Environmental and Experimental Biology (2014) 12: 187–197
Original Paper
Xylem-mediated channeling of nitrogen in broad bean
(Vicia faba)
Bikash Baral1,3*, Jaime A. Teixeira da Silva2, Vimal Narayan Gupta3
1
Biotechnology Unit, Nepal Academy of Science and Technology (NAST), GPO Box: 3323, Kathmandu, Nepal
P.O. Box 7, Miki-cho Post Oice, Ikenobe 3011-2, Kagawa-ken, 761-0799, Japan
3
Biotechnology Unit, Central Department of Botany, Tribhuvan University, Kirtipur, GPO Box: 26429, Kathmandu, Nepal
2
*Corresponding author, E-mail: bikubaral@yahoo.com
Abstract
Nitrogenous compounds in leguminous plants translocate in the form of ureides, allantoin and allantoic acid, the oxidation products
of de novo purine synthesis, from the nodules to the aerial parts. he nodules are the main sites of ureides synthesis through the
coordination of the plant-bacteria association. However, aspects related to the occurrence, localization and properties associated with
the enzymes involved in the assimilation of ureides in shoot tissues have not yet been fully resolved. In this study, a modiied and
simpliied automated analysis was used to determine allantoin concentration in plant xylem exudates. he total amount of ureides
translocated to the aerial parts of faba bean (Vicia faba L.) plants was quantiied by the stem sap extraction method using allantoin as
the internal standard. Other parameters measured at diferent time intervals (from sowing to harvest) included shoot and root length,
symbiotic parameters, plant biomass, and the nitrogen (N) status of the stem, leaves and nodules. Two rhizobial isolates (KR1 and MR2),
isolated from Pisum sativum L. var. ‘Macrocarpon’ and Phaseolus vulgaris L. (cv. ‘Carioca 29’) plants, respectively, were selected from
entirely diferent agro-climatic regions. MR2 accumulated more ureides (587.28 mg L–1) than KR1 (573.33 mg L–1) when assessed at
harvest. Plants were harvested at regular intervals for dry matter and stem-extracted exudates. Results were insigniicant (P > 0.05) for
diferent inocula, shoot and root length and nodule N, but were signiicant (P < 0.05) for both rhizobial isolates during nodulation. he
concentration of ureides, which were compared with total N concentrations in nodules, stems and leaves, were signiicantly diferent
(P < 0.01). We conclude that the percentage of N in the form of ureides, however, does not always indicate the ability of the plant to
symbiotically ix N2.
Key words: allantoin, assimilate partitioning, crop productivity, nitrogen transporters, nodules, regulators, Rhizobium, ureide biogenesis.
Abbreviations: ANOVA, analysis of variance; BNF, biological nitrogen ixation; DM, dry mass; DPG, days post germination; Gln,
glutamine; I+N, Rhizobium inocula with nitrogen; I, inocula without nitrogen; KR1, Rhizobium leguminosarum bv. viciae; LSD, least
signiicant diference; MR2, Rhizobium leguminosarum bv. phaseoli; N, nitrogen only; QC, quality control; SNF, symbiotic N2 ixation;
YEM, yeast extract mannitol agar.
Introduction
Faba bean (Vicia faba L.) is one of the major staple pulse
crops grown for dry seeds and green pods throughout
the world (Telaye et al. 1994). Beans possess high protein
content (26 to 41%) and are thus very important in the
combat of nutrient depletion, especially in economically
disadvantaged countries. Also, this pulse, together with the
microbial symbionts found in the soil in association with
the plant, help to recharge the soil by ixing nitrogen from
the atmosphere. Hence, inding a better rhizobial isolate
of agronomic importance has been a major challenge,
especially for crops growing at high altitudes.
Plants demand a high supply of nitrogen for their growth
and development, and are used for synthesizing amino
acids (the building blocks of proteins) and nucleic acids.
he nitrogen status of legumes is a well-documented theme
but still has ample aspects that still need to be researched.
Environmental and Experimental Biology ISSN 2255-958
he synthesis and translocation of nitrogenous compounds
that form and translocate from the source to the sink (place
of usage) during the nitrogen (N2)-ixation process takes
place by a vascular network (xylem and phloem) that
connects the internal parts of plant organs (Tegeder 2014).
For the synthesis of amino acids, nitrogenous compounds
need to be incorporated into the carbon skeleton of plant
metabolites (Udvardi, Poole 2013). he transport (basically
of N) is mainly achieved and facilitated by sharing and
exchanging signals between plant organs (nodules) moved
by the mass low of solutes (hompson, Holbrook 2004),
via the xylem and phloem (Brenner et al. 2006; Atkins,
Smith 2007). he N2-ixation takes place by the induction
of nodule-speciic plant genes (genes encoding symbiotic
leghemoglobins) with the development of nodules (Ott
et al. 2005). Nodules, which are closed vesicles (outer
protuberance from the roots), are the main starting point
for the synthesis of these amino acids by a close association
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B. Baral, J.A. Teixeira da Silva, V.N. Gupta
with N2-ixing bacteria (Tegeder 2014). he incorporation
of nitrogenous compounds, minerals (both macro and
micro) and other nutrients into the plant metabolic
pathway are a clear sign that plants have accumulated
carbon into its skeleton. his incorporation balances the N
: C ratio in a ixed amount within the plant (Layzell et al.
