SummaryEthylene is a plant hormone that regulates many aspects of growth and development. Despite the well-known association between ethylene and stress signalling, its effects on stomatal movements are largely unexplored. Here, genetic and physiological data are provided that position ethylene into the Arabidopsis guard cell signalling network, and demonstrate a functional link between ethylene and hydrogen peroxide (H 2 O 2 ). In wild-type leaves, ethylene induces stomatal closure that is dependent on H 2 O 2 production in guard cells, generated by the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase AtrbohF. Ethyleneinduced closure is inhibited by the ethylene antagonists 1-MCP and silver. The ethylene receptor mutants etr1-1 and etr1-3 are insensitive to ethylene in terms of stomatal closure and H 2 O 2 production. Stomata of the ethylene signalling ein2-1 and arr2 mutants do not close in response to either ethylene or H 2 O 2 but do generate H 2 O 2 following ethylene challenge. Thus, the data indicate that ethylene and H 2 O 2 signalling in guard cells are mediated by ETR1 via EIN2 and ARR2-dependent pathway(s), and identify AtrbohF as a key mediator of stomatal responses to ethylene.

Stomatal pores of higher plants close in response to decreases in atmospheric relative humidity (RH). This is believed to be a mechanism that prevents the plant from losing excess water when exposed to a dry atmosphere and as such is likely to have been of evolutionary significance during the colonization of terrestrial environments by the embryophytes. We have conducted a genetic screen, based on infrared thermal imaging, to identify Arabidopsis genes involved in the stomatal response to reduced RH. Here we report the characterization of two genes, identified during this screen, which are involved in the guard cell reduced RH signaling pathway. Both genes encode proteins known to be involved in guard cell ABA signaling. OST1 encodes a protein kinase involved in ABA-mediated stomatal closure while ABA2 encodes an enzyme involved in ABA biosynthesis. These results suggest, in contrast to previously published work, that ABA plays a role in the signal transduction pathway connecting decreases in RH to reductions in stomatal aperture. The identification of OST1 as a component required in stomatal RH and ABA signal transduction supports the proposition that guard cell signaling is organized as a network in which some intracellular signaling proteins are shared among different stimuli.

Most thermal methods for the study of drought responses in plant leaves are based on the calculation of 'stress indices'. This paper proposes and compares three main extensions of these for the direct estimation of absolute values of stomatal conductance to water vapour ( g s ) using infrared thermography (IRT). All methods use the measured leaf temperature and two environmental variables (air temperature and boundary layer resistance) as input. Additional variables required, depending on the method, are the temperatures of wet and dry reference surfaces, net radiation and relative humidity. The methods were compared using measured g s data from a vineyard in Southern Portugal. The errors in thermal estimates of conductance were of the same order as the measurement errors using a porometer. Observed variability was also compared with theoretical estimates of errors in estimated g s determined on the basis of the errors in the input variables (leaf temperature, boundary layer resistance, net radiation) and the partial derivatives of the energy balance equations used for the g s calculations. The full energy balance approach requires accurate estimates of net radiation absorbed, which may not be readily available in field conditions, so alternatives using reference surfaces are shown to have advantages. A new approach using a dry reference leaf is particularly robust and recommended for those studies where the specific advantages of thermal imagery, including its noncontact nature and its ability to sample large numbers of leaves, are most apparent. Although the results suggest that estimates of the absolute magnitude of g s are somewhat subjective, depending on the skill of the experimenter at selecting evenly exposed leaves, relative treatment differences in conductance are sensitively detected by different experimenters.

Summary We conducted an infrared thermal imaging‐based genetic screen to identify Arabidopsis mutants displaying aberrant stomatal behavior in response to elevated concentrations of CO 2.This approach resulted in the isolation of a novel allele of the Arabidopsis BIG locus (At3g02260) that we have called CO 2 insensitive 1 (cis1). BIG mutants are compromised in elevated CO 2‐induced stomatal closure and bicarbonate activation of S‐type anion channel currents. In contrast with the wild‐type, they fail to exhibit reductions in stomatal density and index when grown in elevated CO 2. However, like the wild‐type, BIG mutants display inhibition of stomatal opening when exposed to elevated CO 2. BIG mutants also display wild‐type stomatal aperture responses to the closure‐inducing stimulus abscisic acid (ABA).Our results indicate that BIG is a signaling component involved in the elevated CO 2‐mediated control of stomatal development. In the control of stomatal aperture by CO 2, BIG is only required in elevated CO 2‐induced closure and not in the inhibition of stomatal opening by this environmental signal. These data show that, at the molecular level, the CO 2‐mediated inhibition of opening and promotion of stomatal closure signaling pathways are separable and BIG represents a distinguishing element in these two CO 2‐mediated responses.

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