Langmuir 2008, 24, 14261-14268
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Effects of Cadmium on Photosynthetic Oxygen Evolution from Single Stomata in Brassica juncea (L.) Czern Renkang Zhu,† Sheila M. Macfie,‡ and Zhifeng Ding*,† Department of Chemistry and Department of Biology, The UniVersity of Western Ontario, London, ON, N6A 5B7, Canada ReceiVed June 16, 2008. ReVised Manuscript ReceiVed September 21, 2008 Scanning electrochemical microscopy (SECM) was utilized to investigate photosynthetic oxygen evolution from single stomata in leaves of live Brassica juncea (L.) Czern cultured in nutrient solution to which 0.2 or 0.01 mM CdCl2 had been added. The bulk leaf surface serves as an insulator normally; therefore, a typical negative feedback was observed on the probe approach curves (PACs) when the probe approached epidermal cells. When the probe tip approached an open stoma, a higher tip current was detected due to the O2 release from this stoma. Thus, SECM can be used to map the O2 concentration profile near the leaf surface and study stomatal complex structure size and density. The oxygen release from single stomata was also analyzed by comparison of experimental PACs with those simulated by COMSOL multiphysics software (version 3.4). In addition to an increase in the stomatal complex size and a decrease in the complex density, the Cd accumulation caused up to a 26% decrease in photosynthetic rate determined at the level of a single stoma. The O2 evolution was also monitored by recording the tip current vs time when a tip sat above the center of a stoma. Periodic peaks in O2 release-time curves were observed, varying from 400 to 1600 s. The opening and closing activities of single stomata were also imaged by SECM.
Introduction Some plants, such as Brassica juncea (L) Czern (Indian mustard), are able to accumulate toxic metals, including lead (Pb), chromium (Cr), nickel (Ni), copper (Cu), zinc (Zn), and cadmium (Cd)1 from environments polluted by industry and daily life wastes, mining activities, or byproducts of phosphate fertilizers.2 Cd is an important environmental pollutant, and therefore, much research effort has been directed at the physiology and chemistry of Cd-accumulating plant species.3-12 Nonpolluted soil solutions contain Cd concentrations ranging from 0.04 to 0.32 µM.13 Soil solutions that have a Cd concentration varying from 0.32 to about 1 µM can be regarded as polluted to a moderate level.2 Cd in plants causes leaf roll and chlorosis and reduces growth, both in roots and in stems.2 For instance, Cd damaged nucleoli, inhibited ribonuclease activity, reduced the absorption of nitrate and its transport, induced oxidative stress, and seriously affected photosynthesis.2 Many mechanisms to defend against Cd toxicity have been discussed,2 but information about how Cd affects photosynthetic processes is still scarce. Brassica napus L. (oilseed rape) that was grown from seeds on reconstituted soil * To whom correspondence should be addressed. Tel: (519) 661-2111, ext 86161. Fax: (519) 661-3022. E-mail:
[email protected]. † Department of Chemistry. ‡ Department of Biology. (1) Kumar, P. B. A. N.; Dushenkov, V.; Motto, H.; Raskin, I. EnViron. Sci. Technol. 1995, 29, 1232–8. (2) Di Toppi, L. S.; Gabbrielli, R. EnViron. Exp. Bot. 1999, 41, 105–130. (3) Baryla, A.; Carrier, P.; Franck, F.; Coulomb, C.; Sahut, C.; Havaux, M. Planta 2001, 212, 696–709. (4) Yadav, P.; Srivastava, A. K. J. EnViron. Biol. 2000, 21, 259–262. (5) Gupta, M.; Devi, S. B. EnViron. Contam. Toxicol. 1992, 49, 436–43. (6) Clemens, S. Planta 2001, 212, 475–486. (7) Prasad, M. N. V. EnViron. Exp. Bot. 1995, 35, 525–45. (8) Poschenrieder, C.; Barcelo, J. In HeaVy Metal Stress in Plants: From Biomolecules to Ecosystems, 2nd ed.; Prasad, M. N. V., Ed.; Springer: New York, 2004; pp 249-270. (9) Barcelo, J.; Vazquez, M. D.; Poschenrieder, C. New Phytol. 1988, 108, 37–49. (10) Zhu, R.; Macfie, S. M.; Ding, Z. J. Exp. Bot. 2005, 56, 2831–2838. (11) Liu, J.; Cai, G.; Qian, M.; Wang, D.; Xu, J.; Yang, J.; Zhu, Q. J. Sci. Food Agric. 2007, 87, 1088–1095. (12) Mobin, M.; Khan, N. A. J. Plant Physiol. 2007, 164, 601–610. (13) Wagner, G. J.; Donald, L. S. AdV. Agron. 1993, 51, 173–212.
