Physiological evaluation of nitrogen use efficiency of different apple cultivars under various nitrogen and water supply conditions

Journal of Integrative Agriculture 2020, 19(3): 709–720 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Physiological evaluation of nitrogen use efficiency of different apple cultivars under various nitrogen and water supply conditions WANG Qian1*, LIU Chang-hai1*, HUANG Dong1, DONG Qing-long 1, LI Peng-min1, Steve van NOCKER2, MA Feng-wang1 1

State Key Laboratory of Crop Stress Biology for Arid Areas/Shaanxi Key Laboratory of Apple, College of Horticulture, Northwest A&F University, Yangling 712100, P.R.China 2 Department of Horticulture, Michigan State University, East Lansing, MI 48824, USA

Abstract Nitrogen (N) deficiency is a common problem for apple (Malus×domestica) production in arid regions of China. However, N utilization efficiency (NUE) of different apple cultivars grown under low N conditions in arid regions has not been evaluated. In this study, NUE was assessed for one-year-old seedlings of six apple cultivars, Golden Delicious, Qinguan, Jonagold, Honeycrisp, Fuji and Pink Lady, grafted onto Malus hupehensis Rehd. rootstocks. Four treatments were used, including control water with control N (CWCN), limited water with control N (LWCN), control water with low N (CWLN) and limited water with low N (LWLN). Our results showed that growth indices such as biomass, plant height and stem diameter, and photosynthetic rate of all cultivars decreased in the order CWCN>CWLN>LWCN>LWLN. When subjected to LWLN treatment, Qinguan showed better growth and photosynthetic characters than other tested cultivars. Additionally, Qinguan and Pink Lady had higher NUE, while Honeycrisp and Jonagold had lower NUE, based on the determination of biomass, photosynthetic parameters, chlorophyll content, the maximal photochemical efficiency of PSII (Fv/Fm), 15N and N contents. Keywords: apple, drought, N deficiency, physiological indices, 15N-labeling, NUE

1. Introduction In many regions, drought is becoming one of the most important factors limiting plant growth and development. Received 21 August, 2019 Accepted 1 November, 2019 WANG Qian, E-mail: [email protected]; LIU Changhai, E-mail: [email protected]; Correspondence LI Peng-min, Tel: +86-29-87082613, E-mail: [email protected]; MA Fengwang, Tel: +86-29-87082648, E-mail: [email protected]; [email protected] * These authors contributed equally to this study. © 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)62848-0

Moreover, the adverse effects of drought on plants negatively affect the sustainable development of agriculture (Luo et al. 2009; Wang et al. 2016). Plants, which cannot escape from adverse environmental conditions, have evolved two response mechanisms to drought stress: drought avoidance, by enhancing water uptake and reducing water loss by stomatal adjustment or changes in root architecture (Aaltonen et al. 2017) and drought tolerance, by adjusting osmotic potential and homeostasis

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of reactive oxygen species (ROS) (Holland et al. 2016). Liu et al. (2012) showed that apple genotypes and water supply levels had significant effects on tree height, trunk diameter, and dry biomass. The growth traits of different genotypes of Catalpa bungei were significantly suppressed by drought and the intrinsic water use efficiency (WUE) was significantly elevated upon re-watering (Zheng et al. 2017). Under PEG-induced drought stress conditions, the values of shoot dry mass of different rice cultivars were significantly reduced (Ding et al. 2015). Water deficit strongly reduced the photosynthetic activity of winter wheat and apple (Shangguan et al. 2000; Huang et al. 2018a). Nitrogen (N) is essential for plants, and is a major element in proteins, nucleic acids, phospholipids, chlorophyll, hormones, vitamins, and alkaloids (O’Brien et al. 2016). When subjected to low N, plants generally show less growth, lower chlorophyll content, and decreased photosynthetic parameters (Nunes-Nesi et al. 2010; Gan et al. 2016). Insufficient N not only changed the efficiency of the PSII and photochemical quenching (Shangguan et al. 2000), but also led to reduction of effective leaf area, which decreases plant biomass accumulation and yield (Ren et al. 2015). Under salt stress conditions, Populus simonii can increase the absorption of N to adapt salt stress (Zhang et al. 2014). Plants species vary in their preferences for different types of inorganic N sources, and NUE may be mainly controlled by genotype (Huang et al. 2007; Britto and Kronzucker 2013). Water and N availability both limit agricultural production in arid land independently, but their combined effects are synergistic (Xuan et al. 2017). Studies have shown that the uptake of N, including nitrate and ammonium, both decreased under drought stress (Fotelli et al. 2002; Gebler et al. 2005; Meng et al. 2016). Appropriate N application improves plant drought tolerance. For example, Huang et al. (2018b) reported that the negative effect of drought stress on Malus prunifolia was alleviated when more N was available. Similarly, water absorption ability of rice was enhanced under high N supplied conditions (Ren et al. 2015). A decrease in both soil water and N could cause variation in carbon distribution between shoots and roots, e.g., low N and water level both promoted below-ground carbon allocation (Ibrahim et al. 1997). In tomato, dry matter accumulation and WUE were affected by different N forms and concentrations (Claussen 2002). Different wheat genotypes exhibited varied root growth and above-ground biomass under different water and N supply levels (Huang et al. 2007). Apple is an important horticultural crop throughout the world because of its high nutritional and economic value. China dominates global apple production, and the main cultivation regions are arid areas which are N deficient as well (Li et al. 2009; Zhao and Dai 2015). In these

areas, increasing use of chemical fertilizers has led to high production costs and environmental pollution (Ahmad et al. 2008). By contrast, insufficient N supply results in weak growth and yield reduction. Different apple cultivars have varied WUE and N use efficiency (NUE), likely due to differences in leaf morphology, anatomical structure and photosynthetic properties of leaves (Rao et al. 1995; Wang et al. 2018). Therefore, selection of cultivars with high WUE and NUE is an important consideration (Condon et al. 2004; Morison et al. 2008). However, with few exceptions, natural variation in NUE under drought conditions has not been documented in apple (Dijkstra et al. 2016). In this study, we screened six, commercially important apple cultivars in China for NUE under drought and low N conditions, as a first step to identify genes involved in NUE regulation under drought condition and apple genotypes optimal for production in arid, N-deficient regions.

