1
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
9
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
21
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(which was not certified by peer review) is the author/funder.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
2
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
52
53
* Author correspondence: [email protected]
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3
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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4
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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5
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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6
<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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7
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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8
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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10
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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11
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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12
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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13
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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14
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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15
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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16
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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17
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
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18
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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19
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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20
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
(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
21
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
(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
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
(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
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
represents the average of 4 biological replicates consisting of 25 seedlings and error bars 733
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
represents the average of 6 biological replicates consisting of 25 seedlings and error bars 753
(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
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
(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
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
represents the average of 5 biological replicates consisting of 25 seedlings and error bars 794
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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Tanaka H, Toyota M, Tasaka M, et al (2017) The Arabidopsis LAZY1 Family Plays a 961
Key Role in Gravity Signaling within Statocytes and in Branch Angle Control of Roots 962
and Shoots. The Plant Cell tpc.00575.2016 963
Trachsel S, Kaeppler SM, Brown KM, Lynch JP (2013) Maize root growth angles become 964
steeper under low N conditions. Field Crops Research 140: 18–31 965
Urano D, Phan N, Jones JC, Yang J, Huang J, Grigston J, Taylor JP, Jones AM (2012) 966
Endocytosis of the seven-transmembrane RGS1 protein activates G-protein-coupled 967
signalling in Arabidopsis. doi: 10.1038/ncb2568 968
Vleesschauwer D De, Filipe O, Hoffman G, Seifi HS, Haeck A, Canlas P, Bockhaven J 969
Van, Waele E De, Demeestere K, Ronald P, et al (2017) Target of rapamycin 970
signaling orchestrates growth – defense trade-offs in plants. doi: 10.1111/nph.14785 971
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Plant Cell 24: 842–857 973
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and action in plant stress response, growth and development. An update to the 2007 975
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Willemoes JG, Beltrano J, Montalbi R (1988) and Its Reversion By Gibberellic Acid. 3–5 977
Xiong Y, McCormack M, Li L, Hall Q, Xiang C, Sheen J (2013) Glucose-TOR signalling 978
reprograms the transcriptome and activates meristems. Nature. doi: 979
10.1038/nature12030 980
Xiong Y, Sheen J (2015) Novel links in the plant TOR kinase signaling network. Current 981
Opinion in Plant Biology 28: 83–91 982
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signaling in plants. Journal of Biological Chemistry. doi: 10.1074/jbc.M111.300749 984
Yokawa K, Fasano R, Kagenishi T, Baluška F (2014) Light as stress factor to plant roots – 985
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999
(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
**
*
*
*
*
*
*
*
**
*
* **
*
*
*
*
½ MS 1µM MeJA 5µM MeJA 10µM MeJA
Col-0
D
½ M
S
10nM
BA
P
50nM
BA
P
100nM
BA
P
½ M
S
10nM
AC
C
50nM
AC
C
100nM
AC
C
½ M
S
10nM
AB
A
50nM
AB
A
100nM
AB
A
½ M
S
10nM
BR
50nM
BR
100nM
BR
½ M
S
10nM
GA
50nM
GA
100nM
GA0
60
40
100
80
120
20
Num
ber
of
late
ral ro
ots
(%
)
<40° 40°-70° >70°
10µ
M M
eJA
<40° 40°-70°>70°
60
40
100
80
120
20
C
0
Num
ber
of
late
ral ro
ots
(%
)
5µ
M M
eJA
1µ
M M
eJA
½ M
S
*
*
*
* **
0
20
60
40
100
80
120
½ M
S
1µ
M M
eJA
5µ
M M
eJA
Num
ber
of
late
ral ro
ots
(%
)
½ M
S
1µ
M M
eJA
5µ
M M
eJA
½ M
S
1µ
M M
eJA
5µ
M M
eJA
½ M
S
1µ
M M
eJA
5µ
M M
eJA
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
eJA
5µ
M M
eJA
10µ
M M
eJA
10µ
M M
eJA
Num
ber
of
late
ral ro
ots
(%
)
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).
(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
0.5%Glc 3%Glc
0µ
M M
eJA
*
0
20
60
40
100
80
120Ler gin2-1
Num
ber
of
late
ral ro
ots
(%
)
10µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
0µ
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
60
40
100
80
120
Num
ber
of
late
ral ro
ots
(%
)
>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
20
60
40
100
80
120
Num
ber
of
late
ral ro
ots
(%
)
<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
**
**
(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
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
0µ
M M
eJA
3%Glc
0
60
40
100
80
20Num
ber
of
late
ral ro
ots
(%
)
<40° 40°-70°
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
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
120
20
<40° 40°-70° >70°
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
0µ
M M
eJA
eir1-1 Ws mdr1-1Col-0
Num
ber
of
late
ral ro
ots
(%
)
D
* **
*
*
*
* * *
**
*
**
*
* *
* **
* *
*
**
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
Col-0 tir1-1 axr2-1axr1-3
0
60
40
100
80
120
20
Num
ber
of
late
ral ro
ots
(%
)<40° 40°-70° >70°
0µ
M M
eJA
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
0µ
M M
eJA
10µ
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
M M
eJA
1µ
M M
eJA
0µ
M M
eJA
Col-0 aux1-7
0
60
40
100
80
120
20
<40° 40°-70° >70°
Num
ber
of
late
ral ro
ots
(%
)
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).
