基于筛选安全的共低积累基因型评价空心菜对镉和硝酸盐积累的变异

Variations in cadmium and nitrate co-accumulation among water spinach genotypes and implications for screening safe genotypes for human consumption 基于筛选安全的共低积累基因型评价空心菜对镉和硝酸盐积累的变异 1 first-author 1 2 1 1 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:xeyang@zju.edu.cn †E-mail:xeyang@zju.edu.cn xeyang@zju.edu.cn Ministry of Education Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China Ministry of Education Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China Indian River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Fort Pierce, Florida 34945, USA Indian River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Fort Pierce, Florida 34945, USA Vegetables are important constituents of the human diet. Heavy metals and nitrate are among the major contaminants of vegetables. Consumption of vegetables and fruits with accumulated heavy metals and nitrate has the potential to damage different body organs leading to unwanted effects. Breeding vegetables with low heavy metal and nitrate contaminants is a cost-effective approach. We investigated 38 water spinach genotypes for low Cd and nitrate co-accumulation. Four genotypes, i.e. JXDY , GZQL , XGDB , and B888 , were found to have low co-accumulation of Cd ( < 0.71 mg/kg dry weight) and nitrate ( < 3100 mg/kg fresh weight) in the edible parts when grown in soils with moderate contamination of both Cd (1.10 mg/kg) and nitrate (235.2 mg/kg). These genotypes should be appropriate with minimized risk to humans who consume them. The Cd levels in the edible parts of water spinach were positively correlated with the concentration of Pb or Zn, but Cd, Pb, or Zn was negatively correlated with P concentration. These results indicate that these three heavy metals may be absorbed into the plant in similar proportions or in combination, minimizing the influx to aerial parts. Increasing P fertilizer application rates appears to prevent heavy metal and nitrate translocation to shoot tissues and the edible parts of water spinach on co-contaminated soils. 目的 2 筛选镉-硝酸盐共低积累空心菜基因型,并研究降低空心菜重金属含量,提高营养品质的农艺措施. 创新点 2 首次筛选得到镉-硝酸盐共低积累空心菜基因型,并研究空心菜可食部污染物、矿质元素和营养指标之间的相关性,提出进一步降低空心菜可食部镉和硝酸盐含量的农艺措施. 方法 2 共38个空心菜基因型收集于世界各地,种植在连作了7年的中度镉-硝酸盐复合污染土壤上(Cd 1.10 mg/kg,NO 3 − 235.2 mg/kg),4周后收获.用HNO 3 -HClO 4 (体积比5:1)消煮,电感耦合等离子体质谱仪(ICP-MS)测定各种金属元素,水杨酸-硫酸比色法测定硝酸盐含量,钒钼黄比色法测定磷含量,2,6-二氯靛酚滴定法测定维生素C含量,乙醇-丙酮(体积比2:1)比色法测定叶绿素含量. 结论 2 本试验筛选得到镉-硝酸盐共低积累空心菜基因型4个(Cd < 0.71 mg/kg DW,NO 3 − < 3100 mg/kg FW),分别是 JXDY 、 GZQL 、 XGDB 和 B888 ,可以在中轻度镉-硝酸盐复合污染土壤上安全生产.空心菜地上部镉与铅、锌含量呈正相关,而这3种元素均与磷量呈负相关.这些结果表明镉、铅和锌通过相同的途径被空心菜吸收,可以同时被治理.增加磷肥供应率可以抑制复合污染土壤中的镉和硝酸盐向空心菜可食部的转移. Genotypic difference Heavy metal Nitrate Soil pollution Water spinach 基因型变异 重金属 硝酸盐 土壤污染 空心菜 Introduction 1 Cadmium (Cd) is one of the most toxic and mobile heavy metals that affect human health through the food chain. Agricultural practices, intensive industrial activity, and urban expansion have accelerated the release of Cd into soil, water, and air (Lane et al., 2015 2015 ). About 27 860 000 m 2 of agricultural soils in China are polluted with Cd (Liu et al., 2015 2015 ) and Cd contamination has become one of the most important barriers to agricultural sustainability in China (Zhang et al., 2002 2002 ). Cd in agricultural soils is taken up by plants and then enters humans and animals through the food chain (Kirkham, 2006 2006 ). Phytoremediation is the best strategy to reduce the risk of Cd entry into food chain. Fertilizer application is one of the key factors in improving crop productivity. However, excessive use of chemical fertilizers, especially nitrogen (N) fertilizers, results in severe environmental problems (Hakeem et al., 2013 2013 ). China is one of the largest consumers of N fertilizers in the world, but the average N use efficiency is low (only about 35%) (Wang et al., 2014 2014 ), which results in waste of resources and environmental contamination, and also poses a serious hazard to human health (Xu et al., 2012 2012 ; Chen et al., 2014 2014 ). Nitrate is potentially carcinogenic and may increase the incidence of gastric, bladder, and oesophageal cancers (Gulis et al., 2002 2002 ). However, nitrate can decrease blood pressure, thus reducing