Jasmonic acid coordinates with light to regulate branching ... · 8/11/2020 · bPresent address:...

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Short title: Regulation of Arabidopsis root branching angle 1 2 Corresponding author details: 3

Ashverya Laxmi 4

Lab 203, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-5 110067, India 6 Tel: 91-11-26741612, 14, 17 Ext. - 180 7 Email: [email protected] 8

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Jasmonic acid coordinates with light to regulate branching angle of 10

Arabidopsis lateral roots 11

Manvi Sharmaa,1, Mohan Sharmaa,1,#, Muhammed Jamsheer Ka,b & Ashverya Laxmia* 12

aNational Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-110067, India 13

bPresent address: Amity Food & Agriculture Foundation, Amity University, Sector 125, Noida 14

201303, Uttar Pradesh, India 15

1The authors contributed equally to this work. 16

17 One sentence summary: 18

The Jasmonic acid pathway interacts with light, glucose and auxin machinery to fine tune 19

branching angle of Arabidopsis LRs. 20

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1 22

1Author Contributions

M.S. and A.L. conceived and designed the experiments. M.S. performed physiology and

microarray. M.S, M.S# and M.J.K performed confocal experiments. M.S and M.S# performed

real time assays, western blot and ChIP-qPCR. M.S. wrote the article. M.S# and M.J.K. assisted

in microarray analysis and preparing the manuscript; A.L. supervised and complemented the

article.

This work was financially supported by the Core Grant from the National Institute of Plant

Genome Research to A.L., University Grant Commission, Government of India and

Department of Biotechnology, Government of India. M.S. acknowledges University Grant

Commission, Government of India for research fellowship, M.S. acknowledges Department of

Biotechnology, Government of India and MJK acknowledges Department of Science and

Technology (INSPIRE Faculty Programme Grant IFA18-LSPA110).

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 11, 2020. ; https://doi.org/10.1101/2020.08.11.245720doi: bioRxiv preprint

https://doi.org/10.1101/2020.08.11.245720

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Abstract 23

Studies on the role of jasmonic acid (JA) in root growth and development and plant’s response 24

to external stimuli is very well understood. However, its role in post emergence lateral root 25

(LR) development still remains perplexing. Our work identifies methyl jasmonate (MeJA) as a 26

crucial phytohormone involved in determining the branching angle of Arabidopsis LRs. MeJA 27

inclines the LRs to a more vertical orientation which was found to be dependent on JAR1-28

COI1-MYC2, 3, 4 signalling. Our work also highlights the dual role of light acting with MeJA 29

in governing the LR angle. Glucose (Glc), produced by light mediated photosynthesis induces 30

wider branching angles. A combination of physiological, transcriptional and protein stability 31

assays suggest that Glc antagonizes the MeJA response via HEXOKINASE 1 (HXK1) 32

mediated signalling pathway and by stabilizing JAZ9, a negative regulator of JA signalling. 33

Moreover, physiological assays using auxin mutants; ChIP-qPCR showing the direct binding 34

of MYC2 on the promoters of auxin biosynthetic gene CYP79B2 and LAZY2 and asymmetric 35

distribution of DR5::GFP and PIN2::GFP pinpoints the role of an intact auxin machinery 36

required by MeJA to set the vertical growth of LRs. We also demonstrate that light perception 37

through PHYTOCHROME A and B (PHYA and PHYB) and transcription factor LONG 38

HYPOCOTYL5 (HY5) are indispensable for inducing vertical angles by MeJA. Thus, our 39

investigation highlights antagonism between light and Glc signalling and how they interact 40

with JA-auxin signals to optimize the branching angle of LRs which is a key determinant of 41

foraging capacity of roots under natural environmental conditions. 42

43

Biological significance 44

Root branches grow at specific angles with respect to the gravity vector by suppressing positive 45

orthogravitropic forces. Using physiological and molecular approaches, we have identified 46

light mediated activation of jasmonate responses lead to erect root architecture that might not 47

hold anchorage as well as capture resources. Glc produced via light keeps the jasmonate 48

responses at bay, thus, adjusting the overall root architecture. Our findings introduce new 49

players and how they act in concert in the regulation of LR angle. 50

51

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* Author correspondence: [email protected]


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Introduction 54

The plant root system is highly plastic in nature and is influenced by exogenous and 55

endogenous cues to modify its architecture. Because of their sedentary nature, plants are limited 56

to their immediate surroundings for acquisition of water and nutrients (Malamy, 2005; Cuesta 57

et al., 2013). While the primary root maintains a nearly vertical orientation (orthogravitropism), 58

LRs form a non-vertical growth orientation, away from the main root axis. This orientation of 59

organs with respect to the gravity vector is called gravitropic set-point angle (GSA) (Digby and 60

Firn, 1995). After their emergence, LRs adopt a perpendicular angle with respect to gravity, 61

but as the LR continues to develop, their GSA changes over time (Kiss et al., 2002; 2003). The 62

positioning and placement of LRs is of paramount significance as it directs plant anchorage 63

and uptake of water and nutrients. 64

65

In plants, phytohormones have profound effects on determining GSA. Recent findings have 66

identified the fundamental role of auxin in governing GSA of shoot branches and LRs of 67

Arabidopsis and other higher plants such as rice and bean (Rosquete et al., 2013; Roychoudhry 68

et al., 2013; Roychoudhry et al., 2017; Rosquete et al., 2018; Roychoudhry et al., 2019). 69

Brassinosteroids (BR), indole-acetic acid (IAA), and gibberellic acid (GA) are the principal 70

phytohormones that regulate leaf angle formation, and crosstalk among them controls leaf 71

angle development (Luo et al., 2016). However, the exact molecular mechanism governing this 72

response is still elusive. JA and MeJA collectively called as jasmonates (JAs) are 73

cyclopentenone compounds that are known to primarily modulate a number of vital 74

physiological processes such as gravitropism, senescence, stamen and flower development, LR 75

and root hair formation as well as wound responses and defense responses against pathogens 76

and insects (Wasternack and Hause, 2013). Recently, it has been found that JA receptor 77

CORONATINE-INSENSITIVE1 (COI1) is required for JA-mediated Arabidopsis lateral root 78

formation, LR positioning and emergence on root bends (Raya-González et al., 2012). 79

Investigations in rice have revealed the role of MeJA in modifying lamina joint inclination 80

(Gan et al., 2015). Nonetheless, the role of JAs in steering branching angles in dicots has not 81

yet been demonstrated. 82

83

Besides phytohormones, other factors such as temperature, nutrient status and light play a vital 84

role in governing branching angles (Digby and Firn, 2002; Bai et al., 2013; Trachsel et al., 85

2013; Roychoudhry et al., 2017). Previous reports have concluded the role of sucrose (Suc) in 86

influencing the gravitropic behaviour of stolons in Cynodon (Willemoes et al., 1988). Small 87


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sugars such as Glc is not only an important nutrient but also acts as a key signalling molecule 88

(Ramon et al., 2008; Eveland and Jackson, 2012; Li and Sheen, 2016). In Arabidopsis, there 89

are three distinct sugar signalling pathways: (1) the HEXOKINASE1 (HXK1)-dependent 90

pathway governed by HXK1-mediated signalling function in which Glc enters metabolism and 91

gets phosphorylated by HXK1. HXK1 is mainly associated with mitochondria and a specific 92

isoform is also found in plastids. In addition, HXK1 is present in high-molecular-weight 93

complexes with HXK unconventional partners (HUPs) and transcription factors (TFs) in the 94

nucleus where it controls transcription (Moore, 2003); (2) G-protein coupled receptor 95

signalling by REGULATOR OF G-PROTEIN SIGNALING 1 (RGS1) and GPA1 has been 96

implicated in sensing extracellular glucose and signalling through THF1, located in the 97

plastids. (Huang et al., 2006; Urano et al., 2012) and (3) a glycolysis-dependent pathway that 98

works through the antagonistic interaction between SUCROSE NONFERMENTING 99

RELATED KINASE 1 (SnRK1) and TARGET OF RAPAMYCIN (TOR) (Baena-González et 100

al., 2007; Baena-González, 2010; Xiong and Sheen, 2015; Song et al., 2017). The protein 101

kinase activity of KIN10/11 is repressed by glucose (Baena-González et al., 2007), whereas 102

TOR kinase is activated by glucose (Xiong and Sheen, 2012). KIN10/11 and TOR sense 103

opposite energy levels and govern the partially overlapping plant transcriptional networks, 104

which are intimately connected to glucose-derived energy and metabolite signaling tightly 105

associated with glycolysis and mitochondrial bioenergetics, but are mostly uncoupled from the 106

HXK1 actions as a glucose sensor (Baena-González et al., 2007; Xiong et al., 2013). Previous 107

studies have suggested the involvement of Glc in various aspects of early seedling development 108

(Mishra et al., 2009; Kircher and Schopfer, 2012; Yuan et al., 2014). Glc and phytohormones 109

have been extensively shown to interact with one another to bring about changes to enable 110

better fitness of the plants. The interplay of Glc with various phytohormones has shown to 111

modulate root directional responses in Arabidopsis seedlings (Singh et al., 2014a; Singh et al., 112

2014b). Nonetheless, very few reports link JA and sugar signalling (Song et al., 2017; 113

Vleesschauwer et al., 2017; Guo et al., 2018). Based on physiological and pharmacological 114

studies, an intimate cross-talk occurs between TOR and JA signalling pathways at multiple 115

levels of JA signal transduction (Vleesschauwer et al., 2017). However, reports on JA-Glc 116

signal crosstalk in regulating branching angle of Arabidopsis LRs still remain obscure. 117

118

In this study, we have identified the key role of MeJA in altering the branching angle of 119

Arabidopsis LRs. We have also identified the antagonistic crosstalk between Glc and JA in 120

governing this response. Auxin machinery is central to the regulation of growth angle and we 121


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found that MeJA-mediated modulation of branching angle requires auxin transport and TIR1-122

mediated auxin signalling. Collectively, via a series of hierarchical events, we have shown the 123

integration of light as a key environmental signal with energy and hormone signal transduction 124

to modify the shape of the whole root architecture. 125

126

Results 127

MeJA modulates branching angle of Arabidopsis roots. Findings on JAs regulating main 128

root gravitropism and lamina joint inclination angle have already been reported (Gutjahr et al., 129

2005; Staswick, 2009; Gan et al., 2015). To address the role of MeJA in regulating growth 130

angles, the branching angle of Arabidopsis Col-0 LRs was measured by two methods viz. 131

calculating the angle formed by all LRs of WT seedlings with respect to the main root and 132

averaging the values; and distributing the angles of LRs in three categories viz. <40°, 40°-70° 133

and >70°. We observed that MeJA decreases the branching angle in a dose dependent manner. 134

LRs of Col-0 showed an average branching angle of 67.02° on control media, but a more 135

vertical angle of 40.21° when grown on highest MeJA concentration (10 µM) (Fig. 1A-C). 136

Approximately 63% of LRs adopted angles between 0-40° and very few with angles >70° when 137

grown on 0.5X MS supplemented with 10 µM MeJA (Fig. 1C). LRs falling in the category 138

40°-70° remained relatively constant irrespective of the treatment (Fig. 1C). Since the effect of 139

auxin is already explored (Rosquete et al., 2013; Roychoudhry et al., 2013; Rosquete et al., 140

2018; Roychoudhry et al., 2019), we wanted to identify the role of other hormones in 141

modulating this response. The Arabidopsis Col-0 seedlings did not display any changes in 142

branching angle when grown in BR, 6-Benzylaminopurine (BAP), GA, 1-Aminocyclopropane-143

1-carboxylic acid (ACC) and abscisic acid (ABA) (Fig. 1D). Altogether, the above results 144

suggest that exposure to increasing concentration of MeJA influences LR branching angle, 145

indicative of an overall vertical orientation. 146

147

MeJA decreases branching angle in a SCFCOI1 and MYC2, 3, 4 dependent pathway. In 148

order to elucidate how JA biosynthesis and signalling are involved, several mutants defective 149

in the JA-biosynthesis and signalling pathways were assessed. JA perception by 150

