Endogenous Growth Regulation in Carpophores of Agaricus ... · and that pure 10-4M solutions of 10...

PLANT PHYSIOLOGY

4. GOODWIN, T. W. 1954. Carotinoids: Their Com-parative Biochemistry. Chemical Publishing Co.,Inc., N. Y. x plus 356 pp.

5. GOODWIN, T. W. AND S. PHAGPOLNGARM. 1960.Studies in carotenogenesis. 28. The effect of il-lumination on carotenoid synthesis in French-bean(Phaseolus vulgaris) seedlings. Biochem. J. 76:197-99.

6. KARRER, P. AND E. JUCKER. 1950. Carotenoids.Elsevier Publ. Co., Inc., N. Y

7. KAY, R. E. AND B. PHINNEY. 1956. Plastid pig-ment changes in the early seedling leaves of Zeawtiays L. Plant Physiol. 31: 226-31.

8. KHUDAIRI, A. K. 1961. Xanthium leaf pigmentsand their inhibition by streptomycin. Biochim. Bio-phys. Acta. 46: 344-54.

9. LIND, E. F., H. C. LANE, AND L. S. GLEASON.1953. Partial separation of the plastid pigmentsby paper chromatography. Plant Physiol. 28:325-28.

10. 'MOSTER, J. B. AND F. W. QUACKENBUSH. 1952.The effect of temperature and light on the caro-tenoids of seedlings grown from 3 corn hybrids.Arch. Biochem. Biophys. 38: 297-303.

11. MOSTER, J. B., F. W. QUACKENBUSH, AND J. W.PORTER. 1952. The carotenoids of corn seedlings.Arch. Biochem. Biophys. 38: 287-96.

12. R6BBELEN, G. 1957. Untersuchungen an Strahlen-induzierten Blattfarbmutanten von Arabidopsisthaliana (L.) Heynh. Z. Induktive Abstammungs-Vererbungslehre 88: 189-252.

13. SAPOZHNIKOV, D. I., Z. M. EIDEL'MAN, N. V. BAZ-HANOVA, T. G. MASLOVA, AND 0. F. POPOVA. 1962.Participation of carotenoids in photosynthesis.Trans. Botan. Inst. Akad. Nauk SSSR, Ser. 4,Eksperim. Botan. 15: 43-52. (Chem. Abstr. 57:11559, 1962).

14. STRAIN, H. H. 1938. Formation of carotenoidsand chlorophylls in etiolated barley seedlings ex-posed to red light. Plant Physiol. 13: 413-18.

15. STRAIN, H. H. 1938. Leaf xanthophylls. Car-negie Inst. Washington Publ. 490: xi plus 147 pp.

16. YAMAMOTO, H. Y., T. 0. M. NAKAYAMA, AND C.0. CHICHESTER. 1962. Studies on the light anddark interconversions of leaf xanthophylls. Arch.Biochem. Biophys. 97: 168-73.

Endogenous Growth Regulation in Carpophores ofAgaricus bisporus 1,2

Hans E. GruenFarlow Herbarium, Harvard University, Cambridge, Massachusetts

The development of the fruit body or carpophore3in many Agaricales can be subdivided into an initialphase of cell multiplication and differentiation, anda phase of elongation marked by maximum stipegrowth, by expansion of the pileus (cap) and hy-menophore (lamellae), and by sporulation. The ma-jor features of mushroom growth were recognizedby Schmitz (24) who also discovered that elongationwas maximal in the upper stipe portion. Despite de-tailed descriptive work relatively few quantitativedata have been published on carpophore growth.These include growth curves for Panaeolius retirugis

1 Received March 15, 1963.2 This work was supported by National Science Foun-

dation Grants G-7100 to I. M. Lamb and G-14841 toI. M. Lamb and H. E. Gruen.

3 Following Singer (26) and others the term carpo-phore is preferred to sporophore or basidiocarp.

by Douglas (6), for Coprinus lagopus by Borriss(3) and Voderberg (31), and for the cultivatedAgaricus by Sacchi (23), Bonner et al. (2), andHagimoto and Konishi (9). Bonner et al. (2) re-ported that cell divisions ceased in the Agaricus stipewhen the fruit body was less than 2 cm long, and cellelongation was found by Hagimoto and Konishi (9)to parallel roughly the course of carpophore elonga-tion.

The literature contains scattered and contradictoryobservations concerning the effect of decapitationon growth. Schmitz (24) remarked that very youngcarpophores of agarics continued growing after re-moval of a third or even half of the cap, but stoppedif the whole cap was removed. According to Grantz(7) Coprinus stercorarius grew normally if decapitat-ed shortly before the onset of rapid elongation. Thedetached cap or parts of the cap expanded, and eventhe excised zone of the stipe elongation "in theusual nianner". He suggested that the apparent

652

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GRUEN-GROWTH REGULATION IN CARPOPHORES

independence of individual carpophore regions dur-ing elongation was preceded by a state of nutrition-al correlation between stipe and pileus. Accord-ing to Magnus (19) Agaricus campestris4 primordiadid not tolerate operations, and young carpophores(0.6-1.0 cm length) only continued growing if atleast half the cap was retained. He concluded fromdevelopmental studies that the hymenium played acontrolling role in early differentiation. However,his qualitative observations on later stages are in-conclusive, though decapitated stipes grew undersome conditions. Elongation of several centimeterswas reported by Streeter (27) for decapitatedAmanita stipes, though without comparative data forintact specimens, and Knoll (15) observed that de-capitated stipes, longitudinal slices, and isolatedstipe sections of Coprinus continued growing.

The only previous quantitative test of the presenceof an interrelation between cap and stipe, by Borriss(3), revealed that Coprinus lagopus stipes continuedgrowth if decapitated just after the onset of rapidelongation, but soon stopped if decapitated before thattime. It was sufficient to leave a thin cap slice at oneside of the stipe with a few lamellae to insure con-tinued elongation accompanied by weak negativecurvatures. The final length may have been lessthan normal, according to Borriss, but his data wereinsufficient to prove this point. He postulated thata hormone produced by the lamellae acted on the stipebefore rapid elongation began, but he was unable toconfirm this hypothesis either by replacing matureclosed caps on young stipes, or by injecting pressjuice from caps into the stipe cavity. According toJeffreys and Greulach (14) neither agar diffusatesnor extracts from stipes in agar or solution gavesignificant curvatures when applied unilaterally toCoprinus sterquilinus stipes decapitated during rapidelongation. Decapitation and removal of stipe por-tions were said to have no effect on the growth rate,but no comparative data were given on this point noron total growth. Hawker (11) briefly remarkedthat Collybia velutipes stipes curved after removal ofhalf of the cap or after unilateral placement of "fruitbody extract" agar on top of decapitated stipes.However, Banbury (1) could not confirm these ob-servations. More convincing evidence was given byUrayama (30), who removed the lamellae from oneside of a bilateral cap slice of Agaricus bisporus andobserved curvatures away from the remaining lamel-lae. Curvatures also resulted when lamellae wereplaced on one of two agar blocks which were applied

4 There is much confusion in the literature concerningthe nomenclature of the several cultivated species ofAgaricus, and even the commercially grown, whiteAgaricus has been designated by various specific andvarietal names, as well as by the incorrect generic namePsalliota. In this paper Singer's (26) nomenclature isfollowed, but the species names used by earlier authorsfor the cultivated Agaricus are given unchanged whenreferring to their work.

