Journal of Experimental Botany, Vol. 63, No. 14,
2, pp.
2012
pp.695–709,
5093–5104,
2012
doi:10.1093/jxb/err313
AdvanceAccess
Accesspublication
publication12
4 November,
doi:10.1093/jxb/ers155 Advance
July, 2012 2011
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Hydraulic
conductivity
and PAT determine
hierarchical
In
Posidonia
oceanica cadmium
induces changes
in DNA
resource partitioning
and ramet
development along
methylation
and chromatin
patterning
Fragaria stolons
Maria Greco, Adriana Chiappetta, Leonardo Bruno and Maria Beatrice Bitonti*
Department
of Ecology,
University
of Mark
Calabria,
of Plant Cyto-physiology, Ponte Pietro Bucci, I-87036 Arcavacata di Rende,
Christopher
J. Atkinson*
and
A. Laboratory
Else
Cosenza, Italy
East Malling Research, New Road, East Malling, Kent ME19 6BJ, UK
* To whom correspondence should be addressed. E-mail: [email protected]
* To whom correspondence should be addressed. E-mail: [email protected]
Received 29 May 2011; Revised 8 July 2011; Accepted 18 August 2011
Received 24 February 2012; Revised 26 April 2012; Accepted 27 April 2012
Abstract
Abstract
In
mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent
epigenetic
mechanism.
Here,
thephysiological
effects of Cdactivity
treatment
on theplant
DNAparts
methylation
patten
are examined
together
with
Co-ordination
of metabolic
and
between
is key to
the control
of growth
and develits
effect Here
on chromatin
reconfiguration
in Posidonia
methylation
pattern
were
analysed
in
opment.
the movement
of resources
and their oceanica.
allocation DNA
between
mother level
plantsand
and
daughter
ramets
along
actively
under short(6 h) and
(2 d orGradients
4 d) term of
and
low (10 mM)
andleaf
high
(50 mM)
doses(ψ)
of and
Cd,
Fragariagrowing
stolons organs,
was quantified
with respect
tolonghierarchy.
internodal
ramet
water
potential
through
a ramet
Methylation-Sensitive
Amplification
Polymorphism
technique
an immunocytological
approach,
stolon and
hydraulic conductivities
(L) were
measured together
with and
apparent
stolon IAA movement
via the
respectively.
The expression
one These
member
of the CHROMOMETHYLASE
(CMT) family,
a DNA
methyltransferase,
polar auxin transport
pathwayof
(PAT).
processes
are linked with measurements
of stolon
vascular
development.
was
also assessed
qRT-PCR. Nuclear
chromatin
ultrastructure
investigated
bydemonstrated
transmission the
electron
The pattern
of tissue by
differentiation
and lignification
in sequential
stelewas
sections
of stolons
rapid
microscopy.
induced
DNA hypermethylation,
as well
as an up-regulation
of CMT, with
indicating
de
acquisition ofCd
thetreatment
capacity for
wateratransport,
with transpiration
potentially
varying systematically
stolonthat
lignifinovo
did indeed
occur.
Moreover,
a Stolon
high dose
of CdLled
to a progressive
of
cationmethylation
and the acropetal
decline
in stolon
xylem ψ.
and ramet
declined
acropetally, heterochromatinization
with L across older ramets
interphase
nuclei and
apoptotic
figures
were
also observed
afterThe
long-term
treatment.
demonstrate
thatwith
Cd
being significantly
lower
than that
of the
connecting
stolons.
capacity
for polar The
IAA data
transport
increased
perturbs
thethis
DNA
methylation
status transport
through the
involvement
a specific
methyltransferase.
Such was
changes
are
stolon age;
was
due to increased
intensity
in olderof
tissues.
The partitioning
of dry matter
strongly
linked
to
nuclear
chromatin
reconfiguration
likely
to
establish
a
new
balance
of
expressed/repressed
chromatin.
hierarchical with younger ramets smaller than older ramets, while foliar concentrations of N, P, and K were greater for
Overall,
the data
show
anresults
epigenetic
to the mechanism
underlying
Cd at
toxicity
in plants.
the younger
ramets.
The
showbasis
that stolon
anatomy develops
rapidly
the apical
end, facilitating hierarchical
ramet development, which is evident as a basipetal increase in L. The rapid development of transport tissue functionKey
condition,
chromatin
reconfiguration,
CHROMOMETHYLASE,
ality words:
enables5-Methylcytosine-antibody,
young unrooted ramets cadmium-stress
to acquire water,
in order
to supply
an expanding
leaf area, as well as mineral
DNA-methylation,
MethylationSensitive
Amplification
Polymorphism
(MSAP),
Posidonia
oceanica
(L.) Delile.
ions disproportionally with respect to older ramets. This facilitates colonization and self-rooting
of apical ramets. The
unidirectional increase in basipetal PAT along stolons facilitates hierarchical ramet development.
Key words: Auxin, Fragaria, hydraulic conductivity, polar auxin transport, stolons, ramets.
Introduction
In the Mediterranean coastal ecosystem, the endemic
seagrass Posidonia oceanica (L.) Delile plays a relevant role
Introduction
by
ensuring primary production, water oxygenation and
provides
niches forof some
animals,
counteracting
The co-ordination
metabolic
and besides
physiological
activity
coastal
erosion
through
its
widespread
meadows
(Ott,
between individual plant parts is key to the control
of 1980;
plant
Piazzi
1999; Alcoverro
al., 2001).
also
growth et
andal.,
development
(Enquist,et2003;
Leyser,There
2010).isPlants
considerable
evidence
that
P.
oceanica
plants
are
able
to
achieve this co-ordination at the cellular level and between
absorb
and
accumulate
metals
from
sediments
(Sanchiz
tissues and organs; the latter may be separated spatially over some
et al., 1990; Pergent-Martini, 1998; Maserti et al., 2005) thus
distance. Understanding how such co-ordination is achieved, for
influencing metal bioavailability in the marine ecosystem.
example, with respect to water transport, and carbon partitioning,
For this reason, this seagrass is widely considered to be
offers opportunities to manipulate these processes to optimize
a metal bioindicator species (Maserti et al., 1988; Pergent
plant growth.
et al., 1995; Lafabrie et al., 2007). Cd is one of most
The co-coordination and control of organ growth and
widespread heavy metals in both terrestrial and marine
development is widely agreed to be achieved through the
environments.
