nitrogen acquisition in wetland plants edita...
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Nitrogen acquisition in wetland plants
(nutritional aspects of plant performance under eutrophication)
Edita Tylová
Summary of the PhD thesis
Department of Plant Physiology
Faculty of Science
Charles University in Prague
Supervisor: RNDr. Olga Votrubová, CSc., Department of Plant Physiology, Faculty of
Science, Charles University in Prague
2
The table of contents: Introduction and aims of the study……………………………………………………….1
Flooded soil, eutrophication, sources of N in wetland………………………………..7
NH4+ versus NO3
- preferences in plants, ecological consequences …………………10
Uptake of NH4+ and NO3
- in wetland plants…………………………………………12
NH4+ and NO3
- assimilation, amino acids……………………………………………17
NH4+ versus NO3
- nutrition, NH4+ toxicity…………………………………………...24
Consequences of high N availability to wetland plant performance…………………26
The summary of the original findings of the thesis……………………………………...35
References……………………………………………………………………………….38 Acknowledgement: My greatest thanks belong to Dr. Olga Votrubová for her kind supervision of the work. Many
thanks belong also to my colleagues from the laboratory of plant anatomy, especially to Lenka
Steinbachová, Aleš Soukup, and Tomáš Polák; and to members of the laboratory of Prof.
Hans Brix (Aarhus University), particularly to Prof. Hans Brix and Dr. Bent Lorenzen.
Special thanks are reserved for my whole family, especially for Radovan and Štěpánka.
The work was supported by the grants: No.113100003 Regulation and signalling in living systems. Ministry of Education, Youth and
Sports of the Czech Republic.
No.113100004 Ecological and evolution aspects of diversity of organisms, structures and
interactions. Ministry of Education, Youth and Sports of the Czech Republic.
FRVŠ no. 1778/2001 Study of carbohydrate and nitrogen metabolism of selected wetland
plants.
GA AVČR no. B6111306 Nitrate reductase activity in wetland plants.
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Included publications and manuscripts: Tylová-Munzarová E., Lorenzen B., Brix H. and Votrubová O. 2005. The effects of NH4
+ and NO3
- on growth, resource allocation and nitrogen uptake kinetics of Phragmites australis and Glyceria maxima. Aquatic Botany 81: 326-342.
(article 1) Munzarová E., Lorenzen B., Brix H., Vojtíšková L. and Votrubová O. 2006. Effect of
NH4+/NO3
− availability on nitrate reductase activity and nitrogen accumulation in wetland helophytes Phragmites australis and Glyceria maxima. Environmental and Experimental Botany 55: 49-60.
(article 2) Tylová E., Steinbachová L., Votrubová O., Lorenzen B. and Brix H. Different sensitivity of
Phragmites australis and Glyceria maxima to high availability of ammonium-N. Accepted in Aquatic Botany.
(manuscript 1) Tylová E., Steinbachová L., Votrubová O. and Gloser V. Phenology and autumnal
accumulation of N reserves in belowground organs of wetland helophytes Phragmites australis and Glyceria maxima affected by nutrient surplus. Submitted to Environmental and Experimental Botany.
(manuscript 2) Tylová E., Steinbachová L., Soukup A., Votrubová O. and Gloser V. Performance of
Phragmites australis and Glyceria maxima under changing nitrogen and phosphorus availability. I. Plant growth, resource allocation and C/N balance. Submitted to Environmental and Experimental Botany.
(manuscript 3) Other works of the author, related to the topics: Articles and manuscripts:
Vojtíšková L., Munzarová E., Votrubová O., Říhová A. and Juřicová B. 2004. Growth and biomass allocation of sweet flag (Acorus calamus L.) under different nutrient conditions. Hydrobiologia 518: 9-22.
Vojtíšková L., Munzarová E., Votrubová O., Čížková H. and Lipavská H. 2006. The influence of nitrogen nutrition on the carbohydrate and nitrogen status of emergent macrophyte Acorus calamus L. Hydrobiologia 563:73-85.
Steinbachová-Vojtíšková L., Tylová E., Novická H., Soukup A. and Votrubová O. 2006. Lipavská H., Čížková H. Influence of nutrient supply on growth, carbohydrate and nitrogen metabolic relations in Typha angustifolia. Environmental and Experimental Botany 57: 246-257.
Steinbachová L., Lipavská H. Tylová E., Votrubová O. and Čížková H. Performance of Phragmites australis and Glyceria maxima under changing nitrogen and phosphorus availability. II. Carbohydrate metabolism. Submitted to Environmental and Experimental Botany.
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Poster presentations:
Munzarová E., Lorenzen B., Brix H. and Vojtíšková L., Votrubová O. 2004. Nitrate reductase activity in wetland helophytes Phragmites australis and Glyceria maxima. Book of abstracts. 10th days of plant physiology, Bratislava, p. 114.
Vojtíšková L., Munzarová E., Soukup A., Novická H., Votrubová O., Čížková H. and Lipavská H. 2004. Growth, biomass allocation and C:N balance of Typha angustifolia under high nutrient load. Book of abstracts. 10th days of plant physiology, Bratislava, p. 130.
Munzarová E., Lorenzen B., Brix H. and Votrubová O. 2003. Nitrogen nutrition and NH4+-N
and NO3--N kinetics of selected wetland plants. Book of Abstracts. Plant Root
Development and Adaptation to Stresses, Stará Lesná, Slovensko, p. 40.
Vojtíšková L., Munzarová E. and Votrubová O. 2001. Performance of some wetland plants under nitrogen nutrition - influence on C:N balance. Book of Abstracts. Plant Physiology Days of Young Scientist, Praha, p. 45.
Munzarová E., Vojtíšková L., Votrubová O. and Raimanová I. 2001. Ammnoium and nitrate nutrition of selected wetland plant species, Phragmites australis and Glyceria maxima. Book of abstracts. 9th days of plant physiology, České Budějovice. p. 152.
