PATHWAYS OF CELLULAR MORPHOGENESIS : A Diversity in Nitella

Evidence is presented to show that a given change in cell form or size may generally be brought about by a variety of patterns of local surface distortion and expansion. Structural and chemical features of the cell which are important in morphogenesis may thus be expected to relate not to form per se but to the kinetics of surface behavior which establish form. These kinetics evaluate both the rate at which local regions of cell surface expand and the directed character (anisotropy) of this expansion. These variables have been studied in model systems and, through marking experiments, in growing cells of various shapes in Phycomyces, Clypeaster, and particularly Nitella. In the latter plant, prominent "giant internodes" display a well defined longitudinal anisotropic expansion devoid of sizeable gradients in expansion rate. These cells have their origin, however, in apical cells which have a pronounced gradient in area expansion rate (maximal at the tip). The great part of the expansion in the apical cell is apparently isotropic (equal in all directions), but the basal region often shows predominant expansion laterally. This transverse stretching in the apical cell could align cell wall texture and possibly fibrous cytoplasmic constituents, such as microtubules, into configurations significant in later morphogenetic stages, including the elongation of the internodes.     

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.     

Day/night temperature environment affects cell elongation but not division in Lilium longiflorum Thunb

Lilium tongiflorum Thunb. cv. ‘Nellie White’ plantswere grown in different day/night temperature (DT/NT) environmentsto determine the anatomical basis for differential responsesof stem elongation to DT and NT. Lilium plants were forced in1986 and 1987 under 25 and 12 different DT/NT environments,respectively, with temperatures ranging from 14 to 30 °C.Parenchyma and epidermal cell length and width were measuredin stem tissue (1987) and epidermal cell length and width weremeasured in leaf tissue (1986). Total cell number per internodeand vertical cell number per internode were calculated. Stemparenchyma and stem and leaf epidermal cell length increasedlinearly as the difference (DIF) between DT and NT increased(DIF = DT —- NT), i.e. as DT increased relative to NT.DIF had no effect on stem parenchyma width, stem and leaf epidermalcell width, or cell number per internode. Data suggested thatstem elongation responses to DIF are elicited primarily througheffects on cell elongation and not division. Key words: Thermoperiodism, thermomorphogenesis, stem elongation, DIF, cell division, cell elongation, leaf expansion     

The Influence of the Cell Membrane on some Toxicity Effects of Growth Substances in Nitella

When cells of Nitella are placed in a solution of some plantgrowth substances there is a profound increase in the membraneresistance as measured by means of an internal silver/silverchloride micro-electrode. This impedance effect is accompaniedby some marked visible changes within the cell cytoplasm, thesechanges being dependent upon the concentration of undissociatedgrowth substance in the vicinity of the membrane, and upon thepH of the external environment. A detailed study is made of these visible changes and, takinginto account the negatively charged membrane, one possible interpretationof the results is that only undissociated molecules of the growthsubstance may enter the cytoplasm of Nitella from the environment,the diffusion obeying a simple Fick Law relationship. The substances are used in sufficient quantity to kill the Nitellacell, death following the visible effects, and they appear tobe unique in that their toxic effect produces a system of highmembrane impedance.     

ACTION POTENTIAL OF NITELLA INTERNODES

The ionic current during a non-propagating action potentialis analysed from the voltage clamp experiments. The shape ofthe action potential of the Nitella internode can be reconstructedfrom the data of the voltage clamp experiments. The N-shapedcurrent-voltage characteristics (I-V curve) of the Nitella membraneis not constant with time as it is in the tunnel diode, butdecays with time, converging finally into a delayed rectificationcurve. The temporal locus of the potential at which each I-Vcurve crosses the voltage axis coincides almost exactly withthe action potential. The membrane resistance which is calculatedfrom the slope of the I-V curve at each intersection with thevoltage axis also changes in parallel to the action potential.Such correlations are found in the Nitella not only in the pondwater, but also in high Na, high Ca or high Mg medium, wherethe shape of the action potential is modified in various ways.It is highly probable that the action potential is a locus ofthe change of the membrane potential so that the net membranecurrent may be maintained at zero after the transient modificationof the membrane structure by stimulation. (Received June 30, 1966; )     

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; )     

Uptake of Radioactivity by Aquatic Plants and Location in the Cells: II. UPTAKE OF CERIUM-144 BY THE FRESHWATER PLANT NITELLA OPACA

The uptake of cerium-144 by Nitella from pond water at pH 7.7without added carrier was recorded over a period of 207 days,separate measurements being made for each of the main componentsof the cell. Some of the Nitella cells were then transferredto water which contained no radiocerium, for measurements overa further 54 days. A cerium-144 equilibrium was approached afterabout 100 days, with the whole plant having 26,000 times theactivity of the water. Under these conditions most of the ceriumwas located in the cell wall, though small but significant amountsentered the protoplasm and cell sap. It was apparent that cerium,freshly taken up either into the cell wall or into the interior,was freely exchangeable, but after a short period (some 10 days)the cerium became more firmly bound to the plant, and was notreleased again.Outstanding differences noticed between the locationof cerium in Nitella opaca and of yttrium in Ulva lactuca arediscussed in chemical and biological contexts.     

