) and own-rooted seedling Y (Yunyan 87). The K fertilizer used was potassium sulfate with 270 K 2 O ha − 1 . In order to avoid the effects of different sulfur levels on tobacco, superphosphate was applied to provide P 2 O 5 of 135 kg ha − 1 . Nitrogen fertilizer was applied at the rate of 90 kg N ha − 1 using urea. To make sure of a good success rate of grafting, we planted the rootstock seeds 7 d prior to the scion. After 6–8 true leaves grew on the seedlings of the rootstock, grafting was done by the ‘Split grafting’ method . During the grafting process, utilize silicone conduits or grafting clips to secure the junction between the rootstock and scion. The plants were grown at 22.5 °C under a 16-h light/8-h dark cycle using fluorescent lamps with an average photosynthetic photon flux density ( PPFD ) of 300 µmol m –2 s –1 in the greenhouse. The relative humidity ranged from 60 to 95%. After emerging new leaves on the grafted plants, they were transplanted into the field, and after 78 d, samples of flue-cured tobacco growth were obtained. Three tobacco plants with the same growth status under the same treatment were harvested. The obtained samples were divided into two groups for preservation, one part was dried at 80 ℃ and preserve at room temperature, while the other part was frozen with liquid nitrogen before storing in a refrigerator at -80 ℃. 

Experiments were performed at the Western Chongqing Vegetable Research and Development Center, Chongqing Academy of Agricultural Sciences, China (106 ◦ 18′ E, 29 ◦ 47′ N). K-efficient genotype tobacco ‘Wufeng No.2’ ( N.tabacum , Yichang Tobacco Company of Hubei Province, Yichang, China) and the main cultivar ‘Yunyan 87’ ( Nicotiana tabacum , Yunnan Tobacco Research Institute, Yuxi, China) were employed. The soil was a Dystric Regosol (FAO classification with a pH of 6.4) . The experiment consisted of two treatments, grafted seedling Y/W (Y grafted to Wufeng No.2) and own-rooted seedling Y (Yunyan 87). The K fertilizer used was potassium sulfate with 270 K 2 O ha − 1 . In order to avoid the effects of different sulfur levels on tobacco, superphosphate was applied to provide P 2 O 5 of 135 kg ha − 1 . Nitrogen fertilizer was applied at the rate of 90 kg N ha − 1 using urea. To make sure of a good success rate of grafting, we planted the rootstock seeds 7 d prior to the scion. After 6–8 true leaves grew on the seedlings of the rootstock, grafting was done by the ‘Split grafting’ method . During the grafting process, utilize silicone conduits or grafting clips to secure the junction between the rootstock and scion. The plants were grown at 22.5 °C under a 16-h light/8-h dark cycle using fluorescent lamps with an average photosynthetic photon flux density ( PPFD ) of 300 µmol m –2 s –1 in the greenhouse. The relative humidity ranged from 60 to 95%. After emerging new leaves on the grafted plants, they were transplanted into the field, and after 78 d, samples of flue-cured tobacco growth were obtained. Three tobacco plants with the same growth status under the same treatment were harvested. The obtained samples were divided into two groups for preservation, one part was dried at 80 ℃ and preserve at room temperature, while the other part was frozen with liquid nitrogen before storing in a refrigerator at -80 ℃. 

Cured middle leaves (prepared into a composite sample) were used for determining the chemical composition of the probing leaves three times. A continuous flow analyzer (SEAL, Norderstedt, Germany) was employed to determine the quantity of total nitrogen, reducing sugars, total sugars, and chlorine of the samples . K ratios were determined by a flame atomic absorption spectrometer (Varian AA-220FS, Thermo Fisher Scientific, USA) . 

