Research article
Organic nitrogen sources promote andrographolide biosynthesis by reducing nitrogen metabolism and increasing carbon accumulation in Andrographis paniculata

https://doi.org/10.1016/j.plaphy.2021.04.016Get rights and content

Highlights

  • Andrographis paniculata plants grew better in organic N than in inorganic N.

  • Organic N reduced N assimilation activity and depletion of carbohydrates in A. paniculata.

  • Organic N promoted andrographolide biosynthesis by up-regulation of HMGR, DXS, GGPS and CPS.

  • Andrographolide biosynthesis was positive related to starch accumulation.

Abstract

Nitrogen (N) form affects secondary metabolites of medicinal plants, but the physiological and molecular mechanisms remain largely unknown. To fully understand the response of andrographolide biosynthesis to different N forms in Andrographis paniculata, the plants were fed with nutritional solution containing sole N source of nitrate (NO3), ammonium (NH4+), urea or glycine (Gly), and the growth, carbon (C) and N metabolisms and andrographolide biosynthesis were analyzed. We found that plants grown in urea and Gly performed greater photosynthetic rate and photosynthetic N use efficiency (PNUE) than those grown in NO3 and NH4+. Organic N sources reduced the activities of enzymes involving in C and N metabolisms such as glutamine synthase (GS), glutamate synthase (GOGAT) and NADH-dependent glutamate dehydrogenase (NADH-GDH), invertase (INV), isocitrate dehydrogenase (ICDH) and glycolate oxidase (GO), resulting in reduced depletion of carbohydrates and increased starch accumulation. However, they enhanced andrographolide content by up-regulating the key genes in its biosynthetic pathway including HMGR, DXS, GGPS and ApCPS. Besides, NH4+ decreased leaf SPAD value, contents of soluble protein and amino acids and GO activity, but increased photosynthetic rate and contents of soluble sugar and starch in comparison to NO3. Andrographolide biosynthesis was also up-regulated. The results revealed that increasing accumulation of carbohydrates, especially starch, was beneficial to the biosynthesis of andrographolide; organic N sources decreased carbohydrate depletion by reducing N metabolism, and promoted plant growth and andrographolide biosynthesis synergistically.

Introduction

Nitrogen (N) is the most quantitative mineral nutrient required for plant growth and development. Plants can use both inorganic and organic N (Schmidt and Stewart, 1999; Wang et al., 2014; Cao et al., 2015). Nitrate (NO3) and ammonium (NH4+) are the major inorganic N forms in soil available for most terrestrial plants. Plants incorporate inorganic N into amino acids via a series of redox reactions catalyzed by nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase-glutamate synthetase (GS-GOGAT) system (Wang et al., 2014), whereas amino acid N can be utilized directly by plants via transamination and deamination (Cao et al., 2015). Urea has long been used as an important N fertilizer in agricultural system due to its lower production cost and high use efficiency of plants. Since 1940s Virtanen and Linkola (1946) demonstrated that plants can absorb amino acids directly, the absorption characteristics of amino acids N in many species including vegetables, crops, and trees (Khattab et al., 2012; Cao et al., 2016, 2017, 2017; Noroozlo et al., 2019) have been extensively studied in the past decades. It has been revealed that plants had a comparable short-term 15N uptake rate from amino acids and inorganic N (Metcalfe et al., 2011), and that amino acids highly nutritionally contributed to plant growth (Cao et al., 2016). In fact, the amount of Gly, a model N source of amino acids (Lipson et al., 1999), assimilated by plants is comparable to or greater than that of NO3 (Schmidt and Stewart, 1999).

In general, terrestrial plants prefer NO3, while high NH4+ concentration as a sole N source usually depresses plant growth and N metabolism (Jian et al., 2018). Only a few known plant species, such as rice (Oryza sativa L.), tea tree (Camellia sinensis) and hybrid Napier grass, prefer NH4+ and can tolerant a high concentration of NH4+ in surroundings (Ruan et al., 2000; Jampeetong et al., 2014). A large number of studies have demonstrated that N form affects plant growth and physiological responses differentially, depending upon the N preference of species and the developmental stage of plant (Jian et al., 2018), and these studies were focused mainly on the effects of NH4+, NO3 and their ratios (Walch-Liu et al., 2000; Guo et al., 2007).

Medicinal plant is a specific kind of plant group producing a diversity of bioactive molecules with pharmaceutical, cosmetic and nutritional values, many of which are secondary metabolites (Mehta and Dhapte, 2016; Mehta et al., 2017). It has been widely reported that inorganic N forms affect the accumulation of secondary metabolites in medicinal plants, but the responses of different secondary metabolites to N forms (including their ratios) are quite different (Biesiada et al., 2009; Tang et al., 2017; Liang et al., 2018; Sui et al., 2018). An important reason for the differential influences of N forms on secondary metabolites of medicinal plants is that N form regulates the transcription level of genes dominating the biosynthesis and transformation of these compounds (Wei et al., 2018; Yang et al., 2018; Zhang et al., 2018). Nevertheless, little is known about the effect of organic N on secondary metabolism in medicinal plants, and the physiological mechanisms underlying the involvement of N forms in secondary metabolism remain to be addressed.

