Phenotypic plasticity and water flux rates of Citrus root orders under salinity

Abstract

Knowledge about the root system structure and the uptake efficiency of root orders is critical to understand the adaptive plasticity of plants towards salt stress. Thus, this study is describing the phenological and physiological plasticity of Citrus volkameriana rootstocks under severe NaCl stress on the level of root orders.
Phenotypic root traits known to influence uptake processes, e.g. frequency of root orders, specific root area, cortical thickness and xylem traits, did not change homogeneously throughout the root system but changes under stress were root order-specific. Chloride accumulation significantly increased with decreasing root order and the Cl concentration in lower root orders exceeded those in leaves. Water flux densities of first order roots decreased to <20% under salinity and did not recover after stress release. The water flux densities of higher root orders changed marginally under salinity and increased two to six-fold in second and third root orders after short-term stress release.
Changes in root order frequency, morphology and anatomy indicate rapid and major modification of Citrus volkameriana root systems under salt stress. Reduced water uptake under salinity was related to changes of water flux densities among root orders and to reduced root surface areas. The importance of root orders for water uptake changed under salinity from root tips towards higher root orders. The root order-specific changes reflect differences in vulnerability (indicated by the salt accumulation) and ontogenetic status and point to functional differences among root orders under high salinity.

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Journal of Experimental Botany, Page 1 of 11
doi:10.1093/jxb/err457
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Phenotypic plasticity and water flux rates of Citrus root
orders under salinity
Boris Rewald1,*, Eran Raveh2, Tanya Gendler1, Jhonathan E. Ephrath1and Shimon Rachmilevitch1
1French Associates Institute for Agriculture and Biotechnology of Drylands, Ben-Gurion University of the Negev, Israel
2Citriculture, Gilat Research Center, Israel
* To whom correspondence should be addressed. E mail: rewald@rootecology.de
Received 22 November 2011; Revised 16 December 2011; Accepted 19 December 2011
Abstract
Knowledge about the root system structure and the uptake efficiency of root orders is critical to understand the
adaptive plasticity of plants towards salt stress. Thus, this study describes the phenological and physiological
plasticity of Citrus volkameriana rootstocks under severe NaCl stress on the level of root orders. Phenotypic root
traits known to influence uptake processes, for example frequency of root orders, specific root area, cortical
thickness, and xylem traits, did not change homogeneously throughout the root system, but changes after 6 months
under 90 mM NaCl stress were root order specific. Chloride accumulation significantly increased with decreasing
root order, and the Cl2concentration in lower root orders exceeded those in leaves. Water flux densities of first-
order roots decreased to <20% under salinity and did not recover after stress release. The water flux densities of
higher root orders changed marginally under salinity and increased 2- to 6-fold in second and third root orders after
short-term stress release. Changes in root order frequency, morphology, and anatomy indicate rapid and major
modification of C. volkameriana root systems under salt stress. Reduced water uptake under salinity was related to
changes of water flux densities among root orders and to reduced root surface areas. The importance of root orders
for water uptake changed under salinity from root tips towards higher root orders. The root order-specific changes
reflect differences in vulnerability (indicated by the salt accumulation) and ontogenetic status, and point to
functional differences among root orders under high salinity.
Key words: Anatomy, biomass, Citrus volkameriana, miniature depletion chambers, NaCl accumulation, phenotypic plasticity,
root architecture, root order, salt stress, water flux density.
Introduction
Salinity is a major concern for agriculture worldwide; at
least 20% of all irrigated lands are salt affected, with some
estimates being as high as 50% (Pitman and La ¨uchli, 2004).
Secondary salinization is particularly widespread in arid
and semi-arid environments whose agricultural systems are
often associated with cultivation of one of the various
Citrus varieties. With global food production having to
meet the demands of a growing world population, un-
derstanding plant responses to salinity is decisive to
improve the salt tolerance of crops.
Phenotypical or physiological changes in response to
environmental conditions often enhance the fitness of plants
(Sultan, 2000). Root systems can exhibit enormous plastic-
ity on the level of biomass, morphology, and/or physiology
in response to different environmental parameters such as
water and nutrient availability (e.g. Sorgona ` et al., 2007;
Wang et al., 2009; Gruber et al., 2011) or excess ions (Deak
and Malamy, 2005; Zolla et al., 2010; Rewald et al., 2011b,
c). Previous studies addressing salinity effects on tree crops
such as Citrus spp. and Olea europaea found, for example,
increased root:shoot ratios (Zekri and Parsons, 1989),
reduced root branching (Gucci and Tattini, 1997), modified
axial root conductivity (Rewald et al., 2011c), and a well-
developed Casparian strip closer to the root apex (Walker
et al., 1984). While salt exclusion, compartmentation, and
osmoregulation are the mechanisms particularly considered
ª 2012 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-
nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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to increase the salt tolerance of Citrus spp. and other woody
glycophytes, adaptation to salinity is determined by the
integrating effects of several mechanisms (Zekri and
Parsons, 1992; Maas, 1993; Kozlowski, 1997; Munns,
2002). Thus, it is reasonable to speculate that root system
modifications under salinity are a trade-off between the
capacity to exclude excess ions and sustained water or
nutrient uptake. However, studies on the (structural) differ-
ences among salt-stressed root systems that may partially
underlie uptake capacities have received less attention
(Vadez et al., 2007; Rewald et al., 2011b).
