A similar structure of the herbicide binding site in photosystem II of plants and cyanobacteria is demonstrated by site specific mutagenesis of the psbA gene

Many herbicides inhibit the photosynthetic electron transfer in photosystem II by binding to the polypeptide D1. A point mutation in the chloroplast gene psbA, which leads to a change of the amino acid residue 264 of D1 from serine to glycine, is responsible for atrazine resistance in higher plants. We have changed serine 264 to glycine in Synechococcus PCC7942 and compared its phenotype to a mutant with a serine to alanine shift in the same position. The results show that glycine at position 264 in D1 gives rise to a similar phenotype in cyanobacteria and in higher plants, indicating a similar structure of the binding site for herbicides and for the quinone Q_B in the two systems. A possible mode of binding of phenyl-urea herbicides to D1 is predicted from the difference in herbicidal cross-resistance between glycine and alanine substitutions of serine 264.Abbreviations DCPIP 2,6-dichlorophenolindophenol - I₅₀ concentration of herbicide giving 50% inhibition - K_b binding constant - kb kilobase - MES 2(N-morpholino)ethanesulfonic acid - PS II photosystem II     

Genetic analysis of two new mutations resulting in herbicide resistance in the cyanobacterium Synechcoccus sp. PCC 7002

Two herbicide-resistant strains of the cyanobacterium Synechococcus sp. PCC 7002 are compared to the wild-type with respect to the DNA changes which result in herbicide resstance. The mutations have previously been mapped to a region of the cyanobacterial genome which encodes oneof three copies of psbA, the gene which encodes the 32 kDa Q_b-binding protein also known as D1 (Buzby et al. 1987). The DNA sequence of the wild-type gene was first determined and used as a comparison to that of the mutant alleles. A point mutation at codon 211 in the psbA1 coding locus (TTC) to TCC) results in an amino acid change from phenylalanine to serine in the D1 protein. This mutation confers resistance to atrazine and diuron at seven times and at two times the minimal inhibitory concentration (MIC) for the wild-type, respectively. A mutation at codon 211 resulting in herbicide resistance has not previously been described in the literature. A second point mutation at codon 219 in the psbA1 coding locus (GTA to ATA) results in an amino acid change from valine to isoleucine in the D1 protein. This mutation confers resistance to diuron and atrazine at ten times and at two times the MIC for the wild-type, respectively. An identical codon change conferring similar herbicide resistance patterns has previously been described in Chlamydomonas reinhardtii. The atrazine-resistance phenotype in Synechococcus sp. PCC 7002 was shown to be dominant by plasmid segregation analysis.Abbreviations At ^r atrazine resistance - Du ^r diuron resistance - Km ^r kanamycin resistance - Ap ^r ampicillin resistance - MIC Minimum inhibitory concentration     

Over-production of the D1 protein of photosystem II reaction centre in the cyanobacterium Synechococcus sp. PCC 7942

The unicellular cyanobacterium Synechococcus sp. PCC 7942 has three psbA genes encoding two different forms of the photosystem II reaction centre protein D1 (D1:1 and D1:2). The level of expression of these psbA genes and the synthesis of D1:1 and D1:2 are strongly regulated under varying light conditions. In order to better understand the regulatory mechanisms underlying these processes, we have constructed a strain of Synechococcus sp. PCC 7942 capable of over-producing psbA mRNA and D1 protein. In this study, we describe the over-expression of D1:1 using a tac-hybrid promoter in front of the psbAI gene in combination with lacI ^Q repressor system. Over-production of D1:1 was induced by growing cells for 12 h at 50 mol photons m^-2 s^-1 in the presence of 40 or 80 g/ml IPTG. The amount of psbAI mRNA and that of D1:1 protein in cells grown with IPTG was three times and two times higher, respectively. A higher concentration of IPTG (i.e., 150 g/ml) did not further increase the production of the psbAI message or D1:1. The over-production of D1:1 caused a decrease in the level of D1:2 synthesised, resulting in most PSII reaction centres containing D1:1. However, the over-production of D1:1 had no effect on the pigment composition (chlorophyll a or phycocyanin/number of cells) or the light-saturated rate of photosynthesis. This and the fact that the total amounts of D1 and D2 proteins were not affected by IPTG suggest that the number of PSII centres within the membranes remained unchanged. From these results, we conclude that expression of psbAI can be regulated by using the tac promoter and lacI ^Q system. However, the accumulation of D1:1 protein into the membrane is regulated by the number of PSII centres.     

