Phytochrome
Discovery of Phytochrome
Phytochrome is a blue protein pigment responsible for the perception of light in photo-physiological processes. It is possibly the only photoreceptor in photoperiodism and the flowering process.
The discovery of phytochrome is closely associated with studies on flowering. However, many other light controlled plant responses other than photosynthesis, collectively called photo-morphogenesis, are the effects of phytochrome action.
In 1932, Beltsville research group of the USDA headed by Borthwick and Hendricks showed that red light (630 to 680 nm) elicits the germination of lettuce seeds, whereas far-red light (710 to 740 nm) inhibits the process.
It was further observed that when lettuce seeds were exposed to alternating red and far-red light, almost all seeds that received red light as the final treatment germinated, whereas the seeds receiving far-red light as the final treatment did not germinate.
The phytochrome involvement in the flowering process was envisioned when in 1952. Borthwick. Hendricks and Parker demonstrated that red light inhibition of flowering in Xanthium could be reversed by a subsequent far-red light treatment.
The action spectra for inhibition and promotion of flowering shows that the red light near 660 nm and far-red light near 730 nm respectively, are maximally effective. When the plant is subjected to several consecutive irradiations with red and far-red in sequence, the flowering response is determined by the wavelength of the final exposure (Table 1 4.2).
A simple model of phytochrome action can be represented as follows:
In 1959, Butler and his associates first extracted phytochrome from etiolated oat coleoptiles. It occurs as a chromo-protein in which the chromophore is a linear tetrapyrrole similar to C-phycocyanin.
Phytochrome is widely distributed in the plant kingdom. Although green leaves are the organs, which perceive day light most effectively, their phytochromecontent is very low. For this reason, most studies with phytochrome have used etiolated seedlings from which phytochrome has been obtained in highly purified form.
The absorption spectra of the two forms of phytochrome, i.e., P660 and P730 overlap considerably (Fig. 14.1). The overlap is the reason why total photochemical conversion is not possible when irradiated with either red or far-red. When we irradiate the system with red light (660 nm), about 75% of the total phytochrome can be present as P730 (Pfr) at photochemical equilibrium.
Under irradiation with far-red (730 nm), the proportion of Pfr to total phytochrome (Pfr/P) is usually 3% at photo-stationary state.
Besides photochemical conversions, non-photochemical reactions also occur in vivo. Thus, Pfr may undergo dark reversion to Pr. Since natural white light acts like R, phytochrome will remain mainly in the Pfr form at the end of the day. It has been observed that after several hours of .darkness, plants become sensitive to R indicating that Pfr is present in a large amount.
Thus, it is inferred that Pfr is converted spontaneously to Pr in darkness. Phytochrome decay or destruction is also a dominant irreversible process in a seedling, which is the thermo-chemical transformation of Pfr to an inactive form.
Thus, the model of phytochrome action including synthesis, dark reversion and decay can be presented as:
Structure and Biosynthesis of Phytochrome
Phytochrome is a soluble chromo-protein with a molecular mass of 250 kDa it occurs as a dimer made up of two subunits, each of 125 kDa. Each subunit consists of two components — a light-absorbing pigment molecule, chromophore, and a polypeptide chain, Apo protein. The Apo protein monomer has a molecular mass of 125 kDa.
Apo protein and chromophore together make up the holochrome. The chromophore is a linear tetrapyrrole similar to phycocyanin termed phytochromobilin and it is a ring attached to the protein through thioether-linkage to a cysteine residue.
The principal difference between the Pr chromophore and the Pfr chromophore appears to be a cis-trans isomerization of the methane bridge between rings C and D. The absorption of red light provides the energy required to overcome high activation energy for rotation around the double bond. There is further evidence that the protein also undergoes photochemically induced conformational changes.
Phytochromobilin is Synthesized in Plastids
Phytochrome Apo protein alone cannot absorb red or far-red light. Light can be absorbed only when the polypeptide is covalently linked with phytochromobilin to form the holoprotein. Phytochromobilin is synthesized within plastids.
After synthesis, it leaks out of the plastid into the cytosol. Assembly of Apo protein with chromophore is autocatalytic, that is, it occurs spontaneously when purified Apo protein is mixed with purified chromophore in a test tube, for which no cofactors are necessary. Assembly in vivo of these two components is also autocatalytic.
