Water transport in submerged macrophytes - Bio-WEB

Aquatic Botany, 44 (1993) 385-406

385

Elsevier Science Publishers B.V., Amsterdam

Water transport in submerged macrophytes Ole P e d e r s e n a n d K a j S a n d - J e n s e n

FreshwaterBiologicalLaboratory, Universityof Copenhagen, 51 Helsingorsgade, DK.3400 Hillerod, Denmark (Accepted 28 August 1992)

ABSTRACT Pedersen, O. and Sand-Jensen, K., 1993. Water transport in submerged macrophytes. Aquat. Bot., 44: 385-406. Compared with their terrestrial ancestors, submerged angiosperm species have a reduced but, nevertheless, weU.structured vascular tissue containing xylem, phloem and an endodermis with casparian strips, thought to be important for a regulated flow of ions and water. The historical data on acropetal water transport from roots to leaves are inconclusive because ofexperimental deficiencies. However, an analysis of the data suggests that acropetal transport occurs at low rates probably confined to the vascular tissue and that roots as well as leaves are important for the flow through the stem. We applied tritiated water as a sensitive tracer to determine acropetal water transport in nine macrophyte species mounted in two-chamber compartments subjected to light/dark cycles under strict temperature control. We confirmed that acropetal water transport takes place at rates markedly faster than is possible by passive diffusion. Variable transport rates among species showed no simple relationship to the quantity of vascular tissue. Relatively fast isotopic equilibrium of the water pool engaged in acropetal water transport (0.5-2 days) for six oftbe species suggested that only the vascular tissues were involved. Our measurements showed that roots alone could support acropetal flow in the stem, but intact plants generated much faster rates. The transport depended on light, and thus presumably on photosynthetic products and energy, and declined during prolonged darkness. The tritiated water was mainly incorporated into young leaves in active growth. This finding supports the general idea that the main role of acropetal water transport is to translocate inorganic nutrients absorbed by roots in the sediment to above-ground shoots and leaves. The measured water transport could theoretically support the maximum observed growth rates ofsubmergad macrophyte species applying typical concentrations of nitrogen in sediments and reported abilities of roots of water plants to absorb and concentrate nutrient ions.

INTRODUCTION T e r r e s t r i a l v a s c u l a r p l a n t s t r a n s p o r t l a r g e a m o u n t s o f w a t e r in x y l e m d u c t s f r o m r o o t s t o l e a v e s . T h i s m a s s t r a n s p o r t is d r i v e n b y e v a p o r a t i o n f r o m l e a f surfaces and can be supplemented by active transport of ions across the en-

Correspondence to: Ole Pedersen, Freshwater Biological Laboratory, University of Copenhagen, Helsingersgade 51, DK-3400 Hillered, Denmark.

© 1993 Elsevier Science Publishers B.V. All rights reserved 0304-3770/93/$06.00

386

O. PEDERSEN AND K. SAND-JENSEN

dodermis of the roots leading to an osmotic root pressure (Zimmermann, 1983). This, in turn, may result in bleeding from the surface of detopped stems and water release from the distal ends of main veins in leaves growing in a water-saturated atmosphere, a phenomenon known as guttation (Bidwell, 1974). Nutrient ions absorbed by the roots are dissolved in the xylem sap, and translocated to the shoot along with the water. A classical question is whether long distance acropetal transport of water from roots to leaves also takes place in water plants (Gessner, 1956; Sculthorpe, 1967; Raven, 1984) and, if so, which mechanism is responsible for the transport, at which rate it occurs, and in which tissue is it located. This question was examined at the end of the last century and at the beginning of this century (Unger, 1862, Sauvageau, 1891; Hochreutiner, 1896; Pond, 1905) until interest gradually vanished in the 1930-1940s, apparently because of lack of appropriate methods. Reviews of these old experiments (Sculthorpe, 1967; Hutchinson, 1975; Raven, 1984) conclude that submerged macrophytes probably do transport water but that experiments are contradictory and difficult to interpret. Several text books mention that the vascular tissue is reduced in water plants compared with terrestrial species (Arber, 1920; Sculthorpe, 1967; Wetzel, 1983). Although this might be expected and seems likely on the basis of published microscopy work, there are no quantitative comparisons to confirm this statement. For example, whether the number or the cross-sectional area of vascular elements supporting a leaf of given weight or production is smaller in aquatic than in terrestrial species has remained untested. Many aquatic plants are recent invaders from terrestrial environments and some have maintained the ability to grow exposed to air. These amphibious species, at least, have a well-developed vascular system (Sculthorpe, 1967; Hostrup and Wiegleb, 1991 ). Submerged macrophytes have fewer tracheary elements with lignified walls than do terrestrial plants (Sculthorpe, 1967). Because water transport in aquatic plants can not be generated by evaporation, however, there is probably no underpressure in the xylem and investments in lignified walls are consequently not needed. On the other hand, the endodermis with casparian strips is often as well-developed in aquatic as in terrestrial plants (Sculthorpe, 1967; O. Pedersen and K. Sand-Jensen, unpublished data, 1991 ). The endodermis is thought to regulate the flow of ions and the accompanying flow of water from the root medium via the cortex to the vascular bundles located inside the endodermis (BidweU, 1974). Back_flowis only possible through the endodermal cells because of the casparian strips. The structural basis for a well-developed acropetal flow of ions and water is therefore present in aquatic plants. Internal water transport is also needed to explain observed rates of nutrient uptake from the sediments, which have been reported as the main source for uptake of N, P, Fe, Mn and micronutrients (Denny, 1980; Barko and Smart,

WATERTRANSPORT IN SUBMERGEDMACROPHYTES

38?

