Plant Physiol. (1988) 87, 226-231 0032-0889/88/87/0226/06/$01.00/0

Enhancement of the Stomatal Response to Blue Light by Red Light, Reduced Intercellular Concentrations of C02, and Low Vapor Pressure Differences' Received for publication August 14, 1987 and in revised form January 15, 1988 SARAH M. ASSMANN2 Department of Biological Sciences, Stanford University, Stanford, California 94305

ABSTRACT Various hypotheses have been proposed to explain the en- hancement of the blue light response by red light. In the present The effects of environmental parameters on the blue light response study, the plausibility of these hypotheses was investigated, using of stomata were studied by quantifying transient increases in stomatal gas exchange techniques in which both stomatal conductance and conductance in Commelina communis following 15 seconds by 0.100 mil- mesophyll assimilation could be measured. Conductance is a limole per square meter per second pulses of blue light. Because con- more reliable indicator of stomatal responses and guard cell tur- ductance increases were not observed following red light pulses of the gor than is transpiration alone, since transpiration is affected by same or greater (30 seconds by 0.200 millimole per square meter per both stomatal aperture and VPD. The effects of c, and VPD on second) fluences, the responses observed could be reliably attributed to the blue light response were also investigated. The experiments the specific blue light response of the guard cells, rather than to guard were performed using Paphiopedilum harrisianum, an orchid cell chlorophyll. In both Paphiopedilum harrisianum, which lacks guard species which lacks guard cell chloroplasts (5, 12), and Com- cell chloroplasts, and Commelina, the blue light response was enhanced melina communis. As with other nongrasses (9), these species by 0.263 millimole per square meter per second continuous background lack the rapid blue-light-induced transpiration increase, but ex- red light. Thus, the blue light response and its enhancement do not require hibit a less rapid stomatal opening response which has been energy derived from red-light-driven photophosphorylation by the guard clearly attributed to the specific blue light system (8, 17, 26). cell chloroplasts. In Commelina, reduction of the intercellular concentra- The present results provide information on the mechanistic basis tion of CO2 by manipulation of ambient CO2 concentrations resulted in of the enhanced blue light response and are also relevant to an enhanced blue light response. In both Commelina and Paphiopedilum, theories on its ecological role. the blue light response was decreased by an increased vapor pressure difference. The magnitude of blue-light-specific stomatal opening thus appears to be sensitive to environmental conditions that affect the carbon MATERIALS AND METHODS and water status of the plant. Plant Material. Plants of Paphiopedilum harrisianum G.S. Ball and Commelina communis L. were raised as described previously (1, 8) except for a different watering regime. Plants were either misted (P. harrisianum) or drip irrigated (C. communis) 6 d/ Light is one of several environmental parameters which affect week with an automatic irrigation system (Rain Master Irrigation rates of water loss and CO2 uptake in the leaves of higher plants Systems, Simi Valley, CA). Plants were hand-watered 1 d/week, (24). Following illumination, uptake of K+ and Cl- and synthesis at which time they were fertilized with Spoonit Orchid Food of malate by the guard cells results in osmotic swelling and an (Plantsmith, CA). increase in the apertures of the stomatal pores through which Gas Exchange. Leaves were sealed in the cuvette of a null gaseous diffusion occurs (15). balance gas exchange system (7). Leaf temperature was meas- In the grasses, blue light elicits an initial rapid increase in ured with a copper-constantan thermocouple appressed against transpiration followed by a second, slower increase. The initial the lower leaf surface and was maintained within + 0.2°C of the increase requires blue light (4) and is presumably mediated by temperature chosen for a given experiment, typically between a specific blue light response that initiates ion uptake via a chemi- 23.5 and 24°C. Vapor pressure difference was regulated through osmotic mechanism (27). Blue light stimulates H+ extrusion (19) adjustment of both air flow rate (Tylan mass flow controller, and ATP-dependent membrane hyperpolarization (1, 2) in guard Carson, CA), and the dewpoint temperature of a custom built cells, presumably creating an electrical gradient for the influx of humidifier system. Except as noted, temperature and VPD were potassium. The magnitude of the rapid blue light response is maintained constant throughout a given experiment. enhanced by illumination with continuous background red light Incoming relative humidity was calculated from the dewpoint (10, 21) or by red light prepulses (4, 10). The slower increase in temperature of the condensor and air temperature inside the transpiration is evoked by both blue and red light and appears cuvette. Chamber humidity was measured with a Vaisala probe to mediated cell Chl in the so-called PAR3 response (Vaisala US, Woburn, MA) located inside the chamber; thus, be by guard measurements of changes in conductance were essentially in- of stomata (17). stantaneous. ' Research supported by a predoctoral fellowship from the McKnight Except as otherwise noted, air with a CO, content of 346 ,ul Foundation. 2Present address: Department of Organismic and Evolutionary Bi- 3Abbreviations: PAR, photosynthetically active radiation; VPD, va- ology, Harvard University, The Biological Laboratories, 16 Divinity Av- por pressure difference; c., intercellular concentration of CO,; c,, am- enue, Cambridge, MA 02138. bient concentration of CO,; g. stomatal conductance. 226 STOMATAL RESPONSES TO BLUE LIGHT 227

