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Alistair M Hetherington

Publications and source records attributed to Alistair M Hetherington.

14 recordsLinked to original sources
PubMed2006

The identification of genes involved in the stomatal response to reduced atmospheric relative humidity.

Stomatal pores of higher plants close in response to decreases in atmospheric relative humidity (RH). This is believed to be a mechanism that prevents the plant from losing excess water when exposed to a dry atmosphere and as such is likely to have been of evolutionary significance during the colonization of terrestrial environments by the embryophytes. We have conducted a genetic screen, based on infrared thermal imaging, to identify Arabidopsis genes involved in the stomatal response to reduced RH. Here we report the characterization of two genes, identified during this screen, which are involved in the guard cell reduced RH signaling pathway. Both genes encode proteins known to be involved in guard cell ABA signaling. OST1 encodes a protein kinase involved in ABA-mediated stomatal closure while ABA2 encodes an enzyme involved in ABA biosynthesis. These results suggest, in contrast to previously published work, that ABA plays a role in the signal transduction pathway connecting decreases in RH to reductions in stomatal aperture. The identification of OST1 as a component required in stomatal RH and ABA signal transduction supports the proposition that guard cell signaling is organized as a network in which some intracellular signaling proteins are shared among different stimuli.

Abscisic Acid↗
PubMed2006

Biolistic delivery of Ca2+ dyes into plant and algal cells.

In eukaryotes, changes in cytosolic Ca2+ concentrations ([Ca2+]cyt) are associated with a number of environmental and developmental stimuli. However, measuring [Ca2+]cyt changes in single plant or algal cells is often problematic. Although a wide range of Ca2+-sensitive fluorescent dyes is available, they are often difficult to introduce into plant cells. Micro-injection is the most robust method for dye loading, but is time-consuming, technically demanding, and unsuitable in many cell types. To overcome these problems, we have adapted biolistic techniques to load Ca2+-sensitive dyes into guard cells of the flowering plant, Commelina communis, cells of the green alga Chlamydomonas reinhardtii, and zygotes of the brown alga, Fucus serratus. Using this approach, we have been able to monitor [Ca2+]cyt changes in response to various stimuli, including a novel [Ca2+]cyt response in C. reinhardtii. The method allows the use of free acid and dextran-conjugated dyes. Biolistic loading of differentiated plant cells is easier, quicker, and more widely applicable than micro-injection, and should broaden the study of plant signal transduction.

Animals↗
PubMed2005

AtMYB61, an R2R3-MYB transcription factor controlling stomatal aperture in Arabidopsis thaliana.

Stomata, dynamic pores found on the surfaces of plant leaves, control water loss from the plant and regulate the uptake of CO(2) for photosynthesis. Stomatal aperture is controlled by the two guard cells that surround the stomatal pore. When the two guard cells are fully turgid, the pore gapes open, whereas turgor loss results in stomatal closure. In order to set the most appropriate stomatal aperture for the prevailing environmental conditions, guard cells respond to multiple internal and external signals. Although much is known about guard-cell signaling pathways, rather little is known about how changes in gene expression are involved in the control of stomatal aperture. We show here that AtMYB61 (At1g09540), a gene encoding a member of the Arabidopsis thaliana R2R3-MYB family of transcription factors, is specifically expressed in guard cells in a manner consistent with involvement in the control of stomatal aperture. Gain-of-function and loss-of-function mutant analyses reveal that AtMYB61 expression is both sufficient and necessary to bring about reductions in stomatal aperture with consequent effects on gas exchange. Taken together, our data provide evidence that AtMYB61 encodes the first transcription factor implicated in the closure of stomata.

Arabidopsis↗
PubMed2005

The vacuolar Ca2+-activated channel TPC1 regulates germination and stomatal movement.

Cytosolic free calcium ([Ca2+]cyt) is a ubiquitous signalling component in plant cells. Numerous stimuli trigger sustained or transient elevations of [Ca2+]cyt that evoke downstream stimulus-specific responses. Generation of [Ca2+]cyt signals is effected through stimulus-induced opening of Ca2+-permeable ion channels that catalyse a flux of Ca2+ into the cytosol from extracellular or intracellular stores. Many classes of Ca2+ current have been characterized electrophysiologically in plant membranes. However, the identity of the ion channels that underlie these currents has until now remained obscure. Here we show that the TPC1 ('two-pore channel 1') gene of Arabidopsis thaliana encodes a class of Ca2+-dependent Ca2+-release channel that is known from numerous electrophysiological studies as the slow vacuolar channel. Slow vacuolar channels are ubiquitous in plant vacuoles, where they form the dominant conductance at micromolar [Ca2+]cyt. We show that a tpc1 knockout mutant lacks functional slow vacuolar channel activity and is defective in both abscisic acid-induced repression of germination and in the response of stomata to extracellular calcium. These studies unequivocally demonstrate a critical role of intracellular Ca2+-release channels in the physiological processes of plants.