Although not essential for plant growth, in terrestrial
plants, Cd is readily absorbed by roots and translocated into
aerial organs while, in acquatic plants, it is directly taken up
by
leaves. and
In plants,
Cd absorption
induces
complexsignalling
changes
synthesis
transport
of long-distance
chemical
at
the
genetic,
biochemical
and
physiological
levels
which
molecules (Went’s ‘hormone-directed transport’ work of
the
ultimately
account
for
its
toxicity
(Valle
and
Ulmer,
1972;
1930s and, more recently, Uggla et al., 1996). The endogenous
Sanitz
di Toppi
and Gabrielli,
1999; Benavides
2005;
morphogenic
hormone
IAA (indole-3-acetic
acid) isetanal.,
example
Weber
et
al.,
2006;
Liu
et
al.,
2008).
The
most
obvious
of a signalling molecule produced by growing apices, which is
symptom of Cd toxicity is a reduction in plant growth due to
transported basipetally; it is a putative candidate responsible
an inhibition of photosynthesis, respiration, and nitrogen
for controlling apical dominance, leaf and fruit abscission, leaf
metabolism, as well as a reduction in water and mineral
elongation, phyllotaxis and various tropisms, and root growth
uptake (Ouzonidou et al., 1997; Perfus-Barbeoch et al., 2000;
(Sachs, 1981; Muday and DeLong, 2001; Else et al., 2004; Billou
Shukla et al., 2003; Sobkowiak and Deckert, 2003).
et al., 2005). The role of IAA, via its cellular accumulation, is well
At the genetic level, in both animals and plants, Cd
established as a factor in the functional programming of cambial
can induce chromosomal aberrations, abnormalities in
© 2011
The Author
[2012]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
ª
The Author(s).
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5094 | Atkinson and Else
initials and xylem cell differentiation in relation to leaf growth
(‘functional hydraulic architecture’; Tyree and Ewers, 1991).
Developing leaves and active meristems produce IAA and the
extent of its transport out of leaves has been linked to the amount
of transpiring leaf area and the intensity of xylem differentiation
(Digby and Wareing, 1966). Endogenous gradients of signal
molecules, such as IAA, from their source of synthesis (shoot
and root meristems) to sites of cellular activity (differentiation
and expansion) are quantitatively linked with vascularization and
feed-back regulation of the IAA transport pathway (Goldsmith,
1977; Scarpella et al., 2002; Leyser, 2010). More recent evidence
suggests an interactive role for IAA with other phytohormones,
such gibberellins and ethylene, in processes such as root growth
(Fu and Harberd, 2003; Keller et al., 2004).
IAA flows from cell to cell via phloem sieve elements and
is basipetally directed via ‘polar auxin transport’ (PAT; Rubery
and Sheldrake, 1974). PAT is a chemiosmotic process which has
similar attributes to plasma membrane vesicle trafficking and
involves auxin-specific uptake and efflux carrier proteins, such
as AUX1 (IAA influx) and PIN1 (IAA efflux) (Friml and Palme,
2002; Muday and DeLong, 2001; Geldner et al., 2001; Baluska
et al., 2003; Schlicht et al., 2006). A change in the expression
polarity profiles of these carrier proteins provides dynamic
direction and intensity control of auxin transport (Wiśniewska
et al., 2006). PAT is now functionally linked with gene
expression (in shoot and root) via the regulation of factors such
as procambial cell differentiation and root lignin and flavonoid
profiles (Scarpella et al., 2002; Laffont et al., 2010).
The aim of the work presented here was to develop a more
integrated understanding of the processes by which modular
growth and development is co-ordinated in clonal ‘daughter’
plants (‘ramet’ the unit of clonal growth) derived from stolons
(Stuefer et al., 1998). Morphologically, the stolon is a swollen
stem that develops from a leaf axillary bud with its subtending
nodal ramet structures. Stolon growth influences the extent
of intra-clonal neighbourhood competition and colonization/
survivorship through changes in stolon length and extension rate
in response to species associations and environment (Harper,
1977; Dushyantha and Hutchings, 1997).
Understanding the function and development of the stolon has
import implications in defining the growth potential of individual
ramets. This is apparent when connected and competing ramets
can be shown to change their behaviour (Holzapfel and Alpert,
2003). Clonal plant studies show that environmental gradients
or ‘habitat patches’ (variation in intensity of radiation or mineral
ions) above and below ground induce changes in the ‘foraging’
responses of ramet and stolon (Birch and Hutchings, 1994;
Tworkoski et al., 2001). Evidence suggests that stolon-linked
ramets share resources through a process described as ‘division
of labour’ (Alpert and Mooney, 1986; Wijesinghe and Hutchings,
1997). This clonal co-operation of resource sharing must rely
on basipetally- and acropetally-directed resource movement
of ions and photoassimilates (Wijesinghe and Hutchings,
1997; Holzapfel and Alpert, 2003). It is postulated that these
self-regulatory developmental processes are subject to the
control of hormones such as IAA (Nick et al., 2009).
