THE MOLECULAR AND BIOCHEMICAL BASIS OF NITROGEN
TRANSFER BY THE MODEL ARBUSCULAR MYCORRHIZAL FUNGUS
RHIZOPHAGUS IRREGULARIS
By
Taghleb Muhammad Al-Deeb

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Plant Biology-Doctor of Philosophy
2015

ABSTRACT
THE MOLECULAR AND BIOCHEMICAL BASIS OF NITROGEN
TRANSFER BY THE MODEL ARBUSCULAR MYCORRHIZAL FUNGUS
RHIZOPHAGUS IRREGULARIS
By
Taghleb Muhammad Al-Deeb
Plants can increase their effective root length and surface area by investing in
symbioses with soil fungi, forming mycorrhizal associations. In mutualistic mode, the
plant provides photosynthate (fixed carbon) to the fungus, whereas the fungus
provides nutrients to the plant. Arbuscular mycorrhizal (AM) associations are found in
80% of vascular plant families and as a consequence, the AM symbiosis is of
tremendous significance to life on this planet, in both natural and agricultural
ecosystems. Work in recent years has substantially increased our understanding of
nitrogen nutrition in the AM symbiosis. At the molecular level, a working model for
nitrogen uptake, metabolism, and transfer has emerged. In this dissertation,
mechanisms and genes believed to be responsible for nitrogen flows in the AM
symbiosis are described and open questions about the pathway and its regulation are
highlighted. Molecular and biochemical experimental approaches were used to
investigate these unresolved questions. A compartmented microcosm was developed
for aseptic and leakage-free whole-plant mycorrhizal experiments. This was used to
monitor S and N uptake by the fungal extraradical mycelium (ERM) and its transfer to
host plants. Our results show rapid S and N transfer by ERM to the host plants. Using
growth parameter measurements, chlorophyll contents as well as

15N

labeling, we

conclude that nitrogen transfer from an arbuscular mycorrhizal fungus confers growth

benefits on the host plant under nitrogen limiting conditions and that the microcosm
system developed will be useful for future work on AM nutrition and metabolism under
physiologically relevant conditions. Isotopic labeling time course experiments using
different

15N

and

13C

labeled substrates as well as expression analysis of the

expression of key genes were performed using microcosms and an in vitro
monoxenic culture system. The results demonstrated the operation of a new pathway
of N transfer by AMF to the host via nitrate translocation from the extraradical
mycelium to plant roots and shoots. The results also indicate that ornithine is made in
the ERM via pyrroline-5-carboxylate and that some of it is broken down in the IRM to
glutamate and to a lesser extent to putrescine. Labeling analysis strongly suggests
that ornithine is also translocated from the intraradical mycelium to the ERM and is
used to make arginine there. Changes in gene expression are consistent with the
labeling data on N uptake, metabolism and movement. Gene expression analysis of
glutamate dehydrogenase suggests a potential dissimilatory role in the IRM.

Copyright by
TAGHLEB MUHAMMAD AL-DEEB
2015

ACKNOWLEDGEMENTS

I would like to express my warm feeling, deepest appreciation, and deep thanks
to my graduate supervisor Prof. Yair Shachar-Hill, for his supervision, encouragement
and guidance through this work, his kindness and patience were always encouraging
me to go ahead, I couldn’t hope for better advisor.
My deep thanks and appreciation to the Professors; Daniel Jones, Frances Trail
and Robin Buell for encouragement and kindness to be member of my committee and
their help.
I would like to thank the mass-spec facility members especially; Prof. Daniel
Jones and Lijun Chen for teaching and helping me through my work there.
I would like also to thank past and current members of Prof. Shachar-Hill’s lab
who have impact on my research including Beth Kasiborski, Chunjie Tian, Rahul
Deshpande, Matthew Juergens, Lisa Carey, (Wilson) Chen, Leann Lerie Matta, Mike
Pollard, Teresa Clark, Mike Opperman and Bradley Disbrow for their teaching, helping,
teasing and encouraging me.
My Great and Deep thanks are to the colleagues in the Department of Plant
Biology and my friends at Michigan State University.
My infinite, great and unlimited deep thanks to my family members; my father,
mother, brothers and sisters for their continuous support and encouragement.

v

TABLE OF CONTENTS

LIST OF TABLES .......................................................................................................... viii
LIST OF FIGURES .......................................................................................................... ix
Chapter 1 ....................................................................................................................... 1
Introduction and literature review .................................................................................... 1
Introduction .................................................................................................................. 2
Morphology, anatomy and life cycle ............................................................................ 3
Phylogeny ................................................................................................................... 5
Plant benefits from AM symbioses .............................................................................. 6
Phosphorous uptake and transfer ........................................................................ 7
Sulfur uptake and transfer ................................................................................... 8
Nitrogen uptake and transfer ............................................................................... 9
The mechanisms of nitrogen transfer and metabolism in the AM symbiosis ............. 10
The pathway of nitrogen movement through the AM symbiosis ........................ 10
The uptake of nitrogen by the ERM of the AM symbiosis .................................. 11
The assimilation of N in the ERM and release of ammonium in the IRM ............ 12
What is the role of AM networks in the uptake and exchange of nitrogen among plants
in ecosystems? ......................................................................................................... 14
Movement of N from soil to plants via AMF in plant ecosystems ....................... 14
Colonization by AMF depends on the nutrient status of host plants .................. 16
Nitrogen exchange between plants through mycorrhizal networks and its
ecological significance ....................................................................................... 19
Interactions between AMF and symbiotic N2-fixing microbes and their contribution to
N accumulation in host plants ................................................................................... 20
Conclusions and perspectives .................................................................................. 21
APPENDIX ................................................................................................................ 24
REFERENCES ......................................................................................................... 32

Chapter 2 ..................................................................................................................... 45
Nitrogen uptake and transfer in an aseptic whole plant-mycorrhizal culture system ..... 45
Abstract ..................................................................................................................... 46
Introduction ............................................................................................................... 48
Experimental procedures .......................................................................................... 52
Chemicals and reagents .................................................................................... 52
Seed sterilization ............................................................................................... 52
Growth conditions .............................................................................................. 52
Experimental design .......................................................................................... 53
Testing the diffusion between compartments and the capillary transfer ............ 54
Extraction and measurement of 35SO4 from mycorrhizal plants ......................... 54
Analysis of mycorrhizal colonization and ERM crossing to the FC .................... 55
vi

Growth parameters and chlorophyll content measurements ............................. 55
Extraction, isolation and quantification 15N metabolites ..................................... 56
Liquid chromatography and mass spectrometry (LC-MS) .................................. 56
Results ...................................................................................................................... 58
Discussion ................................................................................................................. 62
APPENDIX ................................................................................................................ 66
REFERENCES ......................................................................................................... 83
Chapter 3 ..................................................................................................................... 88
Evidence for additional pathways in the nitrogen transfer network of arbuscular
mycorrhizas .................................................................................................................. 88
Abstract ..................................................................................................................... 89
Introduction ............................................................................................................... 91
Experimental procedures .......................................................................................... 94
Chemicals and reagents .................................................................................... 94
Spore material and mycorrhizal in vitro growth conditions ................................. 94
Isotopic labeling in colonized Ri T-DNA-transformed carrot (Daucus carota clone
DCI) roots culture system .................................................................................. 95
15N labeling ............................................................................................. 95
13C labeling ............................................................................................. 95
15N labeling for 15NO measurements in whole plant-mycorrhizal system ......... 96
3
Extraction and isolation of 15N and 13C metabolites ........................................... 96
Liquid chromatography and mass spectrometry (LC-MS) .................................. 97
15N labeled and unlabeled nitrate measurements .............................................. 99
RNA extraction and putative gene fragment isolation ...................................... 100
Quantitative Real-Time PCR measurements ................................................... 100
Results .................................................................................................................... 101
Nitrate movement through the AM symbiosis .................................................. 101
N movement through the AM symbiosis in transformed root system ............... 102
The origin and fate of the N metabolites ........................................................... 104
13C labeling in the ERM and AM roots ............................................................. 104
Gene identification ........................................................................................... 105
Gene expression in response to N addition ..................................................... 106
Discussion ............................................................................................................... 107
APPENDIX .............................................................................................................. 113
REFERENCES ....................................................................................................... 133
Chapter 4 ................................................................................................................... 137
Conclusions and future research ................................................................................ 137
Conclusions ............................................................................................................ 138
Future research ....................................................................................................... 140
REFERENCES ........................................................................................................... 142

vii

LIST OF TABLES

Table 1-1 Nitrogen metabolic proteins and genes identified in the arbuscular mycorrhizal
symbiosis ...................................................................................................................... 29
Table 2-1 Precursor (unlabeled) compounds and product ions for each analyte .......... 81
Table 2-2 Multiple reaction monitoring (MRM) transitions, optimizing source cone
voltages, collision cell voltages, and analyte retention time (for 15N labeling experiment)
...................................................................................................................................... 82
Table 3-1 Multiple reaction monitoring (MRM) transitions, optimizing source cone
voltages, collision cell voltages, and analyte retention time (for 13C labeling experiment)
.................................................................................................................................... 130
Table 3-2 Primers for real-time PCR ........................................................................... 131
Table 3-3 Nitrogen metabolic genes identified in Rhizophagus irregularis in this study
.................................................................................................................................... 132

viii

LIST OF FIGURES

Figure 1-1 Arbuscular mycorrhizal fungal morphologies in tomato (Solanum
lycopersicum) roots plant. ............................................................................................. 25
Figure 1-2 Phylogeny of fungi based on SSU rRNA sequences. ................................. 26
Figure 1-3 Taxonomy of the Glomeromycota containing the arbuscular mycorrhizal and
related fungi, based on SSU rRNA gene sequences .................................................... 27
Figure 1-4 Working model of N transport and metabolism in the AM symbiosis ........... 28
Figure 1-5 Interactions among arbuscular mycorrhizal fungi, host plants, and N2-fixing
bacteria and other soil microbes in nitrogen cycling ...................................................... 29
Figure 2-1 Whole plant-mycorrhizal two-compartment culture system .......................... 67
Figure 2-2 Sulfur transfer from fungus to plant using the whole plant culture system.... 68
Figure 2-3 Colonization of Medicago truncatula by Rhizophagus irregularis ................. 71
Figure 2-4 Seven-week old Medicago truncatula plants growing in the two-compartment
system ........................................................................................................................... 72
Figure 2-5 Total chlorophyll content and growth parameters measurements ................ 73
Figure 2-6 Free N metabolite concentrations and 15N labeling in the mycorrhizal whole
plant system .................................................................................................................. 76
Figure 3-1 Time course of 15N percentage labeling of nitrate in transformed carrot
(Daucus carota) roots colonized by R. irregularis ....................................................... 114
Figure 3-2 The concentrations of 15N labeled nitrate (light grey bars) and unlabeled
nitrate (black bars) in the roots and shoots of mycorrhizal M. truncatula plants grown in
micosms ...................................................................................................................... 115
Figure 3-3 Nitrogen movement from the ERM to the IRM in mycorrhizal transformed
carrot roots .................................................................................................................. 116
Figure 3-4 Time course of the free N metabolites 15N-Isotopomers (mass isomers) in the
extraradical mycelium (ERM) after the addition of 4 mM K15NO3 to the ERM .............. 120

ix

Figure 3-5 Timing course of 13C labeling of N metabolites in the extraradical mycelium
(ERM) and the mycorrhizal roots after the addition of 0.5 mM 13C6 arginine to the fungal
ERM compartment. ..................................................................................................... 123
Figure 3-6 The expression of primary N metabolic and transport genes in the arbuscular
mycorrhizal symbiosis after the addition of 4 mM KNO3 to the fungal extraradical
mycelium (ERM) .......................................................................................................... 128

x

Chapter 1
Introduction and literature review

1

Introduction
The arbuscular mycorrhizal (AM) symbiosis is the most ancient and widespread
plant-microbe mutualism among land plants. Arbuscular mycorrhizas are formed by the
roots of over 80% of plant species with fungi of the Glomeromycota. Arbuscular
mycorrhizal fungi (AMF) are asexual, obligate symbionts (Smith and Read, 2008) that
propagate by forming multinuclear vegetative underground spores and by the growth of
the underground mycelium to colonize additional roots. After spore germination, hyphal
growth is associated with nuclear divisions as well as the migration of nuclei from the
spore (Bianciotto and Bonfante, 1992; Becard and Pfeffer, 1993; Bianciotto et al. 1995).
The hyphae use carbohydrate and lipid reserves during pre-symbiotic growth (Becard et
al., 1991; Bago et al., 1999). In the absence of a host root, the growth of the mycelia
ceases within several days, and spores are capable of multiple germination events.
Presymbiotic hyphae respond to signaling molecules in plant root exudates that
stimulate branching (Giovannetti et al.,1993; Buee et al. 2000). Akiyama et al., (2005)
identified strigolactones in plant root exudates as increasing germ tube metabolism and
growth. Strogolactone production is stimulated when plants experience mineral
deficiency. Hyphal contact with the root is usually followed by adhesion of the hyphal
surface and after 2-3 days, the formation of appressoria followed by root penetration
and formation of arbuscules around 2 days later (Brunndrett et al., 1985; Becard and
Fortin, 1988; Peterson and Bonfante, 1994). Once the AM symbiosis has been
established, the fungal growth proceeds in both the soil (extraradical mycelium) and in
the root (intraradical mycelium).

2

AMF have so far proven to be unculturable axenically and appear in nature to be unable
to complete their life cycle without forming a symbiosis with plant host roots (Smith and
Read, 2008). AMF take up photosynthetically fixed carbon (C) from the plant and
convert it into triacylglycerols, which are the main nutritional form of carbon stored and
translocated by the fungus for growth and sporulation and utilized during germination
(Bago et al. 2000). The intraradical mycelium imports glucose from the roots and
converts it into trehalose and glycogen and then to triacylglycerol (Shachar-Hill et al.,
1995; Pfeffer et al., 1999). Lipid bodies transport C from the intraradical mycelium (IRM)
to extraradical mycelium (ERM) and are utilized for mycelial growth (Bago et al., 2002;
Lammers et al., 2001)

Morphology, anatomy and life cycle
Details of fungal interactions with plant cells and tissues of the root were first
described by Gallaud (1905) in which he indicated that AM roots can contain a variety of
structures including arbuscules hyphae and vesicles. Gallaud (1905) categorized them
into four distinct structural classes; Arum, Paris, hepatic and orchid types. The most
common forms are found in AM roots: the Paris-type and Arum-type (Figure 1-1). In
Paris type the cortical colonization of the root is characterized by extensive
development of intracellular coiled hyphae with arbusculated coils in root cortex cells.
These coils spread from cell to cell and do not form significant intercellular hyphae.
Paris type morphology occurs in many families of pteridophytes, gymnoserms and
angiosperems (Smith and Smith 1997). By contrast, Arum-type is often described in the

3

fast-growing roots systems of crop plants (Brundrett et al., 1990). Although the plant
species clearly influences the fungal morphology type, with the same fungus forming
Arum morphology on one host species and Paris in another (Smith and Smith, 1997), it
has also been shown that different fungal species form different morphologies in the
same host plant (Cavagnaro et al., 2001). In these associations the fungus spread
rapidly through the root cortex by the growth of intercellular hyphae which extend along
well-developed intercellular air spaces. Side branches penetrate the plant cell walls of
cortical cells to produce highly branched hyphal structures named arbuscules for their
tree-like appearance (Smith and Read, 1997). The arbuscules are formed by
dichotomous branching and are transient structures that degenerate and disappear
about 2-3 weeks after they develop.The arbuscules invaginate root cortical cells without
penetrating the host cell membrane and form a periplasmic space between the root cell
and the arbuscular membrane. The host plant cell resumes its normal appearance after
the arbuscule within it breaks down. The periplasmic space is believed to be the site of
nutrient transfer between the fungus and the plant (Bago, 2000; VanAarle et al., 2005).
Outside the root, AMF form mycelia consisting of runner hyphae that branch at a
frequency that depends on the nutritional state of the external substrate. From these
side branches, arbuscule-like Branched Absorbing Structures (BAS) are formed that are
proposed to be involved in nutrient uptake in the soil (Bago, 2000). The BAS are the site
of formation of the spores, which are vegetative multinuclear Sporangiospores that
develop at the hyphal tips of BAS. A single spore contains several hundred to several
thousand haploid nuclei. These spores germinate by forming a germ tube into which
nuclei emerge from the spore and proliferate (Becard et al., 1990). Germination, germ

4

tube growth and branching of the pre-symbiotic mycelium is stimulated by plant
exudates (Mosse, 1958). Strigolactones have been shown to be major active molecules
in these exudates. At the surface of host roots, the presymbiotic hyphae form
appressoria, commonly particularly at the sites of lateral root emergence (Smith and
Read, 1998), and from which hyphae penetrate the root epidermis to form the
intraradical mycelium.

Phylogeny
It was believed, partly on the basis of their asexual life cycle, that AMF are most
closely related to zygomycota but the use of DNA sequences forced a re-evaluation
their relationship (Smith and read, 2008). Based on small subunit (SSU) rRNA gene
sequences, morphological and ecological characteristics, AMF are now regarded as
separate from all other major fungal groups in a monophyletic clade. Consequently they
were placed into a new phylum, the Glomeromycota (Schußler et al., 2001; Figure 1-2 &
1-3). About 240 species of glomeromycotan fungi have been described to date based
on their spore morphology and molecular phylogenetic data (Schüßler & Walker, 2010;
Redecker et al., 2013). Alves de silva et al., (2006) analyzed the large subunit (LSU)
ribosomal RNA (rRNA) for AM fungal taxonomy and their data indicated that
Archaeosporaceae are a basal group in Glomeromycota, Acaulosporaceae and
Gigasporaceae belong to the same clade, while Glomeraceae are polyphyletic. large
subunit of RNA polymerase II (RPB1; Redecker and Raab 2006; Stockinger et al.,
2014) and β-tubulin gene (Msiska and Morton 2009) were phylogentically analyzed too.

5

The β-tubulin gene phylogeny was similar to the 18S (LSU) rRNA gene phylogeny at the
family and species level, but not at the order level (Msiska and Morton 2009). Based on
all of these studies, AMF are divided into ten families in the phylum Glomeromycota,
order Glomerales. Those families are Gigasporaceae, Glomeraceae, Acaulosporaceae,
Diversispora,

Paralglomaceae,

Geosiphoaceae,

Ambisporaceae,

Eutrophosphosporaceae, and Arcaesporaceae.
Glomus is the largest genus within the phylum with more than 70 species
(Redecker and Raab 2006). Several of the Glomus species, most frequently Glomus
intraradices (now Rhizophagus irregularis), are commonly studied (Smith & Read,
2008). Based on a molecular analysis of ribosomal DNA and a re-evaluation of an early
description of this species, it was recently renamed Rhizophagus irregularis (Krüger et
al., 2012) despite the misleading “root eater” title. Tisserant et al., (2013) assembled
and annotated the genome of Rhizophagus irregularis from high throughput DNA
sequencing in association with transcriptome data. This study provides insight into the
capabilities of this fungus and points to genes involved in mycorrhizal symbioses.

