Genome-wide analysis of aquaporin gene family and their responses to water-deficit stress conditions in cassava

Accepted Manuscript Genome-wide analysis of aquaporin gene family and their responses to water-deficit stress conditions in cassava Pattaranit Putpeerawit, Punchapat Sojikul, Siripong Thitamadee, Jarunya Narangajavana PII:

S0981-9428(17)30360-1

DOI:

10.1016/j.plaphy.2017.10.025

Reference:

PLAPHY 5037

To appear in:

Plant Physiology and Biochemistry

Received Date: 8 September 2017 Revised Date:

13 October 2017

Accepted Date: 26 October 2017

Please cite this article as: P. Putpeerawit, P. Sojikul, S. Thitamadee, J. Narangajavana, Genome-wide analysis of aquaporin gene family and their responses to water-deficit stress conditions in cassava, Plant Physiology et Biochemistry (2017), doi: 10.1016/j.plaphy.2017.10.025. 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.

ACCEPTED MANUSCRIPT Genome-wide analysis of aquaporin gene family and their responses to water-deficit stress conditions in cassava

Pattaranit Putpeerawit1,2, Punchapat Sojikul1, Siripong Thitamadee1, Jarunya Narangajavana1,2* Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand

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Center of Excellence on Agricultural Biotechnology (AG- BIO/PERDO-CHE), Bangkok, Thailand

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* Corresponding author:

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Dr. Jarunya Narangajavana

Department of Biotechnology, Faculty of Science, Mahidol University Rama 6 Rd., Rajthewee, Bangkok 10400, Thailand Tel: +662 201 5319; Fax: +662 354 7160

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E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Cassava (Manihot esculenta Crantz) is an important economic crop in tropical countries. Although cassava is considered a drought-tolerant crop that can grow in arid areas, the impact of drought can significantly reduce the growth and yield of cassava storage roots. The discovery of aquaporin molecules (AQPs) in plants has resulted in a paradigm shift in the understanding of plantwater relationships, whereas the relationship between aquaporin and drought resistance in cassava still

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remains elusive. To investigate the potential role of aquaporin in cassava under water-deficit conditions, 45 putative MeAQPs were identified in the cassava genome. Six members of MeAQPs, containing high numbers of water stress-responsive motifs in their promoter regions, were selected for a gene expression study. Two cassava cultivars, which showed different degrees of responses to waterdeficit stress, were used to test in in vitro and potted plant systems. The differential expression of all

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candidate MeAQPs were found in only leaves from the potted plant system were consistent with the relative water content and with the stomatal closure profile of the two cultivars. MePIP2-1 and MePIP2-10 were up-regulated and this change in their expression might regulate a special signal for

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water efflux out of guard cells, thus inducing stomatal closure under water-deficit conditions. In addition, the expression profiles of genes in the ABA-dependent pathway revealed an essential correlation with stomatal closure. The potential functions of MeAQPs and candidate ABA-dependent pathway genes in response to water deficit in the more tolerant cassava cultivar were discussed.

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Keywords: aquaporin; cassava; stomatal closure; water deficit

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Introduction Drought is one of the abiotic stresses that affects plants on molecular to morphological levels. The two major effects of drought stress on plants are impaired growth and reduced plant yield, a measure of productivity. The severity and duration of stress lead to plants to adjust and adapt via changes at the molecular, cellular, biochemical and physiological levels for theirs survival (Osakabe et al. 2014;

plants to acute water deficit to prevent water loss during transpiration.

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Yamaguchi-Shinozaki et al. 2006). When drought conditions occur, stomatal closure is the first response of

In Arabidopsis thaliana, the regulation of drought-responsive signaling has been evaluated by inducing abscisic acid (ABA)-dependent and ABA-independent pathways. Histidine kinase1 (HK1), which is a receptor on the plasma membrane, has been hypothesized to function as an osmosensor. The athk1

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mutants demonstrated decreased concentrations of endogenous ABA and down-regulation of ABAresponsive genes under water-deficit conditions (Wohlbach et al. 2008). NCED3 (9-cis-epoxycarotenoid dioxygenase 3), a key enzyme in ABA biosynthesis, is rapidly induced to generate endogenous ABA under

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drought conditions (Barrero et al. 2006; Iuchi et al. 2001). To close the stomata to prevent water loss, ABA is bound to with pyrabactin (PYR) to inhibit the function of the protein serine/threonine phosphatase 2C (PP2C) family (Umezawa et al. 2009). Without PP2C inhibition, the Snf1-related protein kinase 2 (SnRK2.6 or OST1), which is the kinase protein that is involved in the ABA-signaling complex, induces the factors for stomatal closure regulation, including transcription factors, slow anion channels (SLACs), potassium transporters (KAT1, KUP6 and GORK) and especially, water transporter aquaporins (Grondin et al. 2015; Osakabe et al. 2013; Sato et al. 2009; Sirichandra et al. 2010). KAT1 and KUP6 are

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phosphorylated for K+ uptake inhibition in guard cells, while GORK is induced to eliminate K+ ions. Anion and K+ efflux out of the guard cells decreases the water potential in the outer membrane to a value lower than that in the inner membrane. The water in guard cells crosses the cell membrane, from the inner membrane to the outer membrane, resulting in a reduction in guard cell turgor and stomatal closure, which

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causes plant tolerance to drought (Farooq et al. 2009; Livne & Vaadia 2012; Osakabe et al. 2014). Aquaporins (AQPs) are a major intrinsic protein family that transports water across the cell membrane via a cell-to-cell pathway. AQPs regulate the bidirectional movement of water in response to

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osmotic gradients (Luang & Hrmova 2017). In addition to water transporter, AQPs also transport some uncharged solutes, such as carbon dioxide, hydrogen peroxide, and glycerol. The six membrane-spanning of AQPs filter water molecules from two specific filter regions, the NPA motif and aromatic/Arg, by size exclusion. Based on sequence similarity, the aquaporin protein family is divided into 5 subfamilies: plasma intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin26-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs) and X intrinsic proteins (XIPs). The expression of individual AQP members varies considerably conditions depending on the growth development, tissue specificity, species and variety of plant and especially the level of stress (Chaumont & Tyerman 2014; Maurel et al. 2008; Šurbanovski et al. 2013). Various publications have reported that aquaporin expressions induces plant tolerance to environmental stresses. Recently, the overexpression of JcPIP2-7 and JcTIP1-3 from Jatropha curcas in

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Arabidopsis thaliana resulted in transgenic plants able to withstand water-deficit conditions and recover after re-watering compared with control plants (Khan et al. 2015). The ortholog of a banana aquaporin gene, MaPIP1-1, in Arabidopsis thaliana increased primary root elongation, the amount of root hairs and the survival rate under drought conditions (Xu et al. 2014). Moreover, plants with a mutation in AtPIP2-1 showed stomatal closure defects in response to ABA (Grondin et al. 2015). Based on its ability to rapidly

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facilitate water uptake up to 3x109 water molecules per second through lipid membranes (Luang & Hrmova 2017), aquaporin has a potential function in plant responses under water-deficit conditions.

