The Geoelectric Effect in Plant Shoots:: IV. INTER-RELATIONSHIP BETWEEN GROWTH, AUXIN CONCENTRATION AND ELECTRICAL POTENTIALS IN ZEA COLEOPTILES

Incubation of Zea coleoptiles in 0.5 M mannitol totally inhibitsgrowth and geotropic curvature, but does not affect the developmentof the geoelectric effect. This pre-treatment also inhibitsthe curvature induced by the asymmetrical application of IAAto the apical end of decapitated vertical coleoptiles, but itdoes not prevent the IAA from giving rise to an electropotentialdifference between the two sides of the coleoptile. Neitherthe normal geoelectric effect, nor the auxin-induced potentialdifference in vertical coleoptiles, can therefore arise as theresult of the different rates of cell extension in the two halvesof the organ. They must be the result of the change of IAA concentrationaffecting some other aspect of the cell's physiology or metabolism. The abolition of the electrical responses in coleoptiles whichhave been plasmolysed in 1.0 M mannitol strongly suggests thatboth longitudinal and lateral transport of IAA are severelydepressed by this degree of plasmolysis. Asymmetrical application of 10^-5 M mersalyl and several othersubstances to the apical end of a decapitated vertical coleoptilegave rise to a marked electropotential difference between thetwo sides of the coleoptile, the side beneath the donor beingpositively charged with respect to the other side. Mersalyldoes not promote the growth of Zea coleoptiles. These resultsprovide additional evidence that the electropotentials do notarise from differential growth, and suggest that such substances,especially the diuretics used in clinical medicine, may provideuseful tools in the further study of the induction of surfaceelectropotentials in plant tissues at the cellular level.     

The Geoelectric Effect in Plant Shoots: II. SENSITIVITY OF CONCENTRATION CHAIN ELECTRODES TO REORIENTATION

A concentration chain, static-drop electrode system has beenused by several investigators to measure the geoelectric effectin plant shoots. This paper describes investigations of theinherent sensitivity of this electrode system to reorientationwith respect to gravity. When the gelatine plug of the electrodeis made up with distilled water, and the contact solution is0.1 mM KCl, a potential difference develops immediately afterelectrodes in direct contact are rotated through 90° intothe vertical plane. A similar response is found when the contactsolution is 5 mM CaCl₂. Increasing the concentration of thecontact solution, or incorporating KCl, K₂SO₄, or ZnSO₄ intothe gelatine plug, drastically reduces the potential differencedeveloped after reorientation. The potential difference acrosselectrodes in direct contact decreases as the electrodes age.The potential difference measured with these electrodes acrossa decapitated, horizontally placed, hypocotyl of Helianthusarmuus also decreases as the electrodes age. The polarity ofthe charge is reversed as compared with that found when theelectrodes are in direct contact. The kinetic characteristicsof the geoelectric potential difference developed across a non-decapitated,horizontal coleoptile of Zea mays change as the electrodes age.With fresh electrodes the potential develops immediately afterreorientation and continues to increase with time. With 4-day-oldelectrodes, however, no potential difference develops until9 min after the moment of reorientation, but then it increaseswith time. The characteristics of the geoelectric potentialdifference developed with the aged concentration-chain, static-dropelectrodes are similar to those found with several other typesof electrodes which do not themselves have an inherent sensitivityto reorientation with respect to gravity. The results supportour earlier suggestion that the potential difference which apparentlydevelops with the static-drop electrode system, immediatelyafter a shoot is turned through 90° in reality developsin the electrode system itself and not in the plant tissue.The geoelectric effect which arises in the living plant shootbegins to develop approximately 10 min after reorientation.     

The Geoelectric Effect in Plant Shoots: V. A DISCUSSION

In earlier papers Woodcock and Wilkins, and Hertz and Grahmreported measurements of the geoelectric effect (GEE) in Zeamays coleoptiles. While Woodcock and Wilkins attached contactelectrodes to the coleoptile directly, Grahm and Hertz employeda vibrating-electrode technique that avoided touching the plant. The results obtained by these two methods agree on all pointsexcept the magnitude of the GEE, where a difference of morethan 100 per cent is reported. In this paper this differenceis investigated and explained by certain properties characteristicof the measuring techniques used as well as by differences inexperimental procedure.     

