1 short title: hvcam1 negatively regulates salt tolerance · 2020-06-18 · 1 1. s. hort. title:...

1

Short Title: HvCaM1 negatively regulates salt tolerance 1

Author for Contact: Guoping Zhang, Email: [email protected], Tel & Fax: 2

(86)-571-88982115 3

Title: Calmodulin HvCaM1 negatively regulates salt tolerance via modulation of 4

HvHKT1s and HvCAMTA4 5

Author Names and Affiliations: 6

Qiufang Shen1, Liangbo Fu

1, Tingting Su

1, Lingzhen Ye

1, Lu Huang

1, Liuhui Kuang

1, 7

Liyuan Wu1, Dezhi Wu

1, Zhong-Hua Chen

2,3 and Guoping Zhang

1,* 8

1Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang 9

University, Hangzhou 310058, P.R. China 10

2School of Science, Hawkesbury Institute for the Environment, Western Sydney 11

University, Penrith, NSW 2751, Australia 12

3Collaborative Innovation Centre for Grain Industry, College of Agriculture, Yangtze 13

University, Jingzhou 434025, China 14

*Corresponding author 15

One-sentence Summary: Barley calmodulin HvCaM1 regulates salt tolerance by 16

mediating the expression of Na+ transport-related HKT1 genes and the transcriptional 17

regulation by a CaM-binding transcription activator HvCAMTA4. 18

Author Contributions: GZ, DW and QS designed the research; DW and ZC guided 19

the research; QS, LF, TS, LY, LH, LK and LW performed experiments; QS and LF 20

analyzed the data; QS drafted the manuscript; GZ, DW and ZC revised the 21

manuscript. 22

Funding Information: This work was supported by the Natural Science Foundation 23

of China (31901429, 31771685 and 3161001022), China Postdoctoral Science 24

Foundation (2019T120520 and 2018M640561), China Agriculture Research System 25

(CARS-05) and Jiangsu Collaborative Innovation Center for Modern Crop 26

Production. 27

Plant Physiology Preview. Published on June 18, 2020, as DOI:10.1104/pp.20.00196

Copyright 2020 by the American Society of Plant Biologists

www.plantphysiol.orgon September 28, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

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2

Abstract 28

Calcium (Ca2+

) signaling modulates sodium (Na+) transport in plants; however, the 29

role of the Ca2+

sensor calmodulin (CaM) in salt tolerance is elusive. We previously 30

identified a salt-responsive calmodulin (HvCaM1) in a proteome study of barley 31

(Hordeum vulgare) roots. Here, we employed bioinformatic, physiological, molecular 32

and biochemical approaches to determine the role of HvCaM1 in barley salt tolerance. 33

CaM1s are highly conserved in green plants and probably originated from ancestors 34

of green algae of the Chlamydomonadales order. HvCaM1 was mainly expressed in 35

roots and was significantly up-regulated in response to long-term salt stress. 36

Localization analyses revealed that HvCaM1 is an intracellular signaling protein that 37

localizes to the root stele and vascular systems of barley. After treatment with 200 38

mM NaCl for 4 weeks, HvCaM1 knock-down (RNAi) lines showed significantly 39

larger biomass but lower Na+ concentration, xylem Na

+ loading, and Na

+ 40

transportation rates in shoots compared with overexpression (OE) lines and wild-type 41

plants. Thus, we propose that HvCaM1 is involved in regulating Na+ transport, 42

probably via certain Class I High-affinity Potassium Transporters (HvHKT1;5 and 43

HvHKT1;1)-mediated Na+ translocation in roots. Moreover, we demonstrated that 44

HvCaM1 interacted with a CaM-binding transcription activator (HvCAMTA4), which 45

may be a critical factor in the regulation of HKT1s in barley. We conclude that 46

HvCaM1 negatively regulates salt tolerance, probably via interaction with 47

HvCaMTA4 to modulate the downregulation of HvHKT1;5 and/or upregulation of 48

HvHKT1;1 to reduce shoot Na+ accumulation under salt stress in barley. 49

Keywords: Ca2+

binding protein, Ca2+

signaling, Na+ transport, salt stress, Hordeum 50

vulgare 51



3

Introduction 52

Salt stress is a major abiotic factor restricting crop growth and productivity, which is 53

exacerbated by global climate change and human activities (Munns and Tester, 2008; 54

Munns et al, 2020). Excess sodium (Na+) causes ion toxicity and also ion imbalance 55

through competitively inhibiting the uptake of some mineral nutrients such as 56

potassium (K+). Maintaining lower Na

+ accumulation in shoots is crucial for salt 57

tolerant plant species and genotypes under salt stress (Munns, 2005; Munns and Tester, 58

2008; Horie et al, 2012; Deinlein et al, 2014; Shen et al, 2016, 2017). Lower shoot 59

Na+ accumulation in plants is attributed to either shoot Na

+ exclusion or vacuolar 60

sequestration, both of which are regulated by membrane transporters, such as some 61

members of High-affinity Potassium Transporters (HKTs), Na+/H

+ Exchanger (NHXs) 62

and Salt Overly Sensitive 1 (SOS1) (Apse et al, 1999; Shi et al, 2000; Zhang and 63

Blumwald, 2001; Davenport et al, 2007; Barragán et al, 2012; Huang et al, 2020). 64

Activation of membrane transport systems in roots plays a key role in reducing shoot 65

Na+ accumulation and enhancing salt tolerance in many plant species (Zhu, 2002; 66

Shabala and Cuin, 2008; Ismail and Horie, 2017). However, the mechanisms 67

underlying Na+ exclusion and translocation and the link to calcium (Ca

2+) signaling 68

require further investigation. 69

Ca2+

-mediated signal transduction plays a regulatory role in plant salt tolerance 70

(Zhu, 2002, 2016; Demidchik et al, 2018). For instance, SOS3 (calcineurin-like 71

protein, CBL4) is a Ca2+

sensor, which perceives the Na+-induced cytosolic Ca

2+ rise 72

via the interaction with SOS2 (calcineurin interacting protein kinase, CIPK24), 73

causing phosphorylation and activation of the plasma membrane SOS1/NHX7 (Shi et 74

al, 2000; Zhu, 2002; Mahi et al, 2019). The activation of SOS1 in root epidermal cells 75

and xylem parenchyma cells leads to the extrusion of Na+ from roots to rhizosphere 76

(Shi et al, 2002; Zhu, 2016). Meanwhile, a rapid elevation of cytosolic Ca2+

under Na+ 77

stress can also be captured by other Ca2+

sensors, including calmodulin (CaM), 78

calmodulin-like proteins (CMLs), and calcium-dependent protein kinases (CDPKs) 79

(Galon et al, 2010; Cho et al, 2016). Multi-omics techniques have revealed that CaM 80

exhibits salt-induced expression changes at the transcriptional and translational levels 81

in seedlings or roots, suggesting that these Ca2+

sensors participate in salt stress 82

signaling (Zhang et al, 2012; Wu et al, 2014; Zhu et al, 2015; Shen et al, 2018; Mahi 83

et al, 2019). However, regulation of the complex Ca2+

signaling by CaMs to function 84

in plant salt tolerance is still elusive. 85



4

CaMs usually encode a single peptide protein consisting of a pair of unique 86

Ca2+

binding EF-hands and have no other functional domains or motifs (Reddy et al, 87

2011). Meanwhile, CaM protein sequences are highly conserved in green plants, with 88

an averaged 90% identity of 65 members detected in 15 representative plant species 89

from Chlorophyceae to angiosperms (Zhu et al, 2015). Moreover, CaMs have less 90

variation and fewer family members than other Ca2+

sensors (Xu et al, 2015; Zhu et al, 91

2015). However, little attention has been paid to the origin and evolution of CaMs in 92

green plants. The recent advancement of assembled genomes (Kersey, 2019) and 93

transcriptome databases (Leebens-Mack et al, 2019) will likely unlock the evolution 94

of CaMs, providing insights into their roles in stress tolerance of plants. 95

CaM may function through post-transcriptional modification similar to that in 96

SOS3 via the regulation of downstream receptors, such as CaM-binding proteins 97

(CBPs) (Galon et al, 2010; Batistič and Kudla, 2012; Mahi et al, 2019). In different 98

plants, CaM can adjust the affinity of Ca2+

/CaM complex for the selection of special 99

receptors (e.g. phosphodiesterase) and targets [e.g. cyclic nucleotide-gated channel 12 100

