Studying some features of maize genetics and developmental biology using electrophysiological techniques --Lysikov, VN In the Republic of Moldova, the possibility of employing electrophysiological techniques was first studied on maize plants. The experimental evidence suggests that precisely these techniques provide new insights into extremely involved processes of maize genetics and developmental biology.

Modern breeding programs are aimed at producing heterotic maize hybrids. This involves identification in parental lines of valuable traits and features associated with their combining ability. All experimental evidence from parental lines of hybrids can be classified into two groups: static parameters recorded as dots on the plotting paper, and dynamic parameters representing temporal variation of the developmental process which are recorded as lines on the plotting paper.

Dynamic indices are known to generally provide more complete and adequate information about the occurring processes and are especially good indicators of the effect of environmental factors on an individual cell, tissue, organ, or a whole organism (plant). Electrophysiological techniques allow, without disturbing the plant's vital activity, the dynamics of the processes occurring in the plant to be demonstrated and the results recorded as plots, curves and diagrammes.

That is why electrophysiological techniques were used in obtaining numerous unambiguous electrophysiological data for generative and vegetative organs of maize lines, mutants and hybrids. The essential feature required of the measurement procedure was that the effect of the recording instruments on the pattern of life activity be minimum, because this is the only way to ensure that the data obtained are as highly informative as possible.

Based on these specific requirements, all the electrophysiological techniques employed can be classed into two groups: (1) intracellular recording of bioelectrical potentials (biopotentials), and (2) extracellular measurement of biopotentials. In our study, the second group was represented by two independent types of experiments: (1) measurements performed in artificial conditions - greenhouse, hothouse, climate chamber, laboratory, etc.; (2) extracellular biopotential measurements on plants in the field.

In addition to these two major methods of studying electrophysiological properties of maize, work has been done on recording dielectric properties of maize plants, measuring the electric resistance of stem and pistil, and the electric charge of pollen grains, as well as some other work which is touched upon in passing in the present study.

The intracellular recording of biopotentials was performed by Dr. A. I. Doukhovny. Not only did he carefully study maize pollination and fertilization processes, but he also developed a number of methods and approaches, being one of the first to carry out investigations like these.

For measuring the bioelectrical potential of an individual plant cell, conditions ensuring minimum alteration of the cell should be observed. This is only possible where the cell wall size is an order of 2 to 3 larger than the area of perforation resulting from the microelectrode introduction into the cell. To this end, glass microelectrodes were made in the form of micropipettes whose tip diameter was no more than one micron. Thin Pirex glass tubes were used to produce micropipettes.

Using a device called microforge, the tubes were fixed at both ends and split in half in the middle. The resulting two microelectrodes were filled, under vacuum, with 2.5 M solution of KCl. A filled microcapillary is a microelectrode. This was connected to a mercurous chloride or silver chloride macroelectrode.

A microelectrode produced in the above manner was introduced, using a micromanipulator under constant visual control in a binocular microscope, into the plant cell. The biopotential recording itself was carried out on DC electrometric amplifiers of the UI-2 type at whose outlet quick - response self-balancing EPP potentiometers were connected which recorded the signals on the diagram paper. In addition, connected to the same electrometric amplifiers were electron oscillographs, enabling visual observation of the rapid processes occurring in the cell.

It should be noted that maize proved to be a very convenient experimental plant for intracellular biopotential measurements. This is due to the fact that the female reproductive organ of maize, the ovary in particular, has the style with an elongated stigma often called the pistil filament.

When expanding in cross section, the maize pistil filament is very similar to an asymmetric eight figure with a slight depression at the centre. Through the centre of each half of the figure of eight run vascular bundles normally composed of 3 to 6 strands extending from the stigma to the ovary.

The maize pollen grains and elongating pollen tubes act on pistil tissues as an effective combined mechanical stimulus. Preliminary experiments showed that it is the vascular strand cells that are capable of receiving the stimulus signals and transforming them into electrical signals, and transmitting the latter over particular distances. In vascular strand cells, the resting potential (i.e. the transmembrane difference of potentials of a nonexcited cell) is several dozen millivolts higher than that in the surrounding stigmatic cells, and is normally in excess of 80 mV. Electrophysiological specificity like this allowed reliable identification of the cells under study.

