DEVELOPMENTAL
BIOLOGY
72,50-61
(1979)
Embryo-Lethal
Mutants of Arabidopsis
thaliana
A Model System for Genetic Analysis of Plant Embryo Development DAVID W. MEINKE AND IAN M. SUSSEX Department
of Biology,
Received November
Yale University,
New Haven, Connecticut
13, 1978; accepted in revised form April
06520
3, 1979
Arabidopsis thaliana (Cruciferae) has been chosen as a model system for mutant analysis of plant embryo development. The isolation and characterization of embryo-lethal mutants of Arabidopsis are simplified by the following genetic and developmental characteristics: (1) Aborted seeds can be easily recognized in both immature and mature fruits; (2) spontaneous or physiological embryo abortion is rare; (3) individual fruits contain 30-60 seeds and show clear segregation of normal and aborted seeds in plants heterozygous for embryo-lethal mutations; (4) mature plants contain hundreds of seeds at every stage of development; (5) the generation time of 5-6 weeks is one of the shortest among flowering plants; (6) the diploid chromosome number is 10, and a large number of morphological mutants are available for linkage studies; (7) plants naturally selfpollinate but can be experimentally cross-pollinated; (8) the pattern of normal embryo development is very similar to that of Capsella, a plant used extensively for descriptive and experimental studies; (9) embryo and ovule culture can be used to study the biochemistry of mutant embryos; and (10) the isolation of temperature-sensitive mutants is possible because mature plants are small and viable over a wide range of temperatures. This report describes normal development in Arabidopsis thaliana, strain “Columbia,” and outlines the system used to identify embryo-lethal mutants. A method of classifying embryo-lethal mutants as cellular, nutritional, or developmental lethals is presented, and the potential anulication of these mutants to the study of normal embryo development is discussed. -
known for many years, and have been described in Arabidopsis (Miiller, 1963), tomato (Huang and Paddock, 1962), and corn (Mangelsdorf, 1926; Neuffer et al., 1978), but their application to the study of normal development has never been fully explored. In contrast, embryo development in Drosophila melanogaster has been studied extensively through the isolation and characterization of embryo-lethal mutants (Wright, 1970). Temperature-sensitive mutants and maternal-effect lethals have been particularly useful in studying the problems of maternal control and gene activation during development (King and Mohler, 1975; Suzuki et al., 1976; Shear-n et al., 1978). A similar approach needs to be taken in the analysis of plant embryo development (see Rice and Carlson, 1975; Dure, 1975).
INTRODUCTION
Embryo development in higher plants has generally been approached through descriptive, experimental, and biochemical studies (Raghavan, 1976). Although these approaches have provided valuable information on morphological, nutritional, and biochemical aspects of plant embryogeny, fundamental questions remain concerning developmental regulation. Early stages of development have been particularly difficult to study in flowering plants because the young embryo is surrounded by maternal tissue, and is too small to analyze using most experimental and biochemical techniques. Embryo-lethal mutants provide an alternative, genetic approach to the study of plant embryo development. Lethal mutations affecting plant embryogeny have been 50 0012-16oS/79/o9oo50-12$~.~/0 Copyright All rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
MEINKE
AND SUSSEX
51
Mutant Analysis of Plant Embryogeny
Plant embryogeny is an ideal developmental pathway for mutant analysis because new gene products must be produced at specific times during the embryonic transition from heterotrophy to autotrophy, and in vitro culture techniques provide a unique opportunity to study the biochemical basis of abnormal development. Furthermore, embryogeny is one of the few developmental pathways in higher plants that is relatively insensitive to environmental factors, and is therefore primarily under genetic control. The purpose of this study was to establish a model system for the isolation and analysis of embryo-lethal mutants in higher plants. Characteristics of such a system should include: (1) the ability to distinguish aborted seeds, normal seeds, and unfertilized ovules in fruits of heterozygotes; (2) a low rate of physiological embryo abortion to avoid confusion with mutant abortion; (3) a large number of seeds per fruit to allow detection of segregation in individual fruits; (4) self-pollination, true diploidy, and a short generation time to aid in the isolation of mutants; (5) a pattern of embryo development that is predictable and histologically characterized to allow detection of developmental abnormalities; (6) a method of embryo or ovule culture for rescue experiments; and (7) the ability to distinguish embryo and endosperm lethals genetically. Arabidopsis meets all but the last of these requirements. Embryo development in this crucifer is similar to that in Capsella, a plant used extensively for descriptive studies (Schulz and Jensen, 1968a,b,c) and embryo culture (Raghavan and Torrey, 1963; Monnier, 1976). The generation time for Arabidopsis thaliana strain “Columbia” is only 5-6 weeks, the diploid chromosome number is 10, average plants produce several thousand seeds, and individual fruits contain 30-60 seeds. Mutagenesis procedures and the identification of embryo lethals have been previously described (Miller, 1963; Usmanov and Muller, 1970;
RCdei, 1970), and Redei (1975a,b) has reviewed the use of Arabidopsis as an experimental organism for plant genetics. This report describes important features of normal development in Arabidopsis thaliana, the basic system for identifying and classifying embryo-lethal mutants, and the potential application of these mutants to the study of normal embryo development. Genetic and developmental information on six embryo-lethal mutants isolated following chemical mutagenesis of mature seeds is presented in a subsequent paper (Meinke and Sussex, 1979). MATERIALS
AND
METHODS
Growth Conditions Wild-type seeds of Arabidopsis thaliana (L.) Heynh strain “Columbia” were kindly provided by Dr. G. P. Redei of the University of Missouri, Columbia. Plants were grown in an environmental growth room at 25°C in 16 hr/8 hr light/dark cycles. Illumination was provided by cool white, very high-output fluorescent lights supplemented by incandescent lights. All plants were grown on a watering table, which periodically pumped nutrients (All Purpose Hyponex, Hyponex Co.) from a storage tank, flooding the table top and watering the plants from below. Optimal growth occurred when plants were grown individually in 2%in. pots containing sandy soil with a basal layer of coarse vermiculite and a thin surface layer of finely sieved soil. Alternatively, plants could be grown in larger pots containing only coarse vermiculite. Seeds were soaked in water for 6-12 hr at room temperature before planting, and transferred to the soil surface with Pasteur pipets. In this way it was possible to plant seeds at known positions in each pot, and identify any unwanted plants that developed from stray seeds. Germination was enhanced by keeping the soil constantly moist during the 3- to 5-day germination period, but it was important to let the pots dry out subsequently to avoid seedling mor-
52
DEVELOPMENTAL BIOLOGY
tality. Reasonable growth was also observed when dry seeds were allowed to germinate on the moist surface of coarse vermiculite. Mature plants grown on either soil or vermiculite were watered once or twice a day. RESULTS
AND
DISCUSSION
Normal Development Seven developmental stages can be recognized in the growth of wild-type plants: (1) germination; (2) formation of rosette leaves; (3) bolting of the main stem; (4) flowering of the primary inflorescence; (5) formation and flowering of lateral and additional branches; (6) silique, seed, and embryo development; and (7) senescence. Flowering in wild-type strain “Columbia” begins 3 weeks after planting, seeds mature 2 weeks after pollination, and freshly harvested seeds germinate without any special treatment. The generation time for plants grown at 25°C and 16 hr/8 hr light/dark cycles is therefore 5-6 weeks. Plants grown at lower temperatures show reduced vegetative growth and require longer times for seed maturation (3 weeks at 18°C). Developmental timetables for reproductive development at 25°C are presented in Table 1 and Fig. 1. Germination is both rapid and reliable; 90-100% of wild-type seeds germinate within 4 days of planting, and vegetative growth over the next 2 weeks results in the TABLE
VOLUME 72, 1878
formation of rosette plants that reach a mature size of 6-8 cm in diameter shortly before flowering. Smaller rosettes with fewer inflorescences are common under less favorable growth conditions. Healthy plants develop two or three lateral branches from the axils of leaves on the main stem, and four to six additional branches from the base of the plant. These main branches develop several further orders of branches,
stage
Pollination Octant embryo Globular embryo Heart embryo Linear cotyledon Curled cotyledon Mature cotyledon Desiccation Mature seed
Days after flower opening o-1 2-3 3-4 4-5 5-6 6-8 8-12 12-14 14-15
n Growth conditions described in text. * Curled embryos at cotyledon stages of development
16
-
14
-
12
-
10
-
t
/-
.= 2
\I
I
I1(,111‘
1
=
~~111ll1
-I :r = 1
) 0
8 c
r-
0 I).i\
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4 (11 I ,‘I
= = =
6
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,'I
10
12
14
O,,~'I1IIIL:
FIG. 1. Reproductive development in wild-type (“Columbia”) plants of Arabidopsis thaliana, measured by the change in length of developing siliques, seeds, and embryos. Curled embryos at cotyledon stages of development (>6 days) were extended before measurement. 1
NORMAL EMBRYO DEVELOPMENT IN Arabidopsis Developmental
-
thaliana
Seed color Colorless Colorless Colorless Trace of green Pale green Greening Green Pale green-brown Brown
STRAIN “COLUMBIA”~ Embryo
length (pm)”
20-30 40-60 70-120 150-400 420-800 850-950 850-950 Not measured
were extended before measurement.
