Determination of Signal Molecules, Proteins, and Low-Molecular-Weight Organic Compounds in Wheat Varieties Infected by Leaf Rust Disease

Abstract

The disease resistance of wheat cultivars varies due to the plants’ defense systems, which include signaling molecules such as ferulic (coniferelic) acid, salicylic, arachidonic, and jasmonic acids. Therefore, the determination of quantitative and qualitative composition of signal molecules, low-molecular organic compounds, and proteins in wheat varieties with different leaf rust disease resistance levels is crucial. In this study, proteins and signal molecules in wheat leaf samples inoculated with fungal pathogen were isolated and identified using LCMS and HPLC chromatography. In addition, changes in the concentration of several organic compounds in infected plants were analyzed. The validity of obtained data on the absence of other components of metabolic cycles after chromatographic separation was discussed.

1. Introduction

Infectious plant diseases cause significant yield loss in agricultural crops. Particularly, the losses caused by fungal pathogens in cereals and legumes can reach up to 30% [1]. Plant resistance to various infections can be determined by a complex of physiological and biochemical reactions, each contributing to protection from pathogens. In plant tissues, a range of signaling molecules has been identified that initiate cascades of responsive biochemical reactions in plant organisms upon contact with pathogens. These signaling molecules may include phytohormones, oligosaccharides, jasmonate, salicylate, nitric oxide, and some other compounds [2, 3, 4, 5]. Cell signaling systems involve cascades of biochemical reactions that participate in the recognition and reception of extracellular signals as well as their transformation, amplification, and transmission to the genome. As a result of these processes, there is a change in the gene expression program, leading to the adaptation of plants to altered environmental conditions. It is presumed that signaling molecules not only participate in the formation of primary response reactions but also determine the nature of subsequent biochemical and physiological processes, influencing the degree of adaptation and resistance of plants to pathogen actions [2].

We conducted studies on regionally adapted spring wheat cultivars in Central Kazakhstan. The resistance of plants to various infections is determined by a complex of physiological and biochemical reactions, each contributing to crop protection. The impact apparatus on plants by phytopathogens includes at least two groups of different substances—hormones and hydrolytic enzymes. The ability of pathogenic fungi to secrete compounds with phytohormonal activity into the cells [6, 7, 8, 9] may disrupt the hormonal status of the plant organism and reduce the effectiveness of its defensive reactions. Among the most important biochemical processes in plant defense reactions, expressed both in mechanical damage to plant tissues and during responsive reactions to pathogen invasion, are lignification and oxidative burst [2, 7, 10].

Small molecular-weight organic compounds play a significant role in plants’ defense mechanism, and their molecular masses do not exceed 2000. These compounds are usually water-soluble, poorly separated, and can be detected by GCMS chromatography [5, 11, 12]. Their metabolism in wheat during the leaf rust infection is also poorly studied because of above-mentioned challenges. Individual resistance to rust disease and metabolism processes during infection may vary among wheat varieties. The aim of our research was to study SOCs in spring wheat varieties with different disease-resistant levels after inoculation with Puccinia recondita to identify potential candidates for a signaling role in pathogenesis.

The effective development of the wheat plant's defensive response to fungal pathogen attack is based on restructuring of the hormonal system, associated with an increase in cytokinin content against a relatively stable balance of auxins and abscisic acid. The molecular mechanisms underlying the development of plant resistance to fungal pathogens are still far from being fully understood. The quantitative and qualitative composition of signaling molecules, low-molecular-weight organic compounds, and proteins during fungal infection is poorly studied. Therefore, main goal of this research was to quantitatively determine the content of signaling molecules, low-molecular-weight organic compounds, and proteins within spring wheat varieties with different disease resistance levels that were inoculated Puccinia recondita, cause of wheat leaf rust.

2. Materials and methods

Firstly, the process of uredospore germination in wheat leaves was studied. This was necessary for increasing the percentage of infection and determining the quality of the spores. Microscopic and visual observations indicated sufficient activity of P. recondita spores (Figures 1 and 2). Notably, there was a high percentage of spore germination and leaf infection. In vegetative experiments, we investigated the impact of leaf rust on regionally distributed spring wheat varieties in the Akmolinsk region of Kazakhstan: Astana (a variety with low resistance to rust) and Akmola (a variety with moderate resistance to rust). For this purpose, seeds were sown in soil containers, with 5–7 plants grown in each. The plants were illuminated for 12 h with a light intensity ranging from 200 to 1,300 μmol m⁻² s⁻¹.

Figure 1.

Germinated spores of Puccinia recondita.

Figure 2.

Leaves of wheat seedlings from Astana (1) and Akmola-2 (2) inoculated with Puccinia recondita.

Experiments on determination of low-molecular-weight organic compounds (SOC) content were conducted using an Agilent 6890 GCMS chromatograph. For that, the following modes were selected:

OVEN: Initial temp: 60°C (On). Maximum temp: 330°C. Initial time:0.00 min. Equilibration time: 0.00 min. Ramps: # Rate Final temp Final time 1 3.00 320 4.00 20.0 (Off). Post temp: 98°C. Post time: 0.00 min. Run time: 90.67 min. FRONT INLET (SPLIT/SPLITLESS). Mode: Split Initial temp:300°C(On). Pressure: 8.31 psi (On). Split ratio: 4:1. Split flow: 6.4 mL/min. Total flow: 10.6 mL/mi. Gas saver: On. Saver flow: 15.0 mL/min. Saver time: 2.00 min. Gas type: Helium. BACK INLET (SPLIT/SPLITLESS). Mode: Split Initial temp: 50°C (Off). Pressure: 0.00 psi (Off). Total flow: 45.0 mL/min. Gas saver: Off. Gas type: Helium.

