A sucrose non-fermenting-1-related protein kinase-1 gene, IbSnRK1,confers salt, drought and cold tolerance in sweet potato

Zhitong Ren, Shozhen He,Yunyun Zhou, Ning Zho, To Jing, Hong Zhi,*,Qingchng Liu,b,*

aKey Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs/Beijing Key Laboratory of Crop Genetic Improvement/Laboratory of Crop Heterosis & Utilization and Joint Laboratory for International Cooperation in Crop Molecular Breeding,Ministry of Education, College of Agronomy& Biotechnology,China Agricultural University, Beijing 100193,China

bCollege of Agronomy, Qingdao Agricultural University,Qingdao 266109,Shandong,China

Keywords:Sweet potato IbSnRK1 Abiotic stress tolerance ABA ROS

A B S T R A C T Sucrose non-fermenting-1-related protein kinase-1 (SnRK1) regulates carbon and nitrogen metabolism in plants. However, its roles and their underlying mechanisms in tolerance to abiotic stresses are little known. The present study indicated that the IbSnRK1 gene was strongly induced by NaCl, polyethylene glycol (PEG) 6000, hydrogen peroxide (H2O2), cold(4 °C), and abscisic acid (ABA). Its overexpression significantly increased salt, drought, and cold tolerance in transgenic sweet potato plants. ABA, proline, and K+ contents were significantly increased, whereas malondialdehyde (MDA), Na+ and H2O2 contents and O2?production rate were significantly decreased in the transgenic plants under salt, drought,and cold stresses. Overexpression of the gene up-regulated genes involved in ABA biosynthesis, stress response, and stomatal closure; increased enzyme activities in the reactive oxygen species (ROS) scavenging system; and controlled stomatal closure under salt, drought, and cold stresses. These results show that the IbSnRK1 gene confers salt,drought, and cold tolerance in sweet potato by activating the ROS scavenging system and controlling stomatal closure via the ABA signaling pathway.

1.Introduction

Plants encounter abiotic stresses that limit their growth and affect crop productivity and quality [1,2]. These stresses include high salinity, drought, osmotic, heat, ion toxicity,high temperature, and low temperature [3]. Abiotic stresses such as drought, salinity, and extreme temperature may combine to cause severe damage to plants [4]. Plants have developed multiple and complex strategies to adapt to these abiotic stresses at the molecular, cellular, physiological, and biochemical levels [3,5,6]. In response to stresses, numerous genes involved in many signaling pathways are induced,including functional and regulatory genes [7]. Functional genes encode many kinds of proteins, such as key enzymes for proline biosythesis,scavengers of antioxidants or reactive oxygen species (ROS), ion channel proteins, late embryogenesis abundant protein (LEA), and molecular chaperones.Regulatory genes include calcium sensors, membranelocalized receptors,transcription factors,and protein kinases,and play important roles in signal transduction and gene expression [7]. These genes are thought to increase plant stress tolerance and control gene expression via signaling pathways[8].

Sucrose non-fermenting-1-related protein kinase-1(SnRK1) is a central component of sugar sensing in plants and plays a vital role in the global control of carbon and nitrogen metabolism [9,10]. SnRK1 regulates starch accumulation and nitrogen uptake by inactivating or activating a wide range of enzymes via phosphorylation or dephosphorylation.This regulation affords a potential means of manipulating energy metabolism to improve crop nutritional value and yield [11–15]. The roles and underlying mechanism of SnRK1 in resistance to abiotic stresses are little known. Antisense repression of StubGAL83, the β-subunit of the StubSNF1 protein kinase complex, increased sensitivity to salt stress in potato [16]. The activity of SnRK1 in Arabidopsis incresed the expression of stress-inducible genes and the induction of stress tolerance[17].

Sweet potato,Ipomoea batatas(L.)Lam.,is a food crop and a source of bioenergy worldwide [18,19]. Expansion of its cultivation is limited by abiotic stresses. Increasing its productivity on marginal lands will depend on improving its resistance to abiotic stresses. Gene engineering offers great potential to achieve this goal [20]. Overexpression of several genes increased abiotic stress tolerance in sweet potato[19–22].

