Sharman D. O'Neill

Identification of a meristem L1 layer-specific gene in Arabidopsis that is expressed during embryonic pattern formation and defines a new class of homeobox genes.

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.

Ovary and Gametophyte Development Are Coordinately Regulated by Auxin and Ethylene following Pollination.

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.

Interorgan regulation of ethylene biosynthetic genes by pollination.

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.

Identification of a 1-aminocyclopropane-1-carboxylic acid synthase gene linked to the female (F) locus that enhances female sex expression in cucumber.

Sex determination in cucumber (Cucumis sativus L.) is controlled largely by three genes: F, m, and a. The 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-ACS1) encoding ACC synthase was amplified from genomic DNA by a polymerase chain reaction using degenerate oligonucleotide primers. Expression of CS-ACS1 is induced by auxin, but not by ACC, in wounded and intact shoot apices. Southern blot hybridization analysis of near-isogneic gynoecious (MMFF) and monoecious (MMff) lines derived from diverse genetic backgrounds revealed the existence of an additional ACC synthase (CS-ACS1G) genomic sequence in the gynoecious lines. Sex phenotype analysis of a segregating F2 population detected a 100% correlation between the CS-ACS1G marker and the presence of the F locus. The CS-ACS1G gene is located in linkage group B coincident with the F locus, and in the population tested there was no recombination between the CS-ACS1G gene and the F locus. Collectively, these data suggest that CS-ACS1G is closely linked to the F locus and may play a pivotal role in the determination of sex in cucumber flowers.

Melatonin in plant 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.

Ovule development: identification of stage-specific and tissue-specific cDNAs.

A differential screening approach was used to identify seven ovule-specific cDNAs representing genes that are expressed in a stage-specific manner during ovule development. The Phalaenopsis orchid takes 80 days to complete the sequence of ovule developmental events, making it a good system to isolate stage-specific ovule genes. We constructed cDNA libraries from orchid ovule tissue during archesporial cell differentiation, megasporocyte formation, and the transition to meiosis, as well as during the final mitotic divisions of female gametophyte development. RNA gel blot hybridization analysis revealed that four clones were stage specific and expressed solely in ovule tissue, whereas one clone was specific to pollen tubes. Two other clones were not ovule specific. Sequence analysis and in situ hybridization revealed the identities and domain of expression of several of the cDNAs. O39 encodes a putative homeobox transcription factor that is expressed early in the differentiation of the ovule primordium; O40 encodes a cytochrome P450 monooxygenase (CYP78A2) that is pollen tube specific. O108 encodes a protein of unknown function that is expressed exclusively in the outer layer of the outer integument and in the female gametophyte of mature ovules. O126 encodes a glycine-rich protein that is expressed in mature ovules, and O141 encodes a cysteine proteinase that is expressed in the outer integument of ovules during seed formation. Sequences homologous to these ovule clones can now be isolated from other organisms, and this should facilitate their functional characterization.

Putative regulatory molecules in plants: evaluating melatonin

Numerous classes of chemicals have been considered as regulators of various aspects of plant growth and development. In evaluating these putative regulatory molecules, plant biologists have encountered a number of challenges, including: the problem of quantifying substances present at trace levels in extremely complex mixtures; difficulty in obtaining and interpreting phenotypic responses to exogenous applications; and, until recently, the inability to selectively alter endogenous levels of these substances. Melatonin (N‐acetyl 5‐methoxytryptamine), a methoxylated indoleamine, is a potential regulatory molecule found in plants. Although no specific phenotype is currently associated with melatonin or its analogs in higher plants, it has important and unique biological activity in many other taxa, from algae to primates. In these organisms, melatonin functions as a night signal, coordinating responses to diurnal and photoperiodic environmental cues. We assess the process by which melatonin has been evaluated in plants so far and find that many of the methods for melatonin analysis, which have been adopted from animal studies, are inappropriate for use with plant materials. Thus, despite some interesting preliminary reports, research supporting the case for melatonin as a plant regulator is still in its infancy.

Arabidopsis SKP1, a homologue of a cell cycle regulator gene, is predominantly expressed in meristematic cells

Abstract. The yeast SKP1 gene and its human homolog p19skp1 encode a kinetochore protein required for cell cycle progression at both the DNA synthesis and mitosis phases of the cell cycle. In orchids we identified a cDNA (O108) that is expressed in early stages of ovule development and is homologous to the yeast SKP1. Based on the orchid O108 cDNA clone, we identified and characterized an Arabidopsis thaliana (L.) Heynh. cDNA designated ATskp1 that also has high sequence similarity to yeast SKP1. The Arabidopsis ATskp1 is a single-copy gene that mapped to chromosome 1. The expression of the ATskp1 gene was highly correlated with meristem activity in that its mRNA accumulated in all of the plant meristems including the vegetative shoot meristem, inflorescence and floral meristems, root meristem, and in the leaf and floral organ primordia. In addition, ATskp1 was also highly expressed in the dividing cells of the developing embryo, and in other cells that become multinucleate or undergo endoreplication events such as the endosperm free nuclei, the tapetum and the endothelium. Based on its spatial pattern of expression, ATskp1 is a marker for cells undergoing division and may be required for meristem activity.

Molecular genetic analysis of chalcone synthase in Lycopersicon esculentum and an anthocyanin-deficient mutant.

SummaryTwelve loci have previously been identified in tomato (Lycopersicon esculentum) that control the intensity and distribution of anthocyanin pigmentation; these are useful genetic markers because they encode phenotypes that are readily visualized in the hypocotyls of emerging seedlings. In order to obtain molecular probes for tomato anthocyanin biosynthesis genes, we isolated two cDNAs which encode chalcone synthase (CHS), one of the key enzymes in anthocyanin biosynthesis, from a tomato hypocotyl cDNA library. By comparing their nucleic acid sequences, we determined that the two CHS cDNAs have an overall similarity of 76% at the nucleotide level and 88% at the amino acid level. We identified hybridization conditions that would distinguish the two clones and by Northern analysis showed that 1.5 kb mRNA species corresponding to each cDNA were expressed in cotyledons, hypocotyls and leaves of wild-type seedlings. Hybridization of the cDNAs at low stringency to genomic blots indicated that in tomato, CHS genes comprise a family of at least three individual members. The two genes that encode the CHS cDNAs were then placed onto the tomato genetic map at unique loci by restriction fragment length polymorphism mapping. We also assayed the activity of CHS and another enzyme in the anthocyanin pathway, flavone 3-hydroxylase, in hypocotyl extracts of wild-type tomato and a number of anthocyanin-deficient mutants. Five mutants had reduced CHS activity when compared to the wildtype controls. Of these, three were also reduce in flavone 3-hydroxylase activity, suggesting a regulatory role for these loci. The other two mutants were preferentially reduced in CHS activity, suggesting a more specific role for these loci in CHS expression.

Characterization of the gene encoding dihydroflavonol 4-reductase in tomato

A cDNA clone (DFR) encoding dihydroflavonol 4-reductase was identified from tomato hypocotyls. Nucleotide and amino acid sequence comparisons to Petunia hybrida, Antirrhinum majus and Zea mays DFR sequences confirmed that the cDNA encodes the structural DFR gene. In tomato, the DFR sequence appeared to be present as a single gene and mapped to a region on chromosome 2 near two loci affecting anthocyanin pigmentation, are and aw. DFR was expressed in both leaf and hypocotyl tissue. Sequencing data from two DFR cDNA clones indicated there are alternative polyadenylation sites on DFR.