Physiology of flowering plant Molecular level

Molecular studies on flowering crops Basic knowledge genes, gene expression profile control of gene expression Practical aspect e.g. breeding/improvement

Flowering At shoot apex Induction to Initiation to Specification Vegetative to Reproductive Indeterminate to Determinate Shoot apical meristem to Inflorescence meristem to Floral meristem (primordia)

Flowering Signal Hormone Temperature Photoperiod Autonomous environment/endogenous

Floral stimulus production following inducing signal flowering switch to turn on florigen Site of flowering commitment shoot apex: require sufficient amount of floral stimulus for continuous flower production leaf: commit to continuously production of floral stimulus (irreversible)

Florigen: shoot apex or leaf Impatiens purple flower Short day for flowering 5 SD: flowering SD to LD: continue flowering SD to remove leaves to LD: leaves with purple petals

Florigen: shoot apex or leaf Impatiens red flower Short day for flowering 5 SD: flowering Return to LD: vegetative stage at inner whorls Require continuous supply of inducing signal

Flowering genes expressed in young leaf Maize: intermediate Arabidopsis: constans

Genes in Flower Development Structural gene Flower organ Flower color Flower scent Regulatory gene Protein product involved in controlling expression of other genes Via protein-DNA interaction

Flowering genes Timing Meristem identity Organ identity

Approaches Flowering mutant Gene identification Transformation Mutant complementation

Evolutionary diversification of organisms Alteration of developmental events Variation in structure and regulation of genes controlling developmental mechanism Why flowering?

Flowers : invariant pattern and organization Perianth/Reproductive organs Varied number, size and position Why flowering?

Study model: Arabidopsis and Snapdragon Common characteristics: Floral-specific expression with different roles Identified as homeotic genes Control specification of meristem and organ identity of flower Flowering genes

2 classes: meristem identity genes eg, LFY CAL AP1 organ identity genes eg, AP2 AP3 PI AG Most genes encode proteins with homologous regions of ~ 260 amino acid sequence similarity : common ancestor Flowering genes

Highly conserved regionabout 57 amino acid called MADS box also found in yeast and human Regulatory gene family: transcription factor MADS box gene in other crops: tomato tobacco potato petunia Flowering genes

Homeotic gene :identity of organs/body parts pattern and position Sequence-specific DNA-binding moiety: animal: homeodomain (homeobox gene) plant leaf: homeodomain protein floral organ: MADS box protein / gene

Meristem identity genes Meristem: SAM (indeterminate) for shoot IM (indeterminate) for inflorescence FM (determinate) for flower

Meristem identity genes Inflorescence meristem Mutant: early flowering in Arabidopsis Conversion of IM to FM Terminal flower tfl TFL protein Negative regulator of LFY and AP genes

Meristem identity genes Floral meristem Mutant: partial conversion of FM to IM Leafy in Arabidopsis Floricaula in Snapdragon LFY and FLO protein Positive regulator of AF3 and PI genes

Meristem identity genes Floral meristem Mutant: indeterminate flower within flower (sepal, petal, petal etc) Agamous (AG)in Arabidopsis Plena (PLE)in Snapdragon Protein: 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 genes Mutant:no meristem (strong allele) Reduced number of meristematic cells No effect of root meristem Shoot meristemless, stm stm-5 mutant: 1-2 leaves then terminate leaf primordia consume central zone

Meristem maintenance genes STM protein : Produced throughout development Maintain shoot and floral meristem Inhibit differentiation in central zone Activate 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 inflorescence Loss of indeterminate growth Inhibit inflorescence program Inhibit leaf, lateral shoot development Initiate specific floral organ Activate perianth development Inhibit reproductive organ development

FLIP genes TFL LFY AP1 AP2 TFL: timing of phase transition Tfl mutant: correct sequence of development early bolting early flowering reduced number of inflorescence internode

LFY/AP1/AP2: required in combination rapid and complete transition Mutant: gradual transition from inflo. to flower flower-like lateral shoot leaf in first whorl reproductive organs in outer whorls etc. Late in flower development Reduce FLIP genes, increase gamete genes

Floral Organ Identity Organs with appropriate identity for their positions ABC model 3 classes of genes: A, B and C working individual and in pair A and C inhibit/antagonize each other (no simultaneous functions)

