J. Cell Sci. 37, I-IO (1979)
Printed in Great Britain © Company of Biologists Limited
BLUE LIGHT-INDUCED, INTRINSIC VACUOLAR
FLUORESCENCE IN ONION GUARD CELLS
EDUARDO ZEIGER AND PETER K. HEPLER*
Department of Biological Sciences, Stanford
Stanford, California 94305, U.S.A.
University,
SUMMARY
Guard cells of onion irradiated with broad-band blue light display a green intrinsic fluorescence. The fluorescence has been found in eleven species of Allium, but it has not been
observed in any other monocot or dicot examined. The fluorescence occurs only in guard cells
and is absent in neighbouring epidermal cells. During development it is first apparent in guard
mother cells soon after the asymmetric division. Microscopic observation reveals that the
fluorescence is associated with the vacuole and examination of vacuoles isolated from guard cell
protoplasts suggests that it may be localized on the tonoplast. Microspectrophotometric
analysis of single cells reveals an emission peak at around 520ran.Our results are consistent
with the view that this blue light receptor is aflavinorflavoproteinand that it might be related
to the blue light-enhanced stomatal opening observed in onion.
INTRODUCTION
During the course of our studies on the development and physiology of stomata we
discovered that when guard cells of onion (Allium cepa L.) were irradiated with blue
light they fluoresced green. This observation seems important because it directly
indicates the presence of a blue-light receptor in guard cells and thus might help us
to understand the basis for the blue light-mediated stomatal opening reported in many
species (see Hsiao, 1976, for review). Blue light has been reported to enhance stomatal
opening in onion (Meidner, 1968), and recent studies in our own laboratory show that
blue light also causes ion and water influx into guard cell protoplasts (Zeiger &
Hepler, 1977).
The presence of the intrinsic fluorescence may be another manifestation of a basic
blue light-dependent system capable of driving ion transport in the guard cells
(Zeiger, Moody, Hepler & Varela, 1977) and thus may provide new avenues for
experimentation on its mechanism of action. In this paper we report on the localization
of the fluorescence in onion guard cells, on some of its spectral properties, and on its
intra- and intergeneric distribution.
• Present address: Department of Botany, University of Massachusetts, Amherst, Massachusetts 01003, U.S.A.
2
E. Zeiger and P. K. Hepler
MATERIALS AND METHODS
Plant material
Seeds of AUium cepa cv. Cima Hybrid (Keystone, Co., Ca., U.S.A.) were germinated as
described previously (Zeiger & Hepler, 1976). Sets from A. sativum were bought from a local
supplier and grown in vermiculite in a greenhouse; leaves of A. bivalve were obtained from the
collection of alpine plants at the University of Massachusetts. Seeds of other AUium species
were kindly supplied by Dr G. D. McCollum (U.S. Department of Agriculture, Beltsville,
Md., U.S.A.). Leaves from mature plants of Tradescantiafluminensis,Agapanthus sp., 7m sp.
and Zantedeschia aethiopica were dissected from ornamental plants growing in the surroundings
of the laboratory. A mature specimen of Aspidistra eliator was bought from a commercial
nursery. Hordeum vulgare cv.' Early Bonus', Zea mays cv. Bear Hybrid, Viciafaba and Nicotiana
tabacum var. Mammoth were grown from seeds in Petri dishes layered with moistened filter
paper at room temperature.
Peels from leaves were obtained by tearing off small portions of the epidermis with a pair of
fine forceps. Peels largely free of underlying mesophyll tissue were required to avoid interference
by the intense red fluorescence of the chlorophyll in mesophyll cells. The peels were uncurled
and mounted in a drop of mannitol solution (0-15 to 0-23 M) on a glass slide under a No. 1
coverglass. The osmoticum was used because, in contrast to preparations in distilled water the
protoplasm of guard cells kept close to isotonic conditions continues to stream for several hours
(Zeiger & Hepler, 1976). Peels and protoplasts of A. cepa used for quantitative measurements
were kept in Sykes Moore culture chambers (Bellco Glass Inc., Vineland, New Jersey, U.S.A.)
as described previously (Zeiger & Hepler, 1976). The solutions were perfused through the
chambers with syringe needles inserted through the o-ring.
