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James A H Murray

Publications and source records attributed to James A H Murray.

At least 19 recordsLinked to original sources
PubMed2006

A greenprint for growth: signalling the pattern of proliferation.

The shoot and root apical meristems (SAM and RAM, respectively) of plants serve both as sites of cell division and as stem cell niches. The SAM is also responsible for the initiation of new leaves, whereas the analogous process of lateral root initiation occurs in the pericycle, a specialized layer of cells that retains organogenic potential within an otherwise non-dividing region of the root. A picture is emerging of how cell division, growth, and differentiation are coordinated in the meristems and lateral organ primordia of plants. This is starting to reveal striking parallels between the control of stem cell maintenance in both shoots and roots, and to provide information on how signalling from developmental processes and the environment impact on cell behaviour within meristems.

Cell Cycle↗
PubMed2006

Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance.

Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow-green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 degrees C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.

Animals↗
PubMed2006

The D-type cyclin CYCD3;1 is limiting for the G1-to-S-phase transition in Arabidopsis.

The G1-to-S-phase transition is a key regulatory point in the cell cycle, but the rate-limiting component in plants is unknown. Overexpression of CYCLIN D3;1 (CYCD3;1) in transgenic plants increases mitotic cycles and reduces endocycles, but its effects on cell cycle progression cannot be unambiguously determined. To analyze the cell cycle roles of plant D-type cyclins, we overexpressed CYCD3;1 in Arabidopsis thaliana cell suspension cultures. Changes in cell number and doubling time were insignificant, but cultures exhibited an increased proportion of G2- over G1-phase cells, as well as increased G2 arrest in response to stationary phase and sucrose starvation. Synchronized cultures confirm that CYCD3;1-expressing (but not CYCD2;1-expressing) cells show increased G2-phase length and delayed activation of mitotic genes such as B-type cyclins, suggesting that CYCD3;1 has a specific G1/S role. Analysis of putative cyclin-dependent kinase phosphorylation sites within CYCD3;1 shows that mutating Ser-343 to Ala enhances CYCD3;1 potency without affecting its rate of turnover and results in a fivefold increase in the level of cell death in response to sucrose removal. We conclude that CYCD3;1 dominantly drives the G1/S transition, and in sucrose-depleted cells the decline in CYCD3;1 levels leads to G1 arrest, which is overcome by ectopic CYCD3;1 expression. Ser-343 is likely a key residue in modulating CYCD3;1 activity in response to sucrose depletion.

Aphidicolin↗
PubMed2006

KNOX gene function in plant stem cell niches.

Homeobox genes encode transcriptional regulators that control development in multicellular eukaryotes. In plants, post-embryonic shoot growth relies on the activity of indeterminate cell populations termed shoot meristems, within which members of the class-1 KNOX sub-family of homeobox genes are expressed. KNOX genes are differentially required for meristem development and function to inhibit cell expansion and differentiation associated with organogenesis. Mechanisms must therefore be employed to prevent KNOX gene expression in developing lateral organs such as leaves. This review focuses on the expression patterns, meristematic functions and regulation of KNOX genes, and how the activities of these genes are integrated within the framework of pathways that control plant development.

Arabidopsis↗
PubMed2006

Cell cycle regulated D3-type cyclins form active complexes with plant-specific B-type cyclin-dependent kinase in vitro.

