Root specific elicitation and exudation of fluorescent β-carbolines in transformed root cultures of Oxalis tuberosa

Abstract

Stable transformation was achieved in oca (Oxalis tuberosa L.) using an Agrobacterium rhizogenes-mediated system. Transformation frequencies varied with the use of different types of strains of A. rhizogenes and the age of explants. The transfer of rol A gene into the oca genome was confirmed by PCR analysis. In vitro transformed root cultures of oca grown in sterile liquid media induced purplish-blue fluorescence of the culture flask medium when irradiated with UV light. We have previously observed a similar phenomenon, the exudation of fluorescent compounds by the roots of in vitro and field-grown oca plants. Hairy root cultures of O. tuberosa transformed with A. rhizogenes (ATCC-15834) exuded constitutive levels of harmine (7-methoxy-1-methyl-β-carboline) and harmaline (3,4-dihydroharmine), the main fluorescent compounds detected from oca’s root exudates. Transformed roots showed better growth and exudation of harmine and harmaline compared to the untransformed normal roots. Upon elicitation with fungal cell wall elicitors from Phytophthora cinnamoni, the production and exudation of harmine/harmaline was enhanced in both transformed and non-transformed roots. Harmine and harmaline showed a wide range of antimicrobial activity against soil-borne microorganisms. Biologically, these findings suggest that in nature β-carbolines are constitutive antimicrobial compounds released into the rhizosphere upon microbial challenge. Transformed root cultures of oca make a simple, reliable and well-defined model system to investigate the molecular and metabolic exudation of fluorescent β-carboline biosynthesis, and to evaluate the biological significance of the phenomenon of root exudation of fluorescent metabolites.

Introduction

The Andean root and tuber crops constitute a unique reservoir of germplasm biodiversity. They have been bred to grow at altitudes of 2400-4000 m; therefore, they have great potential for introduction into other highland areas where crops from the Old World are not well adapted [14], [15]. After potato, oca (Oxalis tuberosa L.) is the most well-known tuber crop from the Andean region [14]. It is an annual, herbaceous plant in the Oxalidaceae family. Despite an assortment of valuable features, including high nutritional value, the Andean root and tuber crops have been largely overlooked and poorly studied at the biological level [21]. As is true for most Andean root and tuber crops, there are very few reports on the basic biology, agronomy and biochemistry of oca. Our laboratory has been involved in the investigation of oca for some time, focusing on its unique root biology. Upon elicitation, plants produce a wide diversity of biologically active secondary metabolites termed phytoalexins [11], [29]. Some of these compounds are synthesized and stored during normal growth and development while others are absent in healthy plants, accumulating only in response to pathogen attack, stress conditions or artificial elicitation. Elicitors are defined as molecules that stimulate defense or stress-induced responses in plants [11], [24]. The exogenous application of elicitors to in vitro cultures is useful for studying plant responses to potential microbe attack as well as for enhanced biotechnological production of value-added secondary metabolites in fermentation systems. Fungal elicitors, mostly derived from the cell walls of fungal pathogens, are known to induce de novo synthesis of antimicrobial phytoalexins. Under specific conditions fungal elicitation operates by induction of methyl jasmonate, leading to phytoalexin production [28], [29]. In this manuscript we explored the induction of root secretion of fluorescent compounds upon elicitation.

Root fluorescence is a phenomenon in which roots of plants/seedlings fluoresce when irradiated with ultraviolet (UV) light. Only two plant species have been documented to exhibit this phenomenon: soybean and rye grass, and both only in germinating seedling roots [10]. The biological significance of this occurrence in plants is unknown. We have previously reported that the Andean tuber crop species O. tuberosa secretes fluorescent compounds as part of its root exudates [5], [7]. The main fluorescent compounds from oca’s root exudates were identified as harmine (7-methoxy-1-methyl-β-carboline) and harmaline (3,4-dihydroharmine) [7]. Harmine and harmaline are widespread photoactive β-carbolines, alkaloids, and well-known central nervous system stimulants, reported as a major component of the seeds of Peganum harmala (Peha) [1]. They also occur in Banisteriopsis caapi, an ingredient in the shamanistic Amazonian hallucinogenic mixture “ayahuasca”, as well as in a number of other plant species [2]. The study of roots, the “hidden half” of the plant, and the identification of compounds they excrete pose special challenges. To better understand the functional significance of β-carbolines secretion by roots, we developed a hairy root system of O. tuberosa for the expression and manipulation of constitutive and inducible secondary metabolites. Hairy roots show stable expression of biosynthetic pathways, and thus have been used as an experimental system to study the biology and biochemistry of underground organs [4], [10], [22], [27].

In this communication, we report the isolation and functional characterization of fluorescent β-carbolines along with methyl paraben in O. tuberosa hairy roots. We discovered that the exudation of these fluorescent metabolites along with methyl paraben was triggered upon fungal cell wall elicitation. Furthermore, harmine and harmaline were found to be highly inhibitory against an array of rhizosphere microorganisms. Our results highlight a novel defense mechanism by which hairy roots of O. tuberosa secrete antimicrobial fluorescent metabolites.