2

3 Dipan Roy¹†*, Poonam Mehra²†*, Lisa Clark¹, Vaishnavi Mukkawar¹, Kevin Bellande³,⁴,

4 Raquel Martin-Arevalillo⁵,⁶, Srayan Ghosh¹, Kishor D. Ingole⁸, Prakash Kumar

5 Bhagat¹, Adrian Brown¹, Kawinnat Sue ob⁷, Andrew Jones⁷, Joop EM Vermeer³, Teva

6 Vernoux⁵, Kathryn Lilley⁸, Phil Mullineaux⁹, Ulrike Bechtold¹, Malcolm J. Bennett² and

7 Ari Sadanandom¹*.

8

9 ¹Department of Biosciences, Durham University, Durham, DH1 3LE, United Kingdom

10 ²Plant & Crop Sciences, School of Biosciences, University of Nottingham, LE12 5RD, United Kingdom

11 ³Laboratory of Plant Molecular and Cellular Biology, University of Neuchâtel, Rue Emile Argand 11,
12 2000, Neuchâtel, Switzerland-IPSiM

13 ⁴University of Montpellier, CNRS, INRAE, Institut Agro, 34060, Montpellier, France

14 ⁵Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, CNRS, INRAE,
15 F-69342, Lyon, France

16 ⁶Laboratoire de Recherche en Sciences Végétales, Université Toulouse, CNRS, INP, Castanet-Tolosan,
17 France

18 ⁷Institute of Systems, Molecular and Integrative Biology, Liverpool, L69 3BX, United Kingdom

19 ⁸Department of Biochemistry, University of Cambridge, Cambridge, CB2 1RX, United Kingdom

20 ⁹School of Life Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, United Kingdom

21

22 † *These authors contributed equally to the work*

23 * *To whom correspondence should be addressed*

24 Email id: ari.sadanandom@durham.ac.uk, dipan.roy@durham.ac.uk, p.mehra@nottingham.ac.uk

25
26 Reactive oxygen species function as key signals in plant adaptation to environmental stresses like drought. Roots respond to transient water unavailability by temporarily ceasing branching through the acclimative response xerobranching. In this study, we report how a xerobranching stimulus triggers rapid changes of ROS levels in root nuclei, triggering redox-dependent multimerization of the auxin repressor protein IAA3.
31 Mutations in specific cysteine residues of IAA3 disrupt redox-mediated multimerization and interaction with co-repressor TPL, thereby attenuating IAA3 mediated target gene repression. Other AUX/IAA proteins also vary in their redox mediated multimerization, revealing a regulatory mechanism that connects dynamic changes in cellular redox status to auxin signalling. Our study reveals how ROS, auxin and water availability intersect and shape root adaptive responses, thereby maintaining phenotypic plasticity in plants.
37

38 **One-Sentence Summary:** Water deficit triggers redox-regulated multimerization of Aux/IAAs, such as IAA3, in root cells to regulate xerobranching response

40 Plants optimise their root growth behaviour according to spatial distribution of the soil resources. For instance, roots respond to heterogenous distribution of water in soils by exhibiting adaptive responses such as xerobranching(1), where lateral root formation is temporarily repressed (e.g. in an air-gap), until root tips reconnect with moist soil. ABA (abscisic acid) serves as a key 'hydrosignal' involved in this response(2). Following a xerobranching stimulus, ABA is co-mobilised with phloem-water towards outer root tissues, where it triggers closure of inter-cellular pores, 'plasmodesmata'. This process blocks the inward radial movement of another hydrosignal, auxin to lateral root 'stem' cells, eventually inhibiting root branching. These findings provide insights into water-driven regulation of root branching. However, ABA-mediated signalling networks build gradually, taking several hours
50 (~12 hrs) after the initial perception of water deficit, suggesting the presence of other rapid signals that
51 regulate root responses to water unavailability.

52
53 In this study, we identify reactive oxygen species (ROS) as a potential early “alarm” signal for
54 fluctuating water availability by modulating the functions of specific Aux/IAAs like IAA3 in a redox
55 dependent manner. Our study reveals ROS responsive multimerization by a specific subset of
56 Aux/IAAs like IAA3 is a critical determinant of their capacity to repress downstream target genes,
57 thereby providing a nexus connecting auxin and ROS signalling. Understanding ROS’s role in
58 microscale root adaptations, such as xerobranching, sheds light on how roots rapidly sense and respond
59 to heterogeneous water availability in complex soil environments. This insight could inform
60 biotechnological and breeding approaches to enhance crop water-use efficiency.

61
62 Xerobranching is dependent on ROS generated by specific RBOHs

63 To determine whether xerobranching is dependent on ROS, roots of *Arabidopsis thaliana* wildtype
64 (WT) Col-0 and ROS mutants were exposed to a xerobranching (air-gap) bioassay(2). Unlike WT
65 controls, roots of *Arabidopsis* RBOH (Respiratory Burst Oxidase Homolog) mutants continued to
66 branch when growing across an air-gap (Fig. 1A, B and Fig S1A). To validate whether the
67 xerobranching phenotype of the *rboh* mutants is a consequence of reduced ROS accumulation, we
68 assessed the spatial distribution of H2O2 in the roots of WT Col-0 and *rboh* mutants subjected to
69 xerobranching stimuli using HPF (Hydroxy phenyl fluorescein) staining. Root tips of *rboh b/f* and *rboh
e/f* mutants stained significantly less with HPF when grown across an air-gap, compared to WT
70 controls (Fig. S1 B-E). Moreover, qRT-PCR analysis revealed enhanced expression of *RBOHB*,
71 *RBOHE* and *RBOHF* in *Arabidopsis* roots exposed to a xerobranching stimulus (Fig. S2 A-E).
72 Interestingly, *RBOHE* expresses in endodermis, cortex and epidermis cells overlying lateral root
73 primordia (LRP) and also in the basal meristem where LR priming occurs(3). Furthermore, treatment
75 of WT seedlings with RBOH inhibitor DPI (Diphenyleneiodonium chloride) resulted in an increased
76 lateral root density compared to control seedlings (Fig. S2 F-H). Collectively, our data reveals that
77 ROS generated by specific RBOHs regulates xerobranching.

78

79 Nuclear ROS burst precedes ABA accumulation during xerobranching

80 To identify where and when the ROS burst occurs following a xerobranching stimulus, we employed
81 subcellular versions of the Hydrogen Peroxide (H₂O₂) sensor roGFP2-Orp1(4) targeted to either
82 cytoplasmic or nuclear compartments(5). Reduced roGFP2-Orp1 proteins display a characteristic
83 single fluorescence intensity peak at ~490 nm excitation, whilst the oxidised protein exhibits two
84 fluorescence intensity peaks at ~400 nm and ~490 nm, thereby allowing quantitative assessment of
85 redox states through the ratio of fluorescence intensity between 400 ±5 nm and 490 ±5 nm. Seedlings
86 expressing the nuclear- or cytosol-localized roGFP2-Orp1 H₂O₂ sensor exhibited a significant increase
87 in 405/488 nm ratio when growing root tips traversed an air gap in the xerobranching bioassay. This
88 revealed enhanced oxidation of nls-roGFP2-Orp1, reflecting a ROS burst in the cytoplasm and nuclei
89 of root elongation zone cells exposed to a xerobranching stimulus (Fig. 1C-F, 1E and 1F). However,
90 once root tips reconnected with agar (recovery phase), cytoplasmic and nuclear ROS levels declined
91 rapidly. Monitoring changes in the spatial distribution of roGFP2-Orp1 while the growing root tip
92 traverses across an air-gap and then reconnects with agar, allowed us to determine the temporal kinetics
93 of the ROS burst during xerobranching and compare this to changes in levels of the water stress
94 hormone ABA (abscisic acid), which also regulates this response(2). Elevated ROS levels were
95 detected in elongation zone cells shortly after the root tips had entered ~1mm into the air-gap (i.e. 3
96 hours of exposure to xerobranching stimuli), whereas ABA accumulation occurred much later, after
97 the roots had grown 3mm into the air-gap (i.e. 10-12 hours of exposure) (Fig. 1G).

98

99
100 **Xerobranching-induced ROS targets a subset of Aux/IAAs auxin response components**

101 The rapid ROS burst induced by xerobranching revealed that roots can sense changes in their local
102 water availability significantly earlier than previously demonstrated(2). The nls-roGFP2-Orp1 H2O2
103 sensor detected higher ROS levels in the elongation zone tissues as root tips traverse the air-gap
104 **(Figure 1E)**. Radial cross-sectional root images further revealed enhanced ROS levels in inner stele
105 tissues where new lateral root primordia are specified **(Figure 2A)**. Together with the disrupted
106 xerobranching phenotypes observed in *rboh* mutants, nls-roGFP2-Orp1 H2O2 sensor data reveals that
107 rapid nuclear ROS production in inner root tissues regulate this adaptive response.

108
109 What are the nuclear targets that ROS modify to trigger a xerobranching response? Given that the
110 xerobranching stimulus triggers a ROS burst before ABA levels are elevated (Fig. 1G), we intuited
111 that this early ROS burst may act as the initial signal to orchestrate cessation of lateral root
112 development in air-gaps, before ABA mediated plasmodesmata (PD) gating of radial symplastic
113 auxin flow is activated(2). This led us to consider whether the xerobranching-induced ROS burst
114 modifies components of the auxin response machinery. A recent study by Lu et. al(6) reported that
115 biologically active ROS can modulate components of the auxin response pathway, such as the auxin
116 -receptor TIR1/AFB2, by influencing their nucleo-cytoplasmic redistribution in root hairs.

117
118 Auxin-mediated transcriptional regulation is dependent on the functions of Aux/IAA
119 proteins(7, 8). In general three discrete domains contribute to the molecular functions of the
120 Aux/IAAs : (i) a type I/II Phox and Bem1 (PB1) domain at the C-terminal region(9) which
121 facilitates interactions with ARF (Auxin Response Factor) proteins(10) necessary for its
122 recruitment to the target locus (ii) a GWPP-core degron motif containing internal domain II
123 that is required for interaction with TIR1/AFB proteins(11-13) and therefore determines
124 Aux/IAA stability and (iii) a leucine repeat EAR motif within domain I(14) that facilitates the
recruitment of transcriptional co-repressors such as TOPLESS (TPL)(15). Given that the xerobranching response involves cessation of lateral root formation in air-gaps, and considering the systems biology of lateral root development where the repressive functions of the auxin signalling modules ( involving an ARF-AUX/IAA pair) are driven by the cognate Aux/IAAs(16), we hypothesised that the Aux/IAAs may be likely candidates to positively influence a xerobranching response in a redox-regulated manner. Redox changes triggered by ROS can result in oxidation of cysteine thiols (RSH/RS⁻) leading to the formation of highly reactive sulfenic acid (RSOH) that can in turn react with another thiol to form a disulfide bond(17) and trigger redox dependent homo/hetero multimerization of proteins.

134

We first assessed the distribution and conservation of cysteine residues among Aux/IAAs (Fig. S3) and observed widespread divergence in the frequency and distribution of cysteine residues with only cysteine 171 conserved amongst Aux/IAAs. Next, we explored whether redox conditions influence Aux/IAA multimerization through disulfide linkages. We selected Aux/IAAs that fulfilled the following criteria: (i) representative of the different clades of Aux/IAAs (ii) exhibits a wide spectrum of cysteine residues (iii) have well defined regulatory functions, and (iv) with roles during lateral root development. Each of the shortlisted Aux/IAAs (IAA3, IAA28, IAA14, IAA13 and IAA19) were recombinantly expressed and purified as N-terminal MBP (maltose binding protein) tagged proteins in non-reducing conditions and profiled by SDS polyacrylamide gel electrophoresis (PAGE). In non-reducing conditions Aux/IAAs IAA3 and IAA19 formed higher order multimers, whereas IAA28, IAA14 and IAA13 were largely monomeric (Fig. S4 A). Moreover, assays with redox modifying chemicals indicated IAA3 and IAA19 are more sensitive to redox changes, forming higher order multimers (Fig. S4 B-G). We assessed AlphaFold structures of IAA3, IAA28, IAA14 and IAA19 to verify whether the observed differences in their capacity to mutimerize through
disulphide linkages amongst these candidate Aux/IAAs is influenced by their secondary structure (Fig. S5 - S6). While there was no obvious difference in the secondary structure of the ordered PB1(Phox and Bem1) domain in the modelled Aux/IAAs, the frequency of the cysteine residues and their three-dimensional location within the Aux/IAAs (both in the PB1 domain and the disordered regions) appears a major driver of redox-dependent multimerization. Aux/IAAs like IAA3 and IAA19, which were more sensitive to redox changes and formed robust higher order multimers, had a higher frequency of cysteine residues in both their PB1 and disordered domains. Taken together, our data reveals the ability of a subset of Aux/IAAs like IAA3 to form higher order multimers, providing a potential mechanism to vary auxin-dependent transcriptional outputs in response to environmental cues sensed through dynamic changes in ROS induced cellular redox status. The *IAA3* gene regulates root branching through expression in pericycle lateral root founder cells and then overlying endodermal cells(18). Radial cross sections of ROS reporter lines revealed H₂O₂ levels are elevated in nuclei of inner root tissues following a xerobranching stimulus (Fig. 2 A). Since expression of IAA3 and elevated H₂O₂ levels during a xerobranching stimulus both occurs in inner root tissues, we created an *Arabidopsis* CRISPR/cas9(19) knockout line disrupting *IAA3* (*iaa3*) (Fig. S7 A-F), revealing a significant xerobranching defect (Fig. 2 B-C). The xerobranching defect was also evident in the * shy2-31* loss of function mutant allele of *IAA3* (Fig. S8 A). Like IAA3, IAA28 encodes an Aux/IAA that regulates the fate of pericycle founder cells implicated in lateral root formation in lateral root formation(16). However, we did not observe a xerobranching defect in the *iaa28-2* loss of function mutant allele (Fig. S8 B). Our results reveal that the IAA3 protein represents a promising target for ROS regulation during a xerobranching response.

173

174 Redox dependent multimerization of IAA3 modulates its repressive functions
175

How does IAA3 redox dependent multimerization affect its repressive function during xerobranching? Oligomerization of Aux/IAAs may be required for efficient ARF mediated repression of gene targets(20, 21). However, how this Aux/IAA oligomerization might be triggered, and the underpinning repressive mechanism remains unclear. Cysteine disulfide linkage mediated multimerization of a subset of Aux/IAAs like IAA3 may provide a mechanism to link auxin-dependent transcriptional outputs with environmental cues (such as a xerobranching), sensed through dynamic changes in ROS induced cellular redox status.

176

177

178

179

180

181

182

183

We first identified the cysteine residues that facilitate IAA3 homo-multimer formation. Multimerization of Aux/IAAs has been postulated to occur through the Aux/IAA PB1 domain (9, 22, 23) in a head-to-tail fashion. Cysteine residues 112, 169, 171 and 187 of IAA3 are part of the PB1 domain (Fig. S9 A), while cysteine residues 23, 31 and 32 are between domains DI and DII(24). Modelled structure of IAA3 in a head-to-tail PB1 dimer (generated by AlphaFold), revealed Cys residues in the PB1 Domain (Cys 112, Cys 169, Cys 171 and Cys 187) are too distant to form disulfide bonds required for ROS responsive multimer formation (Fig. 3A and Fig. S9 B-C). However, by hypothetically merging a pair of dimers, and then rotating one set of IAA dimers 180 degrees, allows cysteine residues 112, 169 and 187 to come close enough to form sulfhydryl bridges in multiple combinations to stabilise multimers through stacks of IAA tetramers (Fig. 3B and Fig. S10). To test the effect of manipulating these cysteine residues on IAA3 multimerization, we generated a series of cysteine to serine substitution mutations. The Cys169Ser mutation of IAA3 resulted in the most significant loss of higher order multimers, while Cys32Ser and Cys112Ser mutations resulted in a drastic reduction of tetrameric forms of IAA3. Cys187Ser mutation resulted in reduction of almost all higher order multimeric forms of the IAA3 protein with a reciprocal increase in monomers under non-reducing conditions (Fig. S11 A (i and ii)). From the structure of a wild-type AUX/IAA PB1

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199
domain K96, D151, D153 and D155 residues were identified as amino acid residues that are involved in directional interaction of monomers (Fig. 3A), largely via hydrogen bonds between conserved basic and acidic surface patches(21, 24). Mutations affecting the positive (K96A) or the negative faces (D155A) of IAA3 domain III/IV did not deplete the redox-dependent multimerization of IAA3 in non-reducing conditions *in vitro* (Fig. S11 A (iii)), thereby indicating that the cysteines are the principal ROS responsive residues that allows alternate routes for IAA3 multimerization apart from the homotypic PB1 domain mediated head to tail interaction. In support of this quadruple Cys32Ser, Cys112Ser, Cys169Ser and Cys187Ser mutations in IAA3 (henceforth as 4C/S-IAA3) blocked formation of multimers in non-reducing (Fig. S11 B) and oxidizing (Fig. S11 C) conditions *in vitro*.

To determine the number of cysteine residues responsive to redox changes and involved in inter- and intra-molecular disulphide bond formation in WT-IAA3, we modified the native protein with N-ethylmaleimide, followed by reduction and alkylation with DTT and iodoacetamide respectively. We then carried out trypsin digestion followed by LC-MS/MS to identify which cysteine residues were modified with N-ethylmaleimide and which with iodoacetamide. Our rationale was that natively reduced cysteines free to be labelled with N-ethylmaleimide would be ROS responsive and thus less likely to be engaged in disulphide bonds, whereas ROS responsive cysteines forming disulphide bridges would be labelled with iodoacetamide. Mass spectrometry-based analysis revealed Cys 32, Cys112, Cys169, Cys171 and Cys187 form disulphide bridges because they were labelled with iodoacetamide and not N-ethylmaleimide (Table S1). Cys23 and Cys112 were observed in both the NEM and iodoacetamide modified forms. This could be indicative of regions of the IAA3 structure that are more dynamically involved in disulphide formation and represent more flexible regions of the multimers. Our N-ethylmaleimide/iodoacetamide labelling based MS analysis data
complements the results from our IAA3 cysteine to serine substitution mutation studies, where we observed a significant loss of redox dependent multimers in the 4C/S mutated form of IAA3 (Fig. S11). Taken together our data reveals a mechanism for ROS responsive multimer formation involving disulfide bonds between specific cysteine residues within and outside of PB1 domain. The data also indicates that multiple forms of redox dependent multimers of IAA3 can exists in nature. Amongst these multiple forms of IAA3 redox dependent multimers, some may be driven by the PB1-PB1 domain interaction mediated pair of head-to-tail IAA3-IAA3 homodimers, where one IAA3-IAA3 homodimer unit interacts with the other by free rotation to allow the experimentally verified cysteine residues within the PB1 domain (cys 112, cys 169 and cys 187) to align in very close proximity to each other (Fig. 3B and Fig. S10). On the other hand, free interactions between cysteine residues outside the PB1 domain may also provide an alternate route for IAA3 redox dependent multimerization. The reduction of higher order multimers due to Cys169Ser, Cys112Ser mutation (within PB1 domain) and Cys32Ser (outside PB1 domain) of IAA3 coupled with the almost complete abolishment of higher order multimers in 4C/S-IAA3 in our in vitro studies, support these modes of multimerization (Fig. S11). To further validate these findings, we assessed the redox dependent multimerization of IAA3 in planta.