(CNGC12)] after recognizing the cytosolic Ca2+

change for a specific stress response 101

(Gifford et al, 2013; Defalco et al, 2016). In Arabidopsis (Arabidopsis thaliana), 102

AtCaM1/4 shows an up-regulated expression under salt stress, resulting in enhanced 103

salt tolerance via binding with S-nitrosoglutathione reductase (GSNOR) in nitric 104

oxide (NO) signaling (Zhou et al, 2016). CaM-binding protein kinases (AtCaM2/3/5) 105

modulate the activity of heat shock factors (AtHSFs) through phosphorylation of a 106

CaM-binding protein kinase (AtCBK3) in heat stress signaling (Zhang et al, 2009), 107

while AtCaM6 and AtCaM7 are involved in the light-dependent stress pathway and 108

with reactive oxygen species (ROS) signaling (Kushwaha，2008; Al-Quraan et al, 109

2011). In rice (Oryza sativa), OsCaM1-1 also shows expression patterns similar to 110

AtCaM1/4 (Chinpongpanich et al, 2012), and its overexpression mitigates salt-induced 111

oxidative damage (Kaewneramit et al, 2019). In addition, overexpression of GmCaM4 112

enhances salt tolerance by regulating the expression of a series of salt-responsive 113

genes, including a key proline biosynthetic enzyme P5CS1 in soybean (Glycine max) 114

(Yoo et al, 2005; Rao et al, 2014). Moreover, many transcription factors, including 115

CaM-binding transcription activators (CAMTAs), CBP60s, MYBs, WRKYs, bZIPs, 116

bHLHs, NACs and GTs, have been identified as CBPs, suggesting that CaM might 117

play important roles in the transcriptional regulation of salt stress tolerance in plants 118



5

(Galon et al, 2010; Xi et al, 2012; Shkolnik et al, 2019). For example, AtCAMTA6 119

regulates the expression of AtHKT1 via the binding sites of CGCG-core or 120

CGTGT-core, contributing to Na+ homeostasis during seed germination (Shkolnik et 121

al, 2019). Therefore, the roles of CaMs in salt stress response have been reported, but 122

their function in the regulation of Na+ transport remains to be revealed. 123

As one of the most salt-tolerant diploid crop species with a large and complex 124

genome, barley (Hordeum vulgare) has been used as a model plant to decipher the 125

physiological and molecular mechanisms of salinity tolerance in plants (Chen et al, 126

2005; Munns and Tester, 2008; Nevo and Chen, 2010; Mascher et al, 2017; Dai et al, 127

2018). However, there are limited reports on CaM members and their functions in 128

barley. We have previous identified one salt-responding CaM protein (Protein ID: 129

A0MMD0) in the root proteome of wild barley genotypes (Wu et al, 2014; Shen et al, 130

2018). 131

We hypothesized that HvCaM1 plays a critical role in the establishment of higher 132

salt tolerance in barley via the regulation of the Ca2+

signaling transduction, 133

transcriptional factors and membrane transport. Here, we cloned HvCaM1 (Gene ID: 134

HORVU0Hr1G001270) and comprehensively analyzed its function through 135

overexpression and RNAi in barley with a range of physiological and molecular 136

techniques. We found that HvCaM1 negatively regulates salt tolerance in barley and 137

participates in Na+ transport from roots to shoots. Furthermore, we also detected that 138

HvCaM1 regulates two HvHKT1 members via the differential interaction with the 139

transcriptional activator HvCAMTA4. 140



6

Results 141

Evolutionary bioinformatics and phylogenetic analysis of HvCaM1 142

We first cloned the full-length cDNA and genome DNA of HvCaM1 from a barley 143

cultivar Golden Promise (GP). HvCaM1 contains 2 exons and 1 intron encoding a 144

peptide consisting of 149 aa, with two typical EF-hands (Fig S1A). No difference in 145

coding sequences (CDS) and protein sequences was found between the Tibetan wild 146

barley accessions and barley cultivars (Table S1). Sequence similarity analysis 147

showed HvCaM1 exhibits, on average, 98.8% identity with the CaM members in rice 148

and Arabidopsis with the four conserved Ca2+

binding sites (Fig S1B). Phylogenetic 149

analysis showed that HvCaM1 is relatively close to OsCaM1-1 and AtCaM1 (Fig 150

S1C). 151

Evolutionary analysis further indicated that CaM1 likely originates from the 152

ancestors of the Volvocales Order of chlorophyte algae (Chlamydomonadaceae) (Fig 153

1A). CaM1s are particularly conserved in angiosperms with up to 87% identity 154

detected in the CaM1 proteins of 60 representative plant species (Table S2). Moreover, 155

the sequence analysis showed that each CaM1 protein contains two conserved 156

EF-hand motifs and the ubiquitous presence of a consensus sequence of DDGDGE 157

and DXDXNE in the protein structure of Ca2+

binding sites in major lineages (Fig 1B). 158

However, the difference of a couple of amino acids in the protein sequences of 159

different chlorophyte algae indicated that CaM1s likely evolved from 160

Chlamydomonadaceae but are conserved from streptophyte alage (e.g. Klebsormidium 161

subtile), and then diversified in their function during adaptation of land plants (Figs 1 162

and S1). 163

HvCaM1 is up-regulated in roots under salt stress and mainly localized in stele 164

and vascular cells of roots 165

We investigated the expression of HvCaM1 in roots and shoots of barley seedlings. 166

qPCR analysis showed that HvCaM1 is expressed in both roots and shoots with 167

significantly higher expression in roots (Fig 2A). Moreover, the expression of 168

HvCaM1 in roots was greatly affected by salt concentration, with the highest 169

up-regulated value occurring at 200 mM NaCl treatment (Fig 2B). Interestingly, the 170

expression of HvCaM1 was significantly up-regulated by 3.04-fold after four weeks 171

of long-term salt stress compared to control value (Fig 2C). 172

The subcellular localization detected by green fluorescent protein (GFP) showed 173



7

that HvCaM1 appears to be expressed in the nucleus and plasma membrane but not in 174

the cytosol of onion (Allium cepa L.) epidermal cells, while it is expressed in the 175

nucleus, cytosol and plasma membrane in barley leaf mesophyll protoplasts, 176

suggesting it may be an intracellular signal protein (Fig 2D-E). We performed in situ 177

PCR to determine the cellular localization of HvCaM1 transcripts in the cross sections 178

of roots and leaves, respectively. In roots, the HvCaM1 transcripts were mainly 179

detected in phloem and xylem parenchyma cells of the stele (Fig 2F). In leaves, the 180

transcripts were found predominately in phloem vessel and mesophyll cells and to a 181

lesser extent in stomatal cells (Fig 2G). Additionally, observation of fluorescence 182

signals from GFP fused with the 2.1 kb promoter of HvCaM1 in the transformed 183

barley seedlings also confirmed that HvCaM1 mainly localizes in the stele of roots 184

(Fig S2A) and in stomatal guard cells, subsidiary cells, and leaf mesophyll cells (Fig 185

S2B). 186

HvCaM1 negatively regulates salt tolerance via the modulation of Na+ transport 187

in barley 188

To examine the function of HvCaM1, we generated 46 and 52 independent 189

knockdown (RNAi) and overexpressed (OE) barley lines, respectively. Three RNAi 190

lines and three OE lines were examined by PCR verification via specific primers (Fig 191

S3A), showing the significant average decrease and increase in the expression levels 192

of HvCaM1 by 59% and 2.41-fold, respectively, in comparison with the wild-type 193

(WT) plants (Fig S3B). The phenotypic difference between the 194

genetically-transformed lines and the WT plant was evident 3 weeks after 200 mM 195

salt exposure (Fig 3A). RNAi lines showed significantly higher salt tolerance than OE 196

and WT seedlings 4 weeks after salt stress as reflected by shoot and root dry weight 197

(Fig 3B). Only dry weight of OE73 was significantly lower than that of WT in 198

response to salt stress. Meanwhile, shoots of RNAi lines had higher relative water 199

content than those of OE lines and WT plants at 4 w of salt stress (Fig S4A). When 200

planted in the saline soil, these RNAi lines were less affected in grain yield traits 201

(spikes per plant, seeds per spike, setting rate and kernel weight) in comparison with 202

WT plants (Fig S5). These results indicate that HvCaM1 may function as a negative 203

regulator of salt tolerance in barley. 204

After 4 weeks of salt treatment, RNAi lines had a significantly lower shoot Na+ 205

concentration (52.0 mg g-1

DW) than OE (81.4 mg g-1

DW) and WT plants (78.6 mg 206



8

g-1

DW), but such differences were not found in roots (Fig 3C). RNAi lines showed a 207

significantly higher K+ concentration in both roots and shoots (1.52-fold and 1.45-fold 208

higher) than in WT plants, respectively. The OE lines showed significant 209

NaCl-induced reduction of shoot K+ in comparison to the WT (Fig 3D). Consistently, 210

