Article Nitrogen Forms Alter Triterpenoid Accumulation and Related Gene Expression in Cyclocarya paliurus (Batalin) Iljinsk. Seedlings Jian Qin 1, Xiliang Yue 1, Xulan Shang 1,2 and Shengzuo Fang 1,2,* 1 College of Forestry, Nanjing Forestry University, Nanjing 210037, China; [email protected] (J.Q.); [email protected] (X.Y.); [email protected] (X.S.) 2 Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China * Correspondence: [email protected]; Tel./Fax: +86-25-85427797 Received: 23 April 2020; Accepted: 25 May 2020; Published: 2 June 2020 Abstract: Cyclocarya paliurus (Batalin) Iljinsk. is a multiple function tree species distributed in subtropical areas, and its leaves have been used in medicine and nutraceutical foods in China. However, little information on the effects of nitrogen (N) forms and ratios on growth and secondary + metabolite accumulation is available for C. paliurus. The impact of five NO3−/NH4 ratios on biomass production, triterpenoid accumulation and related gene expression in C. paliurus seedlings was evaluated at the middle N nutrition supply. Significant differences in seedling growth, triterpenoid + accumulation and relative gene expression were observed among the different NO3−/NH4 ratio + treatments. The highest triterpenoid content was achieved in a sole NO3− or NH4 nutrition, while + the mixed N nutrition with equal ratio of NO3− to NH4 produced the highest biomass production in the seedlings. However, the highest triterpenoid accumulation was achieved at the treatment with + + the ratio of NO3−/NH4 = 2.33. Therefore, the mixed N nutrition of NO3− and NH4 was beneficial to the triterpenoid accumulation per plant. The relative expression of seven genes that are involved + in triterpenoid biosynthesis were all up-regulated under the sole NH4 or NO3− nutrition conditions, and significantly positive correlations between triterpenoid content and relative gene expression of key enzymes were detected in the leaves. Our results indicated that NO3− is the N nutrition preferred + by C. paliurus, but the mixture of NO3− and NH4 at an appropriate ratio would improve the leaf triterpenoid yield per area. Keywords: Cyclocarya paliurus; biomass; nitrogen nutrition; secondary metabolite; trade-off 1. Introduction As one of the important secondary metabolites in plants, triterpenoids allow plant to better adapt to the environment, such as their defence against natural enemies and plant communication [1], but also have been confirmed to contain a variety of health-promoting effects in human beings, such as anti-hyperglycemic, anti-hyperlipidemic and antioxidant effects [2–4]. In plants, triterpenoids are synthesized by the isoprenoid pathway, which is composed of three synthetic stages: (1) 3-isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are formed by mevalonate acid (MVA) or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway [5]; (2) 2,3-oxidosqualene is formed by geranyl diphosphate synthase (GPS), farnesyl diphosphate synthase (FPPS), squalene synthase (SQS) and squalene epoxidase (SE), which is subsequently cyclized to triterpenoid skeletons by 2,3-oxidosqualene cyclases (OSCs). Then, cytochrome P450 monooxygenase (PDMO) catalyzes oxidation of triterpenoid skeletons to produce aglycones; (3) the aglycones are glycosylated to triterpenoid saponins by glycosyltransferase (GT) [6] (Figure1). Forests 2020, 11, 631; doi:10.3390/f11060631 www.mdpi.com/journal/forests Forests 2020, 11, 631 2 of 16 Forests 2020, 11, x FOR PEER REVIEW 2 of 16 FigureFigure 1.1. BiosyntheticBiosynthetic pathway pathway of of triterpenoid triterpenoid in inhigher higher plants. plants. Abbreviations: Abbreviations: AACT: AACT: acetyl acetyl CoA: CoA:acetyl acetylCoA C CoA-acetyltransferase C-acetyltransferase or acetoacetyl or acetoacetyl-CoA-CoA thiolase; thiolase; CAS: cycloartenol CAS: cycloartenol synthase; synthase; CMK: CMK:4-(cytidine 4-(cytidine 5′-diphospho) 50-diphospho)-2-C-methyl-D-erythritol-2-C-methyl-D-erythritol kinase; DS: kinase; dammarenediol DS: dammarenediol synthase; DXPS, synthase; i.e., DXPS,DXS: i.e.,1 DXS:-deoxy 1-deoxy-D-xylulose-5-phosphate-D-xylulose-5-phosphate synthase); synthase); DXR1-deoxy-D-xylulose-5-phosphateDXR1-deoxy-D-xylulose-5-phosphate reductoisomerase;reductoisomerase; FPPS: FPPS: farnesyl farnesyl diphosphate diphosphate synthase; synthase; GPPS: GPPS: geranyl geranyl diphosph diphosphateate synthase; synthase; GT: GT:glycosyltransferases; glycosyltransferases; HDR: HDR: 4-hydroxy 4-hydroxy-3-methyl-3-methyl but but-2-(E)-enyl-2-(E)-enyl diphosphate diphosphate reductase; reductase; HDS:HDS: 4-hydroxy-3-methyl4-hydroxy-3-methyl but-2-(E)-enylbut-2-(E)-enyl diphosphatediphosphate synthase;synthase; HMGR:HMGR: 