Developmental Biology Plants Section Flashcards

Front 1

Arabidopsis Genetic Nomenclature

Back 1

•Wild type allele of a gene; uppercase italics –LEAFY or LFY

•Mutant alleles; lower case italics –leafy orlfy

•Plants with mutant phenotype –leafyplants or leafymutants

•Protein product of the gene uses the same name; uppercase plain text –LEAFY or LFY

•Mutant proteins; lowercase plain text –leafy or lfy

•Mutations that produce similar phenotypes often have similar names –leafy1, leafy2 and leafy3.

•LEAFY1, LEAFY2and LEAFY3define three separate genes.

•Mutant alleles of the same gene are designated by hyphenated numbers, for example leafy2-1 andleafy2-2.

Front 2

Embryogenesis

Back 2

Embryogenesis covers development from the time of fertilization until dormancy occurs.

1. Establish the basic body plan.

Radial patterningproduces three tissue systems, and

axial patterningestablishes the apical-basal (shoot-root) axis.

2. Set aside meristematic tissuefor postembryonic elaboration of the body structure (leaves, roots, flowers, etc.).

3. Establish an accessible food reserve for the germinating embryo until it becomes autotrophic

Front 3

Ovule Structure

Back 3

f -funiculus

oi-outer integument

m -micropyle

Front 4

Mature Embryo Sac

Back 4

Note the polarity of each cell, i.e. the position of the cells within the sac and the position of the nucleus and vacuole within each cell.

Front 5

Stages of Arabidopsis embryo development

Back 5

-Egg

-Two cell

-Octant

-Globular

-Triangular

-Heart

-Torpedo

-Mature embryo

Front 6

Fate map: apical-basal axis

Back 6

Eight cell stage: Four anatomically distinct cell types are present: The upper (green) and lower (yellow-green) tiers of the proembryo, the hypophysis (purple) and the suspensor (grey).

Front 7

Fate map: radial axis

-Zygote

Back 7

Front 8

Fate map: radial axis

-Globular embryo

Back 8

Front 9

Fate map: radial axis

-Heart-shaped embryo

Back 9

Front 10

Fate map: radial axis

-Torpedo-stage embryo

Back 10

Front 11

Fate map: radial axis

-Mature seed

Back 11

Front 12

Seed maturation is characterized by:

Back 12

-accumulation of storage compounds

-acquisition of dormancy

-acquisition of desiccation tolerance

Front 13

Monocot

Back 13

-mature seed is largely composed of endosperm with a small embryo

-Storage reserve usually starch

e.g. wheat, maize

Front 14

Dicot

Back 14

-mature seed is largely the embryo with perhaps a remnant layer of endosperm

Storage reserve usually protein (pea) or oil (brassica, sunflower)

Front 15

Seed structure (dicot)

Back 15

Front 16

Endospermic

Back 16

Front 17

Non-endospermic

Back 17

Front 18

Perispermic

Back 18

Front 19

Seed structure (monocot)

Back 19

A = pericarp (fruit wall) fused with testa (seed coat)

B = endosperm

C = scutellum (or cotyledon)

E = coleoptile (i.e. protective sheath of the plumule)

F = plumule (embryonic shoot)

G = radicle (embryonic root)

H = coleorhiza (protective sheath of the radicle)

Front 20

Endosperm function

Back 20

•Required for embryogenesis and seed germination (nurse tissue –placenta)

•Acts as an intermediary between the embryo and surrounding maternal sporophyte.

•Post-fertilization barrier.

Front 21

Endosperm development

Back 21

•Life cycle is confined to the seed stage

•Different modes of development (e.g. nuclear/helobial/cellular-type)

Front 22

Endosperm Development: free-nuclear phase

Back 22

Front 23

Coenocyte

Back 23

-a cell with multiple nuclei in the same cytoplasm

Front 24

Back 24

The triploid endosperm nucleus (en) is located in the basal cytoplasm

of the central cell. A large central vacuole (cv) fills up most of the volume,

surrounded by a thin line of cytoplasm (cy).

The central cell nucleus divides without the formation of cell wall between sister nuclei.

After three rounds of nuclear divisions, eight endosperm nuclei are

located in the basal endosperm coenocyte.

At the end of this phase the complete endosperm coenocyte contains evenly spaced nuclei in the entire cytoplasm.

Front 25

Back 25

The Arabidopsis endosperm coenocyte has nuclei migrating from the

micropylarregion (mp) toward the chalazalend (cz), eventually covering

the entire periphery of the coenocyte.

As development progresses, the endosperm coenocyte

develops three distinct regions: the region surrounding the embryo

(MCE), the central or peripheral endosperm (PEN), and the region of the

chalazalendosperm (CZE), which contains the chalazalcyst (cz).

At the end of the globular embryo stage, the embryo becomes

completely surrounded by MCE cytoplasm.

Front 26

Endosperm Development: cellularization

Back 26

•Initiated by the formation of radial microtubule system on nuclear surfaces.

•Microtubules from neighboring nuclei eventually meet, forming interzonesin which wall material is deposited.

Front 27

Endosperm balance number

Back 27

•Proper balance of maternal (m) and paternal (p) genomes are needed for development.

•Endosperm ploidy ratio:

Cross two diploids -> 2m:1p -> normal endosperm development

Cross two tetraploids -> 4m:2p -> normal endosperm development

Cross tetraploid (female) with diploid (male)

-> 4m:1p -> aborted seed(unbalanced endosperm genome ratio)

Front 28

Genomic imprinting regulates endosperm development

Back 28

•Maternal-and paternal-specific gene expression in the endosperm.

Front 29

Examples of plant hormones

Back 29

-auxin (IAA)

-cytokinins

-gibberellins (GA)

-Abscisic Acid (ABA)

-Ethylene

-Brassinosteroids

-Salicylates

-Strigolactones

-Jasmonates

Front 30

Plant vs. animal hormones

Back 30

Plants Animals

Number of hormones fewer many

Specificity of action non-specific specific Work together yes no

Specific production site no yes

Specific site of action no yes

Fewer plant hormones, each elicits a variety of responses and often works together with other hormones (cross talk).

Front 31

Mode of action

Back 31

Biological response is determined by ….

1.Hormone concentration.

2.Ability of target tissue/cell to recognize the hormone (concentration of receptor).

3.Condition of the plant ( e.g. plant age/developmental stage)

Site of action x developmental stage x concentration of hormone = response

Front 32

Regulation of plant hormone levels

Back 32

Many tightly regulated biochemical pathways contribute to active hormone accumulation. Conjugation can temporarily store a hormone in an inert form, lead to catabolic breakdown, or be the means for producing the active hormone.

Front 33

Hormone Transport

Back 33

Hormones can move:

•through the xylem or phloem (transport tissue)

Xylem: ABA, GA and cytokinins

Phloem: auxin, GA, ABA and cytokinins

Hormones can move:

•across cellular membranes

•through regulated transport proteins

Front 34

Hormone perception

Back 34

-Receptors can be membrane-bound

-Hormone binding initiates an information relay

-Soluble receptors can facilitate interactions between proteins

-Hormones can act like “molecular glue”

Front 35

Hormones and protein ubiquitination

Back 35

The hormones (red) bind to receptors (green), initiating proteolysis of repressors (yellow) to activate a transcriptional regulator (blue)

Front 36

Abscisic Acid (ABA)

Back 36

• A single compound.

• Discovered as a inhibitor of auxin from plant extracts.

• Synthesis occurs in most tissues, especially leaves and seeds.

• ABA synthesis is tightly regulated. Synthesis strongly induced in response to stress.

• Regulate seed maturation and dormancy, desiccation tolerance, stress response and stomatal aperture.

Front 37

ABA in plant development

Back 37

• Growth Inhibitor (e.g. inhibits root growth, cotyledon greening and expansion).

• Promotes seed maturation and dormancy.

• Prevents vivipary (development of the embryo without a dormant period).

Front 38

Gibberellins (GA)

Back 38

• More than 110 different gibberellins are known - abbreviated GA1...GAn.

• No more than 12 or so different gibberellins occur in any one species.

• Only a few of the GA's have biological activity. GA3 is the most active in Arabidopsis.

• Synthesis occurs in young leaves, roots, and developing seeds and fruits.

• Transport occurs through xylem, phloem, or cell-to-cell. Phloem seems to be most important transport route.

• Regulate growth (e.g. leaf expansion and stem elongation), promote seed germination and promote flowering.

Front 39

How do gibberellins regulate growth?

Back 39

The pea mutant le, studied by Mendel, encodes GA3 oxidase, which produces active GA. Loss of function of le reduces active GA levels and makes plants dwarfed.

Semidwarf rice varieties underproduce GA because of a mutation in a the GA20 oxidase biosynthetic gene. “Green revolution” gene.

Front 40

Ethylene

Back 40

• Single compound.

• A gaseous plant hormone.

• Made by all parts of the plant. Meristematic regions (shoot apex) and senescing tissues are rich sources.

• Production is stimulated during physiological stresses (wounding, flooding, drought), senescence and ripening.