MEINKE
AND SUSSEX
Mutant
each with a terminal inflorescence (see drawing by Ross-Craig, 1948). Siliques are arranged along the axis of each stem in order of their reproductive age; immature buds and open flowers are clustered at the tip and mature siliques are located at the base. The age of a developing silique can therefore be estimated by its distance from the shoot apex. Since each inflorescence produces two to four new flowers per day, and mature plants contain at least 25 active inflorescences, hundreds of seeds at any stage of development can be obtained from a single plant. Individual inflorescences may produce as many as 60 flowers before the apex becomes senescent, and average plants produce a total of 200-400 siliques or lO,OOO-20,000 seeds. Characteristic stages of normal reproductive development are shown in Fig. 2. Flow-
Analysis
of Plant Embryogeny
53
ers contain four sepals and petals, six stamens, and a central ovary, which elongates to form the silique. Arabidopsis flowers naturally self-pollinate; all six anthers remain intact and below the stigma surface throughout flower bud development, but four stamens elongate as the flower opens, pushing their anthers above the stigma surface, where they dehisce and release mature pollen. The other two stamens elongate only slightly and do not contribute to selfpollination. Mature pollen is sticky and not easily dispersed through the air; this accounts for the absence of cross-pollination even when plants are crowded together. Flowers usually remain open for 1 day and then close around the elongating silique. Siliques contain two rows of seeds that are attached to a central septum and covered by two valves. Seeds at any stage of
FIG. 2. Normal reproductive development in Arabidopsis thaliana. Flowers and siliques at different stages of development were removed from wild-type plants and arranged in a developmental sequence. Seeds at an equivalent stage of development are included next to each silique. See also Table 1 and Fig. 5B. (a) Closed buds; (b) open flower at pollination; (c) closed flower with protruding silique; (d,e) elongating silique at preglobular embryo stage; (f) Clique at heart stage; (g) mature green silique; (h) mature brown silique. Scale bar equals 2 mm.
54
DEVELOPMENTAL BIOLOGY
development can be examined under a dissecting microscope by separating these valves with a razor blade. Some mature siliques separate and release seeds when left on developing plants, but most siliques can be harvested without seed loss. Average siliques contain 40-60 seeds and reach a mature length of 12-16 mm 4 days after pollination (Fig. 1). Silique length can be used to approximate developmental time during the first 4 days of development, but the length of mature siliques is determined by seed set (Fig. 3). Siliques containing only unfertilized ovules fail to elongate, and remain 3.0-3.5 mm in length. Most siliques contain very few unfertilized ovules; the number of seeds in a silique is determined primarily by the total number of ovules present at the time of pollination. Seeds develop nearly synchronously within each silique, and reach a mature length of 0.6 mm at the linear cotyledon stage of embryo development, 6-7 days after pollination. Seed length is the most reliable marker for developmental time prior to the heart stage, when embryos can be removed from developing seeds and measured under a dissecting microscope. Seeds are initially colorless, but turn green as the developing embryo becomes visible
70
L .jqp; s.5 * .;:..: . . l
8
.
I* . .-’
l
.
i:*
.*: *.
.
10 . I_./ 2
4
:*
. .i . .
.
I
I
6
6
10
SIIII~U"
I( "~I11
1 12
1 14
) 16
1 16
(111111)
FIG. 3. Relationship between seed set (number of seeds per sfiique) and mature silique length in Arabidopsis thakana. Siliques from plants heterozygous for an embryo-lethal mutation show this same relationship.
VOLUME 72, 1979
through the translucent seed coat. Seeds become increasingly green as the cotyledons develop, and remain green until late in seed maturation, when the seed coat dries out and turns brown. Liquid endosperm is abundant at the globular and heart stages of embryo development, and then gradually diminishes as the embryo grows. Mature seeds contain essentially no endosperm tissue, and are filled by the mature embryo. Identification
of Aborted
Seeds
The isolation and characterization of a large number of embryo-lethal mutants are possible only in plants where physiological abortion is rare, and aborted seeds can be clearly distinguished from normal seeds and unfertilized ovules. Physiological or spontaneous abortion in wild-type plants of Arabidopsis grown under the conditions described above averaged less than 0.5% of screened seeds. This is significantly lower than the abortion rates found in many plants with larger seeds. Moderate changes in temperature, availability of water, and the supply of nutrients did not affect the frequency of physiological abortion in Arabidopsis; most aborted seeds appeared to be the result of physical damage to developing siliques. However, siliques that began development just prior to senescence of the whole plant frequently showed a variety of developmental abnormalities, and were not used in subsequent studies of embryo-lethal mutants. In order to study embryo development in embryo-lethal mutants, it is necessary to identify aborted seeds in immature fruits, shortly after embryo development has been arrested, and before the whole seed degenerates. Further studies with these mutants are then simplified if aborted seeds can also be clearly recognized in mature fruits. All recessive embryo-lethal mutants, with the exception of temperature sensitives, must be isolated and maintained as heterozygotes since homozygotes die as embryos.
MEINKE
AND SUSSEX
Mutant Analysis of Plant Embryogeny
Aborted seeds must therefore be obtained from fruits of heterozygous plants, which contain 25% aborted seeds and 75% phenotypically normal seeds (?& heterozygotes and Y3 wild type), as shown in Fig. 4. Aborted seeds can be studied only after they become phenotypically distinguishable from normal seeds in the same fruit. Aborted seeds of Arabidopsis can be unambiguously identified by their size or color in both immature and mature siliques (Fig. 5). Aborted seeds initially resemble normal seeds, but while normal seeds enlarge and turn green during later stages of embryo development, aborted seeds remain colorless and arrested at a size characteristic of the stage of developmental arrest. Seeds arrested at a preglobular embryo stage are therefore significantly smaller than seeds arrested at a heart stage (see Figs. 1 and 5). All aborted seeds containing embryos arrested before the linear cotyledon stage of development can be clearly recognized in heterozygous siliques 8-12 days after pollination. At this stage, normal seeds are green and have reached mature size, unfertilized ovules are very small white masses, and aborted seeds are intermediate in size and either colorless, milky white, or beginning to degenerate and turn brown. Seeds containing pale green, yellow, or albino embryos that have reached mature size can also be recognized at this stage because the embryo is visible through the translucent seed coat. Aborted seeds containing embryos arrested prior to the linear cotyledon stage
~00.00.000. Q.000.0.000 FIG. 4. Segregation of normal and aborted seeds in a silique heterozygous for a recessive embryo-lethal mutation. Individual siliques contain approximately 25% aborted seeds (black), 25% wild-type seeds (white), and 50% heterozygotes (stippled). Heterozygous and wild-type seeds are both phenotypically normal, but their frequency can be determined by scoring plants in the next generation.
55
become deflated as the silique matures because all of the liquid endosperm is resorbed, and there is nothing left to fii the seed coat. Fortunately, all aborted seeds in Arabidopsis can be identified 2-3 days after developmental arrest, and several days before degeneration begins. Although some aborted seeds are only slightly larger than unfertilized ovules, all abortants turn brown at maturity, whereas unfertilized ovules degenerate into white masses.
