On the Nature of the Physiological Activity of Humate
The influence of physiologically active forms of humic acids and related compounds, such as polyphenols, on oxidative metabolism can be considered a firmly established fact. Different researchers provide various explanations for this phenomenon. For many years, we have maintained the view that the key lies in the quinone groups of these compounds, which act as hydrogen carriers from the oxidized object to oxygen, thereby enhancing the crucial electron transport system and, thus, alleviating oxygen deficiency in cells. Observations show that such deficiency occurs more frequently than commonly believed. All this leads to an increase in the energy potential of plants, which is experimentally confirmed by measurements of bioelectric potential (BEP), indicators of the thermodynamic state of plants, and positively affects the organism's metabolism.
One of the most daring biochemists, Szent-Györgyi, proposed the idea that all biochemical processes represent a gradual descent of an electron elevated to a higher level as a result of absorbing a quantum of light during photosynthesis. Bernard and Alberte Pullman, in turn, stated: "The dynamism of life aligns with the dynamism of the electron cloud in conjugated molecules..." They considered the main feature of these molecules to be the presence of delocalized electrons, which provide the molecule with additional stability, resistance to radiation, the ability to transfer electronic excitation over long distances, and the capacity for electron and energy transfer, among other properties.
Delocalized or unpaired electrons gained such significance because they are excited more easily than paired electrons. The excitation of an electron, i.e., its transfer from a lower to a higher energy level, is determined by the absorption of a quantum of visible or ultraviolet light. The reverse process is accompanied by the emission of a quantum and the release of light, i.e., luminescence. Such electron transfer corresponds, as it were, to the intramolecular level of redox processes. Intermolecular transfer of a delocalized electron is already associated with redox processes at the intermolecular level, i.e., with respiration. It is quite obvious that these two processes are interconnected, and since the influence of humic and fulvic acids on respiration processes is a firmly established fact, it can be assumed that they also affect electron excitation.
There is reason to believe that physiologically active forms of humic and fulvic acids, once absorbed by plants, influence the tuning of energy levels of delocalized electrons in conjugated molecules and, thus, affect vital processes. If this assumption is correct, then, firstly, physiologically active humates should promote better absorption of solar energy, with their effectiveness varying across different parts of the spectrum; secondly, they should influence the paramagnetic properties of plant tissues; and thirdly, humates should enhance their luminescent properties. Without aiming to quantify these processes or uncover their mechanism, we undertook this study solely to experimentally test the proposed hypothesis.
Research Methodology
To address the tasks at hand, short-term microvegetation experiments were conducted under different spectral conditions. The experimental methodology was as follows: seeds of corn and mung beans were placed in germination chambers, soaked in solutions according to the experimental design, and placed in a dark thermostat. Then, five- to seven-day-old etiolated seedlings were transplanted into 500 ml containers, five plants each, onto a medium corresponding to the experimental design, and placed under a light setup in compartments with different colored screens. The setup was illuminated with fluorescent lamps at 3500 lx/m². The screens were prepared by applying colored films, obtained using the Grodzinsky method, onto glass plates measuring 36 × 48 cm. Their spectral characteristics were determined using an SF-4A device.
The experimental design included potassium humate, tested at concentrations of 3.1⋅10⁻⁵ mol/L, and ATP—1.4⋅10⁻⁵ mol/L (the latter as an additional control, being an energy-rich compound). The medium was either a depleted Prianishnikov mixture, where phosphorus was provided in the form of Sorensen's buffer salt, or distilled water. The experiment was conducted in triplicate. It lasted for 7 days, after which the plants were measured, weighed, and analyzed for the following parameters: chlorophyll content (using the Getri method), nucleic substances (using the Netubskaya and Kuramshin method). A total of 27 such experiments were conducted.
To investigate whether soluble humates influence the paramagnetic properties of plant tissues, the electron paramagnetic resonance (EPR) method was employed. As is known, the essence of the EPR method lies in the resonant absorption of high-frequency electromagnetic energy by an object under strictly defined relationships between magnetic field strength and frequency. The EPR method is based on the well-known Zeeman effect, which states that when a paramagnetic particle, characterized by the presence of an unpaired electron and a quantum spin number S, is placed in a constant magnetic field, its primary energy level splits into 2S+1 sublevels separated by energy intervals:
ΔE=hν=gbH, where H is the magnetic field strength, ν is the frequency.
When a high-frequency (HF or microwave) magnetic field with a magnetic vector orientation perpendicular to H is applied to a paramagnetic sample placed in a constant magnetic field "H," transitions between two adjacent levels are induced with equal probability at the frequency:
ν=hΔE=hgbH
where:
- g is the spectroscopic splitting factor;
- b is the Bohr magneton;
- h is Planck's constant.
This leads to a redistribution of the population of energy levels toward equalization, with part of the microwave energy being absorbed by the sample and converted into heat. The goal of the experiment when observing the EPR phenomenon is to accurately register the high-frequency energy absorbed by the sample. Many scientists have studied the phenomenon of electron paramagnetic resonance in a range of biologically active compounds and living tissues and demonstrated its effectiveness for this purpose.