Another important finding of the preliminary experiments is the one-way conductance (transmission) of action potentials in the direction of the ovary, which is probably due to physiological and biochemical polarity of the pistil.

The initial bioelectrical response of the maize pistil prior to pollen germination is manifested in the generation of a single impulse of the action potential, with an amplitude of more than 20 mV and pulse duration of 2.5 to 34 sec in the pistil filament and 1.3 to 2.0 sec in the ovary. Generated 3 to 18 min after pollen application, the first impulse travels in a nondecreasing manner at a rate of 12 mm/sec, such that in 20 to 30 sec, depending on the pistil length, it reaches the ovary. It has been found that prior to generation of action potentials, the resting potential of the excited cells increases by 16 to 27 mV.

As a result of further pollen growth, another two impulses are generated whose characteristics are very similar to the first one. One of these arises in the pistil filament 64 to 83 min and the other 49 to 67 min following the first single impulse. These two impulses differ from the first one in that they show a higher travel speed. What we deal with here is probably the "duration" effect. This process takes about 1.5 h to complete after the start of pollen germination. It is worth noting that the essential difference of these impulses from the first one is that they produce (elicit) in the ovary only a local response of resting potential fluctuation, with an amplitude of several millivolts.

It has been shown that the incoming action potentials are transformed at the pistil base. With the start of more intensive elongation of pollen tubes and of their active penetration into the pistil tissues, the stage of higher electrical activity commences, characterized by the generation of a large number of impulses of action potentials which alternate with the local electrical response.

Impulses with an amplitude of 15 to 39 mV travel, almost nondamped, towards the ovary for nearly 1.5 h. The transformation of the incoming action potentials is most pronounced during the subsequent time interval, which starts 97 to 130 min after pollen application, and lasts for about 70 to 80 min. Generation and transmission of a large number of single impulses of action potentials occurs presumably due to higher metabolic rates. It is these impulses, travelling in a nondecreasing manner, that control rhythmic generation of potentials in the ovary.

The rhythmic electrical activity of the ovary manifested as rhythmic generation of resting potentials is a two-stage process. The duration of the first stage is about 35 min and of the second stage about 20 min, such that the second stage starts, on average, 38 to 41 min after the completion of the first one.

A distinctive feature of the first stage is a lower frequency of action potentials, longer pulse and between-pulse duration with the advance of the stage (i.e. towards its end).

Seven pulse trains have been identified and characterized, each with its particular constant or smoothly varying frequency of potential generation. In transition from one pulse train to another caused by single pulses arriving at the ovary from the pistil filament, the frequency of pulse generation varies in a saltatory manner. Furthermore, the transition between some pulse trains is due to an additional increase in the resting potential.

The second stage of rhythmic activity comes about 40 min after the first one and consists of three pulse trains differing in the potential shape and frequency. Characteristic features of the second stage are an increase in frequency action potentials and a decrease in frequency of resting potentials towards the end of the stage, respectively up and down to the values corresponding to those of a nonexcited cell.

The period of multiple pulse generation in the pistil filament, like that of rhythmic pulse generation in the ovary, appears to be the most functionally loaded one. The subsequent bioelectrical response of maize pistils is characterized by generation of widely separated single action potentials which reach the ovary without being transformed at the style base. The type (pattern) of response remains unchanged throughout the pollen tube elongation period.

By the time of pollen tube penetration into the embryo sac and subsequent fertilization, the generation of single action potentials is terminated, and the resting potentials begin to exhibit wave-like variation characterized by rhythmic fluctuations distorting the smooth shape of the wave. Two wave-like variations are the most characteristic ones: the first, in order of appearance, and the fourth or fifth. The distinctive feature of these patterns of variation is a brief (12 to 45 sec) increase in the potential with an amplitude of 2 to 9 mV.

