• The recent discovery of a strong positive relationship between angiosperm genome size and stomatal guard cell length (GCL) opens the possibility of using plant fossil guard cell size as a proxy for changes in angiosperm genome size over periods of environmental change.
• The responses of GCL to environmental stimuli are currently unknown and may obscure this predictive relationship.
• Here, we investigated the effects of environmental variables (atmospheric CO2, drought, relative humidity, irradiance, ultraviolet radiation and pathogen attack) on GCL in the model plant Arabidopsis thaliana to quantify environmentally induced variation.
• GCL responded to all variables tested, but the changes incurred did not significantly impinge on the predictive capability of the relationship.
The study of stomatal responses to the environment has played a central role in plant science research over the last 80 yr (Salisbury, 1928). One of the most exciting areas of stomatal research to recently emerge suggests that the genome size of angiosperms is a strong, positive predictor of guard cell length (GCL) (Beaulieu et al., 2008). This study, which covered a phylogenetically diverse sample of angiosperms, across varying growth forms (monocots, eudicots, trees, shrubs and herbs), supports the early work of Masterson (1994) whose data suggested that the small guard cell size of Cretaceous angiosperms was indicative of a small genome (Leitch, 2007). These published findings open the intriguing possibility of using the fossil angiosperm GCL record to track evolutionary changes in the size of a plant's genome over geological time, analogous to the studies of Thomson (1972), Conway Morris & Harper (1988) and Organ et al. (2007) in animals.
It is well known that stomatal density (SD) and index (SI) (the ratio of the number of stomata to the number of epidermal cells) are sensitive to atmospheric CO2 concentrations (Woodward, 1987) and primarily controlled by transpiration rate and in vivo abscisic acid concentration (Lake & Woodward, 2008). SD and SI are also sensitive to the intensity of incident radiation (Lake et al., 2001; Thomas et al., 2003). However, to date little is known about how these environmental variables influence GCL.
Atmospheric CO2 is predicted to have varied considerably over geological time since the origin and diversification of land plants (Wellman et al., 2003), from ~4000 to 280 parts per million (ppm) (Berner & Kothavala, 2001), and catastrophic climate change and mass extinction events have been linked to rapid increases in CO2 (Lomax et al., 2000; Beerling et al., 2002). In addition to these long-term changes, analysis of the palaeobotanical record and geochemical modelling indicates that, at certain periods, the Earth's terrestrial flora may have been periodically exposed to prolonged and extreme increases in the flux of ultraviolet-B (UV-B) radiation (Visscher et al., 2004; Beerling et al., 2007) and intense periods of fungal attack (Visscher et al., 1996), both environmental stimuli that have been recently shown to alter GCL (J. A. Lake, unpublished).
Consequently, before direct measurements of fossil guard cell size can be used with confidence as a proxy for the palaeo-plant genome, it is necessary to investigate the role of these environmental stimuli under experimental conditions. This study begins to address these uncertainties by quantifying the effects that environmental stimuli falling within geologically relevant ranges (CO2 concentration, UV-B radiation and pathogen attack) and ecologically relevant ranges (water availability, relative humidity and photosynthetically active radiation (PAR)) have on the GCL of Arabidopsis thaliana. The stomatal density response of A. thaliana to a doubling of atmospheric CO2 is representative of the responses seen in 110 species (Woodward et al., 2002) and A. thaliana is therefore viewed as an ideal model plant with which to investigate the environmental controls of GCL. The most recently revised genome size for A. thaliana is reported to be 1C = 157 Mega basepairs (Mb) (Bennett et al., 2003).
Materials and Methods
CO2 concentration and water availability
Seeds of Arabidopsis thaliana (L.) Heynh. (ecotype Columbia (Col-0)) were sown onto multipurpose compost (Arthur Bowyers, William Sinclair Horticulture Ltd, Lincoln, UK) covered with plastic film and stratified for 3 d at 4°C. They were then transferred into growth cabinets (Fitotron Model SGC097.PPX.F; Sanyo Gallenkamp, Loughborough, UK) and grown under a day:night regime of 8 : 16 h at 25 : 21 ± 0.5°C and 55% relative humidity (RH). Seven separate CO2 experiments were conducted, with CO2 held at geologically relevant (Berner & Kothavala, 2001) concentrations of 400, 760, 1000, 1500, 2000 and 3000 ppm. Nested within each CO2 treatment, plants at the 4-wk stage were also subjected to one of three watering regimes, drought (10 ml d−1) well watered (20–30 ml d−1) and saturated (constantly wet compost).
Plants were germinated and stratified as above, transferred into growth cabinets (Fitotron Model SGC097.PPX.F; Sanyo Gallenkamp) and grown under a day:night regime of 8 : 16 h at 22 : 18 ± 0.5°C and either 45 or 85% RH.
Incident (PAR) radiation
Plants were germinated and stratified as above, transferred to controlled-environment chambers and grown under a day:night cycle of 14 : 10 h, 25 : 18 ± 0.5°C and RH 50 ± 3% at different distances from the light source, producing a range of PAR values: 22, 33, 45, 60 and 83 µmol m−2 s−1 (Fitotron SGC970/P/FL; Sanyo Gallenkamp), and 100, 340 and 500 µmol m−2 s−1 (Conviron BDR16; Conviron, Winnipeg, Canada).
