Physiology of Flowering Plant Molecular Level

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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

Why flowering?

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

Flowering genes
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 region about 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

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:
Mutant:

required in combination rapid and complete transition 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)

B A 1 2 3 C 4

A A+B B+C C

sepals petals stamens carpel and determinacy

whorl 1 whorl 2 whorl 3 whorl 4

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 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

Flower Color

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) Rose Chrysanthemum Carnation Tulip Lily Freesia Snapdragon Anthurium Embryogenic callus Leaf Peduncle Petal Stem

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: Scent: ethylene s-linalool synthase (monoterpene)

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