The Grossman Lab has identified many mutants in Chlamydomonas defective in photosynthetic function. A streamlined platform is being developed to analyze these mutants which involves spectroscopy, fluorometry, CO2 fixation and oxygen evolution. Some of the basic parameters for the putative regulatory mutants that we will monitor include: (1) Growth on minimal medium; (2) Light sensitivity, which will be performed on cells grown on plates exposed to light intensities ranging from 0-150 μmol photon m-2s-1 (as shown in Fig. 4); (3) Pigment analysis, from whole cell spectra and spectra of pigments extracted in 80% acetone (and potentially some HPLC analysis of pigments), from which we will calculate levels of chlorophylls, carotenoids and the chlorophyll a/b ratio (104); (4) Gas exchange measurements, including both O2 evolution (in the light) and uptake (in the dark), using a Clark-type O2 electrode. These experiments coupled with the use of specific inhibitors could yield information on whole chain electron transport and the partial reactions of photosynthesis (e.g. PSII, inter-chain carriers, PSI), and potentially provide some insights into alternate electron flow. Many measurements of other photosynthetic parameters will be facilitated by the use of the Dual PAM 100 fluorometer, the fast repetition rate pulse fluorometer, and the JTS10 spectrophotometer (see Equipment & Facilities). The spectrophotometry and fluorescence analyses are rapid, have high information content and are noninvasive, making them ideal for a primary mutant screen. Many of the specific activities measured in these assays are described below.
The functional state of PSII will be analyzed by measurements of changes in variable Chl a fluorescence using a Pulse Amplitude Modulated (PAM) fluorometer; we will also use fast repetition rate fluorometry to perform some of these measurements.
This parameter reflects the maximal efficiency of PSII photochemistry. Prior to performing this fluorescence measurement, algal samples are incubated for 10 min in the dark and then exposed to 10 min of far-red light (activating PSI photochemistry) in order to make sure that PSII (and the PQ pool) is fully oxidized and that the cells are in state 1 prior to the measurement.
qP and ΦPSII:
qP measures photochemical quenching, providing information about the proportion of PSII reaction centers that are available for photochemistry after exposure of the cells to different light intensities (they are grown at one light intensity and then exposed to various intensities for 5-10 min). If the qP values in mutant cells are comparable to those of wild-type cells, it is likely that the mutants are able to acclimate to the various light conditions used for growth and that the extent to which PSII traps remain opened is optimal. Conversely, low values relative to wild-type cells would suggest that a high proportion of the traps are closed and that the cells are potentially aberrant in their ability to properly acclimate to the light environment. Furthermore, a lack of concordance between qP and CO2 fixation (e.g. CO2 fixation is saturated but qP stays high) would suggest the existence of alternative pathways for extracting electrons from PSII, which would include the Mehler reaction (an electron leak on the reducing side of PSI, probably from the ferredoxin-NADP oxidoreductase, and the generation of H2O2) or potentially the re-reduction of O2 and H+ to regenerate H2O through a plastid terminal oxidase (PTOX). While the latter reaction is not usually prevalent, it can be in certain organisms. ΦPSII is related to qP and indicates the photochemical yield of PSII at different light intensities. It can be viewed as the operating efficiency of PSII, reflecting the proportion of light energy absorbed by PSII that subsequently drives photochemistry.
Furthermore, a number of these and other parameters can be measured using fast repetition rate fluorescence. This technology allows for the application of single or multiple turnover subsaturating excitation pulses at microsecond intervals to induce fluorescence transients, allowing measurements of the photochemical conversion efficiency in PSII (Fv/Fm), the functional absorption cross section of PSII (σPSII) and the energy transfer between PSII units (p).
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).
This represents fluorescence quenching of PSII antenna, but not by photochemistry; the processes has been termed NPQ or nonphotochemical quenching. We generally measure this as Stern-Volmer quenching which reports the level of fluorescence that is quenched relative to the maximal remaining fluorescence (Fm-Fm’/Fm’). This measure, which occurs over seconds to minutes, provides information on how much of the absorbed light energy is dissipated in a nonradiative manner, and is a process critical for alleviating the absorption of excess excitation energy by plants and algae. This parameter requires the formation of a ΔpH and is sensitive to reagents that dissipate the proton gradient across the thylakoid membranes (e.g. nigericin).
