FLOWERING

FLOWER INDUCTION

    As the sporophyte develops it passes through different phases. The embryonic phase begins at fertilization and ends in developmental arrest at seed maturation. Upon germination, the plant enters the juvenile vegetative (non-reproductive) phase, followed by an adult vegetative phase and finally flowering (the reproductive phase). The most obvious and dramatic post-germinative phase change is flowering but vegetative (non-reproductive) phases can be quite distinct in some plants.

Vegetative Phases
    Vegetative development is divided into juvenile and adult phases. Juvenile plants are not competent to flower whereas adult phase plants have gained the competence to flower. This has economic ramifications in some horticultural crops, particularly in fruit trees where trees cannot bear fruit until reaching the adult phase. There are also morphological and physiological differences between juvenile and adult which are not obvious in all species. Some of these represent plant adaptations for survival. For example, some woody shrubs protect their succulent juvenile tissues from grazing by accumulating bitter secondary compounds.
    Juvenile Phase represents the early phase of vegetative development, therefore the juvenile organs are the first organs produced. They will be at the bottom of the plant and will be the oldest part of the plant. Adult phase represents a later stage of development so adult organs will be at the top of the plant and younger in age.
Examples of vegetative phase differences
    Ivy—The juvenile grows as a prostrate vine with alternate phyllotaxy while the adult grows as an upright shrub with spiral phyllotaxy.
    Eucalyptus—the juvenile has opposite phyllotaxy with short broad leaves, the adult has spiral phyllotaxy with long slender leaves.
    Maize—juvenile has short round leaves covered with epicuticular wax and no epidermal hairs, adult has long narrow leaves with no epicuticular wax but with epidermal hairs.


Regulation of vegetative phases
    The hormone gibberellic acid (GA) promotes juvenility in some plants. For example in ivy, if the apical bud is removed from an adult branch, the subtending axillary buds will grow into typical adult branches. However, if GA is applied to the axillary buds they will grow into a juvenile branch. In other plants, GA promotes the adult transition. GA deficient mutants of maize are delayed in their transition to adult phase.
    Dominant gain-of-function maize mutations, called Teopod, prolong the juvenile phase but do not affect the onset of adult characteristics. In other words, there are leaves that normally would display adult characteristics which now display juvenile and adult simultaneously. This indicates that the Teopod genes regulate phase change by promoting juvenility and also that juvenile and adult phases are regulated independently. Genetic mosaic experiments where sectors of wild type tissue were induced in Teopod mutant plants show that the Teopod gene acts through some diffusible substance. In leaves or stems that were half mutant and half wild type, the wild type tissue showed the mutant characteristics.

Floral Induction
    Upon reaching adult phase, plants are competent to flower (enter reproductive phase). The process of floral determination is called floral evocation. Some plants flower because of intrinsic signals while others require environmental cues. The most common environmental cues are temperature and photoperiod. Some plants have an absolute requirement for an environmental stimulus (obligate) while others respond to an environmental stimulus but will eventually flower without one (facultative).
Vernalization
    Many biennial plants (plants that grow one year, overwinter and then flower the second year) and "winter crops" such as winter wheat require a cold period to flower. An exposure to cold that stimulates flowering is called vernalization. Different plants have requirements for different vernalization temperatures and periods. In most plants vernalization can be reversed by warm temperatures and  so the cold period must be relatively uninterrupted.
    The site of cold perception is the SAM. A SAM grafted from an unvernalized plant onto one that has received cold treatment will not flower while a SAM grafted from a vernalized plant onto an un-vernalized one will flower.
Photoperiod
    Plants are classified as short day, long day or day neutral according to their photoperiod requirements. Short day plants flower under regimes of short day, long night while day neutral plants have no specific photoperiod requirements. In addition plants can be classified as obligate or facultative in their photoperiod requirements. Obligate plants will remain vegetative indefinitely without the inductive photoperiod while facultative plants will eventually flower without the inductive photoperiod but can be induced to flower sooner.
    Long day plants have a minimum required light period for photoinduction; anything above that will result in flowering and there is no dark period requirement. Short day plants have a maximum photoperiod over which they will not flower; they require a dark period. These are more accurately described as long night plants because it is the dark period that is inductive. The darkness must be continuous. Interrupting the dark period with a flash of light prevents photoinduction.
    Different plants have different requirements for the number of photoinductive cycles. Some require only one cycle, others require several weeks. Once a plant is induced, placing it in noninductive cycles will usually not reverse the photoinduction.
Photoperiod is perceived through the photoreceptor phytochrome. Phytochrome is a protein with a covalently attached chromophore. Phytochrome exists in 2 interconvertable forms. Pr absorbs light in the red part of the spectrum (~660 nm). Pfr absorbs far red light (~730 nm). Absorption of red light converts Pr to Pfr and absorption of far red light converts Pfr to Pr. Pfr also undergoes spontaneous "dark reversion" to Pr. Daylight has a relatively high proportion of red light and thus most phytochrome is in the Pfr form. In the dark, the equilibrium slowly shifts to Pr. This cycling of phytochrome somehow interacts with the circadian clock to determine the proper seasonal time for flowering.
   

