The intensity of manganese deficiency strongly affects ... · 25 DFF-7017-00082 to SH and DAP), and the ERA-NET Coordinating Action in Plant Sciences 26 (ERA-CAPS) project ERACAPS13.089_RootBarriers,

1

Short title: Manganese affects root endodermal suberization 1

Corresponding author: [email protected] 2

3

Title: The intensity of manganese deficiency strongly affects root endodermal 4

suberization and ion homeostasis 5

6

Author names and affiliations: 7

Anle Chen1, Søren Husted1, David E. Salt2, Jan K. Schjoerring1 and Daniel Pergament 8

Persson1* 9

1Department of Plant and Environmental Sciences & Copenhagen Plant Science Center, Faculty of 10

Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark 11

(DPP, AC, JKS and SH) and 2School of Biosciences, University of Nottingham, Nottingham LE12 12

5RD, United Kingdom (DES) 13

14

*Address corresponding author: [email protected] 15

16

One-sentence summary 17

Suberization of the root endodermis is altered in opposite ways under mild and strong manganese 18

deficiency, which profoundly affects radial ion transport and translocation to the shoot. 19

20

Funding 21

This work was carried out within the project ‘Impacts of Physical Root Barriers and Passage Cells 22

on the Efficiency of Plant Nutrient Uptake and Translocation in Barley’, supported by grants from 23

the Independent Research Fund Denmark, Technology and Production Sciences (grant number 24

DFF-7017-00082 to SH and DAP), and the ERA-NET Coordinating Action in Plant Sciences 25

(ERA-CAPS) project ERACAPS13.089_RootBarriers, with financial support from the participants’ 26

national funding agencies: Innovation Fund Denmark 4084-00001B to JKS, Biotechnology and 27

Plant Physiology Preview. Published on August 9, 2019, as DOI:10.1104/pp.19.00507

Copyright 2019 by the American Society of Plant Biologists

www.plantphysiol.orgon March 24, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

2

Biological Sciences Research Council BB/L027739/1 to DES. Additional support was received 28

from the China Scholarship Council (grant number 201406170047). 29

30

Author contributions 31

DPP, SH, JKS and DES conceived the research plan; AC, DPP and SH designed the experiments; 32

AC and DPP performed all experiments; AC, DPP, SH, DES and JKS analyzed the data; AC, DPP 33

and SH drafted the paper, JKS and DES complemented the writing with contributions from all 34

authors. All authors have read and approved the final manuscript. 35

36

Abstract 37

Manganese (Mn) deficiency affects various processes in plant shoots. However, the functions of Mn 38

in roots and the processes involved in root adaptation to Mn deficiency are largely unresolved. 39

Here, we show that the suberization of endodermal cells in barley (Hordeum vulgare L.) roots is 40

altered in response to Mn deficiency, and that the intensity of Mn deficiency ultimately determines 41

whether suberization increases or decreases. Mild Mn deficiency increased the length of the 42

unsuberized zone close to the root tip, and increased the distance from the root tip at which the fully 43

suberized zone developed. By contrast, strong Mn deficiency increased suberization closer to the 44

root tip. Upon Mn resupply, suberization was identical to that seen on Mn-replete plants. 45

Bioimaging and xylem sap analyses suggest that the reduced suberization in mildly Mn-deficient 46

plants promotes radial Mn transport across the endodermis at a greater distance from the root tip. 47

Less suberin also favors the inwards radial transport of calcium (Ca) and sodium (Na), but 48

negatively affects the potassium (K) concentration in the stele. During strong Mn deficiency, Mn 49

uptake was directed towards the root tip. Enhanced suberization provides a mechanism to prevent 50

absorbed Mn from leaking out of the stele. With more suberin, the inward radial transport of Ca and 51

Na decreases, whereas that of K increases. We conclude that changes in suberization in response to 52

the intensity of Mn deficiency have a strong effect on root ion homeostasis and ion translocation. 53

54

Introduction 55



3

Solutes moving from the soil solution to the vascular tissues in plant roots must cross the concentric 56

root cell layers of the epidermis, cortex and endodermis on their way to the vascular bundles inside 57

the stele. This involves a combination of apoplastic, symplastic and transcellular pathways 58

(Geldner, 2013; Barberon and Geldner, 2014). With the apoplastic pathway being blocked by 59

hydrophobic barriers in the endodermis, solutes are forced to cross membranes to enter the 60

symplastic pathway and reach the vascular cylinder. Thereby, plants can regulate solute uptake via 61

transporter proteins in the plasma membrane of the endodermal and neighboring cortex cells 62

(Geldner, 2013; Andersen et al., 2015). 63

Two physical barriers are present in the endodermis, viz. the Casparian strip (CS) and suberin 64

lamellae (Geldner, 2013). In addition to the endodermal barriers, many angiosperms also have an 65

exodermis with a Casparian strip as well as suberin lamellae (Enstone et al., 2003). In the model 66

plant Arabidopsis (Arabidopsis thaliana), the Casparian strip is formed by deposition of lignin in 67

the radial and transverse (anticlinal) primary cell walls and in the middle lamella. The Casparian 68

strip membrane domain (CSD) mediates the formation of an intact CS by recruiting a lignin 69

polymerization machinery (Roppolo et al., 2011). Localized lignin synthesis is achieved by 70

production of reactive oxygen species (ROS), which are used for peroxidase-mediated lignin 71

polymerization (Lee et al., 2013). The dirigent-like protein Enhanced Suberin1 (ESB1) plays a 72

critical role in mediating the correct deposition of lignin (Baxter et al., 2009; Hosmani et al., 2013), 73

but its exact molecular function remains unknown. Mutants lacking ESB1 develop a patchy and 74

defective CS, leading at the endodermis to the formation of ectopic lignification in cell corners, and 75

enhanced deposition of suberin between the plasma membrane and cell wall (Hosmani et al., 2013; 76

Pfister et al., 2014). In addition, SCHENGEN3 (SGN3) and SCHENGEN1 (SGN1) are key proteins 77

required for proper CS establishment and function (Alassimone et al., 2016). Together, the Leu-rich 78

repeat receptor kinase SGN3, and the receptor-like cytoplasmic kinase SGN1 play key roles in the 79

detection of diffusible vasculature-derived peptides Casparian strip Integrity Factors 1 & 2 (CIF1 & 80

2), providing a surveillance system for CS integrity (Doblas et al., 2017; Nakayama et al., 2017). 81

Suberization of the endodermis in Arabidopsis roots starts a few cell layers further away from the 82

root tip than the CS, and the suberin is deposited between the primary cell wall and the plasma 83

membrane. As the root ages, more and more endodermal cells mature and become suberized, first 84

forming a patchy layer whereupon a fully suberized root zone is established (Naseer et al., 2012; 85

Geldner, 2013). In the fully suberized zone, all endodermal cells are suberized, except for 86



4

occasionally occurring ‘passage cells’ located close to the xylem poles (Andersen et al., 2018). The 87

establishment of passage cells is governed by repression of cytokinin signaling in the root meristem, 88

which ultimately results in non-suberized endodermal cells with a probable, but not fully described, 89

role in nutrient and water transport (Andersen et al., 2018). 90

Suberin is a polyester biopolymer with lipophilic and waxy properties. It has both a polyaliphatic 91

domain, where the monomers are typically ω-hydroxyacids and α-ω-diacids and a polyaromatic 92

domain, where the monomers are typically hydroxycinnamic acids (Zeier et al., 1999). The ω-93

hydrolases HORST and RALPH of the CYP86 subfamily of cytochrome P450, as well as the 94

glycerol-3-phosphate acyltransferase GPAT5 are the main enzymes involved in suberin 95

biosynthesis (Andersen et al., 2015). The aromatic domain is not present in the otherwise 96

structurally similar compound cutin, found on leaf surfaces (Schreiber, 2010). 97

Suberization is influenced by various environmental factors (Baxter et al., 2009; Krishnamurthy et 98

al., 2011; Andersen et al., 2015; Tylova et al., 2017), including various nutrient deficiencies 99

(Barberon et al., 2016). Mn deficiency, as well as iron (Fe) and zinc (Zn) deficiency, triggered a 100

reduction in root suberization in young Arabidopsis plants (Barberon et al., 2016). This was the case 101

both with respect to how far from the root tip the suberin appeared and the distance at which a fully 102

suberized layer could be detected by Fluorol Yellow (FY) staining. In contrast, under K and sulfur 103

(S) deficiency, suberization increased. Hence, young Arabidopsis plants can adapt to a sub-optimal 104

nutrient supply in a highly dynamic and element specific matter, either by an increasing or 105

decreasing endodermal suberization (Barberon et al., 2016). To our knowledge, it is not known 106

whether these responses also occur in plants species other than Arabidopsis. 107

The CS and the suberin lamellae are not performing the same functions in plant roots, i.e. they are 108

not different versions of root barriers performing the same task. Whereas the CS blocks the 109

apoplastic pathway (Naseer et al., 2012; Barberon et al., 2016), suberin appears to affect the 110

coupled trans-cellular pathway, which involves influx and efflux carriers to transport nutrients from 111

one cell to the other (Barberon et al., 2016). This conclusion derived from the physical location in 112

the cell wall of each barrier, as well as from the fact that both the sgn3 and esb1-1 Arabidopsis 113

mutants were unable to control the apoplastic bypass flow, despite normal or even enhanced 114

suberization (Baxter et al., 2009; Pfister et al., 2014). Suberin accumulates between the endodermal 115

cell wall, on top of the plasmalemma, which is where ion uptake occurs via transport proteins. 116

Hence, the suberin deposition may restrict the access to these transporters (Geldner, 2013; Andersen 117



5

et al., 2015). Since both the apoplastic and the coupled trans-cellular pathways are dependent on 118

this access, suberin may severely reduce the cellular uptake at the endodermis of ions following 119

these two pathways. In contrast, the plasmodesmatal connections of endodermal cells are not 120

affected by suberin deposition, which means that ions moving within the symplast can reach the 121

vascular bundles without physical restriction, even in the fully suberized root zone (Robards et al., 122

1973; Ma and Peterson, 2003). Transgenic lines of Arabidopsis expressing the suberin-degrading 123

enzyme CUTICLE DESTRUCTING FACTOR1 (CDEF1), displayed a fully functional CS, but no 124

detectable suberin (Naseer et al. 2012). These plants had lower K concentration in their leaves than 125

control plants, but higher concentrations of Na, Ca and Mn (Barberon et al. 2016, Li et al., 2017). 126

Once delivered to the apoplast of the stele, it has been hypothesized that suberin may restrict a 127

‘back-flush’ of the ions back into the endodermis and cortex tissues (Enstone et al., 2003; Doblas et 128

al., 2017). Since the endodermal plasmalemma facing the vasculature is lined with efflux pumps 129

and carriers, this seems plausible. However, this is still only a hypothesis based on correlations 130

between suberin mutants and the resulting leaf ionome (Enstone et al., 2003; Baxter et al., 2009; 131