1981; Nicolardot et al. 2001).
he biogenesis of ureides takes place in root nodules,
which serve as the source (Rentsch 2007). It involves a
rigorous pathway (Atkins, Smith 2000; Kim et al. 2007) that
starts with the assimilation of ammonia, which is produced
by nitrogenase (EC 1.18.6.1; Eq. 1; Schubert 1995). he
reduction of N2 to ammonia (NH3) mediated by nitrogenase
is labile in oxygen (Udvardi, Poole 2013), and subsequently
gives rise to glutamine (Masalkar et al. 2010; Tegeder 2014)
and other amino acids (glutamate, aspartate and glycine)
within the infected cells. Basically, two enzymes, namely
xanthine dehydrogenase (EC 1.1.1.37) and urate oxidase
(EC 1.7.3.3), seem to play a key role in the biosynthesis
of ureides in legume nodules (Tajima et al. 2004). hese
ureides, the inal dominant nitrogenous products, are then
translocated from the nodules to the roots and aerial parts,
namely the leaves, lowers and pods or collectively the sink
(Rentsch 2007; Collier, Tegeder 2012) where they are inally
catabolized (Atkins, Smith 2007).
he initiation of synthesis of ureides in root nodules
takes place via the intimate coordination and complex
interplay between plant (host) and bacteria (symbiont),
and involves several processes (Atkins, Smith 2000; Rice et
al. 2000). Immediately ater N2 ixation in the bacteroids of
infected root cells, NH3, ammonium ion (NH4+), or amino
acids are released or transported from the symbiosome to
the cytosol, utilizing them for Gln synthesis with the aid of
Gln synthetase (Day et al. 2001; Smith, Atkins 2002; Lodwig
et al. 2003). his process is then followed by a purine
synthesis pathway in plastids or mitochondria (Smith,
Atkins 2002; Werner, Witte 2011) and purine degradation
via xanthine in plastids or the cytosol of infected root cells
(Todd et al. 2006). he ureides are synthesized from purines
as allopurinol (a xanthine dehydrogenase inhibitor),
which inhibits the further production of ureides (Quiles
et al. 2009). he ureide allantoin is inally synthesized in
the peroxisomes of non-infected root cells from a purine
degradation product, uric acid (Brychkova et al. 2008),
through several intermediate steps (Todd et al. 2006;
Werner, Witte 2011).
N2 + 16 ATP + 8 e– + 10 H+
2 NH4+ + H2 + 16 ADP + 16 Pi (Eq. 1)
Following N2 ixation in legume nodules, the products –
especially ureides – are exported to the nodule vasculature,
then to leaves and the shoot via the xylem, generally in the
form of the amides asparagine (Asn) and glycine (Gln)
(Quiles et al. 2009; Sulieman, Tran 2013) or the ureides
allantoin and allantoic acid, which are the oxidation
products of purine synthesis in nodules (Todd et al. 2006).
188
his ixed N is transported all the way to the active sinks
(lowers, pods and leaves) but can leak to adjacent cells of
the xylem stream during transport. Accumulating evidence
suggests that ureides comprise up to 90% of the total N
transported in the xylem of N2-ixing tropical legumes
(Todd et al. 2006) and can be stored in high amounts in
diferent plant organs (Tan et al. 2008). Due to their high
concentrations in the vascular system and in certain plant
tissues (as much as 59 nM in the paraveinal mesophyll;
Matsumoto et al. 1977; Costigan et al. 1987), ureides are
believed to have an important function in N transport (2 to
3 mg C mg–1 symbiotic N ixed; Valentine et al. 2011) and
storage in legumes. he accumulation of ureides in legume
nodules under soil-water deicit might trigger a feedback
mechanism that results in decreased N2 ixation (Sinclair,
Serraj 1995; Serraj et al. 1999a; Goh, Bruce 2005) and might
also be involved in protecting plants against abiotic stress
(Werner, Witte 2011). In addition to N from root nodules,
N assimilated from soil-combined N and from N cycling
within the plant will all be present in the transpiration
stream (Stark, Richards 2008); however, the majority of
carbon exported to the xylem stream from nodules is in
the form of ureides, while this partitioning of nitrogenous
compounds requires the action of integral membrane
transporters (Rentsch et al. 2007).
he objectives of this study were to understand the
extent to which ureides are assimilated and translocated to
the aerial part of a legume crop, broad bean (Vicia faba L.),
at diferent time intervals or developmental stages (from
sowing to harvest) by comparing their concentration to
the total N concentration in leaves, stems and nodules. In
addition, in a bid to increase nitrogen ixation by legumes
and pulse crops growing at high altitudes, which is frequent
in Nepal, two new N2-ixing bacterial strains for broad bean
were unveiled and characterized.
