Rintoul, Page 2
Desiccation in Porphyra perforata
ABSTRACT
A study of whole thallus absorbance was combined
with dircect measurements of photosynthesis in air to
assess the phtosynthetic capacity of desiccated Porphyra
perforata. Fresh P. perforata was found to give a flat
absorbance spectrum of high absorbance. Drying of the
sample produces pigment peaks in the spectrum and a reduc¬
tion in the photosynthetic rate. On rehydration, P. per-
forata resumes full photosynthetic capacity, and again
exhibits a flat spectrum at high absorbance. The suggestion
is made that these spectral results reflect an adaptation
to P..perforata's intertidal habitat, for the subtidal
Porphyra nereocystis gave peaks for both desiccated and
fresh samples. In addition, photosynthetic rates measured
during the day were higher than those measured at night,
introducing the possibility that P. perforata possesses
an endogenous photosynthetic rhythm. Cytological exxamina¬
tion of P. perforata collected in situ over a 24 h period
showed greatest mitotic activity when the plants were
exposed during daylight hours.
Rintoul, Page 3
Desiccation in Porphyra perforata
INTRODUCTION
High intertidal algae experience extremes of desicca¬
tion on a daily basis in relation to the tidal cycle. The
conspicuous zonation in this environment has traditionally
been attributed to the relative abilities of different
species to withstand desiccation (Muenscher, 1915; Zaneveld,
1937; Feldmann, 1951; Doty, 1946; Biebl, 1962). Little
attention has been focused upon the continuation of cellu-
lar processes such as photosynthesis and cell division
while intertidal algae are exposed to the air. Stocker and
Holdheide (1938) investigated photosynthesis in exposed
marine algae but did not make comparisonswith submerged
plants. More recently, Johnson et al. (1974) reported
higher rates of photosynthesis in air than in water for
selected intertidal algae. Other recent papers on marine
algal physiology mention aerial photosynthesis only inci-
dentally (Brown and Johnson, 1964; Chapman, 1965; Imada
et al., 1970; Mathieson and Burns, 1971).
There are also few reports on the relationship
between exposure and cell division. The literature relating
to mitotic periodicity has recently been reviewed (Austin
and Pringle, 1968; Pringle and Austin, 1970). Farmer and
Williams (1898) found bursts of cell division in Fucus
immediately following submergence, and Rao (1956) reports
a higher incidence of mitosis in Polyides caprinus (Gunner)
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Desiccation in Porphyra perforata
Papenf. at high tide. Austin and Pringle (1968) have found
a diurnal periodicity in mitosis for Rhodomela larix
(Turner) C. Agardh, with little indication of a tidal
influence. A periodicity in mitosis has been demonstrated
for Porphyra lanceolata (Setch. and Hus) Smith, with peaks
in nuclear division coinciding with periods of exposure in
the dark (Pringle and Austin, 1970). No cytological work
with regard to mitotic periodicity has been published on
red algae exposed during the daylight hours.
Porphyra perforata J. F. Agardh, a monostromatic red
alga, was chosen as a suitable species with which to study
the effects of desiccation on cellular processes, because
its high intertidal habitat, the +2.0 to +3.5 ft. above
mean lower low water tidal level in the Monterey Bay region
(Smith, 1944), results in exposure to air for 20-60% of
each 24-hr period (Johnson et al., 1974).
The possibility that pigment absorbance may change
with desiccation of algae was prompted by preliminary
observations that Porphyra perforata appears redder when
desiccated. As the red phycobilins have the highest
quantum efficiency for photosynthesis among the pigments
in red algae (Yocum, 1951), and the photosynthetic action
for P. perforata closely parallels the phycobilin absor-
bance spectrum (Haxo and Blinks, 1950), these observations
suggested increased phycobilin absorbance in P. perforata
upon desiccation. Haxo and Blinks (1950) used thalli sub-
merged in seawater to measure whole thallus absorbance for
Desiccation in Porphyra perforata
Rintoul, Page
Porphyra perforata. As yet, whole thallus absorbance in
air has not been measured for littoral algae. My studies
of desiccation effects on Porphyra perforata were attempted
in order to assess the photosynthetic capacities of these
exposed algae.
MATERIALS AND METHODS
Spectral Analysis:
Whole thallus absorbance was measured using a Beckman
DK-2A scanning spectophotometer. Samples were prepared
by cutting sections of an intact thallus using a cork
borer, placing them upon a heavy glass coverslip and taping
them into the sample chamber of the spectophotometer with
black electrician's tape, to minimize leakage of the sample
beam around the sample. Part of the same blade from which
the sample was obtained was bleached in a 1:2 Sodium hypo-
chlorite seawater mixture to remove the pigments and to
obtain a blank with similar light scattering properties..