Because root system traits, such as water uptake rates per
surface area, are defined by the properties of individual root
segments (see Rewald et al., 2011a, and references within),
detailed studies about the abundance, morphology, anat-
omy, and physiology of individual roots are needed. Due to
the fact that traits often vary according to the position of
individual root segments among the root branching hierar-
chy (i.e. ‘root order’; Page `s and Kervella, 1990; Pregitzer
et al., 2002; Valenzuela-Estrada et al., 2008), analysis by root
order is a powerful approach to understand complex woody
root systems under stress. However, the morphological/
anatomical properties and frequencies of the most distal
root orders have been determined to date on <40 woody
species world-wide (e.g. Pregitzer et al., 2002; Wang et al.,
2006; Guo et al., 2008a); even fewer studies have quantified
total number, biomass, and/or surface area of root orders
(Valenzuela-Estrada et al., 2008; Rewald et al., 2011a).
Most previous studies have used indirect, specifically
morphological and anatomical, analyses to estimate differ-
ences in root order functionalities (e.g. Valenzuela-Estrada
et al., 2008; Huang et al., 2010). Direct hydraulic measure-
ments on certain root orders were restricted for a long time to
abscised (e.g. Schulte, 2006; Bramley et al., 2007) or distal
(Zwieniecki and Boersma, 1997) root segments. However,
Rewald et al. (2011a) have recently developed a method to
determine water fluxes among root orders. It was shown that
water flux densities under homogeneous, non-stressed con-
ditions are determined by root order but not by root diameter
or the position of a root segment within a root branch or the
whole root system. Because water uptake is reduced under
salinity and during periods of salt stress release (Cimato et al.,
2010; Rewald et al., 2011b), detailed knowledge on water
uptake capacities within the root branching system is key to
understanding plant functioning under salinity.
To understand the adaptive response of Citrus volkameri-
ana rootstocks under severe NaCl stress, the present work
studies root traits and water uptake on the level of root
orders. Two questions are addressed in detail. (i) Which
architectural, morphological, and anatomical changes occur
in salt-stressed C. volkameriana rootstocks? (2) What
contribution do specific root orders make to water uptake
under salinity and after a rapid release of salt stress? It is
hypothesized that the type of plasticity (e.g. architectural,
morphological, and anatomical) differs among root orders
and that the water uptake by salt-stressed root systems is
highly influenced by changes in abundance and water flux
density among specific root orders.
Materials and methods
Plant material and growth conditions
Citrus volkameriana Ten. & Pasq. rootstocks are of economic
importance because of their resistance to the Citrus tristeza virus
and as medium salt excluders (Levy and Shalhevet, 1990; Ramin
and Alirhezanezhad, 2005). In 2006, 1-year-old Citrus sinensis
Osbeck var. Newhall shoots were grafted on adequately sized
C. volkameriana rootstocks. The plants were grown in fertigated,
soil-filled 10 litre pots in a greenhouse at ambient temperature
until October 2009. As of this time, eight equal sized plants were
selected, roots were rinsed, and plants were moved to constantly
aerated hydroponics (Supplementary Fig. S1 available at JXB
online). Plants were placed into opaque 20 litre pots filled with
;17 litres of either 1.0 strength Long Ashton (LA; Ottow, 2005)
solution (control treatment) or 1.0 strength LA plus 90 mM NaCl
(salt treatment). The osmolalities of the solutions were 2461 mmol
kg?1and 16261 mmol kg?1, respectively (mean 6SE, n¼10;
Vapro 5520, Wescor, Logan, UT, USA). The pots, tightly covered
to prevent light penetration and evaporation, were placed in
a controlled growth room [air temperature <28 ?C (day), 20 ?C
(night); relative humidity 30–40% (day), 70% (night); photosyn-
thetic photon flux density (PPFD) 300–400 lmol m?2s?1(day-
length 12.5 h)]. The temperature of the hydroponic system was
kept at 2060.1 ?C (BL-30, MRC, Holon, Israel); transpired water
was refilled every other day and the entire solution was exchanged
every other week. After 5 months, leaf stomatal conductance
between 12:00 h and 13:00 h was 81.667.4 mmol m?2s?1(control)
and 35.563.5 mmol m?2s?1in plants under salinity (mean 6SE,
n¼22–24; SC-1 Porometer, Decagon, Pullmann, WA, USA).
Analysis of root morphology, surface area, and biomass
After ;6 months in hydroponics, the rootstocks of three Citrus
plants per treatment were severed from the stem above the highest
root. Aboveground biomass was separated into leaves and
branches, dried (70 ?C, 48 h), and weighed. Subsequently 12 ‘large
root branches’ (i.e. branches with 5–6 root orders) and four ‘small
root branches’ (<4 root orders present) attached to the tap root
were randomly severed per individual. This ratio was chosen
according to a visual pre-examination of the control rootstocks.
The 16 root branches per plant were dissected into root orders and
were kept moist constantly. The architectural classification follows
the stream classification approach (Pregitzer et al., 2002), allowing
most distal root segments (root tips) to be defined persistently as
first-order roots even if the total number of orders is subject to
change or unknown. Roots that possess only first-order side roots
were named second-order roots, root segments bearing exclusively
first- and second-order side roots were named third-order roots
(starting at the most distal point along the root axis where two
second-order roots met), and so on (see Supplementary Fig. S1A
at JXB online).
The remaining roots (i.e. roots beside the 16 root branches
analysed in detail) were separated into fourth root order and
higher, while the biomass of the root orders 1–3 was later divided
by the ratios calculated from the detailed dissection of root
branches. Finally, ;60–70% of the root systems were analysed in
detail. Live roots were distinguished from dead roots (Rewald and
Leuschner, 2009); dead roots and any adhering particles were
discarded.
