Metabolic adjustments during compatible interaction between barley genotypes and stripe rust pathogen

Plant Physiology and Biochemistry 147 (2020) 295–302

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Research article

Metabolic adjustments during compatible interaction between barley genotypes and stripe rust pathogen

T

Prabhjot Singlaa, Rachana D. Bhardwaja,∗, Simarjit Kaurb, Jaspal Kaurb, Satvir K. Grewala a b

Department of Biochemistry, Punjab Agricultural University, Ludhiana, 141004, India Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, 141004, India

A R T I C LE I N FO

A B S T R A C T

Keywords: PR proteins Polyamines Osmolytes Secondary metabolites Glutamate dehydrogenase Ornithine transaminase

Stripe rust is a fungal disease that has devastated the barley production for a long time. The present study focused on the role of β-glucan, PR proteins, diamine oxidase (DAO), polyamine oxidase (PAO), key enzymes and metabolites of phenol and proline metabolism in the stripe rust resistance of barley. RD2901 with resistant behavior against stripe rust showed increased levels of PR proteins, phenylalanine ammonia lyase (PAL), tyrosine ammonia lyase (TAL) along with the accumulation of β-glucan and lignin which strengthen the plant cell wall during plant-pathogen interaction. It also depicted the enhanced activities of glutamate dehydrogenase (GDH) and ornithine aminotransferase (OAT) coupled with the increased amounts of proline, glycine betaine and choline after infection with M-race of P. striiformis f. sp. hordei. On the contrary, the sensitive genotype Jyoti was unable to enhance the activities of most of these enzymes except PAL and OAT so that it showed an increase in lignin and choline contents only. Secondly, the increase in lignin content was less as compared to the tolerant genotype. Hence, it can be inferred that these key metabolites and enzymes of various metabolic pathways may contribute to the resistance of barley against stripe rust pathogen. This study suggested that these key enzymes and their metabolites could serve as markers for the characterization of plant defensive state that is essential for crop protection.

1. Introduction Barley (Hordeum vulgare L.), a member of the grass family, is adaptable to a wide range of climates and is relatively tolerant against various abiotic stresses such as salt, drought, alkalinity and extreme temperatures (Wiegmann et al., 2019; Elsawy et al., 2018). However, it is continuously exposed to various biotic stresses that adversely affect its growth and yield. Among the biotic stresses, rusts can cause maximum yield losses to the susceptible cultivars of barley when the environment is favorable for disease development. These rusts had been categorized into four types based upon their causal organisms i.e. stripe rust (Puccinia striiformis), stem rust (Puccinia graminis), leaf rust (Puccinia hordei), and grass stripe rust (causal pathogen currently unnamed) (Waqar et al., 2018). P. striiformis f. sp. hordei is one of the dreadful fungal pathogens of barley that causes damage and leads to huge economic loss in the food sector. Barley grains are rich in a dietary fibre called β-glucan that not only has nutritional value but also acts as the first line of defence against the invading pathogen (Ellinger et al., 2013). Callose, a β-(1,3)-glucan polymer with few β-(1,6) branches in

∗

between deposits in the cell wall during plant-pathogen interaction. Further, it tends to form papillae and acts as a barrier to the invading pathogen (Ali et al., 2018). Papillae serve as the site for the accumulation of various metabolites including reactive oxygen species (ROS), phenolics, polysaccharides, etc and provide time to the plant cell to activate its internal defence system offering innate immunity to plant cells (Voigt, 2014). To inhibit the progression of the disease, calloses are sometimes pervaded with the phenolic compounds forming a sheath around the fungal hyphae. Ellinger et al. (2013) reported that callose formation serves as the foremost defence response against the invading pathogen. The reinforcement of these appositions strengthens the plant cell wall and restricts its accessibility to fungal degrading enzymes. Further, lignin which is an insoluble, rigid and virtually indigestible polymer of polyphenols also serves as the physical barrier during initial pathogen colonization (Miedes et al., 2014). It prevents the transfer of water and nutrients from the host cells to the pathogen that ultimately leads to pathogen death. Plants have evolved many other mechanisms to cope with stresses like the production of pathogenesis-related (PR) proteins, polyamines, antimicrobial substances like phenolics (phytoalexins) and compatible

Corresponding author. E-mail address: [email protected] (R.D. Bhardwaj).

https://doi.org/10.1016/j.plaphy.2019.12.030 Received 3 September 2019; Received in revised form 15 November 2019; Accepted 23 December 2019 Available online 27 December 2019 0981-9428/ © 2019 Elsevier Masson SAS. All rights reserved.

