SUN Yu-ming,HUANG Xiao-lei,ZHANG Ting,YANG Yong-heng,CHENG Xiao-fang,XU Xiao-yang,YUAN Hai-yan

1 Jiangsu Key Laboratory for the Research and Utilization of Plant Resources/The Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm,Institute of Botany,Jiangsu Province and Chinese Academy of Sciences,Nanjing 210014,P.R.China

2 College of Resources and Environment,Shanxi Agricultural University,Taigu 030801,P.R.China

3 College of Arts and Sciences,Shanxi Agricultural University,Taigu 030801,P.R.China

Abstract The steviol glycosides (SGs) in stevia (Stevia rebaudiana Bertoni) leaves are becoming increasingly valuable due to its high sweetness but low calorific value,which is driving the development of stevia commercial cultivation.Optimizing fertilization management can effectively increase SGs productivity,but knowledge on the relationship between potassium(K) fertilization and SGs production is still lacking.In this study,pot experiments were conducted in order to investigate the effect of K deficiency on SGs synthesis in stevia leaves,as well as the underlying mechanisms.Our results showed that when compared with standard K fertilization,K deficiency treatment has no significant effect on the biomass of stevia plant grown in a given soil with high K contents.However,K deficiency critically decreased leaf SGs contents as well as the expression of SGs synthesis-related genes.The contents of different sugar components decreased and the activities of sugar metabolism-related enzymes were inhibited under the K deficiency condition.Moreover,spraying sucrose on the leaves of stevia seedlings diminished the inhibitory effect caused by K deficiency.Our results also revealed the significant positive correlations between sucrose,glucose and SGs contents.Overall,our results suggest that K deficiency would suppress the synthesis of SGs in stevia leaves,and this effect may be mediated by the leaf sugar metabolism.Our findings provide new insights into the improvement of SGs production potential.

Keywords:Stevia rebaudiana Bertoni,steviol glycosides,potassium deficiency,sugar metabolism

1.lntroduction

Stevia (SteviarebaudianaBertoni) is a special kind of economic herb that is widely grown all over the world.Surprisingly,the terpene compound steviol glycosides(SGs) in stevia leaves are much sweeter than sucrose but contains almost no calories.Moreover,SGs have also been demonstrated to have various positive effects on human health,such as reducing blood sugar,preventing hyperlipidemia and improving human immunity (Lemus-Mondacaet al.2012;Ritu and Nandini 2016).These features promoted the application of SGs in food and healthcare.Studies have reported that the total SGs (TSGs)in stevia can account for approximately 4-20% of the leaf dry weight,which differs among species (Tavariniet al.2018).Among these different SGs components,stevioside (STV)and rebaudioside A (Reb A) are the most abundant forms,while some others are the key determiners of the taste of SGs,such as Reb C,Reb F and Reb M (Prakashet al.2014).Several approaches have been developed to effectively increase SGs production,such asinvitrofermentation,molecular breeding and cultivation optimization (Wanget al.2016,2020).Among these,the optimization of agricultural management has proven to be the most promising option with a dual role in stevia production and environmental security (Angeliniet al.2016;Vasilakoglouet al.2016).

Numerous previous studies have investigated the influences of fertilization,especially nitrogen (N) fertilization,on the growth and SGs synthesis of stevia plants.Although the positive effects of N fertilization on both biomass and SGs contents in stevia leaves have been previously reported(Palet al.2015;Tavariniet al.2016),the conflicting results concerning stevia growth and SGs metabolism are more widely acknowledged (Barbet-Massinet al.2015;Karimi and Moradi 2018;Sunet al.2019).Such results highlight the importance of enhancing SGs synthesis while maintaining leaf biomass.Interestingly,our previous studies regarding either N fertilization rates or N topdressing strategies both indicated the adverse effects of leaf N content on leaf SGs synthesis,which were tightly associated with the leaf carbon(C)/N ratio and soluble sugar contents (Sunet al.2019,2020b).Similarly,a meta-analysis also reported a positive correlation between soluble sugar and phenolic compound contents in the case of N deposition (Sunet al.2020a).Physiologically,when considering the metabolic pathway of terpenoid compounds including SGs,pyruvate and glyceraldehyde 3-phosphate are acting as both synthesis precursors of terpenoids and finial products of the glycolysis process (Yadav and Guleria 2012;Wanget al.2016),which link leaf SGs synthesis to sugar metabolism.These findings indicate a central role of leaf sugar metabolism in SGs synthesis.

