argos regulates ethylene signaling and response corresponding

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Running head: 2

ARGOS regulates ethylene signaling and response 3

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Corresponding author: 7

Jinrui Shi 8

DuPont Pioneer 9

7300 NW 62nd Ave. 10

PO Box 1004 11

Johnston, IA 50131 12

Phone: (515) 535-2196 13

Fax: (515) 535-3934 14

E-mail: [email protected] 15

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Plant Physiology Preview. Published on July 28, 2015, as DOI:10.1104/pp.15.00780

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Title: 17

Over-expression of ARGOS genes modifies plant sensitivity to ethylene, leading to 18

improved drought tolerance in both Arabidopsis and maize 19

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Jinrui Shi*, Jeffrey E. Habben, Rayeann L. Archibald, Bruce Drummond, Mark A. Chamberlin, 21

Robert Williams, Renee Lafitte, Ben Weers 22

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DuPont Pioneer; 7300 NW 62nd Avenue; PO Box 1004; Johnston, IA 50131-1004; U.S.A. 24

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One-sentence summary: 27

Over-expressed ARGOS negatively regulates the ethylene signal transduction pathway by 28

targeting the ethylene signaling protein complex localized on the ER and Golgi membrane and 29

transgenic Zm-ARGOS8 maize plants have improved drought tolerance. 30

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Keywords: ethylene, maize, grain yield, drought tolerance, ARGOS, phytohormone 32

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Corresponding author: 34

Jinrui Shi 35

E-mail: [email protected] 36

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Abstract 38 39

Lack of sufficient water is a major limiting factor to crop production worldwide and developing 40

drought tolerant germplasm is needed to improve crop productivity. The phytohormone 41

ethylene modulates plant growth and development as well as plant response to abiotic stress. 42

Recent research has shown that modifying ethylene biosynthesis and signaling can enhance 43

plant drought tolerance. Here we report novel negative regulators of ethylene signal 44

transduction in Arabidopsis and maize. These regulators are encoded by the ARGOS gene 45

family. In Arabidopsis, over-expression of maize ARGOS1 (Zm-ARGOS1), Zm-ARGOS8, 46

Arabidopsis ARGOS-LIKE2 (At-ARL2) and At-ARL3 reduced plant sensitivity to ethylene, leading 47

to enhanced drought tolerance. RNA profiling and genetic analysis suggested that the Zm-48

ARGOS1 transgene acts between an ethylene receptor and CONSTITUTIVE TRIPLE RESPONSE1 49

(CTR1) in the ethylene signaling pathway, affecting ethylene perception or the early stages of 50

ethylene signaling. Over-expressed Zm-ARGOS1 is localized to the ER and Golgi membrane 51

where the ethylene receptors and the ethylene signaling protein ETHYLENE-INSENSITIVE2 and 52

REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) reside. In transgenic maize plants, over-53

expression of ARGOS genes also reduces ethylene sensitivity. Moreover, field testing showed 54

that UBI:Zm-ARGOS8 maize events had a greater grain yield than non-transgenic controls under 55

both drought stress and well-watered conditions. 56

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There is an increasing demand for food and feed due to global population growth, urbanization 58

and rapid middle-class emergence. Lack of water limits crop yields worldwide; Bot et al. (2000) 59

estimated that 45% of the agricultural lands are subject to continuous or frequent drought 60

conditions. Drought tolerant varieties can reduce the impact of drought on crop productivity. 61

The phytohormone ethylene regulates many aspects of plant growth and development from 62

seed germination, leaf expansion and floral transition to organ senescence, fruit ripening, and 63

the response to abiotic stresses, such as drought, high temperature, freezing, shading and 64

nutrient deficiency. Ethylene is one of the most widely used hormones in agriculture to increase 65

yield and reduce production costs. For example, ethylene can reduce lodging in wheat and 66

barley by shortening the stem, therefore improving grain yield and quality. Studies have shown 67

that inhibitors of ethylene biosynthesis and perception can mitigate yield loss by enhancing 68

plant tolerance to abiotic stresses, such as drought, heat and a combination of both (Hay et al., 69

2007; Huberman et al., 2014; Kawakami et al., 2010, 2013). The present study explores the 70

potential to improve crop performance by modifying ethylene sensitivity. 71

At the molecular level, ethylene responses in Arabidopsis are initiated by binding of 72

ethylene to a family of endoplasmic reticulum (ER) and Golgi membrane-localized receptors, 73

including ETHYLENE RESPONSE1 (ETR1), ETR2, ETHYLENE RESPONSE SENSOR1 (ERS1), ERS2, and 74

ETHYLENE-INSENSITIVE4 (EIN4) (Chang et al., 1993; Hua and Meyerowiz, 1998). The ethylene 75

signal is transduced from the receptors to the nuclear protein EIN3 via CONSTITUTIVE TRIPLE 76

RESPONSE1 (CTR1) and EIN2 (Qiao et al., 2012; Ju et al., 2012). CTR1 is a Raf-like kinase and 77

physically interacts with the receptors (Huang et al., 2003). EIN3 and EIN3-LIKE1 (EIL1) are the 78

master transcription factors controlling ethylene responsive gene expression (Chao et al., 79



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1997). EIN2, an ER-tethered protein, functions as a shuttle to transduce the signal from the 80

membrane to the nucleus via its cleavable carboxyl-terminal domain (Qiao et al., 2012; Ju et al., 81

2012). Proteins that modulate ethylene perception include REVERSION-TO-ETHYLENE 82

SENSITIVITY1 (RTE1) and RESPONSIVE-TO-ANTAGONIST1, the former promoting the activity of 83

ETR1 (Resnick et al., 2006, 2008; Dong et al., 2010), and the latter, a transporter, providing the 84

ethylene cofactor copper and also playing a role in the biogenesis of active ethylene receptors 85

(Hirayama et al., 1999; Binder et al., 2010). Many aspects of the ethylene signaling pathway 86

and the sequence of the proteins involved are conserved between dicots and monocots 87

(Guillaume et al., 2008). 88

Beltrano et al. (1999) reported that exogenous application of the ethylene biosynthesis 89

inhibitor aminoethoxyvinylglycine reversed a drought stress syndrome in wheat. Transgenic 90

maize plants with reduced ethylene biosynthesis, via silencing 1-aminocyclopropane-1-91

carboxylate synthase6 (ACS6), have shown enhanced yields compared to non-transgenic 92

controls in water deficit and low nitrogen environments (Habben et al., 2014). Here we report 93

novel, negative regulators of ethylene signal transduction from maize and Arabidopsis. These 94

regulators are encoded by the ARGOS gene family, whose first member was identified in 95

Arabidopsis (Hu et al., 2003). ARGOS genes encode a predicted integral membrane protein 96

(Supplementary Fig. S1), and are known to promote plant organ growth by increasing cell 97

number and/or cell size when over-expressed in Arabidopsis (Hu et al., 2003, 2006; Feng et al., 98

2011; Wang et al., 2009). Previously, the phenotype of enlarged leaves in At-ARGOS 99

overexpression Arabidopsis was interpreted as a result of prolonged expression of 100

AINTEGUMANTA and CycD3;1, and At-ARGOS was proposed to function downstream of AUXIN-101



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RESISTANT1, as a signaling component in the auxin signal transduction pathway (Hu et al., 102

2003). An earlier study indicates that Arabidopsis ARGOS-LIKE1 (At-ARL1) also regulates lateral 103

organ size, but does not affect cell proliferation in Arabidopsis. Instead, At-ARL1 promotes cell 104

expansion. At-ARL1 was hypothesized to act downstream of BRASSINOSTEROID-INSENSITIVE1 in 105

the brassinosteroid signaling pathway (Hu et al., 2006). However, in both cases, the function of 106

the over-expressed genes was not established. We found that ARGOS genes, when over-107

expressed, reduce plant (Arabidopsis and maize) sensitivity to ethylene. Over-expressed ARGOS 108

targets the ethylene receptor complex, possibly affecting ethylene perception as well as 109

ethylene signal transduction. We also found that over-expression of ARGOS in maize results in 110

improved grain yields under both drought stress and well-watered conditions. 111

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Results 114 115

Over-expression of Zm-ARGOS1 confers ethylene insensitivity in Arabidopsis 116

Eight family members of ARGOS have been identified in maize and four in Arabidopsis 117

(Supplementary Fig. S1). We initially focused our attention on one of these members: Zm-118

ARGOS1. To determine its molecular function, Zm-ARGOS1 was over-expressed in Arabidopsis 119

under the control of the cauliflower mosaic virus 35S promoter (35S). Transgene expression 120

was confirmed in eight events by Northern blotting (Supplementary Fig. S2). The 35S:Zm-121

ARGOS1 Arabidopsis plants had wider and longer leaves than wild-type (WT) at bolting time as 122

well as flowering time was delayed 3-10 days dependent on growth conditions (Fig. 1A), similar 123

to Arabidopsis over-expressing At-ARGOS genes (Hu et al., 2003, 2006; Feng et al., 2011) and 124

rice ARGOS (Wang et al., 2009). In WT plants, perianth organs in flowers abscised soon after 125

pollination and inflorescences generally had 3-5 opened flowers. In contrast, petals and sepals 126

of the 35S:Zm-ARGOS1 plants remained turgid and intact for a longer time, and the 127

inflorescences had 7-10 opened flowers (Supplementary Fig. S3). These phenotypes of enlarged 128

leaves, delayed flowering time and delayed flower senescence were also reported in the 129

ethylene insensitive mutant etr1-1 and ein2-1 (Guzman and Ecker, 1990; Ogawara et al., 2003; 130

Patterson and Bleecker, 2004), suggesting that ARGOS may be involved in the ethylene 131

pathway. 132

To investigate the effect of 35S:Zm-ARGOS1 on Arabidopsis responses to ethylene, 133

seeds were germinated in the presence of ethylene or the precursor 1-aminocyclopropane-1-134

carboxylic acid (ACC) (Fig. 1, B and C, Supplementary Fig. S4). Etiolated seedlings of WT showed 135



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inhibition of hypocotyl and root growth, exaggerated curvature of the apical hook and excessive 136

radical swelling of the hypocotyl (Fig. 1B), which is the typical triple response of Arabidopsis to 137

ethylene exposure (Guzman and Ecker, 1990). However, the triple response phenotype was 138



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absent in the etiolated 35S:Zm-ARGOS1 seedlings (Fig. 1, B and C), demonstrating that the 139

35S:Zm-ARGOS1 plants are insensitive to exogenous ethylene. 140

To determine the response to endogenous ethylene, the ethylene over-producer1-1 141

(eto1-1) mutant (Chae et al., 2003) was transformed with 35S:Zm-ARGOS1 and transgene 142

expression confirmed by RT-PCR (Supplementary Fig. S5). In the three independent events 143

examined, overexpression of Zm-ARGOS1 overrode the constitutive ethylene response in the 144

etiolated eto1-1 seedlings (Fig. 1, D and E). The light-grown eto1-1 mutant plants flowered 145

earlier than WT, but the floral transition in 35S:Zm-ARGOS1 eto1-1 plants was delayed (Fig. 1F) 146

relative to eto1-1, similar to that of 35S:Zm-ARGOS1 in a WT background. The delayed flowering 147

time was observed in all 10 independent events tested. These results confirmed that the 148