The sample of each treatment with TRIzol reagent (Invitrogen, Thermo Fisher, MA, USA) was employed to extract total RNA pursuant to the instructions of the manufacturer. The integrity number of RNA (RIN) and its contamination were determined both by the Agilent 2100 Bioanalyzer system (Agilent Technologies, CA, USA) and 1% agarose gels. Sequencing libraries were generated according to the following steps. Firstly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First strand cDNA was synthesized using random oligonucleotides and Super Script II. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. A six-lanes HiSeq 2500 System (Illumina) was used to sequence the libraries according to the SR60 protocol. Poly-N, reads with adapters, and low-quality reads were eliminated from the obtained raw data. To compare our data with the tobacco reference genome sequences, clean data were plotted by HISAT2 software . The fragments per kilobase of exon model per million mapped fragments (FPKM) and gene alignment of each gene regarding its length were determined by FeatureCounts v1.6.2 . The differential expression between the two classes of the samples were analyzed by DESeq2 v1.22.1. The correction of their P values was performed according to the Benjamini & Hochberg method. The thresholds of considerable differential expression were indicated by |log2foldchange| and corrected P values. The false discovery rate (FDR) approach was employed to evaluate the importance of gene expression differences using threshold P value of various tests. FDR < 0.05 and |log2Fold Change|≥1 were the screening conditions for differential genes. Gene Ontology (GO) was analyzed by GOseq R package with DEGs . Overrepresentation analysis approach was employed to detect the DEGs-enriched KEGG pathways (FDR < 0.05) . 

A SYBR Green system (TaKaRa, Dalian, China) was used to conduct the quantitative real-time polymerase chain reaction (qRT-PCR). The samples were cycled 35 times by heating for 5 min for predenaturation at 95 ℃, followed by 0.5 min at 94 ℃, 0.5 min at 56 ℃, and finally 1.5 min at 72 ℃. The primers were designed using Primer Premier 5.0 software (Premier Biosoft, CA, USA). A full list of all the primers is presented in Table . The relative expression levels of the candidate genes were determined according to the 2 −ΔΔCt approach. All procedures were conducted on three independent technical and biological repeats. 

A vacuum desiccator equipped with liquid nitrogen was used to snap-freeze 200 mg of each sample. The vacuum-dried samples were crushed by a mixer mill. The extraction and quantification of the freeze-dried samples was done according to an earlier report . Filters (SCAA-104, ANPEL, Shanghai, China) with 0.22-µm thickness were employed to filtrate the obtained extracts for ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. MS/MS (Applied Biosystems 6500 QTRAP) and UPLC (SHIMADZU Nexera X2) were employed to perform metabolite profiling with the reference conditions reported in . Suzhou Panomix Co. (Suzhou, China) database was used to identify and quantify metabolite. Retention time and ion-current intensity were the criteria for the identification of the metabolites. VIP ≥ 1 and fold change (FC) ≥ 2 or ≤ 0.5 were considered to screen differential metabolites. Statistical analysis Each experiment was performed three times. The average values of replicates ± SD are shown in the figures and tables of this study. A statistical software (SAS version 9.2; SAS Institute, Cary, NC) was employed to analyze the data statistically by variance analysis. The least-significant difference (LSD) test with a P value smaller than 0.05 was used to evaluate statistical significance. 

Each experiment was performed three times. The average values of replicates ± SD are shown in the figures and tables of this study. A statistical software (SAS version 9.2; SAS Institute, Cary, NC) was employed to analyze the data statistically by variance analysis. The least-significant difference (LSD) test with a P value smaller than 0.05 was used to evaluate statistical significance. 

Influence of grafting on the chemical characteristics of flue-cured tobacco leaves As shown in Fig. , grafting dramatically enhanced the total nitrogen and K contents of the flue-cured tobacco leaves (FCTLs). Specifically, the K content of tobacco leaves in grafted tobacco plants increased by almost 90%. The reducing sugar content in FCTLs was significantly higher when not grafted compared to the grafted leaves. The contents of other chemical substances present in the tobacco leaves did not change considerably. 

As shown in Fig. , grafting dramatically enhanced the total nitrogen and K contents of the flue-cured tobacco leaves (FCTLs). Specifically, the K content of tobacco leaves in grafted tobacco plants increased by almost 90%. The reducing sugar content in FCTLs was significantly higher when not grafted compared to the grafted leaves. The contents of other chemical substances present in the tobacco leaves did not change considerably. 