COLUMN 1: Capillary Column Model Number: Agilent 19091S-433. HP-5MS, 0.25 mm * 30 m * 0.25 um. Max temperature: 350°C. Nominal length: 30.0 m. Nominal diameter: 250.00 um. Nominal film thickness: 0.25 um. Mode: constant flow.

Initial flow: 1.0 mL/min. Nominal init pressure: 8.31 psi. Average velocity:37 cm/s Inlet: Front Inlet. Outlet: MSD Outlet pressure: vacuum.

COLUMN 2. Capillary Column Model Number: Agilent 19091S-433. HP-5MS, 0.25 mm * 30 m * 0.25 um. Max temperature: 350°C. Nominal length: 30.0 m Nominal diameter: 250.00 um. Nominal film thickness: 0.25 um. Mode: (see column 1). Nominal initial flow: 0.6 mL/min. Nominal init pressure: 8.31 psi. Average velocity: 17 cm/s Inlet: Front Inlet Outlet: Front Detector. Outlet pressure: ambient.

2.1 Extraction with ethanol

10 ml of ethanol was mixed with 1 g of leave mass, and the extraction was performed in an hour at 50°C in an ultrasonic disperser. The mixture was incubated with shaking for 30 min and then centrifuged.

2.2 Derivatization

For hydrolysis in KOH, 1 ml of ethanol extract was mixed with 0.1 ml of 3M KOH in H₂O. Hydrolysis was carried out for an hour at 80°C, then the solution was cooled, and 4 ml of H₂O +0.1 ml of 5M HCl were added into the mixture. Then, 5 ml of TBME was added into the mixture by shaking for 15 min and centrifuged. The solution was evaporated, and 100 μl of MSTFA (N-Methyl-N-(trimethylsilyl) trifluoroacetamide) was added to the dry residue and dissolved for 30 min at 60°C. After cooling, the solution was used for separating TMS (trimethylsilyl-) complexes using GCMS chromatography. NISTO2.L, Wiley7n.1, PMW_Tox3.1 databases were used for the identification of SOC.

The inoculation was performed manually by treating the leaves with water-20% TWEEN solution that contains uredospores. Before the inoculation, the spores were kept in water at 36°C for 2 h. Samples for analysis were collected at 6 days post-inoculation.

2.3 Isolation of small molecular organic compounds (SOC) from the leaves

The samples were incubated for an hour in ultrasonic bath at 50°C, mixed 30 min and centrifuged. 1 ml of ethanol extraction was added into 9 ml H2O and 5 ml tert-butyl-methyl ether (TBME) and mixed for 15 min. TBME was evaporated and diluted in 0.1 ml acetonitrile.

2.4 Liquid chromatography (LC) conditions

Pump: Thermo Scientific 600 pump; column: Hypersil Gold C-18, 50 × 2.1 mm, 1.9 mcm; injection volume 10 mcl.

2.5 Mass spectrometry (MS) conditions

Mass-spectrometer: Thermo Scientific TSQ Vantage; ion source: HESI in negative mode; capillary temperature: 300°C; vaporizer temperature: 400°C; sheath gas pressure:50Arb; aux gas pressure:15Arb; ion sweep gas pressure:1Arb; spray voltage: 3000 V; collision gas pressure: 1,2 mTorr.

2.6 KOH hydrolysis

1ml of ethanol extraction was mixed with 0.1 ml 3M KOH in H₂O and incubated for an hour at 80°C. The mixture was cooled and then mixed with 4 ml H₂O + 0.1 ml 5M HCl. 5 ml TBME was added and mixed for 15 min, then centrifuged. TBME was evaporated and diluted in 0.1 ml acetonitrile.

3. Results

3.1 Identification of organic compounds in wheat after disease inoculation using GCMS chromatography

Seven-day-old seedlings of spring wheat varieties were treated with uredospores of P. recondita TKT/Y. After 7 days, we analyzed extracts from the leaves of control (non-treated) and experimental (infected with rust) seedlings using GCMS. In the spectrum of SOC from the leaves of weakly rust-resistant Akmola сultivars, 36 compounds were detected in control (non-infected with rust) seedlings and 37 in infected samples (Figure 3). Identified compounds include Acetic acid, Propanoic acid, Propanedioic acid, Benzoic acid, Butanedioic acid, Propanoic acid, Butanedioic acid (ITACONIC ACID-DITMS), Methylmaleic acid, 3,4-Dihydroxybutanoic acid, Malic acid, Pentanedioic acid, 3-methyl-3-[(trimethylsilyl)oxy]-,Benzoic acid, Lauric acid, Phthalic acid, cis-Aconitic acid, 1,4-Benzenedicarboxylic acid, Azelaic acid, Myristic acid, Syringic acid, Ferulic acid, Palmitoleic acid, Palmitic acid, 2-Hydroxysebacic acid, Cinnamic acid, 1-Trimethylsiloxy-3,7,11,15-tetramethyl-2-hexadecene, Linoleic acid, Linolenic acid, Stearic acid, 11,14-Eicosadienoic acid, 11-Eicosenoic acid, Eicosanoic acid, Docosanoic acid, monostearin, 3-[(Trimethylsilyl)oxy]ergost-5-ene or 3-[(Trimethylsilyl)oxy]cholest-5-en-24-one, Stigmasterol, and beta-Sitosterol. After inoculation with rust, the component called methylmaleic acid was disappeared from the SOC spectrum, and instead other components such as Squalene and alpha-Tocopherol (vitamin E) were appeared. Thus, a wide range of carbohydrates, phenolic compounds, fatty acids, phytosterols, simple organic acids, and others are represented.