35S:Zm-ARGOS1 plants are insensitive to ethylene. 149

To further verify the activity of Zm-ARGOS1 in conferring ethylene insensitivity, a 150

mutant version, Zm-ARGOS1(L104D), was generated by substituting the leucine 104 with 151

aspartate in the highly conserved proline-rich motif (Supplementary Fig. S1). Western analysis 152

showed that the leucine substitution did not negatively affect the protein expression level in 153

transgenic plants (Supplementary Fig. S6). The 35S:Zm-ARGOS1(L104D) Arabidopsis plants were 154

sensitive to ACC (Fig. 1G) and flowering time was equivalent to non-transgenic controls (data 155

not shown), indicating that the ethylene insensitivity in the 35S:Zm-ARGOS1 plants is 156

dependent on the Zm-ARGOS1 protein. 157

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Overexpression of Zm-ARGOS8 and Arabidopsis ARGOS genes also decrease ethylene 159

sensitivity in Arabidopsis 160

To determine if other ARGOS genes can modulate ethylene responses as well, Zm-ARGOS8, At- 161

ARGOS-LIKE2 (At-ARL2) and At-ARL3 were overexpressed in Arabidopsis. The 35S:At-ARL2 and 162

35S:At-ARL3 etiolated seedlings displayed the ethylene insensitive phenotype in the triple 163

response assay, as did the 35S:Zm-ARGOS1 plants (Fig. 2). Overexpression of Zm-ARGOS8 164

significantly reduced the ethylene-induced triple response as well, but the phenotype was 165

weaker than that of the 35S:Zm-ARGOS1 plants (Fig. 2; Supplementary Fig. S7). Like Zm-166

ARGOS1(L104D), a mutated version of Zm-ARGOS8, Zm-ARGOS8(L67D), was not able to confer 167

ACC insensitivity (Fig. 2). 168

169

Ethylene biosynthesis is increased, but the expression of ethylene inducible genes is down-170

regulated in Zm-ARGOS1 Arabidopsis plants 171

Previous research has shown that the etr1-1 and ein2-1 mutants have increased ethylene 172

biosynthesis (Guzman and Ecker, 1990). Therefore, we determined ethylene emission in 173

35S:Zm-ARGOS1 leaves and found they released 5 to 7-fold more ethylene than the vector 174

control and WT plants (Fig. 3A). We would predict concomitant induced expression of ethylene-175

inducible genes if the transgenic plant had sensed ethylene normally. Northern analysis, 176

however, showed that the steady-state levels of mRNA for the ethylene inducible EIN3-177

BINDING F-BOX PROTEIN2 (EBF2) and ETHYLENE RESPONSE FACTOR5 (ERF5) were decreased in 178

the 35S:Zm-ARGOS1 plants relative to the vector control (Fig. 3B). In the aerial tissues (rosette 179



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leaves and apical meristem) of 19-day-old plants, transcript levels of the EIN3/EIL1-activated 180

EBF2, PORA, PORB, FLS2 and EDF genes (Konishi and Yanagisawa, 2008; Zhong et al., 2009; 181

Boutrot et al., 2010; Alonso et al., 2003) were down-regulated in the 35S:Zm-ARGOS1 plants 182

while the EIN3/EIL1-repressed SID2 (Chen et al., 2009) were up-regulated, as revealed by RNA-183

seq analysis (Table 1). Expression of nine ERF genes was reduced at least 50% relative to the 184

vector control. Among these genes, ERF1, 2, 4, 5, 9, 11 and 72 were reported inducible by 185

ethylene (Solano et al., 1998; Buttner and Singh, 1997; Fujimoto et al., 2000). ERF3 is not 186



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responsive to ethylene treatments (Fujimoto et al., 2000) and we found that its expression was 187

not changed in the 35S:Zm-ARGOS1 plants. As predicted, expression of the ERF-regulated 188

defensin genes PDF1.2, Chitinase, CHI-B and PR4 (Solano et al., 1998; Fujimoto et al., 2000) was 189

reduced (Table 1). These results confirmed that the 35S:Zm-ARGOS1 transgenic plants were 190

unable to properly sense endogenous elevated ethylene levels and suggested that Zm-ARGOS1 191

may act on the ethylene signaling components upstream of EIN3/EIL1. 192

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Zm-ARGOS1 targets the upstream components of the ethylene signal transduction pathway 194

ARGOS family is composed of small integral membrane proteins containing unconserved N- and 195

C-terminal regions and two transmembrane helices which flank a highly conserved proline-rich 196

motif of 8 amino acids (Supplementary Fig. 1S). As an example, At-ARL3 has only 67 amino acid 197

residues. Protein sequence analysis did not reveal any catalytic sites in ARGOS proteins. There is 198

no evidence indicating that ARGOS is one of the signaling cascade steps to relay the ethylene 199

signal. Therefore, we hypothesized that ARGOS may play a regulatory role by directly, or 200

indirectly, modifying the ethylene signaling components. To determine where Zm-ARGOS1 acts 201

in the pathway, the ctr1-1 Arabidopsis mutant was transformed with 35S:Zm-ARGOS1 and 202

transgene expression confirmed by RT-PCR (Supplementary Fig. S5). Of 15 independent events 203

examined, the light-grown 35S:Zm-ARGOS1 ctr1-1 plants all displayed the characteristic 204

constitutive ethylene response phenotype (Fig. 4A), as did the ctr1-1 mutant (Kieber et al., 205

1993). In addition, the event C2 over-expressing the FLAG-HA epitope-tagged Zm-ARGOS1 206

(Supplementary Fig. S6) in a wild-type background was crossed with the ctr1-1 mutant to 207

generate the 35S:Zm-ARGOS1 ctr1-1 plants. Like those events produced by directly 208

transforming ctr1-1, the 35S:Zm-ARGOS1 ctr1-1 plants also showed the same phenotype as the 209

ctr1-1 mutant (Fig. 4B). 210

Under dark conditions the etiolated seedlings of the 35S:Zm-ARGOS1 ctr1-1 Arabidopsis 211

exhibited the exaggerated curvature of the apical hook and inhibited growth of hypocotyls and 212

roots in the absence of exogenously supplied ethylene (Fig. 4C), similar to the ctr1-1 mutant 213

(Kieber et al., 1993). In the three independent events tested, no difference in hypocotyl or root 214



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lengths was detected between the 35S:Zm-ARGOS1 ctr1-1 Arabidopsis plants and the ctr1-1 215

plants carrying empty vector (Fig. 4D). Since CTR1 acts as a suppressor in the ethylene signal 216



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transduction pathway, these results suggested that the signaling pathway downstream of CTR1 217

functions properly even in the presence of over-expressed Zm-ARGOS1. 218

The 35S:Zm-ARGOS1 plants in a WT background from two events, 2B3 and 2C6, were 219

crossed with the ETR1 null allele etr1-7 and the RTE1 loss-of-function mutant rte1-2. Both 220

mutants have increased ethylene sensitivity (Hua and Meyerowitz, 1998; Resnick et al., 2006). 221

The triple response assay showed that the 35S:Zm-ARGOS1 rte1-2 Arabidopsis plants had 222

reduced ethylene sensitivity relative to WT and rte1-2 (Fig. 4, E and F). When seedlings were 223

grown vertically on agar in the presence of ACC under light (Ruzicka et al., 2007), the rte1-2 224

mutant had shorter roots and more pronounced root hairs due to increased ethylene sensitivity 225

relative to WT (Fig. 4G). Roots of the 35S:Zm-ARGOS1 rte1-2 plants were longer than that of WT 226

and rte1-2 in the absence and presence of 0.2 µM ACC (Fig. 4, G and H), confirming reduced 227

ethylene sensitivity in the rte1-2 mutant over-expressing Zm-ARGOS1. Similarly, the 35S:Zm-228

ARGOS1 etr1-7 plants showed reduced ethylene sensitivity relative to WT and etr1-7 in both 229

assays (Supplementary Figs. S8 and S9). 230

Because RTE1 physically interacts with ETR1 in Arabidopsis and modifies the activity of 231

the ethylene receptor (Resnick et al., 2006, 2008; Dong et al., 2010), we tested Zm-ARGOS1 232

over-expression in an etr1-7 rte1-2 double mutant of Arabidopsis. The 35S:Zm-ARGOS1 etr1-7 233

rte1-2 plants were generated by crossing the 35S:Zm-ARGOS1 event 2B3 with the double 234

mutant followed by self-pollination. Under light growth conditions, the 35S:Zm-ARGOS1 etr1-7 235

rte1-2 plants were less sensitive to 0.2 and 0.5 µM ACC relative to the double mutant (Fig. 5A, 236

Supplementary Fig. S10). However, the ethylene insensitive phenotype caused by Zm-ARGOS1 237



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over-expression is weaker in the double mutant background than the WT background (Fig. 5A). 238

In addition, the root inhibition in the 35S:Zm-ARGOS1 etr1-7 rte1-2 plants was responsive to 239

ACC concentrations, but was not in the WT plants over-expressing Zm-ARGOS1 (Fig. 5A). These 240

results indicate that the etr1-7 rte1-2 double mutation partially suppress the ethylene 241

insensitive phenotype of Zm-ARGOS1 over-expression. 242

Ethylene response of the 35S:Zm-ARGOS1 etr1-7 rte1-2 plants was also tested in the 243

triple response assay under dark. Although the etiolated 35S:Zm-ARGOS1 etr1-7 rte1-2 244



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seedlings had reduced sensitivity to 0.5 µM ACC relative to WT and the double mutant (data 245

not shown), they displayed the typical triple response phenotype under 10 µM ACC (Fig. 5B). To 246

make sure Zm-ARGOS1 is expressed in the transgenic plants and to test additional independent 247

events, we used the FLAG-HA epitope tagged Zm-ARGOS1 construct to transform the etr1-7 248

rte1-2 double mutant. Transgene expression in 10 events was determined by Western blotting 249

(Fig. 5C) and four events (E1, E4, E7 and E9) were chosen for phenotyping. In the triple response 250

assay under dark, etiolated 35S:Zm-ARGOS1 etr1-7 rte1-2 seedlings showed reduced sensitivity 251

to 0.5 µM ACC relative to WT and the double mutant (Fig. 5D). However, they displayed the 252

typical triple response phenotype under 10 µM ACC (Fig. 5D), as the 2B3 event in the double 253

mutant background did (Fig. 5B), confirming that Zm-ARGOS1 is not able to properly function in 254

rte1-2 mutant seedlings when ETR1 is absent and concentrations of exogenously supplied ACC 255

is high. A normal interaction between RTE1 and ETR1 likely is required for Zm-ARGOS1 256

conferring ethylene insensitivity in Arabidopsis seedlings. Taken together, these results 257

suggested that Zm-ARGOS1 may act between the ethylene receptor and CTR1, affecting 258

ethylene perception or the early stages of ethylene signal transduction. 259

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Zm-ARGOS1 is localized in the ER and Golgi membranes 261

Sequence analysis with PRODIV-TMHMM (Hakan and Elofsson, 2004) predicted that Zm-262

ARGOS1 and other family members contain two transmembrane alpha-helices. Cell 263

fractionation analysis showed that the FLAG-HA tagged Zm-ARGOS1 was present in the 264

microsomal fraction, but non-detectable in the soluble fraction (Fig. 6A), confirming that Zm-265



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ARGOS1 is a membrane protein. To determine the subcellular localization, Ac-GFP1 was fused 266

to the C-terminus of Zm-ARGOS1 and the construct transfected into Arabidopsis. In stable 267

transgenic plants, the GFP tag did not affect Zm-ARGOS1 function in conferring ethylene 268

insensitivity (data not shown). Fluorescent microscopy of the hypocotyl cells showed that the 269

ZmARGOS1-GFP fusion protein was localized to moderately fluorescing threads that form a 270

loose web-like pattern within the cell (Supplementary Fig. S11). Also, small bright GFP-positive 271

bodies, in close association with the threads, were observed within the lumen of each cell. The 272

localization pattern of the fusion protein in hypocotyl cells of stable transgenic Arabidopsis is 273