After dissection, root segments were stored in closed Petri dishes
with small amounts of tap water to keep the roots hydrated (4 ?C,
<5 d), separated by treatment, plant, root branch, and order, until
digital images capturing of root orders 1–6 took place on a flatbed
scanner (grey scale, 400 dpi). To determine root diameters and
surface areas, images were analysed with the software WinRhizo
2005c PRO (Re ´gent, Quebec, Canada). Finally, root samples were
dried (70 ?C, 48 h) and weighed to a precision of 60.1 mg using an
analytical scale (CP225D, Sartorius, Go ¨ttingen, Germany). The
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specific root area [SRA, cm2g dry weight (d.wt)?1) and the root
diameter were calculated per root order using data of the 16
dissected root branches per plant. The relative biomass and surface
area of root orders 1–6 within root branches was calculated, using
the 12 ‘large root branches’ per plant. The root to leaf biomass
ratio (‘root:leaf ratio’), the total plant biomass (DMT), and the
rootstock biomass were calculated from dry weights per plant
individual.
Analysis of root anatomy
Tissue sections from root order 1 were obtained close to the basal
site of the root segments. Root orders 2–4 were sampled for tissue
sections in the middle between two side branches. Eight randomly
selected tissue sections of root orders 1–4 in each treatment were
studied; root orders 5 and 6 were not analysed due to the difficult
preparation of heterogeneously dense samples. The sections were
fixed for 48 h by immersion [5% formaldehyde, 5% acetic acid,
90% ethanol (70%)]. Dehydration of the tissue sections was
accomplished in a graded ethanol series (50, 70, 95, and 100%, 30
min each) followed by immersion in tert-butanol (8 h) and
embedding in Paraplast Plus. After hardening, 12 lm thick cross-
sections were cut with a rotation microtome (RM2235, Leica,
Nussloch, Germany). Cross-sections were collected on glass slides
and placed on a warming tray (40 ?C, 3 h). The tissue sections were
deparaffinized in xylene (33 10 min) and rehydrated (ethanol 100,
95, 70, and 50%, 5 min each). The washed sections (H2O, 1 min)
were successively stained with safranin (0.5%) and fast green
(0.5%; Ruzin, 1999, and references within), cleared with 100%
xylene (33 10 min), and air dried. Digital images were taken (Zeiss
AxioImager A1 microscope) and cross-sections were analysed
(AxioVision 4.6, Carl Zeiss, Wetzlar, Germany). Measured param-
eters included the number of exodermal layers, and area and
diameter of the root cross-section, the cortex, the stele, and the
xylem. Relative proportions of the cortex diameter and the xylem
area were calculated. The xylem was analysed in detail by
quantifying the number, radii, and areas of xylem vessels; the
hydraulically weighted average conduit diameter [HWCD, i.e.
2(Rr5(Rr4)?1)] was calculated (Lewis and Boose, 1995). Further-
more the cross-sections were analysed for differences in the
suberization of endodermis and peri-/exodermis after staining with
aniline blue using fluorescence microscopy (Brundrett et al., 1988).
Plant chloride and sodium analysis
Dry materials of leaves and root orders 1–6, separated by plant
individual, were ground to a powder and extracted overnight with
distilled water (0.1 g of dry material in 10 ml of double-distilled
water). Chloride (Cl?) concentration was determined by silver ion
titration (Chloride Analyzer 926, Corning, MA, USA), while
sodium (Na+) analyses were carried out using a Corning Flame
Photometer 410 (Raveh, 2005).
Miniature depletion chamber set-up
Rewald et al. (2011a) constructed ‘miniature depletion chambers’
to measure the water fluxes of C. volkameriana root orders under
fresh water supply (‘control’ treatment). The current study
measured the water flux rates under salinity and after released salt
stress. The measurements took place after the plants were growing
under salinity for >4 months and in parallel to the measurements
on fresh water-grown plants.
In brief, the chambers were manufactured from small plastic
tubes (diameter¼15 mm) which were shortened to 15 mm in length
(Supplementary Fig. S1 at JXB online). Septa (IceBlue, Restek,
Bellefonte, PA, USA) were glued in place (LocTite Super Glue-3,
Henkel, Boulogne, France) on both ends. Both septa and plastic
tubes were cut open on one side, allowing for root insertion by
spreading the chamber open along the section. Septa were pre-
drilled (diameter¼0.3–3.5 mm) to enable sealing of inserted roots
(diamter¼0.5–4.1 mm) while preventing excessive squeezing. For
measuring water fluxes of the first-root order, chambers with only
one pre-drilled septum were used, with the root tip ending within
the chamber.
For chamber placement, the root systems were lifted out of the
hydroponics and fixed in mid-air for <10 min. A root segment was
chosen by the following criteria: lack of side braches on a length of
>17 mm, no signs of decay (e.g. dark-coloured, shrivelled), and
undamaged epi-/peridermis. The segment was gently blotted dry
using a paper towel, placed in the septa holes, and the chamber
was closed by a clamp. Cuts were sealed with either hot glue
(plastic tube) or superglue (septa); the root–septa interfaces were
sealed by the pressure of the septa and a small amount of
superglue.
Two different measurements were performed on four salt-
stressed rootstocks and the first four root orders: (i) 0.5 strength
LA plus 90 mM NaCl (osmolality: 15662 mM kg?1; mean 6SE,
n¼10; Vapro 5520, Wescor) was inserted into the chamber to
measure the water fluxes under salt stress (‘salt’ treatment) or (ii)
0.5 strength LA (osmolality: 1561 mM kg?1) was used to measure
the flux rates after rapid release of salt stress (‘stress release’
treatment). In each case, 1.3 ml of aerated (>18 h) solution was
injected into the chamber at 9:00 h to allow for 2 h of equilibration
before measurement started at 11:00 h.
Determination of water flux rates per root order
In brief, a thin plastic tube attached to a hollow needle was used to
connect the ‘miniature depletion chambers’ to a storage container
placed on an analytical scale (see above; Supplementary Fig. S1 at
JXB online). Both the tube and the storage container were filled
with the type of solution added to the chamber. To prevent bias by
gravimetric force, solution levels in the storage container and the
hydroponic pot were brought to the same height. The weight of the
storage container was recorded every minute (Sartorius Connect
1.0; Sartorius, Go ¨ttingen, Germany). To induce high, measurable
mass flux rates (Fm; g h?1) the period between 11:00 h and 14:00 h
was chosen because transpiration maxima (related to temperature
maxima and relative humidity minima) were expected during this
time.