RNAi lines showed a significant 144% and 109% higher shoot K+/Na

+ ratio than the 211

WT and OE lines under salt stress, respectively (Fig 3E). In addition, RNAi lines had 212

a significantly lower Na+ transport rate from roots to shoots than both OE and WT 213

plants, but there was no difference in K+ transport rate or Ca

2+ concentration among 214

these genetic lines (Fig S4B-D). Thus, we hypothesized that HvCaM1 negatively 215

regulates salt tolerance in barley, probably due to root-to-shoot Na+ translocation. 216

To validate the hypothesis that the involvement of HvCaM1 in the regulation of 217

salt tolerance is related to Na+ transport, we examined the responses of RNAi lines 218

(R1, R2 and R3) and WT seedlings to 200 mM (S200) and 250 mM (S250) salt stress 219

solutions (Fig 4). After 4 weeks of salt stress, three RNAi lines had significantly 220

larger root and shoot dry weight than the WT plant under both S200 and S250 221

treatments, but there was no difference under S300 treatment (Fig 4A-B; Table S3). 222

Furthermore, WT plants had significantly higher shoot Na+ concentrations than RNAi 223

lines under S250 treatment. Such a difference was not detected in roots (Fig 4C). 224

Meanwhile, three RNAi lines and WT plants showed little difference in the total Na+ 225

accumulation per plant (Fig 4D), but the RNAi lines had much lower Na+ transport 226

rates than the WT plants at S200 treatment, while differences at S250 were not 227

significant (Fig 4E). Clearly, the higher salt stress tolerance of RNAi lines is closely 228

associated with less Na+ transport from roots to shoots, which was mediated by 229

HvCaM1. On the other hand, RNAi lines had higher K+ concentration and 230

accumulation in plant tissues in comparison with WT plants at S200 treatment, but no 231

difference was found in root-to-shoot K+ transport rate (Fig 4F-H). Therefore, the 232

current results indicate that HvCaM1 negatively regulates salt tolerance, indirectly 233

resulting in reduced Na+ transport from roots to shoots under salt stress. 234

Silencing of HvCaM1 modulates HKT1s expression in roots 235

To determine whether HvCaM1 regulates Na+ transport, qPCR analysis of the 236

well-known genes associated with Na+ and K

+ transport (HKT1;1-5 except 2, 237

HKT2;1-3, NHX1-6 and SOS1-3 members) was performed for roots of the WT and 238

RNAi lines under normal condition and salt treatment (Fig 5). The relative expression 239



9

(salt stress/control) of all examined Na+ and K

+ transporter genes showed the dramatic 240

difference between the two RNAi lines and WT plants at 3 weeks after salt treatment 241

(Figs 5 and S6). Moreover, all eight genes were greatly down-regulated in the two 242

RNAi lines in comparison with the WT plants, except for HvHKT1;1, which was 243

clearly up-regulated in the RNAi lines. Interestingly, over the 3-week salt treatment, 244

relative expression of HvHKT1;5 showed a similar pattern as that of HvCaM1, while 245

HvHKT1;1 showed a completely opposite response, suggesting involvement of 246

HvCaM1 in regulation of both HvHKT1;1 and HvHKT1;5 expression (Fig 5A-C). On 247

the contrary, four NHX genes (HvSOS1/HvNHX7, HvNHX1, HvNHX3 and HvNHX5) 248

showed the different patterns of relative expression from that of HvCaM1, although 249

these genes were also greatly up-regulated in the WT plants under salt treatment (Fig 250

5D-G). HvSOS2 and HvSOS3 showed a similar expression pattern with that of the 251

NHX genes (Fig. 5H-I). Therefore, involvement of HvCaM1 in regulating salt stress 252

tolerance may influence the expression of HvHKT1;1 and HvHKT1;5 responsible for 253

Na+ transport. 254

Furthermore, the physiological role of Na+ transporters, mainly for HvHKT1s in 255

Na+ transportation from roots to shoots, was validated by xylem sap analysis (Fig 6). 256

Na+ concentrations in the xylem sap of HvCaM1 RNAi lines were significantly lower 257

than in the WT plants after 7 d of 50 mM and 100 mM NaCl treatments (Fig 6B). 258

There was a slight difference in K+ concentration in the xylem sap among these barley 259

lines (Fig 6C-D). 260

HvCaM1 interacts with HvCAMTA4 to regulate the preferential expression of 261

HKT1s 262

To understand how HvCaM1 affects HKT1 expression (i.e. HvHKT1;1 and 263

HvHKT1;5), we performed a yeast two-hybrid assay using the library constructed 264

from barley roots under salt stress. We identified nine candidate proteins that interact 265

with HvCaM1 in the assay (Table S4). Calmodulin-binding transcription activator 4 266

(HvCAMTA4, HORVU2Hr1G069950) was amongst the candidate proteins and 267

contains an IQ domain for CaM binding and a CG-1 domain with specific DNA 268

binding activity (specific CGCG-core and co-recognized CGTCT-core with ABRE) to 269

function as a co-activator of transcription. 270

Bimolecular fluorescence complementation (BiFC) assay confirmed the 271

interaction between HvCaM1 and HvCAMTA4 in both the yeast (Saccharomyces 272



10

cerevisiae) system and barley protoplasts (Fig 7A-B). Compared to the sites for 273

protein-protein interaction between HvSOS2 and HvSOS3 in the nucleus, cytoplasm, 274

and plasma membrane, the interaction between HvCaM1 and HvCAMTA4 was 275

limited to the nucleus, indicating a role for HvCaM1 in regulation of 276

HvCAMTA4-mediated transcriptional activation (Fig 7B). Furthermore, qPCR 277

analysis showed consistent patterns of expression for HvCAMTA4 and HvHKT1;5, 278

with a significant correlation of r=0.97 (P<0.01), in the roots of WT and RNAi lines 279

under salt stress (Figs 5C and 7C). 280

Then we identified binding sites along 3 kb promoter region of HKT genes using 281

the two CAMTA-recognized motifs (CGCG-core and CGTCT-core) (Table S5). 282

Within a 2 kb region, the cis-regulatory elements were detected in all HvHKT1s, but 283

not in HvHKT2s of barley (Fig 7D). Among the members of the barley HKT1 284

subfamily, the CGCG-core was only found in the promoters of HvHKT1;5 and 285

HvHKT1;1, while HvHKT1;3 and HvHKT1;4 specifically had only CGTCT-cores. 286

Moreover, only one CGCG-core site was found in the promoter of HvHKT1;5 287

(-818~-821 bp), which is quite close to the regions in HvHKT1;1 (-837~-840 bp for 288

CGCG-core; -823~-827 bp for CGTCT-core), and might be considered an important 289

cis-regulatory region (Fig 7D). This indicated that HvCaM1 likely regulates the 290

expression of HvHKT1;5 and HvHKT1;1 for Na+ transport through coordinated 291

interaction with HvCAMTA4. 292



11

Discussion 293

Down-regulation of HvCaM1 enhances salt tolerance in barley 294

In plants, CaMs are Ca2+

sensors responding rapidly to early signaling transduction 295

under salt stress (Zhang et al, 2012; Rao et al, 2014; Zhou et al, 2016). However, the 296

roles of CaM in the long-term response to salt stress remain elusive. Previously, we 297

discovered a salt-responding HvCaM1 in the analysis of the barley root proteome (Wu 298

et al, 2014; Shen et al, 2018). The current study shows that HvCaM1 is involved in 299

regulation of salt tolerance via the modulation of gene expression of Na+ transporters 300

HvHKT1;5 and HvHKT1;1 and HvCAMTA4-mediated transcriptional regulation. In 301

Arabidopsis, the knockout mutants Atcam1 and Atcam4 show hypersensitivity to salt 302

stress (Zhou et al, 2016). Similarly, higher expression of both rice OsCaM1 and 303

soybean GmCaM4 enhance salt tolerance (Chinpongpanich et al, 2012; Rao et al, 304