**IAA3 exhibits redox regulated multimerization in planta**

We assessed the redox dependent multimerization pattern of IAA3 in transgenic lines. Both N-terminal HA (hemagglutinin) tagged wildtype and quadruple cysteine mutant forms of IAA3 were expressed under the control of the endogenous *IAA3* native promoter in the *iaa3* mutant background (Fig. S12 A). When expressed under the control of the endogenous *IAA3* promoter, WT-IAA3 and 4C/S-IAA3 exhibited distinctly different patterns of multimerization in non-reducing conditions (Fig. 3 C and D). WT- IAA3 proteins existed as a population of
discrete multimers that included a distinct dimeric fraction, a minor monomeric fraction, and major hexameric and tetrameric fractions. Incremental addition of DTT ranging from 5 mM to 10mM concentration in our titration assays shifted the major hexameric fraction primarily to the tetrameric fraction, with no obviously evident incremental changes in the monomeric fraction and limited shift to the dimeric fraction (Fig. 3 C). On the other hand, endogenous promoter expressed 4C/S-IAA3 lost its ability to form higher order multimers and largely formed a heterogenous mixture of almost equivalent amounts of hexamer, pentamer and tetramer, along with a distinct monomeric fraction (Fig. 3 D). In contrast to WT-IAA3, incremental addition of DTT to 4C/S-IAA3 samples ranging from 5 mM to 10mM shifted the higher order multimeric fractions to the monomeric form (Fig. 3 D). These observations that WT-IAA3 and 4C/S-IAA3 have different sensitivities towards reducing agents, coupled with our MS-based analysis, reveal that IAA3 forms redox regulated multimers. These are distinct from previously verified homotypic head to tail multimers in-vitro and in-vivo, through disulphide linkages involving specific cysteine residues.

264

265 ROS generated by RBOHs drive redox regulated IAA3 multimerization

We previously observed: (i) both *RBOHE* and *RBOHF* genes are transcriptionally upregulated during a xerobranching response (ii) *rbohe/f* mutants exhibit the highest degree of xerobranching defect amongst the RBOH mutants (Fig. 1A). Given the pretext that IAA3 is a promising ROS responsive protein of the auxin signalling machinery that is implicated in xerobranching, we generated transgenic lines expressing N-terminal HA tagged WT-IAA3 in the *rbohe/f* mutant background. When we performed anti-HA immunoprecipitation assays with these transgenic lines in the absence of any reducing agents, we observed that WT-IAA3 predominantly existed as a monomer in the pIAA3::WT-IAA3/*rbohe/f* lines in comparison to the experimental control pIAA3::WT-IAA3/*iaa3* line where WT-IAA3 was predominantly
hexameric along with minor tetrameric and dimeric fractions (Fig. 3E). Taken together, these
results confirmed that cellular H₂O₂ specifically produced by the NADPH oxidases/RBOHs
like RBOHE and RBOHF contributes to the steady state formation of higher order redox
dependent multimers of IAA3.

279

280 ROS influences redox regulated multimerization of WT-IAA3 but not 4C/S-IAA3

281 In the context of the ROS mediated biological effects on redox regulation, H₂O₂ represents a
282 key molecule that can direct thiol-based modification of cysteine residues in target proteins(25-
283 28). To understand the *in vivo* physiological relevance of IAA3 multimerization, we tested the
284 impact of exogeneous H₂O₂ application on IAA3 multimerization (Fig. S13). To establish the
285 robustness of multimeric forms induced due to a ROS burst, all anti-HA immunoprecipitations
286 targeted towards WT-IAA3 and 4C/S -IAA3 were performed for control and treated seedlings
287 in buffer supplemented with 5mM DTT. In control conditions, WT- IAA3 exists as a
288 population of equivalent amounts of tetrameric and hexameric fractions along with a minor
289 monomeric fraction as observed previously (Fig. 3 C). Exogenous H₂O₂ application (29)
290 resulted in almost complete loss of the monomeric fraction and robust enhancement of levels
291 of WT- IAA3 multimers, including a transitional shift of the tetrameric fraction to the
292 hexameric form (Fig. S13 A). In contrast, exogenous H₂O₂ application did not cause any
293 apparent change in the pattern of multimerization of 4C/S -IAA3 compared to control
294 conditions (Fig. S13 B). These results indicate ROS or absence of water stimulates WT-IAA3
295 to multimerize through disulphide linkages involving a specific subset of cysteine residues.

296

297 Disrupting WT-IAA3 multimerization attenuates its repressive function

298 To assess the phenotypic effect of disrupting ROS induced IAA3 multimerisation,
299 xerobranching assays were performed using WT, *iaa3* mutant C1 and C2 alleles and *iaa3* lines
300 expressing WT-IAA3 or 4C/S-IAA3 transgenes. C1 and C2 alleles significantly increased
301 lateral root density (Fig. S7), while xerobranching assays indicated disruption of IAA3 caused
302 mutants to branch in air gaps (Fig. 4 A and Fig. 2 B-C). Next, we tested whether disruption of
303 cysteine residues required for redox-dependent multimerization of IAA3 causes its repressive
304 function to be compromised. To assess this, we performed xerobranching assays in transgenic
305 lines expressing equivalent levels of WT-IAA3 or 4C/S-IAA3 under the native promoter in the
306 *iaa3* background (Fig. S12 G). In contrast to pIAA3::IAA3-WT/iaa3 transgenic seedlings and
307 WT Col-0, pIAA3::IAA3-4C/S/iaa3 seedlings branched in the air gap (Fig. 4 A-B) like the
308 *iaa3* mutant (Fig. 4 A and Fig. 2 B-C). Next, we tested multimerization status of WT-IAA3
309 and 4C/S-IAA3 in the transgenic lines subjected to a xerobranching stimulus. We observed in
310 roots of pIAA3::WT-IAA3/iaa3 seedlings, xerobranching triggered a shift of WT-IAA3
311 tetrameric to the hexameric form along with the unique appearance of a dodecameric and a
312 very higher order multimeric form (Fig. 4 C). In contrast, in pIAA3::4C/S-IAA3/iaa3 seedling
313 roots, 4C/S-IAA3 was largely monomeric in both control and air-gap conditions with no
314 obvious detectable shift to multimeric forms induced by xerobranching (Fig. 4 C). Hence, our
315 assays revealed that disrupting redox dependent IAA3 multimerization induced by low-water
316 stress attenuates its repressive function, which is physiologically critical for xerobranching.
317

318 Our in-vitro and in-planta biochemical studies also indicate WT-IAA3 has an inherent capacity
319 to form robust redox dependent multimers in non-reducing or moderately reducing
320 environments, even without a stimulus like low-water stress. What is the physiological
321 relevance of these unstimulated redox dependent multimers? Own promoter WT-IAA3
322 restored the LR density in *iaa3* mutant line to levels similar to that observed in WT Col-0,
323 however independent transgenic lines expressing equivalent levels of 4C/S-IAA3 exhibit ~2-
324 fold increase in lateral root (LR) density similar to *iaa3* mutants (Fig. S12 H-K). *Arabidopsis*
325

seedlings expressing stabilized form of IAA3 ( *shy2-2* gain of function semi dominant mutant),

326

under the control of the *CASP1* promoter(30) normally fail to develop emerged lateral roots.

327

thereby allowing for a distinct and scorable phenotype for LR development in conducive

328

backgrounds. Similar to the *pIAA3::IAA3-4C/S/iaa3* seedlings, *pCASP1::IAA3-4C/S/iaa3*

329

seedlings also exhibit a ~2-fold increase in emerged LRs compared to *pCASP1::IAA3-

330

WT/iaa3 (Fig. S14).

331

332

**Redox dependent multimerization not stability drives IAA3 mediated auxin responses**

333

As sequences other than the DII degron motif of Aux/IAAs contribute to co-receptor complex

334

formation(31-33), we assessed whether introducing the quadruple cysteine mutations impacts

335

the auxin induced degradation kinetics of IAA3 when expressed under the control of the

336

constitutive 35S promoter in the *iaa3* mutant background. Our logic for choosing transgenic

337

lines expressing WT or 4C/S-IAA3 controlled by the 35S promoter stems from IAA3

338

negatively autoregulating its own promoter(34). Therefore, we chose to perform stability

339

assays in p35S::WT-IAA3/iaa3 or p35S::4C/S-IAA3/iaa3 transgenic lines to decouple

340

negative autoregulation and stability aspects of IAA3. Following cycloheximide treatment, our

341

assays revealed a slight reduction in steady state levels of 4C/S-IAA3 versus WT-IAA3 (in

342

the absence of exogeneous auxin treatment), but the kinetics of auxin induced degradation of

343

WT-IAA3 and 4C/S-IAA3 post exogenous auxin treatment were nearly identical (35) (Fig. S15

344

**A-C**). Our results indicate that attenuation of the repressive function of IAA3 mediated by the

345

introduction of the quadruple cysteine to serine substitution mutations cannot be attributed to

346

alterations in auxin dependant stability of the protein, suggesting that these mutations do not

347

impact IAA3-auxin-co-receptor complex formation.

348

349

**Introduction of quadruple cysteine mutation in shy2-2 ameliorates its repressive function**
We next introduced a proline to serine substitution mutation at the 69th amino acid position(36) in both WT- IAA3 and 4C/S -IAA3 to mimic the effect of the shy2-2 semidominant gain of function mutation(37). We generated transgenic lines expressing HA tagged IAA3-WT$^{P69S}$ and IAA3-4C/S$^{P69S}$ under the control of the endogenous IAA3 promoter in the *iaa3* mutant background. Independent transgenic lines expressing $^{P69S}$ WT-IAA3 and $^{P69S}$ 4C/S-IAA3 in *iaa3* exhibited similar degrees of stabilization against exogenous auxin treatment due to the introduction of the shy2-2 semi-dominant gain of function mutation (Fig. S15 D). However, the repressive effects associated with the shy2-2 semi-dominant gain of function mutation(38) in the context of lateral root emergence and primary root length were largely ameliorated in *pIAA3*::$^{P69S}$4C/S-IAA3 (shy2-2)/*iaa3* transgenic seedlings (Fig. 5 A-B). Moreover, constitutive expression of IAA3-4C/S in *iaa3* also failed to mimic repressive lateral root growth effects associated with shy2-2 semi-dominant gain of function mutants (Fig. S16 A-F). Indeed, the inability of 4C/S -IAA3 to repress LR development irrespective of being expressed under the control of the endogenous, CASP1(tissue specific: endodermis) and 35S promoter in comparison to WT-IAA3 or as a stabilised semi-dominant gain- of -function (shy2-2) mutant version (*pIAA3*::$^{P69S}$4C/S-IAA3) reveals how formation of a specific pattern of redox dependent multimers, rather than auxin dependant modulation of protein stability acts as a key determinant of the inhibitory functions of IAA3 to drive auxin responses.

Introducing 4C/S substitutions into IAA3 impacts interactions with TOPLESS

Our phenotyping-based studies demonstrates redox dependent multimerization augments the repressive function of IAA3, but what is the underlying molecular mechanism by which this is achieved? A key determinant of the molecular functions of Aux/IAAs like IAA3 is their capacity to interact with components of the auxin response pathway. Thus, we assessed the ability of 4C/S-IAA3 to interact with ARF7(39) and TIR1 by transient co-immunoprecipitation
375

experiments in *Nicotiana benthamiana*. Pull-down assays revealed interactions between ARF7-GFP with WT-IAA3 and 4C/S-IAA3 and TIR1-GFP with WT-IAA3 and 4C/S-IAA3) were equivalent (Fig. S17 A-B). Hence, the quadruple cysteine mutations do not disrupt the ability of IAA3 to interact with core ARF and TIR1 components of the nuclear auxin signalling machinery. Indeed, identical levels of interaction between WT or 4C/S-IAA3 with TIR1 validated the similar kinetics of auxin induced degradation between WT-IAA3 and 4C/S-IAA3.

376

377

378

379

380

381

382

383

384

385

386

387

388

389

Furthermore, the interaction of IAA3 with ARF7 is determined by the SUMO-SIM interaction module(39), irrespective of multimeric state of IAA3 (Fig. S17 C-E). Moreover, our transient assays revealed exogenous H₂O₂ treatment does not significantly alter interaction between (i) ARF7 and WT-IAA3 or 4C/S-IAA3 (Fig. S17 F); or (ii) TIR1 and WT-IAA3 or 4C/S-IAA3 (Fig. S17 G). Instead, transient pull-down assays revealed 4C/S-IAA3 was significantly compromised in its capacity to interact with transcriptional co-repressor TOPLESS(TPL)(15) (Fig. 5 C). The reduction in 4C/S-IAA3 interactions with TPL was evident in stable transgenic lines expressing 4C/S-IAA3 under the control of the *IAA3* promoter in the *iaa3* mutant background (Fig. 5 D-E).

390

391

392

393

394

395

396

397

398

399

A recent study by Cho et al., revealed how a higher dose of IAA3 having the relatively lower affinity RG-type EAR motif (required for interaction with TPL/TPR) allows more recruitment of TPL at a target locus like RSL4 and thus more stringent TPL mediated repression(40). In a similar manner, stabilized redox-regulated multimers of IAA3 could provide an enhanced interface for interaction with TPL and capacity to repress downstream target genes. Indeed, consistent with altered repressive ability of 4C/S-IAA3, downstream targets of IAA3(34, 38) exhibit differential expression profiles in WT-IAA3 and 4C/S-IAA3 transgenic lines (Fig. 5F and Fig. S18-S19). For example, expression levels of auxin inducible *LBD16* gene (41, 42) was significantly higher in 4C/S-IAA3 expressing transgenic lines in comparison to WT-IAA3
irrespective of the promoter driving their expression (Fig. 5F and Fig. S19). We also observed higher and faster inducibility of LBD16(42) in 4C/S-IAA3 transgenic lines compared to WT-IAA3 on exogeneous auxin (1-NAA) treatment (Fig. S20). The elevated inducibility of LBD16 in the 4C/S-IAA3 transgenics revealed that the repressive function of IAA3 was impaired by the introduction of the quadruple cysteine to serine substitution mutation. The impairment of IAA3 repressive functions due to the introduction of the quadruple cysteine to serine substitution mutation was further substantiated when we assessed the expression levels of the LBD16 gene in control and air-gap conditions. Unlike WT-Col-0 or pIAA3::WT-IAA3/iaa3 transgenics, pIAA3::4C/S-IAA3/iaa3 and iaa3 did not exhibit any significant down-regulation of LBD16 expression in air-gap conditions (Fig.S21) Our ChIP-qPCR assays using primers that specifically encompass an AuxRE site at LBD16 promoter /upstream region indicate that the 4C/S mutation does not disrupt the capacity of IAA3 to nucleate at AuxRE sites on chromatin (Fig. 5G) as it retains the capacity to interact with sequence-specific ARF transcription factors like ARF7 (Fig. S17 A). Given the expression and localization of TPL and IAA3 remained unaltered due to a xerobranching stimulus (Fig. S22), the data collectively suggests alteration of the capacity of IAA3 to multimerize and interact more robustly with TPL (due to the introduction of the quadruple cysteine to serine mutations) is the major cause for IAA3's loss of capacity to repress target gene expression. Hence, our data revealed how ROS responsive multimerization influences the repressive function of IAA3.

In summary, we have discovered an important new mechanism for ROS regulation of auxin-dependent transcriptional responses. Our study reveals that a xerobranching stimulus enhances ROS accumulation in the cytoplasm and nuclei of root elongation zone cells. This ROS burst is driven by increased activity of specific RBOH genes like RBOHB, RBOHE and RBOHF, which are transcriptionally induced under xerobranching conditions. Supporting this, rbohb/f and rbohe/f mutants fail to repress lateral root formation in air-gaps. Given that RBOHs are
425

primarily localized to the plasma membrane and contribute to apoplastic ROS(43), it is
intriguing how a proportion of apoplast-derived H₂O₂ evades cytosolic scavenging and reaches
the nucleus to initiate signalling. While aquaporins likely facilitate H₂O₂ entry into the
cytosol(44, 45), the precise mechanism by which ROS is transported to and maintained in the
nucleus remains unclear. Future studies using mutants impaired in cytosolic ROS scavenging
will be valuable in resolving the origin and regulation of nuclear ROS during xerobranching.

426

427

428

429

430

431

In this study, we deciphered how changes in cellular redox status can intersect with auxin
perception by targeting selected Aux/IAAs to multimerise and repress the expression of auxin
responsive genes. Transcriptional repression through multimerization of transcriptional
regulators can confer additional layers of control towards the expression of target genes in a
context driven manner (46-49). In this study we observed how cellular redox status can
influence multimerization status of a major class of transcriptional repressors (Aux/IAAs) and
provide an alternate route to efficiently repress the expression of their targets. This potentially
allow plants to decode dynamic changes in its immediate environment and fine tune
phytohormone (in this case, auxin) mediated signalling nodes in a ROS dependent manner.

432

433

434

435

436

437

438

439

440

Moreover, our findings reveal that only a specific pattern of IAA3 redox multimers are able to
function as efficient transcriptional repressors, indicating that this mechanism is non-stochastic
and well-ordered. Since there is a wide spectrum in the propensity for redox dependent
multimerization amongst the Aux/IAAs, they can act as ROS sensors and this variability may
potentially contribute to the remarkable levels of functional diversification that the Aux/IAAs
have attained during the course of plant evolution (7, 50). Such alternate routes of regulation
of gene expression via redox dependent multimerization of transcriptional repressors may also
operate in other phytohormone based signalling pathways. Thus, identification and targeted
manipulation of redox dependent multimerization of transcriptional repressors implicated in
various phytohormone mediated signalling pathways will provide insights on how plant

441

442

443

444

445

446

447

448

449
450

adaptive responses can be fine-tuned to improve phenotypic plasticity and resilience. With
growing climate challenges and water-scarcity, these insights will provide researchers with the
opportunity to leverage new agronomic solutions.

451

452

453

# Materials and methods

454

## DNA constructs

455

456

457

458

459

460

461

462

463

464

465

466

467

468

469

470

471

472

473

474

475

All the constructs used in relation to transient assays-based studies and generation of transgenic materials were generated by utilizing combinations of Gateway cloning technology and restriction site-based cloning. ARF7WT-GFP, ARF74K/R-GFP, HA-IAA3 WT, HA-IAA3 SIM, TIR1-GFP constructs were previously generated by Orosa et al, 2018. In order to generate the quadruple cysteine to serine substitution mutated version of IAA3 (4C/S-IAA3), site directed mutagenesis was performed using WT-IAA3 pDTOPO clone as template. Primers used to introduce substitution mutations are listed in Supplemental table. The introduction of mutation was confirmed by sequencing. 4C/S-IAA3 CDS in pDTOPO vector was further cloned in pEarleyGate 201 destination vector by LR reaction as an in-frame fusion with an N-terminal HA tag like HA-WT IAA3 clone that had been previously generated by Orosa et al, 2018. The 35S promoter driving the expression of HA-WT IAA3 or HA-4C/S IAA3 in the pEarleyGate 201 system was replaced with the *CASP1* and *IAA3* promoter by adopting the cloning strategy previously described by Michneiwicz et al, 2015(51). LR reaction mediated recombinational cloning of the WT-IAA3 or 4C/S-IAA3 CDS in the pEarleyGate 201 vector system is accompanied with the excision of the chloramphenicol resistance (*Cm*<sup>R</sup>) gene and this renders the EcoRI and XhoI restriction sites flanking the CAMV 35S promoter as unique in the LR positive clones. The *CASP1* and *IAA3* promoter was then cloned into these unique EcoRI and XhoI sites by conventional restriction enzyme based cloning methods (as all the promoters lack EcoRI and XhoI sites) to generate a chimera with the aforementioned promoters driving the expression of the WT-IAA3 or 4C/S-IAA3 CDS as an in-frame fusion with an N-terminal HA tag followed by an OCS (Octopine synthase) terminator.

476

## Generating transgenic materials

477

478

DNA constructs expressing N-terminal HA tagged WT-IAA3 and 4C/S-IAA3 under the control of the CAMV 35S, *CASP1* and *IAA3* promoter as described above were transformed
479

into *Agrobacterium tumefaciens* GV3101 strain by electroporation. The expression of the
chimeric proteins (HA-WT IAA3 and HA-4C/S IAA3) were first confirmed by agrobacterium
mediated transient assays followed with anti-HA immunoprecipitation (described below) and
then transformed into *Arabidopsis thaliana* Col-0 plants by the floral dip method(52). Positive
transformants were selected in 20mg/litre BASTA selection media (the pEarleyGate 201
destination vector contained the biolophos/ phosphonithricin resistance gene for *in planta*
selection).

