Precision phenotyping of contrasting potato (Solanum tuberosum L.) varieties in a novel aeroponics system for improving nitrogen use efficiency: In search of key traits and genes

Journal of Integrative Agriculture 2020, 19(1): 51–61 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Precision phenotyping of contrasting potato (Solanum tuberosum L.) varieties in a novel aeroponics system for improving nitrogen use efficiency: In search of key traits and genes Jagesh K. TIWARI, Sapna DEVI, Tanuja BUCKSETH, Nilofer ALI, Rajesh K. SINGH, Rasna ZINTA, Vijay K. DUA, Swarup K. CHAKRABARTI Indian Council of Agricultural Research-Central Potato Research Institute, Shimla 171001, India

Abstract With increasing population, degrading soil health, limited arable land area, and high cost of nitrogen (N) fertilizers, improving nitrogen use efficiency (NUE) of potato is an inevitable approach to save the environment and achieve sufficient tuber yields with less N fertilizer supply. Recently, we have developed an aeroponics system to study NUE in potato using genomics, physiology, and breeding approaches. This study aims on precision phenotyping of plants of two distinct potato varieties (Kufri Gaurav, N efficient; Kufri Jyoti, N inefficient) in the novel aeroponics system. Plants were grown in aeroponics under controlled conditions with low N (0.75 mmol L–1 NO3–) and high N (7.5 mmol L–1 NO3–) levels. Plant biomass, root traits, total chlorophyll content, and plant N were increased with increasing N supply, whereas higher NUE parameters namely NUE, agronomic NUE (AgNUE), N uptake efficiency (NUpE), harvest index (HI), and N harvest index (NHI) were observed at low N. An NUE efficient cv. Kufri Gaurav showed higher tuber dry weight, fresh tuber yield, tuber number per plant, early start of tuber harvesting, root traits, stolon traits, NUE parameters, and higher amino acid (aspartic acid and asparagine) content at low N supply. Higher expression of nitrate reductase (NR), nitrite reductase (NIR), and asparagine synthetase (AS) genes was observed in the leaf tissues of Kufri Gaurav at high N. Thus, aeroponics-based precision phenotyping enables identification of NUE efficient genotypes based on key traits and genes involved in improving NUE in potato. Further, this study suggests that the potential of aeroponics can be utilized to investigate N biology in potato under different N regimes. Keywords: aeroponics, precision phenotyping, potato, nitrogen use efficiency

1. Introduction

Received 21 September, 2018 Accepted 2 November, 2018 Correspondence Jagesh K. TIWARI, E-mail: jageshtiwari@gmail. com © 2020 CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). doi: 10.1016/S2095-3119(19)62625-0

Nitrogen (N) is the most important nutrient for plant growth and development including potato. Potato is the third most important food crop after rice and wheat in terms of human consumption. Potato is a resource intensive crop and requires high N fertilizer (150–240 kg N ha–1) to produce tuber yield (30–50 t ha–1) in India (Trehan et al. 2008). Despite the high cost of N fertilizer, potato crop uptakes nearly 50% of applied N and excess losses in the

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environment (Trehan and Singh 2013). The Indian soils are generally deficient in organic matter thus unable to meet crop N demand. Hence, application of inorganic N fertilizer is inevitable to meet the N requirement of crop. Overall, potato is a shallow rooted crop and irrigated cultivation is followed on sandy-loam soil with excessive application of N fertilizer which together increase chance of nitrate leaching and contamination to the groundwater (Elbl et al. 2014; Ospina et al. 2014; Plošek et al. 2017). Plant N uptake, utilization, and remobilization are complex physiological processes and researchers have attempted to understand the underlying mechanism of nitrogen use efficiency (NUE). Despite the considerable advancement in NUE research in model plants like Arabidopsis, rice, maize, success in releasing NUE genotypes is limited might be due to its complex genetics and interaction with environmental variables (Gutiérrez 2012). Potato crop normally acquires nitrate form of N from soil, even though ammonium-based fertilizers are applied (Zebarth and Milburn 2003). Though, in-season good N management has been followed in potato for improving NUE through soil-agronomic approaches and successful tuber production (Zebarth and Rosen 2007; Goffart et al. 2008; Vos 2009; Musilová et al. 2012). Improving NUE of plant is one of the key options to minimize N losses, save the cost of production and improve the environmental quality to achieve sustainable crop yield with less (or without) N fertilizer. Substantial genetic variation has been observed for NUEassociated traits in potato cultivars (Zebarth et al. 2004), advanced clones (Sharifi et al. 2007), and wild species (Zvomuya et al. 2002; Zebarth et al. 2008). Till now, most studies focussed on agronomic managements for improving NUE in potato, a few have assessed gene expression of nitrogen metabolism genes such as nitrate transporter (NRT), ammonium transporter (AMT), nitrate reductase (NR), nitrite reductase (NIR), and asparagine synthetase (AS). Researchers have measured N sufficiency in potato by gene expression analysis of N metabolism gene such as ammonium transporter AT1 (Zebarth et al. 2011, 2012) and NR (Li et al. 2010). Recently, we have developed an aeroponics system for seed potato production till full crop season (Buckseth et al. 2016) and highlighted its importance to study N biology applying integrated genomics, physiology, and breeding approaches (Tiwari et al. 2018). Moreover, knowledge about precision phenotyping in aeroponics, traits associated with NUE, root traits, genes involved in N metabolism and other parameters are very important to dissect the underlying N mechanism to improve NUE in potato. The aim of this study is to make precision phenotyping of contrasting potato varieties Kufri Gaurav (N efficient) and Kufri Jyoti (N inefficient) in aeroponics with high and

