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Plant ROS Research


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Mandelonitrile-associated salicylic acid biosynthesis in peach under abiotic and biotic stresses

Diaz-Vivancos P; Bernal-Vicente A; Petri C; Cantabella D; Hernández JA

(Poster presented at the SMPR2019 congress (Valencia, 10-11 December 2019)

Abstract

Even though that salicylic acid (SA) is a key regulator of plant stress responses and other biological processes, its biosynthetic pathways have not been fully characterized. The proposed SA synthesis originates from chorismate by two distinct pathways: isochorismate and phenylalanine (Phe) ammonia-lyase (PAL) pathways. Cyanogenesis is the process related to the release of hydrogen cyanide from endogenous cyanogenic glycosides (CNglcs), and it has been linked to plant plasticity improvement. The main CNglcs in peach are prunasin and amygdalin, with mandelonitrile (MD), synthesized from Phe, controlling their turnover.

Using [13C]-labelled compounds and metabolomic analysis, we showed that MD, and hence CNglcs turnover, is involved, at least in part, in SA biosynthesis in peach plants. MD-treated peach plants displayed increased SA levels via benzoic acid (one of the SA precursors within the PAL pathway). Moreover, MD treatment modulated the H2O2 levels and had a pleiotropic effect on abscisic acid (ABA) and jasmonic acid (JA) levels (data not shown). Under the stress conditions, however, the contribution of this SA biosynthetic pathway from MD to the total SA pool does not seem to be important. Nevertheless, MD treatment not only increased SA levels, but also improved peach plants performance under the stressful conditions. In fact, MD provided partial protection against Plum pox virus (PPV) infection and stimulated the accumulation of phytotoxic ions in roots in PPV- and NaCl-stressed peach seedlings respectively.

Thus, we proposed a third pathway, alternative to the PAL pathway, for SA synthesis in peach plants, linking SA biosynthesis and cyanogenesis (Fig. 1). This proposed pathway seems to be functional under stress conditions, although the fact that CNglcs may be operating more broadly than by influencing SA pathways and signaling con not be ruled out.

Material and Methods

Plant material

The assays were performed on GF305 peach (Prunus persica L.) plants, under both greenhouse and in vitro conditions. For ex vitro assays, after submitting the GF305 peach seedlings to an artificial rest period in a cold chamber to ensure uniformity and fast growth, seedlings were grown in 2-L pots in an insect-proof greenhouse and distributed into three batches (control and MD- and Phe-treated) of 15 plants each. Plants were irrigated twice per week with water (control) and 1 mM MD or 1 mM Phe for 7 weeks. None of the treatments affected plant growth and development. For in vitro assays [13C]-labeled compounds were used, and 200 mM MD- or Phe-alpha[13C] (Campro Scientific GmbH, Germany) was added to the micropropagation medium during two subcultures. This concentration was selected after carrying out a preliminary assay using concentrations of both compounds ranging from 50 to 1000 mM; 200 mM was the highest concentration that did not show any deleterious effect on the development of micropropagated peach shoots.

Metabolomic analysis

The levels of Phe, MD, amygdalin, BA and SA were determined in in vitro micropropagated shoots at the Metabolomics Platform at CEBAS-CSIC (Murcia, Spain).

SA levels in peach seedlings

The SA levels in leaves of GF305 seedlings treated with MD or Phe were determined using a UHPLC-mass spectrometer (Q-Exactive, ThermoFisher Scientific) at the Plant Hormone Quantification Platform at IBMCP (Valencia, Spain).

Extraction and enzymatic assays

The activities of ascorbate peroxidase (APX),  peroxidase (POX), catalase (CAT) and superoxide dismutase (SOD) in in vitro shoots and ex vitro leaf samples were assayed as described in Diaz-Vivancos et al. (2017) and Bernal-Vicente et al. (2018; 2019).

Results

FIG 1

Fig. 1. Proposed salicylic acid (SA) biosynthetic pathway in peach plants. Blue arrows indicate the SA biosynthesis in plants described previously. The dotted arrow indicates a putative pathway. Red arrows show the new pathway suggested for peach plants. CYP79 and CYP71, Cyt P450 monooxygenases; MDL1, mandelonitrile lyase.

