Annals of Botany 115: 41–53, 2015
doi:10.1093/aob/mcu212, available online at www.aob.oxfordjournals.org
In situ analysis of foliar zinc absorption and short-distance movement in fresh
and hydrated leaves of tomato and citrus using synchrotron-based X-ray
fluorescence microscopy
Yumei Du1, Peter M. Kopittke2, Barry N. Noller1, Simon A. James3, Hugh H. Harris4, Zhi Ping Xu5,
Peng Li6, David R. Mulligan1 and Longbin Huang1,*
1
Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, The University of Queensland, St Lucia, Queensland
4072, Australia, 2School of Agriculture and Food Sciences, The University of Queensland, St Lucia, Queensland 4072,
Australia, 3Australian Synchrotron, Clayton, Victoria 3168, Australia, 4School of Chemistry and Physics, University of
Adelaide, South Australia 5005, Australia, 5School of Chemical Engineering, The University of Queensland, St Lucia,
Queensland 4072, Australia and 6ARC Centre of Excellence for Functional Nano
materials, The University of Queensland, St Lucia, Queensland 4072, Australia
* For correspondence. E-mail [email protected]
Received: 25 June 2014 Returned for revision: 29 August 2014 Accepted: 12 September 2014 Published electronically: 14 November 2014
Background and Aims Globally, zinc deficiency is one of the most important nutritional factors limiting crop
yield and quality. Despite widespread use of foliar-applied zinc fertilizers, much remains unknown regarding the
movement of zinc from the foliar surface into the vascular structure for translocation into other tissues and the key
factors affecting this diffusion.
Methods Using synchrotron-based X-ray fluorescence microscopy (m-XRF), absorption of foliar-applied zinc nitrate or zinc hydroxide nitrate was examined in fresh leaves of tomato (Solanum lycopersicum) and citrus (Citrus
reticulatus).
Key Results The foliar absorption of zinc increased concentrations in the underlying tissues by up to 600-fold in
tomato but only up to 5-fold in citrus. The magnitude of this absorption was influenced by the form of zinc applied,
the zinc status of the treated leaf and the leaf surface to which it was applied (abaxial or adaxial). Once the zinc had
moved through the leaf surface it appeared to bind strongly, with limited further redistribution. Regardless of this,
in these underlying tissues zinc moved into the lower-order veins, with concentrations 2- to 10-fold higher than in
the adjacent tissues. However, even once in higher-order veins, the movement of zinc was still comparatively limited, with concentrations decreasing to levels similar to the background within 1–10 mm.
Conclusions The results advance our understanding of the factors that influence the efficacy of foliar zinc fertilizers and demonstrate the merits of an innovative methodology for studying foliar zinc translocation mechanisms.
Key words: Nutrient absorption, foliar zinc application, short-distance nutrient transport, veins, X-ray fluorescence
microscopy, XRF, Zn movement, crop nutrition, tomato, Solanum lycopersicum, Citrus reticulatus.
INTRODUCTION
Zinc (Zn) deficiency is one of the five most important micronutrient deficiencies in humans, affecting approximately one-third
of the global population (IZINCG, 2004). The foliar application
of Zn fertilizer is one of the important treatments to safeguard
crop yield and fortify Zn intake for human nutrition (Cakmak,
2008). However, the mechanisms of foliar uptake are comparatively poorly understood and current understanding of the factors that influence the ultimate efficacy of foliar applications
remains incomplete (Fernández et al., 2013). Investigation of
the foliar uptake of Zn is needed for the development of foliar
Zn fertilizers that have improved penetration/movement, are
long-lasting and have low phytotoxicity.
The foliar uptake of Zn first requires the movement/
absorption of the Zn across the cuticle and/or through the stomatal cavity (Fernández and Brown, 2013). Both the cuticle
and the stomata appear to be of importance for the uptake of
inorganic nutrients (Eichert et al., 2008), although many
uncertainties remain. For the cuticle, the penetration of apolar
liphophilic compounds can be described using the dissolution–
diffusion model, but the mechanisms of the penetration of hydrophilic polar solutes are not fully understood (for a review
see Fernández and Brown, 2013). Given that the uptake of nutrients first requires movement across the cuticle and/or through
the stomatal cavity, it is not surprising that this uptake is influenced by the characteristics of the leaf surface, including the
thickness of the wax layer and the distribution of stomata and
trichomes (Schreiber and Schönherr, 1993; Schönherr, 2006;
Eichert and Goldbach, 2008). Interestingly, the densities of trichomes and stomata are typically differentially distributed in
the adaxial and abaxial surfaces and can vary substantially
among species (Schilmiller et al., 2008). It is also known that
leaf age and nutrient deficiency can substantially influence
the surface structure of leaves (Hauke and Schreiber, 1998;
Fernández et al., 2008; Shi and Cai, 2009). However,
C The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
V
For Permissions, please email: [email protected]
42
Du et al. — In situ m-XRF analysis of foliar Zn absorption and short-distance movement
comparatively little is known regarding the relationships among
leaf surface structural characteristics (including those induced
by leaf age or by Zn deficiency) and Zn penetration and diffusion into the leaf (Loneragan et al., 1976; Zhang and Brown,
1999a; Fernández and Brown, 2013).
After the movement (absorption) of foliar-applied Zn
through the leaf surface, the overall efficacy of this Zn depends
upon the subsequent loading of the Zn into the foliar vascular
systems (consisting of interconnected veins of minor to primary
classes) and its subsequent translocation via the phloem of primary veins into the other growing tissues of the plant
(Loneragan et al., 1976; Marschner, 1995; Zhang and Brown,
1999b; Fernández and Brown, 2013). However, there remains
much that is unknown regarding the initial movement of newly
absorbed Zn into the vascular vein system and its subsequent
retranslocation out of sprayed leaves into other plant parts (for
recent reviews see Fernández and Brown, 2013; Fernández
et al., 2013).
Recent advances in synchrotron-based techniques now allow
in situ analysis of the distribution (and speciation) of metals
and metalloids in hydrated and fresh plant tissues with no observable damage (Lombi et al., 2011). The Maia detector system represents a new generation of X-ray fluorescence (XRF)
detector that provides unprecedented capability to investigate
low concentrations of trace elements in hydrated plant samples
(Kirkham et al., 2010). Given that it is approximately 10- to
100-fold faster than traditional XRF detectors, it can be used to
study trace metal uptake and distribution in highly hydrated tissues such as fresh roots (Kopittke et al., 2011, 2012, 2014). For
the examination of foliar-applied Zn, some previous studies
have used autoradiography to provide data on the spatial distribution of Zn following foliar application (Wallihan and
Heymann-Herschberg, 1956; Marešová et al., 2012).
Synchrotron-based m-XRF offers several advantages, including
improved spatial resolution and greater flexibility in regard to
the analysis of the data.
Therefore, the aim of the present study was to use m-XRF to
provide in situ quantitative data on the absorption and shortdistance movement of foliar-applied Zn using hydrated and
fresh leaves. Using both tomato and citrus, Zn diffusion characteristics were compared between (1) plant species, (2) abaxial
and adaxial leaf surfaces, (3) leaves of differing age, (4) leaves
of differing Zn status and (5) leaves of differing Zn forms. The
aim was to gather information on the factors that influence the
efficacy of foliar Zn fertilizers, to assist in improving plant
growth in Zn-deficient conditions.
MATERIALS AND METHODS
Plant growth
Plants were grown in a glasshouse at The University of
Queensland (St Lucia, Australia) during autumn with a diurnal
temperature range of 25–30 C. Seeds of Roma tomato
(Solanum lycopersicum ‘Roma’) were rinsed with deionized
water and then germinated on moistened towel paper which
was soaked with 05 mM CaSO4.2H2O at 25 C in the dark.
