The Physiology of Plant Integrity1 V

TRENDS AND HYPOTHESES

The Physiology of Plant Integrity1 V. V. Polevoi Department of Plant Physiology and Biochemistry, St. Petersburg State University, Universitetskaya nab. 7/9, St. Petersburg, 199034 Russia; fax: 7 (812) 427-7310; e-mail: [email protected] Received December 27, 2000

Abstract—The original concept of the plant integration system is presented and exemplified by the data from the studies of the regulatory controls that mediate the effects of red light (R) on the growth of etiolated maize seedlings. The integrity of higher-plant behavior depends on the functional activity and interaction of the dominant (control center). In vegetating plants as the simplest case, these centers include the shoot apex and the distal part of the root comprising the sensory tissues, the zones of the synthesis of specific hormones, and the zones of high morphogenetic and sink capacities. The system of propagating electric signals is usually devoid of the permanent generation centers. The dominant centers recognize the external and internal signals and induce the development of polarity (the bioelectric and physiological gradients), canalized connections (the conducting bundles), and oscillations. Trophic, hormonal, and electrophysiological signals of intercellular regulation are propagated along the conducting bundles and affect the intracellular membrane, metabolic, and genic control systems. The regulatory controls comprise the receptor cells recognizing the external and internal signals, the tissues of connection channels, the effector cells, and the feedback loop elements. When three-day- old etiolated maize (Zea mays L.) seedlings are treated with red light (RL), the photosignal is recognized by the phytochrome in the cells of the mesocotyl intercalary meristem; as a result, the positive biopotential is prolonged in these cells and in the coleoptilar node. An electric field (the receptor potential) thus produced would hamper, by electroosmosis, IAA transport from the coleoptile into the mesocotyl and in this way, would dras- tically inhibit the growth of the latter and temporarily promote the growth of the former. The primary leaves, also recognize R, as a result R promotes cell growth and the synthesis of gibberellins. The Ukhtomskii’s principle of the dominant is used to interpret the plant ability for switching over its physiological systems in response to specific signals.

Key words: Zea mays - plant integrity - dominant centers - canalized connections - bioelectric potentials - regulatory controls - red light - electroosmosis

INTRODUCTION In higher plants, there are up to 80 specialized cell types, tens of particular tissues, 15 to 25 organs, and Speaking of an organism as a whole, we imply that 11 systems providing such functions as aerial nutrition it is an integral system. The integrity of the organism is (mostly leaves), soil nutrition (roots), respiration, long- the capacity of its parts to coordinate their functions in distance transport, excretion, plant movement and sup- the course of individual development and when inter- port, vegetative and sexual propagation, defense, and acting with the environment. The attribute of integrity hormonal and electrophysiological regulation [1]. All was improved over the course of biological evolution, these parts and functional systems in the whole plant and the progress in the differentiation of organisms (the organism switch over interactively in the course of suc- specialization of their parts) occurs in parallel with the cessive phases and stages of plant ontogeny and con- elaboration of the unifying systems providing for the tribute to plant adaptation to changing environmental integrity. conditions. Some people believe that higher plants are less dif- M.Kh. Chailakhyan specially emphasized the ferentiated than animals and plant parts possess consid- importance of the problem of integrity. In his mono- erable autonomy. This is not quite true. None of the iso- graph Tselostnost’ rasitel’nogo organisma (The Integ- lated plant organs can persist for a long time if they do rity of Plant Organism) published in 1955, Chailakhyan not regenerate the missing parts of the whole organism. was the ﬁrst to unravel this fundamental issue of general biology as applied to higher plants. In this book [2], in the papers to succeed this book [3, 4], and in his sum- marizing monograph Regulyatsiya tsveteniya vysshikh 1 Presented at the Sixth Chailakhyan Lecture (Moscow, April 22, 1999). rastenii (Regulation of Flowering in Higher Plants) [5], particularly in the chapter on integrity in plant develop- Abbreviations: BEP—bioelectric potential; MP—membrane ment, Chailakhyan listed several phenomena related to potential; R—red light. plant integrity: anatomical and morphological entirety,

