Plant Physiol. (1993) 103: 911-917

A Serine-to-Threonine Substitution in the Triazine Herbicide-Binding Protein in Potato Cells Results in Atrazine Resistance without lmpairing Productivity’

Reid j. Smeda’, Paul M. Hasegawa, Peter B. Goldsbrough, Narendra K. Singh3, and Stephen C. Weller*

Department of Horticulture, Purdue University, West Lafayette, indiana 47907-1 165

A mutation of the psbA gene was identified in photoautotrophic potato (Solanum tuberosum 1. cv Superior x U.S. Department of Agriculture line 66-142) cells selected for resistance to 6-chloro- N-ethyl-N‘-(l-methylethyl)-l,3,5-triazine-2,4-diamine (atrazine). Photoaffinity labeling with 6-azido-N-ethyl-N’-(l-methyIethyl)- 1,3,5-triazine-2,4-diamine detected a thylakoid membrane protein with a M. of 32,000 in susceptible, but not in resistant, cells. This protein was identified as the secondary quinone acceptor of pho- tosystem II (QB) protein. Atrazine resistance in selected cells was attributable to a mutation from ACT (serine) to ACT (threonine) in codon 264 of the psbA gene that encodes the Q. protein. Although the mutant cells exhibited extreme levels of resistance to atrazine, no concomitant reductions in photosynthetic electron transport or cell growth rates compared to the unselected cells were detected. This is in contrast with the losses in productivity observed in atrazine-resistant mutants that contain a glycine-264 alteration.

Prolonged agronomic use of PSII inhibitors, specifically s- triazine herbicides, has resulted in the evolution of resistant weed biotypes. The predominant basis for resistance is a single nucleotide substitution in the chloroplast psbA gene (Mazur and Falco, 1989), which encodes the D1 thylakoid protein (termed the Q B protein) and is the target molecule for s-triazine binding. In a11 resistant weed biotypes examined to date, the specific mutation is a G for A substitution that results in a change from Ser to Gly in position 264 of the mature QB protein (Blyden and Gray, 1986; Bettini et al., 1987; Barros and Dyer, 1988; Mazur and Falco, 1989). This mutation effectively eliminates s-triazine binding affinity to the protein.

s-Triazine resistance has been stably transferred from weed to crop species by both interspecific (Ayotte et al., 1987) and somatic cell hybridization (Binding et al., 1982). Transgenic plants that express a nuclear gene encoding a modified Q B

protein with a single peptide for chloroplast targeting of the protein also exhibited increased atrazine tolerance (Cheung et al., 1988). Incorporation of s-triazine resistance into crops

Joumal paper No. 12729 of the Purdue University Agricultura1 Experiment Station.

Present address: Southem Weed Science Laboratory, U.S. De- partment of Agriculture-Agricultura1 Research Service, P.O. Box 350, Stoneville, MS 38776.

Present address: Department of Botany and Microbiology, Au- bum University, Aubum, AL 36849-5319.

* Corresponding author; fax 1-317-494-0391. 91 1

will provide growers new options for designing weed man- agement strategies (Duke et al., 1991). This will facilitate the development of cultural practices that reduce the continuous use of specific herbicides, thereby reducing the selection pressure for herbicide-resistant weeds (Gressel and Segel, 1982). Such a strategy is most likely effective when the rotation involves herbicides with different mechanisms of action.

Development of agriculturally acceptable triazine-resistant crops has been thwarted by the reduced photosynthetic ef- ficiency (Ort et al., 1983; Holt, 1988; Jursinic and Pearcy, 1988) and concomitant reduced plant productivity (Ahrens and Stoller, 1983; Gressel and Ben-Sinai, 1985; Holt, 1988; Jacobs et al., 1988) that accompanies an altered QB protein. The substitution of Ser for Gly at position 264 in the QB protein results in slower electron transfer from QA to QB

(Bowers et al., 1980; Jursinic and Pearcy, 1988; Stowe and Holt, 1988). Amino acid substitutions at other positions in Q B have not been observed to impart triazine resistance in higher plants. In algae (Galloway and Mets, 1984) and cy- anobacteria (Brown et al., 1988), substitutions at positions other than amino acid 264 have resulted in herbicide resist- ance but have not been associated with reduced photosyn- thetic productivity. The development of s-triazine-resistant crops for agriculture will not proceed until mutations are identified that do not impair productivity in higher plants.

