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Agricultural Water Management

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Effects of partial root-zone drying and deficit irrigation on yield, irrigationwater-use efficiency and some potato (Solanum tuberosum L.) quality traitsunder glasshouse conditions

Sliman Elhania,⁎, Maroua Haddadia,1, Edina Csákvárib, Said Zantara, Ahlam Hamima,Vanda Villányib, Ahmed Douaikc, Zsófia Bánfalvib

aNational Agricultural Research Institute (INRA), 78, Bd. Sidi Med. Ben Abdellah, 90010 Tangier, MoroccobNARIC Agricultural Biotechnology Institute, Szent-Györgyi A. u. 4, 2100 Gödöllő, HungarycNational Agricultural Research Institute (INRA), PO Box 6356, Avenue Mohamed Belarbi Alaoui, 10101, Rabat, Morocco

A R T I C L E I N F O

Keywords:Antioxidant activityMetabolitePolyphenolProteinSugarTuber

A B S T R A C T

Agriculture water resources are expected to decline due to increasing water demand and ongoing climatechange. In this context, water-saving irrigation techniques, such as partial-root zone drying (PRD) and deficitirrigation (DI) were assessed under glasshouse conditions on potato cultivar Mondial. Four irrigation levels wereapplied: 50, 70, 80 and 100% of field capacity during 2016 and 2017 growing seasons. The results showed thatthe yield penalty with PRD was similar to that caused by DI. Nevertheless, PRD plants had higher number ofstems and were shorter than DI plants. Sugar and protein contents of tubers gradually decreased with waterrestriction, however, remained higher in PRD than DI tubers. In contrast, the amounts of polyphenols and an-tioxidants increased in tubers with decreasing irrigation levels. Untargeted metabolite analysis revealed highermetabolite content of PRD than DI tubers with less decrease in glucose and fructose concentrations and withdouble amount of mannitol. Transcript level of key-genes involved in carbohydrate metabolism was elevated at20% water-saving in PRD tubers, but not in DI tubers. We assumed that the detected changes in tubers reflectbetter adaptation of plants to water-saving irrigation under PRD than DI.

1. Introduction

Water is an essential resource, it plays a central and critical role inall aspects of life, it is the basic element of any biological function, andit is a precious resource, limited and vulnerable. Sustainable manage-ment of this resource requires a holistic approach that integrates ra-tional use. Climate change is expected to put additional stress on watersupplies, making water management even more complex and difficult(Arnell, 2004; Bates et al., 2008). Deficit irrigation (DI) is a promisingmethod consisting in reducing water inputs compared to a maximumlevel of crop, so that the stress imposed does not significantly affectyield (Costa et al., 2007; Geerts and Raes, 2009). The objective of thisirrigation method is to control vegetative growth and improve irriga-tion water-use efficiency (IWUE). Partial root-zone drying, known asPRD, is an evolution of DI and is one of the promising techniques forsaving irrigation water (Dodd et al., 2015).

Many studies have shown the advantage of PRD by reducing waterinput by 30–50%, without penalizing yield, or even having a positiveeffect on quality. Du et al. (2008), for example, showed in an experi-mental vineyard study under arid conditions that PRD treatment im-proved IWUE and increased the number of grapes, vitamin C con-centration and maturity index. Zegbe and Behboudian (2008) appliedPRD in humid climate on apple trees and showed that it did not ad-versely affect yield and fruit quality but improved IWUE by 20%. Si-milar results were reported by dos Santos et al. (2003) on the grapevinewhen moderate water stress inhibited vegetative growth. Indeed, theyargued that vines and fruit trees are the most suited to the PRD strategy,knowing that the responses were variable, depending on the species,variety and climatic conditions. Nevertheless, PRD could be success-fully applied also to tomato and impacted bioactive compounds andantioxidant activity (Casa and Rouphael, 2014; Sun et al., 2014a;Bogale et al., 2016).

https://doi.org/10.1016/j.agwat.2019.105745Received 26 February 2019; Received in revised form 12 July 2019; Accepted 8 August 2019

⁎ Corresponding author.E-mail address: sliman_elhani@yahoo.fr (S. Elhani).

1 Present address: Département de Sciences de la Vie, Équipe de recherche sur la valorisation biotechnologique, Faculté des Sciences et Techniques, Tangier,Morocco.

Agricultural Water Management 224 (2019) 105745

Available online 23 August 20190378-3774/ © 2019 Elsevier B.V. All rights reserved.

