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Differencesinosmoacclimation
betweensporophytesand
gametophytesofthebrownalga
Ectocarpussiliculosus
ArticleinPhysiologiaPlantarum·April2006
ImpactFactor:3.14·DOI:10.1111/j.1399-3054.1991.tb02154.x
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Availablefrom:DavidN.Thomas
Retrievedon:17May2016
PHYSIOLOGIA PLANTARUM 83: 281-289. C#pei.liagen 1991
Differences in osmoacclimation between sporoptiytes and
gametophytes of the brown alga Ectocarpm siUculosus
David N, Thomas and Gunter O. Kirst
Thomas, D.N. and Kirst, G. O. 1991. Differences in osmoacclimation between
sporophytes and gametophytes of the brown alga Ectocarpus silicutosus. — Physiol.
Plant. 83: 281-289.
The osmoacclimation of Ectocarpus siliculosus isolates known to have different salt
tolerances was investigated. Included were isolates originating from 5 different locations in the northern hemisphere, and sporophyte and gametophyte phases of different ploidy from two of the locations were compared. The effect of salinity treatment
(8-64%,.) on inorganic ions (K+, Na+,, Mg^+, C r , SO|*, phosphate) and the low
molecular weight carbohydrate mannitol was measured, together with complimentary measurements of cell viability. Verj' different responses between isolates were
obtained, both between isolates of different geographic origin and between sporophytes and gametophytes from the same parent material. A similarity in response
between haploid and diploid gametophytes, and diploid and triploid sporophytes
indicates that physiological differences between garoetophyte and sporophyte generations are not necessarily based on ploidy changes alone. There were no identifiable
differences in the responses of male and female gametophytes. K* is the major
osmolyte within the species, and differences in the regulation of K* largely account
for the observed variation in osmoacclimation, both between life history phases and
between isolates from different localities. Isolates with broader salt tolerances had
the higher concentrations of mannitol. There were differences between isolates in the
amounts and regulation of CI~ and phosphate, the latter being present in unusually
high concentrations. There were also isolate differences in the concentrations of Mg^*
and SO5", although these divalent ions were present only in low concentrations.
Key words - Ectocarpus silicutosus. life-history phases, osmoacclimation,, population
variation, ploidy.
D. N. Thomas (corresponding auihor), Alfred Wegener institute for Polar and Marine
Research, Am Handelshafen 12, D-2850 Bremerhaven, Germany; G. O. Kirst, Dept
of Marine Botany (FB2), Univ. of Bremen, D-2800 Bremen 33, Germany.
T ^ , .
Introduction
Russeil and Bolton (1975) first reported the occurrence
of salinity ecotypes within the species Ectocarpus
siUculosus (Dillw.) Lyngb., following growth experiments over a range of salinities using populations from
habitats of different natural salinity. This work was
extended by Thomas and Kirst (1991) using measurements of ceil viability, pliotosyntbesis and dark respiration together with a much larger collection of isolates
from a wider geographic origin in the northern hemisphere. The availability of population clones of this
species of known lineaee and ploidv, that have all been
- . j
u
v ^ r
.
maintained at the same salinity for several years provides a rare opportunity to determine to wbat extent
differences in salt tolerance (and the physiological basis
of such variation) result from genotypic differentiation,
Several groupings were found within the isolates
studied, supporting the view that genotsfpic differences
in respect to salt tolerance do occur within the species.
Very different responses between gametophyte and sporophyte life history phases of certain populations were
also found. The gametophyte and sporophyte generations of E. ;si'/!cuto.sMi are morphologically very similar.
Received 29 April, 1991; revised 19 June, 1991
19* Physiol. Plane. 83, 1991
281
there being only a slight tendency towards heteromorphison. There have been few similar investigations into
the differences between reproductive phases of isomorphic algal species. In some species there are clear differences in physiological or ecological performance of
isomorphic life-history phases, whereas in others there
is no conspicuous differences between tbe haploid and
diploid plants (Clayton 1988, Hannach and Santelices
1985, Littler et al. 1987). Tbere was some evidence that
the variation in the salt tolerances between E. siliculosus gametophytes and sporophytes may not result from
the obvious differences in ploidy level alone (Thomas
and Kirst 1991).
