While the metabolic networks in developing seeds during the period of reserve accumulation have been extensively characterized, much less is known about those present during seed desiccation and subsequent germination. Here we utilized metabolite profiling, in conjunction with selective mRNA and physiological profiling to characterize Arabidopsis (Arabidopsis thaliana) seeds throughout development and germination. Seed maturation was associated with a significant reduction of most sugars, organic acids, and amino acids, suggesting their efficient incorporation into storage reserves. The transition from reserve accumulation to seed desiccation was associated with a major metabolic switch, resulting in the accumulation of distinct sugars, organic acids, nitrogen-rich amino acids, and shikimate-derived metabolites. In contrast, seed vernalization was associated with a decrease in the content of several of the metabolic intermediates accumulated during seed desiccation, implying that these intermediates might support the metabolic reorganization needed for seed germination. Concomitantly, the levels of other metabolites significantly increased during vernalization and were boosted further during germination sensu stricto, implying their importance for germination and seedling establishment. The metabolic switches during seed maturation and germination were also associated with distinct patterns of expression of genes encoding metabolism-associated gene products, as determined by semiquantitative reverse transcription-polymerase chain reaction and analysis of publicly available microarray data. When taken together our results provide a comprehensive picture of the coordinated changes in primary metabolism that underlie seed development and germination in Arabidopsis. They furthermore imply that the metabolic preparation for germination and efficient seedling establishment initiates already during seed desiccation and continues by additional distinct metabolic switches during vernalization and early germination.

O(2) photoreduction by photosynthetic electron transfer, the Mehler reaction, was observed in all groups of oxygenic photosynthetic organisms, but the electron transport chain mediating this reaction remains unidentified. We provide the first evidence for the involvement of A-type flavoproteins that reduce O(2) directly to water in vitro. Synechocystis sp. strain PCC 6803 mutants defective in flv1 and flv3, encoding A-type flavoproteins, failed to exhibit O(2) photoreduction but performed normal photosynthesis and respiration. We show that the light-enhanced O(2) uptake was not due to respiration or photorespiration. After dark acclimation, photooxidation of P(700) was severely depressed in mutants Deltaflv1 and Deltaflv3 but recovered after light activation of CO(2) fixation, which gives P(700) an additional electron acceptor. Inhibition of CO(2) fixation prevented recovery but scarcely affected P(700) oxidation in the wild-type, where the Mehler reaction provides an alternative route for electrons. We conclude that the source of electrons for O(2) photoreduction is PSI and that the highly conserved A-type flavoproteins Flv1 and Flv3 are essential for this process in vivo. We propose that in cyanobacteria, contrary to eukaryotes, the Mehler reaction produces no reactive oxygen species and may be evolutionarily related to the response of anaerobic bacteria to O(2).

Incubation of Chlamydomonas reinhardii cells at light levels that are several times more intense than those at which the cells were grown results in a loss of photosystem II function (termed photoinhibition). The loss of activity corresponded to the disappearance from the chloroplast membranes of a lysine-deficient, herbicide-binding protein of 32,000 daltons which is thought to be the apoprotein of the secondary quinone electron acceptor of photosystem II (the QE; protein). In vivo recovery from the damage only occurred following de novo synthesis (replacement) of the chloroplast-encoded QB protein . We believe that the turnover of this protein is a normal consequence of its enzymatic function in vivo and is a physiological process that is necessary to maintain the photosynthetic integrity of the thylakoid membrane. Photoinhibition occurs when the rate of inactivation and subsequent removal exceeds the rate of resynthesis of the QB protein .A 32,000-dalton integral membrane polypeptide ofphotosystem II (PS 11)' is known to be the binding site for several families of herbicides, including the triazines (1). Competition studies using herbicides and quinone analogues (2) have supported the hypotheses that the herbicides act by displacement of a bound quinone (QB) which functions as the secondary quinone electron acceptor for PS II (3, 4). It has been accepted that the 32-kilodalton (kd) polypeptide shall be designated as the QB protein since it functions as the apoprotein of the bound quinone (formalized at the International Conference on Herbicides That Inhibit Photosynthesis, Wageningen, 1983; see reference 5). Pulse-labeling studies using Spirodella (6) and Chlamydomonas (7) indicate that the QB protein exhibits a very rapid turnover in the light.After transfer of dark-grown maize seedlings to light, the level of the mRNA coding for the QB protein becomes the most abundant message in the chloroplast (8) . For this reason the 32-kd protein has also been referred to as the product of a "photogene" (9) . In mature leaf tissue these high mRNA 'Abbreviations used in this paper: kd, kilodalton ; LDS, lithium dodecyl sulfate ; LHC, light-harvesting chlorophyll a/b-protein complex ; PS II, photosystem 11 .THE JOURNAL OF CELL BIOLOGY -VOLUME 99 AUGUST 1984 481-485 0 The Rockefeller University Press -0021-9525/84/08/0481/05 $1 .00 levels are maintained ; this corresponds to the continued high rate of synthesis (and corresponding turnover) of the QB protein .The reason for the high mRNA levels and rapid turnover rate of the QB protein in chloroplasts has not been known. Matoo et al. (10) have suggested that the rapid turnover may in some way be related to a control mechanism for PS II function. Alternatively, Arntzen et al. (1) hypothesized that the unusually high rate of turnover of the QB protein in the light could be a natural consequence of its in vivo function as the secondary acceptor of PS II. (This enzymatic function involves the stabilization of reactive quinone anions in the formation of the reduced ...