Tobacco (Nicotiana tabacum L.) cv Bright Yellow-2 (BY-2) cells are the most highly synchronizable plant cell culture, and previously we used them to analyze cell cycle regulation of cyclin-dependent kinases (CDKs) containing the cyclin binding motifs PSTAIRE (CDKA) and PPTA/TLRE (CDKB). Here we describe the analysis of tobacco CycD3 cyclins whose transcripts predominantly accumulate during G2 to M phase, which represents a unique feature of this type of cyclin D in plants. Although protein levels of CycD3s fluctuate with different patterns during the cell cycle, kinase assays revealed that the CycD3-associated kinases phosphorylate histone H1 and the tobacco retinoblastoma related protein (NtRBR1) with two peaks at the G1/S and G2/M boundaries. In vitro pull-down assays revealed that cell cycle-regulated CycD3s bind to CDKA, but more weakly than does CycD3;3, and that they also bind to CDKB and the CDK inhibitor NtKIS1a. Mutations in the cyclin box of the CycD3s showed that two amino acids are required for binding with CDKA and NtKIS1a, but no diminished interaction was observed with CDKB. A reconstituted kinase assay was adapted for use with bacterially produced GST-CycD3s, and kinase activity could be activated by incubation of extracts from exponentially growing BY-2 cells. Such activated complexes contained CDKA and CDKB, and the reconstituted GST-CycD3 mutants, retaining binding ability to CDKB, showed kinase activity, suggesting that these cell cycle-regulated CycD3s form active complexes with both A- and B-type CDKs in vitro.

Amino Acid Motifs↗
PubMed2006

Synchronization, transformation, and cryopreservation of suspension-cultured cells.

We have recently described the selection of rapidly dividing Arabidopsis cell suspension cultures MM1 and MM2d that provide a powerful platform for plant cell-cycle research. Here we provide detailed protocols and procedures to achieve high levels of synchronization, either by starving the cell cultures of sucrose or by applying the toxin aphidicolin. Cell-cycle activity during cell-cycle reentry (starvation-induced synchrony) or further cell-cycle progression (aphidicolin-induced synchrony) can be conveniently followed by using various validation procedures, such as determination of labeling index and metaphase/anaphase index or flow cytometry. We also describe a procedure that allows clonal transformed cell-suspension lines to be produced using Agrobacterium-mediated transformation, and an optimized and straightforward method for the cryopreservation and recovery of both parental and transformed lines which is applicable both to Arabidopsis and the tobacco BY2 cell lines. Cell-cycle synchronization capacity of the parental lines is maintained after both transformation and recovery from cryopreservation. The techniques described here require no specialized equipment and are suitable for routine laboratory use, greatly facilitating the handling and maintenance of cell cultures. The ability to store easily large numbers of transformed lines opens the possibility of using Arabidopsis cell suspension cultures for future high-throughput cell-cycle analysis.

Antimetabolites↗
PubMed2005

D-type cyclins activate division in the root apex to promote seed germination in Arabidopsis.

Seeds provide survival and dispersal capabilities by protecting the dormant mature plant embryo. Germination and resumption of development under favourable conditions requires the reinitiation of cell growth and division through poorly understood processes. Here we show that four phases of cell division activation during germination in Arabidopsis are related to external morphological changes. Cell division initiates in the root apical meristem (RAM) before root protrusion, followed by sequential activation of cell division in the cotyledons, shoot apical meristem (SAM), and secondary meristems. Major changes in transcript levels of >2,000 genes precede root emergence, including expression peaks of six D-type (CYCD) and two A-type cyclins. Two further CYCDs are activated later with the SAM. Early activated CYCDs play key roles in regulating the extent of cell division, because loss-of-function alleles of early CYCDs display reduced division activation and consequential delayed root emergence. Conversely, elevation of early CYCDs increases cell cycle activation in the RAM and promotes embryonic root (radicle) protrusion, whereas a later-acting CYCD does not. These phenotypes, together with their overlapping expression domains, support a cumulative action of a subset of CYCDs in cell cycle reactivation, rather than a complete functional redundancy. This analysis reveals a phenotype associated with loss-of-function of a plant cyclin and demonstrates that D-type cyclins regulate cell cycle reentry during meristem activation to promote successful germination and early seedling growth.

Arabidopsis↗
PubMed2005

Transcriptional activation of tobacco E2F is repressed by co-transfection with the retinoblastoma-related protein: cyclin D expression overcomes this repressor activity.

