LEl Bo, BlAN Zhong-hua, YANG Qi-chang, WANG Jun, CHENG Rui-feng, Ll Kun, LlU Wen-ke,ZHANG Yi, FANG Hui, TONG Yun-xin

1 Key Laboratory of Energy Conservation and Waste Treatment of Agricultural Structures, Ministry of Agriculture/Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

2 Nottingham Trent University, Brackenhurst Campus, Nottingham NG25 0QF, UK

1. lntroduction

Lettuce (Lactuca sativaL.) is one of the major greenhousegrown vegetables and consumed worldwide. Nitrate(NO3?) as one of the most important nitrogen sources for plant growth and development, is widely used in vegetable production, especially in hydroponic grow system. Lettuce is a hyperaccumulator of NO3?and has a great ability to accumulate NO3?in their leaves (Eysinga and Meijs 1985).In the human body, approximately 80% of the daily intake NO3?stems from vegetables (Santamaria 2006). Studies have indicated that consumption vegetables with high NO3?content poses threaten to human health because besides leading to methemoglobinemia, ingested NO3?could be converted to nitrite, a toxic carcinogen, causing cancers and methemoglobinemia (Wright and Davison 1964; Prasad and Chetty 2008). European Commission has imposed a maximum limit on NO3?concentration in lettuce for human consumption of 4 500 mg NO3?kg?1fresh weight (FW)(UKMAFF 1997).

Since 1957, selenium (Se) has been demonstrated as an essential trace element for maintaining animal and human health (Schwarz and Foltz 1957). Since Se is a vital important component of the glutathione peroxidase,selenoprotein, and tetraiodothyronine 5-deiodinase (Pappet al. 2007; Messaoudiet al. 2009). Se deficiency not only disturbs metabolism in the human body but also increases the risk of cancers (Diwadkar-Navsariwalaet al.2006). However, Se deficiency in the diet is a worldwide problem, especially in China, the UK, Eastern Europe and Australia (Pedreroet al. 2006). This is due to the low concentrations of Se in plant tissues as the consequence of low bioavailability of Se in some soils (Hawkesford and Zhao 2007). Previous studies proved that exogenous application of Se could substantially increase Se concentration in crops,vegetables, and fruits (Carteset al. 2005; Hartikainen 2005).Elevating Se content in plant food can effectively avoid and prevent Se deficiency in humans. Thus, increasing Se concentration and reducing NO3?content in crops arouse widespread concern for both researchers and farmers (Bianet al. 2015, 2016; Carteset al. 2005; Hartikainen 2005).

In plants, the higher uptake rate of NO3?than its metabolic rate leads to excessive NO3?accumulation. Previous studies found that exogenous Se could affect nitrogen metabolism in plants and this effect depends on the application level of Se (Nowaket al. 2004; Rioset al. 2010). Exogenous Se affects uptake and translocation of some mineral elements,such as inhibiting cadmium and Na+accumulation, and promoting K+uptake (Sunet al. 2013). However, less is known about the effect of exogenous Se on NO3?uptake and translocation in plants.

Exogenous Se application could enhance photosynthetic capacity of plants, especially under different biotic stress,such as cold, drought and salt stress (Fenget al. 2013;Zhanget al. 2014). In plants, photosynthetic capacity affects NO3?metabolism and accumulation. The NO3?reduction positively correlates with photosynthetic products, e.g.,carbohydrates (Bianet al. 2016). Since these products can provide carbon skeleton and energy for nitrogen reduction in plants (Champigny 1995). Therefore, we hypothesize that besides inducing activities of nitrogen metabolism enzymes,exogenous Se may positively promote NO3?reduction in plants through maintaining high photosynthetic capacity and concomitantly regulating NO3?uptake and translocation in plants. Therefore, the aims of this study are to investigate the effects of exogenous Se on NO3?uptake and transport,assimilation enzyme activities and photosynthetic capacity of lettuce grown hydroponically. The results of this study will aid in producing high-quality vegetables in greenhouse.

