Changes of Glycolate Oxidase Activity with Leaf Age in Zea maysL.

Agricultural Chemistry Institute, University of Pis a, Italy

Changes of Glycolate Oxidase Activity with Leaf Age in ZeamaysL. G. F. SOLDATINI With 3 figures Received February 12, 1979 . Accepted March 7, 1979

Summary Glycolate oxidase specific aCtiVity, calculated on a protein basis, increases during leaf ontogeny in Zea mays L., the oldest leaves showing an activity 60 Ofo higher than that of the youngest ones. A 50-60 % increase of glycolate oxidase activity was also obtainable in detached leaves kept in the light for 18 hrs.

Key words: glycolate oxidase, activity, leaf age, Zea mays.

Introduction Glycolate oxidase (E.C. 1.1.3.1) and other photo respiratory enzymes are present in both C 4 and C a species, although they are less active in the C 4 species (ZELITCH, 1975). SALIN and HOMANN (1971) found that the activity of glycolate oxidase in C a plants was significantly lower in young leaves than in mature ones. Little research has been done on the effect of leaf age on glycolate oxidase activity in C 4 plants (REHFELD et aI., 1970). Neverthless there are some indications that such a study can be of interest in understanding phenomena related to photorespiration in C 4 plants. In leaves of several C 4 plants including maize photosynthesis decreases and photorespiration increases with leaf age (LUDLOW and WILSON, 1971; KENNEDY, 1976; WILLIAMS and KENNEDY, 1977). It was found (SOLDATINI and ZIEGLER, 1979) that glycolate oxidase is an inducible enzyme. On this basis we should expect an increase in glycolate oxidase activity during leaf development in maize, although this species presents low levels of photorespiration. The aim of the present work was to test this hypothesis. Materials and Methods Zea mays L. var. Asgrow-ATC 39 was grown in field conditions. Plants approximately 60 days old with 11 to 12 fully expanded leaves were used as a source of leaf material. The oldest leaves used still contained 50-60 Ofo of the chlorophyll present in the mature ones, on a fresh weight basis. Z. Pflanzenphysiol. Bd. 94.

s. 267-271.1979.

268

G. F. SOLDATINI

To study changes in enzyme activity in detached leaves during senescence, leaf disks 1.8 cm in diameter were cut randomly from mature leaves and left floating on water. Tests were conducted simultaneously in the dark and in the light (18,000 lux from an Osram NQL 400 W lamp). Three replicated samples were taken at the beginning and at intervals for enzyme assays.

Assay of glycolate oxidase For enzyme extraction, 1 g of fresh leaf material was homogenized at 4 °C by exhaustive grinding in a mortar with 5 ml of 0.02 M glycylglycine, pH 7.5 and quartz in order to break bundle sheath cells. The homogenate was squeezed through four layers of cheesecloth and centrifuged for 10 min at 4000 X g. The enzyme activity was determined in 0.1 ml of the clear supernatant containing 0.3 to 0.5 mg protein by the glyoxylate-phenylhydrazone procedure as described by BAKER and TOLBERT (1966). Such a procedure was found much more sensitive than that involving determination of glycolic acid by the EEGRIVE-CALKINS method (LORIMER, 1977). The rate of increase in absorbancy at 324 mfl was followed at 25°C for 20 to 30 minutes, against a cell containing all components except glycolic acid. The kinetics obtained were perfectly linear in this interval. Protein and chlorophyll determination was carried out as previously described (SOLDATINI and ZIEGLER, 1979).

Results and Discussion Leaves of a developed maize plant from the top to the bottom provide a very homogeneous leaf material at regularly increasing ontogenetic stages. The possibility of working with tissues from a single plant with different photorespiration rates and with different levels of enzyme activities involved in photorespiration provides useful investigation material. The data reported in this paper for glycolate oxidase are expressed as specific activity on a protein basis. Care was taken to solubilize as much enzyme activity as possible and to avoid errors due to incomplete protein extraction. In preliminary experiments a 60-second grinding in Waring Blendor extracted only 40 Ofo of the total enzyme activity present in the leaves on a fresh weight basis (Fig. 1 A). Besides, the Waring Blendor extract was characterized by a specific activity three times lower than that found in the residue when extracted in a mortar with quartz (Fig. 1 B). These results are consistent with those reported by REHFELD et al. (1970). The specific glycolate oxidase activities in different aged leaves of the same plant are reported in Fig. 2, together with the chlorophyll content calculated on a fresh weight basis. Data represent average values from five different plants with 11 developed leaves. The plants were very homogeneous, because the maize used was a Fl hybrid. Two alternative explications for the increase in glycolate oxidase during leaf development can be put forward: 1. Glycolate oxidase and other peroxisomal enzymes are less active in young leaves and represent a limiting factor for photorespiration; 2. Photo respiration itself is increasing in mature and senescent leaves and consequently a synthesis of glycolate oxidase is induced. It has been formerly postulated that in young C s leaves photo respiration might be limited by the

z. Pflanzenphysiol. Bd. 94. s. 267-271. 1979.

