The effects of light intensity and light period on the development of thallus form in the marine red ALGA Pleonosporium squarrulosum (Harvey) Abbott (Rhodophyta: Ceramiales). II. Cell enlargement

J. e.qo. mcrr. Bial. Ecol., 1975, Vol. 19, pp. 165-l 76; (6, North-Holland

Publishing Company

THE EFFECTS OF LIGHT 1NTENSITY AND LIGHT PERIOD ON THE DEVELOPMENT OF THALLUS FORM IN THE MARINE RED ALGA PLEONOSPORIUM

SQUARRULOSUM

(RHODOPHYTA: CERA~LES).

(Harvey) Abbott

II. CELL ENLARGEMENT

STEVEN N. MURRAY Department of Biulogiccd Science, Cnlijkwzin State University, F44ilertctn, Ct~l~fornia~U.S.A. and PETERS. DIXON Depurtment ofPopulation and Environmentnl Biolog.~, iJniversit.v of California, Irvine, CnliJbmia, U.S.A. Abstract: Analyses of patterns of cell enlargement of regenerates of Ple[~n~sporilint s~tu~rr~~~~)sut~~ (Harvey) Abbott grown under light periods of 16L-8D and SL-16D and light intensities of 269, 538, 1076, and 1614 lux have been made. These show that light intensity, and to a mnch greater degree, light period significantly affect the magnitude of cell elongation for main axes and tirstorder IateraI axes; for main axes, such was primarily by an increase in size of the basipetal parts 01 ceils.

INTRODUCTION

In order to understand the morphological variation of a florideophycean taxon, one must know something concerning the effects of diverse ecological conditions on growth. The form of an algal thalius is determined by the interaction of three processes, namely; cell division, cell enlargement, and cell differentiation, and the effect of environmental conditions on the form of Florideophyceae may be best determined by analyses of the responses of these processes. The general principles of thallus development have been outlined by Dixon (1958, 1960,1963a, b, 1966, 1970,197l) and it has been shown that in the Florideophyceae, particularly the simple uniseriate Ceramiaceae, cell division and cell enlargement exert by far the greatest influence on thallus form. Relatively little is known of the rates of division of apical cells of Florideophyceae under different environmental conditions; however, it appears that apical cells of primary axes undergo l-3 divisions per day (Konrad-Hawkins, 1964a, b; Dixon & Richardson, 1970; Duffield, Waaland & Cleland, 1972; Waaland & Cleland, 1972; Murray & Dixon, 1973) and that the rate of division in certain taxa is influenced by light (Dixon & Richardson, 1970; Murray & Dixon, 1973). The relation of cell enlargement to the development of thallus 165

STEVEN N. MURRAY

166

form has been discussed based largely

by Dixon

on field data,

complex

patterns

Analyses

of cell elongation

patterns

PETER S. DIXON

(1971) for several florideophycean

has treated

of enlargement

AND

in detail

and has provided

the magnitude,

taxa; this work, orientation,

a basis for experimental

under different laboratory

illumination

and study.

treatments

have been given for GrifJithsia pacijica (Waaland & Cleland, 1972). Murray & Dixon (1973) have already determined the effects of illumination on the rates of division of apical cells of main axes of Pleonosporium squarrulosum (Harvey) Abbott. The present contribution represents a sequel to that study and is concerned with the effects of light period and light intensity on cell enlargement.

MATERIALS AND METHODS

Regenerates of P. squarrulosum were grown under experimental light-dark periods of 16 h light-8 h dark (16L-SD) and 8 h light-l 6 h dark (8L-16D) for 2 1 consecutive 24-h days. Light intensities of 269, 538, 1076, and 1614 lux were used at each lightperiod treatment. At the end of experiments, the regenerates were fixed in 3-4 “<, formaldehyde-sea water, made into semi-permanent glycerine-jelly preparations, and analysed with respect to the enlargement of cells of main and first-order lateral axes. Seven to twelve replicates were analysed for each experimental treatment. (See Murray & Dixon, 1973, for details.) Although the enlargement of axial cells of P. squarrufosum is three-dimensional, by far the greatest degree of enlargement is consistently in overall cell length, and so this measure was chosen for comparing the effects of experimental treatments on the enlargement of axial cells. Cell lengths were obtained for each axial cell as the distance between its pit connections with immediately acropetal and basipetal cells; patterns of cell enlargement were then determined for experimental plants by plotting cell length against segment number. Plots were made for both main and lateral axes of the plants. In order to carry out a statistical analysis for all except regenerates grown under 269 lux, the lengths of the 30&h, 40th, and 50th cells from the apex of the main axis were pooled for regenerates receiving a similar experimental treatment. These data were then analysed