Five to 11 flux measurements were performed per solution type
and per root order 1–4; higher replicate numbers were used if the
first five measurements were very heterogeneous. Linear regres-
sions (R2¼0.43–0.99, P < 0.01) were performed to determine Fm
from the 3 h measurement period and it was correlated with the
surface area (cm2) of segments to calculate the water flux density
(Js; g cm?2h?1).
Water flux rates on the level of root branches
The 12 large root branches per plant were used to calculate
‘standardized’ Citrus branches under fresh water and salinity. The
biomass and surface area proportions of root orders 1–4 were used
to determine their absolute surface areas (SAs; cm2) in a root
branch of 1 g dry weight. Root orders 5 and 6 were excluded from
estimates of biomass and surface area proportions owing to their
small surface area (<4% and <6% SA of large root branches under
control treatment and salt stress, respectively) and because they
were not measured for water flux.
To determine the mean water flux densities (J? s) of the
standardized root branches, the water flux densities (Js) under
fresh water, salinity, or stress release conditions were weighted by
the surface area of the respective root order (A) under fresh water
or salt stress, respectively. By setting the total flux rates per branch
as 100% and dividing them by the flux rates of the four root
orders, the relative proportion of different root orders on the total
flux rates of the root branch was calculated.
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Statistics
Statistical calculations were conducted with SAS version 9.2 (SAS
Institute, Cary, NC, USA). Data sets were tested for Gaussian
distribution with the Shapiro–Wilk test and for homogeneity of
variances with the Levene test. Because of unbalanced data,
a general linear model (PROC GLM) was used to test for
significant influences of treatment, order, and interactive effect on
root traits and for root order-specific changes in traits between
treatments. For traits expressed as percentages, the Bliss angular
transformation was applied. To test for salt effect on the root
biomass and the root:leaf ratio, the DMTwas used as a covariate
in PROC GLM. Parametric Tukey test was used for examination
of tissue NaCl concentrations. Analyses of variance comparing
root order, treatment, and their interaction were performed by the
PROC ANOVA procedure for Na+and Cl?contents in roots; the
interaction was removed later because of non-significance. Critical
a for all tests was set at <0.05.
Results
Plant biomass, total root biomass, and root:leaf
biomass ratio
Total plant biomass was reduced by 27% under salinity
(Fig. 1A), caused by a major reduction in leaf biomass,
some dead branches, and a minor reduction in total root
biomass (;2%; Fig. 1B inset, Table 1). While the reduction
in root biomass was marginally significant (P¼0.09), the
root:leaf biomass ratio increased significantly (P < 0.03)
from 0.65 under fresh water to 2.96 after 6 months under
high salinity (Fig. 1B, Table 1).
Root architecture
After 6 months in hydroponics, the C. volkameriana root-
stocks had eight root orders in total under fresh water
(control) and seven root orders when grown under salt
stress (data not shown). In both treatments, the highest root
order formed the tap root; the first root orders were clearly
distinguishable as root tips. The architecture of C. volka-
meriana rootstocks; that is, the proportion of root orders
within the branching root system, changed under salinity in
respect to both biomass and surface area (Fig. 2A, B). Both
biomass and surface area frequencies changed significantly
among root orders 1–6 (P < 0.001) and as an interactive
effect of root order and salinity (P < 0.5; Table 2).
First-order roots (root tips) provided 2562% of the
biomass under fresh water; under salinity this amount was
significantly (P < 0.001) reduced to 1762% (mean 6SE;
Fig. 2A, Table 2). The abundance of the root order-specific
biomass declined markedly with increasing root order under
fresh water supply; biomass was more homogeneously
distributed among root orders 1–5 under salinity. No
significant changes were found in biomass frequencies of
root orders 2–6 under salt stress (Table 2).
The surface area shares of root orders (SA%) varied from
the biomass distribution due to differences in SRAs (see
below). Root orders 1 and 2 showed contrasting changes
under salinity; the relative SA provided by the first-root
order (root tips) decreased significantly (P < 0.05) from
4262% (Ctrl) to 3562% (Salt), while the second-order roots
accounted for a significantly (P < 0.05) higher percentage
(3%) of root branch SA under salinity (2961%; mean 6SE;
Fig. 2B, Table 2). The SA% of root orders 3–6 did not
change significantly between treatments.
Morphology of root orders
Root diameter increased significantly under salinity (P < 0.01)
and with increasing root order (P < 0.001; Fig. 2C, Table 2).
However, when separately analysed by root order, only the
diameter increases in root orders 2, 3, and 4 were significant
(P < 0.05). For example, the root diameter of third order roots
increased from 0.8660.02 mm (control) to 0.9860.04 mm
under salinity (mean 6SE; Fig. 2C).
The SRA decreased significantly (P < 0.001) with in-
creasing root order under both treatments and was signifi-
cantly lower (P < 0.001) under salinity (Table 2). The
interaction effect between treatment and root order was
found to be significant (P < 0.01). Analysed by order, the
SRAs of root orders 1–4 were significantly (P < 0.05)
reduced under salinity while the SRA of root orders 5 and 6
did not change significantly (Fig. 2D). For example, the
SRAs of root orders 1 and 4 were 382610 cm2g?1and
10164 cm2g?1under fresh water and 33866 cm2g?1and
8764 cm2g?1under salt stress. respectively (mean 6SE).