Despite knowledge of aspects of the functional behaviour of
stolons in crops such as clover and potato, the same is not true
in multi-rameted stolons and little is known about the functional
relationships between tissue anatomy, hierarchal position, water
movement, resource allocation, and the controlling influence
of auxin transport. Here Fragaria×ananassa, the cultivated
strawberry, is used as a model system which propagates itself
asexually via stolons (clone). As clonal ramets develop along a
stolon at the internodes, they are subject to differences in hierarchical
and environmental variability, particularly with respect to stolon
position/age and access to stolen-derived ‘resources’ (i.e. water,
mineral ions, and photoassimilates) at least prior to self-rooting.
Basipetal IAA transport is hypothesized to be critically
important in influencing ramet development and growth,
particularly rooting (Billou et al., 2005), as well as the nature of the
interaction/competition between daughter ramets along the stolon
for resources. Our objectives were to quantify the movement of
water and minerals along the stolons of Fragaria in relation to
ramet hierarchy, and to determine how they are linked with stolon
hydraulic conductivity (L), stele anatomy, and IAA movement via
the PAT pathway.
Materials and methods
Plant material
Cold-stored ‘bare rooted’ plants (class A=12–15 mm ‘crown’ diameter
grade) Fragaria×ananassa, the June bearer cv. Elsanta were potted into
‘Richmoor’ base compost and grown within a heated greenhouse compartment (minimum 15 °C and vented at 25 °C) until sufficient stolons of
a uniform length had grown. The number of ramets on each stolon varied
slightly depending on the experiment (see individual experiments below).
A schematic diagram shows the typical format of the plant material used
in these experiments and is annotated with respect to the location of the
anatomy sections (Fig. 1). Visually, the phenological development of the
stolon/ramets varied hierarchically with the older ramets having greater
leaf areas and root production with ramets 5 or 6 being at the stolon tip
and having no significant leaf area or root development.
Quantification of functional anatomy of stolons
Changes in vascular development of the stolons in relation to ramet position were measured in tissue sections removed at a constant position
equidistance between ramets, along entire stolons (Fig. 1). Eight replicate samples per position were taken from stolons of similar length bearing five ramets. Tissue sections were prepared using standard alcoholic
dehydration series and wax embedding prior to sectioning on a rotary
microtome (Reichert, Wien, Austria) and tissue counter staining (Safranin
O, for lignified and suberized tissue and Fast Green for cytoplasm and
cellulose) and microscopic analysis (Horscroft, 1989; Atkinson et al.,
2001). Sections were examined under low power light microscopy using
an image analysis system (Sonata II, Seescan Plc, Cambridge, UK) and
the proportions of various tissues (pith, xylem, phloem, and cortex)
quantified. Phloem tissues were quantified separately based on the intensity of lignification. It was assumed that lignified phloem tissue equates
with a functional transport system while phloem which does not show
lignification and is close to the cambial layer is functionally associated
with auxin transport (Sheldrake, 1972). Xylem tissue was not separated
into lignified and non-lignified tissue as these differences were present
but on a much smaller scale relative to differences seen in phloem lignification, particularly for older stolon sections.
Leaf water potentials (ψ) and stolon hydraulic conductivity (L)
Leaf water potential (ψ) of mother plants and ramets was measured on three occasions around midday, on cloudless days with a
Scholander-type pressure chamber. Leaves, with attached petioles, from
PAT control of stolon development | 5095
Fig. 1. A schematic of the hierarchical arrangement of ramets along a stolon of Fragaria and the location of the tissue analysis points
used. Images of radial tissue sections along the stolon are positionally located. The annotation used is as follows; ‘mother plant’ (MP),
ramet 1 (R1), ramet 2 (R2), ramet 3 (R3), ramet 4 (R4), the stolon tip (R5) and similarly for stolons; stolon 1 (S1), stolon 2 (S2), stolon 3
(S3), stolon 4 (S4), and stolon 5 (S5).
stolons with six ramets were rapidly excised with a razor blade, and
measurements completed within 30 s. Hydraulic conductivity (L) was
measured along excised stolon sections and across individual ramets
(xylem tissue of the stolon passes through the basal tissue of each ramet
on the stolon). Basal tissue is the condensed stem of Fragaria, which
contains apices which can develop into leaves, flowers, and stolons.
Measurements of L were made with an in vitro gravity-fed flow system (described by Atkinson et al., 2003). Analysis of stolon L involved
11 replicates. The experimental system, with slight modification, was
used for stolons and ramets and is similar in principle to that described
by Sperry et al. (1988). L was measured on stolon sections midway
between ramets, along stolons with five ramets per stolon. Prior to each
measurement, total leaf area per mother plant and ramet stolon diameter and stolon section lengths were measured. Ramets were left intact
(leaves and roots) for L measurements, leaving small attached acropetal
and basipetal stubs of stolon which were used to make connections to
the measuring system.
L was measured using a pressure across the stolon/ramet section of
about 6 kPa. Degassed and filtered (0.2 µm mesh Millipore filter) oxalic
acid solution (10 mol m–3) was used as the perfusion solution to prevent any long-term decline in conductivity due to microbial growth.
The mass of solution flowing per unit time through stolon/ramet segments was measured continuously with an electronic balance accurate
to 0.01 mg and the pressure head, the length of the segment (mm) and
the temperatures (approximately 20 °C) were all used to calculate continuous measurements of L, i.e.
