Starch Biosynthesis in Guard Cells But Not in Mesophyll Cells Is Involved in CO2-Induced Stomatal Closing

Abstract

Starch metabolism is involved in stomatal movement regulation. However, it remains unknown whether starch-deficient mutants affect CO2-induced stomatal closing and whether starch biosynthesis in guard cells and/or mesophyll cells is rate limiting for high CO2-induced stomatal closing. Stomatal responses to [CO2] shifts and CO2 assimilation rates were compared in Arabidopsis (Arabidopsis thaliana) mutants that were either starch deficient in all plant tissues (ADP-Glc-pyrophosphorylase [ADGase]) or retain starch accumulation in guard cells but are starch deficient in mesophyll cells (plastidial phosphoglucose isomerase [pPGI]). ADGase mutants exhibited impaired CO2-induced stomatal closure, but pPGI mutants did not, showing that starch biosynthesis in guard cells but not mesophyll functions in CO2-induced stomatal closing. Nevertheless, starch-deficient ADGase mutant alleles exhibited partial CO2 responses, pointing toward a starch biosynthesis-independent component of the response that is likely mediated by anion channels. Furthermore, whole-leaf CO2 assimilation rates of both ADGase and pPGI mutants were lower upon shifts to high [CO2], but only ADGase mutants caused impairments in CO2-induced stomatal closing. These genetic analyses determine the roles of starch biosynthesis for high CO2-induced stomatal closing.

Figure 1.

ADGase mutant (adg1-1 and aps1) leaves show starch deficiency in both mesophyll and guard cells, whereas pgi1-1 mutants accumulate starch in guard cells but not in mesophyll cells. Col-0, ADGase (adg1-1 and aps1; Lin et al., 1988; Ventrigliaet al., 2008), and pPGI (pgi1-1) mutants (Yu et al., 2000) were stained with iodine and imaged to visualize their starch content in whole plants (A–D), mesophyll (E–H), and guard cells (I–L). Bars in E to L = 30 µm (lower right) and in I to L = 5 µm (lower left).

Figure 2.

Leaf starch content in the ADGase and pgi1-1 starch biosynthesis mutants. Rosettes from 6-week-old plants were harvested 2 h after the beginning of the light period and immediately frozen in liquid nitrogen. Starch was extracted and quantified using amylase/amyloglucosidase method. Unpaired Student’s t test investigating statistical significance between mutants and the wild type showed a significant reduction in rosette leaf starch levels of adg1-1, aps1, and pgi1-1 mutant plants (**P ≤ 0.005, wild type, n = 3; adg1-1, n = 4; aps1, n = 4 and pgi1-1, n = 4; leaves, each leaf from different plant). Each bar shows the mean of wild type, adg1-1, aps1, or pgi1-1 starch (µg) ± se. Inset shows the same mutant data with a magnified y axis (starch [µg]/gFW). Student’s t test between the starch-deficient mutants (adg1-1, aps1, and pgi1-1) revealed no significant (ns) differences between adg1-1 (ADGase allele) and pgi1-1 (P = 0.3), while aps1 (ADGase allele) starch levels were slightly but significantly larger than adg1-1 (*P = 0.02).

Figure 3.

Arabidopsis starch-deficient ADGase mutants show impaired stomatal closure in response to CO₂ elevation. Time-resolved stomatal conductance responses and net CO₂ assimilation rates were analyzed at the imposed [CO₂] shifts (bottom in ppm) in the wild-type and in two starch-deficient ADGase Arabidopsis mutant alleles adg1-1 (A–D) and aps1 (E– H). A and E, Stomatal conductance in mol H₂O m⁻² s⁻¹. B and F, Data shown in A and E were normalized to the stomatal conductance after 25 min at 360 ppm [CO₂] exposure, 5 min before the change to 800 ppm [CO₂]. C and G, Net CO₂ assimilation rates (µmol CO₂ m⁻² s⁻¹). D and H, The corresponding intercellular [CO₂] (Ci) levels, calculated based on the stomatal conductance and ambient CO₂ concentration. Data are the mean of the wild type, n = 4, and adg1-1, n = 4 (A–D); wild type, n = 5, aps1, n = 4 (E–H) leaves each from different plants ± se for each genotype.

Figure 4.

Analysis of CO₂-induced conductance kinetics. Single exponential functions were fit to data of high CO₂-induced stomatal closure (red dotted traces). A and B, Data traces and fits of wild-type Col-0 and adg1-1 plants from within the same experiment. C and D, Data traces and fits of wild-type Col-0 and aps1 plants from within the same experiment. E and F, Data traces and fits of wild-type Col-0 and pgi1-1 plants from within the same experiment . R² values shown represent the average R² values for all leaves analyzed within the same experiment. The time constants displayed are means ± se of three to five plants.

Figure 5.

The Arabidopsis pgi1-1 mutant that features starch deficiency in mesophyll cells but not in guard cells shows normal stomatal response to CO₂ elevation. Time-resolved stomatal conductance responses and net CO₂ assimilation rates at the imposed [CO₂] conditions (bottom in ppm) in the wild type and in the Arabidopsis starch-deficient mutant pgi1-1 were analyzed using intact whole-leaf gas exchange. A, Stomatal conductance in mol H₂O m⁻² s⁻¹. B, Data shown in A were normalized to the stomatal conductance after 25 min of 360 ppm [CO₂] exposure, 5 min before the change to 800 ppm [CO₂]. C, CO₂ assimilation rates (µmol CO₂ m⁻² s⁻¹). D, The corresponding intercellular [CO₂] (Ci) levels, calculated based on the stomatal conductance and extracellular CO₂ concentration. Data in A to D are the mean of n = 3 leaves each from different plants ± se for each genotype.

Figure 6.

Stomatal aperture of the starch biosynthesis ADGase mutants show reduced response to CO₂ elevation but not pgi1-1 mutant. Stomatal apertures in response to [CO₂] changes were measured in the wild type, the ADGase mutants adg1-1 (A and B) and aps1 (C and D), and in the pPGI mutant pgi1-1 (E and F). B, D, and F, Relative stomatal apertures (%) for each genotype were calculated by normalization to the average stomatal apertures at low CO₂. A to F, Genotype blind assays, n = 3 plants per each genotype and treatment, ∼135 stomata (A and B) and ∼45 stomata (C–F) were analyzed per genotype and condition. Asterisks on the histograms indicate that the means differ significantly (P ≤ 0.05), whereas “ns” indicates no significant difference (P > 0.1).