Over longer time periods, state transitions, also designated qT, may represent a component of the quenching; this takes minutes to tens of minutes to occur and involves phosphorylation of the light harvesting antenna polypeptides under conditions that result in reduction of the PQ pool, and the movement of the antenna from PSII to PSI (cells in state 2). Under oxidizing conditions the antenna moves back to PSII (cells in state 1). Briefly, state 1 can be achieved in cultures by strong aeration and maintaining the cells in light that mostly excites PSI. In contrast, state 2 can be achieved in cultures by placing the cells in anaerobic conditions or by including glucose and glucose oxidase in the reaction mixture (which removes O2 from the suspension).
This parameter is also called photoinhibition. The degradation and replacement of damaged D1 polypeptides of PSII has been extensively studied. However, molecular mechanisms underlying this important process have not been elucidated, other than that the FtsH protease is responsible for cleavage of D1. Potential GreenCut regulatory mutants deficient in the D1 repair cycle will be identified by examining responses of PSII to photoinhibitory light treatments, and immunological studies to monitor D1 turnover.
PSI to PSII stoichiometry:
Regulating reaction center stoichiometry in response to light quality is an essential photoacclimation response, with little known about its regulation. Growing mutants under PSI and PSII-specific lights to induce maximum changes in reaction center stoichiometry may identify some strains unable to properly alter this stoichiometry, which would allow for the identification of critical assembly or regulatory elements. Photosystem stoichiometry would be monitored using a number of spectroscopic measurements (e.g. 77 K fluorescence), but would also require immunological quantification of PSI and PSII reaction center proteins (PsaA and PsbA, respectively). We have antiobodies to these polypeptides and to other polypeptides integral to the various complexes of the photosynthetic apparatus (from Agrisera).
Estimation of ΔpH:
This technique can be used to estimate the proton gradient formed across the thylakoid membranes under various conditions of illumination (88). To obtain this value, samples are illuminated for 3 min and absorption changes at 520 nm are immediately monitored after the light is extinguished (absorption changes at 550 nm are also monitored and subtracted as a background). A rapid decline in 520 nm in the dark occurs as a consequence of the dissipation of the proton gradient across the thylakoid membrane. Following this initial decrease, the absorbance increases to some extent for ~1 min establishing a new ‘dark’ absorbance baseline that is lower than the level measured in the light (just prior to light extinction). This 520 nm – 550 nm signal provides a measure of the ΔpH. Related to this measure is DIRK (Dark Interval Relaxation Kinetic) analysis (142). In DIRK analysis, illumination is interspersed with short dark intervals to create perturbations in steady-state electron and proton fluxes. In each dark interval the decay of P515, a signal originating from an electrochromic shift associated mainly with carotenoids and Chl b absorbance, corresponds to the flux of protons and electrons across the thylakoid membranes (efflux via ATP-synthase that in the steady-state equals influx via protolytic electron transport steps). In this analysis, Chlamydomonas would be illuminated for 8 s intervals that are framed by 20 ms of dark; optical changes at 520 nm are monitored during multiple light dark cycles.
Once the assays discussed above are completed, we perform other assays, some that simultaneously measure more than one character of electron transport. Simultaneous assay for PSII and PSI photochemical efficiency using the dual PAM; discrepancies with respect to wild-type behavior may reflect increased PSI cyclic flow or alternative electron flow upstream of PSI. Rapid induction kinetics of PSII and PSI, which can be monitored during brief multiple turnover flashes and provide detailed information on the coupling of electron flow between reaction centers. Cytochrome b6f redox kinetics will provide information on the turnover rate of the cytochrome b6f complex. Plastocyanin redox kinetics or the plastocyanin turnover rate can be compared to the rates of PSI, PSII and cytochrome b6f turnover, to get a better idea of the integration of these activities in mutant strains.
The assays listed above, while numerous, are also robust and relatively rapid. A comparative analysis of all of the data acquired from the above results will indicate a potential block in electron transport, aberrations in ATP synthase function or problems in establishing and/or maintaining a pH gradient across the thylakoid membranes. Simultaneous measurements of electron transport for the two photosystems and components of the intersystem electron transport chain, under different growth conditions, will reveal mutants that are potentially impaired in photoacclimation. Such mutants may also be assembly-deficient in one or more key photosynthetic complexes.