Evidence is emerging that the E2F family of transcription factors plays an important role in the regulation of gene expression at the G1/S transition in plants. Here, we show that in the tobacco proliferating cell nuclear antigen (PCNA), whose transcript is specifically expressed at G1/S phase, the two E2F binding sites are synergistically responsible for transcriptional activation at G1/S phase in synchronized tobacco BY-2 cells transformed with promoter constructs fused to a reporter gene. In addition, we have isolated the tobacco DP cDNA (NtDP) and showed that significant activation of the reporter gene was observed in transient expression assays by concomitantly transfecting with plasmids expressing NtE2F and NtDP. This transcriptional activation was repressed by co-transfection with a plasmid expressing NtRBR1; in vitro pull-down assays also revealed that NtRBR1 binds directly to NtE2F, thereby potentially blocking the transcriptional activation of NtE2F. Importantly, this repressor activity was cancelled when NtRBR1 was further co-transfected with a plasmid expressing cyclin D but not with cyclin A or cyclin B. These results are discussed with respect to the repression activity of NtRBR1 on the NtE2F/NtDP complex.

Amino Acid Sequence↗
PubMed2005

The developmental context of cell-cycle control in plants.

Plant growth is characterised both by continued growth and organogenesis throughout development, as well as by environmental influences on the rate and pattern of these processes. This necessitates a close relationship between cell cycle control, differentiation and development that can be readily observed and studied. The sequencing of the Arabidopsis genome has revealed the full complexity of cell cycle regulators in plants, creating a challenge to understand how these genes control plant growth and differentiation, and how they are integrated with intrinsic and external signals. Here, we review the control of the cell cycle and examine how it is integrated with proliferative activity within meristems, and during the differentiation processes leading to leaf and lateral root formation.

Arabidopsis↗
PubMed2005

Global analysis of the core cell cycle regulators of Arabidopsis identifies novel genes, reveals multiple and highly specific profiles of expression and provides a coherent model for plant cell cycle control.

Arabidopsis has over 80 genes encoding conserved and plant-specific core cell cycle regulators, but in most cases neither their timing of expression in the cell cycle is known nor whether they represent redundant and/or tissue-specific functions. Here we identify novel cell cycle regulators, including new cyclin-dependent kinases related to the mammalian galactosyltransferase-associated protein kinase p58, and new classes of cyclin-like and CDK-like proteins showing strong tissue specificity of expression. We analyse expression of all cell cycle regulators in synchronized Arabidopsis cell cultures using multiple approaches including Affymetrix microarrays, massively parallel signature sequencing and real-time reverse transcriptase polymerase chain reaction, and in plant material using the results of over 320 microarray experiments. These global analyses reveal that most core cell cycle regulators are expressed across almost all tissues and more than 85% are expressed at detectable levels in the cell suspension culture, allowing us to present a unified model of transcriptional regulation of the plant cell cycle. Characteristic patterns of D-cyclin expression in early and late G1 phase, either limited to the re-entry cycle or continuously oscillating, suggest that several CYCD genes with strong oscillatory regulation in late G1 may play the role of cyclin E in plants. Alone amongst the six groups of A and B type cyclins, members of CYCA3 peak in S-phase suggest it is a major component of S-phase kinases, whereas others show a peak in G2/M. 82 genes share this G2/M regulatory pattern, about half being new candidate mitotic genes of previously unknown function.

Arabidopsis↗
PubMed2004

Cryopreservation of transformed and wild-type Arabidopsis and tobacco cell suspension cultures.

We have recently described Arabidopsis cell suspension cultures that can be effectively synchronised. Here, we describe procedures that allow clonal-transformed cell suspension lines to be produced using Agrobacterium-mediated transformation, and an optimised and straightforward procedure for the cryopreservation and recovery of both parental and transformed lines. Frozen cultures show 90% viability and rapid re-growth after recovery. We show that the cryopreservation procedure is equally applicable to the frequently used tobacco bright yellow (BY)2 cell suspension culture, and that cell cycle synchronisation capacity of parental lines is maintained after both transformation and recovery from cryopreservation. The techniques require no specialised equipment, and are suitable for routine laboratory use, greatly facilitating the handling and maintenance of cell cultures and providing security against both contamination and cumulative somaclonal variation. Finally, the ability to store easily large numbers of transformed lines opens the possibility of using Arabidopsis cell suspension cultures for high-throughput analysis.