2014). However, we found that the knockdown (RNAi) lines of HvCaM1 showed a 305

large increase of salt tolerance, as reflected by their larger biomass than WT and OE 306

lines under 200 mM and 250 mM salt treatments (Figs 3 and 4). There are many 307

reports showing that gene silencing is beneficial for enhanced resistance and 308

production (e.g. AtWRKY11, OsMADS26 and TaDA1), but overexpressing the same 309

gene has negative effects in transgenic plants (Journot-Catalino et al, 2006; Khong et 310

al, 2015; Liu et al, 2020). One possible explanation is that constitutive overexpression 311

of a regulator gene can modulate the expression of downstream stress-associated 312

genes, which could impose a major energy cost to the plants (Munns et al, 2020; 313

Shabala et al, 2020). For example, drought hypersensitive OsMADS26-overexpressing 314

rice lines modulated 412 differentially-expressed genes (DEGs) as compared with 95 315

DEGs in the RNAi rice lines, which displayed an enhanced drought tolerance (Khong 316

et al, 2015). 317

HvCaM1 appears to be highly conserved in comparison with its closest 318

homologous genes, AtCaM1 and OsCaM1 (Figs 1 and S1). Why does it show a 319

different function in salt tolerance in barley? Our previous studies on the root 320

proteome found that HvCaM1 is significantly down-regulated in the salt-tolerant 321

barley genotypes (XZ16, XZ26 and CM72) under salt stress (Wu et al, 2014; Shen et 322

al, 2018). A plausible explanation is that distinct functions of CaMs among the plants 323

may be driven by natural selection or adaption favoring a complex survival system in 324

responding to external environments (Zhu et al, 2015; Zhao et al, 2019). Barley 325



12

originated from the Middle East where drought and high salinity are natural 326

environmental conditions (Nevo and Chen, 2010), in contrast to the natural habitats of 327

Arabidopsis, rice, and soybean (Hyten et al, 2006; Kovach et al, 2009; Beilstein et al, 328

2010). 329

Moreover, the expression level of HvCaM1 varied greatly with NaCl 330

concentrations and the exposed time in salt treatments (Fig 2B-C). As intracellular 331

Ca2+

sensors, the expression level of CaMs could be changed with cytosolic Ca2+

332

oscillation elicited by Na+ stress (Reddy et al, 2011; Ismail and Horie, 2017). In 333

comparison with barley, Arabidopsis, rice and soybean are less tolerant to salt (Munns 334

and Tester, 2008; Horie et al, 2012) and all CaM1s in these four species are linked to 335

salt tolerance/sensitivity. In addition, there is no difference in the protein sequence of 336

HvCaM1 among those genotypes (Table S1) and high similarity to CaM1s in 337

Arabidopsis, rice and soybean, suggesting its function is probably related to 338

post-transcriptional modification or translational regulatory networks (Batistič and 339

Kudla, 2012; Mahi et al, 2019). Therefore, unlike AtCaM1 and other CaM1s, 340

HvCaM1 may negatively regulate salt tolerance with function of long-term adaption 341

to salinity in barley, which has not been previously identified. 342

HvCaM1 may be involved in the modulation of long distance of Na+ transport 343

from roots to shoots 344

In general, roots have higher salt tolerance than shoots, and lower shoot Na+

345

concentration is mainly associated with less Na+ transport from roots, which is an 346

important characteristic for higher salt tolerance in most plants including barley 347

(Shabala et al, 2010; Zahra et al, 2014; Shen et al, 2016). Here, it is obvious that 348

HvCaM1 knockdown lines had significantly lower shoot Na+ concentration than that 349

of WT or OE lines under both 200 and 250 mM NaCl (Figs 3B and 4C). Lower shoot 350

Na+ concentration likely results from either the increase of root Na

+ exclusion or the 351

decrease of root-to-shoot Na+ transport (Wu et al, 2019; Huang et al, 2020). 352

Interestingly, we confirmed that the lower shoot Na+ concentration in RNAi lines is 353

closely associated with a lower Na+ transportation rate from roots to shoots (Figs 4E 354

and S4B) and lower xylem sap Na+ concentration (Fig 6), but with unchanged Na

+ 355

accumulation per plant (Fig 4D). These results indicated that HvCaM1 is involved in 356

root-to-shoot Na+ transportation, which is further supported by the preferential 357

expression of HvCaM1 in root stele and vascular systems (Fig 2A and 2E). Another 358



13

important factor that affects the amount of Na+ transport to the shoot is the rate of 359

xylem Na+ loading. The xylem Na

+ loading is thermodynamically active (Shabala, 360

2013) and is also mediated by either SOS1 or CCC transporters operating at the xylem 361

parenchyma interface (Colmenero-Flores et al, 2007; Ishikawa et al, 2018). Here we 362

showed that transcript levels of SOS genes are significantly downregulated in RNAi 363

lines (Fig 5G), which may restrict the amounts of xylem Na+ loading, thus reducing 364

Na+ transport between roots and shoots. However, it requires a detailed investigation 365

in the future. 366

High K+ accumulation and K

+/Na

+ ratios are beneficial for plants to cope with 367

salt stress (Chen et al, 2007; Shalala et al, 2010). In this study, RNAi lines had higher 368

K+ concentration and K

+/ Na

+ ratios than WT and OE plants (Figs 3D-E, 4F-G), 369

suggesting HvCaM1 might affect K+ homeostasis. However, there was no difference 370

in K+ transportation rate among these barley genotypes under salt stress (Figs 4H and 371

S4B), indicating that HvCaM1 is not involved in the regulation of K+ transportation. 372

In short, we propose that the preferential expression and cellular localization of 373

HvCaM1 makes it possible for barley to modulate long distance Na+ transport from 374

roots to shoots (Zhu, 2016). 375

HvCaM1 regulates Na+ transport via preferential transcriptional regulation of 376

HvHKT1s 377

CaMs may bind with quantitatively changed Ca2+

signals across membrane systems 378

(Gifford et al, 2013; Defalco et al, 2016; Demidchik et al, 2018). The major 379

membrane transporters conferring cellular and whole-plant Na+ homeostasis are HKTs 380

and NHXs/SOS1 (Zhu, 2002; Mahi et al, 2019; Munns et al, 2020; Shabala et al, 381

2020). Here, only some HKT1 members, in particular HvHKT1;5 and HvHKT1;1, 382

were significantly affected by the knockdown of HvCaM1 (Figs 5A-C, S6A-B). HKTs 383

regulate long distance Na+ delivery, depending on root-to-shoot Na

+ translocation and 384

Na+ exclusion from the reproductive organ (Cao et al, 2020). HvHKT1;5 negatively 385

regulates salt tolerance in barley (Huang et al, 2020). Thus, lower expression of 386

HvHKT1;5 in RNAi roots potentially indicates less Na+ transport from roots to shoots, 387

which is consistent with the function of HvCaM1 in Na+ transport. Correspondingly, 388

lower Na+ loading from roots to shoots via the xylem was confirmed with higher salt 389

tolerance by the down-regulation of HvCaM1 and HvHKT1;5 (Fig 6A-B; Huang et al, 390

2020). By contrast, another Na+ transporter, HvHKT1;1, plays a role in Na

+ retrieval 391



14

from shoots to roots, and overexpression of HvHKT1;1 in Arabidopsis reduces Na+ 392

accumulation (Han et al 2018). However, there is no report on the overexpressing and 393

silencing of HvHKT1;1 in barley, and the level of salt tolerance is very different 394

between barley and Arabidopsis, which requires further investigation. 395

The regulation of HvHKT1s by HvCaM1 probably occurs at transcriptional or 396

post-transcriptional levels (Reddy et al, 2011; Wang et al, 2015). In general, CaM 397

functions in transcriptional regulation through specific CBP transcription factors, such 398

as CAMTAs (Galon et al, 2010). CAMTA binds to Ca2+

/CaM and participates in 399

transcriptional regulation by recognizing and binding to a specific cis-element 400

(G/A/C)CGCG(C/G/T) (Yang and Poovaiah, 2002; Shen et al, 2015). AtCAMTA6 401

negatively regulates salt tolerance via HKT1, probably with the specific CGCG-core 402

or AREB co-operated CGTCT-core motif (Shkolnik et al, 2019). Here, we identified 403

an interacting partner of HvCaM1, HvCAMTA4, through yeast two-hybrid screening, 404

showing transcriptional function in the nucleus (Fig 7A-B; Table S4). Meanwhile, the 405

expressing pattern of HvCAMTA4 was similar to those of HvHKT1;5 and HvCaM1 406

(Fig 7C), indicating its possible role as a linker protein for HvCaM1 to 407

transcriptionally regulate the expression of HvHKT1;5. Interestingly both HvHKT1;1 408

and HvHKT1;5 contain regulatory elements (Figure 7D) for HvCaMT4 (Shkolnik et 409

al., 2019), and there are different cis-regulatory element sites in the promoter region 410

of HvHKT1;5 and HvHKT1;1. Therefore, it may be concluded that HvCAMTA4 411

regulates HvHKT1;5 independently while it modulates HvHKT1;1 together with 412

regulatory proteins for abscisic acid signaling in responses to salt stress (Whalley and 413