• Cell division and elongation; fruit ripening; flowering; senescence (leaf and petal) ; root growth; epinasty (downward bending of leaves); triple response; thigmomorphogenesis (change in growth form in response to touch); promotes germination.

Front 41

Ethylene perception mutants interfere with signalling

Back 41

Mutations that affect ethylene perception and signaling interfere with triple response.

etr mutant fail to undergo triple response in the presence of ethylene

ctr mutant undergoes triple response in the absence of ethylene

Front 42

Crosstalk between hormone signaling pathways

Back 42

Crosstalk (or cross-regulation) occurs when two pathways are not independent. It can be positive and additive or synergistic, or negative.

Crosstalk can affect the synthesis, transport or signaling pathway of another hormone.

Front 43

Seed germination

Back 43

Events initiated upon imbibition of non-dormant seed and leading to radicle protrusion through the seed external envelopes (testa and endosperm).

Front 44

ABA and GA during germination

Back 44

Induction and maintenance of seed dormancy by abscisic acid (ABA) and dormancy release by gibberellin (GA).

ABA mutant - seeds germinate prematurely.

Front 45

Auxin

Back 45

• Five naturally occurring auxins isolated from plants. Indole-3-acetic acid (IAA) is the most abundant.

•Regulate

-Growth

-Phototropism and gravitropism

-Branching

-Embryonic patterning

-Stem cell maintenance

-Organ initiation

Charles Darwin studied the way seedlings bend towards light, a direct effect of auxin action.

Darwin concluded that a signal moves through the plant controlling growth.

Differential cell growth is a result of auxin movement to the shaded side.

Front 46

Polar auxin transport

Back 46

•Auxin is made in actively growing tissue which includes young leaves, fruits, and especially the shoot apex.

•Auxin is transported in a polar manner throughout the plant. Promote the formation of local auxin maxima/gradient.

•Auxin is protonated (IAAH) in the acidic extracellular space (pH 5.5) and enters the cell via diffusion or an influx carrier, AUX1 (blue).

•In the cytoplasm (pH 7), auxin is deprotonated (IAA-) and can only exit the cell via auxin efflux carrier, PINFORMED (PIN; red).

• Asymmetric distribution of the transporters controls polar auxin transport.

Front 47

Auxin regulates plant development

Back 47

-Lateral organ initiation at the shoot apical meristem

-Patterning and vascular development

-Maintain stem cell fate at the root apical meristem

-Embryonic patterning

-Inhibit branching in the shoot

-Promote branching in the root

Front 48

Cytokinin

Back 48

•Family of related adenine-like compounds. Most common form is zeatin which was isolated from corn.

•Synthesized primarily in the meristematic region of the roots. Also produced in developing embryos.

•Root-produced cytokinins are transported acropetally, via xylem, to the shoot tip.

•Regulate cell division, control of leaf senescence, control of nutrient allocation, root nodule development, stem cell maintenance, and regulate auxin action.

Front 49

Cytokinins affect grain production and drought tolerance

Back 49

Rice plants that accumulate more CK can produce more grain per plant because of changes in inflorescence architecture.

Tobacco plants that produce more CK are more drought tolerant because of the delay in leaf senescence conferred by CK.

Front 50

Cytokinin act antagonistically to auxin

Back 50

CK:

Promote stem cell fate at the shoot apical meristem

Promote branching in the shoot

Inhibit branching in the shoot

Promote differentiation at the root apical meristem

Auxin:

Promote lateral organ initiation at the shoot apical meristem

Maintain stem cell fate at the root apical meristem.

Inhibit branching in the shoot

Promote branching in the root

Front 51

Role of cytokinin and auxin in plant regeneration

Back 51

High cytokinin

Low auxin

callus forms shoots

Intermedate levels of

cytokinin + auxin

Undifferentiated callus

No cytokinin

No auxin

Control, no callus

Low cytokinin

High auxin

Callus forms roots

Front 52

Embryogenesis

Back 52

Establish the basic body plan.

Radial patterningproduces three tissue systems, and axial patterningestablishes the apical-basal (shoot-root) axis.

Front 53

Apical-basal pattern formation

Back 53

Zygote expands and divides asymmetrically to produce the apical and basal cells.

Asymmetric division….

-establishes apical-basal polarity of the embryo

-establishes two cell lineages –apical (embryo) and basal (extraembryonicsupport structure, suspensor).

Embryo stages are based on the number of cells in the apical domain only, thus the first zygotic division results in a one-cell stage embryo.

Front 54

Invariant cell division pattern

Back 54

•Asymmetry must occur followed by division in each lineage.

-The apical cell undergoes a series of longitudinal and transverse divisions to generate the eight cell proembryo.

-The basal cell undergoes a series of strictly transverse divisions to generate the suspensor.

Asymmetry must occur to produce to root meristem.

Uppermost cell of the suspensor, hypophysis, becomes part of the embryo.

At the mid-globular stage, the hypophysisundergoes an asymmetric division to yield a smaller lens-shaped cell and a larger basal cell, the precursors for cells within the root meristem.

Front 55

Intrinsic and extrinsic cell fates

Back 55

Asymmetric division produce cells of different shape and size (morphology) and fates.

Two mechanism to produce cells of different fates: intrinsic and extrinsic.

Front 56

YODA (YDA)

Back 56

Required for zygote elongation and asymmetric division

YODA is required for specification of suspensor cell fate.

Front 57

Aberrant division patterns in the basal lineage of yda mutants

Back 57

wild-type embryos: A, C, E, and G

ydamutant embryos: B, D, F, and H

Front 58

YODA is required for specification of suspensor cell fate.

Back 58

A: •Wild type heart stage

•Expression of suspensor specific gene

B: •ydamutant heart stage

•NO expression of suspensor specific gene

Front 59

Pattern formation in yda mutant embryos

Back 59

A-D: Wild type

F-I: YODAmutant

p –protoderm

e –vasculature

l -hypophysis

Front 60

What provides the cue for asymmetry and specification of the first embryonic cell fate decision?

Back 60

Pollen provides patterning cue!

Front 61

SHORT SUSPENSOR (SSP)

Back 61

Required for zygote elongation and asymmetric cell division. Mutation affects development only if its paternally derived.

SSP transcript is made in sperm cells.

SSP protein (green) is detected in developing zygote.

•SHORT SUSPENSOR (SSP)transcripts delivered to the egg by the pollen sperm cell.

•Trigger zygote elongation and the first asymmetric cell division after fertilization.

•Activates YODA (YDA) dependent signaling.

•Establishes apical-basal polarity of the embryo.

Front 62

Does polarity of the ovule/embryo sac determine polarity of the zygote?

Back 62

•Multiple embryos of the same zygote can develop inverted apical-basal axis.

•Uncoupling of apical-basal pattern formation from polarity of ovule.

Front 63

What regulates cell division and cell fate specification in the apical and basal cell lineage?

Back 63

•WUSCHEL-related homeobox(WOX) transcription factors Homeoboxgenes that encode for transcription factors.

•Regulators of cell division and cell fate specification.

WOX2: required for apical cell lineage specification.

WOX8andWOX9: required for basal and apical cell lineage specification.

Front 64

Apical-Basal Pattern Formation

Back 64

Embryo pattern formation require a combination of both intrinsic and extrinsic factors.

Front 65

Monopteros(mp)

Back 65

Defectivein embryonic auxinsignaling, fail to form an embryonic root meristem and therefore yield rootless seedlings.

Front 66

Auxin transport in the developing embryo

Back 66

Green arrows indicate the direction of auxin transport; stippling indicates regions with high auxin levels.

•A flux of auxin is established that first directs auxin towards the apical cell and then switches to direct auxin away from the apical cell towards the embryonic base.

•Polarity of this second flux determines the embryonic shoot–root polarity that remains with the plant for the rest of its life. Basal‘auxin maximum’ in the hypophysisis required for root formation.

Front 67

Model for auxin transport and action in the plant cell.

Back 67

Monopteros (MP) is an auxin response factor (ARF).

ARF = transcription factor

AUX/IAA = transcriptional repressor

SCF-TIR = E3 ubiquitin ligase

Front 68

Auxin plays a pivotal role in establishing apical-basal polarity.

Back 68

•Initially, auxin funnelled toward the developing proembryoby PIN7, which accumulates in the apical membrane of the basal cell and its descendents.

•PIN1, which is expressed in the proembryo, does not show polar distribution at first but becomes preferentially localized to the basal membrane of the provascularcells (the central cells of the lower tier) by the early globular stage, establishing apical–basal auxin flux.

Front 69

Radial pattern

Back 69

The radial pattern is arranged in three concentric layers of tissues: the outer protoderm, the inner mass of ground tissue, and the centrally located procambium.

Front 70

Stereotypical pattern of cell divisions produces each tissue type

Back 70

Protoderm, ground meristem, and procambium are separated as the progenitors of the major tissue types in the embryonic axis by regular periclinal and anticlinal divisions.

•Procambium cells (pc) perform periclinal division to generate the pericycle (p) and vascular (v) primordium (xylem and pholem).