The cyanobacterium Synechococcus modulates Photosystem II function in response to excitation stress through D1 exchange

In this minireview we discuss effects of excitation stress on the molecular organization and function of PS II as induced by high light or low temperature in the cyanobacterium Synechococcus sp. PCC 7942. Synechococcus displays PS II plasticity by transiently replacing the constitutive D1 form (D1:1) with another form (D1:2) upon exposure to excitation stress. The cells thereby counteract photoinhibition by increasing D1 turn over and modulating PS II function. A comparison between the cyanobacterium Synechococcus and plants shows that in cyanobacteria, with their large phycobilisomes, resistance to photoinhibition is mainly through the dynamic properties (D1 turnover and quenching) of the reaction centre. In contrast, plants use antenna quenching in the light-harvesting complex as an important means to protect the reaction center from excessive excitation.Abbreviations D1 reaction center protein of Photosystem II - P₆₈₀ the reaction center of Photosystem II - Q_A the primary quinone acceptor of Photosystem II - Tyr_Z tyrosine electron donor to P₆₈₀     

Interaction of Herbicides and Quinone with the Q(B)-Protein of the Diuron-Resistant Chlamydomonas reinhardtii Mutant Dr2

We have used the diuron-resistant Dr2 mutant of Chlamydomonas reinhardtii which is altered in the 32 kilodalton Q_B-protein at amino acid 219 (valine to isoleucine), to investigate the interactions of herbicides and plastoquinone with the 32 kilodalton Q_B-protein. The data contained in this report demonstrate that the effects of this mutation are different from those of the more completely characterized mutant which confers extreme resistance to triazines in higher plants. The mutation in C. reinhardtii Dr2 confers only slight resistance to a number of inhibitors of photosynthetic electron transport. Extreme triazine resistance results from an increase in the binding constant of the herbicide with the 32 kilodalton Q_B-protein, in contrast the diuron binding constant for chloroplasts isolated from wild-type (sensitive) Chlamydomonas and the resistant Dr2 are indistinguishable. We conclude that the altered structure in the 32 kilodalton Q_B-protein of Dr2 does not directly affect the diuron binding site. This mutation appears to alter the steric properties of the binding protein in such a way that diuron and plastoquinone do not directly compete for binding. This steric perturbation confers mild resistance to other herbicidal inhibitors of photosynthesis and alters the kinetics of Q_A to Q_B electron transfer.     

Characterization of the single psbA gene of Prochlorococcus marinus CCMP 1375 (Prochlorophyta)

DNA sequence, copy number, expression and phylogenetic relevance of the psbA gene from the abundant marine prokaryote P. marinus CCMP 1375 was analyzed. The 7 amino acids near the C-terminus missing in higher plant and in Prochlorothrix hollandica D1 proteins are present in the derived amino acid sequence. P. marinus contains only a single psbA gene. Thus, this organism lacks the ability to adapt its photosystem II by replacement of one type of D1 by another, as several cyanobacteria do. Phylogenetic trees suggested the D1-1 iso-form from Synechococcus PCC 7942 as the next related D1 protein and place P. Marinus separately from Prochlorothrix hollandica among the cyanobacteria.     

The carboxy-terminal extension of the D1-precursor protein is dispensable for a functional photosystem II complex in Chlamydomonas reinhardtii

The D1-precursor protein of the photosystem II reaction centre contains a carboxy-terminal extension whose proteolytic removal is necessary for oxygen-evolving activity. To address the question of the role of the carboxy-terminal extension in the green alga Chlamydomonas reinhardtii, we truncated D1 by converting codon Ser345 of the psbA gene into a stop codon. Particle gun transformation of an in vitro modified psbA gene fragment also carrying mutations conferring herbicide resistance yielded a homoplasmic transformant containing the stop codon. Since oxygen evolution capacity is not affected in this mutant as compared with herbicide-resistant control cells, the carboxy-terminal extension is dispensable for a functional photosystem II complex under normal growth conditions.     