Cassava is an important tropical crop and is a staple food for people in worldwide. Cassava is a plant that is enriched in carbohydrates and is used as a bio-renewable resource and for many applications in industrial processes. As cassava can grow in areas that are arid, where the soil is poor and the

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mechanization of farming is not implemented, the production of this crop has been increasing over time. Although cassava can grow in arid areas, the impact of drought stress also significantly affects the cassava biomass (Alves & Setter 2004a; El-Sharkawy et al. 2012; Vandegeer et al. 2013). To date, the correlation

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between aquaporin and drought stress responses in cassava has not been extensively studied. To investigate the role of cassava aquaporins under water-deficit conditions, aquaporin genes (AQPs) in the cassava genome were computationally identified. The putative cis-regulatory motifs available in the promoter region of each gene were analyzed. Two cassava cultivars that respond differently to water-deficit stress were selected to compare the expression profiles of candidate aquaporin genes and other ABA-responsive genes in in vitro and potted plant systems. The knowledge obtained from this study should provide us with a better understanding of aquaporin responses during water deficit and should be

Materials and methods

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used to facilitate further improvement of drought tolerance in cassava.

Plant materials and the water-deficit treatment

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Two cassava cultivars, Hanatee (HN) and Huay Bong 60 (HB60), were used in this study. HN is a domestic cultivar that contains low HCN and that generally grows in irrigated areas. HB60 contains more

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HCN and is grown extensively in arid areas. Plant growing conditions were determined using in vitro plantlet and potted plant systems. The in vitro plantlets were cultured on cassava basal medium (Murashige & Skoog medium supplemented with 2% sucrose and 0.6% agar; pH 5.6) in 8 oz. glass bottles under a long-day condition of 16 h-light/8 h-dark at 26±1oC and 300 µmol m-2s-1 of light intensity. To initiate water-deficit conditions, eight-week-old cassava plantlets were treated with 40% (w/v) PEG 6000 for 24 and 48 h (D24 and D48, respectively). The control group was not treated with 40% (w/v) PEG 6000 (C0). For potted plants, stem cuttings (30 centimeters) were cultivated in 14-inch soil pots and placed in a greenhouse with a temperature range of 26-30oC and a supplementary light intensity of 300 µmol m-2s-1. Plants under the control condition were well watered with 1 liter of tap water every day for 4 weeks (C0). Then, the drought stress condition was conferred by withholding water for 2 and 4 weeks (D2W and D4W, respectively). The fully expanded leaves from three biological replicates in two systems were separately

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collected at 9:00 am to determine leaf relative water content and stomatal closure in each condition. Leaf and root tissues were collected, frozen immediately in liquid nitrogen, and then stored at -80oC for gene expression analysis.

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Stomatal closure observations and relative water content (RWC) measurements The epidermal peel technique was performed to observe the effect of drought on stomata. The characteristics of guard cells and stomata were visualized by light microscopy (Olympus B202, Japan). To determine stomatal closure, three 0.5-mm2 random spots on each of the three biological replicates of expanded leaves were analyzed to count opened and closed stomata. The percentage of opened stomata was

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calculated from the ratio between opened stomata and total stomata counted ×100. The RWC measurement was performed to determine the leaf water status according to a previous report (Phookaew et al. 2014). The fully expanded leaf was cut into five 1-cm-diameter discs. The fresh weight (FW) of each disc was

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measured immediately after cutting. After that, the discs were rehydrated by floating them on water for 8 h at room temperature, and then, they were thoroughly dried for turgid weight (TW). Next, the leaf discs were oven dried at 80oC for 24 h for dry weight (DW). RWC (%) was calculated as (FW – DW)/(TW – DW) × 100.

Identification of MeAQPs and analysis of cis-regulatory elements in promoter regions The

whole

genome

sequences

of

the

cassava

cultivar

AM560-2

in

Phytozome

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(https://phytozome.jgi.doe.gov/pz/portal.html) was used in this study. The sequences of all Arabidopsis thaliana aquaporin genes, AtAQPs, as complete genome sequences, were blasted against nucleotide sequences of cassava. The scaffolds obtained from the blast results, containing the NPA motif were designated as aquaporins in cassava, or MeAQPs, and were named according to existing nomenclature

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(Diehn et al. 2015; Johanson et al. 2001; Lopez et al. 2012). The sequence alignment of amino acid sequences was constructed using ClustalOmega and a phylogenetic tree was then created with the MEGA7 program.

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The putative cis-regulatory elements in promoter regions were analyzed in order to select the

MeAQPs genes that might play an important role in the drought response in cassava. The 3,000 nucleotide sequences located upstream of the ATG sequence of each MeAQP gene were derived from Phytozome, and the cis-regulatory elements that are involved in drought, ABA and other responses were predicted using the NewPLACE program.

