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PUCHI represses early meristem formation in
developing lateral roots of Arabidopsis thaliana

Kevin Bellande, Duy-Chi Trinh, Anne-Alicia Gonzalez, Emeric Dubois, Anne-Sophie Petitot, Mikaël Lucas, Antony Champion, Pascal Gantet, Laurent Laplaze, Soazig Guyomarc’h

► To cite this version:

Kevin Bellande, Duy-Chi Trinh, Anne-Alicia Gonzalez, Emeric Dubois, Anne-Sophie Petitot, et al. PUCHI represses early meristem formation in developing lateral roots of Arabidopsis thaliana. Journal of Experimental Botany, 2022, 73 (11), pp.3496-3510. 10.1093/jxb/erac079. hal-03775291

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Distributed under a Creative Commons Attribution 4.0 International License
1 **Title of the manuscript:**

2 **PUCHI represses early meristem formation in developing lateral roots of *Arabidopsis***

3 **thaliana**

4

5 **Authors**

6 Kevin Bellande¹, Duy-Chi Trinh¹, #, Anne-Alicia Gonzalez², ³, Emeric Dubois², ³, Anne-Sophie

7 Petitot¹, Mikaël Lucas¹, Antony Champion¹, Pascal Gantet¹, Laurent Laplaze¹*, Soazig

8 Guyomarc'h¹*

9 ¹ DIADE, Univ Montpellier, IRD, Montpellier, France

10 ² Univ Montpellier, CNRS, INSERM, Montpellier France

11 ³ Montpellier GenomiX, France Génomique, Montpellier, France

12 # Present address: Laboratoire RDP, Université de Lyon 1, ENS-Lyon, CNRS, INRAE, UCBL,

13 69364 Lyon, France.

14 * Corresponding authors: Soazig Guyomarc'h and Laurent Laplaze

15 **Email** to whom correspondence may be addressed: [soazig.guyomarch@ird.fr](mailto:soazig.guyomarch@ird.fr)

16

17 **Email** of all authors: [kevin.bellande@ird.fr](mailto:kevin.bellande@ird.fr), [duy-chi.trinh@ens-lyon.fr](mailto:duy-chi.trinh@ens-lyon.fr), [anne-

18 alicia.gonzalez@mgx.cnrs.fr, emeric.dubois@mgx.cnrs.fr, anne-sophie.petitot@ird.fr,

19 mikael.lucas@ird.fr, antony.champion@ird.fr, pascal.gantet@ird.fr, laurent.laplaze@ird.fr,

20 [soazig.guyomarch@ird.fr](mailto:soazig.guyomarch@ird.fr)

21

22 **Running title:** PUCHI represses early lateral root meristem formation

23 **Date of submission:** 28 October 2021

24 **Response to reviewers:** 3 February 2022

25 **Word count:** 5169 words

26 **Number of tables and figures:** 5 figures

27 **Supplementary data:** 7 supplementary figures, 1 supplementary table

28
29 **Highlight**

30 Consistent with the hypothesis that mutual inhibition between gene regulatory sub-networks
31 contributes to lateral root formation, we show that *PUCHI* represses early meristematic gene
32 expression in developing root primordia.

33

34 **Abstract**

35 Lateral root organogenesis is a key process in the development of a plant's root system and its
36 adaptation to the environment. During lateral root formation, an early phase of cell proliferation first
37 produces a four cell-layered primordium and only from this stage onwards is a root meristem-like
38 structure, expressing root stem cell niche marker genes, being established in the developing
39 organ. Previous studies reported that the gene regulatory network controlling lateral root formation
40 is organised into two subnetworks whose mutual inhibition may contribute to organ patterning.
41 *PUCHI* encodes an AP2/ERF-transcription factor expressed early during lateral root primordium
42 development and required for correct lateral root formation. To dissect the molecular events
43 occurring during this early phase, we generated time-series transcriptomic datasets profiling lateral
44 root development in *puchi-1* mutants and wild types. Transcriptomic and reporter analyses
45 revealed that meristem-related genes were expressed ectopically at early stages of lateral root
46 formation in *puchi-1* mutants. We conclude that, consistent with the inhibition of genetic modules
47 contributing to lateral root development, PUCHI represses ectopic establishment of meristematic
48 cell identities at early stages of organ development. These findings shed light on gene network
49 properties that orchestrate correct timing and patterning during lateral root formation.

50

51 **Keywords**

52 Arabidopsis - Auxin - Cytokinin - Lateral root development - Meristem - Organogenesis - Patterning
53 - PLETHORA - PUCHI
54 **Abbreviations**

55 CHX: cycloheximide

56 DEGs: differentially expressed genes

57 DEX: dexamethasone

58 FRIP: fraction of reads in peaks

59 GR: glucocorticoid receptor

60 GO: Gene Ontology

61 GRN: gene regulatory network

62 hpg: hours post gravistimulation

63 LR: lateral root

64 LRP: lateral root primordium

65 MS: Murashige and Skoog medium

66 NAA: 1-naphthalene acetic acid

67 NPA: N-1-naphthylphthalamic acid

68 PI: propidium iodide

69 QC: quiescent centre

70 RAM: root apical meristem

71 VLCFA: very long chain fatty acids

72 WT: wild type

73
74 **Introduction**

75 Dynamic and plastic development evolved in sessile organisms such as plants to adapt to
76 changing environmental conditions (Morris et al., 2017). Root branching, the production of new
77 lateral roots (LR) from existing roots, is key in elaborating root system architecture (Motte et al.,
78 2019). In the model plant *Arabidopsis thaliana*, LR arise from pre-branch sites originating in the
79 basal meristem close to the root apex (Xuan et al., 2020). Primed pericycle cells at these pre-
80 branch sites enter new rounds of cell divisions producing a lateral root primordium (LRP). The
81 developing LRP, while growing through overlying cell layers, progressively organises into a new
82 root apical meristem (RAM) that ultimately emerges from the parent root and whose activity will
83 determine the later growth of the LR (Malamy et al., 1997; Banda et al., 2019). Remarkably, LRP
84 development is a two-step process featuring an early, so-called morphogenetic phase (stages I to
85 IV) during which cell proliferation generates a four-cell-layered, bulge-shaped LRP and a second,
86 so-called meristematic phase (stages V to VIII) when the LRP further develops to acquire key RAM
87 characteristics (Lucas et al., 2013; Lavenus et al., 2015; Voß et al., 2015; Goh et al., 2016).
88 However, the mechanisms controlling the establishment of a new root meristem and especially, the
89 transition from the early phase of LRP development to the meristematic phase, when key root
90 meristem genes start to be expressed in the centre of the developing LRP, remain unclear.

91 RAMs are proliferating tissues located at the tip of growing roots within which cell division and
92 differentiation are regulated sequentially. Maintenance of a stem cell niche at the centre of root
93 meristems ensures continuous production of cells and indeterminate organ growth (van den Berg
94 et al., 1995, 1997; Bennett and Scheres, 2010; Trinh et al., 2018). Multiple regulatory components,
95 including transcriptional regulators and hormones, mediate the regulation of cell identity and
96 division activity in RAMs (Himanen et al., 2002; Goh et al., 2012, 2016; Porco et al., 2016; Du and
97 Scheres, 2017, 2018). In *A. thaliana*, networks of key transcription factors including AP2-domain
98 PLETHORA (PLT), GRAS-family transcription factors SHORT-ROOT (SHR) and SCARECROW
99 (SCR), as well as auxin-dependent factors, have been shown to control the patterning of the RAM
100 stem cell niche and specification of its organizing centre, known as the quiescent centre (QC)
101 (Galinha et al., 2007; Hofhuis et al., 2013; Goh et al., 2016; Du and Scheres, 2017; Shimotohno et al., 2018). Many of these genes are also dynamically expressed during the process of LR formation (Tian et al., 2014; Lavenus et al., 2015; Goh et al., 2016; Du and Scheres, 2017).

104 Several studies have shown that cell fate acquisition in developing LRP is not dependent on cell lineage but relies on tissue-scale mechanisms and positional signals (Lucas et al., 2013; von Wangenheim et al., 2016). The progressive organisation of the organ could thus result from emerging properties of a complex genetic network possibly integrating chemical and mechanical cues (Banda et al., 2019). Two examples of such patterning mechanisms are the definition of inner and outer domains of the early LRP by the interplay of SHR and SCR expression domains, and the correct patterning of cell divisions at early stages and transition of the developing LRP to the meristematic phase by distinct sets of PLT genes (Goh et al., 2016; Du and Scheres, 2017). These data highlight that gene regulatory events during early LRP development are necessary to transition to the second meristem organisation phase.

114 Inference of the gene regulatory network (GRN) during LR formation suggested that two distinct genetic sub-circuits operate and that their mutual inhibition could be instrumental in LRP developmental patterning (Lavenus et al., 2015). The first one includes genes predominantly expressed in the whole primordium in the early stages, but only in its base in the later phase. The second one includes meristematic genes whose expression initiates during the transition from the morphogenetic phase to the meristematic phase in the tip of the developing primordium, where the new root meristem stem cell niche is established. Mutual inhibition between these two groups of genes may explain the bifurcation between cell identities in the central zone and the flanking domain of the developing LRP (Lavenus et al., 2015). In addition, in complex genetic systems, mutual inhibition motifs associated to positive regulatory cascades can generate sequential waves in gene expression dynamics and control cell state switch over time (Alon, 2007).

125 PUCHI is an AP2/ERF-family transcription factor belonging to the first GRN sub-circuit and whose loss of function or perturbation in expression kinetics impairs LRP development (Hirota et al., 2007; Kang et al., 2013; Trinh et al., 2019; Toyokura et al., 2019; Goh et al., 2019). GRN inference and
experimental validation previously revealed that PUCHI acted as a master regulator of very long
chain fatty acid (VLCFA) biosynthesis during the first phase of LRP development (Trinh et al.,
2019). However, the role played by PUCHI-dependent genetic pathways in LRP development
remains elusive.

Here, we show that the AP2/ERF-transcription factor PUCHI, which is predominantly expressed at
early stages of LR development, controls the correct timing and pattern of expression of
transcription factors as well as distribution of hormonal signals that orchestrate meristem formation
during late lateral root organogenesis. Root meristem-related genes are ectopically expressed in
inner cell layers of early-phase LRP in a *puchi-1* mutant background. Thus, these results support
the hypothesis that the inhibition of gene regulatory modules participates in LRP functional
patterning, and show that in the absence of PUCHI, the LR gene regulatory network yields
premature expression of meristematic genes in inner cells of young LRP.

140

## 141 Materials and Methods

142

143 Plant Materials and Constructs, Growth Conditions

A. thaliana plant ecotypes Columbia-0 (Col-0) were used for all experiments and as backgrounds
of transgenic lines in this study. A. thaliana seeds were surface-sterilized and sown on half
strength Murashige and Skoog (1/2 MS) solid medium with 0.7% (w/v) plant agar supplemented with
B5 vitamins. Plates were kept at 4°C for 2 days and then placed in long-day conditions (16-h
light/8-h dark cycle) in a vertical position. The *puchi-1* mutant line has previously been described
(Hirota *et al.*, 2007). The *promPLT:PLT-YFP* lines were reported in (Galinha *et al.*, 2007; Hofhuis *et
al.*, 2013). The *DR5::GFP* synthetic auxin response reporter was described in (Friml *et al.*, 2003),
the synthetic cytokinin response reporter *promTCSn::GFP* was described in (Zürcher *et al.*, 2013),
and the organising centre markers *QC25::CFP* and *promWOX5::nls::GFP* were described in (ten
Hove *et al.*, 2010) and (Goh *et al.*, 2016) respectively.
RNAseq

Col-0 wild type (WT) and *puchi-1* mutant seeds were surface-sterilized and sown on ½ MS solid medium containing 0.7% (w/v) plant agar supplemented with B5 vitamins. Plates were kept at 4°C for 2 days and then placed in continuous light conditions in a vertical position. Gravistimulation were performed on 4-day-old seedlings and root bends were sampled at 12h, 18h, 24h, 30h and 36h after gravistimulation. Three biological replicates (for both Col-0 and *puchi-1*) were used for the RNAseq experiment. For each replicate, root bends of more than 400 seedlings were dissected under a binocular microscope and frozen in liquid nitrogen immediately on harvesting. For reference, approximately 400 mature root segments located between the bend and the shoot were harvested in 4-day-old seedlings at 12h after gravistimulation to be used as a reference of non-gravitropic-stimulated root tissues devoid of developing LRP (termed "No LR" in the data set). Total RNA was extracted using Qiagen RNeasy plant mini kit with an on-column DNAse treatment following the manufacturer's recommendation (RNAse-free DNAse Set, Qiagen, Crawley, UK). RNA samples were quantified using a Nanodrop ND100 spectrophotometer (Nanodrop, Wilimington, USA) and RNA purity and integrity were evaluated using High-Resolution Automated Electrophoresis 2100 Bioanalyzer system from Agilent Technologies (https://www.agilent.com/en/product/automated-electrophoresis).

RNAseq analyses was performed by the MGX-Montpellier GenomiX platform (https://www.mgx.cnrs.fr/). cDNA libraries were constructed using a Stranded mRNA Prep Ligation kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, poly-A RNAs were purified using oligo-d(T) magnetic beads from 310 ng of total RNA. Poly-A RNAs were fragmented and underwent reverse transcription using random hexamers. During the second strand generation step, dUTP substituted dTTP to prevent the second strand being used as a matrix during the final PCR amplification. Double stranded cDNAs were adenylated at their 3' ends and ligated to Illumina's universal anchors. Ligated cDNAs were amplified following 12 PCR cycles. During this PCR, the libraries were indexed, and the adapter's sequences were completed to be
compatible with the cluster generation step. PCR products were purified using AMPure XP Beads (Beckman Coulter Genomics, Brea, CA, USA). Libraries were validated using a High Sensitivity NGS kit on a Fragment Analyzer (Agilent Technologies, Santa Clara, CA, USA) and quantified using a KAPA Library quantification kit (Roche, Basel, Switzerland).

