Overexpression of IbSnRK1 enhances nitrogen uptake and carbon assimilation in transgenic sweetpotato

REN Zhi-tong, ZHAO Hong-yuan, HE Shao-zhen, ZHAI Hong, ZHAO Ning, LIU Qing-chang

Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture/Beijing Key Laboratory of Crop Genetic Improvement/Laboratory of Crop Heterosis and Utilization, Ministry of Education/China Agricultural University, Beijing 100193,P.R.China

1. Introduction

Nitrogen is one of the most important essential macronutrients for growth and development of plants. It is a vital element of DNA, RNA and proteins, and its transport and metabolism are necessary for the survival of living organisms (Chenet al. 2016). The nitrogen uptake efficiency is important for achieving high yield and excellent quality in agricultural production (Zhaoet al. 2016). Therefore, how to improve the nitrogen absorption is the primary issue which breeders concern.

Plant nitrogen metabolism is a complicated process.Plants uptake nitrogen with the help of nitrogen transport proteins (NRT) (Lezhneva 2014). The first enzyme involved in nitrate assimilation is nitrate reductase (NR) which converts nitrate to nitrite (Davenportet al. 2015), and subsequently nitrite is converted to ammonium by the second key enzyme nitrite reductase (NiR), which is redistributed to different tissues of plants (Orselet al. 2002). The nitrogen assimilation is tightly linked to photosynthesis and carbon metabolism (Vincentzet al. 1993; Tobinet al. 2005).

The sucrose non-fermenting 1 (SNF1) protein kinase,which belongs to serine/threonine protein kinase, is one of the most important energy and stress regulators. In higher plants, the SNF1 protein kinase family is divided into three subfamilies, SnRK1, SnRK2, and SnRK3. SnRK1 regulates carbon and nitrogen metabolisms by inactivating the sucrose phosphate synthase (SPS), 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA) and NR, and activating sucrose synthase (SUS) and α-amylase (Halfordet al.2003). Overexpression ofStSnRK1in potato increased the starch content and decreased the glucose level (Mckibbinet al. 2006).AKIN10andAKIN11fromArabidopsiswere found to be involved in starch biosynthesis (Fragosoet al.2009). Liet al. (2010) cloned theSnRK1gene fromMalus hupehensisand found that its overexpression in tomato resulted in the increased starch content. They further found that overexpression of this gene increased carbon assimilation and nitrogen uptake and modified fruit development(Wanget al. 2012).

Sweetpotato,Ipomoea batatas(L.) Lam., is an important food crop. Several genes have been cloned from sweetpotato(Liu 2017). However, nitrogen metabolism related genes have not been reported to date in sweetpotato. Jianget al. (2013)cloned theIbSnRK1gene from sweetpotato and found that its overexpression increased the starch content in tobacco.In this study, we found that its overexpression enhanced nitrogen uptake and carbon assimilation in sweetpotato.

2. Materials and methods

2.1. Plant materials

In our previous study, theIbSnRK1gene was cloned from sweetpotatocv. Lushu No. 3 (Jianget al. 2013). The expression vector pCAMBIA3301 withIbSnRK1andbargenes driven by a CaMV 35S promoter, respectively, was constructed and transformed to sweetpotatocv. Lizixiang according to the method of Yuet al. (2007). The transgenic plants were produced and identified with PCR analysis as described by Wanget al. (2016). The primers were listed in Table 1. The transgenic plants, wild type (WT) and empty vector control (VC) were transferred to soils in a greenhouse and further in a field for subsequent study.

2.2. Expression analysis of IbSnRK1

The 4-week-oldin vitro-grown plants of Lushu No. 3 were treated with 8 mmol L–1Ca(NO3)2in liquid Murashige and Skoog (MS) medium, and sampled at 0, 3, 6, 12, 24, and 48 h after treatment to analyze the expression ofIbSnRK1by real-time quantitative PCR (qRT-PCR) according to themethod of Liuet al. (2014). Specific primers ofIbSnRK1and sweetpotatoβ-actingene (GenBank AY905538) as an internal control for qRT-PCR were listed in Table 1. Quantification of the gene expression was done with comparative CTmethod (Schmittgen and Livak 2008).

Table 1 Primers used in this study

2.3. Analysis of nitrogen uptake efficiency

The cuttings about 20 cm in length of the transgenic plants,WT and VC from a field were grown in a transplanting box with a mixture of soil, vermiculite and humus (1:1:1, v:v) for 1 week. All plants were irrigated with a 200-mL of 8 mmol L–1Ca(15NO3)2(10% atoms15N excess) solution once (Liet al.2010; Fanet al. 2014). After 45 days, their roots, stems, and leaves were dried at 80°C for 72 h and weighed, respectively.The15N concentration was measured with the Stable Isotope Ratio Mass Spectrometer Isoprime 100 (Elementar Analysensysteme GmbH, Germany). The total N was detected with Automatic Kieldahl Apparatus K1100F (Haineng, Jinan,China). The total N content, nitrogen derived from fertilizer(NDFF), and nitrogen uptake efficiency (NUE) were calculated according to the method of Wanget al. (2012).