T

PRD studies in potato goes back to 2005 when Saeed et al. (2005)reported that PRD treatment modifies shoot growth, reduces plant leafarea and increases IWUE. Andersen et al. (2008) conducted experi-ments with cv. Folva in outdoor rain-protected lysimeter facilities inthree soil types: loam, sandy loam, and coarse sand. In PRD, plantsreceived 70% of those plants which were irrigated to field capacity(FC). When plants were fully irrigated till tuber-bulking the total yieldwas essentially the same in PRD and FC. PRD during the tuber-initiationstage of growth, however, decreased the yield and quality of tubers.Several studies indicate that PRD irrigation may cause not only an in-crease of IWUE but also higher nitrogen use efficiency (Shahnazariet al., 2008a; Wang et al., 2009; Jensen et al., 2010; Ahmadi et al.,2011). Moreover, PRD results in a slight reduction in soluble sugarconcentration and an increase in starch, nitrogen and antioxidantcontents of tubers (Jovanovic et al., 2010). Nevertheless, there is aninconsistency on the effects of the PRD on potato yield in the literature.In contrast to Shahnazari et al. (2007) testing cv. Folva in coarse tex-tured meltwater sand, Brocic et al. (2009) detected a reduced yield ofcv. Liseta grown in silty-clay by comparing FC to 70% PRD. An ex-planation for inconsistent results in terms of the effect of PRD on tuberyield was given by Ahmadi et al. (2010) who showed that the inter-action between irrigation treatments and soil textures was significantand water-saving irrigations were not recommended in loamy sand soildue to considerable loss (28%) in yield with PRD receiving 65% of FC.Furthermore, by growing cv. May Queen in experimental plots underplastic rainout shelters it was concluded that early-season PRD treat-ment for eight weeks rather than the whole-season treatment should beapplied (Xu et al., 2011). A similar result was obtained by growing thepotato cv. Unica in sandy loam. In that study, it was found that an earlyPRD, initiated six weeks after planting, with a watering level equivalentto even 50% of FC, increased IWUE with no yield reduction relative toFC (Yactayo et al., 2013). Efficiency of PRD, however, is also cultivardependent as it was detected in the case of Agria and Ramos (Ahmadiet al., 2016). Another critical factor in terms of yield might be thecorrect choice of duration of wet/dry cycling, especially in arid areas aspointed out by Zin El-Abedin et al. (2017).

The above cited publications demonstrated that application of PRDin potatoes is possible but it is critical to identify the conditions wherethis method can be effective and understand the physiological re-sponses behind adaptation of potato to these conditions. The best wayof testing plants’ responses to PRD is under controlled conditions in agreenhouse on plants with a split-root system. Liu et al. (2006) irrigatedone-half of the root system of cv. Folva for nine days and found that thexylem sap abscisic acid (ABA) concentration increased exponentiallywith decreasing root water potential. Later it was demonstrated thatHPRD (two lateral root compartments: one dry, the other wet) bettertriggers a long-distance ABA signal than VPRD (upper root compart-ment dry, lower compartment wet) (Puértolas et al., 2015). It was alsoshown that compared to DI, PRD led to modulating stomatal mor-phology and significantly enhanced plant nitrogen content in relation toDI and increased root to shoot ratio (Yan et al., 2012; Sun et al., 2013,2014b; 2015; Liu et al., 2015). Ramírez et al. (2014) found an increasein chlorophyll content of PRD treated leaves and concluded thatchlorophyll concentration in leaves is an indicator of potato tuber yieldin water-shortage conditions.

In Morocco, as water resources are constantly worsening, water-saving is an essential axis of the new agricultural strategy. In line withthis strategy the aims of our work were: (i) to assess the feasibility ofwater-saving irrigation techniques on potato cv. Mondial, a variety withvery high yield potential, under glasshouse conditions; (ii) to elucidatethe differential effects of PRD and DI on growth parameters and themain quality traits of tubers; (iii) and to study the effects of water-saving irrigation on the metabolite profile and gene expression in tu-bers.

2. Material and methods

2.1. Plant material and growth conditions

The experiments were carried out during the growing seasons of2016 and 2017 in a glasshouse of the National Agricultural ResearchInstitute in Tangier (Northern Morocco). Certified seedlings of Solanumtuberosum L. cv. Mondial from NAK-Nederland with uniform size(35–55mm in diameter) were planted in normal and separated pots(25 cm diameter and 30 cm height), filled with 2/3 of sandy soil and 1/3 of an organic substrate. The sandy soil originated from Loukkos re-gion called “R’mel”, is inorganic and poor in nitrogen, its texture issandy with 80.5%, 11% and 8.5% of sand, silt and clay, respectively.The substrate used contained the NPK fertilizer (14:10:18) 1.5 kg/m3

with a pH of 5.5–6.5 and electrical conductivity of 40mS/m (±25%).The experiments were carried out in a CLINVERTEC glasshouse

(RITEC, Riegos y Tecnología S.L., Águilas, Murcia, Spain) with con-trolled heating and humidification systems, air recirculation, CO2

supply, cooling system, thermal screens and shade control. The tem-perature inside the glasshouse was fixed between 24 and 25 °C duringthe cultivation period from March to June and the relative humiditybetween 60 and 70%.

2.2. Experimental design and irrigation treatments

The experiment was setup in a randomized complete block design(RCBD) with two factors: irrigation method and irrigation amount. Thefirst factor had two levels: either deficit irrigation (DI) or partial root-zone drying (PRD) while the second factor had four levels: 50, 70, 80,or 100% of the field capacity (FC). FC of the mixed soil (2/3 of sandysoil and 1/3 of organic substrate) was determined by gravimetricmethod. Before planting, two control pots in each block were watereduntil the soil was saturated, after free drainage had stopped, the potswere weighted to obtain the volumetric water retained by the soil foreach pot. The volumetric soil moisture content (θv) at FC was 25.1%(on the mass basis), permanent wilting point was estimated to be 11%and available water 14.1%. The factorial arrangement of the levels ofthe two mentioned factors lead to eight treatments, each one of themwas replicated in four blocks. In total, there were 2× 4x4=32 ex-perimental units. Each unit comprised six plants (one plant/pot) re-ceiving the same and a unique one of the eight treatments. However, forthe statistical data analyses, the measurements over the 6 plants wereaveraged such that there would be only one observation per experi-mental unit.

