lsolation of Mutations Affecting the Development of Freezing - NCBI

Plant Physiol. (1996) 1 1 1 : 1 O1 1-1 O1 9

lsolation of Mutations Affecting the Development of Freezing Tolerance in Arabidopsis thaliana (L.) Heynh. Gareth Warren’*, Robert McKown2, Analuisa Marin, and Rita Teutonico3

D N A Plant Technology Corp., 6701 San Pablo Avenue, Oakland, California 94608 The study of QTL offers an alternative method of looking for the genes responsible for freezing tolerance. QTL affecting freezing tolerance have been demonstrated in barley, potato, and oilseed Brassica species (Hayes et al., 1993; Stone et al., 1993; Teutonico et al., 1995). A major determinant of freezing tolerance has been localized to chromosome 5 in wheat (Sutka, 1994). It is clear that the loci detected are agronomically significant, but they are not technically accessible to molecular characterization, since it is difficult to clone genes identified only as QTL. Again, there is also the possibility of missing important genes, in this case because they may not show allelic variation between the cultivars tested. The classical genetic approach would be to identify genes required for freezing tolerance by isolating mutations deleterious for this trait. The strengths of this approach are that it is capable of identifying every gene responsible for freezing tolerance and that the mutants’ phenotypes could suggest specific functions for the cognate genes. The weaknesses of the classical approach lie in the difficulty of cloning genes defined only by conventional genetics and the possibility that some mutants may be uninformative. For example, a mutation that exerted its effect by weakening the plant prior to freezing would be uninformative, but mutations having such an effect could probably be screened out. The cloning of genes defined only by conventional genetics has usually required the very laborious process of ”chromosome walking,” but the size of the Arabidopsis tkaliana genome and the molecular tools and information being developed for this species mitigate this difficulty. There has been one preliminary report of a screen for mutants of Arabidopsis deficient in freezing tolerance (Artus and Thomashow, 1992). A recessive, freezing-sensitive mutation was identified using a screen based on the electrolyte leakage assay (M. Thomashow, personal communication). We wished to isolate mutations that impaired freezing tolerance in A . tkaliana without using an assay (such as electrolyte leakage) that is conducted on excised leaves. We reasoned that such an assay might fail to detect injury restricted to other tissues, and also might be poorly suited for detecting certain types of injury within the leaf. Therefore, we chose apparent health and regrowth of intact plants after a freezing episode as the criteria by which to

We screened for mutations deleterious to the freezing tolerance of Arabidopsis fhaliana (1.) Heynh. ecotype Columbia. lolerance was assayed by the vigor and regrowth of intact plants after cold acclimation and freezing. From a chemically mutagenized population, we obtained 1 3 lines of mutants with highly penetrant phenotypes. In 5 of these, freezing sensitivity was attributable to chilling injury sustained during cold acclimation, but in the remaining 8 lines, the absence of injury prior to freezing suggested that they were affected specifically in the development of freezing tolerance. In backcrosses, freezing sensitivity from each line segregated as a single nuclear mutation. Complementation tests indicated that the 8 lines contained mutations in 7 different genes. l h e mutants’ freezing sensitivity was also detectable in the leakage of electrolytes from frozen leaves. However, 1 mutant line that displayed a strong phenotype at the whole-plant leve1 showed a relatively weak phenotype by the electrolyte leakage assay.

What genes are responsible for the development of freezing tolerance in hardy plants? A number of laboratories address this question by characterizing genes and proteins induced during cold acclimation (e.g. Houde et al., 1992; Neven et al., 1993; Nordin et al., 1993; Wilhelm and Thomashow, 1993; Castonguay et al., 1994; Dunn et al., 1994; Jarillo et al., 1994). Thomashow and co-workers have shown that the product of one such gene can cryoprotect the chloroplast in vivo (Artus et al., 1994); Hincha and colleagues (1990) have shown that cold-induced proteins are cryoprotective to thylakoids. However, it may be difficult to discover whether and how a cold-induced gene contributes to freezing tolerance, e.g. when no function is demonstrable in vitro and suppression of the gene’s translation by antisense RNA (which is usually not absolute) fails to generate a phenotype. Moreover, the usual techniques for identifying cold-induced genes may fail to identify cold-induced genes that share homology with constitutively expressed genes; also, some genes responsible for freezing tolerance may be constitutive rather than cold induced. Present address: Department of Biochemistry, Imperial College of Science Technology and Medicine, London, UK SW7 2AY. Present address: College of Integrated Science and Technology, James Madison University, Harrisonburg, VA 22807. Present address: Technology Forecasters, Inc., 1936 University Avenue, Berkeley, CA 94704. * Corresponding author; e-mail g.warrenQic.ac.uk;fax 44-171-

Abbreviations: LT,,, temperature at which 50% injury is incurred; QTL, quantitative trait loci; SFR, sensitivity to freezing.