bilaterally to the underside of a cap slice withoutlamellae. He concluded that a diffusible growthfactor was produced by the lamellae. Hagimoto andKonishi (9) reported similar results with stipes of3 cm A. bisporus fruit bodies; when the lamellaewere removed partially or completely from half thecap the stipe curved so that the side with more lamel-lae was convex. The growth promoting substancewas said to move into the stipe from a cap portionwith lamellae attached only to the annulus or con-nected to the stipe by one ( ?) lamella. Cap ex-pansion was also said to depend on the presence oflamellae. Decapitated stipes of 3 cm carpophoresgrew only a little, but those of longer specimens"grew considerably". Removal of half of the capgave curvatures in young carpophores of 3 Coprinusspecies and Psathyrella candolleana growing in thefield, but had no effect during rapid growth. Sub-sequently, Hagimoto and Konishi (10) reported thatwhen lamella diffusate in agar was applied to awedge-shaped cap slice remnant, with plain agar atthe opposite side, negative curvatures resulted in 3to 4 days. Pieces of stipe and of cap trama (mainbody of the cap) also gave curvature. Curiously,lamellae on agar applied directly to the stipe causedless curvature than those applied to the cap remnant.Photographs of test specimens in this and the earlierpaper show that the stipe surface was sliced offtangentially along the entire length parallel with thesides of the cap remnant. The active material wasextractable by water, ethanol, and other organicsolvents. It resisted boiling in HCl and NaOH,was light stable, but apparently destroyed by bacterialaction. Ethanol extracts from Coprinus, Hypholo-ma, and Armillaria were also active. NeitherUrayama (30), nor Hagimoto and Konishi (9, 10)gave any quantitative data on the effects of their op-erations and tests on growth or degree of curvature.Furthermore, the information given did not provethe claim that the growth promoting action of thelamellae was due to a hormone. Recently, Konishiand Hagimoto (16) reported that 12 amino acidshad been identified in ethanol extracts of lamellae,and that pure 10-4 M solutions of 10 of these, aswell as inorganic ammonium salts, gave negativestipe curvatures when applied to the cap slice rem-nant (see above). In older specimens the lamellaeand ethanol extracts gave positive curvature.

The work reported below, partially described ina preliminary abstract (8), presents a quantitativestudy of the cap-stipe interrelation in Agaricus asan example of growth regulation in mushrooms, andas a basis for work on endogenous growth factors.

Materials and Methods

Carpophores of the "snow white" strain of thecultivated mushroom Agaricus bisporus var. albidus(Lange) Singer were grown in 40 X 30 X 12 cmhigh flats on horse manure-straw compost whichwas already penetrated by mycelium when obtained

653



PLANT PHYSIOLOGY

from a commercial grower. After humidifying thecompost (depth 7-9 cm), and covering with a casinglayer of soil (depth 1-3 cm) the flats were kept at15 to 17° under indirect light and covered by poly-ethylene sheeting until initials appeared. Each flatwas then fully exposed to continuous overhead lightfrom white fluorescent lamps, and covered with thepolyethylene sheeting at about 15 cm above the com-post. The light intensity under the cover averaged50 ft-c at the soil. It should be noted that A. bi-sporus carpophores do not curve in response to light.Constant air movement in the room was maintainedby a large ceiling fan wlhich was only turned offwhen the flats were uncovered for measurements.The relative humidity under the polyethylene wasabove 90 %, and care was taken to keep the soilmoist but not excessively vet. The first crop (flush)of fruit bodies reached about 2 cm length 20 to 26days after starting the flats, and 2 to 3 additionalcrops were produced. Fruit bodies appear isolatedand in clusters. In the latter case only one was al-lowed to remain at an early stage. Occasional, un-usually large specimens with thick, globular stipes,as well as excessively small ones with very thin stipeswere not used.

Length measurements to the nearest millimeterwere taken daily with a horizontal indicator connect-ing the top of the carpophore or stipe with a scaleplaced lightly on the soil tamped flat at one sideand/or in front of the stipe. Since a shallow cen-tral depression develops in most mature caps themeasured depth of this depression was deducted fromthe total length given by the indicator across the topof the cap in order to obtain consistent readings atthe cap center. After certain operations final lengthmeasurements had to be taken after removing the stipeby a horizontal scalpel cut level with the soil at thereading position. This method was also used in addi-tion to in situ readings for final measurements on intactspecimens with large caps. In situ length measure-ments were reproducible to within 1 mm in mostcases though in intact carpophores with expandedcaps the variation was 1.5 to 2 mm since the ruleris farther from the specimen. Cap diameters weremeasured with a vernier caliper and also with a flex-ible ruler at the end. Curvatures were measuredwith a circular protractor on lines bisecting the baseand curved upper stipe portion drawn on prints ofphotographs taken daily. Operations were carriedout with narrow scalpels taking care not to slice intothe stipe surface. Small dental mirrors (Kerr conesocket No. 4, magnifying and plane) were found in-dispensable for inspection in operations involvingtotal or partial removal of lamellae. Without suchinspection small remnants are easily overlooked, es-pecially at the cap-stipe juncture of young specimens.Several of the operations discussed below were per-formed at different stages of development, whileothers requiring lengthier surgery and inspectionwere carried out only in the 2.6 to 3 cm length rangewhich was found convenient for practical reasons,

and which falls on the ascending part of the growthcurve (see below).

Agar blocks were prepared as follows: the desir-ed amount of granulated Difco Bacto agar was stirredin excess 95 % ethanol and allowed to settle fora few hours before carefully decanting the alcolhol.This was repeated 3 to 4 times, and was followed byseveral washings with Pyrex-distilled water in thecold. The agar was made up to 1.5 %, autoclaved,poured into cooled, flat-edged glass rings> aind cutinto 0.7 x 0.7 x 0.5 cm blocks.

In a few specimens internal damage Nx-as observedat the stipe base due to unidentified microorganisms.However, the eggs or larvae of mushroom flies(sciarid flies) brought in with some of the new flatsproved a more serious menace since they attack thestipes internally. Drying of the compost before cas-ing, and application of an insecticide mixture con-taining DDT gave partial to practically completecontrol in such instances. Damaged specimiens werediscarded with the exception of a few7 carpophoreswhich had been attacked only very slightly N-henchecked after the final reading.

ResultsGrowvth of Intact Carpophores. Figure 1 (solid

line) shows an average growth curve for 25 intact

C,,

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OW O 2 3 4 5 6 7 8 9 10

0 ~~DAYS10 z0O 2 3 4 5 6 7 8 9 IQ

T ME IN DAYSFIG. 1. Time course of growth at 15 to 170 for in-

tact carpophores of A. bisporus var. albidus of 1 to 1.5cm initial length and 0.8 to 1.7 cm initial cap diameter.Solid curve, length in centimeters (25 specimens); dash-ed curve, diameter in centimeters for 24 of the saame spe-cimens. Vertical bars, twice standard error of the mean.Inset, average elongation rate (solid histogram), andaverage rate of diameter increase (dashed histogram)in cm/day followed with time. The numbers in the insetgive the average length on the second to fifth day.The average diameters are the same or only 1 mmin largeron the second and fifth day.