Figure 3.

GCMS (Agilent 6890) chromatography of organic compounds in the leaves of 10-day-old seedlings of spring wheat сultivar Akmola. The control sample is indicated in black, and the experimental sample (7 days after plant infection with spores) is indicated in green. Components for improved separation were derivatized with MSTFA (N-Methyl-N-(trimethylsilyl)trifluoroacetamide). The x-axis represents the separation time (retention time) on the column (0-18-28-38-48-57.5-68-76.50-90 min).

In rust-infected leaves, the content of Propanoic acid, Malic acid, Pentanedioic acid, and Azelaic acid increases. As for the control seedlings, the leaves contain more cis-Aconitic acid, Eicosanoic acid, and Docosanoic acid.

In the spectrum of SOC from the leaves of the rust-resistant Astana сultivar, 29 compounds in control and 31 components in infected seedlings were detected. Identified compounds include Acetic acid, Propanoic acid, Benzoic acid, Octanoic acid, Butanedioic acid, Propanoic acid, Nonanoic acid, Malic acid, Benzoic acid, (Z)- cis-Aconitic acid, Azelaic acid, Myristic acid, Trimethylsilyl 3-methoxy-4-(trimethylsilyloxy)cinnamate, n-Pentadecanoic acid, Palmitoleic acid, Palmitic acid, Ferulic acid, 1-Trimethylsiloxy-3,7,11,15-tetramethyl-2-hexadecene, Linoleic acid, Linolenic acid, Stearic acid, 11,14-Eicosadienoic acid, 11-Eicosenoic acid, Arachidic acid, Docosanoic acid, Monostearin, 3-[(Trimethylsilyl)oxy]ergost-5-ene or 3-[(Trimethylsilyl)oxy]cholest-5-en-24-one, Stigmasterol, and beta-Sitosterol. However, after inoculation with rust, several components such as monostearin, 11,14-Eicosadienoic acid, Arachidic acid, and Nonanoic acid (Pelargonic acid) were disappeared from the SOC spectrum, and new compounds such as Lauric acid, Propanedioic acid, Ferulic acid, Phthalic acid, and Palmitelaidic acid were detected.

In rust-infected leaves, the content of acetic acid, Tetradecanoic acid (Myristic acid), and Eicosanoic acid was increased. In the control leaves, Octanoic acid, azelaic acid, (Z)-cis-Aconitic acid, and Docosanoic acid were more abundant.

3.2 Quantitative content of signaling molecules after wheat infection with rust fungus

For spring wheat varieties, the level of signaling molecules after infection with P. recondita was quantitatively determined. A calibration curve was developed for the target acids, with concentrations ranging from 20 to 800 nanograms per 1 gram of leaf mass. Based on literature data, organic molecules such as jasmonic, salicylic, and arachidonic acids [10, 13, 14, 15, 16, 17, 18] were selected as signaling molecules. They were acquired as liquid chromatography standards: 1. Jasmonic acid, J2500 Sigma, J2500-100MG, 2. Salicylic acid, Fluka 52341-50MG, 3. Arachidonic acid, A9673-50MG Sigma. Due to the separation using a highly sensitive liquid chromatography method, purification level of the target compounds was extremely high. Although an attempt was made to separate compounds using gas chromatography with subsequent mass spectrometry, liquid chromatography provided more effective results.

The content of signaling molecules before and after the rust infection was determined. As a result, in the case of Astana varieties after inoculation, without hydrolysis and derivatization of the extract, the content of salicylic acid increased by almost 50%, whereas it increased by only 8% in the resistant Akmola сultivar. Furthermore, the content of jasmonic acid increased by 10% in the Astana сultivar; however, it decreased by almost 30% in the Akmola cultivar. There was a decrease of arachidonic acid content by almost 50% in the Astana variety. In contrast, this indicator increased almost two-fold in the resistant Akmola сultivar.

After KOH hydrolysis and derivatization of the extract, the content of salicylic acid increased by more than 50% after infection, whereas there was no change in the Akmola сultivar. As for the jasmonic acid content, it decreased by almost 20% in the Astana сultivar and decreased by 60% in the Akmola сultivar. It was not possible to detect arachidonic acid after hydrolysis probably due to its saponification (Tables 1 and 2).

Option	Salicylic acid	Jasmonic acid	Arachidonic acid
Control Astana	46.0	100.9	26.8
Experience Astana	62.3	110.7	12.7
Control Akmola	54.9	160.8	21.7
Experience Akmola	58.9	105.0	35.8

Table 1.

