Present address: Section of Cell and Developmental Biology and Center for Molecular Genetics, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093–0634, USA.
Use of infrared thermal imaging to isolate Arabidopsis mutants defective in stomatal regulation
Article first published online: 30 MAY 2002
The Plant Journal
Volume 30, Issue 5, pages 601–609, June 2002
How to Cite
Merlot, S., Mustilli, A.-C., Genty, B., North, H., Lefebvre, V., Sotta, B., Vavasseur, A. and Giraudat, J. (2002), Use of infrared thermal imaging to isolate Arabidopsis mutants defective in stomatal regulation. The Plant Journal, 30: 601–609. doi: 10.1046/j.1365-313X.2002.01322.x
- Issue published online: 30 MAY 2002
- Article first published online: 30 MAY 2002
- Received 25 January 2002; revised 8 March 2002; accepted 13 March 2002.
- abscisic acid (ABA);
- guard cell;
- infrared thermography;
- signal transduction
In response to drought, plants synthesise the hormone abscisic acid (ABA), which triggers closure of the stomatal pores. This process is vital for plants to conserve water by reducing transpirational water loss. Moreover, recent studies have demonstrated the advantages of the Arabidopsis stomatal guard cell for combining genetic, molecular and biophysical approaches to characterise ABA action. However, genetic dissection of stomatal regulation has been limited by the difficulty of identifying a reliable phenotype for mutant screening. Leaf temperature can be used as an indicator to detect mutants with altered stomatal control, since transpiration causes leaf cooling. In this study, we optimised experimental conditions under which individual Arabidopsis plants with altered stomatal responses to drought can be identified by infrared thermography. These conditions were then used to perform a pilot screen for mutants that displayed a reduced ability to close their stomata and hence appeared colder than the wild type. Some of the mutants recovered were deficient in ABA accumulation, and corresponded to alleles of the ABA biosynthesis loci ABA1, ABA2 and ABA3. Interestingly, two of these novel aba2 alleles were able to intragenically complement the aba2–1 mutation. The remaining mutants showed reduced ABA responsiveness in guard cells. In addition to the previously known abi1–1 mutation, we isolated mutations at two novel loci designated as OST1 (OPEN STOMATA 1) and OST2. Remarkably, ost1 and ost2 represent, to our knowledge, the first Arabidopsis mutations altering ABA responsiveness in stomata and not in seeds.
Abscisic acid (ABA) plays an important role in various aspects of plant growth and development (Koornneef et al., 1998; Leung and Giraudat, 1998; McCourt, 1999). In seeds, ABA promotes the acquisition of nutritive reserves, desiccation tolerance and dormancy, and it inhibits seed germination. In vegetative tissues, ABA mediates adaptive responses to abiotic environmental stresses. In particular, ABA is synthesised in response to drought and ABA-induced stomatal closure is vital for plants to conserve water by reducing transpirational water loss. The closing and opening of the stomatal pore result from the osmotic shrinking and swelling, respectively, of the two surrounding guard cells. ABA acts directly on guard cells and induces stomatal closure via efflux of potassium and anions from guard cells and removal of organic osmolytes (Assmann and Wang, 2001; MacRobbie, 1998; Schroeder et al., 2001).
In various plant species, genetic analysis based primarily on ABA promotion of seed dormancy has yielded a series of mutants that are defective in ABA biosynthesis (Koornneef et al., 1998; Liotenberg et al., 1999). Many of these mutants, including the Arabidopsis aba1, aba2 and aba3 mutants, display increased transpirational water loss in addition to reduced seed dormancy (Koornneef et al., 1982; Léon-Kloosterziel et al., 1996). ABA-deficient mutants have also been instrumental in the recent cloning of genes encoding ABA biosynthetic enzymes (Marin et al., 1996; Seo et al., 2000; Tan et al., 1997), which has led to a better understanding of the regulation of ABA biosynthesis. In particular, available evidence suggests that the 9-cis-epoxycarotenoid cleavage reaction catalysed by 9-cis-epoxycarotenoid dioxygenase (NCED) is an essential regulatory step in drought-induced ABA biosynthesis (Qin and Zeevaart, 1999). However, there is apparently a NCED gene family in plants, and mutations in each of these genes would be useful to elucidate the roles of individual NCED genes in different tissues and under different environmental conditions. Moreover, the upstream signalling cascade that links water stress perception to stimulation of the ABA biosynthetic pathway still awaits elucidation.