654




carpophores of 1.0 to 1.5 cm initial length measureddaily until at least 1 day after cessation of growth.The increase in daily growth rate was gradual (fig1, inset) but became slightly more pronounced atabout 3 cm. The maximum rate of 1.4 cm/day wasreached at 4 cm length. The veil broke completelynear 5 cm. The average cap diameter for 24 of thespecimens used for straight growth readings wasalmost identical to the fruit body length up to nearly6 cm when the cap began to flatten (fig 1, dashedcurve). The rates of cap expansion and stipe elon-gation also decreased roughly in parallel, and com-parison in individual specimens showed -that bothceased at the same time or almost the same time.The loose, vertically oriented hyphae which fill thecore of the young stipe were often torn off entirelyor partially near the underside of the mature cap(fig 2a) leaving an irregular cavity, while the corebelow remained filled with hyphae.

In young carpophores of about 2 cm length, wherespores are still absent, the lamellae are faint reddishpurple, but with increasing age the color becomesstronger and darker purple brown. Buller (5)pointed out that the hyphae of Psalliota campestrislamellae contain an intracellular pigment whichchanges from pink to brown on aging, and whichshould not be confused with the brown color of ad-hering spores. However, this pigment is not re-stricted to the lamellae but extends along a distinct,very thin adjacent layer of cap trama (fig 3), andwith increasing age also accumulates as a dark brownzone in the apical stipe cortex (fig 2b). The youngcap trama is otherwise pure white or only very faintreddish above the pigmented layer where browningcan also be seen in old specimens (fig 2c). Small

amounts of cut lamellae held together by this thinlayer always yielded a bright red pigment whichrapidly diffused through agar, and slowly turnedbrown with partial fading if the blocks were kept fora few days in the cold after removing the lamellae.When diffusing through agar into the stipe the pig-ment gave a brown spot at the surface although theagar remained red.

Young cap trama pieces gave no coloration, or atmost a very faint yellowing in agar, and the samewas true for agar applied at the surface of youngstipes. Jadot et al. (13) isolated an unusual aminoacid derivative, N- (y-L-glutamyl) -4-hydroxyanilinefrom the white cultivated mushroom Agaricus horten-sis, and Heinemann and Casimir (12) postulatedthat the reddening on cutting several Agaricus spe-cies, as well as the spore wall coloration, was due tothe oxidation of this compound. The lamellar tramaproper was not mentioned. A similar compound,agaritine, was isolated by Levenberg (17) from A.bisporus, and was identified as a hydroxymethyl phen-ylhydrazine derivative of glutamic acid. The red,agar-diffusible pigment observed in the present work,as well as the purple pigment concentrated in theregions mentioned above, may be oxidation productsof these glutamic acid derivatives. However, atleast the purple color is present in uninjured tissues,although reddening after cutting or bruising the stipesurface also occurs.

Stipe Growth after Removal of Portions of theCap. Selective operations, such as removal of all orparts of the lamellae, would be practically impossibleon the intact cap. Hence, it was necessary to de-termine to what extent the removal of major capportions influenced stipe growth. The simplest sym-

Table IAverage Total Growth Increments of Carpophores after Operations on the Cap

Treatment* Carpophore length at time of operation (cm)2.0-2.5 2.6-3.0 3.1-3.9 4.0-5.0

Total growth (cm)**Intact*** 6.2 ± 0.20(30) 5.7 ± 0.20(30) 5.0 + 0.21(30) 4.0 ± 0.20(30)Cap slice with lamellae 5.0 ± 0.24(11) 4.9 ± 0.28 (11) ... 3.4 + 0.26(12)Cap stump with lamellae ... 5.3 ± 0.49 (11)Cap slice with lamellae

at outer edges ... 2.8 ± 0.43 (7)Cap slice without lamellae .6 1.8 + 0.19(12)Decapitated stipes 0.6 0.13(13) 1.1 + 0.23(14) 1.4 + 0.13(17) ...Agar blocks on stipe apex+ . 1.6 ± 0.19(16) 1.8 ± 0.19(12) 1.7 ±0.26(12)Stipes with cap center 0.6 ± 0.13(11) 1.5 + 0.27(14) 2.5 ±0.37(12) 2.9 ± 0.23(16)

Carpophore length at time of operation: 2.6-3.0 cmGrowth at stipe middle (cm) Growth at stipe sides (cm)f+Lamellae alone 3.5 ± 0.51(10) 4.3 ± 0.38(10):

Lamellae alone, removedafter 2 days 3.0 ± 0.32 (9) 3.1 ± 0.21 (9):

Lamellae covered by1-2 mm of cap trama 3.8 ± 0.25 (6) 4.6 0.28 (6):

*

All cap remnants are bilaterally symmetrical. Operations described in text and illustrated in figures 3 to 12.Number of specimens in parentheses. Variation as standard error of the mean.Grow,th increments for intact carpophores are averages of the differences between the maximum length of eachof 30 specimens and the midpoint of each length range.Blocks renewed every second day until cessation of growth.Average growth at stipe sides and middle for the same specimens.Two measurements per specimen were obtained at the sides with lamellae.

655



Table IIAverage Total Inicrease in Diamneter and Width of the Tramiia of Cap

Slices after Various Operations on the Cap

Treatment*

Intact capCap slice with lamellaeCap slice with lamellae,

outermost edges removedCap slice with lamellae

Total increase indiameter**

cm

7.6 ± 0.38(24)9.2 + 0.77(11)

5.3 ± 0.49 (5)

Total increase in width***At the cap center Maximum width±

cm cm

0.6 ± 0.06(11)

0.3 ± 0.04 (5)

3.4 ± 0.25(11)

1.4 ± 0.12 (5)

at outer edges 10.6 + 1.16 (7) 0.5 ± 0.12 (6) 3.3 ± 0.26 (6)Cap slice without lamellae 2.7 -- 0.35(11) 0.3 ± 0.04 (6) 0.3 + 0.06 (6)

* All cap slices are bilaterally symmetrical and initially parallel sided. See text for description of operations.** Carpophore length at the time of operation, 2.6 to 3.0 cm. Average initial cap diameter, 2.8 cm. Number of

specimens in parentheses. Variation as standard error of the mean.*** Average initial slice width at center, 0.9 cm; at outer edges, 0.8 cm. Width of the expanded cap trama meas-

ured at the base of the cap, but excluding the lamellae. Width and diameter measured on the same specimens.± Measured at or close to the outer edges of the slices. Two measurements per specimen were obtained.

metrical structure for selective operations is a coimi-plete, parallel-sided cap slice of the same diameter asthe intact cap, and of the same wxidth as the stipeapex (fig 3; fig 16, diagram). The veil was remov-ed, and the initial fresh weight of the lamellae insuch slices from three 2.7 to 2.9 cm sample specimens(0.26; 0.27; 0.30 g) was slightly more than one-thirdlof all lamellae in the same caps (0.72; 0.79; 0.77 g).This operation was performed on 10 to 11 carpo-phores each of 2.0 to 2.5, 2.6 to 3.0, and 4 to 5 cimlength, and elongation and cap slice dimensions weremeasured daily (fig 16). The final appearance isshown in figure 4. The growth curves resemblethose of intact specimens of corresponding initiallength but the maximum growth rates for the 2 lowercategories were less, namely 1.1 and 1.0 cnm/day, andthere w,vas a slight, temporary initial decrease at 2to 2.5 cm (fig 16, insets, comlpared to fig 1). Total

growth for the cap slice operation at 2 to 2.5 andt2.6 to 3 cm was significanitly, though not much lessthan for intact carpophores of the same initial length(P<0.05; t-test for small samples), while at 4 to 5cm it was not significantly different (P>0.05), al-though slightly below nornmal (table I).

