II. 54. Activities of ribulose-1,5-bisphosphate carboxylase of Hordeum vulgare L. var. Karri and a Hordeum bulbosum alloplasmic barley, HA-Hb-l-Karri.
E. Vapaavuori, Department of Botany, University of Helsinki, Unioninkatu 44, 001700 Helsinki 17, Finland. "R"
Ribulose-l,S-bisphosphate (RuP2) carboxylase-oxygenase or Fraction I protein is the major soluble protein in green plants. This protein is located in the chloroplasts and consists of eight large and eight small subunits (Baker et al. 1975), which are synthesized on the 70 S chloroplastic and the 80 S cytoplasmic ribosomes, respectively (Criddle et al. 1970). There is much evidence that the catalytic site of RuP2-carboxylase-oxygenase activity is located in the large subunit (Nishimura et al. 1973, Kung 1976), and it has been suggested that the small subunits contain the regulatory sites (Nishimura et al. 1973, Kung 1976). The dual nature of inheritance and function in this enzyme has been a great advantage to plant breeders and geneticists.
The alloplasmic barley of Hordeum bulbosum has been reported to behave like a mutant sensitive to cool temperatures (Fukuyama 1978). The aim of this study was to investigate whether the stunted growth habit of the alloplasmic barley correlates with the activity of RuP2-carboxylase. Hordeum vulgare L. var. Karri (chloroplasts of Hordeum vulgare orgin) served as a normal barley and HA-Hb-l-Karri as an alloplasmic barley of Hordeum bulbosum (chloroplasts of Hordeum bulbosum origin) (for detailed information on the material see Ahokas 1978).
The enzyme assays were made 17 days after sowing, on 1.5 g of the second or third fully expanded leaf. The enzyme was prepared, activated and assayed as described by Lorimer et al. (1977). Chlorophyll was determined by the method of Arnon (1949) and soluble protein by the method of Lowry et al. (1951).
The specific activity of RuP2-carboxlase (Table 1) calculated on the chlorophyll basis was slightly higher in Hordeum vulgare L. Karri than in HA-Hb-l-Karri, but the difference in activity was less than 10%. The difference between the two barleys was greater when the specific activities were calculated on the soluble protein basis. The chlorophyll and soluble protein contents per unit fresh weight were lower in HA-Hb-l-Karri than in Karri, so that the difference between the mutant and Karri would be greater if the specific activity was calculated on a fresh weight basis. The soluble protein/chlorophyll ratio was higher in HA-Hb-l-Karri, 7.0 versus 6.2. There was practically no difference in the chl a/chl b ratios between the two barleys (2.25 for Karri and 2.19 for HA-Hb-l-Karri).
Table 1. Activities of ribulose-1,5-bisphosphate carboxylase of Hordeum vulgare L. Karri and Hordeum bulbosum alloplasmic barley, HA-Hb-l-Karri. The values are the means of 8 replicates.
These results suggest that (l) chlorophyll and protein synthesis are partly inhibited in HA-Hb-l-Karri and that (2) the growth retardation of Hordeum bulbosum alloplasmic barley cannot be solely explained by the decrease in RuP2-carboxylase activity. On the contrary it appears that the abnormal growth habit caused by Hordeum bulbosum cytoplasm is due to factors other than RuP2-carboxylase.
In this experiment RuP2-oxygenase activities were not assayed so that it remained unknown whether the carboxylase and oxygenase reactions were tightly coupled functions as has been suggested by Lorimer and Andrews (1973) and Kung and Marsho (1977).
References:
Ahokas, H. 1978. Barley Genetics Newsletter 8:12-15.
Arnon, D.I. 1949. Plant Physiol. 24:1-15.
Baker, T.S., D. Eisenberg, F.A. Eiserling, and L. Weissman. 1975. J. Mol. Biol. Biol. 91:391-399.
Criddle, R.S., B. Dau, G.E.Kleinkopf, and R.C. Huffaker. 1970. Biochem. Biophys. Res. Commun. 41:621-627.
Fukuyama, T. 1978. Barley Genetics Newsletter 8:42-44.
Kung, S.D. 1976. Science 191:429-434.
Kung, S.D. and T.V. Marsho. 1976. Nature 259:325-326.
Lorimer, G.H. and T.J. Andrews. 1973. Nature 243:359-360.
Lorimer, G.H., M.R. Badger, and T.J. Andrews. 1977. Anal. Biochem. 78:66-75.
Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951. J. Biol. Chem. 193:265-275.
Nishimura, M., T. Takabe, T. Sukiyama, and T. Akazawa. 1973. J. Biochem. 74:945-954.