A total of 232,920,740 of clean reads, with an average read of 38,820,123 per sample, were gathered after undergoing RNA seq of Y and Y/W. The six samples in the study had the Q30 values of 92.06–94.32%, with a mean value of 93.83%, indicating the high quality of the sequencing data (Table ). The PCA using FPKM values successfully distinguished the two tobacco sample types, indicating the transcriptome data’s strong discriminatory power (Fig. ). Additionally, hierarchical clustering showed that Y and Y/W samples exhibit largely contrasting DEG expression patterns. Transcriptomic changes of grafted flue-cured tobacco leaves Figure shows 2044 differentially expressed genes (DEGs) from treatment Y vs. Y/W, with 1324 upregulated and 720 downregulated in Y/W. Among these, 441 transcription factor genes were identified, belonging to 46 families, with bHLH (43), NAC (38), MYB-related (30), and C2H2 (29) being the most prevalent. To systematically explore the influence of grafting on the biological functions of tobacco leaves, we performed a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis regarding DEGs, which were ascribed to 108 pathways. The top 20 enriched pathways, including hormone signal transduction and MAPK signaling, are shown in Fig. . Anthocyanin biosynthesis had the highest enrichment factor (Fig. A). Key pathways related to cell division, elongation, signaling, and metabolism were prominent. Over 50% of the top 20 KEGG secondary classifications were metabolic, as depicted in Fig. A. To elucidate the global connections of enrichment pathways and illustrate the relationships between pathways and genes, we identified them highly correlated with signal transduction and metabolic alterations post-grafting. The constructed gene-pathway network map is presented in Fig. . The plant hormone signal transduction pathway was dominant in the entire network, followed by the MAPK signaling pathway and plant pathway interaction. The IAA and BSK in the plant hormone signal transmission pathway are important node genes. The GO enrichment analysis showed that DEGs were annotated in 1659 terms. Cell wall (GO: 0005618), the extracellular region (GO: 0005576), and external encapsulating structure (GO: 003012) were the three terms with the most annotated DEGs (Fig. B) and mostly up-regulated. The top 20 GO enriched terms were employed to plot bubble chart and GO enrichment circle map. Of the top 20 terms, most of them are related to molecular function (10), while the same number of terms are related to biological process and cellular component (5) (Fig. A). In the secondary classification of GO enrichment, 18 terms were related to biological processes (Fig. ). Metabolomic analysis of grafted tobacco leaves The PCA and OPLS-DA scores plot clearly differentiated samples of grafted tobacco from those of self-rooted seedlings, indicating significant variations in metabolites between the two treatments (Fig. A-B). At the same time, correlation analysis of metabolites of various samples revealed the reproducibility among them (Fig. C). From the leaves of Y and Y/W treatments, 589 metabolites were recognized, which were classified into 9 groups (Table ). The significantly differentially expressed metabolites (DEMs) between Y and Y/W were identified through OPLS-DA analysis ( P ≤ 0.05 and VIP ≥ 1). Out of the 175 screened DEMs (Fig. ), 101 and 74 of them were up- and down-regulated in Y/W, respectively. Among these DEMs, the top three categories were lipid (62), amino acid (21), and carbohydrate (18) (Fig. A). Further analysis revealed the increment of the level of primary metabolites such as organic acids, sugars in Y/W was significantly increased (Fig. B-D), indicating the close relationship between these primary metabolites and the improvement of K absorption efficiency in grafted tobacco. The KEGG enrichment of the DEMs revealed a significant upregulation of the primary metabolic pathways of the TCA cycle, glycolysis, and amino acid metabolism in Y/W (Fig. E), while the ABC transporters had the riboflavin metabolism pathway with the highest enrichment factor. Comprehensive analysis of the metabolome and transcriptome of grafted tobacco Key metabolic pathways affecting K uptake in grafted tobacco were made according to metabolomics results and transcriptome analysis (Fig. ). In the TCA cycle pathway, four genes associated with substrate phosphorylation, namely ATP citrate (pro-S)-lyase (ACLY) , malate dehydrogenase (MDH1) , succinyl-CoA synthetase alpha subunit (LSC1) and dihydrolipoyl dehydrogenase (DLD) were all up-regulated. Correspondingly, the contents of three metabolites (fumarate, succinate, and α-ketoglutarate) in this pathway were significantly increased in Y/W. 6 homologous DEGs are linked with alanine, aspartate and glutamate metabolism pathway. 83.3% of which were up-regulated in Y/W. In this pathway, compared Y, the expression levels of LOC107801037 , LOC107790076 , LOC107812914 , LOC107827398 and LOC107778990 were higher. Meanwhile, only LOC107782142 homologues were lower. Moreover, metabolome analysis showed that the content of a-Ketoglutaramate significantly increased in Y/W. Through transcriptome and metabolome analysis, this study identifies regulatory factors that impact the K utilization efficiency of grafted tobacco leaves. In order to verify the effects of key metabolites and genes on the change of chemical characteristics of tobacco leaves, and identify the driving factors of each chemical characteristic in the dataset, a Mantel-Pearson correlation analysis was conducted between the main chemical components and key genes and metabolites in tobacco leaves. In this analysis, we selected three indicators with significant differences in chemical characteristics after grafting treatment, including total nitrogen, K, and reducing sugars. As shown in Fig. , the genes and metabolites most strongly correlated with K content in grafted tobacco leaves are DLD , CIP1 , rbcS , D-fructose and fumaric acid. At the same time, no significant correlation was found between the gene in plant hormone signal transduction pathway and K content. The rbcS and the oxoglutaric acid exhibited significant correlations with alterations in total nitrogen levels in tobacco leaves. Similarly, a significant correlation was observed between the BSK and methyl jasmonate, as well as changes in reducing sugar content. It is worth noting that the correlation between key genes and metabolites was generally positive, with the exception of BSK. qRT-PCR validation Based on the results of transcriptome analysis, the reliability of the RNA-seq outcomes were verified by ten randomly opted DEGs. As shown in Supplementary Fig. , the change of relative expression levels (2 −ΔΔCt ) in these DEGs obtained by qPCR were consistent with the transcriptome results (FPKM). Except for LOC107782142, all others were downregulated in expression. In order to verify the correlation between these two sets of data, a linear equation was established and the R 2 value was obtained. The result shows that the linear equation results also show a strong correlation between them(R 2 = 0.9279), which confirms the exceptional reliability of the RNA-seq data collected in our investigation. 

