Notes on Phytochrome | Plant Physiology

In plants, there is a photo reversible pigment which is called phytochrome (P), chromophoric protein, and exists in two forms: one which absorbs red (Pr) and the other one which absorbs far-red light (Pfr).

Bestowed with such a versatility of the molecule, several bio-chemicals, physiological and morphogenetic responses can be regulated in the plants. It was in 1920 that Gardner and Allard demonstrated photoperiodism and the importance of dark period.

Thus, short day plants failed to flower once their dark period was intercepted by a short interval of light. In 1944, Borthwick, Parker and Hendricks at the U.S. Department of Agriculture, Beltsville observed that red light (660 nm) was highly effective in inhibiting flowering of short day plants.

On the contrary, it promoted flowering in long day plants. Same effect caused by red light was seen in stem elongation in barley and leaf growth in pea seedling grown in the dark. Earlier, Flint and Moallister (1935-37) had reported that red light highly promoted lettuce seed germination and the latter was inhibited by far-red light.

That is far-red light exposure following red light, reversed its effects. These observations pointed towards the existence of a single photoreceptive compound which occurred in two inter-convertible forms.

Similar situation was reported in several other phenomena. Butler used the term phytochrome for this photoreversible pigment. Norris (1959) demonstrated the photo reversibility of the pigment using a dual spectrophotometer in the cotyledons of turnip seedling.

However, it was in 1962 that this pigment was extracted from shoots of dark grown maize seedlings and was shown to be a chromoprotein and the chromophore was a cyclic tetrapyrole. Soon after it came to be recognized that the active form was far-red absorbing (P730) and this gradually changed to P660 in the dark. The change in configuration during these reversals was also unravelled (Fig. 12-1, 2).

Correl (1965, 1969) using analytical centrifugation studies revealed the occurrence of phytochrome tetramers which were made up of subunits. It was shown to be of similar absorption spectra. Over the years, several aspects of phytochrome chemistry have attracted attention and these are phototransformation of the pigment at low temperature in relation to subsequent dark reaction at normal temperature; changes in the optical activity during photoreversibility of the pigment.

The pigment is found to be stable between pH 6 and pH 8. The photoreversibility is gradually lost in TFA (trifloroacetic acid), DMS (dimethyl sulfamide), urea and mercapto-binders. The presence of glutaraldehyde seems to inhibit the Pr-Pfr transformation.

Such absorption in all probability is attributed to cross linkages between the peptide chains. In a nutshell, it is imperative that chromophore is surrounded by a specific configuration of the protein.

Indeed, studies relating to optical activity of the two forms have shed sufficient light on the role of protein moiety and also on the mechanism of photoreversibility. It is very interesting to note that in the Pr to Pfr transformation several intermediate photo-isomers are produced which are cold temperatures stable.

Further, Pfr to Pr transformation is very simple but dark reversion of Pfr to Pr is highly temperature dependent in vitro. When oxygen is present, there is destruction. That this destruction is inhibited by EDTA. EM and ultracentrifugation techniques have shown that photoreversible part may have a dimer structure.

Since phytochrome mediates a wide range of responses, (Table 12-2), it is difficult to propose a generalized model. By far, most efforts revolve around gene activity. Several enzyme-systems are regulated by phytochrome (nitrate reductase; invertase; peroxidase).

The inhibition of enzyme synthesis by Actinomycin D or Cycloheximide following phytochrome action points towards transcription and translation. Even though possible for several systems, gene activity is unable toexplain short term responses e.g. orientation of Mougeotia chloroplast, pulvinus movement in Albizzia, etc.

Through polarizing microscope, it is evident that this pigment is membrane localized and by changing its orientation it regulagtes membrane permeability. The general view is that chromophore component acts as a photo-receptor and undergoes cis-trans isomerization and causes change in the conformation of protein moiety.

Thus chain of significant events is altered. However, precise mechanism of its action has been described in an oversimplified way and many questions about the mechanism of its action await detailed answers. A photo-response can be defined as phytochrome-mediated one if it could be induced by a short irradiation of red light (nearly 5 min or so, of medium quantum flux density).

Further, the induction by red light should be reversed by far-red light. The responses may be positive or negative or may be highly complex. Then the responses may be developmental or rapid responses. The developmental responses are mediated by phytochrome but involve other physiological processes e.g., growth, differentiation and periodic phenomena. Such processes take long time for the production of a response.

Such responses include photoperiodism, seed germination, anthocyanin formation, chlorophyll synthesis, unfolding of monocot leaves, etc. On the other hand, rapid responses are manifested in a short time after irradiation with red light and do not interact with complex physiological processes.

