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Article

Integrating Transcriptome and Metabolome Analysis Unveils the Browning Mechanism of Leaf Response to High Temperature Stress in Nicotiana tabacum

by
Chunkai Wang
1,†,
Yongliang Ding
2,†,
Bing He
3,
Mingsheng Qiu
1,
Dongmei Shen
1,
Shuaiwei Chen
1,
Wenjing Song
1,
Weicong Qi
3,
Yuanda Lv
3,* and
Lin Meng
1,*
1
Key Laboratory of Tobacco Biology and Processing, Ministry of Agriculture, Tobacco Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Qingdao 266101, China
2
Tobacco Science Institute of Jiangxi Province, Nanchang 330025, China
3
Institute of Germplasm Resources and Biotechnology, Jiangsu Academy of Agricultural Sciences (JAAS), Nanjing 210014, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(8), 1722; https://doi.org/10.3390/agronomy14081722
Submission received: 17 June 2024 / Revised: 28 July 2024 / Accepted: 1 August 2024 / Published: 5 August 2024
(This article belongs to the Special Issue Environmental Ecological Remediation and Farming Sustainability—Volume II)
Browse Figures
Versions Notes

Abstract

:
During the process of flue-curing and processing, leaves from cash crops such as tea and tobacco frequently undergo a phenomenon of browning, leading to reduced quality and significant economic losses. Despite a variety of approaches developed to suppress browning, little is known about the role that flue-curing of postharvest leaves with stems plays in delaying browning. This study investigated the impact of leaf-with-stem (LWS) flue-curing on the browning of tobacco and its underlying mechanisms. Physiological results indicated that LWS flue-curing effectively delayed browning by enhancing antioxidant capacity and maintaining reactive oxygen species (ROS) levels during the yellowing stage. Comprehensive transcriptome and metabolome analyses showed that LWS flue-curing significantly influenced various metabolic pathways. Specifically, 196, 218, and 402 metabolites, and 65, 131, and 718 genes exhibited significant changes at the 38 °C, 40 °C, and 42 °C stages, respectively, inhibiting membrane lipid degradation and enhancing the supply of reducing hydrogen through the oxidative pentose-phosphate pathway. Additionally, hormone signaling pathways were found to be associated with LWS flue-curing. These findings highlight the complex interplay of metabolic pathways and signaling networks in attenuating browning, providing valuable insights for minimizing postharvest leaf browning during flue-curing and processing.

1. Introduction

Browning is an issue widely known in the storage of fruits and vegetables, as the quality of these perishable commodities is closely linked to the browning index, especially when subjected to various stresses [1,2]. Higher storage temperatures accelerate respiration and transpiration rates, leading to the activation of enzymes, such as NADPH oxidoreductase and lipoxygenase, which increase nutrient consumption and cause cell membrane degradation [3]. In addition, the senescence of plant tissues during storage induces reactive oxygen species (ROS), such as superoxide anions, hydrogen peroxide, and hydroxyl radicals. These oxidants promote the peroxidation of membrane lipids, resulting in changes in the composition and saturation of fatty acids. As a result, membrane permeability is altered, disrupting cellular compartmentalization and leading to the localization of PPO in plastids and other organelles. Additionally, the interaction between PPO and phenolic substrates in the vacuole results in the formation of brown polymers [1,2,4]. The increase in membrane permeability negatively affects various physiological reactions that occur within the cell membrane.
Furthermore, the plasma membrane facilitates nutrient exchange and signal recognition, as well as enzymatic activity [5]. Lipids, as the primary components of the membrane, serve as significant markers in plants’ response to various stresses, including high temperatures [6]. Phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI), are fundamental and highly conserved molecules in biological membranes, distinguished by the presence of choline, ethanolamine, or inositol in their polar head groups [7]. The degradation of membrane lipids leads to a loss of structural integrity in the plasma membrane, with enzymes such as phospholipase D (PLD), lipoxygenase (LOX), and lipase contributing to lipid degradation [8].
Due to its distinct aromatic properties, flue-cured tobacco is a highly valued commodity in the global cigarette market. Flue-cured tobacco’s quality is determined by the curing process of postharvest tobacco leaves. A fine-tuning technology is employed to facilitate the stable transfer of compounds to achieve the desired aromatic character. The flue-curing process of tobacco can be divided into the yellowing, leaf-drying, and vein-drying stages. Among these stages, the yellowing stage is crucial as it facilitates the maturation process of tobacco leaves after harvesting and involves intensive biochemical transformations to produce precursors used in subsequent flue-curing. However, an undesirable phenomenon known as browning often occurs during the flue-curing process, leading to a decline in external leaf grading and improper transformation of internal components [9].
Browning is primarily caused by the oxidation of polyphenols catalyzed by polyphenol oxidase (PPO) [10,11,12]. This reaction negatively impacts the quality of tobacco and diminishes its industrial usability. To address this challenge, several methods have been developed, including optimization of the curing procedure [13,14]. Among these methods, the leaf-with-stem (LWS) approach has emerged as the most effective and user-friendly method for reducing the physiological and biochemical effects of tobacco browning. Extensive research has demonstrated that flue-cured leaves experience minimal browning reactions and exhibit improved quality when the top leaves are cured with intact stems [5].
Browning represents the primary challenge encountered during the yellowing phase of tobacco leaf curing. However, the LWS curing method has proven effective in reducing the occurrence of browning in tobacco leaves. Recent studies have shown that flue-curing leaves with the stem intact can help maintain cellular membrane integrity [13,15]. The current study aims to elucidate the molecular mechanism of LWS curing in tobacco leaves and further provide insights into optimizing curing processes for improved leaf quality. To achieve this objective, a comprehensive transcriptome and metabolome analysis was conducted to identify differentially accumulated metabolites, differentially expressed genes, and the associated metabolic pathways in leaves subjected to LWS and leaf-without-stem (LWOS) curing during the yellowing stage, which is the most active period of changes in physiological activity during the tobacco flue-curing process. Additionally, browning-related indicators, including ROS scavengers, membrane lipid metabolism, and hormone signaling pathways, were investigated to verify the mechanisms by which LWS attenuates browning. The data obtained from these analyses will provide a solid foundation for a detailed dissection of the underlying mechanisms of the LWS curing method.

