WATER PERMEABILITY OF THE CELL WALL IN NITELLA

1. A method has been developed to measure the hydraulic conductivityof the wall of the internodal cell of Nitella flexilis.
2. Therate of water penetration through the cell wall varies linearlywith the hydrostatic pressure difference between the two sidesof the wall, showing that water permeability of the cell wallremains independent of the pressure difference applied.
3. Waterpermeability of the cell wall is inversely proportionalto itsthickness It is 30µµmin^–3{dot}atm^–3when the thickness of the wall is 10 µ.
4. Water permeabilityof the cell wall is the same for inward andoutward water flow.The polar water permeability of the entiremembrane system (walland protoplasmic part) of the living celldemonstrated by KAMIYAand TAZAWA (1) is, therefore, due tothe living protoplasmicpart.
5. The ratio of the inward to outward permeability constantsofthe protoplasmic layer alone is higher than that of the entiremembrane system composed of protoplasmic layer and cell wall.

¹ Dedicated to Prof. H. TAMIYA on the occasion of his 60th birthday.The present work was supported in part by a Grant-in-Aid forFundamental Scientific Research from the Ministry of Education. ² Present address: Sh?in Women's College, Kobe. (Received July 21, 1962; )     

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.     

CELL DIVISION IN SPIROGYRA. I. MITOSIS

Dividing cells of Spirogyra sp. were examined with both the light and electron microscopes. By preprophase many of the typical transverse wall micro-tubules disappeared while others were seen in the thickened cytoplasmic strands. Microtubules appeared in the polar cytoplasm at prophase and by prometaphase they penetrated the nucleus. They were attached to chromosomes at metaphase and early anaphase, and formed a sheath surrounding the spindle during anaphase; they were seen in the interzonal strands and cytoplasmic strands at telophase. The interphase nucleolus, containing 2 distinct zones and chromatinlike material, fragmented at prophase; at metaphase and anaphase nucleolar material coated the chromosomes, obscuring them by late anaphase. The chromosomes condensed in the nucleoplasm at prophase, moving into the nucleolus at prometaphase. The nuclear envelope was finally disrupted at anaphase during spindle elongation; at telophase membrane profiles coated the reforming nuclei. During anaphase and early telophase the interzonal region contained vacuoles, a few micro-tubules, and sometimes eliminated n ucleolar material; most small organelles, including swollen endoplasmic reticulum and tubular membranes, were concentrated in the polar cytoplasm. Quantitative and qualitative cytological observations strongly suggest movement of intact wall rnicrotubules to the spindle at preprophase and then back again at telophase.     

THE BEHAVIOR OF CHLORIDES IN THE CELL SAP OF NITELLA

1. A method is given for determining the chloride content in a drop (less than 0.03 cc.) of the cell sap of Nitella. 2. Chlorides accumulate in the sap to the extent of 0.128 M; this accumulation can be followed during the growth of the cell. The chloride content does not increase when the cell is placed for 2 days in solutions (at pH 6.2) containing chlorides up to 0.128 M. 3. The exosmosis of chlorides from injured cells can be followed quantitatively. When one end of the cell is cut off a wave of injury progresses toward the other end; this is accompanied by a progressive exosmosis of chlorides.     

SOME PROPERTIES OF PROTOPLASMIC GELS : II. CONTRACTION OF CHLOROPLASTS IN CURRENTS OF WATER ENTERING THE CELL AND EXPANSION IN OUTGOING CURRENTS

The chloroplasts of Nitella may contract under natural conditions as well as under the influence of certain reagents. When a sufficient amount of water enters any part of the cell they contract in that region and they expand when the direction of the current is reversed. The current may be produced by placing water at one end of the cell and applying at the other end a solution which withdraws water from the cell. The contraction may be due to the washing out of substances from the chloroplast by the ingoing current. The outgoing current bearing dissolved materials from the sap may restore these substances and cause the chloroplasts to resume their normal shape. When blood or sodium dodecylsulfate is present in the ingoing current the contraction of the chloroplast usually fails to occur.     

SOME PROPERTIES OF PROTOPLASMIC GELS : I. TENSION IN THE CHLOROPLAST OF SPIROGYRA

The chloroplast of Spirogyra is a long, spirally coiled ribbon which may contract to form a short, nearly straight rod. This happens under natural conditions and it can also be produced by a variety of inorganic salts and by some organic substances. It also occurs when the chloroplast is freed by centrifugal force from the clear peripheral protoplasm which is in contact with the cellulose wall. It would therefore seem that the chloroplast may be passively stretched by the action of the clear protoplasm and hence it contracts as soon as it is set free. This contraction happens in dead as well as in living cells. It would be of much interest to know how the protoplasm brings about the coiling of the chloroplast and how the chloroplast is set free by various reagents. Presumably they must penetrate the living protoplasm to produce the effects described. In one species partial contraction without detachment from the peripheral protoplasm can be brought about by lead acetate. This is reversible. Lead nitrate does not produce this result. The attack upon the problem is greatly facilitated by the study of dead cells. Thereby we reduce the number of variables but the chloroplast continues to react to certain chemical and physical agents in much the same manner as in the living cell and the solution surrounding it can be controlled as is not possible in the living cell. We must await further investigation to learn what plant and animal cells contain gels under tension and what functions they perform.     

