Examenvragen Geuten 1) Germination can be divided into three phases corresponding to the phases of water uptake

Under normal conditions, water uptake by seeds is triphasic:

Phase 1: imbibition = the rapid uptake of water by dry seeds

Phase 2: Water uptake by imbibitions declines. Metabolic processes, such as transcription and

translation, are reinitiated. The embryo expands because of cell wall

loosening. The radical emerges from the seed coat.

Phase 3: Water uptake resumes due to a decrease in Ψ

as the seedling grows. Stored food reserves of the seed are fully

mobilized.

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2) The cereal aleurone layer is a specialised digestive tissue surrounding the starchy endospermhoe wordt reservestoffen vrijgezet in een kiemend zaad (ABA vs GA)

1) GAs are synthesized by the embryo and released into the starchy endosperm via the scutellum.2) GAs diffuse to the aleurone layer.3) Aleurone layer cells are induced to synthesize and secrete ⍺-amylases and other hydrolases

into the endosperm.4) Starch and other macromolecules are broken down to small molecules.5) The endosperm solutes are absorbed by the scutellum and transported to the growing

embryo.

Once the DELLA protein has been degraded, transcription of GA-MYB gene is activated. GA-MYB has a positive regulation on ⍺-amylase: GA-MYB activates ⍺-amylase gene expression.

1) GA1 from the embryo enters an aleurone cell.2) GA1 binds to the GID1-receptor in the nucleus.3) GID1 undergoes an change that facilitates its binding to a DELLA-repressor.4) Once the DELLA-protein has bound to the GA1-GID-complex, an F-box protein poly-

ubiquitinates the DELLA-protein.5) The poly-ubiquitinated DELLA-protein is degraded by the 26S proteasome.6) The transcription of an early gene (GA-MYB gene) is activated.

ABA inhibits gibberellin-induced enzyme production2 mechanisms:

1) Direct: ABA regulates transcriptional repression of ⍺-amylase directly through VP1 = a protein originally identified as an activator of ABA-induced gene expression.

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5)4)

3)2)

1)

2) Indirect: ABA negatively regulates GA-MYB = transcription factor that mediates the GA-induction of ⍺-amylase gene expression.

EXTRA: The ABA:GA ratio is the primary determinant of seed dormancy

Hormone balance theory = theory that says that the ratio of ABA and gibberellins serves as the primary determinant of seed dormancy and germination. ABA has an inhibitory effect on seed germination. Gibberellin has a positive influence on seed germination.The relative hormonal activities of ABA and gibberellin in the seed depend on:

1) The amounts of each hormone present within the target tissues. Are regulated by their rates of synthesis versus deactivation.

2) Hormonal sensitivity = the ability of the target tissues to detect and respond to each of the hormones. Is a function of the hormonal signaling pathways in the target tissues.

Model for ABA and gibberellins regulation of dormancy and germination in response to environmental factors:

1) Environmental factors such as temperature affect the ABA:GA-ratio and the responsiveness of the embryo to ABA and GA.

2) In early stages of seed development : GA is catabolised and ABA synthesis and signaling is predominate dormancy.

3) Later in seed development: ABA is catabolised and GA synthesis and signaling is predominate germination.

4) The complex interplay between ABA and GA synthesis, degradation and sensitivity in response to ambient (omringende) environmental conditions can result in cycling between dormant and non-dormant stages = dormancy cycling.

5) Germination can proceed to completion when there is an overlap between favourable environmental conditions and non-dormancy.

The balance between the activities of ABA and GA in seeds is under both developmental and environmental control.

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5)

4)

3)2)

1)

3) Auxin promotes growth in stems and coleoptiles, while inhibiting growth in roots

Auxin control of shoot elongation: Auxin synthesized in the shoot apex is transported toward the tissues below. Synthesis in the shoot apex is required for subapical elongation. Because the level of endogenous auxin in the elongation region of a normal healthy plant is

nearly optimal for growth, spraying the plant with exogenous auxin causes only a modest and short-lived stimulation in growth.

Endogenous source of auxin removed by excision (wegsnijding) of stem or coleoptile sections containing the elongation zone growth rate rapidly decreases to a low basal rate exogenous auxin rapidly increasing their growth rate back to the level in the intact plant.

Auxin control of root elongation: Auxin induces the production of ethylene, which inhibits root growth.

These 2 hormones interact differentially in root tissue to control growth. Sensitive to optimal concentration: if ethylene biosynthesis is blocked:

o Low concentrations of auxin promote the growth of intact roots.o High concentrations of auxin inhibit root growth.

While roots may require a minimum concentration of auxin to grow, root growth is strongly inhibited by auxin concentrations that promote elongation in stems and coleoptiles.

3) Auxin-induced proton extrusion induces cell wall creep and cell elongation

Acid growth hypothesis = hydrogen ions act as an intermediate between auxin and cell wall loosening.

Source of hydrogen ions = H+-ATPase in the plasma membrane. The activity of H+-ATPase increases in response to auxin.

Auxin induces proton extrusion (uitstoting) in the cell wall after 10 to 15 minutes of lag time.

Expansins = cell wall loosening proteins. They loosen the cell wall by weakening the hydrogen bonds between the polysaccharide components of the wall under acidic conditions.

3) According to the starch-statolith hypothesis, specialised amyloplasts serve as gravity sensors in root capsRoot cap has 2 functions:

1) Protect the sensitive cells of the apical meristem as the tip penetrates the soil.2) The site of gravity perception.

the cap supplies auxin (= a root growth inhibitor) to lower side of the root during gravitropic bending.

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The gravity sensors in plants = large, dense amyloplasts that are present in specialized gravity-sensing cells.

PIN proteins determine the basal direction of auxin movement:

PIN1 mediates the vertical transport of auxin from the shoot to the root along the embryonic apical-basal axis and creates an auxin sink.

The auxin sink drives basipetal auxin transport upward from the root apex via PIN2.

Some lateral diffusion of auxin may occur, so PIN3 and PIN7 redirect the auxin back into the vascular parenchymal tissue.

When the root is oriented horizontally, most auxin is redirected to the lower side and inhibits growth The transport of [3H]IAA (= auxin) across a horizontally oriented root cap is polar, with a preferential downward movement. (PIN3 is thought to accelerate auxin transport to the lower side of the cap.

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3) Phototropism is mediated by the lateral redistribution of auxin

When a shoot is growing vertically, auxin is transported polarly from the tip to the elongation zone. The polarity of auxin transport from shoot to root is independent of gravity.