It was the intention of this subsequent study to investigate further differences in salt tolerance between
isolates of different geographic origin and life history
phases of E. siliculosus, with emphasis on the physiological basis of this variation. A reduced set of isolates
from that used by Thomas and Kirst (1991) was selected, including plants with very different salt tolerances, together with gametophytes and sporophytes obtained from parent material originally from two different geographic locations. For one population both
haploid and diploid gatnetophytes, and diploid and trjploid sporophytes derived from tbe same parent material
were available. There was therefore a unique opportunity to measure the effect of ploidy level on salt tolerance.
The physiological responses of marine macro-algae to
changes in external osmotic conditions have been well
studied, and were reviewed io detail by Kirst (1990).
When subjected to increases or decreases in salinity
many aigae are able to regulate intracellular osmotic
presstire, by adjusting either cellular ion concentrations
(mainly K+, Na* and Cl") and/or concentrations of organic solutes. A number of investigations have shown
there to be intraspecific differences in such osmotic
acclimation processes during salt stress between algal
populations from different salinity habitats: Cladophora
rupestris (L.) Kiitz. and C. glomerata (L.) Kutz. (Thomas et al. 1990), Enteromorpha intestinalis (L.) Link.
(Edwards et al. 1988, Young et al. 1987b), Pilayella
littoralis (K.) Kjellm. (Reed and Barron 1983) and Palysiphonia lanosa (L.) Tandy (Reed 1983, 1984). Evidently such variation plays a major role in enabling a
particular species to inhabit a variety of saline habitats,
although clearly population differences in plant morphology atid cell ultra-strticture are also of importance
(Reed 1983, Reed and Barron 1983, Young et al. 1984,
1987b).
The low molecular weight carbohydrate mannitol is
an important osmolyte (compatible solute) in matiy
brown algae including E. siliculosus (Reed et al. 1985,
Wright et al. 1989). Marine Filayella littoralis plants had
very much higher concentrations of mannitol than estuarine material and it contributed a significantly higher
proportion to changes in intracellular osmotic potential
in the marine plants during salinity stress than in estua282
rine material (Reed and Barron 1983). The effect of
hypo- and hypersaline stress on mannitol and several
major intracellular ions within the selected isolates is
reported here.
Materials and methods
Plant material and experimental conditions
All isolates (Tab. 1) came from the culture collection of
D.G. Muller, Univ. of Konstanz, Germany. These
plants and the culture conditions have been described
by Thomas and Kirst (1991). For ease of reference
experimental codes assigned to the isolates are the same
as those used in the previous study. Sporophytes (Ic, If
and 2c) were origioally obtained from the corresponding gametophytes listed from each site. A diploid gametophyte (Ig) not used in the previous study was also
included. This originated from a meiosis of a heterozygous tetraploid sporophyte constructed originally
from the two gametophyles la and lb, and was propagated by filament fragmentation (Muller 1970). Although not axenic,, all cultures were in a healthy condition and growing well. All experimental and culture
media were heat sterilised before use, and there was
little evidence of bacterial contatnination.
Experimental media were based on the artificial seawater media ASP12S (Reed et al. 1980), and the range
of salinity treatments used was 8, 16, 32 and 64%o. Five
tnM NaHCO3 was added to sterilised media to provide
an adequate inorganic carbon supply for photosynthesis, and all media were buffered to pH 7.5 using 5 mM
HEPES/NaOH. Experiments were performed at the
Tab. 1. Experimental isolates used in the present study. The
original sites from which parent material was collected by
D.G. Muller are given. Collection sites and plants are more
fully described in Thomas and Kirst (1991), and the experimental codes are consistant between the two studies. *,
Whether this diploid gametophyte is bomozygous or heterozygous in regard to sex factor is not determined (D. G. Muller,
personal communication).
Isolate
Original
collection site
Status
la
lb
Ig
Ic
If
Naples, Italy
Haploid gametophyte (cS)
Haploid gametophyte (?)
