Biomedical subjects

R S Quatrano

Publications and source records attributed to R S Quatrano.

At least 19 recordsLinked to original sources

PubMed2000

The genes ABI1 and ABI2 are involved in abscisic acid- and drought-inducible expression of the Daucus carota L. Dc3 promoter in guard cells of transgenic Arabidopsis thaliana (L.) Heynh.

The ABA INSENSITIVE1 (ABI1) and ABI2 genes encode homologous type-2C protein phosphatases with redundant yet distinct functions in abscisic acid (ABA) responses. Results from Northern blot analysis showed that ABA- and mannitol-inducible expression of the COR47 and COR78/LTI78/RD29A (COR78) genes was more impaired in the abi2 mutant of Arabidopsis thaliana (L.) Heynh than in the abi1 mutant. Furthermore, ABA-plus-mannitol treatments were additive towards COR47 gene expression; however, the ABA-deficient aba1 mutant showed reduced COR expression relative to the wild type in response to mannitol and ABA-plus-mannitol treatments. These results support the notion that drought- and ABA-signalling pathways are separate yet overlapping. To facilitate quantitative analysis of the genetic control of tissue-specific ABA- and desiccation-response pathways, we analyzed ABA- and mannitol-inducible expression of a carrot (Daucus carota L.) Dc3 promoter:uidA (beta-glucuronidase; GUS) chimaeric reporter (Dc3-GUS) in transgenic wild-type, ABA-deficient aba1, and ABA-insensitive abi1 and abi2 mutants. The Dc3 promoter directed ABA- and mannitol-inducible GUS expression in Arabidopsis guard cells and the two treatments were additive. The aba1, abi1, and abi2 mutant genotypes had reduced GUS expression in guard cells of cotyledons in response to mannitol, whereas abi1 and abi2 mutants were reduced in ABA-inducible GUS expression, consistent with overlapping ABA- and drought-response pathways. Quantitative fluorometric GUS assays of leaf extracts showed that abi2 mutants responded less to exogenous ABA than did abi1 mutants, and abi2 mutants responded more to mannitol than did abi1 mutants. We conclude that Dc3-GUS Arabidopsis is a tractable system in which to study tissue-specific ABA and drought signalling and suggest that ABI2 functions predominantly over ABI1 in COR78 and COR47 gene expression and guard-cell Dc3-GUS expression.

Abscisic Acid↗

PubMed2000

Polarity: the role of localized secretion.

Studies of single cells from brown algae suggest that localized secretions stabilize the polar axis resulting in an asymmetry in the cell wall. This cortical asymmetry appears to play a role in orienting the plane of cell division and in determining the different fates of the resulting daughter cells. Recent studies indicate that similar processes may operate in seed plants.

Cell Division↗

PubMed2000

Maize ROP7 GTPase contains a unique, CaaX box-independent plasma membrane targeting signal.

Signals in the carboxy-terminal hypervariable region (HVR) of Rho and Ras GTPases target these proteins to specific membrane compartments, where they function in signal transduction. ROP6 and ROP7 are closely related maize Rops (a plant-specific Rho subgroup) that share HVR sequences divergent from other Rho HVRs. Both ROPs terminate in CAA, instead of the consensus C-terminal CaaX motif required for membrane association of all characterized Ras and Rho GTPases. The ROP6/7 HVR contains two additional cysteines, potential sites for post-translational modification that leads to membrane association; one is in an internal CaaX motif, which would be at the C-terminus if the final intron in both genes were not removed. Transient expression of a GFP-ROP7 fusion revealed its near-total association with the plasma membrane (PM). Furthermore, the ROP7 HVR is sufficient to target GFP to the PM. Surprisingly, the cysteine in the terminal CAA is not required for PM targeting of GFP-ROP7. In contrast, an internal HVR cysteine is essential for proper targeting of the fusion, and the cysteine in the internal CaaX is required for complete membrane association. Interestingly, this CaaX motif can also direct PM association when placed at the fusion C-terminus by addition of an internal stop codon. Fractionation experiments confirm that maize ROPs associate with membranes in maize seedlings. Our analysis suggests that the ROP7 HVR directs PM localization by a mechanism independent of a C-terminal CaaX motif; this mechanism may have evolved through addition of 3' intron/exon sequences to a rop progenitor.

Amino Acid Sequence↗

PubMed2000

Trivalent ions activate abscisic acid-inducible promoters through an ABI1-dependent pathway in rice protoplasts.