Potassium (K) is one of the critical elements in determining plant growth and metabolism,as it plays essential regulatory roles in multiple physiological activities such as osmotic regulation,stomatal movement and signal transduction (Wanget al.2013;Zörbet al.2014).Notably,K nutrition was also demonstrated to be associated with plant C metabolism,especially sugar metabolism.For example,studies in apple and pear fruits have shown that K application can significantly increase the contents of different sugar components,including sucrose,glucose and fructose,as well the expression of sugar metabolism associated genes (Shenet al.2017;Zhanget al.2017).Similar results were also documented in the leaves of multiple plants such as lettuce (Barickmanet al.2016),ChrysanthemummorifoliumRamat (Liuet al.2011),and Kacip Fatimah (Ibrahimet al.2012).Such a correlation between K nutrition and sugar metabolism may be linked to the leaf photosynthetic capacity and the activities of sugar metabolism-related enzymes (Luet al.2016;Huet al.2018b).Of equal importance,K can regulate the expression of sugar transporters,thereby affecting the allocation of plant carbohydrate (Shenet al.2019).Taken together,these studies clearly showed the positive role of K nutrition in plant sugar metabolism.However,in the case of stevia,previous studies mainly focused on the relationship between K fertilization and plant growth rather than sugar metabolism or SGs synthesis (Maet al.2011;Ma and Shi 2011;Palet al.2015).Systematic studies have not yet ascertained whether K nutrition can regulate sugar or SGs metabolism in stevia leaves,nor their inter-connections.

Therefore,in this study,two pot experiments were conducted on stevia plants to address these questions.First,stevia was treated by either standard or no K fertilization,to determine whether K deficiency can influence SGs and sugar components contents in stevia leaves.Given the negative results of K deficiency in this first experiment,a second experiment involved spraying sucrose on the stevia leaves without K fertilization,to evaluate whether such an operation can restore the leaf SGs contents.Finally,the underlying mechanisms concerning K nutrition,sugar metabolism and SGs production are discussed.

2.Materials and methods

2.1.Plant materials and experimental conditions

Pot experiments were conducted in the greenhouse of the Institute of Botany,Jiangsu Province and Chinese Academy of Sciences (Jiangsu,China,latitude 32°03´N,longitude 118°49´E) in the growing seasons of 2018 and 2019.

In 2018,stevia (SteviarebaudianaBertoni) plants propagated from cuttings of the cultivar Zhongshan 8 were transplanted into pots on June 2.The basic properties of the experimental soil were measured as:pH 7.21(soil:H2O=1:2.5),organic matter 34.24 mg g-1,total N 3.282 mg g-1,alkali-hydrolyzable N 212.88 mg kg-1,Olsen-P 57.84 mg kg-1,and NH4OAc-K 261.72 mg kg-1.Two K treatments,including (1) a sufficient K supply treatment(0.5 g per pot in the form of potassium chloride and each pot filled with 12.5 kg of dry soil) as a control,and (2) K deficiency treatment (KD,without K fertilization),were established in the greenhouse and each treatment had eight pots.The K fertilizer for the control treatment was applied to the soil equally on June 1 (before transplanting) and July 1(branching stage).In addition,the fertilization rates of N in the form of urea and P in the form of calcium superphosphate were 1.6 g per pot and 0.42 g per pot,respectively.Stevia plants were harvested at the flower bud stage (September 1) for further analysis.