Pots were irrigated equally 30 days after planting at FC. The soilmoisture content was measured gravimetrically by weighting each potand replacing the individual loss of water to maintain the originalweight. Shifting of irrigation in PRD treatments was done empirically(Jensen et al., 2010). Depending on pots evapotranspiration, DI plantswere irrigated every second or fourth day, while PRD plants were wa-tered at alternating sites daily or every second day. The same amount ofwater supplied to DI treatments was split twice to PRD treatments oneither two sides of the root system, receiving each compartment the halfamount of water supplied to DI plants in each irrigation event. PRD potswere separated by a rigid and impermeable PVC plate to avoid theinfiltration of water from the watered compartment to the dry one.

2.3. Calculation of irrigation water-use efficiency (IWUE)

At final harvest, tubers were collected, and the tuber biomass wasdetermined for each treatment. IWUE (g/l) was calculated by dividingthe fresh weight of tubers (g/plant) by the amount of water (l/plant)used during treatment period (one month after planting to two weeksbefore physiological maturity).

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2.4. Dry weight and ash content determination

Dry matter and water content were determined by the AOAC 934.06official method by vacuum-oven. Three grams of potato sample wasplaced in a clean, empty and dry moisture-can. The moisture-can con-taining the sample was placed in the drying vacuum-oven (ISCO,Lincoln, NE, USA) that was set at 70 °C for more than 8 h. The dryweight (DW) in % was determined by the formula given as: DW =((MS-WW)/MS) ×100, where WW is mass of the water and MS is totalweight of the material.

To measure the ash content the weight of clean and dry crucible wasdetermined and 5 g of sample were placed in the oven. The cruciblecontaining the sample was placed in an ash oven at 525 °C ± 10 °C for3 h. When the ashing was complete the weight of the sample wasmeasured again. The ash content (AC) in % was determined by thefollowing formula: AC = (W2/W1) ×100, where W2 is weight of theremaining sample and W1 is weight of the original sample.

2.5. Tuber quality trait analysis

The total sugar content of 10 g of tubers was extracted with ethanol.Tubers were placed into a sealed test tube. Fifty ml of 80% ethanol wasadded and the tubers were homogenized with an Ultra-Turrax T50(Janke and Kunkel, IKA Labortechnik, Staufen, Germany) and cen-trifuged at 5000 rpm for 30min in a Centrifuge Z 323 K (HermleLabortechnik GmbH, Wehingen, Germany). Sugars were extracted fromthe pellet with 5ml of 40% ethanol and centrifuged at 15,000 rpm for15min. The extraction was repeated and the two extracts combined.Total sugars content was determined using the phenol-sulfuric acidmethod (Dubois et al., 1956) by adding 0.25ml of 80% aqueous phenoland 25ml of 95.5% aqueous sulfuric acid to the extract. The tubes wereallowed to stand for 10min. Then they were shaken and placed for10min in a water bath at 30 °C. The absorbance was measured at490 nm by an Evolution 201 spectrophotometer (Thermo Scientific,Waltham, MA, USA). The standard curve was prepared from sevenglucose standard solutions (10, 20, 30, 40, 50, 60 and 70 μg/l) toquantify total sugars.

Protein content of tubers was calculated from the total nitrogencontent of 1 g tubers determined by the Kjeldahl method. This methodconsists of digestion of samples in sulfuric acid with a catalyst (Kjeldahltablets), which results in conversion of nitrogen to ammonia, distilla-tion of the ammonia into a trapping solution (1% boric acid) andquantification of the ammonia by titration with a standard solution(0.5 M sulfuric acid). Once the percentage of the total nitrogen wascalculated, the concentration of proteins was determined by the fol-lowing equation: crude protein (%) = nitrogen content (%) × 6.25.

Total polyphenol content of tubers was measured from 25 g tissue.Tubers were cut into small pieces and homogenized in 100ml of dis-tilled water with Ultra-Turrax. The homogenate was filtered and cen-trifuged at 5000 g for 20min. The potato extracts were filtered usingfilter paper No 1 in test tubes and boiled for 5min in a water bath toinactivate the enzymes. Total phenolic content was determined withFolin-Ciocalteu reagent by spectrophotometry according to the methodof Siriwoharn et al. (2004) using gallic acid as standard. 7.5 ml ofdistilled water, 0.5 ml of Folin-Ciocalteu reagent and 0.5ml of gallicacid (10, 50, 100, 200, 400 ppm) were added to each tube for cali-bration, while gallic acid was replaced by addition of 0.5ml of potatoextracts to determine the phenolic content of tubers. The reactionmixtures were vortexed and stood for 10min at room temperature.Then, 3ml of 20% sodium carbonate was added to each tube, vortexed,placed into a water bath at 40 °C for 20min and cooled in an ice bathfor 3min. Absorbance of samples was measured at 755 nm in an Evo-lution 201 spectrophotometer. The total phenolic content was ex-pressed in mg gallic acid equivalent (GAE)/g fresh weight, according tothe following formula:

=× ×

×

Total phenolic content mg GAE g FW As b V Fa Q

[ / ] (( – ) )

Where As= absorbance of the sample at 755 nm; b= ordinate at theorigin of the calibration curve; a= slope of the calibration curve;V= volume of the extract (l); F= dilution factor; Q=mass of homo-genized sample (g).