2. Materials and methods

2.1. Plant materials and treatments

Seeds of lettuce (Rijk Zwaan, De Lier, the Netherlands)were washed and soaked for 4 h using distilled water, then germinated in the dark at 25°C. To avoid root damage during sample preparation, these germinated seeds were sown in sponge dices (3 cm×1.5 cm×1.5 cm) with a density of one seed per disc before grown in a controlled growth chamber.The day/night temperature, light intensity, photoperiod,humidity, and CO2levels in the growth chamber was 25°C/(18±1)°C, 200 μmol m?1s?1, 12 h, (75±5)%, 400 μmol mol–1, respectively. Freshly prepared nutrition solution(Hoagland and Arnon 1950) was added daily to maintain the moistness of the substrate and to supply nutrition for plants. After the end of the dark period at 21 d, plants with similar size were randomly transplanted into 25-L containers of Hoagland solution (pH (6.8±0.2), (1.9±0.1) dS m–1) with six concentrations of Se (0, 0.1, 0.5, 5, 10 and 50 μmol L–1) applied as sodium selenium (Na2SeO3). There were three replicates with a total 48 plants per treatment. The nutrient solutions were replaced with fresh solution every 5 d throughout this experiment.

2.2. Measurement of plant growth

After Se treatment for 30 d, three plants were randomly harvested from each treatment and cut at the thypocotyls to calculate shoot and root FW. The root length was determined according to the method of Yanget al. (2016). Shoots and roots were then dried at 75°C for 3 d in an oven to determine the dry weight (DW) of shoots and roots.

2.3. Measurement of photosynthetic parameters

After Se treatment for 30 d, six plants (two plants per replicate, three replicates per treatment) were randomly selected from each treatment. The second youngest, fully expanded leaf was used to monitor photosynthetic capacity using the portable photosynthetic apparatus (LICOR-6400,Lincoln, NE, USA) with the method described by Yanet al.(2012).

2.4. Measurement of NO3? content

The NO3?content in lettuce leaves was determined according to the method of Cataldoet al. (1975). Leaf tissue(1.0 g) was grounded using a mortar and pestle and then suspended in 10 mL of distilled water. The sample was boiled for 30 min at 100°C in a water bath. After cooling down with tap water, the extracts were filtered and then diluted with distilled water to a final volume of 20 mL. The 5%salicylic acid-concentrated sulfuric acid (0.4 mL) was added to 0.1 mL exact and then incubated at room temperature in the dark. After 20 min, the assay mixture was further diluted with 9.5 mL of 2.0 mol L–1NaOH solution and shaken until yellow color appeared. The absorption monitored at 430 nm was used to calculate NO3?contents on its standard curve.

2.5. Measurement of nitrate reductase and nitrite reductase activity

A spectrophotometric method was used to measure nitrite reductase (NiR, EC 1.6.6.4) activity (Mendez and Vega 1981). Leaf tissue (1.0 g) was grounded with 50 mmol L–1Tris-HCl buffer (pH 7.5) and then centrifuged at 10 000 r min–1for 30 min at 4°C. The supernatant was collected for measuring NiR activity. The assay mixture contained 6.25 mL Tris-HCl buffer (50 mmol L–1, pH 7.5),0.002 mL sodium nitrite (2.5 mmol L–1), 0.002 mL methyl viologen (3 mmol L–1), 0.3 mL extract and 0.2 mL 0.025%dithionate-sodium bicarbonate. After shake and incubation at 30°C for 15 min, the assay mixture was further diluted using 1.0 mL NEDH and 1.0 mL 1% sulphanilamide. The absorbance monitored at 540 nm was used to calculate NiR activity. The amount of nitrite disappeared was estimated using blank as the reference. One unit of NiR activity was de fined as μmol NO2–mg–1min–1FW.