(a) Phytochrome is Encoded by a Multi-gene Family:
(b) Phytochrome Controlled Responses:
Complementary DNA (cDNA) copies of mRNAs were isolated from oat and zucchini (Cucurbita pepo) seedlings. Using these clones as probes, five structurally related phytochrome genes were identified in Arabidopsis. This gene family is known as PHY, and its 5 individual members are PHYA, PHYB, PHYC, PHYD, and PHYE.
The Apo protein without chromophore is also called PHY, and the holoprotein with chromophore is called phy. Phytochrome sequences from other higher plants are named according to their homology with Arabidopsis PHY genes.
Phytochrome is the photoreceptor involved in many developmental responses of plants to light. It is involved as a light detector and also in the measurement of light duration.
The regulatory effects of light on plant growth and development are visualized most prominently at two stages in the life cycle of the plant — firstly, at the stage of seed germination and seedling development, and secondly, at the stage of transition from the vegetative to the flowering phase.
For the sake of convenience, these diverse photo-morphogenetic responses can be classified into the following types:
Type I: Fast Responses:
Type II: Slow Responses:
The type I responses include those processes in which the quantum energy absorbed by the plant is transduced to another form of energy.
Examples of this type of essentially energy-transducing response include the leaf movement of Mimosa and chloroplast movement in Mougeotia, Other examples are surface potential changes, membrane potential changes, and ion fluxes. These phenomena are relatively rapid, occurring on a time scale of seconds and minutes.
The rates and activation of certain aspects of growth and development are switched on or modulated under the influence of the quality of light (red or far-red). Examples of type II responses include stem elongation, seed germination, hook opening, leaf expansion, flower initiation, and pigment biosynthesis.
Type II responses are relatively slow responses occurring on a time scale of hours and days. The phytochrome molecule is thought to act as a photo chrome sensor that controls the photo-morphogenetic machinery of plants.
(c) Variation in Lag Time, Escape Time and Light Quanta for Phytochrome Responses:
(i) Very Low Fluence Responses (VLFRs):
Morphological responses to the photo-activation of phytochrome may be visually observed after a lag time ranging from a few minutes (chloroplast rotation in green alga Mougeotia) to as long as a few weeks (flower initiation). It has further been established that red-light-induced effects are reversible by far-red light only for a limited period of time after which the response escapes from the photo-reversible control.
Not only do the lag and escape times differ in diverse phytochrome responses, but different amounts of light (fluence) are required to induce them. The amount of light is termed “fluence” which is defined as the number of photons per unit surface area. Each phytochrome response is characterized by a specific range of light fluences over which the magnitude of response is proportional to the fluence.
These responses can be categorized into three major groups based on their sensitivities to fluence, viz., (a) very low fluence response (VLFR), (b) low fluence response (LFR), and (c) high irradiation response.
Examples can be provided by Arabidopsis seeds, which can germinate with very low red light. The reciprocal relationship between fluence and time, known as the Bunsen-Roscoe law of reciprocity is valid in the case of VLFRs, which, however, fail to show reversal control by light.
This means that a response can be induced either by a brief pulse of red light that is quite bright or by a very dim light for a longer duration. Another point of interest is that far-red cannot reverse VLFRs. The reason is that about 3% of the total phytochrome remaining after far-red exposure is sufficient to induce VLFRs.
(ii) Low Fluence Responses (LFRs):
(iii) High Irradiation Responses (HIRs):
They exhibit characteristic induction with red light and reversion with far-red light. The law of reciprocity, i.e., the light-induced response is a function of total fluence (fluence rate x irradiation time) and independent of the fluence rate or irradiation time holds for LFRs. Such responses include classic red/far-red photo-reversible responses, such as the promotion of lettuce seed germination and leaf movement.
Some photo-morphogenetic responses require prolonged or continuous exposure to light of high irradiance and are proportional to irradiance, but the reciprocity law is not followed here.
Examples of HIRs are:
(i) Anthocyanin synthesis in dicot seedlings and apple skin
(ii) Inhibition of seedling elongation (hypocotyl)
(iii) Flower induction
(iv) Plumular hook opening
(v) Cotyledon expansion in mustard
(vi) Ethylene production to Sorghum
(vii) De-etiolation of seedlings. However, the effect is not photo-reversible. The reason that these responses are called high irradiance responses (HIR) rather than high fluence responses (HFR) is that they are proportional to irradiance (i.e., the brightness of the source) rather than to fluence.