2. Materials and methods 2.1. Plant materials and experimental treatments All experiments were conducted at Northwest A&F University, Yangling, Shaanxi Province, China (34°20´N, 108°24´E) in a semi-open greenhouse, which had a transparent plastic roof but no walls to keep consistent with the outside air environment. One-year-old seedlings of six apple cultivars, Golden Delicious, Qinguan, Jonagold, Honeycrisp, Fuji and Pink Lady, were grafted onto Malus hupehensis rootstocks. Rootstocks were planted in January 2017 and the grafting was conducted in March 2017. Plants were grown in pots (38 cm×23 cm) in a mixture of two parts sand and one part soil. The soil was loess, which has very low nutrient content and is a common soil type in Northwest China. Four conditions were used in the experiment, with two N levels (control-N and low-N) combined with two water treatments (control-water and low-water). To create different nitrate concentration conditions, pots were treated with modified Hoagland nutrient solution (1.0 mmol L–1 K2HPO4, 2.0  mmol L–1 MgSO4·7H2O, 2.5  mmol L–1 FeSO4·7H2O, 2.5  mmol L–1 EDTA-Na2, 0.046  mmol L–1 H3BO3, 0.0067  mmol L–1 MnCl2·4H2O, 0.00077  mmol L–1 ZnSO4·7H2O, 0.00032 mmol L–1 CuSO4·5H2O, 0.00011 mmol L–1 H2MoO4·H2O, pH 6.0) containing either 6 mmol L–1 nitrate (control-N) or 0.01 mmol L–1 nitrate (low-N). Control water was 65–75% field capacity, and water deficit was 45–55% field capacity. The drought treatment started when the grafted seedlings had 20 fully expanded leaves, and continued for 70 days until harvest. The N treatment started on the 10th day of drought treatment and lasted for 60 days until harvest. Experiments comprised 30 seedlings for each combined treatment of N and water.

WANG Qian et al. Journal of Integrative Agriculture 2020, 19(3): 709–720

Seedlings were watered to saturation and the weight of the pots was recorded prior to water-deficit treatment. The water-limiting treatment was  equivalent to moderate soil drought mimicking the situation in Northwest Loess Plateau apple production areas (Liu et al. 2012). Since the initiation of drought treatment, 20 pots were randomly selected and weighed, and the amount of water equivalent to transpiration and evaporation was supplemented daily. Five seedlings from each treatment were fertilized with 0.4 g 15N-labeled urea (CO(15NH2)2) (Shanghai Research Institute of Chemical Industry, China; abundance of 10.14%), while the other 25 seedlings from each treatment were fertilized with 0.4 g of non-labeled urea. The experiment ended on the 70th day of the drought stress treatment (the 60th day of N stress). At the end of the treatment, 10 leaves from each treatment were selected to evaluate photosynthetic parameters and chlorophyll fluorescence. Afterwards, the leaves were used to determine chlorophyll content. Finally, the fresh weight (FW) and dry weight (DW) of five seedlings in each treatment were determined, and then the fully triturated samples were used for determination of N, P, K and 15N contents.

2.2. Determination of growth indices At the end of the treatment, five seedlings from each treatment were selected for evaluation of growth indicators. Shoot height (SH) was measured from the graft union to terminal bud, and stem diameter (SD) was determined using an electronic caliper 10 cm above the  graft union. FW and DW of the seedlings included root, stem, and leaf. The relative growth rate (RGR) was computed following the method of Radford (1967): RGR=(lnDW2–lnDW1)/(t2–t1), where DW1 is plant dry weight at day 0 (t1), and DW2 is plant dry weight at day 70 (t2).

2.3. Determination of photosynthetic parameters and the maximal photochemical efficiency of PSII (Fv/Fm) in leaves At the end of the treatments, 10 leaves were selected randomly to measure net photosynthetic rate (P n ), transpiration rate (Tr), stomatal conductance (Gs) and intercellular CO 2 concentration (C i) using a portable photosynthetic measurement system (Li6400; LICOR, Huntington Beach, CA, USA) between 09:00 and 11:00 on a sunny day without clouds. The measurement parameters were set as follows: photons, 1 000 µmol m–2 s–1; constant airflow rate of 500 µmol s–1; concentration of cuvette CO2, 400 µmol CO2 mol–1 air. After measurement of photosynthetic parameters, seven mature leaves were used for determination of chlorophyll fluorescence. Fully dark-adapted leaves were used to determine the Fv/Fm,

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using a Multi-Functional Plant Efficiency Analyzer (M-PEA, Hansatech Instruments, UK).

2.4. Determination of chlorophyll content in leaves Fresh leaf samples (100 mg) were immersed in 8 mL of 80% acetone in dark for 24 hours, at which point the leaves were white. The absorbance of the extracting solution at 663 and 645 nm was determined using a UV-1750 ultraviolet spectrophotometer (Shimadzu, Kyoto, Japan). The chlorophyll a, chlorophyll b and total chlorophyll contents were calculated according to the method of Arnon (1949).

