Plant Physiol. (1986) 80, 721-7260032-0889/86/80/0721/06/$01.00/0

Phycomyces: Fine Structure Analysis of the Growing Zone'Received for publication April 5, 1985 and in revised form October 18, 1985

R. IGOR GAMOW*, GEOFFREY ANDREW GEER, AND BARBEL BOTTGERDepartment ofChemical Engineering, University ofColorado, Boulder, Colorado 80309

ABSTRACT

Fine structure analysis of the stage IVb Phycomyces sporangiophoregrowing zone (GZ) was performed during steady-state growth using acomputer-video digitizer and recorder. By simultaneously measuring thetrajectory of two independent particles above and within the GZ, we haveconfirmed the previous findings of R. Cohen and M. Delbruck (1958 JCell Comp Physiol 52: 361-388) that the GZ is not uniform. We havebeen unable to confirm their findings that counterclockwise rotation existsin a mature sporangiophore. The rates of rotation and elongation changeindependently as a function of position in the GZ. This change is notlinear as would be expected if the GZ were uniform. The importance ofthis finding is discussed in terms of the fibril reorientation model.

The discovery of spiral growth in mature Phycomyces spor-angiophores was first made by A. J. Oort (14). Operationally,spiral growth is said to occur if a marker on the GZ2 is displacedboth vertically by elongation occurring below the marker andhorizontally because of the rotation of the GZ also below themarker. There is no effect on growth or rotation from the growingzone above the marker; only below the marker. The rotationaldisplacement can be measured either in respect to some fixedobject on the sporangiophore, such as the sporangium or to afixed observer, i.e. the ground. This growth is two-dimensionalin the sense that the two-vector components of growth, twist andstretch, can be thought of as occurring on a plane that has beenfolded into a conical stalk. Although an early report by Castle(2) suggested that rotation and elongation (twist and stretch) wereproportional to one another, recent reports (4, 5, 15) have clearlyshown that they are not. Cohen and Delbruck (5) compared theGZ to the flame of a candle based on the fact that any onematerial section in the GZ is not constant in its behavior buttwists and stretches to varying degrees as it moves down the GZ.We have carried this analogy even further by showing that whenmeasured on a minute-to-minute scale, both stretch and twistshow independently highly irregular growth velocities similar toa 'flickering candle' (9).

In this report we describe the results obtained from the two-dimensional movement of a single particle as it descends theentire GZ. The movement of this descending particle is com-pared, minute to minute, with a control particle (stationary withrespect to the sporangium) located immediately beneath thesporangium. We have determined that during steady stategrowth, the upper 5 to 10% of the GZ shows very low rates oftwist and stretch. The rates of both rotation and elongationincrease dramatically at a definite location in the GZ. The

'Supported by the National Foundation Grants GB-31039, GB-35597 and CPE-821 186 1.

2Abbreviation: GZ, growing zone.

increase in rotational rate starts slower than the increase inelongational rate and decreases faster. Most of the change inrotational rate thus occurs in a small region of the GZ while thechange in elongation occurs over a larger section.

MATERIALS AND METHODS

Wild-type Phycomyces blakesleeanus sporangiophores, NRRL1555(-), originally obtained from M. Delbruck, were grown inshell vials containing 5.0% potato dextrose agar (PDA) with1.0% yeast extract. The shell vials were incubated under diffuseincandescent light in a high humidity room with a temperaturerange between 22 and 27°C. Before each experiment, the spor-angiophores were dark adapted in red light for at least 20 min.During the experiment, the humidity was kept above 90% RHby surrounding the experimental sporangiophore with openwater dishes. The humidity was not measured during the exper-iment; however, an identical set up was measured by using aGeneral Eastern humidity probe. Unless otherwise stated, allexperiments were carried out with a water-filtered red lightsource.The apparatus used to simultaneously measure minute-by-