Diploid gametophyte (2n*)
Diploid sporophyte (d" 2)
Triploid sporophyte (§ § cf)
Haploid gametophyte (tf)
2a
Port Aransas,
TV
1 TC A
2b
2c
6b
10a
14
Newfoundland,
Canada
Tampa, FL, USA
Kallvik, Sweden
Haploid gametophyte (5)
Diploid sporophyte (cf 9)
Diploid sporophyte (cf ? )
Partheno-sporophyte (cf)
Unknown
PhysioL Plant. 83, 1991
temperature and light conditions used to maintain the
cultures: 19 ± O'.5°C, and a photon flux density of 40
imo\ m-^ S-' (12h light/12h dark).
An experimental incubation period of one week was
chosen since there is evidence that in some isolates
some osmotic adjustment still takes place between 72 h
and one week (Thomas and Kirst 1991). It is felt that
measurements made after one week treatment, therefore, better reflect a dynamic equilibrium state within
the plants.
Effect of salinity on cei viability
The effect of salinity on cell viability of most of these
isolates has already been assessed after 72 h treatment
(Thomas and Kirst 1991). It was necessary to repeat
these measurements after one week treatment, thereby
giving viability data more comparable to other measurements made during this study. Cell viability following
salinity treatment was measured using the mortal stain
Evans Blue, and the experimental design and methods
used have been described by Thomas and Kirst (1991).
water extracts were 91%, and Na*, 93% of those obtained with the acid. It is felt that this level of extraction
was quite adequate for a comparative study such as this.
Mannitol contents were measured by an HPLC
equipped with a refractive index detector (Bio-Rad,
Miinchen, Germany). Separations were performed using a Polysphere® CH-CA column (E. Merck, Darmstadt, Germany), as described by Karsten et al. (1991).
These workers have also shown that the extraction of
tnannitol from E. siliculosus using hot water is highly
efficient.
The cations Na^, K* and Mg,^"^ were measured by
atomic absorbtion spectrophotometry (Perkin-Elmer
2380), following appropriate dilution. C r and SO4"
concentrations were measured using an ion chromatograph (Metrohm 690) equipped with a conductivity
detector. Separations were made on a PRP-XIOO column (Hamilton, Bonaduz, Switzerland), as described
by Karsten and Kirst (1989). Phosphate was measured
using a colorimetric technique (Gerlach and Deuticke
1963).
Results and discussion
Effect of salinit}' on tissue water content
Plant material was incubated in 200 ml of experimental
media. Following treatment adhering surface water was
removed from the samples by vacuum filtration (Hall
1981, Reed et al. 1985), and the fresh weights (FW) of 3
replicate samples measured. After drying at 85°C for
48 h, the dry weights (DW) of the samples were determined. Water content was calculated as 100 x
(FW-DW)/FW.
EUect of salinity on mannitol and inorganic ions
Following careful blotting with paper tissue, plant material was incubated in 30 ml of treatment media. Five
replicate samples at each salinity were used, and media
were changed once during, the experimental period.
Samples were weighed after removal of the adhering
surface water (see above), and then washed two times in
40 ml of ice-cold iso-osmotic Ca(NO3)j for 2.5 min, in
order to remove ions from the extracellular space
(Ritchie and Larkum 1982a,b). Samples were then extracted in hot water (85°C) for 2 h, and the supernatant
stored at either -80°C or - 2 0 ^ until required for
mannitol or inorganic ion analyses.
The efficiency of extracting with hot water was tested
with 4 isolates (la, 2c, 10a and 14) which had been
maintained in 32%o experimental media for one week
and then washed in iso-osmotic CafNO,)^. Four replicate samples of each isolate were digested either as
above, or in HNO3 at 80°C for 1 h. Higher amounts of
K* and Na* were extracted by digesting with HNO3,
although on average mean K* concentrations in hot
Physiol. Plant. 83, t991
The effect of sahnity on the ceil viability, tissue water
content and major osmolyte concentrations in different
experimental isolates is shown in Figs 1 to 3. The viability data support the previous observations (Thomas and
Kirst 1991) that E. siliculosus gametophytes have narrower ranges of salt tolerance compared to sporophytes
derived from the same parent plants (Figs 1 and 2). The
inclusion of the diploid gametophyte Ig, enabled a further testing of the hypothesis that differences in salt
tolerance between gametophyte and sporophytes are
not necessarily based on ploidy, but on some other
difference between life history phases (Thomas and
Kirst 1991). If the enhanced salt tolerance of sporophytes is a result of diploidisation only, some increase in
tolerance of the diploid gametophyte would be expected. However, this diploid gametophyte had a remarkably similar response to salinity treatment to that
of the haploid gametophytes la and Ib (Fig. 1), and
changes in ploidy alone, therefore, do not result in
enhancement of salt tolerance. The fact that the triploid
sporophyte If showed such similar responses to that of
the diploid sporophyte Ic further supports this view
(Fig. 1).