In this paper we report the selection of photoautotrophic potato cells that are resistant to atrazine. The basis for resist- ance is a unique mutation in the psbA gene resulting in a Ser- to-Thr substitution at position 264 of the Q B protein. The resistant cells do not exhibit reduced photosynthetic activityl capacity and cell growth, indicating that this mutation may not adversely affect crop yield potential.

MATERIALS AND METHODS

Chemicals

Atrazine (technical grade, 97.2% purity) was suspended in water or DMSO as a concentrated stock solution and filter sterilized through a 0.2-pm 115-mL filter (Nalge Co., Roch-

~~ ~

Abbreviations: atrazine, 6-chloro-N-ethyl-N’-(l-methylethyl)- 1,3,5-triazine-2,4-diamine; [azido-“Clatrazine, 6-azido-N-ethyl- N’-(l-methylethyl)-1,3,5-triazine-2,4-diamine; PCR, polymerase chain reaction; QA, primary quinone acceptor of photosystem 11; QB, secondary quinone acceptor of photosystem 11.

91 2 Smeda et ai. Plant Physiol. Vol. 103,, 1993

ester, NY) before herbicide addition into autoclave-sterile culture medium. Uniformly ring-labeled [a~ido-'~C]atrazine (Sigma; specific activity 13.2 mCi mmol-') was suspended in methylene chloride. In a11 experiments, the final DMSO and methylene chloride concentrations in culture media did not exceed 0.2 or 0.06% (v/v), respectively, and these con- centrations were not detrimental to cell growth. Atrazine was a kind gift of Dr. H.M. LeBaron (Ciba-Geigy Corp., Greensboro, NC).

Culture Mainteinance and Atrazine Resistance

Cell suspensions of potato were initiated from callus (orig- inating from perimedullary tuber tissue) and induced to grow in a medium without supplemental carbohydrates (photoau- totrophically) as described by LaRosa et al. (1984). Cells were maintained on a Murashige and Skoog salts medium as modified (LaRosa et al., 1984) and grown under conditions described by LaRosa et al. (1984) with the following excep- tions. Supplemental lighting was supplied at a PPFD of 200 to 215 pmol m-' s-' of PAR from cool-white fluorescent lamps, with a daily 16-h light period at 29OC and an 8-h dark period at 26OC. Humidified air enriched with COz (2.0 k 0.1%) was flushed through a plexiglass chamber that housed the cells. Measurements of the COz concentration in the head space of a typical flask indicated a concentra- tion of approximately 1.5%. Cell suspensions utilized for growth, electron transport, and [a~ido-'~C]atrazine photo- affinity labeling experiments were taken from cultures in the midlinear phase of growth.

Atrazine-resistant cells were isolated following a stepwise adaptation of susceptible cells (wild type) to increasing con- centrations of atrazine (R.J. Smeda, P.M. Hasegawa, and S.C. Weller, unpublished data). Resistant cells, hereafter termed variant cells, were routinely maintained on 1.0 p~ atrazine, a concentration lethal to wild-type cells. A11 variant cells utilized in experiments reported here were those maintained on 1.0 p~ atrazine for at least 21 months.

Analysis of Growth

Variant and wild-type cells were inoculated into growth medium (at an inoculation density of 20 mg fresh weight mL-') in the absence of atrazine. Cultures were sampled every 3 to 5 d, and cell fresh weight densities were deter- mined after cells were collected on Whatman No. 4 filter paper by vacuum filtration. Cell dry weights were recorded after 48 h at 5OOC. Each treatment was duplicated, and the experiment was repeated.

lsolation of Stroma-Free Chloroplast Thylakoid Membranes

Intact chloroplasts were isolated from 20 to 30 g fresh weight of cells by homogenization in 200 mL of grinding buffer containing 300 m NaCI, 30 n w Tricine (pH 7.8), 3 mM MgC12, and 0.5 m EDTA. The homogenate was filtered through 10 layers of cheesecloth, and the filtrate was centri- fuged for 1 min at 300g. The supematant was further centri- fuged at 24008 for 4 min, and the pellet was resuspended in

