Accepted Manuscript

Aquaporins in Coffea arabica L.: identification, expression, and impacts on plant water
relations and hydraulics
Matilda Miniussi, Lorenzo Del Terra, Tadeja Savi, Alberto Pallavicini, Andrea Nardini
PII:

S0981-9428(15)30070-X

DOI:

10.1016/j.plaphy.2015.07.024

Reference:

PLAPHY 4242

To appear in:

Plant Physiology and Biochemistry

Received Date: 17 April 2015

Accepted Date: 21 July 2015

Please cite this article as: M. Miniussi, L. Del Terra, T. Savi, A. Pallavicini, A. Nardini, Aquaporins in
Coffea arabica L.: identification, expression, and impacts on plant water relations and hydraulics, Plant
Physiology et Biochemistry (2015), doi: 10.1016/j.plaphy.2015.07.024.
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Aquaporins in Coffea arabica L.: identification, expression, and impacts on plant

water relations and hydraulics

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Matilda MINIUSSI1, Lorenzo DEL TERRA2, Tadeja SAVI1, Alberto PALLAVICINI1*, Andrea

NARDINI1*

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1. Dipartimento di Scienze della Vita, Universit di Trieste, Via L. Giorgieri 10, 34127 Trieste, Italy

2. illycaff Spa, Via Flavia 110, 34147 Trieste, Italy

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* Corresponding authors:

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A. Pallavicini: +39-040-5588736, pallavic@units.it

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A. Nardini: +39-040-5583890; nardini@units.it

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Running title: Coffee aquaporins and hydraulics

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Abstract

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Plant aquaporins (AQPs) are involved in the transport of water and other small solutes across cell

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membranes, and thus play major roles in the regulation of plant water balance and transport, as well as

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in growth regulation and response to abiotic stress factors. Limited information is currently available

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about the presence and role of AQPs in Coffea arabica L., despite the economic importance of the

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species and its vulnerability to drought stress. We identified candidate AQP genes by screening a

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proprietary C. arabica transcriptome database, resulting in the identification of eight putative

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aquaporins. A phylogenetic analysis based on previously characterized AQPs from Arabidopsis

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thaliana and Solanum tuberosum allowed to assign the putative coffee AQP sequences to the Tonoplast

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(TIP) and Plasma membrane (PIP) subfamilies. The possible functional role of coffee AQPs was

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explored by measuring hydraulic conductance and aquaporin gene expression on leaf and root tissues

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of two-year-old plants (C. arabica cv. Pacamara) subjected to different experimental conditions. In a

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first experiment, we tested plants for root and leaf hydraulic conductance both before dawn and at

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mid-day, to check the eventual impact of light on AQP activity and plant hydraulics. In a second

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experiment, we measured plant hydraulic responses to different water stress levels as eventually

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affected by changes in AQPs expression levels. Our results shed light on the possible roles of AQPs in

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the regulation of C. arabica hydraulics and water balance, opening promising research lines to

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improve the sustainability of coffee cultivation under global climate change scenarios.

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Keywords: drought, water relations, plant hydraulics, aquaporin

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1. Introduction

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Coffee is one of the worlds most valuable agricultural export commodities (Talbot 2004). About 60%

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of global coffee production is represented by Coffea arabica L., the remaining being largely accounted

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for by C. canephora Pierre (DaMatta 2004). Global agriculture has been suffering from increasing air

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temperatures and anomalous drought episodes over the last decades. In particular, coffee yield is

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considered at risk due to negative effects of increasing temperatures and decreasing rainfall, further

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complicated by additional negative factors such as the low genetic diversity of cultivated plants, and

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the increasing deforestation rates of the areas of greater natural diversity of wild coffee species

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(Labouisse et al. 2008). In a recent study, Davis et al. (2012) modelled the present and future predicted

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distribution of C. arabica in Ethiopia. The most favourable outcome of this modelling effort was a

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predicted 38% reduction of suitable bioclimatic areas for coffee growth and persistence by 2080, with

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the worst scenario suggesting a dramatic 90% reduction. On this basis, it has been suggested that C.

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arabica is a species facing a serious risk of local decline in a global climate change scenario.

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The impact of climate change on coffee production portends economical and social effects that

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cannot be underestimated. Rising temperatures and unpredictable weather events played a major role

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in the recent dynamics of the coffee market. For example, between 2008 and 2009 the Colombian

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harvest dramatically fell from about 12 million 60-kg bags to around 8 millions bags, an abrupt

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decrease of about 35% from the worlds second-largest C. arabica producing country (www.ico.org).

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Moreover, in 2014 Brazil - the world's largest coffee producer - has experienced an unprecedented

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drought coupled to exceptionally high temperatures, affecting coffee yield.

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Drought stress is one of the most important limiting factors for coffee productivity, in that it

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directly impacts plant growth and has important effects on flower and seed production (DaMatta and

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Ramalho 2006). Stomatal closure is one of the first physiological responses to drought stress (Da

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Matta 2004), leading to transient limitations of photosynthesis and productivity (Da Matta et al. 1997),

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but intense or prolonged drought can severely impair water and carbon metabolism of plants up to

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death (Sevanto et al. 2014). Several studies have addressed possible morphological features and

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physiological mechanisms contributing to the drought tolerance of different Coffea species/cultivars.

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These include changes in plant hydraulic efficiency and safety (Tausend et al. 2000; Pinheiro et al.

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2005; Nardini et al. 2014), osmoregulation processes (DaMatta et al. 2003; Dias et al. 2007),

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hormones production and accumulation (Marraccini et al. 2012), and protection against oxidative

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stress (Pinheiro et al. 2004; Rodrguez-Lpez et al. 2013).