2. Results

2.1. Phenotypic Characteristic of Browning Reaction of Tobacco Leaves by LWS

The browning index (BI) indicated the browning progression in flue-cured tobacco and was observed to be reduced with the LWS flue-curing method. The LWS and LWOS flue-curing methods exhibited varying browning levels in the flue-cured tobacco (Figure 1A,B). Leaves cured using the LWS method displayed a smaller browning surface than those cured using LWOS. The average browning index of LWS leaves, measuring 26.55%, was significantly lower than that of LWOS (41.75%).
Considering that the browning reaction is known to be associated with ROS damage, we further examined the ROS status and scavenging capacity during the yellowing stage of flue-curing using both the LWS and LWOS methods. The production of H2O2 levels in flue-cured tobacco leaves increased during the yellowing stage for both LWOS and LWS methods (Figure 1C). However, the ROS content in LWS leaves exhibited lower levels than LWOS. At the end of the yellowing stage, LWS flue-cured tobacco exhibited a reduction of 32.27% in H2O2 compared to LWOS (Figure 1C). Subsequently, we analyzed the activity of enzymes associated with maintaining ROS homeostasis. The SOD activity of tobacco leaves gradually decreased, but at the 40 °C and 42 °C phases, the SOD activity in LWS was 32.5% and 46.6% higher than in LWOS, respectively (Figure 1D). On the other hand, the CAT activity displayed an increasing trend during the yellowing period, with LWS showing higher activity at the 40 °C phase but lower activity at the 42 °C phase compared to LWOS (Figure 1E).

2.2. Metabolome Profiling of LC-MS/MS Data Impacted by LWS Flue-Curing

To investigate the impact of LWS on tobacco leaf metabolism during the flue-curing period, we conducted a wide-targeted metabolomics analysis, generating quantitative profiles of 1236 metabolites. Leaves cured using LWOS and LWS methods were assessed at CK, 38 °C-end, 40 °C-end, and 42 °C-end stages. Principal component analysis (PCA) was performed (Figure 2A), and the results indicated a distinct separation between samples from each yellowing phase and CK, suggesting significant changes in metabolites during the yellowing stage of flue-curing. Additionally, slight differences were observed between LWS and LWOS at the 38 °C and 40 °C stages, while samples were clearly separated at 42 °C. These findings suggest that flue-curing activates metabolic transformations and that the LWS curing method alters the metabolome during the later stages of the yellowing phase. The PCA analysis also revealed a stable pattern of metabolite changes in LWS, with slight differences among the three curing phases due to overlapping scatter points.
To further understand the pattern of metabolic changes during the yellowing stage, we constructed a heatmap to compare each yellowing phase with CK in both LWS and LWOS flue-cured leaves. Notably, most metabolites exhibited an increase during the yellowing curing period. Furthermore, at the 42 °C-end stage, there was a pronounced accumulation of metabolites in LWOS, particularly lipids (Figure 2C). A comprehensive analysis of three biological replicates revealed significant changes (p < 0.1) in the levels of various metabolites between LWOS and LWS. Specifically, 196, 218, and 402 metabolites exhibited significant alterations at the 38 °C, 40 °C, and 42 °C stages, respectively (Figure 2B). It is worth mentioning that only 2.2% of the metabolites showed consistent changes across all three comparisons.
Figure 3A shows that LWS resulted in the upregulation of 104, 82, and 100 metabolites at the 38 °C-, 40 °C-, and 42 °C-end stages, respectively, while at the same stages 92, 136, and 302 metabolites were downregulated (Table S1). During the 38 °C and 40 °C phases, most flavonoids showed a decrease in LWS, whereas they exhibited an increase at the 42 °C-end stage. Component analysis indicated that flavonoids were the most abundant metabolites affected by LWS at the 38 °C phase, with 16 showing an increase and 35 showing a decrease. A similar variation pattern was observed at the 40 °C phase. However, at the 42 °C-end stage, lipid and phenolic acid-associated metabolites were predominantly influenced by LWS, with a significant proportion being reduced. Interestingly, flavonoids accumulated with LWS, therefore an opposite change pattern was observed.
To better understand the pathways influenced by the differentially expressed metabolites (DEMs), KEGG enrichment analysis was performed to identify specific metabolic pathways potentially affected by LWS (Figure 3B–D). At the 38 °C and 40 °C phases, the DEMs were primarily enriched in amino acid-related metabolism and polysaccharide metabolism, including pathways such as arginine biosynthesis, alanine, aspartate, and glutamate metabolism, as well as sucrose and starch metabolism (Figure 3B,C). At the 42 °C phase, most DEMs were enriched in lipid and sugar metabolic pathways, particularly linoleic acid and galactose metabolism (Figure 3D).