1234 A sepalswhorl 1 A+B petalswhorl 2 B+C stamenswhorl 3 C 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 mutant abnormal in whorl = abnormal in organ = B mutant abnormal in whorl = abnormal in organ = C mutant abnormal in whorl = abnormal in organ =

AP1, SQUA Mutant sepal to leaves and no petal Class = AP2 Mutant sepals to leaves or carpels petals to stamens Class=

AP3, DEF Mutant petals to sepals and stamens to carpels Class= AG, PLE Mutant stamens to petals and carpels to sepals Class=

A-class mutant with different phenotypes Varied from predicted pattern Some floral homeotic genes (MADS box) not follow ABC model: new E-class control 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 genes Transcription factor arabidopsis SEP petunia FBP2 tomato TM5 Mutants: changes in organ identity in 3 inner whorls loss of determinacy

Arabidopsis triple mutant ( sep1 sep2 sep3 ) 4 sepals 6 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 model B Im/E class A and C Other factors sepal petal stamen carpel

Quartet model of floral organ identity interaction between MADS-domain proteins to form DNA binding dimers B-class protein form dimer with each other or with A-class protein C-class protein with E-class protein ternary or quaternary complex B- and C-class protein with A-class and E-class protein

Floral organ identity controlled by 4 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 light phytochrome A responds to red light CONSTANS protein:amount above threshold Light receptors:activated Sunlight:late afternoon time for flowering **hundreds of genes involved to build flower**

Color and Color pattern Flower color: important for pollination Different perception of color red flower – visible to hummingbird -- colorless to bee Changes in petal color : effect on pollinator type Color pattern: differential accumulation of pigment

Color and Color pattern Flower color: Accumulation of flavonoids Major pigments: anthocyanins orange, red and purple Vacuole: site of anthocyanin accumulation Transport as glutathione conjugate

Anthocyanin synthesis pathway Biosynthesis enzymes/genes identified Flower Color

Anthocyanin synthesis pathway regulation at transcriptional level Different colors: different enzyme activities or substrate/precursor availability in different steps Mutations: accumulation of intermediates new color Flower Color

Factors on flower perception co-pigmentation vacuolar pH cell shape Flower Color

Co-pigmentation anthocyanin and flavonols / flavones shift in absorption spectrum differential gene expression: different enzyme activities changes in pigment ratio Flower Color

Vacuolar pH pH increase  blueing seven loci (ph1-ph7) control pH in petunia mutation of the ph loci effect on pH in petal extract but not on anthocyanin composition regulatory genes? Flower Color

Cell shape effect on optical properties conical shape: higher light absorption appear velvet sheen flat shape: faint color Flower Color

Cell-shape controlling gene: mixta homolog of gene for Myb-domain protein proposed function: regulatory gene molecular mechanism: still not known

Color and Color pattern Color pattern cell-specific accumulation of pigments specified by expression pattern of regulatory genes that control anthocyanin-synthesis genes

Color pattern mutant with altered pigment synthesis mutated structural (enzyme) genes mutated regulatory genes Two classes of regulatory genes identified TF with MYB domain TF with bHLH motif

Color pattern Target genes to be regulated specific cis (responsive) elements essential for protein-DNA interaction resulting in transcription activation species-specific sequence spatial / temporal specific sequence

Color and color pattern Many factors still unknown More information leads to applied research Genetic engineered cutflowers with novel color and color pattern

Ornamental crop Improvement Color Fragrance Nectar Shape Vase life Disease resistance

Transformation (cocultivation with Agrobacterium) RoseChrysanthemum CarnationTulip LilyFreesia SnapdragonAnthurium Embryogenic callus Leaf Peduncle PetalStem

Molecular breeding Gene transformation then Selection Flower color Maize dfr to petunia: brick-red petunia Petunia mum gerbera rose chs Cosuppression/Antisense technique Various pattern and color white pale pink cream etc.

Regulatory gene for anthocyanin pathway Maize Lc to petunia: red plant Snapdragon del to gerbera: red leaf and flower scape not in flower Vase life:ethylene Scent: s-linalool synthase (monoterpene)