Mu-induced nuclear mutations affecting late stages of chloroplast biogenesis: gene nomenclature, screening methods, and progress in mapping and allelism testing
--Alice Barkan, Rodger Voelker, Janet Mendel-Hartvig, Michelle Nolasco, Macie Walker and David Johnson

The phenotype of "high chlorophyll fluorescence" (hcf) has been used to identify numerous non-photosynthetic mutants with near normal pigmentation (Miles, CD, Methods in Chloroplast Molecular Biology, 1982). hcf mutations are expected to define genes involved in chloroplast gene expression and assembly, as well as structural genes for the various photosynthetic enzymes. Because such mutants are almost invariably slightly pale green, we find it simpler to screen initially for pale green or yellow green mutants that are lethal at the three leaf stage. Subsequent analysis of seedling leaf proteins usually reveals the absence of one or more photosynthetic complexes.

Since many different nuclear-encoded functions contribute to the biogenesis of the photosynthetic apparatus, it is not surprising that such mutants arise at high frequency. In lines harboring active Mu transposons, we find approximately 1 new mutant in every 200 F2 families. While such mutants usually do exhibit some degree of increased chlorophyll fluorescence, we have chosen to stop using the hcf designation. Instead, we have assigned new gene nomenclatures that reflect more completely the variety of mutant phenotypes. Mutants are grouped into different classes according to the specific nature of the chloroplast defect. The assays used include Western analysis of the major chloroplast proteins, Northern analysis of chloroplast RNAs, and analysis of chloroplast polysomes.

The following mutants arose in the Mutator material generated by the Hake, S and Freeling, M groups at the University of California. These mutations segregate as single, recessive Mendelian traits. Many are unstable, exhibiting small revertant sectors of dark-green tissue.

psb mutants. psb mutants lack the protein components of the photosystem II core complex (PSII). Other chloroplast proteins accumulate to normal levels. We have isolated two independent mutants of this type (psb1 and psb2). Preliminary results suggest that these mutants are not allelic. psb1 was formerly called hcf134.

psa mutants. psa mutants lack the protein components of the photosystem I core complex (PSI). Other chloroplast proteins accumulate to normal levels. We have four independent mutants, psa1, psa2, psa3 , and psa4. Preliminary results suggest that these four mutants are not allelic.

pet mutants. pet mutants lack the protein components of the cytochrome f/b6 complex. Other chloroplast proteins accumulate to normal levels when plants are grown in low intensity light (100µE/m2s). Plants grown in higher intensity light have a five- to ten-fold reduction of PSII core proteins, suggesting that PSII proteins are lost due to photo-induced damage that occurs in the absence of the cytochrome f/b6 complex. pet1: uncovered by TB-8Lc; formerly called hcf121. pet2, pet3, pet4, pet5: preliminary results suggest that these four mutants are not allelic to one another.

crp mutants. crp mutants have unique defects in chloroplast RNA processing. hcf38 also falls into this category (Barkan et al., EMBO J. 5:1421-1427, 1986). crp1: fails to accumulate monocistronic petB and petD mRNAs and lacks cytochrome f/b6 proteins; formerly called hcf136. crp2: fails to degrade excised group II introns; formerly called hcf143.

cps mutants. cps mutants have global defects in chloroplast protein synthesis, as revealed by a decrease in the levels of all thylakoid membrane complexes and RUBISCO, as well as a decreased association of all chloroplast mRNAs with polysomes. Chloroplast mRNAs are unaltered in these mutants. hcf7 (isolated by Don Miles, CD from the Neuffer, MG collection) also falls into this category (AB, manuscript submitted). hcf7, cps1, and cps2 mutants define three separate complementation groups. hcf7: uncovered by TB-1La; this mutation causes a defect in the processing of the 16S rRNA. cps1: two independent alleles were obtained (cps1-1 and cps1-2 ); the phenotype of cps1-2 is the more severe. cps2: uncovered by TB-6Lc; formerly called hcf133; this mutation is unusual in that it causes a 20-fold decrease in RUBISCO and only a two-fold decrease in thylakoid membrane complexes.

cgx mutants. cgx mutants have global defects in chloroplast gene expression. These mutants have reduced levels of all thylakoid membrane complexes as well as RUBISCO, but chloroplast mRNAs and rRNAs are normal in size and abundance. This group is distinguished from cps mutants in that most or all chloroplast mRNAs are associated with polysomes. We have two independent mutants of this type, cgx1 and cgx2.

tha mutants. tha mutants have defects in thylakoid membrane assembly. These mutants lack more than one thylakoid complex but have normal levels of RUBISCO. The missing chloroplast-encoded components appear to be synthesized normally but fail to accumulate. hcf106 also falls into this category (Barkan et al., 1986; Martienssen et al., EMBO J. 8:1633-1639, 1989). tha1: causes a 10-fold reduction in PSII proteins, and a 5-fold reduction in PSI and cytochrome f/b6 proteins; the level of the ATP synthase is unaffected. tha2: causes a 10-fold reduction in the cytochrome f/b6 complex, a four-fold reduction in PSI and PSII, and a three-fold reduction in the ATP synthase. 


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