Vol. 67: 251–263, 2012 AQUATIC MICROBIAL ECOLOGY Published online November 6 doi: 10.3354/ame01596 Aquat Microb Ecol

Rapid regulation of phosphate uptake in freshwater cyanobacterial blooms

Luis Aubriot*, Sylvia Bonilla

Phytoplankton Ecology and Physiology Group, Sección Limnología, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay

ABSTRACT: Cyanobacterial blooms are generally explained by nutrient uptake kinetic constants that may confer competitive capabilities, like high nutrient-uptake rate, affinity, and storage capacity. However, are capable of flexible physiological responses to environmen- tal nutrient fluctuations through adaptation of their phosphate uptake properties. Growth opti- mization is possible if the physiological reaction time of cyanobacteria (tR) matches the duration of nutrient availability. Here, we investigate the tR of this complex physiological process. We per- formed [32P] uptake experiments with filamentous cyanobacterial blooms. The were subjected to different nutrient exposure times (tE) by varying patterns of phosphate addi- tions. After 15 to 25 min of phosphate tE, the cyanobacteria-dominated phytoplankton began their adaptive response by reducing and finally ceasing uptake activity before exhausting their nutrient uptake capacity. The time of onset of phosphate uptake regulation (in min) is an indicator of tR. Rapid adaptive behaviour may be the initial phase of longer-term nutrient acclimation (hours to days), which results in a higher growth rate. The growth response of bloom-forming cyanobacteria may be more dependent on the ability to optimize the uptake of phosphate during the time span of nutrient fluctuation than on the amount of nutrient taken up per se. Freshwater cyanobacterial blooms may therefore be promoted by their short tR during phosphate fluctuations.

KEY WORDS: Physiological adaptation · Physiological reaction time · Phosphate uptake · Cyanobacteria · Growth optimization · Phytoplankton · Environmental fluctuations

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INTRODUCTION definable with kinetic constants like the half satu- ration constant, maximum uptake velocity and spe- Cyanobacterial blooms are frequently stable and cific affinity (e.g. Vadstein & Olsen 1989, Tanaka et resilient in lakes (Scheffer et al. 1997, Bonilla et al. al. 2004, Tambi et al. 2009). However, other studies 2012), even after dissolved nutrient depletion have addressed the complex features of phosphate (Catherine et al. 2008). Cyanobacterial dominance uptake regulation by cyanobacteria. These organ- is generally explained by competitive advantages isms sense phosphate availability in the environ- in ex ploiting low phosphate concentrations, includ- ment by histidine kinases and response regulators, ing high affinity and maximum velocity of cellular which induce the expression of Pho regulon genes uptake systems, and by high nutrient storage that encode high affinity P-binding proteins, alka- capacity (Istvánovics et al. 2000, Moutin et al. 2002, line phosphatase and other relevant proteins Antenucci et al. 2005, Burford & O’Donohue 2006, (Suzuki et al. 2004, Schwarz & Forchhammer 2005, Casey et al. 2009, Posselt et al. 2009). These advan- Juntarajumnong et al. 2007, Orchard et al. 2009, tages are based on the assumption that phyto- Pitt et al. 2010). Additional complexities in phos- plankters have species-specific uptake properties phate regulation are related to changes in the

*Email: [email protected] © Inter-Research 2012 · www.int-res.com 252 Aquat Microb Ecol 67: 251–263, 2012

kinetics and energetics of uptake systems during cant alteration in phosphate uptake properties (LP responses to sudden phosphate fluctuations (Fal - and [Pe]A) can be considered an indicator of tR. The tR kner et al. 1995, Wagner et al. 1995, Falkner & to transient phosphate availability is crucial for Falkner 2003, Falkner et al. 2006, Aubriot et al. understanding the effect of short-term nutrient fluc- 2011). When phosphate is available, the external tuations on cyanobacterial growth under apparently nutrient is removed by phytoplankton down to low suboptimal conditions. However, the tR of cyano - nanomolar threshold values ([Pe]A), below which bacterial populations to available phosphate is still incorporation is not energetically possible (Falkner an undefined subject. et al. 1989, 1994). Phosphate uptake takes place In the present study, we investigated the tR of only if the external concentration surpasses the adaptive phosphate uptake behaviour of cyano- threshold value (Rigler 1956, Falkner et al. 1989, bacteria-dominated phytoplankton in response to

Aubriot et al. 2000). Adaptive kinetic responses are phosphate fluctuations and the role of tR in cyano - characterised by alterations in threshold values and bacterial blooms. The analysis of a wide range of tE uptake rates, which reflect adjustments of cel lular and the effect on phosphate uptake parameters energy conversion (Falkner et al. 1989, 1993). allows the identification of the tR of phytoplankton Molecular processes in which the Pho regulon as the onset of an adaptive response. We performed expression is involved are accompanied by mem- [32P] uptake experiments with a planktonic commu- brane processes that affect the functional organisa- nity from a eutrophic lake composed mainly of fila- tion of proteins. For this reason, it is not appropriate mentous cyanobacteria. The community was sub- to employ the Michaelis kinetic model based on a jected to different patterns of phosphate additions particular enzyme description (Falkner et al. 1995, in order to produce increasing nutrient tE from min- Button 1998, Bonachela et al. 2011). This model is utes to several hours but with the same amount of valid for initial velocities and predicts uptake to added phosphate. The results are discussed in stop at zero substrate concentration, therefore not terms of how the short tR of phosphate uptake reg- accounting for the existence of a (variable) thresh- ulation by bloom-forming cyanobacteria may allow old value (Falkner et al. 1995). Alternatively, phos- for rapid growth optimisation in fluctuating envi- phate uptake can be analyzed with a theoretically ronments. and experimentally validated proportional flow– force relationship (Thellier 1970, Falkner et al. 1989). This model is flexible because it considers MATERIALS AND METHODS the following variable physiological parameters that are altered in response to external conditions: Study area the membrane conductivity coefficient, LP, which is proportional to the maximum velocity of the uptake Six experiments (A to F) were performed during process, and the threshold phosphate concentration summer and autumn (December to May) of 2004,