the risk of cardiovascular disease, myocardial infarction, and stroke. Daily consumption of nitrate-rich vegetables is associated with beneficial effects for patients with gastric ulcer, renal failure, and metabolic syndrome (Habermeyer et al., 2015 2015 ). Vegetables are nutritious and are assumed to be safe to consume; people are unaware that some parts of the vegetable may be contaminated with heavy metals and are a major source of human exposure to Cd and nitrate (Tang et al., 2016 2016 ; Fan et al., 2017 2017 ). Water spinach ( Ipomoea aquatica Forsk.), an important leafy vegetable in eastern and southern Asia, can absorb Cd and nitrate naturally into their vacuoles (Wang et al., 2009 2009 ; Xin et al., 2010 2010 ). Therefore, evaluation of water spinach genotypes for low Cd and nitrate accumulation when grown on contaminated soils has become a research priority in minimizing human exposure to these co-contaminants. Accumulation of Cd by plants varies with species and genotype. New strategies have been applied to breeding and screening heavy metal low accumulator vegetable genotypes, such as Chinese cabbage ( Brassica chinensis L.) (Liu et al., 2010 2010 ; Wang X et al., 2015 2015 ), pakchoi ( Brassica rapa L. ssp. chinensis ) (Chen et al., 2012 2012 ), soybean ( Glycine max Merr.) (Arao et al., 2003 2003 ; Sugiyama et al., 2011 2011 ), welsh onion ( Allium fistulosum L.) (Li et al., 2012 2012 ), and sweet potato ( Ipomoea batatas (L.) Lam.) (Huang et al., 2015 2015 ). Nitrate concentration in high-accumulation genotypes was several times greater than that in low-accumulation genotypes, such as lettuce ( Lactuca sativa L.) (Escobar-Gutiérrez et al., 2002 2002 ; Burns et al., 2011a 2011a ), leaf mustard ( Brassica juncea (L.) Czern.) (Sharma et al., 2010 2010 ), and taro ( Colocasia esculenta (L.) Schott) (Kristl et al., 2016 2016 ). Vegetable genotypes with a lower nitrate concentration need to be identified for agricultural production and improved human health. Soils in southern China are often polluted by multiple contaminants. Among them Cd and nitrate contaminations are attributed to irrigation practices, use of low-grade organic fertilizers, and heavy application of N fertilizers, all of which can affect growth, metal tolerance, and metal accumulation in water spinach. Screening different vegetable genotypes with low absorption of Cd and nitrate provides an opportunity to safeguard human consumption. Previous studies were limited to pot and hydroponics where correlations between different contaminants and nutrients were not observed. This study aimed to investigate co-accumulative remediation capacity of Cd and nitrate in field conditions among 38 genotypes of water spinach. Materials and methods 1 Soil characterization 2 The experiment was performed in greenhouses (Chunyi farm) located at 30°23′37″ N and 120°2′13″ E, Hangzhou, Zhejiang Province, China with an average temperature of 30 °C throughout the day. The soil had been moderately contaminated by Cd and nitrate during several decades of intensive vegetable production (Tang et al., 2016 2016 ). The depth of soil sampling for analysis was 10‒15 cm, and four soil samples were collected to represent the area. The preliminary soil assessment and initial concentrations of metals in the soil (Table 1 1 ) were determined according to the previous methods (Li, 2000 2000 ; Bao, 2008 2008 ; Tang et al., 2016 2016 ). DTPA: diethylene triamine pentaacetic acid. Reprinted from Tang et al. ( 2016 2016 ), Copyright 2016, with permission from Elsevier pH Organic matter (g/kg) Total N (g/kg) Total P (g/kg) Total K (g/kg) Available N (g/kg) Available P (g/kg) 5.39±0.05 23.07±0.69 1.22±0.03 0.60±0.07 1.69±0.14 97.35±6.74 77.59±10.11 7 7 Available K (g/kg) Total Cd (g/kg) Total Pb (g/kg) DTPA Cd (g/kg) DTPA Pb (g/kg) Nitrate-N (g/kg) 7 296.87±5.06 1.10±0.18 32.66±0.33 0.23±0.01 0.96±0.12 235.21±6.64 Sample collection and cultivation 2 Thirty-eight genotypes of water spinach ( I . aquatica F.) were obtained from a local seed market in Hangzhou, China. Their genus, origin, and characteristics are listed in Table 2 2 . Split plot design was used with three replicates for major plots and genotypes for