Summary: Reactive oxygen species (ROS) play fundamental roles in plant responses to pathogen infection, including modulation of cell death processes and defense‐related gene expression. Cell death triggered as part of the hypersensitive response enhances resistance to biotrophic pathogens, but favors the virulence of necrotrophs. Even though the involvement of ROS in the orchestration of defense responses is well established, the relative contribution of specific subcellular ROS sources to plant resistance against microorganisms with different pathogenesis strategies is not completely known. The aim of this work was to investigate the role of chloroplastic ROS in plant defense against a typical necrotrophic fungus, Botrytis cinerea. For this purpose, we used transgenic Nicotiana tabacum (tobacco) lines expressing a plastid‐targeted cyanobacterial flavodoxin (pfld lines), which accumulate lower chloroplastic ROS in response to different stresses. Tissue damage and fungal growth were significantly reduced in infected leaves of pfld plants, as compared with infected wild‐type (WT) counterparts. ROS build‐up triggered by Botrytis infection and associated with chloroplasts was significantly decreased (70–80%) in pfld leaves relative to the wild type. Phytoalexin accumulation and expression of pathogenesis‐related genes were induced to a lower degree in pfld plants than in WT siblings. The impact of fungal infection on photosynthetic activity was also lower in pfld leaves. The results indicate that chloroplast‐generated ROS play a major role in lesion development during Botrytis infection. This work demonstrates that the modulation of chloroplastic ROS levels by the expression of a heterologous antioxidant protein can provide a significant degree of protection against a canonical necrotrophic fungus.

The role of polyamine (PA) metabolism in tobacco (Nicotiana tabacum) defense against pathogens with contrasting pathogenic strategies was evaluated. Infection by the necrotrophic fungus Sclerotinia sclerotiorum resulted in increased arginine decarboxylase expression and activity in host tissues, as well as putrescine and spermine accumulation in leaf apoplast. Enhancement of leaf PA levels, either by using transgenic plants or infiltration with exogenous PAs, led to increased necrosis due to infection by S. sclerotiorum. Specific inhibition of diamine and PA oxidases attenuated the PA-induced enhancement of leaf necrosis during fungal infection. When tobacco responses to infection by the biotrophic bacterium Pseudomonas viridiflava were investigated, an increase of apoplastic spermine levels was detected. Enhancement of host PA levels by the above-described experimental approaches strongly decreased in planta bacterial growth, an effect that was blocked by a PA oxidase inhibitor. It can be concluded that accumulation and further oxidation of free PAs in the leaf apoplast of tobacco plants occurs in a similar, although not identical way during tobacco defense against infection by microorganisms with contrasting pathogenesis strategies. This response affects the pathogen's ability to colonize host tissues and results are detrimental for plant defense against necrotrophic pathogens that feed on necrotic tissue; on the contrary, this response plays a beneficial role in defense against biotrophic pathogens that depend on living tissue for successful host colonization. Thus, apoplastic PAs play important roles in plant-pathogen interactions, and modulation of host PA levels, particularly in the leaf apoplast, may lead to significant changes in host susceptibility to different kinds of pathogens.

The possible involvement of apoplastic reactive oxygen species produced by the oxidation of free polyamines in the leaf growth of salinized maize has been studied here. Salt treatment increased the apoplastic spermine and spermidine levels, mainly in the leaf blade elongation zone. The total activity of polyamine oxidase was up to 20-fold higher than that of the copper-containing amine oxidase. Measurements of H₂O₂, ·O₂⁻, and HO· production in the presence or absence of the polyamine oxidase inhibitors 1,19-bis-(ethylamine)-5,10,15 triazanonadecane and 1,8-diamino-octane suggest that, in salinized plants, the oxidation of free apoplastic polyamines by polyamine oxidase by would be the main source of reactive oxygen species in the elongation zone of maize leaf blades. This effect is probably due to increased substrate availability. Incubation with 200 μM spermine doubled segment elongation, whereas the addition of 1,19-bis-(ethylamine)-5,10,15 triazanonadecane and 1,8-diamino-octane to 200 μM spermine attenuated and reversed the last effect, respectively. Similarly, the addition of MnCl₂ (an ·O₂⁻ dismutating agent) or the HO· scavenger sodium benzoate along with spermine, annulled the elongating effect of the polyamine on the salinized segments. As a whole, the results obtained here demonstrated that, under salinity, polyamine oxidase activity provides a significant production of reactive oxygen species in the apoplast which contributes to 25–30% of the maize leaf blade elongation.