(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
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
(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
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
(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
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.
(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
B >70°
10µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
0µ
M M
eJA
3%Glc0.5%Glc
10µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
0µ
M M
eJA
3%Glc0.5%Glc
10µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
0µ
M M
eJA
3%Glc0.5%Glc
10µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
0µ
M M
eJA
3%Glc0.5%Glc
*
*
* * *
*
*
*
*
*
*
*0
60
100
80
120
20Num
ber
of
late
ral ro
ots
(%
)
40
Ler phyA-201 phyB-5 phyA-201B-5 hy5-1
10µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
0µ
M M
eJA
3%Glc0.5%Glc
<40° 40°-70°
0
5
10
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).
(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
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.
(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
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
eJA
5µ
M M
eJA
1µ
M M
eJA
½ M
S
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
½ M
S
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
½ M
S
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
½ M
S
Col-0 jaz1 jaz4jaz2 jaz6
0
60
40
100
80
120
20Num
ber
of
late
ral ro
ots
(%
)
<40° 40°-70° >70°
*
*
*
*
*
*
*
*
*
**
*
*
*
*
* *
*
*
*
*
*
*
*
*
* *
*
**
*
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).
(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
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
(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
0
20
60
40
100
80
120
Num
ber
of
late
ral ro
ots
(%
)
<40° 40°-70° >70°
rgs1-2Col-0 rgs1-1
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
0
20
60
40
100
80
120 <40° 40°-70° >70°
Num
ber
of
late
ral ro
ots
(%
)
gpa1-4Col-0 gpa1-3*
*
*
*
*
*
*
*
* * *
*
*
*
* *
0
10
20
30
40
50
60
70
80
Bra
nchin
g a
ngle
(°)
*
*
*
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
eJA
0µ
M M
eJA
10µ
M M
eJA
0µ
M M
eJA
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
eJA
0µ
M M
eJA
10µ
M M
eJA
0µ
M M
eJA
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).
**
(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
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.
(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
**
**
*
**
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.
**********
*
(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
0
90
20
10
80
100
Bra
nchin
g a
ngle
(°)
50
30
60
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40
Col-0 Ws-2eir1-1 mdr1.1
*
**
**
*
** *
*
0
10
20
30
40
50
60
70
80
90
Bra
nchin
g a
ngle
(°)
Col-0 tir1-1
** *
** *
D
B CA
E F
0
10
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30
40
50
60
70
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90
Bra
nchin
g a
ngle
(°)
Col-0 axr2-1axr1-3
***
*
*
0
90
20
10
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30
60
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40
Bra
nchin
g a
ngle
(°)
Col-0 aux1-7
**
*
*
0
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20
Num
ber
of
late
ral ro
ots
(%
)
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
0µ
M M
eJA
Col-0 pin4-3 pin7-2
<40° 40°-70° >70°
**
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
* *
**
*
3%Glc
0
60
40
100
80
120
20Num
ber
of
late
ral ro
ots
(%
)
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
5µ
M M
eJA
1µ
M M
eJA
0µ
M M
eJA
Col-0 lax3
<40° 40°-70° >70°
*
*
*
*
*
*
*
*
*
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
(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
0
60
40
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80
120
20
Num
ber
of
late
ral ro
ots
(%
)
3%
Glc
10µ
M M
eJA
+100nM
Lat
B
10µ
M M
eJA
10µ
M M
eJA
+500nM
Lat
B
100nM
Lat
B
500nM
Lat
B
*
* *
*
<40° 40°-70° >70°
3%Glc
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)
0
10
20
30
40
50
60
70
80
Bra
nchin
g a
ngle
(°)
3%
Glc
**
10µ
M M
eJA
+100nM
Lat
B
10µ
M M
eJA
10µ
M M
eJA
+500nM
Lat
B
100nM
Lat
B
500nM
Lat
B
3%Glc
(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
0
60
40
100
80
120
20
10µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
0µ
M M
eJA
3%Glc0.5%Glc 3%Glc0.5%Glc
10µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
0µ
M M
eJA
10µ
M M
eJA
0µ
M M
eJA
3%Glc0.5%Glc 3%Glc0.5%Glc
<40° 40°-70° >70°
Light Dark
Col-0 myc2myc3myc4 Col-0 myc2myc3myc4
* *
* *
*
*
*
* *
*
**
*
**
*
*
*
*
* *
*
* *
Num
ber
of
late
ral ro
ots
(%
)
3%Glc 3%Glc+10MeJALight
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
(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
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
(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
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
(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