Hydrogen sulfide (H2S) is a new endogenous gas signaling molecule alongside nitric oxide (NO) and carbon monoxide (CO). In recent years, many studies have shown that it is involved in a variety of physiological activities, promoting root organogenesis, regulating stomatal movement and photosynthesis, and playing an important role in promoting plant growth and development and improving the stress resistance of the plant Plants can play. Tobacco is a special and an important cash crop whose growth usually determines the economic income of farmers. Tobacco root development directly affects leaf growth, disease resistance, chemical composition and yield. However, to date, there are limited studies on the mechanism of H2S in promoting tobacco growth. In this study, tobacco seedlings were exposed to NaHS (exogenous H2S donor) at concentrations of 0, 200, 400, 600, and 800 mg/L. With the gradual increase of NaHS concentration, the root length, wet weight, root activity and antioxidant enzymatic activities of CAT (catalase), SOD (superoxide dismutase) and POD (peroxidase) of tobacco roots showed an increasing tendency. The transcriptome and metabolome showed that NaHS mainly affected 162 key genes, 44 key enzymes, and two metabolic pathways (the brassinolide synthesis pathway and the aspartic acid biosynthesis pathway) in tobacco seedlings after treatment with 600 mg/L NaHS. On the one hand, the addition of exogenous NaHS can promote the development of tobacco roots as the basis of tobacco planting. On the other hand, the use of NaHS can reduce the use of pesticides and better protect the ecological environment. In conclusion, this study provides new insights into the main metabolic pathway in the response of tobacco roots to NaHS and new genetic clues for further investigation- of plant root development.
Research Article
Metabolomics and transcriptomics combined with physiology reveal key metabolic pathway responses in tobacco roots exposed to NaHS
https://doi.org/10.21203/rs.3.rs-4255211/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 18 Jul, 2024
You are reading this latest preprint version
NaHS
tobacco
physiology
transcriptome
metabolome
brassinolide
aspartic acid
Tobacco plants (Nicotiana tabacum L.) are an economically important crop in many countries and regions [1]. To ensure the productivity of tobacco plants, large amounts of pesticides are usually used to control unwanted plants (weeds), insects, rodents and fungi in tobacco fields [2], especially in the period of seedling growth in the greenhouse and the period of transplanting young plants in these fields [1]. Pesticides may cause acute effects over a short period of time or may cause chronic effects over a longer period of time upon repeated exposure at lower concentrations. Excessive use of pesticides to control and prevent tobacco diseases causes soil, water and air pollution and is harmful to human (including tobacco farmers) and ecosystem health due to their toxicity, residues and accumulation of pathogen resistance in living organisms [1, 3]. Therefore, reducing pesticide use and using highly efficient and environmentally friendly formulations are essential for the future sustainability of agriculture.
Hydrogen sulfide (H2S) is an important endogenous gasotransmitter that maintains a dynamic balance in plants with L-cysteine desulfhydrase, D-cysteine desulfhydrase, sulfite reductase, cyanoalanine synthase, cysteine synthase and O-acetyl-l-serine(thiol)lyase maintained [4]. Although high H2S concentration is a malodorous gas and is harmful to plant growth, the correspinding H2S concentrations act as a signaling molecule to regulate plant development and response to environmental stress [5]. In agricultural production, the application of appropriate H2S concentration is involved in various physiological processes to regulate plant growth and development, such as: seed germination [6, 7], organogenesis [8, 9], the formation of adventitious roots and lateral roots [9, 10], maturation, flower senescence [11], and increased tolerance to environmental stress [5, 12]. Recently, the role of hydrogen sulfide (H2S) as a signaling molecule in regulating physiological processes in plants has become a hot topic. These physiological processes are involved in the interaction with hormones, ROS and some other molecular signals [13]. Zou et al. found that pretreatment with exogenous H2S donor (sodium hydrogen sulfide [NaHS]) increased endogenous H2S concentration, promoted nodulation and nasal activity, and upregulated nitrogen metabolism in soybean roots and nodules [14]. Foliar spray with NaHS significantly increased flavin monooxygenase (FMO) activity and relative expression of FMO-like proteins (YUCCA2), which in turn increased endogenous IAA levels and induced chilling tolerance in cucumber seedlings [13]. NaHS treatment effectively promoted the appearance of adventitious roots by regulating the content of osmotic substances and increasing the antioxidant ability of cucumber [15]. Whether proper application of NaHS can reduce the frequency and amount of pesticide use and improve plant immunity.
Roots are important organs that provide crops with water, nutrients, hormones and mechanical support (anchoring), thereby influencing economic yield [16]. A well-developed root is essential for healthy plant growth and development, although it typically only accounts for 10-20% of the plant’s total weight [16, 17]. Tobacco is a annul model crop and an important cash crop grown on a large scale worldwide [18]. Tobacco root development directly affects leaf growth, disease resistance, chemical composition and yield. However, previous studies have mainly focused on physiological processes. There is little knowledge about the general changes in root, MDA (malondialdehyde) accumulation, enzymatic activities of CAT (catalase), SOD (superoxide dismutase) and POD (peroxidase) and important signaling pathways after H2S treatment in tobacco growth. Furthermore, the underlying physiological, biochemical and molecular mechanisms of H2S regulation of tobacco tolerance are still unclear and require in-depth analysis and exploration.
New approaches are needed to uncover the molecular mechanism of NaHS that promotes tobacco root development. Omics technology provides a unique opportunity to study the regulatory mechanism of complex networks related to growth promotion [19]. With the rapid development of sequencing tools, transcriptome sequencing technology has been widely used to elucidate gene function through high-throughput analysis of genomic information, providing a powerful opportunity to systematically study the relationship between genotype and phenotype in transcriptional regulation [20]. For example, it is widely used to study tobacco disease resistance and growth promotion to reveal differentially expressed genes in various biological processes. However, low abundance and unknown transcripts cannot be distinguished [20]. Compared to traditional chemical analysis, the use of liquid chromatography/mass spectrometry (LC/MS) promotes metabolomics by detecting a large number of compounds [21]. Metabolomics is a concept first introduced by Nicholson et al. was proposed [22]. It is a new omics after transcriptomics, genomics and proteomics research [23]. Recently, comprehensive analysis of the metabolome and transcriptome has been widely used to study the regulatory networks and correlations between genes and metabolites [20, 23-27].
Nevertheless, there are limited studies on the transcriptional and metabolic mechanisms of NaHS that promote tobacco root development. This study investigated the effect of NaHS pretreatment on tobacco seedlings through physiological, transcriptomic and metabolomic analysis of Nicotiana tabacum cultivar Yunyan 87. Targeted metabolomic technology was used to reveal the metabolic changes caused by 600 mg/L NaHS. The results suggest that candidate genes and metabolites involved in the brassinolide synthetic pathway and aspartate metabolic pathway could regulate the development of tobacco seedlings and that a metabolic regulatory network was established in response to NaHS pretreatment. This study deepens the understanding of the molecular mechanism of NaHS on tobacco root development and provides a theoretical basis for the development of effective tobacco seedlings and the development of NaHS-related products for tobacco growth and reduced pesticide dosage and greater protection of the soil environment.
Effects of exogenous NaHS on tobacco seedling growth
To investigate the effects of exogenous NaHS on the growth of tobacco seedlings, tobacco seedlings at the four-leaf stage were exposed to different concentrations NaHS (0, 200 mg/L, 400 mg/L, 600 mg/L and 800 mg/L) for treatments over a period of 10 days. As shown in Fig. 1a, although there was no obvious effect on the stem and leaf of tobacco seedlings, there was obvious difference in root development. With increasing the concentration of exogenous NaHS, a concentration-dependent promotion was observed compared to the untreated control (CK, 0 mg/L). Root length promotion was maximal at 600 mg/L NaHS, whereas a higher concentration of donor (800 mg/L NaHS) was less effective (Fig. S1).
The effect of NaHS treatment on tobacco root activity after 10 days was determined using the TTC staining method. Compared with control, the root activity of NaHS treatment increased by 29.5%, 74.4%, 101.8% and 125.7%, after treatment with 200 mg/L, 400 mg/L, 600 mg/L and respectively 800 mg/L exogenous NaHS. The higher the concentration, the higher the root activity of the tobacco (Fig. 1b). However, the difference in root activity between 600 mg/L and 800 mg/L treatment was not significant, indicating that 600 mg/L reached the optimal concentration to promote root activity. The result showed that exogenous NaHS helped to increase root activity, but the change in root activity was not significant when increased to a certain high concentration.
Meanwhile, the length and wet weight of roots were determined in tobacco treated with different concentrations of NaHS for 10 days. The results showed an increase in wet weight of tobacco roots exposed to increasing concentrations of NaHS. The maximum wet weight of the tobacco root treated with 800 mg/L NaHS was 0.143 g (600 mg/L 0.14 g). The longest tobacco root treated with 800 mg/L NaHS was 12.18 cm (600 mg/L 11.94 cm). Furthermore, the root length and wet weight significantly increased by 190.10% and 296.00% (184.28% and 286.53%, respectively), compared to CK (Fig. 1c, d).
Exogenous NaHS triggers endogenous H2S signaling in tobacco roots
We investigated whether exogenous application of NaHS leads to an increase in endogenous H2S content in tobacco seedlings. Therefore, we measured the endogenous H2S content as well as the enzymatic activities of LCD (L-cysteine desulfhydrase) and DCD (D-cysteine desulfhydrase) in tobacco seedlings exposed to 0, 200, 400, 600, and 800 mg/L NaHS. Pretreatment with NaHS significantly increased endogenous H2S content compared to the untreated control (Fig. 2a). The result showed a significant increase in endogenous H2S content by 333.2%, 587.1%, 801.9% and 874.5% in tobacco seedlings treated with 200, 400, 600 and 800 mg/L for one hour, respectively NaHS treated compared to untreated control (CK). The endogenous H2S content in tobacco stabilized after 24 hours. Furthermore, no significant difference in endogenous H2S content was observed after 24 and 48 h in tobacco treated with 600 and 800 mg/L NaHS, respectively (Fig. 2a). As expected, the results showed a significant increase in LCD and DCD activities in NaHS-treated tobacco. An increase in enzymatic activities was observed in tobacco treated with increasing NaHS concentrations (Fig. 2b, c). However, the difference between the latter two concentrations (600 mg/L and 800 mg/L) was not obvious. Our results showed that NaHS could promote the enzymatic activities of LCD and DCD, which could degrade the two cysteine isomers and produce H2S in the absence of other biotic and abiotic stresses in tobacco.