Content of signaling molecules in wheat leaves after rust disease inoculation. Before KOH hydrolysis, in ng/g (mcg/kg).

Option	Salicylic acid	Jasmonic acid	Arachidonic acid
Control Astana	196.7	65.1	0.0
Experience Astana	319.2	48.3	0.0
Control Akmola	201.7	86.8	0.0
Experience Akmola	202.3	32.0	0.0

Table 2.

Content of signaling molecules in wheat leaves after rust disease inoculation. After KOH hydrolysis, in ng/g (mcg/kg).

Other uredospores were used in another series of experiments, and they were collected from a population near the Gvardeyskiy village in the Zhambyl region, Kazakhstan. The same Astana cultivar from previous experiments as well as a more resistant Agent cultivar were used. The previously applied inoculation procedure with slight modifications was used in this variant: Previously, uredospores were rubbed onto the leaves, while in this case, spraying was performed instead. We attributed a greater difference in the concentration of signaling molecules to these cultivars. Upon the fungus penetration into the leaf: for the Astana cultivar, the content of salicylic acid sharply increased by the 5th day, then started decreasing; the content of jasmonic acid also increased by the 5th day, then sharply decreased; the concentration of arachidonic acid decreased sharply by the 5th day, then slightly increased by the 7th day (Table 3).

Option	Salicylic acid	Jasmonic acid	Arachidonic acid
Astana, control, 1 day	101.3	26.0	627.7
Astana, 2 days	859.0	53.1	63.7
Astana 5 days	4624.7	79.2	47.6
Astana 7 days	1424.4	18.8	128.9
Agent, control, 1 day	207.9	16.9	7875.7
Agent 2 days	307.8	64.6	86.7
Agent 5 days	808.9	64.8	956.1
Agent 7 days	321.8	11.3	71.9

Table 3.

Daily dynamics of the content of signaling molecules in wheat leaves after infection. In ng/g (mcg/kg).

For the highly resistant Agent cultivar, the content of salicylic acid reached its maximum by the 5th day, decreasing by the 7th day; the content of jasmonic acid significantly increased on the second day; however, this concentration is maintained until the 5th day, then decreased sharply; the content of arachidonic acid in this cultivar is simply gigantic – 7875 ng/g; it sharply decreased by the second day, increased again by the 5th day, and then decreased back. Data on the Agent cultivar, especially the content of arachidonic acid in the leaves, requires further research, as unexpected concentration fluctuations were observed. This may be associated with methodological difficulties in chromatographic separation of organic compounds in the presence of an increasing amount of released free fatty acids during infection. According to our data, in the presence of detergents, the quality of chromatographic separation decreased. The possibility of release, rather than synthesis, of arachidonic acid from membranes, which does not occur in uninfected cells, is also possible.

For the susceptible Akmola cultivar, the number of small molecular-weight organic components significantly increased after treatment with rust fungus (Figures 4–9): 33 components in control, 78 in the experiment (with the addition of formic acid). For the rust-resistant Astana cultivar, fungal infection did not lead to a significant complication of the spectrum: 61 components in the control for Astana (after formic acid treatment), without treatment – 72. After treatment with rust fungus, the number of components decreased to 45 (without formic acid treatment). These results are consistent with the findings of Delaney KJ et al. [7, 8, 9, 19], who discovered the induction of phenolic compound synthesis after treating plants with jasmonic acid. We have also shown that the addition of formic acid to the eluent and the treatment of the TBME extract improved the quality of chromatographic separation (compare Figures 4 and 9, Table 4).

Figure 4.

Separation of organic compounds using liquid chromatography. The calibration curve for the content of salicylic, jasmonic, and arachidonic acids (SA, JA, AA).

Figure 5.

Separation of SOC of the uninfected leaf of cultivar from Astana by liquid chromatography.

Figure 6.

Separation of SOC of the infected leaf of cultivar from Astana by liquid chromatography.

Figure 7.

Separation of SOC of the infected leaf of wheat cultivar from Astana by liquid chromatography without adding formic acid into eluent.

Figure 8.

Separation of SOC of the infected leaf of wheat cultivar Akmola-2 by liquid chromatography.

Figure 9.

Separation of SOC of the infected leaf of wheat cultivar Akmola-2 by liquid chromatography.