by analyses

of variance

for cases with disproportionate

subclass size (see, Steel & Torrie, 1960). For regenerates grown under 269 lux illumination, replicates were too few or growth was insufficient for statistical treatment of the data. Intracellular patterns of the enlargement of cells of the main axes were also compared between regenerates grown under different experimental treatments. Measurements of individual axial cells were recorded in terms of basipetal and acropetal components. Basipetal components were measured from the pit connection for,med by each axial cell during the production of its single lateral initial (cf., Murray & Dixon, 1973) and its basipetally-orientated pit connection with the axial cell immediately below. Similarly, acropetal components were determined by measuring from the point of lateral initiation to the acropetally-orientated pit connection. The effects of light intensity

THALLUS

FORM

IN A RED

ALGA

167

and light period on patterns of intracellular enlargement were tested by the same statistical technique after pooling basipetal/acropetal enlargement ratios for the 30th. 40&h, and 50th cells from the apex for regenerates receiving common experimental treatments; results from regenerates receiving 269 lux illumination treatments were again not analysed. I 300 8L-160

16L-8D

t

ioow

200

t

300 -

I076 Ix

200

-

100 1614 Ix Apex IO

20

30

40

50 SEGMENT

Apex to

20

30

40

50

NUMBER

regenerates of Pleonosporium grown under experimental light treatments: A, B, C, D = 8L-16D; E, F, G, H -= 16L-SD; A, E = 269 lux; B, F = 538 lux; C, G = 1076 lux; D, H = 1614 lux.

Fig. 1. The

syuarrdosum

patterns of main axis cell elongation for representative

STEVEN

168

N. MURRAY

AND

PETER

S. DIXON

RESULTS AND DISCUSSION

The pattern of thallus development in many Florideophyceae is highly controlled with respect to cell division and cell enlargement. As has been indicated previously (Dixon, 1971; Murray & Dixon, 1973), cells in florideophycean filaments, with only a few exceptions, are produced in a linear series by successive apical cell divisions, so that in mature thalli the relative positions of axial cells within filaments may be employed for age comparisons. MAIN

AXES

The patterns of elongation of the cells of the main axes of P. ~~~~r~~~~~~~under the experimental light treatments are given in Fig. I for representative regenerates; clearly light period affects cell elongation profiles. Additionally, comparison of the data (~5, Fig. I, Table I) for regenerates grown under 269 lux show a reduction in cell elongation for both 8L-16D and 16L-8D photoperiods. Cell elongation is interpreted to be sensitive to the low light intensity (269 lux) in much the same manner as the rates of apical cell division (Murray & Dixon, 1973); cell elongation is, however, much more clearly affected by light period. A statistical comparison such as outlined above on pooled data does not allow for differences in rates of apical cell division which would affect the ages of equivalently-positioned axial cells of different experimental plants. Differencesin the rates ofdivision ofapical cells of the main axes of experimental P. ~q~ffrr~~~~~~regenerates have been reported earher (Murray & Dixon, 1973); by far the greatest effect of light on cell division was in regenerates grown TABLE

1

Increase in length (pm) of the 30th, 40th, and 50th cells of main axes of regenerates various experimental conditions of illumination: means and 5 S.D. Illumination conditions

30th crll ______ 2

50th cell

40th cell

5.S.D.

.t‘

grown under

+S.D.

.i?

_: S.1).

269 iux* SL-I 6D 16L-8D

44 126

_ .._

SO I.56

_

II0 218

538 Iux 8L-16D l6L-SD

133 144

12 I8

I89 258

22 28

216 339

29 32

1076 lux 8L--16D 16L-8D

122 145

22 34

I88 226

29 62

263 359

28 93

1614 lux 8L-I 60 I H.-SD

130 162

I5 24

216 258

I6 38

288 410

35 57

* Data for 269 lux are taken from representative

regenerates.

THALLUS

FORM

IN

A RED

ALGA

169

500

400

I

,

T

I’ I

<’

50th CELL

f

300

200

100

-C 9 40th

i y

He’ : _I’

300

;---________F

2 x :

CELL

200

0 z W 5

100

-I

300 30th

CELL

200 _--________

__--

___a---

-3

T

3

IOk

I$14

100

5~8

LIGHT INTENSITY (LUX) Fig. 2. Enlargement of the 30th, 40th and 50th axial cells of regenerates of Pleonosporium s9uarrulosun1 grown under experimental light treatments: means and confidence limits at P = 0.05: ~~~ 8L-16D; ---16L-8D.