CORONATINE-INSENSITIVE1 (COI1) is the first committed step of JA signalling. Before 151

perception, JA is converted to its biologically active form jasmonyl-isoleucine (JA-Ile) by the 152

enzyme JASMONATE RESISTANT 1 (JAR1) (Staswick and Tiryaki, 2004; Staswick, 2009). 153

The jar1-11 and coi1 did not respond to MeJA treatment and exhibited an overall horizontal 154

orientation of LRs (Fig. 1E; Fig. S1A-B). Also, very few LRs of these mutants adopted angles 155


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<40° as compared to Col-0 (Fig. 1E; Fig. S1A-B). The average branching angle of jar1-11 and 156

coi1 indicated a complete resistance to the MeJA treatment (Fig. S1B). 157

MYC2 is a bHLH transcription factor that is considered as the master regulator of jasmonate 158

and light responses (Lorenzo et al.; Dombrecht et al., 2007). In addition to MYC2, MYC3 and 159

MYC4 are JAZ-interacting TFs that activate many JA responses (Cheng et al., 2011; 160

Fernández-Calvo et al., 2011; Niu et al., 2011). The jasmonate insensitive 1 (jin1-9), a T-DNA 161

insertion mutant of MYC2 was found to be less responsive to MeJA mediated control of 162

branching angle as compared to WT as its LR showed broader angles (Fig. 1F; Fig. S1A and 163

S1C). T-DNA insertion mutants of MYC3 and MYC4 responded similarly to WT for this 164

physiological response (Fig. 1F; Fig. S1A and S1C). However, myc2myc3myc4 displayed 165

diminished sensitivity as a large percentage of LRs acquired angles >70° as compared to their 166

respective single mutants and WT (Fig. 1F; Fig. S1A and S1C), suggesting that MYC3 and 167

MYC4 act additively with MYC2 in regulating this response. T-DNA mutant lines of 168

JASMONATE-ZIM-DOMAIN (JAZ1, JAZ2, JAZ4, JAZ6 and JAZ11) behaved like WT to 169

increasing MeJA doses which can be attributed to redundancy of JAZ genes (Fig. S1D and 170

S1E). We also performed physiological experiments with 20 DAG jar1-11 and myc2myc3myc4 171

in cylindrical tubes containing 0.5X MS and found broader LR angles as compared to Col-0 172

(Fig. S2A and S2B). Thus, JA perception and signalling machinery is involved in making the 173

LRs grow vertically. 174

175

Light mediated Glc production via photosynthesis negatively influences MeJA-176

modulated branching angle. Digby and Firn, 2002 showed that light effects on GSA of organs 177

can be brought about via the action of both photosynthesis and via phytochrome reception and 178

signalling. Glucose produced by light mediated photosynthesis governs GSA of many plant 179

organs. Previous reports have established the role of Glc in main root gravitropism in 180

Arabidopsis (Singh et al., 2014a; Singh et al., 2014b). In order to explore the role of Glc and 181

its interaction with MeJA in altering branching angle, the Col-0 seedlings were co-treated with 182

MeJA (10 µM) and different concentrations of Glc (0.5%Glc and 3%Glc). The presence of Glc 183

(3%) enhanced the branching angle as compared to low concentration (0.5%) (Fig. 2A and 2B; 184

Fig. S3A). The LRs attained more vertical orientation when treated with a combination of 0.5% 185

Glc and MeJA. However, in the presence of 3%Glc, MeJA could not exert a similar effect and 186

the LRs showed wider angles as compared to 0.5% and 0.5%Glc+MeJA (Fig. 2A and 2B; Fig. 187

S3A). These results suggest an antagonistic interaction between Glc and MeJA in controlling 188

this response. Next, we wanted to assess whether the observed phenotype is due to changes in 189


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the osmoticum in the growth media. To test this hypothesis we used mannitol (Man) in the 190

growth media. Man is a non-metabolizable sugar-alcohol that initiates very few LRs (Gupta et 191

al., 2015). Hence, to induce LR production small amount of Glc (1%) was combined with 192

increasing concentrations of Man (1%, 2%, 3% and 4%). We observed that the effects induced 193

by the supply of Glc were not produced by Man. Increased doses of Man induced a more 194

vertical orientation of LRs as compared to Glc (Fig. 2C). Thus suggesting that osmotic changes 195

in the medium are not solely responsible for the observed response. 196

To understand how this antagonistic interaction of Glc and MeJA occurs at molecular level, 197

we investigated the involvement of different components of Glc signal transduction in this 198

response. The HXK1 signalling mutant glucose insensitive 2 (gin2-1) showed an attenuated 199

response to independent and combined treatments of Glc and MeJA as compared to its wild 200

type Ler (Fig. 2D; Fig. S3B), whereas mutants of RGS1 (rgs1-1 and rgs1-2), G PROTEIN 201

ALPHA SUBUNIT 1 (GPA1) (gpa1-3 and gpa1-4) showed a WT-like response for Glc/JA 202

regulation of branching angle (Fig. S3C and S3D). Thus, HXK1-dependent signal transduction 203

pathway is involved in regulation of branching angle and any perturbation in the same leads to 204

an altered JA response. 205

To further strengthen the opposition between Glc and JA signalling in influencing this 206

phenotype, we examined the stability of JAZ9, a negative regulator of JA signalling in the 207

presence and absence of Glc. For this we used Jas9-VENUS, a fluorescent marker widely used 208

for the perception of bioactive JA (Larrieu et al., 2015). As shown in Figure 2E and 2F, upon 209

Glc (3%) treatment, Jas9-VENUS fusion protein was accumulated more at 3 hours. But, 210

addition of MeJA to 3%Glc led to rapid degradation of the fusion protein. In contrast, there 211

was very less accumulation of Jas9-VENUS in the absence of Glc and MeJA (Fig. 2E & 2F). 212

This suggests that Glc stabilizes Jas9-VENUS fusion protein and the presence of MeJA 213

degrades it, hence, supporting the antagonism between the two signals. Surprisingly, MeJA 214

was unable to degrade Jas9-VENUS in the absence of Glc (Fig. 2E & 2F). This prompted us 215

to check whether any amount of energy is required by MeJA to degrade Jas9-VENUS. For this, 216

we treated Jas9-VENUS with a combination of 1%Suc and MeJA. We observed that MeJA 217

was able to cause degradation of Jas9-VENUS in the presence of sugar (Fig. S4). 218

Microarray analysis was also employed to further elucidate the relationship between JA and 219

Glc signalling at the whole genome transcriptome level. It was observed that most of the core 220

JA signalling genes were downregulated in the presence of 3%Glc and 3%Glc+MeJA when 221

compared with 0%Glc+MeJA (Fig. S5). The microarray analysis fall in agreement with our 222


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physiological and molecular data, confirming that antagonism operates between JA and Glc 223

signalling. 224

225

Auxin transport and signalling lies downstream to MeJA-mediated modulation of 226

branching angle. Recent reports have shed light on the role of PIN auxin efflux activity and 227

the TIR1/AFB-Aux/IAA-ARF-dependent auxin signalling module for the establishment of 228

GSA in young LRs (Rosquete et al., 2013; Roychoudhry et al., 2013; Rosquete et al., 2018; 229

Roychoudhry et al., 2019). We assessed the involvement of the auxin transport and signalling 230

in the interaction between Glc and MeJA to govern this physiological response. For this, polar 231

auxin transport inhibitor 1-N-Naphthylphthalamic acid (NPA) was applied to the Col-0 232

seedlings. NPA increased Glc-induced branching angle (Fig. 3A). Also, when applied in 233

combination, NPA abolished the MeJA response (Fig. 3A). To further support whether a 234

functional auxin transport machinery is needed for MeJA to reduce the branching angle, the 235

auxin transport defective mutants were examined for changes in branching angle. Auxin influx-236

defective mutant auxin resistant 1(aux1-7) responded less to MeJA treatment and displayed 237

expansive branching angles as compared to Col-0 with greater percentage of LRs showing 238

angles >70° (Fig. 3B, Fig. S6A). The lax3 (like aux1)3 mutant showed WT-like response (Fig. 239

S6B). Auxin-efflux defective mutants ethylene insensitive root 1 (eir1-1) and multiple drug 240

resistance 1 (mdr1-1) exhibited LRs with significantly broader angle as compared with WT in 241

the presence of Glc and in combination with MeJA (Fig. 3C, Fig. S6C). Other pinoid mutants 242

(pin4-3 and pin7-2) being weak alleles exerted a WT like response (Fig. S6D). The branching 243

angle distribution of auxin receptor mutant transport inhibitor response 1 (tir1-1) showed more 244

LRs in >70° category as compared to Col-0 (Fig. 3D, Fig. S6E). We used auxin resistant 1 245

(axr1-3) and axr2-1 which show constitutive downregulation of auxin responses (Leyser et al., 246

1993; Nagpal et al., 2000). Seedlings carrying the weak allele of axr1-3 had a near normal 247

response (Fig. S6F). However, the response was completely abolished in axr2-1 (Fig. 3D, Fig. 248

S6F). Altogether, these results indicate that an intact auxin transport and signalling machinery 249

is required for MeJA to set the angular growth of LRs. 250

251

MYC2 controls the transcription of CYP79B2 and LAZY2. A former report has accounted 252

the role of jasmonate-mediated regulation of auxin biosynthesis (Sun et al., 2009). One of them 253

being CYP79B2, a cytochrome P450 mono-oxygenase that forms indole-3-acetaldoxime and 254

acts as a precursor for auxin biosynthesis (Zhao et al., 2002). Additionally, there are other 255

molecular components that are involved in modifying plant architecture. One of them being 256


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the LAZY gene family that is known to control both root and shoot gravitropism (Taniguchi et 257

al., 2017; Yoshihara and Spalding, 2017). LAZY2 has been reported to cause auxin 258

redistribution and transport in the Arabidopsis LRs thus leading to an overall erect root 259

architecture (Taniguchi et al., 2017; Yoshihara and Spalding, 2017). Mutants defective in 260

LAZY2 show agravitropic LRs (Taniguchi et al., 2017; Yoshihara and Spalding, 2017), thus 261

prompted us to study jasmonate effects on LAZY2 mediated LR angle. Given the established 262

role of JA in transcriptionally activating CYP79B2 and that MYC2 is the master regulator of 263

many JA responses, it is reasonable to speculate that MYC2 might control the transcription of 264

these genes which in turn enhances LR response to gravity. For this, we examined the MYC2-265

induced expression of CYP79B2 and LAZY2. As shown in Figure 4A and 4B, CYP79B2 and 266

LAZY2 transcript levels were downregulated in myc2myc3myc4. To understand how MYC2 267

regulates the expression of these genes, we scanned the promoter of CYP79B2 and LAZY2 and 268

found G and E-box elements. Using chromatin immunoprecipitation (ChIP)-quantitative PCR 269

(ChIP-qPCR) we found that CYP79B2 and LAZY2 promoters were highly enriched with MYC2 270

protein in 35S::MYC2-GFP as compared to Col-0 (Figure 4C and 4D). We also checked the 271

binding of MYC2 on the promoters of ORA59, a known MYC2 target that served as a positive 272

control (Zhai et al., 2013). ATXR6 was used as a negative control since it does not possess any 273

MYC2 binding sites in the promoter region tested (Figure 4C and 4D). Thus suggesting that 274

MYC2 binds and controls the transcription of these genes that might influence the vertical 275

orientation of LRs. 276

277

MeJA controls the direction of auxin transport that might regulate LR angle. There are 278

various factors that control GSA of LRs via the regulation of auxin flow from LR tips (Claudia-279

Anahı´ Pèrez-Torres et al., 2008; Roychoudhry et al., 2017; Taniguchi et al., 2017). All these 280

findings intrigued us to discern whether JA signalling influence auxin distribution at the Stage 281

II (SII) LR tips. For this, we analyzed the effect of MeJA on DR5::GFP expression. Most of 282

the 0.5X MS treated LRs displayed near symmetrical DR5::GFP expression with a faint green 283

signal streak towards the lower side (Fig 5A). However, after the addition of MeJA, the 284