Figure shows 2044 differentially expressed genes (DEGs) from treatment Y vs. Y/W, with 1324 upregulated and 720 downregulated in Y/W. Among these, 441 transcription factor genes were identified, belonging to 46 families, with bHLH (43), NAC (38), MYB-related (30), and C2H2 (29) being the most prevalent. To systematically explore the influence of grafting on the biological functions of tobacco leaves, we performed a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis regarding DEGs, which were ascribed to 108 pathways. The top 20 enriched pathways, including hormone signal transduction and MAPK signaling, are shown in Fig. . Anthocyanin biosynthesis had the highest enrichment factor (Fig. A). Key pathways related to cell division, elongation, signaling, and metabolism were prominent. Over 50% of the top 20 KEGG secondary classifications were metabolic, as depicted in Fig. A. To elucidate the global connections of enrichment pathways and illustrate the relationships between pathways and genes, we identified them highly correlated with signal transduction and metabolic alterations post-grafting. The constructed gene-pathway network map is presented in Fig. . The plant hormone signal transduction pathway was dominant in the entire network, followed by the MAPK signaling pathway and plant pathway interaction. The IAA and BSK in the plant hormone signal transmission pathway are important node genes. The GO enrichment analysis showed that DEGs were annotated in 1659 terms. Cell wall (GO: 0005618), the extracellular region (GO: 0005576), and external encapsulating structure (GO: 003012) were the three terms with the most annotated DEGs (Fig. B) and mostly up-regulated. The top 20 GO enriched terms were employed to plot bubble chart and GO enrichment circle map. Of the top 20 terms, most of them are related to molecular function (10), while the same number of terms are related to biological process and cellular component (5) (Fig. A). In the secondary classification of GO enrichment, 18 terms were related to biological processes (Fig. ). 

The PCA and OPLS-DA scores plot clearly differentiated samples of grafted tobacco from those of self-rooted seedlings, indicating significant variations in metabolites between the two treatments (Fig. A-B). At the same time, correlation analysis of metabolites of various samples revealed the reproducibility among them (Fig. C). From the leaves of Y and Y/W treatments, 589 metabolites were recognized, which were classified into 9 groups (Table ). The significantly differentially expressed metabolites (DEMs) between Y and Y/W were identified through OPLS-DA analysis ( P ≤ 0.05 and VIP ≥ 1). Out of the 175 screened DEMs (Fig. ), 101 and 74 of them were up- and down-regulated in Y/W, respectively. Among these DEMs, the top three categories were lipid (62), amino acid (21), and carbohydrate (18) (Fig. A). Further analysis revealed the increment of the level of primary metabolites such as organic acids, sugars in Y/W was significantly increased (Fig. B-D), indicating the close relationship between these primary metabolites and the improvement of K absorption efficiency in grafted tobacco. The KEGG enrichment of the DEMs revealed a significant upregulation of the primary metabolic pathways of the TCA cycle, glycolysis, and amino acid metabolism in Y/W (Fig. E), while the ABC transporters had the riboflavin metabolism pathway with the highest enrichment factor. 