For library sequencing, 36 libraries were pooled in equimolar amounts. The balance between all samples of the pool was assessed by sequencing on a Miniseq (Illumina, San Diego, CA, USA) using a 300 cycle Mid Output Reagent Cartridge. The pool was then sequenced on a Novaseq 6000 (Illumina, San Diego, CA, USA) S1 flow cell in paired end 2\*100 nt mode according to the manufacturer's instructions. This sequencing produced between 36 and 44 million passed filter clusters per library.

For sequencing quality control, image analyses and base calling were performed using the NovaSeq Control Software and Real-Time Analysis component (Illumina, San Diego, CA, USA). Demultiplexing and trimming were performed using Illumina's conversion software (bcl2fastq 2.20). The quality of the raw data was assessed using FastQC from the Babraham Institute and the Illumina software SAV (Sequencing Analysis Viewer). FastqScreen was used to estimate the potential level of contamination.

For alignment and statistical analysis, a splice junction mapper, TopHat 2.1.1 (Kim et al., 2013) (using Bowtie 2.3.5.1 (Langmead et al., 2009)), was used to align the RNA-Seq reads to the *A. thaliana* genome (NCBI TAIR10.1) with a set of gene model annotations (gff file downloaded from NCBI on November 13, 2020). Final read alignments with more than 6 mismatches were discarded. Samtools (v1.9) was used to sort the alignment files. Then, gene counting was performed using Featurecounts 2.0.0 (Liao et al., 2014). As the data is from a strand-specific assay, the reads needed to be mapped to the opposite strand of the gene (-s 2 option). Before statistical analysis, genes with less than 15 reads (cumulating all the analysed samples) were filtered out.

Differentially expressed genes (DEGs) were identified using the Bioconductor (Gentleman et al., 2004) package (http://www.bioconductor.org) DESeq2 1.26.0 (Love et al., 2014) (R version 3.6.1).
Data were normalised using the DESeq2 normalisation method. Genes with an adjusted p-value of less than 5% (according to the False Discovery Rate method by Benjamini-Hochberg) were declared differentially expressed. DEGs between Col-0 and *puchi-1* were then investigated for each time point. The DEGs with a Log2Fold-change ≥1 and a DESeq2 package-Wald Test (p-value: *p*<0.05; *p**<0.02; *p***<0.01) were selected for the gene ontology (GO) analysis. GO biological process enrichment analyses of either, the list of up- or down-regulated DEGs in *puchi-1* were performed using a PANTHER Overrepresentation assay (http://pantherdb.org) and Fisher's test followed by a Bonferroni Correction (*p*<0.05) (Thomas *et al.*, 2003; Mi *et al.*, 2013). Only the individual elementary annotations of the GO biological process are shown.

217

## RT-qPCR analysis

For RT-qPCR analyses, plant material was collected following the lateral root-induction system described in (Himanen *et al.*, 2002). Briefly, square Petri dishes containing ½ MS solid medium, 0.7% (w/v) plant agar supplemented with B5 vitamins and containing N-1-naphthylphthalamic acid (NPA) and/or 1-naphthalene acetic acid (NAA) were used. Col-0 and *puchi-1* seeds were germinated and grown for 72 h on 5 µM NPA before transfer to 5 µM NPA-containing (control) or 10 µM NAA-containing (lateral root induction) medium. Three biological replicates of root segments without the root apical meristem were harvested after 24h of treatment and immediately frozen in liquid nitrogen for each condition. Total RNA was extracted using a Qiagen RNeasy plant mini kit with an on-column DNAse treatment following the manufacturer's recommended protocol (RNAse-free DNAse Set, Qiagen, Crawley, UK). Quantification, purity and contaminations assessment of the RNA samples were tested using a gel electrophoresis and a Nanodrop ND100 spectrophotometer (Nanodrop, Wilimington, USA). cDNA generated from the three biological replicates were synthetised following the manufacturer's recommended protocol (Omniscript RT Kit, Qiagen, Crawley, UK): 1000 ng of RNA in RNAse free water were heated 10min at 70°C, added to the manufacturer mix (up to 20µL) for 1h at 37°C. Generated cDNA diluted 1/10 were added to the Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix with Low ROX reaction mix

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and used to performed qPCR analysis as recommended by the manufacturer (Qiagen, Crawley, UK). All qPCR reactions were carried out and analysed using the Roche® LC480 Lightcycler system and software (qPCR analysis program, Cq determination, curves calibration). Complete thermocycling parameters were as follow: 95°-3'15; 95°-20", 60°-20" x 40 cycles; 95°-1', 60°-30", 95°-30". Normalisation was achieved with the tubulin *TUB3* gene (AT5G62700) and the *UBQ5* gene (AT3G62250) (Lavenus *et al.*, 2015; Trinh *et al.*, 2019). The calibrator cDNA for relative quantification of the effect of each treatment was the control WT treated with NPA. Data are represented as mean ± SEM (standard error of the means). Significance between NAA treated samples was determined using a Student's t test (p*<0.05; p**<0.02; p***<0.01). The primers used for RT-qPCR analysis were designed to generate 75-110 bases amplicon length. Primer's efficiency and specificity was confirmed by qPCR. Primer sequences were as follows:

AT3G20840: PLT1-F caacccttttcaaacacaagagt; PLT1-R ttggaacctctcctccttca; AT1G51190: PLT2-F aggaaaggaagacaagtctacttagg; PLT2-R agaggaccccaatatttaagtg; AT5G10510: PLT3-F gatctttaccttggaacctttgc; PLT3-R gctgctatgtcatacgcttca; AT5G17430: PLT4/BBM-F gagacaataatagtcactcccgagat; PLT4/BBM-R ttgttcggtattgttaatgttattgc; AT5G57390: PLT5-F ctccatgtacagaggcgtca; PLT5-R gcagcttccttgtagtgcta; AT5G65510: PLT7-F aacagctgtaggaggaaggt; PLT7-R tctatcttccttgtcatatccaccta; TUB3-F tgcattggtacacaggtgagggaa; TUB3-R agccgttgcatcttggtattgctg; UBQ5-F cgatggatctggaaaggttc; UBQ5-R agctccacaggttgcgttag

## Confocal Microscopy, Image Processing and Analysis

Confocal microscopy images were obtained with a LEICA SP8 confocal system using a 40X HCX corr Plan Apochromat CS 1.1 NA water objective. Samples were mounted in water or in 15 µM propidium iodide (PI) for 1-2 h or in 15 µM PI supplemented with 0.004% Triton X-100 for 20-40 min as previously described (Du and Scheres, 2017). All combinatorial fluorescence analyses were run as sequential scans. The following excitation/emission settings were used to obtain specific fluorescence signals: for PI, 488/600 to 620 nm; for YFP, 514/520-550 nm, for CFP, 458/475-505
261 nm and for EGFP, 488/500 to 550 nm. All post-acquisition image such as channel merging was
262 performed using Fiji (https://imagej.net/Fiji) (Schindelin et al., 2012).

263

264 **Database mining for PUCHI target genes**

265 Genes showing differential expression (fold change ≥ 1.5, p-value ≤0.05 as determined by Welch
266 two sample t-test) in auxin-treated PUCHI-GR roots upon dexamethasone treatment and in
267 presence of cycloheximide were retrieved from (Trinh et al., 2019). Genes associated to genome
268 sequences bound by the transcription factor PUCHI *in vitro* (ampDAP, FRiP ≥ 5%) were retrieved
269 from the Arabidopsis DAP-seq database published at
270 http://neomorph.salk.edu/dev/pages/shhuang/dap_web/pages/index.php (O'Malley et al., 2016). List
271 contents were compared using the “Calculate and draw custom Venn diagrams” system from VIB /
272 UGent Bioinformatics & Systems (https://bioinformatics.psb.ugent.be/cgi-
273 bin/liste/Venn/calculate_venn) to select genes that had been detected by at least two of the three
274 methods: RNAseq profiling of *puchi-1* roots, DAP-seq using PUCHI as a bait, or inducible
275 complementation of PUCHI-GR *puchi-1* roots. Aliases and description summaries were retrieved
276 for each gene from Thalemine (https://bar.utoronto.ca/thalemine/begin.do). Overrepresentation of
277 Gene Ontologies among the 150 genes that are common between two datasets was assayed
278 using the PANTHER algorithm (http://pantherdb.org) and Fisher's test followed by a Bonferroni
279 Correction (p*<0.05) (Thomas et al., 2003; Mi et al., 2013). Among the list of those 150 genes,
280 transcription factors and genes annotated as related to auxin, cytokinin, VLCFA, LR development,
281 or meristem regulation were selected for closer inspection (see suppl. Table 1 for detailed lists).

282

283
286 **Loss of PUCHI function alters sequential expression patterns of PLETHORA genes in**
287 **developing lateral root primordia**

288 To assess the role played by PUCHI in the genetic regulation of LRP development and identify its
289 targets, either direct or indirect, during this organogenesis process, we performed a time-series
290 transcriptomic analysis following LR induction by gravistimulation in Col-0 wild type (WT) and
291 *puchi-1* mutant plants (Fig.1A). Root segments were sampled at 12 hours post gravistimulation
292 (hpg; stage I), 18 hpg (predominantly stage II in Col-0 and *puchi-1*), 24 hpg (stages II-IV in Col-0,
293 stages II-III in *puchi-1*), 30 hpg (stages III-V in Col-0, stages II-IV in *puchi-1*) and 36 hpg (stages III-
294 VI in Col-0, stages II-V in *puchi-1*), which correspond approximately to LR initiation, the mid and
295 late morphogenetic phases, and the early meristematic phase in a Col-0 background in our
296 experimental conditions (Fig. S1A, S1B and S1C) (Goh *et al.*, 2016; Trinh *et al.*, 2019). A root
297 segment was sampled directly upward of the root bend in both genotypes at 12 hpg to be used as
298 "No LR" reference material. RNAseq profiling of these samples was performed and differentially
299 expressed genes (DEGs) between successive time points or between the 2 genotypes at a single
300 time point were identified (Log2FC > 1 or Log2FC < -1, p*<0.05; see Material and methods
301 section).

302 A total of 5791 genes in WT and 9571 genes in the *puchi-1* mutant background were shown to be
303 differentially expressed during the LRP developmental time course. Noticeably, comparison of WT
304 and *puchi-1* reference "No LR" root segments revealed 361 genes differentially expressed between
305 the two genotypes (Fig. 1B). By contrast, comparison of *puchi-1* to WT LRP transcriptomic
306 datasets at 12, 18, 24, 30, and 36 hpg yielded a minimum of 314 up to a maximum of 1009 DEGs,
307 with the highest divergence between the two genotypes observed at 24 hpg and 36 hpg (Fig.1B).
308 Because we were first interested in uncovering the role of PUCHI in the process of lateral root
309 organogenesis without necessarily distinguishing the primary molecular targets of this transcription
310 factor from the secondary regulatory events influencing LRP formation, we examined all the genes
whose expression dynamics during LRP formation was shown to be dependent on PUCHI function, later termed PUCHI-dependent genes.

