TY - JOUR
AU - Kaldenhoff,, Ralf
AB - Abstract Various Arabidopsis thaliana mutants with defects in phytohormone signal transduction or the reception of light were analysed with regard to their stomatal response in a red, red/blue light irradiation programme. Stomatal response to light was detected with a customized gas exchange measurement device, optimized for the small model plant. Small transpiration‐kinetic variations of the two wild‐type lines Columbia (Col) and Landsberg erecta (Ler) were observed. A comparison of the mutant lines to the respective wild type revealed significant differences for the phytochrome A ( phyA‐103 ), the abscisic acid insensitive ( aba3‐2 ) and the auxin resistant ( axr1‐3 ) mutant. Furthermore, the zeaxanthin‐less mutant line npq1‐2 showed no alterations in stomatal response to light. Dual beam irradiation, gas exchange, light signal transduction, phytohormone, stomatal opening. Introduction Plant leaf stomatal opening or closure is modulated by various environmental and endogenous factors. One of the components of major importance is light. It is perceived by cellular photoreceptor systems stimulating two distinct photosensory pathways. One is the transduction of photosynthetic active radiation (PAR) by photosynthetic reactions in the guard cell chloroplasts, the other is induced by blue light. Participation of blue light in stomatal opening has been anticipated since the red light triggered reaction is weak compared to that of white light of equal fluence rate ( Zeiger, 1990 ). The specific response of guard cell protoplasts to blue light induces a change of guard cell osmotic potential caused by proton pumping and hyperpolarization of the plasma membrane ( Assmann et al ., 1985 ) followed by influx of ions ( Hedrich and Schroeder, 1989 ) as well as starch degradation and malate biosynthesis ( Ogawa et al ., 1978 ; Tallman and Zeiger, 1988 ; Talbott and Zeiger, 1993 ). As a consequence, water enters the cell resulting in stomatal opening. Experimentally, a dual beam strategy was found to be appropriate to separate the specific responses to red light, which is the authoritative component of PAR, and blue light. For the former, a response was usually assayed solely under red light irradiation, whereas the specific blue light response is induced by weak blue light given in a background of continuous red light. Stomata, which have achieved their steady‐state aperture under red light irradiation open wider when exposed to additional weak blue light ( Ogawa et al ., 1978 ; Assmann, 1988 ). This blue light‐dependent opening of stomata has revealed a causal connection to a receptor system with a non‐chlorophyll component. Recently, it has been reported that zeaxanthin, a part of the xanthophyll cycle, is possibly the chromophore of the blue light guard cell photoreceptor ( Zeiger and Zhu, 1998 ; Frechilla et al ., 1999 ). Besides the changes in environmental conditions, internal factors such as phytohormones were found to play a significant role in regulating stomatal movement. Closure, induced by abscisic acid (ABA), is established by an increase of the Ca 2+ ‐concentration in guard cell cytoplasm ( Schroeder and Hagiwara, 1989 ). The effects of gibberellic acid (GA) on stomatal diffusive resistance have been shown previously ( Bishnoi and Krishnamoorthy, 1993 ). Auxins modulate anion channels in the plasma membrane, which may initiate stomatal opening as demonstrated for Vicia faba guard cells ( Marten et al ., 1991 ). Studies of light and phytohormone effects on stomatal behaviour broadened the understanding of guard cell metabolism, transmembrane transport activities and the identity of components in signal transduction pathways. It is evident that for stomatal movement these elements do not act in isolation, but in a concerted manner. Due to the complexity of the corresponding signaling pathways and their interaction, the effect of a single factor or transduction chain might turn out to be difficult to separate by techniques of classical plant physiology. Arabidopsis thaliana is one of the few plant species that offers the chance to combine these established techniques with