# Auxin Response Factors (ARFs) are potential mediators of auxin action in tomato response to biotic and abiotic stress (Solanum lycopersicum)

Sarah Bouzroud, Sandra Gouiaa, Nan Hu, Anne Bernadac, Isabelle Mila, Najib Bendaou, Abdelaziz Smouni, Mondher Bouzayen, Mohammed Zouine

<figure><img src="image_1.png" /></figure>

## To cite this version:

Sarah Bouzroud, Sandra Gouiaa, Nan Hu, Anne Bernadac, Isabelle Mila, et al.. Auxin Response Factors (ARFs) are potential mediators of auxin action in tomato response to biotic and abiotic stress (Solanum lycopersicum). PLoS ONE, 2018, 13 (2), 10.1371/journal.pone.0193517. [DOI: 10.1371/journal.pone.0193517](https://doi.org/10.1371/journal.pone.0193517)

HAL Id: hal-02628522

https://hal.inrae.fr/hal-02628522

Submitted on 26 May 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

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

Distributed under a Creative Commons Attribution 4.0 International License
RESEARCH ARTICLE

# Auxin Response Factors (ARFs) are potential mediators of auxin action in tomato response to biotic and abiotic stress (*Solanum lycopersicum*)

Sarah Bouzroud¹,²*, Sandra Gouiaa¹*, Nan Hu¹*, Anne Bernadac¹, Isabelle Mila¹, Najib Bendaou², AbdelAziz Smouni², Mondher Bouzayen¹, Mohamed Zouine¹*

1. GBF, Université de Toulouse, INRA, Castanet-Tolosan, France, 2. Laboratoire de physiologie et biotechnologie Végétales, Centre de biotechnologie végétale et microbienne biodiversité et environnement, Faculté des Sciences, Université Mohammed V de Rabat, Rabat, Maroc

* These authors contributed equally to this work.

* mohamed.zouine@ensat.fr

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

## Abstract

Survival biomass production and crop yield are heavily constrained by a wide range of environmental stresses. Several phytohormones among which abscisic acid (ABA), ethylene and salicylic acid (SA) are known to mediate plant responses to these stresses. By contrast, the role of the plant hormone auxin in stress responses remains so far poorly studied. Auxin controls many aspects of plant growth and development, and Auxin Response Factors play a key role in the transcriptional activation or repression of auxin-responsive genes through direct binding to their promoters. As a mean to gain more insight on auxin involvement in a set of biotic and abiotic stress responses in tomato, the present study uncovers the expression pattern of *SIARF* genes in tomato plants subjected to biotic and abiotic stresses. *In silico* mining of the RNAseq data available through the public TomExpress web platform, identified several *SIARF*s as responsive to various pathogen infections induced by bacteria and viruses. Accordingly, sequence analysis revealed that 5' regulatory regions of these SIARFs are enriched in biotic and abiotic stress-responsive cis-elements. Moreover, quantitative qPCR expression analysis revealed that many SIARFs were differentially expressed in tomato leaves and roots under salt, drought and flooding stress conditions. Further pointing to the putative role of SIARFs in stress responses, quantitative qPCR expression studies identified some miRNA precursors as potentially involved in the regulation of their SIARF target genes in roots exposed to salt and drought stresses. These data suggest an active regulation of SIARFs at the post-transcriptional level under stress conditions. Based on the substantial change in the transcript accumulation of several SIARF genes, the data presented in this work strongly support the involvement of auxin in stress responses thus enabling to identify a set of candidate SIARFs as potential mediators of biotic and abiotic stress responses.

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

### OPEN ACCESS

**Citation:** Bouzroud S, Gouiaa S, Hu N, Bernadac A, Mila I, Bendaou N, et al. (2018) Auxin Response Factors (ARFs) are potential mediators of auxin action in tomato response to biotic and abiotic stress (*Solanum lycopersicum*). PLoS ONE 13(2): e0193517. https://doi.org/10.1371/journal.pone.0193517

**Editor:** Keqiang Wu, National Taiwan University, TAIWAN

**Received:** July 24, 2017

**Accepted:** January 8, 2018

**Published:** February 28, 2018

**Copyright:** © 2018 Bouzroud et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** All relevant data are within the paper and its Supporting Information files.

**Funding:** This research was supported by the "Laboratoire d'Excellence" (LABEX) entitled TULIP (ANR-10-LABX-41) and benefited from networking activities within the European COST Action FA1106, from the ANR TomEpiSet (ANR-16-CE20-0014) project, the TomGEM H2020 project, by Hubert-Curien partnership program Volubilis (MA/12/280-
27105PL) and CMCU program (08G0906) granted to Dr Habib Khoudi. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing interests:** All authors have read, edited, and approved the final version of the manuscript. They have declared that no competing interests exist.

Introduction

Tomato (Solanum lycopersicum) is one of the major crops with significant economic and scien-
tific interests and as in the case of other crop plants, environmental stresses negatively affect
tomato growth, productivity and quality [1]. Developing new tomato cultivars with enhanced
tolerance to abiotic and biotic stress would have a significant impact on global food production
in many regions. Plant responses to environmental stresses are extremely complex and involve
changes at the transcriptome, cellular and physiological levels in order to prevent damage and
ensure survival [2]. Better understanding of the molecular mechanisms by which plants adapt
to constantly changing environmental conditions is a topic of prime interest in the context of
global climate warming. However, our knowledge of the actors and pathways underlying plant
tolerance or susceptibility to environmental stresses remains scarce. Several phytohormones
are known for their role in the response to abiotic and biotic stresses [1]. Abiotic stress
responses are largely controlled by the hormone ABA while defense against different biotic
constraints is specified by antagonism between the salicylic acid (SA) and jasmonic acid (JA)
or ethylene signaling pathways [3,4]. This creates a complex network of interacting pathways
and hormones cross-talk at different levels [5].

Even though auxin is known to control a wide range of plant developmental processes,
strikingly, its putative role in modulating plant growth under stress responses remains poorly
understood. Auxin is involved in different aspects of plant development throughout the plant
life cycle, including apical dominance, tropic responses, vascular development, organ pattern-
ing, flower and fruit development [6–12]. Significant progress has been made towards under-
standing the mechanisms by which this hormone impacts plant growth and development. By
contrast, our knowledge about auxin implication in biotic and abiotic stress responses remains
quite limited [13]. Recent studies including expression profiling suggested that auxin might
act as a regulator of plant responses to environmental stresses [11,14–18].