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judge freezing tolerance. This presented the challenge of recovering mutations that were identified by injury, possibly lethal, to the plants that carried them. Also, there were severa1 unknowns that could influence the success of our approach: the frequency with which this type of mutation would occur in a mutagenized population, the frequency with which nonheritable factors would produce a phenocopy of the desired mutation (the leve1 of "noise" in the screen), and the relative frequency of mutations that would act by causing injury before freezing. It was not even certain that a whole-plant assay could reflect the proficiency of Arabidopsis to acclimate; if its roots were to cold acclimate poorly, as has been reported for wheat (Zhou et al., 1994), root injury and consequent plant death would obscure differences of freezing tolerance in the leaves. This report addresses these considerations and demonstrates that it is possible to isolate mutations specifically deleterious to freezing tolerance by means of a whole-plant assay.

MATERIALS AND METHODS Plant Crowth Conditions for Screening

The pedigreed mutant set of James and Dooner (1990) derives from the Columbia ecotype of Arabidopsis thaliana. Seeds from this set were planted in a grid pattern with the location of each specifying its pedigree. Seedlings were grown for approximately 5 weeks with 8-h photoperiods at 200 to 400 pmol m-* s-' in a shaded greenhouse at 16 to 20"C, then transferred to a growth chamber at 4°C for 2 weeks with 8-h photoperiods at 220 pmol m-* s-'.

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Recovery and Observation of lnjury

After freezing, plants were returned to a shaded greenhouse at 16 to 20°C. Plants were observed daily for up to 5 d, and those with apparent injury or reduced health relative to wild-type control plants were noted. If still alive, these plants (which represented putative mutants) were transplanted and encouraged to grow to maturity. Gene and Mutant Designations

The unique gene designation SFR (SFR1, SFR2, etc. for different loci; sfrl, sfr2, etc. for corresponding mutant alleles) has been registered with Dr. David Meinke, curator of Arabidopsis genetic nomenclature. SFR was chosen to abbreviate sensitivity to freezing. If future work demonstrates the identity of an SFR gene (e.g. SFR5) with that of a previously published gene, the earlier gene designation will take priority and the allele names of cognate mutations ( e g sfr5-1, sfu5-2) will be changed accordingly. Crosses for Complementation Testing

AI1 crosses were performed using only one of the parents as the pollen donor. The mutant line was the pollen donor in backcrosses. In crosses between mutant lines, the parent to act as the pollen donor was chosen at random. To confirm that the progeny of such crosses were truly F, hybrids (rather than being due to contaminating pollen), we selfed an F, plant that had been scored as freezing tolerant from each cross except FS78 X FS79, and we tested for the expected segregation of freezing sensitivity in a sample of 96 F, plants. Measurements of Electrolyte Leakage

Freezing of Whole Plants

For freezes of 60 min, we used a liquid-nitrogen-cooled, controlled-rate freezer (Cryo-Med, Marietta, GA) with a cooling rate of 2"CJmin. For freezes of 24 h, we employed a domestic food freezer modified by the addition of a 7-W fan on each shelf and controlled by an electronic thermostat. This arrangement resulted in a regular oscillation in the chamber's air temperature, with a period of approximately 5 min, a magnitude of 0.8"C, and a reproducibility of 0.1"C. In "Results" we discuss varying the freezing temperature; this refers to the nadir (lower limit) temperature in these oscillations. Thus, a temperature setting of -6.O"C refers to the condition at which chamber air temperature oscillated between -5.2 and -6.O"C. The initial cooling rate was approximately 0.25"CJ min. Thawing, which was achieved by removal of the plants to ambient temperature (20"C), was rapid (approximately 5 min) for aerial structures, but longer (approximately 30 min) for soil. Probes of soil temperature indicated that equilibrium was reached approximately 4 h after the start of a freezing cycle, and remova1 of samples at this time showed that all plants and a11 soil divisions were frozen (i.e. ice nucleation had already taken place). Therefore, a11 plant structures, including roots, would remain frozen for at least 20 h of the usual 24-h cycle.