The antioxidant activity of tubers was measured from the extractthat was previously prepared for determination of total phenolic con-tent. A 1.5 ml aliquot of 2,2-diphenyl-1-picrylhydrazyl (DPPH) solvedin methanol in a concentration of 0.1 mM was mixed with 0.75ml tuberextract. Mixtures were shaken and incubated at room temperature inthe dark. After 30min, absorbance at 517 nm was measured using anEvolution 201 spectrophotometer. Trolox was used as the referencestandard. Scavenging activity of DPPH radical was calculated as fol-lows:

=× ×

×

Antioxidant activity μmol eq trolox g FW As b V Fa Q

[ /100 ] (( – ) )

Where As= absorbance of the sample at 517 nm; b= ordinate at theorigin of the calibration curve; a= slope of the calibration curve;V= volume of the extract; F= dilution factor; Q=mass of homo-genized sample (g).

2.6. Metabolite analysis

Twelve tubers with 3–4 cm in diameter from each treatment werewashed and divided into four equal portions for four biological re-petitions. The tubers were peeled and chopped using a food processor.The samples were frozen in liquid nitrogen and stored at -70 °C. For theGC–MS analysis, the samples were ground into a fine powder in liquidnitrogen, and an extraction was performed according to Nikiforovaet al. (2005) using 100mg tuber powder. Ribitol was added to thesamples as an internal standard, N-methyl-N-(trimethylsilyl) tri-fluoroacetamide (MSTFA) used for derivatization and the samples wereanalyzed in a quadrupole-type GC–MS system (Finnigan Trace/DSQ,Thermo Electron Corp., Austin, TX, USA) equipped with a 30m capil-lary column (Rxi-5 ms, 0.25mm ID, 0.25 μm df, Restek) in TIC (total ionchromatogram) positive mode as described in detail by Uri et al.(2014). Mass spectra were recorded at 0.8170 scans/sec with an m/z50–650 scanning range. The Thermo Scientific Xcalibur software wasused for exporting the spectra and searching the NIST 11 mass spectraldatabase. In addition, the sugars and amino acids were identified basedon a comparison of the retention time and mass spectrum to an au-thentic standard that was analyzed under identical conditions. Rawdata are available in Supplementary Table 3.

2.7. Gene expression analysis

Semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) was used for gene expression analysis. Total RNA was ex-tracted from 100mg of the same tuber powder used for metaboliteanalysis according to Stiekema et al. (1988) and quantified using aNanoDrop ND1000 spectrophotometer (Thermo Fisher Scientific, Wal-tham, MA, USA). DNaseI-treated total RNA (1 μg) was reverse-tran-scribed with RevertAid M-MuLV Reverse Transcriptase and 10xRTRandom primer (Applied Biosystems, Foster City, CA, USA). The cDNAsobtained were PCR-amplified with specific primers and the resultedPCR fragments tested on agarose gel. Gels were documented by a GeneGenius BioImaging System from Syngene (Syngene Gene Genius, Lan-cashire, UK) and analyzed by the ImageJ software (https://imagej.nih.gov/ij/). The housekeeping gene actin was used for normalization(Nicot et al., 2005). Primer sequences, PCR conditions, PCR fragmentson agarose gels and their intensities presented in graphs are shown inSupplementary Fig. 1.

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2.8. Statistical data analysis

Treatments in glasshouse were arranged in RCBD with four re-plications of each treatment. Data were analyzed using the GLM ofthree-way analysis of variance (ANOVA), the three factors were irri-gation method, irrigation amount and block. This was the case for re-sponse variables measured during 2016 season. However, for the otherresponse variables, the experiment was repeated during two growingseasons (2016 and 2017). Therefore, an analysis of combined experi-ments over years (Dixon et al., 2018) was done including the growingseason as an additional factor leading to a four-way ANOVA. The effectsof the two main factors: irrigation method and irrigation amount wereconsidered fixed. Since we have only two years, we considered the yeareffect as fixed (Vargas et al., 2018), while the block effect was con-sidered random. The combination of fixed as well as random effectsleads to a mixed model (Yang, 2010).

Data were analyzed using the MIXED procedure of SAS version 9.1.3(SAS Institute, Cary: NC, USA) using the REML estimation method andthe Kenward-Roger approximation for computing the degrees offreedom of the denominator for eventually heterogeneous variances(Littell et al., 2006). Normality of data was checked using the Shapiro-Wilk test. Correlation analysis was performed by Pearson’s method.

3. Results

3.1. Effects of PRD and DI on yield, IWUE and growth parameters

Effects of PRD and DI on cv. Mondial grown in glasshouse underwell-controlled conditions in pots were compared in two seasons (2016and 2017). Tuber yields per plant are presented in Fig. 1A. The yieldranged from 120 g/plant for DI-50% to 202 g/plant for PRD at 100% ofFC. The tuber yield was reduced by around 40% when the water supply

was restricted to 50%. The statistical analysis showed significant dif-ferences in tuber yield per plant between 50 and 70%, and between 80and 100% water treatments, but not between 70 and 80% (Supple-mentary Table 1).

The IWUE was significantly higher at 70 and 50% of FC than underwell-watered conditions (Fig. 1B). The highest IWUE values were foundfor PRD-50% with an average of 50.6 g/l and the lowest values werefound in FC treatments with an average of 37.9 g/l and 38.8 g/l for DI-100% and PRD-100%, respectively. In all treatments, the IWUE of PRDplants was slightly higher but not significantly different from DI plants(Supplementary Table 1).