In 2019,another pot experiment was conducted in seedlings of stevia cultivar Zhongshan 8.The soil properties and experimental conditions were the same as those listed above.The four treatments included (1) a sufficient K supply treatment as a control;(2) K deficiency treatment and leaf spraying with deionized H2O (KD);(3) KD and leaf spraying with 25 mmol L-1sucrose solution;and (4) KD and leaf spraying with 50 mmol L-1sucrose solution.Each treatment had eight pots.The purpose of this experiment was to illustrate the effect of leaf sucrose spraying on SGs synthesis under K deficiency conditions.Therefore,the leaf spraying was conducted in the one-month-old stevia seedlings to ensure the evenly spraying on all leaves.Leaf spraying was performed for three consecutive days and all leaves were sprayed evenly at 10:00 am.The sampling was conducted two days after the termination of leaf sucrose spraying.

2.2.Sampling and processing

For half of the plants in each treatment,the aboveground parts of the stevia plants were harvested along the soil interface,and then washed briefly before being divided into leaf and stem.These samples were baked in an oven at 105°C for 30 min and then at 70°C to a constant weight.The leaf samples were separately ground and mixed before storage to avoid the influence of leaf positions (Ceunen and Geuns 2013b).Simultaneously,the fresh leaves of the remaining half of the plants were sampled and evenly mixed and ground in liquid nitrogen before storage in -80°C freezer.The dry samples were used for the measurements of total K,N,C,SGs contents and sugar components while the fresh samples were stored for further analysis of enzyme activities as well as RNA isolation.Four replicates were included for each measurement.

2.3.Total K,N and C contents

Dried stevia samples were digested with H2SO4-H2O2and then the leaf total K content in the digestion solution was determined with a Flame Atomic Absorption Spectrophotometer (Huet al.2018a),while the leaf total N content was measured with the method of Kjeldahl (Nelson and Sommers 1972).The measurement of leaf total C followed the wet-combustion method (Hafsiet al.2003).

2.4.Extraction and determination of leaf SGs

The leaf samples were ground in a mortar and then extracted in 80% ethanol (v/v) at 100°C for 1 h.Afterward,the mixtures were centrifuged and the supernatant was transferred to a new centrifuge tube for rotary evaporation.The residue was dissolved in distilled water for further high-performance liquid chromatography (HPLC) analysis with a Sapphire C18 sorbent column (4.6 mm×250 mm)according to Sunet al.(2020b).The analysis temperature was set to 25°C and the UV wavelength was OD210 nm.An acetonitrile:sodium phosphate buffer (32:68) was used as the mobile phase,with a flow rate of 1 mL min-1.The contents of leaf SGs were determined according to the standard curves of Reb A,STV,and Reb C (99.99% pure,Chroma Dex,USA).

2.5.RNA isolation and quantitative real-time PCR(RT-qPCR)

The total RNA of stevia leaf was extracted with TRIzol(Invitrogen,Carlsbad,CA,USA) and the cDNA was synthesized using the Prime ScriptTMRT reagent Kit with DNA Eraser (TaKaRa,Dalian,China).The expression levels of six SGs synthesis related genes were analyzed by RT-qPCR using the ABI 7500 Real-Time PCR System(Applied Biosystems,Foster City,CA,USA) (Guoet al.2020).The RT-qPCR primers of the analyzed genes are listed in Table 1 and theActingene was used as the internal standard.The relative gene expressions were calculated with the 2-ΔΔCtmethod.

2.6.Total soluble sugar,sucrose,fructose and glucose

Dry leaf powder samples (approximately 0.05 g) were mixed with 3 mL of deionized water,boiled for 30 min at 100°C and then centrifuged at 10 000×g for 10 min.The supernatant was transferred to a new glass tube.These steps were repeated twice.Subsequently,deionized water was added to the three combined supernatants to a final volume of 10 mL,which was used for further analysis.

Leaf total soluble sugar contents were determined by the anthrone-sulfuric acid colorimetric method,according to Tianet al.(2019).The measurements of sucrose,fructose and glucose were conducted following Huet al.(2017).Briefly,the extract was transferred into a microplate and heated at 50°C for 45 min,then the sucrose,fructose and glucose were measured after adding of glucose assay kit,phosphoglucose isomerase and invertase,respectively.