L - flowed continuously through the chamber and was supple- 2). An increased, but still subsaturating, level of background red mented with 1% CO2 in N2 as necessary to avoid CO2 depletion light was not effective in activating the response to pulsed red from photosynthesis. CO2 content of air entering and leaving the light (Fig. 3d). chamber was measured using an ADC Mark 3 infrared gas ana- Comparison of Commelina and Paphiopedilum. Figure 4 shows lyzer in the differential mode. There was a flow-dependent delay characteristic responses of Commelina and Paphiopedilum from in the measurement of assimilation that ranged from less than 1 experiments performed in triplicate. Overall levels of conduct- min under the maximum background red light illumination to ance are low in Paphiopedilum, as has been observed previously about 2 min in the absence of background illumination, when (1, 22). In both species, stomatal conductances following a pulse flow rates were kept low in order to maintain an adequate VPD. of blue light were greater when background red light was present. Because conclusions derived from the results presented here do The results of Figure 4 confirm the enhancement of the blue not require precise kinetic analysis, the delay in measurement light response shown in Figures la and 2, and extend those results of assimilation was not deconvolved (cf. 14). Assimilation was to the achlorophyllous guard cells of Paphiopedilum. Figure 4 calculated from flow rates and CO2 measurements, and ci was shows no response of Paphiopedilum to a blue light pulse in the computed (11). Relevant parameters for the calculation of as- absence of red light; other replicates did show a small response similation and conductance were measured at 60 s intervals through (not shown). computer assisted sampling. Carbon Dioxide. Pressurized air tanks with different partial Photobiology. The gas exchange system was located in a dark- pressures of CO2 were used to investigate the effect of manip- ened room. The illumination protocol was a modification of the ulation of CO2 concentrations on the magnitude of the blue light dual beam technique (8, 13). Background red light was obtained response in Commelina. Increasing ca from 346 ,ul L-1, a level from a 300 W lamp (Sylvania PAR56/2MFL) filtered through a typical of natural conditions, to 810 ,u L-1 resulted in an increase Kodak 1A filter (50% cutoff at 545 nm) and a layer of No. 5A in ci and an inhibition of the blue light response, while decreas- Cinemoid. Background fluence rates were varied using neutral ing ca to 120 ,ul L-I enhanced the response (Fig. lb). Figure 5 density screens. Pulsed light was obtained by passing light from exemplifies a typical experiment. The effect of such manipu- a General Electric Gemini 500 projector lamp through either a lations on the blue light response of Paphiopedilum was not Rohm and Haas 2424 blue Plexiglas filter (maximal transmittance tested. at 470 nm) or a Kodak 2-61 red filter. Heat load on the chamber Vapor Pressure Difference. The effect of VPD on the blue light was reduced by filtering both background and pulsed light through response was determined using the same leaves as in the com- wideband hot mirrors (OCLI, Santa Rosa, CA) which transmit parison of Commelina and Paphiopedilum. Following adminis- minimally in the IR. Unless otherwise noted, red and blue pulses tration of the first and second blue light pulses in that experiment, were 15 s x 0.100 mmol m-2 s-', with intensity adjusted as the background red light intensity of 0.263 mmol m-l s- was required using neutral density filters. Fluence rates were meas- maintained, while the VPD was experimentally increased until ured with a Licor quantum probe (Lincoln, NE) placed on top steady state conductance was reduced to the level previously of the cuvette and were corrected for the transmission coefficient observed in the absence of background