( H o r d e u m vulgare L . ) d e f i c i e n t i n p h o s p h o g l y c o l l a t e phosphatase
NIGEL P. HALL, ALAN C. KENDALL, PETER J. LEA*, JANICE C. TURNER and ROGER M. WALLSGROVE Department of Biochemistry, Rothamsted Experimental Station, Harpenden, Herts, AL5 2JQ, UK * Present address: Department of Biological Sciences, University of Lancaster, Lancaster, LA1 4YQ, UK
(Received: 13 January 1986; in revisedform: 14 April 1986) Key words: photosynthesis, photorespiration, barley, mutants, phosphoglycollate phosphatase Abstract. A barley mutant RPr84/90 has been isolated by selecting for plants which grow poorly in natural air, but normally in air enriched to 0.8% CO 2 . After 5 minutes of photosynthesis in air containing 1'CO2 this mutant incorporated 26% of the 14C carbon into phosphoglycollate, a compound not normally labelled in wild type (cv. Maxis Mink) leaves. The activity of phosphoglycollate phosphatase (EC 22.214.171.124) was 1.2 nkat mg -1 protein at 30°C in RPr 84/90 compared to 19.2nkat mg -a protein in the wild-type leaves. Phosphoglycollate phosphatase activity was not detected after protein separation by electrophoresis of leaf extracts from the mutant on polyacrylamide gels; on linear 5% acrylamide gels three bands with enzyme activity were separated from extracts of wild type plants. Gradient gel electrophoresis followed by activity staining showed two bands in Marls Mink tracks of MW 86,000 and 96,000, but no bands in 84/90. This is the first report of isozymes of phosphoglycoliate phosphatase in barley which were absent in the mutant extracts. Our results confirm an earlier report of isozymes of this phosphatase in Phaseolus vulgaris [18 ]. The photosynthetic rate of RPr 84•90 in 1% O2, 350~1 CO~ 1-1 was 9 - 1 2 m g CO 2 dm -2 h -1 at 20°C, whereas the wild-type rate was 2 7 - 2 9 m g CO 2 dm -2 h -1 at 20°C. In 21% O2,350/~1 CO 2 1-1 the rate was 2 - 3 mg CO 2 dm -2 h -1 in the mutant and 20 mg CO 2 dm-a h-1 in the wild type. Genetic analysis has shown that the mutation segregates as a single recessive nuclear gene.
Introduction P h o t o r e s p i r a t i o n , the e v o l u t i o n o f CO2 in the light, occurs following the reaction o f o x y g e n w i t h R u B P catalysed b y R u B P c a r b o x y l a s e / o x y g e n a s e (EC 126.96.36.199). T h e role o f p h o t o r e s p i r a t i o n is unclear, b u t it has b e e n described as a w a s t e f u l process w h i c h limits plant p r o d u c t i v i t y [12, 14, 19]. Mutants w i t h defects in p h o t o r e s p i r a t o r y m e t a b o l i s m , capable o f n o r m a l g r o w t h in CO2-enriched air, b u t unable to thrive in air, were first isolated in Arabidopsis thaliana [ 1 6 ] . These plants, w h i c h had reduced CO2 e v o l u t i o n in
90 the light, were valuable in resolving controversies about the photorespiratory pathway. The Arabidopsis mutant (CS119) lacking phosphoglycollate phosphatase provided conclusive evidence that phosphoglycollate rather than glycollate is the first metabolite of the pathway . At Rothamsted, using a similar approach with azide - mutagenized barley, we have isolated 60 mutants with the air-sensitive phenotype from approximately 120,000 M2 plants. These mutants can be separated into groups on the basis of their photosynthetic and photorespiratory gas exchange characteristics . Some of the barley mutants have lesions which were not found in Arabidopsis. One such mutant, RPr 79/4, deficient in catalase has been previously described . We report here the isolation of a mutant, RPr 84/90, deficient in the first enzyme of the photorespiratory pathway, phosphoglycollate phosphatase and therefore similar to t h e m u t a n t line CS119 isolated from Arabidopsis. This enzyme, normally present in the chloroplast stroma,  has an important role in removing a potentially toxic metabolite from the chloroplast. Phosphoglycollate is a potent inhibitor of triose phosphate isomerase in vitro  and the effect of this mutation on photosynthesis in vivo is therefore of interest. Materials and methods
Mu tan t selection The mutant-selection procedure was based on that originally described in  for Arabidopsis. Details of the method for barley have been published .
Assimilation of 14C02 Leaves of mutant and wild type plants previously equilibrated in 1% 02, 350#1 CO2 1-1 were exposed to air for 5 rain. They were then fed 14CO2 in air (350tzl CO2 1-1, 21% 02) at a photon flux density of 1000/amol m -2 s-1 . The exposed leaves were immediately dropped into liquid nitrogen. Soluble intermediates were extracted and analysed by thin layer chromatography and autoradiography .
Gas exchange analysis Detached leaf pieces (4 x 0.5 cm) were placed with their bases in distilled water in a water-jacketed leaf chamber held at 20 °C. The internal dimensions of the chamber were 3.7 x 0.7 x 4.4 cm and total internal volume 11.4 cm 3 . The chamber was flushed at 271 h -1 with the appropriate gas mixture from a gas blender (Series 850; Signal Instrument Co., Camberley, Surrey, UK). Depletion of CO2 was measured using an infra-red gas analyser (Model 225; Analytical Development Co., Hoddesdon, Herts, UK).