10 ml- of 10 m Tricine (pH 7.8) and 10 mM NaCl to nipture the chloroplasts. Stroma-free thylakoids were then pelleted at 24009 for 4 min and resuspended to 0.8 to 1.0 mg of Chl mL-' in a medium containing 200 m SUC, 5 mM Hepes (pH 7.5), 2 mM MgC12, 0.5 mg mL-' of BSA, and 5 mM sodium ascorbate.

Photosynthetic Electron Transport Assays

Hill reaction activity in the absence or presence of atrazine was measured for both intact cells and isolated thylakoid membranes with the Chl concentration for each calciilated using the equations of Amon (1949). Wild-type and variant cells in the midlinear phase of growth (approximately 125 mg fresh weight mL-' of growth medium) were resuspended to a fresh weight density of 30 mg mL-' of growth medium (total Chl concentrations of 9.5-13.4 pg mL-'). A Clark-type oxygen electrode (Yellow Springs Instruments, Yellow Springs, OH) was used to measure Hill reaction activity of continuously stirred cells, illuminated with 225 pE m-' s-' of PAR in a water-jacketed reaction vessel at 29OC. Hill reiiction activity was similarly measured for isolated thylakoid mem- branes suspended in reaction medium (40 or 60 pg of Chl mL-') containing 100 m SUC, 20 mM Tricine (pH 7.8), 5 mM MgCI2, and potassium femcyanide as the electron acce:pter.

Photoaffinity Labeling with [a~ido-'~C]Atrazine

Conjugation of [a~ido-'~C]atrazine to proteins in isolated thylakoid membranes was camed out under the following conditions. In Iow light, [a~ido-'~C]atrazine (0.3 PM final concentration) was added to reaction medium (identical with electron transport assay medium). Thylakoid membranes (100 pg of Chl) isolated from wild-type and variant cells were incubated for 5 min in labeled reaction medium and irradiated in a UV Stratalinker (Stratagene) for 8 min (254 nm). Mem- branes were then centrifuged at 24008 for 4 min, walshed with unlabeled reaction medium, and solubilized in an (:qual volume of 1.0 M Tris (pH 6.8), 20% (v/v) glycerol, 20% (w/v) SDS, 10% (v/v) 2-mercaptoethanol, and 0.1% (w/v) bromphenol blue for 4 h at room temperature.

PAGE

Analysis of thylakoid membrane polypeptides by SDS- PAGE was camed out using the discontinuous buffer system of Laemmli (1970). Electrophoresis was performed in a slab gel apparatus using a 12% (w/v) linear polyacrylamidle gel and a 4% (w/v) stacking gel. Protein concentration in each sample was estimated using the Bradford (1976) method. Prestained mo1 wt standards (Bio-Rad) were used to deter- mine thylakoid protein M,. Following electrophoresis, pro- teins were stained with Coomassie brilliant blue, or [a~ido-'~C]atrazine protein complexes were identified after fluorography using a fluor enhancer (Resolution; EM Corp., Chestnut Hill, MA) and exposure of dried gels with Kodak AR-5 x-ray film for 75 d at -8OOC.

lmmunoblot of the Qe Protein

[a~ido-'~C]Atrazine-labeled thylakoid membrane polypep- tides (40 pg) were separated by SDS-PAGE as described

