Arabidopsis Thaliana

Arabidopsis thaliana: Genetic Portrait of a Model Plant
A. thaliana, a tiny weed whose common name is mouse ear cress, grows low to the ground (Fig. B.1) and produces clusters of small white owers in meadows and laboratories around the globe. Like more than 150,000 other species of owering plants, including roses, daisies, tomatoes, peas, beans, and maple trees, Arabidopsis is a dicotyledonous angiospermdicotyledonous because the mature embryo carries two specialized seed leaves (or cotyledons), and angiosperm because its seeds are enclosed in an ovary within a ower. Arabidopsis shares three basic characteristics with other plants: It is an autotroph, that is, a nutritionally self-sufcient organism that produces its own food via photosynthesis; it is nonmotile, that is, rooted to one spot; and it produces new organ systems continuously throughout its life cycle (which consists of alternations between a diploid [2n] sporophyte generation and a haploid [n] gametophyte generation). The impetus for studying a small owering weed with no commercial potential is straightforward: Plants are the chief source of food for life on our planet, and an understanding of how they grow and reproduce may make it possible to grow more nutritious, more copious crops in a wide range of environments. In addition, A. thaliana is one of the premier eukaryotic experimental model organisms. Much of what we have learned from this plant can be applied directly to animals and other eukaryotes. Classied in the same family as mustard and cabbage plants, A. thaliana has several features that make it an excellent experimental model. Its general strategies of growth, development, owering, and seed production are the same as those of other higher plants, including many crop plants. However, it has a shorter generation time than most other plants, requiring only six weeks for seeds to germinate and develop into mature plants that produce more seeds. Moreover, like the peas that Mendel studied, it reproduces mainly by self-fertilization, yet cross-pollinationthrough the removal of stamen from one plants owers and the application of pollen from another plants owers onto the stigma of the stamen-deprived owersis possible. Whether self- or cross-pollinated, wild-type Arabidopsis plants produce a very large number of seedsfrom 10,00040,000 per plantand these seeds have a high rate of germination. Such rapid, abundant reproduction makes it possible for geneticists to screen large populations of seedlings for specic phenotypes. Finally, A. thaliana grows well in the laboratory, requiring relatively little light (illumination from cool, white uorescent bulbs is sufcient) and temperatures in the range of 2226C. Because the plant is small, researchers can grow hundreds of thousands of them a year in a modest-sized laboratory with no special growth facilities. One goal of geneticists studying A. thaliana is to understand the physiology, biochemistry, growth, and development of a plant at the molecular level. They can use this knowledge to produce new varieties of crops and other plants with increased stress tolerance, for example, or containing more of particular parts prized for their nutritional content or oral display. Ornamental plant breeders have already accomplished this second goal with some species. For example, the owers of wild camellias contain stamens (male reproductive organs) and carpels (female reproductive organs) that enable them to produce seeds; but the owers of a cultivated variety known as Pink Perfection lack both male and female reproductive organs and carry many extra petals in their place (Fig. B.2).
Reference
The ower of Arabidopsis thaliana.
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Reference B Arabidopsis thaliana: Genetic Portrait of a Model Plant
(a)
(b)
Figure B.1
a hand.
A. thaliana in the palm of
Figure B.2 The Pink Perfection camellia as a horticultural mutant. (a) Wild-type camellia. (b) Pink Perfection ower.
In our examination of A. thaliana as a model plant, we present: The structure and organization of the genome. The plants anatomy and life cycle. Techniques of mutational analysis, including chemical and radiation procedures, insertional mutagenesis, and the analysis of mutations to identify gene function. Genetic analysis applied to various aspects of development, including embryogenesis, hormonal control systems, and responses to environmental signals. The genetic analysis of owering: a comprehensive example.
B.1
Genome Structure and Organization
The genome of a model organism for genetic studies should be relatively small but still share the functional genomic characteristics of the organisms it represents. A. thaliana lls this bill to perfection. Its genome, composed of 125 Mb, is one of the smallest genomes known in the plant kingdom, notably smaller than the genomes of most other angiosperms. By contrast, the genome of the angiosperm maize (Zea mays) is 45 times larger. The Arabidopsis genome is about as large as the genome of Caenorhabditis elegans (see Portrait C) and is 60% the size of the Drosophila melanogaster genome (see Portrait D). The nuclear DNA of Arabidopsis is carried by ve pairs of small chromosomes with well-dened banding patterns. Researchers completed sequencing the whole genome of Arabidopsis in the year 2000.
alleles controlling developmental and physiological characteristics. Common strains used in Arabidopsis research include Columbia (Col) and Landsberg erecta (Ler, a mutant derivative of the Landsberg strain). Arabidopsis strains differ from each other not only in phenotypes but also in a signicant number of DNA polymorphisms that are identiable by RFLP, PCR, or SNP analysis (as described in Chapter 11 of the main textbook). Different strains have different alleles at many different DNA marker loci. Researchers can use allelic variation at DNA markers to follow the segregation of chromosomal regions carrying the markers in the progeny of self-pollinated plants derived from a cross between different ecotypes. They can also analyze the linkage between DNA markers as well as between DNA markers and morphological loci (dened by two or more alleles conferring alternative phenotypes on the different strains). With the resulting data, researchers can create DNA marker and phenotypic maps, and ultimately they can integrate the two types of maps for all ve chromosomes (Fig. B.3).
Comparing Genetic and Physical Maps

Geneticists have taken advantage of different genetic strains of Arabidopsis derived from natural isolates. Because Arabidopsis is a predominantly self-pollinating plant, each of these isolate-derived strains represents a nearly pure inbred line, homozygous for a distinct set of
Little Repetitive DNA and a Tight Arrangement of Genes

The small size of the A. thaliana genome stems from the fact that it contains much less repetitive DNA than most other angiosperms. In fact, the Arabidopsis genome contains 26,607
distance from centromere (megabases)
linkage mapped DNA markers used for cloning
overlapping genomic clones
protein-coding loci
1M 2M 3M 4M 5M 6M
SM106_27.. F20D22-5..
SM31_385,7 NF21M12 SGCSNP148 SM86_163,5 SGCSNP303
SM35_139,0 7M 8M 9M 10M 11M 12M 13M 14M SM172_87,4 15M 16M 17M 18M 19M 20M 21M 22M 23M 24M 25M 26M 27M 28M 29M 30M SM126_13.. ADH VE011 SGCSNP142 SM254_37.. PAP240 SM234_96,3 SM137_21.. SGCSNP392 SM152_16.. SM195_92,5 SM53_190,6 AF-2 SM252_32.. SM184_37.. SM236_22.. SM131_21.. SM98_146,6 S0392 SM31_205,8 CXC750 SM175_77,1 SM83_254,3 SM218_20.. SM82_81,0
Figure B.3
Physical mapping of Arabidopsis chromosome 1. Distance from the centromere is indicated at the left. DNA markers mapped to chromosome 1 by linkage analysis are shown next (with names shown for a small subset). These markers were used to probe genomic libraries to build the physical map. A contig of overlapping genomic clones is in the next column. DNA sequencing and further analysis revealed all of the protein-coding loci along the chromosome. Genes on the left of the line are encoded within the DNA strand having a 5 end at the telomere; genes on the right are encoded on the opposite strand with a 5 end at the centromere.
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Transcription Metabolism
Cell growth, cell division, and DNA synthesis Cell rescue, defense, cell death, and aging Cellular communication/ signal transduction Protein destination Intracellular transport
B.2
Anatomy and Life Cycle
To understand a plants growth and development at the molecular level, geneticists use mutational analyses to identify genes that inuence the plants anatomy and life cycle. A brief overview of the anatomy and life cycle of A. thaliana prepares the way for examining the techniques of genetic analysis in this model plant.
Unclassified
Cellular biogenesis Transport facilitation Energy Protein synthesis Ionic homeostasis
Anatomy: The Basic Body Plan

Like most higher plants, the adult A. thaliana is composed of only a few relatively simple organs and tissues (Fig. B.5). The aboveground shoot connected to the belowground root system represents the plants main axis. Both root and shoot contain several cell types organized into the outer epidermal layer; the middle ground, or cortical, layer; and the inner vascular cylinder (which contains the water-conducting xylem vessels and the food-conducting phloem elements). Attached to the shoot are mature leaves also composed of three layers: an epidermal layer, containing both hair cells, or trichomes, and small pores, or stomata (singular, stoma) that regulate gas exchange; a mesophyll layer responsible for photosynthesis; and vascular tissues that conduct water and nutrients between the leaf and the stem.
Figure B.4 Functional analysis of Arabidopsis genes. Proportion of predicted Arabidopsis genes in different categories.
protein-coding genes that make up close to 40% of the total genome and can be grouped into functional categories by sequence analysis (Fig. B.4). This means there is one gene for every 4.5 kb of genome, on averagean unusually tight arrangement. In most plants, the vast majority of the genome consists of repetitive DNA. Sixty-ve percent of the proteins in Arabidopsis are encoded by more than one gene; and analysis of large duplications in the genome suggests that Arabidopsis derived from an ancient tetraploid. Taken together, these observations suggest a large extent of functional redundancy in Arabidopsis. Like other species, the A. thaliana genome contains multiple copies of transposable elements, as well as several pieces of DNA that appear to have originated in the mitochondrial and chloroplast genomes. As in animals, the centromeres are hetero-chromatic regions rich in repetitive DNA where recombination is strongly supressed. Comparisons of the genetic and physical maps of the A. thaliana genome reveal that genetic and physical distances vary widely along the plants chromosomes. Within each chromosome, the frequency of recombination varies greatly such that a centimorgan represents anywhere from 301600 kb (or 1.6 Mb).
The Apical Meristem Promotes Continuous Growth

Cells at the growing points of both shoot and root are organized into a tissue called the apical meristem, a group of undifferentiated cells that divide continuously. The root apical meristem, which consists of a central zone of meristem cells and a peripheral zone of differentiated cells, produces the cylindrical root through cell division, cell expansion, and cell differentiation. The shoot apical meristem, which consists of a central zone where cell divisions also maintain the meristem and a peripheral zone where cell divisions give rise to mature organs such as leaves and lateral shoots, generates the entire aboveground plant.
Summary
A. thaliana has a small genome carried by ve chromosomes. Each chromosome contains a limited amount of repetitive DNA outside the centromeric regions and the ribosomal RNA gene clusters. Its genes, though similar in structure to the genes of other plants, are more tightly packed and contain only small introns. As a result, a much higher percentage of the genome is associated with coding and genetic function than is true of most plants. The high percentage of coding sequences and the low abundance of dispersed repetitive DNA are features that make A. thaliana an excellent model for genetic analysis.
Vegetative Versus Reproductive Development

