Differential proteomics of tobacco seedling roots at high and low potassium concentrations

The effects of high potassium and normal potassium treatments on protein expression in roots of flue-cured tobacco plant HKDN-5 at the seedling stage were analyzed by an unlabeled protein quantification technique. The results showed that 555 proteins were differentially expressed (245 proteins were down-regulated and 310 proteins were up-regulated) in high potassium treatment compared with normal potassium treatment. Differentially expressed proteins were involved in 96 metabolic pathways (42 metabolic pathways, 21 synthetic pathways as well as catabolic pathways, including fatty acid metabolism, phenylpropane biosynthesis, ketone body synthesis and degradation, and butyric acid metabolism. Root processing of high potassium concentrations leads to increases in the synthesis of peroxidase, superoxide dismutase and acyl-coenzyme-A synthetase. Additional proteomic differences observed in tobacco roots grown in high potassium include proteins involved with genetic information processing as well as environmental sensing. Examples include RNA helicase, ABC transporters and large subunit GTPases. These up-regulated differentially expressed proteins function mainly in protein translation, ribosome structure and protein synthesis. This indicates that under high potassium treatment, root protein synthetic processes are accelerated and substance metabolism pathways are enhanced; thus, providing the material and energetic basis for root growth.

Potassium is the most important and abundant cation in plant cells. Potassium plays an important role in many physiological processes of plants, such as enzyme activation 1,2 , stomatal opening and closing 3 , membrane transport, charge balance, osmotic adjustment 4,5 . At the same time, as an important nutrient element, potassium also participates in important life activities, including photosynthesis, transportation and distribution of assimilates, which have an important impact on the growth and development of plants. Tobacco is a typical potassium loving crop and potassium plays an important role in improving the maturity, combustibility, smoking quality, and safety of cigarette products (for example, by reducing the production of tar and other harmful compounds) 6 . The root system is an important organ for flue-cured tobacco to absorb water and nutrients from soil and to synthesize hormones. Root system development directly affects the subsequent appearance and growth of tobacco plants, and affects the yield and quality of tobacco 7 . Therefore, it is of great significance to study the changes in root proteins under different potassium levels for practical cultivation applications in flue-cured tobacco.
In recent years, there has been more and more research on proteomics in tobacco, which mainly focused on the study of differential proteins under stress. For example, the protein levels in tobacco were studied after infection with the wildfire pathogen to analyze the wildfire disease resistance mechanism. Differences were detected in ten proteins; six had decreased expression, four had increased expression, and the ways in which the functions of the differentially expressed proteins are involved in the disease-resistance mechanism are discussed by Cui 9 . Another study used Cuibi No.1 tobacco as the test material; root and leaf proteins under low and high nitrogen supply levels were systematically analyzed by comparative proteomics 8 , to understand the changes in protein expression and abundance in roots and leaves under low nitrogen stress. This study further analyzed the response mechanism of multiple metabolic pathways in tobacco to a low nitrogen environment on the basis of protein expression. However, there are few reports that analyze the differences in protein abundance in roots of tobacco seedlings grown at high or low potassium concentrations.
Label free quantitative proteomics technologies can generally be divided into two methods: one is a signal intensity method based on peak height, peak area and peak capacity of the spectrum; the other is a spectrum counting method based on the number of secondary spectrums of matching peptides. In theory, label free Methods. Material planting. The experimental materials were hydroponically grown in the greenhouse in Hunan Agricultural University Yunyuan science and education base at 2018, here the tobacco variety was HKDN-5. The treatments were designated high potassium (K + concentration of 720 mg·L −1 ) and normal potassium (K + concentration of 240 mg·L −1 , control). The concentration of potassium in Hoagland's nutrient solution was increased by potassium nitrate, and the content of nitrogen was decreased by ammonium nitrate; during hydroponics, the ambient temperature was controlled at 25 ± 2 °C.
Sample collection, protein preparation and concentration determination. When the seedlings of HKDN-5 grew to 5-6 leaves (before transplanting), 4-5 plants with normal growth were selected and their roots were combined to make a mixed sample. Each treatment was replicated 3 times. Samples were frozen in liquid nitrogen and kept frozen until use.
Proteins from tobacco leaves were extracted with a phenol method 11 , and protein content was determined by the Bradford method 12 .
Proteolysis. 100 μgs of each sample was transferred to a test tube and adjusted to a constant volume of 100 μL with 8 M urea. 11 μLs of 1 M DTT were added and incubated at 37 °C for 1 h, Next, the samples were transferred to 10 K ultrafiltration tubes and centrifuged at 14,000 g for 10 min. 120 μL of 55 mM iodoacetamide were added after centrifugation, and then the samples were incubated at room temperature for 20 min in the dark. In the same ultrafiltration tube, the samples were centrifuged for three times with 100 mM TEAB to replace the urea system. After this operation, trypsin was added at aprotein: enzyme ratio of 50:1. Enzymolysis was conducted overnight, after which the samples were freeze-dried 13 .
Nano-HPLC-MS/MS analysis. The lyophilized peptides were redissolved in 30 μL formic acid solution and separated by nano-LC, and analyzed by on-line electrospray ionization tandem mass spectrometry. The experiment was carried out on the Nano ACQUITY UPLC system, and the system was connected with a Q-Exactive mass spectrometer equipped with an on-line nano electrospray ion source. A 10 μL peptide sample was loaded into a capture column at a flow rate of 10 μL/min and then separated on a linear gradient: 3% a to 32% a (A: 0.1% formic acid ACN solution) within 120 min. The column was equilibrated under initial conditions for 10 min. The column flow rate was controlled at 300 nL/min, and the electrospray voltage was 2 kV 14 .
Data analysis. The peptides were filtered with a 1% FDR and 1 unique peptide. According to the ANOVA algorithm, proteins with significance greater than 13 (p-value less than 0.05) were selected as differentially expressed proteins.
Go was used to analyze the molecular functions, cellular locations and biological processes of proteins; cog & KOG annotation analysis was used to predict and classify protein functions and metabolic pathways. Metabolic pathways with P value < 0.05 were used to analyze the biological process of proteins 15 .