[Pe]A (Falkner et al. 1989, 1994). The flow–force 2006 and 2007, with water samples from Lago Rodó, model takes a more realistic approach because it a shallow (1.5 m mean depth) hypereutrophic urban allows investigation of the time course of flexible lake in Montevideo, Uruguay (Aubriot et al. 2000). phosphate uptake behaviour of phytoplankton in The water column was characterised by in situ fluctuating environments. measurements of photosynthetically available radia- Cyanobacteria growth optimisation is related to tion (PAR) (using a LI-192SA 4π quantum sensor rapid adaptive responses in fluctuating environ- recorded with a LI-250 LI-COR datalogger) to cal - −1 ments. For this optimisation to occur, the timescale of culate the light extinction coefficient (Kd, m ) as well the physiological response of cyanobacteria (reaction as of temperature, dissolved oxygen and pH (Horiba time [tR]) must match the duration of transient envi- D-25). Subsurface samples of 2 to 5 l were taken with ronmental nutrient fluctuations (exposure time [tE]) dark bottles and immediately transported to the (Stomp et al. 2008, Aubriot et al. 2011). Accordingly, laboratory (within ca. 30 min). Samples were pro- a recent study showed that cyanobacteria-dominated cessed by filtering 200 ml aliquots through Whatman phytoplankton respond to phosphate tE by a physio- glass fibre filters (GF/F) pre-soaked in ultrapure logical adaptation in which organisms reduce the water. The unfiltered sample, the filtrate and the uptake rate and increase the threshold value toward filter with the particulate material were stored at an energetically favourable state (Aubriot et al. −20°C for analyses of total and dissolved nutrients

2011). The minimum tE required to provoke a signifi- and chlorophyll a (chl a). Aubriot & Bonilla: Rapid P-uptake regulation in cyanobacterial blooms 253

Spectrophotometric determinations below) for the alteration of phosphate uptake para-

meters. To achieve a different tE, the same total Orthophosphate and dissolved inorganic nitrogen [32P] labelled phosphate was applied to 2 subsam-

(DIN) were analyzed following Strickland & Parsons ples (S1 and S2) with modifications in the pulse pat- (1972). Total phosphorus (TP) and total nitrogen (TN) tern (Fig. 1). In Expt A, the effect of short tE was concentrations were determined after persulfate oxi- evaluated by applying 2 equal, low external phos- 32 dation according to Valderrama (1981). Chl a extrac- phorus isotope [ Pe] concentrations (1.8 µM) to tion was quantified according to Nusch (1980). each subsample; the second pulse was applied 3.3 h after the first phosphate addition. In Expts B to D,

phytoplankton were exposed to higher tE by apply- Analysis of phytoplankton

Expt Subsamples Imprinting pulses Test pulses Growth Phytoplankton samples were pre- ] A Effect of short tE e 1.8 µM served with acid Lugol’s solution and P 32 kept in darkness for up to 3 mo until pro- [ S1 cessing. Taxonomic identification was ] e 1.8 µM performed with an Olympus optical P 32 [ microscope, using 400 to 1000× magnifi- S2 cation, to the lowest possible taxonomic Effect of long t level (species or genus) or morphotype B E ] (eukaryotic phytoplankton with diame- e 10× 1 µM 2 µM 2 µM P 10 89 32 67 ter <5 µm). Phytoplankton was counted [ 345 S1 12 in random fields according to Guillard ] e 10 µM 2 µM 2 µM (1978), and taxa richness was calculated P 32 [ with rarefaction curves (Gotelli & Col- S2 31 well 2010). Calculation of population and +[ Pe ]

C, D ] 3 −1 e 10× 1 µM Day 1 Day 2 community biovolume (mm l ) was 10 P 9 2 µM 7 8 S1 S1 S1 32 6 based on the geometric shape of organ- [ 3 4 5 S1 1 2

isms by using average sizes of 10 to ] e 10 µM P 30 individuals per taxon (Hillebrand et S2 S2 S2

32 2 µM [ al. 1999). S2

Day 1 2 3

Effect of consecutive tE 31 E +[ Pe ] Experimental design ] 1 µM 3 µM 2 µM 2 µM e P S1 S1 S1 32 [ Phosphate uptake was measured in 2 S1 ] identical subsamples of 50 ml (S1 and S2) e 3 µM 1 µM 2 µM 2 µM P S2 S2 S2 obtained from the original lake sample 32 S2 [ (Fig. 1), which were pre-filtered through Day 1 2 3 80 µm mesh to remove large zooplank- ] Day 1 Day 2 F e 1 µM ton. Subsamples were gently mixed with P 0.5 µM 0.2 µM 32 [ a magnetic stirrer and illuminated S1 −2 −1 (150 µmol photons m s ) immediately ] e

P 1 µM 0.5 µM 0.2 µM

after arrival in the laboratory. Acclima- 32 S [ tion to mean lake temperature (25 ± 1°C) 2 was allowed 30 min before experiments. Time Phosphate deficiency was confirmed by Fig. 1. Schematic experimental design. Imprinting and test [32P] pulse treat- activated phosphate influx rates and ments are summarised for all experiments (A to F). The total [32P] added to threshold values in the nanomolar range. subsamples S1 and S2 was the same at the end of imprinting and test pro - The experiments were designed to cedure. The first cylinder represents the sample obtained from hyper - determine the t of cyanobacteria-dom- eutrophic Lake Rodó, Uruguay. Growth measurements were conducted R on lake water samples for Expts C to E using non-radioactive K HPO inated phytoplankton subjected to a 2 4 ([31Pe]). See ‘Materials and methods’ for further details. Pe: external wide range of tE (see ‘Data analysis’ phosphorus; tE: exposure time 254 Aquat Microb Ecol 67: 251–263, 2012