In all further manipulations the sample and the reference
received identical treatment.
Porphyra perforata was collected from the +2.5 tidal
level at Mussel Point, Pacific Grove, California on the
same day experiments were run. Spectra were taken for fresh
algae, for algae which had been desiccated in a 23° oven
with desiccant for four hours, and for algae which had
been rehydrated in seawater for 5 minutes after a similar
period of desiccation. The experiment was repeated using
Desiccation in Porphyra perforata
Rintoul, page 6
Porphyra nereocystis Anderson, collected as drift from
Asilomar Beach, in Pacific Grove.
To determine whether or not the Porphyra perforata
had been irreversibly damaged from the desiccation method,
photosynthetic capacities (P) of the fresh, dried and
rehydrated material were measured. The same sample could
not be measured, dried and then remeasured because a dry¬
ing period of 4 h is necessary to abtain a good spectrum,
and the possibility exists that P. perforata has an endogen-
ous photosynthetic rhythm similar to that found in Iridaea
flaccida (Harris, 1978). With a lapse of four hr between
photosynthetic measurements, differences due to an endogen¬
ous rhythm could not be distinguished from those due to
desiccation. Freshly collected material was divided into
three equal portions. The fresh sample was kept submerged
in a temperature control room at 15° C. The two others were
treated in the same way as the desiccated and rehydrated
samples for the spectral measurements. Both were oven dried
with desiccant for 4 h and one rehydrated at the end of
the drying period. Spectra were taken for all samples
immediately before measuring the photosynthetic rate. Sam¬
ples for taking spectra were not removed directly from
those used for the photosynthetic measurement, but from
separate samples treated identically. The photosynthetic
rate was determined using a Beckman Model 215 Infrared Gas
Analyzer, with a flow-through sample chamber, at saturating
Desiccation in Porphyra perforata
Rintoul, Page 7
light intensity of 290 uE/m/s as provided by a tungsten¬
iodide lamp for illumination. These measurements were made
at 15° C. Runs were made at 1200 h on May 29 and at 1600 h
on June 3. No photosynthetic determinaions could be obtain¬
ed for P. nereocystis because of insufficient material.
Mitosis:
The sites chosen were two boulders 1.2 m apart and
approximately 30 m west of the Monterey Boatyard launching
rails. Each site supported dense growth of Porphyra
perfor¬
ata. Sample site 1 was a vertical rock face with eastern
exposure, while sample site 2 was on the top of a rounded
boulder. Both sites were situated at the +2.2 ft tidal
level. These sites were chosen for their equal tidal height
and relative ease of accessibility at high tide.
Porphyra perforata from the two sites was sampled
at 2 h intervals over a 24 h period from May 8 to May 9,
1978. Rectangles of 1 x 3 cm were removed from the intact
thalli using iris scissors and were immediately placed in
1 dram vials containing a freshly mixed 1:3 glacial
acetic acid-absolute ethanol mixture. The light intensity
at the sample sites were measured with a Lambda Instrument
Quantum Meter Model 185. Field notes recorded either sub¬
mergence or emergence of the sample sites. Sample prepar¬
ation and storage followed the methods of Pringle and Austin
(1968), which included washing of the fixed material in
absolute ethanol and storage in 70% ethanol. Nuclei were
Desiccation in Porphyra perforata
Rintoul, Page 8
stained using an iron-alum-acetocarmine s sh technique
(Austin, 1959). Five fields of 20-25 cells per field were
examined under oil immersion, with cells scored as dividing
if two nuclei were visible within the cell, or if two cells
were pressed together with nuclei on opposite sides, indi-
cating recent division. Earlier stages of mitosis could not
be counted because the chromosomes in P. perforata are too
small to visualize with a light microscope.
RESULT
Spectral Analysis:
The spectra obtained for fresh, desiccated, and rehy-
drated Porphyra perforata are shown in Figure 1. Note that
ata gives a flat absorbance spectrum with
fresh P. perf
88% absorbance, and that the desiccated sample gives peaks
for hlorophyll a, allophycocyanin, phycoerythrin and a
combined chlorophyll a carotenoid peak at 430 nm, although
total absorbance was lower than for a fresh sample. Rehy-
drated samples give the same flat spectrum and high absor-
bance obtained for fresh algae. Peaks were also obtained
for both fresh and desiccated samples of P. hereocystis
(Fig 2), demonstrating that the flat spectrum obtained for
wet P. perforata is not merely an artifact of the sample
preparation.