Arabidopsis↗
PubMed2004

Differential stability of Arabidopsis D-type cyclins: CYCD3;1 is a highly unstable protein degraded by a proteasome-dependent mechanism.

In Arabidopsis, the D-type cyclin CYCD3 is rate-limiting for transition of the G(1)/S boundary, and is transcriptionally upregulated at this point in cells re-entering the cell cycle in response to plant hormones and sucrose. However, little is known about the regulation of plant cell-cycle regulators at the protein level. We show here that CYCD3;1 is a phosphoprotein highly regulated at the level of protein abundance, whereas another D-type cyclin CYCD2;1 is not. The level of CYCD3;1 protein falls rapidly on sucrose depletion, correlated with the arrest of cells in G(1) phase, suggesting a rapid turnover of CYCD3;1. Treatment of exponentially growing cells with the protein synthesis inhibitor cycloheximide (CHX) confirms that CYCD3;1 is normally a highly unstable protein, with a half-life of approximately 7 min on CHX treatment. In both sucrose-starved and exponentially growing cells, CYCD3;1 protein abundance increases in response to treatment with MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), a reversible proteasome inhibitor, but not in response to the cysteine protease inhibitor E-64 or the calpain inhibitor ALLN (N-acetyl-leucyl-leucyl-norleucinal). The increase on MG132 treatment is because of de novo protein synthesis coupled with the blocking of CYCD3;1 degradation. Longer MG132 treatment leads to C-terminal cleavage of CYCD3;1, accumulation of a hyperphosphorylated form and its subsequent disappearance. We conclude that CYCD3;1 is a highly unstable protein whose proteolysis is mediated by a proteasome-dependent pathway, and whose levels are highly dependent on the rate of CYCD3;1 protein synthesis.

Amino Acid Sequence↗
PubMed2003

Genome-wide gene expression in an Arabidopsis cell suspension.

Plant cell suspension cultures are invaluable models for the study of cellular processes. Here we develop the recently described Arabidopsis suspension culture MM2d as a transcript profiling platform by means of Affymetrix ATH1 microarrays. Analysis of gene expression profiles during normal culture growth, during synchronous cell cycle re-entry and during synchronous cell cycle progression provides a unique integrated view of gene expression responses in a higher-plant system. Particularly striking is that expression of over 14 000 genes belonging to all defined categories can be reliably detected, suggesting that integrated and comparative analysis of data sets derived from transcript profiling of cultures is a powerful approach to identify candidate components involved in a wide range of biological processes. Combinatorial analysis of independent cell cycle synchrony methods allows the identification of genes that are apparently cell-cycle-regulated but are most likely responding to the induction of synchrony. We thus present an integrated genome-wide view of the transcriptional profile of a plant suspension culture and identify a refined set of 1082 cell cycle regulated genes largely independent of synchrony method.

Aphidicolin↗
PubMed2003

Arabidopsis transcript profiling on Affymetrix GeneChip arrays.

DNA microarrays are becoming a frequently used research tool. Whilst several studies have confirmed the reproducibility of analysing the same RNA samples on duplicate arrays, there is little analysis of the reproducibility of the results of transcript profiling between microarrays carrying different probes to a common set of genes. To address this question, we compared the performance and reproducibility of two microarrays commonly used in plant research, the Affymetrix Arabidopsis AG array containing more than 8000 probe sets and the Affymetrix Arabidopsis ATH1 array containing more than 22,000 redesigned probe sets. A total of 21 different RNA samples were labelled and hybridized in parallel to the two microarray types. Focusing on the overlap of more than 7300 targets detected with both arrays, we found a high degree of reproducibility. Despite the use of different probe sets, both signal and signal log ratio were very similar for most genes. However, genes that were called absent or not changed by Affymetrix' statistical algorithm implemented in MAS5.0 showed considerably less conservation of expression patterns. Moreover, we identified about 300 genes that yielded strongly different measurements with the two microarrays, emphasizing that RNA profiling data need careful interpretation. Overall, this study shows that results obtained with ATH1 and AG arrays are very comparable and hence that the analysis is largely independent of probe sets. However, the result emphasize the need for appropriate filtering schemes such as those based on the present and change calls provided by MAS5.0 rather than reliance solely on signal values.

