Biomedical subjects

S Gilroy

Publications and source records attributed to S Gilroy.

At least 19 recordsLinked to original sources

PubMed2006

Spatial coordination of aluminium uptake, production of reactive oxygen species, callose production and wall rigidification in maize roots.

Aluminium (Al) toxicity associated with acid soils represents one of the biggest limitations to crop production worldwide. Although Al specifically inhibits the elongation of root cells, the exact mechanism by which this growth reduction occurs remains controversial. The aim of this study was to investigate the spatial and temporal dynamics of Al migration into roots of maize (Zea mays L.) and the production of the stress response compound callose. Using the Al-specific fluorescent probe morin, we demonstrate the gradual penetration of AI into roots. Al readily accumulates in the root's epidermal and outer cortical cell layers but does not readily penetrate into the inner cortex. After prolonged exposure times (12-24 h), Al had entered all areas of the root apex. The spatial and temporal accumulation of Al within the root is similarly matched by the production of the cell wall polymer callose, which is also highly localized to the epidermis and outer cortical region. Exposure to Al induced the rapid production of reactive oxygen species and induced a significant rigidification of the cell wall. Our results suggest that Al-induced root inhibition in maize occurs by rigidification of the epidermal layers.

Aluminum↗

PubMed2003

Touch and gravitropic set-point angle interact to modulate gravitropic growth in roots.

Plant roots must sense and respond to a variety of environmental stimuli as they grow through the soil. Touch and gravity represent two of the mechanical signals that roots must integrate to elicit the appropriate root growth patterns and root system architecture. Obstacles such as rocks will impede the general downwardly directed gravitropic growth of the root system and so these soil features must be sensed and this information processed for an appropriate alteration in gravitropic growth to allow the root to avoid the obstruction. We show that primary and lateral roots of Arabidopsis do appear to sense and respond to mechanical barriers placed in their path of growth in a qualitatively similar fashion. Both types of roots exhibited a differential growth response upon contacting the obstacle that directed the main axis of elongation parallel to the barrier. This growth habit was maintained until the obstacle was circumvented, at which point normal gravitropic growth was resumed. Thus, the gravitational set-point angle of the primary and lateral roots prior to encountering the barrier were 95 degrees and 136 degrees respectively and after growing off the end of the obstacle identical set-point angles were reinstated. However, whilst tracking across the barrier, quantitative differences in response were observed between these two classes of roots. The root tip of the primary root maintained an angle of 136 degrees to the horizontal as it traversed the barrier whereas the lateral roots adopted an angle of 154 degrees. Thus, this root tip angle appeared dependent on the gravitropic set-point angle of the root type with the difference in tracking angle quantitatively reflecting differences in initial set-point angle. Concave and convex barriers were also used to analyze the response of the root to tracking along a continuously varying surface. The roots maintained the a fairly fixed angle to gravity on the curved surface implying a constant resetting of this tip angle/tracking response as the curve of the surface changed. We propose that the interaction of touch and gravity sensing/response systems combine to strictly control the tropic growth of the root. Such signal integration is likely a critical part of growth control in the stimulus-rich environment of the soil.

Arabidopsis↗

PubMed2001

Arabidopsis thaliana Rop GTPases are localized to tips of root hairs and control polar growth.

Plants contain a novel unique subfamily of Rho GTPases, vital components of cellular signalling networks. Here we report a general role for some members of this family in polarized plant growth processes. We show that Arabidopsis AtRop4 and AtRop6 encode functional GTPases with similar intrinsic GTP hydrolysis rates. We localized AtRop proteins in root meristem cells to the cross-wall and cell plate membranes. Polar localization of AtRops in trichoblasts specifies the growth sites for emerging root hairs. These sites were visible before budding and elongation of the Arabidopsis root hair when AtRops accumulated at their tips. Expression of constitutively active AtRop4 and AtRop6 mutant proteins in root hairs of transgenic Arabidopsis plants abolished polarized growth and delocalized the tip-focused Ca2+ gradient. Polar localization of AtRops was inhibited by brefeldin A, but not by other drugs such as latrunculin B, cytochalasin D or caffeine. Our results demonstrate a general function of AtRop GTPases in tip growth and in polar diffuse growth.

Arabidopsis↗

PubMed2001

Localization of GAR transformylase in Escherichia coli and mammalian cells.