Isolation of Mutants Mutants in higher plants have been recovered following mutagenesis of either pollen grains or mature seeds. Although X irradiation of pollen grains has been reported in Arabidopsis (Usmanov and Muller, 1970), and procedures for chemical mutagenesis of pollen grains have been described in corn (Neuffer et al., 1978), the easiest way to generate mutants in Arabidopsis is to mutagenize mature seeds. Embryo-lethal mutants can then be identified by screening immature siliques of the resulting M-l plants for aborted seeds. Recessive mutations affecting morphological features of the mature plant can be recognized only when they become homozygous in the M-2 generation; embryo-lethal mutants can be recovered more quickly because homozygous mutant embryos can be identified in the siliques of M-l plants following self-pollination. However, since the shoot apical meristem in mature Arabidopsis seeds is a multicellular structure, M-l plants grown from mutagenized seeds will be mericlinal chimeras with heterozygous mutant cells and siliques occupying only a wedge-shaped sector of the primary reproductive inflorescence (Fig. 6). The most efficient way to recover embryo-lethal mutants following seed mutagenesis in Arabidopsis is to screen the first five siliques of each M-l plant. This increases the probability of detecting the presence of a mutant sector because each silique arises from a different part of the meristem. Recessive embryo-lethal mutant
FIG. 5. Identification of normal seeds, aborted seeds, and unfertilized ovules. (A) Immature siliques from a plant heterozygous for an embryo-lethal mutation, Valves from each silique have been separated to reveal two phenotypic classes of seeds: normal, green seeds (n) at a mature cotyledon stage of embryo development; and aborted, white seeds (m) prior to degeneration. 78 normal seeds and 25 abortants are visible. Differences in color between normal and aborted seeds at this stage of development are difficult to photograph, but can be clearly seen under a dissecting microscope. Scale bar equals 2 mm. (B) Stages in normal seed development: (a) fertilized ovule (colorless); (b) preglobular embryo stage (white); (c) globular embryo stage (white); (d) early cotyledon stage (pale green); (e) mature cotyledon stage (green); (f) mature seed (brown). B-E are all at the same magnification; scale bar equals 0.5 mm. (C) Unfertilized ovules several days after pollination. (D) Aborted seeds from mature siliques of several different embryo-lethal mutants. All aborted seeds become brown and deflated at maturity. (E) Aborted seeds from immature siliques of several different embryo-lethal mutants. Aborted seeds are initially either colorless or white, and resemble normal seeds at early stages of development. Note that the size of both immature and mature abortants can differ significantly, corresponding to differences in the stage of developmental arrest. 56
MEINKE
AND SUSSEX
Mutant
Analysis
of Plant Embryogeny
57
peared to be segregating for embryo-lethal mutations following X irradiation of imbibed seeds. Segregation ratios and the approximate stage of developmental arrest were determined for 72 of these mutants analyzed in subsequent generations. Mutants were classified according to the stage of developmental arrest and the size and color of aborted seeds and embryos. Six different types of embryonic lethals were recognized (sicca, brevis, vana, diffusa, murca, and parva) in addition to three types of seedling lethals (albina, xantha, and chlorina). This system of classification is described in more detail by Muller (1963) and Usmanov and Miiller (1970). FIG. 6. Diagrammatic cross section of the primary inflorescence and first five siliques of an M-l chimera1 plant grown from mutagenized seed. Consecutive siliques are separated by an angle of 137’. Cells heterozygous for an induced recessive embryo-lethal mutation (stippled) cover a wedge-shaped sector of the primary M-l inflorescence. Siliques arising from this mutant sector contain approximately 25% aborted seeds (dark circles). Open circles represent phenotypically normal seeds.
chimeras will have one or two of the first five siliques segregating for aborted seeds, and three or four siliques with normal seeds (Fig. 6). If immature siliques (8-12 days after pollination) are used for screening, additional mature siliques must be recovered from the mutant sector of the original plant to provide viable heterozygous seeds for the next generation. Each suspected mutant can then be verified and characterized in the M-2 and subsequent generations. Procedures for the identification of aborted seeds and isolation of embryo-lethal mutants in Arabidopsis were first developed by Muller (1963), and have been referred to previously as the Muller embryo test. Miiller was primarily interested in using embryo-lethal mutants to study the mutagenic activity of potential mutagens in higher plants, In his most extensive study, Muller (1963) identified over 3000 M-l plants of wild-type strain “Dijon” that ap-
Classification
of Mutants
Although the classification system of Muller (1963) is useful in cataloguing large numbers of embryo-lethal mutants of Arabidopsis, it provides relatively little information on the stage and possible cause of developmental arrest, particularly for those mutants that arrest early in development. For example, the term “sicca” applies to all mutant embryos that arrest prior to the torpedo stage, when most of the critical events of early embryo development have been completed. An alternative approach is to catalogue mutants by number, determine the stage of developmental arrest (heart, late globular, early globular, etc.) by examining aborted seeds under dissecting and compound light microscopes, and then attempt to classify each mutant as a cellular, nutritional, or developmental lethal, based upon its pattern of developmental arrest and the response of mutant embryos in culture. Mutations affecting essential cellular processes (e.g., membrane biogenesis or protein synthesis) may result in embryo abortion if they do not become lethal during gametogenesis. Embryo abortion in these cellular lethals should occur at very early stages of development, and arrested embryos should be incapable of further growth in culture.
58
DEVELOPMENTALBIOLOGY
Developmental arrest in nutritional lethals is caused by the absence of an amino acid, vitamin, or other diffusible nutrient required for continued growth and development. Only a few nutritional or auxotrophic mutants have been isolated in higher plants, and all are seedling lethals or semilethals (Redei, 1975c; Gavazzi et al., 1975). However, Langridge (1958) has suggested that nutritional mutants may exhibit embryo abortion if the missing nutrient is required for embryonic growth and cannot be supplied by the surrounding maternal tissue. Arrested embryos from nutritional lethals should resume normal embryonic development when cultured on a medium that supplies the missing nutrient, and should form callus when the enriched medium contains the appropriate growth hormones. Embryo culture can therefore be used to identify nutritional lethals, rescue the arrested embryos, and study the biochemistry of the deficiency. A similar approach is being used to study embryo-lethal mutants of corn (Sheridan et al., 1978), and has been applied at the seedling stage to analyze thiamine auxotrophs of Arabidopsis (Li and Redei, 1969). Arrested embryos from developmental mutants should by definition be unable to complete a specific stage of embryo development (e.g., initiation of cotyledons), even when placed on an enriched nutrient medium. However, since basic cellular functions should not be altered, arrested embryos from developmental mutants should be capable of disorganized cellular growth (callus) on either an enriched or a basal nutrient medium containing the appropriate growth hormones. Developmental embryo lethals will be difficult to analyze biochemically because they are not easily rescuable, but microscopy should provide important information on the stage and possible cause of developmental arrest in many of these mutants. It is expected that some developmental mutations will initially affect only one part of the developing seed (embryo proper, suspensor, or endosperm) ,
VOLUME 12,1979
and will allow further development of the other parts. This pattern of developmental arrest may be helpful in the identification of developmental mutants and the analysis of tissue interactions during normal development. Application
to Study of Development
Embryo-lethal mutations and other genetic factors affecting embryo development have been studied extensively in a variety of animal systems (Hadorn, 1961; Gluecksohn-Waelsch, 1963), including the mouse (Wudl et al., 1977), the Mexican axolotl (Brothers, 1976), and the nematode Caenorhabditis elegans (Vanderslice and Hirsh, 1976). A major challenge with these and other developmental mutants has been to determine why development becomes abnormal, and how this abnormal development can provide new information about normal development. Embryo-lethal mutants of Arabidopsis may be useful in the study of three important areas of plant development: (1) maternal control of early embryo development; (2) changing nutritional requirements and synthetic capacities of developing embryos; and (3) the role of the embryo proper, endosperm, and suspensor in normal seed development. Maternal effects on animal development have been studied for many years (see Davidson, 1976), but little is known about the possible maternal control of plant embryo development. Since normal embryo development has been observed in isolated ovules cultured in vitro (Maheshwari, 1958; Kapoor, 1959; Meinke, unpublished resuits), regulatory functions must be limited to maternal tissues located within the developing seed (nucellus, integuments, and seed coat). One way to study the maternal control of embryo development in Arabidopsis would be to isolate maternal-effect embryo-lethal mutants. Unlike recessive embryo-lethal mutants, maternal-effect lethals would not be identified until the M-2 generation, and would produce fruits with 100% rather than 25% aborted seeds. Em-