2.4. Analyses of nitrate content and NR activity

All plants were irrigated with a 200-mL of 8 mmol L–1Ca(NO3)2solution once. Plants treated without Ca(NO3)2were used as the control. After 45 days, the fresh roots were collected to measure the nitrate content. Roots of 0.1 g were added to 1 mL distilled water and boiled for 30 min to extract the nitrate. The samples were centrifuged at 12 000×g for 15 min,0.02 mL supernatant was mixed with 0.08 mL 5% salicylic acid-sulfuric acid solution and incubated at room temperature for 30 min. The 1.9 mL 8% sodium hydroxide solution was added and nitrate was detected at 410 nm under room temperature. The NR activity was measured according to the method of Ferrario-Méryet al. (1998).

2.5. Analyses of free amino acid and soluble protein content

The fresh leaves of transgenic plants, WT and VC treated with Ca(NO3)2as mentioned above were used to measure the content of free amino acids and soluble proteins.Leaves of 0.1 g were mixed with 1 mL 10% acetum and then boiled for 15 min to extract free amino acids. These samples were centrifuged at 8 000×g for 10 min at 4°C to separate supernatant. And 0.05 mL supernatant was mixed with 0.5 mL phosphate buffer (pH=5.4), 0.5 mL ninhydrin and 0.05 mL 0.3% ascorbic acid and boiled for 15 min, and free amino acids were detected at 580 nm under room temperature. The soluble proteins were measured as described by Brownet al. (1989).

2.6. Measurements of photosynthesis activity

The photosynthetic rate, stomatal conductance, intercellular CO2concentration, and transpiration rate in the leaves of transgenic plants, WT and VC treated with Ca(NO3)2as mentioned above were measured at 13:00 with LI-6400 Portable Photosynthesis System LI-6400 (LI-COR Inc.,Lincoln, NE, USA).

2.7. Analyses of sucrose and starch contents

The sucrose and starch contents in the fresh leaves of transgenic plants, WT and VC treated with Ca(NO3)2as mentioned above were determined according to the methods of Xiaoet al. (2016) and Weiet al. (2015), respectively.

2.8. Assays for SUS and AGPase activities

SUS and ADP-glucose pyrophosphorylase (AGPase) activities in the fresh leaves of transgenic plants, WT and VC treated with Ca(NO3)2as mentioned above were measured according to the methods of Baroja-Fernándezet al. (2012)and Kanget al. (2013), respectively.

2.9. Expression analysis of nitrogen and carbon metabolisms related genes

The fresh roots of transgenic plants, WT and VC treated with Ca(NO3)2as mentioned above were used to analyze the expression of nitrogen transporter (NRT 1.1,NRT 1.3,NRT 2.4, andNRT 3.1) andNRgenes, and their fresh leaves were used to analyze the expression of glutamine synthetase (GS), glutamate synthase (GOGAT),SUSand AGPase large subunit (AGPL1) genes according to the method of Liuet al. (2015). Specific primers were designed from conserved regions of genes (Table 1).

2.10. Statistical analysis

All experiments were independently performed three times and the data were presented as the means±SE. Results were analyzed by Student’st-test in a two-tailed analysis using SPSS 20.0 Statistic Program. Significance was de-fined asP＜0.05 (＊) andP＜0.01 (＊＊), respectively.

3. Results

3.1. Expression analysis of IbSnRK1

qRT-PCR analysis showed that the expression ofIbSnRK1reached the highest level (3.1-fold) at 24 h of Ca(NO3)2treatment (Fig. 1). The results indicated thatIbSnRK1was induced by nitrate and might be involved in the nitrogen metabolism.

3.2. Nitrogen uptake efficiency

A total of 18 transgenic plants, named L1, L2, …, L18,respectively, were obtained and 7 of them exhibited signifi-cantly higher15N atom excess in roots, stems, and leaves compared with WT and VC (Table 2). The 4 transgenic plants, L5, L6, L16, and L18, with the highest15N atom excess were selected to analyze dry weight, root/shoot ratio,15N and total N content, NDFF and NUE. These 4 plants showed significantly higher dry weight, root/shoot ratio,15N and total N content, NDFF and NUE compared with WT and VC (Fig. 2).