•Asymmetric periclinal divisions divide the ground meristem (gm) into cortex (c) and endoderm (ed).

•Protoderm cells divides anticlinally to increase the number of cells in the epidermis (ep).

Front 71

Radial axis root

Back 71

Front 72

Radial Pattern Formation

Back 72

Depends on the two GRAS family transcription factors SHORTROOT (SHR) and SCARECROW (SCR).

Front 73

SHORT-ROOT (SHR) and SCARECROW (SCR) expression in the radicle (embryonic root)

Back 73

SCARECROW is required for asymmetric cell division

A. SCARECROW promoter driving GFP expression in the Arabidopsis root meristem.

B. SHORTROOT promoter driving GFP expression in the Arabidopsis root meristem.

ep=epidermis, co=cortex, en=endodermis,

CEI=cortex/endodermis initial, QC=quiescent centre, st=stele.

Front 74

SHOORTROOT moves across cell layers

Back 74

SHORTROOT promoter driving expression of a SHORTROOT-GFP fusion protein.

Front 75

SHORTROOT is responsible for specifying endodermis

Back 75

Ectopic expression of the SHORTROOT

Front 76

Endodermal cell specification

Back 76

Signal for endodermal cell specification originates from the stele.

SHOORTROOT (SHR) and SCARECROW (SCR) are required for cell division and endodermal cell specification.

Front 77

knolle Mutants

Back 77

•Cell division is abnormal; cytokinesis is disrupted (arrows).

•Multinucleate cells (asterisks).

•Protoderm (epidermis) formation is disrupted.

knolle mutant seedlings….

..lack a well-formed epidermis layer.

..lack functional meristems.

..die after germination.

Knolle gene mutation disrupt the regular pattern of embryogenesis by altering the rate and plane of cell division.

•Knolle gene is expressed in mitotically dividing cells; protein localizes to the cell plate.

•Knolle is required for cell plate formation; mediates vesicle trafficking and fusion.

Front 78

keule mutants

Back 78

•Mutant keule embryos have large multinucleate cells with gapped or incomplete cell walls.

•Cell division appears to be slowed down, and the planes of cell division are often disoriented.

•Morphology of the protoderm (epidermis) cells is affected.

•keule mutants die after germination.

KNOLLE and KEULE genes interact to promote vesicle fusion during cytokinesis.

Front 79

fass mutant and fate cell specification

Back 79

•Distort cell divisions result in irregular cell patterns.

•Radial and longitudinal patterns of tissue and organs are preserved.•Fass mutants show an inability to form microtubule preprophase bands.

•FASS gene product is required for the accurate positioning of cell walls during division.

Front 80

fass plants

Back 80

In fass mutants the regular sequence of cell divisions is dramatically disturbed.

Disorganisation of normally precise cell division patterns during

embryogenesis does not necessarily perturb cell fate patterning.

Specification of the basic

embryonic pattern is largely, if not entirely, based on positional

cues, rather than on cell lineage.• miniature, every organ present i

n reduced size.

•A. Mutant seedling at 5 day old, showing root, hypocotyl, and shoot.

•B. Mutant plant at 58 day old, showing rosette and flowers.

•D. Mutant flower of a 57 day old plant. Floral parts: se, sepals; p, petals; st, stamen; c, carpels; o, ovules.

Front 81

Primary development: stem

Back 81

The dicot internode after primary development (radial cross-section)

Front 82

Primary development: root

Back 82

Front 83

Primary development: leaf

Back 83

Front 84

Secondary development includes...

Back 84

...Cork and phelloderm from the protoderm

Fasicular cambium (from the provascular tissue) and Interfasicular cambium (from the ground meristem)

-These two things together lead to the vascular cambium, which leads to the secondary xylem, and the secondary phloem, which leads to the cork cambium.

Front 85

Secondary development: shoot

Back 85

The vascular cambium between the primary xylem and phloem remain meristematic and produces secondary xylem towards the inside & secondary phloem towards the outside.

This produces concentric rings of secondary vascular tissues with the vascular cambium between them.

Front 86

Meristems

Back 86

•Meristems are organized cellular structures capable of indeterminate growth.

•Each contains core of undifferentiated “stem” cells which can divide and differentiate to produce adult tissues, while maintaining and regenerating the meristem.

•Meristems are self-organizing,

for example, meristems

regenerate after bisection.

• Meristems activity is often

iterative.

Primary development relies on meristems that were made during embryogenesis.

All postembryonic structures of the plant are generated from these small collections of pluripotent cells.

Front 87

Different types of meristems

Back 87

• Root Apical Meristem (RAM)

• Shoot Apical Meristem (SAM)

• Inflorescence meristem (IF)

• Auxiliary meristem (AM)

• Floral Meristem (FM)

Front 88

Shoot Meristems

Back 88

• Entire shoot system is derived from post-embryonic development in the shoot apical mersitem (SAM).

• Growth occurs by the production of primordia which develop at the meristem periphery.

• Vegetative SAMs are indeterminate, and in some species are capable of growth for thousands of years.

• After a phase of vegetative growth, the shoot apex changes to become an inflorescence meristem, which in turn produces many floral meristems (determinate) that produces flowers.

Front 89

Primordia

Back 89

Growth occurs by the production of primordia which develop at the meristem periphery.

Primordia (singular – primordium) undergo cell division and differentiation to develop into structures such as leaves or into additional meristems.

Front 90

Vegetative development in shoot apical meristems

Back 90

Front 91

Inflorescence development in shoot apical meristems

Back 91

Front 92

Meristems activity is iterative

Back 92

The activity of meristem determine plant architecture.

A vegetative meristem will produce modular units (phytomers) each consisting of a leaf, bud, and internode.

Front 93

Auxillary meristems

Back 93

Vegetative meristem produce modular units (phytomers) consisting of a leaf, bud, and internode.

Auxuillay or lateral buds

• Forms at a node between the leaf primordium and the stem.

• Contains a nest of cells that keep a meristematic character.

Vegetative meristem produce modular units (phytomers) consisting of a leaf, bud, and internode.

• Vegetative auxiliary meristems have the potential to develop into lateral shoots.

• Lateral shoots reiterate the pattern of development in the primary shoot.

• Also have the alternative option of remaining quiescent as an auxiliary bud.

• Outgrowth of auxiliary buds into active shoot is regulated by endogenous and environmental cues.

Front 94

Organization of the shoot apical meristem (SAM)

Back 94

Meristem is organized in to functional zones (a) and layers (b).

central zone – CZ; peripheral zone – PZ; rib zone - RZ

Front 95

Meristem functional zones

Back 95

• Central zone (CZ) - very slowly dividing. Contains a core of stem cells. Allocates cells to

peripheral zone and rib zone

• Peripheral zone (PZ) - slightly

more rapidly dividing. Site of organ initiation (e.g. leaf primordia).

• Rib zone (RZ) - allocates cells to the stem.

Front 96

Meristem functional layers

Back 96

• L1- a single layer of cells that gives rise to the epidermis.

• L2- a single layer that gives rise to ground tissue.

• L3 (corpus cells) - forms the

body of new tissues, including vasculature.

• L1 and L2 together are called the tunica.

Front 97

Maintaining the SAM

Back 97

Meristem must…

•promote cell division to replenish cells lost to differentiation.

•restrict cell division to prevent meristem overgrowth. Plants must maintain this homeostasis throughout their life span, sometimes hundreds of years.

Front 98

Shoot apical meristem mutants

Back 98

Front 99

clavata (clv)

Back 99

Mutants possess enlarged shoot meristems.

• clavata mutants show a loss of

control of meristem cell proliferation.

• Three loci: CLAVATA1, CLAVATA2,

CLAVATA3: three separate genes, all

with similar phenotypes.

Front 100

Model for CLAVATA genes function

Back 100

•CLAVATA1 (CLV1) - receptor kinase protein.

•CLAVATA2 (CLV2) - receptor-like protein partner for CLAVATA1.

•CLAVATA3 (CLV3) -a secreted peptide ligand for the CLV1/CLV2 receptor complex.

•CLV1 and CLV2 (receptor complex) acts with CLV3 (ligand) to control the balance between meristem cell proliferation and differentiation (fate specification).

Front 101

CLAVATA expression domains

Back 101

Front 102

WUSCHEL (wus)

Back 102

• WUSCHEL (wus) mutants fail to form a shoot meristem in the embryo, but adventitious meristems form later.

•“Rescued” meristems continually form and abort.

•WUSCHEL is required to maintain the SAM meristem.

•WUS encodes a homeodomain transcription factor.

•Expression is limited to a small domain of cells deep within the meristem.

•WUS expression is inhibited by the activity of the CLV complex.

•WUS expression is greatly expanded in clv mutants (CLV pathway necessary to restrict WUS expression and cell division)

•WUS expression is downregulated in CLV3 overexpressors

Front 103

Model for CLAVATA genes function

Back 103

•WUS is a target for CLV signal transduction.

•CLV signaling inhibit WUS gene expression.

