Integrative proteome and metabolome unveil the central role of IAA alteration in axillary bud development following topping in tobacco

Axillary bud is an important aspect of plant morphology, contributing to the final tobacco yield. However, the mechanisms of axillary bud development in tobacco remain largely unknown. To investigate this aspect of tobacco biology, the metabolome and proteome of the axillary buds before and after topping were compared. A total of 569 metabolites were differentially abundant before and 1, 3, and 5 days after topping. KEGG analyses further revealed that the axillary bud was characterized by a striking enrichment of metabolites involved in flavonoid metabolism, suggesting a strong flavonoid biosynthesis activity in the tobacco axillary bud after topping. Additionally, 9035 differentially expressed proteins (DEPs) were identified before and 1, 3, and 5 days after topping. Subsequent GO and KEGG analyses revealed that the DEPs in the axillary bud were enriched in oxidative stress, hormone signal transduction, MAPK signaling pathway, and starch and sucrose metabolism. The integrated proteome and metabolome analysis revealed that the indole-3-acetic acid (IAA) alteration in buds control dormancy release and sustained growth of axillary bud by regulating proteins involved in carbohydrate metabolism, amino acid metabolism, and lipid metabolism. Notably, the proteins related to reactive oxygen species (ROS) scavenging and flavonoid biosynthesis were strongly negatively correlated with IAA content. These findings shed light on a critical role of IAA alteration in regulating axillary bud outgrowth, and implied a potential crosstalk among IAA alteration, ROS homeostasis, and flavonoid biosynthesis in tobacco axillary bud under topping stress, which could improve our understanding of the IAA alteration in axillary bud as an important regulator of axillary bud development.


Outgrowth of axillary buds in response to topping
We compared the growth of axillary buds in response to untopping and topping in tobacco (Fig. 1A).The growth of the axillary buds was distinctly accelerated after topping, as the length of axillary buds increased about 2, 5, 12 times at 1, 3, 5 days after topping, respectively (Fig. 1B).In contrast, the length of axillary buds showed no significant change within 5 days in untopped tobacco plants (Fig. 1C).It means that topping can significantly accelerate the growth of axillary buds in tobacco.To explore the metabolomic and proteomic changes of axillary

UPLC-MS/MS identification of DAMs associated with axillary bud metabolome following topping
The widely targeted metabolomic analysis of axillary buds in untopped and topped tobacco plants at 1 (T1), 3 (T3), and 5 (T5) days after topping, detected 869 metabolites across all the samples.The detected metabolites were divided into 11 categories.Most of these metabolites were primary lipids (147, 16.92%), phenolic acids (140, 16.11%), flavonoids (105, 12.08%), and alkaloids (104, 11.97%).Interestingly, only five quinones were detected (Fig. 2A).Based on the cluster and correlation analysis, the metabolites in the 12 samples were divided into four groups (Fig. 2B) with significant intragroup correlations (Fig. 2C).Additionally, PCA revealed significant differences among the four groups (Fig. 2D).PC1 and PC2 accounted for 46.62 and 22.85% of the samples, respectively, with a cumulative contribution rate of 69.47%.On the PC1 axis, the control (CK) and T1 samples significantly differed from the T3 and T5 samples, implying metabolite differences between the axillary meristem initiation and axillary bud growth.
The top 10 enriched pathways based on KEGG enrichment analysis are shown in Fig. 4A.The DAMs in the T1 vs. CK, T3 vs.CK, and T5 vs. CK groups were enriched in the flavonoid biosynthesis, amino acids biosynthesis, and 2-Oxocarboxylic acid metabolism pathways.Interestingly, only the pathways related to flavonoid metabolism (flavonoid biosynthesis and flavone and flavonol biosynthesis) were significantly enriched (p-value < 0.05) by DAMs in T3 vs.T1, T5 vs. T1, and T5 vs. T3 comparison groups.A total of 100 differentially accumulated flavonoids were identified across all the comparison groups.These flavonoids accounted for the largest proportion (17.54%) of the DAMs (Table S1).Most of these flavonoids were up-regulated after topping and were highly accumulated at T5 (Fig. 4B), suggesting that the tobacco axillary bud has strong flavonoid biosynthesis activity after topping.Notably, flavonols and flavones accounted for 82% of all differentially accumulated flavonoids; most were identified in the glycosylated form (Table S1).These results revealed that flavonoid metabolism, especially related to flavonols and flavones, is essential in regulating axillary bud development.www.nature.com/scientificreports/

