Somatic embryogenesis requires auxin and establishment of the shoot apical meristem (SAM). WUSCHEL (WUS) is critical for stem cell fate determination in the SAM of higher plants. However, regulation of WUS expression by auxin during somatic embryogenesis is poorly understood. Here, we show that expression of several regulatory genes important in zygotic embryogenesis were up-regulated during somatic embryogenesis of Arabidopsis. Interestingly, WUS expression was induced within the embryonic callus at a time when somatic embryos could not be identified morphologically or molecularly. CorrectWUS expression, regulated by a defined critical level of exogenous auxin, is essential for somatic embryo induction. Furthermore, it was found that auxin gradients were established in specific regions that could then give rise to somatic embryos. The establishment of auxin gradients was correlated with the induced WUS expression. Moreover, the auxin gradients appear to activate PIN1 polar localization within the embryonic callus. Polarized PIN1 is probably responsible for the observed polar auxin transport and auxin accumulation in the SAM and somatic embryo. Suppression of WUS and PIN1 indicated that both genes are necessary for embryo induction through their regulation of downstream gene expression. Our results reveal that establishment of auxin gradients and PIN1-mediated polar auxin transport are essential for WUS induction and somatic embryogenesis. This study sheds new light on how auxin regulates stem cell formation during somatic embryogenesis.

Homeobox genes are master regulatory genes that specify the body plan and control development of many eukaryotic organisms, including plants. We isolated and characterized a cDNA designated ATML1 (for Arabidopsis thaliana meristem L1 layer) that encodes a novel homeodomain protein. The ATML1 protein shares high sequence homology inside and outside of the homeodomain with both the Phalaenopsis O39 and the Arabidopsis GLABRA2 (GL2) homeodomain proteins, which together define a new class of plant homeodomain-containing proteins, designated HD-GL2. The ATML1 gene was first expressed in the apical cell after the first asymmetric division of the zygote and continued to be expressed in all proembryo cells until the eight-cell stage. In the 16-cell proembryo, the ATML1 gene showed a distinct pattern of expression, with its mRNA becoming restricted to the protoderm. In the torpedo stage of embryo development, ATML1 mRNA disappeared altogether but reappeared later only in the L1 layer of the shoot apical meristem in the mature embryo. After germination, this L1 layer-specific pattern of expression was maintained in the vegetative shoot apical meristem, inflorescence, and floral meristems, as well as in the young floral organ primordia. Finally, ATML1 mRNA accumulated in the protoderm of the ovule primordia and integuments and gradually became restricted in its expression to the endothelium surrounding the embryo sac. We propose that ATML1 may be involved in setting up morphogenetic boundaries of positional information necessary for controlling cell specification and pattern formation. In addition, ATML1 provides an early molecular marker for the establishment of both apical-basal and radial patterns during plant embryogenesis.

Pollination regulates a syndrome of developmental responses that contributes to successful sexual reproduction in higher plants. Pollination-regulated developmental events collectively prepare the flower for fertilization and embryogenesis while bringing about the loss of floral organs that have completed their function in pollen dispersal and reception. Components of this process include changes in flower pigmentation, senescence and abscission of floral organs, growth and development of the ovary, and, in certain cases, pollination also triggers ovule and female gametophyte development in anticipation of fertilization. Pollination-regulated development is initiated by the primary pollination event at the stigma surface, but because developmental processes occur in distal floral organs, the activity of interorgan signals that amplify and transmit the primary pollination signal to floral organs is implicated. Interorgan signaling and signal amplification involves the regulation of ethylene biosynthetic gene expression and interorgan transport of hormones and their precursors. The coordination of pollination- regulated flower development including gametophyte, embryo, and ovary development; pollination signaling; the molecular regulation of ethylene biosynthesis; and interorgan communication are presented.

Pollination initiates a syndrome of developmental events that contribute to successful reproduction, including perianth senescence, changes in pigmentation, and ovule differentiation in preparation for impending fertilization. In orchid flowers, initiation of each of these processes in distinct floral organs is strictly and coordinately controlled by pollination, thus providing a unique opportunity to study the signals that coordinate interorgan postpollination development. Because ethylene has been implicated in contributing to regulation of severa1 aspects of postpollination development, we focused on determining the expression of its biosynthetic genes and their possible role in regulation. The abundance of mRNA encoding both 1-aminocyclopropane-l-carboxylic acid (ACC) synthase and ACC oxidase in the stigma, ovary, and labellum was found to be coordinately regulated by emasculation, auxin, and ethylene. Although petals contribute up to 26% of total flower ethylene and accumulate high levels of ACC oxidase mRNA and activity following pollination, no ACC synthase mRNA or activity was detected in this tissue. Together, these results support a model of interorgan regulation of postpollination development that depends on pollination-stimulated accumulation of mRNA encoding ethylene biosynthetic enzymes in a developmentally regulated and tissue-specific manner. This model relies on the translocation of a soluble hormone precursor, ACC, rather than on the translocation of the hormone itself. In this way, ACC serves to actuate the response already initiated by ethylene perceived by other parts of the flower. Thus, ACC may function as a secondary transmissible signal that coordinates postpollination development in diverse floral organs.

The indoleamine melatonin, a well-known animal chemical, has been identified in extracts from several plant species. The function of melatonin in plants is unknown. Two major functions of melatonin in animals are dark signaling and antioxidant protection. Fruit ripening was used as a model physiological process that involves changes in the oxidative status of an organ. Tomato fruits at various stages of ripeness were sampled. Morning glory (Pharbitis nil Choisy, cv. Violet) and tomato (Lycopersicon esculentum Mill., cv. T5 and Castlemart) organs were collected throughout a light/dark cycle to determine whether melatonin levels increased during the night. No consistent evidence was found that melatonin increased significantly in organs of these plants during the night, as it does in many animals. The melatonin content of the fruits generally increased during ripening up to the mature ripe stage and thereafter as the fruit became over ripe.