Front 104

Maintaining the SAM

Back 104

CLV activity restricts size of stem cell population. -loss of CLV, increase SAM size

WUS promotes division of stem cell. -loss of WUS, no SAM

Front 105

Fate of stem cells is regulated by negative feedback.

Back 105

WUS and CLV comprise a regulatory loop, such that WUS promotes CLV3 activity, while the CLV complex negatively regulates WUS.

When meristem gets too small - number of CLV1/ CLV3 expressing cells goes down, WUS is derepressed and activates cell division, increase in SAM.

When meristem gets too big - number of CLV1/CLV3

expressing cells goes up, repressing WUS and repressing cell division, decrease in SAM.

Front 106

Hormonal regulation of SAM activity

Back 106

• Accumulation of auxin triggers organogenesis (differentiation) at the periphery of the SAM.

• Cytokinin (CK) is essential for maintaining meristem activity.

CK deficiency reduces SAM size and activity.

CK stimulates the expression of genes involved in GA catabolism to reinforce the low-GA levels.

Front 107

shoot meristemless (stm)

Back 107

Required for maintenance of undifferentiated cells in Arabidopsis shoot meristems.

STM expression: first observed in a few apical cells at the late globular stage embryo – the future SAM. Expressed throughout the SAM (blue) but not in organ primordia (arrows).

Front 108

Hormonal regulation of SAM activity

Back 108

Shoot Meristemless (STM) controls the balance between the two hormones in the SAM by inducing cytokinin production (high CK), directly inhibiting GA synthesis, and indirectly promoting GA breakdown (low GA).

Front 109

Root Zones

Back 109

Developmental zones of the Arabidopsis root Differentiation zone

•Root hairs begin to develop

Elongation zone

Meristematic zone

• Small cells

• Covered by a root cap

Different types of cell expansion occur in different regions of the root.

Front 110

Root organization

Back 110

Vascular Bundle: 2 protoxylem elements, 2 protophloem elements.

Pericycle: 12 cells on average, source of lateral root Initiation, cells closest to xylem form lateral roots.

Endodermis and cortical cells: each forms a ring of exactly 8 cells.

Epidermis: surrounds the cortical cells. 2 types - those touching two cortical cells will form root hairs, those that touch one will not.

Lateral root cap and columella cells: protects the root as it penetrates soil. Columella cells have amyloplasts—required for gravity response.

Front 111

Root meristem

Back 111

Meristem: Quiescent Center (QC) and Initials

QC

• 4 cells in Arabidopsis

• Surrounded by initials

Initials

• Stem cells that divide to give a rise to root tissue.

• Each cell file originates from an initial cell.

Front 112

Cell division in the root meristem

Back 112

Root tissues are produced from root apical meristems by a highly sterotypical pattern of cell divisions.

• Initials divide to give a new initial and a daughter cell.

• Daughter cells divide further and differentiate.

Quiescent center cells divide infrequently.

Cell division rates increase progressively up the root.

Frequency of observed cell division is represented by different densities of stippling.

Front 113

Quiescent center function

Back 113

Ablation of a single QC cell (arrow) results in cessation of division of the contacting columella initial.

Ablation of a single QC cell (arrowhead) triggers the differentiation of the neighboring columella initial (arrow).

• Inhibit differentiation of neighboring cells so as to maintain the surrounding initial cells as stem cells.

• Differentiation occurs within the range of a single cell of QC.

• Stem cell reservoir- ensures regeneration and persistence of meristematic initial cells if they are lost.

Front 114

When quiescent cell is ablated

Back 114

1)abutting cortical initial cell divides longitudinally, acting like its daughter should.

2)neighboring columella differentiates. Conclusion - QC cells prevent initials from differentiating, not from dividing.

Front 115

Root meristem specification

Back 115

Auxin is required for specification of the root apical meristem.

An auxin maximum in the stem cell niche is required for specification.

Green – auxin accumulation

Front 116

Auxin transport and maxima: embryo

Back 116

•Initially, auxin funnelled toward the developing proembryo by PIN7, which accumulates in the apical membrane of the basal cell and its descendents.

•PIN1, which is expressed in the proembryo, becomes preferentially localized to the basal membrane of the provascular cells (the central cells of the lower tier) by the early globular stage, establishing apical–basal auxin flux.

Front 117

Root meristem specification

Back 117

•Polar auxin-transport concentrates auxin at the basal pole of the embryo (hypophysis), leading to root formation.

•Auxin regulates the expression of genes required to establish the root apical meristem, eg PLETHORA (PTL) genes.

Front 118

Auxin transport and maxima: root

Back 118

Auxin maxima in the root tip

Expression of the auxin reporter DR5::GUS in columella cells, columella initial cells and QC.

Front 119

Root meristem specification

Back 119

Front 120

QC cell-fate identity marker

Back 120

Wuschel-related Homeobox 5 (WOX5)

•Similar to WUS in the shoot apical meristem

•Required for specification and maintenance of QC cells

Front 121

Changes in expression of cell-fate identity marker, WOX5, after QC ablation.

Back 121

J: Expression of WOX5 in 1 to 2 cell layers proximal to the ablated QC.

L: Expression of WOX5 gradually confined to central former provascular cells.

Front 122

Root meristem maintenance

Back 122

Auxin-cytokinin cross-talk controls root meristem size in Arabidopsis.

• Repression of auxin activity by cytokinin enables differentiation of root cells.

• Repression of cytokinin activity by auxin is essential for specifying and maintaining stem cell identity.

Front 123

SAM vs RAM

Back 123

• Number of cells RAM has fewer meristem cells

• Cellular organization In SAM, discrete layers In RAM, cell files

• Location In SAM, meristem is at the apex. In RAM, meristem is found subterminally (root cap is terminal structure).

• Cell lineage In SAM, less strict correlation between meristematic cells and differentiated cell types. In RAM, cell lineage is easily defined.

• Organizing centers RAM quiescent center and SAM o organizing center is necessary to promote the undifferentiated state of neighboring cells (stem cells). RAM quiescent center cells are stable, rarely divides. SAM organizing center cells are replaced continuously. RAM stem cells are a single layer abutting the quiescent center.

Front 124

Auxin and cytokinin in shoot apical meristem (SAM)

Back 124

Auxin: Promote lateral organ initiation at the shoot apical meristem

Cytokinin: Promote stem cell fate at the shoot apical meristem

Front 125

Auxin and cytokinin in root apical meristem (RAM)

Back 125

Auxin: Maintain stem cell fate at the root apical meristem

Cytokinin: Promote differentiation at the root apical meristem

Front 126

Apical Dominance

Back 126

• Outgrowth of auxiliary buds into active shoot is regulated by endogenous and environmental cues.

• Shoot apex inhibits branching (auxiliary bud outgrowth) – Apical Dominance

• Inhibitor of bud outgrowth originates from the shoot apex.

Front 127

Agar block

Back 127

When the upper part of a sheet is removed the lateral buds beneath the cut start to grow.

If the main apex is removed and placed on an agar block, auxin diffuses into the block, which can then inhibit lateral bud growth.

Front 128

Two hormones that regulate branching

Back 128

Auxin and cytokinin.

They're antagonistic, so if one is growing, one is inhibiting.

• Auxin inhibits auxiliary bud outgrowth.

• Root produced cytokinin promote auxiliary bud outgrowth when transported to the shoot.

• To maintain apical dominace shoot produced auxin inhibit the effects of cytokinin from the roots.

Front 129

Positioning of lateral organs on the SAM

Back 129

The vegetative shoot apical meristem give rise to primordia (P) in very regular and predictable temporal and spatial patterns.

Spiral arrangement is characterized by a divergence angle between the organs of 137.5°, the golden angle.

Leaf primordia are numbered P1, P2 etc from youngest to oldest.

The next leaf to form is called the incipient primordium, I1.

Front 130

Phyllotaxis

Back 130

Arrangement of leaves (or other organs) on the stem and in relation to each other.

Front 131

Auxin and organ primordia initiation

Back 131

1: Wildtype Arabidopsis shoot apex

2: pin1 mutant shoot apex – fails to produce lateral organs.

3: Local auxin application (red blob) triggers organogenesis in the pin1 mutant.

Front 132

Local maxima of auxin is required to trigger primordia initiation

Back 132

The expression and orientation of PIN was monitored in the shoot apex of plants expressing PIN1::PIN1-GFP. Leaf initiation is preceded by changes in the distribution of PIN1, contributing to a local auxin maximum.

Front 133

Auxin treatment caused PIN1 upregulation and ectopic primordium induction

Back 133

PIN1 distribution is dynamic during organogenesis.

Green=PIN1 protein.

Red arrows =orientation of direction of auxin flow.

Front 134

Model for auxin transport in the shoot apical meristem

Back 134

A: - AUX1 (black lines) help to concentrate auxin in the L1 surface layer (black arrows).

- PIN1 (gray lines) direct auxin fluxes (gray arrows).

- Creates auxin maxima.

- Induce primordium initiation.

B: After primordium initiation, PIN1 distribution changes, directing auxin flow into the developing veins of the leaf.

