Previously we have learned about responses to red and far-red light and the role of phytochrome. However it has long been known that plants respond to blue light in specific ways too. Only recently have the photoreceptors been clearly identified. Probably the oldest well-studied system of blue-light response is the phototropism of grass coleoptiles. The coleoptile (first leaf) of oats curves toward light as the seedling grows.
Even Darwin studied this response (though in canary grass rather than oats). The action spectrum of this response was developed early-on in these studies. Darwin showed that the site of perception is the tip of the coleoptile. Covering it with foil or removing it resulted in loss of perception and response. Frits Went showed that the actual curving response below the tip was signalled to curve by an asymmetric distribution of the hormone, indole-3-acetic acid (IAA), coming from the site of perception as a signal to the site of response, which we will learn about later.
To determine which photoreceptor might be receiving light to initiate the growth responses, scientists have used different wavelengths of light exposed to coleoptiles unilaterally and measured the curvature responses. This of course creates an action spectrum for phototropism in a coleoptile.
The action spectrum was compared to a range of absorption spectra of known plant pigments that could be candidates as the photoreceptor. Shown here are β-carotene, riboflavin, and zeaxanthin.
Since the action spectrum has a massive blue peak with two shoulders, the photoreceptor should as well. This makes riboflavin not such a good candidate for the photoreceptor. However, the UV peak in the action spectrum is missing in the absorption spectrum of β-carotene, making it also not such a good candidate. Thus it appears that zeaxanthin is the best choice of these three pigments to be the blue-light receptor pigment. It has both the UV peak and the tripartite blue peak. But further validation is needed.
Before we leave this introduction to phototropism in grass coleoptiles, it is also worthy to look at how blue light is distributed across an irradiated coleoptile. In the figure below, you can see that a coleoptile is irradiated from one side with blue light. A photodetector was inserted into the coleoptile tissue with micromanipulation to determine the intensity of the blue light perceived in various depths through the plant tissue. Tissue facing the blue light had much higher intensity of that blue light than tissue on the shaded side. And you can see some reflection enhancement of light as it bounces off of structures in the cell and cell wall environment. The same kind of fall-off and reflection enhancement can be observed when the probe is inserted through the hollow portion of the coleoptile. Could the different intensity of blue light be affecting growth on the lighted vs shaded sides? Is the the basis for the curvature growth? We shall see
A related example of phototropism is shown in the phototropic responses in dicots where several responses are shown here.
In the top panel, the a seedling is grown in darkness and you can see that its growth is straight up (probably a negative-gravitropism response only). When this seedling is moved into unilateral light, the hypocotyl curves toward the source of the light. In the lower panel, the dicot seedlings are growing with equal or unequal lighting on both sides. When the light is equally intense from both sides, the seedling grows straight, but if one source is brighter than the other, the seedling grows toward the brighter source. Also noticeable in this figure is the fact that greening of cotyledons is a light-induced response!
Blue light can also be shown to stimulate the transcription of mRNA for certain genes. In this case, GSA is and enzyme that operates early in the pathway for producing chlorophyll. This is what you were observing above in the greening of the cotyledons exposed to light in a previous figure. Of course the mechanism for turning on this gene expression is probably a cascade of signalling that we may learn more about later.
We have previously learned that light inhibits the elongation of the hypocotyl. And in the figure below you can observe that exposure to blue light causes an immediate depolarization and repolarization of membrane potential followed by an inhibition of growth. Hmm...this might have something to do with the response mechanism.
This depolarization of the membrane and RNA synthesis probably influences transport mechanisms which, of course, are critical for osmotic and pressure potentials in plant cells. Turgor pressure is responsible for the growth of a cell, so this could be leading to changes in growth.
A good example of cells that respond dramatically to osmotic and pressure potentials and light are guard cells. Indeed the guard cells open wider when illuminated with blue light.
Indeed we can observe this opening process graphically. Red light will cause a stoma to open partially, but blue light gives a much wider opening.
It is no surprise that this response to blue light has been characterized with an action spectrum showing the classic tri-partite peak that is similar to that of zeaxanthin.
It has also been shown that the stomatal opening correlates with the concentration of zeaxanthin.
In the natural world, there is a diurnal change in lighting with sunrise in the morning and sunset (and perhaps twilight) in the evening. The guard cells of a plant respond to these changes in sunlight. Morning light opens the stomata and evening darkness results in stomatal closure. The concentration of zeazanthin in guard cells fluctuates in a parallel fashion.
Further evidence for the role of zeaxanthin as the photoreceptor for the blue-light response of stomata is shown in mutant plants. In the panel below a mutant with a defective npq1 gene cannot make zeaxanthin, and its stomata cannot open in response to light even when the light signal is red.
The mechanism of stomata opening involves the movements of ions or solutes across the cell membranes of the guard cells to open or close the stoma between them. The ion involved is potassium and the uncharged solute is sucrose.
As you can see, the light (including blue!) comes on in the early morning. Ion pumps move Potassium into the guard cells. This would reduce their solute potential, and increase their turgor pressure. The guard cells expand as a result, pushing each other apart, and opening the stoma. As photosynthesis begins to function in the guard cell chloroplasts, sucrose is made and is used to keep water coming into the cell, keeping the turgor pressure high, and keeping the stomata open. However, by mid-day potassium is no longer needed for the purpose and the ions can be moved back into accessory cells from the guard cells (to return the next morning!).
Below are photographs of protoplasts (no cell walls) of two guard cells. In the left panel you can see the size of the cells while the cells are in the darkness. Then in the right panel you can see those same two guard cells after exposure to blue light are suddenly larger. Of course with walls shaping and connecting these protoplasts into a stomatal apparatus, the two enlarging cells would push each other apart and bend the cells in the middle forming an expanding stoma between the two cells (see above).
The mechanism for this rapid expansion obviously involves those ions and membrane pumps. The blue light exposure causes an acidification of the cell wall environment by proton pumping. As you can see below, as the intensity of blue light increases, the acidification of the medium around the guard cell protoplasts increases.
The proton pumping observed by blue-light exposure can be mimicked by using fusicoccin, a known proton pump stimulator. The acidification of the wall environment may optimize or degrade the environment for existing cell-wall loosening enzymes. These digest the cross-links between the microfibrils and can perhaps alter growth rates of cells. But in stomatal function, the pumping of protons may alter the way that other ions are moved in or out, and this can cause increases or decreases in turgor pressure, and thus can cause guard cells to open or close the stoma between them depending on the presence or absence of blue light.
The blue light responses have been known at least in general terms for more than 100 years. The identification of zeaxanthin as the blue-light photoreceptor has basically become "fact" only recently. The work with mutants for genes in zeaxanthin biosynthese have been critical in moving this topic from a lot of speculation to something closer to fact.