The cuticle, covering the surface of all primary plant organs, plays important roles in plant development and protection against the biotic and abiotic environment. In contrast to vegetative organs, very little molecular information has been obtained regarding the surfaces of reproductive organs such as fleshy fruit. To broaden our knowledge related to fruit surface, comparative transcriptome and metabolome analyses were carried out on peel and flesh tissues during tomato (Solanum lycopersicum) fruit development. Out of 574 peel-associated transcripts, 17% were classified as putatively belonging to metabolic pathways generating cuticular components, such as wax, cutin, and phenylpropanoids. Orthologs of the Arabidopsis (Arabidopsis thaliana) SHINE2 and MIXTA-LIKE regulatory factors, activating cutin and wax biosynthesis and fruit epidermal cell differentiation, respectively, were also predominantly expressed in the peel. Ultra-performance liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer and gas chromatography-mass spectrometry using a flame ionization detector identified 100 metabolites that are enriched in the peel tissue during development. These included flavonoids, glycoalkaloids, and amyrin-type pentacyclic triterpenoids as well as polar metabolites associated with cuticle and cell wall metabolism and protection against photooxidative stress. Combined results at both transcript and metabolite levels revealed that the formation of cuticular lipids precedes phenylpropanoid and flavonoid biosynthesis. Expression patterns of reporter genes driven by the upstream region of the wax-associated SlCER6 gene indicated progressive activity of this wax biosynthetic gene in both fruit exocarp and endocarp. Peel-associated genes identified in our study, together with comparative analysis of genes enriched in surface tissues of various other plant species, establish a springboard for future investigations of plant surface biology.

In flowering plants, two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus, which replicates to generate the endosperm, a tissue that supports embryo development. The FERTILIZATION-INDEPENDENT ENDOSPERM ( FIE ) and MEDEA ( MEA ) genes encode WD and SET domain polycomb proteins, respectively. In the absence of fertilization, a female gametophyte with a loss-of-function fie or mea allele initiates endosperm development without fertilization. fie and mea mutations also cause parent-of-origin effects, in which the wild-type maternal allele is essential and the paternal allele is dispensable for seed viability. Here, we show that FIE and MEA polycomb proteins interact physically, suggesting that the molecular partnership of WD and SET domain polycomb proteins has been conserved during the evolution of flowering plants. The overlapping expression patterns of FIE and MEA are consistent with their suppression of gene transcription and endosperm development in the central cell as well as their control of seed development after fertilization. Although FIE and MEA interact, differences in maternal versus paternal patterns of expression, as well as the effect of a recessive mutation in the DECREASE IN DNA METHYLATION1 ( DDM1 ) gene on mutant allele transmission, indicate that fie and mea mutations cause parent-of-origin effects on seed development by distinct mechanisms. INTRODUCTIONFlowering plant reproduction involves fertilization of two cells (reviewed in van Went and Willemse, 1984). Within the Arabidopsis ovule, the female gametophyte consists of an egg cell and two synergid cells at the micropylar end, a central cell in the middle, and three antipodal cells at the chalazal end. All are haploid except for the central cell, which contains two polar nuclei that fuse to form a diploid nucleus. Reproduction is initiated when an entering pollen tube discharges two genetically identical haploid sperm cells. Fertilization of the egg generates the diploid embryo, which passes through morphologically defined stages (globular, heart, torpedo, walking stick, early maturation, and maturation) (Goldberg et al., 1994;Jurgens and Mayer, 1994). During embryo development, two organ systems (axis and cotyledon) and three tissue layers (protoderm, procambium, and ground meristem) are specified (Lindsey and Topping, 1993;Jurgens, 1994;Meinke, 1994).Fertilization of the central cell generates the triploid endosperm, for which the pattern of development differs dramatically from that of the embryo. Arabidopsis endosperm development is characteristic of nuclear endosperm development in angiosperms (Mansfield and Briarty, 1990a;Webb and Gunning, 1991;Berger, 1999;Brown et al., 1999). The Arabidopsis primary endosperm nucleus replicates without cytokinesis to form a syncytium of nuclear-cytoplasmic domains that migrate to the periphery of the expanding central cell (Brown et al., 1999). When the embryo is at the globular/heart transi...

