Increasing atmospheric CO2 and canopy temperature induces anatomical and physiological changes in leaves of the C4 forage species Panicum maximum

Abstract

Changes in leaf anatomy and ultrastructure are associated with physiological performance in the context of plant adaptations to climate change. In this study, we investigated the isolated and combined effects of elevated atmospheric CO2 concentration ([CO2]) up to 600 μmol mol-1 (eC) and elevated temperature (eT) to 2°C more than the ambient canopy temperature on the ultrastructure, leaf anatomy, and physiology of Panicum maximum Jacq. grown under field conditions using combined free-air carbon dioxide enrichment (FACE) and temperature free-air controlled enhancement (T-FACE) systems. Plants grown under eC showed reduced stomatal density, stomatal index, stomatal conductance (gs), and leaf transpiration rate (E), increased soil-water content (SWC) conservation and adaxial epidermis thickness were also observed. The net photosynthesis rate (A) and intrinsic water-use efficiency (iWUE) were enhanced by 25% and 71%, respectively, with a concomitant increase in the size of starch grains in bundle sheath cells. Under air warming, we observed an increase in the thickness of the adaxial cuticle and a decrease in the leaf thickness, size of vascular bundles and bulliform cells, and starch content. Under eCeT, air warming offset the eC effects on SWC and E, and no interactions between [CO2] and temperature for leaf anatomy were observed. Elevated [CO2] exerted more effects on external characteristics, such as the epidermis anatomy and leaf gas exchange, while air warming affected mainly the leaf structure. We conclude that differential anatomical and physiological adjustments contributed to the acclimation of P. maximum growing under elevated [CO2] and air warming, improving the leaf biomass production under these conditions.

Fig 1. Daily average soil water content (SWC) registered during the experimental period at the Trop-T-FACE facility.

Stack bars show the standard error. [CO₂] levels: aC (ambient [CO₂], ~400 μmol mol^-1) and eC (elevated [CO₂], ~600 μmol mol^-1). Temperature levels: aT (ambient temperature) and eT (+2°C more than the ambient temperature).

Fig 2. Transmission electron microscopy (TEM) photomicrographs of leaves of P. maximum under ambient [CO₂] and ambient temperature (aCaT).

(A) Overview of a bundle sheath cell (BSC). Cl–chloroplast, V–vacuole. N–nucleus. (B) Overview of a mesophyll cell (MC). Cl–chloroplast, V–vacuole. (C) A chloroplast of a BSC with plastoglobuli (indicated by arrowheads) and starch grains. Arrows indicate plasmodesmata between the BSC and MC. S–starch. (D) A chloroplast of a MC with plastoglobuli (indicated by arrowheads) and thylakoid membranes (indicated by arrows). (E) A chloroplast of a BSC showing details of thylakoid membranes (indicated by arrow) and plastoglobuli (indicated by arrowheads). S–starch. (F) The cell wall between the BSC and MC. S–starch, CW–cell wall. (G) Group of mitochondria of a BSC. M–mitochondria.

Fig 3. Transmission electron microscopy (TEM) photomicrographs of leaves of P. maximum under elevated [CO₂] and ambient temperature (eCaT).

(A) Overview of a bundle sheath cell (BSC). (B) Overview of mesophyll cells (MC). V–vacuole. (C) Chloroplast of a BSC with large starch grains. S–starch. (D) Chloroplast of a MC with conserved thylakoid membranes (indicated by arrows) and communication thought plasmodesmata with a BSC (indicated by an arrowhead). CW–cell wall. (E) A chloroplast of a BSC showing details of thylakoid membranes (indicated by arrow). S–starch. (F) Regular cell wall of a BSC and chloroplasts with starch grains and conserved thylakoid membranes (indicated by arrows). CW–cell wall, S–starch. (G) Group of mitochondria associated with chloroplasts in BSC with conserved thylakoid membranes (indicate by arrows). S–starch, M–mitochondria.

Fig 4. Transmission electron microscopy (TEM) photomicrographs of leaves of P. maximum under ambient [CO₂] and air warming (aCeT).

(A) Overviews of a bundle sheath cell (BSC). V–vacuole. (B) Overview of a mesophyll cell (MC). V–vacuole. (C) Chloroplast of a BSC with spaces between the starch grains and chloroplasts and conserved thylakoid membranes (indicated by arrowheads). CW–cell wall, S–starch. (D) Overview of a chloroplast of a MC with plastoglobuli (indicated by arrows) and conserved thylakoid membranes (indicated by arrowheads). (E) Thylakoid membrane detail of the chloroplast of a BSC and spaces between the starch grains and chloroplasts (indicated by arrows). S–starch. (F) Cytoplasm retraction in a BSC and traffic of vacuoles with an MC through plasmodesmata (indicated by an arrow). CW–cell wall. (G) Association of mitochondria and chloroplasts of a BSC. S–starch, M–mitochondria.