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Several plant physiological responses to water stress are controlled at the molecular level by a

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specific family of membrane channel proteins known as aquaporins (Johanson et al. 2001;

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Alexandersson et al. 2005; Maurel et al. 2008; Ayadi et al. 2011). These proteins facilitate the transport

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of water and other small solutes, including CO2, across cell membranes (Li et al. 2014). Data gathered

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over the last two decades suggest that plant aquaporins play a central role in several physiological

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processes, including regulation of plant water balance (Kaldenhoff et al. 2008), stomatal movements

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(Heinen et al. 2009), modulation of root and leaf hydraulic conductance (Lovisolo et al. 2007;

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McElrone et al. 2007; Hachez et al. 2008; Heinen et al. 2009), xylem embolism repair (Secchi and

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Zwieniecki 2014), cell elongation (Besse et al. 2011) and maintenance of cell turgor (Martre et al.

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2002). In turn, all these physiological processes and responses are affected by environmental factors,

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among which light is one of the most important. In fact, close relationships between light and

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aquaporin expression levels have been shown to underlie diurnal changes in some physiological

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parameters like leaf and root hydraulic conductance (Henzler et al. 1999; Nardini et al. 2005b; Lopez

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et al. 2013; Johnson et al. 2014).

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Aquaporins are members of a large superfamily of conserved proteins called major intrinsic

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proteins (MIPs). Their distribution and diversity greatly varies among different organisms (Venkatesh

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et al. 2013), and in plants the number of known aquaporins is apparently larger than in other taxa (e.g.

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at least three times more abundant than in mammals). For example, 35 aquaporin isoforms have been

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identified in Arabidopsis thaliana (Johanson et al. 2001), while 31 are known in Zea mays (Chaumont

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et al. 2001) and 41 in Solanum tuberosum (Venkatesh et al. 2013). Based on the sequence homology

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and subcellular localization, aquaporins are classified into five subfamilies i.e. plasma membrane

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intrinsic protein (PIP), tonoplast intrinsic protein (TIP), NOD26-like intrinsic protein (NIP), small

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intrinsic protein (SIP) and X-intrinsic protein (XIP) (Kaldenhoff and Fischer 2006; Sade et al. 2009).

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All MIPs have a highly conserved structure, consisting of tandem repeats of three membrane-spanning

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-helical domains. Each tandem repeat is characterized by a highly conserved asparagine-proline-

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alanine (NPA) motif. The overlap of the NPA motifs inside the lipid bilayer forms one of the two

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channel constrictions sites involved in proton exclusion and channel transport activity, with a second

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stage being provided by conserved aromatic/arginine (ar/R) regions that operate as a proton filter.

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Variability at this site is thought to form the basis of the broad spectrum of solute transport specificity

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in plant aquaporins (Maurel et al. 2008).

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Despite their predictable importance in regulating the water balance of coffee plants, there is

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presently very limited information about the aquaporin gene superfamily in the genus Coffea (Santos

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and Mazzafera 2013), and no information at all about relationships between aquaporin expression and

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plant hydraulics in this genus. This knowledge gap is remarkable, taking into account the importance

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of these proteins in plant physiological processes which are expected to influence coffee production,

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including drought stress tolerance and responses to climate changes (Martnez-Ballesta et al. 2009).

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The present study is thus aimed at gathering new data on the presence, abundance and possible

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functional roles of aquaporins in coffee hydraulics and water relations.

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We specifically aimed at testing the following hypotheses: 1) different aquaporin isoforms can

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be identified in C. arabica, and their expression levels are a function of physiological and

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environmental conditions; 2) circadian rhythms and/or changes in light levels induce up-regulation of

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aquaporins, resulting in increased plant hydraulic conductance; 3) drought stress induces changes in

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the expression levels of different aquaporins, with consequent impacts on leaf and root hydraulics.

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2. Materials and Methods

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2.1 Bioinformatic analysis of putative C. arabica aquaporins sequences

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Full length aquaporin sequences from several Nicotiana species were isolated from Swissprot and

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TrEMBL databases. About

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(http://nar.oxfordjournals.org/content/32/5/1792.long) embedded in the CLCbio software package

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(QIAGEN Company) to identify a common conserved region, corresponding to the pore region. This

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region was used to design a probe (ISGGHINPAVTFGL) for the identification of putative aquaporin

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sequences in the C. arabica transcriptome (De Nardi et al. 2006; Viera and Andrade 2006) using

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tBLASTn (Basic Local Alignment Search Tool).

protein

sequences

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aligned

with

Muscle

software

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2.2 Phylogenetic analysis

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The known aquaporin sequences of Arabidopsis thaliana (http//:www.uniprot.org) and Solanum

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tuberosum (Venkatesh et al. 2013), and the putative C. arabica aquaporin sequences were aligned with

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Muscle algorithm and a phylogenetic tree was inferred by using the Maximum Likelihood method and

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the LG model. Maximum Likelihood fits of 48 different amino acid substitution models was initially

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performed. Initial tree for the heuristic search were obtained by applying the Neighbor-Joining method

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to a matrix of pairwise distances estimated using a JTT model. A discrete Gamma distribution was

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used to model evolutionary rate differences among sites (5 categories). All positions with less than

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95% site coverage were eliminated for a total of 202 positions. The tree was supported by 1000

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Bootstrap resampling cycles. All the above analysis was conducted with MEGA software version 6.05.

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2.3 Plant material

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The plants used in this study were two-year-old specimens of Coffea arabica L. cv Pacamara, grown

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in 1.75 liters pots filled with a professional substrate (GEOTEC BRILL, Bolzano, Italy). Plants were

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grown in the tropical greenhouse at the Dept. of Life Sciences of the University of Trieste, Italy. The

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plants were irrigated daily to field capacity, except during the drought experiment when irrigation was

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partially reduced for some experimental groups (see below). During the experiments, midday air

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temperature in the greenhouse was 23.4 4.6C, relative humidity was 67 13% and

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photosynthetically active radiation ranged between 420 and 530 mol m-2 s-1. Vapour pressure deficit

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oscillated between a minimum of 0.43 kPa to a maximum of 0.76 kPa.

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2.4 Experimental treatments

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In May 2013, 60 plants with an height of 20 3 cm were randomly selected and assigned to two

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groups for different experimental treatments. A group of 15 plants was maintained under optimal water

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availability by daily irrigating the pots to field capacity. These plants were used to investigate the

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influence of daily changes in light intensity on aquaporin expression and physiological parameters

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related to hydraulic efficiency (see below). Measurements of physiological parameters and the

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extraction of total RNA from leaf and root tissue were made either before dawn (between 05.30 and

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06.30) and at midday (between 11.00 and 13.00) during 5 consecutive days in June 2013.