We previously isolated an IbSnRK1 gene from sweet potato and found that its overexpression increased nitrogen and carbon assimilation and improved starch content and quality in sweet potato [18,23,24]. The objective of the present study was to investigate the roles of IbSnRK1 in salt, drought and cold tolerance of sweet potato.

2. Materials and methods

2.1. Plant materials and growth conditions

The sweet potato cultivar Lushu 3 was used for measuring the expression of IbSnRK1. In our previous study, IbSnRK1 was introduced into sweet potato cv. Lizixiang and 18 IbSnRK1-overexpressing plants and empty vector control (VC) plants were obtained[24].

2.2. Expression analysis of IbSnRK1 in sweet potato

Four-week-old in vitro-grown plants of Lushu 3 cultured in Murashige and Skoog (MS) liquid medium were treated with 86 mmol L?1NaCl, 20% polyethylene glycol (PEG) 6000,10 μmol L?1hydrogen peroxide(H2O2),cold(4°C)or 100 μmol L?1abscisic acid(ABA)and sampled at 0,3,6,12,24,and 48 h after treatments for extracting total RNA with a Trozol Kit(Transgen, Beijing, China). Expression of IbSnRK1 was measured using special primers of IbSnRK1 and Actin(AY 905538)as internal control(Table S1)as described by Ren et al.[24].

2.3. Assay for salt, drought, and cold tolerance

The expression of IbSnRK1 in in vitro-grown transgenic, wild type (WT) and VC plants under normal conditions was measured by qRT-PCR following Ren et al. [24]. The plants were then cultured on MS medium with 86 mmol L?1NaCl or 20% PEG 6000. After four weeks,their leaf and root formation was investigated and proline and malondialdehyde (MDA)contents and superoxide dismutase (SOD) activity were measured[20].

Cuttings of 25 cm in length were collected from transgenic,WT,and VC plants grown in the field for 6 weeks and treated in Hoagland solution with 86 mmol L?1NaCl for four weeks or 20% PEG 6000 for two weeks followed by two weeks of rewatering, with three cuttings for each line. The Hoagland solution was renewed at a 3-day interval.No-stress treatment was used as a control.Fresh weight(FW)and dry weight(DW)were measured[20].

For further tolerance evaluation, 25-cm-long cuttings of transgenic, WT and VC plants were grown in a transplanting box with soil, vermiculite, and humus (1:1:1, v/v/v) in a greenhouse. After irrigation with water for one week, each plant was treated with 200 mL of 200 mmol L?1NaCl solution at two-day intervals for four weeks, drought stressed for six weeks,or subjected to cold stress(4°C)for two days followed by four weeks of recovery under normal conditions. FW and DW were measured for three plants of each line.

2.4. Measurement of abiotic stress components

Transgenic,WT,and VC plants grown in a transplanting box were treated with 200 mmol L?1NaCl for two weeks,drought for two weeks,or 4°C for two days followed by two weeks of recovery,and plants grown under normal conditions for two weeks were used as control. Photosynthetic rate, intercellular CO2concentration, transpiration rate, and relative chlorophyll content in leaves were measured at 11:00 with a LI-6400 Portable Photosynthesis System (LI-COR Inc., Lincoln,NE, USA) [20]. Contents of ABA, proline and MDA and concentrations of Na+and K+in leaves were determined as described by Liu et al.[20]and Li et al.[25].Activities of SOD,ascorbate peroxidase (APX), catalase 2 (CAT2), and peroxidase(POD)were measured following Zhang et al.[26]and Wu et al. [27].

The accumulations of H2O2and superoxide radical (O2?)were assayed by 3,3′-diaminobenzidine (DAB) and nitro blue tetrazolium(NBT)staining,respectively.Stress-treated leaves were infiltrated with 1 g L?1DAB (pH 3.8) or 0.5 g L?1NBT overnight. The samples were destained by boiling with 95%ethanol for 2 h and then extracted with fixative solution(50%ethanol:glacial acetic acid: methyl aldehyde, 17:1:2). The leaves were photographed and average intensity was estimated with Image J software. H2O2content was measured following Rao et al. [28]. Theproduction rate was determined by monitoring formation from hydroxylamine in the presence of[26].

