Plants regulate a complex array of responses to environmental factors such as physical, chemical, and biological stresses. These general responses include basal resistance, hypersensitive response (Heath, 2000), and systemic acquired resistance [SAR (Ward et al., 1991)]. In addition, in plant–pathogen interactions, different mechanisms are activated such as PPOs, antimicrobial agents, and PR proteins. PPOs are a group of copper-containing enzymes that are able to catalyze the oxidation of o-diphenol compounds to quinones (Steffens et al., 1994). In spite of their well-defined role in ultraviolet protection in animals, the physiological role of PPOs in plants has remained unclear and most studies have focused on the postharvest browning reactions observed in cut fruits and vegetables (Arpita et al., 2010; Walker, 1995). Roles of PPO have been demonstrated in biosynthetic processes (Mesquita and Queiroz, 2013; Mueller et al., 1997; Steiner et al., 1999; Strack et al., 2003), defense against herbivores (Wang and Constabel, 2004a, 2004b), fungal pathogenicity, fungal defense reaction (Jacobson, 2000; Soler-Rivas et al., 2000), and resistance of plant to stress, wounding, pathogenesis, and stress-related hormones such as methyl jasmonate (MeJA) and salicylic acid (Li and Steffens, 2002; Raj et al., 2006; Thaler et al., 1999; Thipyapong et al., 2004). Until now, no clear report of how PPO might affect pathogens has been presented, but several mechanisms for the effect of PPO on pathogens have been suggested, including the toxicity to pathogens of quinones generated by PPO, cross-linking of quinones with phenolic compounds, and proteins that might result in the formation of physical barriers to pathogens attack (Li and Steffens, 2002).
PR proteins are another group of proteins induced by the interaction between plant and pathogen and are biomarkers for the functioning of the inducible plant defense response. Production and accumulation of these proteins in plants are a countermeasure against invading pathogens.
Local accumulation of PR proteins has been shown in infected as well as in surrounding tissues and serves to isolate infected tissues from uninfected tissues. The isolation of uninfected tissue can help localize the infection and prevent systemic spread to the rest of the affected plant (Ryals et al., 1996; van Loon and van Strien, 1999). P14a is a member of the PR-1 family from tomato (Solanum lycopersicum) and the most abundant protein accumulating in tomato leaves in response to plant pathogens (Alexander et al., 1993). This protein has been shown to inhibit programmed cell death and also has been found to accumulate during aging of tomato leaves (Camacho Henriquez and Sanger, 1982). P14a also serves as a molecular marker for SAR response (Ryals et al., 1996).
Many phenolic compounds have been found in walnut (Colaric et al., 2005), some of which have a role in defense against pathogens (Radix et al., 1998; Solar et al., 2006). Therefore, walnut could be used as a model to clarify the roles of PPO. A novel and essential role of PPO in secondary metabolism and as a regulator of cell death in walnut has been recently described (Araji et al., 2014).
Walnut blight, caused by Xaj, is one of the most important diseases of walnut and can cause severe economic loss (Garcin and Duchesne, 2001). Yet, the mechanisms of walnut response to this infection are poorly understood. The relationship between infection of walnut with Xaj and expression of the PPO (enzyme activity and gene expression) is a focus of the present study. On the basis of field observation and previous studies, the walnut cultivar Serr is considered very susceptible to Xaj. Although susceptible, blight generally has been a less serious problem for the cultivar Chandler (Teviotdale et al., 1985; Woeste et al., 1992). For this reason, we chose to work with these two economically important cultivars.
The objective of our study was to determine the relative levels of expression of PPO and P14a transcripts with respect to disease symptoms in the walnut–bacterial blight interaction. Herein, we report PPO enzyme activity in leaves of ‘Serr’ and ‘Chandler’ and additionally, JrPPO-1 and PR1 gene expressions following Xaj infection. The results of this study are potentially important for identifying walnut cultivars resistant to bacterial blight in walnut breeding programs.
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