Results and analysis
Flue-cured tobacco seedling root phenotypic differences. The root length of tobacco seedlings treated with a high concentration of potassium is shorter than the root length of control tobacco seedlings. However, at high potassium, the number of fibrous roots are more than the control treatment and the diameter of the taproots are greater than the control treatment. Meanwhile, the dry weights and fresh weights of the roots of the high potassium grown seedlings is greater than the control treatments (Table 1 and Fig. 1). Therefore, growth in Table 1. Phenotype difference on root of flue-cured tobacco at seedling under two potassium treatment. www.nature.com/scientificreports/ a high concentration of potassium leads to a more developed root system of flue-cured tobacco seedlings than growth in a low concentration of potassium and provides a better material basis for the growth of tobacco plants.

SDS-PAGE electrophoresis of roots at seedling stage. The distribution of total proteins in root cells
of high potassium and normal potassium treated seedlings was uniform, with similar protein bands and no high abundance proteins (Fig. 2), which met the requirements of GC-MS detection for protein abundance and was convenient for differential proteomics analysis.

Differential protein identification and statistics.
Compared with the control treatment, 555 differentially expressed proteins in roots were found under high potassium treatment (differential expression greater than 1.5, P < 0.05). Among these 245 were down regulated and 310 were up regulated. Up regulated and down regulated differentially expressed proteins were mainly concentrated in the difference multiple range of 1.5-2, accounting for 61.2% and 54.2% of the total down regulated and up regulated differentially expressed proteins ( Table 2).
The most up-regulated functional class was (J), the least was (D), and the most down-regulated functional class was (O). Compared with the control treatment, the differentially expressed proteins in the high potassium treatment mainly belonged to translation, ribosome structure and biological origin (J),and post translational modification, protein conversion, and chaperone protein (O) functional classes.
Of the most differentially abundant proteins in the high potassium treatment (≥ fourfold difference) there were15 proteins (13 up-regulated and 2 down-Regulated) that are involved in metabolism related processes such  www.nature.com/scientificreports/ as C, E, I, P, Q and H, 6 proteins (5 up-regulated and 1 down-regulated) that are involved in genetic information processing related processes such as A, B and J, 4 proteins (1 up-regulated and 3 down-regulated) that are involved in cell and environmental information processing related processes such as M, O and T, and 9 proteins (8 up-regulated and 1 down-regulated) that are involved in R and S Processes (Table 4).

KEGG pathway analysis of differentially abundant proteins.
The results showed that compared with the control, the proteins that are differentially expressed under high potassium treatment participated in 96 pathways (42 metabolic pathways, 21 synthetic pathways and other catabolic pathways), including fatty acid metabolism, phenylpropane biosynthesis, ketone synthesis and degradation, butyric acid metabolism, etc. The root proteins differentially expreessed under high potassium treatment mainly participated in the process of material metabolism, which provided the basis for the better growth and development of the roots.