the power of genetics and molecular biology. Here, a broad range of mutants is available, which are impaired in single components of a light or phytohormone signal transduction pathway. In many cases, these mutants are characterized with regard to phenotype, physiology and molecular biology. Due to the effective work at the Arabidopsis stock centres, many of the mutant lines are accessible and could provide a helpful means of analysing a particular plant response in plants lacking one or another signal transduction component. In comparison to wild‐type controls without any defect, the participation of the corresponding signal could become evident. In this work, 15 different Arabidopsis thaliana mutant lines were examined for light‐induced stomatal opening under red‐ and blue light conditions. The selected lines are affected in the signal transduction pathway of a phytohormone or a specific light quality. The light‐induced stomatal opening was monitored via an increase in leaf transpiration, which was registered in a customized gas exchange analysis device. Due to the design of the cuvette, which holds the complete rosette of an Arabidopsis thaliana plant, the difficulties caused by the small size of the plant leaves could be overcome and the sensitivity of the measurements was greatly enhanced. The data obtained provide indications for the participation of one light signal transduction and two phytohormone pathways on light‐induced stomatal opening. Furthermore, the amount of zeaxanthin in planta does not influence the blue light‐dependent stomatal opening as determined by the gas exchange measurements. Materials and methods Plant material and growth conditions Arabidopsis thaliana plants were grown in a growth chamber under short day conditions (8:16 h, day/night). Soil mixture contained 4 vols commercial soil; 3 vols vermiculite; 2 vols perlite, and 1 vol. sand. Growth‐temperature ranged between 15 °C (night) and 22 °C (day). Plants requiring increased humidity were grown in acrylic glass boxes for as long as necessary. The following wild‐type and mutant lines were obtained from the Nottingham Arabidopsis Stock Centre (NASC) and the Arabidopsis Biological Resource Centre (ABRC): Ler (NW20); Col (N908); abi1‐1 (CS22); abi3‐1 (CS24); aba3‐2 (CS158); axr1‐3 (CS3075); axr2 (CS3077); aux1‐7 (CS8040); eto1‐1 (CS3072); ein1‐1 (CS3070); hy1 (CS68); hy3 (CS69); hy4 (CS70); fha‐1 (CS108); npq1‐2 (CS3771). Gas exchange measurement Plants were transferred from soil to a hydroponic vessel containing a mineral nutrient solution (5 mM NH 4 NO 3 , 1 mM CaCl 2 , 4 mM K 2 HPO 4 /KH 2 PO 4 , 2 mM MgSO 4 , 0.2 mM Na‐Fe‐EDTA, and 4 μM H 3 BO 3 , 10 μM MnCl 2 , 1 μM ZnSO 4 , 0.3 μM CuSO 4 , 0.1 μM Na 2 MoO 4 ). Anaerobic conditions were avoided by a permanent air supply for 24 h. The remaining soil was carefully removed from the roots and the plants were placed into a 14 ml Falcon tube with the same nutrient solution. The epicotyl was surrounded with a gas‐tight soft rubber fitting (Fig. 1A ) to a drilled cap, which was placed onto the tube (Fig. 1B ). This arrangement was transferred to a gas exchange cuvette of transparent acrylic glass (Fig. 1C ). Pairs of cuvettes were loaded and placed into a black box equipped with two slide projectors which were aligned in a way that the two beams intersected on the plane of the cuvettes (Fig. 1D ). Each beam was covered with appropriate interferic filters (Balzers Thin Films, FL) producing blue light (B46; 380–470 nm) and red light (R61; >600 nm), respectively, in order to set up the dual beam conditions. Fluence rates were adjusted by dimming the projector beams and determined with a LI‐190 quantum sensor (Li‐Cor, Lincoln, NE, USA). The cuvette gas volume for measurement was 30 cm 3 and air flow was adjusted to 600 ml min −1 . Gas exchange of Arabidopsis thaliana plants was measured with HCM‐1000 open‐flow gas circuit photosynthesis systems (Walz, Effeltrich, Germany). H 2 O and CO 2 concentrations at the inlet and outlet of the cuvette were measured using differential infrared gas analysers (BINOS‐100/4PS, Walz, Effeltrich, Germany). Data were (auto)recorded at 2 min intervals using