L (kg m s −1 MPa −1 ) =
flow rate (kg s −1 ) × sample length (m)
(A)
pressure head (MPa)
Recordings of mass flow were made every 10 s for each sample until
the coefficient of variation had declined to <2%; then the final three
readings were averaged to obtain L. Procedures were adopted prior
to each measurement to ensure the system was leakproof, especially
at the micro-clamp connections between the measuring system and
stolon tissue. Measurements of L were made at one pressure based on
preliminary data and knowledge of stolon anatomy; linearity of L at
higher pressures was assumed. Pressure flow curves for root tissues
with a similar anatomy to stolons, but branched (junctions) pipe ways,
such as the entire root system show a linear flow across the in planta
pressure ranges determined from ramet leaf xylem ψ (Tyree et al.,
1995; Rieger and Litvin, 1999). Brodribb and Hill (2000) also show
a linear relationship over a wide range of applied pressures (which
includes the pressure used here) with L over a range of morphological and genetically differing root systems. Higher pressure perfusion
is avoided because in our experience it enhances in planta L due to
the removal of embolisms or the incorporation of non-conducting tissues [dysfunctional pith (stele) tissue post-secondary xylem thickening] into the measured flow path. These assumptions are considered
appropriate as the comparison envisaged here between L and ramet
leaf ψ gradient are not considered to be simple and necessarily directly
relatable, given the invasive way hydraulic flow resistances are determined. Stolon ψ was measured at midday when leaf ψ gradients were
at their greatest and therefore represent maximal diurnal water flow.
Water potential gradients will be different on a diurnal basis and it is
unclear how these measures integrate to determine, for example, cambial tissue differentiation and ion distribution. Other factors may alter
L dynamically particularly if the transcellular pathway components
are a significant proportion of total root L.
Stolon auxin transport
An active polar auxin transport assay (PAT) for detached stolons (rapidly excised within 2 min of detaching) and ramets was developed
which involved the application of micro-droplets (0.5 µl) of labelled
auxin ([3H]-IAA, 1.5 kBq: specific activity 962 GBq mmol–1, Nycomed,
Amersham Plc, UK), in 10 mM sucrose, to the apical ends of 8 mm-long
tissue sections removed from positions along the stolon (for details see
Else et al., 2004). Analysis of IAA movement involved seven replicates. A second label [14C]-benzoic acid (BA, specific activity 229 MBq
mmol–1, Sigma, St Louis, Missouri, USA) was also used to estimate
non-active passive diffusion (drainage) of IAA. BA is a weak acid with
a similar pKa and polarity to IAA and therefore similar passive diffusion characteristics. Stolon tissue was mounted in agar [0.85% (w/v)
Gelrite (85 g l–1), Sigma-Aldrich Company Ltd Poole, Dorset, UK] in
10 mM MES buffer adjusted to pH 5.2 receiver blocks, which collected
the labelled IAA. During incubation, in the dark at 20 °C, the stolon
tissue was transferred to a fresh agar receiver block at 30 min intervals. After 3 h, the assay was terminated and isotope movement was
quantified using stolon tissue sections. The agar receiving blocks were
transferred to screw lid plastic scintillation vials and then dissolved in
2 ml of 100% MeOH. The [3H]-IAA and [14C]-BA was extracted at room
temperature for 24 h in the dark. Optima Gold scintillant (PerkinElmer,
Cambridge) was added (15 ml) after vortexing and the [3H]-IAA and
[14C]-BA counted simultaneously on the dual label 3H 14C channel
(Beckmann Instruments, Fullerton, CA, USA). Stolon sections were
cut with a razor blade, with extreme care to avoid cross-contamination,
removing a 2 mm section from the application zone from the rest of
the tissue; the remaining 6 mm sub-section was divided equally into
middle and basal tissues. All three tissue sub-sections were counted in
screw top plastic scintillation vials with 2 ml of 100% MeOH after a
24 h extraction at room temperature. Preliminary experiments (data not
5096 | Atkinson and Else
shown), determined that a 3 h incubation period, with 30 min changes
of receiver block, was appropriate to calculate accurately the intercept
(transport velocity or rate of [3H]-IAA movement, as mm h–1) and the
slope (transport intensity or amount of [3H]-IAA moved, as dpm h–1)
using the linear portion of the cumulative analysis of labelled [3H]-IAA
in the agar blocks (see the method of Van der Weij, 1932; Else et al.,
2004). Transport intensities and velocities were calculated after taking
into account passive diffusion. An initial experiment showed that when
[3H]-IAA was applied to the acropetal or basipetal ends of stolon tissue sections, radioactivity was only recovered from agar receiver blocks
after basipetal transport when [3H]-IAA was applied to the acropetal
end of the stolon tissue (data not shown). This confirmed that IAA stolon PAT was unidirectional from the youngest stolon/ramet towards the
mother plant (MP). The integrity of the [3H]-IAA after basipetal transport through the tissue sections was verified by HPLC (Ford et al., 2002;
Else et al., 2004).
Distribution of minerals to ramets and their leaves
The distribution of mineral ions to ramet leaves and crowns along a stolon was examined using plants restricted to a single stolon with none of
the associated developing ramets being allowed to root. For this analysis
only plants that had stolons with five ramets were selected for analysis.
Analysis of stolon ramet leaf area involved 12 replicates. At harvest,
five replicate ramets were separated and divided into leaves and crowns.
Leaf area per ramet and MP were determined and leaves and crowns
dried at 80 °C until constant weight. The concentration of macro- (N, P,
K, Ca, and Mg) and micro-nutrients (Mg, Mn, Na, Zn, and Cu) in ramets
was analysed using inductively coupled plasma analysis.
Statistical analyses
All the statistical analyses (ANOVA) carried out used Genstat (13th
addition) and LSDs quoted are at 5% level. Details of the individual
statistical approaches used are presented within the results for each individual experiment.
Results
Stolon anatomy
Due to the larger values for the cortical tissue, and the resultant
higher variance, stolon anatomy data were log transformed
(natural log) to improve homogeneity prior to analysis (Fig. 1).
