Uclacyanin proteins are required for lignified nanodomain ......2020/05/01 · 67 ated with...

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Uclacyanin proteins are required for lignified nanodomain formation within Casparian 1

strips. 2

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Guilhem Reyta, Zhenfei Chaob, Paulina Flisa, Gabriel Castrilloa, Dai-Yin Chaob, David E. 4

Salta,1 5

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a Future Food Beacon of Excellence & School of Biosciences, University of Nottingham, 7

Nottingham, LE12 5RD, UK 8

b National Key Laboratory of Plant Molecular Genetics, Chinese Academy of Sciences, Cen-9

ter for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, 10

Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China 11

1 Corresponding author: [email protected] 12

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Abstract 14

Casparian strips (CS) are cell wall modifications of vascular plants restricting extracellular 15

free diffusion into and out the vascular system. This barrier plays a critical role in controlling 16

the acquisition of nutrients and water necessary for normal plant development. CS are formed 17

by the precise deposition of a band of lignin approximately 2 µm wide and 150 nm thick 18

spanning the apoplastic space between adjacent endodermal cells. Here, we identified a cop-19

per-containing protein, Uclacyanin1 (UCC1) that is sub-compartmentalised within the CS. 20

UCC1 forms a central CS nanodomain in comparison with other CS-located proteins that are 21

found to be mainly accumulated at the periphery of the CS. We found that loss-of-function of 22

two uclacyanins (UCC1 and UCC2) reduces lignification specifically in this central CS 23

nanodomain, revealing a nano-compartmentalised machinery for lignin polymerisation. This 24

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted May 3, 2020. ; https://doi.org/10.1101/2020.05.01.071738doi: bioRxiv preprint

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lack of lignification leads to increased endodermal permeability, and consequently to a loss 25

of mineral nutrient homeostasis. 26

27

Introduction 28

Plant roots perform the critical function of controlling the uptake of water and mineral nutri-29

ents from the soil essential for plant growth and development. A specialized cell layer in the 30

root called the endodermis plays a key role in the selective uptake of mineral nutrients into 31

the stele for translocation to the shoot [1-4]. Of vital importance to this function are Caspar-32

ian strips (CS), which are belt-like lignin structures surrounding each endodermal cell, that 33

interlock to form a barrier to diffusion in the apoplast [5,6]. This barrier is thought to enable 34

the endodermis to exert control over uptake of water and solutes from the environment into 35

the plant and perhaps also to control biotic interactions [7,8]. 36

The precise deposition of lignin for CS formation requires a signalling pathway involving the 37

kinases SGN1 and SGN3 controlling the spatial production of reactive oxygen species 38

through the activation of NADPH oxidases [2,9-12]. Lignin polymerisation requires the lo-39

calised action of a peroxidase (PER64) [9,13], and a dirigent-like protein (ESB1) [1]. This bi-40

osynthetic machinery is likely placed at the CS deposition site by association with CASPAR-41

IAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) [14]. The CASPs form a highly 42

scaffolded transmembrane domain guiding where the Casparian strip forms. Furthermore, the 43

receptor like kinase SGN3 acts as a sensor of CS integrity by inducing over-lignification of 44

the endodermal cells when the CS is defective [2,15]. 45

A network of transcriptional factors involving SHR, SCR and MYB36 controls endodermal 46

differentiation [16,17]. The MYB36 transcription factor controls the expression of most of 47

the described genes associated with CS formation, including CASPs, ESB1 and PER64 48


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[3,18]. Characterisation of other MYB36-regulated genes could thus lead to the identification 49

of new actors involved in CS formation. 50

Here, we identified a copper-containing protein, Uclacyanin1 (UCC1) among the MYB36-51

regulated genes. UCC1 reveals a nano-compartmentalisation of the machinery required for 52

lignin polymerisation at the CS, where UCC1 occupies a central CS nanodomain in compari-53

son with other CS-located proteins. The loss of function of two uclacyanins (UCC1 and 54

UCC2) leads to an atypical CS formation, where a lack of lignification is observed in this 55

central CS nanodomain. This defect in lignification leads to increased endodermal permeabil-56

ity, and consequently to a loss of mineral nutrient homeostasis. 57

58

Results and discussion 59

Among the genes downregulated in a myb36 loss-of-function mutant [3,18], we identified a 60

Uclacyanin1 gene (UCC1), that belongs to the copper-containing phytocyanins family [19] 61

(Figure 1A). This family is divided in three sub-families according to their copper binding 62

amino acid: the uclacyanins, stellacyanins and plantacyanins. The functional role of these 63

proteins remains unknown. However, biophysical and structural data of several phytocyanins 64

suggest their implication in redox reactions with small molecular weight compounds [19-24]. 65

The expression of several members of this family have been previously shown to be associ-66

ated with lignified tissues [25-27]. We looked at the endodermal spatiotemporal expression 67

pattern of the different members of this family (Figure 1A). We found that UCC1, UCC2 and 68

to a less extent UCC8 and STC1 are expressed in the endodermis similarly to that observed 69

for CASP1 and ESB1. Due to the lack of T-DNA insertional mutants in the UCC1 gene, we 70

generated two loss-of-function mutants, ucc1.1 and ucc1.2, using CRISPR/Cas9 technology 71

(Figure 1B). These mutants bear a single base deletion (ucc1.1) and an insertion (ucc1.2), 72


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leading to shifts in the reading frame in both. The two ucc1 alleles show a slight although sig-73

nificant delay in formation of an apoplastic barrier, as visualized by the uptake of the apo-74

plastic tracer propidium iodide (PI) after 10 min of staining (Figure 1C). The T-DNA mutant 75

ucc2.1 did not show an increase of PI permeability. Notably, in a double mutant ucc1.2 76

ucc2.1, a strong delay of PI permeability is observed similarly to that found in other CS mu-77

tants such as esb1 and casp1 casp3 (Supplementary Figure 1A). However, the number of 78

cells permeable to PI in ucc1.2 ucc2.1 is highly variable in comparison with casp1 casp3 and 79

esb1. When the incubation time with PI is increased to 20 min, ucc1.2 ucc2.1 displays an en-80

hanced permeability in comparison with esb1. Taken together, these results highlight the re-81

dundant role of UCC1 and UCC2 for establishing a functional apoplastic barrier. No additive 82

effect was observed in the triple mutant ucc1.1 casp1 casp3 or in the doubles ucc1.2 esb1 and 83

ucc1.2 sgn3 in comparison with casp1 casp3, esb1 and sgn3 respectively (Supplementary 84

Figure 1A). The loss of CS integrity in esb1, myb36 and casp1 casp3 is accompanied by an 85

increased deposition of suberin [1-3] as a compensatory mechanism under the control of 86

SGN3 [15]. We analysed the pattern of suberin deposition using fluorol yellow 088 staining 87

in the ucc mutants (Figure 1D). Deposition of suberin lamellas did not increase in the single 88

mutants ucc1.1, ucc1.2 and ucc2.1. However, the double mutant ucc1.2 ucc2.1 induces an in-89

crease in endodermal suberization compared with wild type plants (WT). This increase is not 90

as strong as that observed in esb1 or casp1 casp3 (Supplementary Figure 1B). The combina-91

tion of ucc1 mutations in esb1 and casp1 casp3 do not affect the enhanced suberization of 92

esb1 or casp1 casp3. 93

Disruption of CS is known to affect the composition of the leaf ionome as observed in esb1, 94

myb36, casp1 casp3, sgn3 and lotr1 [1-3,28]. In order to determine the contribution of UCC1 95

and UCC2 in maintaining mineral nutrient homeostasis, we analysed the leaf ionome in dif-96


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ferent mutant combinations (Figure1E, Supplementary figure 1C, Supplementary table 1) us-97

ing inductively coupled plasma mass spectrometry (ICP-MS). The ucc2.1 mutant only exhib-98

its minor ionomic changes and is similar to WT in a principal component analysis (PCA; 99

Supplementary figure 1C). The single ucc1 and the double ucc1.2 ucc2.1 mutants present a 100

larger number of elemental changes in the leaf ionome in comparison with ucc2.1. Moreover, 101

the leaf ionome of ucc1.2 ucc2.1 shows similarities to the esb1 ionome in a hierarchical clus-102

tering analysis and in a PCA. Adding the ucc1.2 mutation into a sgn3 mutant background 103

does not strongly affect the leaf ionome in comparison with sgn3. The same effect but to a 104

lesser extent is observed for ucc1.2 esb1 in comparison with esb1 (Supplementary figure 1C). 105

The higher endodermal permeability and the atypical ionomic profile observed (Figure 1 and 106

Supplementary figure 1) strongly suggest that UCC play an important role in CS formation. 107

To identify the cell type in which the UCC1 promoter is activated, we fused the UCC1 pro-108

moter to GFP (Supplementary figure 2A). GFP was accumulated only in the endodermis, 109

from the elongation zone and further up into the zone of differentiated endodermal cells. Ac-110

cording to the literature, the phytocyanin proteins are predicted to be located on the cell sur-111

face and anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor 112

[29]. Several members of this family, including UCC2, have been shown to be GPI-anchored 113

using a proteomic approach [29]. In order to determine the precise UCC1 localization, we 114

generated a line (pUCC1::mCherry-UCC1) expressing a tagged UCC1 (Figure 2A). The 115

mCherry-UCC1 fusion accumulates at the endodermal cell junctions where the CS is located 116

