Arbuscular mycorrhizal fungi: effects on plant ...

Plant Biology ISSN 1435-8603

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Arbuscular mycorrhizal fungi: effects on plant terpenoid accumulation M. T. Welling1, L. Liu1, T. J. Rose1,2, D. L. E. Waters1 & K. Benkendorff3 1 Southern Cross Plant Science, Southern Cross University, Lismore, NSW, Australia 2 Southern Cross GeoScience, Southern Cross University, Lismore, NSW, Australia 3 School of Environment, Science & Engineering, Southern Cross University, Lismore, NSW, Australia

Keywords Gene expression; horticulture; phosphorus; plant morphology; secondary plant metabolites; soil biota. Correspondence M. T. Welling and L. Liu, Southern Cross Plant Science, Southern Cross University, Lismore, 2480 NSW, Australia. E-mails: [email protected]; ben. [email protected] Editor A. Weber Received: 1 September 2015; Accepted: 20 October 2015 doi:10.1111/plb.12408

ABSTRACT Arbuscular mycorrhizal fungi (AMF) are a diverse group of soil-dwelling fungi that form symbiotic associations with land plants. AMF–plant associations promote the accumulation of plant terpenoids beneficial to human health, although how AMF mediate terpenoid accumulation is not fully understood. A critical assessment and discussion of the literature relating to mechanisms by which AMF influence plant terpenoid accumulation, and whether this symbiosis can be harnessed in horticultural ecosystems was performed. Modification of plant morphology, phosphorus availability and gene transcription involved with terpenoid biosynthetic pathways were identified as key mechanisms associated with terpenoid accumulation in AMF-colonised plants. In order to exploit AMF–plant symbioses in horticultural ecosystems it is important to consider the specificity of the AMF–plant association, the predominant factor affecting terpenoid accumulation, as well as the end use application of the harvested plant material. Future research should focus on resolving the relationship between ecologically matched AMF genotypes and terpenoid accumulation in plants to establish if these associations are effective in promoting mechanisms favourable for plant terpenoid accumulation.

INTRODUCTION Arbuscular mycorrhizal fungi (AMF) are a diverse soil-dwelling group that make up the phylum Glomeromycota. AMF are widely distributed in a variety of ecosystems (Rosendahl et al. € 2009; Opik et al. 2010) and associate with over 60% of plant species (Strack et al. 2003; Wang & Qiu 2006). Here, we synthesise the current understanding of the mechanisms underlying plant terpenoid accumulation attributed to improved AMF colonisation and examine the potential to increase the efficiency of these mechanisms within the context of horticultural ecosystems. The AMF–plant symbiosis is a mutualistic relationship that provides the plant with a range of essential nutrients, including phosphorus (P) and nitrogen (N), which are often limiting to plant growth (Kindermans et al. 2007), while the fungi receive fixed carbon from the host (Gianinazzi et al. 2010; Miransari 2010). Associations promote hyphopodium formation and penetration of the fungus into the parenchyma cortex of plant roots (Schmitz & Harrison 2014). The symbiosis then culminates in the development of highly branched intracellular structures referred to as arbuscules, which provide a physical site for nutrient transfer between plants and fungi (Gianinazzi-Pearson 1996). In addition to improving plant nutrient availability, there is increasing evidence that the AMF–plant symbiosis contributes to plant terpenoid accumulation (Table 1). Terpenoids are a class of secondary plant metabolites that occur in most plant species (Bohlmann & Keeling 2008). The terpenoids referred to as apocarotenoids are especially relevant