Plants were germinated and stratified as above, transferred into growth cabinets (Model P61700H/FM/RO-HQI; Sanyo Gallenkamp) and grown under a day:night regime of 8 : 16 h at 21 : 15°C, 60% RH, and vapour pressure deficit (VPD) 0.995 kPa. UV-B light was supplied by a bank of nine Sanolux UV high-pressure lamps (323 1219; Phillips, Guildford, UK) which increased UV-B (measured at 300 nm) radiation to 26 ± 0.01 kJ m−2 d−1 at plant height over the entire daylength; in the control cabinet all UV-B radiation was excluded and UV-A radiation was reduced using using specific UV-B and UV-A cut-off wavelength filters (226 Lee U.V.; Lee Filters, Andover, UK). UV-B levels were measured using a Sola-Check System (Solatell Ltd, Croydon, UK), cross-calibrated with a Macam spectroradiometer (SR9000-V7; Macam Photometrics Ltd, Livingston, UK).
Plants were germinated and stratified as above and transferred to controlled-environment chambers (Fitotron SGC2352/FM/HFL; Sanyo Gallenkamp) and grown under a day:night regime of 8 : 16 h at 22 : 18 ± 0.5°C, 150 µmol m−2 s−1 irradiance, and 55 ± 5% RH. Actively growing cultures of Erysiphe cichoracearum were maintained on marrow (Cucurbita pepo L. cv. Long Green Bush). Inoculum was applied at the 7- to 10-d stage. At the 4-wk stage (10 rosette leaves present) leaf insertions 6–10 were painted with inoculum at a spore concentration of 1 × 105 ml−1 in a suspension of Fluorinert (Sigma-Aldrich, Gillingham, UK); control plants were painted with Fluorinert only. Plants (both infected and control) were enclosed in propagators to isolate inoculated from control plants held within the same controlled-environment chamber and to raise humidity to 80–85% for effective infection. Seven days later, leaf insertions 14–17 were harvested for GCL analyses.
Stomatal guard cell analyses
For all treatments, five replicates were grown under each condition, from which three fully mature leaves were harvested for GCL analysis. Epidermal impressions of the abaxial leaf surface were made using standard techniques developed in Sheffield (Coupe et al., 2005). Twenty-one stomata per leaf were sampled per treatment to derive an average GCL; all measurements were taken using image analysis software (Quantimet 500C image analysis system; Leica Instruments, Cambridge, UK) coupled to a light microscope (Leitz Laborlux S; Leica Microsystems, Milton Keynes, UK) at ×400 magnification.
Results and Discussion
Changes in GCL occurred in response to all environmental stimuli investigated. The smallest change in GCL (an increase of 3%), calculated as a percentage change of the full range (i.e. minimum to maximum) mean value, occurred when RH was raised from 45 to 85%. Increases in CO2 from 380 to 3000 ppm and in water availability from 10 ml d−1 to continually saturated soil both resulted in a 6% increase in GCL. High levels of UV-B caused a 7% increase in GCL, while pathogen challenge increased GCL by 10% compared with uninfected control leaves. Reducing incident PAR to values analogous to those measured in the lowest 20 m of rainforest canopies (22–83 µmol m−2 s−1; Torquebiau, 1988) produced the largest response, an 18% decrease from 83 to 22 µmol m−2 s−1 in GCL (Table 1 , Fig. 1a).
Table 1. The range (minimum and maximum) of guard cell size measured under different environmental conditions together with 95% prediction and confidence intervals from Fig. 1
Minimum (log value)
Maximum (log value)
PAR, photosynthetically active radiation; UV, ultraviolet.
95% prediction intervals
95% confidence intervals
Incident PAR (low) 22–87 µmol m−2 s−1
Incident PAR (high) 100–500 µmol m−2 s−1
Log10 transformation of this data set (Fig. 1b) allows direct comparison with the work of Beaulieu et al. (2008). A C value of 157 Mb for A. thaliana (Bennett et al., 2003) translates into a log10 2C value of –0.503 Giga base pairs (Gbp). The box plot (Fig. 1b) of the log10-transformed GCL shows a close correspondence to the herb data set of Beaulieu et al. (2008) with the median, mean, and 5th, 25th and 75th percentiles plotting inside the confidence limits of the regression, with only the 95th percentile and the upper outlier plotting outside these limits, but well within the prediction limits of the data (Fig. 1b , Table 1).
Both the 95th percentile and the upper outlier are associated with extreme irradiance environments; high chronic UV-B radiation and very low PAR, respectively. The relatively large increases in GCL observed in A. thaliana when grown in experimental conditions simulating deep shade suggest that low light intensities may result in an overestimation of the size of the fossil plant genome. However, as a whole, the leaf fossil record tends to be biased towards the preservation of sun rather than shade leaves (Spicer, 1981); furthermore, leaf epidermal cell features can be used to distinguish between sun and shade leaves (Kurschner, 1997; Thomas et al., 2003). Chronic high levels of UV-B radiation may also lead to an overestimation of the size of the fossil plant genome. However, to date, evidence for a collapse in the Earth's protective ozone layer is restricted to the Permo-Triassic boundary mass extinction event, suggesting that high fluxes of UV-B radiation are relatively rare.
Overall, the narrow range in GCLs, given the wide spread of environmental conditions and the closeness of fit between the herb (Beaulieu et al., 2008) and A. thaliana data sets (Fig. 1b), provides evidence that environmental perturbations do not impinge significantly on the overall predictive quality of the relationship between GCL and genome size in this species. GCL values fall well within the predictive intervals of the linear regression (Table 1). The low sensitivity of GCL to the broad range of atmospheric CO2 concentrations investigated here suggests that changes in fossil GCL may be used as a proxy to track changes in plant genome size over evolutionary time and give an insight into how plant genomes have responded to climatic catastrophe.
BHL was funded through a Leverhulme Trust Early Career Fellowship (ECF/2006/0492) and JAL through a Royal Society Dorothy Hodgkin Fellowship. This study was supported by a University of Sheffield Biology Division Small Grant to BHL and JAL. We thank Nicola Hurst, Sarah Wardell and Judith Foulkes for data collection.