1981; Barko et al., 1991 ). Particularly P, Fe and Mn tend to precipitate and are usually present in extremely low ionic concentrations in the oxygenated water column. Ions of relatively abundant salts (e.g. Ca 2+, Mg2+, Na +, K +, CI-, HCO~ and SO2-) are largely supplied from the water (Denny, 1980; Barko et al., 1991 ). In nutrient-rich riverine systems (e.g. Danish lowland streams with typically 200-500/~M NO~"-N, 10-100/tM NI-L~-N and 2-10 /~M PO 3--P; Kristensen et al., 1990), it is likely that N is also absorbed from the water whereas P may still be supplied mainly from the sediment. For example, Chambers et al. (1989) reported that submerged stream macrophytes obtained more than 70% of total P uptake even from rather infertile coarse sediment. In laboratory experiments, macrophyte biomasses of I kg dry weight (DW) m -2 can be achieved on sediments with practically no N and P in solution in the water phase (Smart and Barko, 1985). Considering further that several elodeid species, in lakes with extremely low water column concentrations of N and P, can reach canopy heights of 3 m, it is likely that active acropetal transport is needed to supply the apical parts. Indeed, the fast nutrient translocation rates reported in some tracer experiments (Welsh and Denny, 19"/9a~b) can not be explained by diffusion alone. Acropetal water flow may also be influenced by the growth form of macrophytes. Isoetid species have large root systems (Raven et al., 1988) and welldeveloped vascular bundles (Hostrup and Wiegleb, 1991 ), apparently able to support high transport rates. On the other hand, isoetids have a low stature and grow slowly (Sand-Jensen and Sendergaard, 1978 ), presumably reducing the need for fast translocation. Elodeid species have long stems, grow fast (Nielsen and Sand-Jensen, 1991 ), but have relatively fewer roots (Westlake, 1963) and vascular tissue ~see later) to supply the shoot. If root uptake predominates, flow velocities in the stem may therefore be fast. We set out to examine the rate of acropetal water transport in nine submerged vascular macrophytes using tritiated water (THO) supplied around the roots in a two-chamber system. Following Raven (1984), we hypothesized that acropetal flow, if present, would result from an osmotic and hydrostatic pressure located primarily inside the root endodermis (Miinch, 1930), a mechanism also used to explain root pressure in terrestrial plants (Bidwell, 1974). We therefore hypothesized that metabolic processes would be needed to sustain the flow and that it consequently would decline under prolonged darkness without the photosynthetic input of carbohydrates. Because the most likely function of acropetal water flow would be to supply nutrients and perhaps hormones produced in the roots (Waisel and Shapira, 1971 ) to tissues in active growth (e.g. young leaves), THO should predominantly be diverted to these tissues. We examined acropetal water flow among species of different growth forms and related it to the cross-sectional area of the vascular tissue acknowledging that, at present, we have not shown which tissues are involved in the transport

388


and that no clear expectations of relationships can be established based on theoretical considerations. Because there is no recent work on water transport and the historical literature is extremely difficult to obtain, read (German and French texts) and digest (obscure units), we feel it justified to review the basic background before presenting our own experimental findings.

Review of historical data Water transport in aquatic macroph~es has been measured in the past by qualitative (dyes) and quantitative techniques. Hochreutiner (1896) followed movement of eosin in stems with the cut bases in the dye solution and the remainder of the shoot in pure water. For Batrachium aquatile, Potamogeton crispus, P. densus and P. pectinatus he observed up to 15 cm ofacropetal transport in the main and lateral stems after 1-2 days (flow velocities 1.32.1 cm h - ~). Basipetal movement was negligible. In P. densus and P. pectinatus the dye was confined to the vascular bundles. Snell (1908) showed that Elodea densa and P. densus absorbed Fe (Cn) 2 and that a blue colour reaction occurred in the vascular tissue following addition of FeCl2. The colour was intense at the nodes where the meristematic activity is high. Thoday and Sykes (1909) found eosin absorption from a cut stem surface of Potamogeton lucens L. and acropetal movement at very high velocities (342-570 cm h "~). Removing the apex or some of the leaves reduced the velocity drastically. Students of these two authors observed much lower flow velocities at 2.2 cm h - ~, in better correspondence with Hochreutmer ( 1896 ). Wilson ( 1947 ) observed eosin movements in stem pieces of Batrachium baudotii at 5-8 mm rain- t over 45 min ( -,, 23-36 cm h-~ ). If stems were prepared 10-15 h before experiments, eosin uptake was very small. These applications of dyes suggest that water transport occurs in the vascular bundles, perhaps at very variable velocities. However, exposing cut stems to a dye may result in uptake as a result of infiltration of gas spaces. Also pressure differences as a result of temperature differences among different parts of the shoots may induce flow. Finally, the dramatically falling velocities over time may either be interpreted as the reduction of the motive force or suggest that the high initial rates were experimental artifacts. Around the turn of the century, Von Minden (1899) and Weinrowsky (1899) described hydathodes and apical openings of large veins at leaf apices in several submerged angiosperms including Callitriche autamnalis Kiitz. and Potamogeton praelongus Wulfen. When leaves were placed in a water-saturated atmosphere droplets formed at the apical openings within a few minutes. We observed the same guttation phenomenon for three of the species included in this investigation (Littorella uniflora, Lobelia dortmanna and Sparganium emersum). These species have vascular bundles which open as

WATERTRANSPORTIN SUBMERGEDMACROPHYTES

389

pores or pappils at the leaf tips (Hostrup and Wiegleb, 1991; O. Pedersen and K. Sand-Jensen, unpublished results, 1991 ). Observations demonstrate that the vascular sap is under pressure, most probably exerted by the below-ground parts. Previous quantitative determinations of water flow in submerged macrophytes have been based on volumetric or gravimetric measurements with intact plants or plant portions. These measurements are summarized in Table I. Several potential errors are involved including gas exchange, infiltration of gas spaces, and flow induced by pressure differences. Unfortunately, the authors do not use a unifying parameter (e.g. plant weight, cross-sectional area of the stem or vascular tissue) which would allow us to scale water transport to the same unit for different species and experimental situations. The variability of transport rates is very large, 0.3-6030/~l per plant h-~. However, we can perhaps exclude the very low rates ( < 0.6/A per plant h - ~) observed by Sauvageau ( 1891 ) because he used abcissed stems, applied an over-pressure to t~e stem surface and reports contradictory results. The high rate (6030/A per plant h -l ) reported for Elodea canadensis by Vardar (1950) is dubious because he used cut stem sections infiltrated with water under vacuum to avoid the influence of gas channels. Perhaps the high rates resulted from O2 production forcing water out of the infiltrated gas channels. High rates for the 'land plant' Nomaphila stricta (2100/~1 per plant h'~; H6hn and Ax, 1961 ) are probably real, but presumably not representive for truly submerged species. TABLE I

Rates of acropetal water transport of submerged macrophytes based on literature values Species

Potamogeton crispusL.

