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Protocols in ecological and environmental plant physiology


Article << Previous     |     Next >>   Contents Vol 31(9)

Hydrogen peroxide is a common signal for darkness- and ABA-induced stomatal closure in Pisum sativum

Radhika Desikan A, Man-Kim Cheung A, Andrew Clarke A, Sarah Golding A, Moshe Sagi B, Robert Fluhr C, Christopher Rock D, John Hancock A, Steven Neill A E

A Centre for Research in Plant Science, Genomics Research Institute, Faculty of Applied Sciences, University of the West of England, Bristol, Coldharbour Lane, Bristol BS16 1QY, UK.
B The Institutes for Applied Research, PO Box 653, Beer Sheava 84105, Israel.
C Department of Plant Science, Weizmann Institute of Science, PO Box 26, Rehovot 76100, Israel.
D Department of Biological Sciences, Texas Tech University, PO Box 43131, Lubbock, TX 79409, USA.
E Corresponding author; email: steven.neill@uwe.ac.uk
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The requirement for hydrogen peroxide (H2O2) generation and action during stomatal closure induced by darkness and abscisic acid (ABA) was investigated in pea (Pisum sativum L.). Stomatal closure induced by darkness or ABA was inhibited by the H2O2-scavenging enzyme catalase or the antioxidant N-acetyl cysteine (NAC), or by diphenylene iodonium (DPI), an inhibitor of the H2O2-generating enzyme NADPH oxidase. Exogenous H2O2 induced stomatal closure in a dose- and time-dependent manner, and H2O2 was also required for ABA-inhibition of stomatal opening in the light. H2O2 accumulation in guard cells was increased by darkness or ABA, as assessed with the fluorescent dye dichlorodihydrofluorescein diacetate (H2-DCFDA) and confocal microscopy. Such increases were inhibited by catalase, NAC or DPI, consistent with the effects of these compounds on stomatal apertures. Employing polymerase chain reaction (PCR) with degenerate oligonucleotide primers, several NADPH oxidase homologues were identified from pea genomic DNA that had substantial identity to the Arabidopsis thaliana (L.) Heynh. rboh (respiratory burst oxidase homologue) genes. Furthermore, an antibody raised against the tomato rboh identified immunoreactive proteins in epidermal, mesophyll and guard cells.

Keywords: abscisic acid, darkness, diphenylene iodonium, guard cells, hydrogen peroxide, NADPH oxidase.

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