MEINKE
AND SUSSEX
Mutant Analysis
bryonic lethality in these plants would be determined by the genotype of the maternal plant rather than the genotype of the embryo; homozygous mutant embryos would develop normally in heterozygous plants, but would arrest at some stage of embryo development in homozygous plants due to the loss of a specific regulatory function that is normally performed by the maternal plant. Maternal-effect lethals may be particularly useful in understanding the role of the chalazal nucellar tissue in embryo and endosperm development (see Schultz and Jensen, 1971). Nutritional embryo-lethal mutants will be isolated only if the missing nutrient is required for embryo development and cannot be supplied by the surrounding maternal tissue. Nutritional mutants can therefore provide information on both the maternal supply of nutrients throughout embryo development and the changing nutritional requirements and synthetic capacities of developing embryos. The thiamine auxotrophs of Arabidopsis, which become lethal at the seedling stage, could also be used to study embryogeny; mutant embryos must either receive thiamine from the maternal plant, synthesize a small but sufficient amount of it with defective enzymes, or produce the vitamin through an alternative biochemical pathway that is active only during embryogeny. Information gained from the analysis of nutritional mutants could supplement the more general picture of changing nutritional requirements obtained from embryo culture studies (see Raghavan, 1976). Auxotrophic embryo lethals may also represent a valuable source of conditional-lethal mutants, which could serve as the basis for hybrid selection schemes in plant somatic cell genetics (see Day, 1977; Power and Cocking, 1977). Developmental mutations may initially affect only the endosperm, embryo proper, or suspensor, and may therefore be useful in studying interactions that normally occur between these different parts of a developing seed. Both the endosperm and the
of Plant Embryogeny
59
suspensor are thought to play a nutritive role in plant embryo development (Brink and Cooper, 1947; Yeung, 1977), but they may also regulate or trigger developmental events in the embryo proper. Alternatively, the embryo proper may regulate not only its own development, but that of the endosperm and suspensor as well. Histological examination of one of the embryo-lethal mutants of Arabidopsis described in the subsequent paper (Meinke and Sussex, 1979) has shown that developmental arrest of the embryo proper in this mutant is followed by abnormal growth of the suspensor (Meinke and Marsden, in preparation). When coupled with evidence from other sources, this suggests that continued growth of the suspensor during normal development is inhibited by the embryo proper, and it further illustrates how embryo-lethal mutants may be applied to the study of normal embryo development. Additional questions could be studied through the isolation of temperature-sensitive embryo-lethal mutants. The advantage of temperature-sensitive lethals is that homozygotes can be maintained at the permissive temperature, and the effect of the mutant gene product can be studied at any stage of development. Arabidopsis is an ideal plant for the isolation of temperaturesensitive mutants; vegetative and reproductive development is normal over a wide range of temperatures, and a large number of plants can be grown in the limited space provided by constant-temperature growth chambers. Temperature-sensitive embryolethal mutants have not been reported in flowering plants, but temperature sensitivity has been detected in several naturally occurring races and vitamin-requiring mutants of Arabidopsis (Langridge and Griffing, 1959; Langridge, 1965; Li and Redei, 1968). If the frequency of temperature-sensitive lethals in higher plants is similar to that observed in Drosophila (Baillie et al., 1968), approximately 10% of all EMS-induced embryo-lethal mutants should have a temperature-sensitive phenotype. The
60
DEVELOPMENTAL BIOLOGY
application of temperature-sensitive mutants to all areas of plant genetics and development remains to be explored. We thank Dr. G. P. Redei for wild-type seeds of Arabidopsis, Ruth Schmitt for an English translation of the Muller (1963) paper, and members of the Sussex lab group, particularly John Duesing, for helpful discussions. This work was supported by NSF Grant PCM76-17222 to I. M. Sussex, and NIH Training Grant HD 00032. REFERENCES BAILLIE, D., SUZUKI, D. T., and TARASOFF, M. (1968). Temperature-sensitive mutations in Drosophila melanogaster. II. Frequency among second chromosome recessive lethals induced by ethyl methanesulfonate. Canad. J. Genet. Cytol. 10,412-420. BRINK, R. A., and COOPER, D. C. (1947). The endosperm in seed development. Sot. Rev. 13, 423-541. BROTHERS, A. J. (1976). Stable nuclear activation dependent on a protein synthesized during oogenesis. Nature 260, 112-115. DAVIDSON, E. H. (1976). “Gene Activity in Early Development,” second ed. Academic Press, New York. DAY, P. R. (1977). Plant genetics: Increasing crop yield. Science 197, 1334-1339. DURE, L. S. (1975). Seed formation. Annu. Rev. Plant Physiol. 26, 259-278. GAVAZZI, G., NAVA-RACCHI, M., and TONELLI, C. (1975). A mutation causing proline requirement in Zea mays. Theor. Appl. Genet. 46,339-345. GLUECKSOHN-WAELSCH, S. (1963). Lethal genes and analysis of differentiation. Science 142, 1269-1276. HADORN, E. (1961). “Developmental Genetics and Lethal Factors.” John Wiley, New York. HUANG, P. C., and PADDOCK, E. F. (1962). The time and site of the semidominant lethal action of “Wo” in Lycopersicon esculentum. Amer. J. Bot. 49,388393. KAPOOR, M. (1959). Influence of growth substances on the ovules of Zephyranthes. Phytomorphology 9, 313-315. KING, R. C., and MOHLER, J. D. (1975). The genetic analysis of oogenesis in Drosophila melanogaster. In “Handbook of Genetics,” Vol. 3, pp. 757-791. Plenum Press, New York. LANGRIDGE, J. (1958). A hypothesis of developmental selection exemplified by lethal and semi-lethal mutants of Arabidopsis. Aust. J. Biol. Sci. 11, 58-68. LANGRIDGE, J. (1965). Temperature-sensitive, vitamin-requiring mutants of Arabidopsis thaliana. Aust. J. Biol. Sci. 18, 311-321. LANGRIDGE, J., and GRIFFINC, B. (1959). A study of high temperature lesions in Arabidopsis thaliana. Aust. J. Biol. Sci. 12, 117-135. LI, S. L., and RI?~EI, G. P. (1968). Temperature-sensitive, thiamine-requiring mutants in Arabidopsis. Arabidopsis Inf Seru. 5, 28-29.
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LI, S. L., and R~DEI, G. P. (1969). Thiamine mutants of the crucifer, Arabidopsis. Biochem. Genet. 3, 163-170. MAHESHWARI, N. (1958). In vitro culture of excised ovules of Papaver somniferum. Science 127, 342. MANGELSDORF, P. C. (1926). The genetics and morphology of some endosperm characters in maize. Conn. Agr. Exp. Sta. Bull. 279, 513-620. MEINKE, D. W., and SUSSEX, I. M. (1979). Isolation and characterization of six embryo-lethal mutants of Arabidopsis thaliana. Develop. Biol. 72, 62-72. MONNIER, M. (1976). Culture in vitro de l’embryon immature de Capsella bursa-pastoris Moench. Rev. Cytol. Biol. Veget. 39, l-120. MILLER, A. J. (1963). Embryonentest zum Nachweis rezessiver Letalfaktoren bei Arabidopsis thaliana. Biol. Zentralbl. 82, 133-163. NEUFFER, M. G., SHERIDAN, W. F., and BENDBOW, E. (1978). Rescue of lethal defective kernel mutants by genetic manipulation. Maize Genet. Coop. Newsl. 52,84-88. POWER, J. B., and COCKING, E. C. (1977). Selection systems for somatic hybrids. In “Applied and Fundamental Aspects of Plant Cell, Tissue, and Organ Culture,” pp. 497-505. Springer-Verlag, New York. RAGHAVAN, V. (1976). “Experimental Embryogenesis in Vascular Plants.” Academic Press, New York. RAGHAVAN, V., and TORREY, J. G. (1963). Growth and morphogenesis of globular and older embryos of Capsella in culture. Amer. J. Bot. 50, 540-551. RI?DEI, G. P. (1970). Arabidopsis thaliana (L.) Heynh. A review of the genetics and biology. Bibliogr. Genet. 20, l-151. R~~DEI, G. P. (1975a). Arabidopsis as a genetic tool. Annu. Rev. Genet. 9, 111-127. R~DEI, G. P. (1975b). Arabidopsis thaliana. In “Handbook of Genetics,” Vol. 2, pp. 151-180. Plenum Press, New York. R~DEI, G. P. (1975c). Induction of auxotrophic mutations in plants, In “Genetic Manipulations with Plant Material,” pp. 329-350. Plenum Press, New York. RICE, T. B., and CAHLSON, P. S. (1975). Genetic analysis and plant improvement. Annu. Rev. Plant Physiol. 26, 279-308. ROSS-CRAIG, S. (1948). “Drawings of British Plants. III. Cruciferae,” plate 37. G. Bell, London. SCHULZ, R., and JENSEN, W. A. (1968a). Capsella embryogenesis: The early embryo. J. Vltrastr. Res. 22,376-392. SCHULZ, R., and JENSEN, W. A. (196813). Capsella embryogenesis: The synergids before and after fertilization. Amer. J. Bot. 55, 541-552. SCHULZ, R., and JENSEN, W. A. (1968c). Capsella embryogenesis: The egg, zygote and young embryo. Amer. J. Bot. 55,807-819. SCHULZ, P., and JENSEN, W. A. (1971). Cupsella embryogenesis: The chalazal proliferating tissue. J. Cell Sci. 8, 201-227.
