| Double fertilisation in GNETUM | Second fertilisation event between sperm nucleus and a female (non-sister egg) nucleus. Yields a normal diploid zygote and a supernumerary diploid zygote. |
| Methods used for Understanding Plant Reproduction and Double Fertilisation | i) Choice of experimental system |
| Phytohormones | Regulate transitions between all stages of the plant life cycle |
| Auxins | Are related to tryptophan, promotes stem elongation, regulate many other aspects of plant
development: Phototropism and gravitropism, Branching, Embryonic patterning, Organ initiation.
At high concentrations, auxin inhibits root elongation. Auxins are used as selective herbicides. At lower concentrations, auxin stimulates root development. Auxin induces root formation in tissue culture. |
| Cytokinins | Are related to adenine. Cytokinins promote shoot formation in tissue culture. Delay senescence. |
| Gibberellins are a family of diterpenoids | Growth, Seed germination, Promote flowering, Promote sex ,determination in some species, Promote fruit growth. Only some GAs are biologically active (GA4 major active in Arabidopsis). Gibberellins regulate growth. Discovered in Japan by Eiichi Kurasawa. Genes that control GA, are green revolution genes. Other green revolution genes affect DELLA proteins. GA present: Degradation to DELLA proteins. All0ows growth promoting genes. |
| Brassinosteroids | Cell elongation, Pollen tube growth, Seed germination, Differentiation of vascular, tissues and root hairs, Stress tolerance. Brassinosteroid (BR) mutants are dwarfed. |
| Control of branching patterns | Auxin plays a role in apical dominance; Auxin inhibits branching. Strigolactones inhibit branch outgrowth. Auxin transported from the shoot to the root induces strigolactone synthesis, which indirectly inhibits bud outgrowth. |
| Ethylene | Control of fruit ripening, Control of leaf and petal, senescence, Control of cell division and cell elongation, Sex determination in some plants, control of root growth, Stress responses. Triple responses: Reduced elongation, hypocotyl swelling and apical hook exaggeration. |
| Abscisic acid | Seed maturation and dormancy, desiccation tolerance, Stress response, Control of stomatal aperture. It acts antagonistically with GA, Tolerance drought stress; changes the stomatal aperture and volume of guard cells. |
| ABA-induced stomatal closure is extremely rapid and involves changes in ion channel (mech.) | ABA triggers an increase in cytosolic calcium (Ca2+), which activates anion channels (A-
) allowing Cl- to leave the cell. ABA also activates channels that move potassium out of the cell (K+out) and inhibits channels that move potassium into the cell (K+ in). The net result is a large movement of ions out of the cell. As ions leave the cell, so does water (by osmosis), causing the cells to lose volume and close over the pore. |
| ABA induces stress-responsive genes | Osmoprotectants , Membrane and protein stabilization, Movement of water and ions, Oxidative stress responses |
| Salicylic Acid | Response to biotrophic pathogens, Induced defences response, Systemic acquired resistance, unduces after pathogen attack. |
| Jasmonates | Response to necrotrophic pathogens.
Induction of anti-herbivory responses.