RNA isolation and reverse transcription Total RNA was separately extracted from leaf and root tissues of each condition using a ConcertTM Plant RNA Reagent Kit (Invitrogen). Contaminated genomic DNA was further eliminated using a DNAfreeTM Kit (Ambion). The RNA was reverse transcribed to first-strand cDNA using a High Capacity cDNA

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Transcription Kit (Applied Biosystems). Expression analysis of target genes by quantitative real-time PCR Based on qRT-PCR, the products of amplification (amplicons) are generally represented by SYBR

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green I fluorescent signals. To confirm the expression of candidate aquaporin and ABA-responsive genes, the primers used in qRT-PCR need to have high specificity. The melting or dissociation curves can be used to identify the specific product of genes of interest by gradually increasing the temperature. The denaturation of dsDNA leads to a decrease in the fluorescence signal. The single type of peak generated by each primer pair was observed and considered suitable for detection. Quantitative RT-PCR was performed

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in nine technical replicates using the CFX96 TouchTM Real-Time PCR Detection System with iTaq Universal SYBR Green Supermix (Bio-Rad). The 20 µL PCR reaction was prepared by mixing 1 µL of first stand cDNA, 10 mM each of forward and reverse primers, and dye. The PCR conditions for the gene

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expression studies was 95oC for 10 minutes as an initial step, followed by 40 cycles of 95oC for 30 sec and 57oC for 1 minute, and 18S rRNA was used as an internal control for normalization of the target gene detection level. The relative expression of target genes was determined by the comparative CT method (2∆∆CT

) and exhibited in a heat map using the RStudio program. Statistical analysis was performed using one-

way ANOVA (Duncan’s multiple-range test) to determine significant differences (p<0.05). Results

Classification of all aquaporin genes in the cassava genome and analysis of possible regulatory

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elements in their promoter regions

To identify and classify all aquaporin gene family members in the cassava genome, the scaffolds containing the NPA motif, typical for aquaporin genes, were retrieved from the cassava genome database (cultivar AM560-2) in Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html), and designated aquaporin

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genes. The 45 members of MeAQPs (Supplement Figure S1) were named according to existing nomenclature (Diehn et al. 2015; Johanson et al. 2001; Lopez et al. 2012) and classified into 5 groups, including PIP, TIP, NIP, SIP, and XIP with 14, 13, 10, 4 and 4 members, respectively (Figure 1).

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Consequently, the regulatory elements in each AQP promoter region were determined to select

candidate MeAQPs that might be involved in the response to water-deficit conditions. The 3-kb upstream region from ATG revealed 244 known cis-regulatory elements, of which 13% were motifs involved in responses to drought and ABA, 20% were development responsive, 23% were other-stress responsive and 44% were other motifs (Figure 2A). In this study, target MeAQPs genes involved in dehydration and ABA regulation were selected for a further gene expression study based on the presence of high numbers of drought-responsive cis-regulatory elements (over 90 elements). The MeAQPs that were reported to respond to drought treatment in cassava (Utsumi et al. 2012), as well as some genes in the ABA-responsive signaling pathways involved in stomatal closure under water deficit condition, were also selected for this study (Figure 2B). The specific primers for 2 groups of genes, aquaporin genes (PIP2-1, PIP2-5, PIP2-10,

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PIP1-3, TIP1-3 and TIP1-5) and ABA responsive genes (HK1, NCED3, PYR4, OST1 and GORK), were designed and validated for further gene expression analysis (Supplement Table S1). Two different cultivation systems were employed to test water-deficit effects on the two cassava cultivars The effect of water deficit was generally determined for leaf physiological changes such as

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wilting, stomatal closure and relative water content (RWC). In this study, in vitro culture and potted plants cultivations of cassava cultivars HN and HB60 were observed for their responses under water-deficit condition. PEG was used to treat in in vitro cultures to imitate water-deficit conditions, and the leaves of both cultivars exhibited clear wilting within 24 h (Supplement Figure S2). The RWC of the HN cultivar decreased to 49% at 24 h but then showed better performance of retaining water by increasing the RWC to

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67% at 48 h, while the RWC of HB60 decreased gradually to 35% from 24 h to 48 h (Figure 3A). When compared with the control plants, most of the stomata in both water-deficit-treated cultivars were completely closed at 24 h. It should be noted that there was a strong correlation between RWC and the

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percentage of stomatal closure in HN and HB60. However, at 48 h, 87% of stomata in HN returned to being open, whereas HB60 still tried to retain water by allowing only 7% of stomata to be opened (Figure 3B). In the potted plants, the water-deficit treatment was performed by withholding water for 4 weeks. The wilting of bottom leaves in HN began the 2nd-week after no watering, then the leaves gradually turned yellow and fell off by the 4th-week. In contrast, HB60 showed wilting of bottom leaves in 4th-week of the water deficit treatment, without any yellow or fallen leaves (Supplement Figure S3). During dehydration, the RWC of HN slightly decreased from 91 to 63%, while the RWC of HB60 had no significant change

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(Figure 4A). Furthermore, the stomatal closure status, shown in Figure 4C suggested that more than 90% of stomata in HN remained open under water-deficit treatment, while HB60 stomata were completely closed beginning at the 2nd-week of treatment (Figure 4B).

deficit conditions

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Expression of aquaporin genes and ABA-responsive genes in two cassava cultivars under water-

To analyze the role of aquaporin and ABA-responsive genes related to water deficit in HN and HB60, the expression of candidate genes in in vitro and potted plants of cassava were determined using

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qRT-PCR.

Expression of candidate genes in the cassava leaf under water-deficit conditions The water-deficit treatment was performed in two cultivation systems, in vitro and potted plants.

The expression of candidate genes in the leaf exhibited several patterns (Figure 5 and Supplement Figure S4, S5). In in vitro cassava plantlets, 4 out of 6 aquaporin genes revealed similar expression pattern between HN and HB60 cultivars after PEG treatment for 48 h (Figure 5A). The expression of PIP2-5, PIP2-10 and TIP1-5 was significantly decreased at 24 h and prolonged to 48 hr. Interestingly, sharp upregulation of PIP2-1 in HN at 24 h was 3-fold higher than that of HB60, and then, the expression of PIP2-1 decreased to control levels in both cultivars. PIP1-3 gene expression in HN was decreased in contrast to

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that in HB60, which gradually increased. The up-regulation of TIP1-3 in HN was constant from 24 h to 48 h, whereas in HB60, the expression slightly increased at 24 h and sharply increased 9-fold at 48 h. To investigate whether endogenous ABA, occurring under water-deficit stress conditions, may be involved in stomatal closure in in vitro plantlets, candidate ABA-responsive genes were analyzed. HK1, NCED3 and OST1 genes showed similar expression patterns between the two cultivars (HN and HB60).

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The expression of HK1 was down-regulated 5-fold at 24 h, while NCED3 was up-regulated over 35-fold under drought conditions. The down-regulation of OST1 was observed at 24 h, and then, it was upregulated at 48 h. On the other hand, the other two genes, PYR4 and GORK, had different expression patterns among the two cultivars under water deficit condition. PYR4 showed constant gene expression up to 48 h in HB60, while the down-regulation of PYR4 was observed at 24 h, and then, up-regulation was

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observed at 48 h in the HN cultivar. The 5-fold up-regulation of GORK was found in both cultivars at 24 h after PEG treatment; however, the high expression was prolonged to 48 h in HN, while down-regulation was found in HB60 (Figure 5A).