The first characteristic wave-like variation gives rise to a wave of excitation, which is recorded in maize stems during extracellular measurement of biopotentials. This wave originates at the base of the pollinated ear and spreads up and down the stem at a rate of 40 to 61 cm/min. The amplitude is not the same at different points in the stem: a maximum amplitude of up to 18 mV was recorded at the base of the stem, and up to 17 mV at the point of ear attachment. The duration of the process in maize lines and hybrids varied between 20 and 50 min.

Thus, the results from the above experiments suggest that the bioelectrical potentials traveling through maize generative organs and detectable during intracellular measurements, and the ones traveling through maize stems and detectable during extracellular measurements, offer a tool for analyzing complex concurrent, but spatially isolated, physical and biochemical reactions in a living body.

It has been suggested that recording ten thoroughly studied pulse trains on a magnetic tape and their subsequent application (presentation), through specially implanted microelectrodes, using electronic instruments and observing the timing, the pattern, the specificity and sequence of pulses may be of interest in studying some issues such as apomixis or other involved processes associated with fertilization.

Based on the method of multichannel measurement of bioelectrical potentials with the aid of macroelectrodes, A. I. Doukhovny designed, using a DC electrometric amplifier and a self-balancing twelve-point potentiometer, a special switch enabling simultaneous measurement of potentials at different points in the plant. He demonstrated that normally stabilized, more or less invariable, frequently straight, and parallel bioelectrical potential lines appearing on the plotting paper prior to pollination start, upon landing of pollen on the stigma, to change their direction such that, not infrequently, they "tremble", occasionally become nonparallel to one another, and even intersect.

Pollination and fertilization have been shown to result in higher electrical activity at various points in the stem. An increase in the number of "intersections" in the stem always occurs more smoothly than does the variation of bioelectrical potentials in the pistil, although in the stem they persist, at a particular level, for 2 to 3 days.

In other words, the maize plant "trembles" in terms of electrophysiological parameters, resulting in a higher number of intersections and nonparallel biopotential lines on the plotting paper.

Application of extracellular biopotential measurement techniques deserves to be considered in greater detail. The largest number of experiments employing the techniques have been carried out by S.N. Maslobrod in the laboratory and by F.G. Oloer in the field.

Based on the topography of maize leaf and plant surface biopotentials, S.N. Maslobrod established the existence of more than one type of bioelectrical polarity (right and left), and outlined the bioelectrical stereopolarity and the dynamics of polarity from the seed stage up to the mature plant stage. Subsequently he studied the spatiotemporal organization of maize surface biopotentials in terms of electrophysiological polarity, oscillation, and signal generation and transmission.

Based on the assumption of endogenous rhythmicity of plant biopotentials being genetically preprogrammed, S.N. Maslobrod suggested that the function of genes responsible for a particular trait be regarded in terms of operation of an electrophysiological oscillator with a given frequency range, and that the structural gene be represented as a "wave gene" sui generis, which is in good agreement with the "wave" function of gene activity proposed by Chirkov (Chirkov, 1994).

S. N. Maslobrod made an electrophysiological evaluation of plant genotypes differing in phenotype, including marker lines, lines with high general combining ability, thermotolerant genotypes, samples showing general (nonspecific) ecological stability, cold-hardy forms, ancestral forms, as well as specimens exhibiting high competitiveness under overcrowding. The results of his studies can be presented briefly as follows:

1. Phenotypic characters of the genotypes: It has been established that maize seedlings with marker pigmentation traits differ in the amplitude of response to light and temperature treatment.

2. Ancestral forms of maize are characterized in terms of general ecological stability of genotypes. They exhibit higher ecological stability as compared with cultivated forms. Their ranges (amplitudes) of electrical response are narrower whereas the electrical response values of the cultivated forms are 2 to 3 orders of magnitude higher.

3. General combining ability of lines: Positive correlation has been established between the level of general combining ability of maize lines (topcross) and positive biopotential values, in particular for coleoptile and leaf biopotentials.

4. Heat tolerance of maize lines: Heat-tolerant maize lines have been shown to differ from heat-sensitive ones in that they exhibit smaller amplitudes of electrical response to alternate light / darkness exposure at 40 C and to variation in temperature between 20 C and 40 C.

