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(American Journal of Botany. 2002;89:366-368.)
© 2002 Botanical Society of America, Inc.

Brief Communication

Green light reversal of blue-light-stimulated stomatal opening is found in a diversity of plant species1

Lawrence D. Talbott, Ganka Nikolova, Arisa Ortiz, Irene Shmayevich and Eduardo Zeiger2

Department of Organismic Biology, Ecology and Evolution, University of California, Los Angeles, 900 Veteran Ave., Los Angeles, California 90024 USA

Received for publication June 21, 2001. Accepted for publication September 7, 2001.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Reversal by green light of blue-light-stimulated stomatal opening was found across a number of plant species, including leguminous and nonleguminous dicots and grass and nongrass monocots. Simultaneous exposure to equal fluence rates of blue and green light resulted in ~50% reversal of normal blue light opening. Complete reversal occurred when the fluence rate of green light was approximately twice that of blue light. These results suggest that blue–green reversibility of stomatal opening is a basic photobiological property of guard cells. The blue–green reversibility of stomatal opening has been hypothesized to ensue from the cycling of two interconvertible, isomeric forms of the blue-light photoreceptor, zeaxanthin. Testing of blue–green reversibility could provide a valuable diagnostic tool for zeaxanthin-mediated blue-light photoperception.

Key Words: blue light • guard cell • photoreversibility • stomata • zeaxanthin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Guard cells have two well-characterized light responses, one mediated by chlorophyll-dependent, guard cell photosynthesis, often called a "red light response" (Sharkey and Ogawa, 1987 ; Tallman, 1992 ), and a second response specific for blue light (Zeiger, 1990 ; Tallman, 1992 ; Assmann, 1993 ). Blue-light-specific stomatal opening has an action spectrum typical of other blue-light responses of plants, showing a maximum at 450 nm and minor peaks at 420 nm and 470 nm (Karlsson, 1986 ). The chloroplastic carotenoid, zeaxanthin has been shown to mediate blue-light photoreception in guard cells (Zeiger and Zhu, 1998 ; Frechilla et al., 1999 ; Zeiger, 2000 ).

Recent work in our laboratory has shown that stomatal opening in epidermal strips of Vicia faba exposed to a short blue-light pulse is reversed when a pulse of green light is applied immediately following the blue-light pulse (Frechilla et al., 2000 ). A subsequent pulse of blue light restores opening in a manner analogous to the well-known red/far red reversibility of phytochrome-dependent responses.

Green light reversal of blue-light-stimulated opening has also been observed when Vicia faba stomata are illuminated simultaneously with continuous blue and green light (Frechilla et al., 2000 ). In these conditions, the extent of green-light reversal is dose dependent, with full reversal achieved at a green-light fluence rate twice that of blue light.

In the present paper, the blue–green reversibility of stomatal opening was studied using narrow bandwidth light centered around the maxima found in the action spectra for blue-light-stimulated stomatal opening (450 nm) and its green reversal (540 nm; Frechilla et al., 2000 ). Reversibility was tested in a wide range of plant species commonly used for stomatal studies in order to determine whether this remarkable photobiological observation is a general phenomenon among these plants.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant material and growth conditions
Seeds of Vicia faba L. cv Windsor Long Pod (Bountiful Gardens Seeds, Willits, California, USA), Commelina communis and Pisum sativum argentium mutant (Dr. T. Mansfield, University of Lancaster, Lancaster, UK), Nicotiana glauca (Dr. G. Tallman, Willamette University, Salem, Oregon, USA), Arabidopsis thaliana ecotype columbia (Dr. K. Niyogi, University of California, Berkeley, California, USA), Nicotiana tabacum (Dr. S. Rodermel, Iowa State University, Ames, Iowa, USA), Allium cepa var. sweet sandwich (W. Atlee Burpee, Warminster, Pennsylvania, USA), and Hordeum vulgare (Weigong Yang, University of California, Los Angeles, California, USA) were planted in pots with commercial potting mix (Sunshine mix #1; American Horticultural Supply, Camarillo, California, USA). Plants were grown in a greenhouse under natural light, 50–75% relative humidity, 25–30°C day/15–20°C night. Plants were watered three times a day with an automatic watering system and fertilized once a week (20-10-20 mix; Grow-More Research and Manufacturing Company, Gardena, California, USA).