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AND
SUSSEX
Mutant
A., HERSPERGER, G., and HERSPERGER, E. (1978). Genetic analysis of two allelic temperaturesensitive mutants of Drosophila melanogaster both of which are zygotic and maternal-effect lethals. Genetics 89.341-353. SHERIDAN, W. F., NEUFFER, M. G., and BENDBOW, E. (1978). Rescue of lethal defective endosperm mutants by culturing immature embryos. Maize Genet. Coop. Newsl. 52.88-90. SUZUKI, D. T., KAUFMAN, T., FALK, D., and the U.B.C. Drosophila RESEARCH GROUP (1976). Conditionally expressed mutations in Drosophila melanogaster. In “The Genetics and Biology of Drosophila,” Vol. la, pp. 207-263. Academic Press, New York. USMANOV, P. D., and MOLLER, A. J. (1970). Use of
&EARN,
Analysis
of Plant Embryogeny
61
the embryo test for the analysis of embryonic lethals induced by the irradiation of the pollen grains of Arabidopsis thaliana (L.) Heynh. Sov. Genet. (Engl. transl.) 6(7), 894-902. VANDERSLICE, R., and HIRSH, D. (1976). Temperature-sensitive zygote defective mutants of Caenorhabditis elegans. Develop. Biol. 49, 236-249. WRIGHT, T. R. F. (1970). The genetics of embryogenesis in Drosophila. Advan. Genet. 15, 261-395. WUDL, L. R., SHERMAN, M. I., and HILLMAN, N. (1977). Nature of lethality of “t” mutations in embryos. Nature 270, 137-140. YEUNG, C. T. (1977). “Embryogeny of Phaseolus: The Role of the Suspensor during Early Development,” Ph.D. Thesis. Yale University, New Haven, Conn.
BIOLOGY
72,50-61
(1979)
Embryo-Lethal
Mutants of Arabidopsis
thaliana
A Model System for Genetic Analysis of Plant Embryo Development DAVID W. MEINKE AND IAN M. SUSSEX Department
of Biology,
Received November
Yale University,
New Haven, Connecticut
13, 1978; accepted in revised form April
06520
3, 1979
Arabidopsis thaliana (Cruciferae) has been chosen as a model system for mutant analysis of plant embryo development. The isolation and characterization of embryo-lethal mutants of Arabidopsis are simplified by the following genetic and developmental characteristics: (1) Aborted seeds can be easily recognized in both immature and mature fruits; (2) spontaneous or physiological embryo abortion is rare; (3) individual fruits contain 30-60 seeds and show clear segregation of normal and aborted seeds in plants heterozygous for embryo-lethal mutations; (4) mature plants contain hundreds of seeds at every stage of development; (5) the generation time of 5-6 weeks is one of the shortest among flowering plants; (6) the diploid chromosome number is 10, and a large number of morphological mutants are available for linkage studies; (7) plants naturally selfpollinate but can be experimentally cross-pollinated; (8) the pattern of normal embryo development is very similar to that of Capsella, a plant used extensively for descriptive and experimental studies; (9) embryo and ovule culture can be used to study the biochemistry of mutant embryos; and (10) the isolation of temperature-sensitive mutants is possible because mature plants are small and viable over a wide range of temperatures. This report describes normal development in Arabidopsis thaliana, strain “Columbia,” and outlines the system used to identify embryo-lethal mutants. A method of classifying embryo-lethal mutants as cellular, nutritional, or developmental lethals is presented, and the potential anulication of these mutants to the study of normal embryo development is discussed. -
known for many years, and have been described in Arabidopsis (Miiller, 1963), tomato (Huang and Paddock, 1962), and corn (Mangelsdorf, 1926; Neuffer et al., 1978), but their application to the study of normal development has never been fully explored. In contrast, embryo development in Drosophila melanogaster has been studied extensively through the isolation and characterization of embryo-lethal mutants (Wright, 1970). Temperature-sensitive mutants and maternal-effect lethals have been particularly useful in studying the problems of maternal control and gene activation during development (King and Mohler, 1975; Suzuki et al., 1976; Shear-n et al., 1978). A similar approach needs to be taken in the analysis of plant embryo development (see Rice and Carlson, 1975; Dure, 1975).
INTRODUCTION
Embryo development in higher plants has generally been approached through descriptive, experimental, and biochemical studies (Raghavan, 1976). Although these approaches have provided valuable information on morphological, nutritional, and biochemical aspects of plant embryogeny, fundamental questions remain concerning developmental regulation. Early stages of development have been particularly difficult to study in flowering plants because the young embryo is surrounded by maternal tissue, and is too small to analyze using most experimental and biochemical techniques. Embryo-lethal mutants provide an alternative, genetic approach to the study of plant embryo development. Lethal mutations affecting plant embryogeny have been 50 0012-16oS/79/o9oo50-12$~.~/0 Copyright All rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
MEINKE
AND SUSSEX
51
Mutant Analysis of Plant Embryogeny
Plant embryogeny is an ideal developmental pathway for mutant analysis because new gene products must be produced at specific times during the embryonic transition from heterotrophy to autotrophy, and in vitro culture techniques provide a unique opportunity to study the biochemical basis of abnormal development. Furthermore, embryogeny is one of the few developmental pathways in higher plants that is relatively insensitive to environmental factors, and is therefore primarily under genetic control. The purpose of this study was to establish a model system for the isolation and analysis of embryo-lethal mutants in higher plants. Characteristics of such a system should include: (1) the ability to distinguish aborted seeds, normal seeds, and unfertilized ovules in fruits of heterozygotes; (2) a low rate of physiological embryo abortion to avoid confusion with mutant abortion; (3) a large number of seeds per fruit to allow detection of segregation in individual fruits; (4) self-pollination, true diploidy, and a short generation time to aid in the isolation of mutants; (5) a pattern of embryo development that is predictable and histologically characterized to allow detection of developmental abnormalities; (6) a method of embryo or ovule culture for rescue experiments; and (7) the ability to distinguish embryo and endosperm lethals genetically. Arabidopsis meets all but the last of these requirements. Embryo development in this crucifer is similar to that in Capsella, a plant used extensively for descriptive studies (Schulz and Jensen, 1968a,b,c) and embryo culture (Raghavan and Torrey, 1963; Monnier, 1976). The generation time for Arabidopsis thaliana strain “Columbia” is only 5-6 weeks, the diploid chromosome number is 10, average plants produce several thousand seeds, and individual fruits contain 30-60 seeds. Mutagenesis procedures and the identification of embryo lethals have been previously described (Miller, 1963; Usmanov and Muller, 1970;
RCdei, 1970), and Redei (1975a,b) has reviewed the use of Arabidopsis as an experimental organism for plant genetics. This report describes important features of normal development in Arabidopsis thaliana, the basic system for identifying and classifying embryo-lethal mutants, and the potential application of these mutants to the study of normal embryo development. Genetic and developmental information on six embryo-lethal mutants isolated following chemical mutagenesis of mature seeds is presented in a subsequent paper (Meinke and Sussex, 1979). MATERIALS
AND
METHODS
Growth Conditions Wild-type seeds of Arabidopsis thaliana (L.) Heynh strain “Columbia” were kindly provided by Dr. G. P. Redei of the University of Missouri, Columbia. Plants were grown in an environmental growth room at 25°C in 16 hr/8 hr light/dark cycles. Illumination was provided by cool white, very high-output fluorescent lights supplemented by incandescent lights. All plants were grown on a watering table, which periodically pumped nutrients (All Purpose Hyponex, Hyponex Co.) from a storage tank, flooding the table top and watering the plants from below. Optimal growth occurred when plants were grown individually in 2%in. pots containing sandy soil with a basal layer of coarse vermiculite and a thin surface layer of finely sieved soil. Alternatively, plants could be grown in larger pots containing only coarse vermiculite. Seeds were soaked in water for 6-12 hr at room temperature before planting, and transferred to the soil surface with Pasteur pipets. In this way it was possible to plant seeds at known positions in each pot, and identify any unwanted plants that developed from stray seeds. Germination was enhanced by keeping the soil constantly moist during the 3- to 5-day germination period, but it was important to let the pots dry out subsequently to avoid seedling mor-
52
DEVELOPMENTAL BIOLOGY
tality. Reasonable growth was also observed when dry seeds were allowed to germinate on the moist surface of coarse vermiculite. Mature plants grown on either soil or vermiculite were watered once or twice a day. RESULTS
AND
DISCUSSION
Normal Development Seven developmental stages can be recognized in the growth of wild-type plants: (1) germination; (2) formation of rosette leaves; (3) bolting of the main stem; (4) flowering of the primary inflorescence; (5) formation and flowering of lateral and additional branches; (6) silique, seed, and embryo development; and (7) senescence. Flowering in wild-type strain “Columbia” begins 3 weeks after planting, seeds mature 2 weeks after pollination, and freshly harvested seeds germinate without any special treatment. The generation time for plants grown at 25°C and 16 hr/8 hr light/dark cycles is therefore 5-6 weeks. Plants grown at lower temperatures show reduced vegetative growth and require longer times for seed maturation (3 weeks at 18°C). Developmental timetables for reproductive development at 25°C are presented in Table 1 and Fig. 1. Germination is both rapid and reliable; 90-100% of wild-type seeds germinate within 4 days of planting, and vegetative growth over the next 2 weeks results in the TABLE
VOLUME 72, 1878
formation of rosette plants that reach a mature size of 6-8 cm in diameter shortly before flowering. Smaller rosettes with fewer inflorescences are common under less favorable growth conditions. Healthy plants develop two or three lateral branches from the axils of leaves on the main stem, and four to six additional branches from the base of the plant. These main branches develop several further orders of branches,
stage
Pollination Octant embryo Globular embryo Heart embryo Linear cotyledon Curled cotyledon Mature cotyledon Desiccation Mature seed
Days after flower opening o-1 2-3 3-4 4-5 5-6 6-8 8-12 12-14 14-15
n Growth conditions described in text. * Curled embryos at cotyledon stages of development
16
-
14
-
12
-
10
-
t
/-
.= 2
\I
I
I1(,111‘
1
=
~~111ll1
-I :r = 1
) 0
8 c
r-
0 I).i\
2 \
4 (11 I ,‘I
= = =
6
8
I lll\S
,'I
10
12
14
O,,~'I1IIIL:
FIG. 1. Reproductive development in wild-type (“Columbia”) plants of Arabidopsis thaliana, measured by the change in length of developing siliques, seeds, and embryos. Curled embryos at cotyledon stages of development (>6 days) were extended before measurement. 1
NORMAL EMBRYO DEVELOPMENT IN Arabidopsis Developmental
-
thaliana
Seed color Colorless Colorless Colorless Trace of green Pale green Greening Green Pale green-brown Brown
STRAIN “COLUMBIA”~ Embryo
length (pm)”
20-30 40-60 70-120 150-400 420-800 850-950 850-950 Not measured
were extended before measurement.