After germination, the tomato seedlings were cultured in onethird strength nutrient solution in the glasshouse. When the
second true leaf was nearly fully expanded, seedlings of
uniform appearance and size were transplanted into continuously aerated full strength nutrient solution in 5-L pots lined
with plain polythene bags for 2 weeks. The full-strength basal
nutrient solution used in the experiments contained (mM):
NH4NO3, 2000; KNO3, 2800; Ca(NO3)2, 1600; MgSO4, 1000;
KH2PO4, 100; K2HPO4, 100; Fe-EDTA, 40; NaCl, 8; MnSO4,
2; CuSO4, 05; Na2MoO4, 008; ZnSO4, 1; H3BO3, 10 (Huang
et al., 2008). For tomato, some plants were grown with Zn included in the basal solution (‘Zn-sufficient’) whilst other plants
were grown without Zn in the basal solution (‘Zn-deficient’).
Macronutrient stocks were purified to remove residual Zn by
complexation with 8-hydroxyquinoline (Oxine) in chloroform
at pH 55 (Hewitt, 1966). Deionized water used in the glasshouse was further purified by consecutively passing it through
a column packed with resin (Cartridge type C114, ELGA
LabWater) to prepare all chemical and nutrient solutions used
in the glasshouse experiments. Solution pH in all pots was
maintained between 55 and 65 using 1 mM HCl.
Citrus (Citrus reticulatus ‘Imperial’) plants were purchased
from a commercial nursery and transferred into 20-L pots filled
with potting mix and basal nutrients for at least 1 month of acclimation in the glasshouse. All citrus plants were grown with
Zn included in the basal nutrients of the soil culture (i.e. Znsufficient).
Leaf incubation and Zn treatment
Four types leaves were selected as follows: (1) the youngest
fully expanded leaf (YFEL) from Zn-deficient tomato; (2) the
YFEL from Zn-sufficient tomato; (3) the YFEL from Zn-sufficient citrus; and (4) the oldest leaf (i.e. above the cotyledon)
from Zn-sufficient tomato. One day prior to sampling, the
leaves were surface-rinsed with deionized water and blotted dry
in order to remove dust from the leaf surface. The following
day, the leaves were cut at the base of the petiole. The petioles
of the leaves were immediately immersed in a Zn-free nutrient
solution in a plastic centrifuge vial (15 mL) that was fixed inside an incubation chamber (Vu et al., 2013). At the same time,
another set of four leaves from the same positions was collected
for digestion and tissue analysis.
The leaves were treated and incubated in the same manner as
described in Vu et al. (2013). After 1 h of acclimation in the
laboratory, the leaves were either (1) treated with 12–16 droplets (5 mL each) of 400 mgZn L1 as Zn(NO3)2 or Zn hydroxide
nitrate (ZnHN) suspensions (30–50 mgL1) in two rows on
the central region of each leaf surface (rows were at right angles
to the midrib vein), or (2) sprayed with Zn(NO3)2 or ZnHN (the
latter only for the YFEL of Zn-sufficient citrus and tomato).
These treatments were applied to either the adaxial or the abaxial leaf surface. Thus, the experiment consisted of combinations
of the following treatment factors: four types of leaves (YFEL
from Zn-deficient tomato, YFEL from Zn-sufficient citrus,
YFEL from Zn-sufficient tomato, and oldest leaf from Zn-sufficient tomato); two forms of Zn [Zn(NO3)2 or ZnHN]; leaf surface side (adaxial or abaxial); and two methods of Zn
application (droplets or spray; Supplementary Data Table S1),
with a total of 22 treatment units (including controls). In relevant controls, no Zn was applied directly to the foliage. After
loading, the droplets on leaf surfaces were allowed to evaporate
Du et al. — In situ m-XRF analysis of foliar Zn absorption and short-distance movement
(while their petioles remained in the bathing solution) before
being placed inside incubation chambers. In all tests, the droplets applied to leaf surfaces remained in position without movement because of the small droplet size and surface tension. The
ZnHN suspension droplets adhered well to the surfaces (Vu
et al., 2013). No free droplets of water/solution were visible on
leaf surfaces during incubation.
The ZnHN suspension was prepared as described by Li et al.
(2012). Leaves were then cultured in an incubator (TRISL-4901VW, Thermoline Scientific, Australia) with controlled temperature and light conditions (12 h at 22 C light/12 h dark at
18 C) for 24 h. The leaves were then transported in Petri dishes
in a cooled (10 C) container to the Australian Synchrotron,
Melbourne. The petioles of the leaves remained in contact with
the Zn-free nutrient solution during transport to maintain
turgor.
substantially, the thickness at a given point was first measured
under a light microscope (400), and then the thickness was
calculated for other points along the vein using the Compton
scatter.
Leaf vein order definition
The vein orders of a leaf in this study was defined as follows:
the midrib is the first vein order, hereafter termed ‘1 vein’,
with smaller veins successively branching from the midrib
termed 2 (major vein), 3 , 4 , 5 , 6 , 7 and so forth (or minor
veins, which are not associated with midribs) (Sack et al.,
2012). Changes in projected Zn concentrations along the selected veins obtained by XRF were fitted using an exponential
regression with Origin (OriginPro 8). When examining changes
in the concentration of Zn within leaf tissues, an equation was
fitted of the general form:
h
i
Zn ¼ b=exp ðcDÞh
Synchrotron-based X-ray fluorescence microscopy
The leaf surface was thoroughly washed with a running jetstream consisting of a mixture of 2 % HNO3 and 3 % ethanol
and rinsed with deionized water to remove the Zn residues
from the leaf surface (Vu et al., 2013). Leaves were analysed at
the X-ray fluorescence microscopy (XFM) beamline using an
in-vacuum undulator to produce a brilliant X-ray beam. A silicon-111 monochromator and Kirkpatrick–Baez mirrors were
used to deliver a monochromatic focused beam of around
2 2 mm2 onto the specimen (Paterson et al., 2011). The XRF
emitted by the specimen was collected using the 384-element
Maia detector placed in backscatter geometry (Kirkham et al.,
2010). For all scans, samples were analysed continuously in the
horizontal direction (on the fly).
The washed leaves were blotted dry using paper tissue before
being cut in half. One half of a leaf (without the midrib) was
placed between two pieces of 4-mm thick Ultralene film, forming a tight seal around the leaf to limit dehydration. For each
sample, two scans were performed. The first scan (‘survey
scan’) was comparatively rapid and aimed to (1) examine the
entire sample and (2) identify the portion of the leaf surface to
which the Zn had been applied (to allow a more detailed scan).
The second scan (‘detailed scan’) was performed on a smaller
area of the tissues surrounding the portion of the leaf to which
the Zn had been applied. This detailed scan was conducted with
a smaller pixel size and a lower dwell (to increase both spatial
resolution and sensitivity). For the survey scan, the transit time
per 100-mm pixel was 12 ms (scanning velocity 82 mms1),
and hence maps of 20 70 mm could generally be collected
within 30 min. For the detailed scan, the transit time per
15-mm pixel was 73 ms (scanning velocity 20 mms1), and
hence maps of 5 10 mm could generally be collected within
90 min. Following the survey scan and the detailed scan, the
leaves were checked for damage using light microscopy. As
found for highly hydrated roots analysed using this technique
(Kopittke et al., 2011), there was no visible evidence of damage
following examination using m-XRF.