480

481

482

483

484

485

486

Antibodies

487

Commercially available antibodies used for this study include Anti-HA High Affinity Roche
3F/10, Sigma-Aldrich (catalog no:12158167001), Anti-HA antibody Abcam (ab9110), Anti-
GFP antibody Abcam (ab6556), anti c-myc antibody, Sigma Aldrich (catalog no M4439), Anti
Histone H3 antibody Abcam (ab1791)

488

489

490

491

Plant material and growth conditions

492

*Arabidopsis thaliana* ecotype, Columbia (Col-0) was used as the wild-type control for all
experiments (referred as WT Col-0 throughout the text). The *iaa3* CRISPR cas9 mutant lines
used for this study was kindly provided by Dr. Kevin Bellande and Prof. Joop Vermeer
(University of Neuchâtel). The nuclear RoGFP2orp1ROS reporter lines were kindly provided
by Dr. Ulrike Bechtold and Prof.Phil Mullineaux. The *rboh* mutant lines were kindly shared
by Prof. Xavier Draye. The *pTPL::TPL-GFP* reporter lines were kindly provided by Prof. Lars
Østergaard. All relevant transgenic *Arabidopsis thaliana* lines expressing WT or mutated
version of IAA3/SHY2 under the control of different promoters were generated by intogressing
the appropriate DNA constructs (elaborated below) into the *iaa3* mutant background.
*Arabidopsis thaliana* seed lines were surface sterilized using 50 % (v/v) bleach for 7 min and
two 0.001 % Triton X-100 washes followed five washes with sterile water and then stratified
at 4 °C for 48 h in the dark. For Lateral Root phenotype-based bioassays, seeds were
germinated on media containing 1/2 MS (2.2 g/L) (Murashige and Skoog media, Sigma), 0.5
g/L MES, and 1 % phyto-agar at pH 5.7-5.8 range. Seedlings were grown vertically for a period
of 8-10 days (depending on the experimental design) under continuous temperature 22 °C with
a 16 h photoperiod (150 µmol m⁻² s⁻¹) before they were scored for their LR phenotype within
a window period of 12-16 days post germination, involving root length, emerged LR number
per plant and LR density as critical parameters.

493

494

495

496

497

498

499

500

501

502

503

504

505

506

507

508

509
## 510 Recombinant protein purification

Fragment corresponding to the coding sequence of full-length Aux/IAAs from *Arabidopsis thaliana* and the 4C/S-IAA3 coding sequence (generated by site directed mutagenesis of AtIAA3/SHY2 coding sequence) was cloned in pMal c5X vector (New England Biolabs) and expressed in BL21 (DE3) cells in the presence of 0.5 mM IPTG at 37 °C as an in-frame fusion with N-terminal Maltose binding protein (MBP) tag. Recombinant proteins were purified using MBP-amylose resin-based affinity chromatography under non-reducing conditions. Briefly pelleted cells were lysed with Bug buster (Merck-Millipore) protein extraction reagent (5mg Bug buster solution per gram of pellet) and the slurry was centrifuge at 5,000 rpm for 30 minutes. The supernatant was diluted one-fold with 2X column buffer (40mM Tris-HCl, 400mM NaCl, 2mM EDTA, 1mM PMSF), so that the final solution is equilibrated as a cellular lysate in Bug buster solution and 1X column buffer. The supernatant was incubated with 750ul of amylose resin that had been pre-calibrated in 1X column buffer for 3 hours. The slurry was passed through a 10ml gravity flow-based column (Fisher scientific) and the column was washed stepwise with 1X column buffer supplemented with 1mM maltose, 3mM maltose, 5mM maltose and eluted with 1X column buffer supplemented with 10mM maltose. The eluted fractions were pooled and dialysed against 20 mM Tris-HCl (pH ranging from 7.2 to 8.5, depending on the pI of the Aux/IAA protein), 150 mM NaCl, 1 mM EDTA, 5% glycerol and 1 mM PMSF. For the generation of exclusively reduced versions of full-length Aux/IAA proteins; the eluted fractions were dialysed in the same buffer containing 7.5 mM DTT. Purified recombinant proteins were analysed on an 8% SDS-PAGE gel using Coomassie Brilliant blue (CBB) staining and/or immunoblotting with anti-MBP antibody.

## 532 Treatment of recombinant proteins with reducing and oxidizing agents

For diamide induced multimerization (DIM) assays, recombinant proteins were prepared as mentioned above in the absence of any reducing agent. Approximately 15µg (~10µM) of recombinant IAA3-WT or IAA3-4C/S was treated with 7.5mM DTT for 30 minutes at room temperature. DTT treated protein samples were then subsequently treated with increasing concentrations of diamide ranging from 3mM to 15mM for 30 minutes at room temperature. The treated proteins were then processed in 1X Lamelli Dye without β-mercaptoethanol and analysed in an 8% SDS-PAGE gel by staining with Coomassie brilliant blue. For comparison of the differences in the multimerization capacity of the candidate Aux/IAAs, recombinant protein samples were first treated with 7.5mM DTT followed by treatment with different
542

concentrations of NEM ranging from 1mM to 10mM and finally with a fixed concentration of
10mM diamide. In each case, the incubation was done at room temperature for 30 minutes. The
treated proteins were then processed in 1X Lamelli Dye without β-mercaptoethanol and
analysed in an 8% SDS-PAGE gel by staining with Coomassie brilliant blue.

543

544

545

546

## Protein sequence analyses and structure modelling

547

548

549

Multiple sequence alignment of IAA proteins was performed using MUSCLE(53) in Jalview (54) and Mulialin(55). IAA3 structure was modelled using Alpha fold. Three-dimensional structures were visualized with PyMOL software.

550

## Solution digestion

551

552

553

554

555

556

557

558

559

560

561

562

563

564

565

566

567

Lyophilised sample was resuspended in 23ul total volume in 5%SDS, 50mM Triethylamine ammonium bicarbonate (TEAB), pH 8.5 and acidified with 2.5ul of 27.5% phosphoric acid. 165ul of binding/wash buffer (90% methanol,100mM TEAB) was added to the sample and mix, transfer to the S-Trap column (Protifi, Fairport, NY, USA) and place in 1.7ml Eppendorf tube. This was then centrifuged at 4000 g to trap proteins for 30sec. The S-Trap column was further washed with 150µl of wash/binding buffer and centrifuged at 4,000g for 30 sec. This process was repeated 3 times in total. For digestion (either Trypsin or GluC) the column was placed in a new collection tubes and 20µl of protease solution (1ug per 10ug of protein in 50mM TEAB) and incubated overnight at 37°C. For elution, 40µl of elution buffer 1 (50mM TEAB) was added and the column centrifuged for 1 minute at 4000g, followed by 40µl of elution buffer2(0.2% Formic Acid), and centrifuged for 1 minute at 4000g, followed by the addition of 40µl of elution buffer 3 (50% Acetonitrile (ACN)) and further centrifugation at 4000g for 1 minute. The eluted peptides were lyophilised prior to mass spectrometric analysis.

568

569

570

571

572

573

574

## In gel digestion

Coomassie stained band were excised from the gel and cut into 1mm³ pieces. The pieces were destained by adding 50% acetonitrile, 50% ammonium bicarbonate solution and incubated for 10minutes at 37°C, followed further destaining with HPLC-grade water until the gel pieces were clear. The gel pieces were dehydrated by the addition of 100% ACN, for 10 mins at 37°C. The supernatant was removed, and the gel pieces were then air dried. Trypsin solution was added in 50mM ammonium bicarbonate, trypsin conc. 0.005µg/µl. The volume of trypsin
575

solution used was sufficient to cover gel pieces. The resulting supernatant was lyophilised prior
to mass spectrometric analysis.

576

577

578

LC-MS/MS

579

580

All LC-MS/MS experiments were performed using a Dionex Ultimate 3000 RSLC nanoUPLC (Thermo Fisher Scientific Inc, Waltham, MA, USA) system and a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific Inc, Waltham, MA, USA). Separation of peptides was performed by reverse-phase chromatography at a flow rate of 300 nL/min and a Thermo Scientific reverse-phase nano Easy-spray column (Thermo Scientific PepMap C18, 2µm particle size, 100A pore size, 75µm i.d. x 50cm length). Peptides were loaded onto a pre-column (Thermo Scientific PepMap 100 C18, 5µm particle size, 100A pore size, 300µm i.d. x 5mm length) from the Ultimate 3000 autosampler with 0.1% formic acid for 3 minutes at a flow rate of 15 µL/min. After this period, the column valve was switched to allow elution of peptides from the pre-column onto the analytical column. Solvent A was water + 0.1% formic acid and solvent B was 80% acetonitrile, 20% water + 0.1% formic acid. The linear gradient employed was 2-40% B in 40 minutes. Further wash and equilibration steps gave a total run time of 60 minutes. The LC eluant was sprayed into the mass spectrometer by means of an Easy-Spray source (Thermo Fisher Scientific Inc.). All m/z values of eluting ions were measured in an Orbitrap mass analyzer, set at a resolution of 35000 and was scanned between m/z 380-1500. Data dependent scans (Top 20) were employed to automatically isolate and generate fragment ions by higher energy collisional dissociation (HCD, NCE:26%) in the HCD collision cell and measurement of the resulting fragment ions was performed in the orbitrap analyser, set at a resolution of 17500. Singly charged ions and ions with unassigned charge states were excluded from being selected for MS/MS and a dynamic exclusion window of 40 seconds was employed.

581

582

583

584

585

586

587

588

589

590

591

592

593

594

595

596

597

598

599

600

601

602

Database searching

603

604

605

606

607

608

609

Post-run, text files generated from the raw data were converted to .mgf files and the files were
submitted to the Mascot search algorithm (Matrix Science, London UK, version 2.7) and
searched against a common contaminants database (cRAP_20190401.fasta) and the TAIR 10.0
database. Variable modifications of oxidation (M), deamidation (NQ) and phosphorylation
(STY) and a fixed modification of carbamidomethyl (C) and N-ethylmaleimide (C) were
applied. The peptide and fragment mass tolerances were set to 20 ppm and 0.1 Da, respectively.
610 Scaffold (version Scaffold_5.1.1, Proteome Software Inc., Portland, OR) was used to validate
611 MS/MS based peptide and protein identifications. Peptide identifications were accepted if they
612 could be established at greater than 95.0% probability by the Scaffold Local FDR algorithm.
613 Protein identifications were accepted if they could be established at greater than 99.0%
614 probability and contained at least 2 identified peptides. Protein probabilities were assigned by
615 the Protein Prophet algorithm(56). Proteins that contained similar peptides and could not be
616 differentiated based on MS/MS analysis alone were grouped to satisfy the principles of
617 parsimony. Proteins sharing significant peptide evidence were grouped into clusters.

618 **Western blot analysis of transgenic plants**

619 Total protein was isolated from Arabidopsis thaliana transgenic seedlings expressing N-
620 terminal HA tagged WT-IAA3 and 4C/S-IAA3 in RIPA buffer (20 mM Tris-HCl (pH 7.5),
621 150 mM NaCl, 1 mM Na₂EDTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium
622 pyrophosphate, 1 mM β-glycerophosphate, 1 mM PMSF and 1 mM β-Mercaptoethanol)
623 maintaining a tissue: buffer ratio of 1:1. Crude protein extracts were processed with 4X
624 Lamelli Dye supplemented with β-Mercaptoethanol and separated by 10% SDS-PAGE gels
625 and immunoblotted with the anti-HA antibody (anti-HA Abcam). For endogenous controls,
626 immunoblotting was performed with Anti-histone H3 antibody (Abcam).

627 <u>Agrobacterium mediated transient assays</u>

628

629 Gene constructs were transiently expressed in *Nicotiana benthamiana* plants using the
630 *Agrobacterium tumefaciens* mediated transformation(57). ARF7-GFP, ARF7-GFP (4K/R),
631 TIR1-GFP, TPL-myc and GFP proteins were used to check the interactions with HA-WT
632 IAA3, HA-4C/S IAA3 and HA-SIM IAA3 in *Nicotiana benthamiana* leaves.3 d post-
633 infiltration, leaves were frozen in liquid nitrogen for sample preparation and co-
634 immunoprecipitation, or immunoprecipitation assays were performed.

635 **<u>Co-immunoprecipitation assay</u>**

636 *Nicotiana Benthamiana* plants were infiltrated with a combination of *Agrobacterium
tumefaciens* cells expressing the proteins of interest along with the anti-silencing factor P19. It
637 was ensured that the total O.D. for the combination of the *Agrobacterium tumefaciens* cells did
638 not exceed 0.6 in all the co-immunoprecipitation (co-IP) assays. 3 days post infiltration, total
639
640

protein was then extracted from *Nicotiana Benthamiana* leaves for co-IP using the extraction buffer containing: 50 mM Tris HCl (pH 8.0), 150mM NaCl, 1mM EDTA, 0.5% Triton-X 100, 2% [v/v] Glycerol and 10 mM DTT (depending on the type of immunoprecipitation performed). Anti-GFP IP and anti-HA IP were performed. Total protein was incubated with either 20 µl anti-GFP or 20 µl anti-HA micro magnetic beads (Miltenyi Biotech) and put on a rotating platform at 4°C for 30 minutes and the slurry was subsequently passed through a micro column (Miltenyi Biotech) attached to a magnetic stand (Miltenyi Biotech) to capture the beads. The beads were then washed four times by passing 1 ml of cold IP buffer through the micro column. After the last wash, 80 µl of pre-heated (98°C) 1× Lamelli buffer was used to elute the immuno-complexes. The immuno-complexes was then analysed on a 10% SDS-PAGE gel using immunoblotting methods with Abcam (Cambridge, UK) anti-GFP, anti-HA antibodies (Anti-HA High Affinity Roche 3F/10, Sigma-Aldrich, and anti-HA Abcam). At least three independent biological replicates were performed per experiment.

641

642

643

644

645

646

647

648

649

650

651

652

653

## Redox dependent Immunoprecipitation (Redox IP) assays

654

Transgenic *Arabidopsis thaliana* seedlings or leaves from *Nicotiana Benthamiana* plants infiltrated with a combination of *Agrobacterium tumefaciens* cells expressing the proteins of interest along with the anti-silencing factor P19 were used for Redox dependent Immunoprecipitation assays. Total protein from samples were isolated for Redox-IP using extraction buffer containing: 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1mM EDTA, 0.25% [w/v] Sodium deoxycholate, 2% [v/v] glycerol, 0.1% [v/v] TritonX-100, 0.7% [v/v] Igepal CA-630 (Nonidet P-40), 50 mM KCl, 20mM MgCl₂ and protease inhibitor cocktail. Depending on the redox state of the extraction buffer intended for the assay, the extraction buffer was supplemented either with 10mM DTT or 10mM Diamide. Anti-HA IP was then performed. Total protein was incubated with 20 µl anti-HA micro magnetic beads (Miltenyi Biotech) and put on a rotating platform at 4°C for 30 minutes and the slurry was subsequently passed through a micro column (Miltenyi Biotech) attached to a magnetic stand (Miltenyi Biotech) to capture the beads. The beads were then washed four times by passing 1 ml of cold Redox-IP buffer through the micro column. After the last wash, immunocomplexes were eluted with 80 µl of pre-heated (98°C) 1× Lamelli buffer that either lacked β-Mercaptoethanol (for redox IPs performed in non-reducing or oxidizing environment) or were supplemented with β-Mercaptoethanol (for redox IPs performed in reducing environment). The immuno-complexes was then analysed on a 10% SDS-PAGE gel using immunoblotting methods with anti-HA

655

656

657

658

659

660

661

662

663

664

665

666

667

668

669

670

671
672

antibodies (Anti-HA High Affinity Roche 3F/10, Sigma-Aldrich, and anti-HA Abcam). At least three independent biological replicates were performed per experiment. For DTT based titration assays, total protein from samples were extracted in the extraction buffer described above and anti HA IP was performed. Elution of the immunocomplexes were eluted with 80 µl of pre-heated (98°C) 1× Lamelli buffer that lacked β-Mercaptoethanol, and the immuno-complexes were analysed by the same methodology as described above.

673

674

675

676

677

678

## Detection of SUMO conjugation of targets

679

p35S::YFP-ARF7 WT and p35S::YFP-ARF7<sup>4K/R</sup> constructs were transformed into *Agrobacterium* strain GV3101 and were coinfiltrated along with p35S::HA-SUMO1 and P19 into *N.benthamiana*. The infiltrated *N.benthamiana* leaf samples were collected after 3 days, and total protein was isolated from the samples in extraction buffer containing: 50 mM Tris-HCl (pH 8.5), 150 mM NaCl, 1mM EDTA, 0.1% [v/v] SDS, 0.5% [w/v] Sodium deoxycholate, 1.0 % [v/v] Igepal CA-630 (Nonidet P40), 50mM KCl, 50mM NEM (N-ethyl maleimide) and protease inhibitor cocktail. Anti-GFP IP was performed with the total protein extract using the methodology as described above, and the immuno-complexes was then analysed on a 10% SDS- PAGE gel using immunoblotting methods with Abcam (Cambridge, UK) anti-GFP and anti-HA antibodies (Anti-HA High Affinity Roche 3F/10, Sigma-Aldrich, and anti-HA Abcam). At least three independent biological replicates were performed per experiment.

680

681

682

683

684

685

686

687

688

689

690

## Agar-based xerobranching assay

691

Agar-based Air-gap Assay was designed for phenotyping *Arabidopsis* thaliana seedlings exposed to transient low-water stress in accordance with Mehra et al, 2022 (2). Briefly, ½ MS medium supplemented with 0.3 % sucrose, 0.5 g/L MES and 1 % Bacto agar was poured into square petri plates (12 cm x 12 cm), and agar slants were created by aligning Petri dishes at a 15° angle with respect to a flat surface. In the middle of the petri dish, a clean cut was made with the help of a 10-cm long scraper blade, and cut agar was carefully removed using a scalpel, to create an air-gap of ~0.5 cm. Five days-old *Arabidopsis* seedlings were placed on plates in a manner that allowed the root tips to be positioned 2-3 mm away from the agar-air interface. Plates were sealed and held vertically under appropriate growth conditions (as described above) for 7 days.

692

693

694

695

696

697

698

699

700

701

## Lateral root density
702
703
704

Images of whole plates were taken using a Canon digital camera. PR length was measured using the NeuronJ plugin in ImageJ. LR number was counted manually and LR density was calculated by dividing LR number by the length of the root.

705

Confocal imaging and image analysis

706
707
708
709
710
711

Imaging of roGFP2-Orp1 was performed as described previously (4). Roots exposed to control conditions, XB stimuli and recovery conditions were imaged using a Leica SP5 confocal laser scanning microscope (Leica Microsystems). The GFP signal was sequentially excited at 405 nm and 488 nm wavelengths and emission was recorded at 505-535 nm. Images were acquired as Z-stacks in 12-bit mode. Images were processed to calculate 405/488 ratios using ImageJ (ImageJ 1.53f51).