546 POLEVOI polarity, trophic and hormonal interactions among trol of enzyme activities), and membrane systems (bar- plant parts, and excitability. The latter is the ability of rier, transport, energy generating, receptor and other an organism as a whole to efficiently respond to exter- functions). As multicellular organisms evolved and dif- nal and internal stimuli. Referring to Ivanovskii [6], ferentiated, the mechanisms for the interactions among Chailakhyan enumerated four successive phases of cells, tissues, and organs emerged and became more excitability: (1) the recognition of the external stimulus and more elaborated. These intercellular control sys- by a receptor organ, (2) the development of specific tems, in the broadest sense of the term, include trophic, metabolites, (3) the translocation of these metabolites hormonal, and electrophysiological (local and propa- to other organs, and (4) the physiological and morpho- gating electric potentials) systems. Intracellular control logical responses of these organs [5]. As early as 1956 systems are closely interactive. Similar interactions are [3] Chailakhyan proclaimed that organ interaction in found at the intercellular level, and the intracellular the course of plant growth and development employs control systems are governed by the signals generated these four phases of excitability. In their numerous and by the intercellular systems [1, 20Ð23]. conclusive experiments, Chailakhyan and his associ- The trophic interactions of plant tissues and organs ates demonstrated that an adequate photoperiod is rec- have been studied in detail. They include the transloca- ognized by the leaves, where a specific hormonal signal tion of assimilates from the leaves into the heterotrophic is synthesized; the signal is transported to shoot apical tissues and organs and also the transport of water, min- meristems and induces the flower development and the eral salts, and specific metabolites from the roots into transition to reproduction [5]. Similar processes lead to the underground and aboveground shoots [12, 14, 19]. tuber initiation on the stolon [7]. The experiments proved that sex differentiation in dioecious plants depends Plant hormonal regulation comprises the synthesis; on the hormonal interaction of leaves and roots [8]. transport; immobilization and release; degradation; the The studies by Schmalhausen [9] on the interaction actions and interactions of such well-known phytohor- of differentiation and integration processes, by Dostal mones as auxin (IAA), cytokinins (zeatin, isopenteny- [10] on organ correlations, by Sabinin [11, 12] and Kur- ladenine, and their derivatives), gibberellins, abscisins sanov [13, 14] on metabolic and trophic interactions (ABA, etc.), ethylene; and other natural growth regula- between shoots and roots, by Gunar [15] and Opritov tors, such as brassinosteroids, jasmonates (jasmonic [16, 17] on plant excitability, and by Mokronosov [18, acid and its derivatives), oligosaccharins, etc. [21, 24Ð 19] on sourceÐsink relations were instrumental for 26]. The organization of the hormonal control has not developing the concept of plant integrity. Chailakhyan been studied systematically. In animals, such a system wrote: “The notion of the organism as an integral whole is built hierarchically, and with plants, a similar princi- is one of the major principles of studying living Nature. ple must hold. However thoroughly the researchers are engaged in the IAA and cytokinins are apparently the major agents study of a particular aspect of structure and physiology of the plant hormonal system. When plant tissues are and however deeply they are involved in unravelling the cultured in vitro, no other phytohormones are usually mysterious puzzles of life using such models as iso- added. IAA and cytokinins are produced in the central- lated organs, tissues, cells, protoplasts, and organelles, ized way, in the shoot and root apices, respectively. all scientists must invest their data and hypotheses into These hormones induce cell division, growth, and dif- the integral pattern of the living organism” [5]. ferentiation and switch on two major genetic programs: Yet the mechanisms of integration in plants have not the development of shoots and roots [22, 27]. The pos- been properly elucidated, and the general systematic itive feedback interactions of two phytohormones pro- theory of plant integrity has not been developed. In ani- vide the progressive development of the whole plant. mals and humans, the coordinating activity of the neu- IAA induces the development of root initials, thus rohumoral system is the basic mechanism providing for enhancing the cytokinin synthesis by the roots; in turn, their integrity, with the neural (electrophysiological) due to the elevated level of cytokinins, new metameres control coupled with the endocrine (hormonal) system are initiated in the shoot apex. We can, therefore, define playing the leading role. these hormones as participants in the central hormonal system, with IAA playing the leading role. Unlike other This review attempts to be a concise description of phytohormones, IAA affects bioelectrogenesis [22, the universal integration system in plants; the descrip- 28]: the hormone induces BEPs and MPs, brings about tion is illustrated by the experimental data, which cell polarization, and moves in polar directions via the exemplify the regulatory controls operating in plants. living cells of conducting bundles in the oscillating regime at the rate of 0.5Ð1.5 cm/h. The low rate of IAA BASIC CONCEPTS AND MECHANISMS movement is the bottleneck of the hormonal system; it OF PLANT INTEGRATION SYSTEM is an additional proof of the principal role of this hormone. Meanwhile, other phytohormones, such as When unicellular organisms evolved, intracellular abscisins and ethylene, called stress hormones, are syn- control systems developed, such as genic (the synthesis thesized in all plant tissues and organs. Mutants defi- of proteins, enzymes in particular), metabolic (the con- cient in ABA or ethylene do not manifest morphoge-

