The Physiology of Flowering Plants - KHAM PHA MOI

HOW DO PLANT HORMONES CAUSE RESPONSES? 199stomata by ABA is extremely rapid and is not thought to involvechanges in gene expression, but many other plant responses to thishormone certainly will. It is thought that elevated ABA concentrationsinitiate a series of rapid ion movements, culminating in themovement of certain ions and solutes out of the vacuole of the guardcells and into the apoplast. This results in the loss of water from thecells via osmosis, a loss of turgor, and the closure of the stomata.When ABA is applied to guard cells, ion channels are activatedwhich result in a rapid influx of Ca 2+ . ABA does not act directly onCa 2+ channels, rather binding of ABA to a receptor is considered toactivate an enzyme which rapidly synthesizes a small signallingmolecule, cyclic adenosine 5 0 -diphosphate ribose (cADPR) (Wu et al.1997, Leckie et al. 1998). The Ca 2+ influx occurs within seconds, and istransient, making measurements difficult. However, the Ca 2+ concentrationwithin the guard cell can be visualized by injecting thecells with a Ca 2+ -sensitive fluorescent dye such as Fura-2. The colourof Fura-2 fluorescence depends upon the Ca 2+ concentration.Alternatively, plants can be genetically modified to express proteinswhich emit light, or whose fluorescent properties change, uponbinding Ca 2+ , allowing the Ca 2+ concentration in the cytoplasm tobe determined. Application of ABA causes a wave of Ca 2+ to appear inthe cytoplasm within seconds, apparently released from the cell wallin some cases and the vacuole in others (McAinsh et al. 1992). Theincrease in cytosolic Ca 2+ causes ion channels which allow K + to enterthe cell to be inactivated whilst channels which allow Cl and malateto leave the cell are activated. The loss of Cl and malate from the cellcauses a depolarization of the guard cell plasma membrane and thiscauses the activation of outward K + channels. The loss of ions fromthe cell results in the loss of turgor leading to stomatal closure.Allen et al. (2001) demonstrated that guard cells respond to precisepatterns of oscillations in intracellular Ca 2+ . Guard cells, isolated fromgenetically modified Arabidopsis plants expressing the Cameleonprotein, were alternately washed in buffers containing high or lowconcentrations of Ca 2+ , allowing the intracellular Ca 2+ content to bealtered in a very precise manner. It was shown that a series of Ca 2+spikes, or transient increases in concentration, were required toclose the stomata, and that these needed to be of a particular lengthand duration (Fig. 7.11). These oscillations can therefore be consideredas a digital signal which controls stomatal aperture.Changes in Ca 2+ concentration are also thought to activate aprotein kinase cascade leading to the activation of gene expression.These genes, although not involved in the control of stomatal aperture,are thought to be responsible for the other effects of ABA such asincreased stress tolerance.Box 7.3Both methods for visualizing theCa 2+ concentration use proteinsisolated from the jellyfish Aequoreavictoria. Aequorin is a luminescentprotein which, in the presence ofthe compound coelenterazine,emits blue light when it binds Ca 2+ .This can be detected using an ultrasensitivecamera. In the living jellyfishthis blue light is absorbed by asecond protein, green fluorescentprotein (GFP), which, as its nameimplies, fluoresces green. GFP itselfhas been genetically modified sothat its fluorescence alters in aCa 2+ -dependent manner. Thisengineered version is evocativelycalled ‘Cameleon’ and allowsintracellular Ca 2+ concentrationsto be determined without havingto apply coelenterazine to theplant. The development of these,and other biosensors, is having adramatic impact on all areas ofbiology.7.4.5 Hormone-induced changes in gene expressionLong-term changes in plant growth and development which occur inresponse to plant growth hormones will require changes in gene