Effects of exogenous NaHS on antioxidant enzymes and MDA content of tobacco roots
Our results showed that exogenous application of NaHS could promote endogenous H2S production (Fig. 2a), which could increase the resistance of plants to various abiotic stresses and thereby promote plant survival [24]. Continuous exposure to abiotic stress could lead to accumulation of H2O2 and ROS, leading to lipid peroxidation, protein oxidation and cell damage [7, 12, 28]. CAT, SOD and POD are important antioxidant enzymes that play a crucial role in eliminating free oxygen radicals and influencing plant stress resistance [15, 24]. To demonstrate the effect of irrigating tobacco roots with NaHS on promoting CAT, SOD, and POD enzymatic activities in the absence of stress, we irrigated tobacco roots with 200, 400, 600, and 800 mg/mL NaHS for 10 days. Compared to CK, CAT activity increased by 5.8%, 15.2%, 19.2%, and 21.8%, respectively. Compared to CK, SOD activity increased by 127.5%, 187.5%, 276.2%, and 325.6%, respectively. Compared to CK, POD activity increased by 99.4%, 239.4%, 436.1%, and 580.5%, respectively. With increasing concentrations of exogenous NaHS compared to CK, a gradual increase in the enzymatic activities of CAT, SOD and POD was observed (Fig. 3a, b, c). Interestingly, no significant difference in catalase activity was observed between tobacco treated with 600 mg/mL and 800 mg/mL NaHS (Fig. 3b). In addition, the results showed a significant increase in the enzymatic activities of CAT, POD and SOD in tobacco treated with 600 mg/mL NaHS, suggesting that 600 mg/mL exogenous NaHS was best for the enzymatic activities of CAT, SOD and POD are suitable in tobacco for stress resistance. Therefore, transcriptomic and metabolomic analyzes were performed in the 600 mg/mL NaHS treatment group and the control group.
Malondialdehyde (MDA) is one of the end products of the peroxidation of polyunsaturated fatty acids in cells. An increase in free radicals leads to an overproduction of MDA. Malondialdehyde levels are commonly known as a marker of oxidative stress and antioxidant status in plants. As shown in Fig. 3d, the MDA (malondialdehyde) content in the roots of tobacco seedlings was significantly reduced compared to CK. With increasing NaHS concentration (200, 400, 600 and 800 mg/L), it decreased by 28.4%, 50.2%, 70.0% and 74.9% compared to CK, respectively. It was demonstrated that exogenous NaHS treatment effectively inhibited the accumulation of MDA content in the roots of tobacco seedlings and improved the immunity of tobacco seedlings.
Overview of transcriptome sequencing of tobacco roots after NaHS treatment
Due to the significant differences in physiological indices between the CK and 600 mg/L NaHS treatments, histological measurements of the root systems of these three treatments were performed to determine the changes in gene expression in roots exposed in the CK and 600 mg/L NaHS treatment grew in NaHS groups. A total of 43.59 Gb nucleotides were generated, including 285,649,662 clean reads, with an average GC content of 43.61%, Q30 > 95.05% and an underlying error rate of 0.02%, indicating the high quality of the transcriptome sequencing data (Table S1). Both reads and uniquely mapped reads exceeded 65% (Table S2), demonstrating the suitability of the chosen reference genome. The correlation heatmap of the samples revealed that the correlation coefficients of the three treatment groups were higher than 0.8 (Fig. 4a). The inter-sample correlation analysis demonstrated the high repeatability of the sequencing data from 6 samples. PCA analysis revealed that the expression of unigenes varied significantly under 600 mg/L NaHS treatment (Fig. 4b). The combination of PCoA and PERMANOVA based on Bray-Curtis dissimilarity showed that PC1 and PC2 explained 72.53% of gene expression frequency, and NaHS significantly influenced the change in gene expression frequency (R = 0.625, P = 0.0296), while NaHS significantly increased gene expression frequency (F1.4 = 11.5, p = 0.027) (Fig. 4b, c). After de novo transcriptome assembly, we used a |log2Fold change| ≥ 1 and a strict FDR value < 0.05 as the threshold, and a total of 85570 unigenes were expressed in 6 samples. Venn diagram analysis showed that CK and NaHS had 3933 and 6681 differentially expressed genes, respectively (Fig. 4d). Subsequently, according to the FPKM (fragments per kilobase per million) value of 3145 differentially expressed genes in the control/treatment group, 970 genes were down-regulated and 2175 genes were up-regulated after NaHS treatment (Fig. 4e, Table S3). Table S4 lists the 10 genes with the highest variance.
GO enrichment and KEGG pathway analyzes of DEGs
We performed enrichment analyzes in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to further investigate the biological functions of DEGs (Differentially Expressed Genes) from the two comparison groups in the three ontologies (biological processes (BPs), molecular functions (MFs), and cellular components (CCs)) (Fig. 5a). The root system of tobacco seedlings mainly improved cell wall biogenesis, oxidoreductase activity, anchored component of membrane and supramolecular polymer to improve plant defense ability after NaHS treatment (Table S5). A total of 85,570 unigenes were annotated, including 50,547 up-regulated and 35,023 down-regulated unigenes in the CK_vs._NaHS-enriched GO term libraries. The subcategories with the highest level of enrichment were all cellular components. Among the eight components of the functional classification of biological processes, the cellular process and the metabolic process were the two components with the most significant enrichment of differential genes. Differential genes were most significantly enriched in the functional classification of cell components such as membrane and cellular part. In the functional classification of molecular function, the enrichment of differential genes related to binding and catalytic activity is most significant. The differential genes are mainly enriched in components related to cells, organelles, catalytic activity and metabolism, suggesting that the differentiation genes are mainly concentrated in the secondary metabolic process.
To classify the DEGs based on related signaling pathways and further investigate the associated differentially expressed genes of NaHS that promote tobacco root development, KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis was performed on these selected differentially expressed genes. The top 20 GO terms with the smallest Q-value were selected for KEGG pathway enrichment analysis (Fig. 5b, Table S6). KEGG annotation analysis revealed 128 DEG pathways associated with two different treatment stages, and the most common 15 KEGG pathways were divided into five categories. It includes metabolism, cellular processes, genetic information processing, environmental information processing and biosystem. The results showed that carbohydrate biosynthesis was very common, followed by energy metabolism, amino acid metabolism, lipid metabolism and plant hormone signal transduction. Furthermore, KEGG enrichment analysis of the gene set showed that it was mainly enriched in 128 pathways, of which 58 pathways were significantly different. The results of KEGG metabolic pathway analysis showed that DEGs were mainly enriched in 10 major metabolic pathways, and the most significant differences were phenylpropanoid biosynthesis, photosynthesis, related metabolic pathways such as linolenic acid metabolites, and tyrosine metabolism (Fig. 5c, Table S7). The results showed that the biosynthesis of the secondary metabolite pathway was highly enriched, followed by plant-metabolism interaction, plant hormone signaling, plant-plant MAPK signaling, phenylpropanoid biosynthesis, and starch and sucrose metabolism. Therefore, the DEGs named “biosynthetic pathway of secondary metabolites”, “plant-metabolism interaction” and plant hormone signal transduction were further investigated in detail.
Transcription factors (TFs) are important regulators of plant root development. A total of 29 transcription factors were predicted, and 138 TFs with significant differences between NaHS600 pretreatment and CK treatment were identified to regulate tobacco root development, including 112 up-regulated transcription factors and 26 down-regulated transcription factors. The expression levels of transcription factors related to plant growth promotion were analyzed. The results showed that the differentially expressed genes were significantly expressed in GRFs (growth-regulating factors), GATAs (transcription factors recognizing the DNA sequence W-G-A-T-R), MYB transcription factors (v-myb avian myeloblastosis viral oncogene homologous) and ERFs (ethylene response-factors) were expressed (Fig. 6a), although not always in the same direction. Regarding the general trend of TF gene expression in DEG between CK and NaHS, GRF was preferentially up-regulated and MYB was preferentially down-regulated (Fig. 6b, Table S8).
Non-targeted metabolite profiles of tobacco roots after NaHS treatment
To comprehensively analyze the effects of NaHS600 treatment on tobacco root development, we simultaneously performed an untargeted metabolomics analysis of the samples treated under the same conditions. A total of 19891 peaks were obtained by UPLC-MS/MS (liquid chromatography-tandem mass spectrometry) analysis. The peak abundance of NaHS treatment was significantly higher than that of CK, and 821 metabolites were identified using the tandem mass spectrometry (MS2) database. The combination of PCoA and PERMANOVA based on the microbial dissimilarity index showed that PC1 and PC2 explained 86.83% of the root sediment changes, indicating that these metabolomic data were highly reliable. NaHS significantly affected the composition of root deposits (F1,10 = 2727, P < 0.001) (Fig. 7a). Two-way analysis showed that NaHS plays an important role in root sediment composition (F1,10 = 17.991, P < 0.001) (Fig. 7b). Through permutation test, R2 = (0, 0.1101), Q2 = (0, -0.533), indicating that there is no overfitting in the PLS-DA model (Fig. 7c). These indicate that there were significant differences in the metabolites in the tobacco roots between the CK group and the NaHS 600 mg/L group.
To determine the different metabolites of CK and NaHS treatment, the variable significance of (VIP) value > 1.5 and FC (fold change) ≥ 2 or FC < 0.05 was used to determine the differentially accumulated metabolites (DAMs). Compared with the CK group, 384 metabolites were differentially enriched in the 600 mg/L NaHS-treated group, of which 122 were up-regulated and 262 were down-regulated (Fig. 7d, Table S9). To identify metabolites with consistent expression patterns upon clustering, we used the K-means clustering algorithm, which grouped metabolites according to the similarity of metabolic profiles. A total of 10 clusters were identified (Fig. S2), which can be divided into CK_vs._NaHS down-regulated (classes 2, 3, 4, 5, 7 and 8), CK_vs._NaHS up-regulated (class 1, 6 and 10), CK_vs._NaHS irregular changes (class 9) three categories.
To identify the different metabolites of NaHS in developing tobacco roots, all metabolite profiles were performed by hierarchical cluster analysis based on Pearson correlation. Significant segregation was observed in two samples (CK and NaHS) in the control or NaHS treatment. In addition, most differential metabolites were found to be fatty acids, carboxylic acids and their derivatives, indoles and their derivatives (Fig. 8a). KEGG analysis of all differentially accumulated metabolites revealed the top 20 KEGG pathway enrichment statistics (Table S10). The differentially accumulated metabolites were mainly enriched in the GPI anchor biosynthesis (Fig. 8b), autophagy, and RNA transport pathways. Under the threshold of VIP > 1.8 and p < 0.05, we identified 50 different metabolites (CK and NaHS) (Fig. 8c, Table S11), including furocoumarinic acid glucoside, 3-hydroxy-5-(3-hydroxyphenyl)-1-methyl-4-phenylpiperidine-2,6-dione, Austin, L-tyrosine, etc.