	Testing options
№	Astana control	Astana control, formic acid	Astana variety	Akmola variety, LC	Akmola variety, formic acid	Akmola control, formic acid	Separation signal molecules of wheat by the LC method
1	0.37	0.37	0.38	0.35	0.35	0.07	0.33
2	0.38	0.38	1.15	0.36	0.35	0.59	0.70
3	0.38	0.40	1.20	0.36	0.35	0.90	1.11
4	0.39	0.79	1.21	0.36	0.36	1.59	1.35
5	0.39	1.06	1.60	0.37	0.36	2.15	2.12
6	0.42	1.41	1.65	0.38	0.37	2.25	2.45
7	0.42	2.06	1.78	0.38	0.38	2.27	2.94
8	0.42	2.08	1.80	0.40	0.39	2.27	2.95
9	0.44	2.31	1.82	0.40	0.39	2.38	2.95
10	0.44	2.31	1.82	0.41	0.41	2.39	2.95
11	0.45	2.32	1.86	0.41	0.41	2.40	2.96
12	0.46	2.33	1.90	0.43	0.46	2.40	2.96
13	0.47	2.34	1.98	0.43	0.48	2.43	2.97
14	0.47	2.40	2.52	0.44	0.57	2.43	2.97
15	0.48	2.41	2.53	0.45	0.66	2.96	2.98
16	0.50	2.42	2.54	0.45	0.69	2.97	2.98
17	0.50	2.43	2.61	0.47	0.82	2.99	2.99
18	0.52	2.43	2.62	0.48	1.36	3.31	3.00
19	0.53	2.44	2.65	0.50	1.64	3.31	3.32
20	0.57	2.45	2.68	0.54	1.99	3.32	3.32
21	0.64	2.46	3.15	0.57	2.10	3.32	3.33
22	0.76	2.46	3.15	0.70	2.26	3.33	3.33
23	0.88	2.89	3.16	0.71	2.26	3.33	3.34
24	0.94	2.98	3.18	0.88	2.28	3.33	3.35
25	1.29	2.99	3.19	1.29	2.28	3.35	3.35
26	1.90	3.00	3.20	1.45	2.30	3.40	3.35
27	1.91	3.00	3.21	1.94	2.30	3.42	3.36
28	1.96	3.02	3.22	2.28	2.36	3.84	3.36
29	2.04	3.32	3.23	2.44	2.38	4.76	3.36
30	2.07	3.33	3.24	2.45	2.39	4.78	3.37
31	2.11	3.33	3.53	2.49	2.41	5.69	3.37
32	2.12	3.34	3.56	2.50	2.42	6.04	3.38
33	2.12	3.34	3.93	2.51	2.43	6.70	3.39
34	2.51	3.34	4.27	2.52	2.43		3.55
35	2.55	3.35	4.76	2.52	2.44		4.74
36	2.55	3.36	4.77	2.53	2.81		4.74
37	2.58	3.38	4.78	2.53	2.88		4.75
38	2.61	3.39	4.79	2.53	2.90		4.75
39	2.63	3.40	4.80	2.55	2.91		4.75
40	2.66	3.41	5.11	2.56	2.92		4.76
41	2.67	3.42	5.53	2.57	2.97		4.77
42	2.68	4.03	5.73	2.58	2.97		4.77
43	2.68	4.38	6.02	2.58	2.97		4.78
44	2.70	4.75	6.20	2.58	2.98		4.78
45	2.72	4.76	6.72	2.97	2.99		4.78
46	2.73	4.76		2.97	3.00		4.79
47	2.96	4.77		2.98	3.31		4.80
48	2.97	4.77		3.03	3.31		4.80
49	3.13	4.78		3.13	3.32		4.81
50	3.79	4.79		4.11	3.32		4.81
51	3.83	4.80		4.40	3.33		4.82
52	3.84	4.80		4.69	3.33		4.82
53	4.13	4.82		4.71	3.34		5.04
54	4.50	5.04		4.72	3.35		5.98
55	4.74	5.07		4.73	3.37		6.24
56	4.75	5.50		4.74	3.37		6.88
57	4.75	5.84		4.74	3.40
58	4.76	6.06		4.75	3.41
59	4.76	6.24		4.76	4.00
60	4.77	6.52		4.77	4.07
61	4.77	6.67		4.77	4.35
62	4.78			4.78	4.62
63	4.78			4.78	4.75
64	4.79			4.79	4.75
65	4.80			4.79	4.76
66	5.01			5.55	4.77
67	5.58			6.16	4.78
68	5.63			6.66	4.79
69	6.09				4.83
70	6.26				5.23
71	6.58				5.28
72	6.99				5.31
73					5.80
74					6.06
75					6.13
76					6.36
77					6.57
78					6.84

Table 4.

Quantitative composition of SOC from the leaves of spring wheat cultivars after exposure to rust fungus via liquid chromatography.

Our data indicated low concentration of SOC in wheat leaf tissue. They are comparable with the concentration of phytohormones in plants. Therefore, SOC are called the plant growth regulators. We have identified changes in the content of these compounds (Tables 1 and 2, Figures 4–9). It was indicated on the continued involvement of SOC in the metabolism of cells. Thus, in some cases, the amount of SOC decreases after leaf rust infection. The function of SOC is a warning of leaf rust infection danger, as well as maintenance of the functioning of certain parts of metabolism in normal and stress conditions, including plant wound healing, tuberization, fruit ripening, roles in biotic/abiotic stress responses, defense, and senescence. It is known that exogenous SA between 10 and 500 μM could induce thermotolerance in mustard seedlings [14]. Authors consider that the endogenous SA levels were about 15–120 μM, which is within the range of concentrations used to induce thermotolerance. 10–50 μMSA potentiates the response of soybean cells to an avirulent Pseudomonas syringae pv glycinea strain [10, 13, 14, 15, 16]. The mechanism of JA action has been documented to be hormonal by regulating the translation of varying genes. SA is involved in endogenous signaling, mediating in plant defense against pathogens. It plays an important role in the resistance to pathogens by inducing the production of pathogenesis-related proteins. This confirms the view [3, 17, 20, 21] on the signaling and metabolic role of examined molecules in vital activity of plant.

