Stomatal Development and Conductance of a Tropical Forage Legume Are Regulated by Elevated [CO2] Under Moderate Warming

Abstract

The opening and closing of stomata are controlled by the integration of environmental and endogenous signals. Here, we show the effects of combining elevated atmospheric carbon dioxide concentration (eCO ₂; 600 μmol mol^-1) and warming (+2°C) on stomatal properties and their consequence to plant function in a Stylosanthes capitata Vogel (C₃) tropical pasture. The eCO ₂ treatment alone reduced stomatal density, stomatal index, and stomatal conductance (g_s ), resulting in reduced transpiration, increased leaf temperature, and leading to maintenance of soil moisture during the growing season. Increased CO₂ concentration inside leaves stimulated photosynthesis, starch content levels, water use efficiency, and PSII photochemistry. Under warming, plants developed leaves with smaller stomata on both leaf surfaces; however, we did not see effects of warming on stomatal conductance, transpiration, or leaf water status. Warming alone enhanced PSII photochemistry and photosynthesis, and likely starch exports from chloroplasts. Under the combination of warming and eCO ₂, leaf temperature was higher than that of leaves from the warming or eCO ₂ treatments. Thus, warming counterbalanced the effects of CO₂ on transpiration and soil water content but not on stomatal functioning, which was independent of temperature treatment. Under warming, and in combination with eCO ₂, leaves also produced more carotenoids and a more efficient heat and fluorescence dissipation. Our combined results suggest that control on stomatal opening under eCO ₂ was not changed by a warmer environment; however, their combination significantly improved whole-plant functioning.

Keywords: elevated CO2; gas exchange; global climate change; stomatal conductance regulation; tropical forage legume; warming.

FIGURE 1

Daily average of relative air humidity (Rh), air temperature (T_air) and average diurnal total solar radiation (Rad), during the experimental period.

FIGURE 2

(A) Daily average of soil water content (SWC) and (B) daily average of soil temperature under all treatments during the entire experimental period. Arrows indicate dates in which the leaf gas exchange parameters were measured. Treatments: aCO₂aT (ambient [CO₂] and ambient temperature), eCO₂aT (elevated [CO₂] and ambient temperature), aCO₂eT (ambient [CO₂] and elevated temperature) and eCO₂eT (elevated [CO₂] and elevated temperature). DOE = day of experiment. Stack bars indicate the standard deviation.

FIGURE 3

Difference in canopy temperature (Δ T_canopy) of Stylosanthes capitata during the experiment. (A) Δ T_canopy between eCO₂aT–aCO₂aT plots. (B) Δ T_canopy between aCO₂eT–aCO₂aT plots. (C) Δ T_canopy between eCO₂eT–aCO₂aT plots. (D) Δ T_canopy between eCO₂eT–aCO₂eT plots. T_canopy was recorded every 15 min by Apogee infrared radiometers. Arrows indicate the point 2°C above the ambient canopy temperature that was set in warmed treatments. The missing values between days 35 and 37 after treatment are due to a dead datalogger battery on these days. Treatments: aCO₂aT (ambient [CO₂] and ambient temperature), eCO₂aT (elevated [CO₂] and ambient temperature), aCO₂eT (ambient [CO₂] and elevated temperature) and eCO₂eT (elevated [CO₂] and elevated temperature).

FIGURE 4

(A,B) Stomatal density (SD). (C,D) Stomatal index (SI). (E,F) Stomatal length (SL) on adaxial leaf surface (left column) and abaxial leaf surface (right column) from leaves of S. capitata grown under different levels of [CO₂] and temperature at 66th day of experiment. [CO₂] levels: ambient (aCO₂) and elevated (eCO₂, 600 μmol mol^-1). Temperature levels: ambient (aT) and elevated (eT, +2°C above ambient canopy temperature). ANOVA p-values are shown for main effects (CO₂ or T) or interactions CO₂ × T. Stack bars indicate the standard error.