2.5. Determination of N, P and K content Fully triturated samples of leaf, stem and root were used for determination of N, P, and K content. Samples (200 mg) were digested with concentrated sulfuric acid (H2SO4, AR, 98%) and H2O2 (GR, ≥30%). After addition of 100 mL of deionized H2O, N and P concentrations were obtained with a continuous flow analyzer (Auto Analyzer 3; SEAL Analytical, Norderstedt, Germany), while the K concentration was analyzed with a flame photometer (M410; Sherwood Scientific Ltd., Cambridge, UK). The total N uptake of the seedling was calculated as the sum of the N absorption by each organ, that is, the N content of each organ multiplied by the dry weight of that organ.

2.6. Determination of 15N Stable N isotope ratio (δ15N) was determined using a Flash 2000HT elemental analyzer, coupled with an isotope ratio mass spectrometer (Finnigan DELTA V Advantage, Thermo Fisher Scientific, Inc.). Ratios were indicated by δ-unit notation and were calculated as δ15N (‰)=[(Rsample/ Rstandard)–1]×1 000, where R is the 15N/14N ratio for N and the Rstandard for the 15N tests was Atm-N2 (Feng et al. 2018). Laboratory standards (protein, glycine, and/or urea) were used routinely to ensure that the analytical precision of δ15N was ±0.2‰. Leaf, stem, and root 15N was calculated by multiplying the N concentration by the DW. 15N utilization efficiency (15NUE) was calculated as the ratio of total 15N content in the seedling to total N in the fertilizer (Zheng et al. 2018).

2.7. Statistical analysis All data were analyzed with SPSS 16.0 Software (SPSS, Inc. Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to analyze the differences of various physiological indices among the cultivars and two-way ANOVA was used to confirm whether the effects of water and N, individually

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and combined, had any significant influence on the results. Tukey’s multiple range tests were used at a significance level of P<0.05, and data were indicated by mean±standard deviation (SD).

3. Results 3.1. Growth parameters of different cultivars under low N and drought conditions The SH, SD, FW, and DW of six cultivars decreased in the order CWCN>CWLN>LWCN>LWLN (Table 1). The SH, SD, FW, and DW showed significant differences between control water and drought, i.e., drought stress significantly reduced the growth parameters for all cultivars. Under LWLN treatment, the SH of Qinguan, Jonagold, Honeycrisp, Fuji, Pink Lady, and Golden Delicious decreased by 26.68, 34.40, 37.71, 32.24, 26.24, and 35.85%, respectively, compared with CWCN. Pink Lady showed the least reduction, whereas Honeycrisp showed the greatest reduction. Similarly, SD under LWLN treatment reduced by 25.40, 32.73, 36.16, 30.32, 27.13, and 25.74%, with Qinguan showing the least reduction, and Honeycrisp the greatest. Additionally, Honeycrisp showed the greatest reduction in FW and DW

(50.21 and 49.20%, respectively). Golden Delicious showed the least reduction in FW (42.76%), while Qinguan showed the least reduction in DW (39.93%). The P-value of twoway ANOVA indicated that the water treatments significantly affected the SH, SD, FW and DW of six cultivars, as did the interactions of water and N treatments (W×N) on SD of Jonagold and SH of Fuji (Appendix A).

3.2. Photosynthetic parameters and Fv/Fm of different cultivars under low N and drought conditions In response to drought stress, gas exchange parameters including Pn, Tr, Ci, and Gs were decreased in all treatments regardless of N condition. Under CWCN treatment, Jonagold had the highest Pn among all tested cultivars. However, under LWLN treatment, the Pn values for Qinguan, Jonagold, Honeycrisp, Fuji, Pink Lady, and Golden Delicious were 7.44, 5.14, 3.77, 3.67, 5.30, and 4.48 µmol m–2 s–1, respectively (Fig. 1-A). Values for three other photosynthetic parameters (Tr, Gs and Ci), were also decreased at the end of the LWLN treatment (Fig. 1-B–D), showing a similar trend with Pn. Under drought stress, the Tr of all cultivars was decreased, but these declines were more severe under low N conditions. Qinguan and Honeycrisp had the highest and

Table 1 Effects of different treatments on SH, SD, FW and DW of six cultivars1) Cultivar Qinguan

Pink Lady

Honeycrisp

Golden Delicious

Fuji

Jonagold

1)

Treatment2) CWCN CWLN LWCN LWLN CWCN CWLN LWCN LWLN CWCN CWLN LWCN LWLN CWCN CWLN LWCN LWLN CWCN CWLN LWCN LWLN CWCN CWLN LWCN LWLN

SH (cm) 125.2±3.70 a 118.0±1.58 b 95.6±2.61 c 91.8±2.17 c 96.8±2.95 a 92.6±1.95 a 75.2±4.97 b 71.4±1.82 b 109.0±2.24 a 98.2±4.82 b 76.2±1.79 c 67.9±0.84 d 95.4±2.19 a 87.0±2.45 b 68.2±1.79 c 61.2±2.28 d 121.6±3.29 a 107.8±1.48 b 87.4±2.07 c 82.4±1.14 d 112.2±4.02 a 102.6±5.18 b 77.0±2.12 c 73.6±1.82 c

SD (mm) 10.04±0.28 a 9.40±0.15 b 7.94±0.15 c 7.49±0.22 c 9.54±0.16 a 9.16±0.41 a 7.29±0.07 b 6.95±0.15 b 9.74±0.19 a 9.16±0.25 b 6.97±0.17 c 6.22±0.26 d 9.31±0.20 a 8.82±0.24 b 7.66±0.15 c 6.91±0.18 d 9.81±0.22 a 9.07±0.21 b 7.48±0.22 c 6.84±0.22 d 9.57±0.18 a 8.93±0.12 b 6.96±0.25 c 6.43±0.25 c