Plant benefits from AM symbioses
Association with AMF is often beneficial to plants by improving their access to
nutrients (Smith and Smith, 2011). AMF also enhance pest and disease suppression
and improve drought tolerance (Smith and Read, 2008). Among the benefits that AMF
provide to host plants, improved phosphorus nutrition has received the most attention.
Previous studies, however, indicated that AMF may also be important for a wide variety
of nutrients (Smith and Smith, 2011). AMF enhance the uptake of nitrogen
6

(Govindarajulu et al., 2005), sulfur (S, Allen & Shachar-Hill, 2009), zinc (Seres et al.,
2006), copper (Toler et al., 2005) and iron (Kim et al., 2010). Hart and Forsythe, (2012)
showed that the identity of AMF can influence the uptake of many nutrients but that the
magnitude and direction of the nutrient-derived growth response is also affected by host
plant characteristics and soil nutrient status.

Phosphorous uptake and transfer
AMF improve the phosphorous (P) nutrition of host plants (Bolan, 1991). The
extraradical mycelium (ERM) explores a larger soil volume than is possible for roots and
reaches zones and soil pores that the lateral roots cannot access. The small hyphal
diameter (typically several microns) leads to an increased P absorbing surface area
(Marschner and Dell, 1994) and estimates of ERM levels around host plants indicate
that these reach several meters of hyphal length per gram of soil (Jakobsen et al.,
1992). In AMF, polyphosphates (polyP) are formed after P uptake thus lowering internal
inorganic P (Pi) concentrations. Organic acids and phosphatases are released by the
ERM which increase the availability of P from organic and inorganic sources. In the
extraradical hyphae, N is transported as arginine (Govindarajulu et al., 2005; Tian et al.,
2010) which may be bound to polyphosphate and therefore be coupled to Pi
translocation (Jin et al., 2005). Several studies have shown that AM specific root
phosphate transporters are induced in the roots of plants colonized by AMF (Javot et
al., 2007; Gomez et al., 2009; Nagy et al., 2009). It was suggested that Pi delivery to
cortical cells was necessary for sustaining the symbiosis because in Medicago
truncatula mutants affected in the AM-specific Pi transporter 4 gene, the arbuscules
7

accumulated polyphosphate and prematurely degenerated (Javot et al., 2007).
Increasing carbohydrate availability stimulates P uptake by the ERM to and
translocation to the mycorrhizal roots as well as altering the metabolic and spatial
distribution of P within the fungus (Bücking and Shachar-Hill, 2005). Previous studies
suggested that there is a cross-talk between P and N nutrition (Blanke et al., 2005,
Bonneau et al, 2013). It was found that low P and N fertilization induced a physiological
state of plants favorable for AM symbiosis despite their higher P status (Bonneau et al,
2013).

Sulfur uptake and transfer
Previous studies have reported the effects of AM colonization on the uptake of S
(Gray and Gerdemann, 1973; Rhodes and Gerdemann, 1978). Gray and Gerdemann
(1973) showed that mycorrhizal colonization in clover (Trifolium pratense) and maize
increased 35S uptake compared to nonmycorrhizal plants. Furthermore, Rhodes and
Gerdemann (1978) found that mycorrhizal colonization increased 35S uptake in onion
compared to nonmycorrhizal plants and they showed that the S uptake was heavily
influenced by P nutritional benefits.
Allen and Shachar-Hill (2009) showed using in vitro cultures of transformed roots
colonized by R. irregularis that the ERM takes up S in the form of sulfate and sulfurcontaining amino acids and transfers it to mycorrhizal roots. They also showed that the
root S contents were increased by 25% in a moderate (not growth-limiting)
concentration of sulfate. Fifty percent of

35SO -2
4

8

uptake from the fungal compartment

was detected in the mycorrhizal roots. Similar quantities of 35S were transferred to
mycorrhizal roots whether 35SO42−, [35S]Cys, or [35S]Met was supplied in the fungal
compartment (Allen and Shachar-Hill, 2009). Sieh et al., (2013) studied the effect of
mycorrhizal colonization on sulfur starvation responses in M. truncatula and they found
that colonization reduced S starvation when the plant's phosphate status is high,
concluding that mycorrhizal sulfur transfer improves plant S nutrition.

Nitrogen uptake and transfer
Plant roots and the AM fungal ERM can absorb both nitrate and ammonium from
soil (George et al., 1995; Smith and Read, 2008) as well as soluble organic nitrogen
which together dominate the soil N pool (Jin et al., 2005, McNeill and Unkovich, 2007).
Nitrate and ammonium are mobile under most soil conditions but this can be restricted
in dry soil (Tobar et al., 1994, Tinker and Nye, 2000). These observations suggest a
possible role for N uptake and transfer, a suggestion that has been strengthened by
tracer studies and field observations.
Haines and Best (1976) found that colonization of Liquidambar styraciflua by the
AM fungus Glomus mosseae retarded the leaching of ammonium and nitrate from soil
suggesting that the AMF might be involved in N uptake. When N-15-labelled fertilizer
was added to mycorrhizal or non mycorrhizal control pots, Azcon-Aguilar et al., (1993)
reported that the 14N/15N ratio was higher in AM onion plants than in uncolonized control
plants, suggesting that AMF were able to access soil N that is less available to non
mycorrhizal plants. Furthermore, Cliquet and Stewart (1993) demonstrated that

15N

translocation from roots to shoots through the xylem was higher in AM plants compared
9

with control plants. Under water-stressed conditions that affect the nitrate availability to
roots, the

15N

enrichment was four times higher in mycorrhizal than in non-mycorrhizal

lettuce plants providing evidence of hyphal transport to the plant of N from labeled
nitrate (Tobar et al., 1994). A mechanism of N transfer from the fungus to the plant in
the AM symbiosis was proposed (Bago et al., 2001) in which N taken up by the fungus
is incorporated into amino acids, translocated from the ERM to the intraradical mycelium
(IRM) as arginine, which is broken down to ammonium, that is released to the host root.
This model has gained support from

15N

and

13C

isotopic labeling experiments,

measurements of enzymatic activities (Govindarajulu et al., 2005; Jin et al., 2005, Cruz
et al., 2007) and from gene expression data (Govindarajulu et al., 2005; Guether et al.,
2009; Gomez et al., 2009; Tian et al., 2010).

The mechanisms of nitrogen transfer and metabolism in the AM
symbiosis
The pathway of nitrogen movement through the AM symbiosis
The current working model of N transfer from the fungi to the plant in the AM
symbiosis was proposed by Bago et al. (2001) based on previous work that
demonstrated fungal N uptake and metabolism and implicated amino acids in N
handling (Johansen et al., 1996; Bago et al., 1996; 2000). This mechanism involves N
uptake up by the fungi which is incorporated into amino acids, translocated from the
extraradical mycelium (ERM) to the intraradical mycelium (IRM) as arginine (Arg), but

10

transferred to the plant without C as inorganic N (Govindarajulu et al., 2005; Jin et al.,
2005).
Gene expression and enzyme activities for plant and fungal proteins involved in the
nitrogen metabolic pathway have been analyzed (Table 1-1). Most of the important
enzymes and nitrate or ammonium transporters involved in nitrogen metabolism were
identified from the AM fungus R. irregularis (Kaldorf et al. 1998; Govindarajulu et al.,
2005; Lopez-Pedrosa et al., 2006; Gomez et al., 2009; Tian et al., 2010). Gene
expression at the transcriptional and post transcriptional levels and

15N

and

13C

labeling

experiments support the proposed model (Govindarajulu et al., 2005; Cruz et al., 2007;
Tian et al., 2010). In Figure 1-4, the current model is illustrated, including the genes
identified and the postulated regulation on the transcripts by the metabolites (Tian et al.,
2010).

The uptake of nitrogen by the ERM of the AM symbiosis
A high affinity ammonium transporter has been identified in R. irregularis and
characterized by Lopez-Pedrosa et al. (2006), which is involved in the ammonium
uptake of ERM. A putative high affinity nitrate transporter which is up-regulated in
response to nitrate addition was identified in the ERM of the same AM fungus (Tian et
al., 2010). Plant and fungal uptake of macronutrient ions involves low affinity as well as
high affinity transporters, and it is expected that low affinity transporters for NO3- and
NH4+ will also be identified. AMF can also obtain N from decomposing organic materials
and store it in the mycelium (Hodge and Fitter, 2010). Amino acids including Gly, Glu,

11

Pro and Arg can be taken up by the ERM (Hawkins et al., 2000; Jin et al., 2005) and
Arg, Gly, Gln and Orn can be taken up by germinating spores (Gachomo et al., 2009).
An amino acid permease, GmosAAP1, that can transport proline through a protoncoupled process has been characterized from Glomus mosseae; its expression is
transcriptionally upregulated by external amino acids (Cappellazzo et al., 2008). It is
likely that AMF express permeases allowing the uptake of other amino acids such as
Arg by the fungus from the environment. Indeed, genome-scale analysis of an
ectomycorrhizal fungus has highlighted the existence of many N transporters (Lucic et
al., 2008). However the uptake rates of amino acids by the ERM measured in AM root
cultures are substantially lower than the rates for nitrate, ammonium and urea (Jin et al.
2005, and this work). It also appears that most of the nitrogen taken up by AMF from
organic patches in the soil is taken up without carbon (Hodge & Fitter, 2010), in
agreement with earlier experiments on the fate of nitrogen and carbon supplied to
mycorrhizal plants as labeled peptides (Persson et al., 2003).

The assimilation of N in the ERM and release of ammonium in the IRM
Kaldorf et al. (1998) reported a partial sequence for a putative nitrate reductase
from R. irregularis. The glutamine synthetase/glutamate synthase (GS/GOGAT)
pathway is important in the assimilation of ammonium produced from nitrate or taken up
directly from the soil. Activities for the GS/GOGAT pathway but not the alternative
assimilatory NADP-dependent glutamate dehydrogenase (GDH) were reported by
Cliquet and Stewart (1993) in mycorrhizal roots, and the application of a GOGAT

12

inhibitor to extraradical mycelium reduced

15N

assimilation. Breuninger et al. (2004)

found that GS activity is upregulated in response to N addition in the ERM of R.
irregularis and G. mossae. The glutamine synthetase from R. irregularis reported by
Breuninger et al. (2004) was recently found to be one of a gene family with at least two
members, and this paralog (named GiGS1, Tian et al. 2010) has a high constitutive
transcriptional level whereas another member (GiGS2) is actively up-regulated upon
exposure to nitrate but has a lower constitutive expression level. This suggests different
roles for the two enzymes at different environmental nitrogen levels. Additionally, both of
them have low Km values for glutamate, which indicates that the GS/GOGAT pathway
could operate even at low nitrogen levels (Tian et al., 2010). Since both GS/GOTAT and
NADP-GDH contribute to N assimilation in ectomycorrhizal fungi, more direct
measurements in AMF would be desirable to determine if and when NADP-GDH might
be involved in the AM symbiosis.
Genes for all the reactions of the urea cycle have been identified in

R.

irregularis. Enzymatic activities associated with the synthesis of Arg, are up-regulated in
the ERM after ammonium addition (Cruz et al., 2007). Soon after this, fungal genes for
breaking down Arg, including arginase (GiCAR1), urease (GiURE), and ornithine
aminotransferase (GiOAT1, 2) are up-regulated in the colonized root tissues (Tian et al.,
2010). Enzymatic activity of glutamine synthetase, arginase and urease were shown to
be up-regulated in the colonized root in response to N addition to the ERM (Cruz et al.,
2007), and later gene expression analyses (Tian et al., 2010, Tisserant et al., 2012)
indicate that this activity is fungal.

13

The interface between fungal arbuscules and plant cortical cells is important for P
transfer from the fungus to the plant (Harrison, 1999; Pumplin & Harrison, 2009).
Localized gene expression analysis indicates that the periarbuscular membrane is also
important for ammonium transfer from the fungus to the plant cells in arbuscular
mycorrhiza (Javelle et al., 2003; Gomez et al., 2009). From Lotus japonicus, a
mycorrhiza-inducible ammonium transporter LjAMT2;2, which is found located in the
apoplastic interfacial compartment, was suggested to bind charged ammonium and
release uncharged NH3 into the plant cytoplasm (Guether at al., 2009). Furthermore,
another mycorrhiza-inducible ammonium transporter GmAMT4.1 from Glycine max was
also found to be located in the branch domain of periarbuscular membranes (Kobae et
al., 2010).

What is the role of AM networks in the uptake and exchange of
nitrogen among plants in ecosystems?
Movement of N from soil to plants via AMF in plant ecosystems
The discovery of an elaborate system for moving N from soil to plant is
complemented by demonstrations under controlled experimental conditions of large N
fluxes through the symbiosis. In different experiments, at least 21%, 30% and 50 % of
the total N present in AM roots came from the fungal ERM in an in vitro mycorrhiza
system where mycorrhizal roots had access to N both by direct uptake and via the
fungus (Toussaint et al., 2004). Tanaka and Yano (2005) found that 75 % of N
measured in the leaves of mycorrhizal maize was taken up by the AM fungus’ ERM.
14

AMF can also obtain nitrogen from organic matter from the soil and transfer it to host
plants in significant quantities (Leigh et al., 2009; Hodge and Fitter, 2001; 2010).
However, by comparison with P, there are very few reports that directly demonstrate Ndependent enhancement of plant growth by AM colonization due to N transfer from the
fungus. In a study by Tanaka and Yano (2005), divided compartments in which nitrogen
was available in a compartment to which only the ERM had access were used. The
presence or absence of an air gap to prevent diffusion between compartments
significantly increased nitrogen contents in host plants when ammonium was used but
did not significantly increase plant biomass. The authors concluded from this and

15N

labeling results that the improved plant N content was due to direct N transfer by the
fungus from soil to plant. However, the apparent absence of N transfer when nitrate was
the form of N supplied, contradicts previous and subsequent reports (Jakobsen 1992;
Johansen et al., 1993; Tobar et al., 1995; Jin et al., 2005, this study) raising
uncertainties about the experimental system used. Although the N content of colonized
plants have been observed to be higher than un-colonized plants in a range of other
studies (reviewed in He et al., 2003; 2009), these have not distinguished N transfer by
the fungus from increased uptake by the plant secondary to improved P status or other
changes in the host and/or soil. Indeed, other studies have indicated that the transfer of
N does not always confer a net growth benefit to host plants. For example, Reynolds et
al. (2005) found that even at low N supply, AM colonization did not increase total N
uptake.
The extent and significance of N movement through the AM symbiosis under
natural conditions is even less clear - partly because of methodological difficulties.
15

Recently, the finding that mycorrhizal fungi discriminate against

15N

during nitrogen

transfer from soil to host plant has shown a potential application to track N flow in Nlimited ecosystems. The discrimination against
transfer will result in relatively enriched
rather than

14N

14N

15N

by mycorrhizal fungi during nitrogen

in plants rather than

15N,

while enriched

15N

in mycorrhizal fungi. Accordingly, the lower 15N:14N in mycorrhizal plants

compared with non-mycorrhizal plants suggests the contribution of N transferred from
fungi to host plants (Hobbie & Macko, 2000; Hobbie & Colpaert, 2003; Hobbie et al.,
2005). In addition, N transfer from fungi to host plants could be quantified by using
15N:14N

values for host plants, mycorrhizal fungi, and soil. However, even though this

approach has proved applicable for evaluating nitrogen transfer from fungus to ectomycorrhizal plants in N-limited ecosystems (Hobbie & Colpaert, 2003; Hobbie et al.,
2005; Hobbie & Hobbie, 2008), no reliable estimates of the contribution of AMF to plant
N contents have yet been made using this approach. This is partly due to uncertainties
of interpretation of such analyses and partly because the 15N abundance in AM plants is
closer to background than in nitrogen fixing or ectomycorrhizal symbioses. Further
improvements for the analysis and interpretation of isotopic discrimination results in
model AM systems and natural settings may enable estimates of the extent of N
movement from AMF to plants in a range of ecosystem types.

Colonization by AMF depends on the nutrient status of host plants
Indirect evidence for AMF having a role in N uptake under natural conditions
comes from the effect of soil N levels on colonization and growth of AMF. As reported

16

by Bago et al. (2004), the AM fungal morphological and developmental changes
subjected to different nutritional conditions, especially nitrogen status, as a strategy to
exploit the substrate efficiently. Low nutrient status including low nitrogen in the soil
induce the development and growth of AMF (Yoneyama et al., 2007), and AM
colonization rates have been shown to be related to N availability in natural and
disturbed ecosystems (Egerton-Warburton & Allen, 2000; Jackson et al., 2001; Jia et
al., 2004; Blanke et al., 2005). For example, Blanke et al. (2005) reported a negative
relationship between percentage root colonization by AMF and both tissue N
concentration and N:P ratio in Artemisia vulgaris growing in high P soils and Johnson et
al., (2003) found that N fertilization lowered AM colonization in grassland sites with low
N:P but not at sites with low P. This inverse relationship between AM colonization rates
and nutrient levels is well documented for P in the AM symbiosis and is also consistent
with the finding that development of N2-fixing nodules is reduced when N levels in the
soil are high (Streeter, 1985). This similarity is consistent with a beneficial role for AMF
when N is limiting. Although significant, the effects of N deposition on colonization are
generally not so marked as for elevated CO2 or P addition (Constable et al., 2001;
Treseder 2004; Gamper et al., 2005).
In an in vitro mycorrhizal symbiosis, Olsson et al. (2005; 2010) found that
increased availability of N or P to host roots reduced carbon allocation to the fungus and
concluded that negative impacts of N high nutrient level on AM abundance are caused
by reduced C allocation from the plant when plant requirements are met by direct
uptake. However, no colonization rates were reported in that work and it should be
followed up in more mycorrhizas. Other evidence suggests that the effects of soil N
17

levels on the colonization of AMF could result in part from the change of fungal
morphology which is influenced by the form and availability of inorganic nitrogen (Bago
et al., 1996). Furthermore, both root exudates and the growth and metabolism of
rhizospheric microorganisms which influence AM colonization are also likely to vary with
soil N (Gryndler et al., 2009). Thus, colonization rates may be due to both direct and
indirect effects on the plant and fungal partners making it unclear whether the
correlation between colonization and N availability is related to a beneficial role for AMF
in plant N nutrition. Direct investigation of the importance of plant N status on the
regulation of the symbiosis through analyses of exudate composition and plant defense
gene expression in response to AMF would be valuable. More recently, the turnover
rate of arbuscules, which is accelerated in plants lacking the AM-specific P transporter,
was shown to return to normal when N levels were limiting to plant growth (Javot et al.,
2011)
Thus, isotopic discrimination analyses, the effects of soil N levels on colonization,
and the increased N contents of AM plants all point towards a significant role for the
symbiosis in N uptake by plants. However, none of these lines of evidence is sufficient
to demonstrate unambiguously that N movement through the symbiosis provides a
direct growth benefit to host plants, especially under natural conditions. It is interesting
in this context that work on P nutrition has shown that a large fraction of plant P can be
taken up through AM fungal partners, even in cases when there is no plant growth
enhancement or even net increase in P uptake (Smith et al., 2003). This may be the
case for N more frequently than for P because the higher mobility of nitrate than

18

phosphate generally gives both partners access to soil N, where P depletion zones can
more easily restrict direct access to P by roots.