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The remaining 45 plants were subjected to a water stress experiment in July-August 2013.

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These plants were further divided into three groups of 15 plants each. Daily plant transpiration was

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initially measured by weighing all irrigated plants (under well watered conditions) before and after a

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24 h time interval (without adding water). Average daily water loss was 50 6 ml per plant. The plants

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were then subjected to different treatments for the following two weeks. The control group was

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irrigated daily with 100 ml of tap water (about two times the value of daily water transpiration) to

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assure maintenance of saturated soil water content. The second plant group was subjected to a mild

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water stress by irrigating plants with 25 ml of water every day, i.e. with an amount of water

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corresponding to 50% of the daily transpiration of controls. The last group was subjected to a severe

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water stress by irrigating plants with only 12.5 ml per day, i.e. 25% of the daily transpiration of

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controls. These irrigation regimes were maintained for 21 days. At the end of the treatment,

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physiological parameters and RNA extraction were conducted between 11.00 and 13.00 during five

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consecutive days.

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2.5 Measurements of plant water status and hydraulics

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In the 'light' experiment performed in June 2013, leaf (KL) and root (KR) hydraulic conductance were

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measured at pre-dawn and midday, in order to highlight eventual plant hydraulic responses to changes

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in light intensity. In the 'water stress' experiment, the same physiological parameters were recorded

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only at midday. Leaf conductance to water vapor (gL, mmol m-2 s-1) and leaf water potential (

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were also measured during the 'water stress' experiment in a subset of five plants per treatments (two

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leaves per plant). In particular, gL was measured using a steady-state porometer (SC-1 Leaf Porometer,

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Decagon Devices Inc., Pullman, WA, USA), while

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moisture mod. 3005, Soilmoisture Equipment Corp., Santa Barbara, CA,USA). Photosynthetically

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active radiation at the time of measurements was also recorded using a quantum sensor (HD 9021,

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L,

MPa)

was measured with a pressure chamber (Soil

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Delta Ohm).
All hydraulic measurements were performed using a High Pressure Flow Meter (Tyree et al.

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1995). Plants were transported to the laboratory and the pot was enclosed in a plastic bag secured with

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tape to the base of the plant. The plant was then immersed in water and the shoot was detached 3-4 cm

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above the soil. The shoot was kept with the cut end immersed in water, while the root system was

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immediately connected to the HPFM using chromatography fittings. Root hydraulic conductance was

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measured in the transient mode by rapidly increasing water pressure (P, from 0.01 to 0.4 MPa in 30 s)

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and measuring the resulting flow rates (F) at 3 s intervals. KR was calculated on the basis of the slope

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of the F to P relationship. Immediately after K measurement, roots were carefully excavated and

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cleaned from soil particles under a gentle jet of water, and 1 g of non-suberized roots with diameter <

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1 mm was collected for total RNA extraction (see below). At the same time, the shoot was connected

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to the HPFM, and fully rehydrated by applying a P = 0.2 MPa for 5 min. Then, whole shoot hydraulic

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conductance (Kshoot) was measured in the transient mode as described above. Finally, all leaves were

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removed and stem hydraulic conductance (Kstem) was measured again in the transient mode. Leaf

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hydraulic conductance (KL) was calculated as 1/KL = (1/Kshoot) (1/Kstem) (Tsuda and Tyree 2000).

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Total leaf surface area (Aleaf) was measured by scanning the leaves and analyzing images using ImageJ

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software. Moreover, 1 g of leaf tissue from the two most apical mature leaves was collected for RNA

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extraction. All K values were corrected to a temperature of 21 C (calibration temperature of the

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HPFM) to take into account changes in water viscosity. All K values were normalized by Aleaf and

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expressed in units of mmol s-1 MPa-1 m-2.

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2.6 Reverse transcription quantitative PCR analysis (RT-qPCR)

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Total RNA was extracted from leaf and root samples using an adaptation of the CTAB protocol by

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Chang et al. (1993). All extraction steps were the same as the reference method, except for the use of 8

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M LiCl instead of 10 M LiCl, and the suspension of the pellet in 100 L of DEPC-treated H2O instead

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of 500 L of SSTE. Total RNA was quantified with a NanoDrop spectrophotometer (Thermo

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Scientific) and its quality and integrity were evaluated by two techniques: denaturing agarose gel

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electrophoresis and 2100 Bioanalyzer Instrument (Agilent, Santa Clara, CA, USA). Complementary

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DNA was synthesized starting from 500 ng of total RNA in a volume of 20 l, using GoScript Reverse

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Transcription System (Promega, Madison, WI, USA) plus random hexamers and oligo (dT) primers.

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Before the quantitative gene expression analysis, primers specificity was tested through conventional

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PCR on the leaf and root tissues. Successful amplification products were sent to BMR Genomics

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(Padova, Italy) for Sanger sequencing. The resulting sequences were aligned with the original contigs

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derived from the bioinformatic analysis, to verify the specificity of the primers. Quantitative PCR was

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performed on a C1000 Thermal Cycler associated with a CFX 96 Real Time System (BioRad,

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Hercules, CA, USA), using SsoAdvancedTM universal SYBR Green supermix (BioRad, Hercules, CA,

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USA) according to the manufacturers instructions. Amplification efficiency were determined by

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LinReg software (Ruijter et al. 2009, v. 11.0). Possible reference genes were evaluated with Bio-Rad

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CFX Manager software version 3.1. Five possible reference genes were considered (GAPDH, S24,

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UBQ10, 14-3-3 and Rpl7), and the best two were used simultaneously, to improve the reliability of the

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analysis. Reference gene stability was evaluated by means of the CFX Manager software and

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RefFinder online application (www.leonixie.com). GAPDH and S24 were selected as the best pair. In

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addition, this pair was chosen because the two genes belong to two different metabolic pathways.