Atrazine Resistance in Photoautotrophic Potato Cells 913

O

16

above and transferred electrophoretically onto nitrocellulose paper (0.45 pm; Schleicher and Schuell) using a semidry electroblotter according to the manufacturer's instructions. Transferred proteins were blocked for 30 min in 20 m Tris (pH 7.5), 500 mM NaC1, and 3% (w/v) milk powder, washed repeatedly in TTBS solution (20 m Tris [pH 7.51, 500 mM NaC1, and 0.15% [v/v] Tween-20), and then incubated for 2 h with polyclonal antiserum ( 1 : l O O O dilution) to the M, 32,000 Q B protein in TTBS solution containing 1% (w/v) milk powder. The blot was washed and incubated for 1 h with goat anti-rabbit immunoglobulin G alkaline phosphatase conjugate (Bio-Rad) in TTBS solution containing 1% (w/v) milk powder. The immunoreactive proteins were visualized in alkaline phosphatase buffer (100 m~ Tris-HC1 [pH 9.51, 100 mM NaC1, 5 mM MgC12) using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as chromogenic sub- strates. The blot was then exposed to Kodak AR-5 x-ray film for 180 d at -8OOC to identify the protein labeled with [a~ido-'~C]atrazine. Antiserum for the M, 32,000 Q B protein was kindly provided by Dr. A.K. Mattoo (U.S. Department of Agriculture-Agricultura1 Research Service, Beltsville, MD).

- ~~ .

B -

Amplification of ctDNA and DNA Sequencing

A portion of the psbA gene (target ctDNA) encoding the proposed atrazine-binding niche on the Q B protein, as sug- gested by Trebst (1987), was amplified by PCR. The se- quences of the oligonucleotide primers (19-mers) used in the amplification process were designed to compliment psbA DNA 57 nucleotides upstream and 174 nucleotides down- stream of the DNA targeted for sequencing. The ctDNAs prepared from variant and wild-type cells were used as templates in PCR. The amplified products of approximately 474 bp were purified by agarose gel electrophoresis, eluted, and cloned (Sambrook et al., 1989) into the SmaI site of pBluescript (Stratagene). The DNA sequences of the cloned ctDNA fragments were detennined (Sanger et al., 1977). DNA primers used in the sequencing reaction included plas- mid primers (Stratagene) as well as oligonucleotide primers used for PCR. Four independent subclones of the variant ctDNA insert and three subclones of the wild-type ctDNA were sequenced to confirm any molecular changes found.

RESULTS

Cell Growth

The rate of dry and fresh weight accumulation for wild- type and variant cells in the absence of atrazine was identical for the first 30 d after inoculation (Fig. 1) and did not differ significantly thereafter. Continued maintenance of variant cells for up to 30 generations in suspension medium lacking atrazine resulted in no appreciable loss of cell resistance to atrazine (R.J. Smeda, P.M. Hasegawa, and S.C. Weller, un- published data).

Photosynthetic Electron Transport

Addition of 1.0 p~ atrazine to wild-type cells resulted in a rapid inhibition of photosynthetic electron transport (Fig. 2). However, this concentration had almost no effect on the

O Wild Type "' lo0 75 t 5o 25 w 14 - 12 - 10 L

a - 6 -

4 -

2 -

O 5 10 15 20 25 30 35 40 45 50 55 60 Doys After lnoculotion

Figure 1. Fresh (A) and dry (6) weight accumulation of wild-type and variant potato cells in growth medium lacking atrazine. Each point is the mean of four observations, and vertical bars represent the SE.

variant cells. The concentration of herbicide required to in- hibit photosynthetic electron transport by 50% was approxi- mately 250-fold greater for variant (103.5 p ~ ) than wild-type (0.4 p ~ ) cells. In the absence of atrazine, electron transport rates were approximately 12% greater for variant than for wild-type cells (141.6 versus 126.6 pmol of O2 mg-' of Chl h-').

Photosynthetic electron transport of thylakoid membranes isolated from wild-type cells was also more sensitive to inhibition by atrazine (Fig. 3). The reduction of electron transport by atrazine was not as pronounced with isolated thylakoid membranes compared to cells. The concentration of herbicide required to inhibit photosynthetic electron trans- port by 50% was 65-fold greater for thylakoid membranes isolated from variant (44.4 p ~ ) compared to wild-type (0.7 p ~ ) cells. Electron transport rates of thylakoid mem- branes were 25% higher for variant than for wild-type cells (143.3 and 114.4 pmol of O2 mg-' of Chl h-', respectively), indicating that atrazine resistance was not associated with a reduction in photosynthetic electron transport efficiency.

