CHEMICAL RESTORATION IN NITELLA : II. RESTORATIVE ACTION OF BLOOD

Cells of Nitella exposed to distilled water lose their ability to produce action currents and to distinguish electrically between sodium and potassium. This ability was quickly restored by exposure to blood plasma deprived of calcium. Human blood and that of the cat, calf, and sheep gave essentially the same results. The active agents appear to be organic substances.     

NATURE OF THE ACTION CURRENT IN NITELLA : II. SPECIAL CASES

The action curve involves four movements each of which shows considerable variation. These variations can be accounted for on the assumption that the action curve is due to the movement of potassium ions accompanied by an increase in permeability.     

SEPARATION OF POTASSIUM ISOTOPES IN VALONIA AND NITELLA

The ratio of K³⁹ ÷ K⁴¹ appears to be lower in the sap of Valonia and Nitella than in the environment, indicating that the living cell can separate these isotopes to some extent. Experiments with a mixture of guaiacol and p-cresol suggest that a similar separation may occur here but further experiments are needed.     

THE DEATH WAVE IN NITELLA : III. TRANSMISSION

Cutting a cell of Nitella sets up a series of rapid electrical responses, transmitted at a rate too rapid to be measured by means of our records. These are followed by slower responses whose speed falls off as the distance from the cut increases, as though they were caused by a mechanical disturbance whose intensity falls off as it travels. The faster responses seem to be due to the motion of sap past protoplasmic surfaces which have suffered little or no alteration (they seem to be similar to the electrical changes following a blow on the end of a soft rubber tube containing Ag-AgCl electrodes). The slower responses appear to be due to alterations in the protoplasm and are usually irreversible.     

HYDROSTATIC PRESSURE AND TEMPERATURE IN RELATION TO STIMULATION AND CYCLOSIS IN NITELLA FLEXILIS

Nitella flexilis cells are not stimulated to "shock stoppage" of cyclosis by suddenly evacuating the air over the water or on sudden readmission of air, or on suddenly striking a piston in the water-filled chamber in which they are kept with a ball whose energy is 7.6 joules, provided the Nitella cell is not moved by currents against the side of the chamber. Sudden increases in hydrostatic pressure from zero to 1000 lbs. or 0 to 5000 lbs. per square inch or 5000 to 9000 lbs. per square inch usually do not stimulate to "shock stoppage" of cyclosis, but some cells are stimulated. Sudden decreases of pressure are more likely to stimulate, again with variation depending on the cell. In the absence of stimulation, the cyclosis velocity at 23°C. slows as the pressure is increased in steps of 1000 lbs. per square inch. In some cells a regular slowing is observed, in others there is little slowing until 4000 to 6000 lbs. per square inch, when a rapid slowing appears, with only 50 per cent to 30 per cent of the original velocity at 9000 lbs. per square inch. The cyclosis does not completely stop at 10000 lbs. per square inch. The pressure effect is reversible unless the cells have been kept too long at the high pressure. At low temperatures (10°C.) and at temperatures near and above (32°–38°C.) the optimum temperature for maximum cyclosis (35–36°C.) pressures of 3000 to 6000 lbs. per square inch cause only further slowing of cyclosis, with no reversal of the temperature effect, such as has been observed in pressure-temperature studies on the luminescence of luminous bacteria. Sudden increase in temperature may cause shock stoppage of cyclosis as well as sudden decrease in temperature.     

NATURE OF THE ACTION CURRENT IN NITELLA : VI. SIMPLE AND COMPLEX ACTION PATTERNS

The experiments indicate that the protoplasm of Nitella consists of an aqueous layer W with an outer non-aqueous surface layer X and an inner non-aqueous surface layer Y. The potential at Y is measured by the magnitude of the action curve and the potential at X by the distance from the top of the action curve to the zero line. These potentials appear to be due chiefly to diffusion potentials caused by the activity gradients of KCl across the non-aqueous layers X and Y. The relative mobilities of K⁺ and Cl^- in X and in Y can be computed and an estimate of the activity of KCl in W can be made. In the complete resting state the mobilities of K⁺ and Cl^- in X are not very different from those in Y. The action curve is due to changes in Y which suddenly becomes very permeable, allowing potassium to move from the sap across Y into W, and thus losing its potential. A gradual loss may be due to changes in ionic mobility in Y. When recovery is incomplete and Y has not yet regained its normal potential a stimulus may cause a loss of the potential at Y giving an action curve of small magnitude. The magnitude may vary in successive action curves giving what is called a complex pattern in contrast to the simple pattern observed when recovery is complete and all the action curves are alike. Complex patterns occur chiefly in cells treated with reagents. Untreated cells usually give simple patterns. A variety of complex action patterns is discussed. It is evident that the cells of Nitella show much more variation than such highly specialized cells as muscle and nerve which give stereotyped responses. In some cases it may be doubtful whether the all-or-none law holds.     

