1428common event along with H2O2 production inresponse to ABA or MJ (Pei etal. 2000; Zhang etal.2001; Suhita etal. 2004). In contrast, a change in CO2partial pressure from 100 to 1000ppm did not evokeany cytoplasmic pH change in V. faba guard cells(Brearley etal. 1997). In accordance, we observed thatthe eVect of bicarbonate seems independent of a cyto-plasmic pH change since butyrate (a permeant weakacid) had no signiWcant eVect on the bicarbonate-induced stomatal closure (data not shown).Sources of bicarbonate-induced H2O2 in guard cellsIn plant cells, H2O2 can be produced in multiple ways(Park etal. 2003; Apel and Hirt 2004). One of the well-known sources of H2O2 in guard cells is the plasmamembrane NAD(P)H oxidase (Murata etal. 2001;Kwak etal. 2003; Suhita etal. 2004). Membrane peroxi-dation occurs during stress conditions leading to a sig-niWcant production of ROS (Montillet etal. 2004). Thebicarbonate-induced H2O2 production in guard cellswas diminished by DPI (Fig.7). The absence of H2O2production as well as stomatal closure in response tobicarbonate in atrbohD/F double mutant of Arabidop-sis (Fig.5) provided convincing genetic evidence thatNAD(P)H oxidase was responsible for bicarbonate-induced H2O2 production in guard cells. These observa-tions suggest that H2O2 production in response to bicar-bonate results from NAD(P)H oxidase. Externaladdition of H2O2 enhanced DCF Xuorescence of guardcells in both wild and Atrhoh D/F mutants (Fig.7) indi-cating that NAD(P)H oxidase was the major source ofinternal production of H2O2. Organelles containingelectron transport systems, such as mitochondria andchloroplasts, can also produce H2O2 (Asada 1999),although the direct involvement of such processes insignaling is not yet clear. Zhang etal. (2001) detectedH2O2 in chloroplasts and in the cytosol of guard cells.There are reports that PP2C, protein kinase and PI3Pcould directly or indirectly regulate ROS production inguard cells, particularly during ABA signaling (Murataetal. 2001; Mustilli etal. 2002; Park etal. 2003). Thereversal of stomatal closure (Fig.4) as well as restric-tion of H2O2 production (Fig.8) by wortmannin andLY 294002 suggests that PI3P is involved in the signal-ing of not only ABA but also bicarbonate in guard cells.H2O2 is a possible secondary messenger during CO2 response
ROS production appears to be a dynamic event duringplant development as well as plant response to bioticand abiotic stresses (Apel and Hirt 2004). We there-
123
Planta (2007) 225:1421–1429
fore suggest that H2O2 could be an important second-ary messenger during the transduction of CO2 signal innot only guard cells but also other plant tissues. TheH2O2 production in guard cells appears to be an earlyevent on exposure to bicarbonate.
AcknowledgmentsThis work was supported by grants fromIndo-French Centre for the promotion of Advanced Research (toA.S.R and A.V, No. 2203-1) and Council of ScientiWc and Indus-trial Research (No. 38(0949)/99/EMR-II) both from New Delhi;and a senior research fellowship (to V.A.K) from Institute of LifeScience Grant to University of Hyderabad. We thank ProfessorJulian Schroeder for providing the atrbohD/F double mutantseeds of Arabidopsis. We thank C. S. Murthy, Sr. ScientiWc OY-cer, Central Instrumentation Laboratory for his constant help inusing confocal microscope.
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