DR5::GFP expression started to disappear from the upper side and the green signal streak 285

became more apparent on the margins of the lower side of the LR tip (Fig 5A). We also found 286

out that the length of the 1st two upper epidermal cells of LR treated with MeJA was longer as 287

compared to control (Fig. 5B), thus, suggesting that MeJA might play a potential role in 288

differential cell elongation. We also checked the expression of PIN2::PIN2-eGFP in SII LRs 289

and observed that MeJA diminishes PIN2 distribution in the upper cells of the LR (Fig 5C and 290


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D). In contrast, the PIN2::PIN2-eGFP signal intensity was strong and intact in MS treated 291

seedlings (Fig 5C and 5D). Altogether, these results demonstrate that JA signalling brings the 292

flow of auxin towards the lower side, which might lead to SII LR bending. 293

294

MeJA alters the cell profile of Arabidopsis LR. MeJA and JA have been shown to disrupt 295

cortical microtubules (MTs) in cultured potato cell suspensions and tobacco BY-2 cells (Abe 296

et al., 1990). However, the effect of MeJA on cytoskeleton organization in Arabidopsis root 297

system has not been studied till date. We performed physiological experiments using a 298

combination of Latrunculin B (Lat B) and MeJA. Although the addition of Lat B to MeJA 299

treated seedlings did make the LRs more vertically orientated as compared to 3%Glc (Fig S7A-300

S7C). But, there was not any difference between seedlings treated with 3%Glc+10µM MeJA 301

and 3%Glc+10µM MeJA +100nM LatB (Fig S7B-S7C). When higher dose of LatB was used, 302

there was no emergence of LRs and after a period of time the seedlings died. This prompted us 303

to study the actin cytoskeleton dynamics of Arabidopsis LR. ABD2 is the actin binding domain 304

of Arabidopsis Fimbrin 1 protein which is involved in actin filament crosslinking (Kovar et al., 305

2000). The 35S::GFP:ABD2::GFP lines were treated with 3%Glc and 3%Glc+ 10 µM MeJA. 306

Treatment with MeJA had a notable effect, abolishing the fluorescent signal by eliminating the 307

expression of ABD2, thus suggesting that MeJA negatively regulates the stability of Fimbrin 308

1 (Fig. 6A-6B). To further understand the physiological consequence, Col-0 seedlings treated 309

with 3%Glc and 3%Glc+ 10 µM MeJA were stained with propidium iodide (PI). Exogenous 310

MeJA application caused changes in the cell morphology of Arabidopsis LR as seedlings 311

grown in 3%Glc exhibited regular cell patterning (Fig.6C) whereas, MeJA caused twisting in 312

LR epidermal cells which possibly resulted in the bending of LRs (Fig. 6D). 313

314

Relevance of branching angle under natural environmental condition. Light can modify 315

the GSA of organs (Digby and Firn, 2002; Roychoudhry et al., 2017). In order to explore how 316

light affects MeJA mediated branching angle, 5-day-old, light grown Col-0 and 317

myc2myc3myc4 seedlings were treated with 0.5%Glc, 0.5%Glc+10µM MeJA, 3%Glc and 318

3%Glc+10µM MeJA under long day regime (16h/8h) and continuous dark. In dark, the 319

seedlings showed elongated hypocotyls and significantly horizontally placed LRs than those 320

grown in light conditions (Fig. S8A & S8B). In both Col-0 and myc2myc3myc4, the effect of 321

3%Glc was more prominent in dark with LRs displaying >70° angles. However, MeJA 322

treatment was unable to decrease the angle in dark. Also, LRs of myc2myc3myc4 showed wider 323


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angles in MeJA in dark as compared to Col-0 (Fig. S8A & S8B), thus suggesting that light is 324

obligatory for an optimal MeJA response. 325

326

Light perception and signalling intersect with the action of jasmonates to affect plant 327

development and defense (Dombrecht et al., 2007). We explored the involvement of 328

phytochrome signalling in regulating this response. Photoreceptor single mutants phyA-201 329

and phyB-5 showed a reduced response whereas, phyA201phyB5 double mutant exhibited 330

complete altered Glc and MeJA sensitivity as their LRs showed horizontal angles at all 331

concentrations (Fig. 7A & 7B). Hence, PHYA and PHYB act redundantly in controlling the 332

phenotype. To further substantiate the role of light signalling in regulating this response, we 333

investigated the involvement of HY5, a major downstream positive regulator of phytochrome 334

signalling (Gangappa and Botto, 2016). The hy5-1 mutant seedlings displayed horizontally 335

positioned LRs as compared to their WT (Fig. 7A & 7B). Previous reports of ChIP-seq data 336

claim the binding of HY5 to the LIPOXYGENASE 3 (LOX3) promotor, involved in JA 337

biosynthesis (Lee et al., 2007). To further understand how light signalling is involved in 338

maintaining an optimal JA response, we treated Ler and hy5-1 seedlings in long day light and 339

continuous dark conditions for 6 days. We also treated 6-day-old continuous dark grown Ler 340

and hy5-1 seedlings in light for 6 hours and then checked for the expression of LOX3. We found 341

a significant increase in the expression of LOX3 in long day grown treatment as compared to 342

total darkness (Fig. 7C) as well as upon dark to light 6 hours transition in Ler (Fig. 7D). In 343

contrast, LOX3 expression was significantly downregulated in hy5-1, suggesting the response 344

to be HY5-dependent. Collectively, the above data suggest that phytochrome signalling is 345

critical for MeJA dependent control of branching angle of Arabidopsis LRs. 346

We also assessed whether any alterations in the roots or shoots change the MeJA modulated 347

GSA. For this, we cut the Ler, hy5-1 and phyA201B5 seedlings at the root shoot junction and 348

observed no emergence of LR even after keeping it for the next 6 days (Fig S9A-S9B), 349

suggesting that shoot to root communication is essential for LR emergence, growth and 350

development, as also reported previously (Bhalerao et al., 2002; Ljung et al., 2005). Whether 351

this signal is essential for LR angle maintenance, we performed micro grafting experiments on 352

Arabidopsis seedlings according to Marsch-Martínez et al. 2013 with minor modifications. For 353

this, 5 day old 0.5X MS grown seedlings were used. Uncut Ler and hy5-1 seedlings were used 354

as controls. Ler shoots were placed on hy5-1, phyA201phyB5 roots and vica versa. However, 355

very few new LRs emerged and we did not observe any difference in the LR angle and the LR 356


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angle remained the same as that of the parent root (Fig S9A-S9B). These results suggest that 357

the genetic composition of the root itself plays a major role in determining the LR angle. 358

Discussion 359

The angular growth of LRs (GSA) represents an important element in the adaptability of the 360

root system to its environment. Both nutrient and hormonal signals act locally to regulate GSA 361

(Bai et al., 2013; Rosquete et al., 2013; Roychoudhry et al., 2013; Trachsel et al., 2013; 362

Roychoudhry et al., 2017; Rosquete et al., 2018; Roychoudhry et al., 2019). The net effect of 363

this adaptive response is to increase the surface area of the plant root system for resource 364

capture (e.g. horizontal LRs for phosphorus uptake) or to secure anchorage (Lynch and Brown, 365

2001; Trachsel et al., 2013). Similarly, shoots with erect lateral branches show enhanced 366

efficiency of light capture, allowing higher density planting and thus higher yields (Sakamoto 367

et al., 2006; Vriet et al., 2012). The main aim of this study is to advance our understanding of 368

GSA regulation and find out interconnections between environmental and hormonal signals in 369

fine-tuning the branching angle of Arabidopsis roots. 370

In this work, we show that MeJA reduced the branching angle of roots in a dose-dependent 371

manner, thus resulting in an overall vertical orientation (Fig. 1A-1C). Analysis of mutants 372

defective in JA biosynthesis and signalling revealed that an intact JA machinery is a 373

prerequisite to bring about changes in the branching angle. Also, the branching angles of jar1-374

11, myc2 and myc2myc3myc4 mutants were defective in perceiving endogenous JA even under 375

control conditions (0.5X MS) (Fig. 1E and 1F; Fig. S1A-S1C; Fig. S2A and S2B), thus 376

suggesting that in nature, JA signalling plays an important developmental role in governing 377

root branching angle. The weaker phenotype of jin1-9 as compared to myc2myc3myc4 suggests 378

that MYC3 and MYC4 act additively with MYC2 in regulating this response. Since MYC3 and 379

MYC4 are weakly expressed in the roots of young seedlings unlike MYC2 (Fernández-Calvo et 380

al., 2011), and that MYC2 is the major regulator of many biological responses (Lorenzo et al.; 381

Dombrecht et al., 2007), we postulate that this response is majorly mediated by MYC2. 382

Apart from affecting various parameters of RSA (Gupta et al., 2009; Mishra et al., 2009; Singh 383

et al., 2014a; Singh et al., 2014b; Gupta et al., 2015), sugars can also influence the gravitropic 384

behaviour of lateral organs (Willemoes et al., 1988). In this study, Glc caused a significant shift 385

in the branching angle towards a more horizontal orientation. High concentration of Glc 386

enhanced the branching angle, hence making the LRs wider (Fig. 2A-2B). This effect of Glc on 387

growth favors plant propagation since it allows plants to explore adjacent territories. The 388


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diagravitropic growth of LRs upon Glc treatment adhere to the reports by Willmoës et al, 389

(1998) suggesting that sugars are responsible for maintaining a non-vertical angular growth of 390

lateral organs. 391

There are reports citing crosstalk between sugars and GA3 to regulate growth direction of 392

organs (Montaldi, 1969; Willemoes et al., 1988). Compared with well-studied interactions 393

between Glc and other hormones, relatively little is known about Glc and JA (Song et al., 2017; 394

Vleesschauwer et al., 2017; Guo et al., 2018). Most of the studies demonstrate an antagonistic 395

interaction between the two signalling pathways in both dicots and monocots. Transcriptome 396

analysis of rice cells treated with the TOR-specific inhibitor rapamycin revealed that TOR apart 397

from dictating transcriptional reprogramming of extensive gene sets involved in central and 398

secondary metabolism, cell cycle and transcription, also suppresses many defense-related 399

genes and TOR antagonizes the action of the JA (Vleesschauwer et al., 2017). Microarray 400

analysis, protein stability assays along with physiological studies have confirmed that Glc and 401

JA are two crucial signals that work antagonistically to regulate the branching angle of 402

Arabidopsis roots (Fig. 2; Fig. S3-S5). We hypothesize that this antagonism occurs in nature 403

in order to fine tune the response and achieve the optimum angle required for growth. However, 404

more detailed molecular dissection is necessary to obtain a deeper understanding of this cross-405

talk and driving forces behind it. 406

407

Amid all the hormones, auxin plays a central role in LR GSA control. Transient expression of 408

PIN3 but strong repression of PIN4 and PIN7 in young LRs limits auxin redistribution and 409

hence explains the reduced gravitropic competence of laterals (Rosquete et al., 2013; Rosquete 410

et al., 2018). JAs contribute to the regulation of transport of IAA by inducing the expression of 411

PIN1 and PIN2 (Sun et al., 2009) and modulating the accumulation of PIN2 in the plasma 412

membrane and its recycling via endocytosis in a dose-dependent manner (Sun et al., 2011). In 413

the light of the above reports claiming recent interconnections between JA and auxin signalling 414

and the modulation of JA homeostasis as well as signal transduction can mimic auxin effects 415

on root development, we assume MeJA requires auxin to control branching angle. Our results 416

suggest that genetic disruption of auxin transport and signalling nullifies the effect of MeJA 417

mediated control of branching angle (Fig. 3A-3D). Also, JA signalling via MYC2 induces the 418

transcription of CYP79B2 and LAZY2 (Fig. 4), which is already known to increase auxin levels 419

and redistribution, respectively. Also, modulation of the asymmetric auxin transport in LR 420

columella cells, the diminished PIN2 activity from the upper epidermal cell profile and the 421