Pharmacological and cell biological studies have led to major advances in understanding the transduction pathway by which ABA regulates stomatal movements (Assmann and Wang, 2001; Blatt, 2000; Hetherington, 2001; Schroeder et al., 2001). This signalling pathway involves oscillations in cytosolic free calcium, increases in cytoplasmic pH, protein kinases and phosphatases, and activation of phospholipase D. In recent years, important advances have been made in developing single-cell techniques applicable to Arabidopsis guard cells. Combining the use of mutants with single cell physiological analyses on this model species thus opens up promising avenues for the identification of the remaining elements of the ABA signalling network in guard cells. Thus far, genetic dissection of ABA action in Arabidopsis guard cells has been primarily limited to pleiotropic mutants, including abi1, abi2, era1, gca2 and abh1, that were all initially selected in screens based on ABA inhibition of seed germination and seedling growth (Allen et al., 2001; Hugouvieux et al., 2001; Murata et al., 2001; Pei et al., 1998). However, the AAPK protein kinase from Vicia faba is activated in guard cells but not in leaf epidermal or mesophyll cells (Li et al., 2000), suggesting that certain elements of the ABA signalling network are specific to guard cells. Hence, in parallel to reverse genetic approaches (Lemichez et al., 2001; Wang et al., 2001), there is a need to develop novel forward genetic screens more specifically targeted towards the identification of mutations affecting stomatal regulation.
The ABA-deficient aba mutants of Arabidopsis, as well as the ABA-insensitive abi1 and abi2 mutants mentioned above display an increased tendency to wilt (Koornneef et al., 1982; Koornneef et al., 1984). However, this visual phenotype is rather variable in intensity and hence not ideally suited for large scale mutant screens. Transpiration causes leaf cooling because evaporation of water is associated with heat loss (latent heat loss). Leaf surface temperature can be measured continuously and non-destructively using infrared thermography, and thus provides a convenient indicator of the transpiration of individual plants (Chaerle and Van Der Straeten, 2000; Hashimoto et al., 1984; Jones, 1999). In particular, infrared thermography was used by Raskin and Ladyman (1988) to isolate the barley mutant cool that is unable to close its stomata in response to ABA treatment. This mutant was not studied further but, interestingly, the cool mutation did not affect ABA sensitivity of embryo germination. Despite these promising results, to our knowledge, thermal imaging has not been used since then to screen for mutants affected in stomatal regulation.
In this study, we tested the suitability of thermal imaging for stomatal mutant screening in Arabidopsis. Leaf temperature depends on stomatal aperture and on a complex range of additional factors, including leaf shape and position, radiation absorption, air humidity, air temperature, and wind speed (Jones, 1999). Using available ABA-deficient and ABA-insensitive mutants as positive controls, we optimised experimental conditions for thermographic detection of individual plants with altered stomatal responses to drought. These conditions were then used to perform a pilot screen for mutants that displayed a reduced ability to close their stomata in response to drought stress and hence appeared colder than the wild type. Some of the mutants recovered were deficient in ABA accumulation, whereas others were affected in ABA responsiveness in guard cells. In particular, we identified mutations at two novel loci that inhibit the ABA regulation of stomatal aperture but do not alter ABA sensitivity in seeds.
Optimisation of the experimental protocol
We investigated whether thermal imaging is appropriate for high-throughput screening of Arabidopsis mutants with a reduced ability to close their stomata in response to water stress. In order to define appropriate experimental conditions for such a screen the ABA-deficient aba1–1 (Koornneef et al., 1982) and ABA-insensitive abi1–1 mutants (Koornneef et al., 1984) were used as positive controls. As in vitro culture has been shown to alter stomatal development and ABA sensitivity (Willmer and Fricker, 1996), we used soil grown plants. Plants were first grown in the greenhouse under well-watered conditions. They were then subjected to drought stress by transferring the pots to a growth cabinet with a drier atmosphere and by withholding watering. The plants were analysed by thermal imaging in this same growth cabinet.