STEVEN

170

N.

MURRAY

AND

under 269 lux and this could not be included ages of axial cells for experimental cell division do not result

data of Murray

material

(1973) discussed

different

S. DIXON

in the present analysis.

P. squarrulosum

& Dixon

in interpretations

PETER

from those

Estimates

may be obtained

of the

from the

earlier. These data, however, given

here and really

do not

represent a significant improvement in methodology because the ages of cells can still not be precisely set. The true ages of cells can only be determined by constant daily monitoring (see, Waaland & Cleland, 1972) or by marking techniques (see, Waaland, Waaland & Cleland, 1972). Such techniques are far more precise but since quantitative data on the effects of environmental factors on cell enlargement are almost totally lacking for Florideophyceae, our results represent a useful starting point for further investigations. The magnitudes of enlargement of the 30th, 40ih, and 50th axial cells of these regenerates grown under experimental illumination conditions are given in Table I and Fig. 2. Analysis of variance (Table II) indicates that light intensity did not affect cell elongation at the 30th cell level (P > 0.05) but became a significant factor at the level ofthe40th (0.05 > P > 0.01) and 50&h(0.01 > P >O.OOl ) cells. Light period was found to significantly (P < 0.001 ) affect cell elongation in comparisons of all levels and it represented by far the most important effect: elongation differences became more pronounced as cells aged (Table II, Fig. 2). Since for the experimental treatments used the effect of light intensity appeared to be minimal for regenerates receiving > 269 lux, the data for these light intensity treatments TABLE

II

Analysis of variance of cell elongation at the 30th. 40th, and 50th cell levels for regenerates of Pleonosporiun~ syuarrulosum grown under experimental conditions of illumination greater than 269 lux: method for disproportionate subclass numbers (Steel & Torrie, 1960): *** P 0.001, ** 0.01 > P > 0.001,* 0.05 3. P 0.01.n.s. P _'0.05. Source of variation

D.f.

Sum of squares

Mean square

30th cell Light period Light intensity Interaction Error

2 2 46

6408 1357 I092 21421

6408 679 546 466

40th cell Light period Light intensity Interaction Error

1 2 2 48

321.52 7983 2425 57565

32152 3991 1212 II99

I 2

150535 39497 I840 115421

150535 19748 920 2815

50th cell Light period Light intensity Interaction Error

L

41

F

13.76***

I .46 n.s. 1.17 n.s.

26.78*** 3.33* 1.01 11.5.

53.47*** 7.01** 0.33 n.s.

THALLUS

FORM

IN A RED

were pooled for each cell ‘age’ and regression treatments

lines calculated

(Fig. 3). There is a greater rate of increase

ates grown under longer 9 = - 173.12+ lO.74Xfor

Ill

ALGA

for the two light-period

in cell size with ageforregener-

(I 6L-8D) illumination. The regression equations 16L-8D and 9 = -60.77+6.36Xfor 8L-16D.whers

the length of axial cells and X the axial segment

are: ?is

number.

16L-80

8L- 16D

1

i 30 SEGMENI-

Fig. 3. Regression and

16L-RD

C----)

lines for regenerates light periods:

I

I

40

50

NUMBER

of Pkonosporirmr

means of pooled

sqmrrulosrotr grown under 8L-16D

light intensity

data

(538,

1076,

1614

(

)

ILIX) Or

each cell ‘age‘.

The significant effects of light period and light intensity on cell e~ongatj~~n in P~~o~us~?~r~u~ ~~~~~~~~~~z/~ contrast with results of D&field, Waaland & Cleland ( 1972) and Waaland & Cleland (1972) for Gri’thsia pacijicn where 300 ft-c ( % 3230 lux) and 4Oft-c (Z 4301~~) and 16L-8D, 12L-12D and SL-16D light periods did not markedly affect the rate of cell elongation. A developmental effect of light intensity on “shoot” cells was also found; under higher light intensities (300 ft-c) these cells became predominantly nodal i.e., they produced lateral branch initials. In Pleonospnrim squarrulosum, each axial shoot cell gives rise to a single lateral branch and so there is no differential development from which the effects of primary lateral production would become apparent. A greater production of 3rd order laterals was, however. apparent for most regenerates receiving the longer light period. Dixon (1971) has given evidence for the effect of a lateral filament on cell enlargement within the parent filament for Florideophyceae such as Gr~~t~~s~~pac(fica where each segment cell does

172

STEVEN

not have the same branching

N.

