Growth Responses of Lemna to Different Levels of Nitrogen Limitation

Department of Botany, Section of Plant Physiology, University of Stockholm, S-106 91 Stockholm, Sweden

Received July 21,1981 . Accepted December 18, 1981

Summary Lemna gibba L. G3, L. minor L. and L. paucicostata Hegelm. 6746 were initially cultured in a

nitrogen-sufficient nutrient medium, where they maintained a relative growth rate, RGR, over

(In W2-ln WI) h ) h f h . h . 50 [RGR = 100 ( ) , were (In W2 - In WI represents teres weIg t Increase t2-tl over the time interval (t2 - tIl], After one week of preculture in nitrogen-sufficient medium, the plants were transferred to a nitrogen-free nutrient medium. Growth-limiting amounts of nitrogen, together with ample amounts of all other essential elements, were added once daily in exponentially increasing amounts. The relative rate of nitrogen addition, RN, was varied

(In N2-ln N I) . . between 15 and 45 [RN = 100 (t ) , where (In N 2-ln N I) represents the Increase In 2-tl nitrogen bound in plant material over the time (t2-tI)). During an adaptation phase of 10 to 15 days, the RGR gradually decreased until it reached the value set by RN. The plants then cont­inued to grow at a rate fluctuating around the rate set by RN. Just prior to the daily nutrient additions, only traces of nitrogen were detected in the nutrient medium, indicating that RN was the decisive factor regulating growth. The concentrations of K, P, Ca and Mg did not fall to growth-limiting values. The contents of nitrogen and chlorophyll in the plants were propor­tional to RN, whereas the surface area of the plants expressed on a dry weight basis was fairly unaffected by RN. It seems that adaptation to nitrogen limitation involves reduced net CO2 assi­milation rate on areal basis, which is also reflected in the decrease in chlorophyll per area unit. It is concluded that daily, exponentially increasing additions of nitrogen is a convenient method to obtain plants with stable growth rates under sub-optimal nitrogen nutrition, where the supply rate, not the concentration, is the decisive factor for growth.

Key words: Lemna gibba, L. minor, L. paucicostata, nitrogen, relative growth rate, relative nitro· gen addition rate.

Introduction

A biphasic or multiphasic relationship between nitrogen concentration and nitro­gen uptake has frequently been demonstrated in various plant material (e.g. Fried et

1) Permanent address: Swedish University of Agricultural Sciences, Section of Forest Eco­physiology, S-750 07 Uppsala, Sweden.

2) To whom reprint requests should be directed. Abbreviations: Chi - chlorophyll; Dw = dry weight; EDTA = ethylene diamine tetraac­

etate; Fw = fresh weight; RGR = relative growth rate; RN = relative nitrogen addition rate.

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332 TOM ERICSSON, CARL-MAGNUS LARSSON and ELISABETH TILLBERG

al., 1965; Berlier et al., 1969; Doddema and Telkamp, 1979; Nissen et al., 1980). If, however, plants are allowed to exhaust the nitrogen content of the nutrient solution, uptake is linear with time despite a decrease in concentration of several orders of mag­

nitude, and the uptake eventually ceases when the concentration reaches a threshold

value of a few micro molar (Olsen, 1950; Edwards and Barber, 1976). Similar results have been reported from flowing culture experiments, where the concentration of a

specific nutrient is limiting for uptake and growth only at low values, provided the

flow rate is high enough to keep pace with the uptake (Asher and Ozanne, 1967; Edwards and Asher, 1974; Clement et al., 1978; Datko et al., 1978). Under such

conditions, the rate of mineral uptake is rather a function of the growth rate than of

nutrient concentration, as discussed by Clarkson and Hanson (1980). In an attempt to elucidate the relations between nitrogen availability and growth,

Ingestad and Lund (1979) developed a method to grow plants under nitrogen limita­tion, where the nitrogen supply rate, not the concentration, was used as the decisive

factor regulating growth. With such a technique, plants with stable growth rates, stable internal nitrogen concentrations, but without most of the deficiency

symptoms traditionally ascribed to nitrogen deficiency were obtained at sub-optimal nitrogen nutrition. Their results for Betula were confirmed by Ericsson (1981), working with Salix cultured with a simplified method, where the growth was con­

trolled by daily, exponentially increasing additions of nitrogen. The present paper

adopts the simplified method of Ericsson (1981) in order to study adaptation of Lemna to sub-optimal nitrogen nutrition, with special regard to growth parameters

and mineral relations.