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GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a part of the glycolysis cycle, while S24 is a

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ribosomal protein.

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2.7 Statistical analysis

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Data were analysed for statistical significance using SigmaStat 2.03. Statistically significant

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differences were highlighted by ANOVA, whereas the significance of correlations was inferred

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through the Pearson Product Moment Correlation test.

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3. Results

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3.1 Isolation and classification of nine CaAQP genes from C. arabica L.

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On the basis of the available databases of expressed sequences of C. arabica, the bioinformatic

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analysis allowed us to identify five full-length and four partial cDNAs encoding putative aquaporins.

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The predicted amino acid sequence of the coffee aquaporins and the known aquaporins sequences of

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Arabidopsis thaliana and Solanum tuberosum were used to perform a phylogenetic analysis (Fig. 1).

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Overall, the coffee aquaporins followed the phylogenetic pattern in a similar manner to their

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Arabidopsis and Solanum counterparts, belonging to the same MIP superfamily. On the basis of the

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phylogenetic analysis and amino acid sequence similarities, the coffee sequences were clustered into

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two distinct families, PIP (comprising 4 transcripts) and TIP (5 sequences). The coffee PIP family was

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further divided into two subfamilies, PIP1 (2 transcripts) and PIP2 (2 transcripts), while TIPs were

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divided into three subfamilies i.e. TIP1 (3 transcripts), TIP2 (1 transcript), and TIP4 (1 transcript). A

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systematic nomenclature was proposed for coffee aquaporins, and the proposed gene names, accession

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numbers and subfamily classifications are summarized in Table 1. All selected sequences were aligned

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and for each one, specific PCR primers were designed on the most conserved and represented areas

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(Tab. 2).

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The end-point PCR analysis revealed a single band indicating successful and specific

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amplification of the target sequence (data not shown). Only the PCR of the Ca-TIP1;3 gene did not

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show any band, so this primer pair was discarded. Furthermore, CaTIP1;2 showed a single band in the

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root sample, while no amplification was apparent in the leaf sample, indicating a possible tissue

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specificity for the root. As a further control, the resulting sequences from Sanger sequencing of the

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PCR amplification products were aligned with the original sequences, confirming that all primer

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successfully amplified the target transcripts.

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3.2 Physiological responses and aquaporin expression to light

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In a first experiment, leaf and root hydraulic conductance values were measured at pre-dawn and

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midday to highlight eventual daily and/or irradiance-induced changes in plant hydraulic properties. At

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pre-dawn, photosynthetically active radiation in the greenhouse was less than 5 mol m-2 s-1, and

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peaked to about 450 mol m-2 s-1 at midday. The KR and KL values recorded at the two daytimes are

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reported in Fig. 2. Data analysis did not reveal any statistically significant differences between pre-

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dawn and midday values of both parameters (t-test, p<0.05).

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Total RNA was extracted from root and leaf samples collected from the same plants used for

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hydraulic measurements, and aquaporin expression was quantified. In a RT-qPCR assay using the

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Cq method, it was possible to compare the expression levels of aquaporins at pre-dawn and midday.

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The results are reported in Fig. 3. In root tissue (Fig. 3a), the expression levels of CaPIP1;1, CaPIP1;2,

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CaTIP2;1, CaTIP1;2 were significantly higher at midday than at pre-dawn. At the leaf level (Fig. 3b),

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the main differences in expression between pre-dawn and midday were observed for CaPIP2;1 that

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decreased at midday compared to pre-dawn, and CaTIP4;1 that showed an opposite pattern.

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3.3 Physiological responses and changes in aquaporin expression under water stress

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In the water stress experiment, KL and KR (Fig. 4) were measured in three different plant groups i.e.

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well-irrigated controls (100) and two drought stress levels (50 and 25). The root hydraulic conductance

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significantly decreased at the most severe stress level (25) compared to controls, while mild water

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stress (50) had apparently no impact on KR. On the contrary, leaf hydraulic conductance appeared to

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progressively decline from control to stressed groups, although differences were not statistically

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significant (Fig. 4).

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In the drought stress experiment, leaf conductance to water vapour (gL, mmol m-2 s-1) (Fig. 5a)

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and leaf water potential (L, MPa) were also measured, and related data are presented in Fig. 5b. Data

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analysis of gL showed statistically significant differences between the control group and severely

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stressed plants, as well as between the two drought stress groups. Data analysis of L showed

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statistically significant differences between control group and the drought stress groups, while there

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were no differences between the two drought stress groups.

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Total RNA was extracted from leaf and root tissue sampled from both controls and water

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stressed plants previously measured for their hydraulic traits, and aquaporin expression was quantified.

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The results are shown in Fig. 6, where the control group (100) has an expression value = 1, and the

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values of the drought stress groups are expressed as relative to the control. For each specific

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aquaporin, different letters denote statistically significant differences in expression level among the

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three groups (one-way ANOVA, Tukey test, p<0.05).

In root tissue, aquaporins gene expression decreased as the drought stress severity increased.

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Only CaTIP1;2 showed a different trend, in that expression of this aquaporin in the mild drought

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treatment was higher than in both control and severe drought stress groups. In the leaf, the expression

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of CaPIP1;2, CaPIP2;1, CaPIP2;2 showed a decrease at increasing water stress. The expression of

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CaTIP1;2 did not show significant changes between the three groups. The values of CaPIP1;1,

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CaTIP1;1, and CaTIP4;1 decreased in the mild drought stress group, but increased in the severe

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drought treatment compared to the controls.

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3.4 Correlations

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Relative changes in water status and hydraulics experienced by plants under water stress conditions

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were plotted versus the corresponding aquaporins' expression levels, in order to highlight eventual

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correlations. All values were expressed as relative to the controls. Significant correlations are reported

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in Figs. 7 and 8. Positive correlations were found between relative leaf hydraulic conductance and

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relative expression level of CaPIP2;1, CaPIP2;2 and CaPIP1;2, while negative correlations emerged

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between relative leaf water potential (L) and relative expression of CaPIP2;1, CaPIP2;2 and

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CaPIP1;2. No significant correlation was observed between changes in root hydraulic properties and

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relative expression levels of the different aquaporin isoforms.