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similar to that observed in onion epidermal cells transiently expressing the Zm-ARGOS1-GFP 274

fusion protein (Fig. 6B). In onion epidermal cells, co-expression of the fusion protein and a 275

fluorescently tagged endoplasmic reticulum (ER) marker, ER-ck CD3-953 (Nelson et al., 2007) 276

showed strong co-localization to a web-like pattern throughout the lumen of the cell (Fig. 6B). 277

Additionally, co-expression of the Zm-ARGOS1-GFP fusion protein and a fluorescently tagged 278

Golgi marker, G-ck CD3-961 (Nelson et al., 2007) indicated that there was a strong association 279

between the strongly fluorescent GFP-positive bodies and the Golgi. These Zm-ARGOS1-GFP 280

fusion protein bodies were less than 1µm in diameter and were closely associated with the ER. 281

Expression of the fluorescent ARGOS1 fusion protein was not observed in nuclei, plastids, 282

vacuoles, cytoplasm, vacuolar membranes and the plasma membranes in the transgenic 283

Arabidopsis hypocotyl or onion epidermal cells. 284

285

ARGOS transgenic Arabidopsis plants have increased drought tolerance 286

ARGOS transgenic Arabidopsis plants were tested along with the etr1-1 and ein2-1 287

mutants for drought tolerance. Drought stress was applied by withholding water at 14 days 288

after germination. Since Arabidopsis leaves wilt during drought stress, maintenance of leaf area 289

was used as a criterion for evaluating the drought tolerance of transgenic plants. LemnaTec 290

HTSBonitUV software was used to capture and segment RGB images. Estimates of the leaf area 291

of the Arabidopsis plants were obtained in terms of the number of green pixels. The data for 292

each image were averaged to obtain estimates of mean and standard deviation for the green 293

pixel counts for transgenic and wild-type plants. Parameters for a noise function were obtained 294



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by straight line regression of the squared deviation versus the mean pixel count using data for 295

all images in a batch. Error estimates for the mean pixel count data were calculated using the fit 296



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parameters for the noise function. The mean pixel counts for transgenic and wild-type plants 297

were summed to obtain an assessment of the overall leaf area for each image. The four-day 298

interval with maximal wilting was obtained by selecting the interval that corresponds to the 299

maximum difference in plant growth. The individual wilting responses of the transgenic and 300

wild-type plants were obtained by normalization of the data using the value of the green pixel 301

count of the first day in the interval. The drought tolerance of the transgenic plant compared to 302

the wild-type plant was scored by summing the weighted difference between the wilting 303

response of transgenic plants and wild-type plants over day two through day four; the weights 304

were estimated by propagating the error in the data. A positive drought tolerance score 305

corresponds to a transgenic plant with slower wilting compared to the wild-type plant. An 306

effect size statistic for the difference in wilting response between transgenic and wild-type 307

plants is obtained from the weighted sum of the squared deviations. When the transgenic 308

replicates show a significant difference (score of greater than 2) from the control replicates, the 309

line is then considered a validated drought tolerant line. In the drought assay, the 35S:Zm-310

ARGOS1, 35S:Zm-ARGOS8, 35S:At-ARL2 and 35S:At-ARL3 plants showed a significant delay in 311

leaf area loss relative to WT controls (Fig. 7, A and B). The increased drought tolerance was also 312

observed in the etr1-1 and ein2-1 mutants. However, the transgenic plants over-expressing the 313

mutated, loss-of-function ARGOS, Zm-ARGOS1(L104D) and Zm-ARGOS8(L67D), were not 314

significantly different from WT plants (Fig. 7B). After establishing the role of ARGOS in 315

Arabidopsis growth and development, we next tested its functionality in maize. 316

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Over-expressing Zm-ARGOS genes reduces ethylene responses in transgenic maize plants 318

Zm-ARGOS1 and Zm-ARGOS8 were overexpressed in maize under the control of the maize 319

ubiquitin1 promoter (UBI1) and the banana streak virus promoter (BSV) (Schenk et al., 2001). 320

Both promoters drive constitutive expression with BSV being stronger than UBI1. To determine 321

transgenic maize response to exogenously supplied ACC, seeds were germinated in the 322

presence of the ethylene precursor. The ACC treatment reduced root elongation and affected 323

root gravitropism in non-transgenic seedlings (Fig. 8A), but did so to a lesser extent in the 324

UBI1:Zm-ARGOS1 events (Fig. 8B). The inhibition of root growth was detectable at 50 μM ACC 325

and the severity of the phenotype intensified with an increase in ACC concentration. The 326

BSV:Zm-ARGOS8 plants were insensitive to ACC with no obvious root inhibition observed at 100 327

μM (Fig. 8B). However, when Zm-ARGOS8 expression was driven by the relatively weaker 328

promoter UBI1, the root length of the transgenic and non-transgenic plants was 329

indistinguishable in this assay (Fig. 8B). In the absence of exogenously supplied ACC, no 330

difference in root growth was detected among the ARGOS transgenic and non-transgenic 331

seedlings (Fig. 8B). The reduced ethylene response suggested that ARGOS overexpression 332

affects ethylene sensitivity in maize, similar to that in transgenic Arabidopsis. 333

334

Maize events over-expressing ZM-ARGOS8 have increased grain yield under field conditions 335

Guo et al. (2013) reported that UBI1:Zm-ARGOS1 has a positive effect on maize grain yield, but 336

only in particular environments. The Zm-ARGOS1 construct reduces ethylene sensitivity in 337

maize (Fig. 8B) and has potential in yield improvement under dry and high temperature 338



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conditions. However, yield reduction as revealed in humid and low temperature conditions 339

limits its practical applications. The strength of ARGOS activity and expression levels of a 340

transgene likely influence its capability in regulating ethylene sensitivity in transgenic plants. 341

Zm-ARGOS8, whose activity is weaker than Zm-ARGOS1, is able to reduce ethylene sensitivity 342

when over-expressed in Arabidopsis (Fig. 2) and maize (Fig. 8B) and enhance drought tolerance 343

in Arabidopsis (Fig. 7B). Aiming at developing maize hybrids that have yield advantage under 344

drought stressed environments with no yield loss in optimal growing conditions, we elected to 345

determine the functionality of Zm-ARGOS8, and therefore, tested eight single-copy UBI1:Zm-346

ARGOS8 events in a hybrid background over a two-year period at multiple locations throughout 347



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the U.S. At the end of each growing season, locations were categorized into either a high 348

drought stress or low drought stress environment based on several drought stress parameters 349

(Loffler et al., 2005). Grain yield was analyzed using a mixed model via ASReml (Habben et al., 350

2014; Gilmour et al., 2009) and Table 2 shows that plants grown under a high drought-stress 351

condition had on average a 56% decrease in yield compared to that under a low stress 352

condition, indicating the severity of water limitation on grain yield. Under the high stress 353

condition all events showed a statistically significant increase in yield relative to the bulk null 354

comparator (Table 2). Interestingly, when these same events were grown in low stress, high-355

yielding conditions, they also showed a significant increase in yield relative to the comparator. 356

Thus, events over-expressing the UBI1:Zm-ARGOS8 transgenic cassette showed yield efficacy 357

not only under drought stress, but also under well-watered conditions. 358

To enhance our understanding of transgene efficacy in other genetic backgrounds, 359

seven of the eight ARGOS8 events were converted into two inbreds, top crossed to two testers, 360

and field yield tested. Similar to results described above, locations were grouped into high and 361

low drought stress environments. In both hybrids, all events with the exception of one (DP-362

E5.03; Hybrid 2) showed a statistically significant increase in grain yield under high drought 363

stress (Table 3). At the construct level, events averaged a 6.6 and 4.5 bu ac-1 increase in Hybrid 364

1 and Hybrid 2, respectively. Yield increases of the best event (DP-E4.17) ranged from 5.0 to 365

10.9 bu ac-1 across the two hybrids under a high drought stress condition (Table 3). In the low 366

stress environment, all events showed either a positive or neutral grain yield increase in both 367

hybrids with there being an overall significant increase in yield at the construct level. To 368

determine the basis of the enhanced grain yield, we measured several ear parameters in both 369



26 | P a g e

hybrids (Table 4). There was a significant increase in kernel number per ear, with both hybrids 370

averaging greater than 20 extra kernels. The ear length was also increased significantly relative 371

to the comparator, but there was no change in ear width (Table 4). Based on these results 372

collected in high-yielding germplasm, UBI1:Zm-ARGOS8 events consistently demonstrated 373

increased yield under both drought stress and well-watered conditions. 374

375

376



27 | P a g e

Discussion 377 378

It has been reported that over-expression of ARGOS genes promotes lateral organ 379

growth by regulating cell number and/or size in Arabidopsis (Hu et al., 2003, 2006; Feng et al., 380

2011). However, the molecular mechanism of this enhanced organ growth is not known. We 381

found that ARGOS negatively modulates the response to ethylene when over-expressed in 382

Arabidopsis and maize. The enhanced ethylene production and reduced expression of a large 383

number of ethylene inducible genes in 35S:Zm-ARGOS1 Arabidopsis events supports the 384

concept of reduced ethylene sensitivity in transgenic plants. Over-expressing Zm-ARGOS1 in the 385

Arabidopsis ctr1-1 mutant suggested that ARGOS may not target the ethylene signaling 386

pathway downstream of CTR1. Further genetic analysis showed that Zm-ARGOS1 can reduce 387

ethylene sensitivity in the loss-of-function mutant rte1-2 or the null mutant etr1-7. However, 388

Zm-ARGOS1 was not able to properly function in Arabidopsis rte1-2 mutant plants when the 389

ethylene receptor ETR1 is absent and the exogenously supplied ACC concentration is high, 390

suggesting that Zm-ARGOS1 likely targets ethylene perception or the initial stage of ethylene 391

signal transduction. Cell fractionation and microscopy results indicated that over-expressed Zm-392

ARGOS1 was localized to the ER and Golgi membrane where the ethylene receptor complex 393

resides. This subcellular localization is consistent with its role in regulating ethylene signaling. 394

According to the current model of ethylene perception and signal transduction (Qiao et 395

al., 2012; Ju et al., 2012), in the absence of ethylene, the ethylene receptors are in an active 396

form which activates CTR1. The activated CTR1 represses downstream signaling by 397

phosphorylating EIN2. The membrane localized RTE1 plays a regulatory role by interacting with 398



28 | P a g e

the ethylene receptor ETR1 (Resnick et al., 2008; Dong et al., 2008; 2010). Over-expression of 399

Arabidopsis RTE1 and tomato homolog Green-Ripe (Barry and Giovannoni, 2006) likely 400

enhances the ethylene receptor activity, reducing plant sensitivity to ethylene. In the presence 401

of ethylene, ethylene binding by receptors causes a decrease in CTR1 kinase activity, 402

subsequently resulting in cleavage and nuclear translocation of the EIN2 C-terminus which 403

activates the EIN3/EIL1-dependent ethylene response (Qiao et al., 2012; Ju et al., 2012). Zm-404

ARGOS1 over-expression confers ethylene insensitivity, possibly by enhancing the activity of 405

ethylene receptors or by keeping CTR1 active even in the presence of ethylene. As a result, the 406

expression of the EIN3/EIL1-activated genes is down-regulated while the EIN3/EIL1-suppressed 407

genes are up-regulated in the Arabidopsis plants over-expressing Zm-ARGOS1 (Table 1). 408

We tested an Arabidopsis ARGOS T-DNA insertion knockdown line (GK-627B07) and an 409

