**4. Conclusions**

Recent studies have highlighted the importance of the melittin as a natural drug for different applications, due to its anti-inflammatory, anti-mutagenic, contraceptive, antimicrobial, and even an anticancer effect. Its mass production is, therefore, of great pharmacological interest and, due to this, obtaining bacteria genetically transformed with this gene becomes very desirable. In other studies, melittin cDNA has been inserted into different plasmids: pBR322 [25], pBV220 [26], and pUC118 [27]. Recently, a gene encoding a hybrid peptide with melittin, called LfcinB (1–15)-Melittin (5–12), has been inserted into the pET-32a vector [28]. In addition, in other study, *E. coli* has been transformed with melittin cDNA from *Apis cerana* [4]. In this work, melittin cDNA from *A. mellifera* has been inserted in *E. coli* using the pGEM-T vector. So, its identification and genetic cloning system have been demonstrated, for its 3'T overhangs at the insertion site, proving a binding successful. Furthermore, the mentioned vector has T7 and SP6 RNA promotors that will ensure its expression in the *E. coli* cells used. Also, another study worked with this vector system [4], suggesting the best way for cloning with these kinds of vectors.

However, it must be remembered that in order to obtain melittin in *E. coli* as a final product, the immature peptide prepromelittin should be posttranslationally modified in some steps. In the first step, the enzyme that catalyzes the hydrolysis of prepromelittin to promilittin is supposed to be widely distributed, since prepromellitin has never been obtained in a cell system. Moreover, promelittin has been obtained in venom glands of honeybees fed with radioactive amino acids [9] and in frog oocytes injected with this mRNA from queen bee [29], but melittin has never been obtained in any tissue that does not come from a species of the genus *Apis*.

For all these reasons, it may be thought the other studies that have cloned melittin cDNA in cell systems that do not belong to species of the genus *Apis*, are likely to give rise to the obtaining of promelittin as a final product, as is the case of the present study. It is necessary to clarify which is the final peptide that has been obtained. If promelittin has been finally obtained, the next focus of study could be to design a protocol to convert it to melittin into *E. coli*.


**4.3 Sonic, ultrasonic and ionic toothbrushes**

Sonic tooth brushes bristles vibrate at lower frequency and ultrasonic, indicates a brush action where the bristles vibrate at ultrasonic frequencies (> 20 kHz). An ionic brush releases an electrical charge to the tooth surface which disrupts the attachment of dental plaque [28].


Feature Papers in *NeuroSci*

From Consciousness to Clinical Neurology

Edited by Lucilla Parnetti, Federico Paolini Paoletti and Xavier Gallart-Palau

www.mdpi.com/journal/neurosci/topical\_collections


**Molecular Mechanism of Oxidation of P700 and Suppression of ROS Production in Photosystem I in Response to Electron-Sink Limitations in C3 Plants**

#### **Chikahiro Miyake 1,2**

Received: 13 February 2020; Accepted: 4 March 2020; Published: 11 March 2020

**Abstract:** Photosynthesis fixes CO2 and converts it to sugar, using chemical-energy compounds of both NADPH and ATP, which are produced in the photosynthetic electron transport system. The photosynthetic electron transport system absorbs photon energy to drive electron flow from Photosystem II (PSII) to Photosystem I (PSI). That is, both PSII and PSI are full of electrons. O2 is easily reduced to a superoxide radical (O2 −) at the reducing side, i.e., the acceptor side, of PSI, which is the main production site of reactive oxygen species (ROS) in photosynthetic organisms. ROS-dependent inactivation of PSI in vivo has been reported, where the electrons are accumulated at the acceptor side of PSI by artificial treatments: exposure to low temperature and repetitive short-pulse (rSP) illumination treatment, and the accumulated electrons flow to O2, producing ROS. Recently, my group found that the redox state of the reaction center of chlorophyll P700 in PSI regulates the production of ROS: P700 oxidation suppresses the production of O2 − and prevents PSI inactivation. This is why P700 in PSI is oxidized upon the exposure of photosynthesis organisms to higher light intensity and/or low CO2 conditions, where photosynthesis efficiency decreases. In this study, I introduce a new molecular mechanism for the oxidation of P700 in PSI and suppression of ROS production from the robust relationship between the light and dark reactions of photosynthesis. The accumulated protons in the lumenal space of the thylakoid membrane and the accumulated electrons in the plastoquinone (PQ) pool drive the rate-determining step of the P700 photo-oxidation reduction cycle in PSI from the photo-excited P700 oxidation to the reduction of the oxidized P700, thereby enhancing P700 oxidation.

**Keywords:** P700; P700 oxidation system; photorespiration; photosynthesis; Photosystem I (PSI); reactive oxygen species (ROS); reduction-induced suppression of electron flow (RISE); repetitive short-pulse (SP) illumination (rSP illumination treatment)

#### **1. Introduction**

Numerous researchers have shown that, in photosynthetic organisms, oxidative damage due to enhanced production of reactive oxygen species (ROS) occurs when environmental stress (e.g., extreme low/high temperatures, high salinity, and oligotrophic inorganic components) decreases the photosynthetic efficiency [1–21]. Much of this research has focused on the Mehler reaction, whereby a superoxide radical (O2 −), one of the most important ROS species, is formed via a one-electron reduction of O2 in Photosystem I (PSI) of the chloroplast thylakoid membranes. In addition to PSI, ROS production in Photosystem II (PSII) has long been studied, and numerous studies have been published regarding its relationships to oxidative damage in PSII across oxygenic photosynthetic organisms [22–29]. This review focuses on our recent findings regarding the molecular mechanisms of the production and suppression of ROS in PSI.

We try to answer the following questions in this review: "Is the excess accumulation of electrons in PSI truly harmful to photosynthetic organisms?" and "Does ROS production occur in vivo?" Driever and Baker [30,31] and Ruuska et al. [30,31] reported that the in vivo activity of the Mehler reaction is too small compared to the electron flux in photosynthesis. Additionally, considering the photosynthetic capacity of many photosynthetic organisms, the photosynthetic rate is saturated at a light intensity level corresponding to approximately 25% of natural sunlight or lower [2,3,31–37]. This indicates that photosynthesis regularly proceeds under conditions of an excess photon supply, which can cause the photosynthetic electron transport system to be full of electrons. To address this issue, we asked "How do photosynthetic organisms manage to escape from the accumulation of electrons in PSI?" We then developed a method for suppressing photosynthesis and imitating the accumulation of electrons in PSI. Using this approach, we succeeded in showing how ROS are produced and suppressed in PSI [38]. In what follows, the robust and universal characteristics of the suppression of ROS production in photosynthetic organisms are discussed.

#### **2. The Accumulation of Electrons in the PSI Acceptor Side Promotes ROS Production and Oxidative Damage**

In the photosynthetic electron transport system, the reaction center chlorophyll P700 in PSI functions in the photo-oxidation reduction cycle (Figure 1). Ground state P700 is photoexcited to produce the excited P700 (P700\*), which then donates an electron to the primary electron carrier (A0) to produce the oxidized P700 (P700+). Subsequently, P700+ accepts an electron from the reduced plastocyanin (PC) to regenerate P700. The oxidized PC in turn is reduced by the electrons coming from PSII. The electron in the reduced A0 flows to ferredoxin (Fd) through the electron carriers: A1, Fx, and FA/FB [39]. If a light pulse (light intensity: 20,000 μmol photons m−2 s−1; pulse duration: 400 ms) is applied to C3-sunflower intact leaves in the dark under atmospheric conditions, all P700 molecules are oxidized to P700+ (Figure 2A). P700+ then rapidly decreases during the light pulse, which is due to the occupation of P700\* in the photo-oxidation reduction cycle of P700, indicating the accumulation of electrons at the acceptor side of PSI. If the light pulse is applied every 10 s on C3-sunflower intact leaves (i.e., repetitive short-pulse (rSP) illumination treatment), PSI is deactivated (Figure 3) [38]. Conversely, PSII is not deactivated at all. PSI deactivation is all suppressed when atmospheric O2 partial pressure drops to 2 kPa. The atmospheric rSP illumination treatment then produces a superoxide radical (O2 −) in PSI [32,40–43]. These results suggest that the electrons accumulated on the PSI acceptor side during one light pulse are used for O2 reduction, which triggers the O2 − production reflected as P700\* accumulation. O2 − accumulation occurring over time with rSP illumination treatment indicates progressive PSI oxidative damage to the photosynthetic organism. PSI inactivation due to rSP illumination treatment represents a case of photosynthetically induced CO2 fixation inactivation [9,35,38,44]. Unlike PSII, PSI requires approximately one week for functional recovery following inactivation [7,9,35,36,45,46]. Therefore, PSI inactivation can be fatal to plant growth [7,35,36,42]. Exposure of plants to low temperatures also inactivates PSI and the recovery of PSI function can take several weeks.

**Figure 1.** The photo-oxidation reduction cycle of P700 in Photosystem I (PSI). The reaction center chlorophyll P700 in PSI absorbs the light energy, and the P700 is excited to P700\*. The P700\* donates electrons to the electron carrier, A0, and concomitantly produces the oxidized P700, P700+. The P700+ is reduced by electrons from Photosystem II (PSII) through plastoquinone (PQ), the cytochrome (Cyt) *b6*/*f*-complex, and plastocyanin (PC). The reduced PC directly donates electrons to P700. There are then P700 turnovers in the photo-oxidation reduction cycle of P700 in PSI. The electron on A0 flows to NADP+ to produce NADPH through the electron carriers A1, Fx, FA/FB, and Fd. In the photosynthetic linear electron flow, electrons extracted from H2O oxidation in PSII flow to NADP+ for the production of NADPH.

