Advanced plant physiology

Photomorphogenesis Light mediated photoresponse processes. * Photosynthesis --- a biochemical process * Photoperiodic control of reproductive behavior

* Nonphotosynthetic photoreceptor ---- phytochrome ---- Protochlorophyll, the synthesis of chlorophyll and development of photosynthetic apparatus.

Phytochrome---as photoreceptor, known for long time. lettuce seed germination of certain variety are sensitive to light, (the grand rapid variety) Did absorbance test of spectro,

Phytochorme --- by giving different wavelength to get 50% germination. showed that main region of germination is in the red region.

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1946 Bonner etc. studied the light interruption by using photographic projector. 1952 Hendrick etc reinvestigate lettuce seed. --found low germination in dark.

give red light --- increase germination.

red, followed by far-red light – inhibit germination. Red -- Far red --- red again --- increase again.

Light bulb

Filter

Camera Shutter

Seedlings

Black Box

a similar effect on Xanthium, a short

day plant.

Long night ---- flowering

red light interrupt --- no flowering Red light interrupt long night followed by far red -- flowering.

1952-1959 try to get some physiological evidence. ---- Butler, norris, Siegleman & Hendrick Irradiate with red light ---- decrease in absorbance at 660 nm---- increase in absorbance at 730 nm Irradiate with far red light---- increase in absorbance at 660 nm---- decrease in absorbance at 730 nm

The compound is sensitive to heat, must be protein in nature, same conclusion as

Red, White Pr Pfr Far red, DarkStructure: Protein + Chromophore, similar to phycobilin in algae.

Effect of light: Act on chromophore first then act on protein. idea: the sequence of event suggest that possibly changes of the conformation (configuration) between cis and trans form of the phytochrome molecules.

Start with 100% Pr then irradiate with red light at 10 sec. interval it begin to convert Pr into Pfr at a first order reaction rate:

at some point, the absorption are due to both Pr and Pfr. ex. 80% convert to pfr, 20% to pr -- it is calledPhotostationary state after that, give more light, still no more effect.

at 698 nm wavelength, Pr=50% and Pfr=50% if, Pfr were irradiated with far-red, it reverse to 98% Pr and 1-2% Pfr a dose response to red or far-red or attainment of photostationary state can be shown. see figure.

OD 730OD 730 OD 660OD 660

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3. FRSat3 4. -3’6984

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Dose response curve

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R 亮

Pr Pfr

FR 暗

What are various light do on phytochromesun light = --- Pfr relatively high fluorescent ---- high Pfr (tube light)Incandescent --- low Pfr (more heat)Dark reversion: Pfr convert to Pr in dark

to many plants, when red irradiated treatment Pr convert to Pfr. then Pfr start to decay or denatured, and no more dark reversion.ex. in corn seedlings, only 20% dark reversion from Pfr, it is a common phenomeon in monocot.

Phytochrome. method of detection --- in vivo, through tissue segment. Sample harvest in dim green light. Sample harvest in green light as safe light, or in total darkness.

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子葉 暗箱

The change from Pr to Pfr transformation reflect changes in protein conformation possibly that a single photoreceptor pigment could exist in two photoconvertible forms:

Pr have an action maximun near 660 nm, and Pfr near 730 nm. absorption of light by either form converted it to the other form.

The pigment acting as a photoreceptor and that its absorption maximum must be close to the action maximum. Predict that the pigment would be blue in colour.

The low energy requirement suggested that the photoaction brings about a change in molecular configuration rather athan a transfer of energy to another system.

* Difficulty of detecting phytochrome.1. Low concentration of the pigment in plant tissue with very high molar extinction coefficients.

A= -logT= abc

Beer’s Law : ln I/Io = -Kbc

Io= Intensity of incidence radiation

I= Change in intensity of Io

T= Transmittance = I/Io

a= extinction coefficient or

= absorbancy index

= K / 2.303 to give base10 log

( 以 log10 為底的消光係數 (沒有單位 ))

單位 =cm3mg-1cm-1 or cm2mg-1

b= optical path = 1cm

A is proportional to c

E= molar extinction coefficient

= M-1cm-1 or liter mole-1 cm-1

可查表得到 E 值

舉例 : A. 1mg/ml solution of BSA in neutral phosphate

buffer has an A280 of 0.6 。 If the molecular

weight of BAS is 66,000 ， what is the molar

extinction coefficient?

依 Beer’s Law concept:

A is proportional to c

定義 : If light is absorbed by a substance in solution,

a (constant) is proportional to the concentration

of the substance in solution.