The potato plants growth was assessed as tuber number per plant,number of stems per plant, plant height, total dry weight and ashcontent of tubers. Table 1 presents the mean data of these growthparameters measured in 2016. We found that the number of tubers perplant was not influenced by water withdrawal as it ranged from 12 to14 tubers per plant in each treatment. Thus, after one month of normalirrigation, the water restriction did not impact the tuber number perplant. There were no significant differences either in number of stemsper plant or plant height between the different irrigation levels. How-ever, the PRD plants had a greater number of stems but were shorterthan DI plants even at 100% irrigation level suggesting that the alter-nate irrigation of root system influences plant growth. The dry weight(DW) and ash content of tubers as a % of fresh weight decreased withthe severity of water withdrawal with similar tendency in PRD and DI,however, with significant differences in ash content between the twowater-saving irrigation strategies.

3.2. Effects of PRD and DI on sugar, protein, polyphenol and antioxidantcontent of tubers

Effects of PRD and DI on quality traits were studied both in 2016and 2017 seasons. It was found that the sugar and protein content oftubers gradually decreased with lowering the level of irrigation (Fig. 2,A and B). For all treatments, however, the sugar and protein content ofPRD tubers was higher than that of the DI tubers. The only exceptionwas in the sugar contents of tubers developed at 50% of FC, which wereequally low independent of the irrigation strategy used (SupplementaryTable 2).

In contrast to sugar and protein contents, the amounts of poly-phenols and antioxidants in tubers increased with decreasing irrigationlevels (Fig. 2C and D). No substantial difference between the effect ofPRD and DI in polyphenol content of tubers was detected except at 80%of FC where 20% more polyphenols were found in PRD than in DI tu-bers. The antioxidant activity (AA) of PRD tubers, however, was sig-nificantly higher in all treatments (Supplementary Table 2).

3.3. Correlation analysis

Table 2 presents the correlation matrix for yield, IWUE, DW, ash,polyphenol, AA, protein and sugar contents. There were significant andpositive correlations between tuber yield, DW, AA, protein and sugars.In addition, significant and positive correlations were detected betweenIWUE, polyphenol concentration and AA, while IWUE correlated ne-gatively with DW, ash, protein and sugar contents. Ash content and AAshowed strong and negative correlation (r=-0.890), whereas proteinand total sugars correlated positively (r= 0.704). Total polyphenolsdid not correlate with AA (r= 0.111).

3.4. Effects of PRD and DI on metabolite composition of tubers

Metabolite composition of tubers was tested from the experimentperformed in 2016. After harvest, the tubers were stored at 4 °C for 3months. An untargeted metabolite analysis was performed by GC–MS.Based on mass spectra and authentic standards 33 metabolites wereidentified. These included mainly sugars, amino acids and organic

Fig. 1. Effects of PRD and DI at 50, 70, 80 and 100% of FC irrigation levels onyield (A) and IWUE of potato plants (B). Means presented within an irrigationmethod with the same letter are not significantly different at P≤ 0.05. SeeSupplementary Table 1 for details.

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acids; glucose, sucrose, fructose and citric acid were present in thelargest amounts (Supplementary Table 3). The heat-map presented inFig. 3A shows that the water withdrawal increased the concentrationsof several metabolites and this was more pronounced in PRD than in DItreatments. There were even some differences between the PRD-100%and DI-100% tubers (e.g. in malic acid content) suggesting that alter-nate watering of roots is sensed by the plant and manifested in a mildalteration of tuber metabolism. The dendrogram in Fig. 3B highlightsthe similarity between DI-100% and PRD-80% tubers indicating thatusing alternate irrigation a very similar metabolite constitution as in DI-100% tubers can be achieved with less water. In line with the decreasein total sugar content the concentration of glucose and fructose waslower in tubers of less-watered plants than in tubers of FC plants andhigher in PRD tubers than in DI tubers (Fig. 4, A and B). Interestingly,the concentration of mannitol in PRD tubers was approximately doublethan in DI tubers under each condition (Fig. 4C). Water withdrawaluntil 70% did not change the proline content of tubers, however, atwofold increase was detected in proline content when the soil water-loss reached 50% (Fig. 4D). A detailed statistical analysis is presented inthe Supplementary Table 4.

3.5. Effects of PRD and DI on gene expression

Since treatment-dependent significant differences in sugar andproline content of tubers were detected the expression of some genescharacteristic for sugar and proline metabolism was tested. These genesincluded: sucrose transporter (SUT4), sucrose synthase (Susy), ADP-glucosepyrophosphorylase (AGPase), granule-bound starch synthase (GBSS),mannitol dehydrogenase (MAD), fumarase (FUMase) and Δ1-pyrroline-5-carboxylate synthase (P5CS). Nevertheless, none of them showedchanges in expression correlating with sugar or proline content (Fig. 4,5 and Supplementary Fig. 1). Although, a highly elevated level of SUT4,Susy, GBSS andMAD expression and with less extent, but also of AGPaseand FUMase expression was detected in PRD-80% tubers (Fig. 5 andSupplementary Fig. 1). This observation suggests that the molecularphysiology of PRD-80% plants differs from the others producing tuberswith elevated expression of some key-genes involved in carbohydratemetabolism when stored for three months at 4 °C.