to a subset of AMF–plant symbioses, because they promote hyphal branching (Akiyama et al. 2005), the proliferation of root plastids and a distinctive yellow phenotype in infected plant roots (Strack & Fester 2006). Terpenoids are classified according to the number of isoprene base structural units, with monoterpenes having two, sesquiterpenes three, diterpenes four and tetraterpenes (carotenoids) eight (Bohlmann & Keeling 2008; Fig. 1). Two intermediate pathways associated with terpenoid biosynthesis exist in plants (Fig. 2). Both the plastidic methylerythritol phosphate (MEP) and cytosolic mevalonate (MVA) pathway provide a pool of isoprenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), required for downstream terpenoid biosynthesis (Kirby & Keasling 2009; Hemmerlin et al. 2012). Subsequent condensation reactions by prenyltransferases fuse isoprene units together to produce terpenoids of increasing complexity (Cheng et al. 2007; Hemmerlin et al. 2012). Terpenoids have a diverse set of structures that perform many roles in plant metabolism (Capell & Christou 2004; Vickers et al. 2009; Ramel et al. 2013), including hormonal regulation of plant growth and allopathic protection of plants from herbivore and microbial attack (Langenheim 1994; Umehara et al. 2008). In addition, a number of terpenoids are also associated with bioactive properties of potential benefit to human health (Chandra et al. 2012; Ku & Lin 2013; Martin et al. 2013). A subset of these terpenoids accumulate in plants colonised with AMF (Table 2). Declines in symbiosis between plants and AMF may limit the accumulation of terpenoids in host plants, and as a result poten-

Plant Biology © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands

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AMF: effects on plant terpenoid accumulation

Welling, Liu, Rose, Waters & Benkendorff

Table 1. Arbuscular mycorrhizal fungi (AMF) associated plant terpenoid increases (%) relative to the non-AMF controlsa.

terpenoid

qualitative accumulation (%)

quantitative accumulation (%)

biomass increases (%)

monoterpene carvone p-cymene geraniol limonene d-linalool

117d 280.5d 408.7d 76.9d 9.1d

n.c. n.c. n.c. n.c. n.c.

65.5d n.c. n.c. 65.5d n.c.

a-pinene

697.5d

n.c.

n.c.

thymol

51.2d

n.c.

35.3d

121.4d

190d

53.9d

diterpene forskolin

carotenoids carotenoids

AM fungus speciesb

plant speciesc

ref.

Glomus macrocarpum G. macrocarpum G. macrocarpum G. macrocarpum G. fasciculatum (Rhizophagus fasciculatus) G. fasciculatum (R. fasciculatus) & G. macrocarpum G. fasciculatum (R. fasciculatus)

Anethum graveolens Coriandrum sativum Coriandrum sativum Anethum graveolens Coriandrum sativum

Kapoor et al. (2002a) Kapoor et al. (2002b) Kapoor et al. (2002b) Kapoor et al. (2002a) Kapoor et al. (2002b)

Coriandrum sativum

Kapoor et al. (2002b)

Trachyspermum ammi

Kapoor et al. (2002a)

189.5d

G. fasciculatum (R. fasciculatus) & G. macrocarpum

Artemisia annua

n.c.

n.c.

G. fasciculatum (R. fasciculatus) & G. macrocarpum

Coriandrum sativum

Awasthi et al. (2011) Chaudhary et al. (2008) Kapoor et al. (2007) Kapoor et al. (2002b)

63.2d

146.7d

51.25d

Glomus bagyarajii, Scutellospora calospora & various AMF

Coleus forskohlii

Sailo & Bagyaraj (2005)

n.c.

30.9d

n.c.

Vitis vinifera

Krishna et al. (2005)

carotene

n.c.

40.6d

n.s.

Ipomoea batatas

Farmer et al. (2007)

ß-carotene

n.c.

415.0d

40.0d

Lactuca sativa

Baslam et al. (2013)

lutein

n.c.

