Histogenic protective reactions of plants and humic substances

When plants are poisoned by various substances that enter the xylem with the ascending current through the roots or directly into cut stems with water, a characteristic histogenic defensive reaction is observed. This consists of the browning of walls and the formation of a brown mass that blocks the conducting system, resulting in the cessation of the absorption of the harmful factor.

Thus, when keeping various plants with their roots in water or a nutrient solution containing allelopathically active substances, high concentrations of salts, or other toxic factors, by the 2nd or 3rd day, browning of the xylem walls occurs in the conducting zone of the roots, and more frequently in the higher parts of the stem's conducting system, especially in the nodes. This leads to the formation of blockages consisting of an amorphous brown substance.

Similar blockages in the xylem have been observed previously by other researchers, though they gave them a different interpretation. For example, we observed the localization of brown deposits in the xylem of wheat previously grown together with wheatgrass; however, we believed that this mass was agropyrene, a physiologically active triterpene secreted by wheatgrass rhizomes.

Blockages often arise when plants are affected by bacteria and fungi causing wilt diseases: wilt in tomatoes and cotton, Dutch elm disease, etc. Moreover, vessel blockage is often explained by the development of pathogenic microflora within them. It is also believed that the cause of blockage may not only be the developing mycelium but also specific wilting substances—marasmines. Some marasmines have been identified and obtained as pure preparations, for example, picolinic acid, lycomarasmin, phytonivein, fusaric acid, etc., although the mechanism of their effect on plants has not yet been fully revealed.

It is probable that, just as under the influence of an allelopathic factor, xylem blockage occurs in this case, accompanied by wilting and dying of the aerial part. Thus, xylem blockages arise during allelopathic, phytopathogenic, purely chemical, and other types of plant poisoning.

We hypothesized that the occurrence of blockages is a universal defensive reaction of the plant to the penetration of harmful substances. Such blocking allows the plant to at least temporarily stop the intake of the toxin and avoid immediate death. Over time, the plant develops lateral roots and additional xylem, acquiring a succulent structure to evaporate less water and thereby reduce the intake of the toxin into the aerial part. For example, an increase in leaf succulence under the influence of allelopathy has been described. Blocking xylem cavities allows the plant to delay immediate death from poisoning and, if the toxic factor acted temporarily, to survive the unfavorable period.

We visualize the mechanism of blockage formation in xylem vessels as follows. The harmful factor, having entered the dead xylem vessels, penetrates through the cells into all tissues of the stems and leaves and, apparently, most severely affects the living parenchyma cells located adjacent to the xylem.

In this process, protective histogenic reactions can be divided into two types. First of all, various protective substances flow from the entire plant, and especially from the leaves, to the site of the strongest irritation of the living tissue adjacent to the xylem. Due to these substances, the respiration intensity of the affected tissues increases and, likely, the neutralization of toxic substances is carried out through their metabolization and oxidation.

If the intake of poison continues, the plant fails to completely inactivate the toxin in this way, and protective substances leak into the xylem vessel cavity, where the second stage of protective reactions begins. Due to a sharp change in environmental conditions and the disruption of the spatial separation of substances in the xylem sap, autolysis occurs, leading to the denaturation and decomposition of proteins and other plant cell components, and to the formation of polymeric, partially oxidized compounds. These precipitate, accumulating in certain parts of the vessels and blocking them.

Blocking the vessel cavities may subsequently cause the inhibition or death of plants if they do not manage to regenerate the vascular-conducting system in time. Thus, the mechanism of protective reactions is dual: initially, the plant attempts to inactivate toxins biochemically by increasing respiration and biological oxidation in the zone of toxin penetration. Subsequently, blockages develop, preventing the intake of poisons that the plant is no longer able to cope with biochemically.

Of course, such a division is conditional, because the blocking mass acts not only as a barrier to the ascending current but also as a means of inactivating toxic substances through adsorption and precipitation. As our histochemical studies have shown, the brown mass consists of poorly soluble or insoluble compounds. Its composition changes during formation: initially, glucose, pectin substances, phenolic compounds, and lignin are detected, and in the final stages, melanins are found in the composition of the brown substance. The full composition of the brown mass has not been studied, but it is likely very complex.

Since vessel blockage is found under the most diverse harmful influences, and as it is known that humic compounds often mitigate the harmful influence of various factors, we became interested in the question of the possible influence of humic substances on the formation of blockages and, through them, on the strengthening or weakening of the plant's defensive reactions. Verifying this assumption was the goal of the present work.

METHODOLOGY AND MATERIALS

Humic substances were obtained as follows: 50 g of peat or humus was poured into 150 ml of 0.1 N KOH and filtered after 16 hours of infusion. To 50 ml of the obtained filtrate, 8 ml of 1 N H2SO4 was added, heated in a water bath to precipitate organic colloids, and filtered. Humic acids remained on the filter.

The residue on the filter in our studies was taken as the full dose of humic acids or was divided into two, four, and eight parts and added to the water containing the test plants. Thus, the concentration of humic substances corresponded to a solution from 50 g of peat (humus) in 200, 400, or 800 ml of water, i.e., it was quite high. We did not aim for precise dosage, as we were interested only in the qualitative result.

