Plant Reproduction BIOL 304 11/21/2008-11/26/2008

Transition to reproduction Flower organ development Gametogenesis and fertilization Plant Reproduction

Transition to reproduction Flower ? Main inflorescence shoot in middle, that can branch into lateral shoot At end of each shoot – place to make flower Each inflorescence shoot can make leaves and lateral organs What regulates transition from vegetative growth (main function to increase biomass of plant) to making plant sexually ready Inflorescence Vegetative phase Reproductive phase

Production of flowers involves two transitions in Arabidopsis Convert SAM to inflorescence meristem (infinite, making lateral organs) 2. Convert inflorescence meristem to floral meristem (terminal, flowers) SAM convert to meristems that can make flowers Influorescence mersitems can produce more lateral branches – can further branch into tertiary branches Infinite branching as long as plant’s age allows – produces branches and leaf organs Convert influorescence to floral mersitem – make final flower (cluster of flowers at each end of inflorescence shoot) Once floral meristem finishes function to make flowers, some cell activity lost at that region – terminal organ SC: stem cell P: organ primordia Se: sepal

Factors regulating the transitions Vegetative meristem Genes (flowering-time genes and floral identity genes) Light (photoperiod) The biological clock Temperature Hormones Many factors regulate transition- Vegetative mersitem can convert into inflorescence meristem Inflorescence meristem Floral meristem

Flowering-time genes affecting the transition of vegetative growth to reproductive growth Flowering-time genes regulate general transition from vegetative to reproductive growth Emf1 and emf2 mutants flower during seedling stage – don’t have to go thru long process of making more shoot, root, etc. Very early on, seedlings can make flowers – genes negative regulator of reproduction (early flowering mutants – negative regulator of reproduction; promote vegetative growth – so under normal conditions, go to vegetative growth) WT emf1 emf2 embryonic flower

Floral identity genes affecting formation of inflorescence and floral meristems Flower (from Floral meristem) Floral identity genes – inflorescence and floral meristem affected Flower from floral meristem and inflorescence from inflorescence meristem – how regulated? Other types of mutants can tell function Inflorescence (from Inflorescence meristem)

Mutations in floral identity genes Terminal flower 1 gene normal function is positive regulator of inflorescence (when mutant, lose inflorescence shoot activity = flower early; SAM (vegetatuve) converted to floral mersitem (for making flowers)) Leafy – delay flowering; thus leafy gene acts as negative regulator of inflorescence meristem terminal flower 1 (tfl1): Convert the inflorescence meristem to the flower meristem. leafy (lfy): produce more inflorescences, delayed flowering

Factors regulating the transition to reproduction Vegetative meristem EMF Inflorescence meristem LFY TFL Floral meristem

The discovery of photoperiodism Garner and Allard (1920’s) Soybeans (Glycine max) planted over a three-month period all flowered about the same time

Many more experiments were followed: Eliminate a variety of environmental conditions: Nutrition, temperature, and light intensity Relative length of day and night decides the flowering time Photoperiodism: ability of an organism to measure the proportion of daylight during a 24-hour period

Photoperiod Varies according to the latitude and seasonal changes.

Critical daylength Critical Daylength (CD) Xanthium: a short day plant, flowers when CD is LESS than 15.5 hours. Hyoscyamus: a long day plant, flowers when CD is MORE than 11 hours.

Plants are induced to flower by different photoperiods short day (SD) : plants are stimulated to flower when the length of day falls below a threshold long day (LD): plants are stimulated to flower when the length of day exceeds a threshold Day neutral (DN): plants flower indifference to the changes of day length. Long-short-day: flowering requires certain number of short days are preceded by a certain number of long days. Short-long-day: flowering requires certain number of long days are preceded by a certain number of short days. Intermediate-daylength: not flowering if the daylength is too short or too long.

Do plants really measure the length of the daylength?

Hamner and Bonner (1938): Xanthium strumarium, a SD plant with CD = 15 Hamner and Bonner (1938): Xanthium strumarium, a SD plant with CD = 15.5 hours Xanthium flowers when the dark period exceeds 8.5 hours. Short interruption of dark period, even by a pulse of light as short as 1 minute delays flowering. The relative length of dark is not the determining factor.

Similar results were obtained with other SD plants. For LD plants A longer dark period inhibits flowering. Light break induces flowering. SD: A longer dark period promotes flowering. Light break inhibits flowering.

What tissues/organs perceive photoperiod?