Discussion
Differentially abundant proteins related to metabolism. Analysis of the high potassium treatment compared with the normal treatment suggested that the main differentially abundant proteins related to metabolism are serine carboxypeptidase, phospholipase, alcohol dehydrogenase, peroxidase, superoxide dismutase, acyl coenzyme A synthetase, etc. Serine carboxypeptidase is a kind of protease belonging to the α/β hydrolase family, which plays a significant role in the biosynthesis of secondary metabolites, the catalysis of acyl transfers and the degradation of seed germination related proteins. This enzyme gene is a kind of stress resistance gene of plants, which plays a significant role in plant growth and development and stress resistance 16,17 . Phospholipase plays roles in various biological and abiotic stress signal transduction pathways. It can hydrolyze phospholipid and participate in Table 3. GO annotation and percentage of differential expressed proteins in high potassium treatment.  www.nature.com/scientificreports/ other lipid metabolism pathways as main raw materials after hydrolysis, or directly participate in stress response signal transduction as signal molecules 18,19 . Our results that these two enzymes are down regulated in the high potassium environment may be due to the fact that these enzymes are more conducive to the distribution and utilization of substances or are not needed in the non-adverse high potassium environment. Alcohol dehydrogenase is the leading enzyme of ethanol fermentation, which plays a vital role in the anaerobic respiration of plants. It is an important hydrolase in the abnormal respiratory chain of plants, and it is also closely related to the stress resistance physiology of plants 20 . Peroxidase is involved in physiological and biochemical processes in plants.It regulates the development process of higher plants, contains iron porphyrin cofactors, and closely participates in cell development. The occurrence and development of adventitious roots in plants are related to the action of catalase 21 .
Superoxide dismutase (SOD) is a kind of metal enzyme widely existing in microorganisms, animal and plant cells. In plant cells, it can protect against reactive oxygen species and other external interference in a high potassium environment 22 . Acyl coenzyme A synthetase is involved in fatty acid metabolism. It can activate free fatty acids to Acyl coenzyme a thiolipids. Sulfur lipids can participate in signaling, transcriptional regulation, transmembrane transport and other metabolic pathways, and they are the substrates of β-oxidation 23 . Our results that these enzymes are more abundant under high potassium conditions indicates that the growth and development of roots are promoted at high potassium levels.
Differentially abundant proteins related to genetic information processing. Histones are related to chromosome replication in the cell cycle or are useful in gene expression regulation 24 . Our results showed that compared with the control treatment, differentially up-regulated proteins at high potassium that play a role in gene expression regulation are histones, ribosomal proteins, RNA helicase, etc (Fig. 2). These enzymes are up-regulated and are involved in genetic information processing of tobacco roots in a high potassium environment. The up regulation of ribosomal protein is very obvious (21.21 times), which means that in the high potassium treatment, the protein synthesis related pathway is strengthened. This is more conducive to the growth of  www.nature.com/scientificreports/ tobacco roots. The up regulation of RNA helicase will promote protein synthesis and accelerate the movement of proteins in the process of root development.
Differentially abundant proteins related to cell processes and environmental information processing. Sucrose synthetase is an important enzyme of sucrose metabolism, which plays a vital role in plant growth. It can control sucrose metabolism and sucrose accumulation, provide energy for plant growth and development, and regulate cell metabolism processes 25 . Our results showed that the inhibitory factors of sucrose synthetase and K + transport growth defect (up-regulated by 2.04 times) were up-regulated, and the expression of apoptosis ATPase was down regulated under high potassium treatment. This may be due to the high affinity of tobacco for potassium, The up-regulation of K + transporter or K + channel related protein, and the up-regulation of K + transport growth defect inhibitory factor indicate that the root system needs better growth and development in an environment with high K + concentration. The down regulation of apoptotic ATPase may be due to the energy demand of tobacco's faster growth and development.
Other differentially abundant proteins. Chitinase is an important plant defense factor; it plays an important role in plant growth and development, stress resistance and defense response 26 . ABC transporter has a transmembrane domain, which can be used as a membrane integrin to transport peptides, sugars, lipids, heavy metal chelates and other organic compounds 27 . Our results showed that compared with the control treatment, the other differentially abundant proteins in the high potassium treatment were chitinase, high glucose regulatory enzyme, conserved protein, ABC transporter and GTP enzyme. All of these enzymes were up-regulated. This indicates that in the high potassium treatment, due to the enhancement of metabolic pathways, the high glucose regulatory enzyme was up-regulated, as well as the up-regulated conserved protein (9.79 times). GTP can promote the development and growth of tobacco roots, and can provide energy for the material metabolism of tobacco growth.

Conclusion
In summary, our findings suggest that in the high potassium environment the root differentially abundant proteins of flue-cured tobacco seedlings are mainly located in the cell component, the organelle component and the membrane organelle. The proteins we identified have molecular functions such as hydrolase activity, ion binding, small molecule binding, heterocyclic and organic ring compound binding, and participate in biological processes such as monomer metabolism, cell metabolism, organic acid metabolism, basic metabolism, monomer cell process and nitrogen component metabolism. The proteins in tobacco plants under high potassium treatment that are involved in transformation and translation are easier to express differentially and mainly participated in material metabolism and other related pathways. Meanwhile they also play a key role in promoting the growth and development of the tobacco root system.