The conditional fluorescent ( flu ) mutant of Arabidopsis accumulates the photosensitizer protochlorophyllide in the dark. After a dark-to-light shift, the generation of singlet oxygen, a nonradical reactive oxygen species, starts within the first minute of illumination and was shown to be confined to plastids. Immediately after the shift, plants stopped growing and developed necrotic lesions. These early stress responses of the flu mutant do not seem to result merely from physicochemical damage. Peroxidation of chloroplast membrane lipids in these plants started rapidly and led to the transient and selective accumulation of a stereospecific and regiospecific isomer of hydroxyoctadecatrieonic acid, free (13 S )-HOTE, that could be attributed almost exclusively to the enzymatic oxidation of linolenic acid. Within the first 15 min of reillumination, distinct sets of genes were activated that were different from those induced by superoxide/hydrogen peroxide. Collectively, these results demonstrate that singlet oxygen does not act primarily as a toxin but rather as a signal that activates several stress-response pathways. Its biological activity in Arabidopsis exhibits a high degree of specificity that seems to be derived from the chemical identity of this reactive oxygen species and/or the intracellular location at which it is generated.

SummaryWe have produced 22 090 primary transgenic rice plants that carry a T-DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel-blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T-DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T-DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless b-glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T-DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature¯owers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6±2.1% of tested organs were GUS-positive in the tested organs, and that their GUS expression patterns were organ-or tissue-speci®c or ubiquitous in all parts of the plant. The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.

Plants under oxidative stress suffer from damages that have been interpreted as unavoidable consequences of injuries inflicted upon plants by toxic levels of reactive oxygen species (ROS). However, this paradigm needs to be modified. Inactivation of a single gene, EXECUTER1, is sufficient to abrogate stress responses of Arabidopsis thaliana caused by the release of singlet oxygen: External conditions under which these stress responses are observed and the amounts of ROS that accumulate in plants exposed to these environmental conditions do not directly cause damages. Instead, seedling lethality and growth inhibition of mature plants result from genetic programs that are activated after the release of singlet oxygen has been perceived by the plant.

Photosynthetic organisms must achieve a delicate balance between the light energy absorbed by chlorophyll and their capacity to channel that energy into productive photochemical reactions. Release of excess absorbed energy in the cell can cause lethal photooxidative damage. We identified a basic helix-loop-helix (bHLH) transcription factor, designated PHYTOCHROME-INTERACTING FACTOR 1 (PIF1), that negatively regulates chlorophyll biosynthesis. pif1 mutant seedlings accumulate excess free protochlorophyllide when grown in the dark, with consequent lethal bleaching upon exposure to light. PIF1 interacts specifically with the photoactivated conformer of phytochromes A and B, suggesting a signaling pathway by which chlorophyll biosynthetic rates are tightly controlled during the critical initial emergence of seedlings from subterranean darkness into sunlight.

Shortly after the release of singlet oxygen ( 1 O2), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of 1 O2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of 1 O2-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of 1 O2-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all 1 O2-responsive genes. Retrograde control of 1 O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment.oxidative stress ͉ retrograde signaling ͉ singlet oxygen ͉ chloroplast

Rice contains several MADS box genes. It has been demonstrated previously that one of these genes, OsMADS1 (for Oryza sativa MADS box gene1 ), is expressed preferentially in flowers and causes early flowering when ectopically expressed in tobacco plants. In this study, we demonstrated that ectopic expression of OsMADS1 in rice also results in early flowering. To further investigate the role of OsMADS1 during rice flower development, we generated transgenic rice plants expressing altered OsMADS1 genes that contain missense mutations in the MADS domain. There was no visible alteration in the transgenic plants during the vegetative stage. However, transgenic panicles typically exhibited phenotypic alterations, including spikelets consisting of elongated leafy paleae and lemmas that exhibit a feature of open hull, two pairs of leafy palea-like and lemma-like lodicules, a decrease in stamen number, and an increase in the number of carpels. In addition, some spikelets generated an additional floret from the same rachilla. These characteristics are very similar to those of leafy hull sterile1 ( lhs1 ). The map position of OsMADS1 is closely linked to that of lhs1 on chromosome 3. Examination of lhs1 revealed that it contains two missense mutations in the OsMADS1 MADS domain. A genetic complementation experiment showed that the 11.9-kb genomic DNA fragment containing the wild-type OsMADS1 gene rescued the mutant phenotypes. In addition, ectopic expression of the OsMADS1 gene isolated from the lhs1 line resulted in lhs1 -conferred phenotypes. These lines of evidence demonstrate that OsMADS1 is the lhs1 gene. INTRODUCTIONIn response to floral induction, the inflorescence meristem becomes committed to flowering. LEAFY ( LFY ) and APE-TALA1 ( AP1 ) in Arabidopsis and FLORICAULA ( FLO ) and SQUAMOSA ( SQUA ) in Antirrhinum are responsible for promoting the specification of floral meristem identity (reviewed in Ma, 1994). The genes required for specifying the fate of floral organ primordia include AP1 , AP2 , AGAMOUS ( AG ), PISTILATA ( PI ), and AP3 in Arabidopsis and SQUA , PLENA ( PLE ), GLOBOSA ( GLO ), and DEFICIENS ( DEF ) in Antirrhinum (reviewed in Weigel and Meyerowitz, 1994). Excluding AP2 , these floral homeotic genes encode MADS box proteins that are highly conserved transcription factors in plants, animals, yeast, and fungi and that are regulated by the floral meristem identity gene LFY (Parcy et al., 1998;Wagner et al., 1999).Several other MADS box genes have more subtle functions associated with floral meristem and floral organ identity. Expression of AG-LIKE2 ( AGL2 ), AGL4 , and AGL9 of Arabidopsis begins after the onset of expression of floral meristem identity genes but before the activation of floral organ identity genes (Flanagan and Ma, 1994;Savidge et al., 1995;Mandel and Yanofsky, 1998). DEFH72 and DEFH200 of Antirrhinum appear to function in mediating interactions between the meristem and organ identity genes through direct interaction with PLE (Davies et al., 1996). FLORAL BINDING PROTEIN2 ( FBP2 ) o...

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.

Formation of singlet oxygen ( 1 O 2 ) has been implicated with damaging photosystem II (PSII) that needs to undergo continuous repair to maintain photosynthetic electron transport. In addition to its damaging effect, 1 O 2 has also been shown to act as a signal that triggers stress acclimation and an enhanced stress resistance. A signaling role of 1 O 2 was first documented in the fluorescent (flu) mutant of Arabidopsis. It strictly depends on the chloroplast protein EXECUTER1 (EX1) and happens under nonphotoinhibitory light conditions. Under severe light stress, signaling is initiated independently of EX1 by 1 O 2 that is thought to be generated at the acceptor side of active PSII within the core of grana stacks. The results of the present study suggest a second source of 1 O 2 formation in grana margins close to the site of chlorophyll synthesis where EX1 is localized and the disassembly of damaged and reassembly of active PSII take place. The initiation of 1 O 2 signaling in grana margins depends on EX1 and the ATP-dependent zinc metalloprotease FtsH. As FtsH cleaves also the D1 protein during the disassembly of damaged PSII, EX1-and 1 O 2 -mediated signaling seems to be not only spatially but also functionally associated with the repair of PSII.