    The site of photoperiod perception is the leaves. Exposure of the SAM to photoinductive cycles has no effect. In cocklebur (a short day plant), exposure of 8 cm2 of leaf surface to short days while the rest of the leaves receive long days induces flowering. Not all leaves are equally receptive. Juvenile leaves cannot perceive or transmit the photoinductive signal. The first adult leaves may require more photoinductive cycles to induce flowering than later adult leaves. Grafting experiments show this is a property of the state of the leaf, not it’s position relative to the SAM. Early adult and late adult leaves grafted onto identical positions of recipient plants will still require different numbers of photoinductive cycles.
The induced state is stable in the leaf. A Perilla leaf that has been photoinduced can be grafted to an uninduced plant and cause flowering. The same leaf can then be removed and regrafted several times to other plants under noninductive conditions and continue to induce flowering.
    SAMs from different phases differ in their competence to respond to floral induction. Grafting juvenile meristems of tobacco onto florally induced plants did not produce flowering until after a period of growth (presumably until after adult phase transition) while grafting adult meristems onto induced plants resulted in rapid flowering. The veg gene in pea is required to confer floral competence to SAMs. veg mutant SAMs grafted onto wild type plants under inductive photoperiods will not flower, however wild type SAMs grafted onto veg mutant plants will.
    Florigen: Because of the hormonal nature of the signal sent from the leaves to the SAM, it was believed that a floral inducing hormone existed. This was called florigen. It has never been isolated and current opinion favors a more complex explanation than just a single substance.
Increased hormone sensitivity is correlated with floral induction
    Several lines of evidence, including culturing meristems with various levels of the hormone GA (gibberellic acid) or with GA inhibitors, suggest that an increased sensitivity of the SAM to GA is associated with floral induction. Recently, a gene called FPF1 (floral promoting factor) was identified in Arabidopsis. FPF1 mRNA is not present in the vegetative SAM but is induced in the SAM at the onset of floral induction. Constitutive expression of FPF1 leads to an early flowering phenotype. In addition, plants show additional phenotypic aspects like elongated internodes that are reminiscent of GA responses. The FPF1 overexpressors do not show early flowering or internode elongation if combined with GA deficient mutants or treatment with paclobutrazol (a GA inhibitor). Therefore these effects of FPF1 are GA dependent indicating that expression of FPF1 increases sensitivity to GA and that this increased sensitivity of the SAM leads to floral induction.

Flowering in Arabidopsis

Arabidopsis is a facultative long day plant. That is, it can be induced to flower by providing long day photoperiods but it will eventually flower even under short days. Similarly it shows a facultative response to vernalization; a cold treatment stimulates flowering but flowering will eventually occur without one. Genetic analyses have identified 3 pathways to floral induction. The “long day pathway” is defined by mutants that flower later than wild type under inductive photoperiods (long days) but which flower at the same time as normal under short days (non-inductive photoperiod). One mutation, frigida, interferes with the vernalization response but does not affect responses to photoperiod. Other mutations delay flowering under non-inductive conditions (short days, warm temps) and are said to act in the “autonomous” or “constitutive” pathway. Because of the relationship of the hormone GA with the flowering response, mutants in GA synthesis or response delay flowering leading some to consider the “GA pathway” as a 4th pathway leading to flowering.