* Difficulty of detecting phytochrome.2. The overriding absorbance

of chlorophyll has obscured the measurement of phytochrome in green tissue.

Isolation and purification of phytochrome.－ methods for extracting proteins were used in the isolation. dark grown seedlings.－ Phytochrome, a protein with a chromophoric (pigment) group, low radiant energy needed to bring about.

－ a physiological response suggested a need for amplication analogous to the functioning of an enzyme.－ trace amount of substance are usually physiologically active by virtue of binding to a protein.

－ attempt to isolate phytochrome as a protein were successful, but subsequent purification require considerable time. － isolate phytochrome chromophere directly from plant tissue by method used in pigment chemistry have not yet been successful.

－ Purification of phytochrome has been satisfactorily achieved to date only from dark grown monocot. seedlings (avena sativa, oat). pH is a key factor for the purification. above pH 7.3 in supernatant contained the maximal extractable pigment. at pH 6.2 or below, only in sediment.

In supernatant

pH 7.3↑ In sediment

pH 6.2↓

－ The intracellular location of phytochrome has not been systematically pursued.

－ New satisfactory procedure for purification is available, mostly monocot, dark grown seedling, spinach, Mesotaenium (green algae)

Sphaerocarpus (a liverwort).

－ Absorption of light by the chromophroe brings about a change in conformation of the protein, which passes through several intermediate to the final form.

Denaturation .... Photoreversibility of phytochrome are lost on denaturation is an indicative of a dependence on interaction of the

chromophore with the native protein. Pfr form is more susceptible to urea, proteolytic enzymes and -SH reagent than Pr.

Physiological responses:

Higher plant is a captive of its environment and must be provided with sensor to appraise variables of its milieu.

light is one of the most important variables. Many aspects of the growth and development of higher plants are under control of phytochrome. ex. flowering, other aspects of phytochrome mediated process are:

1. Etiolation --- an assemblage of several responses, including control of phyocotyl length, leaf expansion and plumular hook opening. red light induced a reduction in hypocotyl length., a growth inhibition and expansion of leaves --- a growth promotion.

2. Internode length control --- mediated by phytochrome. These results are similar to those for reversal of etiolation. The location of the photoreceptor for the regulation of nternode length was in the internode itself.

3. Biochemical reaction under phytochrome control are.

a. coloration of the tomato cuticle is a biochemical synthesis under phytochrome control resulting from the deposition of a yet unknown pigment into the cuticle of the fruit.

b. Flavonoid synthesis in many plants requires light and is under phytochrome control. Pfr enhance the synthesis.Flavonoid 類黃素

c. Anthocyanin in apple skin is a case of phytochrome control. apple skin forms anthocyanin when exposed to light and accumulates aldehyde and alcohol when held in darkness.

precursorlight

darkanthocyaninaldehyde & alcohol

c. substrate for anthocyanin synthesis in light, if not utlized, forms aldehyde and alcohol. pigment is banana pseudo-stem possible phytochrome mdeiated.

d. development and differentiation

of plastids of etiolated leaves of higher plants is light dependent.

protochlorophyll --- chlorophyll

prolamella bodies --- lamella & grana formation.

e. NADP-dependent triose- phosphate dehydrogenase and chloroplast protein. (Marcus, Margulis). Prolognged far-red irradiation == brief red light

effect.

f. One aspect of the phytochrome mediated effects of red light at the cellular level is the induction of plastid enzyme synthesis. By determining the magnitude of increase induced by red light treatment and its reversibility by irradiation with far-red light,

f. it is possible to differentiate phytochrome from protochlorophyllide as the primary photoregulator of the synthesis of many enzymes.

f. Among the Calvin cycle enzymes, show phytochrome mediatedincreases in activities are: Ribulose 1,5-diphosphate carboxylase Alkaline fructose 1,6- diphosphatase Ferridoxin-NADP reductase NADP-glyceraldehyde-3- phosphate dephydrogenase.

g. Lag phase for chlorophyll accumulation in dark grown plants is elimiated by a prior illumination with red light and the

effect is reverseed by far-red light. It takes about 6 hrs before PSI & PSII are connected. brief red irradiation will do the job.

h. Geotropic response of dark grown oat seedling was immediately stimulated after exposure to red light.

i. Closing movement of mimosa leaflet on changing from light to darkness is under phytochrome control it requires Pfr for leaf expansion. When light off, there is no Pfr, it closed.

含羞草閉合運動

j. Light induced chlorophlast movements in algae. In Mougeotia and

Mesotaenium the low intensity movement of the single chloroplast is a phytochrome reaction. --- Haupt found that in Mougeotia, the phytochrome situated at the periphery of the cytoplasm is responsible for the following light response.

