Photomorphogenesis

Light

Before delving into the role of phytochrome in determining plant growth patterns in response to different colours of light, we will look briefly at what we mean by 'colour'. If we split light with a prism, we get a spectrum of different colours. We call these colours pure spectral colours: each has but a single wavelength.

A pure spectrum of colours produced by a prism, with the positions of the three cone receptors at wavelengths corresponding to blue, green, and yellowish-green (usually called red, for no very good reason).

Cone cells in the eye detect 420 ('blue' cones), 534 ('green' cones) and 564 nm ('red' cones: not even close, but chartreuse sounds a little pretentious). However, colour perception is largely done by the brain, not the eye. Red things appear red because they stimulate yellow-green receptors ('red' cones) more than green and blue cones, not necessarily because it is producing light of 670 nm. It is possible to make things appear red by producing other colours of light, and mixing them in the appropriate ratios to stimulate the receptors in the same way as pure spectral light of 670 nm. It is generally impossible to exactly reproduce most pure spectral colours with pigments. To make the situation even more confusing, our brains can also perceive colours (such as white, pink and brown) which do not exist as pure spectral colours, but are the product of stimulation of the cones in a way that the spectral colours cannot manage.

What we can do to make this situation a little easier to understand is to (arbitrarily) define primary colours, and mix them together to approximate spectral colours. If we chose our primaries well, we can get good approximations of most colours in the human colour-space, and of the spectral colours. There are two common schemes: additive and subtractive colours.

With the additive colours, we start with a dark room. Our primary colours are red, green and blue lights (RGB colour), which we shine directly into your eyes. Adding all three together mimics white (red + green + blue = white). TV screens use additive colours because they produce light, and are in a position to shine different mixtures of coloured lights into our eyes.

Additive colours are red, blue and green. Summed together, they produce white light.

With the subtractive colours, we start with something reflecting all colours, such as a piece of white paper. Our primaries are cyan, magenta and yellow (CYMK colours, the K stands for black, which is difficult to simulate with these three primaries in a dense enough way). These are close to the primaries that artists use (blue, red, and yellow are used more commonly, and are probably the primary colours you learnt at school). If we put these pigments in the way of white light, they will subtract certain colours; mixing together all three in theory generates black, but if the colours aren't dead right, you'll get brown, as you know from finger-painting aged 5 (cyan + magenta + yellow = black). These primaries are used by printers: the problem of getting a decent black without soaking the paper is usually solved by simply adding a fourth 'primary' colour of black ink.

Subtractive colours are cyan, magenta and yellow. Summed together, they produce black (an absence of light).

Note that these colour schemes and their primaries are not derived from some Platonic ideal, handed down from heaven. They are pragmatic and arbitrary. For the subtractive colours, we have learnt from experience that red, blue and yellow paints allow us to simulate most of the colours that the human brain can perceive.

Let's apply this knowledge to filters and pigments. A pure yellow filter will subtract all colours from white light except yellow (about 580 nm). However, we can also get a yellow appearance by allowing through red and green lights, the combination of which our brains perceive as yellow. Likewise, a green pigment may be one that only transmits light around 530 nm. However, it is at least as likely that the pigment is a mixture of two chemicals, one of which would appear light blue, and the other yellow, considered alone.

Light may be considered as a stream of energetic photons. Two ways of measuring light are important to consider in relation to photomorphogenesis. Irradiance is the number of photons (or amount of energy) absorbed per unit area per second (units of mol s−1 m−2).  Sunlight's irradiance is 2 mmol s−1 m−2. Fluence is the number of photons per unit area (summed over time), and has units of mol m−2.

Photoperception and photomorphogenesis

Photomorphogenesis is the control of growth and morphology (shape, flowering, etiolation, phototropism, circadian rhythms, etc.), by light. It is mediated by several light receptors: PSII, cryptochrome, phototropin, zeaxanthin and phytochrome, amongst others

Much of the current work on photomorphogenesis is done on Arabidopsis thaliana (thale-cress), the 'botanists' fruit-fly, particularly in the analysis of mutants that re unable to respond to light such as nph (non-phototropic hypocotyl).

Action spectra give clues as to which chromophores are responsible for light detection. To determine an action spectrum for e.g. light stimulation of seed germination, we can measure percentage germination of seedlings exposed to different coloured lights (dashed line) and compare this to phytochrome absorption spectrum (solid line). By doing this, we can identify which pigments are involved in different responses to light.

Seed germination action spectrum and phytochrome absorbance both have peaks at about 300 and 700 nm.

UVB sensors

Ultraviolet light causes damage to plant cells, hence the cells synthesise protectant chemicals such as flavonoids in the epidermis and extra wax in the cuticle. These responses are stimulated by ultra-violet-B light, but it turn out that the light isn't sensed by a 'real' chromophore. Instead, the D2 subunit of photosystem II absorbs and is destroyed by UVB and the degradation products induce these pathways. Flavonoids protectants are yellow (flava Lat.), therefore they mostly absorb red and blue light (including 'far-blue', i.e. UV). Flavonoids are antioxidants (like vitamins A, C and E), mopping up free radicals.

