How can an organism with no eyes sense light?
And how can they change their shape and metabolism in response?
Yet that’s exactly what plants do – detecting the intensity, direction, duration and even colour of light, and using this information to regulate the activity in their cells. Plants use light to direct the growth of their shoots and roots, the way that branches grow off the shoots, and how they reproduce.
Underlying these responses, biologists have found that light influences the activity of thousands of plant genes. Now researchers are uncovering precise chains of events that connect light to gene expression.
When we think of how organisms sense light, we naturally think of vision—animals with advanced eyes and brains constructing mental images of the world, more primitive animals sensing just shades of light and dark. Yet vision is only one response to light: outside our conscious awareness, even humans have others. Light detected by non-visual cells in the eye, for example, regulates our biological clock, influencing our patterns of sleep and activity. Ultra-violet light informs our skin of the intensity of sunlight, and induces skin cells to synthesise melanin.
Plants also detect light – an ability that allows them to adapt their growth and metabolism to their environment. The transition from dark to light at the soil surface, for example, induces germinating seedlings to unfurl their leaves and begin photosynthesis. Shoots maximise the light they capture by bending towards the sun – while bending away keeps roots below the soil. Plants detect the change in the colour of light as it shines through leaves above and around them, and in response often grow rapidly taller, helping them compete for sunshine. Finally, seasonal changes in day-length allow plants to fit cycles of dormancy, growth and flowering to the time of year.
Without anything as obvious as an eye to study, discovering exactly how plants detect and respond to light has been a slow and frustrating problem. Now, however, researchers are piecing together complete biological mechanisms—chains of events that link light perception by plant cells to changes in how plants grow. This booklet focuses on just one of these chains: a mechanism that may control thousands of plant genes in response to light absorbed by proteins called phytochromes.
Plant biologists have isolated three types of lightsensitive proteins – called photoreceptors – that control plant growth and metabolism. These are: phytochromes, which are most sensitive to the red region of the spectrum; and two classes of photoreceptors sensitive to both UV-A and blue light (see ‘Seeing blue’ below). Of these three, phytochromes were discovered first and have been studied most.
Plants make several different phytochromes and all of them respond to light in exactly the same way: to understand how, you need to know a little biological short-hand. Researchers investigating phytochromes divide the red region of the spectrum (approximately 620 – 800 nm) into two. They call wavelengths of 620 – 700 nm red, and those of 700 – 800 nm far red (see fig. 1). The reason for this distinction is that all phytochromes can exist in either of two forms: a form called Pr that absorbs ‘red’ light most strongly, and a form called Pfr that absorbs ‘far red’ light most strongly. The key to phytochromes’ function is that when either Pr or Pfr absorbs light, it is converted into the other form—a process called photoconversion (fig 2). So red light converts Pr to Pfr, and far red light converts Pfr back to Pr (for more detail, see Photoreceptors below)
Biologists believe that Pfr is the active form of phytochromes, transmitting signals to other molecules in the cell. Therefore, red light produces Pfr and switches signals from phytochromes ‘on’, whereas far red light removes Pfr and switches the signals ‘off’. To our eyes, the ecological significance of this is hard to guess. Although our retinas are less sensitive to far red than to red, the two colours look much the same to us. “In us, they are absorbed by the same receptor in the so-called ‘red cone cells’,” says Ferenc Nagy, a researcher on phytochromes at the Hungarian Academy of Sciences in Szeged, Hungary. “It follows that we perceive very intensive far red as red.”
With the aid of a spectrophotometer, however,the importance of red and far red becomes very clear—leaves absorb most of the red light that falls on them, but transmit or reflect most of the far red light. Therefore light shining through, or reflecting off, leaves contains a higher ratio of far red to red light than does pure sunlight. Our eyes don’t detect this change, but phytochromes do. In sunlight the proportions of red and far red light are fairly even and just over 50% of every type of phytochrome exists as Pfr. Under a deep canopy of leaves, however, the excess of far red light converts most Pfr to Pr so the proportion of Pfr falls to about 5%.
The changing concentrations of Pfr allow plants to adapt to sun or shade. For example, far red light often inhibits germination (see Page 6 The
discovery of phytochromes), preventing seedlings from emerging beneath other plants. As a response to low Pfr many plants also undergo ‘shade escape’.
They grow much taller stems, and produce fewer and more upright branches—putting their resources into upward rather than outward growth and making it more likely that they will outgrow the plants that are shading them.