3.3. Nitrate content and NR activity

No differences in nitrate content and NR activity were found between the 4 transgenic plants and WT/VC without Ca(NO3)2treatment. The nitrate content and NR activity in the transgenic plants were significantly increased by 62.5–104.4% and 30.6–61.1%, respectively, compared with WT under Ca(NO3)2treatment (Fig. 3-A and B). These results indicated that the overexpression ofIbSnRK1enhanced the nitrogen uptake of the transgenic plants.

Fig. 1 Expression analysis of IbSnRK1 in sweetpotato cv.Lushu No. 3 by qRT-PCR after different times of 8 mmol L–1 Ca(NO3)2 treatment. Bars are SE. ＊＊, significant difference at P＜0.01.

Table 2 The 15N atom excess in roots, stems, and leaves of the transgenic plants, wild type (WT), and empty vector control(VC) under Ca(15NO3)2 treatment

3.4. Free amino acid and soluble protein content

Free amino acid and soluble protein content in the 4 transgenic plants were significantly increased by 18.3–35.6% and 32.1–49.0%, respectively, compared with WT under Ca(NO3)2treatment (Fig. 3-C and D). No significant differences were observed between the transgenic plants and WT under normal condition. These results suggest thatIbSnRK1is involved in the nitrogen metabolism of sweetpotato.

3.5. Photosynthesis activity

The photosynthetic rate, stomatal conductance, and intercellular CO2concentration were significantly higher,while the transpiration rate was significantly lower in the 4 transgenic plants than in WT and VC under Ca(NO3)2treatment (Fig. 4). No significant differences were observed in these parameters between the transgenic plants and WT/VC under normal condition (Fig. 4). These results showed thatIbSnRK1enhanced the photosynthesis activity of the transgenic plants under Ca(NO3)2treatment.

3.6. Sucrose and starch contents, SUS and AGPase activities

Overexpression of the genes associated with the carbon assimilation can increase the sucrose and starch contents and SUS and AGPase activities in plants under normal and treatment conditions (Mckibbinet al. 2006; Fragosoet al. 2009; Liet al. 2010; Wanget al. 2012). The 4IbSn-RK-overexpressing plants exhibited significantly increased sucrose and starch contents and SUS and AGPase activities compared with WT and VC under normal conditions(Fig. 5). These results suggest thatIbSnRK1is involved in the carbon assimilation of sweetpotato. Furthermore,the increased levels of sucrose, starch, SUS, and AGPase were significantly higher in the transgenic plants than in WT and VC under Ca(NO3)2treatment (Fig. 5). These results showed thatIbSnRK1enhanced the carbon assimilation of sweetpotato.

Fig. 2 Dry weight (DW, A), root/shoot ratio (B), 15N content (C), total N content (D), nitrogen derived from fertilizer (NDFF, E), and nitrogen uptake efficiency (NUE, F) in roots, stems, and leaves of the transgenic plants (L5, L6, L16, and L18), wild type (WT), and empty vector control (VC) grown in a transplanting box under Ca(15NO3)2 treatment for 45 days. Bars are SE. ＊ and ＊＊, significant difference at P＜0.05 and P＜0.01, respectively.

3.7. Expression analysis of nitrogen and carbon metabolisms related genes

Expression levels of several nitrogen and carbon metabolisms related genes were analyzed by qRT-PCR under normal and treatment conditions (Fig. 6). The nitrogen metabolism related genesNRT 1.1,NRT 1.3,NRT 2.4,NRT 3.1,NR,GS, andGOGATdid not show the changes in expression levels under normal condition, but their expression was significantly up-regulated in the transgenic plants compared with WT and VC under treatment condition (Fig. 6).The expression of the carbon metabolism related genesIbSnRK1,SUS, andAGPL1 was significantly increased in the 4 transgenic plants compared with WT and VC under normal and treatment conditions (Fig. 6).

Fig. 3 Nitrate content (A), nitrate reductase (NR) activity (B), free amino acid content (C), and soluble protein content (D) in the roots of transgenic plants (L5, L6, L16, and L18), wild type (WT), and empty vector control (VC) grown in a transplanting box under Ca(NO3)2 treatment for 45 days. FW, fresh weight. Bars are SE. ＊ and ＊＊, significant difference at P＜0.05 and P＜0.01, respectively.

Fig. 4 Photosynthetic rate (A), stomatal conductance (B), intercellular CO2 concentration (C), and transpiration rate (D) in the leaves of transgenic plants (L5, L6, L16, and L18), wild type (WT) and empty vector control (VC) grown in a transplanting box under Ca(NO3)2 treatment for 45 days. FW, fresh weight. Bars are SE. ＊ and ＊＊, significant difference at P＜0.05 and P＜0.01, respectively.