We analysed gene ontology (GO) enrichment among differentially expressed genes focusing specifically on genes whose expression is up-regulated in *puchi-1* as compared to WT or on genes whose expression is downregulated in *puchi-1* as compared to WT. Consistent with previous studies, this analysis revealed that PUCHI-dependent genes are highly enriched in lipid metabolism-related genes (*p*-value < 0,001), and confirmed that PUCHI positively influences the expression of multiple VLCFA, cutin, and suberin biosynthetic genes during LRP formation (Fig. 1C-D, Supp. Fig. S2, Supp. Fig. S3) (Trinh *et al.*, 2019). In addition, transcriptomic profiling in *puchi-1* and WT revealed other biological processes dependent on PUCHI. Interestingly, root development related genes were overrepresented in the *puchi-1* RNAseq dataset as compared to WT (*p*<0.05 when comparing *puchi-1* and WT at 24h). These genes include *PLT1*, *PLT2*, and *PLT4/BBM* genes, which are well known regulators of root apical meristem establishment and maintenance (Fig. 1D) (Aida *et al.*, 2004). Other genes whose function can be related to root meristem organisation or activity (e.g., predominantly expressed in the QC (Nawy *et al.*, 2005)) displayed altered expression dynamics during LRP development in the *puchi-1* background (Supp. Fig. S3). Especially our RNAseq analysis shows robust overexpression of *PLT1* and *PLT4* genes in *puchi-1* root segments throughout the four time points of our dataset and especially at 18h and 24h, which corresponds to root segments harbouring young LRP from stage II to stage III, i.e., before the transition to the meristematic phase (Fig. 1D, Supp. Fig. S1). Conversely other regulators of the PLETHORA family, *PLT5* and *PLT7*, were downregulated in *puchi-1* root segments as compared to WT (Fig. 1D). These latter results are consistent with previously published LR GRN prediction analyses and confirm that both *PLT5* and *PLT7* genes are positively regulated downstream of *PUCHI* in the early morphogenetic phase (Lavenus *et al.*, 2015). Under expression of *PLT5* and *PLT7* were confirmed by independent RT-qPCR analyses in *puchi-1* roots compared to WT 24h after LR induction by auxin (which correspond to stages II-III LRP in Col-0 and stages I-III LRP in *puchi-1*) (Himanen *et al.*, 2002) (Supp. Fig. S4A).
We took advantage of published *in vitro* and *in vivo* datasets to compare identified DEGs to previously reported PUCHI-dependent gene expression or PUCHI genomic binding sites. First, a list of 13 putative direct target genes of PUCHI were previously identified from the transcriptomic response of PUCHI-GR seedlings' roots upon dexamethasone treatment and in presence of cycloheximide, an inhibitor of protein synthesis (Trinh *et al.*, 2019). Second, a total of 2412 putative PUCHI binding sites have been identified in the genome using the DAP-seq technology (O'Malley *et al.*, 2016). Comparison of those lists to the list of PUCHI-dependent genes identified by RNAseq profiling of branching root segments (this work) yielded 1 single gene, namely *CYTOKININ RESPONSE FACTOR 1 (CRF1)*, that is retrieved by these 3 investigation methods, and a total of 150 genes identified by at least two of these three experimental strategies (Supp. Fig. S5A and Supp. Table 1). Because DAP-seq profiles genomic sequences for which the PUCHI transcription factor displays affinity *in vitro*, and because PUCHI-GR complementation assay was performed in presence of cycloheximide, *CRF1* is most likely a direct target of PUCHI, that induces its expression during LRP formation (Supp. Fig. S3, Supp. Fig. S6E). Interestingly, this gene encodes a component of the cytokinin signalling pathway that modulates a wide range of developmental processes (Raines *et al.*, 2016) (Supp. Fig. S5). Next, a Gene Ontology analysis revealed that genes associated to responses to stresses and to chemical stimuli are overrepresented among the 150 genes shared by at least two of these three experimental datasets (Supp. Table 1). Transcription factor genes and genes associated to auxin and cytokinin homeostasis and signalling pathways, LR development, meristem regulation, and VLCFA biosynthesis are also in this list (Suppl. Fig. S5B and Supp. Table 1). Remarkably, *PLT5*, whose expression dynamics in branching root segments is dependent on PUCHI (this work), was associated to PUCHI-binding sites identified by DAP-seq analysis. Thus, altogether these results suggest that the early expressed LRP regulator PLT5 may also be a direct target gene of PUCHI during lateral root development.

LRP development progression in a *puchi-1* mutant background has previously been shown to be delayed (Hirota *et al.*, 2007; Trinh *et al.*, 2019). To assess the role played by this developmental delay in the transcriptomic gap observed between *puchi-1* and WT LRPs, we analysed the expression profiles of selected genes whose time-dependent expression dynamics during LRP
366 development have previously been documented (Lavenus et al., 2015; Nagata et al., 2021). In 367 most cases, observed changes in gene expression kinetics between both genotypes were not 368 consistent with, simply, shifted developmental time courses (Supp. Fig. S6 and S7). Taken 369 together, our data confirm that PUCHI activity is required for correct LRP development progression 370 and suggests this transcription factor induces the expression of *CRF1*, *PLT5* and *PLT7* genes and 371 represses the expression of several meristem-specific genes during the LRP early morphogenetic 372 phase. 373

374 Meristematic PLT genes are induced prematurely in *puchi-1* LRP

375 It was previously reported that the transcription factors PLT3, PLT5 and PLT7 control the induction 376 of *PLT1*, *PLT2* and *PLT4* in developing LRP (Du and Scheres, 2017). Unexpectedly, *PLT1* and 377 *PLT4* were overexpressed and *PLT5* and *PLT7* downregulated in *puchi-1* mutant roots, especially 378 in the early stages of development (Fig. 1D; Supp. Fig. S4A). To analyse the impact of *PUCHI* loss 379 of function on the expression pattern of root meristem-related *PLT* genes, transgenic reporter 380 constructs *promPLT1:PLT1-YFP*, *promPLT2:PLT2-YFP*, *promPLT3:PLT3-YFP* and 381 *promPLT4:PLT4-YFP* were introduced into the *puchi-1* mutant background, and their expression 382 pattern compared to that in the WT background (Galinha *et al.*, 2007; Hofhuis *et al.*, 2013). 383 Consistent with our time-course RNAseq results, we observed earlier *PLT1-YFP* and *PLT4-YFP* 384 expression in stages II-III (18h-24h after gravistimulation) in *puchi-1* LRP compared to WT (Fig. 385 *PLT3-YFP*) expression was weaker in *puchi-1* LRPs, while the *PLT2-YFP* 386 construct did not display major changes in expression in *puchi-1* compared to WT before LRP 387 emergence (Supp. Fig. S4B-G and H-M). Hence, our results reveal that PUCHI inhibits expression 388 of *PLT1* and *PLT4*, two key root meristem transcription factors, during the early stages of LRP 389 formation. 390

391 PUCHI represses expression of QC markers during the early LR development phase
During the first phase of LR organogenesis, anticlinal, periclinal and tangential cell divisions progressively generate a 4-layer LRP that grows against the overlying endodermal layer (Malamy et al., 1997; von Wangenheim et al., 2016). A major developmental transition occurs at this stage, during which the LRP first expresses QC marker genes in inner central cells and acquires a dome shape as it breaks through the endodermis (Lucas et al., 2013; von Wangenheim et al., 2016; Goh et al., 2016). The early expression of meristematic PLT genes suggests the transition from the early, morphogenetic to the second, meristematic phase occurred prematurely in the *puchi-1* background. To test this hypothesis and confirm modifications in QC marker gene expressions observed in the RNAseq data (Supp. Fig. S3) (Nawy et al., 2005), we investigated meristem establishment in *puchi-1* LRP using QC marker constructs QC25::CFP and promWOX5::nls:GFP (ten Hove et al., 2010; Goh et al., 2016). Expression of QC25::CFP in Col-0 LRPs was first detected in a few central cells in the second outermost layer from stages IV-V onwards (Fig. 3C, E, G) (Goh et al., 2016; Du and Scheres, 2017). In contrast, in a *puchi-1* loss-of-function background, QC25::CFP expression could be detected in young LRPs as early as stage II (Fig. 3B and 3D), and later in a wider domain compared to WT (Fig. 3G-H). Similarly, promWOX5::nls:GFP was expressed prematurely in stage II in inner cell layers of *puchi-1* LRPs which correlates with WOX5 upregulation observed in *puchi-1* RNAseq data as compared to WT at 18 h after LR induction (Fig. 3I-J; Supp. Fig. S3). Furthermore, the promWOX5::nls:GFP expression domain stretches to inner cell layers, but also to flanking cells at later stages in *puchi-1* LRPs (Fig. 3K-P). Altogether, our observations show that QC marker gene expression occurs earlier and in a wider domain in *puchi-1* LRPs. We conclude that PUCHI is required to delay the activation of QC marker gene expression during LRP development, possibly via repressing PLT1 and PLT4 expression at early stages.

### Auxin and cytokinin signalling patterns are modified in *puchi* LRP

Auxin distribution regulates PLT gene expression and contributes to LRP organisation (Laskowski et al., 2008; Peret et al., 2014; Mähönen et al., 2014; Du and Scheres, 2017; Scheres and Krizek, 2018). Interestingly, our transcriptomic analysis revealed that genes induced by auxin, such as
IAA2, or encoding auxin transporters, such as PIN1, PIN3, PIN4 and PIN7, were upregulated in the early stages (18 hpg-24 hpg) of LR development in *puchi-1* (Fig. 4A and Supp. Fig. S3) (Bishopp *et al.*, 2011). We used the *DR5::GFP* synthetic auxin response reporter to visualise the pattern of auxin signalling in *puchi-1* LRP compared to WT (Friml *et al.*, 2003). As previously described (Benková *et al.*, 2003), the *DR5::GFP* reporter revealed an auxin response gradient in developing WT LRPs with a maximum close to the LRP tip (Fig. 4B, 4D and 4F). In *puchi-1* mutant LRP too, the *DR5::GFP* reporter signal was non-uniformly distributed and displayed predominant expression in the centre of the primordium in contrast to its flanks (Fig. 4C, 4E and 4G). However, in comparison to the WT situation, *DR5::GFP* expression domain was broader and extended closer to the LRP base in *puchi-1*, especially in primordia from stage IV onwards. Thus, in contrast to previous report based on *DR5::GUS* expression pattern (Hirota *et al.*, 2007), our data suggest that PUCHI is required for the establishment of a WT-like auxin signal gradient during LRP development. We therefore investigated the spatial distribution of PIN1 proteins in Col-0 and *puchi-1* LRP using a *promPIN1:PIN1-GFP* construct (Benková *et al.*, 2003). This revealed PIN1 distribution is strongly impaired in *puchi-1* LRP from stage III onwards, displaying a higher signal in inner cell layers compared to WT (Fig. 4H-O).

Previous studies have shown that cytokinin impacts PIN1 polarization in LRP from stage III onwards by reallocating PIN1 to periclinal membranes (Marhavý *et al.*, 2011, 2014). To test whether *puchi-1* LRP defects are correlated with perturbations in cytokinin signalling patterning, the *promTCSn::GFP* reporter was introduced into the *puchi-1* mutant background and its expression compared to that in WT LRPs (Zürcher *et al.*, 2013). In WT, *promTCSn::GFP* expression was not detected in LRPs before stage VII, and from stage VII onwards the expression of this cytokinin signalling reporter was restricted to pro-vascular cells as previously described (Fig. 4P, 4R and 4T (Bielach *et al.*, 2012). In contrast, *promTCSn::GFP* expression was detected in central cells in the inner layers and extended to flanking regions of *puchi-1* LRP from stage II onwards (Fig. 4Q). In later stages, cytokinin signalling was observed in LRP flanks (Fig. 4S and 4U). We conclude that *PUCHI* loss of function compromises the establishment of auxin and cytokinin response patterns in developing LRPs.
**Discussion**

Two successive phases were previously described during the process of LR organogenesis before the emergence of a new LR. During the first, so-called morphogenetic phase, cell proliferation generates a four-cell-layered primordium that grows against the overlying endodermis layer. Only at this stage, at about mid-term of the full developmental time course of the LRP, do anatomic features reminiscent of the meristem stem cell niche become apparent in the centre of the primordium, where expression of QC marker genes initiates (Lavenus et al., 2015; Voß et al., 2015; Goh et al., 2016). Onset of QC marker gene expression at this transition stage is dependent on the previous establishment of the layered pattern that is controlled by the SHR/SCR pathway (von Wangenheim et al., 2016; Goh et al., 2016). Next, during the following developmental steps, the root meristem organisation in the centre of the growing primordium becomes more and more complex while surrounding cells retain LRP boundary characteristics (Malamy et al., 1997; Lavenus et al., 2015). After LR emergence, the QC may maintain the indeterminate growth of the LR by repressing differentiation of the surrounding meristematic cells as it is described in primary root meristems (van den Berg et al., 1995). Many transcriptional regulators were shown to be dynamically expressed during the process of LR organogenesis and may participate in the control of cell proliferation and the acquisition of new cell identities. Remarkably, inference of the gene regulatory network controlling LR development suggested that it is organised in two genetic subnetworks whose mutual inhibition may explain LRP development patterning (Lavenus et al., 2015; Voß et al., 2015). One genetic sub-circuit gathers genes, including those encoding the transcription factors ARF7 and PUCHI, that are expressed at early stages in the whole primordium and only in its base in later stages. The second genetic module gathers transcriptional factors such as ARF5 and PLT4 whose expression spans in the central domain of the primordium where the meristem stem cell niche is being established, only in the second phase of its development (Fig. 5).
The transcription factor PUCHI belongs to the former genetic module and is expressed very early in the developing LRP from stage I (Hirota et al., 2007; Trinh et al., 2019; Goh et al., 2019). Interestingly, the correct timing of PUCHI induction is essential: the sequential induction of ARF7, LBD16, and only then PUCHI, was shown to be critical for the correct progression of LR formation as premature expression of PUCHI disrupted LRP initiation by repressing auxin-induced root branching (Goh et al., 2019). However, how PUCHI function at early stages influences subsequent LRP development remained unclear. Here, we show that meristematic genes, PLT1 and PLT4, as well as root meristem QC marker constructs, QC25::CFP and promWOX5::nls:GFP, are expressed ectopically at earlier stages and in wider domains in *puchi-1* developing LRP as compared to WT. Conversely PLT5 and PLT7, that belong together with PUCHI to the group of genes expressed at early stages of LRP development (Lavenus et al., 2015), are under-expressed in the *puchi-1* loss-of-function background. Although such perturbations are not observed for all the genes in each gene subnetwork, respective changes in PLT5, PLT7, and root meristem genes expression patterns in *puchi-1* mutant LRPs are consistent with the hypothesis that, at the level of the LR gene regulatory network system, early expressed and root meristematic genes belong to two mutually-exclusive sub-circuits (Lavenus et al., 2015). PUCHI participates in the induction of PLT5 and PLT7 expression during lateral root formation, PLT5 likely being a direct target of the transcription factor PUCHI. It also shows for the first time that PUCHI, which is predominantly expressed at early stages of LRP development, is required for the correct timing of meristem establishment and especially represses premature expression of key meristematic genes at early stages of lateral root development, namely PLT1, PLT4, and WOX5. Remarkably, this is clearly different from the role of PLT3, PLT5 and PLT7 transcription factors which were shown to be required for the correct progression of LRP development and its transition to the meristematic phase (Du and Scheres, 2017). This data points to a complex influence of PUCHI on the gene regulatory network controlling lateral root development, as its control over the expression of its direct target genes may translate into intricate effects on the gene system dynamics.

