iTRAQ-based quantitative proteomic of tobacco (Nicotiana tabacum L.) identified major host proteins involved in photosystems throughout the aging process

Background Leaf senescence is one of the most common manifestations in plant senescence and has an important effect on photosynthesis. However, the molecular regulation of leaf senescence in response to photosynthesis is still poorly understood. To gain insight into the molecular mechanisms underpinning tobacco, we integrated photosynthesis, organelle ultrastructural, and proteomic analyses of tobacco leaves during senescence. Results The photosynthetic rate, intercellular CO 2 concentration, tomatal conductance, transpiration rate showed a downward trend and the stability of organelle decreased with the increasing of tobacco leaves age. iTRAQ and PRM verification were used to analyze the proteins expressed in different periods based on photosynthetic physiology and ultramicroscopic observation. A total of 321, 319, 223 diﬀerentially expressed proteins (DEPs) were identified from over maturity (OM) vs immature (IM), OM vs well maturity (WM) and WM vs IM, respectively, including 122/199, 124/195 and 125/98 up/down proteins, respectively. KEGG analysis of DEPs was significantly enriched in metabolic pathways, biosynthesis of secondary metabolites, microbial metabolism in diverse environments, starch and sucrose metabolism. In addition, down-regulated proteins were also involved in metabolic pathways such as carbon sequestration of photosynthetic organisms and photosynthesis. Furthermore, PRM analysis indicated that iTRAQ is highly reliable. Conclusions Our study provided important technical references for screening photosynthetic host proteins of tobacco leaf senescence and revealing the molecular mechanism during the senescence of tobacco leaves.

decreased, and the grana lamella became loose gradually from compact arrangement. The number of grana lamellae increased firstly and then decreased. The chloroplast membrane also gradually dissolved. The proportion of starch granules was positively correlated with increasing of leaf age.

Identification of Proteins at Different Maturity Stages
The number of secondary spectra produced by mass spectrometry and the number of secondary spectra analyzed for three groups of tobacco leaves at different growth stages (IM, OM and WM) were 435604 and 158095, respectively. The identification rate of spectra was more than 36.29%. The number of identified peptide segments was 23346, and the number of identified proteins was 4747. The number of identified proteins containing at least two specific peptide segments was 3292, which accounted for 69.35% of total protein amount.

Quantitative Analysis of Protein between Samples
If there is no significant change in the amount of the same protein between the two samples in relative quantification, the protein abundance ratio is close to 1. Pairwise comparisons of proteins with P-values < 0.05 and fold-changes > 1.2 or < 0.83 in abundance were regarded as differentially regulated proteins (DRPs). We identified a total of 321 DRPs, including 122 up-regulated and 199 down-regulated proteins, by comparing the OM against the IM (Fig.3A). In contrast, we obtained 319 DRPs, including 124 upregulated and 195 down-regulated proteins, by comparing the OM against the WM (Fig.   3B). There were 223 DRPs, including 125 up-regulated and 98 down-regulated proteins between WM and IM (Fig. 3C). Fig. 4 was a Venn diagram showing significant differences in protein expression between pairwise comparisons. Further comparisons of different proteins between the control groups revealed that there were 86 differentially expressed proteins among OM vs IM, OM vs WM and WM vs IM of the three control groups.

Photosynthetic proteins expression patterns
Photosynthesis is critical for tobacco leaves yield; hence, we investigated the abundance in photosystems (I and II) and ATP synthases (Fig.7C).