Exp. 1: The leaf or apex of Perilla (a short day plant) was exposed to different daylength. When leaf part treated w/ short day (regardless w/ what apex treated) – plants made flower When only treated single leaf w/ short day – plant couldn’t make flower The leaf, not the apex perceives photoperiod

Exp. 2: Grafting experiment with Perilla Only single leaf from one of the plant treated – all 5 plants can make flower Something made in leaf can be transferred Flowering signal is grafting transmittable

The flowering signal: florigen vegetative or reproductive growth? the flowering signal is generated in the leaf the signal goes one way: from the leaf to the apex Grafting transmittable SAM Florigen can be transmitted to other plants thru grafting Flower locus T protein claimed to be florigen – but some disputes If you find signal or molecule that can greatly change flowering time – agriculture revolutionized Florigen ? Florigen Florigen

The biological clock Present in plants, animals, fungi, and some photosynthetic bacteria An internal time measuring system (“clock”) that runs on its own with a periodicity of nearly 24 hours. It can be “reset” by external signals. Temperature Present in many different organisms Very important to control daily activities of organisms Movement of leaves, when to make flowers, etc. Central component of clock is central oscillator – internal time measuring machine in many organisms – can run independently of external factors (intrinsic) – can also be regulated by external factors such as light and temperature Output of clock represented in rhythmic activity – circadian

The Arabidopsis biological clock The central oscillator: CCA1, LHY, and TOC1 (these are transcription factors) and other proteins Three TF regulate expression of each other -TOC1 is activator of CCA1 and LHY -CCA1 and LHY are repressors that inhibit TOC1 In morning, TOC1 accumulate to certain abundant level – w/ more TAC1, other two genes expressed in morning More expression of these two genes, more of those two proteins made – those two proteins bind to each other to form heteodimer – bind to promoter of TOC1 gene to inhibit its expression When have more LHY and CCA1 = less TOC1 protein At end of day, TOC1 protein eventually disappear thru protein degradation and lack of making new TOC1 protein, so at end, lack of TOC1 will make expression of these two genes turned off – so no more LHY and CCA1 protein made In evening, no more genes to expression this point, eventually LHY and CC1 protein disappear – as a result, TOC1 no longer being de-repressed – so expressed and accumulate in evening In morning, CCA1 and LHY more thus TOC1 once again repressed

The Arabidopsis biological clock CCA1 and LHY are expressed during the day and together repress expression of TOC1 during the day These three make up central component – expressed in circadian manner Expression of CCA1 and LHY and TOC1 is circadian regulated Arabidopsis seedling expresses CCA1::luciferase TOC1 is expressed at night and is required for activation of CCA1 and LHY1, beginning just before morning

Mutations in the clock genes Lack of the nyctinastic movement: diurnal rise and fall of leaves Altered flowering time in some mutants cca1: early flowering lhy: early flowering toc1: early flowering Some other clock mutants can be late flowering Different type of mutations – some make clock go fast and others make clock go slower

Temperature: Vernalization Vernalization: low temperature treatment can promote flowering in some plants. The vernalization-effective temperature and duration of low temperature treatment may vary. Vernalization is perceived by the shoot apex. The vernalization state is grafting transmissible. Vernalization—low temperature treatment can promote flowering time in some plants Low temperature treatment at earlier time but can still affect later plant development for plants to make flowers or not Affective temperature can vary and in different plants Some plants don’t need vernalization - ex. plants grown in cold regions – have to go thru winter time so have to be vernalized – in high temp, don’t need vernalization Perceived by shoot apex – if treat dry seeds w cold temp – don’t matter – only when seed germinates, shoot apex active and can perceive cold temperature and affect flowering Not known which molecule induced – induced stage can stay and later on affect flowering Also grafting transmissible

Cold acclimation Cold acclimation is different – couple of days of cold weather before deep winter – very important for plant b/c repeated cold treatment to trees can dramatic increase plant tolerance to freezing temps later on in deep winter time Cold acclimation can be induced quickly (not long term like vernalization) and it doesn’t affect flowering time Can be induced quickly Increases plant resistance to freezing stress Does not affect flowering time.

Hormone GA regulates flowering time GA1: an enzyme involved in GA biosynthesis ga1: In addition to the dwarf phenotype, the mutant flowers late under LD conditions and does not flower under SD conditions. GA treatment promotes flowering time. GA positively regulates flowering time

Inflorescence meristem Flower development in Arabidopsis Vegetative meristem Inflorescence meristem Transition to reproduction: Genes & other factors Flower organ development stage less regulated by environmental factors (light, temp) , but more by intrinsic - genes (organ identity genes) Floral meristem Flower: sepals, petals, stamens, and carpels Flower organ development: Organ identity genes

Flower organs petal stamen carpel sepal Petals - attract polinators Stamen – filament and anther (pollen sac) Carpel – stigma (flower perceives pollen; pollens land) and style – long or short neck – connects stigma to ovary; Ovary houses ovules – contain egg cells and central nuclei for double fertilization Sepal – green leaf like structure; degenerative leaves – can perform limited photosynthesis – protect flower bud before it opens Receptacle – holds the flower

The flower is generated from the floral meristem Floral meristem can give rise to cluster of flowers, but in some one flower meristem can only give rise to one flowers (ex. Roses) the floral meristem

Produced in 4 concentric whorls with the same order Flower organs Produced in 4 concentric whorls with the same order sepal (whorl 1) stamen (whorl 3) petal (whorl 2) carpel (whorl 4) w/ Arabiposis flowers – many mutants identified to understand processes of flower organ formation Four whorls – 3rd whorl – 6 stamens Middle – two carpels

sepal-petal-stamen-carpel stamen-carpel-stamen-carpel Mutants have weird flower structure – puzzle for many years Apetala2-2 mutant = only see stamen, carpel, stamen, etc. Ap1 mutant is similar sepal-petal-stamen-carpel stamen-carpel-stamen-carpel (the ap1 mutant is similar)

