Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56835, enero-diciembre 2024 (Publicado Ago. 22, 2024)

Iron and Manganese concentrations in leaf tissues of Rhizophora mangle

(Rhizophoraceae): implications for energetic metabolism

Sávia Soares Pascoalini1*; https://orcid.org/0000-0003-3236-8304

Dielle Meire de Santana Lopes1; https://orcid.org/0000-0002-9951-0179

Antelmo Ralph Falqueto2; https://orcid.org/0000-0003-3146-1873

Verônica D’Addazio3; https://orcid.org/0000-0003-4496-9238

Adriano Alves Fernandes2; https://orcid.org/0000-0002-5016-0745

Marcelo Barcellos da Rosa4; https://orcid.org/0000-0001-5959-0381

Andreia Barcelos Passos Lima Gontijo2; https://orcid.org/0000-0003-3422-4398

Mário Luiz Gomes Soares5; https://orcid.org/0000-0002-3312-7257

Ivoney Gontijo2; https://orcid.org/0000-0002-4251-4689

Edilson Romais Schmildt2; https://orcid.org/0000-0002-3457-7997

Helia Del Carmen Farías Espinoza6; https://orcid.org/0000-0001-6776-0838

Bryan Brummelhaus de Menezes4; https://orcid.org/0000-0002-3431-7669

Lucas Mironuk Frescura4; https://orcid.org/0000-0002-7906-0254

Raquel Vidal dos Santos Leopoldo1; https://orcid.org/0000-0001-5667-650X

Camila Patricio de Oliveira1; https://orcid.org/0000-0002-4211-6752

Lucas de Almeida Leite2; https://orcid.org/0009-0009-0960-9019

Neilson Victorino de Brites Júnior1; https://orcid.org/0009-0008-8765-9022

Ully Depolo Barcelos1; https://orcid.org/0000-0003-4933-5912

Mônica Maria Pereira Tognella2; https://orcid.org/0000-0002-1521-8251

1. Espírito-Santo Foundation of Technology (FEST), Federal University of Espírito Santo, Vitória, ES CEP 29075-910,

Brazil; savia.pascoalini@gmail.com (*Correspondence), dielle.slopes@gmail.com, vidalquel@gmail.com,

patricio.camila@hotmail.com, neilsonbrites@gmail.com, ullydbarcelos@gmail.com

2. Department of Agrarian and Biological Sciences, Federal University of Espírito Santo, São Mateus, ES CEP 29932-900,

Brazil; antelmofalqueto@gmail.com, afernandesufes@gmail.com, albarcelos@hotmail.com,

ivoneygontijo@yahoo.com.br, e.romais.s@gmail.com, bio.lucasdealmeidaleite@gmail.com,

monica.tognella@gmail.com

3. Department of Oceanography and Ecology, Federal University of Espírito Santo, Vitória, ES CEP 29075-910, Brazil;

veronicadaddazio@yahoo.com

4. Department of Chemistry, Federal University of Santa Maria, Camobi Campus, Santa Maria, RS CEP 97105-900,

Brazil; marcelobdarosa@gmail.com, bryanmenezesqmc@gmail.com, lmironuk15@gmail.com

5. Faculty of Oceanography, Rio de Janeiro State University, Rio de Janeiro, RJ CEP 20550-900, Brazil;

mariolgs@gmail.com

6. Center for Technological Sciences of Land and Sea, Vale do Itajaí University, Itajai, SC CEP 88302-901, Brazil;

heliafarespinoza@gmail.com

Received 28-IX-2023. Corrected 06-V-2024. Accepted 08-VIII-2024.

https://doi.org/10.15517/rev.biol.trop..v72i1.56835

AQUATIC ECOLOGY

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56835, enero-diciembre 2024 (Publicado Ago. 22, 2024)

INTRODUCTION

Anthropogenic activities such as petro-

leum exploration, mining, industrial activi-

ties, and other human urban activities are the

main sources of trace elements in mangroves

(Celis-Hernandez et al, 2020). In Brazil, in

2015, the collapse of the Fundão Dam in

Mariana occurred, releasing 40 to 60 million

m3 of mining tailings into the Doce River,

the sediment plume, containing trace met-

als, reached the ocean and coastal region,

ABSTRACT

Introduction: Iron (Fe) and manganese (Mn) are bioessential micronutrients for plants but can impair the ener-

getic metabolism when present at high levels.

Objective: To assess the photosynthetic performance and oxidative damages in Rhizophora mangle L. leaf tissues

at low and high concentrations of Fe (74 and 195 mg kg-1; Feleaf) and Mn (65 and 414 mg kg-1; Mnleaf).

Methods: Photosynthetic pigments, chlorophyll a fluorescence, leaf CO2 assimilation and gas exchange and

DPPH• radical scavenging capacity were sampled in R. mangle growing in estuarine forests in the North region of

Espírito Santo State and the extreme South of Bahia State (Brazil) showing low and high Feleaf and Mnleaf.

Results: Effects of high Fe and Mn were not identified on pigment levels. The increase in Feleaf and Mnleaf at the

levels observed in this assessment had a positive effect on the number of reaction centers and on the efficiency of

the oxygen-evolving complex, evaluated as K-band, while no changes were found in the parameters related to the

excitation trapping efficiency at the active center of photosystem II. Distinct interference patterns of Fe and Mn

on the functional processes of photosynthesis were identified, especially on CO2 assimilation and reactive oxygen

species metabolism, with major effects on CO2 assimilation and carboxylation efficiency of Rubisco at high Mnleaf.

Conclusion: These findings demonstrate the efficiency of R. mangle in positively regulating the electron transport

chain in response to high Fe and Mn, at least in terms of the preservation of structure and functionality of the

plant photosynthetic apparatus. Moreover, interference of high Mnleaf in R. mangle occurs at non-stomatal and

biochemical levels. There is an antagonistic interference of these trace elements with the physiology of R. mangle,

which is a dominant species in Brazilian mangroves.

Key words: JIP-test; CO2 assimilation; DPPH•; photosynthesis; Doce River.

RESUMEN

Concentraciones de hierro y manganeso en tejidos foliares

de Rhizophora mangle (Rhizophoraceae): implicaciones para el metabolismo energético

Introducción: El hierro (Fe) y el manganeso (Mn) son micronutrientes bioesenciales para las plantas, pero pue-

den afectar el metabolismo energético cuando están presentes en niveles altos.

