The metabolic and cellular changes in source leaves of Nicotiana tabacum L. cv SNN during an incompatible interaction with Phytophthora nicotianae van Breda de Haan were investigated and compared with defence reactions. Hypersensitive cell death was preceded by a rapid and highly localized shift to non-assimilatoric metabolism. During the first 6 h post infection (hpi), reactive oxygen species (ROS) accumulated. Callose was deposited at the interface of adjacent mesophyll cells ( ≥ 1 hpi), the export of sucrose collapsed and its content in the apoplast increased. Stomata closed and photosynthetic flux was reallocated from CO 2 assimilation in favour of photorespiration. This was accompanied by an increase in respiration, glucose-6-phosphate dehydrogenase (G6PDH) activity, apoplastic invertase and hexose content. Later ( > 6 hpi) the photosynthetic electron transport chain was interrupted and photosynthesis completely collapsed. This was accompanied by a further increase in apoplastic invertase and carbohydrates, respiration and oxidative pentose phosphate pathway (OPPP) and followed by further burst in ROS release. Hypersensitive cell death did not appear until photosynthesis completely declined. Photosynthesis was visualized by chlorophyll-a fluorescence imaging on a macro-and microscopic scale. Decline in photosynthesis and defence reactions were highly localized processes, which occur in single mesophyll cells. We propose that in photoautotrophic leaves, photosynthesis and assimilatory metabolism must be switched off to initiate respiration and other processes required for defence. An early blockage of intercellular sugar transportation, due to callose deposition, in conjunction with enhanced apoplastic invertase activity could facilitate this metabolic shift.

Light is an important environmental factor that modulates acclimation strategies and defense responses in plants. We explored the functional role of the regulatory subunit B#g (B#g) of protein phosphatase 2A (PP2A) in light-dependent stress responses of Arabidopsis (Arabidopsis thaliana). The predominant form of PP2A consists of catalytic subunit C, scaffold subunit A, and highly variable regulatory subunit B, which determines the substrate specificity of PP2A holoenzymes. Mutant leaves of knockdown pp2a-b#g plants show disintegration of chloroplasts and premature yellowing conditionally under moderate light intensity. The cell-death phenotype is accompanied by the accumulation of hydrogen peroxide through a pathway that requires CONSTITUTIVE EXPRESSION OF PR GENES5 (CPR5). Moreover, the pp2a-b#g cpr5 double mutant additionally displays growth suppression and malformed trichomes. Similar to cpr5, the pp2a-b#g mutant shows constitutive activation of both salicylic acid-and jasmonic acid-dependent defense pathways. In contrast to cpr5, however, pp2a-b#g leaves do not contain increased levels of salicylic acid or jasmonic acid. Rather, the constitutive defense response associates with hypomethylation of DNA and increased levels of methionine-salvage pathway components in pp2a-b#g leaves. We suggest that the specific B#g subunit of PP2A is functionally connected to CPR5 and operates in the basal repression of defense responses under low irradiance.

In source leaves of resistant tobacco, oxidative burst and subsequent formation of hypersensitive lesions after infection with Phytophthora nicotianae was prevented by inhibition of glucose-6-phosphate dehydrogenase (G6PDH) or NADPH oxidases. This observation indicated that plant defense could benefit from improved NADPH availability due to increased G6PDH activity in the cytosol. A plastidic isoform of the G6PDH-encoding gene, G6PD, displaying high NADPH tolerance was engineered for cytosolic expression (cP2), and introduced into a susceptible cultivar. After infection, transgenic (previously susceptible) lines overexpressing cP2 showed early oxidative bursts, callose deposition, and changes in metabolic parameters. These responses resulted in timely formation of hypersensitive lesions similar to resistant plants, although their extent varied considerably between different transgenic lines. Additional RNAi suppression of endogenous cytosolic G6PD isoforms resulted in highly uniform defense responses and also enhanced drought tolerance and flowering. Cytosolic G6PDH seems to be a crucial factor for the outcome of plant defense responses; thus, representing an important target for modulation of stress resistance. Because isoenzyme replacement of G6PDH in the cytosol was beneficial under various kinds of cues, we propose this strategy as a tool to enhance stress tolerance in general.oxidative burst ͉ source-to-sink transition ͉ NADPH availability ͉ pathogen resistance ͉ tobacco P lants are under continuous threat of being challenged by pathogenic microorganisms, which try to exploit them as a source of carbohydrates and other assimilates. Timely recognition of invading microorganisms and rapid and effective induction of defense responses are presently considered the main difference between pathogen-resistant and susceptible plant lines (1). So-called compatible interactions between pathogens and plants result if the pathogen can overcome plant defense barriers and establish disease symptoms. During an incompatible interaction, a wealth of defense mechanisms [e.g., generation of reactive oxygen species (ROS), synthesis of pathogenesis-related (PR) proteins, cell-wall fortification, and the hypersensitive reaction (HR)] can efficiently limit pathogen growth. The HR, which includes ROS formation and programmed cell death (PCD), represents the most efficient mechanism of plant defense. ROS were previously proposed to orchestrate the establishment of plant defense responses (2, 3). NADPH oxidases at the plasma membrane are considered the main source of extracellular ROS formation during defense, the so-called oxidative burst. Down-regulation or elimination of NADPH oxidase leads to suppression of pathogen-induced oxidative bursts and HR (4).The oxidative burst, an evolutionarily conserved mechanism, is part of the innate immune response, and (besides triggering plant PCD) it is thought to contribute to direct killing of invading microorganisms. It also helps to mount an invasion barrier through radical-mediated polymer...