Annals of Botany 99: 765–776, 2007
doi:10.1093/aob/mcm013, available online at www.aob.oxfordjournals.org
Abnormal Root and Nodule Vasculature in R50 (sym16), a Pea Nodulation Mutant
which Accumulates Cytokinins
AL I C I A N . P E P P E R , AN DR E W P. M O R S E and F R ÉDÉ R IQ UE C . G UI NEL*
Wilfrid Laurier University, 75 University Ave W, Waterloo, ON, Canada N2L 3C5
Received: 26 October 2006 Returned for revision: 4 December 2006 Accepted: 3 January 2007
† Background and Aims R50 (sym16) is a pea nodulation mutant with fewer and shorter lateral roots (LR), fewer
nodules and high levels of cytokinins (CK). Because a link exists between CK imbalance and abnormal vasculature,
the vasculature of the primary root (PR) and LR of R50 was studied and it was compared with that of the wild-type
‘Sparkle’. Also nodule vasculature was investigated to correlate R50 low nodulation phenotype with CK
accumulation.
† Methods PR and first-order LR were hand-sectioned transversely in different locations and at different ages.
Vascular poles were counted and root and stele diameters measured. To evaluate LR primordia number, roots
were cleared. Nodules obtained from inoculated plants were either fixed and sectioned or cleared; numbers of vascular strands and of tracheary elements in the strands were counted.
† Key Results ‘Sparkle’ PR is triarch, whereas that of R50 can be triarch, tetrarch or pentarch. Furthermore, as the
R50 roots developed, supernumerary vascular strands appeared but, as they aged, the new growth of more roots displayed the triarch pattern. LR vasculature differed from that of PR: whereas ‘Sparkle’ LR had three or four poles,
those of R50 had two or three. No differences in PR or PR stele diameters existed between the two lines. Whereas
‘Sparkle’ nodules had two vascular strands, most R50 nodules possessed three; however, because R50 nodules were
variable in size, their vasculature was highly diverse in terms of strand length. A strong correlation was found
between nodule length and number of tracheary elements in strands.
† Conclusions R50 displays an additional number of vascular poles in its PR, a smaller number of vascular poles in
its first-order LR and an altered vasculature in its nodules. It appears that these three characteristics are linked to the
high levels of CKs that the mutant accumulates over its development.
Key words: Cytokinin, heterorhizy, nodulation mutant, nodule vasculature, pea mutant, Pisum sativum, pleiotropic mutant,
R50 (sym16), root vasculature, vascular tissue.
IN TROD UCT IO N
Mutants are great tools for studying the physiology behind
growth and development, and they are especially useful in
understanding the control exercised by hormones during
these processes. One nodulation mutant from pea (Pisum
sativum) proves to be extremely powerful as it displays
several pleiotropic traits. R50 (sym16) was first described
as a low nodulator by Kneen et al. (1994); it was further
characterized by Guinel and Sloetjes (2000) and Ferguson
et al. (2005). It is shorter than the near-isogenic wild-type
(WT) line ‘Sparkle’, with pale leaves caused by a lower
total chlorophyll content and a reduced root system (Guinel
and Sloetjes, 2000). Three weeks after inoculation with
Rhizobium, R50 displays a low number of nodules which
are pale and small. Mostly arrested in the root inner cortex,
the infection threads are convoluted and generally not associated with a nodule primordium (Guinel and Sloetjes, 2000).
R50 seedlings have a total cytokinin (CK) content higher
than that of WT; as the plants mature, the CK levels decrease
in WT shoots whereas they remain high in those of R50
(Ferguson et al., 2005). This CK accumulation was partly
explained by Held et al. (Wilfrid Laurier University,
Canada, unpubl. res.), who provided evidence for an R50
deficiency (biochemical activity and RNA expression) in
cytokinin oxidase/dehydrogenase (CKX), the enzyme
* For correspondence. E-mail [email protected]
responsible for CK degradation. In addition, the mutant exhibits a partial de-etiolation phenotype and its shoots and roots
display a differential sensitivity towards ethylene (Ferguson
et al., 2005). Whereas R50 shoots are less sensitive to
exogenous ethylene than WT shoots, R50 and WT roots
exhibit a similar sensitivity to the gas.
The high levels of CKs in R50 prompted us to study the
vasculature of the mutant; although the mutant shoot was
not altered, the root exhibited a tetrarch or pentarch
pattern instead of the usual triarch seen in WT pea root
(Ferguson, 2002). The arrangement of primary vascular
tissue is determined very early in the development of a
plant. A mature embryo is organized in such a way that it
possesses two meristems which give rise to either the
shoot or the root procambium. As the meristematic cells
are continuously dividing, procambial cells are continuously laid out and will follow a tight differentiation programme to become cells of either xylem or phloem
tissues. In the lifetime of a plant, vascular development is
a constant and predictable process which must, however,
remain flexible as plants are incessantly adapting to new
conditions. For example, mature vascular parenchyma
cells are able to dedifferentiate in response to wounding
(e.g. Hardham and McCully, 1982) and the different vascular tissues will alter their anatomy in response to environmental stimuli [e.g. temperature (Lu et al., 1991) or high
salinity (Junghans et al., 2006)]. The differentiation of the
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766
Pepper et al. — Root and Nodule Vasculature in a Pea Nodulation Mutant
procambial cells into mature cells is a complex process that
involves many players. Thus, recently, brassinosteroids and
an arabinogalactan protein, known as xylogen, have been
implicated (reviewed in Fukuda, 2004). In contrast, for
numerous decades now, two major hormones, auxins and
cytokinins, have been known to play key roles in this developmental step (Aloni, 1995). While polar auxin transport
has been recognized as crucial for the continuous
formation of the vascular bundles, cytokinins have proved
to be essential in the formation and maintenance of the procambial cells (Fukuda, 2004). In addition, different types of
cells in the vascular bundles are apparently active sites of
CK biosynthesis (Miyawaki et al., 2004). Thus AtIPT, the
arabidopsis gene encoding isopentenyltransferase, the first
enzyme in the CK biosynthesis pathway, is differentially
expressed: AtIPT1 is expressed in the xylem progenitor
cells of the root tip and AtIPT3 in the phloem throughout
the plant (Miyawaki et al., 2004).