The XRF event stream was analysed using GeoPIXE (Ryan
and Jamieson, 1993; Ryan, 2000). Projected Zn concentrations
were obtained by correcting the areal concentration using sample thickness. Given that the thickness of the veins varied
43
(1)
where Zn is the tissue Zn concentration, b is the maximum tissue concentration of Zn, c is a strength coefficient, D is distance
and h is a shape coefficient (Kinraide, 1999).
Zn analysis
Leaf samples collected for background Zn analysis were
dried at 68 C for 72 h and digested in concentrated nitric acid
and H2O2 mixture at 125 C with a microwave oven (STARTD Microwave Digestion System, Milestone, Italy) (Huang
et al., 2004). Zinc concentrations in the digested solution were
quantified by inductively coupled optical emission spectrometry (ICP-OES, Vista Liberty Varian). Standard reference material (NIST1515 apple leaves) samples were included in acid
digestion and Zn analysis together with all the experimental
samples for accuracy assurance.
RESULTS
Leaf characterization
The Zn concentration in the YFELs of Zn-sufficient tomato
(257 mg g1, fresh mass basis) was similar to that in the mature
(older) leaves of Zn-sufficient tomato plants (245 mg g1) but
higher than that in the YFELs of Zn-deficient tomato
(177 mg g1; Supplementary Data Table S2). Indeed, this Zn
concentration in the YFELs of Zn-deficient tomato (equivalent
to 10 mg g1 on a dry mass basis) is lower than those reported
to be adequate for unlimited growth (Huett et al., 1997). The
concentration of Zn in citrus YFELs (565 mg g1) was higher
than in tomato (257 mg g1), although values were similar
when expressed on a dry mass basis (Supplementary Data
Table S2).
The density of stomata on the abaxial (18 445 cm2) surface
of YFELs from Zn-sufficient tomato plants was 12-fold
higher than that on the adaxial (1517 cm2) surface, while mature leaves had much lower stomatal density (44 cm2) than
YFELs (Supplementary Data Table S3). The YFELs from
Du et al. — In situ m-XRF analysis of foliar Zn absorption and short-distance movement
44
Zn-deficient tomato plants had stomatal density around
117 cm2 on the adaxial leaf surface. In addition, the abaxial
leaf surface of YFELs from Zn-sufficient tomato leaves had
more than twice as many trichomes as the adaxial surface. Zinc
deficiency and leaf ageing significantly decreased the density
of trichomes (Supplementary Data Table S3). The citrus leaves
did not have stomata on the adaxial leaf surface but had stomata
(493 cm2) on the abaxial leaf surface.
Absorption of Zn through the leaf surface
As demonstrated by the highly elevated Zn concentrations
compared with the baseline, the foliar application of Zn-containing solution resulted in penetration and retention (absorption) of the applied Zn through the leaf surface (e.g. Fig. 1,
Supplementary Data Fig. S1, Table 1). Indeed, analyses
using m-XRF revealed that the average projected volumetric
concentration of Zn in the tissues immediately below where
the droplets had been applied increased from 05–1 to
560 mg cm3 (Table 1). However, substantial differences
were observed between treatments.
Firstly, as expected, differences were observed between the
two plant species. The magnitude of the increase in the concentrations of Zn in the tissues immediately below where the droplets had been applied were substantially greater for tomato than
for citrus. Indeed, e.g. projected volumetric concentrations
increased 600-fold from 088 to 560 mg cm3 for tomato
(Table 1, Fig. 1B, C) but only 5-fold for citrus (from 11 to
60 mg cm3) (Table 1, Fig. 2A, C) when droplets of Zn(NO3)2
were applied to the abaxial surfaces of the YFELs.
Although both Zn(NO3)2 and ZnHN resulted in substantial
increases in Zn concentrations in the tissues underlying the
Tomato, Zn-sufficient, Zn(NO3)2, YFEL, abaxial
High
A
C
A
B
Low
B
B
D
Projected Zn
concentration (µg cm−3)
150
R 2 = 0·973
120
90
60
30
0
0
2
4
6
8
10
Distance (mm)
FIG. 1. Zinc concentration of the youngest fully expanded leaf (YFEL) of tomato (abaxial surface from the Zn-sufficient treatment) to which droplets of Zn(NO3)2
had been applied. (A) Image of half of a tomato leaf captured using a digital camera. (B) Survey (overview) scan of leaf in (A) using m-XRF. (C) Detailed scan of
the area indicated by the white rectangle in (B). (D) Projected Zn concentration along the vein from point A to point B as shown in (C). Brighter colours correspond
to higher Zn concentrations. Scale bars ¼ 1 mm.
Du et al. — In situ m-XRF analysis of foliar Zn absorption and short-distance movement
45
TABLE 1. Changes in projected concentrations of Zn in leaf tissues of tomato and citrus to which droplets of either Zn(NO3)2 or ZnHN
were applied to the abaxial or adaxial surface (for the control there was no foliar-applied Zn). Plants were grown in the presence (Znsufficient) or absence (Zn-deficient) of Zn in the rooting medium, and the leaves examined were either the youngest fully expanded leaf
(YFEL) or a mature leaf. Using m-XRF analysis, average concentrations of Zn were determined either in the tissues underneath where
the droplet was applied or in tissues at least 5 mm from where the droplet was applied. For the strength coefficient (c), eqn 1 was fitted
for projected concentrations along the length of a vein. Concentrations are reported on a fresh mass basis
Zn(NO3)2
Tomato
Citrus
Zn-sufficient
Zn-sufficient
Zn-deficient
Zn-sufficient
Zn-sufficient
Zn-sufficient
YFEL
YFEL
YFEL
Mature
YFEL
YFEL
Abaxial
Adaxial
Adaxial
Adaxial
Abaxial
Adaxial
ZnHN
Concentration
under droplet
(mg cm3),
mean 6 s.d.
Baseline leaf
concentration
(mg cm3),
mean 6 s.d.
c (95 % confidence
interval)
Concentration
under droplet
(mg cm3),
mean 6 s.d.
Baseline leaf
concentration
(mg cm3),
mean 6 s.d.
c (95 % confidence
interval)
560 6 150
230 6 200
310 6 150
320 6 110
60 6 87
37 6 040
088 6 022
088 6 018
<05
<05
11 6 027
057 6 014
081 (071, 091)
049 (046, 051)
54 (43, 66)
32 (28, 36)
–
–
220 6 17
87 6 41
180 6 64
220 6 24
17 6 050
21 6 087
081 6 022
096 6 007
< 05
< 05
10 6 029
090 6 039
067 (062, 071)
045 (043, 046)
16 (15, 17)
038 (032, 044)
–
–
Citrus, Zn-sufficient, Zn(NO3)2, YFEL, abaxial and adaxial
A
B
A
B
A
B
C
D
A
A
B
B
E
F
Projected Zn
concentration (µg cm−3)
25
20
15
10
5
0
0
2
4
6
Distance (mm)
8
10
0
2
4
6
8
10
Distance (mm)
FIG. 2. Zinc concentration of the youngest fully expanded leaf (YFEL) of citrus to which droplets of Zn(NO3)2 had been applied. (A) Image of half a leaf (top, abaxial; bottom, adaxial) captured using a digital camera. (B) Survey scan of the leaf in (A) using m-XRF. A detailed scan of either the (C) abaxial surface as indicated by
the white rectangle or (D) the adaxial surface as indicated by the red rectangle. The projected Zn concentration along the vein from point A to B as shown in (C) for
either (E) the abaxial surface or (F) the adaxial surface. Brighter colours correspond to higher Zn concentrations. Scale bars ¼ 1 mm.