At-ARL2 knockout mutant (GK-436G04). No obvious phenotype was observed in the singles and 410

the argos arl2 double mutant (data not shown). However, the triple mutant of the At-ARGOS 411

knockdown, At-ARL2 knockout and rte1-2 showed an increase in ethylene sensitivity relative to 412

the rte1-2 mutant (unpublished data). Similar results were obtained when the double mutant 413

was moved into an etr1-7 mutant background (unpublished data). There are four ARGOS genes 414

in Arabidopsis (Supplementary Fig. S1) and functional redundancy is evident (Feng et al., 2011). 415

It would be interesting to see how a quadruple knockout Arabidopsis line would respond to 416

ethylene. 417

Arabidopsis plants over-expressing various ARGOS members were found to be tolerant 418

to drought in a wilting assay. This tolerance is likely a consequence of reduced ethylene 419



29 | P a g e

sensitivity because the transgenic plants were insensitive to ethylene and the mutant versions 420

of both Zm-ARGOS1 and Zm-ARGOS8, which cannot reduce ethylene sensitivity when over-421

expressed, were unable to confer drought tolerance. In addition, the ethylene insensitive 422

mutant etr1-1 and ein2-1 also showed increased drought tolerance in the wilting assay. In 423

water deficient conditions, the reduced leaf wilting phenotype is possibly a result of reduced 424

water loss through transpiration in ethylene insensitive plants. Tholen et al. (2008) reported 425

that stomatal conductance of the etr1-1 mutant was 44% lower than WT plants. The finding of 426

ethylene inhibiting ABA-induced stomatal closure in drought-stressed Arabidopsis (Tanaka et 427

al., 2005) also supports the concept of a water conservation effect in ethylene insensitive 428

Arabidopsis. Whether or not this occurs in Arabidopsis plants over-expressing ARGOS genes 429

remains to be determined. 430

Maize plants over-expressing Zm-ARGOS8 also had ethylene sensitivity reduced and 431

concomitant better grain yield relative to non-transgenic controls in water deficient 432

environments, indicating enhanced drought tolerance. This result is consistent with our earlier 433

observation that reduced ethylene production improves drought tolerance in maize (Habben et 434

al., 2014). Grain yield is a complex trait affected by numerous molecular and physiological 435

processes during vegetative and reproductive growth. It is widely recognized that drought has a 436

negative effect on plant growth and ethylene plays an important role in this phenotype. 437

Maintenance of plant growth under stressed conditions could mitigate the yield loss. The 438

UBI1:Zm-ARGOS8 transgenic plants had longer ears and produced more kernels, suggesting that 439

their growth was less affected by drought relative to non-transgenic controls. Previous 440

research also showed that over-expressing ARGOS promotes growth in leaves, flowers and 441



30 | P a g e

seeds in maize (Guo et al., 2014), Arabidopsis (Hu et al., 2003, 2006; Feng et al., 2011) and 442

tobacco (Kuluev et al., 2011). 443

Field yield trials have shown that UBI1:Zm-ARGOS8 events enhance maize grain yields 444

under both drought stress and well-watered conditions. A yield increase in multiple genetic 445

backgrounds suggests the broader applicability of Zm-ARGOS8 in crop production. Although 446

over-expression of Zm-ARGOS1 can improve maize yields in certain drought environments, a 447

negative effect on yields was observed in cool and high humidity conditions (Guo et al., 2014). 448

This performance difference between the two ARGOS genes may reflect a magnitude variation 449

in activity between Zm-ARGOS1 and Zm-ARGOS8 with the former being stronger in reducing 450

ethylene sensitivity (Fig. 2 and Fig. 8). Given the effect of ethylene on a myriad of cellular and 451

developmental processes, it is expected that a drastic modification of ethylene signal 452

transduction would not be beneficial in maximizing grain yields. Instead, obtaining an optimal 453

ethylene level and/or ethylene sensitivity may be more desirable. 454



31 | P a g e

Materials and Methods 455 456

Plant materials and growth conditions 457

The Arabidopsis thaliana mutants eto1-1, etr1-7, rte1-2 and ctr1-1 are in the Columbia ecotype. 458

The eto1-1 and ctr1-1 mutants were obtained from ABRC (Columbus, OH) and rte1-2 and ctr1-1 459

were a gift from Caren Chang (University of Maryland). Plants were grown under fluorescent 460

lamps supplemented with incandescent lights (approximate 120 mE m-2 s-1) in growth chambers 461

with 16 hr light period at 24°C and 8 hr dark period at 23°C and 50% relative humidity. 462

For the Arabidopsis triple-response assay, surface sterilized and stratified seeds were 463

germinated in the presence of ethylene gas (Praxair, Danbury, CT) in an airtight container or on 464

medium (1/2 Murashige and Skoog salts with 1% sucrose and 0.8% agar) containing ACC 465

(Calbiochem, La Jolla, CA) at the stated concentrations. Hypocotyl and root length were 466

measured by photographing the seedlings under a dissection microscope with a digital camera 467

and using image analysis software (ImageJ, National Institutes of Health). 468

For assaying the maize seedling response to ACC, seeds were placed in a row between 469

two layers of filter papers wetted in an ACC aqueous solution at stated concentrations. The 470

filter paper was rolled up with a piece of waxed paper on the outside and set vertically in a 471

beaker containing 1 inch (2.5 cm) of the same solution. The beaker was covered with plastic 472

wrap to prevent evaporation and placed at 24°C in the dark. Seedling phenotypes were scored 473

5 days after seeding. 474

475



32 | P a g e

Transgene constructs and plant transformation 476

Open reading frames of maize or Arabidopsis genes were PCR-amplified, cloned into pENTR/D-477

TOPO vector (Invitrogen, Carlsbad, CA) and confirmed by sequencing. The mutated version of 478

ARGOS genes were created by the PCR method using primers containing desired mutations. To 479

generate the FLAG-HA epitope tagged Zm-ARGOS1, PCR primers was designed to include the 480

Zm-ARGOS1 specific sequence and the coding sequence for the FLAG and HA epitope 481

(DYKDDDDKVKLYPYDVPDYAAA). Using Gateway technology (Invitrogen), the genes in 482

the pENTR/D entry vector were mobilized into the binary vector pBC.Yellow (Gutie´rrez-Nava et 483

al., 2008) which contains the CaMV 35S promoter and the phaseolin terminator. The binary 484

vector has two selectable markers, yellow fluorescent protein (YFP) under the control of 485

desiccation-responsive At-Rd29 promoter for color selection and the BAR gene for herbicide 486

selection. The Agrobacterium strain GV3101 was used to transform Arabidopsis Col-0 with the 487

flower dipping method (Clough & Bent, 1998). 488

GFP and Zm-ARGOS1 fusions were created by joining the PCR product of Ac-GFP1 and 489

Zm-ARGOS1 in a vector containing Gateway attL4/3 recombination sites, the CaMV 35S 490

promoter and the NOS terminator. A linker sequence encoding for GGGSGGGS was placed 491

between the two genes. The recombinant gene was integrated into a binary vector containing 492

Gateway attR4/3 recombination sites and the selectable marker UBI1 PRO:UBI1 493

INTRON1:MoPAT:PinII Term (Unger et al., 2001; Cigan et al., 2005). The Agrobacterium strain 494

LBA4404 that harbors the construct was used to transform Arabidopsis ecotype as stated 495

above. 496



33 | P a g e

For maize transformation, the cDNA sequence of Zm-ARGOS1 and Zm-ARGOS8 was 497

integrated between the maize UBI1 or banana streak virus BSV promoter and the PinII 498

terminator using Gateway technology and cointegrated with plant transformation vectors as 499

described previously (Unger et al., 2001; Cigan et al., 2005). Plasmids were introduced into 500

Agrobacterium strain LBA4404 and used to transform maize embryos from a proprietary inbred. 501

Multiple independent events were generated for each construct. Single-copy T-DNA integration 502

events that expressed the transgene were selected and advanced for crosses to WT plants and 503

further characterization. 504

For subcellular co-localization of Zm-ARGOS1, binary plasmids carrying cyan fluorescent 505

protein-tagged ER and Golgi marker (ER-ck CD3-953 and G-ck CD3-961; Nelson et al., 2007) 506

were obtained from ABRC. Onion (Allium cepa) inner epidermal cell transformation was 507

performed as described previously (Scott et al., 1999). 508

509

Ethylene measurements 510

Whole leaves were excised from 3-week-old Arabidopsis plants. After letting the wound-511

induced ethylene burst subside for two hours, the leaves then were placed in 9.77-mL amber 512

vials containing a filter paper disc wetted with 50 μL of distilled water and sealed with 513

aluminum crimp seals. After a 20-h incubation period, 1-ml samples were taken from the 514

headspace of each sealed vial. The ethylene content was quantified by gas chromatography, as 515

described in (Habben et al., 2014). Ethylene production rate was expressed as nL per hour per 516

gram of fresh weight. 517



34 | P a g e

518

Gene expression analysis by RNA-Seq 519

Total RNAs were isolated from aerial tissues of 19-day-old Arabidopsis plants by use of the 520

Qiagen RNeasy kit for total RNA isolation (Qiagen, Germantown, MD). Sequencing libraries 521

from the resulting total RNAs were prepared using the TruSeq mRNA-Seq kit according to the 522

manufacturer’s instructions (Illumina, San Diego, CA). Briefly, mRNAs were isolated via 523

attachment to oligo(dT) beads, fragmented to a mean size of 150nt, reverse transcribed into 524

cDNA using random primers, end repaired to create blunt end fragments, 3’ A-tailed, and 525

ligated with Illumina indexed TruSeq adapters. Ligated cDNA fragments were PCR amplified 526

using Illumina TruSeq primers and purified PCR products were checked for quality and quantity 527

on the Agilent Bioanalyzer DNA 7500 chip (Agilent Technologies, Santa Clara, CA). Ten 528

nanomolar pools made up of three samples with unique indices were generated. Pools were 529

sequenced using TruSeq Illumina GAIIx indexed sequencing. Each pool of three was hybridized 530

to a single flowcell lane and was amplified, blocked, linearized and primer hybridized using the 531

Illumina cBot. Fifty base pairs of insert sequence and six base pairs of index sequence were 532

generated on the Illumine GAIIx. Sequences were trimmed based on quality scores and de-533

convoluted based on the index identifier. Resulting sequences were bowtie aligned (Langmead 534

et al., 2009) to the Arabidopsis gene set and normalized to Relative Parts Per Kilobase Per Ten 535

Million (RPKtM) (Table S1; Mortazavi et al, 2008). The generated RPKtM data matrix was 536

visualized and analyzed in GeneData Analyst software (Genedata AG, Basel, Switzerland). 537

538



35 | P a g e

RNA analysis 539

For Northern blot analysis, total RNA was extracted from Arabidopsis leaf tissues. Five micro 540

grams of RNA were separated by electrophoresis in a 1% (w/v) agarose/formaldehyde/MOPS 541

gel and blotted to a nylon membrane. Probe labeling, hybridization and washing were carried 542

out according to the manufacturer’s instructions. To determine transgene expression in 543

Arabidopsis with RT-PCR, cDNA was synthesized with oligo(dT) primers using SuperScript II 544

RNase H- reverse transcriptase (Invitrogen Life Technology). PCR was conducted using the 545

Advantage-GC 2 PCR kit (Clontech). The primers used were PF1 (5’-gacacccagcagctgatcaacag-3’) 546

and PR2 (5’-atgtaggtcggtccggttccaccg-3’). 547

548

Cell franctionation and immunoblotting 549

Microsomal membranes and soluble fraction were isolated from 3-week-old Arabidopsis plants 550

according to Chen et al. (2002). Protein was separated by SDS-PAGE, blotted to a PVDF 551

membrane and probed with monoclonal anti-FLAG (Sigma-Aldrich, St. Louis, MO) antibodies. 552