ing a sequence of 10 additions of 1 µM phosphate Measurement of growth rate to subsample S1, with each pulse applied every 6 min. The same amount of phosphate (10 µM) was Samples for growth experiments were obtained given to S2 in 1 addition. This ‘imprinting’ proce- from the same original lake samples used for chemi- dure generated different tE values in S1 and S2 with cal analyses and uptake experiments (Fig. 1). Sub- the same amount of total phosphate (Fig. 1). To samples were acclimated and processed as indicated compare the resulting uptake kinetic properties in ‘Experimental design’. Growth rates were evalu- between S1 and S2, the uptake behaviour was then ated on 4 occasions (26 February 2004; 30 March analysed after applying equal phosphate concen- 2004; data not shown; 16 March 2004; and 6 Febru- trations to each subsample (‘test pulses’) (Fig. 1). ary 2007) and run in parallel to the corresponding The fact that equal amounts of total phosphate [32P] uptake experiments. The imprinting and test were applied to S1 and S2 excludes an interpretation phosphate pulse treatments described in Expts C, D of the effects as exhausting phosphate uptake and E were replicated in triplet subsamples of 350 ml 31 capacity. In all experiments (A to F), test pulses each but using non-radioactive K2HPO4 ([ Pe]). A were applied after the achievement of the [Pe]A blank subsample incubation (in triplet) was used as a threshold value of the imprinting treatment. i.e. at control. Replicates were gently bubbled with humid- constant/ asymptotic external phosphate concentra- ified (Milli-Q water) pre-filtered (GF/F sterilised) air tion. Expts C and D were designed to show the and incubated at the same light and temperature long-term effects of imprinting tE, by applying 1 conditions as in the uptake experiments. Growth test pulse of 2 µM phosphate after approx. 17 h. rate was evaluated by measuring optical density (at Expts E and F were designed to examine how con- 750 nm) twice a day. Chl a concentration was deter- secutive tE result in the progressive adaptive mined in each sub sample at the beginning and end response (Fig. 1). The same amount of phosphate was of incubation. applied by giving a smaller and greater imprinting pulse to each subsample but added in reverse tempo- ral order as follows: in S1, the smaller phosphate Theoretical considerations pulse was added first, then the greater amount, and vice versa in S2 (i.e. 1 and 3 µM in Expt E and 0.5 and The dependence of net uptake rates on external 1 µM in Expt F). In all experiments, test pulses were phosphate concentration is assessed by a relation- applied after the achievement of [Pe]A; 2 test pulses of ship between the uptake rate, JP (given in µmol inter- 2 µM phosphate each in Expt E and 1 test pulse of nal P mg−1 chl a h−1), and the driving force of this pro- 0.2 µM at 24 h after the beginning of imprinting cess (Thellier 1970, Falkner et al. 1989), expressed as treatment in Expt F. follows:

Jp = − d[Pe]/dt = LP (log[Pe] − log[Pe]A) (1)

Measurement of phosphate uptake where [Pe] is the external phosphate concentration, LP is a conductivity coefficient proportional to the Net phosphate uptake was measured using maximum uptake velocity (Falkner et al. 1994, 1995), 32 micromolar [ P] (Aubriot et al. 2000). Phosphate and [Pe]A is the threshold concentration at which removal by phytoplankton was measured in 0.8 to uptake ceases due to energetic reasons (Falkner et 1.5 ml aliquots filtered through Millipore HA filters al. 1989, 1994). (0.45 µm pore size) and following a time sequence The linearity of the flow–force relation can be lost of 0.5, 2, 5, 10, 15, 20 and 35 min, then every 20 to in the transient adaptive operation mode of uptake 30 min, as indicated in the figures. Each sampling systems (Falkner et al. 2006, Aubriot et al. 2011). time point and lags were timed (±2 s). The Under these conditions, uptake behaviour during the absence of prokaryotic cell interference in the fil- self-organisation process can be modelled when non- trate was previously tested by Aubriot et al. (2011). linear terms are added to Eq. (1), leading to the fol- Our study integrated phosphate removal by phyto- lowing relation (Thellier 1970): planktonic prokaryotes, eukaryotes and bacterio- J − d[P ]/dt = L (log[P ] − log[P ] ) plankton. The radioactivity in the filtrate was mea- p = e P e e A + L (log[P ] − log[P ] )m (2) sured in water using a Beckman liquid scintillation e e A counter (LS 6000). The detection limit of this where m > 1; m characterises (in addition to conduc- method is 1 nM phosphate. tivity coefficients L and LP) the dependence of the Aubriot & Bonilla: Rapid P-uptake regulation in cyanobacterial blooms 255

phosphate influx rate on the driving force during an RESULTS adaptive process (Falkner et al. 2006). A satisfactory fit (r2 ≥ 0.9) was found for either m = 3 or 5 (see Table Lake conditions and phytoplankton community 2). A more detailed theoretical explanation is found elsewhere (Falkner et al. 1995, Wagner et al. 1995, Lake temperature did not exhibit large variations Aubriot et al. 2011). among summer samplings (25.2 ± 1.9°C), except in Expt F (performed in autumn) (Table 1). The water column was always mixed, and the euphotic zone −1 Data analysis covered almost the entire depth (Kd = 6.2 ± 2.0 m ). Dissolved oxygen was lower at the lake bottom The flow–force relationships were fitted using (13.2 ± 5.6 and 5.1 ± 3.4 mg l−1 at the surface and bot- MLAB (Mathematical Modelling System, Civilized tom, respectively). The orthophosphate concentra- Software) and following Falkner et al. (2006) and tion was below the limit of spectrophotometric detec-

Aubriot et al. (2011). The replot of JP versus log[Pe] tion (<0.3 µM), except in Expt E (0.6 µM). DIN values was performed according to Thellier (1970) (Thellier were high and variable (27.3 ± 17.8 µM) and con- plot), in which the slope corresponds to LP, expressed sisted of up to 53% nitrate. Low nitrate concen - in µmol internal P mg−1 chl a h−1 (Wagner et al. 1995). trations were determined at the end of the study

