Marine Ecology. ISSN 0173-9565
ORIGINAL ARTICLE
Plant–water relations of intertidal and subtidal seagrasses
~o Silva3, Irene Olive
3, Monya M. Costa3, Juan M. Ruiz1,
Jose M. Sandoval-Gil1,2, Isabel Barrote3, Joa
1
2
3
zaro Marı́n-Guirao , Jose L. Sa
nchez-Lizaso & Rui Santos
La
1 Seagrass Ecology Group, Spanish Institute of Oceanography, Murcia, Spain
2 Department of Marine Sciences and Applied Biology, University of Alicante, Alicante, Spain
3 ALGAE-Marine Plant Ecology Research Group, Centre of Marine Sciences, University of Algarve, Faro, Portugal
Keywords
Desiccation stress; emersion; ion
homeostasis; osmolyte; osmoregulation;
seagrass.
Correspondence
Jose M. Sandoval-Gil, Seagrass Ecology
Group, Spanish Institute of Oceanography,
C/ Varadero s/n, 30740 San Pedro del
Pinatar, Murcia, Spain.
E-mail: [email protected]
Accepted: 1 September 2014
doi: 10.1111/maec.12230
Abstract
This work represents the first contribution to (i) examine the changes in plantwater relations of an inter-tidal seagrass during air exposure (Zostera noltii),
and (ii) compare the water status descriptors between inter-tidal- and subtidaladapted species (Cymodocea nodosa, Zostera marina). Two different morphotypes of Z. noltii that develop in the highest and lowest inter-tidal levels in the
Portuguese lagoon of Ria Formosa were exposed to natural emersion periods
under laboratory conditions, and the evolution of leaf water relations and osmolytes (ions, proline and non-structural carbohydrates) was measured. Both
morphotypes regulated their water potential (Ψw) by reducing the osmotic
potential (Ψp) through osmolyte accumulation, but only high inter-tidal plants
were able to do this by adjusting the turgor pressure through cell wall hardening. This is a conservative mechanism for osmotic acclimation, which occurred
only after long emersion periods (7 h). After a rapid increase in ion concentration under air exposure, the high inter-tidal morphotype replaced them by
more physiologically compatible solutes (proline and non-structural carbohydrates) to maintain the osmotic adjustment. Altered ionic homeostasis was
found in low inter-tidal plants when exposed to such unnatural, long emersion
periods. Osmotic unbalances were also observed during the submerged recovery phase. Descriptors of leaf pressure–volume (P–V) curves and H€
ofler diagrams were derived for seagrasses for the first time. They support the
divergences in water relations observed between inter-tidal and subtidal seagrasses according to their vertical distribution. More negative water and osmotic
potentials and higher rigidity of cell walls (higher elastic modulus, e) were
found to be specific osmotic adaptations of seagrasses to the inter-tidal.
Introduction
Seagrasses show particular plant–water relation properties
in order to be able to acclimate to the marine environment (Tyerman 1989; Kuo & den Hartog 2000; Touchette
2007). To maintain positive water balances and osmotic
homeostasis, seagrasses must have lower leaf osmotic/
water potentials than seawater, achieved mainly through
increased tissue ion concentration and the absence of a
real cuticle (Birch 1975; Tyerman 1989; Kuo & den
Hartog 2000; Sandoval-Gil et al. 2012a). Seagrass populaMarine Ecology (2014) 1–17 ª 2014 Blackwell Verlag GmbH
tions growing in sites of varying osmotic gradients or
along geographic salinity gradients have to develop specific
water relation strategies (Tyerman 1989; Vermaat et al.
2000; Ye & Zhao 2003). One of the cases in which seagrasses must adjust their water status properties to withstand
varying osmotic environments is tidal exposure.
The physiological resistance, plant traits (e.g. downsizing, structural rigidity and leaf turnover) and population
growth strategies (e.g. overlapping and the clamp
formation of thalli) of marine macrophytes have been proposed as potential factors determining the vertical zonation
1
Water relations of intertidal seagrasses
Sandoval-Gil, Barrote, Silva, Oliv
e, Costa, Ruiz, Marı́n-Guirao, S
anchez-Lizaso & Santos
and the structure of seaweed communities within the
inter-tidal (Norton 1991; Bj€
ork et al. 1999; Silva & Santos
2003; Tanaka & Nakaoka 2004; Shafer et al. 2007; Coombes et al. 2013). At the physiological level, it is generally
considered that air exposure (commonly expressed as desiccation stress) causes comparable effects to seawater salinity increments, as both are able to alter water fluxes and
tissue water potential (Ψw; Kirst 1989; Davison & Pearson
1996; Touchette 2007). However, while these effects are
well documented in seaweeds (see review in Bisson & Kirst
1995), seagrass-related works are very scarce.
Considerable attention has been paid to changes in water
relations in response to water deficit stressors in land
(mainly crops) and wetland plants (e.g. drought, salinity;
Verslues et al. 2006; Touchette et al. 2007; Ashraf & Athar
2009). These are mostly based on the stress avoidance/tolerance model (Levitt 1980), which consists of a range of
mechanisms that allow the plant to prevent or to resist
water loss from tissues (Verslues et al. 2006; Touchette
et al. 2007). Based on this model, seaweeds and seagrasses
(as homeohydric plants) are considered to use ‘dehydration
avoidance0 strategies to overcome water stress when
exposed to air (Bisson & Kirst 1995; Lobban & Harrison
1997; Karsten 2012). Contrary to ‘dehydration-tolerant
plants0 that are able to endure tissue dehydration (Oliver
et al. 2000; Sanchez-Dıaz & Aguirreolea 2000), seagrasses
activate physiological responses to reduce cell-tissue Ψw,
thus avoiding the loss of water (Kramer & Boyer 1995;
Verslues et al. 2006; Touchette et al. 2007). These strategies
include osmoregulation and cell-wall hardening processes
(Hsiao 1973; Flowers et al. 1977; Verslues et al. 2006).
Osmoregulation is based on the accumulation of intra-cellular osmotically active solutes (osmolytes) such as ions,
free amino acids or non-structural carbohydrates, which
results in a reduction of the osmotic potential (Ψp; Kramer
& Boyer 1995). By contrast, the regulation of cell-wall
hardening (mainly deformability or rigidity, described by
the elastic modulus, e) has been recognized as a metabolically low cost alternative to adjust Ψw (Kramer & Boyer
1995; Verslues et al. 2006). There is some evidence of the
existence of both osmoacclimative mechanisms in seagrass
species in the context of adaptive mechanisms to salinity
regimes and hyper-saline stress (Pulich 1986; Murphy et al.
2003; Koch et al. 2007; Sandoval-Gil et al. 2012a, 2013;
Marın-Guirao et al. 2013), but their operation during
desiccation stress remains unstudied.