2.7.Enzyme activities

Fresh leaf samples (0.5 g) were ground in a mortar with the extraction buffer (50 mmol L-1Hepes-KOH,pH=7.4,with 1 mmol L-1EDTA,1 mmol L-1EGTA,10% glycerol,1 mmol L-1DTT,12 mmol L-1MgCl2,2 mmol L-1benzamidine and 2 mmol L-1e-amino-n-caproic acid).The homogenate was centrifuged (4°C,12 000×g,20 min) and the supernatant was transferred to a new tube for further analysis.The activities of sucrose phosphate synthase (SPS,E.C.2.4.1.14),soluble acid invertase (AI,EC 3.2.1.26) and neutral invertase (NI,EC 3.2.1.26) were then measured according to Huet al.(2018b).

2.8.Statistical analysis

One-way analysis of variance (ANOVA) was performed to evaluate the significant differences among treatments using the SPSS 16.0 Software.All samples were analyzed in quadruplicate and the mean values and calculated standard deviations (SD) are reported in comparisons,where the significance was tested at the 5% level.

3.Results

3.1.Effects of K deficiency on the biomass and leaf K,N and C contents of stevia plants

The biomass of stevia leaf and stem was not significantly changed by K deficiency compared with the control (Table 2),in a given soil with high K content.However,the leaf K content was markedly decreased (approximately 27.72%)by K deficiency treatment when compared with that of the control.In the context of total N and C contents in the stevia leaf,no significant differences were observed between the control and K deficiency treatments,thus leading to the same result for the leaf C/N ratio.

Table 1 Sequences and accession numbers of primers used for qRT-PCRs in this study

Table 2 Effect of potassium (K) deficiency (KD) on plant biomass and the leaf contents of total potassium (TK),total nitrogen (TN),total carbon (TC) and C/N ratio of stevia plants

3.2.Effect of K deficiency on the SGs contents and the relative expressions of SGs synthesis genes of stevia leaves

Under the K deficiency condition,the leaf SGs components were strikingly reduced when compared with those in control plants.As shown in Fig.1,the contents of Reb A andSTV were significantly decreased by approximately 15.46 and 25.52% respectively,which resulted in a decrease in the total SGs content.However,the Reb C content was not significantly changed by K deficiency.Regarding the expression of SGs synthesis-related genes,ent-KAH,SrKO1andSrKS1-1were markedly down-regulated by K deficiency (Fig.2).In contrast,the transcriptions ofSrUGT74G1,SrUGT76G1andSrUGT85C2were not critically altered by K fertilization.

Fig.1 Effect of potassium (K) deficiency (KD) on the leaf contents of rebaudioside A (Reb A;A),stevioside (STV;B),rebaudioside C (Reb C;C) and total steviol glycosides (TSGs).Control,standard potassium fertilization.Data represent means of four replicates and the bars indicate the SD.Significant differences (P＜0.05) between different treatments are indicated by different letters.

Fig.2 Effect of potassium (K) deficiency (KD) on the relative gene expressions of ent-KAH,SrKO1,SrKS1-1,SrUGT74G1,SrUGT76G1 and SrUGT85C2.Control,standard potassium fertilization.Data represent means of four replicates and the bars indicate the SD.Significant differences (P＜0.05) between different treatments are indicated by different letters.

3.3.Effect of K deficiency on the sugar metabolism of stevia leaves

The contents of leaf soluble carbohydrates and activities of C metabolism-related enzymes were significantly altered by soil K status (Fig.3).When compared with that of the control,K deficiency significantly decreased the leaf total soluble sugar content by 19.32%.In the context of different sugar components,the leaf contents of sucrose,glucose and fructose of stevia plants subjected to K deficiency were respectively 12.56,45.72 and 21.22%lower when compared with the control (Fig.3-A).The levels of enzymes involved in sugar metabolism were similarly influenced by K deficiency.As shown in Fig.3-B,K deficiency dramatically inhibited the activities of sucrose phosphate synthase (SPS),acid invertase (AI) and neutral invertase (NI) in the stevia leaves by approximately 23.61,14.10 and 33.04%,respectively.