This raises the question of how PUCHI regulates cell identity acquisition in developing LRPs. Interestingly, cross analysis of three lists of PUCHI-dependent genes highlighted *CRF1* as a likely
direct target gene of PUCHI. This is also supported by *in silico* predictions of PUCHI target genes based on correlated, but delayed, expression profiles (Lavenus *et al.*, 2015). *CRF1* encodes an AP2/ERF transcription factor that, redundantly with other CRFs, mediates part of the cytokinin signalling pathway modulating various plant development processes and responses to abiotic stresses, in interaction with other hormonal signalling pathways (Rashotte *et al.*, 2006). Interestingly, phenotypical analyses of multiple mutant combinations and overexpressing lines revealed complex and redundant influence of CRFs on primary root growth and suggested that at least some members promote lateral root formation (Raines *et al.*, 2016). CRF-dependent expression of PIN-family, auxin transporter genes was shown to contribute to the regulation of primary root growth and lateral root formation by cytokinins (Šimášková *et al.*, 2015). The expression pattern of the cytokinin reporter construct *promTCSn::GFP* and the distribution of the PIN1 reporter PIN1:GFP were shown to be altered in *puchi-1* mutant LRPs as compared to WT (Fig. 4). In the RNAseq profiling of LR development, *CRF1* is the only *CRF* gene displaying such expression dynamics typical of the early expressed subnetwork and positively controlled by PUCHI (Supp. Fig. S3). Thus, *CRF1* may represent a key target of PUCHI impacting the cytokinin and auxin-dependent pathways that regulate LRP organogenesis and patterning.

Interestingly, a number of other transcription factors of the AP2/ERF family were identified as putative PUCHI targets supported by both the RNAseq dataset and the DAP-seq dataset (*PLT5*, *ERF2*, *ERF12*, *ERF53*, *ERF113*, *DDF1*) (Supp. Fig. 5B and Supp. Table 1). Transcription factors of the AP2/ERF family are involved in the regulation of a wide range of developmental processes, in many cases in relation with hormone or stress signalling networks (Horstman *et al.*, 2014; Feng *et al.*, 2020). Interestingly, *ERF12* was shown to regulate flower development, possibly by orchestrating the sequential induction and stabilisation of reproductive developmental programs in shoot apices together with PUCHI and related transcription factors (Chandler and Werr, 2020). The expression dynamics of *PLT5* during LR development previously suggested that it was indeed controlled by PUCHI (Lavenus *et al.*, 2005). Redundantly with PLT3 and PLT7, PLT5 was shown to control cell division patterning, correct auxin signal distribution and wild type progression of LRP development including the onset of root meristem gene expression such as *PLT1*, *PLT4* and
529 WOX5 (Du and Scheres, 2017). Both PLT5 and PLT7 expression is downregulated in the *puchi-1* mutant background, and auxin signal distribution and PIN1 localization is also impaired (Fig. 4 and 530 531 Supp. Fig S3). Expression of PLT3 and/or modification in expression of PLT5/7-independent genes 532 in *puchi-1* may explain the observed differences between *plt3/5/7* and *puchi-1* LRP phenotypes.

533 Several genes involved in hormone homeostasis or signalling have also been identified among the 534 PUCHI-dependent genes (Supp. Fig S3, S5B and Supp. Table 1). Auxin and cytokinin are key 535 phytohormones whose roles in functional patterning of meristems and organ primordia have been 536 well described, although in many cases the multiple feedback loops connecting gene regulatory 537 networks and hormonal homeostasis and signalling pathways make any sequential order of 538 molecular events difficult to establish (Chandler and Werr, 2015; Salvi et al., 2020). Here, we show 539 that auxin and cytokinin signalling patterns are impaired in *puchi-1* mutant LRPs. Interestingly, 540 previous reports have shown higher auxin contents in the roots of 5-day-old *puchi-1* seedlings 541 compared to WT (Goh et al., 2019). Alternatively, PUCHI may modulate auxin signalling processes 542 and responses downstream of auxin accumulation, as suggested by the observation that ectopic 543 expression of *PUCHI* in LR founder cells compromises the auxin signal maximum establishment 544 without affecting auxin content (Goh et al., 2019). More specifically, modification of auxin signalling 545 distribution in *puchi-1* mutant LRP correlates with perturbed distribution of PIN1 auxin transporters 546 (Fig. 4A and 4H-O). Formation of an auxin response gradient is critical for proper LRP 547 development (Benková et al., 2003). PLT1/2/3/4 are well-identified auxin-responsive genes that 548 regulate the establishment and maintenance of a root stem cell niche and this, in turn, regulates 549 the expression and localization of PIN transporters and therefore auxin distribution in the root 550 apical meristem and LRP (Benková et al., 2003; Galinha et al., 2007; Prasad et al., 2011; Pinon et 551 al., 2013; Mähönen et al., 2014; Du and Scheres, 2017).

552 Cytokinins repress the expression of the PIN auxin efflux carriers in the root meristem and in LRP 553 (Laplaze et al., 2007; Dello Loio et al., 2008; Růžička et al., 2009; Bishop et al., 2011). In addition, 554 in the early stages of LRP development, cytokinins modulate PIN1 targeting to periclinal versus 555 anticlinal domains of cell membranes (Marhavý et al., 2011, 2014). Cytokinin signal distribution,
556 PIN1 expression levels and PIN1-GFP distribution are all modified in *puchi-1* LRP567. As observed 558 for root apical meristem initiation and maintenance, or when an apical root meristem is regenerated 559 after root tip excision, auxin and cytokinin signals define complementary spatial domains that may 560 contribute to the positioning of the new stem cell niche and guide further LRP patterning (Schaller 561 et al., 2015; Efroni et al., 2016). This pattern is not observed in the *puchi-1* background. Altogether, 562 this suggests that compromised auxin and cytokinin response distribution in *puchi-1* LRP may 563 trigger the early ectopic expression of meristematic genes through a PIN1-regulated, auxin-564 dependent mechanism. Alternatively, misexpression of meristematic genes in *puchi-1* LRP may 565 contribute to disorganize auxin and cytokinin signal distribution and perturb LRP morphogenesis 566 and patterning. 567

566 We recently showed that VLCFA biosynthetic enzymes are downstream targets of PUCHI in 567 developing LRP (Trinh et al., 2019). Interestingly, an increasing body of evidence highlights cross-568 talk between VLCFA and hormones in plant development (Boutté and Jaillais, 2020). For instance, 569 VLCFAs are involved in controlling cell proliferation in shoot organs in a cytokinin-dependent, non-570 cell autonomous manner (Nobusawa et al., 2013; Boutté and Jaillais, 2020). Furthermore, in the 571 VLCFA mutant *pasticcino1* (*pas1*), the abnormal polar distribution of PIN1 in specific cells results in 572 local alteration of auxin distribution and disturbs LRP formation (Roudier et al., 2010). Last, fused 573 proliferating bulges generated by roots of *puchi* or VLCFA-defective mutants on auxin- and 574 cytokinin-supplemented medium (callus inducing medium), closely resemble that produced by 575 roots of mutants affected in polar auxin transport in response to exogenous auxin supply, as well 576 as those produced by cytokinin-overproducing pericycle upon treatment with a highly diffusible 577 auxin analog (Benková et al., 2003; Geldner et al., 2004; Laplaze et al., 2007; Shang et al., 2016; 578 Trinh et al., 2019). Altogether this data suggests tight links between VLCFA, cytokinin and auxin 579 distribution and LRP patterning. Strikingly, two distinct AP2/ERF-controlled cascades targeting 580 VLCFA biosynthetic enzymes during LRP development were recently described, suggesting that 581 AP2/ERF transcription factors may act as master regulators of VLCFA biosynthesis during LRP 582 formation (Trinh et al., 2019; Guyomarc'h et al., 2021; Lv et al., 2021). Last, VLCFA-containing 583 ceramides were recently shown to participate in a feedforward loop maintaining preferential
expression of the transcription factor genes *ATML1* and *PDF2*, key regulators of protodermis/epidermis differentiation, in the outer cell layer of developing LRPs (Nagata *et al.*, 2021). This function may involve direct binding of these VLCFA-containing ceramides to the START domain of the transcription factors (Nagata *et al.*, 2021). *ATML1* and *PDF2* expression dynamics is lost in *puchi-1* mutant LRP (Supp. Fig S7G-H). Thus, we hypothesise that PUCHI-dependent VLCFA regulation could participate actively in patterning mechanisms controlling LRP organisation.

In conclusion, by analysing the impact of *PUCHI* loss of function on the progression of LRP development and functional patterning, we showed that PUCHI is required for the correct timing of meristem establishment and especially, represses premature expression of key root meristem genes at early stages of LRP development. These results are consistent with the model of a LR gene regulatory network organized in distinct genetic sub-circuits whose sequential inhibitions or activations contribute to the transition from an early morphogenetic phase to a second, meristem-establishment phase, as well as to the bifurcation between the central meristematic domain and the flanking domain in the developing organ. How VLCFA-, auxin- or cytokinin-dependent patterning mechanisms may interact during this process downstream of PUCHI will require further investigations.

601

602 Supplementary data

603

A supplementary figure S1 shows the LRP phenotype in Col-0 and *puchi-1* LRP during the time-course transcriptomic analysis after gravistimulation.

A supplementary figure S2 shows the list of all DEGs GO-terms enrichment during the time-course transcriptomic analysis in *puchi-1* compared to Col-0 upon gravistimulation

A supplementary figure S3 provides the heat map of selected gene patterns up- and down-regulated in *puchi-1* compared to Col-0 background during the formation of LRP.
610 A supplementary figure S4 investigates PLTs gene expression analysis in *puchi-1* compared to
611 Col-0 using q-RT PCR and confocal microscopy.

612 A supplementary figure S5 identified the PUCHI direct target genes using available datasets.

613 A supplementary figure S6 shows predicted PUCHI target gene expression patterns in *puchi-1* and
614 Col-0 background during the formation of LRP.

615 A supplementary figure S7 shows selected gene expression patterns in *puchi-1* and Col-0
616 background during the formation of LRP.

617

618 Acknowledgments

619

620 We thank Prof. M.J. Bennett for critical reading of the manuscript. We also thank the "Montpellier
621 Ressources Imagerie" imaging facility and Carine Alcon for providing valuable advice for confocal
622 imaging. We thank Julien Lavenus for seminal discussions about this project. This work was
623 supported by the Institute of Research for Development, the University of Montpellier, and the
624 French Agence Nationale de la Recherche through the NewRoot grant (ANR-17-CE13-0004-01).
625 MGX acknowledges financial support from the France Génomique National infrastructure, funded
626 as part of "Investissement d'Avenir" programme at the Agence Nationale de la Recherche (contract
627 ANR-10-INBS-09).

628

629 Author contributions

630

631 Conceptualisation: L.L., S.G.; Investigation, validation, methodology and formal analysis: K.B., T.-
632 D. C., A.-A. G., E. D., A.-S. P.; Visualization: K.B and S.G.; Writing - original draft: K.B.; Writing -
633 review & editing: K.B., T.D.C., A.-A. G., E. D., M.L., A.C., P.G., L.L., S.G.; Supervision: L.L., S.G.;
634 Funding acquisition: M.L., A.C., P.G., L.L., S.G.
635

636 **Conflict of interest**

637 The authors declare no conflict of interest.

638

639 **Funding**

640 This work was supported by the Institute of Research for Development, the University of
641 Montpellier, and the French Agence Nationale de la Recherche through the NewRoot grant (ANR-
642 17-CE13-0004-01). MGX acknowledges financial support from the France Génomique National
643 infrastructure, funded as part of "Investissement d'Avenir" programme at the Agence Nationale de
644 la Recherche (contract ANR-10-INBS-09).

645

646 **Data availability**

647

648 All data to support the conclusions of this manuscript are included in the main text and the
649 supplementary materials. The full RNA-seq data discussed in this publication have been deposited
650 in the NCBI's Gene Expression Omnibus and are accessible through GEO Series accession
651 number GSEXXXXXX.

652
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**Figure 1. Time-course transcriptomic analysis of LRP formation in Col-0 and *puchi-1* after root gravistimulation.** (A) Schematic diagram of the experimental procedure for the RNAseq analysis. (B) Number of differentially expressed genes (DEGs) in *puchi-1* compared to Col-0 at No LR, 12h, 18h, 24h, 30h and 36h after gravistimulation. Genes with a fold-change of up to Log2: +/- 1; *p*<0.05 selected. (C) Distribution of elementary Gene Ontologies (GO) among either, upregulated genes or downregulated genes in *puchi* was analysed using a PANTHER overrepresentation assay and Fisher test followed by a Bonferroni Correction (*p*<0.05). The Heat map shows the GO terms fold enrichment (FE) of DEGs for each time point. (D) Heat map of selected gene patterns up- and down-regulated in *puchi-1* compared to Col-0 background during the formation of LRP. Log 2-fold change (FC) of expression in *puchi-1* compared to WT is given. Statistical analysis on three independent RNAseq replicates were performed using the DESeq2 package and Wald Test: *p*-value: *p*<0.05; *p***<0.02; *p***<0.01. Colour code for the heatmaps in (C) and (D) are red for upregulated genes and blue for downregulated genes. Colour code for gene names in (D) indicates the module classification (Orange: module1, Morphogenetic phase and Purple: module 2, Meristematic phase) as described in Lavenus *et al.*, 2015.

**Figure 2. *PUCHI* loss of function results in distinct expression patterns of meristematic phase regulator *PLT* genes during LRP development.** (A-F) and (G-L) Expression pattern of *promPLT1:PLT1-YFP* and *promPLT4:PLT4-YFP* (green) respectively in *puchi-1* and Col-0 LR primordia. Stars indicate earlier detected signal expression during LRP outgrowth. Percentages and numbers indicate the occurrence of the represented pattern over the total number of observations. Cell walls were stained using propidium iodide (magenta). Scale bar: 25 µm.