2.3. Transcriptome Profiling of Tobacco Leaves Flue-Cured by LWS

To investigate the potential influence of gene expression on the observed metabolite changes in LWS-cured leaves, we compared the transcriptomes of LWS and LWOS-cured leaves during the yellowing phase. In total, 13,945 transcripts were identified consistently among LWS- and LWOS-cured leaves at CK and the three yellowing phases. Like the metabolomic data, principal component analysis (PCA) of the transcriptomes revealed noticeable effects of LWOS at the 42 °C phase, with LWS-cured leaves exhibiting more stable impacts than CK (Figure 4A). These findings aligned with the metabolome analysis, indicating extensive transcriptional and metabolic reprogramming during the 42 °C phase of flue-curing.
Among the two curing method groups, we identified 65, 131, and 718 DEGs at the 38 °C, 40 °C, and 42 °C phases, respectively, with only three transcripts consistently differentially expressed across all three phases (Figure 4B, Table S2). This suggests that a more significant number of transcript-level alterations occurred during the later stages of flue-curing yellowing.
To gain insight into the biological functions of the DEGs, we performed a KEGG pathway enrichment analysis (Figure 4C–E). Overall, most genes involved in metabolic pathways were affected by LWS at the 42 °C phase. At the 38 °C-end stage, the DEGs were primarily enriched in protein processing pathways, such as protein processing in the endoplasmic reticulum and glutathione metabolism. At the 40 °C-end stage, biosynthesis of secondary metabolites and specific primary metabolic pathways, such as sulfur, amino acid, and sugar metabolism, were enriched. Finally, at the 42 °C-end stage, pathways related to energy metabolism, oxidative phosphorylation, and photosynthesis were significantly overrepresented.

2.4. Association Analysis of DEGs and DAMs in Lipid Metabolism

The response of plant tissue to stress signals can be reflected in changes in membrane lipid composition, which in turn affect membrane permeability and fluidity, creating suitable conditions for the browning reaction. To gain a deeper understanding of the role of lipid metabolism in browning affected by LWS, we analyzed lipid-related metabolites and genes. Given the significant alterations in lipids observed at the end of the 42 °C phase, we utilized Cytoscape to organize differentially accumulated lipid-related metabolites between LWOS and LWS. Our findings revealed that lipid breakdown products, such as free fatty acids, lyso-phosphatidylcholine (lyso-PC), and lyso-phosphatidylethanolamine (lyso-PE), were lower in LWS, while intact phosphatidylcholine (PC) accumulated (Figure 5A). The decrease in lipid breakdown products indicated a stable lipid homeostasis, potentially attributed to a slowdown in membrane turnover and associated lipolysis in LWS (Figure 5A,B). For example, two forms of phosphocholine (oxo-11:0/18:2 and 18:3/18:3 + O3) exhibited reduced levels during the curing process, whereas lipid breakdown intermediates such as PI (18:2/0:0), lyso-PC 18:4, 15-Hydroxylinoleic acid, and 9,10,13-trihydroxy-11-octadecenoic acid were significantly elevated in LWS at the 42 °C-end stage (Figure 5C–H). Furthermore, we focused on analyzing the gene expression involved in the membrane lipid degradation pathway (Figure 5B). Phospholipase A2 (PLA2) catalyzes the hydrolysis of phosphatidylcholine, generating lyso-PC and free fatty acids from membrane lipids. The expression of the gene encoding PLA2 was significantly suppressed by LWS during the 42 °C phase. Additionally, the genes encoding subsequent degradative enzymes, including lysophospholipase and glycerophosphodiester phosphodiesterase, were also reduced in LWS during the late yellowing period.