DATA‐1000 software (Walz, Effeltrich, Germany). After measurement, the leaf surface area was determined by cutting the leaves, photostating and measuring area using DIAS II equipment (Delta‐T Devices LTD, Cambridge, UK). Data processing and calculations were done with Microsoft Excel TM . Statistical analysis was performed by using Student's t ‐test. Fluorescence measurements were performed using a TEACHING‐PAM chlorophyll fluorometer (Walz, Effeltrich, Germany). Non‐photochemical quenching was examined as described previously ( Niyogi et al ., 1998 ). Photosynthetic capacity was analysed by measurement of photosystem II fluorescence in the light (0–1400 μmol m −2  s −1 ) ( Schreiber, 1997 ). Fig. 1. Open in new tabDownload slide Experimental design of gas exchange measurement. (A) Seal and (B) mount to a 14 ml Falcon tube. (C) Pair of gas exchange measurement cuvettes and (D) scheme of the irradiation chamber with a dual beam device. Fig. 1. Open in new tabDownload slide Experimental design of gas exchange measurement. (A) Seal and (B) mount to a 14 ml Falcon tube. (C) Pair of gas exchange measurement cuvettes and (D) scheme of the irradiation chamber with a dual beam device. Results Conventional commercially available gas exchange cuvettes were designed to clamp single leaves with a minimum of approximately 5 cm diameter. The anatomy of the small model plant Arabidopsis thaliana does not meet these requirements. Therefore, it was difficult to get accurate data about the reaction of stomata in response to light. In order to circumvent these difficulties, a new type of cuvette was designed that holds the complete leaf rosette. This experimental set‐up has two advantages. On one hand, it increases the detectable signal due to higher total transpiration of the greatly increased leaf surface. On the other, the cuvette volume has an appropriate volume and suitable shape which results in an increase of the signal to noise ratio. Due to the rosette arrangement of Arabidopsis thaliana leaves, it was possible to measure the leaf area but difficult to determine the actually irradiated area. In addition, some of the mutant lines show an altered leaf morphology and variations in the size and age of the plants could not be avoided. Thus, in initial experiments the effect of plant size on transpiration rates and opening kinetics during different light stimuli were determined. Small plants (3 cm in diameter) and large plants (8 cm in diameter) were measured as pairs in parallel. The stomatal response to light was comparable with regard to light‐induced changes in the rate of transpiration, when leaf areas were integrated. The relative stomatal response revealed identical kinetics during irradiation with white light or with light of defined quality (Fig. 2 ). Thus, possible differences between leaf area and irradiated area do not affect the accuracy of the measurements. Accordingly, in a certain range, differences in plant size do not affect the kinetics of the stomatal transpiration. On this premise, the data obtained from differing mutant lines were comparable and thus, a deviation in the response‐kinetic is caused by the genetic defect, modulating the stomatal response to light. Dual beam measurement In order to examine the specific stomatal responses to red and blue light, the dual beam protocol (described by Ogawa et al ., 1978 ) was applied. Data obtained for transpiration rates in the dark were subtracted from actual transpiration rates and the resulting data were considered as light‐induced transpiration. As an initial step, the fluence rate of red light background illumination has been selected. A significant transpiration rate with adequate photosynthetic CO 2 uptake was obtained applying 80 μmol m −2  s − red light, or more (Fig. 3 ). 