Transpiration and ozone uptake rates were measured simultaneously in sunflower leaves at different stomatal openings and various ozone concentrations. Ozone uptake rates were proportional to the ozone concentration up to 1500 nanoliters per liter. The leaf gas phase diffusion resistance (stomatal plus boundary layer) to water vapor was calculated and converted to the resistance to ozone multiplying it by the theoretical ratio of diffusion coefficients for water vapor and ozone in air (1.67). The ozone concentration in intercellular air spaces calculated from the ozone uptake rate and diffusion resistance to ozone scattered around zero. The ozone concentration in intercellular air spaces was measured directly by supplying ozone to the leaf from one side and measuring the equilibrium concentration above the other side, and it was found to be zero. The total leaf resistance to ozone was proportional to the gas phase resistance to water vapor with a coefficient of 1.68. It is concluded that ozone enters the leaf by diffusion through the stomata, and is rapidly decomposed in cell walls and plasmalemma. diffusion resistance of the whole gaseous pathway from cell surfaces to ambient air:where E2 is the transpiration rate (minus cuticular transpiration); rg, the diffusion resistance in the leaf gaseous phase to water vapor; wi, the water vapor concentration at evaporating cell surfaces; and wa, that in the ambient air. CO2 is a heavier gas (M = 44) than water vapor (M = 18); therefore, CO2 moves more slowly than water vapor through the same diffusion pathway and at the same concentration difference. The ratio of the diffusion coefficients of H20 and CO2 in the leaf gaseous pathway was measured to be 1.62 (9).We could not find a value of the diffusion constant for 03 in air, DZ, in the literature. However, diffusion constants for various gas mixtures may be calculated using the molecular parameters of component gases (1) 0.43 X (T)00) X Abbreviations: E, transpiration rate; wa, wi, water vapor concentration in ambient air (a) and on evaporating cell surfaces (i); r8, rg, leaf gas phase diffusion resistance to water vapor (w) and to ozone (z); M, molecular weight; DW, Dz, diffusion constant for water vapor (w) and for ozone (z); T, temperature, Tk, critical temperature; Vk, critical volume; P, atmospheric pressure; Za, zi, ozone concentration in ambient air (a) and in the leaf intercellular air space (i); Q, ozone uptake rate; v, gas flow rate; S, leaf area; gz, total leaf conductance for ozone; rz, total leaf resistance to ozone; gge ggz, leaf gas phase diffusion conductance for water vapor (w) and for ozone (z).

Leaf isoprene emission scales positively with light intensity, is inhibited by high carbon dioxide (CO 2 ) concentrations, and may be enhanced or inhibited by low oxygen (O 2 ) concentrations, but the mechanisms of environmental regulation of isoprene emission are still not fully understood. Emission controls by isoprene synthase, availability of carbon intermediates, or energetic cofactors have been suggested previously. In this study, we asked whether the short-term (tens of minutes) environmental control of isoprene synthesis results from alterations in the immediate isoprene precursor dimethylallyldiphosphate (DMADP) pool size, and to what extent DMADP concentrations are affected by the supply of carbon and energetic metabolites. A novel in vivo method based on postillumination isoprene release was employed to measure the pool size of DMADP simultaneously with the rates of isoprene emission and net assimilation at different light intensities and CO 2 and O 2 concentrations. Both net assimilation and isoprene emission rates increased hyperbolically with light intensity. The photosynthetic response to CO 2 concentration was also hyperbolic, while the CO 2 response curve of isoprene emission exhibited a maximum at close to CO 2 compensation point. Low O 2 positively affected both net assimilation and isoprene emission. In all cases, the variation in isoprene emission was matched with changes in DMADP pool size. The results of these experiments suggest that DMADP pool size controls the response of isoprene emission to light intensity and to CO 2 and O 2 concentrations and that the pool size is determined by the level of energetic metabolites generated in photosynthesis.

The responses of isoprene emission rate to temperature are characterized by complex time-dependent behaviors that are currently not entirely understood. To gain insight into the temperature dependencies of isoprene emission, we studied steadystate and transient responses of isoprene emission from hybrid aspen (Populus tremula 3 Populus tremuloides) leaves using a fast-response gas-exchange system coupled to a proton-transfer reaction mass spectrometer. A method based on postillumination isoprene release after rapid temperature transients was developed to determine the rate constant of isoprene synthase (IspS), the pool size of its substrate dimethylallyldiphosphate (DMADP), and to separate the component processes of the temperature dependence of isoprene emission. Temperature transients indicated that over the temperature range 25°C to 45°C, IspS was thermally stable and operated in the linear range of its substrate DMADP concentration. The in vivo rate constant of IspS obeyed the Arrhenius law, with an activation energy of 42.8 kJ mol 21 . In contrast, steady-state isoprene emission had a significantly lower temperature optimum than IspS and higher activation energy. The reversible temperature-dependent decrease in the rate of isoprene emission between 35°C and 44°C was caused by decreases in DMADP concentration, possibly reflecting reduced pools of energetic metabolites generated in photosynthesis, particularly of ATP. Strong control of isoprene temperature responses by the DMADP pool implies that transient temperature responses under fluctuating conditions in the field are driven by initial DMADP pool size as well as temperature-dependent modifications in DMADP pool size during temperature transients. These results have important implications for the development of process-based models of isoprene emission.