FIG 2

Fig. 2. Percentage (of total amount detected) of [13C]- phenylalanine (Phe), mandelonitrile (MD) and salicylic acid (SA) in non-stressed, NaCl-stressed and Plum pox virus (PPV)-infected peach shoots micropropagated in the presence or absence of [13C]MD or [13C]Phe. Ions with an additional 1.0035 accurate mass and confirmation by isotopic distribution and spacing were defined as ions marked with 13C. Data represent the mean of at least 20 repetitions of each treatment.

FIG 3

Fig. 3 Total levels (mMg–1 FW) of amygdalin, benzoic acid, mandelonitrile, phenylalanine and salicylic acid, and mandelonitrile lyase (MDL) enzymatic activity in micropropagated peach shoots in the presence or absence of [13C]MD or [13C]Phe. Data represent the mean ± SE of at least 12 repetitions of each treatment. In each graph different letters above the columns indicate significant differences according to Duncan’s test (P< 0.05).

FIG 4

Fig. 4. Total SA level (ng g–1 DW) in the leaves of peach seedlings grown in the presence or absence of MD or Phe submitted to 34 mM NaCl (A) or PPV infection (B). Data represent the mean ± SE of at least five repetitions of each treatment. Different letters indicate significant differences according to Duncan’s test (P≤0.05).

Fig-5

Fig. 5. Summary of the differential response of MD treatment in salt-stressed and PPV-infected peach seedlings. Data from Diaz-Vivancos et al. (2017) and Bernal-Vicente et al. (2018; 2019).

TABLE 1

Table 1.- Effect of MD and Phe on H2O2-scavenging activities (APX, POX, CAT)  and SOD (H2O2-producing) activity in GF305 micropropagated shoots and seedlings. APX is expressed as nmol min-1 mg-1 protein. POX and CAT are expressed as µmol min-1 mg-1 protein. SOD as U mg-1 protein. Data represent the mean ± SE of at least four repetitions. It has been previously described that SA led to H2O2 accumulation (Durner & Klessig 1995; Rao et al. 1997).

Conclusions

We provide strong  evidences showing that CNglcs turnover is involved, at least in part, in SA biosynthesis in peach plants under control and stress conditions.

MD seems to act as a hub controlling the CNglcs turnover and the SA and amygdalin biosynthesis. MD-treated peach plants displayed increased SA levels via benzoic acid (one of the SA precursors within the PAL pathway).

We have also found evidence that this new SA biosynthetic pathway also works also under stress conditions. MD treatment improved the plant performance of peach plants under salinity or PPV-infection conditions. However, the contribution of this pathway to the SA pool does not seem to be relevant under these stress conditions.

MD also modulated the content in other stress related hormones as well as the antioxidant defenses, that could activate different redox-related signaling pathways.

Our data agrees with the previously reported role for CNglcs in oxidative stress tolerance (Gleadow & Møller 2014; Gleadow et al. 2016).

References

  • Diaz-Vivancos et al (2017) Plant & Cell Physiology 58(12): 2057–2066
  • Durner J and Klessig DF (1995) Proc. Natl. Acad. Sci. U SA 92(24): 11312-11316
  • Bernal-Vicente et al (2018) Plant Biology 20(6):986-994
  • Bernal-Vicente et al (2019) Plant Biology DOI: 10.1111/plb.13066
  • Gleadow Møller (2014) Annual Review of Plant Biology 65:155–185
  • Gleadow et al (2016) Journal of Experimental Botany 6:403-5413
  • Rao et al (1997) Plant Physiology 115:137–149.

 

This work was supported by the Spanish Ministry of Economy and Competitiveness (projects AGL2014-52563-R; RTA2017-00011-C03-02).


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Environmentally friendly strategies against Plum pox virus: Effect of PPV infection on the salicylic acid biosynthetic pathway from mandelonitrile in peach

José A. Hernándeza; Pedro Díaz-Vivancosa,b
aBiotechnology of Fruit Trees Group, Dept. Plant Breeding, CEBAS-CSIC, Campus Universitario de Espinardo, 25. 30100 Murcia (Spain). bDepartment of Plant Biology, Faculty of Biology, University of Murcia, Campus de Espinardo, E-30100 Murcia, Spain.

