Mechanism of [CO2] Enrichment Alleviated Drought Stress in the Roots of Cucumber Seedlings Revealed via Proteomic and Biochemical Analysis

Abstract

Cucumber is one of the most widely cultivated greenhouse vegetables, and its quality and yield are threatened by drought stress. Studies have shown that carbon dioxide concentration ([CO₂]) enrichment can alleviate drought stress in cucumber seedlings; however the mechanism of this [CO₂] enrichment effect on root drought stress is not clear. In this study, the effects of different drought stresses (simulated with 0, 5% and 10% PEG 6000, i.e., no, moderate, and severe drought stress) and [CO₂] (400 μmol·mol^-1 and 800 ± 40 μmol·mol^-1) on the cucumber seedling root proteome were analyzed using the tandem mass tag (TMT) quantitative proteomics method. The results showed that after [CO₂] enrichment, 346 differentially accumulating proteins (DAPs) were found only under moderate drought stress, 27 DAPs only under severe drought stress, and 34 DAPs under both moderate and severe drought stress. [CO₂] enrichment promoted energy metabolism, amino acid metabolism, and secondary metabolism, induced the expression of proteins related to root cell wall and cytoskeleton metabolism, effectively maintained the balance of protein processing and degradation, and enhanced the cell wall regulation ability. However, the extent to which [CO₂] enrichment alleviated drought stress in cucumber seedling roots was limited under severe drought stress, which may be due to excessive damage to the seedlings.

Keywords: CO2 enrichment; TMT-based quantitative proteomic; amino acid metabolism; carbohydrate synthesis; cucumber roots; drought stress.

Figure 1

Quantitative proteome analysis of MS data and identification of differentially accumulating proteins. (A) The basic statistics of the MS results. (B) Principal component analysis of all samples using quantified proteins. (C) Histogram of the numerical distribution of differentially accumulating proteins in different comparison groups. (D) Venn diagram analysis of differentially accumulating proteins under [CO₂] enrichment. Matched spectrum, number of spectrum matched with alignment protein; Total spectrum, number of spectrum produced by mass spectrometer; Peptides, number of peptides which spectrum hit; Unique peptides, number of identified peptides that only come from this protein group; Identified proteins, number of proteins detected by spectrum search analysis; Quantifiable proteins, number of proteins quantifiable; AC, atmospheric [CO₂] + control condition; EC, [CO₂] enrichment + control condition; AM, atmospheric [CO₂] + moderate drought stress; EM, [CO₂] enrichment + moderate drought stress; AS, atmospheric [CO₂] + severe drought stress; ES, [CO₂] enrichment + severe drought stress. The same definitions hold below.

Figure 2

KOG functional classification chart of differential proteins under [CO₂] enrichment: (I) information storage and processing, (II) cellular processes and signals, (III) metabolism, and (IV) other unknown functions.

Figure 3

Cluster analysis of the enrichment patterns of KEGG pathways of differential accumulating proteins under [CO₂] enrichment. The color blocks corresponding to the functional description of the differentially expressed proteins in different groups indicate the degree of enrichment; red represents strong enrichment and blue represents weak enrichment.

Figure 4

Effects of [CO₂] enrichment on the activities of related enzymes in roots of cucumber seedlings under drought stress. (A) Hexokinase, (B) Alcohol dehydrogenase, (C) Malate dehydrogenase, (D) Nitrate reductase, (E) Glutamate synthase, (F) Glutamate dehydrogenase. All results are expressed as the mean ± standard deviation (SD) of three repeated values; *, difference is significant at the 0.05 level; **, difference is significant at the 0.01 level; ***, difference is significant at the 0.001 level.

Figure 5

Effects of [CO₂] enrichment on the contents of non-structural carbohydrates in roots of cucumber seedlings under drought stress. (A) Starch, (B) Total sugar, (C) Sucrose, (D) Reducing sugar, (E) Glucose, (F) Fructose, (G) Raffinose, (H) Stachyose. All results are expressed as the mean ± standard deviation (SD) of three repeated values; *, difference is significant at the 0.05 level; **, difference is significant at the 0.01 level; ***, difference is significant at the 0.001 level.