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containing 50 μl of extract, 100 μl of laminarin and 350 μl of sodium acetate buffer (pH 5.0) was incubated for 1 h at 37 °C. The reducing sugars formed via β-glucanase were estimated by incubating the reaction mixture with alkaline copper tartrate reagent at 100 °C for 10 min. The bluish green colour developed after the addition of arsenomolybdate reagent was read at 575 nm. Enzyme activity was expressed in terms of mg glucose released/min/mg protein. The chitinase (E.C. 3.2.1.14) was extracted by grinding 0.1 g of leaf sample in 2 ml of ice cold 100 mM sodium acetate buffer (pH 5.0) containing 15 mM of βmercaptoethanol. The extract was centrifuged at 12,000×g for 30 min and the supernatant was collected for the estimation of chitinase activity (Boller and Mauch, 1988). The reaction mixture containing 200 μl of enzyme extract and 200 μl of 0.5% (w/v) of chitin was incubated at 37 °C for 1hr. Then the reaction mixture heated at 100 °C for 5 min. The reducing ends, formed as a result of chitinase action were measured (Reissig et al., 1955).

solutes (Fernandes et al., 2019). PR proteins are a structurally diverse group of plant proteins that are toxic to invading fungal pathogens. The most important PR proteins are β-1,3-glucanases (PR-2) and chitinases (PR-3) that weaken the fungal cell wall by hydrolyzing its β-glucan and chitin respectively (Boccardo et al., 2019). Phenylalanine ammonia lyase is an inducible enzyme that synthesizes several defence-related secondary compounds like flavonoids, lignins, coumarins, etc in response to pathogen attack (Jun et al., 2018). It is also involved in the biosynthesis of salicylic acid (SA), an essential signal involved in plant systemic resistance. Polyphenol oxidase catalyzes the oxidation of phenolic compounds to quinones, which are often more toxic to microorganisms than the original phenols (Taranto et al., 2017). On the other hand, polyamines (PA) which are small aliphatic molecules positively charged at cellular pH, play an essential role in cell proliferation, growth, and differentiation (Chen et al., 2019). Under stressed conditions, positively charged polyamines can interact with negatively charged macromolecules like DNA and help in stabilizing and protecting these compounds (Seifi and Shelp, 2019). When plants are exposed to stress factors, their cells protect themselves from high concentrations of intracellular salts by accumulating a variety of small organic metabolites that are collectively referred to as compatible solutes (Murmu et al., 2017). Compatible solutes are very soluble in water and non-toxic even at higher concentrations. These metabolites allow cells to retain water and help in avoiding disturbances in their normal functions when exposed to stresses (Munns et al., 2019). Compatible solutes include sugars, polyols, amino acids, proline, glycine betaine, choline, and related compounds. Rust fungi are the phytopathogens that pose a serious threat to barley production as they can move longer distances, evolve new variants easily and spread rapidly that makes their eradication extremely difficult (Gangwar et al., 2018). A key agricultural target is to improve the yield of crops grown under periods of such biotic stresses. Growing resistant cultivars is the best approach to control stripe rust. Therefore, the study was formulated to evaluate the contribution of β-glucan, PR proteins, polyamines’ catabolizing enzymes, phenol, and proline metabolism in the stripe rust resistance of barley when exposed to the M and G pathotypes of Puccinia striiformis f. sp. hordei.