That cytokinins are important signalling molecules in the
differentiation of vascular tissues (Fukuda, 2004) is supported by the study of both transgenic plants and mutants.
However, because non-physiological interactions are likely
to occur in the heterologous transgenics (Heyl and
Schmülling, 2003), results using mutants are easier to
analyse. Thus, among many arabidopsis mutants described
by Scheres et al. (1995), woodenleg (wol) stands out
because it exhibits only protoxylem in the stele of its
primary root (Mähönen et al., 2000). Furthermore, its procambial cells are unable to divide periclinally, resulting in
only a few cell files in the stele (Scheres et al., 1995;
Mähönen et al., 2000). The WOL gene, also identified as
CRE1 (Cytokinin Response 1; Inoue et al., 2001), encodes
a histidine kinase which is part of the two-component
signal transduction system involved in CK perception.
Here, the data obtained by Ferguson (2002) on the abnormal root vasculature observed in R50, a CK-accumulating
nodulation mutant, are expanded. Because the CK levels
increase with ageing of the mutant, a developmental study
of the vasculature was undertaken not only in the primary
root but also in the lateral roots. We were interested in
determining the origin of the additional vascular poles of
the primary roots and whether or not the lateral roots exhibited an altered vasculature. Because we were unaware of
any study on the hormonal regulation of nodule vasculature,
we were particularly interested in investigating the vascular
arrangement in R50 nodules to potentially link the low
nodulation phenotype to the high levels of root cytokinins.
M AT E R I A L S A N D M E T H O D S
Growth conditions
Seeds of Pisum sativum L. ‘Sparkle’ and of the mutant R50
(sym16) were surface-sterilized in 8 % household bleach
(5.25 % sodium hypochlorite) for 5 min and rinsed three
times with sterile distilled water. They were left to imbibe
overnight in the dark in sterile water and then planted,
four to a pot, in round pots (155 mm diameter) filled with
sterile Holiday vermiculite (Vil Vermiculite Inc., Toronto,
ON, Canada). Pots were placed in a tray partially filled
with deionized water. Plants were grown in a controlled
growth-chamber (Lab-line Biotronette; Lab-line Instruments Inc., Melrose Park, IL, USA), under cool white fluorescent lights (average light intensity of 380 mmol m22 s21)
and 58 % humidity, with a 23/18 8C, 16/8 h, light/dark
regime.
Plants grown for the root vasculature study were watered,
as needed, by adding deionized water to the tray. Those for
the nodule vasculature study were inoculated 4 d after
planting with 5 mL of either 2 % or 5 % (v/v) Rhizobium
leguminosarum bv. viciae 128C53K (Lipha Tech Inc.,
Milwaukee, WI, USA) grown in a yeast– mannitol broth
to an optical density of 0.5 – 0.6 at 600 nm. Inoculated
plants were watered daily for 1 week after planting
and then every second day, alternating between deionized
water and a low nitrogen nutrient solution (2 mM
KH2PO4, 0.5 mM Ca(NO3)2.4H2O, 2 mM K2SO4, 1 mM
MgSO4.7H2O, 0.2 mM FeIII EDTA, 0.05 mM KCl,
0.025 mM H3BO3, 21023 mM ZnSO4.7H2O, 21023 mM
MnSO4.H2O, 51024 mM CuSO4.5H2O, 51024 mM
Na2MoO4.2H2O).
Tissue preparation and microscopy
Fresh tissue. Primary roots were harvested 5, 9, 13 and 17 d
after planting (DAP). Transverse hand-sections were made
at three locations on the primary root: 0.5 cm from the
root tip (i.e. meristematic zone or MeZ), at the root hair
initiation zone (RhZ) and 1.0 cm from the cotyledons
(i.e. maturation zone or MaZ). Two first-order lateral roots,
one being the closest to the cotyledons and the other
located 3 cm from the cotyledons, were also harvested 13
and 17 DAP. These were hand-sectioned transversely
1.0 cm distal to the site of branching from the primary root.
Some sections were stained with 0.05 % (w/v) toluidine
blue O (TBO; Fisher Scientific Company, Fair Lawn, NJ,
USA) in benzoate buffer ( pH 4.4), rinsed and mounted in
water. These sections were observed with a Reichert
Microstar IV microscope using 10 (NA 0.25) and 40
(NA 0.66) objective lenses to determine the number of vascular poles. For all sections, except those made in the
MeZ, the number of xylem poles was determined based on
lignified vessel elements (VEs) and that of phloem poles
on lignified phloem fibres because such cells were easily
identified with TBO. For sections taken in the MeZ, enlarged
immature VEs or the first lignified VEs were used to determine the number of xylem poles. Photographs were taken
on Pan F (ASA 50) film using a Yashica 108 Multi
Program camera (Kyocera Corporation, Japan) connected
to a Reichert-Jung Photostar camera system. Negatives
were scanned using a CanoScan 4200F scanner (Canon
Inc., Mississauga, ON, Canada) and images were edited
using ArcSoft PhotoStudiow 5.5 (ArcSoft Inc., Fremont,
CA, USA).
Additional sections, prepared for fluorescence
microscopy, were stained with TBO to quench autofluorescence and then with 0.01 % (w/v) ethidium bromide
(BioShop Canada Inc., Burlington, ON, Canada) to stain
specifically lignified tissues (O’Brien and McCully, 1981).
Sections were rinsed in water before being mounted in 0.05
Pepper et al. — Root and Nodule Vasculature in a Pea Nodulation Mutant
% (w/v) aniline blue (Sigma Chemical Company, St Louis,
MO, USA) in phosphate buffer ( pH 8.5) to stain callose
for the localization of phloem sieve plates (Smith and
McCully, 1978). A quick verification of the arrangement of
the vascular tissues was done using a Jenalumar (Jena
Zeiss, Germany) fluorescence microscope with a blue
excitation filter set (two KP490 filters and a B229 filter)
and a G47 barrier filter. The lignified cell walls fluoresce
red and the sieve tubes with their sieve plates yellowish
white.