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.     

PHYLOGENETIC TRANSITIONS IN THE CHLOROPLASTS OF THE ANTHOCEROTALES I. THE NUMBER AND ULTRASTRUCTURE OF THE MATURE PLASTIDS

Representatives of three genera of anthooerotes were examined: Phaeoceros, Notothylas, and Megaceros. Species of the first two genera were found to exemplify the typical anthocerote plastid condition. This condition is characterized by the presence in each cell of the gametophyte of only a single large chloroplast containing a “multiple” pyrenoid. The genus Megaceros, however, proved to be quite different. In two species of Megaceros the pyrenoid was observed to be composed of a highly subdivided thylakoid system of even greater complexity than the “multiple” pyrenoids of Phaeoceros. In another species only an indistinct “pyrenoid-like” area was noted while in a fourth species no evidence was found for any internal differentiation. Associated with these changes in plastid structure there are corresponding alterations in the number and the size of the chloroplasts. Together they indicate an evolutionary trend away from a primitive, algal-like condition to a more advanced land plant form.     

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.     

STUDIES ON NITELLA HAVING ARTIFICIAL CELL SAP I. REPLACEMENT OF THE CELL SAP WITH ARTIFICIAL SOLUTIONS

A method for replacing the cell sap of Nitella with an artificialsolution was introduced. The technique, which is a modificationof KAMIYA and KURODA'S (1, 2), is applicable not only for isotonicbut also for hypertonic or hypotonic solutions. Photometricdeterminations of K⁺, Na⁺, Ca⁺⁺ and Cl^– proved that thereplacement of the cell sap with the present method is satisfactory.The internodal cell of Nitella, whose cell sap was replacedwith an isotonic solution with a simple composition such asa mixture of KCl, NaCl and CaCl₂, can be kept living at leastfor several days, sometimes even for more than one month. (Received September 6, 1963; )     

PHOTOCONTROL OF THE ORIENTATION OF CELL DIVISION IN ADIANTUM. I. EFFECTS OF THE DARK AND RED PERIODS IN THE APICAL CELL OF GAMETOPHYTES

When protonemata of Adiantum capillus-veneris L. which had been grown filamentously under continuous red light were transferred to continuous white light, the apical cell divided transversely twice, but the 3rd division was longitudinal. An intervening period of darkness lasting from 0 to 90 hr either between the 1st and the 2nd cell division or between the 2nd and the 3rd one did not affect the number of protonemata in which the 3rd cell division was longitudinal. The insertion of red light instead of darkness greatly decreased the percentage of 1st longitudinal divisions occurring at the 3rd division, and increased the number of transverse divisions. Fifty percent reduction of induction of 1st longitudinal division was caused by ca. 50 hr exposure to red light between 1st and 2nd division and by ca. 20 hr between 2nd and 3rd division, and total loss was induced by an exposure of ca. 100 hr or longer to red light in the former and by ca. 40 hr longer in the latter. Thus, by using an appropriate intervening dark period or exposure to red light, the orientation and timing of cell division could be controlled in apical cell of the fern protonemata.     

STRUCTURE AND DEVELOPMENT OF THE CHLOROPLAST IN CHLAMYDOMONAS : I. THE NORMAL GREEN CELL

The cytoplasmic organization of a normal green strain of the alga Chlamydomonas reinhardi has been investigated with the electron microscope using thin sections of OsO₄ fixed material. The detailed organization of the chloroplast has been of special interest. The chloroplast, a cup-shaped organelle, surrounded by a double membrane, consists of: (1) discs about 1 micron in diameter, considered to represent the basic structural unit of the chloroplast, and each composed of a pair of membranes joined at their ends to form a flat closed vesicle; the discs are grouped into stacks resembling the grana of higher plants; (2) matrix material of low density in which the discs are embedded; (3) starch grains; (4) the pyrenoid, a non-lamellar region associated with starch synthesis, and containing tubules which connect with the lamellae; (5) the eyespot, a differentiated region containing two or three plates of hexagonally packed, carotenoid-containing granules, located between discs, and associated with phototaxis. In addition to the chloroplast, the cytoplasm contains various membranous and granular components, including mitochondria, endoplasmic reticulum, and dictyosomes, identified on the basis of morphological comparability with structures seen in animal cells. The nucleus, not investigated in detail in this study, contains a large, granular nucleolus and is surrounded by a nuclear envelope which is provided with pores and exhibits instances of continuity with the endoplasmic reticulum of the cytoplasm.