2.3. Extraction and assay of polyamine catabolizing enzymes Diamine oxidase (E.C. 1.4.3.6, DAO) and polyamine oxidase (E.C. 1.4.3.4, PAO) were extracted by homogenizing 0.1 g of leaf tissue in 2 ml of 50 mM potassium phosphate buffer (pH 7.0) and centrifuged at 12,000×g for 30 min. The supernatant was used for assaying DAO and PAO (Holmstead et al., 1961). For the determination of DAO, the reaction mixture contained 100 mM potassium phosphate buffer (pH 7.0), 10 mM putrescine (diaminobutane), and the enzyme extract in a total volume of 4 ml. After incubation at 37 °C for 10 min, the reaction was stopped by adding 500 μl of 10% (w/v) trichloroacetic acid (TCA) followed by 50 μl of 1% (w/v) o-aminobenzaldehyde prepared in ethanol. The absorbance of the complex was measured at 430 nm after the removal of proteins by centrifugation. The assay for PAO was the same as that of DAO except that putrescine was replaced by spermidine in the reaction mixture. 2.4. Extraction and assay of enzymes of phenolic metabolism Extraction and estimation of phenylalanine ammonia lyase (E.C. 4.3.1.24, PAL) and tyrosine ammonia lyase (E.C. 4.3.1.25, TAL) was done by following the method of Burrell and Rees (1974). 0.1 g of leaf tissue was extracted with 2 ml of chilled 100 mM Tris HCl buffer (pH 7.5) containing 5 mM β-mercaptoethanol. Homogenate was centrifuged at 10,000×g at 4 °C for 25 min and the supernatant was used for the determination of PAL and TAL activities. The reaction mixture of PAL containing 2.5 ml of 30 mM phenylalanine prepared in 50 mM sodium borate buffer (pH 8.8) and 100 μl of enzyme extract was kept at 37 °C for 1 h. The reaction was stopped by adding 300 μl of 5 N HCl. In control, the reaction was terminated immediately without incubation. For the assay of TAL, the reaction mixture consisted of 1 ml of 33 μM tyrosine, 1.35 ml of 50 mM sodium borate buffer (pH 8.8) and 50 μl of enzyme extract. The activities of PAL and TAL were determined from the standard curve prepared by using cinnamic acid (5–40 μg) and coumaric acid (5–40 μg) respectively. For extracting polyphenol oxidase (E.C. 1.14.18.1, PPO), 0.2 g of leaf tissue was crushed with 1.5 ml of 100 mM sodium phosphate buffer (pH 6.8) followed by centrifugation at 10,000×g for 20 min. The clear supernatant was used for enzyme assay (Zauberman et al., 1991).

2. Materials and methods 2.1. Plant material, fungal races and disease development The four barley genotypes were screened for stripe rust disease during two consecutive years i.e. 2016–17 and 2017–18 (Singla et al., 2019). The disease data showed varying degree of resistance for these genotypes i.e. RD2901 and RD2552 (resistant), RD2900 (moderately resistant) and Jyoti (susceptible). In the present study, two races of stripe rust namely M and G were used to inoculate the barley genotypes that were sown in the fields of Plant Breeding and Genetics, Punjab Agricultural University Ludhiana during 2018–19. M race was the most prevalent in all states of North India with high frequency in Himachal Pradesh and Uttarakhand whereas G race was observed in low frequency (Prashar et al., 2014). The polychambers corresponding to control, M and G races were maintained at distinct locations to avoid cross-contamination. The first leaf of every plant was inoculated with the urediniospores of M and G-pathotypes of Puccinia striiformis f. sp. hordei. The conditions suitable for disease development were maintained as earlier (Singla et al., 2019). Analysis of various biochemical parameters was done from the leaves of healthy and diseased plants at 22 days after inoculation.

2.5. Extraction and assay of proline metabolism enzymes Proline metabolizing enzymes were extracted by homogenizing leaf tissue with 100 mM potassium phosphate buffer (pH 7.4) containing 10 mM β-mercaptoethanol, 1 mM EDTA, 1% (w/v) PVP, 5 mM MgCl2 and 0.6 M KCl. The homogenate was centrifuged at 10,000×g for 15 min and the supernatant was collected for assays. The reaction mixture for glutamate dehydrogenase (E.C. 1.4.1.2, GDH) consisted of 200 mM Tris-HCl buffer (pH 8.0), 150 mM NH4Cl, 150 mM α-

2.2. Extraction and assay of pathogenesis-related proteins For the estimation of β-glucanase (E.C. 3.2.1.6) a soluble enzyme, 0.2 g of leaf tissue was extracted with 2 ml of 100 mM sodium phosphate buffer (pH 6.0) (Fink et al., 1988). The reaction mixture 296

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mg of glucose released/ min/mg protein

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Fig. 1. The activities of β-glucanase (a); chitinase (b); and β-glucan content (c) in four barley genotypes infected with M and G pathotypes of Puccinia striiformis f. sp. hordei. Data represents mean of triplicates; error bars represent SD of triplicates. Different letters illustrates the significant differences between genotypes and races at p ≤ 0.05.