Unstained sections were used to measure the diameters of
the primary roots and of the first-order lateral roots, and
their stele diameters. For consistency, the longest diameter
was measured since the roots were not always perfectly
round. Root and stele diameter measurements were
made using a graticule (Jena Zeiss, Germany) and the aforementioned light microscope (objectives 4 and 10,
respectively).
Fixed tissue. Nodules were harvested from plants 17 d after
inoculation (DAI) in such a way that each nodule studied
was still attached to the root bearing it. Samples were
fixed in 3 % (v/v) glutaraldehyde (Canemco & Marivac
Inc., Canton de Gore, QC, Canada) in 0.05 M potassium
phosphate buffer ( pH 6.8) under a continuous vacuum for
3.5 h. They were rinsed twice (15 min each) in 0.025 M potassium phosphate buffer ( pH 6.8). An increasing ethanol
gradient (3, 10, 30, 50, 70, 90, 95 and 100 %) was used
to dehydrate the tissue which was then infiltrated with an
increasing gradient of LR Whitew hard-grade acrylic resin
(London Resin Company Ltd, distributed by Canemco &
Marivac Inc.) (1 : 3, 2 : 2, 3 : 1 LR white resin : 100 %
ethanol). Samples were infiltrated with the resin for a
minimum of 1 week, changing it once a day, before being
embedded into fresh resin and cured at 60 8C for 24 h in
an oxygen-free environment.
Fixed nodules were sectioned both longitudinally and
transversely using a Sorvallw Porter-Blum MT-2 ultramicrotome. Sections were heat-fixed to slides and then stained
with TBO (pH 4.4); they were observed and photographed
as previously described for the root sections. To determine
the number of vascular poles in the nodules, serial transverse
sections were performed. Ten consecutive thin sections
(approx. 450 nm thick each) were viewed but the next ten
thick sections (approx. 1500 nm thick each) were discarded.
This process was repeated until the entire nodule was
sectioned from its apex to the connection with the root vasculature. This allowed for an approximate three-dimensional
reconstruction of the nodule vasculature to be made.
Cleared tissue. Entire root systems were excised 7 and 12
DAP. After the number of emerged lateral roots was
counted, the root system was cleared according to the protocol of Luschnig et al. (1998). In short, individual root
systems were placed in a methanol : glacial acetic acid
(3 : 1, v/v) solution, which was replaced 30 min later by a
solution made of 130 mM NaCl, 10 mM sodium phosphate
( pH 7) and 0.1 % Tween 20. After two short baths
(5 min) in that solution, roots were placed within the clearing solution (0.16 g chloral hydrate per millilitre of 20 %
glycerol). The primordia corresponding to the initiation of
767
first-order lateral roots were counted using a dissecting
microscope.
As an additional means to understand the threedimensional arrangement of the nodule vasculature, nodules
at 17 DAI were cleared according to a protocol modified
from Eskew et al. (1993). The samples were placed in
undiluted household bleach for 2 h, the first of which was
under continuous vacuum; they were then rinsed twice
(5 min each) with water before being placed under a
vacuum in 30 % glycerol (1 h), then in 60 % glycerol
(1 h). Nodules were mounted in 60 % glycerol for microscopic analysis and were examined and photographed as
previously described. Several parameters listed below
were recorded: nodule length (i.e. the distance taken perpendicularly from the centre of the root vasculature to the
nodule apex), number of vascular traces linked to the root
vasculature, width of the vasculature at the base of the
nodule (i.e. distance along the root vasculature between
the nodular traces the furthest apart), and the number of
tracheary elements (TEs) across that base. Nodule height
and vasculature width were measured using a graticule. In
addition, the longest vascular strand was chosen in individual nodules and its length assessed by counting the
number of TEs from the root vasculature to the trace tip.
Statistical analysis
In all experiments, the mutant R50 was compared with the
wild-type ‘Sparkle’. Statistical analysis of data was done
using SigmaStatw software (Jandel Scientific, San Rafael,
CA, USA). Student’s t-tests were used to determine significant line-related differences between all parameters, but
in the event of non-normally distributed samples, the
Mann– Whitney Rank Sum test was performed instead. An
ANOVA was used to compare the mean diameters when
measurements were grouped according to vascular patterns.
Correlation was determined using a linear regression to
generate the R values.
R E S U LT S
Primary roots
The wild-type ‘Sparkle’. The tissues making up the primary
root of a young wild-type seedling (5 DAP) were seen
to differentiate as expected for a dicotyledon species
(e.g. Popham, 1955; Torrey, 1967; Esau, 1977), especially
for pea (Rost et al., 1988). The root tissues, including the
vascular tissues, differentiated in a basipetal manner, and
the maturation process occurred centripetally. In the MeZ
(Fig. 1A), while the phloem tissue was difficult to distinguish, non-lignified primary VEs of the xylem were not
because of their obvious enlarged size (Fig. 1J). At
the RhZ (Fig. 1A), all tissues were easily recognized. The
inner cortex was delimited by an endodermis while the
stele was enclosed within the pericycle; as noted by Bond
(1948) and reiterated by Rost et al. (1988), the pericycle
in pea is complex, composed of a monolayer over the
phloem but of two or three cell layers opposite the xylem.
In the RhZ, some VEs had secondary walls; however, the
768
Pepper et al. — Root and Nodule Vasculature in a Pea Nodulation Mutant
A
R50
‘Sparkle’
C
B
D
E
G
H
I
K
L
MaZ
pf
First-order lateral root
Xylem vessel elements
Phloem fibers
Endodermis
Root hair
F
RhZ
J
*
MeZ
F I G . 1. Schematic representation of the root system of a young pea plant (A) showing the relative position of the hand sections taken along the primary
root and the arrangement of the vascular tissue seen in the photomicrographs on the right. Transverse hand sections of 9-d-old ‘Sparkle’ (B, F and J) and
R50 (C– E, G– I, K and L) primary roots were taken from the maturation zone (MaZ, 1.0 cm from the cotyledons) (B–E), from the root hair initiation zone
(RhZ) (F–I), and from the meristematic zone (MeZ, 0.5 cm from the root tip) (J– L). All sections were stained with TBO. For ‘Sparkle’, pf¼phloem
fibres; non-lignified primary vessel elements are indicated by an asterisk. Scale bars in B–L¼50 mm.
central region of the stele was still occupied by nonlignified parenchyma cells (Fig. 1F). Phloem fibres with
their secondary walls were also present; the phloem was
easily located in aniline blue-mounted sections since the
sieve plates appeared fluorescent (data not shown). In the
MaZ (Fig. 1A), lignified VEs and parenchyma cells occupied the central region of the vascular cylinder, and lignified phloem fibres were obvious (Fig. 1B).