Fig. 5 Sucrose content (A), starch content (B), sucrose synthase (SUS) activity (C), and ADP-glucose pyophosphorylase(AGPase) activity in the leaves of transgenic plants (L5, L6, L16, and L18), wild type (WT) and empty vector control (VC) grown in a transplanting box under Ca(NO3)2 treatment for 45 days. FW, fresh weight. Bars are SE. ＊ and ＊＊, significant difference at P＜0.05 and P＜0.01, respectively.

4. Discussion

Nitrogen is a very important nutrient to sweetpotato. Effects of NUE on yield and quality in sweetpotato were widely reported (Martí and Mills 2002; Ankumahet al. 2003; Ukomet al. 2011). Gene engineering is an alterative approach to improve NUE in higher plants. This study has indicated that overexpression of theIbSnRK1gene significantly enhanced nitrogen uptake and carbon assimilation in sweetpotato,similar to the results of Wanget al. (2012) in tomato.

The present results showed that the expression ofIbSnRK1in sweetpotato was induced by Ca(NO3)2(Fig. 1).Under Ca(15NO3)2treatment, theIbSnRK1-overexpressing sweetpotato plants exhibited significantly higher dry weight,root/shoot ratio,15N and total N content, NDFF and NUE than WT (Fig. 2), suggesting that its overexpression increases the nitrogen content by improving NUE.

The nitrogen is absorbed by the roots through nitrate transporters, and then is distributed to roots, stems and leaves. Three types of nitrate transporters, nitrate/peptide transporters (NRT1/PTR), NRT2, and NRT3, have been identified in higher plants (Kibaet al. 2012; Baiet al. 2013).In the present study, the expression levels ofNRT1.1,NRT1.3,NRT2.4, andNRT3.1, the content of nitrate and the activity of NR in the roots of transgenic plants were significantly higher than in those of WT under Ca(NO3)2treatment (Fig. 3-A and B, Fig. 6). These results suggest that overexpression ofIbSnRK1enhances the nitrogen uptake by up-regulating theNRTgenes and increasing the NR activity.

The content of free amino acids and soluble proteins can reflect the nitrogen metabolism in higher plants. The primary nitrate is converted into ammonium by NR, subsequently ammonium is converted into glutamine and glutamic acid by GS and GOGAT in plants (Lamet al. 1996; Masclauxet al.2009). The present study has indicated that overexpression ofIbSnRK1increased the content of free amino acids and soluble proteins (Fig. 3-C and D) and the expression level ofGSandGOGAT(Fig. 6). It is thought thatIbSnRK1might enhance the nitrogen metabolism by up-regulatingGSandGOGAT.

Fig. 6 Relative expression level of IbSnRK1 and nitrogen and carbon metabolisms related genes in the leaves (SnRK1, GS,GOGAT, SUS and AGPL1) and roots (NRT 1.1, NRT 1.3, NRT 2.4, NRT 3.1, and NR) of transgenic plants (L5, L6, L16, and L18),wild type (WT), and empty vector control (VC) grown in a transplanting box under Ca(NO3)2 treatment for 45 days. Bars are SE.＊ and ＊＊, significant difference at P＜0.05 and P＜0.01, respectively.

In plants, nitrogen and carbon metabolisms are tightly linked to physiological functions (Vincentzet al. 1993;Nunes-Nesiet al. 2010). The photosynthetic capacity of leaves is related to the nitrogen content primarily (Evanset al. 1989). Sucrose and starch are the main products of carbon assimilation, and SUS and AGPase are widely recognized as the vital enzyme of sucrose metabolism and starch biosynthesis, respectively (Chenet al. 2012; Senget al. 2016). In this study, the photosynthetic rate, stomatal conductance and intercellular CO2concentration in leaves of the transgenic plants were significantly higher than those of WT under Ca(NO3)2treatment, and the content of sucrose and starch were significantly increased (Figs. 4 and 5).Furthermore, the activities of SUS and AGPase and the expression levels ofSUSandAGPL1 were significantly higher in the transgenic plants than in WT under Ca(NO3)2treatment (Fig. 6).

These results suggest that the increased NUE and nitrogen content promote photosynthesis activity, resulting in more accumulation of carbohydrates in the transgenic plants.

5. Conclusion

TheIbSnRK1gene plays important roles in nitrogen uptake and carbon assimilation of sweetpotato. Its overexpression enhanced the nitrogen and carbon metabolisms by improving NUE and up-regulating theNR,GS,GOGAT,SUS, andAGPL1 genes which resulted in the increased free amino acid, soluble protein, sucrose and starch content. This gene has the potential to be used for improving the yield and quality of sweetpotato.

Acknowledgements

This work was supported by the earmarked fund for China Agriculture Research System (CARS-11), the National Natural Science Foundation of China (31461143017) and the Science and Technology Planning Project of Guangdong Province, China (2015B020202008).

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