Tuning the pores: towards engineering plants for improved water use efficiency L. Chaerle 1,* , N. Saibo 2,* and D. Van Der Straeten 1 1 Unit Plant Hormone Signaling and Bio-imaging, Ghent University, Ledeganckstraat 35, B-9000 Gent, Belgium 2 Present address: Plant Genetic Engineering Laboratory, Instituto de Tecnologia Quı´mica e Biolo ´ gica, Av. da Repu ´ blica, 2781-901 Oeiras, Portugal The management of limited fresh water resources is a major challenge facing society in the 21st century. The agricultural sector accounts for more than two-thirds of human water withdrawal and is therefore a prime area to implement a more rational water use. Environmental stresses are a major factor limiting stable food pro- duction. Given the growing shortage of available water for crops this will be an emerging factor in international agricultural economy. The most environmentally friendly and durable solution to the problem of water shortage is to complement more efficient irrigation approaches with crops with optimal water use effi- ciency, achieved either through genetic engineering or conventional breeding, combined with high yields. Introduction Plant leaves and stems have microscopic epidermal pores flanked by a pair of guard cells, called stomata, which enable gas exchange – mainly of water vapour and carbon dioxide – between inner leaf tissues and surrounding air. Environmental cues, such as light intensity, light quality, water status, temperature and the concentration of atmospheric carbon dioxide (which influences the leaf internal CO 2 concentration C i ), and also endogenous (mainly hormonal) signals, control the development, density and aperture of stomata [1,2]. These factors influence both water loss by transpiration and net photosynthesis. Water use efficiency (WUE) is therefore defined as CO 2 assimila- tion per unit water transpired. Although reducing tran- spiration by stomatal closure is the most prominent response to drought, it could also be optimized through the control of stomatal size and density [3]. Here we present an overview of recent advances in understanding stomatal development and response, which determine the actual gas exchange capacity of a plant and have an important impact on its final yield. In addition, we highlight major achievements in enhancing dehydration tolerance in plants. Finally, we illustrate how imaging technology can help in the process towards engineering high yield crops tolerant to limited water supplies. Environmental control of stomatal development Despite the fact that several genes involved in stomatal development have been characterized (Box 1), little is known about the environmental control thereof. In most species, an increase in CO 2 results in a lower stomatal density [3,4]. The Arabidopsis gene HIC (high carbon dioxide) was the first gene to be identified belonging to a signaling pathway that controls stomatal development in response to an environmental cue [5]. HIC codes for an enzyme involved in the synthesis of very-long-chain fatty acids and is a negative regulator of stomatal development in response to CO 2 concentration. The hic K phenotype is not different to wild type except when grown in elevated CO 2 , in which case the mutant shows a higher stomatal index (relative prevalence of stomatal cells). This suggests that the epidermal wax (very-long-chain fatty acid deriva- tives) composition of the cuticle of the guard cells controls stomatal numbers at elevated CO 2 [4]. Furthermore, plants show a more pronounced response (lower stomatal density) to high CO 2 under drought conditions, compared with well- watered plants [3], suggesting that CO 2 and drought signaling might interact. Whether HIC has a role in the transduction of other external signals towards stomatal development remains to be determined [6]. Plants grown at low humidity have an increased cuticle wax load [6] and a lower stomatal density than those grown at higher humidity [7]. Moreover, it was shown that mutants with enhanced wax load and altered wax composition show a lower stomatal index and higher drought tolerance [8], indicating that cuticle wax composition might also be involved in the control of stomatal development by water status. Other environ- mental cues, such as salt stress, also result in a reduction of stomatal index [9]. Mitogen-activated protein kinases (MAPK) have been suggested to integrate responses to stress, growth and cytokinesis [10]. The requirement for YODA (YDA) in meristemoids (Box 1) might reflect the use of MAPK signaling in response to a changing environment [11]. Hence, YDA might have an important role in drought- induced stomatal development. Furthermore, overexpres- sion of protein kinases (such as SRK2C and NPK1 [12,13]) Corresponding author: Van Der Straeten, D. (dominique.vanderstraeten@ ugent.be). * The first two authors contributed equally to this work. Available online 19 April 2005 Review TRENDS in Biotechnology Vol.23 No.6 June 2005 www.sciencedirect.com 0167-7799/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2005.04.005