During vegetative developmentthe period of growth before oweringthe shoot apical meristem initiates a spiral of leaves in precise sequence. When the plant switches to reproductive development, the shoot apical meristem becomes an inorescence meristem, which, in turn, produces a series of lateral oral meristems. Each oral meristem produces a oral primordium that gives rise to a ower. The inorescence is the ower or group of owers at the tip of a branch. In Arabidopsis, the owers are arranged in a spiral around the inorescence stalk, as depicted in Fig. B.5. Plants do not sequester a germline separate from the somatic tissue early in development, as is the case in
B.2 Anatomy and Life Cycle
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Stigma
Petal (whorl 2)
Carpel (whorl 4) Sepal (whorl 1)
Stamens (whorl 3)
Main axis
Shoot
Shoot apical meristem
SEM of pollen grain Elongation zone
Root
Root meristem Root cap Root apex SEM of flower
Figure B.5
The anatomy of A. thaliana. Components of the plant are labeled.
many animals. Consequently, mutations that arise in the shoot apical meristem can give rise to mutant vegetative tissue before being incorporated into the reproductive cell lineages. These reproductive lineages eventually give rise to the haploid cells that result from meiotic cell divisions in the ower.
The Arabidopsis Flower Is the Most Complex Set of Organs in the Plant
The ower consists of a modied stem with four concentric regions, or whorls, of modied leaves, as shown in Fig. B.5.
The rst whorl (whorl 1) consists of four green leaike sepals; whorl 2 is composed of four white petals that are leaike in shape but contain no photosynthetic cells; whorl 3 is made of six stamens (four long and two short) bearing the male gametophytes in the form of pollen; and whorl 4 is a cylinder composed of two fused carpels housing the female gametophytes in the form of embryo sacs enclosed with ovules. The gametophytes represent the haploid phase of the plants life cycle. (They develop by mitotic cell divisions after meiosis and give rise to the alternation of generations: 1n S 2n S 1n S 2n, and so on). The fused carpels are part of a cylinder known as the pistil that
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Two coupled sperm cells Nucleus of enclosing cell Pollen grain (male gametophyte) (a) Pollination Sperm nucleus Central cell nuclei Endosperm (b)
Binucleate central cell Mononucleate egg cell Embryo sac (female gametophyte)
Carpel Pollen tube Style
Ovule
One sperm nucleus fuses with the two nuclei of the central cell forming endosperm nucleus
Pollen tube grows
Egg
Pollen tube (c)
Sperm nuclei moving toward the ovule
One sperm nucleus fuses with the egg nucleus to form the diploid zygote
Figure B.6 Double fertilization in Arabidopsis. (a) Micrograph of a pollen grain landing on a stigma. (b) Diagram of a pollen grain and an embryo sac. (c) Fertilization and postfertilization events.
consists of pollen-receptive stigma at the top and a short neck, or style, leading to the ovary, which houses roughly 50 gamete-bearing ovules. Because the ower contains both male and female reproductive structures, it is considered a perfect ower. After fertilization, the plant sheds the organs of the outer three whorls, and the pistil develops into the seed-bearing fruit. each with one 1n nucleus and a central cell containing two 1n nuclei, all encased within a common cell wall (Fig. B.6b). The landing of a pollen grain on the receptive tissue of the stigma initiates fertilization by triggering germination of the pollen grain (Fig. B.6c). The emerging pollen tube migrates through the short neck of the pistil, also known as the transmitting tract, to an ovule in the ovary. There, one 1n sperm nucleus from the pollen fuses with a 1n egg nucleus from the embryo sac to form the 2n nucleus of the zygote; the second 1n sperm nucleus fuses with the two 1n nuclei within the embryo sac to form a 3n endosperm nucleus. After fertilization, the zygote divides mitotically to form the embryo, and the endosperm nucleus divides mitotically to form endosperm tissue that, like an animal yolk sac, will nourish the developing embryo. Meanwhile, the remaining 1n cells of the ovule degenerate, and the outer layer of the ovule hardens to form a seed coat.
Life Cycle: From Fertilization to Flowering to Senescence

As we have seen, Arabidopsis, like a hermaphrodite animal, carries male and female gametes; it is thus capable of self-fertilization, although cross-fertilization by articial means is easy to accomplish. Fertilization is the rst step in a life cycle that also includes embryonic development, seed germination and vegetative growth, reproductive development, and senescence (Fig. B.6).
Arabidopsis Undergoes Double Fertilization A Process Unique to Higher Plants

In Arabidopsis, each mature pollen grain contains two coupled haploid (1n) sperm cells plus a 1n vegetative cell, while each embryo sac includes six mononucleate cells,
The Embryo Develops Within the Protective Maternal Seed Coat

Embryogenesis begins with a single fertilized egg cell, or zygote, which elongates. A sequence of well-dened cell divisions then leads the embryo through a series of stages (Fig. B.7). The first zygotic division gives rise to two
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Linear axis
Shoot apical meristem Radial axis Ground tissue Epidermis Root apical meristem Vascular system
Torpedo stage Cotyledons
Heart stage
Transient / Triangular stage
Globular stage
Embryo
First cell division Octant stage
Suspensor
Embryo Cell will form suspensor
SEM of embryo in a seed
Figure B.7
Stages of embryonic development in A. thaliana.
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cells, one small and one larger. The small cell constitutes the embryo proper, while the larger cell will divide only a few more times to produce the suspensor, a structure analogous to the umbilical cord in mammals (whose divisions we will not describe here). The embryo divides following a plane at right angles to the rst plane of division to create a vertical wall and two side-by-side cells. The next division, which is at 90 from the previous one, generates four cells that can be seen end-on from the top. The third embryonic division is horizontal and makes an eight-cell, or octant stage, embryo with four cells above and four below. Additional divisions parallel to the surface of the embryo result in the formation of a discrete outer cell layer that will give rise to the epidermis of the mature embryo, and an innermost group of cells that acquire an elongated shape, the rst evidence of differentiation toward the vasculature found in the mature embryo. The embryo has now reached the globular stage. The globular embryo rapidly loses its globular shape and acquires a more triangular form that denes the transient or triangular stage embryo, at which point the embryo becomes self-sufcient for growth, having used up most of its maternally deposited reserves. Next, the embryo moves through the heart stage to the torpedo stage as two protuberances expand and differentiate into two well-dened, discrete cotyledons. By the end of this progression from octant to torpedo stage, cellular differentiation has established all the major tissues required for the future vegetative development of the plant along two main axes: the linear axis that makes up the shoot/root continuum and a radial axis moving from the center out to the edge of that continuum (Fig. B.7). The shoot/root axis includes two cotyledons, which store nutrients in the mature seed and will differentiate into the rst rudimentary leaves after germination. The axis also includes a hypocotyl, which is a short section of embryonic stem between the two cotyledons and the embryonic root, and the root and shoot meristems. The radial axis carries the central vascular system, a middle layer of ground tissues, and the outer epidermis. As the embryo reaches maturity, growth and development cease and desiccation (water loss) occurs. When the loss of water becomes extreme enough, the embryo enters a dormant state in which virtually all metabolic activity ceases. An embryo in the dormant state can withstand adverse environmental conditions such as winter in Madison, Wisconsin, or Buffalo, New York.
along its vertical axis. At this point, environmental cues inuence the form the seedling will take. Both the root and the shoot can sense gravity; the root responds by bending toward it while the shoot bends away from it. Light provides another developmental cue. If germination occurs in the dark, for example, under the surface of the soil, the hypocotyl, or embryonic stem, elongates rapidly; but development of the leaike cotyledons and activity of the shoot apical meristem are suppressed. The elongating hypocotyl is hooked near its apex, an adaptation that enables the seedling to push its way up through the soil while protecting the delicate meristem. When the elongating hypocotyl senses light, elongation ceases, the apical hook opens, and the cotyledons expand and turn green as chloroplasts begin to develop and establish the potential for self-nourishment through photosynthesis (Fig. B.8a). This light-regulated developmental program is called photomorphogenesis. With the establishment of the self-nourishing autotrophic lifestyle, the shoot apical meristem produces a compact spiral, or rosette, of leaves. Early owering varieties of Arabidopsis may only produce 610 leaves before the shoot apical meristem converts to reproductive development. Laterowering varieties may produce a rosette containing dozens of leaves before owering begins (Fig. B.8b).
Reproductive Development Begins When the Leaf-Producing Apical Meristem Switches to a Flower-Producing Meristem
Under the right conditions, the shoot meristem enlarges and switches from producing only cells that differentiate into leaves to producing oral meristems on its anks. At this point, the shoot apical meristem becomes an inorescence meristem. The oral meristems arise in a spiral around the inorescence meristem and, in turn, give rise to ower primordia that differentiate into the four whorls of organs that make up a ower. Initiated one at a time, the owers develop sequentially. Thus a view from the apex of the inorescence meristem reveals the progression of oral developmental stages spiraling away from the shoot apex. Elongation of the main stem between each ower ultimately produces the inorescence branch containing about 35 owers (Fig. B.9). Both environmental cues and endogenous developmental signals (resulting from the genetic program) control the timing of the switch from vegetative to reproductive development in Arabidopsis. Day length, or photoperiod, is a particularly important regulator of flowering. Laboratory strains, such as Columbia and Landsberg erecta, when grown under constant light or a long, 16-hour day, will ower after producing fewer than 10 rosette leaves. When grown in an 8-hour photoperiod, these same strains will produce over 30 leaves before flowering. Another significant environmental signal is temperature. If exposed to extended periods of low temperature during early vegetative development, normally
Favorable Environmental Conditions Trigger Seed Germination and Vegetative Growth

Under appropriate conditions, the Arabidopsis embryo absorbs water and rehydrates within the seed, which enables growth and development to resume. The enlarging embryo bursts out of the seed coat and begins to grow
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Figure B.9 Reproductive development and senescence in A. thaliana. Young owering plants next to older plants undergoing senescence.
that an endogenous developmental clock is active in this process. However, this endogenous program remains poorly characterized.
(a)
Senescence: The Vegetative Plant Ages and Dies

