J PlantPhysiol. Vol. 141. pp. 633-635 (1993)

Short Communication

Corn Phosphoglycolate Phosphatase: Immunodetection in Kranz Cells

PIERRETTE BALDY

Centre de Biologie et Physiologie Vegetales - U niversite Paul Sabatier, 31 062 Toulouse cedex, France

Received October 13, 1992 . Accepted December 10,1992

Summary

Specific polyclonal antibodies raised against corn phosphoglycolate phosphatase were used for im­munodetection of this protein in the two chlorophyllous cells, mesophyll protoplasts and bundle sheath strands, isolated enzymatically from expanded corn leaves. Western blot analysis clearly showed an im­munoreactive protein band with the bundle sheath cells only; its molecular mass of 31.5 kilodaltons cor­responds to the phosphoglycolate phosphatase subunit. This result is totally in accordance with the cellu­lar localization of this specific phosphatase activity, previously shown in bundle sheath cells only, and means that phosphoglycolate phosphatase gene expression was different in the two photosynthetic cell types of maize leaves, a C4 plant.

Key words: Zea mays L., com, bundle sheath, C4 plants, mesophyll, phosphoglycolate phosphatase, photo­respiration.

Abbreviations: BS = bundle sheath; IgG = immunoglobulin G; M = mesophyll; NADP-MDH = malate dehydrogenase (NADP+); P-glycolate phosphatase = phosphoglycolate phosphatase (EC 3.1.3.18); SDS-PAGE = Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Introduction

The characteristic feature of C4 plants is the «Kranz» type leaf anatomy consisting of mesophyll and bundle sheath cells, which differ in their photosynthetic activities (Edwards and Huber, 1981). This partitionning can be accounted for largely by differential expression in the two leaf cell types generally regulated in maize (Rodermel and Bogorad, 1985; Sheen and Bogorad, 1987 a, b; Nelson and Langdale, 1989) as in other NADP-malic enzyme type-C4 plants (Cretin et aI., 1988) and in NAD-malic enzyme type C4-plants (Wang et aI., 1992) at the transcriptional level.

The present investigation attempts to examine if the spe­cialization of Kranz cells in the first step of photorespiration, shown previously with enzymes activities (Baldy and Cavalie, 1984), could be explained by the absence or by the inactivation of phosphoglycolate phosphatase proteins in mesophyll cells.

© 1993 by Gustav Fischer Verlag. Stuttgart

Materials and Methods

Plant material

Corn (Zea mays L. cv. DEA) plants were grown in a controlled environment chamber (Hardy and Baldy, 1986) and the fully ex­panded 4th leaves of 19-day-old plants were used (Baldy, 1986).

Production of antibodies

Antibodies against maize P-glyeolate phosphatase were obtained as described earlier (Baldy et al., 1989) using the native enzyme. In this work the sera were brought to 33 % saturation in (NH4)2S04; the total immunoglobin G (IgG) obtained was redissolved in phosphate saline buffer (PBS buffer), (25 mM K-phosphate, 140 mM NaC!, pH 7.0) and dialyzed overnight against this buffer. The monospecificity of polyclonal antisera was checked as before (Baldy et aI., 1989).

634 PIERRETIE BAillY

Isolation of bundle sheath strands and mesophyll protoplasts

Corn M protoplasts and BS strands were isolated enzymatically, using a combination of cellulase and macerozyme, and purified as previously described (Baldy, 1984).