To simplify screening procedures, the aim was to be able to identify individual mutants amidst a population of wild-type plants by visualising the differences in leaf temperature between several plants in a given image, rather than by measuring the absolute leaf temperature of each individual plant independently. Hence the parameters required to obtain an accurate image of absolute leaf temperature were not determined. Instead environmental conditions were defined that would maximise the difference in temperature between leaves with closed stomata and leaves with open stomata, while maintaining an homogeneous temperature response for a given transpiration rate in all the plants observed over the image field. The growth cabinet was equipped with fluorescent rather than incandescent lights to minimise the short-wave infrared background that could bias the estimation of leaf temperature when using the 3.4–5 μm infrared band for thermal imaging. After a series of empirical tests, the temperature in the growth cabinet was set to 24°C, the relative humidity to 50%, and the ventilation to low air speed (0.4 m sec−1).
To obtain a homogeneous and progressive drought stress within a few days of withholding watering the suitability of different types of soil were compared. Sand (2–3 mm diameter) was satisfactory in this respect, but plants grew rather poorly on this support during the initial well-watered phase. Commercial compost, which has a high water retention capacity, dried very slowly and furthermore had a tendency to collapse during drying. A mixture composed of 50% sand and 50% compost (v/v) was retained as a good compromise.
Figure 1 shows that after 3 days of drought stress, the leaf temperature of wild-type plants was high and homogeneous, indicating the uniformity of stomatal closing response. The leaves of abi1–1 plants were approximately 1°C colder because this ABA-insensitive mutant failed to close its stomata. Since the sensitivity of the camera in this range of temperature is less than 0.1°C, the abi1–1 mutant plants could be easily and reliably distinguished from the surrounding wild-type individuals on the thermal image. It is noteworthy that, at this stage of the drought stress, the abi1–1 plants did not yet display any sign of wilting. Similar results were obtained with the ABA-deficient aba1 mutant (data not shown).
This protocol could be applied to young plantlets with only 2–4 true leaves, thus allowing a sowing density of approximately one seed per 1.5 cm2, while still permitting individual plantlets to be distinguished on the thermal image. This allowed to minimise the culture surface needed for mutant screens.
An M2 population, derived from ethyl methanesulfonate (EMS) mutagenised wild-type Arabidopsis, was screened by thermal imaging for mutants with a ‘cold’ phenotype under drought stress. Seeds were germinated and grown for 1 week under well-watered conditions. Plantlets were then subjected to drought stress, and analysed by infrared thermography 3–4 days later. Individuals displaying a lower leaf temperature than the other plants in the same pot were selected as candidate mutants. From an estimated total of 85 000 M2 seeds screened (derived from seven parental groups of 1450 M1 plants each), 85 candidates were selected during the primary screen. Out of these, 75 were fertile and 44 showed a heritable ‘cold’ phenotype in the M3 generation. For each of these 44 mutant lines, the amount of ABA present in the leaves of drought-stressed plants was quantified. Thirty-six lines displayed ABA levels that were at least 30% lower than in wild-type controls (data not shown), and further analysis confirmed that these lines corresponded to ABA biosynthesis mutants (see below). The remaining eight mutant lines contained 3–8 times more ABA than the wild type upon drought stress (data not shown), and these lines were subsequently shown to correspond to ABA-insensitive mutants.