Young organ is now an auxin sink.

Front 135

Model for auxin transport and primordia initiation

Back 135

•Polarized localization of PIN1 leads to auxin accumulation primordia initiation (I1).

•Basipteal transport in the primordia (P1) drains auxin into the inner layers, creating a sink, which depletes its surroundings from auxin and prevents the formation of new primordia within its vicinity.

•Another auxin maximum (I1)is created in the PZ away from the developing P1.

•Organ initiation at the shoot apical meristem is determined by auxin distribution and PIN1.

•An auxin maximum is necessary and sufficient to specify the site of primordium formation.

•Developing primordia affect auxin distribution and therefore placement of incipient primordia.

Auxin distribution and redistribution patterns the formation of successive leaves with regular spacing.

Front 136

Auxin and organ primordia initiation

Back 136

Polar auxin transport is required for organogenesis in monocots and dicots and in plant with spiral and alternating patterns of phyllotaxy.

Front 137

Lateral root development

Back 137

Lateral roots originate from pericycle cells of the primary root.

Founder cells in the pericycle (P) undergo transformation and organize into lateral root primordia (LR), which later grow through the endodermis (E), cortex (C) and epidermis (EP) cell layers to emerge from the primary root.

Front 138

Invariant pattern of cell division produces a lateral root primordium (LRP)

Back 138

•Stage I. Founder pericycle (P) cell(s) undergoes a series of anticlinal divisions.

•Stage II. Periclinal divisionproduces two layers, outer layer (OL) and inner layer (IL).

•Stage III-VII. Further anticlinal and periclinal divisions create a dome-shaped primordium.

Front 139

Lateral root organization

Back 139

All the radial pattern elements of the primary root are present in the lateral root primordium.

Front 140

Stages of lateral root development

Back 140

Front 141

Lateral root initiation

Back 141

Lateral roots initiate at regular intervals along the primary root from pericycle cells adjacent to the xylem pole. • Auxin acts as a local morphogenetic trigger to specify lateral root founder cells.

•Accumulation of auxin in xylem pericycle cells is a necessary and sufficient signal to respecify these cells into lateral root founder cells.

Front 142

Auxin and lateral root initiation

Back 142

•Auxin accumulation is a local morphogenic trigger for respecification of pericycle cells into lateral root founder cells.

Lateral root initiation is induced by root curvature.

AUX1-YFP accumulates uniformly in the pericycle cells on the outside of the curve prior to lateral root initiation. (A) 230 min before, (B) 90 min before, (C) 10 min after, and (D) 520 min after the first asymmetric cell division. Left shows a blowup of (A); right, a blowup of (C). White asterisks indicate the pericycle cell files.

Front 143

Auxin and lateral root development

Back 143

• Proper auxin transport is required for lateral root development.

Front 144

Auxin and lateral root development in wild type versus pin protein

Back 144

Front 145

Auxin and lateral root emergence

Back 145

•Lateral root emergence requires separation of overlaying cells.

•Auxin is promotes cell separation.

Radish lateral root primordia treated with auxin. cortical cell layers have been sloughed off leaving only the pericycle surrounding the vascular cylinder.

-Auxin promote cell separation.

Expression of LAX3 (yellow) in cortical and epidermal cells adjacent to emerging lateral root primordia.

LAX3 is an auxin influx carrier.

Front 146

Back 146

(a) Auxin (IAA) originating from dividing pericycle (P) cells induces cell-wallremodelling

(CWR) gene expression in adjacent endodermal (End) cells.

(b) Auxin derived from the developing lateral root primordium also induces

expression of LAX3 in adjacent cortical cells (C).

Front 147

Ethylene in lateral root initation

Back 147

Front 148

Leaf morphology

Back 148

Front 149

Stages of leaf development

Back 149

Stage 1: Organogenesis

a few cells in L1 and L2 divide more rapidly than surrounding cells -> produce outgrowth = leaf primordium, which develops into leaf.

Stage 2: Development of suborgan domains

primordia acquire identity as specific parts of leaf differentiation occurs along three axes:

dorsoventral (adaxial –abaxial)

proximodistal (basal –apical)

lateral (margin –blade –midrib)

Stage 3: Cell and tissue differentiation

tissues and cells differentiate as leaf grows:

L1 -> epidermis with trichomes and guard cells

L2 -> mesophyll cells

L3 -> vascular elements

Front 150

Genetic control of leaf identity

Back 150

•Cells in the meristem are functionally distinct from those in the leaf primordia

•They are indeterminate and serve as a stem-cell population.

•Cells in the primordia are functionally distinct from those in the meristem.

•They become determinate.

Front 151

KNOX-1

Back 151

KNOX-1 genes maintain the meristem in an indeterminate state.

Class I KNOX genes (KNOX-1)

•KNOX means Knotted-like homeobox

•Encode transcription factors

•Expressed in meristem

•Not expressed in leaf primordia

•Help maintain indeterminate growth

STM = SHOOT MERISTEMLESS

KNAT1 = KNOTTED-LIKE ARABIDOPSIS THALIANA 1

KNAT2 = KNOTTED-LIKE ARABIDOPSIS THALIANA 2

KNOX genes, e.g. stm, act in part by stimulating cytokinin synthesis

Front 152

STM

Back 152

KNOX genes, e.g. stm, act in part by stimulating cytokinin synthesis.

-STM is a transcription factor that induces expression of an IPTgene.

-stmmutant can be rescued by CK application or by expression of the cytokinin-biosynthesis IPTgene at the SAM.

Front 153

Overexpression of KNOX-1genes increases leaf complexity and indeterminacy

Back 153

Front 154

ARP genes

Back 154

Primordium-specific genes promote differentiation

•“ARP”is derived from three genes, ASYMMETRIC LEAF1 (AS1),ROUGH SHEATH2 (RS2), and PHANTASTICA

•ARPgenes encode MYB transcription factors

•Expressed in cells of leaf primordiaand cotyledons

•Promote determinate growth and differentiation

Loss of ARPgene function resembles KNOX-1overexpression.Loss of ARPgene function allows KNOX-1genes to remain on in the primordia, leading to an increased indeterminacy.

Lack of AS1 function results in overexpression of KNOX-1genes.

Front 155

The activities of ARP and KNOX-1genes are mutually antagonistic

Back 155

The two classes of transcription factors are mutually repressive, and help establish a separate identity for the emerging leaf primordium.

In the stm mutant, AS1is expressed in the meristem (arrow).

Front 156

Expression of KNOX-1 transcription factors correlates with leaf complexity.

Back 156

Front 157

In plants with compoundleaves, KNOX-1expression turns back on in primordia

Back 157

•KNOX-1genes are repressed at the site of leaf primordium initiation.

•Compound leaves (e.g. tomato) KNOX-1genes turn on again in leaf primordia.

•Simple leaves (e.g. Arabidopsis) KNOX-1genes stay off in the leaf primordia.

Front 158

Control of leaf complexity

Back 158

Cytokinincontributes to compound leaf development.

The effects of KNOX-1 expression can be replicated by increased accumulation of cytokinin.

Front 159

Establishing leaf polarity

Back 159

•Leaf primoridiahave inherent polarity.

•The side that is closer to the meristem is adaxial.

•The side that is farther away from the meristem is abaxial.

Front 160

Incipient primordia

Back 160

Incipient primordia is surgically isolated from the rest of the meristem by a small incision.

The isolated primordium lost polarity (it became entirely abaxialized) and became radially-symmetrical.

Front 161

Laser ablation

Back 161

Laser ablation of only the epidermal cell L1 layer is sufficient for the primordium to lose its adaxial polarity.

•A mobile signal from the meristem moves through the epidermis into the incipient primordium.

•The signal conveys the adaxialpositional information.

•Referred to as the “Sussex signal”. The nature of the signal is not known.

Front 162

Phantastica mutant

Back 162

•The phantastica mutant has radially symmetrical leaves.

•Mutant phan leaves are abaxialized, indicating that PHAN is necessary for adaxialcell fate.

•PHANencodes a transcription factor.

•Three redundant genes, PHABULOSA(PHB),REVOLUTA (REV) andPHAVOLUTA(PHV), function similarly to PHANTASTICA (PHAN).

Front 163

Kanadi mutants

Back 163

Loss of function kanadi mutants have radial, adaxialized leaves.

•A triple mutant kanadi1,2and 3 has radial, adaxialized leaves, showing that KANADI genes promote abaxial cell fate.

•This is the opposite function as PHAN.

•KANADI genes also encode transcription factors.

Front 164

Loss of adaxial or abaxial fate causes radial leaves

Back 164

Front 165

Domain-specific transcription factor expression controls polarity

Back 165

Front 166

microRNA (miRNA)

Back 166

PHBexpression is regulated by a miRNA.

In wild-type plants, miR166 binds to the PHB mRNA and degrades it on the abaxial side of the leaf primordium. Therefore PHB mRNA is only found on the adaxial side.

In phb-1dplants, base changes in the PHB mRNA prevent miR166 from binding to it, allowing it to accumulate throughout the leaf primordium producing a radial, adaxialized leaf.