Comparative analysis of axillary bud proteome following topping
A total of 15,486 credible proteins were identified and quantified by proteomics, and their temporal expression patterns were identified by PCA analysis.The CK group was differentiated from the T1, T3, and T5 groups on the PC1 axis and the T1 group from the T3 and T5 groups on the PC2 axis.Contrary to the metabolomics analysis, the T3 group was clustered with the T5 group, suggesting that the difference in protein expression profiles at these time points was limited (Fig. 5A).
Proteins with an FC > 1.5 or < 1/1.5 and P value < 0.05 were assigned as DEPs.A total of 9035 DEPs were identified from all comparison groups.The largest number of DEPs was detected between T1 and CK, where 6247 DEPs were identified, implying highly dynamic proteome changes within the axillary buds following topping.Additionally, 5675, 5804, 2176, and 2199 DEPs were detected in the T3 vs.CK, T5 vs. CK, T3 vs.T1, and T5 vs. T1 comparison groups, respectively.Only 43 DEPs were identified in the T5 vs. T3 comparison group (Fig. 5B).www.nature.com/scientificreports/Based on the GO enrichment analyses, all the DEPs were assigned to at least one of the three categories: biological process, cellular component, and molecular function.In the biological process, DEPs in all comparison groups were highly enriched in stimulus response-related processes, including response to oxygen-containing compounds, inorganic substances, and oxidative stress (Fig. 6A).In addition, the DEPs between T1 vs. CK were specifically enriched in transport-related processes, including the import into the nucleus, protein import into the nucleus, nucleocytoplasmic transport, and nuclear transport (Fig. 6A).KEGG enrichment analyses revealed that the DEPs in T1 vs. CK were mainly enriched in genetic information processing, consistent with the GO enrichment results.Additionally, the DEPs in T3 vs.CK and T5 vs. CK comparison groups were enriched in the plant hormone signal transduction, MAPK signaling pathway, and starch and sucrose metabolism pathways.At the same time, most DEPs in the T3 vs.T1 and T5 vs. T1 comparison groups were enriched in metabolic pathways and biosynthesis of secondary metabolites (Fig. 6B).

Proteins related to the plant hormone signal transduction pathways following topping in tobacco
A total of 67 DEPs related to plant hormone signal transduction involving IAA, CTK, gibberellin, and abscisic acid (ABA) were identified (Fig. 7, Table S2).Among them, 17 DEPs were involved in IAA signal transduction including auxin transporter protein, transport inhibitor response 1 (TIR1), auxin-responsive protein (AUX/ IAA), auxin response factor (ARF), and the indole-3-acetic acid-amido synthetase.Almost all of them were significantly down-regulated after topping, except AUX/IAA, which was up-regulated in T5 (Fig. 7A).For the cytokinin signal transduction pathway, seven DEPs were identified, including two histidine kinases (CRE1), one histidine-containing phosphotransferase protein (AHP), and four two-component response regulators (B-ARR).CRE1, AHP, and one B-ARR expression were down-regulated in T1, T3, and T5.On the contrary, the other three B-ARRs were up-regulated after topping (Fig. 7B).Additionally, the 25 DEPs involved in the gibberellin signal transduction pathways showed a complex expression pattern, which was particularly obvious with gibberellininsensitive dwarf 1(GID1) (Fig. 7C).There were 7, 4, and 4 GID1 were up-regulated in T1 vs. CK, T3 vs.CK, T5 vs. CK, respectively.At the same time, there were 6, 4, and 4 GID1 down-regulated in T1 vs. CK, T3 vs.CK, T5 vs. CK, respectively.Among them, 3 and 2 GIDs were up-regulated and down-regulated in the three comparison groups, respectively (Fig. 7C).Besides, the DEPs involved in ABA signal transduction pathways had similar expression patterns with those involved in cytokinin signaling.Most of these related proteins were down-regulated after topping (Fig. 7D).