ABA-insensitive mutants, including abi1–1 and abi2–1, contain more ABA than the wild type (Koornneef et al., 1984). As the abi1–1 and abi2–1 mutations lead to ABA-insensitive stomata, the presence of these mutations was examined in the eight mutant lines with enhanced ABA contents. The CAPS assay described by Leung et al. (1997) indicated that line II-38 was homozygous for the abi1-1 mutation, and that lines II-3 and II-33 were heterozygous for this dominant mutation. These results were confirmed by sequencing the ABI1 gene in these three lines. Since lines II-3, II-33 and II-38 originate from the same parental pool, it is likely that they all derived from a single M1 plant (Table 1).
|Isolation number a||Mutation|
|II-3, II-33, II-38||abi1-1|
|II-52, II-56, II-123||ost1-1|
Each of the five remaining lines was crossed with the wild type. Analysis of the resulting F1 progenies by thermal imaging showed that the ‘cold’ phenotype was recessive in lines II-52, II-56, II-123 and VII-149, and was dominant in line VII-8. The lines II-52, II-56, II-123 and VII-149 were crossed with each other, and were found to belong to a single complementation group that was called OST1 for OPEN STOMATA 1. Lines II-52, II-56 and II-123 originating from the same parental pool of M1 plants were assumed to be siblings, and only II-52 was retained for further analysis. Lines II-52 and VII-149 originate from distinct parental pools, and thus correspond to two independent mutant alleles of OST1 which were renamed ost1-1 and ost1-2, respectively. The locus identified by the dominant mutation VII-8 was designated as OST2 (Table 1).
Like abi1–1, drought-stressed ost1 and ost2 plantlets were approximately 1°C colder than wild-type plantlets (Figure 2a,b). The stomatal behaviour of these mutants was analysed further by bioassays on epidermal peels. Like abi1-1, ost1 and ost2 were impaired in their ability to close their stomata in response to applied ABA (Figure 2c). However, the stomata of both mutants did close in response to darkness (data not shown), indicating that the ost1 and ost2 mutations do not lead to a global defect in stomatal functioning but rather alter the ABA-regulation of stomatal aperture. Interestingly, unlike other guard cell ABA signalling mutants, the ost1 and ost2 mutants displayed a wild-type seed dormancy (data not shown) and a wild-type sensitivity of seed germination to applied ABA (Figure 2d).
As mentioned above, the 36 other mutant lines showed reduced levels of ABA compared with wild type. Furthermore, these 36 mutants displayed reduced seed dormancy and, when grown under low relative humidity (50%), were smaller and darker green than wild-type plants (data not shown). All these phenotypes are characteristic of the ABA biosynthesis mutants aba1, aba2, and aba3 (Koornneef et al., 1982; Léon-Kloosterziel et al., 1996). Hence we tested whether the ABA-deficient lines isolated here were allelic to these biosynthetic mutants.
The ABA1 gene encodes zeaxanthin epoxidase (Audran et al., 2001; Duckham et al., 1991; Marin et al., 1996; Rock and Zeevaart, 1991). HPLC analysis of carotenoids in leaf extracts revealed that, like the aba1 mutants, 14 of the 36 ABA-deficient lines lacked the epoxy-carotenoids violaxanthin and neoxanthin and accumulated high levels of their biosynthetic precursor zeaxanthin (data not shown). Complementation tests with the aba1-5 mutant confirmed that all 14 of these mutant lines represent aba1 alleles (Table 2). DNA sequence analysis of the ABA1 gene in two of these lines revealed that in line V-115 (designated as aba1-11) the residue Trp-363 is converted to a TGA, thus generating a premature STOP codon, and that in line VI-137 (designated as aba1-12) the residue Ser-374 is converted into Phe.
|III-52, III-53, III-60, III-61, III-64, III-139|
|VI-32, VI-101, VI-104, VI-137c, VI-142|
|VII-10, VII-46, VII-59, VII-89, VII-108|
|VIII-33, VIII-41, VIII-43, VIII-49|
The ABA3 gene encodes a molybdenum cofactor sulfurase (Bittner et al., 2001; Xiong et al., 2001) that produces the desulfo form of the cofactor required for abscisic aldehyde oxidase activity (Schwartz et al., 1997). Xanthine dehydrogenase requires the same sulfurylated cofactor, and its activity can be easily assayed in plant extracts (Marin and Marion-Poll, 1997). Nine of the present ABA-deficient lines were found to lack xanthine dehydrogenase activity (data not shown). Nitrate reductase uses the dioxo form of the cofactor and, as these lines were able to grow on a medium containing nitrate as sole nitrogen source, the lack of xanthine dehydrogenase activity was not due to a defect in the molybdenum cofactor synthesis prior to its sulfuration (Marin and Marion-Poll, 1997). Hence, these results indicate that the nine mutant lines are most likely aba3 alleles, although direct complementation tests were not performed (Table 2).
