Biomedical subjects

Ralph S Quatrano

Publications and source records attributed to Ralph S Quatrano.

14 recordsLinked to original sources

PubMed2007

The B2 domain of VIVIPAROUS1 is bi-functional and regulates nuclear localization and transactivation.

The transcriptional regulator VIVIPA-ROUS1 (VP1) is composed of four functional domains that control different aspects of gene expression during seed development. The B2 domain is required for its role as a transcriptional activator, functioning at the site of transcription and/or for its transport into the nucleus. Previous work showed that the B2 domain was required for transactivation of the Em promoter. We demonstrate that VP1::GFP localizes to the nucleus of barley (Hordeum vulgare) aleurone cells, but when B2 is deleted, nuclear accumulation is lost. However, the B2 domain itself is not sufficient for nuclear localization of GFP::GUS. Using point mutagenesis on the putative NLS within B2, we show that the VP1::GFP still accumulates in the nucleus. Utilizing a comparative approach, through the alignment of B2 domains from various VP1/ABI3 proteins, oincluding the ABI3 orthologs from Physcomitrella patens, revealed the involvement of other conserved amino acids. Mutating VP1 at the conserved threonine on the N-terminal side of the putative NLS and at a conserved arginine-glutamine-arginine sequence on the C-terminal side prevented nuclear localization of VP1. A single amino acid change, from alanine to threonine, within this NLS found in the Arabidopsis abi3-7 mutant prevents transcription of AtEm1 and AtEm6 in vivo. We show that this same mutation in VP1 prevents transactivation of the Em-GUS reporter in barley aleurone but does not interfere with nuclear localization. Our data demonstrate that the B2 domain of VP1 is bifunctional in nature regulating both nuclear localization and transactivation.

Amino Acid Sequence↗

PubMed2006

Identification and functional characterization of the moss Physcomitrella patens delta5-desaturase gene involved in arachidonic and eicosapentaenoic acid biosynthesis.

The moss Physcomitrella patens contains high levels of arachidonic acid and lesser amounts of eicosapentaenoic acid. Here we report the identification and characterization of a delta5-desaturase from P. patens that is associated with the synthesis of these fatty acids. A full-length cDNA for this desaturase was identified by data base searches based on homology to sequences of known delta5-desaturase cDNAs from fungal and algal species. The resulting P. patens cDNA encodes a 480-amino acid polypeptide that contains a predicted N-terminal cytochrome b5-like domain as well as three histidine-rich domains. Expression of the enzyme in Saccharomyces cerevisiae resulted in the production of the delta5-containing fatty acid arachidonic acid in cells that were provided di-homo-gamma-linolenic acid. In addition, the expressed enzyme generated delta5-desaturation products with the C20 substrates omega-6 eicosadienoic and omega-3 eicosatrienoic acids, but no products were detected with the C18 fatty acid linoleic and alpha-linolenic acids or with the C22 fatty acid adrenic and docosapentaenoic acids. When the corresponding P. patens genomic sequence was disrupted by replacement through homologous recombination, a dramatic alteration in the fatty acid composition was observed, i.e. an increase in di-homo-gamma-linolenic and eicosatetraenoic acids accompanied by a concomitant disappearance of the delta5-fatty acid arachidonic and eicosapentaenoic acids. In addition, overexpression of the P. patens cDNA in protoplasts isolated from a disrupted line resulted in the restoration of arachidonic acid synthesis.

Arachidonic Acid↗

PubMed2006

The role of ARPC4 in tip growth and alignment of the polar axis in filaments of Physcomitrella patens.