The responses of both male and female gametophytes
were very similar (Figs 1 and 2). There is little information about physiological and biochemical differences between the sexes in algae. However, in several
investigations concerned with algal temperature tolerance, significant differences in response of male and
female gametophytes have been recorded (Bolton and
Liining 1982, Lee and Brinkhuis 1988, Wiencke and
tom Dieck 1989).
There was a fairly wide range of salt tolerance exhibited by the sporophytes of isolates from different geo283
1a)G
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Hg. 1. Itie ettect ot one week salinity treatment on the ceil
viability, K" (--•-), Na+ (-O-), Cl" (-•-), phosphate (-Q-),
mannitol (-O—) and percentage water content of gametophytes (G) and sporophytes (S) obtained from parent plants
originally collected from Naples (la-lg). The genetic constitution of plants is indicated. *, Whether this diploid gametophyte
is homozygous or heterozygous in regard to sex factor is not
determined (D. G. Muller, personal communication). All osmolyte concentrations are expressed in mmol (kg FW)"'. The
SE of each sample is indicated. (For viability and osmolyte data
n = 5 unless indicated otherwise; for water content n = 3 unless
indicated otherwise).
graphic locations (Figs 1-3): 2c and 10a had broad tolerances, whereas 6b had a very narrow tolerance. The
Baltic isolate (14) was tolerant of hyposaline treattnents
but intolerant of hypersaline treatment, whereas the
two sporoptiytes Ic and If were only intolerant of extreme hyposaline treatment (8%IJ). Viability data for
some isolates obtained in this study differ from those
reported by Thomas and Kirst (1991) following only a
72h incubation period. A higher proportion of cells
were viable at 64% in la, lb. If, 2a and 2b after the
longer incubation time. These were significant changes
in salt tolerance and values were raised from well below
to above the 50% level. After 72 h treatment in 16%o
experimental media ca 50% of celis of lb were alive
(Thomas and Kirst 1991), whereas after one week the
isolate was dead at this salinity. In 6b there was a
further reduction in viability in 8%o during the increased
284
incubation period. A slight increase in ceil mortality
within 10a incubated in 64%o took place, although viability was still significantly higher than the 50% level. In
2c, however, there was no change in viability between
the two incubation periods. This together with the photosynthetic data (Thotnas and Kirst 1991) confirms that
between 72 h and one week osmoacclimation is still
taking place.
Of the osmolytes measured, K* was present in the
highest concentrations in all isolates, and evidently
osmoacclimation is achieved largely by the regulation of
this ion. Na* concentrations were maintained at low
levels varying little with changes in salinity. Such active
extrusion of Na* and active inclusion of K* is common
to several macroalgae (Kirst 1990).
In estuarine and marine populations of Polysiphonia
lanosa loss of viability in hyposaline conditions was
closely correlated to differences in rates of K* loss between the two populations (Reed 1984). Isolates of E.
siliculosus that were tolerant of hyposaline treatment
certainly maintained higher concentrations of K* than
those that were intolerant. This is particularly noticeable in population 2 at 8% (Fig. 2). The tolerant sporophyte 2c had much higher concentrations of K* than
the two gametophytes which had reduced viabilities.
Also 10a and 14 (Fig. 3) lost relatively low amounts of
K* in both the hyposaline treatments in which the cells
showed no loss in viability.