Barberon and Geldner, 2014; Li et al., 2017). 132

In the esb1 Arabidopsis mutant, which display enhanced suberization, the shoot ionome was shown 133

to be altered in an ion specific way. Leaf concentrations of elements were not unanimously lower 134

because of this enhanced suberin barrier, as some elements were found to be positively and others 135

negatively affected by the increased suberization. The radial transport of elements that are assumed 136

to mainly follow the apoplastic or coupled transcellular pathways (i.e. Ca and Mn) were strongly 137

depressed whereas elements thought to mainly depend on the symplastic pathway (i.e. K and S) 138

were increased (Baxter et al., 2009; Li et al., 2017). Besides physical barriers, the specific pathways 139

that the various elements follow of course also depends on the polarity and regulation of the 140

involved influx and efflux transporters (Che at al., 2018). Thus, there are many uncertainties 141

associated with assigning elements to a specific transport pathway. 142

No matter what, at any given time point in the life of a plant, a root has zones completely without 143

root barriers (i.e. the root tip of both primary and lateral roots), unsuberized zones with only a CS, 144

and zones with both a CS and a varying degree of suberized endodermal cells, which may or may 145

not affect the accessibility of ions to transporter proteins (Geldner, 2013; Barberon et al., 2016). 146

These facts constitute both analytical and mechanistic challenges for deciphering the biological 147

consequences of suberization. It is also clear that a classical analysis of leaves or xylem sap mineral 148



6

profiles provide an integrative value, reflecting the net result of many processes, including 149

contributions from all ion transport pathways occurring along the whole root axis. Particularly, the 150

role of suberin as a barrier that may prevent leakage of already acquired nutrients out of the stele is 151

a hypothesis that is still awaiting solid experimental evidence. 152

To further document and explain how root barriers affect radial ion transport and ion translocation, 153

we here combine measurements of suberin with multi-element analyses of xylem sap and leaves and 154

sensitive, cellular-resolved multi-element bioimaging using Laser Ablation-Inductively Coupled 155

Plasma-Mass Spectrometry (LA-ICP-MS). The analytical procedures and advantages of using this 156

imaging technique have been described elsewhere (Persson et al., 2016). 157

We show that barley, an important graminaceous crop plant, initially responds to insufficient Mn 158

supply by decreasing the suberization of the main root axis, seemingly in an attempt to facilitate 159

better access of Mn ions to the transport proteins located at the plasma membrane of endodermal 160

cells. In doing so, the plants experience problems with maintaining a high K concentration within 161

the stele. As Mn deficiency becomes more severe, the roots become more suberized. Radial ion 162

uptake along the root axis is thereby sacrificed, leading to lower Ca and Na concentrations in both 163

the xylem sap and in the leaves. However, as the suberization increases, so do the concentrations of 164

K in the stele, xylem sap and in the leaves. We suggest that plants subjected to prolonged, strong 165

Mn deficiency favor suberization, forcing Mn uptake towards the apoplastic pathway in the root tip, 166

which lacks suberin. Suberin at greater distance from the root tip then acts as a seal to prevent the 167

acquired Mn and other ions within the vascular tissues from being lost to back flow across the 168

endodermis during translocation to the shoots. 169

170

171



7

Results 172

Plant growth 173

Barley plants were cultivated in hydroponics and their Mn status was analyzed by Chlorophyll a 174

fluorescence spectroscopy, which measures the quantum yield efficiency of PSII (Fv/Fm) as a 175

proxy for the intensity of Mn deficiency (Hebbern et al., 2009; Schmidt et al., 2015; Schmidt et al., 176

2016). Plants cultivated at sufficient Mn supply maintained their Fv/Fm values above 0.8 177

throughout the experimental period (Figure 1). In the insufficient Mn treatment, plants became Mn-178

deficient 17 days after start of the experiment as evidenced by a drop in Fv/Fm to 0.65±0.02 (Figure 179

1). This Mn status was characterized as mild Mn deficiency, which was also reflected by the fact 180

that at this time, the 5th leaf was fully developed without any visual deficiency symptoms. After 7 181

days of mild deficiency (24-day-old plants) the intensity of Mn deficiency increased further as 182

evidenced by a drop in Fv/Fm to 0.55±0.02 (Figure 1). This Mn status was maintained until 30 days 183

after the start of the experiment (Figure 1) and was characterized as strong Mn deficiency (Schmidt 184

et al., 2016). The first visual and characteristic symptoms of Mn deficiency, such as intravenous 185

chlorosis and necrosis in combination with slack leaves, appeared at this stage. Within 3 days after 186

resupplying 30-day-old, strongly Mn-deficient plants with control levels of Mn, the Fv/Fm values 187

reverted to above 0.80 (Figure 1). All newly developed leaves were completely symptom-free until 188

termination of the experiment at day 40. The whole cultivation process was repeated several times 189

and followed a similar pattern for each batch of plants. 190

191

During the period when mild Mn deficiency was induced there were no substantial changes in shoot 192

or root biomass (Figure 2). However, in the following period, when the severity of Mn deficiency 193

was further increased to strong Mn deficiency, both the shoot and root biomass became 194

significantly smaller in the Mn-deficient plants. The largest decrease was seen in the roots, where 195

the biomass decreased by approximately 54%. In the same plants, the shoot biomass decreased by 196

approximately 30% (Figure 2). The 40-day-old plants, which had been resupplied with Mn for 10 197

days, still had smaller shoot and root biomass. The length of the main root axes was similar in the 198

Mn-deficient and in the control plants throughout the experimental period. Hence, the difference in 199

root biomass was mainly caused by differences in lateral root formation. The total lengths of the 200

main root axes were approximately 60 cm after 17 days, 80 cm after 28 days and 100 cm after 40 201

days. 202

203



8

The transpiration was monitored throughout the experimental period with no significant differences 204

between the control, the -Mn plants and the –Mn/+Mn plants (Supplemental Figure 1). 205

Barriers in the main root axis 206

Manganese deficiency has a negative effect on more than 35 enzymes involved in plant metabolism, 207

including phenylalanine ammonia-lyase (PAL), which catalyzes the deamination of phenylalanine 208

to cinnamic acid, a key substrate in lignin biosynthesis (Jones, 1984). Since the CS is mainly 209

composed of lignin (Naseer et al., 2012), we hypothesized that Mn-deficient plants might suffer 210

from altered and/or poor CS integrity, which would influence ion transport via apoplastic by-pass 211

across the endodermis. We investigated this by specific CS staining and fluorescence microscopy 212

along the root axis (see Materials and Methods). The CS consistently appeared approximately 2 cm 213

from the root tip (zone 1) and continued upwards. However, with respect to the CS, no differences 214

between Mn-deficient and sufficient plants were observed (Supplemental Figure 2). Hence, we 215

focused only on suberin in the further work. 216



9

The suberization of endodermal cells in the main axes of 17-, 28- and 40-d-old barley roots were 217

analyzed using the specific suberin stains Sudan red 7B (cross sections) and Fluorol Yellow (FY) 218

(whole root sections). We identified four differentially suberized zones in the endodermis (Figure 219

3), but never recorded an exodermis, in any of the treatments. These four zones consisted of a non-220

suberized zone 0-5 cm from the root tip (zone 1), a patchy suberization zone 5-7 cm from the root 221

tip (zone 2), a phloem-facing suberization zone 7-30 cm from the root tip (zone 3) and a fully 222

suberized zone >30 cm from the root tip (zone 4). The endodermis in zone 1 was composed entirely 223

of cells without suberin lamellae. In zone 2, the endodermis had only a few suberized cells in a 224

scattered pattern, while in zone 3, the suberized endodermal cells were arranged in alternate 225

longitudinal columns, 1-2 cells thick. The suberization pattern here was much more structured than 226

in zone 2, with suberized endodermal cells always appearing between the xylem poles, facing the 227

phloem (Figure 3, middle). In zone 4, all of the endodermal cells were suberized (Figure 3, upper). 228

The suberization was quantified by manually recording the length of the four zones (n=8-10; Figure 229

4). Regardless of treatment and age, the fully suberized zone 4 was always the longest, followed by 230



10

zones 3, 1 and 2. Expressed in relative terms, the non-suberized zone accounted in all treatments for 231

8-12% of the whole root length, the patchy zone for 2-8%, the phloem-facing zone for 30-40%, and 232

the fully suberized zone for roughly half of the root length. 233

234

During mild Mn deficiency (17-day-old plants), the suberization was reduced in the deficient plants 235

compared with the controls. This was specifically seen in the length of the non-suberized zone at 236

the root tip (zone 1), which was about 8 cm long in the -Mn plants, compared to about 5 cm in the 237

control plants (P<0.01). The fully suberized zone appeared further away (≈35 cm) from the root tip 238

in the mildly Mn-deficient plants, while this zone started closer to the root tip (≈28 cm) in the 239

control plants (Figure 4, left; P<0.01). 240

241

Following the development of strong Mn deficiency in 28-d-old plants, the suberization increased 242

and the suberin pattern changed relative to the control. Under these conditions, the non-suberized 243

zone was shorter (4 cm) in the deficient plants, whereas in the control plants this zone was about 8 244



11

cm long (P<0.05). The fully suberized zone of the –Mn plants started 28 cm from the root tip, but 245

only after 37 cm in the control plants. As a results of these changes, the fully suberized zone now 246

accounted for approximately 65% of the whole length of the main root axis in the -Mn plants 247

against only about 50% in the control plants (Figure 4, middle; P<0.05). Ten days after resupply of 248

Mn, the now 40-days-old plants no longer displayed any significant differences in their suberization 249

pattern relative to the control plants (Figure 4, right). 250

251

Bioimaging of roots by LA-ICP-MS 252



12

To visualize the effects of Mn-dependent changes in suberization on ion distribution as determined 253

by transport and translocation, cross sections from 2, 10 and 31 cm behind the root tip were 254

prepared and analyzed by LA-ICP-MS. For both the mildly (17-day-old) and the strongly (28-day-255

old) Mn-deficient plants, the element images at 10 cm from the root tip showed, as expected, that 256

the signal for Mn was much lower in the -Mn barley roots than in the controls. The images also 257

suggested that the deficiency indeed had become more severe after 28 days (Figure 5). This was the 258

case for all of the major tissue types, i.e. the epidermis, the cortex, the endodermis and the stele. 259

260

In the non-suberized zone (2 cm from the root tip), the strongly Mn-deficient plants contained much 261

more Mn than the mildly deficient ones, not only in the stele, but also in the epidermis and cortex 262

(Figure 6). Ten cm from the root tip, i.e. in zone 3 with phloem-facing suberization, the stele 263

concentrations of Mn were in both Mn treatments lower compared to the non-suberized zone (2 264

cm), indicating decreased radial Mn uptake in these root segments. The signal intensity of Mn in the 265

epidermis and cortex in zone 3 (10 cm) was similar at both stages of Mn deficiency (Figure 6). 266



13

However, the inner part of the stele in zone 3 contained more Mn in the strongly deficient plants 267

compared to the mildly deficient (Figure 6). The same was the case 31 cm from the root tip, where 268

the image from the mildly deficient plants represent the phloem-facing zone (zone 3) (Figure 7, 269

left), whereas the image from the strongly deficient plants represent the fully suberized zone (zone 270

4) (Figure 7, right). The major part of Mn was present in the inner part of the stele in the strongly 271

Mn-deficient plants, whereas in the mildly Mn-deficient plants more Mn was located to the outer 272

part of the stele, close to the endodermis (Figure 7). The relatively higher concentration of Mn in 273

the inner stele of the strongly Mn-deficient plants compared to the mildly deficient plants (Figure 274

7), suggests that their stronger suberization promoted a more efficient upwards Mn transport from 275

the root tip. Similarly, the relatively high Mn concentration in the outer part of the stele of the 276

mildly Mn-deficient plants with less suberization indicates higher radial uptake and transport of Mn 277

at this distance from the root tip (see also Supplemental Figure 3). 278

279



14

The effect of suberization on element concentrations in leaves, xylem sap and roots 280

