maternal effects govern variable dominance of two abscisic ... · maternal effects govern variable...

Plant Physiol. (1994) 105: 1203-1208

Maternal Effects Govern Variable Dominance of Two Abscisic Acid Response Mutations in Arabidopsis thaliana'

Ruth R. Finkelstein*

Department of Biological Sciences, University of California, Santa Barbara, California 931 06

Three abscisic acid (ABA)-controlled responses (seed dormancy, inhibition of germination by applied ABA, and stomatal closure) were compared in wild-type versus homo- and heterozygotes of two Arabidopsis fhaliana ABA-insensitive mutants, abil and abi2. We found that sensitivity of seeds to applied ABA is partially maternally controlled but that seed dormancy is determined by the embryonic genotype. The effeds of the abi l and abiZ mutations on ABA sensitivity of seed germination ranged from recessive to nearly fully dominant, depending on the parental source of the mutant allele. This maternal effect disappeared during vegetative growth. Stomatal regulation in heterozygotes showed substantial variability, but the average water loss was intermediate between that of homozygous mutants and wild type.

The phytohormone ABA can regulate many processes in plant growth and development, including embryo matura- tion, seed dormancy, transpiration, and adaptation to environmental stresses (Zeevaart and Creelman, 1988). Genetic analyses, using either ABA biosynthetic or response mutants, have provided support for the view that ABA is the endogenous mediator for many, but not necessarily all, of these processes (reviewed by Finkelstein and Zeevaart, 1994). Two of the best-studied responses to ABA are induction of dormancy during seed development and stomatal closure during water stress. These are, respectively, representative of slow (>30 min) and fast (<5 min) responses, which have been proposed to act through different signal transduction pathways (Zeevaart and Creelman, 1988). As expected, ABA biosynthetic mutants are defective for both of these responses, producing nondormant (in some cases, viviparous) seeds and plants prone to excessive water loss, i.e. "wilty " (Koomneef, 1986). However, ABA response mutants may be defective in either or both of these responses. To date, only two classes of response mutants have been described that are both nondormant and wilty: the abil and abi2 mutants of Arabidopsis (Koomneef et al., 1984). Mutants at either of these two loci also show reduced sensitivity to exogenous ABA for the inhibition of germination. Since these mutations affect elements common to slow and fast response pathways, the A B l l and AB12 loci are thought to be central to ABA signaling. Consequently, these two genes are targets of molecular genetic studies aimed at cloning the genes and char- acterizing their biochemical function.

' This work was supported by National Science Foundation grant

* Fax 1-805-893-4724. DCB-9105241.

We and others have undertaken extensive physiological and genetic characterization of these mutants (reviewed by Finkelstein and Zeevaart, 1994). Such studies should provide a biological context for future molecular studies of ABI gene function. In this paper we compare water loss, seed dormancy, and ABA inhibition of germination in wild type, homozygous mutants, and heterozygotes. We were surprised to find that, although originally characterized as dominant and recessive, respectively, the abil and abi2 mutations tested range from recessive to nearly fully dominant, depending on the ABA-controlled response scored and on the parental origin of the mutant allele.

MATERIALS AND METHODS

Plant Material

Arabidopsis thaliana seed stocks carrying the following mutant alleles were a generous gift of M. Koomneef abil (isolation number A 11) and abi2 (E 11). Both are ethyl methane- sulfonate-induced mutants derived from the Landsberg erecta line (Koomneef et al., 1984). Plants were grown to maturity under continuous fluorescent illumination (100-150 pE m-' s-') at 22OC on a mixture of vermiculite, perlite, and sphagnum (1:l:l) imgated with mineral nutrients (Haughn and Somerville, 1986). A11 seed were incubated at least 20 h at 4OC after sowing before transfer to growth chambers. Crosses were performed using lines canying the visible markers m s l (male sterile), gll (glabrous), or y i (yellow inflores- cence) to simplify verification of outcrossing. A11 markers were in the Landsberg ecotype background and are now available through the Arabidopsis Biological Resource Center at Ohio State University. In cases for which no appropriate marker was available, the ABI genotypes of progeny were detennined by scoring germination in the presence of 3 PM exogenous ABA for F2 families derived from each F1 individual. Crosses used were abil labi l , m s l l m s l X A B I l I A B I l , M S l I M S l ; A B l l I A B I l , gll/gll x abil labi l , G L I / G L l ; A B l l I A B l l X ab i l lab i l ; abi2 ms l lab i2 msl X AB12 MSl IAB12 MS1; abi2 yilabi2 y i X AB12 YIIAB12 Yl; and ABI21AB12, gll/gll X abi21abi2, GLIIGLI . Wild-type alleles are designated by cap- ital letters and mutants are written in lowercase; the female parent is indicated first in each cross.