low N levels. Further aim is to identify traits and genes for improving NUE in potato plants in terms of plant biomass, NUE variables, amino acid profiling, and gene expression analysis.

2. Materials and methods 2.1. Plant materials Two contrasting potato varieties Kufri Gaurav (N efficient) and Kufri Jyoti (N inefficient) were used in the study at Indian Council of Agricultural Research-Central Potato Research Institute, Shimla, Himachal Pradesh, India (31.1048°N and 77.1734°E; and 2 276 m above mean sea level). These varieties were selected based on field studies (Trehan 2009; Trehan and Singh 2013). Healthy in vitro plants of these varieties were maintained at the institute and virus-free plants were used for the study.

2.2. Aeroponic culture An aeroponics system was established for potato crop growth as described by Buckseth et al. (2016). An aeroponic experiment was conducted in controlled growth chamber in winter season under short day conditions having photoperiod of 10 h with day temperature of (23±1)°C with average light intensity ~(200±10) µmol L–1 m–2 s–1 and 14 h with night temperature (18±1)°C. In vitro plants were grown in aeroponics following our standard practices (Buckseth et al. 2016). Each treatment was replicated two times. Potato varieties were grown with nutrient solutions at low N (0.75 mmol L–1 NO3–) and high N (7.5 mmol L–1 NO3–) levels. Nutrients solutions were prepared in reverse osmosis water (maintained pH ~7.0). Stock solutions were prepared using various salts: KNO3 (1 mol L–1), KH2PO4 (0.5 mol L–1), K2SO4 (0.5 mol L–1), Ca(NO3)2·4H2O (1 mol L–1), CaSO4·2H2O (0.5 mol L–1), MgSO4·7H2O (1 mol L–1), MnSO4·H2O (0.125 mol L–1), ZnSO4·7H2O (0.125 mol L–1), CuSO4·5H2O (0.005 mol L–1), Na2MoO4·2H2O (0.005 mol L–1), Fe-EDTA (0.0125 mol L–1), and H3BO3 (0.125 mol L–1). Nutrients composition of working low N solution was: 0.75 mmol L–1 N (NO3–), 0.5 mmol L–1 P, 3 mmol L–1 K, 1 mmol L–1 Mg, 4.38 mmol L–1 S, 0.0016 mmol L–1 Mn, 0.00008 mmol L–1 Zn, 0.004 mmol L–1 B, 0.00004 mmol L–1 Cu, 0.00004 mmol L–1 Mo, 0.0062 mmol L–1 Fe, and 2.5 mmol L–1 Ca; whereas high N (7.5 mmol L–1 NO3–) had same composition except 1 mmol L–1 S. Nutrient solutions were supplied to the plant roots for 30 s at an interval of every 5 min by spraying (mist form) through nozzles with an automated timer and motor-pump attached with pipe under the aeroponics system. Solutions were changed at every 7 days interval and pH of was maintained between 5.8–7.0

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using 1 mol L–1 H2SO4 or 1 mol L–1 NaOH. The crop was grown till 90 days after planting (harvest stage). All traits were measured by pooling of four plants from each treatment combinations and presented here on per plant basis.

2.3. Plant biomass Leaf area and plant height were determined at full vegetative growth stage (65 days after planting (DAP)). Plant (shoot and tuber) fresh weight and dry weight were determined at harvest stage (90 DAP). Plants were divided into aboveground (leaf and stem) and under-ground (root, stolon and tuber) parts (Zebarth and Milburn 2003). Total leaf area of plants was measured using the LI-3100C Area Meter (LICOR Biosciences, Lincoln, Nebraska, USA). Dry weight of samples was determined by drying in an oven at 70°C for 5–6 days until constant weight was achieved.

