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Key-word index: Arabidopsis thaliana, Brassicaceae, genetic, mutant, chilling-sensitivity, cold-sensitivity
In this study chilling sensitive mutants of the resistant plant Arabidopsis thaliana were isolated and genetically characterized in order to ascertain the number of loci that can contribute to the chilling sensitive phenotype.
Growth rate. To determine the critical temperature for growth of the class 1 mutants, plants were grown at 22C until 7 days old. Pots were then transferred to growth chambers set at different temperatures. Five plants were removed and weighed separately every 3 days for 15 days. The average weight was plotted against the number of days at the chilling temperature, then the growth rate was calculated from the linear section of the graph.
The chilling sensitive mutants fall into four phenotypic classes. Class 1 mutants first turn yellow, wilt and eventually die (Hugly, et al 1990). Older leaves of class 2 mutants show the same response, but younger leaves of the rosette are completely healthy. Class 3 mutants develop yellow patches which are not always contiguous and the veins remain green. In class 4, the part of each leaf closest to the center of the rosette turns yellow. Plants in classes 2, 3, and 4 eventually flowered and set seed after several weeks at the chilling temperature. Class 1 mutants die after three days exposure to chilling temperatures and cannot be rescued after this time by a return to the normal growth temperature. Scanned photographs of representatives from each class are available from in the image directory of AAtDB (http://weeds.mgh.harvard.edu): Class 1, chs1-1 (PM11), is in file cs-3097.jpeg; chs1-2 is in file st106.gif; Class 2, chs4-1 (ST13), is in file st13.gif; Class 3, chs5 (ST34), is in file st34.gif; and Class 4, (chs11) (ST36), is in file st36.gif. The URL address is, for example, gopher://weeds.mgh.harvard.edu/I9/arabidopsis/image/st13.gif. In addition, seeds will be available from the Arabidopsis Biological Resource Center (ABRC) at Ohio State (614-292-9371 for seed orders, 614-292-0603 FAX, seeds@genesys.cps.msu.edu).
Mutants were backcrossed to wild type and the phenotype of the heterozygous F1 plants were scored to assess recessiveness or dominance of the mutation (see Table 1). Most of the mutants were recessive; ST117 and PM2 were dominant. All mutants except ST64 and ST119 segregated in a manner (3:1) which indicates that a mutation at a single locus is responsible for the mutant phenotype. Segregation of ST64 and ST119 resulted in more wild type plants than predicted; mutations in two loci may be required for the sensitive phenotype.
Segregation Mutant strain, locus M2 batch dominance resistant sensitive, X^2 (a) Class 1 Plant dies ST106 (chs1-2) 3 R 79 28 0.1 PM11 (chs1-1) 1 R 294 87 0.9 b ST117 (chs2-2) 3 D 55 145 0.7 PM2 (chs2-1) 1 D 19 59 0.0 ST119 (chs3) 3 R 205 45 6.5 Class 2 Old leaves die ST13 (chs4-1) 2 R 185 51 1.5 ST35 (chs4-2) 2 R 137 49 0.2 ST64 (chs4-3) 2 R 405 64 32.2 Class 3 Chlorotic patches ST34 (chs5) 2 R 37 10 0.4 ST39 (chs6-1) 2 R 73 23 0.1 ST48 (chs6-2) 2 R 70 16 1.9 ST83 (chs6-3) 3 R 171 49 0.9 Class 4 Chlorotic middle ST23 (chs7) 2 R 136 44 0.0 ST25 (chs8) 2 R 29 10 0.0 ST27 (chs9) 2 R 162 70 3.3 ST28 (chs10) 2 R 169 69 2.0 ST36 (chs11) 2 R 53 17 0.0 ST45 (chs12) 2 R 14 6 0.3 ST74 (chs13) 2 R 104 31 0.3 ST88 (chs14) 3 R 182 65 0.2 ST115 (chs15) 3 R 28 10 0.0a. Predicted ratio of 3:1 (resistant:sensitive) for recessive and 1:3 (resistant:sensitive)for dominant mutants. At P = 0.05, X^2 of 3.84 or above would lead to rejection ofthe hypothesis of a single recessive nuclear mutation. b. (Hugly, et al 1990).
In order to ascertain the number of complementation groups giving rise to each phenotype, mutants were crossed to each other within classes. Within class 1, PM11 and ST106 are in the same complementation group. These mutants are not siblings, as they were derived from two independent batches of mutagenized seed (RM2 batchS in Table 1). Complementation between Class 1 mutant strains ST117 and PM2 could not be determined since they have dominant mutations. No resistant plants wre found in an F2 population from a cross between PM2 and ST117, thus they are members of a single locus. The other recessive mutant in this class, ST119, complements PM11 and ST106; in addition, resistant plants arise in the F2 populations from crosses between either PM11 or ST119 and PM2 or ST117. Thus, three complementation groups were found for the five mutants showing the Class 1 phenotype.
Crosses between mutants in class 2, or between PM11 or ST106 and class 2 result in sensitive F1 plants. The F1 phenotype implies that Class 2 mutants are in the same complementation group as PM11 and ST106, in which case all F2 progeny should be sensitive. However, resistant plants arise in the F2 generation, although there are fewer than expected for independent segregation of two recessive mutants (data not shown). Class 3 mutants, therefore, are possibly in the same complementation group as PM11 and ST106.
Mutants in class 3 fall into two complementation groups: ST39, ST48 and ST83 are in one and ST34 is in another.
In contrast to the above two groups, in which the same loci were mutated repeatedly to generate the sensitive phenotype, the nine mutants in Class 4 are in different complementation groups.
In order to determine the critical temperature for chilling damage in class 1 mutants, growth rates were measured upon a shift from 22C to a range of temperatures from 10C to 28C. As seen in figure 1, three isolates, PM11, ST119 and ST106, ceased growth when shifted to 18C or below. The growth rate of PM11 at different temperatures has been measured previously (Hugly, et al 1990) and, similarly, was found to decrease at 20C and to cease at 18C. The dominant mutants, PM2 and ST117, grew at 18C but ceased growth at 14C. As with PM11, these mutants could be rescued if transferred back to 22C within three days of exposure to chilling temperatures. The critical temperature for development of a phenotype in the mutants which do not die was harder to assess. In many of the mutants, a weak phenotype appeared at one temperature level and a strong phenotype at the next lower level. Mutants ST13, ST39, ST48 and ST35 developed a strong mutant phenotype at 18C; mutant ST64 at 14C. The other mutants tested (ST23, ST25, ST27, ST28, ST34, ST45, ST36, ST74, ST83, ST88 and ST115) did not develop a strong phenotype unless chilled at 10C.
Previous studies with chs1-1 (PM11) suggested that newly- synthesized chloroplast-localized proteins fail to accumulate after chilling (Schneider, et al, in press). In vitro import of proteins into the chloroplast was normal in mutant cells at low temperatures; an alternate possibility is that there is a defect in chaperonins that assisst folding of proteins after import. Protein synthesis in class 1 and 2 mutants was investigated by labeling leaves at normal temperatures, or after three days of chilling, with 35S-amino acids. The pattern of change in labeled proteins is the same as that previously noted in chilled PM11 leaves for mutants ST106, PM2 and ST117. The pattern in mutants ST64 and ST35 is similar but weaker, and the pattern in ST13 is unchanged in chilling conditions (data not shown). These results suggest that Class 1 and Class 2 mutants affect the same process, to which at least three loci contribute.
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