Proc. Indian Acad. Sci., Vol. 83 B, No. 6, 1976, pp Mehler-type reagents in the enhancement of stomatal opening in epidermal strips

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1 Proc. Indian Acad. Sci., Vol. 83 B, No. 6, 1976, pp Mehler-type reagents in the enhancement of stomatal opening in epidermal strips V. S. RAMA DAS, F.A.Sc. AND M. SANTHAKUMARI Department of Botany, Sri Venkateswara University, Tirupati MS received 14 February 1976 ABSTRACT A substantial enhancement of the width of stomatal aperture was produced by methyl viologen (MV) a Mehler,reagent, in isolated epidermal strips from the leaves of Commelina benghalensis L., and Pisum sativum L., other Mehler reagents, flavin mononueleotide and anthraquinone were also found to stimulate the opening process. Histochemical tests have revealed a much greater influx of K + into the guard cells in presence of MV. Isolated mesophyll chloroplasts possessed appreciable activity of MV-supported photophosphorylation and of oxygen uptake through Mehlcr reaction. 1. INTRODUCTION THE involvement of Photosynthesis in the light-induced opening of stomata has been interpreted in two different ways. The classical view which has recently been reviewed by Raschl~e 1 holds that the participation of light energy in the opening process of stomata is through an indirect means of the depletion of carbon dioxide in the guard cells. Recent results from this Laboratory2, 3 have revealed a direct role of light through the photosynthetic-lly generated energy rather than the carbon metabolism that is responsible for the opening of stomata. Various lines of evidence were thus presented indicating that the energy derived through Cyclic phosphorylaticn is responsible for the stomata opening~,4,l The present work is attempted with a view to distinguish between the effect of energy supply from that of the influence of carbon metabolism during the light induced stomatal movements. Methyl viologen is a well known inhibitor of NADP photoreduction and thus prevents COs uptake and its assimilation. 6-1 Whitehouse et ap 1 have studied severai aspects of the MV-supported Mehler reaction in isolated chloroplasts and observed promotion of K + B2mJan

2 238 V.S. RAMA DAS AND M. SANTHAKUMARI influx associated with a marked increase in the rate of the Mehler reaction Recently Gimmter et al. ~2 also showed an MV-enhanced K ~ influx in intact and brokcn chloroplasts in the presence of light and such an influx was attributed by them to Mehler-reaction. Stimulation of ATP production in the presence of MV through an enhancement of photosynthetic phosphorylation was observed earlier and (Ref ) this stimulation was attributed to a faster electron transport by Newmann et al ~6 and Fujita. 17 Presumably the greater ~nflux of K + could result due to a stimulated energy supply in presence of MV and such other autoxidizable photo-system I acceptors. The present investigation has been attempted to reconfirm the direct effect of light in the stomatal opening through the use of rapidly-autoxidizable Mehler type reagents that block carbon assimilation by competing with NADP photoreduction. 2. MATERIALS AND METHODS Commelina benghalensis L., and Fisum sativum L., experimental garden under natural photopeliod. were grown in the The epidermal strips of about 1.0 0' 5 cm from the freshly excised leaves were removed into and floated on 15 ml of 0.05 M Phosphate buffer at ph 7.0 in watch glasses. Separate watch glasses contained each at a desired final concentration, of the various inhibitors and catalyst of cyclic and non-cyclic photophosphorylation in the same medium as the controls, (2, 4-dinitrophenol, DNP M, Salicylaldoxine M; 3-3, 4-(dichlorophenol)-l, 1-dimethylurea, DCMU 2 10-SM, Antimycin A 10t~M, Carbonyl Cyanide m-chlorophenyl hydroze, CCCP 50 ~M, 2, 6-dichlorophenol indophenol 0"3 mm, ascorbate 0.5 mm. Phenazine methosulphate, PMS ~, Ferricyanide M, Methylviologen M, flavin mono nucleotide FMN 1 and 5.0tzg, anthraquinone, AQ M). The strips were illuminated at room temperature by means of an incandescent bulbs at an intensity of 12 K lux. At intervals of two hours one strip each from the control and from each of the treatments were removed. They were fixed by plunging into absolute ethanol and were stained with Heaths reagent. The stained epidermal strips were examined under the microscope and the width of stomatal aperture was measured by means of a precalibrated occular micrometer, as per Das and Raghavendra.2 Histochemical tests for Potassium were performed by the method of Willmer and Pallas is using sodium cobaltinitrite. Rates of Photophosphorylation were determined with 32p as per the method of Whatley and Arnon. 19