MEINKE
AND SUSSEX
Mutant
each with a terminal inflorescence (see drawing by Ross-Craig, 1948). Siliques are arranged along the axis of each stem in order of their reproductive age; immature buds and open flowers are clustered at the tip and mature siliques are located at the base. The age of a developing silique can therefore be estimated by its distance from the shoot apex. Since each inflorescence produces two to four new flowers per day, and mature plants contain at least 25 active inflorescences, hundreds of seeds at any stage of development can be obtained from a single plant. Individual inflorescences may produce as many as 60 flowers before the apex becomes senescent, and average plants produce a total of 200-400 siliques or lO,OOO-20,000 seeds. Characteristic stages of normal reproductive development are shown in Fig. 2. Flow-
Analysis
of Plant Embryogeny
53
ers contain four sepals and petals, six stamens, and a central ovary, which elongates to form the silique. Arabidopsis flowers naturally self-pollinate; all six anthers remain intact and below the stigma surface throughout flower bud development, but four stamens elongate as the flower opens, pushing their anthers above the stigma surface, where they dehisce and release mature pollen. The other two stamens elongate only slightly and do not contribute to selfpollination. Mature pollen is sticky and not easily dispersed through the air; this accounts for the absence of cross-pollination even when plants are crowded together. Flowers usually remain open for 1 day and then close around the elongating silique. Siliques contain two rows of seeds that are attached to a central septum and covered by two valves. Seeds at any stage of
FIG. 2. Normal reproductive development in Arabidopsis thaliana. Flowers and siliques at different stages of development were removed from wild-type plants and arranged in a developmental sequence. Seeds at an equivalent stage of development are included next to each silique. See also Table 1 and Fig. 5B. (a) Closed buds; (b) open flower at pollination; (c) closed flower with protruding silique; (d,e) elongating silique at preglobular embryo stage; (f) Clique at heart stage; (g) mature green silique; (h) mature brown silique. Scale bar equals 2 mm.
54
DEVELOPMENTAL BIOLOGY
development can be examined under a dissecting microscope by separating these valves with a razor blade. Some mature siliques separate and release seeds when left on developing plants, but most siliques can be harvested without seed loss. Average siliques contain 40-60 seeds and reach a mature length of 12-16 mm 4 days after pollination (Fig. 1). Silique length can be used to approximate developmental time during the first 4 days of development, but the length of mature siliques is determined by seed set (Fig. 3). Siliques containing only unfertilized ovules fail to elongate, and remain 3.0-3.5 mm in length. Most siliques contain very few unfertilized ovules; the number of seeds in a silique is determined primarily by the total number of ovules present at the time of pollination. Seeds develop nearly synchronously within each silique, and reach a mature length of 0.6 mm at the linear cotyledon stage of embryo development, 6-7 days after pollination. Seed length is the most reliable marker for developmental time prior to the heart stage, when embryos can be removed from developing seeds and measured under a dissecting microscope. Seeds are initially colorless, but turn green as the developing embryo becomes visible
70
L .jqp; s.5 * .;:..: . . l
8
.
I* . .-’
l
.
i:*
.*: *.
.
10 . I_./ 2
4
:*
. .i . .
.
I
I
6
6
10
SIIII~U"
I( "~I11
1 12
1 14
) 16
1 16
(111111)
FIG. 3. Relationship between seed set (number of seeds per sfiique) and mature silique length in Arabidopsis thakana. Siliques from plants heterozygous for an embryo-lethal mutation show this same relationship.
VOLUME 72, 1979
through the translucent seed coat. Seeds become increasingly green as the cotyledons develop, and remain green until late in seed maturation, when the seed coat dries out and turns brown. Liquid endosperm is abundant at the globular and heart stages of embryo development, and then gradually diminishes as the embryo grows. Mature seeds contain essentially no endosperm tissue, and are filled by the mature embryo. Identification
of Aborted
Seeds
The isolation and characterization of a large number of embryo-lethal mutants are possible only in plants where physiological abortion is rare, and aborted seeds can be clearly distinguished from normal seeds and unfertilized ovules. Physiological or spontaneous abortion in wild-type plants of Arabidopsis grown under the conditions described above averaged less than 0.5% of screened seeds. This is significantly lower than the abortion rates found in many plants with larger seeds. Moderate changes in temperature, availability of water, and the supply of nutrients did not affect the frequency of physiological abortion in Arabidopsis; most aborted seeds appeared to be the result of physical damage to developing siliques. However, siliques that began development just prior to senescence of the whole plant frequently showed a variety of developmental abnormalities, and were not used in subsequent studies of embryo-lethal mutants. In order to study embryo development in embryo-lethal mutants, it is necessary to identify aborted seeds in immature fruits, shortly after embryo development has been arrested, and before the whole seed degenerates. Further studies with these mutants are then simplified if aborted seeds can also be clearly recognized in mature fruits. All recessive embryo-lethal mutants, with the exception of temperature sensitives, must be isolated and maintained as heterozygotes since homozygotes die as embryos.
MEINKE
AND SUSSEX
Mutant Analysis of Plant Embryogeny
Aborted seeds must therefore be obtained from fruits of heterozygous plants, which contain 25% aborted seeds and 75% phenotypically normal seeds (?& heterozygotes and Y3 wild type), as shown in Fig. 4. Aborted seeds can be studied only after they become phenotypically distinguishable from normal seeds in the same fruit. Aborted seeds of Arabidopsis can be unambiguously identified by their size or color in both immature and mature siliques (Fig. 5). Aborted seeds initially resemble normal seeds, but while normal seeds enlarge and turn green during later stages of embryo development, aborted seeds remain colorless and arrested at a size characteristic of the stage of developmental arrest. Seeds arrested at a preglobular embryo stage are therefore significantly smaller than seeds arrested at a heart stage (see Figs. 1 and 5). All aborted seeds containing embryos arrested before the linear cotyledon stage of development can be clearly recognized in heterozygous siliques 8-12 days after pollination. At this stage, normal seeds are green and have reached mature size, unfertilized ovules are very small white masses, and aborted seeds are intermediate in size and either colorless, milky white, or beginning to degenerate and turn brown. Seeds containing pale green, yellow, or albino embryos that have reached mature size can also be recognized at this stage because the embryo is visible through the translucent seed coat. Aborted seeds containing embryos arrested prior to the linear cotyledon stage
~00.00.000. Q.000.0.000 FIG. 4. Segregation of normal and aborted seeds in a silique heterozygous for a recessive embryo-lethal mutation. Individual siliques contain approximately 25% aborted seeds (black), 25% wild-type seeds (white), and 50% heterozygotes (stippled). Heterozygous and wild-type seeds are both phenotypically normal, but their frequency can be determined by scoring plants in the next generation.
55
become deflated as the silique matures because all of the liquid endosperm is resorbed, and there is nothing left to fii the seed coat. Fortunately, all aborted seeds in Arabidopsis can be identified 2-3 days after developmental arrest, and several days before degeneration begins. Although some aborted seeds are only slightly larger than unfertilized ovules, all abortants turn brown at maturity, whereas unfertilized ovules degenerate into white masses.