80 μmol m −2  s −1 red light was found to have no measurable effect on leaf temperature, when leaves were measured during the experiment with an electronically compensated thermo‐element equipped with a NiCr‐Ni sensor (data not shown). Based on this red light background, the effects of 10 μmol m −2  s −1 additional light have been observed. As shown in (Fig. 4 ), a surplus of red light caused only a slight increase in transpiration rate, whereas blue light promoted a significant increase in transpiration rate. Additional blue light also provoked a slight increase in CO 2 uptake. However, the kinetic of this increase is characteristic for the abolition of a stomatal limitation during red light driven photosynthesis. Thus, 10 μmol blue light given in a background of 80 μmol red light did not significantly influence photosynthesis, but specifically controlled the stomatal aperture (U Heber, University of Würzburg, Germany, personal communication). A time‐course of irradiation was determined on wild‐type plants. After 1 h in the dark, the plants showed a basal level of transpiration. Red light was applied for 50 min, a time period where all wild‐type plants reached a steady‐state level of transpiration. Additional blue light for 30 min further increased the transpiration, thus reflecting the specific blue light response. Fig. 2. Open in new tabDownload slide Transpiration measurement of A . thaliana . Small and large plants ( n =2, respectively) were measured in parallel. Fluence rates in μmol m −2  s −1 were blue light=30, red light=60, white light=400. Fig. 2. Open in new tabDownload slide Transpiration measurement of A . thaliana . Small and large plants ( n =2, respectively) were measured in parallel. Fluence rates in μmol m −2  s −1 were blue light=30, red light=60, white light=400. Fig. 3. Open in new tabDownload slide Light‐induced transpiration and CO 2 uptake of A . thaliana plants (Col). Steady‐state levels of plants in response to red light of increasing fluence rates. Standard errors (±SE) as indicated, n =3. Fig. 3. Open in new tabDownload slide Light‐induced transpiration and CO 2 uptake of A . thaliana plants (Col). Steady‐state levels of plants in response to red light of increasing fluence rates. Standard errors (±SE) as indicated, n =3. Fig. 4. Open in new tabDownload slide Light‐induced transpiration and CO 2 uptake of A . thaliana plants (Col) during the dual beam experiment. In a background of 80 μmol m −2  s −1 red light, additional 10 μmol m −2  s −1 red or blue light were given as indicated. Fig. 4. Open in new tabDownload slide Light‐induced transpiration and CO 2 uptake of A . thaliana plants (Col) during the dual beam experiment. In a background of 80 μmol m −2  s −1 red light, additional 10 μmol m −2  s −1 red or blue light were given as indicated. Analysis of Arabidopsis thaliana mutant lines Two wild‐type ecotypes (Ler and Col), reflecting the genetic background of the mutants were measured by employing the light programme described above and revealed slight differences in stomatal opening (Fig. 5 ). Therefore and in order to provide comparable data, every mutant line was compared with the corresponding wild‐type, Ler respectively Col. Fifteen mutant lines as listed in Table 1 were analysed. Eleven showed no significant deviation in transpiration. Five were different in the extent of stomatal response, in the kinetics of stomatal opening or in the expected stomatal response to additional irradiation. axr1‐3 ( Estelle and Sommerville, 1987 ) showed a faster and increased response in red light as well as in blue light‐induced stomatal opening (Fig. 6 ). The transpiration in red light was significantly higher ( P =0.025) than that of controls and the maximum appeared earlier ( c . 10 min). The blue light‐induced response was elevated and also reached a maximum earlier than the wild type. phyA‐103 ( Parks and Quail, 1993 ) appeared to be reduced in red light‐dependent stomatal opening (Fig. 7 ). At the beginning of red light irradiation the phyA‐103 mutant followed the transpiration course of the control, it reached a steady‐state level much earlier and showed about half of the transpiration than observed for the wild type. The high significance ( P =0.005) denotes a reduced response to red light. In contrast, the blue light‐induced stomatal opening has a similar amplitude as the control. aba3‐2 ( Léon‐Kloosterziel et al ., 1996 ) behaved completely different compared to the corresponding wild‐type line (Ler) (Fig. 8 ). Red light background did not result in a