Water transport Plantsize Reference (/zl per plant h - ~)

0.3 8.3 Potamogetonpusillu~ L. 0.3 Potamogeton perfoliatus L. 0.4 Potamogeton densus L. 0.6 Potamogeton :osterifoliusSchum. 2.6 MyriophylhonspicatumL. 3.5 Batrachium baudotii (Gordon) F. Schultz 4.8 10.0 Batrachium circinatum (Sibth.) Spach 6.3 Elodeadensa (Pianch.) Casp. 6.5 8.6 Potamogeton lucens L. 9.6 11.0 Potamogeton nodosusPoir. 11.0 Batrachiumaquatile(L.)Dum. 210.0 NomaphilastrictaNees 2100.0 Elodea canadensis Michx. 6030.0

ND 126 cm 2 ND ND ND 75 cm 56cm 24 leaves 60cm 59 cm 63cm 65 cm 63 cm 65 cm 69 cm 20era 20cm 50 cm

S~uvageau Unger Sauvageau Sauvageau Sauvageau Thut Thut

( 1891 ) (1862) ( 1891 ) ( 1891 ) ( 1891 ) ( 1932)~ (1932) 1 Unger (1862) Wilson (1947) Thut ( 1932 ) I Thut (1932) t Thut (1932) 2 Thut ( 1932)~ Thut (1932) 2 Thut (1932) i Pond (1905) 2 H6hn and Ax (1961) 2 Vardar (1950) 3

tGravimetric method, 2volumetric method, 3basipetal transport rate, ND, not determined.

390

o. PEDERSEN AND K. SANDoJENSEN

Nomaphila stricta held submerged absorbed more water by the roots in the light than in the dark (HShn and Ax, 1961 ) and diel patterns were smooth and reproducible. Increasing CO2 tension around the shoot doubled all rates whereas continued darkness over 2 days reduced water absorption, suggesting a relationship to energy metabolism and growth. Omitting these references reduces the more likely range for submerged species to 2.6-210/d per plant h - t with 11 of 12 measurements confined to the narrow interval 2.6-11/tl per plant h - i. These rates are actually so small that more sensitive techniques (e.g. tracer technique) should be applied to obtain more accurate measurements. Overall, the old literature suggests that acropetal water transport does take place, coupled to metabolic processes, perhaps via osmotic phenomena in the roots as well as in the shoot. Observation of sustained guttation from apical parts enclosed in a water-saturated atmosphere is a strong indieium for the reality of the phenomenon. Expe~ments with intact plants clearly reduce the number of potential artifacts that can affect the measured transport rates, and close temperature control is evidently needed to avoid pressure differences between shoot and root compartments and following light/dark switches. MATERIALS AND METHODS

Experimental plants and design Actively growing plants were collected from Danish localities between May and September. Plants were collected with great care to avoid tissue damage. Fully intact plants (mainly from coarse-textured sediments) were acclimated to experimental conditions (see below) for l day. Plants with slight damage of the root system were potted in sand and grown under the experimental conditions for more than 1 week to ensure healing of wounds. Water transport was measured in two-compartment Perspex chambers using tritiated water (THO) as a tracer (Fig. 1 ). The upper compartment contained the shoot and the lower compartment contained the root system. Compartment volumes (80-500 ml) were adjusted to plant size. The two compartments were separated by a perforated plate and sealed with waterfree lanoline and cacao butter applied as a fluid at 25 °C and hardening at the experimental temperature ( 12 °C). The seal was tested (without plants over 5 days) and proved impermeable to THO. A thin vertical glass tube was attached to the root compartment (Fig. 1 ). This tube had two functions. If the seal was intact, the water level in tbe tube, shortly after mounting the plant, would be slightly above the seal because of capillary forces. If the seal was leaky, the water level in the tube would initially be above the water level of the shoot compartment (i.e. total water head+capillary forces). Net movement of water between the two compartments during the experiment would

WATERTRANSPORT IN SUBMERGED MACROPHYTES I'

.

~wi,

391 I

IB

"

I _ _ _ _ -~ . . . . I

JMUW,

"

._1

Fig. 1. Experimentalset-up (A) Temperature constant water bath (t) with two-chamberperspex chamber (h) with glass-tubesfor air bubbling (a) and pressure equilibration (o) and the hole for sampling (e). (B) details of the seal between shoot and root compartments showing the perforated perspex plate (p) the stem (s), water-free lanoline (1) and hardened cacao butter (c). result in pressure differences which would be counteracted by changes o f the water level in the tube. The shoot compartment was open to the atmosphere and was slowly bubbled with humidified air to ensure mixing and Oe and COe concentrations close to air equilibrium.

392


Experimental water for the freshwater species (except Lobelia dortmanna and Littorella uniflora) was alkaline tap water (4.6 mM dissolved inorganic carbon) enriched with 480/zM NO£-N and 100/~M PO 3--P. For Lobelia dortmanna and Littorella uniflora the medium was diluted with an equal volume of distilled water. Eelgrass was submerged in filtered brackish water from the collection site and enriched with N and P as above. The chambers were submerged in a temperature-constant water bath (12.0_+0.4°C) and illuminated by fluorescent tubes at 90/zmol m -2 s-~ ( 16 h light/8 h dark cycle).