**Figure 2.** *Cont*.

**Figure 2.** The kinetics of oxidized P700 during short-pulse (SP) illumination in the dark and under actinic light. (**A**) An SP illumination (20,000 μmol photons m−2 s−1, 400 ms) to intact leaves in the dark rapidly oxidizes the reaction center chlorophyll P700 in PSI from the fully reduced state to the maximally oxidized state. Afterwards, oxidized P700 (P700+) is reduced by electrons from PSII, with the accumulation of the excited state of P700 (P700\*), as shown by the increase in shaded area. P700\* can donate an electron to O2, thereby producing a superoxide radical (O2 −) through the one-electron reduction by the electron carriers, A0/A1, Fx, and FA/FB, localized at the acceptor side of PSI. (**B**) In actinic light (AL) illumination, before SP illumination, P700 is already oxidized to P700+. SP illumination further oxidizes P700 to its maximum, which is lower than the oxidation obtained in the dark, because P700\* accumulates under the steady-state AL illumination. P700+ produced by SP illumination is not reduced, because P700 in PSI experiences turnover with the rate-determining step of P700+ reduction in the P700 photo-oxidation reduction cycle. The Y-axis shows the oxidation ratio of P700 from 0% to 100%, and the X-axis shows the time for SP illumination: upward arrow: SP illumination starts; downward arrow: SP illumination stops.

**Figure 3.** Repetitive short-pulse (rSP) illumination treatment inactivates PSI. In angiosperms, rSP illumination treatment of the intact leaves inactivates PSI activity [38]. The inactivation is suppressed under low O2 conditions (2 kPa O2). Short-pulse illumination reduces O2 to a superoxide radical, O2 −, and the accumulated O2 − from rSP illumination treatment oxidatively attacks PSI components. Red line: the PSI activity gradually decreases as rSP illumination treatment proceeds under atmospheric O2 (21 kPa) condition; blue line: PSI inactivation by rSP illumination treatment is suppressed under low O2 (2 kPa) condition.

#### **3. ROS Production Is Suppressed by Oxidation of P700**

As described above, ROS-dependent oxidative damage in PSI was shown in vivo. Next, a new question arose: essentially, how do plants survive under natural sunlight? The answer was quickly obtained. PSI inactivation by rSP illumination treatment was suppressed under illumination by actinic light (AL) [38]. Furthermore, the extent of PSI inactivation decreased with increasing AL intensity. In other words, the light-induced inactivation of PSI was prevented by light itself. The effect of AL illumination was that P700 was oxidized to P700+ (Figure 2B). Increasing AL intensity increased P700+ in the P700 photo-oxidation reduction cycle. Afterwards, P700+ accumulation prevented P700\* accumulation during SP illumination (Figure 2B). In other words, high levels of P700+ were maintained, even during light pulses. Thus, the role of AL illumination from the perspective of the P700 photo-oxidation reduction cycle was made clear: both P700\* accumulation in the dark and P700+ accumulation under AL illumination during an SP illumination, indicate the rate-determining step (RdS) transition in the P700 photo-oxidation reduction cycle. In the dark, P700\* oxidation is rate-limited, whereas under AL illumination, P700+ reduction is rate-limited. AL illumination suppresses rSP-illumination-treatment-induced PSI inactivation through the rate-determining step transition mechanism. That is, AL illumination suppresses the accumulation of P700\*, which can donate electrons to O2 to produce O2 − in PSI. Subsequently, we found the robust effect of AL illumination, which was that P700 was oxidized and suppressed PSI inactivation by rSP illumination treatment [42].

Numerous studies have reported the oxidation of P700 in PSI under high intensity AL or low CO2 conditions [33,34,47–54]. Accumulated P700+ during the P700 photo-oxidation reduction cycle absorbs excess photon energy and dissipates it as heat [55–57]. Concomitantly, P700+ accumulation inhibits O2 reduction, producing O2 − by suppressing P700\* accumulation [38]. This is the molecular mechanism that reduces the rate of the Mehler reaction in intact leaves under AL illumination. If P700 is not subsequently oxidized, ROS production will increase, due to the promotion of the Mehler reaction, and PSI will stop functioning [38,42,58].

#### **4. Molecular Mechanism of Oxidation of P700 in Photosystem I under AL Illumination**

In photosynthesis, a light reaction functions with a dark reaction in a tightly coupled state. For example, the light reaction proceeds in the thylakoid membranes of chloroplasts in C3-plants, where photosynthetic linear electron flow produces NADPH and ATP. The dark reaction proceeds in both photosynthesis and photorespiration, where both NADPH and ATP are used for the regeneration of ribulose 1,5-bisphosphate (RuBP) (one of the substrates for RuBP carboxylase/oxygenase which is consumed in the dark reaction). In photorespiration, reduced Fd is used in chloroplasts. Electron flow, which produces both NADPH and the reduced Fd, and proton flow, which produces ATP in the light reaction, are coupled, with both e- flow and H+ flow required for the consumption of NADPH, the reduced Fd, and ATP in the dark reaction. This tight coupling of the light reaction with the dark reaction can cause a dangerous situation in which ROS are produced in the photosynthetic electron transport system. The limitation of the dark reaction is the limitation of the light reaction, which can lead to the accumulation of electrons in the photosynthetic electron transport system. For example, in the photo-oxidation reduction cycle of P700 in PSI, electrons would accumulate at the acceptor side of PSI, where O2 can be reduced to O2 −, a ROS. As described above, unless P700 in PSI is oxidized, PSI suffers from oxidative damage.

We show below how the light reaction tightly couples with the dark reaction in photosynthesis. We later show how P700 is oxidized in response to the limitation of the dark reaction, for example, in drought, low temperature, and high light situations.

#### *4.1. Robust Relationships Support Tight Coupling between the Light and Dark Reactions in Photosynthesis*

Much evidence has accumulated that clarifies a robust tight coupling between the light and dark reactions in C3 photosynthesis (Figure 4). For instance, Genty et al. [59] reported a positive linear relationship with an origin of zero between the photosynthetic linear electron flow rate (Jf) in the light reaction and the photosynthetic electron consumption rate (Jg) based on NADPH consumption—in the dark reaction [59]. Consistently, a positive linear relationship with an origin of zero was also found between the photosynthetic linear electron flow rate (Jf) in the light reaction and the electron consumption rate (Jg)—based on NADPH consumption and reduced Fd—in the dark reaction, where photosynthesis and photorespiration both occur (Equation (1)) [30,31,60]. These results indicate that photosynthetic linear electron flow is the sole driver of both photosynthesis and photorespiration. In other words,

$$\mathbf{J}\mathbf{f} = \mathbf{J}\mathbf{g} \tag{1}$$

Recently, a positive linear relationship with an origin of zero between the reduced Fd oxidation rate (vFd) and the photosynthetic linear electron flow rate (Jf) was found (Equation (2) [61]). These results indicate that electron flow via Fd is driven by both photosynthesis and photorespiration. In other words,

$$\mathbf{k}\,\mathbf{v}\mathbf{F}\mathbf{d} = \mathbf{k}\_{\text{fd}} \times \mathbf{F}\mathbf{d}^{-} = \mathbf{J}\mathbf{g} \tag{2}$$

The constant kfd is the apparent rate constant of the reduced Fd oxidation rate, while Fd− is the amount of reduced ferredoxin.

A positive linear relationship with an origin of zero was reported between Jf and the pmf production rate ((vH+ = gH+ × pmf (gH+, proton conductance) (Equation (3)) [62–65]). These results indicate that photosynthetic linear electron flow provides the formation of pmf. In other words,

$$\text{k}\_{\text{H}}\text{}^+ \times \text{f} \text{f} = \text{v} \text{H}^+ \tag{3}$$

The rate constant kH+ reflects lumen H+ accumulation efficiency, which depends on H2O oxidation in PSII and on the Q-cycle in the Cyt *b*6/*f* complex. Furthermore, a positive linear relationship with an origin of zero was found between vH+ and the proton usage rate (JgH+), due to the regeneration of ATP required for both photosynthesis and photorespiration (Equation (4)) [66]. These results indicate that vH+ also reflects the pmf usage rate during photosynthesis and photorespiration. In other words,

$$\text{v}\text{v}\text{H}^+ = \text{J}\text{g}\text{H}^+\tag{4}$$

Furthermore, a positive linear relationship with an origin of zero was found between JgH+ and Jg (=Jf) (Equation (5)) [66]. These results indicate that JgH+ was determined by the electron consumption rate (Jg), based on the consumption of both reduced Fd and NADPH. In other words,

$$\rm J\rm g\rm H^{+} = k\_{\rm jg} \times J\rm g = k\_{\rm H}{}^{+} \times J\rm f \tag{5}$$

Equation (5) indicates that there is a tight coupling between the light and dark reactions during photosynthesis and photorespiration. Constant kjg is the coefficient that links e- flow with H+ flow during photosynthesis and photorespiration. These findings indicate that the operation of the light reaction is driven by the dark reaction and vice versa, and provide a concrete explanation for the observed decrease in Jf under environmental stress, when the photosynthetic rate decreases due to a dark reaction rate limitation (i.e., when JgH+ and Jg decrease).

Tight coupling between the light and dark reactions, as shown in Equation (5), can cause the accumulation of electrons at the acceptor side of PSI and the accumulation of protons in the lumenal space of thylakoid membranes under stress conditions, e.g., drought, low temperatures, and high intensity of light, which decrease the photosynthesis efficiency [2,3,8,67–69]. For example, under constant light intensity, decreases in both Jg and JgH+ decrease Jf. These situations cause a reduction in PQ and the non-photochemical quenching of chlorophyll fluorescence, both of which reflect the accumulations of electrons and protons in the photosynthetic electron transport system [32,34,42,58,70–72].

However, the electrons do not accumulate at the acceptor side of PSI; P700 is oxidized under stress conditions [32,34,49,50,53,72]. From the model of the photo-oxidation reduction cycle of P700, the reduction of P700+ must be the rate-determining step to accumulate the oxidized P700 in PSI. The suppression of electron fluxes in both light and dark reactions does not limit the reduction of P700+ in the cycle. That is, the electron flux to the oxidized P700 should be suppressed such that P700 is oxidized.