Raimanová I., Votrubová O. and Munzarová E. 2001. Effect of low pH and Al in combination with nitrogen form on performance of two wetland species Phragmites australis and Glyceria maxima. Book of abstracts. 9th days of plant physiology, České Budějovice. p. 156.
Votrubová O., Vojtíšková L., Munzarová E. and Soukup A. 2001. Effect of high nutrient load on growth and morphology of wetland plants. Book of abstracts. 9th days of plant physiology, České Budějovice. p. 161.
Vojtíšková L., Munzarová E. and Votrubová O. 2001. Effect of nutrient level on C:N balance of wetland plants. Book of abstracts. 9th days of plant physiology, České Budějovice. p. 160.
Votrubová O., Vojtíšková L., Munzarová E., Čížková H., Juřicová B. and Pecháčková A. 1999. Nutrient over-supply effects on the performance of three wetland species. Book of abstracts. International conference on Phragmites-dominated wetlands, their function and sustainable use – Třeboň. p. 75.
Pecháčková A., Votrubová O., Brändle R., Munzarová E. and Juřicová B. 1998. Performance of wetland plants under high nutrient load. Book of abstracts. 8th days of plant physiology, Olomouc. p. 175.
Votrubová O., Soukup A., Vojtíšková L., Paveljic D., Munzarová E. and Pecháčková A. 1998. Effect of high nutrient load on roots and rhizomes of wetland plants. Book of abstracts. 8th days of plant physiology, Olomouc. p. 233.
5
Introduction and aims of the thesis:
This thesis followed basic aspects of N acquisition in two co-
occurring helophytes, Phragmites australis (Cav.) Trin. Ex Steud.
and Glyceria maxima (Hartm.) Holmb., which are understood as the
model species. Selected species differ in the growth
strategy (e.g. dormancy requirement, rooting
depth), and sensitivity to eutrophication. While
Phragmites seems positively respond to early
stages of eutrophication, Glyceria is more tolerant
to highly eutrophic sediments rich in organic
matter, and its spread following reed retreat was repeatedly documented
(e.g. Hroudová and Zákravský (1999)). Therefore, they represent useful
model species for understanding the mechanisms underlying competition
ability in the changing environment. Both species are also commonly used in wastewater-
treating systems (Květ et al. 1999).
The main aim of the study was to follow basic aspects of N acquisition and utilization in
wetland plants, and/or their growth and metabolic responses to N level, N/P ratio, and
NH4+/NO3
- ratio. The reason was to characterise the extent to which trays in N acquisition
affect the response of wetland plants towards eutrophication and to separate the effects of
nutrients per-se from the other factors related to eutrophication. The study is a part of broader
physiological effort of the team of Dr. Olga Votrubová (Department of Plant Physiology,
Charles University in Prague), focused on different aspects of wetland plant performance. The
study was realised in the collaboration with the Department of Biological Sciences, Aarhus
University (the team of Prof. Hans Brix; http://person.au.dk/en/[email protected]) and
with the Department of Plant Physiology and Anatomy, Faculty of Science of Masaryk
University in Brno (the team of Dr. Vít Gloser; http://www.muni.cz/people/2691).
6
Fig. 1. Relative growth rate (mg g-1 fresh wt. day-1) of P. australis (PA) and G. maxima (GM) grown under NH4
+ (open columns) and NO3- (solid
columns) at 34 and 179 µM total N availability. Modified according to Tylová-Munzarová et al. 2005 and Munzarová et al. 2006.
0
10
20
30
40
50
60
70
80
PA GM
RG
R (
mg
g-1
day
-1)
b
a
aa
PA GM
b
a
aa
34 µM 179 µM
The summary of main topics focused in the thesis:
1. NH4+ versus NO3
- preferences in wetland plants
Availability of NH4+ and NO3
- shows significant spatial and temporal variability in
wetlands. NO3- is usually the dominant N form in the bulk water, but in the sediment it
disappears rapidly with soil depth, being almost limited to the hypoxic topsoil layers or to the
rhizosphere of emergent macrophytes. NH4+ seems therefore the dominant N form available
for the rooting macrophytes (Cedergreen and Madsen 2003), as well as it is commonly the
prevailing N form of wastewater. Both NH4+ and NO3
- are important N sources and the
majority of plants show the highest growth potential on their mixture. The preferences for
particular N source may, however, occur in individual plant species in response to long-term
soil chemical conditions of their habitats (Kronzucker et al. 1997). Species colonising habitats
with restricted nitrification frequently display preference for NH4+. In agreement, wetland
plants are generally
supposed to posses NH4+
preference (e.g. Brix et al.
(2002), Cedergreen and
Madsen (2003), Tylová-
Munzarová et al. (2005)),
which is supported by
growth studies, studies of
NH4+ and NO3
- uptake
kinetics or studies of
nitrate reductase activity.
In agreement, the positive
growth responses to NH4+ was found in Glyceria maxima treated with 34µM N availability in
our study (Tylová-Munzarová et al. 2005).
Wetland plants are also generally considered to be tolerant towards high NH4+ doses,
which commonly trigger adverse effects in many NH4+ sensitive terrestrial species. Our
experiments with Glyceria maxima, however, showed that NH4+ tolerance in wetland plants
has its limitations. While Glyceria grew better with NH4+ compared to NO3
- N source at
7
34µM N level (Tylová-Munzarová et al. 2005; Fig. 1 - left), the response turned to opposite at
179 µM N level (Munzarová et al. 2006, Tylová et al. - manuscript 1; Fig. 1 - right).
In addition, the capability of NO3- uptake and utilization in wetland plants is obvious,
particularly in emergent species with ability to oxidise their rhizosphere due to radial oxygen
loss. Among them, the sufficient growth
with sole NO3--N source was described e.g.
in Glyceria maxima and Phragmites
australis (Tylová-Munzarová et al. 2005,
Munzarová et al. 2006, Tylová et al. -
manuscript 1), or Typha latifolia (Brix et al.