4. Discussion

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We identified nine putative aquaporins in C. arabica, and some of them showed significant changes in

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their expression levels according to environmental factors to which plants were exposed, thus

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confirming our first hypothesis. Our second hypothesis found only partial support, in that plant

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hydraulic conductance apparently did not change from pre-dawn to midday, despite significant

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changes in the expression level of some aquaporin isoforms. Finally, in partial support to out third

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hypothesis, root hydraulic conductance was reduced by drought stress, in correlation with down-

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regulation of the expression of some aquaporin isoforms. These findings are in line with the major

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roles played by aquaporins in the regulation of plant water balance and specifically in drought stress

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responses (Moshelion et al. 2014). Taking into account the economical importance of C. arabica and

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its predicted vulnerability to ongoing climate changes (Davis et al. 2012), information gathered in this

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study might represent an important basis for selection of coffee genotypes with enhanced resistance to

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environmental stresses.
Due to the absence of a complete C. arabica trascriptome, it was possible to identify only nine

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aquaporin sequences. This is a step forward with respect to the four sequences identified in the same

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species by Santos and Mazzafera (2013), but remains a surprisingly low number when compared to

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model species such as Arabidopsis thaliana (L.) Heynh. (35 aquaporins; Johanson et al. 2001), Zea

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mays L. (31; Chaumont et al. 2001), Oryza sativa L. (33; Sakurai et al. 2005), Solanum tuberosum L.

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(41; Venkatesh et al. 2013). Hence, it is very likely that several other aquaporin sequences remain to

324

be identified in coffee, and the recent release of the C. canephora genome (Denoeud et al. 2014)

325

represents a promising starting point. The identified CaAQP genes were assigned to two families, PIPs

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and TIPs, on the basis of phylogenetic analysis (Fig. 1). Besides confirming the presence of

327

aquaporins in coffee, the present study showed that one of these (CaTIP2;1) is probably specifically or

328

predominantly expressed in the root tissues. The predominant expression of some aquaporin isoforms

329

in the root was already reported for other plant species such as AtPIP1;1 and AtPIP2;2 in A. thaliana,

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(Alexandersson et al. 2005) and TIP1;1, TIP2;3, TIP3;1, TIP3;2 and TIP5;1 in S. tuberosum

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(Venkatesh et al. 2013).

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Starting from aquaporin identification, we designed experiments aimed at correlating water

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status and hydraulic efficiency of coffee plants with the relative expression levels of different genes

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encoding for aquaporins. To this aim, we focused our attention on the two major environmental factors

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influencing both plant water status and aquaporin expression i.e. light (Lopez et al. 2013) and water

336

availability (Almeida-Rodriguez et al. 2010). In the light experiment, both KL and KR were found to

337

be similar when measured either at pre-dawn or at midday, suggesting that no diurnal up-regulation of

338

plant hydraulic efficiency occurs in coffee, in contrast with previous reports on different species

339

(Lopez et al. 2003; Nardini et al. 2005b). The quantitative gene expression (RT-qPCR) data revealed

340

that most of the aquaporin genes had similar expression levels at pre-dawn and midday in leaf tissues,

341

while some aquaporins increased in abundance during the day in root tissue. In particular, the

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expression of CaPIP2;1, CaPIP2;2, CaTIP4;1 and CaTIP1;1 in the root tissue was not statistically

343

different between pre-dawn and midday (t-test, p<0,05). However, the relative expression levels of

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CaPIP1;1, CaPIP1;2, CaTIP2;1 and CaTIP1;2 significantly increased during the day. These changes at

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the molecular level may suggest that indeed some aquaporin genes respond to changes in light

346

intensity or are regulated by the circadian clock, but these changes are apparently not related to KR.

347

This result is somehow unexpected on the basis of recent literature, where short-term changes in root

348

hydraulics are often reported to be induced by changes in aquaporins expression (Laur and Hacke

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2013). In fact, root hydraulic conductance is known to change diurnally with a peak value when

350

transpiration is maximal, and these variations are generally correlated with PIP transcript and protein

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abundance (Lopez et al. 2003). Maggio and Joly (1995) showed that application of mercury chloride

352

(an aquaporin inhibitor) reduced the hydraulic conductivity of tomato roots by about 60%,

353

demonstrating a correlation between aquaporins' functionality and water transport at the root level. In

354

tobacco, antisense-mediated reduction of NtAQP1 abundance reduced the root hydraulic conductivity

355

by 55% compared with controls (Siefritz et al. 2002). In A. thaliana, the silencing of AtPIP2;2

356

expression reduced the root hydraulic conductivity by 14% (Javot et al. 2003). The finding that some

357

coffee aquaporins change their expression level during the day, while the root hydraulic conductance

358

remains unchanged, suggests that their function is not strictly related to water transport, and the

359

identification of their possible roles deserves future investigations.

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In different crop species, Tsuda and Tyree (2000) showed that leaf hydraulic conductance (KL)

361

increases during the day in response to the increased water demand due to stomatal opening, and

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similar results were obtained in woody plants (unapuu and Sellin 2013). Successive studies

363

demonstrated that the variations in KL may be caused by changes in aquaporins activity (Lopez et al.

364

2013). This suggests that aquaporins act to promote water transport into leaf tissues when transpiration

365

is maximal (Johnson et al. 2014), although direct evidence for this phenomenon is scarce, as diurnal

366

changes in KL and aquaporin expression have been measured in only a few species. In addition to the

367

studies by Tyree et al. (2005), Cochard et al. (2007), and Lopez et al. (2013), Nardini et al. (2005b)

368

showed that sunflower KL changes according to day/night cycles, with values about 30-40% higher

369

during the day than at night. Evidence for a direct role of PIP2 in variable leaf hydraulic conductance

370

in response to light in walnut was suggested by Cochard et al. (2007), while Secchi and Zwieniecki

371

(2013) reported decreased leaf hydraulic conductance in poplar leaves upon down-regulation of PIP1.