Reduction in K* after hypersaline treatment in some
of the isolates is similar to that recorded at high salinities in other studies (Kirst and Bisson 1979, Reed and
Barron 1983, Young et al. 1987a,b). It is often observed
that such reductions in K* are accompanied by a large
increase in Na*, although, in these E. sUiculosus isolates, Na* increases did not always occur together with
decreases in K*, and where concentrations did increase
they rose only slightly (isolates la, Ig, 6b). In these 3
isolates the decreases in K* and increases in Na* were
accompanied by a corresponding large decrease in cell
viability (Figs 1 and 3). However, this was not a strict
response in all cases, and loss in viability in hypersaline
treatment was not always linked to loss of K*: cells in
isolates Ic and If were quite viable even after a large
reduction of K* (Fig. 1). It is evident that the relative
loss in K* is the important factor in determining cell
viability. It is an anomaly that isolate 14 did not lose K*
at 64%o but there was a large decrease in cell viability at
this salinity (Fig. 3).
Lowering of the K*/Na* ratio is considered to be an
indication that the selective permeability of the plasmamembrane has been disturbed (Kirst and Bisson 1979).
Table 2 shows the K*/Na* ratios for the isolates, and in
general where there was a lowering in the ratio there
was a corresponding loss of viability. The exceptions to
this trend are 2a at 64%o, 2b at 8 and 64% and Ic at 16
and 64%o. There is a large variation in K*/Na* ratios
between the populations and evidently there is a substantial difference in the selective permeability of the
PhysioL Plant. S3. W91
2a)
2b) G.O
6b)
2c)
14) US
100
50
A
400I
200
oo-Q
<?
oO-O
O
nO-Q
9
zoo-
5^200o
E
.o-
..o-•O'
-O'
oo-
X:
80
8
32
64
8
32" 64
8
32
64
Salinityy %o
Fig. 2. The effect of one week salinity treatment on the cell
viability, K+ ( - • - ) , Na+ (-O-), Cl" (-•-), phosphate (-D-).
mannitol (-O--) and percentage water content of gametophytes (G) and a sporophyte (S) obtained from parent plants
originally collected from Port Aransas (2a-2c). The genetic
constitution of plants is indicated. All osmolyte concentrations
are expressed in mmol (kg FW)-'. The SE of each sample is
indicated. (For viability and osmolyte data n = 5' unless indicated otherwise; for water content n = 3 unless indicated
otherwise).
membranes between isolates. Thomas et al. (1990) also
reported significant differences in the K*/Na"^ ratios of
field populations of Cladophora spp. from different salinity habitats. Sporophytes and gatnetophytes from the
same population had similar K*/Na* ratios at nortnal
salinity (32%o) even though their salt tolerances were
quite different (compare la-lg and 2a-.-2c). Generally
the plants with higher K'^/Na"^ ratio had wider salt tolerances.
Its, many species which regulate ttirgor pressure the
Physiol, Plant. 83, 1»1
8 32
64
8 32
64
8 32
64
Salinity %o
Fig. 3. The effect of one week salinity treatment on the cell
viability, K+ (-•--), Na+ (-O-), C r (-•-), phosphate (-D-),
mannitol ( - 0 - ) and percentage water content of cultured
isolates originally from Newfoundland (6b), Tampa (10a) and
Kallvik (14). The genetic constitution of plants is indicated
(S-sporophyte, PS-partheno sporophyte, US-status unknown).
AU osmolyte concentrations are expressed in mmol (kg FW)~'.
The SE of each sample is indicated. (For viability and osmolyte
data n = 5 unless indicated otherwise; for water content n = 3
unless indicated otherwise).
main changes are in K* concentration, and Ci as the
major counter anion is regulated in a similar fashion. In
these E. siliculosus isolates C r concentrations, however, were much lower than those of K^ after incubation in all salinity treatments. They were also lower than
concentrations measured in otber macroalgal studies,
and they resemble tnore the concentrations found in
cytoplasm rich microalgae (Dickson and Kirst 1986).
They may be maintained at such low levels because of
toxic effects in the cytoplasm (Ritchie 1988). No partic285
Tab. 2. K+/Na* ratios of isolates after one wfeek incubation at
experimental salinities. The SE of the sample is given in parenthesis, n = 5, except; ' , where n = 4.