2002). Similarly, species colonising NO3-
dominated nitrogen-poor soft waters or
species with roots (or leaves) subjected
directly to NO3- rich bulk water, may take
up considerable amounts of N as NO3-. The
later includes not only various floating
macrophytes (e.g. Lemna minor
(Cedergreen and Madsen 2002, Cedergreen
and Madsen 2004)), but also helophytes
developing finely branched aquatic roots at
the stem nodes, which allow acquiring
nutrients directly from the bulk water or
superficial oxidised sediment layers
(Končalová and Pazourek 1988).
2. Uptake of NH4+ and NO3
- in wetland
plants
The uptake of both ions is
multiphasic, mediated by several transport
systems in dependence on ion external
availability. At low rhizosphere levels
(<0.5mM), the uptake is active carrier-
Fig. 2. Average maximum uptake rate per root dry wt (Vmax) (A), half saturation constant (apparent Km) (B) and affinity (C) of P. australis (PA) and G. maxima (GM) acclimated to NH4
+-N (PA NH4, GM NH4) or NO3
--N (PA NO3, GM NO3) source. Values given are mean ± S.E. (n = 4–6). Results of NH4
+ uptake are illustrated with solid columns, results of NO3
- uptake with open columns (Tylová-Munzarová et al. 2005)
0
20
40
60
80
PA NH4 PA NO3 GM NH4 GM NO3V
max
( µµ µµm
ol g
-1 ro
ot d
ry w
t h-1
)
A
0
2
4
6
8
10
PA NH4 PA NO3 GM NH4 GM NO3
Km
(µµ µµ
M)
B
0
4
8
12
16
PA NH4 PA NO3 GM NH4 GM NO3
afin
ity (
L g
-1 r
oot d
ry w
t h-1)
C
8
mediated process, provided by saturable high-affinity transport system (HATS). HATS
operates with Michaelis-Menten kinetics, displays sensitivity to metabolic inhibitors and to
diminution of membrane H+ gradient (Forde and Clarkson 1999). In higher concentration
range (>0.5 mM), NH4+ and NO3
- are taken up by low-affinity transport systems (LATS); as a
passive non-saturable influx through ion channels with the weak response to metabolic
inhibitors (Forde and Clarkson 1999). The concentrations exceeding 0.5 mM, however, rarely
occur at natural stands, even at eutrophicated sites and thus HATS is supposed to be
responsible for the majority of N uptake in situ. The kinetic parameters of HATS differ
among individual species in correspondence with long-term nutritional status of their habitats.
Species adapted to low-fertility environments display lower root absorption capacity (lower
maximum uptake rate; Vmax) but higher affinity (low half saturation constant; Km), the
opposite is true for fast growing species of fertile habitats (Chapin 1980). The characteristics
of HATS kinetics are therefore signs of plant ecological amplitude towards nutrient
availability.
Previous studies dealing with HATS kinetics in wetland plants estimated Vmax and Km
for NH4+ and NO3
- in different emergent macrophytes. The direct interspecific comparison
based on these results, however, has to be done with care and caution, as the kinetic data
obtained under different experimental conditions, particularly under different N levels applied
prior the onset of measurements, may differ considerably. In our study (Tylová-Munzarová et
al. 2005) we therefore compared NH4+ and NO3
- uptake kinetics in Phragmites and Glyceria
cultivated under similar growth conditions. This study showed comparable Vmax in these two
species (Fig. 2). Both species possessed high Vmax, which is typical for fast growing species
of fertile stands. Phragmites, however, exhibited lower Km and higher affinity compared to
Glyceria (Fig. 2), characteristics advantageous under nutrient limitation. This finding
indicates a higher physiological plasticity to adjust N uptake towards low nutrient
availabilities. In agreement, field observations described the ability of Phragmites to colonise
oligotrophic systems, where Glyceria is not present (Brändle et al. 1996).
In adition, both Phragmites and Glyceria exhibited significantly higher Vmax for NH4+
compared NO3- uptake (Fig. 2). Similarly, the affinity for NH4
+ tended to be higher than those
for NO3-, especially in plants acclimated to NH4
+-N. These results indicate that wetland
species share characteristics of plants colonizing habitats with restricted nitrification, where
the preference for NH4+ over NO3
- was described (Kronzucker et al. 1997). The observed
differences were, however, relatively small. Both species were also able to sufficiently grow
with sole NO3--N source and displayed high NR activity (Munzarová et al. 2006). In littoral
9
helophytes, therefore, the preference for NH4+ seems to be very weak or even absent in
comparison with submerged wetland species (examined e.g. by Cedergreen and Madsen
(2003)). This may correspond to the fact, that NH4+ prevalence in the sediment is not so strict
(particularly in superficial sediment layers) and may change with water level fluctuation.
Moreover, the existence of finely branched aquatic roots allows the uptake of NO3- from the
oxidized water column.
3. Nitrate reductase activity in wetland plants
The activity of nitrate reductase (NR) is the critical step of NO3- assimilation, which
integrates various regulatory signals. In ecological studies, NR activity is therefore used as a
marker of NO3- assimilation intensity, which allows characterisation of NO3
- versus NH4+
importance in nutrition of different species under real ecological circumstances. Our study,
however, showed that the interpretation of NR activity data must always be done with care
and caution. Firstly, individual species might considerably differ in amounts of NO3- stored in
plant tissues in unreduced form. In our study, Phragmites exhibited higher leaf NR activity
compared to Glyceria, which might indicate more conspicuous ability of successful growth on
NO3- N source in Phragmites. The overall growth response to NO3
- was, however, more
positive in Glyceria, in spite of significantly lower NR activity (Munzarová et al. 2006).
Glyceria obviously tended to accumulate NO3- in unreduced form (Table 1), without any
Table 1. Total tissue N and N-NO3- contents of Phragmites and Glyceria grown with NH4
+-N or NO3--N
source (179 µM) in the water culture.
The statistical evaluation using analysis of variance (ANOVA). L – leaves, St – stems, Rh – rhizomes, R –roots. Values given are mean ± S.E., n = 4. Effects of plant species (sp.), N treatments (tr.) and interactions (species x treatment) are expressed by P-values. Data were square root transformed for the statistical analysis (Munzarová et al. 2006).
