Cyclic and linear electron flow:
We will determine the proportion of PSI reaction centers that participate in either cyclic or linear electron transport using the method of Joliot. P700 and plastocyanin oxidation are analyzed under subsaturating illumination at 820 and 740 nm, respectively. This illumination induces P700 and plastocyanin oxidation in multiple phases. These phases are associated with electron flow into PSI from various sources, including linear and cyclic electron flow. To differentiate between linear and cyclic electron flow, P700 oxidation kinetics are monitored individually in the presence of an inhibitor of linear (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and cyclic (methyl viologen) electron flow. The results will allow us to distinguish the contributions of these two processes to the reduction of both P700 and plastocyanin under a number of different conditions.
Y1, Y(ND), and Y(NA):
The parameter Y1, also known as the quantum yield of PSI, corresponds to the fraction of P700 that is reduced under illumination when the acceptor side of this complex is not limiting. This parameter provides information on the functionality of PSI, as well as the proportion of PSI complexes limited for either the input of electrons on the donor side or the extraction of electrons on the acceptor side of the complex, designated Y(ND) and Y(NA), respectively. To determine these parameters, Chlamydomonas cells are excited with a saturating pulse, while the redox state of P700 is monitored at 820 nm. This value, designated Pm, reflects a state of maximum P700 oxidation. The sample is then illuminated with various levels of non-saturating actinic light and the proportion P700 that remains oxidized corresponds to donor side limitation (the absorbance at 820 nm at the various light intensities divided by Pm gives the parameter Y(ND)). At the end of the actinic illumination, the sample is exposed to a saturating pulse and then placed in the dark (this oxidizes all reaction centers for which there is not acceptor side limitation). The absorbance at 820 nm following the application of this pulse in combination with actinic illumination provides information on the proportion of P700 for which there is donor side limitation (Y(ND)) as well as the the fraction of P700 that is neither donor nor acceptor side limited (Y1). Subtracting Y1 plus Y(ND) from Pm yields the proportion of PSI reaction centers that are experiencing acceptor side limitation or Y(NA).
In wt leaves the kcat values varied between 1.5 and 4 s−1 in tobacco (up to 6 s−1 in young sunflower) and increased with decreasing Rubisco content. This upper limit of kcat is somewhat higher than the in planta value obtained for tobacco by as well as the in vitro values for Rubisco from tobacco () and from several C3 plants (at least those from cool habitats; ). The maximum value for kcat of 6–7 s−1 observed in some leaves could be overestimated due to problems of extraction of membrane-bound Rubisco as discussed above. But this is still unlikely since the value of 6 s−1 has been confirmed by the data of for Rubisco in –RBC tobacco. Similar high kcat values (up to 8 s−1) were reported in young sunflower leaves adapting from low to high growth light ().
Assuming that PSI density proportionally characterizes the capacity of other components of the electron transport chain () and that Activase is functionally related to ATP synthase, one may expect that more Rubisco is activated in leaves exhibiting a higher ratio of PSI per Rubisco active site. The content of PSI increased with leaf N (). A plot of Rubisco kcat versus the ratio of PSI per Rubisco active site site exhibited a saturating relationship, where data points for wt plants formed the initial slope (low PSI/Rubisco) and data from the –RBC plants formed the saturation phase (high PSI/Rubisco, ). The hyperbolic relationship of describes the dataset involving the maximal kcat of 8 s−1 recorded for –RBC plants:
This overview shows that in normally photosynthesizing leaves Rubisco is present in high quantities, but the variable kcat values reflect partial enzyme activation. It is likely that the stromal redox state and ATP/ADP ratio are not the only parameters controlling Rubisco activation via Activase; an, as yet, unknown inhibitor controls the number of Rubisco sites that can be activated by carbamylation. In this work, the actual Rubisco activity in planta, as a function of the amount of holoenzyme and in relation to the photosynthetic light reactions, is investigated. It is shown that the activation state of Rubisco is generally low in wild-type plants containing high Rubisco levels. It is still low in N-starved leaves where Rubisco content is low, but PSI content is low as well. It is even lower in FNR-deficient and Cyt b6f-deficient tobacco leaves containing normal amounts of Rubisco, but impaired in electron transport capacity. Rubisco activation state approaches the maximum in transgenic leaves containing little Rubisco but sufficient PSI. In these leaves, the catalytic turnover rate of an active Rubisco site may exceed 6 s−1 at 22.5 °C.
The CO2 response curve of Rubisco was measured in planta at CO2 concentrations up to at least three times Km(CO2) by accumulating RuBP at low CO2 and O2 concentrations and then measuring the initial rate of CO2 fixation after a rapid increase in CO2 concentration (; ). The biological variation of kcat was wide. It decreased from 4 to 2.5 s−1 with increasing Rubisco content in sunflower grown under different conditions (). In mature birch (Betula pendula) leaves the average kcat was about 2 s−1 (). In developing birch leaves Rubisco Vm increased in proportion to the capacity of the developing photosynthetic machinery, kcat varied from 1.35 to 2.24 s−1 (). In Betula pendula and Tilia cordata leaves growing in a natural canopy the apparent kcat values were 2.3 and 1.6 s−1, respectively, independent of sun/shade exposure in the vertical cross-section of the canopy (), all cited measurements at 22.5 °C). Significant correlation was detected between the amount of activated Rubisco (Vm) and PSI density, suggesting that the activity of Activase is related to the photosynthetic electron transport system.