The concentration of a range of elements was analyzed in the youngest fully emerged leaf (YFEL) 281

of 17-, 28- and 40 days-old plants (Figure 8). Since 7-8 days are required for a new leaf to develop, 282

the results in Figure 8 (A, B and C) represent different leaves. In the youngest plants, there were no 283

major differences between any elements other than Mn. The Mn concentration was 8.8 µg g-1 in the 284

-Mn plants and 24.4 µg g-1 in the control plants (P<0.001). After 28 days, when strong Mn 285

deficiency had been induced, the Mn concentration was 6.9 µg g-1 in the -Mn plants and 44.4 µg g-1 286

in the control plants (P<0.001). The critical threshold for Mn deficiency in barley leaves is 18 µg g-1 287

(Reuter and Robinson, 1997). In the strongly Mn-deficient plants, the YFEL had higher K (P<0.05) 288

and Cu concentrations (P<0.01). In the same plants, the Na concentration was only about half 289

compared to the control plants (P<0.001). The 40-d-old plants, which upon resupply with Mn had 290

fully recovered from the strong Mn deficiency (Figure 1), now had a similar Mn concentration in 291



15

the YFEL as the control plants. These plants also had lower Ca and Na concentrations, but slightly 292

higher Cu and Zn concentrations (Figure 8). 293

294

The element concentrations in the xylem sap changed more dynamically and profoundly to the Mn 295

treatments than the foliar concentrations (Figure 9). The Mn concentration in the xylem sap was in 296

all cases significantly lower in the Mn-deficient plants than in the controls (P<0.05 in 17-d-old 297

mildly Mn-deficient plants and P<0.001 in 28-d-old strongly Mn-deficient plants; Figure 9). In 298

addition, the mildly Mn-deficient plants had about 20% higher xylem Ca concentration (P<0.05) 299

and 21% lower xylem K concentration (P<0.001) than the control plants (Figure 9A). As plants 300

became strongly deficient and more suberized, the concentrations became opposite to that observed 301

under mild Mn deficiency. In the strongly Mn-deficient plants, the K concentration in the xylem sap 302

was 40% higher (P>0.001), while the Ca concentration was 30% lower (P<0.01) than in the control 303

plants (Figure 9B). The Na concentration in the xylem sap was also significantly reduced in the 304



16

strongly deficient plants (P<0.05), while the trace elements Fe and Zn had similar xylem 305

concentration in the -Mn and control plants (Figure 9B). 306

307

The Mn concentration in the xylem sap of the 40-d-old plants that had experienced the two stages of 308

Mn deficiency, followed by resupply of Mn, was still about 50% lower than in the control plants 309

(Figure 9C). However, the difference relative to the control plants was much smaller at this stage 310

compared to during Mn deficiency (Figure 9C). The differences in Ca, K and Na concentration had 311

now disappeared, showing that the elimination of Mn deficiency-induced differences in suberization 312

upon resupply of Mn also affected these elements. Magnesium (Mg) showed no differences in 313

xylem sap concentrations between the 17, 28 and 40 days treatments and the same was true for the 314

LA-ICP-MS images obtained for roots of replicate plants harvested on the same day (Supplemental 315

Figure S4). Hence, the Mg results served as a negative control of the analytical procedures. 316

317



17

The changes in Ca, K and Na concentrations that were recorded in the xylem sap were also reflected 318

in the root images (Supplemental Figure 5 and 6). The Ca-images taken 31 cm from the root tip, 319

representing the phloem-facing suberization (zone 3) in the 17-d-old, mildly Mn-deficient plants 320

and the fully suberized zone 4 in the +Mn control plants (Figure 3 and 4), suggest that more Ca was 321

transported radially towards the stele in zone 3 than in zone 4 (Supplemental Figure 5, left). The 322

corresponding images in the 28-d-old plants showed stronger Ca signals in and around the stele as 323

well as more Ca in the cortex of the fully suberized zone of the strongly Mn-deficient plants than in 324

the phloem-facing zone of the +Mn control plants. Thus, a relatively more efficient radial transport 325

in the less suberized zone 3, compared to the fully suberized zone 4, was again indicated. The 326

pattern of K accumulation in these different zones was opposite that of Ca, whereas Na was similar 327

to Ca (Supplemental Figure 6). Apart from strengthening the interpretation of the measured xylem 328

concentrations, these results suggest that especially the length of the fully suberized zone had a 329

strong effect on element translocation. 330

331

332



18

Discussion 333

We here show that the degree of suberization in Mn-deficient plants is highly dependent on the 334

intensity and duration of the deficiency (Figure 1 and 4). Changes in suberization in response to Mn 335

deficiency is not a linear, declining process, but rather decreases and then increases, when going 336

from mild to strong Mn deficiency. The response is reversible, with endodermal suberization in Mn-337

deficient plants reverting to normal levels after Mn re-supply in the growth medium. 338

In numerous cases, transport proteins have been shown to be essential players involved in the 339

adaptation of roots to changing availability of nutrients (Che et al., 2018). Recent research suggests 340

that suberin may also be involved in root adaptation to a low nutrient supply, either directly or 341

indirectly, and seemingly with a dual role (Barberon et al., 2016). Firstly, suberin may determine 342

the degree of accessibility to or the presence of ion transporters in the plasmalemma of the 343

endodermis. This assumption is supported by the fact that Arabidopsis mutants with no or strongly 344

reduced suberization display a higher translocation of elements such as Ca and Mn, which are 345

believed to primarily be transported via the apoplastic or the coupled trans-cellular pathways. 346

Secondly, suberin may restrict the back flush of ions from the apoplast of the xylem into the cortex 347

and outer root tissues. In accordance with this assumption, elements such as K and S, which are 348

believed to primarily follow the symplastic pathway, typically show a decreased translocation in 349

less or unsuberized roots. The assumption that reduced outwards leaking from a less suberin-sealed 350

vascular bundle is also supported by the fact that water transport seems to follow the same pattern. 351

Cell wall suberization markedly reduces the hydraulic conductivity of the apoplastic pathway, 352

whereas the cell-to-cell pathway is unaltered. Upon sealing of the apoplast in the fully suberized 353

root zone, uncontrolled back-flow of water is strongly reduced (Kreszies et al., 2018). 354

Based on the results presented here, we suggest that mildly and strongly Mn-deficient plants use 355

completely different suberization strategies to facilitate transport and translocation of Mn to the 356

shoots. These strategies may be seen as extreme examples of the dual role that suberin may have in 357

affecting radial ion transport. Mildly deficient plants appear to prioritize uptake of Mn along the 358

root axis through a reduction in endodermal suberin, which, in theory, would favor xylem loading. 359

More specifically, our results suggest that reduced suberization serves to facilitate trans-cellular Mn 360

transport across the endodermis via increased accessibility to, or presence of Mn transporters at the 361

plasmalemma of the endodermis. It is not currently known in detail how the expression of different 362

Mn transporters in barley may vary along the root axis, and if such expression is connected to the 363



19

suberization patterns of the roots. IRT1 and Nramp1 are transporters involved in Mn uptake in 364

Arabidopsis. Both of these transporters are mainly expressed in the root (Vert et al., 2002) and 365

AtNramp1 is regulated by Mn availability (Cailliatte et al., 2010). In barley, HvNramp5 is localized 366

to the plasma membrane of the epidermal cells in the first half cm of the root tip, but is also weakly 367

expressed in the central cylinder (Wu et al., 2016). HvIRT1 is expressed in a larger part of the root 368

tip (the first 2 cm), showing predominantly epidermal localization in Mn-sufficient roots, but 369

shifting towards the stele during Mn deficiency (Long et al., 2018). Effects of reduced suberization 370

in mildly Mn-deficient plants will therefore likely be more important for HvIRT1 than for 371

HvNramp5, since the latter is expressed closer to the root tip. Upregulation of HvIRT1 expression 372

in the stele rather than in the epidermis in response to Mn deficiency (Long et al., 2018) may under 373

these conditions provide a more efficient Mn transport to the shoot, rather than promoting Mn 374

compartmentalization in root vacuoles. As already stated, previous studies have observed that 375

increased suberization is associated with a decrease in leaf Mn concentrations and vice versa 376

(Baxter et al., 2009; Li et al., 2017). 377

Based on the findings by Barberon et al. (2016), our expectation was that under strong Mn 378

deficiency plants would further de-suberize their endodermis to facilitate more efficient Mn uptake. 379

However, we observed the opposite, i.e. a stronger suberization (Figure 4). We hypothesize that this 380

unexpected, strong suberization occurring under severe Mn deficiency serves to prevent Mn 381

acquired by the unsuberized root tip from leaking out of the stele during the upwards transport in 382

the xylem, very similar to the effect of reduced back-flow of water under drought conditions 383

(Kreszies et al., 2018). The higher Mn concentration in the stele of the strongly Mn-deficient plants 384

compared to the mildly deficient ones (Figure 7) strongly supports this. Furthermore, the 385

concentration of Mn in the roots was clearly higher close to the root tip in the strongly Mn-deficient 386

plants, compared to the mildly deficient ones (Figure 6 and Supplemental Figure 3). These 387

observations support that more Mn was taken up close to the root tip and that the enhanced 388

suberization reduced further uptake along the root axis. In addition, the enhanced suberization of the 389

endodermis along the root axis during strong Mn deficiency may provide a sealed transport pathway 390

for translocation, preventing Mn from leaking back out of the stele. This would enhance the 391

transport efficiency of the Mn acquired by the root tip growing through unexploited soil with the 392

highest concentration of nutrients. 393

The observed decrease in K translocation during mild Mn deficiency (Figure 9 and Supplemental 394

Figure 6) indicates that the de-suberization induced by mild Mn deficiency in 17-d-old plants 395



20

resulted in K leakage. This could have triggered a K-deficiency response that overruled the Mn-396

deficiency, hence a K-induced rather than a Mn-induced over-suberization in strongly Mn-deficient 397

roots of 28-d-old plants. However, we analyzed the youngest fully emerged leaves (YFEL), both at 398

day 17 and 28, and since it takes 7-8 day for the plant to develop a new leaf, these measurements 399

were from two different leaves. As neither of them indicated K-deficiency (<18 µg g-1 DM), 400

combined with the fact that the plants were always fully supplied with K, it does not seem plausible 401

that the observed changes in suberization was induced by K-deficiency. 402

We have shown that ionomic trade-offs accompany the dynamic suberin remodeling in response to 403

Mn deficiency and affect the translocation efficiency of elements such as Ca, K, Na, copper (Cu) 404

and Zn (Figure 9). Ionomic trade-offs have also been observed during the responses to both Fe and 405

P deficiency, although in these cases they were thought to originate, at least in part, from the poor 406

selectivity of the ion-transporters involved (Baxter et al., 2009). In contrast to transporter-regulated 407

adaptations to sub-optimal growing conditions, suberin-regulated adaptation in response to 408

deficiency or excess of a given element will inevitably also affect other elements (Barberon et al., 409

2016; Doblas et al., 2017). Previous work in our group has indicated that the transpiration rate may 410

increase in strongly Mn-deficient barley plants, because of a decrease in the thickness of the leaf 411

cuticle (Hebbern et al., 2009). Such changes would induce differences in xylem sap with respect to 412

flow and solute composition, hence would complicate the interpretations of the xylem data. We 413

recorded no such differences (Supplemental Figure 1), probably because the plants used in the 414

present study were exposed to Mn deficiency for a much shorter time span (2-4 weeks) compared to 415

10 weeks in the previous study. The observed differences may also be related to the fact that 416

different genotypes were used in the two studies. 417

Mapping techniques used for elemental bioimaging have a number of strengths and weaknesses 418