Xylem is unable to differentiate in the absence of phloem
(Aloni, 1995); thus, it is usual for a root to have as many
xylem poles as phloem poles. Although in the stele of a
root, these poles of different tissues have separate locations,
it is conventional to group them together in what is known
as a vascular pole; the number of vascular poles is equal to
either that of the xylem poles or that of the phloem poles. In
this study, the conventional term of vascular poles was used
for convenience. In a young ‘Sparkle’ seedling as well as in
older plants, generally three vascular poles were present at
all locations (e.g. Fig. 1B, F, J); thus, as reported in the literature for pea (e.g. Torrey, 1967), ‘Sparkle’ had a triarch
pattern as the norm. Occasionally, however, four poles
could be seen (Fig. 2); this occurred most often in the
root MaZ (e.g. four out of 36 in Fig. 2A; one out of 36 in
Fig. 2D), as if one vascular pole were added along the
root length as the tissues differentiated basipetally. The
additional vascular pole was initially composed exclusively
of primary tissue since it developed prior to the formation
of a vascular cambium (data not shown). Surprisingly, as
the plants grew older, fewer of them (as a percentage)
displayed a tetrarch pattern in their vascular tissue
(e.g. compare A with D in Fig. 2); however, the numbers
of vascular poles in ‘Sparkle’ plants of different ages
were not significantly different.
The mutant R50. Primary roots of R50 developed in a
fashion similar to that of ‘Sparkle’ (i.e. basipetal cell differentiation and centripetal cell maturation); however, they
showed a significant amount of variability in their vascular
patterning (Fig. 1). For example 9 DAP, whereas roots displayed mostly a triarch vasculature at the MeZ, they exhibited either a triarch, tetrarch or pentarch vasculature at the
RhZ and MaZ (Fig. 2B). At all days analysed, as roots of
individual plants grew in length, a trend towards increased
vasculature in basal regions was observed with .20 % of
roots having a pentarch stele in the MaZ (Fig. 2), suggesting
that the supernumerary poles are the results of a secondary
stage of primary vascularization. Statistically significant
differences were also seen across the ages, but only in the
RhZ and MeZ. For example, at the RhZ, as the plants
grew older, an increasing percentage of primary roots displayed a triarch stele (Fig. 2A – D), as if the additional vascular poles observed in the seedling 5 DAP were
disappearing during temporal growth. As in ‘Sparkle’, the
additional poles were composed initially only of primary
tissues (e.g. Fig 1H, I).
First-order lateral roots
Because cytokinins have been known to affect the
development of lateral roots, especially their emergence
Pepper et al. — Root and Nodule Vasculature in a Pea Nodulation Mutant
A
‘Sparkle’
R50
RhZ (%)
MaZ (%)
80
MeZ (%)
B
Day 5
C
Day 9
D
Day 13
80
80
80
60
60
60
60
40
40
40
40
20
20
20
20
0
0
0
0
80
80
80
80
60
60
60
60
40
40
40
40
20
20
20
20
0
0
0
0
80
80
80
80
60
60
60
60
40
40
40
40
20
20
20
20
0
3 poles
4 poles
5 poles
0
3 poles
4 poles
5 poles
0
3 poles
4 poles
5 poles
0
3 poles
769
Day 17
4 poles
5 poles
F I G . 2. Percentages of ‘Sparkle’ (black bars) and R50 (open bars) plants having three, four or five vascular poles in their primary roots for day 5 (A), day
9 (B), day 13 (C) and day 17 (D). MaZ represents the maturation zone (1.0 cm from the cotyledons), RhZ the root hair initiation zone and MeZ the meristematic zone (0.5 cm from the root tip). Percentages were chosen for simplicity of presentation; however, statistical tests were done on the raw data, and
significant differences were found between the two pea lines at all locations and all ages, except for the MeZ at 9, 13 and 17 d (n¼30–36).
(Li et al., 2006), we sought to determine using cleared root
systems if there were any line-related differences between
primordium initiation and emergence of first-order lateral
roots. At the two ages studied, ‘Sparkle’ had a significantly
greater number of total lateral root events (i.e. sum of primordia, emerging and emerged roots) on their primary
roots than R50 (Table 1). ‘Sparkle’ also had significantly
more first-order lateral roots (i.e. emerging and emerged)
TA B L E 1. Number (mean+s.e.) of root primordia and of
lateral roots of first-order (emerging and emerged) counted
on the primary roots of 7 and 12 DAP plants (n¼14 –17)
Primordia
7d
12 d
First-order lateral roots
‘Sparkle’
R50
‘Sparkle’
R50
12.29+1.08a
13.31+1.66e
19.14+1.37b
13.29+1.16e
37.43+1.30c
53.44+2.03f
12.64+1.14d
44.07+2.49g
For a specific age, means with different letters indicate significant
differences (P,0.001) between ‘Sparkle’ and R50.
than R50; this was caused partially by a failure of the
R50 primordia to emerge in the young seedlings (Table 1).
The number of vascular poles in the first-order lateral
roots of ‘Sparkle’ was often increased in comparison to
that seen in the primary roots; on the contrary, in R50,
these roots frequently had a decreased number of poles
(Fig. 3). The majority of the ‘Sparkle’ first-order lateral
roots located in the MaZ were either tetrarch (Fig. 3A) or
triarch (Fig. 3B), with the tetrarch pattern being the most
common 13 and 17 DAP (Table 2). In the same region
and at the same age, R50 first-order lateral roots were
either diarch (Fig. 3C) or triarch (Fig. 3D), with the
diarch pattern observed in most cases (Table 2). Similar
vascular patterns were observed in the first-order lateral
roots located 3 cm from the cotyledons (data not shown).