Materials and methods
Chemicals and reagents
All chemicals employed in these experiments were of
analytical grade purchased from Sigma Chemical Co. (St.
Louis, USA).
Experimental design
V. faba (cv. ‘Imposa’) was used as the model plant to study
N2-ixation by microbial symbionts and the localization
of ixed N in the aerial parts. Plants, which were raised in
a greenhouse at 28 ± 1 °C and a 16-h photoperiod under
natural light intensity, were potted at four plants per pot in
5-L pots for a total of 40 days. In total, four treatments were
applied: bacterial inoculum (I); urea [CO(NH2)2] at 50 mg
kg–1 of soil as the source of mineral N; inoculum and urea
combined (I+N); and the control (C). Plants were watered
with N-free water when needed and micronutrients
were added as Hoagland’s solution (Hoagland, Arnon
Xylem-mediated channeling of nitrogen in broad bean
1950). More details about the protocol may be found in
Baral et al. (2012). his experimental design produced 32
combinations for each rhizobial isolate.
Isolation and selection of efective rhizobial strains
Studies on rhizobial isolates with reference to synthesis
of ureides by legumes within Kathmandu and Manang
do not exist. Moreover, these locations lie on completely
diferent geographical terrains with vastly diferent
climatic conditions and may harbour rhizobial strains
with potent and highly efective N2-ixing ability, serving
as the basal impulse for this research. Manang lies in the
trans-Himalayan region from sub-alpine to high Alps with
an arid climate, while Kathmandu, a fertile valley, lies in a
temperate zone with high humidity. Composite soil samples
from a depth of 10 to 15 cm from two diferent agroclimatic regions (Kathmandu: 27° 43’ 06” N, 85° 19’ 02” E;
Manang: 28° 40’ 03” N, 84° 15’ 01” E) were sited through a
5-mm mesh (pH 6.0 to 6.5) in pots 16 × 18 cm in size and
transferred to a glasshouse and were used as the source of
rhizobial isolates. Pisum sativum L. var. ‘Macrocarpon’ and
Phaseolus vulgaris L. (cv. ‘Carioca 29’) plants were raised
from viable (89% germination) seeds in a greenhouse (28
± 1 °C) in 5-L pots, with four plants per pot and nodular
rhizobial isolates were isolated using the trap method
(Vincent 1970). Two diferent plant species were selected
for trapping compatible rhizobial isolates so as to maximize
the success of trapping. Seeds were initially germinated in
sterile Petri dishes on ilter paper (Whatman No. 1; SigmaAldrich) moistened with double distilled water. Only seeds
that appeared healthy and seedlings that grew uniformly
were used for the experiment.
Authentication, acid production and mean generation
time of rhizobial isolates
he two rhizobial isolates (Rhizobium leguminosarum
bv. viciae and R. leguminosarum bv. phaseoli, isolated
from Kathmandu-based P. sativum and Manang-based
P. vulgaris, are respectively designated as KR1 and MR2
throughout the remainder of the manuscript) were used
as four treatment combinations and four replications of
each, as suggested by Boone et al. (2001). Rhizobial isolates
were isolated by gently liting the legumes in soil from
their original sampling sites and the endogenous bacteria
(microsymbionts) were isolated by the trap method using a
protocol devised by Baral et al. (2012).
Collection of stem sap
he irst harvest was performed at 20 days post germination
(DPG) and repeated every 5 d until 45 DPG. Four plants
of each treatment were uprooted i.e., one plant per pot of
each replication every ive days starting from 20 DPG were
harvested. A portion of the stem (approximately 3.0 cm
long) at the base just above the radicle, was ground inely in
liquid N2 by using a mortar and pestle then iltered through
Whatman No. 1 ilter paper and centrifuged (10 000 × g, 20
min, 4 °C). Vials with stems in phosphate bufer solution
were stored at 0 °C until extracted (Herridge 1984). he
supernatant was used to determine the ureide content
(de Silva et al. 1996; Herridge et al. 1996). Phenols were
efectively removed from H-bonded complexes following
the addition of 1.5 g of insoluble polyvinylpyrrolidone,
which was used to adsorb phenols and thus obtain active
soluble enzymes.