**Figure 4.** Tight coupling between the light and the dark reactions in photosynthesis of C3 angiosperms. Blue arrows: electron flow; blue dotted arrows: H+ flow; Hs+: stroma H+; Hl +: lumen H+; [H(l − s) +]: difference in H+ concentration (ΔpH) between the stroma and the lumen, i.e., the proton motive force (pmf). Photosystem II (PSII): electron source. P700 functions in the photo-oxidation reduction cycle to catalyze the electron flow from plastocyanin (PC) to Fd in Photosystem I (PSI). P700 is photoexcited to P700\*, which donates electrons to Fd through electron carriers (A0, A1, FA/FB, and FX) to produce P700+. In turn, P700+ is reduced by electrons from PSII through plastoquinone (PQ), the Cyt *b*6/*f* complex, and PC. The Q-cycle in the Cyt *b*6/*f* complex transfers electrons from reduced PQ along two routes: first, PC; second, oxidized PQ. Enhanced reduction of PQ suppresses the reduction of oxidized PQ in the Cyt *b*6/*f* complex, which slows down the activity of the Q-cycle, and the electron flux from the Cyt *b*6/*f* complex to P700+ in PSI. H+ conductance (gH+) is an apparent rate constant that depends on the concentrations of ADP, Pi, ATPase, and the catalytic constant (k(ATPase)). NPQ, non-photochemical quenching of chlorophyll fluorescence, is activated by the acidification of the lumenal space of the thylakoid membrane, which is reflected by the increase in pmf. NPQ suppresses the quantum yield of PSII, which is reflected by the electron flux in photosynthetic linear electron flow, Jf; the pmf also suppresses the oxidation activity of reduced PQ by the Cyt b6/f complex. Tight coupling of the light reactions (the production of electron and proton flows) to the dark reaction (the consumption of electrons and protons in both photosynthesis and photorespiration) yields a robust relationship whereby electron flux (Jf) is equal to the electron flux in electron sinks (photosynthesis and photorespiration) expressed as Jg. The pmf is the driving force to synthesize ATP by ATP synthase using ADP and Pi, and the pmf-dissipation rate (vH+) is expressed as gH+ × pmf. As a robust model, Jf = vFd = Jg [61]; vFd is the oxidation rate of reduced Fd; kJf × Jf = gH+ × pmf [64,65]. vH+ = gH+ x pmf = JgH+ [66]. Jg and JgH+ mutually determine each other. Meanwhile, pH decreases in the lumenal space of the thylakoid membrane during the photo-oxidation of water in PSII and the Q-cycle in the Cyt *b*6/*f* complex. The decrease in pH (i.e., increased pmf) acts as the driving force for ATP production by ATPase.

#### *4.2. How Do Protons and Electrons Accumulate in Both the Lumenal Space of Thylakoid Membranes and the PQ Pool in Response to the Suppression of the Dark Reaction?*

Generally, it is proposed that the electron flux from PQ to P700 in PSI is regulated by the oxidation activity of the reduced PQ (PQH2) by the cytochrome (Cyt) *b*6/*f*-complex [48,67,70,71,73–81]. The PQH2 oxidation activity by the Cyt *b*6/*f*-complex decreases with a lowering pH. Therefore, the accumulation of protons in the lumenal space of thylakoid membranes suppresses the electron flow to the oxidized P700 in PSI, which is known as photosynthesis control [67,73,74]. Furthermore, the overreduction of PQ inhibits the Q-cycle in the Cyt *b*6/*f*-complex, and contributes to the oxidation of P700 in PSI, which is known as the reduction-induced suppression of electron flow (RISE) [70,71]. We propose that the RISE is also involved in photosynthesis control. To clarify the molecular mechanism of oxidation of P700 in PSI upon exposure to the limitation of photosynthesis, we must understand how protons and electrons accumulate in both the lumenal space and the PQ pool in the photosynthetic electron transport system in response to the activities of both photosynthesis and photorespiration.

The induction mechanism of proton gradient (ΔpH) formation across the thylakoid membrane, the accumulation of protons in the lumenal space of thylakoid membranes, is shown as below: ΔpH formation is observed as a pmf increase [65,82,83]. In general, ΔpH mainly occupies the pmf at low photosynthetic efficiency, e.g., high light and low CO2 conditions. The velocity of change in pmf [d(pmf)/dt] is determined by the difference between the pmf generation rate and the pmf decay rate. The pmf generation rate depends on the photosynthetic linear electron flow rate (Equation (3)), and the pmf decay rate depends on the ATP production rate, which is driven by both photosynthesis and photorespiration (Equation (4)): thus, the velocity of change in pmf is as shown (Equations (6) and (7)).

$$\text{pd(pmf)/dt} = \text{k}\_{\text{H}}{}^{+} \times \text{Jf} - \text{v} \text{H}^{+} \tag{6}$$

$$\mathbf{k} = \mathbf{k}\_{\mathrm{H}}\mathbf{^{+}} \times \mathbf{J}\mathbf{f} - \mathbf{g}\mathbf{H}^{+} \times \text{(pmf)}\tag{7}$$

The rate constant kH+ reflects the H+ accumulation in the luminal space of thylakoid membranes, which is driven by photosynthetic linear electron flow. At the steady state, d(pmf)/dt = 0, and the pmf production rate is kH+ × Jf. This depends on H2O oxidation in PSII and on Q-cycle rotation in the Cyt *b*6/*f* complex. Furthermore, the pmf decay rate, vH+, is expressed as gH+ × (pmf). The gH+, H+ conductance, is a rate constant that reflects the apparent rate constant of pmf decay. The vH+ also reflects the pmf dissipation rate, and vH+ can be replaced with JgH+ as follows (Equation (8)):

$$\text{pd(pmf)/dt} = \text{k}\_{\text{H}}{}^{+} \times \text{Jf} - \text{JgH}^{+} \tag{8}$$

We reveal that, in a steady state where d(pmf)/dt = 0, based on Equations (7) and (8), Equation (9) was obtained.

$$\rm kg\_{H+} \times \rm ff = v \rm H^+ \ (= \rm gH^+ \times \rm pmf) = \rm JgH^+ \tag{9}$$

Equation (9) shows that the photosynthetic linear electron flow activity in the light reaction links photosynthesis and photorespiration activity in the dark reaction through pmf formation and dissipation. The dependence of pmf on Jf, gH+, and JgH is shown as follows (Equations (10) and (11)).

$$\text{pmf} = (\text{k}\_{\text{H}} + \text{(f)}) \text{(gH}^{+} \tag{10}$$

$$\mathbf{g} = \mathbf{J} \mathbf{g} \mathbf{H}^+ / \mathbf{g} \mathbf{H}^+ \tag{11}$$

Based on this model, we will discuss the molecular mechanism of proton accumulation in the lumenal space of thylakoid membranes in response to the activities of photosynthesis and photorespiration. For example, if the extent of the decrease in the dark reaction rate, shown as the decrease in both Jg and JgH+, was smaller than that in gH+, then pmf increased. This is how protons accumulate in the lumenal space of thylakoid membranes.

We consider the accumulation of electrons in the photosynthetic electron transport system, reflected as the redox states of both PQ and Fd. The reduced state of PQ is evaluated by a parameter of chlorophyll fluorescence, 1 − qL [72,84]. The qL shows the oxidation state of PQ in thylakoid membranes. A higher value of 1 − qL shows the high accumulation of electrons in the PQ pool. The oxidation rate of the reduced PQ can be expressed as kqL × (1 – qL). Additionally, the oxidation rate of the reduced Fd (Fd−) is expressed as kFd × Fd−. Both kqL and kFd are the apparent rate constants. In the light reaction, the electron fluxes for the oxidations of both the reduced PQ and the reduced Fd are equal to Jf, as shown in the equation (12).

$$\text{Jf} = \text{kqL} \times (1 - \text{qL}) = \text{k}\_{\text{Fd}} \times \text{Fd}^- = \text{Jg} \tag{12}$$

The kqL is given a positive value and is expected to decrease by two factors: first, kqL decreases with a decrease in pH in the lumenal space of thylakoid membranes (photosynthesis control) [67,85,86]; second, kqL decreases by the RISE [70,71]. Generally, a decrease in Jg increases the value of 1 – qL [34,72]. These facts indicate, in response to the decrease in electron sink activity (both photosynthesis and photorespiration), that the extent of the decrease in kqL is larger than that in Jg. That is, from Equation (12), we can clearly understand why 1 – qL increases. This is how electrons accumulate in the PQ pool of thylakoid membranes.

On the other hand, a decrease in Jg does not induce the reduction of Fd [61]. For example, lowering the intercellular partial pressure of CO2 decreases the photosynthesis rate, and Jg decreases; however, the reduced level of Fd either does not change or decrease. These facts show that the extent of the decrease in kFd was almost the same as that in Jg. The kFd depends on both the activity of Fd−NADP+ oxidoreductase (FNR) and NADP+ regeneration efficiency. The decrease in the photosynthesis rate lowers the NADP+ regeneration rate, which decreases kFd.