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To compare the candidate genes expression levels in leaf compared between in vitro and potted plant under water-deficit conditions, the HN and HB60 cultivars were subjected to a withholding water condition for 2 and 4 weeks (D2W and D4W). Interestingly, the expression profiles of candidate genes in HN and HB60 in potted plants were distinct from those previously observed in the in vitro condition (Figure 5B). Surprisingly, most of the AQP genes (PIP2-1, TIP1-3 and TIP1-5) in HB60 were induced since the 2nd week of treatment, while a decrease in the expression of these genes was found in HN. The similar expression patterns of aquaporin genes, sharply up-regulated at D2W and then down-regulated at

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D4W in both cultivars, was found only in PIP2-10. Interestingly, the PIP2-10 expression profile in potted plants was clearly different from that observed in in vitro plantlets. The expression of ABA-responsive genes was also determined in potted plants. Only the HK1 gene exhibited gene expression similar to the AQP genes, especially PIP2-5 and PIP1-3 (Figure 5B). These

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genes were down-regulated in HB60 after withholding water, whereas they were down-regulated at D2W but followed by up-regulated at D4W in HN. The NCED3 gene in both cultivars was significantly induced more than 15-fold after withholding water, similar to that previously observed in in vitro plantlets after

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PEG treatment. The OST1 gene was down-regulated at D2W and was up-regulated at D4W in HN, while the expression level was maintained at D2W and then gradually down-regulated at D4W in HB60. Furthermore, PYR4 encoding for an ABA receptor was down-regulated at D2W and decreased to no signal of expression at D4W in HN. On the other hand, PYR4 in HB60 decreased at D2W but then increased at D4W in response to drought. The expression of the GORK gene, involved in potassium efflux out of the guard cell, decreased at D2W but then sharply increased to 10-fold compared with the control at D4W in HB60. In contrast, in HN cultivar, 3-fold up-regulation was observed at D2W and down-regulation was observed at D4W (Figure 5B). Expression of candidate genes in the cassava root under water-deficit conditions

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To identify aquaporin genes involved in the response to drought in cassava, the expression of candidate AQP genes was determined in the root under two cultivation systems (Figure 6 and Supplement Figure S6, S7). The expression patterns of candidate genes in the cassava root showed a significant difference between in vitro and potted plants under water-deficit conditions. Most of the AQP genes in roots of in vitro plantlets were down-regulated after PEG treatment. In contrast, in the potted plants, the

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expression of AQP genes was up-regulated when water was withheld. PIP2-1, PIP2-5, PIP2-10, PIP1-3 and TIP1-5 were down-regulated in in vitro roots at 24 h to 48 h after PEG treatment, whereas only TIP1-3 was up-regulated. In the potted plant condition, the expression of PIP2-5 and PIP2-10 was down-regulated at D2W but was then up-regulation at D4W. The results of PIP2-1 in HN showed the same pattern with previous PIP members, but its expression level was increased in HB60. Interestingly, the expression level

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of PIP1-3 immediately increased up to 135-fold in HB60 at D4W. TIP1-3 and TIP1-5 presented similar patterns, for HN, down-regulation was observed from D2W to D4W, but for HB60, down-regulation was

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found at D2W, whereas up-regulation was found at D4W.

Discussion

Cassava is a shrub adapted to survive under prolonged drought (El-Sharkawy et al. 2004). The various cultivars of cassava demonstrate specific characteristics and adaptations to different types of ecology (Howeler et al. 2013). In this study, two cultivars of cassava with 2 different cultivation systems were used to demonstrate their physiological responses in relation to candidate gene expression under a water-deficit treatment. In in vitro cultivation, HN exhibited physiological responses that were considered

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water-deficit-tolerant compared with HB60. The stomata of HN kept opening, and the relative water content was recovered over a long duration of water stress, while the stomata of HB60 quickly closed even though the relative water content still decreased. These results suggested that HB60 is more sensitive to adapt to water-deficit stress, while HN might contain a special mechanism for its recovery against water-

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deficit treatment in the in vitro condition, which has high humidity. However, contrasting results were observed in potted plants. HN showed more sensitivity to water deficit than HB60. The stomata of HN remained open the whole time, which resulted in the decrease in relative water content, whereas HB60

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rapidly closed stomata to control or prevent water loss. These observations suggested that each cassava cultivar may encounter to drought stress by different mechanisms under different conditions. Generally, plants survive to various environmental stresses by evolving their physiological and

biological processes, such as stomatal closure and endogenous ABA induction (Osakabe et al. 2014; Yamaguchi-Shinozaki et al. 2006). One of the responses to these stresses, including drought, is epicuticular wax on plant leaf surfaces (Cameron et al. 2006; Seo & Park 2011). Several studies have reported that plants that accumulated higher amounts of wax, presented improved-drought tolerance (Yang et al. 2011). A previous study found high quantities of epicuticular wax covering and occluding stomatal pores in cassava fields (Zinsou et al. 2006). The physiological and morphological disorders involved in epicuticular wax accumulation have not been reported in in vitro plantlets. Many publications from the 20th century

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observed that plants in in vitro cultures lacked of leaf epicuticular wax and thus decreased water uptake through the roots (Ali-Ahmad et al. 1998). As the high water content of the medium in the closed system leads to high relative humidity in the culture vessels, the epicuticular wax and stomata did not control evaporation to the normal levels (Chen et al. 2004). Therefore, the response of HN and HB60 under in vitro condition might be affected by high humidity, causing their response to be different than that of potted

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plants. Nevertheless, the expression of aquaporin genes and ABA-responsive genes in this study might be involved in different responses in the two cultivation systems under water-deficit conditions. The correlation between the expression of some genes and physiological responses to keep sufficient relative water content in the plants suggested the important role of these genes in the drought stress response of cassava.