When the actin related protein 2/3 (Arp2/3) complex member arpc4 was deleted in Physcomitrella patens (moss), the resulting null mutant (Deltaarpc4) was viable and revealed no gross changes during morphogenesis of filaments into gametophores. However, we observed a striking reduction of filamentous tip growth, resulting in smaller, denser colonies. Although polar responses of Deltaarpc4 filaments to unilateral white light were unaffected, these mutant filaments were defective in their response to polarized white light. These observations strongly suggest a specific role of the Arp2/3 complex as a downstream target for signals regulating oriented tip growth. Insertion of YFP-ARPC4 into Deltaarpc4 rescued the mutant phenotypes and localized ARPC4 exclusively to the tip cell of filaments, the site of actin dynamics and polarized growth. The ability of Deltaarpc4 to perform some but not all cellular responses will allow the study of its function in orientation of tip growth in response to directional cues (e.g. light) in a viable but mutated background.

Actin-Related Protein 2-3 Complex↗

PubMed2006

Use of bacterial quorum-sensing components to regulate gene expression in plants.

We describe an efficient inducible system to regulate gene expression in plants based on quorum-sensing components found in Gram-negative bacteria such as Agrobacterium tumefaciens. These bacteria monitor their own population density by utilizing members of the N-acyl homoserine lactone family as inducers and a transcriptional activator as its receptor. In our study, we utilize the components from A. tumefaciens (i.e. 3-oxooctanyl-l-homoserine lactone [OOHL]) synthesized by the TraI protein and its receptor, TraR. When OOHL binds to TraR, it recognizes its specific cis-element, the tra box. We translationally fused the eukaryotic VP16 activation domain to the N terminus of TraR. In the presence of OOHL, the chimeric VP16:TraR transcriptional regulator induces reporter gene expression in moss (Physcomitrella patens), barley (Hordeum vulgare), and carrot (Daucus carota) cells, as well as in transgenic Arabidopsis (Arabidopsis thaliana) seedlings. The inducible system shows a low level of reporter gene expression in the absence of the inducer. Foliar application and a floating-leaf assay in the presence of the inducer shows a 30- and 200-fold induction, respectively. Induction by foliar application of the inducer to whole seedlings is achieved within 8 h. The VP16:TraR activator also shows specificity for binding to its cognate inducer, OOHL. Based on microarray analyses, endogenous gene expression is not significantly affected due to overexpression of the TraR protein or presence of OOHL in either wild-type or lactone-inducible transgenic plants.

Arabidopsis↗

PubMed2006

Agravitropic mutants of the moss Ceratodon purpureus do not complement mutants having a reversed gravitropic response.

New mutants of the moss Ceratodon purpureus have been isolated, which showed abnormal gravitropic responses. The apical cells of protonemal filaments of wild-type strains respond to gravity by growing upwards and are well aligned to the gravity vector. This response only occurs in darkness. Mutants show a range of phenotypes. Some are insensitive to gravity, showing symmetrical growth, while others align to the gravity vector but orient growth downwards. A further class grows in darkness as though it were in light, showing insensitivity to gravity and continued chlorophyll synthesis. Somatic hybrids between mutants and wild-type strains and between pairs of mutants have been selected using transgenic antibiotic resistance as selective markers. Hybrids between wild-type strains and all of the mutants have a wild-type phenotype, and so all mutants therefore have recessive phenotypes. Mutants comprise three complementation groups. One group has a single member, while another has three members. The third has at least 16 members and shows a complex pattern of complementation consistent with a single gene product functioning in both orientation and alignment to gravity, as well as contributing more than one subunit to the mature product.

Bryopsida↗

PubMed2006

Characterization and functional analysis of ABSCISIC ACID INSENSITIVE3-like genes from Physcomitrella patens.

