Physiology of flowering plantMolecular level

Molecular studies on flowering cropsBasic knowledge

genes, gene expression profilecontrol of gene expression

Practical aspect e.g. breeding/improvement

FloweringAt shoot apex

Induction to Initiation to SpecificationVegetative to Reproductive

Indeterminate to DeterminateShoot apical meristem toInflorescence meristem toFloral meristem (primordia)

Flowering SignalHormoneTemperaturePhotoperiod Autonomous

environment/endogenous

Floral stimulus productionfollowing inducing signalflowering switch to turn onflorigen

Site of flowering commitmentshoot apex: require sufficient amount of

floral stimulus for continuous flower productionleaf: commit to continuously production of

floral stimulus (irreversible)

Florigen: shoot apex or leaf

Impatiens purple flowerShort day for flowering

5 SD: floweringSD to LD: continue flowering

SD to remove leaves to LD:leaves with purple petals

Florigen: shoot apex or leaf

Impatiens red flowerShort day for flowering

5 SD: floweringReturn to LD: vegetative stage at inner whorls

Require continuous supply of inducing signal

Flowering genes expressed in young leaf

Maize: intermediateArabidopsis: constans

Genes in Flower DevelopmentStructural gene

Flower organFlower colorFlower scent

Regulatory geneProtein product involved in controlling

expression of other genesVia protein-DNA interaction

Flowering genesTimingMeristem identityOrgan identity

ApproachesFlowering mutantGene identificationTransformationMutant complementation

Evolutionary diversification of organismsAlteration of developmental events

Variation in structure and regulation of genes controlling developmental mechanism

Why flowering?

Flowers: invariant pattern and organizationPerianth/Reproductive organs

Varied number, size and position

Why flowering?

Study model: Arabidopsis and SnapdragonCommon characteristics:

Floral-specific expression with different roles Identified as homeotic genesControl specification of meristem

and organ identity of flower

Flowering genes

2 classes: meristem identity geneseg, LFY CAL AP1

organ identity geneseg, AP2 AP3 PI AG

Most genes encode proteins with homologous regions of ~ 260 amino acid

sequence similarity : common ancestor

Flowering genes

Highly conserved region about 57 amino acidcalled MADS boxalso found in yeast and human

Regulatory gene family: transcription factorMADS box gene in other crops:

tomato tobacco potato petunia

Flowering genes

Homeotic gene: identity of organs/body partspattern and position

Sequence-specific DNA-binding moiety:animal: homeodomain (homeobox gene)plant leaf: homeodomain proteinfloral organ: MADS box protein / gene

Meristem identity genes

Meristem: SAM (indeterminate) for shootIM (indeterminate) for inflorescence

FM (determinate) for flower

Meristem identity genes

Inflorescence meristemMutant: early flowering in ArabidopsisConversion of IM to FMTerminal flowertflTFL proteinNegative regulator of LFY and AP genes

Meristem identity genesFloral meristem

Mutant: partial conversion of FM to IMLeafy in ArabidopsisFloricaula in SnapdragonLFY and FLO proteinPositive regulator of AF3 and PI genes

Meristem identity genesFloral meristem

Mutant: indeterminate flower within flower(sepal, petal, petal etc)

Agamous (AG) in ArabidopsisPlena (PLE) in SnapdragonProtein: putative transcription factor

Meristem maintenance genes

Meristem: - small, dense, large nuclei- to supply new cells- undifferentiated cells (central)- daughter cells with specific developmental fates (subdistal)

Meristem maintenance genesMutant: no meristem (strong allele)

Reduced number of meristematic cellsNo effect of root meristem

Shoot meristemless, stmstm-5 mutant: 1-2 leaves then terminate

leaf primordia consume central zone

Meristem maintenance genesSTM protein:

Produced throughout developmentMaintain shoot and floral meristem Inhibit differentiation in central zoneActivate cell division/proliferation

Floral Initiation Process (FLIP)Arabidopsis structural development

- rosette leaves with compact internode- elongated internode with cauline leaves

and lateral inflorescence (bolting) - nodes without leaves and flowers

Floral Initiation Process (FLIP)

Transition from early to late inflorescenceLoss of indeterminate growthInhibit inflorescence program

Inhibit leaf, lateral shoot developmentInitiate specific floral organ

Activate perianth developmentInhibit reproductive organ development

FLIP genesTFL LFY AP1 AP2

TFL: timing of phase transitionTfl mutant: correct sequence of development

early boltingearly floweringreduced number of inflorescence internode

LFY/AP1/AP2: required in combinationrapid and complete transition

Mutant: gradual transition from inflo. to flowerflower-like lateral shootleaf in first whorlreproductive organs in outer whorlsetc.