A loss of electron transport capacity in chloroplast membranes was induced by high-light intensities (photoinhibition). The primary site of inhibition was at the reducing side of photosystem II (PSII) with little damage to the oxidiiing side or to the reaction center core of PSII. Addition of herbicides (atrazine or diuron) partially protected the membrane from photoinhibition; these compounds displace the bound plastoquinone (designated as QB), which functions as the secondary electron acceptor on the reducing side of PSII.Loss of function of the 32-kilodalton QB apoprotein was demonstrated by a loss of binding sites for [14C]atrazine. We suggest that quinone anions, which may interact with molecular oxygen to produce an oxygen radical, selectively damage the apoprotein of the secondary acceptor of PSII, thus rendering it inactive and thereby blocking photosynthetic electron flow under conditions of high photon flux densities.Exposure of leaves or chloroplasts of higher plants to supraoptimal light intensities results in a loss of photosynthetic activity (1). This phenomenon is referred to as photoinhibition (reviewed in refs. 2 and 3). Photoinhibitory damage to chloroplast membranes (thylakoids) involves inactivation of photosystem II (PSII), but the molecular mechanism damage has not been established.A thylakoid polypeptide of =32 kDa is synthesized at rates equal to or greater than the most abundant chloroplast proteins, yet it remains as a minor membrane constituent; this has been attributed to a rapid turnover of the polypeptide (4,5). The rate of turnover was recently found to increase in plants grown at increasing light intensities (4). The rapidly turned-over 32-kDa polypeptide is identical to a triazine receptor protein (6) that is a structural component of the PSII core complex. Since atrazine (a commonly used triazine herbicide) is a competitive inhibitor of the binding of plastoquinone (PQ) analogues (7), the rapidly turned-over, herbicidebinding protein is identified as the apoprotein of the secondary electron transport carrier on the reducing side of PSII. The redox cofactor associated with this carrier is a bound PQ designated as QB; the 32-kDa polypeptide is therefore called the QB protein (8).In this manuscript we demonstrate a correlation between photoinhibition (PSII inactivation) and the rate of turnover of the QB protein.MATERIALS payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.(Chl) per ml in the growth medium in clear tubes (3 cm in diameter) and immersed in a temperature-regulated, transparent water bath for photoinhibition experiments. A highintensity quartz-halogen lamp provided a photoinhibitory

The light-harvesting and energy-transducing functions of the chloroplast are performed within an intricate lamellar system of membranes, called thylakoid membranes, which are differentiated into granum and stroma lamellar domains. Using dualaxis electron microscope tomography, we determined the three-dimensional organization of the chloroplast thylakoid membranes within cryo-immobilized, freeze-substituted lettuce (Lactuca sativa) leaves. We found that the grana are built of repeating units that consist of paired layers formed by bifurcations of stroma lamellar sheets, which fuse within the granum body. These units are rotated relative to each other around the axis of the granum cylinder. One of the layers that makes up the pair bends upwards at its edge and fuses with the layer above it, whereas the other layer bends in the opposite direction and merges with the layer below. As a result, each unit in the granum is directly connected to its neighbors as well as to the surrounding stroma lamellae. This highly connected morphology has important consequences for the formation and function of the thylakoid membranes as well as for their stacking/unstacking response to variations in light conditions.