Mode of Action in Phytochrome
Pfr is regarded as the physiologically active form of phytochrome. Conversion of Pr to Pfr by light will produce a particular response depending on the localization of phytochrome and the state of differentiation of the responding cells.
It is also possible that the photo stationary state ratio Pfr/Ptotal acts as a signal perceived by the plant under certain conditions. For example, the HIR response for the inhibition of hypocotyl growth in lettuce can be explained in terms of the ratio Pfr/Ptotal = 0.03, i.e., a 3% Pfr level is necessary for the HIR response.
The first step is the absorption of light by the pigment. Then the absorbed light alters the molecular properties of phytochrome, which induces a sequence of cellular events ultimately leading to a change in the growth, development, or position of an organ. Generally, two consistent lines of shreds of evidence are sought to explain the effects of phytochrome.
One is concerned with the Pfr effect on changes in the properties of cellular membranes. The second theme is that Pfr regulates gene expression.
(i) Phytochrome and Permeability:
A rapid photo response is the phytochrome-controlled dark closure or folding of the leaflets of Mimosa or Albizzia. The response involves differential changes in turgor in the cells of the pulvinus at the base of each leaflet. The turgor change is associated with the movement of K+ and other ions into ventral cells and out of dorsal cells.
This has led to the suggestion that the primary action of phytochrome is on membrane permeability. Tanada presented further evidence of membrane changes following Pfr action. It was observed that excised barley and mungbean root tips exposed to red light would stick to a negatively charged glass surface (Tanada Effect).
Such light-induced adhesion was found to be red/far-red reversible. It was suggested that the apical part of the root became electro-positive relative to the basal part in response to Pfr. Rapid phytochrome-controlled changes in electric potential have also been measured in the coleoptiles of oat seedlings.
These electrical changes are consistent with a phytochrome-induced efflux of ions. Among the different ions transported, Ca2+ helps in transducing the photo-activation of phytochrome into physiological changes.
(ii) Phytochrome and Enzymes:
Seedling photo-morphogenesis is associated with the appearance of enzymes necessary for photosynthesis. NADP-dependent GAP dehydrogenase, a key enzyme associated with leaf chloroplasts, changes in activity in response to red and far-red exposure. An enzyme, which has been extensively studied, is phenylalanine ammonia-lyase (PAL).
It is the enzyme that catalyzes the conversion of phenylalanine to coumaric acid and thus initiates the synthesis of compounds like coumarin, lignin, and flavonoids including anthocyanin pigments belonging to the class of secondary metabolites (Fig. 14.4).
This enzyme is present in very low concentrations in the dark but can be greatly increased on exposure to far-red light. Another enzyme, ascorbic acid oxidase, has been shown to increase by Pfr action.
It is not certain whether Pfr stimulates the synthesis of these enzymes or leads to an activation of already existing enzymes.
Some Pfr Responses are mediated by Calcium and Calmodulin:
Calmodulin is a calcium-binding protein and calcium-calmodulin (Ca2+ -CaM) complex may regulate plant responses which include several enzymes like plasma membrane-localized Ca2+ pump (Ca2+ -ATPase), NAD kinase and enzymes (kinases and phosphatases) that cause phosphorylation and activation of other enzymes.
Several lines of evidence indicate that Ca2+ can mediate phytochrome responses. Calcium uptake into the cells is increased by Pfr and some Pfr-stimulated enzymes are also stimulated by calmodulin (CaM).
By using Ca2+ -ionophore, a chemical agent that promotes Ca2+ uptake into cells, some phytochrome responses can be induced in darkness. It is suggested that chemically induced uptake of Ca2+ into a cell can mimic Pfr responses and act as a substitute for a red light. It is tempting to conclude that calmodulin is the agent that transduces Ca2+ entry into physiological responses.
(iii) Intracellular Localization: Phytochrome Bound to Membrane:
In higher plants, 75% of the total amount of phytochrome is localized in protoplasts, while the remaining 25% is divided equally between the vascular and epidermal tissues. Attempts have been made to locate phytochrome within the cell by microspectrophotometry and immunocytochemical methods.