KEGG analysis of all accumulated metabolites revealed 58 enrichment statistics for the KEGG pathway. There were significant differences in 19 signaling pathways between CK and NaHS treatment groups, with tryptophan metabolism, cyanoamino acid metabolism, and plant signal transduction pathways being the most significant (Fig. 9, Table S10). Among all the different metabolites, lipids and lipid-like molecules are the most abundant categories, followed by organic acids and organic oxygen (Fig. 10, Table S12).
Integrated analyzes of transcriptomic and metabolomic changes involved in vital biological pathways
The combination of metabolomics and transcriptomic data also revealed the effects of CK and NaHS on tobacco roots. The related genes and metabolites between CK and NaHS treatment groups were analyzed and the correlation was calculated using the Spearman correlation method. The results showed that 2175 DEGs were positively correlated with 123 DMs (differentially metabolites) (R2 < − 0.9 and P-value < 0.05) and 970 DEGs were negatively correlated with 261 DMs (R2 > 0.9 and P-value < 0.05). Various cumulative metabolites and DEGs were enriched in 58 KEGG pathways, including map00220 (arginine biosynthesis pathway) and map00250 (alanine, aspartic acid and glutamic acid metabolic pathway), map00260 (glycine, serine and threonine metabolic pathway) and map00270 (cysteine and methionine metabolic pathway), map00360 (phenylalanine metabolic pathway) and map04075 (plant hormone signal transduction pathway) were significantly different in the metabolic enrichment pathway and transcription enrichment pathway, indicating that NaHS treatment group tobacco can promote root development through a series of comprehensive energy metabolism and amino acid biosynthetic mechanisms (Fig. 11, Table S13).
By combining different genes in the transcriptome and different metabolites in the metabolome, a map of network regulation was created. With NaHS treatment, the differential metabolite aspartic acid was found to have a positive regulatory relationship with 73 differential genes and a negative regulatory relationship with 30 differential genes. The differential metabolite L-serine had a positive regulatory relationship with 30 differential genes and a negative regulatory relationship with 6 differential genes. The differential metabolite brassinolide had a positive regulatory relationship with 26 differential genes and a negative regulatory relationship with 2 differential genes. The differential metabolite indoleacetic acid had a positive regulatory relationship with 25 differential genes and a negative regulatory relationship with 2 differential genes. The differential metabolite tyrosine had a positive regulatory relationship with 9 differential genes and a negative regulatory relationship with 1 differential gene. The differential metabolite 5-methylthioribose had a positive regulatory relationship with 24 differential genes and a negative regulatory relationship with 4 differential genes (Fig. 12, Table S14).
2.8. Underlying mechanism of the brassinolide synthesis pathway that promotes tobacco root development
The enrichment pathways between transcriptome and metabolome of NaHS-treated tobacco in the KEGG database were compared and analyzed. Various pathways were enriched between CK and NaHS, including phenylpropanol biosynthesis, brassinolide biosynthesis, aspartic acid and glutamic acid metabolism, plant signal transduction, and arginine and proline metabolism.
As a steroidal plant hormone, brassinolide (BL) is the first discovered and at the same time the most widespread and most active representative of the brassinosteroids (BRs), which are indispensable for various stages of plant development. Brassinolide signaling initially depends on the binding of the bioactive brassinolide to receptors on the cell membrane, triggering a series of cascade amplification effects in the cell, thereby inducing or inhibiting the expression of related plant growth and morphogenesis genes. Brassinolide biosynthesis involves three pathways of early C-6 oxidation, late C-6 oxidation pathway and early C-22 oxidation pathway [29-32]. To further clarify the specific role and regulatory mechanism of NaHS, we compared and analyzed the KEGG enrichment pathways between transcriptome and metabolome of tobacco root treated with CK_vs._NaHS600. Compared to the control group, various metabolic pathways were enriched by NaHS pretreatment, including phenylpropanol biosynthesis, brassinolide (BL) biosynthesis, aspartic acid and glutamic acid metabolism, plant signal transduction, and arginine and proline metabolism. The results showed that two significantly different metabolites encoded by 26 significant DEGs were assigned to brassinolide biosynthesis (C08814; c15793) and BR signaling (Fig. 13).
The synthesis of BL, a C28 BR, begins with the conversion of campestanol (CN) via the early or late C-6 oxidation pathway, depending on whether BRs have a ketone group (the former) or a deoxo form (the latter) at the C-6 position (Fig. 13). With the catalytic help of a C-22 hydroxylase DWF4, these parallel pathways converge at castasterone (CS), the immediate precursor of BL. In addition, CPD (cytochrome P450 superfamily protein), DWF4, DET2 (3-oxo-5-steroid 4-dehydrogenase family protein) and ROT3 (3-epi-6-deoxocathasterone 23-monooxygenase) can also catalyze the multistep C-22 hydroxylation reaction and participants in another BR synthesis pathway (the early C-22 oxidation pathway) [33, 34]. Analysis of KEGG enrichment pathways between transcriptome and metabolome of tobacco root treated with CK_vs._NaHS showed that genes encoding CPD (cytochrome P450 superfamily protein) and DWF4 were up-regulated and genes encoding DET2 (3-oxo-5-steroid 4-dehydrogenase family encoding protein) and ROT3 (3-epi-6-deoxocathasterone-23-monooxygenase) were down-regulated after NaHS pretreatment. Accordingly, the content of end metabolites TY (typhasterol) and BRs up-regulated.
When the BR signal is transmitted, BL is up-regulated. After binding to the extracellular region of the BRI1 protein, it induces phosphorylation of the intracellular kinase region of BRIl and triggers the phosphorylation of the BKI1 protein, thereby activating the BRI1 protein. Subsequently, the activated BRI1 phosphorylates its substrate membrane binding protein BSK (BR signaling kinase), the BSK gene family is down-regulated, the activator protein phosphatase PP1-type phosphatase BSU1 (BRI1 suppressor 1) [35], the activated BSU1 acts on BIN2, and the BIN2gene is down-regulated. The down-regulated transcription factors BZR1 and BZR2/BES1 were rapidly dephosphorylated by PP2A (protein phosphatase 2A) [32] and entered the nucleus to bind the target gene and regulate transcription.
Xyloglucan endotransglucosylase/hydrolase (XTH) is a collection of enzymes, including xyloglucan endotransglucosylase (XET) and xyloglucan endohydrolase (XEH), that play important roles in cell wall modifications. TCH4 (TOUCH4), also known as XTH22, encodes a protein with the ability of an XET enzyme. The expression level of TCH4 is rapidly upregulated by brassinosteroids, auxins and various environmental stimuli, such as touch, temperature shock and darkness [36]. After NaHS treatment, the TCH4 gene was down-regulated, which resulted in cell elongation and greatly promoted root development. Plant development is accompanied by cell division and cell cycle activity can regulate cell division [29]. In plants, the expression level and activity of CYCD (D-type cyclins) are influenced by hormone and carbohydrate concentration, which has an important influence on the cell division process of the plant [37]. Most studies on CYCDs focus on the function of a single CYCD protein based on its influence on plant growth and development. The CYCD3 gene is up-regulated, cell division is accelerated and root elongation is promoted.
Previous studies have shown that the BR signaling pathway frequently interacts with AUXIN to regulate root meristem growth and development. The stem cell niche consists of QC cells (quiescent center) and surrounding stem cells. QC cells are essential for maintaining niche stability and growth status of stem cells. The BR signaling mediated by BZR1 activates ERF115 and the ERF115 gene is up-regulated, which up-regulates the expression of PSK5 and thereby promotes QC cell division [38]. AUXIN can induce PLT expression and regulate QC cell division, thereby regulating root growth.
The root meristem is the key area in determining root development and it continues to reproduce and differentiate. BR regulation has the characteristics of space-time balance and hormone interaction balance [30]. The study also found that the BR signal sensed by BRI1 in the root epidermis can promote the expression of AUXIN in the root meristem and thus the proliferation of the root meristem, while the BR signal in the stele activates the activities of BAK1, BRL1 and BRL3equal to these effect and promotes differentiation [39]. SHY2 (short hypocotyl 2) is a repressor of AUXIN signaling, and BRX (brevis radix) is involved in the BR signaling pathway. SHY2 and BRX play an important role in the interaction of BR with AUXIN and CTK to regulate the growth and development of the root meristem [40, 41]. From Figure 13, it can be seen that the early development of root meristem is regulated by hormonal balance, BR and AUXIN are up-regulated, transcription factors activate SHY2 expression and inhibit PIN expression, thereby regulating the development of root meristem. BRX can be induced by a large amount of IAA but is slightly inhibited by BR. In competition with SHY2, BRX dominates and can temporarily increase the expression of PIN3. Both jointly regulate the development of the root meristem [42, 43]. As shown in the figure, the BRX gene is up-regulated and the SHY2 family is both up-regulated and down-regulated. BRX dominates, enhances PIN3 and promotes root meristem development.
Plant lateral roots can absorb water and nutrients from the environment and play an important role in maintaining plant growth. AUXIN and BR jointly regulate lateral root growth and development (Fig. 13, Fig. 14a). Early studies have shown that BR at low concentrations promotes lateral root development by increasing the polar transport of AUXIN. On the contrary, high BR concentrations inhibit lateral root formation [31, 44]. DWF4 (dwarf 4) is an important regulator of the BR synthesis pathway. In addition, BIN2 (BR-insensitive 2), a negative regulator of BR signal transduction, has a positive effect on lateral root development. After NaHS treatment (Table S15), the BIN2 gene was down-regulated, suggesting that BIN2 may be involved in the regulation of DWF4 and the specific mechanism requires further investigation. Up-regulated BR promotes the degradation of AUX/IAAs (auxin/indole-3-acetic acid) (IAA14/SLR single root, etc.) involved in lateral root development by regulating the transcription levels of the auxin input vectors AUX1 (AUXIN 1), LAX2 (Like-Aux1 2) and LAX3 (Like-Aux1 3) in roots. Depletion of AUX/IAAs further activates ARF7/ARF19, thereby promoting lateral root development.
