Ecophysiological responses to excess iron in lowland and upland rice cultivars

Accepted Manuscript Echophysiological responses to excess iron in lowland and upland rice cultivars Caroline Müller, Solange Ferreira da Silveira Silveira, Danilo de Menezes Daloso, Giselle Camargo Mendes, Andrew Merchant, Kacilda Naomi Kuki, Marco Antonio Oliva, Marcelo Ehlers Loureiro, Andréa Miyasaka Almeida PII:

S0045-6535(17)31447-9

DOI:

10.1016/j.chemosphere.2017.09.033

Reference:

CHEM 19904

To appear in:

ECSN

Received Date: 16 May 2017 Revised Date:

29 August 2017

Accepted Date: 8 September 2017

Please cite this article as: Müller, C., da Silveira Silveira, S.F., de Menezes Daloso, D., Mendes, G.C., Merchant, A., Kuki, K.N., Oliva, M.A., Loureiro, M.E., Almeida, André.Miyasaka., Echophysiological responses to excess iron in lowland and upland rice cultivars, Chemosphere (2017), doi: 10.1016/ j.chemosphere.2017.09.033. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Echophysiological responses to excess iron in lowland and upland rice cultivars

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Caroline Müllera, Solange Ferreira da Silveira Silveirab, Danilo de Menezes Dalosoa, Giselle

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Camargo Mendesa, Andrew Merchantc, Kacilda Naomi Kukia, Marco Antonio Olivaa, Marcelo

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Ehlers Loureiroa, Andréa Miyasaka Almeidaa,d

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Leão-RS, Brazil.

Department of Plant Biology, Federal University of Viçosa, 36570-000, Viçosa-MG, Brazil.

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Australia.

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Plant Genomics and Breeding Center, Federal University of Pelotas, 96010-900 Capão do

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Faculty of Agriculture and the Environment, The University of Sydney, Sydney 2006,

Center of Plant Biotechnology, Universidad Andrés Bello, 8370146, Santiago, Chile.

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*Corresponding author (C. Müller):

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Departamento de Biologia Vegetal - UFV

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Av. PH Rolfs, s/n Campus Universitário

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Viçosa – MG, Brazil 36570-000

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Tel #: 55 64 99956 5905

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E-mail address: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT: Iron (Fe) is an essential nutrient for plants but under high concentrations, such as that found

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naturally in clay and waterlogged soils, its toxic effect can limit production. This study aimed to investigate the

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stress tolerance responses exhibited by different rice cultivars. Both lowland and upland cultivars were grown

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under excess Fe and hypoxic conditions. Lowland cultivars showed higher Fe accumulation in roots compared

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with upland cultivars suggesting the use of different strategies to tolerate excess Fe. The upland Canastra cultivar

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displayed a mechanism to limit iron translocation from roots to the shoots, minimizing leaf oxidative stress

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induced by excess Fe. Conversely, the cultivar Curinga invested in the increase of R1/A, as an alternative drain

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of electrons. However, the higher iron accumulation in the leaves, was not necessarily related to high toxicity.

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Nutrient uptake and/or utilization mechanisms in rice plants are in accordance with their needs, which may be

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defined in relation to crop environments. Alterations in the biochemical parameters of photosynthesis suggest

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that photosynthesis in rice under excess Fe is primarily limited by biochemical processes rather than by

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diffusional limitations, particularly in the upland cultivars. The electron transport rate, carboxylation efficiency

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and electron excess dissipation by photorespiration demonstrate to be good indicators of iron tolerance.

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Altogether, these chemical and molecular patterns suggests that rice plants grown under excess Fe exhibit gene

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expression reprogramming in response to the Fe excess per se and in response to changes in photosynthesis and

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nutrient levels to maintain growth under stress.

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Keywords: Oryza sativa; photosynthesis; iron toxicity; OsPI1; OsFER1.

40 Abbreviations

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EDTA

ethylenediamine tetraacetic acid

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A

net CO2 assimilation rate (µmol m-2 s-1)

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E

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gs

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Ci/Ca

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A/Ci

net photosynthesis, A, vs intercellular CO2 concentration, Ci

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Г*

CO2 compensation point in absence of Rd (µmol mol-1)

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Rd

non-photorespiratory respiratory (µmol m-2 s-1)

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RuBP

ribulose-1,5-diphosphate

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Rubisco

ribulose-1,5-diphosphate carboxylase/oxygenase

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transpiration rate (mmol m-2 s-1) stomatal conductance (µmol m-2 s-1)

ratio of sub-stomatal and ambient CO2 concentration

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ACCEPTED MANUSCRIPT Vcmax

maximum carboxylation rate (µmol m-2 s-1)

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Jmax

maximum electrons transport rate (µmol m-2 s-1)

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VTPU

rate of triose phosphate utilization

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JT

total electron transport rate

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Jc

carboxylase reactions of ribulose-1,5-bisphosphate

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Jo

electron transport rate related to oxygenase reactions of ribulose-1,5-bisphosphate

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R1

rate of CO2 production by photorespiration

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PSII

photosystem II

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F0

minimal chlorophyll fluorescence

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Fm

maximal chlorophyll fluorescence

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Fv/Fm

maximal photochemical efficiency of PSII

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ETR

electron transport rate

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qL

photochemical quenching based on the lake model of PSII antenna

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YII

quantum yield of photochemical energy conversion in PSII

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YNPQ

quantum yield of regulated energy dissipation in PSII

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YNO

quantum yield of non-regulated energy dissipation in PSII

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qRT-PCR quantitative reverse transcriptase polymerase chain reaction

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FER1

ferritin

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psbA

protein chloroplastic Q-B that encodes PSII reaction center protein D1

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GS1x

cytosolic glutamine synthetase

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NAS1

nicotianamine synthetase

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YSL1

Yellow Stripe-Like 1

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PI1

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PTF1

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tonoplast monosaccharide transporter inducible by phosphate 1 limitation inducible bHLH by phosphate 1 limitation transcription factor

1. Introduction

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Rice (Oryza sativa L.) is cereal consumed by more than two thirds of the world’s population.

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Consequently, rice production has important social and economic impacts across a broad scope of socioeconomic

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ACCEPTED MANUSCRIPT groups. Brazil is the second largest rice producer in the world. However, abiotic stress such as salinity, high

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temperature and ion toxicity are widely experienced in Brazil and can negatively affect plant growth and

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development (Barkla et al., 2013). One major factor that can affect wetland rice production is iron toxicity under

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anaerobic condition and low pH (Stein et al., 2009a). Iron (Fe) is a chemical element essential for the growth and

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development of plants as well as being critical for human nutrition. Graminaceous plants such as rice, utilize

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strategies to acquire Fe from rhizosphere, whereby roots secrete phytosiderophores (PSs) and chelate Fe(III). The

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resulting Fe(III)-PS complexes are then taken up into root tissues via specific plasma membrane transporters like

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YSL (Yellow stripe like) (Koike et al., 2004; Inoue et al., 2008; Conte and Walker, 2011). In plant cells, iron is

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an important constituent of enzymes and photosynthetic pigments and participates in electron transfer via redox

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reactions during photosynthesis and respiration (Briat et al., 2007; Kim and Guerinot 2007). However, the high

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availability of iron in rice-growing areas may result in excessive uptake, causing toxicity in plants limiting

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wetland rice production (Audebert and Fofana 2009). Thus, investigating and understanding the stress tolerance

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mechanisms exhibited by rice in response to excess iron will provide a better view of the strategies of tolerance.

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This understanding may better inform breeding programs seeking to optimize rice production in areas where Fe

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concentrations are not optimal.