Delphinidin is a blue flavonoid found in the petals of Delphinium plants.

Blue light sensors

Phototropin senses the direction of light in phototropism, which is the growth of plants (particularly seedlings) towards sources of light. Phototropin possesses two flavin chromophores, which absorb blue light. The action spectrum indicates that the phototropic response in seedlings responds to blue light best. The signalling pathway is unknown, but phototropin is known to be a serine kinase. Further evidence of its involvement is that iodide inhibits flavin synthesis, and also inhibits the phototropic response.

The second main blue light receptor in plants is cryptochrome, which modulates the phytochrome response. This pigment contains one flavin and one pterin cofactor.

Flavins absorbs blue light.
Flavin

The third blue-light sensor in plants is zeaxanthin (a carotenoid), which modulates stomatal opening. Zeaxanthin accumulates in irradiated chloroplasts, which activates an H+ ATPase pump in the guard cell membrane, causing ions to flow in via H+ symports. Water follows the ions by osmosis, and the cells swell, closing the stoma.

Zeaxanthin absorbs blue light.
Zeaxanthin

Phytochromes

Phytochromes are blue-green pigments found in all green plants. They sense red and far-red light. They are all multimeric proteins containing a covalently bound tetrapyrrole chromophore called phytochromobilin. Phytochrome is involved in many responses to light. The two most well studied are the:

Low fluence response

The low fluence response (LFR) was discovered by Flint and McAlister in 1937. Some varieties of lettuce seeds were seen to need light to germinate. Red light stimulated germination but far red reversed the effect of red light and inhibited germination. Thus phytochrome appeared to be measuring the red to far-red ratio, and required only about 1 µmol m−2 of photons. This is useful because shaded plants (and seedlings underneath soil or other plants) receive 10 times more far red light than red. Seeds respond by inhibiting germination until the canopy dies away.

Light beneath a green leaf is enriched in far red light relative to red light.

The mechanism of LFR relies on the fact that phytochrome exists in two forms that are interconverted by red and far-red light. Phytochrome is synthesised as inactive PR which absorbs red light. PFR is the active form, and absorbs far-red.

Active phytochrome Pfr has a trans bilin chromophore.
PFR with trans chromophore

inactive phytochrome Pfr has a cis bilin chromophore.
PR with cis chromophore

The ratio of the two forms is a measure of the ratio of red to far red light. High [PFR] indicates a lack of shading and therefore stimulates germination. High [PR] indicates shading and thus prevents germination.

Pfr is converted to Pr by far-red light, and vice versa.

The situation is not quite this simple (is it ever?): PR absorbs a little far-red light (solid line), and PFR absorbs a little red light (dashed line). We never get pure PR or pure PFR: 100% red light will produce 85% PFR, and 100% far-red light will produce 97% PR. The equilibrium ratio at a given red/far-red ratio is called the photostationary state.

Pfr and Pr absorb maximally at different wavelengths, but their spectra show considerable overlap.

Th adaptiveness of the LFR is that it prevents seeds that are buried or shaded from germinating. Phytochrome is synthesised as the inactive PR form. No red light means little active PFR, and no germination. Note that any residual PFR from previous exposure to light is slowly destroyed by a light-independent reaction. Consequently, brief exposure to red light will slowly reverse itself if the seeds are held in complete darkness.

Phytochrome was first demonstrated in intact plants by changes in the pattern of light absorption on exposure to either red or far-red light. Fluorescent antibodies raised against phytochrome show that it occurs all over the plant and in almost every part of its cells. When the chromophore absorbs light, it flips from cis to trans (Z to E) form; much like retinal in opsins. When PR converts to PFR, a nuclear localisation signal is exposed, and the phytochrome enters the nucleus. PFR has kinase activity, and acts on transcription factors, which regulate gene expression.

High irradiance response

The HIR is used to sense the presence of bright light. It tells the plant about when to perform such things as chlorophyll synthesis, plastid differentiation and de-etiolation of seedlings bursting through the soil surface. The HIR requires several hours of bright light but is insensitive to the red/far-red ratio. The LFR can't do this, because it saturates at low light intensities, and the PR/PFR ratio is determined by the spectrum rather than the intensity of light. the mechanism of the HIR appears to be mediated by phytochrome and cryptochrome acting together. There also appear to be intermediates between PFR and PR, and these may also play a role.

Test yourself

  1. What combination of primary-coloured lights would be perceived as yellow?
  2. What colour is cryptochrome?
  3. Order these percentage lettuce seed germinations. Seeds irradiated with:
    • Pure (spectral) yellow light.
    • Far-red light.
    • Yellow light obtained by mixing spectral red and spectral green light.

Answers

Bibliography

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