Enhanced Stomatal Conductance by a Spontaneous Arabidopsis Tetraploid,Me-0, Results from Increased Stomatal Size and Greater Stomatal Aperture

The rate of gas exchange in plants is regulated mainly by stomatal size and density. Generally, higher densities of smaller stomata are advantageous for gas exchange; however, it is unclear what the effect of an extraordinary change in stomatal size might have on a plant’s gas-exchange capacity. We investigated the stomatal responses to CO₂ concentration changes among 374 Arabidopsis (Arabidopsis thaliana) ecotypes and discovered that Mechtshausen (Me-0), a natural tetraploid ecotype, has significantly larger stomata and can achieve a high stomatal conductance. We surmised that the cause of the increased stomatal conductance is tetraploidization; however, the stomatal conductance of another tetraploid accession, tetraploid Columbia (Col), was not as high as that in Me-0. One difference between these two accessions was the size of their stomatal apertures. Analyses of abscisic acid sensitivity, ion balance, and gene expression profiles suggested that physiological or genetic factors restrict the stomatal opening in tetraploid Col but not in Me-0. Our results show that Me-0 overcomes the handicap of stomatal opening that is typical for tetraploids and achieves higher stomatal conductance compared with the closely related tetraploid Col on account of larger stomatal apertures. This study provides evidence for whether larger stomatal size in tetraploids of higher plants can improve stomatal conductance.Gas exchange is a vital activity for higher plants that take up atmospheric CO₂ and release oxygen and water vapor through epidermal stomatal pores. Gas exchange affects CO₂ uptake, photosynthesis, and biomass production (Horie et al., 2006; Evans et al., 2009; Tanaka et al., 2014). Stomatal conductance (gs) is used as an indicator of gas-exchange capacity (Franks and Farquhar, 2007). Maximum stomatal conductance (gsmax) is controlled mainly by stomatal size and density, two parameters that change with environmental conditions and are negatively correlated with each other (Franks et al., 2009).Given a constant total stomatal pore area, large stomata are generally disadvantageous for gas exchange compared with smaller stomata, because the greater pore depth in larger stomata increases the distance that gas molecules diffuse through. This increased distance is inversely proportional to g_smax (Franks and Beerling, 2009). The fossil record indicates that ancient plants had small numbers of large stomata when atmospheric CO₂ levels were high, and falling atmospheric [CO₂] induced a decrease in stomatal size and an increase in stomatal density to increase g_s for maximum carbon gain (Franks and Beerling, 2009). The positive relationship between a high g_s and numerous small stomata also holds true among plants living today under various environmental conditions (Woodward et al., 2002; Galmés et al., 2007; Franks et al., 2009). Additionally, the large stomata of several plant species (e.g. Vicia faba and Arabidopsis [Arabidopsis thaliana]) are often not effective for achieving rapid changes in g_s, due to slower solute transport to drive movement caused by their lower membrane surface area-to-volume ratios (Lawson and Blatt, 2014).Stomatal size is strongly and positively correlated with genome size (Beaulieu et al., 2008; Franks et al., 2012; Lomax et al., 2014). Notably, polyploidization causes dramatic increases in nucleus size and stomatal size (Masterson, 1994; Kondorosi et al., 2000). In addition to the negative effects of large stomata on gas exchange (Franks et al., 2009), polyploids may have another disadvantage; del Pozo and Ramirez-Parra (2014) showed that artificially induced tetraploids of Arabidopsis have a reduced stomatal density (stomatal number per unit of leaf area) and a lower stomatal index (stomatal number per epidermal cell number). Moreover, tetraploids of Rangpur lime (Citrus limonia) and Arabidopsis have lower transpiration rates and changes in the expression of genes involved in abscisic acid (ABA), a phytohormone that induces stomatal closure (Allario et al., 2011; del Pozo and Ramirez-Parra, 2014). On the other hand, an increase in the ploidy level of Festuca arundinacea results in an increase in the CO₂-exchange rate (Byrne et al., 1981); hence, polyploids may not necessarily have a reduced gas-exchange capacity.Natural accessions provide a wide range of information about mechanisms for adaptation, regulation, and responses to various environmental conditions (Bouchabke et al., 2008; Brosché et al., 2010). Arabidopsis, which is distributed widely throughout the Northern Hemisphere, has great natural variation in stomatal anatomy (Woodward et al., 2002; Delgado et al., 2011). Recently, we investigated leaf temperature changes in response to [CO₂] in a large number of Arabidopsis ecotypes (374 ecotypes; Takahashi et al., 2015) and identified the Mechtshausen (Me-0) ecotype among ecotypes with low CO₂ responsiveness; Me-0 had a comparatively low leaf temperature, implying a high transpiration rate. In this study, we revealed that Me-0 had a higher g_s than the standard ecotype Columbia (Col), despite having tetraploid-dependent larger stomata. Notably, the g_s of Me-0 was also higher than that of tetraploid Col, which has stomata as large as those of Me-0. This finding resulted from Me-0 having a higher g_s-to-g_smax ratio due to more opened stomata than tetraploid Col. In addition, there were differences in ABA responsiveness, ion homeostasis, and gene expression profiles in guard cells between Me-0 and tetraploid Col, which may influence their stomatal opening. Despite the common trend of smaller stomata with higher gas-exchange capacity, the results with Me-0 confirm the theoretical possibility that larger stomata can also achieve higher stomatal conductance if pore area increases sufficiently.