Transcription of a “silent” cyanobacterial psbA gene is induced by microaerobic conditions

Cyanobacteria, contrary to higher plants, have a small psbA gene family encoding the reaction centre D1 protein subunit of photosystem II, the first macromolecular pigment-protein complex of the photosynthetic electron transport chain. Modulation of expression of multiple psbA genes in the family allows cyanobacteria to adapt to changing environmental conditions. To date, two different strategies for regulation of the psbA genes have emerged. One, characterized in Synechocystis PCC6803 and Gloeobacter violaceus PCC7421 involves the increased expression of one type of D1 protein to cope with the increased rate of damage. The other strategy, in Synechococcus PCC7942 and Anabaena PCC7120, is to replace the existing D1 with a new D1 form for the duration of the stress. However, most of the psbA gene families characterized to date contain also a divergent, apparently silent psbA gene of unknown function. This gene, present in Synechocystis, Anabaena and Thermosynechococcus elongatus BP-1 was not induced by any stress condition applied so far. Our data shows a reversible induction of the divergent psbA gene during the onset of argon-induced microaerobic conditions in Synechocystis, Anabaena and Thermosynechococcus elongatus. The unitary functional response of three unrelated cyanobacterial species, namely the induction of the expression of the divergent psbA gene as a reaction to the same environmental cue, indicates that these genes and the protein they encode are part of a specific cellular response to microaerobic conditions. There are no specific primary structure similarities between the different microaerobic inducible D1 forms, designated as D1′. Only three amino acid residues are consistently conserved in D1′. These modifications are: G80 to A, F158 to L and T286 to L. In silico mutation of the published D1 structure from Thermosynechococcus did not reveal major modifications. The point by point effects of the mutations on the local environment of the PSII structure are also discussed.     

Construction of an Obligate Photoheterotrophic Mutant of the Cyanobacterium Synechocystis 6803 : Inactivation of the psbA Gene Family

psbA in Synechocystis 6803 was found to belong to a small multigene family with three copies. The psbA gene family was inactivated in vitro by insertation of bacterial drug resistance markers. Inactivation of all three genes resulted in a transformant that is unable to grow photosynthetically but can be cultured photoheterotrophically. This mutant lacks oxygen evolving capacity but retains photosystem I activity. Room temperature measurements of chlorophyll a fluorescence induction demonstrated that the transformant exhibits a high fluorescence yield with little or no variable fluorescence. Immunoblot analyses showed complete loss of the psbA gene product (the DI polypeptide) from thylakoid membranes in the transformant. However, the extrinsic 33 kilodalton polypeptide of the water-splitting complex of photosystem II, is still present. The results indicate that assembly of a partial photosystem II complex may occur even in the absence of the intrinsic D1 polypeptide, a protein implicated as a crucial component of the photosystem II reaction center.     

The IdiA protein of Synechococcus sp. PCC 7942 functions in protecting the acceptor side of Photosystem II under oxidative stress

Synechococcus sp. strains PCC 7942 and PCC 6301 contain a 35 kDa protein called IdiA (Iron deficiency induced protein A) that is expressed in elevated amounts under Fe deficiency and to a smaller extent also under Mn deficiency. Absence of this protein was shown to mainly damage Photosystem II. To decide whether IdiA has a function in optimizing and/or protecting preferentially either the donor or acceptor side reaction of Photosystem II, a comparative analysis was performed of Synechococcus sp. PCC 7942 wild-type, the IdiA-free mutant, the previously constructed PsbO-free Synechococcus PCC 7942 mutant and a newly constructed Synechococcus PCC 7942 double mutant lacking both PsbO and IdiA. Measurements of the chlorophyll fluorescence and determinations of Photosystem II activity using a variety of electron acceptors gave evidence that IdiA has its main function in protecting the acceptor side of Photosystem II. Especially, the use of dichlorobenzoquinone, preferentially accepting electrons from Q_A, gave a decreased O₂ evolving activity in the IdiA-free mutant. Investigations of the influence of hydrogen peroxide treatment on cells revealed that this treatment caused a significantly higher damage of Photosystem II in the IdiA-free mutant than in wild-type. These results suggest that although the IdiA protein is not absolutely required for Photosystem II activity in Synechococcus PCC 7942, it does play an important role in protecting the acceptor side against oxidative damage. This revised version was published online in June 2006 with corrections to the Cover Date.     