Auxin can also be transported laterally:

In gravitropic bending: auxin from the root tip is redirected to the lower side of the root. Auxin inhibits cell elongation, causing the root to bend downward.In phototropic bending: auxin from the shoot tip is redirected to the shaded side of the axis. Auxin stimulates cell elongation. This results in differential growth causes the shoot to bend toward the light.

1) Dark: auxin primarily moves from shoot to the root through: The vascular tissues in the petioles and hypocotyl. The epidermis.

2) Unidirectional blue light : auxin movement briefly stops at the cotyledonary node (knoop). The seedling stops growing vertically.

3) Auxin is redistributed to the shaded side + polar transport resumes.4) The cells on the shaded side of the hypocotyl elongate differential growth the seedling

bends toward the light source.

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4)3)2)1)

4) overgang van skotomorphogenesis naar photomorphogenesis uitleggen + Phytochroom functie uitleggen

Photomorphogenesis = development in the presence of light:The shoots of light-grown seedlings have shorter thicker hypocotyls, open cotyledons, active chloroplasts the expanded leaves have a green color.

Skotomorphogenesis = development in the dark:The shoots of dark-grown seedlings are etiolated = long spindly (sluik) hypocotyls, an apical hook, closed cotyledons, non-photosynthetic proplastids the unexpanded leaves have a pale yellow color.

When dark-grown seedlings are transferred to the light, photomorphogenesis takes over and the

seedlings are said to be de-etiolated.

The switch between dark- and light-grown development involves genome-wide transcriptional and translational changes triggered by the perception of light by several classes of photoreceptors Despite the complexity of the process, the transition of skotomorphogenesis to photomorphogenesis is very rapid (within minutes).

Phytochrome = protein-pigment photoreceptor that absorbs red and far-red light most strongly, but also blue light. It mediates several aspects of vegetative and reproductive development, including germination, photomorphogenesis and flowering.

Cryptochrome receptors = flavoproteins that mediate many blue-light responses involved in photomorphogenesis, including the inhibition of hypocotyl elongation, cotyledon expansion and petiole elongation.

In the dark: transcription factors that regulate photomorphogenesis are degraded in the nucleus via COP1 no photomorphogenesis.In the light: this process is prevented photomorphogenesis === Negatively regulated

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4) Gibberellins and brassinosteroids suppress photomorphogenesis in darkIn the dark: less Pfr (= phytochrome in the far-red absorbing form) Pfr inhibits hypocotyl sensitivity to GA, so if Pfr is low, endogenous GA promote hypocotyl elongation.In the light: Pr (= phytochrome in the red absorbing form) is converted to Pfr more Pfr makes hypocotyl less sensitive to GA reduction of the hypocotyl elongation.

GA suppress photomorphogenesis in the dark, the suppression is reversed by red light.

DE-ETIOLATED2 (= DET2) gene is a brassinosteroid biosynthetic gene. DET2-mutants have reduced levels of brassinosteroids, resulting in a de-etiolated appearance of the seedling even when grown in the dark. Brassinosteroids, like GA, suppress photomorphogenesis in the dark.

The signal transduction pathways of GA and brassinosteroids interact with the phytochrome pathway

4) Hook opening is regulated by phytochrome and auxin

The hook region is located just behind the shoot apex. The hook is used to push through the soil. Hook formation and maintenance in the dark result from ethylene-induced asymmetric growth. A consequence of more rapid elongation of the outer side of the stem compared with the inner side.

Influence of light: Hook exposed to white light elongation rate of the inner side increases, equalizing the

growth rates on both sides induces opening of the hook. Red light opens the hook and far-red light closes the hook indicating the involvement of

phytochrome.A close interaction between phytochrome and ethylene controls hook opening:

In the dark: ethylene production by the hook tissue elongation in the inner side is inhibited no hook opening.

In the light: red light inhibits ethylene production promoting growth on the inner side hook opening.

Interaction with auxin: Axr1 mutant = auxin-insensitive mutant no apical hook. Treatment of wild-type Arabidopsis seedlings with NPA = inhibitor of polar auxin transport

no apical hook. Auxin plays a role in maintaining the hook structure.

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4) Ethylene induces lateral cell expansion photomorphogenesis

The ethylene triple response:1) short hypocotyl 2) short root3) stronger apical hook

Ethylene changes the growth pattern: Reducing the rate of elongation. Increasing lateral expansion.

Leading to the swelling of the hypocotyl or epicotyls.

Ethylene changes microtubule orientation: In the absence of ethylene horizontal microtubule orientation elongation. In the presence of ethylene vertical microtubule orientation lateral expansion.

Orientation of cell expansion is regulated by microtubules.

(A) Growth rate of wild-type Arabidopsis after exposure and removal of ethylene at the times indicated by the arrows. The reduction in the growth rate following exposure to ethylene occurs in 2 distinct phases.

(B) Growth rate of wild-type, ein2-mutant and ein3/eil1-mutant seedlings following exposure to ethylene at the time indicated by the arrow

Wild-type: reduction in growth rate.Ein2: no reduction in growth rate.Ein3/eil1: phase 1 is identical to the wild-type, but phase 2 is absent.

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ZOU een examenvraag KUNNEN zijn: SHADE AVOIDANCE

Shade avoidance = the enhanced (vermeerderde) stem elongation that occurs in certain plants in response to shading by leaves. The response is specific for shade produced by green leaves, which act as filters for red and blue light, and is not induced by other types of shade. = green leaves absorb red light and are relatively transparent to far-red light.

Phytochrome enables plants to adapt to changes in light quality

The presence of a red/far-red reversible pigment in all green plants suggests that these wavelengths of light provide information that helps plants to adjust to their environment.

Decreasing the R:FR ratio causes elongation in sun plants

An important function of phytochrome: it enables plants to sense shading by other plants.

As shading increases, the R:FR-ratio decreases.

A higher proportion of far-red light more Pfr to Pr Pfr:Ptotal-ratio decreases.

Sun plants = plants adapted to an open-field habitat.Shade plants = plants which grow under leaf canopy.Leaf canopy shading = high levels of far-red light, low Pfr:Ptotal-ratio.

When sun plants were grown in natural light under a system of shades so that R:Fr was controlled:High far-red content lower Pfr:Ptotal-ratio stem extension rates increased.So, simulated canopy shading induced these plants to allocate more of their resources to growing taller.

Shade plants showed less reduction in their stem extension rate when they were exposed to higher R:FR values.

The price for increased internode elongation is usually reduced leaf area and reduced branching. But this adaptation increase plant fitness.

Thus, there appears to be a systematic relationship between phytochrome-cotrolled growth and species habitat.

