Metabolic Pathways Involved in Carbon Dioxide Enhanced Heat Tolerance in Bermudagrass

Abstract

Global climate changes involve elevated temperature and CO₂ concentration, imposing significant impact on plant growth of various plant species. Elevated temperature exacerbates heat damages, but elevated CO₂ has positive effects on promoting plant growth and heat tolerance. The objective of this study was to identify metabolic pathways affected by elevated CO₂ conferring the improvement of heat tolerance in a C₄ perennial grass species, bermudagrass (Cynodon dactylon Pers.). Plants were planted under either ambient CO₂ concentration (400 μmol⋅mol^-1) or elevated CO₂ concentration (800 μmol⋅mol^-1) and subjected to ambient temperature (30/25°C, day/night) or heat stress (45/40°C, day/night). Elevated CO₂ concentration suppressed heat-induced damages and improved heat tolerance in bermudagrass. The enhanced heat tolerance under elevated CO₂ was attributed to some important metabolic pathways during which proteins and metabolites were up-regulated, including light reaction (ATP synthase subunit and photosystem I reaction center subunit) and carbon fixation [(glyceraldehyde-3-phosphate dehydrogenase, GAPDH), fructose-bisphosphate aldolase, phosphoglycerate kinase, sedoheptulose-1,7-bisphosphatase and sugars) of photosynthesis, glycolysis (GAPDH, glucose, fructose, and galactose) and TCA cycle (pyruvic acid, malic acid and malate dehydrogenase) of respiration, amino acid metabolism (aspartic acid, methionine, threonine, isoleucine, lysine, valine, alanine, and isoleucine) as well as the GABA shunt (GABA, glutamic acid, alanine, proline and 5-oxoproline). The up-regulation of those metabolic processes by elevated CO₂ could at least partially contribute to the improvement of heat tolerance in perennial grass species.

Keywords: bermudagrass; elevated CO2; heat stress; metabolites; protein.

FIGURE 1

Effects of elevated CO₂ concentration (800 μmol⋅mol^-1 vs. 400 μmol⋅mol^-1) on net photosynthetic rate (P_n) (A), chlorophyll content (Chl) (B) and (C) photochemical efficiency (F_v/F_m) in response to heat stress in bermudagrass. The treatments symbols are 30 and 45 for normal temperature control and heat stress and 400 and 800 for ambient CO₂ and elevated CO₂ concentrations, respectively. Vertical bars indicate significant difference based on LSD values (P ≤ 0.05) for the comparison among treatments.

FIGURE 2

Heat map analysis of total 53 differentially expressed metabolites in response to different temperatures and CO₂ concentrations. The treatments symbols are 30 and 45 for normal temperature control and heat stress and 400 and 800 for ambient CO₂ and elevated CO₂ concentrations, respectively.

FIGURE 3

Effects of elevated CO₂ concentration on total content of organic acids (A), amino acids (B), sugars (C), and sugar alcohols (D) in response to heat stress in bermudagrass. The treatments symbols are 30 and 45 for normal temperature control and heat stress and 400 and 800 for ambient CO₂ and elevated CO₂ concentrations, respectively.

FIGURE 4

Effects of elevated CO₂ concentration on organic acids in response to heat stress in bermudagrass. The treatments symbols are 30 and 45 for normal temperature control and heat stress and 400 and 800 for ambient CO₂ and elevated CO₂ concentrations, respectively. (A) No changes under 30–800 and down-regulation under 45–800; (B) No changes under 30–800 and up-regulation under 45–800; (C) Down-regulation under 30–800 and up-regulation under 45–800. Columns marked with different letters presented the significant differences based on LSD values (P ≤ 0.05) among treatments.

FIGURE 5

Effects of elevated CO₂ concentration on amino acids in response to heat stress in bermudagrass. The treatments symbols are 30 and 45 for normal temperature control and heat stress and 400 and 800 for ambient CO₂ and elevated CO₂ concentrations, respectively. (A) No changes under 30–800 and down-regulation under 45–800; (B) No changes under 30–800 and up-regulation under 45–800; (C) Down-regulation under 30–800 and up-regulation under 45–800; (D) Up-regulation under both 30–800 and 45–800. Columns marked with different letters presented the significant differences based on LSD values (P ≤ 0.05) among treatments.