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THE PHYSIOLOGY OF PLANT INTEGRITY 547 netic abnormalities under optimum conditions, but are The dominant center in the shoots of vegetating unable to survive under extreme conditions. We there- higher plants comprises the shoot apex, the initiating fore define such phytohormonal systems as adaptive. and developing leaves, buds, and internodes; the dominant center in root includes its apex, root cap, and the ÇÂıÛ¯Í‡Shoot apex ÔÓ·Â„‡ zones of growth and differentiation. Higher plants have ← ⊕ àìäIAA two types of dominant centers, because they dwell –– –– simultaneously in two environments, that is, soil and ñËÚÓÍËÌËÌ˚Cytokinins ⊕ ← atmosphere. Shoot movements (elongation, leaf äÓÌ˜ËÍRoot apex ÍÓÌfl unfolding, and tropisms) aim at the optimization of aerial nutrition, whereas root elongation and tropisms The electrophysiological control system comprises aid better water and nutrient absorption. Shoot and root BEP gradients and oscillations, as well as the propagat- apices are the first to encounter a new environmental ing electric potentials (action potentials and variation situation. We can, therefore, compare the behavior of potentials) [16, 17, 29Ð32]. Plants are usually devoid of the distal root part to that of the head of an earthworm, the permanent centers that generate electric potentials, the sensor and dominant center of its body. and the latter arise everywhere under stress conditions. Thus, the shoot and root dominant centers are the In animals, the electrophysiological and hormonal organs commanding the whole plant to accomplish its systems are in close interaction. Some evidence sug- major strategic tasks: to develop all of its parts and to gests that similar interactions can be found in plants as optimize its nutrition-using sensory mechanisms and well. As already mentioned above, IAA generates slow efficient movement activity. These centers are the bioelectric waves. The gradients of electric potentials growing tissues of high and prolonged morphogenetic affect IAA transport [33]. The electric potentials prop- activities, which produce the specific hormones of local agating from the sites of the local application of a stress and distant action. The dominant centers manifest high agent have been shown to shift the hormonal balance in attracting activities, that is, they are sinks for trophic remote parts of the plant [34, 35]. The problem of the factors and physiologically active compounds; they functional interaction of the hormonal and electrophys- comprise the sensory zones and the receptors for phy- iological systems in plants has been posed, and vast tohormones. By using the hormonal signals and com- experimentation will solve this problem. peting for nutrients, the dominant centers produce diverse control effects on the functional activities of tis- The intracellular and intercellular control systems sues and organs in the whole plant. Among the domi- listed above establish the basis of the organism level of nant centers, we find the apices of various vegetative regulation and integration. In animals, it is the central shoots, the distal root parts, and the developing flowers, nervous system, which functions to control and inte- fruits, tubers, and taproots. The fact that there are the grate all physiological systems for particular tasks. To primary and auxiliary shoots with the apical buds of consider the plant as an integral organism rather than a various orders and the primary and lateral roots with colony of cells, one must first answer the question as to their apices implies the rigorous hierarchic interactions whether or not plants comprise centralized control between the dominant centers of various orders. The organs. Darwin was the first to suggest that such organs mechanism of such interaction apparently involving exist as exemplified by shoot and root apices [36]. hormonal, electrophysiological, and trophic factors has In his experiments, Darwin demonstrated that shoot not been studied sufficiently, and we will discuss it in and root apices are sensory zones that specifically rec- more detail in our next paper. ognize the effects of environmental factors, such as The existing evidence suggests that the integrating light, gravitation, mechanical pressure, etc., and send function of the dominant centers is accomplished by the signals dominant the growth and movements in developing polarity, canalized connections, regulatory shoots and roots. This concept was lost in present-day controls, and oscillations [1, 20Ð23]. At the level of the plant physiology; however, in my opinion, it remains organism, polarity, that is, the biophysical, biochemi- true. To support this concept, we may refer to the evi- cal, and physiological gradients, is one of the ways dence of the specific hormonal signals generated in towards integrity, because the polar changes affect the shoot and root apices, the well-known phenomenon of entire field. However, the addressed distribution of sig- apical dominance, when the apical bud inhibits the nals by canalized connections, that is, through such development of auxiliary buds and the root apex conducting tissues as phloem and xylem, originating in represses the lateral root formation, as well as many the shoot and root apical meristems, provides much other facts (see [1, 21Ð23]). The general cybernetic the- more efficient integration. Via the conducting tissues, ory postulates that a special block to command and nutrients [14] and phytohormones [21, 26] are translo- coordinate the activities of a complex differentiated cated and electric impulses propagated [16]. The polar system is necessary; in this context, the concept that IAA transport along the living phloem cells [37] is shoot and root apices are such commanding organs accomplished in an oscillating regime [38, 39] due to seems persuading enough. We will further define these slow BEP waves [21, 32]. This apparently helps to syn- organs as the dominant (control) centers [1, 20Ð23]. chronize the transport, metabolic, and morphogenetic