(Figure 2B). It first appears in a discontinuous manner in the early stage of endodermal dif-117

ferentiation, and then forms a more continuous band later in the endodermis development. 118

Highly mobile vesicles associated with mCherry-UCC1 are also seen in the endodermis (Fig-119

ure 2B, Supplementary video 1). 120


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When we analysed the localization of mCherry-UCC1 in plants expressing CASP1-GFP, we 121

observed a similar pattern of localization at the CS as seen in the maximum projection view 122

of endodermal cells in Figure 2C. However, at higher magnification, in both the median and 123

surface views we observed mCherry-UCC1 to occupy a more central position in comparison 124

with CASP1-GFP. This was further confirmed using super-resolution structure illumination 125

microscopy (Supplementary figure 2B). Moreover, CASP1-GFP does not form a homogene-126

ous domain, but is found to accumulate more at the periphery of the CS and less in the centre 127

where UCC1 is observed. Subsequently, we checked if mCherry-UCC1 colocalises with lig-128

nin deposition in the CS (Supplementary figure 2C). At the early stage of endodermal differ-129

entiation, lignin presents a nearly perfect colocalization with mCherry-UCC1. However, the 130

line expressing mCherry-UCC1 also presents ectopic lignification later in development. This 131

ectopic lignification is similar to that observed in other CS mutants such as casp1 casp3 and 132

esb1 [1,14]. Expression of mCherry-UCC1 also delayed the formation of a functional barrier 133

to PI and promotes a significant increase of suberin deposition (Supplementary figure 2D-F). 134

These observations taken together show that expressing UCC1 with a fluorescent tag causes 135

disruption of the CS. 136

To establish if the expression of mCherry-UCC1 can affect the pattern of accumulation of 137

CASP1-GFP presented in Figure 2C and Supplementary figure 2B, we examined plants ex-138

pressing CASP1-GFP only (Figure 2D). CASP1-GFP is still more accumulated at the periph-139

ery, and this was also observed for another member of the CS machinery ESB1-mCherry. Im-140

munolocalization confirmed that native UCC1 protein is specifically localized to the CS (Fig-141

ure 2E), and its localization was strongly reduced in ucc1.2 and ucc1.2 ucc2.1 mutants, and 142

abolished in a myb36 mutant (Supplementary figure 2G). Immunolocalization confirmed our 143

previous observations of a central localization of UCC1 in the CS as compared to CASP1-144

GFP (Figure 2E). Quantification of pixel intensity across the CS reveals that UCC1 is highly 145


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accumulated in the middle of the CS where CASP1-GFP is slightly less accumulated com-146

pared to the periphery of the CS domain (Figure 2F). The CASP1 domain of the plasma 147

membrane is defined as a microdomain [30] as its width is around 1.5µm. UCC1 with a more 148

central accumulation has a width equal or slightly smaller than 1µm, UCC1 can then be clas-149

sified as a nanodomain of the CS. This subdomain structure has not been previously reported. 150

However, previous studies for CASP1, ESB1 and Peroxidase64 (PER64) [14,31] do appear to 151

show an enrichment of these proteins at the periphery of the CS. This means that the level of 152

organisation observed at the plasma membrane for CASP1 is conserved for cell wall proteins 153

such as ESB1 and PER64. To date, UCC1 is the only protein found in the central 154

nanodomain of the CS, revealing a new level of internal structure within the CS. 155

To characterise the formation of the UCC1 and CASP1 subdomains, we tracked endodermal 156

differentiation cell-by-cell. CASP1-GFP and mCherry-UCC1 are found to be accumulated in 157

the endodermis concomitantly between 4 and 6 cells after the onset of elongation, at the pe-158

riphery of the cells and not yet at the CS (Figure 3 A-B) which is consistent with a common 159

transcriptional regulation by MYB36. We then tracked the central accumulation of CASP1-160

GFP and mCherry-UCC1 at the CS. We observed that the localization of CASP1-GFP and 161

mCherry-UCC1 at the CS mainly occurs at the eighth endodermal cell after the onset of elon-162

gation (Figure 3B). Although, mCherry-UCC1, but not CASP1-GFP, was observed to be cen-163

trally localised in several independent events at the seventh cell after the onset of elongation. 164

This suggests that CASP1 localization at the CS is not required for the recruitment of UCC1. 165

This was further confirmed by the targeting of UCC1 at the CS in a casp1 casp3 mutant (Fig-166

ure 3C and Supplementary figure 3). However, UCC1 is not able to form a continuous do-167

main in casp1 casp3 as in WT. This was also observed in the esb1, sgn3 and esb1 sgn3 mu-168

tants. We then tested the reciprocity to know if UCC1 and UCC2 are required for CASP1-169

GFP localization (Figure 3D). In the ucc1.1 and ucc1.1 ucc2.1 mutants, CASP1-GFP is able 170


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to localise at the CS domain and form a continuous domain without disruption. This demon-171

strate that UCC1 and UCC2 are not required for CASP1 localization to the CS domain. How-172

ever, the absence of UCC1 and UCC2 does affect CASP1-GFP localization at a nanoscale 173

resolution (Figure 3E). The exclusion of CASP1-GFP from the central nanodomain of the CS 174

is reduced in the ucc1 mutants and tends to disappear in the double ucc1.1 ucc2.1 mutant. 175

This indicates a role for UCC1 and UCC2 in the formation of the central CS nanodomain. 176

Knowing that UCC1 localizes at the CS (Figure 2C-F) and mutations in UCC1 and UCC2 177

cause a strong defect in root apoplastic permeability (Figure 1C, Supplementary figure 1A), 178

we looked at lignin deposition at the CS in ucc mutants (Supplementary figure 4A). Muta-179

tions in UCC1 and/or UCC2 do not cause an obvious disruption in the CS as observed in 180

casp1 casp3, esb1, sgn3 (Supplementary figure 4A), and other previously identified CS mu-181

tants, where clear gaps can be observed [1,2,7,10,12-14,28,32]. Surprisingly thought, muta-182

tions in UCC1 do reduce the amount of lignin in the central nanodomain of the CS, as ob-183

served using confocal imaging (Figure 4A), and super-resolution structure illumination mi-184

croscopy (Supplementary figure 4B). Pixel quantification across the CS reveals that ucc1 mu-185

tants show a lower lignification in the central nanodomain (Figure 4B) where UCC1 accumu-186

lates (Figure 2C-F). This decrease is not observed in ucc2.1. However, a further decrease is 187

observed in the double mutant ucc1.2 ucc2.1 in comparison with ucc1 mutants. This is con-188

sistent with the increased root permeability observed in ucc1.2 ucc2.1 in comparison with the 189

single mutants (Figure 1C, Supplementary figure 1A). This reveals that CS permeability can 190

be strongly affected in the absence of clear gaps in the CS. Furthermore, we observed ectopic 191

lignification on the cortical side of the endodermis on a few occasions in the ucc1 mutants, 192

and at a higher frequency in the double mutant ucc1.2 ucc2.1 (Figure 4A-B, Supplementary 193

figure 4). This is a typical phenotype, along with increased suberin deposition, that is due to 194

the SGN3-dependent compensatory mechanism observed in most mutants with defective CS 195


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[1,2,14,18,33]. However, the ectopic lignification and enhanced suberization are observed to 196

a lesser degree in ucc1.2 ucc2.1 in comparison with other CS mutants such as casp1 casp3 197

and esb1 mutants (Figure 1D and 4, Supplementary figure 1B and 4). This could be explained 198

by a more conditional leakiness of the CS in ucc1 ucc2 mutants. Discontinuities below the 199

resolution of light microscopy could occur in ucc1 ucc2 mutants. This could lead to a full 200

permeability for low molecular weight compounds such as ions and PI but an intermediate 201

permeability for high molecular weight compounds such as the CIF peptides required for ec-202

topic lignification and enhanced suberization [10,32]. 203

In conclusion, this study reveals the first loss-of-function phenotype for members of the 204

plant-specific blue copper protein family of phytocyanins. Several studies suggested their im-205

plication in lignin polymerisation [25-27], but this has never previously been shown. Our 206

analysis indicates a role for the Uclacyanins in the deposition of lignin in a newly discovered 207

nanoscale domain within the CS. Further, the subcellular localization of UCC1, and the phe-208

notype of the ucc(s) mutants, reveals a sub-compartmentalisation of the machinery required 209

for lignin polymerisation at the CS. 210

211

Author contributions 212

Conceptualization, G.R., G.C. and D.E.S.; Formal Analysis, G.R.; Investigation, G.R., Z.C., 213 P.F. and G.C.; Methodology G.R. and Z.C.; Writing – Original Draft Preparation, G.R.; Writ-214 ing – Review & Editing G.R., Z.C., P.F., G.C., D-Y.C. and D.E.S.; Supervision, D.E.S. 215