Objetivo: Evaluar el desempeño fotosintético y los daños oxidativos en tejidos foliares de Rhizophora mangle L. a

bajas y altas concentraciones de Fe (74 y 195 mg kg-1; Feleaf) y Mn (65 y 414 mg kg-1; Mnleaf).

Métodos: Se muestrearon pigmentos fotosintéticos, fluorescencia de clorofila a, asimilación e intercambio de

gases de CO2 foliar y capacidad de eliminación de radicales DPPH• en R. mangle que crece en bosques de estuarios

en la región Norte del estado de Espírito Santo y el extremo Sur del Estado de Bahía (Brasil) mostrando Feleaf y

Mnleaf bajos y altos.

Resultados: No se identificaron efectos de niveles elevados de Fe y Mn en los niveles de pigmento. El aumento

de Feleaf y Mnleaf en los niveles observados en esta evaluación tuvo un efecto positivo en el número de centros de

reacción y en la eficiencia del complejo generador de oxígeno, evaluado como banda K, mientras que no se encon-

traron cambios en los parámetros relacionados con la eficiencia de atrapamiento de excitación en el centro activo

del fotosistema II. Se identificaron distintos patrones de interferencia de Fe y Mn en los procesos funcionales de

la fotosíntesis, especialmente en la asimilación de CO2 y el metabolismo de las especies reactivas de oxígeno, con

efectos importantes en la asimilación de CO2 y la eficiencia de carboxilación de Rubisco a niveles altos de Mnleaf.

Conclusión: Los hallazgos demuestran la eficiencia de R. mangle en la regulación positiva de la cadena de

transporte de electrones en respuesta a los altos niveles de Fe y Mn, al menos en términos de preservación de

la estructura y funcionalidad del aparato fotosintético de la planta. Además, la interferencia de Mnleaf alto en R.

mangle se presenta a niveles no estomáticos y bioquímicos. Hay interferencia antagónica de estos oligoelementos

con la fisiología de R. mangle, que es una especie dominante en los manglares brasileños.

Palabras clave: JIP-test; asimilación de CO2; DPPH•; fotosíntesis; Río Doce.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56835, enero-diciembre 2024 (Publicado Ago. 22, 2024)

potentially affecting mangrove areas (Sá et al.,

2021; Tognella et al., 2022). These compounds

impose an energy drain on plants; at least in

the long-term, persistently high concentrations

in mangrove sediments can cause damage to

environmental health and affect plant growth,

energetic metabolism, and cell structure, thus

inducing changes in the ecosystem structure

(Wang et al., 2003).

Iron (Fe) and manganese (Mn) are bio-

essential micronutrients for plant growth and

development for being directly involved in

metabolic processes (Najafpour et al., 2014;

Taiz et al., 2017; Varma & Jangra, 2021). Both

elements have a direct relationship with plant

photosynthesis. Fe plays an important role as

a component of enzymes that participate in

electron transfer (redox reactions), such as

cytochromes, being reversibly oxidized and

reduced through electron transfer between Fe2+

and Fe3+. Besides, chlorophyll (Chl) biosynthe-

sis and maintenance of the structural integrity

of photosynthetic reaction centers and light-

harvesting compounds (LHC) subunits require

Fe. In the photosynthetic electron transport

chain (ETC), Fe acts as a cofactor in photosys-

tems II and I (PSII and PSI, respectively) and in

the cytochrome (Cyt) b6/f complex (Taiz et al.,

2017). Mn ions (Mn2+), in turn, activate several

plant enzymes of the citric acid cycle (Krebs

cycle), such as decarboxylases and dehydroge-

nases. The best-defined function of Mn2+ is to

constitute the oxygen-evolving complex (OEC)

associated with PSII through which oxygen (O2)

is produced from water. The OEC is a manga-

nese-calcium (Mn4CaO5(H2O)4) cluster housed

in a protein complex (Najafpour et al., 2014).

Despite being important for electron

transfer in chloroplasts, excessive Fe and Mn

accumulation in leaf tissues leads to increased

cellular toxicity due to reactive oxygen species

(ROS) overproduction (Gill & Tuteja, 2010).

The chloroplast, more specifically the elec-

tron acceptor side of PSI associated with the

thylakoid membrane, is the main site of ROS

production in plant cells (Gill & Tuteja, 2010).

In conditions of superreduction of the ETC as

an effect of physiological disorders, part of the

electron flow is diverted from ferredoxin to O2,

which is reduced to O2•- via the Mehler reaction

(Taiz et al., 2017). Due to high reactivity and

toxicity, ROS cause damage to proteins, lipids,

carbohydrates, and DNA, ultimately resulting

in cell death (Bailey-Serres & Mittler, 2006).

O2•- ions can donate electrons to Fe+3 to gener-

ate the reduced form Fe+2, which reduces the

H2O2 formed by dismutation of O2•- into OH•

(Gill & Tuteja, 2010). In this sense, oxidative

stress is triggered by disturbances in the pho-

tosynthetic electron flow through the ETC.

Under normal conditions, electron flow leads

to reduction of NADP+ to NADPH, which

is used in the Calvin Cycle to reduce CO2 to

carbohydrates (Taiz et al., 2017). Oxidative

stress occurs when ROS generation exceeds the

capacity of the plant to maintain cellular redox

homeostasis or to scavenge the toxic O2 mol-

ecules (Gill & Tuteja, 2010).

Nevertheless, plants have evolved a sophis-

ticated antioxidant system comprised of specif-

ic protective mechanisms to defend themselves

against oxidant injury (Gholami et al., 2012).

It is well established that the ability to toler-

ate environmental stress is associated with the

expression of an efficient antioxidative system,

which provides the first line of defense against

the toxic effects of enhanced ROS levels (Gill &

Tuteja, 2010). This antioxidative system is com-

posed of antioxidant enzymes as superoxide

dismutase (SOD), ascorbate peroxidase (APX),

guaiacol peroxidase (GPX), catalase (CAT),

monodehydroascorbate reductase (MDHAR),

dehydroascorbate reductase (DHAR) and glu-

tathione reductase (GR) as well as nonenzy-

matic metabolites of low molecular weight like

ascorbic acid (Vitamin C), glutathione (GSH),

proline (Pro), α-tocopherol (Vitamin E), carot-

enoids and flavonoids (Mittler et al., 2004).

The antioxidant potential can also be eval-

uated in plant tissues by assessing the capacity

to scavenge the 1,1-Diphenyl-2-picrylhydrazyl

free radical (DPPH•) (Menezes et al., 2021).