10
negative consequences to N nutrition,
but low NR activity might cause
underestimation of NO3- importance in
nutrition of this species.
Secondly, the reliable use of NR
activity as a marker of NO3- utilization
in individual plant species is impossible
without the precise verification of the
method, which is, unfortunately, not
often taken into account in ecological
studies. Plant species and even organs
may significantly differ in the content
of chemical compounds interfering
with NR activity estimation (e.g.
phenolics, enzymes inactivating NR,
glycosides etc.). In our study
(Munzarová et al. 2006), Glyceria but
not Phragmites appeared to contain cyanogenic glycosides, potent inhibitors of NR activity
releasing CN- during homogenisation (Maranville 1970), which might lead to serious
underestimation of NR activity when ignored.
Keeping these limitations in mind, NR activity approach is promising in
characterisation of the real importance of NO3- in nutrition of wetland plants. This importance
is generally questioned due to low NO3- availability in the waterlogged sediment. In
agreement, Cedergreen and Madsen (2003) observed low ambient NR activity in different
submerged and amphibious plants and attributed these results to i: general preferences for
NH4+ in these plants, and ii: to NH4
+ suppression of NR activity. In our experimental culture,
however, substantial NR activities were detected in Phragmites as well as Glyceria
(Munzarová et al. 2006; Fig. 3).
Considering the waterlogging stress, NR activity is long-term questioned to be involved
in alleviation of hypoxic/anoxic stress in plants due to consumption of reduced cofactors. This
hypothesis, however, possesses several weaknesses and thus the mechanism is still the matter
of discussion. In relation to this discussion, the localisation of NR activity in wetland plants is
not without interest, but such information is scarce. The localisation of NR activity was
Fig. 3. NR activity in tissues of (A) Phragmites australis and (B) Glyceria maxima grown with NH4
+-N or NO3
--N source (179 µM) in the water culture. Values given are means ± S.E. (n = 4–5). L, leaves; St, stems; Rh-y, young rhizomes; R, roots (Munzarová 2006).
(B)
0
10
20
30
40
L St Rh-y R
mol
NO
2- g-1
dry
wt h
-1
(A)
0
10
20
30
40
mol
NO
2- g-1
dry
wt h
-1
NRmax (NH4+)
NRact (NH4+)
NRmax (NO3-)
NRact (NO3-)
11
therefore studied in
greater details in
Phragmites australis
and Glyceria maxima
(Munzarová et al.
2006). Based on our
results, root locali-
zation was shown to
be relatively impor-
tant in Glyceria,
particularly in the
sand culture (Fig. 4).
In contrast, Phrag-
mites seemed to be
the preferential leaf
NO3- reducer (Fig.
4), similarly to some
terrestrial grasses
studied by Scheurwater et al. (2002).
4. Responses to NH4+ versus NO3
- nutrition, NH 4+ toxicity
The possibility to take up N as two chemically different forms brings several
consequences to plant performance. Although the assimilation of NH4+ has lower energy costs
compared to NO3-, the exclusive NH4
+ nutrition is potentially harmful to many plant species.
Sensitive plants even develop NH4+ toxicity syndrome, particularly when rhizosphere NH4
+
levels exceed 100µM, and pH drops to low values (Marschner 1995, Britto and Kronzucker
2002). Certain sensitivity to excessive NH4+ supply occurs even in generally NH4
+ tolerant
wetland plants (see Fig. 1). This growth and metabolic disorder includes the growth
suppression, low R/S ratio, accumulation of NH4+ in tissues, energy exhaustion, and/or the
diminished uptake of essential cations.
The risk of NH4+ toxicity is obviously related to excessive NH4
+ uptake (Britto and
Kronzucker 2002), which might lead to over-accumulation of NH4+ in plant tissues (Hecht
and Mohr 1990). In agreement, higher contents of NH4+ were detected in NH4
+ compared to
Fig. 4. Relative contribution (%) of individual plant organs in the whole plant reduction of NO3
− expressed on dry wt basis) in (A, C) Phragmites australis and (B, D) Glyceria maxima grown in the (A, B) water and (C, D) sand cultures. Data based on NRmax are shown. Values given are means ± S.E. (n = 3–5). L, leaves; St, stems; Rh, rhizomes; R, roots. NO3
− - nitrate nutrition, NH4
+ - ammonium nutrition; OL, EU, HYP - oligotrophic, eutrophic and hypertrophic growth conditions (Munzarová et al. 2006).
(A)
0
20
40
60
80
100
%
NH4+
NO3-
(B)
0
20
40
60
80
100
(C)
0
20
40
60
80
100
L St Rh R
%OL
EU
HYP
(D)
0
20
40
60
80
100
L St Rh R
12
NO3- fed plants of both Phragmites and Glyceria in our study (Tylová et al. - manuscript 1).
The trend was, however, even more pronounced in Phragmites (Fig. 5) and therefore seems
not being the
cause of the
negative growth
response to NH4+
observed in Gly-
ceria.
Besides excessive NH4+ uptake, the induced deficiency of essential cations (especially
Ca2+, Mg2+, K+) commonly occurs, as their uptake and translocation to shoot are lower under
NH4+ compared to NO3
--N provision. This phenomenon may accompany the sensitivity
towards high level of NH4+ found in Glyceria, as the species showed lower contents of Mg2+
and K+ in roots of NH4+ compared to NO3
- fed plants, while no changes in either parameter
occurred in Phragmites (Fig. 6). In agreement, low K+ contents in rhizomes of Glyceria but
not Phragmites were found under excessive sewage dose (Čížková-Končalová et al. 1996).
0
5
10
15
20
25
30
35
40
45
L R L R L R
Ca,
Mg,
K (
mg
g dr
y w
t-1
)
NH4 NO3
K+Mg2+Ca2+
L R L R L R
K+Mg2+Ca2+
Fig. 6. Contents of Ca2+, Mg2+, K+ in tissues of Phragmites australis and Glyceria maxima grown with NH4
+-N or NO3--N at 179 µM total N level. Values given are means ± STD (n = 3-5). L – leaves, St –
stems, Rh – rhizome, R – roots. Modified according to Tylová et al. - manuscript 1.