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In wetlands, Fe is found in its more available reduced divalent form (Fe+2) due to anaerobic conditions

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and low pH (Guerinot and Yi, 1994). This availability may promote toxic accumulation in plant tissues because

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Fe is highly reactive (Conte and Walker, 2011). Formation of iron plaque is generally considered one of the

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mechanisms that aquatic plants have evolved to acclimate to anaerobic conditions, particularly to very high

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concentrations of Fe2+, Mn2+ and S2+ in the soil (Mirsal, 2008). Iron plaque formation in rice is known to be

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related to metal tolerance and varies among cultivars (Pereira et al., 2014). Yet, rice cultivars adapted to such

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circumstances manage these conditions to some degree. In susceptible cultivars, marginal bronzing of the leaves,

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followed by the advancement of necrosis, are evidence of the effect of excess Fe. However, these visible

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symptoms are a reflection of cellular damage from metal toxicity. Physiochemically, Fe can potentiate oxidative

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stress (Becana et al., 1998; Fang et al., 2001; Müller et al., 2015), leading to degradation of cell membranes,

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disruption of photosynthetic protein complexes such as D1 protein (encoded by PsbA genes) (Spiller and Terry,

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1980; Suh et al., 2002), and decreased net carbon assimilation (Pereira et al., 2013), thereby impairing plant

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development and productivity. The disruption of carbon metabolism is mainly due to feedback inhibition by ATP

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and NADPH which are not used by Calvin-Benson cycle enzymes under Fe stress conditions as reported for

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Phaseolus vulgaris (Siedlecka et al., 1997).

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ACCEPTED MANUSCRIPT For cultivars or species resistant to iron toxicity, growth depends in part on an ability to mitigate

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excessive Fe uptake via exclusion mechanisms and/or by storing/remobilizing absorbed iron (Briat et al., 2007).

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Molecular control of iron homeostasis is regulated by an array of genes, including nicotinamine synthetase

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(OsNAS) and nicotianamine aminotransferase (OsNAAT) (DiDonato Jr et al., 2004; Inoue et al., 2008; Kobayashi

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et al., 2010). Six NAAT gene (Inoue et al., 2008) and three-rice gene named NAS (OsNAS1, OsNAS2 and

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OsNAS3) have been identified to play important roles in Fe uptake (Inoue et al., 2003). These genes are directly

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involved in the Fe absorption method of strategy II plants, which consists of releasing phytosiderophores (PS) to

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produce a PS-Fe+3 complex (Curie et al., 2001). Once taken up by roots, the sequestration of this metal is

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achieved by complexation with ferritin (Vasconcelos et al., 2003; Majerus et al., 2009; Müller et al., 2015).

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Ferritin is a Fe storage protein (encoded by OsFER gene) that forms non-toxic complexes in plant tissues and

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protects them against oxidative stress (Ravet et al., 2009; Stein et al., 2009b). Another significant mechanism for

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Fe homeostasis is storage in vacuoles and is one of the most common responses to excess Fe in living tissues.

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Under conditions of excess Fe, the OsVIT1 and OsVIT2, encode vacuolar transporters, facilitating Fe transport

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into vacuole (Zhang et al., 2012). However, such sequestration has limited scope. Once thresholds of vacuolar

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capacity are overcome, plants must invest in strategies to repair and/or avoid oxidative damage, using enzymatic

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(Becana et al., 1998) or non-enzymatic systems to neutralize reactive oxygen species (Sinha and Saxena, 2006).

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Hence, in order to guarantee carbon assimilation at sustainable rates of carboxylation, tolerance traits must also

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ensure the dissipation of excess of energy to avoid and/or repair of photodamage to protein complexes in the

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chloroplast (Liang et al., 2014). Although these responses are well documented, little is known concerning the

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effects of excess iron on broader mechanisms of plant homeostatic control.

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In general, the tolerance or sensitivity to iron toxicity is a complex phenomenon involving responses at

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molecular, biochemical, and physiological levels (Zheng et al., 2009; Müller et al., 2015). To date, a variety of

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rice cultivars have been introduced to the market in order to sustain and improve growth and yield in areas facing

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excess iron toxicity as well as a range of alternative stress conditions. Modern lowland and upland rice cultivars

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were developed during long periods of human directed selection in different environments through parallel

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breeding programs, leading to a diverse genetic background (Zhang et al., 2013). Prior investigations have

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sought to evaluate the performance of rice cultivars under Fe excess, focusing only on morphological

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characteristics (Crestani et al., 2009), oxidative/nutritional responses (Stein et al., 2009a) or production capacity

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under this condition (Sahrawat, 2010). Our previous studies showed that high doses of iron cause stomatal and

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non-stomatal limitations in rice cultivars (Pereira et al., 2013) and damage to photochemical reactions (Müller et

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ACCEPTED MANUSCRIPT al., 2015) in addition to differential morphoanatomical changes due to the formation of iron plaques in irrigated

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and dry rice plants (Pereira et al., 2014). A better understanding of the physiological responses of different rice

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cultivars to excess iron will be extremely useful for breeding programs, helping to select cultivars that are more

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tolerant to excess iron. Thus, this study aimed to investigate the stress tolerance responses exhibited by different

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rice cultivars (lowland and upland) grown under excess Fe in hypoxic conditions.

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2. Materials and methods

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2.1. Plant material, growth conditions and treatment application

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Four rice (Oryza sativa L.) cultivars that are widely cultivated in Brazil were selected. Two upland

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cultivars: Canastra and BRSMG Curinga (Curinga), and two lowland cultivars: BR-IRGA 409 (409) and IRGA

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419 (419). The canastra cultivar was developed in International Center for Tropical Agriculture (CIAT,

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Colombia) and it is a hybrid between japonica and indica. BRSMG Curinga was developed in Embrapa Rice and

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Beans (Brazil) in collaboration with CIAT. BR-IRGA 409 and IRGA 419 was developed by CIAT and IRGA

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419 selected from the cross between Oryzica1 and BR-IRGA 409. Seeds were sterilized with commercial sodium

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hypochlorite (10%) for 10 min and germinated in rolled paper towel wetted with distilled water in growth

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chambers (27±2 °C) for 7 days in the dark, followed by 7 days of a 12-h photoperiod with light intensity of 200-

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250 µmol m-2 s-1. Seedlings were then transferred to Hoagland solution (pH 4.0, full strength), in 3.8 dm-3 pots,

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under greenhouse conditions. The Hoagland’s nutrient solution used to acclimate the plants and for the control

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treatments was composed by 1 mM NH4H2PO4, 6 mM Ca(NO3)2•4H2O, 4 mM KNO3, 2mM MgSO4•7H2O, 46

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µM H3BO3, 9 µM MnCl2•4H2O, 0.78 µM ZnSO4•7H2O, 0.32 µM CuSO4•5H2O, 0.14 µM MoO3 and 18 µM

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FeSO4-EDTA. After 30 days, plants at the V5 developmental stage (Counce et al., 2000) were exposed to excess

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iron. Iron treatment consisted of of 7 mM FeSO4 (1946 µg g-1), which was conjugated with ethylenediamine

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tetraacetic acid (EDTA) (w/w), concentration previously determined by Pereira et al. (2013), in the same

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conditions. A control treatment consisting of a nutritive solution with chelated iron sulphate at physiological

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concentrations (0.018 mM FeSO4-EDTA, i.e, 5.00 µg g-1) was conducted in parallel. Each treatment consisted of

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four replicates, with one pot per replicate and around eight plants per pot. The pH (4.0) was adjusted every two

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days with NaOH or HCl, and the nutrient solution was renewed weekly. Physiological measurements and tissue

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samples were performed after 7 days of exposure to excess iron.

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2.3. Gas exchange measurements

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Gas exchange from rice cultivars O. sativa plants was measured in fully expanded leaves, on the middle

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of the plant, which developed during the imposition of the treatment. Net photosynthetic rate (A, µmol CO2 m-2 s-

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internal and external CO2 concentration (Ci/Ca) were determined using an infrared gas analyzer (IRGA; model

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LI-6400xt, LI-COR Biosciences Inc., Lincoln, Nebraska, USA). Measurements were performed between 8:00

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and 11:30 am under constant photosynthetically active radiation (PAR, 1000 µmol photons m-2 s-1) and CO2

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concentration (Ca, 400 µmol mol-1), at atmospheric temperature (24-28 °C) and relative humidity (50-68%),

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under greenhouse conditions.