Correlations between the metabolome and proteome during axillary bud development in tobacco
The top 10 metabolites and proteins with the most influence on the model based on O2PLS are shown in Fig. 9A.The proteins with the highest influence on the metabolome were involved in carbohydrate metabolic, oxidation-reduction, and genetic information processes (Table S5).The top 10 metabolites included 3 amino acid derivatives, 3 alkaloids, 2 flavonoids, 1 nucleotide derivative, and 1 coumarin.IAA was among the most influential metabolites that regulate axillary bud development.Notably, chrysoeriol-7-O-(6''-malonyl)-glucoside, piperidine, and γ-glutamyl methionine were significantly positively correlated with IAA and were among the most influential metabolites (Table S5).

Discussion
Topping is a necessary agronomic practice in tobacco.It alters various biological processes by switching the plant from its reproductive to its vegetative phase.Subsequently, this alters nicotine biosynthesis, the hormonal balance, root development, and the source-sink relationship [19][20][21] .The elongation of axillary buds is a typical plant response to topping.This phenomenon was also observed in our study, where the length of axillary buds increased rapidly www.nature.com/scientificreports/after topping (Fig. 1).Omics technology can provide a more comprehensive perspective for understanding of the molecular mechanisms underlying axillary bud growth in response to topping.Previous research have revealed that the growth of axillary bud may be controlled by genes involved in carbohydrate metabolism and in the hormone signal transduction pathway 14 .Consistent with this view, our proteome data showed that DEPs in T3 vs.CK and T5 vs. CK were mainly enriched in plant hormone signal transduction, MAPK signaling pathway, and starch and sucrose metabolism (Fig. 6B).Many phytohormones, including IAA, CTK, gibberellin, and ABA, regulate the axillary bud development and can influence each other in various ways 12,[22][23][24] .IAA is produced by the shoot apex and transported basipetally through the stem, and inhibits growth of axillary bud in indirect ways since it does not enter the buds 7 .The IAA depletion in the main stem following topping control bud outgrowth by inhibiting SL and inducing CTK 8,9 .Both CTK and SL can move into the buds, and control the bud outgrowth by regulating, at least partly, the expression of the the transcription factor BRC1 25 .Gibberellin and ABA are also involved in axillary bud development.However, their role is less investigated than the roles of IAA, CTK, and SL [26][27][28] .In the present study, 67 DEPs related to these hormone signal transduction were identified, with most of them down-regulated after topping (Fig. 7, Table S2), consistent with the findings from previous studies wherein the majority of the hormone-related genes were down-regulated after topping 14 .
On the other hand, IAA depletion from the main stem following topping allows canalization driven IAA export from axillary buds, and this is hypothesized to be required for bud outgrowth [29][30][31][32] .Based on metabolome results, we found the IAA content in axillary buds decreased sharply at day one after topping (Figure S1, Table S1), which was probably caused by IAA export from buds.To date, there is a lot of evidences that the growth arrest of bud often correlate with the elevated IAA content and strong IAA signaling in buds 11,33 .For example, the expression of AUX/IAA and SAUF genes were up-regulated in dormant buds of strawberry 34 .Additionally, most of IAA signaling relevent genes were down-regulated in growing buds of tobacco after topping 14 .Consistently, we also identified several IAA signaling relevent proteins, such as ARF, AUX/IAA, and TIR1, were down-regulated at T1, T3, and T5 in topped plant (Fig. 7).These results suggested that reduction of IAA levels and inhibition of IAA signal transduction were associated with axillary buds' release from dormancy in tobacco following topping.It is noted that the concentration of IAA in buds of topped plant returned to similar levels as controls (CK) at day three and remained stable until day five (Figure S1, Table S1).Several studies have confirmed that the upper axillary buds will become a new source of IAA to restore apical dominance in topped plant, and the sustained growth of buds require both IAA synthesis and transport 32,35 .Therefore, our results suggested that IAA alteration in buds might play a critical role in dormancy release and sustained growth of axillary bud.