Glyceria maxima Phragmites australis
Fig. 5. Contents of NH4+ in tissues of Phragmites australis and Glyceria maxima
grown with NH4+-N or NO3
--N at 179 µM total N level. Values given are means ±STD (n = 3-5). L – leaves, St – stems, Rh – rhizome, R – roots. Modifiedaccording to Tylová et al. - manuscript 1.
13
5. Consequences of high N availability to wetland plant performance
Increasing N availability triggers several responses in plants, including changes in
growth rate, biomass distribution, morphology, chemical composition, phenology, and/or
storage formation (Marschner 1995). In wetland plants, all these responses obviously affect
the susceptibility to different stresses, and thus competition ability at natural habitats.
Furthermore, high oxygen demand of eutrophic sediment may cause the shift in NH4+ versus
NO3- availability in favor of NH4
+, which has further consequences to wetland plant
physiology.
Among
general responses to N
addition, enhanced
biomass production
and favoured alloca-
tion of biomass to
aboveground structu-
res are the most
typical ones. Such
stimulatory effect is
well documented in
several wetland plants,
and was also observed
in our studies of
Phragmites australis,
Glyceria maxima
(Tylová et al. -
manuscript 3), Acorus
calamus (Vojtíšková
et al. 2004), and Typha angustifolia (Steinbachová-Vojtíšková et al. 2006). The enhanced
growth obviously does not endanger plant directly, in fact it favours given plant in the
competition for space, but brings about several side effects, which negatively affect long-term
survival at waterlogged habitats. In addition, when the availability of N is further elevated up
to excessive levels, the positive growth response to N surplus turns to negative. This effect
Fig. 7. Biomass (mean ± SD, n = 5-8) of leaves (L), stems (ST), rhizomes (RH) and roots (R) of Phragmites australis (a, c) and Glyceria maxima (b, d) grown under increasing N plus P (a, b) and increasing N (c, d) supplies(Tylová et al. - manuscript 3)
0
20
40
60
80
100
120
dry
wt (
g)
oligo
eu
hyper
(a) (b)
0
20
40
60
80
100
120
L ST RH R
dry
wt (
g)
low N
medium N
high N
(c)
L ST RH R
(d)
14
seems particularly serious under suboptimal P availability, which is clearly showed in our
study of Phragmites australis and Glyceria maxima (Tylová et al. - manuscript 3; compare
upper and bottom parts of Fig. 7). The importance of P was highly visible particularly in
Glyceria, which agrees with the field observations describing well-developed Glyceria stands
as indicators of local sources of pollutions with high P contents in the sediment (Hroudová
and Zákravský 1999). Thus, the ability of Glyceria to expand into reed die-back sites in
highly eutrophic areas may not necessarily be only due to the sensitivity of Phragmites to
severe eutrophication, but also due to the ability of Glyceria to grow faster than Phragmites
under extreme nutrient load, if enough P is available (Fig. 7).
The further common responses to N or N, P addition include the preferential allocation
of biomass to aboveground structures and changes in root and shoot morphology. Considering
roots, decrease of their average length, suppressed branching, and higher incidence of injuries
in response to nutrient enrichment were demonstrated in our experiments with Phragmites,
Glyceria (Steinbachová-Vojtíšková et al. 2006, Tylová et al. - manuscript 3), Acorus calamus
(Vojtíšková et al. 2004), and Typha angustifolia (Steinbachová-Vojtíšková et al. 2006). Such
morphological changes may significantly affect survival of individual wetland plants at their
natural habitats. As an example, shallow rooting might allow overcoming adverse conditions
of deep, reduced sediment layers, but stands are more susceptible to mechanical stress factors,
e.g. wave action, fish grazing, as the anchorage in the sediment is weakened. Plants in more
fertile conditions also showed higher incidence of young, short, unbranched, intensively
growing roots (Vojtíšková et al. 2004, Vojtíšková et al. 2006, Steinbachová-Vojtíšková et al.
2006), which might be a sign of high biomass turnover, which compensate the loss of
damaged biomass.
Considering shoot morphology, moderate N addition was shown to trigger an increase
in shoot heights and diameters (Lippert et al. 1999, Tylová et al. - manuscript 3), while plants
under excessive N dose usually possessed higher shoot numbers, but lower shoot lengths
(Hardej and Ozimek 2002, Tylová et al. - manuscript 3). Enhanced tillering in response to N
addition is triggered by high level of N rich compounds (particularly FAA) in plant tissues
(Saarinen and Haansuu 2000). In agreement, close correlation between tillering and leaf C/N
ratio and FAA status was observed in Phragmites and Glyceria in our experiments (Tylová et
al. - manuscript 3). Shoot morphology determine convective ventilation efficiency, thin shoots
generally posses lower flow rates (Rolletschek et al. 1999), which increase the risk of oxygen
shortage in belowground organs. Low shoot lengths may also impair plant ability to support
15
belowground parts with atmospheric oxygen under suddenly raised water level - and thus
survive occasional floods.
Besides morphological responses, high N availability triggers significant changes in
plant phenology, particularly the delay in transition from vegetative to generative phase and
the delay in autumnal shoot senescence. Altered phenology has consequences to internal
nutrient cycles, and particularly to formation of storages in over-wintering belowground
organs during autumn. Prolonged vegetative growth results in delayed translocation of
carbohydrates from shoots to belowground organs during autumn (Kohl et al. 1998,
Steinbachová 2006). The carbohydrate levels in belowground organs may also be reduced in
response to high N availability due to simple shift in plant C/N balance in favour of N
assimilation under these conditions (for summary see Steinbachová (2006)). In addition,
similar response may occur under NH4+-N provision, as indicated by field observations of
Phragmites australis (Kubín and Melzer 1996) and proved in our studies of Phragmites,
Glyceria (Tylová et al. - manuscript 1), and Acorus calamus (Vojtíšková et al. 2006). These
experiments showed significantly lower starch content in rhizomes of NH4+ compared to NO3
-
fed plants in Phragmites (Tylová et al. - manuscript 1) as well as Acorus (Vojtíšková et al.