FW (g) 218.26±6.89 a 202.34±7.88 a 133.03±6.70 b 121.36±4.89 b 202.13±4.66 a 190.33±6.58 b 117.40±6.23 c 109.87±3.87 c 200.73±9.34 a 178.88±5.70 b 114.16±5.94 c 99.94±4.55 d 190.24±5.24 a 174.65±6.40 b 118.88±6.78 c 102.89±9.21 c 215.22±7.30 a 196.82±5.94 b 127.81±7.47 c 116.88±4.09 c 205.56±14.72 a 173.28±10.10 b 113.41±9.25 c 104.07±5.39 c

DW (g) 81.18±1.92 a 77.41±4.47 a 55.33±0.25 b 48.77±1.56 c 70.82±3.70 a 66.90±1.33 a 54.54±2.59 b 38.80±3.40 c 72.01±3.50 a 57.29±3.92 b 46.46±3.19 c 36.59±2.86 d 75.32±5.44 a 70.89±4.77 a 49.30±3.32 b 42.61±2.12 b 86.17±2.89 a 68.78±5.18 b 52.39±3.34 c 41.99±5.33 d 62.23±0.66 a 54.56±1.95 b 39.48±2.73 c 34.77±2.01 d

SH, shoot height; SD, stem diameter; FW, fresh weight; DW, dry weight. CWCN, 65–75% field capacity with 6 mmol L–1 nitrate; CWLN, 65–75% field capacity with 0.01 mmol L–1 nitrate; LWCN, 45–55% field capacity with 6 mmol L–1 nitrate; LWLN, 45–55% field capacity with 0.01 mmol L–1 nitrate. Ten biological replicates were used for each assay and data are shown as mean±SD. Within a cultivar, different letters following the means indicate significant differences at P<0.05.

2)

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CWCN Qinguan W: Honeycrisp W: *** Fuji W: *** 20

N: N: *** N: **

***

Pn (µmol m–2 s–1)

18

a

16

W×N: W×N: ns W×N: ns

***

**

a a

b

14

a b

12 10

c

8

Pink Lady W: Golden Delicious W: *** Jonagold W: *** ***

CWLN N: N: *** N: *** a

**

b

b

b c

c

c

6

c d

c d

4

LWCN

B

Qinguan W: Honeycrisp W: *** Fuji W: *** 8

c

c

d

d

2

C

Qinguan

Pink Lady Honeycrisp

Qinguan W: *** Honeycrisp W: *** Fuji W: ***

N: * W×N: ns N: * W×N: ns N: ns W×N: ns

0.25

Gs (mol m–2 s–1)

0.2 0.15 0.1

a b

N: ns W×N: ns N: ns W×N: ns N: ** W×N: ns

b

c

c

b

c

d

c d

Pink Lady Honeycrisp

a

a

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4

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3

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N: ns W×N: ns N: ns W×N: ns N: ** W×N: *

a

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c

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c

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2 1 Qinguan

Pink Lady Honeycrisp

Qinguan W: *** Honeycrisp W: *** Fuji W: ***

N: ns W×N: ns N: ** W×N: ns N: * W×N: ns

Golden Delicious

Fuji

Jonagold

Pink Lady W: ** Golden Delicious W: *** Jonagold W: **

200

a

a b

d

Qinguan

a

5

250

a b

c

Pink Lady W: *** Golden Delicious W: *** Jonagold W: **

a a

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N: ns W×N: ns N: ** W×N: ns N: ns W×N: ns

N: ns W×N: ns N: * W×N: ns N: ns W×N: ns

300

c c

D

b a

6

0

Jonagold

a

c

0.05 0

Fuji

Pink Lady W: *** Golden Delicious W: *** Jonagold W: ***

a a

Golden Delicious

Ci (µm mol–1)

0

LWLN ***

7

a

b

W×N: W×N: * W×N: ns

***

Tr (mmol m–2 s–1)

A

c c

a

b b

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c c

c

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a

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d

c d

150 100 50

Golden Delicious

Fuji

Jonagold

0

Qinguan

Pink Lady Honeycrisp

Golden Delicious

Fuji

Jonagold

Fig. 1 Net photosynthesis rate (Pn; A), transpiration rate (Tr; B), stomatal conductance (Gs; C) and intercellular CO2 concentration (Ci; D) under different treatments. CWCN, 65–75% field capacity with 6 mmol L–1 nitrate; CWLN, 65–75% field capacity with 0.01 mmol L–1 nitrate; LWCN, 45–55% field capacity with 6 mmol L–1 nitrate; LWLN, 45–55% field capacity with 0.01 mmol L–1 nitrate. Ten biological replicates were used for each assay and data are shown as mean±SD. Different letters above the bars within a cultivar indicate significant differences at P<0.05. Significant effects of the main factors water treatment (W), N levels (N), and interactions (W×N) are shown: ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001.

lowest Tr values (2.95 and 1.72, respectively). The Gs for different cultivars showed a similar trend to Pn and Tr. Among all the tested cultivars, under LWLN conditions, Qinguan had the highest values for Ci, while Honeycrisp had the lowest. Different cultivars also showed various Fv/Fm values according to water and N treatments (Fig. 2). Both drought and low N treatments reduced Fv/Fm when compared to CWCN. Under LWLN conditions, Golden Delicious had the maximum Fv/Fm value while Honeycrisp had the lowest.