minute the net rotation and the net elongation of a stage IVbGZ was first described by Cohen and Delbruck (5) and thenmodified by Gamow and Bottger (9). The mature stage IVbsporangiophore in a glass shell vial was firmly secured to a stagethat rotated clockwise once every 60 s. To ensure that the GZ ofthe sporangiophore was vertical (parallel to the axis of rotationof the stage), a double knee was inserted between the stage andthe vial. The rotating stage allowed us to measure the angularvelocity of any particle situated above, below, or in the GZ.Because the net rotation of a mature stage IVb sporangiophoreis in the same direction as the rotating stage (clockwise), a particleeither above or in the GZ takes less than 60 s to complete onerevolution. By determining how much less, we can calculate theangular velocity of a particle placed anywhere on the stalk. Thisrotation is in respect to the observer. For instance, if a particlecompletes one revolution in 58 s, we can easily calculate that theentire region below the particle must have a total angular velocityof(60-58)/58 * 360° = 12.4 1/min. For our present experiments,we used a glass bead approximately 15 um in diameter. The beadwas placed about 50,um below the sporangium in the stalk regionwhich does not stretch or rotate (5). The GZ, with the attachedglass bead, was observed and recorded on video tape through acamera attached to a microscope. The magnification of themicroscope was x 100. An electric timer with a digital printoutwas used to measure the time with a resolution of 10 ms.By measuring the angular velocity of a nontwisting particle, a

particle located below the GZ, we have determined that ourangular velocity measurements are correct to within ±1.77/min(9). Five data points were taken every 5 min and then averaged,yielding a standard deviation of ±0.8°/min.A computer-video digitizer was used to measure the rate of

elongation. The video signal from the video tapes was sent to aTecmar video digitizer connected to a Texas Instruments Profes-

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Plant Physiol. Vol. 80, 1986

sional Computer. The digitizer generates a marker that is super-imposed on the sporangiophore which appears on the TV mon-itor. The computer generated marker can be moved across themonitor with commands entered from the computer's keyboard.It was determined that the marker moves across the image of thesporangiophore in steps of 8.2 lsm (i.e. the marker could moveacross an 82 Am diameter stalk in 10 steps). The glass beadattached to the sporangium can be followed with the marker togive an accurate measurement of its trajectory. The computerrecords the position of the marker. The time is measured usinga Panasonic Auto Search Controller which measures the runningtime of the video tape with a resolution of 1.0 s. This time isinput into the computer which calculates the rate of elongationusing the time and the position of the marker.The video digitizer setup provides the only accurate method

to measure elongation from the video tapes. Because of thecurvature of the video monitor, direct measurements from thescreen using a micrometer give a large error. An additional erroroccurs because the image on the monitor is approximately 5 mmbehind the surface of the screen creating a significant error dueto parallax. The marker created by the digitizer is not subject toparallax or curvature error because it appears directly on theimage of the sporangium. The position of the marker is alwaysreferenced to the left side of the screen. This ensures that meas-urement errors do not compound but instead cancel when av-eraged. By making repeated measurements of a known distanceit was determined that a single measurement is accurate to +8,um One measurement was made every minute and averagedwith the measurements from the previous 2 min and the follow-ing 2 min. This average measurement of elongation has a prec-sion of ±2.0 Am/min.A TV video camera in conjunction with a TV monitor (Fig.