substantial increase in transpiration, whereas the specific blue light response was relative high. In contrast, abi1‐1 and abi3‐1 showed more unaffected responses to the irradiation programme. At least abi3‐1 tracked the course of the wild type, which indicates that the defect caused by the mutation does not affect the stomatal signal transduction pathways. npq1‐2 ( Niyogi et al ., 1998 ) showed a stomatal response which is highly comparable to that of the corresponding wild type (Fig. 9A ). Surprisingly, this is not in line with recent reports describing a lack of specific stomatal response to blue light ( Zeiger and Zhu, 1998 ; Frechilla et al ., 1999 ). Detailed studies on the specific response to additional light were performed and revealed no differences between the wild type and npq1‐2 mutant line (Fig. 9B ). In order to confirm the mutation, each investigated plant was tested for its non‐photochemical quenching (NPQ) capability as described previously ( Niyogi et al ., 1998 ). All npq1‐2 plants subjected to the gas exchange measurements showed impaired induction of NPQ, whereas wild‐type plants followed the time‐course of induction and subsequent relaxation without alterations (Fig. 9C ). In order to examine whether a different transpiration kinetic is caused by aberrant morphology or number of guard cells, leaves of the corresponding mutant line were monitored under a microscope. For none of the lines presented in this paper could a striking difference be observed, except an altered rosette formation of the aba3‐2 mutant. All mutant lines, used for this studies were examined with regard to their photosynthetic capacity. No significant alterations in relative electron transport rate were obtained (data not shown). Fig. 5. Open in new tabDownload slide Light‐induced transpiration of A . thaliana wild‐type, ecotype Columbia (Col) and Landsberg erecta (Ler) during the dual beam experiment. Irradiation was 80 μmol m −2 s −1 red light, additional 10 μmol m −2  s −1 blue light was given at 50 min after the onset of red light irradiation. Bars indicate standard errors (±SE) on expressive time points; n =12, respectively n =10. Fig. 5. Open in new tabDownload slide Light‐induced transpiration of A . thaliana wild‐type, ecotype Columbia (Col) and Landsberg erecta (Ler) during the dual beam experiment. Irradiation was 80 μmol m −2 s −1 red light, additional 10 μmol m −2  s −1 blue light was given at 50 min after the onset of red light irradiation. Bars indicate standard errors (±SE) on expressive time points; n =12, respectively n =10. Fig. 6. Open in new tabDownload slide Transpiration of axr1‐3 mutant and control (Col). Irradiation as indicated and described in Fig. 5 . P ‐value is shown for a single time point. axr1‐3 : n =11, Col: n =12. Fig. 6. Open in new tabDownload slide Transpiration of axr1‐3 mutant and control (Col). Irradiation as indicated and described in Fig. 5 . P ‐value is shown for a single time point. axr1‐3 : n =11, Col: n =12. Fig. 7. Open in new tabDownload slide Transpiration of phyA‐103 mutant and control (Col). Irradiation as indicated and described in Fig. 5 . P ‐value is shown for a single time point. phyA‐103 : n =10, Col: n =12. Fig. 7. Open in new tabDownload slide Transpiration of phyA‐103 mutant and control (Col). Irradiation as indicated and described in Fig. 5 . P ‐value is shown for a single time point. phyA‐103 : n =10, Col: n =12. Fig. 8. Open in new tabDownload slide Transpiration of aba3‐2 , abi1‐1 , abi3‐1 mutants and control (Ler). Irradiation as indicated and described in Fig. 5 : n ( aba3‐2 )=10, n ( abi1‐1 )=8, n ( abi3‐1 )=8, n (Ler)=10. Fig. 8. Open in new tabDownload slide Transpiration of aba3‐2 , abi1‐1 , abi3‐1 mutants and control (Ler). Irradiation as indicated and described in Fig. 5 : n ( aba3‐2 )=10, n ( abi1‐1 )=8, n ( abi3‐1 )=8, n (Ler)=10. Fig. 9. Open in new tabDownload slide (A) Transpiration of npq1‐2 mutant and control (Col). Irradiation as indicated and described in Fig. 5 . npq1‐2 : n =5, Col: n =12. (B) Comparison of the effects of additional light on transpiration of npq1 and control (Col). In a background of 80 μmol m −2  s −1 red light, additional 