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1) In direct sunlight: red light predominates high R:FR active Pfr form of phytochrome moves into the nucleus.

2) Phy causes the turnover of PIF proteins, which act as negative regulators of photomorfogenesis.

3) DELLA repressors bind PIF preventing the transcription of PIF-regulated genes.

4) The absence of PIF-induced gene expression stem growth is limited.

5) Under plant canopy: far red light predominates low R:FR inactive Pr form of phytochrome moves into the nucleus.

6) Absence of Pfr PIFs are not degraded.

7) The sensitivity to GA increases degradation of the DELLA repressor.

8) PIF proteins accumulated PIF-induced gene expression increases

promoting stem elongation.

Reducing shade avoidance responses can improve crop yields

In recent years, yield gains in crops (maize) have come largely through the breeding of new varieties with a higher tolerance to crowding induces shade avoidace responses today’s crops can be grown at higher densities than older varieties without suffering decreases in plan yields.

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6) Absence of WER activity of GL2 is not expressed cell hair specification.

5) trichome ontwikkeling en root hair identity

FIRST: root hair identity

The earliest land plants: lacked roots and used instead rhizome-like structures.The vascular land plants now: roots are present.

The developing root axis can be divided into 3 fundamental zones:

1) Meristematic zone: cell division

2) Elongation zone: cell expansion and elongation

Oscillation zone = transition zone between meristematic and elongation zone

3) Differentiation zone: cell differentiation, marked by the formation of root hairs by trichoblasts

Trichoblast identity is determined by the interaction of transcription factors: JACKDAW (JKD) SCRAMBLED (SCM) CAPRICE (CPC) WEREWOLF (WER)

1) Non-hair cell: WER forms a transcriptional complex with TTG1, GL3 and EGL3 activate GL2 gene resulting in a non-hair cell fate.

2) WER transcriptional complex induces the expression of the CPC gene.

3) The CPC protein moves into the presumptive (vermoedelijke) hair cell prevents WER from forming the transcriptional complex.

4) Cells in the cortex release a signal dependent on the JKD gene activates the SCM proteins of the presumptive hair cell.

5) Activated SCM further represses WER.

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The sites of lateral root emergence (dus niet roothair) have been correlated with regions of high auxin activity lateral root primordial develop at sites where the root bends.

According to 1 hypothesis: a root epidermal cell develops into a trichoblast because it has more surface area in contact with 2 cortical cells, so there is more signaling peptide to bind to the SCM receptor.

Transcription fusion = visualisation of an mRNA-transcript

Translation fusion = visualisation of a protein

Root epidermal development follows three basic patterns

TYPE I: The majority of plant species. Every root epidermal cell can potentially differentiate into a root hair.

TYPE II: The primitive vascular plants. The epidermis consists of a mixture of cells: trichoblasts = cells having the potential to form

root hairs and atrichoblasts = cells that are incapable of forming root hairs. Root hairs arise from smaller cells produced by an asymmetric cell division in the root

meristem.TYPE III:

Only in Brassicaceae. The epidermis consists of a mixture of cells: trichoblasts and atrichoblasts. The root epidermis consists of alternating files of cells that are either trichoblasts or

atrichoblasts. Trichoblast cell fate is specified in the meristem.

EXTRA:

Ethylene-treated roots: Produce root hairs in abnormal locations.

Ethylene inhibitors or ethylene-insensitive mutants:Reduction in root hair formation.

Suggesting that ethylene acts as a positive regulator in the differentiation of root hairs.

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SECOND: Trichoom ontwikkeling

Studied in rosette leaves of Arabidopsis. Arabidopsis trichomes are unicellular and branched, with a tricorn (three-horned) structure. Trichomes develop from single protodermal cells.

From protodermal cell to trichome cell, how? By an increase in nuclear size due to the initiation of endoreduplication (= replication of the nuclear genome).

As the leaf expands, new trichomes are initiated at the leaf base, and the previously formed trichomes are further separated by divisions of the intervening epidermal cells.

MUTATIONS:

TTG1, GL1 and GL3 are positive regulators of trichome formation. When a plant has mutations in one of these genes fewer or no trichomes

TRY is a negative regulator of trichome development. When a plant has mutations in one of these genes more or unevenly spaced trichomes. TRY inactivates the GL1-GL3-TTG1 complex by displacing GL1. This inactivation prevents trichome formation in the surrounding cells and thus establishes the regular spacing of trichomes.

GL2 is activated in trichome cells by the GL1-GG3-TTG1 complex. GL2 promotes trichome formation.

Addition of exogenous jasmonic acid (JA) causes an increase in the number of leaf trichomes. Jasmonate ZIM-domain (JAZ) represses trichome formation.

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6) Bespreek de regulatie en functie van Flowering

Flowering at the correct time of the year is crucial for the reproductive fitness of the plant:Plants that are cross-pollinated, must flower in synchrony with:

Other individuals of their species Their pollinators

At the time of the year that is optimal for seeds set.

Flowering = a highly adaptive process with a 100 of genes and a lot of pathway involved.All the pathways together decide whether the plant will flower or not.

The day length (rather than accumulation of photosynthate) is the determining factor in flowering.

(Original hypothesis: The correlation between long days in summer and flowering = a consequence of the accumulation of photosynthetic products synthesized during the long days.)

Flowering species tend to fall into 1 of 2 main photoperiodic response categories:1) Short-day plant = SDP:

flower only in short days (qualitative SDP) flowering is accelerated by short days (quantitative SDP)

2) Long-day plant = LDP: flower only in long days (qualitative LDP) flowering is accelerated by long days (quantitative LDP)

The essential distinction between LDP and SDP: Flowering in LDPs is promoted only when day length exceeds a certain

duration. Flowering in SDPs is promoted only when day length is less than a certain

duration. The certain duration = critical day length. The absolute value of the critical day length varies widely among species.

Ambiguity (dubbelzinnigheid) of day-length signal:The day length = ambiguous signal, because it cannot distinguish between spring and fall. Plants exhibit several adaptations for avoiding this ambiguity:

The coupling of a temperature requirement to a photoperiodic response. No responding to photoperiod until after a cold period = vernalization =

overwintering. Distinguish between shortening and lengthening days.

Photoperiodism = the ability of an organism to detect day length.The photoperiodic stimulus in both LDPs and SDPs is perceived by the leaves. In response to photoperiod the leaf transmits a signal that regulates the transition to flowering at the shoot apex.

Photoperiodic induction = the photoperiod-regulated processes that occur in the leaves resulting in the transmission of a floral stimulus to the shoot apex.