3.3. Chlorophyll content of different cultivars under low N and drought conditions At the end of treatment, chlorophyll a, chlorophyll  b, and total chlorophyll content of all cultivars were significantly lower than under CWCN (Fig.  3). Under CWCN treatment, the order of chlorophyll a content was Fuji>Jonagold>Honeycrisp>Qinguan>Golden Delicious>Pink Lady (2.29, 2.12, 2.12, 1.99, 1.89, and

1.80 mg g–1 FW, respectively). Under LWLN conditions, Fuji had the highest value for chlorophyll a (1.45 mg g–1 FW), while Honeycrisp had the lowest (1.14 mg g–1 FW). Chlorophyll b content decreased under drought stress for all cultivars, regardless of the N levels. The ranking for chlorophyll b content for the six cultivars was similar to that for chlorophyll a, with Jonagold showing the greatest decrease (54.70%) and Golden Delicious showing the least (38.27%). Total chlorophyll content of different cultivars showed various decreases under drought conditions, with Jonagold showing the greatest decrease (48.16%) and Qinguan showing the least (31.20%) under LWLN treatment compared with CWCN.

3.4. Total N content in different organs of different cultivars under low N and drought conditions The concentrations of N in different organs under different treatments are shown in Table  2. The ranking for N

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CWCN Qinguan Honeycrisp Fuji

0.86 0.85 0.84

a

a a b b

0.83 Fv/Fm

W: *** W: *** W: **

N: ns N: ** N: ns

a

CWLN

W×N: ns W×N: ns W×N: ns

a b

b

LWCN

LWLN

Pink Lady Golden Delicious Jonagold a b c

b

c

W: *** W: ** W: *** a

N: * N: ns N: *

a

a b

c

a b c

c

0.82

W×N: ns W×N: ns W×N: ns

d

0.81 0.80 0.79 0.78 Qinguan

Pink Lady

Honeycrisp

Golden Delicious

Fuji

Jonagold

Fig. 2 The maximal photochemical efficiency of PSII (Fv/Fm) values for the six cultivars under different treatments. CWCN, 65–75% field capacity with 6 mmol L–1 nitrate; CWLN, 65–75% field capacity with 0.01 mmol L–1 nitrate; LWCN, 45–55% field capacity with 6 mmol L–1 nitrate; LWLN, 45–55% field capacity with 0.01 mmol L–1 nitrate. Six biological replicates were used for each assay and data are shown as mean±SD. Different letters above the bars within a cultivar indicate significant differences at P<0.05. Significant effects of the main factors water treatment (W), N levels (N), and interactions (W×N) are shown: ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001.

concentrations in different organs was leaves>roots>stems, regardless of cultivar. Drought conditions significantly reduced the level of N in all three organs. Under CWCN conditions, the leaves of Fuji had the highest N concentration (28.40 mg g–1), while Golden Delicious had the lowest (23.55  mg g –1). Under LWLN conditions, in leaves, Honeycrisp showed the greatest decrease (38.96%), while Qinguan showed the least (33.90%); in stems, Golden Delicious showed the greatest decrease (29.02%), while Qinguan showed the least (22.72%); in roots, Jonagold had the greatest reduction (30.10%), whereas Qinguan had the least (19.19%). The P-value of two-way ANOVA indicates that the water treatments significantly affected the total N content of the six cultivars (Appendix B).

3.5. δ15N content in different organs of different cultivars under low N and drought conditions Similarly, drought stress reduced δ15N content in different organs, and the degree of reduction varied with different cultivars (Table  3). However, under control water or water deficit, the δ15N content was induced by low N treatment, regardless of cultivar. Under LWLN conditions, in leaves, the δ15N concentrations for Qinguan, Jonagold, Honeycrisp, Fuji, Pink Lady, and Golden Delicious were 0.235, 0.227, 0.172, 0.235, 0.204, and 0.209 mg g–1, respectively; in stems, Jonagold and Honeycrisp had the

minimum and maximum reduction of δ15N concentrations, respectively, compared with CWCN, which was 12.22 and 29.39%; in roots, the δ 15N concentration ranked in the order Qinguan>Fuji>Golden Delicious>Pink Lady>Honeycrisp>Jonagold (0.160, 0.144, 0.142, 0.141, 0.133, and 0.132 mg g–1, respectively). The P-value of two-way ANOVA indicates that the water treatments significantly affected the δ15N content for six cultivars in leaves, roots and stems (Appendix C).

3.6. P and K contents of different cultivars under low N and drought conditions The concentration of P and K by treatment decreased in the order: CWCN>CWLN>LWCN>LWLN, regardless of cultivar (Appendix D). Similarly, drought stress decreased P and K contents of different organs, regardless of the N condition. The P contents of different organs decreased in the order: root> leaf>stem, while the K contents decreased in the order: leaf>root>stem. Under LWLN condition, in leaves, Qinguan had the highest P content (1.63 mg g–1 DW) and K content (19.60 mg g–1 DW); in stems, Qinguan also had the highest P content (1.40 mg g–1 DW) whereas Jonagold had the highest K content (6.57 mg g–1 DW); in roots, Pink Lady had the highest P content (3.65 mg g–1 DW), whereas Qinguan had the highest K content (6.60 mg g–1 DW). P and K contents in roots were significantly decreased under

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CWCN

Chlorophyll a (mg g–1 FW)

A

2.5 2.0

Qinguan Honeycrisp Fuji a

b

b

1.5

W: W: *** W: *** ***

N: N: *** N: ** ***

CWLN

W×N: ns W×N: ns W×N: ns a

a a a c

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LWLN

Pink Lady Golden Delicious Jonagold a

b

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a b

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d

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1.0 0.5 0 Qinguan

Chlorophyll b (mg g–1 FW)

B

1.0

Qinguan Honeycrisp Fuji a

0.8

b b

0.6

Pink Lady

Honeycrisp

W: *** N: *** W: *** N: *** W: *** N: *** a b b

W×N: * W×N: ns W×N: ***

Fuji

Pink Lady Golden Delicious Jonagold

a

W: *** W: *** W: *** a a

a a

b b

c

c

Golden Delicious

b

c

Jonagold N: *** N: ns N: ***

W×N: ** W×N: ns W×N: * a

b

b

c

b

c

0.4

d

0.2 0 Qinguan

C

Qinguan Honeycrisp Fuji 3.5 3.0

Total chlorophyll (mg g–1 FW)