Germination Assays

For germination assays scoring ABA sensitivity, 2 0 to 100 seeds per treatment were surface sterilized in 5% hypochlorite

Abbreviation: ABI, ABA insensitive. 1203

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1204 Finkelstein Plant Physiol. Vol. 105, 1994

and 0.02% Triton X-100 and then rinsed four to five times with sterile water before plating on minimal medium (Haughn and Somerville, 1986) containing 0.7% agar and ABA (mixed isomers, Sigma) at O, 1, 3, 5, 10, or 30 p~ in 15- X 100-mm Petri dishes. The dishes were incubated ovemight at 4OC to break any residual dormancy and then transferred to 22OC in continuous light (50-70 pE m-' s-I). Germination was scored daily; averages of 5-d assay results from at least two experimental trials are presented in Figure 1.

For germination assays scoring dormancy, seeds were surface sterilized within 2 d after harvesting and plated on minimal medium as described above. Each genotype was plated in triplicate; dishes were incubated directly in either light or dark or in light following a 4-d incubation at 4OC. Germination was scored daily for light-incubated seeds and after 5 d for dark-grown seeds; combined totals from at least three experimental trials are presented in Table I.

Measurement of Water Loss

Excised stems of bolted individuals were weighed before and after a 3-h incubation at ambient laboratory conditions essentially as described by Koomneef et al. (1982).

Measurement of Stomatal Density

Leaves were excised and left for 10 min at ambient laboratory conditions prior to boiling for 5 min in 95% ethanol. The ethanol was replaced with lactophenol (85% lactic acid:phenol:glycerol:water, l:l:l:l), and leaves were boiled for another 5 min. Cleared leaves were mounted for micros- copy, and the abaxial epidermal surface was photographed at X20 using Nomarski optics.

RESULTS

In the course of mapping studies with the abil and abi2 mutants, we observed a broad range of water loss in excised stsms of the FZ segregants, suggesting that heterozygotes had an intermediate phenotype. Furthermore, significantly greater than 25% of the progeny from self-fertilized abi2 heterozygotes displayed this altered stomatal regulation, a surprising result for a presumed recessive mutation. There- fore, we chose to reexamine the dominance relationships of the available abil and abi2 mutant alleles. We performed reciproca1 crosses between abil or abi2 (alleles A I1 and E 11, respectively) and wild-type plants. The F, progeny were sown, along with self-fertilized seed of each parent used for the crosses, on media containing from O to 30 p~ ABA for germination assays.

The dose responses for ABA inhibition of germination (Fig. 1) indicate that this abil mutation is nearly fully dominant over the wild-type allele when introduced from the matemal parent but only partially dominant when introduced through pollen. For example, nearly 90% of abil seed and almost none of the wild-type seed germinated on 3 PM ABA after 5 d; under these conditions, nb i l lABl1 heterozygotes with matemally inherited abil showed about 70% germination, but those with patemally inherited abil showed only 30% germination. This abi2 mutation is also almost fully dominant

1 O0

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Figure 1. ABA dose response for germination inhibition of wild type and abil homozygotes, and heterozygotes (A) and wild type and abi2 homozygotes and heterozygotes (B). Averaged data from at least two trials are shown. The absolute sensitivities varied slightly among experimental trials, probably reflecting differcmces in seed age, but relative sensitivities were reproducible.

over its wild-type allele in reducing sensitivity to applied ABA when introduced matemally but essentia ily recessive when introduced patemally. Consequently, there is no con- centration of ABA that can unambiguously discriminate between heterozygous and homozygous mutant individuals except for abi21AB12 canying patemally inherited abi2.