4. Discussion

4.1. Tuber yield, IWUE and growth parameters responses to PRD and DI

Potato (Solanum tuberosum L.) is considered a water-stress-sensitivecrop. Nevertheless, many authors found that the effects of water stressdepend on the growth stage of the crop. Iqbal et al. (1999) reported thatthe stress imposed during the early development stage of potato might

cause severe yield reduction and the stress applied during the ripeningstage increases IWUE without adverse effect on yield. Although ourtreatments were applied after tuber initiation, a significant decrease intuber yield, was detected even by water reduction of 20% (Fig. 1A). Inanother study reported by Jensen et al. (2010), 30% was found to be thewater-saving limit compared to FC. However, their experiment wascarried out under field conditions in a different soil type and with adifferent potato variety.

Our data showed that all restricted water supply evoked higherIWUE than the control, regardless of irrigation strategy used (Fig. 1B).In all treatments, and for the same amount of water supply, the IWUE ofPRD plants were slightly higher but not significantly different from DIplants. Similar results were reported by Yactayo et al. (2009) for pottedpotato grown under greenhouse conditions.

The irrigation treatments started four weeks after planting. The lackof differences in number of tubers per plant in response to irrigationlevel or irrigation strategies supports the hypothesis that the tuber in-itiation was completed before the application of treatments. This agreeswith several authors who reported that initiation of tubers usually be-gins after plant emergence and is thought to be complete within two-three weeks after that event (O’Brien et al., 1998). Other authors sug-gested that PRD should be started five-six weeks after tuber initiation(Shahnazari et al., 2008b; Saeed et al., 2008; Yactayo et al., 2013). Ourresults support the theory that water-saving irrigation should start afterthe end of tuber initiation to avoid yield decrease, which could be aconsequence of the reduction in tuber number per plant.

For normal growth and development, potato needs regular watersupply. Reduction in water or deficit irrigation leads to modifications inmany morphophysiological traits. Growth parameters such as thenumber of stems per plant, plant height, DW and ash content arecommonly dependent on the amount of water available in the soil. Inthis study, there were significant differences in plant height betweenPRD and DI. When comparing the number per stems per plant for thesame irrigation level, we found higher number of stems in PRD than inDI plants, suggesting that alternate irrigation of root system influencesplant growth by promoting stem initiation and reducing plant height.

4.2. Quality of tubers under PRD and DI

Potato tuber DW is mainly constituted by starch and there are smallquantities of sugars, fiber, protein and ash. DW often ranges from 16 to28% depending on the development stages of the crop, reaching itmaximum towards the end of the crop growth. Water stress in potatotends to improve quality of chips due to the higher percentage of tuberDW, which makes the chips appropriate for the industry (Jensen et al.,2000; Kumar et al., 2003). In our experiment, the DW decreased withthe severity of water restriction with similar tendency in PRD and DI

Table 1Effect of irrigation management on growth parameters of potato grown under glasshouse conditions in 2016 season, subjected to two irrigation methods: partial root-zone drying (PRD) and deficit irrigation (DI), and four irrigation amounts: 50, 70, 80 and 100% of field capacity (FC).

Variable Tubers/plant Stems/plant Plant height (cm) Dry weight of tubers (%) Ash content of tubers (%)

Irrigation Amount / Method DI PRD Mean DI PRD Mean DI PRD Mean DI PRD Mean DI PRD Mean50% 13.1 a 13.4 a 13.3 a 5.8 a 7.5 a 6.7 a 38.3 a 33.0 a 35.7 a 18.5 c 20.2 c 19.3 d 0.67 c 0.56 d 0.61 d70% 12.4 a 13.2 a 12.8 a 5.9 a 6.8 ab 6.3 a 41.2 a 36.9 a 39.0 a 21.3 b 20.6 c 21.0 c 0.76 bc 0.60 c 0.68 c80% 13.9 a 14.4 a 14.1 a 6.5 a 7.6 a 7.1 a 43.9 a 36.6 a 40.3 a 22.5 a 21.2 b 21.8 b 0.80 b 0.70 b 0.75 b100% 13.8 a 13.3 a 13.6 a 5.8 a 6.9 a 6.4 a 46.1 a 35.1 a 40.6 a 22.7 a 22.6 a 22.7 a 0.90 a 0.91 a 0.90 aMean 13.3 A 13.6 A 13.4 6.0 B 7.2 A 6.6 42.4 A 35.4 B 38.9 21.3 A 21.2 A 21.2 0.78 A 0.69 B 0.74F-value (p-value)Irrigation Method (M) 0.25 (0.6221) NS 15.43 (0.0006) *** 16.63 (0.0005) *** 0.26 (0.6140) NS 33,66 (< 0,0001) ***Irrigation Amount (A) 1.07 (0.3846) NS 1.34 (0.2863) NS 1.75 (0.1882) NS 46.95 (< 0.0001) *** 58,33 (< 0,0001) ***Interaction (M × A) 0.25 (0.8590) NS 0.33 (0.8041) NS 0.78 (0.5166) NS 9.13 (0.0005) *** 5,15 (0,0069) **

NS: not significant; *, **, and ***: significant at the 5%, 1%, and 0.1% levels, respectively. Means presented within each column with the same lowercase letter arenot significantly different at the 5% significance level. Means presented within each mean row with the same uppercase letter are not significantly different at the 5%significance level. Lettering adapted after three way-ANOVA followed by Fisher's Least Significant Difference (LSD) test. Data are given as the mean calculated fromn=16, 8 or 4 for each irrigation method, irrigation amount or their interaction, respectively.