However, the role of the concentration variance of IAA in axillary bud outgrowth is not yet clear.In the present study, we identified many DEPs (476) strongly correlated with IAA contents, including 435 negatively correlated DEPs, which were mainly enriched in carbohydrate, amino acid, and lipid metabolism (Fig. 9, Table S6).Carbohydrate metabolism is involved in many essential processes in plants, as it is a energy source, components of natural architecture and is involved in the transfer of information 16,36 .Evidence is emerging that sucrose and trehalose 6-phosphate play a central role in the control of bud release 24 .Lipid signaling molecules are also involved in axillary bud growth.For example, BnFAX6 mediating fatty acid export from plastids functions in axillary bud dormancy release, possibly through enhancing linoleic acid level in axillary buds of Brassica napus 37 .Amino acids are primary units of proteins, and also serve as energy sources, chemical messengers and precursors of diverse metabolites 38 .Therefore, amino acid metabolism play a fundamental role in plant growth and development.Taken together, our results provide clues for further research on whether IAA in axillary buds regulates bud development by influencing the expression level of proteins associated with these metabolic pathways.Additionally, we also identified another three metabolites, including piperidine, γ-glutamyl methionine and chrysoeriol-7-O-(6''-malonyl) glucoside, which seemed had a synergistic role with IAA in regulating the axillary bud development.All of them were among the most influential metabolites in the O2PLS model, and showed a similar change profile following topping and significantly correlated with the 476 DEPs (Fig. 9A, Figure S2, Table S6).However, how these metabolites interact with IAA to affect axillary bud development remained unclear.Herein, we suggested that these metabolites possibly control buds outgrowth by regulating IAA transport.Chrysoeriol-7-O-(6'-malonyl) glucoside is a flavonoid, this kind of substances have a similar structure to the auxin inhibitor N-1-naphthylphthalamic acid and can help accumulate IAA by inhibiting IAA polar transport 39 .In Arabidopsis, a change in flavonoid profile, level and/or distribution can lead to redistribution of auxin efflux carriers, and results in changing of IAA levels 40 .Current studies have also indicates that glutathione can alter IAA transport and metabolism, and its re-synthesis through the γ-glutamyl cycle requires the participation of γ-glutamyl methionine 41,42 .Therefore, it is possible that the down-regulation of these metabolites induced by topping enhanced the IAA polar transport and then reduced the content of IAA in buds.If this was the case, functional studies of these substances may provide more options for screening natural inhibitors to control axillary bud outgrowth.
In this study, we also found a striking enrichment of metabolites and proteins related to flavonoid biosynthesis and metabolism (Figure S3, Fig. 8).Flavonoids are secondary metabolites widely found in plants, which are involved in various processes of plant growth, such as axillary buds outgrowth, male fertility, root development, and pigmentation 43,44 .Previous observations indicate that a high level of flavonoid and flavonoid pathway genes can repress the outgrowth of axillary buds by inhibiting the IAA transport 10 .On the contrary, most of flavonoid and flavonoid pathway genes identified from our omics data were up-regulated in growing buds.Additionally, several DEPs involved in p-coumaroyl-CoA synthesis, which is a common precursor of flavonoids synthesis, showed a negative correlation with the IAA content (Fig. 8C).Therefore, if the mechanism of flavonoid-dependent axillary bud inhibition also applicable to tobacco, it is likely that only a few flavonoids are responsible for the regulation of IAA transport, such as chrysoeriol-7-O-(6'-malonyl) glucoside.On the other hand, accumulation of flavonoids in buds after topping might have another functions.Flavonoids can protect plants against biotic and abiotic stressors, such as herbivores, bacteria, fungi, and ultraviolet 45,46 .Topping is a form of mechanical damage sustained at the aboveground plant part, which can lead to the accumulation of ROS, including H 2 O 2 , superoxide anion, and hydroxyl radicals, subsequently inducing oxidative stress to the plant [47][48][49][50] .Under stress, plants trigger their oxidative defense system to balance ROS synthesis and scavenging by increasing the levels/ activities of enzymatic and non-enzymatic antioxidants.Flavonoids can act as non-enzymatic antioxidants to reduce ROS levels by direct ROS scavenging, inhibiting the activities of free radical-generating enzymes, and activating enzymatic antioxidants 45 .Besides flavonoids, we also identified large number of enzymatic antioxidants, such as SOD, POD, GST, CAT, and GR, and most of which were up-regulated after topping (Figure S4, Table S3).Among them, several enzymatic antioxidants were negatively correlated with the IAA content (Fig. 9D).Several studies have revealed that stress-induced ROS can alter the IAA gradient and interfere with IAA-mediated signal transduction.In turn, IAA induces ROS production and regulates its homeostasis by influencing antioxidant levels 51 .Anyway, there seems to be an interesting crosstalk of IAA alteration and ROS homeostasis in axillary bud outgrowth of tobacco under topping stress.