Comparison of the photosynthetic capacities of fresh,
dried and reydrated P. perforata (Fig. 3) shows that the
Rintoul, Page 9
Desiccation in Porphyra perforata
photosynthetic capacity of this species decreases markedly
on desiccation, and returns to the original rate and even
somewhat enhanced photosynthetic level upon rehydration.
Lower photosynthetic rates were measured at 0200 h than
were found at 1600 h, suggesting that P. perforata may
possess an endogenous photosynthetic rhythm.
Mitosis:
The highest percentages of dividing cells were found
after the sample sites had been exposed to air for 6 h
and to 4 h of daylight. Values of 77% for site 1 and 98/
for site 2 were found in P.
arorata sampled at 1000 h
(Fig. 4). At that time the sample plants were desiccated
to crispness. Although data are available for only a single
site (site 1) and one sample time (1400 h) for submerged
daylight conditions, only 4% of the counted cells were
observed dividing at this time. With afternoon exposure
plants at site 1 show an increase in mitotic activity, then
falling off in the early evening, and increases again
toward morning. Sample site 2 sustained relatively high
mitotic activity (47 to 63) throughout the late afternoon
and night, decreasing by 0200 h.
Data bars are missing in Figure 4 at those points for
which good squashes showing distinct nuclei could not be
obtained, primarily due to the unpredictable staining
properties of iron-alum-acetocarmine. Unless nuclei were
Desiccation in Porphyra perforata
Rintoul, Page 10
clearly visible the accuracy of the count was question¬
able. In incomplete studies, another technique using
acito-iron-haematoxylin-chloral hydrate stain (Wittman.
1963) was found to be more reliable.
DISCUSSION
The flat spectrum at high absorbance obtained for fresh
perforata was a startling result. The pigments
Porphyr:
must be absorbing light to conduct photosynthesis at the
levels measured for fresh P. perforata in air. The photo-
synthetic rate decreases upon desiccation, when the pig-
ments give more distinct peaks in the spectophotometer. What
accounts for this phenomenon? Perhaps plastids in the cells
released their pigments causing the appearance of more
distinct absorbance peaks in the spectra. A reversible
pigment release is unlikely. Efficient energy transfer in
photosynthesis requires perfect alignment of the pigments
on membranes, and it would seem disadvantageous for the
alga to jeopardize such organization every time the plant
dried out. An irreversible pigment release, while not ex¬
pected to occur in situ, may have resulted from the oven
drying of the sample. The high photosynthetic rates measured
for the rehydrated thalli indicate that this did not
happen. Reversible release remains a possibility
Desiccation in Porphyra perforata
Rintoul, Page 11
So the algae are absorbing light when hydrated, but
appear black to the spectophotometer. The spectrum for
the fresh sample is not flat because the peaks are not
absorbing, but because some effect raises the valleys to
the level of the peaks. Rerunning the spectra with a uni-
formly gray reference, such as a lens tissue, might help
with peak resolution by pushing the spectrum down into the
lower absorbance levels. The optical results indicate
that something happens in hydrated Porphyra perforata as
expressed in the absorbance spectra, which does not occur
in sublittoral P. nereocystis, a species with similar
physical characteristics, thus suggesting an adaptation
of P. perforata to a high intertidal habitat.
The photosynthetic measurements of fresh, dry and re¬
hydrated Porphyra perforata showed conclusively that the
rehydrated sample regained full photosynthetic capacity.
The greater photosynthetic rate attained by the algae after
drying and rehydration was unexpected and may be worthy of
further investigation. In addition, the difference between
rates measured at night and in the day, the latter showing
an increased rate, suggests the possibility of an endogenous
photosynthetic rhythm.
A possible change in plastid orientation within the
desiccated P. perforata cells can be postulated. An endogen-
ous rhythm in chloroplast orientation has been documented
in Ulva (Chlorophyta) (Britz and Briggs, 1976), with the
chloroplast orienting for maximal light absorption during
perforata
Desiccation in Porphyra
Rintoul, Page 12
periods of peak photosynthetic efficiency. Perhaps the
plastids orient to maximize light absorption while the
alga is exposed, thus accounting for the enhanced photo-
synthetic rates in air observed by Johnson et al. (1974).
A recording microphotometer similar to that used to measure
light-induced chromatophore movement in brown algae (Pfau
et al, 1974) might prove useful in documenting desiccation
effects upon plastid orientation.