Purified phytochrome is injected into an animal to produce a highly specific antiserum, which is then used to locate phytochrome in tissue sections. These studies indicate that the Pr form of phytochrome is diffusely distributed, whereas the Pfr form is present in a discretely localized pattern. Phytochrome Pr is a stable protein, whereas Pfr can bind and fuse to a membrane, thus modulating membrane activity.
In contrast to higher plants, many Type-I responses (quick responses) in simpler organisms are mediated by membrane-bound phytochrome. For example, phytochrome in Mougeotia, a filamentous green alga, is membrane-bound. The cells of this alga contain a single ribbon-like chloroplast, which shows movement controlled by phytochrome (Fig. 14.5).
When red light falls perpendicular to the long axis of the cell, it is preferentially absorbed in the front and backsides of the cell converting Pr to Pfr form. As Pfr builds up at the front and back of the cell, the chloroplast can rotate about its long axis and can respond to the incident radiation by orienting perpendicular to the direction of light (face position).
On the other hand, red light falling parallel to the long axis of the cell is not effective for chloroplast movement, which persists in the profile position. In this case, the red light effect can be reversed by far-red light proving that phytochrome is implicated in the response. Rotation can also be induced by far-red light, but the chloroplast should remain parallel to the source of far-red light.
(iv) Inhibition of Internode Extension:
Rapid visible response to the photoactivation of phytochrome is the inhibition of internode extension of growing seedlings. Ca2+ and CaM-dependent cellular events can be linked to red light-induced inhibition of internode extension. When calcium concentration of cell wall increases, cell-wall loosening processes are inhibited leading to wall stiffness, which in turn inhibits extension.
Most of the cellular calcium is bound to cell structures and organelles like vacuole, mitochondria, and endoplasmic reticulum. Initially, Pfr promotes the release of Ca2+ from these structures into the cytoplasm and thus causes a transitory increase in cytosolic calcium. Phytochrome regulation of wall extensibility based on CaM is thought to occur in a stepwise manner.
First, phytochrome is activated by red light converting Pr to Pfr. This is followed by Pfr-induced CaM activation. Then Ca2+ -CaM complex stimulates plasma membrane-bound Ca2+ – ATPase which pumps Ca2+ out from cytosol into the walls.
(v) Phytochrome Stimulates Gene Expression:
It is generally accepted that most living cells of a particular plant contain all the genetic information in the form of DNA characteristic of that plant. Differences amongst cells arise from differential gene activity, genes being ‘turned off’ and ‘turned on’ during development. Role of phytochrome in the major developmental events obviously suggests that changes in gene expression are involved.
Studies on phytochrome regulation of gene expression are so far concerned with nuclear genes encoding for mRNAs of chloroplast proteins. Two such proteins have been investigated. These are — (i) the small subunit of Rubisco, and (ii) the light-harvesting chlorophyll binding protein (LHCP) associated with light-harvesting complex of PS II.
These two proteins play an important role in chloroplast development and greening. Rubisco is the key enzyme in photosynthesis, which catalyses the addition of a CO2 molecule to an acceptor molecule RuBP. It is an oligomeric protein, consisting of eight large subunits (LSU) and eight small subunits (SSU).
The LSU is encoded by the chloroplast genome and synthesized in the chloroplast. The SSU is encoded in the nuclear genes and synthesized in the cytoplasm.
Similar to SSU, the Apo protein of LHCP, known as chlorophyll a/b binding protein is encoded in the nucleus, synthesized on cytoplasmic ribosomes, and transferred into the chloroplast. The corresponding genes for these proteins are rbcS (rubisco Small) and cab (chlorophyll a/b).
In an early work, Rubisco protein of barley seedlings was found to be stimulated by light. Recently, translation studies of mRNA isolated from duckweed (Lemna gibba) have been made by Tobin and her associates in the United States, who have shown that mRNA levels for SSU protein and chlorophyll a/b binding protein increase after exposure of dark-grown seedlings to light.
Only I min light exposure is needed to increase mRNA and the effect is reversed by far-red light. It has been confirmed that Pfr acts to increase the rate of transcription of these two genes for the small subunit of Rubisco and the chlorophyll a/b binding protein.