The Geoelectric Effect in Plant Shoots: I. THE CHARACTERISTICS OF THE EFFECT

A comparative study has been made of the geoelectric effectin Helianthus hypocotyls and Zea coleoptiles using two electrodesystems. With the static-drop electrode system a potential differencedeveloped between the upper and lower surfaces of the shootsimmediately after they were turned into the horizontal position.The lower surface of the shoot became positively charged withrespect to the upper surface. In non-decapitated shoots thispotential difference continued to increase for at least 20 min,whereas in decapitated shoots no further increase occurred afterabout 10 min from the moment of reorientation. In contrast,with a flowing-solution electrode system no potential differencedeveloped in non-decapitated shoots until about 12 min afterthey were placed in the horizontal position. Thereafter thelower surface became increasingly positively charged with respectto the upper surface for at least a further 12 min. In decapitatedshoots there was no tendency whatsoever for the lower surfaceof the horizontal shoot to become positively charged with respectto the upper surface, even after 25 min in the horizontal position.The static-drop electrode system has an inherent sensitivityto reorientation in a gravitational field; a potential differencedevelops immediately after reorientation, and increases to amaximum value within the first 10 min of reorientation, regardlessof whether or not the electrodes are in contact with plant tissue.The continued increase in the potential difference measuredacross the shoots with the static-drop electrode system, andthe entire development of the potential difference measuredwith the flowing-solution electrode system, are both dependentupon the shoot apex being intact. These facts have enabled usto show that the electro-potential difference measured acrosshorizontally placed non-decapitated tissues with the static-dropelectrode system is the resultant of two distinct processes:(a) an immediate, purely physical electrical effect generatedin the electrodes themselves, and (b) a delayed geoelectriceffect which arises solely in the living tissues of the shootand which is dependent upon the apex of the shoot being intact.     

THE EFFECT OF HYDROGEN ION CONCENTRATION UPON THE INDUCTION OF POLARITY IN FUCUS EGGS : III. GRADIENTS OF HYDROGEN ION CONCENTRATION

1. Gradients of hydrogen ion concentration across Fucus eggs growing in sea water determine the developmental polarity of the embryo. 2. Gradients may determine polarity even if removed before the morphological response begins. 3. The rhizoid forms on the acid side of the egg unless this is too acid, in which case it develops on the basic side of the egg. 4. Since gradients of hydrogen ion concentration in sea water produce gradients of CO₂ tension, as a result of chemical action on the carbonate buffer system, it is not proven whether the physiological effects are due to the hydrogen ions, or to the CO₂ which they produce in the medium. 5. The developmental response of the eggs to gradients of hydrogen ion (or CO₂) concentration provides an adequate but not an exclusive explanation of the group effect in Fucus. 6. Hydrogen ions may exert their effect by activating growth substance. Hydrogen ions or CO₂ probably also affect the underlying rhizoid forming processes in other ways as well.     

The Role of Boron in Plant Growth: III. THE EFFECTS OF DIFFERENTIATION AND DEFICIENCY ON RADICLE METABOLISM

The effects of boron deficiency on certain components of cellswas followed by tracing the incorporation of ¹⁴C. While a preliminaryexperiment showed an enhanced incorporation of activity intocellulose and a decreased incorporation of activity into pectins,later results, from frequent observations during the onset ofdeficiency and the study of the metabolism of segments fromdifferent regions of normal roots, showed that differences betweendeficient and normal root apices were largely due to the greaterage of the constituent cells of the former. Differentiationcontinued during boron deficiency and the changes of incorporationinto pectic substances, holocellulose, lignins, sugars, andamino-acids paralleled those found during differentiation ofnormal roots. Two results, namely an increased incorporationof activity into pectic substances and an increased acid resistanceoccurred sufficiently early to be possible primary effects ofdeficiency although they are difficult to relate to each other.The results, with other published evidence, suggest that boronis concerned with cell-wall bonding.     

Gravitropism in Higher Plant Shoots : III. Cell Dimensions during Gravitropic Bending; Perception of Gravity

Cross and longitudinal sections were prepared for light microscopy from vertical control plants (Xanthium strumarium L. Chicago strain), free-bending horizontal stems, plants restrained 48 hours in a horizontal position, and plants restrained 48 hours and then released, bending immediately about 130°. Top cells of free-bending stems shrink or elongate little; bottom cells continue to elongate. In restrained stems, bottom cells elongate some and increase in diameter; top cells elongate about as much but decrease in diameter. Upon release, bottom cells elongate more and decrease in diameter, while top cells shorten and increase in diameter, accounting for the bend. During restraint, bottom cells take up water while tissue pressures increase; top cells fail to take up water although tissue pressures are decreasing.