1) has an enormous advantage over the conventional 35 mmphotography used previously (9). First, since an entire experi-ment can be stored on a video cassette and thus become part ofa permanent Phycomyces library, we can rerun any given exper-iment, many lasting several hours, at any future date. Second,since the video tape can be rerun as often as desired, we cansimultaneously follow both the rotation and elongation of manyparticles located on a single GZ. In our present set of experi-ments, we have measured both the angular and vertical velocities,minute by minute, for 4 h of a single particle located immediatelybeneath the sporangium. Since this region neither rotates norelongates (neither twists nor stretches) with respect to the ground,it serves as an ideal control for our experiment in which we tracethe trajectory of a second particle, the test particle. This particleis placed on the very upper edge of the GZ at the beginning ofeach experiment. As this experimental particle descends the GZ,as a result of stretch, we measured its position, minute by minute,and compared its trajectory to our control particle. The experi-ments were run for 120 min. A single particle was followed for115 min during which time it descended from 100 to 200 ,um.The tape was then rewound and a second particle was followed.The second particle was selected so that it started at 200 smbelow the head-just where the first particle had stopped. It wasnecessary to use two particles because a single particle wouldtake much longer than 2 h. A sporangiophore could not be keptgrowing straight for more than about 2 h which limited the totaltime of the experiment. Without the upper control particle, wewould not be able to determine whether the experimental particlewas slowing down or speeding up because it was passing throughthe GZ, or because the entire GZ is either increasing or decreasingin its twist and stretch rate.

RESULTS

On the TV monior screen shown in Figure 1, one can see amature stage IVb sporangiophore, sporangium, and upper part

of the GZ, with several attached glass beads. Figure 1 is includedin order to document the high optical resolution obtained fromboth the sporangiophore and the attached glass beads. Figure 2Ashows a typical experiment in which we measured the angularvelocity of both a particle attached directly beneath the sporan-gium (upper curve) and the angular velocity of a second particle(lower curve) which was attached to the upper region of the GZ.The angular velocities of both particles are plotted as functionsof time. Below the time axis is an axis which represents thedistance from the head of the sporangium to the particle. Thethree lines between the two axes give a correlation between thescales for three important regions. The distances on the loweraxis are given in ,um from the sporangium. Because there is solittle GZ available for growth or rotation above the particle, thetime scale is greatly expanded in the upper GZ (first 300 Arm).As the particle descends down the GZ, leaving more and moreGZ above, the rate of descent naturally increases. The controlparticle represents the sum of all the rotation occurring below it.It is the total rate of rotation.

Figure 2B is the same as 2A except the growth rates of the twoparticles are plotted instead ofthe angular velocities. The controlparticle here reflects the overall growth rate of the sporangiop-hore. The double time-distance axis is the same as used torepresent angular velocities.

Figure 3 represents both the rotation and elongation rates as afunction of location in the GZ for the same representativeexperiment. Both curves are estimated best fits through the datapoints. A straight line cannot be fitted through either set of datawithout an error much larger than the error inherent in themeasurements. A good linear fit would be required if the GZwere uniform throughout. The maximum slope, or maximumrate of change, of the elongation curve occurs around 700 Ambelow the sporangium. The maximum slope of the rotationalcurve occurs around 650 ,um. Most of the change in rotationalrate occurs in a section from 400 to 900 ,um below the sporan-gium. This is evident from the steep slope of the rotational curvein this region. The elongation, in contrast, changes much moreconsistently and continues to change past 1500 ,m.Subzone I. That region of the stalk extending from immedi-

ately below the sporangium to about 100 ,Am below it. SubzoneI shows no stretch or twist, i.e. a particle placed in subzone Idoes not change its relative position with respect to the sporan-gium. This is also the region that has been mechanically deter-mined to be the softest section (8).Subzone II. This zone constitutes the vast majority of the

growing (stretching and twisting) region of the GZ. Twist alwaysoccurs here in a clockise direction and it rarely extends morethan 2000 ,um below the sporangium. In the upper section ofzone II (230 to 600 ,m) the rate of change of both elongationand rotation is monotonically increasing. In the lower sectionthe rate ofchange is monotonicaly decreasing. This is representedin Figure 3 by the inflection points in both curves around 650Am, which represents the location of maximum growth androtation.Subzone III. This zone comprises the rest of the stalk. It is not

part of the GZ. Subzone III neither twists nor stretches. It ismechanically quite stiff, showing no extensibility when loadedup to 500 mg using the Instron technique (1). This zone appearsto serve little if any metabolic support function for the growingand stretching regions found above it (10).