Because plants can produce new organ systems continuously, they do not necessarily display the nite life spans characteristic of animal species. However, some plants, including Arabidopsis, do senesce and die after a single reproductive effort. In Arabidopsis, somatic tissues such as leaves and stems senesce a few weeks after they are initiated at the meristem. In leaves, senescence is a genetically programmed process in which yellowing occurs as salvage systems convert cellular constituents into mobile nutrients for transport out of the aging leaf (Fig. B.9). The limited longevity of individual leaves does not limit the life span of the plant in the vegetative phase because the shoot apical meristem continuously produces new leaves. Rather, it is the conversion of the shoot apical meristem to reproductive development that delimits the production of new photosynthetic organs. In addition, the inorescence meristem ceases proliferative activity after a set number of seeds have been produced. Seeds produced by self-pollination are shed from the parent plant, which undergoes senescence. Quick reproduction, the production of seeds by self-pollination, and the lack of wide seed dispersal serve the plants strategy in natural settings: Arabidopsis is an ephemeral that exploits disturbed soils and completes its life cycle quickly to reseed the soil near the parent plant.
(b)
Figure B.8 Emerging shoot in A. thaliana. (a) The embryonic stem reaches for the surface of the soil (right seedling), where the apical hook will open, allowing the greening of the cotyledons (left seedling). (b) In early-owering varieties, the shoot apical meristem may produce only a few leaves before developing owers (left). Late-owering varieties may produce dozens of leaves before owering (right). No owers are visible here, but the stem growing up at the center of each plant represents the primary inorescence, or owering stem. The late-owering plant on the right is considerably older than the early-owering plant on the left.
late-owering varieties of Arabidopsis will ower early. This phenomenon of exposing plants to the cold early in vegetative development is known as vernalization. Since neither photoperiod stimulation nor vernalization is strictly necessary for owering, researchers inferred
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B.3
Techniques of Mutational Analysis
In the T-DNA Approach, the Bacterium Agrobacterium tumefaciens, Which Causes a Type of Benign Tumor in Plants, Is the Agent of Transformation
A. tumefaciens can transfer a piece of plasmid DNA known as the T-DNA into the genome of wounded plant cells (Fig. B.10a). Experimenters can modify the T-DNA to eliminate its oncogenes and insert transformational marker genes that make the plant resistant to antibiotics (such as kanamycin and hygromycin) or herbicides (such as chlorsulfuron). In one protocol using modied T-DNA, Arabidopsis is transformed by soaking the shoots of mature three-week-old seedlings or germinating seedlings in an Agrobacterium culture to which a strong vacuum or a weak detergent has been applied. The vacuum or detergent enable the bacteria to inltrate the shoots. As the bacterium-infiltrated plants grow and eventually selfpollinate, a few gametes or zygotes are transformed by Agrobacterium, and these produce a transformed seed that is resistant to antibiotics or herbicides. This resistance provides a means of selecting for transformed plants that have inserted the transgenic DNA into their genomes. Because the A. tumefaciens T-DNA integrates more or less randomly into the plant genome, it has the potential to serve as a mutagenic agent for any gene. Initially, researchers used insertional T-DNA mutagenesis to create collections of independent transgenic Arabidopsis lines, which were screened for specic morphological, developmental, or biochemical phenotypes. Subsequently, several large-scale projects sought to assemble sequence-indexed T-DNA mutant collections by determining the nucleotide sequence of regions adjacent to individual insertions. As of 2006, there were over 350,000 different T-DNA insertions mapped at the nucleotide level, covering a large majority of the genes in the Arabidopsis genome. Arabidopsis geneticists can now order from public stock centers T-DNA insertion alleles in almost any gene to begin reverse genetic analyses. Researchers have also developed a second generation of mutagenesis tools based on T-DNA insertions. One variation uses a T-DNA loaded with strong transcriptional enhancer elements to amplify the expression of genes close to the insertion site. This approach, called activation tagging, allows the identication gain-of-function alleles of redundant genes that may go undetected in a traditional loss-of-function mutation screen. Another T-DNA mutagenesis scheme uses a reporter gene, such as GUS, with an incomplete promoter lacking an upstream enhancer element. GUS encodes the enzyme -glucuronidase, which converts a colorless soluble compound (X-Glu) into a blue insoluble product. Cells expressing GUS stain blue. Because the GUS gene in the modied T-DNA has an incomplete promoter, its expression depends on the insertion of the T-DNA into a plantcoding sequence; when that happens, the regulatory region
Researchers have used various techniques to identify mutations affecting many aspects of Arabidopsis anatomy and development. Some of the mutations characterized occur spontaneously in natural populations, but as described in Chapter 7 of the main textbook, these spontaneous mutations appear at a low rate. Geneticists use mutagens to increase their frequency. The means of inducing mutations in Arabidopsis include exposure to mutagenic chemicals, exposure to radiation, and insertional mutagenesis.
Mutagenesis by Chemical and Irradiation Procedures

A mature seed carries a dormant embryo in which one to three cells from the apical meristem are destined to form the germ line of the mature plant, which will give rise to the gametophytes. If a mutation is to be transmitted to progeny, it has to affect one of the two alleles carried by any of these cells. The phenotype derived from a recessive mutation induced by seed mutagenesis in Arabidopsis will segregate in different ratios depending on how many gametophyte progenitor cells are present in the embryo at the time of mutagenesis. To be recovered, induced mutations must pass through the male and female gametophytes, which are multicellular, genetically active, haploid structures. The gametophyte stage lters out mutations in genes that are required for gametophyte viability. For this reason, most large deletions cannot by transmitted through a plants gemetophytes. In contrast, deletions are often transmitted through animal gametes. This difference explains why the use of deletion mutation panels, a common practice in animal genetics, is not possible in plant genetics. In practice, screens or selections to identify Arabidopsis mutants generally focus on investigation of the M2 generation that is derived from self-pollinated seeds harvested from plants that develop from mutagenized seed (M1). In the M2 generation, the induced alleles are segregating, which allows homozygotes to be identied and recovered.
Arabidopsis Researchers Use Two Types of Insertional Mutagenesis: Transformation by T-DNA and Transposon Tagging
Strategies for insertional mutagenesis depend on the ability of certain classes of DNA molecules to integrate into the Arabidopsis genome. If an integration disrupts a gene, it represents a mutation.
B.3 Techniques of Mutational Analysis
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(a)
Agrobacterium tumefaciens Ti plasmid T-DNA
Chromosomes
+
Bacterial chromosome T-DNA transfer Wounded Arabidopsis cell Nucleus
T-DNA integration
T-DNA is integrated into genome of plant cell
(b)
Figure B.10
GUS gene.
The natural process of A. tumefaciens transformation can be exploited for use as a method of gene transfer into A. thaliana. (a) Natural integration of T-DNA from Agrobacterium tumefaciens into A. thaliana. (b) Blue-staining cells expressing
of the disrupted gene controls the expression of GUS. The expression of GUS thus reports the expression pattern of the disrupted gene (Fig. B.10b). The use of the GUS reporter allows not only the mutation of genes but also the determination of the disrupted genes expression pattern (see the discussion of enhancer-trapping in Drosophila in Portrait D).
Transposon-Tagging Strategies Rely on Transposable Elements from Corn (Zea mays)

Transposable elements, originally identied and characterized in corn, can transpose to a new position in the
Arabidopsis genome by the Agrobacterium-mediated DNA transformation just described. The majority of transposonbased mutagenesis systems used in Arabidopsis are based on the corn transposable element (TE) families known as Ac/Ds and Spm/dSpm; early transgenesis experiments demonstrated that these TE families transpose in Arabidopsis. Most of the current mutagenesis strategies use a twoelement scheme: one transgene encodes a transposase required to mobilize a derivative transposon carrying a selectable or screenable marker in between the transposon end sequences. Once mobilized, the transposon derivative carrying the marker gene is stabilized by segregation of the transposase source. Transposons have been used to create
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populations carrying simple insertional alleles. In addition, modied transposon elements have been engineered to generate alleles in which the reporter gene incorporated within the mobile element falls under the control of local enhancer elements (enhancer trap) or lands within a gene to generate a transcriptional fusion (gene trap).
I/R transgenes
Hairpin RNA
Insertional Mutagenesis Not Only Allows the Generation of Mutations but Also Facilitates Their Molecular Characterization
One advantage of insertional mutagenesis over chemical and radiation procedures is that insertional mutagenesis produces mutated alleles carrying a specic DNA insert (the T-DNA or the transposable element). A copy of the specic T-DNA or transposable element that became the insert can therefore serve as a probe to identify and clone the gene the insert disrupts. With the use of enhancer or gene trap systems, the insertions can also indicate the expression pattern of the tagged gene.
Dicer
Dicing P OH Small RNA
Assembly
From Gene to Phenotype: Analyzing Mutations to Identify Gene Function

The genome sequencing project has identied many genes with specic sequences, but the sequence alone is often not sufcient to attribute a specic function to a particular gene. One way to solve this problem is to mutate the gene under study and analyze the phenotype of plants homozygous for the mutation. Since knocking out genes by homologous recombination is not very efcient in plants, Arabidopsis researchers increasingly rely on sequenceindexed insertional mutant libraries. Another approach is the use of gene silencing to suppress the expression of genes of interest.
RISC Slicing
7mG Target mRNA substrate
....A A A A
Figure B.11
The natural RNA interference system can be exploited to achieve experimental gene silencing. The
double-stranded RNA produced by folding of a transgenic mRNA containing inverted repeats is recognized by the Dicer ribonuclease and cleaved into 21 bp fragments, which are loaded onto RISC. Target mRNAs are recognized by the RISCcarrying RNA fragments and cleaved into smaller, noncoding fragments. (See Chapter 18 Tools of Genetics boxed essay for more details.)
Silencing Specic Genes with Antisense siRNA