Euglena sp.

Low light High light

k. Tree hightness varies between east and west side of a forest. Due to difference on the quantity of quality light between morning and evening sunshines.

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l. Points: The synthesis of chloroplast protein and some enzymes can be obtained without chlorophyll synthesis.

l. by exposing leaves to red or prolonged far-red light, which is absorbed by phytochrome but not

by protochlorophyll

m. Tanada. found that root tip

sticks on beaker. numbers of tips stick on beaker varies each time. When red treatment sticks again but the number of root sticks decreased.

m. indicates that there must be

some sort of ion inside and outside of the cell to control

this mechanism, ie. charge effect.

根尖吸附在燒杯壁上

Some explaination on the consequence of reaction.* Osmotic change causes change of turgor, and the wall property too.* Red light -- cause stiffer wall, and auxin loosen the wall.

Down's effect.---at the end of the day

Far-red light --- induces longer stem

red light --- induces shorter stem both receive the same light (quantity and quality) during the day.

Elongation of stem is proportional to the time of the pigment in the inactive form --- ie Pfr.Pine tree treated with 8 hrs light then followed by Incandescent irradiance (low Pfr) --- higher stem elongation.Fluorescent light (high P fr)---Shorter stem

Mechanism of phytochrome action: Wide varieties in types of phytochrome responses of plants do not immediately provide useful clues to mechanism of action. Two mechanisms of action have been suggested.

1. Hendrick and borthwick (1965).control over a diversity of xpressions can be achievedby regulation of a basic metabolic reaction or of cell permeability.Ex. K+ in mimosa leaflet.

2. Mohr (1966) proposes control by gene repression or activation. In neither case is it clear how phytochrome interacts with the control system proposed.

Hypothesis on the mode of primary action, currently under consideration.

1. Phytochrome functioning through gene activation.2. Phytochrome induced changes in membrane properties.

Mode of action:－ Phytochrome localized in the membrane, its transformation affect the state of that membrane's permeability.

－ All subsequent morphogenetic transformations would be secondary derivatives of this primary effect.

－ How membrane permeability is changed is uncertain. one of the results appears to be the induction of a localized electrochemical gradient that manifest itself as a bioelectric potenial.

the evidence is: excised mung bean root tip developed the ability to adhere to a negatively charged glass surface when irradiated for 4 mins with red light, and this was reversed by far-red light.

examination of the electrical potentials on the root tip showed that red light caused the induction of a small positive change and that this was reversed by far-red light.

Photoperoidism ---The general response of plants to the relative length of day and night are refered. To collectively as photoperiodism.

Long day plant ----plant flower in long days. ex. many spinach and many cereals

Short-day plant --- plant flower under short days. 例如菊花。

Day-neutral plant --- examplified by the tomato. flowering is relatively unaffected by the day length of day, but is dependent on internal or structural features, such as node number on the stem.

Day length also affect many aspects of vegetative development, such as hairyness, anthocyanin production, leaf fall, onset of dormancy. formation of underground food storage organs.

Flowering metabolism.Florigen ---- hypothetical flowering hormone of plants. it has never been isolated and chemically identified. but its presence can be shown in a variety of ways.

A. Grafting experiment ---- A plant is induced to flower, being grafted to B will induce B plant to flowering too. Even a single leaf from the donor plant is grafted to the receptor may suffice.

B. Steam girdling technique ---- kill cells and block florigen transfer. flow of hormone occur in the vascular tissue, presumally in the living cells of the

phloem, at about the rate of transport of bulk organic substances in plants.

Application of leaf extracts from the induced plant to receptor plants, resulted in flowering.

---- Extracts of flowering <Xanthium> initiate the flowering in Lemna

(duckweed).

---- Extracts of vegetative Xanthium fail to induce flowering.

---- People in AEC lab. Michigan, use CO2 as tracer, try to find some

evidence of flowering stimulant from leaf, has no success.

In grafting exp. --- a plant under inductive condition can cause another plant to flower ex. LDP vs SDP. Presumally --- under short day --- daficiency of X under long day --- deficiency of Y.

It needs X+Y to flower. grafting, puts X and Y together to flowering.

Chailakhyan (Russian) propose that GA and anthesin are required in order to flower (similar to X & Y in above exp.)

LDP under LD have plenty of GA but lack of anthesin.

LDP under SD are limiting of GA.

SDP under short day have planty of anthesin, but lack of GA. put together--fit the grafting experiment.Anthesin as hypothetical substance

The grafting exp. cause complementary effect between SDP & LDP.