(Becker et al., 2010; Husted et al., 2011; Zhao et al., 2014; Persson et al., 2016). The advantage of 419

the LA-ICP-MS technique used in the present work is the low detection limits and the wide range of 420

elements that can be detected simultaneously, including most of the essential plant nutrients and Na 421

(Persson et al., 2016). However, the strong matrix effects in LA-ICP-MS make it challenging to 422

quantify the obtained signals. This challenge has been addressed in various ways in the LA-ICP-MS 423

community (Pozebon et al., 2017). Comparison of biological samples requires that parameters such 424

as signal strength and differences in sample surfaces and tissue hardness (i.e. ablation efficiency) 425

can be controlled and monitored. In the present work, we used 13C as a continuously monitored 426



21

internal reference to correct for such differences, and we observed only minor differences between 427

the different treatments (Pozebon et al., 2017). Furthermore, the results obtained for magnesium 428

(Mg), showing no significant differences in the xylem sap (Supplemental Figure 4a) or the LA-ICP-429

MS images (Supplemental Figure 4b), provided a negative control supporting the validity of our 430

analytical procedures. Our work show that the extraordinary sensitivity of ICP-MS and LA-ICP-MS 431

translates into an ability to obtain useful data from roots and xylem sap, even though the plants were 432

cultivated under nutrient deficient conditions (20-fold lower than normal levels). If only the leaves 433

had been analyzed, it would not have been possible to decipher the underlying reasons for the 434

observed differences. This was especially obvious in the 17-d-old mildly Mn-deficient plants, 435

showing no differences in their shoot ionome (apart from Mn), but with clear differences both in 436

xylem sap composition (Figure 9) and in the element distribution of the roots (Figure 6, 7 and 437

Supplemental Figure 5 and 6). 438

439

A model for the effects of suberization on radial ion transport and translocation is presented in 440

Figure 10. According to this model, not only the inwards radial ion transport in non-suberized and 441

in suberized root zones is important, but also the loss of ions from the apoplast of the xylem out of 442

the stele (Figure 10). Such outwards leakage would also be important for elements recycled from 443

the shoots to the roots via the phloem, particularly K (White, 1997). Another important 444

consideration for this model is the observations that the localization of IRT1 may change under Mn 445

deficiency, compared to under control conditions, as was observed by (Long et al., 2018). This 446

would implicate that more Mn could be taken up in the epidermis in the control plants, which 447

potentially would lead to more Mn following the symplastic pathway, which is unaffected by 448

suberin. Once inside the stele, however, in the apoplast of the xylem or xylem parenchyma, local 449

concentration gradients may force ions out of the xylem back towards the pericycle (the cylinder of 450

cells that lies just inside the endodermis and is the outer most part of the stele). Since the pericycle 451

does not have a Casparian strip, ions expelled from the xylem may travel between the cells of the 452

pericycle to the inwards-facing plasmalemma of the endodermis. Here, suberin, which is also 453

covering the inwards facing side of the endodermis (Figure 3, top) may block outwards movement 454

of the ions into the symplast of the endodermal cells, while the Casparian strip will block apoplastic 455

efflux out of the stele (Figure 10). These assumptions are supported by the fact that Arabidopsis 456

mutant plants with a perforated CS (sgn3) (Pfister et al., 2014), as well as transgenic lines lacking 457

suberin (cdef1) (Li et al., 2017), both appear to suffer from a loss of ions from the xylem. It must be 458



22

noted, however, that in these plants certain ions can also leak inwards, which is assumed to depend 459

on the electrochemical gradient across the endodermis (Baxter et al., 2012). We are also aware that 460

passage cells occurring in the fully suberized zone, possibly allowing both inwards and outwards 461

ion transport, may be important for the resulting ion translocation, which is a factor not covered by 462

our model. 463

464

Conclusions 465

Our work demonstrates that nutrient deficiency has a more complex effect on root suberization than 466

previously shown in the model plant Arabidopsis. We show that root suberization is altered in 467

response to Mn deficiency, also in barley. More importantly, however, we show that it is the 468

intensity of the deficiency that ultimately determines whether the roots become more or less 469

suberized. During mild Mn deficiency, suberization decreases to promote Mn uptake over a more 470

extensive part of the root. In contrast, during strong Mn deficiency, suberization is increased, 471

thereby directing Mn acquisition towards the root tip and optimizing root-to-shoot Mn 472



23

translocation. By use of multi-element ionomic analyses of xylem sap and cross sections from roots, 473

we document that this plasticity in suberization has trade-offs also with respect to the acquisition of 474

nutrients other than Mn, both under mild and strong Mn deficiency. We conclude that it is the 475

intensity of the deficiency that ultimately determines the degree of suberization, and that such 476

changes have strong and previously unknown effects on the ion homeostasis of in plants. Further 477

work is required to obtain full understanding of the underlying mechanisms and their implications. 478

479

Materials and Methods 480

481

Plant material and growth conditions 482

Barley (Hordeum vulgare cv. Irina) seeds were germinated in vermiculite for 7 days, and then 483

cultivated at a 16-hour day length (natural and/or artificial light) and an ~18°C/~15°C day/night 484

cycle in a greenhouse. Uniform seedlings were transferred to light-impermeable, black 5 L 485

cultivation units and grown hydroponically in aerated nutrient solution. Each unit contained three 486

plants. The units were filled with nutrient solution containing 200 µM KH2PO4, 200 µM K2SO4, 487

300 µM MgSO4·7H2O, 100 µM NaCl, 300 µM Mg(NO3)2·6H2O, 900 µM Ca(NO3)2·4H2O, 600 µM 488

KNO3, 50 µM Fe(III)-EDTANa, 0.8 µM Na2MoO4·2H2O, 0.7 µM ZnCl2, 0.8 µM CuSO4·5H2O, 0.8 489

µM NiCl2, 2 µM H3BO4. The nutrient solutions were replaced once per week during the first 490

4 weeks and 2 times per week subsequently. The pH was adjusted daily with HCl to 6.0 ± 0.2. All 491

plants harvested for xylem sap collection and imaging by LA-ICP-MS, were harvested exactly 3 492

days after replacement of the nutrient solutions. 493

The plants were grown with different Mn additions, either i) maintained at control Mn level 494

(adjusted to a start concentration of 200 nM MnCl2 on a weekly basis during the first 17 days, then 495

supplemented with 800 nmol MnCl2 per cultivation unit every 2nd day until harvest), or ii) 496

maintained at a low Mn level (adjusted to a start concentration of 10 nM MnCl2 on a weekly basis 497

during the first 17 days, then supplemented with 40 nmol MnCl2 per cultivation unit every 2nd day 498

until harvest). An additional batch of plants were exposed to the same low Mn treatment for 30 499

days, and then re-supplied to the control Mn level (800 nmol MnCl2 supplemented every 2nd day) 500

until 40 days old. The Mn statuses of the plants were continuously analyzed using chlorophyll a 501

fluorescence as described below and according to Schmidt et al. (Schmidt et al., 2016). The 502

procedure used for Mn addition allowed the quantum yield efficiency of photosystem II (Fv/Fm) to 503



24

be maintained at 0.65±0.02 for mild Mn deficiency and at 0.55±0.02 for strong Mn deficiency 504

(Figure 1). 505

506

Chlorophyll a fluorescence 507

Chlorophyll a fluorescence was recorded with a Handy PEA (Plant Efficiency Analyzer, Hansatech 508

Instruments, King’s Lynn, UK). The measurements were performed on the youngest fully emerged 509

leaves (YFEL). A flash of saturating light (3.000 µmol photons m-2 s-1) lasting two seconds was 510

applied to leaves, previously dark adapted for 25 min using leaf clips. The fluorescence data were 511

analyzed and the Fv/Fm was calculated using the Handy PEA software (version 1.30). The quantum 512

yield efficiency of photosystem II (Fv/Fm) was monitored daily and maintained at approximately 513

0.6-0.7 (mild Mn deficiency) by the Mn additions listed above for the first 17 days and then allowed 514

to drop to 0.5-0.6 (strong Mn deficiency) until 28 days after planting. Mn-deficient plants were re-515

supplied with Mn after 30 days and harvested 10 days later. After re-supply of Mn, these plants 516

reverted to normal Fv/Fm values (~0.8) after three days (Figure 1). 517

518

Transpiration 519

The measurement of transpiration (E) was made on the YFEL, using a PP Systems instrument 520

(CIRAS-3, Portable Photosynthesis Systems, Amesbury, USA). The transpiration (E) was measured 521

five times for each leaf, always between 10 and 11 AM. During each measurement, the CO2 522

concentration was maintained at 390 µmol mol-1, the relative humidity at 65% and the light 523

intensity at 300 µmol m-2 s-1. For the 40 days old plants, the measurement of E was made on 524

similar-sized leaves from the central part of the shoot. 525

526

Casparian strip staining 527

One cm long segments of the primary root axis of seminal roots were cut at 1, 2, 3, 4, 5, 10 and 30 528

cm distance from the tip. The fresh root segments were embedded in 5% agar in petri dishes. A 529

vibratome (HM 650 V, Zeiss, MICROM GmbH, Germany) was used to obtain 300 µm thick cross-530

sections. The staining of CS was performed according to Lux et al. (2005), where the cross sections 531

were stained with berberine hemisulphate solution dissolved in lactic acid (1 g L-1), followed by 532

staining in berberine (1 g L-1) for 30 min (Lux et al., 2005). Finally, the cross-sections were washed 533

and post-stained with toluidine blue O (1 g L-1) for 30 min. After staining, the sections were washed 534

in water and the CS was detected with a microscope (Leica DM 5000B, Leica Microsystems, 535



25

Wetzlar, Germany) under ultraviolet light, using the following settings: filter cube: A4, excitation: 536

340-380 nm, beam filter: 400 and emission: 450-490 nm. 537

538

Suberin staining of cross-sections and root main axes 539

Suberin staining with Sudan Red 7B was performed according to Brundrett et al. (1991). A 540

sufficient amount of Sudan Red 7B to make a 0.2 g L-1 final solution was dissolved in polyethylene 541

glycol (average Mw 400 Da) by heating at 90℃ for 1 hour. An equal volume of 90% (v/v) glycerol 542

in water was added to the Sudan Red-containing polyethylene glycol solution (Brundrett et al., 543

1991). One cm root segments were cut at 2, 5, 10, 15 and 30 cm distance from the tip of the main 544

root axis. These fresh root segments were embedded in 5% (w/v) agarose in petri dishes, which 545

were left to cool down at 4oC overnight. A vibratome (HM 650 V, Zeiss, Oberkochen, Germany) 546

was used to obtain 300 µm thick cross-sections, which were then stained with Sudan Red 7B for 1 547

hour at room temperature. Each cross-section was rinsed three times (10 min each time) in 50% 548

(v/v) glycerol and mounted on glass slides with a cover slip. The sections were checked with a 549

microscope (Leica DM 5000B) under bright field. 550

Suberin staining of the whole root axis was done with Fluorol Yellow 088 (FY) (Santa Cruz 551