#### *4.3. How Do the Accumulated Protons in the Lumenal Space and Electrons in the PQ Pool Oxidize P700 in PSI?*

The decrease in Jf (=Jg) shows the negative linear relationship with P700+ (Figure 5) [42,87]. The decrease in photosynthesis efficiency induces the oxidation of P700 in PSI. As described above, the suppression of photosynthesis enhances both the pmf and the RISE, which would cause the reduction of P700+ in the photo-oxidation reduction cycle of P700 in PSI to be the rate-determining step. The decrease in Jg lowers Jf in the tight coupling of the light reaction with the dark reaction. Here, we have to pay an attention to the magnitude of the decrease in Jf. The extent of the decreases in gH+ and kqL are larger than that in Jg, so the pmf and (1 – qL) increase. We also must consider that the magnitude of Jf would be downregulated by both the pmf and the RISE, such that the reduction of P700+ should be the rate-determining step in the photo-oxidation reduction cycle of P700 in PSI. Jf must be lower than the theoretical value of Jg obtained by the suppression of photosynthesis. For example, if Jf decreases to the same extent as the decrease in Jg in the response to the decrease in Ci, the photo-excited P700 (P700\*) would accumulate, because the efficiency of photo-excitation of P700 to P700\* does not change at a constant intensity of AL. This deduction contradicts the experimental facts; that is, that suppressed photosynthesis induces the oxidation of P700.

The RISE was evidenced to have the potential to stop the electron flow in the light reaction and to oxidize P700 in PSI of cyanobacteria [70,71]. Flavodiiron protein (FLV) catalyzes the reduction of O2 to H2O using NADPH in the cyanobacteria [88,89]. The electron flux in the FLV reaction is so high that it alternates the electron flux in photosynthesis under CO2-deficient conditions [88,90]. The cyanobacteria deficient in FLV show suppressed photosynthesis, with the accumulation of electrons in the PQ pool [70]. Furthermore, illumination by SP light reduces PQ almost transiently with the suppression of O2 evolution under steady-state conditions [70]. FLV can oxidize PQ even though photosynthesis can function at a higher activity. FLV keeps PQ in an oxidized state so as not to induce the RISE for the full activity of photosynthesis, if enough CO2 is supplied to cyanobacteria.

**Figure 5.** Dependence of oxidized P700 (P700+) induction on electron flux in photosynthetic linear electron flow in intact leaves. Generally, P700 is oxidized with a decreasing electron flux of photosynthetic linear electron flow (Jf) by lowering CO2 partial pressure. At 21 kPa O2, when both photorespiration and photosynthetic CO2 fixation are functioning, P700+ shows the maximum value at the CO2 compensation point, while Jf shows the minimum value (blue dotted line). The horizontal arrow at 21 kPa O2 shows the range over which Jf is driven by both photosynthetic CO2 fixation and photorespiration. At 2 kPa O2, when no detectable photorespiration occurs and photosynthetic CO2 fixation functions normally, P700+ starts to decrease to the minimum at the CO2 compensation point after reaching its maximum value, while Jf reaches its minimum value (red dotted line). The horizontal arrow at 2 kPa O2 shows the range over which Jf is driven by photosynthetic CO2 fixation.

Furthermore, we found that inductions of the pmf and RISE oxidize P700 in PSI of the C3-plant wheat leaves [42,87]. A decrease in the intercellular partial pressure of CO2 lowers the photosynthetic rate and increases the photorespiration rate. However, the photorespiration rate cannot reach the value theoretically derived from Rubisco kinetics at lower CO2 [37,91]. The induction of the pmf and RISE lowers the activity of the light reaction, which causes the activity of the dark reaction, the photorespiration rate, to be suppressed, even though the potential activity of the dark reaction exceeds the suppressed activity of the light reaction. These results show that the oxidation of P700 requires the suppression of electron flux in the light reaction much more so than in the dark reaction; thus, the suppressed activity in the dark reaction is lower than the theoretical potential activity. As a result, the reduction of P700+ becomes the rate-determining step in the P700 photo-oxidation reduction cycle. This is the essence of the molecular mechanism of P700 oxidation.

On the other hand, the induction of the pmf and RISE does not always oxidize P700 in response to the limitation of photosynthesis. Unless the electron flux of the light reaction dips below the potential electron flux of the dark reaction, which is, theoretically, expected from the limited activity of photosynthesis, the limitation of the PSI acceptor side is so strong that P700 is not oxidized (Figure 5). When photosynthesis is severely limited, where both photosynthesis and photorespiration functions are suppressed, P700 cannot be oxidized because the oxidation of P700\* becomes the rate-determining step in the P700 photo-oxidation reduction cycle.

#### *4.4. Suppression of the Production of ROS in PSI by the P700 Oxidation System*

We summarize the molecular mechanism of P700 oxidation in PSI of chloroplasts (Figure 6). Both PSI and PSII absorb photon energy to excite the reaction center chlorophylls P680 (PSII) and P700 (PSI). Similarly to P700 in PSI, P680 undergoes a photo-oxidation reduction cycle. The ground state of P680 is photo-excited to P680\*. The P680\* donates electrons to PQ through electron carriers in PSII. Additionally, then, the electrons start to flow from the reduced PQ to PSI, through the Cyt *b*6/*f*-complex, and plastocyanin (PC). During the electron flow, H+ from both H2O oxidation in PSII and the reduced PQ (PQH2, plastoquinol) oxidation of the Cyt *b*6/*f*-complex accumulate in the lumenal space of thylakoid membranes. The accumulated protons are the pmf to drive ATP synthase to produce ATP in the stroma. On the other hand, NADPH is produced by ferredoxin-NADP oxidoreductase (FNR) at the acceptor side of PSI. ATP, NADPH, and the reduced Fd are used for the regeneration of RuBP in both photosynthesis and photorespiration. The regeneration rates of NADP+, the oxidized Fd, and ADP are determined by the activities of both photosynthesis and photorespiration.

As shown in Equations (5), (9), and (12), the light reaction tightly couples with the dark reaction. The suppression of photosynthesis decreases Jg or JgH+. Afterwards, the regeneration rates of ADP, Pi, and NADP+ decrease. That is, gH+ decreases to increase the pmf, and kqL decreases to increase (1 – qL), which induces the RISE. Both the pmf and the RISE lower kqL, as reflected in the suppressed activity of the reduced PQ oxidation by the Cyt *b*6/*f*-complex. NPQ induced by the pmf also downregulates the quantum yield of PSII. The suppressed electron flow from PSII to the oxidized P700 also causes the reduction of P700+ in the photo-oxidation reduction cycle to be the rate-determining step, which leads to the oxidation of P700.

We compared the rate-determining step in the photo-oxidation reduction cycle of P700 in PSI in response to the limitation of electron sink activities and the photosynthesis and photorespiration activities at high light conditions (Figure 6). In Case (I), we set the electron sink to a large size. The black arrows show the electron flow in the photosynthetic electron transport system from the redox reaction of PQ to the electron sink, including both photosynthesis and photorespiration, through the Cyt *b*6/*f*-complex, PC, and P700 in PSI and Fd. The width of the black arrows reflects the magnitude of the apparent rate constant in each elementary reaction. The rate-determining step of the P700 photo-oxidation reduction cycle in PSI is the reduction of P700+, leading toward to the oxidation of P700+. Both the pmf and RISE contribute to the suppression of the Q-cycle activity of the Cyt *b*6/*f*-complex, which causes the reduction of P700+ to be the rate-determining step.

In Case (II), the electron sink is set to a medium size. Environmental stress, e.g., drought and/or low temperatures, can suppress photosynthetic activities. That is, both the pmf and RISE increase in response to the decrease in both gH+ and kqL, as determined by Equations (11) and (12). The apparent rate constant for the reduction of P700+ further decreases, compared to Case (I). The magnitude of the apparent rate constant for P700+ reduction is lower than the apparent constant for P700\* oxidation. The increase in the suppression of the Q-cycle in the Cyt *b*6/*f*-complex enhances the rate-determining step, the reduction of P700+, in the P700 photo-oxidation reduction cycle of PSI.

In Case (III), the electron sink is smaller than the medium size in Case (II). The activities of both photosynthesis and photorespiration are further suppressed, compared to Case (II). However, the rate-determining step leads to the elementary reaction of the oxidation of the photo-excited P700, P700\*, and P700\* then accumulates, although both the pmf and RISE are kept to the same or a slightly greater extent or as in Case (II). That is, the suppression of the apparent rate constant of the oxidation of P700\* is larger than that of the oxidation activity of the reduced PQ in the Cyt *b*6/*f*-complex in response to the limitation of the electron sink. Electrons accumulate at the acceptor side of PSI, and the electrons flow to O2 to produce O2 −, the production of ROS. The transition of the rate-determining step from the reduction of P700+ to the oxidation of P700\* is a dangerous condition that induces oxidative damage to PSI.

**Figure 6.** The P700 oxidation system and ROS production mechanism. The photosynthetic linear electron flow in the light reaction is tightly coupled to the dark reaction, photosynthesis and photorespiration (electron sinks). The P700 oxidation system enhances the rate-determining step of P700+ reduction to stimulate the oxidation of P700 in PSI, in response to the decrease in electron sinks. Once exposed to a further decrease in electron sinks, P700+ reduction cannot remain the rate-determining step, and the rate-determining step transitions from P700+ reduction to the oxidation of P700\*. Three distinct scenarios of the rate-determining step can take place under saturated light intensity: In Case (I) with the large electron sink, both the pmf and RISE are induced by the two electron sinks: photosynthesis and photorespiration. Afterwards, the oxidation activity of the reduced PQ by the Cyt *b*6/*f*-complex is suppressed to decrease the reduction rate of P700+. As a result, the photo-oxidation reduction cycle of P700 in PSI turns over with the rate-determining step of P700+ reduction. In Case (II) with the medium-sized electron sink, PQ is further reduced, and the pmf is further accumulated, compared to Case (I), by the decreased gH+ and kqL. The electron flux from the reduced PQ to the oxidized P700 is then further suppressed. The oxidation of P700 becomes the rate-determining step in the photo-oxidation reduction cycle of P700 in PSI. In Case (III) with the small electron sink, the further induced pmf and RISE cannot decrease the reduction rate more than the suppression of the oxidation of P700\* due to the smaller electron sink, compared to Case (II). Afterwards, P700\* accumulates. As a result, the photo-oxidation reduction cycle of P700 in PSI turns over with the rate-determining step of P700\* oxidation. From Case (II) to Case (III), the rate-determining step transitions from the reduction of P700+ to the oxidation of P700\* in the photo-oxidation reduction cycle of P700 in PSI. The accumulated P700\* increases the chance of the reduction of O2 to O2 −, the production of ROS at the acceptor side of PSI. The reduced electron carriers in PSI—A0, A1, Fx, FA/FB, and the reduced Fd—can react with O2 to produce O2 − [2,39–41,92,93].