Changes of glycolate oxidase activity

269

B

A

r

Fig. 1: Glycolate oxidase actlvltles in leaf extracts of Zea mays. Open columns: supernatant from 60 s homogenization in Waring Blendor. Closed columns: extract in mortar with quartz of the Blendor residue. A: enzyme activity on fresh weight basis. B: specific activity on a protein basis. 22

4

.~

..,.

~ 20

l:'

~

~

I

~ 't1

~

,""

18

I

3~

.~

"

~

" 16 ~

~

, I

..!!!

'"

~ 14

~

~

~

:::::

~

~

~ 12

2 ~

<::

0

~

\S 11th

9 th

7th

5th

3th

1st

Fig. 2: Columns represent specific glycolate-oxidase aCtlVlty from the 11th (youngest) to the 1s t (oldest) leaves in maize. (e-e) Chlorophyll content.

low activity of peroxisomal enzymes, as an alternative hypothesis to that involving a sluggish formation of glycolate (SALIN and HOMANN, 1971). This hypothesis is however in apparent contrast with the finding that all the species appear to have glycolate oxidase and other photorespiratory enzymes in excess of what is needed for photorespiration (ZELITCH, 1973; TOLBERT, 1971). The minimum rate of glycolate oxidase we found in young maize leaves (10 nmoles glycolate X min-1 X mg- 1 protein at 25°C) is enough to support the scarce photorespiration of maize.

z. Pflanzenphysiol. Bd. 94. S. 267-271.1979.

270

G. F. SOLDATINI

The enhancing in glycolate oxidase aCtIVIty during leaf ontogeny is probably a consequence of increased photorespiration, i. e. glycolate formation. In a previous work (SOLDA TIN! and ZIEGLER, 1979) was reported that glycolate oxidase is indeed inducible after fumigation with S02 in light over a period of 18 hrs, under which condition also photo respiration was probably stimulated. The idea that an increase in glycolate oxidase activity may represent an adaptation to the increased photorespiration is supported also by the data obtained in the experiments with detached leaves (Fig. 3). In these experiments, increase in glycolate oxidase occurred only in leaf disks submitted to light treatment, i. e. under conditions stimulating photo respiration. 35,----------------------------------, .'>

~

~ ~"'- 25

'~

~"

:~

" 15

~

..!!!

~

~

<>,

OJ

<5

~ 5 ~__~~--~-----L-----L~--~----~ 10 20 30 40 50 60

It

Fig. 3: Glycolate oxidase activity in maize leaf disks incubated in light (0-0) and dark (e-e).

The results reported in this paper are consistent with the idea that photorespiration, as identified with glycolate oxidation, is present in both leaves of C 4 plants and Ca plants, although the effect on net CO 2 fixation in C 4 ones is small. The data presented make it imperative for future publications on glycolate oxidase activity and photorespiration to state the age of the plant material. References BAKER, A. L. and N. E. TOLBERT: Methods in Enzymology 9, 338-347 (1966). KENNEDY, R. A.: Planta 128, 149-154 (1976). LORIMER, G. H.: Analyt. Biochem. 83, 785 (1977). LUDLOW, M. M. and G. L. WILSON: Aust. J. BioI. Sci. 24, 1077-1087 (1971). REHFELD, D. W., D. D. RANDALL, and N. E. TOLBERT: Can. J. of Bot. 48, 1219-1226

(1970).

SALIN, M. L. and P. H. HOMANN: Plant Physiol. 48,193-196 (1971). SOLDATINI, G. F. and ZIEGLER, I.: Phytochemistry 18. 21-22 (1979). TOLBERT, N. E.: Ann. Rev. Plant Physiol. 22, 45-74 (1977).

Z. PJlanzenphysiol. Bd. 94. S. 267-271.1979.

Changes of glycolate oxidase activity

271

WILLIAMS, L. E. and R. A. KENNEDY: Z. Pflanzenphysiol. 81, 314-322 (1977). ZELITCH, I.: Proc. Natl. Acad. Sci. U.S.A. 70, 579 (1973). - Ann. Rev. Biochem. 44, 123-145 (1975). G. F. SOLDATINI, Universita degli Studi, Istituto di Chimica Agraria, Via S. Michele degli Scalzi, 2, 56100 Pisa, Italy.