The aspartate metabolic pathway may also play an important role in tobacco root development
Aspartate is the starting compound of the aspartic acid metabolic pathway, which is formed by transamination of phthalic acid. Aspartate is a common precursor of two major pathways. One pathway forms aspartic acid, which is used for nitrogen transport and storage in plants. Aspartic acid is synthesized by transferring the amino compound nitrogen from glutamate to aspartic acid salt. This process is catalyzed by aspartic acid synthase, another way to synthesize lysine, threonine, methionine and isoleucine.
After NaHS treatment, plants modify some amino acids through changes in amino acid metabolic pathways, thereby maintaining the stability of functional proteins and metabolic enzymes. The amino acids that make up plant proteins and enzymes can be divided into five categories: 3-phosphoglycerate, α-ketoglutarate, pyruvate, oxaloacetate and aromatic amino acids from the source of the synthetic carbon framework [45]. The results of the joint analysis showed that the application of hydrogen sulfide significantly changed the content of various amino acids in tobacco roots (Fig. 15). The amino acids with different tobacco contents in different treatments were efficiently distinguished: the content of α-ketoglutarate-arginine in tobacco roots was significantly up-regulated; The content of asparagine of the α-ketoglutarate group, serine of 3-phosphoglycerate group and aromatic tyrosine were significantly down-regulated. Arginine is an amino acid with the highest nitrogen to carbon ratio. In addition to being a protein component, it is also a precursor to the biosynthesis of nitric oxide, polyamines and proline [46]. The large accumulation of arginine in tobacco roots can make the development of tobacco roots more vigorous, effectively promoting the growth and development of tobacco.
At the same time, due to the complexity of the physiological and biochemical activities of amino acids, amino acids in plants can be assimilated into other amino acids through transamination, deamination and other reaction processes. They can also produce carbohydrates and organic acids through the citric acid cycle, glycolysis and other forms of metabolic pathways, and ultimately convert them into proteins or other plant cellular components. Therefore, the relationship between amino acid content and gene expression is complex and needs to be further investigated. Based on the expression of genes related to amino acid synthesis (Fig. 14b, Table S16), it was found that the expression of DEGs related to aspartic acid changed greatly, including GOT1 (glutamate oxaloacetate transaminase 1), ansA (aspartic acid synthesis- gene, L-asparaginase), etc., which may be due to the important role of aspartic acid. Aspartate is the common precursor of the two main pathways and forms aspartic acid, which affects glutamine production after subsequent ammonia. It can provide amino donors for the synthesis of other amino acids, purines and pyrimidines, promote the synthesis of other amino acids, and play an active role in growth and development. At the same time, the transcriptome data were verified by quantitative experiments on genes related to the synthesis and metabolism of amino acids and organic acids. The results showed that the change trend of qRT-PCR (quantitative real-time PCR) results was consistent with the change trend of transcriptome data (Fig. 16).
Preliminary review of some key genes involved in the brassinolide synthetic pathway and the aspartate metabolic pathway
To verify the reliability of expression levels in RNA sequence data, quantitative real-time PCR (qRT-PCR) was used to check the related genes of 12 differential metabolites brassinolide, aspartic acid and phydroxyphenylalanine (Fig. 16). The qRT-PCR results confirmed that the expression trends of 12 DEGs were highly consistent with the transcriptome data and supported the credibility of the RNA sequence data.
The key genes of the brassinolide metabolites CPD (cytochrome P450 superfamily protein, Gen_70248), NAGS1 (N-acetyl-L-glutamate synthase 1, Gen_54060), BZR1 (brassinazole-resistant 1, Gen_27329) and BRI1 (brassinosteroid-insensitive 1, Gen_76304, leucine-rich receptor-like protein kinase family) showed a trend towards upregulation after treatment. The expression level of NaHS treatment was significantly higher than that of CK, DET2 (3-oxo-5-steroid 4-dehydrogenase family protein, Gen_30735) and BIN2 (BR-insensitive 2, Gen_16737) showed a trend of downregulation and the expression level of NaHS treatment was significantly lower than that of CK. The key genes APA1 (Aspartic Protease, Gen_34238) and PCS1 (Phytochelatin Synthase 1, Gen_58153, eukaryotic Aspartyl Protease family protein) showed an upward trend after treatment, and the expression level of NaHS treatment was significantly higher than that of CK. The key genes of tyrosine metabolites ACR12 (ACT domain-containing protein, Gen_58257) and PPC3 (phosphoenolpyruvate carboxylase 3, Gen_1854) showed a downward trend, and the expression level of NaHS treatment was significantly lower than that of CK. AT1G (phosphoenolpyruvate carboxylase family protein, Gen_7059) and TAT (tyrosine transaminase family protein, Gen_12807) showed an upward trend after treatment, and the expression level of NaHS treatment was significantly higher than that of CK.
Many reports demonstrate that H2S is involved as a signaling molecule in several physiological processes in plants. An appropriate H2S concentration plays a crucial role in various processes including the formation and growth of plant root, the growth and development of plants, and their responses to biotic and abiotic stresses [4, 7, 9, 12, 14, 15, 17, 28]. Many reports have indicated that H2S inhibits ROS burst and promotes antioxidant activity under various environmental stresses including heavy metals, cold, drought and salinity [5-7, 12, 15, 28]. For example, NaHS dose-dependently promoted the formation and growth of lateral roots of sweet potato (Ipomoea batatas L.) [9]. Li reported that pretreatment with NaHS significantly increased the endogenous H2S content in the roots of Malus hupehensis seedlings, also increased the root activity, root length and activities of antioxidant enzymes (CAT and POD), and significantly decreased the MDA content in roots, decreased detoxified ROS generated by alkaline salt stress [47]. NaHS pretreatment significantly increased the activities of antioxidant enzymes (CAT, POD and SOD), which resulted in a decrease in MDA and H2O2 contents in cucumber roots under salt stress [48]. H2S maintains and promotes the activities of SOD and POD in tomato plant [28]. In this study, tobacco seedlings treated with exogenous NaHS were able to increase their root length, root activity, root wet weight, D/L-CD enzyme activities and endogenous H2S concentration and their CAT, SOD and POD activities without promoting stress and at the same time improve their acclimation to different stresses.
After NaHS treatment, a large number of DEGs (970 up-regulated and 2175 down-regulated) were found in the roots of the treatment group. GO enrichment analysis showed that these DEGs are related to large-scale biological processes and molecular functions, and that cellular processes and metabolic processes are the two most important components of differential gene enrichment. The differentially expressed genes were mainly enriched in components related to cells, organelles, catalytic activity and metabolism, suggesting that the differentially expressed genes were mainly concentrated in the secondary metabolic process, which was similar to the results of previous research [49-52]. Furthermore, these DEGs were mapped to KEGG pathways. The most significant differences concerned phenylpropanoid biosynthesis, photosynthesis, related metabolic pathways such as linolenic acid metabolites, and tyrosine metabolism (Fig. 5c). These results were also similar to previous studies on biological processes [4, 24, 27]. However, our study found that treating tobacco seedlings with sodium hydrosulfide is to improve their immunity and not to respond to various biological stresses.
Transcription factors (TFs) also play important roles in the developmental responses of plant roots, such as MYB, EFR (EF-TU RECEPTOR), bZIP (basic region/leucine zipper motif), GRF and MADS. Several transcription factors for root development, such as GRF, MYB and GATA, have also been identified. These NaHS-treated transcription factors may play a key role in tobacco root development. Several important transcription factors directly regulate tobacco root development in different ways. However, the connection between transcription factors has not been thoroughly investigated. In this study, transcriptome sequencing data provide a basis for distinguishing regulatory networks and novel regulatory factors involved in promoting NaHS treatment (Fig. 6). The GRF transcription factor is a special transcription factor in plants. It mainly regulates plant cell size, is involved in chloroplast proliferation and pistil development, and regulates plant growth and developmental processes such as osmotic stress[53, 54]. GRF transcription factors mainly contain two conserved domains, QLQ (Gln, Leu, Gln) and WRC (Trp, Arg, Cys) [53]. Studies have shown that the miR396-GRF regulatory module plays an important regulatory role in root development. The balance between miR396 and GRFs in Arabidopsis mainly influences the size of the meristem [55, 56]. Excessive accumulation of miR396 will seriously affect the entire longitudinal structure of roots. MicroRNA miR396 regulates the switch between stem cells and transit enhancing cells in Arabidopsis roots. The members of the MYB transcription factor family in plants are diverse and numerous and are involved in the regulation of various physiological processes such as plant development, metabolism and response to biotic and abiotic stresses [57, 58]. This finding is a vivid example of the cooperation of various transcription factors in regulating plant growth and development. Under NaHS treatment, the expression of MYB, GRF and GATA was significantly up-regulated, indicating that these three genes are closely related to the physiological process of NaHS treatment to promote root development and the ability to defend against various biological and abiotic stresses.
Metabolomics can define and quantify small-molecule metabolites in biological cells and is widely used to study the accumulation patterns of metabolites [24]. In addition, the technology also revealed changes in genes and metabolites in various tissues. Metabolites are intermediate and end products that regulate plant growth and development [19]. The total number of metabolites in plants is up to 200,000, making them an ideal target for studying biosynthetic regulation [22]. In recent years, the genetic mechanism of changes in plant metabolites has been investigated [20, 27]. We identified 384 metabolites that can be annotated in the MS2 database using a comprehensive targeted metabolomics approach. The KEGG categories related to the expression of different metabolites were “amino acid metabolism”, “carbohydrate metabolism”, “glycosylphosphatidylinositol (GPI) anchored biosynthesis” and “plant hormone signal transduction”. These results are similar to previous studies on biological processes of plant resistance [23, 24, 27, 50].