216

Competing interests 217

The authors declare no competing interests. 218

219

220


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Figures legends 221 222 Figure 1. Uclacyanins UCC1 and UCC2 are required for a functional Casparian strip. 223 A. (left) Figure shows a phylogenetic analysis of phytocyanins protein family in A. thaliana. 224 The tree was built using the full-length amino acid sequences for all proteins. Different col-225 ours represent the three phytocyanins subfamilies: uclacyanins, stellacyanins and plantacya-226 nins (39). In the tree, branch lengths are proportional to the number of substitutions per site. 227 AT3G17675 have been previously annotated as a stellacyanin (STC4), however the signal 228 peptide for the secretion pathway and the hydrophobic extension for Glycosylphosphatidylin-229 ositol (GPI) anchoring are missing. (right) Heatmap showing the endodermal expression of 230 the phytocyanins family in A. thaliana across the different root zones (Meristematic, Elonga-231 tion, Maturation). For the analysis, expression data was collected from the Bio-Analytic Re-232 source database, AtGenExpress Consortium. The expression of two endodermal localised 233 proteins, CASP1 and ESB1, were added to the analysis as a reference. Asterisks indicate a 234 significant downregulation in a myb36 mutant according to [18]. B. Schematic representation 235 of the UCC1 and UCC2 proteins showing the different protein domains and the types of mu-236 tations. Domains were defined according to [19]. C. Boxplot analysis showing the number of 237 the cells from the onset of elongation permeable to propidium iodide in wild type plants 238 (WT), ucc1 mutants (ucc1.1 and ucc1.2), ucc2 mutant (ucc2.1), and the double mutant ucc1.2 239 ucc2.1. Data were collected from two independent experiments (n ≥ 29). Different letters rep-240 resent significant differences between genotypes in a Mann-Whitney test (p < 0.01). D. Dia-241 gram shows the quantification analysis of the endodermal suberization in the plant genotypes 242 used in C. Each colour in the graph represents the percentage of the root length (percentage 243 of root length (%)) that is unsuberised (white), discontinuously suberised (yellow), continu-244 ously suberised (orange). Suberin was staining with Fluorol yellow 088. (n ≥ 18). Error bars 245 in the figure are the standard deviation (SD). Different letters represent significant differences 246 between genotypes using a Mann-Whitney test (p < 0.01). E. Heatmap representing the io-247 nomic profiles (z-scores) of wild type plants (WT), and a collection of mutants with a defec-248 tive casparian strip: ucc2.1, ucc1.1, ucc1.2, ucc1.2 ucc2.1, esb1, ucc1.2 esb1, sgn3 and ucc1.2 249 sgn3 grown in full nutrient conditions on agar plate for 2 weeks (n=10). Elements concentra-250 tion were determined by ICP-MS and the raw data is available in the supplementary Table 1. 251 Asterisks indicate a significant difference in comparison with WT using a t-test (p<0.01). 252 Columns (Genotypes) were subjected to hierarchical clustering analysis. 253


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Figure 2. UCC1 defines a new central sub-domain in the Casparian strip. 254 A. Diagram representing the construct pUCC1::mCherry-UCC1 (UTR: Untranslated region, 255 SP: Signal peptide, H Cter: Hydrophobic C-terminus for GPI anchoring). B. Maximum inten-256 sity projection, orthogonal, median and surface views of confocal sections of plants express-257 ing pUCC1::mCherry-UCC1 (red) in cleared roots. In the case of maximum intensity projec-258 tion (Maximum projection) figure represents different regions of the root measured as num-259 ber of cells after the onset of elongation. White arrow heads point to vesicles containing 260 mCherry-UCC1. For the orthogonal, median and surface views, cell walls were stained with 261 Calcofluor white (grey in the figures). Scale bar = 20 µm for the maximum projection and or-262 thogonal views. Scale bar = 5 µm for the median and surface views (Ep: epidermis, Co: cor-263 tex, End: endodermis). C. Maximum intensity projection, median and surface view of confo-264 cal sections of plants expressing CASP1-GFP (cyan) and mCherry-UCC1 (red). Signal was 265 capture at the 10thendodermal cell after the onset of elongation observed in vivo. Scale bar = 266 20 µm for maximum projection and 3 µm for median and surface view. D. In vivo observa-267 tion of the surface view of an endodermal cell expressing pESB1::ESB1-268 mCherry or pCASP1::CASP1-GFP. Scale bar = 2 µm. E. Immunolocalization assay of UCC1 269 protein (red) in plant expressing pCASP1::CASP1-GFP (cyan). A primary polyclonal anti-270 body targeting UCC1 was used in combination with a secondary antibody conjugated with 271 Dylight 633. Scale bar = 2 µm. F. Graph presenting the distribution of normalised pixels in-272 tensity (Relative Pixel Intensity, 0 to 1) across the Casparian strip (Distance µm) for CASP1-273 GFP fluorescence (cyan) and UCC1 immunofluorescence (red, Dylight 633). Light curves 274 represent individual replicates coming from individual plants (n = 4). Each replicate is the av-275 erage pixel intensity across a segment of 25 µm along the Casparian strip axis. Dark curves 276 represent the mean values for CASP1-GFP and UCC1 immunofluorescence. 277


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Figure 3. Relations between UCC1 positioning and other components of the Casparian 278 strips machinery. 279

A. Analysis of the spatial distribution of CASP1 and UCC1 at the endodermal cell junctions. 280 Images were generated from the same plant co-expressing CASP1-GFP and mCherry-UCC1 281 using confocal microscopy. The numbers at the bottom of the figure indicate the number of 282 cells after the onset of elongation. White arrows indicate the central accumulation for 283 CASP1-GFP or mCherry-UCC1. Scale bar = 6 µm. B. Histograms showing the frequency 284 distribution (Frequency (%)) of the onset of expression (upper plot, n = 18) and the onset of 285 localization at the Casparian strip of CASP1-GFP and mCherry-UCC1 (lower plot, n = 28). 286 C. (left panel) Maximum intensity projection and (right panel) surface view of UCC1 immu-287 nolocalization (red) at 10 cells after the onset of elongation in wild type plants (WT) and a 288 collection of Casparian strips mutants: casp1 casp3, esb1, sgn3, esb1 sgn3. White arrows 289 show gaps in the UCC1 localization. Scale bar = 20 µm for the maximum projections and 2 290 µm the surface views. D. Maximum intensity projection of CASP1-GFP localization in 291 cleared root of wild type plants (WT), and the mutants ucc1.1 and ucc1.1 ucc2.1. Scale bar = 292 20 µm. E. Surface view of the localization of CASP1-GFP in cleared root of wild type plants 293 (WT), and the mutants: ucc1.1, ucc1.2 and ucc1.1 ucc2.1. Scale bar = 2 µm. F. Quantification 294 of normalised pixels intensity (Relative Pixel Intensity; 0 to 1) across the Casparian strip in 295 plants expressing CASP1-GFP. The plots are showing the intensity profile for individual rep-296 licates (n ≥ 10), the mean value (black line) and the 95% confidence interval (grey interval). 297 Each replicate corresponds to the quantification of one picture containing a Casparian strip 298 segment of approximately 25µm long. The pictures were generated at the 15th cells after the 299 onset of elongation from at least 8 individual plants per genotype. Intensity profiles across the 300 Casparian strip were always measured in the same orientation, from the cortical side toward 301 the pericycle side of the endodermis. Letters indicate statistically significant differences be-302 tween genotypes for the intensity values comprised between the dashed lines using an 303 ANOVA and Tukey’s test as post hoc analysis (p<0.01). 304


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Figure 4. UCC1 and UCC2 are necessary for the central lignification of the Casparian 305 strip. 306 A. Surface view of the Casparian strip lignin stained with Basic fuchsin in wild type plants 307 (WT), and the mutants ucc2.1, ucc1.1, ucc1.2 and ucc1.2 ucc2.1. Whites arrows show lack of 308 lignification in the central domain of the Casparian strip across the different genotypes. Scale 309 bar = 2 µm. B. Quantification of normalised pixels intensity (0 to 1) (Relative pixel intensity) 310 across the Casparian strip using surface views as shown in A. The plots show the intensity 311 profile for individual plants (n ≥ 13). In the figure, the mean value is represented by a black 312 line and the 95% confidence interval is in grey. The data were generated using individual pic-313 tures containing a Casparian strip segment of approximately 25 µm long. The pictures were 314 taken at the 15th cells after the onset of elongation and the intensity profiles were measured in 315 the same direction, from the cortex side toward the pericycle side of the endodermis. Letters 316 indicate statistically significant differences between genotypes using an ANOVA and 317 Tukey’s test as post hoc analysis (p<0.01). 318