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External signal ing sensory zones different from the dominant centers. Like in animals, the sensory zones can be located in Perception various regions of the plant. Thus, the leaves perceive Endogenous the photoperiodic signals and translate this information Transduction signal to the central meristem of the shoot apices to induce ﬂowering or to the distal part of stolons to initiate tuber The receptor cell Signal formation [5]. translo- Endogenous signal This model of the regulatory control looks rather cation schematic, because relevant experimental evidence for The cell in the plants is very scarce. In our laboratory, we attempted to connection channel Perception study the regulatory control using as a model the pho- tomorphogenetic processes that occur under the inﬂu- EFFECT Feedback Transduction ence of R. The effector cell

Fig. 1. The basic scheme of the regulatory controls in plants. PHOTOMORPHOGENESIS AS A MODEL OF THE REGULATORY CONTROL processes in the adjacent tissues. If this suggestion In etiolated maize seedlings, R is known to dramat- stands true, the IAA-dependent BEP oscillations ically inhibit mesocotyl growth, which primarily should be seen as the plant pacemaker tool. depends on IAA transport from the coleoptile [44, 45]. Indeed, the content of free IAA in the mesocotyl The regulatory controls [1, 20Ð23] highly essential growth zone (10 mm below the node) decreased follow- for plant integrity consist of the following components: ing the R treatment [46, 47]. Interpretations of this phe- (1) the sensory zones, where the external signals are nomenon vary: some researchers believe that the phy- recognized by the receptor cell and transduced; (2) the tochrome-mediated effect of R results in lower rates of tissues, which accomplish the long-distance transport IAA synthesis or transport [46], and another group has of the endogenous signals (the communication chan- reported an increase in the conjugated IAA content in nels); (3) the effector tissues and organs, which respond this zone [48]. to these signals with efficient (adaptive) changes in the functional activities; and (4) the feedback mechanisms For our study, we used three-day-old etiolated seed- (Fig. 1). lings of maize (Zea mays) hybrid Moldavskii 215. The seedlings were grown on a 0.1-strong Knop solution at As already mentioned, the shoot and root dominant 26°C in complete darkness. The coleoptile, leaf, meso- centers comprise the greatest number of sensory zones. cotyl, and root of the three-day-old seedlings reached The shoot apex reacts to the gravitation field, light average lengths of 14 ± 0.5, 13 ± 0.5, 38 ± 2.0, and 69 ± direction, and its spectrum. Thus, in the distal part of 2.9 mm, respectively. Segments including the 5-mm- the etiolated maize seedling comprising the coleoptile, long basal part of the coleoptile with the primary primary leaves, shoot apex, coleoptilar node, and inter- leaves, the coleoptilar node, and the 5-mm-long upper calary mesocotyl meristem, the subepidermal coleop- (growing) part of mesocotyl were used in the experi- tile cells recognize the gravitation [40], the apical ments. Batches of 12 segments were held in the vertical coleoptile cells recognize unilateral blue light [41], and position 2-mm-deep in solutions of IAA, kinetin, GA the intercalary mesocotyl meristem [42] and leaf tis- (each 1 mg/l), and their mixture; distilled water was the sues [43] recognize R (Fig. 2). The root tip recognizes control treatment. All these operations were carried out the gravitation, light, mechanical pressure, as well as under dim green light. R irradiation (640Ð690 nm, the moisture and nutrient gradients [41]. 34 J/(m2 s)) was carried out at 26°C, while the Reception and transduction of the external signals untreated segments were kept in darkness at the same by sensory cells leads to the generation of endogenous temperature. The lengths of the segments were assessed receptor signals, such as phytohormones and BEPs. at the beginning of the experiment and following 5, 24, The cells of conducting bundles translocate the endog- and 48 h, using a Microfot 5P01 device for reading enous signals. When the latter arrive at the effector microfilms (Russia), at 16× magnification. The experi- cells, the signals are recognized and transduced into ments were replicated eight times. The figures present efficient functional responses. At all successive steps of mean arithmetic values and their standard errors. this informational pathway of recognizing, transduc- In darkness, the mesocotyl elongated much more rap- ing, and translating signals, the negative feedback loops idly than the coleoptile and leaf, whereas R inhibited must exist to correct the efficient adaptive responses, twofold the mesocotyl growth without any significant and, therefore, we may consider such a pathway as a effect on the growth of the coleoptile and leaf (Fig. 3). regulatory control. Within the following 19-h-interval, the mesocotyl A similar model of the regulatory control can be growth under R also slowed down when compared to extended to other parts of the plant organism compris- the dark control, while the growth of the coleoptile and