Root and stele diameter
There was no significant difference between the two pea
lines in either the primary root diameter or the stele diameter of that root at all locations and all ages studied; the
data for MaZ at 17 DAP are shown in Table 3. When the
770
Pepper et al. — Root and Nodule Vasculature in a Pea Nodulation Mutant
‘Sparkle’
A
R50
B
pf
C
D
VE
VE
pf
F I G . 3. Transverse hand-sections of ‘Sparkle’ (A and B) and R50 (C and D) first-order lateral roots located the closest to the cotyledons. These lateral
roots were from 17-d-old plants and sections were made 1.0 cm from the site of branching from the primary root. Whereas most first-order lateral roots
were tetrarch (A) and diarch (C) for ‘Sparkle’ and R50 respectively, there were triarch roots (B and D) in both lines. All sections were stained with TBO.
For ‘Sparkle’, VE¼vessel elements; pf¼phloem fibres. Scale bars¼20 mm.
TA B L E 2. Percentage of plants having different vascular
patterns in the first-order lateral roots located in the MaZ
(n¼27– 29)
13 DAP
Vascular pattern
Diarch
Triarch
Tetrarch
Pentarch
17 DAP
‘Sparkle’
R50
‘Sparkle’
R50
7.1
32.1
57.1
3.6
63.0
37.0
0.0
0.0
0.0
44.8
55.2
0.0
72.4
24.1
3.4
0.0
All comparisons are statistically significant (P,0.001) between the
two pea lines on both days.
root or stele diameters of R50 plants with a triarch vasculature were compared with those of R50 roots having
increased vasculature (four or five poles), the differences
were not significant at most locations and for plants of
different ages (data not shown), suggesting that the
increased vasculature was not related to a larger vascular
cylinder. This contrasts with the first-order lateral roots
where the root and stele diameters of ‘Sparkle’ were significantly greater than those of R50, as shown in Table 3 for the
oldest first-order lateral roots. This increase in lateral root
and stele diameters was correlated with a higher number
of vascular poles (as determined through an ANOVA).
The nodule
Gross anatomy. ‘Sparkle’ mature nodules appeared as
described in the literature (e.g. Bond, 1948; Newcomb
et al., 1979). In short, as seen in a median longitudinal
section (data not shown) as well as in a cleared mature
nodule (Fig. 4A), the central infected zone comprising
infected and non-infected cells was surrounded by a
cortical zone, which was divided by the common nodule
endodermis into outer and inner cortex. Two main vascular
strands connected to the root vasculature were seen running
within the inner cortex, parallel to the nodule epidermis
(Fig. 4A). The differentiation of these strands was acropetal,
i.e. from the base of the nodule to its apex, in contrast to the
basipetal differentiation of the root vasculature.
When a reconstruction of the ‘Sparkle’ nodule was
accomplished through serial transverse sectioning
(Fig. 5A), the two main vascular strands appeared to
branch dichotomously; thus, in cross-sections of a
‘Sparkle’ nodule, there were two vascular bundles at the
base of the nodule, but four at mid-height and above
(right of Fig. 5A). There were never more than four
bundles observed at the apex of each nodule, suggesting
that there was no more than one dichotomous branching
per bundle. In a cross-section, the individual bundles were
simple in their making (as described by Bond, 1948);
each had its own vascular endodermis enclosing a pericycle
layer surrounding a few small TEs and a few phloem cells
(data not shown).
At 17 DAI, R50 nodules were smaller than those of
‘Sparkle’; for a meaningful comparison between pea
lines, care had to be taken to section nodules of similar
size. When the nodule size was thus taken into consideration, R50 nodules had an anatomy similar to those of
‘Sparkle’, with an infected central zone and a cortex including vascular traces in its inner region. However, R50 vasculature was more complex than that of ‘Sparkle’; the number
of bundles within nodules was highly variable. The variability was most obvious in the three-dimensional reconstruction of the nodules where, most of the time, two
main bundles existed but one very quickly bifurcated to
give an additional main bundle (Fig. 5B). Furthermore,
these main bundles did branch in a dichotomous manner
but not necessarily at the same height (Fig. 5). Thus,
overall, the vasculature of the nodule was not uniform in
contrast to what was seen in the WT (compare B with A
TA B L E 3. Root diameter and stele diameter of the primary root in the MaZ (n¼18) and of the oldest first-order lateral root
(n¼12) of ‘Sparkle’ and R50 plants 17 DAP
Primary root
‘Sparkle’
R50
First-order lateral root
Poles
Root diameter (mm)
Stele diameter (mm)
Poles
Root diameter (mm)
Stele diameter (mm)
3
3–5
1.90+0.18a
2.01+0.13a
0.65+0.07b
0.69+0.08b
3 –4
2 –3
0.67+0.02c
0.54+0.03d
0.23+0.01e
0.18+0.01f
Different lower-case letters indicate significant line-related differences (P,0.001) between ‘Sparkle’ and R50.
Pepper et al. — Root and Nodule Vasculature in a Pea Nodulation Mutant
771
in Fig. 5); this lack of symmetry was evident in crosssections where three to five vascular bundles were observed
in the inner cortex of most nodules (left of Fig. 5B).
A
MZ
En
CZ
RC
RV
B
C
D
E
Morphology of cleared nodules. To confirm and to complement the previous findings, and to increase the sample
size, cleared nodules were observed. WT at 17 DAI displayed a homogeneous population of nodules; these were
mostly mature and nitrogen-fixing (indicated by their pink
hue). Generally, cleared nodules of ‘Sparkle’ possessed
two main strands (Fig. 4A); a few had three or four main
vascular strands (Table 4) which had been missed via thin
sectioning. By continually re-adjusting the fine focusing
on the cleared nodule and by following the longest vascular strand along the nodule length, the number of TEs
making that trace could be counted. The strands comprised,
on average, 16 TEs positioned end-to-end (Table 5);
however, this number was quite variable ranging from
nine to 32. There was a positive correlation between the
number of TEs and the length of the nodule (Fig. 6);
longer nodules had strands with a greater number of TEs.
R50 nodules borne by 17 DAI plants were significantly
smaller than ‘Sparkle’ nodules (Table 5) with a greater
variability in size and shape than previously reported
(Guinel and Sloetjes, 2000); few of these nodules were
pink. With the clearing technique, the vasculature appeared
especially variable (Table 4): some nodules had only a
single strand while a few had up to five different main
strands. However, most had three strands and further variation was observed within this population (Fig. 4B – E).