Settling of amyloplasts was observed in cells of the starch sheath.

Removal of different amounts of stem (Xanthium; Lycopersicon esculentum Miller, cv Bonny Best; Ricinus communis L. cv Yolo Wonder) showed that perception of gravity occurs in the bending (elongation) zone, although bending of fourth and fifth internodes from the top was less than in uncut controls. Uniform application of 1% indoleacetic acid in lanolin to cut stem surfaces partially restored bending. Reversing the gradient in tension/compression in horizontal stems (top under compression, bottom under tension) did not affect gravitropic bending.

     

THE EFFECT OF HYDROGEN ION CONCENTRATION UPON THE INDUCTION OF POLARITY IN FUCUS EGGS : II. THE EFFECT OF DIFFUSION GRADIENTS BROUGHT ABOUT BY EGGS IN CAPILLARY TUBES

1. When a Fucus egg develops near one end in a close fitting capillary tube of pyrex glass or silica (quartz), diffusion of substances passing to and from the egg is more impeded on the side of the egg toward the far end of the tube. 2. The egg therefore develops in a gradient of its own diffusion products, and of oxygen tension. 3. More than 600 eggs have been reared, each near one end in a capillary, in sea water at various regulated and measured pH values. 4. When the medium, which is initially homogeneous inside and outside the capillary, is initially at pH 6.5 to 7.6, nearly all of the eggs develop rhizoid protuberances on the sides of the eggs toward the far ends of the capillaries. This is on the sides of the eggs where the concentration of substances diffusing from the eggs is greatest. 5. The polarity and developmental pattern of the egg is thus determined either by a concentration gradient of products diffusing from it, or by a gradient of oxygen tension. The former interpretation is favored. 6. This is regarded as an extension of earlier observations that rhizoid protuberances form on the sides of two neighboring eggs in the direction of the neighbor if the sea water is acidified. 7. It appears hardly possible that a mitogenetic effect could be responsible for the response of an egg to its own diffusion gradients. 8. When the medium is made more basic, the percentage of the eggs which form rhizoid protuberances toward the far end of the tube decreases to about 20 or 25 per cent between pH 8.1 and 8.6. The response of the egg to the gradients which it produces is thus statistically reversed. The determination of the polarity of the eggs by the diffusion gradients does not become as complete in alkalinized as in acidified sea water. 9. When the pH of the sea water is elevated to 9.1 or 9.2, salts precipitate out. The type of development is altered and the control of the diffusion gradients over the polarity of the eggs decreases.     

THE SELECTIVE ABSORPTION OF POTASSIUM BY ANIMAL CELLS : III. THE EFFECT OF HYDROGEN ION CONCENTRATION UPON THE RETENTION OF POTASSIUM.

By perfusing frogs for varying periods with potassium-free Ringer solutions having a pH ranging from 6.0 to 8.0, it has been determined that such solutions have little or no effect upon the retention of potassium by muscle cells.     

Gravitropism in Higher Plant Shoots : V. Changing Sensitivity to Auxin

An alternative to the Cholodny-Went, auxin-transport hypothesis of gravitropic stem bending was proposed as early as 1958, suggesting that gravistimulation induces changes in sensitivity to auxin, accounting for differential growth and bending. To test the sensitivity hypothesis, we immersed marked, decapitated sunflower (Helianthus annuus L.) hypocotyl sections in buffered auxin solutions over a wide concentration range (0, 10⁻⁸ to 10⁻² molar IAA), photographed them at half-hour intervals, analyzed the negatives with a digitizer/computer, and evaluated surface-length changes in terms of Michaelis-Menten enzyme kinetics. Bending decreases with increasing auxin concentration; above about 10⁻⁴ molar IAA the hypocotyls bend down; increasing auxin inhibits elongation growth of lower surfaces (which is high at zero or relatively low auxin levels) but promotes upper-surface growth (which is low at low auxin levels). Thus, lower surfaces have a greater K_m sensitivity to applied auxin than upper surfaces. At optimum auxin levels (maximum growth), growth of bottom surfaces exceeds that of top surfaces, so bottom tissues have a greater V_max sensitivity. V_max sensitivity of vertical controls is slightly lower than it is for either horizontal surface; K_m sensitivity is intermediate. Clearly, gravistimulation leads to significant changes in tissue sensitivity to applied auxin. Perhaps these changes are also important in normal gravitropism.     