Anatomy of root orders
The cortex thickness increased significantly (P < 0.01) in
higher root orders but showed no homogeneous change
under salinity (Fig. 2E, Table 2; Supplementary Fig. S2 at
JXB online). Split up into root orders, the relative cortex
thickness decreased significantly in root order 3 and in-
creased significantly in root order 1 (root tips) and 4 under
salinity (P < 0.05).
The proportion of the stele increased significantly (P <
0.001) in higher root orders (Table 2). However, the direction
of change differed among orders under salinity: stele propor-
tions were unchanged in first-order roots, increased signifi-
cantly in third-order roots (;39%; P < 0.01), and decreased
significantly in fourth-order roots (;32%; P < 0.05; Fig. 2F,
Table 2).
Table 1. GLM results for the effect of salt stress on the root:leaf
biomass ratio and root biomass (n¼3)
The total plant dry mass (DMT) was used as covariate for root:leaf
ratio and root biomass.
ParameterClass Covariate
SalinityDMT
Root:leaf biomass ratio
F
P
F
P
16.24
0.026
6.34
0.086
8.62
0.061
13.12
0.036
Root biomass
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The xylem density was significantly lower in higher root
orders (P < 0.001) and a significant interaction effect
between salinity and root order was found (P < 0.05;
Table 2). Fourth-order roots of salt-stressed plants had
a significantly (P < 0.01) lower xylem vessel density
(2.160.2 n mm?2) than control plants (3.360.5 n mm?2;
mean 6SE, Fig. 2G). No significant changes in xylem
density associated with salinity were found for root orders
1–3 (Table 2); however, the xylem vessel density tended to
decrease (P¼0.06) with salinity in third-order roots.
HWCD increased significantly (P < 0.001) in higher root
orders (Fig. 2H; Table 2). Changes in HWCD were also
significant (P < 0.05) between treatments and a significant
(P < 0.05) interaction effect between salt stress and root
order was found. Analysed per root order, the HWCD of
root order 3 increased significantly (P < 0.01) from
11.460.7 lm under control treatment to 14.860.7 lm
(mean 6SE) under salt stress, and the HWCD of root order
4 tended to increase (P¼0.09) by the same magnitude
(Table 2).
No differences in the number of peri- and exodermal
layers and the suberinization of the endodermis and the
peri-/exodermis were found between treatments (data not
shown). However, in higher root orders, larger areas of
both endo- and exodermis were suberized compared with
low root orders (data not shown).
Na and Cl ion accumulation in leaves and roots
Sodium and chloride ion concentrations increased signifi-
cantly (P < 0.01) in salt-stressed leaves (7- and 4-fold,
respectively; Table 3). In roots, the concentration of both
ions differed significantly (P < 0.001) between root orders,
with lower concentrations in higher root orders (Table 3,
Supplementary Table S1 at JXB online). For example, the
Cl?concentration in salt-stressed root tips (root order 1)
was 13.6564.08 mg g d.wt?1, while sixth-order roots had
a Cl?concentration of 3.6860.27 mg g d.wt?1(mean 6SE).
Ion concentrations in low root orders were often signifi-
cantly (P < 0.05) higher than in leaves (Table 3). Between
treatments, concentrations of both ions increased in roots
under salinity; while the increase was marginally significant
(P ¼ 0.07) for sodium, possibly due to low Na+concen-
trations in higher root orders, the increase in chloride
concentrations was highly significant (P <0.001; Supple-
mentary Table S1).
Surface areas of root orders within root branches
When root systems were analysed on the level of root orders
1–4 (which were measured for water flux density, see
below), 1 g of root biomass (d.wt) built 261 cm2root SA
under control conditions and 201 cm2under salt stress.
Approximateloy 39–50% of the SA was provided by root
order 1 (root tips); that is, a SA of 12265 cm2under fresh
water in contrast to 7865 cm2under salinity (mean6SE;
Fig. 3). Root order 2 accounted for 28–32%, root order
3 for 18%, and the fourth root order for 7–12% of root
branch SA. While the SA generally decreased with in-
creasing root order under both control and salinity, no
significant difference was found between SA of first and
second root orders under salinity. Significant differences
were found between the root SA built by root orders 1 and
3 between the fresh water and saline treatment, with
significantly lower SA in first- and third-order roots under
salinity (P < 0.05).
Table 2. Frequency of root orders (in respect to biomass and surface area; n¼36), root diameter, specific root area (n¼36–197), cortex
and stele dimensions, xylem density, and the hydraulically weighed conduit diameter (HWCD; n¼8) were analysed by two-way GLM
either pooled or separated by root order
Parameter Salinity effectRoot order effectSalinity 3 root orderSalinity effect by root order
123456
Frequency of root order (biomass)a
F
P
F
P
F
P
F
P
F
P
F
P
F
P
F
P
0.76
0.385
1.47
0.226
12.21
0.001
62.54
<0.001
0.67
0.416
0.89
0.349
0.23
0.636
4.57
0.037
135.65
<0.001
1780.67
<0.001
1194.64
<0.001
2247.65
<0.001
41.14
<0.001
115.36
<0.001
33.03
<0.001
86.00
<0.001
10.31
0.001
4.47
0.035
0.20
0.652
10.76
0.001
0.81
0.372
2.63
0.110
5.23
0.026
6.73
0.012
14.08
<0.001
6.16
0.015
2.01
0.157
13.63
<0.001
5.23
0.026
0.44
0.519
1.34
0.267
0.00
0.976
0.01
0.925
4.58
0.035
6.39
0.012
28.75
<0.001
0.20
0.664
0.12
0.731
0.23
0.636
1.38
0.300
0.68
0.412
0.02
0.089
6.34
0.012
26.55
<0.001
5.10
0.041
9.78
0.007
4.38
0.055
11.56
0.004
2.29
0.134
0.90
0.345
6.45
0.012
4.62
0.032
10.44
0.006
6.49
0.023
11.70
0.004
3.40
0.086
3.16
0.079
3.49
0.065
0.25
0.621
0.41
0.524
n.d.