Figure 2A shows the cross-sectional area at different positions
along a stolon. Younger stolon tissue, particularly between
ramets 3, 4, and 5, had less tissue of all types compared with older
inter-ramet positions. Stolons at these positions also had smaller
diameters. In general, stolon anatomy, irrespective of sampling
position and, therefore, age, was similar. There was evidence,
however, of cortical cell disintegration in older stolon sections
and the presence of non-lignified and lignifying xylem tissue
in younger stolon sections. To remove possible compositional
differences due to variation in stolon diameter, tissue areas were
also expressed as a percentage of the total stolon cross-sectional
area (Fig. 2B). Proportional analysis of stolon anatomy data was
not transformed, despite the large values for the cortex; these data
were excluded from the analysis as the cortex differences were
marked (Fig. 2B). These data show little difference in percentage
of any tissue type with respect to stolon position (Table 1). Even
the lignified tissue outside the phloem, which strengthens the
extending stolon, developed rapidly in relation to hierarchy and
secondary diametric growth close to the stolon tip.
Fig. 2. Anatomical composition of vascular tissue types (pith, xylem,
phloem, lignified phloem, and cortex) from different positions along
the stolons of Fragaria x ananassa cultivar Elsanta plants. Tissue
composition is shown for stolon samples removed mid-way between
ramets and is presented on a cross-sectional area basis (mm2) (A) and
as a proportion (%) of the total stolon area (B). MP refers to the rooted
mother plant (see Fig. 1). Each point is the mean of a single stolon
taken from 12 plants and the standard error of each measurement is
shown. The calculated LSD (5% level) for tissues types not including
the cortex are for stolons, 0.984 with df of 28; tissue type, 0.412 with
df of 105, and stolon × type, 1.191 with df of 79.
Ramet and stolon leaf water ψ and hydraulic
conductivity
Midday leaf xylem ψ for ramets along a stolon are shown in
Fig. 3. Analysis of stolon ψ involved six replicates. Initially,
mother plants were compared with ramet, while a second
ANOVA examined the trend along the stolon. This analysis
showed that there was a strong linear trend of increasing ψ with
increasing ramet number (P <0.001), with this trend accounting
for all significant variation between ramets (deviations from
linear P=0.701). Leaves of the mother plant had the highest ψ,
with a gradual decrease in ψ with ramet position along the stolon
from the oldest ramet to the youngest.
PAT control of stolon development | 5097
Table 1. Statistical analysis of anatomical composition of vascular
tissue types (pith, xylem, phloem, lignified phloem, and cortex)
from different positions along the stolons of Fragaria×ananassa
cultivar Elsanta
Stolon position Tissue type
Cross-sectional area basis (Fig. 1A)
Including cortical tissue
Excluding cortical tissue
Proportional area basis (Fig. 1B)
Including cortical tissue
Excluding cortical tissue
LSD
d.f.
LSD
d.f.
0.276
28
0.259
28
0.140
140
0.558
105
LSD
d.f.
LSD
d.f.
0.092
28
0.908
28
1.04
140
0.412
105
The analyses are for the data presented in Fig. 1A [cross-sectional area basis
(mm2)]) and Fig. 1B [proportion (%) of the total stolon area]. LSDs are shown for
separate analyses, including and excluding the predominant cortical tissue.
Stolon L was highest (3.96 kg m s–1 MPa–1×10–6) between
the mother plant and ramet 1 (Fig. 4). Stolon L declined with
distance away from the mother plant until ramet 4 (2.26 kg m
s–1 MPa–1×10–6), after which L rapidly declined and was much
lower between ramets 4 and 5 (9.17 kg m s–1 MPa–1×10–8) and the
developing stolon (S6) beyond ramet 5 (2.23 kg m s–1 MPa–1×10–8).
In the latter case no developing ramet 6 was evident on these
stolons. Measurement of stolon L involved 11 replicates. There
were large differences (P <0.001) between measures of stolon
L with a highly significant (P <0.001) linear trend decreasing
with increasing stolon number; deviations from linear were
significant (P=0.002) and the decline was greater with higher
stolon positions (Fig. 4).
The mean L value of 1.01 kg m s–1 MPa–1×10–6 for ramet 1
declined by two-thirds with increasing distance from the mother
Fig. 3. Midday leaf water potential measurements of
Fragaria×ananassa cultivar Elsanta ‘mother plants’ (MP) and
unrooted daughter ramets (R1–R6) along the stolon (see Fig. 1).
Ramet 1 is the oldest plant along the stolon while ramet 6 is the
youngest. Each point is the mean of two leaves from three plants
and the standard error of each measurement is shown. The
calculated LSD (5% level) for comparing mother plant with ramets
is shown with 33 df
Fig. 4. Stolon and ramet hydraulic conductivity of
Fragaria×ananassa cultivar Elsanta plants. Stolon hydraulic
conductivity (S1–S6) was measured mid-way between each pair
of ramets and across each individual ramet (R) along the stolon
(see Fig. 1). S1 is the stolon sampled between the ‘mother plant’
(MP) and the oldest ramet R1; the same nomenclature is used
with S5 being the stolon between ramets R4 and R5. R1 is the
oldest ramet and R5 the youngest. The calculated LSD (5% level)
for stolons (1) with 49 df and for ramets (2) with 39 df.
plant so that L for ramet 4 was 0.33 kg m s–1 MPa–1×10–6.
Measurements of ramet L involved 12 replicates. The L ANOVA
showed strong differences between ramets (P <0.001) with
a linear trend with ramet number being highly significant (P
<0.001) decreasing with increasing ramet number (deviations
from linear P=0.945).