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differential cell elongation caused by MeJA might correlate with the vertical bending of the 422

LRs (Fig.5A-5D). 423

424

Light is one of the many diverse signals that changes the sugar/energy status in plants. Previous 425

reports demonstrate that light quality govern the gravitropic behaviour of lateral organs. 426

Arabidopsis LRs show negative phototropism in the presence of white light, and positive 427

phototropism in response to red light (Kiss et al., 2002). In the present study, we observed that 428

light acting via the redundant function of PHYA-PHYB and HY5 induces a more vertical 429

orientation of LRs (Fig. 7A-7B), which fall in line with results of Kiss and co-workers (Kiss 430

et al., 2002). In our experiments, exogenous application of MeJA could not induce a vertical 431

branching angle of LRs in dark. Thus, it suggests that light is a prerequisite to MeJA induced 432

change in branching angle. We suppose that the alteration in the angle is not just achieved by 433

the biosynthesis of JA but by the influence of some unknown factor or signalling event which 434

is currently unknown. ChIP-seq data showed the binding of HY5 at the promoter of LOX3 (Lee 435

et al., 2007). Consistent with this observation, we found decreased expression level of LOX3 436

in hy5-1 in light conditions (Fig. 7C-7D). Thus it suggests the likelihood of HY5 regulating JA 437

levels in light. Another study shows that light environment and circadian clock are crucially 438

involved to modulate plant’s response to JA biosynthesis (Radhika et al., 2010; Goodspeed et 439

al., 2012). JA levels have been shown to go up during the day, reaching a maximum at midday 440

and then declining again in the afternoon (Goodspeed et al., 2012). MYC2 protein levels were 441

also up during the day and in continuous light conditions (Shin et al., 2012). Moreover, ChIP-442

seq analyses show the binding of HY5 on the promoter of MYC2 (Lee et al., 2007). Together, 443

these findings suggest an intimate crosstalk in which light is a key environmental factor for JA 444

biosynthesis and signalling. 445

446

Based on our investigation and previous findings, we propose a testable model in Figure 8. 447

Light works via two branches to optimize the branching angle of Arabidopsis LRs. Contrary to 448

the general notion that light and sugars should have the same influence on developmental 449

outputs, there are many reports that suggest that light and glucose signalling have opposite 450

effect on the growth and development of Arabidopsis seedlings. Reports by Moore et al, 2003 451

and Eckstein and co-workers (Moore, 2003; Eckstein et al., 2012) show that the expression of 452

CAB genes that encode CHLOROPHYLL A/B-BINDING PROTEINS is increased in the 453

presence of light, but is repressed in the presence of Glc, respectively. Light working via 454

phytochrome signalling (PHYA/PHYB-HY5) increases JA levels which ultimately leads to the 455


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activation of JA signalling and an overall vertical orientation of the roots and this physiological 456

response is shown to be mainly mediated by MYC2. Our results also suggest that auxin works 457

further downstream as auxin transport and signalling mutants displayed abrogated responses to 458

JA regulation of branching angle. Additionally, MYC2 regulates the expression of CYP79B2 459

and LAZY2 by binding to their promoters that might promote auxin biosynthesis and its 460

transport and redistribution, ultimately leading to vertical angles. Glc on the other hand, 461

produced by photosynthesis promotes radial expansion of the root architecture and 462

antagonistically interacts with JA signalling via the HXK1-mediated pathway as well as by 463

affecting the stability of JAZ9 protein to regulate this developmental aspect. Moreover, 464

previous findings on light and Glc-mediated changes in actin cytoskeleton dynamics in root 465

growth (Kushwah et al., 2011; Yokawa et al., 2014) along with our study of MeJA modulation 466

of actin filament organization might govern LR angle. 467

468

The overall root architecture plays an important role in defining plant anchorage. Studies have 469

shown that horizontal branches show an insignificant role in root anchorage but are capable of 470

exploring adjacent territories better, meanwhile, the angular branches play a more effective 471

role in root anchorage due to higher soil column weight and therefore more pulling out 472

resistance (Khalilnejad et al.). These angled branches act as guy ropes holding the root that acts 473

as a fixed pole in position (Ennos, 2000), thus providing maximum anchorage. Apart from the 474

root architecture, signals from the environment as well as endogenous cues act in concert to 475

optimize growth angles of lateral branches. Depending upon the strength of the signal, the angle 476

of the laterals are decided. In the present study, using physiological and molecular approaches, 477

we have identified light mediated activation of jasmonate responses lead to erect root 478

architecture that might not hold anchorage as well as capture resources. Glc produced via light 479

keeps the jasmonate responses at bay, thus, adjusting the overall root architecture. Future work 480

will be directed at identifying substantial mechanistic insights into hormonal-environmental 481

crosstalk and characterizing novel molecular components that are involved in shaping the 482

overall root architecture. 483

484

Materials and methods 485

Plant materials. Arabidopsis thaliana ecotypes of Col-0, Ws and Ler were used as wild-type 486

controls. The following seed stocks were obtained from the Arabidopsis Biological Resource 487

Center (ABRC) at Ohio State University (http://www.arabidopsis.org/abrc/): DR5::GFP; phyA-488


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201 (CS6291); phyB-5 (CS6213); phyA201B5 (CS6224); hy5-1 (CS71); gin2-1 (CS6383); 489

rgs1-1 (CS6537); rgs1-2 (CS6538); gpa1-3 (CS6533); gpa1-4 (CS6534); tir1-1 (CS3798); 490

axr1-3 (CS3075); aux1-7 (CS3074); axr2-1 (CS3077); eir1-1 (CS8058); myc2/jin1-9 491

(SALK_017005C); myc3 (SALK_012763C); myc4 (SALK_052158C); coi1 492

(SALK_095916C); jar1-11 (CS67935); jaz1 (SALK_011957C); jaz2 (SALK_012782C); jaz4 493

(SALK_141628C); jaz6 (SALK_017531). Following lines were obtained from the original 494

published source as: myc2myc3myc4 (Schweizer et al., 2013) mdr1-1 (Noh et al., 2001); lax3 495

(Swarup et al., 2008); pin4-3 (Friml et al., 2002b); pin7-2 (Friml et al., 2003); 496

35S::GFP:ABD2::GFP (Wang et al., 2008); p35S::Jas9-N7-VENUS (Larrieu et al., 2015); 497

35S::MYC2::GFP (Jung et al., 2105); PIN2::PIN2-eGFP (NASC). All mutant lines were in 498

the Col-0 background except the following: mdr1-1 was derived from Ws background; and 499

gin2-1, hy5-1, phyA-201, phyB-5, phyA201B5 were in the Ler background. 500

Growth conditions. Seeds were surface sterilized and stratified at 4°C for 48 hours. The 501

imbibed seeds were grown vertically on square petri dishes containing 0.5X Murashige and 502

Skoog (MS) medium supplemented with 1% sucrose (29.13mM; w/v) and solidified with 0.8% 503

agar (w/v). Seed germination and plant growth were carried out in climate-controlled growth 504

rooms under long day conditions (16 hr light and 8 hr darkness), with 22°C ± 2°C temperature 505

and 60 µmol/sec/m2 light intensity. To study branching angle of LRs, five-day-old MS grown 506

grown seedlings were transferred to hormone/inhibitor/sugar treatment media with their root 507

tips marked and grown vertically under above mentioned growth conditions, unless otherwise 508

stated. 509

All chemicals were purchased from Sigma (St. Louis, MO, USA) except specified otherwise. 510

MeJA was prepared as 50 mM stock solution in 100% (v/v) ethanol. Epibrassinolide was 511

prepared as 10-2 M stock solution in 50% (v/v) ethanol. The following were prepared as 10-2 512

M stock solutions in dimethyl sulfoxide: NPA, BAP, ABA, and GA3. ACC was prepared as a 513

sterile 10-2 M aqueous stock solution. Propidium iodide (PI) was prepared as a sterile 10 mg 514

mL−1 aqueous stock solution. 515

Physiological analyses. Five-day-old light grown seedlings on 0.5X MS, 0.8% agar, and 516

1%Suc-containing medium were transferred to 0.5X MS medium, solidified with 0.8% agar 517

and supplemented with different combinations of Glc [0.5% (27.75 mM; w/v) and 3% 518

(166.52mM; w/v)] and MeJA (10 µM) or 0.5X MS medium, solidified with 0.8% agar, 519

supplemented with 1% sucrose and MeJA (1 µM, 5 µM, 10 µM) or combinations of Glc (1%) 520


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and Man (1%, 2%, 3%, 4%) with and without 10 µM MeJA and their root tips were marked. 521

Dark experiments were carried out by transferring light grown, five-day-old Col-0 and 522

myc2myc3myc4 seedlings to treatment media in light for 24 hours, followed by complete 523

darkness for the next 5 days. For measurement of branching angle, the angle formed between 524

each LR and primary root was measured. The average branching angle was determined by 525

summing the angle formed by each LR of all the seedlings divided by the total number of LRs. 526

Also, the angular response was measured by distributing the branching angle in three categories 527

viz. <40°, 40°-70°, >70° and expressed as percentage of LR. ImageJ program from NIH was 528

used to quantify the branching angle as well epidermal cell length of LRs 529

(http://rsb.info.nih.gov/ij/). Micro-grafting experiments on Arabidopsis seedlings were 530

performed according to (Marsch-Martínez et al., 2013) with minor modifications. Digital 531

images were captured from Nikon Coolpix camera on 12th day of seedling growth. 532

Laser Confocal Scanning Microscopy. To observe auxin distribution, PIN2 expression and 533

the actin filament organization in LRs, five-day-old light grown, DR5::GFP, PIN2::PIN2-534

eGFP and 35S::GFP-ABD2-GFP-expressing seedlings were transferred to treatment medium 535

for 6 days. GFP fluorescence was imaged under a Leica TCS SP2 AOBS Laser Confocal 536

Scanning Microscope (Leica Microsystems). To image GFP, the 488 nm line of the argon laser 537

was used for excitation and emission was detected at 520 nm. Cell profile of LRs was checked 538

by staining the seedlings with 10 µg/ml PI solution for 30 sec before confocal image analysis. 539

For imaging PI, 514 nm line of the argon laser was used for excitation and emission was 540

detected at 600 nm. The laser, pinhole and gain settings of the confocal microscope were kept 541

identical among different treatments. Images were assembled using Photoshop (Adobe 542

Systems). At least two biological replicates, with each replicate having 15 seedlings, were 543

performed for all the experiments. 544

Gene Expression Analysis. For gene expression analysis, RT-qPCR was performed. Imbibed 545

seeds of the wild type (Col-0 and Ler), myc2myc3myc4, and hy5-1 were sown on 0.5X MS 546

medium supplemented with 1% (w/v) Suc and 0.8% (w/v) agar and grown vertically in culture 547

room conditions. Five-day-old light grown seedlings of Col-0 and myc2myc3myc4 were 548

harvested and stored in -80°C. Ler and hy5-1 were grown in light (long day regime) and 549

continuous dark for 5 days. For dark to light transition, Ler and hy5-1 were grown in continuous 550

dark for 5 days followed by light treatment for 6 hours. Afterwards, whole seedlings were flash 551

frozen in liquid nitrogen and stored at -80°C. Total RNA was isolated from frozen tissue using 552

the RNeasy Plant Mini Kit (Qiagen) following the manufacturer’s protocol. RNA was 553


http://rsb.info.nih.gov/ij/

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quantified and tested for quality before it was used for subsequent analyses. First-strand cDNA 554

was synthesized by reverse transcription using 2 µg of total RNA with a high-capacity cDNA 555

Reverse Transcription Kit (Applied Biosystems). Primers were designed by identifying a 556

sequence stretch with a unique sequence. All candidate gene primers were designed using the 557

software Primer Express (version 3.0; Applied Biosystems). An ABI Prism 7900 HP fast real-558

time PCR system (Applied Biosystems) was used. For the normalization of variance among 559

samples, UBIQUITIN10 (UBQ10) was used as a reference control. The fold-change for each 560

candidate gene in different experimental conditions was determined using the quantitative 561