The Relationship Between the Distribution of Periclinal Cell Divisions in the Shoot Apex and Leaf Initiation

Periclinal cell divisions in vegetative shoot apices of Pisumand Silene were recorded from serial thin sections by mappingall the periclinal cell walls formed less than one cell cyclepreviously. The distribution of periclinal divisions in theapical domes corresponded to the distributions subsequentlyoccurring in the apices when the young leaf primordia were forming.In Pisum, periclinal divisions were almost entirely absent fromthe I₁ region of the apical dome for half a plastochron justafter the formation of a leaf primordium and appeared, simultaneouslyover the whole of the next potential leaf site, about half aplastochron before the primordium formed. In Silene periclinaldivisions seemed to always present in the apical dome at thepotential leaf sites and also round the sides of the dome wherethe ensheathing leaf bases were to form. Periclinal divisionstherefore anticipated the formation of leaf primordia by occuring,in Pisum about one cell cycle and in Silene two or more cellcycles, before the change in the direction of growth or deformationof the surface associated with primordial initiation. Pisum, Silene, planes of cell division, orientation of cell walls, leaf primordia, shoot apical meristem, plastochron     

Oscillations in Extracellular Current, External pH and Membrane Potential and Conductance in the Alkaline Bands of Nitella and Chara

Oscillations with a period of approximately 2 min were observedin the membrane potential of Chara and Nitella upon illuminationof dark-treated cells, as well as in the extracellular currentpattern and pH values. A 2-min oscillation in the membrane potentialwas observed when the voltage electrode was placed close tothe border of an alkaline and acid region. Comparison of resultsfrom Chara and Nitella revealed an identical control mechanismfor external pattern stabilization in the effect of light onmembrane potential and conductance. Vibrating probe experimentsindicated that oscillations in the extracellular current occurredonly at the border of the alkaline band. Ion-specific pH micro-electrodesplaced within the alkaline band detected oscillations associatedwith light reactivation of the banding phenomenon. These resultsindicate that the oscillations represent a localized phenomenoninvolving spatially-dependent time-constants. More evidencefor the spatial dependence of time constants is gained fromsingle active acid regions in Nitella. Using this combinationof techniques, we have established that a light-dependent H⁺transporter is involved in this oscillation. Current-voltagecurves taken during these oscillations and relaxation, afterchanging the light intensity, confirmed this identification. Key words: Oscillation, vibrating probe, pH micro-electrode, time-constant. I/V curve, Chara, Nitella     

A Sequential Study of Cell Divisions and Expansion Patterns on a Single Developing Shoot Apex of Vinca major

During the growth of a single developing vegetative apex ofVinca major, both the orientation and frequency of cell divisions,and the pattern of cell expansion, were observed using a non-destructivereplica technique. Micrographs taken at daily intervals illustratethat the central region of the apical dome remains relativelyinactive, except for a phase of cell division which occurs after2 d of growth. The majority of growth takes place at the proximalregions of the dome from which develop the successive pairsof leaves. The developing leaf primordia are initiated by aseries of divisions which occur at the periphery of the centraldome and are oriented parallel to the axis of the subsequentleaves. The cells which develop into the outer leaf surfaceof the new leaves undergo expansion and these cells divide allowingfor the formation of the new leaf. This paper describes thefirst high-resolution sequential study of cell patterns in asingle developing plant apex. Sequential development, cell division, expansion patterns, SEM, Vinca major, apical dome, leaf primordium, leaf initiation     

The Role of Cell Expansion in the Abscission of Impatiens sultani leaves

The histological events occurring during the latter stages ofabscission were followed continuously in longitudinal slicesthrough the petiole base of Impatiens sultani Hook. It appearsthat the middle lamella of the cortical parenchyma cells isdegraded first. This is followed by an expansion of these cellsand a concomitant stretching and separation of the collenchymaand vascular trace. The parenchyma cells continue to enlargeuntil they become virtually spherical, a process which finallyruptures the last restraining xylem vessels. The increased volumeof the parenchyma cells appears to be principally due to a conformationalchange in cell shape from a near regular hexagonal prism toa sphere of similar surface area. The dimensions of the prismaticcells are such that most of them change into spheres whose diametersare the same as the transverse distance between the oppositesides of the six axially orientated faces of the prisms. Cellularexpansion is thus entirely directed along the axis of the petiole.The significance of these observations to the general anatomyand mechanism of fracture of abscission zones is discussed. Impatiens sultani Hook., abscission, cell expansion, cell wall degradation, cell shape     