In contrast to the inheritance of reduced sensitivity to applied ABA, the abi effects on dormancy showcd at most a slight matemal effect (Table I). Both of the abi niutants used in these studies have reduced requirements for light and chilling, evidenced by their enhanced germination (relative to wild type) when incubated in either light or clark without prechilling (Koomneef et al., 1984; Table I). H eterozygous seed of either genotype were nearly as nondormant as the homozygous mutant parents, regardless of the parenta1 source of the mutant alleles.

Follo wing the germination assays, seedlings of each genotype were transplanted to soil and subsequentl y scored for control of water loss in excised stems left at room tempera- ture. Wild-type plants close their stomata during this imposed stress, thereby limiting their water loss to 25 to 35% of the initial fresh weight. In contrast, ABA-deficient inutants lose nearly :70% of their initial fresh weight during a comparable period, indicating that stomatal closure is an ABA-dependent response (Koomneef et al., 1984). Both abi mutations ap-



Maternal Effects of Arabidopsis ABA Response Mutations 1205

Table 1. Seed dormancy in wild type, abil homo- and heterozygotes and abi2 homo- and heterozygotes

Percentage of germination after 3 or 5 d in light or 5 d in dark are shown; prechilling resulted in 100% germination of all genotypes within 2 d in light. Numbers in parentheses are total numbers of seeds scored.

Percentage of Cermination

Seed Cenotype Dark Female Male Parent Parent

Light

3 d 5 d 5 d

ABII/AB/I (wild type) abil labil abi l /AB/ l abi I /AB/I

AB/2/AB/2 (wild type) a bi2/abi2 abi2/AB/2 abi2/AB/2

AB/I /AB/I abi I/abi I AB/I /AB/ I abi l labi l

AB/2/AB/2 a bi2/a bi2 AB/2/AB/2 a bi2/a bi2

AB/ 1/AB/I abi l labi l abi l labi l AB/ l /AB/ I

AB/Z/AB/2 abiZ/a bi2 a biZ/a bi2 AB/Z/AB/2

7 99 71 88

7 92 75 85

75 (349) 100 (179) 1 O0 (42) 1 O0 (43)

75 (349) 100 (264) 98 (52)

100 (59)

O (241) 28 (194) 20 (41) 31 (35)

O (241) 50 ( 1 50) 42 (31) 34 (50)

peared incompletely dominant in this response, regardless of the source of the mutant allele (Table 11). The range of water loss by heterozygotes overlapped those of both wild-type and mutant homozygotes (Fig. 2). In repeated water loss assays with newly grown shoots of individual plants, we found that single abi2 heterozygous individuals could span the entire range observed within the population (data not shown). There was no obvious correlation between the degree of water loss and variables such as time of day or RH during the assay. Furthermore, the variability observed in repeated assays on individual plants indicates that the range within a population probably does not reflect segre- gation of another mutation within that population.

To determine whether the intermediate wilting of the abi heterozygotes reflected a difference in the rate of water loss, we assayed the kinetics of water loss. Data in Figure 3 show that, for both mutations, the heterozygotes lost water at a rate intermediate between homozygous wild type and mutant.

An increased transpiration rate in excised stems could reflect a decreased ability to close stomatal pores in response to water stress or an increase in stomatal density. Measure-

ments of stomatal density showed substantial variability but no significant difference between wild-type and abi mutants in comparably expanded leaves (Table 111). However, following a brief incubation in the laboratory, excised leaves of both mutants had a consistently higher proportion of open stomata (>90% in the mutants versus <20% in wild type) and the stomata of abil mutants had larger apertures than wild-type plants (Fig. 4).

DISCUSSION

abil (alleles A I1 and C VI) and abi2 (allele E 11) were originally described as dominant and recessive mutations, respectively, based on the relative sensitivities of heterozygotes and homozygotes for ABA inhibition of seedling growth (Koomneef et al., 1984). In these experiments, heterozygotes were produced by wild type X mutant crosses, and growth was scored in terms of fresh weight following 12 d of culture on media containing ABA, a value that can reflect differences in either germination rate or seedling growth. We reexamined the dominance relationships of abil (allele A 11) and abi2 (allele E 11) by comparing the dose response for ABA inhibi-

Table II. Water loss in excised plants during 3-h incubation of wild type, abil homo- and heterozygotes and abi2 homo- and heterozygotes

Plant Cenotype Female Parent

Water Loss (percentage of initial

fresh wt)