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), stomatal conductance (gs, mol H2O m-2 s-1), transpiration rate (E, mmol H2O m-2 s-1) and the ratio between

A/Ci curves were obtained by measuring A under various CO2 concentrations using an Infra-red gas

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analyser (IRGA, LI-6400xt portable photosynthesis system LI-COR, USA) with a high-pressure CO2 cartridge.

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Measurements of the A/Ci curves were conducted using nine CO2 levels (50, 100, 200, 400, 700, 1000, 1300,

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1600 and 2000 µmol mol-1), with 2 to 3 min between readings, under the same conditions as described above.

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2.4. Rubisco carboxylation and RuBP regeneration

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The A/Ci curves were analysed to estimate the in vivo maximum rate of RuPB carboxylation (Vcmax), the

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in vivo maximum rate of electron transport driving RuBP regeneration (Jmax) and the rate of triose-phosphate

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utilization (VTPU). Vcmax, which represents the slope of the A/Ci curve, were calculated according to Long and

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Bernacchi (2003) and to Sharkey et al. (2007), using gas exchange, leaf temperature, atmospheric pressure and

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A/Ci curve parameters:

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Ac = Vc,max [ (Cc - Γ*) / (Cc + Kc(1 + O / Ko) ] - Rd

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where Ac is the photosynthethic rate limited by Rubisco activity, Cc was obtained from C c = C i − A / g m

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(Sharkey et al., 2007) is the intercellular CO2 concentration, Г* is the CO2 compensation point in the absence of

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day respiration, Kc and Ko are Michaelis-Menten constants of Rubisco activity for CO2 and O2 for rice plants

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from (Makino et al., 1988), respectively, and Rd is the dark respiration.

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When A is limited by RuBP regeneration, then the following equation is used:

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A = ((J (Cc – Γ*) / (4Cc + 8 Γ*)) / -Rd

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where J is the electron transport rate. This equation assumes that four electrons are required for each

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carboxylation or oxygenation cycle.

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When A is limited by TPU, then the following equation is used:

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A = 3TPU - Rd

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where TPU is the triose phosphate utilization.

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Rates of photorespiration were calculated by gas exchange and chlorophyll fluorescence parameters

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according to Epron et al. (1995) and to Valentini et al. (1995). For these calculations, linear electron flow was

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assumed to be driven by the carboxylation and oxygenation of ribulose-1,5-bisphosphate (i.e., all other processes

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consuming light-driven electrons are negligible). With four electrons required for each carboxylation or

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oxygenation cycle, and one CO2 molecule is released every two oxygenation cycles by glycine decarboxylation

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in the photorespiration pathway. Thus,

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JT = YII x PAR x 0.5

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Jc = 1/3 x (JT + 8 x (A + Rd))

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JO = 2/3 x (JT - 4 x (A + Rd))

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R1 = (JT - 4 x (A + Rd) / 12)

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where JT is the total rate of electron transport through PSII to photosynthesis and photorespiration; YII is the

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quantum efficiency of linear electron flow through PSII; PAR is the photosynthetically active radiation (photon

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flow, µmol m-2 s-1) incident on the leaf; 0.5 represents the proportional quanta used by PSII centers (Melis et al.,

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1987); Jc and Jo are the electron costs attributable to the carboxylation and oxygenase RuBP reactions,

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respectively; A is the net CO2 assimilation rate; Rd is the dark respiration and Rl is the rate of CO2 production by

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photorespiration.

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2.5. Chlorophyll fluorescence measurements

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Variables of chlorophyll fluorescence were obtained using a leaf chamber fluorometer (6400-40, LI-

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COR Biosciences Inc., Lincoln, Nebraska, USA) coupled to an infrared gas analyzer (IRGA; LI-6400xt, LI-

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COR) at the same region where gas exchange data was collected. Leaves were initially overnight dark-

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ACCEPTED MANUSCRIPT acclimated so that the reaction centers were fully opened to obtain the minimal (F0) and maximal (Fm)

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chlorophyll fluorescence. From these values, the maximum photosystem II (PSII) quantum yield was calculated

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(Fv/Fm = (Fm-F0)/Fm) (Genty et al., 1989). After sample illumination, saturation pulses were applied to determine

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the light-acclimated maximal fluorescence (Fmʹ) and the steady-state fluorescence yield (Fs), which allowed to

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calculate the minimal fluorescence on light-acclimated leaves (F0’= F0/(((Fm-F0/Fm)+(F0/Fm’))) (Oxborough and

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Baker, 1997). The coefficient of photochemical quenching was calculated using the lake model, which estimates

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the fraction of PSII centers in open states, as (qL = (Fm’-Fs)/(Fm’-F0’) x (F0’/Fs)) (Kramer et al., 2004). The

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quantum yield of photochemical energy conversion in PSII (YII = (Fm’-Fs)/Fm’), and the quantum yields of

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regulated ((YNPQ = (Fs/Fm’) – (F/Fm)) and non-regulated (YNO = Fs/Fm) energy dissipation in PSII were calculated

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according to Genty et al. (1989) and to Hendrickson et al. (2004). The YII was also used to estimate the apparent

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electron transport rate (ETR = YIIxPARx0.84x0.5) (Bilger et al., 1995), where PAR is photosynthetically active

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radiation (µmol m-2 s-1), 0.84 is the assumed irradiance absorbed by the leaf (Ehleringer, 1981), and 0.5 is the

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assumed proportion of absorbed quanta used by PSII reaction centers (Laisk and Loreto, 1996). Dark-acclimated

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measurements were performed pre-dawn and the light-acclimated (1000 µmol photons m-2 s-1) variables obtained

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between 8:00 and 11:30 am at greenhouse conditions.

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2.7. Gene expression

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Total RNA was extracted from fresh leaf and root materials using homemade TRIzol reagent [38%

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phenolic acid (pH 4.5), 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate (pH

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5.0), 5.0% glycerol, and diethylpyrocarbonate-treated water] (modified from Chomczynski and Sacchi, 1987).

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After extraction, sodium chloride solution (150 µL, 5 N) and chloroform (450 µL) were added and the mixture

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was centrifuged (16 000 × g for 10 min at 4 °C). RNA was precipitated from the aqueous phase using an equal

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volume of isopropanol and washed in ethanol (75%). Total RNA was quantified in a nanocell coupled to a

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spectrophotometer and analyzed by agarose gel electrophoresis (0.8%, w/v) stained with GelRed (BiotiumTM).

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cDNA synthesis was performed with 2 µg of total RNA, pre-treated with DNase (1 µL, 50 U µL-1, Amplification

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Grade DNase I, InvitrogenTM) and incubated at 37 °C for 15 min. The first-strand cDNA was synthetized using

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the kit SuperScriptTM First-Strand Synthesis System for RT-PCR (InvitrogenTM), in a thermomixer, and stored at

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-20 °C.