To silence the expression of specic plant genes or groups of genes, researchers can take advantage of the RNA interference system present naturally in all eukaryotic cells (see Chapter 18 of the main textbook). As described in the Chapter 18 Tools of Genetics boxed essay, it is possible to elicit the production of siRNAs with specic antisense sequences from specially designed transgenic constructs inserted into a plants genome. Alternatively, double-stranded siRNA can be introduced directly. The siRNA is processed by the cytoplasmic ribonuclease DICER, and then loaded onto RISC. The loaded RISC degrades mRNAs that have been transcribed from the complementary gene targeted for silencing (Fig. B.11). The siRNA silencing strategy, heavily used in plants and animals offers two key advantages. It is not restricted by the types of mutations available in current collections; and it allows the isolation of transgenic lines in which the endogene under investigation is not completely silenced. This partial silencing is quite similar to the effect of hypomorphic mutations, which only partially alter the function of a particular gene product (see Chapter 20 of the main textbook). Hypomorphs or partially silenced lines can bypass the early developmental arrests associated with the total silencing of a gene required for early development; they thereby permit a functional characterization of the gene of interest at later phases of development. Another advantage of the silencing strategy over more classical knockout methods is that the same transgene can simultaneously knock down the expression of several endogenes. Indeed, if several homologous genes encode similar proteins, it is possible to construct a transgene that
B.4 The Genetic Analysis of Development in Arabidopsis
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expresses an inverted duplication of the DNA fragment conserved between these genes. When introduced into plants, such a transgene will silence all genes that encode mRNAs sharing nucleotide similarity with it. This eliminates the need to isolate numerous mutations in each one of these genes, and to develop a complex scheme of crosses aimed at bringing together in the same plant multiple mutations in members of the gene family under investigation. This strategy makes it possible to determine the phenotype associated with the disappearance or reduction of a specic protein function that is encoded by multiple, functionally redundant genes. Insertional mutagenesis and gene-silencing strategies for moving from gene to phenotype have found wide use now that the Arabidopsis genome has been completely sequenced. Indeed, geneticists currently not only identify the genes involved in specic functions, they also use their knowledge of gene sequences and mutation-induced phenotypes to dene what specic genes do.
B.4
The Genetic Analysis of Development in Arabidopsis
Although geneticists only partially understand the mechanisms of development in plants and animals, they do know that instructions for the formation of a body plan and the elaboration of specic functions are ultimately encoded in the DNA. Mutations can thus change the position, structure, or function of an organ or alter an individuals orderly passage through the life cycle. We now look at the use of genetic analysis in the study of embryogenesis, hormone control systems, and responses to environmental signals.
Maternal-effect mutations disrupt embryogenesis in all progeny of a mutant mother, regardless of the zygotes genotype. The small number of maternal-effect mutations constitutes a major difference between plants and certain kinds of animals, such as Drosophila, that develop from large eggs. (In Drosophila, numerous maternal-effect mutations alter the early stages of embryogenesis, as described in Portrait D.) A major reason for the near absence of maternal effects in Arabidopsis is that the plants embryonic development does not depend on a large cytoplasm lled with maternal products (as happens in Drosophila). Instead, it depends from a very early stage on a cytoplasm newly produced from the zygotes own genome (as in mammals). Also unlike what happens in Drosophila, in Arabidopsis, very few zygotic mutations (that is, mutations in the zygotes own genes) affect embryogenesis before the globular stage. At least part of this difference between Arabidopsis and Drosophila may derive from the fact that in plants many zygotic genes are expressed very early in embryogenesis (as they are in mammals), and these zygotic genes may encode protein products that are functionally redundant with those of maternal origin. In addition, many plant genes are members of multigene families in which each member encodes a protein whose function duplicates that of some of the other members. Hence, as in mammals, the scarcity of maternal-effect and early zygotic mutations in plants may simply reect a large amount of functional redundancy between maternally deposited and zygotically derived products.
Screenings for Mutations That Arrest Development at a Specic Stage of Embryogenesis Enable the Identication of Important Regulatory Processes in Plant Development
A large number of mutations affecting Arabidopsis embryo development have been isolated. We will highlight just two of these mutations to give a avor of range of phenotypes and mechanisms involved. One type of embryodefective mutation known as leafy cotyledon (lec) results in embryos with cotyledons resembling mature leaves rather than wild-type cotyledons (Fig. B.12a). As described earlier, cotyledons are embryonic structures that rst appear late in the globular stage; leaves, by contrast, are products of the apical meristem that appear only after germination. Unlike cotyledons, leaves have trichomes small, hairlike projections from the epidermisand a leafspecic pattern of vascularization. In addition to causing the conversion of cotyledons into leaves, lec mutations produce seeds unable to germinate because they cannot withstand the desiccation that accompanies seed maturation. These observations suggest that the wild-type LEC
The Genetic Analysis of Embryogenesis

Mutations that are lethal to the embryo or that arrest embryonic development have provided a basis for understanding embryonic development in Arabidopsis. Screenings for mutations resulting in developmental arrest at a specic stage of embryogenesis have led to the identication of more than 500 embryo-lethal and embryo-defective mutations. A signicant portion of the embryo-lethal mutants are arrested at the transient stage. Many of them carry mutations in housekeeping genes.
Researchers Have Found Very Few Maternal-Effect Mutations in Arabidopsis

Such mutations occur in maternal genes whose protein products are deposited in the egg during oogenesis.
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the hypothesis that a general perturbation of embryo development can derepress development in suspensor cells.
The Genetic Analysis of Hormonal Control Systems