Protoplasts were obtained from slices of 6- to 10-day-old cotyledons by digestion with 4 %
Cellulysin (Calbiochem, Los Angeles, Ca.) in 0-23 M mannitol for 10-12 h (Zeiger & Hepler,
1976). At the end of the digestion, the enzyme solution was replaced by 0-4-0-5 M mannitol
and 0-5 mM CaCl,. Changing solutions washed away most of the mesophyll protoplasts and
thus eliminated background red fluorescence. For the release of isolated vacuoles, chambers
with the isolated protoplasts were perfused with 5 to 10 ml of a 0-23 M mannitol solution.
Swelling and bursting of the protoplasts occurred within a few minutes.
Fluorescence microscopy
Two optical systems were used for the fluorescence microscopy: (1) a Reichert Zetopan
microscope with a dark-field fluorescence condenser, a 200-W Hg lamp, a BG12 exciting filter
(maximum at 410 nm) and a GG9/OG53O barrier filter, 50% cutoff point at 50011m; and
(2) an American Optical H10TU-VF4 microscope with an AO 2070C vertical illuminator for
incident fluorescence and a 50-W Hg lamp, with either (a) a BG12 exciting filter, a 500-nm
cutoff dichroic beamsplitter and a OG515 barrier filter, or (b) a 436-nm blue exciting filter and
a 450-nm cutoff dichroic beamsplitter with or without an OG515 barrier filter. Transmission
curves for the latter combination are shown in Fig. 8, p. 6. Light intensities under the
conditions described in (a), measured with the blue-light window of a Plant Growth Photometer (International Light, Newburyport, Massachusetts) at the level of the preparation were
0-55 mW cm"1 with the 40 x objective and 3 mW cm"1 with the 100 X objective.
Fluoromicrospectrophotometry
Emission spectra and fluorescence decay of single cells were studied with a NanoSpec/10
microfluorospectrophotometer (Nanometrics, Sunnyvale, Ca., U.S.A.) attached to the AO
optical system. The NanoSpec has a high sensitivity gallium-arsenide photomultiplier and a
motor-driven diffraction grating monochromator allowing readings to the nearest 01 nm. The
relative fluorescence intensity is displayed in a digital read-out and a chart recorder. Preparations on microscope slides or in chambers were placed on the stage of the microscope and
observed under dim green light (green interference filter, A = 546 nm, Reichert) using the
built-in halogen lamp. Cells selected for active protoplasmic streaming and normal cytological
Vacuolar fluorescence of guard cells
3
appearance were centred in the optical field of the slit viewing system of the spectrophotometer
head and the slit was adjusted to expose about 50 /im' of cell area. The halogen lamp was then
switched off and the exciting beam from the Hg lamp was unblocked. Emission spectra were
obtained by mechanically advancing the motor-driven monochromator between 2 preselected
wavelengths. The relative fluorescence intensity at each wavelength was registered by a chart
recorder. For the measurement of fluorescence decay, the monochromator was set at 530 nra,
and the changes in intensity with time were followed by the chart recorder with zero time set
at the instant the cells were exposed to the exciting beam.
RESULTS
Cytology of fluorescing guard cells and their isolated protoplasts and vacuoles
Onion guard cells show a green, intrinsic fluorescence (Fig. 2) when excited with
broad-band blue light. Examination of epidermal peels and paradermal slices reveals
that the green fluorescence is restricted to the guard cells; epidermal cells do not
fluoresce (Figs. 1, 2). In paradermal slices that include mesophyll cells there is a
pronounced red fluorescence due to chlorophyll. Red-fluorescing chloroplasts are also
observed in the guard cells (Fig. 2).
In order to determine the intracellular location of the fluorescing compound, cells
were examined at high magnification, and direct comparisons were made on the same
cells with both fluorescence and Nomarski differential interference-contrast optics.