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Normally, water is transported from the root to shoot following the water potential. Aquaporin is a water transporter protein that facilitates water transport via a cell-to-cell pathway in response to osmotic gradients. There are several methods to study the function of aquaporins in plants. Studying of stomatal

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closure and the relative water content are simple and direct methods representing the activity of aquaporin in the leaf. Guard cells are symplastically isolated from their neighboring cells, implying that the regulation of transmembrane water movement is central to the control of their aperture/closure mechanism. The results of stomatal closure are a main part of water loss prevention in plants and certainly involve aquaporin expression and function. Moreover, a publication by Grondin et al. in 2015 showed that a lack of aquaporin member PIP2-1 in Arabidopsis thaliana leads to a defect in stomatal closure specifically in response to ABA. In this case, water in guard cells cannot efflux out to subsidiary cells or neighboring cells because

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PIP2-1 causes the stomata to stay open. Therefore, under water-deficit conditions, which cause plants to have higher ABA content, stomatal closure can represent the activity of aquaporins. Aquaporin genes have been identified in the genomes of approximate 14 plant species and are reported to be involved in drought tolerance (Maurel et al. 2008). However, the connection between drought tolerance and aquaporins in

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cassava has remained unknown.

In 2014, a number of dehydration-responsive genes in cassava were reviewed using a microarray (Utsumi et al. 2012), but the relation between water deficit and aquaporins was not clarified. To determine

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the potential functions of MeAQPs in response to water deficit, 45 putative MeAQP genes were retrieved from the cassava genome database and classified into five subfamilies (PIP, TIP, NIP, SIP and XIP). A total of 244 regulatory motifs were determined in the promoter regions of 45 MeAQPs members. Interestingly, the presence of dehydration-responsive element (DRE) and ABA-responsive element (ABRE) was found to be associated with osmotic stress. The expression of osmotic stress responsive genes was reported to be induced by ABA-dependent and ABA-independent pathways when osmotic stress damaged plants (Yoshida et al. 2014). ABRE is the most conserved cis-regulatory element in dehydrationinducible promoters in three plant species, Arabidopsis, rice and soybean (Maruyama et al. 2012). This element is bound with ABA-responsive element binding factors (AREB) for ABA-dependent pathway induction. On the other hand, DRE is bound to dehydration-responsive binding factor for ABA-independent

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pathway induction (Yoshida et al. 2014). It should be noted that others cis-elements involved in osmotic stress, such as MYBR, CRT and HSEs, were also observed in MeAQPs promoters. This result suggested that each aquaporin member in cassava is regulated by various factors, including drought and ABA. In addition to aquaporin, ABA signaling pathways are also of interest when studying the drought response in cassava. There were reports of the involvement of ABA in the regulation of plant hydraulic

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conductance and stomatal closure under drought conditions in various plant species (Fujita et al. 2011; Grondin et al. 2015; Mahdieh & Mostajeran 2009; Osakabe et al. 2014; Parent et al. 2009; Qian et al. 2015). Therefore, to elucidate the role of aquaporin gene expression in cassava under a water-deficit treatment, six candidate MeAQP genes and some genes in the ABA-responsive signaling pathways were selected for the present study. The expression of AQP genes under drought conditions has been reviewed in

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various plant species in which their expression profiles increase or decrease depending on the severity and duration of the stress (Claeys & Inzé 2013). Different tissues, especially the leaf and root, express different members of AQP genes (Prado & Maurel 2013). In this study, the expression of candidate AQP genes in

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cassava showed different patterns in the leaf and root among HN and HB60 cultivars and in two cultivation systems. When comparing the expression of aquaporin genes in in vitro and potted plants, the in vitro cultivation showed the same expression pattern for most MeAQP genes between both cultivars, while the expression patterns were different in potted plants. This result suggested that different water-deficit responses might be involved in the expression of AQP genes in potted plants, but less so in in vitro cultures, which were affected by high humidity.

Although most of the MeAQP genes in in vitro leaves exhibited the same expression pattern

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between HN and HB60, the expression levels of PIP2-1 and PIP1-3 showed different patterns in this regard. MePIP2-1 is 81% homologous to AtPIP2-1, which induces stomatal closure (Grondin et al. 2015). MePIP2-1 was up-regulated at 24 h and then down-regulated at 48 h. The up-regulation of PIP2-1 in the HN leaf might regulate a special signal for water efflux out of the guard cell, resulting in the stomata

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closing. In contrast, the decrease in the PIP2-1 gene inhibited the water efflux out of the guard cell, resulting in the stomata opening. Remarkably, the expression of PIP2-1 in HB60 showed the same pattern with HN, but the stomata still closed at 48 h. This result suggested that the defective stomatal closure in

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HB60 might be involved in the post-transcriptional regulation of PIP2-1. The down-regulation of PIP1-3, PIP2-5, PIP2-10 and TIP1-5 in the leaf might be involved in water loss prevention. Several studies demonstrated that the reduction of many AQP gene expression levels under stress led to reduced leaf hydraulic conductance (KLeaf) to keep water in the cell. The expression of TIP2-1 and PIP2-1 in Vitis vinifera L. was decreased consistent with KLeaf under water stress condition (Pou et al. 2013). Moreover, a report using artificial miRNAs stated that specific PIP1 subfamilies decreased the expression of the PIP1 transcript and translation level in Arabisdopsis thaliana. This result led to a decrease in water permeability (Pf) in the mesophyll and bundle sheath as well as in KLeaf (Sade et al. 2014). Interestingly, PIP1-3, which is 92.3% homologous to AtPIP1-2, was up-regulated in HB60 and was consequently correlated with the decrease in RWC. The overexpression of AtPIP1-2 in tobacco plants under water deficit conditions led to a

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reduction in drought resistance (Aharon et al. 2003). We found that the down-regulation of MePIP1-3 was important for water loss prevention in the leaves of cassava plantlets. The activity of TIP was reported to regulate the cellular mechanism for water balance, as the overexpression of TIP was demonstrated to lead to increased water permeability of mesophyll cells in the leaf. (Sade et al. 2009). Recently, the expression of PeTIP4-1 was reported to be up-regulated under drought condition in leaf and root tissues. The PeTIP4-

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1 transgenic Arabidopsis thaliana exhibited more resistance to drought by increasing water content (Sun et al. 2017). In this study, the increase in MeTIP1-3 in in vitro leaves and roots suggested the involvement of water content adjustment in the cytoplasm of cells under stress conditions. Moreover, in vitro roots exhibited the same expression pattern for all candidate AQP genes in both cultivars after PEG treatment. PIP subfamilies were down-regulated while some TIP genes were up-regulated. A similar AQP gene

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expression pattern was reported in tea roots after exposure to imitated-drought with 10% PEG. These results suggested that down-regulation of aquaporin in the root might be due to a reduction in root hydraulic conductance during drought stress (Yue et al. 2014).