In the Portuguese Natural Park of Ria Formosa, most
of the inter-tidal is dominated by beds of the seagrass
Zostera noltii Hornem, whereas Cymodocea nodosa (Ucria) Ascherson and Zostera marina L. co-occur in the
subtidal. Two Z. noltii morphotypes/ecotypes, which
show distinct morphometric and metabolic traits (Mercado et al. 2003; Alexandre et al. 2004; Cabacßo et al. 2009),
2
develop in different levels of the inter-tidal, and thus
experience different air-exposure periods. Different architectural-vegetative features, as well as photosyntheticrelated divergences between the morphotypes, seem to
reflect distinct acclimation to emersion (Perez-Llorens &
Niell 1993; Peralta et al. 2000, 2005). Cymodocea nodosa
and Z. marina are also considered eurybiontic, highly
plastic species able to grow within a wide range of marine
environments including hyper-saline confined waters and
inter-tidal flats (Moore & Short 2007; Fernandez-Torquemada & Sanchez-Lizaso 2011; Sandoval-Gil et al. 2012b,
2013). Despite their high plasticity, these seagrass ecotypes and species maintain a characteristic, stable vertical
distribution in Ria Formosa lagoon. Here, we tested
whether this differential distribution is determined, at
least in part, by particular adaptive/acclimation mechanisms of plant-water relations.
This work represents the first contribution to the
assessment of the water relation strategies of seagrasses in
the inter-tidal/subtidal continuum, combining an experimental approach under controlled laboratory conditions
and comparative measures of plants collected in the field.
The responses of the high and low inter-tidal morphotypes of Z. noltii to contrasting emersion regimes were
compared in the laboratory. Water relations descriptors
were assessed at different points along a simulated tidal
cycle (i.e. before air exposure, after emersion and at the
end of a recovery phase) by measuring leaf osmolality
and osmolyte concentration (ions, non-structural carbohydrates and proline). Then, we compared the in situ
water status properties of the two inter-tidal Z. noltii
morphotypes and of the subtidal species C. nodosa and
Z. marina. Adding to the above-mentioned descriptors,
leaf pressure–volume curves (P–V curves) and H€
ofler diagrams (Sun 2002; Kirkham 2005) were obtained. These
are useful tools to assess plant responses under water
stress, which have provided valuable insights into the
eco-physiological strategies followed by land plants, wetland herbs and poikilohydric species subjected to water
deficits (Youngman & Heckathorn 1992; Beckett 1997;
Touchette et al. 2007). To the best of our knowledge this
is the first time that these techniques have been applied
to seagrasses.
Material and Methods
Plant collection and study area
This study was performed with two Zostera noltii morphotypes growing in the inter-tidal of Ria Formosa (a
shallow mesotidal coastal lagoon located in southern
Portugal), and with Zostera marina and Cymodocea
nodosa populations, which occupy the subtidal zone.
Marine Ecology (2014) 1–17 ª 2014 Blackwell Verlag GmbH
Sandoval-Gil, Barrote, Silva, Olive, Costa, Ruiz, Marı́n-Guirao, S
anchez-Lizaso & Santos
Plants were collected by hand during spring tides from
sites widely separated from each other, in order to
obtain experimental plant pools with representative local
genotypic variability. The Z. noltii morphotypes from the
high and low inter-tidal (Zn-HI and Zn-LI, respectively)
were collected after ~1 h of emersion in the extreme
upper and lower vertical distribution of the species, corresponding to emersion periods of 6–8 h for Zn-HI and
1–3 h for Zn-LI, depending on the tidal amplitude.
Zostera noltii plants were collected near the Ramalhete
channel (37°00 10.300 N, 7°580 40.4600 W; Praia de Faro),
while C. nodosa (Cn) and Z. marina (Zm) plants were sampled at Culatra Island (36°590 47.3700 N, 7°490 40.5100 W).
Care was taken to keep plants intact during collection
(i.e. maintaining the shoots–rhizome–root and within
ramets connectivities) because of the importance of clonal
integrity in the eco-physiological responses of seagrasses
(Marba et al. 2002). Plants were rapidly transported in
large coolers to the laboratory (within 1 h). In the case
of the comparative analyses between subtidal and intertidal species, Z. noltii plants were collected during submersion. Measurements of plant–water status and the
separation of plant material for the analysis of osmolytes
were carried out immediately. For the experimental tide
simulations, 40–50 shoots of each Z. noltii morphotype
were placed fully submersed in separate aquaria until the
day after collection, when tide phase simulations were
performed (see below).
Experimental tide simulation
Plants of both Zostera noltii morphotypes (Zn-HI and
Zn-LI) were separately exposed to simulated tidal cycles
under laboratory conditions. For each morphotype, plants
were distributed in 10 independent aquaria of 15 l that
were filled with seawater from the sampling site, provided
with independent air-pumps and situated in a plant culture/walk-in climatic chamber (FITOCLIMA 10000
THIM) within the facilities of the Centro de Ci^encias do
Mar (CCMAR, University of Algarve). Temperature,
salinity (absolute salinity, SA gkg1) and humidity were
controlled and adjusted following mean field values
(15 0.1 °C, 36.6 0.4 gkg1 and 65–70%, respectively). Irradiance was adjusted to ~300 lmols
quantam2s1 on a 12:12 h photoperiod. This irradiance value is lower than the much higher irradiances to
which plants are exposed during emersion in the field,
but such high irradiance can induce confounding additional stress (Silva et al. 2005), which we wanted to
avoid.
To simulate emersion during low tide, Z. noltii plants
were removed from the aquaria the day after sampling
and transferred to pots filled with sediment from the
Marine Ecology (2014) 1–17 ª 2014 Blackwell Verlag GmbH
Water relations of intertidal seagrasses
sampling site soaked with seawater. The plants were then
disposed simulating their natural habit during emersion,
i.e. maintaining the seawater surface film in the interface
plant canopy-sediment. This simulation is a key aspect
that has to be considered to obtain accurate water relation responses to emersion. In fact, Z. noltii do not completely dehydrate in the field as the thin, highly dense
leaves of the plants overlap and trap seawater (Silva &
Santos 2003; Silva et al. 2005). In addition, this community strategy to avoid plant dehydration has been considered as one the main causes of the increasing
photosynthesis of emerged seagrasses, compared with the
photosynthetic reductions closely related to tissue dehydration under controlled experimental conditions (Leuschner & Rees 1993; Perez-Llorens & Niell 1993;
Leuschner et al. 1998; Seddon & Cheshire 2001; Shafer
et al. 2007). It was not possible to measure the tissue
osmolality in situ using a thermocouple psychrometer
(i.e. osmometer; see Plant–water relations below) due to
technical limiting factors (e.g. accurate measurements
need strictly constant temperature and humidity conditions; Boyer 1995; Kirkham 2005).
Following the emersion period, plants were transferred
again to aquaria and re-submerged in order to complete
the tidal cycle. The different phases corresponding to
the full experimental tidal cycle were then carried out in
1-day experiments: (i) the S phase (corresponding to
the initial submerged plants), (ii) E phase (i.e. emerged
plants) and (iii) SR phase (i.e. re-submersed plants for
10 h). Recovery from emersion was tested after this 10h period, the mean of the time between low tides in the
field (see Silva & Santos 2003). To study the effect of
the duration of air exposure on plant–water status
descriptors, plants were exposed to three distinct emersion
periods (30 min, and 3 and 7 h). Forced by logistical constraints (e.g. longer times required to the osmometer stabilization between following measurements; Sandoval-Gil
et al. 2012a), three different experimental tidal cycles, in
which plants were exposed to the three distinct times of
emersion, were performed on three consecutive days for
each Z. noltii morphotype. The measurements of water
relation descriptors and the collection of plant material for
the analysis of osmolytes were carried out in the S phase,
and at the end of the E and SR phases. The change of these
descriptors was compared with an experimental control
cycle for each morphotype that consisted of the maintenance of permanently submerged plants (PS) in 10 similar
aquaria under the same experimental conditions. In this
case, the measurements of water relations and collection of
plant material were performed at an initial, intermediate
and final phase (PS1, PS2 and PS3, respectively) at the
same time as the measurements in the S, E and SR experimental phases.