Analytical techniques for the estimation of ureides
he experiment used the Herridge and Peoples (1990)
protocol with minor modiications, where needed. Briely,
Vicia faba L. plants were grown under aseptic conditions,
subjected with diferent treatments (I+N, I, N and C). he
plants were uprooted at diferent time intervals starting
from 20 DPG. A portion of the stem (approximately 3.0
cm long) just above ground level was sampled and inely
ground with a mortar and pestle with the aid of liquid
N2. One mL of each plant sample (the ground mixture),
a quality control (QC) sample (i.e., the test sample for
measuring consistency), and standard or distilled water
(blank) were pipetted out into 15-mL tubes, treated with
5 mL of distilled water and 1 mL of 0.5 M NaOH followed
by vigorous shaking for a few seconds. NaOH was used for
color development at room temperature. he tubes were
then placed in a boiling water bath for approximately 7
min, then cooled in cold water. To each tube, 1 mL of HCl
(0.5 M) was added, adjusting the pH to between 2 and 3
(at higher pH, uric acid forms the duly charged full urate
ion) followed by the addition of 1 mL of phenylhydrazine
solution. he tubes were again transferred to a boiling water
bath for 7 min then let to cool in an ice-cold alcohol bath
for 10 min. Fresh solutions of phenylhydrazine HCl and
potassium ferricyanide were applied to get better results.
he cooled tubes were treated with 3 mL of precooled
HCl (conc.) and 1 mL of potassium ferricyanide. his was
performed in a fume cupboard within the shortest possible
time span. he solutions were then thoroughly mixed and
transferred to 4.5-mL cuvettes at room temperature. he
absorbance with a blank (or distilled water) was adjusted
at 522 nm. he absorbance for each tube was read exactly
20 min ater the addition of potassium ferricyanide. An
allantoin standard curve was then plotted against allantoin
concentration (mg L–1) on the X-axis versus optical density
(at 522 nm) on the Y-axis.
For accurate weighing on a balance, a large quantity of
each working standard solution with pH 6.2 (by the dropwise addition of 0.1N HCl and 0.1N NaOH) was made. For
this, 50 mg of allantoin (used as an internal standard) was
weighed and then transferred to a 500-mL volumetric lask.
his was dissolved in 100 mL of 0.01 M NaOH, and made
up to volume with distilled water (dH2O). he addition
of NaOH served only to dissolve allantoin. For preparing
50 mL of the working standards (10, 20, 30, 40, 50 and 60
189
B. Baral, J.A. Teixeira da Silva, V.N. Gupta
mgL–1), a stock solution of weight 5, 10, 15, 20, 25 and 30 g
was accurately weighed into a 50-mL volumetric lask and
made up to volume with dH2O. Each working standard was
stored at 0 °C as a small aliquot in a freezer and only the
necessary quantities were thawed while the remainder was
discarded.
he analysis of ureides was based on a colorimetric
method (Herridge, Peoples 1990), which is a rapid and
inexpensive method in which allantoin is converted to
allantoic acid followed by hydroxylation to urea and glyoxylic
acid in a weak solution. Glyoxylic acid thus formed reacts
with phenylhydrazine-HCl producing a phenylhydrazone
derivative of the acid which is an unstable chromophore
in reaction with potassium ferricyanide; the color obtained
was read at 522 nm using a UV/Vis spectrophotometer
(6715, Jenway; Bibby Scientiic Ltd., Stone, UK).
Spectrophotometric methods used were based on the
Rimini-Schryver reaction, which is the condensation of
a hydrazine derivative with keto-acids to give a colored
product. he absorbance of standard and samples was
taken as quickly as possible since the colour fades out
gradually and the absorbance decreases with time and
thus the process cannot be interrupted in the middle of
the experiment. herefore, a few samples in duplicate were
processed in each run using a set of standards and a blank
(dH2O) in parallel. For consistency between the diferent
time periods in the same laboratory, a QC sample was
performed at each run with a 20X dilution (Kaito et al.
1977).
he concentration of the QC and the other samples
was determined using the following formula (de Silva et al.
1996; Herridge et al. 1996):
C = (Y – a) / (b × F),
where C and Y are the concentration and absorbance of
the unknown, a and b are the intercept and slope of the
standard curve respectively, and F is the dilution factor.
Statistical analysis
he experiment was arranged in a randomized complete
block design. Two rhizobial isolates (KR1 and MR2) with
four treatment combinations and four replications of each
block were employed for the experiment giving a total
of 32 combinations. All statistical analyses were carried
out using SPSS version 16.0 (IBM SPSS, NY, USA) and
Excel 2007 (Microsot Oice; Microsot Corp., WA, USA).
All comparisons were performed by applying t-tests for
independent samples and Duncan’s multiple range test at
α = 0.05 (Somasegaran, Hoben 1994). he F-ratio between
variables was employed. ANOVA with post-hoc LSD (least
signiicant diference; Baye’s LSD) was used to compare the
values of two adjacent means. he independent samples
t-test was employed for comparing nodule induction
and biomass, shoot and root length and total N ixed.
Similarly, the F-test was employed for comparing ureide
concentration with diferent treatments (bacteria, nitrogen
and control). he error bars in the igures represent the
standard error (± SE).
Experimental parameters
Plants were harvested ater 20 DPG at 10-day intervals
lasting for a total period of 40 days. Ater each harvest (i.e.,
every 10 day), multiple experimental parameters [total
number of nodules, shoot and root length (longest root),
shoot dry biomass (dried in an incubator at 30 °C for three
days; Bod Incubator, Proliic instruments, Mumbai, India),
stem, leaf and nodule N were determined.