2.4. Estimation of total chlorophyll, N content, and NUE variables Fresh leaf tissue (100 mg) was processed for estimation of total chlorophyll content using the method described by Anderson and Boardman (1964). The absorbance of the chlorophyll fraction was measured at 645 and 663 nm against 80% acetone as a blank using the UV-1700 Spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Plant total N content of the samples was estimated based on dry weight basis using the modified Kjeldahl method (Chapman and Pratt 1961). All plant parts (shoot including leaf and stem, root including stolon, and tuber) were estimated separately. NUE variables were calculated for NUE (plant dry matter accumulation/crop N supply); N uptake efficiency (NUpE; plant N accumulation/crop N supply); N utilisation efficiency (NUtE; plant dry matter accumulation/ plant N accumulation); harvest index (HI; tuber dry matter accumulation/plant dry matter accumulation); N harvest index (NHI; tuber N accumulation/plant N accumulation); and agronomic NUE (AgNUE: fresh tuber yield/crop N supply) as described elsewhere (Zebarth and Milburn 2003; Zebarth et al. 2004, 2008).

2.5. Root traits Root traits were measured at full growth stage of root (60 DAP) using the EPSON Perfection V700 Photo root scanner (Seiko Epson Corporation, Suwa, Nagano, Japan) and root system architecture was analyzed using WinRHIZO Regular V 2009 Software (Arsenault et al. 1995). Root length (m), stolon length (m), root surface area (m2), stolon surface area (m2), root volume (cm3), stolon volume (cm3), root average diameter (mm), and stolon average diameter

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(mm) per plant were measured and reported in this study.

2.6. Amino acid profiling Leaf and tuber samples (1 mg of each on fresh weight basis) were processed for HPLC analysis following the procedures described by Gonzalez-Castro et al. (1997). A volume of 20 µL of the sample was loaded with setting of flow rate at 1 mL min–1 and detected at 254 nm using the reverse phase HPLC model 1200 series (Agilent Technologies, USA) following the run method as summarized in Appendix A. Amino acid standards (SIGMA catalogue nos. A6407 and A6282) were used to estimate the amino acid content in the samples.

2.7. Gene expression analysis Gene expression pattern was measured using the RT-PCR analysis. Root and leaf samples were collected from the aeroponics-grown plants at three stages: 30 DAP (prestolon initiation stage), 45 DAP (stolon initiation stage), and 60 DAP (tuber growth stage) and samples were snap frozen in liquid nitrogen till further use. Total RNA of each sample was isolated using RNeasy Plant Mini Kit following manufacturer’s instructions (Qiagen, Venlo, Limburg, the Netherlands) and quantified (260/280 nm>1.8) by a ND-1000 NanoDrop (Wilmington, USA) and verified by agarose gel electrophoresis. The RT-PCR primers were used from Li et al. (2010). cDNA synthesis was performed using 1 μg total RNA which was reverse-transcribed using TaqMan® Reverse Transcription Reagent (Applied Biosystems, New Jersey, USA). cDNA was used for RTPCR analysis using Power SYBR Green PCR Master Mix (Applied Biosystems Warrington, UK) in the ABI PRISM HT7900 following thermal cycler profiles 50°C for 2 min; 95°C for 10 min; and 40 cycles of 95°C for 15 s, 60°C for 1 min, 72°C for 30 s using internal standard of mitochondrial cytochrome oxidase gene (coxI) (X83206.1, Li et al. 2010) as described by Tiwari et al. (2015).

2.8. Data analysis Data were analysed with analysis of variance (ANOVA) using the XLSTAT 2018.5 (www.xlstat.com) and treatment means were compared using the Tukey’s test at least significant difference (P≤0.05).

3. Results 3.1. Phenotype and plant biomass Potato crop was grown successfully in aeroponics and

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an overview is depicted in Fig. 1. Significant differences (P≤0.05) were observed in most traits studied in potato varieties (Kufri Jyoti and Kufri Gaurav) grown in aeroponics with two N levels (low N: 0.75 mmol L–1 NO3–; high N, 7.5 mmol L –1 NO 3–) (Fig. 2). Both varieties differed significantly in terms of plant height and recorded the maximum in Kufri Jyoti at high N and the minimum in Kufri Gaurav at low N. Leaf area increased with increasing N and differed significantly in both varieties. Significant difference was observed for shoot dry weight at high N in both varieties and observed the maximum shoot dry weight in Kufri Jyoti at high N and the minimum in Kufri Gaurav at low N. Root dry weight varied significantly in both varieties at both N levels. High N resulted in significantly higher tuber yield of both fresh weight and dry weight in both varieties. Tuber number per plant differed significantly in both varieties at both N levels and recorded the maximum in Kufri Gaurav and the minimum in Kufri Jyoti at low N. Start of tuber harvesting was significantly different at low N-fed plants in both varieties, which had early harvesting than high N-fed plants.