In another series of experiments, we performed protein analysis using LC-MS/MS chromatography to isolate and identify proteins after treating plant leaves with leaf rust. This is because the responsibility for the efficient and coordinated system of plant defense against stress factors, particularly infection by phytopathogens, and the subsequent restoration of plant cell integrity are attributed to protective proteins [10, 17, 18, 20, 21, 22, 23]. These proteins also play a significant role in the effective regulation of signal intensity in tissues and cells of the plant (various protein kinases, phosphatases, lectins, and acetylases), in the synthesis of the final protective product (phenylalanine ammonia-lyase, enzymes involved in the synthesis of active oxygen forms (AOF), phytoalexins, and lignin), or in disrupting the normal functioning of the pathogen (chitinases, glucanases, protease inhibitors, and proteins that inactivate ribosomes). With the discovery of the signaling role of AOF in plants, special attention has been given to oxidoreductases (peroxidases and catalases) that regulate the AOF level, thus maintaining their content in the cell at a relatively safe level. Therefore, we studied the composition of proteins when wheat is infected with rust fungi because of the activity of signaling molecules in the form of SOC, as well as the proteins themselves as components of the signaling system.

The composition of proteins and their identification were carried out according to the accepted proteomics sequence of methods: extraction, SDS-electrophoresis, and subsequent extraction of proteins from the two-dimensional gel. Analysis on the mass spectrometer was performed as well. The obtained peptides were analyzed using nano-HPLC (Agilent Technologies 1200), directly connected to the ion trap mass spectrometer (Bruker 6300 series), equipped with a nano-electrospray source. The separating gradient of acetonitrile ranged from 5% to 90%, with a duration of 25 min. Fragmentation voltage was set at 1.3 V. The ion trap has sequential sets of four scanning modes, consisting of fully scanning MS in ranges above 200–2000 m/z, accompanied by three data-dependent MS/MS scanners of the three most abundant ions during full scanning. Protein identification was performed using the Mill Spectrum software package. Quantitative analysis of the spectrum and chromatogram was carried out using data analysis for the 6300 Ion Trap LC/MS series, software version 3.4. The relative content of each peptide in different fractions was determined by comparing the peak areas with the total area of the ion chromatogram (TIC) for that peptide [6, 7, 8, 9, 18, 22].

We studied, in a comparative inter-varietal cross-section, the composition of leaf and root polypeptides of wheat after treatment with uredospores of rust fungus (Figures 10 and 11). The electropherogram showed 25–35 polypeptide components. Differences in the number of components were observed in leaves and roots, experimental and control variants, with some reduction in the staining of certain components in the experimental variant (leaves of wheat seedlings treated with rust fungal spores). The Astana cultivar is less resistant than the Akmola. This is reflected in the greater variability of the polypeptide composition in the Astana after rust infection.

Figure 10.

Influence of rust (Puccinia recondita) on the polypeptide composition of Astana wheat variety leaves. Coomassie staining. Lines: 1-marker, 2–5 five-component peptide composition of control plants, protein concentration per lane was – 10, 15, 20, 25 μl; 7–10 polypeptides of wheat leaves infected with rust, 20, 25, 30, 35 μl.

Figure 11.

Influence of rust (Puccinia recondita) on the polypeptide composition of Astana wheat variety leaves. Staining with silver. Lines: 1-marker, 2-control leaves of wheat variety Akmola 2, 3-control roots of wheat variety Akmola 2, 4-control leaves of wheat variety Astana, 5-control roots of wheat variety Astana, 8-infected leaves of wheat variety Akmola 2, 9-infected roots of wheat variety Akmola 2, 10-infected leaves of wheat variety Astana, and 11-infected roots of wheat variety Astana.

Subsequent analysis of peptides by LS-MS/MS allowed the identification of 104 proteins in infected and control leaf samples. There were overall 74 common proteins. In the control, there were 17 specific proteins, and in the experiment – 13. We conducted the determination of the protein composition in control and experimental plants. Only in control plants, the following proteins were identified: Fructan 1-exohydrolase w1; Fructan 1-exohydrolase w2; Fructan 1-exohydrolase w3; Histone H1; NAD(P)H-quinone oxidoreductase subunit 1, chloroplastic; NAD(P)H-quinone oxidoreductase subunit H, chloroplastic; NADP-dependent glyceraldehyde-3-phosphate dehydrogenase; mitochondrial outer membrane porin; cytochrome c oxidase subunit 2; Arf-GAP with Rho-GAP domain; elongation factor 1-beta; 30S ribosomal protein S8, chloroplastic; 30S ribosomal protein S7, chloroplastic; eukaryotic initiation factor iso-4F subunit p82-34; retinoblastoma-related protein 1; ubiquitin-activating enzyme E1; cysteine synthase. Only in experimental plants, the following proteins were identified: рeroxidase; ATP synthase subunit 9, mitochondrial; 50S ribosomal protein L23, chloroplastic; сytochrome b6-f complex subunit 4; ubiquitin; 50S ribosomal protein L16, chloroplastic; 30S ribosomal protein S3, chloroplastic; DNA-directed RNA polymerase subunit beta; DNA-directed RNA polymerase subunit alpha; glutathione S-transferase 1; small heat shock protein, chloroplastic; non-specific lipid-transfer protein 3.

