Cell and Developmental BiologyModule BS1003

Plant Cell and Developmental BiologyPlant Cell and Developmental Biology

Pat Heslop-HarrisonPat Heslop-Harrison

phh4@le.ac.uk

• http://tinyurl.com/seedsBS1003

Cell and Developmental BiologyModule BS1003

Plant Cell and Developmental BiologyPlant Cell and Developmental Biology

Pat Heslop-HarrisonPat Heslop-Harrison

phh4@le.ac.uk

Practical Friday 25 OctoberLab coats/ruler/pencil ...

• Watch YouTube videos• Reminder how you set up tissue culture:• tinyurl.com/bs1003

• Results from carrot – Agrobacterium infection• tinyurl.com/carrotbs1003

• Or search YouTube for BS1003

Aim: To develop your knowledge & understanding of the cell and developmental biology of plants

Objectives: You should be able to describe….• The role of light in regulating growth and reproduction

• Next lecture: The transition to flowering

• End next week: the mechanisms involved in transferring foreign genes into plant cells

In my lectures, I will try to bring up issues to think about: you will need to read up more in the textbooks (‘flip-teaching’ – wiki).NB: flowering hormones (next lecture) is new since 2009 and you need to look in recent textbooks; wiki is poor on this too!

By the end of this lecture you will:

1. Have thought about active learning and how you are learning at University

2. Know about the information in light3. Understand plant responses to light4. Apply knowledge from Prof Twell’s

lectures to a developmental question

In my lectures, I will try to bring up issues to think about: you will need to read up more in the textbooks (‘flip-teaching’ – wiki).NB: flowering hormones (next lecture) is new since 2009 and you need to look in recent textbooks; wiki is poor on this too!

‘Growth’ and light

CELL EXPANSION

CELL DIVISION

PHH: My use of Powerpoint Slides• In general, I will talk about slides with

illustrations• Slides with more bullet points

– Review what I have said– Remind ME if I have got away from the points I want

to make– Help YOU with notes

• Learning is active – I try to interact – so• Be ready to answer questions or discuss with

your neighbours

Sources of information

• Text books:– Reece et al. 2011 ‘Campbell Biology’ 9th edition– Raven et al. 2009 ‘Biology’ 9th edition– Brooker et al. 2013 ‘Biology’ 3rd edition– Sadava et al. 2013 ‘Life’ 10th edition

Learning from textbooks• Excellent presentation of facts

• How can you make your learning from books active?

Learning from textbooks• How can you make your learning from

books active?• Look at small parts• Look up parts from lectures• Ask yourself questions• Make notes• Design your own ‘exam’ questions

– (often better than reading the given ones)

Learning and reflection

• What have you found difficult so far?

• Scheduling and time planning• Don’t get left behind!• Notes, quizzing yourself• How-to-study books / websites

What have you found difficult so far?

• Scheduling and time planning• Don’t get left behind!• Notes, quizzing yourself• How-to-study books / websites

What have you found difficult so far?

What new skills have you learnt?

• Scheduling and time planning• Don’t get left behind!• Notes, quizzing yourself• How-to-study books / websites

• Learning from sources with more detail than you need

• Learning from multiple sources• Coping with information overload

What have you found difficult so far?

What new skills have you learnt?

Overview – 8 LecturesProf Dave Twell8. Pattern Formation in Plants (Embryogenesis)9. Meristems & Organogenesis10. Chemical Communication Systems in Plants

Prof Pat Heslop-Harrison11. The Role of Light in Plant Development12. The Transition to FloweringDr Trude Schwarzacher13. The Biology of Crown Gall14. Genetic Engineering of Plant Development15. Genes, Genomes & Genomics in Plants

Module Booklet

Lectures on SlideshareTinyurl.com/phhlight

Light important to plants for two reasons

Photosynthesis and Photomorphogenesis

• Photosynthesis - BS1013, Animal and Plant Physiology

• Light in eliciting developmental programmes• Role of phytochrome, a light receptor molecule, in

determining the responses of plants

In the absence of light we see: ETIOLATION

Photomorphogenesis

Etiolation orSkotomorphogene

sis

LONG HYPOCOTYLREDUCED LEAF EXPANSIONAPICAL HOOKPALE IN COLOUR

skoto- & photo-morphogenesis

in potato

DE-ETIOLATION IS DRAMATIC Developmental response to the light signal!

Ecological significance of Etiolation

• TO MAXIMISE THE CHANCES OF REACHING THE LIGHT BEFORE FOOD RESERVES ARE EXHAUSTED

• ALL RESOURCES DIVERTED INTO 'VERTICAL' GROWTH

D + 10 min WW

D

HOW IS LIGHT INVOLVED?