2.6. Measurement of glutamate synthase and glutamine synthetase activity

Fresh tissue was homogenized with 50 mmol L–1KH2PO4buffer (pH 7.5), containing 2 mmol L–1EDTA, 1.5 % (w/v)soluble casein, 2.0 mmol L–1dithiothreitol, and 1% (w/v)insoluble polyvinylpyrrolidone. The extract sample was centrifuged at 3 000×g for 5 min at 4°C. The supernatant was collected and centrifuged at 12 000 r min–1for 20 min at 2°C. The supernatant, referred as ‘crude enzyme’, was used for glutamate synthase (GOGAT, EC 1.4.1.13) and glutamine synthetase (GS, EC 6.3.1.2) activity assay.

The activity of GOGAT was measured according to the method described by Chen and Cullimore (1988). A 3-mL reaction mixture containing 0.2 mL enzyme extract,40 mmol L–1KH2PO4buffer (pH 7.5), 10 mmol L–1L-glutamine,10 mmol L–12-oxoglutarate, and 0.14 mmol L–1NADH. The reaction was started by the addition of enzyme. The rate of oxidation of NADH was observed at 340 nm, and the activity of GOGAT was de fined as μmol γ-glutamylhydroxamate g–1min–1FW.

A spectrophotometric method was used to calculate GS activity in lettuce leaves (Canovaset al. 1991). An aliquot of extract was added to a reaction mix containing 100 mmol L–1Tris-HCl, 20 mmol L–1MgSO4, 30 mmol L–1glutamate,6 mmol L–1NH2OH, 6 mmol L–1aspartate, 4 mmol L–1EDTA,and 12 mmol L–1ATP (pH 7.6). After incubation for 10 min at 26°C, the reaction was terminated by the addition of stop solution containing 0.37 mol L–1FeCl3, 0.2 mol L–1TCA, and 0.67 mol L–1HCl. After centrifugation for 15 min at 16 000×g,the supernatants were used for the spectrophotometric determination of c-glutamylhydroxamate formed at 540 nm. The enzyme activity was expressed as μmol NADH oxidised g–1min–1FW.

2.7. Measurement of net NO3? fluxes

Net NO3–fluxes were measured using the non-invasive scanning lon-selective electrode technique (SIET) (Younger USA LLC, Amherst, MA01002, USA) with ASET 2.0(Sciencewares, Falmouth, MA 02540, USA) and iFluxes 1.0(Younger USA, LLC, Amherst, MA 01002, USA) Software(Kochianet al. 1992; Yueet al. 2006; Sunet al. 2009).Pre-pulled and silanized glass micropipettes ((5±1) μm)(XY-DJ-01, Younger, USA) were filled with a backfilling solution to a length of 1 cm from the tip. The micropipettes were front-filled with approximately 15 to 50 μm columns of selective liquid ion exchanger cocktails (NO3–: XY-SJ-NO3; Younger). An Ag/AgCl wire electrode holder(XY-DJGD, Younger) was inserted into the back of the electrode to make electrical contact with the electrolyte solution. YG003-Y05 (Younger) was used as the reference electrode. Ion-selective electrodes were calibrated before and after flux measurements in the following solutions:0.05 and 0.5 mmol L–1NO3–(concentration for NO3–in the measuring solution was usually 0.1 mmol L–1). Only electrodes with nernstian slopes >26 mV per decade were used in this experiments. The same microelectrodes were calibrated again according to the same procedure and standards after each test.

2.8. Measurement of flux NO3– excised stems and flux NO3? in roots

The fluxes of NO3–in excised stems were measured to get the direct evidence of ion transport from the root to the shoot. The plants at the six-leaf stage were treated with 0, 0.1, 0.5, 5, 10 and 50 μmol L–1Na2SeO3for 12 d before the measurement of NO3–flux of the stem (with hypocotyls,Fig. 1-C and D). The plants were cut from the cotyledon,and the bottom part with the roots was fixed with a belt. The transverse section of the stem was immediately incubated in the measuring solution to equilibrate for 30 min (a rapid and large ef flux of NO3–occurred after the plants were cut). The flux rate gradually decreased and reached a steady level within 30 min. The transport of ions from the root to the shoot was measured in the transverse sections of the stems. The electrode was fixed at the center of the stems.