Estimation of ureides
he allantoin curve, which was used as an internal standard
by which the ureide (allantoin) content of the plant was
determined at various intervals, showed that allantoin
concentration decreased over time. Experiments were
carried out separately for both isolates from 20 DPG every
ive days until 45 DPG. he concentration of ureides in
plants inoculated with KR1 and MR2 isolates at 20 DPG was
128.175 and 210.85 mg L–l, respectively, which increased by
40 DPG (KR1 = 573.333 mg L–l, MR2 = 587.237 mg L–l).
In other words, the translocation of ureides was higher
when the P. sativum inoculum was used. he MR2 ureide
content declined slightly at 40 DPG, which was unexpected.
he F-ratio (P-value: 0.202) and correlation coeicient,
assessed separately for the two rhizobial isolates (0.9613 for
KR1; 0.971 for MR2), showed highly signiicant diferences
(P < 0.01). he concentration of ureides increased in the
presence of urea as the N source more than in the control
(Fig. 2).
Total nitrogen
he total N concentration (% of dry mass) in plant samples
(nodules, stem and leaves) was determined at 20, 30 and
40 DPG with four replications of each treatment using
a Micro-Kjeldahl method in three diferential steps:
wet digestion, distillation and titration for ammonium
estimation (ammonia concentration is proportional to
nitrogen content in the sample; PCARR 1980; Baral et al.
2012; Muñoz-Huerta et al. 2013).
190
Results
Characterization of microbial symbionts
Our analyses reveal that both isolates were Gram-negative,
as expected, fast growing, and acid-producing with no
signiicantly diferent doubling time.
Shoot and root length
MR2 inoculum had a signiicantly greater efect on shoot
length than KR1 (treatments = 8.788 (signiicant); blocks
= 48.326 (signiicant); P < 0.01; LSD0.05 = 11.49 cm). he
root length of test plants inoculated with either rhizobial
inoculum increased (treatments = 3.956 (signiicant);
blocks = 153.646 (signiicant); P < 0.05; LSD0.05 = 6.906 cm)
(Fig. 1).
Xylem-mediated channeling of nitrogen in broad bean
Fig. 1. Shoot length (SL) and root length (RL) of Vicia faba on diferent days ater infection by rhizobial isolates KR1 and MR2. DPG =
days post germination. n = 16 (biological replicates 4 × analytical replicates 4), mean ± SE. Diferent letters within treatments for a single
rhizobial isolate are signiicantly diferent according to DMRT at α = 0.05. White bars = shoot length, black bars = root length. A, C and E
were inoculated by KR1 and B, D and F were inoculated by MR2 and sampled at 20, 30 and 40 days ater sowing, respectively. Treatments:
I+N, inocula with N; I, without N; N, nitrogen only; C, control.
he concentration of ureides in young seedlings
increased from 2 to 5 DPG, suggesting that leguminous
plants are capable of producing ureides without the aid or
presence of a microbial symbiont. he N2-ixing ability of
KR1 and MR2 at 40 DPG showed that the latter was more
efective in ixing atmospheric N and translocating it in
the form of ureides to the aerial parts. However, plant age
signiicantly afected symbiotic efectiveness, peaking at the
fourth week of plant growth. he translocation of ureides
was higher in those plants inoculated with MR2 at 40 DPG
(587.237 mg L–1), while plants inoculated with KR1 could
only translocate 573.333 mg L–1 of ureides at 40 DPG (Fig.
2).
Estimation of nitrogen content
he N (%) of nodules, stems and leaves was estimated
using the Micro-Kjeldahl method (Fig. 3). Diferences
were insigniicant for nodule N (%) (treatments =
0.870 (insigniicant); blocks = 0.164 (insigniicant); P
> 0.05; LSD0.05 = 1.022%), stem N (treatments = 1.230
(insigniicant); blocks = 0.747 (insigniicant); P >
0.05; LSD0.05 = 0.898%) and leaf N (treatments = 6.390
Fig. 2. Concentration of ureides in Vicia faba with the application of bacterial inoculum, nitrogen and with control. DPG = days post
germination. n = 16 (biological replicates 4 × analytical replicates 4), mean ± SE. (A) Inoculated by KR1 and (B) inoculated by MR2 at
20, 30 and 40 days ater sowing (DAS). Diferent letters indicate signiicant diferences for each isolate separately according to DMRT at
α = 0.05. Treatments: I+N, inocula with N; I, without N; N, nitrogen only; C, control.