535.7d

40.0d

Acaulospora laevis, Acaulospora scrobiculata, Entrophospora colombiana, Gigaspora gigantea, Glomus manihotis (Rhizophagus manihotis) & Scutellospora heterogama Mixed inoculant; G. manihotis (R. manihotis), Glomus mosseae (Funneliformis mosseae) & G. gigantea G. intraradices (Rhizophagus intraradices) & G. mosseae (F. mosseae) Mixed inoculant; G. etunicatum (Claroideoglomus etunicatum), G. intraradices (R. intraradices) & G. mosseae (F. mosseae) G. fasciculatum (R. fasciculatus) Mixed inoculant; G. intraradices (R. intraradices) & G. mosseae (F. mosseae) G. fasciculatum (R. fasciculatus) Mixed inoculant; G. intraradices (R. intraradices) & G. mosseae (F. mosseae)

Lactuca sativa

Baslam et al. (2013)

sesquiterpenes artemisinin

ß-caryophyllene

(continued)

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Table 1. (Continued) qualitative accumulation (%)

quantitative accumulation (%)

biomass increases (%)

lycopene

n.c.

18.5e

18.3e

zeaxanthin

n.c.

747.7d

40.0d

terpenoid

AM fungus speciesb

plant speciesc

ref.

G. intraradices (R. intraradices) G. fasciculatum (R. fasciculatus) Mixed inoculant; G. intraradices (R. intraradices) & G. mosseae (F. mosseae)

Solanum lycopersicum

Giovannetti et al. (2012) Baslam et al. (2013)

Lactuca sativa

n.c., not calculated; n.s., not-significant. Qualitative terpenoid increases: percentage increase in terpenoid n/within the terpenoid fraction relative to the non-AMF control; Quantitative increase: percentage increase in terpenoid n/within the DW (w/w) or FW (w/w) of plant material relative to the non-AMF control; Biomass increases: percentage increase in shoot or root biomass relative to the non-AMF control. a Table summarises highest percentage increases in qualitative and quantitative terpenoid concentration and biomass in AMF-colonised plants compared with non-AMF controls. b Arbuscular mycorrhizal fungal species associated with terpenoid accumulation identified in column one. c Plant species in which the terpenoid identified in column one was accumulated. d P < 0.05 eP < 0.01.

tially limit the human health-promoting properties of some plant-based foods (Liu 2003; Garcıa-Mier et al. 2013). AMF– plant associations should therefore be an important consideration in horticultural production systems where cultivating such associations could be utilised to improve yield and quality of terpenoids in crops. This review summarises the current knowledge of plant terpenoid accumulation in response to AMF–plant symbiotic relationships and explores whether this can be harnessed in horticultural ecosystems. In addition, we identify key areas for future research into AMFmediated plant terpenoid accumulation. PLANT TERPENOID ACCUMULATION ASSOCIATED WITH ARBUSCULAR MYCORRHIZAL FUNGI Qualitative (Kapoor et al. 2002a,b, 2004; Sailo & Bagyaraj 2005) and quantitative (Sailo & Bagyaraj 2005; Farmer et al. 2007; Kapoor et al. 2007; Chaudhary et al. 2008; Awasthi et al. 2011; Baslam et al. 2013) changes in plant terpenoid accumulation associated with AMF symbiosis have been described (Table 1). However, the terms qualitative and quantitative are rarely defined and are in some instances used interchangeably (Kapoor et al. 2002a; Sailo & Bagyaraj 2005; Copetta et al. 2006; Khaosaad et al. 2006; Mandal et al. 2014). In this review, qualitative terpenoid accumulation refers to an increase in the proportion of a given terpenoid (relative %) within the terpenoid extract/fraction and is synonymous with changes in terpenoid composition (Kapoor et al. 2002a,b; Khaosaad et al. 2006), while quantitative terpenoid accumulation refers to an increase in the amount of a given terpenoid as a proportion of the dry weight (w/w, DW) or fresh weight (FW) of plant material and is synonymous with increases in terpenoid content (Kapoor et al. 2007; Baslam et al. 2013). Both qualitative and quantitative terpenoid accumulation are calculated as a concentration of a specific terpenoid relative to either the terpenoid extract/fraction (qualitative) or to a given plant tissue (quantitative). Qualitative terpenoid concentration is an important consideration when the terpenoid extract/fraction is utilised in its