**Figure 3. *PUCHI* loss of function impacts QC marker gene expression patterns during LRP formation.** Confocal microscopy images of expression of QC reporters (A-H) QC25::CFP and (I-P) *promWOX5::nls:GFP* (green) in LRP in Col-0 and *puchi-1*. Stars indicate earlier detected signal expression during LRP outgrowth. Percentages and numbers indicate the occurrence of the
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represented pattern over the total number of observations. Cell walls were stained using propidium iodide (magenta). Scale bar: 25 µm.

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**Figure 4.** *PUCHI* loss of function alters auxin and cytokinin signal distribution in developing LRP. (A) Heat map of selected auxin transporter gene patterns upregulated in *puchi-1* compared to Col-0 background during LRP formation. Log 2-fold change (FC) of expression in *puchi-1* compared to WT is given. Statistical analysis on the three independent RNAseq replicates were performed using the DESeq2 package and Wald Test: *p*-value: *p*<0.05; *p**<0.02; *p***<0.01. (B-G) *DR5::GFP* (green), (H-O) *promPIN1::PIN1:GFP* (green) and (P-U) *promTCSn::GFP* (green)

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expression during LR development. Percentages and numbers indicate the occurrence of the represented pattern over the total number of observations. (B-M) Cell walls were stained using propidium iodide (magenta). (N-U) (Inset) Signal intensity monitor. (blue) low intensity; (red) high intensity. Scale bar: 25 µm.

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**Fig. 5.** *PUCHI*-dependent early repression of meristem establishment. (A) Hypothetical model showing the role of *PUCHI* at early stages of LRP formation, *PUCHI* acts as a key regulator of spatiotemporal distribution of auxin balance and represses specific meristematic module genes.

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**Fig. S1: LRP phenotype of Col-0 and *puchi-1* LRP during the time-course transcriptomic analysis after gravistimulation (hpg).** (A) and (B) Brightfield images showing the LRP stages observed for each timepoint. Time points corresponds to phenotype of the LRP observed in the RNAseq samples. The percentages of occurrence for each LRP stages per time point are shown.

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(C) Distribution of LRP developmental stages after 18, 24, 30 hours post-gravistimulation (hpg) in Col-0 and *puchi-1* roots. Data are one biological replicate for Col-0 and two biological replicates for *puchi-1*. The number of observed seedlings is indicated (n=X).

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**Fig. S2. GO-terms enrichment during the time-course transcriptomic analysis of LR formation in *puchi-1* compared to Col-0 upon gravistimulation.** (A) Distribution of Gene
709 Ontologies among DEGs in *puchi* was analysed using a PANTHER overrepresentation assay and
710 Fisher test followed by a Bonferroni Correction (p*<0.05). The Heat map shows the GO terms fold
711 enrichment of DEGs for each time point.

712

713 **Figure S3. Time-course transcriptomic analysis in Col-0 and *puchi-1* after gravistimulation.**
714 Heat map of selected gene patterns up- and down-regulated in *puchi-1* compared to Col-0
715 background during the formation of LRP. Statistical analysis on three independent RNAseq
716 replicates were performed using the DESeq2 package and Wald Test: p-value: *<0.05; **<0.02;
717 ***<0.01. Colour code for the heatmap is red for upregulated genes and blue for downregulated
718 genes. Colour code for gene names and AGI numbers indicates the module classification (Orange:
719 module1, Morphogenetic phase and Purple: module 2, Meristematic phase) as described in
720 Lavenus *et al.*, 2015.

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722 **Fig. S4. Time-course transcriptomic analysis in Col-0 and *puchi-1* upon gravistimulation.** (A)
723 RT-qPCR analysis of *PLT1*, *PLT2*, *PLT3*, *PLT4*, *PLT5* and *PLT7* gene expression levels 24h after
724 lateral root induction NPA/NAA treatment (Himanen *et al.*, 2002) in *puchi-1* and Col-0. Data are
725 represented as mean ± SEM (standard error of the means), n=3 independent biological replicates.
726 Significance was determined using a Student's t test (* p< 0.05, ** p < 0.01, *** p < 0.001).
727 Expression pattern of (B-G) *promPLT2:PLT2-YFP* and (H-M) *promPLT3:PLT3-YFP* (green) in
728 *puchi-1* and Col-0 LR primordia. Percentages and numbers indicate the occurrence of the
729 represented pattern over the total number of observations. (B-M) Cell membranes were stained
730 using propidium iodide (magenta). Scale bar: 25 µm.

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732 **Figure S5. Identified PUCHI target genes in different datasets.** (A) Venn diagram showing the
733 number of targeted genes by PUCHI using DAPseq (Ronan *et al.*, 2016;
734 http://neomorph.salk.edu/dap_web/pages/index.php), RNAseq analysis in *puchi-1* background
735 after gravistimulation (This paper) and PUCHI-GR inducible system (Trinh *et al.*, 2019). Genes in
736 pPUCHI::PUCHI:GR in *puchi-1* seedlings showed an upregulation (fold change ≥ 1.5, p-value
≤0.05 as determined by Welch two sample t-test) in the following conditions were used: NAA CHX
DEX vs NAA CHX. (GR: GLUCOCORTICOID RECEPTOR; DEX: dexamethasone; CHX:
cycloheximide). (B) The common gene (red) between the three datasets and 25 genes (Auxin-;
cytokinin-; VLCFA-; LR-; meristematic- related and transcription factors) that were common
between two datasets (among a full list of 150 genes, Supp. Table 1) were selected and their AGI
number, aliases and TAIR summary are described in the table.

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Figure S6. Expression patterns of the 8 predicted PUCHI target genes. (A-H) Gene expression
patterns in *puchi-1* and Col-0 background during the formation of LRP. Data are means +/- SE of
three replicates per time point from the transcriptomic dataset. Statistical analysis on RNAseq
replicates were performed using DESeq2 package and Wald Test: p-value: *<0.05; **<0.02;
***<0.01.

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Fig. S7: Selected gene expression analysis in Col-0 and *puchi-1* after gravistimulation. (A-H)
Gene expression patterns in *puchi-1* and Col-0 background during the formation of LRP. Data are
means +/- SE of three replicates per time point from the transcriptomic dataset. Statistical analysis
on RNAseq replicates were performed using DESeq2 package and Wald Test: p-value: *<0.05;
**<0.02; ***<0.01.

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A

Transcriptomic analysis

Col-0 and *puchi-1*

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

B

DEGs in *puchi-1* compared to Col-0

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

C

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

GO biological process in *puchi-1* Upregulated genes

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

D

<figure><img src="image_7.png" /><figcaption>**Figure 1.** Time-course transcriptomic analysis of LRP formation in Col-0 and *puchi-1* after root gravistimulation. (A) Schematic diagram of the experimental procedure for the RNAseq analysis. (B) Number of differentially expressed genes (DEGs) in *puchi-1* compared to Col-0 at No LR, 12h, 18h, 24h, 30h and 36h after gravistimulation. Genes with a fold-change of up to Log2: +/-1; *p*<0.05 selected. (C) Distribution of elementary Gene Ontologies (GO) among either, upregulated genes or downregulated genes in *puchi* was analysed using a PANTHER overrepresentation assay and Fisher test followed by a Bonferroni Correction (p*<0.05). The Heat map shows the GO terms fold enrichment (FE) of DEGs for each time point. (D) Heat map of selected gene patterns up- and down-regulated in *puchi-1* compared to Col-0 background during the formation of LRP. Log 2-fold change (FC) of expression in *puchi-1* compared to WT is given. Statistical analysis on three independent RNAseq replicates were performed using the DESeq2 package and Wald Test: p-value: *p*<0.05; *p*<0.02; *p***<0.01. Colour code for the heatmaps in (C) and (D) are red for upregulated genes and blue for downregulated genes. Colour code for gene names in (D) indicates the module classification (Orange: module1, Morphogenetic phase and Purple: module 2, Meristematic phase) as described in Lavenus *et al.*, 2015.</figcaption></figure>
promPLT1:PLT1-YFP

<figure><img src="image_8.png" /></figure>

promPLT4:PLT4-YFP

<figure><img src="image_9.png" /><figcaption>**Figure 2.** *PUCHI* loss of function results in distinct expression patterns of meristematic phase regulator *PLT* genes during LRP development. (A-F) and (G-L) Expression pattern of *promPLT1:PLT1-YFP* and *promPLT4:PLT4-YFP* (green) respectively in *puchi-1* and Col-0 LR primordia. Stars indicate earlier detected signal expression during LRP outgrowth. Percentages and numbers indicate the occurrence of the represented pattern over the total number of observations. Cell walls were stained using propidium iodide (magenta). Scale bar: 25 µm.</figcaption></figure>
QC25::CFP

promWOX5::nls:GFP

<figure><img src="image_10.png" /><figcaption>**Figure 3.** *PUCHI* loss of function impacts QC marker gene expression patterns during LRP formation. Confocal microscopy images of expression of QC reporters (A-H) *QC25::CFP* and (I-P) *promWOX5::nls:GFP* (green) in LRP in Col-0 and *puchi-1*. Stars indicate earlier detected signal expression during LRP outgrowth. Percentages and numbers indicate the occurrence of the represented pattern over the total number of observations. Cell walls were stained using propidium iodide (magenta). Scale bar: 25 µm.</figcaption></figure>
A

Log2 (FC)

0 3

<table><thead><tr><th rowspan="2">Gene Name</th><th rowspan="2">AGI Number</th><th colspan="4">Log2 FC</th><th colspan="4">p-value</th></tr><tr><th>12h</th><th>18h</th><th>24h</th><th>30h</th><th>12h</th><th>18h</th><th>24h</th><th>30h</th></tr></thead><tbody><tr><td><strong>PIN1</strong></td><td>AT1G73590</td><td>0.24</td><td>0.49</td><td>0.88</td><td>0.65</td><td>*</td><td>***</td><td>***</td><td>***</td></tr><tr><td><strong>PIN4</strong></td><td>AT2G01420</td><td>0.28</td><td>0.17</td><td>0.90</td><td>0.74</td><td>2.9E-01</td><td>3.9E-01</td><td>***</td><td>***</td></tr><tr><td><strong>PIN7</strong></td><td>AT1G23080</td><td>0.22</td><td>0.50</td><td>0.45</td><td>0.54</td><td>2.3E-01</td><td>***</td><td>***</td><td>***</td></tr><tr><td><strong>PIN3</strong></td><td>AT1G70940</td><td>0.47</td><td>0.59</td><td>0.75</td><td>0.67</td><td>***</td><td>***</td><td>***</td><td>***</td></tr><tr><td><strong>LAX3</strong></td><td>AT1G77690</td><td>0.44</td><td>0.42</td><td>0.71</td><td>0.63</td><td>**</td><td>***</td><td>***</td><td>***</td></tr><tr><td><strong>IAA2</strong></td><td>AT3G23030</td><td>0.21</td><td>0.41</td><td>0.34</td><td>0.76</td><td>1.7E-01</td><td>***</td><td>*</td><td>***</td></tr><tr><td><strong>IAA29</strong></td><td>AT4G32280</td><td>1.14</td><td>0.87</td><td>0.60</td><td>1.01</td><td>***</td><td>***</td><td>*</td><td>***</td></tr></tbody></table>

DR5::GFP

promTCSn::GFP

<figure><img src="image_11.png" /></figure>

promPIN1::PIN1:GFP

<figure><img src="image_12.png" /><figcaption>**Figure 4.** *PUCHI* loss of function alters auxin and cytokinin signal distribution in developing LRP. (A) Heat map of selected auxin transporter gene patterns upregulated in *puchi-1* compared to Col-0 background during LRP formation. Log 2-fold change (FC) of expression in *puchi-1* compared to WT is given. Statistical analysis on the three independent RNAseq replicates were performed using the DESeq2 package and Wald Test: *p*-value: *p*<0.05; *p**<0.02; *p***<0.01. (B-G) *DR5::GFP* (green), (H-O) *promPIN1::PIN1:GFP* (green) and (P-U) *promTCSn::GFP* (green) expression during LR development. Percentages and numbers indicate the occurrence of the represented pattern over the total number of observations. (B-U) Cell walls were stained using propidium iodide (magenta). (H-O) (Inset) Signal intensity monitor. (blue) low intensity; (red) high intensity. Scale bar: 25 µm.</figcaption></figure>
A

<figure><img src="image_13.png" /><figcaption>**Fig. 5.** *PUCHI-dependent early repression of meristem establishment.* (A) Hypothetical model showing the role of *PUCHI* at early stages of LRP formation, *PUCHI* acts as a key regulator of spatiotemporal distribution of auxin balance and represses specific meristematic module genes.</figcaption></figure>
A
30hpg (IV: 38; V:54%)

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

B
30hpg (II:54%; III:38%)
24hpg (II:72%; III:28%)

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

<figure><img src="image_16.png" /><figcaption>**Fig. S1:** LRP phenotype of Col-0 and *puchi-1* LRP during the time-course transcriptomic analysis after gravistimulation (hpg). (A) and (B) Brightfield images showing the LRP stages observed for each timepoint. Time points corresponds to phenotype of the LRP observed in the RNAseq samples. The percentages of occurrence for each LRP stages per time point are shown. (C) Distribution of LRP developmental stages after 18, 24, 30 hours post-gravistimulation (hpg) in Col-0 and *puchi-1* roots. Data are one biological replicate for Col-0 and two biological replicates for *puchi-1*. The number of observed seedlings is indicated (n=X).</figcaption></figure>
A