#### *4.5. Relationship between Photorespiration- and Flavodiiron Protein (FLV)-Dependent Electron Flows and Their Contribution to P700 Oxidation in PSI*

Following the model of the P700 oxidation system, photosynthetic linear electron flow contributes to the induction of either the pmf or RISE, which suppresses the electron flow from the Cyt *b*6/*f*-complex to the oxidized P700 through PC. Afterwards, P700 is oxidized, as shown in Figures 5 and 6. From cyanobacteria to gymnosperms, in the evolution of green lineage oxygenic photosynthetic organisms, flavodiiron protein (FLV) drives O2-dependent electron flow in the photosynthetic electron transport system, in addition to the photorespiration pathway [88–90,94–96]. FLV reduces O2 to H2O using NADPH as the electron donor [88]. Different from photorespiration, FLV-dependent electron flow does

not consume ATP; i.e., it only induces the pmf. Therefore, FLV-dependent electron flow has a higher capacity to oxidize P700. In fact, in the cyanobacteria *Synechocystis* sp. PCC6803 and *Synechococcus* sp. PCC7002, the deletion of *flv* genes caused the photoinhibition of PSI, which was enhanced by the reduction of P700 in PSI [58]. Furthermore, the liverwort *Marchantia polymorpha*, which was deficient in flv genes, suffered from the photoinhibition of PSI due to the reduction of P700 [58]. Angiosperms, however, do not have FLV genes in their genomes [87,97]. Among land plants, the electron flux of photorespiration-dependent photosynthetic linear electron flow was compared to FLV-dependent electron flux at the CO2 compensation point, where photosynthetic activity was suppressed to zero. The maximum activities of both FLV-dependent and photorespiration-dependent photosynthetic linear electron flow were then evaluated among land plants [98].

Photorespiration activities at the CO2 compensation point increased with the evolution of photosynthetic organisms from liverworts to C3 angiosperms to ferns and gymnosperms (Figure 7A). C3-C4 and C4 angiosperms show lower activities of photorespiration, compared to C3 angiosperms. C3 angiosperms show the maximum activities of photorespiration [98]. In C3 angiosperms, photosynthetic linear electron flow was mainly driven by photorespiration, which was the main electron sink in photosynthesis, at the CO2 compensation point (Figure 7B). An electron sink other than photorespiration was found in liverworts, ferns, and angiosperms. FLV could be an electron sink at the CO2 compensation point. C3-C4 and C4 angiosperms also had an electron sink other than photorespiration and FLV-dependent photosynthetic linear electron flows, but its mechanism is unknown.

What do C3-angiosperms achieve by discarding FLV genes in their genomes? Different from photorespiration, FLV-dependent electron flow does not consume ATP; it produces it, as described above. FLV-dependent electron flow induces the pmf, which can be a self-regulating electron flow and suppress photosynthesis, because an enhanced pmf suppresses the activity of the Cyt *b*6/*f*-complex, as shown in Figures 5 and 6. In liverworts, ferns, and gymnosperms, FLV-dependent photosynthetic linear electron flow functions immediately after the actinic light illumination starts, and its flux is suppressed at the steady state of photosynthesis [98]. This indicates that FLV-dependent photosynthetic linear electron flow is generally not essential to photosynthesis organisms, and that there is a molecular mechanism to reduce FLV-dependent photosynthetic linear electron flow under a steady state of photosynthesis. On the other hand, photorespiration drives both the light reaction and the dark reaction, and inevitably functions with photosynthesis. This situation implies that a tight coupling of the light reaction with the dark reaction driven by both photosynthesis and photorespiration can regulate both the pmf and RISE in response to their activities, because the dark reaction induces and dissipates the pmf and RISE simultaneously. This could be a sensitive trigger for the regulation of P700 oxidation. If FLV-dependent photosynthetic linear electron flow participates in the photosynthetic linear electron flow driven by both photosynthesis and photorespiration, the automatic regulation of photosynthetic linear electron flow will collapse. Therefore, C3 angiosperms do not require FLV-dependent photosynthetic linear electron flow, and discard FLV genes in their genomes. These conclusions explain why angiosperms can have a high activity of photosynthesis, if photosynthetic organisms having FLV genes in their genomes have lower photosynthetic activity.

**Figure 7.** Evolution of tight coupling between the light and the dark reactions in photosynthesis. Upon spreading on the surface of the earth, oxygenic photosynthetic organisms inevitably metabolized 2-phosphoglycolate to regenerate 3-phosphoglycerate via the photorespiration pathway, because ribulose 1,5-bisphophate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes both RuBP carboxylation and oxygenation under atmospheric conditions. Consequently, photorespiration inevitably accompanies photosynthetic CO2 fixation. In C3 angiosperms, both photosynthetic CO2 fixation and photorespiration have become the main electron sinks in the photosynthetic process. Hanawa et al. [98] found that photorespiration activity increased with the evolution of oxygenic photosynthetic organisms. Photorespiration activity is evaluated as a photorespiration-dependent O2-uptake at the CO2 compensation point, at which point its activity peaks [66]. (**A**) Photorespiration activity increases with the evolution of oxygenic photosynthetic organisms from liverworts to C3 angiosperms through ferns and gymnosperms. (**B**) The electron sink capacity of photorespiration reaches its maximum in C3 angiosperms. The activities of flavodiiron (FLV)-dependent photosynthetic linear electron flow and other alternative photosynthetic linear electron flow (AEF) decrease upon the evolution of oxygenic photosynthetic organisms from liverworts to C3 angiosperms through ferns and gymnosperms. On the other hand, C3-C4 and C4 plants show lower photorespiration activity, and other electron sinks function at the CO2 compensation point. Data are from Hanawa et al. [98].

#### **5. Conclusions**

Oxygenic photosynthetic organisms have a robust, common molecular mechanism to suppress the production of reactive oxygen species (ROS), the P700 oxidation system, because the ROS-dependent oxidative inactivation of PSI is lethal to these organisms. When cyanobacteria first appeared on the Earth about 2–3 billion years ago, they already had a P700 oxidation system [58]. Photosynthetic organisms regulate the activity of plastoquinol oxidation by the cytochrome (Cyt) *b*6/*f*-complex using two strategies: pmf formation and the reduction-induced suppression of electron flow (RISE), both of which are used to suppress the electron flux from the Cyt *b*6/*f*-complex to the reaction center chlorophyll P700 in PSI. The induction of both the pmf and RISE is sensitive to changes in the activities of both photosynthesis and photorespiration in land plants, which drive photosynthetic linear electron flow. The magnitudes of both the pmf and RISE are determined by the activity of photosynthetic linear electron flow. Upon exposure to environmental stress, e.g., low temperature, and/or drought, suppressed activities of both photosynthesis and photorespiration cause both the pmf and RISE to oxidize P700 in the manner described in the text for the suppression of ROS production in PSI.

**Funding:** This work was supported by JST CREST Grant Number JPMJCR15O3, Japan. **Acknowledgments:** The author is thankful to MDPI for English language editing.

**Conflicts of Interest:** The authors declare that there is no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**

© 2020 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*


**3.4 Methods**

These reservoirs have been monitored for standard physical, chemical and microbiological water quality, including, since 2003, determination of phytoplankton biomass and composition. Monthly surface and bottom samples were taken, from 2003 to 2009, at Choça Queimada tower for Odeleite reservoir, and at the extraction tower of the Beliche reservoir. During 2009 and 2010, a new sampling site in the middle of the lake, 500m upstream from the dam wall, was added according to new guidelines from the European Med GIG (INAG, 2009). At each of these sampling sites, vertical profiles were determined *in situ* using a YSI

Ecological Tools for the Management of Cyanobacteria

http://www.drapalg.min-agricultura.pt/

**Month/year Bottom discharge**

source: http://snirh.pt/)

**(dam3)**

Blooms in the Guadiana River Watershed, Southwest Iberia 177

Fig. 9. Evolution of stored water volume (hm3) and mean monthly rainfall from July 2003 to November 2010 in Odeleite and Beliche reservoirs. Data source: http://snirh.pt/ and

> **Surface discharge (dam3)**

Jul/2003 0 0 13 0 Aug/2003 0 0 26 0 Sep/2003 0 0 13 0 Oct/2003 0 0 1 0 Nov/2003 1234 8722 1818 0 Dec/2003 3894 9821 382 0 Feb/2004 6648 0 1562 0 Mar/2006 5970 0 1409 0 Nov/2006 15196 19868 3536 1776 Dec/2006 8685 0 543 0 Feb/2007 2364 0 0 0 Aug/2007 74 0 0 0 Mar/2008 0 261 0 0 Apr/2008 6095 18388 0 1012 Feb/2009 8495 0 0 3502 Dec/2009 0 22450 0 5105 Dec/2010 0 29821 40 6714 Jan/2011 22528 0 0 2186 Mar/2011 50139 0 0 12577 Table 2. Surface and bottom water discharged from Odeleite and Beliche since 2003 (Data

**Bottom discharge (dam3)**

**Surface discharge (dam3)**

**Reservoir Odeleite Beliche**

650 MDS probe, for water temperature, dissolved oxygen, pH and conductivity. Nutrient concentrations analysis were performed at the accredited (EN 17025) water analysis laboratory from Administração da Região Hidrográfica do Algarve (ARH Algarve), who was also responsible for all the sampling campaigns. Phytopigments including chlorophyll *a* were analyzed at Huelva University (Forján et al., 2008) through HPLC, according to Young et al. (1997). Microcystin detection and quantification was performed according to Carmichael & An (1999) using the micro-ELISA kit Microcystin Plate Kit from Adgen – Agrifood Diagnostic. Phytoplankton composition was determined by the same methods referred in 2.1. Phytoplankton biovolumes following European guidelines were calculated on the basis of predefined 3-dimensional shapes and their respective stereometric formulas as recommended by Edler (1979a, 1979b) and Hillebrand et al. (1999), according to the CEN/TC230/WG2/TG3 N108 Water Quality and Olenina et al. (2006).