Auxin regulates the cell-specific transcription of auxin response genes via three types of
transcriptional regulators, Auxin/Indole-Acetic Acid (Aux/IAAs), Auxin Response Factors
(ARFs) and TOPLESS proteins (TPS) [19–21]. In *Arabidopsis*, 23 ARFs were identified based
on the presence of a conserved N-terminal DNA-binding domain (DBD), a variable central
transcriptional regulatory region which can function as activator or a repressor domain, and a
carboxy-terminal dimerization domain (CTD) that contributes to the formation of either
ARF/ARF homo- and hetero-dimers or ARF/Aux/IAA hetero-dimers [21–23]. The DBD
enables ARFs to specifically bind the conserved Auxin Response Element (AuxRE, 5' TGTCTC
3') present in the promoters of various Auxin-regulated genes and in this way to control gene
transcription associated with plant responses to auxin [23–26].

ARF proteins orchestrate several biological and physiological processes such as embryogen-
esis, leaf expansion and senescence, lateral root development and fruit development by regulat-
ing the expression of auxin response genes [27-30]. The ARF gene family has been identified
and well characterized in many crop species, such as Arabidopsis (Arabidopsis thaliana) [31],
Maize (Zea mays) [32], Rice (Oryza sativa) [11,15], Poplar (Populus trichocarpa) [33], Tomato
(Solanum lycopersicum) [21,34,35], Chinese cabbage (Brassica rapa) [36], Sorgho (Sorghum
bicolor) [18], and Banana (Musa acuminata) [37]. In recent years, small RNA, known as
miRNA, have been shown to substantially contribute to the regulation of plant development,
physiology and stress responses [38]. To date, 872 miRNAs, belonging to 42 families, have
been identified in 71 plant species by genetic screening, direct cloning, computational strate-
gies and EST analysis [38,39]. Noteworthy, among the first miRNAs to be identified and char-
acterized, miR160 has sequence complementarities with ARF10, ARF16 and ARF17 [21,40] and
miR167 potentially regulates ARF6A, ARF6B and ARF8A [21,41]. Therefore, ARF expression in
tomato is likely to be regulated at the post-transcriptional level by a large family of miRNAs that are non-coding RNAs playing a critical role in regulating auxin-dependent gene expression at the post-transcriptional level [42,43].

To address the putative involvement of tomato *SlARF* genes in plant responses to stresses we performed in the present study *in silico* analyses of the 5' regulatory regions of using PLACE web plateform (https://sogo.dna.affrc.go.jp/cgi-bin/sogo.cgi?lang=en) [44] and established their expression profiles under biotic stress conditions using TomExpress web platform [45]. We then, assessed the transcript accumulation of *SlARF* genes under salt, drought and flooding stresses using a qPCR experimental approach. We also examined the expression of miRNA precursor genes to check whether their expression is regulated under stress conditions and whether they are involved in post-transcriptional regulation of their target *ARF* genes under these conditions. Overall, the study provides guidance on the implication of auxin signaling pathway in plant responses to biotic and abiotic stresses and defines new targets towards engineering tomato plants better adapted to adverse environmental conditions.

# Materials and methods

## Plant materials

Tomato (*Solanum lycopersicum* cv Micro-Tom) plants of Wild type and transgenic DR5::GUS, pARF8A::GUS and pARF10::GUS lines generated in house (GBF laboratory) were used in this study.

## Histochemical analysis of GUS expression

Transgenic plants expressing pARF8A::GUS and pARF10A::GUS were generated by *A. tume-faciens*-mediated transformation according to Wang et al. (2005) [46]. For that, PCR was performed on the genomic DNA of tomato 'Micro-Tom' (10 ng.ml⁻¹) using specific primers. The corresponding amplified fragment was cloned into the pMDC162 vector containing the GUS reporter gene using Gateway technology (Invitrogen). The cloned *SlARF* promoter was sequenced from both sides using vector primers in order to see whether the end of the promoter is matching with the beginning of the reporter gene. Sequence results were carried out using the Vector NTI (Invitrogen) and ContigExpress software by referring to ARF promoter sequences.

GUS assays were conducted on DR5::GUS, pARF8A::GUS and pARF10::GUS tomato lines. After being surface sterilized, seeds were cultivated in Petri dishes containing half strength Murashige & Skoog medium for 7 days in a growth chamber at 25°C with 16h light/ 8h dark cycle. One week plants were then grown hydroponically for two weeks in BD (BROUGHTON & DILLWORTH) liquid medium [47]. Three week-old plants were subjected to salt and drought treatment. Salt stress was performed by adding 250 mM of NaCl to the culture medium. After 24 hours of the salt stress application, plants were incubated in GUS solution. Drought stress was conducted by adding 15% of PEG 20000 to the liquid culture solution. Plants were collected after 5 days of stress application and were incubated in the GUS solution. For each stress condition, control plants were cultivated in BD liquid medium for the same period.

GUS staining was performed overnight at 37°C in 3 mM XGluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide (Duchefa Biochemie, Haarlem, The Netherlands), 0.1% (v/v) Triton X-100 Sigma, Steinheim, Germany, 8 mM β-mercaptoethanol and 50 mM Na₂HPO₄/NaH₂PO₄ (pH 7.2), then followed by a destaining in EtOH.
## Plant growth and stress conditions

Wild type tomato seeds were sterilized for 10 min in 50% sodium hypochlorite, rinsed four times with sterile distilled water and sown in pots containing peat. Then they were incubated in a culture room with 16h light/ 8h dark photoperiod and 25±2°C temperature. After 3 weeks, plants were subjected to salt, drought and flooding stresses. Salt stress was performed by watering daily the plants with 250mM of NaCl solution. Control plants were daily watered with distilled water. Leaves and roots samples were harvested after 2 and 24 hours of salt stress application. Drought stress was performed on three week-old plants by water holding for 48 hours and for 5 days. Watering continued normally throughout for control plants. Leave and root samples were collected after drought stress application. For flooding stress, three week-old plants were flooded with deionized water which was maintained at the level of cotyledonary node throughout the experiment. After 48 hours, the leaves and roots were harvested. Control plants were daily watered. Three biological replicates were done for each condition and three independent biological replicates were done for each experiment.