Enzymes of the de novo purine biosynthetic pathway may form a multienzyme complex to facilitate substrate flux through the ten serial steps constituting the pathway. One likely strategy for complex formation is the use of a structural scaffold such as the cytoskeletal network or subcellular membrane of the cell to mediate protein-protein interactions. To ascertain whether this strategy pertains to the de novo purine enzymes, the localization pattern of the third purine enzyme, glycinamide ribonucleotide transformylase (GAR Tfase) was monitored in live Escherichia coli and mammalian cells. Genes encoding human as well as E. coli GAR Tfase fused with green fluorescent protein (GFP) were introduced into their respective cells with regulated expression of proteins and localization patterns monitored by using confocal fluorescence microscopy. In both instances images showed proteins to be diffused throughout the cytoplasm. Thus, GAR Tfase is not localized to an existing cellular architecture, so this device is probably not used to concentrate the members of the pathway. However, discrete clusters of the pathway may still exist throughout the cytoplasm.

Animals↗

PubMed2001

Signal processing and transduction in plant cells: the end of the beginning?

Plants have a very different lifestyle to animals, and one might expect that unique molecules and processes would underpin plant-cell signal transduction. But, with a few notable exceptions, the list is remarkably familiar and could have been constructed from animal studies. Wherein, then, does lifestyle specificity emerge?

Animals↗

PubMed2001

A 90-kD phospholipase D from tobacco binds to microtubules and the plasma membrane.

The organization of microtubule arrays in the plant cell cortex involves interactions with the plasma membrane, presumably through protein bridges. We have used immunochemistry and monoclonal antibody 6G5 against a candidate bridge protein, a 90-kD tubulin binding protein (p90) from tobacco BY-2 membranes, to characterize the protein and isolate the corresponding gene. Screening an Arabidopsis cDNA expression library with the antibody 6G5 produced a partial clone encoding phospholipase D (PLD), and a full-length gene was obtained by sequencing a corresponding expressed sequence tag clone. The predicted protein of 857 amino acids contains the active sites of a phospholipid-metabolizing enzyme and a Ca(2+)-dependent lipid binding domain and is identical to Arabidopsis PLD delta. Two amino acid sequences obtained by Edman degradation of the tobacco p90 are identical to corresponding segments of a PLD sequence from tobacco. Moreover, immunoprecipitation using the antibody 6G5 and tobacco BY-2 protein extracts gave significant PLD activity, and PLD activity of tobacco BY-2 membrane proteins was enriched 6.7-fold by tubulin-affinity chromatography. In a cosedimentation assay, p90 bound and decorated microtubules. In immunofluorescence microscopy of intact tobacco BY-2 cells or lysed protoplasts, p90 colocalized with cortical microtubules, and taxol-induced microtubule bundling was accompanied by corresponding reorganization of p90. Labeling of p90 remained along the plasma membrane when microtubules were depolymerized, although detergent extraction abolished the labeling. Therefore, p90 is a specialized PLD that associates with membranes and microtubules, possibly conveying hormonal and environmental signals to the microtubule cytoskeleton.

Amino Acid Sequence↗

PubMed2001

Changes in root cap pH are required for the gravity response of the Arabidopsis root.

Although the columella cells of the root cap have been identified as the site of gravity perception, the cellular events that mediate gravity signaling remain poorly understood. To determine if cytoplasmic and/or wall pH mediates the initial stages of root gravitropism, we combined a novel cell wall pH sensor (a cellulose binding domain peptide-Oregon green conjugate) and a cytoplasmic pH sensor (plants expressing pH-sensitive green fluorescent protein) to monitor pH dynamics throughout the graviresponding Arabidopsis root. The root cap apoplast acidified from pH 5.5 to 4.5 within 2 min of gravistimulation. Concomitantly, cytoplasmic pH increased in columella cells from 7.2 to 7.6 but was unchanged elsewhere in the root. These changes in cap pH preceded detectable tropic growth or growth-related pH changes in the elongation zone cell wall by 10 min. Altering the gravity-related columella cytoplasmic pH shift with caged protons delayed the gravitropic response. Together, these results suggest that alterations in root cap pH likely are involved in the initial events that mediate root gravity perception or signal transduction.

Arabidopsis↗

PubMed2000

Increases in cytosolic Ca2+ are not required for abscisic acid-inhibition of inward K+ currents in guard cells of Vicia faba L.