Biotechnology, Dallas, USA), combined with ClearSee solution. ClearSee solutions were prepared 552

according to Kurihara et al. (2015), by mixing the powder of xylitol (100 g L-1), sodium 553

deoxycholate (150 g L-1) and urea (250 g L-1) in water (Kurihara et al., 2015). Uniform roots were 554

fixed with PFA (paraformaldehyde, 40 g L-1) for 60 min in phosphate buffer saline (PBS) under 555

vacuum, at room temperature. These fixed tissues were washed twice for 1 min in PBS and then 556

incubated in ClearSee solution for one week. After this, the roots were again rinsed in distilled 557

water three times and suberin was stained with (FY) (Lux et al., 2005). The whole roots were 558

incubated in a freshly prepared solution of FY (0.1 g L-1, in lactic acid) at 70oC for 1 hour, washed 559

in water three times (5 min each time) and then counterstained with aniline blue (5 g L-1, in water) 560

at room temperature for 30 min in darkness. After the last step, they were again rinsed in water 561

three times (10 min each time). The whole root was cut into 5 cm segments from the root tip and 562

upwards, and all lateral roots were cut off from these segments. A 5 cm scale was drawn on the 563

glass slide and two or three segments were placed on the glass slide with root tips pointing in the 564

same direction. The segments were mounted on slides in 50% glycerol (v/v) and observed with GFP 565

filter using fluorescence microscopy (Leica DM 5000B) using the following settings: filter cube: 566

L5, excitation: 460-500 nm, beam filter: 505 and emission: 510-540 nm. Based on the suberin 567



26

pattern seen with FY staining, the start and end of each zone was determined and the suberization 568

was quantified by manually recording the length of these four zones. The length of each zone is 569

displayed in centimeters (cm). 570

571

Multi-element analysis of leaves 572

The YFEL were collected and freeze-dried (-45℃, 1 mbar) for 36 hours. Hereafter, each sample 573

was weighted. All samples were digested using a pressurized microwave digestion system 574

(UltraWAVE, Anton Paar GmbH, Graz, Austria) with 2.5 mL HNO3 (70 %) and 1 mL H2O2 (15%), 575

and subsequently diluted to 50 mL (Hansen et al., 2013). Elemental analysis was performed with an 576

ICP-OES (Agilent 5100, Agilent technologies, Manchester, UK), using external calibration, drift 577

check samples and certified reference material for optimal data quality (Olsen et al., 2016). For the 578

40 days old plants, leaves were taken from the same position on the plants, and similar sized leaves 579

were selected for the elemental analysis. Data was tested with a one-way ANOVA t-test. 580

581

Multi-element analysis of xylem sap 582

Xylem sap exudate was collected half an hour before the onset of light in the green house, and the 583

plant stem was cut approximately 2 cm above the roots with a stainless steel razor (Kawai et al., 584

2001). A pipette set at 10 µL was used to collect the xylem. The first drop of xylem sap was 585

discarded, hereafter 20-50 µL were collected in 1.5 mL tubes from each replicate during 30 586

minutes, keeping the tubes on ice during sampling. The samples were stored at -20ºC until analysis. 587

Each sample was diluted 1:1 with 7% HNO3 (2 µL xylem sap + 2 µL 7% HNO3), producing a final 588

volume of 4 µL with an acid concentration of 3.5%. Two µL of this sample was analyzed by flow 589

injection analysis (FIA) mode by ICP-MS using an inert HPLC (Ultimate 3000, Thermo Scientific, 590

Waltham (MA), USA) as auto sampler and injector. HNO3 (3.5 %) was used as mobile phase with a 591

flow rate of 0.3 mL min-1. The concentration was measured using external calibration and data 592

quality was checked with certified reference material, according to Persson et al. (2016), and the 593

data was tested with a one-way ANOVA t-test. 594

595

Sample preparation for LA-ICP-MS analysis 596

Samples were prepared for LA-ICP-MS according to Persson et al. (2016). The main axis of 597

uniform seminal roots of 17 and 28 days old plants were selected and harvested on the same day as 598

the plants from which xylem sap was collected. The quantum yield efficiency (Fv/Fm) of PSII was 599



27

used as a proxy to classify the degree of Mn deficiency (Schmidt et al., 2015). Plants with Fv/Fm 600

values ranging from 0.6 to 0.7 (mildly Mn-deficient, 17 days old) and 0.5-0.6 (strongly Mn 601

deficiency, 28 days old) were selected for LA-ICP-MS analysis. Similar to the suberin staining, root 602

samples for LA-ICP-MS analysis were cut 2, 10 and 31 cm behind the root tip, corresponding to 603

zone 1, 3 and 4, respectively (based on previous data on suberin staining). The length of each root 604

piece was approximately 1 cm, from which all lateral roots, if any, were cut off. Each root segment 605

was then gently dried on a napkin, encapsulated in melted paraffin, embedded in OCT (Optimal 606

Cutting Temperature, Tissue-Tek, Sakura Finetek, Tokyo, Japan) and finally frozen in liquid 607

nitrogen (N2). After freezing, the solid OCT block was transferred to a cryotome (Leica CM050S, 608

Leica, St. Gallen, Switzerland), precooled to -23oC, where it was mounted for sectioning. The first 2 609

mm of the root segment was discarded and then 30 µm thick cross-sections were cut and transferred 610

onto plastic microscopy slides (Permanox Microscope Slide, Electron Microscopy Sciences, 611

Hatfield (PA), USA). These plastic slides gave very low background signal on the elements of 612

interest. The sections were kept frozen (-23oC) overnight inside the cryotome, allowing for a slow 613

and gentle drying for 16-18 hours. The dry sections were checked with bright field microscopy, and 614

sections with perfect structural integrity were selected and marked for LA-ICP-MS analysis. This 615

sample preparation technique has proven to preserve the native ion distribution (Persson et al., 616

2016). Three cross-sections (from different plants) from each treatment and root zone were 617

analyzed. 618

619

LA-ICP-MS analysis 620

A nanosecond laser ablation unit (NWR193, New Wave Research, Fremont (CA), USA) equipped 621

with an ArF excimer laser source operating at 193 nm wavelength was used for all LA-ICP-MS 622

analyses. For improved sample transfer from the ablation chamber to the ICP-MS, a Dual 623

Concentric Injector (DCI, New Wave Research, Fremont (CA), USA) was employed. The following 624

settings were used: Energy: 1.4-2.3 J cm-2 (30-40 % of maximum energy), Scan speed: 20 µm s-1, 625

Repetition rate: 40 Hz and spot size 10 µm. Helium was used as transfer gas (from LA unit to ICP-626

MS) with a flow of 750 mL min-1. All elemental signals were obtained with an Agilent 7900 ICP-627

MS (7900 ICP-MS, Agilent technologies, Manchester, UK), operated in H2-mode (3 mL min-1). 628

Sample cone depth in the ICP-MS was 3.5 mm and the ICP-carrier gas (Ar) was set to 0.9 mL min-629 1. The isotopes analyzed were 13C, 39K, 24Mg, 44Ca, 23Na 55Mn, 56Fe and 66Zn, using integration 630



28

times of 0.05 (13C), 0.04 (55Mn), 0.03 (44Ca) and 0.02 (23Na, 24Mg, 39K, 56Fe and 66Zn) seconds. The 631

scan cycle was 0.235 seconds. All data was processed with SigmaPlot version 13 (Systat Software 632

Inc., London, UK) and normalized against endogenous carbon, analyzed as 13C. A spot size of 10 633

µm in diameter was enough to get sufficient signal strength of all elements that were included in the 634

analysis and provided sufficient resolution to visualize the ion distribution at the single cell level in 635

the main root tissues, i.e. the epidermis, cortex and the stele. Every LA-ICP-MS analysis was 636

repeated three times on cross-sections from different plants. 637

638

639

640

Supplemental data 641

Supplemental Figure S1. Transpiration measurements of manganese-deficient and control plants 642

from day 17 to 40. 643

Supplemental Figure S2. Staining of the Casparian strip in control and manganese-deficient plants. 644

Supplemental Figure S3. LA-ICP-MS images of the manganese (55Mn) distribution in root cross 645

sections from Mn-deficient plants. 646

Supplemental Figure S4. Magnesium concentrations in xylem sap and the magnesium (24Mg) 647

distribution in root cross-sections from Mn-deficient plants. 648

Supplemental Figure S5. Distribution of Ca in cross-sections from Mn-deficient plants; sampled 2, 649

10 and 31 cm from the root tip. 650

Supplemental Figure S6. Distribution of potassium and sodium in cross-sections from Mn-deficient 651

plants; sampled 31 cm from the root tip. 652

653

Figure Legends 654

Figure 1. The quantum yield efficiency (Fv/Fm) of PSII was used as a proxy for the intensity of Mn 655

deficiency. Mild Mn deficiency (yellow box) was induced 17 days after the start of the experiment, 656

where Fv/Fm was allowed to drop to 0.65±0.02 by adjusting the Mn addition rate. Mild Mn 657

deficiency was continued until day 24, where strong Mn deficiency (red box) was induced by 658

allowing Fv/Fm to drop to 0.55±0.02 until Mn was re-supplied at day 30. Re-supplying plants with 659



29

Mn induced a rapid correction of Mn deficiency and from day 34 until the termination of the 660

experiment and at day 40 these plants had the same Fv/Fm as the control (green box; Fv/Fm > 0.8). 661

662

Figure 2. The dry weight biomass of 17, 28 and 40-d-old plants. Data represents mean values ± SE 663

(n = 4-6) for control, deficient (-Mn) and resupplied (-Mn/+Mn) plants. Data was tested with a one-664

way ANOVA t-test, and the asterisks indicate significant differences (* p ≤ 0.05; ** p ≤ 0.01; *** p 665

≤ 0.001) between control and -Mn treatments. 666

667

Fig. 3. Typical suberin patterns in the main root axis of 17-d-old barley roots under control Mn 668

conditions. Suberin was visualized using a GFP filter mounted on a fluorescence microscope after 669

staining whole root segments with Fluorol Yellow (FY) (left lane) or visualized under bright field 670

microscopy after staining with Sudan Red 7B (right lane). Four zones were identified, viz. a 671

nonsuberized zone (zone 1; 0-5 cm), a patchy zone (zone 2; 5-7 cm), a phloem-facing zone (zone 3; 672

7-30 cm) and a fully suberized zone (zone 4; >30 cm). The images from zone 1 had no visual 673

suberized cells, whereas in zone 4, all endodermal cells were suberized. Suberized cells in the right 674

lane are indicated by arrows. Scale bars: 250 μm. Mx: metaxylem, Xp: xylem poles, Ph: phloem 675

676

Figure 4. Quantification of suberin deposition along the main root axis of 17, 28 and 40-d-old 677

plants. The plants were exposed to control (+Mn) or Mn deficient conditions (-Mn) and the latter 678

resupplied with Mn at day 30 (-Mn/+Mn). The 17-d-old -Mn plants represent mild Mn deficiency 679

and the 28-d-old -Mn plants represent strong Mn deficiency. Four different root zones were 680

identified in 8-10 biological replicates: zone 1 (non-suberized zone), zone 2 (patchy zone), zone 3 681

(phloem-facing suberization zone) and zone 4 (fully suberized zone). The length of each zone is 682

expressed in cm. Data was analyzed with a one-way ANOVA ttest, where asterisks indicate 683

significant differences (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001) between control and Mn treatments. 684

The suberin quantification was performed on two different batches of plants, with similar results. 685

686

Figure 5. Distribution of Mn in cross-sections sampled 10 cm from the root tip (zone 3) and 687

analyzed by LA-ICP-MS with a spot size of 10 μm, monitoring the 55Mn isotope. The 17-d-old -688

Mn plants represent mild Mn deficiency and the 28-d-old -Mn plants represent strong Mn 689



30

deficiency. Signal intensities are displayed as a heat map where red represents the strongest 690

intensities and 691

purple the weakest (background). All ion intensities were normalized to endogenous carbon 692

(measured as 13C). Three cross sections with intact tissue structures from each root zone were 693

analyzed. Ep: epidermis, co: cortex and st: stele. Scale bars represent 100 μm. 694