10 μmol m −2 s −1 red or blue light were given as indicated. Bars represent the mean of five experiments and include standard errors (+SE). RL: red light, BL: blue light. (C) Representative time‐course for induction and relaxation of NPQ in leaves of npq1 and wild type (Col). Actinic light was given at time point zero and switched off after 5 min. Pretreatment of plants and measurement of fluorescence as described previously ( Niyogi et al ., 1998 ). Fig. 9. Open in new tabDownload slide (A) Transpiration of npq1‐2 mutant and control (Col). Irradiation as indicated and described in Fig. 5 . npq1‐2 : n =5, Col: n =12. (B) Comparison of the effects of additional light on transpiration of npq1 and control (Col). In a background of 80 μmol m −2  s −1 red light, additional 10 μmol m −2 s −1 red or blue light were given as indicated. Bars represent the mean of five experiments and include standard errors (+SE). RL: red light, BL: blue light. (C) Representative time‐course for induction and relaxation of NPQ in leaves of npq1 and wild type (Col). Actinic light was given at time point zero and switched off after 5 min. Pretreatment of plants and measurement of fluorescence as described previously ( Niyogi et al ., 1998 ). Table 1. List of mutant lines analysed for light‐induced transpiration Alternative line descriptions are given in brackets; + indicates a wild‐type like transpiration kinetic, – an aberrant kinetic. Numbers of investigated plants were given as n . Pathway/ Mutant line and WT‐like receptor background transpiration ABA abi1‐1 /Ler − / n =8 abi3‐1 /Ler + / n =8 aba3‐2 /Ler − / n =10 Auxin axr1‐3 /Col − / n =11 axr2 /Col + / n =10 aux1‐7 /Col + / n =12 Ethylene eto1‐1 /Ler + / n =10 ein1‐1 /Ler + / n =10 Phytochrome hy1 /Ler + / n =10 hy3 (phyB) /Ler + / n =12 phyA‐103 /Col − / n =10 Blue light hy4(cry 1) /Ler + / n =12 nph1‐5 /Col + / n =13 fha‐1 (cry2) /Ler + / n =10 npq1‐2 /Col + / n =5 Pathway/ Mutant line and WT‐like receptor background transpiration ABA abi1‐1 /Ler − / n =8 abi3‐1 /Ler + / n =8 aba3‐2 /Ler − / n =10 Auxin axr1‐3 /Col − / n =11 axr2 /Col + / n =10 aux1‐7 /Col + / n =12 Ethylene eto1‐1 /Ler + / n =10 ein1‐1 /Ler + / n =10 Phytochrome hy1 /Ler + / n =10 hy3 (phyB) /Ler + / n =12 phyA‐103 /Col − / n =10 Blue light hy4(cry 1) /Ler + / n =12 nph1‐5 /Col + / n =13 fha‐1 (cry2) /Ler + / n =10 npq1‐2 /Col + / n =5 Open in new tab Table 1. List of mutant lines analysed for light‐induced transpiration Alternative line descriptions are given in brackets; + indicates a wild‐type like transpiration kinetic, – an aberrant kinetic. Numbers of investigated plants were given as n . Pathway/ Mutant line and WT‐like receptor background transpiration ABA abi1‐1 /Ler − / n =8 abi3‐1 /Ler + / n =8 aba3‐2 /Ler − / n =10 Auxin axr1‐3 /Col − / n =11 axr2 /Col + / n =10 aux1‐7 /Col + / n =12 Ethylene eto1‐1 /Ler + / n =10 ein1‐1 /Ler + / n =10 Phytochrome hy1 /Ler + / n =10 hy3 (phyB) /Ler + / n =12 phyA‐103 /Col − / n =10 Blue light hy4(cry 1) /Ler + / n =12 nph1‐5 /Col + / n =13 fha‐1 (cry2) /Ler + / n =10 npq1‐2 /Col + / n =5 Pathway/ Mutant line and WT‐like receptor background transpiration ABA abi1‐1 /Ler − / n =8 abi3‐1 /Ler + / n =8 aba3‐2 /Ler − / n =10 Auxin axr1‐3 /Col − / n =11 axr2 /Col + / n =10 aux1‐7 /Col + / n =12 Ethylene eto1‐1 /Ler + / n =10 ein1‐1 /Ler + / n =10 Phytochrome hy1 /Ler + / n =10 hy3 (phyB) /Ler + / n =12 phyA‐103 /Col − / n =10 Blue light hy4(cry 1) /Ler + / n =12 nph1‐5 /Col + / n =13 fha‐1 (cry2) /Ler + / n =10 npq1‐2 /Col + / n =5 Open in new tab Discussion Mutants in light signal transduction Blue light: The analysis of mutants in a light signal transduction pathway was of particular interest because, as outlined above, light is one of the primary inducers and in the plants' natural environment is of major importance for stomatal opening. Interestingly, none of the four mutant lines, which most likely are defective in different blue light receptors, showed stomatal behaviour different from the wild type (Table 1 ). Consequently, the two CRY gene products and NPH1 are not involved in the regulation of stomatal movement, which is in agreement with measurements from