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ACCEPTED MANUSCRIPT Four biological replicates with three technical replicates were performed for the quantitative reverse

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transcriptase polymerase chain reaction (qRT-PCR) experiment. Gene expression was analyzed using a

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StepOnePlusTM Real-Time PCR System (Applied Biosystems®, CA, USA), with a fluorescence detection system

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for SYBR Green I Mix (Applied Biosystems). The reaction was performed with first-strand cDNA solution (0.5

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µL), primers (forward and reverse, 10 µM each), dNTPs (5 mM), 10X PCR buffer (Invitrogen), MgCl2 (50 mM),

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SYBR Green I (1:10 000; Invitrogen), Platinum Taq DNA polymerase (5 U µL-1), ROX (Invitrogen) and sterile

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water. The amplification conditions were as follows: hot start at 95 °C; 40 cycles of 95 °C for denaturation;

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annealing temperature between 58 °C and 60 °C, depending on the primer Tm and extension temperature of 72

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°C. Ct values were calculated from the values obtained from the fluorescence reactions using Real-time PCR

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Miner v 2.2 software (Zhao and Fernald 2005; http://www.miner.ewindup.info/). The expression of the following

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genes were evaluated: ferritin (OsFER1), chloroplastic protein Q-B (OsPSBA), cytosolic glutamine synthetase

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(OsGS1), nicotianamine synthetase (OsNAS1), Yellow Stripe-Like 1 (OsYSL1), tonoplast monosaccharide

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transporter inducible by phosphate 1 limitation (OsPI1), inducible bHLH by phosphate 1 limitation transcription

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factor (OsPTF1), nicotianamine transferase (OsNAAT1). The data were normalized to the following

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housekeeping genes selected from the rice genome using geNorm v 3.5 software: actin (OsACT) and β-1,3-

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glucanase (OsGLU) for roots and tubulin (OsTUB) and ubiquitin (OsUBQ5) for shoots. The comparative

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algorithm proposed by Zhao and Fernald (2005) was used to compare RNA transcript abundance. All forward

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and reverse primer sequences are provided in the supplementary material (Table SM1).

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2.2. Iron uptake, mineral quantification and roots and shoots dry matter

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To minimize interference with the apoplastic quantification of nutrients in the roots, the fresh organ

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samples were washed for 70 min in a solution containing sodium dithionite (3%, w/v), sodium citrate (0.3 M)

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and sodium bicarbonate (1.0 M) to remove traces of iron plaque (Taylor and Crowder, 1983). The plant material

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was separated in shoots and roots, dried and weighted to obtain the root (RDM) and shoot (SDM) dry matter. To

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quantify the nutrient concentration in shoot and roots, the dried plant material was ground in a stainless steel mill

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Wiley type. Samples of dried roots and shoots were digested in concentrated HNO3 at 118 °C for 4 h and then

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diluted with water. The mineral concentration analysis was performed using mass spectrometry with an

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inductively coupled plasma source (ICP-MS) according to Lahner et al. (2003). The mineral concentration (mg

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kg-1) was determined for each of the following elements: iron (Fe), calcium (Ca), magnesium (Mg), phosphorus

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(P), potassium (K), sulfur (S) and zinc (Zn).

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2.8. Statistical analysis

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exchange, chlorophyll fluorescence and mineral concentration values were analyzed by ANOVA and Duncan’s

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test, with a 5% level of significance, using SAS v 9.0 software. A/Ci curves were analyzed by non-linear

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regression obtained from Sigma Plot 10.0 software. The relative levels of gene expression were expressed as fold

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changes and grouped by the Scott-Knott test using Assistat v 7.7 software.

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300 3. Results

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3.1. Leaf gas exchange was reduced in rice plants grown under excess Fe

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The net photosynthetic rate (A), stomatal conductance (gs) and transpiration rate (E) were substantially

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reduced in all four rice cultivars cultivated under elevated Fe compared with the respective controls. However,

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the ratios of sub-stomatal and ambient CO2 concentrations (Ci/Ca) were not altered under excess Fe. Although

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clear tendencies of higher gs and E were observed in the absence of stress in the lowland cultivars 409 and 419

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compared with the upland cultivars (Table 1).

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The maximum carboxylation velocity (Vcmax, Table 2) obtained from the A/Ci curves (Fig. SM1) was

311

reduced in almost all cultivars, except in 409. The Curinga and 419 cultivars showed lower values of Vcmax, with

312

reductions of 70% and 64% relative to their appropriate control values, respectively (Table 2). The Canastra

313

cultivar showed reduction of 39% compared with their control values. Although all cultivars displayed

314

reductions in the maximum rate of electron transport driving RuBP regeneration (Jmax), these reductions were

315

only statistically significant for the Canastra and Curinga cultivars. Similarly, the rate of triose-phosphate

316

utilization (VTPU) was reduced in all cultivars; however, the value was significantly reduced only in the Curinga

317

cultivar (Table 2).

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Total electron transport rates (JT) were reduced by 31% in the Canastra and 409 cultivars and by 38% in

319

the Curinga and 419 cultivars under Fe excess compared with their control values (Table 3). The reduction in JT

12

ACCEPTED MANUSCRIPT was followed by a reduction in the electron transport rate, which is related to the carboxylase reactions of

321

ribulose-1,5-bisphosphate (Jc) and to the Jc/JT ratio. Jc was reduced by 40% in the Canastra and 409 cultivars and

322

by 46% in Curinga and 419 cultivars under excess Fe compared with their control values. However, no

323

differences in JT, Jc or the Jc/JT ratio were detected between the cultivars. Electron transport rates are related to

324

oxygenase reactions of ribulose-1,5-bisphosphate (Jo) and to the rate of CO2 production by photorespiration (R1)

325

were not affected by the excess Fe. Although the R1/A ratios increased by at least 2-fold in all cultivars under

326

excess Fe compared with their control ratios, no statistically significant differences were detected (Table 3).

327 328

3.2. Variations in chlorophyll a fluorescence parameters induced by excess Fe

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Chlorophyll a fluorescence was analyzed to determine whether excess Fe alters the photochemical

331

parameters of rice plants. The linear electron transport rate (ETR) and photochemical quenching (qL) were

332

significantly reduced in all cultivars (Table 4). However, no differences were detected in the potential quantum

333

yield of photosystem II (PSII) (Fv/Fm) under Fe excess. The minimal fluorescence in dark-adapted leaves (F0)

334

was reduced under excess Fe only in the 409 cultivar (Table 4). Energy dissipation through PSII, via chlorophyll

335

a fluorescence, provides information regarding the destination of light energy absorbed in PSII. YII was reduced

336

in all cultivars grown under iron excess, up to 42% for the Curinga cultivar compared with control values (Table

337

4). An increase in YNPQ was observed for all cultivars, mainly in 419. YNO was reduced in the lowland cultivars

338

(409 and 419) compared with their control values (Table 4).

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3.3. Gene expression analysis

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Gene expression analyses were performed and the data are shown as relative expression normalized to

343

the control values of the Canastra cultivar. Several changes in Fe-related gene expression were observed under

344

excess Fe. In leaves, no differences in OsFER1 expression, which codifies for the iron complexing protein

345

ferritin, were observed among the cultivars under control conditions but increased under excess Fe; this increase

346

was less pronounced in 409, which is a lowland cultivar (Table 5). This increase was also observed in roots,

347

primarily in the 419 (lowland) cultivar, when exposed to excess Fe. Under control conditions, OsFER1 is highly

348

expressed in the 409 cultivar compared with the other cultivars (Table 5). OsPSBA, which codifies the

349

chlorophyll binding proteins D1 on the reaction center core of photosystem II (Marder et al., 1987), and OsGS1,

13

ACCEPTED MANUSCRIPT which are important for the photorespiration process (Oliveira et al., 2002) These are expressed only in leaves,

351

showing increased expression in the Canastra cultivar under control conditions compared with the other

352

cultivars. Moreover, under excess Fe, OsPSBA showed decreased expression in the lowland (409 and 419)

353

cultivars, whereas OsGS1 showed increased expression in the 419 cultivar compared with control conditions.

354

The lowland cultivars (409 and 419) showed higher expression values of OsGS1 under control conditions (Table

355

5). The expression level of OsNAS1, involved in iron uptake/long-distance transport (Inoue et al., 2003), varied

356

among the cultivars in both shoot and roots where a slight increase in OsNAS1 expression was observed in the

357

leaves of plants cultivated under excess Fe. In the roots, the relative expression of OsNAS1 was higher in the

358

upland cultivars (Canastra and Curinga) under control conditions (Table 5) and decreased under excess Fe. For

359

the lowland cultivars (409 and 419), there are no differences in OsNAS1 expression levels under control and Fe

360

excess conditions. OsNAAT1, an iron relocalization-related gene (Inoue et al., 2008), which is expressed only in

361

roots had the highest expression level in the Canasta cultivar under control conditions whereas under excess Fe,

362

decreased OsNAAT1 expression was observed in all cultivars (Table 5).