From these observations it is readily apparent that the green fluorescence does not
emit uniformly from throughout the cell; rather it is confined to those regions occupied
by the vacuole. The nucleus, usually centrally located, is always opaque. The cytoplasm
and plasmalemma also appear to lack the green fluorescence since the chloroplasts,
which can be distinguished by their characteristic redfluorescence,are observed at the
outer boundary of the green emission (Fig. 2). Fully mature guard cells show, in
addition, a faint green fluorescence in the ridge surrounding the stomatal pore. Its
slightly different colour from the fluorescence of the vacuole and its position in the
ridge suggest to us that it originates from a different molecular species, probably
lignin within the thickened cell wall.
Wall-less protoplasts from onion guard cells, kept in a 0-4 M mannitol and 0-5 niM
CaCL, also exhibit the intrinsic fluorescence (Fig. 3). Because of the rearrangement of
the subcellular organelles in the spherical protoplasts, the green fluorescence is
sometimes masked by bleached, yellowish chlorophyll.
Exposure of the protoplasts to an hypotonic medium like a 0-23 M mannitol solution
causes them to swell and burst. By alternately observing such a preparation under
bright-field and incident-fluorescent illumination, it can be seen that the intact
swollen vacuole emerges through the broken plasmalemma and that it exhibits all of
the green fluorescence (Fig. 4). An isolated fluorescing vacuole is seen in Fig. 5.
Prolonged exposure to the hypotonic medium leads, in turn, to the bursting of the
isolated vacuole. Unlike the plasmalemma, which seems to collapse upon rupture, the
tonoplast reforms into smaller vesicles of varying sizes. Qualitative observations
indicate that most, if not all, of the greenfluorescenceremains confined to the vesicles,
but quantitative measurements remain to be done.
E. Zeiger and P. K. Hepler
Vacuolar fluorescence of guard cells
5
The fluorescing properties of developing guard cells
Developing guard cells in epidermal peels from 5- to 8-day-old onion cotyledons
(Zeiger & Cardemil, 1973) were tested for their ability to fluoresce when excited with
blue light. Guard cells showed the characteristic fluorescence at all stages of development. The fluorescence was faint in newly formed guard cells and increased at later
stages of development. Guard cells from peels of leaves dissected from older plants
growing in a garden also fluoresced, although the intensity was weaker than the one
exhibited by mature guard cells from young cotyledons. The green fluorescence was
also seen in a dividing guard mother cell found in prophase; it was found in the
corners of the cell, areas where the vacuoles would be expected to be located. At an even
earlier developmental stage very young guard mother cells, observed soon after the
completion of the asymmetrical division at which they originated (Zeiger & Cardemil,
1973), showed a faint, yet unequivocal green fluorescence. On the other hand, their
sister cells which are to remain epidermal, did not fluoresce. These observations
indicate that the fluorescing compound was synthesized or became physiologically
competent at a very early stage of guard cell differentiation.
Observations made on etiolated preparations suggest that the ability of the guard
cells to fluoresce is not dependent upon illumination during growth. Seeds were
germinated in complete darkness, and peels from 5- to 8-day-old cotyledons were
made under dim green light. The preparation was observed immediately after illumination with blue light, and the fluorescing pattern was indistinguishable from that of
peels grown in the light.
Fig. 1. Nomarski phase-interference micrograph of a pair of onion guard cells in an
epidermal peel from a young cotyledon, x 1350 approx.
Figs. 2-5. Green intrinsic fluorescence in the onion guard cells, their protoplasts and
vacuoles. AO optical system with incident fluorescence, 100 x oil immersion objective,
BG12 exciting filter, 500-nm dichroic beamsplitter, OG515 barrier filter.
Fig. 2. Fluorescence in the guard cells, occupying most of the cell volume, except
for the nuclei and the ridges surrounding the pore. The red-fluorescing organelles
are chloroplasts. Epidermal cells in the optical field do not fluoresce. x 1250 approx.
Fig. 3. Fluorescing guard cell protoplast in 0-5 M mannitol and 0-5 min CaCl,.