## RNA extraction

Total RNA was extracted from leaves and roots samples by using the Plant RNeasy extraction kit (RNeasy Plant Mini Kit, Qiagen, Valencia, CA, USA). To remove any residual genomic DNA, the RNA was treated with an RNase-Free DNase according to the manufacturer's instruction (Ambion® DNA-free™ DNase). The concentration of RNA was accurately quantified by spectrophotometric measurement and 1µg of total RNA was separated on 2% agarose gel to monitor its integrity. DNase-treated RNA (2µg) was then reverse-transcribed in a total volume of 20µl using the Omniscript Reverse Transcription Kit (Qiagen).

## Real time PCR

The real-time quantification of cDNA corresponding to 2µg of total RNA was performed in the ABI PRISM 7900HT sequence detection system (Applied biosystems) using the Quanti-Tech SYBR Green RT-PCR kit (Qiagen). The Gene-specific primers used are listed in S1 Table. The reaction mixture (10µl) contained 2µg of total RNA, 1,2 µM of each primer and appropriate amounts of enzymes and fluorescent dyes as recommended by the manufacturer. Actin gene was used as reference. Real-Time PCR conditions were as follow: 50°C for 2 min, 95°C for 10 min, then 40 cycles of 95°C for 15 s and 60°C for 1 min, and finally one cycle at 95°C for 15 s and 60°C for 15 s. Three independent biological replicates and three technical replicates of each sample were used for real-time PCR analysis. For each data point, the C~T~ value was the average of C~T~ values obtained from the three biological replicates and three technical replicates.

Three stress related genes were used as markers in these experiments. *CI7* gene was used in both leaves and roots for salt and drought stresses [48,49]. For flooding, *ACO1* gene was used as a marker in leaves and *PDC* gene in roots [50].

## Tomato ARF gene expressions under biotic stress conditions

The expression pattern of 22 *SlARF* genes was analyzed by RNAseq technology in several studies and the results presented here were extracted from the TOMEXPRESS database (published online on October 2014) (S2 Table). TOMEXPRESS database includes 16 RNA-seq projects and 124 unique global conditions [45].
## Analysis of Cis-acting regulatory elements

Using database associated search tools, 2 kb of 5' regulatory region of each ARF transcription factor gene in tomato was scanned for the presence of putative cis-acting regulatory elements and motifs registered in Plant PLACE database (https://sogo.dna.affrc.go.jp/cgi-bin/sogo.cgi?lang=en) (consulted in March 22nd, 2015) [44]. The cis-acting elements analyzed are listed in S3 Table.

## Statistical analysis

Data presented in this work are expressed as arithmetic means +/- SD of replicate plants within an experiment. The data shown are representative of a total of three independent biological replicates. The results were statistically analyzed using Student's t test; Statistica software version 10 (Statsoft, Tulsa, USA). A p value of <0,05 was considered statistically significant. In all the figures presented, error bars indicate standard deviation.

## Results

### Analysis of SlARF promoter genes to search for stress response cis-acting regulatory elements

PLACE signal scan analysis revealed the presence of several cis-regulatory elements that are putatively associated with plant response to stress in the 5' regulatory region (2kb upstream region from the translation start codon) of SlARFs (S1 Fig). The names of the identified cis-acting elements and their predicted functions are listed in S3 Table. Some of these elements (CCATBOX1, WBOXNTERF3, RAV1AAT, MYCCONSENSUSAT, GT1GMSCAM4, MYB 2A; MYBCORE and MYBCONCENSUSAT) are common to the majority of the analyzed SlARFs. A high number of these conserved motifs is identified for SlARF1, SlARF2A, SlARF4, SlARF6B, SlARF9A, SlARF16A and SlARF16B whereas a low number are found for SlARF10A and SlARF10B genes.

Abiotic stress associated cis-acting regulatory elements were found for all SlARFs (S1 Fig). MYBCORE cis-acting element related to drought stress response [51] was detected in most of the 22 SlARFs except for SlARF5, SlARF10A and SlARF24 while the MYB1AT [52] motif was only located in 13 of the 22 SlARFs. Noteworthy, MYCONSENSUSAT motif [53] was found in all the 22 SlARFs genes with varying occurrence. MYCATERD1 motif [52] was only found in SlARF1, SlARF6A, SlARF7B, SlARF8A, SlARF8B, SlARF9B, SlARF16A and SlARF24 while the presence of the other MYC binding site "MYCATRD22" [51] was specific to SlARF4, SlARF5, SlARF7A, SlARF7B, SlARF8B, SlARF16A and SlARF16B. Screening for motifs involved in temperature variation showed that with the exception of SlARF8A and SlARF18, all other SlARFs promoter regions were enriched with the two cis-acting elements RAV1AAT and CCAAT BOX1 boxes [54,55]. Cis-acting element associated with biotic stress response such as WBOX NTCHN48, WBOXNTERF3 and GT1GMSCAM4 were located in most SlARF promoters with differences in their occurrence. The WBOXNTERF3 motif, involved in wounding [56], was found in the SlARFs gene promoters except for SlARF3, SlARF7A and SlARF9B while the elicitor binding motif WBOXNTCHN48 [57] was only detected in SlARF2A, SlARF5 and SlARF6B. Finally, the GT1GMSCAM4 motif [58] was present in all the SlARFs promoter regions.

### Tomato ARF responsiveness to biotic stress

We mined the public tomato RNA-Seq web platform TomExpress [59] to examine the expression profile of tomato SlARF genes upon biotic stress. Among the expression data available in the TomExpress platform, we focused on those related to flagellin, bacteria (Pseudomonas
<figure><img src="image_5.png" /><figcaption>**Fig 1.** *SLARFs* gene expression in tomato plant leaves exposed to various biotic stresses: *Flagellin*, *Pseudomonas putida*, *Pseudomonas syringae*, *Pseudomonas fluorescens*, *Agrobacterium tumefaciens* and *Yellow curl virus*. All the data presented here were extracted from TOMPEXPRESS database (http://gbf.toulouse.inra.fr/tomexpress).</figcaption></figure>

https://doi.org/10.1371/journal.pone.0193517.g001

*putida*, *Pseudomonas fluorescens*, *Pseudomonas syringae* DC3000 and *Agrobacterium tumefaciens*) and Yellow curl leaf virus YCV infection (Fig 1). When tomato leaves were exposed six hours to *Pseudomonas* strains (*Pseudomonas putida*, *Pseudomonas fluorescens* or *Pseudomonas syringae* DC3000), most *SLARF* genes were down-regulated.

Flagellin is considered as the main building unit of the bacterial motility organ and constitutes an important microbial pattern for virulence through its use by plants to recognize bacterial pathogens. When tomato leaves are exposed six hours to flagellin [60], the expression of almost all members of the *SLARF* family is repressed except for *SLARF6A* and *SLARF18* that showed a twofold induction.