Using modern molecular techniques, it has been established that the promoter region of rbcS gene is light-regulated.
Promoters are DNA sequences located upstream on 5′ flanking side of a gene, which function in the regulation of transcription. Such regulatory sequences in the promoter region are called c/s-acting DNA. Recently, promoter regions of rbcS gene from peas have been shown to contain c/s-acting elements involved in light regulation.
It has been proposed that red light converts Pr to Pfr. Then Pfr activates one or more regulatory proteins. It is curious that phytochrome itself has no DNA-binding capacity. So, the activated regulatory proteins behave as trans-acting factors, which are DNA-binding proteins that bind to c/s-acting DNA sequences and regulate gene transcription.
Such DNA sequences known as light regulated elements (LREs) have been identified in the promoter region for two genes — rbcS abd cab.
Thus, the activated regulatory protein then binds to specific light regulated element (LRE) and stimulates transcription of the gene, leading to an enhanced synthesis of gene products, SSU of Rubisco and light harvesting chlorophyll protein (LHCP).
These proteins contain transit peptides that facilitate their entry into chloroplasts. After entering the chloroplast, SSU combines with LSU to form holoenzyme. The other protein LHCP is associated with PS II in thylakoid membrane.
(vi) Phytochrome Inhibits Gene Expression:
Contrary to the genes stimulated by phytochrome, there are at least two genes where Pfr causes a decrease in transcription. One such negatively regulated gene encodes for NADPH-protochlorophyllide oxidoreductase, the enzyme that catalyzes reduction of protochlorophyllide to chlorophyllide. A decrease in the level of translatable mRNA is thought to be the cause of decrease in activity of this enzyme in light.
The other negatively regulated gene is the gene that encodes for phytochrome itself, meaning thereby that phytochrome regulates the expression of its own gene. When Pr is converted to Pfr by red light, phytochrome mRNA decreases by decline in the rate of transcription.
Thus, phytochrome genes are auto-regulated by some form of feedback inhibition. Such phytochrome-induced repression of phytochrome gene expression is a factor, which possibly explains a rapid decrease in total phytochrome when dark-grown plants are transferred to light.
(vii) Crypto Chrome: Blue-Light Responses:
A large number of plant responses to blue light have been known for a long time. Blue-light signals are generally used by a plant in many responses that provide means to sense the presence of light and its direction. The specific blue light responses of higher plants include phototropism, stomatal movement, inhibition of hypocotyl elongation, pigment biosynthesis and gene activation.
Such plant responses to blue light are quite distinct from phytochrome-induced responses.
These responses have a characteristic action spectrum showing a “three-finger” structure in 400 to 500 nm that is not observed in the absorption properties of either phytochrome or chlorophyll (Fig. 14.8).
For example, phototropism, which involves bending towards light, cannot be induced by red light. Likewise, inhibition of hypocotyl elongation by blue light is a specific blue light response in 400-500 nm, independent of phytochrome activity (Fig. 14.9).
It is true, however, similar response in 700-750 nm indicates phytochrome-dependent response. Another distinction between red light (i.e., phytochrome) and blue light inducing the same response is based on the relative rapidity. The inhibitory effect of phytochrome on hypocotyl elongation occurs within 15 to 90 min, whereas the blue light response is quite fast requiring only 15 sec.
(viii) Phytochrome and Flowering Response in Short-Day Plants (SDP):
In several SDP like Xanthium, soybean, Amaranthus, Chrysanthemum, Pharbitis and Chenopodium, night break by R inhibits flowering and FR is effective in reversing a red light (R) night break. So, it is assumed that Pfr which is produced by a R night break reacts to inhibit flowering in SDP (Table 13.2).
It has also been observed that irradiation with R or FR establishes widely different photo stationary states of 0.75 and 0.03, respectively, yet both when applied singly prevent flowering.
Thus it is clear that inhibition of flowering is not proportional to the amount of Pfr. but it depends on the maintenance of the Pfr level for sufficient time above some threshold level.
A relatively short exposure with R is effective in preventing flowering in SDP because it induces a high photo stationary state (Pfr/P = 0.75). Thus, when the plants are returned to darkness, it takes a relatively long time for Pfr to decay below the threshold level.