Some of the strands appeared vestigial because they were
made of very few TEs (Fig. 4E); others were well developed with up to 18 TEs lengthwise (Fig. 4B). However,
on average, they were only six TEs long (Fig. 4C). In
R50 too, there was a strong correlation between length of
the nodule and number of TEs within a strand (Fig. 6),
but because R50 nodules rarely reached sizes as large as
those of ‘Sparkle’ nodules, they never displayed as many
TEs. To determine whether or not there was a correlation
between nodule length and nodule width, the width of the
nodule was measured at its base, and the number of TEs
which were in direct contact with the root vasculature was
counted (Table 5). Both parameters were significantly
different between WT and R50, but no correlation existed
between these two parameters (data not show).
DISCUSSION
F I G . 4. Cleared nodules harvested from 17 DAI ‘Sparkle’ (A) and R50
(B–E) plants. (A) is considered to be a representative of the ‘Sparkle’
nodule; this nodule is used here to describe the organ gross anatomy.
The cortex protects the central zone (CZ) which comprises infected and
non-infected cells; it is traversed longitudinally by the common endodermis (En) which separates outer (triangle) from inner (diamond) cortex.
Each vascular strand (white arrows) is connected to the root vasculature
(RV); it possesses its own pericycle and vascular endodermis (not indicated). RC¼Root cortex; MZ¼meristematic zone. Nodules depicted in
(B) to (E) illustrate the large diversity of size and shape observed in
R50. The vascular traces (white arrows) were highly variable in R50
nodules with numbers of tracheary elements (TEs) ranging from 1 (E) to
18 (B). Refer to Materials and methods for assessment of the number of
TEs. Scale bars¼250 mm.
Variability in the vasculature of the underground
organs of ‘Sparkle’ and the mutant R50
Primary root. The establishment of the vascular tissue of a
primary root occurs under genetic control during embryogenesis; in an older plant, the pre-existing pattern is continuously
perpetuated by the root meristematic activity (e.g. Raghavan,
1999). Thus, in a ‘Sparkle’ seedling, the radicle possesses
three vascular poles ‘inherited’ from the embryo. As it
develops into a primary root, the triarch pattern, described
elsewhere for pea roots (e.g. Popham, 1955; Torrey, 1967),
is propagated in the stele. Variations in this triarch
pattern were seldom seen in ‘Sparkle’ primary roots; some
772
Pepper et al. — Root and Nodule Vasculature in a Pea Nodulation Mutant
A ‘Sparkle’
B R50
N
N
NV
NV
RV
RV
R
R
F I G . 5. Schematic 3D-reconstruction of the vasculature of mature nodules of ‘Sparkle’ (A) and R50 (B) plants obtained via serial transverse sectioning.
In the centre of the figure are schematics displaying the arrangement of the vascular bundles in the cross-sections taken at the relative positions indicated
by the dashed lines. Only the position and the shape of the vascular bundles are comparable between ‘Sparkle’ and R50, not their sizes. N¼nodule;
NV¼nodule vasculature; RV¼root vasculature; R¼root.
variability was also reported by Bond (1948) and Popham
(1955) in the pea cultivars they observed but not by Torrey
and Wallace (1975). In contrast, variability was high in
R50 primary roots which often had an increased vasculature
in the MaZ. As R50 primary roots were similar to those of
‘Sparkle’ in the MeZ and displayed a triarch pattern, the
increase of vasculature is likely to be a post-embryonic
event. These alterations from the embryonic template are
likely to be occurring via either the spontaneous appearance
or the loss of a vascular pole, as had been observed
previously in pea by Barlow (1984).
of the vascular pattern seen in lateral roots which emerged
at different locations along the main axis; accordingly, a
young lateral root could display a diarch pattern whereas
an older one could exhibit a pentarch pattern or vice versa
(Torrey and Wallace, 1975). Additionally, they found significant differences in the stelar arrangement within a
single lateral root through the addition or deletion of a
strand. The first-order lateral roots of R50 were more homogeneous in their vascular pattern than those of ‘Sparkle’ as
they displayed steles with two or three vascular poles (omitting one plant out of 29 which had four poles).
First-order lateral root. Contrary to what is traditionally
reported in the literature (i.e. the primordium of a lateral
root displays a pattern similar to that of the embryonic
root; e.g. Raghavan, 1999), the stelar arrangement in the
first-order lateral roots of both pea lines was different
from that of their primary roots. In contrast to ‘Sparkle’
lateral roots which often had an increased vasculature compared with the primary root, those of R50 exhibited a
decreased vasculature. The first-order lateral roots of
‘Sparkle’ exhibited more extensive variations in their vascular pattern than the primary roots because their number
of vascular poles could range from two to five. ‘Sparkle’
variability was reminiscent of that observed by Torrey
and Wallace (1975) who studied hundreds of pea root
systems obtained through sub-cultures of a single root.
These authors reported no obvious order in the arrangement
Diameter of root stele. Torrey (1957) demonstrated that the
number of vascular poles could be altered through the
manipulation of the growth conditions of excised pea root
tips. By adding auxin, the subsequent new growth of
those roots which previously displayed a triarch pattern
had changed to exhibit a hexarch stele. When these roots
were transferred back to an auxin-free medium, the subsequent new growth displayed the normal triarch pattern
(Torrey, 1957). Torrey proposed that auxin influenced the
procambium cylinder by making it larger; and to accommodate this enlargement the number of vascular poles
increased. From these experiments, a linear correlation
between the stele diameter and the number of vascular
poles was proposed and is still cited in the literature
TA B L E
TA B L E 4. Percentage of nodules having 1, 2, 3, 4 or 5 traces
of vasculature in ‘Sparkle’ (n¼53) and R50 (n¼71)
Trace
1
2
3
4
5
‘Sparkle’
R50
0
94.3
3.8
1.9
0
12.7
26.8
53.5
5.6
1.4
5. Mean measurements (+ s.e.) from cleared
nodules of ‘Sparkle’ (n¼53) and R50 (n¼71)
Parameter
Length (mm)
Width (mm)
Number of TEs across nodule base
Number of TEs along one trace
Number of traces in one nodule
‘Sparkle’
R50
1039.5+37.2
481.4+14.1
18.5+0.6
16.5+0.7
2.1+0.04
651.9+15.5
401.3+10.6
14.6+0.4
6+0.3
2.6+0.1
There is a statistically significant difference between ‘Sparkle’ and R50
for all parameters (P,0.001).