A

3.2. Estimation of total chlorophyll, plant N, and NUE variables Results on total chlorophyll, plant N content, and NUE variables per plant are summarized in Fig. 2. Total chlorophyll content was significantly different except nonsignificant in Kufri Gaurav at both N levels and recorded the maximum value in Kufri Jyoti at high N. High N resulted in more total plant N content (shoot N, root N, and tuber N). Shoot N content was significantly different in both varieties at both N levels and estimated the maximum in Kufri Jyoti at high N and the minimum in Kufri Gaurav at low N. Similarly, significant difference was observed for root N content in both varieties, and the maximum root N content was recorded in Kufri Jyoti at high N and the mininum in Kufri Gaurav at low N. Tuber N content also differed significantly in both varieties at both N levels, which observed the maximum tuber N content at high N and the minimum at low N in Kufri Jyoti. Significant differences were observed for NUE variables such as AgNUE, NUE, NUpE, NUtE, HI, and NHI except non-significance of AgNUE at high N in both varieties. Significantly higher AgNUE, NUE, and NUpE were observed in Kufri Gaurav than in Kufri Jyoti at low N. In other words, significantly lower AgNUE, NUE, and NUpE were recorded at low N than at high N in both varieties. However, Kufri Jyoti recorded significantly higher NUtE than Kufri Gaurav at low N. Significantly higher HI was observed in Kufri Gaurav at low N and the minimum in Kufri Jyoti at high N. Similarly, significant differences were observed for NHI in both varieties at both N levels, and the maximum NHI was observed in Kufri Gaurav at low N. Overall, Kufri Gaurav recorded the maximum NUE variables such as AgNUE, NUE, NUpE, HI and NHI than Kufri Jyoti at low N.

3.3. Root traits B KG7.5N

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Fig. 1 Potato growth in aeroponics system with low nitrogen (N) and high N supplies. A, crop view at 60 days after planting. B, root biomass with tuber growth. KJ, Kufri Jyoti; KG, Kufri Gaurav. Low N, 0.75 mmol L–1 NO3–; high N, 7.5 mmol L–1 NO3–.

Root and stolon traits (length, surface area, average diameter, and volume) were measured on per plant basis in both varieties (Fig. 2). Significant differences were observed in terms of root length in both varieties, except non-significance between Kufri Jyoti at high N and Kufri Gaurav at low N. Significant differences were observed for root surface area in both varieties, except non-significance in Kufri Jyoti at both N levels. Both root length and root surface area were observed the maximum in Kufri Gaurav at high N. Root average diameter was non-significant in all treatment combinations except Kufri Jyoti at low N. Root volume was significantly different except non-significance between Kufri Gaurav at high N and Kufri Jyoti at low N. Significant differences were observed for stolon length and stolon surface area at both N levels, except nonsignificant stolon length between Kufri Jyoti at high N and

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Fig. 2 Performance of potato varieties (Kufri Jyoti (KJ) and Kufri Gaurav (KG)) grown under aeroponics with low N (nitrogen) 250 –1 400 (0.75 mmol L–1 NO3– (0.75N)) and high N (7.5 mmol L NO3– (7.5N)) levels for phenotype, NUE use efficiency)-associated 20 000(nitrogen a a a 350 traits, and root traits. All traits are measured per 200 plant basis. NUtE, N utilisation efficiency;18NUpE, N uptake efficiency. DAP, days 000 300 after planting. Different letters on bar indicate significant difference (P<0.05) between the16treatments, whereas same letter is not 000 250 significantly different. Bars indicate NUE related 150 traits studied in potato. Bars mean SE. b 14 000 12 000 10 000 8 000 6 000 4 000

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Kufri Gaurav at low N. Maximum stolon length and stolon surface area values were observed in Kufri Gaurav at high N and the minimum in Kufri Jyoti at low N. Stolon average diameter was significant in Kufri Gaurav and non-significant in Kufri Jyoti at both N levels. Stolon volume was observed significantly different in both varieties at both N and recorded the maximum in Kufri Jyoti at high N.