DISCUSSIONIn recent years our laboratory has studied the variety ofgrowth

patterns in terms of both the magnitude and the direction ofstretch and twist that occurs in the stage IVb GZ. We have shownthat these patterns not only change as a function ofGZ positionbut also after the living cell is mechanically deformed (7). These

722 GAMOW ET AL.

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PHYCOMYCES: FINE STRUCTURE ANALYSIS

FIG. 1. A video monitor during a typical experiment is shown. On the monitor screen are seen the sporangiophore's head (the sporangium) andthe upper region of the GZ with some attached beads.

changes in the growth patterns have led to testable molecularmodels that appear to account for some aspects of the structure,growth, and regulation of the living cell wall (8, 19). All theseexperiments have yielded data that are consistent with the modelof cell wall growth first developed by Roelofsen and Houwink(18) and recenty expanded by Gertel and Green (1 1), called themultinet theory of growth. The multinet theory is also entirelyconsistent with the fibril reorientation model that was developedto explain the spiral growth ofa mature stage IVb sporangiophore(15).

Multinet growth states that the microfibrils are deposited onthe inner surface of the cell wall in a transverse manner and thenpassively reoriented towards the longitudinal direction as a resultof cell wall expansion; the driving force ofthis cell wall expansionis turgor pressure. Gertel and Green ( 11) working with Nitellahave reported that changing the direction of cell wall strain doesindeed change the direction of cell wall orientation as predictedby theory, but in no case could they influence initial transverseorientation which results directly from fibril synthesis. The roleof turgor pressure in the rate of cell wall extension, althoughqualitatively related, has presented a problem that has onlyrecently been solved. The problem was that by using uniaxialextension and matching the cell's longitudinal stresses, the rateof extension of isolated cell walls was significantly greater than

the normal growth rate (16). When multiaxial extension stresswas induced by filling a nongrowing cell with mercury (12), itwas found that these cells were much less extensible than uniax-ially stressed cells. This result directly follows the work of Probineand Preston (16) who reported that the transverse modulus ismany times greater than the longitudinal modulus. Recently,Metraux et al. (13), using a 'growing Nitella' and an imposedmultiaxial stress, have concluded that cell wall extension canonly occur by the addition of some 'metabolic event'; this met-abolic event may be the laying down of a new primary wall. Thisis consistent with the known behavior of subzone I; although itis known that the cell wall region in this region is the mostextensible one of the entire stalk (8, 17), it neither stretches nortwists, presumably because of the absence of some metabolicevent. We would expect that this metabolic event first becomespresent in subzone II, although at a low rate.

If the entire growing zone was uniform in structure andmetabolism it would be expected that the rate of rotation andelongation would vary directy with the location in the GZ. Thetwo curves of Figure 3 are decidedly nonlinear showing that theGZ is nonuniform in structure, metabolism, or both.Although the rotation and elongation curves of Figure 3 are

subject to the error of fitting a curve to the data set, it is clearthat the rotation stops higher in the GZ than does the elongation.

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GAMOW ET AL. Plant Physiol. Vol. 80, 1986

zI

I I _-I510 1050 1 100 1 150 200

A 1000 3000 5000

DISTANCE FROM HEAD (microns)

50

40 <t\_\*controlportile

30~~~~ ~ ~ ~~~~~~~~N

20t \ moving particle

10

0 1100 150 200 250

250

i- i a . i i

B 1000 3000

DISTANCE FRCM HEAD (microns)