entirety without compositional alteration. For example, AMFcolonised plants have exhibited an increase in qualitative concentration of a-pinene, a monoterpene with potential antioxidant health benefits (Table 2). These changes are as high as 697.5% (P < 0.05) in Coriandrum sativum (coriander) plants colonised with Glomus fasciculatum (now Rhizophagus fasciculatus; (RF; Kapoor et al. 2002b; Table 1). This provides evidence that AMF can improve the composition of specific bioactive terpenoids, and thereby potentially improve the antioxidant or health-promoting properties of a given plant extract/fraction. Increasing the intake of a specific terpenoid, per unit of food, is possible by increasing quantitative terpenoid concentration. For example, quantitative increases of zeaxanthin concentration, as high as 747.7% (P < 0.05) in Glomus intraradices- (now Rhizophagus intraradices; RI) and Glomus mosseae- (now Funneliformis mosseae; FM) colonised Lactuca sativa (lettuce) have been observed (Baslam et al. 2013; Table 1). Zeaxanthin is a dietary carotenoid (Fig. 1) with potential anti-lipid peroxidation capabilities (Table 2). Quantitative enhancement of zeaxanthin concentration through AMF colonisation is therefore capable of improving zeaxanthin intake, without the need for increasing L. sativa consumption. Quantitative terpenoid concentration is also an important variable capable of influencing the content or yield of a specific terpenoid per plant or per hectare. FM-colonised Ipomoea batatas (sweet potato) was found to have a concentration of 2.8 lgg1 carotene/DW (P < 0.05) more than the non-AMF control (40.6%; Farmer et al. 2007; Table 1), equating to approximately 20.5 gha1 increase in carotene yield. However, AMF symbiosis has the potential to negatively affect terpenoid yield by reducing plant biomass. This problem was observed in AMF-colonised Artemisia annua, where AMF colonisation did not significantly affect total quantitative monoterpene and sesquiterpene concentrations, but did significantly reduce leaf biomass by 32% (P < 0.05; Rapparini et al. 2008), thereby resulting in an overall reduction in terpenoid yield from AMF. The use of both quantitative terpenoid concentration and plant biomass provides an estimate as to the potential contri-


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Fig. 1. Structures of plant terpenoids stimulated by arbuscular mycorrhizal fungi.

bution of AMF symbiosis to plant terpenoid content or yield (Table 1). However, some caution is required when extrapolating terpenoid content from quantitative terpenoid concentration and plant biomass data, as terpenoids may not be equally distributed. Tissue-specific terpenoid quantification or differentiation of plant tissue beyond the classification of shoots and roots has been limited in AMF-associated terpenoid accumulation studies (Kapoor et al. 2002a; Krishna et al. 2005; Sailo & Bagyaraj 2005; Chaudhary et al. 2008; Awasthi et al. 2011; Giovannetti et al. 2012). For example, RI- and FM-colonised L. sativa exhibited an increase in zeaxanthin concentration in the outer leaves of 3.29 lgg1 zeaxanthin/FW more than the nonAMF control (Baslam et al. 2013). However, zeaxanthin concentration was not significantly higher within the inner leaves compared with the non-AMF control, and no reference to the 4

degree to which outer and inner leaves contributed to shoot biomass was given (Baslam et al. 2013). Tissue-specific terpenoid quantification should therefore be a focus for future studies in order to accurately determine AMF-associated plant terpenoid accumulation. Variability in AMF-associated terpenoid accumulation and biomass has been found to be subject to both the specificity of the AMF (Kapoor et al. 2002a,b, 2007; Sailo & Bagyaraj 2005; Copetta et al. 2006; Rapparini et al. 2008) and the plant genotype involved in the symbiotic relationship (Khaosaad et al. 2006; Bagheri et al. 2014). Plants used to examine AMF terpenoid accumulation have been largely propagated from seed (Kapoor et al. 2002a,b; Chaudhary et al. 2008; Rapparini et al. 2008; Awasthi et al. 2011; Giovannetti et al. 2012), and therefore heterogeneity between plant individuals cannot be ruled