contaminated with Cd had a marked chlorosis of the leaves, which was investigated using a combination of biochemical, biophysical, and physiological methods.3 The obtained results3 indicated that chlorosis was not due to a direct interaction of Cd with the chlorophyll biosynthetic pathway but to an apparent decrease in the chloroplast density caused by a reduction in the number of chloroplasts per cell and a change in cell size. It was suggested that Cd interfered with chloroplast replication and cell division.3 Mobin and Khan12 also observed that Cd caused a decline in total chlorophyll (a + b) and chlorophyll a content in two mustard cultivars. Chloroplast structure was also affected by Cd in bush bean plants Phaseolus Vulgaris as reported by Barcelo et al.,9 and the water-splitting apparatus of photosystem II and photosynthetic electron transport can be altered by Cd in the green microalga Scenedesmus.14 Cd was also found to decrease stomatal density10 by our group and conductance to CO2 by Baryla et al.3 A reduced number of open stomata9,10 would further affect photosynthetic rates. Because many functions of living plant cells and tissues are tightly related to photosynthesis, it is desirable to determine noninvasively the effects of Cd on photosynthetic reactions. While net photosynthesis can be estimated via gas exchange and photosynthetic electron transport can be estimated from noninvasive measurements of chlorophyll fluorescence,3,14 the scale of measurement is relatively large, yielding gross measurements from several square centimeters of plant tissue or several cubic milliliters of algal culture. Havaux utilized Clark electrodes to detect oxygen evolution from weed leaf disks in comparison with photoacoustic measurements, which were in vitro and determined the bulk oxygen concentrations released from the disks.15 In the case of plant leaves, the density of chloroplasts may influence the measurements taken. One can either normalize the readings to a “per chloroplast” measurement or one can seek alternative methods that are less affected by organelle density. Scanning electrochemical microscopy (SECM) has shown great applications in the area of chemical and biochemical kine(14) Mallick, N.; Mohn, F. H. Ecotoxicol. EnViron. Saf. 2003, 55, 64–69. (15) Havaux, M. Photosynth. Res. 1989, 21, 51–9.
10.1021/la8018875 CCC: $40.75 2008 American Chemical Society Published on Web 11/11/2008
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tics,16-20 chemical activity imaging,10,21-33 and micrometer scale structuring and reading34-37 at liquid/liquid, liquid/solid, and liquid/membrane interfaces. The most significant advantage offered by SECM is its capability to probe chemical processes and to map concentrations in a small domain (micrometer scale or submicrometer scale). SECM is an ideal tool for mapping the O2 concentration above single stomatal complexes10,29 and for studying the effect of toxic metals, such as Cd, on the oxygen evolution rate and the stomatal complex structures.10 Tsionsky et al.29 reported photoelectrochemistry and in vivo topography of single stomata in unstressed Tradescantia fluminensis Vell Variegata using SECM. Mancuso et al.38 reported the detection of oxygen from normal leaves and roots using a vibrating microelectrode, which measured oxygen flux from the current difference at two distances above the specimen. A contour map showing the oxygen evolution was constructed by recording the flux along an x-y plane. The concept of this scanning probe microscopy is very similar to SECM. In our previous paper,10 we investigated Cd-induced stress by using SECM to probe oxygen evolution (oxygen