712

RNA isolation and Quantitative real time PCR (q-RT PCR)

713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732

Total RNA was extracted from 12 to 14-day old seedlings grown in 1/2 MS media grown vertically for a period of 8-10 days (depending on the experimental design) under continuous temperature 22 °C with a 16 h photoperiod (150 µmol m⁻² s⁻¹). A Direct-zol RNA Miniprep kit (ZYMO research) was used to extract total RNA following the manufacturer's recommendations. Cross contaminating DNA was removed from the samples by on-column DNase I digestion following the manufacturer's recommendations. The RNA was then quantified by measuring the absorbance (260 and 280 nm) using a NanoDropTM 1000 Spectrophotometer (Thermo Scientific), and cDNA was synthesized with Invitrogen SuperScript® II Reverse Transcriptase. The synthesized cDNA was subsequently used as template for quantitative real time PCR based analysis of the expression levels of the target genes using Brilliant III Ultra-Fast SYBR QPCR MM (Agilent) in conjunction with Rotor-Gene® Q (Qiagen) system. In order to assess the expression levels of the transgene (HA-IAA3 CDS expressed under the control of different promoters) and the auxin responsive downstream targets of IAA3 in the transgenic lines generated in this study, we adopted the absolute quantification-based approach for our quantitative real time PCR based analysis in accordance with Paul et al, 2017(58). Briefly, forward and reverse primers corresponding to the HA tag and 3' end of the IAA3 CDS was used to uniquely amplify the transgene (HA-WT IAA3 or HA-4C/S IAA3) intogressed in the *iaa3* mutant background and expressed under the control of the CAMV 35S, *CASP1* and *IAA3* promoter. Logarithmic fold dilutions of plasmids cloned with HA-WT *IAA3*, HA-4C/S *IAA3* CDS and an amplicon corresponding to the CDS of the
housekeeping gene ACTIN7 (At5g09810) were used as template for real time PCR reaction and Ct values were plotted against the dilutions to generate a standard curve for both IAA3/SHY2 CDS and actin. The levels of the transgene in the different transgenic lines were then calculated by extrapolation of the Ct value against the generated standard curve followed by normalisation with the actin levels. WT Col-0 and *iaa3* mutant seedlings were used as negative controls for the analysis. Based on this approach, transgenic lines showing similar levels of HA-WT IAA3 and HA-4C/S IAA3 expression at the transcriptional level were selected and the abundance of the HA-WT IAA3 and HA-4C/S IAA3 proteins were further verified. Post verification, multiple optimised transgenic lines were used to assess the expression levels of the auxin responsive downstream targets of IAA3 in the absence of exogeneous auxin treatment, using the same approach as described above with the exception that the standard curves for each target gene tested was generated from gel extracted DNA fragments corresponding to the amplicon of the target gene. To assess difference in the inducibility of LBD16 expression on exogeneous auxin treatment between the transgenic lines, conventional ΔΔCt method(59) was adopted with the housekeeping gene ACTIN7 (At5g09810) used for normalisation. Fold change was calculated relative to untreated control.

## Chromatin Immunoprecipitation and qPCR

For ChIP assays nuclei were isolated from 1% formaldehyde fixed plant samples using Nuclei extraction kit (CelLytic PN isolation/extraction kit, Merck) in accordance with the manufacturer's protocol. The chromatin isolation and subsequent immunoprecipitation was performed according to Srivastava et al 2021(60) using anti-HA magnetic microbeads (Miltenyi Biotech). Quantitative real-time PCR of the ChIPed DNA was performed in the Rotor- Gene® Q system (Qiagen) in a total reaction volume of 10 µl, using Brilliant III Ultra-Fast SYBR qPCR master mix (Agilent). *At GYPSY* (gene code: AT4G07700) retrotransposon was used as an internal reference gene control. Primers used for ChIP-qPCR are documented in supplementary table S3. The log fold change was calculated as percentage input for each sample normalized with input samples.

## Immunoprecipitation, Sample preparation and LC-MS/MS Analysis for WT-IAA3 and 4C/S-IAA3 interaction with TOPLESS in stable transgenic lines

762
763 Proteins were extracted from the root tissues of pIAA3:IAA3 wild type (WT)/iaa3 and 764 pIAA3:IAA3 4C/S /iaa3 plants for immunoprecipitation. The extraction buffer (EB) used 765 contained 50 mM Tris-Cl (pH 7.5), 125 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 5% (v/v) 766 glycerol, 0.1% (v/v) Tween-20, 0.2% (v/v) Igepal CA-630, and 0.5% (w/v) digitonin, along 767 with freshly added protease inhibitors (Complete Mini EDTA-free from Roche – 1 tablet per 768 10 mL of solution). Root tissues (2 g) were ground to a fine powder using a mortar and pestle 769 with liquid nitrogen. Four ml of extraction buffer was added, and the lysate was cleared by 770 centrifugation at 14,000g for 10 minutes. The cleared lysates were incubated with 20 µL of 771 anti-HA magnetic beads (Miltenyi Biotec, Cat No. 130-091-122) on an end-to-end rotator for 772 2 hours in a cold room. The lysates with the beads were then passed through micro-columns 773 (Miltenyi Biotec, Cat No. 130-042-701) placed on the µMACSTM Separator. The beads were 774 washed three times with cold IP wash buffer (EB without digitonin). After the final wash, 50 775 µL of pre-heated (95°C) 1x SDS-loading buffer was used to elute the immunocomplex. The 776 eluted proteins were analyzed by 4-15% gradient sodium dodecyl-sulfate polyacrylamide gel 777 electrophoresis (SDS-PAGE) and immunoblotting with an anti-HA antibody (Roche 3F/10) to 778 visualize the higher-order multimer formation by IAA3 WT and IAA3 4CS mutant proteins. 779

The immunoprecipitated eluates were loaded onto a 12% SDS-PAGE gel and electrophoresed 780 for 20 minutes to allow the proteins to enter the resolving gel. The gel was then stained with 781 colloidal Coomassie brilliant blue for 30 minutes and subsequently destained with a solution 782 containing 50% water, 45% methanol, and 5% glacial acetic acid for another 30 minutes. 783 Protein bands were excised into 1 mm³ pieces using a sterile blade and collected into fresh 1.5 784 mL tubes. To remove the Coomassie stain, the gel pieces were washed three times with a 785 solution of 50% acetonitrile (ACN) and 50 mM ammonium bicarbonate (ABC). The gel pieces 786 were then dehydrated with 500 µL of 100% ACN for 10 minutes. Disulfide bonds were reduced 787 by incubating the gel pieces in 10 mM dithiothreitol (DTT) in 50 mM ABC at 37°C for 1 hour. 788

Following this, 100 µL of 55 mM iodoacetamide in 50 mM ABC was added, and the samples 789 were incubated at room temperature for 45 minutes in the dark for alkylation. The alkylation 790 solution was removed, and the gel pieces were again dehydrated with 500 µL of 100% ACN 791 for 10 minutes. After removing residual ACN, the samples were incubated with trypsin 792 (Promega, USA) at a protease-to-protein ratio of 1:25 (w/w) for in-gel digestion at 37°C for 16 793 hours. The resulting peptides were extracted in 0.1% formic acid (FA) and dried using a speed-794 vac. Desalting of the peptides was performed using Pierce™ Peptide Desalting Spin Columns 795 (Thermo Fisher Scientific, Cat No. 89851) as per the manufacturer's instructions. The desalted 796 peptides were dissolved in 0.1% FA and analyzed on a tims-TOF mass spectrometer (Bruker). 797
797
798
799

The MS/MS data were analyzed using PEAKS Studio 11 software to identify potential
interactors. The area under the curve (AUC) was used to quantify the corresponding peptides
for TPL protein in both IAA WT and IAA 4CS samples.

800

**<u>Statistical Analysis</u>**

801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820

Both Student's t-test and ANOVA was used to perform the statistical analysis of the data. The Student's t-test determined if the means of two data sets differed significantly from each other in relation to the null hypothesis. A different letter indicated a significant difference from that of WT (Col-0) roots based on the Student t-test *P* value (*P*<0.05). ANOVA was used when there were more than two data sets to compare. The analysis of variance, one-way ANOVA considered the total number of observations, means, standard deviations (or standard error) within each data set. Using GenStat, multiple comparisons, Tukey HSD (Honest Significant Difference) test was applied at the *P*-value 0.05 significance level (95% confidence interval) to each data set. Asterisk notation was used to indicate significance levels between all the data sets in relation of analysis of LR phenotype and quantitative real time PCR data. Additionally, for analysis of parameters related to LR phenotype (root length, emerged lateral root per plant and lateral root density) in normal ambient water supplied conditions, Welch's unequal variance t-test was also performed to consider unequal population variances in a two-sample location test. Transgenic seedlings expressing WT-IAA3 and 4C/S-IAA3 under the control of different promoters were used as the two different populations to test the null hypothesis that these populations have equal means. Statistical analysis was done using software Prism-GraphPad. In relation to the data corresponding to xerobranching assays, letters were used to indicate significance levels between all the data sets, where similar letters stated that there were no significant differences observed. Whereas different letters indicated that there was a significant difference observed between the data sets (*P*<0.05).

821

**References and notes:**

822
823
824
825
826
827
828

1. B. Orman-Ligeza *et al.*, The xerobranching response represses lateral root formation when roots are not in contact with water. *Current Biology* **28**, 3165-3173. e3165 (2018).

2. P. Mehra *et al.*, Hydraulic flux-responsive hormone redistribution determines root branching. *Science* **378**, 762-768 (2022).

3. B. Orman-Ligeza *et al.*, RBOH-mediated ROS production facilitates lateral root emergence in Arabidopsis. *Development* **143**, 3328-3339 (2016).
829 4. T. Nietzel *et al.*, The fluorescent protein sensor ro GFP 2-Orp1 monitors in vivo H2O2 and thiol redox integration and elucidates intracellular H2O2 dynamics during elicitor-induced oxidative burst in Arabidopsis. *New Phytologist* **221**, 1649-1664 (2019).

833 5. D. Arnaud, M. J. Deeks, N. Smirnoff, Organelle-targeted biosensors reveal distinct oxidative events during pattern-triggered immune responses. *Plant Physiology* **191**, 2551-2569 (2023).

836 6. B. Lu *et al.*, FERONIA-mediated TIR1/AFB2 oxidation stimulates auxin signaling in Arabidopsis. *Molecular Plant* **17**, 772-787 (2024).

838 7. J. Luo, J.-J. Zhou, J.-Z. Zhang, Aux/IAA gene family in plants: molecular structure, regulation, and function. *International journal of molecular sciences* **19**, 259 (2018).

840 8. M. Lavy, M. Estelle, Mechanisms of auxin signaling. *Development* **143**, 3226-3229 (2016).

842 9. T. J. Guilfoyle, The PB1 domain in auxin response factor and Aux/IAA proteins: a versatile protein interaction module in the auxin response. *The Plant Cell* **27**, 33-43 (2015).

845 10. T. Vernoux *et al.*, The auxin signalling network translates dynamic input into robust patterning at the shoot apex. *Molecular systems biology* **7**, 508 (2011).

847 11. N. Dharmasiri, S. Dharmasiri, M. Estelle, The F-box protein TIR1 is an auxin receptor. *Nature* **435**, 441-445 (2005).

849 12. S. Kepinski, O. Leyser, The Arabidopsis F-box protein TIR1 is an auxin receptor. *Nature* **435**, 446-451 (2005).

851 13. X. Tan *et al.*, Mechanism of auxin perception by the TIR1 ubiquitin ligase. *Nature* **446**, 640-645 (2007).

853 14. S. Kagale, K. Rozwadowski, EAR motif-mediated transcriptional repression in plants: an underlying mechanism for epigenetic regulation of gene expression. *Epigenetics* **6**, 141-146 (2011).

856 15. H. Szemenyei, M. Hannon, J. A. Long, TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis. *Science* **319**, 1384-1386 (2008).

859 16. J. S. Teixeira, K. Ten Tusscher, The systems biology of lateral root formation: connecting the dots. *Molecular Plant* **12**, 784-803 (2019).

861 17. C. M. Cremers, U. Jakob, Oxidant sensing by reversible disulfide bond formation. *Journal of Biological Chemistry* **288**, 26489-26496 (2013).

863 18. J. R. Ramos, B. J. Reyes-Hernández, K. Alim, A. Maizel, Auxin-mediated stress relaxation in pericycle and endoderm remodelling drive lateral root initiation. *Biophysical Journal*, (2024).

866 19. R. Ursache, S. Fujita, V. Dénervaud Tendon, N. Geldner, Combined fluorescent seed selection and multiplex CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants. *Plant Methods* **17**, 1-14 (2021).

869 20. D. A. Korasick *et al.*, Molecular basis for AUXIN RESPONSE FACTOR protein interaction and the control of auxin response repression. *Proceedings of the National Academy of Sciences* **111**, 5427-5432 (2014).

872 21. M. H. Nanao *et al.*, Structural basis for oligomerization of auxin transcriptional regulators. *Nature communications* **5**, 3617 (2014).

874 22. M. I. Wilson, D. J. Gill, O. Perisic, M. T. Quinn, R. L. Williams, PB1 domain-mediated heterodimerization in NADPH oxidase and signaling complexes of atypical protein kinase C with Par6 and p62. *Molecular cell* **12**, 39-50 (2003).

877 23. M. L. Seibenhener, T. Geetha, M. W. Wooten, Sequestosome 1/p62-more than just a scaffold. *FEBS letters* **581**, 175-179 (2007).
879 24. D. C. Dinesh et al., Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response. *Proceedings of the National Academy of Sciences* **112**, 6230-6235 (2015).

882 25. C. E. Paulsen, K. S. Carroll, Orchestrating redox signaling networks through regulatory cysteine switches. *ACS chemical biology* **5**, 47-62 (2010).

884 26. L. B. Poole, The basics of thiols and cysteines in redox biology and chemistry. *Free Radical Biology and Medicine* **80**, 148-157 (2015).

886 27. A. Zeida et al., Catalysis of peroxide reduction by fast reacting protein thiols: focus review. *Chemical reviews* **119**, 10829-10855 (2019).

888 28. C. H. Foyer, G. Noctor, Redox homeostasis and signaling in a higher-CO2 world. *Annual Review of Plant Biology* **71**, 157-182 (2020).

890 29. X. Huang et al., ROS regulated reversible protein phase separation synchronizes plant flowering. *Nature Chemical Biology* **17**, 549-557 (2021).

892 30. J. E. Vermeer et al., A spatial accommodation by neighboring cells is required for organ initiation in Arabidopsis. *Science* **343**, 178-183 (2014).

894 31. L. I. A. Calderón Villalobos et al., A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin. *Nature chemical biology* **8**, 477-485 (2012).

896 32. K. A. Dreher, J. Brown, R. E. Saw, J. Callis, The Arabidopsis Aux/IAA protein family has diversified in degradation and auxin responsiveness. *The Plant Cell* **18**, 699-714 (2006).

899 33. B. L. Moss et al., Rate motifs tune auxin/indole-3-acetic acid degradation dynamics. *Plant physiology* **169**, 803-813 (2015).

901 34. Q. Tian, N. J. Uhlir, J. W. Reed, Arabidopsis SHY2/IAA3 inhibits auxin-regulated gene expression. *The Plant Cell* **14**, 301-319 (2002).

903 35. S. H. Kim et al., A gain-of-function mutant of IAA15 inhibits lateral root development by transcriptional repression of LBD genes in Arabidopsis. *Frontiers in Plant Science* **11**, 1239 (2020).

906 36. T. Goh, H. Kasahara, T. Mimura, Y. Kamiya, H. Fukaki, Multiple AUX/IAA-ARF modules regulate lateral root formation: the role of Arabidopsis SHY2/IAA3-mediated auxin signalling. *Philosophical Transactions of the Royal Society B: Biological Sciences* **367**, 1461-1468 (2012).

910 37. A. Colón-Carmona, D. L. Chen, K.-C. Yeh, S. Abel, Aux/IAA proteins are phosphorylated by phytochrome in vitro. *Plant physiology* **124**, 1728-1738 (2000).

912 38. Q. Tian, J. W. Reed, Control of auxin-regulated root development by the Arabidopsis thaliana SHY2/IAA3 gene. *Development* **126**, 711-721 (1999).

914 39. B. Orosa-Puente et al., Root branching toward water involves posttranslational modification of transcription factor ARF7. *Science* **362**, 1407-1410 (2018).

916 40. H.-T. Cho et al., A dose-dependent bimodal switch by homologous Aux/IAA transcriptional repressors. *Molecular Plant* **17**, 1407-1422 (2024).

918 41. Y. Okushima, H. Fukaki, M. Onoda, A. Theologis, M. Tasaka, ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis. *The Plant Cell* **19**, 118-130 (2007).

921 42. T. Goh et al., Lateral root initiation requires the sequential induction of transcription factors LBD16 and PUCHI in Arabidopsis thaliana. *New Phytologist* **224**, 749-760 (2019).

924 43. A. Shapiguzov, J. P. Vainonen, M. Wrzaczek, J. Kangasjärvi, ROS-talk-how the apoplast, the chloroplast, and the nucleus get the message through. *Frontiers in plant science* **3**, 292 (2012).
<table>
  <tbody>
    <tr>
      <td>927</td>
      <td>44.</td>
      <td>O. Rodrigues et al., Aquaporins facilitate hydrogen peroxide entry into guard cells to mediate ABA-and pathogen-triggered stomatal closure. Proceedings of the National Academy of Sciences 114, 9200-9205 (2017).</td>
    </tr>
    <tr>
      <td>928</td>
      <td>45.</td>
      <td>G. P. Bienert, F. Chaumont, Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide. Biochimica et Biophysica Acta (BBA)-General Subjects 1840, 1596-1604 (2014).</td>
    </tr>
    <tr>
      <td>929</td>
      <td>46.</td>
      <td>M. Chambers et al., Mechanisms of Groucho-mediated repression revealed by genome-wide analysis of Groucho binding and activity. BMC genomics 18, 1-17 (2017).</td>
    </tr>
    <tr>
      <td>930</td>
      <td>47.</td>
      <td>C. R. Horne et al., Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism. Nature communications 12, 1988 (2021).</td>
    </tr>
    <tr>
      <td>931</td>
      <td>48.</td>
      <td>D. Moazed, Common themes in mechanisms of gene silencing. Molecular cell 8, 489-498 (2001).</td>
    </tr>
    <tr>
      <td>932</td>
      <td>49.</td>
      <td>S. Payankaulam, L. M. Li, D. N. Arnosti, Transcriptional repression: conserved and evolved features. Current biology 20, R764-R771 (2010).</td>
    </tr>
    <tr>
      <td>933</td>
      <td>50.</td>
      <td>W. Wu et al., Evolution analysis of the Aux/IAA gene family in plants shows dual origins and variable nuclear localization signals. International journal of molecular sciences 18, 2107 (2017).</td>
    </tr>
    <tr>
      <td>934</td>
      <td>51.</td>
      <td>M. Michniewicz, E. M. Frick, L. C. Strader, Gateway-compatible tissue-specific vectors for plant transformation. BMC research notes 8, 1-8 (2015).</td>
    </tr>
    <tr>
      <td>935</td>
      <td>52.</td>
      <td>S. J. Clough, A. F. Bent, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The plant journal 16, 735-743 (1998).</td>
    </tr>
    <tr>
      <td>936</td>
      <td>53.</td>
      <td>R. C. Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic acids research 32, 1792-1797 (2004).</td>
    </tr>
    <tr>
      <td>937</td>
      <td>54.</td>
      <td>A. M. Waterhouse, J. B. Procter, D. M. Martin, M. Clamp, G. J. Barton, Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189-1191 (2009).</td>
    </tr>
    <tr>
      <td>938</td>
      <td>55.</td>
      <td>F. Corpet, Multiple sequence alignment with hierarchical clustering. Nucleic acids research 16, 10881-10890 (1988).</td>
    </tr>
    <tr>
      <td>939</td>
      <td>56.</td>
      <td>A. I. Nesvizhskii, A. Keller, E. Kolker, R. Aebersold, A statistical model for identifying proteins by tandem mass spectrometry. Analytical chemistry 75, 4646-4658 (2003).</td>
    </tr>
    <tr>
      <td>940</td>
      <td>57.</td>
      <td>M. M. Goodin, R. G. Dietzgen, D. Schichnes, S. Ruzin, A. O. Jackson, pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves. The Plant Journal 31, 375-383 (2002).</td>
    </tr>
    <tr>
      <td>941</td>
      <td>58.</td>
      <td>A. Paul, P. Dasgupta, D. Roy, S. Chaudhuri, Comparative analysis of Histone modifications and DNA methylation at OsBZ8 locus under salinity stress in IR64 and Nonabokra rice varieties. Plant molecular biology 95, 63-88 (2017).</td>
    </tr>
    <tr>
      <td>942</td>
      <td>59.</td>
      <td>K. J. Livak, T. D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. methods 25, 402-408 (2001).</td>
    </tr>
    <tr>
      <td>943</td>
      <td>60.</td>
      <td>M. Srivastava et al., The conjugation of SUMO to the transcription factor MYC2 functions in blue light-mediated seedling development in Arabidopsis. The Plant Cell 34, 2892-2906 (2022).</td>
    </tr>
    <tr>
      <td>944</td>
      <td>61.</td>
      <td></td>
    </tr>
    <tr>
      <td>945</td>
      <td>62.</td>
      <td></td>
    </tr>
    <tr>
      <td>946</td>
      <td>63.</td>
      <td></td>
    </tr>
    <tr>
      <td>947</td>
      <td>64.</td>
      <td></td>
    </tr>
    <tr>
      <td>948</td>
      <td>65.</td>
      <td></td>
    </tr>
    <tr>
      <td>949</td>
      <td>66.</td>
      <td></td>
    </tr>
    <tr>
      <td>950</td>
      <td>67.</td>
      <td></td>
    </tr>
    <tr>
      <td>951</td>
      <td>68.</td>
      <td></td>
    </tr>
    <tr>
      <td>952</td>
      <td>69.</td>
      <td></td>
    </tr>
    <tr>
      <td>953</td>
      <td>70.</td>
      <td></td>
    </tr>
    <tr>
      <td>954</td>
      <td>71.</td>
      <td></td>
    </tr>
    <tr>
      <td>955</td>
      <td>72.</td>
      <td></td>
    </tr>
    <tr>
      <td>956</td>
      <td>73.</td>
      <td></td>
    </tr>
    <tr>
      <td>957</td>
      <td>74.</td>
      <td></td>
    </tr>
    <tr>
      <td>958</td>
      <td>75.</td>
      <td></td>
    </tr>
    <tr>
      <td>959</td>
      <td>76.</td>
      <td></td>
    </tr>
    <tr>
      <td>960</td>
      <td>77.</td>
      <td></td>
    </tr>
    <tr>
      <td>961</td>
      <td>78.</td>
      <td></td>
    </tr>
    <tr>
      <td>962</td>
      <td>79.</td>
      <td></td>
    </tr>
    <tr>
      <td>963</td>
      <td>80.</td>
      <td></td>
    </tr>
    <tr>
      <td>964</td>
      <td>81.</td>
      <td></td>
    </tr>
    <tr>
      <td>965</td>
      <td>82.</td>
      <td></td>
    </tr>
    <tr>
      <td>966</td>
      <td>83.</td>
      <td></td>
    </tr>
    <tr>
      <td>967</td>
      <td>84.</td>
      <td></td>
    </tr>
    <tr>
      <td>968</td>
      <td>85.</td>
      <td></td>
    </tr>
    <tr>
      <td>969</td>
      <td>86.</td>
      <td></td>
    </tr>
    <tr>
      <td>970</td>
      <td>87.</td>
      <td></td>
    </tr>
  </tbody>
</table>
972