others ( Lascève et al ., 1999 ; RP Hangarter, Indiana University, USA, WR Briggs Carnegie Institution of Washington, USA; personal communications). Recent findings which demonstrate that guard cells of the Arabidopsis mutant npq1 ( Niyogi et al ., 1998 ) lack the specific response to blue light in a background of red light ( Zeiger and Zhu, 1998 ; Frechilla et al ., 1999 ), indicate a defect in a component of stomatal blue light‐dependent signal transduction. Npq1 plants are defective in violaxanthin de‐epoxidase activity and therefore unable to accumulate zeaxanthin. Results presented in this work do not confirm these findings. Although all plants of the mutant line npq1‐2 showed strongly reduced non‐photochemical quenching, none of the plants subjected to gas exchange measurement exhibited deviations in stomatal responses, neither to red light, nor to blue light in a red light background. However, the experimental approaches were different, since Frechilla et al . studied stomatal responses of epidermal strips. The apparent differences might well be due to isolated guard cells on the one hand and intact leaves on the other. Red light: Several studies clearly revealed that the red light response of stomata is caused by photosynthetic action ( Wu and Assmann, 1993 ) and not by the red, far‐red light sensitive phytochrome systems A or B. While the measurements with various phytochrome B mutants could confirm these results, the Arabidopsis line with a defective phytochrome A seems to be different. The mutant line used in the gas exchange measurements with allele phyA‐103 exhibits almost wild‐type levels of detectable phytochrome protein. However, due to a missense mutation, it is defective in its regulatory activity. Although in red light the kinetic of the response was identical with that of the wild type, the amplitude of stomatal opening was significantly reduced. This effect was not recorded during additional blue light irradiation, but restricted to the red light response of the phyA‐103 mutant. In contrast, the mutant in phytochrome B ( hy3 ) as well as the mutant in phytochrome chromophore biosynthesis ( hy1 ) did not show any deviation in the dual beam experiment. The reason for this difference remains unclear and further studies are required to discuss this issue. Nevertheless, the highly significant aberrant response to red light ( P =0.005) may be taken into consideration in reflections on phytochrome action. Mutants in phytohormone signal transduction Auxin: Auxin influences plant growth and differentiation. The axr1 plants show morphological defects, which are consistent with a reduction in auxin sensitivity ( Estelle and Sommerville, 1987 ). The AXR1 gene product is required for normal auxin response in the whole plant ( Lincoln et al ., 1990 ). The mutant line axr1‐3 shows less severe morphological alterations than other axr1 mutants. Because of the morphological similarity to a wild type this line is most suitable for comparison of transpiration kinetics. Molecular genetic studies revealed that the function of AXR1 protein might be involved in activation of ubiquitin (UBQ) or UBQ‐related proteins ( Leyser et al ., 1993 ; Pozo et al ., 1998 ). In the context of stomatal movement, the enhanced light‐induced response of axr1‐3 can not be directly related to mechanisms requiring activated UBQ‐related proteins. However, the obtained data indicate an accelerated stomatal response which was unique for the axr1‐3 mutant line and seems not to be specific for a certain light quality. Abscisic acid: aba3 mutants display a strong phenotype, caused by the disruption of aldehyde oxidase activities, which require a sulphur‐containing molybdenum cofactor (MOCO). The most severe impairment is established by the lack of ABA‐aldehyde to ABA conversion ( Schwartz et al ., 1997 ). The strongly reduced internal ABA content leads to a wilty phenotype, due to a constitutive high stomatal aperture ( Léon‐Kloosterziel et al ., 1996 ). The regulation of the aba3‐2 stomatal aperture was strongly affected by the lack of ABA, not only in response to water stress but also in the dark, where high transpiration rates were