Acropetal transport in nine species To measure acropetal transport the plants were mounted in the chambers and acclimated for 6 h in the light before THO (1.1 × 105 to 1.2× 106 dpm ml- ~ final activity) was added to the root compartment before the dark period. Every 16 or 8 h, at the onset of light or dark, triplicate 1.00 ml water samples were retrieved from the shoot compartment and radioassayed. All experiments were in triplicate and continued for 5 days. Plants were then rinsed, weighed and homogenized in distilled water 20 times the volume of water in the plant. Water content and dry weight of plants were measured on parallel samples. In some experiments, plants were separated into roots, stem and leaves of different ages. Water samples and homogenates ( 1 ml) were mixed with 10 ml of Aqualyte® (Baker Chemicals, UK) and counted in a liquid scintillation counter (Wallac, Rack Beta 1219). Separate quench curves were constructed for water samples (quench agent CC14) and for coloured plant homogenates (chlorophyll extracts).

Acro- and basipetal transport and light dependency To evaluate the role of roots or shoot as driving force for acropetal THO transport in Batrachium trichophyllum the stem was cut at the base. In one experiment, the roots were mounted with 1 cm of the stem sticking into the shoot compartment. In the other, the shoot was mounted with 1 cm of the stem exposed to the root compartment. In both experiments, THO was added to the root compartment and THO release to the shoot compartment was followed over 5 days. Basipetal and acropetal transport of Sparganium emersum were compared in intact plants over 5 days. The role of light for acropetal water transport was also evaluated by keeping the plant in the usual light/dark cycle for 96 h, while frequent THO samples were retrieved from the shoot compartment. The plant was then kept in the dark for 48 h and finally shifted to continuous light for another 61 h.

WATER TRANSPORT IN SUBMERGED MACROPHYTES

393

Microscopy The stem ~ ~'aetransition between above- and below-ground parts was fixed under vacuum in 3% glutaraldehyde (24 h) buffered to pH 7.0 (0.1 M phosphate buffer). The material was rinsed in the phosphate buffer and dehydrated sequentially over 3 days in 2-methoxyethanol, 99% ethanol, n-propanol and n-butanol. Then the tissue was infiltrated with glycol methacrylate and cut on a rotation microtome (Reichert) at a thickness of 3 _+1/an, stained with toluidine blue and mounted in DePeX (BDH Chemicals, France). The material was photographed at 40-400 × magnification under a microscope (Reichert). Cross-sectional areas of the stem and the vascular bundles were quantified on the photographs.

Calculations Water transport rates (Q) to the shoot compartment via the plant (M, dry mass) were calculated as

Q Vt(Sst(Sst-IFt)) where V.. is the time-dependent water volume in the shoot compartment, Ss, and Sst_ ~ is the time-dependent specific activity of THO in the water of the shoot compartment at time t and t - 1, respectively, Ft is the dilution factor (close to 1 ) caused by water addition to the compartment after sampling to maintain a constant volume and Sr is the specific activity of THO as added to the root compartment. Cumulative uptake and transport of water after 5 days was calculated from the activity of THO in the plant when harvested and the final activity in the shoot compartment corrected for removal of THO during the experiment. RESULTS

Acropetal water transport in nine species Acropetal water transport and subsequent release to the shoot compartment was calculated on the basis of movements of THO. All nine species showed a clear time lag of THO release to the shoot compartment from the initially unlabelled plant (Fig. 2). Species with high transport rates reached constant release of THO after 0.5-2 days whereas species with low transport rates showed linearly increasing THO release over the 5 days of the experiment. Constant rates of release should be obtained when the specific activity of THO is the same in the released water as in the root compartment and this should occur more quickly when transport rates are high. This situation is

394

O. PEDERSEN

AND

K. SAND-JENSEN A

~o

oO

~t

~

•~

.~

o ,g

, g, , -~

~

o

F

,~

g

~~ _ g_• )

o

iv.

g

g

~

g

,o

i ~~-~ O,.cl

E ~o O

=

=ram

L i

,

|

,

i

i

!

,

L-q M(] ),ueld¢_60ZH Irl

|

,

|

2~ •= ~

N

WATERTRANSPORTIN SUBMERGEDMACROPHYTES

395

also reached much more quickly than the time needed to obtain homogeneous labelling of the entire water pool in the plant, strongly supporting the idea that activated water transport is confined to specific parts of the plant (i.e. the vascular tissue). For Batrachium trichophyllum, for example, constant release rates were about 50/~l H20 g- l plant DW h - ' and equal labelling ofthe water pool engaged in water transport rakes about 12 h. Without any dilution this water pool would be about 600/zl and this is just a small fraction of the entire water volume of about 9000/d g- ' plant DW. Because THO probably does not move as a sharp front up through the plant, the water pool engaged in transport is actually smaller. The mean acropetal water transport rate was calculated when maximum rates of THO release to the shoot compartment had been attained. Thereby, we avoided the initial lag phase when isotope dilution was large. Values were averaged for the three individual plants. To facilitate comparisons, transport rates were expressed per dry mass of the entire plant, the shoot and the root system (Fig. 3). Differences among species were pronounced. Water trans-

o.3

0.1 0 ~ o~

i 0.4 0.3 0.2 0.1 0 T= 3.0 ~ 2.o ~ 1.0 0

Fig. 3. Acropetai water transport from root to shoot compartment recorded at the time of maximum rates ( Fig. 2 ). "values are based on total plant (A), shoot ( B ) and root DW (C) and were averaged ( _+SD) over three individual plants. Release in the light ( [] ) and dark periods (ll).

396

O. PEDERSENAND K. SAND-JENSEN

TABLE 2 Rates ofacropetal water transport of nine submerged macrophytes based on plant, shoot and root dry weight Species

Zostera marina L. Myriophylhon rerticillatum L. Elodea canadensis Michx. Littorella uniflora (L.) Ascherson Batrachium trichophylhon (Chaix)

/d H20 g - ~ plant DW h - ~

#1 H , O g - ~ shoot DW h - ~

/zl H , O g root DW h - i

7+ 1 16_+ 3 11 .+_4 41 +21 56 + 28

10_+ 1 18 + 3 12 _+4 181 +93 63 + 31

21 + 8 165 + 30 146 + 12 53+28 491 + 264

109 _+87 122 + 25 143 _+.40 127 + 55

123 _+98 145 + 30 195 _+52 ! 47 + 64

910 + 771 + 672 + 949 +

Van den Bosch

Potamogeton pectinatus L. Sparganium emersum Rehmann Lobelia dortmanna L. Potamogeton crispus L.