The fruit body lenigth includes the cap but the in-crease in cap thickness nmeasured at the stipe surfacecontributed only 0.4 cm to the total growth incre-ment of 6 cm during nearly the entire elongationphase of intact specimens (from 2-8 cm), and thefinal cap thickness after the slice operation was thesame as in intact carpophores.

In contrast to stipe growtlh the diameter of thecap slice increased at a greater rate than that of theintact cap, and attaine(d a final value nearly 2 cimwvider (fig 16; table II). Nevertheless, the rates

FIG. 2. Median longitudinal section through an intact carpophore at the termination of growth; a, cavity in thestipe core where the hyphae are partially torn off from the cap; b, apical stipe regions with dark brown pigment; c,faint brown pigmentation in the main body of cap trama. The deeply pigmented thin layer adjacent to the lamellae isnot distinguishable.

FIG. 3-15. Various operations performed on carpopheres at 2.6 to 3 cm length.FIG. 3. Parallel-sided complete cap slice showing the thin purple layer just above the lamellae.FIG. 4. Stipe with complete cap slice at 7 days after the operation.FIG. 5. Cap stump operation.FIG. 6. Decapitated stipe.FIG. 7. Cap center operation with the cap-stipe juncture faintly visible.FIG. 8. Cap trama slice after removal of all lamellae.FIG. 9. Stipe with lamellae alone.FIG. 10. Stipe with lamellae alone at 7 days after the operation.FIG. 11. Stipe with lamellae covered by 1 to 2 mm of cap trama.FIG. 12. Cap slice with lamellae only at the outer edges; a, initial view, b, and c, after 3 and 5 days, photograph-

ed at an angle from below.FIG. 13. Top view of a complete cap slice 8 days after the operation showing the pronounced expansion towards

the periphery in contrast to the center which expanded little beyond the initial width of the slice. The difference ingrowth between the upper layers and those adjacent to the lamellae is also observable.

FIG. 14. Negative stipe curvature (950) induced by a unilateral cap slice half in 5 days. The stipe apex and captrama (which also covers the apex) are hidden by the expanded lamellae.

FIG. 15. Stipe with lamellae alone at one side; a, initial view, b, after 1 day (-12°), c, after 3 days (-40°).FIG. 3, 5-9, 11, 12 a, natural size. FIGs. 2, 4, 10, 12 b-c, 14, 15 a-c, half natural size. FIG.. 13, one-fourtli natural

size.

656 PLA\NT PIIYSIOLOGY



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FIG. 16. Time course of elongation and cap expan-sion after the cap slice operation (diagram) at 2.0 to 2.5cm (curves A, A'; 10 specimens), 2.6 to 3.0 cm (curvesB, B'; 10 specimens), and at 4 to 5 cm length (curvesC, C'; 11 specimens). Ordinate, length or cap diameterincentimeters. Elongation (solid curves) and cap

diameter increase (dashed curves) mieasured simultane-ously on every specimen were followed for intact carpo-phores in each length category for 1 day (-1 on abscis-sa) before the operation (arrows), and thereafter for thesame specimens with the cap slice. Vertical bars; twicestandard error of the mean. Inset, stipe elongation rate(solid histogram) and rate of cap diameter increase(das,hed histogram) in cm/day during 1 day before theoperation (arrows), and for the cap slice thereafter.Number of specimens and ranges of initial length are thesame as in the main graph.

for cap expansion and elongation began to decreaseat about the same time, and measurements on flatspecimens showed that both terminated simultaneous-ly. The lamellae of the cap slice also expanded con-siderably, and their fresh weight was 20 and 21 g in2 specimens, or about two-thirds of the lamellae inthe mature intact cap (31 g average for 4 samples).The fresh weight increase of the lamellae in the capslice was close to 75-fold as compared to 40-fold forthe intact cap.

An even nmore radical operation consisted of theremoval of all marginal portions of the cap sliceleaving merely a parallel-sided cap stump with nar-row bilateral fringes of lamellar remnants adjoiningthe stipe (fig 5; diagram, fig 17). The operationwas performed at 2.6 to 3 cm length, and the drasticreduction in the cap is indicated by the stippled ar-row in figure 17. The average fresh weight of the

remaining lamellae was 0.07 g (5 samples), or onlyone-eleventh of all lamellae in the cap, and one-fourth of those in the cap slice. Nevertheless, thetotal growth (though more variable) was intermedi-ate between the complete cap slice and intact carpo-phores of comparable initial length (table I). How-ever, in contrast to the cap slice there was a tempo-rary decrease and disturbance of growth after theoperation (fig 17, inset), while on the other han(delongation continued for a day longer. The diameterof the cap stump increased only slightly, but thelamellae expanded, attaining an average fresh weightof 3.5 g (4 samples) after 8 to 9 days (50-fold in-crease).

Stipe Growth after Decapitation anid after t(le CapCenter Operation. Stipes were decapitated (fig 6) bycutting across the cap-stipe juncture after removalof all of the cap except the center. The veil was alsoremoved. Figure 18A shows how the daily growthrates decreased without recovery after decapitation(arrows) at the lengths indicated in each histogram.Below the 4 cm stage this decrease contrasts marked-ly with the increase in growth rate of comparable in-tact carpophores (fig 1, inset) and of stipes with capslices (fig 16, inset). Even decapitation at 4 to 5cm gave a faster decrease in growth than with capportions. However, growth after (lecapitation,which will be called residual growth, (lid not ceaseuntil 3 to 4 days after the operation, except for the1.3 to 1.9 cm specimens which stopped during thefirst day. Total residual growth was determined fornearly the entire growth period, and individual valueswere averaged for successive 0.5 and 1 cm intervalsof carpophore length at decapitation (fig 18C). Se-lected averages are included also in table I for com-

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FIG. 17. Time course of elongation and cap diameterincrease after the cap stump operation (diagram) at 2.6to 3 cm initial length (11 specimens). The shorteningof the cap diameter by the operation at time zero is in-dicated by the dashed arrow. Inset, stipe elongation rate(solid histogram) and rate of diameter increase (dashedhistogram) for the same specimens as in the main graph.

658

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02 3 4 5 6 7 8

CARPOPHORE LENGTH IN CENTIMETERS

FIG. 18 A. Growth rate of stipes after decapitation(diagram) of carpophores of various lengths. FIG. 18B. Same after the cap center operation (diagram). Inboth figures: Growth rates in cm/day (ordinate) for in-tact carpophores during 1 day (-1 on abscissa) beforethe operation (arrows), and thereafter for the same spe-cimens following decapitation or the cap center operation.Carpophore lengths at the time of operation are indicatednext to each histogram. Number of specimens in paren-theses. Distance between horizontal bars indicates twicethe standard error of the mean.

FIG. 18 C. Total growth increments (ordinate) afterdecapitation (open circles), and after the cap center(crosses) and cap slice (squares) operations, carried outat various carpophore lengths (abscissa). Each pointis plotted for the mean carpophore length of the testspecimens at the time of operation in the intervals: 1 to1.5, 1.6 to 1.9 (decapitation: 1.3 to 1.9), 2 to 2.5, 2.6 to3, 3.1 to 3.9, 4 to 5, 5.1 to 5.9, 6 to 6.9, 7 to 7.9 cm. Forcomparison, growth increments for intact carpophores

parison with other operations. Residual growth in-creased to a plateau at about 4 cm where the normalgrowth rate also reached maximum (fig 1). Al-though measurements were not extended beyond 7to 7.5 cm the residual growth would decline to zeroat little over 8 cm. The curves for residual growthand for the total remaining growth of intact speci-mens converge at 6cm fruit body length which alsomarks the beginning of the terminal phase of normalgrowth. In longer specimens the influence of thecap is no longer detectable by decapitation, but be-low this length the contrast between the 2 curves, aswell as the data for stipes with cap slices, demon-strate clearly that the presence of the cap, or partsof it, is essential for stipe elongation.