MURRAY

AND

characteristics;

PETER

analysis

S. DIXON

of the effects of environmental

parameters on cell elongation in the main axes of G. pacijica could be complicated the additional variable of differential branching of shoot cells. LATERAL

Analysis

by

AXES

of enlargement

of cells of first order laterals

(Fig. 4) show comparable

patterns to those described above for main axes. Elongation was uniformly less pronounced under the 8L-16D light period as compared with 16L-8D. Jn addition, reduction in cell size is apparent when comparisons are made between regenerates

150 1 16L_

269 Ix

-

-’

1

1

x

a

k 150 E P- 100i

SEGMENT

NUMBER

Fig. 4. The patterns of first-order lateral axis cell elongation for representative regenerates of Pleonosporium squarrulosum grown under experimental light treatments: A, B, C, D : SL-16D: E, F, G, H = 16L-SD; A, E = 269 lux; B, F : 538 lux; C, G = 1076 lux; D, H 7 1614 lux.

THALLUS

receiving

FORM

IN

A RED

173

ALGA

269 lux and those grown under higher light intensities;

variations

in enlarge-

ment profiles as a function among regenerates receiving

of light intensity were minimal for each light period light intensities greater than 269 lux.

PATTERNS

ELONGATION

Analysis

OF INTRACELLULAR

of intracellular

elongation

patterns

for a number

of Florideophyceae.

as indicated by using pit connections formed between parent axial cells and their pericentral-cell derivatives, have shown asymmetric patterns of intracellular growth (Dixon, 1971). In such thalli, pericentral cells are formed when axial cells are young and in a position just a few cells removed from their apical cell origin. At the time of pericentral cell formation, the pit connection is positioned approximately midway along the length of the parent axial cell, so that differential patterns of intracellular elongation with cell age may be determined by treating separately the elonpaSEGMENT

NO

LENGTH

./,, :. P

;.

j ,.

: j

: i

Total cell

,:’

39

106

259

IN MICRONS

Acropetol

17

Basipetal

22

29

77

36

223

100 urn

Fig. 5. The

intracellular

pattern

of elongation

of main

axis cells of Pleonosporium

syuarrulosurn.

174

STEVEN

N.

MURRAY

AND

PETER

S. DIXON

TABLE 111 Ratio

of basipetal/acropetal grown under

for the 30th, 40th, and 50th cells of main axes of regenerates conditions of illumination; means and LS.D. 40th cell

30th cell

Illumination conditions 269

elongation experimental

F

_AS.D.

.i

SOth cell S.D.

.r

S.,).

lux* 2.0 3.3

SL--lbD l6L-8D

2.6 3.1

3.5 3 .:

5.38 lux 8L -16D 16L-8D 1076

0.5 0.7

3.4 4.9

0.5 0.6

3.9 4.5

0.5 0 .6

3.0 ?.I

0.4 0.6

3.6 4.2

0.5 0.6

3.7 4.6

0.5 0.5

3.0 3.5

0.5 0.7

3.9 4.3

0.6 0.6

4. I 4.x

0.5

lux

XL-16D 16l..-8D I614

3.1 3.5

lux

XL-l6D l6L-8D * Data

for 269 Iux are taken

from

representative

0.x

plants

Analysis of variance of basipetalKacropetal elongation ratios at the 30th, 40th, and 50th cell levels for regenerates of Pleonosporiu~r~ sqrrarrrrlmrm grown under experimental conditions of illumination greater than 269 Iux: method for disproportionate subclass numbers (Steel & Torric. 1960): ratios have been inverted and transformed to arcsine values in order to stabilirc the variance\. *** p 0.05. 0.001, ** 0.01 -. P ’ 0.001, * 0.05 ’ P 0.01, 1l.S. P Source ot variation

D.f

Sum oi squares

Mean square

30th cell Light period Light intensity Interaction Error

2 2

46

49. IO 18.96 17.84 593.15

49. IO 9.48 8.92 12.89

3.81

1.b.

0.74 n.5. 0.69 I1.S.

40th cell Light period Light intensity Interaction Error

145.56 14.31 24.04 5.71

25.49*** 2.51 n.5.

2 48

145.56 28.61 48.07 274. I9

2 2 41

90.79 14.01 7.09 188.59

90.79 7.01 3.55 4.60

19.74*** I .52 n.s. 0.77 n.s.

2

3.21*

50th cell Light period Light intensity Interaction Error

THALLUS

tion

of cell portions

acropetal

‘marker’.