Du et al. — In situ m-XRF analysis of foliar Zn absorption and short-distance movement
46
droplets (particularly in tomato), the extent to which this Zn
moved through the leaf surface varied. Across all treatments, it
was observed that the concentration of Zn in the underlying tissues was approximately 2-fold higher for Zn(NO3)2 than for
ZnHN (Table 1). For example, in the YFELs of Zn-sufficient
tomato, the application of droplets of Zn(NO3)2 to the abaxial
leaf surface resulted in an increase in average projected volumetric concentrations of Zn from 088 to 560 mg cm3 (Table 1,
Fig. 1B, C) but ZnHN resulted in an increase from 081 to
220 mg cm3 (Table 1, Fig. 3B, C). Similarly, for the adaxial
surface of the YFELs of tomato, the application of Zn(NO3)2
increased projected volumetric concentrations of Zn to
230 mg cm3 (Table 1, Fig. 4B, C) whilst ZnHN increased tissue concentrations to 87 mg cm3 (Table 1, Supplementary Data
Fig. S2).
The surface of the leaf to which the Zn was applied was also
important in influencing the extent to which Zn was absorbed
(particularly for tomato), with more Zn moving through abaxial
surfaces than through adaxial surfaces. For tomato, the concentration of Zn in the underlying tissues was 2-fold higher
when the Zn was applied to the abaxial surface (560 mg cm3;
Table 1, Fig. 1B, C) than to the adaxial surface (230 mg cm3;
Table 1, Fig. 4B, C). This effect appeared to be independent of
whether the Zn was supplied as Zn(NO3)2 or ZnHN, with the
tissues containing 220 mg cm3 when supplied with ZnHN to
the abaxial surface (Table 1, Fig. 3B, C) but only 87 mg cm3
when supplied to the adaxial surface (Table 1; Supplementary
Data Figs S2 and S3). The relative importance of abaxial surfaces versus adaxial surfaces in citrus was less clear due to the
much lower absorption of Zn in these leaves (Table 1).
Finally, neither the Zn status of the plant nor the age of the
leaf appeared to influence the magnitude of the movement of
Zn through the leaf surface. For example, when Zn(NO3)2 was
supplied to the adaxial surfaces of tomato leaves, the projected
concentration of Zn in the underlying tissues was similar in the
Zn-sufficient YFEL (230 mg cm3; Table 1, Fig. 4B, C) and in
the Zn-sufficient mature leaf (320 mg cm3; Table 1, Fig. 5B, C)
and the Zn-deficient YFEL (310 mg cm3; Table 1, Fig. 6B, C).
Redistribution of Zn within the leaf tissues
Although the foliar application of Zn often increased projected concentrations in the underlying tissues in tomato
Tomato, Zn-sufficient, ZnHN, YFEL, abaxial
A
C
B
A
B
B
A
D
Projected Zn
concentration (µg cm−3)
60
R 2 = 0·957
40
20
0
0
2
4
6
8
10
Distance (mm)
FIG. 3. Zinc concentration of the youngest fully expanded leaf (YFEL) of tomato (abaxial surface from the Zn-sufficient treatment) to which droplets of ZnHN had
been applied. (A) Image of half a leaf captured using a digital camera. (B) Survey (overview) scan of the leaf in (A) using m-XRF. (C) Detailed scan of the area indicated by the white rectangle in (B). (D) Projected Zn concentration along the vein from point A to point B as shown in (C). Brighter colours correspond to higher Zn
concentrations. Scale bars ¼ 1 mm.
Du et al. — In situ m-XRF analysis of foliar Zn absorption and short-distance movement
47
Tomato, Zn-sufficient, Zn(NO3)2, YFEL, adaxial
A
C
A
A
B
B
B
D
60
Projected Zn
concentration (µg cm−3)
R 2 = 0·890
40
20
0
0
2
4
6
8
10
Distance (mm)
FIG. 4. Zinc concentration of the youngest fully expanded leaf (YFEL) of tomato (adaxial surface from the Zn-sufficient treatment) to which droplets of Zn(NO3)2
had been applied. (A) Image of half a leaf captured using a digital camera. (B) Survey (overview) scan of the leaf in (A) using m-XRF. (C) Detailed scan of the area
indicated by the white rectangle in (B). (D) Projected Zn concentration along the vein from A to point B as shown in (C). Brighter colours correspond to higher Zn
concentrations. Scale bars ¼ 1 mm.
>100-fold (and 2- to 5-fold in citrus) (Table 1), the subsequent redistribution of this Zn within the leaf tissues appeared
to be limited.
Firstly, we considered how the concentration of Zn within
the interveinal tissues changed as the distance from the droplet
increased. This is important given that it is this Zn in the interveinal tissues that could potentially be loaded into the vascular
tissues for further transport. Therefore, whilst avoiding higherorder veins (2 or 3 ), projected volumetric concentrations
were examined across a transect that encompassed the tissues
to which the Zn-containing droplet had been applied (for the
purposes of this analysis, these tissues are termed ‘interveinal’
although they still include lower-order (4–7 ) veins (Fig. 7).
For example, consider the YFEL of tomato supplied with
Zn(NO3)2 on the adaxial surface (Fig. 7, and also see Fig. 4B,
C). As expected, projected volumetric concentrations underneath the droplet were typically 100 mg cm3 (Fig. 7B, C).
However, it was noted that the tissue concentration of Zn decreased rapidly with increasing distance from the site of application. Indeed, assuming that this Zn droplet was 2 mm in
diameter (data not shown, but see Supplementary Data Figs S1
and S4 for an example), the concentration of Zn in the
interveinal tissues decreased to background levels within
15–3 mm of the edge of the droplet (Fig. 7, Supplementary
Data Fig. S4). A similar trend was observed for YFELs of tomato supplied with ZnHN (Supplementary Data Fig. S5).
Although the movement of Zn within the interveinal tissues
was highly limited, it was also influenced by the treatment.
Specifically, in leaves of Zn-deficient plants, the movement of
Zn was restricted to an even greater extent (Fig. 8). In these
Zn-deficient leaves, projected volumetric concentrations of Zn
returned to background levels within 05–1 mm of the edge of
the droplet [for Zn(NO3)2 see Fig. 8; for ZnHN see
Supplementary Data Fig. S6].
Secondly, it was possible to examine the concentration of Zn
within the vascular tissues immediately below the Zn-containing droplet. This analysis is important because it is the movement of Zn into these vascular tissues that allows subsequent
loading into the higher-order veins for the wider movement of
Zn. In this regard, it was noted that these lower-order (4–7 )
veins typically contained a projected volumetric Zn concentration that was 2–10-fold higher than that of the surrounding
tissues. For example, when Zn(NO3)2 was applied to the adaxial surface of YFELs of tomato, projected volumetric
Du et al. — In situ m-XRF analysis of foliar Zn absorption and short-distance movement
48
Tomato, Zn-sufficient, Zn(NO3)2, mature leaf, adaxial
A
C
B
A
B
B
A
D
R 2 = 0·857
Projected Zn
concentration (µg cm−3)
180
150
120
90
60
30
0
0
2
4
6
8
10
Distance (mm)
FIG. 5. Zinc concentration of a mature leaf of tomato (adaxial surface from the Zn-sufficient treatment) to which droplets of Zn(NO3)2 had been applied. (A) Image
of half a leaf captured using a digital camera. (B) Survey (overview) scan of the leaf in (A) using m-XRF. (C) Detailed scan of the area indicated by the white rectangle in (B). (D) Projected Zn concentration along the vein from point A to point B as shown in (C). Brighter colours correspond to higher Zn concentrations. Scale
bars ¼ 1 mm.
concentrations in the lower-order veins underneath the droplet
were 2-fold higher than in the tissues immediately adjacent to
them (Figs 7 and 8). Similarly, when Zn(NO3)2 (or ZnHN) was
applied to the abaxial surface of Zn-sufficient YFELs of tomato, projected volumetric concentrations in the lower-order
veins underneath the droplet (1000–2000 mg cm3) were
10-fold higher than those in the tissues immediately adjacent
to them (100–200 mg cm3; Supplementary Data Figs S4, S5
and S6).