Male Parent

AB/ l /AB/ I (wild type) ABI I /AS/ l AB/I/AB/ I 22.6 k 6.2 a b i l / a bi I a bi 1 /abil abiI/a bi 1 62.2 k 10.0 abiI/AB/ I A51 I/ABII abi l /a bi I 38.8 f 10.3 abiI/AB/ 1 abi l labi l AB/I/ABI 1 39.6 f 10.0

AB/2/AB/2 (wild type) AB/2/AB/2 AB/2/AB/2 29.5 f 5.5 64.6 f 7.0 a bi2/a bi2

abi21AB12 abiZlabi2 AB/Z/AB/2 51.5 k 1.3

a bi2/a bi2 a bi2/a bi2 a bi2/AB/2 AB/2/AB/2 a biZ/a bi2 53.7 f 3.3




3 0 c] abil/+

20

10

n " 1 5 2 5 35 4 5 55 6 5

Water Loss (% lnitial FW)

abi2 abi2/+

2 0

1 0

O 15 2 5 3 5 4 5 5 5 6 5

Water Loss (% lnitial FW)

Figure 2. Range of water loss in excised plants during a 3-h incubation of wild type and abil homozygotes and heterozygotes (A) and wild type and abi2 homozygotes and heterozygotes (B). Fre- quency distribution for each genotype was determined from assays on 16 to 43 individuals, depending on the genotype, and are expressed as percentages of individuals per genotype within each 5% interval of water loss. FW, Fresh weight.

tion of germination in wild-type, homozygous mutants and heterozygotes produced by reciproca1 crosses between wild- type and mutant plants. Although our results are consistent with the original observation, we also find that the ABA sensitivity of heterozygous seed shows a maternal effect. For both loci, the mutations tested appear more dominant (nearly fully versus incompletely for abi l , nearly fully versus recessive for abi2) when introduced through the matemal plant. In contrast, both the abil and abi2 alleles appeared incompletely to nearly fully dominant in their effects on seed dormancy, showing at most a slight maternal effect. Our results support the conclusion from studies with ABA biosynthetic mutants that ABA regulation of dormancy is primarily controlled by the embryo (Karssen et al., 1983).

Although induction of dormancy is dependent on ABA produced in Arabidopsis embryos, the degree of dormancy is strongly influenced by environmental factors (reviewed by Koomneef and Karssen, 1994), resulting in much variation in dormancy among seed lots of the same genotype. Such variability could account for the observation that a11 genotypes have lower requirements for light and after-ripening in our experiments than in those of Koomneef et al. (1984). Genetic evidence for additional ABA-independent mechanisms of dormancy regulation is provided by the recent

6 0

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60

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40

30

2 0

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O 1 2 3 Hours After Excision

O 1 2 :i

Hours After Excision

Figure 3. Kinetics of water loss in excised plants during a 3-h incubation of wild type and abil homozygotes and heterozygotes (A) and wild type and abi2 homozygotes and heterozygotes (B). Error bars represent SE of measurements on 3 to 18 irdividuals per genotype. FW, Fresh weight.

isolatiori of nondormant Arabidopsis mutants (uts, aberrant testa shape, and fus3, fusca3) with wild-type AB.4 sensitivity and no symptoms of excessive water loss, implying normal ABA biosynthetic capacity (Keith et al., 1994; Koomneef and Karssen, 1994).

Maternal effects on seed germinability have been docu- mented in severa1 species, including Arabidopsis, i obacco, and tomato (Kasperbauer, 1968; Taylor, 1979; Koomneef and Karssen, 1994). They can be due to factors prl2sent in the mature seed coat, since this is matemally deriwd tissue, or to differences in materials supplied to the developing seeds via the matemal vascular connection. For example, matemal ABA erthances, but is not required for, dormancy in Arabi- dopsis (Karssen et al., 1983). In this case, ABA may act by stimulating development of a mucilage layer around the

Table 111. Stomatal density on the abaxial surface of wild-type, abil, and abi2 leaves

aooroxirnatelv 0.22 mm2 each. Values are the means of densities in at least lour fields of

Genotype No. Stomatalmm'

Wild type abil abi2

214.8 f 73.5 21 7.5 f 43 21 7.8 f 42.6



Maternal Effects of Arabidopsis ABA Response Mutations 1207

Figure 4. Abaxial epidermal surfaces of wild type (A), abil (B), and ab/2 (C) leaves. Magnification, X260.

seeds; nondormancy is correlated with a reduced mucilagelayer. However, differences in mucilage layer thickness areunlikely to be the basis of the reduced ABA sensitivity of abiseeds, since homozygous abi seeds are surrounded by amucilage layer of wild-type thickness (Koornneef et al., 1984).Mutations at several other loci indicate a correlation betweenseed coat characteristics and dormancy (Leon-Kloosterziel etal., 1993), but it is not known whether these are causalrelationships.