The green fluorescence is somewhat masked by yellow bleached chlorophyll from
the chloroplasts. x 1050 approx.
Fig. 4. Swelling protoplasts in 0-23 M mannitol. Only the vacuoles emerging from
the bursting protoplasts exhibit green fluorescence, x 1050 approx.
Fig. 5. Isolated fluorescing vacuole and a single red fluorescing chloroplast.
x 1050 approx.
Fig. 6. Stomatal complex in an epidermal peel from Tradescantiafluminensisshowing
bright, red-fluorescing chloroplasts and a faint yellow-green fluorescence in the
ridges around the pore. No green fluorescence can be seen in the vacuoles. Optical
conditions as in Figs. 2-5. x 1250 approx.
E. Zeiger and P. K. Hepler
Emission spectra and fluorescence decay
The availability of a sensitive microfluorospectrophotometer allowed us to make
some quantitative measurements of the green fluorescence in single cells. Cells
scanned between 400 and 600 nm showed peaks in the blue and in the green regions
of the spectrum. Qualitative observations made with different filter combinations
indicated that the blue component resulted from residual transmission of the exciting
beam and from light scattered by the cell walls.
e
D
f
90 - . ]Lc-~r~
70
r 50
30
a? 10
WO 500 600 700
0 SSIL
r
tra
520
Wavelength. i m
527
500
520
540
560
580
600
Wavelength, nm
Fig. 7. Emission spectra from a so-/tms area of a guard cell vacuole, measured in a
Nanospec microfluorospectrophotometer with an AO optical system, 100 x oilimmersion objective, 436-nm blue exciting filter, 450-nm dichroic beamsplitter, with
(lower) or without (upper) a BG515 barrier filter. Cells from an epidermal peel in a
0-23 M mannitol solution kept in a sealed microchamber.
Fig. 8 (inset to Fig. 7). Transmission spectra of the filters as provided by the
manufacturer: E, exciter, D, dichroic, and B, barrier filters.
Vacuolarfluorescenceof guard cells
7
Spectra from the 500 to 600 nm region, obtained with a narrow-band, exciting blue
filter, a dichroic beamsplitter and a barrier filter, resolved a single peak in the green,
with a maximum at 525 to 530 nm (Fig. 7). Exclusion of the barrier filter increased
the blue background substantially but also caused a shift of the green peak to around
520 nm (Fig. 7), indicating that the transmission properties of the barrier filter (Fig. 8,
inset to Fig. 7) produced an artifactual displacement of the green peak toward a
longer wavelength.
It was also found that the relative intensity of the fluorescence decreased with time
of exposure of the preparation to the exciting beam. Because of that fluorescence
decay, successive spectra from the same cell had decreasing overall intensities, but the
position of the green peak was always the same. That the kinetics of decay do not
influence the emission spectrum is also apparent from the observation that scans of
cells done from 500 to 600 or from 600 to 500 nm gave identical peaks.
Some quantitative parameters of the fluorescence decay were also obtained. Guard
cells from epidermal peels kept in a 0-23 M mannitol solution in sealed microchambers
(chamber volume: 0-65 ml) were irradiated with blue light (BG12 exciting filter
500-nm dichroic beamsplitter and OG515 barrier filter, 100 x oil-immersion objective)
and the relative fluorescence intensity was measured at 530 nm. The fluorescence
reached a maximum in less than 0-5 s, the time lag of the chart recorder, and decayed
continuously thereafter with approximately exponential kinetics. Typically, 50% of
the initial intensity was lost within 1 min. Exposed cells, kept in the dark for periods
of up to 5 h, failed to recover any of the lost intensity in spite of their continuous
protoplasmic streaming. The rate of decay decreased substantially with decreased
intensity of exciting light attained by using either a 40 x objective or a double BG12
exciting filter.
The intragenic and intergeneric distribution of the green fluorescence
All eleven species of the genus Allium tested {A. ascalonicum, bivalve, cepa,fistulosum,
galanthum, pskemense, roylei, sativum, schoenoprasum, vavilovii, and tuberosum) showed
the green fluorescence in their guard cells. We therefore conclude that the fluorescence
is a common characteristic of the Allium genus.