ketoglutaric acid and 200 μl of enzyme extract (Kanamori et al., 1972). Then 3 mM of NADH was added to initiate the reaction. GDH activity was determined as a decrease of absorbance at 340 nm Δ1-pyrollinecarboxylate synthetase (E.C. 2.7.2.11, 1.2.1.41, P5CS) was assayed by measuring the rate of utilization of NADPH at 340 nm (Filippou et al., 2013). The reaction for ornithine transaminase (E.C. 2.6.1.13, OAT) was initiated by the addition of 0.125 mM NADH to the reaction mixture containing 200 mM Tris-HCl buffer (pH 8.0), 46.8 mM L-ornithine, 12.5 mM α-ketoglutaric acid and 0.2 ml of enzyme extract (Mazelis and Fowder, 1969). PDH (E.C. 1.5.5.2) was assayed according to the method of Chen et al. (2001) following the NADH generation at 340 nm. For determining the specific activity of various enzymes soluble protein content was measured by the method of Lowry et al. (1951).

betaine were solubilized in 9 ml of 1,2-dichloroethane and absorbance was measured at 365 nm. For choline determination, 500 μl of the extract was mixed with 500 μl of 200 mM potassium phosphate buffer (pH 6.8) instead of 2 N H2SO4. The other steps followed were the same as used for GB estimation. 2.8. Data analysis Two-way analysis of variance (ANOVA) was employed to find out biochemical responses of the plants against M and G-races of stripe rust pathogen. Then, Tukey's test was used to test significant differences between mean values using SPSS 16.0 software (p ≤ 0.05). Further, the correlation between various biochemical parameters was calculated using MS Excel 2007. The data presented in bar diagrams and tables depict the Mean ± SD of triplicate samples.

2.6. Determination of cell wall components 3. Results and discussion The extraction and estimation of β-glucan were carried out according to the method earlier described (Kaur et al., 2019). For the extraction and estimation of lignin, 0.1 g of leaf tissue was crushed with 2 ml of 95% (v/v) ethanol and centrifuged at 10,000×g for 20 min (Lee et al., 2007). The pellet was washed 3 times with 95% (v/v) ethanol and two times with ethanol:hexane (1:2) and was dried at 45 °C. The dried pellet was dissolved in 1 ml of 25% acetyl bromide prepared in acetic acid. The test tubes were incubated at 70 °C for 30 min and cooled at room temperature. The reaction mixture containing 30 μl of extract, 180 μl of NaOH, 20 μl of hydroxylamine HCl and 1.6 ml of acetic acid was centrifuged for 5 min. Then the brown color obtained was read at 280 nm. Lignin content was calculated from the standard curve of lignin (0–50 μg) which was run simultaneously.

Stripe rust pathogen adversely affects the growth and yield of the barley crop. The foremost reaction of a plant towards any stress is the production of defence-related compounds that counteract the deleterious effects of stress. So, the aim of the present study was to decipher the role of PR proteins and various biomolecules of primary and secondary metabolism in imparting the resistance against stripe rust pathogen in barley. 3.1. Status of pathogenesis-related proteins Pathogenesis-related proteins are induced by pathogen attack and get accumulated during the systemic acquired resistance and hypersensitive response (HR). β-glucanases and chitinases are the proteins that break down the fungal cell wall and hence result in cell lysis and death (Boccardo et al., 2019). After infection with M-pathotype, the resistant genotype (RD2901) showed a significant increase in β-glucanase activity whereas the other genotypes exhibited a decrease in its activity (Fig. 1a). However, all the barley genotypes depicted decreased activity of β-glucanase on inoculation with G-race. An increase in chitinase activity was observed in RD2901 and RD2552 after infection with M-pathotype whereas, Jyoti and RD2900 were unable to enhance this enzyme (Fig. 1b). However, plants of all the genotypes infected by G-race exhibited increased activity of chitinases. Gupta et al. (2013)

2.7. Determination of osmolytes Proline was extracted from the healthy and diseased leaves using 3% (w/v) sulphosalicylic acid and estimated based upon its reaction with acidic ninhydrin reagent (Bates et al., 1973). Glycine betaine (GB) and choline were determined by the standard protocol of Grieve and Grattan (1983). The extraction of GB and choline was done from leaf tissue ground in deionized water. The extract was mixed with 2 N H2SO4 followed by cooling. 200 μl of KI3 solution was added to the extract, vortexed and cooled in ice-bath. Periodide crystals of glycine 297

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(GABA) which enters the Kreb's cycle via the formation of succinic acid.

also observed the increased levels of these PR proteins in the plants of Eruca sativa when infected with Alternaria brassicicola. The cleavage of fungal cell wall causes the release of oligomeric products that act as elicitors which further stimulates the plant defence responses viz. deposition of lignin (Tian et al., 2019). It will set up a local barricade that slows the movement of pathogens to other parts of the plants. Indeed, PAL was found to be positively correlated to PR proteins i.e. β-glucanase (r = 0.772) and chitinase (r = 0.381) at 1% and 5% levels of significance respectively.