2.5. Association Analysis of Reducing Hydrogen with Antioxidative Activity Increased by LWS

NADPH plays a crucial role as the primary reducing power in maintaining antioxidative activity in plant cells [16]. It is recycled from NADP+ through the oxidative pentose-phosphate pathway [17,18,19,20,21]. The enzymes glucose-6-phosphate 1-dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH) facilitate the oxidation of sugars, providing the necessary NADPH for subsequent reducing reactions. Table 1 indicates that the genes encoding G6PDH and 6PGDH were significantly upregulated by LWS during the 42 °C phase. Conversely, the gene encoding transketolase, which catalyzes the non-oxidative phase of the pentose-phosphate pathway, was suppressed by LWS during the same phase.
Generally, oxidative phosphorylation is a direct mechanism for ATP production and a source of ROS generation through the transmembrane protein complex of the electron transport chain [22]. In LWS-treated samples during the 42 °C phase, the catalytic center of Complex I (NADH dehydrogenase), specifically NADH-ubiquinone oxidoreductase chain 5, exhibited a 3.93-fold downregulation (Table 1). Additionally, the subunit cytochrome b of Complex III was downregulated 4.88-fold, and ATP synthase subunit 9 was downregulated 4.35-fold. These findings suggest that LWS flue-curing modifies the utilization of reducing hydrogen during the yellowing stage (Table 1).

2.6. Alteration of Hormone Signaling Transduction by LWS

Phytohormones play a crucial role in regulating the biochemical and physiological processes involved in the browning of postharvest crops, attracting significant attention [23]. Our study examined the expression patterns of differentially expressed genes (DEGs) related to plant hormone signal transduction. Our findings revealed significant variations in the signaling transduction factors of several hormones, including abscisic acid (ABA), auxin (Aux), brassinosteroids (BR), ethylene (ET), and jasmonic acid (JA), during the 42 °C phase (Table 2).
In the ABA signaling pathway, the genes PPC2 and ABF were down-regulated by LWS, while the SnRK2 genes were up-regulated during the 42 °C phase. Regarding auxin signaling, the expression of IAA genes increased, while the expression of SAUR genes decreased in LWS compared to LWOS at the 42 °C phase. Genes related to BR signaling, such as BKI1, and genes related to ET signaling, such as ETR, were upregulated in LWS at the 42 °C phase. Conversely, genes associated with JA signaling, specifically JAZ genes, were downregulated in LWS during the 42 °C phase (Table 2).