The DPPH• assay provides rapid results to eval-

uate the scavenging efficiency of extracts from

Rhizophora mangle leaf tissues against ROS,

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56835, enero-diciembre 2024 (Publicado Ago. 22, 2024)

maintaining the flow of electrons along the

ETC as well as CO2 assimilation at high rates.

This work aims to evaluate the photosyn-

thetic performance and the integrity of the

antioxidant defense mechanisms of the true

mangrove species R. mangle in response to high

levels of Fe and Mn in leaf tissues. The present

findings contribute to better understand the

role of Fe and Mn in carbon assimilation and

evidence how beneficial these elements are.

Thus, carbon assimilation, Chl a fluorescence,

photosynthetic pigments, and DPPH• radical

scavenging activity were compared in plants

from different mangrove areas containing the

highest and lowest leaf concentrations of Fe and

Mn. Such evaluations allowed to demonstrate

how these elements drain energy from plants,

and that their persistence in the sediment

can induce a long-term loss of environmental

health. These approaches are highly relevant in

the current century, since coastal eutrophica-

tion and the increase in atmospheric carbon

have been widely discussed (Gilman et al.,

2008; Sanders et al., 2014). Our experiment

was conducted in several mangrove systems in

the states of Espírito Santo and Bahia, Brazil,

following the failure of the Fundão tailings

dam in 2015 (Instituto Brasileiro do Meio

Ambiente e dos Recursos Naturais Renováveis

[IBAMA], 2019).

MATERIALS AND METHODS

Study area, plant material, and sampling:

The sampling locations are shown in Fig. 1 and

further described in Tognella et al. (2022). The

areas were selected according to the concentra-

tions of Fe and Mn in leaf tissues (hereafter,

“Feleaf” and “Mnleaf”, respectively) of R. mangle,

which is the dominant species in the stud-

ied plots. Thus, four estuaries (Aracruz, Barra

Nova, São Mateus and Caravelas, within the

geographic coordinates (19°93’97”-17°72’70”

S & 40°21’32”-39°28’32” W) showing low and

high Feleaf and Mnleaf were selected in fringe

and basin forests in the North region of Espírito

Santo State and the extreme South of Bahia

State (Brazil).

Five plants were randomly selected and

marked in each plot. Sampling and measure-

ments were performed in fully expanded but

not visibly senescent leaves of the second pair of

the branch from the apex. Measurements were

made in the morning (between 8:00 and 12:00).

Plant height varied from 0.68 to 1.64 m and

0.88 to 1.54 m at the low and high Feleaf sites,

respectively. All sites were inserted within pre-

defined areas (fixed plots). The sampling loca-

tions showed statistical similarities regarding

the minimum and maximum values of salinity

(3.7-33.4 psu and 3.7-29.6 psu at the low and

high Fe and Mn sites, respectively). Mean annu-

al rainfall ranges between 1 100 and 1 400 mm

at the study sites (Alvarez et al., 2014; Instituto

Nacional de Meteorologia, 2019). In addition,

precipitation and relative air humidity in the

source areas ranged from 115.76 to 119.17 mm

and 75.5 to 87.0 %, respectively between April

and August 2019 (Instituto Nacional de Meteo-

rologia, 2019), characterized as dry period.

Sampling was conducted at low tide.

Sediment and leaf sampling: Sediment

samples were collected in the intertidal area

using collectors built with a 50 cm PVC pipe.

The material was obtained in two depths of

0 to 5 cm (surface) and 5 to 15 cm. In each

plot, simple samples were randomly collected

(for each depth) and homogenized to remove

roots, shell fragments, leaves and branches and

thus form a composite sample for each depth.

Next, the sediment samples were placed into

previously identified plastic bags, kept in a cool

box with ice and taken to the laboratory for

freezer storage.

To quantify the concentration of metals in

leaf tissues of R. mangle, healthy mature leaves

showing no signs of herbivory were sourced

from five trees in each location, stored in paper

bags and maintained under refrigeration until

processing. In the laboratory, leaves were dried

at 60 °C until constant weight was attained.

Sediment and leaf chemical analysis:

Analysis of metals in sediments was performed

according to the United States Environmental

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56835, enero-diciembre 2024 (Publicado Ago. 22, 2024)

Fig. 1. Location of the sampling sites in Northern Espírito Santo and Southern Bahia. Triangle, square, circle and hexagon

identify the sites with high and low leaf Fe concentration and high and low leaf Mn concentration, respectively.

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56835, enero-diciembre 2024 (Publicado Ago. 22, 2024)

Protection Agency (USEPA) Method 3051A

(USEPA, 2013). Dry and homogenized sedi-

ment (about 0.5 g) was digested with HNO3:HCl

(3:1; 12 ml) in Teflon tubes in a microwave oven

(CEM, Marx X-Press) using the following pro-

gram: 1st ramp 25-175 °C in 5:30 min; and 2nd

ramp 25-175 °C in 4:30 min (both with a power

setting of 1 600 W). Afterwards, the solution

was cooled, filtered through a Whatman n° 1

filter, diluted to 100 ml in a volumetric flask

and analyzed by ICP-MS (Inductively Coupled

Plasma Mass Spectrometry; Agilent, CX7 500).

The plant material was dried, ground

and submitted to nitroperchloric digestion.

Feleaf and Mnleaf were subsequently quanti-

fied by atomic absorption spectrophotometry

(Silva, 2009).

Pigment quantification: Leaf samples of 5

g (fresh mass) were frozen at -30 °C and ground

in liquid nitrogen with a mortar and pestle

to form a fine powder, which was transferred

to test tubes and homogenized in 15 ml of 90

% acetone solutions and 0.5 g l-1 of calcium

carbonate (CaCO3). Next, the test tubes were

immediately stored at 2 °C for 24 h for com-

plete pigment extraction (modified by Arar,

1997). The samples were then filtered, and the

supernatant was collected and stored in amber

flasks at -30 °C pending analysis by spectropho-

tometry. The extraction procedure was carried

out at room temperature in dark conditions

to minimize Chl degradation by enzymatic

action. Ice cold extraction solvents were used.

The extraction time was kept to a minimum to

reduce degradation of the analyzed pigments.

Subsequently, the optical density read-

ings were determined on a spectrophotometer

(Genesys 10S UV-Vis, Thermo Fisher Scien-

tific, Waltham, USA) at 470, 645 and 663 nm.