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2 B H2O

1 GRAVI 2 0

2 IAA R Growth rate, mm/5 h Growth 1 3

0coplme coplme coplme 4 coplme coplme coplme R D RRDD 5 h 24 h 48 h Fig. 2. The sensory zones in the distal part of the three-day- Fig. 3. The effects of R and (D) darkness on the growth rate old etiolated maize seedling. of (co) the coleoptile, (pl) the primary leaf, and (me) the meso- (1) Coleoptile; (2) primary leaves; (3) coleoptilar node; cotyl in the segments of the three-day-old etiolated maize (4) mesocotyl. Arrows indicate the zones recognizing (B) seedling within (5 h) the initial 5-h period, (24 h) the follow- blue light, (R) red light, and (GRAVI) the gravitation ing 19 h, and (48 h) the second day of the experiment [49]. ﬁeld. The incubation medium contained either H2O or 1 mg/l IAA. leaf enhanced. On the second day of the experiment, lin activity was evaluated as GA equivalents using a the leaf growth enhanced dramatically compared to the highly sensitive biotest (the growth of the maize seed- dark control. ling leaf base). The contents of free IAA, zeatin + When treated with IAA, the mesocotyl growth rate zeatin riboside, and ABA and their alcohol-soluble con- during the initial 5 h under R became as high as in dark- jugates were assayed in the coleoptile, leaf, coleoptilar ness (Fig. 3), that is, IAA alleviated the inhibitory node, mesocotyl (10 mm below the node), and root effect of R on the mesocotyl growth. We therefore con- (10-mm-long distal end). After R irradiation for clude that R irradiation decreased the IAA content in 30 min, the contents of free IAA, zeatin + zeatin ribo- the mesocotyl growth zone [49]. Within the initial 5 h, side, gibberellins, and ABA in the coleoptilar node con- kinetin and GA did not considerably affect the meso- siderably decreased (by 36, 38, 39, and 25%, respec- cotyl growth under light and in darkness. The effect of tively). Meanwhile, the contents of free IAA, zeatin + the mixture of hormones depended mostly on the IAA zeatin riboside, and gibberellins in the coleoptile component (the data are not shown). Previously, we increased by 32, 40, and 61%, respectively (Fig. 4). In demonstrated [50] that R did not affect the growth of contrast, the contents of alcohol-soluble IAA conju- the isolated mesocotyl segments within the 3-h-long gates did not change in the coleoptilar node and slightly exposure. These data corroborate the published evi- increased in the coleoptile by 29% (data not shown). dence that relates to the R-induced inhibition of the These data support the idea that R inhibited IAA trans- mesocotyl growth to the inhibition of IAA transport port from the coleoptile to the node. In addition to IAA, from the coleoptile to the mesocotyl [45, 46]. R decreased the content of other phytohormones in the node. It is noteworthy that R evoked a dramatic To determine the contents of phytohormones in the increase in the content of free gibberellins (by 165%) in organs of the three-day-old etiolated maize seedlings the leaves and roots [54]. irradiated with R for 30 min and kept in darkness, we used the immunoenzyme method [51, 52] and the Apart from the phytohormone analysis, we followed already published protocol for phytohormone extrac- the R effects on BEP in the coleoptilar node and adjoin- tion, puriﬁcation, and fractionation [53, 54]. Gibberel- ing zone (1 mm) of the mesocotyl intercalary meristem