Six-week-old seedlings were incubated in growth chambers with 8 h of light per day at 220 p E mp2, at either 16°C (to provide nonacclimated plants) or 4°C (for cold acclimation). After 14 d, young leaves between 40 and 80% of the size of fully expanded leaves were harvested. The leaves were washed and placed in 3-mL aliquots of distilled water, two leaves to a tube. Tubes were equilibrated to -2"C, nucleated with ice chips, and allowed to remain for 1 h at -2°C to re-equilibrate. The bath temperature was now ramped down to -10°C at a rate of 2°C h-l. Two tubes of each type were withdrawn every half-hour (including two withdrawn at the start) to provide a series of duplicate samples that had experienced nadir temperatures of -2, -3, -4"C, etc. The withdrawn tubes were held at 4°C for 16 h and then warmed to room temperature and shaken for 15 min. Electrical conductivity was measured. The tubes were then heated to 65°C for 30 min to release all electrolytes, cooled to room temperature, and shaken. A second measurement of conductivity was made to determine the total content of electrolytes in each sample. Division of the first measurement by the second indicated the proportion of total electrolytes that had leaked from the leaves in each sample during the freezing protocol. Means were taken from duplicate samples and plotted with estimates of SE. Logistic curves were fitted to each set

Mutations Affecting Freezing Tolerance in Arabidopsis of data. The logistic function (Causton and Venus, 1981) may be written:

f ( t ) = c + A(l

+ e(KP--Kt))-l

with asymptotes at f(t) = c and f(t) = c + A, and inflection at t = p. We adjusted parameters c, A, K , and p to minimize the sum of squares of differences between f(t) and the sample means for each data set. We did not follow the recommendation of Von Fircks and Verwijst (1993) to use a derivative of the logistic function (the Richards function) because the small improvements in curve fit do not appear to justify the introduction of another parameter, unless data with very small SE values are available. Mapping

The sfr2, sfr4, and sfr5-1 lines (FS61, FS67, and FS68, respectively), which have a Columbia background, were crossed with Landsberg evecta. The F, and F, generations were allowed to self, and the progeny of individual F, plants were freeze-tested to reveal (by the segregation of phenotypes) the sfr genotypes of their respective parents. DNA was isolated from families (pooling DNA from 18 or more F, plants per family to assure complete representation of each family's parenta1 genome), digested with EcoRI, and subjected to a Southern blot. Probes were nonradioactively labeled by the digoxigenin method using the Genius System V2.0 kit from Boehringer Mannheim. Hybridization and detection methods followed the manufacturer's recommendations. Recombination was estimated by the maximum likelihood method (Mather, 1951) and converted to map distances by the mapping function of Kosambi (1944). RESULTS Defining the Conditions for Screening

It was first desirable to find conditions for freezing intact specimens of A. thaliana (L.) Heynh. that would distinguish cold-acclimated from nonacclimated plants. Our tests utilized wild-type, vegetatively growing seedlings of ecotype Columbia, the ecotype that would be screened subsequently for mutants. Initially, we cooled plants to various temperatures at a rate of 2°C min-' and then held temperature constant for 60 min before rapid thawing. A holding temperature of -8.O"C gave the clearest distinction (by differences in visible damage, observed 2 d later) between cold-acclimated and nonacclimated plants. However, damage to nonacclimated plants increased considerably from the center to the outside of a tray; we ascribed this to slower temperature equilibration at the center. To circumvent this effect, we extended the plants' low-temperature exposure to 24 h and reduced the cooling rate to 0.25"C min-'. We varied the holding temperature under this new regime and found that temperatures between -4.9 and -6.O"C clearly differentiated cold-acclimated from nonacclimated plants. Below -6.0°C, partia1 damage to coldacclimated plants reduced the clarity of the distinction.

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Above -4.9"C, the incompleteness of the damage to nonacclimated plants made their sensitivity less obvious. We tried reducing the holding period to 12 h, but this reintroduced variability according to position within a tray, and so we abandoned this modification. For screening we chose the lowest temperature compatible with clear detection of the cold-acclimated state (-6.O"C) because we wanted to be able to detect mutants only slightly less hardy than the wild type. lsolating Mutants