Determination of photosynthetic pigment

concentrations was performed according to

the equations proposed by Wellburn (1994):

chlorophyll a (Chla) a = (12.25 x A663 − 2.79 x

A645), chlorophyll b (Chlb) = (21.5 x A645 − 5.1

x A663), and carotenoids (Car) = (1 000 x A470

− 1.82 x Chla − 85.02 Chlb/198); values were

expressed in mg ml-1 of fresh mass, where A470,

A645 and A663 represent the absorbance at 470,

645 and 663 nm, respectively.

Chlorophyll a fluorescence and leaf gas

exchange: Chl a fluorescence was measured

at room temperature using a plant efficiency

analyzer (Handy-PEA, Hanstech Instruments

Ltd., King’s Lynn, Norkfolk, UK), as in Strasser

& Govindjee (1992). Previously, leaves were

adapted to the dark during 30 min using leaf

clips (Hansatech Instruments Ltd.). Fluores-

cence rise OJIP trace was induced by 1 s pulses

of red light (650 nm, 3 000 μmol (photon) m–2

s–1). O and P refer to the initial and maximum

fluorescence intensity considered here at 50 μs

(F0) and 300 ms, respectively. J (≅ 2 to 3 ms)

and I (≅ 30 ms) are inflection points between O

and P levels. The fluorescence transients OJIP

curves were analyzed according to the JIP-test

(Strasser et al., 2004). L and K-bands were

calculated as VOK = (F100μs − F0)/(F300μs − F0)

and VOJ [(F300μs − F0)/(F2ms − F0)] (Srivastava

& Strasser 1997; Strasser & Stirbet, 2001). A

detailed description of parameters and their

meaning can be found elsewhere (Strasser et al.,

2004) and briefly addressed in Table 1.

Leaf CO2 assimilation (A [μmol m–2 s–1]),

stomatal conductance (gs [mol m–2 s–1]), inter-

cellular CO2 concentration (Ci [μmol m–2 s–1])

and leaf transpiration rate (E [mmol m–2 s–1])

were estimated in the same leaves used to mea-

sure Chl a fluorescence; for that, portable infra-

red gas analyzers (models Lci, Lci T and Lcpro T,

ADC, BioScientific Ltd., Hoddesdon, England)

were used. The gas chamber was maintained

at ambient conditions, the average photon flux

density in the chamber was 200 ± 28.7 and

316 ± 46.9 μmol m–2 s–1, with the average leaf

temperature reaching 29.5 ± 0.4 and 31.0 ±

0.5 °C for the Fe and Mn treatments, respec-

tively. Estimation of water-use efficiency was

calculated and determined as intrinsic water-

use efficiency (WUEint = A/gs [μmol (CO2)

mol–1(H2O)]) and instantaneous water-use

efficiency (WUEins = A/E [μmol (CO2) mmol–

1(H2O)]) (Krauss, et al, 2006). A and Ci were

used to estimate the carboxylation efficiency

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56835, enero-diciembre 2024 (Publicado Ago. 22, 2024)

of ribulose-1,5-bisphosphate-carboxylase/oxy-

genase (Rubisco) (A/Ci) (Zhang et al., 2001).

DPPH• radical scavenging assay: The free

radical scavenging ability of the extracts was

determined using the DPPH• method (Dal Prá

et al., 2013; Huang et al., 2005). To obtain the

extracts, which were referred to as working

solutions (WS), plant samples were dried in an

oven and then diluted in methanol (two, five,

ten or twenty times). The tests were carried out

by adding aliquots of 15, 25 and 35 µL of each

WS into cuvettes containing 3.0 mL of DPPH

methanolic solution (0.2 mmol l−1), which were

kept protected from light for 60 min. Tests

were made in triplicate. Measurements were

performed on a Perkin Elmer Lambda 16 spec-

trophotometer, monitoring the absorbance of

the samples at 517 nm. The blank consisted of

3.0 ml of methanol containing 15, 25 or 35 µl of

the respective WS. A solution containing DPPH

served as negative control. The percentage of

DPPH• scavenging was calculated as [1 − (As −

Ab)/ (Ac − Ab)] x 100, where As, Ab and Ac are

the absorbance of samples, blank and negative

control, respectively.

The IC50 values (concentration necessary

to inhibit 50 % of the DPPH• radical) were

determined by means of linear regression. As

analyses were performed in triplicate, the mean

and the standard deviation were used to repre-

sent the IC50 of each sample.

Tabl e 1

Abbreviations of the JIP-test parameters, formulas, and description of the data derived from the transient of Chl a

fluorescence.

Fluorescence Parameters Description

FtFluorescence at time t after onset of actinic illumination

Fo ≅ F20ms Minimal fluorescence, 11cep all PSII RCs are open

FK ≅ F0.3ms Fluorescence intensity at the K-step (0.3 ms) of OJIP

FJ ≅ F2ms Fluorescence intensity at the J-step (2 ms) of OJIP

FI ≅ F30ms Fluorescence intensity at the I-step (30 ms) of OJIP

FP (=Fm)Maximal fluorescence at the peak P, 11cep all PSII RCs are closed

Fv ≅ Fm − F0Maximal 11ceptor11 fluorescence

Area Total complementary 11cep between the fluorescence induction curve and

F0 and Fm

VJ = (FJ − F0)/(Fm − F0)Relative 11ceptor11 fluorescence at the J-step

L-band = VOK = (Ft − F0)/(FK − F0)Variable fluorescence between steps O (50 µs) and K (300 µs), indicative of

the energetic connectivity between the subunits associated with PSII

K-band = VOJ = (Ft − F0)/(FJ − F0)Variable fluorescence between steps O (50 µs) and J (2 ms), which is an

indicative of stability of oxygen 11ceptor11n complex (OEC)

ABS/RC = M0.(1/VJ).(1/ φP0)Absorption flux per active reaction center (RC) at t = 0.

TR0/RC = M0.(1/VJ)Trapped energy flux per RC (at t = 0)

ET0/RC = M0.(1/VJ).ψЕ0 Electron transport flux per RC (at t = 0)

DI0/RC = [(ABS/RC) − (TR0/RC)] Dissipated energy flux per RC at t = 0.

RC/CS0 = φP0·(VJ/M0)·(ABS/CS) Total number of active reaction center per cross section

φP0 = TR0/ABS = [1 − (F0/Fm)] = Fv/FmMaximum quantum yield of primary photochemistry at t = 0).

φD0 = 1 − φP0 = (F0/Fm)Quantum yield of energy dissipation (at t = 0).