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140 measures with EVL-1MZ extracellular silver chloride 120 IAA electrodes (Russia) coupled to a pH-340 direct-current 100 ampliﬁer using the techniques described previously 80 60 [55]. The segments were held in the vertical positions, 40 with their apical ends upward. The whole segment 20 before the experiment (for one hour) and its basal end 0 during the experiment were kept in 0.1 mM CaCl2 solution. The segments were prepared and mounted, and 140 120 Z + ZR BEPs were measured initially (during the ﬁrst hour of 100 the experiment) under dim green light. Irradiation with 80 R induced dramatic electronegative changes in the node 60 region comprising the intercalary meristem. Then, two 40 minutes later, an electropositive BEP wave developed 20 0 and reached its maximum at the 35th minute (Fig. 5). Our previous experiments showed that R did not pro- 40 duce an electrophysiological response in the middle 35 GA 30 part of the coleoptile [32]. We, therefore, interpret the 25 oscillations in the potential difference shown in Fig. 5 20 as the BEP changes in the node with the adjacent meso-

Phytohormone contents, pM/g fr wt Phytohormone 15 10 cotyl intercalary meristem. These data and the previ- 5 ously published evidence [56] led us to the conclusion 0 that R generates an electropositive BEP wave in the zone of the coleoptilar node and the adjoining tissues. 350 300 ABA The next series of experiments focused on the 250 effects of an externally applied potential difference on 200 the mesocotyl growth in the three-day-old etiolated 150 maize seedlings [57]. The rate of elongation was 100 recorded with a 6MKh1S mechanotron (an angular 50 0 converter of linear displacement, Russia) along with an Coleoptile Leaf Node Mesocotyl Root impedance-matching transformer and recording potentiometer KSP-4 [58]. Segments comprising the coleop- Fig. 4. The contents of free IAA (Z + ZR) zeatin plus zeatin tile, coleoptilar node, and mesocotyl were loaded verti- riboside, (GA) gibberellins, and ABA in various tissues of the three-day-old etiolated maize seedling (filled column) cally into a special Plexiglas cell (Fig. 6c). A soft rub- following 30-min incubation in darkness or (open column) ber ring was slipped on the basal part of the coleoptile after irradiation with R [54]. to fix a plastic funnel, and the wider end of the latter propped against the mechanotron probe. The potential R difference between the coleoptile tip and the node was mV generated by platinum electrodes, which contacted 5 with cotton-wool plugs attached to the shoot surface. 0 020 40 6080 100 120 The filter paper in the cell and the cotton wool were –5 min moistened with a 0.1-strong Knop solution. An ISE-01 –10 electronic stimulator (Russia) fed the appliance, which co produced a potential difference of 4, 6 or 15 V at a current –15 pl of 3Ð12 µA. With the anode at the node, the mesocotyl –20 cn growth was inhibited following a lag-phase of 1Ð EP me –25 1.5 min, and the extent of inhibition was proportional to the applied potential. In reverse polarity, the cathode Fig. 5. The effect of R on the BEP changes in the zone of the was in contact with the node that promoted the meso- coleoptilar node of the segment of the three-day-old eti- cotyl growth (Figs. 6a, 6b). It follows that the electrop- olated maize seedling (unpublished data obtained with the participation of K.B. Frolov). During the initial 60 min, ositive changes in the node and adjacent intercalary BEP was measured under weak green light. (co) Coleoptile; meristem inhibited the mesocotyl growth. We have (pl) primary leaves; (cn) coleoptilar node; (me) mesocotyl, already mentioned that R irradiation of the segments or (EP) measuring electrode. the intact etiolated maize seedlings induced electropositive changes in the node with the adjoining intercalary (Fig. 5). The reference electrode was placed at the mid- meristem (Fig. 5). These changes were accompanied by dle of the coleoptile. The experiments used segments of a decrease in the phytohormone content (Fig. 4) and the three-day-old etiolated maize seedlings comprising the inhibition of the mesocotyl growth (Fig. 3). coleoptile with leaves, the coleoptilar node, and the To interpret the effects of the potential difference, upper 7-mm-long section of the mesocotyl. BEPs were both endogenous and externally applied, on plant-tis-

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+ 6V (a) 10 Control segment 6 –6V (c) 10 2 11 12 0 9 m/min µ (b) 2 13 18 14 4 14 8 Control 3 + 6V segment Growth rate, Growth 10 1 –6V 6 5 7 2 6 0 5 1015202530 Exposure, min