The tE is defined as the period of time between period (<7.1 µM). The mean TP concentration was 32 [ Pe] phosphate addition and the moment at which 6.2 ± 1.3 µM, and the concentration of TN varied 32 [Pe]A is attained (when 99% of the added [ Pe] phos- between 130.5 and 308.2 µM. The chl a concentration phate is incorporated by phytoplankton after a ranged from 107.8 ± 4.4 µg l−1 at the end of the study −1 pulse). The cumulative tE (∑tE) is the sum of tE values (Expt F) to the highest value of 411.6 ± 9.8 µg l on determined in each phosphate pulse. tR is the mini- the sampling date of Expt D (Table 1). mum tE required to provoke a significant alteration in The highest number of taxa belonged to cyanobac- the phosphate uptake properties LP and [Pe]A. [Pe]A teria, followed by diatoms, cryptophytes, eugleno- increase was calculated as the amount of change phytes, dinoflagellates and chlorophytes (Fig. 2). The from the [Pe]A of 1 pulse to the [Pe]A obtained after the high phytoplankton biomass is indicated by the chl a next phosphate addition. and biovolume values (chl a: >100 µg l−1; biovolume: The reliability of the method has been validated 27 to 130 mm3 l−1). Filamentous cyanobacteria were previously (Aubriot et al. 2000, 2011). Significant the dominant group (>83% of total biovolume) in all

differences of [Pe]A between subsamples were deter- samplings, consisting of Planktothrix agardhii, Raphid- mined due to a lack of overlap of the 95% confidence iopsis mediterranea and Planktolyngbya sp. (Fig. 2). intervals (CI95%) calculated from the flow–force fit- ting of each time course of phosphate removal. LP and growth rate differences among treatments were Influence of increasing phosphate tE on tested with a general linear model combined with uptake parameters analysis of covariance (GLM-ANCOVA) and

Kruskal-Wallis 1-way analysis of variance, respec- A short tE of 15 to 24 min was obtained after 2 con- tively (Statistica 6.0, StatSoft). secutive phosphate pulses, which resulted in practi-

Table 1. Physicochemical characteristics of Lago Rodó at each corresponding sampling date. Expt: experiment; T: water temperature; DO: dissolved oxygen at surface and bottom, respectively; TN: total nitrogen; TP: total phosphorus; Chl a: + − − 3− chlorophyll a; −: below detection limits of standard methods (NH4 : 0.7 µM N; NO2 : 0.2 µM N; NO3 : 7.1 µM N; PO4 : 0.3 µM P; TN: 7.1 µM N; TP: 0.3 µM P; chl a: 0.1 µg l−1)

+ − − 3− Expt Date T DO NH4 NO2 NO3 PO4 TN TP Chl a (d.mo.yr) (°C) (mg l−1) (µM N) (µM N) (µM N) (µM P) (µM N) (µM P) (µg l−1)

A 11.01.06 24.0 9.3−6.7 2.1 3.8 43.2 − 308.2 6.2 240.3 ± 1.0 B 29.01.04 26.8 10.0−1.0 2.2 1.4 38.5 − 130.5 4.7 220.0 ± 6.2 C 26.02.04 26.8 20.0−10.1 4.4 0.7 13.8 − 161.9 6.1 308.5 ± 10.9 D 16.03.04 24.8 20.0−5.0 2.5 0.8 16.4 − 209.8 6.3 411.6 ± 9.8 E 06.02.07 27.5 13.6−1.6 16.9 − − 0.6 170.4 8.6 161.5 ± 7.8 F 11.05.06 16.0 6.6−6.4 3.6 − − − 237.3 5.3 107.8 ± 4.4 256 Aquat Microb Ecol 67: 251–263, 2012

140 cally identical uptake properties between subsam- a

) 120 ples S1 and S2 (Expt A) (Fig. 3, Table 2). Phytoplank- –1

l ton dominated by Planktothrix agardhii achieved 3 100 nanomolar threshold values ([Pe]A: 50 and 55 nM for 80 the first and second pulse, respectively). The maxi-

60 mum uptake rate (LP) was lower after the second 40 pulse, indicating the beginning of uptake kinetic alterations (Fig. 3 inset) (mean Lp = 45 and 31 nM

biovolume (mm 20 Total phytoplankton Total min−1 for the first and second pulse, respectively; 0 GLM-ANCOVA, F = 26.4, p < 0.001). 100 b In Expt B, the imprinting pulse sequence applied to S1 (10 additions of 1 µM phosphate) provoked an 80 extension of tE relative to S2, which had received a single phosphate pulse of 10 µM (tE = 91 and 43 min 60 for S1 and S2, respectively) (Fig. 4a). As a result, the uptake rate in S decreased, as evidenced by a >2- 40 1

biovolume fold LP reduction (Table 2). The initial differences

20 between S1 and S2 were amplified after applying the

% total phytoplankton test pulses. Phytoplankton of S showed a progres- 1 0 sive, significant alteration of uptake properties (Fig. A B CDE F 4). The phytoplankton that experienced a longer t Expt E (S1) decreased LP 4-fold after the first test pulse. The Planktothrix agardhii Other cyanobacteria Planktolyngbya sp. Eukaryotes Raphidiopsis mediterranea t impr test Fig. 2. Phytoplankton from Lago Rodó identified in each E experiment (A to F). (a) Total phytoplankton biovolume. 2.5 a (b) Relative contribution of main taxa to total phytoplankton biovolume 2.0 1.5 ] (µM) tE e 1.0

50 [P a 0.5 1.5 40 30 0.0 P impr test J t 20 E b 10.0 1.0 b 10 2.0

] (µM) 0 e –7.5 –7.0 –6.5 –6.0 1.5 [P log [P ] 0.5 e ] (µM)

e 1.0 [P 0.5 0.0 0 12345678 0.0 Time (h) 0 2 4 6 8 10 12 25 Fig. 3. Expt A. Time course of [32P] phosphate removal in Time (h) 32 phytoplankton subsamples from Lago Rodó (d: S1; s: S2) Fig. 4. Expt B. Time course of [ P] phosphate removal in 2 during 2 consecutive phosphate pulses. Top horizontal bars identical phytoplankton subsamples (d: S1; s: S2) of Lago represent the exposure time (tE) to external phosphate ([Pe]) Rodó. (a) S1 was exposed to 10 imprinting pulses (impr) of 32 during each pulse (black bars: S1; white bars: S2). Curves 1 µM and (b) S2 to 1 pulse of 10 µM [ P] phosphate. Two test represent the best fit linear equation applied to time-courses pulses of 2 µM phosphate (test) were applied to each sub- of each subsample. Kinetic parameters are shown in Table 2. sample immediately after the attainment of the correspond- Inset: Thellier plot of the concentration dependence of in - ing threshold values. Curves represent the best fit obtained corporation rates (JP) obtained from the time course of for each subsample with linear and nonlinear equations phosphate removal in the (a) first and (b) second pulses. JP (Eqs. 1 & 2, respectively). Kinetic parameters are shown in −1 −1 is given in µmol Pi mg chl a h ; log[Pe] is expressed Table 2. Top horizontal bars represent the exposure time (tE) relative to 1 M to external phosphate ([Pe]) during each pulse Aubriot & Bonilla: Rapid P-uptake regulation in cyanobacterial blooms 257