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Supplementary figure 1. Uclacyanins UCC1 and UCC2 are required for the correct 319 function of the Casparian strips. 320 A. Boxplot showing the number of cells, from the onset of elongation, permeable to propid-321 ium iodide (PI) after 10 min or 20 min of root staining in wild type plants (WT) and a collec-322 tion of mutants (ucc1.1, ucc1.2, ucc2.1, ucc1.2 ucc2.1, casp1 casp3, esb1, sgn3, ucc1.1 casp1 323 casp3, ucc1.2 esb1, ucc1.2 sgn3). The number of plants analysed was n ≥ 16 and n ≥ 10 for 324 the 10 min and 20 min of PI exposition, respectively. Different letters represent significant 325 differences between genotypes using a Mann-Whitney test (p < 0.01). B. Quantification of 326 the endodermal suberization using the suberin specific staining Fluorol yellow 088 in the 327 genotypes from A. The results (Percentage of root length (%)) represent the percentage of the 328 root that remains unsuberised (white), discontinuously suberised (yellow) or continuously su-329 berised (orange). In all cases the number of plants analysed was n ≥ 15, error bars mean 330 standards deviation (SD). Different letters represent significant differences between geno-331 types using a Mann-Whitney test (p < 0.01). C. Principal component analysis (PCA) of the 332 shoot ionomic profiles of wild type plants (WT) and the defective CS mutants ucc2.1, ucc1.1, 333 ucc1.2, ucc1.1 ucc2.1, ucc1.2 ucc2.1, esb1, ucc1.1 esb1, ucc1.2 esb1, sgn3, ucc1.2 sgn3 and 334 ucc1.1 sgn3 grown in agar plates. The PCA was performed using the average of the elemental 335 profiles from each genotype (n=10) available in the Supplementary Table 1. 336


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Supplementary figure 2. UCC1 accumulates in the root endodermis and defines a cen-337 tral sub-domain at the Casparian strip. 338 A. (Top) Schematic representation of the construct used to study the expression of UCC1 in 339 the root. Approximately 3 Kb of the promoter region of the gene UCC1 (pUCC1 (3Kb)) was 340 used to drive the expression of theb-glucuronidase (GUS) and the green fluorescent protein 341 (GFP). (Bottom) Pictures showing the GFP accumulation in the root of plants expressing the 342 construct pUCC1::GUS-GFP. The fluorescence (GFP, green) and brightfield (grey) pictures 343 are merged. The different zones of the root (meristematic, elongation and differentiation) are 344 highlighted in the figure. Scale bars = 100 µm (left) and 20 µm (right). (Ep: Epidermis; Co: 345 Cortex; En: Endodermis). B. Structure illumination microscopy of a surface view of plants 346 expressing CASP1-GFP (cyan) and mCherry-UCC1 (red) at the 10th endodermal cell after the 347 onset of elongation in cleared root. Scale bar = 2 µm. C. Surface view of Casparian strip lig-348 nin stained with Auramine-O (cyan) in wild type plants (WT) and in a line expressing the 349 construct pUCC1::mCherry-UCC1 (red) in cleared roots at 10th and 20th cells after the onset 350 of elongation. In the figure the white arrow shows ectopic lignification. Scale bar = 351 2µm. D. Boxplot showing the number of the cells from the onset of elongation permeable to 352 propidium iodide in wild type plants (WT) and a line expressing pUCC1::mCherry-UCC1. 353 For both genotypes 10 plants were analysed. Asterisk indicates a significant difference with 354 WT using a Mann-Whitney test (p < 0.01). E. Brightfield and suberin staining using Fluorol 355 yellow 088 of roots in WT and a line expressing pUCC1::mCherry-UCC1. Scale bar = 2 356 mm. F. Quantification of suberin staining with Fluorol yellow 088 along the root. The results 357 are expressed in percentage of root length that is unsuberised (white), discontinuously suber-358 ised (yellow), and continuously suberised (orange). In all cases n ≥ 10 plants were used, error 359 bars: SD. Asterisks indicates significant differences in comparison with WT for the same 360 zone using a Mann-Whitney test (p < 0.01). G. The anti UCC1 antibody generated in this 361 work is functional and specifically recognises the UCC1 protein. Maximum intensity projec-362 tion of UCC1 immunolocalization (red) in wild type plants (WT), the mutants myb36, ucc1.2, 363 ucc1.2 ucc2.1 in the root at the 10th cell after the onset of elongation. As a control the primary 364 polyclonal antibody anti UCC1 was used or not (“No primary Ab”) in combination with a 365 secondary antibody conjugated with Dylight 633. Scale bar = 20 µm. 366 367 368 369 370 371


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Supplementary figure 3. Other Casparian strip molecular players are required for 372 UCC1 to fuse into a continuous band. 373 Surface view of mCherry-UCC1 at the 15th cell after the onset of elongation in cleared root 374 of WT, casp1 casp3, esb1 and sgn3. White arrows represent discontinuous mCherry-UCC1 375 localization. Scale bar = 2 µm. 376 377 378


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Supplementary figure 4. UCC1 and UCC2 are necessary for the central lignification of 379 the Casparian strip. 380 A. Figures show the maximum intensity projection of confocal sections of plant roots stained 381 with basic fuchsin for visualising lignin. Pictures were taken using cleared roots at the 10th 382 and 15th cells after the onset of elongation of wild type plants (WT), and a collections of mu-383 tants: ucc1.1, ucc1.2, ucc2.1, ucc1.2 ucc2.1, casp1 casp3, ucc1.1 casp1 casp3, esb1, ucc1.2 384 esb1, sgn3 and ucc1.2 sgn3. Spiral structures in the centre of the root are the xylem. Blue ar-385 rows indicate gaps in the lignin deposition at the Casparian strip. Green triangles show ec-386 topic lignification. Scale bar = 20 µm. 387 B. Structure illumination microscopy of a surface view of Casparian strip lignin stained with 388 basic fuchsine in cleared roots of WT, ucc1.2 and ucc1.2 ucc2.1. Blue arrows show lack of 389 lignification in the central domain of the Casparian strip. Scale bar = 2 µm. 390 391


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Supplementary table 1. UCC1 and UCC2 are required for maintaining ion homeostasis 392 in the shoot. 393 Elemental content in shoot of ucc2.1, ucc1.1, ucc1.2, ucc1. 1 ucc2.1, ucc1.2 ucc2.1, esb1, 394 ucc1.1 esb1, ucc1.2 esb1, sgn3, ucc1.2 sgn3 and ucc1.1 sgn3 mutants compared to WT grown 395 in agar plates (long day, n=10). Elements concentration were determined by ICP-MS. Data 396 are presented as mean ± standard deviation (SD). t-tests were performed to determine the sig-397 nificant differences to WT and the corresponding p-values are presented. 398 399 400


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Supplementary Video1. 401 Time-lapse video showing vesicles of mCherry-UCC1 in the endodermis of 402 pUCC1::mCherry-UCC1. 403 404 405


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Materials and methods 406 407

Plant material 408

Arabidopsis thaliana ecotype Columbia (Col_0) and the following mutants and transgenic 409

lines were used in this study: sgn3 (sgn3.3; SALK_043282[2]), myb36 (myb36.2; GK-410

543B11 [3]), casp1 casp3 (casp1.1 casp3.1, [14]), pCASP1::CASP1-GFP [14], 411

pESB1::ESB1-mCherry [1], ucc2.1 (GK_250F04). 412

The corresponding gene AGI are: UCC1, AT2G32300; UCC2, AT2G44790; 413

SGN3, At4g20140; MYB36, At5g57620; CASP1, At2g36100; CASP3, AT2G27370 414

ESB1, At2g28670. 415

416

Generation of transgenic lines and CRISPR/Cas9 mutants 417

The line pUCC1::mCherry-UCC1 was obtained using Gateway Cloning Technology (Invitro-418

gen). A genomic DNA fragment containing the UCC1 promoter (-3083bp before ATG), the 419

UCC1 5’UTR and the UCC1 signal peptide (+78bp after ATG) was amplified by PCR using 420

the following primers: F: GGGGACAACTTTGTATAGAAAAGTTGGTTGAATTTCG-421

TAAGAGTTAGG and R: GGGGACTGCTTTTTTGTACAAACTTGCATGGTCAG-422

TAGCTACTGTTAAACC); and cloned in a pDONR P4-P1R. A second fragment contain-423

ing the rest of the genomic UCC1 sequence (from +79 to +1081 from ATG) was amplified by 424

PCR with the primers: F: GGGGACAGCTTTCTTGTACAAAGTGGTAACCATT-425

GGTGGTCCTAGTGGTTGG and R: GGGGACAACTTTGTATAATAAAGTT-426

GACCCATATAAATTGTAATAATGTATTATAAAC and cloned in a pDONR P2R-P3. 427

Both fragments and a pEN-L1-mCherry-L2 vector were assembled in the expression vector 428

pB7m34GW,3 [34]. The construct pUCC1::GFP-GUS was generated using Gateway cloning 429

technology. The UCC1 promoter (-3083bp to -83bp before ATG) was amplified by PCR with 430


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these primers: F: GGGGACAAGTTTGTACAAAAAAGCAGGCTGTTGAATTTCGTAA-431

GAGTTAGG; Primer R : GGGGACCACTTTGTACAAGAAAGCTGGGTATGACAT-432

ATGGTGTCAAATGTGTG and cloned in pBGWFS7 [35]. Following Agrobacterium 433

strain GV3101 transformation with the resulting vector, transgenic plants were generated by 434

floral dipping [36]. 435

The ucc1.1 and ucc1.2 mutants were obtained using CRISPR/Cas9 according to [37]. Two 436

sgRNA targeting UCC1 coding sequence (GTCCTCGCTACTACACTCA and GGTCCTAG-437

TGGTTGGACTG) were inserted in to the pHEE401 vector following Agrobacterium strain 438