3. Discussion

Browning during the flue-curing process poses a significant challenge, leading to quality deterioration and substantial economic losses in the tobacco industry [5,24,25]. Our study investigated the effectiveness of leaf-with-stem (LWS) flue-curing in mitigating browning and elucidated the underlying mechanisms through multi-omics analysis. The results highlighted several key findings that contribute to our understanding of how LWS flue-curing delays browning and enhances the quality of flue-cured tobacco leaves.
Previous studies have emphasized the importance of antioxidative components in suppressing browning reactions during postharvest storage and processing [26,27]. Our findings demonstrate that LWS flue-curing significantly delays the browning of tobacco leaves compared to the traditional leaf-without-stem (LWOS) method (Figure 1A,B). The presence of the stem appears to induce distinct metabolic and signaling changes that are absent in detached leaves. The enhancement of antioxidant capacity and the maintenance of reactive oxygen species (ROS) balance during the yellowing stage were notable in the LWS method (Figure 1C–E). This suggests that the attached stem plays a critical role in activating antioxidative pathways, thereby attenuating browning.
Multi-omics analysis could provide a comprehensive perspective for studying the molecular mechanisms of plant tissue browning [3,28,29]. In our study, multi-omics analysis revealed comprehensive changes in the transcriptome and metabolome of tobacco leaves under LWS flue-curing, compared with the traditional leaf-without-stem (LWOS) curing method during the three critical curing-temperature phases of the yellowing stage. The regulation of phenolic acid metabolism, lipid breakdown, and the accumulation of antioxidative flavonoids were particularly pronounced (Figure 2C and Figure 3A). Compared to LWOS, LWS flue-curing significantly upregulated genes involved in antioxidant defense and repressed the expression of genes associated with lipid degradation (Figure 4, Table 1). These findings are consistent with previous studies highlighting the importance of antioxidative components in suppressing browning reactions during postharvest storage and processing [5,9,30].
The cell membrane system plays a crucial role in maintaining the physiological metabolism of plants [31]. Compartmental distribution within cells prevents enzymatic browning by separating browning substrates and polyphenol oxidase into vacuoles and the cytoplasm [10,27]. Previous studies have shown that hydrolysis of phospholipids and alterations in fatty acid composition influence plasma lipid metabolism [11,32,33,34]. Our study showed that LWS flue-curing reduces lipid degradation, as evidenced by the accumulation of intact phospholipids and the decrease in lipolysis products such as fatty acids and partially degraded lipids (Figure 5). The repression of phospholipid degradation enzymes at the transcript level further supports this observation. In addition, the content of lysophosphatides is closely associated with the browning level, increasing dramatically under extreme external conditions [31,35]. Our study demonstrated a lower content of lyso-PC and lyso-PE in LWS flue-curing during the late yellowing stage, providing evidence for the suppression of membrane lipid breakdown contributing to the attenuation of tobacco leaf browning (Figure 5).
The pentose-phosphate pathway is an alternative sugar degradation route that serves as a primary source of NADPH in plants [36]. This pathway provides intermediate products required for synthesizing aromatic acids and nucleotides and is closely associated with various environmental stresses [24,37,38,39,40]. G6PDH and 6PGDH are essential in catalyzing NADPH production and enhancing antioxidant capacity during postharvest storage [17]. Our study showed increased expression of G6PDH and 6PGDH in LWS flue-curing, indicating a stable NADPH supply during the late yellowing stage. This enhanced NADPH production correlates with improved antioxidative capacity, similar to findings in other postharvest studies where increased NADPH levels were associated with reduced ROS accumulation [41,42]. The generation of ROS accompanies the discharge of hydrogen carriers through the respiratory electron-transport chain. Oxidative phosphorylation is a vital energy-generating pathway that consumes reducing hydrogen [43]. In our study, NADH dehydrogenase, cytochrome b, and ATP synthase were found to be reduced in LWS during the late yellowing stage. Consequently, another mechanism for suppressing browning and reducing ROS content in LWS could be the reduction of electron leakage in the electron-transport chain, primarily occurring in complex I and III [44].
Hormone signaling pathways play a vital role in regulating oxidative stress responses [23]. Our study found that LWS flue-curing increased the expression of genes involved in ABA and BR signaling pathways, such as SnRK2 and BKI1, which are known to enhance ROS scavenging ability. SnRK2 was found to play a central role in the ABA signaling pathway and improves the antioxidant system to enhance ROS scavenging ability [45,46]. BKI1, a negative regulator of the BR signaling pathway, has been shown to potentially promote ROS scavenger activity [47]. Additionally, alterations in the expression of genes related to JA, Auxin, and ET signaling pathways were observed, suggesting a complex hormonal regulatory network in response to LWS flue-curing. These findings align with previous reports on the role of hormones in modulating ROS homeostasis during postharvest crop storage. However, further studies are required to elucidate the mechanisms, such as individual hormone effects and crosstalk among multiple hormones, in regulating the browning of tobacco leaves during the yellowing stage of flue-curing, And to explore the potential of hormone-based treatments to improve the curing process. For example, the exogenous application of certain hormones or hormone inhibitors could be measured to determine their ability to modulate ROS levels and reduce browning during flue-curing.

4. Materials and Methods

4.1. Plant Material and Sampling

The tobacco variety “Yunyan87”, a popular and widely cultivated variety of tobacco in China, was selected for the study, and was cultivated according to the established local production standards in Yunnan, China. Matured tobacco plants exhibiting uniform growth and size were carefully selected as the experimental subjects for this experiment. An experimental design based on randomized blocks was used to ensure robustness. The upper four leaves from each tobacco plant were harvested, and two treatments were applied: individual leaves without stems (referred to as LWOS) and leaves with stems (referred to as LWS). Following the harvest, both sets of leaves underwent the flue-curing process, adhering to a standardized schedule as outlined in the previous study [5]. Leaves were collected at two specific time points: before initiating the flue-curing process (designated as CK) and after the 38 °C-, 40 °C-, and 42 °C-curing phases. These temperature points were strategically selected due to their significance in the subsequent analyses. Each sample group was collected with three biological replicates, with leaves pooled from five plants. The collected leaves were immediately frozen in liquid nitrogen and stored at −80 °C until they were processed and analyzed. For the evaluation of the browning index, flue-cured leaves from both the LWOS and LWS treatments were collected.

4.2. Measurement of Browning Index

To determine the level of tobacco browning, the browning index was measured by evaluating the extent of surface browning on the leaves, following a method adapted from the previous study with minor adjustments [48]. Browning index was calculated as the ratio of browning area to leaf area.

4.3. Measurement of H2O2 Contents

The quantification of H2O2 production was carried out using a previously established method [5]. Commercially available assay kits H2O2-1-Y (Comin Biotechnology, Suzhou, China) were employed to determine H2O2 contents. In this process, 0.1 g of the samples was finely powdered using liquid nitrogen and prepared according to the instructions provided by the manufacturer. Subsequently, the content of H2O2 was assessed using a microplate reader, allowing for accurate measurement and analysis. The H2O2 level was expressed as μmol·g−1.