2.5. Observation of stomatal morphology

Leaves were detached and immediately fixed with 2.5% (v/v)glutaraldehyde for 2.5 h. After being rinsed three times with 0.1 mol L?1phosphoric acid buffer,they were fixed in 1%(v/v)osmic acid for 2 h, followed by dehydration with a pyruvate series (30, 50, 70, 80, 90, and 100%) and drying with LEICA EM CPD 300(Leica,Wetzlar,Germany)critical point dryer.EIKO IB 3(Variable Electron Microscope,Hitachi S3400N,Tokyo,Japan)was used to sputter gold on each sample and the stomatal images were collected using a scanning electron microscope.FW, turgid weight (TW, weight of leaves submerged in water for 6 h),and DW(weight of leaves dried at 80°C for 96 h)were recorded.Relative water content(RWC)was calculated as(FW–DW)/(TW–DW)× 100%.

2.6. Expression measurement of abiotic stress-responsive genes

Transgenic, WT, and VC plants grown in a transplanting box were treated with no stress(normal)for two weeks as a control,200 mmol L?1NaCl for two weeks, drought for two weeks, or 4 °C for two days followed by two weeks of recovery. Their leaves were used to measure the expression of the genes encoding zeaxanthine peroxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), aldehyde oxidase (AAO), pyrroline-5-carboxylate synthase (P5CS), phosphoribulokinase (PRK), SOD,APX, POD, CAT2, vacuolar Na+/K+antiporters 2 (NHX2), S-type anion channel protein 1 (SLAC1), and protein kinase open stomatal 1(OST1/SnRK2.6)with specific primers(Table S1).

2.7. Statistical analysis

All experiments were performed with three biological replicates. Means were compared by Student's t-test (two-tailed analysis) at P < 0.05 (* or lowercase letters) and P < 0.01 (** or capital letters).

3.Results

3.1. Expression of IbSnRK1 in sweet potato

Expression of IbSnRK1 in Lushu 3 was highly induced by NaCl,PEG 6000, H2O2, and ABA with respective expression peaks at 6,12,24,and 6 h after treatment(Fig.1).Under 4°C stress,the transcript level of IbSnRK1 kept going up over 48 h compared to control(Fig.1).

3.2. Tolerance to salt, drought, and cold

Expression of IbSnRK1 was significantly higher in the 18 transgenic plants than in WT and VC plants (Fig. S1).Transgenic, WT, and VC sweet potato plants cultured on MS medium without stress showed no differences in growth and rooting (Fig. S2). In contrast to the poorly growing WT and VC,the transgenic plants showed vigorous growth and rooting on MS medium with 86 mmol L?1NaCl or 20% PEG6000. Proline content and SOD activity were significantly higher and MDA content was significantly lower in the transgenic plants than in WT and VC (Tables S2 and S3). These results indicated increased salt and drought tolerance of the transgenic plants compared with WT and VC. There was a significant positive correlation between the expression level of IbSnRK1 and salt and drought tolerance of the transgenic plants(Fig.S1;Tables S2 and S3).

Fig.1–Expression of IbSnRK1 in sweet potato cv.Lushu 3 at six time points in response to water(control), 86 mmol L?1 NaCl,20%PEG 6000,10 μmol L?1 H2O2,4°C, or 100 μmol L?1 ABA.Values are means±SD(n=3).**indicates a significant difference compared with 0 h at P<0.01 by Student's t-test.

Four transgenic sweet potato lines, L5, L8, L11, and L18,which showed the highest tolerance to salt and drought by in vitro assay, were selected for further evaluation of their tolerance to abiotic stresses. The transgenic, WT, and VC plants incubated in the Hoagland solution without stress showed no differences in growth and rooting (Fig. 2A; Table S4). Under 86 mmol?1NaCl or 20% PEG 6000 stresses, the transgenic plants produced new leaves and roots and their FW and DW were significantly increased, while WT and VC almost died(Fig.2B,C;Table S4).

For further evaluation of salt, drought, and cold tolerance, transgenic, WT, and VC plants were grown in a transplanting box, and then treated with 200 mmol L?1NaCl, drought, or 4 °C. There were no differences in growth and rooting among the transgenic and WT and VC plants under normal conditions (Fig. 3A). Under salt, drought, or cold stresses, WT and VC gradually withered and died, but the transgenic plants showed more growth, greater FW and DW, and higher photosynthetic rate, intercellular CO2concentration, transpiration rate, and relative chlorophyll content (Fig. 3B–H).