illumination. of the glass cuvette lid. In both Commelina and Paphiopedilum, the increased VPD diminished the magnitude of the blue light response (Fig. 6). RESULTS This result was observed, even though the increased VPD low- ered ci (not shown), by decreasing baseline levels of stomatal Red and Blue Light Pulses. In four separate experiments, a conductance. single attached leaf of Commelina was allowed to attain steady Responses ofAssimilation and c, to Blue and Red Pulses. Figures state levels of assimilation and conductance at each of four dif- 7 and 8 show responses of assimilation and calculated values of ferent background red light intensities. Responses to a red and ci from the experiment providing the conductance responses shown to a blue light pulse were recorded at each background light in Figure 2. There was a short pulse of assimilation directly intensity. associated with both the blue (Fig. 7a) and the red pulses (Fig. Responses are summarized in Figure la, and typical results 7b). At 0.263 mmol m-2 s-1 background red light, both assim- are illustrated in Figure 2. At all background fluence rates of ilation (Fig. 7a) and ci (Fig. 8a) increased after the blue light red light, stomata responded to a blue light pulse, resulting in a pulse. Otherwise, no significant increases in assimilation (Fig. 7) transient increase in conductance (Figs. la and 2a). In all four or c, (Fig. 8) were observed following either blue or red light experiments, the magnitude of the response was enhanced at pulses. 0.263 mmol m-2 s-1 background red light (Fig. la). Figure 2b illustrates the absence of a conductance response following the DISCUSSION administration of red pulses to the same leaf and in the same experiment as in Figure 2a. An occasional gradual increase in The data reported in Figures 1 and 2 indicate that, in Com- conductance to a new steady state level after application of a red melina, pulses of blue light result in a transient increase in sto- light pulse at a background light intensity of 0.043 mmol m-2 matal conductance (cf. 8) that is enhapced by background red s I was seen (not shown) but could not be consistently observed. light, This result is consistent with previous reports on red and Additional Red Pulses. The absence of response to pulses of blue light effects on transpiration in grasses (4, 10, 21). Com- red light in the previous experiment was unexpected, given that parable pulses of red light failed to elicit an increase in con- stomata do respond to red light under continuous illumination ductance (Fig. 2b) even though the background red light in- (cf. baseline levels of conductance in Figs. la and 2). Additional tensities used here were below the saturation levels for stomatal experiments were performed in duplicate to confirm the absence opening mediated by guard cell Chl (Figs. la and 2; 17). Altering of a response to pulses of red light. Data shown in Figure 3 typify the duration (Fig. 3a) or fluence rate (Fig. 3b) of the red pulses, results from these experiments, indicating a lack of response increasing their total fluence (Fig. 3, c and d), or increasing the under a range of pulse lengths and intensities comprising three background fluence rate (Fig. 3d) were also ineffective. These different total pulse fluences: 1.5 mmol m-2 S-1 (Fig. 3, a and results confirm that the conductance responses reported here are b), 3.0 mmol m-2 S-1 (Fig. 3d), and 6.0 mmol m-2 s-1 (Fig. specific to blue light. The results also provide information on the 3c). Results were obtained under 0.090 mmol m-2 s-1 back- PAR response of stomata. They suggest that, in addition to a ground red light (Fig. 3, a, b, and c), where the PAR response fluence rate threshold (17), there may also be a threshold to the is clearly not saturated (cf. baseline conductance in Figs. la and duration, or to the total fluence required to elicit a PAR response. 228 ASSMANN Plant Physiol. Vol. 87, 1988