ΔΔCT method. All primers used are mentioned in the Supplemental Table S1. Gene expression 562

analysis was performed 3 times unless otherwise stated. 563

Chromatin Immunoprecipitation. ChIP assays were performed by following the protocol of 564

Saleh et al. (2008) with minor modifications. For this, chromatin from 7 day old MS grown 565

Arabidopsis 35S::MYC2::GFP seedlings was isolated. The resuspended chromatin was 566

sonicated in a 4°C water sonicator (Diagenode Bioruptor Plus). All primers used are mentioned 567

in the Supplementary Table S1. 568

Protein extraction and immunoblot assay. Extraction of soluble proteins was performed 569

on seven day old seedlings of Jas9::VENUS. Light and grown seedlings of p35S::Jas9-N7-570

VENUS were grown in sugar-free liquid 0.5X MS in dark for 24h to deplete internal sugars. 571

This was followed by priming the seedlings with 0% (w/v) Glc and 3% (w/v) Glc containing 572

½ MS liquid medium for 3h. Following Glc treatment, 10µM MeJA was added to all 573

seedlings for 3h in dark. For another experiment, 5 day old ½ MS grown Jas9::VENUS 574

seedlings were treated with liq 0.5X MS supplemented with 10 µM MeJA for 3h in light. 575

Seedlings were harvested, frozen in liquid nitrogen and then ground in pre chilled mortar 576

pestle using liquid nitrogen. The powder was resuspended in cold extraction buffer (137 mM 577

of NaCl, 2.7 mM of KCl, 4.3 mM of Na2HPO4, 1.47 mM of KH2PO4, 10% glycerol, and 1 mM 578

of phenylmethylsulfonyl fluoride) supplemented with plant protease inhibitor cocktail (Sigma-579

Aldrich, http://www.sigmaald rich.com/). This was followed by two rounds of centrifugation 580

(15 min, 13,000 rpm at 4 °C) to remove cell debris. The samples were boiled for 5 min at 581

95 °C before being loaded onto a gel (30 μl per lane). SDS–PAGE was performed on 10% 582

polyacrylamide gel. After transfer onto a nitrocellulose membrane, protein amounts in each 583

lane were checked using Ponceau staining (0.1%, 1.5 mM). Immunoblots were detected 584

using a primary rabbit polyclonal anti-GFP antibody (ab290, Abcam, diluted 1:5,000), 585

primary rabbit polyclonal anti HSP90-2 antibody (diluted 1:5,000) and a secondary anti-586


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rabbit IgG-HRP (diluted 1:10,000). Proteins were visualized using the enhanced 587

chemiluminescence kit. 588

589

Microarray analysis. For microarray of Arabidopsis Col-0 seedlings under Glc and MeJA 590

treatment, 5-d–old seedlings were first starved under 0% Glc for 24 h in the dark. After 591

starvation, seedlings were treated with and without Glc and a combination of 10µM MeJA. 592

Harvested samples were outsourced for transcriptome profiling. The data were analyzed by 593

the Transcriptome Analysis Console (v3.0; Affymetrix) with default parameters. Two 594

biological replicates were used for the microarray experiment. 595

596

Statistical analyses. All physiological experiments yielding similar results were repeated as 597

mentioned in the figure legends, in which each experiment was considered as an independent 598

biological replicate consisting of at least 25 seedlings. Immunoblot assays were performed 3 599

times unless otherwise stated. ChIP assays were performed as mentioned in the figure legends. 600

Statistical differences between control/treatment and WT/mutant pair were analyzed using 601

Student’s t test with paired two-tailed distribution. P-value cutoff was taken at P <0.05. All 602

data were managed and analyzed using Microsoft Excel. The graphs were made using 603

Microsoft Excel and Instant Clue. End point analyses were carried out on 12th day of seedling 604

growth. 605

Accession Numbers 606

Arabidopsis Genome Initiative locus identifiers for the genes mentioned in this article are: 607

phyA-201, AT1G09570; phyB-5, AT2G18790; hy5-1, AT5G11260; gin2-1, AT4G29130; 608

rgs1-1, AT3G26090; rgs1-2 AT3G26090; gpa1-3 AT2G26300; gpa1-4, AT2G26300; tir1-1, 609

AT3G62980; axr1-3, AT1G05180; aux1-7, AT2G38120; axr2-1, AT3G23050; eir1-1, 610

AT5G57090; myc2/jin1-9, AT1G32640; myc3, AT5G46760; myc4, AT4G17880; coi1, 611

AT2G39940; jar1-11, AT2G46370; jaz1, AT1G19180; jaz2, AT1G74950; jaz4, AT1G48500; 612

jaz6, AT1G72450; mdr1-1, AT3G28860; lax3, AT1G77690; pin4-3, AT2G01420; pin7-2, 613

AT1G23080. 614

615

Supplemental data 616

Figure S1. MeJA regulation of branching angle of Arabidopsis roots. (A) Phenotype of 617

Col-0 and JA signalling mutant seedlings grown on different doses of MeJA. (B and C) 618

Average branching angle of 12-day-old Col-0 and JA signalling mutants grown in different 619


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doses of MeJA. The data represents the average of 4 biological replicates consisting of 25 620

seedlings and error bars represent SE. (D and E) Distribution and average branching angle of 621

seedlings of Col-0 and different jaz mutants grown in different doses of MeJA. The data 622

represents the average of 3 biological replicates consisting of 25 seedlings and error bars 623

represent SE. Asterisks indicate a significant difference in the studied parameter 624

(P<0.05,Student’s t-test; *control vs treatment and WT vs mutant). 625

626

Figure S2. MeJA regulation of branching angle of Arabidopsis roots. (A) A representative 627

image of 20 DAG (days after germination) of Col-0 and JA biosynthesis and signalling mutants 628

jar1-11 and myc2myc3myc4 grown in 0.5X MS medium in cylindrical tubes. (B) Average 629

branching angle of 20 DAG (days after germination) of Col-0 and JA biosynthesis and 630

signalling mutants jar1-11 and myc2myc3myc4 grown in 0.5X MS medium in cylindrical tubes. 631

The data represents images from 2 biological replicates (P<0.05, Student’s t-test; * WT vs 632

mutant.) 633

634

Figure S3. MeJA-Glc regulation of branching angle of Arabidopsis roots. (A) Average 635

branching angle of 12-day-old Col-0 seedlings grown in different concentrations of Glc (0.5%, 636

3%) and in combination with MeJA. The data represents the average of 6 biological replicates 637

consisting of 25 seedlings and error bars represent SE. (B) Average branching angle of 12 day-638

old Ler and HXK1-dependent Glc signalling mutant gin2-1 grown in different concentrations 639

of Glc (0.5%, 3%) and in combination with MeJA. The data represents the average of 4 640

biological replicates consisting of 25 seedlings and error bars represent SE. (C and D) 641

Distribution of branching angle in seedlings of Col-0 and HXK1-independent signalling 642

mutants rgs1-1, rgs1-2, gpa1-3 and gpa1-4 grown in different concentrations of Glc in 643

combination with MeJA. The data represents the average of 4 biological replicates consisting 644

of 25 seedlings and error bars represent SE. (P <0.05, Student’s t-test; * control vs treatment 645

** 0.5%Glc vs 3%Glc+10MeJA and wild-type vs mutant). 646

647

Figure S4. Western blot depicting the stability of Jas9-VENUS. Immuno-blot detection of 648

Jas9-VENUS in seven-day-old Jas9-VENUS expressing seedlings treated with 1%Suc without 649

and with 10µM MeJA. Jas9-VENUS protein was detected using anti-GFP–specific antibody. 650

Ponceau S stained RUBISCO and Anti-HSP90 detecting HSP90 were used as loading controls. 651

The data represents average of 3 biological replicates. 652

653


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Figure S5. Heat map of JA signalling genes upon treatment with 0%Glc+10µM MeJA, 3%Glc 654

and 3%Glc+10µM MeJA. The expression value of genes treated with 0%Glc+10µM MeJA is 655

taken as 1. The expression values of genes in 3%Glc and 3%Glc+10µM MeJA is compared 656

with those of 0%Glc+10µM MeJA. * indicates expression values of genes that are significantly 657

altered. The data is the average of 2 biological replicates. Heat map was generated using MeV. 658

659

Figure S6. Role of auxin machinery in controlling JA-mediated branching angle. (A) 660

Average branching angle of auxin influx defective mutant aux1-7 grown in 3%Glc in 661

combination with MeJA. (B) Distribution of branching angle of auxin influx defective mutant 662

lax3 grown in 3%Glc in combination with different doses of MeJA (C) Average branching 663

angle of auxin efflux defective mutants eir1-1 and mdr1.1 grown in 3%Glc in combination 664

with MeJA. (D) Distribution of branching angle of auxin efflux defective mutants pin4-3 and 665

pin7-2 grown in 3%Glc in combination with different doses of MeJA. (E and F) Average 666

branching angle of auxin signalling mutants tir1-1, axr1-3 and axr2-1 grown in 3%Glc in 667

combination with MeJA. The data represents the average of 4 biological replicates consisting 668

of 25 seedlings and error bars represent SE. Asterisks indicate a significant difference in the 669

studied parameter (P <0.05, Student’s t-test; * control vs treatment and WT vs mutant). 670

671

Figure S7. Effect of MeJA and Lat B on branching angle of Arabidopsis roots. (A) 672

Phenotype of Col-0 seedlings treated with 3%Glc and in combination with 100nM and 500nM 673

Lat B and 10µM MeJA. (B) Distribution of branching angle of Col-0 seedlings treated with 674

3%Glc and in combination with 100nM and 500nM Lat B and 10µM MeJA. (C) Average 675

branching angle of Col-0 seedlings treated with 3%Glc and in combination with 100nM and 676

500nM Lat B and 10µM MeJA. The data represents the average of 4 biological replicates 677

consisting of 25 seedlings and error bars represent SE. Asterisks indicate a significant 678

difference in the studied parameter (P <0.05, Student’s t-test; * control vs treatment) 679

680

Figure S8. Role of light signalling in controlling JA-mediated branching angle. (A) 681

Phenotype of light and dark adapted Col-0 and myc2myc3myc4 seedlings in different 682

concentrations of Glc (0.5%, 3%) and in combination with MeJA (10µM). (B) Distribution of 683

branching angle in light and dark adapted Col-0 and myc2myc3myc4 seedlings in different 684

concentrations of Glc (0.5%, 3%) and in combination with MeJA (10µM). The data represents 685

the average of 6 biological replicates consisting of 25 seedlings and error bars represent SE 686


https://doi.org/10.1101/2020.08.11.245720

22

Asterisks indicate a significant difference in the studied parameter (P <0.05, Student’s t-test; * 687

control vs treatment and WT vs mutant). 688

689

Figure S9. Alterations in shoot or root affects branching angle of LRs. (A) Phenotypes of 690

Ler and hy5-1 (uncut) micrografted Ler, hy5-1 and stalkless hy5-1 and Ler. (B) Phenotypes of 691

Ler and phyA201phyB5 (uncut) micrografted Ler, phya201phyb205 and stalkless 692

phyA201phyB5 and Ler. The data represents the average of 1 biological replicates consisting 693

of 15 seedling. The experiment was repeated twice. 694

695

Supplementary Table 1: the primers used in the study. 696

Acknowledgements 697

The authors would like to thank Dr. Aditi Gupta for her advice and discussions and Ms Harshita 698

Bharti Saksena for reading the manuscript. The authors acknowledge NIPGR Confocal Facility 699

for their assistance, Prof. Philippe Reymond for providing seeds of myc2myc3myc4, Dr. Nam-700

Hai Chua for providing seeds of 35S-MYC2-GFP and Dr. Laurent Laplaze for providing 701

seeds of p35S::Jas9-N7-VENUS. The authors are thankful to DBT-eLibrary Consortium 702

(DeLCON) for providing access to e-resources. This work was financially supported by the 703

Core Grant from the National Institute of Plant Genome Research to A.L., University Grant 704