The control of plant development requires complex communications within cells, between neighboring cells, and over long distances between different organ systems. Plant hormones are the main internal chemical signals that inuence plant development. One group of hormones, the auxins (from the Greek auxein, to increase or augment), promotes shoot growth through cell elongation and lateral root formation and helps prevent senescence. The auxins also inhibit growth in lateral buds in favor of growth in the apical meristem (apical dominance), and they inhibit cell elongation in roots. A second set of hormones, the cytokinins, promotes growth by stimulating cell division, including cytokinesis (hence their name). The cytokinins also play a role in the prevention of senescence but, unlike the auxins, promote the growth of lateral buds. Together, the auxins and cytokinins help regulate a range of processes including meristem induction, apical dominance, and root-shoot communication. A third type of hormone, abscisic acid, controls aspects of seed development and dormancy; it also mediates responses to water stress (too much or too little water). Yet another group of plant hormones, the gibberellins, regulates seed germination and stem elongation. The brassinosteroids enhance cell growth and expansion, and they suppress leaf organogenesis in dark-grown seedlings. Ethylene promotes maturation of the seedling and inuences the timing of leaf senescence. Finally, salicylic acid, jasmonic acid, and methyl-jasmonate are associated with disease resistance. Jasmonic acid also inhibits seed and pollen germination, inhibits seedling growth, and induces fruit ripening and abscission (detachment) of owers.
(a)
(b)
Figure B.12
Examples of two mutations that arrest development. (a) Micrograph of lec mutant (right) versus a plant
carrying LEC wild type (left). The cotyledons in a lec mutant are abnormally expanded. (b) Micrograph of twin (right) versus wildtype TWIN embryos (left). a axis; c cotyledons.
genes encode molecules that help regulate the process of seed maturation in Arabidopsis. Two LEC genes, LEC1 and LEC2, have been cloned and shown to encode transcription factors that play fundamental roles in the differentiation of cells into an embryonic state. Another class of embryo-defective phenotypes is characterized by the abnormal development of suspensor cells. In one subset of suspensor mutants, called twin, ectopic secondary embryos form from the suspensor (Fig. B.12b). In many of the suspensor mutants, the embryo proper shows defects prior to the abnormal proliferation of the suspensor. From this observation, Arabidopsis developmental geneticists infer that the embryo signals back to the suspensor to repress embryonic development. Based on this model, genes affected by suspensor mutations are predicted to encode molecules necessary for sustained embryo development or molecules that are directly involved in the repression of a default embryo development program in the suspensor cells. Several of these genes have been identied and shown to encode components necessary for basic cellular functions, consistent with
Mutations That Disrupt Hormone Activity Can Clarify a Hormones Biological Signicance and the Biosynthetic Pathways Involved in Its Production
Researchers originally identied the gal-1 mutation, an Xray-induced deletion, through a dwarf phenotype that could be rescued by gibberellin application (Fig. B.13). Later, analysis of the cloned gene indicated that it encodes an enzyme that operates early in the gibberellin biosynthetic pathway. A second mutation, the semi-dominant gai mutation, produces a phenotype similar to that produced by gal-1, but gai mutants are not rescued by gibberellin application. The inference from this observation is that gai most likely affects a plants response to gibberellin rather than the hormones biosynthesis. This inference was supported by the isolation of the GAI gene and genes encoding related proteins that act as repressors of the GA signaling
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Mutations That Render Arabidopsis Insensitive to a Hormone Help Reveal the Mechanisms by Which Plants Perceive and Transduce Hormone Signals
In the most advanced studies of this type, investigators look at ethylene signaling, using ethylenes effects on early seedling development as the basis for mutant screens. The exposure to ethylene of seedlings grown in the dark produces an inhibition of shoot and root elongation and an accentuation of the apical hook. Mutant seedlings that are insensitive to ethylene grow tall in the presence of ethylene. Several loci that affect responsiveness to ethylene have been identied in Arabidopsis (Fig. B.14). For example, dominant mutations at the ETR1 locus (including Ein1) cause insensitivity to ethylene, as do mutations at EIN2 and EIN3. By contrast, mutations at the CTR1 locus result in the constitutive activation of ethylene responses. All these mutations affect a wide range of ethylene responses in various tissues, suggesting that the genes inuence the primary steps of ethylene signal processing. Analysis of various combinations of double mutants reveals that CTR1 is epistatic to ETR1, while EIN2 and EIN3 are epistatic to CTR1 (Fig. B.15). The genes involved in ethylene signaling have been identied by gene tagging and map-based cloning strategies. The ETR1 gene is a member of a small family of Arabidopsis genes that are related to the genes for receptor protein kinases typical of bacterial signaling systems. Expression of the ETR1 gene in yeast confers ethylene-binding activity on the cells, demonstrating the ETR1 protein serves as the ethylene receptor. CTR1, by contrast, is related to eukaryotic genes for protein kinases that initiate MAP kinase cascades in yeast and mammals (see Chapter 18 of the main textbook), while EIN2 encodes a membrane protein related to metal transporters and EIN3 encodes a member of a small family of plant-specic transcription factors. The genetic
Figure B.13
The gal-1 mutation. The wild-type plant on the left compared to a dwarf plant with the gal-1 mutation (right).
pathway. GA negatively regulates these proteins to induce signaling. Researchers are currently using similar mutational analyses to identify the structural and regulatory genes contributing to the biosynthesis of other plant hormones.
Figure B.14 The effects of ethylene exposure on wild-type and mutant plants. Ethylene inhibits shoot and root elongation in wild-type seedlings. Single- and double-mutant analyses allow the ordering of genes in the signal pathway. Ein1 is a mutation at the ETR1 locus. The EIN3 protein acts before CTR1, which acts before ETR1.
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C2H4
ETR1 ETR2 ERS1 ERS2 EIN4
CTR1
EIN2
EIN3 EIL1 EIL2
ERF1
Responses
Map-kinase cascade ETR-FAMILY Two-component regulators CTR1 RAF-like kinase
EIN2 Nramp metal transporter
EIN3/EIL transcription factors
AAA
ERF1 EREBP transcription factor AAA
Responses
Figure B.15
The genetic interactions and biochemical identities of components of the ethylene signal transduction pathway.
Progressing from left to right, ethylene is thought to regulate negatively a family of membrane-associated receptors. The histidinekinase transmitter domains of members of the receptor family interact with the regulatory domain of the Raf-like kinase CTR1. The receptor/CTR1 complex negatively regulates a membrane protein (EIN2) related to a superfamily of metal transporters. The cytoplasmic C-terminal domain of EIN2 positively signals downstream to the EIN3 family of transcription factors. ERF1 is rapidly induced in response to ethylene and is capable of activating a subset of ethylene responses when expressed.
analysis of this pathway has identied components of a complex signaling pathway that leads from signal perception at the membrane to a transcriptional cascade in the nucleus. Arabidopsis mutants that are insensitive to other plant hormones have also been isolated; the gai mutant discussed earlier is an example. The isolation of genes affected in the various mutants demonstrates that protein phosphatases, farnesyl transferases, transcription factors, and the ubiquitin protein degradation pathway play a role in plant hormone signaling, while genes for membrane-based permeases and transporters contribute to hormone transport. The fact that many of the genes identied are homologous to genes in yeast and mammals is evidence that the basic information-processing systems arose very early in the evolution of eukaryotes. On the other hand, receptors for ethylene, cytokinins, and red light (the phytochromes) are related to bacterial sensory receptors. These systems were likely inherited from the bacterial endosymbiont ancestor of the chloroplast.
place when the seedling grows up through the soil and encounters light. Collectively known as photomorphogenesis, these changes include the opening of the apical hook and the generation of chloroplasts. Two classes of photoreceptorsphytochromes and cryptochromesmediate the changes. Receptors in the phytochrome family sense light primarily in the red and far-red wavelengths. Receptors in the cryptochrome family perceive blue wavelengths of light.
Mutational Analyses Have Helped Characterize the Photoreceptor Molecules by Which Plants Receive Light Signals
White light inhibits the elongation of the hypocotyl, or seedling stem, in Arabidopsis. A class of photomorphogenic mutants referred to as hy (for long hypocotyl) show reduced sensitivity to white light (Fig. B.16a). Linkage map locations for two of the hy mutations, hy3 and hy8, coincide with the locations of phyA and phyB members of the phytochrome gene family. The hy3 mutant has an altered response to red wavelengths of light, while the hy8 mutant has an altered response to far-red wavelengths of light, indicating that these two genes have different roles in mediating photomorphogenesis. Two other mutations, hy1 and hy2, block the biosynthesis of the tetrapyrolle prosthetic group associated with all phytochromes. The hy4 mutant in Arabidopsis shows a decreased sensitivity to blue light, suggesting that the normal HY4 gene
The Genetic Analysis of PhotomorphogenesisThe Regulation of Growth and Development in Response to Lighting Cues
During growth and development, plants continuously alter their responses to changing environmental signals. One of the most dramatic series of developmental changes takes
37
Mutational Studies Have Also Claried How Plants Process Light Signals
While Arabidopsis researchers have analyzed hy mutants for information on light perception, they have studied other sets of mutants known as cop (for constitutive photomorphogenesis) and det (for de-etiolated, that is, released from development without light) to understand how plants process light signals into the multitude of changes that characterize morphogenesis. Mutant cop and det seedlings grown in the dark behave as though they have received the light signaltheir apical hook opens, the cotyledons expand, light-regulated genes are derepressed, and chloroplasts develop, all in the absence of light (Fig. B.16b). The det and cop mutations are recessive. From characterizing the phenotypes generated by these recessive mutations, researchers have inferred that the normal COP and DET gene products somehow suppress all or some photomorphogenic responses until a light signal is received. The COP genes affect all morphogenic responses. Several COP loci have been cloned, and biochemical analyses of the gene products reveal that some COP polypeptides associate to form a large protein complex (the signalosome) in the nucleus. After its discovery in Arabidopsis, the COP signalosome was shown to be present in other eukaryotes and to function in the ubiquitin-proteosome protein degradation pathway. Interestingly, in the dark, the COP1 gene product localizes in the nucleus, where it directly interacts with the HY5 and related transcription factors, targeting them for degradation. In this capacity, COP1 functions as a negative regulator, mediating the degradation of transcription factors that are required for photomorphogenesis. COP1 also directly interacts with the cryptochrome bluelight photoreceptors, raising the possibility that a short signal transduction pathway, through which light promotes a change in cryptochrome conformation, helps inactivate the cryptochrome-associated COP1 protein. With this inactivation, HY5 and related transcription factors accumulate in the nucleus, where they turn on the expression of downstream genes whose protein products promote photomorphogenesis. The DET genes affect only some photomorphogenic responses. The det2 mutant, for example, shows derepression of some light-regulated genes, but it does not develop chloroplasts in the dark. This mutant develops into a dwarf adult, indicating that the wild-type gene inuences more than morphogenesis. The cloning of DET2 led to the discovery that the gene is related to mammalian genes that operate in steroid biosynthetic pathways. Later, researchers found that the application of brassinosteroids, a class of steroidlike compounds found in plants, can rescue det2 mutants. While botanists had long suspected that brassinosteroids contribute to the regulation of plant growth and development, genetic analysis clarified that they play a role in the regulation of gene expression by light.
(a)
(b)
Figure B.16
Mutations that affect the response to light.
(a) Comparison of the response of a wild-type seedling to light, versus seedlings with the hy mutation in one of ve different long hypocotyl loci. (b) Comparison of the response of wildtype and mutant seedlings to dark. The three different mutant seedlings shown behave as if they have been exposed to light when they have not.
product plays a role in the reception of blue light. Cloned using a T-DNA-tagged allele and then sequenced, the HY4 locus exhibits homology with a bacterial enzyme that uses absorbed light energy to repair thymidine dimers in DNA. This conservation of a light-absorbing function is compelling evidence that the HY4 gene product is one of two blue-light receptors active in photomorphogenic responses. Mutations in the HY5 locus also block photomorphogenesis. Molecular cloning of the HY5 gene revealed that it encodes a transcription factor that appears to control expression of a number of genes contributing to morphogenesis.
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B.5
The Genetic Analysis of Flowering: A Comprehensive Example
One of the major life events in the development of an angiosperm is the switch to reproductive growth that culminates in owering. During the switch, the reprogramming of the apical meristem from a leaf-producing to a owerproducing tissue gives rise to an astonishing series of events: The meristem no longer generates mature leaves in a specic spiral pattern. Instead, it becomes an inorescence meristem (IM) that produces smaller (cauline) leaves, an elongated stem, and many side shoots. The
meristems of the side shoots develop into oral meristems (FMs) that produce a single ower of specic design. The IM is indeterminate because it produces FMs indenitely. By contrast, the FMs are determinate because they normally produce a xed number of oral organs in a precise pattern of concentric rings, and in the process, they lose the potential for meristematic activity as cells of the meristem become incorporated into the maturing pistil (Fig. B.17). Arabidopsis accomplishes the reprogramming of the meristem through regulation of a complex network of intricately overlapping genetic pathways in response to environmental cues. Some of these determine the body plan of the ower; others specify the identity of the oral meristem; still others control the timing of oral meristem formation.
(a) Sepal primordium (1 of 4)
(c)
Sepal primordium (others have been removed to reveal inner organs)
Late-stage flower primordium showing early steps in sepal formation Shoot apical meristem Early-stage flower primordium
Developing gynoecium (ovary) Developing stamen Petal primordium
(b)
The single remaining sepal primordium
(d) Style Gynoecium (ovary) Anther
Stigma Remaining sepal (other three have been removed) Petal Anther of another developing stamen (there are six) Filament and anther = stamen Scar of removed sepal
Ovary primordium One of the six stamen primordium
Petal Lateral stamen Developing petal
Petal primordium
Differentiation in whorls of oral organs. This series of SEMs shows the earliest stage of development of the A. thaliana ower. (a) Sepals are just beginning to form. Three of the four developing sepals have been removed to (b) reveal the other organ primordia. Petal and stamen formation are noticeable in (c). The structure of the stamen and anthers become more dened in (d). Again, three outer sepals have been removed to show the developing ower parts; mature ower carrying four sepals, six stamen, and one gynoecium.
Figure B.17
B.5 The Genetic Analysis of Flowering: A Comprehensive Example
39
How Genes Determine the Body Plan of a Flower