Front 167

Meristem-derived signal

Back 167

In some cases domain-specific expression is maintained by miRNAs.

Different transcription factors specify adaxial and abaxial domains.

The inner surface of a leaf is specified by a signal derived from the meristem.

Front 168

Control of leaf size and shape

Back 168

Size is determined by growth, shape is determined by differential growth.

Leaf blade growth in dicots, e.g. tobacco.

Leaf expansion stops first at the tip, and later at the base.

The shape of the leaf is caused by more growth at the base than at the tip.

Patterns of cell division correlate with blade expansion.

Cell division arrests first at the tip and later at the base.

Cell division is determined by transcription of a cyclin gene, assayed using a reporter construct.

Darker blue indicates higher levels of gene expression.

Front 169

Leaf margin in dicots

Back 169

1. Smooth(entire). 2.Serrate. 3.Double serrate. 4.Saw-shaped. 5.Toothed. 6.Crenate. 7.Lobed. 8.Parted. 9. Pinnately (like a feather) incised. 10.Palmately (like a hand) incised. 11.Palmately (like a hand) lobbed.

Front 170

Back 170

Cell divisions and expansion contribute to the shape of the blade. Complex leaf shapes (e.g. lobbed or serrate) are formed by persistent growth in isolated regions of the developing blade.

Front 171

Vein Order Designation

Back 171

Primary vein or mid vein are in red and the secondary veins are blue. Higher order tertiary and quaternary veins have not been colored.

Front 172

Control of venation or vascular patterning

Back 172

Auxin is required for proper vein patterning. Inhibition of auxin transport disrupt patterning.

A: Leaf from a plant grown in normal growth medium.

B: Leaf from a plant grown in the presence of a polar auxin transport inhibitor.

Front 173

After a leaf primordium is initiated, auxin moves basipetally and specifies the site of the midvein

Back 173

Picture on the left: Auxin redistributing in a very young primordium –arrows indicate direction of auxin transport

Picture on the left: In an older primordium the future midveinis clearly demarcated. (Inset shows “top down” view)

Front 174

Second-order veins are formed by further auxin redistribution

Back 174

Pattern of PIN1 expression within the leaf predicts the pattern of vascular differentiation.

Auxin transport inhibitors disrupt the pattern and connectivity of the vasculature in

Arabidopsis leaves.

Front 175

The leaf epidermis comprises pavement cells, trichomes and guard cells

Back 175

Front 176

Guard cell structure

Back 176

Guard cells regulate leaf pores through which water vapor and gasses move.

Front 177

Stomatal patterning in Arabidposis

Back 177

Distribution of stomata is not random; they are evenly distributed.

Each stomata is typically surrounded by 3-4 pavement cells.

Front 178

Stomatal patterning in grasses

Back 178

Grass leaves have parallel veins (indicated by black arrows) separated by mesophyll cells. Guard cells form in certain cell files (indicated by red arrows) between the veins.

Front 179

“One cell spacing rule”

Back 179

Although the overall pattern of stomata on the leaf surface is variable among species, Stomata are almost universally patterned according to a one-cell spacing rule, such that at least one intervening epidermal cell separates nearby stomata from one another.

Front 180

Guard cell (GC) development in grasses

Back 180

Guard cells (light blue) are supported by subsidiary cells (yellow) which form from adjacent cell files.

Front 181

Asymmetric and symmetrical cell division

Back 181

Asymmetric division in GC file produces Guard Mother Cell (GMC).

GMC induces division in adjoining cells.

GMC divides to produce guard cells.

Front 182

Guard cell (GC) development in Arabidopsis

Back 182

Both the patterned distribution of stomatal complexes and the differentiation of the guard cells themselves are associated with asymmetric, oriented divisions.

Front 183

Cell division in the stomatal lineage

Back 183

Front 184

Meritsemoid cell

Back 184

Mesritemoid cell (P) behave as stem cell for three or four rounds of division, after which they become a GMC (A).

Meristemoid cell undergo two or three rounds of division in alternating orientations creating a spiral arrangement of 3 cells with an internalized meristemoid cell.

Front 185

Cell division in the stomatal lineage

Back 185

Front 186

SLGC cells

Back 186

Mesritemoid cell (P) divide asymmetrically, with the smaller cell retaining P cell identity.

Stomatal-lineage ground cell (SLGC) (N) cells may also divide, but such divisions are usually symmetric.

SLGC (N) cells may divide asymmetrically, these cells have reacquired mesritemoid cell P identity.

Front 187

basl

Back 187

Asymmetric distribution of BASL precedes asymmetric cell division.

Loss of baslfunction leads to more symmetric cell divisions. How BASL affect division polarity is not yet known.

Front 188

Three genes (basic-helix-loop-helix (bHLH) transcription factors) direct three sequential steps of cell-fate transition during stomatal development

Back 188

Front 189

SPCH = SPEECHLESS

Back 189

SPCH = SPEECHLESS is required for initial asymmetric division.

SPCHis expressed transiently in epidermal cells, which undergo divisions to produce meristemoids.

spch mutations block stomatal lineage initiation.

Front 190

MUTE

Back 190

MUTE acts to terminate meristemoid asymmetric divisions and ensure transition to GMC.

MUTE mutations results inwardly spiraling meristemoid cell divisions with aborted meristemoids in the center.

Overexpression of MUTE produces an epidermis made up of mainly guard cells.

Front 191

FAMA

Back 191

FAMA = many “false mouths” of the Roman goddess of Rumor.

FAMAis expressed in guard

cells.

FAMAcontrols the switch from the GMC to GC identity. Halt division and promote differnetiation.

FAMAmutations result in reiterative symmetric divisions of GMCs, forming abnormal rows of cells (“fake mouths”) but no mature guard cells.

Front 192

Stomatal patterning is determined by inhibition of differentiation

Back 192

A proposed genetic pathway for stomatal patterning and development showing positive and negative regulators

Front 193

TOO MANY MOUTHS (TMM)

Back 193

TOO MANY MOUTHS (TMM) repress asymmetric divisions in cells that are next to guard cells. Required to enforce the one cell spacing rule. TMM is a leucine-rich repeat (LRR) receptor-like protein.

Clusters result from disruption of the number, plane and polarity of asymmetric divisions.

TMM prevents Stomatal-lineage ground cells (N) from dividing to produce a satellite meristemoid (S) adjacent to previously formed meristemoids (M).

Front 194

Mutation in ERECTA-family and YODA

Back 194

Mutation in ERECTA-family(ERf; LRR receptor-like kinases) and YODA(MAP protein kinase) genes result in elevated numbers of stomata, many of which are clustered.

Required to enforce the one cell spacing rule.

Required to maintain both the number and orientation of asymmetric cell divisions.

Front 195

EPIDERMAL PATTERNING FACTOR 1 and EPF2

Back 195

Loss-of-function mutations in two related genes encoding putative ligands,EPIDERMAL PATTERNING FACTOR 1andEPF2also confer defects in stomatal patterning.

EPF1 and EPF2 regulates the orientation and frequency of division in neighboring cells (i.e. inhibits the formation of adjacent satellite meristemoid).

Front 196

Guard cell development is sensitive to exogenous factors

Back 196

Light affects the activity of SPCH and other regulators of GC differentiation.

Stomatal density is also determined by environmental CO2 levels.

Transferring a plant to a higher-CO2 environment causes the newly initiating leaves to produce fewer stomata.

Front 197

Generating stomatal complex diversity

Back 197

No spiral arrangemt suggests a lack of amplifying divisions of a meristemoid.

Surrounded by a spiral arrangement of cells due to a large number of amplifying divisions.

Stomata arise in groups, can be explained by early symmetrical division of the MMC.

Front 198

Trichomes

Back 198

Trichomes are epidermal hairs.

Some trichomesprotect plants by deterring insects and herbivores.

Some trichomes reflect light to reduce UV and heat absorption. Seed trichomes such as cotton fibers aid seed dispersal.

Trichomes can be unicellular or multicellular, branched or unbranched, glandular or non-glandular.

Front 199

Glandular trichomes

Back 199

Glandular trichomes produce chemicals that can have antimicrobial, insecticidal or medicinal properties.

Modifying gene expression in trichomes can increase the production of chemical compounds. The plant in the center makes more of an insecticidal compound (cembratriene-ol(CBT-ol)) and is resistant to aphids.

Front 200

Arabidopsis trichomes

Back 200

•Found on upper or adaxialside of leaves, and on stems but not on hypocotyl and cotyledons. Appear as soon as the leaf primordia begins to develop.

•Trichomeson leaves are branched, stem trichomesare unbranched.

•Spacing between trichomesis regulated (not usually found adjacent to one another).

Front 201

Trichome morphogenesis

Back 201

Stage 1: radial expansion of the trichome precursor cell in the plane of the leaf.

Stage 2: stalk emergence and expansion.

Stage 3: formation of branch structures (primary branching).

Stage 4: branching (secondary branching), expansion of the stalk and branches which have blunt tips. Stage 5: continued expansion of the stalk and branches, which develop pointed tips.