Although dormancy and inhibition of germination by ap-plied ABA have the same phenotype, i.e. nongermination,several lines of evidence suggest that they are likely to bedifferent phenomena. First, the lack of germination observedin dormancy occurs even though endogenous ABA levels inmature Arabidopsis seeds are low (Karssen et al., 1983).Furthermore, as discussed above, dormancy induction ap-pears to involve both ABA-requiring and ABA-independentmechanisms (Koornneef and Karssen, 1994). Finally, theArabidopsis ABA response mutants show a significant mater-nal effect on exogenous ABA regulation of germination butat most a slight maternal effect on dormancy induction.

We have also tested stomatal regulation and found that,during vegetative growth, the maternal effect disappears: allheterozygotes display an intermediate phenotype. The wilt-ing behavior appears to reflect differences in stomatal phys-iology, since the stomatal density on mutant leaves isunchanged. This is not surprising, since mutants with anincreased stomatal density are not wilty (Yang and Sack,1993) and the ABA-insensitive "cool" mutant of barley iswilty despite a normal stomatal density (Raskin and Lady-man, 1988).

The results presented in this paper indicate that it is difficultto determine unambiguously whether an individual is het-erozygous or homozygous for the abil and abil alleles testedon the basis of either germination on ABA or resistance towater loss. This has significant implications for moleculargenetic studies of these loci, since the restriction fragmentlength polymorphism mapping needed for chromosomewalking requires accurate scoring of the ABI genotype ofindividual F2 segregants (or F3 families). Furthermore, thestrategy used for assaying genetic function within a clonedsegment of DNA by construction of transgenic plants de-pends on whether the available mutations at the locus ofinterest are dominant or recessive. Our studies indicate thatfunctional assays for both the ABI1 and ABI2 loci could usemutant DNA to confer ABA insensitivity on wild-type plants.

In summary, we have found that seed sensitivity to exog-enous ABA is partly determined by maternally controlledfactors. We have observed that the effects of the availableabi mutations may range from recessive to dominant depend-ing on the phenotypic aspect tested. Many plant hormoneresponse mutations have been described as at least partiallydominant, but few have been extensively characterized inheterozygotes. At least one, the axr2 (auxin resistant) muta-tion of Arabidopsis, ranges from incompletely to fully domi-nant, depending on the characteristic assayed (Wilson et al.,1990). The response-specific variability in the axr2, abil, andabil mutants may reflect similar mechanisms in plant hor-mone signal transduction. The interpretation of our resultsdepends on whether the abil and abil mutants carry loss- orgain-of-function mutations. Assuming loss-of-function, twopossible models consistent with our observations are (a) theABI gene products interact with different signal transductionelements for the responses tested and (b) the ratio of availableABI gene product to the amount required for each responsediffers. If these are gain-of-function mutations, as describedfor the incompletely dominant GA-insensitive mutants ofArabidopsis, maize, and wheat (gai, D8/Mpll, and Rht, re-spectively) (Gale and Marshall, 1975; Harberd and Freeling,1989; Peng and Harberd, 1993), the mutant abi gene productsmay actually interfere with ABA signal transduction mecha-nism^). In this case, identification of the ABI gene productsmight not be very informative, since wild-type ABI functionswould not necessarily be required for ABA response. Futuremolecular and genetic experiments should allow us to distin-guish among these possibilities.

ACKNOWLEDGMENTS

I thank Steve Poole for use of his Zeiss Axioskop microscope andJames Cooper, Tim Lynch, and Joanna Werner for critical reading ofthe manuscript.

Received October 18, 1993; accepted April 26, 1994.Copyright Clearance Center: 0032-0889/94/105/1203/06.

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