724 175 257 353

Mean values + SD of triplicates. Rates are calculated from T H O released to the shoot chamber plus the internal pools of T H O in the plant at the end of 5 days incubation.

port was particularly low for Zostera marina and varied between 3 and 308 /zl H20 g- t plant DW h - t for the remaining species. The variability among species was equally large on the basis of shoot and root dry weight. However, the position of the individual species in the comparison changed somewhat. We did not find significant differences in water release between the light and the dark for seven of the nine species in the 16 h light/8 h dark cycles. Sparganium emersum, however, showed higher release in light and P. crispus showed higher release in dark. Water transport rates were lower when calculated on the basis of the cumulated amount of THO transferred to the shoot compartment plus the amount absorbed in the plant over 5 days (Table 2) because of the time lag of THO release in the beginning of the experiment (Fig. 2). The only exception was Zostera marina because it released very small amounts of THO and thus had most THO within the plant.

Role of shoot and roots in acropetal transport Acropetal transport in Batrachium trichophyllum was much higher for separate root than shoot systems (Fig. 4) suggesting that roots are particularly important for the flow. Transport rates for the root system declined over the 5 days, perhaps because of reduced energy status, whereas the shoot maintained constant but low rates (the shoot can produce energy via photosynthesis). Furthermore, damaged tissue may have an increasingly impeding effect over experimental time. As mentioned previously, however, experiments with plant sections are problematic. Here, for example, the separate shoot

397

WATER TRANSPORT IN SUBMERGED MACROPHYTES

Ta:: 1.2

~''"~"

° ' L"~ ~

A

0.4 0.8

o

~ )',= 400

B

"3

o "3

" 1:2 I¢~ e.,

_"°:Oo. ~,oo [ o 2

3 Time (d)

4

5

Fig. 4. Batrachium trichophyllum: the role of shoot and roots for acropetal water transport. (A) Release of water (based on T H O ) to the shoot chamber during 5 days of incubation for the root system only ( I"1, shoot removed ) and the shoot only ( I , roots removed ). (B) Same as (A) but expressed as release per g root or shoot DW. (C) Same as (B) but values are for intact plants. Values are means + SD of three plants.

system is only exposed to THO via the cut basal stem surfaces and this may contribute to the small rates. Experiments with intact plants (Fig. 4) showed 19-times higl~er transport rates than the separate shoot system (both expressed per unit of shoot DW) and about three times higher rates than the separate root system (both based on root DW).

Internal distribution of THO After 5 days incubation of Lobelia dortmanna with the roots exposed to THO, all leaves had a significantly higher specific activity of THO than did the water surrounding them (Fig. 5). The leaves also had a markedly higher specific activity than the stem did. The young, actively growing leaves contained more THO than the mature, non-growing, leaves did. The specific activity in the young leaves after 5 days was 30% of that in the root compartment to which THO was initially added.

398


1.0 •~ 0.8

g

0

~

0.6 0.4

"~ o.2

w

Fig. 5. Lobetia dortmanna: relative specific activity o f T H O in the stem and young and old leaves after 5 days o f incubation to that in the root compartment where T H O was added. Values are m e a n s + SD o f three plants.

A similar pattern was found for Spargamum e~:ersum (Fig. 6). The specific activity of THO in the youngest leaf after 5 days was 55% of that in the root compartment and it was lower in older leaves. When THO was added to the shoot compartment we found lower specific activities in the leaves, no dependency of leaf location, and no measurable activity in the root. This shows that basipetal flow of THO was negligible.

Acropetal transport during prolonged light or darkness We examined the influence of prolonged light and darkness on acropetal water transport in Sparganium emersum by following the THO concentration in the shoot compartment (Fig. 7). At the end of the initial 3 day period in the 16 light/8 h dark cycle, the plant released 7.7/tl H20 h-~. After the 72 h dark period the release had dropped significantly to 2.9/d H20 h - ~. During the final 48 h of continuous light, water release again increased significantly to 6.0/zl H20 h - ~.

Development of the vascular system and relationships to waterflow The nine species are markedly different in growth form and quantitative development of the vascular tissue. Littorella uniflora, Lobelia dortmanna and Sparganium emersum are amphibious species which have short thick stems

399

WATER TRANSPORT IN SUBMERGED MACROPHY'rES

1.0

A

.~o.s

L

~

o

'~

o

~ 1.0

~0.5

0 o•~

o

Fig. 6. Sparganium emersum: relative specific activity o f T H O in the stem and leaves of different ages after 5 days of incubation to that in the source. (A) THO added to the root compartment. (B) THO added to the shoot compartment. Values are means + SD of three plants.

1.0

6.0 pl HaOplant'~ h°V ~ / /

o.e

2.9 tJl H

2

~

_f.

0.6

Z ~¢ 0.4 •~ o~

s (3

0

J 0

~ - - 7.7 pl I~0 plant"1 h"! I

I

I

I

50

100

150

200

Time(hi

Fig. 7. Sparganium emersum: cumulated water release to the shoot compartment over 8 days o f incubation with THO added to the root compartment. Three periods with different treatments are shown: ( l ) light/dark cycle ( o • ), (2) darkness ( • • ), ( 3 ) light ( o o ). The water transport rate is shown at each arrow.