Production of herbivore-induced volatiles to prime other tissues and attract predatory insects. Warn other plants |
| Most auxin is transported in a polar fashion | Auxin is mostly transported basipetally (from the apical part of the plant towards the root). Polar auxin transport is independent of gravity. Auxin becomes redistributed in the root tip in response
to gravitropic signals. It enables rapid changes in growth and development patterns in response to the environment. |
| Monitoring auxin response by reporter gene transcription | The DR5 reporter construct is widely used to monitor auxin-response level. DR5 consists of seven repeats of an auxin-response element (AuxRE) which is a binding site for auxin-responsive
transcription factors (ARFs). |
| Auxin is transported from cell to cell | Outside of the cell the pH is acidic (about 5.5) and auxin is in its uncharged form (IAAH). This uncharged form is lipid soluble and can cross the plasma membrane into the cell. In the cytoplasm at pH 7 Indole-3-acetic acid is deprotonated (IAA-). This charged ion cannot cross
the membrane and is unable to exit other than through specific transporters. |
| Most auxin movement is through efflux and influx carriers | PIN family move IAAH out of the cell, ABCB transporters also export auxin. The AUX1/LAX
influx carriers move IAAH into the cytoplasm. PIN1 mediates long-distance, basipetal transport from the shoot apex to the root. |
| The PIN proteins are named for the pin-formed mutant | PIN1 is expressed in the vascular tissue. Asymmetric distribution on the plasma membrane allows
directional transport of auxin. The distribution of PIN proteins determines the direction of auxin fluxes in different tissues. Auxin redistribution triggers developmental responses: Leaf formation, Gravitropism, Establishment of the apical-basal axis during embryonic development. PIN1 cycle between the plasma membrane and endosome not in gnom mutant. PIN1 is required to transport auxin to the periphery of the apical meristem and to induce leaf formation. Polarised distribution of the PIN1 protein directs auxin to the organ primordial. |
| Establishment of the apical-basal axis during embryogenesis | Auxin gets in the embryo, change of polarisation, apical-basal then established. |
| Gravitropism | Perception of gravity is mediated by specialised cells called statocysts: Starch sheath cells, Collumella cells (Both cell types contain large starch grains). |
| PIN3 | PIN3 is expressed within statocysts. PIN3 changes polarity in response to a change in the gravity vector. Root and shoot elongation are opposite. |
| How does auxin control responses at the cellular level? | Auxin has two receptors: APB1 (involved in auxin responses at the cell surface), SCF^TIR1 (controls responses in the nucleus). |
| ABP1 | Mediates rapid auxin responses at the plasma membrane. Perception of auxin by ABP1 at the outer face of the plasma membrane initiates signals that lead to proton-pump activation, wall
acidification and wall loosening. |
| Wall acidification | Activates wall loosening enzymes called expansins. Purified expansins are sufficient to induce cell
elongation. |
| TIR1 | Is an F-box protein, part of the SCF^TIR1 ubiquitin ligase complex. TIR1 forms a coreceptor
complex with Aux/IAA proteins which are inhibitors of downstream responses. TIR1 targets AUX/IAA proteins for ubiquitination and degradation by the proteasome. |
| The auxin signaling pathway | Low levels: Aux/IAA proteins and ARF proteins associate and interfere with ARF action.
High levels: Auxin promotes the association of Aux/IAA proteins and the SCFTIR1ubiquitin ligase complex. |
| Plants actively seek light for photosynthesis. | Plants adopt different developmental programmes when germinated in the light or in darkness. Shade avoidance responses allow plants to escape competition for light from neighbouring plants. Plants sense the reduced ratio of red to far-red light caused by shading from other plants. |
| Plants sense different light qualities | Red / far red light, Blue light, UV A, UV B |
| There are multiple photoreceptors of each type | In Arabidopsis there are: – 5 phytochromes – 3 cryptochromes – 2 phototropins – 1 UVB photoreceptor. Respond to different light intensities. Control different aspects of plant
physiology and development. |
| Different photoreceptors are involved in different responses | PhyA is responsible for seedling de-etiolation. PhyB is involved in the shade avoidance response. |
| Photoreversible response. | Effects of red light pulses can be reversed by immediate exposure to far-red. Red light: activation. Far-red light inactivation: inhibition. Red or far-red light absorption causes cis-trans isomerisation of the chromophore at carbon 15. |
| Phytochrome structure | Phytochrome is a soluble homodimer consisting of a polypeptide apoprotein (each subunit ~120kDa) covalently attached to a light absorbing pigment called the chromophore. PAS domains: protein interaction domains. The phytochrome chromophore is a linear tetrapyrole
which is synthesized in the chloroplast. |
| Phytochrome conformational change | The change between Pr and Pfr is associated with both a conformational change in protein structure and a change in absorption maxima from 666 nm (Pr) to 730 nm (Pfr). |
| Light regulates expression of many plant genes | LHCB or CAB-a component of light-harvesting complexes in chloroplasts. RBCS- small subunit of RUBISCO, the key enzyme in carbon fixation. Chalcone synthase – a key enzyme in the flavonoid pathway. |
| Phytochrome-interacting proteins | PIF = Phytochrome interacting factors. PIFs regulate photomorphogenesis and promote hypocotyl elongation. Basic Helix-Loop-Helix (bHLH) transcription factors. |
| PIFs dark vs light | PIFs bind active phytochrome (Pfr) and are targeted to the proteasome in response to light.