3-hydroxy-3-methylglutaryl3-hydroxy-3-methylglutaryl CoACoA reductase;reductase; HMGS:HMGS: 3-hydroxy-3-methylglutaryl3-hydroxy-3-methylglutaryl CoACoA synthase;synthase; IPPI:IPPI: isopentenylisopentenyl diphosphatediphosphate isomerase;isomerase; LAS:LAS: lanosterollanosterol synthase;synthase; LS,LS, i.e.,i.e., LUS:LUS: lupeollupeol synthase;synthase; MCT:MCT: 2-C-methyl-D-erythritol2-C-methyl-D-erythritol 4-phosphate4-phosphate cytidylyl cytidylyl transferase transferase or 4-(cytidineor 4-(cytidine 50-diphospho)-2-C-methyl-D-erythritol 5′-diphospho)-2-C-methyl-D-erythritol synthase; synthase; MDC: mevalonate-5-pyrophosphateMDC: mevalonate-5-pyrophospha decarboxylase;te decarboxylase; MECPS: 2-C-methyl-D-erythritol-2, MECPS: 2-C-methyl 4-cyclodiphosphate-D-erythritol-2, synthase;4-cyclodiphosphate MK: mevalonate synthase; kinase; MK: OSCs:mevalonate 2, 3-oxidosqualene kinase; OSCs: 2, cyclases; 3-oxidosqualene PDMO: cytochrome cyclases; PDMO: P 450 -dependentcytochrome monooxygenases; P 450 -dependent PMK: monooxygenases; phosphomevalonate PMK: kinase; phosphomevalonate SE: squalene epoxidase; kinase; SE: SS: squalenesqualene synthase;epoxidase;β -AS:SS: squaleneβ-amyrin synthase; synthase β (quoted-AS: β-amyrin from Zhao synthase et al. (quoted [7]). from Zhao et al. [7]). Cyclocarya paliurus (Batalin) Iljinsk., a member of Juglandaceae, is mainly scattered in Cyclocarya paliurus (Batalin) Iljinsk., a member of Juglandaceae, is mainly scattered in the C. paliurus themountainous mountainous across across sub sub-tropical-tropical regions regions of ofChina China [8 []8. ].Leaves Leaves of of C. paliurus havehave been made intointo nutraceutical tea for a long time in China, while its leaf, bark and root are used in traditional Chinese nutraceutical tea for a long time in China, while its leaf, bark and root are used in traditional Chinese C. paliurus medicinemedicine [9,,10].]. Moreover, Moreover, the the leaves leaves of of C. paliurus havehave been been listed listed as asnew new food food raw raw material material by the by theNational National Health Health and and Family Family Planning Planning Commiss Commissionion of of China China since since 2013 2013 [ [1111]].. Previous Previous studies havehave C. paliurus foundfound that that C. paliurusleaves leaves contain contain various various bioactive bioactive constituents, constituents including, polyphenolics,including polyphenolics, flavonoids, C. paliurus triterpenoids,flavonoids, triterpenoids, polysaccharides polysaccharides [4,11,12]. However, [4,11,12] triterpenoids. However, triterpenoids in in playC. paliurus a very play important a very roleimportant in the health-promotingrole in the health e-promotingffect, such aseffect, enhancing such as antihyperlipidemic enhancing antihyperlipidemic activity and ameliorating activity and diabetesameliorating [13,14 diabetes]. Moreover, [13, there14]. Moreover, are several specificthere are triterpenoids several specific that have triterpenoids been identified that and have isolated been C. paliurus fromidentified and isolatedleaves from [15,16 C.]. paliurus Due to leaves its various [15,16 health-promoting]. Due to its various eff ects,health a- hugepromoting production effects of, a C. paliurus C. paliurus huge productionleaves isof requiredC. paliurus for leaves tea production is required and for for tea medical production use. and However, for medical use. However,is mainly distributedC. paliurus is in mainly natural distribut forests anded in there natural are notforests enough and C.there paliurus are notplantations enough C. to paliurus produce plantations the leaves. to produce the leaves. Therefore, there is an urgent need to develop C. paliurus plantations for meeting the strong market demand for high-quality raw material. Forests 2020, 11, 631 3 of 16 Therefore, there is an urgent need to develop C. paliurus plantations for meeting the strong market demand for high-quality raw material. Environmental factors can affect growth, secondary metabolite biosynthesis and accumulation in plants. It was reported that the concentration of CO2 in the Earth’s atmospheric would increase to between 600 and 1000 µmol/mol by the end of 21st century [17]. This will affect the ratio of carboxylation to oxygenation, and consequently decrease photorespiration. Nitrogen (N), as an important environmental factor for plant growth, is primarily absorbed by plants in the form of ammonium + (NH4 ) and nitrate (NO3−)[18]. Many studies have indicated that the response of higher plants to elevated CO2 is determined by nitrogen availability, and is primarily related to the different forms of + nitrogen nutrition [17,19–21]. Modification of the NO3−/NH4 ratio can modulate the relative uptake of other anion and cations,