Knight, 2013). Moreover, HvHKT1;1 is an important Na+ transporter that confers salt 414

tolerance (Han et al., 2018), but HvHKT1;5 is a negative regulator of salt tolerance in 415

barley (Huang et al., 2020). Overall expression of the key salt-responsive genes 416

(HKTs, NHXs, SOSs) is down-regulated in the HvCaM1 RNAi lines with the 417

exception of HvHKT1;1. Interestingly, HvHKT1;1 and HvHKT1;5 in the R1 and R2 418

RNAi lines are similarly highly expressed after salt-induced upregulation and 419

downregulation at Week 3, respectively (Fig 5). This could be the explanation for the 420

differential expressions of HvHKT1;1 and HvHKT1;5 in HvCaM1 knockdown lines. 421

However, the possibility of other HvCaM1 interacting-proteins, such as CBP60b, 422

participating in the regulation of HKT1 transportation activity by post-transcriptional 423

modification requires future experimentation (Lin et al, 2014). 424

In summary, we found a transcriptional activator HvCAMTA4 interacts with 425



15

HvCaM1 under salt stress to regulate the expression of HvHKT1;5 and HvHKT1;1 at 426

different sites of cis-regulatory elements of the promoter regions. However, the 427

inherent interaction and signal transduction between HvCaM1 and HvCAMTA4 in 428

mediating HvHKT1 activity remains unknown. Meanwhile, the protein sequence of 429

HvCaM1 is quite conserved among plant species. Thus, RNAi or gene editing of 430

CaM1s may be used for improving salt tolerance of different crops. 431



16

Materials and methods 432

Growth condition and sampling 433

Barley (Hordeum vulgare) genotype c.v. Golden Promise (GP) was used for the 434

experiment. Barley germination and growth conditions were performed as described 435

in previous studies (Wu et al, 2014; Shen et al, 2016). In the hydroponic experiments, 436

salt treatment began at 14-d-old seedlings by adding 100 mM NaCl per day to reach a 437

final salt concentration of 100, 200, 250 or 300 mM, according to experimental 438

purposes. For a screening assay, the seedlings were exposed to 200 mM salt condition 439

and photographed every week. For gene expression analysis, roots and shoots were 440

sampled under normal conditions. Moreover, roots were sampled under 0, 100, 200 441

and 300 mM salt treatments, and at 0, 1, 2, 3 and 4 w under 200 mM salt level, 442

respectively. Normal condition without salt addition was used as the control. The 443

modified 1/5 Hoagland solution was renewed every 3 d. In the soil experiment, 444

seedlings were transplanted into 10 L containers with mixed artificial soil, supplied 445

with the same conditions as in Shen et al (2018). Soil salt treatment began at heading 446

stage by applying 1 L of 200 mM NaCl solution to the bottom of the pots every 3 d to 447

a final content of 2% NaCl (w/w). Seedlings supplied with tap water were used as the 448

control. Yield traits, including spikes per plant, seeds per spike, setting rate and kernel 449

weight were recorded. There were six biological replicates in both hydroponic and 450

soil experiments. 451

Gene cloning and bioinformatics analysis 452

HvCaM1 (HORVU0Hr1G001270) was cloned from GP. Single nucleotide 453

polymorphism and amino acid variation of HvCaM1 were validated among barley 454

genotypes (c.v. Morex, CM72 and GP, Tibetan wild barley accessions XZ16, XZ26 455

and XZ169) (Wu et al, 2014; Shen et al, 2018). Evolutionary bioinformatics were 456

performed according to Zhao et al. (2019) with some modifications as in Feng et al 457

(2020). The candidate sequences of CaM1 members among 60 representative plants 458

were selected from the 1000 plant Transcriptome (OneKP) and EnsemblPlants 459

databases by blast-p (Zhu et al, 2015; Leebens-Mack et al, 2019) (Table S2). Multiple 460

sequence alignment and phylogenetic analysis were performed by MAFFT and 461

FastTree via the maximum-likelihood method, respectively. The phylogenetic tree was 462

annotated by Interactive Tree of Life resource (http://itol.embl.de). Jalview was used 463

to perform the alignment of protein sequences. The primers used are listed in 464



17

Supplemental Table S6. 465

Plant transformation and verification 466

Genetic transformation was mediated by Agrobacterium tumefaciens strain AGL1 467

according to Harwood (2014) with some modification. To generate the RNAi 468

construct, a 388 bp specific fragment (primers RNAi_HvCaM1_F/R) of HvCaM1 469

transcript was cloned into pDONR-Zeo cloning vector by Gateway BP ClonaseTM

II 470

enzyme mix kit (11789, Invitrogen, USA), according to Miki et al (2004; 2005). The 471

positive cloning plasmid was then recombined with pANDA vector by Gateway LR 472

reaction (11791, Invitrogen, USA). To generate the overexpression plasmid, a 450 bp 473

complete coding region (primers OE_HvCaM1_F/R) of HvCaM1 was amplified and 474

then constructed into pBract214 vector with the similar method of RNAi construction. 475

There were more than 10 independent transgenic events of RNAi and overexpression. 476

Among them, 3 RNAi lines (R1, R2 and R3), 3 OE lines and 1 transgenically-negative 477

line (WTTN) were used as the materials. Only PCR-positive plants with specific test 478

primers (R_Test_F/R for RNAi and OE_Test_F/R for OE) were used in the following 479

experiments. 480

Gene expression analysis 481

The transcript levels of HvCaM1 and transporter-associated genes were detected by 482

qPCR. Briefly, total RNA of samples was extracted by MiniBEST kit and reversed by 483

PrimeScript™ RT reagent kit (Takara, Japan). Then qPCR was performed with SYBR 484

Green Supermix (Bio-Rad, USA) on a Roche LightCycler 480 instrument. For 485

absolute qPCR analysis, a pDONR plasmid containing the complete CDS region of 486

HvCaM1 was used to quantify the transcript concentration by diluting to 100, 10

-1, 487

10-2

, 10-3

, 10-4

, 10-5

ng μg-1

(a standard curve of R2 >0.999), as described by Wu et al 488

(2015). For relative gene expression analysis, the comparative 2-△△Ct

method was 489

performed using a reference gene α-Tublin (Wu et al, 2014). The transcripts of WT 490

(GP) roots under normal condition were regarded as the controls. The primers of HKT, 491

NHX and SOS genes were designed following Fu et al (2018) and Huang et al (2020). 492

There were four biological replicates and three technical repeats for gene expression 493

analysis. 494

Element determination and analysis 495



18

After 4 w salt exposure (200 or 250 mM), fresh weight (FW) of roots and shoots was 496

determined, and then tissues were dried in an oven at 80 °C for 2 d to obtain dry 497

weight (DW). Relative water content (RWC) was calculated using the formula: RWC 498

(%)= (FW-DW)/FW×100%. Dried samples were digested by HNO3 using microwave 499

digestion equipment (Multiwave 3000, Anton Paar GmbH, Australia) according to 500

Shen et al (2018). For element analysis, the concentrations of Na+, K

+ and Ca

2+ were 501

determined by an inductively coupled plasma-optical emission spectrometer 502

(ICP-OES, iCAP 6000 series, Thermo Fisher Scientific, USA). In addition, Na+ or K

+ 503

content was calculated based on concentration and DW, while the transportation rates 504

were also calculated as (shoot content) / (total content per plant) × 100%. There 505

were four biological replicates for element analysis. 506

Xylem sap analysis 507

Na+ and K

+ concentrations in the xylem sap were measured according to Huang et al 508

(2020). Four-week-old seedlings of the WT and RNAi lines (R1-R3) were exposed to 509

0 (control), 50 and 100 mM NaCl. After 2 and 7 d, the stems were cut 1-2 cm above 510

the root and shoot junction. The cut surface was cleaned using soft filter papers to 511

avoid contamination. Xylem sap exudates were collected within 1 h with a 512

micropipette and stored in ice-chilled Eppendorf tubes. About 10 µl sap was collected 513

for each replicate pooled from two to six plants and then diluted with 3-10 ml 2%(v/v) 514