695

Figure 6. Manganese (55Mn) distribution in root cross-sections 2 (bottom), 10 (middle) and 31 696

(upper) cm from the root tip, analyzed by LA-ICP-MS with spot size of 10 μm. All signal 697

intensities were normalized to endogenous carbon (measured as 13C). The images to the left 698

represent 17-d-old, mildly Mn-deficient plants, which had less suberin than the control plants. The 699

images to the right 700

represent 28-d-old, strongly Mn-deficient plants, which have more suberin than non-deficient 701

control plants. The images from 31 cm reflect the differences in suberization, where the -Mn image 702

from the 17-d-old plants represent the phloem-facing (i.e. less suberized) zone 3, whereas the -Mn 703

image from the 28-d-old plants represent the fully suberized zone 4. The signal intensities are 704

displayed as heat maps, where red represents the strongest intensities and purple the weakest 705

intensities. Three cross sections with intact tissue structures from each root zone were analyzed and 706

representative data were selected. Ep: epidermis, co: cortex and st: stele. Scale bars represent 100 707

μm. 708

709

Figure 7. LA-ICP-MS images of the 55Mn distribution in the stele of root cross-sections sampled, 31 710

cm from the tip. The image to the left represents a mildly Mn deficient plant, which had less suberin 711

than non-deficient, control plants. The image to the right represents a strongly Mn deficient plant, 712

which had more suberin than control plants. The images reflect the differences in suberization, 713

where the -Mn image from the 17-d-old plants is from the phloem-facing (i.e. less suberized) zone 714

3, whereas the -Mn image from the 28-d-old plants is from the fully suberized zone 4. The signal 715

intensities were normalized to endogenous carbon (13C) and are displayed as heat maps, where red 716

represents the strongest intensities and purple the weakest intensities. The scale bar represents 50 717

μm. 718

719



31

Fig. 8. Element concentrations in the dry matter of the youngest fully emerged leaves (YFEL) of 17 720

(A), 28 (B) and 40-d-old (C) barley plants. The 17-d-old -Mn plants represent mild Mn deficiency 721

and the 28-d-old -Mn plants represent strong Mn deficiency. Since it takes 7-8 days for the YFEL to 722

develop, the results in A, B and C are from different leaves. The plants were cultivated in a 723

hydroponic system with control (+Mn), Mn deficient conditions (-Mn) or first Mn-deficient, then 724

resupplied with Mn at day 30 (-Mn/+Mn). Data represents mean values ± SE (n = 4), which was 725

tested with a one-way ANOVA t- test. Asterisks indicate significant differences (* p ≤ 0.05; ** p ≤ 726

0.01; *** p ≤ 0.001) between control and -Mn treatments. 727

728

Fig. 9. Element concentrations in xylem sap from 17 (A), 28 (B) and 40-d-old (C) barley plants. 729

The plants were cultivated in a hydroponic system and the xylem sap was collected from control 730

(+Mn), deficient (-Mn) and first Mn-deficient, then resupplied with Mn at day 30 (-Mn/+Mn). The 731

17-d-old -Mn plants represent mild Mn deficiency and the 28-d-old -Mn plants represent strong Mn 732

deficiency. Data represents mean values ± SE (n=4), which was tested with a one-way ANOVA t-733

test. Asterisks indicate significant differences (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001) between 734

control and -Mn treatments. 735

736

Figure 10. Schematic model of ionic movements from the soil solution towards the xylem in a fully 737

suberized (a) and a nonsuberized (b) stele of barley plants. 738

739

740



Parsed CitationsAlassimone J, Fujita S, Doblas VG, van Dop M, Barberon M, Kalmbach L, Vermeer JE, Rojas-Murcia N, Santuari L, Hardtke CS, GeldnerN (2016) Polarly localized kinase SGN1 is required for Casparian strip integrity and positioning. Nat Plants 2: 16113

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Andersen TG, Barberon M, Geldner N (2015) Suberization - the second life of an endodermal cell. Curr Opin Plant Biol 28: 9-15Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Andersen TG, Naseer S, Ursache R, Wybouw B, Smet W, De Rybel B, Vermeer JEM, Geldner N (2018) Diffusible repression of cytokininsignalling produces endodermal symmetry and passage cells. Nature 555: 529-533


Barberon M, Geldner N (2014) Radial Transport of Nutrients: The Plant Root as a Polarized Epithelium. Plant Physiol 166: 528-537Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Barberon M, Vermeer JE, De Bellis D, Wang P, Naseer S, Andersen TG, Humbel BM, Nawrath C, Takano J, Salt DE, Geldner N (2016)Adaptation of root function by nutrient-induced plasticity of endodermal differentiation. Cell 164: 447-459


Baxter I, Hermans C, Lahner B, Yakubova E, Tikhonova M, Verbruggen N, Chao DY, Salt DE (2012) Biodiversity of mineral nutrient andtrace element accumulation in Arabidopsis thaliana. PLoS One 7: e35121


Baxter I, Hosmani PS, Rus A, Lahner B, Borevitz JO, Muthukumar B, Mickelbart MV, Schreiber L, Franke RB, Salt DE (2009) Rootsuberin forms an extracellular barrier that affects water relations and mineral nutrition in Arabidopsis. PLoS Genet 5: e1000492


Becker JS, Zoriy M, Matusch A, Wu B, Salber D, Palm C, Becker JS (2010) Bioimaging of metals by laser ablation inductively coupledplasma mass spectrometry (LA-ICP-MS). Mass Spectrom Rev 29: 156-175


Brundrett MC, Kendrick B, Peterson CA (1991) Efficient Lipid Staining in Plant-Material with Sudan Red-7b or Fluoral Yellow-088 inPolyethylene Glycol-Glycerol. Biotechnic & Histochemistry 66: 111-116


Cailliatte R, Schikora A, Briat JF, Mari S, Curie C (2010) High-affinity manganese uptake by the metal transporter NRAMP1 is essentialfor Arabidopsis growth in low manganese conditions. Plant Cell 22: 904-917


Che J, Yamaji N, Ma JF (2018) Efficient and flexible uptake system for mineral elements in plants. New Phytologist 219: 513-517Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Doblas VG, Geldner N, Barberon M (2017) The endodermis, a tightly controlled barrier for nutrients. Current Opinion in Plant Biology39: 136-143


Doblas VG, Smakowska-Luzan E, Fujita S, Alassimone J, Barberon M, Madalinski M, Belkhadir Y, Geldner N (2017) Root diffusionbarrier control by a vasculature derived peptide binding to the SGN3 receptor. Science 355: 280-284


Enstone DE, Peterson CA, Ma F (2003) Root Endodermis and Exodermis: Structure, Function, and Responses to the Environment. JPlant Growth Regul 21: 335-351


Geldner N (2013) The endodermis. Annu Rev Plant Biol 64: 531-558Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Hansen TH, de Bang TC, Laursen KH, Pedas P, Husted S, Schjoerring JK (2013) Multielement Plant Tissue Analysis Using ICP www.plantphysiol.orgon March 24, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.

https://www.ncbi.nlm.nih.gov/pubmed?term=Alassimone J%2C Fujita S%2C Doblas VG%2C van Dop M%2C Barberon M%2C Kalmbach L%2C Vermeer JE%2C Rojas%2DMurcia N%2C Santuari L%2C Hardtke CS%2C Geldner N %282016%29 Polarly localized kinase SGN1 is required for Casparian strip integrity and positioning%2E Nat Plants&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Alassimone&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Polarly localized kinase SGN1 is required for Casparian strip integrity and positioning.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Polarly localized kinase SGN1 is required for Casparian strip integrity and positioning.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Alassimone&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Andersen TG%2C Barberon M%2C Geldner N %282015%29 Suberization %2D the second life of an endodermal cell%2E Curr Opin Plant Biol&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Andersen&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Suberization - the second life of an endodermal cell&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Suberization - the second life of an endodermal cell&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Andersen&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Andersen TG%2C Naseer S%2C Ursache R%2C Wybouw B%2C Smet W%2C De Rybel B%2C Vermeer JEM%2C Geldner N %282018%29 Diffusible repression of cytokinin signalling produces endodermal symmetry and passage cells%2E Nature&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Andersen&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Diffusible repression of cytokinin signalling produces endodermal symmetry and passage cells&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Diffusible repression of cytokinin signalling produces endodermal symmetry and passage cells&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Andersen&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Barberon M%2C Geldner N %282014%29 Radial Transport of Nutrients%3A The Plant Root as a Polarized Epithelium%2E Plant Physiol&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Barberon&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Radial Transport of Nutrients: The Plant Root as a Polarized Epithelium&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Radial Transport of Nutrients: The Plant Root as a Polarized Epithelium&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Barberon&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Barberon M%2C Vermeer JE%2C De Bellis D%2C Wang P%2C Naseer S%2C Andersen TG%2C Humbel BM%2C Nawrath C%2C Takano J%2C Salt DE%2C Geldner N %282016%29 Adaptation of root function by nutrient%2Dinduced plasticity of endodermal differentiation%2E Cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Barberon&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Adaptation of root function by nutrient-induced plasticity of endodermal differentiation&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Adaptation of root function by nutrient-induced plasticity of endodermal differentiation&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Barberon&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Baxter I%2C Hermans C%2C Lahner B%2C Yakubova E%2C Tikhonova M%2C Verbruggen N%2C Chao DY%2C Salt DE %282012%29 Biodiversity of mineral nutrient and trace element accumulation in Arabidopsis thaliana%2E PLoS One 7%3A e&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Baxter&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Biodiversity of mineral nutrient and trace element accumulation in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Biodiversity of mineral nutrient and trace element accumulation in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Baxter&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Baxter I%2C Hosmani PS%2C Rus A%2C Lahner B%2C Borevitz JO%2C Muthukumar B%2C Mickelbart MV%2C Schreiber L%2C Franke RB%2C Salt DE %282009%29 Root suberin forms an extracellular barrier that affects water relations and mineral nutrition in Arabidopsis%2E PLoS Genet 5%3A e&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Baxter&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Root suberin forms an extracellular barrier that affects water relations and mineral nutrition in Arabidopsis.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Root suberin forms an extracellular barrier that affects water relations and mineral nutrition in Arabidopsis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Baxter&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Becker JS%2C Zoriy M%2C Matusch A%2C Wu B%2C Salber D%2C Palm C%2C Becker JS %282010%29 Bioimaging of metals by laser ablation inductively coupled plasma mass spectrometry %28LA%2DICP%2DMS%29%2E Mass Spectrom Rev&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Becker&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Bioimaging of metals by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Bioimaging of metals by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Becker&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Brundrett MC%2C Kendrick B%2C Peterson CA %281991%29 Efficient Lipid Staining in Plant%2DMaterial with Sudan Red%2D7b or Fluoral Yellow%2D088 in Polyethylene Glycol%2DGlycerol%2E Biotechnic %26 Histochemistry&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Brundrett&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Efficient Lipid Staining in Plant-Material with Sudan Red-7b or Fluoral Yellow-088 in Polyethylene Glycol-Glycerol&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Efficient Lipid Staining in Plant-Material with Sudan Red-7b or Fluoral Yellow-088 in Polyethylene Glycol-Glycerol&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Brundrett&as_ylo=1991&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Cailliatte R%2C Schikora A%2C Briat JF%2C Mari S%2C Curie C %282010%29 High%2Daffinity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions%2E Plant Cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Cailliatte&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=High-affinity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=High-affinity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Cailliatte&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Che J%2C Yamaji N%2C Ma JF %282018%29 Efficient and flexible uptake system for mineral elements in plants%2E New Phytologist&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Che&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Efficient and flexible uptake system for mineral elements in plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Efficient and flexible uptake system for mineral elements in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Che&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Doblas VG%2C Geldner N%2C Barberon M %282017%29 The endodermis%2C a tightly controlled barrier for nutrients%2E Current Opinion in Plant Biology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Doblas&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The endodermis, a tightly controlled barrier for nutrients&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The endodermis, a tightly controlled barrier for nutrients&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Doblas&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Doblas VG%2C Smakowska%2DLuzan E%2C Fujita S%2C Alassimone J%2C Barberon M%2C Madalinski M%2C Belkhadir Y%2C Geldner N %282017%29 Root diffusion barrier control by a vasculature derived peptide binding to the SGN3 receptor%2E Science&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Doblas&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Root diffusion barrier control by a vasculature derived peptide binding to the SGN3 receptor&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Root diffusion barrier control by a vasculature derived peptide binding to the SGN3 receptor&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Doblas&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Enstone DE%2C Peterson CA%2C Ma F %282003%29 Root Endodermis and Exodermis%3A Structure%2C Function%2C and Responses to the Environment%2E J Plant Growth Regul&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Enstone&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Root Endodermis and Exodermis: Structure, Function, and Responses to the Environment&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Root Endodermis and Exodermis: Structure, Function, and Responses to the Environment&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Enstone&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Geldner N %282013%29 The endodermis%2E Annu Rev Plant Biol&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Geldner&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The endodermis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The endodermis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Geldner&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1