The control of foliar isoprene emission is shared between the activity of isoprene synthase, the terminal enzyme catalyzing isoprene formation from dimethylallyldiphosphate (DMADP), and the pool size of DMADP. Due to limited in vivo information of isoprene synthase kinetic characteristics and DMADP pool sizes, the relative importance of these controls is under debate. In this study, the phenomenon of postillumination isoprene release was employed to develop an in vivo method for estimation of the DMADP pool size and to determine isoprene synthase kinetic characteristics in hybrid aspen (Populus tremula 3 Populus tremuloides) leaves. The method is based on observations that after switching off the light, isoprene emission continues for 250 to 300 s and that the integral of the postillumination isoprene emission is strongly correlated with the isoprene emission rate before leaf darkening, thus quantitatively estimating the DMADP pool size associated with leaf isoprene emission. In vitro estimates demonstrated that overall leaf DMADP pool was very large, almost an order of magnitude larger than the in vivo pool. Yet, the difference between total DMADP pools in light and in darkness (light-dependent DMADP pool) was tightly correlated with the in vivo estimates of the DMADP pool size that is responsible for isoprene emission. Variation in in vivo DMADP pool size was obtained by varying light intensity and atmospheric CO 2 and O 2 concentrations. From these experiments, the in vivo kinetic constants of isoprene synthase were determined. In vivo isoprene synthase kinetic characteristics suggested that isoprene synthase mainly operates under substrate limitation and that short-term light, CO 2 , and O 2 dependencies of isoprene emission result from variation in DMADP pool size rather than from modifications in isoprene synthase activity.

Photosynthesis is a complex process whose rate is affected by many biochemical and biophysical factors. Fortunately, it is possible to determine, or at least estimate, many of the most important parameters using a combination of optical methods and gas transient analyses. We describe here a computer-operated routine that has been developed to make detailed assessments of photosynthesis at a comprehensive level. The routine comprised the following measurements: steady-state light and CO 2 response curves of net CO 2 assimilation at 21 and 2 kPa O 2 ; transients from limiting to different saturating CO 2 concentrations at 2 kPa O 2 ; post-illumination CO 2 fixation transient; dark-light induction of O 2 evolution; O 2 yield from one saturating single-turnover flash; chlorophyll fluorescence F 0 , F s and F m during the light and CO 2 response curves; leaf transmission at 820 nm (P700 + ) during the light and CO 2 response curves; post-illumination re-reduction time of P700 + . The routine was executed on a two-channel fast-response gas exchange measurement system (A. Laisk and V. Oja: Dynamic Gas Exchange of Leaf Photosynthesis. CSIRO, Canberra, Australia). Thirty-six intrinsic characteristics of the photosynthetic machinery were derived, including quantum yield of CO 2 fixation ( Y CO2 Key-words : Betula pendula Roth; leaf; methods; photosynthesis.)Abbreviations : A , net CO 2 assimilation rate; AC, assimilatory charge; a II , a I , a 0 , relative optical cross-sections of PSII and PSI antenna and of non-photosynthetic absorption; C a , C i , C w , C c , CO 2 concentrations: ambient, intercellular space, cell wall liquid and carboxylation site, respectively; CRC, carbon reduction cycle; Cyt, b 6 f, cytochrome b 6 f; DCMU, dichlorophenol-dimethyl urea; ETR, and J , electron transport rate; F 0 , F s , F m , F md , fluorescence yields, minimum, steady state, maximum (all in the light) and maximum F m in the dark, respectively; FRL, far-red light; Γ , CO 2 compensation point; K s , Rubisco CO 2 /O 2 specificity; k N , relative rate constant for regulatory non-photochemical excitation quenching; k P0 , relative rate constant for photochemical excitation quenching at open PSII centres; P s , P m , P o , 820 nm signal difference from the dark level, steady state, maximum and corresponding to oxidizable P700; PAD, ( I in equations), PFD, photon flux density, absorbed and incident, respectively; PC, plastocyanin; PGA, 3-phosphoglyceric acid; P i , inorganic phosphate; PSI, PSII, photosystems I and II; PQ, plastoquinone; P700, donor pigment of PSI; q E , q I , non-photochemical quenching, energy dependent and inhibitory; R d , R K , Krebs cycle CO 2 evolution rate in the dark and in the light, respectively; r gw , r m , r md , leaf diffusion resistances, in gas phase, mesophyll total and mesophyll diffusional; RuBP, ribulose 1,5-bisphosphate; Rubisco, ribulose 1,5-bisphosphate carboxylase-oxygenase; SCE, specific carboxylation efficiency; VPD, water