Apical hook unfoldsLeaves expandHypocotyl shows reduced elongation

Only 10 min of white light is sufficient to initiate (or signal) partial de-etiolation

Is photosynthesis involved in the de-etiolation response?

Evidence?DE-ETIOLATION CAN BE INDUCED BY VERY SHORT BURSTS OF LIGHT

ETIOLATED PLANTS DO NOT CONTAIN CHLOROPHYLL AND SO CANNOT ABSORB LIGHT EFFICIENTLY

Are all wavelengths of light effective at inducing de-

etiolation?

• Expt. Test response to specific wavelengths

• Result• Red-light (650-680 nm) alone found to

be sufficient to induce de-etiolation

• Suggested that a red light absorbing photoreceptor existed

LETTUCE SEED GERMINATION

RED RED>FAR-REDDARK

Germination response of lettuce seeds to RED (650-680 nm) and FAR-RED (710-750nm) wavelengths of light

NB: Modern varieties of lettuce are selected NOT to show this response: It is inconvenient for farmers and gardeners!

http://tinyurl.com/lightbs1003

(or search YouTube – BS1003)

LETTUCE SEED GERMINATION

• Response of lettuce seeds to RED and FAR-RED

• LIGHT TREATMENT GERMINATION RESPONSE• R +• FR -• R>FR -• R>FR>R +• R>FR>R>FR -• R>FR>R>FR>R +

Germination is promoted by RedFar-red reverses the responseResponse depends on the last light treatment Can operate over multiple cycles > SWITCH

• HOW DO PLANTS DETECT LIGHT?

• ABSORBING IT VIA PHOTORECEPTOR MOLECULES!

PLANTS MUST BE ABLE TO DETECT

BOTH RED AND FAR-RED LIGHT INDEPENDENTLY

Chlorophyll absorption spectrum

selective

• Chlorophyll A absorption peaks at 432 nm and 663 nm

• Chlorophyll B absorption peaks at 453 nm and 643 nm

BLUE RED

• Following the discovery of the effects of red and far red light……….

• the search was on for photoreceptors that absorb at those wavelengths.

• 1964: A COMPOUND THAT SHOWED DIFFERENT ABSORPTION CHARACTERISTICS IN RED AND FAR-RED LIGHT WAS PURIFIED FROM ETIOLATED OAT (Avena sativa) SEEDLINGS

• PHYTOCHROME

H. William Siegelman & Firer USDA > 1964Richard Vierstra & Peter Quail > 1983

Pr Pfrred

far red

Phytochromes are reversibly photochromic

Phytochrome absorption spectrum

FAR-RED

Ab

sorb

an

ce

Wavelength (nm)

666

730

MODEL

PR PFRRED >>>>BIOLOGICAL ACTIVITY

• Phytochrome– Red- and far-red-light receptor– Flips back and forth between 2

conformations

– Pfr – conformation that only absorbs far-red light and activates cellular responses

– When left in the dark, Pfr transforms to red light absorbing Pr

• Pr can only absorb red light and cannot activate cellular responses

– Lettuce seed germination experiments

THE PHYTOCHROME MOLECULE

PROTEIN (124kd) + (CHROMOPHORE) via Thioether linkage

TWO IDENTICAL MONOMERS MAKE THE PHYTOCHROME DIMER

ONE CHROMOPHORE TETRAPYRROLE PER MONOMER

CHROMOPHORE CHANGES CONFORMATION UPON ILLUMINATION (cis-trans isomerization at Carbon15)

C15

Slow (some plants)

PHYTOCHROME MODE OF ACTION

http://tinyurl.com/lightbs1003

WHERE IS PHYTOCHROME LOCALISED?

• APICAL REGIONS of the root and epicotyl, where most of the dramatic developmental changes occur

Subcellular localisation of Phytochrome?

• IMMUNOLOCALIZATION: In etiolated seedlings, DIFFUSE

• On illumination by red light (i.e. conversion to the active Pfr) LOCALISED to multiple sites within the cell >> nucleus!!!

• ASSOCIATION WITH INTRACELLULAR RECEPTOR MOLECULES?