Isolate
la
Ib
l£
Ic
If
2a
2b
2c
6b
lOa
14
Salinity treatment (%o)
8
16
32
64
Dead
Dead
Dead
1.0 (O'.l)
3.5 (1.0)
7.5 (3.2)
12.8 (2.6)
18.3 (2.3)
0.5 (0.3)
14.5 (2.3)
16.9 (2.0)
0.3 (0.2)
Dead
Dead
3.6 (0.2)
5.2 (0.7)
11.4(1.9)
11.7(2.4)
13.7 (1.9)
7.4 (1.4)
13.9 (1.6)
15.1 (3.0)
6.7 (1.4)
5.6 (1.4)
6.5 (1.8)
7.7(1.4)
6.6 (0.4)
14.1 (1.2)*
14.1 (2.0)
14.2 (l.o)
9.6 (2.4)
14.6 (2.9)
13.6(1.8)
2.3 (0.7)
2.8 (O.9)
2.5 (O.9)
3.6 (0.6)
5.9(1.2)
19.7 (1.8)
14.2(1.1)
13.8 (3.2)
0.5 (0.3)
11.4 (1.9)
9.5 (3.5)
creasing salinity in the three isolates 2a, 2b and 2c, the
increases in sporopbyte 2c were very much larger than
in the two gametophytes (2a and 2b), especially following treatment with 64%o media (Fig. 2).
Table 3 shows the concentrations of Mg-** and SO;" of
all experimental isolates and the effect of salinity on
these two ions. The concentrations of these two ions
were very low in most isolates, although isolates 10a, 2c,
and very noticeably 14 bad a higher concentration of
SOJ". The sigtiificance of the differences in SO4" beTab. 3. Concentrations of Mg^+ and SO5" in experimenlal
isolates following one week incubation in salinity treatment
media. All values are expressed as mmol (kg FW)"'. The SE of
the sample is given in parenthesis. *, Samples dead. n = 5.
Isolate
Salinity
(%„)
la
ular trend in the regulation of Cl that was common to
all isolates was discernable: In isolates la—lg and 2a it
varied in a similar way to that of K* (Figs 1 and 2),
whereas there was little or no variation in Cl" concentration in 2b, 2c, 6b, 10a and 14 (Figs 2 and 3). Similar
concentrations of C r were measured in the sporophytes
and gametophytes of both populations 1 and 2 at 32%o.
An exception was the sporophyte Ic which had very
slightly higher concentrations of Cl" than corresponding
gametophytes (Figs 1 and 2), but in ali cases concentrations were never sufficient to balance cations.
Relatively high concentrations of phosphate were
measured in all isolates, and generally these were of a
similar magnitude to Cl" at 32%». Usually phosphate
contents of algae are reported to be very low aod/or not
to vary with changes in external salinity (Karsten and
Kirst 1989, Reed et al. 1981, Wiencke and Lauchli 1981,
Young et al. 1987a). Phosphate has not always been
measured in salt tolerance studies and maybe its importance as an osmolyte has been overlooked. However,
high internal phosphate oniy occurs in the presence of
high concentrations in the medium, which is usual in
enriched culture media as used in this study. Therefore
these high levels may reflect a luxury uptake of phosphate by these isolates, and it is interesting to note that
they occur here in conjunction with low concentrations
of Cl".
As in the case of C r , there was no definite pattern for
all isolates regarding the variation in phosphate with
salinity change. However, in isolates Ib, lg, 2a-2c, 10a
there were proportional increases of phosphate with
rising salinity, across the whole salinity range investigated (Figs 1-3). This was particularly marked in the
very salt tolerant isolate 2c (Fig. 2). In sporophytes Ic
and If there were increases in phosphate between 8 and
32%o only (Fig. 1). Tbere were much higher concentrations of phosphate in the two sporophytes (Ic and If)
compared with the corresponditig gametophytes (Fig.