We thank Prof. Xavier Draye and Prof. Tom Beeckman for kindly providing the *rboh* mutants
and Prof. Lars Østergaard for kindly providing the *pTPL:TPL-GFP* reporter lines.

973

974

FUNDING

975

976

977

978

979

980

981

982

983

984

985

986

987

This study was supported by Biotechnology and Biological Sciences Research Council (BBSRC) grant (BB/T001437/1 to D.R.) and (BB/V003534/1 to D.R.); The BBSRC Discovery Fellowship (BB/Z514482/1 to P.M.) and the Horizon Europe ERC Starting Grant (101161820-WATER-BLIND to P.M.); Global Challenge Research Fund (GCRF) fellowship (UKRI GCRF Durham PhD scholarship award to V.M.); Biotechnology and Biological Sciences Research Council (BBSRC) grant (BB/V003534/1 to L.C.); the Biotechnology and Biological Sciences Research Council grant (BB/T001437/1 to M.J.B. and A.S).; (BB/V003534/1 to M.J.B. and A.S).; (BB/W008874/1 and BB/W015080/1 to M.J.B); European Research Council grant HYDROSENSING (101118769 to M.J.B.); European Research Council, SUMOrice (grant no. 310235 to A.S.); ChromAuxi grant from the Agence Nationale de la Recherche (ANR-18-CE12-0014-02 to T.V. and R.M.A); Biotechnology and Biological Sciences Research Council (BBSRC) grant (BB/V003534/1 to K.S. and A.J.); Biotechnology and Biological Sciences Research Council (BBSRC) grant (BB/V003534/1 to K.L).

988

989

AUTHOR CONTRIBUTIONS

990

Conceptualization: DR, AS, PM, and MJB.

991

Methodology: DR, PM, VM, LC, KB, RMA, KS, SG, KDI, PKB

992

Investigation: DR, PM, VM, LC, KB, RMA, KS, AB, SG, KDI, PKB.

993

Funding acquisition: AS and MJB

994

Supervision: AS and MJB

995

Writing – original draft: DR, PM, AS and MJB

996

997

Writing – review & editing: DR, PM, VM, LC, KB, JV, RMA, TV, KS, SG, KDI, PKB, AJ, UB, PM, KL, AB, MJB and AS

998

**Competing interests:** The authors declare no competing interests.

999

1000

**Data and materials availability:** No restrictions are placed on materials, such as materials transfer agreements. All data are available in the main text or the supplementary materials.
1001

1002 **Supplementary Materials:**

1003 References (51-60)

1004 Figs. S1 to S22

1005 Tables S1 to S3

1006 Data S1 to S3

1007 **Other Supplementary Material for this manuscript includes the following:**

1008 MDAR Reproducibility Checklist

1009

1010 * *To whom correspondence should be addressed*

1011 **Email id:** ari.sadanandom@durham.ac.uk, dipan.roy@durham.ac.uk, p.mehra@nottingham.ac.uk

1012

1013 **Fig. 1.** Xerobranching response is dependent on ROS (A,B) Agar-based air-gap assay system
1014 showing xerobranching response in *Arabidopsis thaliana* (air-gap ~5mm). Representative
1015 images (A) and bar graphs (B) of agar-based air-gap assay show xerobranching defects in *rboh*
1016 mutants compared to WT Col-0. The red arrowheads in (A) denote the 5 mm zones used for
1017 analysis of lateral roots in control moist agar plates and 5mm air-gap xerobranching plates.
1018 Lateral roots specifically growing in the air-gaps are marked with yellow arrows. Schematics
1019 of the air-gap plates are illustrated at the bottom panel for representation of the xerobranching
1020 phenotype. Grey rectangles indicate the agar interface encompassing the air-gaps, and lateral
1021 roots that initiate and emerge in the air-gap are marked with red arrows. Lateral root numbers
1022 are scored corresponding to the zones in (A) for a population of seedlings denoted as n in (B).
1023 Different letters denote statistical significance for one-way ANOVA tests followed by post-
1024 hoc Tukey HSD test at p<0.05. Xerobranching stimulus triggers ROS production in cytoplasm
1025 and nuclei of ROS sensor lines, where roGFP2-Orp1 oxidation (ratio of 405/488 nm
1026 fluorescence intensity) shows Hydrogen peroxide levels in cytoplasm (C and D) and nucleus
1027 (E and F). (C), (E) Ratio (405nm/488nm) images of cyt-roGFP2-Orp1 and nls-roGFP2-Orp1
1028 roots. Color scale represents 405/488 nm ratios. Higher ratios indicate higher H₂O₂ levels. (D),
1029 (F) corresponds to boxplots that reveal the dynamic changes in the endogenous ROS levels as
1030 root tips grow across an air-gap and after recovery. n denotes the number of seedlings. At least
three biological replicate experiments were performed. (G) roGFP2-Orp1 shows increased oxidation as soon as root tips grows 1mm across an air-gap in comparison to nls-ABACUS2-400n that peaks as the root tips traverses across 4-5mm of the air-gap. Error bars represent standard error. ns, not significant, *P ≤ 0.05; **P≤ 0.01 ***P ≤ 0.001.

1035

Fig. 2. Xerobranching response involves ROS accumulation in inner root tissues and is regulated by the AUX/IAA repressor IAA3 (A) Cross-sectional images of nls-roGFP2-Orp1 reveal increased ROS accumulation in the inner tissues of roots growing across the air-gap. (B) Agar-based air gap assay showing xerobranching defect in the CRISPR/Cas9 knockout line of *Arabidopsis* IAA3 (referred as *iaa3*) mutants compared to WT Col-0. (C) Frequency plots showing the percentage of WT Col-0 and *iaa3* mutant plants that develop lateral roots (LR) across an air gap within a given population of plants. One-way ANOVA analysis between WT Col-0 and the *iaa3* mutant was performed. ns, not significant, *P* ≤ 0.05; **P**≤ 0.01. n denotes the number of individual seedlings used in the study to define the population of plants. At least three biological replicate experiments were performed with 20-25 seedlings per replicate.

1046

Fig. 3. Specific cysteine residues orchestrate IAA3 redox-dependent multimerization driven by RBOHs in planta (A) Model of the IAA3 PB1 dimer (generated by AlphaFold), where one monomer is shown in blue and the other in yellow. The mutated Cys residues are indicated in light blue and orange, respectively. On the right, a zoom on the PB1 head to tail interaction interface is shown, where both the residues involved in these interactions and the position of the Cys residues are indicated to illustrate that Cys are far away from the head-to-tail interaction interface. (B) A proposed hypothetical model indicating one of the possible modes of IAA3 redox-dependent multimerization. The model illustrates how cys 112 and cys169 residues of IAA3 in one IAA3 PB1-IAA3 PB1 homodimer unit can come in close proximity to cys 112 and cys169 residues of a second IAA3 PB1-IAA3 PB1 homodimer unit to form disulphide bonds, if one unit is rotated freely by 180 degrees. Redox dependent
1058 multimerization pattern of (C) IAA3-WT and (D) IAA3-4C/S expressed under the control of
1059 the IAA3 promoter in the *iaa3* mutant background. (E) IAA3-WT cannot form redox
1060 dependent multimers in *rbohef* mutant background. IAA3-WT expressed under the control of
1061 the endogenous promoter in the *rbohef* mutant background does not form redox dependent
1062 multimers and is primarily monomeric. All immunoprecipitation and elution steps were
1063 performed in the absence of reducing agents. IP: immunoprecipitation, IB: immunoblotting.
1064 Three independent experiments were performed with similar results.

1065

1066 **Fig.4.** IAA3 regulates xerobranching by sensing changes in cellular redox status via
1067 specific cysteine residues (A-B) Representative images (A) and bar graph (B) of agar-based
1068 air gap assay showing transgenic lines expressing IAA3-4C/S under the control of the *IAA3*
1069 promoter in the *iaa3* mutant background cannot rescue the xerobranching defect of *iaa3* plants
1070 in comparison to IAA3-WT. Statistical significance for the bar graphs in (B) was analysed by
1071 one-way ANOVA followed by post-hoc Tukey HSD test at p<0.05. Different letters indicate
1072 statistically significant. Yellow arrows in (A) highlight the lateral roots that develop in air-gap
1073 and the red arrowheads in (A) denotes the zone taken in control moist agar plates that
1074 correspond to the 5mm air-gap in the test samples undergoing a xerobranching stimulus.
1075 Lateral root numbers are scored corresponding to the zones in (A) for a population of seedlings
1076 denoted as “n”. At least three biological replicate experiments were performed with 20-25
1077 seedlings per replicate. (C) Redox dependent multimerization pattern of IAA3-WT and IAA3-
1078 4C/S expressed under the control of the IAA3 promoter in control and air-gap conditions.
1079 IP:anti HA and IB: anti HA denotations are the same as described in Figure 3.

1080

1081 **Fig. 5.** IAA3 multimerization enhances interactions with transcriptional regulator TPL
1082 to repress downstream targets. (A) Representative images highlighting the difference in the
number of emerged lateral roots between pIAA3::shy2-2-WT and pIAA3::shy2-2 4C/S lines
expressing the respective dominant gain of function mutant forms in the iaa3 mutant
background. (B) One-way ANOVA analysis followed by post-hoc Tukey HSD test at p<0.05
for (i) root length, (ii) number of emerged lateral roots /plant (iii) lateral root density between
WT Col-0, iaa3, IAA3::shy2-2-WT/iaa3 and pIAA3::shy2-2 4C/S/iaa3 lines. Different letters
indicate statistically significant. (C) CO-IPs indicate that IAA3-4C/S interacts with
TPL(TOPLESS) at significantly lower levels in comparison to that of WT-IAA3. (D,E) Mass
spectrometry-based quantification of the peptides of TPL protein interacting with IAA3-WT
and IAA3-4C/S by Area under the curve method. Transgenics expressing IAA3-WT and IAA3-
4C/S driven by the IAA3 promoter in the iaa3 mutant background was used. (F) Expression
profile of LBD16 as a representative downstream target of IAA3 without exogeneous auxin
treatment. qRT PCR analysis was done for three biological replicates by absolute quantification
method. Statistical significance was analysed by one-way ANOVA followed by post-hoc
Tukey HSD test at p<0.05. Different letters indicate statistically significant. (G) ChIP-qPCR
analysis of WT-IAA3 and 4C/S-IAA3 occupancy at the LBD16 promoter AuxRE sites. The
AtGYPSY (AT4G07700) retrotransposon was used as an internal negative reference control and
untransformed WT Col-0 plants were used as a refernce control for non- specific antibody
binding.ChIP experiments were repeated three times with similar results. Statistical
significance was analysed by one-way ANOVA followed by post-hoc Tukey HSD test at
p<0.05. Different letters indicate statistically significant.

1103

1104

1105

1106

1107

1108

1109

1110

**Fig.0. Model of the proposed mechanism for ROS modulated IAA3 multimerization in**
**mediating repression of lateral root development during Xerobranching.** Redox dependent
multimerization of WT-IAA3 results in the formation of a specific pattern of higher order
1110 multimers, where they are recruited at target promoter sites like LBD16 through their
1111 interaction with ARFs like ARF7, via the SUMO-SIM interaction. IAA3-WT multimers
1112 provide an increased interface for interaction with TOPLESS (TPL), leading to robust
1113 repression of auxin dependant gene expression during Xerobranching in the air gap (double
1114 headed red arrow) curtailing lateral root development. IAA3-4C/S mutant forms cannot form
1115 higher order multimers therefore lose ROS flux perception resulting in significantly reduced
1116 recruitment of TPL co-repressor molecules at the target locus, thereby leading to de-repression
1117 of gene expression promoting lateral root development.

1118

1119

1120

1121
<figure><img src="image_1.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_2.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_7.png" /></figure>
**Fig.1. Xerobranching response is dependent on ROS (A,B)** Agar-based air-gap assay system showing xerobranching response in *Arabidopsis thaliana* (air-gap ~5mm). Representative images (A) and bar graphs (B) of agar-based air-gap assay show xerobranching defects in *rboh* mutants compared to WT Col-0. The red arrowheads in (A) denote the 5 mm zones used for analysis of lateral roots in control moist agar plates and 5mm air-gap xerobranching plates. Lateral roots specifically growing in the air-gaps are marked with yellow arrows. Schematics of the air-gap plates are illustrated at the bottom panel for representation of the xerobranching phenotype. Grey rectangles indicate the agar interface encompassing the air-gaps, and lateral roots that initiate and emerge in the air-gap are marked with red arrows. Lateral root numbers are scored corresponding to the zones in (A) for a population of seedlings denoted as n in (B). Different letters denote statistical significance for one-way ANOVA tests followed by post-hoc Tukey HSD test at p<0.05. Xerobranching stimulus triggers ROS production in cytoplasm and nuclei of ROS sensor lines, where roGFP2-Orp1 oxidation (ratio of 405/488 nm fluorescence intensity) shows Hydrogen peroxide levels in cytoplasm (C and D) and nucleus (E and F). (C), (E) Ratio (405nm/488nm) images of cyt-roGFP2-Orp1 and nls-roGFP2-Orp1 roots. Color scale represents 405/488 nm ratios. Higher ratios indicate higher H₂O₂ levels. (D), (F) corresponds to boxplots that reveal the dynamic changes in the endogenous ROS levels as root tips grow across an air-gap and after recovery. n denotes the number of seedlings. At least three biological replicate experiments were performed. (G) roGFP2-Orp1 shows increased oxidation as soon as root tips grows 1mm across an air-gap in comparison to nls-ABACUS2-400n that peaks as the root tips traverses across 4-5mm of the air-gap. Error bars represent standard error. ns, not significant, \*P \le 0.05; \*\*P \le 0.01; \*\*\*P \le 0.001.
<figure><img src="image_8.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 2. Xerobranching response involves ROS accumulation in inner root tissues and is regulated by the AUX/IAA repressor IAA3 (A) Cross-sectional images of nls-roGFP2-Orp1 reveal increased ROS accumulation in the inner tissues of roots growing across the air-gap. (B) Agar-based air gap assay showing xerobranching defect in the CRISPR/Cas9 knockout line of *Arabidopsis* IAA3 (referred as *iaa3*) mutants compared to WT Col-0. (C) Frequency plots showing the percentage of WT Col-0 and *iaa3* mutant plants that develop lateral roots (LR) across an air gap within a given population of plants. One-way ANOVA analysis between WT Col-0 and the *iaa3* mutant was performed. ns, not significant, *P* ≤ 0.05; **P**≤ 0.01. n denotes the number of individual seedlings used in the study to define the population of plants. At least three biological replicate experiments were performed with 20-25 seedlings per replicate.</figcaption></figure>
A

<figure><img src="image_12.png" /></figure>

B

<figure><img src="image_13.png" /></figure>

C

pIAA3::HA-IAA3-WT

<figure><img src="image_14.png" /></figure>

D

pIAA3::HA-IAA3-4C/S

<figure><img src="image_15.png" /></figure>

E

<figure><img src="image_16.png" /><figcaption>**Fig. 3.** Specific cysteine residues orchestrate IAA3 redox-dependent multimerization driven by RBOHs in planta (A) Model of the IAA3 PB1 dimer (generated by AlphaFold), where one monomer is shown in blue and the other in yellow. The mutated Cys residues are indicated in light blue and orange, respectively. On the right, a zoom on the PB1 head to tail interaction interface is shown, where both the residues involved in these interactions and the</figcaption></figure>
1163 position of the Cys residues are indicated to illustrate that Cys are far away from the head-to-
1164 tail interaction interface. (B) A proposed hypothetical model indicating one of the possible
1165 modes of IAA3 redox-dependent multimerization. The model illustrates how cys 112 and
1166 cys169 residues of IAA3 in one IAA3 PB1-IAA3 PB1 homodimer unit can come in close
1167 proximity to cys 112 and cys169 residues of a second IAA3 PB1-IAA3 PB1 homodimer unit
1168 to form disulphide bonds, if one unit is rotated freely by 180 degrees. Redox dependent
1169 multimerization pattern of (C) IAA3-WT and (D) IAA3-4C/S expressed under the control of
1170 the IAA3 promoter in the *iaa3* mutant background. (E) IAA3-WT cannot form redox
1171 dependent multimers in *rbohe/f* mutant background. IAA3-WT expressed under the control of
1172 the endogenous promoter in the *rbohe/f* mutant background does not form redox dependent
1173 multimers and is primarily monomeric. All immunoprecipitation and elution steps were
1174 performed in the absence of reducing agents. IP: immunoprecipitation, IB: immunoblotting.
1175 Three independent experiments were performed with similar results.