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and ranged from 18.5% for stressed plants (DI-50%) to 22.7% for well-watered plants (DI-100%). Similar results were reported by Deblondeet al. (1999) on six potato cultivars grown under temperate climateconditions and three irrigation treatments. They showed that tuber DWwas significantly reduced under drought treatment in comparison withFC. Levy (1983) and later Schafleitner (2009) found that tuber DW is

cultivar dependent and is a trait of adaptation of potato to water stress.Total sugars, mainly sucrose, glucose and fructose, are important

factors affecting quality in potatoes, especially color of processed pro-ducts (Smith, 1987). In tubers, they are conditioned by several para-meters, which include variety, the environmental conditions, cropmanagement especially irrigation, and several post-harvest factors likestorage period and temperature (Kumar et al., 2004). There are di-vergent opinions on the impact of water stress on sugars accumulationin potatoes. In this study, it was found that the sugar content of tubers,particularly glucose and fructose contents, gradually decreased withlowering the water supply (Figs. 3A, 4 A and B). These results wereconsistent with those found by Wegener et al. (2017) who reported thatdrought stressed tubers presented lower concentration of total sugarsand also glucose and fructose than the well-watered control tubers. Incontrast, Eldredge et al. (1996) demonstrated that drought stress in-creases the sugar content in tubers. The contradiction was solved by theobservation of André et al. (2009) and Muttucumaru et al. (2015) whofound that the different genotypes were affected in dissimilar fashion bythe same treatment, indicating that there is no single, unifying potatotuber drought stress response. For all treatments, the sugar content,including glucose and fructose contents, was higher in PRD than in DItubers. The only exception was in the total sugar contents of tubersdeveloped at 50% of FC, which were equally low independent of theirrigation strategy used. This increase in sugars under PRD compared toDI was also observed by Battilani et al. (2014) in one year on potatogrown under field conditions in a sub-humid area, however, not inanother year which was particularly dry.

Potato tuber proteins are considered of important nutritional value(Knorr, 1978). In our study the protein content ranged from 0.57 to3.13%, while van Niekerk (2015) reported 1.49% in Mondial tubers.Nevertheless, water stress and irrigation management play an im-portant role in the determination of protein content in potato tubers.Drought stress generally decreases plant nitrogen concentration (Heand Dijkstra, 2014). Our results are consistent with this observation aswe showed that protein contents of tubers gradually decreased withlowering the level of irrigation (Fig. 2B). In addition, tubers from PRDtreatments presented higher protein content compared to DI for all ir-rigation levels. Similar results were reported by Wang et al. (2009) andSun et al. (2013) who demonstrated that PRD plants grown in pots hadsignificantly higher nitrogen contents in tubers compared to DI plants.This finding was confirmed also for potato plants grown under fieldconditions (Jovanovic et al., 2010; Ahmadi et al., 2016). The rise innitrogen uptake under PRD treatments may be explained either by theincrease in nitrogen mineralization rate due to the high frequency ofwetting/drying cycles (Jensen et al., 2010; Wang et al., 2010; Sun et al.,2013), or by an increase in root nitrogen absorption (Hu et al., 2009).

Polyphenols represent a large family of plant secondary metabolitesmainly hydroxycinnamic acid derivatives and flavonoids, implicated inthe prevention of various diseases such as cancers and cardiovasculardiseases (Evers and Deußer, 2012). In this study, we examined the ef-fect of PRD and DI on the total polyphenols of potato tubers. The resultsobtained (Fig. 2C) showed a significant increase in polyphenol contentin response to water restriction. Our results are similar to those ob-tained by Hamouz et al. (2010) who reported higher total polyphenolcontents in potatoes grown under drought and extreme temperatures.Highly cultivar-dependent variations of polyphenol content were ob-served by André et al. (2009), who proposed that the altered sucroseflux induced by the drought stress is partly responsible for the changesin expression of genes involved in polyphenol synthesis.

Water stress leads to increased accumulation of ROS, to detoxifythese reactive molecules, plants can intrinsically develop different typesof antioxidants reducing oxidative damage and conferring droughttolerance, they have both non-enzymatic antioxidants such as car-otenoid, flavonoids, α- tocopherol, ascorbic acid and glutathione, andenzymatic antioxidants such as catalase and peroxidase (Choudhuryet al., 2017). Antioxidant capacity depends on the variety,

Fig. 2. Effects of PRD and DI at 50, 70, 80 and 100% of FC irrigation levels onsugar (A), protein (B), polyphenol (C) and antioxidant (D) contents. FW-freshweight, DW-dry weight. Means presented within an irrigation method with thesame letter are not significantly different at P≤ 0.05. See SupplementaryTable 2 for details.

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environmental growth conditions, crop management, and post-harveststorage conditions (Dumas et al., 2003). Previous work highlighted thatpotato responses to drought stress are complex with levels of anti-oxidants showing increases, decreases, or remaining stable, dependingon the genotype and kind of antioxidant (André et al., 2009). Our datapresented in Fig. 2D, show a clear effect of water stress and irrigationstrategy on AA. For each irrigation level, AA was significantly higher inwater-stressed tubers and elevated under PRD compared to DI. Similarresults were obtained by Jovanovic et al. (2010) from a field experi-ment on potato showing that PRD treatment increased AA in potatotubers. They presumed that the increased AA was due to an increase oftotal phenolic content. Though, we also measured the total phenoliccontent of tubers we did not detect significant correlation between AAand phenolics (Table 2). Therefore, in our case, the high AA foundunder PRD could not be explained just by an increase in phenolics.