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Plants monitor day length by measuring the length of the nightNight break = the interruption of the night with a short exposure to light. The dark period can be made ineffective by a night break. But the interruption of a long day with a brief dark period does not cancel the effect of the long day.

Conclusion:These observations underlie the importance of the dark period instead of the light period.So WRONG TERMINOLOGY:

SDPs = long-night plants LDPs = short-night plants

Observations: Night-break treatments of only a few minutes are effective in preventing

flowering in many SDPs. ↔ Much longer exposures are often required to promote flowering in LDPs. A night break given near the middle of a dark period of 16h was found to

be the most effective in both LDPs and SDPs.

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7) Plant development has 3 phases + verandering juveniel naar adult bij Arabidopsis (hij wou dat van miRNA weten)

The timing of these transitions often depends on environmental conditions, allowing the plant to adapt to a changing environment. The transitions between different phases are tightly regulated developmentally, since plants must integrate:

Information from the environment Autonomous signals

to maximize its reproductive fitness.

Postembryonic development in plants can be divided into 3 phases:1) juvenile phase2) adult vegetative phase3) adult reproductive phase

Phase change = the transition from one phase to another.

Phase changes:1) Distinction between the juvenile and the adult vegetative phase:

Ability to form reproductive structures: Angiosperm: flowers Gymnosperms: cones (kegel)

The transition from juvenile to adult is frequently accompanied by vegetative changes.Often a gradual transition involving intermediate forms.

!!! Note: the absence of flowering is not a reliable indicator of juvenility. (+ conditions that promote vigorous growth accelerate the transition to the adult phase).

2) Distinction between adult vegetative and reproductive phase: Flowering = the expression of the reproductive competence of the adult phase.Often depends on specific environmental en developmental signals.Abrupt transition

Juvenile tissues are produced first and are located at the base of the shoot Results in a spatial gradient of juvenility along the shoot axis. (Because growth in height is restricted to the apical meristem, the juvenile tissues are located at the base (because they form first).

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Phase changes can be influenced by nutrients, gibberellins and other signals

Carbohydrates:In many plants, exposure to low-light conditions prolongs juvenility or causes reversion to juvenility.

Carbohydrates (especially sucrose) may play a role in transition between juvenility and maturity.

Gibberellins:Experimental evidence:

The application of GA causes reproductive structures to form in young juvenile plants of several conifer families.

Regulation of phase change in Arabidopsis by microRNAs:

miRNA156:• Some of his target genes promote the transition to flowering so miRNA156 inhibits flowering.• Level decreases over time and once below a threshold the targeted genes are expressed and phase change becomes possible (so flowering).• Overexpression is sufficient to greatly delay phase changes.

miRNA172:• Target genes include several transcripts that encode TF involved in the repression of flowering so miRNA172 promotes flowering phase change from adult vegetative to reproductive growth.• Level increases during development as miRNA156 decline.• Expression appears to be

under photoperiodic control (↔ miRNA156: plant age).Juvenile leaves Adult vegetative leavesSmall Larger

The decline in adult leaf size reflects the gradual shift in the allocation of sugars from leaves to developing reproductive structures.

Round More elongatedTrichomes only on adaxial side

Abaxial trichomes

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9) Welke mechanismen zijn essentieel voor het functioneren van een oscillator bij de circadiane klok? (negatieve feedback-loop)

Circadian clock = showing rhythmic behaviour with a period of 24 hours; especially of a biological process while diurnal is happening or occurring during daylight, or primarily active during that time.

Organisms are normally subjected to daily cycles of light and darkness, both plants and animals often exhibit rhythmic behavior in association with these changes (ex. leaf and petal movement, stomatal opening and closing, ...).

When organisms are transferred from daily light-dark cycles to continuous dark/light, many of these rhythms continue to be expressed, at least for several days. Under such uniform conditions the period of the rhythm is close to 24h = circadian rhythm

Because they continue in a constant light/dark environment these circadian rhythms cannot be direct responses to presence/absence of light, but must be based on an internal pacemaker = endogenous oscillator

Under natural conditions: endogenous oscillator is entrained = synchronized to a true 24h period by environmental signals. The most important are:

Dusk (schemering): light dark Dawn (zonsopgang): dark light

Such environmental signals = zeitgebers (German for time givers). When such signals are removed (ex. by transfer to continuous dark) the rhythm is said to be free-running and the rhythm reverts to the circadian period that is characteristic of the particular organism.

In addition: many rhythms damp out (= amplitude decreases) when the organism is subjected to a constant environment for several cycles. An environmental zeitgeber (such as transfer from light to dark or temperature change) is needed to restart the rhythm.

If a light pulse is given during the first few hours of the subjective night the organism interprets the light pulse as the end of the previous day the rhythm is delayed. If a light pulse is given towards the end of the subjective night the organism interprets the light pulse as the beginning of the following day the phase of the rhythm is advanced.

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The circadian clock is driven by a circadian oscillator

Transcriptional-translational feedback loop:

The central oscillator is based on a self-regulating principle of a negative feedback loop:

1) Gene expression of CCA1 (Circadian and Clock-Associated 1) and LHY (Late Elongated hypocotyl) varies and has a maximum in the morning. They are activated by light via both phytochromes and cryptochromes. These genes encode MYB TF that regulate circadian rhythms in Arabidopsis.

2) CCA1 and LHY activate the expression of LHCB and other “morning genes”.(ex. transcription of CO mRNA)

3) CCA1 and LHY also provide the repression of TOC1 (Time Of CAB -expression, CAB: chlorophyll a, b binding proteins) and other “evening genes”.

4) The expression of CCA1 and LHY decreases in the day so that by the evening, there is a maximum expression of TOC1 .

5) In the night, stimulates TOC1 the expression of CCA1 and LHY so that in the morning CCA1 and LHY can reach a maximum.

Day and night factors influence each other.

The presence of this negative feedback loop (stimulation of LHY to TOC1 and CCA1 and inhibition in the opposite direction) ensures that the system continues so that the plant possesses an internal oscillating clock system.

Geuten: “LHY & CCA1 inhibit their own activator, this creates a negative feedback loop which intuitively leads to an oscillation.”

Phytochromes and cryptochromes entrain the clock

Light is sensed by phytochromes and cryptochromes: Phytochrome = a photoreceptor, a pigment that plants, and some bacteria

and fungi, use to detect light in the red and far-red region of the visible spectrum. 

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Italic = genesNo italic = proteins

Arabidopsis had 5 phytochromes: all but one of them (phytochromes C) have been implicated in clock entrainment.