Fig. 6. The effects of the external electric field applied between the coleoptile tip and the coleoptilar node on the growth of the mesocotyl in the segments of the three-day-old etiolated maize seedling [57]. The polarity at the node is shown. The treatments (a) and (b) differed in the exposure to direct and reverse polarity and the applied voltage; (c) the schematic drawing of the appliance used in the study of the effect of the electric current on the growth of maize mesocotyl. (1) Mesocotyl; (2) coleoptile; (3) coleoptilar node; (4) primary leaf; (5) Plexiglas cell; (6) paper filter; (7) nutrient solution; (8) rubber band; (9) plastic funnel; (10) mechanotron probe; (11) mechanotron; (12) impedance-matching transformer; (13) a KSP-4 recording potentiometer; (14) cotton-wool plug moistened with the nutrient solution. sue growth, we employ the phenomenon of electroos- Based on the ideas stated above, we suggest the fol- mosis. The nature of this phenomenon lies in the move- lowing model for the contour regulating the effect of R ment, from the positive to negative pole, of an electro- on etiolated maize seedlings. The maximum phyto- lyte solution in capillary or capillaryÐporous bodies the chrome contents reported in the maize seedlings that solid phase of which carries fixed negative charges and were grown in the dark were found in the mesocotyl a counter-ion layer, when a potential difference is intercalary meristem [42] and leaves [43]. Therefore, applied [59]. In plant tissues, the conducting vessels are these tissues are the R sensory zones (see Fig. 2). R capillaries, and intercellular and apoplast spaces are reception by the intercalary meristematic cells results capillaryÐporous bodies. Our experiments with carrot in the generation of an electropositive BEP, which we root disks [60] and maize mesocotyl segments (Fig. 7) define as the receptor potential. The transformation of unquestionably demonstrated the phenomenon of elec- the light (R) signal into the receptor electric potential is probably related to an increase in the cytoplasmic cal- troosmosis. Figure 7 shows that with a potential differ- 2+ ence of 10 and 15 V applied (the corresponding cur- cium concentration [62] followed by Ca ion exchange rents were 37 and 50 µA), the electrolyte solution for protons across the tonoplast. The resulting acidifica- tion of the cytoplasm activates H+-ATPase in the plas- moved through the mesocotyl segment toward the neg- malemma; by pumping H+ ions out, ATPase induces ative pole. In reverse polarity, the direction of the liquid MP hyperpolarization, which is recorded by the exter- movement was the opposite. These experiments imply nal electrode as BEP electropositivation. Such a mech- that when the node zone including the mesocotyl inter- anism for the generation of the electropositive BEP calary meristem is charged positively in reference to wave was put forward and verified in our laboratory the coleoptile tip, the solutions in the conducting ves- when we investigated the IAA effects on competent sels and the apoplast move acropetally and deplete the cells [63, 64]. I believe that the electropositivation of node zone with the adjoining intercalary meristem of the zone of mesocotyl intercalary meristem and node IAA and other phytohormones. The direction of the involves the electroosmotic mechanism, which water movement itself is also essential as the shoot tis- restrains IAA transport from the coleoptile to the meso- sues are under moisture deficit. Such a situation may cotyl growth zone. As a result, cell division and elonga- hinder cell division and elongation in the mesocotyl tion in this zone are dramatically arrested. BEPs, growth zone [61], while the coleoptile growth tempo- osmotic phenomena, mechanical tissue tensions, and rarily enhances due to the enrichment with phytohor- the competition for nutrients and phytohormones work mones and water. as the feedback factors in the regulatory control (see

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(b) Cu 5 II I 5 Cu 2.5 CuSO4 4 2 4 CuSO4 (a) agar agar 1 +10 3 3 2.0 0 I 0 0 +15

l 1.5 –15 µ –10 –10 0 0 1.0 Volume, Volume, +10

+15 0 II 0.5 0 0 –15

010203040 50 60 70 80 90 100 110 Time, min

Fig. 7. The effect of the electric ﬁeld on the transport of electrolyte solution (0.01 M KCl) across the mesocotyl segment of the four- day-old etiolated maize seedling (unpublished data obtained with the participation of T.E. Bilova). (a) A typical experiment demonstrating the changes in the liquid volume in (I) the right and (II) left measuring capillaries when the voltage was switched on, (0) switched off, changed (values at the curves are volts), and (+ or Ð) when the polarity was reversed. (b) The basic scheme of the appliance to measure electroosmosis in the segments of plant axial organs. (1) 10-mm-long mesocotyl segment; (2) connecting rubber couplings; (3) vials with 0.01 M KCl; (4) measuring capillaries; (5) nonpolarizing electrodes (Cu/CuSO4Ðagar).