This category includes orientation of chloroplast in Maugeotia filaments, leaflet, and movements in Mimosa pudica, increased permeability of water on the basis of permeability changes affected by red phytochrome, whereas developmental responses indicate an effect at gene, enzyme or hormonal level. In the following some of the phytochrome-mediated phenomenon are briefly discussed (See Table 12-2).

(i) Phytochrome and flowering:

The inhibition of flowering in short day plants by a red (R)- break indicates the existence of some important reactions which cause synthesis of floral stimulus. This is completed in dark. It was shown that there was involvement of a ‘light- Pfr’—’high Pfr’ reaction and a ‘low Pfr’ reaction but their sequence varies.

During the formation of floral stimulus, there is GA-like compound synthesized. Thus, distinct ratios of P660/P730 are essential to induce flower formation.

(ii) Chloroplast development:

The effect of light on chloroplast development is surely mediated by phytochrome, since red illumination promotes chloroplast development and synthesis of photosynthetic enzymes.

(iii) DNA- and Protein synthesis:

Red light is also shown to induce DNA and protein synthesis in the cells of etiolated pea stem apices.

In dwarf peas, R-light induced proteins which were complexed with GA₃ and suggestively this complex prevented normal growth of the dwarf peas.

(iv) Water uptake:

Another significant effect of R-light consists of its role in regulating the uptake of different substances such as water, acetate and also exogenously applied auxins.

(v) Seed germination/dormancy:

In the air dried seeds of Cucurbitapepo, whole of the phytochrome exists as Pfr. On moistening of the seeds, phytochrome increased in steps as below:

(vi) Pollen germination:

Studies in Arachishypogaea pollen have shown that short exposure to R-light caused early tube emergence and its enhanced elongation, and that this effect was annulled by FR exposure. Obviously, the effects of R and FR were mutually reversible. Furthermore, acetylcholine and GA3 could replace the R-light effect.

In apple, anthocyanin synthesis is regulated by the phytochrome system. M.J. Jaffe has proposed how phytochrome might affect changes in membrane permeability. His group also demonstrated that acetylcholine, the animal neurohumor, could mimic the effect of far-red light.

These workers further proposed that acetylcholine possibly mediated several phytochrome responses in roots. There is a good possibility to believe that Red-light results in the synthesis of acetylcholine and the latter affects membranes and mitochondria and regulates the transport as well as oxygen uptake, etc.

It is only recently that much attention is being devoted to the distribution and functions of acetylcholine in plants. H.Mohr highlighted the role of phytochrome in chloroplast development.

From his studies a few points may be summarized below:

The rate, at which grana appear under continuous white light saturating with respect to chlorophyll formation, is controlled by red light pulse pre-treatment.

i. Chlorophyll a which is a characteristic marker-molecule of the plastid compartment, its formation is controlled by phytochrome.

ii. The level of Calvin cycle enzymes is also controlled by phytochrome.

iii. Phytochrome has also been shown to regulate photophosphorylation.

iv. Phytochrome has also been shown to control chlorophyll b appearance.

It is still debatable whether or not the multiple controls exerted by phytochrome during pattern realisation in plastogenesisis the result of a single initial action of Pfr or not. However, one fact is obvious that Pfr controls chloroplast development at different levels and through several independent initial actions.

There is evidence that many of the blue-green algae (Nostocales) contain photochromic (photoreversible) pigments regulating morphogenesis, mobility and pigment synthesis. These photochromic pigments resemble the phytochrome of higher plants but have their absorption peaks at shorter wavelengths.

In blue-green algae there is green vs. red antagonism instead of red vs far-red. As analogues of phytochrome they are referred to as cynophyceanphycochromes. Recently phycochromes a, b and c have been described. A pigment system sensing blue light (400-450 nm) and not reversible has been located in several higher and lower plants (e.g., Neurosporacrassa. Dictyostelium sp.).

This photoreceptor may be a flavoprotein, which absorbs blue light. The reduced cytochrome gets reoxidized in dark. In Arabidopsis there are five phytochrome genes encoding five species of phytochrome (PHYA- E). Of these PhytochromeA (PHYA) accumulates in darkgrown seedlings as PrA which is stable. PfrA is unstable and is destroyed with a half-life of 1 to 1.5 hours. PHYB is expressed at low levels in both light and dark.

PfrB is stable, with a half-life of 8-hours or more. A mixture of red and FR light will establish a photoequilibrium mixture of Pr and Pfr. Phytochrome-mediated effects are conveniently grouped into three categories on the basis of their energy requirements: very low fluence responses (VLFR), low fluence responses (LFR), and high irradiance reactions (HIR).

LFR includes seed germination and deetiolation. VLFR are not photoreversible, is HIR requires prolonged exposure to high irradiance, are time dependent, and are not photoreversible. It seems that PHYB is the sensor that detects changes in R/FR fleucne ratio.