Fig. 2. Biosynthetic pathway of plant terpenoids. Plastidic methylerythritol phosphate (MEP) and cytosolic mevalonate (MVA) pathways provide a pool of the isoprenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) required for downstream terpenoid biosynthesis (Kirby & Keasling 2009; Hemmerlin et al. 2012). AACT, Acetyl-CoA C-acyltransferase (EC 2.3.1.16); ATP, Adenosine triphosphate; CTP, Cytidine triphosphate; DMAPP, Dimethylallyl diphosphate; DXR, 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (EC 1.1.1.267); DXS, 1-Deoxy-D-xylulose 5 phosphate synthase (EC 2.2.1.7); CMK, 4-(Cytidine 50 -diphospho)-2-C-methyl-D-erythritol kinase (EC 2.7.1.148); FDS, (2E,6E) Farnesyl diphosphate synthase (EC 2.5.1.10); FPP, Farnesyl diphosphate; GDS, Dimethylallyltranstransferase (EC 2.5.1.1); GGDS, Geranylgeranyl diphosphate synthase (EC 2.5.1.29); GGPP, Geranylgeranyl diphosphate; GPP, Geranyl diphosphate; HDR, 1-Hydroxy-2-methyl-2-butenyl 4-diphosphate reductase; HDS, 1-Hydroxy-2-methyl-2-butenyl 4-diphosphate synthase; HMGR, Hydroxymethylglutaryl-CoA reductase (NADPH) (EC 1.1.1.34); HMGS, Hydroxymethylglutaryl-CoA synthase (EC 2.3.3.10); IDI, Isopentenyl diphosphate D-isomerase (EC 5.3.3.2); NADPH, Nicotinamide adenine dinucleotide phosphate; PP, Isopentenyl diphosphate; PPMD, Diphosphomevalonate decarboxylase (EC 4.1.1.33); PMK, Phosphomevalonate kinase (EC 2.7.4.2); MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (EC 2.7.7.60); MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (EC 4.6.1.12); MEP, 2-C-methyl-D-erythritol-4-phosphate; MK, Mevalonate kinase (EC 2.7.1.36); MVA, Mevalonic acid; Adapted from (Aharoni et al. 2005; Zhi-lin et al. 2007; Floß et al. 2008; Vranova et al. 2012; Kumari et al. 2013).

out as a possible explanation for plant terpenoid variability (Khaosaad et al. 2006). It is suggested that future studies should propagate plants from cuttings or use cloned replicate plants to eliminate the contribution of plant genetic variability to AMF-associated plant terpenoid accumulation.

MECHANISMS OF ARBUSCULAR MYCORRHIZAL FUNGI ASSOCIATED WITH PLANT TERPENOID ACCUMULATION Several potential mechanisms by which AMF promote terpenoid accumulation have been reported in the literature.


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Monoterpene

Monoterpene

Geraniol

Thymol

Carotenoid Monoterpene

Monoterpene Carotenoid

Thymol Zeaxanthin

Carotenoid

Zeaxanthin

Lutein a-Pinene

Monoterpene

Thymol

Monoterpene

Monoterpene

d-Linalool

Geraniol

Carotenoid

Lutein

antioxidant

Monoterpene Monoterpene

Geraniol Limonene

Sesquiterpene

Monoterpene

p-Cymene

Artemisinin

In vitro

Sesquiterpene

Caryophyllene

anti-inflammatory

antimalarial

In vivo

Carotenoid Diterpene

Lycopene Forskolin

antiglaucoma

In vitro In vitro

In vivo In vitro

In vitro

In vivo

In vivo

In vitro

In vitro In vitro

In vivo

In vitro

In vivo In vivo

In vivo

Monoterpene

Limonene

In vivo

Monoterpene

In vitro

In vitro

In vitro

In vivo

in vivo/ in vitro

Geraniol

anticancer

Monoterpene

Carvone

antimicrobial

Monoterpene

p-Cymene

analgesic

class

terpenoid

potential biological activity

>45% in superoxide dismutase activity in tertiary -Butyl hydroperoxide stressed rat alveolar macrophages Antioxidant activity in 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay & bcarotene bleaching test