The plant hormone abscisic acid (ABA) mediates many vital processes in plant growth and development, including seed dormancy, cell division, water use efficiency, and adaptation to drought, salinity, chilling, pathogen attack, and UV light. Our understanding of ABA signal transduction is fragmentary and would benefit from specific and facile probes of the process. Protoplasts from rice (Oryza sativa L. cv IR54) embryonic suspension cultures cotransformed with effector plasmids encoding the maize (Zea mays) VIVIPAROUS1 cDNA and/or the Arabidopsis dominant negative mutant (abi1-1) ABA-insensitive cDNA demonstrated genetic interactions of VIVIPAROUS1 and abi1-1 in transactivation of the ABA-inducible HVA1 promoter from barley (Hordeum vulgare), suggesting the mechanisms of these effectors are conserved among monocots and dicots. Trivalent ions have been shown to act as an effector of gene expression in plants and animals, although the mechanism of action is unknown. We show in two complementary transient ABA-inducible gene expression assays (beta-glucuronidase and luciferase enzymatic activities and quantitative flow cytometry of green fluorescent protein) that trivalent ions specifically interact with an ABI1-dependent ABA-signaling pathway leading to gene expression. Trivalent ions mimic ABA effects on gene expression and may be a useful tool to study ABA signaling.

Abscisic Acid↗

PubMed1999

Isolation and characterization of a cDNA clone from Arabidopsis thaliana with partial sequence similarity to integrins.

An Arabidopsis thaliana cDNA, called At14a, was isolated by immunoscreening an expression library with an anti-integrin antibody. The At14a cDNA is 1459 nucleotides and has an open reading frame encoding a protein of 385 amino acids and a predicted molecular weight of 43kDa. At14a has a small domain that has sequence similarities to integrins from fungi, insects and humans. Transcripts of At14a are found in all Arabidopsis tissues examined, and when expressed as an epitope tagged fusion protein in transgenic plants, At14a localizes partly to the plasma membrane.

Amino Acid Sequence↗

PubMed1998

Genomics

Explore the source record for details and available documents.

Journal Article↗

PubMed1998

14-3-3 proteins are part of an abscisic acid-VIVIPAROUS1 (VP1) response complex in the Em promoter and interact with VP1 and EmBP1.

Protein-DNA complexes were formed when nuclear extracts from embryogenic rice suspension cultures or maize embryos were incubated with an abscisic acid-VIVIPAROUS1 (VP1) response element (Em1a) from the Em promoter. Monoclonal antibodies generated to GF14, a 14-3-3 protein from plants, resulted in gel retardation of the Em1a-protein complexes. Antibodies generated to the C and N termini of GF14 detected protein isoforms in rice nuclear and cytoplasmic extracts, but no differences in distribution of the GF14 isoforms were recognized between the nucleus and cytoplasm or when abscisic acid-treated and untreated tissues were compared. When recombinant GF14 fusion proteins from rice were added to nuclear extracts, novel complexes were formed that required the dimerization domain of GF14. Chemical cross-linking showed that GF-14 interacted with the basic leucine zipper factor EmBP1, which binds specifically to Em1a, and with VP1, which transactivates Em through Em1a. GF14 proteins from rice were shown to interact with VP1 in yeast through the dimerization domain of GF14. Our results indicated that GF14 interacts with both site-specific DNA binding proteins (i.e., EmBP1) and tissue-specific regulatory factors (i.e., VP1) and may provide a structural link between VP1 and the Em1a transcriptional complex.

14-3-3 Proteins↗

PubMed1997

Characterization and expression of a rice RAD23 gene.

In order to identify proteins that interact with plant transcriptional complexes, we performed a two-hybrid screen in yeast using a cDNA library from embryogenic rice suspension cultures and the plant transcriptional activator viviparous-1 (vp1) as 'bait'. In this screen, we detected an interaction between VP1 and a rice homologue of the Saccharomyces cerevisiae RAD23 gene (osRAD23). The RAD23 protein is associated with the general transcriptional machinery in yeast, and is believed to play a role in the processes of nucleotide excision repair in yeast and mammalian cells. This report is the first identification of a RAD23 homolog in plants. The osRAD23 amino acid sequence shares 50-60% similarity throughout its length with RAD23 sequences from yeast, mice, and man. osRAD23 contains a characteristic ubiquitin-like domain at its N-terminus, which is similar to other RAD23 genes. Analysis of the expressed sequence tag database identifies two different classes of RAD23 genes in both Arabidopsis and rice. Southern analysis of rice genomic DNA indicated the presence of at least two RAD23-like genes. A single transcript (1.5 kb) of osRAD23 was detected in total RNA from rice embryonic tissue, while three transcripts (1.8, 1.5 and 1.0 kb) were observed in total RNA from vegetative tissues of rice.