Aptala3 and Pistillata = see sepal, sepal, carpel, carpel (lose petal and stamen; 2nd and 3rd whorl are lost) wt apetala3 (ap3) pistillata (pi) sepal-petal-stamen-carpel sepal-sepal-carpel-carpel

sepal-petal-stamen-carpel sepal-petal-petal-sepal Agamous 1 – see sepal and petal only; so lost two inner whorls sepal-petal-stamen-carpel sepal-petal-petal-sepal

The “ABC” model for flower development AP1, AP2 B AP3, PI C AG ABC model used to explain weird flower structure A, B, and C genes function to specify different structures in diff. whorls A+B = 2nd, B+C = stamen, and C alone = carpel = only when functioning in all three categories, have normal flower A and C genes are antagonizing each other in their function – only in certain physical region The ABC genes function in the distinct fields. The A and C genes are mutually exclusive in their expression.

WT Ap1 and ap2 mutants are A genes – if knock them out, C will expand to A territory – don’t have sepal and petal, but will have carpel in sepal position b/c C expanded into A region and C only specifies carpel So, get carpel-stamen-stamen-carpel ap1 or ap2 The A genes: ap1 or ap2 mutants should (and do) make carpel-stamen-stamen-carpel

WT Ap3 and pi are B genes, if lost them, lost combination of AB and BC, so lose petal and stamen and have sepal-sepal-carpel-carpel ap3 or pi The B genes: ap3 or pi mutants should (and do) make sepal-sepal-carpel-carpel

The C genes: ag mutants should (and do) make sepal-petal-petal-sepal WT If don’t have C genes, loss of C, make A expand to C’s territory and lose stamen and carpel So have sepal-petal-petal-sepal ABC genes are all transcription factors that act as master regulators to control downstream genes – all together to regulate floral organ formation All ABC genes function within physical restriction space The ABC genes are TF that set up the boundary of gene expression to determine which organ will be formed, and where Additional downstream genes are required to ensure the property development of each organ ag The C genes: ag mutants should (and do) make sepal-petal-petal-sepal

Flower organ function: Gametogenesis and Fertilization Gametogenesis involves 2 meiosis – takes place in two different locations

Male gametogenesis Diploid pollen mother cells undergo meiosis to produce a tetrad of haploid microspores. Each microspore develops into a pollen grain containing two haploid cells (mitosis I): Pollen mother cells undergo meiosis to produce tetrad of haploid microspores – attach to each other Later separate into individual pollen cells – undergo mitosis to give rise to two cells for each pollen cell One cell engulfes the other cell (small = generative cell – lives inside the large, vegatative cell) the generative cell (small) The vegetative cell (large) generative cell

Vegetative cell grows to produce pollen tube Goal of pollen tube is to deliver male sex cells to female Generative cell produce 2 sperm cells via mitosis II – 2 sex cells for double fertilization the vegetative cell grows to produce the pollen tube the generative cell produce 2 sperm cells (mitosis II)

Female gametogenesis an ovule primordium emerges as a bump on the inner wall (placenta) of the ovary the megasporocyte undergoes meiosis to produce 4 haploid cells, only one of which (the megaspore) survives. Ovule inner wall can bud to form ovule primordium – one of the cells undergo meiosis to produce 4 cells – only one survives Further divides three rounds to make 8 cells

Female gametogenesis placental wall the megaspore undergoes 3 mitotic divisions to produce 8 cells: 3 antipodal cells 2 synergid cells 2 central cell nuclei 1 egg cell (EC) 3 antipodal cells (die eventually) 2 synergid cells located right at entrance of ovule – micropyle Pollen tube navigate all the way to micropyle and deliver sperm cell In end, synergid cells die – only 3 cells survive

Female gametogenesis placental wall

Double fertilization Pollens land on the stigma, hydrate, and begin to germinate the pollen tube

Pollen tubes grow, by tip growth, down through the stigma and style and into the ovary, toward the ovules. The pollen tube navigates to the micropyle and discharges the two sperm cells. Pollen tube doesn’t penetrate but navigates in b/w of cells

Double fertilization One sperm fertilizes the egg cell to develop into the embryo. the other sperm fertilizes the diploid central cell nucleus to develop into the endosperm. Ovule Antipodal cells Central cell nuclei One sperm cell will fuse w/ egg – into embryo and other will fuse w/ central nuclei – into endosperm Egg Synergids Pollen tube Micropyle Sperms

Plant reproduction Ovule (1 to many) Ovary Silique

Fruit development The ovary and other tissue together produce a fruit. Fruit is important for seed dispersal in many species Many foods are also called “vegetables”: tomatoes, pea pods, squash Fruit size, texture, and sugar content are determined by genes. Ethylene stimulates fruit ripening.

Life cycle of a flowering plant