φE0 = (1 − F0/Fm) (1 − VJ)Quantum yield for PSII electron transport (ET)

δR0 = (1 − VI)/(1 − VJ)Quantum yield for reduction of the end electron acceptors at the PSI

11ceptor side

PIABS = RC/ABS.φP0/(1 − φP0).ψE0/(1 − ψE0)Performance index based on absorption

PITot a l = PI(ABS) × [δR0/(1 − δR0)] Performance index (potential) for energy conservation from photons

absorbed by PSII to the reduction of PSI end acceptors

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56835, enero-diciembre 2024 (Publicado Ago. 22, 2024)

Statistical analysis: Data were submitted

to normality (Shapiro-Wilk) and homogene-

ity (Barttlet) tests. To determine differences

between the physiological parameters in rela-

tion to the concentration of Fe and Mn (low

x high), the non-normal data were analyzed

via the non-parametric Mann-Whitney U test,

while normally distributed data were assessed

through the t-student test. In the basic statisti-

cal analyses, Excel or the statistical treatment

package Statistica (STATSOFT®) were used. The

significant threshold was set at 0.05 for all tests.

RESULTS

Fe and Mn concentration: In R. mangle,

the low and high Feleaf were 74 and 195 mg kg-1,

while the low and high Mnleaf were 65 and 414

mg kg-1, respectively. The concentration of Mn

in the sediment was 101 and 239 mg kg-1 at

the sites with the lowest and highest concen-

trations, respectively, while no variation in Fe

concentration was observed among the sites (≅

22 252 mg kg-1) (Fig. 2).

Pigment quantification: There was no

significant difference in the concentrations of

Chla, Chlb and Car in the leaves of R. mangle

growing in both high and low Fe and Mn sites

(Table 2).

Chl a fluorescence: The OJIP transients

of samples exposed to high and low Feleaf and

Mnleaf showed a typical polyphasic rise with the

fluorescence signal rising from the initial fluo-

rescence level (F0) to the maximal fluorescence

level (Fm), with well-defined intermediate J

and I steps (Fig. 3). High Feleaf increased the

fluorescence yield (F0 and Fm) and the area

beneath the fluorescence curve between F0 and

Fm (Table 3). In contrast, high Fe did not affect

quantum yield for primary photochemistry,

for electron transport and for energy dissi-

pation, φP0, φE0, and φD0, respectively. The

Fig. 2. Concentration (mean ± SE) of Fe and leaves (A) and sediments (B); Mn in leaves (C) and in sediments (D) of the

Rhizophora mangle, referring to sampling sites (Low and High concentration). Different letters indicate significant difference

between sites (P < 0.05; n.s.: non-significative; U or t: Mann-Whitney U test or t-student, respectively).

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56835, enero-diciembre 2024 (Publicado Ago. 22, 2024)

specific energy fluxes per reaction center (RC)

for absorption (ABS/RC), trapping (TR0/RC)

and electron transport (ET0/RC) were lower at

high Fe without variation in the specific energy

flux for dissipation (DI0/RC). Furthermore,

the total number of active reaction centers per

cross section (RC/CS0) and the quantum yield

for reduction of the end electron acceptors at

the PSI acceptor side (δR0) increased about

15.3 % and 4.51 % at high Fe, and the perfor-

mance indexes (PIABS and PITot al ) were statisti-

cally similar among sites, with average values

of 20.92 and 13.19, respectively. Moreover, the

increase in Feleaf increased the energetic con-

nectivity between the subunits associated with

PSII and the stability of OEC, which can be

visualized by lower values of L and K-bands,

respectively, while no changes were registered

in the oxidoreduction capacity of QA by elec-

trons originated from P680 (VJ or J-step values)

among sites (Table 3).

The increase in Mnleaf did not affect the

basal fluorescence yield (F0) in R. mangle,

but increased Fm and Area. Contrastingly, R.

mangle high in Mnleaf showed enhanced val-

ues of quantum yield for photochemistry and

electron transport (φP0 and φE0, respectively),

and decreases the quantum yield of energy dis-

sipation (φD0) at high Mnleaf. A greater number

of active reaction centers was recorded at high

Mnleaf, the absorption (ABS/RC) and dissipa-

tion (DI0/RC) energy flux were lower, although

the capture (TR0/RC) and electron transport

flux (further than QA-) or ET0/RC did not vary.

As for the performance indexes (PIABS and

PITotal), only PIABS showed a statistically signifi-

cant difference among sites, with higher values

(23.33) being detected at the sites where Mnleaf

was higher. The opposite occurred with the the

quantum yield for reduction of the end electron

acceptors at the PSI acceptor side (δR0), and

L-band at high Mnleaf. No changes were seen in

the K-band or VJ (Table 3).

Leaf gas exchange: Leaf CO2 assimilation

and gas-exchange variables were analyzed to

better characterize the effects of Feleaf and Mnleaf

on R. mangle photosynthetic performance (Fig.

4). The present results indicate that photosyn-

thesis was differently influenced by Fe and Mn

in leaf tissue. E, gs, A, and CE (carboxylation

efficiency = A/Ci) values increased in leaf tis-

sue of R. mangle containing high Fe, while Ci

Tabl e 2

Concentration of pigments (mean ± SE) in Rhizophora mangle, referring to sampling sites (low and high concentration of Fe

and Mn in the leaf) in Northern Espírito Santo and Southern Bahia.

Feleaf Mnleaf

Low High U or t value P-value Low High U or t value P-value

Chla (μg mL-1)297.65 ± 23.21 A238.94 ± 139. 00 A12.00 Un.s. 249.20 ± 31.77 A331.09 ± 62.58 A50.00 Un.s.

Chlb (μg mL-1)212.75 ± 16.70 A147.21 ± 76.90A-1.36 tn.s. 165.74 ± 19.22 A231.59 ± 43.25 A46.00 Un.s.

Car (μg mL-1)589.64 ± 58.78 A576.89 ± 189.00 A-0.08 tn.s. 597.92 ± 72.67 A628.86 ± 68.39 A-0.31 tn.s.

Chla (chlorophyll a- μg ml-1), Chlb (chlorophyll b- μg ml-1), Car (carotenoids- μg ml-1). Different letters indicate significant

differences between sites (P < 0.05; n.s.: non-significative; U or t: Mann-Whitney U test or t-student, respectively).

Fig. 3. The OJIP chlorophyll a fluorescence transient curve

in Rhizophora mangle, referring to sampling sites (low and

high concentration of Fe and Mn in the leaf).