In response to *P. putida*, the expression of *SLARFs* was also repressed except for *SLARF1* and *SLARF18*. Likewise, upon *P. syringae* infection the expression of the overwhelming majority of *SLARF* genes was downregulated although *SLARF8A* was induced. *P. fluorescens* infection also resulted in down-regulation of most *ARF* genes, regulation that is highly marked for *SLARF4*. Noteworthy, none of the 22 *SLARFs* showed a significant change in their expression at the transcription level upon *Agrobacterium tumefaciens* infection.

Tomato yellow leaf curl virus (TYLCV) designates a complex of geminiviruses infecting tomato cultures worldwide. This virus is transmitted by a single insect species, the whitefly *Bemisia tabaci*. In response to TYLCV, the expression of *SLARF4*, *SLARF5*, *SLARF6A*, *SLARF8B* and *SLARF9A* genes was downregulated in leaf tissues whereas the expression of the remaining *SLARFs* did not exhibit significant change. Overall, down-regulation seems to be clearly the main trend for *SLARF* genes upon bacterial infection.
## Tomato ARF responsiveness upon abiotic stress

**Distribution of auxin response activated upon abiotic stress.** The extent of changes in auxin distribution and auxin signaling upon application of salt and drought stresses, were assessed using tomato lines expressing the GUS reported gene driven by the DR5 auxin-responsive promoter [61]. DR5::GUS tomato transgenic lines were subjected to salt and drought stress by adding 250 mM of NaCl and 15% PEG20000 to the nutrient solution, respectively (Fig 2). In leaf organs, GUS activity appears after 24 hours of salt stress and is localized in leaf veins and petioles while it is barely detectable in untreated plants. In salt stressed roots, the distribution of GUS expression is observed in lateral root primordia and in primary root tip. The control plants showed also a blue coloration in primary and lateral root tips. After 5 days of PEG treatment, a slight increase in GUS activity was detected in leaf veins and primary and lateral root tips while it scarcely appears in lateral root primordia of the untreated plants.

**Expression profiling of SlARF genes in response to salt stress.** We analyzed the expression profiles of *SlARF* genes in tomato leaves and roots after 24 hours of salt treatment. The expression of *Cold Inducible 7* gene (*CI7*), a known marker for salt stress [48], was significantly induced in both leaves and roots as expected, thus validating the efficiency of the applied stress in our condition. The data reveal that almost all *SlARF* genes were downregulated in leaves except *SlARF1*, *SlARF4* and *SlARF19* whose expression was significantly induced (Fig 3). Among the down-regulated genes, *SlARF3*, *SlARF5*, *SlARF8A* and *SlARF18* were highly repressed. In roots, 9 *SlARF* genes were up regulated among which *SlARF3*, *SlARF4*, *SlARF8A* and *SlARF9A* showed high induction (Fig 3). The expression of *SlARF2A*, *SlARF5* and *SlARF7A* were slightly repressed though not statistically significant while the expression of *SlARF19* was significantly repressed.

<figure><img src="image_6.png" /><figcaption>**Fig 2.** GUS activity in DR5::GUS tomato lines in salt or drought stress conditions. Salt and drought stresses were performed on three week-old tomato plants by adding 250 mM of NaCl or 15% PEG 20000 to the nutrient solution. Black arrows show the location of the GUS activity in the different tissues analyzed.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0193517.g002
<figure><img src="image_7.png" /><figcaption>**Fig 3.** *SIARF*s gene expression under salt, drought and flooding conditions. Values are mean ± SD of three biological replicates. White bars represent the expression of stress marker: *CI7* for salt and drought stresses (Leaves & Roots), *ACO1* in flooded leaves and PDC1 in flooded roots. Stars (*) indicate the statistical significance (*p*<0,05) using Student's t-test.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0193517.g003

**Expression profiling of SIARF genes in response to drought stress.** The responsiveness of *SIARF* members under water deficit condition was investigated by qPCR after five days of drought stress application. The drought marker gene *C17*, unsurprisingly, showed the same pattern (high induction) in leaves and roots as previously reported by Kirch *et al.*, (1997) [48]. In leaves, solely, *SIARF1*, *SIARF4*, *SIARF6B*, *SIARF10A* and *SIARF18* showed significant increase in expression, while the expression of *SIARF3*, *SIARF5*, *SIARF6A*, *SIARF7A* and *SIARF19* was significantly repressed (Fig 3).

In roots, the expression of *SIARF2A*, *SIARF2B*, *SIARF4*, *SIARF7A*, *SIARF8A* and *SIARF9A* was significantly increased upon drought stress while *SIARF3*, *SIARF10A*, *SIARF19* displayed significant down-regulation. Of particular note, *SIARF7A* with 32 fold induction displayed the most dramatic up-regulation compared to *CI7* control gene which showed only four times increase in the stressed roots (Fig 3).

**Expression profiling of SIARF genes in response to flooding stress.** After 48 hours of flooding, 3 *SIARFs* (*SIARF2B*, *SIARF7A* and *SIARF9A*) were significantly downregulated and 4 were upregulated (*SIARF5*, *SIARF6B*, *SIARF17* and *SIARF24*) in leaves (Fig 3). The highest changes were observed within *SIARF7* and *SIARF17* whose expression was three times more repressed or ten times more induced respectively. As validation of the flooding stress, the expression of the *amino-cyclopropane-1-carboxylate oxidase (ACO1)* and *pyruvate decarboxylase 1 (PDC1)* flooding marker genes was substantially increased in both leaves and roots as reported by Nie *et al.*, (2002) [50] and Gharbi *et al.*, (2007) [62], respectively. In root parts, *SIARF7A* was strongly repressed (512 times) (Fig 3).
Investigating the expression of SlARF8A gene family in salt, drought and flooding conditions have shown that several members of these transcriptional factors are regulated. Among the candidate genes identified, SlARF8A and SlARF10A were significantly regulated in salt and water stresses. Moreover, for these two genes, we were able to generate transgenic lines expressing promoter-GUS construct (pARF8A::GUS or pARF10As::GUS). All this prompt us to check SlARF8A and SlARF10A genes expressions in planta in response to salt and drought stresses and also to examine a possible involvement of related miRNA in their regulation.