3
Water relations of intertidal seagrasses
Sandoval-Gil, Barrote, Silva, Oliv
e, Costa, Ruiz, Marı́n-Guirao, S
anchez-Lizaso & Santos
Plant–water relations
Descriptors of leaf–water relations (water potential, Ψw;
osmotic potential, Ψp; turgor pressure, Ψp) were determined by thermocouple psychrometry (Wescor Vapour
Pressure Osmometer 5520, Logan, Utah, USA) in one
shoot from each aquarium (n = 10) and per morphotype
during each of the different experimental tidal phases and
of the submerged control. Ten shoots of each morphotype were also collected in the natural populations of the
inter-tidal Zostera noltii and the subtidal Cymodocea nodosa and Zostera marina. Measurements of leaf-tissue
osmolality [mmolkg1 fresh weight (FW)] were performed in fresh leaf tissues and in freeze (in liquid N2)thawed leaf tissues following the methods adapted by
Sandoval-Gil et al. (2012a) from Tyerman et al. (1984)
and Boyer (1995). Osmolality measurements were
expressed in megapascals (MPa), using the van’t Hoff
equation (Tyerman et al. 1984; Nobel 2009).
Leaf relative water content (RWC, h; Slatyer 1967) was
determined by the equation:
RWCðhÞ ¼ ðWf Wd Þ=ðWt Wd Þ
(1)
where Wf is the fresh weight and Wd is the oven-dried
weight (60 °C). For the experimental tide simulations,
RWC was measured in two different shoots in each aquaria and averaged, so that the total number of replicates
per Z. noltii morphotype was n = 10. The turgid weight
(Wt) was obtained by placing small Z. noltii leaf pieces
(~0.6 cm2) in closed 2.5-ml vials containing tissue paper
soaked with de-ionized water. These were maintained in
darkness for ~4 h at 4 °C in order to inhibit physiological activity and potential dry weight alterations (Gonzalez
& Gonzalez-Vilar 2001).
Pressure–volume curves (Tyree & Hammel 1972) were
only used for the comparative analysis among the natural
populations. P–V curves were constructed for five single
leaf pieces for each morphotype and species using thermocouple psychrometry, and following the methods
described for bryophytes by Beckett (1997) and Proctor
et al. (1998) under stable humidity and temperature. For
each curve, a fully hydrated leaf piece (i.e. at Wt, obtained
as described above) of ~2 mg (for Z. noltii) or ~5 mg
(for C. nodosa and Z. marina) was placed in the osmometer sample holder coated with VaselineTM (see method
specifications in Boyer 1995). Between measurements of
Ψw, the sample holder was opened and the leaf tissue was
exposed to the room atmosphere for a few seconds (5–
10) to cause leaf-tissue dehydration. The change in the
weight due to dehydration was measured using a highresolution (0.001 mg) microbalance. At every weight
loss of ~0.02 mg (in the case of Z. noltii) or ~0.05 mg
4
(for C. nodosa and Z. marina), successive measurements
of Ψw (until 19–25 measurements) were carried out at
constant time intervals on each sample to construct the
curve. There was a time delay of 10 min between the successive measurements in order to equilibrate the osmometer sample chamber, which was calibrated with standard
solutions before each entire run (Sandoval-Gil et al.
2012a). After these measurements, the leaf samples were
oven-dried at 60 °C for 24 h to obtain Wd. P–V isotherms were constructed by plotting the reciprocal of
water potential (i.e. 1/Ψw) against RWC (Fig. 1A). As
the first reading of Ψw typically never equals zero at full
turgor, the RWC = 1 theoretically corresponding to
Ψw = 0 was determined by linear extrapolation from the
first three or four Ψw readings, following Turner (1988).
From the P–V isotherm, Ψp was calculated as the difference between the extrapolated linear portion of the curve
and the actual curve (Fig. 1A). When Ψp was plotted as a
function of RWC, it appeared not to change during the
initial RWC reductions in either Z. noltii or Z. marina
leaves (see Figs 2B and 1B). As explained in previous
works (Beckett 1997; Sun 2002; Hajek & Beckett 2008),
this may be related to the initial loss of inter-cellular
water, which must be excluded from the RWC corresponded to symplastic (i.e. intracellular) water (Fig. 1B).
The corrections proposed in these previous studies were
then applied [RWC = (Wf Wd)/(Wt Wd weight
of inter-cellular water)] and RWC was recalculated for all
the data, assuming that RWC equals 1 when Ψp starts to
decline. Water status parameters [i.e. osmotic potential at
full turgor (w100
p ), osmotic potential at turgor loss point
(w0p ), symplastic water content (hsym), RWC at turgor
loss point (htlp)] and Hofl€er diagrams were calculated
from corrected P–V curves (Kirkham 2005; Tyree 2007).
was determined by the y-intercept either by
w100
p
extrapolating the linear portion of the P–V isotherms or
it was taken as the osmotic potential when Ψp reaches its
maximum values (RWC = 1) in the Hoflër diagrams;
w0p was the osmotic potential when Ψp = 0 in the
Hofl€er diagrams; htlp is the RWC at Ψp = 0; and hsym
was determined by the x-intercept by extrapolating the
linear portion of the P–V isotherm (Figs 2C–F and 1A).
The bulk elastic modulus (e) was also obtained from the
slope of the initial part of the curve as e = (dΨp/
dh) 9 hsym (Nobel 2009).
Leaf osmolyte content
Five leaf-tissue pools of each Zostera noltii morphotype
were collected from five randomly selected aquaria from
the tidal experiment to measure leaf osmolyte content
(n = 5). The same number of samples was also collected
from inter-tidal morphotypes and subtidal natural popuMarine Ecology (2014) 1–17 ª 2014 Blackwell Verlag GmbH
Sandoval-Gil, Barrote, Silva, Olive, Costa, Ruiz, Marı́n-Guirao, S
anchez-Lizaso & Santos
A
Water relations of intertidal seagrasses
B
Fig. 1. Schematic representation of a pressure–volume curve (A) and a derived graph of turgor pressure (Ψp) as a function of relative water
content (RWC) (B, uncorrected for extra-cellular water), corresponding to the Zostera noltii low inter-tidal morphotype (Zn-LI). The water status
0
descriptors presented are: htlp, RWC at turgor loss point; hsym, symplastic water fraction; w100
p , osmotic potential at full saturation; wp ,
osmotic potential at the turgor loss point. Plotted values are means and the error bars are SEs. Ψw, water potential.
lations (Zn-HI, Zn-LI, Zostera marina and Cymodocea
nodosa).
The plant material was cleaned (to remove surface epiphytes and salts) and stored at 80 °C until further
analysis. The non-structural carbohydrates [mgg1 dry
weight (DW)] were analysed following the methods
described in Dubois et al. (1956) and Burke et al. (1996).