The inward K+ channels (IKin) of guard cells are inhibited upon application of abscisic acid (ABA). It has been postulated that I(Kin) inhibition requires an elevation in cytosolic free Ca2+ levels ([Ca2+]c) because: (i) experimental increases in [Ca2+]c can mimic the ABA effect, and; (ii) ABA can trigger an elevation of [Ca2+]c in guard cells. However, not all guard cells respond to ABA with a [Ca2+]c increase, and the magnitude of the increases that do occur is variable. Therefore, an obligate role for Ca2+ in the regulation of downstream effectors of ABA response, such as the I(Kin) channels, remains in question. In this study, we developed a methodology for simultaneous patch clamping and confocal ratiometric Ca2+ imaging of Vicia faba L. guard-cell protoplasts. This allowed us to directly assess the relationship between ABA-induced changes in [Ca2+]c and I(Kin) inhibition. In the presence of extracellular Ca2+, the extent of [Ca2+]c elevation correlated with the extent of I(Kin) inhibition. However, upon chelation of either extracellular Ca2+, [Ca2+]c or both, extracellular Ca2+ and [Ca2+]c, [Ca2+]c elevation did not occur in response to ABA yet I(Kin) currents were still strongly inhibited. These data illustrate that Ca2+-independent regulation is involved in ABA-inhibition of stomatal opening processes.

Abscisic Acid↗

PubMed2000

Through form to function: root hair development and nutrient uptake.

Root hairs project from the surface of the root to aid nutrient and water uptake and to anchor the plant in the soil. Their formation involves the precise control of cell fate and localized cell growth. We are now beginning to unravel the complexities of the molecular interactions that underlie this developmental regulation. In addition, after years of speculation, nutrient transport by root hairs has been demonstrated clearly at the physiological and molecular level, with evidence for root hairs being intense sites of H(+)-ATPase activity and involved in the uptake of Ca(2+), K(+), NH(4)(+), NO(3)(-), Mn(2+), Zn(2+), Cl(-) and H(2)PO(4)(-).

Nitrogen↗

PubMed2000

Plant Signaling 2000. Cross talk among geneticists, physiologists, and ecologists.

Plants respond in complex ways to their environment, to their internal physiological status, and to the activity of other plants, pathogens, herbivores, and organisms. Plant Signaling 2000, a symposium sponsored by the Penn State Intercollege Graduate Program in Plant Physiology (May 18-20, 2000), explored the machinery underlying these responses and their potential for cross talk. We recount here some of the major themes emerging from this interdisciplinary symposium, which ranged from genetic and biochemical analyses of signaling pathways in Arabidopsis and other model plants to field studies of plants responding to insect damage.

Animals↗

PubMed2000

Abscisic acid stimulation of phospholipase D in the barley aleurone is G-protein-mediated and localized to the plasma membrane.

We have previously determined that phospholipase D (PLD) is activated by abscisic acid (ABA), and this activation is required for the ABA response of the cereal aleurone cell. In this study, ABA-stimulated PLD activity was reconstituted in vitro in microsomal membranes prepared from aleurone protoplasts. The transient nature (20 min) and degree (1.5- to 2-fold) of activation in vitro were similar to that measured in vivo. Stimulation by ABA was only apparent in the membrane fraction and was associated with a fraction enriched in plasma membrane. These results suggest that an ABA receptor system and elements linking it to PLD activation are associated with the aleurone plasma membrane. The activation of PLD in vitro by ABA was dependent on the presence of GTP. Addition of GTPgammaS transiently stimulated PLD in an ABA-independent manner, whereas treatment with GDPbetaS or pertussis toxin blocked the PLD activation by ABA. Application of pertussis toxin to intact aleurone protoplasts inhibited the ability of ABA to activate PLD as well as antagonizing the ability of ABA to down-regulate gibberellic acid-stimulated alpha-amylase production. All of these data support the hypothesis that ABA stimulation of PLD activity occurs at the plasma membrane and is mediated by G-protein activity.

Abscisic Acid↗

PubMed2000

Plant cell biology in the new millennium: new tools and new insights.