0.00
0.954
0.64
0.424
2.33
0.125
1.31
0.260
n.d.
Frequency of root order (surface area)a
Root diameter
Specific root area
Cortex diameter:root diameter ratio
Stele area : root area ration.d.n.d.
Xylem density n.d.n.d.
HWCDn.d.n.d.
aBliss angular transformed. n.d., not determined.
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Water flux density
Water flux densities (Js) differed significantly between C.
volkameriana root orders and treatments (Fig. 4). Water
uptake under fresh water (control) declined significantly (P <
0.05) from root order 1 (root tips) to root orders 2 and 3
(0.7160.15, 0.1660.04, and 0.1160.02 g cm?2h?1, respec-
tively; mean 6SE); fourth root orders possessed water excess
(–2.4960.69 g cm?2h?1; Fig. 4A). Under salinity and after
stress release, Jsof first-order roots was significantly reduced
by >80% to 0.1260.25 g cm?2h?1and 0.1360.09 g cm?2
h?1, respectively (Fig. 4B, C). Mean water flux densities (J? s)
in root orders 1–4 were generally low under salinity (–0.07 g
cm?2h?1to 0.18 g cm?2h?1) while the variability in water
flux densities increased significantly as compared with the
control (data not shown). Under stress release conditions (i.e.
0.5 LA solution in the miniature depletion chamber, root
system placed in 1.0 LA + 90 mM NaCl) all root orders took
up water (Fig. 4C). The highest Jsvalues under stress release
conditions were found in second- (0.3160.06 g cm?2h?1)
and third-order roots (0.6760.49 g cm?2h?1); second-order
roots had significantly (P < 0.05) higher uptake rates of fresh
water after stress release than under continuous fresh water
treatment (control). The fluxes of stress-released third-order
roots were significantly higher than under salinity (P < 0.05;
Fig. 4B, C).
Up-scaled, using the surface area and Jsof root orders,
a root branch with four root orders and 1 g d.wt (Fig. 3)
had mean water flux densities (J? s) of 0.208, 0.099, and 0.283
g cm?2h?1under control conditions, salinity, and stress
release, respectively (Fig. 4, insets).
The relative contribution of root orders to the branch
water flux density differed among treatments (see Supple-
mentary Fig. S3 at JXB online). For example, 57% of the
water fluxes were mediated by first-order roots under fresh
water (control), in contrast to 38% under salinity and 18%
after stress release conditions. Second-order roots mediated
48% of the water fluxes under salinity and 35% after salt
release in contrast to 7% under fresh water supply
(Supplementary Fig. S3).
Discussion
Changes in phenotype in response to salinity are often
adaptive by enhancing the fitness of plants. For example,
increased root:shoot ratios are thought to improve the
‘source:sink ratio’ for water and nutrients under salinity
(Zekri and Parsons, 1989). In this study, the root:leaf ratio
of Citrus increased significantly while the rootstock biomass
was marginally reduced under salinity (Fig. 1, Table 1).
However, because root functions, such as water uptake, are
strongly related to root tissue differentiation (Doussan
et al., 1998) and root order (Pregitzer et al., 2002; Comas
and Eissenstat, 2009; Rewald et al., 2011a), total root mass
and root:shoot ratios cannot effectively determine the
functionality of woody root systems under stress. Thus, to
predict uptake, knowledge of the active root surface area
and the flux density is needed (Hinsinger et al., 2011)
Structural, morphological, and anatomical changes
under salinity
Several previous studies on woody species found reduced
numbers of lateral roots under salinity (e.g. Reinhard and
Rost, 1995; Eshel and Waisel, 1996; Croser et al., 2001);
similarly, this study provides evidence that severe NaCl
stress reduces the number of Citrus root orders from eight
to seven. However, more importantly, the current study
shows that the frequency of root orders and morphological
and anatomical traits known to influence uptake processes,
for example root system branching (Dunabin et al., 2004),
SRA (Trubat et al., 2006), cortex thickness (Rieger and
Litvin, 1999), and xylem traits (Rodrı ´guez-Gamir et al.,
2010), do not change homogenously throughout C. volka-
meriana root systems under salinity but that changes are
often root order specific (Table 2).
Changes among order frequencies of Citrus roots were
previously reported under altered nitrate supply (Sorgona `
et al., 2007, 2011). Similarly, in this study, lower root
orders, especially root tips, were most plastic in frequency,
expressed as both relative biomass and surface area per root
Table 3. Sodium (Na+) and chloride (Cl?) ion concentration in Citrus spp. leaf and root (root orders 1–6) tissues under fresh water
(Control) and salinity (90 mM NaCl)
OrganIon concentration in tissues (mg g d.wt?1)
ControlSalt
Na+
Cl?
Na+
Cl?
Leaves
First-order roots
Second-order roots
Third-order roots
Fouth-order roots
Fifth-order roots
Sixth-order roots
1.0260.10 a
7.9561.35 b
8.1961.64 b
5.7961.31 b
2.1260.39 a
1.0860.32 a
0.9960.18 a
1.8260.10 a
6.8462.07 b
7.1461.22 b
6.2160.38 b
3.8660.36 ab
2.1960.22 a
2.0060.16 a
7.4960.09 a,b
10.8662.98 a
9.3961.92 a
6.0861.48 a,b
3.3660.22 b
2.5160.04 b
2.1260.03 b
6.0460.03 b,c
13.6564.08 a
12.8462.48 a,b
10.3861.70 a,b,c
6.4560.28 a,b
4.6260.31 c
3.6860.27 c
Different lower letters denote differences within columns (mean 6SE; Tukey, P < 0.05, n¼3–10); see Supplementary Table S1 at JXB online for
ANOVA results on the influence of treatment and root order on the ion concentrations.