Conclusion
A total of 9035 DEPs and 569 DAMs were identified from proteomic and metabolomic data of the axillary bud following topping in tobacco.Our work highlight the role of IAA alteration in axillary bud outgrowth, and implied a potential crosstalk among IAA alteration, flavonoids accumulation profile and ROS homeostasis.Additionally, three metabolites (piperidine, γ-glutamyl methionine and chrysoeriol-7-O-(6''-malonyl) glucoside) were newly identified from axillary buds in tobacco, which might act as inhibitor of IAA transport.The findings in this study will substantially expand the current spectrum of how to define the axillary bud at the proteomic and metabolomic levels, providing valuable metabolites coupled with proteins for further functional characterization.

Plant materials and cultivation
K326, the main tobacco cultivar in China, characterized by strong axillary growth, was used as the model crop in this study.The use of plants in study complies with international, national and/or institutional guidelines.K326 seeds were germinated in a mixture of 60% (w/w) peat culture substrate, 20% (w/w) ground maize stalk, and 20% (w/w) perlite in a naturally illuminated glasshouse.Two months from sowing, the seedlings were transferred into 4 L pots (one plant per pot) containing paddy soil.The plants were watered daily.
Five weeks after transplanting, the head and two leaves below the flag leaf were topped.The upper axillary buds were collected before and 1, 3, and 5 days after topping.Upper axillary buds from seven individual tobacco plants were collected as a biological replicate and three biological replicates were harvested 66 , giving a total of 12 samples.Samples were immediately frozen in liquid nitrogen and stored at − 80℃, awaiting further analysis.