Each of the remaining 13 ABA-deficient lines was crossed to the aba2-1 mutant. The resulting F1 progenies were subjected to drought stress and analysed by thermal imaging. For nine lines, the F1 plants displayed a ‘cold’ phenotype like the parents, indicating that they were aba2 alleles (Table 2). In contrast, the F1 plants derived from crosses between aba2-1 and lines II-47, II-51, III-20 and IV-64 displayed a wild-type ‘warm’ phenotype (data not shown), suggesting that these four lines were not allelic to aba2. The mutant lines II-47, II-51, III-20 and IV-64 were crossed with each other. The resulting F1 plants showed a ‘cold’ phenotype (data not shown), indicating that the four lines belonged to the same complementation group. Using molecular markers, the mutation IV-64 was mapped between the BAC clones F2J6 and F14J6 on chromosome 1 (data not shown). This region contains the BAC clone F19K6 that harbours the ABA2 gene (Rook et al., 2001). Sequencing the ABA2 gene revealed that line IV-64 contains a point mutation converting Asp-271 into Asn, and that all three lines II-47, II-51 and III-20 contain another point mutation that converts Gly-27 into Glu. These data suggest that these four lines represent aba2 alleles that can intragenically complement the aba2-1 mutation. Like other aba2 alleles (Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000; Rook et al., 2001), lines II-51 and IV-64 displayed sucrose-insensitive seedling development (Table 3). The F2 progenies of crosses between aba2-1 and II-51 or IV-64 showed a 1 : 1 segregation of this phenotype (Table 3), which is consistent with intragenic complementation. Intragenic complementation between aba2-1 and another aba2 allele (isi4) has been reported recently (Rook et al., 2001). Interestingly, the aba2-1 (S-264 to V), IV-64 (D-271 to N) and isi4 (A-236 to V) mutations map to the same region of the ABA2 protein. This region is highly conserved in the ABA2-like short-chain dehydrogenase/reductase superfamily and corresponds to one of the b-strand containing domains at the C-terminus. In contrast the II-51 (G-27 to E) mutation concerns the first G in a GxxxGxG motif proposed to be the fixation site for the NAD(H) or NADP(H) coenzyme (Jornvall et al., 1995). The mutations identified in the ABA2 protein thus highlight the importance of these regions in the functional properties of this multimeric enzyme.
|Genotype||Number of seeds analysed||Number of seedlings with first leaves||Percentage of seedlings with first leaves|
|aba2–1 × ler F2||295||76||26a|
|IV-64 × II-51 F2||273||243||89|
|aba2–1 × II-51 F2||300||161||54b|
|aba2–1 × IV-64 F2||300||166||55b|
Leaf temperature depends not only on stomatal conductance to water vapour but also on a range of other environmental and plant variables, including absorbed net radiation, air humidity, air temperature and boundary layer conductance, which also determine the leaf energy balance (Jones, 1999; Nobel, 1999). In this context, there is an ongoing debate as to whether cooling in leaves derives predominantly from convection or from transpiration (Beerling et al., 2001; Hedrich and Steinmeyer, 2001). Moreover, changes in evaporative cooling might be brought about not only by changes in stomatal aperture but also by changes in stomatal density or in water loss through the cuticle. It is thus noteworthy that all the mutations isolated in the present screen are related to ABA biosynthesis or responsiveness in guard cells. This indicates that under these experimental conditions leaf temperature depends mainly on stomatal conductance, and it confirms that ABA plays a prominent role in controlling stomatal aperture in response to drought.