## Spatio-temporal analysis of SlARF8A and SlARF10A expression under salt and drought stress conditions

Since SlARF8A and SlARF10A were significantly regulated by salt and drought stresses, we sought to examine their spatiotemporal expression in planta. Tomato transgenic lines harboring pARF8A::GUS or pARF10A::GUS constructs were therefore generated and GUS expression was analyzed under stress conditions. GUS staining performed on three week-old plants expressing pARF8A::GUS fusion construct and exposed to 24 hours salt stress reveals a strong expression in leaves and lateral roots compared to untreated plants showing GUS expression only in leaves top and leaves veins (Fig 4). After 5 days of drought stress, GUS activity was detected in the primary root and in the middle part of leaves while the control plants showed a uniform blue coloration in leaf veins, primary root and lateral root tips (Fig 4).

The GUS activity in pARF10A::GUS transgenic plants remained similar to the control plants after 24 hours on salt stress application in leaf tissues while it becomes intense in stressed roots. The control showed a uniform blue coloration all over the leaf. Five days

<figure><img src="image_8.png" /><figcaption>**Fig 4.** GUS activity in pARF8A::GUS tomato lines in salt or drought stress conditions. Salt and drought stresses were performed on three week-old tomato plants by adding 250 mM of NaCl or 15% PEG 20000 to the nutrient solution. Black arrows show the location of the GUS activity in the different tissues analyzed.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0193517.g004
<figure><img src="image_9.png" /><figcaption>**Fig 5.** GUS activity in pARF10A::GUS tomato lines in salt or drought stress conditions. Salt and drought stresses were performed on three week-old tomato plants by adding 250 mM of NaCl or 15% PEG 20000 to the nutrient solution. Black arrows show the location of the GUS activity in the different tissues analyzed.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0193517.g005

exposure to drought restricted the GUS expression to the apical part of the leaf while the control plants displayed a blue coloration in the whole leaf and in the primary and lateral root tips (Fig 5).

## SlARF specific miRNAs responsiveness under abiotic stress conditions

miRNAs are small non-coding RNA molecules that negatively regulate their target genes at the post-transcriptional level [38,42]. ARF family members are known to be subject to this type of regulation. *SlARF8* and *SlARF6* are known to be targeted by *miR167* and *SlARF10* is specifically regulated by *miR160* [41,63]. Since the data described above (Fig 3) indicated that the expression of *SlARF8A* and *SlARF10A* was altered by both salt and drought stress, we checked whether the modification in the expression of these SlARF was correlated with the expression of their specific miRNA regulators. qPCR analysis showed that the expression pattern of *miR160* and its target gene *SlARF10A* changed significantly in response to salt stress (Fig 6). In leaves, *miR160* was two times induced after 24 hours of salt treatment while *ARF10A* gene was concomitantly downregulated. In roots, *SlARF10A* was up-regulated during the first two hours of salt treatment while the expression of *miR160* remained unchanged. Tomato *miR160* was significantly repressed in leaves after 48 hours of drought stress whereas the expression of *ARF10A* showed a high induction in 5 days drought stressed roots while the expression of *miR160* remained similar to the control.

The expression of miR167 precursors and their target gene *SlARF8A* were modified by salt and drought stresses (Fig 7). In salt stressed leaves, *SlARF8A* was highly induced up to 16 times after 24 hours of salt treatment while the expression *miR167b* was in a concurring way
<figure><img src="image_10.png" /><figcaption>**Fig 6.** *SLARF10A and miR160* expression under salt and drought stress conditions. Values are mean ± SD of three biological replicates. Stars (*) indicate the statistical significance (p<0,05) using Student's t-test.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0193517.g006

highly repressed. In roots, *SLARF8A* was highly up-regulated after 24 hours of salt treatment while no strong changes occurred in the expression of *miR167* precursors. After 48 hours of drought stress exposure, *SLARF8A* gene expression in leaves was not strongly affected while

<figure><img src="image_11.png" /><figcaption>**Fig 7.** *SLARF8A and miR167s (miR167a, miR167b, miR167c, miR167d)* expression under salt and drought stress conditions. Values are mean ± SD of three biological replicates. Stars (*) indicate the statistical significance (p<0,05) using Student's t-test.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0193517.g007
*miR167a* and *miR167c* genes were highly induced when *miR167b* was repressed. In roots, *miR167a* was highly repressed when *miR167c* was induced while *SLARF8A* expression did not change. Five days of drought stress exposure resulted in an over-expression of *ARF8A* gene and down expression of *miR167a* in roots.

Discussion

Plants are constantly exposed to various abiotic and biotic stresses in their immediate environ-
ment, limiting considerably their growth rate and productivity. Plants have evolved complex
signaling pathways in response to various stimuli such as salt, drought, cold, wounding, or
pathogen invasion in order to minimize damages while conserving valuable resources for
growth and reproduction. These responses were found to be mediated by plant growth regula-
tors [64]. Several phytohormones such as abscisic acid, ethylene, salicylic acid and jasmonate
were identified as key regulators in various stress responses. Auxin is implicated in many plant
developmental processes and recent studies suggest its involvement in stress [65,66]. It has
been reported that the endogenous auxin level decreased substantially upon drought stress
conditions but increased in response to cold and heat stresses in Rice and during pathogen
infection in *Arabidopsis thaliana* [67–69]. The expression of *AuxIAA* and *ARF* genes is altered
during cold acclimation in *Arabidopsis thaliana* and during salt, water stresses and biotic
stresses in *Oryza sativa* [16,70,71]. In this work, we investigated the implication of some actors
of auxin signalling pathway in tomato responses to biotic and abiotic stresses.

Auxin signaling pathway is altered by pathogen infection in tomato

The auxin response has emerged recently as an active actor in plant defense against pathogens [72]. Auxin coordinates plant development essentially through two transcriptional regulators Aux/IAA and ARFs [24]. In tomato, 22 *SLARFs* were previously isolated and characterized [21]. *In silico* promoter analysis of *SLARFs* using Plant PLACE database have revealed that at least one copy of the pathogenesis induced element (GT1GMSCAM4) was found in *SLARFs* 5' regulatory regions. Furthermore, the expression of almost the entire *SLARF* gene family was altered in response to some of the pathogen tested based on the results related to biotic stresses that are available in TOMEXPRESS platform [59]. This suggests that auxin might be involved in biotic stress responses through ARFs. Other previous studies have underlined the involve- ment of auxin responsive genes in biotic stress responses. Auxin responsive genes are downre- gulated in *Arabidopsis thaliana* upon Botrytis cinerea infection [73]. In cotton, gene expression profiling in response to infection with *Fusarium oxysporum f. sp. vasinfectum* also revealed dif- ferential expression of auxin responsive genes [74]. Studies conducted on rice had revealed that only two *OsARFs* were responsive to *Magnaporthe grisea* (ascomycete fungus) and *Striga hermonthica* (obligate root hemiparasite) infection [71]. The present results revealed that the expression of the *SLARF* gene family was altered by pathogen infections. Our findings suggest that the regulation of auxin pathway might be an important aspect of the defense response through the regulation of the expression of *ARF* gene family in tomato.