On the other hand, we were unable to find the greenfluorescencein any other species
of several monocotyledons and dicotyledons tested. These include: Hordeum vulgare,
Zea mays (Gramineae); Zantedeschia aethiopica (Araceae), Tradescantia fluminensis
(Commelinaceae; kindly identified by Dr J. Thomas, Stanford University); Aspidistra
eliator (Liliaceae), Agapanthus sp. (Amaryllidaceae), Iris sp. (Iridaceae), Vicia faba
(Leguminosae) and Nicotiana tabacum (Solanaceae). With the exception of the two
Gramineae, all guard cells observed under the established optical conditions lacked
the green fluorescence characteristic of Allium while exhibiting large, bright redfluorescing chloroplasts and a faint fluorescence on the ridges surrounding the pore
(Fig. 6). Guard cells from barley and corn, on the other hand, did show an abundant,
green fluorescence. It was clear, however, that most, if not all, of the fluorescence
originated from the thickened walls of the guard cells. Because of the special morphology of the gramineous stomata, with their thick walls occupying most of the cell
8
E. Zeiger and P. K. Hepler
volume, it was especially difficult to ascertain whether a protoplasmic green fluorescence
nresent.
was present.
DISCUSSION
The green, intrinsic fluorescence found specifically in onion guard cells directly
indicates the existence of a blue light photoreceptor in these cells. The location of the
fluorescent compound in the vacuole, and perhaps in the tonoplast, suggests that it
might be associated with membrane-mediated active ion transport in the guard cells
and thus offers a new approach to the study of some basic aspects of stomatal function.
The spectral properties of the fluorescing compound are suggestive of a flavin, thus
raising the possibility that it could be a mediator of electron transport in the guard
cells and be connected with blue light-enhanced stomatal opening.
The evidence for the vacuolar localization of the fluorescing compound is compelling. First, direct observations reveal fluorescence in the region occupied by the
vacuole, and not within the nucleus, or cytoplasm in general; second, isolated protoplasts fluoresce, thus eliminating the cell wall as a source for the blue light receptor;
finally, rupture of the protoplasts shows that the isolated, intact vacuole retains all the
fluorescence. The observations showing that burst vacuoles reform into many smaller
vesicles all of which appear fluorescent, indicate that the fluorescing compound is
likely to be located on the tonoplast.
The cellular specificity of the fluorescence is also clear. The fluorescence is not only
absent from neighbouring epidermal cells, but can be detected in guard mother cells
just emerging from the asymmetrical division at which they originate. Both the
cellular specificity and its appearance at a very early stage of development are suggestive of a physiological role of the fluorescing compound in connexion with stomatal
function.
The emission characteristics of the green fluorescence agree with the spectral
properties of flavins and flavoproteins. Furthermore, recent experiments have provided information on the excitation properties of the fluorescing compound which
show a distinct peak around 450 run (Zeiger, unpublished). Similar spectra have been
reported for the flavoprotein flavodoxin (Ghisla, Massey, Lhoste & Mayhew, 1975)
and for presumed flavins from Avena coleoptiles (Zenk, 1967) and the eyespot of
Euglena (Sperling-Pagni, Walne & Wehry, 1976). In both Avena and Euglena, the
fluorescing compounds have absorption spectra in the blue that closely correlate with
action spectra for phototropism (Zenk, 1967) and phototaxis (Sperling-Pagni et al.
1976) indicating that they could be primary photoreceptors. It should be noted,
however, that some flavonoids also fluoresce in the yellow green (Geissman, 1955)
and glycoside derivatives of the flavonol quercetin have been reported in Alliutn
leaves (Harborne, 1965). However, in contrast to the compound described here, the
fluorescing flavonoids do not seem to absorb at wavelengths longer than 400 nm.
Furthermore, quercetin can be irradiated for a few hours without considerable photodestruction (Kaneta & Sugiyama, 1971), while the fluorescing substance in onion
guard cells decays to about one half of its initial fluorescence intensity within 1 min.