Thirdly, the movement of Zn away from the droplet within
higher-order (2 and 3 ) veins was examined. Although Zn
moved a greater distance in the vascular tissues than in the
interveinal tissues (e.g. Fig. 1), it could be seen that the concentration of Zn in the vascular tissues also decreased comparatively rapidly (in contrast, examine the movement of Zn within
the vascular tissue of a root, shown in Supplementary Data Fig.
S7). To further examine the movement of Zn within vascular
tissues, regressions were fitted using eqn 1, where the strength
coefficient (c) indicates how quickly the concentration of Zn in
the vein decreases (higher values of c indicating a more rapid
decrease in Zn). It was noted in all instances that the concentration of Zn within the vascular tissues decreased substantially
within <5–10 mm to concentrations similar to background levels for the vascular tissues (Figs 1–6). Although the movement
of Zn within the vascular tissues was limited, differences were
observed between treatments. Examination of the values for
c in Table 1 showed that the Zn status of the plant was the most
important factor influencing the distance moved by Zn within
the vascular tissues. For example, for YFELs of tomato that
had had Zn(NO3)2 applied to the adaxial surface, the concentration of Zn within the vascular tissues decreased by 50 % after
14 mm for the Zn-sufficient plant (c ¼ 049) but by 50 % after 01 mm for the Zn-deficient plant (c ¼ 54) (Table 1, Figs 4
and 6).
Finally, we also examined the distribution of Zn within
YFELs that had been sprayed with Zn(NO3)2 or ZnHN across
the entire abaxial or adaxial surface (Supplementary Data Fig.
S8). As expected, the movement of Zn into the leaf tissues was
greater than when only single droplets were applied, with uptake of Zn greater for tomato than for citrus (Supplementary
Data Fig. S8). Again, it was noted that the concentration of Zn
was higher in the vascular tissues than in the interveinal tissues.
DISCUSSION
To the best of our knowledge, this is the first study to utilize mXRF for the in situ examination of Zn in hydrated and fresh
leaves to which Zn in soluble and suspension forms has been applied. Foliar uptake of Zn uptake is considered to be a twophase process: (1) passive diffusion through the leaf surface
(penetration across the cuticle and cell walls of the epidermis);
and (2) uptake of solutes in the leaf cells and/or phloem for
Du et al. — In situ m-XRF analysis of foliar Zn absorption and short-distance movement
49
Tomato, Zn-deficient, Zn(NO3)2, YFEL, adaxial
A
C
B
B
A
A
B
D
Projected Zn
concentration (µg cm−3)
30
R 2 = 0·911
20
10
0
0
2
4
6
8
10
Distance (mm)
FIG. 6. Zinc concentration of the youngest fully expanded leaf (YFEL) of tomato (adaxial surface from the Zn-deficient treatment) to which droplets of Zn(NO3)2
had been applied. (A) Image of half a leaf captured using a digital camera. (B) Survey (overview) scan of leaf in (A) using m-XRF. (C) Detailed scan of the area indicated by the white rectangle in (B). (D) Projected Zn concentration along the vein from point A to point B as shown in (C). Brighter colours correspond to higher Zn
concentrations. At a distance >2 mm, concentrations were below the detection limit. Scale bars ¼ 1 mm.
transport (Fernández and Eichert, 2009). In the present study,
the use of synchrotron-based m-XRF has allowed an assessment
of both the movement of Zn through the leaf surface into the underlying tissues and its subsequent redistribution within the leaf.
The overall pattern of the behaviour of foliar-applied Zn was
similar regardless of the treatment imposed, although the different treatments did influence the extent to which these various
processes occurred. Following its foliar application, Zn moved
through the leaf surface, with the concentrations in the underlying tissues increasing by up to 5-fold in citrus or 600-fold in tomato (Table 1). Next, once the Zn had moved through the leaf
surface, we then examined three processes that are of importance for the subsequent redistribution of Zn within the leaf: (1)
the redistribution of Zn within the interveinal tissues themselves; (2) the movement of Zn from the interveinal tissues into
the adjacent lower-order veins; and (3) the movement of Zn
into the higher-order veins for its subsequent redistribution
through the leaf. Firstly, the redistribution of Zn (away from the
location of the foliar-applied Zn) through the interveinal tissues
was found to be highly restricted, with projected volumetric
concentrations of Zn decreasing to background levels within
3 mm of the edge of the Zn-containing droplet (Figs 7 and 8).
Secondly, although this redistribution of Zn through the
interveinal tissues was highly restricted, Zn was moved into
the lower-order veins underlying the foliar-applied Zn:
concentrations of Zn in these lower-order veins were up to 2to 10-fold higher than in the surrounding interveinal tissues
(Figs 7 and 8). Finally, as expected, after the Zn had been
moved into the higher-order veins, the distance moved by the
Zn was greater than that observed in the surrounding interveinal
tissues (e.g. see Fig. 1). However, even within higher-order
veins, the movement of Zn was comparatively restricted, with
concentrations in these veins decreasing to concentrations similar to the background within 1–10 mm (Figs 1–6). Interestingly,
Zn concentration peaks coincided with portions of the veins
that intersect with other veins, which may be entry points for increased Zn loading into the vein. Further tracing examination is
required to confirm this assumption.
Redistribution of Zn within tomato and citrus leaves is highly
limited
As noted above, following its movement through the leaf surface, the movement of the foliar-applied Zn was highly restricted. This pattern of Zn movement in leaves is in contrast to
that observed for Zn in roots (Supplementary Data Fig. S7) or
in leaves for some other foliar-applied nutrients. For example,
using autoradiography to study 32P applied to leaves of bean
(Phaseolus vulgaris), Koontz and Biddulph (1957) found
50
Du et al. — In situ m-XRF analysis of foliar Zn absorption and short-distance movement
Tomato, Zn-sufficient, Zn(NO3)2, YFEL, adaxial
A
1 mm
B
1 mm
Projected Zn
concentration (µg cm−3)
1000
C
100
10
1
0
1
2
3
4
Distance (mm)
5
6
7
FIG. 7. Zinc concentration across an area of a tomato leaf (adaxial surface from the Zn-sufficient treatment) to which droplets of Zn(NO3)2 had been applied (see Fig.