5. Cold tolerance of maize lines: It has been demonstrated that maize lines whose cold tolerance is due to the genotype or environmental factors exhibit smaller amplitudes of electrical response to a sharp drop in temperature (5 C and more) and retention of amplitude during repeated exposure.

6. Competitiveness of genotypes: Viewing competitiveness as tolerance of maize lines and hybrids to overcrowding, the author distinguishes competitive and less competitive lines by: 1) lower electrical resistances of root contacts of seedlings within the group, and 2) better synchronization, i.e. by the uniformity of morphological, physiological and electrical variables; normally, higher absolute values of these variables are observed in plants belonging to the group.

Based on the above theoretical points, S.N. Maslobrod devised a number of express methods of practical importance for which he obtained author's certificates. Among these, the following should be mentioned first of all:

1. Express methods for estimating stimulative doses of gamma and laser irradiation in presowing treatment of maize seeds.
2. Express methods for producing bioisomeres (right and left) in maize plants.
3. Electrophysiological express methods for evaluating economic traits in maize such as heat tolerance, cold tolerance and competitiveness.
4. Electrophysiological methods for evaluating genotypes of evolutionarily different plant forms (wild, cultivated and segregating).
5. Electrophysiological methods for estimating the effects of physical factors: rhythmic light, weak current, etc.

Studies by S.N. Maslobrod yielded numerous data characterized by a high degree of novelty. Among these, the following should be mentioned.

A. Maize plants have been found to possess two induced electrophysiological stereopolarities of the mirror type, the so-called "flat structure" and "hollow structure". They depend on the environmental factors and on the pattern of maize plant architectonics. Structural and functional elements of stereopolarity have been identified.

B. The existence of an intimate relationship between maize plant electrophysiological stereopolarity and disymmetry (left and right symmetry) has been established, and the morphological role of the former with respect to the latter demonstrated.

C. It has been shown that plants, as well as plant communities (phytocenoses), represent ensembles of electrical and physiological oscillators whose degree of adjustment to one another determines competitiveness of the components of a unified oscillatory system.

D. Using maize as an experimental object, the ability of action potentials to propagate from one plant to another through the mechanism of electromagnetic induction has been demonstrated.

E. Maize plants have been found to be capable of responding electrically to unconventional stimulants (gamma and laser irradiation) and of assuming the state of total electrical excitation via the mechanism of spatial synchronization of action potentials.

F. Action potentials of maize plants have been shown to be able to transmit information about stereoscopic structure of the object and to coordinate functions of the underground and above ground plant parts upon exposure to light.

In light of the above, the use of stationary (time-independent)potentials to test maize growing capacity and productivity, as well as ecological stability, is justifiable.

It is also logically justifiable to employ the method of electrical control of adaptive potential of maize plants by simulation of their electrophysiological parameter adjustment and optimization.

Summarizing S. N. Maslobrod's studies, we can say that the formation and maintenance of spatiotemporal organization of maize plant surface biopotentials is ensured by electrophysiological control systems, performing in a plant body the functions of nutrient and energy transfer, and transmission of information, including that of plant body stereoscopic structure.

An extensive and interesting study of electrophysiological methods directly in the field has been carried out by F.G. Oloer. To this end, nine mutant lines derived by maize experimental mutagenesis from a single VIR-44 line, and one single-cross hybrid synthesized from lines derived from mutants, were examined. The above mutant lines differed considerably from their original line in morphological and agronomic traits.

For experiments in the field, a special experimental plot consisting of 100 test strips whose area totaled 700 sq.m, was established. Each strip was sown to 12 plants including 9 mutant lines, 1 hybrid and 2 control plants (VIR-44 line). For randomization, each strip had its own, differing from the others, order of positioning of mutants and controls.

Measurement of electrical parameters of maize mutants was performed using a specially re-equipped unit MTL-62(magneto-telluric laboratory) mounted on a bus. The additional equipment included: (a) DC electrometric amplifiers A1-2, EPP-09 potentiometers, a PSR-1 potentiometer, an S1-1 oscillograph, a set of meteo instruments, a transportable electric power station with power generating capacity of 1 kWt and a knockdown screening box 200x150x150 cm in size.