191
B. Baral, J.A. Teixeira da Silva, V.N. Gupta
Fig. 3. Total ixed N by Vicia faba using an inoculum of two rhizobial isolates KR1 and MR2. DPG = days post germination, n = 16
(biological replicates 4 × analytical replicates 4), mean ± SE. Diferent letters within treatments for a single rhizobial isolate within
each treatment are signiicantly diferent according to DMRT at α = 0.05. White bars = leaf N (LN), black bars = shoot N (SN), grey
bars = nodule N (NN). A, C and E were inoculated by KR1 and B, D and F were inoculated by MR2 at 20, 30 and 40 days ater sowing,
respectively. Treatments: I+N, inocula with N; I, without N; N, nitrogen only; C, control.
(signiicant); P < 0.01); blocks = 0.213 (insigniicant); P >
0.05; LSD0.05 = 0.822%). MR2 had a higher total N content
(2.88%) than KR1 (2.78%). Similarly, the total N content
of the stem was highest for MR2 following the application
of urea (2.8%). he percentage of N in leaf blades was
maximum for I+N for both isolates (1.78 and 1.96% N for
KR1 and MR2, respectively; Fig. 3).
Nodulation
Inoculated plants had many efective nodules (determinate
nodules) on their root systems at 40 DPG. Visible nodules
were observed at 20 DPG but were only counted ater 20
DPG (Fig. 3). Treatments I, I and I+N showed maximum
number of nodules for both isolates (KR1 and MR2) at 20,
30 and 40 DPG (P > 0.05).
Symbiotic efectiveness
he efectiveness of the two inocula tested (KR1 and MR2)
at 40 DPG could be ranked (I+N > I > N > C), although
diferences were insigniicant (P > 0.05; LSD0.05 = 0.479). he
formation of nodules (i.e., the number of nodules) by both
rhizobial isolates was signiicantly diferent on diferent
days of the experiment (treatments = 34.992 (signiicant;
P < 0.01); blocks = 19.438 (signiicant; P < 0.01); LSD0.05
= 13.99). Shoot biomass was recorded every 10 d starting
192
from 20 DPG. For KR1, the highest value recorded was for
treatments I+N, N and N at 20, 30, and 40 DPG, respectively
and for MR2, highest values were for I+N, C and C at 20,
30 and 40 DPG, respectively, although diferences were
insigniicant (treatments = 0.492; blocks = 1.368; P > 0.05;
LSD0.05 = 0.6418 g; Fig. 4). he nodules formed by MR2 were
two-fold larger than the KR1-induced nodules, suggesting
that MR2 was a highly efective strain.
Discussion
he Rhizobium-legume symbiosis is the only and
pivotal source for injecting a bulk amount of nutrient N
into agricultural systems (ca. 40 million t of N year–1;
Udvardi, Poole 2013), representing an economical and
environmentally friendly alternative to chemical fertilizers.
his biological nitrogen ixation (BNF) provides about 65%
of the total biosphere’s available N (Lodwig 2003). Legumes
contribute approximately 30,000 t of N annually in Nepal
(Maskey et al., 2001), recharging the soil N pool every year.
Plant productivity in regions with extreme climates, such
as in Nepal, are limited by very low soil fertility (Maskey et
al. 2001). he eicient N supply through microsymbionts
(by inducing nod genes; Rolfe, Gresshof 1988; Gage
2004) may help in stabilizing food production, recharging
Xylem-mediated channeling of nitrogen in broad bean
Fig. 4. Plant dry biomass and nodule dry biomass on diferent days ater infection by rhizobial isolates KR1 and MR2. DPG = days post
germination. n = 16 (biological replicates 4 × analytical replicates 4), mean ± SE. Diferent letters within treatments for a single rhizobial
isolate are signiicantly diferent according to DMRT at α = 0.05. White bars = plant biomass (PB), black bars = nodule biomass (NB).
A, C and E were inoculated by KR1 and B, D and F were inoculated by MR2 at 20, 30 and 40 days ater sowing, respectively. Treatments:
I+N, inocula with N; I, without N; N, nitrogen only; C, control.
soil N reserves, and increased yield of agricultural crops
(Dahal, Dahal 1998; Hartwig 1998). hus, the soil nutrient
content (micro- and-macro nutrients) together with
other characteristics should be considered in conjunction
with the selection of appropriate plant and symbiotic
microorganisms (Maskey et al. 2001). Consequently, some
symbiotic characteristics of rhizobial isolates obtained from
the ield soils of Kathmandu and Manang were compared.
he carbon sink is related to nodule growth and legume
BNF, especially in faba bean (Lawrie, Wheeler 1975). he
N2 ixing kinetics based on nodule biomass (0.05 g N g–1
nodule DM day–1; Boote et al. 2002, 2008), provides evidence
that C is allocated to and used by nodules, incorporating
these processes for potential legume BNF. Legume BNF is
less sensitive to ammonium than nitrate (Bollman, Vessey
2006), and the N2 ixing rate of nodules depends on the
amount of C supplied to the nodules (Haase et al. 2007;
Voisin et al. 2007). In the present study, plant height was
maximum when mineral N was supplied, followed by
bacterial inocula, suggesting that the plants readily absorbed
available N in the soil (until exhaustion) rather than ixing
it through an energy-consuming process (Fig. 1). However,
the longer roots of the experimental plants when N was
supplied can be explained by the healthy growth of these
plants and active mineral absorption activity that could
be used to maintain physiological activities of the plant.