2006), Glyceria reduced rather the content of soluble sugars in roots (Tylová et al. -
manuscript 1). From these results, we can conclude, that the shift in NH4+/NO3
- pore water
ratio in favour of NH4+ may per se participate on weakening of carbohydrate reserves in
plants at eutrophic habitats. These reserves are important prerequisite for over-wintering and
spring outgrowth of perennial wetland species. The reduction seems, however, not enough
conspicuous to endanger plants directly, but it increases plant susceptibility to occasional
stresses e.g. flood event or mechanical disturbance (Čížková-Končalová et al. 1992, Čížková
et al. 2001, Steinbachová 2006, Steinbachová et al. - submitted).
Similarly to carbohydrates, the delayed retranslocation of N-rich compounds
(particularly FAA) from senescing shoots and thus formation of N storages in belowground
organs was repeatedly observed at eutrophic stands (Kühl and Kohl 1993, Lippert et al. 1999).
Similar response was also found in Phragmites australis and Glyceria maxima treated with
high N, P availability in our experimental cultures (Tylová et al. - manuscript 2, Tylová et al. -
manuscript 3). It is, however, not completely clear, whether this delay really disrupts rhizome
N storage function, as plants generally respond to N enrichment by the increase of their N
status, which may compensate weak retranslocation under these conditions. The formation of
16
belowground N storages was therefore studied in more detail in our experiments, using
Phragmites australis and Glyceria maxima as the model species. The obtained data confirmed
the delayed onset of retranslocation in more fertile conditions, as the majority of N was
translocated to belowground organs very late in the season. The total amount of resorbed N
was, however, high compared to plants in oligotrophic treatment due to high aboveground N
standing stock accumulated during previous period of growth (Tylová et al. - manuscript 2).
These results indicate that the delay is not critical feature, at least under normal
circumstances. It may, however, cause a loss of aboveground N pool in situation of early
coming winter.
Furthermore, the efficiency of N retranslocation did not differ between oligotrophic
and eutrophic growth conditions in Phragmites or Glyceria in our study in spite of the fact,
that the process started significantly later in more nutritive treatment (Tylová et al. -
manuscript 2). The dead shoots, however, contained significantly higher amounts of N in
more nutritive treatment (Tylová et al. - manuscript 2) as similarly documented in different
wetland species at natural habitats (Kühl and Kohl 1993, Lippert et al. 1999). From this point
of view, N conservation is less efficient in plants grown under more fertile growth conditions,
as they do not realize their maximum (potential) resorption (Kohl et al. 1998, Lippert et al.
1999). N remaining in the litter also affects the quality and decomposability of the litter, and
thus nutrient cycles in wetlands. The decay of N-rich litter facilitates eutrophication, being the
substrate for intensive fungal and microbial colonization.
17
The summary of main original findings of the thesis:
Following basic aspects of N acquisition and utilization in Phragmites australis and
Glyceria maxima, this study showed several “primary nutritional” factors, which might affect
the response of individual wetland species to multiple change of habitat characteristics
triggered by eutrophication.
1. Preference for NH4+ indicated by NH4
+ and NO3- uptake kinetics.
Higher uptake capacity and affinity for NH4+ uptake observed in both species
indicated preference for this N source. Sufficient growth on NO3--N and high
activity of nitrate reductase, however, showed that the preference is obviously very
weak. This may correspond to the fact, that NH4+ prevalence in the littoral sediment
is not so strict and may change with water level fluctuation. Moreover, the
existence of finely branched aquatic roots allows the uptake of NO3- from the
oxidized water column (Tylová-Munzarová et al. - article 1; Munzarová et al.
2006 - article 2).
2. NH4+ and NO3
- uptake kinetics correspond with the growth strategy of the given
species.
High uptake capacity found in Phragmites and Glyceria is the typical feature of fast
growing species colonising fertile stands. Phragmites, however, exhibited lower
Km and higher affinity compared to Glyceria, characteristics advantageous under
nutrient limitation. This finding indicates a higher physiological plasticity to adjust
N uptake towards low nutrient availabilities, which agrees with field observations
describing the ability of Phragmites to colonise oligotrophic systems, where
Glyceria is not present (Tylová-Munzarová et al. - article 1).
3. Differences in the response to source of N (NH4+ versus NO3
-).
Glyceria but not Phragmites exhibited sensitivity towards higher levels of NH4+.
Commonly supposed high NH4+ tolerance of wetland plants as species colonising
NH4+ dominated habitats, is therefore not a completely general feature. Phragmites,
species rooting deeply into hypoxic NH4+ dominated sediment layers, seems more
tolerant to high external NH4+ availability. In contrast, Glyceria’s ability to colonise
fertile habitats rich in NH4+ is related rather to the avoidance strategy due to
18
shallow rooting, or the ability to cope with high NH4+ levels when enough P is
available or NO3- is co-provided. (Tylová et al. - manuscript 1).
4. High NH4+-N supply decreases cabohydrate levels in plants.
NH4+-N nutrition triggered the decline of internal carbohydrate levels in plants
(starch in rhizomes of Phragmites; soluble sugars in roots of Glyceria). NH4+
prevalence may thus per se participate on carbohydrate shortage of plants at
eutrophic habitats (Tylová et al. - manuscript 1).
5. N/P ratio determinates plant response to N surplus.
High N load (N/P ratio 100/1) caused negative growth response in both Phragmites
and Glyceria, similar N dose applied in N/P ratio 10/1 affected plant growth
positively. The importance of P was especially visible in Glyceria, which agrees
with the field observations describing well-developed Glyceria stands as indicators
of local sources of pollutions with high P contents in the sediment (Tylová et al. -
manuscript 3).
6. Nitrogen addition alters morphology of both species in the manner that could increase
plant susceptibility to stresses.
High N load (N/P ratio 100/1) triggered changes in biomass distribution and
morphology, which might affect ventilation efficiency, ability to survive high water
levels, plant anchorage or belowground storage capacity. The changes resembled
those observed at stand affected by eutrophication (Tylová et al. - manuscript 3).
