Secret Message at the Plant Surface

Abstract

In general, stomata open during the day and close at night. This behavior has a crucial importance because it maximizes the update of CO₂ for photosynthesis and minimizes the water loss. Blue light is one of the environmental factors that regulates this process. Certainly, when either entire plants or epidermal strips adapted to the dark are exposed to blue light, the stomata open widely their pores. But, what does happen if we illuminate individual stomata instead of peels or entire plants? In the inaugural issue of PLoS ONE, we have answered this question by irradiating individual stomata with a laser attached to a confocal microscope. Our study not only demonstrates that the stomata function independently from the behavior of their neighbors, and illuminates the implication of the blue light receptors PHOTOTROPIN1 and PHOTOTROPIN2 in such response. It also gives clues about the physiological relevancy of this behavior.

Illuminating Individual Cells

Driven by the desire to image biological events occurring in living organisms, Marvin Minsky, a pioneer in artificial intelligence and robotics, developed the basic concept of confocal microscopy in the mid-1950s. From its invention, we have experienced a tremendous explosion in its number of applications. Some of the most conventional ones include live cell imaging by attaching fluorochromes to subcellular components.^1–3 Confocal microscopy is also used to analyze functions, such as pH gradients and membrane potentials, and to measure intracellular changes in ion concentrations of molecules such as calcium, sodium, magnesium, zinc and potassium.^3–6 Imaging protein-protein interactions is also a target, among many others, of this versatile technique.^3,7

But confocal microscopy can be used not only to visualize the structural details and the dynamics of cellular processes, and to create images in three dimensions, it can also be used to modify these dynamics and/or structures. For example, twelve years ago, an unfiltered laser beam coupled to a confocal microscope was used to modify the cellular structure of the Arabidopsis root by ablating individual cells.⁸ The response of the root to the ablation experiments allowed uncovering the cell signalling underlying its development.⁸ Obviously, confocal microscopy can also be used to irradiate individual cells with a laser bean of defined wavelength and intensity. Because both light quality and quantity modulate many developmental and physiological processes at a cellular (stomatal opening) and even subcellular level (chloroplast movement), laser confocal microscopy can be used to deep into the cell and/or sub-cell signalling mechanisms underlying such as processes. For example, it is known that when either entire plants or epidermal strips adapted to dark are exposed to blue light, the stomata open their pores.^9–11 But, does anything happen if we illuminate individual stomata? Given that blue light induces stomatal opening exhibiting an action spectrum with a maximum at 450-nm and two minor peaks at 420-nm and 470-nm (ref. ¹²), specific confocal laser lines can be selected as light source of individual cells to study the cell signalling underlying this response.

The Autonomy of the Stomatal Response

Plants open stomata to allow carbon dioxide uptake. However, when they do it, tissues lose part of their precious water. Opening the stomata is then a crucial physiological decision, which is tightly controlled by both endogenous and environmental signals.¹³ The environmental factors that regulate stomatal movements include both blue and red light, carbon dioxide concentrations and atmospheric humidity.¹³ Little is known about the mechanisms by which stomata sense red light, carbon dioxide or atmospheric humidity. In contrast, blue-light stomatal perception is a well-characterized process. The blue-light receptors PHOTOTROPIN1 (PHOT1) and PHOTOTROPIN2 (PHOT2) mediate stomatal opening in a redundant manner:⁹ when peels of single mutants are illuminated with blue light, they retain the wild type response by opening their pores, but phot1phot2 double mutant lack such response. It is known that in the dark, phototropins locate to the plasma membrane.^14–20 However, blue light illumination induces the release of PHOT1 to the cytoplasm²¹ and the association of PHOT2 with the Golgi apparatus.²⁰

Equipped with both a 458 nm line and a 476 nm line of an argon laser attached to a confocal microscope, we addressed the cell signalling mechanism underlying the stomatal response to the blue-light. When individual stomata were illuminated, they opened their pores (Fig. 1). However, their nonirradiated neighbors remained unaltered (Fig. 1). This is telling us that stomata function autonomously in the blue light response, and that the signal that triggers stomatal opening does not transmit among stomata. The later conclusion contrasts with elegant work done by others, which highlights that stomatal responses to several stimuli seem to be dictated by the behavior of neighbor stomata.^22–25 The fact that the irradiation of neighbor epidermal cells did not induce opening of the adjacent stoma, extends the absence of cell signalling from nonstomatal cells to stomatal ones.

Figure 1.

Stoma autonomy in its blue light-induced response. When individual stomata of wild-type seedlings were blue-light irradiated with a laser coupled to a confocal microscope, they opened they pores. Unexpectedly, their dark-adapted neighbors experienced no change. This finding uncovers the stomatal autonomy in its blue-light opening, which contrasts with other works suggesting that stomatal opening is dictated by that of neighbor stomata. In addition, this induction of the stomatal opening in the wild type was disrupted in the phot1phot2 double mutant, which indicates that the stomatal autonomy depends on phototropins.

To unravel the hypothetical implications of both PHOT1 and PHOT2 in the autonomous stomatal opening to the blue light response, we took a combinatorial approach by illuminating individual cells in the phot1phot2 double mutant. We found that the induction of the stomatal opening in the wild type was disrupted in the double mutant (Fig. 1), which led us to propose that the stomatal autonomy depends on PHOT1 and/or PHOT2. Certainly, PHOT1 was released from the cell membrane to the cytoplasm in irradiated cells, illuminating the cellular mechanism that underlies this response.

The Physiological Relevancy

But, why did nature invent this behavior? While penetrating thought a forest, light suffers a reduction in quantity but, given the heterogeneous nature of the canopies, highly illuminated areas receiving sunflecks and deeply shaded areas can be observed at different scales on the soil surface (Fig. 2). This pattern is dynamic thanks to the movement of the foliage by wind and the changes in solar elevation throughout the photoperiod. Leaf surface is then a mobile mosaic constituted by lighted and shadowed patches. By measuring irradiance levels in transects across the boundary between shaded and light leaf areas, we observed that the transition can be relatively abrupt. This leads to large differences in light in short distances, which are in the range of the average distance between stomata. Then, the autonomous stomata behavior would contribute to optimize the balance between water loss and CO₂ acquisition, allowing the opening only of the illuminated paired guard cells.

Figure 2.

Photosynthetic active radiation (PAR) reaching the soil surface of understorey patches of (A) 20 × 20 cm2, (B) 4 × 4 cm2 and (C) 0.4 × 0.4 cm2. PAR reaching the patch containing the light grey bar in (A), and the dark grey in (B), are detailed in (B and C) respectively. PAR was measured with a radiometer Licor 250. The punctual sensor was placed in the centre of every patch. PAR measurements in (C) were obtained covering the sensor with an aluminium foil containing a 1 mm diameter-hole in the centre to perform milimetric measurements. PAR determinations in (C) were corrected multiplying the PAR values by a factor, which was obtained from the quotient between the PAR value determined under direct solar radiation with the uncovered sensor and he PAR value obtained with the cover. Sensor was mounted on a microscope to make sharply movements.

Addendum to: Cañamero RC, Boccalandro H, Casal J, Serna L. Use of Confocal Laser as Light Source Reveals Stomata-Autonomous Function. PLoS ONE. 2006;1:e36. doi: 10.1371/journal.pone.0000036.