When extracted Rubisco protein was precipitated with sulphate ions and redissolved and carbamylated in a phosphate-free medium, the resulting ‘maximal activity’ was greater than the ‘total activity’ of the carbamylated enzyme before the precipitation. Precipitation evidently released an unknown inhibitor from carbamylated or uncarbamylated sites (). When the Activase content was gradually decreased in anti-sense transgenic tobacco, the number (or in planta turnover rate) of carbamylated sites decreased finally about 10-fold (; ). Similar impairment of the function of Rubisco was observed under the influence of moderately high temperatures in these anti-sense plants (). Early experiments on lysed chloroplasts (, ), as well as Cyt b6f-deficient and GAPDH-deficient tobacco (), revealed that activation of the Activase is not a simple function of the ATP/ADP ratio, but is somehow related to Cyt b6f content and PSI electron transport, demonstrating the importance of further in planta investigations.
Site turnover rate (kcat) of Rubisco was measured in intact leaves of different plants. Potato (Solanum tuberosum L.) and birch (Betula pendula Roth.) leaves were taken from field-growing plants. Sunflower (Helianthus annuus L.), wild type (wt), Rubisco-deficient (–RBC), FNR-deficient (–FNR), and Cyt b6f deficient (–CBF) transgenic tobacco (Nicotiana tabacum L.) were grown in a growth chamber. Rubisco protein was measured with quantitative SDS-PAGE and FNR protein content with quantitative immunoblotting. The Cyt b6f level was measured in planta by maximum electron transport rate and the photosystem I (PSI) content was assessed by titration with far-red light. The CO2 response of Rubisco was measured in planta with a fast-response gas exchange system at maximum ribulose 1,5-bisphosphate concentration. Reaction site kcat was calculated from Vm and Rubisco content. Biological variation of kcat was significant, ranging from 1.5 to 4 s−1 in wt, but was >6 s−1 at 23 °C in –RBC leaves. The lowest kcat of 0.5 s−1 was measured in –FNR and –CBF plants containing sufficient Rubisco but having slow electron transport rates. Plotting kcat against PSI per Rubisco site resulted in a hyperbolic relationship where wt plants are on the initial slope. A model is suggested in which Rubisco Activase is converted into an active ATP-form on thylakoid membranes with the help of a factor related to electron transport. The activation of Rubisco is accompanied by the conversion of the ATP-form into an inactive ADP-form. The ATP and ADP forms of Activase shuttle between thylakoid membranes and stromally-located Rubisco. In normal wt plants the electron transport-related activation of Activase is rate-limiting, maintaining 50–70% Rubisco sites in the inactive state.
The coral was allowed to dark-acclimate overnight before minimum chlorophyll fluorescence (Fo) and maximum fluorescence (Fm - through application of a photosynthetically-saturating pulse of light) were determined. The coral was then exposed to light intensities that were incrementally adjusted upwards, and Fm' (maximum chlorophyll fluorescence while illuminated) measurements were made 15-20 minutes after light intensity was increased. Yields of Photochemistry, Non-Photochemical Quenching (of chlorophyll fluorescence) and NO (other energy dissipation pathways) were determined. When the Yield of any of these processes is multiplied by the light intensity level (PPFD, or Photosynthetic Photon Flux Density, as determined by a PAR meter), we can arrive at an estimate of the relative electron flow from Photosystem II to Photosystem I. The electron flow is called ETR for Electron Transport Rate. Since we did not measure the amount of light actually absorbed by the coral and its symbionts, this is called the Relative ETR, or rETR. The coral's zooxanthellae were allowed to acclimate to darkness for 20-30 minutes after each round of testing.
A novel result of this work is the relationship between Rubisco activation state and the activity of the photosynthetic electron transport chain. Expression of kcat as a function of the ratio of PSI per Rubisco site revealed a hyperbolic dependence with a K0.5 of about 0.1 PSI per Rubisco active site. In wt plants the PSI/Rubisco active site ratio varied from 0.02 to 0.1 (10 to 50 sites per PSI) with a median value of about 0.03 (33 sites per PSI). Therefore, typically the measured kcat was only 20–30% of the theoretical maximum. Important new information came from the experiments with –FNR and –CBF tobacco. In control leaves grown together with the –FNR series kcat was about 1.3 s−1, typical for leaves with a relatively low PSI/Rubisco ratio in wt plants. In the –FNR plants Rubisco activity decreased, as was noticed by . Quantitatively, in the –FNR plants the in planta apparent kcat was suppressed to 0.5 s−1 and correlated with the relative expression of FNR (). Similarly, as soon as the expression of Cyt b6f was low enough to limit electron transport, kcat values decreased in strong correlation with the Cyt b6f-limited electron transport rate (). It is unlikely that in the –FNR case the redox control of Activase was observed, since FNR deficiency must have caused the increased reduction of ferredoxin and correspondingly more activation of Rubisco. Instead, the opposite was observed.