(A.L.); and lstituto di Biochimica ed Ecofisiologia Vegetali, 0001 6 Monterotondo Scalo, Rome, ltaly (F.L.)Using simultaneous measurements of leaf gas exchange and chlorophyll fluorescence, we determined the excitation partitioning to photosystem I1 (PSII), the CO,/O, specificity of ribulose-l,5-bisphosphate carboxylase/oxygenase, the dark respiration in the light, and the alternative electron transport rate to acceptors other than bisphosphoglycerate, and the transport resistance for CO, in the mesophyll cells for individual leaves of herbaceous and tree species. The specificity of ribulose-1,5-bisphosphate carboxylase/ oxygenase for CO, was determined from the slope of the O , dependente of the CO, compensation point between 1.5 and 21 ?& O,. Its value, on the basis of dissolved CO, and O , concentrations at 25.5"C, varied between 86 and 89. Dark respiration i n the light, estimated from the difference between the CO, compensation point and the CO, photocompensation point, was about 20 to 50% of the respiration rate in the dark. The excitation distribution to PSll was estimated from the extrapolation of the dependence of the PSll quantum yield on F/F, to F = O, where F is steady-state and F,,, is pulse-saturated fluorescence, and varied between 0.45 and 0.6. The alternative electron transport rate was found as the difference between the electron transport rates calculated from fluorescence and from gas exchange, and at low CO, concentrations and 10 to 21 % O*, it was 25 to 30% of the maximum electron transport. l h e calculated mesophyll diffusion resistance accounted for about 20 to 30% of the total mesophyll resistance, which also includes carboxylation resistance. Whole-leaf photosynthesis is limited by gas phase, mesophyll diffusion, and carboxylation resistances in nearly the same proportion in both herbaceous species and trees.

After darkening, isoprene emission continues for 20 to 30 min following biphasic kinetics. The initial dark release of isoprene (postillumination emission), for 200 to 300 s, occurs mainly at the expense of its immediate substrate, dimethylallyldiphosphate (DMADP), but the origin and controls of the secondary burst of isoprene release (dark-induced emission) between approximately 300 and 1,500 s, are not entirely understood. We used a fast-response gas-exchange system to characterize the controls of dark-induced isoprene emission by light, temperature, and CO 2 and oxygen concentrations preceding leaf darkening and the effects of short light pulses and changing gas concentrations during dark-induced isoprene release in hybrid aspen (Populus tremula 3 Populus tremuloides). The effect of the 2-C-methyl-D-erythritol-4-phosphate pathway inhibitor fosmidomycin was also investigated. The integral of postillumination isoprene release was considered to constitute the DMADP pool size, while the integral of dark-induced emission was defined as the "dark" pool. Overall, the steady-state emission rate in light and the maximum dark-induced emission rate responded similarly to variations in preceding environmental drivers and atmospheric composition, increasing with increasing light, having maxima at approximately 40°C and close to the CO 2 compensation point, and were suppressed by lack of oxygen. The DMADP and dark pool sizes were also similar through their environmental dependencies, except for high temperatures, where the dark pool significantly exceeded the DMADP pool. Isoprene release could be enhanced by short lightflecks early during dark-induced isoprene release, but not at later stages. Fosmidomycin strongly suppressed both the isoprene emission rates in light and in the dark, but the dark pool was only moderately affected. These results demonstrate a strong correspondence between the steady-state isoprene emission in light and the dark-induced emission and suggest that the dark pool reflects the total pool size of 2-C-methyl-D-erythritol-4-phosphate pathway metabolites upstream of DMADP. These metabolites are converted to isoprene as soon as ATP and NADPH become available, likely by dark activation of chloroplastic glycolysis and chlororespiration.

A gas-exchange system with a rapid response time was used to study the interplay between rate-limiting processes of C3 photosynthesis in wild-type tobacco (Nicotiana tabacum L. cv. W38) and transgenic tobaccos with antisense DNAs directed against the Rubisco small subunit (anti-SSu plants) or the chloroplast glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH plants). High ribulosebisphos-phate (RuBP) pools were generated in leaves by exposing them briefly to very low CO2, after which they were transferred to varying CO2 concentrations, and transient CO2 assimilation rates were measured within the first 2–3 s. Comparison of the transient (RuBP-saturated) and steady-state rates confirmed that the CO2 assimilation rate in anti-SSu plants was RuBP-saturated (i.e. Rubisco limited) at all intercellular CO2 partial pressures (Ci), and that, in anti-GAPDH plants, the transition from RuBP-saturation to RuBP-limitation occurred at lower assimilation rates and lower Ci as GAPDH activity was decreased. In addition, we investigated whether the integrated post-illumination CO2 uptake could be used as a non-destructive means of estimating RuBP pools in leaves. In wild-type plants there was generally a good agreement between RuBP pools extracted from leaves after rapid freeze-clamping and estimates made from post-illumination CO2 uptake. However, in the anti-SSu plants, the post- illumination CO2 uptake underestimated the actual RuBP content and the discrepancy became larger as the Rubisco content decreased. Possible explanations for this are discussed.