Photoaffinity Labeling with [a~ido-'~C]Atrazine

[a~ido-'~C]Atrazine is an analog of atrazine that can also bind to the active site on the Q B polypeptide and becomes covalently linked to the M, 32,000 to 34,000 Q B chloroplast membrane polypeptide following UV irradiation (Pfister et al., 1981). [a~ido-'~C]Atrazine specifically photoaffinity la- beled a peptide of M, 32,000 from thylakoid membranes of

914 Smeda et al. Plant Physiol. Vol. 103, 1993

zo

o

LUQL

0.01 1.0

[ATRAZINE]

10.0 100.0

Figure 2. Atrazine inhibition of photosynthetic electron transportin wild-type and variant potato cells. The rate of electron transportwas measured within 90 s of herbicide addition. Each point is themean of four observations, and vertical bars represent the SE. Ratesof oxygen evolution for wild-type and variant cells in mediumlacking atrazine were 126.6 and 141.1 jimo! of O2 mg~' of Chl h~',respectively.

wild-type but not of variant cells (Fig. 4). No apparentdifference in the amount of protein loaded onto the gels wasevident, nor was there any visible evidence for a differencein the amount of Mr 32,000 protein in the cells. This resultsuggests that the QB protein in variant cells can no longerbind atrazine.

Western Blot of the QB Protein

Abolition of atrazine binding to the QB protein from thy-lakoids of variant cells was inferred from the absence of an[azido-14C]atrazine-labeled protein; however, confirmationthat the QB protein was present in membrane preparations

and selectively labeled by [azido-uC]atrazine was demon-strated immunologically (Fig. 5). Antiserum to the Mr 32,000QB protein recognized a protein of identical electrophoreticmobility in thylakoid membranes from both types of cells(Fig. 5A). This protein comigrated with the protein labeledby [azido-14C]atrazine in wild-type thylakoids (Fig. 5B).

Sequence Analysis of Chloroplast psbA GeneAll reported mutations resulting in atrazine resistance in

all organisms have occurred in a 168-bp region of the psbAgene (Mazur and Falco, 1989). A 474-bp fragment of thepsbA gene was cloned from both wild-type and variant cells(Fig. 6). DNA sequence analysis indicated a single nucleotidedifference between the psbA genes of wild-type and variantcells (Fig. 6). This mutation changed the codon from ACT inthe wild type to ACT in the variant, resulting in a Ser-to-Thrsubstitution. These results were confirmed from the sequenceof three and four independent psbA gene fragments fromwild-type and variant cells, respectively.

DISCUSSION

The high level of atrazine resistance exhibited by culturedvariant potato cells (Figs. 2 and 3) is due to a single aminoacid substitution from Ser to Thr at position 264 in the matureQB protein (Fig. 6). The same substitution has been isolatedin atrazine-resistant tobacco cells grown in vitro (Sato et al.,1988). In other atrazine-resistant higher plants that have beenexamined, the Ser at position 264 is altered to either Gly(Blyden and Gray, 1986; Betrini et al., 1987; Barros and Dyer,1988; Mazur and Falco, 1989) or Asn (Pay et al., 1988).Definitive confirmation that point mutations to the 264thcodon in the psbA gene result in atrazine resistance has beenprovided in cyanobacteria (Golden and Haselkorn, 1985;Ohad and Hirschberg, 1992) and tobacco (Cheung et al.,1988). Anacystis nudulans was transformed with a psbA gene

0.01 0.1 1.0 10.0

[ATRAZINE] 0-iM)

100.0

Figure 3. Relative rates of photosynthetic electron transport instroma-free chloroplast thylakoid membranes isolated from wild-type and variant potato cells in response to atrazine. Electrontransport was measured within 90 s of herbicide addition. Eachpoint is the mean of four observations, and vertical bars representthe SEM. Rates of O2 evolution for wild-type and variant thylakoidsin medium lacking atrazine were 114.4 and 143.4 /imol of O2 mg"'of Chl h"', respectively.

