Regulation of guard cell anion channels

Wang, Yizhou (2013) Regulation of guard cell anion channels. PhD thesis, University of Glasgow.

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Stomata account for much of the 70% of global water usage associated with agriculture, and have a profound impact on the water and carbon cycles of the world. Anion channels at the plasma membrane of the guard cell are thought to comprise a major pathway for anion efflux essential for driving stomatal closure. The activity of these channels is therefore tightly linked to abscisic acid (ABA)-dependent stomatal movements.

Both the inorganic anion Cl- and the organic acid anion malate (Mal) are transported during stomatal movements. The metabolism of organic acids, primarily Mal plays an especially important role in these processes. However, little is known of the regulation of anion channel current (ICl) or its connection with cytosolic Mal and its immediate metabolite oxaloacetate (OAA). Work described here focuses on the relation of ICl and its connections with metabolism and signals controlling other transport at the guard cell plasma membrane. Thus the impact of Mal, OAA and of acetate in Vicia guard cells was examined, and results shown that all three organic acids affect ICl with different characteristics. Most prominent, the suppression of ICl by OAA within the physiological range in vivo indicates a capacity for OAA to co-ordinate organic acid metabolism with ICl through a direct effect of organic acid pool size.

In a second set of studies, the ABA pathway that elevates cytosolic free Ca2+ ([Ca2+]i) in order to activate ICl was explored. These studies build on the discovery of PYR/PRL ABA receptors. Previous analysis of the pyr1/pyl1/pyl2/pyl4 mutant suggested that [Ca2+]i increases are suppressed. However, direct evidence had not been forthcoming. Thus a combination of voltage clamp and fluorescent ratio analysis with the Ca2+-sensitive dye Fura2 was used to show that the activity of Ca2+ channels (ICa) at the plasma membrane in the pyr1/pyl1/pyl2/pyl4 mutant is not activated in ABA, with the effect that [Ca2+]i increases were suppressed. Further studies showed that the normal action of ABA in promoting reactive oxygen species (ROS) was impaired, although adding H2O2 was sufficient to activate the ICa and trigger stomatal closure in the mutant. These results offer direct evidence that the PYR/PYL receptor proteins contribute to the activation by ABA of ICa through ROS, thus affecting [Ca2+]i and its regulation of osmotic solute flux for stomatal closure.

Finally, the connection between ICl and the K+ channel currents in the slac1 mutant of Arabidopsis was studied. These studies employed systems dynamic modelling to explain the paradoxical suppression of the inward-rectifying K+ channel current, IK,in and slowing of stomatal opening, by mutation that eliminated the SLAC1 anion channel. Experimental results confirmed the model predictions that the abnormal cytosolic pH (pHi) and [Ca2+]i suppresses IK,in in the slac1 mutant, through measuring pHi and [Ca2+]i in vivo, and experimental manipulation of pHi and [Ca2+]i is sufficient to recover IK,in and stomatal opening. These data uncover a previously unrecognised signaling network that minimises the effects of the slac1 mutant on transpiration, and they underscore the importance of H+-coupled anion transport for pHi homeostasis.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Guard Cell; Ion Channels; Organic Acid; ABA; cytosolic pH and Ca2+
Subjects: Q Science > QK Botany
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Supervisor's Name: Blatt, Professor Michael
Date of Award: January 2013
Depositing User: Mr Yizhou Wang
Unique ID: glathesis:2013-3859
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 07 Feb 2013 16:18
Last Modified: 09 Feb 2016 16:38

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