Vacuolarfluorescenceof guard cells
9
This latter characteristic is also consistent with flavins which are known to photodestruct rapidly in the absence of adequate levels of electron donors (Schmidt &
Butler, 1976).
If the fluorescing compound is, indeed, a physiologically functional flavin, its
presence in the guard cells could have important implications. We have postulated a
light-sensitive proton motive force as the basic energy transducing mechanism driving
ion transport in the guard cells (Zeiger & Hepler, 1977; Zeiger et al. 1977). Flavins
can be reduced by blue light (Schmidt & Butler, 1976) and could mediate the initial
photoreduction in a series of electron transfers that would generate an electrochemical
gradient driving the uptake of potassium associated with stomatal opening. Such a
possibility certainly warrants further investigation of the photochemical properties
of the fluorescing compound which, if located in the tonoplast, would also provide
another means of studying potential electrogenic activities at that membrane (Moody
& Zeiger, 1978).
The restriction of the green fluorescence to the Allium genus is intriguing. The
compound might be absent from guard cells of the other genera tested, or the cellular
milieu in the latter might preclude its fluorescence under the experimental conditions
used. Allium guard cells are unusual in their lack of starch (Schnabl & Ziegler, 1977),
their small chloroplasts (compare Figs. 2 and 6) and their seemingly extreme requirement for Cl~ as a counter-ion for K+ (Schnabl & Ziegler, 1977). On the other hand,
it is well documented that Allium guard cells share many basic physiological properties
classically associated with stomatal function, such as sensitivity to light and CO2
(Heath, 1952; Meidner & Heath, 1959), and the use of K + as the main osmotic cation
(Schnabl & Ziegler, 1977). Even in their special response to blue light (Meidner, 1968;
Zeiger & Hepler, 1977), a phenomenon to which the green fluorescence could well be
related, Allium stomata are not unique, since blue light-enhanced stomatal responses
have been reported in a significant number of species, including Vicia (Hsiao,
Allaway & Evans, 1973), many grasses (Johnsson, Issaias, Brogardh & Johnsson, 1976;
Skaar & Johnsson, 1978; Raschke, Hanebuth & Farquhar, 1978) and Aspidistra
(Voskresenskaya & Polyakov, 1976).
Hence, while it is clear that Allium guard cells show some unique characteristics
that probably reflect specific variations in their functioning, the fact that they exhibit
many basic responses common to stomata of most of the higher plants suggests to us
a single, basic cellular mechanism driving stomatal function in all of them. If that is
the case, the further characterization of the green-fluorescing compound in onion
provides us with a unique opportunity to enrich our understanding of the photobiological properties of the guard cells.
We are grateful to Vince Coates of Nanometrics and Steve Westrate of Westrate Scientific
for generous help with equipment, to Drs E. J. Stadelmann (University of Minnesota) and
S. Malkin (Weizmann Institute, Israel) for valuable suggestions, to Dr G. D. McCollum
(U.S. Department of Agriculture at Beltsville, Maryland) for the supply of onion seeds, to
Dr J. Brown, Carnegie Institution, Department of Plant Biology, for her generous help with
the fluorospectrophotometer and to Eleanor Crump for editing the manuscript. Supported
by NSF Grants PCM 74-15243 to P.K.H. and PCM 77-17642 to H. Mooney.
io
E. Zeiger and P. K. Hepler
REFERENCES
GEISSMAN, T. A. (1955). Anthocyanins, chalcon.es, auron.es, flavones and related water-soluble
plant pigments. In Moderne Methoden der Pflanzenanalyse, vol. 3 (ed. K. Paech & M. V.
Tracey), pp. 450-498. Berlin: Springer.
GHISLA, S., MASSEY, V., LHOSTE, J. & MAYHEW, S. G. (1975). Fluorescence characteristics of
reduced flavins and flavoproteins. In Reactivity of Flavins (ed. K. Yagi,) pp. 15-24. Baltimore : University Park.
