# Chloroplasts at the Crossroad of Photosynthesis, Pathogen Infection and Plant Defense
## 3 The Chloroplast is a Major Site of Free Radical Production  
### The Photosynthetic Electron Transport Chain is a Major Site of ROS Production  
ROS are reactive molecules and free radicals derived from O\({}_{2}\). Examples of ROS include O\({}_{2}\)'\({}^{-}\), H\({}_{2}\)O\({}_{2}\), 'OH (hydroxyl radical), and \({}^{1}\)O\({}_{2}\). Different ROS have different half-lives and specificities [192]. ROS can be produced in the apoplast, chloroplasts, mitochondria, and peroxis of a plant cell [21, 192, 193]. Increases in ROS accumulation have been detected in both PTI and ETI [21]. The biphasic accumulation of ROS in different subcellular compartments is the hallmark of successful recognition of pathogens by plants [193]. The first, low-amplitude, and transitory phase occurs within minutes after infection and is mostly apoplastic and tightly linked to the activities of plasma-membrane Rboh NADPH oxidases (e.g., RbohD and RbohF) and cell-wall peroxididases [21, 193, 194]. O\({}_{2}\)'\({}^{-}\) produced by NADPH oxidases is rapidly converted to H\({}_{2}\)O\({}_{2}\) either spontaneously or by superoxide dismutase (SOD) [193]. The resulting H\({}_{2}\)O\({}_{2}\), along with H\({}_{2}\)O\({}_{2}\) generated by cell-wall peroxididases, crosses the plasma membrane and enters the cell via free diffusion or aquaporin-facilitated diffusion [195, 196, 197]. The second, high-amplitude, and sustained phase takes place a few hours after infection and is typically associated with the establishment of defense responses and the hypersensitive response [21, 193, 194]. The second phase happens in multiple compartments, including the apoplast, chloroplasts, mitochondria, and peroxisomes [193]. In the chloroplast, PSI and PSII are two main sources of ROS production [15, 16]. PSI is a primary generator of O\({}_{2}\)'\({}^{-}\)[198, 199]. During photosynthesis, O\({}_{2}\) is continuously reduced to O\({}_{2}\)'\({}^{-}\) by PSI, and O\({}_{2}\)'\({}^{-}\) is quickly converted to H\({}_{2}\)O\({}_{2}\) and O\({}_{2}\) by the Cu-Zn-SOD attached to PSI [15, 16]. PSII is a major generator of \({}^{1}\)O\({}_{2}\)[200, 201]. During photosynthesis, ground-state oxygen (\({}^{3}\)O\({}_{2}\)) is continuously excited to \({}^{1}\)O\({}_{2}\) by triplet-excited-state chlorophyll (\({}^{3}\)P680\({}^{\star}\)) in the PSII reaction center. ROS production has both positive and negative effects on photosynthesis. On the one hand, ROS production by the two photosystems acts as alternative electron sinks, alleviating the negative impacts of over-reduction and photo-inactivation of the photosynthetic apparatus [15, 202]. In this regard, chloroplastic ROS production protects the photosynthetic apparatus, especially when the absorbed excitation energy exceeds the energy consumed by the photosynthetic electron transport chain [15; 16]. On the other hand, non-physiological concentrations of chloroplastic ROS may cause irreversible damage to thylakoid membranes and photosynthetic components, by lipid peroxidation, protein damage, membrane destruction, and ion leakage [203; 204]. During plant defense against pathogens, ROS have a variety of functions, including (1) killing pathogens directly [205], (2) strengthening cell walls [206], (3) activating defense gene expression [165; 207], (4) mediating lipid peroxidation (to execute localized cell death) [208], (5) causing phytoelexin accumulation (to inhibit pathogen growth) [209], (6) inducing the hypersensitive response [210], (7) modulating vesicle trafficking (to mediate signaling) [211], and (8) being required for the internalization of pattern recognition receptors [212]. Similar to ROS produced in other cellular compartments, chloroplastic ROS are essential for the hypersensitive response in plants [213; 214; 215; 216]. For example, infiltration of wild-type tobacco leaves with a non-host pathogen _Xanthomonas campestris_ pv. _vesicatoria_ (_Xcv_) resulted in increased ROS accumulation, preceding the appearance of localized cell death [215].