It was necessary to determine whether this depend-ence of the stipe on the cap was due primarily to an un-known growth-promoting compound produced by thecap, or if nonspecific factors such as injury or inter-ference with water movement played a significantrole. In order to test whether the cut surface at thestipe apex was responsible for growth inhibition afterdecapitation another series of measurements wasmade on stipes without lamellae, but with the centralcap portion of the same diameter as the stipe apex(fig 7). Growth rate changes after this cap centeroperation are shown in figure 18B for the same car-pophore lengths as decapitation. Below 2.6 cm thebehavior was the same as in decapitated stipes, andat 2.6 to 3 cm total growth was slightly, but notsignificantly greater than after decapitation, althoughit ceased a day later. Hence, injury at the stipe apexcannot be a major factor in limiting growth of de-capitated stipes. However, stipes with cap centerabove 3 cm length showed practically no decrease ingrowth rate until 1 day after the operation, andgrowth ceased 1 to 2 days later than after decapita-tion. Also total growth was significantly more(P<0.05) than for decapitated stipes (table I), butless than for comparable intact, or cap slice speci-mens up to 4.5 to 5 cm length (fig 18C). Thisgrowth promotion by the cap center suggests that itreleases additional amounts of a growth factor to thestipe (see Discussion).

No visible symptoms of drying were evident indecapitated stipes until residual growth had stoppedor nearly stopped, and often not even then. Nosignificant longitudinal shrinking of more than 1 mmwas observed although several stipes were measuredfor 3 days after cessation of growth. Borriss (3)remarked that Coprinus stipes decapitated before

(solid circles) were obtained by averaging the differ-ences between the maximum length obtained by eachspecimen and the midpoint of each of these intervals.Number of specimens per point: intact, 30; cap slice, 11to 12; cap center, 11 to 16; decapitation, 8 below 2 cm,10 to 17 at 2 to 5 cm, and 7 to 9 above 5 cm. Verticalbars indicate twice standard error of the mean.

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PLANT PHYSIOLOGY

rapid elongation grew slightly more if surrounded bywet cotton than without, but still did not enter theelongation phase. Jeffreys and Greulach (14) men-tioned transitory slight curvatures in Coprinus stipessprayed unilaterally with water. More strikingly,the unicellular sporangiophores of Phycomycescurved strongly in response to localized applicationof water in a nearly saturated atmosphere (29). Inorder to test for analogous reactions in Agaricus1.5 % agar blocks were applied symmetrically to thecut surface immediately after decapitation, and re-newed every 2 days. These blocks shrink to varyingextents, mainly because of water absorption by thestipe and not because of evaporation. Total growthafter this treatment (table I) was slightly greaterthan without agar blocks but the differences are notsignificant (P>0.05). When the agar block wasfirst applied 2 days after decapitation there was nosubsequent increase in growth. Despite these nega-tive results for straight growth, unilateral applica-tion of agar blocks to the stipe side or apical surfacegave weak negative curvatures which will be con-sidered in detail in a subsequent publication on stipecurvatures.

The experiments described in this section showthat there is no regeneration of a physiological apexin Agaricus stipes proper, nor in the cap center.

Localization of the Growth-promioting Regionwithin the Cap. Since the presence of the cap withlamellae is necessary for normal stipe growth theregion within the cap which is responsible for thegrowth promotion had to be located. The mere factthat the cap stump operation (see above) gave farmore growth than the cap center can only be at-tributed to the narrow fringes of lamellar remnantspresent on the cap stump since the amount of captrama is nearly the same. However, the presenceof cap trama and lamellae together does not excludethe possibility that interaction between the 2 couldbe the determining factor. Accordingly, all lamellaewere removed from the cap slice at 2.6 to 3 cmlength, leaving only the cap trama (fig 8; fig 19A,diagram), and on the other hand the cap trama wascut off leaving only bilateral masses of lamellae heldtogether by a very thin layer of cap trama (fig 9;fig 19A, diagram). Complete removal of the lamel-lae also involved cutting off a little of the adjacentcap trama and a slight portion of the incurved, outer-most edges. Nevertheless, the amount of cutting re-quired for the 2 operations is comparable, or evenslightly more pronounced during removal of captrama, which exposes the stipe apex, than duringremoval of the lamellae. The growth rate after thelatter operation declined without recovery (fig 19A,curve A, and inset), and total growth was not evensignificantly greater than with the cap center (tableI).

Measurements on stipes with lamellae alone pre-sent special problems since the middle of the stipeelongates less than the sides to which the lamellaeare attached. While this (lifference in growth aver-

aged only 1 mm by the second day, it increased fin-ally to 8 mm. Moreover, measurements at the sideswere only possible for a limited time because accesswas prevented eventually by the pronounced expan-sion of the lamellae which assume a vertical positionand spread out in front and back (figs 10, 15).Hence the complete growth curve can only be givenfor the stipe center (fig 19A, curve B), and finalmeasurements at the sides (point B') were takenafter removing the specimens (see Methods) w-hengrowth had ceased at the center for 1 or 2(days.The operation caused an initial temporary decreasein growth rate followed by an increase to 0.7 cm/ 'dayat the stipe center, and presumably more at the sides.The total growth increment was 3.5 cim at the centerbut amounted to 4.3 cm at the stipe sides, which isthe correct estimate of the effect of the lamellae, andwhich closely approaches the value for the completeslice (4.9 cm). The contrast between stipes withcap trania alone, and -,vith lamellae demon-strates thatthe lamellae must be the sole source of a grow-th pro-moting factor acting on the stipe.

In another series of 2.6 to 3 cm carpophores thecap trama was removed from the lamlellae as before,but the lamellae were also removed 2 davs later.Figure 19A, curve C showTs that the final growth clif-ferential across the stipe was abolishe(d (point C'),and that total growtlh was significantlIy less tlhan atthe stipe sides where lamlellae were present throughI-out the growth period. The average stipe lengtlh atthe time of lamella removal (at R) correspoinds to acarpophore length of 3.5 cm including the cap thick-ness. Growth after lamella removal w-as 2.1 cmwhile decapitation of intact 3.1 to 3.9 cim carpophioresgave only 1.4 cm (table I). Furthermore, the growthrate still increased (fig 19A, inset at R) in conitrastto the decrease during the first day after decapitation(fig 18A). This discrepancy requires furtlher in-vestigation.