Spectrometry. In FJM Maathuis, ed, Plant Mineral Nutrients: Methods and Protocols. Humana Press, Totowa, NJ, pp 121-141Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Hebbern CA, Laursen KH, Ladegaard AH, Schmidt SB, Pedas P, Bruhn D, Schjoerring JK, Wulfsohn D, Husted S (2009) Latentmanganese deficiency increases transpiration in barley (Hordeum vulgare). Physiologia Plantarum 135: 307-316


Hosmani PS, Kamiya T, Danku J, Naseer S, Geldner N, Guerinot ML, Salt DE (2013) Dirigent domain-containing protein is part of themachinery required for formation of the lignin-based Casparian strip in the root. Proc Natl Acad Sci U S A 110: 16283-16283


Husted S, Persson DP, Laursen KH, Hansen TH, Pedas P, Schiller M, Hegelund JN, Schjoerring JK (2011) Review: The role of atomicspectrometry in plant science. Journal of Analytical Atomic Spectrometry 26: 52-79


Jones DH (1984) Phenylalanine Ammonia-Lyase - Regulation of Its Induction, and Its Role in Plant Development. Phytochemistry 23:1349-1359


Kawai S, Kamei S, Matsuda Y, Ando R, Kondo S, Ishizawa A, Alam S (2001) Concentrations of iron and phytosiderophores in xylem sapof iron-deficient barley plants. Soil Science and Plant Nutrition 47: 265-272


Kreszies T, Schreiber L, Ranathunge K (2018) Suberized transport barriers in Arabidopsis, barley and rice roots: From the model plantto crop species. Journal of Plant Physiology 227: 75-83


Krishnamurthy P, Ranathunge K, Nayak S, Schreiber L, Mathew MK (2011) Root apoplastic barriers block Na+ transport to shoots inrice (Oryza sativa L.). J Exp Bot 62: 4215-4228


Kurihara D, Mizuta Y, Sato Y, Higashiyama T (2015) ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging.Development 142: 4168-4179


Lee Y, Rubio MC, Alassimone J, Geldner N (2013) A mechanism for localized lignin deposition in the endodermis. Cell 153: 402-412Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Li B, Kamiya T, Kalmbach L, Yamagami M, Yamaguchi K, Shigenobu S, Sawa S, Danku JM, Salt DE, Geldner N, Fujiwara T (2017) Role ofLOTR1 in Nutrient Transport through Organization of Spatial Distribution of Root Endodermal Barriers. Curr Biol 27: 758-765


Long LZ, Persson DP, Duan FY, Jorgensen K, Yuan LX, Schjoerring JK, Pedas PR (2018) The iron-regulated transporter 1 plays anessential role in uptake, translocation and grain-loading of manganese, but not iron, in barley. New Phytologist 217: 1640-1653


Lux A, Morita S, Abe J, Ito K (2005) An improved method for clearing and staining free-hand sections and whole-mount samples. AnnBot 96: 989-996


Ma FS, Peterson CA (2003) Current insights into the development, structure, and chemistry of the endodermis and exodermis of roots.Canadian Journal of Botany-Revue Canadienne De Botanique 81: 405-421


Nakayama T, Shinohara H, Tanaka M, Baba K, Ogawa-Ohnishi M, Matsubayashi Y (2017) A peptide hormone required for Casparianstrip diffusion barrier formation in Arabidopsis roots. Science 355: 284-286


Naseer S, Lee Y, Lapierre C, Franke R, Nawrath C, Geldner N (2012) Casparian strip diffusion barrier in Arabidopsis is made of a ligninpolymer without suberin. Proc Natl Acad Sci USA 109: 10101-10106 www.plantphysiol.orgon March 24, 2020 - Published by Downloaded from

Copyright © 2019 American Society of Plant Biologists. All rights reserved.

https://www.ncbi.nlm.nih.gov/pubmed?term=Hansen TH%2C de Bang TC%2C Laursen KH%2C Pedas P%2C Husted S%2C Schjoerring JK %282013%29 Multielement Plant Tissue Analysis Using ICP Spectrometry%2E In FJM Maathuis%2C ed%2C Plant Mineral Nutrients%3A Methods and Protocols%2E Humana Press%2C Totowa%2C NJ%2C pp&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hansen&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Multielement Plant Tissue Analysis Using ICP Spectrometry.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Multielement Plant Tissue Analysis Using ICP Spectrometry.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hansen&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Hebbern CA%2C Laursen KH%2C Ladegaard AH%2C Schmidt SB%2C Pedas P%2C Bruhn D%2C Schjoerring JK%2C Wulfsohn D%2C Husted S %282009%29 Latent manganese deficiency increases transpiration in barley %28Hordeum vulgare%29%2E Physiologia Plantarum&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hebbern&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Latent manganese deficiency increases transpiration in barley (Hordeum vulgare)&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Latent manganese deficiency increases transpiration in barley (Hordeum vulgare)&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hebbern&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Hosmani PS%2C Kamiya T%2C Danku J%2C Naseer S%2C Geldner N%2C Guerinot ML%2C Salt DE %282013%29 Dirigent domain%2Dcontaining protein is part of the machinery required for formation of the lignin%2Dbased Casparian strip in the root%2E Proc Natl Acad Sci U S A&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hosmani&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based Casparian strip in the root&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based Casparian strip in the root&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hosmani&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Husted S%2C Persson DP%2C Laursen KH%2C Hansen TH%2C Pedas P%2C Schiller M%2C Hegelund JN%2C Schjoerring JK %282011%29 Review%3A The role of atomic spectrometry in plant science%2E Journal of Analytical Atomic Spectrometry&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Husted&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Review: The role of atomic spectrometry in plant science&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Review: The role of atomic spectrometry in plant science&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Husted&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Jones DH %281984%29 Phenylalanine Ammonia%2DLyase %2D Regulation of Its Induction%2C and Its Role in Plant Development%2E Phytochemistry&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Jones&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Phenylalanine Ammonia-Lyase - Regulation of Its Induction, and Its Role in Plant Development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Phenylalanine Ammonia-Lyase - Regulation of Its Induction, and Its Role in Plant Development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Jones&as_ylo=1984&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Kawai S%2C Kamei S%2C Matsuda Y%2C Ando R%2C Kondo S%2C Ishizawa A%2C Alam S %282001%29 Concentrations of iron and phytosiderophores in xylem sap of iron%2Ddeficient barley plants%2E Soil Science and Plant Nutrition&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kawai&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Concentrations of iron and phytosiderophores in xylem sap of iron-deficient barley plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Concentrations of iron and phytosiderophores in xylem sap of iron-deficient barley plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kawai&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Kreszies T%2C Schreiber L%2C Ranathunge K %282018%29 Suberized transport barriers in Arabidopsis%2C barley and rice roots%3A From the model plant to crop species%2E Journal of Plant Physiology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kreszies&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Suberized transport barriers in Arabidopsis, barley and rice roots: From the model plant to crop species&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Suberized transport barriers in Arabidopsis, barley and rice roots: From the model plant to crop species&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kreszies&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Krishnamurthy P%2C Ranathunge K%2C Nayak S%2C Schreiber L%2C Mathew MK %282011%29 Root apoplastic barriers block Na%2B transport to shoots in rice %28Oryza sativa L%2E%29%2E J Exp Bot&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Krishnamurthy&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Root apoplastic barriers block Na+ transport to shoots in rice (Oryza sativa L&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Root apoplastic barriers block Na+ transport to shoots in rice (Oryza sativa L&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Krishnamurthy&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Kurihara D%2C Mizuta Y%2C Sato Y%2C Higashiyama T %282015%29 ClearSee%3A a rapid optical clearing reagent for whole%2Dplant fluorescence imaging%2E Development&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kurihara&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kurihara&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Lee Y%2C Rubio MC%2C Alassimone J%2C Geldner N %282013%29 A mechanism for localized lignin deposition in the endodermis%2E Cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lee&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=A mechanism for localized lignin deposition in the endodermis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A mechanism for localized lignin deposition in the endodermis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lee&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Li B%2C Kamiya T%2C Kalmbach L%2C Yamagami M%2C Yamaguchi K%2C Shigenobu S%2C Sawa S%2C Danku JM%2C Salt DE%2C Geldner N%2C Fujiwara T %282017%29 Role of LOTR1 in Nutrient Transport through Organization of Spatial Distribution of Root Endodermal Barriers%2E Curr Biol&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Li&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Role of LOTR1 in Nutrient Transport through Organization of Spatial Distribution of Root Endodermal Barriers&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Role of LOTR1 in Nutrient Transport through Organization of Spatial Distribution of Root Endodermal Barriers&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Li&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Long LZ%2C Persson DP%2C Duan FY%2C Jorgensen K%2C Yuan LX%2C Schjoerring JK%2C Pedas PR %282018%29 The iron%2Dregulated transporter 1 plays an essential role in uptake%2C translocation and grain%2Dloading of manganese%2C but not iron%2C in barley%2E New Phytologist&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Long&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The iron-regulated transporter 1 plays an essential role in uptake, translocation and grain-loading of manganese, but not iron, in barley&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The iron-regulated transporter 1 plays an essential role in uptake, translocation and grain-loading of manganese, but not iron, in barley&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Long&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Lux A%2C Morita S%2C Abe J%2C Ito K %282005%29 An improved method for clearing and staining free%2Dhand sections and whole%2Dmount samples%2E Ann Bot&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lux&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=An improved method for clearing and staining free-hand sections and whole-mount samples&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=An improved method for clearing and staining free-hand sections and whole-mount samples&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lux&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Ma FS%2C Peterson CA %282003%29 Current insights into the development%2C structure%2C and chemistry of the endodermis and exodermis of roots%2E Canadian Journal of Botany%2DRevue Canadienne De Botanique&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Ma&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Current insights into the development, structure, and chemistry of the endodermis and exodermis of roots&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Current insights into the development, structure, and chemistry of the endodermis and exodermis of roots&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ma&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Nakayama T%2C Shinohara H%2C Tanaka M%2C Baba K%2C Ogawa%2DOhnishi M%2C Matsubayashi Y %282017%29 A peptide hormone required for Casparian strip diffusion barrier formation in Arabidopsis roots%2E Science&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Nakayama&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=A peptide hormone required for Casparian strip diffusion barrier formation in Arabidopsis roots&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A peptide hormone required for Casparian strip diffusion barrier formation in Arabidopsis roots&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Nakayama&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1