The highly regulated structural components of the plant cell form the basis of its function. It is becoming increasingly recognized that cellular components are ordered into regulatory units ranging from the multienzyme complexes that allow metabolic channeling during primary metabolism to the "transducon" complexes of signal transduction elements that allow for the highly efficient transfer of information within the cell. Against this structural background the highly dynamic processes regulating cell function are played out. Recent technological advances in three areas have driven our understanding of the complexities of the structural and functional dynamics of the plant cell. First, microscope and digital camera technology has seen not only improvements in the resolution of the optics and sensitivity of detectors, but also the development of novel microscopy applications such as confocal and multiphoton microscopy. These technologies are allowing cell biologists to image the dynamics of living cells with unparalleled three-dimensional resolution. The second advance has been in the availability of increasingly powerful and affordable computers. The computer control/analysis required for many of the new microscopy techniques was simply unavailable until recently. Third, there have been dramatic advances in the available probes to use with these new microscopy approaches. Thus the plant cell biologist now has available a vast array of fluorescent probes that will report cell parameters as diverse as the pH of the cytosol, the oxygen level in a tissue, or the dynamics of the cytoskeleton. The combination of these new approaches has led to an increasingly detailed picture of how plant cells regulate their activities.

Journal Article↗

PubMed1999

Abscisic acid signal transduction in guard cells is mediated by phospholipase D activity.

In guard cells, the plant hormone abscisic acid (ABA) inhibits stomatal opening and induces stomatal closure through the coordinated regulation of ion transport. Despite this central role of ABA in regulating stomatal function, the signal transduction events leading to altered ion fluxes remain incompletely understood. We report that the activity of the enzyme phospholipase D (PLD) transiently increased in guard cell protoplasts at 2.5 and 25 min after ABA application. Treatment of guard cell protoplasts with phosphatidic acid (PtdOH), one of the products of PLD activity, led to an inhibition of the activity of the inward K+ channel. PtdOH also induced stomatal closure and inhibited stomatal opening when added to epidermal peels. Application of 1-butanol (1-buOH), a selective inhibitor of PtdOH production by PLD, inhibited the increase in PtdOH production elicited by ABA. 1-BuOH treatment also partially prevented ABA-induced stomatal closure and ABA-induced inhibition of stomatal opening. This inhibitory effect of buOH was enhanced by simultaneous application of nicotinamide, an inhibitor of cADP ribose action. These results suggest that in the guard cell, ABA activates the enzyme PLD, which leads to the production of PtdOH. This PtdOH is then involved in triggering subsequent ABA responses of the cell via a pathway operating in parallel to cADP ribose-mediated events.

Journal Article↗

PubMed1999

Laser ablation of root cap cells: implications for models of graviperception.

The initial event of gravity perception by plants is generally thought to occur through sedimentation of amyloplasts in specialized sensory cells. In the root, these cells are the columella which are located toward the center of the root cap. To define more precisely the contribution of columella cells to root gravitropism, we used laser ablation to remove single columella cells or groups of these cells and observed the effect of their removal on gravity sensing and response. Complete removal of the cap or all the columella cells (leaving peripheral cap cells intact) abolishes the gravity response of the root. Removal of stories of columella revealed differences between regions of the columella with respect to gravity sensing (presentation time) versus graviresponse (final tropic growth response of the root). This fine mapping revealed that ablating the central columella located in story 2 had the greatest effect on presentation time whereas ablating columella cells in story 3 had a smaller or no effect. However, when removed by ablation the columella cells in story 3 did inhibit gravitropic bending, suggesting an effect on translocation of the gravitropic signal from the cap rather than initial gravity perception. Mapping the in vivo statolith sedimentation rates in these cells revealed that the amyloplasts of the central columella cells sedimented more rapidly than those on the flanks do. These results show that cells with the most freely mobile amyloplasts generate the largest gravisensing signal consistent with the starch statolith hypothesis of gravity sensing in roots.

Arabidopsis↗

PubMed1999

Microtubules regulate tip growth and orientation in root hairs of Arabidopsis thaliana.

The polarized growth of cells as diverse as fungal hyphae, pollen tubes, algal rhizoids and root hairs is characterized by a highly localized regulation of cell expansion confined to the growing tip. In apically growing plant cells, a tip-focused [Ca2+]c gradient and the cytoskeleton have been associated with growth. Although actin has been established to be essential for the maintenance of elongation, the role of microtubules remains unclear. To address whether the microtubule cytoskeleton is involved in root hair growth and orientation, we applied microtubule antagonists to root hairs of Arabidopsis. In this report, we show that depolymerizing or stabilizing the microtubule cytoskeleton of these apically growing root hairs led to a loss of directionality of growth and the formation of multiple, independent growth points in a single root hair. Each growing point contained a tip-focused gradient of [Ca2+]c. Experimental generation of a new [Ca2+]c gradient in root hairs pre-treated with microtubule antagonists, using the caged-calcium ionophore Br-A23187, was capable of inducing the formation of a new growth point at the site of elevated calcium influx. These data indicate a role for microtubules in regulating the directionality and stability of apical growth in root hairs. In addition, these results suggest that the action of the microtubules may be mediated through interactions with the cellular machinery that maintains the [Ca2+]c gradient at the tip.