In addition, legumes harbour small bio-factories on their
roots, the nodules, which discharge N2 through a complex
process. In the legume-Rhizobium symbiosis, plants supply
amino-acids to bacteroids, while bacteria reciprocally
supply ammonium to the plants enabling the synthesis
of amino acids, making both reliant on each other in the
amino-acid cycle (Lodwig et al. 2003). hus, legumes enjoy
a competitive advantage in the trade of reduced nitrogen
provided by microbial symbionts over other plants.
he level of ureides difers between drought-sensitive
and drought-tolerant lines of legumes, especially soybean,
with greater levels in the former (Vadez, Sinclair 2001).
During scarce water deicit, these ureidic legumes hoard
ureides in plant tissues and N2 ixation is inhibited
(Alamillo et al. 2010). Exogenously applied ureides may
increase ureide concentration 5- to 8-fold in legumes
(King, Purcell 2005). However, experiments that employ
drought-sensitive or drought-tolerant faba bean lines are
scarce. he concentration of ureides in a plant’s aerial parts
(basically the leaves) is dependent on the water conditions
of the soil during N2 ixation (inhibition of nitrogenase
activity restricts N2 translocation; Serraj et al. 1999b). he
exact determination of ureides in N-fed crops sometimes
becomes diicult, rendering false data. hus, in such a
prevailing scenario, sap nitrate status is a proven method
to detect over-fertilized plants (Muñoz-Huerta et al. 2013).
193
B. Baral, J.A. Teixeira da Silva, V.N. Gupta
Moreover, the factors that inhibit the low of nutrients and
other products from phloem to nodules also inhibit the
low rate of ixed N2 from nodules to the aerial parts (Serraj
et al. 1999b), causing the accumulation of nitrogenous
products in the nodules themselves. Our study conirms this
observation. Also, when the aerial translocation of ureides
was analyzed, the treatment combination of inoculum
and nitrogen (i.e., I+N) and inoculum alone (i.e., I) were
eicient for both isolates, suggesting that the broad bean
plants were able to channel N in the form of ureides (Fig.
2). Moreover, MR2 was a more efective rhizobial symbiont,
as it ixed more total ureides in broad bean when seed were
inoculated (Baral et al. 2012).
Translocation of total ureides was higher in plants
inoculated with MR2 than in KR1. Moreover, ureide
content continued to increase with plant development
until plant harvest (40 days), which indicates that plants
actively ixed N2 and that N was translocated as ureides to
active sinks, i.e., aerial parts, including leaves. Such elevated
expression of ureides in plants shows the active ixation of
N2, which may be regarded as a speciic metabolic feature
of symbiosis in such ureide-forming legumes (Smith,
Atkins 2002). A similar experiment on soybean shoots
found elevated levels of ureides in sinks (pods and leaves)
during the reproductive stage and also in response to the
application of boric acid (Vadez et al. 2000). It has now been
widely accepted that nitrogenous compounds are stored
mostly as ureides in seeds, while they break down into other
products in germinating seeds (Duran, Todd 2012). Further
research is needed to ascertain how long these plants
continue to channel N to the sinks (aerial parts), while
the ureide content of pods also needs to be analyzed. his
experiment suggests that these two Himalayan isolates are
somewhat efective in inluxing or injecting atmospheric
N2 into the biological cycle. However, substantial evidence
using various microbial symbionts from higher elevations
and analyzing several physiological parameters is desired to
further validify this claim.
In a plant-bacterium symbiosis, the plant supplies
amino acids to the microbial symbiont, which helps them
to shut down their ammonium assimilation while the
microbial symbiont helps to recycle the amino acids back
to the plant for asparagine synthesis (Lodwig et al. 2003).