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In 2016, a report on sequencing wild and cultivated cassava revealed extensive interspecific hybridization and genetic diversity, which suggested that there was common genetic information among related species of cassava (Bredeson et al. 2016). HB60 is derived from a cross between the breeding populations R5 (27-77-10 x R3) and KU50 (R1 (from Malaysia) x R90 (CMC76 x V43)), while HN is a local Thai cultivar. Although genome-wide analysis of HN and HB60, including their ancestors, have not been available, these two cultivars are genetically closer on the basis of homology of their orthologues such as PIP2-1 and PIP1-3. The difference in the expression profiles of PIP2-1 and PIP2-10 genes in HN and

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HB60, is supposed to be a key factor for drought tolerance in HB60 on the assumption that there are common genetic backgrounds in these cultivars. When focusing on AQP genes expression in potted plants after withholding water, most of the AQPs in the leaf showed up-regulation in HB60 but down-regulation in HN. Interestingly, PIP2-1 was increased in HB60 in parallel with stomatal closure while its expression

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decreased in HN with stomatal opening. This result suggested that the up-regulation of MePIP2-1 might regulate stomatal closure under water deficit treatment. As mentioned above, the up-regulation of TIP and down-regulation of PIP in the leaf were involved in the drought response in cassava. The up-regulation of

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TIP1-3 and TIP1-5 in HB60 may control water balance in the cell, while PIP2-5 and PIP1-3 were downregulated to decrease KLeaf to prevent water loss. However, the up-regulation of PIP2-1 and PIP2-10 in both cultivars might play prominent roles in increasing KLeaf after water-deficit stress. PIP2-1 and PIP2-10 may efflux water out of the guard cell, which leads to stomatal closure in HB60 (Figure 7). Furthermore, the expression of MeAQPs in the root was also interesting. Most of the AQP genes were down-regulated at D2W but were up-regulate at D4W. Similar results were reported in rice aquaporin genes under drought condition (Ding et al. 2016). PIPs and TIPs were down-regulated in the root for leaf ABA accumulation and stomatal closure induction. After that, the expression increased for root hydraulic induction and shoot growth sustainability. It should be noted that MePIPs and MeTIPs in HB60 were more highly expressed

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than those in HN. These results suggested that HB60 might more efficiently manage water movement in the cell than HN through the expression of PIPs and TIPs. It has been reported that there are discrepancies between the mRNA aquaporin expression and the amount of aquaporin proteins in Arabidopsis, maize and broccoli (Boursiac et al. 2005; Aroca et al. 2005; Muries et al. 2011). These findings suggested the possibility that mRNA synthesis is modulated by the

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accumulation of the corresponding encoded protein. In Arabidopsis, an increase in the abundance of proteins in roots under salinity stress has been widely described as being uncorrelated with an increase in the expression level of their corresponding mRNA. The exposure of roots to salt induced changes in aquaporin expression at multiple levels including the coordinated transcriptional down-regulation and subcellular relocalization of both PIPs and TIPs. However, there were also previous reports that revealed

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the correlation of aquaporin at the mRNA level and the abundance of proteins in some plants. The upregulation of aquaporin genes in tobacco was correlated with protein abundance under ABA treatment and seemed to increase the sap flow rate, the hydraulic conductance of tobacco roots, for drought resistance

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(Mahdieh et al. 2008). The different responses of HN and HB60 to stomatal closure and relative water content under drought condition may have occurred from defective phosphorylation sites on PIP2-1 and PIP1-3 proteins in HN. To prove this possibility, the full-length cDNA of PIP2-1 and PIP1-3 was cloned from HN and HB60 for nucleotide sequences comparison. Interestingly, the same deduced amino acid sequences of PIP2-1 and PIP1-3 proteins were found in both cassava cultivars (data not shown). Therefore, the phosphorylation sites on the protein of these genes did not affect to the different responses. Another possibility that may play an important role in drought response is the differences in transcription factors

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that bind to cis-regulatory elements at each aquaporin gene promoter in each cultivar. There were also many results that supported the possibility that the level of aquaporin transcripts was correlated with the ability to adapt to drought conditions (Khan et al. 2015, Qian et al. 2015). The expression of TIP4;1 in bamboo was sharply up-regulated in leaves and roots after drought treatment. When this gene was over-

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expressed in Arabidopsis, the higher water content could enhance stress tolerance after exposure to drought (Sun et al. 2017). Therefore, another possible cause of the differential expression of aquaporin genes may be the differences in transcription factors that bind to cis-regulatory elements on each aquaporin gene

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promoter in each cassava cultivar. In reference to the available sequences of the genome of the cassava cultivar AM560-2, the type and number of each cis-regulatory motif found in promoter regions of the PIP2-1 and PIP2-10 genes are shown in Supplementary Table S2. The responsive-types of drought 10 elements were found in only PIP1-2 but not in PIP2-10 promoter regions, whereas only 2 types were found in only the PIP2-10 promoter region. The 12 cis-regulatory elements, related to water stress, dehydration, and the response to ABA, may be involved in increasing or decreasing gene transcription and exhibiting different expression levels in these two genes in response to water deficit. It is now well documented that the concentration of ABA will increase in the leaf in response to water deficit in the plant root (Wilkinson and Davies, 2002). The regulatory role of ABA was also reported to affect the water status in cassava (Alves and Setter, 2004b). ABA accumulation in cassava was observed to increase rapidly and

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substantially under rather mild water-deficit conditions. ABA then induces an internal signal transduction cascade, which eventually reduces guard cell osmotic potential to cause stomatal closure (Duque and Setter, 2013). However, to elucidate a key event for conferring drought tolerant in HB60, the promoter sequences of target aquaporin genes should be compared between HB60 and HN.