Auxin accumulation is modified within high salinity and water deficit in tomato

Auxin and its gradients are found to be closely associated to some morphological changes
observed in plants exposed to environmental stresses. These changes are likely associated with
lateral root formation and axillary branching and represent one of the most specific responses
to abiotic stresses [75,76]. In tomato, we showed that auxin distribution is modified by the
stress application. Auxin was accumulated only in lateral root primordia and tips of DR5::GUS
tomato transgenic plants exposed to salt and drought stress conditions. In support to these
results, Zolla et al., (2009) [77] reported that salt stress stimulates the development of a larger
number of lateral root primodia in *Arabidopsis*. This stimulation was associated with auxin
accumulation in the lateral root primordia [78–80].

Auxin response factors are potential key actors in abiotic stress responses
in tomato

In *silico*, *SLARF*5 promoter analysis has shown that *SLARF* regulatory regions were enriched
with stress associated motifs. Drought and salt related *cis-acting* elements were found in all the
22 *SLARF* promoter regions. *SLARF1*, *SLARF4*, *SLARF8A*, *SLARF19* and *SLARF24* regulatory
regions were more enriched in the salt induced element (GT1GMSCAM4). The promoter
regions of *SLARF3* and *SLARF9A* although present respectively one copy of the GT1GMSCAM4
salt induced element. The MYC (MYCATERD1, MYCATRD22 and MYCCONSENSUSAT)
were found in several copies *SLARF1*, *SLARF4*, *SLARF6A*, *SLARF7A*, *SLARF8A*, *SLARF9A* and
*SLARF18* regulatory regions while the MYB motifs (MYBCORE, MYB1AT, MYB2AT and
MYB2CONSENSUSAT) was highly present in *SLARF2A*, *SLARF2B*, *SLARF4*, *SLARF6B*, *SLARF9A*
and *SLARF18* promoter regions. All these finding suggest that the functions of these transcrip-
tional factors may be associated with environmental stresses response. Moreover, investigating
the expression of Auxin Response Factor family under abiotic stress conditions showed that
most of the tomato ARFs were responsive and some of them were significantly regulated.
Among the regulated genes, *SLARF1*, *SLARF4*, *SLARF8A*, *SLARF19* and *SLARF24* showed a signifi-
cant upregulation in response to salt stress while *SLARF1*, *SLARF2A*, *SLARF2B*, *SLARF4*, *SLARF6A*,
*SLARF6B*, *SLARF7A*, *SLARF8A*, *SLARF9A* and *SLARF18* were substantially induced in drought
stress conditions. In rice, several OsARFs were shown to be involved in salt and drought stress
responses. Jain and Khurana (2009) [11] had reported that both *OsARF11* and *OsARF15* genes
had shown differential expression in salt stress conditions and suggested that they are involved
in rice response to stress. Zhou et al., (2007) [81] had shown the involvement of nine *ARFs*
genes in response to water deficit (*OsARF2*, *OsARF4*, *OsARF10*, *OsARF14*, *OsARF16*, *OsARF18*,
*OsARF19*, *OsARF22* and *OsARF23*). In *Sorghum bicolor*, Wang et al., (2010) [17] reported that
*SbARF10*, *SbARF16*, and *SbARF21* genes were significantly induced in leaves and roots tissues
exposed to drought conditions. However, among the 50 *ARF* isolated and identified in Soy-
bean (*Glycine max*), only *GmARF33* and *GmARF50* were responsive (induced) to water deficit
and were suggested as excellent candidates for drought stress responses in this plant [18].

The expression of some SLARFs showed different pattern in leaf and root under the same
treatment. This difference might be associated with the function of these SLARFs. Some ARF
functions are more related to the underground part more than to the aerial part. This is the
case for *SLARF7A*, whose expression was significantly repressed or induced respectively in
shoot and root parts under drought treatment. This gene seems to act in the underground part
of the plant. This assumption is supported by Okushima et al., (2007) [82] and Goh et al.,
(2012) [83] who had shown that *ARF7* is involved in the control of lateral root formation in
*Arabidopsis*.

Functional studies have shown that ARF genes are involved in the control of plant growth
and development. Actually, Okushima et al., (2007) [82] and had reported that *AtARF7* acts
synergistically with *AtARF19* in the control of lateral root formation and hypocotyl gravito-
tropism. Otherwise, *Solanum lycopersicum* *ARF2* has been proposed as a key regulator of apical
hook formation while, in *Arabidopsis thaliana*, the same gene seems to act as a positive activa-
tor of flowering senescence and abscission and as a repressor of cell growth in the presence or
absence of light and differential hypocotyl growth [84,85]. All these results suggest that auxin,
through *ARF* gene family, activates stress specific auxin response genes in order to mitigate the
negative effects of abiotic stresses.

## ARF8A and ARF10 are good indicators for salt and drought response in tomato

Our data have shown that *SLARF* gene family expression is modified by salinity and water deficit. Among these genes, *SLARF8A* and *SLARF10A* expressions are altered by the abiotic stress conditions. Moreover, their regulatory regions contain several copies of salt and/or drought induced elements. Investigating their expression *in planta* had revealed that their expression is clearly modified by salt and drought stress conditions. We found that *SLARF8A* promoter is especially expressed in lateral roots after 24 hours of exposure to both stresses. Previous studies confirmed the involvement of *ARF8* in abiotic stress response. Tian *et al.*, (2004) [86] have suggested that *AtARF8* is stably expressed in lateral roots of *Arabidopsis thaliana* under light conditions and appears to control hypocotyl elongation.

Histochemical analysis of pARF10A::GUS tomato lines in salt stress conditions revealed that *ARF10A* gene is particularly more expressed in primary and lateral roots. The *SLARF10A* regulatory region presents four different salt/drought related motifs, which suggests that this gene might be implicated in root growth and development under stress conditions. Wang *et al.*, 2005 [87] have found that *Arabidopsis thaliana* *ARF10A* is implicated in root formation and architecture by restricting cell division and promoting cell differentiation in the distal region which might suggest its implication in root development under stress conditions.