Sex determination in cucumber (Cucumis safivus 1.) is controlled largely by three genes: F, m, and a. l h e F and m loci interact to produce monoecious (M-f_) or gynoecious (M-F-) sex phenotypes. Ethylene and factors that induce ethylene biosynthesis, such as 1 -aminocyclopropane-1 -carboxylate (ACC) and auxin, also enhance female sex expression. A genomic sequence (CS-ACSI) encoding ACC synthase was amplified from genomic DNA by a polymerase chain reaction using degenerate oligonucleotide primers. Expression of CS-ACSI is induced by auxin, but not by ACC, in wounded and intact shoot apices. Southern blot hybridization analysis of near-isogenic gynoecious (MMFF) and monoecious (MMff) lines derived from diverse genetic backgrounds revealed the existence of an additional ACC synthase (CS-ACSIC) genomic sequence in the gynoecious lines. Sex phenotype analysis of a segregating F, population detected a 100% correlation between the CS-ACSIG marker and the presence of the F locus. The CS-ACSIG gene is located in linkage group B coincident with the F locus, and in the population tested there was no recombination between the CS-ACSIG gene and the F locus. Collectively, these data suggest that CS-ACSIC is closely linked to the F locus and may play a pivotal role in the determination of sex in cucumber flowers.Sex determination in flowering plants is a developmentally regulated process that has been the topic of much research (Dellaporta and Calderon-Urrea, 1993;Grant et al., 1994). Dioecious and monoecious species of flowering plants present an excellent opportunity to study the diverse, developmental pathways that give rise to unisexual flowers. In monoecious wild-type cucumber (Cucumis sativus L. var sativus), flowers are produced in a preset, developmental sequence along the main stem, with a first phase of staminate flowers, followed by a mixed phase of staminate and pistillate flowers, and terminated by a pistillate flower phase (Galun, 1961;Shifriss, 1961). The develop-

Microsomal membes isolated from red beet (Beta ulgatns L.) storage tissue were found to contain high levels of ioophore-stimulated ATPase activity. The distribution of this ATPase activity on a continuous sucrose gradient showed a low density peak (1.09 grams per cubic centimeter) that was stimulated over 400% by gramicdin and coincded with a peak of NO3-sensitive ATPase activity. At In the last few years several groups have reported the existence of an anion-sensitive, proton-translocating ATPase in a microsomal membrane fraction from plant cells (3,8,11,13,16,21). There have also been several reports describing an ATPase associated with isolated plant vacuoles (1,15,(26)(27)(28) been suggested that this anion-sensitive H+-ATPase is of tonoplast origin (11,16). In order to confirm a tonoplast origin for the anion-sensitive H+-ATPase, this study used membrane isolation procedures and H+-transport assays developed for studying the corn root, anion-sensitive H+-ATPase (3, 1 1) to identify a similar ATPase in red beet microsomal membranes. This tissue was chosen for several reasons: (a) Briskin and Poole (7) have recently shown that microsomal membranes can be readily isolated from this tissue in high yields and substantially free of nonspecific phosphatase activity, (b) intact vacuoles can be easily isolated from this tissue (14) and, (c) a tonoplast ATPase has already been characterized in this tissue (1, 28). These advantages allowed the identification ofan anion-sensitive H+-translocating ATPase in red beet microsomal membranes which could be compared directly to the ATPase activity associated with isolated vacuoles. The results support a tonoplast origin for the anionsensitive H+-ATPase and further identify a second, vanadatesensitive H+-translocating ATPase in the red beet microsomal membranes, which is presumably of plasma membrane origin. MATERIALS AND METHODSPlant Material. Red beets (Beta vulgaris L.) were obtained commercially. Care was taken to purchase beets with fresh leafy tops to ensure that they were freshly harvested. The tops were removed and the storage tissue (swollen hypocotyl) stored at 4°C until use, but not longer than 1 month.Membrane Preparation. Microsomal membranes were prepared essentially as described by Briskin and Poole (7)

The differentiation and development of ovules in orchid flowers are pollination dependent. To define the developmental signals and timing of critical events associated with ovule differentiation, we have examined factors that regulate the initial events in megasporogenesis and female gametophyte development and characterized its progression toward maturity and fertilization. Two days after pollination, ovary wall epidermal cells begin to elongate and form hair cells; this is the earliest visible morphological change, and it occurs at least 3 days prior to pollen germination, indicating that signals associated with pollination itself trigger these early events. The effects of inhibitors of ethylene biosynthesis on early morphological changes indicated that ethylene, in the presence of auxin, is required to initiate ovary development and, indirectly, subsequent ovule differentiation. Surprisingly, pollen germination and growth were also strongly inhibited by inhibitors of ethylene biosynthesis, indicating that male gametophyte development is also regulated by ethylene. Detailed characterization of the development of both the female and male gametophyte in pollinated orchid flowers indicated that pollen tubes entered the ovary and grew along the ovary wall for 10 to 35 days, at which time growth was arrested. Approximately 40 days after pollination, coincident with ovule differentiation as indicated by the presence of a single archesporial cell, the direction of pollen tube growth became redirected toward the ovule, suggesting a chemical signaling between the developing ovule and male gametophyte. Taken together, these results indicate that both auxin and ethylene contribute to the regulation of both ovary and ovule development and to the coordination of development of male and female gametophytes.