Even in intense red light, some of every type of phytochrome exists as Pr. Referring to the absorption spectra of Pr and Pfr shown in fig. 2, suggest why this is so.
Mutant plants that are deficient in phytochromes display ‘shade escape’ (elongated stems and reduced branching) even in full sunlight. a) What does this suggest about the function of phytochromes in regulating shade escape? b) Does this support the theory that Pfr is the active form of phytochromes?
Light switches for genes
Phytochromes are always synthesised in their inactive Pr form, which only converts to Pfr after absorbing light. In seedlings grown in darkness, therefore, cells contain only Pr and so phytochromes are inactive. Shining light on such seedlings allows researchers to study what happens when signals from phytochromes begin. In this way, Nagy and his colleagues, together with researchers in Germany and Japan, have found that light does more than convert Pr to Pfr. It also makes phytochromes move.
The researchers fused genes encoding phytochromes to the gene for green fluorescent protein (GFP—a jellyfish protein with the useful property of glowing green after absorbing blue light) to form hybrid genes. They then inserted these hybrid genes into plant DNA, creating plants that synthesise hybrid ‘phytochrome-GFP’ proteins. Observing fluorescence in the cells of these plants let the researchers see the location of phytochromes under different conditions. In seedlings kept in darkness, fluorescence spread uniformly through the cytoplasm, suggesting this is the site of newly synthesised Pr. However, shining red light onto seedlings to convert Pr to Pfr caused fluorescence to move into the nucleus where it collected in discrete speckles (fig. 3a).
The speckles probably mark the sites at which phytochromes act in the nucleus and, according to Nagy, their size suggests that they contain large groups of proteins. “We do not have a definitive answer,” he says, “but we think that they represent large regulatory complexes containing about 50 protein molecules.” Recently, a separate line of research pointed to the function of these protein complexes—they may be involved in switching genes ‘on’.
Biologists have identified a nuclear protein called PIF3 (for Phytochrome-Interacting Factor 3) that can bind to phytochromes. The amino acid sequence of PIF3 shows that it is a transcription factor—a protein that binds to DNA to control gene transcription (the synthesis of messenger RNA). Completing the picture, researchers have now shown that PIF3 only activates gene transcription when it is also bound to a phytochrome, and that phytochromes only binds to PIF3 in the Pfr form (fig. 3b). This suggests that Pfr and PIF3 are two halves of a molecular switch that allows light to activate genes. Therefore, the speckles of fluorescence may be visible signs of Pfr binding to PIF3 and other proteins to activate gene transcription.
“At least in part, it would seem that this is what’s going on,” says Karen Halliday, a researcher at Bristol University who has studied PIF3. However, she warns that this has yet to be proved. “No-one’s done the biochemistry yet,” she says. “Until someone purifies the complexes, we do not really know.”
The effects of far red light on the fluorescent speckles support the hypothesis that they mark the sites of genes controlled by phytochromes. If PIF3 and Pfr do form the core of an ‘on’ switch for genes, then far red light should flip this switch ‘off’—far red light converts Pfr to Pr, which cannot bind to PIF3, and so PIF3 will stop promoting gene transcription (fig. 3b). Using GFP attached to a type of phytochrome called phytochrome B (phyB), Nagy has shown that far red light makes the fluorescent speckles vanish. “Far red does not induce phyB-GFP containing speckle formation,” he says “and far red treatment leads to the disappearance of red induced phyB-GFP speckles.”
Tobacco plants synthesising high concentrations of a hybrid phytochrome-GFP protein in their cells produce leaves at the normal rate but their stems are only about half the normal height. Given your answer to question 2, suggest an explanation for this.
|BOX 1 – MICROARRAYSMicroarrays—sometimes called ‘gene chips’—allow researchers to monitor the activity of thousands of genes in a single experiment. Microarrays consist of an ordered pattern of unique DNA samples dotted onto a glass chip: each sample corresponds to a particular gene in the organism under investigation.
Gene activity is monitored by extracting mRNA from cells and using it to construct labelled probes that bind to DNA on the chip. If a gene is active, its mRNA is present in the cell and the probe made from that mRNA binds to the corresponding DNA dot. The chip is then scanned by computer and the amount of probe attached to each dot is recorded to give an estimate of the activity of each gene represented on the chip.