 

Reeves and George Coupland (2001) Plant Physiol. 126:1085

 

Floral Determination
    Day-neutral tobacco consistently forms about 40 nodes and then a terminal flower. Undetermined vegetative SAMs can be cut off and rerooted indefinitely. Removal of leaves from plants has no effect on the number of nodes to flowering, but induction of adventitious roots by piling up soil as the stem grows increases the number of nodes produced. This suggests that the distance between the SAM and roots is important for floral determination. SAMs become florally determined prior to actual floral development. Determined SAMs that are cut off and rerooted will produce 4 more nodes, then a flower (the same as if they had remained on the plant).
    Subtending axillary buds also form floral branches in tobacco. The axillary buds form over a period of 5 days following SAM floral determination. The earliest the axillary buds are large enough to remove and root is 9 days after. At this time most but not all axillary buds are florally determined. Thus floral determination in lower axillary buds occurs later than in the terminal meristem.
    The florally determined state is not restricted to tissues that will actually form flowers. Internode segments will produce shoots when cultured on hormone free medium. Vegetative state segments will produce vegetative shoots while segments from the upper nodes of flowering plants will form floral shoots.
    Sunflower and maize represent extremes in the timing of floral determination. Rooting experiments showed that the sunflower SAM is determined to flower during the seedling stage, approximately 14 nodes prior to morphological differentiation. The apical bud was removed and rooted at different times. Whether the apex was removed from the seedling or after producing 9 nodes, the total number of nodes produced before flowering remained about 16.
In maize, floral determination does not occur until after vegetative development is complete and it appears to be a stepwise process. Cultured SAMs form vegetative shoots until floral transition. Early tassel meristems develop vegetative shoots from their branch primordia. Later tassel primordia develop sterile flowers with leaf like organs. Only flowers that are initiating floral organs at the time of culture will go on to complete normal floral development.


Measuring developmental time
Because of the nature of plant development, spatial relationships change over time. For example the SAM becomes increasingly separated from the root system over time. This makes it difficult to tell whether the mechanisms regulating the duration of developmental phases are based on temporal or spatial considerations. Thus there is debate over whether plant homeotic mutants are really heterochronic. Homeotic is when structures form in an inappropriate place. Heterochronic is when something occurs at an inappropriate time. This dilemma is particularly evident in the control of phase changes. Phase change could be based on spatial relationships (distance between the SAM and the root system for example) or on temporal considerations or counting mechanisms. The phenotype of one mutant in arabidopsis called paused has been interpreted to suggest there might be a "developmental clock." paused plants initiate leaves more slowly than normal. The first seedling leaf after germination is delayed by several days relative to wild type. The first leaf that forms in the mutant resembles the leaf that the normal plant would be making at the same time although it is not in the same position. Furthermore, this mutant flowers at the same time as normal even though it possesses fewer total leaves. Thus it appears to enter phase transitions at the proper time, regardless of position. There are other possible explanations and the majority of the evidence (from rooting experiments etc.) suggests that spatial relationships are more important for determining phase.

FLOWER DEVELOPMENT

SAM changes
    Flowers exist in a variety of arrangements on plants but basically they can be considered as occurring singly or clustered in inflorescences. When they occur singly the flower usually arises by the conversion of a vegetative SAM into a flower meristem. In the case of inflorescences, the vegetative SAM is converted to an inflorescence meristem and then flower meristems can arise directly on the inflorescence with no preceding vegetative period. Thus these meristems go through no change to become flower meristems.
Cytological changes and Morphological changes
    Despite the developmental differences between single flowers and inflorescences, the changes in the SAM from vegetative to floral are remarkably similar. One of the earliest events in the transition from vegetative to floral is an increase in mitotic activity, particularly in the central zone. The distinction between the central zone and peripheral zone is lost as mitotic activity becomes uniformly high. Associated with the increased mitotic activity is an increase in meristem size and usually a change in shape.
    The phyllotaxy changes as the meristem becomes floral. Commonly the phyllotaxy becomes whorled (ie. >2 primordia are initiated at the same node). Sometimes the first set of organs (the sepals) initiates sequentially, but in subsequent organs (petals and stamens) organs of a whorl initiate simultaneously. In maize the meristem shifts from alternate to spiral phyllotaxy as it assumes inflorescence identity.
    Internode elongation is minimal in flowers resulting in the compact arrangement of organs.
In flowers, the meristem changes from an indeterminate to determinate mode of growth. In other words the meristem produces a set number of organs and then stops. Inflorescence meristems can be determinate or indeterminate depending on the species.