<table><thead><tr><th colspan="7">GO biological process enrichment in <i>puchi-1</i> Downregulated genes</th></tr><tr><th></th><th>No LR</th><th>12h</th><th>18h</th><th>24h</th><th>30h</th><th>36h</th></tr></thead><tbody><tr><td>wax biosynthetic process (GO:0010025)</td><td>0</td><td>0</td><td>36</td><td>0</td><td>27</td><td>23</td></tr><tr><td>cuticle development (GO:0042335)</td><td>43</td><td>0</td><td>41</td><td>13</td><td>37</td><td>22</td></tr><tr><td>suberin biosynthetic process (GO:0010345)</td><td>0</td><td>48</td><td>46</td><td>0</td><td>0</td><td>0</td></tr><tr><td>cutin biosynthetic process (GO:0010143)</td><td>0</td><td>49</td><td>0</td><td>0</td><td>0</td><td>25</td></tr><tr><td>fatty acid biosynthetic process (GO:0006633)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>10</td><td>0</td></tr><tr><td>fatty acid metabolic process (GO:0006631)</td><td>0</td><td>9</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>very long-chain fatty acid metabolic process (GO:0000038)</td><td>0</td><td>0</td><td>25</td><td>0</td><td>19</td><td>13</td></tr><tr><td>lipid transport (GO:0006869)</td><td>9</td><td>0</td><td>15</td><td>5</td><td>10</td><td>7</td></tr><tr><td>response to fatty acid (GO:0070542)</td><td>0</td><td>0</td><td>0</td><td>5</td><td>0</td><td>0</td></tr><tr><td>lipid biosynthetic process (GO:0008610)</td><td>5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>triterpenoid metabolic process (GO:0006722)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>22</td></tr><tr><td>phyllome development (GO:0048827)</td><td>5</td><td>0</td><td>0</td><td>0</td><td>0</td><td>3</td></tr><tr><td>anther development (GO:0048653)</td><td>0</td><td>14</td><td>13</td><td>0</td><td>14</td><td>0</td></tr><tr><td>secondary metabolic process (GO:0019748)</td><td>0</td><td>0</td><td>0</td><td>5</td><td>0</td><td>0</td></tr><tr><td>response to stress (GO:0006950)</td><td>2</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>cellular response to hypoxia (GO:0071456)</td><td>0</td><td>0</td><td>0</td><td>8</td><td>0</td><td>0</td></tr><tr><td>response to salicylic acid (GO:0009751)</td><td>0</td><td>0</td><td>0</td><td>5</td><td>0</td><td>0</td></tr><tr><td>cell differentiation (GO:0030154)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>3</td></tr></tbody><tbody><tr><th colspan="7">GO biological process enrichment in <i>puchi-1</i> Upregulated genes</th></tr><tr><td>root development (GO:0048364)</td><td>0</td><td>0</td><td>0</td><td>5</td><td>0</td><td>0</td></tr><tr><td>anatomical structure morphogenesis (GO:0009653)</td><td>0</td><td>0</td><td>0</td><td>4</td><td>0</td><td>0</td></tr><tr><td>cell growth (GO:0016049)</td><td>0</td><td>0</td><td>0</td><td>6</td><td>0</td><td>0</td></tr><tr><td>response to hormone (GO:0009725)</td><td>0</td><td>0</td><td>0</td><td>4</td><td>0</td><td>0</td></tr><tr><td>response to auxin (GO:0009733)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>6</td></tr><tr><td>regulation of hormone levels (GO:0010817)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>6</td></tr><tr><td>response to organic substance (GO:0010033)</td><td>4</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>response to wounding (GO:0009611)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>7</td><td>0</td></tr><tr><td>cellular response to endogenous stimulus (GO:0071495)</td><td>0</td><td>0</td><td>0</td><td>5</td><td>0</td><td>0</td></tr><tr><td>response to jasmonic acid (GO:0009753)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>9</td><td>0</td></tr><tr><td>response to fatty acid (GO:0070542)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>9</td><td>0</td></tr><tr><td>regulation of transcription, DNA-templated (GO:0006355)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>3</td></tr></tbody></table>