3.4. Amino acid profiling Amino acid content was analysed in leaf and tuber samples of both varieties grown in aeroponics at high and low N levels. Results of amino acid content are summarized in Table 1. Standard retention time (min) used in HPLC analysis and chromatogram of standard amino acid mixture are given in Appendices B and C. Amino acids are categorised into various categories like i) essential and semi-essential amino acids (cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryphtophan, tyrosine, and valine), ii) non-essential amino acid (alanine, arginine, aspargine, aspartic acid, glutamic acid, glycine, proline, and serine), and iii) amino acid mixture compounds (amino adipic acid, anserine, carnosine, cystathionine, OH lysine, OH proline, ornithine, phoshoserine, phosphoenolamine, taurine, 1-methyl histidine, 3-methyl histidine and β-amino

butyric acid). In shoots, Kufri Jyoti showed higher content (>10 mg 100–1 mg fresh weight) of threonine, glutamic acid, and carnosine at high N; and aspartic acid, carnosine, and phoshoserine at low N; whereas, Kufri Gaurav showed higher values of threonine, aspartic acid, glutamic acid, amino adipic acid, carnosine, and phoshoserine at high N; and histidine, threonine, aspartic acid, glutamic acid, carnosine, and phoshoserine at low N. In tubers, Kufri Jyoti recorded higher amino acid content (>10 mg 100 mg–1 fresh weight) of methionine, phenylalanine, threonine, tyrosine, arginine, aspargine, aspartic acid, glutamic acid, serine, anserine, carnosine, OH proline, and phoshoserine at high N; and cysteine, histidine, tyrosine, aspargine, aspartic acid, glutamic acid, serine, carnosine, phoshoserine, and taurine at low N; whereas, Kufri Gaurav showed higher contents of histidine, threonine, tyrosine, aspargine, aspartic acid, glutamic acid, serine, carnosine, OH proline, and phoshoserine at high N; and threonine, aspargine, aspartic acid, glutamic acid, amino adipic acid, carnosine, and phoshoserine at low N. Overall, Kufri Gaurav, an N use efficient potato variety, showed higher content of essential amino acid threonine, and non-essential amino acid like asparagines, aspartic acid, and glutamic acid than Kufri Jyoti at both N levels.

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Table 1 Amino acid content (mg 100 mg–1 fresh weight±standard deviation) of potato varieties grown in aeroponics with low N (nitrogen) (0.75 mmol L–1 NO3– (0.75N)) and high N (7.5 mmol L–1 NO3– (7.5N)) supply Shoot Kufri Jyoti Kufri Gaurav 7.5N 0.75N 7.5N 0.75N Essential and semi-essential amino acids 1 Cysteine 0 8.78±1.08 5.16±0.58 0 2 Histidine 0 1.24±0.5 0 14.71±0.82 3 Isoleucine 0 0 0 1.50±0.57 4 Leucine 5.06±1.05 3.21±0.59 4.75±0.12 0 5 Lysine 0 0 0 0 6 Methionine 0 0 0 3.88±0.58 7 Phenylalanine 2.56±0.52 0 0 1.30±0.12 8 Threonine 80.01±2.51 6.79±0.92 17.12±1.5 26.74±1.62 9 Tryphtophan 0 0 0 0 10 Tyrosine 0 0 9.32±0.54 5.20±0.47 11 Valine 0 0 0 0 Non-essential amino acids 12 Alanine 7.53±0.27 4.57±1.06 7.30±0.54 0 13 Arginine 0 0 0 0 14 Aspargine 5.27±1.01 5.90±0.81 2.36±0.5 1.20±0.5 15 Aspartic acid 0 129.35±11.55 57.36±1.57 51.291±5.48 16 Glutamic acid 19.24±1.52 0 11.42±0.56 11.16±1.58 17 Glycine 1.32±0.26 0.76±0.05 0.45±0.11 0.43±0.04 18 Proline 9.276±0.5 2.57±0.5 0 0 19 Serine 2.35±0.13 1.24±0.15 0 0 Amino acid mixture compounds 20 Amino adipic acid 8.03±0.12 0 14.471±0.77 3.37±0.84 21 Anserine 0 0 0 2.20±1.30 22 Carnosine 77.08±3.56 48.86±2.57 74.16±1.57 37.01±2.32 23 Cystathionine 0 0 0 3.89±0.32 24 OH lysine 0 0 0 0 25 OH proline 0 1.77±0.27 0.84±0.02 0.893±0.05 26 Ornithine 0 0 0 1.87±0.17 27 Phoshoserine 0 10.66±1.56 56.53±2.53 24.62±2.72 28 Phosphoenolamine 0 0 0 0 29 Taurine 0 0 1.63±0.58 0 30 1-Methyl histidine 0 0 0 0 31 3-Methyl histidine 0.78±0.14 0.45±0.08 0 0 32 β-Amino butyric acid 1.18±0.18 0.36±0.25 0.68±0.08 0

No.