Commission, Government of India and Department of Biotechnology, Government of India. 705

M.S. acknowledges University Grant Commission, Government of India for research 706

fellowship, M.S. acknowledges Department of Biotechnology, Government of India and MJK 707

acknowledges Department of Science and Technology (INSPIRE Faculty Programme Grant 708

IFA18-LSPA110). 709

710

Figure Legends 711

Figure 1. MeJA decreases branching angle of Arabidopsis roots. (A) Phenotype of light 712

grown 12-day-old Arabidopsis Col-0 seedlings grown on different doses of MeJA. (B) Average 713

branching angle of 12-day-old Col-0 seedlings on different doses of MeJA. (C) Distribution of 714

branching angle of 12-day-old Col-0 Arabidopsis seedlings on different doses of MeJA. (D) 715

Comparison of the effects of different phytohormones on Col-0 seedlings to determine their 716

roles in root branching angle. (E) Distribution of branching angle in Col-0, jar1-11 and coi1 717

seedlings grown on different concentrations of MeJA. (F) Distribution of branching angle in 718

seedlings of Col-0, jin1-9, myc3, myc4 and myc2myc3myc4 mutants grown on different 719


https://doi.org/10.1101/2020.08.11.245720

23

concentrations of MeJA. Data are mean-SE of 4 biological replicates with 25 seedlings. 720

Asterisks indicate a significant difference in the studied parameter (P <0.05, Student’s t-test; * 721

control vs treatment and WT vs mutant (P <0.05, Student’s t-test). 722

723

Figure 2. Glc antagonizes MeJA-induced branching angle in Arabidopsis roots. (A) 724

Phenotype of Col-0 seedlings grown in different concentrations of Glc in combination with 725

MeJA. (B) Distribution of branching angle in Col-0 seedlings grown in different concentrations 726

of Glc in combination with MeJA. The graph represents the average of 7 biological replicates 727

and error bars represent SE. (C) Distribution of branching angle in Col-0 seedlings grown in 728

different concentrations of Man in combination with 1%Glc. The graph represents the average 729

of 3 biological replicates consisting of 25 seedlings and error bars represent SE. (D) 730

Distribution of branching angle in seedlings of Ler and HXK1-dependent signalling mutant 731

gin2-1 grown in different concentrations of Glc in combination with MeJA. The graph 732


represent SE. (E & F) Western blot analysis of total protein extracts of Jas9-VENUS seedlings 734

treated for 3 hours with 3% and without (0%) Glc in combination with 10µM MeJA and probed 735

with an anti-GFP antibody. The graph represents the average of 3 biological replicates and 736

error bar represent SE. Asterisks indicate a significant difference in the studied parameter 737

(P <0.05, Student’s t-test; * control vs treatment, ** 3%Glc vs 3%Glc+10µM MeJA and WT 738

vs mutant). 739

740

Figure 3. Components of auxin biosynthesis, transport and signalling lie downstream to 741

MeJA-mediated branching angle. (A) Distribution of branching angle in Col-0 seedlings 742

grown in 3%Glc in combination with MeJA (10µM) and different doses of NPA. The graph 743

represents the average of 4 biological replicates consisting of 25 seedlings and error bar 744

represent SE. (B) Distribution of branching angle in seedlings of auxin influx defective mutant 745

aux1-7 grown in 3%Glc in combination with different doses of MeJA. The graph represents 746

the average of 5 biological replicates consisting of 25 seedlings and error bars represent SE. 747

(C) Distribution of branching angle in seedlings of auxin efflux defective mutants eir1-1 and 748

mdr1-1 grown in 3%Glc in combination with different doses of MeJA. The graph represents 749

the average of 5 biological replicates consisting of 25 seedlings and error bars represent SE. 750

(D) Distribution of branching angle in seedlings of auxin signalling defective mutants tir1-1, 751

axr1-3 and axr2-1 grown in 3%Glc in combination with different doses of MeJA. The graph 752



https://doi.org/10.1101/2020.08.11.245720

24

represent SE. Asterisks indicate a significant difference in the studied parameter (P <0.05, 754

Student’s t-test; * control vs treatment and WT vs mutant). 755

756

Figure 4. Transcriptional activation of CYP79B2 and LAZY2 by MYC2. (A & B) RT-qPCR 757

expression of Col-0 and myc2myc3myc4 seedlings showing the expression CYP79B2 and 758

LAZY2 in 0.5X MS media. The graph represents the average of 3 biological replicates 759

consisting of 25 seedlings and error bars represent SE. (C) ChIP-qPCR showing the enrichment 760

of CYP79B2 promoter fragments with 35S::MYC2::GFP in all two regions under 0.5X MS 761

grown condition. The plot shows average of 2 biological replicates (D) ChIP-qPCR showing 762

the enrichment of LAZY2 promoter fragments with MYC2 in all two regions under 0.5X MS 763

grown condition. Fold enrichment of promoter fragments was calculated by comparing samples 764

treated without or with anti-GFP antibody. Untransformed Col-0 was taken as a negative 765

genetic control. The plot shows average of 3 biological replicates respectively. ORA59 and 766

ATXR6 are used as positive and negative controls, respectively. Asterisks indicate a significant 767

difference in the studied parameter. Error bars represent SE. (P <0.05, Student’s t-test; * WT 768

vs mutant). 769

Figure 5. MeJA controls lateral auxin redistribution in LRs. (A) LR of 12-day-old 770

DR5::GFP seedling treated with 0.5X MS and in combination with MeJA (10 µM). Scale bar: 771

50 µm. The data was repeated three times, with 8-9 roots imaged every time. The figure depicts 772

3 representative images. (B) The graph represents the cell length of 1st two upper and lower 773

epidermal cells of LR treated with 0.5X MS and in combination with 10µM MeJA. The data is 774

average of two biological replicates. (P <0.05, Student’s t-test; * Control vs treatment; ** 775

difference within MeJA treatment. (C) LR of 12-day-old PIN2::PIN2-eGFP LRs treated with 776

1/2MS and in combination with MeJA. (D) Enlarged view of 0.5X MS and MeJA treated 777

PIN2::PIN2-eGFP LR showing diminished PIN2 activity from the upper epidermal cell 778

profile. Solid white arrowheads depict strong GFP signal at the LR tip and dotted white 779

arrowheads depict reduced GFP signal. The data was repeated two times with 10-12 roots 780

imaged. 781

782

Figure 6. Effect of MeJA on cell profile and actin cytoskeleton in Arabidopsis seedlings. 783

(A & B) LR of 12-day-old ABD2::GFP treated with 3%Glc and in combination with MeJA 784

(10µM). (C & D) LR cell profile of 12-day-old Col-0 treated with 3%Glc and in combination 785


https://doi.org/10.1101/2020.08.11.245720

25

with MeJA (10 µM). Scale bar: 50µm. MR: Main root, LR: lateral root, solid arrowheads 786

indicate direction of seedling. The data was repeated two times with similar results. 787

788

Figure 7. Light modulation of Arabidopsis root branching angle. (A) Phenotype of light 789

grown 12-day-old Arabidopsis light signalling mutants hy5-1 and phyA201phyB5 treated with 790

0.5%Glc and 3%Glc alone and in combination with 10µM MeJA. (B) Distribution of branching 791

angle in seedlings of light signalling mutants phyA-201, phyB-5, phyA201B5 and hy5-1 grown 792

in different concentrations of Glc (0.5%, 3%) in combination with MeJA (10µM). The graph 793


represent SE. Asterisks indicate a significant difference in the studied parameter (P <0.05, 795

Student’s t-test; * control vs treatment and WT vs mutant. (C) RT-qPCR expression of Ler 796

and hy5-1 seedlings showing the expression LOX3 in 0.5X MS media under continuous light 797

and dark for 6 days. (D) RT-qPCR expression of Ler and hy5-1 seedlings showing the 798

expression LOX3 in 0.5X MS media under dark and dark to light transition for 6 hours. The 799

graph represents the average of 3 biological replicates, error bars represent SE. Asterisks 800

indicate a significant difference in the studied parameter (P <0.05, Student’s t-test; * control vs 801

treatment and ** WT vs mutant). 802

803

Figure 8. A testable model explaining the various interactions among different signals involved 804

in MeJA-modulated branching angle of Arabidopsis roots based on current and previous 805

findings. 806

807

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10µ

M M

eJA

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60

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20

C

0

Num

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)

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eJA

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S

*

*

*

* **

0

20

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S

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)

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S

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5µ

M M

eJA

½ M

S

1µ

M M

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5µ

M M

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½ M

S1µ

M M

eJA

5µ

M M

eJA

<40° 40°-70° >70°

jin1-9 myc3 myc4Col-0

F

10µ

M M

eJA

10µ

M M

eJA

10µ

M M

eJA

10µ

M M

eJA

10µ

M M

eJA

B

Bra

nchin

g a

ngle

( °)

0

60

40

70

80

50

20

10

30

*

**

5µ

M M

eJA

10µ

M M

eJA

1 µ

M M

eJA

½ M

S

A

myc234

*

*

*

*

*

*

½ M

S

1µ

M M

eJA

5µ

M M

eJA

10µ

M M

eJA

½ M

S

1µ

M M

eJA

5µ

M M

eJA

½ M

S

1µ

M M

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M M

eJA

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eJA

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M M

eJA

Num

ber

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ots

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)

0

20

60

40

100

80

120<40° 40°-70° >70°

Col-0 coi1-1

E

jar1-11

*

*

** *

*

*

*

Figure 1. MeJA decreases branching angle of Arabidopsis roots. (A) Phenotype of light grown 12-day-old

Arabidopsis Col-0 seedlings grown on different doses of MeJA. (B) Average branching angle of 12-day-old Col-

0 seedlings on different doses of MeJA. (C) Distribution of branching angle of 12-day-old Col-0 Arabidopsis

seedlings on different doses of MeJA. (D) Comparison of the effects of different phytohormones on Col-0

seedlings to determine their roles in root branching angle. (E) Distribution of branching angle in Col-0, jar1-11

and coi1 seedlings grown on different concentrations of MeJA. (F) Distribution of branching angle in seedlings

of Col-0, jin1-9, myc3, myc4 and myc2myc3myc4 mutants grown on different concentrations of MeJA. Data are

mean-SE of 4 biological replicates with 25 seedlings. Asterisks indicate a significant difference in the studied

parameter (P <0.05, Student’s t-test; * control vs treatment and WT vs mutant (P <0.05, Student’s t-test).


https://doi.org/10.1101/2020.08.11.245720

0.5%Glc 3%Glc

0µ

M M

eJA

*

0

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120Ler gin2-1

Num

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M M

eJA

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M M

eJA

0.5%Glc 3%Glc

<40° 40°-70° >70°

**

**

*

*

*

10µ

M M

eJA

10µ

M M

eJA

0µ

M M

eJA

Sig

nal In

tensity

3 hours

***

10µ

M M

eJA

10µ

M M

eJA

0µ

M M

eJA

0µ

M M

eJA

0%Glc 3%Glc

0

0.5

1

1.5

A

10µM MeJA

3%

Glc

0.5

%G

lc

Col-0

B

0

20

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Num

ber

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ots

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)

>70°

40°-70°<40°

Col-0

*

*

*

*

* *

*

10µ

M M

eJA

0µ

M M

eJA

10µ

M M

eJA

0µ

M M

eJA

0.5%Glc 3%Glc

C

Glc MeJA

- - + +- - ++

HSP90

Jas9-VENUS

3 hours

RUBISCO

D FE

0

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ots

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<40° 40°-70° >70°

1%

Glc

1%

Man

2%

Man

4%

Man

3%

Man

*

*

*

*

*

*

*

1%Glc

Col-0

*

Figure 2. Glc antagonizes MeJA-induced branching angle in Arabidopsis roots. (A) Phenotype of Col-0

seedlings grown in different concentrations of Glc in combination with MeJA. (B) Distribution of branching angle in

Col-0 seedlings grown in different concentrations of Glc in combination with MeJA. The graph represents the

average of 7 biological replicates and error bars represent SE. (C) Distribution of branching angle in Col-0

seedlings grown in different concentrations of Man in combination with 1%Glc. The graph represents the average

of 3 biological replicates consisting of 25 seedlings and error bars represent SE. (D) Distribution of branching

angle in seedlings of Ler and HXK1-dependent signalling mutant gin2-1 grown in different concentrations of Glc in

combination with MeJA. The graph represents the average of 4 biological replicates consisting of 25 seedlings and

error bars represent SE. (E & F) Western blot analysis of total protein extracts of Jas9-VENUS seedlings treated

for 3 hours with 3% and without (0%) Glc in combination with 10µM MeJA and probed with an anti-GFP antibody.