A biopotential measurement procedure suitable for field studies was developed. Thus, measurements were carried out simultaneously on 6 plants, 5 of which were mutants and 1 control. Measurements were performed using nonpolarizing silver chloride electrodes of the 5268-AgCl-180 type. A total of 36 electrodes with agar-agar adapters were used. Biopotentials were measured on the lower leaf surface of maize plants at points located 3/4 of the leaf length from the stem. Reference electrodes were placed at the base of the plant.

The biopotentials measured were transmitted by wires to 6 specially designed arithmetic units, automatically calculating arithmetic means of biopotentials for each particular mutant. While still measuring biopotentials, each arithmetic unit was, one by one, connected in a certain order to the amplifier general input via a specially designed automatic switch. Connected to the amplifier output via a compatible voltage divider was a self-balancing potentiometer of the EPP-09 type, on which recorder chart averaged values of biopotentials were recorded for each mutant.

To control the magnitude and pattern of electromagnetic disturbance, connected to the A1-1 amplifier output was an S1-1 oscillograph offering visualization of the disturbances on the screen.

In order to overcome the adverse effects of electromagnetic disturbance in measuring biopotentials, a combination of measures was taken including: (a) balancing of electric parameters of input measuring circuits with respect to earth, (b) use of braided (shielded) wires, (c) employment of high-frequency filters, and (d) compensation of variable and constant disturbances. The above combination of measures allowed measurements of biopotentials in the field without shielding boxes. However, in a few isolated instances, a knockdown shielding box was used.

Another peculiarity of measuring biopotentials on plants in the field is the need for simultaneous recording of environmental quantitative indices such as light intensity, soil and air temperature, soil moisture and air humidity, etc.

To examine the sensitivity of mutants to environmental factors, abrupt alternate switches from light to darkness were performed by obscuring the box or using a light-proof screen, or by employing a red or blue light filter. The results from studies of bioelectrical indices of mutant sensitivity were processed on the BSM-4 computer by solving a multiple regression equation by the least squares method.

In addition to general regularities in the topography of distribution of biopotentials on the plant vegetative organs (leaf, stem), F.G. Oloer proposed an ingenious technique for characterization of mutants by: (a) bioelectrical light sensitivity, (b) bioelectrical moisture sensitivity, and (c) bioelectrical thermosensitivity.

It was demonstrated that when the degree of exposure to one or another meteorological factor is varied, mutants tend to adjust (adapt) to new environmental conditions by changing their bioelectrical sensitivity to a particular environmental factor. Differences among mutants in bioelectrical sensitivity are readily detectable under low light intensity. Thus, mutants with dark-green leaf color (No 61 and No 67) exhibited high values of bioelectrical light sensitivity whereas low values were observed in mutants with light-green leaf color (No 35 and No 149).

With varying air humidity, mutants change their bioelectrical moisture sensitivity. Thus, with relative air humidity ranging from 70 to 90%, bioelectrical moisture sensitivity of both mutants and the control is insignificant or even negative in sign. Outside this range, bioelectrical moisture sensitivity changes abruptly and reverses its sign.

Of particular interest here is mutant No154, whose absolute value of moisture sensitivity is relatively small over a wide range of air humidities. Morphological features of this mutant are its long and narrow leaves which do not twist or wilt even under drought conditions. The opposite is observed in the control: its leaves exhibit severe wilting and twisting under drought.

Of considerable interest is also mutant No56. It has very high bioelectrical moisture sensitivity of negative sign. During the morning dew, it folds its leaves in a peculiar way, like a closed book, thus probably retaining moisture for a longer period of time, resulting in that its leaves do not wilt or twist during drought periods.

Under temperatures ranging from 20 to 30 C, bioelectrical thermosensitivity is relatively low in both mutants and the control. With temperature decrease below 20 C, bioelectrical sensitivity rises sharply. Thus, in mutant No149, bioelectrical thermosensitivity exhibits a sharp increase in the direction of positive sign.