The great majority

basipetal

intracellular

Pleonosporizrm

manifested

IN

A RED

and basipetal

to the pericentral

of Florideophyceae

elongation

as axial

I7

ALGA

show a marked

cells mature

(Dixon,

cell pit-connection predominance 1971,

1973).

of For

squarrulosum,

basipetally

were determined

FORM

the elongation of axial cells with age is also large11 (Fig. 5). Basipetal/acropetal ratios of intracellular elongation

(Table

III) for the 30th, 40th, and 50th axial cells of regenerates

subjected to experimental transformation to arcsin-

light treatments. Analyses of variance of the data after ’ values for all plants receiving > 269 lux were made to test whether the previously observed effects of light on the magnitude of axial cell elongation affected the established basipetal to acropetal growth pattern; these analyses (Table IV) indicate that the longer I6 h light period significantly increz\ed the relative basipetal elongation; the effects of light period on cell elongation art‘. therefore, primarily manifested in an increased rate of basipetal elongation.

ACKNOWLEDGEMFNTS

Partial assistance for this work was gained from a California State University. Fullerton, Faculty Research stipend (SNM). We are grateful to Kathryn Heath for preparation of the illustrations.

REFERENCES DIXON, I’. S., 1958. The morpholcgy, ecology and taxonomy of certain Florideae. Bv. $?~.ujI. Buli.. Vol. I, pp. 32-33. DIXON, P. S., 1960. Studies on marine algae of the British Isles: Crrtr,uirorr sll//rrlel~o~!/fi~i,rul,r (Kutr.) Silva. J. MT. hiol. Ass. U.K., Vol. 39, pp. 375-390. DIXON, P. S., 1963a. The Rhodophyta: some aspects of their hiologq. Ocrtrno/z iu tlrc, YPCI. edited by J. P. Harding and N. Tebble, Systematic Association, London, pp. 51 -62. DIXON, P. S., 1966. On the form of the thallus in the Florideophyceae. In, Tre&s i/l /~/trut /r~orplu~qenesis, edited by E. G. Cutter, Longmans, Green & Co., London, pp. 45-63. DIXON, P. S., 1970. The Rhodophyta, some aspects of their biology. II. Ocrtrr~oyr. ,2lc1r. Bfr~l. ,l/~/r. Rer.. Vol. 8, pp. 307-352. DIXON, P. S.. 1971. Cell enlargement in relation to the development of thallus form in Florldeophyceae. Br. ph~~col. J., Vol. 6, pp. l95--205. DIXON, P. S., 1973. Biology qf’the Rhodophyrtr. Oliver and Boyd, Edinburgh, 285 pp. DIXON, P. S. & W. N. RICHARDSON, 1970. Growth and reproduction in red algae in relation to light and dark cycles. Ann. N.Y. Acod. Sri., Vol. 175, pp. 764-777. DUFFIELU, E. C. S., S. D. WAALAND & R. CLELAND, 1972. Morphogcnesis in the red alga. Gritirthsilr pcrcificu. Regeneration from single cells. Plntzfcr, Vol. 105, pp. lSSLl95. KONRAD-HAWKINS, E., 1964a. Developmental studies on regenerates of Ctr/[ithrrrtrnion YOS~II~~I Harvey. Part. 1. The development of a typical regenerate. Proroplasmtr, Vol. 58, pp. 42-59. KONRAD-HAWKINS, E., 1964b. Developmental studies on regenerates of Ctrllithtrr~~nir~z roscuvr Harvey. Part II. Analysis of apical growth. Regulation of growth and form. Profopkrsnro, Vol. 58. pp. 60-74.

176

STEVEN N. MURRAY

AND

PETER S. DIXON

MURRAY, S. N. & P. S. DIXON, 1973. The effect of light intensity and light period on the development of thallus form in the marine red alga Pleonosporium squnrrulosurn (Harvey) Abbott (Rhodophyta: Ceramiales). 1. Apical cell division main axes. J. exp. mar. Biol. Ecol., Vol. 13, pp. 15-27. STEEL, R. C. D. & J. H. TORRIE, 1960. Principles and procedures qfstatistics. McGraw-Hill Book Co. Inc., New York, 481 pp. WAALAND, S. D. & R. CLELAND, 1972. Development in the red alga Grififh’rhsicr ptrcificcr. Control by internal and external factors. Planta, Vol. 105, pp. 196-204. WAALAND, S. D., J. R. WAALAND & R. CLELAND, 1972. A new pattern of plant cell elongation: bipolar band growth. J. Cell Biol., Vol. 172, pp. 184-190.