A Ser–Gly substitution in plastid-encoded photosystem II D1 protein is responsible for atrazine resistance in foxtail millet (<Emphasis Type="Italic">Setaria italica</Emphasis>)

A foxtail millet (Setaria italica L. Beauv.) line resistant to atrazine was obtained through interspecific hybridization between wild S. viridis L. Beauv. and cultivated S. italica. The resistance was proved to be controlled by a chloroplast-inherited gene and it has further been utilized in foxtail millet production. However, the sequence information of the putative atrazine resistance gene, psbA in foxtail millet’s chloroplast genome encoding photosystem II D1 protein (32 kDa thylakoid membrane protein) (photosystem Q_B protein) and the mutation site responsible for the resistance are not known. In this paper the psbA sequences of six atrazine susceptible/resistant foxtail millet varieties were obtained and compared. The results indicated that there was only one amino acid difference between susceptible and resistance gene, resulting from a single base substitution. It was concluded that a mutant allele of photosystem II protein D1 encoding a Gly residue instead of a Ser residue at position 264 is a major gene of resistance to atrazine. Moreover, the phylogenetic tree based on the psbA coding region of thirty-five plant species was carried out. The phylogenetic relationship between S. italica and other plants and the related evolutionary issues were discussed and it was suggested that psbA sequences could be used in phylogenetic studies in plants. Xiaoping Jia and Jincheng Yuan have equal contribution.     

Over-production of the D1:2 protein makes Synechococcus cells more tolerant to photoinhibition of photosystem II

Over-expression of the psbAIII gene encoding for the D1 protein (form II; D1:2) of the photosystem II reaction centre in the Synechococcus sp. PCC 7942 was studied using a tac promoter and the lacI ^Q system. Over-expression was induced with 40 g/ml IPTG in the growth medium for either 6 or 12 h at growth irradiance (50 mol photons m^-2 s^-1). This treatment doubled the amount of psbAII/III mRNA and the D1:2 protein in membranes but decreased the amount of psbAI messages and the D1:1 protein. The total amount of both heterodimeric reaction centre proteins, D1 and D2, remained constant under growth light conditions, indicating that the number of PSII centres in the membranes was not affected, only the form of the D1 protein was changed from D1:1 to D1:2 in most centres. When the cells were photoinhibited either at 500 or 1000 mol photons m^-2 s^-1, in the presence or absence of the protein synthesis inhibitor lincomycin, the D1:2 protein remained at a higher level in cells in which over-expression had been induced by IPTG. These cells were also less prone to photoinhibition of PSII. It is suggested that the tolerance of cells to photoinhibition increases when most PSII reaction centres contain the D1:2 protein at the beginning of high irradiance. This tolerance is further strengthened by maintaining psbAIII gene over-expression during the photoinhibitory treatment.     

Herbicide binding and thermal stability of photosystem II isolated from Thermosynechococcus elongatus

Binding of herbicides to photosystem II inhibits the electron transfer from Q_A to Q_B due to competition of herbicides with plastoquinone bound at the Q_B site. We investigated herbicide binding to monomeric and dimeric photosystem II core complexes (PSIIcc) isolated from Thermosynechococcus elongatus by a combination of different methods (isothermal titration and differential scanning calorimetry, CD spectroscopy and measurements of the oxygen evolution) yielding binding constants, enthalpies and stoichiometries for various herbicides as well as information regarding stabilization/destabilization of the complex. Herbicide binding to detergent-solubilized PSIIcc can be described by a model of single independent binding sites present on this important membrane protein. Interestingly, binding stoichiometries herbicide:PSIIcc are lower than 1:1 and vary depending on the herbicide under study. Strong binding herbicides such as terbutryn stabilize PSIIcc in thermal unfolding experiments and endothermically binding herbicides like ioxynil probably cause large structural changes accompanied with the binding process as shown by differential scanning calorimetry experiments of the unfolding reaction of PSIIcc monomer in the presence of ioxynil. In addition we studied the occupancy of the Q_B sites with plastoquinone (PQ9) by measuring flash induced fluorescence relaxation yielding a possible explanation for the deviations of herbicide binding from a 1:1 herbicide/binding site model.     