Although the moss Physcomitrella patens is known to respond to abscisic acid (ABA) by activating gene expression, the transcriptional components involved have not been characterized. Initially, we used the ABA-responsive Em promoter from wheat linked to beta-glucuronidase (GUS) to determine whether ABI3/VP1, transcriptional regulators in the ABA-signaling pathway in angiosperms, were similarly active in the ABA response of P. patens. We show by particle bombardment that ABI3 and VP1 affect Em-GUS expression in P. patens in a manner similar to angiosperms. We also show the involvement of ABI1 in the pathway, utilizing the abi1-1 mutant allele. We isolated three ABI3-like genes from P. patens. Using an Em-like ABA-responsive promoter from P. patens (PpLea1), we demonstrate that PpABI3A, only in the presence of ABA, strongly enhances PpLea1-GUS expression in P. patens. PpABI3A also enhances ABA-induced Em-GUS expression in P. patens. In barley aleurone, PpABI3A transactivates Em-GUS but to a lesser extent than VP1 and ABI3. PpABI3A:GFP is localized to the nucleus of both protonemal cells and barley aleurone, indicating that the nuclear localization signals are conserved. We show that at least a part of the inability of PpABI3A to fully complement the phenotypes of the Arabidopsis abi3-6 mutant is due to a weak interaction between PpABI3A and the bZIP transcription factor ABI5, as assayed functionally in barley aleurone and physically in the yeast-two-hybrid assay. Our data clearly demonstrate that P. patens will be useful for comparative structural and functional studies of components in the ABA-response pathway such as ABI3.

Abscisic Acid↗

PubMed2005

Arabinogalactan proteins are required for apical cell extension in the moss Physcomitrella patens.

Cell biological, structural, and genetic approaches have demonstrated the presence of arabinogalactan proteins (AGPs) in the moss Physcomitrella patens and provided evidence for their function in cell expansion and specifically in the extension of apical tip-growing cells. Inhibitor studies indicated that apical cell expansion in P. patens is blocked by synthetic AGP binding beta-glucosyl Yariv reagent (betaGlcYR). The anti-(1-->5)-alpha-L-arabinan monoclonal antibody LM6 binds to some AGPs in P. patens, to all plasma membranes, and to the cell wall surface at the most apical region of growing protonemal filaments. Moreover, LM6 labeling of cell walls at the tips of apical cells of P. patens was abolished in the presence of betaGlcYR, suggesting that the localized movement of AGPs from the plasma membrane to the cell wall is a component of the mechanism of tip growth. Biochemical and bioinformatic analyses were used to identify seven P. patens ESTs encoding putative AGP core proteins from homology with Arabidopsis thaliana, Brassica napus, and Oryza sativa sequences and from peptide fragments isolated from betaGlcYR-precipitated AGPs. Gene knockout by homologous recombination of one of these genes, P. patens AGP1, encoding a classical AGP core protein, resulted in reduced cell lengths in protonemal filaments, indicating a role for AGP1 in apical cell expansion in P. patens.

Arabidopsis↗

PubMed2005

Actin-related protein2/3 complex component ARPC1 is required for proper cell morphogenesis and polarized cell growth in Physcomitrella patens.

The actin-related protein2/3 (Arp2/3) complex functions as a regulator of actin filament dynamics in a wide array of eukaryotic cells. Here, we focus on the role of the Arp2/3 complex subunit ARPC1 in elongating tip cells of protonemal filaments of the moss Physcomitrella patens. Using RNA interference (RNAi) to generate loss-of-function mutants, we show dramatic defects in cell morphology manifested as short, irregularly shaped cells with abnormal division patterns. The arpc1 RNAi plants lack the rapidly elongating caulonemal cell type found in wild-type protonemal tissue. The absence of this cell type prevents normal bud formation even in response to cytokinin treatment and results in filamentous colonies lacking leafy gametophores. In addition, arpc1 protoplasts show an increased sensitivity to osmotic shock and are defective in their ability to properly establish a polarized outgrowth during regeneration from a single cell. This failure of arpc1 protoplasts to undergo proper tip growth is rescued by ARPC1 overexpression and is phenocopied in wild-type protoplasts treated with Latrunculin B, a potent inhibitor of actin polymerization. We show in moss that ARPC1, and by inference the Arp2/3 complex, plays a critical role in controlling polarized growth and cell division patterning through its regulation of actin dynamics at the cell apex.