The following proteins were detected in both control and experimental plants: ATP synthase subunit beta, chloroplastic; ATP synthase subunit alpha, chloroplastic; ribulose bisphosphate carboxylase large chain; RuBisCO large subunit-binding protein subunit alpha, chloroplastic; рhosphoglycerate kinase, cytosolic; photosystem II CP47 chlorophyll apoprotein; оxygen-evolving enhancer protein 2, chloroplastic; рhosphoribulosokinase, chloroplastic; аpocytochrome f; Sedoheptulose-1,7-bisphosphatase, chloroplastic; рhotosystem II CP43 chlorophyll apoprotein; рhotosystem I P700 chlorophyll a apoprotein A1; ADP, ATP carrier protein 1, mitochondrial; ADP, ATP carrier protein 2, mitochondrial; photosystem II D2 protein; photosystem I P700 chlorophyll a apoprotein A2 ; оxygen-evolving enhancer protein 1, chloroplastic; fructose-1,6-bisphosphatase, chloroplastic; сytochrome b6-f complex iron-sulfur subunit, chloroplastic, 2-Cys peroxiredoxin BAS1, chloroplastic, ribulose bisphosphate carboxylase small chain PW9; ribulose bisphosphate carboxylase small chain clone 512 chloroplastic; ribulose bisphosphate carboxylase small chain PWS4.3, chloroplastic; ATP synthase subunit alpha, mitochondrial; рeroxiredoxin Q , chloroplastic; сatalase; ATP synthase subunit b, chloroplastic; сytochrome b6; сhlorophyll a-b binding protein chloroplastic; сytochrome b559 subunit alpha; 50S ribosomal protein L9, chloroplastic; рhotosystem Q(B) protein; histone H4 variant TH091, histone H4 variant TH011, elongation factor 1-alpha, oxalate oxidase GF-3.8, oxalate oxidase GF-2.8, histone H2A.2.2, protein H2A.6, histone H2A.1, protein H2A.5, histone H2A.2.1, histone H2A.4; protein H2A.7; eukaryotic initiation factor 4A; adenosylhomocysteinase; calmodulin; beta-amylase; histone H2B.1; histone H2B.2; histone H2B.3; histone H2B.5; histone H2B.4 ; ATP synthase epsilon chain, chloroplastic; 30S ribosomal protein S2, chloroplastic; photosystem II reaction center protein H; 50S ribosomal protein L2, chloroplastic; 30S ribosomal protein S4, chloroplastic ; histone H3.250S; ribosomal protein L14, chloroplastic; ribosomal protein S13, mitochondrial; 1-Cys peroxiredoxin PER1; thioredoxin M-type, chloroplastic; translationally-controlled tumor protein homolog; ubiquitin-activating enzyme E1; 3 Arf-GAP with SH3 domain; serine–glyoxylate aminotransferase; photosystem I iron-sulfur center; Sushi, von Willebrand factor type A; Alpha-amylase/trypsin inhibitor CM1; Arf-GAP with Rho-GAP domain 1; Arf-GAP with Rho-GAP domain 2. In this way, only 17 proteins were identified in the control, only 13 in the experiment, and 74 proteins were common.

In the affected leaves, there was an increase in the concentration of apocytochrome, reflecting the tendency of cytochrome complex degradation. Additionally, there was an increase in the concentration of ADP, ATP carrier protein 1, mitochondrial; ADP, ATP carrier protein 2, mitochondrial; oxygen-evolving enhancer protein 1, chloroplastic; fructose-1,6-bisphosphatase, chloroplastic; elongation factor 1-alpha; photosystem II reaction center protein H. Of interest is the increase in catalase concentration, as we have detected an increase in hydrogen peroxide content in rust-infected leaves by 3.2 times. The concentration of hydrogen peroxide in the roots remained unchanged. In addition, in the infected leaves, there was a decrease in the content of phosphoglycerate kinase, chloroplastic; sedoheptulose-1,7-bisphosphatase, chloroplastic; photosystem II D2 protein; cytochrome b6-f complex iron-sulfur subunit, chloroplastic; 2-Cys peroxiredoxin BAS1, chloroplastic; cytochrome b6; Arf-GAP with Rho-GAP domain. These compounds are localized in chloroplasts, mitochondria. Their decrease reflects the process of cell and organelle decay in infected leaves. Of interest is the observed increase in the concentration of ubiquitin protein in plants infected with rust fungi. This protein is involved in the regulation of metabolism. With its direct participation, certain proteins are destroyed, and responsive reactions to stress and pathogen action [10, 14, 17, 21, 23] are formed. The observed decay of leaves and cells during fungal infection is accompanied by protein degradation. Therefore, the increase in ubiquitin content is quite explainable. Thus, when infected with rust fungi in the leaves of spring wheat, changes in the concentration of 17 proteins out of 104 identified, an increase in hydrogen peroxide content, peroxidase activity, and RNA polymerase activity are observed, reflecting the process of tissue and cell decay.

4. Discussion

The main goal of the research was the application of previously developed methods to study signaling molecules. The emphasis was placed on the qualitative and quantitative determination of jasmonic, arachidonic, and salicylic acids, low-molecular-weight organic compounds, and specific proteins. Both gas and liquid chromatography were used for the research.

