Update on a putative Mutator-induced chloroplast mutant

Some time ago, we described a chloroplast mutant that seemed to have been induced in one of our Mutator lines. This mutant (which we call 3366) was first found as a single striped plant in the outcross progeny of a male parent Mutator plant and a female standard Q60 line. This plant was crossed reciprocally to a B70 standard line, and 50 seeds from each of the outcrosses planted. In the female outcross progeny, we found that 17 plants were dead or dying by the fourth or fifth leaf stage, one plant was severely striped, while the remainder appeared normally green. The viable plant tissue that could be seen in the dying plants suggested that this class consisted of wholly yellow-green or heavily striped plants. The progeny from the reciprocal cross, in which the striped plant was used as a male parent, consisted entirely of green plants. Curiously enough, we have found recently that in crosses involving a modified B73 line with the WSP type of cytoplasm (kindly provided by Don Duvick of Pioneer Hi-Bred International), which has a moderate mutator activity and contains Mu1-like elements (unpublished observations), a striped plant was also recovered. The striping pattern we saw in this case, we must emphasize, was not similar to that seen in plants with the WSP type of cytoplasm, but was very much like that found in the apparently cytoplasmically-inherited striping pattern found in the Mutator stock.

In both of these mutants, further reciprocal crosses have confirmed that the yellow-green striping condition is maternally inherited. The first mutant line, 3366, has been crossed reciprocally for four generations, and has always given the same pattern of inheritance (i.e., the striped condition was transmitted only through the female). Normal green sibling plants, when reciprocally crossed, have never produced striped seedlings. Furthermore, no striped seedlings have been seen in the progeny of selfs of the male outcross progeny or the self progeny of outcrosses of green sibling plants. In progenies of striped plants which were pollinated by standard lines, various numbers of striped plants were found with considerable variation in regard to the degree of striping exhibited by the plants. In such crosses, at one extreme there were some cases that gave no striped seedlings (i.e., they were totally green); at the other, there were ears that produced seedlings consisting entirely of mutant tissue (i.e., they showed no stripes). We also noted that the ears giving striped seedlings formed a continuous spectrum of types between these two extremes. There was some correlation, however, between the degree of plant striping of the female parent and the frequency of striped, entirely green or entirely yellow-green seedlings present in the outcross progeny. Generally, the more intense the striping in the female parent, the more striped, and entirely yellow-green, were the seedlings obtained. The Pioneer striped mutator plant has only been crossed reciprocally for two generations, and so far is behaving like the original striped line.

We have carried out a comparative morphological examination by electron microscopy of the plastids in normal and in yellow-green plantlets of line 3366. Transverse sections were taken from about the first third of young leaves, and prepared and stained using conventional techniques. We found that bundle-sheath chloroplasts were quite normal in appearance in the mutant line; mesophyll chloroplasts, on the other hand, appeared abnormal. They were similar in abundance and size to those seen in the tissues of a normal plant; however, gross abnormalities were apparent in the grana stacks. These were few in number compared to normal mesophyll chloroplasts, and seemed to be either very large or very small in size, the intrathylakoidal membranes appearing compressed and often convoluted and distorted. In some cases partial disintegration of the membranes had occurred, no doubt increasing as development proceeded and leading, ultimately, to plant death. We suspect that these mesophyll chloroplasts are also largely unfunctional. We found that when the leaves were removed from wholly yellow-green plantlets, new leaf growth occurred from the cut bases. If the young leaves were not harvested, however, the plants died. This cycle could be repeated until the endosperm was depleted.

We have examined the leaf chlorophyll fluorescence induction kinetics of mutant 3366. The yellow-green mutant leaf regions of both the completely yellow-green leaves and the leaves with yellow-green and green sectors displayed a two- to three-fold higher initial yield of fluorescence and kinetics, indicative of a lesion in electron transport on the reducing side of photosystem II. Blocks in photosystem I, plastocyanin, cytochrome f/b563 or plastoquinone mediated electron transport would generate this fluorescence data.