7. Nitrogen addition delays but not disrupts formation of N storages in belowground
organs.
Phragmites and Glyceria showed delayed shoot senescence and retranslocation of
N-rich compounds into storage organs under N surplus. In spite of this fact, both
species accumulated higher N levels (and particularly higher N standing stock) in
belowground organs prior the winter in more nutritive treatment (Tylová et al. -
manuscript 2).
In summary, the study showed that many trays of plant performance at natural stands
and their responses towards eutrophication are underlined by physiological characteristics of
19
N acquisition and utilization. Although secondary impacts of eutrophication (litter
accumulation, phytotoxin occurrence) were repeatedly considered as more important factors
triggering plant damage at eutrophic stands, the primary nutritional aspects of N addition and
changes in NH4+ versus NO3
- occurrence have to be taken into account.
20
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Lippert I., Rolletschek H., Kühl H. and Kohl J.G. 1999. Internal and external nutrient cycles in stands of Phragmites australis - a model of two ecotypes. Hydrobiologia 408/409, 343-348.
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22
Abstracts of the included publications and manuscripts: The effects of NH4
+ and NO3- on growth, resource allocation and nitrogen uptake kinetics of
Phragmites australis and Glyceria maxima.
Tylová-Munzarová E., Lorenzen B., Brix H., Votrubová O. 2005.
Aquatic Botany 81: 326-342.
(article 1)
Abstract
The effects of NH4+ or NO3
- on growth, resource allocation and nitrogen (N) uptake
kinetics of two common helophytes Phragmites australis (Cav.) Trin. ex Steudel and Glyceria
maxima (Hartm.) Holmb. were studied in semi steady-state hydroponic cultures. At a steady-
state nitrogen availability of 34 µM the growth rate of Phragmites was not affected by the N
form (mean RGR = 35.4 mg g-1 d-1), whereas the growth rate of Glyceria was 16 % higher in
NH4+-N cultures than in NO3
--N cultures (mean = 66.7 and 57.4 mg g-1 d-1 of NH4+ and NO3
-
treated plants, respectively). Phragmites and Glyceria had higher S/R ratio in NH4+ cultures
than in NO3- cultures, 123.5% and 129.7%, respectively.
Species differed in the nitrogen utilisation. In Glyceria, the relative tissue N content was
higher than in Phragmites and was increased in NH4+ treated plants by 16%. The tissue NH4
+
concentration (mean = 1.6 µmol g fresh wt –1) was not affected by N treatment, whereas NO3-
contents were higher in NO3- (mean = 1.5 µmol g fresh wt –1) than in NH4
+ (mean = 0.4 µmol
g fresh wt –1) treated plants. In Phragmites, NH4+ (mean = 1.6 µmol g fresh wt –1) and NO3
-
(mean = 0.2 µmol g fresh wt –1) contents were not affected by the N regime. Species did not
differ in NH4+ (mean = 56.5 µmol g-1 root dry wt h-1) and NO3
- (mean = 34.5 µmol g-1 root
dry wt h-1) maximum uptake rates (Vmax), and Vmax for NH4+ uptake was not affected by N
treatment. The uptake rate of NO3- was low in NH4
+ treated plants, and an induction phase for
NO3- was observed in NH4
+ treated Phragmites but not in Glyceria. Phragmites had low Km
(mean = 4.5 µM) and high affinity (10.3 l g-1 root dry wt h-1) for both ions compared to
Glyceria (Km = 6.3 µM, affinity = 8.0 l g-1 root dry wt h-1). The results showed different
plasticity of Phragmites and Glyceria toward N source. The positive response to NH4+-N
source may participates in the observed success of Glyceria at NH4+ rich sites, although other
factors have to be considered. Higher plasticity of Phragmites toward low nutrient availability
may favour this species at oligotrophic sites.
23
Effect of NH4+/NO3
− availability on nitrate reductase activity and nitrogen accumulation in wetland helophytes Phragmites australis and Glyceria maxima.
Munzarová E., Lorenzen B., Brix H., Vojtíšková L., Votrubová O. 2006.
Environmental and Experimental Botany 55: 49-60.
(article 2)
Abstract
The effect of NH4+/ NO3
- availability on nitrate reductase (NR) activity in Phragmites
australis and Glyceria maxima was studied in sand and water cultures with the goal to
characterise the relationship between NR activity and NO3- availability in the rhizosphere and
to describe the extent to which NH4+ suppresses the utilization of NO3
- in wetland plants.
The NR activity data showed that both wetland helophytes are able to utilize NO3-. This
finding was further supported by sufficient growth observed under the strict NO3- nutrition.
The distribution of NR activity within plant tissues differed between species. Phragmites was
proved to be preferential leaf NO3- reducer with high NR activity in leaves (NRmax > 6.5 µmol
NO2- g dry wt –1 h-1) under all N treatments, and therefore Phragmites seems to be good
indicator of NO3- availability in flooded sediment. In the case of Glyceria the contribution of
roots to plant NO3- reduction was higher, especially in sand culture. Glyceria also tended to
accumulate NO3- in non-reduced form, showing generally lower leaf NR activity levels. Thus,
the NR activity does not necessarily correspond with plant ability to take up NO3- and grow
under NO3--N sources. Moreover, the species differed significantly in the content of
compounds interfering with NR activity estimation. Glyceria, but not Phragmites, contained
cyanogenic glycosides releasing cyanide, the potent NR inhibitor. It clearly shows that the use
of NR activity as a marker of NO3- utilization in individual plant species is impossible without
the precise method optimisation.
24
Different sensitivity of Phragmites australis and Glyceria maxima to high availability of ammonium-N.
Tylová E., Steinbachová L., Votrubová O., Lorenzen B., Brix H.
Acceptad in Aquatic Botany.