HNO3 for ion measurement. The concentrations of Na+ and K

+ in the solution were 515

determined by an inductively coupled plasma mass spectrometry (ICP-MS, ELAN 516

DRC-e, PerkinElmer, USA). Four to six biological replicates for each treatment and 517

time point were used. 518

Tissue-specific and Subcellular localization 519

To generate the stable expression plasmid for tissue-specific localization, 520

approximately 2.1 kb upstream fragments of the HvCaM1 start was amplified with the 521

primers Pro_HvCaM1_F/R and then cloned into pCAMBIA1300 (deleted 35S 522

promoter) fused with the GFP coding region. The recombinant plasmid was 523

transferred into AGL1 to obtain genetically modified barley materials. Fresh root and 524

leaf samples of 14-d-old seedlings (verified by PCR with Pro_Test_F/R) were used to 525

observe GFP fluorescence under the confocal laser microscope at 488/490-540 nm. 526

To generate the transiently-expressing plasmid for subcellular localization, the 527



19

447 bp coding region of HvCaM1 without the stop code was amplified and fused with 528

the 5’ terminus of the sGFP gene into pCAMBIA1300 vector after linearization 529

digestion using the homologous recombination cloning kit (C112, Vazyme, Nanjing). 530

The recombinant and empty vectors driven by the 35S promoter were transformed 531

separately into onion (Allium cepa L.) epidermis cells by a biolistic PDS-1000/He 532

particle device (Bio-Rad, USA), along with a cell-localized RFP marker, as described 533

previously (Huang et al, 2020). GFP and RFP fluorescence signals were measured 534

under a confocal laser microscope (LSM780, Zeiss, Germany) at 488/490-540 nm and 535

561/580-630 nm, respectively. 536

in situ PCR 537

For in situ PCR analysis, a 388 bp sequence of the HvCaM1 transcript, same as in 538

RNA silencing, was amplified and performed according to Athman et al (2014) with 539

some modification, as described by Long et al (2018). In brief, 1-cm-length barley 540

leaf and root sections from 14-d-old seedlings were immersed into fixative solution 541

(63% ethanol, 5% acetic acid and 2% formaldehyde (v/v)) for 4 h at 4 °C. The 542

samples were then embedded in 5% (w/v) low melting-temperature agarose and cut 543

into 60~70 μm cross sections. After DNase I treatment (Takara, Japan), reverse 544

transcription was performed with gene specific reverse primers using DIG labelled 545

dUTP and KOD FX enzyme (Toyobo, Japan) for PCR amplification. The tested 546

samples were then incubated with anti-DIG-AP antibody, stained with BM purple 547

substrate (Roche), and mounted in 40% (v/v) glycerol after washing. The ribosomal 548

18 S transcript was amplified by in situ PCR as the positive control, while only water 549

without primer addition was used as a negative control. A Leica microscope was used 550

for observation and imaging, according to the color reaction between alkaline 551

phosphate and DIG-labeled antibody. 552

Yeast two-hybrid interaction assay 553

The candidate gene HvCAMTA4 (HORVU2Hr1G069950) was originally screened 554

from a yeast (Saccharomyces cerevisiae)library constructed with the total cDNA of 555

salt-treated barley roots by CloneMiner II kit (A11180, Invitrogen, USA). To verify 556

the interaction between HvCaM1 and HvCAMTA4, the cDNA of HvCAMTA4 was 557

amplified into pGADT7 as prey, while the coding region of HvCaM1 cloned into 558

pGBKT7 was used as a bait according to Cho et al (2016). The combination of 559

pGBKT7-53 and pGADT7-T was used as the positive control, while empty vector of 560



20

pGBKT7-Lam and pGADT7-T was used as the negative control. The yeast 561

two-hybrid interaction assay was performed after co-transformation of both the bait 562

and prey constructs into Y2HGold yeast competent cells (Clontech). The transformed 563

yeast cells were then screened on SD double dropout media (lacking Leu and Trp) 564

with or without Aureobasidin A (AbA) addition. 565

BIFC assay 566

For the BIFC interaction assay, the coding regions of HvCaM1 and HvCAMTA4 were 567

separately cloned into pSAT1-cEYFP/nEYFP vectors to generate HvCaM1-cEYFP 568

and nEYFP-HvCAMTA4 vectors by Vazyme homologous recombination kit. Barley 569

protoplast isolation and transformation were performed as described by Ye et al. 570

(2019). Seven-d-old barley seedlings were used to isolate the protoplast. The genes 571

HvSOS3 (HORVU1Hr1G080820, CBL4) and HvSOS2 (HORVU7Hr1G090260, 572

CIPK24) involved in the SOS pathway were used to generate HvCBL4-cEYFP and 573

nEYFP-HvCIPK24 vectors as the positive control, while HvCaM1-cEYFP and 574

nEYFP were used as the negative control. A total of 10 mg plasmid DNA of each 575

construct was transformed by polyethylene glycol into the barley protoplast. The YFP 576

fluorescence signals were detected by a confocal laser microscope at 514/520-550 nm. 577

Statistical analysis 578

The significant difference of dry weight, relative water content, element content, yield 579

and gene expression among genotypes, treatments or tissues were analyzed by 580

one-way ANOVA or independent t-test using SPSS 20.0 software (IBM SPSS 581

Statistics). The significance level at P<0.05 and P<0.01 were defined as significant 582

and highly significant, respectively. 583

Accession Numbers 584

All accession numbers and species for sequence aliment and gene expression are 585

listed in Supplemental Table S2. 586

Supplemental Data 587

Supplemental Figure S1. Cloning and sequence analysis of HvCaM1. 588

Supplemental Figure S2. Tissue-specific localization of HvCaM1. 589

Supplemental Figure S3. PCR verification in RNAi and OE lines. 590



21

Supplemental Figure S4. Physiological indexes of WT, RNAi and OE seedlings 591

under salt stress. 592

Supplemental Figure S5. Growth performance and yield traits of WT and RNAi 593

plants in the saline soil (n=6). 594

Supplemental Figure S6. Expression analysis of Na+ and K

+ transporters under salt 595

stress (remaining). 596

Supplemental Table S1. Sequence variation in the HvCaM1 CDS region of barley 597

genotypes. 598

Supplemental Table S2. Accession numbers for sequence alignment and gene 599

expression. 600

Supplemental Table S3. Dry weight of WT and RNAi seedlings under salt conditions 601

for 4 w (n=4). 602

Supplemental Table S4. Positive interaction candidates with HvCaM1 by yeast two 603

hybrid screening assay. 604

Supplemental Table S5. CAMTA cis-regulatory elements in the 3 kb promoters of 605

HKT genes. 606

Supplemental Table S6. The primer list used in this study. 607

Acknowledgements 608

We thank Dr. Peter Dominay (University of Glasgow, UK.) and Dr. Hiroyuki Tsuji 609

(Nara Institute of Science and Technology, Japan) for providing the vector pBract214 610

and pANDA, respectively. We also thank Dr. Yong Han (Murdoch University, 611

Australia), Dr. Zhonchang Wu and Dr. Jiming Xu (Zhejiang University, China) for the 612

technical support. 613



22

Figure legends 614

Figure 1. Phylogenetic tree and sequence analysis of HvCaM1. (A) Evolution 615

analysis of predicted CaM1 candidates in 60 representative species of the major 616

lineage of green plants. All sequences were downloaded from OneKP and 617

EnsemblPlants databases. (B) Sequence alignment of the two conserved EF-hand 618

domains among nine representative species of major lineages. Hv, Horduem vulgare; 619

Mt, Microcachrys tetragona; Pg, Polypodium glycyrrhiza; Sm, Selaginella 620

moellendorffii; Tl, Takakia lepidozioides; Ph, Paraphymatoceros hallii; Mp, 621

Marchantia polymorpha; Ks, Klebsormidium subtile; Pp, Pyramimonas parkeae. 622

Arrows indicate conserved amino acids in the four Ca2+

binding sites. 623

Figure 2. Expression pattern analysis of HvCaM1. (A) Absolute quantification of 624

HvCaM1 transcript in roots and shoots under normal condition. (B) Relative 625

expression of HvCaM1 in roots exposed to salt concentration gradients within 2 h. (C) 626