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branch (Fig. 2A, B, Table 2). Because low root orders have
a high SRA, the 3% loss in total root system biomass under
salinity caused a major reduction of active root surface area
by ;23% (Figs 1–3). Because uptake is strongly coupled to
root SA (e.g. Korn, 2004), the reduced SA provided by root
tips under salinity is indicative of a decrease in functional-
ity, in terms of uptake, of this order. In contrast, the
relative importance of root orders >2 for water and/or
nutrient uptake should increase accordingly. The high
plasticity of first (second) root orders in respect of biomass
(SA) frequency and morphology was anticipated as lower
root orders have relatively high turnover rates (Guo et al.,
2008b, and references within) and are considered most
vulnerable to environmental stresses. However, in addition
to results from Citrus spp., seedlings under varied nitrate
supply, in which the morphology of second and third (i.e.
tap roots of these seedlings) root orders were considerably
less plastic than those of root tips (Sorgona ` et al., 2007,
2011), the present result showed that even intermediate,
third and fourth, root orders of more mature plants are able
to undergo significant morphological changes within 6
months (Table 2).
Similar to earlier reports, the cortex diameter decreased
and the proportion of the stele increased in higher root
orders under fresh water supply (Fig. 2E, F; Guo et al.,
2008a). The increase in root diameter under salinity was
expected to be caused by increasing cortex dimensions as
reported, for example, for cotton roots (Casenave et al.,
1999). However, under salinity, significant changes of cortex
and stele dimensions were found in third and fourth root
orders only, and reaction norms differed in direction
(Fig. 2E, F, Table 2). It is hypothesized that the contrasting
changes are related to the different functions of these two
root orders for water uptake under fresh water; in brief,
third-order roots of C. volkameriana were found to perform
water uptake under fresh water supply, while fourth-order
roots showed water excess (Fig. 4A). The outflow of water
in fourth-order roots was related to changes in chamber
solute osmolalities, possible caused by exudation (for
details, see Rewald et al., 2011a).
The lack of significant changes in gross root anatomy (i.e.
cortex and stele dimension) and xylem traits in first and
second root orders was surprising because these root orders
have a larger number of passage cells (Eissenstat and
Achor, 1999), and are the preferred sites of water and
nutrient uptake under fresh water (Fig. 4; Supplementary
S2 at JXB online; Peterson and Enstone, 1996; Rewald
et al., 2011a). Previous studies on cotton radicles and roots
of herbaceous plants found smaller xylem vessels at higher
frequencies under salinity, probably caused by a repression
in the development of metaxylem vessels and altered
cambial activity (Reinhardt and Rost, 1995; Casenave
et al., 1999; Boughalleb et al., 2009). However, wider and
Fig. 1. (A) Total plant biomass and (B) root:leaf biomass ratio of
Citrus spp. after 6 months under fresh water (Ctrl, open bars) and
salinity (Salt, filled bars). The inset in B shows the root biomass per
treatment (mean 6SE, n¼3). See Table 1 for statistics.
Fig. 2. Structural, morphological, and anatomical traits of Citrus
volkameriana root orders 1–4/6 after 6 months under fresh water
(ctrl, open bars) or salt stress (salt, filled bars). (A and B) Relative
root biomass and root surface area of orders 1–6 in root branches
(mean 6SE, n¼36). (C and D) Root diameter and specific root
area (mean 6SE, n¼36–197). (E–H) Cortex diameter relative to
root diameter, percentage of the stele on the root cross-section
area (CSA), xylem vessel density related to the area of the stele,
and hydraulically weighed conduit diameter (HWCD; mean 6SE,
n¼8). See Table 2 for statistics.
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fewer xylem vessels have been found in both stems and
some coarse roots after exposure to salinity (e.g. Eckstein
et al., 1978, Rewald et al., 2011c), thus the present results
might indicate a different reaction norm in ephemeral
roots (such as root order 1 and 2) and more persistent
woody roots (such as root order 3 and 4) under salinity.
While the underlying molecular mechanisms and the
functional significance of these changes in the xylem
structure remain open, the differences between root orders
under salt stress are probably related to the varied
accumulation of sodium and chloride ions (Table 3).
Lower salt concentrations are suggested to impair the
cambial activity and metaxylem differentiation in higher root
orders to a lesser extent. Suberization of the endo- and
exodermis increased in older (higher) Citrus root orders (data
not shown) as found elsewhere (e.g. Eissenstat and Volder,
2005) and is likely to be one factor underlying the
significantly lower Na+and Cl?accumulation in higher root
orders of C. volkameriana (Table 3, Supplementary Table S1
at JXB online; Krishnamurthy et al., 2009). In contrast to
findings in herbaceous plants, which often have lower Na+
and Cl?concentrations in roots than the external solution
(Munns, 2002), salt accumulation in Citrus root orders 1–5
was explicitly higher than those of the surrounding solution
and partially higher than those in leaves (see also Arbona
et al., 2005). Because physiological damage in Citrus spp. is
associated with tissue chloride build-up rather than with
sodium accumulation (Romero-Aranda et al., 1998), the high
Cl?concentration in lower root orders backs up the
hypothesis that many structural, morphological, and ana-
tomical changes were driven by accumulating salt.