Isolation of proteins and protein gel blot analysis

The purified M protoplasts were pelleted (150 g for 4 min) and then osmotically burst in 0.01 M Na+ -cacodylate buffer, pH 6.8, whereas the purified BS strands were broken in a mortar with acid­washed sand using the same buffer. The M and BS proteins, obtain­ed in the soluble fractions (15,000 g, 20 min), were dissociated in the denaturing buffer (8 min at 100°C) and subjected to SDS-PAGE using a 12 % polyacrylamide slab gel (Hardy and Baldy, 1986). Fol­lowing SDS-PAGE, proteins were stained for 3 h with 0.2 % Coomassie Brillant blue R-250 in 7% (v/v) acetic acid and destained with acetic acid: methanol: water (10 : 20 : 70, by vol.) or were trans­ferred passively at 34°C for 36 h onto 0.45 ~m nitrocellulose mem­branes. Immunoreaction was carried out, essentially, as described in Bio-Rad's instructions; nitrocellulose blots were blocked for 2h with 3% (w/v) gelatin in TBS buffer (20mM Tris, 500mM NaCl, pH 7.5), then washed and incubated for 1 h with first and second antibodies, respectively, diluted 1: 300 and 1: 3,000 in TBS buffer containing 1 % (w/v) gelatin. Horseradish peroxidase conjugated second antibody was used to visualize cross-reactivity on the mem­brane and peroxidase activity was detected with 4-chloro-1-naphthol as reagent.

Assays of enzymic activity and protein determination

P-glycolate phosphatase (EC 3.1.3.18) (Baldy et aI., 1989), PEP­carboxylase (EC 4.1.1.31) and RuBP-carboxylase (EC 4.1.1.39) (Baldy, 1986) activity measurements were based on published methods and protein level was determined according to Lowry et al. (1951).

Results and Discussion

Based on cross-contamination, respectively of the BS prep­aration by PEP carboxylase activity, the M cytosolic marker and of M cells by RuBP carboxylase activity, the BS chloro­plastic marker, results showed that the M protoplast prepara­tions were obtained pure and that contamination of the BS strands was less than 1 % as obtained previously (Baldy, 1984). The high purity of these preparations allowed to interpret the results related in this paper.

Western blot analysis of purified corn P-glycolate phos­phatase showed that anti-P-glycolate phosphatase IgG recog­nizes a single polypeptide of 31.5 kDa (Fig. 1, lane 3), corre­sponding to the subunit of corn P-glycolate phosphatase, a homodimeric protein (Hardy and Baldy, 1986); hence, the antigenic determinants were preserved in spite of SDS dena­turation of the protein.

In BS crude extracts (Fig. 1, lane 2), visualization of im­munoreactive protein after SDS-PAGE and immunoblotting revealed the presence of the 31.5 kDa anti-P-glycolate phos­phatase antibody reactive protein; this single band was also visualized when the amount of protein loaded onto the gel was as low as 10 Ilg (data not shown). In contrast to these re­sults, with a similar amount of M proteins (37Ilg), immuno-

M

-

M

-.. ...

2

Fig. 1: Western blot analysis of corn P-glycolate phosphatase. SDS­PAGE (left) and immunoblot (right) of soluble proteins from M protoplasts (lane 1; 37.0 ~g) and BS strands (lane 2; 32.0 ~g), and of the purified corn P-glycolate phosphatase subunit of 31.5 kDa (lane 3; 7.5 ~g). Coomassie staining and immunoblot were carried out as described. Molecular size markers, lane M: ovalbumin, 45 kDa; pepsin, 34.7 kDa; trypsinogen, 24 kDa; i3-lactoglobulin, 18.4 kDa; lysozyme, 14.3 kDa. Arrows indicate P-glycolate phosphatase sub­unit (31.5 kDa).

reactive protein was not detectable on the immunoblots (Fig. 1, lane 1).

In conclusion, the lack of the specific P-glycolate phos­phatase activity, previously shown in M cells (Baldy and Cavalie, 1984), could not be inherent to an inactive protein or to an inhibition of the activity in this one cell type; it must be ascribed to the absence of the protein since anti-P­glycolate phosphatase IgG, used as a probe, did not recognize any M proteins.

Thus, as many other C 4 enzymes (Nelson et al., 1989) this specific phosphatase is differentially expressed in the two dis­tinct photosynthetic cell types of mature corn leaves. Fur­thermore, it is of interest to determine if the cell-specific ex­pression of P-glycolate phosphatase gene is light dependent.

References

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