The obtained data indicated low concentrations of signaling molecules in wheat leaf tissues, comparable to the concentration of phytohormones in plants. Therefore, they are referred to as plant growth regulators. Changes observed in the content of these compounds suggest their constant involvement in cell metabolism, as in some cases, their concentration decreased after inoculation with rust fungi. Their function extends beyond signaling the presence of a rust threat, including the maintenance of specific metabolic pathways under normal and stress conditions, such as plant wound healing, tuberization, fruit ripening, roles in biotic/abiotic stress responses, defense, and senescence.

Signal transmission through a series of mediators involves the following process: 1) receptor interaction with the stimulus; 2) activation of the membrane-bound effector molecule responsible for generating secondary mediators; 3) formation of secondary mediators; 4) activation of mediator proteins, primarily protein kinases, triggering the generation of subsequent mediators or activation of specialized cellular structures leading to a physiological response; 5) disappearance of the mediator.

Low-molecular-weight organic compounds with a molecular mass up to 2000 were studied to identify new signaling molecules. Despite the limited set of signaling molecules known during pathogen infection, the discovery of hundreds of low-molecular-weight compounds in wheat leaves with unknown functions makes testing them as signaling molecules intriguing. Proteomics methods have been proven to be promising, allowing the identification of individual proteins and peptides with the quantitative determination of their content. We have identified the induction of peroxidase and catalase synthesis, which is logical since their synthesis intensifies during infection.

We studied small-molecular-weight organic compounds (SOC) in spring wheat cultivars differing in resistance to brown rust after infection with rust fungi. A technology for extraction, derivatization, and separation of SOC from leaves was developed, including a detailed protocol for obtaining gas chromatography-mass spectrometry (GC-MS)-based metabolite profiling. Infected rust leaves showed an increase in Propanoic acid, Malic acid, Pentanedioic acid, Azelaic acid, and a decrease in cis-Aconitic acid, Eicosanoic acid, and Docosanoic acid. In the weakly resistant Astana cultivars, 29 components were detected in the spectrum of SOC in control, non-infected leaves of seedlings, and 31 components in infected leaves.

We investigated the quantitative content of signaling molecules—salicylic, jasmonic, and arachidonic acids during the infection of spring wheat varieties differing in resistance to Puccinia recondita rust. For determining the concentration of signaling molecules, liquid chromatograph was used. The direction of changes in the molecule content during wheat infection with rust fungi was shown to depend on the cultivars's resistance. The studied signaling molecules not only signal the threat of rust infection but also stimulate the functioning of specific metabolic pathways under normal and stress conditions.

Systemic acquired resistance (SAR) to Puccinia recondita in wheat was studied using proteomic analysis. A total of 104 proteins were identified using a combination of peptide fingerprinting (PMF) and MSMS fragmentation of peptides. Most of the identified proteins are enzymes related to photosynthesis, metabolism, biosynthesis, and defense reactions, the properties of which change depending on the susceptibility/resistance of varieties. The results indicate that plants may reduce the level of photosynthesis and other energy processes and increase the synthesis of protective proteins to cope with infection. We hypothesized that resistance to Puccinia recondita is mediated by defense and stress-related proteins. Compounds with proven signaling molecule properties or physiological activity, the mechanisms of which are poorly studied, were identified. Based on this, all these compounds, especially Cholest-5-en-3-ol (3.beta.), stigmasterol, beta-Sitosterol—precursors of brassinosteroids, eicosanoids, indole-3-acetic acid, acetoacetic acid, azelaic acid, squalene, pelargonic acid, 11,14-eicosanoic acid, linolenic acid, palmitoleic acid, coumaric acid, ferulic or coniferyl acid, salicylic, arachidonic, and jasmonic acids, can be considered candidates for participation in wheat defense reactions during rust infection.

In this study, we isolated simple and complex molecules, many of which are involved in various metabolic cycles. The absence of even more components of metabolic pathways does not mean the absence of these compounds in plant cells, because without them, metabolism is not possible. This means we are talking about the complexity of extraction and chromatographic separation of these components. Therefore, we analyze the overall picture of the separation of available organic compounds. The whole picture of cell damage by a pathogenic fungus is not available to us. We have a picture of the process on the level of labile compounds. We know this and talk about a limited understanding of the infectious process. The validity of analyzing the obtained data in the absence of other components of their metabolic cycles after a single extraction and chromatographic separation is discussed.

5. Conclusions

1. The composition of low-molecular-weight organic compounds was determined in wheat cultivars with different resistance levels to various pathotypes of rust fungus. Over 100 compounds were identified, and some of them were formed in response to pathogen exposure.

2. Using LCMS chromatography (proteomic analysis), proteins from wheat leaves were isolated and identified. For the first time, the impact of fungal pathogen of leaf rust disease on the synthesis of specific proteins, particularly peroxidases and catalases, was demonstrated.

3. Signal molecules were isolated and identified using HPLC chromatography. The quantitative content of signal molecules in wheat during fungal pathogenesis was determined.

4. Around 200 organic compounds were identified in wheat during rust infection using GCMS chromatography. Moreover, the synthesis of several organic compounds including phenols in wheat during rust infection was demonstrated.