GV3101 transformation with the resulting vector, transgenic plants were generated by floral 439

dipping [36]. Two individual homozygous lines (ucc1.1; ucc1.2) were identified at the gener-440

ation T1. 441

442

Growth Conditions 443

All seeds were surface sterilised, and then stratified for two days at 4°C. Seeds were directly 444

germinated on plates containing ½ MS medium (Murashige and Skoog, Sigma) solidified 445

with 0.8% agar, pH 5.7, and grown in a vertical position in a growth chamber under long-day 446

conditions (16h light 22°C/8h dark 19°C, light intensity 100µE). Seedlings were analysed at 447

6-day-old for microscopy analysis, and 2-week-old for ionomic analysis. 448

449

Phylogeny 450

A BLASTP was performed using UCC1 amino acid sequence against the Araport11 protein 451

sequences dataset [38]. Only the matches with an Expect value (E-value) lower than 0.05 452

were considered. A first alignment was performed to select the protein sequence containing 453

the amino acid required for copper binding as described in [19]. Genes were named and clas-454


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sified as stellacyanin, uclacyanin and plantacyanin according to the amino acid binding cop-455

per as described in [39]. A second alignment and tree assembling were performed using Clus-456

tal Omega with amino acid sequence of the putative copper containing protein [40]. 457

458

Endodermal spatio-temporal expression pattern 459

The spatio-temporal endodermal expression pattern of the corresponding genes was checked 460

using the Bio-Analytic Resource database from the AtGenExpress Consortium [41]. 461

462

Ionome analysis 463

The shoot elemental content was measured using Inductively Coupled Plasma Mass Spec-464

trometry (ICP-MS) and the analysis was performed as described [42]. Briefly, shoots of 2-465

week-old plants grown on agar plates were harvested into Pyrex test tubes (16 x 100 mm) to 466

be then dried at 88 oC for 20h. After weighing the appropriate number of samples (these 467

weights were used to calculate the weights of rest of the sample), the trace metal grade nitric 468

acid Primar Plus (Fisher Chemicals) spiked with indium internal standard was added to the 469

tubes (1 mL per tube). The samples were then digested in a dry block heater (DigiPREP MS, 470

SCP Science; QMX Laboratories, Essex, UK) at 115˚C for 4 hours. The digested samples 471

were diluted to 10 mL with 18.2 MΩcm Milli-Q Direct water (Merck Millipore). Elemental 472

analysis was performed using an ICP-MS, PerkinElmer NexION 2000 equipped with Ele-473

mental Scientific Inc. autosampler, in the collision mode (He). Twenty-three elements (Li, B, 474

Na, Mg, P, S, K, Ca, Mn, Fe, Co, Ni, Cu, Zn, As, Rb, Sr, Mo, Cd) were monitored. Liquid 475

reference material composed of pooled samples was prepared before the beginning of sample 476

run and was used throughout the whole samples run. It was run after every ninth sample to 477

correct for variation within ICP-MS analysis run. The calibration standards (with indium in-478

ternal standard and blanks) were prepared from single element standards (Inorganic Ventures; 479


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Essex Scientific Laboratory Supplies Ltd, Essex, UK) solutions. Sample concentrations were 480

calculated using external calibration method within the instrument software. Further data pro-481

cessing was performed using Microsoft Excel spreadsheet. Principal component and heatmap 482

were generated using ClustVis [43]. 483

484

Histological staining of roots 485

Propidium iodide staining was performed as previously described [5]. Root clearing and 486

staining with Calcofluor White, Basic Fuchsin and Auramine-O was performed according to 487

[44]. Fluorol Yellow 088 staining of the suberin was performed as previously described 488

[4,6]. 489

490

UCC1 immunolocalization 491

Custom affinity-purified polyclonal anti-UCC1 antibodies were produced by Genscript, USA 492

and were used as a primary antibody. Anti-UCC1 antibodies were generated in rabbits 493

against a recombinant truncated UCC1 494

(TDHTIGGPSGWTVGASLRTWAAGQTFAVGDNLVFSYPAAFHD VVEVTKPEFDSCQAVKPLIT-495

FANGNSLVPLTTPGKRYFICGMPGHCSQGMKLEVNVVPTATVAPTA) produced in E. coli. 496

UCC1-immunolocalization was performed according to [45]. 6-day-old seedlings were vac-497

uum infiltrated and fixed with 2% formaldehyde in MTSB buffer (microtubule-stabilizing 498

buffer) supplemented with 0.1 % Triton for 1h. A stock solution of 2x MTSB was prepared 499

with: 15 g PIPES, 1.90 g EGTA, 1.22 g MgSO4·7H2O and 2.5 g KOH and dissolved in a total 500

volume of 500 mL water at pH 7.0 (adjusted with 10 M KOH). Seedlings were washed twice 501

in water that was then replaced by 100% methanol. Methanol content in the wash was gradu-502

ally decreased until its final concentration reached ~20 %. Seedlings were then washed twice 503

in water and incubated in a cell wall digestion solution (0.2% Driselase, 0.15% Driselase in 504


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2mM MES, pH 5.0) for 30 min at 37°C. After washing with MTSB buffer, the seedlings were 505

incubated in a solution containing 3% IGEPAL CA-630, 10% DMSO in MTSB buffer for 506

15min at 37°C in order to permeabilise the cell membranes. Seedling were then washed 4 507

times with MTSB buffer and blocked using a blocking solution containing 2% albumin frac-508

tion V BSA in MTSB buffer for 20 min at room temperature. The primary anti-UCC1 anti-509

body was diluted (1/500) in the blocking solution and added to the seedlings for 1h incuba-510

tion at37°C.Then seedlings were washed twice with MTSB buffer and incubated with the 511

secondary antibody goat anti-Rabbit IgG DyLight 633 (Invitrogen) in MTSB buffer for 1h at 512

37°C. After washing three times in MTSB buffer, the seedlings were mounted on micro-513

scopic slides in MTSB buffer for the microscopy analysis. 514

515

Microscopy 516

Laser scanning confocal microscopy was performed with a Zeiss LSM500 and a Leica SP8. 517

Structure illumination microscopy was performed with a Zeiss PS1 Super Resolution Micro-518

scope. The following excitation and emission detection settings were applied: Calcofluor 519

White, 405 nm/425-475 nm; GFP, 488 nm/500-550 nm; Fluorol yellow 088, 488 nm/500-550 520

nm; Auramine-O 488nm/505-530nm; propidium iodide, 561 nm/600-620 nm; Basic Fuchsin, 521

561 nm/570-650 nm; mCherry, 561 nm/ 570-620 nm; Dylight 633, 633 nm/640-670 nm. 522

523

Pixel quantification across the Casparian strip 524

Images of CS surface view were analysed for CASP1-GFP, lignin (Basic fuchsin), mCherry-525

UCC1 and DyLight 633 using Fiji [46]. A segmented line with a width of 3.8µm was traced 526

along the central part of the CS. This selection was then straighten using the “Straighten…” 527

function. A profile of pixels intensity along the y-axis was then generated from the pericycle 528

side toward the cortical side. Each replicate represents the average value of pixels intensity 529


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across 13 µm approximately and it was generated from one picture. The pixels intensity val-530

ues were normalised to compare the profile of pixel intensity across the different pictures. 531

The intensity values were scaled from 0 to 1 using the following formula: (x-xMIN)/(xMAX-532

xMIN). In order to obtain a normalisation based solely on CS signal and not on ectopic lignifi-533

cation, the values corresponding to ectopic lignification were intentionally omitted, in the 534

cases the fluorescence signal intensity coming from ectopic lignification (corner of the endo-535

dermal cells) was higher than the one coming from the CS itself in the single picture. The 536

profiles of normalised pixel intensity were plotted using PlotTwist [47] and RStudio. Statisti-537

cal analysis was performed on normalised data in the ranges defined in the figures. 538

539

Timing of expression and localization of CASP1 and UCC1 540

The timing of CASP1 and UCC1 accumulation and localization at the CS domain were quan-541

tified as number of cells after the onset of elongation as previously described [12,31]. The 542

cell number for the endodermal accumulation of CASP1 and UCC1 was determined in the 543

first cell after the onset of elongation where GFP or mCherry signal were detectable using 544

confocal microscopy. 545

546

Acknowledgements. 547

We thank Dr Robert Markus (Super Resolution Microscopy Facility, University of Notting-548

ham), the Microscopy and Histology Facility of the University of Aberdeen and John Danku 549

for ICP-MS analysis. We thank Dr Inês Barbosa and Prof Niko Geldner for critical reading of 550

the manuscript. This work was supported by grants from the UK Biotechnology and Biologi-551

cal Sciences Research Council Grant (grant no. BB/N023927/1 to D.E.S.), the Coordinating 552

Action in Plant Sciences Promoting sustainable collaboration in plant sciences (grant no. 553

ERACAPS13.089_RootBarriers to DES) and a Nottingham Research Fellowship to GC. 554


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[37] Wang Z-P, Xing H-L, Dong L, Zhang H-Y, Han C-Y, Wang X-C, et al. Egg cell-spe-667 cific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants 668 for multiple target genes in Arabidopsis in a single generation. Genome Biol 2015:1–669 12. doi:10.1186/s13059-015-0715-0. 670