Cryptochrome = are a class of flavoproteins that are sensitive to blue light. Photo-activated CRY2 is able to activate flowering in response to blue light by directly up-regulating the expression of a key flowering gene: FLOWERING LOCUS T (FT).

10) Bespreek de regulatie en functie van Flowering Locus T

Cryptochrome = are a class of flavoproteins that are sensitive to blue light. Photo-activated CRY2 is able to activate flowering in response to blue light by directly up-regulating the expression of a key flowering gene: FLOWERING LOCUS T (FT).

Zie pag 22

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11) Coincidence model (Arabidopsis en rijst) uitleggen

Coincide model= model in which is proposed that control of flowering by photoperiodism is achieved by an oscillation of phases with different sensitivities to light.= model in which the circadian oscillator controls the timing of light-sensitivity and light-insensitive phases (red line in picture). Made by E. Bünning.

The ability of light to promote/inhibit flowering depends on the phase in which the light is given. When a light signal is administered during the light sensitive phase of the rhythm, the effect can be:

Light promotes flowering in LDPs. Light prevents flowering in SDPs.

The phases of sensitivity and insensitivity to light continue to oscillate in the darkness.Flowering in both SDPs and LDPs is induced when the light exposure is coincident with the appropriate phase of the rhythm:

Flowering in SDPs is induced only when the light signal is given after completion of the light-sensitive phase of the rhythm.

Flowering in LDPs is induced only when the light signal is given during the light-sensitive phase of the rhythm.

Plant flowering responses are sensitive to light only at certain times of day-night cycle.The coincidence of CONSTANTS expression and light promotes flowering in LDPs

CONSTANS (CO)= key component of a regulatory pathway that promotes flowering of Arabidopsis (LDP) in long days. It encodes a zinc finger protein that regulates transcription of other genes. The coincidence model relies on protein stability of CONSTANS. The expression of CO is controlled by the circadian clock.

(A) Short day:

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A little overlap between CO mRNA expression and daylight. CO protein does not accumulate to sufficient levels in the phloem. CO protein will not promote the FT protein expression. Plant remains vegetative.

(B) Long day: A large overlap between CO mRNA expression and daylight. Sensed by phyA and cry. CO protein accumulates to sufficient levels in the phloem. CO protein promotes the FT protein expression. FT protein is translocated to the apical meristem Plant will flower.

The expression of CO mRNA is controlled by circadian clock, with the peak of activity 12h after dawn. mRNA transcription of the CO gene is INDEPENDENT of light, but the translation of the mRNA into the CO protein is DEPENDENT of light.

CO protein accumulates in response to long days which accelerates flowering:Long days expression of CO mRNA in light CO protein accumulation acceleration of flowering:

Short days: CO expression entirely in the dark increase in CO mRNA will not lead to an increase in CO protein noninductive for flowering.

Long days: CO gene expression overlaps with the light period increase in CO mRNA increase in CO protein inductive for flowering.

So there must be overlap (coincidence) between CO mRNA synthesis and daylight so that the daylight can permit the accumulation of CO protein that promotes flowering.

How does daylight bring about the accumulation of CO protein?Experiment: CO expression from a constitutive promoter CO mRNA was expressed continuously level remained constant throughout day-night cycle.

Observation: CO protein continued to cycle, suggesting that CO protein abundance is regulated by a posttranscriptional mechanism. The posttranscriptional mechanism is based in part on difference in the rates of CO degradation in light vs dark:

Dark : CO is ubiquitinated + degraded rapidly by 26S proteasome no accumulation.

Day : light enhances the stability of the CO protein accumulation. This explains why CO mRNA promotes flowering only when its mRNA expression coincides with light.

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It's a bit more complex:The effect of light on the CO stability depends on the photoreceptor involved:

Contribute to setting the phase of the circadian rhythm. Can affect CO accumulation and flowering.

In the morning: phyB signaling enhances CO degradation. In the evening: cryptochromes and phyA antagonize degradation + allow CO protein accumulation.

How does CO protein stimulate flowering in LDP?CO is a transcriptional regulator that stimulates the expression of FLOWERING LOCUS T (FT).= a key floral signal.= the phloem-mobile signal that stimulates flower evocation in the meristem.SDPs use a coincidence mechanism to inhibit flowering in long days

Rice = SDP

(C) Short day:Lack of coincidence between Hd1 mRNA expression and daylight no accumulation of the Hd1 protein no repression of the Hd3a gene Hd3a mRNA is expressed Hd3a protein is translocated to the apical meristem Plant will flower.

(B) Long day: Coincidence between Hd1 mRNA expression and daylight accumulation of the Hd1 protein repression of the Hd3a gene Hd3a

mRNA is not expressed Hd3a protein is not translocated to the apical meristem Plant remains vegetative.

Similarity between Rice and Arabidopsis:The Rice genes Heading-date1 (Hd1) and Heading- date3a (Hd3a) encode proteins homologous to Arabidopsis proteins CO and FT respectively.

Difference between Rice and Arabidopsis:In Rice Hd1 protein acts as a inhibitor of Hd3a mRNA expression.↔ In Arabidopsis CO protein promotes the FT mRNA expression.

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12) ZOU een vraag KUNNEN zijn: VERNALIZATION

Vernalization= promoting flowering with cold.= process whereby repression of flowering is alleviated (verlichten) by a cold treatment given to a hydrated seed (= a seed that has imbibed (opnemen) water) or to a growing plant. Dry seed do not respond because vernalization is an active metabolic process.

Without the cold treatment: Not competent to respond to flowering signals such as inductive

photoperiods. Delayed flowering. Remain vegetative.

Grow as rosettes with no elongation of the stem.

The effective temperature range for vernalization: Just below freezing to about 10°C, with a broad optimum usually between 1-7°C. The effect of cold increases with the duration of the cold treatment until the response is saturated.Vernalization loss: Vernalization can be lost as a result of exposure to devernalization conditions (such as high T). But the longer the exposure to low temperature, the more permanent the vernalization effect(The longer the exposure to a cold treatment, the greater the number of plants that remain vernalized when the cold treatment is followed by a devernalizing treatment)

Place of vernalization:Vernalization appears to take place primarily in the SAM. Localized cooling causes flowering when only the stem apex is chilled and this effect appears to be largely independent of the temperature experienced by the rest of the plant.

A vernalization requirement is often linked to a requirement for a particular photoperiod. The most common combination: requirement for a cold treatment followed by a requirement for long days (lead to flowering in early summer at high latitudes).