Form II of D1 is important during transition from standard to high light intensity in Synechococcus sp. strain PCC 7942

In Synechococcus sp. strain PCC 7942 the D1 protein of Photosystem II is encoded by a multigene family; psbAI encodes Form I of D1 whereas both psbAII and psbAIII encode Form II. The psbA genes are differentially regulated in response to changes in light intensity, such that psbAI expression and Form I predominate at standard light intensity, whereas psbAII and psbAIII are induced at high light intensity, causing insertion of Form II into the thylakoids. The present study addressed whether high-light induced Form II is important for Synechococcus cells during adaptation to high light intensity. Wild-type Synechococcus, and mutants which produce only Form I (R2S2C3) or only Form II (R2K1), were co-cultured at standard light (130 E · m^–2 · s^–1) and then shifted to high light (750 E·m^–2·s^–1). Measurement of the proportion of each cell type at various time intervals revealed that the growth of R2S2C3, which has psbAII and psbAIII inactive, and thus lacks Form II, is transiently impaired upon shift to high light. Both mutants R2S2C3 and R2K1 maintained normal levels of psbA messages and D1 protein under standard and high light through an unknown mechanism that compensates for the inactive psbA genes. Thus, the impairment of R2S2C3 at high light is not due to a deficiency of D1 protein, but results from lack of Form II. We discounted the influence of possible secondary mutations by re-creating the psbA-inactivated mutants and testing the newly isolated strains. We conclude that Form II of D1 is intrinsically important for Synechococcus cells during a critical transition period after exposure to high light intensities.     

Two forms of the Photosystem II D1 protein alter energy dissipation and state transitions in the cyanobacterium Synechococcus sp. PCC 7942

Synechococcus sp. PCC 7942 (Anacystis nidulans R2) contains two forms of the Photosystem II reaction centre protein D1, which differ in 25 of 360 amino acids. D1: 1 predominates under low light but is transiently replaced by D1:2 upon shifts to higher light. Mutant cells containing only D1:1 have lower photochemical energy capture efficiency and decreased resistance to photoinhibition, compared to cells containing D1:2. We show that when dark-adapted or under low to moderate light, cells with D1:1 have higher non-photochemical quenching of PS II fluorescence (higher q_N) than do cells with D1:2. This is reflected in the 77 K chlorophyll emission spectra, with lower Photosystem II fluorescence at 697–698 nm in cells containing D1:1 than in cells with D1:2. This difference in quenching of Photosystem II fluorescence occurs upon excitation of both chlorophyll at 435 nm and phycobilisomes at 570 nm. Measurement of time-resolved room temperature fluorescence shows that Photosystem II fluorescence related to charge stabilization is quenched more rapidly in cells containing D1:1 than in those with D1:2. Cells containing D1:1 appear generally shifted towards State II, with PS II down-regulated, while cells with D1:2 tend towards State I. In these cyanobacteria electron transport away from PS II remains non-saturated even under photoinhibitory levels of light. Therefore, the higher activity of D1:2 Photosystem II centres may allow more rapid photochemical dissipation of excess energy into the electron transport chain. D1:1 confers capacity for extreme State II which may be of benefit under low and variable light.Abbreviations D1 the atrazine-binding 32 kDa protein of the PS II reaction centre core - D1:1 the D1 protein constitutively expressed during acclimated growth in Synechococcus sp. PCC 7942 - D1:2 an alternate form of the D1 protein induced under excess excitation in Synechococcus sp. PCC 7942 - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - Fo minimal fluorescence in the dark-adapted state - Fo minimal fluorescence in a light-adapted state - F_M maximum fluorescence with all quenching mechanisms at a minimum, measured in presence of DCMU - F_M maximal fluorescence in a light-adapted state, measured with a saturating flash - F_Mdark maximal fluorescence in the dark-adapted state - F_V variable fluorescence in a light-adapted state (F_M-Fo) - PAM pulse amplitude modulated fluorometer - q_N non-photochemical quenching of PS II fluorescence - q_N (dark) q_N in the dark adapted state - q_P photochemical quenching of fluorescence     

Effects of Photosystem II Herbicides on the Photosynthetic Membranes of the Cyanobacterium Aphanocapsa 6308