The peaks for mitosis found in the desiccated P. per-
forata exposed in the daytime were surprising at first,
because this is thought to be a time of maximum stress for
the plant. Pringle and Austin (1970) felt that the diurnal
rhythmicity they found for P. lanceolata was controlled
by fluctuations Win light intensity, independent of the tidal
cycle. Closer analysis of their data reveals that peak
mitotic activity ocurred during periods of exposure, as well
as at night. The investigators did not examine any collections
made from exposed plants during daylight hours.
One would expect mitosis to commence after a period
of high photosynthesis, for cellular products must accumu-
late before division can proceed. Johnson et al. (1974)
report that P. perforata reaches peak photosynthetic
capacity after a period of drying, but that photosynthesis
decreases upon further desiccation. Ability to conduct
mitosis while extremely desiccated would be advantageous
to an alga which must partition alternating periods of
Desiccation in Porphyra perforata
Rintoul, Page 13
carbon fixation and cell division within the diurnal and
tidal cycles. The optimal time for mitosis would be those
periods that the plant is unable to conduct photosynthesis,
i.e., during the night and when the plant is desiccated.
Porphyra perforata would be an interesting algal species
with which to document and describe an endogenous photo¬
synthetic rhythm, with particular attention to a possible
tidal component. Studies of mitotic rates of plants kept
in constant laboratory conditions might reveal an endogenous
mitotic rhythm as well, further elucidating the relation¬
ship between photosynthesis, cell division and desiccation
in littoral algae.
Desiccation in Porphyra perforata
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SUMMARY
1. Fresh Porphyra perforata gives a flat spectrum of high
absorbance when whole thallus absorbance is measured.
Peaks appear in the spectrum when the sample is desic-
cated, and total absorbance is lowered. This behavior
is thought to reflect P. perforata's intertidal habitat,
for the subtidal Porphyra nereocystis gives peaks in
whole thallus absorbance for both fresh and desiccated
samples.
2.
Photosynthesis decreases when P. perforata is desiccated,
to resume to original levels when the sample is
rehydrated. Values recorded at night are lower than
those measured during the day, suggesting a rhythm in
photosynthesis.
3. Cytological examination of a 24 h collection showed the
greatest percentages of cells dividing during daylight
hours when the plants were exposed.
Desiccation in Porphyra per
forat
Rintoul, Page 15
ACKNOWLEDGEMENTS
Thanks are extended to the whole Hopkins faculty
associated with our class for their encouragement and
contagious fascination with the organisms. Special
thanks are due to Drs. Larry Harding and Isabella
Abbott for their special attention and care, and to
Dr. Blinks for helping me to ask the right questions.
Desiccation in Porphyra perforata
Rintoul, Page 16
LITERATURE CITED
Austin, A. P., 1959. Iron-alum aceto-carmine staining for
chromosomes and other anatomical features of Rhodophy-
ceae. Stain Technol., 34:69-75.
Austin, A.P. and J. D. Pringle, 1968. Mitotic index in sel-
ected red algae in situ. I. Preliminary report. J. Mar.
Biol. Ass. UK, 48:609-635.
Biebl, R., 1962. Seaweeds, pp. 799-815. In R. A. Lewin (ed.)
Physiology and biochemistry of algae. Academic Press.
New York.
Brown, J. M. A. and A. Johnson, 1964. Preliminary studies.
on the ecology and physiology of Scytothenmus australis
(J. Agardh Hook. et Harv. 1845. Bot. Mar., 6:233-246.
Chapman, V. J., 1965. The physiological ecology of some New
England seaweeds. Proc. Fifth Int. Seaweed Symp..
pp. 29-54.
Doty, M. S., 1946. Critical tide factors that are correlated
with the vertical distribution of marine algae and
other organisms along the Pacific coast. Ecol., 27: 315-
328.
Desiccation in Porp
a perforata
Rintoul, Page 17
Farmer, J. B. and J. L. L. Williams, 1898. Contributions to
our knowledge of the Fucaceae: their life history and
cytology. Phil. Trans. Roy. Soc. B, 190:623-645.
Feldmann J., 1951. Ecology of marine algae, pp. 313-334.
In G. M. Smith (ed.) Manual of phycology. Chronica
Botanica, Waltham, Mass.
Harris, J., 1978. Endogenous circadian rhthmicity of photo-
synthesis in the marine alga Iridaea flaccida (Rhodophyta)
of the central California coast. Unpublished MS, Spring
class paper, Biology 175 H, Hopkins Marine Station,
Stanford University.