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The 419 cultivar showed higher OsYSL1 expression in leaves and roots under control conditions. In

364

roots, the expression of this gene decreased in all cultivars in response to excess Fe compared with control

365

conditions (Table 5). The opposite effect was observed in leaves where OsYSL1 expression increased in all

366

cultivars except 419 in response to excess Fe compared with control conditions (Table 3). Similar expression

367

patterns were observed for OsPI1 and OsPTF1 among leaves, with increased expression in the Canastra, Curinga

368

and 419 cultivars in response to excess Fe compared with control conditions (Table 5). The gene OsPI1 and the

369

transcription factor OsPTF1 are induced under Pi deficiency in rice (Wasaki et al., 2003; Yi et al., 2005). OsPI1

370

expression in the roots decreased under excess Fe in all cultivars, and the expression of OsPTF1 decreased under

371

excess Fe only in the Canastra and 409 cultivars. For the expression of OsPI1 and OsPTF1 in leaves or roots

372

under control conditions, differences were detected primarily in the 409 cultivar (Table 5).

374 375

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3.4. Nutrient concentration and dry matter of shoot and roots were altered in plants grown under excess Fe

376

All cultivars showed increased Fe concentration in shoot and roots under elevated Fe treatments. Higher

377

accumulation was observed in the roots than in the shoot tissues (Table 6). No significant differences in the total

378

Fe concentration in shoots were detected between the cultivars (Table 6). However, Canastra showed higher Fe

379

levels in the roots, compared to the respective control, followed by 419, 409 and Curinga (Table 6).

14

ACCEPTED MANUSCRIPT 380

The levels of several nutrients were significantly different in the roots and shoots of rice cultivars grown

381

under excess Fe (Table 7). The level of Mg, K and S were reduced, whereas P and Zn increased in the roots of all

382

cultivars grown under excess Fe (Table 7). In shoot tissues, the levels of P, K and Mg were reduced in all rice

383

cultivars grown under excess Fe (Table 7). The level of Ca was reduced only in the cultivars Canastra and 409.

384

Canastra showed significant increases in Zn levels in the shoots (Table 7).

386

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385 4. Discussion

387 388

4.1. Gas-exchange analysis suggests a biochemical limitation for CO2 fixation in plants grown under excess Fe

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The flux of CO2 from the atmosphere to the site of carboxylation inside the chloroplasts of mesophyll

391

cells has both physical (diffusive) and biochemical limitations. The stomatal pore is the first barrier for the influx

392

of CO2 for photosynthesis therefore the magnitude of stomatal opening, (expressed here as stomatal conductance

393

gs), can limit net photosynthetic rate (A) (Jones, 1998). Although the observed reduction in A in the rice cultivars

394

grown under excess Fe was followed by a reduction in gs, minor changes were observed in the ratio of sub-

395

stomatal and ambient CO2 concentrations (Ci/Ca). Stomatal limitations have been observed previously in rice

396

cultivars at moderate Fe doses (Pereira et al., 2013). Once CO2 overcomes the diffusive limitations, such as

397

stomatal and mesophyll resistance, the next limiting step are the carboxylation reactions, which are commonly

398

used to infer the cumulative effects of biochemical limitations. Plants grown under excess Fe presented lower

399

photosynthetic rates under saturating levels of CO2, suggesting that the lower CO2 fixation capacity in these

400

plants may be related to biochemical limitation rather than to diffusional limitation.

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The observed reduction in Vcmax in all cultivars except 409 may be due to degradation of the major

402

subunit of Rubisco by reactive oxygen species (Desimone et al., 1996), or deleterious effects on the Calvin cycle

403

(RuBP regeneration) caused by iron toxicity. Slight changes in VTPU was observed under excess Fe, whilst Jmax

404

was significantly reduced only in the upland cultivars Canastra and Curinga.. These results together with the fact

405

that there was no difference at Ci/Ca ratio between control and treated plants suggest that the reduced net

406

photosynthetic rate in plants grown under excess Fe is due to a biochemical limitation rather than a diffusive

407

limitation. Furthermore, despite the fact that ETR reduced in all cultivars under excess Fe, substantial differences

408

were found in YNO and Jmax between the lowland and upland cultivars. The lowland cultivars (409 and 419)

409

showed no changes in Jmax and significant reduction in the non-regulated energy loss (YNO) under excess Fe,

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15

ACCEPTED MANUSCRIPT 410

suggesting that these cultivars have a remarkable capacity to minimize the constitutive loss of energy at the

411

PSII, which may aid to maintain the Jmax values at levels similar to the control conditions. However, the

412

mechanisms by which lowland cultivars present to maintain Jmax under excess Fe remain to be elucidated and

413

therefore requires further investigation. The data presented in this study suggest that reductions in Vcmax and A in leaves of the rice cultivars

415

grown under excess Fe may be due to three mechanisms. First, reduction in P concentration in leaves could limit

416

ATP production rates in the chloroplasts. In turn, given that RuBP regeneration and CO2 fixation during the

417

Calvin-Benson cycle fundamentally depend on ATP and that NADPH originates from photochemical reactions

418

in the chloroplast (Farazdaghi, 2011; Lawlor, 2002), the reductions in Jmax, JT and Jc provide important evidence

419

that the reductions in both Vcmax and A are most likely due to a lack of RuBP regeneration. Indeed, this

420

mechanism has previously been observed in Beta vulgaris grown under excess Fe (Terry, 1980). Moreover, the

421

reduced use of ATP and NADPH by Calvin-Benson cycle enzymes under Fe stress conditions can lead to

422

feedback inhibition of the electron transport chain (ETC) (Siedlecka et al., 1997, Siedlecka and Krupa, 2004). In

423

fact, reductions in the electron transport rate (ETR), with simultaneous decreases in qL, were observed in the

424

Curinga, 419 and 409 rice cultivars (Figure 4). Excess electrons in the chloroplast ETC can also inhibit the repair

425

mechanisms in the reaction center of photosystem II (PSII), such as D1 protein turnover (Gong and Ohad, 1991).

426

In this context, decreases in the expression of OsPSBA, (the gene encoding the D1 protein), in the 409 and 419

427

cultivars grown under excess Fe suggests that damage in the reaction center of PSII has occurred, as previously

428

observed in pea plants under excess Fe (Suh et al., 2002). Furthermore, D1 protein turnover can also be inhibited

429

by the loss of activity of the Calvin-Benson cycle (Nishiyama et al., 2006; Takahashi and Murata, 2005) and by

430

oxidative stress (Nishiyama et al., 2001).

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414

Significant reductions in A suggest that photorespiratory processes may be an important alternative sink

432

for excess electrons and reductive potential generated by photochemical reactions. With no alterations in the rate

433

of electron transport related to the reaction of Rubisco oxygenase (Jo), the relation between photorespiratory and

434

photosynthesis rates (R1/A) increased significantly in leaves of the Curinga cultivar grown under excess Fe.

435

Photorespiration has been described as a potential method by which inhibition of D1 protein synthesis can be

436

avoided (Takahashi et al., 2007), in addition to being activated by the photosynthetic electron flux regulation

437

(Huang et al., 2015), in order to favor CO2 fixation. Supporting this notion, the expression level of OsGS1, which

438

is a photorespiratory gene involved in the GS/GOGAT pathway, increased in the Canastra and 419 cultivars.