372

However, Kaldenhoff et al. (2008) showed that leaf hydraulic conductance did not differ between

373

controls and NtAQP1 or NtPIP2 antisense plants. Martre et al. (2002) found no differences in leaf

374

hydraulic conductance between wild-type A. thaliana and double antisense plants with reduced

375

expression of PIP1 and PIP2. In our case, the expression of CaTIP4;1 increased during the day in the

376

leaves, with no apparent effect on KL, again suggesting a functional role different from water transport

377

for this aquaporin. Clearly, more physiological studies using aquaporin knockouts are needed to

378

establish the role that aquaporins play in the regulation of leaf hydraulic conductance in different

379

species, including C. arabica.

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The lack of hydraulic responses to light intensity recorded in coffee might not be surprising

381

from an adaptive point of view, considering that the species is original from shaded habitats where

382

light-driven modulation of plant hydraulics would probably confer no competitive advantage (Araujo

383

et al. 2008). Indeed, KL values recorded in our plants as well as in other studies on coffee (Gasc et al.

384

2004; Martins et al. 2014; Nardini et al. 2014) were low when compared with several other woody

385

species (Nardini and Luglio 2014) and close to values typically recorded in shade-adapted plants

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(Nardini et al. 2005a). However, coffee can be exposed to drought stress even in its native habitat. In

387

other plant species, such as A. thaliana, S. tuberosum, and Malus sp, some of the plant responses to

388

drought stress are controlled at the molecular level by aquaporins (Johanson et al. 2001;

389

Alexandersson et al. 2005; Venkatesh et al. 2013; Laur and Hacke 2014). In our study, the values of L

390

progressively decreased from controls to severely water stressed plants, confirming that irrigation

391

volumes supplied were effective in exposing the three groups to different water stress levels. The

392

decrease of L is known to affect stomatal aperture, as a first plant response to water stress. Stomatal

393

closure reduces leaf conductance to water vapour and transpiration, thus helping plants to prevent or

394

delay cellular dehydration. In our experiment, gL significantly decreased only in severely drought

395

stressed plants with respect to controls, suggesting that coffee plants did not reduce leaf conductance

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to water vapour under mild water stress condition. This suggests that the species has a moderate

397

tolerance to drought stress, as also suggested by observed changes of leaf and root hydraulic

398

conductance values.

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Water stress is known to impact both stomatal aperture and plant hydraulic properties

400

(Cochard et al. 2002). Mechanisms underlying modulation of plant hydraulic conductance under

401

drought stress are still not well understood (Javot et al. 2002; Nardini et al. 2011), but likely involve

402

several modifications of both xylem efficiency and membrane permeability. In the case of coffee

403

plants analysed in this study, root hydraulic conductance slightly increased under mild drought stress,

404

and then drastically declined under severe drought. Leaf hydraulic conductance decreased as well from

405

controls to severely water stressed plants, although differences recorded were not statistically

406

significant. Tyerman et al. (2002) and Galmes et al. (2007) showed that drought-induced changes in

407

hydraulic conductance might be influenced by aquaporins, which can help plants to maintain

408

homeostasis of water balance under water limitation. Using antisense techniques, it was shown that

409

PIPs can play an important role during the early phase of water stress, by acting on root water

410

transport, or during recovery from water stress, by favouring water mobilization in dehydrated leaves

411

(Siefritz et al. 2002) and/or refilling of embolized conduits (Laur and Hacke 2014). In coffee root

412

tissues, the aquaporin gene expression steadily decreased under water stress, except for CaTIP1;2. The

413

expression level of this aquaporin paralleled the trend of root hydraulic conductance, i.e. an increase in

414

the mild water stress group compared to the other groups. This might suggest an early compensation

415

response to mild drought in coffee, based on aquaporin-mediated enhancement of root hydraulic

416

conductance.

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At the leaf level, different trends of expression were observed for the different putative

418

aquaporins. In particular, CaPIP2;1, CaPIP2;2 and CaPIP1;2 showed a significant decrease of the

419

expression values from the control group to 25 drought stress group. The opposite trend was observed

420

in CaPIP1;1, CaTIP4;1 and CaTIP1;1, showing a statistically significant increase under severe water

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stress with respect to the other groups. In particular, there was a decrease from control group to 50

422

group, like for the other aquaporins, but under severe drought the gene expression value was higher

423

than in both control and 50 groups. Finally, in the case of CaTIP1;2, the aquaporin most expressed in

424

leaves, expression values showed no differences between the three treatments. The CaPIP2;1,

425

CaPIP2;2, CaPIP1;2 and CaTIP1;2 showed the same trend in the root and leaf tissue, indicating that

426

they probably have the same role in different tissues. Instead, CaPIP1;1, CaTIP4;1 and CaTIP1;1

427

showed different trends indicating that these isoforms were differentially regulated on the basis of the

428

specific tissue localization. The increase of these aquaporins during severe drought stress may be

429

related to processes of leaf hydraulic recovery (Laur and Hacke 2014), and might explain why leaf

430

hydraulic conductance showed no statistically significant differences between the three groups.

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The expression level of the aquaporins constantly decreasing in the three different groups was

432

correlated with the values of KR, KL and L. This might suggest that CaPIP2;1, CaPIP2;2 and

433

CaPIP1;2 have an influence on these physiological parameters. Our data would be in accordance with

434

the fact that different tissue-specific expression of AQPs in response to drought have been reported in

435

other plant species, like A. thaliana, S, tuberosum, Malus sp and Camellia sinensis (Venkatesh et al.

436

2013; Alexandersson et al. 2005; Liu et al. 2013; Yue et al. 2014). Liu et al. (2013) compared the

437

aquaporins gene expression in two species of Malus, i.e. the drought-sensitive M. hupehensis and the

438

drought-tolerant M. prunifolia, suggesting that differences in expression levels may be related to the

439

ability to respond to water stress.

