Stomatal and pavement cell density linked to leaf internal CO2 concentration

Abstract

Background and aims: Stomatal density (SD) generally decreases with rising atmospheric CO2 concentration, Ca. However, SD is also affected by light, air humidity and drought, all under systemic signalling from older leaves. This makes our understanding of how Ca controls SD incomplete. This study tested the hypotheses that SD is affected by the internal CO2 concentration of the leaf, Ci, rather than Ca, and that cotyledons, as the first plant assimilation organs, lack the systemic signal.

Methods: Sunflower (Helianthus annuus), beech (Fagus sylvatica), arabidopsis (Arabidopsis thaliana) and garden cress (Lepidium sativum) were grown under contrasting environmental conditions that affected Ci while Ca was kept constant. The SD, pavement cell density (PCD) and stomatal index (SI) responses to Ci in cotyledons and the first leaves of garden cress were compared. (13)C abundance (δ(13)C) in leaf dry matter was used to estimate the effective Ci during leaf development. The SD was estimated from leaf imprints.

Key results: SD correlated negatively with Ci in leaves of all four species and under three different treatments (irradiance, abscisic acid and osmotic stress). PCD in arabidopsis and garden cress responded similarly, so that SI was largely unaffected. However, SD and PCD of cotyledons were insensitive to Ci, indicating an essential role for systemic signalling.

Conclusions: It is proposed that Ci or a Ci-linked factor plays an important role in modulating SD and PCD during epidermis development and leaf expansion. The absence of a Ci-SD relationship in the cotyledons of garden cress indicates the key role of lower-insertion CO2 assimilation organs in signal perception and its long-distance transport.

Keywords: 13C discrimination; Arabidopsis thaliana; Fagus sylvatica; Helianthus annuus; Lepidium sativum; Stomatal density; cotyledons; leaf internal CO2; pavement cells; stomata development.

Fig. 1.

Effects of shading and water stress treatments on internal CO₂ concentration of the leaf, C_i, as inferred from the δ¹³C in leaf biomass, and on stomatal density SD. (A) Circles indicate increased (1, 2, 3) and decreased (4, 5) C_i, and concomitant changes in SD, under low light in sunflower grown in nutrient solution (1), with ABA (2) or PEG (3) added, and under high light in sunflower fed with ABA (4) or PEG (5). Values are differences between treatments and control, i.e. plants grown under high light in plain nutrient solution; n_ci = n_SD = 6–8. The triangle shows the effect of shading on beech leaves (single tree, sampled during the 2007 and 2009 seasons, n_ci = 18, n_SD = 20). Squares depict the shading effect in Arabidopsis thaliana rosette leaves grown for 18 d at irradiances of 200 or 80 μmol m^–2 s^–1, respectively (ecotypes Columbia and C24, n_ci = 6, n_SD = 18). Diamonds indicate the ABA effect in cress plants (n_ci = 5, n_SD = 48). All plants experienced an atmospheric CO₂ concentration close to 390 μmol mol^–1. (B) The changes in SD from (A) expressed as a percentage of control. The points sharing the same C_i co-ordinate in (A) and (B) represent identical treatments. Standard errors of the mean (bars), regression lines and 95 % confidence intervals are shown.

Fig. 2.

Changes in internal CO₂ concentration (C_i) of the leaf in Lepidium sativum plants grown under sub-ambient or super-ambient CO₂ concentrations, as related to changes in stomatal density (SD; A), pavement cell density (PCD; B) and stomatal index (SI; C). The negative values of the C_i difference show by how much C_i was lowered when C_a was reduced from 400 to 180 μmol mol^–1; the positive values indicate the increase of C_i in plants grown at 800 compared with controls kept at 400 μmol mol^–1. Stomatal and pavement cell density in the first leaves (light green triangles and light green regression line) was much more sensitive to CO₂ concentration than in cotyledons (dark green circles and dark green regression line). Each point represents a difference between the C_a treatment (180 or 800) and C_a control (400) for one of three sub-treatments (two air humidities and hypobaric plants), each grown either in air or in helox gas mixture. Data for 14-day-old leaves and 7- and 14-day-old cotyledons are shown. The SD and C_i values used in calculation of the differences were means from three independent measurements (three plants per treatment). The C_i values were calculated from δ¹³C in plant dry matter (see Appendix). Bars show the standard error of the mean, for SI calculated from the primary SD and PCD data [see eqn (6) in ‘Statistical evaluation, meta-analysis’].

Fig. 3.

Response to internal CO₂ (C_i) of stomatal density (SD; A), pavement cell density (PCD; B) and stomatal index (SI; C) of Arabidopsis thaliana leaves grown at two irradiances. Two wild ecotypes, Columbia (Col) and C24, were grown under low light (25 μmol m^–2 s^–1) or high light (250 μmol m^–2 s^–1) conditions in growth chambers. Mean values of SD, PCD and SI (symbols) for the adaxial and abaxial sides of mature first rosette leaves are shown as well as the standard error of the mean (bars, n_Ci = 6 and n_SD,PCD,SI = 16) and regression lines (thick solid line and equation) for information about leaf-averaged sensitivity (slope) of the relationship. The circles group the low and high light data.

Fig. 4.

Response of stomatal density (SD; A), pavement cell density (PCD; B) and stomatal index (SI; C) of Lepidium sativum leaves and cotyledons to the internal CO₂ (C_i) of leaves grown at various irradiances. The light green triangles and light green regression lines represent the first leaves; the dark green circles and dark green regression lines show the cotyledons' response. The plants were grown for 21 d under 7–8 photosynthetic photon flux densities (PPFDs), ranging from 100 to 590 μmol m^–2 s^–1, in 100 mL glass cuvettes ventilated with ambient air. The points represent averages from six independent runs of the experiment. Mean values and the standard error of the mean are indicated. Linear or hyperbolic fits with 95 % confidence intervals are shown.

Fig. 5.

The effect of various environmental factors on concomitant changes in the internal CO₂ concentration (C_i) and stomatal density (SD) of leaves. The C_i values were calculated from carbon isotope discrimination data extracted together with SD values from 17 publications presenting factorial experiments. The differences between treatment and control plants in SD values (A) and in SD normalized to SD of control (B) are shown together with the best fits and 95 % confidence intervals. The compiled data are shown in Supplementary Data Table S1, and come from the following studies: Bradford et al. (1983), Van de Water et al. (1994), Beerling (1997), Sun et al. (2003), Gitz et al. (2005), Takahashi and Mikami (2006), Aucour et al. (2008), He et al. (2008), Sekiya and Yano (2008), Lake et al. (2009), Yan et al. (2009), Craven et al. (2010), Gorsuch et al. (2010), He et al. (2012), Sun et al. (2012), Yan et al. (2012) and Rogiers and Clarke (2013).