1). Although coticentrations of phosphate rose with in286
Ib
lg
16
32
64
8
16
32
64
8
16
32
64
14.8 (6.4)
8.3 (1.2)
13.6 (2.0)
*
0.0 ( 0.0)
16.4 ( 6.7)
8.9 ( 5.3)
14.2 (2.7)
23.5 (6.5)
*
8.5 (2.2)
16.1 (l.2)
5.3 ( 1.7)
4.2 ( 6.0)
*
*
5.5 ( 0.4)
10.3 (l9.3)
*
Ic
8
16
32
64
17.3
16.5
17.2
58.7
(2.6)
(4.0)
(3.2)
(7.2)
4.6
8.4
10.0
4.1
(
(
(
(
1.2)
2.5)
0.3)
1.8)
If
8
16
32
64
18.9 (3.1)
17.4(1.0)
21.7 (2.4)
24.1 (2.3)
8.2
8.5
9.5
8.2
(
(
{
(
2.1)
2.2)
3.0)
5.0)
2a
8
16
32
64
6.9 (2.0)
9.9(1.1)
11.8 (2.0)
13.9 (2.7)
2.4 ( 2.3)
3.5 ( 1.3)
4.4 ( 1.0)
1.8(3.1)
2b
8
16
,32
64
6.9
9.4
11.8
19.7
(0.9)
(l.o)
(2.0)
(4.O)
13.0
9.7
10.4
17.5
(
(
(
(
3.9)
2.4)
1.8)
5.2)
2c
8
16
32
64
5.3
7.5
10.4
23.6
(0.3)
(1.5)
(0.6)
(3.8)
29.5
19.6
8.9
20.3
(
(
(
(
5.3)
8.5)
4.0)
2.2).
6b
8
16
32
64
17.0 (6.1)
12.7 (2.8)
12.6 (3.8)
28.8(7.4)
Trace
9.7 (
10.4 (
7.4 (
only
2.2)
1.9)
4.5)
10a
8
16
32
64
15.3
17.2
17,7
27.3
(0.9)
(0.7)
(2.3)
(5.4)
20.9
9.7
10.4
7.4
(
(
(
(
6.4)
2.2)
1.9)
4.5)
14
8
16
32
64
8.2(1.5)
15.9 (3.7)
22.6 (3.6)
25.1 (6.7).
52.2
58.9
67.3
63.1
(16.5)
( 3.2)
( 3.9)
(21.5)
Physiol. Plant. S3. 1991
tween isolates is unclear, but clearly there is a differetitial uptake of this anion. In several isolates there was a
significant increase of Mg^* following incubation in 64%D
treatment media. Since the influx of Mg^* into algal
cells is thought to be passive (Raven 1976), these rises
may indicate that slight changes in membrane permeability to this ion might have occurred. The low concentrations of Mg-* and SO4" are similar to concentrations
found in other studies, and it is evident that these ions
have little role in osmoacclimation within this species
(Kirst 1990).
There was a deficit in anionic charge compared to
that of the cations within all isolates. Such an imbalance
is often reported in salt tolerance studies, and is accounted for by fixed negative charges of cellular proteins or organic acids (Kirst 1990, Raven 1976). However, anion deficits are not universal, and Karsten and
Kirst (1989) report a cation deficit in Blidingia minima.
The concentrations of mannitol were in the same
range as those given by Reed et al. (1985) for E. siliculosus at normai salinity. However, it should be noted
that cultured Pilayella littoralis plants had significantly
less mannitol than field isolates (Reed et al. 1985), and
it is possible that the concentrations recorded in the
present study are lower than would have been recorded
in original field material. The highest concentrations (ca
70 mmol kg"' at 32%o) were found in those isolates that
had wider salt tolerances, 2c (Fig. 2) having the most. In
all isolates except 6b (Fig. 3), there was an increase in
mannitol with increases in salinity, and clearly it is regulated as an osmolyte. The differences in mannitol content between the isolates, however, were not as pronounced as those between marine and estuarine populations of Pilayella littoralis (Reed and Barron 1983).
Mannitol concentrations were of a similar magnitude in
both gametophytes and sporophytes of population 1,
although slightly lower concentrations were found in the
gametophyte la (Fig. 1). Mannitol concentrations in the
two gametophytes 2a and 2b were similar, although
obviously lower than those of the sporophyte 2c in all
salinity treatments (Fig. 2).
Mannitol is a major photoassimilatory product in
brown algae. However, the high concentrations of mannitol in the hypersaline treatment occur when net photosynthetic rates are very iow (Thomas and Kirst 1991).