Berger Parker dominance index was determined by calculating the proportion of the most abundant species over the total phytoplankton cell density (Magurran, 1988). Carlson Trophic State Index (TSI) was calculated for chlorophyll *a* values and both for total phosphorus (TP) and soluble reactive phosphorus (SRP) concentrations, according to Carlson (1977). Contribution of cyanobacteria to total phytoplankton is given by the percentage of total biovolume attributed to cyanobacteria. Catalán Index for Algal Groups (InGA) was determined by using biovolume proportions of colonial and non-colonial algal groups (Catalán et al., 2003). The MedPTI index was calculated according to Marchetto (2009).

## **3.5 Results and discussion**

## **3.5.1 Hydrometric features**

Monitoring data for the last 7 years included a severe 18 months drought from 2004 to 2006, and an exceptional rainy year in 2010. As seen in Fig. 9, water level at Odeleite reservoir had to be lowered in November 2006 for maintenance works, originating intense bottom and surface discharges. Surface overflow was released through the stream bed into the Guadiana estuary, but bottom discharged water flowed through the underground channel into Beliche reservoir, causing more surface and bottom discharges at this reservoir. Water volumes discharges at surface and at bottom of both reservoirs are enlisted in Table 2, revealing how water level regulation in both reservoirs is interconnected. Apart from water extraction for municipal consumption, no water outflow occurred from February 2004 to March 2006, during the drought (see Table 2). In fact, there is no ecological flow stipulated for these streams, such that, downstream from the dam walls, only estuarine water flows in during high tide.

Bottom discharges from Odeleite to Beliche, through the underground channel, induced mixing of water column, with ressuspension of sediment and nutrients. Indeed, both reservoirs did not behave as warm monomyctic lakes, but rather as artificially polimyctic, since water column mixed in the winter and also partially, whenever channel sluices were opened. Water level regulation and Beliche water withdrawal for drinking water were seemingly the main impacts on the water quality of these reservoirs. Nevertheless, sharp shifts in water level also affected margin vegetation, contributing to increased nutrient leaching from soils.

650 MDS probe, for water temperature, dissolved oxygen, pH and conductivity. Nutrient concentrations analysis were performed at the accredited (EN 17025) water analysis laboratory from Administração da Região Hidrográfica do Algarve (ARH Algarve), who was also responsible for all the sampling campaigns. Phytopigments including chlorophyll *a* were analyzed at Huelva University (Forján et al., 2008) through HPLC, according to Young et al. (1997). Microcystin detection and quantification was performed according to Carmichael & An (1999) using the micro-ELISA kit Microcystin Plate Kit from Adgen – Agrifood Diagnostic. Phytoplankton composition was determined by the same methods referred in 2.1. Phytoplankton biovolumes following European guidelines were calculated on the basis of predefined 3-dimensional shapes and their respective stereometric formulas as recommended by Edler (1979a, 1979b) and Hillebrand et al. (1999), according to the

Berger Parker dominance index was determined by calculating the proportion of the most abundant species over the total phytoplankton cell density (Magurran, 1988). Carlson Trophic State Index (TSI) was calculated for chlorophyll *a* values and both for total phosphorus (TP) and soluble reactive phosphorus (SRP) concentrations, according to Carlson (1977). Contribution of cyanobacteria to total phytoplankton is given by the percentage of total biovolume attributed to cyanobacteria. Catalán Index for Algal Groups (InGA) was determined by using biovolume proportions of colonial and non-colonial algal groups (Catalán et al., 2003). The MedPTI index was calculated according to Marchetto

Monitoring data for the last 7 years included a severe 18 months drought from 2004 to 2006, and an exceptional rainy year in 2010. As seen in Fig. 9, water level at Odeleite reservoir had to be lowered in November 2006 for maintenance works, originating intense bottom and surface discharges. Surface overflow was released through the stream bed into the Guadiana estuary, but bottom discharged water flowed through the underground channel into Beliche reservoir, causing more surface and bottom discharges at this reservoir. Water volumes discharges at surface and at bottom of both reservoirs are enlisted in Table 2, revealing how water level regulation in both reservoirs is interconnected. Apart from water extraction for municipal consumption, no water outflow occurred from February 2004 to March 2006, during the drought (see Table 2). In fact, there is no ecological flow stipulated for these streams, such that, downstream from the dam walls, only estuarine water flows in during

Bottom discharges from Odeleite to Beliche, through the underground channel, induced mixing of water column, with ressuspension of sediment and nutrients. Indeed, both reservoirs did not behave as warm monomyctic lakes, but rather as artificially polimyctic, since water column mixed in the winter and also partially, whenever channel sluices were opened. Water level regulation and Beliche water withdrawal for drinking water were seemingly the main impacts on the water quality of these reservoirs. Nevertheless, sharp shifts in water level also affected margin vegetation, contributing to increased nutrient

CEN/TC230/WG2/TG3 N108 Water Quality and Olenina et al. (2006).

(2009).

high tide.

leaching from soils.

**3.5 Results and discussion 3.5.1 Hydrometric features**

Table 2. Surface and bottom water discharged from Odeleite and Beliche since 2003 (Data source: http://snirh.pt/)

Ecological Tools for the Management of Cyanobacteria

biovolume proportion (Fig. 12) in at least 50% of the samples.

Blooms in the Guadiana River Watershed, Southwest Iberia 179

gathered during 2009 and 2010 showed diatom (Bacillariophycea) dominance in terms of

Fig. 11. Cyanobacteria dynamics in Odeleite and Beliche reservoirs. Total phytoplankton

In terms of cell abundance, *Microcystis* spp. dominated both reservoirs until spring 2008, but Oscillatoriales dominated in terms of biovolume. Cyanobacteria cell densities above WHO alert level 1 of 2000 cells mL-1, occurred in 62 to 63% of all samples, with episodes of *Microcystis* blooms in June 2004 for Odeleite and July 2004 for Beliche. In summer 2006, a *Microcystis* spp. bloom was toxic with microystin concentrations at the bottom of Beliche reservoir reaching 3.5 µg L-1. Despite high cyanobacteria abundances, no significant levels of

Biovolume proportions for main algal groups (Fig. 12) confirmed cyanobacterial dominance from August to October 2009 in Beliche and from October to December 2009 in Odeleite.

abundance (Log cells mL-1) compared with total Cyanobacteria, *Microcystis* and

*Oscillatoria*/*Planktothryx* spp. abundances

microcystins were detected under other bloom situations.

#### **3.5.2 Nutrient dynamics**

Despite these hydrographical fluctuations, no nutrient accumulation or eutrophication trend was detected. Yearly turn-over of Dissolved Inorganic Nitrogen (DIN) and Soluble Reactive Phosphorus (SRP) was clear in Fig. 10, where water temperature at the surface can be used as reference for seasonal changes in nutrient dynamics in Beliche reservoir.

Fig. 10. Dissolved Inorganic Nitrogen (DIN; mg\*L-1), Soluble Reactive Phosphorus (SRP; mg\*L-1) and water temperature (ºC) during 2003 -2010 in Beliche reservoir. Drought months are highlighted in light grey. Months were unusual Odeleite to Beliche discharges occurred are highlighted in darker grey

The increase in DIN levels during the drought years was fictitious (see light grey box Fig. 10), since water level was so low that surface and bottom samples were almost undistinguishable. Shallower depth allowed for oxygen diffusion to the bottom inhibiting deep summer denitrification. Unusual surface and bottom DIN levels occurred in November 2006 due to exceptional water transfer from Odeleite to Beliche. As stated in Galvão et al, 2008, management of the underground channel between the two reservoirs has been associated with conditions favoring blooms through bottom sediment and nutrient resuspension. Consequent water column mixing was revealed by similar bottom and surface temperatures. Comparing Fig. 9 with Fig. 10 also indicated that the increase in DIN concentration in March 2010 was linked to high precipitation levels. Overall low nutrient concentrations in both reservoirs were associated with oligotrophic conditions. In spite of phosphorus limitations and low median and mode values for DIN:SRP ratios, high average N:P ratios were observed, due to outlier values observed in 2005, 2006 and March 2010.

#### **3.5.3 Phytoplankton dynamics**

In terms of cell abundance, more than 80% of monthly water samples during last eight years from Odeleite and Beliche reservoirs, were dominated by cyanobacteria (Fig. 11), but data

Despite these hydrographical fluctuations, no nutrient accumulation or eutrophication trend was detected. Yearly turn-over of Dissolved Inorganic Nitrogen (DIN) and Soluble Reactive Phosphorus (SRP) was clear in Fig. 10, where water temperature at the surface can be used

Fig. 10. Dissolved Inorganic Nitrogen (DIN; mg\*L-1), Soluble Reactive Phosphorus (SRP; mg\*L-1) and water temperature (ºC) during 2003 -2010 in Beliche reservoir. Drought months are highlighted in light grey. Months were unusual Odeleite to Beliche discharges occurred

The increase in DIN levels during the drought years was fictitious (see light grey box Fig. 10), since water level was so low that surface and bottom samples were almost undistinguishable. Shallower depth allowed for oxygen diffusion to the bottom inhibiting deep summer denitrification. Unusual surface and bottom DIN levels occurred in November 2006 due to exceptional water transfer from Odeleite to Beliche. As stated in Galvão et al, 2008, management of the underground channel between the two reservoirs has been associated with conditions favoring blooms through bottom sediment and nutrient resuspension. Consequent water column mixing was revealed by similar bottom and surface temperatures. Comparing Fig. 9 with Fig. 10 also indicated that the increase in DIN concentration in March 2010 was linked to high precipitation levels. Overall low nutrient concentrations in both reservoirs were associated with oligotrophic conditions. In spite of phosphorus limitations and low median and mode values for DIN:SRP ratios, high average N:P ratios were observed, due to outlier values observed in 2005, 2006 and March 2010.