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ACCEPTED MANUSCRIPT Because Fe is an element that is highly reactive at toxic concentrations, non-compartmentation or non-

440

complexing Fe can result in oxidative stress (Souza-Pinto et al., 2016; Suh et al., 2002). Under oxidative stress,

441

plants respond by activating enzymatic and non-enzymatic antioxidant defense pathways (Fang et al., 2001). The

442

potentially oxidative stress suggests that this rice cultivar was not able to completely dissipate excess light

443

energy absorbed in LHCII via the quantum yield of regulated energy dissipation of PSII (YNPQ). The increase in

444

the YNPQ contributes to the avoidance of oxidative stress, despite reductions in A, which is the primary sink for

445

electrons and reductive potential derived from photochemical reactions. Increases in YNPQ have been observed

446

previously, such as an initial response to abiotic stress conditions when CO2 fixation is inhibited, occurring

447

through the formation of a pH range (∆pH) in the chloroplast lumen in a xanthophyll-regulated mechanism

448

(Golding and Johnson, 2003; Joliot and Joliot, 2006). Combined, evidence suggests that excess Fe primarily

449

imparts a biochemical limitation to carbon assimilation in rice under the conditions experienced in this study.

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451

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4.2. Excess iron alters the mineral composition of rice tissues, leading to the reprogramming of gene expression.

452

Rice plants grown in anaerobic environments are exposed to high availability of Fe+2. All roots and

454

shoots of the excess Fe-treated rice cultivars showed increased iron concentrations that are considered toxic

455

according to the recommendation for rice plants (above 300 mg Fe kg-1 dry weight) (Dobermann and Fairhurst,

456

2000). The lowland cultivars (409 and 419), which are commonly exposed to higher Fe concentrations in the

457

soil, showed higher Fe accumulation in roots compared with upland cultivar Curinga.

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In addition to the increase in Fe in leaf tissues, excess Fe influenced the growth of rice plants with both

459

decreased biomass, and radical changes observed in the concentrations of other elements in leaf tissues. P and Zn

460

showed increased accumulation in roots of rice plants under excess Fe (Table S2). Moreover, Fe transporters,

461

such as IRT transporters (Iron Regulated Transporter) (Hell and Stephan, 2003) and ZIP transporters (ZRT/IRT-

462

related Proteins; ZRT, Zinc Regulated Transporter) (Guerinot, 2000; Ramesh et al., 2003) can also absorb Zn.

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Genes encoding Fe and P transporters are highly co-expressed (Hirsch et al., 2006; Zheng et al., 2009),

464

and the high capacity for Fe transport, which was demonstrated in this study by the high Fe levels in shoot, can

465

restrict P translocation between apoplasts and symplasts, leading to P deficiency in rice leaves (Misson et al.,

466

2005; Suriyagoda et al., 2017). Consistent with this observation, a decrease in the P concentration was observed

467

in the shoot of almost all cultivars grown under excess Fe, with the exception of 409. The leaf P starvation

468

induced the expression of OsPI1 and OsPTF1, which encode two P transporters that are known to be expressed

17

ACCEPTED MANUSCRIPT under phosphate deficiency (Wasaki et al., 2003; Yi et al., 2005). By contrast, increased P concentration in the

470

roots resulted in decreased OsPI1 and OsPTF1 expression in this organ. Altogether, these patterns indicated that

471

the expression of Fe and P transporter genes are dependent on balancing the accumulation of these nutrients,

472

suggesting the presence of a feedback mechanism to maintain cellular homeostasis during excess Fe.

473

Consequently, P deficiency and toxic Fe levels have many destructive effects on the photosynthetic process, as

474

discussed below.

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The Fe transporter YSL1 (Yellow stripe-like 1) is responsible for transporting Fe+2 from the soil to roots

476

(Curie et al., 2009; Schaaf et al., 2004) and is transcriptionally responsive to changes in Fe in ysl1ysl3 mutants

477

(Waters et al. (2006)) analyzing. Although higher Fe levels were observed in the roots of the lowland cultivars,

478

these increases did not correspond with OsYSL1 expression. Thus, other Fe transporters may be related to the

479

higher Fe absorption observed in these cultivars. By contrast, the high translocation of Fe led to increased

480

OsYSL1 expression in leaves of rice plants grown under excess Fe perhaps as a mechanism for remobilizing this

481

element within leaf tissues (Kim and Guerinot, 2007). Thus, the higher iron accumulation in shoot was not

482

necessarily related to high toxicity.

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The nicotianamine synthetase (OsNAS) and nicotianamine transferase (OsNAAT) genes are important

484

for Fe homeostasis in rice tissues (Inoue et al., 2003, 2008; Kobayashi et al., 2010), directly involved in the Fe+3

485

uptake by PS-Fe+3 complex. This strategy is particularly important for upland cultivars, which are usually grown

486

in soil in which Fe is predominantly available as Fe+3 (Curie et al., 2001). OsNAS1 and expression in roots was

487

shown to be highly responsive to excess Fe. OsNAS1 was several times higher in the upland cultivars compared

488

to lowland cultivars in control conditions, indicating that this gene is a good marker for discriminating between

489

lowland and upland cultivars. Both genes showed strongly downregulated expression in the roots of the upland

490

cultivars exposed to Fe excess most likely as a mechanism to mitigate the excessive Fe uptake. In the roots of the

491

lowland cultivars, the OsNAS1 expression level was lower than in the roots of the upland cultivars and did not

492

change under Fe excess, indicating that this gene most likely does not participate in any tolerance mechanisms

493

against excess Fe in the roots of the lowland cultivars.

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Given that all mechanisms for minimizing the excessive Fe uptake described above were insufficient to

495

avoid toxic Fe concentrations, other mechanisms, such as Fe sequestration in the form of ferritin, are most likely

496

used by rice plants to tolerate high Fe concentrations in these tissues (Briat et al., 2010a; Majerus et al., 2009).

497

Iron stored in ferritin molecules cannot react with oxygen therefore ferritins are important for plant defenses

498

against oxidative stress induced by iron (Briat et al., 2010b; Ravet et al., 2009). The data shown in this study

18

ACCEPTED MANUSCRIPT suggest that this mechanism may contribute to growth and survival under excess Fe conditions because a strong

500

increase in OsFER1 expression was observed in the leaves of the rice cultivars under Fe stress and particularly

501

strongly in the 419 cultivar. However, the 409 cultivar control plants showed high levels of OsFER1 expression

502

compared with the 419 cultivar control plants. In addition to the other mechanisms already discussed, the 409

503

cultivar is likely to have a greater capacity for avoiding high levels of iron in the plant, which contributes to the

504

maintenance of growth under excess Fe at the initial stages.

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505 506

5. Conclusions

507

Here we show that rice cultivars commercially grown in lowland and upland culture systems in Brazil

509

show differential physiological, molecular and chemical responses upon exposure to iron excess. The upland

510

Canastra cultivar, with higher iron uptake in the roots, displayed a mechanism to limit iron translocation from

511

roots to the shoots, minimizing the leaf oxidative stress induced by excess Fe. Combined with the increase in

512

OsPSBA and decrease of qL, Vcmax, Jmax Canastra cultivar showed more tolerance to excess of iron than cultivar

513

Curinga. Conversely, the cultivar Curinga invested in the increase of R1/A, as an alternative drain of electrons.

514

The higher iron accumulation in the shoot, however, was not necessarily related to high toxicity. Nutrient uptake

515

and/or utilization mechanisms in rice plants are in accordance with their needs, which may be defined in relation

516

to crop environments.

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Altogether, the physiological, molecular and nutritional data suggest that the upland Canastra cultivar is

518

more tolerant to iron excess compared to Curinga cultivar. These results assist in understanding the responses of

519

different rice cultivars, contributing to the development of cultivars tolerant to excess iron. The decrease in the

520

net photosynthetic rate upon exposure to excess Fe is due to biochemical limitation and may be a response to P

521

starvation. In addition, the OsNAS gene, Jmax and VTPU are good markers for discriminating between lowland (409

522

and 419) and upland (Canastra and Curinga) cultivars in anaerobic condition.

524

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Acknowledgments

525

The authors thank the Brazilian Agricultural Research Corporation (EMBRAPA) and the Agricultural

526

Research Company of Minas Gerais (EPAMIG) for providing us with rice seeds. The authors also thank Dr.