Amino acid mixture

Tuber Kufri Jyoti 7.5N 0.75N

Kufri Gaurav 7.5N 0.75N

4.06±1.25 16.54±2.32 3.57±1.32 0 0 32.08±5.12 24.05±2.65 0 3.01±1.02 2.91±1.32 0.74±0.12 1.85±0.35 0 0 5.54±0.82 0 0 0 0 0 19.36±3.21 4.60±1.25 4.17±2.04 3.77±1.32 11.24±1.52 8.63±2.56 2.18±0.82 2.15±0.81 494.43±14.82 0 648.67±12.57331.14±22.62 0 0 0 0 61.14±2.21 26.28±2.65 14.17±3.20 7.56±2.01 6.31±1.38 5.90±1.32 0 0 0 0 0 0 25.41±4.42 0 0 0 53.57±3.62 28.11±2.84 61.66±7.21 43.59±2.43 21.66±1.64 103.54±7.42 287.34±11.21175.74±15.02 34.93±1.32 42.24±5.82 17.17±1.20 13.22±2.03 2.34±0.21 1.74±0.32 1.97±1.21 0.96±0.20 0 0 0 1.89±1.32 19.64±2.32 10.26±2.56 14.09±2.54 2.39±0.21 0 0 0 14.41±1.92 11.06±1.72 2.65±1.22 0 0 165.30±10.26 63.73±8.21 157.94±10.32102.54±11.82 0 7.53±0.62 0.94±0.32 0.85±0.42 0 0 0 0 19.93±0.72 1.45±1.04 17.73±2.32 0.93±0.32 0 0 4.12±1.02 2.07±0.32 49.64±2.43 17.08±3.32 43.81±3.32 35.11±4.82 0 3.17±0.11 6.86±1.25 1.95±0.82 0 33.67±3.25 0 0 0 1.78±1.01 8.02±2.02 5.48±0.79 5.91±0.21 3.10±1.43 2.96±1.32 0 4.21±0.82 2.70±1.21 3.13±1.32 1.88±1.32

Values are mean±SE.

3.5. Gene expression analysis Gene expression was analysed in the leaf and root tissues by real time polymerase chain reaction (RT-PCR) using candidate genes-specific primers involved in N metabolism in potato (Fig. 3). NR, NIR, and AS genes were monitored in shoot, and ammonium transporter and nitrate transporter genes were analyzed in root samples at three stages (30, 45, and 60 DAP). Relative up-regulation of NR, NIR, and AS genes was observed in the leaf tissues of high N than low N in both varieties at all stages. In particular, higher expression of NR, NIR, and AS genes was observed in shoots of Kufri Gaurav than Kufri Jyoti at both N levels. Similarly, higher expression of NRT and AMT genes was observed in high N-fed plants than in the corresponding varieties at low N. Moreover, higher expression of AMT was observed than

NRT throughout the stages, especially at later growth stage (60 DAP). Relative expression of NRT gene was higher in Kufri Gaurav than in Kufri Jyoti at high N during the early growth (30 DAP) and stolon formation stages (45 DAP).

4. Discussion 4.1. Plant phenotype and NUE traits Precision phenotyping is essential to identify key traits involved in improving NUE of potato plants. In this study, two contrasting potato varieties (Kufri Gaurav and Kufri Jyoti) were evaluated under the novel aeroponics system at high and low N in terms of plant biomass, plant N content, NUE variables, root traits, amino acid content, and gene expression analysis. The varieties showed significant

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Jagesh K. TIWARI et al. Journal of Integrative Agriculture 2020, 19(1): 51–61

Relative gene expression (fold change)

14 12

NR

10

NIR

8

AS

Shoot

6 4 2 0

KJ0.75N KJ7.5N KG0.75N KG7.5N KJ0.75N KJ7.5N KG0.75N KG7.5N KJ0.75N KJ7.5N KG0.75N KG7.5N 30 DAP

60 50

NRT

40

AMT

45 DAP

60 DAP

Root

30 20 10 0

KJ0.75N KJ7.5N KG0.75N KG7.5N KJ0.75N KJ7.5N KG0.75N KG7.5N KJ0.75N KJ7.5N KG0.75N KG7.5N 30 DAP 45 DAP 60 DAP

Fig. 3 Relative gene expression (fold change) of candidate genes involved in nitrogen (N) metabolism in potato leaf and tuber samples grown in aeroponics with low and high N supply. NR, nitrate reductase; NIR, nitrite reductase; AS, asparagine synthetase. KJ, Kufri Jyoti; KG, Kufri Gaurav. 0.75N, 0.75 mmol L–1 NO3–; 7.5N, 7.5 mmol L–1 NO3–. DAP, days after planting. Bars indicate relative gene expression in varieties at both N levels. Bars mean SE.