his process helps the bacteria remain associated with the
plant, and also prevents the plants from being dominant
over the rhizobium in symbiosis (Lodwig et al. 2003). he
membrane proteins (transporters) involved in the transport
of N in faba bean have not yet been characterized, although
three transporters (GmUPS1-1, GmUPS1-2 and PvUPS1)
in common bean involved in the transport of ureides are
localized in the plasma-membranes of nodules (Collier,
Tegeder 2012). Moreover, the xylem loading of these N
compounds from the vasculature of nodules remains an
unsolved issue (Collier, Tegeder 2012). In this study, nodule
N was signiicantly higher in most plants at 30 and 40
194
DAS (Fig. 3A, B, C, E) with both rhizobial inocula, which
might be a good indicator of hoarding suicient N as
reserve by the nodules before channeling to the aerial parts
(Fig. 3). Regarding N content of nodules, shoot and stem,
plants inoculated with MR2 showed a higher percentage
of total N which indicates a relatively higher degree of
efectiveness. In most cases in this study, nodules possessed
more N than other parts (shoot or leaves) (Fig 3B, C, E),
suggesting that nodules play a vital role in N2 ixation. he
amount of N in a shoot can be used to indicate that the
ixed N2 is channeled through the xylem to pods, which are
an active N sink (Atkins, Smith 2007). hus, the nodules
(in this case) may act as a transient storage pool of the
ixed N2 and translocated N (Diaz-Leal et al. 2012). his N
test was done using the Micro-Kjeldahl digestion method
(PCARR 1980). However, the N measurement employing
Kjeldahl digestion method only measures nitrogen bound
to organic components (proteins, amino acids, nucleic
acids) and ammonium in the sample, while other forms of
nitrogen (nitrate and nitrite) cannot be measured through
this method (Muñoz-Huerta et al. 2013). hus, for a clear
picture of actual N content, a modiied standard Kjeldahl
procedure employing the addition of salicylic acid prior
to digestion followed by sodium thiosulfate for nitrate
reduction is desired (Lee et al. 1996; Labconco 1998).
Moreover, the lower N2-ixation values represent the lower
biomass production of plants and the nodules (compare
Fig. 3 and Fig. 4) as suggested by Maskey et al. (2001). he
use of these two rhizobial isolates resulted in signiicant
diferences in plant N, plant dry biomass, nodule N and
nodule dry biomass (Baral et al. 2012) (Fig. 4).
Isolate MR2 was more eicient than KR1 in terms of
inluxing N under environmentally stressed conditions
(MR2 was isolated from the high Himalayan region), as
assessed by longer shoots and roots. his might suggest
that the oxygen status rather than the nitrogen status is
responsible for the induction of genes needed for nitrogen
ixation (nif genes) and associated processes, the ix genes
(Dixon, Kahn 2004). At a high oxygen concentration
and the absence of leghaemoglobin, the nitrogenase
in bacteroids might be damaged by oxygen difusion,
limiting the N2-ixing ability and the carbohydrate supply
(Sheehy et al. 1984). he distribution of N may be altered
in the long-distance transport pathway and its symplasmic
discontinuity (Tegeder 2014). hus, such alterations during
the transport of N to the shoot as observed in this study,
rather than to the root, might also be possible (comparing
Fig. 1 and Fig. 3). Ureide catabolism, although equally
responsible for every organism, responds to manganese
(Mn) fertility levels, especially in soybean leaves (Vadez,
Sinclair 2000; Todd et al. 2006). Genotypic variations were
found in soybean cultivars to which Mn was applied to
soil and this Mn apparently had stimulatory efects on
N2-ixation in soybean plants grown under water deicit
(Vadez et al. 2000; Sinclair et al. 2003). However, a detailed
Xylem-mediated channeling of nitrogen in broad bean
molecular pathway regarding the catabolism of ureides and
ureides transporters (other than GmUPS1-1, GmUPS1-2
and PvUPS1 in common bean; Collier, Tegeder 2012) has
yet to be identiied. Moreover, a partitioning in C and N
distribution in the vegetative and reproductive parts of a
legume occurs, rendering the developing seeds a potential
sink for assimilates (horne 1985). Our results indicate that
ureide-N concentration varies with the Rhizobium strain
used and also depends on the plant part analyzed. Also, the
soil mineral N is a potent inhibitor of N2-ixation, hence,
the assessment of soil N before and ater plant harvest is
highly recommended (Hartwig 1998; Herridge et al. 1998).
Conclusions and future research directives
his study presents proof of the symbiotic efectiveness of
rhizobial strains isolated from two entirely diferent regions,
with more eicient N2 ixing ability of the rhizobial strain
isolated from the higher elevation. he reliable and versatile
technique used in our study may have great potential for
ield studies in legumes of diferent geographical terrains,
and may be an important asset for a irst-assessment
of the N2-ixing capability of numerous legume species
grown under ield conditions. he N2-ixing ability of
the micro-symbiont from this high altitude could also be
further justiied by employing other legumes restricted
to the high Himalayas. hus, the present investigation
opens up an array of recent advances in studies on high
altitude legumes and their microbial symbionts. Additional
research is needed for the genetic discourse of rhizobia
inhabiting higher altitudinal soils, to elicit the potential
of the Himalayan rhizobial strains discovered in this
study, and to document the organization of rhizobia in
the rooting zones of legumes. Hence, to better understand
the symbiosis between a plant and bacteria, molecular,
cellular, biochemical and physiological methods need to be
integrated to develop a holistic approach with predictive
models of BNF systems in legumes.
Acknowledgements
he authors thank Mr. Sishir Panthi, Mr. Trilok hakur and Mr.
Deepak Mahat of the Central Department of Botany, Tribhuvan
University for their supportive role during the research period.
he authors declare no conlicts of interest.
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Received 1 August 2014; received in revised form 29 September 2014; accepted 24 November 2014
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