The graph represents the average of 3 biological replicates and error bar represent SE. Asterisks indicate a

significant difference in the studied parameter (P <0.05, Student’s t-test; * control vs treatment, ** 3%Glc vs

3%Glc+10µM MeJA and WT vs mutant).

0µM MeJA

**

**


https://doi.org/10.1101/2020.08.11.245720

10µ

M M

eJA

5µ

M M

eJA

1µ

M M

eJA

0µ

M M

eJA

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eJA

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M M

eJA

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eJA

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eJA

3%Glc

0

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20Num

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eJA

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eJA

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eJA

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eJA

5µ

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eJA

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eJA

0µ

M M

eJA

10µ

M M

eJA

5µ

M M

eJA

1µ

M M

eJA

0µ

M M

eJA

** * * * * * * * *

* * * ** *

**

*****

*

*

*

*

*

**

**

*

*

*

*

3%Glc+500nM

NPA

3%Glc+250nM

NPA

3%Glc+100nM

NPA3%Glc+1µM NPA

120>70°

A

0

60

40

100

80

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20

<40° 40°-70° >70°

10µ

M M

eJA

5µ

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eJA

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eJA

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M M

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eir1-1 Ws mdr1-1Col-0

Num

ber

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ral ro

ots

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)

D

* **

*

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**

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*

* *

* **

* *

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5µ

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M M

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M M

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5µ

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eJA

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eJA

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eJA

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M M

eJA

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M M

eJA

1µ

M M

eJA

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eJA

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eJA

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eJA

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M M

eJA

Col-0 tir1-1 axr2-1axr1-3

0

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ots

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)<40° 40°-70° >70°

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eJA

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eJA

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eJA

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eJA

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eJA

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M M

eJA

5µ

M M

eJA

1µ

M M

eJA

0µ

M M

eJA

* * *

*

**

* *

* * *

*

**

*

*

*

* *

**

**

**

**

C

10µ

M M

eJA

5µ

M M

eJA

1µ

M M

eJA

0µ

M M

eJA

10µ

M M

eJA

5µ

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eJA

1µ

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eJA

0µ

M M

eJA

Col-0 aux1-7

0

60

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<40° 40°-70° >70°

Num

ber

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ots

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)

B

3%Glc 3%Glc

3%Glc

* *

*** * *

*

**

**

Figure 3. Components of auxin biosynthesis, transport and signalling lie downstream to MeJA

mediated branching angle. (A) Distribution of branching angle in Col-0 seedlings grown in 3%Glc in

combination with MeJA (10µM) and different doses of NPA. The graph represents the average of 4 biological

replicates consisting of 25 seedlings and error bar represent SE. (B) Distribution of branching angle in seedlings

of auxin influx defective mutant aux1-7 grown in 3%Glc in combination with different doses of MeJA. The graph

represents the average of 5 biological replicates consisting of 25 seedlings and error bars represent SE. (C)

Distribution of branching angle in seedlings of auxin efflux defective mutants eir1-1 and mdr1-1 grown in 3%Glc

in combination with different doses of MeJA. The graph represents the average of 5 biological replicates

consisting of 25 seedlings and error bars represent SE. (D) Distribution of branching angle in seedlings of auxin

signalling defective mutants tir1-1, axr1-3 and axr2-1 grown in 3%Glc in combination with different doses of

MeJA. The graph represents the average of 6 biological replicates consisting of 25 seedlings and error bars

represent SE. Asterisks indicate a significant difference in the studied parameter (P <0.05, Student’s t-test; *

control vs treatment and WT vs mutant).


https://doi.org/10.1101/2020.08.11.245720

Fo

ld E

nrichm

ent (I

P/I

nput)

A CATGE-box+1

-2162

-1926

G-box

-1386

-1597

R2

Col R

1

MY

C2ox R

1

Col R

2

MY

C2ox R

2

Col

MY

C2ox

Col

MY

C2ox

*

*

*

2

CYP79B2

4

6

ORA59 ATXR6

R1

CYP79B2

Fold

expre

ssio

n 0.8

1.2

1.0

0.2

0.6

0.4

myc2myc3myc4

Col-0

0

*

* *

*

LAZY2

Col R

1

MY

C2ox R

1

Col R

2

MY

C2ox R

2

Col

MY

C2ox

Col

MY

C2ox

0

6

4

2

8

Fo

ld E

nrichm

ent (I

P/I

nput)

ATG

+1

-155 +94

-76

R1 R2

-337

CAACTG

CATCTG

CAATTG

CAAGTG

ORA59 ATXR6

LAZY2

Fo

ld e

xpre

ssio

n *0.8

1.2

1.0

0.2

0.6

0.4

0

myc2myc3myc4Col-0

Figure 4. Transcriptional activation of CYP79B2 and LAZY2 by MYC2. (A & B) RT-qPCR expression of

Col-0 and myc2myc3myc4 seedlings showing the expression CYP79B2 and LAZY2 in 0.5X MS media. The

graph represents the average of 3 biological replicates consisting of 25 seedlings and error bars represent

SE. (C) ChIP-qPCR showing the enrichment of CYP79B2 promoter fragments with 35S::MYC2::GFP in all

two regions under 0.5X MS grown condition. The plot shows average of 2 biological replicates (D) ChIP-

qPCR showing the enrichment of LAZY2 promoter fragments with MYC2 in all two regions under 0.5X MS

grown condition. Fold enrichment of promoter fragments was calculated by comparing samples treated

without or with anti-GFP antibody. Untransformed Col-0 was taken as a negative genetic control. The plot

shows average of 3 biological replicates respectively. ORA59 and ATXR6 are used as positive and negative

controls, respectively. Asterisks indicate a significant difference in the studied parameter. Error bars

represent SE. (P <0.05, Student’s t-test; * WT vs mutant).

B D


https://doi.org/10.1101/2020.08.11.245720

DR5::GFP½ MS 10µM MeJA

A B

½ MS PIN2::PIN2-eGFP (enlarged view)

10µM MeJA PIN2::PIN2-eGFP (enlarged view)

Cell

length

(µ

m)

20

40

80

1st two upper

epidermal

cells

½ MS 10µM MeJA

1st two lower

epidermal cells

1st two upper

epidermal cells1st two lower

epidermal

cells

**

*

*

*

C

D

Figure 5. MeJA controls lateral auxin redistribution in LRs. (A) LR of 12-day-old DR5::GFP seedling treated

with 0.5X MS and in combination with MeJA (10 µM). Scale bar: 50 µm. The data was repeated three times, with 8-

9 roots imaged every time. The figure depicts 3 representative images. (B) The graph represents the cell length of

1st two upper and lower epidermal cells of LR treated with 0.5X MS and in combination with 10µM MeJA. The data

is average of two biological replicates. (P <0.05, Student’s t-test; * Control vs treatment; ** difference within MeJA

treatment. (C) LR of 12-day-old PIN2::PIN2-eGFP LRs treated with 1/2MS and in combination with MeJA. (D)

Enlarged view of 0.5X MS and MeJA treated PIN2::PIN2-eGFP LR showing diminished PIN2 activity from the

upper epidermal cell profile. Solid white arrowheads depict strong GFP signal at the LR tip and dotted white

arrowheads depict reduced GFP signal. The data was repeated two times with 10-12 roots imaged.

g

g g

g

½ M

S10µ

M M

eJA

PIN2::PIN2-eGFP

g

g g

g


https://doi.org/10.1101/2020.08.11.245720

LR

MR

LR

MR

3%Glc 3%Glc+10 µM MeJA

3%Glc 3%Glc+10 µM MeJA

LR

MR

LR

MR

A B

C D

Col-0

35S::GFP:ABD2::GFP

Figure 6. Effect of MeJA on cell profile and actin cytoskeleton in Arabidopsis seedlings. (A & B) LR of

12-day-old ABD2::GFP treated with 3%Glc and in combination with MeJA (10µM). (C & D) LR cell profile of

12-day-old Col-0 treated with 3%Glc and in combination with MeJA (10 µM). Scale bar: 50µm. MR: Main

root, LR: lateral root, solid arrowheads indicate direction of seedling. The data was repeated two times with

similar results.


https://doi.org/10.1101/2020.08.11.245720

B >70°

10µ

M M

eJA

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eJA

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eJA

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eJA

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eJA

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eJA

3%Glc0.5%Glc

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eJA

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eJA

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eJA

3%Glc0.5%Glc

10µ

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eJA

0µ

M M

eJA

10µ

M M

eJA

0µ

M M

eJA

3%Glc0.5%Glc

*

*

* * *

*

*

*

*

*

*

*0

60

100

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20Num

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ots

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40

Ler phyA-201 phyB-5 phyA-201B-5 hy5-1

10µ

M M

eJA

0µ

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eJA

10µ

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eJA

0µ

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eJA

3%Glc0.5%Glc

<40° 40°-70°

0

5

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15

20

25

30

Ler

Dark

hy5-1

lightLer

Light

hy5-1

Dark

Fo

ld e

xpre

ssio

n

***

*

LOX3DC

0

2

4

6

8

10

Ler

Darkhy5-1

Dark

Ler Dark

to Light hy5-1 Dark

to Light

Fo

ld e

xpre

ssio

n

*

***

LOX3

A0.5%Glc 0.5%Glc+10µM MeJA 3%Glc 3%Glc+10µM MeJA

Ler

phyA

-201phyB

-5hy5-1

Figure 7. Light modulation of Arabidopsis root branching angle. (A) Phenotype of light grown 12-day-old

Arabidopsis light signalling mutants hy5-1 and phyA201phyB5 treated with 0.5%Glc and 3%Glc alone and in

combination with 10µM MeJA. (B) Distribution of branching angle in seedlings of light signalling mutants phyA-201,

phyB-5, phyA201B5 and hy5-1 grown in different concentrations of Glc (0.5%, 3%) in combination with MeJA

(10µM). The graph represents the average of 5 biological replicates consisting of 25 seedlings and error bars

represent SE. Asterisks indicate a significant difference in the studied parameter (P <0.05, Student’s t-test; * control

vs treatment and WT vs mutant. (C) RT-qPCR expression of Ler and hy5-1 seedlings showing the expression

LOX3 in 0.5X MS media under continuous light and dark for 6 days. (D) RT-qPCR expression of Ler and hy5-1

seedlings showing the expression LOX3 in 0.5X MS media under dark and dark to light transition for 6 hours. The

graph represents the average of 3 biological replicates, error bars represent SE. Asterisks indicate a significant

difference in the studied parameter (P <0.05, Student’s t-test; * control vs treatment and ** WT vs mutant).


https://doi.org/10.1101/2020.08.11.245720

Sugars

Glucose

HXK1

PHYA/PHYB

HY5

JA-Ile

COI1

Actin filament reorganization

Cellular organization

Branching angle

JA Sign

alling

Light sign

alling

Glu

cose

sig

nal

ling

JAZ9

MYC2

CYP79B2

Auxin Homeostasis

LAZY2

JA levels (LOX3)

Factor X?