The experiments also investigated aqueous extracts from humus and peat; 80 g of air-dry humus or peat was poured into water and the volume brought to 500 ml. After 24 hours of infusion, the extracts were used for the experiment.

To study the influence of humic substances on the defensive reactions of a plant organism under the influence of allelopathically active substances, we prepared aqueous extracts from dry rhizomes of quackgrass (wheatgrass) Agropyron repens L. and dry aerial mass of red clover in a ratio of 1:10. Humic substances were added to 200 ml of the wheatgrass and clover extract, and plants dug up with roots from the field were placed in them.

For the study, we used plants of winter rape Brassica napus var. oleifera D. variety Mytishchinsky No. 1, oats Avena sativa L. variety Lgovsky 1023, and strawberry Fragaria ananassa Duch. variety Kievskaya Early. Control plants were placed in tap water, and experimental ones in extracts according to the scheme presented in Table 1.

The plants were kept in the extracts under laboratory conditions and studied on the 5th and 7th day after the start of the experiment. Microscopic sections were made manually at the sites where the brown substance blocking the vessels appeared: in winter rape—in roots, stems, and leaf petioles; in oats—in tillering nodes and stem nodes; in strawberries—in stems and leaf petioles. The presence of pathological changes in the vessels was studied on microscopic sections. For the study, 5 plants were taken, and 30–50 sections were viewed under the microscope.

Results and Discussion

The results of the experiments are given in Table 1. In control rape plants, on the fifth day after the start of the experiment, a substance blocking the vessels is detected. This phenomenon can obviously be explained by the fact that rape is an allelopathically very active plant, and harmful substances accumulated under it in the water, causing self-poisoning.

It should be noted, however, that the amount of vessel-blocking substances and the extent of blockages in control rape plants are incomparably smaller than in those kept in the experimental extracts. The introduction of humic substances obtained from humus and peat into the water did not have any effect on the formation of blockages in rape.

In strawberries, on the 5th–8th day under these conditions, browning of xylem vessels occurred in petioles and stems; however, the full dose of humic acids from humus somewhat mitigated this process, and no browning was detected in the petioles. In stems, browning of xylem walls and blockages appeared only by the end of the experiment.

In oats, conversely, the full dose of humic acids from humus increased the formation of blockages in xylem cavities. Thus, humic acids diluted in water increased the formation of blockages by the end of the experiment.

Humic acids obtained from humus and added to wheatgrass extracts did not have a special effect on the plants, and the vessel-blocking substance appeared in approximately the same amount as in the pure wheatgrass extract without humic substances. At the same time, humic acids obtained from peat significantly softened the allelopathic influence of the wheatgrass extract on oat and strawberry plants, as a result of which only traces of a yellowish mass blocking the vessels were noted on the seventh day of the experiment. In rape, in the wheatgrass extract, even diluted with water (1:1), humic acids obtained from peat did not have a positive effect.

In the concentrations we studied, they significantly softened the allelopathic influence of wheatgrass extracts on the studied plants. Clover extracts are more toxic to plants than wheatgrass extracts. Humic acids from humus and peat enhance the allelopathic effect of clover.

In these variants, in all plants studied by us, the substance blocking the vessels or causing the browning of their walls is detected in larger quantities than in the pure clover extract. Strawberry is a very sensitive plant. On the 4th–5th day of the experiment, strawberry leaves partially turn brown and dry out while green, while in oats and rape, leaves gradually turn yellow and lose color: chloroplasts are completely destroyed in them. This phenomenon is obviously related to the fact that in rape and oats, harmful substances, having penetrated the plant, act more softly and poison it gradually.

Reserve substances of the leaves are consumed to increase respiration intensity and play an important role in the formation of protective reactions. Allelopathically active substances contained in the clover extract, having penetrated strawberry leaves, exert a sharp toxic effect on chloroplasts, resulting in leaves turning brown and drying out. It is likely that humic acids in the clover extract intensify the pathological process. Diluting the extract with water did not matter for the strawberry.

Aqueous extracts from humus and peat had the same effect on plants as pure humic acids. Thus, humic acids obtained from humus and especially from peat reduce the allelopathic influence of wheatgrass on plants to a certain extent (the number of blockages decreases) and enhance it in extracts from red clover (blockage intensifies).

These studies confirm our assumption that the brown substance arising in xylem vessels is a defensive reaction of the plant organism to the harmful element that has penetrated it. Moreover, humic acids manifest their effect in two ways: in one case, the number of blockages decreases and, consequently, the biochemical intoxication of wheatgrass secretions intensifies; in the other, blocking increases, which is apparently associated with the strengthening of the defensive function under the influence of humic acids.

The data we obtained allow us to conclude that humic substances are capable of increasing the resistance of plants to the effects of allelopathic factors.

Table 1. Influence of Humic Substances (HS) on Xylem Damage Caused by Allelopathic Factor

Note: — damage absent; + vessels damaged; ++ vessels damaged heavily; +++ vessels damaged in very large numbers; X vessel damage is weak; 0 study was not conducted.