Var WtA A

Var WtB B

21.5»

14.4»

Figure 4. SDS-PACE of thylakoid membrane polypeptides fromwild-type (Wt) and variant (Var) cells. Proteins were visualized afterstaining with Coomassie brilliant blue (A lanes) and after fluorogra-phy (B lanes). Membranes from wild-type and variant cells wereincubated with 0.3 MM [azido-14C]atrazine under UV light for 8 minbefore SDS solubilization. The center lane contains mol wt proteinstandards, with the M, of each depicted on the left.

Atrazine Resistance in Photoautotrophic Potato Cells 915

A BW V W V

75*-

50"

Figure 5. Fluorogram (B) and immunoblot (A) of thylakoid mem-brane polypeptides from wild-type (W) and variant (V) cells. Mem-branes from wild-type and variant cells were incubated with 0.3 IIM[azido-14C]atrazine under UV light for 8 min before SDS solubiliza-tion. Immunoblot was reacted with anti-M, 32,000 QB polypeptide.Mol wt standards are in the center lane, and M, of each is depictedon the left.

mutated at the 264th codon and exhibited resistance toatrazine (Golden and Haselkorn, 1985). In tobacco, a chimericgene constructed with a nuclear promoter, mutant psbA gene,and chloroplast transit peptide-encoding sequence was incor-porated into the nuclear genome of atrazine-susceptible to-bacco, with recovery of atrazine-tolerant transgenic plants(Cheung et al., 1988).

Atrazine resistance in two weed biotypes (Pfister et al.,1981; Barros and Dyer, 1988) was linked to the inability of[azido-14C]atrazine to bind to the QB protein, and this alteredbinding was based on the conversion of Ser264 to Gly. Thesubstitution of Ser by Thr in the QB protein of our variantpotato cell thylakoids also precludes the binding of[azido-14C]atrazine to this protein (Figs. 3 and 5) and is thefirst direct evidence that an amino acid substitution otherthan Gly abolishes [azido-14C]atrazine binding.

Atrazine may be unable to bind to a mutated QB proteinbecause of conformational changes that sterically inhibit in-teraction with atrazine or the absence of a chemical grouprequired for bond formation with atrazine. The Ser264 andPhe255 amino acids of the QB protein are considered essentialfor binding of terbutryn (a triazine) to the correspondingamino acids in the L subunit of Rhodopseudomonas viridisreaction centers (Michel et al., 1986). Substitution of Ser264

with some other amino acid may abolish association of ter-butryn to its binding site (Michel et al., 1986). In the thylakoidQB protein of higher plants, a hydrogen bond is suggested toform between Ser264 and His252 (Tietjen et al., 1991), arrang-ing a series of amino acids around Ser264 to form a parallelhelix between the fourth and fifth transmembrane helicalspans of the QB protein (Trebst, 1987). This parallel helixpartially constitutes the QB-binding niche, because many ofthe mutations in this region result in resistance to PSII her-bicides (Trebst, 1987). Substitution of Gly or Ala (Mazur andFalco, 1989) for Ser264 weakens s-triazine herbicide bindingto the QB protein due to the loss of a side chain hydroxylgroup, which may be important in formation of a hydrogen

bond with s-triazine herbicides (Tietjen et al., 1991). Alter-natively, this mutation may destabilize the parallel helixmentioned above, changing the conformation of the bindingniche and sterically preventing s-triazine binding. Substitu-tion of Ser264 with Thr maintains the side chain hydroxylgroup but introduces an extra methyl group. This subtlechange has been postulated to alter the conformation of theQB protein, resulting in a steric hindrance to herbicide binding(Shigematsu et al., 1989). Such a steric hindrance could beresponsible for the resultant cross-resistance of our potatocells to DCMU (approximately 13- to 21-fold), which bindsnear the same site in the QB protein (data not shown).