The behavior of stipes with lamellae alone slhowsthat movement of the growth factor (s) inlto the stipe,and of water and possibly other materials into thestrongly expanding lamellae can take place in theabsence of practically all cap trama. Tw-o furtherpreliminary experiments were made in conniectionwith translocation. All parts of lamellae ad-jacentto the stipe, as well as the thin basal layer ot captrama, were removed bilaterally, leaving lam ellarremnants only at the outer margins of the cap slice(fig 12; fig 19B, diagram). Consequently. all sub-stances moving between stipe and lamiiellae had topass through the bridge of cap trama. Total -rowthwas more than wvith cap trama alone. but far lessthan for all other operations with lamellae adjacentto the stipe (table I). The growth curve and grovthrate histogram (fig 19B, curve D) resemble the ef-fects of complete removal of lamellae from the slice(curve A) while the cap trama itself expanded asmuch in diameter and width as in the complete slice(table II), and the (listance between the innler edgesof the remaining lamiiellae and the stipe increased

660




F20S .XRL,,0RID 'I

~~~~~~~R 07 .

1X1 ~~~-I .2 3 4 5 6 7 8_DAYS AFTER OPERATION

17 ~ ~ 4Q5

2$ x -W0D3._~~~ o0.1 -

-- -IuOl A2 L-456z 01~~~

DAYS AFTER OPERATION

I 12 7~~~DAYS AFTER OPERATION

FIG. 19 A and B. Time course of stipe growth after

selective removal of lamellae or cap trama from 2.6 to

3 cm carpophores. Each structure is illustrated by a

diagram. Elongation (ordinate) for intact carpophores

during 1 day before (-1 on abscissa) each operation

(arrows), and then for the same specimens after the op-

erations. In curves B, C, and E the arrows also in-

dicate the shortening in length due to removal of all or

part of the cap trama. Vertical bars, twice standard

error of the mean. Insets, growth rates in cm/day be-

fore and after each operation (arrows). The histograms

and corresponding curves in the main graph are des-

ignated by the same letters. Curve A (12 specimens),

cap slice without lamellae. Curve B (10 specimens),

lamellae alone, stipe length measured at the middle; point

B', final length at stipe sides. Curve C (shifted 1 day

to the right; 9 specimens), lamellae alone but removed 2

days after the start (at R) ; stipe length at middle and

sides identical or differing only by 1 mm; point C', final

length at stipe sides. Curve D (7 specimens), lamellae

only at outer edges of cap slice. Curve E (6 specimens),

lamellae and stipe apex covered by 1 to 2 mm of cap

trama.

from 4 to 5 mm to 1 to 2 cm (fig 12a-c). Thus,water and other substances necessary for cap expan-sion can move freely through the main portions ofthe cap trama, but transmission of the growth pro-moting action of the lamellae through the main bodyof the cap seems to be impeded though not complete-ly prevented.

In the other operation a continuous 1 to 2 mmthick layer of cap trama was left on the lamellae andstipe apex (fig 11; fig 19B, diagram). This layerincreased slightly in thickness to a maximum of 5 to6 mm above the lamellae but the overall behaviorwas essentially the same as without the cover, in-cluding the uplifting of the lamellae, and the growthdifferential between stipe middle and sides. Thegrowth pattern was the same (fig 19B, curve E),and so was the total increment (table I). As far asthe stipe is concerned, the initial temporary declinein growth rate, reflected in a small decrease in totalelongation, is the only noteworthy difference betweenthese 2 operations and the complete slice which lacksthe extensive cut surface across the top. No signifi-cant role in the transport of the growth promotingmaterial to the stipe can be attributed to portions ofthe cap other than the layer of hyphae immediatelyadjacent to the lamellae.

Cap Trama Expansion. Changes in diameter andwidth of initially parallel-sided cap slices on the stipewere used to study the effect of lamellae on cap tramaexpansion. Slices expanded at a greater rate thanthe intact cap (fig 16, 1), presumably because thetrama is released from the restraint of adjacent re-gions. Similar qualitative observations were madeby Magnus (19) after single cuts into the cap.Table II gives the total diameter increases for theintact cap, and for slices after various operations,as well as the maximum increase in width at, or closeto the outer edges, and at the center of the slices.All data are for 2.6 to 3 cm carpophores. Unfor-tunately, the slices with and without lamellae couldnot be made entirely comparable since the latternecessitated removal of an adjacent thin layer of captrama, and in most cases of a small portion of theincurved outer edges. When these edges were cutoff from complete slices in a similar manner, total ex-pansion was reduced but was still double that with-out lamellae (table II). There was practically noincrease in width in the cap trama alone, whileslices with lamellae showed marked enlargement,especially near the periphery (fig 13), and appearedroughly trapezoidal in cross section since the layersnearer the hymenophore grew more than the toplayers. This vertical difference in expansion leadsto the flattening of the cap.

The limited cap expansion after removal oflamellae at 2.6 to 3 cm was followed with time andresembles residual stipe growth after decapitation.However, unlike the latter, the daily expansion ratestill increased from 0.7 to 1.3 cm (7 specimens) dur-

661



PLANT PHYSIOLOGY

ing the first day following the operation before de-clining to zero on the fourth day. In contrast, theexpansion rate of the complete slice (fig 16), or ofthe slice with lamellae at the outer edges increasedstrongly for several days. The difference in totaldiameter increase between the latter is not signifi-cant considering the variability, and the increase inwidth w-as the same (table II). Provided the capmargin is retained the trama expands normally withless than the full complement of lamellae. Notice-able, but only slight cap trama enlargement occurredeven with very small patches of lamellae remnantsnear the periphery of the slice.

Stipe Curvatuire. Strong negative curvatures de-veloped away from half cap slices with the stipe apexleft covered by cap trama (fig 14). For instance,a 2.9 cm11 stipe gave 1350 curvature after 8 days, whilea comparable specimen from the same crop withlamellae restricted to the outer edge gave only 550maximuml, and stopped curving after 5 days. Growthof the sides with the cap portions was 5.5 and 3.2cim respectively, similar to the difference in straightgrowth for the 2 treatments (table I). Curvaturesaway from cap slice halves left at 1 side of the stipeare not restricted to the early stage of elongation.Wheni the operation was performed at 4.6 to 5.3 cmlength (6 specimens) negative curvatures averaging300 were measured after 6 (lays although curvingstoppe(l mostly at 4 to 5 days. The curved zone wasshorter than in younger specimens, and was restrict-ed to the upper third or less of the stipe. A fewtests withimarkers showed that the growth zone atthis stage of development is also restricted to theupper half of the stipe. It is not surprising that thecurvatures are smaller than those at 2.6 to 3 cm lengthsince the longer specimens are closer to the end oftheir growtlh period. When all lamellae were remov-e(l froml unilateral cap slice halves at 2.6 to 3 cmlength small negative curvatures averaging only 150were observed, but not in every case. At around 5 cmthere was nlo significant curvature without lamellae.On the other hand, lamellae alone left on one sideat 2.6 to 3 cim gave pronounced negative stipe curva-tures (fig 15 a-c) which reachedl over 900. In alloperations wN-here lamellae were left adjacent to thestipe their expansion blocks the view of the stipeapex after the first few days, andl the curvature can-not be followed accurately by photography. Hence,thle maximumi could only be determined at the estimat-ed en(d of the growth period, and no detailed quantita-tive stu(lies were made of the timle course of curva-ture dlevelopment.

Distinct curvatures occurred when larger portionsof the stipe surface split off due to unilateral cuts, orspontaneously. A few specimens curved more than900 towvards the injured sidle, but not enough in-formation is available as yet to predict generally thedirection of such curvatures. However, the splitsurface laver itself always curved outNwards from thestipe. Surface injury of this type can modify the