The flux NO3–in root was determined as the method described by Sunet al. (2009). Plants were placed in the middle of poly-L-lysine-pretreated coverslips (2 cm×2 cm)in the measuring chamber. For steady-state NO3–flux measurements, roots were rinsed with redistilled water and immediately incubated in the measuring solution to equilibrate for 15 min (a rapid ef flux of NO3–occurred after the roots were immediately sampled). Flux rate gradually decreased and reached a steady level within 15 min for roots. The measuring root site was 600 μm from the root tip (Fig. 1-A and B). NO3–was monitored by solutions containing 0.1 mmol L–1NO3–, 0.1 mmol L–1KCl, 0.1 mmol L–1CaCl2, 0.1 mmol L–1KNO3, and 0.3 mmol L–1MES(2-(N-morpholine)-ethane sulphonic acid) buffer with pH level at 6.0, which was adjusted by choline and HCl.

Fig. 1 Net flux of NO3? in roots (A and B) and excised stems (C and D) of lettuce under supplementation of different concentrations of sodium selenium (Na2SeNO3). Values are mean±standard error (n=4). Different letters are significantly different at the P＜0.05 level according to Duncan’s multiple range test.

2.9. Statistical analysis

Three-dimensional ionic fluxes were calculated using MageFlux developed by Younger, USA (http://www.youngerusa.com/mageflux). All of the data were subjected to variance analysis by SAS 8.1 Software (SAS Institute,Cary, NC, USA). Significant differences between treatments were assayed by Duncan’s multiple range test atP＜0.05.

3. Results

3.1. Plant growth

The effect of exogenous Se on lettuce growth depends on its application levels (Table 1). In the present study, the highest and the lowest plant biomass were obtained under 0.5 and 50 μmol L–1Se treatments, respectively. Compared with control (0 μmol L–1Se), FW of shoots for 0.1 and 0.5 μmol L–1Se treatments was increased by 23 and 43%,respectively. However, compared with control, a further increase in Se concentrations (i.e., beyond 5 μmol L–1)showed negative on shoot growth, leading to decreases by 22% for 10 μmol L–1Se and by 41% for 50 μmol L–1Se.Furthermore, exogenous Se concentration higher than 5 μmol L–1led to decreases in the shoot-to-root ratio (S/R),but the S/R for other Se treatments was comparable to that of control. Root FW under 0.5 μmol L–1Se treatment was higher than that of control, while 10 and 50 μmol L–1Se application caused significant decreases in this parameter.

3.2. Photosynthetic parameters

The photosynthetic parameters of lettuce plants under different Se treatments were summered in Table 2. There was an increase trend inPnwith increasing in exogenous Se concentration and peaked under 0.5 μmol L–1Se treatment,but higher Se concentration led to decrease constantly inPn. It is notable that exogenous Se higher than 10 μmol L–1resulted in a significant reduction inPn, as shown by the lowestPnunder 50 μmol L–1Se treatment. Interestingly,similar change trends were also observed inTrandGsunder exogenous Se treatments: Exogenous Se concentration lower than 10 μmol L–1showed positive effects, while higher concentration led to negative effects on these parameters.The values ofLsshowed opposite change pattern ofPn. The WUE was significantly elevated under 10 and 50 μmol L–1Se, but this parameter for other treatments was comparable to that of control (0 μmol L–1Se).