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In a recent review Moshelion et al. (2014) highlighted the links between aquaporin activity

441

and crop water-use efficiency and yield under drought conditions. Apparently, differential regulation

442

of different aquaporins might allow some plants to switch from an anisohydric strategy maximizing

443

net assimilation and growth under mild drought, to a marked isohydric strategy favoring water saving

444

at the expense of productivity under severe water stress (Zhang et al. 2011). The transient increase and

445

the subsequent drop of KR in coffee plants subjected to mild and severe drought, respectively,

446

paralleled by similar changes in expression levels of some aquaporins isoform, might suggest that this

447

strategy can be achieved also by coffee plants, and selecting for genotypes where this functional trait is

448

maximized might turn out to be an interesting strategy to assure coffee productivity/survival under

449

future scenarios of increased intensity/severity of drought events.

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In conclusion, our study showed that some of the identified putative coffee aquaporins might

451

play important roles in the response of this species to water stress. However, it has to be noted that

452

correlations between aquaporins expression and root/leaf hydraulics does not allow to conclude about

453

the underlying mechanistic relationships, and the role of these proteins in regulating coffee plant

454

hydraulics deserves further studies. In particular, future work is needed to identify other aquaporin

455

isoforms in C. arabica, confirm their water-channel nature, and highlight their possible roles in

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456

modulating plant drought tolerance. These studies are urgently needed in order to identify possible

457

molecular targets for selection of coffee genotypes better adapted to future global-change drought

458

scenarios in the cultivation areas.

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5. Acknowledgements

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M. Miniussi was supported by EU and Regione Friuli Venezia Giulia (Fondo Sociale Europeo,

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Programma Operativo Regionale 2007-2013) in the frame of the project S.H.A.R.M. (Supporting

464

Human Assets of Research and Mobility).

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trees is detrimental to recovery from embolism. Plant Physiology 164:1789-1799

Sevanto S, Mcdowell NG, Dickman LT, Pangle R, Pockman WT (2014) How do trees die? A test of the

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hydraulic failure and carbon starvation hypotheses. Plant, Cell and Environment 37:153-161
Siefritz F, Tyree MT, Lovisolo C, Schubert A, Kaldenhoff R (2002) PIP1 plasma membrane

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aquaporins in tobacco: from cellular effects to function in plants. Plant Cell 14:869-876
Talbot JM (2004) The political economy of the coffee commodity chain. Rowman & Littlefield

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Publishers, Inc., New York.

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vulnerability in three contrasting coffee (Coffea arabica) cultivars. Tree Physiology 20:159-168

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Tyree MT, Nardini A, Salleo S, Sack L, El Omari B (2005) The dependence of leaf hydraulic

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conductance on irradiance during HPFM measurements: any role for stomatal response? Journal of

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Experimental Botany 56:737-744

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tuberosum aquaporins. Plant Physiology and Biochemistry 73:392-404

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expression analysis of tea plant aquaporin (AQP) gene family. Plant Physiology and Biochemistry

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83:65-76
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arid Vitis vinifera L. cv. Merlot vineyard: direct effects of hydraulic properties and indirect effects

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of canopy leaf area. Tree Physiology 32:262-279

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Gene
Ca-PIP2;1
Ca-PIP2;2
Ca-PIP1;1
Ca-PIP1;2
Ca-TIP4;1
Ca-TIP2;1
Ca-TIP1;1
Ca-TIP1;2
Ca-TIP1;3

Nucleotide database
accession number*
LM654169
LM654170/GAJT01000002
LM654171/GAJT01000001
LM654172
LM654173
LM654174
LM654175
LM654176/GAJT01000004
LM654177

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Subfamily
classification
PIP2
PIP2
PIP1
PIP1
TIP4
TIP2
TIP1
TIP1
TIP1

RI
PT

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Table 1: Proposed nomenclature for identified coffee aquaporin genes and related accession numbers

649

and subfamily classification. *The second accession code points to related sequences described by

650

Santos and Mazzaferra (2013).

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651
FORWARD PRIMER
ATGTCACATACGGCGGAG
TTCTACAACAGGTACGGT
AAGGGATTTGAGAAGGGA
GGTGCTGAAATTGTTGGT
AAGGCGTGATAATGGAGA
ACACATAACGTTGCCTCA
AGCATTTTCCCTTTCATCC
GTGTGGGATGCGTTTATT
CTTCTCAAACTCGCTACC

REVERSE PRIMER
TTGGGATCAGTGGCAGAG
TCTGGCATTTCTCTTGGG
GAAGAGGAGCCAAAATAG
AAAAACACAGCGAACCCA
TTGCACCAACAACTAGCC
CTCCAACAATGAACCCAA
AGCACCTACAATGAAACC
CGCCACAATCAAACCAAT
CCCTTCTTGGGATCAACT

652

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Table 2: Sequences of primers used for quantitative Real-Time PCR.

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GENE
Ca-PIP2;1
Ca-PIP2;2
Ca-PIP1;1
Ca-PIP1;2
Ca-TIP4;1
Ca-TIP2;1
Ca-TIP1;1
Ca-TIP1;2
Ca-TIP1;3

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Figure legends

655

Fig. 1: Phylogenetic analysis of aquaporin sequences of C. arabica and all the known aquaporin of

656

Arabidopsis thaliana and Solanum tuberosum. Peptide sequences were aligned using Muscle sequence

657

alignment program and the phylogenetic tree was constructed using Maximum Likelihood method as

658

detailed in Material and methods. The Uniprot accession name is indicated for each protein. The name

659

of each subfamily is indicated next to the corresponding subfamilies. Families without an analyzed C.

660

arabica representative were collapsed.

661

Fig. 2: Means values ( SD) of root and leaf hydraulic conductance scaled by total leaf surface area as

662

measured in coffee plants at pre-dawn (black column) or at midday (grey column). n.s.: not significant.

663

Fig. 3: Relative expression levels of genes encoding putative aquaporins as measured in roots (a) and

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leaves (b) of coffee plants sampled at pre-dawn (black) or midday (grey), as obtained by Real Time

665

analysis ((Ct) method). Asterisks indicate a statistically significant difference (t-test, p<0.05).