Rosette plant (LDP) --- induced to flowering under GA treatment and long day. induced bolting followed by flowering. -- examples are Samolus, silene and spinach.

AMO 1618 or CCC inhibit GA synthesis but not GA action. In Samolus --- GA more associate with flower bud initiation than elongation.

In Silene---2 LD ---- 80% flowering 4 LD --- 100% flowering6 LD --- microscopic detectable flowering bud7 LD --- stem elongation. in this case, GA more associate with bud elongation than flower initiation.

In spinach ---- elongation needs GA but not necessary associated with flowering.

SDP --- Bryophyllum.景天科，多肉植物 LD+GA --- no floweringGA+SD --- flowering.

Application of GA to long day plant under short days --- stimulate stem elongation, bolting and frequently follwed by flowering.

possibly, that GA function as the flowering hormone in certain long day plant, but it has no effect on short day plant.

* New evidence showed that ABA can promote flowering in some short-day plants.

*Both GA and ABA are made of isoprenoid( 異帖類 )

unit, some people speculated that phytochrome conversion controls some aspects of isoprenoid biosynthesis.

* Florigen would then be a different compound depending upon the nature of plant's response to day length.

We also know that ---* GA are synthesized in response to long days.* ABA are synthesized in response to decreasing day length.

* The effects of these

two hormones are however, far from universal.

•The exact nature of florigen still remains a mystery. 1.GA 2. ABA 3.Rubber 4.Carotenoids 5.Steroids 6.β-Carotene 7.Vitamine A

The interaction of phytochrome with other controlling factors:

Not all plants show photoperiodic control of flowering. day neutral plants, does not necessary means lack of phytochrome.

one can demonstrate red-far-red reversible control of some aspects of their vegetative morphogenesis.

or some other aspect of responese apparatus would appear to be undeveloped.

There are various anatomical stages involved as the plants progress from vegetative stage to complete flowering.

If the stages are arranged in order they constitute a quantitative expression of the degree to which flowering has progressed.

Using this tool, showed that increasing lengths of dark period beyond the critical period leads to steadily higher scores in flowering assay.

Some reaction proceeding progressively as the length of the critical dark period is exceeded. Point of no return

How many cycle are required to cause flowering ? In the Cocklebur, it appears that only one such period suffice.

Circadian ---- RhythmsMany experiments have

suggested that all higher plants contain in their cells endogenously oscillating systems or biological

clocks that govern rhythmic alteration in behavior, some of these rhythms, being approximately 24 hrs.

long are refered to as circadian. one example is the sleep movement of leaves.

Phytochrome mediated flowering, may involve circadian rhythum. make the explaination much more complicated.

Low temperature requirement for flowering.

---- Biennal plants, must over winter before they are ready to receive a photoperiodic stimulus ---receive 5

for 4 to 6 weeks℃ will the plant flower in long day.

Physiological basis for action of low temp. still very mysterious.

GA can replace some cold treatment.GA treated plant become sensitive to long photoperiod.--bolting.

cold treatment perhaps may produce GA or its precursors. that are convert to GA in long days.

Biennal: Sown late summer or fall --- pass winter as some size and

receive cold treatment --- through summer long day --- harvest in Fall.

annual: Sown in spring---harvest at Fall, no cold required.

harvest

Days

The practice of spring or winter rye are dependent on location. for spring type --- end of growing season(late fall) hit early frost.

For winter type ---winter are so severe that the seedlings could not withstand the temperatures that exist in the field.

under this condition. seeds can be allowed to begin germination indoor in early spring and then can be exposed to low temp.(2-5 ) for a period of 4-6 ℃weeks.

When spring condition permit sowing --- they are planted in usual fashion. harvest by the late summer.

The artificial administration of low-temperature treatments in an attmpt to hasten flowering is refered to as VERNALIZATION.

Vernalization furnished the basis for the claims of the Russian agronomist LYSENKO to transformed the genetic nature of plants by environmental treatments. --- a false claim.

The progeny of vernalized plants are not temp. independent. They still need low temp to initiate flowering. as did the vernalized seed from which they arose.

Photoperiodism in animals:Animals also sensitive to environment. examples are:

* migration patterns and reproduction in birds, Makings and ..... Response to day length. No evidence in any of these forms of the existence of or control by phytochrome.

* Pigment used by animals for this control is not phytochrome.

* In common with plants, the photoperiodic stimulus is translated into hormonal production to accomplish the onset of reproduction.

In conclusion.--- LDP are promoted by Pfr it needs longer day length.

--- Short day plant are inhibited by Pfr. it needs longer dark to decrease the inhibition of Pfr.