the minor plots. Each minor plot was 10 m 2 . Approximately 900 seeds of each genotype were soaked overnight in aerated deionized water at (23±0.3) °C, disinfected with 0.7 g/L NaClO for 30 min, drained and sown in the field in June 2014. Planting density and field management were the same as in conventional farming practice. Region (province or city of China) or country (Thailand, Indonesia) 2 Serial No. 2 Accession name 2 Origin * * 2 Horticultural characteristics Petioles Blades 1 JXDY Jiangxi Green Extra-big 2 JADY Jiangxi Green Big 3 GDLY Jiangxi White Small 4 GZTB Guangdong White Extra-big 5 G501 Guangdong Light yellow Small 6 GZQL Hong Kong Green Small 7 XGDB Fujian White Big 8 G268 Fujian Green Small 9 TWBD Jiangxi White Big 10 TWLY Thailand Green Small 11 TWBL Guangdong White Small 12 TWYX Taiwan Green Small 13 TWZY Indonesia Green Small 14 T311 Taiwan White Small 15 TAIG Thailand Green Small 16 TGLY Thailand Green Small 17 T221 Guangxi Green Small 18 TGJY Thailand Green Small 19 TGLL Jiangsu Green Small 20 YXBL Guangdong White Small 21 X606 Guangdong Light green Small 22 Y601 Beijing Green Small 23 CHFE Hebei Green Extra-big 24 QCUI Tianjin Green Extra-big 25 XIAO Jiangsu Green Small 26 LZHU Thailand Green Small 27 GDZY Thailand Green Small 28 GLCQ Jiangxi Green Small 29 DAYE Jiangxi Green Extra-big 30 BQBA Fujian Green Small 31 CBDY Jiangxi White Extra-big 32 LIUY Jiangxi Green Small 33 BGDY Hubei White Big 34 LYQG Fujian Green Small 35 HSZY Shanghai Green Small 36 B888 Jiangxi White Small 37 DQGU Jiangxi Light green Small 38 QGZY Hebei Green Small Fresh weight and biomass determination 2 The experiment was performed according to the previous method (Tang et al., 2017 2017 ). All genotypes were grown in green house for four weeks without application of fertilizer. Plants excised at 0.5 cm above the base were used for fresh weight (FW) and biomass determination. Mature and immature leaves (leaf blades and petioles) were separated and FWs were estimated. After rinsing with tap water, ten representative plants selected from each genotype were combined together to make a composite sample. Subsamples were dried in an oven at 105 °C for 30 min, and then at 65 °C until a constant weight was attained and the biomass of shoots was then recorded. Heavy metal analysis and nitrate determination of plant samples 2 The concentrations of Cd and other metals (K, Ca, Mg, Fe, Zn, Cu, Pb, Mn, and Se) of plant samples, which were digested with HNO 3 and HClO 4 (5:1, v/v), were determined using inductively coupled plasma mass spectrometer (ICP-MS, 7500a, Aligent, USA), according to the previous method (Bao, 2008 2008 ). Fresh samples were placed in 10 ml deionized water and heated in a boiling water bath for 30 min. Then the nitrate concentration in the water extract was determined using an ultraviolet spectrophotometer (Lambda 350V-vis, Perkin Elmer, Singapore), according to salicylic acid colorimetric method described by Li ( 2000 2000 ). Analyses of other mineral elements (N and P) and nutritional indices (chlorophyll, protein, vitamin C, and cellulose) were performed according to the pervious methods (Tang et al., 2016 2016 ). Statistical analysis 2 The statistical evaluation was represented as the mean±standard error (SE) of four replicates. The differences between 38 genotypes of water spinach were estimated by the least significant difference (LSD) method and the correlation was analyzed by Bivariate analysis (SPSS 20.0) and graphical representation was generated by Origin Pro 8.0. Results 1 Fresh weight and biomass yields 2 FWs and biomass of 38 water spinach genotypes were determined (Figs. 1 1 and 2 2 ) after growth for four weeks. FW ranged from 16.62 g/plant ( LIUY ) to 10.22 g/plant ( BQBA ), approximately a 1.6-fold difference between the highest and the lowest levels with a mean value of 13.30 g/plant (Fig. 1 1 ). Biomass values ranged from 1.24 g/plant ( LIUY ) to 0.74 g/plant ( BQBA ), approximately a 1.7-fold difference with a mean value of 0.98 g/plant (Fig. 2 2 ). Bars represent standard error of the mean with four replicates. Data analysis was performed using LSD method Bars represent standard error of the mean with four replicates. Data