The Role of the Coleoptile Apex in Controlling Organ Elongation: II. EFFECTS OF AUXIN SUBSTITUTION AND AUXIN TRANSPORT INHIBITORS ON DECAPITATED COLEOPTILES

The most widely quoted evidence that auxin controls coleoptileelongation is that auxin applied to the cut surface of a coleoptilecan obviate the effects of decapitation. A quantitative studyof this old observation shows that exogenously applied auxinonly overcomes the effects of decapitation if supplied at unphysiologicallyhigh concentrations. Furthermore, when a ring of an auxin transportinhibitor is applied just beneath the apex of an intact coleoptile,so that auxin supplied from the apex is abolished, the growthrate of the coleoptile is not reduced significantly. These experimentssuggest that coleoptile elongation is not dependent on auxintransported from the apex. Key words: Coleoptile, auxin, transport inhibitors     

Gravitropism in Higher Plant Shoots: I. A ROLE FOR ETHYLENE

It has long been known that applied ethylene can redirect the gravitropic response, but only occasionally has it been suggested that ethylene normally plays a role in gravitropism. Two inhibitors of ethylene synthesis [Co²⁺ and aminoethoxyvinylglycine (AVG)] and two inhibitors of ethylene action (Ag⁺ and CO₂) were shown to delay the gravitropic response of cocklebur (Xanthium strumarium L.), tomato (Lycopersicon esculentum Mill.), and castor bean (Ricinus communis L.) stems. Gentle shaking on a mechanical shaker does not inhibit the gravitropic response, but vigorous hand shaking for 120 seconds delays the response somewhat. AVG and Ag⁺ further delay the response of mechanically stimulated plants. AVG delays the response of defoliated and of decapitated plants. Plants laid on their side and restricted so that they cannot bend upward store both bending energy and gravitropic stimulus; they bend immediately when released from restriction (stored energy) and continue to bend for some hours after (stored stimulus). AVG retards the storage of bending energy but not of stimulus. In gravitropism, graviperception may first stimulate ethylene evolution, which may then influence bending directly, or responses involving ethylene could be more indirect.     

ION SERIES AND THE PHYSICAL PROPERTIES OF PROTEINS : III. THE ACTION OF SALTS IN LOW CONCENTRATION.

1. Ions with the opposite sign of charge as that of a protein ion diminish the swelling, osmotic pressure, and viscosity of the protein. Ions with the same sign of charge as the protein ion (with the exception of H and OH ions) seem to have no effect on these properties as long as the concentrations of electrolytes used are not too high. 2. The relative depressing effect of different ions on the physical properties of proteins is a function only of the valency and sign of charge of the ion, ions of the same sign of charge and the same valency having practically the same depressing effect on gelatin solutions of the same pH while the depressing effect increases rapidly with an increase in the valency of the ion. 3. The Hofmeister series of ions are the result of an error due to the failure to notice the influence of the addition of a salt upon the hydrogen ion concentration of the protein solution. As a consequence of this failure, effects caused by a variation in the hydrogen ion concentration of the solution were erroneously attributed to differences in the nature of the ions of the salts used. 4. It is not safe to draw conclusions concerning specific effects of ions on the swelling, osmotic pressure, or viscosity of gelatin when the concentration of electrolytes in the solution exceeds M/16, since at that concentration the values of these properties are near the minimum characteristic of the isoelectric point.     

Action Potentials in Lupinus angustifolius L. Shoots: III. DETERMINATION OF THE REFRACTORY PERIODS

The refractory periods for action potentials (AP) were investigatedin Lupinus shoots by application of pairs of electrical stimuli(d.c). The response (AP) for the second stimulus is characterized.The second response differs in amplitude, range, and propagationvelocity from the first, depending on the strength of the stimuliand the time interval between them. The absolute refractoryperiod determined on this basis lasts 12 min, and the relativeone from 12 to about 90 min. The character of the refractoryphenomenon is similar in the case of nerves, only the processesin Lupinus are 10³ to 10⁵ times slower. The possibility of transmission of excitation from cell to cellis discussed.     