Preparation of detached epidermal strips and aperture measurements
Abaxial epidermis was carefully stripped by hand from the interveinal regions of young, fully expanded leaves into an incubation solution containing 0.1 mMol/L CaCl2 and a species-dependent KCl concentration empirically determined to provide optimal blue-light-stimulated opening (V. faba, 1 mMol/L; Arabidopsis thaliana, N. tabacum, P. sativum, Allium cepa, H. vulgare, 20 mMol/L; C. communis, 30 mMol/L; N. glauca 50 mMol/L). Detached peels were dark adapted for 60 min, after which baseline stomatal apertures were measured. The epidermal strips were then transferred to individual treatment dishes containing the same incubation medium and illuminated with different light treatments for 90 min (45 min for H. vulgare). Final aperture measurements were obtained at the end of the light treatment. The incubation solution was aerated with compressed air and maintained at 23°C throughout the dark adaptation and light treatments.

Average apertures (width of the short axis of the stomatal pore) for each experiment were determined from measurements of 30 digitized video images of stomata in epidermal peels (Talbott and Zeiger, 1993 ) using an Olympus BH-2 microscope connected to a Javelin JE2362A digital imaging camera (Javelin systems, Torrance, California, USA). Image processing was handled with an IBM PC-based MV-1 image analysis board (Metrabyte Corporation, Taunton, Massachusetts, USA) and JAVA image analysis software (Jandel Scientific, Corte Madera, California, USA).

Blue and green light was provided by optical fibers (Oriel Instruments, Stratford, Connecticut, USA) using Dolen-Jenner fiber optic illuminators (Edmund Scientific, Barrington, New Jersey, USA) as light sources. Monochromatic blue (450 nm) and green (540 nm) light was provided by interference filters (Nos. 53830 and 53880, 10 ± 2.5 nm bandwidth, Oriel Instruments). Light fluence rates were measured with a quantum sensor (LI-COR, Lincoln, Nebraska, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Blue-light-specific stomatal opening in detached epidermis of broadbean (V. faba), illuminated with 10 µmol·m–2·s–1 broadband blue light is reversed by simultaneous illumination with 20 µmol·m–2·s–1 broadband green light (Frechilla et al., 2000 ). Similar results were obtained in the present study (Fig. 1) using narrow bandwidth illumination centered at the action spectra maxima for blue-light-stimulated opening (450 ± 5 nm; Karlsson, 1986 ) and green-light-dependent reversal (540 ± 5 nm; Frechilla et al., 2000 ). A 90-min illumination with blue light resulted in a 1.9 µm increase in aperture. Simultaneous exposure to 10 µmol·m–2·s–1 green light completely reversed blue-light-stimulated opening, while exposure to 5 µmol·m–2·s–1 green light resulted in ~50% reversal of opening.



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  		Fig. 1. Green light reversal of blue-light-stimulated stomatal opening in six dicotyledonous species. Stomata were illuminated with continuous 10 µmol·m–2·s–1 green light (Green), 10 µmol·m–2·s–1 green and 5 µmol·m–2·s–1 blue light (2 : 1), 5 µmol·m–2·s–1 green and 5 µmol·m–2·s–1 blue light (1 : 1), or 5 µmol·m–2·s–1 blue light (Blue). The change in aperture from the initial value resulting from 90 min of illumination is shown ± SE of the measurement. Results are the average of three experiments

 
This same experimental protocol was used to test green reversibility of the blue-light opening response of stomata from a number of other dicotyledonous species (Fig. 1). Reversibility was found in another legume species, the argentium mutant of pea (Pisum sativum), which has occasionally been used for stomatal studies because of its easily removable epidermis (Jewer, Neales, and Incoll, 1985 ). Reversibility was also observed in the herbaceous dayflower (Commelina communis), another commonly used subject of stomatal research (Meidner and Mansfield, 1968 ; Assmann and Schwartz, 1992 ). Other dicot species showing the blue–green reversibility were Arabidopsis thaliana and two species of tobacco (commercial tobacco, Nicotiana tabacum, and native California tree tobacco, N. glauca). In all cases, simultaneous illumination with a 2 : 1 ratio of green to blue light produced an ~100% reversal of the opening caused by blue light alone. As found in the previous study of V. faba, a 1 : 1 ratio of green to blue illumination produced an ~50% reversal of blue-light opening in all species (Fig. 1).