We saw in Section 3 that phytochromes are always synthesised in their inactive Pr form, which only converts to Pfr after the first exposure to light. This event—the first exposure of a seedling to light—is more than a convenient moment for researchers to study: it also triggers the most dramatic change of growth and metabolism of the plant’s life.
Below ground, seedlings rely on nutrients in the seed and water from the soil to grow upwards by rapid stem elongation. They keep their leaves and/or cotyledons (seed leaves) tightly furled and hooked over (a pattern of growth called etiolation). After breaking into the light, stem elongation slows; the shoot straightens; and the leaves or cotyledons unfurl, synthesise chlorophyll and begin photosynthesis (a process called photomorphogenesis, or ‘de-etiolation’) (fig. 4, see also Web resources in Teachers’ Guide).
Researchers have used microarrays (see below) to investigate the changes in gene expression that underlie photomorphogenesis. Working with the weed Arabidopsis thaliana (thale cress)—a favourite subject for genetic experiments—they found, as expected, that light activates genes required for photosynthesis and inhibits genes needed to mobilise food stores in the seed. However, photomorphogenesis also affects genes involved in almost every other aspect of the seedling’s growth and metabolism, from hormone production to cell wall synthesis. In total, about one third of the seedling’s genes change activity. For Arabidopsis, this means that light regulates over 8,000 genes.
How many of these 8000 genes could be controlled by the phytochrome-PIF3 switch? “It’s a big, unanswered question,” says Halliday. According to Nagy, only 50 – 100 fluorescent speckles are visible in the nuclei of their plants. However, identifying genes regulated by PIF3 shows that the effects of phytochromes won’t be limited to 50 – 100 genes. Researchers have discovered that PIF3 activates the transcription of at least two genes that encode transcription factors—suggesting that phytochromes may trigger a domino effect known as a transcriptional cascade.
As the first step in the cascade, phytochromes bind to and activate PIF3 and probably several similar transcription factors, forming the protein complexes revealed by the fluorescent speckles. As the second step, each of these ‘primary’ transcription factors regulates the genes for several or many ‘secondary’ transcription factors. The secondary transcription factors do not have to bind to phytochromes to act and therefore do not show up as ‘speckles’. The cascade could then continue to ‘tertiary’ transcription factors, and so on. Between them, all the transcription factors in the cascade regulate the thousands of genes needed to control plant growth and metabolism.
According to Halliday, we are still a long way from understanding the full consequences of this cascade, and she stresses the phytochromes may also act in other ways. “It’s still unclear whether all the activity goes on in the nucleus,” she says. “There’s evidence of phytochrome action in the cytoplasm.” By interacting with proteins in the cytoplasm, she points out the phytochromes could trigger signalling pathways that control gene transcription indirectly
A seedling growing in total darkness initially displays rapid stem elongation, but after several days its growth slows and then stops. Suggest what is limiting the seedling’s growth?
Although the image of falling dominoes is very useful, it is already clear that the domino analogy is far too simple to explain plant responses to light. As described in Section 4, researchers have identified two transcription factor genes that are regulated by PIF3—genes in the ‘second layer’ of the transcriptional cascade. Other research, however, shows that these same two genes form part of the plant internal clock (the circadian clock: from Latin,circa; ‘about’, dies; ‘day’).
The two genes are most active at dawn and least active at dusk and this oscillating pattern continues for several days even if plants are moved to continual light or darkness. As a result, the two transcription factors encoded by these genes fluctuate in concentration and so control the plant’s gene expression according to the time of day. For example, helping to ensure that genes required for photosynthesis are most active around noon, whereas genes needed to protect the plant from cold have their highest activity just before dark. By controlling these clock-related genes in response to light, phytochromes provide one way in which the plant keeps its internal clock in time with the real cycle of nights and days. However, even this description is too simple. Nagy has shown that the speckles formed by phytochromes in the nucleus are themselves controlled by the internal clock. Shortly after nightfall, speckles start to disappear and shortly before dawn they begin to reappear. “We hypothesise that the circadian clock regulates the assembly and disassembly of the speckles,” he says.