Leaf area, was larger in older ramets with the mean area
ranging from 1700 cm2 for ramet 1 to 29 cm2 for ramet 5
(Fig. 5). Due to the strong increase in variance with increasing
mean values data were log transformed for the complete
ANOVA which showed highly significant (P <0.001) differ­
ence between mother plants and ramets. For the ramets alone,
there was a highly significant (P <0.001) linear trend with
decreasing leaf area and increasing ramet number. Deviations
from linear were significant (P=0.009) particularly with
smaller ramets.
Expressing ramet L relative to ramet leaf area gave a positive
linear relationship and therefore also ramet position (Fig. 6A).
Stolon L also increased initially with stolon cross-sectional area
reaching a maximum at about 4 mm2, beyond which there was
no further increase (Fig. 6B). This increase in L, in relations
to cross-sectional area, follows the increase in stolon age
characterized by the increases in stolon diameter from left to
right on Fig. 6B.
Stolon IAA transport
Curve fitting has not been attempted with the cumulative
data; however, IAA proportional distribution showed highly
significant interactions between positions (stolon location) and
tissue sampling points (application point, middle tissue etc.,
5098 | Atkinson and Else
Fig. 5. Total plant leaf area of Fragaria×ananassa cultivar Elsanta
plants for each ‘mother plant’ (MP) and daughter ramets (R1–R5)
(see Fig. 1). Each point is the mean of 12 plants and the standard
error of each measurement is shown. The calculated LSD (5% level)
for ramets alone, with 45 df, without including the MP leaf area.
Fig. 7. Cumulative labelled IAA (dpm) in receiver blocks from
Fragaria×ananassa cultivar Elsanta stolons of different age (A)
and distribution of tritium-labelled IAA in the tissue segments
and receiver blocks (B). Stolon sections were sampled from
between; the ‘mother plant’ (MP) and ramet 1 (R1), ramet 1
(R1) and ramet 2 (R2), ramet 2 (R2) and ramet 3 (R3) and ramet
3 (R3) and the stolon tip (see Fig. 1). The calculated LSD (5%
level) including all stolon positions was 3.798 with 105 df.
While for separate within sampling positions LSDs were for
‘application point’ (1), ‘middle tissue’ (2), ‘basal tissue’ (3), and
‘receiver tissue’ (4) all with 8 df.
Fig. 6. The relationship between hydraulic conductivity across the
ramet and leaf area per ramet (A) and the relationship between
stolon hydraulic conductivity and stolon cross-sectional area
(B) for Fragaria×ananassa cultivar Elsanta plants. A sigmoidal
Gompertz, 3 parameter fit was used for (B), f=a*exp(–exp(–
(x–x0)/b)). Each point is the mean of 12 plants and the standard
error of each measurement is shown.
Fig. 7A). There were differences in variability between tissue
points and so these were analysed separately. The application
point varied slightly (P=0.055), with no difference between
middle and basal (‘middle’ P=0.251 and ‘basal’ P=0.267), with
differences in receiver blocks (P=0.021). Between 40–55% of
applied [3H]-IAA remained in the apical 1 mm of stolon tissue
section, while about 10% of the [3H]-IAA was transported to
the receiver blocks over 4 h (Fig. 7B). There were differences
in the cumulative amounts of [3H]-IAA in the receiver blocks of
stolon sections of different ages, with higher amounts measured
in tissue sections taken from stolons between the mother plant
and the first ramet. The proportion of [3H]-IAA label recorded in
PAT control of stolon development | 5099
Fig. 8. Labelled benzoic acid (dpm) in receiver blocks from
Fragaria×ananassa cultivar Elsanta plant stolons of different age
and distribution of labelled benzoic acid in the tissue segments
and receiver blocks. Stolon sections were sampled from
between; the ‘mother plant’ (MP) and ramet 1 (R1), ramet 1 (R1)
and ramet 2 (R2), ramet 2 (R2) and ramet 3 (R3), and ramet 3
(R3) and the stolon tip (see Fig. 1). The calculated LSD (5% level)
including all stolon positions was 1.078 with 32 df. While for
separate within sampling positions LSDs are shown ‘application
point’ (1), ‘middle tissue’ (2), ‘basal tissue’ (3), and ‘receiver
tissue’ (4) all with 21 df.
the receiver blocks decreased as tissue was sampled further away
from the mother plant (Fig. 7B).
Analysis of benzoic acid movement involved eight replicates
(Fig. 8) and a similar approach to that used in Fig. 7. There
were interactions between position and tissue sample and strong
differences between sampling point and variability. Analysis
of sampling points only showed differences with ‘basal’ tissue
(P=0.010), while ‘apical’ (P=0.026), ‘middle’ (P=0.209), and
‘receiver block’ (P=0.159) were not different. The [14C]-benzoic
acid label applied remained in the apical 1 mm (>70%) with <2%
reaching the basal tissue section, and virtually nothing detected
in the receiver blocks (Fig. 8).
Further analysis of the [3H]-IAA label was carried out
to determine the cause of the decline in [3H]-IAA stolon
transport; this analysis involved eight replicates (Fig. 9). The
amount of IAA label moved declined with decreasing stolon
age. Differences with respect to stolon position were highly
significant (P <0.001) showing a highly significant linear
trend (P <0.001). Analysis of velocity had missing values but
despite an apparent decline in speed of label movement there
was no evidence of significant positional differences overall
(P=0.289).
Distribution of minerals and dry matter to ramets
Nitrogen, P, and K concentrations decreased in leaves and
crown tissue with ramet age and were lowest in the mother plant
Fig. 9. The calculated intensities (A) and velocities (B) of [3H]-IAA
PAT movement in tissues taken from Fragaria×ananassa cultivar
Elsanta stolons with four ramets. Stolon sections were sampled
from between; the ‘mother plant’ (MP) and ramet 1 (R1), ramet 1
(R1) and ramet 2 (R2), ramet 2 (R2) and ramet 3 (R3), and ramet 3
(R3) and the stolon tip (see Fig. 1). The calculated LSD (5% level)
for the intensity and velocity measurements are shown above with
8 df and 15 df.