Gravitropism in Higher Plant Shoots : IV. Further Studies on Participation of Ethylene

Ethylene at 1.0 and 10.0 cubic centimeters per cubic meter decreased the rate of gravitropic bending in stems of cocklebur (Xanthium strumarium L.) and tomato (Lycopersicon esculentum Mill), but 0.1 cubic centimeter per cubic meter ethylene had little effect. Treating cocklebur plants with 1.0 millimolar aminoethoxyvinylglycine (AVG) (ethylene synthesis inhibitor) delayed stem bending compared with controls, but adding 0.1 cubic centimeter per cubic meter ethylene in the surrounding atmosphere (or applying 0.1% ethephon solution) partially restored the rate of bending of AVG-treated plants. Ethylene increases in bending stems, and AVG inhibits this. Virtually all newly synthesized ethylene appeared in bottom halves of horizontal stems, where ethylene concentrations were as much as 100 times those in upright stems or in top halves of horizontal stems. This was especially true when horizontal stems were physically restrained from bending. Ethylene might promote cell elongation in bottom tissues of a horizontal stem or indicate other factors there (e.g. a large amount of `functioning' auxin). Or top and bottom tissues may become differentially sensitive to ethylene. Auxin applied to one side of a vertical stem caused extreme bending away from that side; gibberellic acid, kinetin, and abscisic acid were without effect. Acidic ethephon solutions applied to one side of young seedlings of cocklebur, tomato, sunflower (Helianthus annuus L.), and soybean (Glycine max [L.] Merr.) caused bending away from that side, but neutral ethephon solutions did not cause bending. Buffered or unbuffered acid (HCl) caused similar bending. Neutral ethephon solutions produced typical ethylene symptoms (i.e. epinasty, inhibition of stem elongation). HCl or acidic ethephon applied to the top of horizontal stems caused downward bending, but these substances applied to the bottom of such stems inhibited growth and upward bending—an unexpected result.     

Gravitropism in Higher Plant Shoots : II. Dimensional and Pressure Changes during Stem Bending

Dimensional changes during gravitropic bending of cocklebur (Xanthium strumarium L.) dicot stems were measured using techniques of stereo photogrammetry. The differential growth is from an increased growth rate on the bottom of the stem and a stopping or contraction of the top.

Contraction of the top was especially evident upon release and immediate bending of horizontal stems that had been restrained between stiff wires for 36 hours. The energy for this could have been stored in both the top and bottom, since the bottom elongated, and the top contracted.

Forces developed during bending were measured by fastening a stem tip to the end of a bar with attached strain gauges and recording electrical output from the strain gauges. Restrained mature cocklebur stems continued to accumulate potential energy for bending for about 120 hours, after which the recorded force reached a maximum.

Pressures within castor bean (Ricinus communis L.) stems were also measured with 3.5-millimeter diameter pressure transducers. As expected, the pressure on the bottom of the restrained plants increased with time; pressures decreased in vertical controls, tops of restrained stems, and bottoms of free-bending stems. Pressures increased in tops of free-bending stems. When restrained plants were released, pressure on the bottom decreased and pressure on the top increased. Results suggest a possible role for cell contraction in the top of stems bending upward in response to gravity.

     

The Role of Boron in Plant Growth: IV. INTERRELATIONSHIPS BETWEEN BORON AND INDOL-3YL-ACETIC ACID IN THE METABOLISM OF BEAN RADICLES

The hypothesis that boron deficiency is equivalent to a stateof IAA toxicity was explored. Bioassays showed that extractsof substances similar to IAA taken from boron-deficient rootswere significantly more inhibitory to the growth of bean-rootsegments than those from normal roots. Supplied IAA and borondeficiency together restricted root growth to a greater extentthan either deficiency or IAA treatment separately. Roots werefound to recover more quickly from the inhibitory effects ofsupplied IAA if boron was present at high (0.5 ppm) rather thanlow (0.01 ppm) concentrations. Experiments with ¹⁴C-labelled IAA showed that deficient rootsabsorbed ¹⁴C more slowly than boron-fed roots and there wasalso a lower rate of decarboxylation in the deficient tissue.Consideration of the published evidence showed that many ofthe effects of boron deficiency could follow from an upset inIAA metabolism. It is suggested that boron-deficient tissuesuffers from excess auxin either because the element is necessaryfor some growth process, such as cell wall formation or nucleicacid synthesis, which, when impaired, results in the accumulationof auxin, or because the IAA-oxidation system is affected byphenolic inhibitors which boron normally inactivates by complexformation.     