3.3. Contents of abiotic stress components

The transgenic plants showed increased contents of ABA and proline,increased activities of SOD,APX,POD,and CAT2,and the decreased content of MDA compared with WT and VC under 200 mmol L?1NaCl, drought and 4 °C stresses (Fig.4A–G). Under salt stress, higher K+and lower Na+accumulation was found in the transgenic plants, resulting in a higher K+/Na+ratio in the transgenic plants than in WT and VC (Fig. 4H–J).

The transgenic,WT and VC plants were stained with DAB and NBT to assess the levels of ROS accumulation (Fig. 5).The brown and blue color of the WT and VC leaves were darker than those of the leaves of transgenic plants(Fig.5 A1 and B1). The mean intensities were significantly higher in WT and VC than in the transgenic plants (Fig. 5 A2 and B2).H2O2andproduction rates were consistent with the staining assay(Fig.5 A3 and B3).Thus,the transgenic plants showed lower ROS levels than WT and VC under salt,drought, and cold stresses.

3.4. Stomatal movement in leaves

Three types of stomata(completely closed,partially open,and completely open) are shown in Fig. 6A. Completely closed stomata accounted for 48.3–55.6%, 66.9–73.8% and 37.1%–44.1% in the transgenic plants, but only 20.2%, 12.4%, and 20.9% in WT under salt, drought and cold stresses, respectively(Fig.6B;Table S5).In contrast,completely open stomata at 15.4%–21.7%, 8.7%–13.2%, and 17.8%–24.1% were observed only in the transgenic plants,but at 50.4%,70.5%,and 53.8%in WT under salt, drought, and cold stresses, respectively (Fig.6B; Table S5). Stomatal aperture (width/length ratio) was significantly decreased in the transgenic plants compared with WT and VC under salt, drought, and cold stresses (Fig.6C). higher RWC was found in leaves of the transgenic plants than in those of WT and VC(Fig.6D).These results suggested that IbSnRK1 might play an important role in reducing water evaporation by controlling stomatal movement.

3.5. Expression of the abiotic stress-responsive genes

Genes involved in ABA biosynthesis (ZEP, NCED, and AAO),proline biosynthesis (P5CS), photosynthesis (PRK), ROSscavenging system (SOD, APX, POD, and CAT2), ion transportation (NHX2) and stomatal movement (SLAC1 and OST1/SnRK2.6) were significantly up-regulated in the transgenic sweet potato plants compared with WT and VC under salt,drought, and cold stresses (Fig. 7). These results suggested that IbSnRK1 regulates tolerance to salt, drought, and cold stresses in sweet potato via the ABA signaling pathway.

4. Discussion

4.1. Overexpression of IbSnRK1 increases salt, drought and cold tolerance

SnRK1 is important in plant carbon and nitrogen metabolism[24,29]. The activity of SnRK1 in Arabidopsis may affect the induction of stress tolerance[17].However,its roles in abiotic stress tolerance have remained unclear. Thus, its functional elucidation is desirable for advancing our understanding of its roles and underlying mechanisms.Expression of IbSnRK1 was highly induced by NaCl, PEG 6000, H2O2, 4 °C, or ABA (Fig. 1)and its overexpression increased salt, drought, and cold tolerance of transgenic sweet potato plants (Figs. S2, 2 and 3;Tables S2, S3 and S4). These findings suggest that IbSnRK1 is involved in the ABA-dependent pathway in response to these abiotic stresses. The finding that the tolerance of some transgenic sweet potato plants (L1 and L3) was lower than that of WT and VC was similar to those of Liu et al. [20] and Wang et al. [30].The explanation awaits further study.

Fig.4–Contents of the abiotic stress-associated components under normal conditions or 200 mmol L?1 NaCl,drought,or cold(4°C)stresses.(A)ABA content.(B)Proline content.(C)MDA content.(D)SOD activity.(E)APX activity.(F)POD activity.(G)CAT2 activity.(H)Na+content.(I)K+content.(J)Na+/K+ratio.Transgenic,WT,and VC sweet potato plants grown in a transplanting box were treated with no stress(normal)for 2 weeks,200 mmol L?1 NaCl for 2 weeks,drought for 2 weeks,or 4°C for 2 days followed by 2 weeks of recovery.Values are means± SD(n= 3). *and** indicate significant difference compared with WT at P< 0.05 and P<0.01, respectively,by Student's t-test.

