We have produced several photosynthesis mutants by crossing stock bearing Robertson's Mutator to standard lines W23 and Mo17. Families with lesions in the cytochrome b6/f complex of the chloroplast thylakoid membrane (Taylor, 1986, in press), PSI (Hunt & Miles, MNL 58:68,1984), and coupling factor (Hunt, personal communication) have been identified previously. This communication is to report the characterization of a Mutator-induced PSII mutant.
The mutant was identified as segregating for a marked increase in chlorophyll fluorescence compared with wild type plants of the same family. Figure 1 shows a typical fluorescence induction trace of an excised leaf from a mutant plant. The fluorescence yield among individual mutant plants ranges from 2.8-5.5 times the wild type. In Figure 1 the fluorescence trace is not square as would be expected from a complete block of electron transport, but retains about 10% variable fluorescence. Traces from other mutant individuals ranged from just over 0% to 22% variable fluorescence.
LDS polyacrylamide gel electrophoresis of thylakoid polypeptides from the mutant shows that all of the major polypeptides associated with PSII (49, 45, 32, 23, 16 & 9 kDa) are decreased or missing. An unidentified band with an apparent molecular mass of 20.1 kDa is present among the polypeptides from the mutants but not among those from normal plants (see Figure 2). Of the polypeptides associated with PSII only the peripheral water-splitting components are encoded by the nuclear genome. Nevertheless, all of the chloroplast encoded core proteins of PSII are missing as well. At this point we have no evidence to suggest whether the gene which is disrupted by the Mutator element codes for a regulatory, structural or enzymatic polypeptide.
Chloroplasts isolated from the mutant showed greater oxygen consumption than wild type when incubated in the light with methyl viologen, ascorbate, DCMU (diuron) and diaminodurene (DAD). This requires normal electron flow through PSI. When incubated with p-phenylenediamine and ferricyanide the mutant chloroplasts produced only 10% as much oxygen as those from normal siblings. This is consistent with a block of electron transport through PSII (Table 1).
Leaves from the mutant plants were excised above the sheath at the three-leaf stage for the preceding tests. Following excision the plants generally died without producing any further growth. One individual plant continued to grow following the second defoliation, producing several leaves. Subsequent checks of the fluorescence induction kinetics and polypeptide composition of the plant showed that the characteristics described above did not change over time. The plant continued to grow for 90 days before it died. Among the fluorescence induction traces of the mutant individuals the surviving plant had the highest percentage of variable fluorescence. It appears that in this plant electron transport through PSII, though impaired, was sufficient to support growth for several weeks.
The oxygen evolution experiment was carried out using a sample of chloroplasts isolated from several plants. While the sample evolved only 11.8% as much oxygen as the wild type sample this value represents an average. The plant that survived must have had a rate of oxygen evolution (and so electron transport) significantly higher than the average.
Other hcf mutants identified by this lab are able to grow to as much as 25 cm before they die, in spite of a partial block in photosynthetic electron transport. Apparently, the surviving mutant was able to carry out electron transport at a level adequate to support growth but was not able to mature fully.
We conclude from the variability that we have observed among these mutant individuals and from the extended growth of a single plant that the putative Mutator mutation is variable in its effect. It may be that other Mutator-induced effects cause the variability we see among the hcf mutants. Or it may be that the actual mutation which causes the hcf phenotype is variable in its effect.
The identification of a Mutator-induced PSII mutant adds to the accumulating evidence that Mutator (Mu1) causes all of the types of photosynthetic mutations previously induced by chemical mutagens.
Figure 2. LDS-PAGE of thylakoid membrane polypeptides from mutant 1218-20 and wild type plants from the same family. Samples in lanes 1 and 3 were heated before being loaded onto the gel. ¨ PSII-associated polypeptides missing from 1218-20. Ð Unidentified band not present in wild type.
Bill Cook and Don Miles
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