FIGURE 6

Effects of elevated CO₂ concentration on sugars in response to heat stress in bermudagrass. The treatments symbols are 30 and 45 for normal temperature control and heat stress and 400 and 800 for ambient CO₂ and elevated CO₂ concentrations, respectively. (A) Down-regulation or no changes under 30–800 and down-regulation under 45–800; (B) Up-regulation under both 30–800 and 45–800; (C) No changes under 30–800 and up-regulation under 45–800. Columns marked with different letters presented the significant differences based on LSD values (P ≤ 0.05) among treatments.

FIGURE 7

Effects of elevated CO₂ concentration on sugar alcohols in response to heat stress in bermudagrass. The treatments symbols are 30 and 45 for normal temperature control and heat stress and 400 and 800 for ambient CO₂ and elevated CO₂ concentrations, respectively. Columns marked with different letters presented the significant differences based on LSD values (P ≤ 0.05) among treatments.

FIGURE 8

The metabolic pathways associated with differentially expressed metabolites. The treatments symbols are 30 and 45 for normal temperature control and heat stress and 400 and 800 for ambient CO₂ and elevated CO₂ concentrations, respectively.

FIGURE 9

Representative gels of 2-D with differentially expressed proteins identified in bermudagrass grown under normal temperature (A) and heat stress (B) at 28 days of treatments. Labels of spots in each gel were consistent with Table 2.

FIGURE 10

Subcellular location of identified proteins in response to different CO₂ concentrations and temperatures. The treatments symbols are 30 and 45 for normal temperature control and heat stress and 400 and 800 for ambient CO₂ and elevated CO₂ concentrations, respectively.

FIGURE 11

Cluster analysis from gene ontology (GO) analysis of differentially expressed proteins in response to different CO₂ concentrations under normal temperature (A) and heat stress (B) in leaves of bermudagrass. The treatments symbols are 30 and 45 for normal temperature control and heat stress and 400 and 800 for ambient CO₂ and elevated CO₂ concentrations, respectively. BP, biological process; MF, molecular function; CC, cellular component.

FIGURE 12

Venn analysis of up-regulated proteins (A) and down-regulated proteins (B) identified in bermudagrass at 28 days of treatments. The treatments symbols are 30 and 45 for normal temperature control and heat stress and 400 and 800 for ambient CO₂ and elevated CO₂ concentrations, respectively.

FIGURE 13

Functional classification of CO₂ responsive proteins identified in bermudagrass grown under normal temperature (A) and heat stress (B) at 28 days of treatments.

FIGURE 14

Comparison of protein abundance caused by elevated CO₂ (800 μmol⋅mol^-1) with ambient CO₂ (400 μmol⋅mol^-1) under normal temperature control (30°C). Charts are organized by the functional category of proteins involved in photosynthesis, protein synthesis and degradation, oxidative pentose phosphate and mitochondrial electron transport as shown in (A) as well as amino acid metabolism, glycolysis, stress defense, nucleotide metabolism, N-metabolism, TCA cycle, miscellaneous, transport and unknown proteins as shown in (B). The values of the mean ± SE represent the relative expression fold change of proteins in response to elevated CO₂ under normal temperature. Labels with ‘n’ in X-axle were same as Table 2.

FIGURE 15

Comparison of protein abundance caused by elevated CO₂ (800 μmol⋅mol^-1) with ambient CO₂ (400 μmol⋅mol^-1) under heat stress (45°C). Charts are organized by the functional category of proteins involved in photosynthesis and protein synthesis as shown in (A) as well as amino acid metabolism, glycolysis, stress defense, TCA cycle, metal handing, major CHO metabolism, cell and transport as shown in (B). The values of the mean ± SE represent the relative expression fold change of proteins in response to elevated CO₂ under heat stress. Labels with ‘h’ in X-axle were same as Table 2.

FIGURE 16

The metabolic pathways associated with differentially expressed proteins. The treatments symbols are 30 and 45 for normal temperature control and heat stress and 400 and 800 for ambient CO₂ and elevated CO₂ concentrations, respectively. Labels with ‘n’ or ‘h’ were same as Table 2. RuBP, Ribulose 1, 5-bisphosphate; R5P, Ribulose 5-phosphate; Rubisco, Ribulose 1, 5-bisphosphate carboxylase/oxygenase; PGA, 3-phosphoglyceric acid; G3P, Glyceraldehyde 3-phosphate; PC, Plastocyanin; PQ, Plastoquinone; Fd, Ferredoxin; Cyt, Cytochrome complex.