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56835, enero-diciembre 2024 (Publicado Ago. 22, 2024)

Tabl e 3

Mean values (± SE) of the parameters calculated from the JIP-test obtained from Rhizophora mangle plants in the sampling of Fe and Mn (low and high concentration of Fe and Mn

in the leaf) sites in Northern Espírito Santo and Southern Bahia.

Parameters

Feleaf Mnleaf

Low High

Percentage of

change in

reduction

U or t value P-value Low High

Percentage of

change in

reduction

U or t value P-value

Area 65 073.51 ± 1 305.51 B71 012.28 ± 1573.50 A-- 2.92 t** 66 382.32 ± 1136.52 A67 127.65 ± 1257.73 An. s. 1 759.00 Un. s.

F0434.37 ± 4.41 B474.93 ± 3.65 A-- 444.00 U*** 438.10 ± 4.15 A447.01 ± 4.22 An. s. 1.50 tn. s.

Fm2 644.97 ± 26.51 B2 768.17 ± 31.95 A-- 2.98 t** 2 497.76 ± 29.73 B2 751.91 ± 40.58 A--- 4.64 U***

ϕP00.802 ± 0.001 A0.798 ± 0.006 An. s. -1.56 tn. s. 0.792 ± 0.002 B0.806 ± 0.001 A--- 830.00 U***

ϕE00.377 ± 0.004 A0.361 ± 0.006 An. s. 1 123.00 Un. s. 0.357 ± 0.007 B0.374 ± 0.005 A--- 2.04 t*

ϕD00.197 ± 0.002 A0.202 ± 0.002 An. s. 1.50 tn. s. 0.207 ± 0.002 A0.193 ± 0.001 B6.76 % 832.00 U***

J-Step 0.459 ± 0.005 A0.441 ± 0.008 An. s. 1 257.5 Un. s. 0.435 ± 0.008 A0.454 ± 0.006 An. s. 1.79 tn. s.

ABS/RC 1.89 ± 0.037 A1.75 ± 0.034 B7.40 % -2.68 t** 1.85 ± 0.037 A1.74 ± 0.038 B5.94 % -2.11 t*

TR0/RC 1.51 ± 0.026 A1.39 ± 0.025 B7.94 % -3.06 t** 1.46 ± 0.026 A1.40 ± 0.030 An. s. 1 559.00 Un. s.

ET0/RC 0.704 ± 0.011 A0.629 ± 0.014 B10.65 % 688.00 U*** 0.654 ± 0.016 A0.642 ± 0.012 An. s. 1 728.00 Un. s.

DI0/RC 0.382 ± 0.011 A0.356 ± 0.009 An. s. 1 138.00 Un. s. 0.390 ± 0.011 A0.340 ± 0.009 B12.82 % 1 150.00 U***

RC/CS0279.12 ± 3.81 B321.85 ± 5.26 A--- 6.74 t*** 284.89 ± 5.69 B310.68 ± 5.69 A--- 1 156.00 U***

PIABS 21.43 ± 0.871 A20.41 ± 0.989 An. s. 0.77 tn. s. 19.37 ± 1.00 B23.33 ± 1.04 A--- 1 255.00 U**

PITot a l 13.02 ± 0.628 A13.36 ± 0.652 An. s. 1 224.00 Un. s. 12.42 ± 0.621 A13.86 ± 0.661 An. s. 1 476.00 Un. s.

δR00.377 ± 0.004 B0.394 ± 0.005 A--- 2.55 t*0.396 ± 0.005 A0.372 ± 0.004 B6.06 % 1 137.00 U***

L-Band 0.167 ± 0.001 A0.164 ± 0.001 B1.79 % -2.37 t*0.171 ± 0.001 A0.165 ± 0.0008 B3.50 % 1 354.00 U*

K-Band 0.365 ± 0.006 A0.340 ± 0.006 B6.84 % -3.20 t** 0.367 ± 0.001 A0.348 ± 0.007 An. s. -1.82 tn. s.

Different letters indicate significant differences between sites (P < 0.05; n.s.: non-significative; U or t: Mann-Whitney U test or t-student, respectively). Parameters expressed in relative

units.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56835, enero-diciembre 2024 (Publicado Ago. 22, 2024)

and WUEint (intrinsic water use efficiency =

A/gs) and WUEins (instantaneous water use

efficiency = A/E) remained unchanged. Con-

versely, despite exhibiting better photochemical

performance, the rise in Mnleaf increased Ci

and decreased E, A, WUEint, WUEins and CE

(Fig. 4).

DPPH• radical scavenging assay: Higher

Feleaf in R. mangle reduced the concentration of

the extract needed to scavenge DPPH• by 50 %

(IC50). For Mnleaf, however, the opposite effect

was observed (Fig. 5).

DISCUSSION

The effects triggered by Fe and Mn expo-

sure were seen in all evaluated parameters

and resulted in physiological changes in R.

mangle. We understand that, although iron

and manganese concentrations in leaf tissues

have a great influence on photosynthetic pro-

cesses, other factors also act as regulators.

Fig. 4. Gas exchange parameters (mean ± SE) in Rhizophora mangle, referring to sampling sites (Low and High concentration

of Fe and Mn in the leaf). A. Ci (intercellular CO2 concentration), B. A (net carbon assimilation rate), C. gs (stomatal

conductance), D. E (transpiration rate), E. WUEint (intrinsic water use efficiency - A/gs), F. WUEins (instantaneous water use

efficiency) and G. CE (carboxylation efficiency). Different letters indicate significant differences between sites (P < 0.05; n.s.:

non-significative; U or t: Mann-Whitney U test or t-student, respectively).

Fig. 5. Antiradical activity IC50 against DPPH• (mean ± SE)

in Rhizophora mangle, referring to sampling sites (Low and

High concentration of Fe and Mn in the leaf). Different

letters indicate significant differences between sites (P <

0.05; U: Mann-Whitney U test).

12 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56835, enero-diciembre 2024 (Publicado Ago. 22, 2024)

Nevertheless, we observed alterations occurred

among locations where Feleaf and Mnleaf were

significantly different, and involved analyses

of photosynthetic pigments, transient Chl a

fluorescence, gas exchange, CO2 assimilation,

oxidative stress, and the detoxification capac-

ity of plants, evaluated here as DPPH• radical

scavenging activity. The achieved results will

certainly allow to understand the successful

strategies of the R. mangle tree to cope with the

altered environment as well as its capacity to

cleanse sediment and water.