## miRNAs contribute to ARF gene regulation under stress conditions

miRNA are post transcriptional regulators of a large number of target genes by guiding target mRNAs for degradation or by repressing translation. Zhao and Srivastava (2007) [88] reported that some miRNAs are regulated and could be involved in cell responses to abiotic stresses such as salinity, cold and dehydration. Some miRNAs, including *miR160* and *miR167*, known to regulate the levels of transcription factor transcripts and protein abundance showed altered expression profiles in salt and drought conditions. Our results show that the downregulation of *miR167* precursors (*miR167a*, *miR167b*, *miR167c* and *miR167d*) was negatively correlated with the accumulation of *SLARF8A* transcripts in leaves after 24 hours of salt exposure. This finding was also observed in roots after 5 days of drought treatment. Knowing that *ARF8A* is implicated in control and development of vegetative and floral organs in Dicots, the downregulation of *miR167* gene precursors might enhance the auxin response and thus enhance shoot and leaf development. In cassava (*Manihot esculenta*), Xia *et al.*, (2014) [89] showed that *miR167* expression was modified in response to extreme temperature and could induce the cleavage of its target gene, *ARF8*, due to the presence of the *miR167* cleavage sites on it. The expression of *miR160* was also negatively correlated with the expression of its target gene *SLARF10A* in roots exposed to drought stress. This finding suggests that *miR160* might be implicated in the post-transcriptional regulation of the expression of *ARF10A* gene under stress conditions.

## Conclusion

Auxin plays crucial roles in various aspects of plant growth and development. This phytohor-
mone acts on the transcriptional regulation of target genes, mainly through Auxin Response
Factors. The current study provide several clues on the potential involvement of many ARF
genes as mediators of the auxin action in biotic and abiotic stress responses in tomato. The 5'
regulatory regions analysis of the *SLARFs* genes indicates the presence of several biotic and
abiotic stress-responsive cis-elements. Moreover, transcriptome analysis reveals the respon-
siveness of SlARF genes to a wide range of biotic and abiotic stresses. Additionally, SlARF8A
and SlARF10A specific miRNA were involved in the regulation of these genes suggesting the
importance of post-transcriptional regulation in auxin signaling pathway and plant response
to abiotic stresses. Taking together, the data presented in this work brings new elements and
open new ways to explore the molecular mechanisms associated with stress tolerance in
tomato. This provides new insights into tomato selection and breeding for environmental
stress-tolerant.

**Supporting information**

S1 Table. Quantitative RT-PCR primers of SlARFs genes, stress marker genes and miRNA gene precursors.

(PDF)

S2 Table. SlARFs RNA levels in tomato leaves in the control plants and upon pathogen infections. The data presented in this table were extracted from TOMEXPRESS database [44].

(PDF)

S3 Table. Potential Cis-acting regulatory elements identified in the 5' regulatory sequences of tomato Auxin response factor gene family. The 2 Kb of 5' regulatory region were analyzed using the Place software.

(PDF)

S1 Fig. Map of the 5' regulatory sequences of *Solanum lycopersicum* auxin response factors gene family. The consensus sequences corresponding to the various putative cis-elements are described in S2 Table. Positions are with respect to the first base of translation start site.

(PDF)

Acknowledgments

The authors are grateful to L. Lemonnier, D. Saint-Martin and O. Berseille for genetic transfor-
mation and culture of tomato plants. This research was supported by the “Laboratoire d’Excel-
lence” (LABEX) entitled TULIP (ANR-10-LABX-41) and benefited from networking activities
within the European COST Action FA1106, from the ANR TomEpiSet (ANR-16-CE20-0014)
project, the TomGEM H2020 project, by Hubert-Curien partnership program Volubilis (MA/
12/280-27105PL) and CMCU program (08G0906) granted to Dr Habib Khoudi.

Author Contributions

**Conceptualization:** Anne Bernadac, Najib Bendaou, AbdelAziz Smouni, Mondher Bouzayen, Mohamed Zouine.

**Formal analysis:** Sarah Bouzroud, Sandra Gouiaa, Nan Hu.

**Investigation:** Sarah Bouzroud, Sandra Gouiaa, Nan Hu.

**Methodology:** Sarah Bouzroud, Sandra Gouiaa, Nan Hu, Isabelle Mila.

**Resources:** Isabelle Mila.

**Supervision:** Anne Bernadac, AbdelAziz Smouni, Mondher Bouzayen, Mohamed Zouine.

**Validation:** Anne Bernadac, AbdelAziz Smouni, Mondher Bouzayen, Mohamed Zouine.
**Writing – original draft:** Sarah Bouzroud, Anne Bernadac, AbdelAziz Smouni, Mondher Bouzayen, Mohamed Zouine.

**Writing – review & editing:** Anne Bernadac, Isabelle Mila, Najib Bendaou, AbdelAziz Smouni, Mondher Bouzayen, Mohamed Zouine.

## References

1. Popko J, Hänsch R, Mendel R, Polle A, Teichmann T. The role of abscisic acid and auxin in the response of poplar to abiotic stress. Plant Biol. 2010; 12: 242–258. https://doi.org/10.1111/j.1438-8677.2009.00305.x PMID: 20398232

2. Atkinson NJ, Urwin PE. The interaction of plant biotic and abiotic stresses: from genes to the field. J Exp Bot. 2012; 63: 3523–3543. https://doi.org/10.1093/jxb/ers100 PMID: 22467407

3. Tuteja N. Abscisic Acid and Abiotic Stress Signaling. Plant Signal Behav. 2007; 2: 135–138. PMID: 19516981

4. Dorantes-Acosta AE, Sánchez-Hernández CV, Arteaga-Vazquez MA. Biotic stress in plants: life lessons from your parents and grandparents. Front Genet. 2012; 3.