1176
<figure><img src="image_17.png" /><figcaption>**Fig.4.** IAA3 regulates xerobranching by sensing changes in cellular redox status via specific cysteine residues (A-B) Representative images (A) and bar graph (B) of agar-based air gap assay showing transgenic lines expressing IAA3-4C/S under the control of the *IAA3* promoter in the *iaa3* mutant background cannot rescue the xerobranching defect of *iaa3* plants in comparison to IAA3-WT. Statistical significance for the bar graphs in (B) was analysed by one-way ANOVA followed by post-hoc Tukey HSD test at p<0.05. Different letters indicate</figcaption></figure>

1177

1178

1179

1180

1181

1182

1183
1184 statistically significant. Yellow arrows in (A) highlight the lateral roots that develop in air-gap

1185 and the red arrowheads in (A) denotes the zone taken in control moist agar plates that

1186 correspond to the 5mm air-gap in the test samples undergoing a xerobranching stimulus.

1187 Lateral root numbers are scored corresponding to the zones in (A) for a population of seedlings

1188 denoted as “n”. At least three biological replicate experiments were performed with 20-25

1189 seedlings per replicate. (C) Redox dependent multimerization pattern of IAA3-WT and IAA3-

1190 4C/S expressed under the control of the IAA3 promoter in control and air-gap conditions.

1191 IP:anti HA and IB: anti HA denotations are the same as described in Figure 3.

1192

1193

1194
<figure><img src="image_18.png" /><figcaption>ii)</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>iii)</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>iv)</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>v)</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>vi)</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>B(i)</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>ii)</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>iii)</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_32.png" /></figure>
**Fig. 5.** IAA3 multimerization enhances interactions with transcriptional regulator TPL to repress downstream targets. (A) Representative images highlighting the difference in the number of emerged lateral roots between pIAA3::shy2-2-WT and pIAA3::shy2-2 4C/S lines expressing the respective dominant gain of function mutant forms in the iaa3 mutant background. (B) One-way ANOVA analysis followed by post-hoc Tukey HSD test at p<0.05 for (i) root length, (ii) number of emerged lateral roots/plant (iii) lateral root density between WT Col-0, *iaa3*, IAA3::shy2-2-WT/iaa3 and pIAA3::shy2-2 4C/S/iaa3 lines. Different letters indicate statistically significant. (C) CO-IPs indicate that IAA3-4C/S interacts with TPL(TOPLESS) at significantly lower levels in comparison to that of WT-IAA3. (D,E) Mass spectrometry-based quantification of the peptides of TPL protein interacting with IAA3-WT and IAA3-4C/S by Area under the curve method. Transgenics expressing IAA3-WT and IAA3-4C/S driven by the IAA3 promoter in the *iaa3* mutant background was used. (F) Expression profile of LBD16 as a representative downstream target of IAA3 without exogeneous auxin treatment. qRT PCR analysis was done for three biological replicates by absolute quantification method. Statistical significance was analysed by one-way ANOVA followed by post-hoc Tukey HSD test at p<0.05. Different letters indicate statistically significant. (G) ChIP-qPCR analysis of WT-IAA3 and 4C/S-IAA3 occupancy at the *LBD16* promoter AuxRE sites. The *AtGYPSY* (AT4G07700) retrotransposon was used as an internal negative reference control and untransformed WT Col-0 plants were used as a refernce control for non- specific antibody binding.ChIP experiments were repeated three times with similar results. Statistical significance was analysed by one-way ANOVA followed by post-hoc Tukey HSD test at p<0.05. Different letters indicate statistically significant.

1219
1220
1221
1222
<figure><img src="image_33.png" /><figcaption>**Fig.0.** Model of the proposed mechanism for ROS modulated IAA3 multimerization in mediating repression of lateral root development during Xerobranching. Redox dependent multimerization of WT-IAA3 results in the formation of a specific pattern of higher order multimers, where they are recruited at target promoter sites like LBD16 through their interaction with ARFs like ARF7, via the SUMO-SIM interaction. IAA3-WT multimers provide an increased interface for interaction with TOPLESS (TPL), leading to robust repression of auxin dependant gene expression during Xerobranching in the air gap (double headed red arrow) curtailing lateral root development. IAA3-4C/S mutant forms cannot form higher order multimers therefore lose ROS flux perception resulting in significantly reduced recruitment of TPL co-repressor molecules at the target locus, thereby leading to de-repression of gene expression promoting lateral root development.</figcaption></figure>

1223

1224

1225

1226

1227

1228

1229

1230

1231

1232

1233

1234

1235

1236

1237

1238

1239
Supplementary Materials for

Redox regulated Aux/IAA multimerization modulates auxin responses

Dipan Roy¹†*, Poonam Mehra²†*, Lisa Clark¹, Vaishnavi Mukkawar¹, Kevin

Bellande³,⁴, Raquel Martin-Arevalillo⁵,⁶, Srayan Ghosh¹, Kishor D. Ingole⁸, Prakash

Kumar Bhagat¹, Adrian Brown¹, Kawinnat Sue ob⁷, Andrew Jones⁷, Joop EM

Vermeer³, Teva Vernoux⁵, Kathryn Lilley⁸, Phil Mullineaux⁹, Ulrike Bechtold¹,

Malcolm J. Bennett² and Ari Sadanandom¹*.

¹Department of Biosciences, Durham University, Durham, DH1 3LE, United Kingdom

²Plant & Crop Sciences, School of Biosciences, University of Nottingham, LE12 5RD, United Kingdom

³Laboratory of Plant Molecular and Cellular Biology, University of Neuchâtel, Rue Emile Argand 11,
2000, Neuchâtel, Switzerland-IPSiM

⁴University of Montpellier, CNRS, INRAE, Institut Agro, 34060, Montpellier, France

⁵Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, CNRS, INRAE,
F-69342, Lyon, France

⁶Laboratoire de Recherche en Sciences Végétales, Université Toulouse, CNRS, INP, Castanet-Tolosan,
France

⁷Institute of Systems, Molecular and Integrative Biology, Liverpool, L69 3BX, United Kingdom

⁸Department of Biochemistry, University of Cambridge, Cambridge, CB2 1RX, United Kingdom

⁹School of Life Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, United Kingdom
1264 † These authors contributed equally to the work

1265 * To whom correspondence should be addressed

1266 **Corresponding authors:** ari.sadanandom@durham.ac.uk, dipan.roy@duham.ac.uk,

1267 p.mehra@nottingham.ac.uk

1268 **The PDF file includes:**

1269

1270 Figs. S1 to S22

1271 Tables S1 to S3

1272 References (55-64)

1273 **Other Supplementary Material for this manuscript includes the following:**

1274 MDAR Reproducibility Checklist

1275

1276

1277

1278

1279

1280

1281

1282

1283

1284

1285

1286

1287

1288

1289

1290

1291

1292

1293

1294
A

<figure><img src="image_34.png" /></figure>

B

Control

Air-gap

<figure><img src="image_35.png" /></figure>

WT Col-0

C

Control

Air-gap

<figure><img src="image_36.png" /></figure>

rbohb/f

D

Control

Air-gap

<figure><img src="image_37.png" /></figure>

rbohe/f

E

<figure><img src="image_38.png" /></figure>
1296

1297

Fig. S1. rboh b/f and rboh e/f mutants are impaired in xerobranching response. (A)

1298

1299

1300

1301

1302

1303

1304

1305

1306

1307

1308

1309

1310

Frequency plots showing the percentage of WT Col-0 and rboh mutant plants that develop lateral roots (LR) across an air gap within a given population of plants. One-way ANOVA analysis between WT Col-0 and the respective rboh mutants was performed. ns, not significant, *P ≤ 0.05; **P≤ 0.01. n denotes the number of individual seedlings used in the study to define the population of plants. At least three biological replicate experiments were performed with 35-50 seedlings per replicate. A significant fraction of *Arabidopsis rboh* mutants (> 20% for rbohb/f and rbohe/f) continue to branch when growing across an air-gap in contrast to WT Col-0. HPF staining reveals a higher accumulation of ROS in the air gap (highlighted by a white line) in (B) WT Col-0. In contrast, (C) rhohb/f and (D) rbohe/f exhibit limited ROS accumulation, as indicated by the reduced staining in their respective air gaps. (E) Quantification data of the Fluorescence intensity of the HPF staining normalized to the area corresponding to the zone of the air gap. ‘n’ denotes the number of seedlings. ns, not significant, *P ≤ 0.05; ***P≤ 0.001, ****P ≤ 0.0001.

1311

1312

1313

1314

1315

1316

1317

1318

1319

1320

1321

1322
<figure><img src="image_39.png" /><figcaption>Fig. S2. RBOHB, RBOHE and RBOHF are transcriptionally induced by xerobranching stimulus. qRT PCR analysis of the expression of RBOH genes in control and air-gap conditions using the 2^Δ-Ct^ based quantification method, where ΔCt is Ct<sub>RBOH</sub> - Ct<sub>Actin</sub>. The dots in the box-plots represent individual 2^Δ-Ct^ values. (A) RBOHB (B) RBOHC (C) RBOHD (D) RBOHE (E) RBOHF. The y-axis denotes the level of RBOH expression after normalization to Actin. The experiments were performed with four independent biological replicates. Statistical significance was assessed by Student's t test at p<0.05. **P ≤ 0.01; ***P≤ 0.005; ****.</figcaption></figure>
<figure><img src="image_40.png" /><figcaption>**A**</figcaption></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /><figcaption>**B**</figcaption></figure>

<figure><img src="image_46.png" /><figcaption>**C**</figcaption></figure>

<figure><img src="image_47.png" /><figcaption>**D**</figcaption></figure>

<figure><img src="image_48.png" /><figcaption>**E**</figcaption></figure>

<figure><img src="image_49.png" /><figcaption>**F**</figcaption></figure>

<figure><img src="image_50.png" /><figcaption>**G**</figcaption></figure>

<figure><img src="image_51.png" /></figure>

**Fig. S4.** Aux/IAAs can sub-functionalize via their varying propensities to multimerize in a redox regulated manner (A) Redox modulated cysteine disulphide linkage dependent multimerization of candidate Aux/IAAs (i)IAA3 (ii) IAA28 (iii) IAA13 (iv) IAA19 (v) IAA14 (vi) MBP (Maltose binding protein) tag control. Assays determining the varying sensitivities towards Redox dependent multimerization of (B) IAA3 (C) IAA28 (D) IAA13 (E) IAA19 (F) IAA14 and (G) MBP. Full length native protein was treated with 7.5mM DTT alone (lane3), or with 10mM Diamide after treatment of the full-length protein with 7.5mM DTT (lane4), or
1348 with 10-mM diamide after treatment of the full-length protein first with 7.5mM DTT, followed
1349 by treatment with gradually increasing concentrations of NEM (N-ethyl maleimide) ranging
1350 from 1mM to 10mM NEM (lane5-8). Treatment of the different full-length Aux/IAAs with
1351 10mM diamide alone (lane 9) is used to assess the sensitivity of the Aux/IAAs to an exclusively
1352 oxidizing environment. Electrophoresis of the treated protein samples were done in SDS PAGE
1353 gels in the absence of β-Me except for the samples loaded in the indicated lane as experimental
1354 control. Direction of the arrowhead indicates the sequence in which the redox reagents are
1355 added in the assay. Aux/IAAs exhibiting higher order multimers even at higher threshold
1356 concentrations of NEM indicate that these Aux/IAAs have a higher frequency of cysteine
1357 residues that are likely to be engaged in disulphide linkages.

1358

1359
<figure><img src="image_52.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_53.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_54.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_55.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_56.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_57.png" /></figure>
<figure><img src="image_58.png" /></figure>
<figure><img src="image_59.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_60.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_61.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_62.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_63.png" /></figure>
Fig. S6. AlphaFold predicted structures of the PB1 domain of the biochemically examined Aux/IAAs with varying propensities of redox dependent multimerization (A)

Sequence alignment of Aux/IAAs highlighting the conservation of the cysteine residues. The position of the conserved cysteine residue is highlighted with a red star. Modelled structures of the dimers of the PB1 domain of (B) IAA3 (C) IAA19 and (D) IAA14 predicted using AlphaFold. The cysteine residues are highlighted in orange and a 90° rotation of the structure is shown immediately below. The 90° rotations of the structure of the dimer pair of the Aux/IAAs indicate that the conserved cysteine residue 171 of IAA3 (cysteine residue 173 for IAA19 and cysteine residue 201 for IAA14) is inaccessible to the solution to potentially form disulphide linkages. (E) Overlap of the structure of the PB1 domain from the three examined Aux/IAAs (IAA3, IAA19 and IAA14), highlighting that there is minimal secondary structural variation in the PB1 domain of these Aux/IAAs.
<figure><img src="image_64.png" /><figcaption>A</figcaption></figure>

protospacer 1 ggaaaaaaagctaagcttgt

protospacer 2 gaatctgagcatgaggtgca

protospacer 3 acatttgaattttaccgatg

<figure><img src="image_65.png" /><figcaption>D i)</figcaption></figure>

<figure><img src="image_66.png" /><figcaption>ii)</figcaption></figure>

<figure><img src="image_67.png" /><figcaption>iii)</figcaption></figure>

<figure><img src="image_68.png" /><figcaption>E i)</figcaption></figure>

<figure><img src="image_69.png" /><figcaption>ii)</figcaption></figure>

<figure><img src="image_70.png" /><figcaption>iii)</figcaption></figure>

<figure><img src="image_71.png" /><figcaption>F i)</figcaption></figure>

<figure><img src="image_72.png" /><figcaption>ii)</figcaption></figure>

<figure><img src="image_73.png" /><figcaption>iii)</figcaption></figure>

<figure><img src="image_74.png" /></figure>
**Fig. S7. IAA3 CRISPR cas9 mutant fails to repress lateral root emergence (A) Schematic**
representing CRISPR cas9 mediated deletion at the IAA3 locus. *iaa3* deletion mutant lines
(two independent alleles C1 and C2) are affected in lateral root development in comparison to
WT Col-0 as indicated by **(B)** Representative images and **(C)** stereomicroscope images. **(D)**
Comparative analysis of the differences in (i) root length, (ii) number of emerged lateral roots
per plant, (iii) lateral root density between WT Col-0 and *iaa3* lines C1 and C2. *P* ≤ 0.05;
***P* ≤ 0.0001; ns, non-significant changes. **(E-F)** Welch's unequal variance t-test based
Estimation plots showing the difference in the means of (i) root length, (ii) number of emerged
lateral roots per plant, and (iii) lateral root density between WT Col-0 and *iaa3* lines.
<figure><img src="image_75.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_76.png" /><figcaption>Fig. S8. Response of loss-of-function mutants of IAA genes to xerobranching conditions.</figcaption></figure>

(A) Phenotypes of WT Col-0, *iaa28-2*, *iaa3*, *Ler* and *shy2-31* under control and air-gap conditions (B) Frequency plot of knock-out mutants reveals *iaa3* and *shy2-31* loss-of-function mutants are defective in xerobranching response as compared to their wild-type controls, WT
1436

Col-0 and Ler, respectively. Other IAA knock-out (iaa28-2) behave similar to wildtype (WT

1437

Col-0) in air-gap conditions. Yellow arrows mark LRs (Lateral Roots) in air-gap

1438

<figure><img src="image_77.png" /></figure>

<figure><img src="image_78.png" /></figure>

1439

<caption>Fig. S9. Modelled structures of IAA3 PB1, IAA3 PB1 dimer and ARF7-IAA3 PB1</caption>

1440

**complex. (A)** Alignment of Aux/IAAs and ARFs with known crystal structures of their PB1

1441

domain (B-C). The structure of the monomer IAA3 PB1 (generated by AlphaFold) is shown in

1442

blue overlapped to the structures of PsIAA4 (2M1M, Dinesh 2015) in sand (C) and AtIAA17
1443 (2MUK, Han 2014) in light blue (D). For each comparison, a zoom is shown on the right
1444 illustrating the residues for the basic and acidic interfaces. These residues are conserved
1445 between the three proteins and structure model predicts that their position is also conserved.
1446 (E) (Model of the ARF7-IAA3 PB1 complex (generated by AlphaFold), where the Cys residues
1447 are shown. On the right, superposition of this model with the ARF5-IAA17 structure (6L5K,
1448 Kim 2020, light pink-blue). Please note that in this structure a helix of the IAA17 PB1 domain
1449 is missing compared to the structure presented in panel C
1450
1451
1452
1453
1454
1455
1456
1457
1458
1459
1460
1461
1462
1463
1464
1465
1466
A

<figure><img src="image_79.png" /></figure>

B

<figure><img src="image_80.png" /></figure>

C

<figure><img src="image_81.png" /></figure>

**Fig. S10.** Proposed hypothetical models indicating the possible modes of IAA3 redox-dependent multimerization. The models illustrate that if one IAA3 PB1-IAA3 PB1 homodimer unit can rotate freely then it can form cysteine disulphide linkages with cysteine residues in close proximity from a second IAA3 PB1-IAA3 PB1 homodimer unit. This may result in the formation of a more stable tetramer. The possible disulphide bonds include : ( A ) cys 169-cys169 and cys112-cys 112 (B) cys 169- cys187 and (C) cys 187-cys187
<figure><img src="image_82.png" /><figcaption>Fig. S11. Key cysteine residues drive the redox dependent multimerization of IAA3 (A)</figcaption></figure>

Identification of the key cysteine residues responsible for redox dependent IAA3 multimerization, where the impact on the multimerization of IAA3 due to (i, ii) single or double cysteine to serine substitutions and (iii) Lysine 96 to alanine and Aspartic acid 155 to Alanine substitution mutations is assessed by the redox dependent multimerization assays described previously. Note that the WT- IAA3 control for this experiment is represented in main fig. S4 A (i). **(B)** Impact on the multimerization of IAA3 due to combinatorial cysteine to serine substitutions (4C/S-IAA3) **(C)** Reoxidation of DTT reduced 4C/S-IAA3 by the thiol specific oxidizing agent, diamide, results in an alternate pattern of multimerization compared to WT-IAA3. Major fraction of 4C/S-IAA3 is monomeric even in the presence of 15mM diamide. + or - β-Me indicates the presence or absence of β-mercaptoethanol in Lamelli dye.