was used as a redox mediator), hence net photosynthetic activity, in B. juncea. In this paper, we present a more detailed study of the effect of Cd on the release of O2 from single stomata on the leaves of three plants: the control plant and plants with 0.2 or 0.01 mM CdCl2 dosages during their growth in hydroponic culture. While these concentrations are higher than one would find in soil solution,2 B. juncea does not show signs of photosynthetic stress with up to 0.2 mM CdCl2 in hydroponic solution.39 The single stomtal complex was found by using SECM images, with a closed-loop instrument where the tip was easily moved to the center of the opened stomatal center. The oxygen evolution was evaluated by fitting the experimental probe approach curves (PACs) with the ones simulated by COMSOL software (version 3.4). Periodicity in oxygen evolution as well as the stomatal opening and closure was also examined. (16) Ding, Z.; Quinn, B. M.; Bard, A. J. J. Phys. Chem. B 2001, 105, 6367– 6374. (17) Amemiya, S.; Ding, Z.; Zhou, J.; Bard, A. J. J. Electroanal. Chem. 2000, 483, 7–17. (18) Fonseca, S. M.; Barker, A. L.; Ahmed, S.; Kemp, T. J.; Unwin, P. R. Chem. Commun. 2003, 1002, 1003. (19) Sun, P.; Zhang, Z.; Gao, Z.; Shao, Y. Angew. Chem., Int. Ed. 2002, 41, 3445–3448. (20) Sun, P.; Liu, Z.; Yu, H.; Mirkin, M. V. Langmuir 2008, 24, 9941–9944. (21) Zhu, R.; Qin, Z.; Noeel, J. J.; Shoesmith, D. W.; Ding, Z. Anal. Chem. 2008, 80, 1437–1447. (22) Zhao, X.; Petersen, N. O.; Ding, Z. Can. J. Chem. 2007, 85, 175–183. (23) Diakowski, P. M.; Ding, Z. Phys. Chem. Chem. Phys. 2007, 9, 5966– 5974. (24) Diakowski, P. M.; Ding, Z. Electrochem. Commun. 2007, 9, 2617–2621. (25) Lee, Y.; Ding, Z.; Bard, A. J. Anal. Chem. 2002, 74, 3634–3643. (26) Liu, B.; Rotenberg, S. A.; Mirkin, M. V. Anal. Chem. 2002, 74, 6340– 6348. (27) Macpherson, J. V.; Jones, C. E.; Barker, A. L.; Unwin, P. R. Anal. Chem. 2002, 74, 1841–8. (28) Mauzeroll, J.; Bard, A. J. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7862– 7867. (29) Tsionsky, M.; Cardon, Z. G.; Bard, A. J.; Jackson, R. B. Plant Physiol. 1997, 113, 895–901. (30) Yasukawa, T.; Kaya, T.; Matsue, T. Electroanalysis 2000, 12, 653–659. (31) Zhao, C.; Wittstock, G. Angew. Chem., Int. Ed. 2004, 43, 4170–4172. (32) Turcu, F.; Schulte, A.; Hartwich, G.; Schuhmann, W. Angew. Chem., Int. Ed. 2004, 43, 3482–3485. (33) Fernandez, J. L.; Mano, N.; Heller, A.; Bard, A. J. Angew. Chem., Int. Ed. 2004, 43, 6355–6357. (34) El-Giar, E. M.; Said, R. A.; Bridges, G. E.; Thomson, D. J. J. Electrochem. Soc. 2000, 147, 586–591. (35) Wittstock, G. Fresen. Anal. Chem. 2001, 370, 303–315. (36) Katemann, B. B.; Schulte, A.; Schuhmann, W. Chem.-Eur. J. 2003, 9, 2025–2033. (37) Zhu, R.; Xu, S.; Podoprygorina, G.; Bohmer, V.; Mittler, S.; Ding, Z. J. Phys. Chem. C 2008, 112, 15562–15569. (38) Mancuso, S.; Papeschi, G.; Marras, A. M. Planta 2000, 211, 384–389. (39) Gadapati, W. R.; Macfie, S. M. Plant Sci. 2006, 170, 471–480.
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Simulation Model General Considerations. The COMSOL multiphysics software (version 3.4) was used to investigate the O2 evolution from a single stoma by simulating PACs to the center of a stoma. In the bulk solution, the dissolved O2 was used as the redox mediator. The mediator, O2, is reduced to hydroxide (eq 1) at the SECM probe biased more negative than -0.600 V versus a Ag/AgCl reference electrode:10
O2 + 2H2O + 4e-f4OH-
(1)
The tip electrochemical process is at the diffusion control of the oxygen. The solution was air-saturated and the O2 concentration in bulk solution does not change with the O2 release from the leaves, since a stoma is small (