Actin-Related Protein 2↗

PubMed2005

Cis-regulatory element based targeted gene finding: genome-wide identification of abscisic acid- and abiotic stress-responsive genes in Arabidopsis thaliana.

MOTIVATION: A fundamental problem of computational genomics is identifying the genes that respond to certain endogenous cues and environmental stimuli. This problem can be referred to as targeted gene finding. Since gene regulation is mainly determined by the binding of transcription factors and cis-regulatory DNA sequences, most existing gene annotation methods, which exploit the conservation of open reading frames, are not effective in finding target genes. RESULTS: A viable approach to targeted gene finding is to exploit the cis-regulatory elements that are known to be responsible for the transcription of target genes. Given such cis-elements, putative target genes whose promoters contain the elements can be identified. As a case study, we apply the above approach to predict the genes in model plant Arabidopsis thaliana which are inducible by a phytohormone, abscisic acid (ABA), and abiotic stress, such as drought, cold and salinity. We first construct and analyze two ABA specific cis-elements, ABA-responsive element (ABRE) and its coupling element (CE), in A.thaliana, based on their conservation in rice and other cereal plants. We then use the ABRE-CE module to identify putative ABA-responsive genes in A.thaliana. Based on RT-PCR verification and the results from literature, this method has an accuracy rate of 67.5% for the top 40 predictions. The cis-element based targeted gene finding approach is expected to be widely applicable since a large number of cis-elements in many species are available.

Abscisic Acid↗

PubMed2004

Localization to the rhizoid tip implicates a Fucus distichus Rho family GTPase in a conserved cell polarity pathway.

Generation and expression of cell polarity in brown algal zygotes of the Fucales involve regulation of the actin cytoskeleton and localized secretion. We used degenerate PCR to isolate cDNAs that encode two small GTPases, FdRac1 and FdRab8, from zygotes of Fucus distichus (L.) Powell. Sequence analysis placed FdRac1 in the Rho family, which regulates actin, and FdRab8 in the Rab family, which regulates vesicle transport. As expected, bacterially expressed forms of both proteins bound GTP in vitro. When expressed in budding yeast, FdRac1 showed some functional overlap with CDC42, the Saccharomyces cerevisiae Rho family gene required for yeast cell polarity. Immunolocalization revealed an asymmetric distribution of FdRac1 in polarized zygotes and embryos, with FdRac1 concentrated at or near the growing tip of the algal rhizoid. Our data support the hypothesis that FdRac1 regulates algal cell polarity, possibly via the actin cytoskeleton. Because brown algae belong to the heterokont group, which diverged from other groups early in eukaryotic evolution, we argue that the Rho family function of regulating cell polarity is ancient and may extend throughout the eukaryotes.

Actins↗

PubMed2003

Conserved subgroups and developmental regulation in the monocot rop gene family.

Rop small GTPases are plant-specific signaling proteins with roles in pollen and vegetative cell growth, abscisic acid signal transduction, stress responses, and pathogen resistance. We have characterized the rop family in the monocots maize (Zea mays) and rice (Oryza sativa). The maize genome contains at least nine expressed rops, and the fully sequenced rice genome has seven. Based on phylogenetic analyses of all available Rops, the family can be subdivided into four groups that predate the divergence of monocots and dicots; at least three have been maintained in both lineages. However, the Rop family has evolved differently in the two lineages, with each exhibiting apparent expansion in different groups. These analyses, together with genetic mapping and identification of conserved non-coding sequences, predict orthology for specific rice and maize rops. We also identified consensus protein sequence elements specific to each Rop group. A survey of ROP-mRNA expression in maize, based on multiplex reverse transcriptase-polymerase chain reaction and a massively parallel signature sequencing database, showed significant spatial and temporal overlap of the nine transcripts, with high levels of all nine in tissues in which cells are actively dividing and expanding. However, only a subset of rops was highly expressed in mature leaves and pollen. Intriguingly, the grouping of maize rops based on hierarchical clustering of expression profiles was remarkably similar to that obtained by phylogenetic analysis. We hypothesize that the Rop groups represent classes with distinct functions, which are specified by the unique protein sequence elements in each group and by their distinct expression patterns.