400

O. PEDERSEN AND IC SAND-JENSEN

L. ¢ ~ r t ~

• P. crls~s

• B. tHc~hyr~m

O

! $. emersum &

Q E. canl~nsl: m L. ~flora

• P. pecttnerus 9) M. vertici#at~m

& Z. marina

0.5

I

I

10

100

Cross-sectional area of the vascular bundles (10 -s ma) Fig. 8. Acropetal water transport as a function of the cross-sectional area of the vascular tissue in the stem for nine macrophyte species with isoetid ( I ) , basal rosette ( A ) and elodeid ( • ) growth form. Calculations are based on maximum rates (Fig. 3).

with many vascular bundles, a high cross-sectional area of the vascular tissues (20-100 × 10- s m 2) and thus ~,~well-developed route for water transport. These species display spiral-formed thickenings of the xylem walls probably to resist the negative tension in the xylem ducts when the plants grow exposed to air and water evaporates from leaf surfaces. The truly submerged species ;,ad one to three vascular bundles and a cross-sectional area of the vascular tissue between I and 7 × 10- s m 2. Because we do not know with which tissue acropetal water transport is associated, we quantified the entire cross-sectional area of the vascular bundles in the stem including phloem and parenchymatic cells as well as intercellular spaces which may be air or water filled. Also, in the permanently submerged species it was difficult to define xylem vessels because of weak lignification. There was no simple relationship between acropetal water transport and crosssectional area ofthe vascular tissue of the stem (Fig. 8). Elodeid species with relatively small vascular systems had highly variable transport rates including the highest measured. DISCUSSION

Activated water transport

Our findings demonstrate activated water transport in the submerged macrophyte because of ( 1 ) the marked variability of transport rates among spe-

WATERTRANSPORT IN SUBMERGED MACROPHYTES

40 |

cies, (2) the short time lag to reach constant release of THO from the plant to the shoot compartment for species with high transport rates, (3) the higher transport rates in the light than the dark for Sparganium emersum, and (4) the uneven distribution of absorbed THO in the different plant parts. Moreover, the observed water transport exceeded the possible diffusive flux of water from the root to the shoot compartment via the vascular tissue or via the entire cross-section of the stem for most species, calculated by assuming the diffusive path to be half the length of the plant and the diffusive coefficient to be that of water, ignoring the effect of necessary passages through membranes and cell walls (HaUett et al., 1982). Measured water fluxes are 600fold higher for Lobelia dortmanna and 2 X 106 fold higher for B. trichophyllum than those predicted by diffusion through an area equal to that of the vascular bundles alone, and 30 (Lobelia dortmanna) and 9000-fold higher (B. trichophyllum) than the possible diffusive flux through the entire crosssection of the stem. For Zostera marina, however, the very small acropetal transport rates measured could be accounted for by diffusion through the stem. Given that the water transport is restricted to the vascular bundles alone, activated transport needs to be involved. Decline of transport rates for Sparganium emersum in prolonged darkness and stimulation by light suggest that photosynthetic production of organics and energy is needed to sustain transport. Declining transport over time for abeissed roGt systems ofB. trichophyllum supports this conclusion. The main motive force for acropetal transport in B. trichophyllum appears to be the root system, but intact plants transported more water than abcissed root and shoot systems perhaps because of larger combined osmotic forces. Acropetal water flow is clearly restricted to only part of the plant tissue, most likely the vascular bundles or even the xylem alone. Thus. isotopic equilibrium of the released water, or rather constant rates of THO release, was reached relatively fast ( 1-2 days) for most species. Moreover, the water absorbed was clearly diverted to the young leaves in active growth, suggesting that acropetal water flow mainly serves to supply nutrients to above-ground parts. Nutrients absorbed by roots are also translocated to actively growing tissue (Welsh and Denny, 1979a,b; Lynghy et al., 1982; Brix and Lynghy, 1985). Wilson (1947) noted that, when he introduced hydrostatic pressure to the cut stem surface of a shoot of B. baudotii, water with eosin moved up the stem and out through the youngest leaves. Occasionally he also found water flow through leaves of medium age, but never through old leaves. Microscopy showed that the hydathodes with the underlying epithem in the old leaves were occluded by a brown gummy mass described earlier for other species by Von Minden (1899). Hostrup and Wiegleb ( 1991 ) also found vascular xylem ducts ofLittorella uniflora opening directly into a small cavity at the leaf tip. They did not examine whether young and old leaves differed. Clogging of the hydathodes of old leaves should ensure an easy and appropriate distribu-

402


tion of water and ions, absorbed by roots, to young leaves in active growth. Alternatively, intensive use of dissolved nutrients in regions of active growth should result in an osmotic under-pressure in the growth active regions and thus a suction of water and dissolved ions from the roots (the 'sink hypothesis'; Miinch, 1930; Bidwell 1974). When large quantities of water are transported there must be a drain for filtered water, and the flow will probably be regulated by a combination of the two proposed mechanisms. Alternatively, the water received may be released from the leaves as ions (H + and O H - ) actively involved in several membrane transport processes as previously proposed by Hutchinson (1975).

Water transport in different species We observed large variability in acropetal water transport among different species. This variability was not significantly coupled to the size of the vascular system. This is perhaps not surprising considering the variability of growth rates, growth forms and relative size of root systems. Isoetid species have a large vascular system (Sculthorpe, 1967; Hostrup and Wiegleb, 1991 ) and many roots (Raven et al., 1988), but display slow growth rates (SandJensen and Sondergaard. 1978) and low tissue concentrations of N and P, indicative of a small nutrient demand relative to a wide transport route. Elodeid species, on the other hand, have a relatively large nutrient demand and a more narrow transport route. Also, because we only examined the species in one experimental trial, the observed species differences may not be consistent, but may reflect the characteristics of the organisms collected from only one locality at one time of the year. If the main purpose of the acropetal water transport is to supply nutrients to above-ground parts, we would expect large intraspecies variability of water transport, depending on the growth rate before and during the experiment, the relative supply of nutrients absorbed by roots and leaves, and internal nutrient " r~centrations in the tissue. It is hard to compare water transpo:, rates measured here with previous results because of use of various units in the past. For 1". crispus, however, Unger (1862) found an acropetal transport rate at 8.3/tl H20 (126 cm 2 leaf) "~ h -~ and we can recalculate our transport rates to 20.4/tl H20 (126 cm 2 leaf) - ~h - ~which is not grossly different. Considering the measured cross-sectional areas of vascular bundles in the stems we could have estimated the mean flow velocity. However, this value may grossly underestimate the actual flow velocity in xylem ducts which ovly constitute part of the tissue inside the endodermis. Moreover, flow velocity in the xylem will presumably vary greatly from vessel to vessel because of the nature of liquid flow in capillaries. For equal pressures, flow velocity in the capillaries increases with the second power of the radius (Zimmermann, 1983). Thus, a main objective of future work would be to define the struc-

WATERTRANSPORT IN SUBMERGEDMACROPHYTES

403

tures actively engaged in acropetal water transport and the flow velocities in these tissues.