The effects of the photosystem II herbicides diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) on the photosynthetic membranes of a cyanobacterium, Aphanocapsa 6308, were compared to the effects on a higher plant, Spinacia oleracea. The inhibition of photosystem II electron transport by these herbicides was investigated by measuring the photoreduction of the dye 2,6-dichlorophenol-indophenol spectrophotometrically using isolated membranes. The concentration of herbicide that caused 50% inhibition of electron transport (I₅₀ value) in Aphanocapsa membranes for diuron was 6.8 × 10⁻⁹ molar and the I₅₀ value for atrazine was 8.8 × 10⁻⁸ molar. ¹⁴C-labeled diuron and atrazine were used to investigate herbicide binding with calculated binding constants (K) being 8.2 × 10⁻⁸ molar for atrazine and 1.7 × 10⁻⁷ molar for diuron. Competitive binding studies carried out on Aphanocapsa membranes using radiolabeled [¹⁴C]atrazine and unlabeled diuron revealed that diuron competed with atrazine for the herbicide-binding site. Experiments involving the photoaffinity label [¹⁴C]azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-2-triazine) and autoradiography of polyacrylamide gels indicated that the herbicide atrazine binds to a 32-kilodalton protein in Aphanocapsa 6308 cell extracts.     

Energetics in Photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA1 or psbA3 gene

The main cofactors involved in the function of Photosystem II (PSII) are borne by the D1 and D2 proteins. In some cyanobacteria, the D1 protein is encoded by different psbA genes. In Thermosynechococcus elongatus the amino acid sequence deduced from the psbA₃ gene compared to that deduced from the psbA₁ gene points a difference of 21 residues. In this work, PSII isolated from a wild type T. elongatus strain expressing PsbA1 or from a strain in which both the psbA₁ and psbA₂ genes have been deleted were studied by a range of spectroscopies in the absence or the presence of either a urea type herbicide, DCMU, or a phenolic type herbicide, bromoxynil. Spectro-electrochemical measurements show that the redox potential of Pheo_D1 is increased by 17 mV from −522 mV in PsbA1-PSII to −505 mV in PsbA3-PSII. This increase is about half that found upon the D1-Q130E single site directed mutagenesis in Synechocystis PCC 6803. This suggests that the effects of the D1-Q130E substitution are, at least partly, compensated for by some of the additional amino-acid changes associated with the PsbA3 for PsbA1 substitution. The thermoluminescence from the S₂Q_A^−• charge recombination and the C ≡ N vibrational modes of bromoxynil detected in the non-heme iron FTIR difference spectra support two binding sites (or one site with two conformations) for bromoxynil in PsbA3-PSII instead of one in PsbA1-PSII which suggests differences in the Q_B pocket. The temperature dependences of the S₂Q_A^−• charge recombination show that the strength of the H-bond to Pheo_D1 is not the only functionally relevant difference between the PsbA3-PSII and PsbA1-PSII and that the environment of Q_A (and, as a consequence, its redox potential) is modified as well. The electron transfer rate between P₆₈₀^+• and Y_Z is found faster in PsbA3 than in PsbA1 which suggests that the redox potential of the P₆₈₀/P₆₈₀^+• couple (and hence that of ¹P₆₈₀^*/P₆₈₀^+•) is tuned as well when shifting from PsbA1 to PsbA3. In addition to D1-Q130E, the non-conservative amongst the 21 amino acid substitutions, D1-S270A and D1-S153A, are proposed to be involved in some of the observed changes.     

Determination of the Rate Limiting Step for Photosynthesis in a Nearly Isonuclear Rapeseed (Brassica napus L.) Biotype Resistant to Atrazine

Plant biotypes that are resistant to S-triazines under most conditions often grow less vigorously and have lower quantum yields and lower maximum rates of photosynthesis. The photosynthetic reactions responsible for these effects were identified in whole leaves and thylakoids of nearly isonuclear lines of oilseed rape (Brassica napus L.). The lower quantum yield was a result of poor efficiency in the use of separated charge at the photosystem II reaction center. Charge separation occurred normally, but over 30% of the charges recombined instead of being used for oxygen evolution and for reduction capacity in photosystem I. The lower maximum rate of photosynthesis in the resistant biotype was set by the transfer of electrons between the primary, Q_A, and secondary, Q_B, acceptors of photosystem II. This charge transfer reaction became rate limiting in resistant biotypes. The decreased quantum yield and decreased maximum rate of photosynthesis are both believed to be consequences of changes in the 32 kilodalton herbicide binding protein. As such, it is likely that these traits will not be genetically separable.