Organ initiation
    The general sequence of events in a flower meristem is that the meristem initiates sepal primordia, either sequentially or simultaneously. Then petal primordia are initiated, usually simultaneously, in positions that alternate with the sepals. Stamen primordia arise shortly after or simultaneous with the petals, and in alternate positions to them. Finally the carpels initiate in the center of the meristem to give rise to the pistil. As the different organs initiate the meristem gets progressively smaller. After the carpels initiate from the center, the meristem is "used up".


SAM cell layer contributions to floral organs
    Periclinal chimera fate mapping experiments have shown that the cell layers of the floral meristem have the same basic relationships with floral organs as with leaves. That is L1 gives rise to epidermis, L3 to core tissues and L2 to intermediate tissues of sepals, petals etc. The key point about flower lineage studies is that in majority of cases, germ cells are derived from the L2. Thus sexually produced progeny from a G-W-G type chimera (with an albino L2 layer) will all carry the albino marker. (Note: because a gamete is a single cell, it is not possible to propagate the chimeral arrangement of cells sexually) This has implications for genetic engineering because for a gene to be heritably introduced into a plant it must be incorporated into the L2.

Genetic control of flower development
    A collection of mutants has been generated that provides considerable information about the genetic control of flower morphogenesis. The 2 plants that have been most informative are arabidopsis and Antirrhinum (snapdragon). Essentially 2 classes of genes have been identified: meristem identity and organ identity genes. The same genes are involved in both plants. In addition, snapdragon has genes regulating the bilateral symmetry of the flowers (arabidopsis flowers are radially symmetrical). For simplicity we will only discuss arabidopsis except for the bilateral symmetry.
Meristem identity
    There are three types of meristems involved in flower development: vegetative, inflorescence and flower. As such there are 2 different transitions involved (veg to infl., infl. to flower). Genes have been identified that control both these transitions.
    The phenotype of a mutant called embryonic flower (emf) suggest that a floral meristem is a default pathway. emf mutants form an inflorescence meristem during embryogenesis. Upon germination they form an inflorescence with no vegetative stage. Because this is a recessive loss of function mutation, it suggests the normal gene function is to suppress floral identity and allow vegetative meristem identity. This gene has not been cloned yet. Presumably the transition of a normal vegetative meristem to an inflorescence involves the suppression of the normal EMF gene.
    The identity of floral meristems is controlled by the opposing action of 2 sets of genes. A mutation called terminal flower (tfl) results in the conversion of the inflorescence meristem to a flower. Thus TFL is required to promote inflorescence or suppress flower meristem identity in the inflorescence meristem. Two mutations called leafy (lfy) and apetala1 (ap1) result in the production of inflorescences in place of flowers. These genes are normally required to promote flower or suppress inflorescence identity. The balance between these 2 sets of genes controls meristem identity.
    TFL is expressed in the corpus of inflorescence meristems. LFY is normally expressed throughout flower meristems. In the tfl mutant LFY is expressed throughout the apical meristem (which should be an inflorescence meristem but has been converted to a flower meristem). Therefore TFL in the corpus normally represses LFY expression in the tunica and corpus.
    LEAFY is required for the expression of downstream flower organ identity genes.


Organ identity
    Mutations in genes controlling the identity of flower organs results in homeotic conversions of one organ to another. There are 3 classes of homeotic genes involved in flower organ identity and each class affects the identity of 2 adjacent whorls of organs. The classes of genes have been designated A, B and C. The A class gene is called apetala2 and ap2 mutations cause the sepals and petals to form carpels and stamens respectively. 2 genes, apetala3 and pistillata belong to class B and mutations in either ap3 or pi cause petals and anthers to form sepals and carpels respectively. The class C gene is called agamous and ag mutations cause the stamens and carpels to form petals and sepals respectively. In addition, ag mutant flowers are indeterminate and reiterate this pattern of floral organs.
    A model was proposed where the overlapping action of these genes would specify organ identity. A function would be present in the first 2 whorls, B function in whorls 2 and 3, and C function in 3 and 4. A alone would specify sepals, A+B=petals, B+C=stamens and C alone specifies carpels. In addition, A and C would negatively regulate each other (be mutually exclusive) so that in the absence of A function, C function would be expressed in whorls 1 and 2, and in C mutants, A function would be expressed in whorls 3 and 4. This model accounts for all the basic mutant phenotypes observed. Recent models also include a D function which is involved in ovule development.
    Most of these genes (except TFL, AP2 and LFY) contain a conserved DNA-binding motif called the MADS box. This motif appears to have evolved a similar function as the homeobox which is important in specifying organ identity in animals.