Conductance (mmol mr2S1) Eu Intercellular [ CO 2 ] (JJLlV1) Blue light-stimulated conductance increase (cm 2) 28 700r 0.28r a b N 25 600H T

20 - 500- 0.2 F

15 _ 400F 300H iok 0.1p- 2001 5 look OL oL oL I i - 0 6 43 263- 0 6 43 263- 0 6 43 263- O1U 'e1lU 1JU 3'bQ 810 346 120 298 298 298 RED LIGHT FLUENCE RATE (pmolm-2s 1) AMBIENT CONCENTRATION OF C02 (PttaV)

FIG. 1. Effects of the background fluence rate of red light (a) and the ambient concentration of CO2 (b) on baseline levels of conductance in mmol m-2 1-' (g), baseline intercellular concentrations of CO2 in Al L- 1 (ci), and the magnitude of the conductance response in cm2 (Ag) to a 15 s x 0.100 mmol m -2 s- 1 pulse of blue light. In both (a) and (b), the magnitude of Ag is positively correlated with g and negatively correlated with ci. Results are averages ± 1 SE (depicted on one side only for clarity); n = 4 for (a), n = 3 for (b), except for values recorded at 120 and 346 /,l L - ambient C02, where n =2. The magnitude of Ag was measured as the area under a graph of blue light-stimulated conductance (cf. Fig. 2); all experiments were graphed on the same scale.

a b 21 ^ - background red ; 0. 090 mmo m 2s- background red%.. .18 .19.>* TL = 25.0°C VPD -0.55kPa 0.O90mmol m s , . .1 T, - 25.0°C .17 .18 . VPD 0.65kPo In red pulse A e\ .16 .E 7.5s XO.2 mmolmr5 .15 - red l16 E pulse 2 ~0Os X 0.015mmol m s I lOmin 10min. 7 a) .14 background red- 0O..090 mmol m 2 background red TL = 25.0°C VPD = 0.65kPa*...0.200ommol m 2 E .14 blue pulse cI TL = 23.8°C 0 pulse .12 = o ,1 .I* VPD 0.60kPo E . . .. . J2.0.043 oL red pulse -A* . . . 3OsXO.100 . .. 0.043 .. .10 Ms .. C3 .09 .09 mmol .08 ~blue red red pulse -3 Ipulse pulse 30sX 0.200mmolm s- l0 min 10 min C d 0 06l.006 l llllred FIG. 3. Absence of a response.. of conductance in C. communis to four blue .08 Ipulse different red light illumination protocols. In a, b, and c, the background .6.08jpulse>'*"-.08 0 000 red light was kept at the same fluence rate and the fluence x time combination of a red pulse (a, b) or the total fluence of the pulse (c) .08 .~.08 was altered. In d, the intensity of the background red light was increased. ble 'fred are puse -pulse Leaf temperature (T1_) and VPD indicated for each experiment. .06 0 20 40 60 80 100 0 20 40 60 Time (min) Additional experiments addressed four hypotheses concerning the mechanistic basis of the enhanced blue light response. One FIG. 2. Response of conductance in C. communis to 15 s x 0.100 possibility is that the enhancement reflects increased baseline mmol m -2 - pulses of blue (a) or red (b) light. Pulses were administered levels of guard cell turgor under red illumination (4, 10). Upon under four different intensities of background red light, whose fluence stomatal opening from a completely closed state, there is an rates, in mmol m-2 s- , are indicated by the numbers to the right of initial phase, the 'Spannungsphase,' during which ion and water each response trace. Leaf temperature (TL) and VPD are as indicated. movement contribute to guard cell inflation and changes in guard The shaded region exemplifies the area of the conductance curves used cell shape, but there is little change in pore aperture (18) and in calculations of the magnitude of the blue light response for Figure 1 consequently little change in stomatal conductance. The presence and Table I. of background red light might increase turgor beyond this initial STOMATAL RESPONSES TO BLUE LIGHT 229

a .40- . a Commelina an_- VPD = 0.35kPa Commelino .38 - . TL=24.0°C .28- *-%< *..> background red= 0. 263 mmol rri 2S1 .36- . * .J2 .26- I * TL= 24.9C . _ *~~~~~~34-*1% * .10 .24- ? 7N .14- .32- ~-.~'..,2a'>** .#.* background red:

.- 0263mmo1 2 lblue pulse E *' 0. VPD=0.34kPa . I,, jIbLuepulsed 2rkness0mi F1VPDO0.83kPa cm .101 .28--. IE L) Tblue pulse Paphiopedilum 20mi blue pulse VPD=0.59kPa .16- V 0~~~~~~~~~~~~~~~~~Pophiopedilum .16- 00 1.-U E0 TL= 23.4°C .14- o background red c 0 0 0. 263mmol r2s .14-