3 Enhancement of stomatal opening in epidermal strips 239 Oxygen uptake by isolated chloroplasts was measured using an oxygen electrode (Rank Bros., Bottisham, Cambridge) of the type described by Delieu and Walker 2 and by the method of Whitehouse, Ludwig and Walker. n The experiments were conducted on five different days and the average of the results were presented. 3. RESULTS Methyl viologen has produced maximum stomatal opening only in presence of light. It had no effect on the stomatal aperture in darkness (figures 1 and 2 and table 2). When the incubation medium contained DNP or salicylaldoxime a complete closure of stomata has resulted without or with MV. DCMU, Antimycin A or CCCP when included in the medium have produced only partial closure. DCMU showed a slight initial stimulation of stomatal opening over the control. Ferricyanide by itself could not bring appreciable stimulation of stomatal opening whereas PMS could produce substantial enhancement. In presence of DCMU, the alternate electron donating system the DCPIP ascorbate couple has nearly reversed the DCMU COMMELINA BENGHALENSIS PISUM SATIVUM 24-0 oj z_,6 / 4- O 6-0/0 } o TIME IN H OUP.S O MV, & PMS ~X DCMU, CONTROL~& Fe3CN 6, ~'CCCP~ E] ANTIMYCtN A ~ IJ DNP ~ (I) SALICYLALDOXlME. Figure 1. Influence of various catalysts and inhibitors of photophosphorylatioa oa the width of stomatal aperture,

4 240 V.S. RAMA DAS AND M. SANTHAKUMARI COMMELtNA E.E NGNALENtsI5 PtSUM SATI VUM 24 0 _~4 E :L Z O / X o.////x /:Y.z- -~0 "- "r t- Q J I,.,- < t2 8 i 12 - /O'.'~x/~ /J: o O l-- 4 O TIME IN HOURS O MV,p X DCPIP+A5C+ OCMU+MV, ~ MV+OCMU, ~ MV+CCCP~ q~ MV+ANTIMYCIN A, & /,.lv'+dnp, D MV-f-SALI- CYLAL DOX'IME. Figure 2. Influence of MV in combination with various catalysts and inhibitors of photo phosphor~lation on stomatal width. inhibition of MV-induced opening (figure 2) other readily autoxidizane Mehler reagents like FMN and AQ could al~o produce enhanced stomatal opening but comparatively less than that induced by MV (table 2). Histochemical tests by qualitative observations have revealed greater K + influx by the presence of MV or AQ in the incubation medium than in the controls. Isolated mesophyll chloroplasts exhibited lower rates of cyclic (PMS-- supported), non-cyclic (FczCN~) and whole chain (H20--+ MV) photophos phorylation than with MV--supported Photo-system I (DCMU and DCPIP ---Ascorbate). ATP formed in the H~O--MV system has been severely inhibited by the addition of DNP er Salicylaldoxime. The inhibition caused by the addition of DCMU, Antimycin A or CCCP of the same whole chain system is comparat,vely less (table I) The rate of oxygen upt~.ke by MV was well correlated with ATP production, The oxygen uptake was highest in the DCPIP q- Asc --MV system,

5 Enhancement of stomatal opening in epidermal strips 241 Table 1. Photophosphorylation and oxygen uptake by isolated chloroplasts in presertce of inhibitors and catalysts (t~ mol/mg Chl/hr ) Commelina benghalensis Pisum s~tivum ATP O~ ATP O,~ PMS Catalyzed cyclic Photophosphorylation FeuCN~ Catalyzed noncyclic Photophosphorylation ,. MV + H20 (Whole chain) MV + DCPIP + Asc + DCMU MV + DNP I MV + DCMU CCCP + MV MV + Salicylaldoxime MV + Antimycin A Table 2. Influence of Mehler reagents on the Stomatal opening of Comrnelina benghalensis in Light and Darkness. Stomatal Width in/zm after Treatment 2 hours 4 hours 6 hours Light Dark Light Dark Light Dark Control (Initial 6.0 gm) 4-0' Methylviologen , Anthraquinone ~ Flavin mono nucleotide 1 gg " "0 20'0 2.0 ± 0' ' /*g ' " '25 -I DISCUSSION A significant finding of this investigation is the marked stimulation of the stomatal opening process by Mehlel reagents, which inhibit the carbon assimilation through the blockage of PhotoreductioI~ of NADP. In the