phox -independent NADPH oxidase activation by prenylated Rac1 is inhibited by Rho GDP dissociation inhibitor and by phosphatidylcholine vesicles, both competing with membrane for prenylated Rac1. We conclude that, in vitro, targeting of Rac to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly, suggesting that the principal or, possibly, the only role of Rac is to recruit cytosolic p67 phox to the membrane environment, to be followed by the interaction of p67 phox with cytochrome b 559 .The production of reactive oxygen species represents the major microbicidal mechanism of professional phagocytes. The primordial oxygen radical is superoxide (O 2 . ), 1 and it is produced by the NADPH-derived one-electron reduction of molecular oxygen, by an enzyme complex known as the NADPH oxidase (referred to here as "oxidase"; reviewed in Refs. 1 and 2 phox , and Rac to a critical concentration of an anionic amphiphile (3, 4).Rac1 or -2 is absolutely required for oxidase assembly in the amphiphile-activated cell-free system (5, 6). It is also clearly involved in O 2. production in intact phagocytes, as shown by the inhibitory effect of Rac antisense oligonucleotides (7) and by selective defects in O 2 . production by neutrophils of Rac2-deficient mice (8) and of a patient with an inhibitory mutation in Rac2 (9). It is the consensus opinion that, in the course of oxidase assembly, Rac is translocated to the membrane (10), although lack of translocation (11) or the lack of relevance of translocation to assembly (12) was also claimed. In the cytosol, Rac is found as a C-terminally prenylated (geranylgeranylated) protein (13), forming a heterodimer with the regulatory protein GDP dissociation inhibitor for Rho (Rho GDI) (5). Dissociation of prenylated Rac from Rho GDI was proposed to be an obligatory step preceding translocation of Rac from the cytosol to the membrane (14 -16). The role of Rac in oxidase assembly was studied extensively in the semirecombinant amphiphile-activated cell-free system (4). Both nonprenylated (5, 17) and prenylated (17-19) Rac are capable of supporting oxidase activation in vitro. It was recently suggested (20) that, whereas membrane association of prenylated Rac (Rac1 or -2) is mediated principally by hydrophobic interaction between the geranylgeranyl tail and membrane lipids, nonprenylated Rac1 binds by electrostatic interaction between a C-terminal polybasic region (21) and negative charges on the membrane. We intended to test the hypothesis that binding of Rac to the membrane is a crucial event in the initiation of oxidase assembly. We found indeed that prenylated, but not nonprenylated, Rac1 initiates oxidase assembly and NADPH-dependent O 2 .production in a cell-free system, containing phagocyte membranes and p67 phox , in the absence of an amphiphilic activator

The elicitation of an oxidative burst in phagocytes rests on the assembly of a multicomponental complex (NADPH oxidase) consisting of a membrane-associated flavocytochrome (cytochrome b 559 ), representing the redox element responsible for the NADPH-dependent reduction of oxygen to superoxide (O 2 . ), two cytosolic components (p47 phox , p67 phox ), and the small GTPase Rac (1 or 2). We found that 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), an irreversible serine protease inhibitor, prevented the elicitation of O 2 . production in intact macrophages and the amphiphile-dependent activation of NADPH oxidase in a cell-free system, consisting of solubilized membrane or purified cytochrome b 559 combined with total cytosol or a mixture of recombinant p47 phox , p67 phox , and Rac1. AEBSF acted at the activation step and did not interfere with the ensuing electron flow. It did not scavenge oxygen radicals and did not affect assay reagents. Five other serine protease inhibitors (three irreversible and two reversible) were found to lack an inhibitory effect on cell-free activation of NADPH oxidase. A structure-function study of AEBSF analogues demonstrated that the presence of a sulfonyl fluoride group was essential for inhibitory activity and that compounds containing an aminoalkylbenzene moiety were more active than amidinobenzene derivatives. Exposure of the membrane fraction or of purified cytochrome b 559 , but not of cytosol or recombinant cytosolic components, to AEBSF, in the presence of a critical concentration of the activating amphiphile lithium dodecyl sulfate, resulted in a marked impairment of their ability to support cell-free NADPH oxidase activation upon complementation with untreated cytosol or cytosolic components. Kinetic analysis of the effect of varying the concentration of each of the three cytosolic components on the inhibitory potency of AEBSF indicated that this was inversely related to the concentrations of p47 phox and, to a lesser degree, p67phox . AEBSF also prevented the amphiphile-elicited translocation of p47 phox and p67 phox to the membrane. These results are interpreted as indicating that AEBSF interferes with the binding of p47 phox and/or p67 phox to cytochrome b 559 , probably by a direct effect on cytochrome b 559 .The production of reactive oxygen radicals represents the major microbicidal mechanism of phagocytes (1). Oxygen radicals are also generated, in lesser amounts, by some nonphagocytic cells, sharing the enzymatic machinery characteristic of phagocytes (2), and, under certain conditions, by plant cells (3). Interest in reactive oxygen species has also been stimulated by accumulating evidence for their involvement in the pathogenesis of diseases, ranging from respiratory distress syndrome to ischemia-reperfusion injury in several organs (4).The primordial oxygen radical produced by phagocytes is superoxide (O 2 . ). 1 It is generated, in response to appropriate stimuli, by NADPH-derived one-electron reduction of molecular oxygen, a reaction catalyzed by a membr...

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Very short exposures of embryogenic calli of Vitis vinifera cv. Superior Seedless grape plants to diluted cultures of Agrobacterium resulted in plant tissue necrosis and subsequent cell death. Antibiotics used for Agrobacterium elimination or as plant selectable markers were not responsible for this necrotic response. Rather, cell death seemed to be oxygen-dependent and correlated with elevated levels of peroxides. Therefore, we studied the effects on necrosis of various combinations of antioxidants during and after grape-Agrobacterium cocultivation. The combination of polyvinylpolypyrrolidone and dithiothreitol was found to improve plant viability. Tissue necrosis was completely inhibited by these antioxidants while Agrobacterium virulence was not effected. These treatments enabled the recovery of stable transgenic grape plants resistant to hygromycin.