Isolation of Mutants Mutants in higher plants have been recovered following mutagenesis of either pollen grains or mature seeds. Although X irradiation of pollen grains has been reported in Arabidopsis (Usmanov and Muller, 1970), and procedures for chemical mutagenesis of pollen grains have been described in corn (Neuffer et al., 1978), the easiest way to generate mutants in Arabidopsis is to mutagenize mature seeds. Embryo-lethal mutants can then be identified by screening immature siliques of the resulting M-l plants for aborted seeds. Recessive mutations affecting morphological features of the mature plant can be recognized only when they become homozygous in the M-2 generation; embryo-lethal mutants can be recovered more quickly because homozygous mutant embryos can be identified in the siliques of M-l plants following self-pollination. However, since the shoot apical meristem in mature Arabidopsis seeds is a multicellular structure, M-l plants grown from mutagenized seeds will be mericlinal chimeras with heterozygous mutant cells and siliques occupying only a wedge-shaped sector of the primary reproductive inflorescence (Fig. 6). The most efficient way to recover embryo-lethal mutants following seed mutagenesis in Arabidopsis is to screen the first five siliques of each M-l plant. This increases the probability of detecting the presence of a mutant sector because each silique arises from a different part of the meristem. Recessive embryo-lethal mutant
FIG. 5. Identification of normal seeds, aborted seeds, and unfertilized ovules. (A) Immature siliques from a plant heterozygous for an embryo-lethal mutation, Valves from each silique have been separated to reveal two phenotypic classes of seeds: normal, green seeds (n) at a mature cotyledon stage of embryo development; and aborted, white seeds (m) prior to degeneration. 78 normal seeds and 25 abortants are visible. Differences in color between normal and aborted seeds at this stage of development are difficult to photograph, but can be clearly seen under a dissecting microscope. Scale bar equals 2 mm. (B) Stages in normal seed development: (a) fertilized ovule (colorless); (b) preglobular embryo stage (white); (c) globular embryo stage (white); (d) early cotyledon stage (pale green); (e) mature cotyledon stage (green); (f) mature seed (brown). B-E are all at the same magnification; scale bar equals 0.5 mm. (C) Unfertilized ovules several days after pollination. (D) Aborted seeds from mature siliques of several different embryo-lethal mutants. All aborted seeds become brown and deflated at maturity. (E) Aborted seeds from immature siliques of several different embryo-lethal mutants. Aborted seeds are initially either colorless or white, and resemble normal seeds at early stages of development. Note that the size of both immature and mature abortants can differ significantly, corresponding to differences in the stage of developmental arrest. 56
MEINKE
AND SUSSEX
Mutant
Analysis
of Plant Embryogeny
57
peared to be segregating for embryo-lethal mutations following X irradiation of imbibed seeds. Segregation ratios and the approximate stage of developmental arrest were determined for 72 of these mutants analyzed in subsequent generations. Mutants were classified according to the stage of developmental arrest and the size and color of aborted seeds and embryos. Six different types of embryonic lethals were recognized (sicca, brevis, vana, diffusa, murca, and parva) in addition to three types of seedling lethals (albina, xantha, and chlorina). This system of classification is described in more detail by Muller (1963) and Usmanov and Miiller (1970). FIG. 6. Diagrammatic cross section of the primary inflorescence and first five siliques of an M-l chimera1 plant grown from mutagenized seed. Consecutive siliques are separated by an angle of 137’. Cells heterozygous for an induced recessive embryo-lethal mutation (stippled) cover a wedge-shaped sector of the primary M-l inflorescence. Siliques arising from this mutant sector contain approximately 25% aborted seeds (dark circles). Open circles represent phenotypically normal seeds.
chimeras will have one or two of the first five siliques segregating for aborted seeds, and three or four siliques with normal seeds (Fig. 6). If immature siliques (8-12 days after pollination) are used for screening, additional mature siliques must be recovered from the mutant sector of the original plant to provide viable heterozygous seeds for the next generation. Each suspected mutant can then be verified and characterized in the M-2 and subsequent generations. Procedures for the identification of aborted seeds and isolation of embryo-lethal mutants in Arabidopsis were first developed by Muller (1963), and have been referred to previously as the Muller embryo test. Miiller was primarily interested in using embryo-lethal mutants to study the mutagenic activity of potential mutagens in higher plants, In his most extensive study, Muller (1963) identified over 3000 M-l plants of wild-type strain “Dijon” that ap-
Classification
of Mutants
Although the classification system of Muller (1963) is useful in cataloguing large numbers of embryo-lethal mutants of Arabidopsis, it provides relatively little information on the stage and possible cause of developmental arrest, particularly for those mutants that arrest early in development. For example, the term “sicca” applies to all mutant embryos that arrest prior to the torpedo stage, when most of the critical events of early embryo development have been completed. An alternative approach is to catalogue mutants by number, determine the stage of developmental arrest (heart, late globular, early globular, etc.) by examining aborted seeds under dissecting and compound light microscopes, and then attempt to classify each mutant as a cellular, nutritional, or developmental lethal, based upon its pattern of developmental arrest and the response of mutant embryos in culture. Mutations affecting essential cellular processes (e.g., membrane biogenesis or protein synthesis) may result in embryo abortion if they do not become lethal during gametogenesis. Embryo abortion in these cellular lethals should occur at very early stages of development, and arrested embryos should be incapable of further growth in culture.
58
DEVELOPMENTALBIOLOGY
Developmental arrest in nutritional lethals is caused by the absence of an amino acid, vitamin, or other diffusible nutrient required for continued growth and development. Only a few nutritional or auxotrophic mutants have been isolated in higher plants, and all are seedling lethals or semilethals (Redei, 1975c; Gavazzi et al., 1975). However, Langridge (1958) has suggested that nutritional mutants may exhibit embryo abortion if the missing nutrient is required for embryonic growth and cannot be supplied by the surrounding maternal tissue. Arrested embryos from nutritional lethals should resume normal embryonic development when cultured on a medium that supplies the missing nutrient, and should form callus when the enriched medium contains the appropriate growth hormones. Embryo culture can therefore be used to identify nutritional lethals, rescue the arrested embryos, and study the biochemistry of the deficiency. A similar approach is being used to study embryo-lethal mutants of corn (Sheridan et al., 1978), and has been applied at the seedling stage to analyze thiamine auxotrophs of Arabidopsis (Li and Redei, 1969). Arrested embryos from developmental mutants should by definition be unable to complete a specific stage of embryo development (e.g., initiation of cotyledons), even when placed on an enriched nutrient medium. However, since basic cellular functions should not be altered, arrested embryos from developmental mutants should be capable of disorganized cellular growth (callus) on either an enriched or a basal nutrient medium containing the appropriate growth hormones. Developmental embryo lethals will be difficult to analyze biochemically because they are not easily rescuable, but microscopy should provide important information on the stage and possible cause of developmental arrest in many of these mutants. It is expected that some developmental mutations will initially affect only one part of the developing seed (embryo proper, suspensor, or endosperm) ,
VOLUME 12,1979
and will allow further development of the other parts. This pattern of developmental arrest may be helpful in the identification of developmental mutants and the analysis of tissue interactions during normal development. Application
to Study of Development
Embryo-lethal mutations and other genetic factors affecting embryo development have been studied extensively in a variety of animal systems (Hadorn, 1961; Gluecksohn-Waelsch, 1963), including the mouse (Wudl et al., 1977), the Mexican axolotl (Brothers, 1976), and the nematode Caenorhabditis elegans (Vanderslice and Hirsh, 1976). A major challenge with these and other developmental mutants has been to determine why development becomes abnormal, and how this abnormal development can provide new information about normal development. Embryo-lethal mutants of Arabidopsis may be useful in the study of three important areas of plant development: (1) maternal control of early embryo development; (2) changing nutritional requirements and synthetic capacities of developing embryos; and (3) the role of the embryo proper, endosperm, and suspensor in normal seed development. Maternal effects on animal development have been studied for many years (see Davidson, 1976), but little is known about the possible maternal control of plant embryo development. Since normal embryo development has been observed in isolated ovules cultured in vitro (Maheshwari, 1958; Kapoor, 1959; Meinke, unpublished resuits), regulatory functions must be limited to maternal tissues located within the developing seed (nucellus, integuments, and seed coat). One way to study the maternal control of embryo development in Arabidopsis would be to isolate maternal-effect embryo-lethal mutants. Unlike recessive embryo-lethal mutants, maternal-effect lethals would not be identified until the M-2 generation, and would produce fruits with 100% rather than 25% aborted seeds. Em-