FIGURE 5

Leaf gas exchange of S. capitata performed at 44th (left column) and 66th DOE (right column) under different levels of [CO₂] and temperature. (A,B) Stomatal conductance (g_s). (C,D) Transpiration rate (E). (E,F) Net photosynthesis rate (A). (G,H) Instantaneous water-use efficiency (WUE). [CO₂] levels: ambient (aCO₂) and elevated (eCO₂, 600 μmol mol^-1). Temperature levels: ambient (aT) and elevated (eT, +2°C above ambient canopy temperature). DOE = day of experiment. ANOVA p-values are shown for main effects (CO₂ or T) or interactions CO₂ × T. Stack bars indicate the standard error.

FIGURE 6

Transmission electron micrograph of S. capitata leaves grown under different levels of [CO₂] and temperature at 66th day of experiment. (A–D) aCO₂aT samples. (A,B) Longitudinal section of chloroplast showing large and numerous starch grains. Scale bar of 10 μm in panels (A,B). (C) Longitudinal section of the terminal portion of a chloroplast associated with mitochondria. Arrowheads show intact thylakoid membranes. Scale bar 3 μm. (D) Details of thylakoid membranes of an intact chloroplast (arrowhead). Scale bar 2 μm. (E–G) eCO₂aT samples. (E) Overview of a chloroplast in longitudinal section showing a large number of starch grains. Scale bar 10 μm. (F) Enlarged cell. Scale bar 4 μm. (G) Details of a chloroplast showing the integrity of thylakoid membranes (arrowhead). Scale bar 3 μm. (H–J) aCO₂eT samples (H) Overview of a chloroplast from leaf mesophyll with a reduced number and size of starch grains and the presence of plastoglobuli in the stroma of chloroplast. Scale bar 3 μm. (I,J) Detail of chloroplasts with a greater association with mitochondria. (J) Evidence that the internal membranes are less organized in the thylakoid (arrowhead). Scale bars 3 μm in (I) and 2 μm in (J). (K–O) eCO₂eT samples. (K) Overview of the mesophyll cell with chloroplasts in association with mitochondria and vacuole with presence of electrodense amorphous material. Scale bar 5 μm. (L) Detail of a chloroplast in longitudinal section. Arrowhead – internal membranes of chloroplasts. Scale bars 2 μm. (M) Detail of chloroplasts in cross section showing the large number of plastoglobuli in the stroma. Scale bar 2 μm. (N,O) Details of chloroplasts showing plastoglobuli, starch grains, and internal membranes not associated in thylakoids (arrowhead). Scale bar 3 μm in N and O. Abbreviations: S, starch grain; M, mitochondria; CW, cell wall; N, nucleus; L, plastoglobuli; C, chloroplasts; V, vacuole.

FIGURE 7

Maximum electron transport rate (ETR_max) of S. capitata leaves under different levels of atmospheric [CO₂] and temperature at 66th day of experiment at maximum PPFD (784 μmol photons m^-2 s^-1). [CO₂] levels: ambient (aCO₂) and elevated (eCO₂, 600 μmol mol^-1). Temperature levels: ambient (aT) and elevated (eT, +2°C above ambient canopy temperature). ANOVA p-values are shown for main effects (CO₂ or T) or interactions CO₂ × T. Stack bars indicate the standard error.

FIGURE 8

Images of chlorophyll fluorescence parameters made in leaves of S. capitata exposed to different levels of atmospheric [CO₂] and temperature at maximum PPFD (784 μmol photons m^-2 s^-1). (A–D) Coefficient of photochemical quenching (lake model) (qL). (E–H) Coefficient of photochemical quenching (puddle model) (qP). (I–L) Effective PSII quantum yield Y(II). (M–P) Quantum yield of non-regulated energy dissipation Y(NO). (Q–T) Quantum yield of regulated energy dissipation Y(NPQ). Measurement was performed at 66th day of experiment. [CO₂] levels: ambient (aCO₂) and elevated (eCO₂, 600 μmol mol^-1). Temperature levels: ambient (aT) and elevated (eT, +2°C above ambient canopy temperature). Different letters in the same row indicate statistical differences between treatments determined by t-test. Relative values of each parameter in the images have been mapped to a color palette ranging from 0 to 1 and displayed using an identical false color scale (bar is at the bottom of image).