Fig.5–ROS accumulation in transgenic,WT,and VC sweet potato plants under normal conditions or 200 mmol L?1 NaCl,drought,or cold(4°C)stresses.(A1,B1)DAB and NBT staining in the leaves of transgenic,WT,and VC plants.(A2,B2)Average intensities of DAB and NBT staining in leaves after converting to 256-level grayscale images.(A3,B3)Quantification of H2O2 content and O2?production rate.Transgenic plants,WT,and VC grown in a transplanting box were treated with no stress(normal)for 2 weeks,200 mmol L?1 NaCl for 2 weeks,drought for 2 weeks,or 4°C for 2 days followed by 2 weeks of recovery.Values are means±SD(n=3).**indicates a significant difference compared with WT at P<0.01 by Student's t-test.

Fig.6–Stomatal movement in the transgenic,WT and VC sweet potato plants under normal conditions and 200 mmol L?1 NaCl,drought,or cold(4°C)stresses.(A)Scanning electron microscopy images of three types of stomata.(B)Percentage of three types of stomata(n>150).(C)Width/length ratio of stomata(n=3).(D)Relative water content(n=3).Transgenic plants,WT,and VC grown in a transplanting box were treated with no stress(normal)for 2 weeks,200 mmol L?1 NaCl for 2 weeks,drought for 2 weeks,or 4°C for 2 days followed by 2 weeks of recovery.**indicates a significant difference compared with WT at P<0.01 by Student's t-test.

4.2. Overexpression of IbSnRK1 up-regulates abiotic stressresponsive genes via the ABA signaling pathway

Fig.7–Expression of stress-responsive genes in transgenic,WT,and VC sweet potato plants.Plants grown in a transplanting box were treated with no stress(normal)for 2 weeks,200 mmol L?1 NaCl for 2 weeks,drought for 2 weeks,ord cold(4°C)stresses for 2 days followed by 2 weeks of recovery. Values are means±SD(n =3). *and** indicate a significant difference compared with WT at P< 0.05 and P<0.01, respectively,by Student's t-test.

ABA serves as a chemical signal in response to environmental stimuli in plants [6,31–33]. Stresses induce the synthesis of ABA and its accumulation can also feed back to stimulate the expression of biosynthetic genes, including ZEP, NCED, and AAO [31–34]. Studies [21,35,36] in several plant species have indicated that ABA regulates the expression of abiotic stressresponsive genes. In the present study, the expression of IbSnRK1 in sweet potato was strongly induced by ABA(Fig.1).The transcript levels of ZEP,NCED,and AAO and the content of ABA were significantly increased in transgenic sweet potato plants under salt, drought, or cold stresses (Figs. 4A and 7).The transgenic plants also showed increased expression of P5CS, SOD, APX, POD, CAT2, and NHX2 (Fig. 7). These results suggest that IbSnRK1 confers tolerance to salt, drought, and cold in transgenic sweet potato plants by activating stressresponsive genes via the ABA signaling pathway(Fig.8).

4.3. Overexpression of IbSnRK1 changes abiotic stress components

Plants have evolved a range of mechanisms to sense abiotic stresses and adapt their physiology, growth and development. Proline, one of the most common osmolytes and osmoprotectants, promotes plant stress tolerance by maintaining cell membrane integrity [37]. MDA is a well-known lipid oxidation product and its content indicates the extent of membrance lipid peroxidation [38]. In the present study,high levels of proline and lower levels of MDA were found in IbSnRK1-overexpressing sweet potato plants under salt,drought, and cold stresses (Fig. 4B, C). These compounds might maintain cell membrane integrity and protect cells from oxidative damage [20].