In a previous work, we reported that the cyanogenic glycosides (CNglcs) pathway can be involved in a new salicylic acid (SA) biosynthetic pathway in peach, with mandelonitrile (MD) linking both pathways (Diaz-Vivancos et al. 2017). In this pathway, MD acts as an intermediary molecule between CNglcs turnover and SA biosynthesis (Diaz-Vivancos et al. 2017). The plant hormone SA plays multiple roles in plants and acts as an endogenous signal mediating plant defense responses against both biotic and abiotic stimuli. In that regards, we study the effect of MD and phenylalanine (Phe), a known SA-precursor, treatments on stress-related plant hormone contents [(SA, abscisic acid (ABA), and jasmonic acid (JA)] and symptomatology in Plum pox virus-infected peach seedlings.

            The PPV-infected peach seedlings were treated with 1 mM MD or Phe for six weeks and then submitted to an artificial rest period again, which was necessary to ensure the later multiplication of the virus. Samples were taken six weeks after the second artificial rest period; the seedlings were inspected for sharka symptoms and were irrigated with either 1 mM MD or Phe during these six weeks. For all the conditions, 12 seedlings were assayed, and another 12 plants were kept as control.

            In PPV-infected seedlings, an increase of about 1.5-fold in total SA content was observed in control and MD- and Phe-treated plants due to the infection (Fig.1), suggesting that the SA biosynthetic pathway from MD is also functional under biotic (Plum pox virus infection) stress conditions, although the contribution of this pathway to the total SA pool does not seem to be important under such condition. We also analyzed the effect of MD and Phe treatments on abcisic (ABA) and jasmonic acids (JA) levels in control and PPV-infected seedlings. The MD treatment produced a drop in ABA levels in control plants. However, PPV-infection induced an increase in ABA content in MD-treated plants, whereas JA levels strongly increased by both MD and Phe treatments (Fig 1).

Fig 1 color

Figure 1.- Total ABA , JA and SA levels in the leaves of peach seedlings grown in the presence or absence of MD or Phe submitted PPV infection . Data represent the mean ± SE of at least five repetitions of each treatment. Different letters indicate significant differences according to Duncan’s test (P≤0.05).

            Regarding PPV symptoms, venal chlorosis and leaf deformation were observed in non-treated seedlings. The mean intensity of PPV symptoms observed in non-treated plants, around 3.0 on a scale of 0 to 5, confirmed the high susceptibility described for this cultivar. Both MD and Phe treatments reduced the severity of symptoms, although Phe in a lesser extent than MD (Fig 2). This response correlated with higher levels of SA and JA in peach leaves, as well as with enhanced ABA levels in MD-treated seedlings.

 

Fig 2 color

Fig. 2.- Phenotypic scoring for evaluating sharka symptoms in peach seedlings. Data represent the mean ± SE of at least 10 repetitions. Different letters indicate significant differences according to Duncan’s test (P≤0.05).

            As a conclusion, based on our previous results suggesting that the CNgls pathway can be involved in SA biosynthesis via MD, we have found evidences that this new SA biosynthetic pathway also works also under stress conditions. These data suggest that SA biosynthesis from MD could have a positive effect in the response of peach plants to PPV infection, reducing Sharka symptomatology, even though that the contribution of this pathway to the total SA pool does not seem to be relevant.

For more information, please see: Euphresco_success_story_Epi-PPV_2

Project ID: 2015-E-147 Determine different Plum pox virus strains in wild hosts and in stone fruit cultivars with different susceptibility as a part of improved control and surveillance strategies


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Cytosolic antioxidant machinery and resistance to biotic stress

José A. Hernández Cortés, Senior Researcher (CEBAS-CSIC, Biotechnology and Fruit Trees Group, Murcia)

The effect of over-expression of cytosolic Cu,Zn-superoxide dismutase (cytsod) and ascorbate peroxidase (cytapx) alone, or in combination, in tobacco plants, increased the resistance to bacterial wild fire, caused by Pseudomonas syringae pv. Tabaci.

