Biosynthesis and tissue-specific partitioning of camphor and eugenol in Ocimum kilimandscharicum

Highlights

• Integrating metabolomics and NGS revealed tissue-specific metabolite partitioning.

• In planta bioassays showed the involvement of bdh and gpps in camphor biosynthesis.

• Camphor partitioning in tissues is governed by differential expression of bdh and gpps.

• Tissue-specific eugenol partitioning potentially involves transporter proteins.

Abstract

In Ocimum kilimandscharicum, the relative volatile composition of camphor in leaves was as high as 55%, while that of eugenol in roots was 57%. These metabolites were differentially partitioned between the aerial and root tissues. Global metabolomics revealed tissue-specific biochemical specialization, evident by the differential distribution of 2588 putative metabolites across nine tissues. Next-generation sequencing analysis indicated differential expression of 51 phenylpropanoid and 55 terpenoid pathway genes in aerial and root tissues. By integrating metabolomics with transcriptomics, the camphor biosynthesis pathway in O. kilimandscharicum was elucidated. In planta bioassays revealed the role of geranyl diphosphate synthase (gpps) and borneol dehydrogenase (bdh) in camphor biosynthesis. Further, the partitioning of camphor was attributed to tissue-specific gene expression of both the pathway entry point (gpps) and terminal (bdh) enzyme. Unlike camphor, eugenol accumulated more in roots; however, absence of the eugenol synthase gene in roots indicated long distance transport from aerial tissues. In silico co-expression analysis indicated the potential involvement of ATP-binding cassette, multidrug and toxic compound extrusion, and sugar transporters in eugenol transport. Similar partitioning was evident across five other Ocimum species. Overall, our work indicates that metabolite partitioning maybe a finely regulated process, which may have implications on plant growth, development, and defense.

Introduction

Functional metabolic specialization in organisms occurs as a result of metabolite partitioning, which refers to the vectorial transport and accumulation of primary and/or specialized metabolites in an organ-, tissue-, cell-, or organelle-specific manner. Plants are known to harbor several interwoven metabolic pathways sharing substrates, intermediates, and products. In view of this, such strategic partitioning and compartmentalization may circumvent metabolic interference between diverse classes of metabolites for their optimal synthesis (Lunn, 2007; Tiessen and Padilla Chacon, 2013; Nagegowda, 2010; Ikonen, 2008; Gerdes et al., 2012). It also facilitates proper allocation of resources towards growth and/or defense (Coley et al., 1985; Coley, 1988; Lavinsky et al., 2015; Havko et al., 2016). It occurs during normal growth and development (Kopriva et al., 2012; Osorio et al., 2014; Gao et al., 1998; De Groot et al., 2001), as well as being a mechanism acting against stress (Lavinsky et al., 2015; Ibrahim and Jaafar, 2012).

Metabolite partitioning can occur via the following two major underlying mechanisms: (1) differential or tissue-specific gene expression, and (2) transport of metabolites from the source to sink tissue. Tissues accumulating higher amounts of a metabolite mostly show a higher expression of biosynthetic pathway genes, and thus, a more active pathway for the production of that metabolite. In several cases, the expression of pathway entry point, rate-limiting, or committed step catalyzing enzymes play a critical role in manipulating metabolic flux (Lorence et al., 2004; Howles et al., 1996; Xiang et al., 2012). However, some tissues tend to accumulate certain metabolites in enormous quantities, even though pathway enzymes for their biosynthesis are absent. Such metabolites are synthesized in the source tissue and transported to the sink tissue (Ludewig and Flügge, 2013; Shoji et al., 2000). Several ATP-binding cassette (ABC) transporters have been identified in transportation of specialized metabolites such as alkaloids, terpenoids, and phenolics (Yazaki, 2006). The phenomenon of metabolite partitioning can be understood at the level of genes as well as metabolites. Integrating metabolomics with transcriptomics helps us gain a holistic view of the pathway genes, intermediates, transporters, and transcription factors which may be involved in the synthesis, transport, and storage of the said metabolite(s).

Ocimum species contain numerous specialized metabolites including terpenes, phenylpropanoids, flavonoids, and phenolics (Singh et al., 2015). These metabolites are distributed in a highly tissue-specific manner. Thus, these species provide an attractive model system for studying the phenomenon of metabolite partitioning and its underlying biological significance. The biosynthesis, transport, and storage of most metabolites remains unknown; however, our understanding of this genus has increased in the past few years due to the availability of genomic, transcriptomic, and metabolomic data sets on different species (Upadhyay et al., 2015; Gang et al., 2001; Rastogi et al., 2014; Mandoulakani et al., 2017). Here, we used Ocimum kilimandscharicum Gürke (Lamiaceae) as a representative member of genus Ocimum and investigated the biosynthesis of two major metabolites, namely camphor and eugenol, by performing and integrating next-generation sequencing (NGS)-based transcriptomics with global untargeted metabolomics. To the best of our knowledge, this is the first study reporting global metabolomics using a high-throughput high-resolution technique like Orbitrap for different vegetative and reproductive tissues of an Ocimum spp. Further, the potential mechanism(s) underlying differential partitioning of these metabolites between the aerial and underground system was also proposed.