The small molecular weight GTP-binding protein Rac (1 or 2) is an obligatory participant in the activation of the superoxide-generating NADPH oxidase. Active NADPH oxidase can be reconstituted in a cell-free system, consisting of phagocyte-derived membranes, containing cytochrome b559, and the recombinant cytosolic proteins p47-phox, p67-phox, and Rac, supplemented with an anionic amphiphile as an activator. The cell-free system was used before for the analysis of structural requirements of individual components participating in the assembly of NADPH oxidase. In earlier work, we mapped four previously unidentified domains in Rac1, encompassing residues 73-81 (a), 103-107 (b), 123-133 (c), and 163-169 (d), as important for cell-free NADPH oxidase activation. The domains were defined by assessing the activation inhibitory effect of a series of overlapping peptides, spanning the entire length of Rac1 [Joseph, G., and Pick, E. (1995) J. Biol. Chem. 270, 29079-29082]. We now used the construction of Rac1/H-Ras chimeras, domain deletion, and point mutations, to ascertain the functional relevance of three domains (b, c, and d) predicted by "peptide walking" and to determine the importance of specific residues within these domains. This methodology firmly establishes the involvement of domains b and d in the activation of NADPH oxidase by Rac1 and identifies H103 and K166, respectively, as residues critical for the effector function of these two domains. The functional significance of domain c (insert region) could not be confirmed, as shown by the minor effect of deleting this domain on NADPH oxidase activation. Analysis of the three-dimensional structure of Rac1 reveals that residues H103 and K166 are exposed on the surface of the molecule. Modeling of the activity-impairing point mutations suggests that the effect on the ability to activate NADPH oxidase depends on the side chains of the mutated amino acids and not on changes in the global structure of the protein. In conclusion, we demonstrate the existence of two novel effector sites in Rac1, necessary for supporting NADPH oxidase activation, supplementing the canonical N-terminal effector region.

The superoxide generating NADPH oxidase of phagocytes consists, in resting cells, of a membrane-associated electron transporting flavocytochrome (cytochrome b 559 ) and four cytosolic proteins as follows: p47 phox , p67 phox , p40 phox , and the small GTPase, Rac(1 or 2). Activation of the oxidase is consequent to the assembly of a membrane-localized multimolecular complex consisting of cytochrome b 559 and the cytosolic components. We used "peptide walking" (Joseph, G., and Pick, E. (1995) J. Biol. Chem. 270, 29079 -29082) for mapping domains in the amino acid sequence of p47 phox participating in the molecular events leading to the activation of NADPH oxidase. Ninety-five overlapping pentadecapeptides, with a four-residue offset between neighboring peptides, spanning the complete p47 phox sequence, were tested for the ability to inhibit NADPH oxidase activation in a cell-free system. This consisted of solubilized macrophage membranes, recombinant p47 phox , p67 phox , and Rac1, and lithium dodecyl sulfate, as the activator. Eight functional domains were identified and labeled a-h. phox , and domains f and h, in the C-terminal half, represent newly identified entities, for which there is no earlier experimental evidence of involvement in NADPH oxidase activation. "Peptide walking" was also applied to the identification of domains in p47 phox mediating binding to p67 phox . This was done by quantifying, by enzyme-linked immunosorbent assay, the binding of p67 phox , in solution, to a series of 95 overlapping biotinylated p47 phox peptides, attached to streptavidin-coated 96-well plates. A single proline-rich domain (residues 357-371) was found to bind p67 phox in the absence and presence of lithium dodecyl sulfate.Phagocytic cells produce, in response to appropriate stimuli, a variety of oxygen-derived toxic radicals, all of which are derived from superoxide (O 2 . ). 1 O 2 . is generated by the NADPHderived one-electron reduction of molecular oxygen, catalyzed by a membrane-associated heterodimeric flavocytochrome (cytochrome b 559 ), composed of a 91-kDa glycoprotein (gp91 phox ) and a 22-kDa protein (p22 phox ), and incorporating two redox centers, FAD and two hemes (reviewed in Refs. 1-3). The conversion of cytochrome b 559 from the resting to the activated state is, most likely, the result of a conformational change leading to a high turnover electron flow from NADPH to oxygen, through the redox centers. This change in conformation is brought about by protein-protein interactions involving the two cytochrome b 559 subunits and four cytosolic regulatory proteins, p47phox , p67 phox , p40 phox , and the small GTPase Rac(1 or 2) (reviewed in Refs. 4 and 5). It is commonly assumed that activation leads to the formation of a membrane-localized multimolecular structure, known as the NADPH oxidase complex. The physical expression of this, in the intact cell, is the translocation of parts of p47 phox and p67 phox to the plasma membrane (6) associated with the phosphorylation of p47 phox at multiple sites (7). Whe...