3.4. Profile of enzymes of secondary metabolism PAL catalyzes the first step of the phenylpropanoid pathway which is a key regulatory point between primary and secondary metabolism (Jun et al., 2018). In general, PAL activity was reported to be minimum in sensitive genotype (Jyoti) under control as well as diseased conditions. The susceptible genotype (Jyoti) was able to maintain the PAL activity during plant-pathogen interaction. An increase in PAL activity was recorded in the leaves of RD2901 on inoculation with both the races of P. striiformis f. sp. hordei whereas, RD2900 exhibited decrease in its activity (Fig. 3a). RD2552 behaved differentially in terms of PAL activity towards both the races of stripe rust pathogen. PAL activity increased by 1.7 folds on inoculation with M-race whereas it remained constant after infection with G-race. Similar results were obtained when tomato plants were affected by bacterial wilt disease (Vanitha et al., 2009). In Gramineae family, there is a predominantly occurring enzyme i.e. tyrosine ammonia lyase (TAL) which deaminates L-tyrosine to coumaric acid. TAL activity was found to be less in all the genotypes after infection with G-race of stripe rust pathogen (Fig. 3b). A maximum decrease was observed in Jyoti followed by RD2900, RD2552 and RD2901. However, M-race infected plants of RD2901 showed increased activity of TAL by 1.5 folds whereas the rest of the genotypes exhibited a decrease in its activity. Polyphenol oxidase catalyzes the oxidation of phenols to highly reactive quinones by utilizing molecular oxygen. PPO-generated quinones are toxic to invading pathogens as they alter dietary proteins in plants making them indigestible by pathogens (Araji et al., 2014). Jyoti exhibited maintained levels of PPO when infected with M and G-races of P. striiformis f. sp. hordei (Fig. 3c). However, plants of resistant genotype RD2552 depicted the increased activity of PPO by more than 1.4 folds in response to M and G-pathotypes while RD2901 behaved differentially towards both the races. The diseased plants of moderately resistant genotype (RD2900) showed the decrease in PPO enzyme. The induced PPO activity in many plants was found to be involved in resistance against bacterial, fungal and insect pathogens (Taranto et al., 2017).

3.2. Status of β-glucan β-glucan is a linear homopolysaccharide of D-glucopyranosyl residues occurring in the cell wall of plants. The deposition of β-glucan is one of the first steps in the plant's defence against pathogens that prevents the penetration of pathogens into the tissues (Granato et al., 2019). The β-glucan content of four barley genotypes ranged from 903.2 to 1080.8 μg/g FW. After infection with M-race, RD2900, RD2901 and RD2552 showed maintained levels of β-glucan content while 14.4% decrease was reported in sensitive genotype i.e. Jyoti (Fig. 1c). On the other hand, G-pathotype infected plants of all the genotypes were able to maintain β-glucan content when compared to control. Fernandes et al. (2019) observed the callose deposition induced by indole-3-carboxylic acid in response to Plectosphaerella cucumerina in Arabidopsis. 3.3. Profile of polyamine catabolizing enzymes Polyamines, phytohormone-like aliphatic amines such as putrescine, spermidine, and spermine that are involved in many processes like plant growth and development, anti-senescence, anti-stress, antioxidants and cell wall stabilizing (Liu et al., 2019). So, the biosynthesis and accumulation of polyamines during stress conditions is necessary. Polyamine oxidase (PAO) catalyzes the oxidation of spermidine and spermine to putrescine. The putrescine so formed gets converted to Δ1pyrroline, NH3, and H2O2 by the action of diamine oxidase (DAO). The DAO and PAO were found to be decreased in all the genotypes after infection with M-race (Fig. 2a and b). RD2901 exhibited a maximum decrease of about 3.5 and 6 folds in DAO and PAO activities respectively. After infection with G-pathotype, the increased activities of DAO and PAO were recorded in RD2900 whereas RD2552 was able to maintain these activities. On the contrary, Jyoti and RD2901 depicted a decrease in both the activities when compared with control conditions. Similar results were reported by Seifi et al. (2019) where spermine activates defence response against gray mold disease on Solanum lycopericum, Phaseolus vulgaris, and Arabidopsis thaliana. However, RD2900 that showed increased activities of both the enzymes on infection with G-race tends to cope with the disease by generating H2O2 during polyamine degradation that further may help in lignification and crosslinking reactions during unfavorable environment (Gill and Tuteja, 2010). Secondly, Δ1-pyrolline is converted to γ-aminobutyric acid