Pink Lady

2.5 2.0

a

a

W: *** W: *** W: *** a

b

b

a

N: ** N: *** N: *

Honeycrisp

Golden Delicious

W×N: ns W×N: ** W×N: ns

Pink Lady Golden Delicious Jonagold

b c

1.5

b

W: * W: *** W: *** a

b c

Jonagold N: ** N: ns N: ***

b

a a

a b

Fuji

W×N: ns W×N: ns W×N: ns a

c

b d

c

c

d

1.0 0.5 0.0 Qinguan

Pink Lady

Honeycrisp

Golden Delicious

Fuji

Jonagold

Fig. 3 Chlorophyll a (A), chlorophyll b (B), and total (C) chlorophyll contents under different treatments. CWCN, 65–75% field capacity with 6 mmol L–1 nitrate; CWLN, 65–75% field capacity with 0.01 mmol L–1 nitrate; LWCN, 45–55% field capacity with 6 mmol L–1 nitrate; and LWLN, 45–55% field capacity with 0.01 mmol L–1 nitrate. Five biological replicates were used for each assay and data are shown as mean±SD. Different letters above the bars within a cultivar indicate significant differences at P<0.05. Significant effects of the main factors water treatment (W), N levels (N), and interactions (W×N) are shown: ns, not significant; *, P<0.05; **, P<0.01; and ***, P<0.001.

LWLN conditions compared with CWCN, and all cultivars showed a consistent trend. The P-value of two-way ANOVA indicates that the water treatments significantly affected P and K contents of the six cultivars in leaves, roots, and stems, and significantly affected N content of some cultivars (Appendix E). A correlation of total N, P and K after drought and low N

treatment is shown in Appendix F. Total N and P contents were significantly correlated (correlation coefficient, 0.903). However, there was no significant correlation between N and K (0.752), or between P and total K (0.45). This result suggests that the uptake of P and N in apple might be synergistic, whereas this effect might not exist between P and K uptake.

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WANG Qian et al. Journal of Integrative Agriculture 2020, 19(3): 709–720

Table 2 Effects of different treatments on total N content of six cultivars Cultivar Qinguan

Pink Lady

Honeycrisp

Golden Delicious

Fuji

Jonagold

Treatment1) CWCN CWLN LWCN LWLN CWCN CWLN LWCN LWLN CWCN CWLN LWCN LWLN CWCN CWLN LWCN LWLN CWCN CWLN LWCN LWLN CWCN CWLN LWCN LWLN

Leaf (mg g–1 DW) 27.16±0.42 a 25.46±0.68 b 19.89±0.61 c 17.95±0.44 d 26.00±0.96 a 22.70±0.41 b 18.40±0.89 c 16.30±0.66 d 27.05±1.16 a 22.34±0.23 b 19.50±0.44 c 16.51±0.72 d 23.55±0.43 a 22.86±0.66 a 17.85±0.67 b 15.12±0.86 c 28.40±0.67 a 25.90±0.90 b 20.95±0.50 c 18.30±0.86 d 25.59±0.49 a 23.83±0.64 b 18.14±0.78 c 16.09±0.44 d

Stem (mg g–1 DW) 12.99±0.31 a 12.10±0.22 b 11.53±0.28 c 10.04±0.56 d 13.86±0.37 a 12.10±0.20 b 11.53±0.42 c 10.04±0.50 c 12.70±0.74 a 12.06±0.24 a 9.93±0.35 b 9.05±0.23 c 13.38±0.24 a 12.89±0.52 a 11.42±0.36 b 10.55±0.48 c 14.73±0.32 a 13.85±0.27 b 11.42±0.32 c 10.55±0.16 d 13.63±0.43 a 12.75±0.32 b 10.83±0.56 c 9.81±0.36 d

Root (mg g–1 DW) 16.44±0.36 a 15.40±0.23 b 14.35±0.22 c 13.28±0.56 d 16.17±0.35 a 14.74±0.61 b 13.01±0.15 c 11.70±0.78 d 14.81±0.47 a 12.74±0.59 b 11.63±0.22 c 10.54±0.68 d 14.51±0.34 a 13.21±0.40 b 12.34±0.30 c 11.32±0.26 d 13.54±0.23 a 12.59±0.47 b 10.97±0.55 c 9.89±0.25 d 13.41±0.38 a 12.76±0.50 a 11.55±0.29 b 9.38±0.66 c

1)

CWCN, 65–75% field capacity with 6 mmol L–1 nitrate; CWLN, 65–75% field capacity with 0.01 mmol L–1 nitrate; LWCN, 45–55% field capacity with 6 mmol L–1 nitrate; LWLN, 45–55% field capacity with 0.01 mmol L–1 nitrate. Four biological replicates were used for each assay and data are shown as mean±SD. Within a cultivar, different letters following the means indicate significant differences at P<0.05.