Table 2. Kinetic parameters of phosphate removal by phytoplankton from Lago Rodó. Expt: experiment, Sbs: subsample, [32P]: initial phosphate concentration labelled with the radioactive 32P isotope. Kinetic parameters of the linear and nonlinear curve fitting of flow–force relationship. LP and L: conductivity coefficients; [Pe]A: threshold value ± CI95% of phosphate uptake; tE: exposure time to external phosphate concentrations; r2: determination coefficient. Values of m for Eq. (2) are in brackets in col- + umn L. *Significant differences between subsamples S1 and S2 within the same pulse treatment according to CI95%. Signifi- x cant differences between pulses within the same subsample according to CI95%. LP: significant differences between subsam- xx ples S1 and S2 within the same pulse treatment, LP: significant differences between pulses within the same subsample (GLM- ANCOVA, p < 0.01)

32 2 Expt Pulse treatment Sbs [ P] pulse LP L [Pe]A tE r (µM) (nM min−1) (nM min−1) (nM) (min)

xx A First S1 1.8 45.9 − 48 ± 33 15 0.999 xx S2 1.8 45.0 − 52 ± 19 17 1.000 xx Second S1 1.8 30.8 − 56 ± 17 24 1.000 xx S2 1.8 31.1 − 54 ± 24 24 1.000 x + B Imprinting S1 1.0 (10 times) 17.3 0.05 (5) 41 ± 26 91 1.000 x S2 10.0 (once) 37.0 0.05 (5) 46 ± 27 43 1.000 x + First test S1 2.0 4.0 0.10 (5) 69 ± 64 142 0.998 x S2 2.0 26.0 − 40 ± 79 27 0.997 x + Second test S1 2.0 23.0 0.10 (5) 1201 ± 121* 152 0.962 x S2 2.0 19.0 − 62 ± 83* 44 0.997 x + C Imprinting S1 1.0 (10 times) 1.9 0.05 (3) 63 ± 46 352 0.998 x S2 10.0 (once) 3.9 2.00 (5) 35 ± 43 60 1.000 x + First test S1 2.0 0.7 1.00 (3) 269 ± 127 1644 0.980 x S2 2.0 1.3 0.25 (5) 91 ± 73 338 0.997 x + D Imprinting S1 1.0 (10 times) 1.0 0.10 (3) 62 ± 54 435 0.998 x S2 10.0 (once) 3.3 2.25 (5) 51 ± 30 74 1.000 x + First test S1 2.0 2.1 9.00 (3) 501 ± 90* 950 0.991 x S2 2.0 0.7 0.20 (5) 86 ± 99* 543 0.993 x + E First imprinting S1 1.0 25.7 − 25 ± 33 15 0.998 x + S2 3.0 19.1 − 27 ± 98 40 0.998 x + Second imprinting S1 3.0 18.3 0.50 (3) 39 ± 22 39 1.000 x + S2 1.0 15.8 0.50 (3) 44 ± 14 29 1.000 x + First test S1 2.0 12.2 0.60 (3) 70 ± 121 62 0.994 x + S2 2.0 9.3 0.50 (3) 90 ± 40 91 0.999 x + Second test S1 2.0 5.7 1.20 (5) 343 ± 34* 347 0.999 x + S2 2.0 5.2 1.50 (5) 493 ± 34* 505 0.998 x F First imprinting S1 0.5 1.8 0.10 (3) 11 ± 20 75 0.997 x + S2 1.0 0.9 0.01 (3) 20 ± 18 223 0.999 x Second imprinting S1 1.0 0.5 0.10 (5) 76 ± 47 − 0.993 x + S2 0.5 0.1 0.10 (5) 57 ± 43 − 1.000 x Test S1 0.2 5.8 − 89 ± 4* 129 0.999 x + S2 0.2 3.2 − 186 ± 32* 159 0.822 effect increased after the second test addition, when and D, respectively) (Table 2). The test pulses uptake ceased at a threshold value 20-fold higher revealed significant differences in uptake behaviour than the parallel subsample S2 (Table 2). Conse- between subsamples the next day (Fig. 5). S1, which quently, organisms from S1 incorporated less phos- experienced a longer tE the day before, ceased phate than those of S2, as indicated by the threshold uptake at a threshold 10-fold higher than the parallel values ([Pe]A = 1201 ± 121 nM and 62 ± 83 nM phos- subsample S2 (Fig. 5b, Table 2). This higher threshold phate, ±1 CI95%, for S1 and S2, respectively), which value was stable for several hours, as confirmed on remained constantly higher for >14 h (Fig. 4). Phyto- the third day. plankton was again dominated by Planktothrix The adaptive response of cyanobacteria, indicated agardhii (61% of biovolume) (Fig. 2). by [Pe]A increase and LP decrease, had consequences The effect of several hours of tE was tested with a for growth (Expts C, D and E; Table 3). The higher less uptake-active community than previous experi- threshold levels determined in S1 (Expts C and D) ments. In this case, tE was 352 to 435 min (S1 Expts C and S2 (Expt E) were followed by a significant higher 258 Aquat Microb Ecol 67: 251–263, 2012