Figure 6

Effects of [CO₂] enrichment on the contents of metabolism-related compounds in roots of cucumber seedlings under drought stress. (A) Total nitrogen, (B) NH₄⁺-N, (C) NO₃⁻-N, (D) Free amino acid, (E) Pyruvic acid, (F) Citric acid, (G) Total phenols, (H) Flavonoid. All results are expressed as the mean ± standard deviation (SD) of three repeated values; *, difference is significant at the 0.05 level; **, difference is significant at the 0.01 level; ***, difference is significant at the 0.001 level.

Figure 7

Correlation analysis between biochemical indicators; red indicates positive correlation, blue indicates negative correlation.

Figure 8

Schematic diagram of experimental design. Cucumber seedlings were placed in four open-top tunnels for hydroponics and two of them were treated with [CO₂] enrichment using gas cylinders. There were six treatments in total, with 96 biological replicates per treatment.

Figure 9

The main differentially accumulating proteins (DAPs) of cucumber seedling root response to [CO₂] enrichment under drought stress. Black words indicate metabolites, arrows indicate metabolic processes, and omitted processes are indicated by dashed lines. Red words indicate the upregulated DAPs under moderate drought stress after [CO₂] enrichment, green words indicate the downregulated DAPs under moderate drought stress after [CO₂] enrichment, and purple words indicate the upregulated DAPs under both moderate and severe drought stress after [CO₂] enrichment. Abbreviations: STP: sugar transport protein; HK: hexokinase; G6P: glucose 6-phosphate; F6P: fructose 6-phosphate; FK: fructokinase; PFP: pyrophosphate-fructose 6-phosphate 1-phosphotransferase; FBPase: fructose-1,6-bisphosphatase; FBP: fructose 1,6-bisphosphate; ALDO: fructose-bisphosphate aldolase; TPI: triosephosphate isomerase; G3P: glyceraldehyde 3-phosphate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; 1,3-bis-PGA: 1,3-bisphospho-D-glycerate; PGK: phosphoglycerate kinase; 3-PGA: 3-phosphoglycerate; gpmI: 2,3-bisphosphoglycerate-independent phosphoglycerate mutase; 2-PGA: 2-phospho-D-glycerate; PEP: phosphoenolpyruvate; PDC: pyruvate decarboxylase; ADH: alcohol dehydrogenase; MDH: malate dehydrogenase; tktA: transketolase; aroB: 3-dehydroquinate synthase; trpD: anthranilate phosphoribosyltransferase; PAL: phenylalanine ammonia-lyase; C4H: cinnamate 4-hydroxylase; serA: D-3-phosphoglycerate dehydrogenase; serC: phosphoserine aminotransferase; cysk: cysteine synthase; SDH: serine dehydratase; ilvC: ketol-acid reductoisomerase; leuC: 3-isopropylmalate dehydratase large subunit; lysA: diaminopimelate decarboxylase; hom: homoserine dehydrogenase; thrB: homoserine kinase; metB: cystathionine gamma-synthase; SAM: S-adenosyl-L-methionine; ACC: 1-aminocyclopropane-1-carboxylate; ACS: ACC synthase; ACO: ACC oxidase; NR: nitrate reductase; GOGAT: glutamate synthase; GDH: glutamate dehydrogenase; proB: glutamate 5-kinase/delta-1-pyrroline-5-carboxylate synthase; HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA; HMGS: HMG-CoA synthase; DMAPP: dimethylallyl diphosphate; DXP: 1-deoxy-D-xylulose 5-phosphate; DXR: DXP reductoisomerase; HMBPP: 4-hydroxy-3-methylbut-2-enyldiphosphate; HDS: HMBPP synthase.