Infiltration – Penetrating the Leaf
| Polish version is here |
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.
You do not need complicated or expensive equipment to observe these structures. Even a regular school microscope is enough, or a homemade device built from a webcam, as described in an earlier issue of Biologia w Szkole [2]. In Photo 1, you can see stomata on the lower (abaxial) 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
For our experiments, we can use leaves from many plant species, such as small-leaved lime Tilia cordata, large-leaved lime Tilia platyphyllos, or common columbine Aquilegia vulgaris. Since 2014, the columbine has been under partial species protection (in Poland), so experiments should only involve cultivated ornamental varieties. We can also use many species from the genus Rosa [4]. In my own experiments, I used leaves from 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. They have petioles that typically measure between 4 and 8 cm (approximately 1.6 to 3.1 inches) in length and a palmate, hand-like shape [6]. Stipules appear at the base of the petiole but fall off quickly. The leaf blade is usually 5 to 15 cm (about 2 to 6 inches) long and wide, with both dimensions being roughly equal.
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 capable of wetting the cuticle, which is the thin layer covering the outer cell walls of epidermal cells found on the surface of all aerial parts of a plant, except woody stems. The cuticle forms a continuous coating across the plant’s surface, interrupted only by the stomata. Because of its availability, low cost, and relatively low toxicity, kerosene will be used as the infiltrating liquid (Photo 4). However, it is important to remember that kerosene can irritate the skin, its vapors are harmful if inhaled, and it is also highly flammable.
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.
You can also conduct observations using transmitted light by placing the leaf against a piece of tracing paper illuminated from behind with 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 effectively prevents water loss, and even kerosene does not penetrate through its layer. Instead, the liquid reaches deeper tissues only by entering through the stomatal pores. After passing through the stomatal complexes, kerosene fills the nearby intercellular spaces and seeps into the crevices between adjacent cells, which increases the transparency of the tissue. The difference in the experiment’s outcome, depending on which side of the leaf is moistened, results from the uneven distribution of stomata. In grapevine, as in many other plants, these structures are primarily located on the lower (abaxial) 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.
Plants have the ability to regulate the opening of their stomatal complexes which helps limit water loss during high temperatures. Using the method described above we can indirectly and fairly easily observe how open the stomata are whether at night, in the morning, during the hottest part of the day, or in the afternoon. Factors to investigate include the light intensity on the leaves and the surrounding temperature. In every case the indicator of whether the stomata are open, closed, or partially open is the presence and speed of the agent infiltrating the leaf. The observations mentioned here were conducted in the late morning when temperatures were relatively mild.
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