Therefore, not only does signalling by phytochromes affect the clock, but the clock affects phytochromesignalling. This complex interaction may help explain the other major function of the clock—to measure the lengths of days and nights and so help the plant match its growth to the time of year. Such measurement requires the plant to distinguish between light perceived at different ‘times’ on its internal clock, and regulating phytochrome-signalling according to the time of day could be one way to achieve this.
|BOX 2 – PHOTORECEPTORSLiving organisms detect light using photoreceptors.These are signalling proteins bound to light-absorbing pigments called chromophores that regulate the protein’s activity. For example, light-sensitive cells in the human eye (‘rods’ and ‘cones’) contain photoreceptors called opsins (such as rhodopsin in rod cells). Each opsin is bound to a chromophore called retinal (a derivative of vitamin A) which blocks the opsin’s activity. However, when retinal absorbs light the retinal molecule changes shape and the bond between opsin and retinal breaks. This allows opsins to initiate signals within the cell, causing neurotransmitter release and a nerve impulse from the eye to the brain. Opsin signals are switched ‘off’ either when additional light absorption reverts retinal to its original shape,or when a new retinal molecule binds to the protein.
Photoreceptors in plants operate on similar principles. Phytochromes, for example, contain a chromophore called ‘phytochromobilin’ which is bound to the Pr form of phytochromes (see text) in a way that blocks phytochromesignalling. When the chromophore absorbs light, it changes shape and allows phytochromes to initiate signals to the rest of the cell. The change in shape also alters the sensitivity of the chromophore to light, favouring the absorption of far red rather than red wavelengths. Hence light absorption converts inactive Pr to active Pfr (see text). Like retinal, additional light absorption can convert phytochromobilin back to its original form—converting Pfr back to Pr and stopping signals from phytochromes.
In many plants, leaves are held horizontally during the day but drop to a vertical position at night. Experiments on beans in the 1920s found that these movements persisted even when plants were transferred to continual darkness. However, in these conditions the 24-hour rhythm became a 25.4-hour rhythm (a ‘circadian’ rhythm, see above).
Why did the fact that the rhythm was inaccurate in continual darkness (in this case, running slowly) demonstrate that the leaves were moving to an internal rather than external measure of time?
The Discovery Of Phytochromes
In the 1940s and 1950s, Harry Borthwick and Sterling Hendricks at the U.S. Department of Agriculture Research Station in Beltsville, Maryland investigated how seeds responded to different colours of light. Using the ‘Grand Rapids’ variety of lettuce seeds, they produced an action spectrum showing the extent to which light of different wavelengths promoted germination. This demonstrated that seeds germinated most readily when exposed to red light of wavelengths of 620 – 700 nm, and that the highest germination rate was given by wavelengths of around 660 nm. Borthwick and Hendricks speculated that seeds contained a light-sensitive pigment that promoted germination. Furthermore, they suggested that the action spectrum for germination corresponded to the absorption spectrum of this pigment, i.e. the proportions of light of different wavelengths that the pigment absorbed.
At what wavelength did Borthwick and Hendricks conclude that the pigment absorbed light most strongly?
In these experiments, about 20% of lettuce seeds germinated even in complete darkness. Although red light promoted germination, Borthwick and Hendricks found that seeds exposed to far red light – wavelengths of 700 – 775 nm – germinated at less than 20%. This suggested that seeds contained two pigments: a red absorbing pigment that promoted germination, and a far red absorbing pigment that inhibited it. The explanation to this puzzle came in 1952 when the researchers tried exposing seeds first to a flash of red and then immediately after to a flash of far red. They found that far red light reversed the effects of the red light. Red promoted germination: red followed by far red did not. What’s more, red and far red could be alternated many times and the germination rate always depended on the last colour given (fig. 5).
If the red-absorbing pigment that promoted germination and the far red-absorbing pigment that inhibited germin-ation acted independently, what would you expect to be the result of alternating red and far red illumination?
Red and far red acted as two halves of an on/off switch for germination. Red was ‘on’; far red was ‘off’. To explain this effect, Borthwick and Hendricks proposed that seeds did not have two independent pigments, but one pigment that existed in either a red absorbing or a far red absorbing form. They called the pigment ‘phytochrome’ (meaning ‘plant colour’), the red-absorbing form Pr, and the far red-absorbing form Pfr. They predicted that light absorbed by either form converted phytochrome into its other form: hence red light converted Pr to Pfr, and far red light converted Pfr to Pr (see fig 2).
Borthwick and Hendricks explained the on/off effect by assuming that Pfr, but not Pr, promoted germination. Seeds exposed to red light converted Pr to Pfr and germinated. However, seeds exposed to red light and then to far red light converted the Pfr back to Pr and so did not germinate. To complete the picture, the researchers assumed that lettuce seeds contained some Pfr even in darkness (made when the seeds were maturing) -so that a small number of seeds germinated without light. Seeds exposed to only far red light converted this original Pfr to Pr, reducing germination to below that occurring in darkness.