The oral primordium is an outgrowth of undifferentiated cells near the tip of an inorescence meristem. As cells in the primordium divide, differentiate, and elongate according to genetic instructions, a ower emerges. A wild-type Arabidopsis flower, we have seen, carries four green sepals in the outside whorl, four white petals just inside the sepals, six stamens in the next-to-center whorl, and a cylinder of two fused carpels in the center (review Fig. B.5). Cascades of genetic signals alert cells to their position within the developing ower and thereby direct the proper growth and differentiation of the organs that make up a flower. Mutants can have an abnormal arrangement of organs.
The Radial Pattern Model Suggests How Three Classes of Genes Could Determine the Identity of Floral Organs
For the sake of simplicity, consider that class A mutants are missing class A gene activity, B mutants are missing B gene activity, and C mutants are missing C gene activity. The radial pattern model makes three sets of assumptions about these three classes of genes (Fig. B.18b): 1. A genes are active early in the emergence of the rst and second whorls; B genes are active early in the denition of the second and third whorls; and the C gene is active early in the differentiation of the third and fourth whorls. 2. The gene products of these three types of genes, alone and in combination, determine the identity of the oral organs as they develop from the ower primordium. The A product determines sepals, A and B together determine petals, the combination of B plus C determines stamens, and the C product alone determines carpels. 3. The activities of A and C are mutually exclusive; that is, in areas where A is active, C is repressed, and in areas where C is active, A is repressed. Moreover, if a mutation incapacitates A, C is abnormally active and vice versa. The model provides an elegant explanation of the mutational evidence. To conrm its predictions, researchers carried out further genetic investigations. In one line of experiments, they bred double and triple mutants and predicted what they would look like. For example, they bred plants with mutations in both AP2 (the A gene) and AP3 (a B gene) and predicted that with only normal C activity in the ower primordium, the emerging ower would consist of all carpels. The resulting owers conrmed the prediction. Similarly, they eliminated both B and C activity by breeding plants with mutations in the AP3 and AG genes, and as predicted, the resulting mutants bore owers consisting of only sepals (Fig. B.18c). In triple mutants lacking A, B, and C gene activity, the ower organs differentiated as leaves (Fig. B.18d). Parallel studies in snapdragons support the ABC radial pattern model of angiosperm ower development. The cloning and sequencing of the homeotic genes for oral identity have further validated the radial pattern model. All of the genes produce DNA-binding transcription factors. In situ hybridizations using DNA fragments complementary to A-, B-, and C-type mRNAs have monitored the activity of the normal genes and shown that the B and C genes (AP3, PI, and AG) are expressed specically in the whorls predicted by the model (Fig. B.18e). The A gene, AP2, however, is expressed in all four whorls of the ower although its product affects organ identity only in the rst and second whorls. In ap2 mutants, AG RNA appears in all four whorls of the ower, as predicted by the model. In these ap2 mutants, however, only the rst and
Analyses of Homeotic Mutations Reveal That in Arabidopsis, Three Types of Single-Gene Products Inuence Floral Pattern Formation
A homeotic gene is one that plays a role in determining a tissues identity during development. A homeotic mutation causes cells to misinterpret their position in the blueprint and become normal organs in inappropriate positions. Homeotic mutations thus alter the overall body plan. In one study, researchers looked at mutants with an abnormal order and selection of oral organs. They obtained some of the mutants for their study from T-DNA mutagenized laboratory populations, and they used the mutagen ethyl methane sulfonate to generate more. Phenotypic screens of a large number of mutants showed they fell into three classes (Fig. B.18a). Class A displayed carpels instead of sepals in the rst whorl, and stamens instead of petals in the second whorl, for an overall radial pattern of carpels, stamens, stamens, carpels. In controlled crosses, the ratio of normal to abnormal phenotypes indicated that one gene was responsible for this abnormal radial pattern. Researchers named the gene carrying the mutations that caused this aberrant pattern APETALA2 (AP2) for the lack of petals arising as a result of the mutation. Class B mutants carried sepals in the rst and second whorls, and carpels in the third and fourth whorls. Mutations in either APETALA3 (AP3) or PISTILLATA (PI) caused this deviation from the normal pattern (in botanical terminology, pistillate owers lack stamen). Class C mutants had an abnormal radial pattern of sepals, petals, petals, sepals. Mutations in the AGAMOUS (AG) gene, named for the lack of gamete-forming tissues resulting from the mutations, determined these mutant phenotypes. Loss-of-function ag mutations also lead to the production of additional whorls of structures at the center of the flower, indicating that the wild-type AGAMOUS gene is required for the determinate nature of oral meristems.
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(a) Class A, B, and C mutants Wild type
Class A mutants
Lack of activity of APETALA2 (AP2)
Class B mutants
Lack of activity of APETALA3 (AP3) or PISTILLATA (PI) Class C mutants Schematic key: Sepal Petal Carpel Stamen Lack of activity of AGAMOUS (AG)
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(b)
(c)
(d)
(e)
se pe st
ca
st
pe se
B A C C
B A
Figure B.18 Homeotic mutants and ower formation in Arabidopsis. (a) Class A, B, and C mutants. (b) Diagram showing how the activities of gene types A, B, and C can produce the four types of oral organs: sepals (se), petals (pe), stamens (st), and carpels (ca). The spatial and temporal expression of each gene type is represented by rings containing the three primary colors at the bottom of the diagram. Following this color-coded representation forward, A (blue) expression alone gives rise to sepals, while C (red) expression alone gives rise to carpels. A and B expression gives rise to petals (blue yellow green), while C and B expression gives rise to stamens (red yellow orange). (c) ap2 / ap3, ap2 / ag, and ap3 / ag double mutants. (d) ap2 / ap3 / ag triple mutant. (e) Hybridization probe visualizing AP3 activity in the ower primordium.
second whorls have an altered phenotype. The explanation is as follows: AP1 (one of the A-function genes) is on everywhere in the early ower primordium as is AP2. AG turns on in whorls 3 and 4, repressing AP1 and thus excluding A function from whorls 3 and 4. In ap2 mutants, AG comes on everywhere and represses all AP1 activity, so there is C function in all whorls, but no A function. Thus in ap2 mutants, you expect to see only whorl 1 and whorl 2 phenotypes. The data discussed here indicate that the A, B, and C genes are necessary for radial patterning in owers. However, it appears that they are not sufcient to determine ower organ identity. For instance, the expression of C genes in leaves does not convert them into carpels. Subsequent genetic analysis of ower patterning in Arabidopsis has demonstrated that three functionally redundant genes (SEPALLATA1, 2, and 3, abbreviated sep1, 2, and 3) are essential for the activity of the B- and C-type genes. Indeed, triple sep1 sep2 sep3 mutants form owers that carry an indeterminate number of whorls. Interestingly, single sep1, sep2, or sep3 mutants, or double mutants carrying different combinations of these genes, have wild-type owers, indicating that the three genes are functionally redundant. Molecular analyses of these genes have provided an explanation for these striking observations. Such analyses show that sep1, 2, and 3 encode putative transcription factors that are highly similar to each other, as well as to AG. These three proteins can bind directly to type B and C proteins, forming heteropolymers that are likely to regulate the expression of different sets of target genes. In short, the A, B, C model explains one part of a complicated genetic processthe interpretation of cellular position within the ower primordiumbut leaves unanswered the question of what establishes the positions. As we see next, the differentiation of oral organs, in addition to the appropriate expression of the A, B, C, and sep genes, requires
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(a)
(b)
(c)
Figure B.19 Identifying genes that inuence development of the oral meristem. (a) Single mutants Ify-6 (left), ap11 (middle), and t-2 (right). (b) Double mutant ap11 / cal-1. (c) Hybridization probes visualize the activity of AG RNA in the center of the ower primordium.
the activity of many other interacting genes at the proper time and place in an immense hierarchy of genetic control. wild-type Arabidopsis plants. Table B.1 describes six of the oral meristem development genes uncovered by these loss-of-function and overexpression strategies. By comparing the effects of each genes activity with that of the others, the researchers were able to suggest how they interact.
Earlier-Acting Genes Specify the Identity of the Floral Meristem

The oral meristem produces the ower primordium that differentiates into four whorls of organs, two of which contain the gametes. But what tells cells in the side shoots of an inorescence meristem to become a oral meristem? To answer this question, researchers analyzed loss-of-function single and double mutants (Fig. B.19a and b) and overexpression transgenic plants. They constructed the transgenic plants by rst fusing a strong constitutive promoter called 35S with the DNA of the cloned gene under study and then inserting the promoter/gene fusion product into otherwise
Model of the Progressive Specication of the Floral Fate

The rst sign of oral development at the molecular level is expression of the LEAFY (LFY) gene. Activation of LFY marks the position of a future oral meristem and takes place before a visible FM appears. With inadequate LFY function, the meristem remains an inorescence meristem. Next comes the expression APETALA1 (AP1) and CAULIFLOWER (CAL), whose activities overlap in newly formed meristems. The orderly expression of LFY, AP1, and CAL
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TABLE B.1
Six of the Arabidopsis Genes That Control Meristem Identity

Loss of Function (Mutant Phenotypes in Meristem Identity)
Conversion of ower to shoot or shoot-like structures; oral reversion Conversion of ower to shoot; branched owers Enhances ap1 phenotypes Enhances ap1 and Ify phenotypes Indeterminate owers; axillary buds; oral reversion Terminal ower; early owering
Arabidopsis Gene
LEAFY (LFY ) APETALA1 (AP1) CAULIFLOWER (CAL) APETALA2 (AP2) AGAMOUS (AG)
Gain of Function (Phenotypes of Transgenic Plants)

Conversion of lateral shoots to ower; terminal ower; early owering Conversion of lateral shoots to ower; terminal ower; early owering Not tested Not tested Conversion of lateral shoots to ower; terminal ower; early owering; suppression of late owering mutants Not tested
TERMINAL FLOWER (TFL1)
Reference: TIG January 1998 Vol. 14 No. 1, To Be, Or Not To Be, A FlowerControl of Floral Meristem Identity by H. Ma.
promotes emergence of an unbranched owering structure with four whorls of organs. Loss-of-function mutations in any of these genes prevents the shoot from owering. Interestingly, mutations in the AP2 gene enhance the effects of lfy and ap1 mutations. Expression of AG occurs at the center of the oral meristem where its product represses AP1 expression and further commits the meristem to the determinate production of owers (Fig. B.19c). A lack of AG activity can cause the developing FM to revert to an IM. With the expression of LFY, AP1, and AG, cells in the lateral, oral meristems become increasingly committed to reproductive development. Wild-type Arabidopsis plants produce only lateral owers because the TFL1 gene prevents the expression of FM-producing genes in the center of the IM. As a result the indeterminate inorescence meristem continues to grow and produce new side shoots even as older lateral shoots are activating the genetic program for owering.
Some Genes Control the Timing of FM Formation and Flowering