SAM cell layer interactions in flower development
    Periclinal chimeras in which different cell layers contain genotypes that confer different floral characteristics have shown that cell layers interact and cooperate in the development of flowers. In a classical work the flowers of a periclinal chimera between 2 species of camellia were examined. The L1 consisted of a species with normal flowers ( fertile and containing all the normal organs) while the L2 and L3 consisted of a species with sterile flowers containing no stamens or carpels. The chimera formed functional pollen even though the pollen is derived from the L2 of the genotype that normally does not form reproductive structures. Thus the fertile L1 induced reproductive development in tissues that lacked the genetic capacity to induce their own reproductive development.
    Another example is in tomato where periclinal chimeras were made with a mutant called fasciated. This mutant makes too many carpels. All chimeras with fas in the L3 produced extra carpels but the presence of fas in the L1 or L2 had no effect. Thus L3 specified carpel number.
    A third example involves the floricaula mutation of snapdragon (which corresponds to the leafy mutation of arabidopsis). Wild type Flo+ is required for expression of a downstream organ identity gene called deficiens (def). In chimeras where Flo+ was only present in one of the apical cell layers, def mRNA was expressed throughout the meristem indicating that somehow Flo+ expression in one cell layer was inducing def expression in other cell layers.
    Finally there is the example of TFL that we already discussed.
Protein trafficking is likely to be involved in some of these interactions. Two snapdragon genes called GLO and DEF (homologs of PI and AG3) function cell autonomously in the L1 of periclinal chimeras but non-cell autonomously in the L2. That is that wild type L2 can rescue mutant L1 but NOT vice versa. These transcription factors can apparently be transported between cells through the plasmodesmata (similar to gap junctions of animal cells). GLO can traffic from L2 to L1 but cannot from the L1 to L2. Thus trafficking is polar.
Sex determination
    Flowers that contain both male and female reproductive organs are called perfect (also called hermaphroditic or bisexual). Flowers that lack one or the other are called imperfect (unisexual). The developmental decision of which sexual organs to form (sex determination) can occur at several points. Some plants contain all male or all female flowers and therefore the sex determination event occurs at the whole plant level (these plants are called dioecious). Other plants such as maize have male and female flowers located on separate inflorescences of the same plant (these plants are called monoecious). In the majority of cases, unisexuality arises by the selective abortion of one type of reproductive organ. For example, flowers on the maize ear initiate both stamens and a pistil but at an early stage the stamens abort. Conversely on the tassel the pistil aborts. There are several mutations called tasselseed that result in female flowers developing on the tassel. ts2 has been cloned and encodes a short chain alcohol dehydrogenase. Normal expression of ts2 in the pistil primordium of male flowers induces apoptosis (programmed cell death) leading to pistil abortion.
    No mutants are known that convert the ear to completely male flowers. GA deficient mutants form bisexual flowers on the ear indicating that GA is important for suppressing stamen development on the female inflorescence.
Organ fusions
    Many flowers have fused floral organs. It is common for petals or sepals to be fused into a tubular structure and the pistils of many flowers consist of fused carpels. The fusion events can be either congenital (occurring before or during organ initiation) or postgenital (occurring after organ initiation).
    Postgenital carpel fusion has been studied in a plant called Catharanthus roseus (Matagascar periwinkle). The process involves the dedifferentiation of the carpel epidermis and is mediated my a diffusible substance. Plant epidermis is usually resistant to the formation of organ fusions so this represents a highly specialized process. Initially the separate carpels are completely covered with an epidermis. As the growing organs come in contact, the epidermis in the region of contact dedifferentiates. Insertion of a permeable barrier between the organs allows the dedifferentiation event to proceed but a non-permeable barrier will block the event. Therefore a diffusible signal mediated the dedifferentiation. If an agar impregnated porous barrier is inserted between the carpels, the agar can be removed and applied to another portion of the carpel epidermis not involved in the contact. Therefore the whole carpel epidermis is competent to respond to the dedifferentiation signal, but only the area of contact receives or produces the signal. Application of the agar to other organs such as anthers does not cause epidermal dedifferentiation so competence to respond to this signal is specific to carpels. The molecular basis of this process is not known.