4.3. Metabolites and gene expression under PRD and DI

One of the novelties of our findings was the detection of doubleamount of mannitol in PRD than in DI tubers irrespective of soil watercontent. Mannitol is an osmoprotectant providing salt, osmotic, andoxidative stress tolerance to plants and is proposed to have a role(s) inprotecting cells and cellular structures against damage by ROS (Pateland Williamson, 2016). The difference in mannitol level and in con-centration of some other metabolites (Fig. 3A and 4C), as well as theslight, but statistically significant differences in sugar, protein and AAlevels (Fig. 2A, B, D and Supplementary Table 2) indicate that alternatewatering of root system is sensed by the plant even at 100% of FC.

Proline, like mannitol, has important roles in adaptation to osmotic

and dehydration stresses and also in redox control (Fichman et al.,2015). However, unlike mannitol that had an elevated level only in PRDtubers (Fig. 4C), proline concentration was increased in both PRD andDI tubers, however, only at 50% of FC (Fig. 4D). 50% soil water-loss is astrong stress for plants irrespective of the way of irrigation and that isreflected in higher proline concentration of tubers. The bifunctionalenzyme, P5CS catalyzes the first two steps of proline synthesis(Fichman et al., 2015). Nevertheless, expression of P5CS, like that of thegenes encoding enzymes involved in carbohydrate metabolism, was notin line with the level of corresponding metabolite. It is very likely thatthis is due to the three months storage of tubers at 4 °C that might re-sulted in degradation of mRNAs synthesized in the growing stage, whiledue to the very low activity of transcription factors at 4 °C only a lownumber of new mRNAs could be generated.

Despite of the presumed low activity of transcription factors largeamounts of mRNAs derived from SUT4, Susy, GBSS and MAD weredetected in PRD-80% tubers and also AGPase and FUMase expressionwas elevated in them to some extent (Fig. 5). This observation togetherwith the general picture presented in Fig. 3A on the elevated level ofmetabolites in PRD-80% tubers compared to DI-80% tubers suggeststhat there were more changes in PRD-80% than in DI-80% plantscompared to FC-100% plants both at transcriptome and metabolomelevels.

5. Conclusions

In conclusion, our results showed that the yield penalty with partialroot-zone drying (PRD) was similar to that caused by deficit irrigation(DI). The irrigation water-use efficiency (IWUE) was higher for all

Table 2Correlation coefficients among yield per plant, irrigation water-use efficiency (IWUE), dry weight (DW), ash content, total polyphenols content (TPC), antioxidantactivity (AA), protein content and total sugars of potato tubers grown under glasshouse conditions in 2016 and 2017.

Yield IWUE DW Ash TPC AA Protein Sugars

Yield 1.000IWUE – 0.144 1.000DW 0.573*** – 0.671*** 1.000Ash – 0.223 – 0.726*** 0.642*** 1.000TPC – 0.555*** 0.698*** 0.756*** – 0.494** 1.000AA 0.574*** 0.722*** – 0.462** – 0.890*** 0.111 1.000Protein 0.561*** – 0.568*** 0.690*** 0.490** – 0.565*** – 0.398* 1.000Sugars 0.624*** – 0.522*** 0.734*** 0.157 – 0.584*** – 0.443* 0.704*** 1.000

Bolds values represent significant correlations. ⁎, ⁎⁎, *** Significant at 0.05, 0.01 and 0.001 probability level, respectively.

Fig. 3. Heat-map (A) and dendrogram (B) ofthe metabolite changes in tubers related to PRDand DI at 50, 70, 80 and 100% of FC irrigationlevels. Means of data in log2 derived from 4groups of 12 tubers of 3–4 cm in diameter fromeach treatment are presented as colored boxes.The exception is DI 50% from which 9 tuberswere analyzed in 3 groups. See SupplementaryTable 3 for details. The heat-map and dendro-gram were generated by Heatmapper (http://www.heatmapper.ca).

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restricted water treatments compared to control, while no substantialdifferences in IWUE between PRD and DI occurred.

Regardless of the irrigation strategy used, water restriction de-creased tuber dry weight, sugar and protein contents, however, it

increased the amount of polyphenols and the antioxidant activity. Ourresults revealed that PRD influences plant growth by promoting steminitiation and reducing plant height.

Another important finding of the present work is the detection ofdouble amount of an osmoprotectant, mannitol, in PRD tubers irre-spective of water shortage level. The elevated concentration of man-nitol and some other metabolites, as well as the slight, but statisticallysignificant differences in sugar, protein and antioxidant activity levels,indicate that alternate watering of root system induces importantchanges at metabolome levels in tubers and is sensed by the plant evenat full irrigation. Transcript level of some key-genes involved in car-bohydrate metabolism was elevated at 20% water-saving in PRD, butnot in DI tubers confirming that the mode of irrigation is sensed by theplant.

Acknowledgements

This work was supported by MESRSFC/CNRST [grant numberPPR2/2016/23] and partially by the bilateral Hungarian-Moroccanresearch program [grant numbers TÉT_16-1-2016-0112 for theHungarian partner; CNRST/NRDIO 2017-2018 for the Moroccanpartner].

Appendix A. Supplementary data

Supplementary material related to this article can be found, in theonline version, at doi:https://doi.org/10.1016/j.agwat.2019.105745.

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