Leaf extracts were prepared by grinding 0.2g tissue with 1.8ml 50mMTris HC1 (pH 8.3 at 4 °C) for several minutes in a pestle and mortar. The homogenate was centrifuged at 12,000 rpm for 4 mins at 4°C. Phosphoglycollate phosphatase (EC 188.8.131.52) in the homogenates was assayed at 30°C according to . The Pi released was measured using stabilized stannous chloride, by reading the absorbance of the phosphomolybdate complex at 700nm . RuBP carboxylase (EC 4.1.1..39) was measured at 13°C immediately after extraction from the leaf, and again after a 10 minute activation period with CO2 and Mg2÷ . GlycoUate oxidase (EC 184.108.40.206.) was measured using a spectrophotometric assay . Protein was measured by Coomassie Blue dye binding .
Gel electrophoresis Phosphoglycollate phosphatase activity on 5% acrylamide gels was detected by the formation of a white band of lead phosphate at the site of enzymatic activity . Maris Mink and 84/90 leaf extracts were also run on 4-20% non-denaturing gradient gels with 2.7-5.3% bis-acrylamide . Gradient gels were stained for phosphoglycollate phosphatase activity  or for protein using Coomassie Blue. Molecular weight standards for gradient gels were ferritin (440,000) and dimer (880,000); serum albumin (66,000) and dimer (132,000); egg albumin (45,000). Results and discussion
In high (0.8%) CO2, plants of RPr 84[90 were indistinguishable from the wildtype. In air and light, however, they turned pale within 24 hours, and the leaf tips became bleached. Prolonged exposure resulted in bleaching and death of the whole plant. The distribution of radioactivity in products of 14CO2 assimilation in leaves of the mutant RPr 84/90 was very different to that of Marls Mink (Table 1). In particular, over 25% of the 14C accumulated in phosphoglycollate, a compound not normally labelled in wild-type leaves. The incorporation into phosphoglycollate was accompanied by a decrease of label in other photorespiratory metabolites, notably glycine, serine and glycerate. The decreased incorporation of label into phosphoglycerate (3.3% against 10.4% in Marls Mink) was probably due to lack of carbon recycling through the glycollate pathway. The decreased incorporation of 14C into glucoseand fructose- phosphates again suggests a shortage of carbon in the Calvin cycle when RPr 84/90 is exposed to air. The total 14C incorporated in RPr 84•90 was about a third of the 14C fixed by Marls Mink leaves (Table 1). This probably reflects the lower and declining rate of photosynthesis of the mutant during the 5 minutes feeding in air.
92 Table 1. Radioactivity in the products of 1"CO2 assimilation in leaves of mutant RPr 84/90 and wild type barley. Leaves were extracted after photosynthesis for 5 rain ~*CO~ in air (350 ~11-1 CO 2 , 21% O~ ; specific activity 4 mCi mmo1-1 ). Previously, leaves were in air for 5rain following 60min in 350tfl CO~ and 1% O 2 . Photon flux density was 1000 t~mol m -2 s -1 . Values are a mean of five ~4C feeding experiments. The standard deviation (based on five experiments) of the % of ~4C incorporated into phosphoglycollate in RPr 84/90 was ± 0.9% 14C (% of total extracted) RPr 84/90 Glucose - 1 phosphate + Glucose - 6 phosphate Fructose - 6 phosphate Phosphoglyceric acid Phosphoglycollate Aspartate Sucrose Serine Glycine Glycerate Alanine Glyeollate Others Total dpm/cm 2 leaf
Activities were measured at 30°C except RuBP carboxylase which was measured at 13 oC. Values shown are the means of measurements ± standard errors on four extracts. RPr 8 4 / 9 0 was deficient in the activity o f the e n z y m e p h o s p h o g l y c o l l a t e phosphatase: the specific activity o f phosphoglycollate phosphatase in leaf extracts o f RPr 8 4 / 9 0 was less than 10% o f that assayed in similar extracts o f cv. Marls Mink (Table 2). F u r t h e r m o r e , n o phosphoglycollate phosphatase activity could be d e t e c t e d in m u t a n t leaf extracts separated b y electrophoresis on non-denaturing p o l y a c r y l a m i d e gels (Figure 1). Decreased p h o s p h o g l y c o l l a t e phosphatase activity in the m u t a n t was in contrast to activities o f o t h e r e n z y m e s assayed; these had activities similar to the wild t y p e (Table 2). PhosphoglycoUate phosphatase was assayed at pH 6.3, w h i c h has been r e p o r t e d as the o p t i m u m for the t o b a c c o e n z y m e [5, 15] whilst the e n z y m e f r o m pea leaves has a pH o p t i m u m o f 8.3 [ 1 1 ] . The pH o p t i m u m o f phosphoglycollate phosphatase i n barley leaf h o m o g e n a t e s in the presence o f Mg 2÷ was b e t w e e n 6.0 and 6.5, b u t the pH response curve was fairly broad.
Figure 1. Phosphoglycollate phosphatase activity in proteins separated on potyacrytamide gels by electrophoresis. Phosphoglycollate phosphatase was located by incubating 5% acrytamide gels in a buffered solution containing magnesium acetate, lead acetate and the substrate . Tracks 1, 2, 3 and 4 contain 41 (84•90); 41 (wild-type); 21.5 (84/90); 20 (wild-type) Cg of protein respectively. Track 5 blank. Electrophoresis on linear 5% acrylamide gels revealed three bands o f phosphoglycoUate phosphatase activity in Marls Mink extracts, suggesting the presence o f isozymes (Figure 1): two bands were of similar intensity whilst the third was distinctly fainter. As all the bands were absent in extracts from RPr 84/90, it is probable that they represent phosphoglycoUate
1% 02 I
Characteristics of a photorespiratory mutant of barley (Hordeum vulgare L.) deficient in phosphogly collate phosphatase.
A barley mutant RPr84/90 has been isolated by selecting for plants which grow poorly in natural air, but normally in air enriched to 0.8% CO2. After 5...