Soluble carbohydrates were solubilized from lyophilized
(finely ground) tissues by sequential extractions with 80%
(v/v) ethanol at 80 °C (10 min) and centrifugation
(2000 rpm, 5 min). Starch was extracted from the ethanol-insoluble residue after washing in Milli-Q water and
centrifuged (12,000 rpm, 2 min). The washed pellet was
re-suspended in Milli-Q water and incubated at 100 °C
(5 min), after which it was digested by enzymatic (aamylase and amyloglucosidase) reactions. Sugar and
starch concentration in the extractants were measured
using the phenol-sulphuric colorimetric method and
standardized to sucrose.
Proline (lmolg1 FW) was analysed spectrophotometrically using the acidic ninhydrin reagent colorimetric
assay described in Sandoval-Gil et al. (2012a) that was
based on Bates (1973).
Ions (Cl, K+, Na+, Ca2+ and Mg2+) (mgg1 DW)
were determined by ionic chromatography according to
the methods detailed in Marın-Guirao et al. (2013).
Statistical analysis
One-way analysis of variance (ANOVA) was used to test
for significant differences (P < 0.05) of response variables
among the different phases of the simulated tidal cycle (i.e.
S, E, SR and PS1, PS2 and PS3 controls). Two-way ANOVA
was applied to test whether differences in the response variables between plants in the E and S phases depend upon
the duration of the air-exposure period. One-way ANOVA
Marine Ecology (2014) 1–17 ª 2014 Blackwell Verlag GmbH
was used to test for significant differences among the
response variables measured in inter-tidal morphotypes
and subtidal species (Zn-HI, Zn-LI, Zostera marina and
Cymodocea nodosa). When significant differences were
found, a post hoc mean comparisons test (Student–Newman–Keuls test; Zar 1984) was performed. A non-parametric Kruskal–Wallis test (Quinn & Keough 2002) was
applied if the assumptions of ANOVA were not met. The
statistical analyses were performed using the SIGMAPLOT
11 statistical package (Systat Software Inc, USA).
Results
Experimental tide simulation
The Zostera noltii morphotype from the high inter-tidal,
Zn-HI, showed significant decreases in both water and
osmotic potentials, Ψw and Ψp (30–55%) after both
30 min and 3 h of emersion (Fig. 3A and B). This
response was reduced to 18% after 7 h of air exposure as
a consequence of a notable significant reduction in turgor
pressure, Ψp (72%, Fig. 3C). Consequently, the response
of these descriptors was significantly dependent upon the
emersion time. In the case of the Z. noltii morphotype
from the low inter-tidal, Zn-LI, Ψw and Ψp decreased
progressively along the air-exposure period (from 16–
18% after 30 min to 31–42% after 7 h). The extent of air
exposure also affected Ψp significantly but only after 7 h,
when turgor pressure drastically increased up to 207%
(Fig. 3C). After re-immersion (SR phase), all variables
measured in the air-exposed plants of both morphotypes
recovered to the initial values measured in the S phase.
As expected, there were no significant differences between
plants in the S phase and PS1 control plants, indicating
that the experimental set-up did not affect the water status of plants. The water status of plants (Ψw and Ψp, but
5
Sandoval-Gil, Barrote, Silva, Oliv
e, Costa, Ruiz, Marı́n-Guirao, S
anchez-Lizaso & Santos
Water relations of intertidal seagrasses
A
B
C
D
E
F
not Ψp) varied significantly with continuous submergence
time (i.e. from PS1 to PS3), particularly those from the
high inter-tidal morphotype (Fig. 3D). Interestingly, the
observed variation of Ψw and Ψp under continuous submersion followed the rhythm of the submerged–
emerged–re-submerged plants shown in Fig. 3A–C,
although with a lower level of variation (5–11%). When
comparing the permanently submerged control plants
(Fig. 3D) with those exposed to simulated tidal cycles
(Fig. 3A–C), the only significant differences found in Ψw
and Ψp were between E and PS2, indicating a significant
effect of emersion on Z. noltii water relations. The only
significant difference found in Ψp between E and PS2 was
for Zn-LI after 7 h of desiccation, which was significantly
higher than in PS2 control plants (Fig. 3C and D).
6
Fig. 2. Pressure–volume (P–V) curves (A) and
derived graphs of turgor pressure (Ψp) as a
function of relative water content (RWC) (B,
uncorrected for extra-cellular water) and
€fler diagrams (C–F). Plotted values are
Ho
reported as the mean (n = 5) and the error
bars are SEs. Statistical analyses of water
status descriptors derived from P–V isotherms
are shown in Table 2. Zn-HI, Zostera noltii
high inter-tidal morphotype; Zn-LI, Z. noltii
low inter-tidal morphotype; Cn,
Cymodocea nodosa; Zm, Zostera marina; Ψw,
water potential; Ψp, osmotic potential; Ψp,
turgor pressure; htlp, RWC at turgor loss
point; w100
p , osmotic potential at full
saturation; w0p , osmotic potential at the
turgor loss point; e, elastic modulus.
Emersion caused slight but significant RWC reductions
(~1%) in plants from the low inter-tidal after both
30 min and 3 h (Zn-LI, Table 1), whereas after 7 h the
reduction was up to 3%, and RWC did not recover afterwards to the initial values measured during the SR phase.
By contrast, the RWCs of plants from the high inter-tidal
did not vary after either 30 min or 3 h of emersion but
after 7 h they increased (Zn-HI, Table 1).
The total ionic content of Zn-LI increased significantly
under air exposure (Fig. 4A–C), but in Zn-HI this increment was only detected after 30 min of emersion
(Fig. 4A). The total ionic content of the controls did not
vary (Fig. 4D), which indicates that the exposure to the
air triggered the regulation of ionic content. After reimmersion (SR), a strong ionic decrease was observed in
Marine Ecology (2014) 1–17 ª 2014 Blackwell Verlag GmbH
Sandoval-Gil, Barrote, Silva, Olive, Costa, Ruiz, Marı́n-Guirao, S
anchez-Lizaso & Santos
A
B
C
D
Marine Ecology (2014) 1–17 ª 2014 Blackwell Verlag GmbH
Water relations of intertidal seagrasses
low inter-tidal plants that were air exposed for 7 h,
whereas in the rest of cases, the total ionic content
returned to initial S values.
Both morphotypes showed similar significant increases
(17–25%) in Na+, Cl and Mg2+ concentrations after the
30-min air-exposure treatment (Fig. 5A and D). Longer
emersion exposures caused significant ionic increases only
in Zn-LI (Fig. 5B, C, E and F), with the maximum
percentage increments (32–41%) being detected after 7 h
(Fig. 5C and F). A significant effect of emersion time on
Na+, Cl and Mg2+ concentrations was also observed in
this morphotype. These ionic species recovered after
re-submersion (SR) to levels comparable to the initial S,
except for Zn-LI plants exposed to severe desiccation
(7 h), in which Ca2+ did not recover (Fig. 5F), and
Na+, Cl and K+ concentrations significantly decreased
(45–65%) below mean initial values (Fig. 5C).