Fig.3 Effect of potassium (K) deficiency (KD) on the contents of sugar components (A) and the activities of sucrose phosphate synthase (SPS),acid invertase (AI) and neutral invertase (NI) (B) of the stevia leaf.Control,standard potassium fertilization.Data represent means of four replicates and the bars indicate the SD.Significant differences (P＜0.05) between different treatments are indicated by different letters.

3.4.Effect of exogenous sucrose spraying on the contents of leaf K,N,C and sugar components

Leaf total K and N contents were not significantly changed by leaf sucrose spraying (Table 3).However,leaf spraying with sucrose solutions of 25 and 50 mmol L-1significantly increased the total C content of stevia leaves by 7.84 and 9.08%,respectively,compared to that of the K deficiency treatment.This difference,therefore,produced the higher leaf C/N ratio in the sucrose sprayed leaves.

Table 3 Effect of exogenous sucrose spraying on the leaf contents of total potassium (TK),total nitrogen (TN),total carbon (TC)and C/N ratio of stevia plants

The contents of different sugar components in the leaves of stevia seedlings were significantly increased by exogenous sucrose spraying (Fig.4).Compared with that of the K deficiency treatment,spraying with 25 and 50 mmol L-1sucrose strikingly increased leaf total soluble sugar content by 27.59 and 34.43%,respectively (Fig.4-A).Compared with the K deficiency treatment,the contents of sucrose,fructose and glucose in the stevia leaves sprayed with exogenous sucrose were also significantly increased by 34.39-42.53%,142.81-322.08% and 43.43-61.72%respectively (Fig.4-B-D).The exogenous sucrose spraying also resulted in the significantly higher leaf sucrose,fructose and glucose contents when compared with the K fertilized stevia plants.

Fig.4 Effect of exogenous sucrose spraying on the content of total soluble sugar (A),sucrose (B),fructose (C) and glucose (D)in the stevia leaves.Control,standard potassium fertilization;KD,potassium deficiency;KDS1,potassium deficiency with leaf spraying of 25 mmol L-1 sucrose;KDS2,potassium deficiency with leaf spraying of 50 mmol L-1 sucrose.Data represent means of four replicates and the bars indicate the SD.Significant differences (P＜0.05) between different treatments are indicated by different letters.

3.5.Effect of exogenous sucrose spraying on the SGs contents and the relative expressions of SGs synthesis genes of stevia leaves

Leaf spraying with sucrose significantly increased the contents of the SGs.As shown in Fig.5,compared with those of stevia plants suffering from K deficiency,exogenous spraying of sucrose resulted in approximately 23.44,11.77 and 28.65% increases of Reb A,STV and Reb C contents in the stevia leaves,respectively.Likewise,the average TSGs content was increased by 20.46 and 23.25% by the 25 and 50 mmol L-1sucrose spraying,respectively.However,when comparing the contents of the various SGs in the leaves of stevia plants with K fertilization and stevia plants without K fertilization but with sucrose spray,no significant difference was observed.

In the context of SGs synthesis-related genes,all these genes were again significantly down-regulated by K deficiency when compared with those of the control (Fig.6).Strikingly,the leaf spraying with sucrose induced upregulation of the expressions ofent-KAH,SrKO1andSrKS1-1compared to those of the K deficient plants.However,the expression ofSrUGT74G1was not changed by the 25 mmol L-1sucrose spraying but it was significantly increased by leaf spraying of 50 mmol L-1sucrose compared to that under the K-deficient condition (Fig.6-D).No significant changes in the expressions ofSrUGT76G1andSrUGT85C2were exhibited by leaf sucrose spraying.

3.6.Correlations between the contents of leaf SGs and TK,TN,TC,C/N ratio and sugar components

The leaf TK and TN were not significantly associated with the contents of different leaf SGs components (Fig.7).Changes in leaf TC and C/N ratio were consistent with those of Reb A,STV and TSGs,while no significant correlations were observed between Reb C,TC and C/N ratio.Interestingly,significant correlations were found between the contents of leaf sugar components and SGs components.From the perspective of leaf TSGs contents,total soluble sugar (TSS)sucrose and glucose each exhibited a significant positive correlation.