Effect of salinity on root water uptake
It was hypothesized that changes in water flux rates in salt-
stressed Citrus rootstocks are related to changes in both
root surface area and root anatomy/physiology, as origi-
nally suggested by Storey and Walker (1999). As mentioned
above, changes were observed in abundance and SRA of
root orders 1–4 that resulted in a 23% reduction of root
branch surface area under salinity compared with fresh
water (Fig. 3). Changes among anatomical (see above) and
physiological parameters (e.g. membrane properties) are
also known to modify root hydraulic conductivity (Lpr;
Peterson and Enstone, 1996; Steudle, 2000; Vandeleur et al.,
2009). In the present study we did not find major changes in
gross root anatomy; thus, besides the lower water potential
of the salt solution, fine-scale anatomical or physiological
changes and damage caused by high salt accumulation (see
above) have probably contributed to the 50% reduction of
root branch mean water flux densities (J? s) in this study
(Fig. 4A, B). This is supported by the finding that Jsdid not
increase in first-order roots after release of the salt stress
(Fig. 4C) as expected if only temporarily caused by the
lower water potential of the salt solution. Because water
flow rates among Citrus root systems follow the same trend
as root conductivities (Zekri and Parsons, 1989), it is valid
to hypothesize that the reduction of J? sin salt-stressed Citrus
rootstocks is related to changes in both root surface area
and root physiology.
Fig. 4. Water flux density (Js) of Citrus volkameriana root orders 1–4
under (A) fresh water (control), (B) salinity, and (C) stress release
conditions. Plants were placed either in 1.0 strength LA (A) or 1.0
strength LA + 90 mM NaCl (B, C). The miniature depletion
chambers were filled with either 0.5 strength LA (A, C) or 0.5 LA +
90 mM NaCl (B), respectively. Different lower case letters denote
significant differences between root order-specific flux densities;
different upper case letters denote significant differences between
treatments (mean 6SE; Mann–Whitney U-test, P < 0.05, n¼5–11).
Mean water flux densities (J? s) of root branches of 1 g d.wt. are given.
Fig. 3. Surface area of root orders 1–4 under fresh water (Ctrl,
open bars) and salinity (Salt, filled bars) in a standardized root
branch of 1 g d.wt. Different lower/upper case letters denote
significant differences between root orders within control and
saline treatments respectively; Asterisks denote significant differ-
ences between treatments (mean 6SE; Mann–Whitney U-test,
P < 0.05, n¼189–214).
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Interestingly, the water flow densities (Js) of root orders
changed differently under salinity (Fig. 4A, B). The
decrease in J? s under salinity was mainly caused by
a significant reduction (>80%) of the water uptake by
first-order roots. The degree of Jsreduction is in accor-
dance with measurements on apical segments of corn
roots, among which Lprwas reduced by 80% under salinity
(Evlagon et al., 1990). The water flux densities of the other
three Citrus root orders did not change significantly under
salinity (Fig. 4B). Together with the different reduction in
surface areas (see above), the varying water flux densities
are changing the contribution of root orders to the overall
water flux density of the root system (Fig. 4, Supplemen-
tary Fig. S3A, B at JXB online). For example, second-
order roots contributed only 7% to the water fluxes under
fresh water but nearly 50% under salinity. The increased
importance of higher root orders for water uptake under
salinity may help to explain previous results of Zekri and
Parsons (1989) who found the highest reductions of root
length under salinity in Citrus species which were most
tolerant to salinity in terms of water flow rate or root
conductivity. This has been thought to be in contrast to
studies which found that Citrus rootstocks with high
specific root lengths tend to exhibit high hydraulic
conductivities under fresh water supply (Graham and
Syvertsen, 1985; Eissenstat, 1997). However, the overall
root length density might cause an overestimation of the
water uptake capacity under salt stress if thin, first-order
roots contribute less to water uptake (as seen in the current
study). This might be true as well if parts of the root
system are temporarily released from salt stress; in this
study, second-order and third-orders roots contributed
more to water uptake under stress relief than under both
control and salt treatments (Fig. 4, Supplementary Fig.
S3C). A temporal and spatial release of roots from salinity
might occur in situ, for example in saline water-irrigated
orchards (during ‘salt leaching’) or after rainfall events
(Cimato et al., 2010).
While Sorgona ` et al. (2007) stated that more distal root
orders have a prominent adaptive significance in Citrus,
possibly due to their high number of passage cells (Eissenstat
and Achor, 1999), this study demonstrated that the impor-
tance of specific root orders for water uptake and tolerance
of the whole root system is subject to changes in response to
environmental conditions. The underlying anatomical and
physiological factors still remain open but root order-specific
changes in the development of Casparian bands, suberin
lamellae, passage cells (Peterson et al., 1993; Peterson and
Enstone, 1996), aquaporin expression (Vandeleur et al.,
2009), or a different susceptibility to reactive oxygen
species (Li et al., 2009) or salt accumulation (this study)
might have resulted in the observed differences. Further
investigation is needed to determine the parameters un-
derlying the (i) different susceptibility of root tissues to salt
stress and ion accumulation and (ii) the different water flux
densities among root orders, and should seek to compare
the function of different salt-tolerant rootstocks on the
level of root orders.
Supplementary data
Supplementary data are available at JXB online.
Figure S1. Schematic side view of a ‘miniature depletion
chamber’ (A), attached to a second-order root, and a
drawing of the experimental set-up (B).
Figure S2. Photographs of Citrus volkameriana root
orders 1–4 after 6 months under fresh water (A–D) and
salinity (E–H).
Figure S3. Relative contribution of root orders 1–4 to the
total water flux (100%) under (A) fresh water (control), (B)
salt stress, and (C) after stress release.
Table S1. Influence of treatment and root order on the
Na+and Cl?concentrations in Citrus volkameriana roots.
Acknowledgements
The authors wish to thank L. Summerfield and O. Shelef for
their help regarding root dissection and image analyses.
L. Rose and two anonymous reviewers provided helpful
comments on earlier drafts of the manuscript. BR was
partially supported by a post-doctoral fellowship awarded
by the Jacob Blaustein Center for Scientific Cooperation
(BCSC), Israel.
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