0 .02- T:= 234C 0 o C 0 .00~ VPD=1.89kPa 0 VPD=0.59kPa 0 background red = 0.263mmo1 m2 s 20min blue pulse 2'-4 blue pulse FIG. 4. Response of conductance in C. communis (a) and P. harri- FIG. 6. Effect of VPD on the magnitude of the conductance response -2 I sianum (b) to a 15 s x 0.100 mmol m s- blue pulse administered on in C. communis (a) and P. harrisianum (b) to a 15 s X 0.100 mmol m-2 a background of either darkness or 0.263 mmol m-2 s red light. s- pulse of blue light. The background red light intensity was kept constant at 0.263 mmol m-2 s -. Leaf temperature (TL) and VPD are indicated for each experiment. Commelina VPD = 0. 56kPa a a8K8 ' background red = 0.263mmol m2 s -5 8.6 8.8 0.263 cqE 0.263 8.4 I .215- > 'i,, 8.6 - fred N blue 8.2 _ pulse _0 pulse 0 .1 -o -5'E i.0.043' X8 4 z 1.4 i.4 c .16 ~.175- 0.043 0 blue pulse blue pulse o 1.2 - blue 1.2 - 6red c pulse pulse C, =7717,u Q 20min c1=114,uQQ' a -E .2 .2 L 0.006 FIG. 5. Effect of manipulation of ci on the response of conductance 0.006 - Fred in C. communis to a 15 s x 0.100 mmol m-2 s - blue pulse. The back- - blue pulse ground fluence rate of red light was kept constant at 0.263 mmol m-2 C pulse 0 0 s-I. -.1 0000 0.000. -.2 blue state. Consequently, all subsequent solute uptake stimulated by pulse 2 redpulse a blue light pulse would be effective in driving aperture and 0 20 40 60 80 l00 0 20 40 60 conductance increases. The magnitude of response to a given Time (min) blue light stimulus would then appear larger in the presence of FIG. 7. Response of assimilation in C. communis to the blue (a) and red light than in its absence. However, if this explanation is red (b) pulses which resulted in the conductance responses shown in correct, once the Spannungsphase has been surpassed, as indi- Figure 2. Background fluence rates of red light in mmol m-2 s are cated by an increase in baseline levels of conductance, no effect indicated to the right of each response trace. on the blue light response of a further increase in red light fluence rate should be observed. In contrast, the data of Figures la and A second possible explanation for the enhanced blue light 2a show a clear enhancement of the blue light response between response is that red-light-driven photophosphorylation in the guard 0.043 mmol m-2 s-1 background red light, where the Span- cell chloroplasts (20) provides energy for stomatal opening trig- nungsphase has already been exceeded (cf. Figs. 1 and 2, baseline gered by blue light (10, 21). However, chloroplasts must not be conductance levels), and 0.263 mmol m-2 s-I red light. These required for red light enhancement of the blue light response, results, and previous results of Skaar and Johnsson (21), do not since the response of the achlorophyllous guard cells of Paphio- support the hypothesis that the interaction with red light is an pedilum can also be enhanced (Fig. 4). The results cannot, of effect dependent on guard cell turgor. The relationship that is course, rule out an interaction between the blue light and PAR observed between baseline levels of conductance and the mag- responses of chlorophyllous guard cells, but do suggest that an- nitude of the blue light response (Table I) may be a secondary other red-light absorbing pigment may be present in guard cells. correlation. An obvious candidate is phytochrome (16), and it is of interest 230 ASSMANN Plant Physiol. Vol. 87, 1988

