•  DOI: 10.3390/biotech9040019
• Published online 2020 Sep 23

Emily Amor Stander,¹ Wesley Williams,^1,2 Yamkela Mgwatyu,¹ Peter van Heusden,¹ Fanie Rautenbach,³ Jeanine Marnewick,³ Marilize Le Roes-Hill,³ and Uljana Hesse^1,2,

Abstract

Rooibos (Aspalathus linearis), widely known as a herbal tea, is endemic to the Cape Floristic Region of South Africa (SA). It produces a wide range of phenolic compounds that have been associated with diverse health promoting properties of the plant. The species comprises several growth forms that differ in their morphology and biochemical composition, only one of which is cultivated and used commercially. Here, we established methodologies for non-invasive transcriptome research of wild-growing South African plant species, including (1) harvesting and transport of plant material suitable for RNA sequencing; (2) inexpensive, high-throughput biochemical sample screening; (3) extraction of high-quality RNA from recalcitrant, polysaccharide- and polyphenol rich plant material; and (4) biocomputational analysis of Illumina sequencing data, together with the evaluation of programs for transcriptome assembly (Trinity, IDBA-Trans, SOAPdenovo-Trans, CLC), protein prediction, as well as functional and taxonomic transcript annotation. In the process, we established a biochemically characterized sample pool from 44 distinct rooibos ecotypes (1–5 harvests) and generated four in-depth annotated transcriptomes (each comprising on average ≈86,000 transcripts) from rooibos plants that represent distinct growth forms and differ in their biochemical profiles. These resources will serve future rooibos research and plant breeding endeavours.

Keywords: rooibos, Aspalathus linearis, medicinal plants, non-model organism, transcriptomics, method evaluation, NGS, RNA-Seq, biochemical screening, bioinformatics

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1. Introduction

Rooibos (Aspalathus linearis) is an indigenous South African shrub widely used to brew the popular rooibos herbal tea. The genus Aspalathus (Fabaceae) includes more than 270 species, most of which are endemic to the Cape Floristic Region of South Africa. Eight distinct A. linearis growth types have been described [1], which vary in their geographic distribution as well as in their morphological, chemical and genetic characteristics [2,3,4]. The Southern and Northern sprouters are prostrate shrublets (max 50 cm high). The Grey sprouters, Nieuwoudtville sprouters, and Wupperthal type plants are medium sized densely branched shrubs. The Red type, Black type, and Tree type plants are erect, slender bushes that can reach up to 2 m in height. The rooibos growth types can be further categorized based on their fire survival strategies: sprouters regrow after fire from an underground lignotuber while seeders are destroyed by veld fires and repopulate from seeds [3,5,6]. The commercially cultivated rooibos plants (Nortier/Rocklands type) descend from successively selected wild Red type plants originally collected from the northern parts of the Cederberg Mountains and the Pakhuis Pass areas [1]. An increasing body of literature provides scientific evidence for beneficial health effects of rooibos, including anti-inflammatory [7,8,9,10], beneficial health effects of rooibos, including anti-inflammatory[11,12,13,14,15,16], anti-diabetic [17,18,19,20], and anti-diabetic [21,22,23] properties (for reviews see [24,25,26,27]). These bio-activities have been associated with the antioxidant properties of diverse phenolic compounds produced by the rooibos plants. Rooibos herbal tea is caffeine-free, low in tannins, high in volatile compounds, and rich in a unique combination of polyphenols [28]. The primary phenolic compound found in commercial unprocessed/green rooibos plant material is a C-glucosyl dihydrochalcone known as aspalathin. In commercial rooibos plants, it represents 4–12% of the plant dry weight [29,30,31]. Other major rooibos compounds include the flavones iso-orientin and orientin (the two oxidation products of aspalathin), luteolin, and chrysoeriol; as well as the flavonols rutin, hyperoside, iso-quercetin, and quercetin [32,33,34]. Wild rooibos plants were shown to have distinct chemical profiles, which differed between populations but were very similar within the same population. Some wild rooibos plants were found not to produce aspalathin at all. In these plants, orientin, iso-orientin, and rutin were found to be the main phenolic compounds [2]. The biosynthesis pathway for rooibos dihydrochalcones and flavonoids has recently been proposed [4].

The field of plant transcriptomics has been revolutionized by Next Generation Sequencing (NGS) technologies [35,36]. Transcriptomics provides a wealth of information on plant genes without prior knowledge on the underlying genome sequence, which greatly facilitates research on non-model organisms (such as rooibos). Medicinal plants usually belong to taxonomic groups that do not have high quality reference genomes [37]. In most cases, research focuses exclusively on identification of genes involved in secondary metabolite biosynthesis. On average, plants encode between 20,000 to 60,000 genes, of which only 15–25% contribute to secondary metabolite production. For these plant species, RNA-Seq is considered the method of choice to gather information on secondary metabolism associated plant genes, as whole genome analysis is considered redundant [38]. Knowledge on the genes and biosynthetic pathways involved in the production of economically important metabolites is increasingly exploited in synthetic biology and genetic engineering programs. Transcriptome data of non-model plants are already employed to optimize in vitro and in vivo biosynthesis of medicinal compounds [39,40,41], biodiesel feedstock’s [42,43,44], and essential oils [45,46,47].

South Africa is home to the unique Cape Floristic Region, where more than 70% of the plants are endemic [48]. The country has a rich traditional history in the application of diverse plant species for medicinal purposes. Yet, agricultural production systems for the nearly 3000 plant species used for traditional medicine are all but missing, and most plants are collected from the wild [49]. To promote research on the endemic medicinal plants of South Africa, this study aimed to locally establish all procedures essential for plant transcriptome analysis, including sample collection and biochemical screening methods geared at identifying interesting ecotypes from distant geographic locations, as well as laboratorial procedures and biocomputational methods for plant transcriptome analysis. The second aim of this study was to generate rooibos transcriptomes that would allow research on genes and biosynthetic pathways associated with diverse important plant traits (e.g., medicinal compound production, stress tolerance, growth form). To facilitate gene discovery, transcriptomes were sequenced from four rooibos plants that represent different growth types with distinct morphological traits and contrasting biochemical profiles (including aspalathin producers and non-producers).

Reference:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9258316/