Mutations in the QB protein resulting in resistance to atra-zine in plants have also affected photosynthetic performance.Reduced vigor of atrazine-resistant biotypes has been attrib-uted to lower efficiency of charge transfer from QA to QB(Holt, 1988; Jursinic and Pearcy, 1988). Ultimately, maximumrates of photosynthesis were lower in atrazine-resistant ver-sus atrazine-sensitive plants and were manifested in lowerproductivity. This was true for atrazine-sensitive plants fromnearly nuclear-isogenic lines of Brassica napus (Gressel andBen-Sinai, 1985) and Senecio vulgaris (Holt, 1988), whichproduced more total plant dry weight and seed than atrazine-resistant plants. McCloskey and Holt (1991) have shown thatreduced photosynthetic efficiency, reduced overall plant bi-omass, and triazine resistance are genetically linked to thechloroplast genome. Discrepancies in the literature regardingreports of similar or increased photosynthetic rates for tria-zine-resistant versus -sensitive weed biotypes may be relatedto changes in traits under nuclear control (Ort et al., 1983).Therefore, although the specific contribution of a Ser264 toGly mutation to reduced vigor for atrazine-resistant speciesis not known, arguably this mutation influences plant per-formance. To our knowledge, this is the first demonstration

VARIANT

AA JL AAP <•

/A Qln (261) Oln A\.' A________A '«

T T 'A Tyr (262) Tyr A

WILD TYPE

«c c /•Q Arg (269) Arg G/T T'

Figure 6. Autoradiogram of psbA DMA-sequencing gels for wild-type and variant cells. The only site for divergence was at the aminoacid Ser at position 264 where a G residue was substituted by a Cresidue. This observation was confirmed by sequencing four uniqueplasmid target ctDNA inserts.

91 6 Smeda et al. Plant Physiol. Vol. 103, 1993

that the alteration of Ser264 to Thr264 results in atrazine resistance with no reduction in cell growth or photosynthetic productivity. In the absence of atrazine, photosynthetic elec- tron transport rates for variant cells (Fig. 2) and isolated thylakoid membranes (Fig. 3) were slightly, but consistently, higher than in wild-type cells. Additionally, no significant differences in the'rates of fresh or dry weight accumulation (Fig. 1) were measured.

The explanation for similar photosynthetic efficiency and biomass accumulation for variant and wild-type potato cells rather than the reduced productivity of atrazine-resistant versus atrazine-sensitive biotypes of S. vulgaris may be based on the specific amino acid substituted for Ser at position 264. The loss of a side chain hydroxyl group at position 264 of the QB protein with a Gly or Ala substitution may directly impair electron transfer efficiency from Q A to QB in resistant weed biotypes. Jursinic and Pearcy (1988) have suggested that the charge transfer reaction from Q A to QB is rate limiting in weed biotypes with Ser264 to Gly mutations. In contrast, the substitution Iof Thr for Ser264 maintains the side chain hydroxyl group and the capacity to form a hydrogen bond between HisZ5' alnd Thr264 and thus allows the amino acids around position 264 to form the parallel helix. This may result in relatively normal rates of electron transfer from Q A

to QB, but further experiments will be necessary to specifically measure the efficiency of electron transfer from QA to QB.

Both temperature and light intensity influence the magni- tude of the growth and photosynthetic productivity differ- ences between atrazine-resistant and atrazine-sensitive plants (Ort et al., 1983; Jursinic and Pearcy, 1988; Stowe and Holt, 1988). However, in our study, photosynthetic measurements from both intact cells and isolated thylakoids were not ap- preciably different between variant and wild-type potatoes under light (215 PE m-' s-l) and temperature levels (29OC) that were sufficient to measure differences.

We conclude that mutations in the psbA gene that confer atrazine resistance need not necessarily reduce photosyn- thetic efficiency and plant productivity. The influence of an altered Q B protein on productivity and the leve1 of s-triazine resistance may depend on the specific amino acid substitution in the QB protein. It is possible that the loss of a hydroxyl group with the substitution of Ser by Gly gives both s-triazine resistance and a reduction in the rate of electron transfer. However, the substitution of Thr for Ser at position 264 imparts s-triazine resistance without apparent detrimental effects on photosynthesis or productivity. This mohfication should be considered in future efforts to incorporate atrazine resistance in crop plants. In further experiments we will examine the regeneration of plants from wild-type and var- iant cells and estimate the response of variant plants chal- lenged with atrazine.

Received March 8, 1!393; accepted July 2, 1993. Copyright Clearance Center: 0032-0889/93/103/09 11/07.

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