527

David Salt for the ICP-MS analysis of rice tissues. This work was supported by the Coordination for the

528

Improvement of Higher Education Personnel - PROCAD/CAPES [grant no. 0361054/2005 to MAO] and Minas

19

ACCEPTED MANUSCRIPT 529

Gerais State Agency for Research and Development - FAPEMIG [grant no. APQ-1011-3.08/07 to AMA], Brazil.

530

CM and DMD are grateful to the CNPq and to FAPEMIG for scholarships.

531 532

Conflicts of interest The authors declare that they have no conflict of interest.

533

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ACCEPTED MANUSCRIPT Table 1 Net photosynthetic assimilation rate (A, µmol m-2 s-1); stomatal conductance (gs, µmol m-2 s-1); transpiration rate (E, mmol m-2 s-1) and the ratio between internal and external CO2 concentration (Ci/Ca) of the upland (Canastra and Curinga) and lowland (409 and 419) rice cultivars, after 7 d exposure to adequate (0.018 mM, control) or excessive level (7 mM) of iron sulfate (Fe excess) in a hydroponic system.

Curinga 409 419

A 24.3±2.1 a 11.2±1.3 b 19.6±2.5 a 6.7±0.8 b 24.5±1.5 a 12.4±3.2 b 24.9±1.7 a 10.6±0.7 b

gS 0.366±0.094 bc 0.169±0.029 de 0.306±0.056 cd 0.102±0.003 e 0.555±0.036 a 0.217±0.049 de 0.489±0.071 ab 0.161±0.017 de

E 3.60±0.71 bc 2.32±0.17 d 3.59±0.41 bc 1.65±0.02 d 4.77±0.35 a 2.64±0.26 cd 4.40±0.32 ab 2.28±0.23 d

Ci/Ca 0.72±0.027 b 0.70±0.015 b 0.69±0.023 b 0.71±0.026 b 0.79±0.014 a 0.75±0.021 ab 0.75±0.015 ab 0.70±0.012 b

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Canastra

Treatment Control Fe excess Control Fe excess Control Fe excess Control Fe excess

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Cultivar

The data are presented as the means (n = 4) ± SE. Means in a single column and followed by the same letter

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do not differ significantly, as determined by Duncan’s test (p > 0.05).

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ACCEPTED MANUSCRIPT Table 2 Maximum carboxylation rate (Vcmax); maximum capacity for photosynthetic electron transport (Jmax) ans rate of triose phosphate utilization (VTPU) in upland (Canastra and Curinga) and lowland (409 and 419) rice cultivars, after 7 d exposure to adequate (0.018 mM, control) or excessive level (7 mM) of iron sulfate (FeSO4EDTA) in a hydroponic system.

Canastra Curinga 409

Vcmaxa 113.5±10.3 a 68.9±2.4 bc 106.5±13.5 a 32.3±8.9 d 104.5±12.0 a 84.4±12.3 ab 105.1±15.9 a 37.7±4.0 dc

Jmaxb 157.2±13.5 a 100.9±9.5 bc 120.3±16.8 abc 69.2±14.6 d 154.6±11.1 a 124.6±25.9 ab 149.5±18.1 ab 114.7±1.5 abc

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Treatment Control Fe excess Control Fe excess Control Fe excess Control Fe excess

VTPUc 10.3±0.38 ab 7.5±0.34 bc 8.8±0.87 ab 5.9±0.85 c 11.6±0.78 a 9.9±1.54 ab 11.5±0.83 a 9.5±0.54 ab

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The data are presented as the means (n = 3) ± SE. Means in a single column and followed by the same letter

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do not differ significantly, as determined by Duncan’s test (p > 0.05).

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3 Table 3 Total electron transport rate through PSII (JT; µmol m-2 s-1), electron flow attributable to the carboxylation (Jc; µmol m-2 s-1) and oxygenation (Jo; µmol m-2 s-1) reactions of

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RuBP, the Jc/JT ratio, the rate of CO2 production by photorespiration (Rl; µmol m-2 s-1) and the R1/A ratio of the upland (Canastra and Curinga) and lowland (409 and 419) rice cultivars, after 7 d exposure to adequate (0.018 mM, control) or excessive level (7 mM) of iron sulfate (FeSO4-EDTA) in a hydroponic system.

Curinga 409 419

JT 172±8.1 a 118±5.6 bcd 161±6.7 abc 97±3.2 d 165±7.4 ab 114±15.5 bcd 176±3.9 a 111±2.8 cd

Jc 126.3±8.4 ab 74.0±5.2 cd 108.7±8.9 abc 53.8±3.3 d 124.7±6.3 ab 76.8±13.6 bcd 129.3±5.8 a 70.3±1.2 cd

Jo 45.8±0.6 ab 43.6±1.2 bc 52.3±2.2 a 43.5±0.8 bc 40.1±1.1 bc 37.0±2.4 c 46.3±2.4 ab 40.6±3.5 bc

Jc/JT 0.73±0.02 ab 0.63±0.02 bc 0.67±0.04 abc 0.55±0.02 c 0.76±0.01 a 0.66±0.05 abc 0.74±0.02 ab 0.64±0.03 abc

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Treatment Control Fe excess Control Fe excess Control Fe excess Control Fe excess

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R1 5.73±0.07 ab 5.45±0.16 bc 6.54±0.28 a 5.44±0.10 bc 5.01±0.13 bc 4.62±0.30 c 5.79±0.30 ab 5.08±0.44 bc

R1/A 0.24±0.02 b 0.48±0.05 ab 0.36±0.07 b 0.79±0.09 a 0.20±0.01 b 0.48±0.13 ab 0.23±0.03 b 0.47±0.07 ab

The data are presented as the means (n = 4) ± SE. Means in a single column and followed by the same letter do not differ significantly, as determined by Duncan’s test (p >

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0.05).

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4 Table 4 Minimal fluorescence (F0), maximal PSII quantum yield (Fv/Fm), electron transport rate (ETR), photochemical quenching based on the lake model of PSII antenna (qL),

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quantum yield of photochemical energy conversion in PSII (YII), quantum yield of regulated (YNPQ) and non-regulated (YNO) energy dissipation in PSII of the upland (Canastra and Curinga) and lowland (409 and 419) rice cultivars, after 7 d exposure to adequate (0.018 mM, control) or excessive level (7 mM) of iron sulfate ( Fe excess) in a

Canastra Curinga 409 419

Treatment Control Fe excess Control Fe excess Control Fe excess Control Fe excess

F0 569±17.9 d 593±6.7 bcd 636±6.1 a 645±17.0 a 623±11.2 ab 583±11.1 cd 640±3.4 a 614±6.3 abc

Fv/Fm 0.81±0.013 a 0.80±0.011 ab 0.80±0.003 ab 0.79±0.005 b 0.80±0.007 ab 0.80±0.008 ab 0.80±0.004 ab 0.79±0.004 b

ETR 151.4±8.4 a 103.7±5.3 b 144.6±6.3 a 84.6±3.0 b 148.0±6.9 a 100.1±14.5 b 158.2±3.6 a 97.4±2.7 b

qL 0.32±0.024 ab 0.27±0.019 bc 0.35±0.003 a 0.23±0.009 c 0.32±0.013 ab 0.25±0.024 c 0.37±0.017 a 0.27±0.011 bc

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hydroponic system.

YII 0.363±0.020 a 0.241±0.013 b 0.347±0.015 a 0.203±0.007 b 0.355±0.017 a 0.240±0.035 b 0.379±0.009 a 0.234±0.007 b

YNPQ 0.37±0.027 b 0.53±0.017 a 0.41±0.024 b 0.56±0.014 a 0.37±0.011 b 0.52±0.039 a 0.36±0.019 b 0.54±0.002 a

YNO 0.27±0.008 ab 0.23±0.022 d 0.24±0.009 bcd 0.23±0.008 cd 0.28±0.011 a 0.24±0.008 bcd 0.26±0.014 abc 0.22±0.007 d

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The data are presented as the means (n = 4) ± SE. Means in a single column and followed by the same letter do not differ significantly, as determined by Duncan’s test (p >

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5 Table 5 qRT-PCR analysis of the relative mRNA levels (fold changes) of several genes in the roots of the upland (Canastra and Curinga) and lowland (409 and 419) rice cultivars, after 7 d exposure to adequate (0.018 mM, control) or excessive level (7 mM) of iron sulfate (FeSO4-EDTA) in a hydroponic system.