Laboratory-grown, wild-type Arabidopsis plants adjust their owering time in response to the amount of light they receive. Those that receive 16 hours of light a day begin to ower three weeks after germination, while those that receive only 810 hours of light do not initiate owering until at least six weeks after germination. Moreover, the shorter days increase the duration of all phases of development, causing the production of more leaves in the rosette and more owers on the inorescence. Exposure of hydrated seeds (which have taken up water and are ready to germinate) or young plants to low temperatures for three to six weeks after germination accelerates the owering of wild-type plants. As Table B.2 shows, two types of mutations inuence owering time: those that delay it, and those that precipi-
tate it. Researchers identied many of the mutations that disrupt normal owering by screening laboratory populations for their owering responses. They also found that some mutations identied on the basis of other phenotypes affect owering. For example, phyB mutants, which have an altered response to far-red wavelengths of light, and cop1 mutants, which grow in the dark as if they have received a light signal, both ower early. To understand the function of genes that affect owering time, researchers have analyzed single mutants, double mutants, and transgenic plants containing extra copies of a particular gene. Among their ndings is the observation that cop1, phyB double mutants show the Cop phenotype of short hypocotyls (rather than the phyB phenotype of expanded hypocotyls). This suggests that COP1 acts downstream of PHYB. Since both mutations accelerate owering, the normal gene products may be part of a pathway required to repress owering, and several overlapping pathways most likely lead to owering. Another nding is that environmental inuences on owering time affect the interactions among oral meristem identity genes. The lfy mutants, for example, show a stronger phenotype under short days than under long days; and there is more COP1 RNA in young seedlings grown under long days than under short days, suggesting that the COP1 gene helps promote owering.
Hundreds of Mutational Analyses Have Generated a Preliminary Model to Guide Future Research on Flowering
Since there are probably hundreds of genes that contribute to the control of oral development, the genetic analysis of owering is a work in progress. Nevertheless, certain observations enable us to build a preliminary genetic model of the process. It appears that at least four pathways promote owering in Arabidopsis: a pathway regulated by day length (photoperiod-dependent pathway), an autonomous
44
TABLE B.2
Full Name
Some of the Arabidopsis Mutations That Affect Flowering Time Probable Function of Gene Product
Abbreviated Name
Mutations causing late owering gibberillic acid insensitive gigantea fca fpa fve fd ft Mutations causing early owering Constitutive photomorphogenic 1 early owering 3 early short day 4 embryonic ower 2 cop1 elf3 esd4 emf2 An E3 ubiquitin ligase; protein degradation Nuclear protein, mechanism unknown SUMO-specic protease Polycomb group protein gai gi fca fpa fve fd ft Transcription factor Nuclear protein, mechanism unknown RNA binding protein RNA binding protein WD-40 protein involved in histone deacetylation bZIP Transcription factor Interacts with FD; paralogous with TFL1
pathway, a vernalization-dependent (cold-responsive) pathway, and a gibberellin-dependent pathway (Fig. B.20). Genetic analysis of late-owering mutants revealed that mutations in one group of genes (PHYTOCHROME A, CRYPTOCHROMES A and B, CONSTANS [CO], CCA1, ELF3, FD, FKF1, FE, FHA, FT, FWA, GIGANTEA [GI], LHY, TOC1, ZTL) delay owering in periods of long, but not short, days. Hence these late-owering plants do not respond to photoperiod (in both long and short days, they behave as if they are in short days); and the corresponding genes appear to promote owering in response to long days. CONSTANS is a key regulator in the photoperiodic pathway.
(1) Autonomous pathway
Photoperiod-Dependent Pathway.
Researchers have observed that its overexpression in plants under the control of the strong 35S promoter is sufcient to promote early owering; thus plants with CO overexpression are insensitive to day length. The CO gene encodes a transcription factor that most likely regulates expression of downstream genes.
Autonomous Pathway. Mutations in a second group of genes (FCA, FLOWERING LOCUS D [FLD], FPA, FVE, FY, LUMINIDEPENDENS [LD]) result in late owering in both long and short days. This suggests that these genes promote owering in an autonomous fashion, independent of photoperiod. Interestingly, autonomous-pathway mutants respond to vernalization (they ower early when exposed to long cold treatments), which implies that a third pathway is responsible for ower promotion by vernalization. Vernalization-Dependent Pathway. To look at the vernalization-dependent pathway, scientists early-owering plants with plants belonging to late-owering winter-annual ecotypes (which ower early if exposed to vernalization), and they showed that the late-owering phenotype was due to dominant mutations at two loci: FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). When they set up to clone these loci, they found that most of the early-owering ecotypes carry loss-of-function mutations. They cloned FRI and FLC by chromosome walking and showed that FRI encodes a novel protein of unknown function while FLC encodes a putative transcription factor in shoot and root tip tissues. Analysis of FLC expression in plants subjected to vernalization, as well as in plants carrying mutations in
FRI
FLC
VRN2
(2) Vernalization pathway Flowering
Vegetative development
(4) Gibberellin-dependent pathway
(3) Photoperiodic pathway
Figure B.20
Flowering time is determined by at least four different pathways: (1) the autonomous pathway, (2) the vernalization-dependent (cold-responsive) pathway, (3) the photoperiod-dependent pathway, and (4) the gibberellindependent pathway. Green arrows indicate positive regulation. Red lines ending with a T represent negative regulation.
Connections
45
FRI or in some of the genes belonging to the autonomous pathway, increased researchers understanding of the mode of action of vernalization pathway genes. FLC expression appears to decrease to undetectable levels in fri mutants and autonomous-pathway mutants, suggesting that FRI helps promote FLC expression. Furthermore, vernalization suppresses expression of FLC, and this FLC suppression is maintained over the entire life cycle of the plant. It is not, however, transmitted to the next generation. Thus, FLC expression correlates with an earlyflowering phenotype, suggesting that FLC is a key negative regulator of the transition between the vegetative and reproductive phases in Arabidopsis (Fig. B.20). To test this hypothesis, researchers created plants that overexpress FLC under the control of the strong 35S promoter. These plants flowered late and could not be induced to ower early by mutations in FRI and in the autonomous pathway, nor by vernalization. Conversely, loss-of-function mutations at FLC resulted in an earlyowering phenotype, as discussed previously. It is interesting that, upon vernalization, FLC is silenced not only during cold treatment but also during the entire life cycle of the plant. This means that the plant maintains a memory of the cold treatment, but, as we saw, that memory is not transmitted to the next generation. To understand this long-term memorization of vernalization, scientists isolated mutations that eliminate a plants responsiveness to vernalization. One such mutation, named vrn2, gave rise to a plant that still responded to vernalization by decreasing FLC expression but that did not maintain FLC suppression after vernalization ended. Instead, the gene became active again, and the plant owered late despite having been subjected to vernalization. When these researchers cloned the VRN2 gene by chromosome walking, they found that it encodes a regulatory protein with homology to animal proteins of the Polycomb-complex. Polycomb-complex proteins suppress the expression of multiple developmental genes by modifying the chromatin structure at their periphery.
Gibberellin-Dependent Pathway. Gibberellins also promote owering, through a fourth pathway. Mutations
that affect gibberellin biosynthesis or response often result in later-owering phenotypes (Fig. B.20). Genetic analyses of owering in Arabidopsis have uncovered a number of mechanisms that help control the transition from vegetative to reproductive development in plants. Some of the key regulators identied to date also appear to contribute to the control of owering in other plants, including crops. For instance, FRI and FLC homologs seem to confer a winter-annual habit (in which cold treatment gives rise to an early-owering phenotype) or a biennial habit (in which cold treatment is required for owering) on cultivars of many different types of domesticated plants. Further research will lead to a better understanding of owering time in plants. It will also aid in the design of strategies for engineering different owering habits in crops and horticultural species.
To Flesh out the Multiple Pathway Model, It Will Be Necessary to Identify More of the Genes That Inuence Flowering
There are several reasons why some of these genes have not yet been identied: The genome is not yet saturated with mutations; some genes have redundant functions; and mutations in regulatory genes controlling earlier stages of oral development do not necessarily cause unique oral phenotypes. To nd these hard-to-identify genes that inuence owering, researchers can look for new mutants, try to isolate suppressors and enhancers of existing mutations, and devise protocols that test for the late function of early-acting genes (see Chapter 20 of the main textbook). In addition to identifying more of the genes active in the pathways that promote flowering, researchers will continue to analyze the mechanisms by which these genes work. To this end they will seek to demonstrate the activation of transcription by encoded proteins and to discover the target genes of the transcription factors. Because the food consumed by people around the globe consists largely of grains, fruits, and other ower products, an understanding of owering at the molecular level may increase the chances of feeding an ever-expanding human population.
Connections
Plants are fundamentally different from animals at the level of the whole organism. In contrast to animals, plants continue to produce new organ systems throughout their life cycles, are autotrophic, and do not move about. Despite these differences, the cellular machinery and genetic mechanisms of plants and animals are surprisingly similar. In fact, many of the genes involved in basic metabolic functions and information processing show a striking degree of homology between plants and animals. For example, the AGAMOUS gene species a DNA-binding protein that most likely regulates downstream genes. A human relative of the AGAMOUS gene makes a DNA-binding protein, known as serum response factor, that regulates the c-fos oncogene. The remaining genetic portraits (C, D, and E) look at genetic mechanisms that propel development in three model animals: the nematode C. elegans, the fruit y Drosophila, and the house mouse Mus musculus.
46
Essential Concepts
1. Arabidopsis makes an excellent model organism for genetic analysis because of its diploid nature, its capacity for prolic reproduction, and the relatively small size of its genome. The genome is carried in ve chromosomes that contain little repetitive DNA and a tight arrangement of genes with a few small introns. 2. Arabidopsis switches from vegetative to reproductive development when the leaf-producing apical meristem becomes a ower-producing meristem. After owering, it undergoes double fertilization to produce an embryo and an endosperm that develops within a protective maternal seed coat. In response to proper conditions, including favorable light and temperature, the seed germinates and vegetative growth begins again. The vegetative plant eventually ages and dies. 3. Researchers use several techniques of genetic analysis in Arabidopsis to generate mutations and characterize them at the molecular level. Mutagenesis by treatment with chemicals or X-rays produces a variety of mutations for study. Insertional mutagenesis by A. tumefaciens and transposons produces mutated alleles carrying a specic DNA insert that can serve as a probe for identifying and cloning the gene of interest. Researchers have also assembled large collections of sequence-indexed insertion mutants. The ability to generate and obtain mutations makes it possible to move from the identication of a gene to the phenotype it produces. It is also possible to determine gene function with the use of novel RNA-based silencing techniques, which do not depend on the recovery of mutations in the gene of interest. 4. The genetic analysis of mutations that affect embryogenesis has revealed that in one respect, Arabidopsis is more similar to mammals than to arthropods such as ies. Like mammals and unlike ies, plants have very few maternal-effects mutations and few zygotic mutations before the globular stage. Mutational analyses have also helped identify some of the regulatory processes involved in embryogenesis. Future research will try to pin down the molecular mechanisms that control embryogenesis. 5. The genetic analysis of hormonal control systems has claried the biological signicance of hormones and the biosynthetic pathways that produce them. It has also shown how Arabidopsis perceives and transduces hormone signals. Some of the genes involved in hormone production and hormone responses are homologous to those found in yeast and mammals. 6. The genetic analysis of growth and development in response to light conditions, known as photomorphogenesis, has claried how plants receive and process light signals. Some of the genes that contribute to photomorphogenesis are related to mammalian genes that operate in steroid biosynthetic pathways. 7. From the genetic analysis of owering, researchers have proposed that three types of genes determine the identity of oral organs. These genes operate in the middle of a regulatory cascade in which earlieracting genes specify the identity of the oral meristem, while other genes control the timing of both oral meristem formation and owering.
Solved Problems
I. The St strain of Arabidopsis switches from vegeta-
tive to reproductive (ower) development late in the season compared to the Limburg-5 strain, which owers early. How could you determine if owering time is based on the alleles of a single or multiple genes? Describe the results you would expect for the two different cases.
either two or three phenotypes, depending on whether there was complete or incomplete dominance. If the trait is determined by several genes, there would be a spectrum of phenotypes in the F2 generation ranging from early to late owering.
II. HY5 is a gene that encodes a positive regulator of gene
Answer
This problem requires an understanding of single- and multigene inheritance. The two different strains could be crossed and the hybrid selfed to produce an F2 generation. If the phenotype is due to a single gene, you would expect discrete phenotypes in the F2 generation
expression in response to light in Arabidopsis. a. What do you predict would happen to the expression of light-regulated genes in a loss-of-function hy5 mutant? b. To examine the action of the Hy5 protein at the molecular level, the expression of promoterGUS reporter gene fusions was monitored. One of the fusions contained a light response element
Problems
47
(G box) and the other lacked the light response element. Results are shown here. (A indicates an increase in expression.)
Promoter elements Light-activated GUS expression HY5 G box No G box No light Light hy5 No light Light
b.
c.
c.
What would you conclude about the action of Hy5? If you wanted to determine if HY5 regulates expression in nonphotosynthetic as well as photosynthetic tissues of the plant, how would you design the experiment?
Answer
This question requires an understanding of gene regulation. a. Loss of function of a positive regulator would result in no expression of genes in response to light.
Both the G box in the promoter and the HY5 gene product are needed to get increased gene expression in response to light. (It is reasonable to hypothesize that HY5 interacts directly or indirectly with the G box sequence.) There are several ways in which you could examine the tissue specicity of light-regulated, HY5dependent transcription. In all cases, the wild type (HY5) and hy5 mutant should be compared with and without the light stimulation. RNA can be isolated from photosynthetic and nonphotosynthetic tissues in the two strains, with and without light, and hybridized using a probe from a light-regulated, HY5-dependent gene; or DNA from a HY5-dependent gene can be hybridized to xed tissues in the different plants (in situ hybridization) or a reporter gene linked to a promoter of an HY5-regulated gene could be used to monitor expression in tissues.
Problems
B-1 For each of the terms in the left column, choose the
best matching phrase in the right column.