These conclusions were confirmed when phytochrome was isolated from seedlings in the early 1960s. Purified phytochrome existed as either bright blue Pr or olive green Pfr – colours invisible in the plant because the concentration of phytochrome was too low. We now know that plants contain several different phytochromes (called phytochrome A, phytochrome B, etc.), but all convert between Pr and Pfr as Borthwick and Hendricks predicted.
Why are Pr and Pfr different colours?
Further experiments on phytochromes have shown that when damp seeds are kept in darkness for several days, the Pr form of phytochrome A accumulates to concentrations about 100 times higher than those measured in the light. This makes such seeds extremely light sensitive: they can be induced to germinate by a brief, dim flash of light containing just 0.1 μmol photons m-2 (by comparison, full sunlight provides about 2000 μmol photons m-2 s-1).
a) How long would such a seed have to be exposed to sunlight to trigger germination?
b) How could this phenomenon be exploited to reduce the germination of weed seeds?
c) The epidermal cells of a plant embryo have a roughly rectangular external surface and are relatively small. Given cells with external surface dimensions of 20 μm x 10 μm, how many photons—as a number—will pass through an epidermal cell in an embryo exposed to a total of 0.1 μmol photons m-2 (assume that the cell is perpendicular to the light, and that the seed coat is perfectly transparent)?
Note: 1 mole (1 mol) is approximately 6.023 x 1023 (Avogadro’s constant).
This article was written by Stephen Day
|Absorption spectrum||A diagram illustrating the amount of light absorbed by a pigment across a spectrum of wavelengths.|
|Action spectrum||A diagram that illustrates how effectively different wavelengths of light induce a biological response, normally by plotting the amplitude of the response against the wavelength of illumination.|
|Chromophore||The light-absorbing pigment attached to a photoreceptor.|
|Circadian clock||An internal clock in a cell or organism that maintains an approximately 24 hour rhythm in the absence of external information about time. ‘Circadian’ is derived from the Latin, circa: ‘about’ and dies: ‘day’.|
|Cryptochrome||A member of a class of UV-A/blue absorbing photoreceptors that regulate several features of plant development, cf. phototropin.|
|Etiolation||The pattern of growth of seedlings in continual darkness, characterised by rapid elongation of a hook-shaped shoot with folded leaves and/or cotyledons (seed leaves), cf. photomorphogenesis.|
|Green fluorescent protein (GFP)||A protein originally isolated from the jellyfish Aequorea victoria. GFP fluoresces green after absorbing blue light.|
|Photoreceptor||A protein that initiates signalling pathways within the cell as a result of absorbing light.|
|Phototropin||A member of a class of UV-A/blue absorbing photoreceptors dedicated to phototropism and other light induced movements, cf. cryptochrome.|
|Photoconversion||The conversion of Pr to Pfr and vice versa by light.|
|Photomorphogenesis||The pattern of growth displayed by seedlings exposed to light, characterised by slow stem elongation, stem straightening, unfolding of leaves, synthesis of chlorophyll, and the initiation of photosynthesis, cf. etiolation.|
|Phototropism||Bending in response to the direction of illumination.|
|Phytochrome||A member of a class of red/far red absorbing photoreceptors. Phytochromes exist in two forms, the red absorbing Pr form and the far red absorbing Pfr form. Light absorption by Pr converts it to Pfr, and vice versa.|
|PIF3||A transcription factor that induces transcription when bound to the Pfr form of phytochrome.|
A form of phytochrome that absorbs red wavelengths most strongly.
|Pfr||A form of phytochrome that absorbs far red wavelengths most strongly.|
|Far red||The region of the spectrum with wavelengths of 700 – 800 nm.|
|Red||The region of the spectrum with wavelengths of 620 – 700 nm.|
|Spectrophotometer||A light meter that measures the intensity of light at different wavelengths.|
|Transcription factor||A protein that binds to the regulatory region of a gene to control gene transcription.|
|Transcriptional cascade||A process in which ‘primary’ transcription factors regulate the transcription of genes encoding ‘secondary’ transcription factors, and so on. In this way, the signal that regulates the primary transcription factors is amplified to control the activity of a much larger number of secondary and tertiary transcription factors.|