[38] Cheng C-Y, Krishnakumar V, Chan AP, Thibaud-Nissen F, Schobel S, Town CD. 671 Araport11: a complete reannotation of the Arabidopsis thaliana reference genome. 672 Plant J 2017;89:789–804. doi:10.1111/tpj.13415. 673

[39] Nersissian AM, Shipp EL. Blue copper-binding domains. Adv Protein Chem 674 2002;60:271–340. 675

[40] Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, et al. The EMBL-676 EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res 677 2019;47:W636–41. 678

[41] Toufighi K, Brady SM, Austin R, Ly E, Provart NJ. The Botany Array Resource: e-679 Northerns, Expression Angling, and promoter analyses. Plant J 2005;43:153–63. 680 doi:10.1111/j.1365-313X.2005.02437.x. 681

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[43] Metsalu T, Vilo J. ClustVis: a web tool for visualizing clustering of multivariate data 685 using Principal Component Analysis and heatmap. Nucleic Acids Res 686 2015;43:W566–70. doi:10.1093/nar/gkv468. 687

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https://doi.org/10.1101/2020.05.01.071738

Tree scale: 0.1

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AT2G32300 UCC1

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Figure 1. Uclacyanins UCC1 and UCC2 are required for a functional Casparian strip.


https://doi.org/10.1101/2020.05.01.071738

Figure 1. Uclacyanins UCC1 and UCC2 are required for a functional Casparian strip. A. (left) Figure shows a phylogenetic analysis of phytocyanins protein family in A. thaliana. The tree was built using the full-length amino acid sequences for all proteins. Different colours represent the three phytocyanins subfamilies: uclacyanins, stellacyanins and plantacyanins (39). In the tree, branch lengths are proportional to the number of substitutions per site. AT3G17675 have been previously annotated as a stellacyanin (STC4), however the signal peptide for the secretion pathway and the hydrophobic extension for Glycosylphosphatidylinositol (GPI) anchoring are missing. (right) Heatmap showing the endodermal expression of the phytocyanins family in A. thaliana across the different root zones (Meristematic, Elongation, Maturation). For the analysis, expression data was collected from the Bio-Analytic Resource database, AtGenExpress Consortium. The expression of two endodermal localised proteins, CASP1 and ESB1, were added to the analysis as a reference. Asterisks indicate a significant downregulation in a myb36 mutant according to [18]. B. Schematic representation of the UCC1 and UCC2 proteins showing the different protein domains and the types of mutations. Domains were defined according to [19]. C. Boxplot analysis showing the number of the cells from the onset of elongation permeable to propidium iodide in wild type plants (WT), ucc1 mutants (ucc1.1 and ucc1.2), ucc2 mutant (ucc2.1), and the double mutant ucc1.2 ucc2.1. Data were collected from two independent experiments (n ≥ 29). Different letters represent significant differences between genotypes in a Mann-Whitney test (p < 0.01). D. Diagram shows the quantification analysis of the endodermal suberization in the plant genotypes used in C. Each colour in the graph represents the percentage of the root length (percentage of root length (%)) that is unsuberised (white), discontinuously suberised (yellow), continuously suberised (orange). Suberin was staining with Fluorol yellow 088. (n ≥ 18). Error bars in the figure are the standard deviation (SD). Different letters represent significant differences between genotypes using a Mann-Whitney test (p < 0.01). E. Heatmap representing the ionomic profiles (z-scores) of wild type plants (WT), and a collection of mutants with a defective casparian strip: ucc2.1, ucc1.1, ucc1.2, ucc1.2 ucc2.1, esb1, ucc1.2 esb1, sgn3 and ucc1.2 sgn3 grown in full nutrient conditions on agar plate for 2 weeks (n=10). Elements concentration were determined by ICP-MS and the raw data is available in the supplementary Table 1. Asterisks indicate a significant difference in comparison with WT using a t-test (p<0.01). Columns (Genotypes) were subjected to hierarchical clustering analysis.


https://doi.org/10.1101/2020.05.01.071738

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Figure 2. UCC1 defines a new central sub-domain in the Casparian strip.


https://doi.org/10.1101/2020.05.01.071738

Figure 2. UCC1 defines a new central sub-domain in the Casparian strip. A. Diagram representing the construct pUCC1::mCherry-UCC1 (UTR: Untranslated region, SP: Signal peptide, H Cter: Hydrophobic C-terminus for GPI anchoring). B. Maximum intensity projection, orthogonal, median and surface views of confocal sections of plants expressing pUCC1::mCherry-UCC1 (red) in cleared roots. In the case of maximum intensity projection (Maximum projection) figure represents different regions of the root measured as number of cells after the onset of elongation. White arrow heads point to vesicles containing mCherry-UCC1. For the orthogonal, median and surface views, cell walls were stained with Calcofluor white (grey in the figures). Scale bar = 20 µm for the maximum projection and orthogonal views. Scale bar = 5 µm for the median and surface views (Ep: epidermis, Co: cortex, End: endodermis). C. Maximum intensity projection, median and surface view of confocal sections of plants expressing CASP1-GFP (cyan) and mCherry-UCC1 (red). Signal was capture at the 10thendodermal cell after the onset of elongation observed in vivo. Scale bar = 20 µm for maximum projection and 3 µm for median and surface view. D. In vivo observation of the surface view of an endodermal cell expressing pESB1::ESB1-mCherry or pCASP1::CASP1-GFP. Scale bar = 2 µm. E. Immunolocalization assay of UCC1 protein (red) in plant expressing pCASP1::CASP1-GFP (cyan). A primary polyclonal antibody targeting UCC1 was used in combination with a secondary antibody conjugated with Dylight 633. Scale bar = 2 µm. F. Graph presenting the distribution of normalised pixels intensity (Relative Pixel Intensity, 0 to 1) across the Casparian strip (Distance µm) for CASP1-GFP fluorescence (cyan) and UCC1 immunofluorescence (red, Dylight 633). Light curves represent individual replicates coming from individual plants (n = 4). Each replicate is the average pixel intensity across a segment of 25 µm along the Casparian strip axis. Dark curves represent the mean values for CASP1-GFP and UCC1 immunofluorescence.


https://doi.org/10.1101/2020.05.01.071738

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Figure 3. Relations between UCC1 positioning and other components of the Casparian strips machinery.


https://doi.org/10.1101/2020.05.01.071738

Figure 3. Relations between UCC1 positioning and other components of the Casparian strips machinery.

A. Analysis of the spatial distribution of CASP1 and UCC1 at the endodermal cell junctions. Images were generated from the same plant co-expressing CASP1-GFP and mCherry-UCC1 using confocal microscopy. The numbers at the bottom of the figure indicate the number of cells after the onset of elongation. White arrows indicate the central accumulation for CASP1-GFP or mCherry-UCC1. Scale bar = 6 µm. B. Histograms showing the frequency distribution (Frequency (%)) of the onset of expression (upper plot, n = 18) and the onset of localization at the Casparian strip of CASP1-GFP and mCherry-UCC1 (lower plot, n = 28). C. (left panel) Maximum intensity projection and (right panel) surface view of UCC1 immunolocalization (red) at 10 cells after the onset of elongation in wild type plants (WT) and a collection of Casparian strips mutants: casp1 casp3, esb1, sgn3, esb1 sgn3. White arrows show gaps in the UCC1 localization. Scale bar = 20 µm for the maximum projections and 2 µm the surface views. D. Maximum intensity projection of CASP1-GFP localization in cleared root of wild type plants (WT), and the mutants ucc1.1 and ucc1.1 ucc2.1. Scale bar = 20 µm. E. Surface view of the localization of CASP1-GFP in cleared root of wild type plants (WT), and the mutants: ucc1.1, ucc1.2 and ucc1.1 ucc2.1. Scale bar = 2 µm. F. Quantification of normalised pixels intensity (Relative Pixel Intensity; 0 to 1) across the Casparian strip in plants expressing CASP1-GFP. The plots are showing the intensity profile for individual replicates (n ≥ 10), the mean value (black line) and the 95% confidence interval (grey interval). Each replicate corresponds to the quantification of one picture containing a Casparian strip segment of approximately 25µm long. The pictures were generated at the 15th cells after the onset of elongation from at least 8 individual plants per genotype. Intensity profiles across the Casparian strip were always measured in the same orientation, from the cortical side toward the pericycle side of the endodermis. Letters indicate statistically significant differences between genotypes for the intensity values comprised between the dashed lines using an ANOVA and Tukey’s test as post hoc analysis (p<0.01).


https://doi.org/10.1101/2020.05.01.071738

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Figure 4. UCC1 and UCC2 are necessary for the central lignification of the Casparian strip.A. Surface view of the Casparian strip lignin stained with Basic fuchsin in wild type plants (WT), and the mutants ucc2.1, ucc1.1, ucc1.2 and ucc1.2 ucc2.1. Whites arrows show lack of lignification in the central domain of the Casparian strip across the different genotypes. Scale bar = 2 µm. B. Quantification of normalised pixels intensity (0 to 1) (Relative pixel intensity) across the Casparian strip using surface views as shown in A. The plots show the intensity profile for individual plants (n ≥ 13). In the figure, the mean value is represented by a black line and the 95% confidence interval is in grey. The data were generated using individual pictures containing a Casparian strip segment of approximately 25 µm long. The pictures were taken at the 15th cells after the onset of elongation and the intensity profiles were measured in the same direction, from the cortex side toward the pericycle side of the endodermis. Letters indicate statistically significant differences between genotypes using an ANOVA and Tukey’s test as post hoc analysis (p<0.01).