Because the testing process might be lethal for the desired mutants, we screened a collection of mutagenized germ plasm that was organized so that siblings of a putative mutant could be found. The collection of James and Dooner (1990) consists of M, seed pools derived from 1804 M, plants from ethyl methanesulfonate mutagenesis of the ecotype Columbia. We screened 2 plants from each M, pool. In 30 of the 1804 pools, just 1 of the 2 screened plants appeared sensitive to freezing, whereas in 2 pools, both plants were injured. (Thus, we observed a total of 34 plants injured out of approximately 3600 tested.) Injury was not always lethal: 14 of the injured plants survived and grew to maturity. Their M, progeny were subsequently tested for freezing sensitivity. However, this left 18 pools from which the putative mutation had to be sought among siblings of a killed plant. Eight siblings from each such pool were grown to maturity, and their M, progeny were tested for freezing sensitivity. By testing the M, families we could identify M, parents that had bred true for freezing sensitivity, because all M, members of such families would be freezing sensitive. This was true in 13 pedigrees; we were thus left with 13 truebreeding lines, discounting duplicate M, lines from the same pedigree. The other 19 pedigrees failed to yield truebreeding M, plants. Some gave no freezing sensitives at a11 among the M, plants; this is attributable to purely coincidental injury (from some other cause) to the pedigree's representative in the original screen. However, some pedigrees gave apparent segregation of freezing sensitivity among M, families without yielding any M, families in which all members displayed the trait. Severa1 explanations for this are plausible: (a) among a pedigree's M, plants, mutant homozygotes were inviable or infertile, so that only heterozygotes gave rise to progeny; (b) the mutation had incomplete penetrance, therefore giving the appearance of segregation even among progeny of homozygotes; or (c) by chance, none of the M, plants that had grown out were homozygous. Because of the last possibility, we repeated our sampling of M, siblings, grew them out, and tested their M, families. However, no more truebreeding lines were obtained. We decided not to pursue mutations likely to be (a) homozygous inviable or sterile or (b) incompletely penetrant, and therefore abandoned all pedigrees that had failed to yield a true-breeding M, plant. Since our goal was to isolate mutations causing a specific deficiency in the process of cold acclimation, we wished to identify and eliminate mutant lines in which freezing sensitivity was due to injury incurred before freezing (during

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the period of cold acclimation). Mutations of the latter type (i.e. chilling-sensitive mutants) have been previously observed by Hugly et al. (1990) and Schneider et al. (1995). Seedlings of the 13 lines were cold acclimated and then examined for chilling injury by two criteria: visible damage during cold acclimation and stunted growth upon a return to normal temperature. Five mutant lines showed injury during acclimation, in every case by both criteria. These five lines were therefore eliminated from further consideration. We tentatively concluded that the remaining eight lines contained mutations causing more specific deficiencies in the processes that confer freezing tolerance on the wild-type plant. The freezing-sensitive phenotypes by which these lines were recognized are illustrated in Figure 1. The controls shown in this figure, which are plants of the mutant lines grown and cold acclimated in parallel but not frozen, illustrate the absence of chilling injury in these lines. Penetrance, Dominance, and Mendelian Inheritance

We assessed the penetrance of each mutant line's phenotype with a minimum of three separate tests. We measured not only the frequency with which mutant phenotypes were distinct from the typical wild type, but also the frequency with which wild-type controls, tested in parallel, were distinct from the typical mutant phenotype. The effective penetrance is given as the product of both frequencies (Table I). Penetrance was high enough in each case to permit genetic analysis. To assess dominance, backcrosses were made to wild types of the same genetic background (Columbia). Twelve F] plants from each cross, divided between two separately conducted tests, were frozen alongside wild-type and homozygous mutant control plants, and the F5 plants' freezing tolerance was compared with both. As reported in Table I, F, phenotypes were in most cases similar to wild type, indicating that the respective mutations are recessive. The Fj plants derived from backcrosses of lines FS68 and FS79 were exceptional, having intermediate levels of freezing tolerance, and thus showing codominance. Figure 1. Phenotypes of mutant lines in comparison to wild type (wt). Three plants of each line (Tests) are shown 9 d after freezing under standard conditions (24 h at -6.0°C). Two plants of each line (Controls), with a parallel history except for freezing, are shown as controls for any injurious effects of the coldacclimation period.

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F, plants from the backcrosses were allowed to self and the F2 plants were screened to observe segregation of the mutant phenotype. In all cases the segregation ratios differed significantly from 15:1 but not from 3:1. This is consistent with single-gene segregation and refutes the hypothesis that freezing sensitivity is caused by the combined effects of two unlinked recessive mutations (Table II). Another two-gene hypothesis is that freezing sensitivity is caused by the combined action of a recessive mutation and a dominant mutation at unlinked loci. This predicts an F2 segregation ratio of 13:3, which is experimentally difficult to distinguish from 3:1. However, according to this hypothesis (or any hypothesis requiring two unlinked genes), only 1 in 16 F2 progeny will breed true for the mutant phenotype. Therefore, we examined the genotypes of F2 individuals by freezing families of their F3 progeny. For every mutant line, the ratio of F2 individuals breeding true for freezing sensitivity to all other F2 individuals differed significantly from 1:15 but not from 1:3 (Table II). This indicates that in each of the 7 mutant lines tested (FS79 was omitted), freezing sensitivity was caused by mutation in a single nuclear gene. Complementation