Haxo, F. and L. R. Blinks, 1950. Photosynthetic action spectra
of marine algae. J. gen. physiol., 33:389-422.
Imada, 0., Y. Saito, and S. Maeki, 1970. Relationships between
the growth of Porphyra tenera and its culturing condi¬
tion in the sea. II. Influence of atmospheric exposure
on photosynthesis, growth and others on Porphyra fronds.
Bull. Jap. Soc. Fish., 36:369-376.
Johnson, W. S., Andreas Gigon, S. L. Gulmon, and H. A. Mooney,
1974. Comparative photgsynthetic capacities of inter¬
tidal algae under exposed and submerged conditions.
Ecol., 55:450-453.
Desiccation in Porphyra perforata
Rintoul, Page 18
Mathieson, A. E., and R. L. Burns, 1971. Ecological studies
of economic red algae. I. Photosynthesis and respira¬
tion of Chondrus er
ispus Stackhouse and Gigartina
stellata (Stackhouse) Batters. J. exp. mar. biol..
ecol., 7:197-206.
Muenscher, W. L. C., 1915. Ability of seaweeds to withstand
desiccation. Puget Sound Mar. Stat. Publ, 1:19-23.
Rao, C. S. P., 1956. The life history and reproduction of
Polyides coprinus (Gunn.) Papenf., Ann. bot., N. S.,
20:211-230.
Smith, G. M., 1944. Marine algae of the Monterey Penninsula,
Stanford University Press, Stanford, 622 pp.
Pfau, J., G. Throm and W. Nultch, 1974.Recording micropho¬
tometer for determinaion of light-induced chromatophore
movements in brown algae. Z. Pflanzenphysiol., 71:242-260.
Pringle, J. D. and A. P. Austin, 1970. Mitotic index in selected
red algae in situ. II. A supralittoral species, Por
rhyra
lanceolata (Setchell and Hus) G. M. Smith. J. exp. mar.
biol. ecol., 5:113-137..
Desiccation in Porphyra perforata
Rintoul, Page 19
Stocker, 0., and W. Holdheide, 1938. Die Assimilation helgo-
lande Gezeitenalgen wahrend der Ebbezit. Z. bot., 32:1-59.
Wittman, W., 1965. Aceto-iron-haematoxylin- chloral hydrate
for chromosome staining. Stain technol., 40:161-164.
Yocum, C. S., 1951. Some experiments on photosynthesis in
marine algae (Diss. Abstr.)in Abstracts of disserta¬
tions, Stanford University, 1951.
Zaneveld, J. S., 1937. The littoral zonation of some Fucaceae
in relation to desiccation. J. ecol., 25:431-468.3
forata
Desiccation in Porphyra pe
Rintoul, Page 20
FIGURE LENGENDS
Figure 1: Whole thallus absorbance spectra for fresh, dried
and rehydrated Porphyra pe
orata, with the
curves labelled appropriately. All readings were
taken with the samples exposed to air.
Figure 2: Whole thallus absorbance spectra for fresh, dried
and rehydrated Porphyra nereocystis. All readings
were taken with the samples exposed to air.
Figure 3: Photosynthetic rates for fresh,dry and rehydrated
P. per
rata as measured in an Infrared Gas
Analyzer. Each rate is labelled withtan expression
for the water content of the sample measured, calcu-
lated as %fresh weight. Two runs were made, at
1600 h on 6/3 and at 0200 h on 5/9, which are
shown as gray and dotted bars, respectively.
Figure 4: The percentage of cells dividing in Porphyra per¬
forata observed for collections taken at 2 h
intervals over a 24 h period from May 8 to May
1978, as represented by the vertical gray bars..
At each sample time the left bar represents
Zmitosis for site 1, and the right bar for site
2. The dotted line labelled"
represents the
light intensity in uE/m/s as measured for each
daylight collection time. The top horizontal bar
is solid
those times
S.
?

10
upqosqy
0
8
9

unqosqu
5





S
L
24.0
3.0
22
O
2
210
a
1.0
FRESH
6/3:1600
1.10
exptl. wt.
„fresh wt.
111
1.0
0.56
0.69
REHVORATEI
DRY
5/9:0200
-
-
.
Fig. 3
O
L
O

Z
0

2
LIGHT INTENSITY TPE/m2/sec)
—
—



.


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ONIdIAId S1139 %
88
88
LL
—
ap; ap;
.