normal direction of curvature due to unilateral capportions left on the stipe to the extent that curvingtakes place at right angles to the expected direction.Spontaneous surface splitting is uncommon in un-damaged stipes, even during strong curving, but theincidence increased when agar blocks were applied.No curvatures resulted if only small localized sliverssplit from the stipe surface, nor if the loose whitehyphal cover was scraped or bruised without cuttingthe main hyphae. After vertical median cuts intothe apex of decapitated stipes the 2 halves curvedoutwards, illustrating the fact that differential tissuetensions mentioned by earlier workers (4, 15) are in-volved in these injury effects. Cap portions withlamellae which promote growth in the outer stipelayers can reverse the outward curvature caused bytissue tensions. Thus, a median cut through the capslice and upper part of the stipe at right angles to theslice led to an inward curving of the stipe halveswhich spread apart below the apex. This response,like the one obtained by Hagimoto and Konishi (9)by insertion of mica plates, is similar to the curvatureof whole stipes with cap portions left at one si(le (seeabove).

Discussion

During the elongation phase the lanmellae of A.bisporus are the center of stipe growth regulation aswell as of cap trama expansion, and no synergisticeffect on stipe growth could be detected between thecap trama and the lamellae. Borriss (3) thought thatthe lamellae of Coprinu(s lagopus acted as a trigger inthe sense that they had to be present on the stipe onlyuntil the onset of elongation, but not thereafter, sincedecapitation was without effect during that stage.In Agaricus there is no such evidence since the stipedepends oIn the cap over a wide range of carpophorelengths. In effect, total stipe growtlh after (lecapita-tion (residual growth) only equals that of intactspecimens at 6 to 7 cm carpophore length (fig 18C)when normal growth begins to decline (fig 1).Moreover, up to 4 cm, hence after the onset of rapidelongation, the growth rate decreases following de-capitation (fig 18A) in contrast to the increase inintact specimens (fig 1, inset), or in those withlamellae alone (fig 19A, curve B). The negativecurvatures obtained when cap slice halves with lamel-lae were left on stipes at around 5 cm length furnishadditional evidence for the continuing growth pro-moting effect of the lamellae. The lamellae veryprobably act on the stipe and the cap traimia by pro-ducing a growth factor (or more than one) whichdoes not seem to be synthesized elsewhere in the fruitbody since there is no regeneration of growth promot-ing activity in decapitated stipes or in those with captrama alone. The remaining growth after these op-erations is merely cdue to residutal aimiolits presentin the stipe.

662




It could be argued that growth decreases afterdecapitation because the operation eliminates or re-duces a transpiration stream through the stipe whichmay be necessary for the supply of essential mate-rials from the mycelium. Even during the elonga-tion phase substances other than water move into theintact carpophore as shown by the increase in dryweight in Agaricus (2), and in other mushrooms(18,22), although the significance of these findingsfor stipe elongation is unknown. However, the argu-ment that reduced transpiration could account for themarked inhibition of growth after decapitation is un-tenable since the presence on the stipe of wholeslices of cap trama without lamellae only led to prac-tically the same growth as the small amount of captrama at the cap center, despite a much larger sur-face, and since neither of these operations (at 2.6-3cm) promoted growth significantly over decapitatedcontrols (table I). Also, similar growth was obtain-ed with large differences in cap portions with lamel-lae (cap stump, cap slice). Schiitte's (25) findingthat the lamellae of certain agarics accounted onlyfor half or less of the total water loss from the fruitbody is further evidence against the view that a rolein transpiration may be the primary function of thelamellae as centers of growth regulation. Recently,1Ioser (20) also remarked that water loss was great-er from the cap and even stipe surfaces than from thelamellae. \Water loss by itself can equally be ruledout as a causal factor in the decline in stipe growthafter decapitation since agar blocks gave only slightpromotion although they yield some water to thestipe. Borriss (3) mentioned that Coprinus stipesdecapitated before the onset of rapid elongation stilldid not enter that stage even when surrounded bywet cotton, and showed only slightly more growththan untreated controls. Water supply to the stipe,which is attached to the mycelium, appears to be theresult of the primary growth regulation mechanism,and external application or reduction of transpirationact only secondarily, if at all significantly under theconditions of the present work.

Provided some lamellae were retained, removalof large portions of the cap above 2 cm only resultedin a relatively slight decrease in total growth (tableI), and in a lower maximum growth rate (1.0-1.1 in-stead of 1.4 cm/day), possibly in part because theremaining cap portions are bilateral instead of sur-rounding the stipe. Interestingly, there is no pro-portionality between elongation and the amount oflamellae left on the stipe. Thus, an initial averagefresh weight of 0.07 g of lamellae present on the capstump at 2.6 to 3 cm length gave nearly the sametotal growth as 0.27 g of lamellae for the completeslice, and even 0.75 g for the intact cap. The dis-crepancy in weight persists after expansion of thelamellae since specimens of the same initial lengthgave an average of 3.5 g of lamellae for the capstump, and 31 g for the intact cap at the terminationof growtth. The minimum weight of lamellae giv-

ing normal or almost normal growth was not deter-mined, but preliminary observations showed thatgrowth was clearly far below normal if much smalleramounts than in the cap stump were left at the cap-stipe juncture, although they were sufficient to in-duce curvature. However, the available data fordifferent cap portions with lamellae adjacent to thestipe cover a sufficiently wide range to show that amere fraction of the total complement can almosttake over the function of all lamellae with regard tostipe growth. It is quite unlikely that these resultscould be explained merely by a gross nutritional role ofthe lamellae. However, a quantitative interpretationin terms of a growth factor acting at low concentra-tion will only be possible when more information isavailable concerning the site of production withinthe expanding, complex lamellae, and when theamounts present in different regions of the cap andstipe can be evaluated. Only a fraction of all lamel-lae extends from the periphery of the cap to thestipe, while the others extend only part of the wayinwards. Buller (5) counted 5 times more lamellaeat the margin than at the stipe, and it is not knownwhether these marginal lamellae normally contributeto stipe elongation or merely to cap expansion whichis most pronounced at the periphery (2). This latterpossibility could account for the lack of proportion-ality between weight of lamellae and stipe elongation.The designation of hormone for the factor responsiblefor growth promotion by the lamellae is prematuresince only one property characteristic of phytohor-mones (28) was proven, namely action at a distancefrom the region of production. All reports in theliterature claiming the presence of growth hormonesin mushrooms lack adequate evidence (see Introduc-tion).

The characteristics of the residual growth of de-capitated A. bisporus stipes suggest an explanationfor the contradictory reports in the literature on cap-stipe interrelations (see Introduction). Statementsconcerning independence of the stipe from the capwere practically all based on qualitative observa-tions or isolated measurements without data for com-parable intact specimens, and without reference tothe whole growth curve. Consequently, the mere ex-istence of residual growth could give the misleadingimpression that the stipe is independent. EvenCoprinus lagopus requires reexamination in that con-nection. Work from this laboratory on Collybiaveliutipes in pure culture will be presented elsewhere,but it may be noted here that stipe growth in thismushroom is also dependent on the presence of thecap in the light, and that there is residual growthafter decapitation. In Agaricus the total residualgrowth increased from zero for decapitation below2 cm to a plateau at 4 cm (fig 18C). However, theaverage growth rate of intact 4 to 5 cm carpophoresis twice as high as at 2 to 3 cm (fig 1, inset and fig18A at time zero), the growth zone of the stipereaches its maximum length, which is nearly twice aslong as for the 2 to 3 cm stage (2), and total residual

663



PLANT PHYSIOLOGY

