Infiltration – Penetrating the Leaf
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The following article was originally published in the journal for educators Biologia w Szkole (eng. Biology in School) (6/2019):
Plants, like animals, must exchange gases with their surrounding environment. Due to physiological differences, the mechanism underlying this process is, of course, different, but the common challenge remains the regulation of overall gas exchange. Plants, whose aerial parts are covered by a gas-impermeable cuticle, overcome this problem by employing specialized structures called stomata or stomatal complexes.
In dicots, some monocots, gymnosperms, ferns, and the sporophyte stage of mosses, the basic stomatal complex consists of two guard cells directly surrounded by epidermal cells. Typically, these guard cells are kidney-shaped and form an oval opening, also known as the stomatal pore. The thickening of their cell walls enables stomata to open and close efficiently, regulating gas exchange and transpiration. In other plant groups, stomatal complexes may exhibit structural variations, often incorporating additional subsidiary cells. Based on these variations, we can distinguish the following types of stomatal complexes:
- Anisocytic – featuring three subsidiary cells, one of which is smaller than the others,
- Diacytic – with two subsidiary cells arranged perpendicular to the stoma’s axis,
- Paracytic – with two subsidiary cells arranged parallel to the stoma’s axis,
- Tetracytic – featuring four subsidiary cells,
- Anomocytic – where the cells surrounding the stomatal complex are indistinguishable from the surrounding epidermal cells [1].
A distinction is sometimes made between stomata composed solely of two guard cells and stomatal complexes that include additional subsidiary cells.
Simple observations of these structures do not require complicated or expensive equipment. A standard school microscope—or even a homemade device built from a webcam, as described in an earlier issue of Biologia w Szkole [2]—suffices. In Photo 1, you can observe the stomata on the abaxial (lower) surface of grapevine leaves.
Even if we do not possess a microscope, can we examine the distribution of stomatal complexes on plant leaves and determine the influence of various factors on their aperture? The answer is yes, because such observations can be carried out using a brilliantly simple infiltration method developed by botanist Hans Molisch [3].
Experiment
In our experiments, we can use leaves from many plant species, for example, small-leaved lime Tilia cordata or large-leaved lime Tilia platyphyllos, common columbine Aquilegia vulgaris (which, since 2014, has been under partial species protection—therefore, experiments can only be performed on cultivated ornamental varieties of this plant), as well as many species from the genus Rosa [4]. In my experiments, I used leaves of the common grapevine Vitis vinifera, growing in my garden (Photo 2).
The common grapevine—often simply called grapevine or wine grape—is a species in the Vitaceae family. The natural range of the wild subspecies once spanned vast areas of the Mediterranean basin and southwestern Asia [5]. The cultivated grapevine, which constitutes a separate subspecies, has spread worldwide. The fruits are used to make wine, consumed directly (fresh or dried as raisins), and processed into juices, jams, and jellies. It’s also worth noting that a valuable oil is extracted from grape seeds.
Individual grapevine leaves are arranged in a spiral (or alternate) pattern, possess petioles measuring 4–8 cm (approximately 1.6–3.1 inches) in length, and have a palmate (hand-like) shape [6]. At their base, stipules appear and quickly fall off. Both dimensions of the lamina—that is, its length and width—are similar, usually ranging from 5 to 15 cm (about 2–6 inches).
For the experiment, we should select leaves that are undamaged, free of discoloration, and without signs of insect feeding. The experiment is best conducted on live leaves that remain attached to the plant and are well-exposed to sunlight. Of course, for the purpose of observation or photography, the leaves can be removed and relocated as needed.
Photo 3 shows a leaf selected for the experiment. It has been divided into two parts, labeled A and B. For clarity, the boundary between the areas has been marked with a black marker. We will apply the infiltrating agent to both parts accordingly.