LONGITUDINAL MICROFIBRILLAR ALIGNMENT IN THE WALL OF CYLINDRICAL NITELLA RHIZOIDAL CELLS: OBSERVATION UNDER POLARIZING AND INTERFERENCE MICROSCOPES

Polarizing and interference microscopes were employed to measure overall orientation of microfibrils and dry matter (expressed in optical thickness) in the cell wall of Nitella rhizoids. The microfibrils are aligned predominantly parallel to the cell's long axis (positively birefrengent), except in the apical dome where the arrangement appears to be random. The optical thickness, however, is greater at the very tip and the base region. The wall contains about 50-60% of acid extractable amorphous, noncrystalline substance. This extraction does not make a significant change in the alignment, but the remaining dry matter becomes less at the tip and increases slightly toward the base. The alignment parallel to the direction of cell growth in the rhizoidal cell is different from that of the elongating Nitella internodal cell where the alignment is transverse.     

The role of apical development around the time of leaf initiation in determining leaf width at maturity in wheat seedlings (Triticum aestivum L.) with impeded roots

High soil resistance to root penetration (measured as penetrometerresistance, R_s slows down leaf growth and reduces mature leafsize in wheat seedlings {Triticum aestivum L.). Underlying changesin the kinetics of cell partitioning and expansion and in thesize and organization of mature cells were reported in companionpapers (Beemster and Masle, 1996; Beemster et al., 1996). Inthe present study, the relationships between apex growth, primordiuminitiation and expansion were analysed for plants grown at contrastingR_s, focusing on a leaf whose whole development proceeded afterthe onset of root impedance (leaf 5). High R_s reduced the rates of apex and leaf development, butdid not appear to have immediate effects on the pattern of developmentof the newly initiated phytomers. During an initial short period,the rate of development of a leaf primordium and associatednode were related to plastochronic age, according to similarrelationships (slopes) at the two R_s. Effects on developmentalpatterns were first detected on phytomer radial expansion duringplastochron 2. The ontogenetic pattern of leaf elongation wasaffected later, during the next few plastochrons preceding leafemergence (‘post-primordial stage’). It is concludedthat a reduction in the number of formative divisions and inthe number of proliferative cells along the intercalary mer-istemreported earlier (Beemster and Masle, 1996; Beemster et al.,1996) is not related to the size of the apical dome at leafinitiation nor to the size and number of meristematic cellsinitially recruited to the leaf primordium, which were all unaffectedby R_s. Rather they are generated at the primordial and post-primordialstages. Key words: Wheat, apex development, leaf primodium development, mature leaf width, root impedance     

Effects of soil resistance to root penetration on leaf expansion in wheat (Triticum aestivum L.): kinematic analysis of leaf elongation

Wheat leaves (Triticum aestivum L.) elongated 50% more slowlywhen plants were grown in soils with high mechanical resistanceto penetration (R_s. The profiles of epidermal cell lengths alongthe growth zone of expanding leaves and the locations of newlyformed walls were recorded in order to compare the kineticsof elongation and partitioning of both meristematic and non-meristematiccells. In leaf 5, which completely developed under stress, highR_s, did not affect the flux of mature cells through the elongationzone; leaf elongation was reduced only because these cells wereshorter. This reduced size reflected a reduction in cell lengthat partitioning, associated with shorter cycling time. The relativerates of cell elongation before and after partitioning wereunchanged. Cell fluxes were similar because the population ofmeristematic cells was reduced, offsetting their increased partitioningrate. In contrast, in leaf 1, high R_s, had no effect on thenumber of dividing cells; elongation rate was reduced becauseof slower relative cell expansion rate and slower cell partitioningrate. These differences could reflect differences in the stageat which successive leaves perceived root stress and also time-dependentchanges in the responsiveness of leaf development to stress-inducedroot signals or in the nature of these signals. The data reveal that cell cycling time may in fact be decreasedby unfavourable growth conditions and is not directly relatedto cell expansion rates; they also show that the elongationrate of meristematic cells is partly independently controlledfrom that of non-meristematic cells. Key words: Wheat, kinematics of leaf expansion, cell partitioning, cell elongation, root impedance     

