Putrescine Promotes Betulin Accumulation in Suspension Cell Cultures of Betula platyphylla by Regulating NO and NH4+ Production

Yaguang Zhan,Guizhi Fan,Baojiang Zheng,Tingting Zhang,Yingtian Liu

doi:10.3390/f11121336

Abstract

Putrescine (Put) can enhance secondary metabolite production, but its intrinsic regulatory mechanism remains unclear. In this study, Put treatment promoted betulin production and gene expression of lupeol synthase (LUS), one of betulin synthetic enzymes. The maximum betulin content and gene expression level of LUS was 4.25 mg·g−1 DW and 8.25 at 12 h after 1 mmol·L−1 Put treatment, approximately two- and four-times that in the control, respectively. Put treatment increased the content of nitric oxide (NO) and its biosynthetic enzyme activity of nitrate reductase (NR) and NO synthase (NOS). Pretreatment of the birch suspension cells with NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline- 1-oxyl-3-oxide (cPTIO), NR inhibitor sodium azide (NaN3), and NOS inhibitor NG-nitro-L-Arg methyl ester (L-NAME) decreased Put-triggered NO generation and blocked Put-induced betulin production. Put treatment improved the content of NH4+ and its assimilation enzyme activity of glutamate synthase and glutamate dehydrogenase. NH4+ supplementation also promoted NO and betulin production. Thus, the above data indicated that Put-induced NO was essential for betulin production. NO derived from NR, NOS, and NH4+ mediated betulin production in birch suspension cell cultures under Put treatment.

Highlights

Polyamines (PAs), including putrescine (Put), spermidine (Spd), and spermine (Spm), are low-molecular-weight aliphatic polycations that are quite common in living organisms [1]
Our results showed that nitric oxide (NO) scavenger cPTIO reduced NH4 + -triggered NO generation and decreased NH4 + -induced betulin production; Put treatment enhanced NH4 + production and the activities of glutamine-2-oxoglutarate aminotransferase (GOGAT) and glutamate dehydrogenase (GDH), which are the NH4 +
The current study allowed us to conclude that Put-induced NO is essential to betulin production in birch suspension cell cultures, and it may have been derived from nitrate reductase (NR) and NO synthase (NOS) biosynthetic pathways and nonenzymatic pathways in NH4 +

Summary

Introduction

Polyamines (PAs), including putrescine (Put), spermidine (Spd), and spermine (Spm), are low-molecular-weight aliphatic polycations that are quite common in living organisms [1]. PAs have been suggested to play important roles in morphogenesis, growth, embryogenesis, organ development, leaf senescence, and abiotic and biotic stress responses [3,4]. PAs regulate various fundamental cellular processes as signaling molecules [5]. The major links in PA signaling may be hydrogen peroxide (H2 O2 ) and nitric oxide (NO) [6,7]. Like PAs, NO participates in various cell processes such as growth and development, respiratory metabolism, senescence, and maturation, as well as plant response to abiotic and biotic stressors [8,9]. Nitric oxide synthase (NOS) and nitrate reductase (NR) are known as two major sources of NO production in plants [10]. NO is produced via nonenzymatic pathways, such as NO induced by the presence of hydrogen sulfide, abscisic acid, PAs, and ammonium cation (NH4 + ) [11,12]

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