(Fig. 10). Ca and Mg (data not shown) concentrations showed the
opposite, with concentrations increasing with ramet age, with
the exception of the crown tissue where concentrations were
higher in the older ramets (Fig. 10). Manganese concentration
in the crown tissue was not influenced by ramet position, while
within leaves it increased in the primary and MP tissues (data
not shown). Concentrations of Na, Zn, and Cu were similar in
leaves of both ramets and the MP. However, Na concentration
in the crown tissues was lowest in the youngest ramet but
increased significantly in ramets 3 and 4 and then declined with
ramet age. Zn concentration was highest in ramet 5, lowest in
ramet 4, and increased again with ramet age (data not shown).
Cu concentrations were similar between ramets of different
ages and between leaves and crowns; only in the crowns from
the MP was the concentration much greater. Dry matter of ramet
leaves was, as expected, smaller in younger ramets, as was
5100 | Atkinson and Else
Fig. 10. Concentration and amount of nitrogen (A), phosphorus (B), potassium (C), and calcium (D) in leaves and crowns of
Fragaria×ananassa cultivar Elsanta ‘mother plants’ (MP) and ramets along the stolon (see Fig. 1). The amount of N was obtained by
multiplying nutrient concentration by the organ part dry weight. Each mother plant was allowed to produce a single stolon, which after
the production of five ramets was harvested for analysis. The bars on the concentrations reflect the LSD (at 5% level) for differences in
leaf (first) and crown (second) measurements, respectively.
PAT control of stolon development | 5101
leaf area (data not shown). There was a similar pattern of
decline in crown tissue mass but at a lower magnitude than
with leaves.
Discussion
Differences in stolon cellular anatomy were generally
small. The quantification of vascular differentiation on a
cross-sectional area basis showed greater proportions of pith
and lignified phloem cells (i.e. Safranin O staining) in sections
taken closer to the mother plant compared with those closer
to the apical meristem (Blanke and Cooke, 2004). The pattern
of differentiation and lignification in sequential stele tissue
sections indicated rapid stolon functionality. The acquisition of
a functional water transport system in the stolon is necessary
because the transpiring leaf area of ramets expands rapidly and
systematically with stolon position. Ramets in apical positions
had no fully expanded leaves (ramet 5 had <30 cm2) and
therefore little actively transpiring leaf area compared with the
oldest ramets and the leaf areas of the MP (1700 cm2). Potential
‘transpirational demand’ varied systematically with the spatial
pattern of stolon vascularization and lignification (Peter
and Neale, 2004). To support the developing transpirational
requirements of the ramets requires xylem conduits that are
functionally capable of withstanding the imposed negative
xylem ψ. Phloem transport develops rapidly as shown by the
pattern of radial differentiation along the stolon which support
its suggested importance as a transport system (van Bel, 2003).
Direct evidence of rapidly acquired stolon functionality, with
respect to the control of ramet development, is supported by
measurements of stolon PAT.
The colonizing behaviour and functional morphology of
stolons indicates that ramet survival, prior to rooting, is achieved
through plasticity of intra-stolon ramet competition for resources
such as water, ions, and photoassimilates (Alpert and Mooney,
1986; Wijesinghe and Hutchings, 1997). For a distally located
ramet these resources are derived initially only from the mother
plant and then progressively via hierarchical competition with
older ramet siblings. This notion is corroborated by the measured
systematic acropetal decline in stolon xylem ψ, providing a
gradient for water flux to the youngest ramet. A similar systematic
ψ gradient is seen with Trifolium repens stolons (Turner, 1990).
The approach reported here did not allow ramets to root and this
will influence the rate of stolon stele maturation and the intensity
of intra-ramet competition for resources, as they switch from
sinks to sources of water and mineral ions.
Support for this suggestion of rapid stolon functionality was
apparent from the measurements of hydraulic conductivity.
Preliminary examination of ramet tissue sections showed that
vascularization across the ramet itself would probably provide
the highest resistance to water movement and not the stolon
per se. This follows the Ohm’s law analogy frequently used to
quantify potential water transport capacities (Tyree and Ewers,
1991), where ramet L is a determinant of stolon water flux as
described by ‘pipe theory’ and evident from hydraulic flow in
other plant systems (Shinozaki et al., 1964). Stolon and ramet
L progressively declined acropetally, with L across all the older
ramets being considerably lower than that of its connecting
acropetal or basipetal stolon. Measurements of L across the
ramet and along the stolon conform to the descriptive changes
in stele anatomy and the speed with which newly formed stolon
tissue achieves functional water transport. The point of greatest
hydraulic resistance declined progressively acropetally with
distance from the mother plant. Initial comparative differences
in L between subtending stolon sections and ramets declined
acropetally. At ramet 4, measured L in stolon sections S5 and
S6 were similar to those across ramet 5. Functional support is
provided by the close correlations determined between ramet
L and ramet leaf area and stolon cross-sectional area. Such
allometric relationships are well established between yield
potential and stem cross-sectional area for perennial crops and
are closely correlated to total canopy area (transpiring surface
area) and factors such as flower number and crop yield (Lauri
et al., 1996; Atkinson et al., 2003). The allometric relationship
between developing leaf area and the production of a water
transport system is implicit in the relationship between supply
(xylem transport) and use (transpiration) (Digby and Wareing,
1966; Niklas, 1993).