Late in flower developmentReduce FLIP genes, increase gamete genes

Floral Organ IdentityOrgans with appropriate identity for their positions

ABC model3 classes of genes: A, B and Cworking individual and in pairA and C inhibit/antagonize each other

(no simultaneous functions)

1 2 3 4

A sepals whorl 1A+B petals whorl 2B+C stamens whorl 3C carpel and determinacy whorl 4

A C

B

ABC model: Developed from floral homeotic mutants

of Arabidopsis and Antirrhinum(flowers with abnormal organ pattern)

Genes identified: MADS-box family(transcription factor with conserved domain) Also work well in petunia, tomato and maize

A mutantabnormal in whorl =abnormal in organ =

B mutant abnormal in whorl = abnormal in organ =

C mutant abnormal in whorl = abnormal in organ =

AP1, SQUAMutant

sepal to leaves and no petalClass =

AP2Mutant

sepals to leaves or carpelspetals to stamens

Class=

AP3, DEFMutant

petals to sepals and stamens to carpelsClass=

AG, PLEMutant

stamens to petals and carpels to sepalsClass=

A-class mutant with different phenotypesVaried from predicted pattern

Some floral homeotic genes (MADS box)not follow ABC model: new E-classcontrol 3 inner whorls and determinacy

ABC model necessary but not sufficient- ** **D class for ovule identity

E-class or Identity mediating factors

Im genes: MADS box genesTranscription factor

arabidopsis SEPpetunia FBP2tomato TM5Mutants: changes in organ identity

in 3 inner whorlsloss of determinacy

Arabidopsis triple mutant (sep1 sep2 sep3)4 sepals 4 sepals6 sepals new mutant flower

petunia FBP2: functional equivalent to SEP protein(complementation of sep mutant)

E-class essential for function of B and C class

Revised ABC modelBIm/E classA and COther factors sepal petal stamen carpel

Quartet model of floral organ identityinteraction between MADS-domain proteins

to form DNA binding dimersB-class protein form dimer with each other

or with A-class proteinC-class protein with E-class protein

ternary or quaternary complexB- and C-class protein with A-class and E-class protein

Floral organ identity controlled by4 different combinations of

4 floral homeotic proteins

e.g. Arabidopsis whorl 1: A- class AP1 homodimer whorl 2: A- class AP1, B- class AP3 and PI, E- class SEP whorl 3: B- class AP3 and PI, C- class AG, E- class SEP whorl 4: C- class AG, E- class SEP heterodimer

Blooming gene When to flower

winter spring summer too early: no pollinating insect too late: not enough time to make seed (winter)

one gene: CONSTANS in Arabidopsis control flowering time

CONSTANS protein helps measure day length

Quality of light perceived by 2 light receptors

cryptochrome 2 responds to blue lightph ytochrome A responds to red light

CONSTANS protein: amount above threshold Light receptors: activated

Sunlight: late afternoontime for flowering

** **hundreds of genes involved to build f lower

Color and Color patternFlower color: important for pollinationDifferent perception of color

red flower – visible to hummingbird-- colorless to bee

Changes in petal color : effect on pollinator typeColor pattern: differential accumulation of pigment

Color and Color patternFlower color:

Accumulation of flavonoidsMajor pigments: anthocyanins

orange, red and purpleVacuole: site of anthocyanin accumulationTransport as glutathione conjugate

Anthocyanin synthesis pathway

Biosynthesisenzymes/genes identified

Flower Color

Anthocyanin synthesis pathwayregulation at transcriptional level

Different colors: different enzyme activities or substrate/precursor availability in different steps

Mutations: accumulation of intermediatesnew color

Flower Color

Factors on flower perceptionco-pigmentationvacuolar pHcell shape

Flower Color

Co-pigmentationanthocyanin and flavonols / flavonesshift in absorption spectrum

differential gene expression:different enzyme activitieschanges in pigment ratio

Flower Color

Vacuolar pHpH increase blueingseven loci (ph1-ph7) control pH in petuniamutation of the ph loci

effect on pH in petal extractbut not on anthocyanin compositionregulatory genes?

Flower Color

Cell shapeeffect on optical propertiesconical shape: higher light absorption

appear velvet sheenflat shape: faint color

Flower Color

Flower Color

Cell-shape controlling gene: mixtahomolog of gene for Myb-domain proteinproposed function: regulatory genemolecular mechanism: still not known

Color and Color pattern

Color patterncell-specific accumulation of pigmentsspecified by expression pattern of

regulatory genes that control anthocyanin-synthesis genes

Color patternmutant with altered pigment synthesis

mutated structural (enzyme) genesmutated regulatory genes

Two classes of regulatory genes identifiedTF with MYB domainTF with bHLH motif

Color patternTarget genes to be regulated

specific cis (responsive) elements essential for protein-DNA interactionresulting in transcription activation

species-specific sequencespatial / temporal specific sequence

Color and color patternMany factors still unknownMore information leads to applied researchGenetic engineered cutflowers

with novel color and color pattern

Ornamental crop ImprovementColor

FragranceNectarShape

Vase lifeDisease resistance

Transformation (cocultivation with Agrobacterium)Rose ChrysanthemumCarnation TulipLily FreesiaSnapdragon Anthurium

Embryogenic callusLeaf Peduncle PetalStem

Molecular breedingGene transformation then Selection

Flower colorMaize dfr to petunia: brick-red petuniaPetunia mum gerbera rose chs

Cosuppression/Antisense techniqueVarious pattern and color

white pale pink cream etc.

Regulatory gene for anthocyanin pathwayMaize Lc to petunia: red plantSnapdragon del to gerbera:

red leaf and flower scape not in flower

Vase life: ethylene

Scent: s-linalool synthase (monoterpene)