Acropetal water transport and nutrient supply The main purpose of acropetal water transport is supposedly the translocation of nutrients to above-ground meristems. The question is, therefore, whether the measured acropetal water transport alone can support a given growth rate, applying reasonable mean nutrient contents of the tissues. Performing this evaluation for nitrogen (N), which is commonly the limiting nutrient element to submerged macrophyte growth (Barko et al., 1991 ), we find that the necessary concentration (CN) in the water flow is given by CN= RGRNc where RGR is the relative growth rate, Nc is the N content in the plant tissue, Wd is the daily acropetal water transport and MN is the atomic weight of N. For P. crispusthe typical mean values of 0.05 day-' (RGR; Nielsen and SandJensen, 1991 ), 0.03 g N g-~ DW (Nc; K. Sand-Jensen, unpublished data, 1991 ) and 5.64 ml H20 g- ~DW day- ' ( Wd; Fig. 3 ) would yield a minimum N content in the acropetal water flow of 19 mM. Concerning nitrate alone, this level is never reached in the pore water of natural sediments where maximum values are reported to be 0.5 mM in lowland streams (Christensen and Serensen, 1988), i.e. 38 times lower than 19 mM. However, Shepherd and Bowling (1973) have shown that Potamogeton natans L. is able to elevate its nitrate concentration in the root cells 140 times relative to the interstitial water. If the same applies to P. crispus, the observed water transport should be more than adequate to translocate nitrate from roots to shoots to sustain maximum growth. The observed ability ofP. natans to concentrate nitrate in the root cells becomes less critical when including ammonium as an additional N source. Ammonium concentrations in the pore water of sediments with rooted macrophytes were up to 2 mM in Lake Wingra (Nichols and Keeney, 1976), which reduces the necessary concentrating factor to eight or less. When we perform the same estimate for the isoetids Littorella uniflora and Lobelia dortmanna, applying a maximum growth rate of 0.01 day- ' (SandJensen and Sendergaard, 1978; Nielsen and Sand-Jensen, 1991 ), a N content of 0.02 g N g-~ DW (Christiansen et al., 1985; K. Sand-Jensen and O. Pedersen, unpublished data, 1991 ), and an acropetal water transport of 6 ml H20 g - ' DW d a y - ' (Fig. 3), the estimated minimum N content in the acropetal water flow is 2 mM. For these isoetids, the necessity to concentrate N ions in the root cells is even less demanding than for P. crispus. Concerning nitrate, levels of 0.3 mM are often reached in nutrient-poor sediments inhabited by Littorella and Lobelia (Christensen and Serensen, 1986). Including the am-

404

(3.PEDERSENANDK.SAND-JENSEN

m o n i u m N source (typical levels o f 0.4 m M in Littorella sediments; P.B. Christensen, unpublished data, 1988) reduces the necessary concentrating factor to 2.9. Our work therefore shows that submerged macrophytes generate sufficient acropetal water transport rates to meet nutrient requirements for m a x i m u m plant growth at ambient sediment nutrient levels. ACKNOWLEDGEMENTS This work was supported by Grant 91-0454/20 from the Carlsberg Foundation to the project Coupling o f Gas, Water and Nutrient Transport in Submerged Macrophytes, and Grant SNF-I 1-7795 from the Danish Natural Science Research Council to the project Physiology and Ecology o f Macrophytes. We thank L.B. Jorgensen for helpful assistance, Carlos M. Duarte and Sven Beer for critique o f the manuscript and P. Bondo Christensen for supplying a m m o n i u m data. REFERENCES Arber, A., 1920. Water Plants: A Study of Aquatic Angiosperms.University Press, Cambridge. (Reprinted 1963 with an introduction by W.T. Steam, as Historiae Naturalis Classica, Vol. 23. Gramcr, Weinheim.) Barko,J.W. and Smart, R.M., 1981. Sediment-basednutritionof submergedmacrophytes.Aquat. Bot., 10: 339-352. Barko, J.W., Gunnison, D. and Carpenter, S.R., 199 I. Sediment interactions with submersed macrophytegrowthand communitydynamics.Aquat. Bot., 41: 41-66. Bidwell, R.G.S., 1974. Plant Physiology.Macmillan,London, 577 pp. Brix, H. and Lyngby,J.E., 1985. Uptake and translocation of phosphorus in eelgrass (Zostera marina ). Mar. Biol., 90: I I l-l 16. Chambers, P.A., Prepas, E.E., Bothwell, M.L. and Hamilton, H.R., 1989. Roots versus shoots in nutrient uptake by aquatic macrophytesin flowingwaters. Can. J. Fish. Aquat. Sci., 46: 435-439. Christensen, P.B. and Sorensen,J., 1986. Temporalvariation ofdenitrifieation activityin plantcovered, littoral sediment from Lake Hampen, Denmark. Appl. Exp. Microbiol., 51: 11741179. Christensen, P.B. and S~rensen, J., 1988. Denitrification in sediment of lowland streams: regional and seasonal variation in Gelb~k and Rabis B~k, Denmark. Microbiol. Ecol., 53: 335-344. Christiansen, R., Friis, N.J.S. and Sondergaard, M., 1985. Leaf production and nitrogen and phosphorus tissue content of Littorella uniflora (L.) Asch~rs. in relation to nitrogen and phosphorus enrichment in oligotrophic Lake Hampen, Denmark. Aquat. Bot., 23: l-l I. Denny, P., 1980. Solutemovement in submergedangiosperms.Biol. Rev., 55: 65-92. Gessner, F., 1956. Der Wasserhaushaltder Hydrophytenund Helophyten.In: O. Stocker (Editor), H~,ndbuchder PflanzenphysiologieIII, Springer, Berlin. Hallett, F.R., Speight, P.A. and Stinson, R.H., 1982. Physicsfor the BiologicalSciences:A Topical Approachto BiophysicalConcepts. Methuen,Toronto, 255 pp. Hochreutiner,G., 1896. l~tudessur les phanrrogames aquatiques du Rhrne et du Port de Gen~ve. Rev. Grn. Bot., 8: 158-167.