Soil salinization leads to excessive accumulation of toxic Na+in the cytosol,disrupts K+homeostasis,and impairs plant growth by inhibiting metabolic processes and decreasing photosynthetic efficiency [39]. The most important mechanism for maintaining ion balance is minimizing Na+influx and reducing K+efflux for a high K+/Na+ratio, which is beneficial for cell metabolism and salinity tolerance in plants[40,41]. In the present study, higher K+and lower Na+accumulation,resulting in a higher K+/Na+ratio,was observed(Fig. 4H–J), and higher expression of NHX2 was found in transgenic sweet potato plants than in WT and VC under salt stress (Fig. 7). Na+flux into and out of cytosols in plants is mediated by the transporters of NHX2 [30]. These results suggest that a higher K+/Na+ratio maintained osmotic balance and protected membrane integrity, resulting in increased tolerance of the transgenic plants(Fig.8).

Photosynthesis, necessary for plant growth, is sensitive to abiotic stresses [42]. In the present study, photosynthesis capacity was increased and the gene PRK was also upregulated in transgenic sweet potato plants (Figs. 3 and 7).The higher photosynthesis in the transgenic plants could be due to greater accumulation of proline, which provides protection against photoinhibition under abiotic stresses[20,43].

4.4. Overexpression of IbSnRK1 reduces ROS production by activating related enzymes

ROS as signaling molecules play a key role in the regulation of numerous biological processes in plants [44]. Various environment stresses lead to the excessive production of ROS,and the increase of H2O2andcontents is regarded as a marker of all kinds of stresses that result in increased oxidative injury[4,45]. To cope with such damage, plants have developed complex redox homeostatic antioxidative mechanisms [46].The ROS-scavenging system protects plant cells from oxidative damage[47].In our study,transgenic sweet potato plants accumulated less H2O2andthan the WT and VC under salt,drought, and cold stresses (Fig. 5). In agreement with these results,the relevant enzyme activities(of SOD,APX,POD,and CAT2) and gene expressions were significantly higher in the transgenic sweet potato plants (Figs. 4D–G and 7). These results suggest that a high level of proline activates ROSscavenging system,resulting in lower oxidative damage in the transgenic plants under salt,drought,and cold stresses(Fig.8)[20,21].

4.5.Overexpression of IbSnRK1 regulates stomatal closure via an ABA-dependent pathway

Stomata are located in the plant epidermis.Stomatal aperture controls gas exchange, leaf temperature, pathogen invasion,and water status, thus playing important roles in both yield gain and stress tolerance of crops[48].ABA plays a critical role in the regulation of plant stomatal behavior.When plants are exposed to abiotic stress conditions, leaves lose water primarily through stomata, and ABA controls water status and stomatal function by triggering stomatal pore closure[32,49]. In the present study, transgenic sweet potato plants showed a higher frequency of closed stomata and a decreased stomata aperture,resulting in higher RWC and greater growth of the transgenic plants under salt,drought,and cold stresses(Fig. 6). In contrast, WT and VC plants gradually dehydrated,owing to their higher frequency of open stomata and lower RWC(Fig.6).The overexpression of IbSnRK1 also up-regulated several positive regulators of ABA-mediated stomatal closure,including OST1/SnRK2.6 and SLAC1 (Fig. 7). These results suggest that IbSnRK1 might modulate stomatal closure through an ABA-dependent pathway under abiotic stresses.Yoshida et al. [50] found that OST1/SnRK2.6 encoded an Arabidopsis SnRK2 protein kinase and acted as a positive regulator in the ABA-induced stomatal closure. We speculate that the activation of OST1/SnRK2.6 and SLAC1 played an important role in the stomatal closure of the transgenic plants(Fig. 8).

This is the first report that SnRK1 confers tolerance to abiotic stresses in plants. Its overexpression up-regulated stress-responsive genes, increased proline content, K+/Na+ratio, and antioxidative capacity, and reduced stomatal aperture via the ABA signaling pathway, leading to increased salt, drought, and cold tolerance in transgenic sweet potato plants. IbSnRK1 is a potentially useful gene for improvement of abiotic stress tolerance in sweet potato and other plants.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.04.010.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2019YFD1001303 and 2019YFD1001300) and China Agriculture Research System(CARS-10,Sweetpotato).

Author contributions

Qingchang Liu, Zhitong Ren, and Hong Zhai designed the experiments; Zhitong Ren, Shaozhen He, Yuanyuan Zhou, Ning Zhao, and Tao Jiang conducted the experiments; Qingchang Liu and Zhitong Ren wrote the paper.