Amino Acid Sequence↗

PubMed2003

A signal peptide secretion screen in Fucus distichus embryos reveals expression of glucanase, EGF domain-containing, and LRR receptor kinase-like polypeptides during asymmetric cell growth.

Zygotes of the brown alga Fucus distichus (L.) Powell develop polarity prior to the first embryonic cell division and retain a pattern of asymmetric growth during early embryogenesis. In order to identify F. distichus polypeptides secreted during asymmetric cell growth, we used a functional assay in Saccharomyces cerevisiae to screen a cDNA library generated from asymmetrically growing Fucus embryos for sequences encoding polypeptides that function as signal peptides for secretion. We isolated and sequenced 222 plasmids containing Fucus cDNAs encoding signal peptide activity. The cDNA inserts from these plasmids were translated in silico into 244 potential polypeptide sequences, 169 of which are predicted to contain signal peptides. BlastP analysis of the Fucus sequences revealed similarity between many Fucus proteins and cell surface proteins that function in development in other eukaryotes, including epidermal growth factor (EGF)-like repeat-containing proteins, plant leucine-rich repeat (LRR)-receptor kinases, and algal beta-1, 3-exoglucanase. However, most of the isolated Fucus polypeptides lack similarity to known proteins. The isolation of cDNAs encoding secreted Fucus proteins provides an important step toward characterizing cell surface proteins important for asymmetric organization and growth in fucoid embryos.

Amino Acid Sequence↗

PubMed2003

Phylogenetic analysis of new plant myosin sequences.

We have sampled a large number of plant taxa, ranging from brown algae to angiosperms, for the presence of myosin sequences. Using phylogenetic analysis, we show that all but two of the new plant myosin sequences fall into two of three preexisting myosin classes. We identified two outlying sequences, which do not fall into any preexisting myosin class. Additionally, all genomic sequences encoding class XI myosins contain an intron in the region studied, suggesting that this genomic region has been conserved over at least 1 billion years of plant evolution. With these data, we can rapidly and consistently classify partial myosin sequences from plants. Our data show that plant myosins do not have clear orthologues in other kingdoms, providing interesting insights into the diversification of myosins.

Evolution, Molecular↗

PubMed2003

RNA interference in the moss Physcomitrella patens.

The moss Physcomitrella patens performs efficient homologous recombination, which allows for the study of individual gene function by generating gene disruptions. Yet, if the gene of study is essential, gene disruptions cannot be isolated in the predominantly haploid P. patens. Additionally, disruption of a gene does not always generate observable phenotypes due to redundant functions from related genes. However, RNA interference (RNAi) can provide mutants for both of these situations. We show that RNAi disrupts gene expression in P. patens, adding a significant tool for the study of plant gene function. To assay for RNAi in moss, we constructed a line (NLS-4) expressing a nuclearly localized green fluorescent protein (GFP):beta-glucuronidase (GUS) fusion reporter protein. We targeted the reporter protein with two RNAi constructs, GUS-RNAi and GFP-RNAi, expressed transiently by particle bombardment. Transformed protonemal cells are marked by cobombardment with dsRed2, which diffuses between the nucleus and cytoplasm. Cells transformed with control constructs have nuclear/cytoplasmic red fluorescence and nuclear green fluorescence. In cells transformed with GUS-RNAi or GFP-RNAi constructs, the nuclear green fluorescence was reduced on average 9-fold as soon as 48 h after transformation. Moreover, isolated lines of NLS-4 stably transformed with GUS-RNAi construct have silenced nuclear GFP, indicating that RNAi is propagated stably. Thus, RNAi adds a powerful tool for functional analysis of plant genes in moss.

Bryopsida↗