WATERTRANSPORTINSUBMERGEDMACROPHYTES

405

HShn, K. and Ax, V~., 1961. Untersuchungen fiber Wasserbewegung und Wachstum submerser Pflanzen. Beitr. Biol. Pfl., 36: 273-298. Hostrup, O. and Wiegleb, G., 1991. Anatomy of leaves of submerged and emergent forms of Littorella uniflora (L.) Ascberson. Aquat. Bot., 39:195-210. Hutchinson, G.E., 1975. A Treatise on Limnology. Vol. III, Limnological Botany. John Wiley, New York, 660 pp. Kristensen, P., Kronvang, B., Jeppesen, E., Gnesboli, P., Erlandsen, M., Rebsdorf, Aa., Bruhn, A. and Sondergaard, M., 1990. Ferske vandomrlder--vandlob, kilder og seer. Vandmiljoplanens Overvigningsprogram--Danmarks Miljoundersogelser. Faglig Rapport fra DMU m.f. Lynghy, J.E., Brix, H. and Schierup, H.H., 1982. Absorption and translocation of zinc in eelgrass (Zostera marina L.). J. Exp. Mar. Biol. Ecol., 58: 259-270. Miinch, E., 1930. Die Stoffbewegungen in der Pflanze. Gustav Fischer, Jena, 245 pp. Nichols, D.S. and Keeney, D.R., 1976. Nitrogen nutrition in Myriophyilum spicatum: variation of plant tissue nitrogen concentration with season and site in Lake Wingra. Freshwater Biol., 6: 137-144. Nielsen, S.L. and Sand-Jensen, K., 1991. Variation in growth rates of submerged aquatic macrophytes. Aquat. Bot., 39: 109-120. Pond, R.H., 1905. Contributions to the biology of the Great Lakes. The biological relation of aquatic plants to the substratum. Report of Commissioner of Fish and Fisheries, University of Michigan, Ann Arbor. Raven, J.A., 1984. Energetics and Transport in Aquatic Plants. Liss, New York, 580 pp. Raven, J.A., Handley, L.L., MacFarlane, J.J., Mclnroy, S., McKenzie, L., Richards, J.H. and Samuelsson, G., 1988. The role of CO, uptake by roots and CAM in acquisition of inorganic C by plants of the i~etid life form: a review, with new data on Eriocaulon decangulare L. New Phytol., 108: 125-148. Sand-Jensen, K. and Sondergaard, M., 1978. Growth and production ofiseetids in oligotrophic Lake Kalgaard, Denmark. Verb. Int. Ver. Limnol., 20: 659-666. Sauvageau, C., 1891. Sur les feuilles de quelques monocotyl~dones aquatiques. Ann. Sci. Nat. Vll S6r. Bot., 13: 280-292. Sculthorpe, C.D., 1967. The Biology of Aquatic Vascular Plants. Edward Arnold, London, 610 pp. Shepherd, U.H. and Bowling, D.J.F., 1973. Active accumulation of sodium by roots of five aquatic species. New Phytol., 72: 1075-1080. Smart, R.M. and Barko, J.W., 1985. Laboratory culture of submersed freshwater macrophytes on natural sediments. Aquat. Bot., 21:251-263. Snell, K., 1908. Untersuchungen fiber die Nahrungaufnahme der Wasserpflanzen. Flora, 98: 213-249. Thoday, D. and Sykes, M.G., 1909. Preliminary observations on the transpiration current in submerged water-plants. Ann. Bot., 23: 635-637. Thut, H.F., 1932. The movement of water through some submerged water- plants. Am. J. Bot., 19: 693-709. Unger, F., 1862. Beitr~igezur Anatomie und Physiologie der Pflanzen. IX. Neue Untersuchungen fi0er die Transspiration der Gew~ichse.Sitzungsber. Wien. Akad. Wiss., 44:181-217. Vardar, Y., 1950. Untersuchungen fiber die Wasserbewegungen in untergetauchten Pfianzen. Rev. Fac. Sci. Univ. Istanbul, S~r. B: Sci. Nat., 15: 1-59. Von Minden, M., 1899. Beitr~ige zur anatomischen und physiologischen Kenntnis Wasser-secernierender Organe. Biblio. Bot., 46: 1-72. Waisel, Y. and Shapira, Z., 1971. Functions performed by roots of some submerged hydrophytes. Isr. J. Bot., 20: 69-77.

406

o. PEDERSENANDK. SAND-JENSEN

Weinrowsky, P., 1899. Unt~.rsuchungen fiber die Scheiteltiffning die Wasserpflanzen. Beitr. Wissen~chaf. Bot., 3: 205-247. Welsh, R.P.H. and Denny, P., 1979a. The translocation of lead and copper in two submerged aquatic angiosperm species° J. Exp. Bot., 30: 339-345. Welsh, R.P.H. and Denny~ F., 1979b. The translocation of 32p in two submerged aquatic angiosperm species. New Phytol., 82: 645-656. Westlake, D.F., 1963. Comparisons of plant productivity. Biol. Rev., 38: 385-425. Wetzel, R.G., 1983. Limnology, 2rid edn. Saunders~ Philadelphia, ~67 pp. Wilson, K., 1947. Water movement in submerged aquatic plants, with special reference to cut shoots of Ranunculusfluitans. Ann. Bot., I l: 91-122. Zimmermann, M.H., 1983. Xylem structure and the ascent of sap. Springer, Berlin, 143 pp.