We have examined the polypeptide composition of thylakoids isolated from fully yellow-green 3366 plantlets and green siblings. On performing lithium dodecylsulfate polyacrylamide electrophoresis of gently solubilized membranes, we found no cytochrome f and no cytochrome b563 when gels were stained for heme. Heme-staining works well for cytochrome f, but poorly for cytochromes such as cytochrome b563, where the heme is not covalently bound. We also observed an approximate 50 percent reduction of the CP1-protein, believed to be the reaction center of photosystem 1. Two nonallelic nuclear maize mutants are known that block cytochrome f/b563 assembly, hcf*-2 and hcf*-6. Neither of these reduces the CP1-protein (Metz et al., Plant Physiol. 73:452-459, 1983). The cytochrome f/b563 complex can be isolated, and is known to comprise nuclear-encoded Rieske Fe-S proteins and three plastome-encoded polypeptides: apo-cytochrome f, apo-cytochrome b563 and subunit 4. We compared Western blots of mutants and normal thylakoids (antisera were kindly provided by W. Taylor, Berkeley) and found no cytochrome f or subunit 4 cross-reacting material and a reduced level of the Rieske Fe-S polypeptides. In the mutant we observed two polypeptides that bound anti-cytochrome b563, which were apparently 2-4 kD greater in molecular weight than the normal cytochrome b563. There was cross-reacting material of this apparent size class in normal thylakoids. We found no reaction in the mutant with a polypeptide corresponding to the normal cytochrome b563. This anti-cytochrome b563 serum also cross-reacts strongly with the Rieske Fe-S polypeptides. However, because we find no interaction of any of the other three antisera with additional polypeptides of either mutant or normal thylakoids, we believe the new polypeptides in the mutant are related to cytochrome b563 and not the Rieske Fe-S polypeptides. They may be unprocessed and partially processed apo-cytochrome b563.

We have compared chloroplast DNAs of mutant 3366 and wild type lines. Chloroplast DNAs were isolated by the method of Kolander and Tewari (Biochim. Biophys. Acta 402:372, 1975). Restriction endonucleases HindIII, PstI and BamHI were used to digest the DNAs. The fragments were fractionated by electrophoresis on agarose gels which were used subsequently in Southern blot analyses. Visual examination of the gels revealed no apparent differences between the restriction fragment patterns of wild type and mutant 3366 lines. Several Southern hybridizations, using an internal fragment of the cloned Mu1 element as a probe to blots of these gels, failed to detect any homology with chloroplast DNA restriction fragments of both wild type and mutant lines. However, we did occasionally observe a faintly hybridizing band at a location between chloroplast DNA fragments in the mutant line that was about 4 kb in size in the HindIII and BamHI profiles. We cannot, of course, rule out the possibility that this may be due to contamination of chloroplast DNA by genomic DNA. However, we have demonstrated that the mutant line has about 20 to 30 copies of Mu1-like elements in the genome, and these are randomly distributed in BamHI and HindIII hybridization profiles. This is not consistent with the appearance of a single band in chloroplast DNA hybridization profiles should genomic DNA contamination have occurred. We have no explanation for this intriguing, and somewhat perplexing, result at the present. Our observation that the mutant line is deficient in cytochrome f led us to examine, again by Southern blot hybridization, these gene sequences in the chloroplast DNA of the mutant line. We used a spinach cytochrome f gene (kindly provided by R. Herrmann) as a probe to wild type and mutant chloroplast DNAs digested with HinfI and PstI. We found no discernible differences in the hybridization patterns of both lines.

We cannot rule out the possibility that Mutator and Mu1 elements are involved in the induction of this mutant. The deficiency in the chloroplast-encoded polypeptides of the cytochrome complex raises the possibility of a Mu1 insertion that occurred at one or another structural genes, or alternatively, at a locus specifying a protein essential for their incorporation into the membrane system of the chloroplasts. Genetic exchanges between nuclear and organellar DNAs are, by no means, improbable (see, for example, J.N. Timmis and N. Steele Scott, Nature 305:65, 1983). Perhaps the strong conservation of chloroplast DNA size led to the rapid excision of a Mu1 element from one of these sites which, nevertheless, left a "footprint" resulting in a mutation. A detailed analysis, possibly involving DNA sequencing, would characterize the putative mutations at these loci.

In conclusion, we contend that the appearance of striped leaf mutants, similar in phenotype, in two lines (i.e., 3366 and B73 with the WSP type of cytoplasm), obtained from entirely different sources but having in common mutator activity and Mu1-like elements, strongly suggests that this unique system may be playing a role in the generation of chloroplast mutations.

Robert V. Masterson, David W. Morris, Donald S. Robertson, Mary-Jane Skogen-Hagenson, Judith G. Wheeler and Mary L. Polacco

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