4 also). (A) Detailed scan. (B) Close-up of the area indicated by the red rectangle in (A). (C) Concentration of Zn across the region indicated by the white dashed
rectangle in (B). In (C) the horizontal dashed line represents the background concentration of Zn (Table 1). Brighter colours correspond to higher Zn concentrations.
substantial translocation of P within 24 h (e.g. see Fig. 11 of
Koontz and Biddulph, 1957). However, the limited movement
of foliar-applied Zn observed in the present study is similar to
that observed previously for Zn, with only 5–20 % of the Zn
taken up following foliar fertilization typically remobilized
within the phloem (Fernández et al., 2013). For example,
Zhang and Brown (1999b) found that only 54 % of the Zn absorbed by the leaf was transported out of the treated leaves in
pistachio (Pistachio vera). Similarly, Swietlik and LaDuke
(1991) found no evidence of Zn movement from leaves of
Citrus aurantium. A total of 120 d after foliar application to citrus, Sartori et al. (2008) reported that only 14 % of the absorbed
Zn had been translocated from the applied leaves to other plant
tissues. In a manner similar to the present study, other authors
have used autoradiography to examine the spatial distribution
of foliar-applied Zn. For example, Marešová et al. (2012) studied Zn in leaves of tobacco (Nicotiana tabacum) and hop
(Humulus lupulus) and found that >99 % of the foliar-applied
Zn was retained within tissues to which it had been directly applied (by immersion in a ZnCl2 solution) and that <1 % was
transported to non-immersed tissues. Similarly, when Wallihan
and Heymann-Herschberg (1956) applied 65Zn as single drops
to surfaces of citrus leaves, their autoradiograms indicate that
there was comparatively little movement of the Zn, but the
greatest movement occurred when the Zn-containing droplet
was applied to the midrib. It is recognized that there would be
high proportions of Zn droplets directly located on veins of
leaves fully sprayed in the field. As a result, it may be hypothesized that the transport effectiveness of foliar absorbed Zn out
of a treated leaf may be rate-limited by the Zn concentration
gradient in the midrib, which is the cumulative product of the
spatial distribution of the Zn uptake intensity and internal diffusivity of the Zn specific to leaf physiology/biochemistry and Zn
chemical forms. This should be tested in further studies.
This very limited movement of foliar-applied Zn (observed
in the present study but also previously by other authors) occurs for two reasons: (1) high binding capacity of leaf cells for
Zn (Zhang and Brown, 1999a, b) and/or (2) limited/conditional
mobility in the phloem (Marschner, 1995). Indeed, it is known
that the negative charges of the cell wall and cuticle have a
high affinity for Zn2þ and that, following its foliar application,
much of the absorbed Zn is in exchangeable forms rather than
soluble forms (Zhang and Brown, 1999a; Fernández et al.,
2013). This is in contrast to Zn taken up by roots, which is
largely complexed with ligands, including organic acids or
amino acids (Cakmak, 2000; Haydon and Cobbett, 2007;
Du et al. — In situ m-XRF analysis of foliar Zn absorption and short-distance movement
51
Tomato, Zn-deficient, Zn(NO3)2, YFEL, adaxial
A
Projected Zn
concentration (µg cm−3)
1 mm
10 000
B
1000
100
10
1
0·1
0
1
2
3
5
4
Distance (mm)
6
7
8
9
FIG. 8. Zinc concentration across a traverse of a tomato leaf (adaxial surface from the Zn-deficient treatment) to which droplets of Zn(NO3)2 had been applied (see
also Fig. 6). (A) Detailed scan. (B) Concentration of Zn across the region indicated by the white dashed rectangle in (A). In (B) the horizontal dashed line represents
the background concentration of Zn (Table 1). Brighter colours correspond to higher Zn concentrations.
Kopittke et al., 2011). While this strong binding of foliar-absorbed Zn limits its subsequent movement, this observation
does not necessarily imply that foliar application of Zn cannot
be effective in improving growth. Indeed, even though the redistribution of Zn observed in the present study was highly restricted, our experimental data for tomato indicate that it is still
sufficeint to increase both shoot and root mass substantially in
Zn-deficient plants when the whole leaf surfaces of two or
three mature leaves were fully covered with foliar spray of
ZnHN droplets in a follow-up glasshouse experiment (Y. Du,
unpubl. data). Similarly, for wheat (Triticum aestivum) it was
reported that whilst the translocation of foliar-applied Zn was
small it was sufficient to improve growth (Haslett et al., 2001).
Regardless, whilst translocation of Zn would indeed otherwise
increase the potential for a whole-plant benefit, the application
of foliar Zn would still have local benefits (e.g. see Zn concentrations when applied as a spray, shown in Supplementary
Data Fig. S8). These may suggest that repeated application of
soluble Zn fertilizers is necessary to achieve meaningful
growth and agronomic effects in the field, through increasing
spatial coverage of canopy or foliar surface area and/or temporal re-application across extended developmental stages, such
as the flowering stage (Fernández et al., 2013).
Factors that influence the absorption of Zn and its redistribution
The plant species had a marked effect on the absorption of
foliar-applied Zn, with the concentrations in the underlying tissues increased by an average of up to only 5-fold in citrus but
up to 600-fold in tomato (Table 1). Indeed, in contrast to that
observed with tomato (Table 1, Fig. 1), there were generally no
significant effects on the movement of Zn in the higher-order
veins of citrus leaves, regardless of the treated leaf surface or
the form of the Zn that was applied (Table 1, Fig. 2). Reduced
absorption of Zn into leaves of citrus results from the thickness
of the cuticular wax layer on citrus leaves, this being an important barrier (Riederer and Schreiber, 1995).
The surface to which the Zn was applied (abaxial or adaxial)
was also one of the important factors influencing the absorption
of Zn, with the concentration of Zn in the underlying tissues
2-fold higher when applied to the abaxial leaf surface regardless of the Zn chemical form applied or the species (Table 1,
Figs 1 and 4). In tomato, although stomata and trichomes are
present on both the adaxial and abaxial surfaces, their density is
substantially higher on the abaxial than the adaxial surface
(Supplementary Data Table S3). This is particularly important
given that the aqueous pathways (or polar pathways) for the
penetration of inorganic ions (such as Zn2þ) through the cuticle
are located in the cuticular ledges at the base of trichomes, guard
cells and cuticles over anticlinal cell walls of stomata
(Schönherr, 2006). Similar observations have been reported by
other authors, with the absorption of Ca and Na on the surface
of stomatous leaf surfaces significantly higher than on astomatous leaf surfaces of apple (Malus domestica) (Schlegel and
Schonherr, 2002; Burkhardt et al., 2012). For leaves of citrus,
stomata are only present on the abaxial surface. However, in the
present study the relative importance of application to adaxial
surfaces versus abaxial surfaces in citrus was unclear due to the
52
Du et al. — In situ m-XRF analysis of foliar Zn absorption and short-distance movement
comparatively low movement of Zn through the surfaces of
these leaves (Table 1).
As reported previously, plant Zn status was found to influence the movement of Zn within the leaf tissue, although it did
not influence the initial movement of Zn through the leaf surface (Table 1). For example, in Zn-deficient plants the redistribution of Zn was restricted both in the interveinal tissues
(Figs 7 and 8) but also in the higher-order veins (Figs 4 and 6).
Firstly, the observation that Zn deficiency did not influence the
initial absorption of Zn is in agreement with the findings of
Erenoglu et al. (2002), who stated that the foliar absorption of
65
Zn was not influenced by Zn nutritional status. The finding
that the Zn status of the plant did impact upon the subsequent
redistribution of the foliar-absorbed Zn is also in agreement
with the previous observations that Zn is more easily mobilized
in Zn-sufficient leaves than in Zn-deficient leaves (Longnecker
and Robson, 1993). This limitation of foliar Zn movement in
Zn-deficient leaves may be caused by the effects of Zn deficiency on cellular membrane structure and functions, such as
the density and functionality of ion pumps. Zinc deficiency disrupts the integrity of leaf cell membranes and their permeability
to inorganic ions (Cakmak, 2000). The impairments in the integrity of leaf cell membranes of Zn-deficient plants may also
cause alterations in the activity of membrane-bound protonpumping enzymes and ion channels (Cakmak, 2000), thus
affecting nutrients across the plasma membranes. Zinc transporters such as ZIP4, cation diffusion facilitator (MTP1), heavy
metal ATPase (HMA2, HMA4) and yellow stripe-like (YSL1,
YSL3) proteins, which are primarily located in leaf cell membranes (Grotz and Guerinot, 2006; Waters et al., 2006), may be
affected structurally and functionally in Zn-deficient leaves,
thus limiting Zn loading into the phloem. In addition, cuticles
and cell walls of leaves are strong sinks for Zn binding, resulting in low Zn mobilization out of the treated leaves, particularly
under Zn deficiency conditions (Kannan and Charnel, 1986;
Marschner, 1995; Zhang and Brown, 1999b).