Arabidopsis↗
PubMed2005

ROS perception in Arabidopsis thaliana: the ozone-induced calcium response.

Ozone is responsible for more crop losses than any other air pollutant. The changes in gene expression, which occur in plants in response to ozone, have been well characterized, yet little is known about how ozone is perceived or the signal transduction steps that follow. The earliest characterized response to ozone is an elevation in cytosolic-free calcium, which takes place within seconds of exposure. In this study, the calcium response to ozone was investigated in Arabidopsis thaliana seedlings using a variety of fumigation protocols. Ozone elicited distinct calcium responses in the aerial tissue and roots of seedlings. The calcium response in the cotyledons and leaves was biphasic and sensitive to the rate at which the ozone concentration increased. The response in the root was monophasic and insensitive to the rate of increase in ozone concentration. Experiments utilizing inhibitors of antioxidant metabolism demonstrated that the magnitude of the first peak in calcium in the aerial tissues was dependent upon the redox status of the plant. Seedlings were shown to be able to distinguish between ozone and hydrogen peroxide, producing a calcium signal in response to one of these reactive oxygen species (ROS) when they had become refractory to the other. Pre-treatment with ozone altered the calcium response to hydrogen peroxide and vice versa, indicating that the calcium response to a given ROS may reflect the stress history of the plant. These data suggest ROS signalling is more sophisticated than previously realized and raise questions over current models of ozone perception.

Arabidopsis↗
PubMed2004

Plant development: YODA the stomatal switch.

The appearance of stomatal pores during plant evolution is believed to have been a crucial step in land colonisation. A recent screen for genes involved in stomatal development has identified for the first time a mutant plant with no stomata; the results implicate a MAP kinase cascade in stomatal development.

Adaptation, Physiological↗
PubMed2004

Seeing 'cool' and 'hot'--infrared thermography as a tool for non-invasive, high-throughput screening of Arabidopsis guard cell signalling mutants.

The use of Arabidopsis mutants defective in abscisic acid (ABA) perception has been instrumental in the understanding of stomatal function, in particular, ABA signalling in guard cells. The considerable attention devoted to ABA signalling in guard cells is due in part to (1) the fundamental role of ABA in drought stress and (2) the use of a screening protocol based on the sensitivity of seed germination to ABA. Such a screen has facilitated the isolation of ABA signalling mutants with genetic lesions that exert pleiotropic effects at the whole plant level. As such, there is a requirement for new approaches to complement the seed germination screen. The recent advances made in the use of infrared thermography as a non-invasive, high-throughput tool are reviewed here and the versatility of this technique for screening Arabidopsis defective in stomatal regulation is highlighted.

Abscisic Acid↗
PubMed2004

The generation of Ca(2+) signals in plants.

The calcium ion is firmly established as a ubiquitous intracellular second messenger in plants. At their simplest, Ca(2+)-based signaling systems are composed of a receptor, a system for generating the increase in [Ca(2+)]cyt, downstream components that are capable of reacting to the increase in [Ca(2+)]cyt, and other cellular systems responsible for returning [Ca(2+)]cyt to its prestimulus level. Here we review the various mechanisms responsible for generating the stimulus-induced increases in [Ca(2+)]cyt known as Ca(2+) signals. We focus particularly on the mechanisms responsible for generating [Ca(2+)]cyt oscillations and transients and use Nod Factor signaling in legume root hairs and stimulus-response coupling in guard cells to assess the physiological significance of these classes of Ca(2+) signals.

Calcium↗
PubMed2003

The effects of manipulating phospholipase C on guard cell ABA-signalling.