impr test between S1 and S2 resulted in similar uptake kinetics tE (data not shown). In Expts C and D the phytoplank- 10.0 a 2.5 ton community was dominated by Raphidiopsis mediterranea (84 and 89% of biovolume, respec- 2.0 tively) and co-dominated with Planktothrix agardhii 1.5 in Expt E (35 and 44% of biovolume, respectively) ] (µM) e 1.0 (Fig. 2). [P 0.5 0.0 tE Cumulative effect of tE on the adaptive response 10.0 2.5 b The accumulated tE during a sequence of phos- 2.0 phate pulses resulted in a progressive adaptive 1.5 response (Expts E and F). The same imprinting phos- ] (µM) e 1.0 phate concentrations applied in reverse order (S1: [P smaller then greater and S : greater then smaller) led 0.5 2 to a differentiation between subsamples (Fig. 6). S2, 0.0 0 51015 20 25 30 35 40 45 50 which first received the greater pulse, responded Time (h) with a gradual reduction of phosphate uptake activ- Fig. 5. Expts C and D. Time course of [32P] phosphate re- ity. In Expt E (Fig. 6a), the resulting tE was 2-fold moval in 2 identical phytoplankton subsamples (d: S1; s: S2) of Lago Rodó in (a) Expt C and (b) Expt D. S1 was exposed to impr test 10 imprinting pulses of 1 µM, and S2 was exposed to 1 pulse t of 10 µM [32P] phosphate (impr). Initial and final concentra- E 3.0 tions of external phosphate ([Pe]) are shown after each pulse a of 1 µM. At 17 h after initiation, 1 pulse of 2 µM phosphate 2.5 was applied to S and S (test). Curves represent the best fit 1 2 2.0 obtained for each subsample with the nonlinear Eq. (2) (Table 2). Top horizontal bars represent the exposure time 1.5 ] (µM) (tE) to external phosphate during each pulse (black bars: S1; e 1.0 white bars: S2) [P 0.5 0.0 0 1 23 10 11 μ 456789 growth rate (Expt C: = 0.48 ± 0.03 and 0.11 ± impr test −1 −1 t 0.05 d , Expt D: μ = 0.73 ± 0.04 and 0.56 ± 0.02 d E −1 and Expt E: μ = 0.09 ± 0.01 and 0.12 ± 0.01 d for S1 1.0 b and S2, respectively; Table 3) and chl a production 0.8 in Expts C and D (Table 3). Conversely, differences 0.6 in growth rate were not found when the same tE ] (µM) e 0.4 [P 0.2 Table 3. Comparison of growth rates (μ) and chlorophyll a production (chl a) between subsamples (Sbs) S1 and S2 of 0.0 Expts C, D and E (means ± SD). Chl a was measured at the 0 2 468 10 23 24 25 26 27 28 end of incubation (49 h). Differences between subsamples Time (h) were all significant (Kruskal-Wallis test, p < 0.05) except for Fig. 6. Expts E and F. Time course of [32P] phosphate removal chl a values of Expt E in 2 identical phytoplankton subsamples (d: S1; s: S2) from Lago Rodó. (a) Expt E: S1 was exposed to 1 pulse of 1 µM fol- Expt Date Sbs μ Chl a lowed by the addition of 3 µM; S2 was exposed to 1 pulse of 32 (d.mo.yr) (d−1) (µg l−1) 3 µM followed by the application of 1 µM [ P] phosphate (impr). Then, both subsamples were subjected to 2 test phos- phate pulses of 2 µM (test). (b) Expt F: S was exposed to 1 C 26.02.04 S1 0.48 ± 0.03 524.5 ± 35.5 1 pulse of 0.5 µM followed by 1 µM addition, and S had 1 S2 0.11 ± 0.05 391.3 ± 11.8 2 pulse of 1 µM applied followed by 0.5 µM phosphate (impr). D 16.03.04 S1 0.73 ± 0.04 475.2 ± 15.6 At 24 h after the first phosphate addition, both subsamples S2 0.56 ± 0.02 375.7 ± 31.3 were subjected to 1 phosphate pulse of 0.2 µM (test). Curves E 06.02.07 S1 0.09 ± 0.01 220.0 ± 67.4 represent the best fit using linear and nonlinear equations S2 0.12 ± 0.01 250.1 ± 63.0 (Table 2). See Fig. 5 for further details Aubriot & Bonilla: Rapid P-uptake regulation in cyanobacterial blooms 259

higher in the greater-then-smaller imprinting pattern eral, initial [Pe]A values were 42 ± 16 nM, with a min- (S2) than in the smaller-then-greater one (S1) (tE: 15 imum value of 11 nM phosphate, before the adaptive and 29 min in S1 and S2, respectively, after 1 µM response took place. After the adaptive response, phosphate). Also, the LP values obtained from the [Pe]A values increased up to 3 orders of magnitude imprinting pulses of each subsample were different and remained high for >15 h. The general tendency

(Table 2), evidenced in the cumulative tE (∑tE: 54 and shown in Fig. 7a illustrates that the adaptive 69 min for the S1 and S2 imprinting pulses, respec- response found in cyanobacteria-dominated phyto- tively). This difference was amplified after the test plankton begins after ca. 25 min of exposure to phos- pulses, and as a consequence, S2 showed the higher phate since all threshold values are higher after that threshold value (Fig. 6a). The low phosphate uptake time. The fitted equation intercepts the tE axis at activity shown by phytoplankton in Expt F resulted in 18 min (no signifi cant effect), and an almost 2-fold an extended tE (Fig. 6b). The high [Pe]A values were increase of [Pe]A was determined at a tE of 29 min. stable for >24 h after the imprinting procedure was The maximum [Pe]A was 16.4-fold higher after a tE of initiated. Phytoplankton of both subsamples again 150 min. Fig. 7b shows the significant decrease of LP 2 showed significant differences in threshold values with tE (r = 0.70, n = 30, F = 66, p < 0.0001). The max- and LP. The community was again dominated by imum LP values were obtained within 15 to 17 min of Planktothrix agardhii. tE. All values of uptake rate were lower after this tE The tR is obtained when the effect of tE on thresh- range. old values and maximum uptake rates from all of the experiments is analyzed (Fig. 7). The increase of threshold values positively correlated with tE DISCUSSION (Fig. 7a) (r2 = 0.89, n = 18, F = 135, p < 0.0001). In gen- Reaction time of bloom-forming cyanobacteria 1.6 a to available phosphate

1.2 Our results revealed that bloom-forming cyano- bacteria have a rapid reaction to available phos- phate, which begins within 15 to 25 min of nutrient

increase 0.8 A

] exposure. When the tE to external phosphate sur- e passes tR, cyanobacteria progressively adjust their 0.4 up take properties (e.g. Fig. 3). The LP and [Pe]A are log [P inverse and directly proportional to tE, respectively. 0.0 This proportional effect of tE on the adaptive re- 2.5 sponse allowed the attainment of new uptake proper- b ties that were stable for several hours, which in turn 2.0 affected growth rate. It is remarkable that the higher growth rate found with longer t was achieved when 1.5 E