Stage 6: mature trichome with papillate surface; cell walls thicken, accessory cells differentiate around the base.

As trichomes differentiate, they endoreduplicate, causing their ploidy(DNA content) to increase from 2n to 32n. The cell cycle of endoreduplicatingcells skips mitosis.

Number, spacing and shape of trichomesis tightly regulated.

Front 202

Trichome spacing

Back 202

Some mutants under produce trichomes (like gl1).

Some mutants overproduce trichomes (like cpc try).

Front 203

TRIPTYCHON (TRY)

Back 203

TRIPTYCHON (TRY) mutation cause clusters of trichomes.

TRYis an inhibitor of trichome development.

Front 204

Trichome shape

Back 204

GLABRA(GL) and TRANSPARENT TESTA GLABRA1 (TTG1 ) mutants

GL1and TTG1mutation: complete absence of trichomes

GL2 mutation: small trichomes with little to no branching

GL3mutation: reduced number of trichomes that are smaller and have fewer branches.

Front 205

Trichome mutants

Back 205

Front 206

GL1, GL3 and TTG complex regulate trichomecell fate specification

Back 206

•GL1encodes a MYB transcription factor.

•GL3encodes helix-loop-helix protein –transcriptional regulator.

•TTG1 encodes a WD40 domainprotein-transcriptional co-regulator.

•Complex activates the expression of GL2, a homeoboxtranscription factor and TRY, a MYB-like protein.

TRY inhibits the activity of the complex.

GL2 turn on trichomespecific genes.

Front 207

Trichome patterning

Back 207

•All epidermal cells have equivalent developmental potential.

•Each use TRY to inhibit its neighbor –lateral inhibition.

Front 208

Expression of TTG1, GL1 and GL3 increases some cells. Result…

Back 208

-Increase in TRY activity.

-Inhibition of differentiation in adjacent cells.

-trichome development.

Front 209

In Arabidopsis, trichome spacing is controlled by an inhibitory signal

Back 209

A transcription complex activates expression of the trichome inducer GLABRA2 (GL2).....

and a mobile inhibitor moves into adjacent cells to inhibit GL2 expression –lateral inhibition.

GL2expressing shoot epidermal cells adopt a trichomecell fate.

Front 210

Root hair

Back 210

Root hair is an extension of an epidermal cells.

Trichoblasts root epidermal cells that produce a root hair.

Front 211

Root hair patterning

Back 211

•Determined by position relative to neighboring cortical cell.

•Trichoblasts: root hair cell, H position, forms at cortical cell junction.

•Atrichoblasts: non-root hair cell, N position, all the cells between.

Front 212

Back 212

•Three mutants, werewolf (wer), ttg, and gl2, produce root hairs on essentially every root epidermal cell.

•CAPRICE (CPC) mutation reduce the number of root hairs.

Front 213

Back 213

WER, GL3 and TTG complex regulate root hair cell fate specification.

•WERencodes a MYB transcription factor, similar to GL1.

•CPCencodes a protein similar to TRY.

•Complex activates the expression of GL2 andCPC.

CPC inhbit the activity of the complex.

GL2 turns OFF root hair genes.

GL3

Front 214

Back 214

•All epidermal cells have equivalent developmental potential.

•Each use CPC to inhibit their neighbors –lateral inhibition.

•Expression of WER increases in N cells.

•Result….

-Increased CPC activity (N cells).

-CPC inhibit of GL2 expression in adjacent cell (H cell).

-Adjacent cell forms root hair (H cell).

Front 215

In Arabidopsis, root hair spacing is controlled by an inhibitory signal

Back 215

A transcription complex activates expression of the root hair inhibitorGLABRA2 (GL2).....

and a mobile inhibitor moves into adjacent cells to inhibit GL2 expression –lateral inhibition.

GL2expressing root epidermal cells (N position) adopt a non-root-hair cell fate.

Front 216

How does the hair cell form only at the cortical cell junction???

Back 216

Inhibition of GL2 is biased towards the epidermal cell located at the cortical cell junction.

Inhibit GL2 expression –root hair develops.

Look for a mutant where root hairs are randomly formed = SCRAMBLED

Front 217

Scrambled (SCM) mutant

Back 217

N position cells produce root hairs.

Blue = GL2 expression , expressed in N position -NO root hair.

•SCRAMBLED (SCM) is a epidermal cell-localized receptor.

•High levels of an unknown signal produced by the root cortical cells activate SCM.

•SCM negatively regulates WERexpression.

•SCM activity in H cell position bias the outcome of lateral inhibition.

Front 218

Spores, gametophytes, gametes

Back 218

Spores

•Megaspore (female)

•Microspore (male)

Gametophytes

•Embryo sac (female)

•Pollen grain (male)

Gametes

•Egg cell (female)

•Sperm cell (male)

Front 219

Pollen Grain Structure: Germinated Pollen Grain

Back 219

Pollen grain (gametophyte)

Front 220

Pollen Grain Structure: Pollen grain (gametophyte)

Back 220

Front 221

Ovule Structure - Arabidopsis

Back 221

Female reproductive organ (pistil) contains an ovary with two fused carpels (gynoecium). Each carpel contain ovules.

Trt –transmiting tract

Ovu –ovule

Stg-stigma

Sty –style

f -funiculus

oi-outer integument

m -micropyle

O –ovary

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Back 222

cn, central cell nuclei; en, egg cell nucleus; ii, inner integument; n, nucellus; oi, outer integument; sn, synergid nucleus.

Front 223

Ovule Development

Back 223

op –ovule primordium

ov–ovule

fu-funiculus

oip-outer integument primordium

iip –inner integument primordium

nu -nucellus

mp-micropyle

Pt –pollen tube

Front 224

ino mutant

Back 224

Front 225

Auxin (IAA) dependent cell specification

Back 225

Front 226

Pollination and fertilization

Back 226

1. Pollen lands on the stigma, where it hydrates, and germinates.

2. Growing pollen tube penetrates the cuticle and cell wall of papillarcell and grows down the transmitting tract (septum).

3. In the ovary, tube emerges from the transmitting tract , grows along funiculusand enters ovule, where it fertilizes the egg and central cells.

Front 227

Pollination

Back 227

1.Pollen adhesion

2.Pollen coat “foot” formation and pollen hydration

3.Pollen tube emergence (germination)

4.Pollen tube invasion of the papillae cell wall and extension towards the style.

Front 228

Compatible vs. Incompatible Pollination

Back 228

• A genetic “checkpoint” for

the rejection of self pollen.

•Pollen germination only

occurs on a compatible stigma.

• Incompatibility promotes

cross-pollination/outcrossing.

Front 229

Types of Self-incompatibility: Gametophytic incompatibility

Back 229

Front 230

Types of Self-incompatibility: Sporophytic incompatibility

Back 230

Front 231

Protein Ubiquitination

Back 231

Front 232

Pollen Tube Growth

Back 232

Front 233

Double Fertilization

Back 233

Results from the discharge of two sperm cells from the pollen tube into the embryo sac.

1) sperm cell + egg cell ->diploid zygote

2) sperm cell + central cell ->triploid endosperm

Front 234

Double Fertilization Steps

Back 234

Step 1 is the pollen tube reception and discharge phase. Sperm cells are delivered from the pollen tube into the female gametophyte. The receptive synergidcell breakdowns (cell death) just after the start of pollen tube discharge.

Step 2 is the immobility phase. After delivery, sperm cells remain at the boundary region between the egg cell and the central.

Step 3 is the double-fertilization phase. Sperm cells fuse with each target cell.

Front 235

Pollen Tube Reception and Discharge

Back 235

The synergidcell (SC) wall forms a highly thickened structure called the filiform apparatus (FA) at the micropylarend, consisting of finger-like projections that extended into the synergidcell cytoplasm.

Pollen tube (PT) grows through the filiform apparatus (FA), stops growth, and ruptures to release the two sperm cells. The synergidcell (SY) that receives the PT undergoes cell death (DSY). Cell death facilitates the fertilization process.

Front 236

Arabidopsis lyrata x Cardamine flexuosa

Back 236

In crosses with Arabidopsis lyrata, (closely related) and Cardamineflexuosa, (distantly related) pollen tubes that entered a wild-type A. thalianaembryo sac continued to grow inside the female gametophyte -reception failed.

Front 237

ANXUR (ANX)1and ANXUR2

Back 237

ANXUR (ANX)1and ANXUR2

•Male components controlling pollen tube reception

•Expressed within the growing pollen tube

•Mutants are male sterile

•Pollen tube discharges prematurely

Front 238

Gamete Fusion

Back 238

• Generative Cell Specific1

(GCS1/HAP2)

• Wid type ovules following

targeting by wild type (D,E) or

hap2 (F,G) pollen tubes.

• Two sperms are released into the

degenerating synergid but

fertilization does not occur.

Front 239

Pollen Tube Guidance

Back 239

•Pollen-tube growth involves attraction and guidance mechanisms acting along the apical-basal axis andalong lateral axes toward ovules.