Green reversibility was also tested in two monocotyledonous species, onion (Allium cepa) a species widely studied because of the lack of starch in the chloroplasts of its guard cells (Ogawa, 1981 ), and oat (Hordeum vulgare), a member of the grass family (Fig. 2). In both species, a 2 : 1 ratio of green to blue light resulted in nearly complete reversal of the opening obtained with blue light alone, while a 1 : 1 ratio resulted in an intermediate opening.



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  		Fig. 2. Green light reversal of blue-light-stimulated opening in two monocotyledonous species. Results are shown as in Fig. 1

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The reversal of blue-light-stimulated stomatal opening by green light discovered in V. faba stomata (Frechilla et al., 2000 ) was found in all species examined in the present study, which represent a broad range of plant genera. The conservation of an approximate 2 : 1 green to blue light ratio for full reversal suggests that the reversal of blue-light-stimulated stomatal opening by green light is a basic photobiological property of guard cells, rather than an adaptation to specialized environmental conditions.

The green-light reversal of the stomatal response to blue light is distinctly different from previously reported green-light responses in plants. The action spectra for the typical blue-light responses of phototropism and inhibition of stem elongation in Arabidopsis and lettuce extend into the green region (Steinitz, Ren, and Poff, 1985 ; Lin, Ahmad, and Cashmore, 1996 ; Liscum and Briggs, 1996 ). However, green light has a stimulatory effect on these responses, in contrast to its antagonistic effect on blue-light-stimulated stomatal opening. Furthermore, the green-light effect reported here cannot be observed in the absence of blue light (Frechilla et al., 2000 ).

The action spectrum for the reversal of blue-light-stimulated stomatal opening by green light shows peaks at 490 nm, 540 nm (maximum), and 580 nm and does not extend into the red or blue portion of the spectrum (Frechilla et al., 2000 ). This action spectrum rules out chlorophyll or phytochrome as the primary photosensor for the green light. Furthermore, red light does not reverse blue-light-stimulated opening (Frechilla et al., 2000 ), as would be expected if chlorophylls or phytochrome were the primary photoreceptors.

Compelling available evidence indicates that the chloroplastic carotenoid, zeaxanthin, is a blue-light photoreceptor in guard cells (Zeiger and Zhu, 1998 ; Frechilla et al., 1999 ). It has recently been found that the zeaxanthin-less mutant, npql, fails to show a green-light reversal of stomatal opening (L. Taiz, University of California, Santa Cruz, personal communication). These findings implicate zeaxanthin in the blue-green reversal response.

An interconversion of the blue-light photoreceptor between active and inactive forms was previously inferred from kinetic studies of the stomatal responses to light pulses (Iino, Ogawa, and Zeiger, 1985 ). The dominant photochemical reactions of carotenoids such as zeaxanthin are isomerizations, which can cause large shifts in absorption spectrum of carotenoids in a protein environment (Britton et al., 1997 ). The action spectrum for green-light reversal of stomatal opening resembles the spectrum for blue-light opening, red shifted by ~90 nm. The interpeak distance of the red-shifted spectrum shows a broadening that is characteristic of spectral shifts resulting from isomerizations of polyene chromophores. Thus, the blue–green reversibility of stomatal opening could result from the cycling of the zeaxanthin photoreceptor between a physiologically inactive, blue-light-absorbing isomer and a physiologically active, green-light-absorbing isomer. This process is analogous to the isomerizations occurring in the phytochromobilin chromophore resulting in red–far red reversibility of phytochrome responses.

A recent study of chloroplast movement in Lemna has implicated zeaxanthin as a photoreceptor mediating the sensory transduction of blue light in that well-characterized blue-light response (Tlalka, Runquist, and Fricker, 1999 ). Testing of blue-green reversibility in this and other blue-light responses could be a valuable diagnostic tool for zeaxanthin involvement.