Comparative analysis of metabolomics and transcriptome is helpful to explain the biological processes and mechanisms involved in the regulatory response. In plant roots, the regulation of H2S at plant roots is generally concentration-dependent. With the increase of NaHS concentration (as H2S donor), the length and number of cucumber roots showed a trend that first increased and then decreased [27], and the length and density of lateral roots of tomatoes also showed the same trend [8]. Furthermore, the regulation of H2S in tomato lateral root growth is related to hydrogen peroxide and methane signaling pathways [59, 60]. During the course of NaHS treatment, plants mobilize a significant number of defense factors, especially metabolites. Brassinolide biosynthesis, indispensable for different phases of plant development, was one of the primary enrichment pathways between CK and NaHS groups. The BRI1 receptor (Brassinosteroid Insensitive 1) receives BR signals on the cell surface. It is currently believed that there are two mechanisms for BR signaling to activate BRI1 activity. One mechanism is that BAK1 (BRI1-associated receptor kinase 1) is a BRI1 co-receptor that is similar in structure. Both can interact in vivo and in vitro. BR first induces BRI1 and BAK1 to form heterodimers, and then mutual phosphorylation occurs between BRI1 and BAK1 [61-64], thereby regulating root cell development. Another mechanism is that BR binds to the BRI1 homodimer and induces its conformational change, activates its activity and then phosphorylates BAK1 [65]. Phosphorylated BRI1 and BAK1 act on the downstream components BES1 (BRI1-EMS-suppressor 1) and BZR1 (brassinazole-resistant 1) to receive upstream signals in the nucleus [65-68]. After their activities are activated, they further regulate the division or differentiation of root cells. This study showed that the metabolite BL was significantly up-regulated after NaHS treatment. However, it is not clear whether H2S regulates BR-induced root development. Heyman et al. showed that BR can promote the division of QC cells by inducing the expression of ERF115 and regulating the expression of PSK5 (a gene encoding the peptide hormone) [69]. In this study, the ERF115 gene was significantly up-regulated, promoted QC cell division, and induced tobacco growth.
AUXIN can induce PLT expression, but BZR1-mediated BR signaling can inhibit its expression, suggesting that BR and AUXIN have antagonistic effects on QC cell division [65], which is an important reason for NaHS regulation of the BR-induced root development could be. Recent studies have revealed that BR signaling can inhibit SHY2 expression by inducing BES1 expression and that BES1 can also act directly on PIN7 to regulate root meristem development [70]. These above results showed that the interaction between BR signaling pathway transcription factors and auxin regulates the mechanism of root stem cell niche, root apical meristem, root cell growth and lateral root. After NaHS treatment, increasing BR and decreasing auxin can better promote root development. At the same time, xyloglucan endotransglucosylase encoded by the TCH4 gene was also significantly down-regulated, which was involved in the integration of new xyloglucan into the cell wall and cell wall degradation in plant morphogenesis [71, 72]. Therefore, NaHS regulation of BR-induced downregulation of TCH4 gene might be involved in the regulation of cell wall relaxation. However, the specific mechanism of action can further investigate the target genes downstream of BR in NaHS response.
Amino acids are essential for protein synthesis. Previous studies have shown that amino acids, as intermediate or final metabolites of certain metabolic pathways, are also involved in the regulation of several metabolic pathways, which are connected to other physiological and biochemical metabolic pathways in plants, and influence many physiological processes in plants [24]. In this study, the levels of aspartic acid, lysine, threonine, methionine and isoleucine were significantly higher than CK after NaHS treatment. The aspartate family pathway includes four major essential amino acids (EAA), including lysine, methionine, threonine and isoleucine, and is also closely related to homoserine, glutamic acid, glycine and proline. After NaHS treatment, plants modify some amino acids through changes in amino acid metabolic pathways, thereby maintaining the stability of functional proteins and metabolic enzymes. The amino acids that make up plant proteins and enzymes can be divided into five categories: 3-phosphoglycerate, α-ketoglutarate, pyruvate, oxaloacetate and aromatic amino acids from the source of the synthetic carbon framework [45]. The results of the joint analysis showed that the application of hydrogen sulfide significantly changed the content of various amino acids in tobacco roots (Fig. 15). After NaHS treatment, the amino acids were efficiently differentiated: the content of α-ketoglutarate-arginine in tobacco roots was significantly up-regulated; The asparagine content of α-ketoglutarate group, 3-phosphoglycerate group serine and aromatic tyrosine was significantly down-regulated. Arginine is an amino acid with the highest nitrogen to carbon ratio. In addition to being a protein component, it is also a precursor of biosynthesis of nitric oxide, polyamines and proline [46]. The large accumulation of arginine in tobacco roots can make the development of tobacco roots more vigorous, effectively promoting the growth and development of tobacco.
NaHS treatment can positively and negatively regulate the metabolism of amino acids through different pathways in tobacco (Fig. 13). NaHS treatment significantly down-regulated the content of AnsA gene and asparagine in tobacco roots. The reason may be that hydrogen sulfide promotes the deamination of asparagine to aspartic acid, which is converted into glutamic acid and glutamine via the tricarboxylic acid cycle [73]. After NaHS treatment, the content of arginine and aromatic tyrosine in the α-ketoglutarate family in tobacco roots was significantly increased, and the content of serine in 3-phosphoglycerate family was significantly decreased. Arginine and aspartic acid are important intermediates in the urea cycle. NaHS treatment can promote the conversion of aspartic acid to arginine by accelerating the urea cycle, thereby significantly increasing arginine content. Arginine can regulate the growth and development of plants through its metabolites and associated enzymes (Fig. 14c). For example, arginine produces NO under the catalysis of NOS and regulates root growth and differentiation, flower induction, dormancy disruption and gene expression through NO [70, 74, 75]. In addition, arginine can also be catalyzed by enzymes such as ADC to form PA, which can induce lateral root formation, regulate fruit development, promote flower bud differentiation and pollen germination through PA [76]. The synthesis of aromatic amino acids requires the synthesis of shikimic acid via the shikimic acid pathway using phosphoenolpyruvate in the glycolysis pathway and 4-phosphoerythritol produced by the pentose phosphate pathway as substrates. Subsequently, the synthetic chorismate is converted into tryptophan, phenylalanine and tyrosine [77]. The significant up-regulation of tyrosine content in tobacco after NaHS treatment suggested that it may be involved in the coordinated regulation of the pentose phosphate pathway, glycolysis and shikimate pathway. The reason may lie in the fact that H2S can increase the expression of enzyme proteins related to the chorismate synthetic pathway in plants [78, 79]. However, the mechanism of coordinated regulation of H2S on plant carbon and nitrogen metabolism is complex, and it is still necessary to analyze the nitrogen metabolism mechanism of plants using molecular biology.
This study provides valuable molecular information and new genetic engineering clues for further research to promote plant growth. However, the functions of most of the DEGs or DAMs found in this study are still largely unknown, and we will continue to investigate the functions of these genes and metabolites.
Plant materials, growing conditions and treatments with NaHS and other chemicals
Seeds of tobacco Yunyan87 (Nicotiana tabacum cv. Yunyan87) seedlings were provided by the Tobacco Research Institute of Hubei in Wuhan, China. Healthy seeds were surface sterilized alternately with 70% (v/v) ethanol solution for 10 min and 10% (v/v) sodium hypochlorite solution for 5 min, followed by repeated washing with distilled water. Thereafter, seeds were sown on Murashige and Skoog (MS) medium and germinated in the dark at 26 ± 1 °C in a plant growth chamber with 16/8 h day/night regime and 65-70% relative humidity at 26 ± 1 °C.
Seedlings were able to grow in the tray and developed fourth leaves. Seedlings with fourth leaves were carefully rooted and roots were washed with tap water. Seedlings of the same size were then acclimatized in tap water for 24 h. The seedlings were then administered different concentrations of the H2S donor sodium hydrogen sulfide (NaHS) (CK, 200 mg/mL, 400 mg/mL, 600 mg/mL, 800 mg/mL). Normal deionized water was used as a control (CK). All these solutions were renewed once and twice every 5 days. The experiment was conducted under natural conditions in the greenhouse of Hubei University with an air temperature of 25 °C during the day (14 hours) and 22 °C at night (10 hours).
For each omics analysis, tobacco seedlings were treated with CK, NaHS (H2S) for 10 days and tobacco roots were collected from 3 plants (n = 3), immediately frozen in liquid nitrogen after treatment and stored at -80 °C until use.
Measurement of growth and root vigor
The length of plant roots was measured directly with a ruler after 10 days of treatment. Morphological characteristics such as growth were determined based on fresh weight. Tobacco plants were photographed after 10 days of treatment. For this purpose, treated and untreated seedlings were harvested and their fresh weight was determined using a digital scale.
We measured root vigor using the 2,3,5-triphenyl tetrazolium chloride (TTC) method [80, 81]. Briefly, we added 0.5 g of root samples to 10 mL equal volumes of phosphate buffer and 0.4% TTC in a tube, mixed and incubated the mixture for 2 h at 37 °C in the dark in a temperature chamber until the root tip sections redeveloped red color. Then we stopped the reaction with 2 mL of 1 mol/L sulfuric acid. Next, we cut the root tip with red color, completely immersed it in 10 mL of methanol in a sealed graduated test tube, and incubated it at a temperature of 30-40 °C until the root tip turned completely white. Finally, we measured the OD of the above extract at 485 nm using the spectrophotometric method (SP-756PC). The blind test served as a reference. All experiments were performed in triplicate. Root strength was calculated as follows:
Root vigor ACT=CmW×h [μg TTF/(g⋅h)]
Where C is the extent of tetrazolium reduction (μg), W is the root weight (g), h is the time in h and m is the Dilute the extracted solution.
Determination of the endogenous H2S contents and the activities of L/D-CD
We determined the level of endogenous H2S as previously described [7]; However, the protocol has been slightly changed. First, about 0.1 g of tobacco roots were ground in 0.9 mL of 20 mmol/L Tris-HCl (pH 8.0) with a mortar and centrifuged. The supernatant was then collected in a flask and 1% Zn(AC)2 was added to the flask and sealed with a rubber stopper. H2S released during the reaction was absorbed by Zn(AC)2. After reaction at 37 °C for 40 min, we added 100 μL of 20 mmol/L DPD in 7.2 mol/L HCl to 100 μL of 30 mmol/L FeCl3 in 1.2 mol/L HCl and left it for 5 min react. Finally, the absorbance values were measured at 670 nm, and we constructed a standard curve based on the Na2S concentration gradient. All experiments were performed three times and the final data were presented as the mean ± standard error of the three measurements.
The activity of the L/D-CD enzyme was measured by determining the amount of H2S released per minute by the degradation of L/D-Cys by L/D-CD enzymes as previously described with minor modifications. First, we ground 0.1 g of tobacco roots with 0.9 mL of 20 mmol/L Tris-HCl (pH 8.0) in a mortar and centrifuged. Next, we added 100 μL of the supernatant to 100 μL of 0.8 mmol/L L/D-cysteine, 400 μL of 2.5 mmol/L DTT, and 400 μL of 100 mmol/L Tris-HCl. The pH was adjusted to 9 to measure LCD activity and to 8 to measure DCD activity. The H2S content is shown in Section 1. After reaction at 37 °C for 40 min, we added 100 μL of 20 mmol/L DPD in 7.2 mol/L HCl to 100 μL of 30 mmol/L FeCl3 in 1.2 mol/L HCl. Finally, we measured the absorbance at 670 nm after 5 min.