4.4. Measurement of SOD and CAT Activities

We used a commercial assay kit from Comin Biotechnology, Suzhou, China, to determine superoxide dismutase (SOD) and catalase (CAT) activity. The assay kits SOD-1-W and CAT-1-W provided specific reagents and protocols for accurately measuring SOD and CAT activities, respectively. In order to ensure reliable and consistent results, these enzymatic activities were assessed using the manufacturer’s instructions. The activities of SOD and CAT were expressed as U·mg−1 protein.

4.5. RNA Extraction and Sequencing

Total RNA extraction from the samples was performed using the TRIzol reagent, a widely used method for RNA extraction. Following RNA extraction, the RNA Seq transcriptome cDNA libraries were explicitly constructed from poly(A) using the Illumina TruseqTM RNA Sample Prep kit. The sequencing was conducted by the Illumina NovaSeq 6000 platform in PE150 mode from Annoroad Gene Technology Co., Ltd., Beijing, China.

4.6. RNA-seq Data Processing

First, the sequencing quality of raw data was evaluated by the FastQC v0.12.0 program (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 1 May 2023). Subsequently, Trimmomatic v0.36 [49] was utilized to eliminate adapters, low-quality bases, and reads shorter than 50 bases. The cleaned reads were then aligned to the tobacco reference genome [50] using Tophat v2.1.1 [51]. Gene expression abundances were quantified and normalized by cuffquant and cuffnorm, respectively [51]. Fragments per kilobase of transcripts per million fragments mapped (FPKM) represented the gene expression abundance. Genes were considered expressed if they had an FPKM value greater than 1 in all three biological replicates.
Differentially expressed genes (DEGs) were identified using the DESeq2 package [52], employing the following criteria: Log2(fold change) ≥ 1 and false discovery rate (FDR) < 0.05. The identified DEGs were then subjected to KEGG pathway enrichment analysis using the R package clusterProfiler v4 [53]. Significantly enriched pathways were defined as those with a p-value less than 0.05.

4.7. Widely Targeted Metabolomic Analysis

Firstly, 100 mg of powdered frozen leaf samples was mixed with 1.2 mL of 70% methanol overnight at 4 °C and ground into powder. The resulting supernatants were then centrifuged at 10,000 rpm for 10 min for filtration. Finally, UPLC-ESI-MS/MS analysis was performed on the sample extracts using a UPLC-ESI-MS/MS system (UPLC, SHIMADZU Nexera X2; MS, Applied Biosystems 6500 QTRAP) equipped with an SB-C18 column (18 mm, 2.1 mm, 100 mm, Agilent Technologies, Santa Clara, CA, USA). For the QQQ scan, multiple reaction monitoring (MRM) was used with medium collision gas. Secondary mass-spectrometry data were utilized to identify metabolites using the Metware database (MWDB) created by MetWare Biotechnology Co., Ltd. (Wuhan, China).
Differentially accumulated metabolites were determined based on a p-value threshold of less than 0.1. KEGG enrichment analysis for the metabolome data was conducted using the online software MetaboAnalyst 5.0 (https://www.metaboanalyst.ca, accessed on 1 May 2023). Metabolites that exhibited significant differences during the yellowing period were normalized to the control (CK) and used to generate a heat map using the R package. Metabolites related to lipids were classified into subclasses based on their function using Cytoscape v3.9.1 [54] for clustering. Student’s t-tests were performed in GraphPad 9. The Spearman correlation matrix was calculated using the R package. The network was visualized by Cytoscape v3.9.1 [54].

5. Conclusions

The findings of this study indicate that leaves attached to the stem (LWS) exhibited a lower incidence of browning compared to leaves without the stem (LWOS), making it a suitable method for tobacco flue-curing. LWS flue-curing reduces browning in tobacco leaves through a complex interplay of metabolic pathways and signaling networks. The method also enhances antioxidative capacity, maintains lipid homeostasis, and modulates hormone signaling. Our study will provide a fundamental framework for enhancing curing processes and improving tobacco leaf quality.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14081722/s1, Table S1: Differentially accumulated metabolites by wide-targeted metabolomics analysis; Table S2: Differentially expressed genes across all three phases (38 °C,40 °C and 42 °C) with/without stem.

Author Contributions

L.M. and Y.L.: Conceptualization, Methodology, Software, Writing. C.W., Y.D. and Y.L.: Data curation, Investigation, Software and Writing. Y.L., B.H., M.Q., D.S., S.C., W.Q. and W.S.: Software, Visualization, Review. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the China National Tobacco Corporation Program (110202201013(JY-13)) and the Agricultural Science and Technology Innovation Program (ASTIP-TRIC03).