A peculiar feature of this mutation (No149) is that on exposure to lower temperatures (15-19 C) its biopotentials decrease, such that its leaves begin to show anthocyan in coloration. In another mutant (No56),under lower temperatures (15-19 C), bioelectrical thermosensitivity continues to be high, but is of negative sign. Biopotentials in this mutant are, nevertheless, higher than in the control.

Analysis of biopotential variation during ontogenesis revealed that maize plants (both mutants and the control) are capable, in the course of their development, of increasing their biopotentials from 5-30 mV to 50-70 mV at the stages of 3-4 leaves through flowering. Following the flowering stage, biopotentials start to decrease, being reduced to zero at full maturity.

Most significant differences in the magnitude of biopotentials between the control and mutants are readily observable in the wax stage. Admittedly, some mutants (tall, polyphyllous and multi-ear) compare favorably with the control in that their biopotentials are higher throughout the growth season.

Interestingly, while analyzing coefficients of correlation between bioelectrical parameters and some breeding characteristics, F. G. Oloer established correlation between biopotentials of mutants during grain filling and their general combining ability (r=0.75+0.13) determined by the topcross method.

It proved possible to make early prediction of general combining ability of mutant lines at early stages of ontogenesis. Thus, correlation was established between bioelectrical thermosensitivity at the stage of 5-7 leaves and general combining ability (r=0.44+0.29).

Direct correlation was also established between biopotentials of mutants at the grain filling stage and their yielding capacity at full maturity (r=0.51+0.26).

Very interesting data were obtained while examining bioelectrical characteristics of maize mutants following artificial stimulation. Thus, sharp changes in light intensity, temperature or air humidity result in plant biopotential variations. Sunlight is the most powerful stimulant for maize mutants. As short as 10 min shading of maize mutants with an opaque screen against the sunlight induces biopotential oscillations which are not damped until 10 to 15 min later.

It was found that at the beginning of exposure to light (following shading), biopotentials are shifted towards positive values. Most commonly, the first positive amplitude of biopotential oscillations is reached as soon as the first minute of exposure to light. It is at minutes 2 to 7 of light exposure that the biopotential reaches a maximum negative value. It was established that at this point in time the stomata show intensive opening, the biopotential is shifted towards positive values and exhibits a few damped oscillations, equivalent to the original ("shaded") level.

It should be noted that in some mutants biopotential oscillations may last for as long as a few hours. Interestingly, in cases like this, the frequency of biopotential oscillations coincides with the frequency of stomatal pore oscillations.

It must be emphasized that the most important parameter of mutant biopotential variation in the shade-light transition is the first amplitude, which is an indicator of the magnitude and sign of electrical charges formed on leaves in the first minute of light exposure, and the slope of the curve which represents biopotential variation precisely at the initial moment of light exposure.

With increasing light intensity, the first positive amplitude increases, but its growth stops under very high light intensity, and there sets in, as it were, the saturation effect. The rate of biopotential variation is also increased, but without saturation.

It is in the above two parameters that nearly all mutants differ among themselves. They can be classed, as it were, into two groups: (1) mutants superior to the control in these two parameters and exhibiting high intensity of photosynthesis (No61 and No122), and (2) mutants inferior to the control in the above two parameters (No149, No700, and No67).

Of particular interest here is that the curves representing decreasing biopotentials under shading and those representing increasing biopotentials after removal of shading may be regarded as mutant specific.

Ingenious studies with a view to developing techniques for identifying the characteristics of maize mutant lines by kernels were carried out by M.E. Volinsky using the method of extracellular biopotential measurement. He succeeded in demonstrating the possibility of identifying maize mutant lines by sprouting kernels, such that particularly clear-cut results were obtained on exposure of the sprouted kernels to extreme factors, such as temperature (hot water). Curves representing the damping of biopotentials due to mortality of plants from exposure to superhigh doses of extreme factors also proved to be mutant specific.

Acknowledgments: The author is deeply grateful to G.K. Lakhman for translating the text into English.

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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