Metabolomics and differentially accumulated metabolite (DAM) analysis
The sample preparation and metabolome profiling were performed by Metware Biotechnology Co., Ltd.(Wuhan, China) following their standard procedures.All samples were freeze-dried using a vacuum freeze-dryer (Scientz-100F) and then ground into powders.Dissolve 50 mg of powder with 1.2 mL 70% methanol solution, vortex 30 s every 30 min for 6 times in total.Following centrifugation at 12,000 rpm for 3 min, the extracts were filtrated before UPLC-MS/MS analysis.All sample extracts were mixed in equal proportions to generate samples for quality control.
The sample extracts were analyzed using an UPLC system (SHIMADZU Nexera X2) coupled with tandem mass spectrometry (MS/MS, Applied Biosystems 4500 Q TRAP), running in electrospray ionization mode.The column in the UPLC system was Agilent SB-C18 (1.8 µm, 2.1 mm × 100 mm).The mobile phase was consisted of solvent A (pure water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid).Sample measurements were performed with a gradient program that employed the starting conditions of 95% A, 5% B. Within 9 min, a linear gradient to 5% A, 95% B was programmed, and a composition of 5% A, 95% B was kept for 1 min.Subsequently, a composition of 95% A, 5.0% B was adjusted within 1.1 min and kept for 2.9 min.The flow velocity was set as 0.35 mL per minute; The column oven was set to 40 °C and the injection volume was 4 μL.The ESI source operation parameters were as follows: source temperature 550 °C; ion spray voltage 5500 V (positive ion mode)/− 4500 V (negative ion mode); ion source gas I, gas II, curtain gas were set at 50, 60, and 25 psi, respectively; the collision-activated dissociation was high.
Analyst 1.6.3software was used for data filtering, peak detection, alignment and calculations.Qualitative analysis was performed base on the Metware Database, the substance was characterized according to the secondary spectral information.Metabolite quantification was conducted through multiple reaction monitoring analysis using triple quadrupole mass spectrometry.Unsupervised principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were carried out with the package MetaboAna-lystR.The variable importance in projection (VIP) score was obtained by the OPLS-DA model, and metabolites with VIP ≥ 1, absolute log2 fold change (FC) ≥ 1, and p-value ≤ 0.05 were defined as DAMs.Hierarchical cluster analysis and higher Venn diagram were visualized by TBtool v2.080.Moreover, R software analyzed Pearson correlation coefficients between treatments and metabolites.The DAM functions were characterized using GO/ KEGG enrichment analyses.

Proteomics and DEP analysis
The proteins in the 12 samples were quantified using the BCA Kit (P0010S) according to the manufacturer's instructions.Half of each sample was used for SDS-PAGE detection and to determine the protein concentration, while the other half was used for trypsin hydrolysis.After desalting the enzymatic hydrolysis peptide, the proteins in the samples were analyzed by LC-MS/MS.

Figure 1 .
Figure 1.Axillary buds outgrowth of untopped and topped tobacco plants after 1, 3 and 5 days.(A) Phenotypes of axillary buds in different time points after topping.Axillary buds were showed as red box.(B) The length of lateral buds in topped and untopped plants.The data shown are two-tail t-test: * P < 0.05; ** P < 0.01; ns P > 0.05.

Figure 2 .
Figure 2. Qualitative and quantitative analysis of the metabolomics data.(A) Classification of metabolites with annotated structures.(B) Hierarchical cluster analysis of metabolite profiles.(C) Heat map of samples obtained from correlation analysis.(D) PCA analysis plots of the four group samples.

Figure 3 .
Figure 3. Identification and characterization of DAMs.(A) Advanced Venn diagram and Upset plots showing the DAMs in different comparison groups.(B) Accumulation profiles of 20 DAMs common in six comparison groups.(C) Accumulation profiles of 37 DAMs specific to T1 vs. CK group.

Figure 4 .
Figure 4. Flavonoid metabolism in the axillary bud after topping.(A) Top 10 enriched KEGG terms in each comparison group.(B) Hierarchical cluster analysis of all DAMs.The accumulation profiles of flavonoids were highlighted with the red box.

Figure 5 .
Figure 5. Proteome divergence among the four group samples.(A) PCA analysis plots of the four group samples.(B) DEPs statistics in six comparison groups.

Figure 6 .
Figure 6.Enrichment analysis of DEPs in five comparison groups.(A) Top 10 enriched biological process GO terms in each comparison group.(B) Top 10 enriched KEGG terms in each comparison group.

Figure 8 .
Figure 8. Expression patterns of the DEPs involved in flavonols and flavones biosynthesis.(A) Flavonols and flavones biosynthesis pathway in tobacco.The proteins identified in the proteome are shown in red, and the main flavonoid products in the metabolome are shown in bold.(B, C) Heat map of DEPs expression patterns during flavonols and flavones biosynthesis.(D) Heat map of UGTs differential expression patterns after topping.

Figure 9 .
Figure 9. Joint metabolome and proteome analysis.(A) The top 10 influential metabolites and proteins based on O2PLS analysis.(B) The top 20 enriched KEGG terms of 435 DEPs.(C, D) Correlation network analysis between DEPs and metabolites.Metabolites and proteins are indicated as rhombus and circles, respectively.