Data Availability Statement

The raw sequence reads have been deposited at the NCBI sequence read archive (SRA) database with the accession number (Bioproject ID: PRJNA855915). All other data generated or analyzed during this study are included in this published article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 14 01722 g001
Figure 1. LWS treatment reduced the browning of tobacco leaves during flue-curing by increasing the antioxidative activity and reducing ROS content. (A) Tobacco leaves browning phenotype after flue-curing process; (B) browning index of flue-cured tobacco leaves; (C) H2O2 content; (D) SOD activity; (E) CAT activity of tobacco leaves of LWOS and LWS during the yellowing stage of flue-curing process. Stars between treatments and control indicate significant (*, p < 0.05) differences according to the t-test. CK indicates fresh leaves before the initiation of the flue-curing process.
Figure 1. LWS treatment reduced the browning of tobacco leaves during flue-curing by increasing the antioxidative activity and reducing ROS content. (A) Tobacco leaves browning phenotype after flue-curing process; (B) browning index of flue-cured tobacco leaves; (C) H2O2 content; (D) SOD activity; (E) CAT activity of tobacco leaves of LWOS and LWS during the yellowing stage of flue-curing process. Stars between treatments and control indicate significant (*, p < 0.05) differences according to the t-test. CK indicates fresh leaves before the initiation of the flue-curing process.
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Figure 2. Metabolomics analysis of tobacco leaves of LWS and LWOS at the yellowing stage. (A) Principal component analysis (PCA) of metabolites identified in tobacco leaves between LWS and LWOS at CK, 38 °C, 40 °C and 42 °C. (B) Venn diagram of differentially accumulated metabolites (DAMs) between LWS and LWOS at 38 °C, 40 °C and 42 °C. (C) Heatmap of metabolites showing relative abundance to CK at 38 °C, 40 °C and 42 °C.
Figure 2. Metabolomics analysis of tobacco leaves of LWS and LWOS at the yellowing stage. (A) Principal component analysis (PCA) of metabolites identified in tobacco leaves between LWS and LWOS at CK, 38 °C, 40 °C and 42 °C. (B) Venn diagram of differentially accumulated metabolites (DAMs) between LWS and LWOS at 38 °C, 40 °C and 42 °C. (C) Heatmap of metabolites showing relative abundance to CK at 38 °C, 40 °C and 42 °C.
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Figure 3. Function annotation and enrichment of DAMs at 38 °C, 40 °C and 42 °C. (A) Annotation of DAMs according to metabolic function. (B–D) KEGG enrichment of DAMs between LWS and LWOS at 38 °C (B), 40 °C (C) and 42 °C (D).
Figure 3. Function annotation and enrichment of DAMs at 38 °C, 40 °C and 42 °C. (A) Annotation of DAMs according to metabolic function. (B–D) KEGG enrichment of DAMs between LWS and LWOS at 38 °C (B), 40 °C (C) and 42 °C (D).
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Figure 4. Gene expression profiles of LWS and LWOS during the yellowing stage. (A) PCA of transcripts identified in tobacco leaves between LWS and LWOS at CK, 38 °C, 40 °C and 42 °C. (B) Venn diagram of differentially expressed genes (DEGs) between LWS and LWOS at 38 °C, 40 °C and 42 °C. (C) Heatmap of selected genes related to phosphate pentose pathway and hormone signaling transduction. (D–F) KEGG enrichment of DEGs between LWS and LWOS at 38 °C (D), 40 °C (E) and 42 °C (F).
Figure 4. Gene expression profiles of LWS and LWOS during the yellowing stage. (A) PCA of transcripts identified in tobacco leaves between LWS and LWOS at CK, 38 °C, 40 °C and 42 °C. (B) Venn diagram of differentially expressed genes (DEGs) between LWS and LWOS at 38 °C, 40 °C and 42 °C. (C) Heatmap of selected genes related to phosphate pentose pathway and hormone signaling transduction. (D–F) KEGG enrichment of DEGs between LWS and LWOS at 38 °C (D), 40 °C (E) and 42 °C (F).
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Figure 5. Effects of the LWS treatment on membrane-lipid degradation. (A) Effects of LWS treatment on the lipid-related metabolic profile of the tobacco leaves at 42 °C. (B) Phospholipid degradation pathways were affected by LWS at the yellowing stage. (C–H) Examples of compounds related to lipid metabolism that LWS impacts. Stars between treatments and control indicate significant (*, p < 0.05; **, p < 0.01) differences according to the t-test.