Amino Acid Sequence↗

PubMed1997

Effect of the nuclear factors EmBP1 and viviparous1 on the transcription of the Em gene in HeLa nuclear extracts.

Templates constructed from the wheat Em and maize rab28 promoters are efficiently and accurately transcribed in the well-characterized cell-free transcription system prepared from HeLa nuclei. Deletion analysis of the Em promoter indicates that a G-box (CACGTG) element (Em1b) is required for transcription. USF, a Myc transcription factor in HeLa nuclear extracts, activates transcription by binding to Em1b, as shown by the ability of an antibody raised against USF to inhibit transcription and to interfere with Em1b complex formation in an electrophoretic mobility shift assay. The addition of the recombinant Viviparous1 protein from maize to HeLa nuclear extracts specifically stimulated transcription of the Em promoter but was dependent on the presence of USF in the extract. In USF-depleted extracts, the addition of recombinant EmBP1, a basic leucine zipper transcription factor from wheat, activated transcription through Em1b as well as from a similar G-box in the adenovirus major late promoter. Our study demonstrates that the basic transcriptional apparatus in HeLa nuclear extract supports transcription from plant promoters and can be used to assay the function of certain plant nuclear proteins, thereby helping to determine their effects on transcription.

Cell Nucleus↗

PubMed1997

Plant cell morphogenesis: plasma membrane interactions with the cytoskeleton and cell wall.

Because plants are composed of immobile cells, plant morphogenesis requires mechanisms allowing precise control of cell expansion and cell division patterns. Cortical domains, localized in response to directional cues, are of central importance in establishing cell polarity, orienting cell division, and determining daughter cell fates in a wide variety of prokaryotic and eukaryotic organisms. Such domains consist of localized macromolecular complexes that, in plant cells, provide spatial control of cell expansion and cell division functions. The role of the cytoskeleton, plasma membrane, and targeted secretion to the cell wall in the spatial regulation of cell morphogenesis in plants is discussed in light of recent results from model organisms, including brown algal zygotes (e.g. Fucus). A general model, emphasizing the importance of cortical sites and targeted secretion, is proposed for morphogenesis in higher plant cells based on current knowledge and principles derived from analysis of the establishment of a stable cortical asymmetry in Fucus. The model illustrates mechanisms to direct the orientation of an asymmetric division resulting in daughter cells with different fates.

Animals↗

PubMed1997

Cortical asymmetries direct the establishment of cell polarity and the plane of cell division in the Fucus embryo.

External gradients, such as unilateral light, applied to apolar zygotes of Fucus result in a cortical asymmetry expressed as the actin-dependent translocation of existing plasma membrane molecules (e.g., DHP receptors) to the shaded side (Fig. 1a). This process corresponds to the alignment of the polar axis. The localized cortical domain identified by the accumulation of DHP receptors, F-actin, and free calcium forms a target site for Golgi vesicle (F granule) secretion. Localized secretion of F granules is essential to stabilize the polar axis (Fig. 1b), and to complete a structural complex at the site for polar growth, postulated to span the plasma membrane, from the actin cytoskeleton to the cell wall (Fig. 2). Furthermore, targeted secretion of the contents of F granules into the plasma membrane and/or cell wall appears to provide localized positional information required to orient the first cell division plane and to differentiate the rhizoid and thallus cells of the two-celled embryo (Fig. 1c). Our cytological approaches using Fucus zygotes point to the importance of directed vesicle movement and secretion in creating asymmetries in the plasma membrane/cell wall during embryogenesis, which appear to have a critical role in cell morphogenesis. Conclusions drawn from these results may provide a useful paradigm for the study of cell morphogenesis and pattern formation in higher plant embryos and vegetative tissues.

Cell Division↗

PubMed1996

Characterization of three rice basic/leucine zipper factors, including two inhibitors of EmBP-1 DNA binding activity.