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Model for how vernalization stably effects competence of the meristem to flower: There are changes in the pattern of gene expression in the meristem after cold treatment that persist into the spring and throughout the remainder of the lifecycle. Epigenetic changes = stable changes in gene expression that do not involve alterations in the DNA sequence and which can be passed on to descendant cells through mitosis and meiosis. These changes are stable even after the signal (in this case cold) that induced the change is no longer present.

Experiment in LDP Arabidopsis:Winter-annual types of Arabidopsis require vernalization and long days for flowering. Identification of FLOWERING LOCUS C (FLC) = a gene that acts as a repressor of flowering. FLC is highly expressed in nonvernalized shoot apical regions.

Process:1) Vernalization2) FLC is epigenetically switched off for the remainder of the plant's life cycle3) Flowering occurs in response to long days4) In the next generation: FLC is switched on again, restoring the requirement

for cold

Working mechanism in Arabidopsis:FLC directly represses the expression of:

The key floral signal FT in the leaves. The TFs SOC1 and FD at the SAM.

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13) SOC1, FT en leg hiermee flowering uit (dus dat schematje vooral he, met Locus C ook uitleggen)

Florigen (or flowering hormone) is the hypothesized hormone-like molecule responsible for controlling and/or triggering flowering in plants. Florigen is produced in the leaves, and acts in the shoot apical meristem of buds and growing tips = protein that serves as a long-distance signal during flowering

The floral stimulus is translocated along with photoassimilates in the phloem.

The Arabidopsis protein FLOWERING LOCUS T (FT) is florigen

Coincidence model: flowering in LDP occurs when the CONSTANS gene is expressed during the light. CO gene expression seems to be highest in the companion cells of phloem of leaves and stems.

FLOWERING LOCUS T (FT) = the downstream target gene of CO. FT is also specifically expressed in the companion cells.

In Arabidopsis: CO expression during long days results in an increase of FT mRNA.

Current model:The FT protein moves via phloem from leaves to the meristem under inductive photoperiods. There are 2 critical steps in this process:

1) Export of FT from the companion cells to the sieve tube elements.2) Activation of FT target genes at shoot apex, which triggers flower

development.

zie volgende pagina!!

LFY was activated by SOC1 and AP1, but LFY also directly activates the expression of AP1 and FD, forming 2 positive feedback loops. These positive feedback loops keep the meristem in a flowering state. Because of these feedback loops, floral initiation in Arabidopsis is irreversible. The meristems of some species lack such positive feedback loops and revert to producing leaves in absence of a continuous inductive photoperiod (reversible).Gibberellins and ethylene can induce flowering

Gibberellin appears to promote flowering in Arabidopsis by activating the expression of the LFY genes. This expression is mediated by the GA-MYB TF. (the levels of gibberllins are relatively low in short days)

Ethylene appears to promote flowering in the pineapple family

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Multiple factors regulate flowering in Arabidopsis:

Process:1) FT mRNA is expressed in the companion cells of the leaf vein in response to

multiple signals: day length, light quality and temperature.

2) FTIP1 mediates the transport of the FT protein through a continuous ER network between the companion cell and the sieve tube elements. The endoplasmatic reticulum (ER) is one of the major routes for transport of proteins from the companion cells to the sieve tube elements. FT INTERACTING PROTEIN1 (FTIP1) = the ER-localized protein that is required for FT movement into phloem translocation stream, which takes it to the meristem.

3) FT moves in the phloem from the leaves to the apical meristem.

4) FT is unloaded from the phloem in the meristem and interacts with FD.Once in the floral meristem, the FT protein enters the nucleus and forms a complex with FLOWERING D (FD) = a basic leucine zipper (bZIP) TF that is expressed in the meristem.

5) The FT-FD-complex activates: Suppressor of Overexpression of CONSTANTS1 (SOC1) in the

inflorescence meristem APETALA1 (AP1) = floral identity gene in the floral meristem

This triggers LEAFY (LFY) gene expression = a floral identity gene.

6) LFY and AP1 trigger the expression of the floral homeotic genes.

7) The autonomous and vernalization pathways negatively regulate FLC, which act as a negative regulator of SOC1 in the meristem and FT in the leaves.

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.14) Bespreek de genetische en biochemische aspecten bij floral organ identity development

The shoot apical meristem in Arabidopsis changes with development

Size: floral meristems > vegetative meristems. In vegetative meristems: cells of the central zone complete their division cycles slowly. The transition from vegetative to reproductive development: increase in frequency of cell divisions within the central zone of the SAM resulting in an increase in size of the SAM.

Flowering Arabidopsis plant:

Different organs at different stages of development.

The SAM develops different primordiaon its flanks throughout its development.

During the vegetative phase of growth: Arabidopsis SAM produces leaves with very short internodes resulting in a basal rosette of leaves.

When reproductive development is initiated:The vegetative meristem is transformed into the primary inflorescence meristem. This meristem produces an elongated inflorescence axis bearing 2 types of lateral organs:

1) Stem-borne/inflorescence leaves The axillary buds of the stem borne leaves develop secondary inflorescence meristems and their activity repeats the pattern of development of primary inflorescence meristem. The Arabidopsis inflorescence meristem has the potential to grow indefinitely and thus exhibit indeterminate growth.

2) Flowers They arise from floral meristems that form on the flanks of the inflorescence meristem. The floral meristem is determinate.

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The 4 different types of floral organs are initiated as separate whorls

Floral organs are initiated sequentially by the floral meristem of Arabidopsis:

(C) Combinatorial model: the functions of each whorl are determined by 3 overlapping developmental fields these fields correspond to the expression patterns of specific floral organ identity genes.

Floral meristems initiate 4 different types of floral organs:1) Sepals = kelkbladen2) Petals = kroon-/bloembladen3) Stamens = meeldraden4) Carpels = stampers

These sets of organs are initiated in whorls = concentric rings around the flank of the meristem.

The initiation of the innermost organs (the carpels) consumes all of the meristemetic cells in the apical dome and only the floral organ primordia (localized region of cell division) are present as the floral bud develops.

Arrangement of the whorls in Arabidopsis:1) First (outermost) whorl = 4 sepals which are green at maturity.2) Second whorl = 4 petals which are white at maturity.3) Third whorl = 6 stamens (2 short + 4 long) = male reproductive structures4) Fourth (innermost) whorl = 1 gynoecium/pistil = female reproductive

structures composed of: an ovary with 2 fused carpels, each containing numerous ovules short style capped with a stigma

In the formation of the whorls starts outside and progresses inward.