The primary antibodies were detected with the Pierce Fast Western Blot Kit, ECL Substrate 553

(Thermo Fisher Scientific, Rockford, IL). Monoclonal anti-HA antibodies (Thermo Fisher 554

Scientific, Rockford, IL) were also used to analyze expression of FLAG-HA epitope-tagged ARGOS 555

proteins. 556

557

Fluorescence microscopy 558



36 | P a g e

Hypocotyls of Arabidopsis seedlings and onion inner epidermal peels were harvested and 559

immediately placed in PBS (pH7.2) on glass slides for microscopic observations. Observations 560

and images were taken with a Leica (Wetzlar, Germany) DMRXA epi-fluorescence microscope 561

with a mercury light source. Two different fluorescent filter sets were used to monitor Ac-GFP1 562

fluorescence; Alexa 488 #MF-105 (exc. 486-500nm, dichroic 505LP, em. 510-530) and Red-563

Shifted GFP #41001 (exc. 460-500nm, dichroic 505LP, em. 510-560). Cyan fluorescence was 564

monitored using an Aqua filter set #31036v2 (exc. 426-446nm, dichroic 455LP, em. 465-495). 565

All filter sets were from Chroma Technology (Bellows Falls, VT). Images were captured with a 566

Photometrics (Tucson, AZ) CoolSNAP HQ CCD. Camera and microscope were controlled, and 567

images manipulated by Molecular Devices (Downingtown, PA) MetaMorph imaging software. 568

569

Quantitative drought assay for Arabidopsis plants 570

YFP positive transgenic seed and YFP negative non-transgenic sibs from a segregating T2 571

population were sown in a single flat on Scotts® Metro-Mix® 360 soil supplemented with Peters 572

fertilizer and Osmocote®. Flats were configured with 8 square pots each. Each of the square 573

pots was filled with soil. Each pot (or cell) was sown to produce 9 seedlings in a 3x3 array. 574

Within a flat, 4 pots consisted of transgenic plants and 4 pots consisted of non-transgenic 575

control plants. The soil was watered to saturation and then plants were grown in conditions of 576

16 hr light/22°C and 8 hr dark cycle/20°C at ~65% relative humidity. Plants were given a normal 577

watering regime until day 14 after germination. At day 14, plants were given a final saturating 578

watering. Digital images of the plants were taken once a day at the same time of day, starting at 579



37 | P a g e

the onset of visible drought stress symptoms, approximately 14 days after the last watering, 580

until the plants appear desiccated. Typically, four consecutive days of data was captured. 581

582

Maize hybrid yield testing 583

To evaluate the grain yield of the UBI::Zm-ARGOS8 transgenic events, field trials were 584

conducted with a hybrid over a range of environments in two years. Using standard 585

backcrossing techniques, the insertion was backcrossed from a donor inbred line into two 586

inbred lines, one in each of two major complimentary heterotic groups. Two hybrids 587

originating from these converted inbred lines were evaluated over a similar range of 588

environments. Hybrid seed for these trials was generated in a winter nursery and sent back to 589

North America for the subsequent growing season. Hybrid seed segregated 1:1 for the 590

transgene and selectable markers linked to the transgene were used to identify and separate 591

the transgene positive F1 seed from the transgene negative F1 seed (event nulls). A subsample 592

of each of the event nulls was combined to create a bulk null control. All subsequent yield 593

comparisons were made between the F1 transgene positive hybrid and the bulk null. In some 594

cases, other transgenic de-regulated traits including herbicide tolerance and/or insect 595

protection were already included in these hybrids. When this occurred, the same de-regulated 596

genes were present in both the experimental and bulk null entries of a hybrid. Multiple 597

individual events were backcrossed to determine effect of insertion site on efficacy. 598

Experimental events and bulk null controls were grown in field environments at research 599

centers in Woodland, CA; Garden City, KS; Plainview, TX; York NE; Fruitland, IA; Marion, IA, 600



38 | P a g e

Johnston, IA; Windfall, IN; Princeton, IN; Sciota, IL; and San Jose, IL. Some environments were 601

managed to impose various levels of drought stress while others were managed for optimum 602

yield/non-stress conditions. Experimental designs were set up as a randomized complete block 603

or split-plot arrangement with hybrids as main plots and transgene status (positive or wild type) 604

as the split plot. Three to four replicates were established at each location and the plant 605

population density used was typical for growers in that particular region. Harvest weight and 606

grain moisture from each plot were used to calculate yield area-1 at a constant moisture. 607

Statistical analysis was conducted within environment to eliminate plot level outliers before 608

analyzing across environments. Individual growing locations were classified as stressed or low-609

stressed based on management practices and yield levels attained at those locations (Loffler et 610

al., 2005). Statistical models accounting for environments effects were used to eliminate within 611

location spatial variation. Best linear unbiased predictions (BLUPs) were generated at the 612

individual event level as well as across events (construct level) for both the stress and low-613

stress environmental groupings (Gilmour et al., 2009; Habben et al., 2014). At one low stress 614

environment, ten preselected and consecutive plants within each plot had ears removed before 615

combine harvest in order to evaluate individual ear characteristics such as ear length, ear width 616

and kernels per ear via image analysis. The ten ears were imaged, shelled for grain weight and 617

a moisture sample was taken. After combine harvest, the grain weight of the imaged ears was 618

adjusted to the combine moisture for that plot and added to the grain weight of the remaining 619

plot. Average ear length, ear width and kernels per ear values were calculated from the ten 620

ears in each plot and submitted as one plot value per trait. 621

622



39 | P a g e

Accession numbers 623 624

Sequence data for the genes described in this article can be found in the Arabidopsis Genome 625

Initiative or GenBank/EMBL databases under following accession numbers: Zm-ARGOS1 626

(JN252297), Zm-ARGOS8 (JN252302), At-ARL2 (At2g41230) and At-ARL3 (At2g41225). 627

628

629

Acknowledgements 630 631

We thank Caren Chang for kindly providing rte1-2 and etr1-7 Arabidopsis mutants. We 632

acknowledge Kathleen Schellin, Hongyu Wang, Jim Saylor and Jason Brothers for assistance 633

with phenotyping and genotyping, Karen Kratky for measuring ethylene, Wally Marsh for onion 634

transformation, Mary Beatty and Gina Zastrow-Hayes for RNA-seq analysis, as well as Brooke 635

Peterson-Burch and Stanley Luck for bioinformatics and statistics support. We are grateful to 636

Salim Hakimi, Mary Trimnell, Hua Mo and Weiguo Cai for their excellent contributions to the 637

field studies, as well as our colleagues at outlying breeding stations who conducted first-rate 638

yield trials. We also thank Tom Greene, Mike Lassner and Mark Cooper for their organizational 639

leadership and helpful input. 640

641

642



40 | P a g e

Figure Legends 643 644

Figure 1. Over-expression of ZM-ARGOS1 reduces ethylene responses in Arabidopsis plants 645

A. Bolting time is delayed in transgenic Arabidopsis plants over-expressing Zm-ARGOS1 relative 646

to wild-type plants. Two 35S:Zm-ARGOS1 transgenic events (2B3 and 2C6) and wild-type (WT) 647

controls were grown under a regime of 16 hr of light (approximately 120 mE m-2 s-1) at 24°C and 648

8 hr of darkness at 23°C. Means are shown for bolting time, days after planting (DAP). Error 649

bars, + stdev; n = 18. Student’s t-test was performed to compare the transgenic events and the 650

WT control (*p < 0.01). 651

B. The 35S:Zm-ARGOS1 Arabidopsis plants are insensitive to the ethylene precursor ACC. 652

Seedlings were germinated in the dark in the presence or absence of 10 μM ACC for 3 days. 653

Composite images of representative seedlings of 35S:Zm-ARGOS1 transgenic, etr1-1 mutant 654

and wild-type (WT) Col-0 plants are shown. 655

C. The hypocotyl and root lengths of 3-day-old etiolated Arabidopsis seedlings. The triple 656

response assay was conducted with 10 μM ACC. The data represent the mean of 10 to 20 657

seedlings from two 35S:ZM-ARGOS1 events (2B3 and 2C6), an etr1-1 mutant and WT controls. 658

Error bars, + stdev. Student’s t-test was performed to compare the transgenic events and the 659

etr1-1 mutant to the WT control (*p < 0.01). 660

D. Overexpression of Zm-ARGOS1 in the Arabidopsis eto1-1 mutant background. The 3-day-old, 661

etiolated 35S:Zm-ARGOS1 eto1-1 seedlings lack the ethylene triple response phenotype in the 662



41 | P a g e

absence of exogenously supplied ethylene. A composite image of representative seedlings of 663

wild-type (WT) plants and the transgenic Arabidopsis eto1-1 plants carrying Zm-ARGOS1 and 664

empty vector is shown. 665

E. The hypocotyl and root lengths of etiolated Arabidopsis eto1-1 mutant plants over-666

expressing Zm-ARGOS1. Seeds were germinated under dark in the absence of exogenously 667

supplied ethylene for 3 days. Three 35S:Zm-ARGOS1 events (Zm-ARGOS1; E1, E2 and E3) and 668

three empty vector events (Vector; E11, E12, E13) in the eto1-1 mutant background are shown. 669

Wild-type (WT) Arabidopsis and the eto1-1 mutant serve as controls. The hypocotyls and roots 670

in the 35S:Zm-ARGOS1 eto1-1 plants are significantly (* p < 0.01) longer than those in the 671

vector eto1-1 control events and the eto1-1 mutant. Error bars, + stdev; n = 20. 672

F. Flowering time was delayed in the 35S:Zm-ARGOS1 eto1-1 Arabidopsis plants relative to the 673

eto1-1 mutant. The delayed flowering time was observed in all 10 independent events tested. 674

Representative 34-day-old transgenic eto1-1 plants carrying 35S:Zm-ARGOS1 and empty vector 675

as well as the eto1-1 mutant plants are shown. Bar = 20 mm. 676

G. Zm-ARGOS1 (L104D), a mutated version of Zm-ARGOS1, was unable to confer ethylene 677

insensitivity in Arabidopsis plants. Root and hypocotyl lengths were determined in transgenic 678

Arabidopsis plants carrying empty vector, 35S:Zm-ARGOS1, and 35S:Zm-ARGOS1(L104D). 679

Twelve T1 seeds per construct, each representing an independent event, were randomly 680

selected based on the YFP marker and germinated in the dark in the presence or absence of 10 681

µM ACC. The means of the hypocotyl and root lengths are shown for 3-day-old seedlings. Error 682



42 | P a g e

bars, + stdev. Student’s t-test was performed to compare the transgenic events and empty 683

vector controls (*p < 0.01). 684

685

Figure 2. The 35S:At-ARL2, 35S:At-ARL3 and 35S:Zm-ARGOS8 transgenic Arabidopsis plants 686

have reduced ethylene sensitivity. The root and hypocotyl lengths are shown for the 3-day-old 687

etiolated Arabidopsis T1 seedlings germinated in the presence of 10 µM ACC. The data 688

represent the mean of 12 to 20 independent events randomly selected for each construct 689

based on YFP marker expression in T1 seeds. The 35S:Zm-ARGOS1 events, ethylene insensitive 690

mutant etr1-1 and ein2-1 and wild-type (WT) plants served as controls. Error bars, + stdev. 691

Student’s t-test was performed to compare the transgenic plants and the mutants to the WT 692

control (*p < 0.01). 693

694

Figure 3. Increased ethylene production and reduced expression of ethylene-inducible genes in 695