Number of elements along one
trace
Pepper et al. — Root and Nodule Vasculature in a Pea Nodulation Mutant
35
30
25
20
15
10
5
0
‘Sparkle’
R50
heterogeneous in their size, their strand length was highly
variable. This correlation could be a reflection of the
nodule function, i.e. larger nodules would need longer vascular traces to accommodate the export of nitrogenous compounds. However, the present data do not agree with this
explanation for a nodule such as that seen in Fig. 4C had
a large central infected zone but little differentiation of
vascular strands.
y = 0·0148x + 1·1074
R2 = 0·6499
y = 0·0132x – 2·7204
R2 = 0·4502
0
500
1000
1500
773
2000
Nodule length (µm)
F I G . 6. Correlation between the number of TEs along a single nodule vascular strand and the nodule length, and lines of best fit for ‘Sparkle’ (open
circles; n¼53) and R50 (closed circles; n¼71).
(e.g. Aloni et al., 2006). In hindsight (see below), this
relationship should perhaps not be applied indiscriminately
to any type of root as it was drawn from excised roots.
In support of this point, the first-order lateral roots of
‘Sparkle’ had a tetrarch arrangement compared with the
primary roots, despite having smaller diameters (Table 3).
Consequently, in the present analysis, we decided to separate the two types of roots (i.e. primary and first-order lateral
roots). For the primary roots of ‘Sparkle’, a link between
stele diameter and number of poles could not be verified
because only a few plants displayed variations in their vasculature. On the contrary, the large variability in the vascular pattern of R50 primary roots allowed such a correlation
to be tested; however, there was no linear relationship
between stele diameter and number of poles. This suggests
that the increase in poles was not in response to the presence of a larger stele. To test a correlation between stele
diameter and number of poles in first-order lateral roots,
the data for the two lines had to be grouped. When this
was done, a relationship between the two parameters was
evident; there were a larger number of poles in the roots
with a larger stele diameter.
Nodule. ‘Sparkle’ nodule vasculature with its two main
strands was consistent with what has been described in the
literature (Bond, 1948; Dart, 1977). However, contrarily to
what is reported in the literature whereby up to ten strands
could be counted in the apical region of a mature pea
nodule (e.g. Bond, 1948), ‘Sparkle’ displayed only one
dichotomy for each of its strands. It is possible that the
difference lies in the age of the nodule, as Bond (1948)
did not indicate a precise time for her observations. R50
nodules were unique in that they commonly had three vascular strands which were linked to the root vasculature; as
far as is known, such abnormal nodule vasculature has
never been reported in legume mutants. In the literature,
two mutants of Lotus japonicus [alb1, Imaizumi-Anraku
et al. (2000); crinkle, Tansengco et al. (2003)] have been
shown to exhibit incompletely developed vascular
bundles. These mutants differ from R50 in that their
nodule vasculature is uniform; in contrast R50 displays
large variations in the number of TEs present along an individual strand. In R50 as in ‘Sparkle’, the TEs number (along
the strand length) and nodule length were strongly correlated (Fig. 6). However, because R50 nodules were
R50 root system and cytokinins
The R50 root system differs from that of ‘Sparkle’ by a
shorter primary root, shorter lateral roots (Guinel and
Sloetjes, 2000), a reduced number of first-order lateral root
primordia and an abnormal vasculature (this study).
Furthermore, R50 roots have a higher total CK concentration
than those of ‘Sparkle’ (Ferguson et al., 2005). We postulate
that these traits are linked to the accumulation of CKs. It
could be a direct effect or more likely the result of an
altered auxin : CK ratio (Aloni et al., 2006). Although it is
likely to be a simplistic view (e.g. Nordström et al., 2004),
it is thought that there are two opposite hormonal gradients
along the root length. The auxin gradient is acropetal as a
continuous polar flow of auxin moves from the aerial parts
of the plant (e.g. Sachs, 1968) to the root tip which acts as
a sink; the CK gradient is basipetal since CKs are synthesized
in the root tip (Miyawaki et al., 2004) and travel apoplastically towards the root base (e.g. Aloni, 1995). Accordingly,
it is the ratio of auxin : CK which determines the fate of
the cells and influences the differentiation of the tissues
imprinted in the embryo.
CK and the initiation of lateral root primordium. Some of the
target cells responsive to the hormonal gradient are the progenitor cells of the lateral roots (i.e. the pericycle cells;
Li et al., 2006); when triggered by the appropriate signals,
these cells divide first anticlinally then periclinally to initiate
the formation of a lateral root primordium (Raghavan,
1999). The spatial location where the lateral roots develop
(Torrey, 1957; Stirk and van Staden, 2001), the primordium
initiation (Li et al., 2006) and the root emergence (Hinchee
and Rost, 1986; Raghavan, 1999) are all controlled tightly
by auxin and CK. Specifically, CKs have been shown to
inhibit lateral root growth. For example, CKX over-expressing
plants (i.e. CK-deficient) exhibit an increased number of
lateral roots (Werner et al., 2003; Lohar et al., 2004)
whereas exogenous treatment of root systems by CKs
reduces that number (Lorteau et al., 2001; Li et al., 2006).
The lower number is caused by an inhibition of the
primordium formation whereby CKs act through the regulation of the cell cycle as shown in arabidopsis (Li et al.,
2006). In addition, a link between the number of primordia
and CK perception (via CRE1 in combination with the
shoot CK-receptor AHK3) has been drawn in arabidopsis
(Li et al., 2006). Consequently, the accumulation of CKs
could be the cause of the reduced number of lateral root
primordia in R50.