• Phytochome interacting protein (PIF)

RED LIGHT

DARK

Shading responses

• Mediated by phytochrome • Responses include the extension of leaves

from shady portions of a dense tree canopy into the light, and growth that allows plants to avoid being shaded by neighboring plants

• Occur by the elongation of branch internodes

• Leaves detect shade as an increased proportion of far-red light to red light

Phytochrome regulates growth & development through gene

activation

• CAB = CHLOROPHYLL A/B BINDING PROTEIN

• RUBISCO = (RIBULOSE 1,5-BIS PHOSPHATE CARBOXYLASE-OXYGENASE)

RED PR PFR >>>> BIOLOGICAL ACTIVITY

FAR-RED

MODEL

Gene activation

A model of phytochrome regulation of rbcS & cab genes

Pr Pfrred

far red

Nucleus

Pfr

PfrPIF

Cell surface

Cytoplasm

Chloroplast

CAB

RBCS

Photoperiodism

• Phytochromes play a critical role • Influences the timing of dormancy

and flowering.• Flowering plants can be classified as

long-day, short-day, or day-neutral plants according to the way their flowering responds to night length

• Plants measure night length

• Long-day plants – flower in spring or early summer, when the night period is shorter (and thus the day length is longer) than a defined period

• Short-day plants – flower only when the night length is longer than a defined period such as in late summer, autumn or winter, when days are short

• Day-neutral plants – flower regardless of the night length, as long as day length meets the minimal requirements for plant growth

Rafflesia arnoldii

THE TRANSITION TO FLOWERING

1 meter11 kg

Light and other environmental factors influence not only vegetative aspects of higher plant development, contributing to the plant's overall shape, but also the transition to reproductive development, i.e. flowering. In particular we consider interactions of three factors, namely plant age, light (especially day length) and temperature in determining the transition to flowering

VEGETATIVE VERSUS REPRODUCTIVE GROWTH

Flower development involves a dramatic change in the STRUCTURE and ACTIVITY of

the SHOOT APEX

Vegetative meristem

Leafprimordia

Inflorescence meristem

Flowerprimordia

Floral meristem

Floral organprimordia

Apical meristem transformations

• Shoot apical meristem

Inflorescence & floral meristems

• VEGETATIVE SHOOT APEX - simple structure

• 1. LEAF PRIMORDIA EMERGE IN A SPIRAL ARRANGEMENT (PHYLLOTAXY)

• 2. REPETITIVE• 3. INDETERMINATE

SUMMARY

FLORAL APEX - more complex1. SHOOT STOPS ELONGATION GROWTH2. INITIATES MULTIPLE FLORAL ORGANS3. NON-REPETITIVE4. DETERMINATE

Development of a single flower bud of Arabidopsis

Coordinated growth of different organs

P

P

C

PLANT AGE LIGHTTEMPERATURE

FACTORS THAT INFLUENCE FLOWERING

Flowering Signals

• 1. PLANT AGE - JUVENILE TO ADULT FORM

• “RIPENESS-TO-FLOWER”

• eg. Tobacco will only flower after 15-20 nodes

• eg. Many tree species flower only after >10 years

Development of competence to flower

• ENDOGENOUS TIMING MECHANISM?• DIFFUSIBLE FACTORS?• TEST IN GRAFTING EXPERIMENTS • Eg.MANGO

juvenile mature

If the juvenile shoots, which normally fail to flower, are grafted on to a mature plant, they will flower

• Two GENERAL CHARACTERISTICS that could be required for the ability to flower:

• THE CHRONOLOGICAL AGE OF THE PLANT

• THE LARGER SIZE OF THE PLANT

Century plant(Agave americana)

Botanic GardensUniversity of Leicester

LATE FLOWERING MUTANTS of Arabidopsis

Both OLD and LARGEBut still flower late

Genetic Control

2. LIGHT: PHOTOPERIOD

• SECOND MAJOR FACTOR• INFLUENCING THE • 'DECISION' TO FLOWER • IS LIGHT (DAYLENGTH)

• 1. LONG DAY PLANTS

• 2. SHORT DAY PLANTS

• 3. DAY-NEUTRAL PLANTS • eg. tobacco, tomato, sunflower• dandelions, cucumbers, roses,

snapdragons, carnations, cotton

LDP

SDP

Day Neutral

FloweringResponse

Photoperiod(h)

CDL = Critical Daylength

Poinsettia

KalanchoeSHORT DAY PLANTCoffea arabica SoybeanStrawberryChrysanthemum Christmas cactusDahliasLate summer/autumn

LONG DAY PLANTSWheat/SpinachLettuce/RadishBeet/CloverGladiolus/IrisArabidopsisLate spring/Summer

SHORT DAYS (<8h)

LONG DAYS (>12h)

WHY USE DAYLENGTH OR OTHER ENVIRONMENTAL SIGNAL?

• PROVIDES A MEANS OF SYNCHRONISING GROWTH AND REPRODUCTION

• - WITH EACH OTHER• - WITH THE ENVIRONMENT

Harry Allard photoperiod experiments

LIG H T T R E A T M E N T F L O W E RIN G R E S P O N S E

S D P L D P

Relationship between photoperiod and flowering response

Length of the DARK PERIOD determines the flowering responseIn both SDP & LDP

VegetativeFlower

Vegetative Flower

Vegetative Flower

Vegetative Flower

Promotes flowering in LDP

Day break no effect

Night break inhibits flowering in SDP

HOW DO PLANTS DETECT THE LENGTH OF DARKNESS?