The promoter of the wheat Em gene contains elements with a CACGTG core sequence (G-boxes), which are recognized by EmBP-1, a wheat basic/leucine zipper (bZIP) protein. G-boxes are required for Em expression in response to the phytohormone abscisic acid and for transactivation by the Viviparous-1 protein (VP1) using transient expression systems. In order to identify other factors that are part of the transcriptional complex that associates with G-boxes, we have screened a rice (Oryza sativa) cDNA library with biotinylated EmBP-1. We have isolated osZIP-1a, a homolog of EmBP-1 and other plant G-box-binding factors. We show that EmBP-1 and osZIP-1a will preferentially heterodimerize in vitro. Overexpression of osZIP-1a in rice protoplasts can enhance expression from the Em promoter only in the presence of abscisic acid. Two other clones have been identified by screening with EmBP-1: osZIP-2a and osZIP-2b. These osZIP-2 factors represent a novel class of bZIP proteins with an unusual DNA-binding domain that does not recognize G-boxes. The osZIP-2 factors can heterodimerize with EmBP-1 and prevent it from binding to the Em promoter. Interestingly, osZIP-1a does not heterodimerize with the osZIP-2 factors and its DNA binding activity is unaffected by their presence. Thus, osZIP-2 factors may be involved in sequestering a particular group of G-box-binding factors into inactive heterodimers.

Amino Acid Sequence↗

PubMed1996

Histone H1 enhances the DNA binding activity of the transcription factor EmBP-1.

Previous work indicated that nuclear extracts isolated from embryogenic rice suspension cells treated with the phytohormone abscisic acid (ABA) have enhanced binding activity to an ABA response element (Em1a) in the promoter of the Em gene from wheat. We identified an activity in wheat and maize nuclear extracts that enhances binding of the recombinant transcription factor EmBP-1 to Em1a by 80-fold. Fractionation of nuclear extracts led us to identify histone H1 and HMGb (but not HMGc or -d) as two factors that can enhance the ability of EmBP-1 to bind to Em1a and account for at least a part of this activity of nuclear extracts. Our results, which indicate for the first time that histone H1 possesses this type of activity, lend further support to the model that positively charged proteins can drastically affect the DNA binding activity of specific transcription factors. Furthermore, our study points to these chromosomal proteins as potential targets of an ABA-mediated modification (e.g. acetylation) that could affect the regulation of Em gene expression.

Basic-Leucine Zipper Transcription Factors↗

PubMed1996

A conserved domain of the viviparous-1 gene product enhances the DNA binding activity of the bZIP protein EmBP-1 and other transcription factors.

The maize VP1 protein is a seed-specific regulator of gene expression that effects the expression of a subset of abscisic acid (ABA)-regulated genes that are expressed during the maturation program of the seed. In addition, VP1 has pleiotropic effects on seed development that are not related to ABA. In transient expression assays, VP1 has been shown to transactivate gene expression through at least two distinct promoter elements: the G boxes from the ABA-inducible wheat Em gene and the SphI box from the maize C1 gene. We have investigated how VP1 can transactivate gene expression through diverse promoter elements by analyzing its association in vitro with EmBP-1, a factor that binds the Em promoter. We demonstrate that VP1 can greatly enhance the DNA binding activity of EmBP-1 in a gel retardation assay. This enhancing activity has also been observed on transcription factors as diverse as Opaque-2, Max, Sp1, and NF-kappaB. Deletion of a small but highly conserved region (BR2) in VP1 eliminates the enhancement in vitro as well as the ability of VP1 to transactivate Em gene expression in a transient expression assay. A 40-amino acid fragment from VP1 sandwiched between the maltose-binding protein and LacZ can confer the enhancement function to this fusion protein in vitro. A weak and relatively nonspecific interaction between BR2 and DNA is demonstrated by UV cross-linking. The in vitro properties we observe for VP1 might explain the regulatory effects of VP1 on a diverse set of genes and why mutations in the vp1 locus have pleiotropic effects.

Base Sequence↗

PubMed1996

The alignment of the axis of asymmetry in regenerating protoplasts of the moss, Ceratodon purpureus, is determined independently of axis polarity.