Molar ratios of K+:Na+ decreased significantly in Zn-LI
air-exposed plants, increasing again as emersion progressed (from 16% to 46%, Fig. 5I–K). In Zn-HI, this
reduction was only observed in plants subjected to
30 min of air-exposure (Fig. 5I). A significant increase of
Ca2+:Na+ (176%) was observed in Zn-LI leaves during
the recovery phase, after 7 h of emersion (Fig. 5K).
Both organic solutes, the free amino-acid proline and
soluble sugars, increased significantly (33–44% and 22%,
respectively) in Zn-HI plants after both the 30-min and
3-h emersion periods, but not after 7 h (Fig. 6A–C). Similar significant effects were detected in Zn-LI plants
exposed to 3 and 7 h of desiccation (Fig. 6A–F). The concentrations of proline in both morphotypes and soluble
sugars only in Zn-HI were significantly dependent upon
emersion time. Starch content did not vary during emersion–immersion cycles except for Zn-LI plants emerged
after 3 h, which followed the same significant variation
Fig. 3. Effects of air exposure and recovery on leaf-tissue water
relations of Zostera noltii morphotypes from the high and low intertidal (Zn-HI and Zn-LI, respectively). See Material and Methods for
experimental design details. Different emersion periods are
represented in different graphs [E = 30 min, 3 h and 7 h in (A), (B)
and (C), respectively]. The responses of permanently submerged
control plants are shown in (D). Statistical differences [one-way
ANOVA and post hoc analysis (Student–Newman–Keuls test)] among
the simulated tidal phases [fully submerged plants (S), emerged plants
(E), re-submerged plants (SR)] and among controls [initial phase (PS1),
intermediate phase (PS2), final phase (PS3)] are indicated by different
letters. For simplicity, when only one of the three cases was
significantly different an asterisk (*) is used instead of letters. Lines
inside the bars represent the seawater osmolality expressed in
megapascals (MPa). Plotted values are means (n = 10) and the error
bars are SEs. Ψw, water potential; Ψp, osmotic potential; Ψp, turgor
pressure.
7
Sandoval-Gil, Barrote, Silva, Oliv
e, Costa, Ruiz, Marı́n-Guirao, S
anchez-Lizaso & Santos
Water relations of intertidal seagrasses
Table 1. Change of leaf relative water content (RWC) in both Zostera noltii morphotypes (Zn-HI, high inter-tidal; Zn-LI, low inter-tidal) during
emersion–immersion cycles in aquaria (S, fully submerged plants; E, emerged plants; SR, re-submerged plants) with different air-exposure periods
(E = 30 min, 3 and 7 h) and in permanently submerged control plants. Statistical differences (one-way ANOVA, df = 2, 27) and the post hoc
analysis (Student–Newman–Keuls test) between homogeneous mean values in the different phases of the simulated tidal cycle (S, E, SR) and the
control (initial, intermediate and final phase, PS1–PS3) are indicated by different letters. Values are means (n = 20) with SEs in parentheses.
Leaf RWC
Morphotype
Tidal cycle
E = 30 min
E=3h
E=7h
Control cycle
Control
Zn-HI
S
E
SR
S
E
SR
0.833
0.835
0.848
0.82
0.813
0.818
0.805
0.801
0.849
0.825
0.819
0.824
0.859
0.927
0.907
0.882
0.853
0.831
PS1
PS2
PS3
PS1
PS2
PS3
0.877
0.887
0.898
0.795
0.823
0.851
Zn-LI
(0.002)a
(0.002)a
(0.002)b
(0.001)a
(0.0008)b
(0.001)a
(0.018)
(0.015)
(0.015)
(0.001)a
(0.001)b
(0.001)a
pattern observed for the submerged control plants
(Fig. 6B and G). This was the only significant effect
observed for the submerged control plants.
Comparisons among natural populations
Both the water and the osmotic potentials of Zostera noltii plants collected in the field were significantly lower
(7% and 11%, respectively) in the upper than in the
lower inter-tidal, whereas the turgor potential was 47%
higher (Fig. 7). The subtidal species Cymodocea nodosa
and Zostera marina showed higher Ψw and Ψp and lower
Ψp than the inter-tidal Zn morphotypes, although these
differences were only significant with respect to Zn-HI.
Thus, Ψw and Ψp mean values ranked in the order
Zn-HI < Zn-LI < Cn < Zm, and the same rank order but
with the opposite sign was obtained for Ψp.
All species showed similar concentrations of the ionic
species analysed, which ranked in the order
Cl > K+ > Na+ > Mg2+ > Ca2+ (Fig. 8A). Cl, K+ and
Na+ had the highest concentrations, accounting for 91–
93% of the total leaf ion content. The total ion concentration was significantly lower (5–7%) in both inter-tidal
morphotypes of Z. noltii than in the subtidal species,
mostly due to the lower Cl content. Ca2+:Na+ was significantly higher (10–22%) in both inter-tidal morphotypes relative to the subtidal species, and K+:Na+ was
greater (7–14%) only in the case of Zn-HI (Fig. 8D).
The proline concentration followed the same rank
order as the turgor potential, Zn-HI > Zn-LI > Cn > Zm
(Fig. 8B). Starch was significantly higher only in Zn-HI,
whereas significantly higher concentrations of soluble sugars were detected in Zn-HI and Z. marina (Fig. 8C).
In general, the water relation descriptors (Fig. 7), the
total ion content and the concentration of osmolytes of
both morphotypes of Z. noltii collected in the field
(Fig. 8) were similar to those observed in aquaria for initially submerged plants (S phase, Figs 3–6), indicating
8
(0.018)a
(0.014)b
(0.014)a
(0.006)a
(0.011)b
(0.01)b
(0.015)
(0.014)
(0.018)
(0.012)a
(0.01)ab
(0.007)b
that the experimental set-up had no major effects on the
variables measured.
In order to improve the clarity of how the water status
descriptors were determined from the P–V curves and
H€
ofler diagrams, a schematic representation is presented
in Fig. 1, which shows the data corresponding to Zn-LI
0
as an example; some parameters (i.e. w100
p , wp , htlp, e)
are also indicated in Fig. 2C–F. The plant water status
descriptors were significantly different between the Z. noltii
morphotypes and the subtidal species (Table 2; Fig. 2).
The osmotic potentials at full saturation (w100
p ) and at
the turgor loss point (i.e. when the hydrostatic pressure
in the cell sap is equal to the external pressure, w0p )
were significantly higher in both inter-tidal morphotypes
and w0p ,
of Z. noltii (14–25% and 6–21% for w100
p
respectively) than in the subtidal species Z. marina and
C. nodosa. Their ranking order was Zn-HI > ZnLI > Cn = Zm. Likewise, the bulk modulus of elasticity
(e) was highest in Zn-HI (17%), and higher in inter-tidal
than in subtidal species (23–50%). The ranking order of
the relative water content at the turgor loss point (htlp)
was Zn-HI > Zn-LI = Zm > Cn whereas that of the
symplastic cell volume (hsym) was Zn-LI > Cn = Zm >
Zn-HI.