A probable source of mannitol would be the degradation of the reserve carbohydrate laminaran (Davison
and Reed 1985). This provides further evidence that
changes in mannitol concentrations in response to salinity stress cannot arise solely due to an inhibition or
stimulation of photosynthetic activity (Reed and Barron
1983, Reed et al. 1985).
Variation in tissue water with salinity is indicative of
changes in cell volume, and the trend of a linear decrease in tissue water content with increasing salinity is
ootnmonly reported (Karsten and Kirst 1989, Reed et
al. 1981, Young et al. 1987a,b). Betweeti 8 and 32%,,
there were only slight changes in the water content of
Physiol. PJanl. 83. l
most isolates (ca 5%), whereas in the 64%o treatment
media there was a substantial water ioss (10 to 20%)
from that at 32%o (Figs 1-3). In those isolates in which
there were reductions in viability in the hyposaline
treatments a linear response of water content with salinity (8 to 32%o) was not maintained. The water contents
at 32%D varied slightiy betwen the isolates, 2c, 10a, Ic
and If having lower concentrations than the others.
These differences, and differences in response to saiinity, may reflect slight differences in cell volume, and
differing resistance to volume change between isolates.
Estuarine Pilayella littoralis had thinner cell walis than
its marine counterpart, which permitted the greater
changes in cell volume indicated by changes in water
content (Reed and Barron 1983). In future studies it
would be necessary to look for such differences between
these Ectocarpus isolates.
Conclusions
1) Genotypic differences in response to changes in external salinity between E. siliculosus isolates from different geographic locations were confirmed. Such genetic diversity can explain why this species is found in a
wide range of salinity habitats. In some isolates chatjges
in cell viability between 72 h and one week incubation
periods were evident, especially in extreme salinity
treatments. Ciearly caution is needed when interpreting
reponses meastired during short-term incubations in experiments designed to establish macroalgal salt tolerance.
2) Differences in the responses to sait stress between
gametophytes and sporophytes originating from the
same parent plants were also confirmed. The sporophytes are evidently osmotically more robust than
gametophytes, having a broader sait tolerance than
their corresponding gametophytes. However, the degree of differentiation between life history phases varied between the two sets of clones where sporophyte
and gametophyte generations were available.
3) Differences between gametophyte and sporophyte
generations are independent of pioidy level, since hapioid and diploid gametophytes had very similar responses to salt stress, as did diploid and triploid sporophytes.
4) K*, in common with many other marine algae, was
the major osmolyte in all isolates, and osmoacclimation
is probably largely controlled by the regulation of this
ion. Mannitol and to iesser extent CV and phosphate
concentrations were also regulated with changes in external salinity. The phosphate concentrations were unusually high, and C r lower than often found in marine
macroalgae.
5) Differences in salt tolerance between both isolates
of different geographic origin and sporophyte and
gametophyte generations were largely governed by differential abilities to regulate K*. However, those isolates with the broader osmoacclimation also had the
287
higher concentrations of mannitol. There was evidence
for isolate differences in phosphate and C r regulation.
6) There were differences between isolates in the
concentrations of the divalent ions Mg-"^ and SO}'.
These, however, were only present in low concentrations and probably have little function in osmoacclimation processes.
7) Differences in tissue water content indicate that
there may be slight differences in the cell volumes of
isolates and in the resistance to cell voiume changes.
This study has served to identify some physiological
differences in the osmoacciimation between isolates
from different geographic origin and life-history phases
of £. siliculosus. The results clearly point to a need for
more detailed investigations into the regulation of intracellular osmolytes between the contrasting isolates if
the basis of the variation in salt tolerance within this
species is to be understood.
Acknowledgements - Once again we wish to thank D.G.
Muller for his advice and for providing the experimental isolates. We are grateful to U. Winter for her help with the cation
analysis, and R. Ulmke for carefui technical assistance
throughout. We also thank G. Russell, U. Karsten and C.
Wiencke for their support during the investigation and help
during the preparation of the manuscript. The study was supported by a Research Fellowship awarded to D.N.T. by the
Royal Society (UK) through the European Exchange Programme.
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Edited by S. Pettersson
Physio!. Plant. 83. J99I
289