Key metabolic pathways affecting K uptake in grafted tobacco were made according to metabolomics results and transcriptome analysis (Fig. ). In the TCA cycle pathway, four genes associated with substrate phosphorylation, namely ATP citrate (pro-S)-lyase (ACLY) , malate dehydrogenase (MDH1) , succinyl-CoA synthetase alpha subunit (LSC1) and dihydrolipoyl dehydrogenase (DLD) were all up-regulated. Correspondingly, the contents of three metabolites (fumarate, succinate, and α-ketoglutarate) in this pathway were significantly increased in Y/W. 6 homologous DEGs are linked with alanine, aspartate and glutamate metabolism pathway. 83.3% of which were up-regulated in Y/W. In this pathway, compared Y, the expression levels of LOC107801037 , LOC107790076 , LOC107812914 , LOC107827398 and LOC107778990 were higher. Meanwhile, only LOC107782142 homologues were lower. Moreover, metabolome analysis showed that the content of a-Ketoglutaramate significantly increased in Y/W. Through transcriptome and metabolome analysis, this study identifies regulatory factors that impact the K utilization efficiency of grafted tobacco leaves. In order to verify the effects of key metabolites and genes on the change of chemical characteristics of tobacco leaves, and identify the driving factors of each chemical characteristic in the dataset, a Mantel-Pearson correlation analysis was conducted between the main chemical components and key genes and metabolites in tobacco leaves. In this analysis, we selected three indicators with significant differences in chemical characteristics after grafting treatment, including total nitrogen, K, and reducing sugars. As shown in Fig. , the genes and metabolites most strongly correlated with K content in grafted tobacco leaves are DLD , CIP1 , rbcS , D-fructose and fumaric acid. At the same time, no significant correlation was found between the gene in plant hormone signal transduction pathway and K content. The rbcS and the oxoglutaric acid exhibited significant correlations with alterations in total nitrogen levels in tobacco leaves. Similarly, a significant correlation was observed between the BSK and methyl jasmonate, as well as changes in reducing sugar content. It is worth noting that the correlation between key genes and metabolites was generally positive, with the exception of BSK. 

Based on the results of transcriptome analysis, the reliability of the RNA-seq outcomes were verified by ten randomly opted DEGs. As shown in Supplementary Fig. , the change of relative expression levels (2 −ΔΔCt ) in these DEGs obtained by qPCR were consistent with the transcriptome results (FPKM). Except for LOC107782142, all others were downregulated in expression. In order to verify the correlation between these two sets of data, a linear equation was established and the R 2 value was obtained. The result shows that the linear equation results also show a strong correlation between them(R 2 = 0.9279), which confirms the exceptional reliability of the RNA-seq data collected in our investigation. 