Cell Division and Expansion and Resultant Tissue 3

By following the movement of carbon particle markers on theexposed surface of a cultured tomato apex it has been shownthat a leaf primordium is formed by growth on the flank of theapex raising the tissue upwards and outwards to form the leafbuttress. The whole of the apical surface is in an active stateof cell division and expansion except in the axillary regionabove the primordium. The data provide direct estimates of therates of division in the outer layer of cells. The distribution of blocked metaphase figures following treatmentwith colchicine, shows that in the early stages of primordiumformation cell divisions are concentrated in what appears tobo a ‘growth centre’ in the corpus to one side ofthe apical dome. As the bulge of the primordium develops, thegrowth centre spreads out and splits into two parts continuingthe growth of the dome (proximal side) and the primordium (distalside). Between these two regions of active division there arisesa small pocket of cells in the axil, whose rate of divisionrapidly declines. Cuts made in the apical surface in the early stages of primordiumformation immediately gape widely, apparently as a result ofpressure exerted on the outer layers from within by divisionsin the corpus. Once the upper surface of the primordium becomesraised above the dome, the axillary cells seem to become compressedbetween the two zones of active division. In the axil at thisstage (a) cuts do not gape but close up after exuding cell sapand (b) the carbon particle markers move slightly together.     

Regulation of lettuce hypocotyl elongation by gibberellic acid. Correlation between cell elongation, stress-relaxation properties of the cell wall and wall polysaccharide content

Lettuce hypocotyl elongation caused by gibberellic acid wasstrongly inhibited by coumarin and dichlobenil, known inhibitorsof cellulose biosyndiesis. Stress-relaxation analysis of thecell wall revealed that gibberellic acid induces a decreasein both minimum relaxation time (To) and relaxation rate (b)and an increase in maximum relaxation time (Tm), when gibberellicacid stimulates hypocotyl elongation. Both coumarin and dichlobenilnullified the effect of gibberellic acid on changes in To, Tmand b values. The content of pectic, hemicellulosic and cellulosic substancesin the cell wall increased per hypocotyl but decreased per unithypocotyl length, in response to gibberellic acid treatment.Particularly, gibberellic acid caused a substantial increasein cellulose content per hypocotyl but a decrease per unit length.A good correlation existed between the decrease in To and thedecrease in hemicellulose content per unit lengdi of the cellwall. The increase in Tm was correlated with the decrease incellulose content per unit length of the cell wall. The decreasein b was correlated with the decrease in the content of bothcellulose and hemicellulose per unit length. Based on these results, we discuss the role of polysaccharidemetabolism of the cell wall in gibberellic acid-induced lettucehypocotyl elongation and the nature of gibberellic acid-inducedbiochemical modifications of the cell wall, which are representedby changes in stress-relaxation properties of the cell wall. ¹Present address: Department of Anatomy, Aichi Medical University,Nagakutecho, Aichigun, Aichi 480-11, Japan. (Received September 22, 1975; )     

Oriented structure of matrix polysaccharides in and extension growth of Nitella cell wall

Infrared evidence indicated that the oriented structure of matrixpolysaccharides changed on mechanical extension as well as duringextension growth of Nitella cell walls. The importance of theultrastructure of polysaccharide gels in controlling extensiongrowth is discussed. (Received July 3, 1974; )     

Respiration-Dependent Membrane Hyperpolarization in Tonoplast-Free Cells of Nitella axilliformis

Cells of Nitella axilliformis were made tonoplast-free by intracellularperfusion of media containing ethyleneglycol-bis-(ß-aminoethylether)N,N'-tetraaceticacid (EGTA). When the perfusion medium contained ADP as wellas ATP, the membrane hyperpolarized in darkness in a mannersimilar to light-induced hyperpolarization. This light-independenthyperpolarization seems to be due to activation of the electrogenicion pump in the plasma membrane because the hyperpolarized valueof the membrane potential was more negative than the equilibriumpotential for K^$, the most negative ion equilibrium potentialin Nitella. The hyperpolarization was inhibited by the respiratory chaininhibitors NaCN (1 mM), antimycin A (10 µM) and rotenone(10 µM). NaCN slightly decreased the ATP concentrationin the cell perfused with medium containing 1 mM ATP and 1 mMADP; but, even after treatment with NaCN, the cell had about80% of the ATP value for the control. ^* This study is dedicated to the late Professor J. Ashida. (Received June 24, 1982; Accepted October 15, 1982)     

Thigmomorphogenesis: Changes in Cell Division and Elongation in the Internodes of Mechanically-perturbed or Ethrel-treated Bean Plants

Examination of first internodes of young Phaseolus vulgarisL. plants which have been subjected to mechanical perturbationshows decreased elongation and increased radial growth. Thedecreased elongation can be attributed to both reduced cellelongation of epidermal and cortical cells and a reduced numberof cells in the vascular and pith tissues. The increased radialenlargement is due to increased cortical cell expansion andincreased secondary xylem production resulting from increasedcambial activity. All of these responses are observable withina few hours of a single mechanical perturbation. Treatment ofplants with ethrel mimics all of these effects of mechanicalperturbation. Phaseolus vulgarisL, Kidney bean, thigmomorphogenesis, mechanical perturbation, ethrel, (2-chloroethyl phosphonic acid), cell division, internode elongation