HARBORNE, J. B. (1965). Plant polyphenols. XIV. Characterization of flavonoid glycosides by
acidic and enzymic hydrolyses. Phytochemistry 4, 107-120.
HEATH, O. V. S. (1952). The role of starch in the light response of stomata. II. The light
response of stomata of AUium cepa L., together with some preliminary observations on the
temperature response. New Phytol. 51, 30-47.
HSIAO, T . C. (1976). Stomatal ion transport. In Encyclopedia of Plant Physiology (ed. U. Liittge
& M. G. Pitman), N.S. vol. 2, pt. B, pp. 195-221. Berlin: Springer.
HSIAO, T . C , ALLAWAY, W. G. & EVANS, L. T . (1973). Action spectra for guard cell Rb+
uptake and stomatal opening in Vicia faba. PI. Physiol., Lancaster 51, 82-88.
JOHNSSON, M., ISSAIAS, S., BROCARDH, T . & JOHNSSON, A. (1976). Rapid, blue-light-induced
transpiration response restricted to plants with grass-like stomata. Physiol. PL 36, 229-232.
KANETA, M. & SUGIYAMA, N . (1971). The light resistance of the flavones and the ftavonols.
JB. Chem. Soc.Jap. 44, 3211.
MEIDNER, H. (1968). The comparative effects of blue and red light on the stomata of AUium
cepa L. and Xanthium pennsylvanicum. J. exp. Bot. 19, 146—151.
MEIDNER, H. & HEATH, O. V. S. (1959). Stomatal responses to temperature and carbon dioxide
concentration in AUium cepa L. and their relevance to mid-day closure, jf. exp. Bot. io,
206-219.
MOODY, W. & ZEIGER, E. (1978). Electrophysiological properties of onion guard cells. Planta
139, 159-165RASCHKE, K., HANEBUTH, W. F . & FARQUHAR, G. D. (1978). Relationship between stomatal
conductance and light intensity in leaves of Zea mays L., derived from experiments using the
mesophyll as shade. Planta 139, 73-77.
SCHMIDT, W. & BUTLER, W. L. (1976). Flavin-mediated photoreactions in artificial systems: a
possible model for the blue-light photoreceptor pigment in living systems. Photochem.
Photobiol. 24, 71-75.
SCHNABL, H. & ZIEGLER, H. (1977). The mechanism of stomatal movement in Allium cepa L.
Planta 136, 37-43.
SKAAR, H. & JOHNSSON, A. (1978). Rapid, blue-light-induced transpiration in Avena. Physiol.
PI- 43. 39O-396.
SPERLING-PAGNI, P. G., WALNE, P. L. & WEHRY, E. L. (1976). Fluorometric evidence for
flavins in isolated eyespots of Euglena gracilis var. bacillaris. Photochem. Photobiol. 24,
373-375VOSKRESENSKAYA, N. P. & POLYAKOV, A. M. (1976). Regulatory action of blue light on photosynthetic gas exchange: action spectrum of light saturation of CO, exchange in lily of the
valley leaves. Soviet PI. Physiol. 23, 6—11.
ZEIGER, E. & CARDEMIL, L. (1973). Cell kinetics, stomatal differentiation and diurnal rhythm
in AUium cepa. Devi Biol. 32, 179-188.
ZEIGER, E. & HEPLER, P. K. (1076). Production of guard cell protoplasts from onion and
tobacco. PI. Physiol., Lancaster 58, 492-498.
ZEIGER, E. & HEPLER, P. K. (1977). Light and stomatal function: blue light stimulates swelling
of guard cell protoplasts. Science, N. Y. 196, 887-889.
ZEIGER, E., MOODY, W., HEPLER, P. & VARELA, F. (1977). Light sensitive membrane potentials
in onion guard cells. Nature, Lond. 270, 270—271.
ZENK, M. H. (1967). Untersuchungen zum Phototropismus der ^4w«na-Koleoptile: II. Pigmente. Z. PflPhysiol. 56, 122-140.
{Received 24 July 1978)