Fig. 1). The development of new functionally active UKHTOMSKII’S PRINCIPLE OF THE DOMINANT sites is the major factor that triggered the feedback AS APPLIED TO HIGHER PLANTS mechanism. As seen in Fig. 4, the 30-min R irradiation of the primary leaves of the etiolated maize seedlings The animal and plant organism can switch from one dramatically increased the gibberellin activity and in particular activity to another and concentrate all of its this way promoted rapid leaf growth. functional systems on this new activity. Many such examples are found among plants. Thus, all the A similar mechanism regulating the root behavior resources of a seedling grown in the dark (e.g., in soil) must exist in the root apex under the conditions of gravi- are focused on axial elongation. In this way, a plant tropic induction. Konigs [65] reported that the root guided by the gravitation field grows to the light by the deviation from the vertical position induced a trans- shortest way possible. As shown above, a light signal verse electric polarization in the root cap columella. induces drastic changes in the seedling behavior: the Such polarization can modify the direction of IAA stem elongation is restrained, the leaves start unfolding transport and in this way, result in asymmetric cell and expanding, and the photosynthetic apparatus is elongation in the root growth zone and produce a gravi- developed and brought to action. From this point on, tropic bending. the functional activity of the whole plant is focused on photosynthesis. The experimental data and the published evidence To explain the mechanism that focuses all the func- presented above were combined in a comprehensive tional systems of the organism on the particular activ- model of the regulation and integration in plants shown ity, Ukhtomskii advanced the principle of the dominant in Fig. 8. This model combines two concepts, namely [66]: a specific signal evokes a particular constellation hormonal regulation and sourceÐsink relations. The of nerve centers, and their coordinated activities control model is still tentative and schematic; it does not cover the exact activity of the organism. Such steady activa- such items as the functions of some phytohormones tion (excitation) of the particular nerve centers inhibits (gibberellins, ABA, ethylene), some electrophysiologi- other nerve centers. Ukhtomskii called such transient cal phenomena, and the driving forces of the long-dis- constellation of activated centers “a dominant organ.” tance transport. Yet this model can serve as the basis for He commented that the principle of the dominant can the further discussion of the mechanisms of plant integ- be applied to the control via not only the electric rity and promote research in this field. impulses generated by the nervous system, but via the

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 48 No. 4 2001 THE PHYSIOLOGY OF PLANT INTEGRITY 553

External signals

Sensory cells

Shoot dominant IAA synthesis and transport center (sink) Cell attracting activity

Morphogenesis

Growth

IAA External signals

Root absorbtion Mature leaves zone Nutritive organs (assimilates, (H2O, (sources) hormones) mineral nutrients)

Cytokinins

Growth

Morphogenesis

Cell attracting activity Root dominant center (sink) Cytokinin IAA transport synthesis Sensory cells

External signals Fig. 8. A simpliﬁed scheme of the integration system in a growing higher plant. humoral (hormonal) mechanisms, particularly in vegetating plant as a whole. When the environmental embryogenesis, as well (see [67]). changes are regular, e.g., with the arrival of a short-day I believe that plant behavior is also governed photoperiod, an adequate signal coming from the according to Ukhtomskii’s principle of the dominant. leaves to the distal part of potato stolons invokes the In a vegetating plant, the actively growing tip of the pri- active (excited) state in the competent zones of these mary shoot evoked by the gravitation ﬁeld and the illu- stolons. A new constellation of the dominant centers— mination gradient maintains the strategic task of the the distal regions of stolons and roots—evokes a new whole plant: to move (grow) towards the light. At the behavioral pattern. It is evident that the developing same time, the apical bud inhibits the neighboring aux- potato tubers become the focus of the coordinated activ- iliary buds (the phenomenon of apical dominance). A ities of the whole plant. similar mechanism of moving along the vectors of gravitation and/or water and mineral nutrients governs the root behavior. The interaction of two activated ACKNOWLEDGMENTS (dominant) centers, that is, the shoot apex and the distal This work was supported by the Russian Founda- part of the root, employs the hormonal signals and tion for Basic Research, projects nos. 96-04-49007 trophic factors and molds the behavior pattern of the and 99-04-49700.

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 48 No. 4 2001 554 POLEVOI

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