Nitrogen exchange between plants through mycorrhizal networks and its
ecological significance
Transfer of N among different plants via AMF has been reported in experimental
systems indicating that AMF can play an important role in N transfer between plants,
especially from legumes to non-legumes (Bethlenfalvay et al., 1991; Frey & Schuepp,
1993; Johansen & Jensen, 1996). Recent work using

15N

labeling and natural

abundance measurements has reported modest nitrogen transfer between plants via
common arbuscular mycorrhizal networks. For example, Jalonen et al. (2009) deduced
that 2.5% of the total N of grass was transferred by the common mycorrhizal network
from neighboring leguminous trees. Such studies (reviewed by He et al., 2003; 2009),
have resulted in estimates of nitrogen transfer between plants through mycorrhizal
networks, (especially from N fixing legumes to non-legumes) that range widely, between
0% and 80% of N in recipient plants coming from donor plants. AMF probably also
influence the allocation of N at the community level through effects on rhizospheric
functioning; plant growth, uptake and release of N; sequestration of N in soil organic
matter (Rillig et al., 2001); soil structure, affecting N mobility (Rillig, 2004; Wilson et al.,
2009); as well as transport through common mycorrhizal networks. The relative
importance of many of these factors in natural, disturbed and agricultural settings has
been reviewed by He et al. (2003). Because of the challenges of assigning the relative

19

importance of different mechanisms and the wide variation among findings no clear
consensus has yet emerged about the occurrence or significant direct plant-to-plant N
transfer via AMF in natural settings.

Interactions between AMF and symbiotic N2-fixing microbes and their
contribution to N accumulation in host plants
Nitrogen-fixing bacteria colonizing mycorrhizal plants play important roles in N
metabolism and movement in the symbiosis (Spriggs and Dakora, 2009). Figure 1-5
illustrates the network of nitrogen movement and recycling in ecosystems from a
mycorrhizal perspective. It has been found that the N2-fixing activity of the bacteria can
be improved when they are inoculated together with AMF because of the more available
nutrients for bacteria by AMF in mycorrhizal than non-mycorrhizal plants (Barea et al.,
1980; 2005). Indeed, AMF have been found in root nodules (Scheublin et al., 2004)
making it possible that nutrients are transferred between the symbionts without being
translocated within the plant vasculature.
Nitrogen transferred from symbionts to plants is believed to be predominantly in the
form of ammonium in both N2-fixing bacteria and AMF (Day et al., 2001; Rosendahl et
al., 2001; Govindarajulu et al., 2005, Jin et al. 2005), although uncertain amounts of Ala
and/or other amino acids can also be exported by bacteroids (Waters et al., 1998).
Photosynthate in the form of sucrose is the major form of carbon translocated from
source leaves to symbiotic roots, although neither AMF nor N-fixing bacteria utilize this
directly. Sucrose synthase seems to be involved in making C available to the micro20

symbiont in both arbuscular mycorrhizas and nodules - being activated in both tissues
(Hohnjec et al., 2003) - although the AM IRM takes up hexose (Shachar-Hill et al., 1995,
Solaiman and Saito, 1997) whereas bacteroids take up organic acids produced by their
host cells. Thus, N and C exchange with host plants by the two symbioses have
common metabolic intermediates, making it tempting to speculate about the relative
costs for plants of obtaining N from one or the other. The metabolic cost of converting
N2 to ammonium is one and a half reducing equivalents and eight ATP’s per N atom
versus four reducing equivalents for nitrate reduction and none for ammonium taken up
directly by AMF. Adding the costs of assimilating and then releasing ammonium in AMF
in the pathway described earlier and estimating the additional costs of long distance
translocation, appear to make N acquisition via nodules significantly less expensive
than via arbuscular mycorrhizas. However, a complete accounting should include the
respective costs of development and maintenance of the two symbioses, which is much
harder to do (Leake et al., 2004; Kaschuk et al., 2009).

Conclusions and perspectives
The transformation of inorganic nitrogen taken up from the soil into organic form
by the fungus and its translocation within the mycelium as arginine and subsequent
conversion to ammonium followed by the release of this inorganic N in colonized root
tissues seems to constitute the main pathway of nitrogen movement through the AM
symbiosis. Many of the molecular mechanisms involved in this pathway have been
identified in recent years lending support to the current model and providing detailed

21

information on genes and proteins involved. Future work in this area may be expected
to address significant remaining questions about the regulation, undetermined
components of the metabolism and transport machinery, and possible additional routes
of N movement in both plant and fungal partners. The work of this thesis contributes to
this effort.
AMF can increase the uptake of N by host plants from the soil under natural and
perturbed conditions and nutrient transfer from AMF can account for significant
proportions of N in plants in controlled model systems. Plant growth may be improved
by this N transfer although the extent to which this N-transfer-dependent growth
enhancement occurs outside the laboratory or greenhouse is unclear. Natural
abundance isotope fractionation studies indicate that some N is acquired by plants from
AMF and it is to be hoped that experts in this methodology will be able to obtain
quantitative estimates of N transfer. The effect of elevated soil N levels on suppressing
AM colonization rates is also indicative of a beneficial role for the symbiosis in plant N
acquisition. This implies control by the plant host through C allocation, for which there is
some support or via altered defense and/or signaling mechanisms which have not
apparently been explored.
The AM symbiosis can increase the exchange of N between plants and common
AM mycelial networks have been shown to contribute to this flow. The extent of this AM
mediated exchange seems to be quite variable and its ecological implications in natural,
agricultural and disturbed systems remain to be determined. More detailed analyses
that go beyond demonstrating exchange to quantify net transfer would be valuable in
this context. The interaction between AMF and N2-fixing bacteria can increase both N
22

fixation and colonization by AMF (Stancheva et al., 2008), and the interactions in the
tripartite association may play a role in the N cycle.
The uptake of N through AMF from the soil to the host plants and the exchange
of N between plants via the mycorrhizal networks have potential implications for the
application of AMF in sustainable agriculture. Horticultural and on-farm experiments
have shown that substantial benefits in yields can be realized from inoculation with AMF
(Johansson et al. 2004, Artursson et al., 2006). The extent to which N transfer is
important in these cases has been little studied, and it is generally believed that P
movement is more important. It may be that more attention to this question would
increase the range of settings where AMF are usefully applied. The interaction of AMF
and beneficial soil bacteria (both N fixing and non-N fixing) contributes to plant fitness
and soil quality, which has been argued to be important for a sustainable agricultural
development and ecosystems (Jeffries et al., 2003). Exploration of different
combinations of AMF and bacterial inocula in greenhouse and on-farm experiments
show promise for increased productivity (Zaidi et al., 2003). Optimal combinations will
depend on the particular soil, crop, and cultivation methods (Jeffries et al., 2003) and
finding them is likely to benefit from investment in systematically identifying AMF strains
in a wide range of different environments.

23

APPENDIX

24

APPENDIX

A

B

Figure 1-1 Arbuscular mycorrhizal fungal morphologies in tomato (Solanum
lycopersicum) roots plant.
A) Arum type morphology formed by Glomus mosseae, showing an intercellular hypha
(IH) and arbuscules (A) formed in adjacent host cells. B) Paris type morphology formed
by Glomus coronatum, showing a hyphal coil and growth through rather than between
adjacent plant cells. Bars indicate 160um. Modified from Caravagnaro et al., (2001).

25

Figure 1-2 Phylogeny of fungi based on SSU rRNA sequences. Thick lines delineate
clades supported by bootstrap values above 90%. Modified from Schussler et al,.
(2001)

26

Figure 1-3 Taxonomy of the Glomeromycota containing the arbuscular
mycorrhizal and related fungi, based on SSU rRNA gene sequences. Thick lines
delineate bootstrap support above 95%, lower bootstrap support values are given on
the branches. The four orders for the Glomeromycota are shown in tan. Many of the
classical, better studied AM species are in the order Glomerales. The families are
shown by pink ovals. Rhizophagus irregularis (previously Glomus intraradices) is in the
Glomus group A family. Modified from Schussler et al., (2001).

27

-

NO3

Glu
-

NO3

Putrescine
+

+

NH4

NH4

+

NH4

+

NH4

Glu

Orn

+

NH4

Gln

Arg
Urea
AA +

Arg
ERM

IRM
INTERFACIAL APOPLAST

SOIL
HOST

Figure 1-4 Working model of N transport and metabolism in the AM symbiosis.
Inorganic N is taken up by the fungal extraradical mycelium (ERM), the nitrogen is then
incorporated into arginine (Arg) in the urea cycle which is translocated to the fungal
intraradical mycelium (IRM) in colonized root tissues. Arg is broken down to release
ammonium which is exported from the fungus and imported by the host into the root
cortical cells. In addition, other forms of nitrogen may be transferred from the ERM to
the IRM and transferred to or exchanged with the host. Glu, glutamate; Gln, glutamine,
Orn, ornithine; AA, amino acid Modified from Tian et al., (2010).
28

Figure 1-5 Interactions among arbuscular mycorrhizal fungi, host plants, and
N2-fixing bacteria and other soil microbes in nitrogen cycling. arbuscular
mycorrhizal fungi take up inorganic nitrogen from the soil through the extraradical
mycelium, assimilate and translocate it into colonized roots where it is transferred to
the host. Nitrogen fixed by symbiotic N2-fixing bacteria in root nodules and nitrogen
taken up directly by the plant root itself from the soil also contribute to plant nitrogen
nutrition. N2-fixing and non-N2-fixing plants are connected by common mycorrhizal
networks, allowing exchange and net movement of N between plants.

29

Table 1-1 Nitrogen metabolic proteins and genes identified in the arbuscular
mycorrhizal symbiosis.

Proteins and/or genes
reported
Fungal amino acid permease
(GmosAAP1 from G.
mosseae)
Fungal nitrate transporter
(GiNT from R. irregularis)

Fungal ammonium
transporters (GiAMT and
GintAMT1 from R. irregularis)

Plant ammonium transporters
(MtAMT from M. truncatula;
LjAMT2;2 from Lotus
japonicus; GmAMT4.1 from
Glycine max)

Fungal (R. irregularis) and
Plant (Zea mays) Nitrate
reductases.

Potential function for N transfer in the AM
symbiosis
Amino acid acquisition by fungus from the
soil. Cappellazzo et al., (2008) characterized
a permease that facilitates the uptake of
proline and is induced in the presence of
several N sources.
Nitrate uptake from the soil. Transcript levels
upregulated by nitrate addition to the ERM
(Tian et al., 2010).
Ammonium uptake from soil and transfer to
host.
Two
fungal
AMT
ammonium
transporters reported: one is expressed more
highly in the IRM (GiAMT, Govindarajulu et
al., 2005) the other (GiAMT1, López-Pedrosa
et al., 2006) is induced in the ERM in
response to NH4+.
Ammonium/ammonia uptake by plant from the
host/fungus interface. Plant AMT transporters
are induced in mycorrhizal roots of three
species (MtAMT, Gomez et al., 2009;
LjAMT2;2, Guether et al., 2009; GmAMT4.1,
Kobae et al., 2010). LjAMT2;2 and
GmAMT4.1
were
localized
to
the
periarbuscular membrane of arbusculated
cortical cells.
Nitrate assimilation in mycorrhizal roots. The
mRNA level of maize NR was lower in roots
and shoots of mycorrhizal plants than in
noncolonized controls, and fungal NR
transcripts were localized in the IRM.
Suggests that the fungal nitrate reductase
assimilated nitrate in AM roots (Kaldorf et al.,
1998).

29

Table 1-1 (cont’d)

Fungal (R. irregularis) and
Plant (Zea mays) Nitrate
reductases.

Fungal glutamine synthetase
(GmGln1 from G. mosseae;
GiGln1 (same as GiGS1) and
GiGS2 from R. irregularis)
and glutamate synthase
(GiGluS from R. irregularis)

Plant (Daucus carota)
glutamine synthetase genes

Nitrate assimilation in mycorrhizal roots. The
mRNA level of maize NR was lower in roots
and shoots of mycorrhizal plants than in
noncolonized controls, and fungal NR
transcripts were localized in the IRM.
Suggests that the fungal nitrate reductase
assimilated nitrate in AM roots (Kaldorf et al.,
1998).
N assimilation in the ERM. Breuninger et al.,
(2004) identified GS homologs (GiGln1 and
GmGln1) in two fungal species expressed in
all AM tissues. Activity was elevated after N
addition to the ERM. Govindarajulu et al.,
(2005) reported induction of the R. irregularis
gene (GiGS1) in the ERM in response to N.
Gomez et al., (2009) reported the expression
of GiGS1 in mycorrhizal roots. Tian et al.,
(2010) found a second GS (GiGS2) in R.
irregularis and showed that the two GiGS
genes are differently upregulated by N
addition to the ERM and have different kinetic
properties.
Assimilation by plant of fungal-derived N. One
of three D. carota GS’s reported by Higashi et
al., (1998) is upregulated following the import
of N into mycorrhizal roots by the fungus (Tian
et al., 2010).

30

Table 1-1 (cont’d)

Fungal Arginine synthesis:
Carbamoyl-phosphate
synthase glutamine chain
(GiCPS); Argininosuccinate
synthase (GiASS);
Arginosuccinate lyase
(GiAL); (from R.
irregularis)

Fungal Arginine breakdown
Arginase (GiCAR1);
Urease accessory protein
(GiUAP); Urease (GiURE);
Ornithine
aminotransferases
(GiOAT1 and GiOAT2);
Ornithine decarboxylase
(GiODC); from R.
irregularis)

Arginine synthesis in the ERM. Gomez et al.,
(2009) reported the expression of GiASS in
mycorrhizal roots. Tian et al., (2010) showed
that these arginine synthesis genes are induced
in the ERM but not in the IRM after N addition
to the ERM.

N release in the IRM. Govindarajulu et al.,
(2005) reported higher expression of GiUAP in
the IRM than the ERM. Gomez et al., (2009)
showed transcription of R. irregularis arginase,
and GiOAT1 in cortical cells of mycorrhizal M.
truncatula. Tian et al., (2010) reported the
upregulation of GiCAR1, GiURE, GiOAT1,
GiOAT2 and GiODC in the IRM but not ERM
after N addition to the ERM.

31

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land plants. Mycorrhiza 16: 299-363.
Yoneyama K, Xie XN, Kusumoto D, Sekimoto H, Sugimoto Y, Takeuchi Y,
Yoneyama K. 2007. Nitrogen deficiency as well as phosphorus deficiency in
sorghum promotes the production and exudation of 5-deoxystrigol, the host
recognition signal for arbuscular mycorrhizal fungi and root parasites. Planta 227:
125-132.
Zaidi A, Khan MS, Amil M. 2003. Interactive effect of rhizotrophic microorganisms
on yield and nutrient uptake of chickpea (Cicer arietinum L.). European Journal of
Agronomy 19: 15-21.

44

Chapter 2
Nitrogen uptake and transfer in an aseptic whole plant-mycorrhizal
culture system

45

Abstract
Arbuscular mycorrhizal fungi (AMF) can increase the uptake of N by their host
plants and play a significant role in the transformation and movement of N in plant
communities. It is widely assumed that N transfer to host plant roots, which has been
observed in tracer experiments, can improve host plant growth, but no unambiguous
demonstration of this has been reported. To test whether N uptake via an AM fungus
can enhance plant growth and fitness, a compartmented microcosm was developed
for aseptic and leakage free whole-plant mycorrhizal experiments. This was used to
monitor S uptake by the fungal extraradical mycelium (ERM) and its transfer to host
plants. Our results indicated rapid S transfer by ERM to the host plants as indicated
by high

35S

levels in roots and shoots. To assess the contribution of the AM fungal

mycelium to plant nitrogen nutrition, N was added to the fungal compartment of
mycorrhizal plants. Controls were used to ensure that N transfer was responsible for
any benefits (mycorrhizal plants with P but without N addition to the fungal
compartment) and that transfer was entirely via the fungus (non-mycorrhizal plants
with N added to the empty compartment). Biomass, shoot length, number of pods,
chlorophyll content, and

15N

labeling were used to investigate N transfer from the

fungus as well as nutritional benefits to the plant. Mycorrhizal plants with N available
to the fungal ERM had higher weight, longer shoot length, higher number of pods as
well as higher chlorophyll content than the controls which showed N deficiency
symptoms. Furthermore, mycorrhizal plants with N had high levels of, and fractional
15N

labeling in, N metabolites (glutamine, glutamate, pyrroline-5-carboxylate,

ornithine and arginine) in both roots and shoots while the control plants showed no
labeling and low level of those metabolites. We conclude that N transfer from an
AMF confers growth benefits on the host plant under nitrogen limiting conditions and
46

that the microcosm system developed will be useful for future work on AM nutrition
and metabolism under physiologically relevant conditions.

47

Introduction
Plant-microbial mutualistic associations play an important role in global
nutrient cycles as well as in the ecology and physiology of plants (Read and PerezMoreno, 2003). Plant growth depends on mineral nutrients whose availability is
frequently limiting. Phosphorous and nitrogen availability most commonly limits
plant growth in ecosystems (Reich et al., 2006). Hayman and Mosse, (1971) first
documented that the arbuscular mycorrhizal (AM) symbiosis can increase plant
growth in P-deficient soils dramatically.
Plants gain several benefits from AMF. For example AMF take up and transfer
to their host nutrients including phosphorus, nitrogen (Govindarajulu et al., 2005),
sulfur (Allen and Shachar-Hill, 2009), and zinc (Clark & Zeto, 2000), improve drought
resistance (George et al., 1992), protection from pathogens has also been
demonstrated

(Newsham

et

al.,

1995).