In the practice of crop cultivation, the use of high-affinity materials of the same species or even different species as the rootstock for grafting can not only enhance resistance to plant diseases, insect pests and adverse conditions, but also effectively improve yield and crop quality . As for tobacco, it has been shown that the absorption of various nutrient elements can be influenced by grafting . In this study, an evident rise in K levels was observed in grafted tobacco leaves, almost doubling in comparison to the control sample. More importantly, the levels of nicotine did not change significantly. This means that, through grafting, the K(≥ 2.5%, w/w) and nicotine content(2–3%, w/w) of Y/W tobacco leaves reach the international standards for tobacco leaves with high quality . The findings of this experiment indicate that grafting, as an agronomic intervention, positively influences the quality of tobacco leaves, compatible with our prior reports . Besides, the content of reducing sugars in the leaves of grafted tobacco has decreased. This may be due to the influence of grafting on the sugar metabolism pathway of the scion materials . The mechanism of grafting to improve plant nutrient utilization efficiency is very complex, and it may involve multiple aspects such as root growth, nutrient transport, and hormone regulation. Previous studies have shown that grafting can regulate plant growth and nutrient absorption by affecting the hormone balance within the plant, such as cytokinins and auxins . Meanwhile, grafting can also regulate the development and function of xylem, promoting the transport of nutrients from roots to shoot parts . The KEGG results revealed that the DEGs between Y/W and Y mainly augment in pathways associated with cell division, elongation, signaling, and metabolism. Furthermore, analysis of the gene-pathway network map identified plant hormone signal transduction and the MAPK signaling pathway as key players in the overall network, with IAA and BSK being pivotal node genes. The MAPK pathway and plant hormone signal transduction are crucial systems for transmitting signals in plants . IAA was an early-response gene to auxin, and its protein products can specifically bind to auxin response factors, thereby regulating the expression of auxin-responsive genes . BSK serves as a vital receptor kinase in brassinosteroid signal transduction and greatly affects plant development, immunity, and abiotic stress response . Herein, Y/W was upregulated in a series of genes involved in signal transduction, indicating that these genes regulate callus formation and regeneration by sensing mechanical stimulation from phloem treated by grafting. Cell wall (GO:005618), the extracellular region (GO:0005576), and envelope structure (GO:0003012) are the top three most annotated DEGs items in the GO enrichment analysis. This finding suggests that metabolic pathways and biological functions associated with callus regeneration were significantly activated by grafting treatment. In addition, genes related to carbon and nitrogen metabolism were also up-regulated in Y/W, including rbcS and GS . These results are consistent with our earlier reports, indicating that grafting has the potential to improve the photosynthetic efficiency and nutrient utilization of tobacco leaves through synergistic regulation of carbon and nitrogen metabolism, thereby indirectly promoting the absorption and utilization of K nutrients . Among the DEMs identified in this experiment, lipids (62), amino acids (21), and carbohydrates (18) were identified as the top three metabolites. Among them, primary metabolites, including organic acids and sugars significantly increased in Y/W than Y. For example, the |log2FC| (Y vs. Y/W) for 4-Oxoglutaramate, D-Fructose, and Alpha-D-Glucose was 17.95, 20.72, and 17.09, respectively. Sugars serve as crucial energy sources and metabolic intermediates in plants. Their increased content aids in maintaining cell osmotic pressure and resisting temporary water shortages caused by the formation of xylem ducts in callus . It has already been reported that sucrose can induce the expression of K transporter genes , thereby promoting the absorption and transport of K + . Organic acids are vital plant metabolites involved in various physiological processes such as cellular pH balance, osmotic equilibrium, and chelation of metal-ions . The levels of citric acid, malic acid, and oxalic acid in Y/W leaves were significantly higher than those in Y leaves. These organic acids can form chelates with K + to facilitate long-distance transport within plants while enhancing the fluidity and availability of K . For instance, reducing soil pH with fumaric acid may help diminish the fixation of potassium ions by soil colloids . These metabolites are important for energy metabolism and osmotic regulation of plants, and increasing their content is beneficial for enhancing K absorption and utilization efficiency in tobacco. These findings suggest that grafting promotes K uptake and utilization by regulating both primary and secondary metabolism in tobacco. By conducting a comprehensive analysis of the transcriptome and metabolome, DLD , CIP1 , rbcS , D-fructose, and fumaric acid were identified as closely associated with the impacts of grafting on the increase of K content in tobacco leaves (Fig. ). Specifically, CIP1 was implicated in K signal transduction , while rbcS was linked to photosynthesis . Through coordinated regulation of carbon metabolism, it is possible to enhance the photosynthetic efficiency and nutrient utilization in tobacco leaves, indirectly facilitating the absorption and utilization of K nutrients. As a primary metabolite, fructose significantly affects plant energy metabolism and osmotic regulation, and the rise of its content helps to enhance the K absorption and utilization efficiency of tobacco . These results suggest that carbon metabolism and signal transduction pathways regulate the main processes of K metabolism in grafted tobacco leaves. 

In conclusion, the up-regulation of Y/W genes existing in K signal transduction (e.g. IAA , CIP1 ) and the significant increase in primary metabolites such as organic acids and sugars (including D-fructose, Fumaric acid, Oxoglutaric acid) were key factors contributing to the increase in K content in grafted tobacco leaf. A comprehensive analysis of metabolite and transcriptome data exhibited that grafting enhances the synergistic regulation ability of transcription and metabolism levels in plants, providing ample energy for K absorption while improving the fluidity and availability of K + within the plant body. This ultimately leads to efficient K utilization. This study offers a new perspective and experimental foundation for further elucidating the physiological and biochemical mechanisms underlying improved crop nutrient use efficiency by grafting. 

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 2 