In the non-transformed controls, the inoculated areas with P. syringae pv tabaci became initially chlorotic at 2 days post-inoculation (dpi), then subsequently water soaked and necrotic. At 6 dpi, the necrotic areas extended to the border of the leaves and finally the necrotic area appeared surrounded by a chlorotic yellow area. In transgenic lines harboring cytsod (line 17) or cytapx (line 51) necrotic areas were confined within the infiltrated sites, mimicking symptoms of hypersensitive reaction (HR) observed during an incompatible plant-pathogen interaction. In contrast, in the transgenic lines harboring both cytsod and cytapx genes (lines 35 and 39), infected leaf tissue presented only a chlorotic area and localized necrosis occurred only occasionally (Fig. 1).

Disease resistance against Pseudomonas syringae pv. tabaci in transgenic tobacco lines. Fully expanded leaves of 8-weeks-old tobacco plants were inoculated with 3 x 105 cfu/ml of P. syringae pv. tabaci. Photos were taken 10 days after infection

Disease resistance against Pseudomonas syringae pv. tabaci in transgenic tobacco lines. Fully expanded leaves of 8-weeks-old tobacco plants were inoculated with 3 x 105 cfu/ml of P. syringae pv. tabaci. Photos were taken 10 days after infection

In addition, the symptoms observations correlated with reduced bacterial growth, mainly in lines showing fewer symptoms (Lines 35 and 39).  The reduced necrotic lesions observed in the transgenic lines are parallel with increased antioxidant enzyme levels (SOD, APX, Catalase, and Glutathione Reductase). In this sense, the balance between SOD (O2.--scavenger and H2O2-producer enzyme) and H2O2-scavenger enzymes (APX, Catalase and Peroxidases) is crucial to control ROS accumulation and the triggering of cell death (Fig. 2). The observed resistance of transgenic lines to bacterial wild fire seemed to be independent of tissue necrosis, as observed in other plant-pathogens interactions (Kiraly et al. 1999; 2008). These authors suggested that the induction of antioxidant enzymes might reduce plant cell necrosis after infection (Kiraly et al., 2002; 2008).

Fig. 2.- Schema showing the pathogen-induced ROS generation in the apoplastic space from tobacco leaves. Bacterial pathogens stimulated the superoxide (O2.-) generation in the apoplastic space by activating the plasma membrane NADPH oxidase (Lamb & Dixon 1997). Superoxide is subsequently dismutated to H2O2 by the apoplastic SOD. There are potential sources of apoplastic H2O2 generation including peroxidases, amine oxidases and oxalate oxidases (Bollwell & Wojtaszek 1997). Hydrogen peroxide can participate in the lignification process (Ros Barceló 1998) as well as in the direct killing of the pathogens. ROS, such as the protonated form of HO2.- (perhydroxyl radical) or H2O2, can permeate biological membranes. Therefore, an important part of the apoplastic ROS can also originate an oxidative stress in the cytosol, in addition to that produced by ROS originated from chloroplasts, mitochondria and peroxisomes (Diaz-Vivancos et al., 2008; Amirsadeghi et al., 2007; Van Breusegem & Dat, 2006) and can contribute to the induction of an oxidative stress in the cytosolic compartment. This ROS accumulation can induce the HR, manifested as cell death and tissue necrotization. Transgenic lines overexpressing both cytsod and cytapx (lines 35 and 39), can cope with ROS accumulation and avoid tissue necrotization.