3.3. Net flux of NO3? and flux of NO3? in root and excised stem

Net fluxes of NO3?at the root tips of lettuce showed substantial variation under different concentrations of Se,ranging from 33.89 to 356.75 pmol cm?2s?1. The NO3?ef flux in roots increased with exogenous Se concentration, and the highest NO3–ef flux was observed under 50 μmol L–1Se,which was 10.9 times greater than that of control (0 μmol L–1Se) (Fig. 1-A and B).

The flux of NO3?, measured at the center of the excised stem, was also affected by exogenous Se concentration.Exogenous Se resulted in markedly decreases in NO3?ef flux. The ef flux of NO3?in stems decreased to 37.7,45.8, 37.6, 42.6 and 46.9% of control (0 μmol L–1Se) after treated with 0.1, 0.5, 5, 10 and 50 μmol L–1exogenous Se, respectively. However, no signi ficant difference was observed in NO3?ef flux under exogenous Se treatments with concentrations from 0.1 to 50 μmol L–1(Fig. 1-C and D).

3.4. NO3? content and nitrogen reduction enzyme activities

Exogenous Se application showed a positive effect on reducing NO3?content. The NO3?content was the lowest under 0.5 μmol L–1Se treatment (1 276.26 μg g–1FW),followed by 0.1, 5, 10, 50 and then 0 μmol L–1Se treatment.Compared with 0 μmol L–1Se treatment (2 069.86 μg g–1FW),NO3–content for 0.1-50 μmol L–1Se treatments decreased by 49.7, 53.4, 43.1, 35.3, and 27.65%, respectively.Furthermore, the NO3–content signi ficantly differed amongdifferent exogenous Se treatments (Table 3).

Table 1 The fresh weight (FW), dry weight (DW), shoot-to-root ratio (S/R, DW) and root length in lettuce in response to exogenous selenium (Se) treatment

The reduction of NO3?to nitrite is the first step in NO3?metabolism, which is catalyzed by NR. There were signi ficant differences in NR activity in leaves of the exogenous Se treated lettuce. Compared with control, NR activities increased by 73.1, 142.3, 76.9, 48.1 and 28.8%after treated with 0.1, 5, 10 and 50 μmol L–1Se, respectively.The response of NiR to exogenous Se treatments was similar to that of NR. The highest activity of NiR was observed under 0.5 μmol L–1Se treatment. The NiR activity for 5 μmol L–1Se was the second highest, followed by 0.1,10, 0 and 50 μmol L–1Se treatment (Table 3).

Similarly, the activities of GS and GOGAT in lettuce plants showed an increased trend and peaked at 0.5 μmol L–1Se supplementation (2.2- and 1.5-fold higher than that of control). Exogenous Se concentrations higher than 0.5 μmol L–1led to decreases in GS and GOGAT activities,but these values were greater than that of control (0 μmol L–1Se). These data indicate that exogenous Se could modulate nitrogen reduction enzyme activities and this function depends on its application concentration. In the present study, the most suitable exogenous Se concentration was 0.5 μmol L–1(Table 3).

4. Discussion

Increasing evidence shows that Se at low concentrations exerts a positive effect on crop growth and stress tolerance, although Se is not yet confirmed as an essential micronutrient in higher plants (Turakainenet al. 2004;Raniet al. 2005). However, Golobet al. (2016) found that exogenous Se had little effect on grain mass and grain yield of tartary buckwheat. In the present study, lettuce plants treated with the low concentration of selenium(≤0.5 μmol L–1) resulted in a does-dependent increase in shoot weight. This result is consistent with previous reports that exogenous Se increased shoot and root biomass production in lettuce (Simojokiet al. 2003; Liuet al. 2017), cucumber seedlings (Jozwiaket al. 2016)and pepper (Shekariet al. 2017). Hawrylak-Nowak (2008)found that exogenous Se treatments with concentrations between 5-50 μmol L–1stimulated maize seedling growth by 11-44%. Furthermore, the positive effect of higher Se concentration (50-100 μmol L–1) on seedling length and dry weight of broccoli sprout was reported by Takedaet al.(2016). However, in our study, 10-50 μmol L–1Se treatment can cause significant decreases in plant biomass and root length (Table 1). Similar observation was made by Penget al. (2000) and ascribed that low selenium concentrations induce the development of hydroponically-grown wheat,whereas excess Se inhibits plant growth in a non-linear/dose-response relationship.