In

return,

plants

translocate

photosynthetically fixed carbon to the fungal symbiont (Smith and Read, 2008).
While P is generally believed to be the most important nutrient taken up by plants via
AM networks, other macronutrients can be moved in large amounts via AMF. After it
was shown that mycorrhizal colonization increased plant

35S

uptake compared to

nonmycorrhizal plants (Rhodes and Gerdemann, 1978; Gray and Gerdemann,
1973 ), Allen and Shachar-Hill, (2009) reported that 50% of

35SO 2
4

uptake by the

fungus was detected in the mycorrhizal roots of an in vitro mycorrhizal system.
Similar quantities of 35S were transferred to mycorrhizal roots whether 35SO42−,
[35S]Cys, or [35S]Met was supplied in the fungal compartment (Allen and ShacharHill, 2009).

48

The AM fungal hyphae take up N from the soil in different forms and transfer it
to the plant, however nitrogen is mainly captured by the AM extraradical mycelium in
inorganic form (i.e. as nitrate or ammonium) (Bago et al., 1996; Hawkins et al., 2000;
Azcon et al., 2001; Hodge et al., 2001; Vazquez et al., 2001). Although nitrogen
transfer from AMF to the host plant has been demonstrated (Ames et al., 1983;
Johansen et al., 1993; Hawkins et al., 2000; Azcon et al., 2001; Hodge et al., 2001;
Vazquez et al., 2001) and a working model of the metabolism and transport
processes has been proposed (Bago et al., 2001) and supported ( Jin et al., 2005;
Cruz et al., 2007; Tian et al., 2010), nitrogen transfer has never been rigorously
demonstrated to confer a growth benefit. Indeed, much of the work in recent years
has been on the molecular mechanisms and on ecological aspects in the field. A
monoxenic culture system consisting of transformed roots in symbiosis with the
mycorrhizal fungus R. irregularis has been used to study the transfer and
metabolism of nitrogen (Govindarajulu et al., 2005; Jin et al., 2005; Tian et al., 2010).
This transformed root system was used because it is aseptic and provides easy
access to the ERM. However, it is not a whole plant system, which casts doubt on
the relevance of the plant metabolic results obtained. Unlike phosphate, which is
immobile in soils (Gahoonia and Nielsen, 1991) inorganic nitrogen is mobile in most
soils which makes it hard to determine the role of nitrogen transfer via AMF on plant
growth and reproduction.
In order to test whether nitrogen transfer confers a nutritional benefit in
plants, an aseptic and leakage –free system is required which will prevent diffusion
between compartments and which minimizes physical restrictions on plant growth
that are typically seen in closed systems. Such a system should allow rapid and
efficient fungal mycelial growth into a fungal compartment separated from the growth
49

zone of the colonized plant roots. This is important so that nutrients can be made
available to the fungal extraradical mycelia (ERM) without the plant having access to
them other than the fungus.
Previous studies reported compartmented pot systems based on the same
concept of having a root and fungal compartments (Ames et al., 1983; Frey and
Schuepp, 1992; Ma¨der et al., 1993; Schweiger and Jakobsen, 2000; Smith et al.,
2000; Jansa et al., 2003; Smith et al., 2003). Several researchers have used
different variants of the pot system to demonstrate nutrient transfer in arbuscular
mycorrhizal symbionts. Johansen et al., (1993) used containers divided by a fine
nylon mesh into a root compartment (RC) and a root-free hyphal compartment (HC)
to demonstrate phosphorous nutrient transfer between Subterranean clover
(Trifolium subterraneum L. cv. Nuba) and R. irregularis. Nylon mesh was used also
in a container system to separate the main soil compartment from the fungal
compartment in experiments studying P acquisition by two AMF and its transfer to
host plants (Smith et al., 2000; Hodge et al., (2001) found that the arbuscular
mycorrhizal symbiosis can enhance the decomposition of, and increase nitrogen
capture from, complex organic material in soil. They grew plants of Plantago
lanceolata inoculated with the mycorrhizal fungus G. hoi in microcosms in which the
compartments were separated by a double layer of 20-µm mesh, which was
permeable to hyphae but not roots. In studying nitrogen and phosphorous transfer
and regulation, Fellbaum et al., (2014) used a double membrane with an air gap (two
sheets of 50-μm nylon mesh with a wire spiral between then) to prevent the diffusion
of nutrients from the fungal compartment (FC) to the root compartment (RC), but
allow fungal hyphae to cross from the RCs into the FCs has been used.

50

All of these systems have some drawbacks such as lack of control over the
presence and growth of other microorganisms which could influence element bioavailability, especially when dealing with organic compounds, and whose levels and
behavior is likely to be influenced by the fungal ERM. In other systems without
complete isolation via an uninterrupted air gap, diffusion of mobile nutrients or
tracers allows direct uptake by the roots due to leakage into the root compartment
caused by diffusion or mass-flow due to transpiration. Another system type was used
by Dupre´ de Boulois et al. (2006), an arbuscular mycorrhizal–plant (AM–P) in vitro
culture system. In this system, the root compartment and hyphal compartments
consist of a bi-compartmented Petri plate and a shoot compartment consists of a 50
ml Falcon tube and a membrane filter fixed onto the shoot compartment to allow gas
exchange. This system is aseptic but the drawbacks are media drying and
substantial physical restrictions on plant growth. Our aim in this study was to
overcome the limitations of previous systems by developing an aseptic, leakage-free
system that allows longer and more physiologically relevant experiments in a reusable, inexpensive microcosm of modest footprint, with separate reservoirs for
supplying nutrients and water independently to the two compartments with sufficient
capacity for multi-week long experiments.
Here we report such a system. This system was used to investigate growth
parameters, chlorophyll content and nitrogen metabolite 15N labeling and levels when
N was made available to the fungal ERM at N levels that limited plant growth and
reproduction. We observed that nitrogen taken up by the fungal extraradical
mycelium and transferred to the plant accounts for much of the nitrogen entering the
plant under N-limited conditions and confers growth and reproductive benefits.

51

Experimental procedures
Chemicals and reagents
Gelzan, (MP Biomedical, Solon, OH) was used for solidification of the minimal
(M) media. Radioactive labeled sulfate was obtained as Na2
Biomedicals, (Solon, OH).

15N-Labeled

35SO

4

from MP

nitrate was obtained as K15NO3 from (MP

Biomedicals, (Solon, OH)).

Seed sterilization
Medicago truncatula seeds were scarified with concentrated sulfuric acid for
5-10 minutes. After that, seeds were rinsed with sterile water 4-5 times. Surface
sterilization was done in concentrated Clorox for 2 minutes, then seeds were rinsed
8 times with water.

Growth conditions
Seeds were germinated at 22.5 °C for 48 hours on M-medium (Fortin et al.,
2002) following cold treatment at 4 °C for 36 hours. After that, M. truncatula
seedlings and old fragments of Daucus carota roots (Ri T-DNA transformed)
colonized with R. irregularis were transferred to the root compartments filled with
bacto:perlite (2:1) soils in the two-compartment system (Figure 2-1). Alternatively,
the fungal compartment was filled with medium grain sand. This system was
constructed using 50 µm woven double mesh glued to metal frame with an air gap to
separate the two compartments and prevent the crossing of roots, yet allow fungal

52

crossover into the fungal compartment (RCs). The lower compartments were used
as reservoir to supply both compartments with 0.5x Hoagland’s solution (Hoagland
and Arnon, 1950). Linen cotton rope wicks were used for capillary transfer of
Hoagland’s solution to both compartments.
The plants were grown in a growth chamber under the following conditions:
16 h photoperiod, 22.5°C, photosynthetically active radiation of 200 µmol m-2 s-1, and
30% humidity.

Experimental design
Three experiments were conducted to demonstrate the role of arbuscular
mycorrhizal in nutrition transfer. Whole plant two compartments system were used to
test the transfer of

35SO
4

from AMF to the host plants. After growing plants for 5

weeks, plants were deprived of sulfur for a week, then 100 µCi of

35SO

4

was added

directly to the fungal compartment for a week. Plant leaves were sampled 1, 3, 5, 7
days after addition of

35SO

4.

Plant shoots and roots were collected to measure

radioactivity. Two controls were used, mycorrhizal colonized plants with no
added and non-mycorrhizal plants with

35SO

4

35SO

4

added to FC to test leakage. Five

biological replicates were used for each.
The second experiment was conducted to demonstrate the nutritional benefit
of arbuscular mycorrhizal colonization on N transfer. After growing plants for five
weeks, plants were deprived from N for 4 days then 10 mM KNO3 was added to the
reservoir of fungal compartment for two weeks. Plant shoots and roots were
collected for growth metrics and chlorophyll measurements. Two controls were used,

53

mycorrhizal colonized plants with no N added and non-mycorrhizal plants with 10
mM KNO3 added to the FC to test leakage. Total of 10 biological replicates in two
different experiments were used.
The third experiment was done to test the uptake and transfer of

15N

in the

whole plant system. After growing plants for 5 weeks, plants were deprived from N
for 4 days then 10 mM K15NO3 was added to the reservoir of the fungal compartment
for two weeks. Plant shoots and roots were collected for N metabolite analysis. Two
controls were used, mycorrhizal colonized plants with no

15N

added and non-

mycorrhizal plants with 10 mM K15NO3 added to FC reservoir to test leakage. Total
of 10 biological replicates in two different experiments were used.

Testing the diffusion between compartments and the capillary transfer
In order to make sure that there was no diffusion of nutrients from the FC into
the RC,

35SO
4

was added to FC reservoir, Aliquots of the soil and the reservoir

solution of the RC were collected (3, 5, 7 days after adding

35SO

4).

Aliquots of the

sand of FC were collected to investigate the rate of nutrients transfer using linen
cotton rope wicks. The

35S

content was extracted from all samples and measured by

liquid scintillation counting.

Extraction and measurement of 35SO4 from mycorrhizal plants
Plant leaves were sampled 1, 3, 5, 7 days after addition of

35SO
4

to the FC.

Also plant roots and shoots were sampled after 7 days. 100 mg leaves of each time
point as well as 100 mg of each plants roots and shoots(stems and leaves) were
54

ground in a mortar and pestle with a 100mg acid washed sand and extracted three
times with a mixture of cold methanol : water (70 : 20). then the solution were
vortexed for 5 min. While keeping particulates suspended, a 1-mL aliquot of the
solution was transferred to a microcentrifuge tube and centrifuged and 0.5 mL of the
supernatant solution was counted in scintillation counter after adding to 5 mL of
BioSafe II (MP Biomedicals) scintillation cocktail.

Analysis of mycorrhizal colonization and ERM crossing to the FC
Fraction colonization were checked for plants 1, 2, 3 and 4 weeks after
transferring the seedlings with the spores to the system. Three biological replicates
were used for each time point. Thirty root fragments of each biological replicates
were stained with trypan blue and the percentage root length colonized by R.
irregularis was estimated using the gridline intersect method (Newman, 1966). In
order to check crossing, ERM from the FCs of 5 week old plants were collected,
cleared in KOH, and stained with Trypan blue.

Growth parameters and chlorophyll content measurements
Experimental plants were collected two weeks after adding KNO3 to the FC
reservoir. The two control plants were collected. The plants were weighed and the
length of the shoots were measured. The fruits were collected and counted. Leaf
chlorophyll

was

extracted

using

80%

spectrophotometer according to Ni et al., (2009).

55

acetone

and

quantified

using

Extraction, isolation and quantification of 15N metabolites
Three hundred mg of each plants roots and shoots were ground in a mortar
and pestle with a 100mg acid washed sand and extracted three times with a mixture
of methanol : chloroform : water (12 : 5 : 3, v/v/v). Methylene chloride and water were
added to the extraction solution to facilitate the separation of chloroform and the
methanol–water phases. The methanol–water phase containing the amino acids
(AAs) was collected and evaporated in a rotary evaporator at 50°C, and the residues
containing the AAs were dissolved in 1 ml of 0.01 M HCl and loaded onto a cation
exchange column (0.3 ml of DOWEX 50 X8-200– hydrogen form; Sigma-Aldrich, St
Louis, MO, USA), which was previously washed with 1 M NH4OH, deionized H2O
and 1 M HCl, and followed by deionized H2O. The neutral compounds, principally
carbohydrates washed off the column with 5 ml of water and the free amino acids
were eluted with 5 ml of 1 M NH4OH (Bengtsson & Odham, 1979). This eluent was
collected and dried then resuspended in 70 µl of Milli-Q water. N metabolite levels
and labeling were measured using Liquid chromatography (LC)-MS analyses.

Liquid chromatography and mass spectrometry (LC-MS)
A Waters (Milford, MA) Quattro micro mass spectrometer coupled to a
Shimadzu (Columbia, MD) LC-20AD HPLC system and SIL-5000 autosampler was
used. A Waters Symmetry C18 column (2.1 × 100 mm, 3 μm particle size) was used
with column oven temperature at 30 °C. The injection volume was 10 μL, and the
HPLC flow rate was 0.3 mL/min using 1 mM perfluroheptanoic acid in a
water/acetonitrile (A/B) gradient at ambient temperature, The intial gradient

56

(A/B)=99/1, held until 2 minutes, followed by a linear gradient to 60/40 at 4 minutes,
held at 60/40 until 8 minutes, followed by a ramp to 99/1 at 8.01
Mass spectra were acquired using electrospray ionization in positive ion mode
and MRM. The capillary voltage, extractor voltage, and rf lens setting were set at
3.17 kV, 4 V, and 0.3, respectively. The flow rates of cone gas and desolvation gas
were 20 and 400 L/h, respectively. The source temperature and desolvation
temperature were 110 and 350 °C, respectively. Collision-induced dissociation
employed argon as collision gas at a manifold pressure of 2 × 10-3mbar, and collision
energies and source cone potentials were optimized for each transition using Waters
QuanOptimize software. This method was composed of two ESI+ functions (0−1.8
and 1.8−6.0 min) covering full run time to allow for adequate dwell time for each
analyte. Data were acquired with MassLynx 4.0 and processed for calibration and for
quantification of the analytes with QuanLynx software. Table (2-1) of precursor and
product ions for each compound is given in appendix.
To determine concentrations, standard curves were run for authentic
unlabeled standards of each compound in the table above with concentrations range
from 0 to 100 μM together with 10uM Phenylalanine-D8 as internal standard, which
was also added to each biological sample during extraction to control for recovery
efficiency and as internal standard. Integrated peak areas were obtained from
daughter ion chromatograms using the MassLynx 4.0. Total levels for each
compound were obtained from the sum of all the labeled and unlabeled molecules.
For 15N labeled samples, each product ion mass isomer for each precursor
mass isomer was quantified. Thus for example for 15N labeling in Arginine the
precursor ions have mass-to-charge ratios (m/z) of 175 (15N0 = M+0), 176 (15N1 =

57

M+1), 177 (15N2 = M+2), 178 (15N3 = M+3), and 179 (15N4 = M+4) with the product ion
CH6N3+ have m/z of 60 (15N0 = M+0), 60 and 61 (15N1 = M+1), 61 and 62 (15N2 =
M+2), 62 and 63 (15N3 = M+3), and 63 (15N4=M+4) (see Table 2-1 and 2-2) .

Results
To establish conditions for studying N transfer between the AM fungus and
the host plants, a whole plant mycorrhizal two-compartment culture system was
developed (Figure 2-1). The final model has three compartments. The top
compartment allows room for shoot growth and is separated from the outside
atmosphere using a 0.22 µM pore size breathe-EASIER membrane across the entire
top of the microcosm to allow for gas exchange, including water vapor, and to
maintain the sterility of the system. The central compartment contains soil or other
solid medium for root and fungal growth. This is divided into two subcompartments: a
soil and a sand sub-compartment separated by two layers of metal woven mesh
(Dutch weave 40 µm exclusion size) that are glued together with autoclavable epoxy
to a metal frame. The divider is fixed vertically in the middle of the central
compartment using autoclavable silicone rubber. This creates a robust air gap of
~1.5mm and an area for hyphal penetration that spans most of the area of the
divider. A seedling is inoculated with R. irregularis spores and grown in the soil
compartment. The woven mesh was found to allow the fungal ERM to cross from
plant to fungal compartment at a high frequency while preventing plant roots from
doing so. By sticking two stiff mesh sheets to a metal frame to form the divider, the
air gap consistently prevented diffusion of nutrients between compartments, which
occurs for diffusible nutrients or tracers when single mesh barriers are used. To
58

create air gaps, flexible double mesh barriers were tested with smaller area dividers
to prevent the mesh sheets from touching. These had low hyphal connectivities
between compartments. Using a course mesh as a spacer between the rootexcluding mesh layers can facilitate diffusion between compartments by forming a
continuous diffusion pathway. The design shown in Figure 2-1 prevents leakage
between compartments as indicated by the absence of

35

SO42- in the plant

compartment when this diffusible tracer was added to the distal compartment of nonmycorrhizal plants (Figure 2-2A). The third, lower compartment contains liquid
reservoirs to supply the root and fungal compartments with water and nutrients via
wicks. The reservoirs in the lower compartment were separated by attaching a 50100 mL glass beaker to the floor using autoclavable silicone gel under the fungal
compartment (FC) while the rest of the chamber was used as a root compartment
(RC) reservoir. In order to supply each compartment of nutrients from reservoirs,
different kinds of ropes were tested as wicks. Some of them allowed too little
capillary transfer capacity, resulting in water stress for the plants while other
materials allowed too much transfer, saturating the RC and FC and causing
diffusional tracer leakage between compartments and excessive soil moisture that
reduced plant growth. Linen cotton rope wicks were found to provide efficient but not
excessive transfer of water and nutrients as indicated by rapid

35SO 24

movement

into the fungal (sand) compartment (Figure 2-2A) without leakage into the root
compartment or soil water saturation. In order to eliminate complications arising from
the uptake and metabolism of nitrogen, carbon, or other tracers by other
microorganisms, the maintenance of aseptic conditions over several weeks was
required. By selection of appropriate materials for wicks, dividers, and adhesives, the
microcosms can be autoclaved after assembly, with the easy breath membranes

59

being added under sterile conditions (laminar flow hood). The sterility of the system
was investigated using periodic sampling of all compartments and inoculation onto
enrichment media (trypticase soy agar and potato dextrose agar) for bacteria and
fungi. This culture system was found to be reproducibly aseptic with no growth of any
microbes seen on the rich media throughout the time frame of the experiments.
The level of colonization per root length of M. truncatula plants (Figure 2-3)
reached approximately 90% for internal hyphae and 62% for arbuscules and vesicles
in four-week old plants. These high and reproducible colonization levels and
establishment rate (most plants were substantially colonized within a week) allow
observations of nutrient exchange between the fungal mycelium and the plant roots
to be interpreted without uncertainties due to variable or slow colonization rates.
We used the culture system described above to monitor S uptake by the
fungal extraradical mycelium and its transfer to host plants. By supplying

35SO

4

to

the FC, It was found that leaves had measurable 35S after one day and reaching over
60 fmol/g (FW) counts after 7 days (Figure 2-2B). Furthermore,

35S

levels were

measured in whole plant roots and shoots after 7 days and compared with two
controls (mycorrhizal plants with no
plants with

35SO

4

35SO

4

added to the FC and non-mycorrhizal

provided to the FC) (Figure 2-2C) . High

shoots were found. However, plant shoots had higher

35S

35S

levels of roots and

level than roots. The two

control roots and shoots showed no counts above background (comparable with the
background counts) proving that this culture system had no leakage between
compartments.
By adding nitrogen to the fungal compartment reservoir (FCR) of the
mycorrhizal plants, it was found that nitrogen transfer confers growth benefit as

60

plants were larger and greener with multiple stems compared with the other two
controls (mycorrhizal plants with nutrients but no nitrogen added to the FCR and
non-mycorrhizal plants with N provided to the FCR, see Figure 2-4). In the two
controls, plants stopped growing and senesced. Shoots dried out and had low
numbers of leaves as many dehisced with most of the residual leaves were visibly
chlorotic (a symptom of nitrogen deficiency, Figure 2-4). Chlorophyll content of
mycorrhizal plants (2-5) with nitrogen supplied to the FCR were significantly higher
than for plants from the two control treatments (p<0.01). In the two controls, no
nitrogen was available to the plant through the fungus and this presumably led to
chlorophyll degradation, and/or reduced biosynthesis (Figure 2-5A). Mycorrhizal
plants with nitrogen added to their fungal compartment reservoirs had significantly
longer shoots, higher biomasses and higher numbers of fruits compared with the two
control treatments (Figure 2-5 B,C, and D) (p<0.01). Mycorrhizal plants with nitrogen
added to the fungal compartment reservoir had significantly higher levels of the
soluble

nitrogen-containing

metabolites

glutamate,

glutamine,

carboxylate, ornithine and arginine (p<0.05) as well as a significant

15

pyrroline-5N percentage

labeling in both root and shoot tissues (p<0.01) . This confirms that nitrogen transfer
from the fungal compartment via the fungal mycelium drives substantial fluxes
through N metabolism (Figure 2-6). The control plants showed low levels of N
metabolite and no detectable

15N

labeling, consistent with the

35S

results that

indicated no significant nutrient movement between compartments not connected by
fungal mycelium. Mycorrhizal plants with P and other nutrients but not N supplied to
the FCR had similar N metabolite levels and growth parameters to non-mycorrhizal
plants. Thus, the nutritional benefit to plants is due to direct fungal-mediated N
transfer. The high percentage labeling of intermediary metabolites in shoots as well

61

as roots demonstrates directly, in a way that the use of transformed roots cannot,
that N transfer to mycorrhizal roots benefits the N status of the whole plant.