Determination of the enzymatic activity of antioxidant enzymes and analysis of MDA content
We determined CAT activity by monitoring the decomposition of H2O2 at 240 nm for 3 min with a slight modification [11, 82]. The reaction mixture contained 2 mL of 50 mmol/L sodium phosphate buffer (pH 7.0), 1 mL of 0.2% H2O2, and 0.2 mL of enzyme extract. The change in optical density (OD) at 240 nm was recorded with a spectrophotometer after 45 s and 60 s at the time of adding the extract into the cuvettes. The difference in optical density between the 45-s and 60-s readings was used to calculate the CAT activity. One unit (U) was defined as the amount of enzyme that resulted in a change in absorbance of 0.01 per minute.
We determined POD activity previously described [24, 83]. Briefly, 5.0 g of washed tobacco roots were treated with 0.0, 200, 400, 600, and 800 mg/L NaHS for 10 days, weighed, and homogenized separately using a mortar and pestle. The roots were stored in 4 mL of 50 mM potassium phosphate buffer (pH 7.6) at 4 °C. We transferred the homogenate to a tube and centrifuged it for 10 min at 3000 g. We then transferred the supernatant to a 25 mL volumetric flask. Next, we extracted the precipitate twice with 5 mL of phosphate buffer and added the supernatant to the volumetric flask. The volume was attached to the balance and stored at low temperature. For the enzyme activity assay, we added 0.1 mL of the enzyme extract, 2.9 mL in 0.05 mol/L phosphate buffer to 1.0 mL of 2% H2O2 and 0.05 mol/L guaiacol and incubated it in a water bath for 15 min at 37 °C protocol. As a control, the enzyme extract was boiled for 5 min. Next, the reaction system was transferred to an ice bath. The reaction was stopped with 2.0 mL of 20% trichloroacetic acid, filtered or centrifuged at a rate of 5000 g for 10 min, diluted and the absorbance measured at 470 nm. POD activity was calculated using the following formula:
The change value of A470 by 0.01 per minute was used as 1 unit of POD activity (U). Where △A470 is the change value of the absorption during the reaction, W is the fresh weight of the tobacco in g, t is the reaction time in minutes, Vt is the total volume of the enzyme extract in mL and Vs is the volume of the test enzyme extract, determined in mL.
We determined the SOD activity previously described [84]. First, we treated 0.5 g of fresh tobacco roots with 0.0, 200, 400, 600, and 800 mg/L NaHS for 10 days. Next, we homogenized and ground the leaves into a slurry with 5 mL of prechilled phosphate buffer in a prechilled mortar on an ice bath. The homogenate was then filtered with cheesecloth and centrifuged at 1000 g for 20 min. Finally, the supernatant served as crude enzyme extract. All steps were performed at 0-4 °C. SOD activity was measured as follows: We added 0.05 mL of the enzymatic extract to 0.3 mL of 130 mmol/L methionine, 750 μmol/L nitroblue tetrazolium chloride (NBT), and 100 μmol/L EDTA-Na2 and 0.32 mL of 20 μmol/L riboflavin in a 0.05 mol/L potassium phosphate buffer (pH 7.8). The reaction mixture was subjected to incubation for 20 minutes in a chamber under a 4000 LX fluorescent lamp. The reaction was started by turning on the fluorescent lamp and ended by turning off the lamp after 5 minutes. The increase in absorbance at 560 nm was used to measure blue formazan using NBT photoreduction. The reaction mixture without enzyme extract was used as a control and incubated in the dark. One unit of SOD was the amount of enzyme required to suppress 50% of NBT photoreduction compared to the reaction mixture without plant extract.
We calculated the SOD activity as follows:
Where ACK and AE are the absorptions of the illuminated control and sample tubes respectively, V is the total volume of the sample tube in mL, Vt is the determined amount of the test sample in mL and W is the fresh sample weight in g.
We determined MDA (malondialdehyde) content using the thiobarbituric acid reactive substances (TBARS) method [24, 85]. 1 g of treated tobacco roots was weighed, a small amount of quartz sand and 2 mL of 10% trichloroacetic acid were added, ground for homogenization, and then 8 mL of 10% trichloroacetic acid was added for further grinding. The homogenate was centrifuged at 4000 rpm for 10 min and the supernatant was malondialdehyde extract. 2 mL of extract was added to each clean test tube, 2 mL of distilled water was added to the control tube, and then 2 mL of 0.6% thiobarbituric acid solution was added to each tube. Shake well, let the mixture react in a boiling water bath for 15 minutes, cool quickly and centrifuge. The absorbance values(A) of the supernatant were measured at 532 nm, 600 nm and 450 nm, respectively. There were three replicates for each treatment. We calculated the MDA content using the following formula:
Where A532, A600 and A450 are the absorptions of the sample tubes at 532 nm, 600 nm and 450 nm, respectively, VT is the total volume of the extract in mL, V0 is the measured liquid in mL and W is the fresh weight of tobacco tissue in g.
Transcriptomics analysis
Tobacco seedlings were collected for hydroponic experiments. After 10 days of treatment with 600 mg/L NaHS, these roots were frozen in liquid nitrogen and sent to Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China) for transcriptome sequencing with three biological replicates per treatment. For sequencing, RNA was extracted from tobacco roots and after screening the raw data, the sequencing error rate and GC content distribution were examined to obtain clean measurements for subsequent analyses. The FPKM (fragments per kilobase per million) values were used to indicate transcript or gene expression levels. The specific steps of data processing were as described by Meng et al. [84] carried out ad described. DEGs between the two groups were measured using DESeq2 software under the conditions of |log2Fold Change| screened ≥ 1 and false discovery rate (FDR) < 0.05 [84, 86]. The hypergeometric distributions of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms were tested. A heatmap was created to represent the bidirectional hierarchical grouping of the selected DEGs and samples. Gene ontology (GO) pathway analysis of biological process (BP), molecular function (MF), and cellular component (CC) was performed using TopGO to clarify the relationships between DEGs and pathways related to plant development.
Metabolomics analysis
Tobacco seedlings were collected for hydroponic experiments. After 10 days of treatment with 600 mg/L NaHS, these roots were frozen in liquid nitrogen. Sample preparation for metabolic analysis and data analysis were carried out at Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China) using standard procedures and analyzed six biological replicates of each treatment. The frozen root is used for metabolite extraction using a 400 µL solution of methanol/water (4:1). The ground materials were treated with a high-throughput tissue grinder at 50 Hz for 6 min at -20 °C, followed by ultrasonication at 40 kHz for 30 min at 4 °C and 30 min at -20 °C to precipitate proteins and finally centrifuged at 13000 g and 4 °C for 15 min. The supernatants obtained were carefully transferred to sample vials for LC-MS/MS analysis. A pooled quality control (QC) sample was prepared by mixing equal volumes of all samples and tested in the same manner as the analytical samples.
Quantitative PCR analysis
Quantitative fluorescence PCR was used to validate the twelve differentially expressed unigene transcript expression levels using the same RNA samples used for sequencing. The specific primers were designed using Primer Premier 6.0 and are listed in Table S18. The housekeeping gene was ubiquitin. According to the user manual, the first cDNA strand fragments were synthesized from total RNA using the PrimeScript™ RT Master Mix Kit (Takara, Japan). qRT-PCR was performed on an ABI7500 real-time fluorescence quantitative PCR machine (Applied Biosystems, Foster City, USA). Three replicates were performed for each sample. The relative expression levels of the candidate genes were measured using the comparative threshold cycle method (2-ΔΔCT) [36].
Statistical analysis
All data analyses and graphics drawings were accomplished in R 4.1.2. The statistical significance was calculated by Student’s t-test. The gene expression values of FPKM were scaled to Z-score to draw the heatmap of transcriptome and metabolome. Graphics and Heatmaps were plotted with the ggplot and the heatmap packages of R language respectively. Each experiment was repeated three times.
In summary, the differences between CK and NaHS treatment groups were analyzed using physiology, transcriptomics and metabolomics. NaHS could promote the root growth of tobacco seedlings and provide tobacco seedlings with some ability to defend against harmful biological and abiotic stresses. The possible mechanism between gene expression and metabolite biosynthesis was discussed. It is believed that the biosynthesis of brassinolide and the metabolism of aspartic acid may play an important role in promoting root development in tobacco seedlings. The key to the biosynthesis of brassinolide and the metabolic regulation genes of aspartic acid and their role in promoting root development need to be further explored and verified. This study provides valuable molecular information on the growth-promoting mechanism of tobacco roots and will significantly promote tobacco growth. It is possible for tobacco farmers to reduce pesticide dosage and maximize the protection of the soil environment.
Availability of data and materials
Acknowledgments
The authors are grateful to Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) for helping with RNA-sequencing and metabolomic analysis.
Authors’ Contributions
W.Y.: Data curation, Investigation, Writing-review & editing. D.W.: Formal analysis, Data curation, Investigation, Writing-original draft, Visualization. Y.Y.: Investigation, Writing-review. H.L.: Investigation, Data curation. C.Y.: Investigation, Data curation. J.Y.: Conceptualization, Supervision, Project administration, Funding acquisition. H.X.: Conceptualization, Methodology, Project administration, Supervision, Writing–review & editing.
Funding
This work was supported by the key technology projects of China National Tobacco Corporation (CNTC) [No.110202101059 (XJ-08); 110202201040 (XJ-11); 110202201035 (XJ-06)].
Availability of data and materials
Data supporting this study are included within the article and/or supporting materials. The RNA-seq data has been deposited into the Sequence Read Archive (SRA) at NCBI (BioProject ID: PRJNA1103126).
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Conflicts of Interest
The fundings from CNTC were used for the purchase of experimental materials, the cost of instrument testing and the cost of transportation. The experimental design and conduct, data collection, analysis and interpretation, patents related to this article, and the potential benefits of this article are all unrelated to CNTC. The authors declare no competing interests with CNTC.
Author details
1State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Science, Hubei University, Wuhan 430062, China
2Tobacco Research Institute of Hubei Province, Wuhan 430030, China
- Zhou Y, Jiang S, Jiao Y, Wang H. Synergistic effects of nanochitin on inhibition of tobacco root rot disease. Int J Biol. Macromol. 2017;99:205-12.
- Li Z. Modeling pesticide residues in tobacco leaves for improving life cycle inventory analysis of pesticides in the cigarette industry. Sci Total Environ. 2022;845:157267.
- Wang J, Wen G, Ding J, Li Y. The research progress of the residues of pesticides of tobacco and the discussion of reduce the residues of pesticides of tobacco leaf. J Yunnan Agric Univ. 2006;21:329-32.