The difference in Fe uptake and accumula-

tion allowed us to select the study areas. Such

differences in absorption, given the invari-

ability of the element in the sediment, may

be associated with the presence of other trace

elements at higher concentrations, e.g. Mn,

(data not shown), as observed in the sedi-

ment of the site showing lower Feleaf (data not

shown). Conversely, low Mn sediment concen-

tration was found under high Feleaf (data not

shown). The content of clay and soil organic

matter also influences the availability of Fe

to plants, since there is a tendency to retain

Fe in muddy soils. Adequate levels of organic

matter improve Fe uptake due to its acidify-

ing and reducing properties; moreover, certain

humic substances could form chelates under

adverse pH conditions (Dechen & Nachti-

gall, 2006). Besides, Fe absorption decreases

with increased concentrations of Ca, Mg, Cu,

Zn and especially Mn (Jones-Junior, 2012).

High concentrations of Mn in acidic soils can

competitively inhibit Fe absorption (Malavolta,

1980). Such Mn values are independent of the

results observed in this study, considering the

different sampling sites. Like Fe, Mn in the

sediment tends to form stable and insoluble

compounds, suitable for the adsorption of other

metals (Förstner & Witmann, 1981). How-

ever, upon contact with mangrove sediments,

imported particulate manganese is reduced,

due to the physical-chemical characteristics of

this compartment. This process makes the ele-

ment soluble, facilitating its export to adjacent

environments in the form of Mn (II) (Vidal &

Becker, 2006). Therefore, it is possible that the

variation in Mn concentration in the sediment

observed in this study is associated with areas

of export (low concentrations) and import

(high concentrations).

Chl a fluorescence transient in R. mangle

under low and high Feleaf and Mnleaf revealed

the three typical OJIP phases (O-J, J-I and I-P),

indicating that all samples remained photosyn-

thetically active (Strasser & Stirbet, 2001) inde-

pendently of metal concentration. The increase

in F0 and Fm at the sites with higher Feleaf

resulted in higher area above the fluorescence

curve (Area) between F0 and Fm. As stated by

Joliot and Joliot (2002), the area represents the

electron acceptor pool sizes of PSII, including

QA and QB. In this investigation, the area over

the fluorescence curve was slightly but sig-

nificantly (P ≤ 0.05) increased by 9.12 % under

high Feleaf compared to low Feleaf, showing that

the elevation in Feleaf improves the electron

transfer rates at the donor side of PSII and

increases QA pool size (Gao et al., 2022; Mehta

et al., 2010). Furthermore, despite the increase

in Fm at high Mnleaf, there were no changes in

F0 or Area. It has been suggested that high Fm

occurs when thylakoid membranes are pre-

served, thus leading to the clustering of light-

harvesting complexes (LHCII) associated with

PSII (Schreiber & Neubauer, 1987).

The levels of F0 were only increased by

about 2.03 % (P ≥ 0.05) at high Mnleaf, while

at high Feleaf they were 9.3 % (P ≤ 0.05) higher

than at low Feleaf. Nonetheless, although F0 val-

ues showed a significant rise under high Feleaf,

no alterations were registered in parameters

related to efficiency, as similarly observed at

high Mnleaf. F0 is associated with the donor

side of PSII, with the adjustment capacity of

antenna pigment level or with the excitation

trapping efficiency at the active center of PSII.

In this regard, the current findings indicate that

the increased F0 values observed at high Feleaf

are demonstrated at antenna pigment level, as

verified by decreased levels of Chlb, since the

values of L and K-bands were reduced and the

parameters related to the excitation trapping

efficiency at the active center of PSII (φP0,

φE0, φR0, TR0/RC, PIABS and PITot al) remained

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56835, enero-diciembre 2024 (Publicado Ago. 22, 2024)

unchanged. K-band and L-band are associated

with the donation of electrons from the OEC

to PSII and with the connection/disconnec-

tion (or energetic connectivity) of the PSII core

antenna (LHC), respectively (Strasser et al.,

2004). The observed decreases/invariability of

K-band and L-band values evidence that con-

centrations of Fe and Mn, as those registered

in leaf tissues of R. mangle grown under in

situ conditions, exert some protective effect on

electron donor and acceptor sides of PSII, thus

maintaining the integrity of the OEC and LHC.

It is worth noting that the OEC uses Mn as

an essential cofactor in the oxidation of water

(Strasser et al., 2004); moreover, as well as the

ferredoxin protein, the end electron acceptor of

PSI contains Fe in its molecular structure.

Analysis of the efficiency of the energy

flow per reaction center (RC) revealed reduc-

tion in absorption (ABS/RC), trapping (TR0/

RC) and transport (ET0/RC) and no change in

dissipation (DI0/RC) in plants growing under

high Feleaf. Similar results were obtained for

R. mangle from sites with high Mnleaf; there

were significant decreases in DI0/RC and ABS/

RC and no alterations in TR0/RC or ET0/

RC. The energy flow parameters per RC are

calculated as the total number of photons

absorbed, captured, transported, and dissipated

from all RCs divided by the total number of

active RCs (Mehta et al., 2010). Thus, the ratio

of active/inactive RCs influences the values

of energy fluxes. Consequently, as disclosed

herein, reductions in all energy fluxes occur

when the number of RCs is improved; increases

in RC/CS0 of 15.3 % (P ≤ 0.05) and 9.05 % (P

< 0.05) were found in R. mangle grown under

high Feleaf and Mnleaf, respectively. Increased

RC/CS0 in R. mangle can reduce the effects of

photoinhibition and thus result in lower energy

dissipation. These statements are reinforced by

the decreases seen in ABS/RC values, which

indicate that both metals increased the antenna

size of active RCs; such observations were more

important in R. mangle growing under high

Feleaf and Mnleaf conditions.

Collectively, these results indicate the effi-

ciency of the photosynthetic apparatus, since the

excitation energy was absorbed and captured by

the Chl molecules and directed towards elec-

tron transport to reduce pheophytin, QA and

the other electron acceptors in the ETC (Taiz et

al., 2017). The increase in Fm and the reduction

and/or invariability of parameters connected to

energy dissipation mechanisms, as DI0/RC and

φD0, are thus explained. In general, although it

is widely accepted that PSII is extremely suscep-

tible to several types of environmental stresses,

the observed increases in R. mangle Feleaf and

Mnleaf have a positive regulatory effect, at least

in terms of preservation of structure and func-

tionality of the plant photosynthetic apparatus.

Mechanisms of photosynthetic regulation relat-

ed to the ability to dissipate excitation energy

leading to photoprotection have been suggested

and discussed (Wang et al., 2016). Carotenoids

participate in the photoprotection system, and

the invariability of this pigment is consistent

with the results found for the increase in Feleaf

and Mnleaf (Szabó et al., 2005).

