Tetrapyrroles such as chlorophylls and bacteriochlorophylls play a fundamental role in the energy absorption and transduction activities of photosynthetic organisms. Because of these molecules, however, photosynthetic organisms are also prone to photooxidative damage. They had to evolve highly efficient strategies to control tetrapyrrole biosynthesis and to prevent the accumulation of free intermediates that potentially are extremely destructive when illuminated. In higher plants, the metabolic flow of tetrapyrrole biosynthesis is regulated at the step of ␦-aminolevulinic acid synthesis. This regulation previously has been attributed to feedback control of Glu tRNA reductase, the first enzyme committed to tetrapyrrole biosynthesis, by heme. With the recent discovery of chlorophyll intermediates acting as signals that control both nuclear gene activities and tetrapyrrole biosynthesis, it seems likely that heme is not the only regulator of this pathway. A genetic approach was used to identify additional factors involved in the control of tetrapyrrole biosynthesis. In Arabidopsis thaliana, we have found a negative regulator of tetrapyrrole biosynthesis, FLU, which operates independently of heme and seems to selectively affect only the Mg 2؉ branch of tetrapyrrole biosynthesis. The identity of this protein was established by map-based cloning and sequencing the FLU gene. FLU is a nuclear-encoded plastid protein that, after import and processing, becomes tightly associated with plastid membranes. It is unrelated to any of the enzymes known to be involved in tetrapyrrole biosynthesis. Its predicted features suggest that FLU mediates its regulatory effect through interaction with enzymes involved in chlorophyll synthesis.

Enhanced levels of singlet oxygen ( 1 O 2 ) in chloroplasts trigger programmed cell death. The impact of 1 O 2 production in chloroplasts was monitored first in the conditional fluorescent (flu) mutant of Arabidopsis thaliana that accumulates 1 O 2 upon a dark/light shift. The onset of 1 O 2 production is rapidly followed by a loss of chloroplast integrity that precedes the rupture of the central vacuole and the final collapse of the cell. Inactivation of the two plastid proteins EXECUTER (EX1) and EX2 in the flu mutant abrogates these responses, indicating that disintegration of chloroplasts is due to EX-dependent signaling rather than 1 O 2 directly. In flu seedlings, 1 O 2 -mediated cell death signaling operates as a default pathway that results in seedlings committing suicide. By contrast, EX-dependent signaling in the wild type induces the formation of microlesions without decreasing the viability of seedlings. 1 O 2 -mediated and EX-dependent loss of plastid integrity and cell death in these plants occurs only in cells containing fully developed chloroplasts. Our findings support an as yet unreported signaling role of 1 O 2 in the wild type exposed to mild light stress that invokes photoinhibition of photosystem II without causing photooxidative damage of the plant.

SummaryThe regulation of tetrapyrrole biosynthesis in higher plants has been attributed to metabolic feedback inhibition of Glu tRNA reductase by heme. Recently, another negative regulator of tetrapyrrole biosynthesis has been discovered, the FLU protein. During an extensive second site screen of mutagenized flu seedlings a suppressor of flu, ulf3, was identified that is allelic to hy1 and encodes a heme oxygenase. Increased levels of heme in the hy1 mutant have been implicated with inhibiting Glu tRNA reductase and suppressing the synthesis of d-aminolevulinic acid (ALA) and Pchlide accumulation. When combined with hy1 or ulf3 upregulation of ALA synthesis and overaccumulation of protochlorophyllide in the flu mutants were severely suppressed supporting the notion that heme antagonizes the effect of the flu mutation by inhibiting Glu tRNA reductase independently of FLU. The coiled-coil domain at the C-terminal end of Glu tRNA reductase interacts with FLU, whereas the N-terminal site of Glu tRNA reductase that is necessary for the inhibition of the enzyme by heme is not required for this interaction. The interaction with FLU is specific for the Glu tRNA reductase encoded by HEMA1 that is expressed in photosynthetically active tissues. FLU seems to be part of a second regulatory circuit that controls chlorophyll biosynthesis by interacting directly with Glu tRNA reductase not only in etiolated seedlings but also in light-adapted green plants.

SUMMARYThe conditional flu mutant of Arabidopsis thaliana generates singlet oxygen ( 1 O 2 ) in plastids during a darkto-light shift. Seedlings of flu bleach and die, whereas mature plants stop growing and develop macroscopic necrotic lesions. Several suppressor mutants, dubbed singlet oxygen-linked death activator (soldat), were identified that abrogate 1 O 2 -mediated cell death of flu seedlings. One of the soldat mutations, soldat10, affects a gene encoding a plastid-localized protein related to the human mitochondrial transcription termination factor mTERF. As a consequence of this mutation, plastid-specific rRNA levels decrease and protein synthesis in plastids of soldat10 is attenuated. This disruption of chloroplast homeostasis in soldat10 seedlings affects communication between chloroplasts and the nucleus and leads to changes in the steady-state concentration of nuclear gene transcripts. The soldat10 seedlings suffer from mild photo-oxidative stress, as indicated by the constitutive up-regulation of stress-related genes. Even though soldat10/flu seedlings overaccumulate the photosensitizer protochlorophyllide in the dark and activate the expression of

When plant cells are under environmental stress, several chemically distinct reactive oxygen species (ROS) are generated simultaneously in various intracellular compartments and these can cause oxidative damage or act as signals. The conditional flu mutant of Arabidopsis, which generates singlet oxygen in plastids during a dark-to-light transition, has allowed the biological activity of singlet oxygen to be determined, and the criteria to distinguish between cytotoxicity and signalling of this particular ROS to be defined. The genetic basis of singlet-oxygen-mediated signalling has been revealed by the mutation of two nuclear genes encoding the plastid proteins EXECUTER (EX)1 and EX2, which are sufficient to abrogate singlet-oxygendependent stress responses. Conversely, responses due to higher cytotoxic levels of singlet oxygen are not suppressed in the ex1/ex2 background. Whether singlet oxygen levels lower than those that trigger genetically controlled cell death activate acclimation is now under investigation.