variation for the traits studied. Plant biomass (plant height, leaf area, shoot dry weight, root dry weight, tuber dry weight, and fresh tuber yield) per plant increased with high N. On contrary, NUE variables (AgNUE, NUE, NUpE, NUtE, HI, and NHI) were observed higher with low N-fed plants. Total plant N increased with high N supply. It has been shown in many studies that plant biomass increases with increasing N and higher NUE is observed with low N supplied plants (Errebhi et al. 1998, 1999; Zebarth et al. 2004). In our study, Kufri Gaurav showed higher tuber dry weight, fresh tuber yield, and tuber number per plant than Kufri Jyoti at low N, and start of tuber harvesting was also early at low N in both the varieties. High N promotes higher plant biomass, more shoot growth, higher growth rate of leaf area, taller plants, high N content in leaf chlorophyll and hence delays tuber induction and tuber growth processes (Krauss 1985; Biemond 1995), where N reallocation in plant affects ~20–40% tuber dry matter content (Vos 1999). Thus, Kufri Gaurav had significantly higher plant biomass (root dry weight per plant, tuber dry weight per plant, fresh tuber yield per plant, and tuber number per plant) and higher NUE variables (AgNUE, NUE, NUpE, HI, and NHI) than Kufri Jyoti at low N. Overall, based on aeroponics phenotyping, Kufri Gaurav was observed an NUE potato variety. Our results are consistent with earlier findings. Potato

varieties show significant differences for NUE parameters and selection based on tuber yield under low N is suggested to screen genotypes for improving NUE (Errebhi et al. 1998, 1999; Zebarth et al. 2004). Previous researchers observed significant effect of N supply on total dry weight and N content with high regression coefficient (Errebhi 1998). Zebarth (2004) observed more variation in N uptake capacity (plant N accumulation in abundant N supply) than NUpE (plant N content per unit crop N supply) in potato varieties. Plant N accumulation and root dry weight traits are indicator of N uptake efficiency under low N. Since, plant N accumulation and plant total dry weight are highly correlated, total dry weight is suggested as an alternative criterion for screening for NUE (Vos 1997). Significant difference was observed in potato varieties in terms of N concentration in tuber dry matter, which increases with N uptake, on contrary harvest index for dry matter and N decease with N uptake (Vos 1997). Low N supply causes low leaf area index, low leaf N concentration, and low tuber yield (Hu et al. 2014). In other studies, an in vitro method of screening of potato genotypes at early stage, showed increased root growth and high N utilization capacity under low N (Schum and Jansen 2014). Besides, hydroponics system has also been demonstrated to screen potato genotypes in 30-day crop (Sharifi et al. 2007).

Jagesh K. TIWARI et al. Journal of Integrative Agriculture 2020, 19(1): 51–61

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4.2 Root traits

4.4. Gene expression analysis

In this study, root biomass (root length, root surface area, root volume, stolon length, stolon surface area, and stolon volume) increased with increasing N. The NUE variety, Kufri Gaurav, showed higher root length, root average diameter, root volume, stolon length, stolon surface area, and stolon average diameter than Kufri Jyoti at low N. Moreover, as above discussed higher root dry weight and tuber dry weight were observed in Kufri Gaurav at low N than in Kufri Jyoti. Moreover, it has been suggested that basal roots are primarily involved in plant anchorage and water uptake, where as stolon roots are more associated with nutrient uptake and tuber growth processes (Villordon et al. 2014) and thus improving NUE of Kufri Gaurav. Variation in root (basal and stolon) traits has been measured earlier in potato in both glasshouse and field, and developed screening technique based on root phenotype (Wishart et al. 2013) and root biomass (Sattelmacher et al. 1990; Iwama 2008). In our aeroponics system, there is no stress on root growth and hence larger root biomass was observed unlike soil-based field studies (Wishart et al. 2013), which might be helpful in various environments like drought (Iwama 2008). Thus, based on higher root length, root volume, stolon length, and stolon surface area at low N, NUE variety genotype could be identified like cv. Kufri Gaurav.