On the contrary, relatively long exposure to FR is required to prevent flowering because the proportion of Pfr to total phytochrome (Pfr/P) is low, and so on return to darkness, decay below the threshold value occurs rapidly, it is also interesting to note that the possibility of re-promotion of flowering by FR is reduced with each successive R/FR cycle (Table 13.2).
In these treatments, the dark intervals between the light flashes are sufficiently long to permit Pfr to fall below the threshold value leading to the maintenance of Pfr above the optimum level required for flowering.
There is evidence that phytochrome controls flowering response in SDP not only in the dark period but also during the day period. In Xanthium and Pharbitis, the light quality during a light period intervening between a subcritical dark period and an inductive dark period strongly favours flower formation. Action spectra indicate that the maximum promotion is at 660 nm and inhibition at 730 nm.
It is likely that phytochrome is the photoreceptor since there is evidence of R/FR reversibility. Again, in dark-grown a seedling of Pharbitis, prior exposure to light with either long or brief R irradiation is required if a single inductive dark period is to be effective for flowering. The effect of R irradiation is reversible with FR, hence it indicates that phytochrome is the photoreceptor.
(ix) Phytochrome and Flowering Response in Long-Day Plants (LDP):
It is generally believed that phytochrome controls opposite reactions in SDP and LDP. This means that light interruptions of long nights which inhibit flowering in SDP should promote flowering in LDP. Basically this is true but very brief interruptions of long nights do not always promote flowering in many LDP. Generally, a long night break is effective for LDP.
The light quality during prolonged night breaks shows that either FR or R and sometimes a mixture of R and FR is effective in promoting flowering in LDP. It has further been demonstrated that flowering in various LDP is more enhanced with incandescent lamp with a high proportion of FR energy than with fluorescent lamps having less FR energy.
In crucifers, neither R nor FR shows marked acceleration of flowering. Here blue light is most effective either as night interruption or as day extension as initially shown by early reports of Funke and Wassink et al.
Just as dark interruption with light is effective in promoting flowering, a simple day length extension has also been found to be effective. In this case, R alone or fluorescent light given during the extension period is not as effective as FR or a mixture of R and FR or incandescent light.
The exact identity of the blue light photoreceptor is not yet known hence the name crypto chrome has been given which implies a “hidden pigment”. In view of the similarity between the absorption spectra of β-carotene and riboflavin and the action spectrum for phototropism.
It has been proposed that carotenoids and flavins are the probable photoreceptors or crypto chrome for blue light responses. Carotenoid zeaxanthin has recently been implicated as a blue light photoreceptor.
It has a photo-protective role and a role in signal transduction. The absorption spectrum of zeaxanthin closely matches the action spectrum for blue light-stimulated stomatal opening. The most common flavins occurring widely in living organisms are riboflavin and its two nucleotide derivatives, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).
Blue light has been shown to regulate gene expression. The gene that codes for the enzyme chalcone synthase (for flavonoid biosynthesis) is blue light-regulated, while the gene for the small subunit of Rubisco and for the proteins that bind chlorophylls a and b are not only sensitive to red light (i.e., phytochrome response), but also sensitive to blue light.
Another enzyme, glutamyl semialdehyde aminotransferase, a key enzyme in chlorophyll biosynthetic pathway, is encoded by GSA gene which is sensitive to blue light. Recently, a gene has been isolated from Arabidopsis, which has been shown to encode the blue photoreceptor that causes inhibition of hypocotyl elongation.
The gene and the corresponding protein have been named CRY 1 (for crypto chrome 1). Two chromophores are likely to be associated with CRY 1. One of them is a flavin (FAD) and the other is possibly a pterin.
In view of the above observations, several hypotheses have been put forward to account for the promotive effect of FR and the requirement of prolonged exposure to light in LDP. It has been argued whether phytochrome is the only photoreceptor involved in photo induction in LDP. Thus, the existence of another pigment, possibly a flavoprotein. has also been postulated to explain the effectiveness of blue light.
It is, however, suggested that flowering in LDP is a type of ‘high irradiation response’ (HIR) and it is now widely accepted that phytochrome is the photoreceptor in HIR. Even the responses to blue light may reflect different Pfr requirements and thus no pigment other than phytochrome may be involved.