Discussion
Many of the detailed studies of mycorrhizal metabolism and nutrient transfer
(e.g. Bago et al., 1996, Jin et al., 2005, Govindarajulu et al., 2005) were performed
using transformed roots cultured on bicompartmental petri plates as the model
mycorrhizal system (St. Arnaud et al., 1996). This system was used because of ease
of handling, maintenance of sterility, the advantages of defined media and ease of
ERM tissue isolation. However, these studies did not investigate nutrient transfer in
whole-plant mycorrhizal systems which are likely to have different physiological
characteristics and regulatory dynamics.
The studies of N nutrition and metabolism in the AM symbiosis that have been
reported using whole plants were not conducted under aseptic conditions (e.g.
Johansen et al., 1993, Hodge et al., 2001; Fellbaum et al., 2014). Although such
studies have been important in establishing for example the contribution of AMF to
the mobilization of soil N (Tobar et al. 1995, Hodge et al. 2001) and are
physiologically substantially more realistic than transformed root systems, the
impractibility of maintaining sterility complicates the interpretation of metabolic and
translocation experimental results. In this study, we developed a sterile and leakagefree (Figure 2-2A) whole plant two-compartment culture system (Figure 2-1) allowing
the exclusion of other microbes and avoidance of diffusion between compartments.
Low cost, easy to use, reproducible and autoclavable, these microcosms make
feasible investigations of mycorrhizal transport, gene expression, metabolism and
62

nutrition that have hitherto been challenging or impossible. In particular, the sterility
of this system will enable the study of the role of organic C, P, N, and S compounds
in AM symbiosis. The culture system provided M. truncatula plants with optimized
levels of nutrients and water, including low phosphorous levels to stimulate
colonization. Within two weeks of seedling planting, the plants were associated with
higher levels of colonization and numbers of arbuscules (Figure 2-3) than is
commonly observed, and significantly, also with well-developed external hyphal
mycelia of R. irregularis. This allows experiments on uptake and transfer to be
conducted on plants that, while mature, are not senescent or growth-limited by
physical space constraints.
The uptake and transfer of 35SO42- by the fungal partner to the host plant was
demonstrated in the microcosms (Figure 2-2B) with
host plants after one day. Shoots had higher

35S

35S

detected in the leaves of

concentration than roots (Figure 2-

2C) which is consistent with the fact that plants transport sulfate to the aerial parts,
where the majority is stored as a vacuolar sulfate pool or metabolized in reductive
sulfur assimilation (Kataoka et al.,2004).
We conclude that sulfate is transferred by R. irregularis to host plants, as has
been reported for AM transformed roots (Allen and Shachar-Hill, 2009). Allen and
Shachar-Hill (2009) demonstrated the transfer of sulfate by R. irregularis to
transformed roots and found that the fungus can uptake and transfer reduced forms
of S at rates comparable to sulfate. Since 95% of S is in organic form in soil
(Tabatabai, 1986; Scherer, 2001), this observation points to a wider role for AMF for
S plant nutrition in nature. This example also highlights the need for aseptic whole
plant AM experiments to determine whether this capacity may be significant for S
nutrition of AM plants.
63

It has been reported that AMF are able to substantially increase the uptake of
N by host plant roots (Ames et al., 1983; Johansen et al., 1993; Bago et al., 1996;
Johansen et al., 1996; Tobar et al., 2004; Hodge et al., 2010). Nevertheless, the
literature lacks a demonstration of growth or reproductive benefits to plants of N
transfer from AMF. The microcosm system developed in this study provides
sufficient room for plant growth with minimal physical restriction, sufficient gas
exchange, and high rates of hyphal crossing to examine this important question. We
observed that N transfer by AMF conferred growth and reproductive benefits to the
host plant.
After supplying the fungal compartment with N, the mycorrhizal plants
appeared healthier, greener and larger than the controls. Mycorrhizal plants with N
had longer shoots, higher biomass and increased chlorophyll contents compared to
the controls. In contrast, control plants showed N deficiency symptoms. N deficiency
in plants results in a breakdown of chlorophyll (Gaude et al., 2007) and also affects
the abundance of thylakoid membranes in chloroplasts (Malavolta et al., 2004).
Furthermore, N deficiency causes severe consequences for N and C metabolism
(Wang et al., 2003).
15N

labeling and concentrations of N metabolites (glutamine, glutamate,

pyrroline-5-carboxylate, ornithine, and arginine) showed that

15N

taken up by the

ERM in the FC arrives in significant amounts (p<0.01) at the roots and shoots of host
plants (Figure 2-6). Control plants have low levels of N metabolites and no

15N

labeling, proving that there is no diffusion between compartments. It has been
reported that N deficiency affects the abundance of amino acids in plants (Scheible
et al., 2004).

64

The high abundance of N metabolites and
mycorrhizal plants after adding

15NO

3

15

N percentage labeling in

coincides with N transfer (model in which

Inorganic N is taken up by the fungal ERM, and assimilated by GS-GOGAT system,
raising the levels of glutamate and glutamine then nitrogen is incorporated into
arginine in the urea cycle which is translocated to the fungal IRM in colonized root
tissues and then broken down into ornithine and urea that is in turn is broken down
to release ammonium which is exported from the fungus and imported by the host
into the root cortical cells. Plants then assimilate ammonium to produce free amino
acids (Tian et al., 2010) and as a consequence, the

15N

labeled metabolites in the

root are in fungal IRM and plant roots. The presence of high levels of

15N

labeled

metabolites in shoots indicated a significant amount of N being transferred to the
host plants.

65

APPENDIX

66

APPENDIX

A

B
0.22um gas permeable

Double mesh
barrier with air

Fungal
compartmen

Wick

FC
Reservoir

Plant
compartment

Wick

RC
Reservoir

Figure 2-1 Whole plant-mycorrhizal two-compartment culture system.
A) Diagrammatic representation of the culture system that is composed of two
compartments: root (RC) and fungal compartments (FC). RC and FC are separated
by double woven mesh glued to metal, thus air gap is created between the two
layers of mesh. The upper compartment is sealed with breathe-EASIER membrane
and the lower compartments are used as reservoirs; root compartment reservoir and
fungal compartment reservoir. Nutrients were transferred using linen cotton wicks. B)
Four-week old Medicago truncatula plant growing in the system.

67

A
30

0.1

d

A

Fungal
compartment

0.09
25

0.08
Root
compartment

0.07
20

15

fmol/g

fmol/g

0.06

c

Root
compartment
reservoir

0.05
0.04
0.03

10

ee ee eeee

0.02
5

a

b

0.01
0
1 day

0
1 day

3 days

5 days

7 days

5
7
3
days days days

Figure 2-2 Sulfur transfer from fungus to plant using the whole plant culture
system.
A) 35SO4 concentration (fmol/g) in root compartment soil and reservoir at the days 1,
3, 5, and 7 days after applying 35SO4 to fungal compartment reservoir to investigate
diffusion between compartments. 35SO4 concentration in fungal compartment sand
wa measured to investigate the rate of transfer of nutrients by the cotton wicks. The
right panel is the same figure as the left but with lower maximum limits to show
smaller values. Values are reported as mean ± SEM (n=3 biological replicates). The
transfer of 35SO4 was significantly increasing with time (ANOVA single factor analysis
(alpha=0.05). the 35SO4 concentration in root compartment soil and reservoir was
comparable with the background.
B) 35S concentration (fmol/g (FW)) in leaves at 1, 3, 5, and 7 days after applying
35SO4 to Fungal compartment. Values are reported as mean ± SEM (n=5 biological
replicates).
C) 35S concentration (fmol/g (FW)) in whole plant shoots (above ground stem and
leaves) and roots at 7 days after applying 35SO4 to Fungal compartment. Values are
reported as mean ± SEM (n=5 biological replicates).
Statistical analyses were done by ANOVA single factor analysis, alpha = 0.05.
Letters (a, b, ...) above the bar graphs designate statistically significant difference
between means.

68

Figure 2-2 (cont’d)

B

d

80
70

fmol/g (FW) leaves

60
50

c

40
30

b
20

a
10
0
1 day

3 days

5 days

69

7 days

Figure 2-2 (cont’d)

C

110

b

100
90
80
70

a
60

fmol/g (FW)

50
40
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2

c

0.1

c

c

c

Roots

Shoots

0
Roots

Shoots

Myc plants + 35S

Roots

Shoots

nonmyc plants
+35S

myc plants -35S

70

A
b

1
0.9

b

Fractional Colonization
(internal hypha)

0.8
0.7
0.6

a

0.5
0.4
0.3
0.2
0.1
0

2wks

3wks

4wks

B
0.8

b

Fractional colonization
(Arbuscules and vesicles)

0.7

b

0.6
0.5

a

0.4
0.3
0.2
0.1
0
2wks

3wks

4wks

Figure 2-3 Colonization of Medicago truncatula by R. irregularis. Plants were
raised in the two-compartments system under low phosphate and in the presence of
old Daucus carota colonized roots. Values are fraction of root length associated with
(A) internal hyphae and (B) arbuscules and vesicles. Values are reported as mean ±
SEM (n=3 biological replicates (30 technical replicates)). Statistical analyses were
done by ANOVA single factor analysis, alpha = 0.05. Letters (a, b, ...) above the bar
graphs designate statistically significant difference between means.

71

Non-myc plants

Myc plants +N

Myc plants –N

Figure 2-4 Seven-week old Medicago truncatula plants growing in the twocompartment system. Mycorrhizal plants with KNO3 (Myc plants +N) added to the
fungal compartment reservoir for two weeks. Two controls were used; nonmycorrhizal (non-myc) plant with KNO3 added to fungal compartment reservoir to
check for leakage and mycorrhizal plants with no N added (Myc –N).

72

A

0.9
Total chlorophyll content (mg/g)

0.8

a

0.7
0.6
0.5
0.4
0.3

b

0.2

b

0.1
0

Figure 2-5 Total chlorophyll content and growth parameter measurements.
10mM KNO3 was added to the fungal compartment of Mycorrhizal plants (Myc plants
+N) reservoir for two weeks. Two controls were used; non-mycorrhizal (non-myc)
plant with KNO3 added to fungal compartment reservoir to check for leakage and
mycorrhizal plants with no N added (Myc –N). A) Total chlorophyll content, B)
number of pods, C) Fresh weight, and D) shoot length were measured. Values are
reported as mean ± SEM (n=10 biological replicates). Statistical analyses were done
by ANOVA single factor analysis, alpha = 0.05. Letters (a, b, ...) above the bar
graphs designate statistically significant difference between means.

73

Figure 2-5 (cont’d)

B
4.5

a
4

Fresh weight (g)

3.5
3
2.5

b

b

2
1.5
1
0.5
0

C

12

a
Number of pods

10
8

b

b

6
4
2
0

74

Figure 2-5 (cont’d)

D

35

Shoot length (cm)

30
25

a
b

b

20
15
10
5
0

75

A
Glutamate

Unlabeled
N labeled

15

15

Non-mycorrhizal plant + N

Mycorrhizal plant +15N

120

450

nmol/g (FW)

350
300
250

*

100
nmol/g (FW)

*

400

80
60
40
20

200

0

150

Root

Shoot

100
50

Mycorrhizal plant with no

0
Root

15

N

Shoot
120
nmol/g (FW)

100
80
60
40
20
0
Root

Shoot

Figure 2-6 Free N metabolite concentrations and 15N labeling in the whole
mycorrhizal plant system. Total of 10 mM K15NO3 was added to the fungal
compartment reservoir of mycorrhizal plants (Myc plants +N) for two weeks. Two
controls: non-mycorrhizal plant with 15N added nonplant compartment to the and
mycorrhizal plants with no 15N added. N metabolites are glutamate (A), glutamine (B),
pyrroline-5-carboxylate (C), ornithine (D) and arginine (E). The labeling was calculated
by adding the concentrations of all 15N labeled isotopomers for each N metabolite.
Values are reported as mean ± SEM (n=10 biological replicates). Statistical analyses
were done by ANOVA single factor analysis, alpha = 0.01. Stars show statistically
significant 15N labeling (p<0.01).
76

Figure 2-6 (cont’d)

B
Glutamine

Unlabeled
N labeled

15

Non-mycorrhizal plant +15N
50

Mycorrhizal Plant +15N
40

*

200

*

30
20
10

150

0
Root

Shoot

100
50

50

Mycorrhizal plant with no 15N

45
40

0
Root

Shoot

nmol/g (FW)

nmol/g (FW)

250

nmol/g (FW)

300

35
30
25
20
15
10
5
0
Root

77

Shoot

Figure 2-6 (cont’d)

C
Pyrroline-5-Carboxylate

Unlabeled
N labeled

15

Non-mycorrhizal plant +15N
Mycorrhizal plant +15N

7
6

*

nmol/g (FW)

25
20
15

*

10

nmol/g (FW)

30

Mycorrhizal plant with no 15N

5
4
3
2
1

5

0
Root

0

Shoot

Shoot

Mycorrhizal plant with no 15N
5
4
nmol/g (FW)

Root

3
2
1
0
Root

78

Shoot

Figure 2-6 (cont’d)

D
Ornithine

Unlabeled
N labeled

15

Non-mycorrhizal plant +15N

70

300

Mycorrhizal plant +15N
*

200

50
40
30
20
10

150

0
100

Root

*
50

70

0

Shoot

Mycorrhizal plant with no 15N

60
Root

Shoot
nmol/g (FW)

nmol/g (FW)

250

nmol/g (FW)

60

50
40
30
20
10
0
Root

79

Shoot

Figure 2-6 (cont’d)

E
Arginine

Unlabeled
N labeled

15

14

Non-mycorrhizal plant +15N

12
nmol/g (FW)

Mycorrhizal plant

+15N

160
140

10
8
6
4
2

*

100

0

80
60

Root

*

14

Shoot

Mycorrhizal plant with no 15N

40
12
20
10
0
Root

nmol/g (FW)

nmol/g (FW)

120

Shoot

8
6
4
2
0
Root

80

Shoot

Table 2-1 Precursor (unlabeled) compounds and product ions for each analyte.
Compound

Precursor compound
molecular formula

Daughter ion(s) quantified
Molecular formula

Arginine

C6H14N4O2

CH6N3+

Glutamate

C5H9NO4

C4H6NO+

Glutamine

C5H10N2O3

C5H7NO3+

Ornithine

C5H12N2O2

C4H8N+

Putrescine

C4H12N2

C4H10N+

Pyrroline-5-Carboxylate

C5H7NO2

C4H6N+

81

Table 2-2 Multiple reaction monitoring (MRM) transitions, optimizing source cone
voltages, collision cell voltages, and analyte retention time (for 15N labeling
experiment).
Compound

Arginine

Glutamate
Glutamine

Ornithine

Putrescine

Pyrroline 5Carboxylate
Phenylalanine
-D8

Precursor ion>
product ion
(m/z)
M+0: 175.1>60
M+1: 176.1>60
M+1: 176.1>61
M+2: 177.1>62
M+2: 177.1>62
M+3: 178.1>62
M+3:178.1>63
M+4:179.1>63
M+0: 148.02>83.84
M+1: 149.02>84.84
M+0: 147.06>129.95
M+1: 148.06>129.95
M+1: 148.06>130.95
M+2: 149.06>130.95
M+0: 133.1>70
M+1: 134.1>70
M+1: 134.1>71
M+2: 135.1>71
M+0: 89.03>71.79
M+1: 90.03>71.79
M+1: 90.03>72.79
M+2: 91.03>72.79
M+0: 113.98>67.75
M+1: 114.98>68.75
174.04>128.06

Cone
Voltage
(V)
22

Collision
Voltage
(V)
22

Retention
time (min)

Function
no.