•Transmitting tissue provides and

nutrient support and chemical gradients or guidance cues for pollen tube growth.

•Ovule also provides guidance cues.

Front 240

Pollen Tube Guidance: nttmutant

Back 240

•A properly developed tract is required for tube growth.

•no transmitting tract (ntt)abnormal tract.

•Pollen tubes grow slowly and exit the tract early (decrease in apical-basal growth)

Front 241

Pollen Tube Attraction

Back 241

Stigma an style confer competence. Pollen tube that did not go through stigma cannot target ovule. That is pollen tubes acquire the ability from pistil tissue to perceive ovule guidance signals.

Pollen-tube guidance to the ovule is governed by the targeted female gametophyte.

In vitro assay: GFP-tagged pollen tubes make a committed turn (arrows) before entering a ovule and lysing (arrow heads).

Attraction of the pollen tube to the naked embryo sac.

Embryo sac emits a diffusible signal.

Front 242

Back 242

•Embryo sac contains 7 cell types.

•Which cell type provides the guidance cue?

•Most likely candidates are the egg cell and synergid cells located next to the micropyle.

Front 243

Back 243

Science 2001. 293(5534): 1480 - 1483

• Laser ablation of each gametophytic cell in the embryo sac.

• Egg/central cell ablated - attraction not affected.

• Two synergid cells ablated - no pollen tube attraction.

The synergid cells attracts to the growing pollen tube.

Front 244

LURE

Back 244

Pollen tubes grown through a cut style (competent) is attracted to LURE coated bead.

Pollen tubes germinated in vitro(noncompetent pollen tubes) is not attracted to LURE coated bead.

LUREs (LURE1 and LURE2) are expressed specifically in the synergid cell.

•secreted toward the micropylar end of the embryo sac.

•localized in the filiform apparatus (structure at the base the synergid cells.

Cell

Front 245

Block to polytubey

Back 245

Only one pollen tube enters each micropyle.

Front 246

Fertilization recovery mechanism

Back 246

A “block to polytubey” is triggered by gamete fusion.

If defective sperms are deposited, additional pollen tubes are attracted.

The second synergidcell accepts a pollen tube.

Fertilization is recovered.

Front 247

Flower architecture

Back 247

•Arabidopsis flowers are arranged in whorls

-Four concentric whorl of organs

-Regular pattern of number and position

Flowers always have radial or bilateral symmetry

Front 248

Modifications when making a flower

Back 248

Shoot -> Flower

Modifications:

•No leaves below flowers

•Change in phyllotaxy

(whorl instead of spiral)

•Very short internodes

•No branching

•Change in organ identity from leaves to floral organs

•Determinate growth instead of indeterminate growth (organ production stops after carpels are made)

Overall, all a plant does is convert a shoot into a flower, have whorls instead of shoots, and very short internodes.

Front 249

Four class of genes are involved in flower development

Back 249

Flowering time genes: regulate the transition from vegetative (when it is just producing leaves) to reproductive growth (when it produces flowers).

Meristem identity genes: specify and maintain floral meristem identity.

Flower organ identity genes: determine the fate of organ primordia. Now having different organs instead of just primordia

Late-acting genes: control ovule development

Front 250

Regulatory cascade of flower development

Back 250

It's a cascade, one step turns on the first and those turn on the next.

Front 251

Control of flowering time

Back 251

•Multiple signals regulate flowering in Arabidopsis.

•Ultimate result of signals: turn on floral homeotic genes.

SOC1 has to be expressed first, with conditions such as leaf number, temperature, photoperiod, etc.

The outcome is SOC1 gets turned on, and that turns on LFY and AP1, and that causes flower homeotic genes. Once you turn on LFY and AP1,

Front 252

Once you turn on LFY and AP1

Back 252

As plants initiate reproductive development, the vegetative meristem is transformed into an intermediate inflorescence meristem that produces floral meristems on its flanks.

Front 253

Floral meristem

Back 253

•Floral meristem are determinate, produce a defined number of organ primordia.

•Floral organs are:

-initiated sequentially by the floral meristem

-produced as successive whorls (concentric circles)

Once you've initiated floral meristem, you star determining what is going to be sepal, petal, etc.

Front 254

Two distinct classes of genes for floral meristem identity

Back 254

1.Promotes floral meristem identity. Two key regulators LEAFY(LFY) and APETALA1 (AP1). Ensures that primordiainitiated along the periphery of an inflorescence meristem adopt a flower fate.

2.Maintains the identity of inflorescence shoot meristems. Includes TERMINAL FLOWER.

Front 255

leafy (lfy) and apelata1 (ap1) single mutants

Back 255

•Partial conversion of flowers into shoot

•lfymutant: spiral phyllotaxy in flowers (not perfect whorl) and floral organs look like leaves, instead of having petals and primordium.

•ap1mutant: branches are formed in flower and leaves instead of sepals, no petals.

Front 256

Environmental factors such as age of plant, etc turn on LFY and AP1

Back 256

which turn on organ primordoa

Front 257

apetala 1 leafy doublemutant

Back 257

•“Flowers” lack most flower characteristics (still have short internodes).

•Lots of leaves, no floral organs, spiral phyllotaxy (no whorls).

•Indeterminate growth.

Front 258

agamous mutants

Back 258

•agamousmutants show indeterminacy.

•In the flower LEAFY (LFY) and WUSCHEL (WUS)activate AGAMOUS (AG).

•AGAMOUS then turns off WUSCHEL, causing cessation of floral meristem growth.

So if you lose LFY, you lose determinancy, so the flower keeps growing.

Front 259

What genes are involved in specification of particular organs?

Back 259

•Find plants that have organs incorrectly specified for their position (e.g. petals converted to sepals).

•These mutations represent a defect in the ability of cells to correctly asses their position, so they differentiate incorrectly.

Front 260

Mutations in the floral identity genes: 5 genes identifies

Back 260

Each mutant effects two whorls.

The 5 genes are apetala1(ap1), apetala2(ap2), pistillata (pi), apetala 3 (ap3), agamous (ag)

Front 261

The ABC model of flower development

Back 261

•3 classes of genes A, B and C.

•Each class act on two whorls (w). Function combinatoriallyto specify organs.

•A alone = sepals (w1)

•A+B = petals (w2)

•B+C = stamens (w3)

•C alone = carpels(w4)

Front 262

3 classes of genes A, B and C

Back 262

If you lose agamous the expression of A expands into whorls 1 and 2.

•A function: AP2, AP1

•B function: AP3, PI

•C function: AG

•A and C are mutually exclusive.

•C function genes are required for the production of stamens and carpel .

•agamous (ag) = no gametes

ag mutants: AP1/2 is expressed in all four whorls. stamen replaced by petals, carpels replaced by sepals . Indeterminacy –more whorls are formed.

Front 263

A function genes

Back 263

•A function genes are required for the production of sepals and petals.

•apetala2 (ap2), apetala (ap1)

•ap2 mutant: AG is expressed in all four whorls. Sepals are converted to carpels and petals are converted to stamens.

Front 264

B function genes

Back 264

•B function genes are required for the production of petals and stamens.

•apetala3 (ap3), pistillata (pi) mutants: both mutations give the same phenotype.

Petals replaced by sepals, stamens replaced by carpels

Front 265

C function genes

Back 265

•C function genes are required for the production of stamens and carpel .

•agamous (ag) = no gametes

ag mutants: AP1/2 is expressed in all four whorls. stamen replaced by petals, carpels replaced by sepals . Indeterminacy –more whorls are formed

Front 266

MADS box proteins

Back 266

•MADS box proteins are required for floral development but they are not sufficient.

Modifying the ABC model

•Plants lacking all three classes of ABC gene activities formed flowers that had only leaf-like organs (default identity).

•Overexpression of ABC genes, alone or in combination, failed to convert leaves into floral organs.

Front 267

SEPALLATA (SEP) genes

Back 267

•A fourth class, the SEPALLATA (SEP) genes, is also required organ identity.

•SEPALLATA 1-4 (SEP1-4) genes are expressed in all four whorls.

•SEPgenes combination loss of function -produces leaf like organs.

•Overexpression of SEPgenes in combination with ABC genes (AP1, AP3, PI ) lead to transformation of vegetative leaves into floral organs.

Front 268

Floral homeotic genes

Back 268

•All encode MADS box transcription factors.

•Activate organ-specific gene expression in a combinatorial manner.

•Dimerization of MADS box proteins supports the ABCE model.

Front 269

agamous pistillata double mutant

Back 269

•No B and C function

•A function spreads throughout the flower –all sepals

•Indeterminacy due to loss of AGAMOUS

Front 270

apetala2 pistillata double mutant

Back 270

•No A and B function

•C function spreads throughout the flower

•All carpels

Front 271

apetala2 agamous double mutant

Back 271

•No A and C function

•Model does not predict what organs may form when only B function is present.

Front 272

Overexpression of AGAMOUS (AG) in all four whorls

Back 272

•C function in all four whorls.

•Loss of A function in whorl 1 and 2.

•Whorl arrangement is maintained.