Gene expression analysis showed up-regulation of NR, NIR, and AS transcripts in the shoots of high N-supplied plants than those of low N. Kufri Gaurav showed higher expression of all genes than Kufri Jyoti in shoot tissues. In roots, NRT expressed higher during the early stages (30 and 45 DAP) in Kufri Gaurav than in Kufri Jyoti, while AMT expressed higher in all stages in both varieties. Earlier studies showed the reduction in gene expression of nitrate reductase, nitrite reductase, and asparagine synthetase was observed under low nitrate fed plants, except a few cultivars (Li et al. 2010). Stems and leaves are responsive to N supply and accumulate more nitrate, and nitrate reduction is observed in the canopy of potato (Mäck and Schjoerring 2002). Low nitrate level is observed in the leaves formed later in the season might cause low response of NR and NiR genes in later season. However, expression of AT1 genes was relatively constant across the season (Zebarth et al. 2011) and less influenced by N form, and expression of AT1 was increased under low N supply and independent of growth stages and hence, it is suggested to use for measuring plant N status in potato (Zebarth et al. 2012). Expression of NR transcripts was detected only in leaf and stem at low N, while they could also be detected in root and stolon at high N (Harris et al. 2000). In other crop, BraNRT2.1 gene is a high-affinity nitrate transporter and its highest expression was observed at 5 mmol L–1 N in non-heading Chinese cabbage (Liu et al. 2014). Transcription levels of NRT1 and NRT2 gene families (TaNRT1.1, TaNRT1.3, TaNRT1.4, TaNRT1.7, TaNRT1.8, TaNRT2.1, TaNRT2.2, and TaNRT2.3) were significantly increased during 2 to 6 days after N starvation in wheat roots, whilst TaNRT1.5 and TaNRT2.4 genes were inhibited, and TaNRT1.2 and TaNRT2.5 genes were induced (Guo et al. 2014). Taken together, contrasting potato varieties were evaluated in aeroponics to identify traits and genes associated in improving NUE in terms of plant biomass, total chlorophyll, plant N content, NUE variables, root traits, gene expression, and amino acid content. This aeroponics study demonstrates that NUE potato genotypes (for example, cv. Kufri Gaurav) can be identified based on the traits such as higher plant biomass (root dry weight, tuber dry weight, fresh tuber yield, tuber number per plant) at high N; higher NUE variables (AgNUE, NUE, NUpE, HI, and NHI) at low N; higher root traits (root length and root volume); and stolon traits (stolon length and stolon surface area) at low N; higher content of threonine (essential amino acid), and asparagine as well as aspartic acid (non-essential amino acid) in tubers at low N; and higher expression of NR, NIR, AS, NRT, and AMT genes at both high and low N levels. The aeroponics system is a soil-less cultivation and has added advantages

4.3. Amino acid analysis Amino acid profiling showed variable response to low and high N in leaf and tuber samples. Threonine (essential amino acid), and asparagine as well as aspartic acid (nonessential amino acid) were found higher in the tubers of Kufri Gaurav than those of Kufri Jyoti at both N levels. Whereas, in amino acid mixture compounds, carnosine (consists of alanine and histidine) and phoshoserine (ester of serine and phosphoric acid) were found relatively higher in leaf and tuber samples of both varieties grown at both N levels. It has been studied earlier that among amino acid and amides, aspartic acid and asparagines predominate in potato tubers (Qsaki et al. 1995). The free amino acid pool in potato contains 40–60% of total N that includes mainly amides, glutamine, and asparagines (Desborough 1985). Amino acid composition and nutritional value were analyzed in potato (Galdón et al. 2010; Ježek et al. 2011; Bártová et al. 2015) and sulphur-containing amino acid (cysteine and methionine) were found limiting in all potato cultivars and lysine was limiting in a few varieties (Galdón et al. 2010). Bártová et al. (2015) observed high level of lysine, phenylalanine, tyrosine, leucine, valine, histidine, and threonine in potato cultivars.

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over conventional method (e.g., soil or sand-based screening in pot) such year the round potato cultivation with controlled conditions (mainly light and temperature), more precision and accuracy on N supply through nutrient solutions, no restriction of plant growth, no N leaching loss and evaluation of more genotypes within limited time using in vitro plants and most importantly, aeroponics enables better study of root system architecture (Sharifi et al. 2007; Wishart et al. 2013; Tiwari et al. 2018).

5. Conclusion This study provides insights of the novel aeroponics system for precision phenotyping of potato to identify traits (roots and shoots) to improve NUE. Precise phenotyping for plant biomass, NUE variables, root traits, amino acid profiling, and gene expression analysis showed implication of aeroponics in improving NUE by genetic interventions in target traits. Further, studies on more genotypes evaluation under aeroponics, 15N-labelled flux, RNA-sequencing based transcriptomes, and other physiological cum biochemical parameters are in progress. Moreover, validation of aeroponics findings in the field would also be required to improve NUE in potato.

Acknowledgements The authors are grateful to the Competent Authority, Indian Council of Agricultural Research (ICAR)-Central Potato Research Institute (CPRI), Shimla, Himachal Pradesh, India for necessary supports under the Biotechnology Program and the CABin Scheme (ICAR) (HORTCPRICIL 201500300131). Appendices associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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