6.08

1

22

16

1.23

2

16

10

1.07

2

16

16

5.52

2

50

10

6.05

2

16

10

1.15

2

18

15

5.26

2

82

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87

Chapter 3
Evidence for additional pathways in the nitrogen transfer network of
arbuscular mycorrhizas

88

Abstract
In recent years, our understanding of nitrogen nutrition in the arbuscular
mycorrhizal (AM) symbiosis has substantially increased. Work at the ecological level
has highlighted interactions among soil nitrogen, mycorrhizal fungi, and plant
communities; and at the molecular level a working model for nitrogen uptake,
metabolism, and transfer has been established. However, significant questions
remain about potential additional N fluxes through the metabolic network, including
nitrate transport and the origins and fate of ornithine. In

15N

labeling experiments,

using cultured mycorrhizal transformed roots as well as with mycorrhizal plant
microcosms, we observed that nitrate taken up by the AM fungal extraradical
mycelium (ERM) is translocated to the intraradical mycelium (IRM) and transferred to
the host roots. Labeled nitrate was measured in substantial quantities in shoots,
showing translocation within the plant after transfer from the fungus. After providing
15N

labeled nitrate or

13C
6

arginine to the fungal ERM, the levels and labeling of

metabolic intermediates as well as the expression of fungal N metabolism genes were
measured over a 3-day time course in the ERM and colonized roots. The results are
consistent with ornithine biosynthesis in the ERM via pyrroline-5-carboxylate and
indicate that that some ornithine is broken down in the IRM to glutamate and to a
lesser extent to putrescine. The timing of ornithine labeling in the IRM and ERM
indicates that a significant proportion of ornithine in the ERM is derived from arginine
breakdown in the IRM and translocated back to ERM where it is recycled to make
arginine. The results extend the working model of the N metabolic and transport

89

network in ways that point to significantly greater flexibility and energetic efficiency by
the fungal partner than appeared possible in the network structure.

90

Introduction
More than 80% of land plant species are mycorrhizal. Of the different mycorrhizal
types, the arbuscular mycorrhizal symbiosis is the predominant one (Wang & Qiu,
2006). Arbuscular mycorrhizal fungi (AMF) take up and transfer nutrients including P, N,
S, and Zn to their hosts (Clark & Zeto, 2000; Hamel, 2004; He et al. 2005). In return,
AMF depend on fixed carbon received from host roots, which can consume up to 20%
of photosynthate (Jakobsen & Rosendahl, 1990).
Nitrogen availability limits plant growth in many ecosystems (Reich et al., 2006;
Jackson et al., 2008; Marschner, 1995). The arbuscular mycorrhiza creates a well
distributed and extensive absorption network, which has been shown to be able to
substantially increase the uptake of N by host plant roots (Ames et al., 1983; Johansen
et al., 1993; Bago et al., 1996; Johansen et al., 1996; Tobar et al., 2004; Hodge et al.,
2010). In the current model for N transfer in the symbiosis (Bago et al., 2001,
Govindarajulu et al. 2005), nitrogen taken up by the ERM is assimilated via the
GS/GOGAT pathway and used to synthesize arginine, which is translocated to the
intraradical mycelium (IRM), and broken down to ammonium, which is released to the
host inside the colonized root without nutritionally significant amounts of carbon (see
Figure 1-5 in Chapter 1). Since it was proposed by Bago et al. (2001), substantial
biochemical and molecular biological evidence has accumulated to support this scheme
(Govindarajulu et al., 2005, Cruz et al., 2007; Tian et al., 2010). However, important
questions remain to be answered about the mechanisms and regulation of nitrogen
handling and about N transfer from the fungus to the host plant.

91

In the current model, arginine translocation is responsible for all or almost all N
movement from ERM to IRM (Bago et al., 2001, Tian et al., 2010)), but a possible role
for nitrate in translocation is unknown. Kaldorf et al. (1998) reported that the expression
of plant nitrate reductase is lower in mycorrhizal than non-mycorrhizal maize plants,
suggesting that N transfer is predominantly not in this form. However, Faure et al.
(1998) reported that nitrate reductase is increased in leaves of mycorrhizal plants
independently of P status, and this was interpreted as indicating that nitrate is
transferred from fungus to plant. Direct assessment of nitrate movement within the
fungus and/or between IRM and the plant would be valuable in answering this question,
and has not been addressed in previous studies that tracked

15N

and

13C

labeling using

LC/MS methods that do not detect inorganic nitrogen molecules. Likewise, the role if
any of ammonium in the translocation of N within the fungus is not known although it
can accumulate to millimolar levels in some fungi (Jennings, 1995), and Chalot et al.
(2006) suggested that ammonium might be taken up into vesicles within the mycorrhizal
fungi and released to the host by exocytosis.
Although AM, as well as ectomycorrhizal fungi, have the enzymes of the
GS/GOGAT pathway for N assimilation (Chalot et al., 1994; Johansen et al., 1996; Tian
et al. 2010), the role, if any, of glutamate dehydrogenase (GDH) in nitrogen assimilation
in the ERM of the arbuscular mycorrhizal is still unknown. In Tilia platyphyllos-Tuber
borchii ectomycorrhizae, N assimilation in the mycelium is the combined function of
NADPH dependent GDH and GS (Pierleoni et al., 2001). However, Morel et al. (2005)
found that NADP-GDH is dispensable for ammonium assimilation by ECM fungi. The
expression of a putative GDH gene from R. irregularis was reported (Govindarajulu et
92

al., 2005) although this now appears not to be a GDH (Tian and Shachar-Hill
unpublished) and another gene was annotated with this function (Tisserant et al. 2012,
2013) but none of the published data provide evidence for its function.
The breakdown of ornithine (a product of arginine hydrolysis) in the IRM yields
glutamate, apparently via ornithine aminotransferase, and ornithine may also be
converted to putrescine via ornithine decarboxylase, but the importance and fates of
these products is unclear. Glutamate in the IRM might be broken down to release
ammonium or metabolized by amino-transferase reactions to produce other amino
acids. Also, the origin of ornithine in ERM is still not well understood, although enzymes
that can be involved in its synthesis, including ornithine amino-transferase and other
urea cycle enzymes are upregulated in the ERM upon N addition. Specifically, since
carbon is not transferred to the plant upon arginine breakdown (Jin et al. 2005) in the
IRM, the carbon skeleton may return to the ERM for re-use in ornithine and arginine or
their precursors.
The expression of N transport and metabolism genes in fungi is commonly
regulated by the levels of nitrogenous metabolites such as Arg, Gln and Orn (nitrogen
metabolite control, Hinnebusch, 1988; ter Schure et al., 2000) and the levels of amino
acids in presymbiotic AMF tissue are increased substantially in the presence of
inorganic N (Gachomo et al., 2009). However R. irregularis, which is the primary model
AM fungus in molecular genetics and biochemical studies, does not down-regulate the
genes for Arg synthesis in the ERM when intracellular Arg levels are high, as occurs for
example in S. cerevisiae suggesting either that Arg is sequestered, or perhaps that host
demands for N are communicated to maintain N flow. Thus studying the gene
93

expression of the key genes of the pathway coupled with

15N

labeling can help to

understand how the genes involved in nitrogen metabolism and transport are regulated
(Jin et al., 2010).
In this study, isotopic labeling time course experiments using different
13C

15N

and

substrates were performed and the levels and labeling patterns of putrescine,

pyrroline-5-carboxylate and nitrate in addition to the amino acids glutamate, glutamine,
ornithine and arginine were measured. The identification and expression time courses
of several new N metabolic enzyme transcripts were measured over the same time
course. The results extend our understanding of the N transfer network, including the
origin and fate of key intermediates and shed light on its regulation.

Experimental procedures
Chemicals and reagents
Gelzan, (MP Biomedical, Solon, OH) was used for solidification of M media.

13C-

argiinine and 15N labeled potassium nitrate were obtained from MP Biomedicals, (Solon,
OH).
Spore material and mycorrhizal in vitro growth conditions
The spore material of R. irregularis (DAOM 181602) was purchased from
Premier Tech Biotechnologies in units of 106 and was stored at 4°C until further use. Ri
T-DNA-transformed carrot (Daucus carota clone DCI) roots were grown at 25°C in
modified medium (Bécard and Fortin, 1988) with 3.5 g L−1 Phytagel (Sigma) using the
94

split-plate method of St-Arnaud et al. (1996). The roots and fungus were allowed to
proliferate on both sides of bicompartmented petri plates at 25°C until the fungal ERM
was well developed (approximately 6 weeks). The colonized roots and media in each
compartment were transferred to empty compartments of new plates in which the other
compartment contained new medium with no nitrogen. The fungal ERM typically grew
over the barrier within 2 week of the transfer, colonizing the empty compartment. Root
growth over the barrier after transplantation was prevented by pruning.

Isotopic labeling in colonized Ri T-DNA-transformed carrot (Daucus carota clone
DCI) roots culture system
15N

labeling
The medium of the root compartment was modified to limit the nitrogen

concentration to 1 mM. The ERM was allowed to cross over the divider into the fungal
compartment that contained modified medium with no N or Sucrose added and only 2 g
L−1 Phytagel. After 2-3 weeks, the ERM were supplied with 4 mM KNO3 for gene
expression studies or K15NO3 to determine the labeling percentage of the free N
metabolites and

15NO

3

in the IRM part and plant tissues. The colonized roots and ERM

samples were collected after 0, 2, 4, 8, 16, 24, and 72 h, rinsed with sterilized water,
and immediately frozen in liquid N and stored at −80°C
13C

labeling
[13C6]arginine (0.5 mM) was supplied to ERM. ERM and AM roots samples were

collected after 0, 3, 6, 12, 24, and 48 h for metabolite analysis, rinsed with sterilized
water, immediately frozen in liquid N and stored at −80°C
95

15N

labeling for 15NO3 measurements in whole plant-mycorrhizal system
Medicago truncatula plants were grown for 5 weeks in two-compartment culture

system (growth condition and the culture system mentioned in the methods of Chapter
2) after that plants were deprived from N for 4 days at which time 10 mM K15NO3 was
added to the reservoir of fungal compartment for two weeks. Plant shoots and roots
were collected for N metabolite analysis. Two controls were used, mycorrhizal colonized
plants with no

15N

added and non-mycorrhizal plants with 10 mM K15NO3 added to

fungal compartment (FC) reservoir to test leakage. Total of 10 biological replicates in
two different experiments were used.

Extraction and isolation of 15N and 13C metabolites
A total of 300 mg of AM roots (or plant roots and shoots in whole plant system)
were ground in a mortar and pestle with a pinch of acid-washed sand and extracted
three times with a mixture of methanol:chloroform:water (12:5:3, v/v/v). Methylene
chloride and water were added to the extraction solution to facilitate the separation of
chloroform and the methanol–water phases. The methanol–water phase containing the
amino acids (AAs) was collected and evaporated in a rotary evaporator at 50°C, and the
residue containing the AAs and N metabolites were dissolved in 1 ml of 0.01 M HCl.
Lyophilized fungal mycelium was pulverized with two 3-mm stainless steel beads using
a bead mill (Retsch MM301). Cold methanol:water (70:30, 0.2ml) was added to aid in
disruption. The samples were shaken at 30 Hz for 4 min, and 2-μL samples were
analyzed by dissecting microscope to ensure that hyphae and any spores had been
96

broken. After disruption, 0.8 mL of cold methanol:water (70:30) was added, and the
sample was vortexed for 5 min. Samples were then centrifuged and the supernatants
collected. The cold aqueous methanol extraction was repeated twice more using 1 mL
each time and the supernatants pooled. Then the methanol-water mixer were
evaporated in a rotary evaporator at 50°C, and the residue containing the AAs and N
metabolites were dissolved in 1 ml of 0.01 M HCl.
ERM and AM root samples in 1 ml of 0.01 M HCl were loaded onto a cation
exchange column (0.3 ml of DOWEX 50 X8-200– hydrogen form; Sigma-Aldrich, St
Louis, MO, USA), which was previously washed with 1 M NH4OH, deionized H2O and 1
M HCl, and followed by deionized H2O. after loading the sample, the columns were
washed 5 times with water so the neutral compounds and anions were eluted so that
they were collected for nitrate measurements and the free amino acids were eluted with
5 ml of 1 M NH4OH (Bengtsson & Odham, 1979). This eluent was collected and dried
then resuspended in 70 µl of Milli-Q water.

Liquid chromatography and mass spectrometry (LC-MS)
A Waters (Milford, MA) Quattro micro mass spectrometer coupled to a Shimadzu
(Columbia, MD) LC-20AD HPLC system and SIL-5000 autosampler was used. A
Waters Symmetry C18 column (2.1 × 100 mm, 3 μm particle size) was used with
column oven temperature at 30 °C. The injection volume was 10 μL, and the HPLC flow
rate was 0.3 mL/min using 1 mM perfluroheptanoic acid in a water/acetonitrile (A/B)
gradient at ambient temperature, The intial gradient (A/B)=99/1, held until 2 minutes,
97

followed by a linear gradient to 60/40 at 4 minutes, held at 60/40 until 8 minutes,
followed by a ramp to 99/1 at 8.01.
Mass spectra were acquired using electrospray ionization in positive ion mode
and MRM. The capillary voltage, extractor voltage, and rf lens setting were set at 3.17
kV, 4 V, and 0.3, respectively. The flow rates of cone gas and desolvation gas were 20
and 400 L/h, respectively. The source temperature and desolvation temperature were
110 and 350 °C, respectively. Collision-induced dissociation employed argon as
collision gas at a manifold pressure of 2 × 10-3mbar, and collision energies and source
cone potentials were optimized for each transition using Waters QuanOptimize
software. This method was composed of two ESI+ functions (0−1.8 and 1.8−6.0 min)
covering full run time to allow for adequate dwell time for each analyte. Data were
acquired with MassLynx 4.0 and processed for calibration and for quantification of the
analytes with QuanLynx software. A table (2-1) of (unlabeled) precursor and product
ions for each compound is given in chapter 2. To determine concentrations, standard
curves were run for authentic unlabeled standards of each compound in the table above
with concentrations range from 0 to 100 μM together with 10uM Phenylalanine-D8 as
internal standard, which was also added to each biological sample during extraction to
control for recovery efficiency and as internal standard. Integrated peak areas were
obtained from daughter ion chromatograms using the MassLynx 4.0. Total levels for
each compound were obtained from the sum of all the labeled and unlabeled molecules.
For

15N

and

13C

labeled samples, each product ion mass isomer for each

precursor mass isomer was quantified. Thus for example for 15N labeling in Arginine
the precursor ions have mass-to-charge ratios (m/z) of 175 (15N0 = M+0), 176 (15N1 =
98

M1), 177 (15N2 = M+2), 178 (15N3 = M+3), and 179 (15N4 = M+4) with the product ion
CH6N3+ have m/z of 60 (15N0 = M+0), 60 and 61 (15N1 = M+1), 61 and 62 (15N2 = M+2),
62 and 63 (15N3 = M+3), and 63 (15N4=M+4) (see Table 1-2, 2-2, 3-1) . The proportion of
N atoms which were

15N

labeled was calculated ( calculation is described in Figure 3-3)

to shows how much of the total N in that metabolite pools is new. All calculations was
done after correction for natural abundance, mainly 13C.

15N

labeled and unlabeled nitrate measurements
3200 QTRAP® LC/MS/MS coupled to a Shimadzu (Columbia, MD) LC-20ADvp

HPLC system and SIL-HTC autosampler was used. ZIC®-pHILIC column (50 x 2.1 mm,
5 µm particle size) was used with column oven temperature at 40 °C. The injection
volume was 1 μL, and the flow rate was 0.2 mL/min using water/acetonitrile (A/B)
gradient at ambient temperature. The initial gradient (A/B)=10/90, held until 2 minutes,
followed by a linear gradient to 30/70 at 3 minutes, held at 30/70 until 4 minutes,
followed by a ramp to 10/90 at 4.01.
Mass spectra were acquired using turbo VTM ion source and multiple ion
monitoring scan type in negative ion mode. Masses of 62 (unlabeled nitrate) and 63 (15N
labeled nitrate) were measured at retention time of 1.3 min. To determine the
concentration of
authentic

15N

15N

labeled and unlabeled nitrate, standard curve were run for

labeled nitrate standard with concentration range from 0 to 100 μM. Data

were processed for calibration and for quantification of the analytes with QTRAP analyst
software.
99

RNA extraction and putative gene fragment isolation
Sequences

of

nitrite

reductase,

ornithine

transcarbamylase,

pyrroline-5-

carboxylate dehydrogenase, and glutamate dehydrogenase gene fragments were
identified from EST sequences obtained by high throughput RNAseq of R. irregularis
transcriptome performed at MSU (Tian et al. 2010). To confirm their identity total RNA
was extracted using the RNeasy Plant Mini kit (Qiagen, Valencia, CA) from R.
irregularis germinating spore tissue that was disrupted using a bead mill, followed by
DNA removal using RNase-free DNase (Turbo DNA-free; Ambion, Austin, TX). cDNA
was synthesized using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA).
Primer sets were developed from EST sequence and designed using the primer3
website (http://frodo.wi.mit.edu/primer3/). PCR was performed and the products were
separated by agarose gel electrophoresis then extracted using the QIAquick Gel
Extraction kit (Qiagen) and sequenced at the MSU RTSF on an ABI PRISM 3100
Genetic Analyzer (Applied Biosystems, CA, USA). Sequences were compared with the
R. irregularis genomic database to confirm identity.

Quantitative Real-Time PCR measurements
Mycorrhizal split plates were grown until the fungal compartment was
approximately one-half colonized. To the fungal compartment, 1 ml of sterile KNO3
solution was applied to give a final concentration of 4 mM KNO3. Plates were incubated
for 0, 2, 4, 8, 16, 24 and 72 h before tissue from 9-12 plates was collected and
immediately frozen in liquid N. RNA was extracted and converted to cDNA as described

100

above. The initial quantitative real-time PCR (qRT-PCR) reaction mixture containing
primers at a concentration of 300 nM and 1 ng of cDNA template. The PCR reactions
were monitored using an ABI Prism 7900 HT Sequence Detection system (Applied
Biosystems, CA, USA) with the following cycling program: 50°C for 2 min, 95°C for 10
min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. Power SYBR Green 2Step Master mix (Applied Biosystems, CA, USA) was used for all real-time PCR assays.
The ΔΔCT, and comparative CT, methods were utilized for the determination of relative
gene expression (Livak and Schmittgen, 2001). The expression of an S4 ribosomal
protein was used to normalize relative gene expression data as described
by Govindarajulu et al. (2005). Primers of the four genes above and previously used
primers (Table 3-1) and the closest homology of the newly identified genes are listed in
Table 3-2.

Results
Nitrate movement through the AM symbiosis

The fungal ERM of transformed carrot roots was exposed to 4 mM K15NO3 in the
fungal compartment of split plate in vitro mycorrhizal cultures (Fortin et al. 1996) so that
the ERM but not the roots had access to labeled nitrate. The levels of labeled and
unlabeled nitrate (15NO3- and

14NO -)
3

in the mycorrhizal roots were measured over the

next 3 d using LC-MS/MS and the percentage labeling was calculated by dividing the
concentration of

15N

nitrate by the total concentration of labeled plus unlabeled nitrate.