All the ABA-deficient mutants isolated during this screen correspond to alleles of the ABA1, ABA2 and ABA3 loci, which control ABA biosynthesis in both seeds and vegetative tissues. The same range of ABA-deficient mutants was recovered in a screen for germination in the presence of the gibberellin biosynthesis inhibitor paclobutrazol (Léon-Kloosterziel et al., 1996). It is surprising that we did not isolate additional ABA biosynthetic mutants affected more specifically in stomatal regulation. In particular, loss-of-function mutations in the Arabidopsis ABA-aldehyde oxidase AAO3 and 9-cis-epoxycarotenoid dioxygenase AtNCED3 genes have been shown to increase transpiration rates (Iuchi et al., 2001; Seo et al., 2000). However, these two mutations produce milder symptoms than the aba mutations mentioned above (Iuchi et al., 2001; Seo et al., 2000). It is plausible that, during our first screen by thermal imaging, such mutants exhibiting more subtle temperature differences were discarded in favour of individuals with most conspicuous phenotypes. We have verified that under our experimental conditions the aao3 mutant displays a lower leaf temperature than the wild type (data not shown). It should thus be possible to recover such mutants in more extensive screens by thermal imaging. An attractive possibility is that, in addition to ABA biosynthesis enzymes, this type of screen may identify elements of the putative signalling cascade between the perception of water stress and the stimulation of ABA biosynthesis.
The ABA-insensitive mutants isolated during this screen correspond to three different loci: ABI1, OST1 and OST2. The mutation recovered in ABI1 is identical to the well-characterised abi1-1 mutation that reduces ABA sensitivity in both seed and vegetative tissues (Koornneef et al., 1984; Leung et al., 1994; Meyer et al., 1994). In contrast, ost1 and ost2 mutant seeds displayed a wild-type sensitivity to ABA. Several mutations, including abi3, abi4 and abi5, which inhibit ABA action in seed without affecting ABA responsiveness in guard cells have been described (Finkelstein, 1994; Koornneef et al., 1984). Conversely, to our knowledge, ost1 and ost2 represent the first Arabidopsis mutations altering ABA responsiveness in stomata and not in seeds. This result thus illustrates the usefulness of thermal imaging for the genetic dissection of ABA signalling in Arabidopsis guard cells. The present screen was most likely not at saturation because gca2 and abi2-1 mutations, which markedly inhibit ABA-induced stomatal closure (Koornneef et al., 1984; Pei et al., 2000), were not recovered. Hence, more extensive screening for mutants with a ‘cold’ phenotype under drought will presumably reveal additional elements of the ABA signalling network.
The era1 and abh1 mutants show ABA-supersensitive stomatal closing and reduced transpirational water loss during drought (Hugouvieux et al., 2001; Pei et al., 1998). However, under our experimental conditions, we did not detect any significant difference between the leaf temperatures of era1 and wild-type plants (data not shown). It thus seems that ABA-supersensitive mutants cannot be readily identified by thermal imaging amidst a population of wild-type plants. ABA-supersensitive mutations may be more easily isolated as suppressors of the ‘cold’ phenotype of ABA-deficient or ABA-insensitive mutants. An intragenic revertant of the ABA-insensitive abi2-1 mutant displayed a clear warmer leaf phenotype than the abi2-1 parental line (Merlot et al., 2001), indicating that such a suppressor screen is technically feasible. In addition to ABA-supersensitive mutations, this type of screen may also identify mutations leading to enhanced ABA accumulation under drought, as a result of increased biosynthesis or reduced catabolism.
Guard cells integrate a variety of environmental and endogenous signals to tightly regulate the stomatal pore aperture. In most cases the corresponding signalling cascades are still poorly understood (Assmann and Wang, 2001; MacRobbie, 1998; Schroeder et al., 2001). The approach described here for ABA should be effective for the genetic dissection of some of these pathways. For instance, infrared thermal imaging should be able to identify Arabidopsis mutants with a reduced ability to open their stomata in response to light or to atmospheric CO2 depletion. Conversely, mutants can be screened for their reduced ability to close their stomata in response to darkness or to CO2 enrichment. An automated infrared imaging system would clearly facilitate screenings in darkness or under confined atmospheric conditions, and would also permit to design screens based on the kinetics of the stomatal response to a given signal (Chaerle et al., 1999).