Arabidopsis overexpressing Zm-ARGOS1 696

A. Ethylene production in rosette leaves of 20-day-old Arabidopsis plants is shown for three 697

35S:Zm-ARGOS1 events (2C6, 2B3 and 1C6), vector controls (Vec) and wild-type (WT) Col-0 698

plants. Ethylene was collected for a period of 22 hr and subsequently measured using a gas 699

chromatograph. Error bars, + stdev; n = 4. Student’s t-test was performed to compare the 700

transgenic events and empty vector controls to WT plants (*p < 0.01). 701

702



43 | P a g e

B. Reduced expression of ethylene inducible genes in transgenic Arabidopsis plants 703

overexpressing Zm-ARGOS1. Total RNA was extracted from rosette leaves of 3-week-old plants. 704

Northern blotting analysis of three 35S:Zm-ARGOS1 events (2C6, 2B3 and 1C6) and the vector 705

control (Vec) were performed using 10 µg of RNA per lane and probed with ethylene-inducible 706

genes At-EBF2 and At-ERF5. The gel stained with ethidium bromide is shown at the bottom as a 707

control for loading. 708

709

Figure 4. Over-expression of Zm-ARGOS1 in Arabidopsis ctr1-1 and rte1-2 mutant plants 710

A. The constitutive ethylene response phenotype in the transgenic Arabidopsis ctr1-1 plants 711

carrying 35S:Zm-ARGOS1 (Zm-ARGOS1 ctr1-1) and empty vector (Vector ctr1-1). Wild-type (WT) 712

Col-0 plants grown in the same conditions serve as controls. Representative 23-day-old plants 713

are shown. Bar = 10 mm. 714

B. Rosette sizes measured in the 35S:Zm-ARGOS1 ctr1-1 Arabidopsis plants, the ctr1-1 mutant 715

and the wild-type (WT) control. The 35S:Zm-ARGOS1 ctr1-1 plants were generated by crossing 716

the 35S:Zm-ARGOS1-FLAG-HA event C2 with the ctr1-1 mutant followed by self-pollination. The 717

plants were grown for 20 days. The data represent the mean of 13 to 17 plants. Error bars, + 718

stdev. Student’s t-test was performed to compare the transgenic plants and the ctr1-1 mutant 719

to WT plants (*p < 0.01). No significant difference was found between the 35S:Zm-ARGOS1 720

ctr1-1 plants and the ctr1-1 mutant (p > 0.05, t-test). 721



44 | P a g e

C. Overexpression of Zm-ARGOS1 in Arabidopsis ctr1-1 mutant background. Etiolated ctr1-1 722

seedlings over-expressing Zm-ARGOS1 (Zm-ARGOS1 ctr1-1) displays the constitutive triple 723

response in the absence of exogenously supplied ethylene, similar to the transgenic ctr1-1 724

plants carrying empty vector (Vector ctr1-1). Wild-type (WT) Col-0 seedlings grown in the same 725

conditions serve as controls. A composite image of representative 3-day-old etiolated seedlings 726

is shown. 727

D. The hypocotyl and root lengths of etiolated Arabidopsis ctr1-1 mutant plants over-expressing 728

Zm-ARGOS1. Seeds were germinated under dark in the absence of exogenously supplied 729

ethylene for 3 days. Three 35S:ZM-ARGOS1 events (Zm-ARGOS1; E1, E2 and E3) and three 730

empty vector events (Vector; E11, E12, E13) in the ctr1-1 mutant background are shown. Wild-731

type (WT) Arabidopsis and the ctr1-1 mutant serve as controls. Error bars, + stdev; n = 20. 732

Student’s t-test was performed to compare the 35S:Zm-ARGOS1 and empty vector constructs, 733

and no difference was found. The hypocotyl and root lengths in WT are significantly longer than 734

those in the ctr1-1 transgenic plants (* p < 0.05). 735

E. Over-expressing Zm-ARGOS1 in Arabidopsis rte1-2 mutant background. The 35S:Zm-ARGOS1 736

rte1-2 plants were generated by crossing the Zm-ARGOS1 event 2B3 and 2C6 with the rte1-2 737

mutant followed by self-pollination. A composite image of representative 3-day-old etiolated 738

seedlings is shown. The triple response assay was conducted in the presence or absence of 10 739

µM ACC. 740

F. The root and hypocotyl lengths of 3-day-old, etiolated Arabidopsis seedlings are presented 741

for the 35S:Zm-ARGOS1 rte1-2 plants, rte1-2 mutant, 35S:Zm-ARGOS1 transgenic plants and 742



45 | P a g e

wild-type (WT) controls. The 35S:Zm-ARGOS1 rte1-2 plants were generated by crossing the Zm-743

ARGOS1 event 2B3 and 2C6 with the rte1-2 mutant followed by self-pollination. The triple 744

response assay was conducted in the presence and absence of 10 µM ACC. Measurements are 745

shown for the event 2B3 cross. Error bars, + stdev; n = 10; Student’s t-test was performed to 746

compare the plants with or without the Zm-ARGOS1 transgene. * p < 0.01. 747

G. Root phenotypes are shown for the 35S:Zm-ARGOS1 rte1-2 Arabidopsis plants, the rte1-2 748

mutant, 35S:Zm-ARGOS1 transgenic plants and wild-type (WT) controls. Plants were grown for 749

5 days in agar that contained half-strength MS medium with 0 or 0.2 µM ACC and were set 750

vertically in a growth chamber under a regime of 16 hr of light (approximately 120 mE m-2 s-1) at 751

24°C and 8 hr of darkness at 23°C. 752

H. The root lengths of the 5-day-old Arabidopsis seedlings are shown for the 35S:Zm-ARGOS1 753

rte1-2 plants, rte1-2 mutant, 35S:Zm-ARGOS1 transgenic plants and WT controls grown in the 754

light in the presence of 0 or 0.2 µM ACC. Error bars, + stdev; n = 15. Student’s t-test was 755

performed to compare the plants in the presence and absence of ACC within each genotype. * 756

P < 0.01. 757

758

Figure 5. Over-expression of Zm-ARGOS1 in Arabidopsis etr1-7 rte1-2 double mutant 759

A. The root lengths in the 35S:Zm-ARGOS1 etr1-7 rte1-2 Arabidopsis plants compared to the 760

etr1-7 rte1-2 double mutant. The 35S:Zm-ARGOS1 etr1-7 rte1-2 plant was generated by 761

crossing the Zm-ARGOS1 event 2B3 and the etr1-7 rte1-2 double mutant followed by self-762



46 | P a g e

pollination. Plants were grown for 5 days in agar that contained half-strength MS medium with 763

0, 0.2 or 0.5 µM ACC and were set vertically in a growth chamber under a regime of 16 hr of 764

light (approximately 120 mE m-2 s-1) at 24°C and 8 hr of darkness at 23°C. The root lengths of 765

the 35S:Zm-ARGOS1 plants in a WT background have no significant difference under 0, 0.2 and 766

0.5 µM ACC (P > 0.05, ANOVA), but the 35S:Zm-ARGOS1 etr1-7 rte1-2 plants are significantly 767

different (P < 0.05, ANOVA). Significant differences are denoted by different letters. Data are 768

means + stdev. n = 10 to 20. 769

B. The hypocotyl and root lengths of 3-day-old, etiolated Arabidopsis seedlings are presented 770

for the 35S:Zm-ARGOS1 etr1-7 rte1-2 plants and the etr1-7 rte1-2 double mutant. The 35S:Zm-771

ARGOS1 etr1-7 rte1-2 plant was generated by crossing the Zm-ARGOS1 event 2B3 and the etr1-772

7 rte1-2 double mutant followed by self-pollination. The triple response assay was conducted in 773

the presence and absence of 10 µM ACC in the dark. The wild-type (WT) Arabidopsis transgenic 774

plants carrying 35S:Zm-ARGOS1 and non-transgenic WT serve as controls. Error bars, + stdev; n 775

= 10; Student’s t-test was performed to compare the plants with or without the Zm-ARGOS1 776

transgene. * p < 0.01. 777

C. Immunoblotting analysis of Zm-ARGOS1 over-expression in the etr1-7 rte1-2 Arabidopsis 778

mutant plants. A FLAG-HA epitope-tagged Zm-ARGOS1 was over-expressed in the double 779

mutant (etr1-7 rte1-2) or wild-type (WT) background under the control of the cauliflower 780

mosaic virus 35S promoter. Ten events (E1 to E10) are shown. The event C2 of 35S:Zm-ARGOS1-781

FLAG-HA in the WT background serves as a positive control. Western blotting analysis was 782

performed with anti-FLAG antibodies. 783



47 | P a g e

D. The hypocotyl and root lengths of 3-day-old, etiolated Arabidopsis seedlings are presented 784

for the 35S:Zm-ARGOS1 etr1-7 rte1-2 plants and the etr1-7 rte1-2 double mutant. The 35S:Zm-785

ARGOS1 etr1-7 rte1-2 plant was generated by transforming the etr1-7 rte1-2 double mutant 786

plants with 35S:Zm-ARGOS1-FLAG-HA. The triple response assay was conducted with four 787

independent events (E1, E4, E7 and E9 in Figure 5C) in the presence of 0, 0.5 or 10 µM ACC in 788

the dark. Results from the event E1 are shown. The wild-type (WT) Arabidopsis transgenic 789

plants carrying 35S:Zm-ARGOS1 and non-transgenic WT serve as controls. Error bars, + stdev; n 790

= 10; Student’s t-test was performed to compare the plants with or without the Zm-ARGOS1 791

transgene. * p < 0.01. 792

793

Figure 6. ER and Golgi membrane localization of over-expressed Zm-ARGOS1 794

A. Immunoblotting analysis of cellular fractions of Arabidopsis plants overexpressing FLAG-HA 795

epitope-tagged Zm-ARGOS1 (Zm-ARGOS1) and untagged Zm-ARGOS1 control (CK). Total (T) 796

homogenates were ultracentrifuged to separate the soluble (S) and microsomal membranes 797

(M) fraction. Western blotting analysis was performed with anti-FLAG antibodies. 798

B. Over-expressed Zm-ARGOS1 protein is localized to the ER and Golgi in transiently 799

transformed onion epidermal cells. Upper panel - fluorescent microscopy images of the interior 800

of single cells displaying co-localization of the GFP-tagged Zm-ARGOS1 (middle) with an ER 801

marker, the cyan fluorescent protein CFP-tagged ER CD3-953 (left). At right is the merged 802

image of Zm-ARGOS1 (green) and ER CD3-953 (red). Lower panel – images of GFP-tagged Zm-803

ARGOS1 (middle) and a Golgi marker, the CFP-tagged Golgi CD3-961 (left). The merged image 804



48 | P a g e

of Zm-ARGOS1 (green) and Golgi CD3-961 (red) at right indicates that there is strong association 805

between the strongly fluorescent Zm-ARGOS1-GFP fusion bodies and the Golgi marker. Bars = 806

10 µm. 807

Figure 7. Over-expression of ARGOS genes in Arabidopsis increased drought tolerance in a 808

wilting assay 809

A. Increased drought tolerance in the ARGOS transgenic Arabidopsis plants. A representative 810

phenotype is shown for the 35S:At-ARL2 plants and wild-type (WT) controls 15 days after the 811

last watering. 812

B. Over-expression of multiple ARGOS genes increases drought tolerance in Arabidopsis as well 813

as that of etr1-1 and ein2-1 mutants. Maintenance of green leaf area under drought stress was 814

used as a criterion for evaluating the drought tolerance of transgenic plants. A drought score 815

greater than 2 indicates drought tolerant plants. 816

817

Figure 8. Over-expression of ARGOS genes reduced ethylene sensitivity in maize 818

A. The ethylene precursor ACC inhibits root growth and affects gravitropism in maize seedlings. 819

Representative 5-day-old seedlings are shown for the wild-type maize plants grown in filter 820

paper rolls set vertically in the dark in the presence of 0, 50 and 100 µM ACC. Bar = 2 cm. 821