666

Fig. 4: Mean values ( SD) of root and leaf hydraulic conductance scaled by leaf surface area, as

667

measured in coffee plants either well irrigated (100) or subjected to progressively more intense

668

drought stress (50 and 25, see text for details about experimental treatments). Different letters indicate

669

statistically significant differences among groups (p<0.05).

670

Fig. 5: Mean values ( SD) of leaf conductance to water vapor (a) and leaf water potential (b), as

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measured in coffee plants either well irrigated (100) or subjected to progressively more intense

672

drought stress (50 and 25, see text for details about experimental treatments). Different letters indicate

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statistically significant differences among groups (p<0.05).

674

Fig. 6: Relative expression levels of genes encoding putative aquaporins, as measured in roots (a) and

675

leaves (b) of coffee plants either well irrigated (100) or subjected to progressively more intense

676

drought stress (50 and 25, see text for details about experimental treatments). Different letters indicate

677

statistically significant differences among groups (p<0.05).

678

Fig. 7: Relationships between relative leaf hydraulic conductance values and relative expression levels

679

of three genes encoding putative aquaporins, as measured in leaves of coffee plants either well

680

irrigated (100) or subjected to progressively more intense drought stress (50 and 25, see text for details

681

about experimental treatments). Correlation coefficients (r) and P values are also reported.

682

Fig. 8: Relationships between relative leaf water potential and relative expression levels of three genes

683

encoding putative aquaporins, as measured in leaves of coffee plants either well irrigated (100) or

684

subjected to progressively more intense drought stress (50 and 25, see text for details about

685

experimental treatments). Correlation coefficients (r) and P values are also reported.

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Figure 1

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689

Figure 2

0.8
0.6
0.4
0.2

3.5

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691

26

3.0
2.5
2.0
1.5
1.0
0.5
0.0

0.0

690

n.s.

SC

1.0

(a)

Leaf hydraulic conductance,

-1 -1
-2
mmol MPa s m

n.s.

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Root hydraulic conductance,

-1 -1
-2
mmol MPa s m

1.2

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pre-dawn
midday

(b)

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692

Figure 3

(a)

*
*

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PIP
Ca

27

(b)

EP

Relative Normalized Expression

leaf pre-dawn
leaf midday

694

RI
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0
5

693

M
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Relative Normalized Expression

root pre-dawn
root midday

1;1

PIP
Ca

1;2

PIP
Ca

2;1

PIP
Ca

2;2

1;1
TIP
a
C

1;2
TIP
a
C

4;1
TIP
a
C

696

697

EP
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Leaf hydraulic conductance,

mmol MPa-1 s -1 m -2

28
1.0

0.5

0.0

100

100
50

50

Treatment
25

25

RI
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1.5

SC

2.0

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Root hydraulic conductance,

mmol MPa-1 s -1 m -2

695

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Figure 4

2.5

ab
b

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Figure 5

(a)
a

250

200

150

100

50

100

-0.4
-0.6

-0.8
-1.0

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Leaf water potential,

MPa

-0.2

-1.2
-1.4

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700

29

b
b

(b)

EP

-1.6

699

25

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0.0

50

100

RI
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Leaf conductance to water vapor,

mmol m -2 s -1

300

SC

698

50
Treatment

25

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701

Figure 6

1.6

(a)
b
a

a
1.0

a
b

b
c

0.8
0.6

0.4

0.2

0.0
1;1

PIP
Ca

1.8

1;2

PIP
Ca

2;1

PIP
Ca

1.4

a
a

0.8
0.6

0.4

0.2
0.0

1;1

AC
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PIP
Ca

703

30

PIP
Ca

1;2

PIP
Ca

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1.0

(b)

1.2

1;1
1;2
2;1
4;1
TIP
TIP
TIP
TIP
a
a
a
a
C
C
C
C

EP

Relative Normalized Expression

1.6

2;2

M
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PIP
Ca

702

RI
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1.2

SC

Relative Normalized Expression

1.4

100_R
50_R
25_R

c
b
b

bb

2;1

PIP
Ca

2;2

1;1
1;2
4;1
TIP
TIP
TIP
a
a
a
C
C
C

100_L
50_L
25_L

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704

Figure 7

705

1.1

100

r= 0.99
P=0.11

1.0
0.9

RI
PT

0.8
0.7
0.6

50

0.5
0.4 25

0.2

0.4

0.6

SC

0.3
0.8

1.0

1.2

Relative CaPIP2;1 Expression

0.9
0.8
0.7
0.6

100

M
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r= 0.99
P=0.03

1.0

50

0.5

25

0.4

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R elative leaf hydraulic co nd uctance

1.1

0.3

0.2

0.4

0.6

0.8

1.0

1.2

Relative CaPIP2;2 Expression

1.1

r= 0.99
P=0.04

100

EP

1.0
0.9
0.8

AC
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0.7
0.6

50

0.5
0.4

25

0.3
0.0

0.2

0.4

0.6

0.8

Relative CaPIP1;2 Expression

706
707

31

1.0

1.2

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708

Figure 8

2.2

r= -0.97
P= 0.17

25
2.0
1.8

1.4
1.2
1.0

100

0.8
0.2

0.4

0.6

0.8

1.0

1.2

SC

Relative CaPIP2;1 Expression

r= -0.99
P= 0.03

25
2.0

50

1.8

M
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Relative leaf water potential

2.2

1.6
1.4
1.2

100

1.0
0.8
0.2

0.4

0.6

0.8

1.0

1.2

TE
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Relative CaPIP2;2 Expression

2.2

25

2.0
1.8

r= -0.99
P= 0.09

50

EP

1.6
1.4
1.2

100

AC
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1.0
0.8

0.0

709
710

32

RI
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50

1.6

0.2

0.4

0.6

0.8

1.0

Relative CaPIP1;2 Expression

1.2

Author contribution statement

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AN and AP conceived and designed the research. All authors contributed to conduct experiments and to
analyse data. MM and LDT wrote the first draft of the manuscript. AN and AP revised and finalized the

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manuscript. All authors read and approved the manuscript.