Materials and Methods Plant material: Axenic stock cultures of Lemna gibba L. strain G3, L. paucicostata Hegelm.

6746 (formerly L. perpusilla Torr. 6746) and a locally collected strain of L. minor L., were maintained in the «K» medium of Maeng and Khudairi (1973).

Preculture and growth conditions: Before the experiments on nitrogen limitation, the plants were precultured for one week in a nitrogen-sufficient medium. The composition of this medium is given in Table 1. The plants were transferred from the stock cultures to open plastic vessels with a surface area of 18 x 25 em, containing 1 litre of nitrogen-sufficient medium. The cultures were kept in a climate chamber at 21°C and 75 % relative humidity and continuous illumination from a bank of fluorescent tubes (General Electric «Power Groove de Luxe Cool White F 96 PG 17) giving a photon flux density of 200 /lmol m- 2s- I

). Aeration and stirring were provided by compressed air filtered through activated charcoal. Bacterial or algal infec­tions were negligable.

Growth under nitrogen limitation: Samples of 50 to 200 mg fresh weight of plant material were transferred from the nitrogen-sufficient medium to 1 litre of nitrogen-free medium. This medium contained ample amounts of all other essential elements (see Table 1). The conditions were otherwise the same as above. The daily requirements of nitrogen and all other elements were given as an addition solution, composed as outlined in Table 1. The amount of nutrients added, increased exponentially from day to day. The nitrogen source in the addition solution was a mixture of ammonium and nitrate in the molar ratio 2 : 3. The pH was initially 5.8 but the nutrient consumption slowly acidified the medium, which had to be regularly adjusted back

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Nitrogen-limited growth of Lernna 333

Table 1: Composition of the nutrient solutions. Concentrations in mM (N to S) or 11M (Fe to EDTA).

Element Nitrogen Nitrogen-deficient

sufficient Initial Addition

N 7.1 0 7.1 K 1.7 1.7 1.7 P 0.5 0.5 0.4 Ca 1.6 0.2 0.2 Mg 1.7 0.4 0.4 S 0.3 0.4 0.3 Fe 12.5 12.5 12.5 Mn 7.2 7.2 7.2 B 18.5 18.5 18.5 Cu 0.5 0.5 0.5 Zn 0.5 0.5 0.5 Cl 0.03 1.0 1.0 Mo 0.1 0.1 0.1 Na 14.1 14.1 0.1 EDTA 14.0 14.0 0

to its original pH by additions of NaOH. Provided the pH was kept above 4.3, the acidification did not affect growth.

The growth was determined by measuring fresh weight increase. The relative growth rate. RGR, expressed as per cent fresh weight increase per day, was calculated from the formula:

RGR = 100 x .;..(I_n--,W_2_-_ln.,...W--,-I) (t2 -tl)

where WI and W2, respectively, are the fresh weights at start (tl) and harvest (t2)' The relative nitrogen addition rate, RN, can be expressed in a similar manner:

RN 100 x (In N2-ln N 1)

(t2 -h)

(1)

(2)

where NI and N2 stand for the amount of nitrogen bound in the plants at start and harvest, respectively.

Since, for practical reasons, the nitrogen content of the plants could not be checked daily, the nitrogen additions were calculated with a constant rate of exponential increase. This means that N 1 and N2 in practice stand for the nitrogen content in plant + medium, and RN for the rate of increase in nitrogen in plant + medium. However, as will be shown in the results, the uptake of nitrogen by the plants is effective enough to justify the approximation that all nitrogen added is bound in the plants within 24 h.

In all experiments, four different RN values were employed: 15, 25, 35 and 45. The experi­mental periods varied from 6 to 40 days. The cultures were diluted before the surface of the culturing vessel was completely covered with plants, i.e. when the fresh weight of the culture had increased to approximately 10 g. Fresh and dry weights of the plants were determined, and the mineral contents (N, K, P. Ca, Mg) of the plants were analysed according to Ingestad (1979). Chlorophyll was extracted with hot methanol and determined according to Senger (1970). Plant area was measured photometrically.