Canastra

a

OsPTF1g 1.00 c 4.86 a

1.28 b 3.89 a 1.92 b 2.60 a

0.54 c 1.54 b 0.50 c 0.90 c

0.37 c 1.69 b 0.21 c 0.82 c

0.98 b 3.08 a 1.16 a 1.95 b Root expression OsNAAT1h OsYSL1e 1.00 a 1.00 b 0.07 c 0.17 c

0.78 c 1.85 b

0.64 c 1.71 b

Control Fe excess Control Fe excess Control Fe excess

0.31 c 19.75 a 1.68 c 5.39 b

1.71 a 1.42 a 1.72 a 1.28 b

1.22 c 1.67 c 2.60 b 2.22 b

Control Fe excess

0.32 c 17.78 a

2.02 a 0.59 b

2.70 b 3.39 a

Treatment

Canastra

Control Fe excess

Curinga

Control Fe excess

409

Control Fe excess

419

Control

0.52 b 0.91 b 0.74 b 1.52 a

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Leaf expression OsNAS1d OsYSL1e 1.00 b 1.00 b 1.41 a 3.37 a

OsPI1f 1.00 c 4.99 a

OsGS1 1.00 c 3.15 a

OsFER1 a 1.00 c 2.02 c

OsNAS1d 1.00 a 0.57 b

2.47 c 5.83 b 5.79 b 6.33 b

0.88 a 0.03 c 0.06 c 0.02 c

0.67 b 0.18 c 0.49 b 0.07 c

2.69 c 12.71 a

0.07 c 0.01 c

0.51 b 0.12 c

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c

OsPSBA 1.00 b 1.89 a

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b

OsFER1 1.00 c 19.02 a

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a

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OsPI1f 1.00 a 0.05 c

OsPTF1g 1.00 a 0.64 b

1.06 b 0.36 c 0.94 b 0.46 c

0.70 b 0.07 c 0.22 c 0.04 c

0.95 a 0.94 a 1.06 a 0.61 b

3.88 a 0.54 c

0.64 b 0.08 c

1.16 a 1.22 a

ferritin, bchloroplastic protein Q-B, ccytosolic glutamine synthetase, dnicotianamine synthetase, eYellow Stripe-Like 1, ftonoplast monosaccharide transporter inducible by

phosphate 1 limitation, ginducible bHLH by phosphate 1 limitation transcription factor, h nicotianamine transferase. Relative levels of leaves gene expression (fold changes) were normalized to the expression levels of the housekeeping genes ubiquitin 5 (OsUBQ5) and tubulin (OsTUB) and relative values of roots genes expression were normalized to the the expression levels of the housekeeping genes actin (OsACT) and glutamine (OsGLU). Means followed by the same letters are grouped by the Scott-Knott

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6 test. The data are presented as fold changes compared with the control from the Canastra cultivar. Fold values were derived from averages of four biological and three

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technical replicates.

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ACCEPTED MANUSCRIPT Table 6 Iron concentration (mg kg-1 DW) in roots (Feroot) and in shoot (Feshoot) and root (RDM) and shoot (SDM) dry matter of the upland (Canastra and Curinga) and lowland (409 and 419) rice cultivars, after 7 d exposure to adequate (0.018 mM, control) or excessive level (7 mM) of iron sulfate (Fe excess) in a hydroponic system. The data are presented as the means (n=4) ± SE. Means followed by the same letter do not differ significantly from each other, as determined by Duncan’s test (p>0.05)

Curinga 409 419

Feroot 585±81 d 16,071±297 b 616±65 d 12,646±797 c 796±31 d 18,958±1,525 a 769±18 d 18,474±1,148 a

Feshoot 191±28 b 506±90 a 76±8.8 b 469±42 a 72±3.3 b 492±59 a 69±2.7 b 499±41 a

RDM 1.64±0.07 ab 1.14±0.14 b 2.13±0.15 a 1.27±0.11 b 2.27±0.20 a 1.50±0.17 ab 1.59±0.14 ab 1.26±0.22 b

SDM 8.43±0.45 ab 6.49±0.26 b 11.62±1.18 a 7.53±0.93 b 11.39±1.05 a 7.69±0.33 b 9.06±0.28 ab 7.20±0.52 b

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Canastra

Treatment Control Fe excess Control Fe excess Control Fe excess Control Fe excess

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Cultivar

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not differ significantly, as determined by Duncan’s test (p > 0.05).

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8 Table 7 Levels (mg kg-1) of elements in the roots and shoot of the upland (Canastra and Curinga) and lowland (409 and 419) rice cultivars, after 7 d exposure to adequate (0.018

419

Canastra Curinga 409 419

Control Fe excess Control Fe excess Control Fe excess Control Fe excess

P 9982±673 a 5871±222 b 8713±522 a 6205± 84 b 9277±489 a 6389±169 b 9640±620 a 5808±329 b

K 24959±2561 c 8873±310 d 22426±923 c 10125±920 d 30469±1957 b 11772±725 d 40450±3036 a 10018±444 d

Ca 1709±512 ab 1666±65 ab 1680±455 ab 1351±51 b 3144±1205 a 2032±106 ab 2104±346 ab 1739±144 ab

Zn 32.80±6.56 b 60.03±2.03 a 27.90±6.85 b 54.22±1.35 a 38.58±4.75 b 62.65±1.42 a 39.70±1.41 b 61.40±5.26 a

K 81062±5248 a 59242±4028 b 73482±6227 a 49079±3266 b 81718±3919 a 56702±1440 b 83942±3065 a 60432±3112 b

Ca 5694±239 a 3796±504 bc 3791±705 bc 2837±401 c 5596±658 a 3915±180 bc 4977±338 ab 4512±331 ab

Zn 49.99±4.42 bc 86.68±17.9 a 39.95±2.38 c 58.77±4.52 bc 46.96±4.44 bc 68.35±2.65 ab 50.56±4.26 bc 54.68±2.13 bc

SC

Mg 9015±566 a 5646±583 c 5635±561 c 3980±304 d 6977±14 b 4712±92 cd 7472±27 b 5527±29 c

M AN U

409

Control Fe excess Control Fe excess Control Fe excess

P 4431±434 c 11910±447 b 4107±326 c 12097±728 ab 4602±673 c 13883±861 a 4270±295 c 11543±712 b

Roots (mg kg-1) S 15470± 2047 b 6517±549 c 15230±2467 b 7244±762 c 28628±2851 a 10973±1580 bc 28610±2639 a 11036±1162 bc Shoot (mg kg-1) S 6973±428 a 6947±513 a 6285±67 a 5899±39 a 6784±67 a 7365±24 a 6386±17 a 7502±55 a

TE D

Curinga

Control Fe excess

Mg 1669±220 b 715±49 c 1593±207 b 580±43 c 2545±480 b 932±25 bc 3047±402 a 795±37 c

EP

Canastra

Treatment

AC C

Cultivar

RI PT

mM, control) or excessive level (7 mM) of iron sulfate (FeSO4-EDTA) in a hydroponic system.

The data are presented as the means (n = 4) ± SE. Means in a single column and followed by the same letter do not differ significantly, as determined by Duncan’s test (p > 0.05.

ACCEPTED MANUSCRIPT

Highlights Excess iron leads to substantial changes in nutrient accumulation in plants

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Photosynthesis in rice under excess Fe ir biochemically limited

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Iron tolerance mechanisms differ between the upland and lowland rice cultivars

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Jmax appears to be a good marker to discriminate upland from lowland rice cultivars under Fe excess

AC C

EP

TE D

M AN U

SC

RI PT

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