The chemical form of the Zn was found to influence its absorption through the leaf surface, with tissue concentrations
2-fold higher when Zn(NO3)2 was used rather than ZnHN
(Table 1). This is not unexpected given that the solubility of
ZnHN in saturation phase is 30–50 mg Zn L1 (Li et al.,
2012), whilst Zn(NO3)2 was applied at 400 mgL1. Given that
foliar Zn absorption is a passive diffusion process driven by a
concentration gradient (Fernández and Eichert, 2009), it is not
surprising that Zn concentrations in leaves treated with
Zn(NO3)2 were higher than in leaves to which the ZnHN suspension was applied. Importantly, the permeability of polar
pathways in the cuticle is closely related to ambient humidity
(Schönherr, 2001; Schönherr and Luber, 2001; Schreiber et al.,
2001) and the dissolution of ZnHN would have only occurred
at a relative humidity above its point of deliquescence (87 %).
Given that the relative humidity during incubation was maintained >95 % in the present study, cuticle permeability would
not have become a limiting factor for the penetration of Zn if it
was available in aqueous phase on the leaves, and the dissolution of ZnHN would have been at its maximal rate.
In conclusion, in the present study we have provided quantitative data on the spatial distribution (absorption and shortdistance movement) of Zn in hydrated and fresh leaves of
tomato. Due to physiological differences in the leaves
(particularly the cuticles), the absorption of the foliar-applied
Zn differed substantially between tomato and citrus, with Zn
concentrations in tissues underlying the Zn-containing droplet
up to 600-fold higher than in the surrounding tissues for tomato but only 5-fold in citrus. Regardless of the treatment,
once the Zn had been absorbed, there was comparatively little
subsequent movement of the Zn within the leaf tissue. Indeed,
within the interveinal tissues, Zn concentrations decreased to
background levels within <3 mm from the Zn source. Despite
the loading of Zn into the lower-order veins in the tissues underlying the Zn-containing droplet (with Zn concentrations in
these lower-order veins being up to 10-fold higher than in the
surrounding interveinal tissues), the movement of Zn within
the higher-order veins was also limited (reducing to concentrations similar to the background within <5–10 mm). Movement
of foliar-applied Zn within the leaf tissues was reduced even
further in Zn-deficient leaves. The information provided here
advances our understanding of the factors that influence the efficacy of foliar Zn fertilizers and their interactions with plant
foliage characteristics.
SUPPLEMENTARY DATA
Supplementary data are available online at www.aob.oxfordjournals.org and consist of the following. Table S1: treatments
used to investigate the foliar absorption and short-distance
movement of Zn. Table S2: bulk concentration of Zn in leaves
of tomato and citrus. Table S3: Densities of stomata and trichomes on leaves of tomato and citrus. Fig. S1: abaxial surface
of a youngest fully expanded leaf of Zn-sufficient tomato to
which Zn(NO3)2 was applied. Fig. S2: adaxial surface of a
youngest fully expanded leaf of Zn-sufficient tomato to which
ZnHN was applied. Fig. S3: adaxial surface of a youngest fully
expanded leaf of Zn-deficient tomato to which ZnHN was applied. Fig. S4: transect across the abaxial surface of a youngest
fully expanded leaf of Zn-sufficient tomato to which Zn(NO3)2
was applied. Fig. S5: transect across the abaxial surface of a
youngest fully expanded leaf of Zn-sufficient tomato to which
ZnHN was applied. Fig. S6: Transect across the adaxial surface
of a youngest fully expanded leaf of Zn-deficient tomato to
which ZnHN was applied. Fig. S7: concentrations of Zn in the
root of cowpea (Vigna unguiculata) exposed to 40 mM Zn in solution culture. Fig. S8: abaxial and adaxial surfaces of tomato
and citrus leaves sprayed with Zn(NO3)2 and ZnHN.
ACKNOWLEDGEMENTS
We thank the Australian Synchrotron for technique support
of XFM beamline (AS131/XFMFI/5835) and Dr Lu Zhao
for help with ICP analysis of Zn in tomato leaves. This work
was supported by Australian Research Council (ARC) and
AgriChem Liquid Fertilizers Pty Ltd (LP0989217) and the
ARC Future Fellowship scheme (FT120100277 and
LP130100741).
LITERATURE CITED
Burkhardt J, Basi S, Pariyar S, Hunsche M. 2012. Stomatal penetration by
aqueous solutions - an update involving leaf surface particles. New
Phytologist 196: 774–787.
Du et al. — In situ m-XRF analysis of foliar Zn absorption and short-distance movement
Cakmak I. 2000. Tansley Review No. 111. Possible roles of zinc in protecting
plant cells from damage by reactive oxygen species. New Phytologist 146:
185–205.
Cakmak I. 2008. Enrichment of cereal grains with zinc: agronomic or genetic
biofortification? Plant and Soil 302: 1–17.
Eichert T, Goldbach GD. 2008. Equivalent pore radii of hydrophilic foliar uptake routes in stomatous and astomatous leaf surfaces – further evidence for
a stomatal pathway. Physiologia Plantarum 132: 491–502.
Eichert T, Kurtz A, Steiner U, Goldbach HE. 2008. Size exclusion limits and
lateral heterogeneity of the stomatal foliar uptake pathway for aqueous solutes
and water-suspended nanoparticles. Physiologia Plantarum 134: 151–160.
Erenoglu B, Nikolic M, Romheld V, Cakmak I. 2002. Uptake and transport of
foliar applied zinc (65Zn) in bread and durum wheat cultivars differing in
zinc efficiency. Plant and Soil 241: 25–57.
Fernández V, Brown PH. 2013. From plant surface to plant metabolism: the uncertain fate of foliar-applied nutrients. Frontiers in Plant Science 4: 1–5.
Fernández V, Eichert T. 2009. Uptake of hydrophilic solutes through plant
leaves: current state of knowledge and perspectives of foliar fertilization.
Critical Reviews in Plant Sciences 28: 36–38.
Fernández V, Eichert T, Del Rio V, et al. 2008. Leaf structural changes associated with iron deficiency chlorosis in field-grown pear and peach: physiological implications. Plant and Soil 311: 161–172.
Fernández V, Sotiropoulos T, Brown P. 2013. Foliar fertilization: scientific
principles and field practices. Paris: International Fertilizer Association.
Grotz N, Guerinot ML. 2006. Molecular aspects of Cu, Fe and Zn homeostasis
in plants. Biochimica et Biophysica Acta 1763: 595–608.
Haslett BS, Reid RJ, Rengel Z. 2001. Zinc mobility in wheat: uptake and distribution of zinc applied to leaves or roots. Annals of Botany 87: 379–386.
Hauke V, Schreiber L. 1998. Ontogenetic and seasonal development of wax
composition and cuticular transpiration of ivy (Hedera helix L.) sun and
shade leaves. Planta 207: 67–75.