growth after decapitation is also maximal. Accord-ing to these observations larger absolute amounts oflamellar growth factor seem to be present in stipesdecapitated at 4 to 5 cm than at 2 to 3 cm. On theother hand, the daily growth rate after the operationdecreased about twice as rapidly in the longer thanin the shorter stipes (fig 18A), which suggests thatthe growth factor remaining in the stipe is used upfaster in the former. Nevertheless, growth does notcease until 3 to 4 days after decapitation at 2 to 4cm, indicating that other internal conditions alsoparticipate secondarily in stipe growth regulation,and probably determine the rate at which the lamellargrowth factor is used up. The reason for the plateauin the residual growvth above 4 cm is not yet clear.Although when plants have been decapitated at 5 to7 cm length growth still ceased only on the fourthday, the average growth rate decreased by 0.3 cmduring the first day, and then by 0.7 cm during thesecond day, hence much faster than after decapita-tion at 4 to 5 cm length (compare with fig 18A).Near 7 cm the stipe becomes independent of thelamellae since the total renmaining growth is the samewith or without the cap.

The cap center (fig 7) only increased elongationsignificantly over that of decapitated stipes when thecarpophor length was 3 cm or more (fig 18C).This promotion cannot be explained merely by theabsence of a cut surface at the stipe apex since pro-tection from injury could not be greater above thanbelow 3 cm. The opposite would be expected.Neither can it be due only to conserving water sinceafter decapitation at 3.1 to 3.9 cm agar blocks placedon the stipe caused only limited growth promotionwhich was less than the growth of comparable stipeswith the cap center (table I). The data suggestthat these stipes received additional growth factorthat still remained in the nongrowing trama at thecap center after removal of the rest of the cap. Thefact that elongation with the whole cap trama slicewas almost the same as with the cap center does notcontradict this hypothesis since the former includesperipheral regions undergoing limited residual ex-pansion (table II).

In further studies on translocation of the growthfactor to the stipe special attention will have to begiven to the basal layer of hyphae of the cap, par-ticularly the pigmented layer to which the lamellaeare attached. When the lamellae were held togetherby this layer and hardly any additional cap trama thetotal stipe growth approached that given by the com-plete cap slice (table I) despite the initial decline ingrowth (Fig 19A, curve B), which was probably caus-ed by the fairly extensive cutting required for removalof the cap trama. This experiment shows that thegrowth promoting substance is translocated into thestipe either through the basal layer of the cap orthrough the lamellae themselves which are appressedto the stipe surface (fig 2,3), and also attach to thestipe apex along a very narrow strip although in

taxonomy they are called free (26). On the otherhand, the results of removing all portions ot thelamellae and of the basal cap layer adjacent to thestipe (fig 12 a-c) suggest that the growth factor isnot translocated readily through the main body ofcap trama towards the stipe since elongation w-as re-duced nearly 50 % compared to specimens w\ith ad-jacent lamellae (cap slice, cap stump), anid despitevery strong expansion of the cap. Part of thelamellae left on the cap after this operation extendedinitially to the stipe, and are at least comiiparable inamount to those on the cap stump.

The movement of dyes need have no bearing onintracellular translocation of other materials, anddiffuse penetration of several dyes into agaric fruitbodies is well known (20, 21). However, Schifitte(25) reported that fluorescein and a red dye, RoseEdicol, moved very rapidly upwards alonig a zonelining the inner surface of the stipe cylinder, andthen along the basal layer of the cap, including thezone considered important in connectioni x-ith thepresent studies. The dye-translocation hIyphae xx erenot distinguishable anatomically, and xv-lile the zoneNvas a little broadler in the cultivated iuThslirooimi thalniin other species tested, these (lyes did nut movethrough other regions.

The large curvatures which develop in responseto unilateral cap portions or lamellae alonie (eventhough the cap trama completely covered the stipeapex in the former case) indicate that the growvthfactor is not readily transported across the stipe.This is also shown by the growth differential betweenstipe sides and middle when lamellae alone x-ere lefton bilaterally, or even with a thin cover of cap tramaacross the stipe. (table I). SUch1 a (liffereince isless pronounced with the complete cap slice altlhoughin some cases separation of the stipe lavers fromii thecap occurs at the uncovered juncture. Trai-sportthrough the core hyphae can be ruled out since theyare torn apart during elongation.

The observations reported above concel-rninig theeffects of injury must be taken into account in de-veloping a test based on stipe curvature. Significantasymmetrical surface injury would interfere serious-ly with such a test, and even procedures involvingsymmetrical or near symmetrical cutting- into thestipe surface may introduce secondary factors diffi-cult to control in practice. Problems relatinig tostipe curvature and test procedures will be discussedin a later publication.

Summary

A growth curve was obtained at 15 to 170 forcarpophores of Agaricus bisporuts var. albidus(Lange) Singer from 1 to 1.5 cm initial length untilcessation of growth. The average maximumii growthrate was 1.4 cm/day. The average increase in capdiameter wvas numiierically equal to the carpopliorelength up to 5 cm vwhen the cap began- to flatten.

664




Growth rate, both in stipe length and cap diameter,declined and ceased at the same time. Observationson pigmentation of the lamellae and the basal caplayer are included.

The effects of removal of cap portions on elonga-tion and cap expansion were studied by daily meas-urements before and after leaving only parallel-sidedcap slices on stipes at 3 ranges of initial length, andalso an even smaller cap remnant (cap stump) at onelength range. These operations resulted only in asmall decrease in total growth compared to intactspecimens, although the maximum growth rate wasreduced to 1.0 to 1.1 cm/day for both operations.At the same carpophore length the average initialfresh weights of lamellae sampled for the cap stump,cap slice, and intact cap were not proportional to thetotal growth increments.

Decapitation decreased the stipe growth up to 6to 7 cm carpophore length where growth began todecline in intact fruit bodies. The growth rate de-creased during the first day after decapitation, butgrowth did not stop until 3 to 4 days after the op-eration, except below 2 cm carpophore length whereit stopped during the first day. This residual growthwas studied in detail. When the trama at the capcenter was left on the stipe apex after removal of therest of the cap with all lamellae, stipe growth waspromoted significantly compared to decapitated stipes,but only at or above 3 cm carpophore length. Thispromotion suggests the release of residual amounts ofa growth factor to the stipe. There is no regener-ation of a physiological apex either in the stipe itselfor in the cap trama.

Growth studies after selective removal of the captrama or lamellae demonstrated that the lamellae arethe regulatory center of stipe growth and also of captrama expansion, and that there is no synergistic ef-fect on stipe growth between the lamellae and thecap trama. The action of the lamellae cannot be at-tributed to water relations, and must be due to oneor more growth factors which are not synthesizedelsewhere in the fruit body. Transmission of thegrowth promoting effect from the lamellae to thestipe seems to take place either through hyphae atthe base of the cap adjacent to the lamellae, or direct-ly through the lamellae to the cap-stipe juncture.Transmission through the main body seems to beimpeded though not completely prevented.

When the cap slice or the lamellae alone were leftat one side of the stipe during the early phase ofelongation strong negative stipe curvatures resulted,while such curvatures were small or absent when thelamellae were removed from the cap slice. During alater phase of elongation stipes bearing cap sliceswith lamellae at one side also gave negative curva-tures, but these were smaller than in younger speci-mens, and restricted to the upper third or less of thestipe. Significant unilateral injury due to cuttingor splitting of the stipe surface layers caused curva-

tures which can modify the direction of curving ex-pected in response to unilateral cap remnants bear-ing lamellae, and which must be taken into accountin the development of a stipe curvature test.

Acknowledgments

The author wishes to express his appreciation to Mrs.Nobuko Kamecke for her conscientious assistance in thecourse of this work and in drawing the illustrations. Heis grateful to Professor K. V. Thimann for his criticalcomments and discussion on reading the manuscript.

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