Arabidopsis↗

PubMed1999

The sensitivity of barley aleurone tissue to gibberellin is heterogeneous and may Be spatially determined

In cereals, gibberellin (GA) enhances the synthesis and secretion of hydrolytic enzymes from aleurone cells. These enzymes then mobilize the endosperm storage reserves that fuel germination. The dose-response curve of aleurone protoplasts to GA extends over a range of concentrations from 10(-11) to more than 10(-6) M. One hypothesis is that subpopulations of cells have different sensitivities to GA, with each cell having a threshold concentration of GA above which it is switched on. The dose-response curve therefore reflects a gradual recruitment of cells to the pool exhibiting a full GA response. Alternatively, all cells may gradually increase their responses as the GA level is increased. In the present study we found that at increasing GA concentrations, increasing numbers of barley (Hordeum vulgare) cells showed the enhanced amylase secretion and vacuolation characteristic of the GA response. We also observed that the region of aleurone tissue closest to the embryo contains the highest proportion of cells activated at the GA concentrations thought to occur naturally in germinating grain. These data indicate that an aleurone layer contains cells of varying sensitivities to GA and that recruitment of these differentially responding pools of cells may explain the broad dose response to GA.

Journal Article↗

PubMed1998

Abscisic acid signal transduction in the barley aleurone is mediated by phospholipase D activity.

The plant hormones abscisic acid (ABA) and gibberellic acid (GA) are important regulators of the dormancy and germination of seeds. In cereals, GA enhances the synthesis and secretion of enzymes (principally alpha-amylases) in the aleurone cells of the endosperm, which then mobilize the storage reserves that fuel germination. ABA inhibits this enhanced secretory activity and delays germination. Despite the central role of ABA in regulating germination, the signal transduction events leading to altered gene expression and cellular activity are essentially unknown. We report that the application of ABA to aleurone protoplasts increased the activity of the enzyme phospholipase D (PLD) 10 min after treatment. The product of PLD activity, phosphatidic acid (PPA), also increased transiently at this time. The application of PPA to aleurone protoplasts led to an ABA-like inhibition of alpha-amylase production, and induction of the ABA up-regulated proteins ASI (amylase subtilisin inhibitor) and RAB (responsive to ABA). Inhibition of PLD activity by 0.1% 1-butanol during the initial 20 min of ABA treatment resulted in inhibition of ABA-regulated processes. This inhibition coincided with the timing of PLD activation by ABA and was overcome by simultaneous addition of PPA. These results suggest that ABA activates the enzyme PLD to produce PPA that is involved in triggering the subsequent ABA responses of the aleurone cell.

1-Butanol↗

PubMed1998

Calcium-Dependent Protein Phosphorylation May Mediate the Gibberellic Acid Response in Barley Aleurone

Peptide substrates of well-defined protein kinases were microinjected into aleurone protoplasts of barley (Hordeum vulgare L. cv Himalaya) to inhibit, and therefore identify, protein kinase-regulated events in the transduction of the gibberellin (GA) and abscisic acid signals. Syntide-2, a substrate designed for Ca2+- and calmodulin (CaM)-dependent kinases, selectively inhibited the GA response, leaving constitutive and abscisic acid-regulated events unaffected. Microinjection of syntide did not affect the GA-induced increase in cytosolic [Ca2+], suggesting that it inhibited GA action downstream of the Ca2+ signal. When photoaffinity-labeled syntide-2 was electroporated into protoplasts and cross-linked to interacting proteins in situ, it selectively labeled proteins of approximately 30 and 55 kD. A 54-kD, soluble syntide-2 phosphorylating protein kinase was detected in aleurone cells. This kinase was activated by Ca2+ and was CaM independent, but was inhibited by the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (250 mum), suggesting that it was a CaM-domain protein kinase-like activity. These results suggest that syntide-2 inhibits the GA response of the aleurone via an interaction with this kinase, implicating the 54-kD kinase as a Ca2+-dependent regulator of the GA response in these cells.

Journal Article↗