- Zhu C, Wei Q, Hu W, Kong Y, Xiang X, Zhang H, Cao X, Zhu L, Liu J, Tian W, Jin Q, Zhang J. Unearthing the alleviatory mechanisms of hydrogen sulfide in aluminum toxicity in rice. Plant Physiol Bioch. 2022;182:133-44.
- Kharbech O, Ben Massoud M, Sakouhi L, Djebali W, Jose Mur LA, Chaoui A. Exogenous application of hydrogen sulfide reduces chromium toxicity in maize seedlings by suppressing NADPH oxidase activities and methylglyoxal accumulation. Plant Physiol Bioch. 2020;154:646-56.
- Chen Z, Yang B, Hao Z, Zhu J, Zhang Y, Xu T. Exogenous hydrogen sulfide ameliorates seed germination and seedling growth of cauliflower under lead stress and its antioxidant role. J Plant Growth Regul. 2017;37:5-15.
- Zhang H, Hu L, Hu K, He Y, Wang S, Luo J. Hydrogen sulfide promotes wheat seed germination and alleviates oxidative damage against copper stress. J Integr Plant Biol. 2008;50:1518-29.
- Fang T, Cao Z, Li J, Shen W, Huang L. Auxin-induced hydrogen sulfide generation is involved in lateral root formation in tomato. Plant Physiol Bioch. 2014;76:44-51.
- Zhang H, Tang J, Liu X, Wang Y, Yu W, Peng W, Fang F, Ma D, Wei Z, Hu L. Hydrogen sulfide promotes root organogenesis inIpomoea batatas, salix matsudana and glycine max. J Integr Plant Biol. 2009;51:1086-94.
- Lin Y, Li M, Cui W, Lu W, Shen W. Haem oxygenase-1 is involved in hydrogen sulfide-induced cucumber adventitious root formation. J Plant Growth Regul. 2012;31:519-28.
- Liu D, Xu S, Hu H, Pan J, Li P, Shen W. Endogenous hydrogen sulfide homeostasis is responsible for the alleviation of senescence of postharvest daylily flower via increasing antioxidant capacity and maintained energy status. J Agric Food Chem. 2017;65:718-26.
- Zhang X, Fu X, Liu F, Wang Y, Bi H, Ai X. Hydrogen sulfide improves the cold stress resistance through the CsARF5-CsDREB3 module in cucumber. Int J Mol Sci. 2021;22:13229.
- Zhang X, Liu F, Zhai J, Li F, Bi H, Ai X. Auxin acts as a downstream signaling molecule involved in hydrogen sulfide-induced chilling tolerance in cucumber. Planta. 2020;251:69.
- Zou H, Zhang N, Pan Q, Zhang J, Chen J, Wei G. Hydrogen sulfide promotes nodulation and nitrogen fixation in soybean-rhizobia symbiotic system. Mol Plant Microbe In. 2019;32:972-85.
- Liu Y, Wei L, Feng L, Zhang M, Hu D, Tie J, Liao W. Hydrogen Sulfide Promotes Adventitious Root Development in Cucumber under Salt Stress by Enhancing Antioxidant Ability. Plants. 2022;11:935.
- Fageria NK, Moreira A. The role of mineral nutrition on root growth of crop plants. Advances in agronomy. 2011;110:251-331.
- Li J, Zhang Q, Li M, Yang X, Ding J, Huang J, Yao P, Zhang X, Li X, Yang L. Multi-factor correlation analysis of the effect of root-promoting practices on tobacco rhizosphere microecology in growth stages. Microbiol Res. 2023;270:127349.
- Schaeffer S, Koepke T, Dhingra A. Tobacco: a model plant for understanding the mechanism of abiotic stress tolerance. Improv Crop Resist Abiotic Stress. 2012:1169-201.
- Alotaibi F, Alharbi S, Alotaibi M, Al Mosallam M, Motawei M, Alrajhi A. Wheat omics: Classical breeding to new breeding technologies. Saudi J Biol Sci. 2021;28:1433-44.
- He L, Liu X, Wu Z, Teng N. Transcriptome and metabolome analyses provide insights into the stomium degeneration mechanism in Lily. Int J Mol Sci. 2021;22:12124.
- Büscher JM, Czernik D, Ewald JC, Sauer U, Zamboni N. Cross-platform comparison of methods for quantitative metabolomics of primary metabolism. Anal Chem. 2009;81:2135-43.
- Nicholson JK, Lindon JC, Holmes E. 'Metabonomics': understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica. 1999;29:1181-89.
- Hao Z, Liu S, Hu L, Shi J, Chen J. Transcriptome analysis and metabolic profiling reveal the key role of carotenoids in the petal coloration of Liriodendron tulipifera. Hortic Res. 2020;7:70.
- Lv L, Dong C, Liu Y, Zhao A, Zhang Y, Li H, Chen X. Transcription-associated metabolomic profiling reveals the critical role of frost tolerance in wheat. BMC Plant Biol. 2022;22:333.
- Chen Z, Yan J, Qian L, Zhu W, Qiu J, Lu Q, Wang X, Wu Q, Ruan S, Huang Y. Integrated analysis of the transcriptome and metabolome of purple and green leaves of Tetrastigma hemsleyanum reveals gene expression patterns involved in anthocyanin biosynthesis. Plos One. 2020;15:e0230154.
- Guo J, Wu Y, Wang G, Wang T, Cao F. Integrated analysis of the transcriptome and metabolome in young and mature leaves of Ginkgo biloba L. Ind Crop Prod. 2020;143:111906.
- Ji X, Ren J, Zhang Y, Lang S, Wang D, Song X. Integrated analysis of the metabolome and transcriptome on anthocyanin biosynthesis in four developmental stages of Cerasus humilis peel coloration. Int J Mol Sci. 2021;22:11880.
- Li J, Shi C, Wang X, Liu C, Ding X, Ma P, Wang X, Jia H. Hydrogen sulfide regulates the activity of antioxidant enzymes through persulfidation and improves the resistance of tomato seedling to Copper Oxide nanoparticles (CuO NPs)-induced oxidative stress. Plant Physiol Bioch. 2020;156:257-66.
- Inzé D, De Veylder L. Cell cycle regulation in plant development. Annu Rev Genet. 2006;40:77-105.
- Ackerman-Lavert M, Fridman Y, Matosevich R, Khandal H, Friedlander-Shani L, Vragović K, Ben El R, Horev G, Tarkowská D, Efroni I, Savaldi-Goldstein S. Auxin requirements for a meristematic state in roots depend on a dual brassinosteroid function. Curr Bio. 2021;31:4462-72.e6.
- Bao F, Shen J, Brady SR, Muday GK, Asami T, Yang Z. Brassinosteroids interact with auxin to promote lateral root development in Arabidopsis. Plant Physiol. 2004;134:1624-31.
- Planas-Riverola A, Gupta A, Betegón-Putze I, Bosch N, Ibañes M, Caño-Delgado AI. Brassinosteroid signaling in plant development and adaptation to stress. Development. 2019;146:dev151894.
- Fujioka S, Takatsuto S, Yoshida S. An early C-22 oxidation branch in the brassinosteroid biosynthetic pathway. Plant Physiol. 2002;130:930-39.
- Ohnishi T, Godza B, Watanabe B, Fujioka S, Hategan L, Ide K, Shibata K, Yokota T, Szekeres M, Mizutani M. CYP90A1/CPD, a brassinosteroid biosynthetic cytochrome P450 of Arabidopsis, catalyzes C-3 oxidation. J Biol Chem. 2012;287:31551-60.
- CañO-Delgado A, Yin Y, Yu C, Vafeados D, Mora-GarcíA S, Cheng J, Nam KH, Li J, Chory J. BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis. Development. 2004;131:5341-51.
- Zhang C, He M, Jiang Z, Liu L, Pu J, Zhang W, Wang S, Xu F. The xyloglucan endotransglucosylase/hydrolase gene XTH22/TCH4 regulates plant growth by disrupting the cell wall homeostasis in Arabidopsis under boron deficiency. Int J Mol Sci. 2022;23:1250.
- He C, Liang J, Wu Z, Zhuge X, Xu N, Yang H. Study on the interaction preference between CYCD subclass and CDK family members at the poplar genome level. Sci Rep. 2022;12:16805.
- Heyman J, Cools T, Vandenbussche F, Heyndrickx K, Van Leene J, Vercauteren I, Vanderauwera S, Vandepoele K, De Jaeger G, D. VDS, L. DV. ERF115 controls root quiescent center cell division and stem cell replenishment. Science. 2013;342:860-63.
- Vragović K, Sela A, Friedlander-Shani L, Fridman Y, Hacham Y, Holland N, Bartom E, Mockler TC, Savaldi-Goldstein S. Translatome analyses capture of opposing tissue-specific brassinosteroid signals orchestrating root meristem differentiation. Pro Natl Acad Sci USA. 2015;112:923-28.
- Dello Ioio R, Linhares FS, Scacchi E, Casamitjana-Martinez E, Heidstra R, Costantino P, Sabatini S. Cytokinins determine Arabidopsis root-meristem size by controlling cell differentiation. Curr Biol. 2007;17:678-82.
- Moubayidin L, Perilli S, Dello Ioio R, Di Mambro R, Costantino P, Sabatini S. The rate of cell differentiation controls the Arabidopsis root meristem growth phase. Curr Biol. 2010;20:1138-43.
- Mouchel CF, Osmont KS, Hardtke CS. BRX mediates feedback between brassinosteroid levels and auxin signalling in root growth. Nature. 2006;443:458-61.
- Scacchi E, Salinas P, Gujas B, Santuari L, Krogan N, Ragni L, Berleth T, Hardtke CS. Spatio-temporal sequence of cross-regulatory events in root meristem growth. Proc Natl Acad Sci U S A. 2010;107:22734-39.
- Gupta A, Singh M, Laxmi A. Interaction between glucose and brassinosteroid during the regulation of lateral root development in Arabidopsis. Plant Physiol. 2015;168:307-20.
- Rossi S, Chapman C, Yuan B, Huang B. Improved heat tolerance in creeping bentgrass by γ-aminobutyric acid, proline, and inorganic nitrogen associated with differential regulation of amino acid metabolism. Plant Growth Regul. 2021;93:231-42.
- Bokhary SUF, Wang L, Zheng Y, Jin P. Pre-storage hot water treatment enhances chilling tolerance of zucchini (Cucurbita pepo L.) squash by regulating arginine metabolism. Postharvest Biol Tec. 2020;166:111229.