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2 major categories of genes regulate floral development

Mutation studies identify of 2 key categories of genes that regulate flower development:

1) Floral meristem identity genes: Encode TF necessary for the initial induction of floral organ identity

genes. They are positive regulators of floral organ identity in the developing

floral meristem. FLO (protein) controls the determination step that establishes floral

meristem identity LFY, FD, SOC1, AP1 are critical genes in the genetic pathway that

must be activated to establish floral meristem identity. 2) Floral organ identity genes:

Directly control floral organ identity. Encoded proteins that are TF that interact with other protein

cofactors to control expression of downstream genes whose products are involved in the formation or function of floral organs.

!!! Floral development involves complex, nonlinear gene networks. In these networks individual genes often play multiple roles (so one gene can be in the 2 categories):ex. APETALA2 first regulates floral meristem identity and then floral organ identity.Floral meristem identity genes regulate meristem function

Floral meristem identity genes must be active for: The immature primordia formed at the flanks of the SAM. The inflorescence meristem becomes floral meristems.

Inflorescence meristem = an apical meristem that is forming floral meristems on its flanks.

floral organ = any of the modified leaves comprising the calyx, corolla, androecium, and gynoecium of a flower.

When plants begin the developmental process known as flowering, the shoot apical meristem is transformed into an inflorescence meristem, which goes on to produce the floral meristem, which produces the sepals, petals, stamens, and carpels of the flower.

The transition from shoot meristem to floral meristem requires floral meristem identity genes, that both specify the floral organs and cause the termination of the production of stem cells. (Floral organ identity genes zijn dan degene die effectief het floral organ uit het floral meristem produceren denk ik)

Homeotic mutation led to the identification of floral organ identity genes

The genes that determine floral organ identity were discovered as floral homeotic mutants.

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Homeotic genes act as major developmental switches that activate the entire genetic program for a particular structure. The expression of homeotic genes thus gives organs their identity.

5 key genes initially were identified in Arabidopsis that specify floral organ identity:

1) APETALA1 (AP1) 2) APETALA2 (AP2) 3) APETALA3 (AP3)

4) PISTILLATA (PI)5) AGAMOUS (AG)

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Role of organ identity genes:Dramatically illustrated in experiments in which 2 or 3 activities are eliminated by loss-of-function mutations:

Quadruple-mutant (ap1 + ap2 + ap3/pi + ag): Floral meristems no longer produce floral organs, but produce green leaflike structures. These leaflike structures are produced with a whorled phyllotaxy typical of normal flowers.

Absence of floral organ identity transforms reproductive structures into leaves. The same happens in combined E-class mutants (SEPALLATA).

Conclusion: The leaves are the 'ground state' of organs produced by shoot meristems.The activity of additional genes such as AP1 and AP2 are required to convert the leaflike 'ground state' organs into petals, sepals, stamens and pistils (stampers).

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14) The ABC model

The 5 floral organ identity genes fall into 3 classes: A, B and C. Defining 3 different kinds of activities encoded by 3 different types of genes:

1) Class A activity: - Encoded by AP1 and AP2- Controls organ identity in whorls 1 and 2- Loss of class A: carpels instead of sepals (whorl 1) and stamens instead of petals (whorl 2)

2) Class B activity: - Encoded by AP3 and PI- Controls organ identity in whorls 2 and 3- Loss of class B: sepals instead of petals (whorl 2) and carpels instead of stamens (whorl 3)

3) Class C activity: - Encoded by AG- Controls organ identity in whorls 3 and 4- Loss of class C: petals instead of stamens (whorl 3) and sepals instead of carpels (whorl 4) In the absence of class C activity whorl 4 is replaced by a new flower. The floral meristem is no longer determinate.

The ABC model= model that postulates organ identity in each whorl is determined by a unique combination of the 3 organ identity gene activities:

Class A alone: sepals Class A + B: petals Class B + C: stamens Class C alone: carpels

The model also proposes that class A and C activities mutually repress each other: both A and C exclude each other from their expression domains in addition to their function in determining organ identity.

A represses C in whorls 1 & 2 and C represses A in whorls 3 & 4.

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But not all observations can be accounted for by ABC genes alone.Expression of ABC genes throughout the plant does not transform vegetative leaves into floral organs. Thus ABC genes are necessary but not sufficient to impose floral organ identity onto a leaf developmental program.

Arabidopsis class E genes are required for the activities of A,B and C genes

Mutations in AGAMOUS-LIKE1 (AGL1), AGAMOUS-LIKE2 (AGL2) and AGAMOUS-LIKE3 (AGL3):

Individual mutants: subtle phenotypes Due to functional redundancy.

agl1/agl2/agl3 triple mutants: only sepal-like structures Renamed: SEPALLATA1-3 (SEP1-3 + 4) and added to the ABC model as class E genes.

sep quadruple mutants: all 4 floral organ types convert into leaflike structures, similar to the ap1 + ap2 + ap3/pi + ag quadruple-mutant.

Class D genes are required for ovule formation

Class D genes = a 3th group of MADS box genes required for ovule formation. Closely related to the class C genes. Since the ovule structure is within the carpel, class D genes are not strictly speaking “organ identity genes”, although they function in much the same way in specifying ovules.

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15) Stomata differentatiation and patterning

There are three main types of epidermal cells: pavement cells, trichomes and guard cells.

Pavement cells are unspecialized epidermal cells and are the default developmental fate of protoderm

Trichomes = extensions of the shoot epidermis Guard cells = pairs of cells that surround the stomata. These regulate gas exchange between

the leaf and the atmosphere (by turgor pressure changes in respons to light)

Leaf has an “tip-to-base developmental gradient”: cell division happens at the base of the leaf, differentiation occurring near the tip.

Each MMC divides asymmetrically (entry division) to give rise to a larger stomatal lineage ground cell (SLGC) and a smaller meristemoid. An SLGC can either differentiate into a pavement cell (3) or become an MMC. The meristemoid will differentiate into a guard mother cell (GMC). The GMC undergoes one symmetrical division, forming a pair of guard cells surrounding a pore. Stomata must be at least one cell length apart to maximize gas exchange. SPCH drives MMC formation and the asymmetric entry division of these cells. MUTE promotes the differentiation of meristemoids into GMC’s. FAMA promotes differentiation of GMCs into guard cells. EXTRA: The meristemoid may undergo additional asymmetric divisions called the ‘amplifying divisions’, giving rise to three SLGCs. Ultimately the meristemoid differentiates into a guard mother cell.

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The receptor ERECTA restricts asymmetric entry division in MMCs, inhibits stomatal development. A receptor-like protein, TOO MANY MOUTHS (TMM) is also required for stomatal pattering. TMM provides specifity for the ERECTA and guides spacing divisions in leaves. The EPIDERMAL PATTERING FACTOR-LIKE (EPFL-family) proteints like EPF1 and EPF2 repress stomatal development. EPF2 is synthesized and secreted by meristemoid mother cells (MMCs) and early meristemoids. EPF1 is synthesized and secreted by GMCs. Together with TMM, the EPF2-ERECTA complex activates an intracellular signalling cascade that represses the production of new meristemoids.