CK and vasculature morphogenesis. CKs are also known to
affect the development of the root vascular tissue; they
are thought to increase the sensitivity of the tissue to
774
Pepper et al. — Root and Nodule Vasculature in a Pea Nodulation Mutant
auxin (Aloni, 1995) and thus these two hormones act
together to specify the root vascular pattern (Raghavan,
1999; Aloni et al., 2006). That auxin is not the sole actor
playing in the differentiation of vascular tissue and that it
acts with CKs have been demonstrated elegantly using
in vitro xylogenic cultures of Zinnia cells; if one of the
two hormones is omitted from the medium, no TEs differentiate from the mesophyll cells (e.g. Pesquet et al., 2005).
Other examples of the synergistic action of auxin and CK in
the vasculature formation are found in several mutants. For
example, the rice mutant ral1 exhibits fewer vascular poles
in its roots than the WT seedlings and metaxylem elements
with smaller diameters (Scarpella et al., 2003). In this
mutant, it appears difficult to dissociate a hypersensitivity
to CK and a defective auxin response (Scarpella et al.,
2003). Another mutant, the pls arabidopsis mutant
(Casson et al., 2002), displays a short root phenotype and
a surprisingly reduced vascularization in its leaf. In this
mutant, Casson et al. (2002) were unable to uncouple the
effects of auxin from those of CK.
Recently, CKs alone have been placed at the centre stage
of root vasculature morphogenesis. Our interpretation of the
recent literature is that CK perception plays a larger role in
this process than CK homeostasis. In arabidopsis transgenic
plants over-expressing the CKX gene, there was no alteration in the root vascular pattern despite low levels of CK
(Werner et al., 2003). However, other effects on root
anatomy, such as an enlarged meristem, increased number
of cells in some files, and occasional increase in cell size,
were observed (Werner et al., 2003). In contrast, as demonstrated by Helariutta’s group with arabidopsis mutants,
appropriate CK signalling is essential for the correct maintenance of the root procambium. The WOL/CRE1/AHK4
root CK-receptor was shown to be required for controlling
the number of cells composing the procambial root cylinder
(Mahönen et al., 2000). The receptor is required for the specification of the protoxylem cells because in the roots of the
wol mutant, all procambial cells differentiate into protoxylem cells (Mahönen et al., 2000). Recently, Mahönen et al.
(2006) demonstrated that this differentiation event was the
result of the effect of AHP6, a pseudo-phosphotransfer
protein inhibiting the action of CK.
R50 is unique in that the alteration of its root vasculature
is post-embryonic. The high CK levels measured in the R50
root system (Ferguson et al., 2005) must create an imbalance in the auxin : CK ratio. The effects of such an imbalance are likely to be observed anywhere along the length of
the root. An optimal ratio of auxin : CK will provoke xylogenesis which requires the re-entry of some parenchyma
cells of the mature vascular tissue into the cell cycle.
CK and nodules. It is known that CK regulates nodule
number; however, this effect appears to be concentrationdependent with the existence of an optimum (Lorteau
et al., 2001). This could explain seemingly contradictory
results where exogenous CK-treatment (Lorteau et al.,
2001) and CK-deficiency obtained through the overexpression of CKX (Lohar et al., 2004) both lead to a
reduction in nodule formation. As far as is known, anomalous
vasculature of nodules as seen in R50 has never been
described. In view of what has been written above, regarding
a relationship between abnormal vasculature and CK influences, we suggest that the increased vasculature observed
in R50 nodules is also a result of the high levels of CK
found in R50 (Ferguson et al., 2005).
Existence of heterorhizy in pea
The discrepancy of the vascular pattern in primary and
first-order lateral roots of ‘Sparkle’ and R50 further
supports the need for questioning the ‘a root is a root’
concept (Zobel, 2003). The present data add further
evidence to the existence of heterorhizy demonstrated by
Hochholdinger et al. (2004a, b) with mutants of Zea mays
and Oryza sativa. If we are correct in our assumptions
that R50 abnormal vasculature is caused by high levels of
CKs, then R50 primary roots with their increased vasculature and R50 first-order lateral roots with their decreased
vasculature are regulated differently, suggesting the existence of multiple components in the complex organ which
is the root. Further evidence for separate controls for the
two types of roots can be seen in the recent work of
Riefler et al. (2006), whose results indicate that primary
root growth and lateral root branching are under different
regulation. Whereas the former process appears to be
under the control of the root CK-receptor WOL/CRE1/
AHK4, the latter process is regulated by a combination of
the two shoot CK-receptors AHK2 and AHK3 (Riefler
et al., 2006). The concept of ‘a root is a root’ has been
long-lived, but it is obviously too simplistic to match the
complexity of the hormonal interactions and the intricacy
of molecular control. We hope that researchers in the
future will take into account the sophistication of the
root system and carefully distinguish the different types
of roots.
Conclusions
In summary, in this study, the R50 phenotype has been
refined by demonstrating that the mutant generally displays
an additional number of vascular poles in its primary root, a
smaller number of vascular poles in its first-order lateral
roots, and an altered vasculature in its nodules. We trust
that these three characteristics are linked to the high
levels of CKs that the mutant accumulates over its development (Ferguson et al., 2005). In view of the literature,
altered root vasculature is not caused simply by CK but
rather by abnormal auxin/CK levels; furthermore, it
appears to be more an effect of CK perception and signalling (Mahönen et al., 2000, 2006) than of CK metabolism
(Werner et al., 2003; Takei et al., 2004). Because of the surprisingly strong similarity existing between the phenotype
of R50 and those of the CK receptor-double and -triple
mutants of arabidopsis (Riefler et al., 2006), we hypothesize that R50 is a CK-receptor mutant; however, at
present the product of the gene sym16 remains unknown.
Regardless of the sym16 identity, R50 should be considered
as a useful tool for studying vasculature regulation,
especially in the nodule.
Pepper et al. — Root and Nodule Vasculature in a Pea Nodulation Mutant
AC KN OW L E DG E M E N T S
The research was supported by the Natural Sciences and
Engineering Research Council of Canada operating grants to
FCG, and three Natural Sciences and Engineering Research
Council of Canada Undergraduate Student Research Awards
to A.N.P. The authors wish to thank Dr T. A. LaRue for his
gift of R50 seeds, and Dr M. E. McCully for her critical
reading of the manuscript.
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