For a deeper understanding of the role of

Fe and Mn as elements in carbon assimilation in

R. mangle, a detailed gas-exchange analysis was

carried out associated with oxidative stress and

antiradical activity. It is generally agreed that

environmental alterations directly affect carbon

assimilation in plants, considering that photo-

synthesis is highly vulnerable to metal toxicity

(Ahmad et al., 2008; Barcelos et al., 2022); the

effects are multi-dimensional and influence

photosynthetic CO2 fixation under controlled

conditions as well as in situ. The current results

evidenced distinct interference patterns of Fe

and Mn with the functional processes of photo-

synthesis, including the antioxidant capacity of

R. mangle, despite the improved performance of

the ETC of chloroplast.

Unlike the results obtained at high Mnleaf,

the antioxidant system of R. mangle, evalu-

ated here through the free radical scavenging

activity, undoubtedly functioned as a protec-

tion system under high Feleaf. As a result, high

CO2 assimilation capacity and carboxylation

efficiency (CE) of ribulose-1.5-bisphosphate

carboxylase oxygenase (Rubisco) were main-

tained. Thus, increased Ci as well as decreased

14 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56835, enero-diciembre 2024 (Publicado Ago. 22, 2024)

CO2 assimilation, WUEint, WUEins and CE

under excessive Mn are linked to oxidative

damage buildup in R. mangle leaf tissue, despite

its plasticity in light energy utilization. These

are alarming results, since such estuarine sys-

tems undergo great climatic variability (Lopes

et al., 2019; Pascoalini et al., 2022). Addition-

ally, the high nutrient content intensifies plant

vulnerability to drought, especially under low

atmospheric humidity and/or limited fresh-

water input, thus compromising the use of

water by the vegetation (Lovelock et al., 2009;

Oliveira et al., 2022).

The increases in A and gs under high Feleaf

conditions, which were associated with the

invariability of Ci, suggest that the enhanced

CO2 flux into R. mangle leaves caused by sto-

matal opening was a relevant factor for the

increase in A. In contrast, the lower A, gs and

E values at high Mnleaf, which were associ-

ated with higher Ci levels, imply that R. mangle

photosynthesis was limited by non-stomatal

factors, besides indicating that Mn interfered

with stomatal regulation. Reductions in CO2

assimilation is likely to be associated not only

with lower CO2 entry into leaves, but also some

biochemical limitation in CO2 fixation within

the chloroplasts, which is related to lower

Rubisco activity and changes in the capacity for

ribulose-1,5-bisphosphate regeneration (Alves

et al., 2011; Wang et al., 2022). Investigations

about the role of Mn on photosynthesis have

evidenced repression of specific genes, particu-

larly of those nuclear-encoded small subunits of

Rubisco (Sheen, 1994). Consequently, Rubisco

content and CO2 assimilation are reduced.

According to Li et al. (2010), the regulatory

role of Mn-excess appears to be linked with

the accumulation of soluble sugars, as sucrose,

glucose, and fructose, but it remains to be fur-

ther clarified. The authors also report that the

reduced CO2 assimilation in Mn-excess leaves

was not accompanied by Chl, since there were

no differences in the contents of Chla or Chlb

between the tested Mn treatments. Such finding

is consistent with the unchanged concentrations

of photosynthetic pigments described here for

R. mangle under high Mnleaf conditions.

Globally, mangroves play a particularly

important role in maintaining biodiversity

hotspots. However, despite the environmen-

tal and economic values attributed to man-

groves, these ecosystems are constantly prone

to anthropogenic or natural actions that result

in increased heavy metal concentrations in

sediments, which often act as sinks for these

toxic elements. Our results showed that Fe and

Mn affected the physiological performance of

R. mangle in a different manner. Neverthe-

less, when taking all physiological parameters

into consideration, we verified that the effects

of high Feleaf at antenna pigment level did not

impair the carbon gain of R. mangle trees at

the evaluated sites, since K-band values were

reduced and there were no changes in param-

eters related to the excitation trapping effi-

ciency at the active center of PSII. Rises in Feleaf

and Mnleaf in R. mangle at the levels recorded

in this evaluation increased RC/CS0, thus sug-

gesting a positive regulatory effect at least in

terms of preservation of structure and func-

tionality of the plant photosynthetic apparatus.

This study also identified distinct interference

patterns of Fe and Mn with the functional

processes of photosynthesis, especially with

CO2 assimilation and ROS metabolism. The

most pronounced effects were observed in CO2

assimilation and carboxylation efficiency of

Rubisco at high Mnleaf. Thus, interference of

high Mnleaf in R. mangle occurs at non-stomatal

and biochemical levels. Further research should

be conducted under controlled conditions and

including higher doses of the metals to expand

our understanding of the regulatory role of

excessive Fe and Mn on the metabolism of

R. mangle at the whole-tree level. The cur-

rent findings may aid in predicting alterations

resulting from the effects of environmental

changes, which make the mangrove forest even

more vulnerable.

This assessment described the antagonism

between Fe and Mn regarding the physiology

of R. mangle, which is a dominant species in

Brazilian mangroves. Moreover, attention is

drawn to the coastal eutrophication processes

currently taking place in the global mangrove

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56835, enero-diciembre 2024 (Publicado Ago. 22, 2024)

area. R. mangle appears to be participating in

bioremediation, but the physiological responses

disclosed herein raise concern about the inter-

ference of long-term eutrophication in ecosys-

tem productivity.

Ethical statement: the authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledgments

section. A signed document has been filed in

the journal archives. Funding: This research

was financially supported by Renova Founda-

tion via its Technical-Scientific Cooperation

Agreement nº 30/2018 with Espírito Santo

Foundation of Technology (FEST). Declaration

of Competing Interest: The authors declare no

conflict of interest.

ACKNOWLEDGMENTS

This research was developed under the

Aquatic Biodiversity Monitoring Program,

Environmental Area I, established by the Tech-

nical-Scientific Cooperation Agreement nº

30/2018 between Espírito Santo Foundation of

Technology (FEST) and Renova Foundation,

published in Brazil’s Official Gazette (Diário

Oficial da União). The authors would like to

thank the CAPES/FAPES Cooperation—Post-

graduate Development Program—PDPG and

the Laboratory of Environmental Geochemis-

try and the Laboratory of Geological Ocean-

ography of the University of Espírito Santo for

their analyses of metals and sediment.

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