analysis was performed using LSD method Cd and nitrate uptake 2 Cd uptake ranged from 2.75 mg/kg dry weight (DW) ( GDZY ) to 0.07 mg/kg DW ( TWBL ), a difference of more than 39-fold with a mean value of 1.15 mg/kg DW (Fig. 3 3 ). Nitrate concentrations among all genotypes ranged from 10 983 mg/kg FW ( LIUY ) to 1809 mg/kg FW ( JXDY ), a 6-fold difference with a mean value of 5177 mg/kg FW (Fig. 4 4 ). Bars represent standard error of the mean with four replicates. Data analysis was performed using LSD method Bars represent standard error of the mean with four replicates. Data analysis was performed using LSD method Correlations between Cd, nitrate, and other elements 2 No correlation was observed between Cd and nitrate concentrations (Fig. 5a 5a ). However, there was a significant positive correlation between Cd, Pb, and Zn concentrations (Fig. 6 6 ) and a significant negative correlation between Cd or Pb and P (Figs. 7a and 7b 7a and 7b ), and between Zn and P (Fig. 7c 7c ). There was a positive correlation between nitrate and chlorophyll concentrations (Fig. 8b 8b ), and a significant positive correlation between nitrate and vitamin C (Fig. 8a 8a ). However, no significant correlation between Cd, nitrate and biomass yield in water spinach (Figs. 5b and 5c 5b and 5c ) was observed. (a) Nitrate vs. Cd; (b) Plant biomass vs. Cd; (c) Plant biomass vs. nitrate (a) Pb vs. Cd; (b) Zn vs. Cd; (c) Zn vs. Pb. * Significance at P < 0.05; ** Significance at P < 0.01 (a) Cd vs. P; (b) Pb vs. P; (c) Zn vs. P. * Significance at P < 0.05; ** Significance at P < 0.01 (a) Vitamin C vs. nitrate; (b) Chlorophyll vs. nitrate. * Significance at P < 0.05; ** Significance at P < 0.01 Discussion 1 Water spinach is a common leafy vegetable which provides rich nutrients to the diet. All the selected water spinach showed successive growth in Cd and nitrate co-contaminated soil. The FW or biomass yield is close to the mean value. Water spinach tolerates high Cd and nitrate stress relatively well. The results were consistent with previous studies (Wang et al., 2007 2007 ). Since there is no visible symptom of toxicity when water spinach is grown in Cd and nitrate co-contaminated soils, the potential health risk may be high from inadvertently consuming contaminated water spinach. The National Food Safety Standard of China GB 2762-2012 (Ministry of Health of the People’s Republic of China, 2012 2012 ) sets the safe limit for Cd contamination of fresh leafy vegetables at 0.05 mg/kg FW. Since the water content of water spinach is 93%, it can be calculated that the safe consumption level of Cd in water spinach shoot is 0.71 mg/kg DW. Given this standard, 17 water spinach genotypes tested in our study can be safe for consumption. The maximum permissible concentration (MPC) of nitrate is 3100 mg/kg FW (Zhou et al., 2000 2000 ), which means that only five water spinach genotypes can be safe for consumption. Using the combined standards for Cd and nitrate, four genotypes were recognized as safe, i.e. JXDY , GZQL , XGDB , and B888 (Table 3 3 ), which are low Cd and nitrate co-accumulators and suitable to grow in slightly or moderately contaminated soils without any risk to human health. Data are expressed as mean±standard error with four replicates Genotypes Cd (mg/kg DW) Nitrate (mg/kg FW)   JXDY 0.50±0.08 1809.6±96.4   GZQL 0.11±0.02 2154.9±236.2   XGDB 0.41±0.04 2723.7±443.6   B888 0.57±0.03 2549.3±668.0 Previous studies showed that low doses of heavy metals may promote plant hormone secretion and regulate plant growth and development (Liu et al., 2010 2010 ). However, the distinct tolerance mechanisms of water spinach genotypes to Cd are not fully understood. Variations in Cd accumulation among genotypes may be related to uptake, transfer, and bioaccumulation of Cd in shoots (Uraguchi et al., 2009 2009 ) and roots (Lux et al., 2011 2011 ) and are influenced by several environmental factors such as light, cold, and humidity (Cheng et al., 2006 2006 ; Li et al., 2015 2015 ), including properties of the rhizosphere (Arao et al., 2009 2009 ; Zheng and Zhang, 2011 2011 ). The accumulation of Cd in plants varies greatly not only among plant species but also among genotypes or cultivars