All the Arabidopsis lines used in this study are in the ecotype Landsberg erecta (Ler). The abi1-1 (Koornneef et al., 1984), aba1-1 (Koornneef et al., 1982), aba1-5 (Léon-Kloosterziel et al., 1996), and aba2-1 (Léon-Kloosterziel et al., 1996) mutants were provided by M. Koornneef. EMS-mutagenised Ler M2 seeds were purchased from Lehle Seeds (Round Rock, TX, USA).
Approximately 50 M2 seeds were uniformly sown on 9 cm × 9 cm pots containing a mixture of 50% sand (2–3 mm particles) and 50% horticultural compost (v/v). The pots were incubated in the dark at 4°C for 3 days to break seed dormancy. Plants were then grown for 1 week under well-watered conditions in the greenhouse (21°C, 70% RH, 16 h light photoperiod). The pots were then incubated without watering in a growth cabinet (24°C, 50% RH, 16 h light photoperiod). The plants were examined 3 and 4 days later by thermal imaging. Individuals with a lower leaf temperature than the wild type were selected in real time using the live infrared image visualised on a colour monitor. These candidates were grown to maturity under well-watered conditions, and their phenotype was retested in the next (M3) generation.
Thermal images were obtained using a Thermacam PM250 infrared camera (Inframetrics, FLIR Systems, North Billerica, MA, USA) equipped with a 16° lens. The camera used a cooled 256 × 256 PtSi array detector responsive to the short wave infrared (3.4–5μm band). Specified temperature resolution was below 0.1°C at room temperature. Straight temperature images generated by the camera software on the basis of manufacturer calibration were used. Leaf emissivity was set to 1 since an accurate absolute measurement of leaf temperature was not required. The camera was mounted vertically at approximately 40 cm above the leaf canopy for observations, and was connected to a colour monitor to facilitate visualisation of individual plants. Images were saved as 8-bit TIFF files on the PMCIA memory card of the camera, and were subsequently analysed for temperature determination on a Macintosh using version 1.56 of the public domain image analysis program NIH Image (available on the Internet at http://rsb.info.nih.gov/nih-image/).
The aerial parts from 20 to 30 drought-stressed plantlets were frozen in liquid nitrogen, lyophilised and ground into powder. ABA was then extracted, HPLC purified and quantified by ELISA as previously described (Kraepiel et al., 1994), using a mouse monoclonal anti-ABA antibody (LPDP 229, Jussieu, France) and a peroxidase-conjugated goat antibody to mouse immunoglobulins (Sigma, Saint Quentin Fallaries, France). For each sample, the ABA content was determined five times.
Stomatal aperture bioassays
Germination and seedling development assays
Seed dormancy and sensitivity of seed germination to inhibition by exogenous ABA were assayed as described previously (Merlot et al., 2001). Sucrose sensitivity of seedling development was assayed by plating surface-sterilised seeds onto solid Gamborg B5 medium (Duchefa Biochemie BV, Haarlem, The Netherlands) supplemented with 0.3M sucrose. The plates were then incubated at 20°C with a 16 : 8 h light/dark photoperiod for 3 weeks, and seedlings that had formed true first leaves were scored as sucrose resistant.
Carotenoids were extracted from leaf discs with acetone:methanol:petroleum ether (50 : 30 : 20, v:v:v) and analysed by reverse phase HPLC on a C18 column (Audran et al., 2001).
Xanthine dehydrogenase activity was detected after native gel electrophoresis as previously described (Marin and Marion-Poll, 1997), except that crude leaf extracts were used directly.
We thank Jeffrey Leung, François Parcy and Annie Marion-Poll for discussions and comments on the manuscript. We are grateful to Magda Bonnet for her assistance in ABA analysis. This work was supported by the Centre National de la Recherche Scientifique, a joint grant from CNRS and Université Paris-Sud for the purchase of the infrared camera, an EMBO long-term Fellowship to A.-C. M., a fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche to S.M., and an IFCPAR grant to A.V.
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