Arabidopsis↗
PubMed2003

The ethanol switch: a tool for tissue-specific gene induction during plant development.

Controlled gene expression in time and space is a powerful tool for the analysis of gene function during plant development. Here, we report ethanol inducible gene expression in defined sub-domains of the shoot apical and floral meristems. For this, expression of an ethanol-regulated transcription factor, ALCR, is restricted to precise domains using specific promoters. Gene expression activation is followed using reporters under the control of the alcA promoter, which responds to ALCR only in the presence of the ethanol. We demonstrate that precise control of spatially limited gene expression can be achieved. The kinetics of reporter gene activation and inactivation following a pulse of ethanol induction shows that the system is dynamic and suitable for precise temporal control of expression. The system is both flexible and robust, permitting simultaneous expression of two genes in a given domain or, conversely, the expression of a gene in two separate domains. We also show that this strategy can be applied to mis-express genes with developmental roles, by manipulating expression of the SHOOT MERISTEMLESS (STM) and CYCLIN D3;1 (CYCD3;1) genes during plant development.

Arabidopsis↗
PubMed2003

Isolation, characterization and expression of cyclin and cyclin-dependent kinase genes in Jerusalem artichoke (Helianthus tuberosus L.).

Tuber explants of Jerusalem artichoke (Helianthus tuberosus L.) are a model system for cell-cycle re-entry from a quiescent state, involving the activation of division of tuber parenchyma cells in response to exogenous auxin. To enable molecular studies of this system, two cyclin (Heltu;CYCD1;1 and Heltu; CYCD3;1) and two cyclin-dependent kinase (Heltu; CDKA;1 and Heltu;CDKB1;1) genes have been isolated from a Jerusalem artichoke cDNA library and their expression demonstrated during the activation of cell division. It was found that CDKA;1 transcripts are present in quiescent tubers, whereas CYCD1;1, CYCD3;1 and CDKB1;1 transcripts are induced during cell-cycle re-entry as well as during bud growth of whole tubers. Both CYCD1;1 and CYCD3;1 transcripts appear shortly before, or coincident with, the onset of S phase.

Cyclin D↗
PubMed2003

Altered cell cycle distribution, hyperplasia, and inhibited differentiation in Arabidopsis caused by the D-type cyclin CYCD3.

CYCD3;1 expression in Arabidopsis is associated with proliferating tissues such as meristems and developing leaves but not with differentiated tissues. Constitutive overexpression of CYCD3;1 increases CYCD3;1-associated kinase activity and reduces the proportion of cells in the G1-phase of the cell cycle. Moreover, CYCD3;1 overexpression leads to striking alterations in development. Leaf architecture in overexpressing plants is altered radically, with a failure to develop distinct spongy and palisade mesophyll layers. Associated with this, we observe hyperproliferation of leaf cells; in particular, the epidermis consists of large numbers of small, incompletely differentiated polygonal cells. Endoreduplication, a marker for differentiated cells that have exited from the mitotic cell cycle, is inhibited strongly in CYCD3;1-overexpressing plants. Transcript analysis reveals an activation of putative compensatory mechanisms upon CYCD3;1 overexpression or subsequent cell cycle activation. These results demonstrate that cell cycle exit in the G1-phase is required for normal cellular differentiation processes during plant development and suggest a critical role for CYCD3 in the switch from cell proliferation to the final stages of differentiation.

Arabidopsis↗