5000

FIG. 2. A, In the upper curve (0), the angular velocity of a glass bead attached directly below the sporangium is measured minute by minute.Each point is an average of five consecutive measurements. The lower curve (AL) represents then angular velocity of a second particle that was

initially attached to the upper portion of the GZ. This curve is averaged in the same manner as the top curve. The variations in the rate of rotationare not experimental error. They are real fluctuations that have been described by Gamow and Bottger (9). In this representative experiment, theupper (control) particle was placed 64 gm below the sporangium; the second particle started 119 ,Am below the sporangium and descended to 1500,um in 230 min. At the bottom of each figure a schematic of a mature sporangiophore is shown indicating the various zones of the GZ. A length axisis given below the schematic. The three lines connect the two different scales to show the locations of the edges of the various zones. Figure B, Theupper curve (0) represents the growth rate with respect to the ground of the control particle. This particle is placed in the nongrowing region near

the sporangium and therefore represents the overall growth rate of the sporangiophore. The lower curve (E) is the growth rate of a test particle whichis moving down the GZ. The growth rates of both particles were measured once every minute and averaged over a period of 5 min. The schematicsporangium and length scale are the same as those in A. The variations in the rate of elongation are not experimental error; they are real fluctuationsthat have been described by Gamow and Bottger (9).

724

20

181.

16-

14-

12-1-

E

z0

0

10-I

8-

6-

4

2-

control particle

*a4"

N\ moving particle

I ta lMF (min,)^snfa- -1

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PHYCOMYCES: FINE STRUCTURE ANALYSIS

E

21

-0

!iix

20

18-

16-

14-

12-

10-

8-

4-12

0

i i* lI

500 1000 1500

DISTANCE FROM HEAD (microns)

725

-40

30 >

_E"i

-20 Q

- z0

-10 <

z

eu

- 9

FIG. 3. The rates of rotation and elongation of the same representative experiment of Figure 2, A and B, are plotted as a function of distancefrom the sporangium. The upper (A) curve represents the elongational rate in microns per minute of the test particle. The lower (0) curve is therotational rate in degrees per minute of the same particle. The distance from the sporangium (head), rate of rotation, and rate of elongation were allmeasured every minute. All the elongational rates that occurred in 20 ,um sections were averaged together and appear as a single point. The rotationalrates were handled in the same fashion. The curves drawn are estimates of the 'best' fit.

This indicates that two or more separate growth mechanismsoccur simultaneously. In 1974 we proposed (15) that the left-handed spiral growth of stage IVb and the right-handed spiralgrowth of stage IVa could be explained via a fibril reorientationmechanism and a fibril slippage mechanism, respectively. If weassume a multinet mechanism in which new fibrils are first laiddown in a horizontal position and then passively reorientedtowards the vertical during growth, we would expect a muchlarger rotation to extension ratio in the lower region of subzoneII than we have experimentally found. The relatively high exten-sion rates in the lower part of the subzone II can be explained iffibril slippage is assumed to occur there. In general, if a fibrilangle at a position in the growing zone is assumed, one can

deduce from the data given in Figure 3 the relative amounts offibril reorientation and fibril slippage at that point. A computeranalysis based on the growth curves shown in Figure 3 has shownthat the fibril slippage plays an important role in Phycomycescell wall growth, especially in the lower region of subzone II (MWold, unpublished data). Wold has shown that in the upper 52%of the GZ the majority of elongation and rotation must occur byfibril reorientation. The lower 48% of the GZ undergoes elon-gation and rotation via the fibril slippage mechanism.

It would be ofgreat interest to know whether the entire subzoneII responds to sensory stimuli. The data needed to answer thisquestion are in conflict; Cohen and Delbruck (5, 6) reported thatthe upper region of the GZ is devoid of a light response, whereasCastle (3) reported that this region is light sensitive. We havemeasured the rate of vertical descent of a test particle located insubzone II away from the sporangium, both before and after asaturating light stimulus and, qualitatively, it appears to us thatsubzone II is light sensitive. The response is small, but at bestone must expect a small response, since we are only observingsome 5 to 10% of the entire GZ.Another discrepancy concerns the existence of 'negative twist'

in the upper GZ reported by Cohen and Delbruck (5); we havenever observed this negative twist in the mature GZ although wehave carefully looked for it. If a region of the GZ did indeedshow negative twist, then necessarily we would observe an in-crease in the angular velocity as the test particle descendedthrough this region because the control particle is rotating in the

opposite direction.It is clear that the GZ of Phycomyces is complex; we feel that

this complexity may be more of an advantage than a disadvan-tage in terms of unraveling the molecular architecture of theliving cell wall. The advantage stems from the fact that specificmolecular mechanisms can be rigorously tested experimentallyand then acepted or rejected.