6 242 V.S. RAMA DAS AND M. SANTHAKUMARI normal course of events one should expect a stomatal closure under the influence of such compounds since they should tend to increase the carbon dioxide concentration in the substomatal cavity and intercellular space system of the leaf due to an inhibited photosynthetic cacbon fixation, in Vivo as evidenced from unpublished data has indicated that stomata! behaviour of intact plants sprays with paraqueat (methyl viologen) was similar to that of isolated epidermis. On the contrary a substantial stimulation indicates clearly that the stomatal opening process is independent of the changes in the CO2 content of guard cells but perhaps it is an energy based reaction. Light was essential for the MV-induced opening of stomata. It became obvious that this role of light could, be ascribed only to photophosphorylation since the possibility of light induced changes in the CO2 concentration wele excluded. That MV is a powerful stimulant of photophosphorylation including the cyclic and pseudocyclic types has been reported by Shavit, lz Baldwin ~4 and Forti and Rosa. 15 The present results with the mesophyll chloroplasts also confirm this enhanced ATP production. Although the guard cell chloroplasts were not studied in respect of the influence of MV on photophosphorylation it is very likely their behaviour would be similar to those of mesophyll chloroplasts from the same leaves. In the present observation that Mehler reagents produced greater K + influx into guard cells together with earlier observation of Gimmler et al 12 in this respect suggest that the stimulation of stomatal opening is promoted by the potassium ion uptake. It was previously shown from this laboratory 3 that the K + uptake by guard cells was under the control of cyclic photophosphorylation. Recently Meidner and Willmer 5 also made a few speculations in terms of guard cell ion fluxes and carbon metabolism. They also suggested that energy cycling within guard ceils might be the basic control of stomatal endogenous rhythms. Therefore the increased K + influx of guard cells could presumably have been brought ~bout by MV-enhanced photophosphorylation. The endogenous stomatal opening is insensitive to DCMU, but strongly inhibited by the cyclic inhibitors like salicylaldoxime while MV-induced component is inhibited by DCMU. This might indicale the involvement of two different types of phosphorylations. But the total absence of any influence of MV in the presence of inhibitors of cyclic photophosphorylation and not in presence of DCMU together with the observation that the whole chain system was more susceptible to DNP or saticylaldoxime makes the MV-operated reaction is photosystem I based. Presumably some endogenous electron donors which permit bypassing of the water oxidation step might exist.

7 Enhancement of stomatal opening in epidermal strips 243 It is believed from the worle presented here that the stomatal opening is more intricately connected with the photosynthetic energy source rather than perhaps with the carbon metabolism in light. Further work on the detailed characterisation of the MV-induced enhancement of stomatal opening is in progress. ACKNOWLEDGEMENT M. Santakumari wa~ a recipient of Senior Research Fellowship from the Department of Atomic Energy, Government of India. REFERENCES 1. Raschke, K., Annu. Rev. Plant. PhysioL (1975). 2. Das, V. S. R. and Raghavendra, A. S., Bull. R. Soe (1974 a). 3. Das, V. S. R. and Raghavendra, A. S., Indian J. Exp. Biol. 12 (1974 b). 4. Fujino, M., Sei. Ball. Fae. Edu. Nagasakhi Univ. 1 (I967). 5. Meidner, H. and Wil!mer, C., Curr. Adv. Pl. Sei (1975). 6. Van Oorschot, J. L. P., P~vc. 7th Br. Weed. Control. Conf 321 (19,54). 7. Couch, R. W. and Davis, D. E., Weeds (1966). 8. Zwig, G., Residue Rev (1969). 9. Dodge, A. D., Endeavour, (1971). 10. Dodge, A. D., SeL Prog (1975) Whitehouse, D. G., Ludwig, L. J. and Walker, D.A., J. Exp. Bot (1971). 12. Gimmler, H., Schafer, G. and Heber, U., Proe. Third Inter. Cong. Photosyn~.h. Elsevier Scientific Publishing Co., Netherlands (1974). 13. Shavit, N., II International Cong. Photosynth. Stresa 1221 (1971). 14. Baldwin, B. C., Progress in Photosynthesis. Research (1969). 15. Forti, G. and Rosa, L., H International Cong. on Photosynthesis, Stresa 1251 (1971). 16. Newmann, J., Yannai, Y., Arntzen, C. J. and Dilley, R.A., IIInternational C')a,~. Phgtosyr, th. Stresa 1271 (1971). 17. Fujita, Y., Plant Cell PhysioL (1974). 18. Willmer, C. W. and Pallas, J. E., Can. J. Bot (1973). 19. Whatley, F.R. and Arnon, D.I., Methods Enzymol. 6 Academic Press New York, 308 (1963). co. Delieu, T and Walker, D. A.~ New Phytol (1972).