Lignin, a component of middle lamella and secondary cell wall makes the cell wall less accessible to degrading enzymes (Miedes et al., 2014). The maximum increase in lignin content was reported in RD2901 followed by Jyoti after infection with the M-pathotype of stripe rust pathogen (Fig. 3d). However, it decreased by more than 1.6 folds in RD2900 and RD2552. On inoculation with G-pathotype, all the genotypes except RD2901 exhibited a decrease in lignin content. Thus, diseased plants of RD2901 were able to maintain the lignin content when compared with healthy plants. Indeed, the correlation studies also revealed the positive correlation between PAL activity and lignin

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Fig. 2. The activities of diamine oxidase (a); and polyamine oxidase (b) in four barley genotypes infected with M and G pathotypes of Puccinia striiformis f. sp. hordei. Data represents mean of triplicates; error bars represent SD of triplicates. Different letters illustrates the significant differences between genotypes and races at p ≤ 0.05. 298

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Fig. 3. The activities of phenylalanine ammonia lyase (a); tyrosine ammonia lyase (b); polyphenol oxidase (c) and lignin content (d) in four barley genotypes infected with M and G pathotypes of Puccinia striiformis f. sp. hordei. Data represents mean of triplicates; error bars represent SD of triplicates. Different letters illustrates the significant differences between genotypes and races at p ≤ 0.05.

specific activity of P5CS decreased in Jyoti and RD2900 after inoculation with both the races of stripe rust pathogen (Fig. 4b). The resistant genotypes namely RD2901 and RD2552 showed a maintained level of P5CS after infection with P. striiformis f. sp. hordei. In mitochondria, an alternative ornithine pathway operates for proline biosynthesis that utilizes ornithine and produces P5C and glutamate by the action of ornithine aminotransferase (OAT) (Hayat et al., 2012). Ornithine aminotransferase catalyzes the conversion of ornithine into glutamyl-5semi-aldehyde and vice versa, using α-ketoglutarate and glutamate as cosubstrates. P5C formed in mitochondria can be converted back to proline in the cytosol by P5C reductase and thus stimulates the Pro/P5C cycle. In comparison to control, all the four barley genotypes showed significantly higher OAT activity on inoculation with M-race (Fig. 4c). A similar trend was reported with G-race except for Jyoti where it remained comparable to control. Indeed, the negative correlation between P5CS and OAT (r = 0.427) at a 1% level of significance indicated

content (r = 0.552) at a 1% level of significance. Similar observations were recorded by Giberti et al. (2012) when phenylalanine ammonia lyase was induced in rice plants in response to fungal pathogen Magnaporthe oryzae. 3.6. Profile of enzymes of proline metabolism In the plant's cytosol, the precursor for proline biosynthesis is Lglutamic acid (Murmu et al., 2017). Glutamate dehydrogenase (GDH) catalyzes the conversion of glutamate to α-ketoglutarate and vice versa. Its activity declined in Jyoti and RD2552 whereas, RD2901 showed enhanced activity by more than 1.4 folds on infection with M and Graces of stripe rust pathogen (Fig. 4a). When compared with control, RD2900 was able to maintain GDH activity after infection with stripe rust pathogen. A bifunctional enzyme, Δ-pyrroline-5-carboxylate synthetase (P5CS) catalyzes the first two steps in proline biosynthesis. The a) GDH activity

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Fig. 4. The activities of glutamate dehydrogenase (a); Δ-pyrroline-5-carboxylate synthetase (b); ornithine aminotransferase (c); proline dehydrogenase (d) in four barley genotypes infected with M and G pathotypes of Puccinia striiformis f. sp. hordei. Data represents mean of triplicates; error bars represent SD of triplicates. Different letters illustrates the significant differences between genotypes and races at p ≤ 0.05. 299