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NCED3 encodes a key enzyme for ABA biosynthesis and ABA signaling pathways induce stomatal closure under water-deficit conditions. In Arabidopsis thaliana, overexpression of NCED3 led to decreased transpiration rate and drought tolerance improvement while the disruption of this gene revealed drought sensitivity (Iuchi et al. 2001). In this study, the expression of MeNCED3 was sharply up-regulated in both cultivation systems after water-deficit treatment. A consistency was found in the cassava cultivar

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KU50 after potted plants were treated with 20%PEG (Fu et al. 2016). These results suggested that the ABA-dependent pathway is also essential to the water-deficit response in cassava. HK1 was suggested to act as an osmosensor in Arabidopsis thaliana (Wohlbach et al. 2008). The MeHK1 gene revealed different

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expression patterns among in vitro and potted plant systems in cassava. Normally, HK1 should be upregulated under water-deficit treatment conditions for ABA-dependent pathway induction. As the results of this study indicate, the MeHK1 gene was down-regulated while the MeNCED3 was dramatically upregulated. These results suggested that this MeHK1 gene might not be involved in the ABA-inducing pathway. However, the expression pattern of MeHK1 revealed similarities to PIP2-5 and PIP1-3 in potted plants, thus, it is possible that HK1 may induce PIP2-5 and PIP1-3 through the ABA-independent pathway. Moreover, PYR4, OST1 and GORK, which are ABA-responsive genes, also revealed inconsistencies with

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aquaporin expression and relative water content/stomatal closure in cassava. The results suggested that these three genes might be involved in the drought response in cassava through other regulatory pathways. In conclusion, this study demonstrated that two cultivars of cassava, HN and HB60, have their own specific characteristics of adaptation to water deficit treatment. The expression of aquaporin genes revealed essential roles for these genes in water-deficit responses in both cultivars. When drought

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conditions occurred, most aquaporin genes in the leaf were down-regulated in order to reduce the rate of water loss. The expression of MePIP2-1 and MePIP2-10 was identified in guard cells that efflux water to control stomatal closure. In addition, the NCED3 gene was dramatically up-regulated to induce the ABA-

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responsive pathway under water-deficit conditions. The expression of all of these genes led to the induction of stomatal closure and relative water content maintenance, improving the drought tolerance of cassava.

Acknowledgements

This research was partially supported by the Center of Excellence on Agricultural Biotechnology, Science and Technology Postgraduate Education and Research Development Office, Office of Higher Education Commission, Ministry of Education (AG-BIO/PERDO-CHE) and a grant from Mahidol University.

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Figure Legends Figure 1 Phylogenetic tree of putative MeAQPs available in the cassava genome. Amino acid sequences from 45 members of the MeAQPs were aligned using ClustalOmega, and a phylogenetic tree was then constructed using MEGA7 program with the maximum likelyhood method. MeAQPs were then classified

members; and XIPs, with 4 members.

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into 5 subfamilies: PIPs, with 14 members; TIPs, with 13 members; SIPs, with 4 members; NIPs with 10

Figure 2 Putative cis-regulatory elements determined in the aquaoporin promoter regions. A) Percentage of all regulatory motifs in MeAQPs promoters, B) Number of putative cis-regulatory elements involved in

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dehydration and ABA in the 3-kb predicted promoter sequences of MeAQPs.

Figure 3 Water-deficit responses of two cassava cultivars in the in vitro plantlet system. A) Relative water content. Data are the mean of three samples from three independent experiments (n=9), and the error bars

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indicate SE. The results were considered statistically significant when the P-value < 0.05, which is marked by different letters. B) Stomatal closure after 40%PEG (w/v) treatment at 0, 24 and 48 h. The number indicates the percentage of opened stomata and the inset shows a selected representative stoma in each condition.

Figure 4 Water-deficit responses of two cassava cultivars in the potted plant system. A) Relative water content. Data are the means of three samples from three independent experiments (n=9), and the error bars

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indicate SE. The results were considered statistically significant when the P-value < 0.05, and these results are marked by different letters. B) Stomatal closure after 40%PEG (w/v) treatment at 0, 24 and 48 h. The number indicates the percentage of opened stomata and the inset shows a selected representative stoma in each condition.

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Figure 5 Expression patterns of candidate aquaporin and abscisic acid response genes in the cassava leaf under water-deficit conditions. Heat map depicts the relative expression of target genes determined by the comparative CT method (2-∆∆CT). The 18S rRNA was used as an internal control for the normalization of the

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target gene detection levels. Statistical analysis was performed using one-way ANOVA (Duncan’s multiple-range test) to determine significant differences (p<0.05). Dark shading represents higher expression levels compared with the control, while gray shading represents lower expression levels. A) Expression patterns of candidate genes in HN and HB60 in the in vitro system after 40%(w/v) PEG6000 treatments at 0, 24 and 48 h. B) Expression patterns of candidate genes in HN and HB60 in the potted plant system after withholding water for 0, 2 and 4 weeks (see color version in supplementary data). Figure 6 Expression patterns of candidate aquaporin genes in the cassava root under water-deficit conditions. Heat map depicts the relative expression of target genes determined by the comparative CT method (2-∆∆CT). The expression of 18S rRNA was used as an internal control for the normalization of the target gene expression levels. Statistical analysis was performed using one-way ANOVA (Duncan’s

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multiple-range test) to determine significant differences (p<0.05). Dark shading represents higher expression levels compared with the control, while gray shading represents lower expression levels. A) Expression patterns of candidate genes in HN and HB60 in the in vitro system after 40% (w/v) PEG6000 treatments at 0, 24 and 48 h. B) Expression patterns of candidate genes in HN and HB60 in the potted plant

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system after withholding water for 0, 2 and 4 weeks (see color version in supplementary data). Figure 7 Schematic of the hypothesis of water movement in the cassava leaf under normal conditions and water-deficit conditions. A) Under normal condition, water moves into guard cells through aquaporins leads to stomata opening. B) Water movement in the cassava leaf under normal condition; water moves from mesophyll cells to the stomatal cavity and moves out of the leaf through stomata pores following the

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water potential. C) Under water deficit condition, water move out of the guard cell through the upregulation of MeAQPs (PIP2-1 and PIP2-10) to subsidiary cells and the epidermis, which leads to stomata closure. D) Water movement in the cassava leaf under water-deficit conditions; PIP2-1 and PIP2-10 efflux

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water out of the guard cell, which leads to stomata closure. The other PIP members decrease the rate of water movement to prevent water loss, while TIP members regulate the water balance in the cell. The dark thick arrow represents the up-regulation of MeAQPs, whereas the gray thin arrow represents the downregulation of MeAQPs. BS, Bundle sheath; MP, Mesophyll cell; GC, Guard cell; SC, Subsidiary cell; EP,

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Epidermis; TP, Tonoplast or Vacuole. (see color version in supplementary data)

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Supplementary data

Figure S1 Sequence alignment of 45 MeAQPs. The amino acid sequences of MeAQPs were aligned using ClustalOmega and reviewed in A-E: A) PIPs, B) TIPs, C) NIPs, D) SIPs and E) XIPs. The blue highlight represents the predicted NPA conserved motifs in the amino acid sequences. NPL and NPV are present

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instead to NPA motif. The asterisk denotes conserved region.