Fig.7 Correlation coefficients between leaf steviol glycosides (SGs) components and leaf total potassium (TK),total nitrogen(TN),total carbon (TC),C/N ratio and sugar components.TSS,total soluble sugar.Reb A,rebaudioside A;STV,stevioside;Reb C,rebaudioside C;TSGs,total steviol glycosides.The number in each square represents the correlation coefficient while the superscript represents the significance.＊ and ＊＊ indicate significant differences at 0.05 and 0.01 probability levels,respectively;ns means difference not significant.

4.Discussion

With the increasing importance of SGs in the food andmedical fields,the improvement of SGs productivity by optimizing fertilization management has become increasingly important (Rameshet al.2006;Sunet al.2020b).K is a crucial nutrient element that affects crop quality in general,but a causal relationship between K fertilization and stevia production is still largely unclear.In the current study,the effect of K fertilization on SGs synthesis as well as the underlying mechanisms was investigated.We found that K deficiency has no significant influence on stevia growth,which may be related to the high background K content of the tested soil.However,the synthesis of various SGs was severely inhibited by the K deficiency treatment (Fig.1).Further analysis showed that this effect is related to leaf sugar metabolism,as the exogenous spraying of sucrose rescued the SGs synthesis which was inhibited by K deficiency (Fig.5).Crucially,such ametabolic connection provides an effective strategy for future stevia cultivation.

Fig.5 Effect of exogenous sucrose spraying on leaf contents of rebaudioside A (Reb A;A),stevioside (STV;B),rebaudioside C (Reb C;C) and total steviol glycosides (TSGs;D).Control,standard potassium fertilization;KD,potassium deficiency;KDS1,potassium deficiency with leaf spraying of 25 mmol L-1 sucrose;KDS2,potassium deficiency with leaf spraying of 50 mmol L-1 sucrose.Data represent means of four replicates and the bars indicate the SD.Significant differences (P＜0.05) between different treatments are indicated by different letters.

K is one of the necessary nutrients for plant life,which can account for 1-5% of the plant dry weight and play vital roles in plant growth,physiological metabolism and pathogen/pest resistance (Römheld and Kirkby 2010;Wanget al.2013).The stevia plant has a high demand for K nutrition during the growth process,however,K fertilization has always been disregarded by farmers in actual management due to the high fertilizer prices (Rameshet al.2006;Brunelleet al.2015).Such management results in a physiological K deficiency in the crops,and thereby influence plant metabolism.Our current study showed the negative effect of K deficiency on SGs contents and the SGs-synthesis related genes (Figs.1 and 2).Consistent with our results,Santoset al.(2000) reported a positive linear correlation between K fertilization rates and leaf SGs contents.In the context of other terpenoid compounds,such negative responses to K deficiency were also documented in the contents of lycopene in tomato (Taberet al.2008) and tanshiones inSalviamiltiorrhizaB.(Luet al.2013).Besides,the positive effects of K fertilization on the contents of essential oils,which consist of various terpenoid compounds,have been extensively emphasized inMenthaspicataL.(Chrysargyriset al.2017b),LavandulaangustifoliaM.(Chrysargyriset al.2017a),OcimumbasilicumL.(Esetliliet al.2015) andArtemisiadracunculusL.(Heidariet al.2014).All these findings suggest an inhibitory effect of K deficiency on the synthesis of terpenoids,including SGs.

