Internal conductance to CO(2) diffusion and C(18)OO discrimination in C(3) leaves

Abstract

(18)O discrimination in CO(2) stems from the oxygen exchange between (18)O-enriched water and CO(2) in the chloroplast, a process catalyzed by carbonic anhydrase (CA). A proportion of this (18)O-labeled CO(2) escapes back to the atmosphere, resulting in an effective discrimination against C(18)OO during photosynthesis (Delta(18)O). By constraining the delta(18)O of chloroplast water (delta(e)) by analysis of transpired water and the extent of CO(2)-H(2)O isotopic equilibrium (theta(eq)) by measurements of CA activity (theta(eq) = 0.75-1.0 for tobacco, soybean, and oak), we could apply measured Delta(18)O in a leaf cuvette attached to a mass spectrometer to derive the CO(2) concentration at the physical limit of CA activity, i.e. the chloroplast surface (c(cs)). From the CO(2) drawdown sequence between stomatal cavities from gas exchange (c(i)), from Delta(18)O (c(cs)), and at Rubisco sites from Delta(13)C (c(c)), the internal CO(2) conductance (g(i)) was partitioned into cell wall (g(w)) and chloroplast (g(ch)) components. The results indicated that g(ch) is variable (0.42-1.13 mol m(-2) s(-1)) and proportional to CA activity. We suggest that the influence of CA activity on the CO(2) assimilation rate should be important mainly in plants with low internal conductances.

Figure 1

Diagram showing the ¹⁸O content in fluxes of CO₂ (F_out) and H₂O (E) from leaf to atmosphere. H₂O enters the leaf with isotopic composition δ_s, evaporates from the cell surfaces, and diffuses from the leaf, experiencing both phase-change (ε*) and diffusional (ε_k) fractionation, giving rise to depleted transpiring water (δ_t) and enriched evaporating surfaces (δ_e). Similarly, CO₂ from the atmosphere (F_in) dissolves in the chloroplast, equilibrates (ε_eq) to composition δ_c depending on the δ¹⁸O of water in the chloroplast and the extent of isotopic equilibrium (θ_eq), and then approximately two-thirds retro-diffuses outward (F_out) with fractionation during diffusion (ā). This can be observed as an ¹⁸O enrichment in outgoing CO₂ (δ_out) relative to incoming CO₂ (δ_in), which is proportional to discrimination against C¹⁸OO, termed Δ¹⁸O. CO₂ reference points along the leaf-atmosphere pathway are marked (with average values in μmol mol⁻¹) as c_c, c_cs, c_i, and c_a, referring to the CO₂ concentration in the chloroplast, chloroplast surface, substomatal cavity, and air, respectively.

Figure 2

Discrimination against C¹⁸OO (Δ¹⁸O) as a function of chloroplast CO₂ concentration (calculated from Δ¹³C) and expressed as c_c/c_a for tobacco (a), soy (b), and oak (c). In a and b, experiments were conducted under depleted source CO₂ (−30‰, white symbols) and ambient CO₂ (0‰, black symbols). For oak, experiments were conducted all in depleted CO₂, but at 2% (squares), 21% (circles), and 35% (triangles) oxygen.

Figure 3

The difference between δ_e from the Craig and Gordon equation and bulk leaf water (δ_LW) as a function of evaporation rate (E) for soy (triangles), tobacco (circles), maize (diamonds), and sorghum (squares). White symbols are data from the last measurement of the light response study; black symbols are additional points from the leaf water heterogeneity test. The three marked points excluded from statistical analysis are thought to represent non-steady-state conditions.

Figure 4

Diagram showing the dynamics of oxygen isotope exchange between atmospheric CO₂ (δ_a) and leaf water (δ_e) and the resulting δ¹⁸O of CO₂ (δ_c). Isotopic equilibrium (θ_eq) from Equation 4, solid line was calculated from the CA activity and CO₂ residence time (kτ), which represents the number of hydrations per CO₂ molecule and is related to Δ_ca/Δ_ea.

Figure 5

The number of hydration reactions per CO₂ molecule (kτ) calculated from CA_leaf/F_in as a function of the CO₂ assimilation rate. Shown on the second axis is the equivalent extent of isotopic equilibrium from Equation 4, in which full equilibrium (>99.5%) occurs above kτ = 15. White symbols, Soy; black symbols, tobacco (different symbols refer to different light responses). All values for oak were above kτ = 15 because of the assumed high CA rates.

Figure 6

Data shown for all light response curves, showing internal CO₂ concentration (μmol mol⁻¹) as a function of CO₂ assimilation rate (A) (μmol m⁻² s⁻¹). Symbols refer to CO₂ concentration in internal air space (c_i) (from gas exchange measurements, ▵), c_cs (from Δ¹⁸O, δ_e, and CA activity; ♦), and c_c (from Δ¹³C; ○). Species are tobacco (a–c), soy (d–e), and oak (f–h). Light responses in c and e were conducted using ambient δ¹⁸O CO₂, whereas the rest used CO₂ depleted in ¹⁸O. f through h, Experiments in 40%, 20%, and 2% ) O₂, respectively.

Figure 7

Diagram representing the backflux of CO₂ from sites of CO₂ fixation (c_c) and sites of oxygen exchange (c_cs) in the chloroplast, showing the partitioning of total internal conductance (g_i) (relevant to Δ¹³C) into chloroplast (g_ch) and wall (g_w) conductance (from Δ¹⁸O).

Figure 8

The potential change in CO₂ assimilation rate (A) as a function of g_ch (oak and tobacco, solid lines). g_ch is normalized relative to a constant wall conductance (g_w) (0.35 and 1.12 mol m⁻² s⁻¹ for oak and tobacco, respectively). The changes in A are expressed relative to measured assimilation rates at actual conductance values, g_ch/g_w = 0.8 and 3.2 for tobacco (A = 12.7 μmol mol⁻¹) and oak (A = 13.7 μmol mol⁻¹), respectively. Also marked is the estimated g_ch/g_w (see text) of tobacco mutants lacking CA (Price et al., 1994; Williams et al., 1996), indicating only a small effect on assimilation relative to the wild-type tobacco.

Figure 9

Arrangement of on-line CO₂ trapping and off-line H₂O trapping apparatus for continuous flow CO₂ isotopic analysis, in conjunction with leaf chamber and gas exchange system.