II. 6. Chloroplast membrane protein differences between uzu, uzu-wst3, and a normal barley cultivar.
H. Ahokas, Department of Genetics, University of Helsinki, P. Rautatiekatu 13, 00100 Helsinki 10, Finland. "R"
The recessive uzu gene of barley is responsible for a special type of dwarfism (Tsuchiya 1976). A white streak mutant (wst3) in the uzu cultivar Akashinriki has been induced and described (Takahashi and Moriya 1969). The wst3 gene shows absolute linkages with uzu loci (Takahashi and Moriya 1969, Tsuchiya and Haus 1973). In conjunction with another study, I observed that the organization of the plastid lamellae in the wst3 mutant chloroplasts of the green tissue was somewhat different from that of a Finnish non-uzu cultivar.
The following questions were the subject of this preliminary study. First, is there any difference in the chloroplast membrane proteins between uzu and non-uzu genotypes? Second, could the wst3 mutant be detected in the chloroplastid membrane polypeptides? Third, can the putative differences in membrane polypeptides be detected in heterozygotes? Several reports of differences in barley chloroplast membrane proteins or polypeptides have been described in pigment mutants (Henriques and Park 1975, Machold and Høyer-Hansen 1976, Machold et al. 1977, Apel and Kloppstech 1978, Burke et al. 1979), while uzu phenotype is green.
Cv. Akashinriki (OUJ 659) was obtained from Dr. T. Konishi (Barley Germplasm Center, Okayama University, Kurashiki, Japan) and its wst3 mutant (BGS 0103) from Dr. T. Tsuchiya (Barley Genetic Stock Center, Colorado State University, Fort Collins, Colorado USA). The Finnish six rowed non-uzu cultivar Tammi is from the Hankkija Plant Breeding Institute (Hyryla, Finland). The plants and their Fl hybrids were grown under the same conditions in a greenhouse on fertilized Finnpeat B2 without any pesticide treatment and were free from diseases. Cv. Tammi and F1 hybrids with cv. Tammi as one parent were planted respectively 16 and 23 days later than the Akashinriki material in order to synchronize the development of the later stages when leaf material was prepared. Under our conditions, F1 hybrids of Tammi x Akashinriki head about one week earlier than cv. Tammi.
Fully expanded upper laminas were collected at the anthesis stage, and their chloroplasts isolated and the membrane proteins prepared and run on SDS-polyacrylamide slab gels as described in Ahokas (1980). Molecular weights were estimated on the gels as described elsewhere (Ahokas 1982).
The SDS-PAGE polypeptide distributions of the six different genotypes are shown in Fig. 1. Since much more leaf tissue is needed from the wst3 mutant for the preparations, contaminants of small molecules tend to remain in its tracks as background staining. The following differences were observed. The major polypeptide of Mr 13 kdalton had a distinctly higher concentration in the uzu cultivar and all their F1 hybrids. This band moves about as fast as the cytochrome c standard, and has a blue color after the Brilliant blue R staining, while the zone above it is violet.
Figure 1. SDS-PAGE slab gel of chloroplastid membrane polypeptides from the following materials: (a) cv. Tammi; (b) Tammi (female) x Akashinriki (uzu/ + ); (c) Tammi (female) x white streak Akashinriki (uzu wst3/ + + ); (d) cv. Akashinriki (uzu/uzu); (e) Akashinriki (female) x white streak Akashinriki (uzu wst3/uzu + ); (f) white streak Akashinriki (uzu wst3/uzu wst3). The zones showing apparent differences are indicated with arrows. Each track was loaded with protein corresponding to 112 µg of chlorophyll detected in the lipid extraction step before drying the protein sampLes.
At Mr 17 kdalton there is a wide band (one or more polypeptides) which is less intensive in cv. Tammi (Fig. la versus b, c, d, e, f).
At Mr 33 kdalton the content in cv. Tammi is lower than in the uzu cultivars or the F1 hybrids (Fig. la versus b, c, d, e, f).
At Mr 41 kdalton cv. Tammi has a relatively strong staining zone among the minor polypeptides (Fig. la). This zone is weak or lacking in the uzu types (Fig. ld, e, f). The Tammi x uzu F1 hybrids show an intermediate situation: their 41 kdalton band is weaker than that of cv. Tammi (Fig. lb, c).
A band that is evidently more intensive in the wst3 mutant only, appears at Mr 62 kdalton (Fig. lf).
The questions presented in the beginning can be answered as follows: in the uzu (Akashinriki) and non-uzu (Tammi) pair there are at least four differences in the chloroplastid membrane proteins. A single increase was evident for the homozygous wst3 mutant. Heterozygosity seems to be expressed in an intermediate level in the 41 kdalton polypeptide. To my knowledge, the Tammi versus Akashinriki pair is the first report of differences in chloroplastid membrane proteins in normal green barleys.
Acknowledgment:
The work was supported by the Emil Aaltonen Foundation.
References:
Ahokas, H. 1980. Cytoplasmic male sterility in barley. V. Physiological characterization of the msml - Rfmla system. Physiol. Plant. 48:231-238.
Ahokas, H. 1982. Cytoplasmic male sterility in barley. VIII. Lipozygenase activity and anther amino nitrogen in the msml - Rfmla system. Plant Physiol. 69:268-272.
Apel, K. and K. Kloppstech. 1978. The plastid membranes of barley (Hordeum vulgare). Light-induced appearance of mRNA coding for the apoprotein of the light-harvesting chlorophyll a/b protein. Eur. J. Biochem. 85:581-588.
Burke, J. J., K. E. Steinback and C. J. Arntzen. 1979. Analysis of the light-harvesting pigment-protein complex of wild type and a chlorophyllb-less mutant of barley. Plant Physiol. 63:237-243.
Henriques, F. and R. B. Park. 1975. Further chemical and morphological characterization of chloroplast membranes from a chlorophyll b-less mutant of Hordeum vulgare. Plant Physiol. 55:763-767.
Machold, O. and G. Høyer-Hansen. 1976. Polypeptide composition of thylakoids from viridis and xantha mutants in barley. Carlsberg Res. Commun. 41:359-366.
Machold, O., A. Meister, H. Sagromsky, G. Hoyer-Hansen and D. von Wettstein. 1977. Composition of photosynthetic membranes of wild-type barley and chlorophyll b-less mutants. Photosynthetica 11:200-206.
Takahashi, R. and I. Moriya. 1969. Inheritance and linkage studies in barley. IV. Linkages of four variegated mutants. Ber. Ohara Inst. Landwirtsch. Biol. 15:35-46.
Tsuchiya, T. 1976. Allelism testing in barley. II. Allelic relationships of three uzu genes. Crop Sci. 16:496-499.
Tsuchiya, T. and T. E. Haus. 1973. Allelism testing in barley. I. Analysis of ten mapped genes. J. Hered. 64:282-284.