Fig. 2.- Schema showing the pathogen-induced ROS generation in the apoplastic space from tobacco leaves. Bacterial pathogens stimulated the superoxide (O2.-) generation in the apoplastic space by activating the plasma membrane NADPH oxidase (Lamb & Dixon 1997). Superoxide is subsequently dismutated to H2O2 by the apoplastic SOD. There are potential sources of apoplastic H2O2 generation including peroxidases, amine oxidases and oxalate oxidases (Bollwell & Wojtaszek 1997). Hydrogen peroxide can participate in the lignification process (Ros Barceló 1998) as well as in the direct killing of the pathogens. ROS, such as the protonated form of HO2.- (perhydroxyl radical) or H2O2, can permeate biological membranes. Therefore, an important part of the apoplastic ROS can also originate an oxidative stress in the cytosol, in addition to that produced by ROS originated from chloroplasts, mitochondria and peroxisomes (Diaz-Vivancos et al., 2008; Amirsadeghi et al., 2007; Van Breusegem & Dat, 2006) and can contribute to the induction of an oxidative stress in the cytosolic compartment. This ROS accumulation can induce the HR, manifested as cell death and tissue necrotization. Transgenic lines overexpressing both cytsod and cytapx (lines 35 and 39), can cope with ROS accumulation and avoid tissue necrotization.

The results of this work showed that the overexpression of CuZn-SOD and APX in the cytosol of tobacco plants enhances resistance against P. syringae pv tabaci, highlighting the importance of the cytosolic antioxidant machinery in the response to biotic stress, similar to the results showing the importance of this cell compartment in the response to abiotic stress, such as salinity or drought stress (Hernández et al., 2000; Faize et al., 2011).

For more information, please consult:

Faize, M., Burgos, L., Faize, L., Petri, C., Barba-Espin, G., Diaz-Vivancos, P., Clemente-Moreno, M.J., Alburquerque, N. and Hernández, J.A. (2012) Modulation of tobacco bacterial disease resistance using cytosolic ascorbate peroxidase and Cu, Zn-superoxide dismutase. Plant Pathol., 61, 858-866.

References

Amirsadeghi S, Robson CA, Vanlerberghe GC, 2007. The role of mitochondrion in plant response to biotic stress. Physiologia Plantarum 129, 253-266.

Bolwell GP, Wojtaszek P, 1997. Mechanisms for the generation of reactive oxygen species in defence – a broad perspective. Physiological and Molecular Plant Pathology 51, 347-366

Díaz-Vivancos P, Clemente-Moreno MJ, Rubio M, Olmos E, García JA, Martínez-Gómez P, Hernández JA, 2008. Alteration in the chloroplastic metabolism leads to ROS accumulation in pea plants in response to Plum pox virus. Journal of Experimental Botany 59, 2147-2160.

Faize, M., Burgos, L., Faize, L., Piqueras, A., Nicolas, E., Barba-Espin, G., Clemente-Moreno, M.J., Alcobendas, R., Artlip, T. and Hernández, J.A. (2011) Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought. J. Exp. Bot., 62, 2599-2613.

Hernández, J.A., Jiménez, A., Mullineaux, P.M. and Sevilla, F. (2000) Tolerance of pea (Pisum sativum L.) to long-term salt stress is associated with induction of antioxidant defences. Plant Cell Environ., 23, 853-862.

Király L, Cole AB, Bourque JE, Schoelz JE, 1999. Systemic cell death is elicited by the interaction of a single gene in Nicotiana and gene VI from cauliflower mosaic virus. Molecular Plant-Microbe Interaction 12, 919-925.

Király Z, Barna B, Kecskés A, Fodor J, 2002. Down-regulation of antioxidative capacity in a transgenic tobacco which fails to develop acquired resistance to necrotization caused by TMV. Free Radical Research 36, 981-991.

Király L, Hafez YM, Fodor J, Király Z, 2008. Suppression of tobacco mosaic virus-induced hypersensitive-type necrotization in tobacco at high temperature is associated with downregulation of NADPH oxidase and superoxide and stimulation of dehydroascorbate reductase. Journal of General Virology 89, 799-808.

Lamb C, Dixon RA, 1997. The oxidative burst in plant disease resistance. Annual Review of Plant Physiology and Plant Molecular Biology 48, 251-275.

Ros Barceló A, 1998. The generation of H2O2 in the xylem of Zinnia elegans is mediated by an NADPH-oxidase-like enzyme. Planta 207, 207-216.

Van Breusegem F, Dat JF, 2006. Reactive oxygen species in plant cell death. Plant Physiology 141, 384-390.