B. Over-expressing ARGOS genes reduces the ethylene response in maize seedlings. Four 822

UBI1:Zm-ARGOS1 and UBI1:Zm-ARGOS8 events and three BSV:Zm-ARGOS8 events were 823

germinated in the dark in the presence of 0 and 100 µM ACC. The data represent the mean of 824



49 | P a g e

the root lengths of 5-day-old seedlings. Student’s t-test was performed to compare the 825

transgenic and non-transgenic segregant (null) (* P < 0.05). Error bars, + stdev. 826

827



50 | P a g e

Table 1. Ethylene responsive gene expression in the aerial tissues (rosette leaves and apical 828

meristem) of 19-day-old 35S:Zm-ARGOS1 Arabidopsis plants, as measured by RNA-Seq analysis. 829

Sequence reads were normalized to relative parts per kilobase per ten million (RPKtM). 830

Gene Locus TR (RPKtM) Ve (RPKtM) TR/Ve Ratio

t-test p-value

EBF2 At5g25350 305.8+25.2 737.8+43.0 0.41 0.0000 PORA At5g54190 2.2+0.5 9.3+5.1 0.23 0.0091 PORB At4g27440 1618+124.5 2172.7+169.6 0.74 0.0038 FLS2 At5g46330 113+17.7 298+13.3 0.38 0.0001 SID2 At1g74710 10.2+0.9 6.5+2.2 1.57 0.0475 EDF1 At1g25560 416.5+29.7 733.3+37.6 0.57 0.0001 EDF2 At1g68840 490.3+34.8 1200.1+36.0 0.41 0.0000 EDF4 At1g13260 795.6+15.8 1339.5+34.6 0.59 0.0000 ERF1 At4g17500 112.5+8.7 211.3+13.2 0.53 0.0001 ERF2 At5g47220 186.1+8.8 347.9+24.2 0.53 0.0000 ERF3 At1g50640 481.9+14.4 478.0+19.2 1.01 0.7744 ERF4 At3g15210 419.7+19.9 649.9+31.5 0.65 0.0001 ERF5 At5g47230 69.4+4.6 270.5+33.0 0.26 0.0000 ERF6 At4g17490 88.7+10.2 236.9+17.0 0.37 0.0000 ERF9 At5g44210 17.4+4.9 53.9+11.9 0.32 0.0019 ERF11 At1g28370 30.2+4.2 74.9+13.6 0.40 0.0010 ERF13 At2g44840 11.7+5.8 26.4+7.4 0.45 0.0524 ERF72 At3g16770 1079.2+196.3 2541.1+263.7 0.42 0.0004 ERF104 At5g61600 233.6+8.6 556.1+50.1 0.42 0.0000 PDF1.2 At5g44420 147.7+51.5 564.9+77.7 0.26 0.0009 PDF1.2c At5g44430 31.7+15.1 222.0+43.5 0.14 0.0005 PDF1.2b At2g26020 26.1+8.8 209.8+26.8 0.12 0.0001 Chitinase At2g43590 52.6+9.3 127.5+40.8 0.41 0.0109 CHI-B At3g12500 37.2+5.7 57.8+11.8 0.64 0.0376 PR4 At3g04720 779.0+44.8 1175.1+117.0 0.66 0.0014

Values are mean + standard deviation, three replications for transgenic plants (TR) and four 831

replications for vector controls (Ve). p: t-test statistic (two-sided) p-value. 832



51 | P a g e

Table 2. Grain yield of maize Ubi:Zm-ARGOS8 transgenic events and bulk null in a hybrid under 833

high and low drought stress conditions. 834

835

High Drought Stress Low Drought Stress

Entry Yield Prediction Mg ha-1(Bu ac-1)

Predicted Difference Mg ha-1(Bu ac-1)

Yield Prediction Mg ha-1(Bu ac-1)


DP-E3.10 7.83 (124.4) 0.32 (5.1)* 13.55 (215.5) 0.26 (4.1)*

DP-E3.12 7.83 (124.6) 0.33 (5.3)* 13.55 (215.4) 0.25 (4.1)*

DP-E4.13 7.83 (124.5) 0.33 (5.3)* 13.55 (215.4) 0.25 (4.0)*

DP-E4.15 7.84 (124.6) 0.33 (5.3)* 13.54 (215.3) 0.24 (3.9)*

DP-E4.16 7.83 (124.4) 0.32 (5.2)* 13.55 (215.5) 0.26 (4.1)*

DP-E4.17 7.84 (124.6) 0.33 (5.3)* 13.55 (215.5) 0.26 (4.1)*

DP-E5.10 7.83 (124.5) 0.33 (5.2)* 13.55 (215.4) 0.25 (4.0)*

DP-E5.03 7.83 (124.5) 0.33 (5.2)* 13.55 (215.5) 0.26 (4.1)*

Constructⱡ 7.83 (124.5) 0.33 (5.2)* 13.55 (215.4) 0.26 (4.1)*

Bulk Null 7.50 (119.3) ---- 13.29 (211.4) ---- 836

Data are from eight individual transgenic maize events (plus construct mean) and bulk null at 837 high and low drought stress locations in 2012 and 2013. 838

Predicted difference for each transgenic entry is compared to the bulk null. 839

All analyses were implemented using ASReml with output of the model presented as Best Linear 840 Unbiased Predictions (see Methods). 841

* Predicted difference significant at P < 0.05. 842

ⱡ Construct = the mean of transgene positive events. 843

844

845



52 | P a g e

Table 3. Grain yield of maize Ubi:ZM-ARGOS8 transgenic events and bulk null in two hybrids 846 under high and low drought stress conditions. 847 High Drought Stress Low Drought Stress

Entry Yield Prediction Mg ha-1(Bu ac-1)


Yield Prediction Mg ha-1(Bu ac-1)


Hybrid 1 DP-E3.10 8.87 (141.0) 0.52 (8.3)* 14.34 (228.0) 0.29 (4.6)* DP-E3.12 8.83 (140.5) 0.49 (7.8)* 14.29 (227.2) 0.24 (3.8)* DP-E4.13 8.75 (139.1) 0.41 (6.5)* 14.19 (225.6) 0.14 (2.3) DP-E4.15 8.81 (140.1) 0.47 (7.4)* 14.24 (226.5) 0.20 (3.1) DP-E4.16 8.61 (136.8) 0.26 (4.2)* 14.08 (223.9) 0.03 (0.5) DP-E4.17 9.03 (143.6) 0.69 (10.9)* 14.49 (230.4) 0.44 (7.0)* DP-E5.03 8.81 (140.0) 0.46 (7.4)* 14.29 (227.2) 0.24 (3.8)* Constructⱡ 8.76 (139.3) 0.42 (6.6)* 14.22 (226.1) 0.17 (2.7)* Bulk Null 8.34 (132.7) --- 14.05 (223.4) ---

Hybrid 2 DP-E3.10 7.62 (121.2) 0.21 (3.4)* 12.80 (203.5) 0.14 (2.3) DP-E3.12 7.63 (121.3) 0.22 (3.6)* 12.80 (203.6) 0.15 (2.4) DP-E4.13 7.80 (124.0) 0.39 (6.3)* 12.94 (205.8) 0.29 (4.6)* DP-E4.15 7.63 (121.3) 0.22 (3.5)* 12.78 (203.1) 0.12 (1.9) DP-E4.16 7.86 (125.1) 0.46 (7.3)* 13.03 (207.2) 0.38 (6.0)* DP-E4.17 7.72 (122.8) 0.32 (5.0)* 12.89 (204.9) 0.23 (3.7)* DP-E5.03 7.39 (117.5) -0.02 (-0.3) 12.56 (199.7) -0.10 (-1.5) Constructⱡ 7.69 (122.2) 0.28 (4.5)* 12.85 (204.3) 0.20 (3.1)* Bulk Null 7.41 (117.8) --- 12.65 (201.2) --- 848

Data are from eight individual transgenic maize events (plus construct mean) and bulk null 849 converted into two hybrids and grown at high and low drought stress locations in 2013. 850

Predicted difference for each transgenic entry is compared to the bulk null. 851

All analyses were implemented using ASReml with output of the model presented as Best Linear 852 Unbiased Predictions (see Methods). 853

* Predicted difference significant at P < 0.05. 854

ⱡ Construct = the mean of transgene positive events. 855

856



53 | P a g e

Table 4. Ear parameters of maize Ubi:Zm-ARGOS8 transgenic events and bulk null in two 857 hybrids. 858

859

Hybrid 1 Hybrid 2

Entry Kernels per ear number

Ear length cm

(inches)

Ear Width cm

(inches) Kernels per ear number

Ear length cm (inches)

Ear Width cm

(inches) Constructⱡ 559* 19.4 (7.6)* 5.3 (2.1) 559* 19.5 (7.7)* 5.3 (2.1) Bulk Null 538 19.0 (7.5) 5.3 (2.1) 537 19.0 (7.5) 5.3 (2.1)

860

Data are presented at the construct level compared to the bulk null in two hybrids at a low drought 861 stress location in 2013. 862 863 All analyses were implemented using ASReml with output of the model presented as Best Linear 864 Unbiased Predictions (see Experimental procedures). 865 866 * Predicted difference significant at P < 0.05. 867 868 ⱡ Construct = the mean of transgene positive events. 869



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http://search.crossref.org/?page=1&rows=2&q=Binder BM, Rodr�guez FI, Bleecker AB (2010) The copper transporter RAN1 is essential for biogenesis of ethylene receptors in Arabidopsis. J Biol Chem 285: 37263-37270

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http://scholar.google.com/scholar?as_q=Exploiting the triple response of Arabidopsis to identify ethylene-related mutants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Guzm�n&as_ylo=1990&as_allsubj=all&hl=en&c2coff=1

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Huberman M, Riov J, Goldschmidt EE, Apelbaum A, Goren R (2014) The novel ethylene antagonist, 3-cyclopropyl-1-enyl-propanoicacid sodium salt (CPAS), increases grain yield in wheat by delaying leaf senescence. Plant Growth Regul 73: 249-255


Ju C, Yoon GM, Shemansky JM, Lin DY, Ying ZI, Chang J, Garrett WM, Kessenbrock M, Groth G, Tucker ML, Cooper B, Kieber JJ,Chang C (2012) CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane tothe nucleus in Arabidopsis. Proc Natl Acad Sci USA 109: 19486-19491


Kawakami EM, Oosterhuis DM, Snider JL (2010) Physiological effects of 1-methylcyclopropene on well-watered and water-stressedcotton plants. J Plant Growth Regul 29: 280-288


Kawakami EM, Oosterhuis DM, Snider J (2013) High temperature and the ethylene antagonist 1-methylcyclopropene alter ethyleneevolution patterns, antioxidant responses, and boll growth in Gossypium hirsutum. Am J Plant Sci 4: 1400-1408


Kieber JJ, Rothenburg M, Roman G, Feldmann KA. Ecker JR (1993) CTR1, a negative regulator of the ethylene response pathwayin Arabidopsis encodes a member of the Raf family of protein kinases. Cell 72: 427-441


Konishi M, Yanagisawa S (2008) Ethylene signaling in Arabidopsis involves feedback regulation via the elaborate control of EBF2expression by EIN3. Plant J 55: 821-831


Kuluev BR, Knyazev AV, Iljassowa A A, Chemeris AV (2011) Constitutive expression of the ARGOS gene driven by dahlia mosaicvirus promoter in tobacco plants. Rus J Plant Physiol 58: 507-515