The percentage labeling of

15NO

3

increased rapidly in extracts of AM roots reaching
101

approximately 20% within 8 h (Figure 3-1). The percentage labeling of

15NO

3

reached

50% after 24 h with no significant increase between 24 and 72 h (Figure 3-1). The
mycorrhizal carrot roots contained 1-2% fungal biomass, so it is uncertain whether the
labeled nitrate remained in the fungal tissue or was transferred to plant cells.

To assess transfer of nitrate from the fungus to the host plant, an aseptic
microcosm (see Chapter 2) system was used to perform a similar experiments with
mycorrhizal M. truncatula plants. Levels of

15NO

3

and

14NO

3

were measured in root and

shoot tissues after the ERM in the fungal compartment was exposed to 10 mM K15NO3
for two weeks. The mycorrhizal plants showed high

15NO

3

concentrations in both roots

and shoots (Figure 3-2). The control plants (non-mycorrhizal plants with
the second compartment or mycorrhizal plants with no
15NO

3

15N

15N

added to

added) showed no

labeling and low levels of 14NO3 – consistent with the N deprivation pretreatment.

N movement through the AM symbiosis in transformed root system
The levels and labeling of the free N metabolites glutamine, glutamate, ornithine,
arginine, pyrroline-5-carboxylate, and putrescine in the ERM and AM roots were
measured using LC-MS/MS after the ERM was exposed to 4 mM K15NO3. The fraction
of N in each metabolite pool that was labelled with
15N

15N

is expressed as the amount of

in molecules of that metabolite divided by the total amount of N (both labeled and

unlabeled) in molecules of that metabolite. The amount of

15N

in a metabolite pool was

calculated from the sum of the MS signals from each mass isomer multiplied by the
number of

15N

atoms in that isomer: (0xM0 + 1xM1 + 2xM2 …), and the total nitrogen in
102

the molecule was calculated as the sum of all the mass isomers multiplied by the
number of N atoms in the one metabolite molecule (one for glutamate, two for ornithine,
four for arginine etc). Fractional labeling levels are shown as a function of time after 15N
addition in Figure 3-3. Significant labeling levels were observed in free N metabolites
within 2 h after 15N addition, and within 8 h about 40% of the arginine molecules were
15N

labeled in the ERM. Pyrroline-5-carboxylate, glutamate, glutamine, ornithine also

became rapidly labeled within the ERM, consistent with their serving as precursors for
arginine biosynthesis. After 24 h, more than 25% of N in glutamate, glutamine, ornithine
and arginine molecules were labeled within the ERM. On the other hand, about 25% of
N in pyrroline-5-carboxylate molecules were labeled after 24 h within the ERM and the
labeling levels rose in a hyperbolic manner indicating saturation of the labeling levels
significantly below full labeling. No

15N

labeling of putrescine was detectable in the

ERM. After 72 hours more than 80% labeling of the amino acid molecules contained
one or more 15N atoms.
In the colonized roots (IRM plus plant cells), the labeling of metabolites showed a
lag of ~2h and was slower than in the ERM over the first 8 h. The fractional labeling of N
in arginine molecules reached approximately 15% after 8 h, and rose to about 80% by
72h. A total of 30% of N in ornithine molecules were labeled within the colonized roots
at 16 h. The other metabolites showed similar patterns of
AM roots. The

15N

15N

fractional labeling in the

fractional labeling of arginine and ornithine and other metabolites is

consistent with arginine was transport from ERM to the IRM and its breakdown there via
arginase.

15N-

putrescine was detectable in IRM after 8 h and reaching more than 15%

fractional labeling after 72 h (Figure 3-3).
103

The origin and fate of the N metabolites
In order to study the origin and fate of the metabolites in the ERM,
isotopomers (chemically identical molecules containing different numbers of

15N

15N

atoms

in different positions) of glutamate, ornithine and arginine were measured using intact
molecular ions and fragment ions to obtain the number of

15N

atoms and information on

positional labeling from mass spectra. M+1 isotopomers were detectable first and higher
mass isotopomer levels increased after that. M+2 ornithine molecules were detected at
significant levels (p<0.01) after 8 h while the fully labeled M+4 arginine mass isomer
was detectable after 4 h (Figure 3-4).

13C

labeling in the ERM and AM roots
The levels and

13C

labeling of free N metabolites (glutamate, pyrroline-5-

carboxylate, glutamine, ornithine, arginine and putrescine) in the ERM and AM roots of
mycorrhizal transformed carrot roots were measured after the ERM was exposed to 0.5
mM [13C6]arginine. [13C6]arginine was found in the ERM within 3 h (Figure 3-5E).
[13C5]Arginine was detected in the ERM at 12 h and then rapidly increased. On the other
hand, glutamine

13C ,
5

glutamate

13C ,
5

putrescine

13C
4

were not detectable at any time in the ERM. Ornithine

and pyrroline-5-carboxylate
13C
5

13C
5

was detectable in the ERM

at 12 h and then rapidly increased (Figure 3-5C).
In the AM roots, [13C6]arginine appeared first at 3 h and its level rapidly reached
over 30 nmol/g at 12 h. No significant increase of [13C6]arginine levels were observed
between 12 and 24 h, levels then doubled between 24 and 48 h. [13C5]Arginine was
104

detectable at 24h in AM roots and reached over 50 nmol/g. [13C5]Arginine level rose
between 24 and 48 h (Figure 3-5E). Glutamine was not detectable at any time.
Ornithine 13C5 was detectable in AM roots within 3 h while glutamate 13C5, and pyrroline5-carboxylate

13C
5

and putrescine

were detectable in AM roots after 6 h and completely absent in ERM

13C
4

was detectable after 12h in AM roots but not detectable in ERM

(Figure 3-5).

Gene identification
Based on sequence data previously deposited in public databases (GenBank),
sequences from R. irregularis for four putative enzymes of N metabolism were
identified.

Partial

transcarbamylase

coding
(OTC),

sequences

of

nitrite

pyrroline-5-carboxylate

reductase

(NiR),

dehydrogenase

ornithine

(P5CD)

and

glutamate dehydrogenase (GDH) were obtained that show high sequence similarities to
known genes involved in N uptake and metabolism in fungi and bacteria (Table 3-3).
Nitrite reductase was identified with 83% similarity at the amino acid level to the
Laccaria bicolor NADPH-nitrite reductase (EC 1.7.1.4), an assimilatory nitrite
reductase which catalyzes the NADPH-dependent formation of ammonia from nitrite.
Ornithine carbamoyltransferase was identified with 81% similarity to the Cryptococcus
gattii (WM276) Ornithine carbamoyltransferase, mitochondrial precursor (OTCase; EC
2.1.3.3) which catalyze catalyzes the reaction between carbamoyl phosphate
and ornithine to form citrulline and phosphate. Pyrroline-5-carboxylate dehydrogenase
was identified with 63% similarity to Cryptococcus neoformans var. neoformans
105

JEC21 1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12) which catalyze the
reversible conversion of pyrroline-5-carboxylate to glutamate. Furthermore, glutamate
dehydrogenase was identified with 63% similarity with glutamate dehydrogenase NAD+
(EC 1.4.1.2) of Sphingobacterium spiritivorum (ATCC 33861), which catalyzes the
reversible oxidative deamination of glutamate to alpha-ketoglutarate and ammonia.

Gene expression in response to N addition

Based on the current model and previous results (Cruz et al., 2007; Tian et al.,
2010) we hypothesized that the expression of the genes involved in N movement and
metabolism from soil through the fungus and into the host is temporally and spatially
coordinated with the flux of N. The transcriptional levels for eight genes from R.
irregularis (4 of them identified here, the others identified and functionally confirmed
previously) were measured in ERM and IRM tissues by quantitative real-time PCR.
The transcript levels of the putative assimilatory nitrite reductase (NiR) and
ornithine transcarbamylase (OTC) increased strongly in the ERM beginning by 2 h after
4 mM KNO3 was added to the fungal compartment. NiR and OTC were highly
upregulated after 4 h of nitrate addition. By contrast ornithine aminotransferase 1
(OAT1) and pyrroline-5-carboxylase (P5CD) were modestly upregulated and reached
over 2folds expression after 4 h in the ERM. Arginase (CAR1), ornithine
aminotransferase

2

(OAT2),

glutamate

dehydrogenase

(GDH),

and

ornithine

decarboxylase (ODC) transcript levels in the ERM were little affected by the supply of
nitrate. By contrast, the expression of CAR1, OAT1, OAT2, P5CD and GDH was

106

substantially up-regulated in the IRM within 24 h. The transcript level of ODC increased
modestly in the IRM by 24 h. NiR transcript level was low in the IRM and not
upregulated over the time course.

Discussion
The current working model of N transfer from the fungi to the plant in the AM
symbiosis was proposed by Bago et al. (2001) based on previous work that
demonstrated fungal N uptake and metabolism and implicated amino acids in N
handling (Johansen et al., 1996; Bago et al., 1996; 2000). Supporting evidence for the
model (Govindarajulu et al., 2005, Cruz et at., 2007, Jin et al., 2005, Tian et al., 2010)
has led to its widespread acceptance (Smith and Read 2008, Parniske et al., 2009, He
et al., 2011). However, the network is incomplete on the question of the fate of carbon
translocated from ERM to IRM in the form of arginine and the potential remains for other
forms of N besides arginine to be involved in N movement from ERM to the IRM and
roots.
The detection of high

15N

labeling of nitrate in AM carrot roots after

15N

nitrate

was supplied to the ERM (Figure 3-1) indicates that not all of the nitrate taken up by the
ERM is reduced and assimilated there. Because total nitrate levels are not high, it may
be that the nitrate translocated to the colonized roots remains in the IRM. To address
this question, we conducted gene expression analysis of the fungal putative assimilatory
nitrite reductase in the colonized transformed roots and ERM as well as a

15N

nitrate

labeling experiment using the whole plant microcosm described in Chapter 2. NiR
107

expression is upregulated in the ERM but not IRM in response to nitrate addition (Figure
3-6) suggesting that the nitrate that reaches the IRM is not converted to ammonia. Tian
et al., (2010) reported that other genes involved in N assimilation, such as GS/GOGAT
were not upregulated in the IRM. The results of the whole plant experiment showed that
the roots and shoots both had high

15N

nitrate labeling levels while control treatments

showed no labeling (Figure 3-2). We conclude that nitrate also contributes to N transfer
by AMF to host plants so that arginine synthesis and translocation within the fungus
followed by its breakdown and the transfer of ammonium to the plant is not the only
route for N transfer in AM symbiosis.
After supplying K15NO3 to the ERM, rapid

15N

labeling was found in N

metabolites in the ERM and in the AM roots (Figure 3-3). This is consistent with
previous observations of amino acid labeling by Tian et al. (2010). The increase of the
ornithine

15N

fractional labeling in the ERM and in the mycorrhizal roots coincided with

the increase of labeled arginine (Figure 3-3 C,D), which is consistent with their close
location in the metabolic network. These results are consistent with previous
observations (Jin et al. 2008) and with the synthesis of arginine in ERM and its
breakdown in the IRM as previously proposed (Bago et al. 2001).
Neither the data shown in Figures 3-1 - 3-3, nor previous studies provide a
complete picture of the origins of ornithine in the ERM or its fate in the IRM
(Govindarajulu et al., 2005; Jin et al., 2005; Cruz et al., 2007). Ornithine released during
arginine breakdown in the IRM might be stored, transferred to the host, broken down or
translocated to the ERM. Govindarajulu et al., (2005) reported that ornithine was neither
stored in substantial quantities in the IRM nor is transferred to the host in detectable
108

amounts. Breakdown of ornithine in the IRM with the release of ammonium would seem
to be more efficient for N transfer to the host compared with its translocation back to the
ERM, which would return half of the N in arginine to its original location. However, the
simultaneous de novo synthesis of ornithine in the ERM and its catabolism in the IRM
also consumes energy and carbon.
The presence of

15N

labeled pyrroline-5-carboxylate as well as

15N

glutamate in

the ERM (Figure 3-3) is consistent with de novo ornithine biosynthesis, since pyrroline5-carboxylate is the intermediate metabolite for the formation of ornithine from
glutamate via P5CD. Gene expression analysis of OAT1 and P5CD showed 3-fold
increases in gene expression for these genes 4 h after adding nitrate to the ERM. Both
of those enzymes catalyze the reversible interconversion of ornithine and glutamate.
However, OAT1 and P5CD were not highly upregulated in the ERM compared with the
IRM (Figure 3-6). Enzyme activity analyses will be required to determine which of the
routes is more active.
The absence of detectable levels of

15N

putrescine in the ERM is consistent with

the gene expression analysis of ODC, which was not upregulated in the ERM after N
addition. By contrast, significant levels of

15N

putrescine were found in the IRM later

during the time course, which coincided with an approximately 3-fold ODC gene
expression increase in IRM 24 h after nitrate addition to the ERM (Figure 3-6).
Significant amounts of

15N

glutamate and

15N

pyrroline-5-carboxylate were found in the

IRM coinciding with the upregulation of OAT1, OAT2 and P5CD. Based on the levels
and fractional labeling of putrescine, pyrroline-5-carboxylate and glutamate as well as
OAT1, OAT2 and P5CD gene expression analysis, we conclude that significant
109

amounts of ornithine are converted to glutamate and that lower quantities are converted
to putrescine in the IRM.
The levels of

15N

isotopomers of glutamate, ornithine and arginine were

measured in the ERM after adding K15NO3 to the ERM (Figure 3-4). The increase in
M+1 (Non R group N(NR) ornithine in the ERM coincided with the increase in M+1

15N-

glutamate is consistent with the formation of ornithine from glutamate. M+2 ornithine
levels increased with the increase of

15N

arginine isotopomers containing 2 or more

atoms and these increased more rapidly when
of

13C
6

15N

arginine M+4 levels rise. Application

arginine to the ERM resulted in detectable level of arginine

ornithine

13C
5

15N

13C
6

within 3 h while

was detectable in the ERM only after 12 h (Figure 3-5). The absence of

ornithine 13C5 before 12 h and the appearance of ornithine 13C5 in AM roots indicate that
arginine was broken down in the IRM but not in the ERM (consistent with low arginase
expression there). The absence in the ERM of

13C

labeled N metabolites other than

ornithine indicates that ornithine is not broken down in the ERM.
Several observations are consistent with translocation of ornithine from IRM to
ERM. The appearance of

13C
5

ornithine in the ERM is substantially delayed compared

to the IRM. This could be due to either the return of

13C
5

ornithine from IRM to ERM or

to a delayed induction of arginase in the ERM. The total levels of arginine, ornithine and
other amino acids in the ERM are not affected by arginine uptake (which is much slower
than the uptake of inorganic N substrates) so that a delayed induction of arginase
activity in the ERM is improbable as an explanation. Indeed arginase expression and
activity were not induced in the ERM by N addition, (this study, Tian et al., 2010 and
Cruz et al,. 2007).

13C
5

arginine, which is made from
110

13C
5

ornithine, was detected in the

ERM much later than
13C
5

of

13C
6

arginine, which is consistent with the delayed appearance of

ornithine in the ERM as a precursor not a product of

13C
5

13C
5

arginine. The appearance

arginine in ERM preceded its appearance in the IRM, consistent with the

unidirectional translocation of arginine from ERM to IRM (Figure 3-5). The fractional
labeling of ornithine in the IRM is higher as well as rising faster than that in the ERM
(since carbon is not transferred to the plant, ornithine in the plant cells is unlabeled so
that the measured fractional labeling of ornithine in the AM roots represents a still higher
labeling level in the IRM). This is necessary if ornithine in the IRM is the precursor of
ornithine in the ERM. The presence of glutamate
putrescine

13C
4

13C ,
5

pyrroline-5-carboxylate

13C
5

and

indicated that some ornithine is being converted to them in the IRM of

AM roots. The absence in the ERM of

13C

labeled glutamate, glutamine, pyrroline-5-

carboxylate or putrescine despite their accumulation in the AM roots indicates that none
of these metabolites returns to the ERM from the IRM.
The possible role of glutamate dehydrogenase in N movement is still unknown.
Cliquet and Stewart (1993) reported the activities of the GS/GOGAT pathway but not
assimilatory NADP-dependent glutamate dehydrogenase (GDH) in mycorrhizal roots,
and the application of a GOGAT inhibitor to extraradical mycelium reduced

15N

assimilation. A putative GDH gene was identified and it is most probably dissimilatory
GDH. The gene expression of GDH in slightly upregulated in ERM and highly
unregulated in IRM indicating that GDH breaks down glutamate to release more
ammonium to the host in IRM.
Within 2 h of nitrate addition to the ERM, the expression of the OTC transcript
was upregulated in the ERM, coinciding with the building up of arginine since OTC is
111

part of urea cycle. On the other hand, OTC was not upregulated in the IRM, which is
consistent with the previously reported down regulation of other arginine biosynthesis
genes in the IRM (Tian et al., 2010). The up regulation in the IRM of the expression of
fungal genes involved in arginine and ornithine breakdown (Arginase: CAR1, OAT1,
OAT2 as well as P5CD and ODC) coincides with the arrival of

15N

label and a rise in

arginine levels within AM roots. Thus, it is likely that this is the signal for regulatory gene
expression in the IRM.

112

APPENDIX

113

APPENDIX

70

50

40

30

15NO

3

Percentage labeling

60

20

10

0
0

10

20

30

40

50

60

70

80

Time (h)

Figure 3-1 Time course of 15N percentage labeling of nitrate in transformed carrot
(Daucus carota) roots colonized by R. irregularis . 15N labled and unlabeld nitrate
were measured after supplying 4 mM K15NO3 to the fungal extraradical mycelium (ERM)
compartment in divided petri dishes. Values are reported as mean ± SEM (n=3
biological replicates).

114

15

Unlabeled nitrate
15
N labeled nitrate

Non-mycorrhizal plant + N
nmol/g (FW)

2

15

Mycorrhizal plant + N
20

*

1.5
1
0.5

nmol/g(FW)

15

*

0
Root

10

Shoot

Mycorrhizal plant with no

5

Root

nmol/g (FW)

0

Shoot

15

N

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

Root

Shoot

Figure 3-2 The concentrations of 15N labeled nitrate (light grey bars) and unlabeled
nitrate (black bars) in the roots and shoots of mycorrhizal M. truncatula plants
grown in micosms. Mycorhizal plant +15N: 10mM K15NO3 was supplied to the fungal
compartment reservoir (FCR) for two weeks before tissue collection and analysis. Nonmycorrhizal plant +15N: non-mycorrhizal plants supplied with 10mM 15N nitrate in the
FCR– controlling for non-mycorrhizal N levels and for non-fungally mediated N label
movement. Mycorrhizal plant with no 15N: mycorrhizal plants with no 15N added –
controlling for N deprived N levels in myc plants and for natural abundance isotope
levels. Values are reported as mean ± SEM (n=10 biological replicates). Statistical
analyses were done by ANOVA single factor analysis, alpha = 0.01. Stars show
statistically significant 15N labeling (p<0.01).