https://doi.org/10.1101/2020.05.01.071738

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Supplementary figure 1. Uclacyanins UCC1 and UCC2 are required for the correct function of the Casparian strips.A. Boxplot showing the number of cells, from the onset of elongation, permeable to propidium iodide (PI) after 10 min or 20 min of root staining in wild type plants (WT) and a collection of mutants (ucc1.1, ucc1.2, ucc2.1, ucc1.2 ucc2.1, casp1 casp3, esb1, sgn3, ucc1.1 casp1 casp3, ucc1.2 esb1, ucc1.2 sgn3). The number of plants analysed was n ≥ 16 and n ≥ 10 for the 10 min and 20 min of PI exposition, respectively. Different letters represent significant differences between genotypes using a Mann-Whitney test (p < 0.01). B. Quantification of the endodermal suberization using the suberin specific staining Fluorol yellow 088 in the genotypes from A. The results (Percentage of root length (%)) represent the percentage of the root that remains unsuberised (white), discontinuously suberised (yellow) or continuously suberised (orange). In all cases the number of plants analysed was n ≥ 15, error bars mean standards deviation (SD). Different letters represent significant differences between genotypes using a Mann-Whitney test (p < 0.01). C. Principal component analysis (PCA) of the shoot ionomic profiles of wild type plants (WT) and the defective CS mutants ucc2.1, ucc1.1, ucc1.2, ucc1.1 ucc2.1, ucc1.2 ucc2.1, esb1, ucc1.1 esb1, ucc1.2 esb1, sgn3, ucc1.2 sgn3 and ucc1.1 sgn3 grown in agar plates. The PCA was performed using the average of the elemental profiles from each genotype (n=10) available in the Supplementary Table 1.


https://doi.org/10.1101/2020.05.01.071738

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Supplementary figure 2. UCC1 accumulates in the root endodermis and defines a centralsub-domain at the Casparian strips


https://doi.org/10.1101/2020.05.01.071738

Supplementary figure 2. UCC1 accumulates in the root endodermis and defines a central sub-domain at the Casparian strip. A. (Top) Schematic representation of the construct used to study the expression of UCC1 in the root. Approximately 3 Kb of the promoter region of the gene UCC1 (pUCC1 (3Kb)) was used to drive the expression of theb-glucuronidase (GUS) and the green fluorescent protein (GFP). (Bottom) Pictures showing the GFP accumulation in the root of plants expressing the construct pUCC1::GUS-GFP. The fluorescence (GFP, green) and brightfield (grey) pictures are merged. The different zones of the root (meristematic, elongation and differentiation) are highlighted in the figure. Scale bars = 100 µm (left) and 20 µm (right). (Ep: Epidermis; Co: Cortex; En: Endodermis). B. Structure illumination microscopy of a surface view of plants expressing CASP1-GFP (cyan) and mCherry-UCC1 (red) at the 10th endodermal cell after the onset of elongation in cleared root. Scale bar = 2 µm. C. Surface view of Casparian strip lignin stained with Auramine-O (cyan) in wild type plants (WT) and in a line expressing the construct pUCC1::mCherry-UCC1 (red) in cleared roots at 10th and 20th cells after the onset of elongation. In the figure the white arrow shows ectopic lignification. Scale bar = 2µm. D. Boxplot showing the number of the cells from the onset of elongation permeable to propidium iodide in wild type plants (WT) and a line expressing pUCC1::mCherry-UCC1. For both genotypes 10 plants were analysed. Asterisk indicates a significant difference with WT using a Mann-Whitney test (p < 0.01). E. Brightfield and suberin staining using Fluorol yellow 088 of roots in WT and a line expressing pUCC1::mCherry-UCC1. Scale bar = 2 mm. F. Quantification of suberin staining with Fluorol yellow 088 along the root. The results are expressed in percentage of root length that is unsuberised (white), discontinuously suberised (yellow), and continuously suberised (orange). In all cases n ≥ 10 plants were used, error bars: SD. Asterisks indicates significant differences in comparison with WT for the same zone using a Mann-Whitney test (p < 0.01). G. The anti UCC1 antibody generated in this work is functional and specifically recognises the UCC1 protein. Maximum intensity projection of UCC1 immunolocalization (red) in wild type plants (WT), the mutants myb36, ucc1.2, ucc1.2 ucc2.1 in the root at the 10th cell after the onset of elongation. As a control the primary polyclonal antibody anti UCC1 was used or not (“No primary Ab”) in combination with a secondary antibody conjugated with Dylight 633. Scale bar = 20 µm.


https://doi.org/10.1101/2020.05.01.071738

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Supplementary figure 3. Other Casparian strip molecular players are required for UCC1 to fuse into a continuous band. Surface view of mCherry-UCC1 at the 15th cell after the onset of elongation in cleared root of WT, casp1 casp3, esb1 and sgn3. White arrows represent discontinuous mCherry-UCC1 localization. Scale bar = 2 µm.


https://doi.org/10.1101/2020.05.01.071738

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Supplementary figure 4. UCC1 and UCC2 are necessary for the central lignification of the Casparian strip.A. Figures show the maximum intensity projection of confocal sections of plant roots stained with basic fuchsin for visualising lignin. Pictures were taken using cleared roots at the 10th and 15th cells after the onset of elongation of wild type plants (WT), and a collections of mutants: ucc1.1, ucc1.2, ucc2.1, ucc1.2 ucc2.1, casp1 casp3, ucc1.1 casp1 casp3, esb1, ucc1.2 esb1, sgn3 and ucc1.2 sgn3. Spiral structures in the centre of the root are the xylem. Blue arrows indicate gaps in the lignin deposition at the Casparian strip. Green triangles show ectopic lignification. Scale bar = 20 µm. B. Structure illumination microscopy of a surface view of Casparian strip lignin stained with basic fuchsine in cleared roots of WT, ucc1.2 and ucc1.2 ucc2.1. Blue arrows show lack of lignification in the central domain of the Casparian strip. Scale bar = 2 µm.


https://doi.org/10.1101/2020.05.01.071738

Element

(ppm) Mean ± SD Mean ± SD p-value Mean ± SD p-value Mean ± SD p-value Mean ± SD p-value Mean ± SD p-value Mean ± SD p-value Mean ± SD p-value Mean ± SD p-value Mean ± SD p-value Mean ± SD p-value Mean ± SD p-value

Li 0.11 ± 0.01 0.14 ± 0.01 2.1E-09 0.15 ± 0.02 1.1E-07 0.11 ± 0.01 3.3E-01 0.13 ± 0.01 9.3E-06 0.12 ± 0.01 4.2E-04 0.14 ± 0.02 7.6E-06 0.15 ± 0.01 1.9E-09 0.14 ± 0.01 1.3E-08 0.14 ± 0.01 6.8E-07 0.13 ± 0.02 1.6E-03 0.13 ± 0.02 6.5E-03

B 65.14 ± 3.30 72.04 ± 4.41 9.1E-04 64.03 ± 5.53 5.9E-01 69.12 ± 4.24 3.1E-02 74.75 ± 5.00 7.9E-05 67.98 ± 4.30 1.2E-01 71.03 ± 10.05 9.6E-02 68.56 ± 7.14 1.2E-01 63.75 ± 3.61 3.8E-01 59.28 ± 4.33 3.2E-03 57.87 ± 4.66 7.9E-04 61.56 ± 5.00 7.5E-02

Na 7018.04 ± 584.97 9015.89 ± 767.67 3.8E-06 9078.37 ± 799.61 3.5E-06 7081.35 ± 801.72 8.4E-01 7619.27 ± 836.58 7.9E-02 7437.08 ± 653.06 1.5E-01 10049.03 ± 1600.53 2.5E-05 8594.43 ± 528.46 2.0E-05 8629.81 ± 436.91 1.6E-06 9222.20 ± 463.35 2.5E-08 7646.20 ± 545.95 2.3E-02 8669.27 ± 865.04 9.3E-05

Mg 2979.44 ± 57.30 3111.87 ± 76.53 3.6E-04 2982.05 ± 58.66 9.2E-01 3161.74 ± 115.59 6.8E-01 3144.32 ± 89.25 1.1E-04 3002.79 ± 85.41 4.8E-01 2966.03 ± 77.54 6.7E-01 3101.96 ± 72.32 9.2E-04 3026.98 ± 79.52 1.4E-01 2951.54 ± 105.05 4.7E-01 3010.08 ± 142.89 5.4E-01 3044.64 ± 96.97 3.6E-03

P 21520.59 ± 864.21 21936.86 ± 1167.83 3.8E-01 21399.07 ± 1676.84 8.4E-01 23074.83 ± 657.36 2.6E-04 23630.50 ± 1472.38 1.0E-03 24688.07 ± 1533.57 2.1E-05 22676.96 ± 1654.24 6.6E-02 22440.81 ± 1244.45 9.2E-02 24186.78 ± 1679.71 3.0E-04 24679.85 ± 1483.26 1.6E-05 24443.92 ± 1002.17 1.6E-06 24867.09 ± 768.28 3.4E-08