• RED/FAR RED REVERSIBILITY OF THE PHOTOPERIODIC RESPONSE

• MODELS:• SD PLANTS - REQUIRE LONG NIGHTS • - PFR IS DEGRADED TO PR• - PFR INHIBITS FLOWERING • - LOW PFR SIGNALS FLOWERING

• RED LIGHT NIGHT BREAK PREVENTS FLOWERING BY CONVERTING PR TO PFR - inhibitor

RED PR PFR >>>> BIOLOGICAL ACTIVITY

INHIBIT FLOWERINGFAR-RED/DARK

LONG DAY PLANTS > REQUIRE SHORT NIGHTS

• PFR PROMOTES FLOWERING• INSUFFICIENT DEGRADATION OF PFR TO

PR

• RED LIGHT BREAK IN A LONG DARK PERIOD INDUCES FLOWERING BY PREVENTING DEGRADATION OF PFR TO PR RED PR PFR >>>> BIOLOGICAL ACTIVITY

PROMOTE FLOWERINGFAR-RED/DARK

• BUT DAYLENGTH CANNOT BE USED TO DISTINGUISH BETWEEN AUTUMN & SPRING

Both have short nights, but very different outcomes!

3. TEMPERATURE

• SOME PLANTS FLOWER MORE RAPIDLY WHEN SEEDLINGS ARE GIVEN A COLD TREATMENT:

• The promotion of flowering by cold is known as

• VERNALIZATION

• EFFECTIVE TEMPERATURE -2 to +120C• Eg. Autumn sown, Winter wheat/Winter rye• Long term Winter ‘memory’ winter > summer

(~200 days)• Many biennials > rosette form over winter >

flower spring/early summer

Vernalization• Cabbage (biennial) • Requires exposure to the

environmental cue of prolonged winter cold to flower the second spring after planting.

Cabbage grown in the greenhouse for 5 years without vernalization.

WHAT ABOUT INTERNAL (CELLULAR) PHOTOPERIOD SIGNALLING

MECHANISMS?

• APPROPRIATE LIGHT IS DETECTED, AND THE SIGNAL TRANSDUCED INTO A RESPONSE AT THE SHOOT APEX

• LEAF (not the apical meristem) IS THE SITE OF DETECTION OF PHOTOPERIOD

EVIDENCE?

• ‘BAGGING’

• GRAFTING

•

Cocklebur (Xanthium)

SDP

BAGGING EXPERIMENTS (Cocklebur= SDP)

Bagging of apical leaf on plant grown in LONG DAYS (un-induced) leads to flowering

Signal

LD LD

GRAFTING EXPERIMENTS (Cocklebur = SDP)

• graft SD-induced leaf onto LD-uninduced stock

induces flowering

repeat cell memory Signal moves

leaf to apex

LD

SD

LD LD

LD LD

• FLOWERING SIGNAL MUST TRAVEL FROM LEAF TO THE SHOOT APEX?

• MICHAEL CHAILAKHYAN (1930) POSTULATED A CHEMICAL SIGNAL

OR FLOWERING HORMONE? FLORIGEN• 2007: FT-protein is +/- florigen (George Coupland)• mRNA and protein made in leaf phloem companion cells in

response to light perception• Protein travels to shoot apical meristem• In SAM, FT protein combines with another protein and acts as

transcription factor for flower induction genes

• (NB: Big change: need post-2010 textbook; Wikipedia is poor!)

• http://faculty.washington.edu/takato/i.html• http://www.mpipz.mpg.de/25240/coupland_2008_01

_31_part1.pdf• And part 2• And

www2.ju.edu.jo/sites/Academic/tamimi/Material/Fflower.ppt&ei=qPNnUoflDbSg0wWzjYHQCA&usg=AFQjCNGb1dG34gBOdILat7ZfRIcSQQrpag&sig2=Icn04C7ZjwyC0lDjAhbmbg&bvm=bv.55123115,d.d2khttps://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=7&cad=rja&ved=0CGEQFjAG&url=http%3A%2F%2Fwww2.ju.edu.jo%2Fsites%2FAcademic%2Ftamimi%2FMaterial%2Fflower.ppt&ei=qPNnUoflDbSg0wWzjYHQCA&usg=AFQjCNGb1dG34gBOdILat7ZfRIcSQQrpag&sig2=Icn04C7ZjwyC0lDjAhbmbg&bvm=bv.55123115,d.d2k