P relatively less phosphate was taken up in comparison L 1.0 with the shorter tE treatment, which confirms the

log energetic benefit of the adaptive response. There- 0.5 fore, rapid phosphate fluctuations on the timescale of 0.0 minutes have direct consequences for further growth. –0.5 The sequence of small pulses applied within a 10 100 1000 short time interval determined that the cyanobacte- tE (min) ria-dominated community was not able to achieve Fig. 7. Adaptive response of phytoplankton, assessed as (a) the increase of threshold value for uptake of external phos- the threshold value after each pulse, so the organ- phate ([Pe]A) as a function of the exposure time (tE) during isms experienced a persistent elevated external the same phosphate addition. The best fit was obtained with phosphate concentration over time. This relatively − 2 the equation y = 0.6002 + 0.2074ln(x); r = 0.89, F = 135, n = long exposure to phosphate may occur in a lake 18, p < 0.0001. h: outliers. (b) Decrease of L as a function P when phosphate dilutes in the water column (Aubriot of tE. Best fit was obtained with the equation y = 2.5946 − 0.3805ln(x); r2 = 0.70, F = 66, n = 30, p < 0.0001. Dashed lines et al. 2000), for example during nutrient release from represent 95% confidence interval sediments or inflow events after rainfall (Glibert et al. 260 Aquat Microb Ecol 67: 251–263, 2012

2008). In contrast, very short nutrient pulse events the same range as that of cyanobacteria. Phytoplank- may occur during local small inputs in a lake (e.g. ton composition and biomass in our study was typical excretion by ), in which the uptake prop- of hypereutrophic ecosystems, dominated by filamen- erties may remain relatively unchanged if tE ≤ tR (e.g. tous cyanobacteria (>83% of biovolume), al though Fig. 3). Therefore, our results offer a new perspective bacterioplankton and eukaryote phytoplankton were on what can be considered as a significant input of present. Bacterioplankton biomass in Lago Rodó is phosphate into the water column, enough to induce a low in comparison with other hypertrophic lakes physiological and growth response of cyanobacteria. (3.6% of total phytoplankton and bacterioplankton

The tR of cyanobacterial populations and its propor- biovolume) (Sommaruga 1995, Sommaruga & Robarts tional response are crucial variables for future mod- 1997). The adaptive response was found either with els of flexible nutrient uptake that attempt to predict Planktothrix agardhii dominance (Expts A, B and F), bloom formation. Raphidiopsis mediterranea dominance (Expts C and

The adaptive response to tE may be driven by the D) or co-dominance (Expt E). For example, LP was 1 biomass-dominant populations; however, the tR of order of magnitude higher in Expt B than in Expt F, other coexisting planktonic members may be within both under the dominance of P. agardhii. Phytoplank- ton of Lago Rodó was previously found to exhibit a common threshold value that precludes direct competi- Case Increase of exposure time (tE ) Adaptive response Phase 1 Phase 2 tion (Aubriot et al. 2011). The in- 1 tE shorter or equal than tR (min) (hours–days) creased threshold value, which is in- deed available phosphate, persists for Previous threshold No effect on ] a long-term period (days) because no e value is maintained. growth rate Slight decrease in additional uptake takes place. Our uptake rate if t = t E R present and previous results (Aubriot Pre[P ] = new [P ] et al. 2011) suggest that this adaptive Pre[P ] e A e A e A behaviour is paradoxical in the con- text of resource competition theory 2 New uptake Slight effect tE longer than tR properties: uptake on growth rate since potential competitors would rate decrease, slight increase in have more phosphate available in the ] [P

e threshold value environment for their growth. We identified a higher growth rate when relatively less phosphate was taken New [Pe]A Pre[P ] up, which can be explained by a sig- e A nificant increase in the efficiency of t several times longer than t New uptake Stable new energy conversion during the ac- 3 E R properties: uptake uptake rate properties. High quired adapted state (Falkner et al. New [P ] decrease, high growth rate 1989, 1994, Wagner et al. 2000, Plaet-

] [P e A

e threshold value zer et al. 2005). Growth response may [P therefore be more dependent on the Previous [P ] [P ] increase e A e A ability of cyanobacteria to optimise

External[Pe] LP decrease Pre[Pe]A the uptake of phosphate during the New [P ] [P ] = [P ] e A e e A time span of nutrient fluctuation than 0 12Exposure time t E on the amount of nutrient taken up Time (h) Reaction time tR per se. Fig. 8. Conceptual diagram of the influence of exposure time (tE) and reaction time (tR) on adaptive phosphate uptake. Case 1: tE ≤ tR, uptake of a low concen- tration phosphate pulse or by highly uptake-active cyanobac terial populations. Influence of tR on growth Case 2: tE > tR, uptake of higher concentration phosphate pulse or by less uptake-active cyanobacteria. New uptake properties are observed during optimisation Phase 1 and slight effect on growth rate can be determined (Phase 2). Case 3: long tE (>>tR) provoked by a previous phosphate pulse exposure (i.e. sequence Cyanobacteria may reach higher of 10 pulses) or during a persistent pulse addition. New uptake properties are long-term stable (Phases 1 and 2) with resulting growth optimisation (Phase 2). nutrient exploitation efficiency in 2 [Pe]: external phosphate concentration; [Pe]A: threshold concentration at which consecutive phases. The rapid (min- phosphate uptake ceases utes) adaptive response is the initial Aubriot & Bonilla: Rapid P-uptake regulation in cyanobacterial blooms 261

physiological response (Phase 1) to the ongoing 2007, Burut-Archanai et al. 2009, Kimura et al. 2009). nutrient fluctuation that affects further growth Changes in gene expression of phosphate binding (Phase 2). In the first phase, the adaptive response proteins in Synechocystis (Pitt et al. 2010) and Tricho - will not take place if tE is shorter than tR; thus, desmium (Orchard et al. 2009) as well as membrane cyanobacteria maintain the same uptake properties protein patterns in Synechococcus (Wagner et al.