For our research, we used leaves and roots of five- to six-day-old mung bean seedlings grown under specific conditions, which were subjected to lyophilic drying after freezing to −70°C. They were dried for 24 hours to a residual pressure of 10⁻³ mmHg. For EPR studies, samples of dried leaves and roots weighing 3-5 mg were placed in thin-walled quartz tubes with a diameter of 4-5 cm, which were annealed in an acetylene-oxygen flame. EPR spectra were recorded as the first derivative of the resonance absorption curve. The content of free radicals in the sample was determined by comparing the standard and the areas under the integral curves of resonance absorption of the test substance, which were constructed from differential curves I′=f(H) at ν=const. The areas were calculated using the double graphical integration method. For measuring the line widths of the studied spectra, γ-irradiated alanine served as the standard, and for determining the areas, γ-irradiated sucrose was used. To determine the g-factor, evacuated tubes with a standard of Mn++ in MgO were used, for which the g-factor values are precisely known. For approximate calculations of the areas under the integral curves, Simpson's formulas were employed.
To assess the influence of humic acid on the luminescence of plants grown under different spectral conditions, the following procedure was used: a sample of leaf tissue was ground with water in a 2:100 ratio and left for some time. Then, 1 ml of fluorescein (10⁻³ mol/L) was added to a specific volume of the filtered extract, irradiated with ultraviolet light (Wood's glass) for 5 minutes, after which the relative fluorescence intensity was determined.
Research Results
First, let us analyze the most characteristic data showing the influence of humic acids and ATP on the accumulation of fresh mass under different spectral conditions. These data indicate that the accumulation of fresh mass in corn seedlings was maximal in the red-orange and blue-violet parts of the spectrum and minimal in the green part. The response of plants to physiologically active forms of humic acids and ATP was similar.
It is easy to see that the response of corn seedlings to physiologically active substances in terms of fresh mass production mirrors the light absorption curve of chlorophyll, suggesting that physiologically active substances influence the photochemical reactions of photosynthesis and promote better absorption of solar energy. This experiment was repeated multiple times, yielding similar results with some shifts in the peak of mass accumulation within the violet part of the spectrum. Mung beans were less responsive in this regard.
The influence of physiologically active substances affects not only fresh mass production but also plant morphogenesis, and this effect differs from that on weight. It was found that roots respond relatively better to humates in the blue-violet part of the spectrum, while leaves respond better in the red-orange part.
Observations on the influence of the studied factors on chlorophyll content showed that the peak response in corn shifts from the short-wavelength to the long-wavelength part of the spectrum. In some cases, the peak occurs in the yellow-green part of the spectrum. The response of plants to changes in spectral composition in terms of chlorophyll content is quite consistent and more related to light intensity. Observations on nucleic acid content indicate that the action of physiologically active substances under different light qualities undoubtedly affects this apparatus of the plant cell. However, discrepancies in data across experiments do not yet allow for a clear determination of the direction of this process.
EPR measurement data and corresponding calculations are presented in Tables 1 and 2. These data show that the EPR of lyophilized mung bean tissues is represented by single lines with a g-factor close to that of a free electron (for leaves, gavg is 2.00388±0.0005 and ΔHavg−18.1±0.2 G; for roots, gavg−2.00349±0.0005 and ΔHavg−13.5±0.2 G).
| Indicators | Seeds germinated on | |||||
|---|---|---|---|---|---|---|
| Water | Potassium humate | ATP | 2,4-DNF | |||
| Seedlings transplanted to | Water | Potassium humate | ATP | 2,4-DNF | ||
| ΔН G | 15.9 | 18.2 | 16.4 | 19.0 | ||
| g-factor | 2.00441 | 2.00279 | 2.00489 | 2.00438 | ||
| S-integral (mm²) | 150 | 220 | 120 | 175 | ||
| Note. For the γ-sucrose standard, containing 10¹⁷ spins/g, the S-integral is 455 mm². | ||||||
| Indicators | Seeds germinated on | |||||
|---|---|---|---|---|---|---|
| water | potassium humate | ATP | 2,4-DNF | |||
| Seedlings transplanted to | water | potassium humate | ATP | 2,4-DNF | ||
| ΔН G | 11.2 | 14.5 | 16.3 | 9.6 | ||
| g-factor | 2.00461 | 2.00440 | 2.00473 | 2.00380 | ||
| S-integral (mm²) | 20 | 40 | 60 | 12 | ||
Comparing the differential curves reveals that potassium humates and ATP increase the amplitude of the EPR signal, while 2,4-dinitrophenol quenches it. Importantly, additional transplantation of plants from this inhibitor to physiologically active substances partially restores the EPR signal, which correlates with the physiological response of plants to these substances. Their influence on the integral curve was similar, suggesting an increase in the number of free radicals under the influence of physiologically active substances. It is also noteworthy that the number of free radicals in leaves was significantly higher than in roots.
The physiological action of humic acids should be linked to the presence of free radicals in them. The presence of free radicals in soil humic acids was later demonstrated by EPR studies conducted in our laboratory.
The degree of luminescence in extracts from plants grown under different spectral conditions and treated or untreated with physiologically active substances varies. A trend toward increased luminescence effects from potassium humates in the red-orange part of the spectrum was observed. The influence of ATP remained unclear.
Conclusions
- The obtained results first and foremost demonstrate that the response of plants to physiologically active forms of humates varies across different parts of the spectrum: it is greater in the red-orange and blue-violet regions, i.e., in the parts of the spectrum that are better absorbed by chlorophyll.
- EPR signal measurements in mung bean tissues showed that physiologically active substances increase the number of paramagnetic centers in them.
- The above facts, along with changes in the luminescence intensity of plant extracts in the presence of fluorescein, suggest that physiologically active humates promote the absorption of light quanta and influence the tuning of energy levels of delocalized electrons in conjugated molecules that constitute plant tissues.