a. apical meristem b. photomorphogenesis c. whorls d. inorescence e. strain 1. light-regulated developmental program 2. variety of plant 3. growing point of a plant containing undifferentiated cells 4. concentric portions of a ower 5. group of owers at the tip of a branch
related genetically to the plant on which they are found? a. endosperm b. zygote c. embryo sac cells
B-6 After germinating mutagenized seed, there may be
B-2 Why are the following considered to be advantages for
some mutant phenotypes in the rst generation. However, these are not usually the plants that a geneticist will choose to study. Why not?
B-7 Match the type of mutagenic agent with the best use
the geneticist or molecular biologist using Arabidopsis as a model plant system compared to corn or peas? a. generation time of six weeks b. 10,00040,000 seeds per plant c. genome size similar to that of C. elegans
B-3 Arabidopsis has a small genome yet carries out the
(assume there is only one match for each agent).

a. promoterless GUS-containing vector b. EMS c. kanr Ti 1. generate a collection of plants with different loss-of-function mutations 2. study expression of genes 3. generate temperature-sensitive mutants
same functions as plants such as tobacco and pea, which have genomes 50100 times larger. How can Arabidopsis accomplish similar physiology with so little DNA?
B-4 Different strains produce different numbers of seeds
B-8 An alternative to the use of sequence-indexed mu-
(seed sets). a. If you wanted to determine whether this trait was due to a single gene or multiple genes, how would you test this? b. If the trait was due to multiple genes, what characteristic of the different strains would you use to locate genes involved?
B-5 What is the ploidy of each of the following tissues,
tant libraries is an RNA-based silencing protocol for determining gene function. Explain how the phenotype associated with a loss-of-function mutation at a particular gene can be ascertained even when no mutations in that gene are actually available to the researcher.
B-9 A sterile mutant was identied in which there was a
what nuclei combined to form them, and how are they
random instead of directed growth of pollen tubes toward the ovary. The mutant was discovered after mutagenesis of seeds and self-fertilization of the progeny plants. It was not known therefore whether the sterility defect was a male or female defect. How could you determine that?
48
B-10 How could you nd out if an APETALA homolog ex-
b.
ists in petunias, snapdragons, or potatoes?

B-11 The gravitropic response in plants includes upward
curve of the hypocotyl and owering stalk and downward growth of the root. a. It has been postulated that this response is mediated by auxin. What is a genetic experiment to test this hypothesis? b. Auxin-regulated mRNAs have been identied in Arabidopsis. How would you determine the distribution of these auxin-regulated mRNAs in the part of the plant responding to gravity?
B-12 Ethylene affects plant growth and development in
many ways including inhibition of stem and root elongation, stimulation of seed germination, and ripening of fruits. Ethylene biosynthesis occurs in the plant at several different developmental times. Mutants that are defective in ethylene stimulation might be defective in the biosynthesis of ethylene by the plant or in the signaling pathway in cells that receive and process the signal. a. The ctr1 mutant has a defect in the signaling pathway resulting in a constitutive ethylene response. If you treat this mutant with an inhibitor of ethylene biosynthesis, what effect do you expect?
Ethylene overproduction mutants eto1, eto2, and eto3 are sensitive to inhibitors of ethylene biosynthesis. Would you conclude that the ETO genes are biosynthetic or signaling mutants? c. No loss-of-function mutations have been isolated in the ETR1 gene. This could indicate that the gene is required for growth or that there is redundancy of this gene. How could you look for redundancy of this gene in the genome? d. A series of bases in the promoter region of several ethylene responsive genes have been identied that are essential for proper DNA binding to specic transcription factors in the ethyleneresponsive element-binding protein (EREBP) family. The APETALA2 gene product has a polypeptide domain that is homologous to a polypeptide domain found in the EREBPs. Why would these proteins show evidence of homology in one of their domains? e. Genetic engineering of ETR1 and/or CTR1 genes may prevent or modify the ethylene response and could be very helpful in regulating the ripening of fruits and vegetables. If you need to control expression of the genes, what would you want to be part of the DNA construct that you injected into the plant?

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Arabidopsis thaliana: Genetic Portrait of a Model Plant

The ower of Arabidopsis thaliana.

Reference B Arabidopsis thaliana: Genetic Portrait of a Model Plant

A. thaliana in the palm of

Genome Structure and Organization

Comparing Genetic and Physical Maps

Little Repetitive DNA and a Tight Arrangement of Genes

distance from centromere (megabases)

linkage mapped DNA markers used for cloning

overlapping genomic clones

SM31_385,7 NF21M12 SGCSNP148 SM86_163,5 SGCSNP303

Reference B Arabidopsis thaliana: Genetic Portrait of a Model Plant

Anatomy and Life Cycle

Cellular biogenesis Transport facilitation Energy Protein synthesis Ionic homeostasis

Anatomy: The Basic Body Plan

The Apical Meristem Promotes Continuous Growth

Vegetative Versus Reproductive Development

B.2 Anatomy and Life Cycle

Carpel (whorl 4) Sepal (whorl 1)

Shoot apical meristem

SEM of pollen grain Elongation zone

Root meristem Root cap Root apex SEM of flower

The anatomy of A. thaliana. Components of the plant are labeled.

Reference B Arabidopsis thaliana: Genetic Portrait of a Model Plant

Carpel Pollen tube Style

Pollen tube grows

Pollen tube (c)

Sperm nuclei moving toward the ovule

Life Cycle: From Fertilization to Flowering to Senescence

Arabidopsis Undergoes Double Fertilization A Process Unique to Higher Plants

The Embryo Develops Within the Protective Maternal Seed Coat

B.2 Anatomy and Life Cycle

Torpedo stage Cotyledons

Transient / Triangular stage

First cell division Octant stage

Embryo Cell will form suspensor

SEM of embryo in a seed

Stages of embryonic development in A. thaliana.

Reference B Arabidopsis thaliana: Genetic Portrait of a Model Plant

Favorable Environmental Conditions Trigger Seed Germination and Vegetative Growth

B.2 Anatomy and Life Cycle

Senescence: The Vegetative Plant Ages and Dies

Reference B Arabidopsis thaliana: Genetic Portrait of a Model Plant

Techniques of Mutational Analysis

Mutagenesis by Chemical and Irradiation Procedures

B.3 Techniques of Mutational Analysis

Agrobacterium tumefaciens Ti plasmid T-DNA

T-DNA is integrated into genome of plant cell

Transposon-Tagging Strategies Rely on Transposable Elements from Corn (Zea mays)

Reference B Arabidopsis thaliana: Genetic Portrait of a Model Plant

Dicing P OH Small RNA

From Gene to Phenotype: Analyzing Mutations to Identify Gene Function

7mG Target mRNA substrate

Silencing Specic Genes with Antisense siRNA

B.4 The Genetic Analysis of Development in Arabidopsis

The Genetic Analysis of Development in Arabidopsis

The Genetic Analysis of Embryogenesis

Researchers Have Found Very Few Maternal-Effect Mutations in Arabidopsis

Reference B Arabidopsis thaliana: Genetic Portrait of a Model Plant

The Genetic Analysis of Hormonal Control Systems

B.4 The Genetic Analysis of Development in Arabidopsis

Reference B Arabidopsis thaliana: Genetic Portrait of a Model Plant

ETR1 ETR2 ERS1 ERS2 EIN4