(Fig. 8, Case 1). The adaptive response starts when tE 1994, Scanlan et al. 1997) occurred on 1 to several meets tR with an LP decrease while the threshold days following phosphate enrichment, despite the −1 −1 value is maintained relatively constant (tE < 20 min) wide range of growth rates (~0.2 d and up to 2.0 d (Fig. 8, Case 1). If tE is extended, cyanobacteria of Trichodesmium sp. and Synechococcus sp., increase the threshold value concomitantly with a respectively). Gene expression of alkaline phos- new adjustment of LP (tE > 20 min) (Fig. 8, Case 2). phatases drops faster (by 4 h) than enzyme activity, This kinetic and energetic adaptation takes place which decreases more slowly after phosphate supply toward the optimum efficiency of energy conversion (Orchard et al. 2009). Since the reaction of cyanobac-

(Falkner et al. 1989, Wagner et al. 1995, Plaetzer et teria takes minutes, tR may be attributed to the regu- al. 2005). In the initial phase (tE ≤ tR), uptake proper- lation of the existing array of uptake systems. We ties follow the linear flow–force equation. When this propose that the rapid physiological adaptation stable state is perturbed by longer tE, it results in a allows a ‘real-time’ response within the timescale of transient shift from linear to nonlinear kinetic uptake environmental nutrient fluctuation. This adaptive behaviour. A state of optimum efficiency is achieved process may occur in connection to the second phase, when the linear relationship between phosphate flow by which cyanobacteria are able to acclimate to and the driving force of this process is re-established experienced nutrient fluctuations by the reconstruc- at a higher threshold value (Fig. 8, Case 3) (Falkner tion of uptake systems. It remains to be established if et al. 1989, 1993). This complex regulation of cellular cyanobacteria growing under rapidly fluctuating uptake systems can be explained in single cyanobac- nutrient conditions (i.e. very active phosphate teria by concurrently operating binding sites of mem- removal by dense populations in eutrophic freshwa- brane proteins with different phosphate affinity ters that results in rapid fluctuation) may differ in tR (Wagner et al. 1994, Pitt et al. 2010). The absence of 1 from those that are dominant in more stable environ- of the high-affinity phosphate-binding proteins ments (i.e. oligotrophic seas). (sphX) disables the capacity of a Synechococcus The rapid adaptive behaviour may explain the mutant to adapt to phosphate pulses, although it can predominance of bloom-forming cyanobacteria in still incorporate phosphate down to nanomolar levels freshwaters under apparently suboptimal conditions. (Falkner et al. 1998). In the case of a community, the As shown here and in other studies, Planktothrix transient nonlinear behaviour may be the result of agardhii is able to endure several months with phos- small specific differences in tR during adaptation, for phate concentrations below detection limits (Cather- example between filamentous cyanobacteria and ine et al. 2008). The predominance of bloom forming bacterioplankton. In this short-term process, envi- cyanobacteria must be maintained by phosphate ronmental information is stored in the newly fluctuations that are invisible to lake monitoring acquired kinetic and energetic properties, which programs due to their short timescales (Glibert et al. result in a novel adapted state (Wagner et al. 1995, 2008) and low cyanobacterial threshold values. For Falkner et al. 2006, Aubriot et al. 2011). We hypothe- instance, the minimum threshold value found in our sise, therefore, that this initial response may mark study was 11 nM phosphate, which is below the the onset of more stable, long-lasting acclimated detection limit of standard methods (>100 nM). In our states (hours to days). experiments, the external phosphate concentration In a second longer-term phase (hours to days), the was raised to levels sufficient to double the biomass. biochemical reconstruction of the uptake systems is After such additions, phosphate concentrations expected (i.e. a new array of phosphate binding pro- decreased to nanomolar levels in <1 h. Similarly, the teins). This nutrient acclimated state may be the case dominance of the invasive Cylindrospermopsis raci- in Expts C, D and F, in which the new energetic and borskii is achieved by sporadic (periodic) large or kinetic properties were stable for >24 h with result- small phosphate additions (Posselt et al. 2009), ing growth optimisation (Fig. 8, Case 3). The long- besides its advantageous phosphate uptake proper- term effects observed in these experiments suggest a ties (Isváno vics et al. 2000). Furthermore, a recent new functional organisation of membrane proteins worldwide lake dataset study suggested that C. raci- via Pho regulon expression (Juntarajumnong et al. borskii is physiologically more flexible than P. agard- 262 Aquat Microb Ecol 67: 251–263, 2012

hii, which may explain its ongoing expansion (Piccini Button DK (1998) Nutrient uptake by microorganisms et al. 2011, Bonilla et al. 2012). We suggest that according to kinetic parameters from theory as related to cytoarchitecture. Microbiol Mol Biol Rev 62: 636−645 resilience of cyanobacteria populations in eutrophic Casey JR, Lomas MW, Michelou VK, Dyhrman ST, Orchard fresh waters may be related to their capability to ED, Ammerman JW, Sylvan JB (2009) Phytoplankton respond rapidly in order to match the timescale of taxon-specific orthophosphate (Pi) and ATP utilization in phosphate fluctuations. the western subtropical North Atlantic. Aquat Microb Ecol 58: 31−44 We have shown here the t of adaptive phosphate R Catherine A, Quiblier C, Yéprémian C, Got P and others uptake by natural cyanobacterial blooms. These (2008) Collapse of a Planktothrix agardhii perennial organisms are able to adjust the energetic and bloom and microcystin dynamics in response to reduced kinetic properties of phosphate uptake within min- phosphate concentrations in a temperate lake. FEMS utes. This short t allows for growth optimisation. Microbiol Ecol 65: 61−73 R Falkner R, Falkner G (2003) Distinct adaptivity during phos- Further studies of flexible physiological responses of phate uptake by the cyanobacterium Anabaena vari- cyanobacteria to environmental fluctuations may abilis reflects information processing about preceding identify early stages of bloom formation. The time phosphate supply. J Trace Microprobe Tech 21: 363−375 required for physiological adaptive responses and Falkner G, Falkner R, Schwab A (1989) Bioenergetic charac- terization of transient state phosphate uptake by the the concomitant energetic and kinetic adjustments of cyanobacterium Anacystis nidulans. 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Editorial responsibility: Patricia Glibert, Submitted: May 30, 2012; Accepted: August 28, 2012 Cambridge, Maryland, USA Proofs received from author(s): October 29, 2012