Fig 5. Transmission electron microscopy (TEM) photomicrographs of leaves of P. maximum under elevated [CO₂] and air warming (eCeT).

(A) Overview of a bundle sheath cell (BSC). V–vacuole, N–nucleus. (B) Overview of mesophyll cells (MC). V–vacuole. (C) Chloroplast of a BSC with starch grains. S–starch. (D) Chloroplast of a MC with plastoglobuli (indicated by arrowheads) and conserved thylakoid membranes (indicated by arrows). (E) Details of the thylakoid membrane and external membrane of chloroplast of a BSC. S–starch. (F) Plasmodesmata (indicated by an arrow) between a BSC and a MC with regular cell wall. CW–cell wall. (G) Group of mitochondria with conspicuous cristae. M–mitochondria, CW–cell wall.

Fig 6. Stomatal parameters measured in leaves of P. maximum.

(A) Adaxial and (B) abaxial stomatal density–SD. (C) Adaxial and (D) abaxial stomatal index–SI. (E) Adaxial and (F) abaxial stomatal length–SL. Measurements are shown for both leaf surfaces, and statistical analysis was performed between treatments on same leaf surface. Stack bars show the standard error. [CO₂] levels: aC (ambient [CO₂], ~400 μmol mol^-1) and eC (elevated [CO₂], ~600 μmol mol^-1). Temperature levels: aT (ambient temperature) and eT (2°C more than the ambient temperature). ANOVA p-values are shown and significant effects (p < 0.05) are detailed in bold. [CO₂] (isolated effect of elevated [CO₂]), Temp. (isolated effect of air warming) and [CO₂] × Temp. (interaction of elevated [CO₂] × Temp.).

Fig 7. Leaf gas exchange parameters measured during the growing season of P. maximum at the Trop-T-FACE facility.

(A) Net photosynthesis rate (A). (B) Stomatal conductance (g_s). (C) Transpiration rate (E). (D) Intrinsic water-use efficiency (iWUE). Stack bars show the standard error. [CO₂] levels: aC (ambient [CO₂], ~400 μmol mol^-1) and eC (elevated [CO₂], ~600 μmol mol^-1). Temperature levels: aT (ambient temperature) and eT (2°C more than the ambient temperature). ANOVA p-values are shown and significant effects (p < 0.05) are detailed in bold. [CO₂] (isolated effect of elevated [CO₂]), Temp. (isolated effect of air warming), and [CO₂] × Temp. (interaction of elevated [CO₂] × Temp.).

Fig 8. Starch content in leaves of P. maximum at Trop-T-FACE facility.

Stack bars show the standard error. [CO₂] levels: aC (ambient [CO₂], ~400 μmol mol^-1) and eC (elevated [CO₂], ~600 μmol mol^-1). Temperature levels: aT (ambient temperature) and eT (2°C more than the ambient temperature). ANOVA p-values are show and significant effects (p <0.05) are detailed in bold. [CO₂] (isolated effect of elevated [CO₂]), Temp. (isolated effect of air warming) and [CO₂] × Temp. (interaction of elevated [CO₂] × Temp.).

Fig 9. Dry mass of P. maximum at Trop-T-FACE facility.

(A) Leaf dry mass. (B) Stem dry mass. (C) Total aboveground biomass. Stack bars show the standard error. [CO₂] levels: aC (ambient [CO₂], ~400 μmol mol^-1) and eC (elevated [CO₂], ~600 μmol mol^-1). Temperature levels: aT (ambient temperature) and eT (2°C more than the ambient temperature). ANOVA p-values are show and significant effects (p <0.05) are detailed in bold. [CO₂] (isolated effect of elevated [CO₂]), Temp. (isolated effect of air warming) and [CO₂] × Temp. (interaction of elevated [CO₂] × Temp.).

Fig 10. Main anatomical and physiological acclimation mechanisms of P. maximum developed under elevated [CO₂] and warming.

Created with BioRender. Elevated [CO₂] (eC, green circle, isolated effect of CO₂) exerted more pronounced effects on epidermis anatomy and leaf gas exchange. A CO₂-enriched atmosphere reduced the differentiation of epidermal cells to stomata on both leaf surfaces, reducing stomatal density and index. In addition, stomatal aperture and transpiration were also decreased. Therefore, water use efficiency, photosynthesis and starch content increased. Due to low transpiration flux, soil water content was conserved during the experiment. Warming (eT, red circle, isolated effect of temperature) affected leaf structure and starch metabolism. Leaves developed protection mechanisms against the effects of a warmer environment with a thicker adaxial cuticle and reduced size of vascular bundles and bulliform cells. Under the combination of elevated [CO₂] and warming (eCeT, purple circle, interaction of CO₂ × temperature), warming cancelled the CO₂ effect on soil water content and transpiration. However, when combined, these two environmental factors produced a set of anatomical adjustments that contributed to the acclimation of this species to future conditions increasing leaf biomass production. Down arrow: decrease. Up arrow: increase.