280 _ a light response and c, (Table I). Previous experiments with blue . . i ,,. 280 light also support such a correlation (4, 10). Although the causal _..... %- * *Z 260 0''. 263 basis for an interaction remains unknown, recent experiments 260 ,,, 0.263 suggest that both blue light (2) and CO2 (6) may directly affect 240 Ablue ~ - puse ion at the N 2401 transport plasmalemma of guard cells. Ipulse The intercellular concentration of CO2 is not the sole modu- C= lator of the blue light response, because combining the data from C 340 340 0.043. red light and ca manipulations does not increase the total cor- 0 relation coefficient with respect to blue light (Table I). In ad- 320 0 - I blue 0.043 320 dition, increasing the VPD results in a decrease in ci (not shown) pulse T pulse 0 yet decreases the blue light response (Fig. 6). One important 340 340 0.006. aspect of future experiments will be to determine the relative a) rv- 0.006 magnitudes of the effects of red light, c' and VPD on the blue a1) 320 i red 0 pblue 320 I pulse light response. pulse The data reported here have several ecological implications. Ii) 35(o _ 0.000 350 0.000 The observation of conductance changes in response to blue but 34(C) 340 ! not red light pulses (Figs. 2 and 3) supports the suggestion bl red (25) 33( I ue 330 that the blue light response plays a significant role in conductance increases during sunflecks. At 0.263 mmol m-2 s - red light, 0 20 40 60 80 00 0o 20 40 60 Time (min) there is an increase in assimilation (Fig. 7a) that is correlated with blue light-stimulated increases in conductance and c; (Figs. FIG. 8. Changes in ci resulting from the conductance (Fig. 2) and 7a and 8a). However, at all other background light levels, as- assinmilation (Fig. 7) responses of C. communis to blue and red pulses. similation is limited by light rather than by C02, as indicated by Background fluence rates of red light in mmol m-2 s-I are indicated to the short pulse of assimilation associated directly with the light the right of each response trace. pulse (Fig. 7). Thus, the level of background or 'shade' illumi- nation in the natural environment may determine whether tran- that red light, apparently acting via phytochrome, shifts the flu- sient, blue light-stimulated increases in conductance have a sig- ence response curve of blue light-stimulated phototrophic bend- nificant effect on carbon gain. ing (3, 28). The results shown here also illustrate a property of the blue A third possibility is that the magnitude of the blue light re- light response of stomata that has not yet been demonstrated for sponse is affected by a 'messenger' from the mesophyll whose the red light, C02, or VPD responses of guard cells. That prop- concentration is proportional to the rate of carbon assimilation erty is that the blue light response is directly sensitive both to (23). In most of the experiments reported here (cf. Figs. 2a and factors which affect carbon gain, such as overall illumination 7a), the magnitude of the blue light response is positively cor- levels and CO2 concentrations, and to factors affecting rates of related with the baseline level of mesophyll assimilation. But, in water loss, such as VPD. The blue light response is greatest the experiments of Figures lb and 5, increasing ca resulted in a precisely under those conditions where CO2 is most likely to limit greater ci and a greater assimilation rate (not shown), yet de- carbon gain, namely conditions of high background irradiance pressed the blue light response. These results speak against a (Figs. 1, 2, and 4) or low ci (Fig. 5). Conversely, under conditions control of the blue light response by mesophyll assimilation. promoting water stress, such as an increased VPD, the magnitude A fourth suggestion is that the 'red light' enhancement may of the blue light response is diminished (Fig. 6). These results be explicable as an indirect effect, mediated by the intercellular suggest that the blue light response may be the modulator re- concentration of CO2. Both in experiments where ci is altered sponsible for 'fine-tuning' the ratio of carbon gain to water loss using light intensity (Figs. la, 2, 4) and in experiments where ci such that for a given steady state fluence rate, ca, and VPD, a is manipulated by changing c0 (Figs. lb and 5), there is a con- precise compromise is achieved between conflicting requirements sistent negative correlation between the magnitude of the blue for CO2 uptake and water conservation.

Table I. Correlation Coefficients (r) for the Magnitude of the Conductance Response to a 15 s x 0.100 mmol m-2 5-1 Pulse of Blue Light (Ag) versus Baseline Levels of Conductance (g), or ci, from the Experiments of Figure I Type of Experiment Experiment Number 1 2 3 4 1-4 combined Correlation coefficient (r) (a) Experiments altering the red light fluence rate Ag and g 0.98 0.76 0.95 0.99 0.63 Ag and c, -0.94 -0.91 -0.99 - 0.998 -0.77 (b) Experiments altering the ambient concentration of CO2 Ag and g 0.94 0.94 _a -a 0.41 Ag and ci -0.95 -0.91 a a -0.73 Data points from (a) and (b) combined Ag and g 0.37 Ag and ci 0.45 a These experiments were performed twice. STOMATAL RESPONSES TO BLUE LIGHT 231

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