PRM verification
Three proteins, including A0A1S4DCM1, A0A140G1R2 and A0A140G1P2 which were significantly expressed and related to photosynthesis were selected and their expression was examined by PRM. Skyline can not only visualize the target peptide ion signal which is collected by mass spectrometry, but also judge the quality and content of the peptide signal by observing the peak shape, peak intensity, and retention time consistency [24].
The effect is immediately visible when the ratios are presented in a density plot ( There were researches indicated that higher intercellular CO 2 concentration and tomatal conductance were factors that improved photosynthesis. Photosynthesis was inseparable from photosynthesis rate, intercellular CO 2 concentration, tomatal conductance and transpiration rate [27]. During the senescence of tobacco leaves, the decrease of tomatal conductance resulted in the decrease of gas exchange, which decreased the intercellular CO 2 concentration and transpiration rate, then resulted in the decrease of photosynthetic rate. The decline of photosynthesis related index indicated that the photosynthesis of tobacco leaves decreased significantly during senescence.
The number of chloroplasts showed a downward trend with the increasing of tobacco leaf age. The grana lamellae gradually became loose from close arrangement, and its amount increased first and then decreased, and the chloroplast membrane gradually dissolved. Up-regulated proteins were also involved in the metabolic pathways including "Endoplasmic reticulum protein processing" and "Phenylpropanoid biosynthesis".
Phenylpropanoid was a kind of secondary metabolite, and its protein expression was up- indicated that photosynthesis and sugar synthesis were weakened during tobacco leaves senescence. Plaxton et al. [40] found that plant glycolytic enzymes were multifunctional proteins involved in the process, rather than carbohydrate metabolism. ATP was produced in glycolysis pathway [41], the down-regulation of protein expression related to glycolysis and glycogenesis indicated that ATP synthesis was decreased and sugar synthesis weakened during tobacco leaves senescence.
According to the enrichment of 86 proteins, they were mainly involved in metabolic pathways, including metabolic pathways, biosynthesis of secondary metabolites, metabolism of microorganisms in different environments, carbon sequestration of photosynthetic organisms, photosynthesis and metabolism of starch and sucrose.
Photosystem I and II were photosynthetic electron transport centers [42]. According to the search results of 8 proteins related to photosynthesis, 3 proteins with significant differences between the comparison groups were screened for PRM verification. In the PRM validation process, three differentially expressed proteins were consistent with the quantitative results of iTRAQ in pairwise comparisons. Therefore, three differentially expressed proteins including A0A1S4DCM1, A0A140G1R2 and A0A140G1P2 were likely to be key regulators of tobacco leaves senescence, and can be used as marker protein for tobacco leaves senescence.
Photoreaction of photosynthesis is driven by protein complexes such as photosystem I, photosystem II, cytochrome b6/f complex, ATP synthase, and so on [43]. Oxygen-evolving enhancer protein 3-2, chloroplastic-like was a protein related to photosystem II, which participated in oxygen release during photosynthesis and maintained the stability of photosynthesis system [44,45,46]. In this study, the expression of A0A1S4DCM1 was upregulated during tobacco leaves senescence, which was consistent with previous research results [47,48,49]. It is indicated that during the senescence of tobacco leaves, attributing to the decline of photosynthesis, the A0A1S4DCM1 is increased in order to maintain the stability of the photosynthetic system. In addition, oxygen-evolving enhancer proteins are products of degradation of the photosynthetic system, the oxygen-evolving

Conclusion
In this study, tobacco leaf photosynthetic rate, intercellular CO 2 concentration, body surface conductivity, transpiration rate, and organelle ultrastructure all changed significantly. Combining proteomics technology and physiological changes for the first time, photosynthesis during tobacco senescence was described. Physiological changes revealed the mature metabolism of tobacco leaves; and dynamic protein changes obtained using iTRAQ revealed important metabolic pathways during tobacco senescence from the protein level, including "metabolic pathways", "biosynthesis of secondary metabolites", Metabolism in different environments "," starch and sucrose metabolism "and" photosynthesis "and so on. Finally, through PRM verification of photosynthesis-related differential expressed proteins, three differential expressed proteins including A0A1S4DCM1, A0A140G1R2 and A0A140G1P2 were different at three growth stages.
Therefore, they can be used as protein biomarkers in the process of tobacco leaves senescence.
All iTRAQ dataset related to this study has been public available on iProX (www.iprox.org) with ID IPX0001668000/ IPX0001668001/ IPX0001668002.

Plant Materials
The seeds of the tobacco variety K326 were acquired from Zhongyan Tobacco Seed Co.,  10).

Determination of Photosynthetic Rate
LI-6400 portable photosynthetic system (Li-Cor Inc., Lincoln, NE, USA) was used to measure the photosynthetic indexes of tobacco leaves at different maturity stages [54].
The photosynthetic rate, intercellular carbon dioxide concentration, tomatal conductance and transpiration rate of tobacco leaves were measured at IM, WM and OM stages, respectively. The determination time was once every two hours from 8:00 to 18:00 and five times in total.Then calculated the average value. During the measurements, the leaf temperature was adjusted to 25 °C. Sigma Plot is used to plot according to the data.

Electron Microscope Scanning
Refer

Separation and Identification of Proteins
Protein extraction and separation, iTRAQ quantification, bioinformatics analysis and PRM validation were carried out according to the following procedures (Fig. 11).

Enzymatic Hydrolysis and Desalination
100 μg of protein from each sample was taken for trypsin digestion and then diluted 5 times with 100 mM Triethylamine borane (TEAB). Trypsin was added at a mass ratio of 1:50 (trypsin: protein) and theenzymatic hydrolysis was digested overnight at 37 °C. After enzymatic hydrolysis, the peptide segments were desalted by AAA-C18 column (150 mm×4.6 mm I.D., 5! M, AB SCIEX), and then frozen and dried in vacuum [56].

LC-MS/MS Analysis
Referring to the method of Zhu et al.     The top ten metabolic pathways involved by three groups of differential proteins.
(A) OM vs IM metabolic pathways involved by differential proteins. (B) OM vs WM metabolic pathways involved by differential proteins. (C) WM vs IM metabolic pathways involved by differential proteins.   Column map of PRM quantitative protein differences among different samples.