Olsen LI, Hansen TH, Larue C, Osterberg JT, Hoffmann RD, Liesche J, Kramer U, Surble S, Cadarsi S, Samson VA, Grolimund D,Husted S, Palmgren M (2016) Mother-plant-mediated pumping of zinc into the developing seed. Nat Plants 2: 16036


Persson DP, Chen A, Aarts MG, Salt DE, Schjoerring JK, Husted S (2016) Multi-Element Bioimaging of Arabidopsis thaliana Roots. PlantPhysiol 172: 835-847


Pfister A, Barberon M, Alassimone J, Kalmbach L, Lee Y, Vermeer JE, Yamazaki M, Li G, Maurel C, Takano J, Kamiya T, Salt DE,Roppolo D, Geldner N (2014) A receptor-like kinase mutant with absent endodermal diffusion barrier displays selective nutrienthomeostasis defects. Elife 3: e03115


Pozebon D, Scheffler GL, Dressler VL (2017) Recent applications of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for biological sample analysis: a follow-up review. Journal of Analytical Atomic Spectrometry 32: 890-919


Robards AW, Jackson SM, Clarkson DT, Sanderson J (1973) Structure of Barley Roots in Relation to Transport of Ions into Stele.Protoplasma 77: 291-311


Roppolo D, De Rybel B, Denervaud Tendon V, Pfister A, Alassimone J, Vermeer JE, Yamazaki M, Stierhof YD, Beeckman T, Geldner N(2011) A novel protein family mediates Casparian strip formation in the endodermis. Nature 473: 380-383


Schmidt SB, Persson DP, Powikrowska M, Frydenvang J, Schjoerring JK, Jensen PE, Husted S (2015) Metal Binding in Photosystem IISuper- and Subcomplexes from Barley Thylakoids. Plant Physiol 168: 1490-1502


Schmidt SB, Powikrowska M, Krogholm KS, Naumann-Busch B, Schjoerring JK, Husted S, Jensen PE, Pedas PR (2016) Photosystem IIFunctionality in Barley Responds Dynamically to Changes in Leaf Manganese Status. Front Plant Sci 7: 1772


Schreiber L (2010) Transport barriers made of cutin, suberin and associated waxes. Trends Plant Sci 15: 546-553Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Tylova E, Peckova E, Blascheova Z, Soukup A (2017) Casparian bands and suberin lamellae in exodermis of lateral roots: an importanttrait of roots system response to abiotic stress factors. Ann Bot 120: 71-85


Vert G, Grotz N, Dedaldechamp F, Gaymard F, Guerinot ML, Briat JF, Curie C (2002) IRT1, an Arabidopsis transporter essential for ironuptake from the soil and for plant growth. Plant Cell 14: 1223-1233


White PJ (1997) The regulation of K+ influx into roots of rye (Secale cereale L.) seedlings by negative feedback via the K+ flux fromshoot to root in the phloem. Journal of Experimental Botany 48: 2063-2073


Wu D, Yamaji N, Yamane M, Kashino-Fujii M, Sato K, Feng Ma J (2016) The HvNramp5 Transporter Mediates Uptake of Cadmium andManganese, But Not Iron. Plant Physiol 172: 1899-1910


Zeier J, Ruel K, Ryser U, Schreiber L (1999) Chemical analysis and immunolocalisation of lignin and suberin in endodermal andhypodermal/rhizodermal cell walls of developing maize (Zea mays L.) primary roots. Planta 209: 1-12


Zhao FJ, Moore KL, Lombi E, Zhu YG (2014) Imaging element distribution and speciation in plant cells. Trends Plant Sci 19: 183-192Pubmed: Author and Title www.plantphysiol.orgon March 24, 2020 - Published by Downloaded from

Copyright © 2019 American Society of Plant Biologists. All rights reserved.

https://www.ncbi.nlm.nih.gov/pubmed?term=Naseer S%2C Lee Y%2C Lapierre C%2C Franke R%2C Nawrath C%2C Geldner N %282012%29 Casparian strip diffusion barrier in Arabidopsis is made of a lignin polymer without suberin%2E Proc Natl Acad Sci USA&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Naseer&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Casparian strip diffusion barrier in Arabidopsis is made of a lignin polymer without suberin&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Casparian strip diffusion barrier in Arabidopsis is made of a lignin polymer without suberin&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Naseer&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Olsen LI%2C Hansen TH%2C Larue C%2C Osterberg JT%2C Hoffmann RD%2C Liesche J%2C Kramer U%2C Surble S%2C Cadarsi S%2C Samson VA%2C Grolimund D%2C Husted S%2C Palmgren M %282016%29 Mother%2Dplant%2Dmediated pumping of zinc into the developing seed%2E Nat Plants&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Olsen&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Mother-plant-mediated pumping of zinc into the developing seed.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Mother-plant-mediated pumping of zinc into the developing seed.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Olsen&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Persson DP%2C Chen A%2C Aarts MG%2C Salt DE%2C Schjoerring JK%2C Husted S %282016%29 Multi%2DElement Bioimaging of Arabidopsis thaliana Roots%2E Plant Physiol&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Persson&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Multi-Element Bioimaging of Arabidopsis thaliana Roots&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Multi-Element Bioimaging of Arabidopsis thaliana Roots&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Persson&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Pfister A%2C Barberon M%2C Alassimone J%2C Kalmbach L%2C Lee Y%2C Vermeer JE%2C Yamazaki M%2C Li G%2C Maurel C%2C Takano J%2C Kamiya T%2C Salt DE%2C Roppolo D%2C Geldner N %282014%29 A receptor%2Dlike kinase mutant with absent endodermal diffusion barrier displays selective nutrient homeostasis defects%2E Elife 3%3A e&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Pfister&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=A receptor-like kinase mutant with absent endodermal diffusion barrier displays selective nutrient homeostasis defects.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A receptor-like kinase mutant with absent endodermal diffusion barrier displays selective nutrient homeostasis defects.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pfister&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Pozebon D%2C Scheffler GL%2C Dressler VL %282017%29 Recent applications of laser ablation inductively coupled plasma mass spectrometry %28LA%2DICP%2DMS%29 for biological sample analysis%3A a follow%2Dup review%2E Journal of Analytical Atomic Spectrometry&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Pozebon&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Recent applications of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for biological sample analysis: a follow-up review&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Recent applications of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for biological sample analysis: a follow-up review&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pozebon&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Robards AW%2C Jackson SM%2C Clarkson DT%2C Sanderson J %281973%29 Structure of Barley Roots in Relation to Transport of Ions into Stele%2E Protoplasma&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Robards&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Structure of Barley Roots in Relation to Transport of Ions into Stele&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Structure of Barley Roots in Relation to Transport of Ions into Stele&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Robards&as_ylo=1973&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Roppolo D%2C De Rybel B%2C Denervaud Tendon V%2C Pfister A%2C Alassimone J%2C Vermeer JE%2C Yamazaki M%2C Stierhof YD%2C Beeckman T%2C Geldner N %282011%29 A novel protein family mediates Casparian strip formation in the endodermis%2E Nature&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Roppolo&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=A novel protein family mediates Casparian strip formation in the endodermis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A novel protein family mediates Casparian strip formation in the endodermis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Roppolo&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Schmidt SB%2C Persson DP%2C Powikrowska M%2C Frydenvang J%2C Schjoerring JK%2C Jensen PE%2C Husted S %282015%29 Metal Binding in Photosystem II Super%2D and Subcomplexes from Barley Thylakoids%2E Plant Physiol&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Schmidt&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Metal Binding in Photosystem II Super- and Subcomplexes from Barley Thylakoids&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Metal Binding in Photosystem II Super- and Subcomplexes from Barley Thylakoids&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Schmidt&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Schmidt SB%2C Powikrowska M%2C Krogholm KS%2C Naumann%2DBusch B%2C Schjoerring JK%2C Husted S%2C Jensen PE%2C Pedas PR %282016%29 Photosystem II Functionality in Barley Responds Dynamically to Changes in Leaf Manganese Status%2E Front Plant Sci&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Schmidt&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Photosystem II Functionality in Barley Responds Dynamically to Changes in Leaf Manganese Status.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Photosystem II Functionality in Barley Responds Dynamically to Changes in Leaf Manganese Status.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Schmidt&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Schreiber L %282010%29 Transport barriers made of cutin%2C suberin and associated waxes%2E Trends Plant Sci&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Schreiber&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Transport barriers made of cutin, suberin and associated waxes&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Transport barriers made of cutin, suberin and associated waxes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Schreiber&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Tylova E%2C Peckova E%2C Blascheova Z%2C Soukup A %282017%29 Casparian bands and suberin lamellae in exodermis of lateral roots%3A an important trait of roots system response to abiotic stress factors%2E Ann Bot&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Tylova&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Casparian bands and suberin lamellae in exodermis of lateral roots: an important trait of roots system response to abiotic stress factors&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Casparian bands and suberin lamellae in exodermis of lateral roots: an important trait of roots system response to abiotic stress factors&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Tylova&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Vert G%2C Grotz N%2C Dedaldechamp F%2C Gaymard F%2C Guerinot ML%2C Briat JF%2C Curie C %282002%29 IRT1%2C an Arabidopsis transporter essential for iron uptake from the soil and for plant growth%2E Plant Cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Vert&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Vert&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=White PJ %281997%29 The regulation of K%2B influx into roots of rye %28Secale cereale L%2E%29 seedlings by negative feedback via the K%2B flux from shoot to root in the phloem%2E Journal of Experimental Botany&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=White&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The regulation of K+ influx into roots of rye (Secale cereale L&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The regulation of K+ influx into roots of rye (Secale cereale L&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=White&as_ylo=1997&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wu D%2C Yamaji N%2C Yamane M%2C Kashino%2DFujii M%2C Sato K%2C Feng Ma J %282016%29 The HvNramp5 Transporter Mediates Uptake of Cadmium and Manganese%2C But Not Iron%2E Plant Physiol&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wu&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The HvNramp5 Transporter Mediates Uptake of Cadmium and Manganese, But Not Iron&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The HvNramp5 Transporter Mediates Uptake of Cadmium and Manganese, But Not Iron&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wu&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zeier J%2C Ruel K%2C Ryser U%2C Schreiber L %281999%29 Chemical analysis and immunolocalisation of lignin and suberin in endodermal and hypodermal%2Frhizodermal cell walls of developing maize %28Zea mays L%2E%29 primary roots%2E Planta&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zeier&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Chemical analysis and immunolocalisation of lignin and suberin in endodermal and hypodermal/rhizodermal cell walls of developing maize (Zea mays L&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Chemical analysis and immunolocalisation of lignin and suberin in endodermal and hypodermal/rhizodermal cell walls of developing maize (Zea mays L&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zeier&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhao FJ%2C Moore KL%2C Lombi E%2C Zhu YG %282014%29 Imaging element distribution and speciation in plant cells%2E Trends Plant Sci&dopt=abstract


Google Scholar: Author Only Title Only Author and Title


https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhao&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Imaging element distribution and speciation in plant cells&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Imaging element distribution and speciation in plant cells&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhao&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1


Documents

The intensity of manganese deficiency strongly affects ... · 25 DFF-7017-00082 to SH and DAP), and the ERA-NET Coordinating Action in Plant Sciences 26 (ERA-CAPS) project ERACAPS13.089_RootBarriers,