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levels in the chloroplast regulate the defence response of plants in response to pathogens including programmed cell death and systematic acquired resistance (Upchurch, 2008). It is a well-known fact that the accumulation of these compatible solutes helps in counteracting the abiotic stresses by stabilizing the native structure of various biomolecules (Annunziata et al., 2019). During the pathogen attack, in addition to other antioxidants, the increased levels of GB and choline in barley could also assist in reducing stress damage.

that the major pathway for proline biosynthesis under stress conditions was the ornithine pathway. Proline dehydrogenase (PDH) is a flavoprotein that catalyzes the conversion of proline into Δ-pyrroline-5carboxylate (P5C). In the present study, the specific activity of PDH was increased in the G-race infected plants of RD2900, RD2901 and RD2552 as compared to healthy plants (Fig. 4d). However, resistant genotypes (RD2901 and RD2552) showed maintained levels of PDH after infection with M-pathotype while it increased by 3.2 folds in RD2900. Jyoti with sensitive behavior towards stripe rust showed either declined or maintained levels of PDH on infection with both the races. Similar results had been observed where proline dehydrogenase contributes to pathogen defence in Arabidopsis (Cecchini et al., 2011) as it may provide energy and nutrients during stress recovery.

4. Conclusion The present study illustrates the pivotal role of metabolites and metabolic pathways in barley during the stripe rust pathogen attack. Metabolic adjustments in plants in response to pathogen are dynamic and multifaceted. The M-race infected plants of RD2901 were able to maintain the β-glucan content that forms the first line of defence to the invading pathogen as can be visualized from Fig. 5. It also depicted the enhanced levels of PR proteins i.e. β-glucanase and chitinase along with the increase in the activities of key enzymes of phenylpropanoid pathway namely PAL and TAL which in turn increase the lignin content that strengthens the cell wall of plant cells (Fig. 5). The scenario was different in the sensitive genotype Jyoti where there was a decrease in β-glucan content and PR proteins which may be responsible for its sensitive behavior towards the stripe rust pathogen. Although, this cultivar was able to increase the lignin content but the increase was less as compared to RD2901 which may be attributed to the increased activity of PAL only. Proline can be synthesized by ornithine and glutamate pathways (Fig. 6). In RD2901, enhanced GDH activity may lead to the accumulation of glutamate which can enter glutamate pathway for the synthesis of proline in the cytosol as well as in the chloroplast. Indeed, proline content was higher in this genotype after infection with M race of stripe rust pathogen whereas the sensitive genotype showed the opposite trend. Alternatively, glutamate may be involved in the synthesis of choline which can be further used for the generation of glycine betaine or fatty acids. In mitochondria, increased OAT activity may lead to the synthesis of P5C which can convert back to proline in the cytosol by P5C reductase. The scrutiny of data showed that the ornithine pathway plays an important role in the biosynthesis of proline which may act as ROS scavenger during compatible interaction between the plant and stripe rust pathogen. Further, polyamine degrading enzymes namely DAO and PAO showed decreased activities in both the genotypes irrespective of their behavior towards the pathogen. Correlation studies helped to analyze the interaction between various metabolic responses that might be of great help in our better understanding of the tolerance mechanisms. Additionally, this study might be helpful to comprehend our knowledge about the contribution of metabolites and metabolic pathways that may help the breeder to select the resistant genotypes and boost crop production.

3.7. Status of compatible solutes Proline accumulation can influence stress tolerance in many ways such as it can act as an osmolyte, ROS scavenger and molecular chaperone stabilizing the structure of proteins, thereby protecting cells from damage caused by stress (Munns et al., 2019). Proline is a proteinogenic amino acid with exceptional conformational rigidity and is essential for primary metabolism. In the present study, Jyoti the susceptible genotype showed decreased content of proline on infection with the G-pathotype of stripe rust pathogen (Table 1). The moderately resistant (RD2900) and resistant (RD2901 and RD2552) genotypes exhibited differential behavior on infection with both the races as compared to control. RD2901 showed 2.1 folds increase in proline content on inoculation with M-race whereas it showed 1.3 folds decrease after infection with G-race. Various studies had been done to reveal the role of proline under stress conditions viz. proline treatment can diminish ROS levels in fungi and yeast, thus preventing programmed cell death (Chen and Dickman, 2005) and can also inhibit peroxidation of lipids in alga cells facing heavy metals toxicity (Mehta and Gaur, 1999). The damaging effects of singlet oxygen and hydroxyl radicals on Photosystem II (PSII) can be reduced by proline in isolated thylakoid membranes (PSII) (Alia and Mohanty, 1997). Further, reduced levels of ROS were noticed in the transgenic algae and tobacco plants engineered for proline hyperaccumulation via overexpression of P5CS (Siripornadulsil et al., 2002). Glycine betaine (GB) a quaternary ammonium compound is one of the most efficient osmolytes found in a wide range of organisms (Choudhury et al., 2017). GB is synthesized via two different pathways that use different precursors i.e. choline and glycine. All the genotypes showed maintained levels of GB content on infection with both the races of stripe rust pathogen. However, choline content either remained unaffected or increased in diseased plants of different barley genotypes after inoculation with P. striiformis f. sp. hordei. Choline instead of generating GB during stress conditions, it enters the fatty acid biosynthesis pathway. As, different studies suggest that free oleic acid