Figure 5. Effects of the LWS treatment on membrane-lipid degradation. (A) Effects of LWS treatment on the lipid-related metabolic profile of the tobacco leaves at 42 °C. (B) Phospholipid degradation pathways were affected by LWS at the yellowing stage. (C–H) Examples of compounds related to lipid metabolism that LWS impacts. Stars between treatments and control indicate significant (*, p < 0.05; **, p < 0.01) differences according to the t-test.
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Table 1. Fold change of DEGs related to the pentose-phosphate pathway and oxidative phosphorylation between LWS and LWOS.
Table 1. Fold change of DEGs related to the pentose-phosphate pathway and oxidative phosphorylation between LWS and LWOS.
Metabolic PathwayGene IDDescriptionLog2FC (LWS/LWOS)
38 °C40 °C42 °C
Pentose-phosphate pathwayNitab4.5_0000861g0020Glucose-6-phosphate 1-dehydrogenase0.00−0.331.03 *
Nitab4.5_0000071g0180Transketolase−0.98−1.12−2.85 *
Nitab4.5_0001309g0010ATP-dependent 6-phosphofructokinase 3-like−0.160.061.33 *
Nitab4.5_0002381g00406-phosphogluconate dehydrogenase decarboxylating 2−0.22−0.031.39 *
Oxidative
phosphorylation		Nitab4.5_0003945g0040ATP synthase subunit 9−0.180.12−4.35 *
Nitab4.5_0000192g0020V-type proton ATPase 16 kDa proteolipid subunit−0.040.061.09 *
Nitab4.5_0002776g0060V-type proton ATPase subunit F−0.11−0.011.46 *
Nitab4.5_0000120g0110Cytochrome b--−4.88 *
Nitab4.5_0006854g0040Putative cytochrome c oxidase subunit 5b-like0.190.70 *1.41 *
Nitab4.5_0006854g0070Cytochrome c1-2, heme protein−0.25−0.581.22 *
Nitab4.5_0001869g0010Plasma membrane ATPase 40.05−0.110.99 *
Nitab4.5_0001788g0200NADH-ubiquinone oxidoreductase chain 50.40−0.62−3.93 *
* means that the significant variation of p-value < 0.05.
Table 2. Fold change of DEGs related to hormone signaling transduction between LWS and LWOS.
Table 2. Fold change of DEGs related to hormone signaling transduction between LWS and LWOS.
ClassificationGene IDSymbolDescriptionLog2FC (LWS/LWOS)
38 °C40 °C42 °C
Abscisic acidNitab4.5_0001231g0090PPC2Probable protein phosphatase 2C 51-−0.26−2.44 *
Nitab4.5_0001725g0110ABFProtein ABSCISIC ACID-INSENSITIVE 5-like-−1.40−2.62 *
Nitab4.5_0000743g0010SnRK2Serine/threonine-protein kinase SAPK2-like−0.55 *−0.57 *1.18 *
Nitab4.5_0001896g0090SnRK2Serine/threonine-protein kinase SAPK2-like−0.140.191.46 *
AuxinNitab4.5_0001556g0100IAAAuxin-induced protein 22D-like0.460.921.32 *
Nitab4.5_0002424g0010IAAAuxin-responsive protein IAA17-like0.170.221.22 *
Nitab4.5_0000319g0270SAURAuxin-responsive protein SAUR32-like−0.30−0.44−1.64 *
BrassinosteroidsNitab4.5_0000790g0070BKI1Protein BRASSINAZOLE-RESISTANT 1-like0.09−0.131.27 *
Nitab4.5_0001580g0010BKI1BRI1 kinase inhibitor 1-like−0.11−0.411.74 *
EthyleneNitab4.5_0000856g0210ETRProtein EIN4-like0.030.130.97 *
Nitab4.5_0000342g0270ETREthylene receptor 2-like0.440.601.44 *
Jasmonic acidNitab4.5_0004234g0080JAZProtein TIFY 10A-like0.05−1.04−1.51 *
Nitab4.5_0000073g0270JAZProtein TIFY 10A-like0.49−0.16−1.71 *
* means that the significant variation of p-value < 0.05.
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Wang, C.; Ding, Y.; He, B.; Qiu, M.; Shen, D.; Chen, S.; Song, W.; Qi, W.; Lv, Y.; Meng, L. Integrating Transcriptome and Metabolome Analysis Unveils the Browning Mechanism of Leaf Response to High Temperature Stress in Nicotiana tabacum. Agronomy 2024, 14, 1722. https://doi.org/10.3390/agronomy14081722

AMA Style

Wang C, Ding Y, He B, Qiu M, Shen D, Chen S, Song W, Qi W, Lv Y, Meng L. Integrating Transcriptome and Metabolome Analysis Unveils the Browning Mechanism of Leaf Response to High Temperature Stress in Nicotiana tabacum. Agronomy. 2024; 14(8):1722. https://doi.org/10.3390/agronomy14081722

Chicago/Turabian Style

Wang, Chunkai, Yongliang Ding, Bing He, Mingsheng Qiu, Dongmei Shen, Shuaiwei Chen, Wenjing Song, Weicong Qi, Yuanda Lv, and Lin Meng. 2024. "Integrating Transcriptome and Metabolome Analysis Unveils the Browning Mechanism of Leaf Response to High Temperature Stress in Nicotiana tabacum" Agronomy 14, no. 8: 1722. https://doi.org/10.3390/agronomy14081722

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