Discussion
Osmoacclimation of Z. noltii to emersion
We have shown here the different water relations
responses between the two morphotypes of Zostera noltii,
reflecting acclimation to different levels of the inter-tidal
of Ria Formosa lagoon. When exposed to the air, both
morphotypes were able to reduce the leaf-water potential
(Ψw), a primary response to cope with water stress as
described for terrestrial plants (Bisson & Kirst 1995;
Kramer & Boyer 1995; Verslues et al. 2006), and also
documented in seagrass species subjected to hyper-saline
Marine Ecology (2014) 1–17 ª 2014 Blackwell Verlag GmbH
Sandoval-Gil, Barrote, Silva, Olive, Costa, Ruiz, Marı́n-Guirao, S
anchez-Lizaso & Santos
A
B
C
D
Water relations of intertidal seagrasses
stress (Tyerman 1989; Sandoval-Gil et al. 2012a, 2013;
Marın-Guirao et al. 2013). In contrast to the low intertidal morphotype, which took longer (3–7 h) to reach the
minimum values of Ψw and Ψp in the acclimation to
emersion, the high inter-tidal morphotype rapidly osmoregulated after 30 min of air exposure. Leaf soluble sugars
and proline also increased faster in Zn-HI (30 min–3 h
compared with 3–7 h for Zn-LI). Evidence of ion accumulation related to osmotic adjustment in Zn-HI was
only detected in the short-term response to emersion
(30 min), contrary to their general progressive increase in
Zn-LI. This suggests that the fast accumulation of ions
and organic solutes was essential for the high inter-tidal
morphotype of Z. noltii to adjust its water status in the
initial phase of osmotic stress. As the osmotic stress continued (3 h), some ions were entirely replaced by those
more compatible organic solutes (sugars, proline), probably to prevent the metabolic toxicity of excessive ion
accumulation (e.g. membrane potential alteration, enzymatic inhibition or disruption of photosynthetic electron
transport; Beer et al. 1980; Niu et al. 1995; Yeo 1998;
Munns 2002; Yancey 2005). Osmotic regulation demands
a large amount of energy, which may be compensated by
mobilization of internal resources and increments in photosynthesis (Munns 2002). It has been demonstrated that
emerged plants of Z. noltii show higher photosynthetic
capacities than submerged plants under natural (in situ)
conditions, but these are greater in Zn-HI compared with
Zn-LI (Perez-Llorens & Niell 1993; Peralta et al. 2000;
Silva & Santos 2003; Silva et al. 2005). This indicates the
key role of the enhancement of photosynthesis in acclimation to emersion of these species, but also strongly
supports the differing efficiencies of the osmoacclimation
mechanisms between the morphotypes in this study.
The reduction in the water potential of Z. noltii leaves
in the low inter-tidal was achieved mainly by Ψp reduction, as opposed to the high inter-tidal morphotype in
which the reduction of Ψw was also accomplished by a
drastic decrease in turgor pressure after 7 h of exposure.
Although turgor reduction is typically related to the loss of
the cell symplastic water (Kirst 1989; Tyerman 1989; Kramer & Boyer 1995), our data on leaf dehydration (RWC,
Table 1) do not support this process. These particular
responses of Ψp have been described as dependent upon
Fig. 4. Effects of air exposure and recovery on the total ionic
content of leaf tissue of Zostera noltii morphotypes from the high
and low inter-tidal (Zn-HI and Zn-LI, respectively). See Fig. 3 for
further information on symbols and statistical analysis. Plotted values
are means (n = 6) and the error bars are SEs. S, fully submerged
plants; E, emerged plants; RS, re-submerged plants; PS1, initial phase;
PS2, intermediate phase; PS3, final phase.
Marine Ecology (2014) 1–17 ª 2014 Blackwell Verlag GmbH
9
Sandoval-Gil, Barrote, Silva, Oliv
e, Costa, Ruiz, Marı́n-Guirao, S
anchez-Lizaso & Santos
Water relations of intertidal seagrasses
A
D
I
B
E
J
C
F
K
G
H
L
Fig. 5. Effects of air exposure and recovery on leaf ionic concentrations and ion molar ratios of Zostera noltii morphotypes from the high and
low inter-tidal (Zn-HI and Zn-LI, respectively). See Fig. 3 for further information on symbols and statistical analysis. Na+, K+ and Cl are shown in
the left column, Ca2+ and Mg2+ in the middle column and molar ratios (K+:Na+, Ca2+:Na+) in the right column. Plotted values are means (n = 5)
and the error bars are SEs. DW, dry weight; S, fully submerged plants; E, emerged plants; RS, re-submerged plants; PS1, initial phase; PS2,
intermediate phase; PS3, final phase.
10
Marine Ecology (2014) 1–17 ª 2014 Blackwell Verlag GmbH
Sandoval-Gil, Barrote, Silva, Olive, Costa, Ruiz, Marı́n-Guirao, S
anchez-Lizaso & Santos
Fig. 6. Effects of air exposure and recovery
on leaf non-structural carbohydrates (NSCs;
soluble fraction, starch) and proline of
Zostera noltii morphotypes from the high and
low inter-tidal (Zn-HI and Zn-LI, respectively).
See Fig. 3 for further information on symbols
and statistical analysis. NSCs are shown in the
left column and proline in the right column.
Plotted values are means (n = 5) and the
error bars are SEs. DW, dry weight; FW, fresh
weight; S, fully submerged plants; E,
emerged plants; RS, re-submerged plants;
PS1, initial phase; PS2, intermediate phase;
PS3, final phase.
A
D
B
E
C
F
G
H
the cell wall properties (i.e. elasticity or rigidity; SandovalGil et al. 2012a). Plants that can adjust the level of hardness of their cell walls to become more rigid and/or thick
(i.e. higher elastic modulus, e) can experience severe
changes in Ψp without substantial cell-volume alterations
(Kramer & Boyer 1995; Verslues et al. 2006; Nobel 2009).
Marine Ecology (2014) 1–17 ª 2014 Blackwell Verlag GmbH
Water relations of intertidal seagrasses
This is a more energy-conservative mechanism than osmoregulation, which is common in species more tolerant to
water or salinity stress conditions, or adapted to environments characterized by fluctuating saline regimes (Bisson
& Kirst 1995; Lambers et al. 2008; Sandoval-Gil et al.
2012a). The activation of these processes in Zn-HI stron11
Sandoval-Gil, Barrote, Silva, Oliv
e, Costa, Ruiz, Marı́n-Guirao, S
anchez-Lizaso & Santos
Water relations of intertidal seagrasses
Fig. 7. Comparison of leaf water relations (Ψw, water potential; Ψp,
osmotic potential; Ψp, turgor pressure) among inter-tidal Zostera noltii
morphotypes (Zn-HI, high inter-tidal; Zn-LI, low inter-tidal) and
subtidal seagrass species (Cn, Cymodocea nodosa; Zm, Zostera
marina) of Ria Formosa. Lines within the bars represent seawater
osmolality expressed in megapascals (MPa). Statistically significant
differences [one-way ANOVA (df = 3, 39) and post hoc analysis
(Student–Newman–Keuls test)] are indicated by different letters.
Plotted values are means (n = 10) and the error bars are SEs.
gly suggests that this morphotype has developed specific
acclimatative capacities to respond more efficiently to
prolonged emersion periods than Zn-LI. This apparently
distinct resistance to emersion is also supported by the fact
that the RWC decreased in Zn-LI air-exposed plants, and
did not recover after 7 h of emersion.