within the same species (Yu et al., 2006 2006 ; Liu et al., 2007 2007 ; Wang et al., 2007 2007 ; Martin et al., 2012 2012 ). The results of the present study showed that different genotypes of water spinach had different Cd retention abilities, resulting in different Cd accumulations. Genotype-dependent Cd accumulation of water spinach largely depends on bioprocesses occurring in shoots and roots (Xin et al., 2013a 2013a ). Some evidence indicates that Cd subcellular distribution may be associated with Cd tolerance and detoxification in plants. Distribution of Cd in plants is influenced by cross-membrane transport systems, the existence of intracellular binding sites, vacuole sequestration, xylem and phloem transport, and root retention (Grant et al., 2008 2008 ). In most cases heavy metals are retained in plant root cell wall, restricting their translocation to shoot and minimizing plant damage (Wang Y et al., 2015 2015 ). Similar results were reported by Xin et al. ( 2013a 2013a , 2013b 2013b ) and Huang et al. ( 2016 2016 ). Retention of Cd in the cell wall is the effective and most important mechanism for Cd detoxification in all water spinach tissues, especially in young leaves (Xin et al., 2013b 2013b ), though this may vary between genotypes (Huang et al., 2016 2016 ). The presence of thicker phloem and outer cortex cell walls in the low Cd cultivars may explain why low Cd cultivar roots were able to retain more Cd, thus reducing Cd translocation to shoots (Xin et al., 2013b 2013b ). Nitrate is taken up by plants from fertilizer, but this differs depending on the genotype and environment (Burns et al., 2011b 2011b ). Nitrate concentration in plants depends mainly on environmental, genetic, and nutritional factors (Anjana et al., 2009 2009 ). When plants are provided with excess nitrate, only a small portion taken up by roots may be immediately translocated to and assimilated by shoots, while the majority of the absorbed nitrate is stored in vacuoles in both roots and shoots (Luo et al., 2006 2006 ). Nitrate accumulation in plant tissues acts not only as a temporary store, but also as a replacement osmoticum for other plant solutes for maintaining turgor and driving leaf expansion (Burns et al., 2010 2010 ). Excessive N accumulation in plant tissues is attributed to the imbalance between uptake and assimilation (Cárdenas-Navarro et al., 1999 1999 ). As a result, the uptake of nitrate may reduce osmatic pressure that limits the storage of organic solutes in vacuole (Wojciechowska and Kołton, 2014 2014 ). Although there is strong evidence for the genotypical effects on Cd or nitrate accumulation in vegetable plants (Arao et al., 2003 2003 ; Burns et al., 2011a 2011a ; Sugiyama et al., 2011 2011 ; Wang X et al., 2015 2015 ; Kristl et al., 2016 2016 ), less is known about the higher specificity. The present study indicated that no correlation occurred between plant Cd, nitrate, and biomass, suggesting that yield, Cd and nitrate levels may be independent of each other. This suggests that these three traits could be improved separately or in combination for high yield with low concentrations of Cd and nitrate. Heavy metals, such as Cd, Pb, and Zn, are phytotoxic when present in excessive amounts and can interfere with photosynthetic and respiratory activities, mineral nutrition, enzymatic activity, membrane functions, and hormone balance (Clijsters and van Assche, 1985 1985 ). The significant positive correlation between Cd or Pb and Zn in water spinach genotypes may indicate that they have similar uptake mechanisms. Similar results were reported in previous studies (Wu and Zhang, 2002 2002 ; Liu et al., 2003 2003 ; Dong et al., 2006 2006 ; Li et al., 2015 2015 ). These heavy metals are probably transported by similar transporters in the form of compounds or chelate complexes, and the mobilizing function of root exudates is effective not only for Cd but also for Pb and Zn (Kabata-Pendias, 2011 2011 ). Cohen et al. ( 1998 1998 ) reported that IRT1 may facilitate the transport of heavy metals in the form of divalent cations such as Cd 2+ , Pb 2+ , and Zn 2+ . Almost all Cd hyper-accumulation plants could accumulate high concentrations of Pb and Zn (He et al., 2002 2002 ). All of these suggest that these three metals may have similar transport mechanisms, although the interaction of Cd with some nutrients in soil is not fully understood. P is a macronutrient and has an important role in plant physiology including metabolism. When P is deficient, plant growth and crop yield are reduced, as P is an essential element for the synthesis of nucleic acids, phospholipids, and adenosine triphosphate (ATP) (Yin et al., 2016 2016 ). Our results indicated that there was a significant negative correlation between Cd, Pb, or Zn and P concentration in water spinach plants (Fig. 7 7 ). Similar results have also been reported (Keller and Römer, 2001 2001 ; Zhang et al., 2002 2002 ; Dheri et al., 2007 2007 ). Application of P-containing materials influences the bioavailability of heavy metals such as Cd, Pb, and Zn in soil (Qiu et al., 2011 2011 ). Jiang et al. ( 2007 2007 ) reported that increased P in soil resulted in substantial precipitation of heavy metal-P complexes in the cell wall and vacuoles in maize ( Zea mays L.), and a similar effect was reported in strawberry ( Fragaia ananassa D.) (Nuzahath et al., 2013 2013 ). P-heavy metal interactions reduce the availability of heavy metals in soil and limit their mobility in plants (Clemens, 2006 2006 ). The supply of adequate and balanced mineral nutrients to crops has the potential to improve plant tolerance, growth, development, and productivity under stress environments (Mitchell et al., 2000 2000 ; Astolfi et al., 2004 2004 ). However, the proportion of macronutrient fertilizers in agriculture is often out of balance in China. Our present study points out that increasing the P supply to heavy metal (Cd, Pb, or Zn)-contaminated soils reduces Cd, Pb, or Zn concentration in water spinach. Chlorophyll concentration is influenced by environmental factors and N fertilization (Barickman and Kopsell, 2016 2016 ) and the form and ratio of N in plants (Borowski and Michalek, 2008 2008 ). Previous research demonstrated that higher ratios of NO 3 − -N to NH 4 + -N positively influenced chlorophyll concentrations in the leaf tissue of kale ( Brassica oleracea L. var. acephala ) (Kopsell et al., 2007 2007 ) and leaf lettuce ( L . sativa L.) (Stagnari et al., 2015 2015 ). Chlorophyll concentration in low nitrate accumulation genotypes oilseed rape ( Brassica napus L.) was reported to be significantly lower than that in high nitrate accumulation genotypes (Han et al., 2016 2016 ). In the present study, there was a positive correlation between nitrate and chlorophyll concentration (Fig. 8b 8b ), which was consistent with previous studies. There was a significant positive correlation between nitrate and vitamin C in water spinach genotypes (Fig. 8a 8a ). Similar results in spinach ( Spinacia oleracea L.) were previously reported by Conesa et al. ( 2009 2009 ) and Koh et al. ( 2012 2012 ). The levels of vitamin C and nitrate in leafy vegetables are two key indices for evaluating their nutritional quality (Konstantopoulou et al., 2010 2010 ), and vitamin C content in leafy vegetables is mainly controlled by the form and ratio of N (Sørensen et al., 1994 1994 ). The significant positive correlation between nitrate and vitamin C concentration in water spinach genotypes suggests that it would be difficult to reduce nitrate and increase vitamin C concentration simultaneously, so it is necessary to improve these two traits separately. Conclusions 1 We have identified four genotypes of water spinach, JXDY , GZQL , XGDB , and B888 , as low co-accumulators for Cd and nitrate, indicating that they should be the preferred genotypes for growing in slightly or moderately contaminated soils, minimizing risk to human health. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1700017 Project supported by the Key Projects from Ministry of Science and Technology of China (No. 2016YFD0800805), the Zhejiang Provincial Science and Technology Bureau (Nos. 2015C02011-3 and 2015C03020-2), and the Fundamental Research Funds for the Central University, China 2017-01-12 2017-04-09 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2018 2018 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 19