**Fig. S2. GO-terms enrichment during the time-course transcriptomic analysis of LR formation in *puchi-1* compared to Col-0 upon gravistimulation.** (A) Distribution of Gene Ontologies among DEGs in *puchi* was analysed using a PANTHER overrepresentation assay and Fisher test followed by a Bonferroni Correction (p*<0.05). The Heat map shows the GO terms fold enrichment of DEGs for each time point.
<table><thead><tr><th rowspan="2">Gene Name</th><th rowspan="2">AGI Number</th><th colspan="5">Log2 FC</th><th colspan="5">p-value</th></tr><tr><th>No LR</th><th>12h</th><th>18h</th><th>24h</th><th>30h</th><th>36h</th><th>No LR</th><th>12h</th><th>18h</th><th>24h</th><th>30h</th><th>36h</th></tr></thead><tbody><tr><td colspan="14" style="text-align: left; font-weight: bold;">QC specific</td></tr><tr><td>NAP</td><td>AT1G69490</td><td>2,33</td><td>-0,06</td><td>1,08</td><td>0,14</td><td>2,98</td><td>2,02</td><td>***</td><td>9,4E-01</td><td>9,4E-02</td><td>7,9E-01</td><td>***</td><td>***</td></tr><tr><td>WIP4</td><td>AT3G20880</td><td>0,78</td><td>0,83</td><td>5,13</td><td>4,41</td><td>1,24</td><td>0,68</td><td>3,9E-01</td><td>5,6E-01</td><td>***</td><td>***</td><td>*</td><td>8,1E-02</td></tr><tr><td>DUF9</td><td>AT5G23780</td><td>0,48</td><td>0,36</td><td>1,10</td><td>0,48</td><td>0,61</td><td>0,14</td><td>3,7E-01</td><td>4,2E-01</td><td>***</td><td>1,1E-01</td><td>1,3E-01</td><td>6,4E-01</td></tr><tr><td>NPY4</td><td>AT2G23050</td><td>0,48</td><td>0,39</td><td>0,88</td><td>1,96</td><td>1,19</td><td>0,72</td><td>5,6E-01</td><td>6,1E-01</td><td>***</td><td>***</td><td>***</td><td>***</td></tr><tr><td>APL4</td><td>AT2G21590</td><td>-0,29</td><td>0,34</td><td>1,12</td><td>1,18</td><td>1,06</td><td>0,11</td><td>7,6E-01</td><td>5,4E-01</td><td>*</td><td>***</td><td>***</td><td>8,0E-01</td></tr><tr><td>WOX5</td><td>AT3G11260</td><td>0,10</td><td>0,55</td><td>0,64</td><td>-0,50</td><td>-0,75</td><td>-0,98</td><td>7,8E-01</td><td>1,1E-01</td><td>***</td><td>*</td><td>***</td><td>***</td></tr><tr><td colspan="14" style="text-align: left; font-weight: bold;">Morphogenesis</td></tr><tr><td>FEZ</td><td>AT1G26870</td><td>0,18</td><td>0,39</td><td>-0,18</td><td>2,13</td><td>1,22</td><td>-0,70</td><td>8,6E-01</td><td>6,3E-01</td><td>8,0E-01</td><td>*</td><td>***</td><td>*</td></tr><tr><td>SHR</td><td>AT4G37650</td><td>0,14</td><td>0,06</td><td>0,31</td><td>0,46</td><td>0,33</td><td>0,36</td><td>5,3E-01</td><td>6,0E-01</td><td>***</td><td>***</td><td>***</td><td>***</td></tr><tr><td>SCR</td><td>AT3G54220</td><td>-0,07</td><td>0,06</td><td>-0,07</td><td>0,27</td><td>-0,06</td><td>-0,04</td><td>8,2E-01</td><td>6,5E-01</td><td>5,3E-01</td><td>6,4E-02</td><td>5,7E-01</td><td>7,3E-01</td></tr><tr><td>WER/MYB66</td><td>AT5G14750</td><td>-1,06</td><td>0,55</td><td>-0,17</td><td>0,65</td><td>-1,38</td><td>-2,44</td><td>4,3E-01</td><td>2,7E-01</td><td>6,2E-01</td><td>3,1E-01</td><td>***</td><td>***</td></tr><tr><td>MIR166b</td><td>AT3G61897</td><td>0,08</td><td>0,04</td><td>0,55</td><td>-0,18</td><td>-1,11</td><td>1,39</td><td>9,6E-01</td><td>9,7E-01</td><td>4,5E-01</td><td>8,4E-01</td><td>3,1E-01</td><td>7,4E-02</td></tr><tr><td>KAN4/ATS</td><td>AT5G42630</td><td>-1,27</td><td>0,10</td><td>-0,63</td><td>-0,98</td><td>-2,02</td><td>-1,64</td><td>**</td><td>7,6E-01</td><td>*</td><td>***</td><td>***</td><td>***</td></tr><tr><td colspan="14" style="text-align: left; font-weight: bold;">Auxin transport</td></tr><tr><td>LAX3</td><td>AT1G77690</td><td>0,41</td><td>0,44</td><td>0,42</td><td>0,71</td><td>0,63</td><td>0,48</td><td>2,7E-01</td><td>**</td><td>***</td><td>***</td><td>***</td><td>***</td></tr><tr><td colspan="14" style="text-align: left; font-weight: bold;">Auxin signalling</td></tr><tr><td>ARF2</td><td>AT5G62000</td><td>0,36</td><td>0,06</td><td>0,18</td><td>0,09</td><td>0,22</td><td>0,37</td><td>***</td><td>5,9E-01</td><td>1,6E-01</td><td>4,7E-01</td><td>***</td><td>***</td></tr><tr><td>MP/ARF5</td><td>AT1G19850</td><td>-0,13</td><td>-0,10</td><td>0,08</td><td>0,11</td><td>0,44</td><td>0,24</td><td>7,0E-01</td><td>6,7E-01</td><td>5,6E-01</td><td>3,2E-01</td><td>***</td><td>1,1E-01</td></tr><tr><td>ARF7</td><td>AT5G20730</td><td>-0,11</td><td>-0,07</td><td>-0,06</td><td>-0,02</td><td>-0,02</td><td>0,00</td><td>5,5E-01</td><td>5,6E-01</td><td>5,2E-01</td><td>7,6E-01</td><td>8,2E-01</td><td>9,9E-01</td></tr><tr><td>ARF9</td><td>AT4G23980</td><td>0,24</td><td>0,10</td><td>0,24</td><td>0,05</td><td>0,15</td><td>0,22</td><td>2,1E-01</td><td>3,2E-01</td><td>6,6E-02</td><td>6,3E-01</td><td>1,6E-01</td><td>6,8E-02</td></tr><tr><td>ARF11</td><td>AT2G46530</td><td>0,14</td><td>0,09</td><td>0,31</td><td>-0,07</td><td>0,20</td><td>0,18</td><td>8,2E-01</td><td>7,1E-01</td><td>2,1E-01</td><td>7,2E-01</td><td>3,4E-01</td><td>3,9E-01</td></tr><tr><td>ARF19</td><td>AT1G19220</td><td>0,13</td><td>0,14</td><td>0,17</td><td>0,35</td><td>0,17</td><td>0,27</td><td>4,8E-01</td><td>2,4E-01</td><td>8,9E-02</td><td>***</td><td>9,5E-02</td><td>*</td></tr><tr><td>ETT</td><td>AT2G33860</td><td>-0,03</td><td>0,08</td><td>0,05</td><td>-0,28</td><td>-0,06</td><td>-0,06</td><td>8,6E-01</td><td>5,7E-01</td><td>7,2E-01</td><td>4,3E-02</td><td>***</td><td>4,7E-01</td></tr><tr><td>ARF4</td><td>AT5G60450</td><td>-0,11</td><td>0,52</td><td>0,49</td><td>-0,02</td><td>0,24</td><td>0,10</td><td>3,8E-01</td><td>***</td><td>***</td><td>8,7E-01</td><td>*</td><td>4,2E-01</td></tr><tr><td>ARF6</td><td>AT1G30330</td><td>0,06</td><td>0,21</td><td>0,24</td><td>0,26</td><td>0,09</td><td>-0,07</td><td>8,7E-01</td><td>1,6E-01</td><td>*</td><td>*</td><td>3,8E-01</td><td>5,4E-01</td></tr><tr><td>ARF17</td><td>AT1G77850</td><td>0,16</td><td>0,25</td><td>0,24</td><td>-0,12</td><td>-0,06</td><td>0,05</td><td>3,7E-01</td><td>8,2E-02</td><td>8,1E-02</td><td>3,5E-01</td><td>7,3E-01</td><td>7,3E-01</td></tr><tr><td>IAA11</td><td>AT4G28640</td><td>0,09</td><td>0,14</td><td>0,22</td><td>0,25</td><td>0,43</td><td>0,58</td><td>7,5E-01</td><td>2,8E-01</td><td>1,9E-01</td><td>*</td><td>***</td><td>***</td></tr><tr><td>IAA13</td><td>AT2G33310</td><td>0,51</td><td>0,32</td><td>0,60</td><td>0,55</td><td>0,53</td><td>0,51</td><td>***</td><td>***</td><td>***</td><td>***</td><td>***</td><td>***</td></tr><tr><td>IAA2</td><td>AT3G23030</td><td>0,86</td><td>0,21</td><td>0,41</td><td>0,34</td><td>0,76</td><td>1,06</td><td>***</td><td>1,7E-01</td><td>***</td><td>*</td><td>***</td><td>***</td></tr><tr><td>IAA29</td><td>AT4G32280</td><td>1,21</td><td>1,14</td><td>0,87</td><td>0,60</td><td>1,01</td><td>1,71</td><td>1,0E-01</td><td>***</td><td>***</td><td>*</td><td>***</td><td>***</td></tr><tr><td>IAA5</td><td>AT1G15580</td><td>-0,32</td><td>0,84</td><td>1,04</td><td>1,33</td><td>0,53</td><td>1,06</td><td>6,3E-01</td><td>1,1E-01</td><td>8,7E-02</td><td>8,6E-02</td><td>3,4E-01</td><td>*</td></tr><tr><td>SLR/IAA14</td><td>AT4G14550</td><td>0,19</td><td>0,23</td><td>-0,01</td><td>0,50</td><td>0,32</td><td>-0,03</td><td>4,4E-01</td><td>1,0E-01</td><td>9,3E-01</td><td>***</td><td>*</td><td>8,9E-01</td></tr><tr><td>IAA19</td><td>AT3G15540</td><td>0,36</td><td>0,30</td><td>0,54</td><td>-0,17</td><td>0,25</td><td>0,12</td><td>5,1E-01</td><td>1,5E-01</td><td>***</td><td>1,2E-01</td><td>2,4E-01</td><td>5,4E-01</td></tr><tr><td>SHY2</td><td>AT1G04240</td><td>0,80</td><td>0,15</td><td>0,32</td><td>0,41</td><td>0,59</td><td>0,71</td><td>***</td><td>3,8E-01</td><td>6,2E-02</td><td>***</td><td>***</td><td>***</td></tr><tr><td colspan="14" style="text-align: left; font-weight: bold;">Auxin homeostasis</td></tr><tr><td>GH3.1</td><td>AT2G14960</td><td>1,19</td><td>0,94</td><td>1,51</td><td>0,49</td><td>0,42</td><td>0,12</td><td>***</td><td>***</td><td>***</td><td>***</td><td>*</td><td>5,0E-01</td></tr><tr><td>YDK1/GH3.2</td><td>AT4G37390</td><td>1,53</td><td>0,47</td><td>0,77</td><td>0,55</td><td>0,59</td><td>1,17</td><td>***</td><td>***</td><td>*</td><td>***</td><td>1,1E-01</td><td>***</td></tr><tr><td>GH3.3</td><td>AT2G23170</td><td>2,32</td><td>0,61</td><td>0,88</td><td>1,09</td><td>1,60</td><td>2,08</td><td>***</td><td>4,0E-01</td><td>***</td><td>***</td><td>***</td><td>***</td></tr><tr><td>GH3.5</td><td>AT4G27260</td><td>1,24</td><td>0,85</td><td>1,17</td><td>0,87</td><td>0,80</td><td>1,09</td><td>***</td><td>***</td><td>***</td><td>***</td><td>***</td><td>***</td></tr><tr><td>DA01</td><td>AT1G14130</td><td>0,13</td><td>-0,57</td><td>-0,87</td><td>-0,39</td><td>-0,36</td><td>-0,19</td><td>3,9E-01</td><td>***</td><td>***</td><td>**</td><td>***</td><td>1,3E-01</td></tr><tr><td colspan="14" style="text-align: left; font-weight: bold;">VLCFA and Cuticle</td></tr><tr><td>CDEF1</td><td>AT4G30140</td><td>1,07</td><td>-0,71</td><td>0,43</td><td>0,20</td><td>0,49</td><td>1,19</td><td>4,6E-01</td><td>*</td><td>5,2E-01</td><td>8,2E-01</td><td>**</td><td>***</td></tr><tr><td>MPK14</td><td>AT4G36450</td><td>0,29</td><td>0,46</td><td>0,62</td><td>0,71</td><td>1,15</td><td>0,56</td><td>4,8E-01</td><td>1,8E-01</td><td>1,2E-01</td><td>*</td><td>*</td><td>2,1E-01</td></tr><tr><td>ERF13</td><td>AT2G44840</td><td>0,51</td><td>-0,57</td><td>-0,31</td><td>-1,81</td><td>0,92</td><td>0,12</td><td>3,3E-01</td><td>6,0E-01</td><td>8,3E-01</td><td>1,6E-01</td><td>1,6E-01</td><td>9,4E-01</td></tr><tr><td colspan="14" style="text-align: left; font-weight: bold;">Signalling</td></tr><tr><td>GLV6</td><td>AT2G03830</td><td>-0,16</td><td>0,77</td><td>1,11</td><td>1,11</td><td>0,11</td><td>-0,72</td><td>8,5E-01</td><td>**</td><td>***</td><td>***</td><td>***</td><td>7,6E-01</td></tr><tr><td>MAKR4</td><td>AT2G39370</td><td>0,88</td><td>0,64</td><td>0,90</td><td>1,20</td><td>1,00</td><td>1,99</td><td>1,5E-01</td><td>***</td><td>***</td><td>***</td><td>***</td><td>***</td></tr><tr><td>MKK4</td><td>AT1G51660</td><td>-0,03</td><td>-0,27</td><td>-0,08</td><td>-0,52</td><td>-0,04</td><td>0,06</td><td>9,4E-01</td><td>2,3E-01</td><td>8,0E-01</td><td>*</td><td>8,0E-01</td><td>8,2E-01</td></tr><tr><td>MKK5</td><td>AT3G21220</td><td>-0,37</td><td>-0,19</td><td>-0,21</td><td>-0,34</td><td>-0,01</td><td>-0,31</td><td>7,6E-02</td><td>2,7E-01</td><td>1,4E-01</td><td>*</td><td>9,7E-01</td><td>***</td></tr><tr><td>MPK3</td><td>AT3G45640</td><td>0,32</td><td>-0,56</td><td>-0,25</td><td>-0,99</td><td>0,51</td><td>0,06</td><td>5,4E-01</td><td>3,7E-01</td><td>7,3E-01</td><td>***</td><td>8,9E-02</td><td>9,2E-01</td></tr><tr><td>TOLS2</td><td>AT4G37295</td><td>0,32</td><td>0,56</td><td>0,67</td><td>0,34</td><td>0,22</td><td>0,55</td><td>1,9E-01</td><td>**</td><td>***</td><td>*</td><td>1,5E-01</td><td>***</td></tr><tr><td colspan="14" style="text-align: left; font-weight: bold;">Transcription factors</td></tr><tr><td>FLP/MYB124</td><td>AT1G14350</td><td>-0,65</td><td>-0,77</td><td>-0,68</td><td>-0,71</td><td>-0,39</td><td>-0,54</td><td>1,5E-01</td><td>***</td><td>***</td><td>***</td><td>4,3E-02</td><td>***</td></tr><tr><td>GATA23</td><td>AT5G26930</td><td>1,08</td><td>1,55</td><td>1,79</td><td>2,80</td><td>2,38</td><td>3,57</td><td>4,5E-01</td><td>***</td><td>***</td><td>***</td><td>***</td><td>***</td></tr><tr><td>LBD16</td><td>AT2G42430</td><td>-0,09</td><td>-0,93</td><td>-0,89</td><td>-1,11</td><td>-0,86</td><td>-0,44</td><td>8,3E-01</td><td>***</td><td>***</td><td>***</td><td>***</td><td>***</td></tr><tr><td>LBD18</td><td>AT2G45420</td><td>0,09</td><td>0,27</td><td>0,59</td><td>0,20</td><td>0,42</td><td>0,20</td><td>8,5E-01</td><td>1,5E-01</td><td>***</td><td>3,5E-01</td><td>*</td><td>4,8E-01</td></tr><tr><td>LBD29</td><td>AT3G58190</td><td>0,94</td><td>0,39</td><td>0,62</td><td>0,82</td><td>0,99</td><td>1,13</td><td>***</td><td>1,7E-01</td><td>***</td><td>***</td><td>***</td><td>***</td></tr><tr><td colspan="14" style="text-align: left; font-weight: bold;">Cytokinins</td></tr><tr><td>CRF1</td><td>AT4G11140</td><td>1,68</td><td>-2,64</td><td>-2,88</td><td>2,40</td><td>2,50</td><td>-2,24</td><td>***</td><td>***</td><td>***</td><td>***</td><td>***</td><td>***</td></tr><tr><td>CRF3</td><td>AT5G53290</td><td>0,09</td><td>0,58</td><td>0,71</td><td>0,54</td><td>0,65</td><td>0,38</td><td>7,0E-01</td><td>***</td><td>***</td><td>***</td><td>***</td><td>***</td></tr><tr><td>CRF2</td><td>AT4G23750</td><td>0,56</td><td>0,48</td><td>0,67</td><td>0,40</td><td>0,16</td><td>-0,18</td><td>1,9E-01</td><td>5,7E-02</td><td>***</td><td>***</td><td>2,5E-01</td><td>2,8E-01</td></tr><tr><td>CRF4</td><td>AT4G27950</td><td>-0,51</td><td>0,29</td><td>0,22</td><td>0,66</td><td>0,38</td><td>-0,21</td><td>2,4E-01</td><td>4,9E-01</td><td>4,2E-01</td><td>*</td><td>7,7E-02</td><td>5,4E-01</td></tr><tr><td>APT5</td><td>AT5G11160</td><td>-1,05</td><td>0,10</td><td>0,23</td><td>-0,14</td><td>-0,14</td><td>-0,52</td><td>1,4E-01</td><td>9,1E-01</td><td>6,3E-01</td><td>6,3E-01</td><td>6,1E-01</td><td>6,9E-02</td></tr><tr><td>AHP6</td><td>AT1G80100</td><td>-1,59</td><td>-1,24</td><td>-1,02</td><td>-0,87</td><td>-0,37</td><td>-1,56</td><td>***</td><td>5,6E-02</td><td>*</td><td>7,9E-02</td><td>4,5E-01</td><td>***</td></tr><tr><td>ARR5</td><td>AT3G48100</td><td>-0,73</td><td>-0,53</td><td>-0,12</td><td>0,10</td><td>0,02</td><td>-0,85</td><td>8,3E-02</td><td>3,7E-01</td><td>5,2E-01</td><td>9,5E-01</td><td>***</td><td>***</td></tr><tr><td>ARR14</td><td>AT2G01760</td><td>-0,04</td><td>-0,51</td><td>0,18</td><td>0,09</td><td>0,38</td><td>0,03</td><td>9,6E-01</td><td>3,0E-01</td><td>7,5E-01</td><td>8,0E-01</td><td>2,5E-01</td><td>8,9E-01</td></tr><tr><td>ARR18</td><td>AT5G58080</td><td>1,38</td><td>N/A</td><td>N/A</td><td>-0,46</td><td>-0,04</td><td>N/A</td><td>2,7E-01</td><td>N/A</td><td>N/A</td><td>7,1E-01</td><td>9,8E-01</td><td>N/A</td></tr><tr><td>ARR15</td><td>AT1G74890</td><td>-2,12</td><td>0,42</td><td>0,58</td><td>1,15</td><td>-0,99</td><td>-0,45</td><td>4,3E-01</td><td>2,1E-01</td><td>1,6E-02</td><td>1,9E-01</td><td>7,0E-01</td><td>***</td></tr><tr><td>ARR7</td><td>AT1G19050</td><td>-0,15</td><td>-0,07</td><td>-0,07</td><td>-0,13</td><td>-0,15</td><td>-0,67</td><td>8,6E-01</td><td>7,5E-01</td><td>7,2E-01</td><td>4,4E-01</td><td>4,4E-01</td><td>***</td></tr><tr><td>IPPT7</td><td>AT3G23630</td><td>-0,60</td><td>-0,45</td><td>-0,50</td><td>-0,64</td><td>-0,56</td><td>0,61</td><td>1,7E-01</td><td>*</td><td>***</td><td>***</td><td>***</td><td>***</td></tr><tr><td>IPPT1</td><td>AT1G68460</td><td>-2,00</td><td>0,62</td><td>1,47</td><td>0,40</td><td>-1,01</td><td>2,40</td><td>2,4E-01</td><td>6,8E-01</td><td>2,3E-01</td><td>7,4E-01</td><td>4,4E-01</td><td>*</td></tr><tr><td>IPPT3</td><td>AT3G63110</td><td>-0,62</td><td>0,06</td><td>-0,06</td><td>-0,34</td><td>-0,42</td><td>-0,27</td><td>6,7E-02</td><td>7,3E-01</td><td>7,0E-01</td><td>1,0E-01</td><td>**</td><td>2,7E-01</td></tr><tr><td>CKK1</td><td>AT2G41510</td><td>0,24</td><td>-0,24</td><td>0,01</td><td>0,89</td><td>0,82</td><td>1,31</td><td>6,8E-01</td><td>4,3E-01</td><td>9,6E-01</td><td>***</td><td>***</td><td>***</td></tr><tr><td>CKK6</td><td>AT3G63440</td><td>0,72</td><td>0,94</td><td>1,23</td><td>1,21</td><td>1,21</td><td>1,18</td><td>***</td><td>***</td><td>***</td><td>***</td><td>***</td><td>***</td></tr><tr><td>CKK5</td><td>AT1G75450</td><td>-0,71</td><td>0,12</td><td>0,16</td><td>-0,26</td><td>-0,09</td><td>-0,47</td><td>**</td><td>6,7E-01</td><td>2,5E-01</td><td>1,7E-01</td><td>7,1E-01</td><td>**</td></tr><tr><td>CKK2</td><td>AT2G19500</td><td>-0,37</td><td>N/A</td><td>N/A</td><td>N/A</td><td>-1,75</td><td>1,77</td><td>8,7E-01</td><td>N/A</td><td>N/A</td><td>N/A</td><td>4,0E-01</td><td>1,2E-01</td></tr><tr><td>LOG3</td><td>AT2G37210</td><td>1,32</td><td>0,29</td><td>1,80</td><td>1,42</td><td>1,74</td><td>0,86</td><td>*</td><td>4,9E-01</td><td>***</td><td>***</td><td>***</td><td>***</td></tr></tbody></table>