Figure 8: A testable model explaining the various interactions among

different signals involved in MeJA-modulated branching angle of

Arabidopsis roots based on current and previous findings.


https://doi.org/10.1101/2020.08.11.245720

1

11

21

31

41

51

61

71

jin1-9

½ M

S1µ

M M

eJA

5µ

M M

eJA

10µ

M M

eJA

jar1-11 coi1 myc2myc3myc4Col-0

½ MS10µM

MeJA

5µM

MeJA

1µM

MeJA

10µ

M M

eJA

5µ

M M

eJA

1µ

M M

eJA

½ M

S

10µ

M M

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5µ

M M

eJA

1µ

M M

eJA

½ M

S

10µ

M M

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5µ

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1µ

M M

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½ M

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10µ

M M

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5µ

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M M

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½ M

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10µ

M M

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5µ

M M

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1µ

M M

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½ M

S

Col-0 jaz1 jaz4jaz2 jaz6

0

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20Num

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ots

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*

*

*

*

*

*

*

*

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*

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* *

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*

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0

60

40

80

20

10

50

70

30

Bra

nchin

g a

ngle

(°)

Col-0 jaz1 jaz4jaz2 jaz6

5µM

MeJA½ MS 1µM

MeJA

10µM

MeJA

*

*

*

*

*

***

*

*

*

A B

C

DE

0

10

20

30

40

50

60

70

80

jin1-9 myc3 myc4 myc2myc3myc4Col-0

Col-0 jar1-11 coi1-1

*

*

***

*

*

* *

*

***

*

**

*

*

* *

Bra

nchin

g a

ngle

(°)

Bra

nchin

g a

ngle

(°)

Figure S1. MeJA regulation of branching angle of Arabidopsis roots. (A) Phenotype of Col-0 and JA

signalling mutant seedlings grown on different doses of MeJA. (B and C) Average branching angle of 12-day-old

Col-0 and JA signalling mutants grown in different doses of MeJA. The data represents the average of 4

biological replicates consisting of 25 seedlings and error bars represent SE. (D and E) Distribution and average

branching angle of seedlings of Col-0 and different jaz mutants grown in different doses of MeJA. The data

represents the average of 3 biological replicates consisting of 25 seedlings and error bars represent SE.

Asterisks indicate a significant difference in the studied parameter (P<0.05,Student’st-test;*control vs treatment

and WT vs mutant).


https://doi.org/10.1101/2020.08.11.245720

Col-0 jar1-11 myc2myc3myc4

Figure S2. MeJA regulation of branching angle of Arabidopsis roots. (A) A representative image of 20

DAG (days after germination) of Col-0 and JA biosynthesis and signalling mutants jar1-11 and myc2myc3myc4

grown in 0.5X MS medium in cylindrical tubes. (B) Average branching angle of 20 DAG (days after germination)

of Col-0 and JA biosynthesis and signalling mutants jar1-11 and myc2myc3myc4 grown in 0.5X MS medium in

cylindrical tubes. The data represents images from 2 biological replicates (P<0.05,Student’st-test; * WT vs

mutant.

0.5X MS

0

10

20

30

40

50

60

70

80

90

100

Bra

nchin

g a

ngle

(°)

Col-0 jar1-11 myc2myc3myc4

* *

A

B


https://doi.org/10.1101/2020.08.11.245720

0

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rgs1-2Col-0 rgs1-1

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

0

20

60

40

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80

120 <40° 40°-70° >70°

Num

ber

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late

ral ro

ots

(%

)

gpa1-4Col-0 gpa1-3*

*

*

*

*

*

*

*

* * *

*

*

*

* *

0

10

20

30

40

50

60

70

80

Bra

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(°)

*

*

*

0.5

% G

lc

0.5

%G

lc+

10µ

M M

eJA

3%

Glc

3%

Glc

+

10µ

M M

eJA

0

10

20

30

40

50

60

70

80

90

Ler gin2-1

*

*

*

Bra

nchin

g a

ngle

(°)

0.5% Glc

3% Glc+10µM MeJA3%Glc

0.5% Glc+10µM MeJA

0.5%Glc 3%Glc

0µ

M M

eJA

10µ

M M

eJA

0µ

M M

eJA

0.5%Glc 3%Glc

10µ

M M

eJA

0µ

M M

eJA

10µ

M M

eJA

0µ

M M

eJA

10µ

M M

eJA

0.5%Glc 3%Glc

10µ

M M

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A B

C D

Figure S3. MeJA-Glc regulation of branching angle of Arabidopsis roots. (A) Average branching angle

of 12-day-old Col-0 seedlings grown in different concentrations of Glc (0.5%, 3%) and in combination with

MeJA. The data represents the average of 6 biological replicates consisting of 25 seedlings and error bars

represent SE. (B) Average branching angle of 12 day-old Ler and HXK1-dependent Glc signalling mutant

gin2-1 grown in different concentrations of Glc (0.5%, 3%) and in combination with MeJA. The data

represents the average of 4 biological replicates consisting of 25 seedlings and error bars represent SE. (C

and D) Distribution of branching angle in seedlings of Col-0 and HXK1-independent signalling mutants rgs1-

1, rgs1-2, gpa1-3 and gpa1-4 grown in different concentrations of Glc in combination with MeJA. The data

represents the average of 4 biological replicates consisting of 25 seedlings and error bars represent SE.

(P <0.05, Student’s t-test; * control vs treatment ** 0.5%Glc vs 3%Glc+10MeJA and wild-type vs mutant).

**


https://doi.org/10.1101/2020.08.11.245720

Jas9-VENUS

MeJA - +

HSP90

RUBISCO

1% Suc + +

3 hours

Figure S4. Western blot depicting the stability of Jas9-VENUS. Immuno-blot detection of Jas9-VENUS in

seven-day-old Jas9-VENUS expressing seedlings treated with 1%Suc without and with 10µM MeJA. Jas9-

VENUS protein was detected using anti-GFP–specific antibody. Ponceau S stained RUBISCO and Anti-

HSP90 detecting HSP90 were used as loading controls. The data represents average of 3 biological replicates.


https://doi.org/10.1101/2020.08.11.245720

**

**

*

**

Figure S5. Heat map of JA signalling genes upon treatment with 0%Glc+10µM MeJA, 3%Glc and

3%Glc+10µM MeJA. The expression value of genes treated with 0%Glc+10µM MeJA is taken as 1. The

expression values of genes in 3%Glc and 3%Glc+10µM MeJA is compared with those of 0%Glc+10µM MeJA.

* indicates expression values of genes that are significantly altered. The data is the average of 2 biological

replicates. Heat map was generated using MeV.

**********

*


https://doi.org/10.1101/2020.08.11.245720

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*

3%Glc

Figure S6. Role of auxin machinery in controlling JA-mediated branching angle. (A) Average branching

angle of auxin influx defective mutant aux1-7 grown in 3%Glc in combination with MeJA. (B) Distribution of

branching angle of auxin influx defective mutant lax3 grown in 3%Glc in combination with different doses of MeJA

(C) Average branching angle of auxin efflux defective mutants eir1-1 and mdr1.1 grown in 3%Glc in combination

with MeJA. (D) Distribution of branching angle of auxin efflux defective mutants pin4-3 and pin7-2 grown in 3%Glc

in combination with different doses of MeJA. (E and F) Average branching angle of auxin signalling mutants tir1-

1, axr1-3 and axr2-1 grown in 3%Glc in combination with MeJA. The data represents the average of 4 biological

replicates consisting of 25 seedlings and error bars represent SE. Asterisks indicate a significant difference in the

studied parameter (P <0.05, Student’s t-test; * control vs treatment and WT vs mutant).

3% Glc

3% Glc+10µM MeJA3% Glc+5µM MeJA

3% Glc+1µM MeJA


https://doi.org/10.1101/2020.08.11.245720

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3%Glc 3%Glc+100nM LatB 3%Glc+ 500nM LatB

3%Glc+ 100nM LatB

+10µM MeJA

3%Glc+ 500nM LatB

+10µM MeJA

3%Glc+10µM MeJA

B C

A

Figure S7. Effect of MeJA and Lat B on branching angle of Arabidopsis roots. (A) Phenotype of Col-0

seedlings treated with 3%Glc and in combination with 100nM and 500nM Lat B and 10µM MeJA. (B) Distribution

of branching angle of Col-0 seedlings treated with 3%Glc and in combination with 100nM and 500nM Lat B and

10µM MeJA. (C) Average branching angle of Col-0 seedlings treated with 3%Glc and in combination with

100nM and 500nM Lat B and 10µM MeJA. The data represents the average of 4 biological replicates consisting

of 25 seedlings and error bars represent SE. Asterisks indicate a significant difference in the studied parameter

(P <0.05, Student’s t-test; * control vs treatment)

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https://doi.org/10.1101/2020.08.11.245720

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Light Dark

Col-0 myc2myc3myc4 Col-0 myc2myc3myc4

* *

* *

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Dark

3%Glc 3%Glc+10MeJA

Col-0 myc234

A

B

Figure S8. Role of light signalling in controlling JA-mediated branching angle. (A) Phenotype of light

and dark adapted Col-0 and myc2myc3myc4 seedlings in different concentrations of Glc (0.5%, 3%) and in

combination with MeJA (10µM). (B) Distribution of branching angle in light and dark adapted Col-0 and

myc2myc3myc4 seedlings in different concentrations of Glc (0.5%, 3%) and in combination with MeJA

(10µM). The data represents the average of 6 biological replicates consisting of 25 seedlings and error bars

represent SE Asterisks indicate a significant difference in the studied parameter (P <0.05, Student’s t-test; *

control vs treatment and WT vs mutant).

myc2myc3myc4Col-0


https://doi.org/10.1101/2020.08.11.245720

Ler control Ler root-hy5-1 shoot Ler (root only) Ler control Ler root-

phyA201phyB5 shootLer (root only)

hy5-1 control hy5-1 root-Ler shoot hy5-1(root only) phyA201phyB5 control phyA201phyB5 root-

Ler shoot

phyA201phyB5

(root only)

Figure S9. Alterations in shoot or root affects branching angle of LRs. (A) Phenotypes of Ler and hy5-1

(uncut) micrografted Ler, hy5-1 and stalkless hy5-1 and Ler. (B) Phenotypes of Ler and phyA201phyB5

(uncut) micrografted Ler, phya201phyb205 and stalkless phyA201phyB5 and Ler. The data represents the

average of 1 biological replicates consisting of 15 seedling. The experiment was repeated twice.

A B


https://doi.org/10.1101/2020.08.11.245720

Supplementary Table 1: The primers used in the study.

Oligo name 5'-3' Sequence

MYC2_ChIP_CYP79B2 R1-F CGTAGTCGTGTCATATAGCGATTAAC

MYC2_ChIP_CYP79B2 R1-R GGATTGGTTTGTAATACTTTGCAAT

MYC2_ChIP_CYP79B2 R2-F CATGGTGAAAAACATTTTGCTAGC

MYC2_ChIP_CYP79B2 R2-R CCCAAATGTTTTCGCCTTCT

MYC2_ChIP_LZY2 R1-F GGCCATAGAAGAATGTTGGTGGA

MYC2_ChIP_LZY2 R1-R CAAGTTCCAAACTTTGACTACTTAAAAGAGA

MYC2_ChIP_LZY2 R2-F GATTCATCAATAACTCGAGTTGAGAAAT

MYC2_ChIP_LZY2 R2-R GAAAACAAAAGCCAGAAGAAAACACTT

ATXR6_ChIP FP CCGAACCGAACAACCAAAATATATG

ATXR6_ChIP RP CCAGAGAAAGAGAGAGAGTGAGAGATT

ORA59_ChIP FP GTACGTCATACACTCAACCTG

ORA59_ChIP RP CAATTAGGCTGCCTCCGAATA

LOX3 RT-qPCR-F ACGCTGATCCTGACCGTAGAA

LOX3 RT-qPCR-R GCTCAGAACTCGGAACCAACA

LAZY2 RT-qPCR-F AGAGGAATTTTTTTGGTGCATCTG

LAZY2 RT-qPCR-R CAGATCAAATCATGCAAAAAAAGAAG

CYP79B2 RT-qPCR-F GGCTCCGGCGCTAGGACYP79B2 RT-qPCR-R TTGAAGAAGTCTCGCGAGCAT


https://doi.org/10.1101/2020.08.11.245720

Jasmonic acid coordinates with light to regulate branching ... · 8/11/2020 · bPresent address:...

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