115

A
15N-Glutamate

ERM

1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

0.8
0.7
Fractional labeling

Fractional labeling

AM roots

0.6
0.5
0.4
0.3
0.2
0.1
0

0

8 16 24 32 40 48 56 64 72

0

Time (h)

8

16 24 32 40 48 56 64 72
Time (h)

Figure 3-3 Nitrogen movement from the ERM to the IRM in mycorrhizal
transformed carrot roots . These resullts are based on the timing of labeling in N
metabolites in the ERM and AM roots after the addition of 4 mM K15NO3 to the fungal
ERM. The fraction of N in each metabolite pool that is labelled with 15N is expressed as
the amount of 15N in molecules of that metabolite divided by the total amount of N (both
labeled and unlabeled) in molecules of that metabolite. The amount of 15N in a
metabolite pool is calculated from the sum of the MS signals from each mass isomer
multiplied by the number of 15N atoms in that isomer: (0xM0 + 1xM1 + 2xM2 …), and
the total nitrogen in the molecule is calculated as the sum of all the mass isomers
multiplied by the number of N atoms in the one metabolite molecule (one for glutamate,
two for ornithine, four for arginine etc). Fractional labeling of A) glutamate, B) pyrroline5-carboxylate, C) glutamine, D) ornithine, E) arginine in ERM and AM roots, and F)
putrescine in AM roots only as 15N-labeled putrescine was not detectable in ERM.
Means and standard error of the means of three replicates.

116

Figure 3-3 (cont’d)

B
15N-1-Pyrroline-5-carboxylate

ERM

AM roots

0.55
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

0.5
0.45
Fractional labeling

Fractional labeling

0.4
0.35
0.3

0.25
0.2

0.15
0.1
0.05
0

0

8

0

16 24 32 40 48 56 64 72

8

16 24 32 40 48 56 64 72
Time (h)

Time (h)

C
15N-Glutamine
0.7

0.7

ERM

AM roots

0.6

0.5

0.5
Fractional labeling

Fractional labeling

0.6

0.4
0.3
0.2
0.1

0.4
0.3
0.2
0.1

0
0

0

8 16 24 32 40 48 56 64 72

0

Time (h)

8 16 24 32 40 48 56 64 72
Time (h)

117

Figure 3-3 (cont’d)
D
15N–Ornithine

AM roots
0.6

ERM

0.5

Fractional labeling

E

Fractional labeling

0.6

0.4
0.3
0.2
0.1

0.5
0.4
0.3
0.2
0.1

15N Arginine

0

0
0

8

0

16 24 32 40 48 56 64 72

8

16 24 32 40 48 56 64 72
Time (h)

Time (h)

E
15N-Arginine

1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

0.7

AM roots

0.6
Fractional labeling

Fractional labeling

ERM

0.5
0.4
0.3
0.2
0.1

0

8

0

16 24 32 40 48 56 64 72

0

Time (h)

8

16 24 32 40 48 56 64 72
Time (h)

118

Figure 3-3 (cont’d)

F
15N-Putrescine

AM roots
Fractional labeling

0.25
0.2
0.15
0.1
0.05
0
0

8

16

24

32

40

Time (h)

119

48

56

64

72

A

Glutamate (M+1) 15N isotopomer

160
140
nmol/g (DW)

120
100
80
60
40
20
0
0

10

20

30

40
Time (h)

50

60

70

80

Figure 3-4 Time course of the free N metabolites 15N-Isotopomers (mass isomers)
in the extraradical mycelium (ERM) after the addition of 4 mM K15NO3 to the ERM.
M+1, singly labeled, M+2 doubly labeled, etc. position of label within a molecule is
indicated with inset chemical structures. The levels of 15N isotopomers of glutamate (A)
ornithine (B) and arginine (C). Means and standard errors of means of three replicates.
R, nitrogen is in the R-group; NR, nitrogen is not in the R-group; G, nitrogen is in the
Guanidine group; NG, nitrogen is not in the Guanidine group.

120

Figure 3-4 (cont’d)

B

15N

Onithine isotopomers

1600
1400

nmol/g (DW)

1200
1000
M+1 (NR)
800

M+1 (R)

600

M+2

400
200
0
0

10

20

30

40
Time (h)

121

50

60

70

80

Figure 3-4 (cont’d)

C

NG
15N

Arginine isotopomers

G
3250

M+1 NG

3000
M+1 G

2750

M+2(NG&G)

2500

M+2 (2G)
M+3 (2G&1NG)

2000

nmol/g (DW)

nmol/g (DW)

2250

M+4

1750
1500
1250
1000
750
500
250
0
0

8

16

24

32

40

48

56

64

72

M+1 NG

200
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0

M+1 G
M+2(NG&G)
M+2 (2G)
M+3 (2G&1NG)
M+4

0

80

2

4

6

8

10 12 14 16 18

Time (h)

Time (h)

122

A
60

Glutamate 13C5

50

nmol/g (FW)

40
30

ERM
AM roots

20
10
0
0

6

12

18

24
30
Time (h)

36

42

48

Figure 3-5 Timing course of 13C labeling of N metabolites in the extraradical
mycelium (ERM) and the mycorrhizal roots after the addition of 0.5
mM 13C6 arginine to the fungal ERM compartment. Levels of A) glutamate 13C5, B)
pyrroline-5-carboxylate 13C5, C) ornithine 13C5 (the right panel is the earlier time points
of the left panel), D) arginine 13C6 (the right panel is the earlier time points of the left
panel) and arginine 13C5 , and E) putrescine 13C4. Symbols show the means and
standard errors of the means of three biological replicates.

123

Figure 3-5 (cont’d)

B

Pyrroline-5-carboxylate

13C5

45

nmol/g (FW)

40
35
30
25

ERM

20

AM roots

15
10
5
0
0

6

12

18

24
30
Time (h)

124

36

42

48

Figure 3-5 (cont’d)

C

Ornithine 13C5

1800

ERM
AM roots

1500

Ornithine

300

13C
5

nmol/g (FW)

nmol/g (FW)

250
1200
900
600
300

200
150
100
50

0
0

6

12

18 24 30
Time(h)

36

42

48

0
0

125

6

12

18 24 30
Time (h)

36

42

48

Figure 3-5 (cont’d)
Arginine

13C
6

13C
6

nmol/g (FW)

Arginine

10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0

ERM
AM roots

0

6

12 18 24 30 36 42 48
Time (h)
900

1200
1100
1000
900
800
700
600
500
400
300
200
100
0

ERM
AM roots

0

6

12

18

24
Time (h)

Arginine

800

13C
5

700
nmol/g (FW)

nmol/g (FW)

D

600
500
ERM

400

AM roos

300
200
100
0
0

6

12

18

24 30
Time (h)

126

36

42

48

30

36

42

48

Figure 3-5 (cont’d)

E

12

Putrescine 13C4

nmol/g (FW)

10
8
6

ERM
AM roots

4
2
0
0h

3h

6h

12h
Time

127

24h

48h

A
ERM

27
24

Relative expression

21
18
15
12
9
6
3

NiR

OTC

CAR1

OAT1

OAT2

P5CD

GDH

2h
4h
8h
24h
72h

2h
4h
8h
24h
72h

2h
4h
8h
24h
72h

2h
4h
8h
24h
72h

2h
4h
8h
24h
72h

2h
4h
8h
24h
72h

2h
4h
8h
24h
72h

2h
4h
8h
24h
72h

0

ODC

Figure 3-6 The expression of primary N metabolic and transport genes in the arbuscular mycorrhizal symbiosis
after the addition of 4 mM KNO3 to the fungal extraradical mycelium (ERM). Bars are as follows: 2 h (white bars), 4 h
(gray bars), 8 h (hatched bars), 24 h ( black bars), and 72 h (striped bars). Gene expression of nitrite reductase (NiR),
ornithine transcarbamylase (OTC), arginase (CAR1), ornithine aminotransferase 1 and 2 (OAT1, 2), pyrroline-5carboxylate dehydrogenase (P5CD), glutamate dehydrogenase (GDH), and ornithine decarboxylase (ODC) was
measured by quantitative real-time PCR. Gene expression was measured in ERM (A) and intraradical mycelium (IRM) (B)
with fungal ribosomal protein S4 gene as the reference. Means and standard errors of means of three biological replicates
and each had three technical replicates.
128

NiR
OTC
CAR1
OAT1

129
OAT2
P5CD
GDH

2h
4h
8h
24h
72h

2h
4h
8h
24h
72h

2h
4h
8h
24h
72h

2h
4h
8h
24h
72h

24

2h
4h
8h
24h
72h

2h
4h
8h
24h
72h

2h
4h
8h
24h
72h

2h
4h
8h
24h
72h

Relative expression

Figure 3-6 (cont’d)

B

IRM

21

18

15

12

9

6

3

0

ODC

Table 3-1 Multiple reaction monitoring (MRM) transitions, optimizing source cone
voltages, collision cell voltages, and analyte retention time (for 13C labeling
experiment).
Compound
Precursor
13
ion>
( C
isotopomers) product ion
(m/z)
Arginine
M+0: 175.1>60
M+5: 180.1>60
M+6: 181.1>61
Glutamate
M+0: 148.02>83.84
M+5: 153.02>87.84
Glutamine
M+0: 147.06>129.95
M+5: 152.06>134.95
Ornithine
M+0: 133.1>70
M+5:138.1>74
Putrescine
M+0: 89.03>71.79
M+4:93.03>75.79
Pyrroline-5M+0: 113.98>67.75
Carboxylate M+5: 118.98>71.75

Cone
Voltage
(V)

Collision
Voltage
(V)

Retention
time (min)

Function
no.

22

22

6.08

1, 3

22

16

1.23

2, 3

16

10

1.07

2, 3

16

16

5.52

2, 3

50

10

6.05

2

16

10

1.15

2, 3

130

Table 3-2 Primers for real-time PCR.

Primer

Forward (5’-3’)

Reverse (5’-3’)

Nitrite reductase
(GiNiR)
Ornithine
transcarbamylase
(OTC)
1-Pyrroline-5carboxylate
dehydrogenase
(P5CD)
Glutamate
dehydrogenase
(GDH)
Ornithine
aminotransferase
1 (GiOAT1)
Ornithine
aminotransferase
2 (GiOAT2)
S4 ribosomal
protein (GiSR4)

CCAGCTATACGCGTCA
ATTTT
GCTCAACGTATAAAAG
ATTTTGCTG

AGGCGTAATTTCA
CCTCCAG
CATTAGTGCATCA
ATAACGGCTA

TTAAGACCGGTCCTCC
TGAA

AAACTTGGGCTTC
CTGCTTT

This study

TTCCCTTTACACCATAA
TACACACC

CTGCGCCAGATAT
GGGTACT

This study

GGTTCGAGCGGATATT
GTCATAC

AGGACTGCTGATA
TTGGGTAAACG

Tian et al.,
(2010)

CGGGTAAGATGCTTTG
TCAAGA

Tian et al.,
(2010)

AAGCCGCCTACGTGTC
GTT

GCCTGAAAGTGCT
TTACCAAGTATAA
C
AACAGGTGGTAGA
AATATGGGAAG

TGATGCGGTGAATCCT
AAGAGA
TTGATTGCGTTACCAA
AAATGG

GATCAAGTGCATC
AACGTCAAAG
TCGAAATACAACC
AGTCACCAAGA

Arginase
(GiCAR1)
Ornithine
decarboxylase
(GiODC)

131

Referenc
e
This study
This study

Govindara
julu et al.,
(2005)
Tian et al.,
(2010)
Tian et al.,
(2010)

Table 3-3 Nitrogen metabolic genes identified in Rhizophagus irregularis in this
study.
Genes (encodes Closest
the enzymes
homolog
below)
(Nucleotides
similarity)
Blastn
Accession:
Nitrite reductase ZP_01854725
(Planctomyces
(NiR)
maris DSM
8797)
Identities=51%
Accession:
Glutamate
Q54KB7
(Dictyostelium
dehydrogenase
discoideum)
(GDH)
Identities= 61%
Accession:
DQ662599
Ornithine trans(Neocallimastix
carbamylase
frontalis)
(OTC)
Identities=76%

Closest homolog R. irregularis genome
(amino acids
reference**
similarity)
(gene model name)
Blastx
Accession:
ZP_01854725
(Planctomyces
maris DSM 8797)
Identities=56%

Accession:
EFA84685
(Polysphondylium
pallidum PN500)
Identities= 61%
Accession:
KFH69100
(Mortierella
verticillata NRRL
6337)
Identities= 68%
Accession:
Accession:
KFH71447
KFH71447
Pyrroline-5(Mortierella
(Mortierella
carboxylate
verticillata NRRL verticillata NRRL
dehydrogenase
6337)
6337)
(P5CD)
Identities= 67% Identities= 67%
** http://genome.jgi-psf.org/Gloin1/Gloin1.home.html

132

CE150538_1056

e_gw1.12044.3.1
(accession: EXX78633)

MIX6106_366_99
(accession: EXX65553)

fgenesh1_kg.1256_#_3
_#_ACTTGA_L001_R1
_(paired)_contig_4977

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Chapter 4
Conclusions and future research

137

Conclusions
Nitrogen nutrition has been a topic of considerable interest and growing
importance in mycorrhizal research. AMF can increase the uptake of N by host plants
from the soil under natural and perturbed conditions, and transfer from AMF can
account for significant proportions of N in plants in controlled model systems. However,
our knowledge of the nutritional importance of, as well as of the enzymes and
transporters involved in, N transfer is still limited. Furthermore, important questions
remain to be answered about the mechanism and regulation of nitrogen handling and its
transfer from fungus to the host plant. In this study, I highlighted recent findings about
nitrogen transfer and metabolism in AM symbiosis and report my findings concerning
the remaining unanswered questions. We developed a sterile and leakage-free whole
plant two-compartment culture system allowing the exclusion of other microbes and the
avoidance of nutrient diffusion between compartments. Low cost, easy to use,
reproducible and autoclavable, these microcosms make feasible investigations of
mycorrhizal transport, gene expression, metabolisms and nutrition that have hitherto
been challenging or impossible. The uptake and transfer of 35SO42- by the fungal partner
to the host plant was demonstrated in the microcosms. Based on our data, we conclude
that sulfate is transferred by R. irregularis to host plants leaves within a day as has been
reported for AM transformed roots (Allen and Shachar-Hill, 2009). Using this microcosm
to study the role of N transfer by AMF to host plants indicated, that N transfer by AMF
conferred growth and reproductive benefits to the host plant.
15N
15N

isotopic labeling results showed increased levels of N metabolites with high

percentage labeling in mycorrhizal plants after adding
138

15NO

3.

Labeling time course

data are consistent with the current N transfer model in which inorganic N is taken up by
the fungal ERM and assimilated via the GS-GOGAT pathway raising the levels of
glutamate and glutamine. Nitrogen is then incorporated into arginine via enzymes of the
urea cycle and is translocated to the fungal IRM in colonized root tissues and broken
down into ornithine and urea that is in turn is broken down to release ammonium which
is exported from the fungus and imported by the host into the root cortical cells. The
presence of high levels of
amount of

15N

15N

labeled metabolites in shoots indicated that a significant

being transferred to the host plants. One of the major questions in N

transfer in the AM symbiosis was the role, if any, of nitrate translocation. Based on

15N

labeling experiments in microcosms (described in Chapter 2) and monoxenic culture
system (mycorrhizal transformed roots) we conclude that arginine translocation is not
the only pathway for N movement and transfer in AM symbiosis, and that nitrate is also
directly transferred by the AM fungus to host plants. Gene expression of a putative
fungal nitrite reductase indicated that a significant amount of nitrate was assimilated in
the ERM but not IRM, which is consistent with the labeling and the revised N transfer
model.
Investigating the origin and fate of ornithine was another objective of this study.
The presence of

15N

labeled pyrroline-5-carboxylate as well as

15N-glutamate

in the

ERM suggests a role for these metabolites in making ornithine there since pyrroline-5carboxylate is a biosynthetic intermediate between glutamate and ornithine. This
conclusion was also supported by gene expression analysis of OAT1 and P5CD which
showed a 3-fold increase in gene expression 4h after adding nitrate to ERM. The
presence of

15N

glutamate,

15N

pyrroline-5-carboxylate and low levels of
139

15N

putrescine

in

15N

labeling experiments as well as of glutamate

and putrescine

13C
4

13C ,
5

pyrroline-5-carboxylate

in the IRM after labeling the ERM with arginine

13C
6

13C
5

coincided with

the upregulation of OAT1, OAT2 and P5CD. This suggests that ornithine is converted to
glutamate and to a lesser extent to putrescine but that a significant proportion is
translocated back to the ERM. Together, with the translocation and transfer of nitrate,
this additional flux in the N transfer network provides increased flexibility and potential
for improved efficiency. The expression of a putative GDH gene is slightly upregulated
in the ERM and highly upregulated in IRM indicating that GDH may play a dissimilatory
role in breaking down glutamate to release more ammonium to the host.

Future research
In this dissertation, I report experimental data on the metabolism and transfer
of N from the fungus to the host plant in the AM symbiosis and its role plant growth.
We extended our understanding of the N transfer network, including the origins and
fate of key intermediates and shed light on its regulation. However, there are some
unresolved questions that need investigation. The way that arginine is transferred
from the ERM to the IRM is still not well understood. Passive diffusion is too slow for
the long distances from ERM to IRM. It has been found that arginine is bound to
polyphosphate in ectomycorrhizal symbiosis (Martin, 1985), and polyphosphate is
believed to move in vesicles or tubular vacuoles (Dürr et al., 1979). Accordingly,
where polyphosphate from and how arginine is bond with polyphosphate are the key
points for illuminating the transfer of arginine.

140

R. irregularis does not down-regulate the genes for arginine synthesis in the
ERM when intracellular arginine levels are high, as occurs for example in
Saccharomyces cerevisiae suggesting either that arginine is sequestered, or perhaps
that host demands for N are communicated to maintain N flow. How the genes
involved in nitrogen metabolism and transport are regulated need further studies.
Studying the enzymatic activity of the putative genes which were identified to
date, will confirm their role in N transfer. Enzyme assays of glutamate dehydrogenase
will clarify its role in N transfer as well as its specificity and regulation. On the other
hand, the possibility of transforming R. irregularis shall lead to a better understanding
of the role of genes in nutrients transfer through studying knockout mutants.
Cross talk between C and N as well as N and P are not well understood and
need further investigation. Furthermore, the incorporation of Medicago truncatula
mutants impaired in N assimilation pathway genes would help reveal the extent to
which AMF contribute to plant N metabolism and the possibility of plant-to-plant N
transfer.

141

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through

an

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143