Ceratodon protoplasts regenerate by polar outgrowth to form cell filaments. The kinetics of regeneration show that some cellular event has to be completed before regeneration can be initiated. The development of the regeneration axis is strongly influenced by light, with axis alignment and axis polarity being fixed independently. We define axis alignment as the relationship of the regeneration axis to the incident light, independent of polarity. Thus protoplasts regenerating directly towards, or directly away from the light source are defined as being similarly aligned but with opposite polarity. Protoplasts that regenerate in unidirectional red light form axes that are aligned parallel to the light direction, with about 70% being polarised towards the light and about 30% away. In unidirectional blue or white light, almost all protoplasts regenerate towards the light but axis alignment is determined less stringently. Re-orientation of protoplasts regenerating in unidirectional light shows that axis alignment is fixed between 8 and 9 hours before protoplasts regenerate and that axis polarity is fixed later. When protoplasts are removed from directional light to either non-directional light or to darkness, regeneration axes continue to be aligned by the earlier directional stimulus for at least 24 hours. Thus although axis alignment is fixed only about 8 hours before regeneration, in the absence of contradictory information about directionality in the light environment, protoplasts retain a memory of light direction for much longer. However, both reorientation and removal from a directional light field have profound effects on axis polarity; the pattern observed in undisturbed protoplasts being lost. To account for these observations, we propose that separate gradients are established independently to determine the alignment and polarity of the regeneration axis respectively. The alignment gradient is established rapidly and is steeper in red than in blue or white light, the polarity gradient is established slowly and is steeper in white or blue light than in red. These studies will now allow a genetic dissection of these processes in moss.

Bryopsida↗

PubMed1996

The role of targeted secretion in the establishment of cell polarity and the orientation of the division plane in Fucus zygotes.

In this study, we investigate the role of polar secretion and the resulting asymmetry in the cell wall in establishing polarity in Fucus zygotes. We have utilized brefeldin-A to selectively interrupt secretion of Golgi-derived material into the cell wall as assayed by toluidine blue O staining of sulfated fucoidin. We show that the polar secretion of Golgi-derived material is targeted to a cortical site of the zygote identified by the localization of actin filaments and dihydropyridine receptors. The deposition of Golgi-derived material into the cell wall at this target site is temporally coincident with and required for polar axis fixation. We propose that local secretion of Golgi-derived material into the cell wall transforms the target site into the fixed site of polar growth. We also found that polar secretion of Golgi-derived material at the fixed site is essential for growth and differentiation of the rhizoid, as well as for the proper positioning of the first plane of cell division. We propose that the resulting asymmetry in the cell wall serves as positional information for the underlying cortex to initiate these polar events. Our data supports the hypothesis that cell wall factors in embryos, previously shown to be responsible for induction of rhizoid cell differentiation, are deposited simultaneously with and are responsible for polar axis fixation. Furthermore, the pattern of polar growth is attributable to a positional signal at the fixed site and appears to be independent of the orientation of the first cell division plane. Thus, the establishment of zygotic cell polarity and not the position of the first division plane, is critical for the formation of the initial embryonic pattern in Fucus.

Actins↗

PubMed1996

Polar localization of a dihydropyridine receptor on living Fucus zygotes.

We have used a fluorescently-labeled dihydropyridine (FL-DHP) to vitally stain living Fucus zygotes during the establishment of cell polarity. Localization of FL-DHP is primarily at the plasma membrane and FL-DHP binding is competitively blocked by an unlabeled dihydropyridine. Distribution of FL-DHP is initially symmetrical before fixation of the polar axis, but becomes asymmetrical in response to a unilateral light gradient. The distribution of FL-DHP receptors can be relocalized when the direction of the photopolarizing stimulus is changed. Treatment of cells with cytochalasin B prior to axis fixation reversibly prevents localization of FL-DHP receptors. Observation of FL-DHP labeling by time-lapse fluorescence microscopy indicates that the existing receptors are redistributed during polar axis formation. The asymmetric distribution of FL-DHP receptors coincides temporally and spatially with increased local intracellular calcium ion concentrations, as measured by calcium green dextran. Based on the site, timing, photo-reversibility, and actin dependence of the asymmetric localization of FL-DHP receptors, we conclude that FL-DHP is a vital probe for the later stage of polar axis formation in Fucus zygotes. Furthermore, we propose that FL-DHP receptors correspond to ion channels that are transported to the future site of polar growth to create the changes in local calcium concentration required for polarity establishment.

Calcium↗

PubMed1995

Spatial redistribution of poly(A)+ RNA during polarization of the Fucus zygote is dependent upon microfilaments.

Asymmetrical distribution of mRNA has been associated with polarization and cell fate determination during early development of animal embryos. In this report we determine the distribution pattern of poly(A)+ RNA during early embryogenesis of the brown alga Fucus. Poly(A)+ RNA is symmetrically distributed in the egg and early zygote. Shortly after the polar axis is established, poly(A)+ RNA becomes segregated to the thallus pole of the zygote. Following cytokinesis, most of poly(A)+ RNA is partitioned into the thallus cell. We show that the spatial redistribution of poly(A)+ RNA requires intact microfilaments and the fixation of the polar axis, but is not dependent upon polarized growth of the rhizoid, intact microtubules, or orientation of the division plane.

Cell Division↗