1476

1477

1478

1479

1480

1481

1482

1483

1484

1485

1486
<figure><img src="image_83.png" /><figcaption>B i)</figcaption></figure>

<figure><img src="image_84.png" /></figure>

p/AA3::IAA3-WT-4/iaa3

<figure><img src="image_85.png" /><figcaption>ii)</figcaption></figure>

p/AA3::IAA3-WT-6/iaa3

<figure><img src="image_86.png" /><figcaption>iii)</figcaption></figure>

p/AA3::IAA3-4C/S-10/iaa3

<figure><img src="image_87.png" /><figcaption>iv)</figcaption></figure>

p/AA3::IAA3-4C/S-5/iaa3

<figure><img src="image_88.png" /><figcaption>C</figcaption></figure>

WT Col-0

<figure><img src="image_89.png" /></figure>

iaa3-C1

<figure><img src="image_90.png" /></figure>

iaa3-C2

<figure><img src="image_91.png" /></figure>

p/AA3::IAA3-WT-4/iaa3

<figure><img src="image_92.png" /></figure>

p/AA3::IAA3-WT-6/iaa3

<figure><img src="image_93.png" /></figure>

p/AA3::IAA3-4C/S-10/iaa3

<figure><img src="image_94.png" /></figure>

p/AA3::IAA3-4C/S-5/iaa3

<figure><img src="image_95.png" /><figcaption>D i)</figcaption></figure>

p/AA3::IAA3 WT 4/iaa3
p/AA3::IAA3 WT 6/iaa3
p/AA3::IAA3 4C/S 10/iaa3
p/AA3::IAA3 4C/S 5/iaa3

<figure><img src="image_96.png" /><figcaption>ii)</figcaption></figure>

p/AA3::IAA3 WT 4/iaa3
p/AA3::IAA3 WT 6/iaa3
p/AA3::IAA3 4C/S 10/iaa3
p/AA3::IAA3 4C/S 5/iaa3

<figure><img src="image_97.png" /><figcaption>iii)</figcaption></figure>

p/AA3::IAA3 WT 4/iaa3
p/AA3::IAA3 WT 6/iaa3
p/AA3::IAA3 4C/S 10/iaa3
p/AA3::IAA3 4C/S 5/iaa3

<figure><img src="image_98.png" /><figcaption>E i)</figcaption></figure>

Root length_Estimation Plot

<figure><img src="image_99.png" /><figcaption>ii)</figcaption></figure>

Emerged lateral root/plant_Estimation Plot

<figure><img src="image_100.png" /><figcaption>iii)</figcaption></figure>

Lateral root density_Estimation Plot
Fig. S12. Endogeneous promoter driven IAA3 requires key cysteines involved in multimerization to repress lateral root emergence (A) Analysis of the protein abundance levels of HA-tagged WT-IAA3 and 4C/S- IAA3 expressed in the *iaa3* mutant background under the control of the native IAA3 promoter. Protein extracts from transgenic seedlings with anti-HA antibody (Roche 3F/10). Histone H3 and Rubisco were used as loading controls. (B) Representative images and (C) Stereomicroscope images highlighting the difference in the number of emerged lateral roots per plant between pIAA3:: WT -IAA3 /iaa3 and pIAA3:: 4C/S- IAA3/iaa3 lines. Analysis was performed with two independent transgenic lines. WT Col-0 and two independent alleles C1 and C2 of the *iaa3* mutant lines were used as reference controls (as indicated in panel B-F of this figure). (D) One-way ANOVA analysis followed by post-hoc Tukey HSD test at p<0.05 of the differences in (i) root length,(ii) number of emerged lateral roots per plant,(iii) lateral root density between WT Col-0, *iaa3*, pIAA3::IAA3-WT/iaa3 and pIAA3::IAA3-4C/S/iaa3 lines. Different letters indicate statistically significant. (E) Welch's t-test (unequal variance t-test) based Estimation plots showing the difference in the means of (i) root length,(ii) number of emerged lateral roots per plant, and (iii) lateral root density between transgenic lines expressing WT -IAA3 and 4C/S -IAA3 under the control of IAA3 promoter.
A

pIAA3::HA-IAA3-WT

<figure><img src="image_101.png" /></figure>

B

pIAA3::HA-IAA3-4C/S

<figure><img src="image_102.png" /></figure>

Fig.S13. Exogenous H₂O₂ application induces IAA3 multimerization. Redox dependent multimerization pattern of (A) WT-IAA3 and (B) 4C/S-IAA3 expressed under the control of the IAA3 promoter in control conditions and when subjected to 1mM Hydrogen peroxide treatment. IP:anti HA indicates that the immunoprecipitation experiments were carried out with anti-HA magnetic microbeads (Miltenyi Biotech) and IB:anti HA indicates that the immunoprecipitated proteins were probed with anti HA antibody (Roche 3F/10) by standard immunoblotting techniques.

1535

1536
<figure><img src="image_103.png" /><figcaption>iv</figcaption></figure>

pCASP1::IAA3-WT-2/iaa3

<figure><img src="image_104.png" /><figcaption>B i)</figcaption></figure>

<figure><img src="image_105.png" /><figcaption>ii)</figcaption></figure>

<figure><img src="image_106.png" /><figcaption>v)</figcaption></figure>

<figure><img src="image_107.png" /><figcaption>iii)</figcaption></figure>

<figure><img src="image_108.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_109.png" /></figure>

pCASP1::IAA3-4C/S-12/iaa3

<figure><img src="image_110.png" /><figcaption>ii)</figcaption></figure>

<figure><img src="image_111.png" /><figcaption>iii)</figcaption></figure>

<figure><img src="image_112.png" /><figcaption>E i)</figcaption></figure>

<figure><img src="image_113.png" /><figcaption>ii)</figcaption></figure>

<figure><img src="image_114.png" /><figcaption>iii)</figcaption></figure>

<figure><img src="image_115.png" /></figure>
Fig. S14. CASP1 promoter driven IAA3 requires key cysteines involved in multimerization to repress lateral root emergence (A) analysis of the protein abundance levels of HA-tagged IAA3 WT and IAA3 4C/S mutant transgenic lines expressed in the *iaa3* mutant background under the control of the CASP1 promoter. Lines having comparatively equal protein abundance were taken forward for subsequent analysis. (B) Representative and (C) stereomicroscope images highlighting the difference in the number of emerged lateral roots per plant between pCASP1::IAA3-WT and pCASP1::IAA3-4C/S lines. Analysis was performed with two independent transgenic lines each of pCASP1::IAA3-WT/iaa3 and pCASP1::IAA3-4C/S/iaa3 that were showing equivalent expression of IAA3 WT and IAA3 4C/S. Transgenic lines were generated by intogressing constructs expressing HA-IAA3-WT and HA-IAA3 4C/S in the *iaa3* mutant background. WT Col-0 and two independent alleles C1 and C2 of the *iaa3* lines were used as reference controls (D) One-way ANOVA analysis followed by post-hoc Tukey HSD test at p<0.05 for (i) root length, (ii) number of emerged lateral roots per plant, and(iii) lateral root density between WT Col-0, *iaa3* mutants (allele C1 and C2), and two independent lines expressing IAA3-WT and IAA3-4C/S under the control of the CASP1 promoter in the *iaa3* mutant background. Different letters indicate statistically significant. (e) Welch's t-test (unequal variance t-test) based Estimation plots showing the difference in the means of (i) root length,(ii) number of emerged lateral roots per plant, and (iii) lateral root density between transgenic lines expressing IAA3-WT and IAA3-4C/S under the control of the CASP1 promoter.
<figure><img src="image_116.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_117.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_118.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_119.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_120.png" /></figure>
Fig. S15. Redox dependent multimerization rather than stability is the key driver of IAA3 mediated auxin responses. (A) Auxin (1-NAA) induced degradation of HA-IAA3 WT expressed under the control of 35S promoter. Transgenic Arabidopsis thaliana seedlings expressing HA-IAA3 WT under the control of 35S promoter were grown in Gamborg B5 media liquid cultures and HA-IAA3 WT expressing transgenic seedlings were treated with increasing concentrations of 1-NAA for three hours post treatment with cycloheximide (CHX) and the degradation kinetics were assessed by standard Western Blotting techniques. (B) Auxin (1-NAA) induced degradation of HA-IAA3 4C/S expressed under the control of 35S promoter. Transgenic Arabidopsis thaliana seedlings expressing HA-IAA3 4C/S under the control of 35S promoter were grown in Gamborg B5 media liquid cultures and were treated with increasing concentrations of 1-NAA for three hours post treatment with cycloheximide and their degradation kinetics were assessed by standard Western Blotting techniques. Histone H3 was used as loading control (C) Quantification of the rate of degradation of IAA3-WT and IAA3 4C/S plotted as normalised signal intensity relative to histone H3. Three independent experiments were performed with similar results. Error bars represent standard error (SE). The results indicate that IAA3-WT and IAA3-4C/S have similar rates of auxin induced degradation (D) Stability assays of HA tagged IAA3 WT and IAA3-4C/S after introduction of the shy2-2 semidominant (P69S) gain of function mutation. Three-week-old seedlings of pIAA3:: IAA3-WTP69S and pIAA3:: IAA3-4C/SP69S were treated with 100µM cycloheximide(CHX) with or without 10µM NAA for 180 minutes. Total protein was isolated and the relative abundance of (i)IAA3-WTP69S and (ii) IAA3-4C/SP69S was assessed by standard Western blotting techniques.
<figure><img src="image_121.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_122.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_123.png" /><figcaption>D i) ii)</figcaption></figure>

<figure><img src="image_124.png" /></figure>

<figure><img src="image_125.png" /></figure>

<figure><img src="image_126.png" /><figcaption>E i) ii) iii)</figcaption></figure>

<figure><img src="image_127.png" /></figure>

<figure><img src="image_128.png" /></figure>

<figure><img src="image_129.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_130.png" /></figure>
**Fig. S16.** CAMV 35S promoter driven IAA3 requires key cysteines involved in multimerization to repress lateral root emergence (A) Analysis of the protein abundance levels of HA-tagged IAA3 WT and IAA3 4C/S mutant transgenic lines expressed in the *iaa3* mutant background under the control of the 35S promoter. Lines having comparatively equal protein abundance were taken forward for subsequent analysis. (B) Representative and (C) stereomicroscope images highlighting the difference in the number of emerged lateral roots per plant between p35S:: IAA3-WT/iaa3 and p35S::IAA3-4C/S /iaa3 lines (D) One-way ANOVA analysis followed by post-hoc Tukey HSD test at p<0.05 of the differences in (i) root length,(ii) number of emerged lateral roots per plant, (iii) lateral root density between WT Col-0, iaa3, p35S::IAA3-WT/iaa3 and p35S::IAA3-4C/S /iaa3 lines. Different letters denote statistically significant. (E) Welch's t-test (unequal variance t-test) based Estimation plots showing the difference in the means of (i) root length,(ii) number of emerged lateral roots per plant, and (iii) lateral root density between transgenic lines expressing IAA3-WT and IAA3-4C/S under the control of 35S promoter in the iaa3 mutant background. Circles within the plots represent data for individual plants taken from at least two independent lines expressing equivalent levels of IAA3-WT and IAA3-4C/S. (F) Representative images highlighting the difference in the rosette diameter between p35S::IAA3-WT/ iaa3 and p35S::IAA3-4C/S /iaa3 lines.

1613
1614
1615
1616
1617
1618
1619
1620
1621
1622
1623
<figure><img src="image_131.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_132.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_133.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_134.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_135.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_136.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_137.png" /></figure>
Fig. S17. Redox regulated multimerization of IAA3 does not determine its capacity to interact with ARF7 or TIR1 Quadruple Cysteine to Serine substitutions does not impact the capacity of IAA3 to interact with ARF7 or TIR1, since both WT and 4C/S version of IAA3 remains able to interact with (A) ARF7-WT and (B) TIR1-WT. (C) ARF7 is SUMOylated in planta as indicated in transient assays in N. benthamiana leaves co-expressing ARF7 WT-GFP or ARF7 4K/R-GFP and SUMO-HA. The SIM (SUMO Interacting Motif) of IAA3 rather than its redox dependent multimerization status is a key determinant of its ability to interaction with ARF7 as both (D) exclusively multimeric form of IAA3 and (E) a heterogeneous population of monomeric and multimeric forms of IAA3 remains able to interact with SUMOylatable ARF7 WT through the SUMO-SIM interaction module. Exogeneous Hydrogen Peroxide treatment does not impact IAA3 WT or IAA3 4C/S interaction with (F) ARF7-WT or (G) TIR1-WT. Transient assays were performed in Nicotiana benthamiana leaves where agrobacterium cells expressing the respective constructs were infiltered in appropriate combinations. Two days post infiltration, the infiltered leaves were further treated with 10mM Hydrogen Peroxide for two hours and then harvested for further co-immunoprecipitation assays. Immunoprecipitation reactions were performed using anti-GFP beads and the levels of HA tagged IAA3 WT or IAA3-4C/S pulled down in the presence or absence of hydrogen peroxide were assessed by standard immunoblotting methods.

1643
1644
1645
1646
1647
1648
1649
1650
1651
1652
<figure><img src="image_138.png" /></figure>

<figure><img src="image_139.png" /></figure>

<figure><img src="image_140.png" /></figure>

<figure><img src="image_141.png" /></figure>

<figure><img src="image_142.png" /></figure>

<figure><img src="image_143.png" /></figure>

<figure><img src="image_144.png" /></figure>

<figure><img src="image_145.png" /></figure>

<figure><img src="image_146.png" /></figure>

<figure><img src="image_147.png" /></figure>

<figure><img src="image_148.png" /></figure>

<figure><img src="image_149.png" /></figure>

<figure><img src="image_150.png" /></figure>

<figure><img src="image_151.png" /></figure>

<figure><img src="image_152.png" /></figure>

<figure><img src="image_153.png" /></figure>

<figure><img src="image_154.png" /></figure>

<figure><img src="image_155.png" /></figure>
1654

1655

Fig. S18. Expression profile of downstream target genes of IAA3 without exogeneous auxin treatment in pIAA3::IAA3-WT/iaa3 and pIAA3::IAA3-4C/S/iaa3 transgenic lines.

1656

1657

qRT PCR analysis was done by absolute quantification method for at least three biological replicates of two independent transgenic lines each expressing equivalent levels of WT-IAA3 or 4C/S-IAA3 and their respective shy2-2 semi dominant gain of function mutant forms under the control of the native IAA3 promoter in the *iaa3* mutant background. WT Col-0 and *iaa3* mutant was used as reference controls.

1658

1659

1660

Statistical significance was analysed by one-way ANOVA followed by post-hoc Tukey HSD test at p<0.05. Different letters indicate statistically significant. (A, B) LBD29 (C, D) SAUR13 (E, F) SAUR15 (G,H) GH3.1 (I, J) GH3.3 and (K, L) GH3.5 (M, N) IAA1 (O, P) IAA5 (Q, R) IAA6

1661

1662

1663

1664

1665

1666

1667

1668

1669

1670

1671

1672

1673

1674

1675
<figure><img src="image_156.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_157.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_158.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_159.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_160.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_161.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_162.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_163.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_164.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_165.png" /><figcaption>K</figcaption></figure>

<figure><img src="image_166.png" /><figcaption>L</figcaption></figure>

<figure><img src="image_167.png" /><figcaption>M</figcaption></figure>

<figure><img src="image_168.png" /><figcaption>N</figcaption></figure>

<figure><img src="image_169.png" /><figcaption>O</figcaption></figure>

<figure><img src="image_170.png" /><figcaption>P</figcaption></figure>

<figure><img src="image_171.png" /><figcaption>Q</figcaption></figure>

<figure><img src="image_172.png" /><figcaption>R</figcaption></figure>

<figure><img src="image_173.png" /><figcaption>S</figcaption></figure>

<figure><img src="image_174.png" /><figcaption>T</figcaption></figure>

<figure><img src="image_175.png" /></figure>
1677

1678

1679

1680

1681

1682

1683

1684

1685

1686

Fig. S19. Expression profile of downstream target genes of IAA3 without exogeneous auxin treatment in (A-J) p35S::IAA3-WT/iaa3, p35S::IAA3-4C/S/iaa3 and (K-T) pCASP1::IAA3-WT/iaa3, pCASP1::IAA3-4C/S/iaa3 transgenic lines. qRT PCR analysis was done by absolute quantification method for at least three biological replicates of at least two independent transgenic lines each expressing equivalent amounts of WT-IAA3 or 4C/S-IAA3 under the control of the respective promoters in the iaa3 mutant background. (A, K) LBD16 (B, L) LBD29 (C, M) SAUR13 (D, N) SAUR15 (E, O) GH3.1 (F, P) GH3.3 and (G, Q) GH3.5 (H, R) IAA1 (I, S) IAA5 (J, T) IAA6. Statistical significance was assessed by Student's t test at $p<0.05$. $P \le 0.05$; ***$P \le 0.001$ ****$P \le 0.0001$

<figure><img src="image_176.png" /></figure>

<figure><img src="image_177.png" /></figure>

<figure><img src="image_178.png" /></figure>

<figure><img src="image_179.png" /></figure>

1687

1688

1689

1690

Fig. S20. Inducibility of LBD16 on exogeneous auxin (1-NAA) treatment in IAA3WT and IAA3-4C/S expressing lines. (A) Expression of LBD16 in WT Col-0, iaa3, p35S::IAA3-WT, p35S::IAA3-4C/S, pCASP1::IAA3WT and pCASP1::IAA3-4C/S lines. Comparative analysis
1691 of the inducibility of LBD16 between (B) WT Col-0 and *iaa3* (C) p35S::IAA3-WT and
1692 p35S::IAA3-4C/S (D) pCASP1::IAA3WT and pCASP1::IAA3-4C/S lines upon exogeneous
1693 auxin (1-NAA) treatment. Error bars indicate standard error mean for three independent
1694 replicates.

<figure><img src="image_180.png" /></figure>

1695 **Fig.S21. Air-gap condition does not result in LBD16 downregulation in iaa3 mutant or in**
1696 **pIAA3::IAA3-4C/S/iaa3.** Expression profile of LBD16 as a representative downstream target
1697 of IAA3 in control and air-gap conditions. qRT PCR analysis was done for three biological
1698 replicates by using absolute quantification method. Statistical significance was analysed by
1699 one-way ANOVA followed by post-hoc Tukey HSD test at p<0.05. Different letters indicate
1700 statistically significant.
<figure><img src="image_181.png" /></figure>

<figure><img src="image_182.png" /><figcaption>**Fig.S22.** Subcellular localization of pTPL:TPL-GFP. (A) Confocal images of *pTPL:TPL:GFP* showing nuclear localization of GFP tagged TPL protein in *Arabidopsis* root tips exposed to control and air-gap conditions. Cell boundaries were stained with propidium iodide (magenta). Bar =100 µm (B) Quantification of the changes in the fluorescence intensity of the GFP signal in elongation zone nuclei of pTPL::TPL-GFP lines in control and air-gap conditions.</figcaption></figure>

1702
1703
1704
1705
1706
1707
1708
1709
1710
1711
1712
1713
1714

1715

<caption>Table. S1. Identification of ROS responsive cysteines of WT-IAA3 by Mass spectrometry.</caption>

1716

Cysteine residues of the native protein that were not labelled by NEM were labelled with carbamidomethyl group post reduction with DTT.