Acknowledgment-We are grateful to Matt Wold for the many fruitful discus-sions and for critically reading this manuscript.

LITERATURE CITED

1. AHLQUIST CN, RI GAMOW 1973 Phycomyces: mechanical behavior of stage II

and stage IV. Plant Physiol 51: 586-5872. CASTLE ES 1937 The distribution ofelongation and of twist in the growth zone

of Phycomyces in relation to spiral growth. J Cell Comp Physiol 9: 477-4893. CASTLE ES 1959 Growth distribution in the light growth response in Phyco-

myces. J Gen Physiol 42: 697-7024. CLOUGH DE, RI GAMOW 1983 Stochastic growth patterns generated by Phy-

comyces Sporangiophores. Foundation of Biochemical Engineering; Kineticsand Thermodynamics in Biological Systems, ACS Symposium Series 207,403-420

5. COHEN R, M DELBRUCK 1958 System analysis for the light-growth response ofPhycomyces. II. Distribution of stretch and twist along the growing zone andthe distribution of their responses to a periodic illumination program. J CellComp Physiol 52: 361-388

6. COHEN R, M DELBRUCK 1959 Photoreactions in Phycomyces growth andtropic responses to the stimulation of narrow test areas. J Gen Physiol 42:677-695

7. GAMOw RI 1980 Phycomyces: mechanical analysis of the living cell wall. JExp Bot 31: 947-956

8. GAMOw RI, B BOTTGER 1980 The extensibility of the cell wall above thegrowing zone. Phycomyces 4-Publicaciones de la Universidad de Sevilla 4:42-43

9. GAMOw RI, B BOTTGER 1981 Phycomyces: irregular growth patterns in stageIV sporangiophores. J Gen Physiol 77: 65-75

10. GAMOw RI, W GOODELL 1969 Local metabolic autonomy in Phycomycessporangiophores. Plant Physiol 44: 15-20

11. GERTEL ET, PB GREEN 1977 Cell growth pattern and wall microfibrillararrangement. Plant Physiol 60: 247-254

12. KAMIYA N, M TAZAWA, T TAKATA 1963 The relation of turgor pressure tocell volume in Nitella with special reference to the mechanical properties ofthe wall. Protoplasm 57: 501-521

13. METRAUX J-P, PA RICHMOND, L TAIZ 1980 Control of cell elongation inNitella by endogenous cell wall pH gradients. Plant Physiol 65: 204-210

14. OORT AJ 1931 The spiral growth of Phycomyces. Koninkl Nederland AkadWetenschap Proc 34:564-575

15. ORTEGA JKE, RI GAMOW 1974 The problem of handedness reversal during

- .- elongation; =-- - - eogto

rotation

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726 GAMOV

the spiral growth of Phycomyces. J Theor Biol 47:317-33216. PROBINE MC, RD PRESTON 1962 Cell growth and the structure and mechanical

properties of the wall in internodal cells of Nitella opaca. II. Mechanicalproperties of the walls. J Exp Bot 13: 111-127

17. ROELOFSEN PA 1950 The origin of spiral growth in Phycomyces sporangio-phores. Rec Trav Bot Neerl 42: 73-110

V ET AL. Plant Physiol. Vol. 80, 1986

18. ROELOFSEN PA, AL HouWINK 1953 Architecture ofgrowth ofthe primary cellwall in some plant hairs and in Phycomyces sporangiophores. Acta Bot Neerl2: 218-225

19. YOSHIDA K, T OOTAKI, JKE ORTEGA 1980 Spiral growth in the radially-expanding piloboloid mutants of Phycomyces blakesleeanus. Planta 149:370-375

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