In terms of cell abundance, more than 80% of monthly water samples during last eight years from Odeleite and Beliche reservoirs, were dominated by cyanobacteria (Fig. 11), but data

as reference for seasonal changes in nutrient dynamics in Beliche reservoir.

**3.5.2 Nutrient dynamics**

are highlighted in darker grey

**3.5.3 Phytoplankton dynamics**

gathered during 2009 and 2010 showed diatom (Bacillariophycea) dominance in terms of biovolume proportion (Fig. 12) in at least 50% of the samples.

Fig. 11. Cyanobacteria dynamics in Odeleite and Beliche reservoirs. Total phytoplankton abundance (Log cells mL-1) compared with total Cyanobacteria, *Microcystis* and *Oscillatoria*/*Planktothryx* spp. abundances

In terms of cell abundance, *Microcystis* spp. dominated both reservoirs until spring 2008, but Oscillatoriales dominated in terms of biovolume. Cyanobacteria cell densities above WHO alert level 1 of 2000 cells mL-1, occurred in 62 to 63% of all samples, with episodes of *Microcystis* blooms in June 2004 for Odeleite and July 2004 for Beliche. In summer 2006, a *Microcystis* spp. bloom was toxic with microystin concentrations at the bottom of Beliche reservoir reaching 3.5 µg L-1. Despite high cyanobacteria abundances, no significant levels of microcystins were detected under other bloom situations.

Biovolume proportions for main algal groups (Fig. 12) confirmed cyanobacterial dominance from August to October 2009 in Beliche and from October to December 2009 in Odeleite.

Ecological Tools for the Management of Cyanobacteria

index applied to Beliche and Odeleite.

**B)**

**MedPTI classes and upper limits**

interpretation

**MedPTI**

Blooms in the Guadiana River Watershed, Southwest Iberia 181

Monthly values for the MedPTI index are illustrated in Fig. 13, with open circles and boxes representing non-valid values based on lower than 70% species contribution to total phytoplankton biovolume. This figure constituted a test to the robustness of the MedPTI

**Beliche** 30 47 3,05

**Odeleite** 19 47 2,90

good – moderate (<2.45)

moderate – poor (<2.13)

poor – bad (1.81)

**TSI classes** <30 30-40 40-50 50-60 60-70

(<2.77)

Fig. 13. Monthly values for MedPTI. Dashed lines indicate lower boundaries of "excellent" and "good" classifications. Open circles and boxes correspond to non-valid values for

Odeleite and Beliche, respectively. See text for explanation

Table 3. A) Determined values for Carlson Trophic State Index (TSI) based on total P concentrations and on chlorophyll a content, and for phytoplankton composition MedPTI index. B) Classification boundary values for TSI and MedPTI with a color code to facilitate

excelent high – good

**A) Reservoir/Index TSI (Tota P) TSI (Chl-a) MedPTI**

Summer bloom absence in 2010 could be linked to high water discharges in consequence of an exceptional rainy winter and spring. (see Fig. 9 and Table 2).Thus, during the study period both Beliche and Odeleite reservoirs were susceptible to CHABs.

Fig. 12. Relative contribution (%) of main algal groups to total phytoplankton biovolume

### **3.5.4 Ecological indices**

Table 3, compiles Carlson Trophic State Indices (TSI) calculated for Beliche and Odeleite based on Chlorophyll *a* (Chl-a) and on total phosphorus (TP) as well as for phytoplankton ecological index (MedPTI) proposed by Marchetto et al. (2009) for Italian deep lakes in the Mediterranean region. This index is based on the proportion of biovolumes of species, listed in Italian lakes, and should not be applied in situations where the biovolume of listed existing species does not exceed 70% of total phytoplankton biovolume. Since the contribution of Marchetto species to total phytoplankton biovolume reached 77% in Beliche during 2010 and 71% in Odeleite, the MedPTI index was also calculated for comparison. TSI based on transparency measured by Secchi depth was not calculated, since it has long been established that torrential hydrographic regimes promote high values for this index without any correlation with eutrophication. Total Phosphorus concentrations were also misleading, since bottom sediment resuspension promoted by artificially induced polimyctic behavior, released organic phosphorus unavailable for phytoplankton into the water column. Low chlorophyll *a* values in spite of high cellular cyanobacteria abundance, as referred previously, was due to low chlorophyll content of cyanobacteria.

Summer bloom absence in 2010 could be linked to high water discharges in consequence of an exceptional rainy winter and spring. (see Fig. 9 and Table 2).Thus, during the study

> Bacillariophyceae Chlorophyceae Chrysopyceae Conjugatophyceae Cryptophyceae Cyanobacteria Dinophyceae Euglenophyceae Klebsormidiophyceae

Trebouxiophyceae Unidentified phytoplankton

previously, was due to low chlorophyll content of cyanobacteria.

Fig. 12. Relative contribution (%) of main algal groups to total phytoplankton biovolume

Table 3, compiles Carlson Trophic State Indices (TSI) calculated for Beliche and Odeleite based on Chlorophyll *a* (Chl-a) and on total phosphorus (TP) as well as for phytoplankton ecological index (MedPTI) proposed by Marchetto et al. (2009) for Italian deep lakes in the Mediterranean region. This index is based on the proportion of biovolumes of species, listed in Italian lakes, and should not be applied in situations where the biovolume of listed existing species does not exceed 70% of total phytoplankton biovolume. Since the contribution of Marchetto species to total phytoplankton biovolume reached 77% in Beliche during 2010 and 71% in Odeleite, the MedPTI index was also calculated for comparison. TSI based on transparency measured by Secchi depth was not calculated, since it has long been established that torrential hydrographic regimes promote high values for this index without any correlation with eutrophication. Total Phosphorus concentrations were also misleading, since bottom sediment resuspension promoted by artificially induced polimyctic behavior, released organic phosphorus unavailable for phytoplankton into the water column. Low chlorophyll *a* values in spite of high cellular cyanobacteria abundance, as referred

period both Beliche and Odeleite reservoirs were susceptible to CHABs.

**0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%**

**% Biovolume of algal groups**

**0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%**

**3.5.4 Ecological indices**

**Odeleite**

**Beliche**

Monthly values for the MedPTI index are illustrated in Fig. 13, with open circles and boxes representing non-valid values based on lower than 70% species contribution to total phytoplankton biovolume. This figure constituted a test to the robustness of the MedPTI index applied to Beliche and Odeleite.

Table 3. A) Determined values for Carlson Trophic State Index (TSI) based on total P concentrations and on chlorophyll a content, and for phytoplankton composition MedPTI index. B) Classification boundary values for TSI and MedPTI with a color code to facilitate interpretation

Fig. 13. Monthly values for MedPTI. Dashed lines indicate lower boundaries of "excellent" and "good" classifications. Open circles and boxes correspond to non-valid values for Odeleite and Beliche, respectively. See text for explanation

Ecological Tools for the Management of Cyanobacteria

allowance of natural variability and (v) reliability.

should be reevaluated, at least for warm semi-arid regions.

contribution of cyanobacteria blooms to phytoplankton biomass.

(Eq. 2)

phytoplankton biovolume.

Blooms in the Guadiana River Watershed, Southwest Iberia 183

As discussed by Jackson et al. (2000), an index that does not allow for temporal and spatial variability, might lose reliability. The decreased InGA value calculated for summer 2009 (Fig. 14), reflected the weight of 4 given to cyanobacterial biovolumes in the index formula

> 1 0.1 \* Cr Cc 2 \* (Dc Chc) 3 \* Vc 4 \* Cia + ++ + <sup>=</sup> + ++ + + +

In fact, whenever a lake or reservoir has a cyanobacterial bloom, InGA value will indicate it, but so will simply calculating the relative contribution of cyanobacteria to total

Another problem emerging from the application of multi-metric indices is the loss of interpretability. With InGA, high contributions to total biovolumes of Dinophyceae and non- colonial phytoplankton groups are assumed to improve the ecological status, whereas colonial forms and cyanobacteria worsen it. Thus, high biovolume proportions of non-toxic Chroococcales, are given the same negative weight as toxic filamentous cyanobacteria.

It is well known and accepted that different metrics applied to the same ecological condition can attribute different classifications. This is obviously linked to the information provided by the variables selected in each metric analysis. Criteria for the selection of these metrics should consider the prerequisites previously mentioned (see section 1.), such as: (i) obtainability, (ii) relevance in term of specific objectives, (iii) discriminant capacity, (iv)

Results obtained for dominance (Fig. 11) and cyanobacterial contribution (> 9%) to total phytoplankton biovolume, (Fig. 12), indicate eutrophication under WFD guidelines (JRC EC, 2009). Considering the oligotrophic state of both reservoirs, the reliability of such indicators

Solimini et al. (2006) considered the contribution of cyanobacteria to total phytoplankton biomass, as a reliable and simple indicator of trophic state, based on the following assumptions: (i) most cyanobacteria species show a strong preference for eutrophic conditions, (ii) due to toxicity of some taxa, blooms can pose serious water quality, animal and human health risks, as well as environmental problems, and finally, (iii) the large

This study contradicts the first assumption, since the genera and species typically linked to eutrophication were found associated to oligotrophy. The second assumption was partially verified, but toxin production seemed to be limited in oligotrophic conditions, despite high cell abundances. Potentially toxic cyanobacteria do not always produce cyanotoxins, so toxicity needs to be confirmed. Finally, the last assumption which links eutrophication to

(2)

1 2 \* (D Cnc) Chnc Dnc InGa

D Dinophyceae Cc Chrysophyceae colonial Cnc Chrysophyceae non colonial Dc Bacillariophyceae colonial Chnc Chlorococcales non colonial Chc Chlorococcales colonial DnC Bacillariophyceae non colonial Vc Volvocales colonial Cr Cryptophyceae Cia Cyanobacteria

Since MedPTI was specifically developed for deep natural lakes in Italy, this index should probably not be applied to reservoirs without adjustments to existing species lists. Nevertheless, MedPT1 classifications obtained in this study seemed more consistent than those obtained using General Algal Group Index (InGA) proposed by Catalàn et al (2003).