In the dark: PIF transcription factors activate genes involved in hypocotyl elongation. Light-activated phytochrome B moves to the nucleus where it binds PIF transcription factors. PIF proteins are targeted for degradation and hypocotyl elongation is inhibited. PIFs act to upregulate expression of auxin (IAA) biosynthesis genes, YUCCA (YUC). |
| Mutants | cop1 mutant: fails to adopt an etiolated morphology in darkness. The transcription factor HY5 is a positive regulator of photomorphogenesis. HY5 is a nuclear, DNA-binding molecule. It binds a DNA sequence motif called the G-box (CACGTG). HY5 is not degraded in the dark so the plant develops as if it were in the light. |
| COP1 | COP1 is an E3 ligase that ubiquitinates the transcription factor HY5 targeting it for degradation.
Dark: translocate the nucleus. Light: is located in the cytoplasm and is inactive. |
| Cryptochromes | Evolved from DNA-photolyases (repair UV-induced DNA damage). Contain 2 chromophores |
| Photoexitation of crytochromes | 2 mechanisms for CRY regulated gene expression: By CIBs and COP1 |
| UV-B photoreceptor (UVR8) | Β-propeller protein with 7 blade-shaped β-sheets. No attached chromophore. Forms homodimers in the dark. Dimers dissociate in response to UV-B light (280-315 nm). |
| The UVR8-COP1 complex activates HY5 | PHY, CRY and UVR8 signaling pathways converge onto COP1. Results in the down regulation, allows HY5 expression and allows growth. |
| Female development in Arabidopsis | 1. Megaspore 2. 1st, 2nd, 3rd mitotic division 3. Cellularisation and formation of the embryo sac. |
| The mature female gametophyte | The eight-nuclei/ seven-cell embryo sac: Gametes: 1 egg cell (1n), 1 central cell (2n)
Accessory cells: 2 sinergid cells, 3 antipodal cells |
| Pollination and pollen growth | THE MEETING OF THE SPOROPHYTE AND GAMETOPHYTE GENERATIONS. Matrix forms at stigma for male gametophyte. gametophytic selection: pistil selects |
| Incompatibility systems | 1. Heteromorphic and 2. Homormoprhic |
| Pollen Tube Growth | The male gametophyte contains signals required for correct pollen tube guidance to the female gametophyte. Signals from the ovary involved in initial stages of Pollen Tube guidance and
bidirectional growth along the funiculus |
| Pollen Tube Guidance | Signals from the haploid female gametophyte (embryo sac) involved in latter stages of Pollen Tube guidance toward the micropyle of the ovule. Evidence from the identification of mutations in genes affecting embryo sac development. |
| Pollen tube guidance by the embryo sac | The embryo sac attracts the pollen tube: Ovules are able to attract pollen tubes –even when
repositioned. The “One Tube-One Ovule” rule: Evidence for a repulsive cue that directs supernumerary Pollen Tubes. |
| Gametophytic Signals involved in Double Fertilisation. | Pollen tube rupture, sperm cell release and synergid degeneration. During or shortly before fertilisation one of the two synergids degenerates. The PT penetrates this synergid and ruptures
to release its contents. Each sperm is guided towards one of the two female gametes. |
| Key Historical Events Leading to the Discovery of Double Fertilisation in Angiosperms | 1884 - Proof of a fusion event between male and female gametes to produce an embryo.
1898 - Each male nucleus fuses with a female one within the embryo sac.
1899 - Double fertilisation was independently confirmed by Leon Guignard. |
| Methods used for Understanding Plant Reproduction and Double Fertilisation | i) Choice of experimental system. Choices: Lilium and Fritillaria genera, Torenia fournieri, Arabidopsis thaliana. |
| Is double fertilisation characteristic of the earliest angiosperms (flowering plants)? | Link between earliest seed plants. Ginkos, cycads and conifers lack double fertilisation. Ephedra and Gnetum undergo double fertilisation –but no endosperm produced. |
| Double fertilisation in EPHEDRA | Second fertilisation event between sperm nucleus and ventral canal (sister egg) nucleus. Yields a normal diploid zygote and a supernumerary diploid zygote. |