obtained. When aba3‐2 plants were transferred from dark to red, red/blue light, the initially widely opened stomata opened further, indicating that light signal transduction pathways at least for blue light are unaffected. The stomatal opening induced by red light was very weak. If this was due to a masking effect caused by the high initial stomatal aperture, the same should be expected for the also constitutively high transpirating abi1‐1 mutant line, but not for abi3‐2 ( Roelfsema and Prins, 1995 ). However, the responses of both lines are more comparable to the wild type than to the response of aba3‐2 . Thus, an initial high stomatal aperture did not lead to any uncertainty regarding the gas exchange measurements. The comparable transpiration courses of wild type and abi3‐1 are consistent with reports describing the involvement of the ABI3 gene product in seed development ( Koornneef et al ., 1989 ). Accordingly, this mutation in ABA signal transduction seems not to affect the stoma mechanisms. The differences between abi1‐1 and aba3‐2 could possibly account for the sequence of ABA signal transduction. For red light, the absence of ABA seems to abolish the response ( aba3‐2 ), whereas the disrupted ABI1‐1 gene product seems not to be essential for light‐induced stomatal opening ( abi1‐1 ). The fact that blue light opens the stomata further indicates that the aba3‐2 stomatal opening mechanism is still intact and is an argument in favour of a blue light‐signalling completely independent from ABA. In summary, the investigations presented in this paper indicate an impairment of the phyA mutant with respect to red light‐driven stomatal opening as well as a close interaction between red light and ABA signal transduction pathways. Support was also found for the independence of the blue light‐induced stomatal opening from a blocked ABA signal transduction during the dual beam programme. Furthermore, a deficiency in ubiquitin activation, as it is supposed for the axr1‐3 mutant line, enhances the stomatal response to light, which leads to the idea that ubiquitin could participate on the signal transduction or mechanism for light‐induced stomatal opening. Previous work on gas exchange of mutants and model plants revealed interesting data and point to new aspects in plant research ( Lascève et al ., 1997 , 1999 ; Leymarie et al ., 1998 ; Karlsson, 1986 ; Vavasseur et al ., 1988 ). The described gas exchange measurement established for this work improved the physiological analysis of genetically well‐defined Arabidopsis thaliana mutants. 1 To whom correspondence should be addressed. Fax: +49 931 8886158. E‐mail: kaldenhoff@botanik.uni‐wuerzburg.de We are grateful to R Hedrich (Molekulare Pflanzenphysiologie und Biophysik, Universität Würzburg, Germany) for providing the gas exchange apparatus and his help in the design of the cuvettes. We thank the NASC and ABRC for shipping the bulk of Arabidopsis thaliana mutants and WR Briggs (Department of Plant Biology, Carnegie Institution of Washington, USA) for the generous gift of the nph1‐5 mutant as well as Peter Quail (Department of Plant and Microbial Biology, University of California, Berkeley, USA) and Brian Parks (Department of Botany, University of Wisconsin, Madison, USA) for supplying phyA‐103 seeds. We thank U Heber (University of Würzburg, Germany) for helpful discussions and critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft. References Assmann SM. 1988 . Enhancement of the stomatal response to blue light by red light, reduced intercellular concentrations of CO 2 and low vapor pressure differences. Plant Physiology 87 , 226 –231. Assmann SM, Simoncini L, Schroeder JI. 1985 . Blue light activates electrogenic ion pumping in guard cell protoplasts of Vicia faba . Nature 318 , 285 –287. 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TI -  Light‐induced stomatal movement of selected Arabidopsis thaliana mutants 
JF - Journal of Experimental Botany
DO - 10.1093/jxb/51.349.1435
DA - 2000-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/light-induced-stomatal-movement-of-selected-arabidopsis-thaliana-UX726xMkzE
SP - 1435
VL - 51
IS - 349
DP - DeepDyve
ER -