<figure><img src="image_17.png" /></figure>

Module 1 (Lavenus *et al.*, 2015)

Module 2 (Lavenus *et al.*, 2015)

<caption>**Figure S3.** Time-course transcriptomic analysis in Col-0 and *puchi-1* after gravistimulation. Heat map of selected gene patterns up- and down-regulated in *puchi-1* compared to Col-0 background during the formation of LRP. Statistical analysis on three independent RNAseq replicates were performed using the DESeq2 package and Wald Test: p-value: *<0.05; **<0.02; ***<0.01. Colour code for the heatmap is red for upregulated genes and blue for downregulated genes. Colour code for gene names and AGI numbers indicates the module classification (Orange: module1, Morphogenetic phase and Purple: module 2, Meristematic phase) as described in Lavenus *et al.*, 2015.</caption>
A

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /><figcaption>**Fig. S4. Time-course transcriptomic analysis in Col-0 and *puchi-1* upon gravistimulation.** (A) RT-qPCR analysis of *PLT1*, *PLT2*, *PLT3*, *PLT4*, *PLT5* and *PLT7* gene expression levels 24h after lateral root induction NPA/NAA treatment (Himanen *et al.*, 2002) in *puchi-1* and Col-0. Data are represented as mean ± SEM (standard error of the means), n=3 independent biological replicates. Significance was determined using a Student's t test (*p* < 0.05, **p** < 0.01, ***p** < 0.001). Expression pattern of (B-G) *promPLT2:PLT2-YFP* and (H-M) *promPLT3:PLT3-YFP* (green) in *puchi-1* and Col-0 LR primordia. Percentages and numbers indicate the occurrence of the represented pattern over the total number of observations. (B-M) Cell membranes were stained using propidium iodide (magenta). Scale bar: 25 µm.</figcaption></figure>
A

<figure><img src="image_20.png" /></figure>

DEG RNAseq PUCHI_12h, 18h, 24h, 30h FC ≥ Log2: +/-1; p-value<0.05
(Bellande et al., this paper)

PUCHI-GR (Trinh et al., 2019)

B

<table><thead><tr><th>AGI number</th><th>TAIR Aliases</th><th>TAIR Summary</th></tr></thead><tbody><tr><td>AT4G11140</td><td>CRF1</td><td>Encodes a member of the ERF (ethylene response factor) subfamily B-5 of ERF/AP2 transcription factor family. The protein contains one AP2 domain. There are 7 members in this subfamily. CRF proteins relocalize to the nucleus in response to cytokinin.</td></tr><tr><td>AT3G51060</td><td>SRS1, STY1</td><td>A member of SHI gene family. Arabidopsis thaliana has ten members that encode proteins with a RING finger-like zinc finger motif. Despite being highly divergent in sequence, many of the SHI-related genes are partially redundant in function and synergistically promote gynoecium, stamen and leaf development in Arabidopsis. STY1/STY2 double mutants showed defective style, stigma as well as serrated leaves. Binds to the promoter of YUC4 and YUC8 (binding site ACTCTAC)</td></tr><tr><td>AT1G19790</td><td>SRS7</td><td>A member of SHI gene family. Arabidopsis thaliana has ten members that encode proteins with a RING finger-like zinc finger motif. Despite being highly divergent in sequence, many of the SHI-related genes are partially redundant in function and synergistically promote gynoecium, stamen and leaf development in Arabidopsis.</td></tr><tr><td>AT2G28500</td><td>LBD11</td><td></td></tr><tr><td>AT3G56400</td><td>WRKY70</td><td>Member of WRKY Transcription Factor</td></tr><tr><td>AT4G29230</td><td>NAC075</td><td>NAC domain protein involved in negative regulation of flowering.</td></tr><tr><td>AT5G53950</td><td>ANAC098, CUC2</td><td>Transcriptional activator of the NAC gene family, with CUC1 redundantly required for embryonic apical meristem formation, cotyledon separation and expression of STM. Proper timing of CUC2 expression is required to maintain the phyllotactic pattern initiated in the meristem. CUC2 expression in leaf sinus region is required for serration and the extent of serration is modulated by mir164A mediated repression of CUC2. Together with CUC3-DA1-UBP15 part of a regulatory module which controls the initiation of axillary meristems, thereby determining plant architecture. Regulates the axillary meristem initiation, directly binding to the DA1 promoter.</td></tr><tr><td>AT1G28360</td><td>ERF12</td><td>Encodes a member of the ERF (ethylene response factor) subfamily B-1 of ERF/AP2 transcription factor family (ERF12). The protein contains one AP2 domain. There are 15 members in this subfamily including ATERF-3, ATERF-4, ATERF-7, and leafy petiole. Regulates floral development.</td></tr><tr><td>AT1G12610</td><td>DDF1</td><td>Encodes a member of the DREB subfamily A-1 of ERF/AP2 transcription factor family (DDF1). The protein contains one AP2 domain. There are six members in this subfamily, including CBF1, CBF2, and CBF3. Overexpression of this gene results in delayed flowering and dwarfism, reduction of gibberellic acid biosynthesis, and increased tolerance to high levels of salt. This gene is expressed in all tissues examined, but most abundantly expressed in upper stems. Overexpression of this gene is also correlated with increased expression of GA biosynthetic genes and RD29A (a cold and drought responsive gene). Under salt stress it induces the expression of GAOX7, which encodes ad C20-GA inhibitor.</td></tr><tr><td>AT2G20880</td><td>ERF53</td><td>Encodes ERF53, a drought-induced transcription factor. Belongs to the AP2/ERF superfamily, and has a highly conserved AP2 domain. Regulates drought-responsive gene expressions by binding to the GCC box and/or dehydration-responsive element (DRE) in the promoter of downstream genes. Overexpression of ATERF53 driven by the CaMV35S promoter resulted in an unstable drought-tolerant phenotype in T2 transgenic plants. Involved in heat shock response.</td></tr><tr><td>AT5G24110</td><td>WRKY30</td><td>Member of WRKY Transcription Factor</td></tr><tr><td>AT5G47220</td><td>ERF2</td><td>Encodes a member of the ERF (ethylene response factor) subfamily B-3 of ERF/AP2 transcription factor family (ATERF-2). The protein contains one AP2 domain. Functions as activator of GCC box&Aring;dependent transcription. Positive regulator of JA-responsive defense genes and resistance to F. oxysporum and enhances JA inhibition of root elongation.</td></tr><tr><td>AT1G29670</td><td>GDSL1</td><td>GDSL-motif esterase/acyltransferase/lipase. Enzyme group with broad substrate specificity that may catalyze acyltransfer or hydrolase reactions with lipid and non-lipid substrates. The mRNA is cell-to-cell mobile.</td></tr><tr><td>AT2G38110</td><td>GPAT6</td><td>bifunctional sn-glycerol-3-phosphate 2-O-acyltransferase/phosphatase. Involved in cutin assembly.</td></tr><tr><td>AT3G16370</td><td></td><td>GDSL-motif esterase/acyltransferase/lipase. Enzyme group with broad substrate specificity that may catalyze acyltransfer or hydrolase reactions with lipid and non-lipid substrates. The mRNA is cell-to-cell mobile.</td></tr><tr><td>AT5G56970</td><td>CKX3</td><td>It encodes a protein whose sequence is similar to cytokinin oxidase/dehydrogenase, which catalyzes the degradation of cytokinins.</td></tr><tr><td>AT4G26200</td><td>ACCS7, ACS7</td><td>Member of a family of proteins in Arabidopsis that encode 1-Amino-cyclopropane-1-carboxylate synthase, an enzyme involved in ethylene biosynthesis. Not expressed in response to IAA.</td></tr><tr><td>AT1G51500</td><td>ABCG12, CER5</td><td>Encodes an ABC transporter involved in cuticular wax biosynthesis. Lines carrying recessive mutations in this locus have weakly glaucous stem surface, and relative elevated secondary alcohols and ketones.</td></tr><tr><td>AT1G43160</td><td>ERF113, RAP2.6</td><td>Encodes a member of the ERF (ethylene response factor) subfamily B-4 of ERF/AP2 transcription factor family (RAP2.6). The protein contains one AP2 domain. There are 7 members in this subfamily.</td></tr><tr><td>AT5G57390</td><td>AIL5, PLT5</td><td>Encodes a member of the AP2 family of transcriptional regulators. May be involved in germination and seedling growth. Mutants are resistant to ABA analogs and are resistant to high nitrogen concentrations, essential for the developmental transition between the embryonic and vegetative phases in plants. Overexpression results in the formation of somatic embryos on cotyledons. It is also required to maintain high levels of PIN1 expression at the periphery of the meristem and modulate local auxin production in the central region of the SAM which underlies phyllotactic transitions. Acts redundantly with PLT3 and 7 in lateral root pattern formation.</td></tr><tr><td>AT2G14960</td><td>GH3.1</td><td>Encodes a protein similar to IAA-amido synthases. Lines carrying an insertion in this gene are hypersensitive to auxin.</td></tr><tr><td>AT1G24470</td><td>KCR2</td><td>Encodes one of the two Arabidopsis homologues to YBR159w encoding a S. cerevisiae beta-ketoacyl reductase (KCR), which catalyzes the first reduction during VLCFA (very long chain fatty acids, >18 carbon) elongation: KCR1 (At1g67730), KCR2 (At1g24470). Complementation of the yeast ybr159Delta mutant demonstrated that the two KCR proteins are divergent and that only AtKCR1 can restore heterologous elongase activity similar to the native yeast KCR gene.</td></tr><tr><td>AT1G46264</td><td>HSFB4, SCZ</td><td>Encodes SCHORIZZA, a member of Heat Shock Transcription Factor (Hsf) family. Functions as a nuclear factor regulating asymmetry of stem cell divisions.</td></tr><tr><td>AT1G71030</td><td>MYBL2</td><td>Encodes a putative myb family transcription factor. In contrast to most other myb-like proteins its myb domain consists of a single repeat. A proline-rich region potentially involved in transactivation is found in the C-terminal part of the protein. Its transcript accumulates mainly in leaves.</td></tr><tr><td>AT4G36260</td><td>SRS2, STY2</td><td>A member of SHI gene family. Arabidopsis thaliana has ten members that encode proteins with a RING finger-like zinc finger motif. Despite being highly divergent in sequence, many of the SHI-related genes are partially redundant in function and synergistically promote gynoecium, stamen and leaf development in Arabidopsis. Encodes protein with a single zinc finger motif and a members of a small gene family of putative transcription factors in which the SHORT INTERNODES (SHI) gene is found. STY2/STY1 double mutants showed defective style, stigma as well as serrated leaves.</td></tr></tbody></table>

**Figure S5. Identified PUCHI target genes in different datasets.** (A) Venn diagram showing the number of targeted genes by PUCHI using DAPseq (Ronan et al., 2016; http://neomorph.salk.edu/dap_web/pages/index.php), RNAseq analysis in *puchi-1* background after gravistimulation (This paper) and PUCHI-GR inducible system (Trinh et al., 2019). Genes in *pUCHI::PUCHI:GR* in *puchi-1* seedlings showed an upregulation (fold change ≥ 1.5, p-value ≤0.05 as determined by Welch two sample t-test) in the following conditions: NAA CHX DEX vs NAA CHX. (GR: GLUCOCORTICOID RECEPTOR; DEX: dexamethasone; CHX: cycloheximide). (B) The common gene (red) between the three datasets and 25 genes (Auxin-; cytokinin-; VLCFA-; LR-; meristematic- related and transcription factors) that were common between two datasets (among the 150 genes, see suppl. Table 1 for details) were selected and their AGI number, aliases and TAIR summary are shown in the table.
A

AT1G28360-ERF12

<figure><img src="image_21.png" /></figure>

E

AT4G11140-CRF1

<figure><img src="image_22.png" /></figure>

B

AT1G29670-GDSL1

<figure><img src="image_23.png" /></figure>

F

AT5G56970-CKX3

<figure><img src="image_24.png" /></figure>

C

AT5G57390-PLT5

<figure><img src="image_25.png" /></figure>

G

AT3G51060-STY1

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

D

AT5G53950-CUC2

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

H

AT2G20880-ERF53

<figure><img src="image_28.png" /><figcaption>**Figure S6. Expression patterns of the 8 predicted PUCHI target genes.** (A-H) Gene expression patterns in *puchi-1* and Col-0 background during the formation of LRP. Data are means +/- SE of three replicates per time point from the transcriptomic dataset. Statistical analysis on RNAseq replicates were performed using DESeq2 package and Wald Test: p-value: *<0.05; **<0.02; ***<0.01.</figcaption></figure>
A

PHV

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

B

PHB

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

C

GATA23

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

D

SHI

<figure><img src="image_32.png" /></figure>

E

MYB52

<figure><img src="image_33.png" /></figure>

F

MYB56

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

G

ATML1

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

H

PDF2

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

**Fig. S7: Selected gene expression analysis in Col-0 and *puchi-1* after gravistimulation. (A-H) Gene expression patterns in *puchi-1* and Col-0 background during the formation of LRP. Data are means +/- SE of three replicates per time point from the transcriptomic dataset. Statistical analysis on RNAseq replicates were performed using DESeq2 package and Wald Test: p-value: *<0.05; **<0.02; ***<0.01.**