EXTRA: STOMAGEN results in a decrease in stomatal numbers.

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16) Bespreek ontwikkeling van bladprimordia, bladpolariteit, blad rand vorming en blad complexiteit.

All leaves and modified leaves begin as primordia, on the flanks of the Shoot Apical Meristem (SAM). SAM is surrounded by serveral emerging leaf primordia (or in the case of inflorescence meristem, flower primordia). Leaf primordia develop from a group of cells on the flank of the SAM.

Polar auxin transport in the L1 layer of the SAM is essential for leaf primordia emergence. When shoot apices are cultured in the presence of auxin transport inhibitors, they fail to form primordia.

The initiation of leaf primordia is light-dependent, independent of photosynthesis. When grown in the dark, loss of polar PIN1 localization.

Communication between the SAM and the leaf primordium is required for the establishment of leaf adaxial-abaxial polarity (unkown) signal from the SAM is required for specification of adaxial identity.

ARP genes promote adaxial identity and repress the KNOX1 gene

Phan mutants produce leaves with altered adaxial-abaxial symmetry. Auxin and ARP (AS1, RS2 and phan) repress KNOX1.The down-regulation of KNOX genes in leaf primordia, which occurs initially in response to the focused accumulation of auxin at leaf initiation sites, is required for adaxial development and is essential for normal adaxial-abaxial patterning of the leaf in many, but not all, species. Mutations in AS1 alone do not affect abaxial-adaxial polarity.

Developing leaf primordia can be divided into four main zones: boundary meristem, lower-leaf zone, petiole and blade.

The boundary meristem expresses CUC. This regulates cotyledon formation. Lower-leaf zone forms stipules or leaf sheaths by recruiting additional cells. This recruitment

is dependent on WOX and PRS Petiole expresses BOP. This suppresses laminar outgrowth in the petiole region.

EXTRA:

In plants with simple leaves, KNOX1 expression remains off in leaf primordia.

In plants with compound leaves, KNOX1 expression turns back on in primordia

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Adaxial leaf development requires HD-ZIP III transcription factors

Expression of the HD-ZIP III genes (i.e. PHB and PHV) is limited to the adaxial side of the leaf primordia. They promote adaxial identities in tissues where they are expressed. Supression of these genes in the abaxial regions by miRNAs inhibits the epression of his target gene by base pairing with a complementary sequence (miR166 in abaxial regions).Antagonism between KANADI and HDZIP III is a key determinant of adaxial- abaxial leaf polarity

A triple mutant kanadi 1,2 and 3 has radial,adaxialized leaves, showing that KANADIgenes promote abaxial cell fate.

also ARF3 and ARF4 are required for the normal establishment of abaxial fate.

YABBY genes are upregulated by KANADI, ARF3 and ARF4. YABBY promotes also KAN1 and ARF4, forming a positive feedback (not shown in picture). What does YABBY? Well, in the absence of all YABBY gene activity, leaf primordia establish adaxial-abaxial polarity BUT fail in blad outgrowth (=lamina outgrowth). YABBY positively regulate PRS and WOX1 KLU promotes cell division activity.

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ALSO auxin participates in blade outgrowth.

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17) Bespreek de kanalisering van Auxin + geef het experiment waarmee dit is aangetoond.

The leaf vascular system consists of two main conducting tissues: xylem and phloem. The spatial organization of the leaf vascular system = venation pattern. Venation patterns fall into two categories: reticulate venation (eudicots) and parallel venation (monocots).

Veins are organized into size classes: primary, secondary and tertiary. Larger veins function in the bulk transport (of water, minerals, sugars, …). Smaller veins function in phloem loading.

The primary leaf vein is initiated discontinuously from the pre-existing vascular system

The leaf vascular bundles arise from vascular precursor cells called the procambium. The portin of the vascular bundle that enters the leaf is called the leaf trace.

Auxin canalization initiates development of the leaf trace

Auxin stimulates formation of vascular tissues. Experiment: Wound regeneration experiment (removing the leaf and shoot above the wound). Vascular bundle regeneration is prevented! But the vascular bundle can be restored by the application of auxin (above the wound). The upper end of the cut vascular bundle acts as the auxin source and the lower cut end as the auxin sink.

New vasculature usually develops toward, and unites with pre-existing vascular strands, resulting in a connected vascular network. The developing leaf trace acts as an “auxin source” and the existing vascular bundle as an auxin sink. This source-sink model = canalization model

The distribution of PIN1 can be used to predict the direction of auxin flow within a tissue. The canalization of auxin toward the tip of the leaf primordium (P1) in the L1 layer via PIN1 transporters leads to an accumulation of auxin at the tip. Auxin efflux from this region of high auxin concentration toward the older leaf trace directly below it.

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The existing vasculature guides the growth of the leaf trace

Dit is aangetoond door dit experiment. The leaf trace emerging from the existing leaf primordium initial (P0) has connected tot he existing leaf trace of the leaf primordium below it (P3). Hower, if P3 is surgically removed, the leaf trace from P0 connects instead to the vascular bundle of the leaf primordium on the other side (P2).

Primary phloem is the first vascular tissue to form from procambium cells. Primary xylem differentiation lags behind primary phloem.

In general, vein development and pattering progress in the basipetal direction = venation is generally at a more advanced stage at the tip of a developing leaf than is at the base.

Under normal conditions of normal adaxial,abaxial polarity, xylem develops on the adaxial side and phloem on the abaxial side.

Auxin also regulates higher-order vein formation

PIN1 directs auxin to convergence points along the leaf margin (bladrand). These convergence points correspond to locations where water pores (=hydathodes) can develop. As the auxin concentration builds up in these regions, auxin efflux induces PIN1-mediated auxin flow away from the convergence points toward the primary vein, which in turn causes the differentiation of pre-procambium along the path of auxin flow, eventually forming a sec vein.

Localized auxin biosynthesis is critical for higher-order venation patterns.

An additional cause of auxin accumulation on the leaf margin, in addition to canalization by PIN1, is based on localized auxin synthesis. Auxin production at the leaf margins is thought to stimulate the expansion of the lamina. Auxin accumulation is concentrated in the hydathode regions along the leaf margin, where YUCCA genes are expressed Auxin is synthesized by YUCCA proteins and accumulates in the hydathode regions.

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