The eight mutant lines were crossed together in all pairwise combinations, except for some involving FS79 (see below). Approximately 12 F, progeny from each of these crosses were frozen alongside control plants of both parental lines and wild type. In the majority of such tests (Table III), the F, progeny appeared less freezing sensitive than either parent and usually no more sensitive than the wild type (an example is shown in Fig. 2a). Such a result indicates complementation and therefore suggests that the respective mutations affect different genes. The only exception involved the F, progeny from the cross FS68 x FS79, which were as freezing sensitive as either parent and clearly distinct from wild type (Fig. 2b). The lack of complementation here suggests that the mutations in the FS68 and FS79 lines are allelic. To confirm their allelism, we allowed the F] progeny from FS68 X FS79 to self, and froze

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Table 1. Penetrance and dominance of mutations Mutant Line FS59 FS61 FS65 FS67 FS68 FS69 FS78 FS79 a

Mutants Distinct from Wild Type

Wild Type Distinct from Mutant

59/61 6 816 8 69/69 5615 6 56/57 44/44 5615 6 5 815 8

69/72 73/73 68/69 5 715 7 56/56 44/44 5 715 7 63/63

Effective

Phenotypes of Fla

lnference

Penetrance

Mutant

lntermediate

Wild type

92% 100% 98% 100% 98% 100%

O O O O 3 O O 1

O O O 4 8 O O 6

12 9 12 20 1 12 13 6

100% 100%

Recessive Recessive Recessive Recessive Codominant

Recessive Recessive Codominant

F, plants derived from backcrosses to ecotype Columbia.

the F, plants. A11 108 of the F, plants examined were freezing sensitive. The absence of segregants with wildtype freezing tolerance refutes other possible explanations for the lack of complementation in the F, progeny and confirms allelism. After reaching this conclusion, we performed no additional crosses with FS79.

Electrolyte Leakage Assays The plasmalemma may be the primary site of freezing injury in nonacclimated plants (Steponkus, 1984).The leakage of electrolytes from frozen and thawed tissues is a sensitive indicator of loss of integrity by the plasmalemma, and it has been commonly used to assay freezing injury (reviewed by Calkins and Swanson, 1990). We applied this assay to the mutants to determine whether their freezing sensitivity was manifested by increased sensitivity of the plasmalemma in leaf tissues. We also wished to assess the applicability of the electrolyte leakage assay as a screening method for mutants. The electrolyte leakage assay was applied both to nonacclimated and cold-acclimated leaf tissue of each genotype. It was not technically feasible to conduct a11 the tests simultaneously; therefore, leaf tissue from wild-type plants, grown and treated in parallel, was included as a control each time the assay was conducted. For each data set, a best fit to the sigmoid logistic function was calculated. The inflection point of each fitted curve was taken as the best estimate of LT,,, and confidence limits for this temperature were calculated based on the goodness of fit of the curve. The data and estimates of LT,, are presented in Figure 3a. The freezing tolerance of nonacclimated leaf tissue is depicted in the data identified by open symbols. In most cases, the inferred LT,, value for mutant tissue was very close to that for the wild-type control. The inferred LT,,

Description of Freezing Sensitivity and Pleiotropic Effects Since only two of the eight mutant lines contained mutually allelic mutations, the eight lines defined seven genes that were required for freezing tolerance after cold acclimation. In accord with convention, we named these genes for the mutant phenotype (sensitivity to freezing): they are SFRl to SFR7. The correspondence of mutant alleles (sfvl, etc.) to mutant lines is given in Table IV. As is apparent from Figure 1, the mutant lines do not manifest freezing sensitivity in identical ways. The differences we observed are summarized in Table IV (third column). In three of the lines we also observed other heritable characteristics that distinguished them from wild type (Table IV, fourth column). A11 such characteristics were recessive. To determine whether these characteristics were pleiotropic effects of the sfr mutations, we examined their cosegregation with freezing sensitivity. The F, populations described in Table I1 were used. There was complete cosegregation in each case (Table IV, fifth column), indicating that the other phenotypes were indeed pleiotropic effects of the sfr mutations.

Table II. Segregation of phenotypes and genotypes Phenotypes of F,a lndividuals Mutant Line

FS59 FS61 FS65 FS67 FS68 FS69 FS78 a

Cenotypes of F, lndividuals

Probability of chance deviation from

Observed segregation

Observed segregation

Probability of chance deviation from

Wild type/intermediate

Mutant

15:l

3:l

Other

m/m

15:l

80 54 88 67 88 74 75

22 22 39 27 39 20 17