(manuscript 1)
Abstract
The ability to cope with NH4
+-N was studied in the littoral helophytes Phragmites
australis and Glyceria maxima, species commonly occupying fertile habitats rich in NH4+ and
often used in artificial wetlands. In the present study, Glyceria growth rate was reduced by
16% at 179 µM NH4+-N, and the biomass production was reduced by 47% at 3700 µM NH4
+-
N compared to NO3--N. Similar responses were not found in Phragmites. The amounts (mg g-
1 dry wt) of starch and total non-structural carbohydrates (TNC) in rhizomes were
significantly lower in NH4+ (8.9; 12.2 starch; 20.1; 41.9 TNC) compared to NO3
- treated
plants (28.0; 15.6 starch; 58.5; 56.3 TNC) in Phragmites and Glyceria, respectively. In
addition, Glyceria showed lower amounts (mg g-1 dry wt) of soluble sugars, TNC, K+, and
Mg2+ in roots under NH4+ (5.6; 14.3; 20.6; 1.9) compared to NO3
- nutrition (11.6; 19.9; 37.9;
2.9, for soluble sugars, TNC, K+, and Mg2+, respectively), while root internal levels of NH4+
and Ca2+ (0.29; 4.6 mg g-1 dry wt, mean of both treatments) were only slightly affected. In
Phragmites, no changes in soluble sugars, TNC, Ca2+, K+, and Mg2+ contents of roots (7.3;
14.9; 5.1; 17.3; 2.6 mg g-1 dry wt, means of both treatments) were found in response to
treatments. The results, therefore, indicate a more pronounced tolerance towards high NH4+
supply in Phragmites compared to Glyceria, although the former may be susceptible to starch
exhaustion in NH4+-N nutrition. In contrast, Glyceria’s ability to colonise fertile habitats rich
in NH4+ is probably related to the avoidance strategy due to shallow rooting or to the
previously described ability to cope with high NH4+ levels when P availability is high and
NO3- is also provided.
25
Phenology and autumnal accumulation of N reserves in belowground organs of wetland helophytes Phragmites australis and Glyceria maxima affected by nutrient surplus.
Tylová E., Steinbachová L., Votrubová O., Gloser V.
Submitted to Environmental and Experimental Botany.
(manuscript 2)
Abstract
Two co-occurring dominant wetland helophytes and potential competitors, Phragmites
australis and Glyceria maxima, were cultivated under N, P availabilities simulating the
trophic status of wetlands with different fertility (oligo- and eutrophic). The long-term
outdoor cultivation was performed with the goal to characterise the extent to which the
nutrient enrichment affects plant growth, phenology, and particularly, the accumulation of N
storage compounds in belowground organs of wetland rhizomateous plants prior the onset of
winter dormancy. Selected species differ in their dormancy requirement and sensitivity
towards severe eutrophication, but in the present study, they responded similarly towards
nutrient surplus. The enhanced growth, delayed shoot senescence, and delayed retranslocation
of N into belowground organs were found in more fertile treatment. Furthermore, N levels
remaining in dry leaves were proportionally related to those of living ones, being significantly
higher in eutrophic treatment. The efficiency of N retranslocation from senescing leaves was
60% in both species and treatments. The formation of N reserves was, however, not disrupted
in either species. Although plants in eutrophic treatment accumulated N in their belowground
organs significantly later in the season (in period September-December), the amount of
accumulated N was sufficient to reach high belowground N standing stock. Therefore, the lost
of N with N-rich litterfall, observed repeatedly at eutrophic habitats, obviously does not
increase risk of N shortage in over-wintering organs in these particular species, but rather
endangers plants indirectly, as the decay of N-rich litter facilitates eutrophication of the given
habitat.
Considering formation of N reserves, the differences in species response to treatments
were negligible. Phragmites and Glyceria accumulated similar belowground N standing stock
prior the winter. Glyceria may, however, additionally profit from N-standing stock of over-
wintering green leaves and from the potential of growth and N assimilation during mild
winter period, which is not possible in fully dormant Phragmites.
26
Performance of Phragmites australis and Glyceria maxima under changing nitrogen and phosphorus availability. I. Plant growth, resource allocation and C/N balance.
Tylová E., Steinbachová L., Soukup A., Votrubová O., Gloser V.
Submitted to Environmental and Experimental Botany.
(manuscript 3)
Abstract
Worldwide, freshwater wetlands undergo changes in species composition, being
affected by eutrophication. The complexity of the process makes the importance of individual
causal factors difficult to discern in nature. The present study follows the nutritional aspects
of eutrophication in long-term sand cultures of two co-occurring littoral grasses. These
grasses, Phragmites australis and Glyceria maxima, are fast-growing helophytes of fertile
habitats, with different sensitivities towards highly nutrient-loaded habitats. Both species
showed enhanced growth in response to eutrophic, and even hypertrophic, growth conditions
(substrate N/P ratios 26/1 and 10/1, respectively), but displayed considerable sensitivity to an
unbalanced high N load (substrate N/P ratio 100/1). The lower biomass production, along
with the appearance of signs of stress, changes in resource allocation and morphology (e.g.
high shoot density; low shoot diameters and heights; reduced root and rhizome growth)
observed in this treatment, resemble those found at reed die-back sites. Changes in
morphology and resource allocation were correlated with biomass C/N ratios, but low free
amino acids levels in the belowground organs of eutrophically treated plants in late summer
indicated a delayed accumulation of storage N compounds. The effects of nutrient
availabilities on carbohydrate levels were followed, in detail, in a separate study
(Steinbachová et al., submitted as Part II).
Our findings show that the response of littoral vegetation to eutrophication,
considering nutritional aspects per se, is highly dependent on the sediment N/P ratio, and that
the unbalanced high N load alters plant morphology and performance in a manner that could
increase plant susceptibility to stresses (altering e.g. ventilation efficiency and ability to
survive high water levels; plant anchorage and belowground storage capacity). As the positive
effect of high P supply was especially visible in Glyceria, this species may be favoured in
competition with Phragmites at highly fertile habitats rich in P. But generally, the ability of
G. maxima to colonise heavily loaded sites is related to the avoidance strategy of shallow
rooting, rather than to the tolerance of high N load, per se.