Other results support the distinct tolerance to emersion
of the Z. noltii morphotypes. Leaves of Zn-LI plants
A
C
12
underwent much higher increases in potentially toxic
ionic species (Na+ and Cl) than Zn-HI in all emersion
periods. This led to greater alterations in ion homeostasis,
as indicated by the consistent reduction in the K+:Na+
ratio in Zn-LI during emersion. The maintenance of the
elevated ion molar ratios K+:Na+ and Ca2+:Na+ is one of
the major factors conditioning the resistance of plants to
water stress, also evidenced in seagrass species under
hyper-saline conditions (Marın-Guirao et al. 2013) as K+
and Ca2+ are co-factors of cellular processes essential for
plant growth and survival (e.g. enzymatic activity, signal
transduction pathways, ionic transport and compartmentalization, membrane potential stabilization; Maathuis &
Amtmann 1999; Hasegawa et al. 2000; Cramer 2002; Zhu
2003). Thus, the significant reduction (below initial values in submerged plants; S phase) of K+, Na+ and Cl
observed in the recovery phase of Zn-LI plants exposed
to 7 h of emersion (Fig. 5C) was indicative of severe metabolic alterations related to the transport, exclusion and
compartmentalization of ions at the cell, leaf tissue and
whole plant levels (Maathuis & Amtmann 1999). This
was not observed in Z. noltii plants from the high intertidal and demonstrated that the osmotic capacities of
Zn-LI were limited after the most severe emersion period.
As described above, after prolonged emersion (7 h) Ψp
increased in Zn-LI as opposed to Zn-HI. Similar exceptional increases in turgor in response to emersion have
been documented in some land plants (e.g. Citrus), seaweeds and, more recently, in the Mediterranean seagrasses
Posidonia oceanica and Cymodocea nodosa (Lloyd et al.
1987; Bisson & Kirst 1995; Marın-Guirao et al. 2013;
B
D
Fig. 8. Comparison of leaf osmolyte content
ions (A), proline (B), non-structural
carbohydrates (C) and ionic molar ratios (D)
among inter-tidal Zostera noltii morphotypes
(Zn-HI, high inter-tidal; Zn-LI, low inter-tidal)
and subtidal seagrass species (Cn,
Cymodocea nodosa; Zm, Zostera marina) of
Ria Formosa. Statistically significant
differences [one-way ANOVA (df = 3, 19) and
post hoc analysis (Student–Newman–Keuls
test)] are indicated by different letters. For
simplicity, statistical differences for only one
species or Z. noltii morphotype are indicated
by an asterisk (*). Plotted values are means
and the error bars are SEs. DW, dry weight;
FW, fresh weight; NSCs, non-structural
carbohydrates.
Marine Ecology (2014) 1–17 ª 2014 Blackwell Verlag GmbH
Sandoval-Gil, Barrote, Silva, Olive, Costa, Ruiz, Marı́n-Guirao, S
anchez-Lizaso & Santos
Water relations of intertidal seagrasses
Table 2. Water status descriptors derived from pressure–volume (P–V) curves: htlp, relative water content at the turgor loss point; hsym, symplastic
water fraction; w0p , osmotic potential at the turgor loss point (MPa); w100
p , osmotic potential at full saturation (MPa) and e, bulk modulus of
€fler diagrams in Fig. 2C–F.
elasticity (MPa). These descriptors are also indicated in the Ho
Water status descriptors derived from P–V curves
Descriptors
Zn-HI
htlp
hsym
w0p
w0p
e
0.857
0.420
5.018
3.349
11.16
Zn-LI
(0.004)a
(0.004)a
(0.108)a
(0.071)a
(0.4853)a
0.833
0.507
4.185
2.894
9.264
Cn
(0.00009)b
(0.002)b
(0.074)b
(0.062)b
(0.397)b
0.798
0.492
3.956
2.303
5.578
Zm
(0.002)c
(0.003)c
(0.034)c
(0.038)c
(0.083)c
0.832
0.492
3.906
2.494
7.129
(0.00004)b
(0.00002)c
(0.054)c
(0.088)c
(0.252)d
F
P
84.82
141.9
49.55
47.11
51.36
***
***
***
***
***
Cn, Cymodocea nodosa; Zm, Zostera marina; Zn-HI, high inter-tidal Zostera noltii morphotype; Zn-LI, low inter-tidal Zostera noltii morphotype.
Significant differences between species are indicated by asterisks (***P < 0.001; one-way ANOVA, df = 2, 12). Significant differences in the post
hoc analysis (Student–Newman–Keuls test) are indicated by different letters. Values are means (n = 5) with SEs in parentheses.
Sandoval-Gil et al. 2013). We are not able to explain this
observation, which may be related to some turgor-dependent process. In previous studies (Zimmermann 1978;
Kirst 1989; Bisson & Kirst 1995) it has been argued that
the increment in Ψp is an essential factor conditioning
the activation of some mechanoreceptor-operated ion
channels and calcium-dependent transduction cascades
that have leading roles within the osmotic acclimation
response (Kirst 1989). The potential activation of these
specific ion channels may be related to the ionic unbalances detected in the recovery phase of the Zn-LI plants
emerged for 7 h (Fig. 5C), as well as the increment in
Ca2+ detected in these plants (Fig. 5F). However, this
interpretation must be treated with caution, as more specific experimental studies are needed.
With the exception of the Zn-LI plants exposed to a
emersion period of 7 h, the high physiological plasticity
developed by Z. noltii to overcome the severe water stress
at low tide must be highlighted. Strategies that involve
high physiological plasticity have been repeatedly documented in plants exposed to heterogeneous environmental alterations at different temporal and spatial scales (e.g.
incident irradiance; Lichtenthaler 1996; DeWitt & Scheiner 2004; Dudley 2004; Pearcy 2007). With respect to
plant water relations, there is some evidence of changes
in water relations descriptors during daily cycles due to
plants’ adjustments (e.g. leaf conductance and transpiration) to such cycles of environmental parameters (e.g.
light, temperature, humidity) (Bowman & Roberts 1985;
Gordon 1993). In addition to the responses to emersion
of Z. noltii, our results suggest some daily adjustment of
water relations in permanently submerged plants, as Ψw
and Ψp were significantly reduced (although to a lesser
extent than in air-exposed plants) in PS2 compared with
PS1 and/or PS3. This was also supported by slight (but
non-significant) increments observed consistently in some
osmolytes (ions, proline) in PS2 plants.
Marine Ecology (2014) 1–17 ª 2014 Blackwell Verlag GmbH
Water status properties of inter-tidal and subtidal species
As described for terrestrial and wetland plants (Touchette
2006; Lambers et al. 2008), the differences in the water
relations observed here among seagrass species and
between Zostera noltii morphotypes may reflect distinct
ecological strategies and stress tolerance capacities. In
studies carried out in other geographic locations, the
euryhaline Cymodocea nodosa consistently showed more
negative values of leaf Ψw and Ψp (and higher Ψp) than
the more stenohaline Posidonia oceanica (Sandoval-Gil
et al. 2012a, 2013). Similar divergences have been
described between C. nodosa populations growing in
open waters and in a hyper-saline coastal lagoon (Sandoval-Gil 2012). Our results show that the more negative
values of Ψw and Ψp were related to a the position of the
plants in the intertidal-subtidal continuum. In effect, leaf
Ψw and Ψp measured in Z. noltii morphotypes yielded
more negative values than those exhibited by C. nodosa
and Zostera marina, more adapted to subtidal conditions
in Ria Formosa. Such variation was also evident between
Z. noltii morphotypes, with the minimum values of Ψw
and Ψp being observed in Zn-HI populations exposed to
the maximum periods of air exposure during tidal cycles.