S 15553.96 ± 833.67 18932.37 ± 1109.73 4.2E-07 17309.21 ± 588.08 6.6E-05 16883.46 ± 409.35 2.6E-04 16785.15 ± 600.05 1.3E-03 16625.63 ± 629.99 4.5E-03 17857.27 ± 480.26 5.3E-07 17510.79 ± 800.07 2.4E-05 19755.62 ± 581.02 1.3E-10 18657.41 ± 555.65 1.2E-08 16425.43 ± 698.49 2.1E-02 17377.82 ± 829.98 1.1E-04

K 77759.28 ± 2915.45 67147.58 ± 4155.35 3.3E-06 64064.70 ± 2636.41 2.0E-09 78925.57 ± 4475.87 5.0E-01 80474.03 ± 3742.59 8.7E-02 81133.74 ± 3002.28 2.0E-02 67342.75 ± 3476.22 9.5E-07 72141.51 ± 3123.34 1.1E-03 66933.09 ± 2848.20 1.2E-07 63052.01 ± 4192.43 3.7E-08 75299.37 ± 1906.76 3.8E-02 72225.48 ± 2545.79 2.6E-04

Ca 8059.54 ± 299.72 7425.74 ± 219.58 4.0E-05 8114.52 ± 562.32 7.9E-01 8176.90 ± 361.28 4.4E-01 8362.83 ± 350.28 5.2E-02 8150.55 ± 362.33 5.5E-01 8211.02 ± 603.54 4.9E-01 8505.45 ± 506.64 6.1E-04 7187.76 ± 323.14 6.7E-06 7229.80 ± 322.73 1.2E-05 7781.09 ± 342.60 6.9E-02 7773.89 ± 382.88 8.0E-02

Mn 349.57 ± 8.84 356.24 ± 8.80 1.1E-01 385.08 ± 19.76 6.2E-05 353.17 ± 14.01 5.0E-01 364.31 ± 13.59 1.0E-02 360.03 ± 7.64 1.1E-02 381.73 ± 30.27 4.7E-03 398.98 ± 24.44 5.9E-08 347.75 ± 11.61 7.0E-01 338.40 ± 15.67 6.5E-02 344.45 ± 14.70 3.6E-01 346.29 ± 16.67 5.9E-01

Fe 178.05 ± 12.58 160.21 ± 6.48 8.7E-04 177.32 ± 12.87 9.0E-01 167.57 ± 16.13 1.2E-01 155.96 ± 10.16 4.1E-04 155.43 ± 9.05 2.1E-04 159.33 ± 13.61 5.0E-03 154.35 ± 9.94 3.1E-04 148.86 ± 5.81 3.0E-06 157.73 ± 17.15 7.4E-03 213.84 ± 66.68 9.8E-02 159.83 ± 15.88 1.1E-02

Co 0.26 ± 0.04 0.23 ± 0.04 9.6E-02 0.22 ± 0.03 2.6E-02 0.20 ± 0.03 1.4E-03 0.19 ± 0.03 3.4E-04 0.20 ± 0.04 2.0E-03 0.17 ± 0.03 5.3E-05 0.20 ± 0.04 3.1E-04 0.25 ± 0.02 4.8E-01 0.23 ± 0.03 6.6E-02 0.24 ± 0.13 6.1E-01 0.15 ± 0.01 1.8E-07

Ni 0.10 ± 0.02 0.20 ± 0.09 2.6E-03 0.20 ± 0.10 3.9E-03 0.16 ± 0.17 2.3E-01 0.17 ± 0.09 2.1E-02 0.20 ± 0.09 1.8E-03 0.16 ± 0.09 5.1E-02 0.15 ± 0.08 5.4E-02 0.16 ± 0.05 7.4E-04 0.13 ± 0.03 6.8E-03 0.16 ± 0.07 2.6E-02 0.19 ± 0.16 2.3E-02

Cu 2.09 ± 0.17 2.21 ± 0.10 7.1E-02 2.31 ± 0.14 5.9E-03 2.25 ± 0.21 8.0E-02 2.22 ± 0.16 1.0E-01 2.21 ± 0.06 5.5E-02 2.31 ± 0.26 4.2E-02 2.40 ± 0.12 1.3E-04 2.24 ± 0.11 3.1E-02 2.35 ± 0.34 4.9E-02 2.41 ± 0.80 2.4E-01 2.12 ± 0.15 7.2E-01

Zn 293.02 ± 27.65 252.98 ± 25.86 3.6E-03 268.22 ± 24.79 4.9E-02 284.04 ± 25.48 4.6E-01 282.03 ± 24.62 3.6E-01 282.40 ± 24.47 3.8E-01 252.59 ± 37.79 1.4E-02 246.48 ± 26.43 5.5E-04 278.17 ± 19.99 1.9E-01 301.20 ± 30.13 5.3E-01 276.27 ± 14.86 1.1E-01 264.13 ± 23.52 2.2E-02

As 0.30 ± 0.02 0.45 ± 0.18 1.6E-02 0.51 ± 0.16 7.0E-04 0.34 ± 0.08 2.6E-01 0.33 ± 0.06 1.3E-01 0.35 ± 0.05 1.6E-02 0.58 ± 0.25 2.6E-03 0.37 ± 0.03 1.2E-04 0.40 ± 0.07 8.6E-04 0.41 ± 0.05 1.0E-05 0.32 ± 0.03 1.1E-01 0.41 ± 0.05 2.2E-05

Se 0.02 ± 1.02 0.03 ± 1.73 9.9E-01 -0.03 ± 0.92 9.0E-01 0.19 ± 0.38 6.3E-01 0.01 ± 0.82 9.7E-01 0.00 ± 0.26 9.4E-01 -0.15 ± 0.34 6.2E-01 -0.12 ± 1.13 8.1E-01 0.15 ± 0.34 7.2E-01 0.23 ± 0.73 6.2E-01 0.02 ± 0.39 9.8E-01 -0.31 ± 0.54 3.7E-01

Rb 1.42 ± 0.07 1.24 ± 0.06 1.1E-05 1.16 ± 0.03 7.1E-09 1.41 ± 0.07 8.9E-01 1.42 ± 0.06 7.6E-01 1.43 ± 0.04 6.9E-01 1.22 ± 0.06 5.3E-06 1.26 ± 0.03 1.4E-05 1.22 ± 0.04 8.0E-07 1.17 ± 0.07 3.6E-07 1.40 ± 0.06 5.7E-01 1.36 ± 0.10 2.0E-02

Sr 4.11 ± 0.15 4.00 ± 0.16 1.4E-01 4.52 ± 0.08 4.5E-07 4.08 ± 0.15 6.1E-01 4.16 ± 0.13 4.5E-01 4.12 ± 0.17 9.3E-01 4.63 ± 0.24 1.7E-05 4.40 ± 0.20 1.1E-05 3.82 ± 0.18 1.0E-03 3.95 ± 0.09 9.1E-03 4.06 ± 0.11 3.6E-01 4.28 ± 0.14 1.7E-02

Mo 3.61 ± 0.33 4.61 ± 0.52 7.4E-05 3.84 ± 0.31 1.4E-01 3.85 ± 0.40 1.7E-01 3.60 ± 0.57 9.3E-01 3.85 ± 0.34 1.3E-01 4.59 ± 1.13 1.7E-02 3.76 ± 0.28 2.8E-01 4.29 ± 0.36 4.0E-04 4.31 ± 0.27 6.7E-05 3.93 ± 0.36 5.8E-02 5.07 ± 0.49 3.7E-07

Cd 0.07 ± 0.01 0.05 ± 0.01 3.1E-05 0.04 ± 0.005 5.5E-06 0.07 ± 0.01 9.3E-01 0.07 ± 0.01 1.2E-01 0.07 ± 0.01 4.5E-01 0.10 ± 0.13 4.3E-01 0.06 ± 0.04 8.9E-01 0.07 ± 0.02 9.9E-01 0.07 ± 0.02 7.1E-01 0.08 ± 0.02 1.3E-01 0.06 ± 0.01 3.3E-01

ucc1.2*ucc2.1ucc1.2 ucc1.1sgn3 ucc1.2sgn3.3 ucc1.1esb1 ucc1.2esb1 ucc1.1ucc2.1ucc1.1WT esb1 sgn3 ucc2.1

Supplementary table 1. UCC1 and UCC2 are required for maintaining ion homeostasis in the shoot.Elemental content in shoot of ucc2.1, ucc1.1, ucc1.2, ucc1.1 ucc2.1, ucc1.2 ucc2.1, esb1, ucc1.1 esb1, ucc1.2 esb1, sgn3, ucc1.2 sgn3 and ucc1.1 sgn3 mutants compared to WT grown in agar plates (long day, n=10). Elements concentration were determined by ICP-MS. Data are presented as mean ± standard deviation (SD). t-tests were performed to determine the significant differences to WT and the corresponding p-values are presented.


https://doi.org/10.1101/2020.05.01.071738

Uclacyanin proteins are required for lignified nanodomain ......2020/05/01 · 67 ated with...

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