Haydon MJ, Cobbett CS. 2007. Transporters of ligands for essential metal ions
in plants. New Phytologist 174: 499–506.
Hewitt EJ. 1966. Sand and water culture methods used in the study of plant nutrition. Farnham Royal, UK: Commonwealth Agricultural Bureaux.
Huang L, Bell RW, Dell B, Woodward J. 2004. Rapid nitric acid digestion of
plant material with an open-vessel microwave system. Communications in
Soil Science and Plant Analysis 35: 427–440.
Huang LB, Bell RW, Dell B. 2008. Evidence of phloem boron transport in response to interrupted boron supply in white lupin (Lupinus albus L. cv. Kiev
Mutant) at the reproductive stage. Journal of Experimental Botany 59:
575–583.
Huett DO, Maier NA, Sparrow LA, Piggott TJ 1997. Vegetables. In:
DJ Reuter, JBD Robinson. eds. Plant analysis: an interpretation manual. Collingwood, Australia: CSIRO, 385–464.
IZiNCG 2004. Assessment of the risk of zinc deficiency in populations and options for its control. Food Nutrition Bulletin 25 (1 Suppl 2): S99–S203.
Kannan S, Charnel A. 1986. Foliar absorption and transport of inorganic nutrients. Critical Reviews in Plant Sciences 4: 341–375.
Kinraide TB. 1999. Interactions among Ca2þ, Naþ and Kþ in salinity toxicity:
quantitative resolution of multiple toxic and ameliorative effects. Journal of
Experimental Botany 50: 1495–1505.
Kirkham R, Dunn PA, Kuczewski AJ, et al. 2010. The Maia spectroscopy detector system: engineering for integrated pulse capture, low-latency scanning and real-time processing. AIP Conference Proceedings 1234: 240–243.
Koontz H, Biddulph O. 1957. Factors affecting absorption and translocation of
foliar applied phosphorus. Plant Physiology 32: 463–470.
Kopittke PM, Menzies NW, de Jonge MD, et al. 2011. in situ distribution and
speciation of toxic Cu, Ni and Zn in hydrated roots of cowpea. Plant
Physiology 156: 663–673.
Kopittke PM, de Jonge MD, Menzies NW, et al. 2012. Examination of the distribution of arsenic in hydrated and fresh cowpea roots using two- and threedimensional techniques. Plant Physiology 159: 1149–1158.
Kopittke PM, de Jonge MD, Wang P, et al. 2014. Laterally resolved speciation
of arsenic in roots of wheat and rice using fluorescence-XANES imaging.
New Phytologist 201: 1251–1262.
Li P, Xu ZP, Hampton MA, et al. 2012. Control preparation of zinc hydroxide
nitrate nanocrystals and examination of the chemical and structural stability.
Journal of Physical Chemistry C 116: 10325–10332.
Lombi E, de Jonge MD, Donner E, et al. 2011. Fast x-ray fluorescence microtomography of hydrated biological samples. PLoS One 6: e20626.
53
Loneragan JF, Snowgall K, Robson AD. 1976. Remobilization of nutrients and
its significance in plant nutrition. In: IF Wardlaw, JB Passioura. eds.
Transport and transfer process in plants. New York: Academic Press,
463–469.
Longnecker NE, Robson AD. 1993. Distribution and transport of zinc in plants.
In: AD Robson. ed. Zinc in soils and plants: Dordrecht, The Netherlands:
Springer, 79–91.
Marešová J, Remenárová L, Hornı́k M, Pipı́ška M, Augustı́n J, Lesný J.
2012. Foliar uptake of zinc by vascular plants: radiometric study. Journal of
Radioanalytical and Nuclear Chemistry 292: 1329–1337.
Marschner H. 1995. Mineral nutrition of higher plants. London: Academic
Press.
Paterson DJ, de Jonge MD, Howard DL, et al. 2011. The X-ray fluorescence
microscopy beamline at the Australian Synchrotron. AIP Conference
Proceedings 1365: 219–222.
Riederer M, Schreiber L 1995. Waxes: the transport barriers of plant cuticles.
In: RJ Hamilton. ed. Waxes: chemistry, molecular biology and functions.
Dundee, UK: Oily Press, 130–156.
Ryan CG. 2000. Quantitative trace element imaging using PIXE and the nuclear
microprobe. International Journal of Imaging Systems and Technology 11:
219–230.
Ryan CG, Jamieson DN. 1993. Dynamic analysis: on-line quantitative PIXE
microanalysis and its use in overlap-resolved elemental mapping. Nuclear
Instruments and Methods in Physics Research Section B: Beam Interactions
with Materials and Atoms 77: 203–214.
Sack L, Scoffoni C, McKown AD, et al. 2012. Developmentally based scaling
of leaf venation architecture explains global ecological patterns. Nature
Communications 3: 837.
Sartori RH, Boaretto AE, Villanueva FCA, Fernandes HMG. 2008. Foliar
and radicular absorption of 65Zn and its redistribution in citrus plant. Revista
Brasileira De Fruticultura 30: 523–527.
Schilmiller AL, Last RL, Pichersky E. 2008. Harnessing plant trichome biochemistry for the production of useful compounds. Plant Journal 54:
702–711.
Schlegel TK, Schonherr J. 2002. Selective permeability of cuticles over
stomata and trichomes to calcium chloride. Acta Horticulturae (ISHS)
594: 91–96.
Schönherr J. 2001. Cuticular penetration of calcium salts: effects of humidity,
anions, and adjuvants. Journal of Plant Nutrition and Soil Science 164:
225–231.
Schönherr J. 2006. Characterization of aqueous pores in plant cuticles and permeation of ionic solutes. Journal of Experimental Botany 57: 2471–2491.
Schönherr J, Luber M. 2001. Cuticular penetration of potassium salts: effects
of humidity, anions, and temperature. Plant and Soil 236: 117–122.
Schreiber L, Schönherr J. 1993. Determination of foliar uptake of chemicals:
influence of leaf surface microflora. Plant, Cell and Environment 16:
743–748.
Schreiber L, Skrabs M, Hartmann KD, Diamantopoulos P, Simanova E,
Santrucek J. 2001. Effect of humidity on cuticular water permeability of
isolated cuticular membranes and leaf disks. Planta 214: 274–282.
Shi GR, Cai QS. 2009. Photosynthetic and anatomic responses of peanut leaves
to zinc stress. Biologia Plantarum 53: 391–394.
Swietlik D, LaDuke JV. 1991. Productivity, growth, and leaf mineral composition of orange and grapefruit trees foliar-sprayed with zinc and manganese.
Journal of Plant Nutrition 14: 129–142.
Vu DT, Huang LB, Nguyen AV, et al. 2013. Quantitative methods for estimating foliar uptake of zinc from suspension-based Zn chemicals. Journal of
Plant Nutrition and Soil Science 176: 764–775.
Wallihan EF, Heymann-Herschberg L. 1956. Some factors affecting absorption and translocation of zinc in citrus plants. Plant Physiology 31:
294–299.
Waters BM, Chu HH, DiDonato RJ, et al. 2006. Mutations in Arabidopsis
Yellow Stripe-Like1 and Yellow Stripe-Like3 reveal their roles in metal ion
homeostasis and loading of metal ions in seeds. Plant Physiology 141:
1446–1458.
Zhang QL, Brown PH. 1999a. The mechanism of foliar zinc absorption in pistachio and walnut. Journal of the American Society for Horticultural Science
124: 312–317.
Zhang QL, Brown PH. 1999b. Distribution and transport of foliar applied zinc
in pistachio. Journal of the American Society for Horticultural Science 124:
433–436.