- Li H, Shi J, Wang Z, Zhang W, Yang H. H2S pretreatment mitigates the alkaline salt stress on Malus hupehensis roots by regulating Na+/K+ homeostasis and oxidative stress. Plant Physiol Bioch. 2020;156:233-41.
- Jiang J, Tian Y, Li L, Yu M, Hou R, Ren X. H2S alleviates salinity stress in cucumber by maintaining the Na+/K+ balance and regulating H2S metabolism and oxidative stress response. Front Plant Sci. 2019;10:678.
- Li C, Nong Q, Solanki MK, Liang Q, Xie J, Liu X, Li Y, Wang W, Yang L, Li Y. Differential expression profiles and pathways of genes in sugarcane leaf at elongation stage in response to drought stress. Sci Rep. 2016;6:25698.
- Tian Y, Feng F, Zhang B, Li M, Wang F, Gu L, Chen A, Li Z, Shan W, Wang X, Chen X, Zhang Z. Transcriptome analysis reveals metabolic alteration due to consecutive monoculture and abiotic stress stimuli in Rehamannia glutinosa Libosch. Plant Cell Rep. 2017;36:859-75.
- Wang Y, Hao X, Lu Q, Wang L, Qian W, Li N, Ding C, Wang X, Yang Y. Transcriptional analysis and histochemistry reveal that hypersensitive cell death and H2O2 have crucial roles in the resistance of tea plant (Camellia sinensis (L.) O. Kuntze) to anthracnose. Hortic Res. 2018;5:18.
- Wang R, Xu S, Wang N, Xia B, Jiang Y, Wang R. Transcriptome analysis of secondary metabolism pathway, transcription factors, and transporters in response to methyl jasmonate in Lycoris aurea. Front Plant Sci. 2017;7:1971.
- Weidemüller P, Kholmatov M, Petsalaki E, Zaugg JB. Transcription factors: bridge between cell signaling and gene regulation. Proteomics. 2021;21:e2000034.
- Huang Y, Chen J, Li J, Li Y, Zeng X. Genome-wide identification and analysis of the growth-regulating factor family in Zanthoxylum armatum DC and functional analysis of ZaGRF6 in leaf size and longevity regulation. Int J Mol Sci. 2022;23:9043.
- Rodriguez RE, Mecchia MA, Debernardi JM, Schommer C, Weigel D, Palatnik JF. Control of cell proliferation in Arabidopsis thaliana by microRNA miR396. Development. 2010;137:103-12.
- Liebsch D, Palatnik JF. MicroRNA miR396, GRF transcription factors and GIF co-regulators: a conserved plant growth regulatory module with potential for breeding and biotechnology. Curr Opin Plant Biol. 2020;53:31-42.
- Li C, Ng CK-Y, Fan L. MYB transcription factors, active players in abiotic stress signaling. Environ Exp Bot. 2015;114:80-91.
- Smita S, Katiyar A, Chinnusamy V, Pandey DM, Bansal KC. Transcriptional regulatory network analysis of MYB transcription factor family genes in rice. Front Plant Sci. 2015;6:1157.
- Mei Y, Chen H, Shen W, Shen W, Huang L. Hydrogen peroxide is involved in hydrogen sulfide-induced lateral root formation in tomato seedlings. BMC Plant Biol. 2017;17:162.
- Mei Y, Zhao Y, Jin X, Wang R, Xu N, Hu J, Huang L, Guan R, Shen W. L-cysteine desulfhydrase-dependent hydrogen sulfide is required for methane-induced lateral root formation. Plant Mol Biol. 2019;99:283-98.
- Wang X, Goshe MB, Soderblom EJ, Phinney BS, Kuchar JA, Li J, Asami T, Yoshida S, Huber SC, Clouse SD. Identification and Functional Analysis of in Vivo Phosphorylation Sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 Receptor Kinase. The Plant Cell. 2005;17:1685-703.
- Nam KH, Li J. BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Cell. 2002;110:203-12.
- Whippo CW, Hangarter RP. A brassinosteroid-hypersensitive mutant of BAK1 indicates that a convergence of photomorphogenic and hormonal signaling modulates phototropism. Plant Physiol. 2005;139:448-57.
- Karlova R, De Vries SC. Advances in understanding brassinosteroid signaling. Sci STKE. 2006;2006:pe36.
- Wang X, Li X, Meisenhelder J, Hunter T, Yoshida S, Asami T, Chory J. Autoregulation and homodimerization are involved in the activation of the plant steroid receptor BRI1. Dev Cell. 2005;8:855-65.
- Yin Y, Wang Z, Mora-Garcia S, Li J, Yoshida S, Asami T, Chory J. BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell. 2002;109:181-91.
- He J, Gendron JM, Sun Y, Gampala SSL, Gendron N, Sun C, Wang Z. BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses. Science. 2005;308:1743.
- Li J, Wen J, Lease KA, Doke JT, Tax FE, Walker JC. BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell. 2002;110:213-22.
- Yu X, Li L, Li L, Guo M, Chory J, Yin Y. Modulation of brassinosteroid-regulated gene expression by Jumonji domain-containing proteins ELF6 and REF6 in Arabidopsis. Proc Natl Acad Sci U S A. 2008;105:7618-23.
- Bethke PC, Libourel IGL, Jones RL. Nitric oxide reduces seed dormancy in Arabidopsis. J Exp Bot. 2006;57:517-26.
- Xu W, Campbell P, Vargheese AK, Braam J. The Arabidopsis XET-related gene family environmental and hormonal regulation of expression. Plant J. 1996;9:879-89.
- Antosiewicz DM, Purugganan MM, Polisensky DH, Braam J. Cellular localization of Arabidopsis xyloglucan endotransglycosylase-related proteins during development and after wind stimulation. Plant Physiol. 1997;115:1319-28.
- Kovács Z, Simon-Sarkadi L, Sovány C, Kirsch K, Galiba G, Kocsy G. Differential effects of cold acclimation and abscisic acid on free amino acid composition in wheat. Plant Sci. 2011;180:61-68.
- Yamasaki H. The NO world for plants: achieving balancein an open system. Plant, Cell & Environment. 2005;28:78-84.
- Grün S, Lindermayr C, Sell S, Durner J. Nitric oxide and gene regulation in plants. J Exp Bot 2006;57:507-16.
- Kalac̆ P, Krausová P. A review of dietary polyamines: Formation, implications for growth and health and occurrence in foods. Food Chem. 2005;90:219-30.
- Heinemann B, Hildebrandt TM. The role of amino acid metabolism in signaling and metabolic adaptation to stress-induced energy deficiency in plants. J Exp Bot. 2021;72:4634-45.
- Ren X, Jiang J, Sun W, Li L. Proteomic analysis of cucumber seedling response to high salt stress by exogenous H2S. Acta Botanica Boreali-Occidentalia Sinica. 2018;38:2236-48.
- Qian Y, Lynch JH, Guo L, Rhodes D, Morgan JA, Dudareva N. Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants. Nat Commun. 2019;10:15.
- Liu C, Wang X, Tu B, Li Y, Chen H, Zhang Q, Liu X. Root K affinity drivers and photosynthetic characteristics in response to low potassium stress in K high-efficiency vegetable soybean. Front Plant Sci. 2021;12:732164.
- Duncan DR, Widholm JM. Osmotic induced stimulation of the reduction of the viability dye 2,3,5-triphenyltetrazolium chloride by maize roots and callus cultures. J Plant Physiol. 2004;161:397-403.
- Tareen MJ, Abbasi NA, Hafiz IA. Postharvest application of salicylic acid enhanced antioxidant enzyme activity and maintained quality of peach cv. ‘Flordaking’ fruit during storage. Sci Hortic-Amsterdam. 2012;142:221-28.
- Wang Y, Liang C, Meng Z, Li Y, Abid MA, Askari M, Wang P, Wang Y, Sun G, Cai Y, Chen S, Lin Y, Zhang R, Guo S. Leveraging Atriplex hortensis choline monooxygenase to improve chilling tolerance in cotton. Environ Exp Bot. 2019;162:364-73.
- Meng L, Yang Y, Ma Z, Jiang J, Zhang X, Chen Z, Cui G, Yin X. Integrated physiological, transcriptomic and metabolomic analysis of the response of Trifolium pratense L. to Pb toxicity. J Hazard Mater. 2022;436:129128.
- Haghighi M, Ramezani MR, Rajaii N. Improving oxidative damage, photosynthesis traits, growth and flower dropping of pepper under high temperature stress by selenium. Mol Biol Rep. 2018;46:497-503.
- Liu C, Zhou G, Qin H, Guan Y, Wang T, Ni W, Xie H, Xing Y, Tian G, Lyu M, Liu J, Wang F, Xu X, Zhu Z, Jiang Y, Ge S. Metabolomics combined with physiology and transcriptomics reveal key metabolic pathway responses in apple plants exposure to different selenium concentrations. J Hazard Mater. 2024;464:132953.
No competing interests reported.
- SupplementaryData2024417.docx
- SupplementaryTableS1Evaluationstatisticsofsequencingdata.xlsx
- SupplementaryTableS2Comparisonefficiencybetweensampleandreferencegenome.xlsx
- SupplementaryTableS3StatisticsofupregulatedanddownregulatedDEGs.xlsx
- SupplementaryTableS4Top10updownDEGsstatistics.xlsx
- SupplementaryTableS5DEGsGOanalysis.xlsx
- SupplementaryTableS6KEGGenrichmentanalysisofDEGs.xlsx
- SupplementaryTableS7KEGGannotationanalysisofDEGs.xlsx
- SupplementaryTableS8ExpressionprofileofrelatedtranscriptionfactorsfromDEGs.xlsx
- SupplementaryTableS9UpdownDAMsstatistics.xlsx
- SupplementaryTableS10Thetop20statisticaldataofKEGGpathway.xlsx
- SupplementaryTableS1150differentialmetabolitesDAMs.xlsx
- SupplementaryTableS12ThecategoriesofDAMs.xlsx
- SupplementaryTableS13TheKEGGpathwaysofDEGsandDAMs.xlsx
- SupplementaryTableS14CorrelationNetworkanalysisofDEGsandDAMs.xlsx
- SupplementaryTableS15DEGsenrichedbrassinosteroidbiosynthesispathway.xlsx
- SupplementaryTableS16DEGsenrichedAspartatemetabolicpathway.xlsx
- SupplementaryTableS17SimultaneousenrichedDAMsandDEGs.xlsx
- SupplementaryTableS18Primesequences.xlsx