Table 2 The net photosynthetic rate (Pn), stomatal conductance (Gs), stomatal limitation (Ls), transpiration rate (Tr), and water use efficiency (WUE) under different concentration of exogenous selenium (Se) treatments

Table 3 Nitrate (NO3–) content, enzymatic activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS),and glutamate synthase (GOGAT) in lettuce under under different concentration of exogenous selenium (Se) treatments

The NO3?accumulation in plants depends on its uptake and assimilation (Comettiet al. 2011). NO3?is mainly assimilated in plant leaves, since NO3?assimilation needs photosynthetic products to provide energy and carbon skeleton (Champigny 1995). NO3?transported into leaves not only can be assimilated but also could be re-transported to root and redirected out of the root cellviathe xylem (Forde and Clarkson 1999). Therefore, the net flux of NO3?in roots is a major factor for NO3?accumulation in plants. In this study, net NO3?ef flux in roots was increased from 33.89 to 356.75 pmol cm?2s?1under different exogenous Se concentrations (Fig. 1-A and B). This ef flux rate was relatively higher than those reported in eucalyptus roots (around 28 nmol m?2s?1) by Garnettet al. (2001) and Hawkinset al.(2008). Ingemarssonet al. (1987) reported that internal NO3?negatively correlates with the NO3?ef flux inLemna.Thus, the positive effect of exogenous Se on reducing NO3?accumulation in lettuce may lie in the high net ef flux of NO3?in lettuce roots aroused by Se supplementation.

NO3?assimilation in leaves involves the operation of nitrite and NiR to generate ammonium and then assimilatedviathe GOGAT pathway. The operation of the GS/GOGAT pathway leads to the production of glutamate, which is the source of C and N in the biosynthesis of most other amino acids (Forde and Lea 2007). Besides being regulated by NR activity,NO3?assimilation in plants is subjected to the negative feedback regulation of the amount of NO3?metabolites, such as nitrite and ammonium (Mi flin and Lea 1976). Therefore,NO3?assimilation is indirectly affected by the activitieses of NiR, GS and GOGAT (Templeet al. 1998; Ruizet al. 1999;Barneix 2007). In our experiments, the relationship between the NR activity and the activities of NiR, GS and GOGAT in NO3?assimilation was positive and signi ficant in leaves,indicating NR activity was also regulated by the metabolic intermediates of NO3?in exogenous Se treated lettuce. In plants, NR and NiR are induced by the same factors (Ruizet al. 1999), but the response of NiR to the supplementation of Se resembled that of NR in the leaves. Ruizet al. (2007)reported that exogenous Se applied at high concentration has a toxic effect on NiR resulting in a decline in its activity.In the present study, the positive effects of exogenous Se on NiR, GS and GOGAT activities were eliminated under 50 μmol L?1Se indicating higher Se application (>50 μmol L?1) might have toxic effect on nitrogen metabolism enzymes.However, Rioset al. (2010) reported that exogenous Se application even at 120 μmol L?1signi ficantly increased NiR, GS, and GOGAT activities. To reveal the mechanism of exogenous Se on NO3?metabolism, future studies and genetic analyses, together with clone NRT and analysis of Se levels in different parts of lettuce plants will shed light to the regulation of NO3?metabolism by exogenous Se.

5. Conclusion

Acknowledgements

This work was supported by the National High-Tech R&D Program of China (863 Program, 2013AA103004),the International S&T Cooperation Program of China(2014DFG32110), the National Key Research and Development Program of China (2014BAD08B020106).The authors are grateful to the reviewers and editorial team for comments that greatly improved the manuscript.

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