Relative HvCaM1 expression within 4 w of salt exposure (200 mM). * and ** 627

represent highly significant and significant at P<0.05 and P<0.01 level determined by 628

independent t-test, respectively (n=4, ±SE). (D) Subcellular localization of HvCaM1 629

in onion epidermal cells. Scale bars=50 μm. in situ PCR analysis of HvCaM1 630

transcript in (E) root and (F) leaf cross section of 14-d-old seedlings. s: stomatal guard 631

cell; pv: phloem vessel; xv: xylem vessel; bs: bundle sheath; ms: mesophyll; en: endodermis; 632

pe: pericycle; p: phloem; xp: xylem parenchyma; c: cortex. Scale bars=100 μm. 633

Figure 3. Phenotypic difference and ion responses of WT, RNAi and OE seedlings 634

under salt stress. (A) Phenotypes under 200 mM salt treatment and control condition. 635

Observation was conducted every week since 14-day-old seedling (referred as the ‘0’ 636

time point) until 4 w. Scale bar=10 cm. (B) Dry weight, (C) Na+ concentration, (D) K

+ 637

concentration and (E) K+/Na

+ ratio of barley genotypes under salt stress for 4 w. WT: 638

wild type; RNAi lines: R1, R2, R3; OE lines: OE73, OE74, OE15; WTTN: negative 639

lines; 4C: 4 week under control condition since 14-day-old seedlings. Data were 640

means of six replicates (n=6, ±SE). Different letters in each column represent 641

significant difference at P<0.05 determined by one-way ANOVA. 642

Figure 4. Na and K element analysis of WT and RNAi plants under salt 643

conditions. (A, B), WT and RNAi seedlings exposed to 200 mM (S200) and 250 mM 644

(S250) salt solutions for 4 w, respectively. Scale bar=10 cm. (C) Na+ concentration, 645



23

(D) Na+ content per plant, (E) Na

+ transportation rate, (F) K

+ concentration, (G) K

+ 646

content per plant and (H) K+ transportation rate of WT and RNAi plants under salt 647

stress. WT: wild type; RNAi lines: R1, R2, R3. Data used were means of four 648

replicates (n=4, ±SE). * and ** represent significant and highly significant at P<0.05 649

and P<0.01 determined by independent t-test, respectively. 650

Figure 5. Expression analysis of Na+ and K

+ transporters under salt stress. The 651

expression levels were investigated in WT and RNAi (R1 and R2) roots when 652

exposed to 200 mM NaCl for 0, 1, 2, and 3 weeks. (A) HvCaM1, (B) HvHKT1;1, (C) 653

HvHKT1;5, (D) HvNHX1, (E) HvNHX3, (F) HvNHX5, (G) HvSOS1, (H) HvSOS2 and 654

(I) HvSOS3. Data used were means of four replicates with standard deviation. 655

Figure 6. Na+ and K

+ concentrations in xylem sap of WT and RNAi plants under 656

control and salt conditions. Na+ (A, B) and K

+ (C, D) concentrations of xylem sap in 657

four-week-old WT and RNAi seedlings exposed to control, 50 mM and 100 mM NaCl 658

solutions for 2d (A,C) and 7 d (B, D) (n=4-6, ±SE). WT: wild type; RNAi lines: R1, 659

R2, R3. Different lowercase letters represent significant difference at P<0.05 660

determined by one-way ANOVA. 661

Figure 7. HvCaM1 interacts with HvCAMTA4 via specific binding sites in the 662

promoters of HKT1 genes. (A) Yeast interaction assay between HvCaM1 and 663

HvCAMTA4. (B) BiFC assay between HvCaM1 and HvCAMTA4 in barley protoplasts. 664

Scale bar=5μm. (C) The expression level of HvCAMTA4 in roots of WT and RNAi 665

under 200 mM NaCl for 3 w. * and ** represent the expression level is significant and 666

highly significant at P<0.05 and P<0.01 determined by independent t-test in roots of 667

WT and RNAi, respectively. Data used were means of four replicates with standard 668

deviation. (D) The cis-regulatory elements of CGCG-core and CGTGT-core along 3 669

kb promoter regions of HKT genes, marked as red and blue, respectively. The specific 670

sequences are enlarged in the promoter of HvHKT1;5 and HvHKT1;1. 671



24

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https://www.ncbi.nlm.nih.gov/pubmed?term=Apse MP%2C Aharon GS%2C Snedden WA%2C Blumwald E %281999%29 Salt tolerance conferred by overexpression of a vacuolar Na%2B%2FH%2B antiport in Arabidopsis%2E Science&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Barrag�n V%2C Leidi EO%2C Andr�s Z%2C Rubio L%2C De Luca A%2C Fern�ndez JA%2C Cubero B%2C Pardo JM %282012%29 Ion exchangers NHX1 and NHX2 mediate active potassium uptake into vacuoles to regulate cell turgor and stomatal function in Arabidopsis%2E Plant Cell&dopt=abstract

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https://scholar.google.com/scholar?as_q=Time-course of ionic responses and proteomic analysis of a Tibetan wild barley at early stage under salt stress&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Shen&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Shen QF%2C Yu JH%2C Fu LB%2C Wu LY%2C Dai F%2C Jiang LX%2C Wu DZ%2C Zhang GP %282018%29 Ionomic%2C metabolomic and proteomic analyses reveal molecular mechanisms of root adaption to salt stress in Tibetan wild barley%2E Plant Physiol Bioch&dopt=abstract


https://scholar.google.com/scholar?as_q=Ionomic, metabolomic and proteomic analyses reveal molecular mechanisms of root adaption to salt stress in Tibetan wild 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=Ionomic, metabolomic and proteomic analyses reveal molecular mechanisms of root adaption to salt stress in Tibetan wild barley&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Shen&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Shi H%2C Ishitani M%2C Kim C%2C Zhu JK %282000%29 The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na%2B%2FH%2B antiporter%2E Proc Natl Acad Sci USA&dopt=abstract

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https://scholar.google.com/scholar?as_q=The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/H+ antiporter&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 Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/H+ antiporter&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Shi&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Shi H%2C Quintero FJ%2C Pardo JM%2C Zhu JK %282002%29 The putative plasma membrane Na%2B%2FH%2B antiporter SOS1 controls long%2Ddistance Na%2B transport in plants%2E Plant Cell&dopt=abstract

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https://scholar.google.com/scholar?as_q=The putative plasma membrane Na+/H+ antiporter SOS1 controls long-distance Na+ transport 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=The putative plasma membrane Na+/H+ antiporter SOS1 controls long-distance Na+ transport in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Shi&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Shkolnik D%2C Finkler A%2C Pasmanik%2DChor M%2C Fromm H %282019%29 CALMODULIN%2DBINDING TRANSCRIPTION ACTIVATOR 6%3A A key regulator of Na%2B homeostasis during germination%2E Plant Physiol&dopt=abstract

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


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Shkolnik&as_ylo=2019&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wang R%2C Jing W%2C Xiao LY%2C Jin TK%2C Shen LK%2C Zhang WH %282015%29 The rice high%2Daffinity potassium transporter1%3B1 is involved in salt tolerance and regulated by an MYB%2Dtype transcription factor%2E Plant Physiol&dopt=abstract

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

https://scholar.google.com/scholar?as_q=The rice high-affinity potassium transporter1;1 is involved in salt tolerance and regulated by an MYB-type transcription factor&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 rice high-affinity potassium transporter1;1 is involved in salt tolerance and regulated by an MYB-type transcription factor&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wang&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Whalley HJ and Knight MR %282013%29 Calcium signatures are decoded by plants to give specific gene responses%2E New Phytol&dopt=abstract

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https://scholar.google.com/scholar?as_q=Calcium signatures are decoded by plants to give specific gene responses&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=Calcium signatures are decoded by plants to give specific gene responses&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Whalley&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wu DZ%2C Shen QF%2C Qiu L%2C Han Y%2C Ye LZ%2C Jabben Z%2C Shu QY%2C Zhang GP %282014%29 Identification of proteins associated with ion homeostasis and salt tolerance in barley%2E Proteomics&dopt=abstract

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https://scholar.google.com/scholar?as_q=Identification of proteins associated with ion homeostasis and salt tolerance 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=Identification of proteins associated with ion homeostasis and salt tolerance in barley&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wu&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1


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Xu XW, Liu M, Lu L, He M, Qu WQ, Xu Q, Qi XH, Chen XH (2015) Genome-wide analysis and expression of the calcium-dependentprotein kinase gene family in cucumber. Mol Genet Genomics 290: 1403-14


Yang T and Poovaiah BW (2002) A calmodulin-binding/CGCG box DNA binding protein family involved in multiple signaling pathways inplants. J Biol Chem 277: 45049-58