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334 TOM ERICSSON, CARL-MAGNUS LARSSON and ELISABETH TILLBERG

Results

1. Preculture: Growth and mineral content under nitrogen-sufficient conditions

The RGR values for L. gibba, L. minor and L. paucicostata cultured in a medium with a surplus of nitrogen (composition given in Table 1) were above 50 (Table 2) i.e. the doubling time was less than 1.4 day. The nitrogen contents of the plants varied between 4.1 and 4.7 percent of dry weight (Table 2), and these values were used for computations of nitrogen additions in the subsequent cultivation under nitrogen limitation.

Table 2: Growth (RGR) and mineral content (% of DW) of Lemna grown under nitrogen-suffi­cient conditions. RGR represents overall growth during one week.

Species RGR Mineral content, % of Dw

N K P Ca Mg

L. gibba 56 4.6 5.3 1.1 0.57 0.35 L. minor 65 4.7 5.7 0.86 0.47 0.40 L. paucicostata 53 4.1 4.9 0.74 0.33 0.30

RGR

40 t t

20 t RN= 45 t

0

40 RN = 35

20 t t

0

40 '. RN = 25

20 t

0

40 RN = 15

20 " t.

00 5 10 15 20 25 Time, d

Fig. 1: Time course of growth of Lemna gibba at four different RN. Arrows indicate points of dilution of the cultures, i.e. when the surface of the culturing vessels were completely covered with plants.

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Nitrogen-limited growth of Lemna 335

2. Growth under nitrogen limitation

The plants were precultured in nitrogen-sufficient medium for one week, and were then transferred to nitrogen-deficient medium (Table 1). After the transfer to nitro­gen-limited conditions, the RGR was initially higher than RN but eventually decreased to values fluctuating around the value set by RN (Fig. 1). Towards the end of the adaptation phase, the cultures tended to grow slower than RN . Figure 2 is a plot of RGR versus RN for L. gibba after adaptation of the plants to nitrogen limitation. The RGR of adapted plants showed a linear relationship to RN with a slope close to 1, and with extrapolated intercepts close to origo. Similar adaptation characteristics were obtained with L. minor and L. paucicostata (not shown).

3. Mineral content in media and plants after adaptation to nitrogen limitation

The mineral contents of the nutrient media, sampled when the surface of the culturing vessels were completely covered with plants, are presented in Table 3. Both

RGR

50

40

30

20

.----------0-1

Fig. 2: RGR plotted against RN for L. gibba. The data represent RGR 10 for plants adapted to different RN for more than 13 days. Based on the data given in Figure 1. Vertical bars give standard deviations. °0~/----C15:--2:'::5--:'35=--4-'::'5

RN

Table 3: Final contents of N (as nitrate and ammonium), K, P, Ca and Mg in the nutrient media after adaptation to nitrogen limitation. Samples taken 24 h after last nutrient addition, and at the time when the surface was completely covered with plants. N.D. = not detectable. Results given for one representative experiments.

Concentration in solution, mM

Species RN N N03--N NH4+-N K P Ca Mg

L. gibba 15 0.064 N.D. 0.064 1.4 0.43 0.23 0.52 25 0.009 N.D. 0.009 1.0 0.28 0.19 0.47 35 0.006 0.004 0.002 1.3 0.49 0.23 0.57 45 0.917 0.892 0.025 2.0 0.62 0.22 0.63

L. minor 15 0.001 <0.001 0.001 1.8 0.39 0.11 0.30 25 <0.001 <0.001 <0.001 1.8 0.43 0.11 0.30 35 <0.001 N.D. <0.001 1.9 0.46 0.11 0.30 45 <0.001 N.D. <0.001 2.0 0.47 0.12 0.29

L. paucicostata 15 0.001 <0.001 0.001 1.1 0.32 0.08 0.20 25 0.001 <0.001 <0.001 1.3 0.40 0.09 0.30 35 0.001 <0.001 <0.001 1.6 0.45 0.11 0.30 45 0.002 <0.001 0.002 1.7 0.49 0.11 0.30