   FOOTNOTES
 
1 The authors thank the National Science Foundation and the U.S. Department of Energy for funding this work. Back

2 Author for reprint requests (zeiger{at}biology.ucla.edu ). Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Assmann S. M. 1993 Signal transduction in guard cells. Annual Review of Cell Biology 9: 345-375[CrossRef][ISI]

Assmann S. M. A. Schwartz 1992 Synergistic effect of light and fusicoccin on stomatal opening: epidermal peel and patch clamp experiments. Plant Physiology 98: 1349-1355[Abstract/Free Full Text]

Britton G. R. J. Weesie D. Askin J. D. Warburton L. Gallardo-Guerrero F. J. Jansen H. J. M. de Groot J. Lugtenburg J. P. Cornard J. C. Merlin 1997 Carotenoid blues: structural studies on carotenoproteins. Pure and Applied Chemistry 69: 2075-2084

Frechilla S. L. D. Talbott R. A. Bogomolni E. Zeiger 2000 Reversal of blue light-stimulated stomatal opening by green light. Plant and Cell Physiology 41: 171-176

Frechilla S. J. Zhu L. D. Talbott E. Zeiger 1999 Stomata from npq1, a zeaxanthin-less Arabidopsis mutant, lack a specific response to blue light. Plant and Cell Physiology 40: 949-954[Abstract/Free Full Text]

Iino M. T. Ogawa E. Zeiger 1985 Kinetic properties of the blue-light response of stomata. Proceedings of the National Academy of Sciences, USA 82: 8019-8023[Abstract/Free Full Text]

Jewer P. C. T. F. Neales L. D. Incoll 1985 Stomatal responses to carbon dioxide of isolated epidermis from a 3-carbon pathway plant, the Argenteum mutant of Pisum sativum, and a crassulacean acid metabolism plant Kalanchoe daigremontiana. Planta 164: 495-500[CrossRef][ISI]

Karlsson P. E. 1986 Blue light regulation of stomata in wheat seedlings. II. Action spectrum and search for action dichroism. Physiologia Plantarum 66: 207-210[CrossRef]

Lin C. M. Ahmad A. R. Cashmore 1996 Arabidopsis cryptochrome 1 is a soluble protein mediating blue light-dependent regulation of plant growth and development. Plant Journal 10: 893-902[CrossRef][ISI][Medline]

Liscum E. W. R. Briggs 1996 Mutations of Arabidopsis in potential transduction and response components of the phototropic signaling pathway. Plant Physiology 112: 291-296[Abstract]

Meidner H. T. Mansfield 1968 Physiology of stomata. McGraw-Hill, London, UK

Ogawa T. 1981 Blue light response of stomata with starch-containing (Vicia faba) and starch-deficient (Allium cepa) guard cells under background illumination with red light. Plant Science Letters 22: 103-108[CrossRef][ISI]

Sharkey T. D. T. Ogawa 1987 Stomatal responses to light. In E. Zeiger, G. D. Farquhar, and I. R. Cowan [eds.], Stomatal function, 195–208. Stanford University Press, Stanford, California, USA

Steinitz B. Z. Ren K. L. Poff 1985 Blue and green light-induced phototropism in Arabidopsis thaliana and Lactuca sativa L. seedlings. Plant Physiology 77: 248-251[Abstract/Free Full Text]

Talbott L. D. E. Zeiger 1993 Sugar and organic acid accumulation in guard cells of Vicia faba in response to red and blue light. Plant Physiology 102: 1163-1169[Abstract]

Tallman G. 1992 The chemiosmotic model of stomatal opening revisited. Critical Reviews in Plant Science 11: 35-57

Tlalka M. M. Runquist M. Fricker 1999 Light perception and the role of the xanthophyll cycle in blue-light-dependent chloroplast movements in Lemna trisulca L. Plant Journal 20: 447-459[CrossRef][ISI][Medline]

Zeiger E. 1990 Light perception in guard cells. Plant, Cell and Environment 13: 739-747[CrossRef]

Zeiger E. 2000 Sensory transduction of blue light in guard cells. Trends in Plant Science 5: 183-185[CrossRef][ISI][Medline]

Zeiger E. J. Zhu 1998 Role of zeaxanthin in blue light photoreception and the modulation of light-CO2 interactions in guard cells. Journal of Experimental Botany 49: 433-442[Abstract]




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