Table 3 Effects of different treatments on δ15N content of six cultivars Cultivar Qinguan

Pink Lady

Honeycrisp

Golden Delicious

Fuji

Jonagold

1)

Treatment1) CWCN CWLN LWCN LWLN CWCN CWLN LWCN LWLN CWCN CWLN LWCN LWLN CWCN CWLN LWCN LWLN CWCN CWLN LWCN LWLN CWCN CWLN LWCN LWLN

Leaf (mg g–1 DW) 0.259±0.008 b 0.299±0.007 a 0.209±0.008 d 0.231±0.009 c 0.250±0.008 b 0.274±0.007 a 0.182±0.004 d 0.204±0.006 c 0.242±0.005 b 0.257±0.002 a 0.164±0.002 d 0.172±0.002 c 0.233±0.008 b 0.269±0.006 a 0.197±0.005 c 0.209±0.010 c 0.323±0.005 a 0.331±0.013 a 0.218±0.003 c 0.235±0.005 b 0.261±0.006 b 0.280±0.005 a 0.210±0.004 d 0.235±0.005 c

Stem (mg g–1 DW) 0.159±0.003 b 0.174±0.005 a 0.117±0.003 d 0.128±0.005 c 0.147±0.003 b 0.154±0.002 a 0.097±0.003 d 0.104±0.006 c 0.147±0.005 a 0.154±0.006 a 0.097±0.004 b 0.117±0.004 b 0.156±0.004 b 0.166±0.004 a 0.106±0.002 d 0.117±0.004 c 0.168±0.004 a 0.176±0.007 a 0.132±0.004 b 0.134±0.003 b 0.151±0.007 a 0.159±0.005 a 0.119±0.004 c 0.132±0.004 b

Root (mg g–1 DW) 0.163±0.007 a 0.172±0.004 a 0.145±0.008 c 0.160±0.003 ab 0.144±0.004 b 0.157±0.006 a 0.127±0.005 c 0.141±0.007 b 0.158±0.005 a 0.163±0.003 a 0.113±0.007 c 0.133±0.006 b 0.155±0.002 b 0.163±0.003 a 0.134±0.005 c 0.142±0.006 c 0.163±0.010 a 0.167±0.005 a 0.124±0.002 c 0.144±0.006 b 0.159±0.003 a 0.165±0.006 a 0.117±0.004 c 0.132±0.004 b

CWCN, 65–75% field capacity with 6 mmol L–1 nitrate; CWLN, 65–75% field capacity with 0.01 mmol L–1 nitrate; LWCN, 45–55% field capacity with 6 mmol L–1 nitrate; LWLN, 45–55% field capacity with 0.01 mmol L–1 nitrate. Four biological replicates were used for each assay and data are shown as mean±SD. Within a cultivar, different letters following the means indicate significant differences at P<0.05.

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WANG Qian et al. Journal of Integrative Agriculture 2020, 19(3): 709–720

3.7. Relative growth rate (RGR) and 15N use efficiency (15NUE) of different cultivars under low N and drought conditions The RGR of different cultivars showed a descending order according to reduction of water and N level, which was consistent with the trend for Pn (Fig. 4-A). Under drought stress, RGR of all cultivars showed significant difference from control-watered condition, regardless of N level. Under CWCN condition, Qinguan had the highest RGR of all cultivars (15.88 mg d–1), while Honeycrisp had the lowest (13.12 mg d–1). Under LWLN treatment, Qinguan and Honeycrisp still had the highest and lowest RGR values, respectively, among all tested cultivars. Drought stress reduced 15NUE while low N enhanced 15NUE (Fig.  4-B). Under different treatments, the order of 15NUE of different cultivars was CWLN>LWLN>CWCN>LWCN. Under CWCN treatment, Pink Lady had the highest 15NUE among all cultivars (1.20%), while Honeycrisp had the lowest (0.94%). However, under LWLN conditions, Qinguan had the highest and Honeycrisp had the lowest. We used 15N content, 15NUE, Pn and total N content to create a cluster analysis chart (Fig.  5). Under LWLN conditions, Jonagold and Honeycrisp belonged to the same cluster. Golden Delicious and Fuji fell into the same category, while Qinguan and Pink Lady belonged to the rest of the class.

4. Discussion To improve drought resistance, plants can form strong root CWCN A

Qinguan W: *** Honeycrisp W: *** Fuji W: ***

N: ** W×N: ns N: ** W×N: ns N: ns W×N: ns

CWLN

systems and maintain high root activity, thereby increasing the ability to absorb water from the soil. Previous results showed that moderate application of N under drought conditions alleviated drought stress, while excessive application of N aggravated drought stress. When N is deficient, the application of appropriate amounts of N positively regulated plant growth (Yang et al. 2012; Xu et al. 2015). Our results showed that in all six cultivars, SH, SD and seedling biomass were significantly suppressed by drought stress under both N conditions, and the inhibition was eased when moderate N was applied, which was consistent with previous studies (Shi et al. 2017). Drought reduces photosynthetic performance of plants (Sapeta et al. 2013). When plants suffer from drought stress, a reduction in photosynthesis enables plants to enhance drought tolerance and redistribute the limited resources (Skirycz et al. 2011). Under drought stress, the reduction of photosynthesis is linked with many factors including changes in stomata number and chlorophyll content (Warren et al. 2011). When suffering from drought stress, stomatal apertures decrease, which leads to an increase in stomatal resistance and decrease in intercellular CO2 concentration. This in turn reduces photosynthesis, an important indicator of CO2 assimilation ability (Karimi et al. 2015). Carbohydrates generated through photosynthesis are the main raw material and energy source for biomass formation, and inhibition of photosynthesis can explain the reduced biomass of apple seedlings observed in our study. Drought stress also reduced the Fv/Fm value of apple seedlings (Shangguan et al. 2000; Liang et al. 2017). Photosynthesis can also be limited by chlorophyll content (Pagter et al. 2005). Under LWCN

Pink Lady W: *** N: *** W×N: ** Golden Delicious W: *** N: ns W×N: ns Jonagold W: *** N: ** W×N: ns

B

LWLN Qinguan W: *** Honeycrisp W: *** Fuji W: ***

18.0

4.00 a

RGR (mg d–1)