WATER RELATIONS IN THE CELL : I. THE CHLOROPLASTS OF NITELLA AND OF SPIROGYRA

Chloroplasts may contract under natural conditions and give up water to the rest of the cell, thus indicating changes in metabolism or constitution. Such contractions may be produced experimentally. In Nitella the chloroplasts are ellipsoid bodies which, under natural conditions, may contract to spheres with a loss of volume. This may be brought about by lead acetate, ferric chloride, and digitonin: the contraction may occur while the cell is alive. The contraction in lead acetate is reversible (in lead nitrate little or no contraction occurs). In Spirogyra the chloroplast is a long, spirally coiled ribbon which may contract under natural conditions to a short nearly straight rod with a loss of volume. This can be brought about by inorganic salts and in other ways while the cell is still alive.     

CELL WALL POTENTIAL IN NITELLA

In the process of inserting a microelectrode into the vacuoleof Nitella three potential levels were recorded. The first onewas at a water phase outside the cell wall, the second one inthe cell wall and the third one across the plasmalemma. Thefirst potential was variable with the distance from the surfaceof the cell wall. When the external solution was 10^–4M KCl, the second potential level was –90 mv and the thirdone –170 mv against an external reference electrode. Thesepotentials were less negative (more negative) with the increase(decrease) of the external KCl concentration and varied to someextent among samples. The vacuolar potential measured againstthe cell wall phase was, therefore, –80 mv inside negativeto outside. A large potential change such as action potentialwas observed only across the plasmalemma. An overshoot of theaction potential of Nitella flexilis was observed very often,when the vacuolar potential was measured against the cell wallphase. This work was supported by a Research Grant from the Ministryof Education of Japan. Part of this work was performed whenR. NAGAI was a Yukawa Research Grant fellow.     

CHEMICAL RESTORATION IN NITELLA : IV. EFFECTS OF GUANIDINE

Leaching in distilled water may remove irritability and the potassium effect in Nitella but both of these may be restored by appropriate treatment with guanidine.     

CYTOTAXONOMY OF THE CHARACEAE: KARYOTYPE ANALYSIS OF NITELLA OPACA AND NITELLA FLEXILIS

The haploid chromosome complement of Nitella flexilis (n = 12) is composed of two quite different basic karyotypes. One of these is symmetrical and appears to be identical to the karyotype of N. opaca (n = 6), and the other is quite asymmetrical and seems to be identical to that of an apparently undescribed dioecious Nitella from Kansas (n = 6). This may indicate that the monoecious N. flexilis has arisen through hybridization between two dioecious species. Although heteromorphic sex chromosomes were not observed in either species, it appears that female and male potentialities are confined to separate basic chromosome complements and mechanisms determining the monoecious and dioecious states are in close relation to ploidy.     

HYPERPOLARIZING RESPONSE IN NITELLA INTERNODES

The ionic aspect of the hyperpolarizing response in the internodalcell of Nitella is reported in some detail. The response wasobserved by passing a large inward current through the Nitellamembrane, the resistance of which had been decreased by a concentratedalkali metal ion. It was not possible to demonstrate the responsein a concentrated solution of CaCl₂, MgCl₂, BaCl₂, ZnCl₂ orAlCl₂ or AlCl₃. After hundreds of the spontaneous repetitiveaction potentials, which occurred in a single solution of concentratedNaCl or LiCl or caused by an application of 1–2 mM EDTAin the artificial pond water, the Nitella cell showed the hyperpolarizingresponse. Almost the same size of the response was observedfor change in pH of the external KC1 solution from 6.7 to 10.0,but it decreased markedly for pH lower than 4.7. It seems tobe an essential condition for the response to remove the divalentcations from the cell surface, having a concentrated monovalentcation in the external medium. (Received April 22, 1966; )     

POSITIVE VARIATIONS IN NITELLA

The reversible electrical variations hitherto described for plants and animals consist in a reversible loss of positive potential at a stimulated spot by which it becomes more negative. In this paper we describe changes which consist in a reversible loss of negative potential at a stimulated spot whereby it becomes more positive. We suggest that this be called a positive variation. The stimulation was produced in all cases by pinching or bending the cell. This produced a compression wave which traveled along the cell, producing a negative variation at a spot which was positive and a positive variation at a spot which was negative (due to application of 0.1 M KCl). The response produced by the compression wave differs in several respects from an ordinary propagated negative variation and may be termed a positive mechanical variation.     

REPETITIVE ACTION POTENTIALS IN NITELLA INTERNODES

Typical spontaneous action potentials can be elicited in 10–100mM NaCl or LiCl solution. The period of repetition is 0.5–2seconds and the action potential generally consists of a rapidspike alone. Similar spontaneous action potentials are alsodemonstrated by adding either 1 mM EDTA (pH 6.6) or 2 mM ATP(pH 6.6) to the artificial pond water. In these cases, however,the period of repetition is much longer and the action potentialis of a normal shape, a rapid spike being followed by a slowtransient depolarization. The period of repetition and the sizeof the action potential decrease with the elevation of the vacuolarpotential level. The cause of the spontaneous firing is supposedto be the removal of Ca⁺⁺ from the outer surface of the Nitellamembrane. (Received May 18, 1966; )     

THE DEATH WAVE IN NITELLA : I. APPLICATIONS OF LIKE SOLUTIONS.