 Table 4 compiles values for InGA Catalàn et al. 2003). These authors recommended that the use of this multi-metric index of phytoplankton composition for ecological status classification should be calculated for late summer and fall samples. Trophic state metrics, as recommended by several water authorities in Portugal and in Europe, apply a color code for easy comparison of different classifications, which was used in Tables 3 and 4. In the case of Beliche and Odeleite reservoirs, fall values represented a worst case scenario and artificially attributed a good or moderate classification to waters that would otherwise be recognized as very good. Fig. 14 illustrates monthly variability of Catalàn InGA values.

Table 4. Catalàn InGA values determined for Beliche and Odeleite reservoirs for several groups of samples

Fig. 14. Monthly values of the General Algal Groups Index (InGA) determined for Beliche and Odeleite Reservoir 2009-2010 data. Dashed lines indicate the lower limits for Very Good (0,1) and Good (0,01) classification

Since MedPTI was specifically developed for deep natural lakes in Italy, this index should probably not be applied to reservoirs without adjustments to existing species lists. Nevertheless, MedPT1 classifications obtained in this study seemed more consistent than those obtained using General Algal Group Index (InGA) proposed by Catalàn et al (2003). Table 4 compiles values for InGA Catalàn et al. 2003). These authors recommended that the use of this multi-metric index of phytoplankton composition for ecological status classification should be calculated for late summer and fall samples. Trophic state metrics, as recommended by several water authorities in Portugal and in Europe, apply a color code for easy comparison of different classifications, which was used in Tables 3 and 4. In the case of Beliche and Odeleite reservoirs, fall values represented a worst case scenario and artificially attributed a good or moderate classification to waters that would otherwise be recognized as very good. Fig. 14 illustrates monthly variability of Catalàn InGA values.

**Period Beliche Odeleite InGA limits InGA classes 2009-2010** 0.167 0.237 >0.1 very good **2009** 0.098 0.067 0.01-0.1 good **2010** 0.196 0.403 0.005-0.01 moderate

**October 2009** 0.022 0.008 0.003-0.005 poor **October 2010** 0.536 1.864 <0.003 bad

Table 4. Catalàn InGA values determined for Beliche and Odeleite reservoirs for several

Fig. 14. Monthly values of the General Algal Groups Index (InGA) determined for Beliche and Odeleite Reservoir 2009-2010 data. Dashed lines indicate the lower limits for Very Good

groups of samples

**InGA**

(0,1) and Good (0,01) classification

As discussed by Jackson et al. (2000), an index that does not allow for temporal and spatial variability, might lose reliability. The decreased InGA value calculated for summer 2009 (Fig. 14), reflected the weight of 4 given to cyanobacterial biovolumes in the index formula (Eq. 2)

$$\text{InGa} = \frac{1 + 2 \,\text{\*} \,(\text{D} + \text{Cnc}) + \text{Chnc} + \text{Dnc}}{1 + 0.1 \,\text{\*} \,\text{Cr} + \text{Cc} + 2 \,\text{\*} \,(\text{Dc} + \text{Chc}) + 3 \,\text{\*} \,\text{Vc} + 4 \,\text{\*} \,\text{Ca}} \tag{2}$$

In fact, whenever a lake or reservoir has a cyanobacterial bloom, InGA value will indicate it, but so will simply calculating the relative contribution of cyanobacteria to total phytoplankton biovolume.

Another problem emerging from the application of multi-metric indices is the loss of interpretability. With InGA, high contributions to total biovolumes of Dinophyceae and non- colonial phytoplankton groups are assumed to improve the ecological status, whereas colonial forms and cyanobacteria worsen it. Thus, high biovolume proportions of non-toxic Chroococcales, are given the same negative weight as toxic filamentous cyanobacteria.

It is well known and accepted that different metrics applied to the same ecological condition can attribute different classifications. This is obviously linked to the information provided by the variables selected in each metric analysis. Criteria for the selection of these metrics should consider the prerequisites previously mentioned (see section 1.), such as: (i) obtainability, (ii) relevance in term of specific objectives, (iii) discriminant capacity, (iv) allowance of natural variability and (v) reliability.

Results obtained for dominance (Fig. 11) and cyanobacterial contribution (> 9%) to total phytoplankton biovolume, (Fig. 12), indicate eutrophication under WFD guidelines (JRC EC, 2009). Considering the oligotrophic state of both reservoirs, the reliability of such indicators should be reevaluated, at least for warm semi-arid regions.

Solimini et al. (2006) considered the contribution of cyanobacteria to total phytoplankton biomass, as a reliable and simple indicator of trophic state, based on the following assumptions: (i) most cyanobacteria species show a strong preference for eutrophic conditions, (ii) due to toxicity of some taxa, blooms can pose serious water quality, animal and human health risks, as well as environmental problems, and finally, (iii) the large contribution of cyanobacteria blooms to phytoplankton biomass.

This study contradicts the first assumption, since the genera and species typically linked to eutrophication were found associated to oligotrophy. The second assumption was partially verified, but toxin production seemed to be limited in oligotrophic conditions, despite high cell abundances. Potentially toxic cyanobacteria do not always produce cyanotoxins, so toxicity needs to be confirmed. Finally, the last assumption which links eutrophication to

Ecological Tools for the Management of Cyanobacteria

Vasconcelos was greatly appreciated.

**6. References**

Blooms in the Guadiana River Watershed, Southwest Iberia 185

45 ("Risk evaluation of toxic blooms in lower Guadiana") from LEADER+ program, project DYNCYANO (PTDC/AMB/64747/2006) funded by the Portuguese Science and Technology Foundation (FCT). We also thank past post-doctoral fellows Dr. Alexandre Matthiensen, Dr. Carlos Rocha, and Dr. Cristina Sobrino for contributing to different parts of this study at different times, and the following Master´s and PhD students Rute Miguel, Pedro Mendes, Vânia Sousa and Teresa Cecílio, as well as research assistants Cátia Luis, Tânia Anselmo, Erika Almeida and Cátia Guerra. Collaboration from Dr. Wayne Carmichael and Prof. Vitor

R. Domingues acknowledges PhD (SFRH/BD/27536/2006) and Post-Doctoral (SFRH/BPD/ 68688/2010) fellowships from FCT, and S. Mesquita acknowledges PhD grant (SFHR/BD/ 18921/2004) and the research grant financed in the scope of the Southwest European

Algarve reservoirs were studied in the scope of 4 research projects, namely CIANOALERTA I, II and III (2003-2008) funded through INTERREG IIIA, contracts nº SP5/P35/01, SP5/P19/02 and SP5/P138/03 and CIANOTOOLS financed in the scope of the Southwest European Research Net (RISE) through the POCTEP UE Program in collaboration with

Admnistração da Região Hidrográfica do Alentejo (ARH Alentejo), (2011). Planos de gestão

http://www.arhalentejo.pt/downloads/part\_publi\_pgrh/fase\_final/RH7/t09122\_

Alves, M. H. & Bernardo, J. M. (1998). Novas perspectivas para a determination do caudal

Alves, M. H. & Gonçalves, H. (1994). O caudal ecológico como medida de minimização dos

Barbosa, A.B., Domingues, R.B. & Galvão, H.M. (2010). Environmental forcing of

Berg, K., Carmichael, W.W., Skulber, O.M., Benestad, C. & Underdal, B. (1987). Investigation

Blaha, L., Babica, P. & Maršálek, B. (2009). Toxins produced in cyanobacterial water blooms – toxicity and risks. *Intersisciplinary Toxicology*, Vol. 2, No. 2, (May 2009), pp.36-41

das bacias hidrográficas integradas nas regiões hidrográficas 5, 6 e 7-região hidrográfica 7. In: Relatório técnico para efeitos de participação pública, t09122/01,

ecológico em regiões semi-áridas. Proceedings of *Seminário sobre barragens e ambiente*. Comissão nacional Portuguesa da Grandes barragens, ISBN 92-894-5122-

impactes nos ecossitemas lóticos. Métodos para a sua determinação e aplicações. Proceedings of: "*Actas do 6ºSILUSB/1ºSILUSBA, Simpósio de Hidráulica e Recursos dos*

phytoplankton in a semi-arid estuary (Guadiana estuary, south-western Iberia): a decadal study of climatic and anthropogenic influences. *Estuaries and Coasts*, Vol.

of a toxic-water bloom of *Microcystis aeruginosa* (Cyanophyceae) in Lake Akersvatn,

Research Net (RISE) through the I2TEP UE Program- subprogram CIANOTOOLS.

Algarve Regional Hydrographycal Administration and University of Huelva.

(Junho 2011), Acessed in August 1st 2011, Available from

*Países de Língua oficial Portuguesa*", (Abril de 1994), Lisboa

33, No. 2, (March 2010), pp. 324-341, ISSN 1559-2723

Norway. *Hydrobiologia*, Vol. 144, pp. 97–103

01\_PGBH\_RH7\_Relatorio\_Tecnico.pdf

X, Porto, Maio de 1998

large contribution of cyanobacteria to phytoplankton biomass, should be re-assessed in Mediterranean regions, where, even under oligotrophic conditions, cyanobacteria are favoured in naturally warm waters.


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