1717

<table><thead><tr><td>Tryptic peptide sequence</td><td>NEM</td><td>Cysteine residue number</td></tr></thead><tbody><tr><td>LGLPGTDNVCEAK</td><td>Yes</td><td>23</td></tr><tr><td>RVSCCNNNNKR</td><td>Yes</td><td>31</td></tr><tr><td>RVSCCNNNNKR</td><td>No</td><td>32</td></tr><tr><td>(K)IDLSCYK</td><td>No</td><td>112</td></tr><tr><td>DGDWMLIDVPWEMFICTCK</td><td>No</td><td>169</td></tr><tr><td>DGDWMLIDVPWEMFICTCK</td><td>No</td><td>171</td></tr><tr><td>(K)GLGCGV</td><td>No</td><td>187</td></tr></tbody></table>

1718

1719

1720

1721

1722

1723

1724

1725

1726

1727
<table><caption>**Table. S2. DNA constructs used for this study.** List of constructs generated by either conventional restriction enzyme-based cloning or Gateway recombination cloning technologies for performing various experiments related to this study.</caption><thead><tr><td>Genes</td><td>Gene Constructs</td><td>Background Vectors</td><td>Experiments</td></tr></thead><tbody><tr><td>WT IAA3</td><td>MBP-WT IAA3</td><td>pMal c5X</td><td>In vitro redox assays</td></tr><tr><td>4C/S IAA3</td><td>MBP-4C/S IAA3</td><td>pMal c5X</td><td>In vitro redox assays</td></tr><tr><td>WT IAA28</td><td>MBP-WT IAA3</td><td>pMal c5X</td><td>In vitro redox assays</td></tr><tr><td>WT IAA13</td><td>MBP-WT IAA3</td><td>pMal c5X</td><td>In vitro redox assays</td></tr><tr><td>WT IAA14</td><td>MBP-WT IAA3</td><td>pMal c5X</td><td>In vitro redox assays</td></tr><tr><td>WT IAA19</td><td>MBP-WT IAA3</td><td>pMal c5X</td><td>In vitro redox assays</td></tr><tr><td>MBP</td><td>MBP</td><td>pMal c5X</td><td>Control for experiments</td></tr><tr><td>ARF7</td><td>ARF7-GFP</td><td>pEG 103</td><td>Co-IP and SUMO IP</td></tr><tr><td>ARF7 4K/R</td><td>ARF7<sup>4K/R</sup>-GFP</td><td>pEG 103</td><td>Co-IP and SUMO IP</td></tr><tr><td>WT IAA3</td><td>HA-WT IAA3</td><td>pEG 201</td><td>Co-IP and Redox IP</td></tr><tr><td>IAA3-SIM</td><td>HA-SIM IAA3</td><td>pEG 201</td><td>Co-IP</td></tr><tr><td>4C/S-IAA3</td><td>HA-4C/S IAA3</td><td>pEG 201</td><td>Co-IP and Redox IP</td></tr><tr><td>TIR1</td><td>GFP-TIR1</td><td>pEG 104</td><td>Co-IP</td></tr><tr><td>TPL</td><td>TPL-myc</td><td>pGWB18</td><td>Co-IP</td></tr><tr><td>NINJA</td><td>NINJA-myc</td><td>pGWB18</td><td>Co-IP</td></tr><tr><td>SUMO1</td><td>HA-SUMO1</td><td>pEG 201</td><td>SUMO IP</td></tr><tr><td>GFP</td><td>GFP</td><td>pEG103/pEG104</td><td>Control for experiments</td></tr><tr><td>WT IAA3</td><td>p35S::HA-WT IAA3</td><td>pEG 201</td><td>Generation of transgenic plants</td></tr><tr><td>WT IAA3</td><td>pCASP1::HA-WT IAA3</td><td>pEG 201</td><td>Generation of transgenic plants</td></tr><tr><td>WT IAA3</td><td>pIAA3::HA-WT IAA3</td><td>pEG 201</td><td>Generation of transgenic plants</td></tr><tr><td>4C/S IAA3</td><td>p35S::HA-4C/S IAA3</td><td>pEG 201</td><td>Generation of transgenic plants</td></tr><tr><td>4C/S IAA3</td><td>pCASP1::HA-4C/S IAA3</td><td>pEG 201</td><td>Generation of transgenic plants</td></tr><tr><td>4C/S IAA3</td><td>pIAA3::4C/S-WT IAA3</td><td>pEG 201</td><td>Generation of transgenic plants</td></tr></tbody></table>
1732 Table. S3. Primers used for this study. Variety of primers used for site-directed mutagenesis,

1733 Gateway recombination cloning technology, ChIP qPCR and qRT PCR.

<table><thead><tr><td>Gene</td><td>Forward primer</td><td>Reverse Primer</td><td>Experiment</td></tr></thead><tbody><tr><td>IAA1</td><td>GTCGGTGATGTTCCATGGGA</td><td>TGCCTCGACCAAAAGGTGTT</td><td>qRT PCR</td></tr><tr><td>IAA2</td><td>GTAAGAGACTCAGAATCATGAAGG</td><td>GTCAAACTCCGATCCGAATC</td><td>qRT PCR</td></tr><tr><td>IAA5</td><td>CGTGAAAGTGAGTGTAGATGGAG</td><td>GAACATCTCCAGCAAGCATCC</td><td>qRT PCR</td></tr><tr><td>IAA6</td><td>CTGTCTTGGCATAGGAGTGG</td><td>GAGACCAAAACCAGTTGCATC</td><td>qRT PCR</td></tr><tr><td>IAA19</td><td>GCTATGATGATCTAGCCTTTG</td><td>AACCTCTTGCATGACTCTAG</td><td>qRT PCR</td></tr><tr><td>LBD 16</td><td>GCTTTGGCTCTTAGAATGATG</td><td>TATTGATGATAGCATGTGAACAT</td><td>qRT PCR</td></tr><tr><td>LBD 29</td><td>GAGCTAGCAATGCTTCTAAG</td><td>CCAAGTCAGAGTAGATGGAG</td><td>qRT PCR</td></tr><tr><td>GH3.1</td><td>CCATGTTTTTTCCTTGGAGTTC</td><td>CACACAGAGATCCCACAG</td><td>qRT PCR</td></tr><tr><td>GH3.3</td><td>CGCATCGATCAATCAGTACAAGG</td><td>CAGACAAAGGAGGGACAGAG</td><td>qRT PCR</td></tr><tr><td>GH3.5</td><td>CTCTTTGAGCTAGAAGGTTTGGGAC</td><td>TTGGAGGCTCGAGAAAGAGTG</td><td>qRT PCR</td></tr><tr><td>SAUR 13</td><td>TTCTTGCAGTGTACGTTGGAG</td><td>GCTGAGAAGTGACGGTGAT</td><td>qRT PCR</td></tr><tr><td>SAUR 15</td><td>GTTAGCTAATTTCCCCTGGAGA</td><td>ATTCTGCCGTTTCACTGTAA</td><td>qRT PCR</td></tr><tr><td>RBOH B</td><td>CCGGGGATGATTACCTCAGC</td><td>AGGGAAGTTGACAAACCTTGGA</td><td>qRT PCR</td></tr><tr><td>RBOH C</td><td>CGGCAGGAGTTAGTGGTCTG</td><td>ATTGGTGTGGCTCCAATCCC</td><td>qRT PCR</td></tr><tr><td>RBOH D</td><td>ACTCTCCGCTGATTCCAACG</td><td>ATCGCCGGAGACGTTATTCC</td><td>qRT PCR</td></tr><tr><td>RBOH E</td><td>AAGACCTCGTCATGTGGTTCA</td><td>AGAACCCAGCTTCTTTGCCA</td><td>qRT PCR</td></tr><tr><td>RBOH F</td><td>CTTGGCATTGGTGCAACTCC</td><td>TCTTTCGTCTTGGCGTGTCA</td><td>qRT PCR</td></tr><tr><td>ACT7</td><td>CCATCGCTCATCGGAATGGA</td><td>TGGAACCACCACTGAGAACG</td><td>qRT PCR</td></tr><tr><td>IAA3</td><td>CACCATGGATGAGTTTGTTAACC</td><td>TCATACACCACAGCCTAAAC</td><td>Cloning into gateway vector</td></tr><tr><td>IAA3</td><td>CGCGGATCCATGGATGAGTTTGTTAACC</td><td>AAAACTGCAGTCATACACCACAGCCTAAAC</td><td>Cloning into pMal vector</td></tr><tr><td>IAA3 C187S</td><td>AGGTTTAGGCTCTGGTGTATGAATG</td><td>CATTCATACACCAGAGCCTAAACCT</td><td>Site directed mutagenesis (SDM)</td></tr><tr><td>IAA3 C169S</td><td>GATGTTCATATCTACGTGCAAGAGAC</td><td>GTCTCTTGCACGTAGATATGAACATC</td><td>SDM</td></tr><tr><td>IAA3 C112S</td><td>AGATCTGAGTTCTTACAAAGGATAC</td><td>GTATCCTTTGTAAGAACTCAGATCT</td><td>SDM</td></tr><tr><td>IAA3 C32S</td><td>AGTTTCTTGCTCTAATAACAACAATAAG</td><td>CTTATTGTTGTTATTAGAGCAAGAAACT</td><td>SDM</td></tr><tr><td>IAA3 P69S</td><td>TGTTGGATGGAGCCCAAGTTAGATCTTAC</td><td>GTAAGATCTAACTGGGCTCCATCCAACA</td><td>SDM</td></tr><tr><td>IAA3 K96A</td><td>AATCTATGTGGCGGTAAGTATGGATGGTG</td><td>CACCATCCATACTTACCGCCACATAGATT</td><td>SDM</td></tr><tr><td>IAA3 D155A</td><td>CAAAGATGGTGCGTGGATGCTCATTG</td><td>CAATGAGCATCCACGCACCATCTTTG</td><td>SDM</td></tr><tr><td>CASP1promoter</td><td>CCGGAATTCTTAATCTGCATAAAAGTGAGTA</td><td>CCGCTCGAGTTTCTCTTGCAATTGGGGT</td><td>Cloning of promoter</td></tr><tr><td>HA-IAA3</td><td>CCATGTACCCATACGATGTTCCAGA</td><td>GCCATCCAACAATCTGAGC</td><td>Confirmation of transgenic plants</td></tr><tr><td>IAA28</td><td>CGCGGATCCATGGAAGAAGAAAAGAGATTG</td><td>AAAACTGCAGCTATTCCTTGCCATGTTTTCT</td><td>Cloning into pMal vector</td></tr><tr><td>IAA13</td><td>CGCGGATCCATGATTACTGAACTTGAGATG</td><td>AAAACTGCAGCTAAACCGGCTGCTTTCG</td><td>Cloning into pMal vector</td></tr><tr><td>IAA19</td><td>CGGCGGATATCATGGAGAAGGAAGGACTCG</td><td>CGCGGATCCTCACTCGTCTACTCCTCTAGG</td><td>Cloning into pMal vector</td></tr><tr><td>IAA14</td><td>CGGCGGATATCATGAACCTTAAGGAGACGG</td><td>AAAACTGCAGTCATGATCTGTTCTTGAACTTCTCC</td><td>Cloning into pMal vector</td></tr><tr><td>LBD 16</td><td>AGAAGTCTCATGTTGCAGTCTCC</td><td>TTATCGAGTGAGCCAAAGGGTGTG</td><td>ChIP-qPCR</td></tr><tr><td>GYPSY</td><td>GATGAGAGTGTCAATGCAAC</td><td>GTCCATTGAAGTGTATAGG</td><td>ChIP-qPCR</td></tr></tbody></table>
1734 1. B. Orman-Ligeza et al., The xerobranching response represses lateral root formation when roots are not in contact with water. *Current Biology* **28**, 3165-3173. e3165 (2018).

1735 2. P. Mehra et al., Hydraulic flux-responsive hormone redistribution determines root branching. *Science* **378**, 762-768 (2022).

1739 3. B. Orman-Ligeza et al., RBOH-mediated ROS production facilitates lateral root emergence in Arabidopsis. *Development* **143**, 3328-3339 (2016).

1741 4. T. Nietzel et al., The fluorescent protein sensor ro GFP 2-Orp1 monitors in vivo H2O2 and thiol redox integration and elucidates intracellular H2O2 dynamics during elicitor-induced oxidative burst in Arabidopsis. *New Phytologist* **221**, 1649-1664 (2019).

1745 5. D. Arnaud, M. J. Deeks, N. Smirnoff, Organelle-targeted biosensors reveal distinct oxidative events during pattern-triggered immune responses. *Plant Physiology* **191**, 2551-2569 (2023).

1748 6. B. Lu et al., FERONIA-mediated TIR1/AFB2 oxidation stimulates auxin signaling in Arabidopsis. *Molecular Plant* **17**, 772-787 (2024).

1750 7. J. Luo, J.-J. Zhou, J.-Z. Zhang, Aux/IAA gene family in plants: molecular structure, regulation, and function. *International journal of molecular sciences* **19**, 259 (2018).

1752 8. M. Lavy, M. Estelle, Mechanisms of auxin signaling. *Development* **143**, 3226-3229 (2016).

1754 9. T. J. Guilfoyle, The PB1 domain in auxin response factor and Aux/IAA proteins: a versatile protein interaction module in the auxin response. *The Plant Cell* **27**, 33-43 (2015).

1757 10. T. Vernoux et al., The auxin signalling network translates dynamic input into robust patterning at the shoot apex. *Molecular systems biology* **7**, 508 (2011).

1759 11. N. Dharmasiri, S. Dharmasiri, M. Estelle, The F-box protein TIR1 is an auxin receptor. *Nature* **435**, 441-445 (2005).

1761 12. S. Kepinski, O. Leyser, The Arabidopsis F-box protein TIR1 is an auxin receptor. *Nature* **435**, 446-451 (2005).

1763 13. X. Tan et al., Mechanism of auxin perception by the TIR1 ubiquitin ligase. *Nature* **446**, 640-645 (2007).

1765 14. S. Kagale, K. Rozwadowski, EAR motif-mediated transcriptional repression in plants: an underlying mechanism for epigenetic regulation of gene expression. *Epigenetics* **6**, 141-146 (2011).

1768 15. H. Szemenyei, M. Hannon, J. A. Long, TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis. *Science* **319**, 1384-1386 (2008).

1771 16. J. S. Teixeira, K. Ten Tusscher, The systems biology of lateral root formation: connecting the dots. *Molecular Plant* **12**, 784-803 (2019).

1773 17. C. M. Cremers, U. Jakob, Oxidant sensing by reversible disulfide bond formation. *Journal of Biological Chemistry* **288**, 26489-26496 (2013).

1775 18. J. R. Ramos, B. J. Reyes-Hernández, K. Alim, A. Maizel, Auxin-mediated stress relaxation in pericycle and endoderm remodelling drive lateral root initiation. *Biophysical Journal*, (2024).

1778 19. R. Ursache, S. Fujita, V. Dénervaud Tendon, N. Geldner, Combined fluorescent seed selection and multiplex CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants. *Plant Methods* **17**, 1-14 (2021).

1781 20. D. A. Korasick et al., Molecular basis for AUXIN RESPONSE FACTOR protein interaction and the control of auxin response repression. *Proceedings of the National Academy of Sciences* **111**, 5427-5432 (2014).
1784 21. M. H. Nanao et al., Structural basis for oligomerization of auxin transcriptional regulators. *Nature communications* **5**, 3617 (2014).

1785 22. M. I. Wilson, D. J. Gill, O. Perisic, M. T. Quinn, R. L. Williams, PB1 domain-mediated heterodimerization in NADPH oxidase and signaling complexes of atypical protein kinase C with Par6 and p62. *Molecular cell* **12**, 39-50 (2003).

1786 23. M. L. Seibenhener, T. Geetha, M. W. Wooten, Sequestosome 1/p62-more than just a scaffold. *FEBS letters* **581**, 175-179 (2007).

1787 24. D. C. Dinesh et al., Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response. *Proceedings of the National Academy of Sciences* **112**, 6230-6235 (2015).

1788 25. C. E. Paulsen, K. S. Carroll, Orchestrating redox signaling networks through regulatory cysteine switches. *ACS chemical biology* **5**, 47-62 (2010).

1789 26. L. B. Poole, The basics of thiols and cysteines in redox biology and chemistry. *Free Radical Biology and Medicine* **80**, 148-157 (2015).

1790 27. A. Zeida et al., Catalysis of peroxide reduction by fast reacting protein thiols: focus review. *Chemical reviews* **119**, 10829-10855 (2019).

1791 28. C. H. Foyer, G. Noctor, Redox homeostasis and signaling in a higher-CO2 world. *Annual Review of Plant Biology* **71**, 157-182 (2020).

1792 29. X. Huang et al., ROS regulated reversible protein phase separation synchronizes plant flowering. *Nature Chemical Biology* **17**, 549-557 (2021).

1793 30. J. E. Vermeer et al., A spatial accommodation by neighboring cells is required for organ initiation in Arabidopsis. *Science* **343**, 178-183 (2014).

1794 31. L. I. A. Calderón Villalobos et al., A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin. *Nature chemical biology* **8**, 477-485 (2012).

1795 32. K. A. Dreher, J. Brown, R. E. Saw, J. Callis, The Arabidopsis Aux/IAA protein family has diversified in degradation and auxin responsiveness. *The Plant Cell* **18**, 699-714 (2006).

1796 33. B. L. Moss et al., Rate motifs tune auxin/indole-3-acetic acid degradation dynamics. *Plant physiology* **169**, 803-813 (2015).

1797 34. Q. Tian, N. J. Uhlir, J. W. Reed, Arabidopsis SHY2/IAA3 inhibits auxin-regulated gene expression. *The Plant Cell* **14**, 301-319 (2002).

1798 35. S. H. Kim et al., A gain-of-function mutant of IAA15 inhibits lateral root development by transcriptional repression of LBD genes in Arabidopsis. *Frontiers in Plant Science* **11**, 1239 (2020).

1799 36. T. Goh, H. Kasahara, T. Mimura, Y. Kamiya, H. Fukaki, Multiple AUX/IAA-ARF modules regulate lateral root formation: the role of Arabidopsis SHY2/IAA3-mediated auxin signalling. *Philosophical Transactions of the Royal Society B: Biological Sciences* **367**, 1461-1468 (2012).

1800 37. A. Colón-Carmona, D. L. Chen, K.-C. Yeh, S. Abel, Aux/IAA proteins are phosphorylated by phytochrome in vitro. *Plant physiology* **124**, 1728-1738 (2000).

1801 38. Q. Tian, J. W. Reed, Control of auxin-regulated root development by the Arabidopsis thaliana SHY2/IAA3 gene. *Development* **126**, 711-721 (1999).

1802 39. B. Orosa-Puente et al., Root branching toward water involves posttranslational modification of transcription factor ARF7. *Science* **362**, 1407-1410 (2018).

1803 40. H.-T. Cho et al., A dose-dependent bimodal switch by homologous Aux/IAA transcriptional repressors. *Molecular Plant* **17**, 1407-1422 (2024).

1804 41. Y. Okushima, H. Fukaki, M. Onoda, A. Theologis, M. Tasaka, ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis. *The Plant Cell* **19**, 118-130 (2007).
1833 42. T. Goh et al., Lateral root initiation requires the sequential induction of transcription factors LBD16 and PUCHI in Arabidopsis thaliana. *New Phytologist* **224**, 749-760 (2019).

1836 43. A. Shapiguzov, J. P. Vainonen, M. Wrzaczek, J. Kangasjärvi, ROS-talk-how the apoplast, the chloroplast, and the nucleus get the message through. *Frontiers in plant science* **3**, 292 (2012).

1839 44. O. Rodrigues et al., Aquaporins facilitate hydrogen peroxide entry into guard cells to mediate ABA-and pathogen-triggered stomatal closure. *Proceedings of the National Academy of Sciences* **114**, 9200-9205 (2017).

1842 45. G. P. Bienert, F. Chaumont, Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide. *Biochimica et Biophysica Acta (BBA)-General Subjects* **1840**, 1596-1604 (2014).

1845 46. M. Chambers et al., Mechanisms of Groucho-mediated repression revealed by genome-wide analysis of Groucho binding and activity. *BMC genomics* **18**, 1-17 (2017).

1848 47. C. R. Horne et al., Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism. *Nature communications* **12**, 1988 (2021).

1850 48. D. Moazed, Common themes in mechanisms of gene silencing. *Molecular cell* **8**, 489-498 (2001).

1852 49. S. Payankaulam, L. M. Li, D. N. Arnosti, Transcriptional repression: conserved and evolved features. *Current biology* **20**, R764-R771 (2010).

1854 50. W. Wu et al., Evolution analysis of the Aux/IAA gene family in plants shows dual origins and variable nuclear localization signals. *International journal of molecular sciences* **18**, 2107 (2017).

1857 51. M. Michniewicz, E. M. Frick, L. C. Strader, Gateway-compatible tissue-specific vectors for plant transformation. *BMC research notes* **8**, 1-8 (2015).

1859 52. S. J. Clough, A. F. Bent, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. *The plant journal* **16**, 735-743 (1998).

1861 53. R. C. Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput. *Nucleic acids research* **32**, 1792-1797 (2004).

1863 54. A. M. Waterhouse, J. B. Procter, D. M. Martin, M. Clamp, G. J. Barton, Jalview Version 2—a multiple sequence alignment editor and analysis workbench. *Bioinformatics* **25**, 1189-1191 (2009).

1866 55. F. Corpet, Multiple sequence alignment with hierarchical clustering. *Nucleic acids research* **16**, 10881-10890 (1988).

1868 56. A. I. Nesvizhskii, A. Keller, E. Kolker, R. Aebersold, A statistical model for identifying proteins by tandem mass spectrometry. *Analytical chemistry* **75**, 4646-4658 (2003).

1871 57. M. M. Goodin, R. G. Dietzgen, D. Schichnes, S. Ruzin, A. O. Jackson, pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves. *The Plant Journal* **31**, 375-383 (2002).

1874 58. A. Paul, P. Dasgupta, D. Roy, S. Chaudhuri, Comparative analysis of Histone modifications and DNA methylation at OsBZ8 locus under salinity stress in IR64 and Nonabokra rice varieties. *Plant molecular biology* **95**, 63-88 (2017).

1877 59. K. J. Livak, T. D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2⁻ΔΔCT method. *methods* **25**, 402-408 (2001).

1879 60. M. Srivastava et al., The conjugation of SUMO to the transcription factor MYC2 functions in blue light-mediated seedling development in Arabidopsis. *The Plant Cell* **34**, 2892-2906 (2022).
1883

1884

1885

1886

1887

1888

1889

1890

1891

1892

1893

1894

1895

1896

1897

1898