Gravitropism in Higher Plant Shoots : VI. Changing Sensitivity to Auxin in Gravistimulated Soybean Hypocotyls

Although the Cholodny-Went model of auxin redistribution has been used to explain the transduction phase of gravitropism for over 60 years, problems are apparent, especially with dicot stems. An alternative to an auxin gradient is a physiological gradient in which lower tissues of a horizontal stem become more sensitive than upper tissues to auxin already present. Changes in tissue sensitivity to auxin were tested by immersing marked Glycine max Merrill (soybean) hypocotyl sections in buffered auxin solutions (0, 10⁻⁸ to 10⁻² molar indoleacetic acid) and observing bending and growth of upper and lower surfaces. The two surfaces of horizontal hypocotyl sections responded differently to the same applied auxin stimulus; hypocotyls bent up (lower half grew more) in buffer alone or in low auxin levels, but bent down (upper half grew more) in high auxin. Dose-response curves were evaluated with Michaelis-Menten kinetics, with auxin-receptor binding analogous to enzyme-substrate binding. V_max for the lower half was usually greater than that for the upper half, which could indicate more binding sites in the lower half. K_m of the upper half was always greater than that of the lower half (unmeasurably low), which could indicate that upper-half binding sites had a much lower affinity for auxin than lower-half sites. Dose-response curves were also obtained for sections `scrubbed' (cuticle abraded) on top or bottom before immersion in auxin, and `gravitropic memory' experiments of L. Brauner and A. Hagar (1958 Planta 51: 115-147) were duplicated. [1-¹⁴C]Indoleacetic acid penetration was equal into the two halves, and endogenous plus exogenously supplied (not radiolabeled) free auxin in the two halves (by gas chromatography-selected ion monitoring-mass spectrometry) was also equal. Thus, differential growth occurred without free auxin redistribution, contrary to Cholodny-Went but in agreement with a sensitivity model.     

Regulators of Cell Division in Plant Tissues1: XIX. THE METABOLISM OF 6-BENZYLAMINOPURINE IN RADISH COTYLEDONS AND SEEDLINGS

Excised immature radish cotyledons were allowed to take up [³H]6-benzylaminopurinefor up to 5 h and were then transferred to cytokinin.free mediumfor various intervals, after which they were extracted. Thecytokinin was rapidly metabolized by the tissue to two mainproducts which accounted for 20 per cent of the extracted radioactivityafter 1 h of uptake, and for 95 per cent after transfer to thecytokinin-free medium. These metabolites were coincident withcytokinin activity on chromatograms of the cotyledon extractand were also formed when 6-benzylaminopurine was supplied to9-d-old derooted seedlings through the transpiration stream.The metabolites were identified by mass spectrometry, u.v. spectroscopy and chemical degradation as 6-benzylarnino-7-glucosylpurineand 6-benzylamino-9. glucosylpurine. Several minor metaboliteswere detected in the derooted seedlings; one was 6-benzylaminopurineriboside while another appeared to possess high cytokinin activity.     

THE EFFECT OF HYDROGEN ION CONCENTRATION UPON THE INDUCTION OF POLARITY IN FUCUS EGGS : I. INCREASED HYDROGEN ION CONCENTRATION AND THE INTENSITY OF MUTUAL INDUCTIONS BY NEIGHBORING EGGS OF FUCUS FURCATUS

1. The eggs of Fucus furcatus develop perfectly in sea water acidified to pH 6.0. They are retarded at pH 5.5. At pH 5.0 they do not develop, nor do they cytolize. 2. In normal sea water in the dark at 15°C., eggs develop rhizoids on the sides in the resultant direction of a mass of neighboring eggs. The polarity and the whole developmental pattern of the embryo is thereby induced. This inductive effect does not operate, however, unless the directing mass is an appreciable aggregation of cells (10 or more), or unless there are numerous other eggs in the dish. A group of five eggs alone in a dish do not carry out mutual inductions. Two eggs alone in a dish do not develop rhizoids toward each other. 3. When the sea water is acidified to pH 6.0 all sizes of aggregations carry out mutual inductions. Two eggs alone in a dish now develop rhizoids on the sides toward each other, provided they are not more than about 4 egg diameters apart. 4. Increased hydrogen ion concentration thus augments or intensifies the mutual inductive effect. 5. This may explain why only larger masses of eggs show inductions in normal sea water, since presumably the larger masses considerably increase the hydrogen ion concentration locally. 6. The nature of the inductive action is discussed. 7. In acidified sea water at pH 6.0, compared with normal sea water at pH 7.8–8.0, the rhizoids originate and extend with a strongly increased downward component. The substrate then forces further extension or growth of the rhizoid to be in the plane of the substrate.