The infiltrating agent can be any liquid that wets the cuticle—that is, the thin layer covering the outer cell wall of the epidermal cells present on the surface of all aerial organs of the plant, except for woody stems. The cuticle forms a continuous layer over the entire plant surface, except at the stomata. Due to its ready availability, low cost, and relatively low toxicity, kerosene will be used as the infiltrating liquid (Photo 4). We must remember, however, that kerosene can irritate the skin, its vapors are harmful when inhaled, and its flammability must also be taken into account.
The selected leaf should be moistened with a small amount of kerosene, for example, using a brush. In the case described, area A was moistened only on the adaxial (upper, typically sun-facing) surface of the leaf, while area B was moistened only on the abaxial (lower) surface.
After 10–15 minutes, the leaf was excised and placed against a dark, uniform background to enhance visibility (Photo 5).
In this way, we can observe that the areas of the leaf moistened with kerosene on the abaxial side (B) appear noticeably darker than those moistened on the adaxial side (A), regardless of the angle from which the leaf is viewed.
Observations can also be conducted using transmitted light—for example, by placing the leaf against a piece of tracing paper backlit by a lamp (Photo 6).
In this case, the opposite effect is observed: Area A appears darker than Area B.
From these observations, one can conclude that, for some reason, under the influence of kerosene the abaxial side (Area B) becomes more transparent, whereas no such change is observed on the adaxial side (Area A). Why is that?
Explanation
The cuticle is highly effective at preventing water loss, and even kerosene—which can wet it—does not penetrate through its layer. Instead, the liquid enters deeper tissues exclusively through the stomatal pores. Once kerosene passes through the stomatal complexes, it fills the intercellular spaces in their vicinity and further seeps into the crevices between adjacent cells, thereby increasing the transparency of the tissue. The difference in the experimental outcome depending on which surface of the leaf is moistened is due to the uneven distribution of stomatal complexes. In grapevine—as in many other plants—these structures are predominantly located on the abaxial (lower) surface of the leaves.
It is also possible to test other liquids that differ in their ability to wet the cuticle and penetrate the leaf, such as alcohol, petroleum ether, and other solvents. In each case, the proper safety protocols should be followed.
We know that plants possess the ability to regulate the aperture of their stomatal complexes, thereby limiting water loss under high-temperature conditions. By employing the method described above, we can indirectly observe the degree of stomatal opening relatively easily—whether at night, in the morning, during the peak heat of the day, or in the afternoon. Factors worth investigating include the intensity of light on the leaves and the ambient temperature. In every instance, the indicator of stomatal opening or closure—and any intermediate state—is the presence (or absence) and the rate of infiltration of the agent into the leaf. The observations described in the text were carried out in the late morning under relatively mild temperatures.
This method is straightforward and uncomplicated, making it ideally suited for use in school or hobbyist biology laboratories. I encourage readers to try their own experiments!
References:
- [1] Broda B., Zarys botaniki farmaceutycznej, Państwowy Zakład Wydawnictw Lekarskich, Warszawa, 1975, pp. 86-87 back
- [2] Ples M., Nieprzyzwoicie tani mikroskop (eng. Incredibly cheap microscope), Biologia w Szkole (eng. Biology in School), 4 (2015), Forum Media Polska Sp. z o.o., pp. 55-60 back
- [3] Grosse E., Z biologią za pan brat – eksperymenty biologiczne, Państwowe Wydawnictwo „Iskry”, Warszawa, 1969, pp. 74-75 back
- [4] Rozporządzenie Ministra Środowiska z dnia 9 października 2014 r. w sprawie ochrony gatunkowej roślin (Dz.U. z 2014 r. nr 0, poz. 1409) back
- [5] Pipia I., Gamkrelidze M., Gogniashvili M., Tabidze V., Genetic diversity of Georgian varieties of Vitis vinifera subsp. sylvestris, Genetic Resources and Crop Evolution, 61, 2014, pp. 1507-1502 back
- [6] Godet J.-D., Drzewa i krzewy, Multico Oficyna Wydawnicza, Warszawa, 1997, pp. 154 back
All photographs and illustrations were created by the author.
Marek Ples