Mechanistically the link between transpiring leaf area
demand and vascular supply is credited predominantly with the
production and movement of IAA. The process of long-distance
transport through the vascular system and its allometric scaling
with leaf area has physiological and evolutionary significance
(Enquist, 2003). Expanding leaves provide a source of IAA
which, when transported basipetally, regulates the fate of cambial
initials, vascular differentiation and the development of xylem
(Digby and Wareing, 1966; Uggla et al., 1996). Measurements
of Fragaria stolon IAA transport show unequivocally that IAA
transport (PAT) was an active process and exclusively basipetal.
The movement of applied [3H]-IAA was not due to simple
diffusion as ‘surrogate’ movement of labelled benzoic acid, a
molecule of similar chemistry, was non-existent. Importantly,
measurements of PAT can be linked to proportional changes in
stolon tissue anatomy, as is the case with other similar transport
systems (Leyser, 2010). IAA immunolocalization of functional
(number and activity) cell types may provide a more direct
link between changes in PAT activity and its transport tissues
(cambial cell initials and their derivatives; Scarpella et al.,
2002). Active stolon IAA transport increased with stolon age
with younger tissue having reduced transport capacity. Increased
capacity in older tissue was primarily due to increased transport
intensity (the amount of IAA moved). Lomax et al. (1995)
suggests the most likely explanation for an increase in intensity
would be an enhancement in the numbers of IAA-specific carrier
cells relative to those in more juvenile stolon tissue. It is unclear
whether, with stolons, this was due to differences in absolute
cell number or the acquisition of efflux/influx carrier proteins to
existing PAT transport cells. Systematic variations in the pattern
of respiratory and ATPase activity between the stolon tip and
the mother plant supports the notion of hierarchical patterning
of PAT transport cells (Blanke and Cooke, 2000). The smaller
changes in IAA transport velocity with stolon age, if significant,
imply a modification to specific carrier characteristics (‘upregulation’) associated with cellular IAA uptake and efflux
(Leyser, 2010). An ability to regulate the basipetal flow of IAA
5102 | Atkinson and Else
through a modification of carrier cell number and activity during
growth and in hte intensity response to environmental change is
known (Delbarre et al., 1996; Lomax et al., 1995).
Functionality of an organ, or meristem, through its ability
to export auxin (source) via PAT provides a mechanistic and
quantifiable measure of ‘sink strength’ to attract photoassimilates
and mineral ions. For example, the regulation of sink metabolism
occurs because, in many cases, tissue growth stimulates the
synthesis or activity of sucrose-degrading enzymes such as
acid invertase (Morris and Arthur, 1984). Failure or a decline
in a meristem’s capacity to export IAA, or the presence of other
competing auxin sources can cause ‘correlative inhibition’,
resulting in a decline in sink strength (Leyser, 2010). Seed-derived
IAA regulates sink activity and resource partitioning particularly
photoassimilates and a decline in PAT capacity is closely linked
with abscission (Patrick, 1979; Else et al., 2004). The correlative
inhibition described by Prusinkiewicz et al. (2009) may not only
determine fruit retention but may also be relevant where sibling
ramets, with shoot and root apical meristems, provide competing
sources of auxin within the basipetal complex of interacting
stolon PAT transport routes.
As expected, dry matter distribution was hierarchically/age
determined with younger ramets being smaller. Concentrations
of N, P, and K were greater in younger ramets. This suggests
differential allocation, with respect to that seen with the decline
in bulk leaf concentrations, and total leaf contents, for Ca, Mn,
and Mg. Mechanistically, bulk xylem solute delivery, linked
to ion accumulation in younger ramets can only account for
the pattern seen with, for example, Ca. Despite potential
differences in the water flux to ramets based on leaf area,
assumptions of passive distribution of xylem mobile ions (or
phloem immobile) being quantitatively linked to xylem sap
flux (Else et al., 1995) appear simplistic and lacks experimental
support (Canny, 1990; Atkinson, 1991). However, more is
known about the complexity of ion distribution within tissues,
such as leaves, if predominantly only for monocots, than we do
between organs along stolons (Karley et al., 2000). Transport
independent of the transpirational flux and basipetal auxin flow
in stolons is better linked with N, P, and K movement than
with calcium (Banuelos et al., 1987). The importance of the
speed with which phloem development occurs on these stolons
provides an alternative transport route when transpirational
demand is low, as would be the case with young ramets (Tanner
and Beevers, 2001).
Maintaining viable distal ramets which colonize suitable
‘niches’ (vegetation gaps with a favourable micro-environment)
will require, at least initially, resources for root growth and
establishment prior to autonomy, i.e. rooting (Tworkoski et al.,
2001). Potentially, ion distribution, which is not transpirationdependent, but sustained through an IAA-directed hierarchical,
phloem-mobile, ion flow along stolons, is a necessity (De Guzman
and Dela Fuente, 1984; Banuelos et al., 1987), as would be the
accumulation of ions, such as nitrate and phosphate, in ramet
tissues (Wijesinghe and Hutchings, 1997; Tworkoski et al., 2001).
Plasticity in the stolon hierarchy and function as described here
provides the means by which ramets root independently from
competing siblings where IAA flow through autoinhibition acts
as a control (Bangerth, 1989; Chun-Jian and Bangerth, 1999).
Regulation, turnover, and distribution of endocytic trafficking
proteins, such as PIN2, and their link with IAA action on stolon
root development suggests how environmental variation in
irradiance and below-ground resources (ions and water) would
induce plasticity in stolon mineral and carbon partitioning
(Laxmi et al., 2008).
Acknowledgements
This work was financially supported by the East Malling Trust.
Thanks go to L Taylor for preparing the microscopic stolon tissue
for analysis, to P Greeves for the graphics, to G Arnold for the
statistical analysis, and to P Gregory for comments on an earlier
version of this manuscript.
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