When examining the common mechanisms that determine SGs synthesis,previous studies have attributed this to variations of the leaf C/N ratio or soluble sugar contents(Sunet al.2019,2020a).Interestingly,our present study seems to exclude the effect of the C/N ratio but highlights the changes in leaf sugar metabolism (Table 2;Fig.3).Studies have demonstrated that K can regulate the activities of C metabolism-related enzymes,such as pyruvate kinase,starch synthase and Rubisco,by acting as an activator or cofactor (Armengaudet al.2009;Zörbet al.2014).Such functions directly determine the sugar metabolism in the plant,while the regulatory effects are diverse.For instance,transcriptome analysis in rice and pear plants found that K deficiency significantly decreases the leaf K and reducing sugar contents together with the differential expressions of genes involved in carbohydrate metabolism and glycolysisrelated enzyme synthesis (Zhanget al.2013;Shenet al.2017).However,metabolomic profiling analysis in tomato indicated that the relationship between K nutrition and plant sugar metabolism has tissue specificity (Sunget al.2015).In this study,K deficiency induced significant decreases of leaf carbohydrate and organic acid contents,while the opposite result was observed in root tissue.In this context,plant growth stages or source-sink relationships should also be highlighted.A study in cotton seedlings found that K fertilization significantly promoted leaf sucrose accumulation,as well as the activities of sugar metabolismrelated enzymes (Zahooret al.2017).However,during the reproductive growth period of cotton plants,K deficiency inhibited the leaf carbohydrate export to sink organs and therefore resulted in leaf sugar accumulation (Huet al.2018b).The above findings indicate that K can mediate leaf sugar metabolism at the enzyme and transcription levels,and such effects depend on the feedback signals from other tissues.These studies again place leaf sugar metabolism at the centre of the mechanisms for K regulated SGs accumulation in stevia leaves at the vegetative growth stage.

From the insight of the SG biosynthesis pathway,leaf sugar metabolism can affect the C flux to terpenoidsviathe 2-methyl-D-erythritol 4-phosphate (MEP) pathway,because the glycolysis products are acting as terpenoid synthetic precursors (Yadav and Guleria 2012;Wanget al.2016).Webbet al.(2013) indicated that the expression of genes in the entire terpene synthesis pathway would be significantly up-regulated when the C flux through the MEP or mevalonate pathway was enhanced.Indeed,previous studies in medium cultures showed that an increase of sucrose content could significantly enhance SGs content and the expression of SGs synthesis related genes in stevia leaves (Bondarevet al.2003;Ghorbaniet al.2017).Similar effects were also recorded inArtemisiaannuaL.,where exogenous addition of sucrose or glucose can significantly increase the content of artemisinin together with the expression of terpenoid synthesis related genes(Wang and Weathers 2007;Arsenaultet al.2010).The above results seem to support our ongoing study which shows that sucrose spraying can compensate for the inhibition of leaf SGs synthesis under K deficiency (Figs.5 and 6).Furthermore,when considering the relationship between leaf sugar and SGs contents in stevia plants under diverse environments,the importance of sugar metabolism is again illuminated.For example,the leaf soluble sugar and SGs were simultaneously accumulated under osmotic stresses (Cantabellaet al.2017;Aghighiet al.2019).Although these studies attribute such a connection to the osmotic adjustment role of soluble sugars and terpenoids,it also implies the close link between soluble sugar and SGs synthesis.In addition,Ceunen and Geuns (2013a) and Karimiet al.(2015) indicated that among these reducing sugar components,sucrose and glucose might be the key factors affecting SGs,which is similar to our results (Table 3)and suggests that the glycolysis pathway is the determiner of SGs synthesis.When combing all these components together,we can produce a model in which leaf sugar metabolism is the determining link between K and SGs synthesis.Consistent with this proposition are the findings in Kacip Fatimah (Ibrahimet al.2012),Douglas-fir (Shawet al.1998) andChrysanthemummorifoliumR.(Liuet al.2011) that emphasize the positive correlation between K,leaf soluble sugar content and C-based secondary metabolism.

5.Conclusion

Our results show that K deficiency could significantly inhibit the contents of leaf carbohydrates as well as the activities of sugar metabolism-related enzymes,thus negatively regulating SGs synthesis.However,exogenous spraying of sucrose solution can effectively eliminate the inhibitory effect caused by K deficiency.This work stresses the importance of K fertilization and sugar metabolism in SGs synthesis and also provides implications for further SGs productivity from the perspectives of plant physiology and agronomy.

AcknowledgementsThis work was supported by the Natural Science Foundation of Jiangsu Province,China (BK20180312),the Jiangsu Key Laboratory for the Research and Utilization of Plant Resources,China (JSPKLB201810) and the Natural Science Foundation of Shanxi Province,China (201901D111230).

Declaration of competing interest

The authors declare that they have no conflict of interest.