In this work, Z. noltii showed more negative osmotic
potentials at full turgor (w100
p ) and at the turgor loss
point (w0p ) than the subtidal species, particularly the
high inter-tidal plants, which were able to concentrate
intra-cellular osmolytes, mainly proline, at high levels. Similar relationships between high leaf proline
concentrations and low osmotic potentials have also been
documented in other seagrasses (Brock 1981; Tyerman
1989 Sandoval-Gil et al. 2012a, 2013; Marın-Guirao et al.
2013). The ability to regulate the concentration of compatible solutes such as proline (and non-structural carbohydrates) is a key trait determining the osmotic resistance
of this morphotype (Drew 1978; Sandoval-Gil et al.
13
Water relations of intertidal seagrasses
Sandoval-Gil, Barrote, Silva, Oliv
e, Costa, Ruiz, Marı́n-Guirao, S
anchez-Lizaso & Santos
2012a; Marın-Guirao et al. 2013). The role of starch in
the regulation of osmotic stability and cell volume has
been also suggested elsewhere (Ackerson & Hebert 1981;
Sandoval-Gil et al. 2013). In addition, the differences in
ionic ratios reflected distinct capacities to withstand water
stress conditions. Our analysis showed that the Ca2+:Na+
molar ratio was higher in the inter-tidal than in the subtidal species, and greater values of K+:Na+ were found in
Zn-HI. These differences may be related to specific adaptation properties based on the ability of discrimination of
Na+ over K+ and Ca2+ by means of different metabolic
adjustments (e.g. selective transport, excretion or compartmentalization), as recently reported in seagrasses with
contrasting salinity tolerance ranges (Garrote et al. 2014).
In marine macrophytes, it has been proposed that
higher e values (i.e. more rigid/thick cell walls) reflect an
effective adaptation to fluctuating salinity regimes (Tyerman 1989; Bisson & Kirst 1995). High cell wall rigidity
allows the maintenance of reduced Ψw without an appreciable loss of symplastic water (Kramer & Boyer 1995;
Verslues et al. 2006). By contrast, changes in cell wall
elasticity (i.e. reduced e) allow the maintenance of Ψp at
lower RWC (Touchette et al. 2007, 2009). Our study
showed a gradient in cell wall rigidity (and thus decreased
RWC at the turgor loss point, htlp), with the highest values seen in the high inter-tidal morphotype of Z. noltii
and the lowest in the subtidal C. nodosa. This is consistent with previous observations, for example on plant
halophytes of tidal marshes (e.g. Juncus roemerianus),
which endure higher salinities than plants developing in
zones more influenced by freshwater inputs (Touchette
2006). Increasing cell wall rigidity was also reported in
marsh halophytes and mangroves exposed to salinity
increments (Paliyavuth et al. 2004; Touchette et al. 2009).
Cell turgescence is expected to decrease as plant tissue
dehydrates (Sun 2002). However, the P–V curves of Z.
noltii and Z. marina show that Ψp remained constant
during the initial decline in RWC. This anomalous
response has also been reported in poikilohydric plants
that initially lose inter-cellular water, which contrary to
intra-cellular water, it does not interfere directly with Ψp
(Beckett 1995, 1997; Proctor et al. 1998; Hajek & Beckett
2008). The specific advantages of these processes are still
unknown (Beckett 1995, 1997). As some Z. marina populations are also able to grow in the inter-tidal (e.g. in
Northern Europe), we speculate that this trait is related
to that ability.
Conclusions
Our results provide evidence that the inter-tidal morphotypes of the seagrass Zostera noltii can exhibit different
osmoacclimation strategies (i.e. reduction of Ψw) in
14
response to emersion, which may explain the differences
in the maximum periods of emersion that they can tolerate and thus, their vertical distribution in the inter-tidal.
These can be summarized as:
1 Zn-HI showed the most negative Ψw and Ψp values,
which allowed more positive water balances.
2 After a rapid increase in ion concentration under air
exposure, Zn-HI replaces these ions by more physiologically compatible solutes (proline and non-soluble carbohydrates) in order to maintain the osmotic adjustment.
3 Zn-HI developed cell-wall hardening processes in the
most severe emersion treatment, which is considered
to be a more conservative acclimation strategy.
4 The maximum increases in ion concentrations and
greatest alterations in homeostasis of ions (i.e. reduction in K+:Na+) were detected in air-exposed plants of
Zn-LI, indicating less potential for enduring air exposure than in the other groups.
The differences in the plant water status properties of
the inter-tidal adapted Z. noltii and the subtidal-adapted
species Zostera marina and Cymodocea nodosa are also
noteworthy. Both morphotypes of Z. noltii showed more
and w0p than the subtidal spenegative values of w100
p
cies, and Zn-HI exhibited the most negative values of Ψp
and Ψw. These properties and others, such as the higher
K+:Na+ found in Zn-HI and the greater Ca+2:Na+ ratios
observed in both morphotypes, are related to higher resistance to water stress. Zostera noltii, particularly in the
high inter-tidal, adjusted the osmolyte concentration and
activated cell wall hardening strategies to achieve osmotic
stability. This is supported by greater values of e.
This study highlights that water relations and osmoadaptation are key traits determining the zonation patterns
of seagrass species. These results are supported by the
parameters derived from the P–V isotherms and H€
ofler
diagrams, never before derived for seagrasses. Valuable
information can be obtained with these techniques, either
to elucidate seagrass adaptive ecological strategies (e.g.
ecophysiological/functional groups) or local acclimation
capabilities (e.g. environmental tolerance ranges), as
already widely described in land and wetland plants.
Acknowledgements
This research was accomplished within the framework of
the European Cooperation in Science and Technology
(COST) Action ES0906 ‘Seagrass productivity from genes
to ecosystem management’. A Short Term Scientific Mission (STSM) grant from the COST Action and a grant
from the University of Alicante (Department of Marine
Sciences and Applied Biology) were awarded to J. M.
Sandoval-Gil. The OSMOGRASS II project (ref. no.
CTM2009-08413MAR) funded by the National Plan of
Marine Ecology (2014) 1–17 ª 2014 Blackwell Verlag GmbH
Sandoval-Gil, Barrote, Silva, Olive, Costa, Ruiz, Marı́n-Guirao, S
anchez-Lizaso & Santos
Scientific Research of the Spanish Government also supported this research. A Fundacß~ao para a Ciência e a Tecnologia (FCT) post-doctoral fellowship SFRH/BPD/
71129/2010 was awarded to I. Olive. The authors are
especially grateful to Silvia Albano and Bruno Fragoso
from the ALGAE group and the Instituto Espa~
nol de
Oceanografıa (IEO) technician Rocıo Garcıa Mu~
noz for
their invaluable field and laboratory logistical support.
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