Ye LZ, Wang Y, Long LZ, Luo H, Shen QF, Sue B, Wu DX, Shu XL, Dai F, Li CD, Zhang GP (2019) A trypsin family protein gene controlstillering and leaf shape in barley. Plant Physiol DOI: 10.1104/pp.19.00717


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Zhang H, Han B, Wang T, Chen S, Li HY, Zhang YH, Dai SJ (2012) Mechanisms of plant salt response: insights from proteomics. JProteome Res 11: 49-67


Zhang HX and Blumwald E (2001) Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nature Biotech 19:765-8


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Zhao CC, Wang YY, Chan KX, Marchant DB, Franks PJ, Randall D, Tee EE, Chen G, et al. (2019) Evolution of chloroplast retrogradesignaling facilitates green plant adaptation to land. Proc Natl Acad Sci USA 116(11): 5015-20


Zhou S, Jia LX, Chu HY, Wu D, Peng X, Liu X, Zhang JJ, Zhao JF, Chen KM, Zhao LQ (2016) Arabidopsis CaM1 and CaM4 promote nitricoxide production and salt resistance by inhibiting S-nitrosoglutathione reductase via direct binding. PLoS Genet 12: e1006255


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https://www.ncbi.nlm.nih.gov/pubmed?term=Wu DZ%2C Yamaji N%2C Yamane M%2C Kashino M%2C Sato K%2C Ma JF %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=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=Wu HH%2C Shabala L%2C Zhou MX%2C Su N%2C Wu Q%2C UI%2DHaq T%2C Zhu J%2C Mancuso S%2C Azzarello E%2C Shabala S %282019%29 Root vacuolar Na%2B sequestration but not exclusion from uptake correlates with barley salt tolerance%2E Plant J&dopt=abstract


https://scholar.google.com/scholar?as_q=Root vacuolar Na+ sequestration but not exclusion from uptake correlates with barley salt tolerance&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 vacuolar Na+ sequestration but not exclusion from uptake correlates with barley salt tolerance&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wu&as_ylo=2019&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Xi J%2C Qiu YJ%2C Du LQ%2C Poovaiah BW %282012%29 Plant%2Dspecific trihelix transcription factor AtGT2L interacts with calcium%2Fcalmodulin and responds to cold and salt stresses%2E Plant Sci&dopt=abstract

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

https://scholar.google.com/scholar?as_q=Plant-specific trihelix transcription factor AtGT2L interacts with calcium/calmodulin and responds to cold and salt stresses&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=Plant-specific trihelix transcription factor AtGT2L interacts with calcium/calmodulin and responds to cold and salt stresses&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xi&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Xu XW%2C Liu M%2C Lu L%2C He M%2C Qu WQ%2C Xu Q%2C Qi XH%2C Chen XH %282015%29 Genome%2Dwide analysis and expression of the calcium%2Ddependent protein kinase gene family in cucumber%2E Mol Genet Genomics&dopt=abstract

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

https://scholar.google.com/scholar?as_q=Genome-wide analysis and expression of the calcium-dependent protein kinase gene family in cucumber&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=Genome-wide analysis and expression of the calcium-dependent protein kinase gene family in cucumber&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xu&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yang T and Poovaiah BW %282002%29 A calmodulin%2Dbinding%2FCGCG box DNA binding protein family involved in multiple signaling pathways in plants%2E J Biol Chem&dopt=abstract

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

https://scholar.google.com/scholar?as_q=A calmodulin-binding/CGCG box DNA binding protein family involved in multiple signaling pathways 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=A calmodulin-binding/CGCG box DNA binding protein family involved in multiple signaling pathways in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yang&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Ye LZ%2C Wang Y%2C Long LZ%2C Luo H%2C Shen QF%2C Sue B%2C Wu DX%2C Shu XL%2C Dai F%2C Li CD%2C Zhang GP %282019%29 A trypsin family protein gene controls tillering and leaf shape in barley%2E Plant Physiol DOI%3A 10%2E1104%2Fpp&dopt=abstract

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

https://scholar.google.com/scholar?as_q=A trypsin family protein gene controls tillering and leaf shape 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=A trypsin family protein gene controls tillering and leaf shape in barley.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ye&as_ylo=2019&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yoo JH%2C Park CY%2C Kim JC%2C Heo WD%2C Cheong MS%2C Park HC%2C et al%2E %282005%29 Direct interaction of a divergent CaM isoform and the transcription factor%2C MYB2%2C enhances salt tolerance in Arabidopsis%2E J Biol Chem&dopt=abstract

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https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yoo&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zahra J%2C Nazim H%2C Cai SG%2C Han Y%2C Wu DZ%2C Zhang BL%2C Haider SI%2C Zhang GP %282014%29 The influence of salinity on cell ultrastructures and photosynthetic apparatus of barley genotypes differing in salt stress tolerance%2E Acta Physiologiae Plantarum&dopt=abstract

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

https://scholar.google.com/scholar?as_q=The influence of salinity on cell ultrastructures and photosynthetic apparatus of barley genotypes differing in salt stress tolerance&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 influence of salinity on cell ultrastructures and photosynthetic apparatus of barley genotypes differing in salt stress tolerance&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zahra&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhang H%2C Han B%2C Wang T%2C Chen S%2C Li HY%2C Zhang YH%2C Dai SJ %282012%29 Mechanisms of plant salt response%3A insights from proteomics%2E J Proteome Res&dopt=abstract

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https://scholar.google.com/scholar?as_q=Mechanisms of plant salt response: insights from proteomics&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=Mechanisms of plant salt response: insights from proteomics&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhang HX and Blumwald E %282001%29 Transgenic salt%2Dtolerant tomato plants accumulate salt in foliage but not in fruit%2E Nature Biotech&dopt=abstract


https://scholar.google.com/scholar?as_q=Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit&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=Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhang W%2C Zhou RG%2C Gao YJ%2C Zheng SZ%2C Xu P%2C Zhang SQ%2C Sun DY %282009%29 Molecular and genetic evidence for the key role of AtCaM3 in heat%2Dshock signal transduction in Arabidopsis%2E Plant Physiol&dopt=abstract


https://scholar.google.com/scholar?as_q=Molecular and genetic evidence for the key role of AtCaM3 in heat-shock signal transduction 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=Molecular and genetic evidence for the key role of AtCaM3 in heat-shock signal transduction in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhao CC%2C Wang YY%2C Chan KX%2C Marchant DB%2C Franks PJ%2C Randall D%2C Tee EE%2C Chen G%2C et al%2E %282019%29 Evolution of chloroplast retrograde signaling facilitates green plant adaptation to land%2E Proc Natl Acad Sci USA&dopt=abstract

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=Evolution of chloroplast retrograde signaling facilitates green plant adaptation to land&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=Evolution of chloroplast retrograde signaling facilitates green plant adaptation to land&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhao&as_ylo=2019&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhou S%2C Jia LX%2C Chu HY%2C Wu D%2C Peng X%2C Liu X%2C Zhang JJ%2C Zhao JF%2C Chen KM%2C Zhao LQ %282016%29 Arabidopsis CaM1 and CaM4 promote nitric oxide production and salt resistance by inhibiting S%2Dnitrosoglutathione reductase via direct binding%2E PLoS Genet 12%3A e&dopt=abstract

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

https://scholar.google.com/scholar?as_q=Arabidopsis CaM1 and CaM4 promote nitric oxide production and salt resistance by inhibiting S-nitrosoglutathione reductase via direct binding.&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=Arabidopsis CaM1 and CaM4 promote nitric oxide production and salt resistance by inhibiting S-nitrosoglutathione reductase via direct binding.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhou&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhu JK %282002%29 Salt and drought stress signal transduction in plants%2E Annu Rev Plant Biol&dopt=abstract

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

https://scholar.google.com/scholar?as_q=Salt and drought stress signal transduction 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=Salt and drought stress signal transduction in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhu&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhu JK %282016%29 Abiotic stress signaling and responses in plants%2E Cell&dopt=abstract


https://scholar.google.com/scholar?as_q=Abiotic stress signaling and responses 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=Abiotic stress signaling and responses in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhu&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhu XY%2C Dunand C%2C Snedden W%2C Galaud JP %282015%29 CAM and CML emergence in the green lineage%2E Trends Plant Sci&dopt=abstract


Google Scholar: Author Only Title Only Author and Title



https://scholar.google.com/scholar?as_q=CAM and CML emergence in the green lineage&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=CAM and CML emergence in the green lineage&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhu&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1


1 short title: hvcam1 negatively regulates salt tolerance · 2020-06-18 · 1 1. s. hort. title:...

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