Figure S2 The effect of water deficit on cassava plantlets of two cultivars (HN and HB60). The PEG was used to treat in in vitro cultures to imitate water deficit conditions. The leaves of both cultivars clearly

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exhibited wilting within 24 h.

Figure S3 The effect of water deficit on potted plants of cassava of two cultivars. Watering was withheld

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from HN (left) and HB60 (right) for 4 weeks (D4W). During drought imitation, the wilting of leaves was observed in the two cultivars. HN showed yellow and fallen leaves, while HB60 did not show either of those characteristics.

Figure S4 Expression patterns of candidate genes in HN and HB60 leaves in the in vitro system after 40% (w/v) PEG6000 treatments at 0, 24 and 48 h (CL0, DL24, and DL48, respectively). The blue line represents the expression patterns of the candidate genes in HN, and the red line represents the expression patterns of

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the candidate genes in HB60. Statistical analysis was performed using one-way ANOVA (Duncan’s multiple-range test) to determine significant differences (p<0.05). Figure S5 Expression patterns of candidate genes in HN and HB60 leaves in the potted plant system after withholding water at 0, 2 and 4 weeks (C0, D2W, and D4W, respectively). The blue line represents the

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expression patterns of the candidate genes in HN and the red line represents the expression patterns of the candidate genes in HB60. Statistical analysis was performed using one-way ANOVA (Duncan’s multiple-

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range test) to determine significant differences (p<0.05). Figure S6 Expression patterns of candidate genes in HN and HB60 roots in the in vitro system after 40% (w/v) PEG6000 treatments at 0, 24 and 48 h (CR0, DR24, and DR48, respectively). The blue line represents the expression patterns of the candidate genes in HN, and the red line represents the expression patterns of the candidate genes in HB60. Statistical analysis was performed using one-way ANOVA (Duncan’s multiple-range test) to determine significant differences (p<0.05). Figure S7 Expression patterns of candidate genes in HN and HB60 roots in the potted plant system after withholding water for 0, 2 and 4 weeks (C0, D2W, and D4W, respectively). The blue line represents the expression patterns of the candidate genes in HN and the red line represents expression pattern of candidate genes in HB60. Statistical analysis was performed using one-way ANOVA (Duncan’s multiple-range test)

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to determine significant differences (p<0.05). Color version of Figure 5 Expression patterns of candidate aquaporin and abscisic acid response genes in the cassava leaf under water-deficit conditions. Heat map depicts the relative expression of target genes determined by the comparative CT method (2-∆∆CT). The 18S rRNA was used as an internal control for the

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normalization of the target gene detection levels. Statistical analysis was performed using one-way ANOVA (Duncan’s multiple-range test) to determine significant differences (p<0.05). Red color represents higher expression levels compared with the control, while green color represents lower expression levels. A) Expression patterns of candidate genes in HN and HB60 in the in vitro system after 40%(w/v) PEG6000

system after withholding water for 0, 2 and 4 weeks.

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treatments at 0, 24 and 48 h. B) Expression patterns of candidate genes in HN and HB60 in the potted plant

Color version of Figure 6 Expression patterns of candidate aquaporin genes in the cassava root under

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water-deficit conditions. Heat map depicts the relative expression of target genes determined by the comparative CT method (2-∆∆CT). The expression of 18S rRNA was used as an internal control for the normalization of the target gene expression levels. Statistical analysis was performed using one-way ANOVA (Duncan’s multiple-range test) to determine significant differences (p<0.05). Red color represents higher expression levels compared with the control, while green color represents lower expression levels. A) Expression patterns of candidate genes in HN and HB60 in the in vitro system after 40% (w/v) PEG6000 treatments at 0, 24 and 48 h. B) Expression patterns of candidate genes in HN and HB60 in the

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potted plant system after withholding water for 0, 2 and 4 weeks.

Color version of Figure 7 Schematic of the hypothesis of water movement in the cassava leaf under normal conditions and water-deficit conditions. A) Under normal condition, water moves into guard cells through aquaporins leads to stomata opening. B) Water movement in the cassava leaf under normal

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condition; water moves from mesophyll cells to the stomatal cavity and moves out of the leaf through stomata pores following the water potential. C) Under water deficit condition, water move out of the guard cell through the up-regulation of MeAQPs (PIP2-1 and PIP2-10) to subsidiary cells and the epidermis,

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which leads to stomata closure. D) Water movement in the cassava leaf under water-deficit conditions; PIP2-1 and PIP2-10 efflux water out of the guard cell, which leads to stomata closure. The other PIP members decrease the rate of water movement to prevent water loss, while TIP members regulate the water balance in the cell. The black arrow represents the up-regulation of MeAQPs, whereas the red arrow represents the down-regulation of MeAQPs. BS, Bundle sheath; MP, Mesophyll cell; GC, Guard cell; SC, Subsidiary cell; EP, Epidermis; TP, Tonoplast or Vacuole. Supplement Table S1 List of candidate gene-specific primers used in this study Supplement Table S2 List of the type and number of putative cis-regulatory motifs found in the promoter regions of the PIP2-1 and PIP2-10 genes of cassava cultivar AM560-2

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ACCEPTED MANUSCRIPT Highlights •

Forty-five members of MeAQPs were identified in the cassava genome.

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Drought-responsive motifs were found in each MeAQP promoter region.

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Differential expression of MeAQPs was consistent with relative water content.

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PIP2-1 and PIP2-10 in guard cells was involved in water efflux controlling

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stomata. NCED3 and ABA-responsive genes were up-regulated under water-deficit

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ACCEPTED MANUSCRIPT Contributions P.P. performed the experiments, data analysis and drafted the manuscript. All authors suggested on the experimental plan and discussed the results. J.N. conceived the project, supervised the research and provided support for the experiments. All

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authors have read and approved the final manuscript.