Experiments on cutting confirm the prediction that the current of injury will be positive when the cell is in contact with concentrated solutions and negative with dilute solutions. They support the idea that the protoplasm is made up of layers differing considerably in their properties, each having a death curve of simple and regular form, the more rapid alteration of the outer layer making the protoplasm more positive and the more rapid alteration of the inner making it more negative. From the point where the cell is cut a wave of some sort, which we may for convenience call a death wave, passes along the cell, setting up at each point it touches a death process which has the greater speed and intensity the nearer it is to the cut.     

CINEMATIC OBSERVATIONS ON THE GROWTH AND DIVISION OF CHLOROPLASTS IN NITELLA

As it elongates from about 0.2 to 80 mm, the Nitella internodal cell shows an increase in plastid number from a few thousand to about 4 million. The increase takes place by plastid division. A continuous motion picture record followed a population of 8 plastids in an elongating cell until their progeny numbered 18, a span longer than 1 fission cycle for some of the plastids. One complete fission-fission cycle was about 22 hr. The highly directed nature of chloroplast expansion (elongation) is lost when cell wall strain (expansion) is mechanically inhibited by pressing the cell between glass plates. The plastids then expand about equally in all directions in the plane of the cell surface. When a new direction of maximum strain is introduced by the mechanical induction of a lateral in the cell, the plastids elongate in this new direction. The direction of the protoplasmic stream does not show this striking response to strain but tends to follow the lines of the chloroplast chains, not the long axis of individual plastids.     

STABILITY AND COLCHICINE EFFECT OF WALL MICROFIBRILLAR ALIGNMENT IN THE NITELLA RHIZOID

The cell wall of the Nitella rhizoid was stripped to make wedges of various thicknesses. Polarizing and interference microscopes were used to examine the post-deposition orientation of wall microfibrils. The fibrils appeared to maintain alignment after they were deposited. Since during growth the rhizoid wall elements are static in the cylindrical part or extend isotropically in the dome (Chen, 1973), these observations provide indirect evidence that the fibrillar reorientation observed in the Nitella internode is due to a passive reorientation during the predominant longitudinal cell elongation (Gertel and Green, 1977). The static microfibrils of the secondary wall of rhizoid, however, reoriented under the influence of colchicine, the alignment becoming almost random after 48 hrs. The disturbance of alignment started in the region adjacent to the plasma membrane, increasing in thickness with prolonged treatment.     

CHANGES OF APPARENT IONIC MOBILITIES IN PROTOPLASM : IV. INFLUENCE OF GUAIACOL ON THE EFFECTS OF SODIUM AND POTASSIUM IN NITELLA

In Nitella, as in Halicystis, guaiacol increases the mobility of Na⁺ in the outer protoplasmic surface but leaves the mobility of K⁺ unaffected. This differs from the situation in Valonia where the mobility of Na⁺ is increased and that of K⁺ is decreased. The partition coefficient of Na⁺ in the outer protoplasmic surface is increased and that of K⁺ left unchanged. Recovery after the action current is delayed in the presence of guaiacol and the action curves are "square topped."     

Ultrastructural Localization of Ions: IV. LOCALIZATION OF CHLORIDE AND BROMIDE IN NITELLA TRANSLUCENS AND X-RAY ENERGY SPECTROSCOPY OF SILVER PRECIPITATION PRODUCTS

The cytoplasmic distribution of Cl^– in Nitella translucenswas assessed by means of two contrasting approaches. The firstinvolved the histochemical precipitation of Cl^– with Ag⁺followed by X-ray analytical verification of the silver precipitationproducts, and the second, quench-freezing of whole Nitella cellsfollowed by freeze-substitution in the presence of silver underanhydrous conditions. Both methods produced identical evidencefor Cl^– distribution, showing that a large proportionof the Cl^– is present in the stationary cortical gel layerwhich includes the chloroplasts. However, the chloroplasts appearedto be low in Cl^– content while the bulk of the Cl^–appeared to be situated between the chloroplasts and plasmalemma. Experiments were carried out in other to detect the pathwayof the ‘fast component’ of halide ion transfer tothe vacuole. Br^– was supplied for various time intervalsto low Cl^– Nitella cells, followed by attempts to differentiatebetween AgCl and AgBr deposits. Solutions of various strengthof NH₄OH or ammonium carbonate were used to remove AgCl (butnot AgBr) deposits by formation of a Ag(NH₃)₂⁺ complex. AlthoughX-ray analytical verification showed that the method had somepotential usefulness it could not be carried out successfullybecause of loss of structural detail caused by NH⁺₄. The distribution and density of deposits near the plasmalemmasuggested the occurrence of a process in which cytoplasmic loadingis achieved by a sequential rupture and repair of the plasmalemmamembrane. Vesicles and reticulate structures in the streamingcytoplasmic phase generally showed very little deposit, butthese structures, together with the tonoplast, became greatlyenriched with deposits when cells had been given a brief exposers(3 min) to a Cl^– or Br^– solution. These rapid changesmay possibly be related to the ‘fast component’of halide ion transfer to the vacuole.