14.0 12.0 10.0

a a

c

8.0 6.0

b

b c

c

c d

d

d

0.0

Qinguan

Pink Lady Honeycrisp Golden Delicious

Fuji

Jonagold

a

a

a

2.00

a

1.50

0.00

b

b

c

c d

0.50

2.0

W×N: * W×N: * W×N: ns

2.50

1.00

4.0

Pink Lady W: ** N: ** Golden Delicious W: *** N: ** Jonagold W: *** N: *

a

3.00 c

W×N: ** W×N: ns W×N: *

a

3.50

b b

c

a

a

a

b

b

a

NUE (%)

aa

15

16.0

N: ** N: * N: *

Qinguan

b

c d

d

b

c d

Pink Lady Honeycrisp Golden Delicious

b

c

b c

d

Fuji

d

Jonagold

Fig. 4 Relative growth rate (RGR) and 15N utilization efficiency (15NUE) under different treatments. CWCN, 65–75% field capacity with 6 mmol L–1 nitrate; CWLN, 65–75% field capacity with 0.01 mmol L–1 nitrate; LWCN, 45–55% field capacity with 6 mmol L–1 nitrate; LWLN, 45–55% field capacity with 0.01 mmol L–1 nitrate. Four biological replicates were used for each assay and data are shown as mean±SD. Different letters above the bars within a cultivar indicate significant differences at P<0.05. Significant effects of the main factors including water treatment (W), N levels (N), and interactions (W×N) are shown: ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001.

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WANG Qian et al. Journal of Integrative Agriculture 2020, 19(3): 709–720

Golden Delicious Fuji Pink Lady Qinguan Honeycrisp Jonagold Label no.

0

5

10

15

20

25

Fig. 5 Cluster analysis for different cultivars under limited water with low N (LWLN) condition. Label number is shown under the chart.

drought conditions, the content of reactive oxygen species (ROS) in leaves increases, which causes chlorophyll degradation (Liang et al. 2018). The pigments in leaves including chlorophyll a and chlorophyll b play an important role in electron transport during photosynthesis, and are often used as an index of plant drought resistance. Drought stress reduced the content of chlorophyll in leaves (Meng et al. 2016; Liang et al. 2018), regardless of cultivar. The chlorophyll content in leaves was the lowest under LWLN conditions, and varied by cultivar. Under drought conditions, when appropriate amount of N was supplied, osmolyte synthesis was increased (Shi et al. 2017). This could maintain the stability of membranes and protein conformation, thus increasing carbon assimilation. Drought stress also inhibits absorption of minerals (Huang et al. 2007). The mechanisms of water and nutrient absorption by plants are distinct, yet interdependent. The absorption of nutrients by the root system in the form of ions requires water as medium. The NO3– absorbed by the root system is converted to ammonium by NR and NiR, and then is assimilated to glutamine and glutamate via the activities of glutamate synthase (GS) and glutamine oxoglutarate aminotransferase (GOGAT). Changes in N metabolism-related enzyme activity and gene expression under different N levels cause the discrepant absorption under different N conditions. Many studies have shown that applying appropriate N under drought conditions alleviates the inhibition of root growth by drought, especially for fine roots with a diameter <0.2 mm, which are important for water and mineral element adsorption (Zhang et al. 2013; Wang et al. 2016). Our results indicated that N supply enhanced N content and promoted root system development, which increased the capacity for water uptake under drought stress. Previous studies also showed that drought stress reduced the absorption of mineral elements, including macro- and micro-elements (Liang et al. 2018). Proper N supply under drought conditions maintained root activity and increased the amount of osmotic substances (Shi

et al. 2017), which promoted the absorption of mineral elements and water. Jiang et al. (2019) showed that N coexisting with P significantly increased above-ground biomass, and that N supply promoted P uptake, which to some extent were consistent with our results about the correlation between N and P. A recent study showed that nitrate signal simultaneously stimulated the activation of nitrate and P response genes through NRT1.1B-SPX4 complex, so as to achieve homeostatic balance of N and P (Hu et al. 2019). Our data revealed that drought stress significantly diminished the uptake of nutritional elements by apple plants, but the extent of that inhibition was significantly mitigated by moderate N application. We showed that δ15N was obviously increased under low N conditions, irrespective of water levels. Similarly, the absorption of δ15N and the 15 NUE were also inhibited under drought stress (Liang et al. 2018). Low N and drought differentially affect the growth of different apple cultivars. Under LWLN conditions, Qinguan and Pink Lady had higher NUE, indicating that they could maintain better growth when N was limiting. Previous studies showed that Qinguan had higher WUE under longterm drought conditions (Liu et al. 2012), implying that Qinguan had higher water absorption ability, which also benefits the absorption of mineral elements including N. In contrast, in Catalpa bungei, it has been reported that NUE was improved under drought conditions compared with well-watered conditions, and NUE was also improved under sufficient-N supply conditions compared with low N conditions (Shi et al. 2017). The differences between our work and previous studies were probably due to several factors, including differences of experimental conditions, inherent differences between plant species, and the method used to compute NUE.

5. Conclusion Our data indicated that different apple cultivars showed varied responses to low N and drought stress and application of appropriate N under drought conditions had positive effects on growth and drought resistance. Under LWLN conditions, Qinguan and Pink Lady had the best tolerance, followed by Fuji and Golden Delicious, while Honeycrisp and Jonagold had the least tolerance, among all tested cultivars. The comparison of different cultivars under drought and low N conditions provides a basis for revealing the mechanism of high NUE under drought stress and breeding new apple cultivars with drought and low N resistance.

Acknowledgements The authors are grateful to Mr. Ma Zhengwei from

WANG Qian et al. Journal of Integrative Agriculture 2020, 19(3): 709–720

Northwest A&F University, China for management of the apple seedlings and members of Stress Biology of Fruit Trees Group for assistance in successfully completing this study. This work was financially supported by the National Key Research and Development Program of China (2018YFD1000300) and the earmarked fund for the China Agriculture Research System (CARS-27). Appendices associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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