MEINKE
AND SUSSEX
Mutant Analysis
bryonic lethality in these plants would be determined by the genotype of the maternal plant rather than the genotype of the embryo; homozygous mutant embryos would develop normally in heterozygous plants, but would arrest at some stage of embryo development in homozygous plants due to the loss of a specific regulatory function that is normally performed by the maternal plant. Maternal-effect lethals may be particularly useful in understanding the role of the chalazal nucellar tissue in embryo and endosperm development (see Schultz and Jensen, 1971). Nutritional embryo-lethal mutants will be isolated only if the missing nutrient is required for embryo development and cannot be supplied by the surrounding maternal tissue. Nutritional mutants can therefore provide information on both the maternal supply of nutrients throughout embryo development and the changing nutritional requirements and synthetic capacities of developing embryos. The thiamine auxotrophs of Arabidopsis, which become lethal at the seedling stage, could also be used to study embryogeny; mutant embryos must either receive thiamine from the maternal plant, synthesize a small but sufficient amount of it with defective enzymes, or produce the vitamin through an alternative biochemical pathway that is active only during embryogeny. Information gained from the analysis of nutritional mutants could supplement the more general picture of changing nutritional requirements obtained from embryo culture studies (see Raghavan, 1976). Auxotrophic embryo lethals may also represent a valuable source of conditional-lethal mutants, which could serve as the basis for hybrid selection schemes in plant somatic cell genetics (see Day, 1977; Power and Cocking, 1977). Developmental mutations may initially affect only the endosperm, embryo proper, or suspensor, and may therefore be useful in studying interactions that normally occur between these different parts of a developing seed. Both the endosperm and the
of Plant Embryogeny
59
suspensor are thought to play a nutritive role in plant embryo development (Brink and Cooper, 1947; Yeung, 1977), but they may also regulate or trigger developmental events in the embryo proper. Alternatively, the embryo proper may regulate not only its own development, but that of the endosperm and suspensor as well. Histological examination of one of the embryo-lethal mutants of Arabidopsis described in the subsequent paper (Meinke and Sussex, 1979) has shown that developmental arrest of the embryo proper in this mutant is followed by abnormal growth of the suspensor (Meinke and Marsden, in preparation). When coupled with evidence from other sources, this suggests that continued growth of the suspensor during normal development is inhibited by the embryo proper, and it further illustrates how embryo-lethal mutants may be applied to the study of normal embryo development. Additional questions could be studied through the isolation of temperature-sensitive embryo-lethal mutants. The advantage of temperature-sensitive lethals is that homozygotes can be maintained at the permissive temperature, and the effect of the mutant gene product can be studied at any stage of development. Arabidopsis is an ideal plant for the isolation of temperaturesensitive mutants; vegetative and reproductive development is normal over a wide range of temperatures, and a large number of plants can be grown in the limited space provided by constant-temperature growth chambers. Temperature-sensitive embryolethal mutants have not been reported in flowering plants, but temperature sensitivity has been detected in several naturally occurring races and vitamin-requiring mutants of Arabidopsis (Langridge and Griffing, 1959; Langridge, 1965; Li and Redei, 1968). If the frequency of temperature-sensitive lethals in higher plants is similar to that observed in Drosophila (Baillie et al., 1968), approximately 10% of all EMS-induced embryo-lethal mutants should have a temperature-sensitive phenotype. The
60
DEVELOPMENTAL BIOLOGY
application of temperature-sensitive mutants to all areas of plant genetics and development remains to be explored. We thank Dr. G. P. Redei for wild-type seeds of Arabidopsis, Ruth Schmitt for an English translation of the Muller (1963) paper, and members of the Sussex lab group, particularly John Duesing, for helpful discussions. This work was supported by NSF Grant PCM76-17222 to I. M. Sussex, and NIH Training Grant HD 00032. REFERENCES BAILLIE, D., SUZUKI, D. T., and TARASOFF, M. (1968). Temperature-sensitive mutations in Drosophila melanogaster. II. Frequency among second chromosome recessive lethals induced by ethyl methanesulfonate. Canad. J. Genet. Cytol. 10,412-420. BRINK, R. A., and COOPER, D. C. (1947). The endosperm in seed development. Sot. Rev. 13, 423-541. BROTHERS, A. J. (1976). Stable nuclear activation dependent on a protein synthesized during oogenesis. Nature 260, 112-115. DAVIDSON, E. H. (1976). “Gene Activity in Early Development,” second ed. Academic Press, New York. DAY, P. R. (1977). Plant genetics: Increasing crop yield. Science 197, 1334-1339. DURE, L. S. (1975). Seed formation. Annu. Rev. Plant Physiol. 26, 259-278. GAVAZZI, G., NAVA-RACCHI, M., and TONELLI, C. (1975). A mutation causing proline requirement in Zea mays. Theor. Appl. Genet. 46,339-345. GLUECKSOHN-WAELSCH, S. (1963). Lethal genes and analysis of differentiation. Science 142, 1269-1276. HADORN, E. (1961). “Developmental Genetics and Lethal Factors.” John Wiley, New York. HUANG, P. C., and PADDOCK, E. F. (1962). The time and site of the semidominant lethal action of “Wo” in Lycopersicon esculentum. Amer. J. Bot. 49,388393. KAPOOR, M. (1959). Influence of growth substances on the ovules of Zephyranthes. Phytomorphology 9, 313-315. KING, R. C., and MOHLER, J. D. (1975). The genetic analysis of oogenesis in Drosophila melanogaster. In “Handbook of Genetics,” Vol. 3, pp. 757-791. Plenum Press, New York. LANGRIDGE, J. (1958). A hypothesis of developmental selection exemplified by lethal and semi-lethal mutants of Arabidopsis. Aust. J. Biol. Sci. 11, 58-68. LANGRIDGE, J. (1965). Temperature-sensitive, vitamin-requiring mutants of Arabidopsis thaliana. Aust. J. Biol. Sci. 18, 311-321. LANGRIDGE, J., and GRIFFINC, B. (1959). A study of high temperature lesions in Arabidopsis thaliana. Aust. J. Biol. Sci. 12, 117-135. LI, S. L., and RI?~EI, G. P. (1968). Temperature-sensitive, thiamine-requiring mutants in Arabidopsis. Arabidopsis Inf Seru. 5, 28-29.
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LI, S. L., and R~DEI, G. P. (1969). Thiamine mutants of the crucifer, Arabidopsis. Biochem. Genet. 3, 163-170. MAHESHWARI, N. (1958). In vitro culture of excised ovules of Papaver somniferum. Science 127, 342. MANGELSDORF, P. C. (1926). The genetics and morphology of some endosperm characters in maize. Conn. Agr. Exp. Sta. Bull. 279, 513-620. MEINKE, D. W., and SUSSEX, I. M. (1979). Isolation and characterization of six embryo-lethal mutants of Arabidopsis thaliana. Develop. Biol. 72, 62-72. MONNIER, M. (1976). Culture in vitro de l’embryon immature de Capsella bursa-pastoris Moench. Rev. Cytol. Biol. Veget. 39, l-120. MILLER, A. J. (1963). Embryonentest zum Nachweis rezessiver Letalfaktoren bei Arabidopsis thaliana. Biol. Zentralbl. 82, 133-163. NEUFFER, M. G., SHERIDAN, W. F., and BENDBOW, E. (1978). Rescue of lethal defective kernel mutants by genetic manipulation. Maize Genet. Coop. Newsl. 52,84-88. POWER, J. B., and COCKING, E. C. (1977). Selection systems for somatic hybrids. In “Applied and Fundamental Aspects of Plant Cell, Tissue, and Organ Culture,” pp. 497-505. Springer-Verlag, New York. RAGHAVAN, V. (1976). “Experimental Embryogenesis in Vascular Plants.” Academic Press, New York. RAGHAVAN, V., and TORREY, J. G. (1963). Growth and morphogenesis of globular and older embryos of Capsella in culture. Amer. J. Bot. 50, 540-551. RI?DEI, G. P. (1970). Arabidopsis thaliana (L.) Heynh. A review of the genetics and biology. Bibliogr. Genet. 20, l-151. R~~DEI, G. P. (1975a). Arabidopsis as a genetic tool. Annu. Rev. Genet. 9, 111-127. R~DEI, G. P. (1975b). Arabidopsis thaliana. In “Handbook of Genetics,” Vol. 2, pp. 151-180. Plenum Press, New York. R~DEI, G. P. (1975c). Induction of auxotrophic mutations in plants, In “Genetic Manipulations with Plant Material,” pp. 329-350. Plenum Press, New York. RICE, T. B., and CAHLSON, P. S. (1975). Genetic analysis and plant improvement. Annu. Rev. Plant Physiol. 26, 279-308. ROSS-CRAIG, S. (1948). “Drawings of British Plants. III. Cruciferae,” plate 37. G. Bell, London. SCHULZ, R., and JENSEN, W. A. (1968a). Capsella embryogenesis: The early embryo. J. Vltrastr. Res. 22,376-392. SCHULZ, R., and JENSEN, W. A. (196813). Capsella embryogenesis: The synergids before and after fertilization. Amer. J. Bot. 55, 541-552. SCHULZ, R., and JENSEN, W. A. (1968c). Capsella embryogenesis: The egg, zygote and young embryo. Amer. J. Bot. 55,807-819. SCHULZ, P., and JENSEN, W. A. (1971). Cupsella embryogenesis: The chalazal proliferating tissue. J. Cell Sci. 8, 201-227.
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