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

http://scholar.google.com/scholar?as_q=Heat stress induced ethylene production in developing wheat grains induces kernel abortion and increased maturation in a susceptible cultivar&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

http://scholar.google.com/scholar?as_q=Heat stress induced ethylene production in developing wheat grains induces kernel abortion and increased maturation in a susceptible cultivar&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hays&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hirayama&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Cell%5BTitle%5D%29 AND 15%5BVolume%5D AND 1951%5BPagination%5D AND Hu Y%2C Xie Q%2C Chua NH%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Hu Y, Xie Q, Chua NH (2003) The Arabidopsis auxin-inducible gene ARGOS controls lateral organ size. Plant Cell 15: 1951-1961

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

http://scholar.google.com/scholar?as_q=The Arabidopsis auxin-inducible gene ARGOS controls lateral organ size&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

http://scholar.google.com/scholar?as_q=The Arabidopsis auxin-inducible gene ARGOS controls lateral organ size&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hu&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant J%5BTitle%5D%29 AND 47%5BVolume%5D AND 1%5BPagination%5D AND Hu Y%2C Poh HM%2C Chua NH%5BAuthor%5D&dopt=abstract

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

http://scholar.google.com/scholar?as_q=The Arabidopsis ARGOS-LIKE gene regulates cell expansion during organ growth&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

http://scholar.google.com/scholar?as_q=The Arabidopsis ARGOS-LIKE gene regulates cell expansion during organ growth&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hu&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Cell%5BTitle%5D%29 AND 94%5BVolume%5D AND 261%5BPagination%5D AND Hua J%2C Meyerowitz EM%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Hua J, Meyerowitz EM (1998) Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana. Cell 94: 261-271

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

http://scholar.google.com/scholar?as_q=Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hua&as_ylo=1998&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Biochemical%5BTitle%5D%29 AND 1%5BVolume%5D AND a protein kinase that negatively regulates ethylene signaling in Arabidopsis%2E Plant J 33%3A 221%5BPagination%5D AND Huang Y%2C Li H%2C Hutchiso%2C C E%2C Laskey J%2C Kieber JJ%5BAuthor%5D&dopt=abstract

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


http://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Huang&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

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

http://scholar.google.com/scholar?as_q=The novel ethylene antagonist, 3-cyclopropyl-1-enyl-propanoic acid sodium salt (CPAS), increases grain yield in wheat by delaying leaf senescence&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

http://scholar.google.com/scholar?as_q=The novel ethylene antagonist, 3-cyclopropyl-1-enyl-propanoic acid sodium salt (CPAS), increases grain yield in wheat by delaying leaf senescence&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Huberman&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Proc Natl Acad Sci USA%5BTitle%5D%29 AND 109%5BVolume%5D AND 19486%5BPagination%5D AND Ju C%2C Yoon GM%2C Shemansky JM%2C Lin DY%2C Ying ZI%2C Chang J%2C Garrett WM%2C Kessenbrock M%2C Groth G%2C Tucker ML%2C Cooper B%2C Kieber JJ%2C Chang C%5BAuthor%5D&dopt=abstract

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

http://scholar.google.com/scholar?as_q=CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus 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

http://scholar.google.com/scholar?as_q=CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ju&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28J Plant Growth Regul%5BTitle%5D%29 AND 29%5BVolume%5D AND 280%5BPagination%5D AND Kawakami EM%2C Oosterhuis DM%2C Snider JL%5BAuthor%5D&dopt=abstract

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

http://scholar.google.com/scholar?as_q=Physiological effects of 1-methylcyclopropene on well-watered and water-stressed cotton 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

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Am J Plant Sci%5BTitle%5D%29 AND 4%5BVolume%5D AND 1400%5BPagination%5D AND Kawakami EM%2C Oosterhuis DM%2C Snider J%5BAuthor%5D&dopt=abstract

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

http://scholar.google.com/scholar?as_q=High temperature and the ethylene antagonist 1-methylcyclopropene alter ethylene evolution patterns, antioxidant responses, and boll growth in Gossypium hirsutum&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

http://scholar.google.com/scholar?as_q=High temperature and the ethylene antagonist 1-methylcyclopropene alter ethylene evolution patterns, antioxidant responses, and boll growth in Gossypium hirsutum&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kawakami&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28CTR%5BTitle%5D%29 AND 1%5BVolume%5D AND a negative regulator of the ethylene response pathway in Arabidopsis encodes a member of the Raf family of protein kinases%2E Cell 72%3A 427%5BPagination%5D AND Kieber JJ%2C Rothenburg M%2C Roman G%2C Feldmann KA%2E Ecker JR%5BAuthor%5D&dopt=abstract

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

http://scholar.google.com/scholar?as_q=Ethylene signaling in Arabidopsis involves feedback regulation via the elaborate control of EBF2 expression by EIN3&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

http://scholar.google.com/scholar?as_q=Ethylene signaling in Arabidopsis involves feedback regulation via the elaborate control of EBF2 expression by EIN3&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Konishi&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Rus J Plant Physiol%5BTitle%5D%29 AND 58%5BVolume%5D AND 507%5BPagination%5D AND Kuluev BR%2C Knyazev AV%2C Iljassowa A A%2C Chemeris AV%5BAuthor%5D&dopt=abstract

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Mortazavi A, Williams B A, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq.Nature Methods 5: 621-628


Nelson BK, Cai X, Nebenführ A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis andother plants. Plant J 51: 1126-36


Ogawara T, Higashi K, Kamada H, Ezura H (2003) Ethylene advances the transition from vegetative growth to flowering inArabidopsis thaliana. J Plant Physiol 160: 1335-1340


Patterson SE, Bleecker AB (2004) Ethylene-dependent and -independent processes associated with floral organ abscission inArabidopsis. Plant Physiol 134: 194-203


Qiao H, Shen Z, Huang SS, Schmitz RJ, Urich MA, Briggs SP, Ecker JR (2012) Processing and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas. Science 338: 390-393


Resnick JS, Wen CK, Shockey JA, Chang C (2006) REVERSION-TO-ETHYLENE SENSITIVITY1, a conserved gene that regulatesethylene receptor function in Arabidopsis. Proc Natl Acad Sci USA 103: 7917-7922


Resnick JS, Rivarola M, Chang C (2008) Involvement of RTE1 in conformational changes promoting ETR1 ethylene receptorsignaling in Arabidopsis. Plant J 56: 423-431


Ruzicka K, Ljung K, Vanneste S, Podhorska R, Beeckman T, Friml J, Benkova E (2007) Ethylene regulates root growth througheffects on auxin biosynthesis and transport-dependent auxin distribution. Plant Cell 19: 2197-2212


Schenk PM, Remans T, Sági L, Elliott AR, Dietzgen RG, Swennen R, Ebert PR, Grof CP, Manners JM (2001) Promoters forpregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants. Plant Mol Biol 47:399-412


Scott A, Wyatt S, Tsou PL, Robertson D, Allen NS (1999) Model system for plant cell biology, GFP imaging in living onion epidermalcells. BioTechniques 26:1125-1132


Solano R, Stepanova A, Chao QM, Ecker JR (1998) Nuclear events in ethylene signaling, a transcriptional cascade mediated byETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. Genes Dev 12: 3703-3714


Tanaka Y, Sano T, Tamaoki M, Nakajima N, Kondo N, Hasezawa S (2005) Ethylene inhibits abscisic acid-induced stomatal closure in www.plantphysiol.orgon February 2, 2018 - Published by Downloaded from

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http://search.crossref.org/?page=1&rows=2&q=Schenk PM, Remans T, S�gi L, Elliott AR, Dietzgen RG, Swennen R, Ebert PR, Grof CP, Manners JM (2001) Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants. Plant Mol Biol 47: 399-412

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Arabidopsis. Plant Physiol 138: 2337-2343Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Tholen D, Pons TL, Voesenek LACJ, Poorter H (2008) The role of ethylene perception in the control of photosynthesis. PlantSignal Behav 3: 108-109


Unger E, Betz S, Xu R, Cigan AM (2001) Selection and orientation of adjacent genes influences DAM-mediated male sterility intransformed maize. Transgenic Research 10: 409-422


Wang B, Sang Y, Song J, Gao XQ, Zhang X (2009) Expression of a rice OsARGOS gene in Arabidopsis promotes cell division andexpansion and increases organ size. J Genet Genomics 36: 31-40


Zhong S, Zhao M, Shi T, Shi H, An F, Zhao Q, Guo H (2009) EIN3/EIL1 cooperate with PIF1 to prevent photo-oxidation and topromote greening of Arabidopsis seedlings. Proc Natl Acad Sci USA 106: 21431-21436



http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Physiol%5BTitle%5D%29 AND 138%5BVolume%5D AND 2337%5BPagination%5D AND Tanaka Y%2C Sano T%2C Tamaoki M%2C Nakajima N%2C Kondo N%2C Hasezawa S%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Tanaka Y, Sano T, Tamaoki M, Nakajima N, Kondo N, Hasezawa S (2005) Ethylene inhibits abscisic acid-induced stomatal closure in Arabidopsis. Plant Physiol 138: 2337-2343

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

http://scholar.google.com/scholar?as_q=Ethylene inhibits abscisic acid-induced stomatal closure 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

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http://search.crossref.org/?page=1&rows=2&q=Tholen D, Pons TL, Voesenek LACJ, Poorter H (2008) The role of ethylene perception in the control of photosynthesis. Plant Signal Behav 3: 108-109

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

http://scholar.google.com/scholar?as_q=The role of ethylene perception in the control of photosynthesis&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

http://scholar.google.com/scholar?as_q=The role of ethylene perception in the control of photosynthesis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Tholen&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

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http://search.crossref.org/?page=1&rows=2&q=Unger E, Betz S, Xu R, Cigan AM (2001) Selection and orientation of adjacent genes influences DAM-mediated male sterility in transformed maize. Transgenic Research 10: 409-422

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

http://scholar.google.com/scholar?as_q=Selection and orientation of adjacent genes influences DAM-mediated male sterility in transformed maize&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

http://scholar.google.com/scholar?as_q=Selection and orientation of adjacent genes influences DAM-mediated male sterility in transformed maize&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Unger&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28J Genet Genomics%5BTitle%5D%29 AND 36%5BVolume%5D AND 31%5BPagination%5D AND Wang B%2C Sang Y%2C Song J%2C Gao XQ%2C Zhang X%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Wang B, Sang Y, Song J, Gao XQ, Zhang X (2009) Expression of a rice OsARGOS gene in Arabidopsis promotes cell division and expansion and increases organ size. J Genet Genomics 36: 31-40

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

http://scholar.google.com/scholar?as_q=Expression of a rice OsARGOS gene in Arabidopsis promotes cell division and expansion and increases organ size&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

http://scholar.google.com/scholar?as_q=Expression of a rice OsARGOS gene in Arabidopsis promotes cell division and expansion and increases organ size&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wang&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Proc Natl Acad Sci USA%5BTitle%5D%29 AND 106%5BVolume%5D AND 21431%5BPagination%5D AND Zhong S%2C Zhao M%2C Shi T%2C Shi H%2C An F%2C Zhao Q%2C Guo H%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zhong S, Zhao M, Shi T, Shi H, An F, Zhao Q, Guo H (2009) EIN3/EIL1 cooperate with PIF1 to prevent photo-oxidation and to promote greening of Arabidopsis seedlings. Proc Natl Acad Sci USA 106: 21431-21436

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

http://scholar.google.com/scholar?as_q=EIN3/EIL1 cooperate with PIF1 to prevent photo-oxidation and to promote greening of Arabidopsis seedlings&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

http://scholar.google.com/scholar?as_q=EIN3/EIL1 cooperate with PIF1 to prevent photo-oxidation and to promote greening of Arabidopsis seedlings&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhong&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1


argos regulates ethylene signaling and response corresponding

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