Studies using stably transformed tobacco plants containing very low levels of PI-PLC in their guard cells show that this enzyme plays a role in the events associated with the inhibition of stomatal opening by ABA, but not in the cellular reactions that are responsible for ABA-induced stomatal closure. However, Commelina communis guard cells microinjected with the InsP3 antagonist, heparin, fail to close on addition of ABA. There are three possible explanations for this apparent data mismatch. The differences may be indicative of species-specific signalling pathways, the presence of a PI-PLC isoform(s) that is not down-regulated in these transgenic lines and/or they may reflect differences between short-term (acute) administration of an inhibitor and long-term (chronic) effects of gene manipulation. It is possible that the guard cell is a robust signalling system that is able to adapt or compensate for the chronic loss of PI-PLC, but which is unable to adjust quickly to acute loss of this component. It would be interesting to investigate this possibility further using either transient manipulation of gene expression or through the use of an inducible promoter.

Abscisic Acid↗
PubMed2003

Mitochondria provide the main source of cytosolic ATP for activation of outward-rectifying K+ channels in mesophyll protoplast of chlorophyll-deficient mutant rice (OsCHLH) seedlings.

The role of mitochondria in providing intracellular ATP that controls the activity of plasma membrane outward-rectifying K+ channels was evaluated. The OsCHLH rice mutant, which lacks chlorophyll in the thylakoids, was isolated by T-DNA gene trapping (Jung, K.-H., Hur, J., Ryu, C.-H., Choi, Y., Chung, Y.-Y., Miyao, A., Hirochika, H., and An, G. (2003) Plant Cell Physiol. 44, 463-472). The OsCHLH mutant is unable to fix CO2 and exhibits reduced growth. Wild type and mutant plants exhibit similar rates of respiratory O2 uptake in the dark, whereas the rate of photosynthetic O2 evolution by the mutant was negligible during illumination. During dark respiration the wild type and mutant exhibited similar levels of cytoplasmic ATP. In the mutant oligomycin treatment (an inhibitor of mitochondrial F1F0-ATPase) drastically reduced ATP production. The fact that this was reversed by the addition of glucose suggested that the mutant produced ATP exclusively from mitochondria but not from chloroplasts. In whole cell patch clamp experiments, the activity of outward-rectifying K+ channels of rice mesophyll cells showed ATP-dependent currents, which were 1.5-fold greater in wild type than in mutant cells. Channels in both wild type and mutant cells were deactivated by the removal of cytosolic ATP, whereas in the presence of ATP the channels remained active. We conclude that mesophyll cells in the OsCHLH rice mutant derive ATP from mitochondrial respiration, and that this is critical for the normal function of plasma membrane outward-rectifying K+ channels.

Adenosine Triphosphate↗
PubMed2003

The role of stomata in sensing and driving environmental change.

Stomata, the small pores on the surfaces of leaves and stalks, regulate the flow of gases in and out of leaves and thus plants as a whole. They adapt to local and global changes on all timescales from minutes to millennia. Recent data from diverse fields are establishing their central importance to plant physiology, evolution and global ecology. Stomatal morphology, distribution and behaviour respond to a spectrum of signals, from intracellular signalling to global climatic change. Such concerted adaptation results from a web of control systems, reminiscent of a 'scale-free' network, whose untangling requires integrated approaches beyond those currently used.

Acclimatization↗
PubMed2003

Sphingolipids, new players in plant signaling.

Sphingolipids are a diverse group of compounds, some of which play important signaling roles in animals and yeast. Results from recent research suggest that not only do plants contain components present in animal sphingolipid signaling pathways but that they might also possess novel plant-specific sphingolipid signaling systems. We suggest that the time is ripe for an in depth investigation of the role of this enigmatic group of compounds in plants.

Animals↗
PubMed2003

Phospholipase C is required for the control of stomatal aperture by ABA.

The calcium-releasing second messenger inositol 1,4,5-trisphosphate is involved in the regulation of stomatal aperture by ABA. In other signalling pathways, inositol 1,4,5-trisphosphate is generated by the action of phospholipase C. We have studied the importance of phospholipase C in guard cell ABA-signalling pathways. Immunolocalisation of a calcium-activated phospholipase C confirmed the presence of phospholipase C in tobacco guard cells. Transgenic tobacco plants with considerably reduced levels of phospholipase C in their guard cells were only partially able to regulate their stomatal apertures in response to ABA. These results suggest that phospholipase C is involved in the amplification of the calcium signal responsible for reductions in stomatal aperture in response to ABA. As full ABA-induced inhibition of stomatal opening was not observed, our results support a role for the action of other calcium-releasing second messengers in the guard cell ABA-signalling pathway. It is not known whether these different calcium-releasing second messengers act in the same or parallel ABA-signalling pathways.

Abscisic Acid↗