# Chloroplasts at the Crossroad of Photosynthesis, Pathogen Infection and Plant Defense
## 3 The Chloroplast is a Major Site of Free Radical Production
### The Chloroplast is a \(\cdot\)NO Generator  
\(\cdot\)NO is a gaseous free radical. \(\cdot\)NO is primarily produced in the apoplast, chloroplasts, mitochondria, and peroxisomes of a plant cell [242]. There are two independent \(\cdot\)NO production pathways in the chloroplast: (1) reductive \(\cdot\)NO generation from nitrite catalyzed by the thylakoid membrane-associated nitrate reductase; and (2) oxidative \(\cdot\)NO synthesis from arginine (Arg) catalyzed by a \(\cdot\)NO synthase-like enzyme [243, 244]. Nitrate reductase generally reduces nitrate to nitrite, but this enzyme can also reduce nitrite to \(\cdot\)NO [245, 246]. It is worth mentioning that the production of \(\cdot\)NO by nitrate reductase and the \(\cdot\)NO synthase-like enzyme requires NAD(P)H [246]. \(\cdot\)NO modulates various aspects of plant growth, development, and stress responses [242, 247, 248]. For example, exogenous \(\cdot\)NO has been shown to inhibit photosynthesis in intact leaves and the \(\cdot\)NO concentration causing inhibition to net photosynthesis is much lower than those required for visible injury [249, 250]. \(\cdot\)NO may regulate the activities of target proteins via metal center-binding, tyrosine (Tyr) nitration, and Cys S-nitrosylation [248]. \(\cdot\)NO inhibits PSII electron transfer by binding reversibly to three sites in PSII [251]: the non-heme iron between Q\({}_{\text{A}}\) and Q\({}_{\text{B}}\)[252, 253], the Mn cluster of the oxygen-evolving complex [254], and the redox-active Tyr residue in D2 [255]. The first two sites involve interaction with metal centers while the third site involves Tyr nitration [252, 255, 256]. \(\cdot\)NO could also inhibit photosynthetic carbon fixation (i.e., Rubisco activity) via S-nitrosylation of Cys [65] in the Rubisco small subunit [257]. \(\cdot\)NO plays a variety of roles in plant defense against pathogens. As mentioned previously, a concerted production of ROS and \(\cdot\)NO and the interaction between H\({}_{2}\)O\({}_{2}\) and \(\cdot\)NO are required for the development of the hypersensitive cell death [236]. On the one hand, \(\cdot\)NO potentiates ROS-induced localized cell death and induces defense gene expression [258]. On the other hand, as the concentration of S-nitrosothiols increases during the oxidative and nitrosative bursts, \(\cdot\)NO induces S-nitrosylation of the RbohD NADPH oxidase at Cys [890], limiting further ROS production and cell death [259]. In addition, \(\cdot\)NO may enable local resistance and SAR, by inducing SA accumulation [260]. Furthermore, \(\cdot\)NO may induce the expression of defense-related genes via Ca\({}^{2+}\)-dependent (or \(-\)independent) pathways [261]. \(\cdot\)NO influences the production and signaling of phytohormones (Figure 1). \(\cdot\)NO participates in the regulation of stomatal movements: ABA and \(\cdot\)NO both induce stomatal closure; ABA promotes \(\cdot\)NO production [76, 78, 262]. Conceivably, \(\cdot\)NO scavenging and impaired \(\cdot\)NO generation inhibit ABA-induced stomatal closure [76, 78, 262]. However, \(\cdot\)NO was recently found to inhibit the activity of OST1 (a positive regulator of H\({}_{2}\)O\({}_{2}\) production) in guard cells via S-nitrosylation, suggesting that \(\cdot\)NO may also act as a negative regulator of ABA signaling [263]. \(\cdot\)NO counteracts ABA in seed dormancy release and germination as well [264]. For example, \(\cdot\)NO is involved in H\({}_{2}\)O\({}_{2}\)-mediated up-regulation of ABA catabolism during seed imbibition [231]. \(\cdot\)NO is necessary for biphasic ET generation during the hypersensitive response [265, 266]. The positive effect of \(\cdot\)NO on ET production is also seen under hypoxia, during which over-accumulation of \(\cdot\)NO triggers ET synthesis, possibly via S-nitrosylation of ET biosynthetic enzymes, e.g., ACC synthase and ACC oxidase [267]. \(\cdot\)NO could also have negative effects on ET production.