Acanthaceae plant Andrographis paniculata is a vital medicinal plant widely used as a pain killer, anti-inflammatory, antibacterial and anti-viral agent in Indian, Malaysian, Thai, and Chinese traditional medicine (Tang and Eisenbrand, 1992; Aromdee, 2012; Xu et al., 2012; Talei et al., 2014; Valdiani et al., 2014). Modern medical studies have revealed that andrographolide compounds, the major bioactive ingredients of A. paniculata (Pholphana et al., 2013), have great potential medical values in anti-HIV (Reddy et al., 2005), anti-cancer (Chun et al., 2010), anti-H1N1 (Yu et al., 2014), etc. However, they are difficult to be synthesized artificially. To date, it mainly relies on artificial cultivation to meet the growing demand for commercial herbs of A. paniculata due to the exhausting of wild A. paniculata resources worldwide. To improve the content of andrographolide has long been the main target of breeding and cultivation of A. paniculata (Valdiani et al., 2012; Raina et al., 2013). Andrographolide compounds belong to diterpenoid lactones. There are two independent pathways for terpenoids biosynthesis in higher plants: cytosolic mevalonic acid (MVA) pathway and plastidial methylerythritol phosphate (MEP) pathway (Tholl, 2015). It has been demonstrated that the expression of genes in the MVA and MEP pathways such as HMGR, DXS and ISPH, were well correlated with the accumulation of andrographolide (Jha et al., 2011; Sharma et al., 2015; Shen et al., 2016).

A. paniculata is an N-intensive medicinal plant, but there is limited information on the responses of that plant to N forms. Given that C and N metabolisms are fundamental biochemical pathways in plants that differentially response to N forms, we hypothesized that N form could affect andrographolide biosynthesis by regulating the coordination of C and N metabolisms. In this study, we aimed to compare the effects of inorganic and organic N forms on the growth, photosynthesis, C and N metabolism and andrographolide biosynthesis of A. paniculata plants, and to reveal the mechanism of N forms on andrographolide biosynthesis. We found that N form-dependent coordination of C and N metabolisms plays a critical regulatory role in andrographolide biosynthesis. The results of this study could be of great reference significance for N management in A. paniculata cultivation.

Section snippets

Cultivation and treatment of plant materials

A. paniculata seeds were provided by the seed bank of Guangxi Botanical Garden of Medicinal Plants. The seeds were sown on a wet filter paper and placed at room temperature (about 28 °C) for a week for germination. Then, the seedlings were transferred to seedling trays filling with nutrient soil. When the seedlings at the age of four-pair true leaves, they were transplanted to the pots containing 4 L mixtures of roseite and pearlstone, and grown in a greenhouse. There were three seedlings in

Plant growth and photosynthesis

As shown in Fig. 1a and c, plants grown in inorganic N sources (NO3 and NH4+) showed growth depression in comparison to those grown in organic N sources (urea and Gly), although the biomass was not different among N forms (Fig. 1b). However, plants grown in NO3 displayed a darker leaf color and a higher SPAD value (P < 0.05) than those in other N forms (Fig. 1c and d). Specific leaf mass (SLM) of NH4+-fed plants was higher than that of NO3- and Gly-fed plants (P < 0.05), but not different

Plant growth performed better in organic nitrogen conditions

The performance of plant growth, C and N accumulation, and photosynthesis varied greatly in response to different N sources (Ruan et al., 2000; Wang et al., 2012; Hong et al., 2016). Our results showed that A. paniculata plants grown in organic N sources displayed relatively higher biomass, photosynthetic rate and PNUE in comparison to those grown in inorganic N sources. These differences were considered to be resulted from the differential physiological effects of N forms on plants (Wang et

Conclusion

Our results indicated that N form affected the balance between C and N via N metabolism, thus regulating andrographolide biosynthesis in A. paniculata. Inorganic N increased N assimilation activity of the plant, leading to increase in the depletion of carbohydrates. In contrast, plants grown in organic N had a higher photosynthetic rate and maintained relatively lower N metabolic activity, thus increased carbohydrates accumulation. Starch content was closely related to andrographolide

Author contributions

C.Z. designed the experiment, analyzed the data and drafted the manuscript. S.F.J., D.L.C. and X.J.H. conducted the experiments. J.H.M. provided the experimental platform for this study. All authors read and approved the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This work was financially supported by the Guangxi Natural Science Foundation of China (2020GXNSFAA159025), the China Agriculture Research System (CARS-21) and the Guangxi Innovation-Driven Development Project (GuiKe AA18242040).

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