Table 1 Status of proline, glycine betaine and choline in four barley genotypes infected with M and G pathotypes of Puccinia striiformis f. sp. hordei. Data represent Mean ± SD of triplicates. Different letters presented in superscript depict significant differences between the genotypes and races at p ≤ 0.05. Genotypes JYOTI

RD2900

RD2901

RD2552

Treatments Control Patho M Patho G Control Patho M Patho G Control Patho M Patho G Control Patho M Patho G

Proline (μg/g fw) 587.1 484.2 407.0 362.5 624.5 354.7 348.5 725.9 276.8 322.8 316.8 591.8

± ± ± ± ± ± ± ± ± ± ± ±

Glycine betaine (mg/g fw)

abc

18.2 15.9 16.8 15.9 16.2 17.9 16.4 12.2 10.8 14.6 14.8 14.0

2.3 55.8bcd 26.0cde 48.0de 91.2ab 22.5de 86.6de 99.8a 17.9e 28.1de 71.6de 79.7ab

300

± ± ± ± ± ± ± ± ± ± ± ±

a

4.4 1.9a 2.0a 1.5a 1.8a 3.7a 1.1a 2.7a 0.9a 2.7a 3.3a 3.0a

Choline (μg/g fw) 526.5 ± 59.5d 887.4 ± 76.2abc 1044.4 ± 135.3abc 812.6 ± 144.1cd 881.8 ± 62.7abc 1140.2 ± 145.3ab 849.4 ± 112.2bc 981.8 ± 28.8abc 1104.2 ± 130.9ab 878.8 ± 44.9abc 1140.8 ± 48.6a 1066.7 ± 93.3abc

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Fig. 5. Changes in callose content, PR proteins and phenylpropanoid metabolism in contrasting barley genotypes on inoculation with M-race of stripe rust pathogen. depicts increase in specific enzyme or content in RD2901 and Jyoti respectively depicts decrease in specific enzyme or content in RD2901 and Jyoti respectively depicts comparable amounts of specific enzyme or content in RD2901 and Jyoti respectively.

Authors contributions

➢ ➢ ➢ ➢ ➢

P.S. performed the experiments, analysed the data, wrote the paper and critically revised the manuscript. R.D.B. conceived the idea and designed the experiments, supervised the work with data evaluation and critically revised the manuscript; S.K. and J.K. provided the germplasm and inoculum and supervised the experimental design in the field. S.K.G. assisted in designing the experiments and critical analysis of the data.

TCA- Tricarboxylic acid α-KG- α-Ketoglutarate ODC- Ornithine decarboxylase BADH- Betaine aldehyde dehydrogenase GSA- Glutaratesemialdehyde

➢ ➢ ➢ ➢ ➢

BAD- Betaine aldehyde CMO- Choline monooxygenase ADC- Arginine decarboxylase GB- Glycine betaine P5C- Pyrroline-5-carboxylate

Declaration of competing interest The authors declare that they have no known competing financial

Fig. 6. Changes in proline metabolism, glycine betaine, choline, DAO and PAO in contrasting barley genotypes on inoculation with M-race of stripe rust pathogen. depicts increase in specific enzyme or content in RD2901 and Jyoti respectively depicts decrease in specific enzyme or content in RD2901 and Jyoti respectively depicts comparable amounts of specific enzyme or content in RD2901 and Jyoti respectively. 301

Plant Physiology and Biochemistry 147 (2020) 295–302

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interests or personal relationships that could have appeared to influence the work reported in this paper.

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