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336 TOM ERICSSON, CARL-MAGNUS LARSSON and ELISABETH TILLBERG

Mineral content. % of Dw

N .. ,..,.P,c:.K ___ ---"C.:::;a .. Mg N,;:...P"-.K>--__ ---"'C""'a.Mg N.P.K Ca.Mg

K A K B K C

5 055~0.5

3 ::~: 4 Ca

2 0.2 2

N 0.1

~ p 01 1 E... __ -- 0.1

o 0 O~~-~~-~O O~~-~~-~O 15 25 35 45 15 25 35 45 15 25 35 45

RN RN RN

Fig. 3: Contents of N, P, K, Mg, and Ca in percent of dry weight as a function of RN • (A) L gibba, (B) L minor, and (C) L paucicostata. Results from one representative experiment.

L. minor and L. paucicostata efficiently exhausted the nitrogen content of the medium, which lead to final nitrogen concentrations of the order of 1 IlM. L. gibba was less efficient, especially with regard to the RN 45 treatment, where the final con­centration was almost 1 mM. With this exception, the results show that the added nitrogen was almost completely consumed. The other investigated elements (K, P, Ca, Mg) remained roughly in the same proportions and concentrations as in the addi­tion solution (compare Table 1 with Table 3).

The nitrogen content of the three investigated Lemna species increased linearly with RN (Figs. 3 A-C). Also other elements showed variations at different RN, although these responses were less regular than for nitrogen: e.g. K. decreased with increasing RN in L. gibba and L. minor, and, similarly, the Ca content decreased in L. minor and L. paucicostata.

4. Chlorophyll content, dry matter content, and areal development after adaptation to nitrogen limitation

The data are compiled in Figures 4 A and B for L. gibba, 5 A and B for L. minor, and 6 A and B for L. paucicostata. The chlorophyll content increased on both fresh weight, dry weight and areal basis with increasing RN in all three species. The dry matter content, expressed as per cent of fresh weight decreased with increased RN in L. gibba, whereas it in L. minor showed the opposite tendency and in L. paucicostata was more constant. The surface area of the plants expressed as cm2 frond per g dry weight was fairly constant in L. gibba and L. paucicostata, while in L. minor the RN 15 culture exhibited a higher surface area per g dry weight than at higher RN .

Discussion

The principle for the culturing method employed in the present paper is that growth-limiting amounts of nitrogen are added daily in exponentially increasing

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Nitrogen-limited growth of Lemna 337

amounts, calculated to sustain different exponential growth rates of the cultures. No attention has been paid to maintain constant external concentrations of nitrogen or any other element. This means that the daily nitrogen additions to 1 litre medium range approximately from 15 J.lmoles to 1 mmol from the start of a culture to the point of time when the surface is completely covered with plants. The cumulative nitrogen addition to a culture is approximately 3 mmoles. The data in Table 3 show that the nitrogen concentration in the medium at the point of surface covering is in the order of 1 J.lM for L. minor and L. paucicostata and somewhat higher for L. gibba. Thus, the added nitrogen is almost completely consumed. Since the other elements investigated (K, P, Ca, Mg) remain essentially in their initial concentrations

mg Chl m~Chl gl5W g, w Ow.Ofo of Fw

o 0 ~' _~'=--_-=-_~. 0 15 25 35 45

RN

cm' cm' ugChl 9 Ow gr-,F-'-'w _____ --'c"-;n:"

o ~_-:':' _--:":--~ 15 25 35 ~

o

RN

Fig. 4: A: Chlorophyll content and dry matter content of L. gibba adapted to different RN . • ,

mg Chi . g -1 fresh weight; ., mg Chi . g -1 dry weight; A, dry matter content in % of fresh weight. Vertical bars indicate standard deviations. Three to five measurements. - B: Surface area per wei!}ht unit and chlorophyll content per area unit of L. gibba adapted to different RN .

• , cm2 • g - fresh weight; ., cm2

• g -1 dry weight; A, I1g Chi . cm - . Vertical bars indi­cate standard deviations. Three to five measurements.

mg Chl mg Chl Ow."Io of Fw gOw g~Fwu-_____ ~

cmZ cmt

gOw g,~F~w~ ____ ~~

8

6

4

2 0.2 5 10

o 0 '=-_'-:':-_--:':' ,..-----:" 0 0. 0 =-_'-:':-_-:':,..-----:' 0 ~ ~ ~ ~ ~ ~ ~ ~

RN RN

Fig. 5: Legend as for Figure 4, but data given for L. minor.

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338 TOM ERICSSON, CARL-MAGNUS LARSSON and ELISABETH TILLBERG

cm2 cm2 mg Chl mg Chl Dw.%of Fw gDw g~,FWll-__________ ~ 9 Dw g,,.!-, F-"w'---________ ---.!<!"

5 05

10 2

5 5 1 01

0 o I , , 0 o , , , , 0 15 25 35 45 15 25 35 45

RN RN

Fig. 6: Legend as for Figure 4, but data given for L. paucicostata.

(Table 3), or show internal concentrations that do not drastically deviate from the values obtained with nutrient-sufficient cultures (compare Table 2 with Fig. 3), it is reasonable to assume that the rate of nitrogen addition has been decisive for growth. The RN 45 treatment of L. gibba is an exception in this case, where nitrogen has been non-limiting for growth. This is also demonstrated in the flat growth curve (Fig. 1).

The apparent difference in affinity for nitrogen between L. gibba and the other two species may reflect species differences in the uptake mechanism. However, L. gibba responds to nitrogen limitation with excessive root growth, which may prevent free communication between the medium and the lower surface of the fronds, thus decreasing the efficiency of uptake. The importance of the root itself as an absorptive and ion-translocating organ in the Lemnaceae is unclear (Hillman, 1961). In those cases when substantial amounts of nitrogen can be detected in the medium, most of it is recovered as nitrate, although the initial ammonium to nitrate ratio was 2 : 3. This indicates preferential absorption of ammonia, probably caused by low nitrate reductase activity (Stewart, 1972; Orebamjo and Stewart, 1976).

During adaptation to reduced RN, the growth rate is initially higher than RN (Fig. 1) which leads to reduced nitrogen content of the plants. After adaptation, the nitrogen content is proportional to RN (Figs. 3 A-C). The growth rate after adapta­tion is also linearly related to RN (Fig. 2), although the rate fluctuates (Fig. 1). This confirms results obtained for different tree species, cultured in a similar manner (Ingestad and Lund, 1979; Ingestad, 1980; Ericsson, 1981). Adapted cultures of Lemna have been kept in our laboratory for several months without any visible sign of deterioration. Lemna cultures are in this respect a suitable experimental material, since RGR represents horizontal population growth, and effects on RGR caused by differentiation, senescence and self-shading do not interfere.

The surface area of the plants expressed on a dry weight basis was fairly unaffected by RN (Figs. 4 B, 5 B, and 6 B). This leads to the conclusion that growth (weight increase) per unit area increases with increasing RN . Also in birch seedlings cultured in a similar manner, the leaf area efficiency (expressed as seedling weight increase per

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Nitrogen-limited growth of Lemna 339

day and cm2 of leaf area) increased linearly with RN (Ingest ad and Lund, 1979). This is reflected in the plots of chlorophyll per area (Figs. 4 B, 5 B, and 6 B), which increases with increasing RN . Other adaptations of the photosynthetic machinery are probably also involved, e.g. variations in the levels of enzymes associated with C02 fixation and intermediary carbon metabolism (Humphrey et aI., 1977; Cooke et aI., 1979).

In conclusion, the present results show that stable growth rates of Lemna colonies under nitrogen limitation can be maintained by daily, exponentially increasing addi­tions of nitrogen. The growth rate is controlled by the rate of nitrogen supply, and not by the concentration. The method seems convenient for long-term studies of adaptation to nitrogen limitation since the Lemna plants grow steadily at the desired rate. The results indicate that the adaptation to reduced nitrogen supply involves reductions in the net CO2 assimilation rate. The details and essential steps of the adaptation of the productivity apparatus to limited nitrogen supply remain, however, to be investigated.

Acknowledgement

Support from the Swedish Natural Science Research Council is greatfully acknowledged.

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