5. Tuteja N, Sopory SK. Plant signaling in stress: G-protein coupled receptors, heterotrimeric G-proteins and signal coupling via phospholipases. Plant Signal Behav. 2008; 3: 79–86. PMID: 19516978

6. Leyser H, Pickett FB, Dharmasiri S, Estelle M. Mutations in the AXR3 gene of Arabidopsis result in altered auxin response including ectopic expression from the SAUR-AC1 promoter. Plant J. 1996; 10: 403–413. PMID: 8811856

7. Reinhardt D, Pesce E-R, Stieger P, Mandel T, Baltensperger K, Bennett M, et al. Regulation of phyllo-taxis by polar auxin transport. Nature. 2003; 426: 255–260. https://doi.org/10.1038/nature02081 PMID: 14628043

8. Mattsson J, Ckurshumova W, Berleth T. Auxin signaling in Arabidopsis leaf vascular development. Plant Physiol. 2003; 131: 1327–1339. https://doi.org/10.1104/pp.013623 PMID: 12644682

9. Esmon CA, Tinsley AG, Ljung K, Sandberg G, Hearne LB, Liscum E. A gradient of auxin and auxin-dependent transcription precedes tropic growth responses. Proc Natl Acad Sci U S A. 2006; 103: 236–241. https://doi.org/10.1073/pnas.0507127103 PMID: 16371470

10. Cheng Y, Zhao Y. A role for auxin in flower development. J Integr Plant Biol. 2007; 49: 99–104.

11. Jain M, Khurana JP. Transcript profiling reveals diverse roles of auxin-responsive genes during reproductive development and abiotic stress in rice. Febs J. 2009; 276: 3148–3162. https://doi.org/10.1111/j.1742-4658.2009.07033.x PMID: 19490115

12. Krizek BA. Auxin regulation of Arabidopsis flower development involves members of the AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) family. J Exp Bot. 2011; 62: 3311–3319. https://doi.org/10.1093/jxb/err127 PMID: 21511900

13. Kazan K. Auxin and the integration of environmental signals into plant root development. Ann Bot. 2013; 112: 1655–1665. https://doi.org/10.1093/aob/mct229 PMID: 24136877

14. Cheong YH, Chang H-S, Gupta R, Wang X, Zhu T, Luan S. Transcriptional profiling reveals novel interactions between wounding, pathogen, abiotic stress, and hormonal responses in Arabidopsis. Plant Physiol. 2002; 129: 661–677. https://doi.org/10.1104/pp.002857 PMID: 12068110

15. Wang D, Pei K, Fu Y, Sun Z, Li S, Liu H, et al. Genome-wide analysis of the auxin response factors (ARF) gene family in rice (Oryza sativa). Gene. 2007; 394: 13–24. https://doi.org/10.1016/j.gene.2007.01.006 PMID: 17408882

16. Song Y, Wang L, Xiong L. Comprehensive expression profiling analysis of OsIAA gene family in developmental processes and in response to phytohormone and stress treatments. Planta. 2009; 229: 577–591. https://doi.org/10.1007/s00425-008-0853-7 PMID: 19034497

17. Wang S, Bai Y, Shen C, Wu Y, Zhang S, Jiang D, et al. Auxin-related gene families in abiotic stress response in Sorghum bicolor. Funct Integr Genomics. 2010; 10: 533–546. https://doi.org/10.1007/s10142-010-0174-3 PMID: 20499123

18. Van Ha C, Le DT, Nishiyama R, Watanabe Y, Sulieman S, Tran UT, et al. The Auxin Response Factor Transcription Factor Family in Soybean: Genome-Wide Identification and Expression Analyses During Development and Water Stress. DNA Res Int J Rapid Publ Rep Genes Genomes. 2013; 20: 511–524. https://doi.org/10.1093/dnares/dst027 PMID: 23810914

19. Szemenyei H, Hannon M, Long JA. TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis. Science. 2008; 319: 1384–1386. https://doi.org/10.1126/science.1151461 PMID: 18258861
20. Causier B, Lloyd J, Stevens L, Davies B. TOPLESS co-repressor interactions and their evolutionary conservation in plants. Plant Signal Behav. 2012; 7: 325–328. https://doi.org/10.4161/psb.19283 PMID: 22476455

21. Zouine M, Fu Y, Chateigner-Boutin A-L, Mila I, Frasse P, Wang H, et al. Characterization of the tomato ARF gene family uncovers a multi-levels post-transcriptional regulation including alternative splicing. PloS One. 2014; 9: e84203. https://doi.org/10.1371/journal.pone.0084203 PMID: 24427281

22. Ulmasov T, Hagen G, Guilfoyle TJ. Activation and repression of transcription by auxin-response factors. Proc Natl Acad Sci. 1999; 96: 5844–5849. PMID: 10318972

23. Ulmasov T, Hagen G, Guilfoyle TJ. Dimerization and DNA binding of auxin response factors. Plant J. 1999; 19: 309–319. PMID: 10476078

24. Guilfoyle TJ, Hagen G. Auxin response factors. Curr Opin Plant Biol. 2007; 10: 453–460. https://doi.org/10.1016/j.pbi.2007.08.014 PMID: 17900969

25. Chandler JW. Auxin response factors. Plant Cell Environ. 2016; 39: 1014–1028. https://doi.org/10.1111/pce.12662 PMID: 26487015

26. Wang D, Pajerowska-Mukhtar K, Culler AH, Dong X. Salicylic acid inhibits pathogen growth in plants through repression of the auxin signaling pathway. Curr Biol. 2007; 17: 1784–1790. https://doi.org/10.1016/j.cub.2007.09.025 PMID: 17919906

27. Wilmoth JC, Wang S, Tiwari SB, Joshi AD, Hagen G, Guilfoyle TJ, et al. NPH4/ARF7 and ARF19 promote leaf expansion and auxin-induced lateral root formation. Plant J. 2005; 43: 118–130. https://doi.org/10.1111/j.1365-313X.2005.02432.x PMID: 15960621

28. De Jong M, Mariani C, Vriezen WH. The role of auxin and gibberellin in tomato fruit set. J Exp Bot. 2009; erp094.

29. Lim PO, Lee IC, Kim J, Kim HJ, Ryu JS, Woo HR, et al. Auxin response factor 2 (ARF2) plays a major role in regulating auxin-mediated leaf longevity. J Exp Bot. 2010; 61: 1419–1430. https://doi.org/10.1093/jxb/erq010 PMID: 20164142

30. Sagar M, Chervin C, Mila I, Hao Y, Roustan J-P, Benichou M, et al. SIARF4, an auxin response factor involved in the control of sugar metabolism during tomato fruit development. Plant Physiol. 2013; 161: 1362–1374. https://doi.org/10.1104/pp.113.213843 PMID: 23341361

31. Liscum E, Reed J. Genetics of Aux/IAA and ARF action in plant growth and development. Plant Mol Biol. 2002; 49: 387–400. PMID: 12036262

32. Xing H, Pudake RN, Guo G, Xing G, Hu Z, Zhang Y, et al. Genome-wide identification and expression profiling of auxin response factor (ARF) gene family in maize. BMC Genomics. 2011; 12: 178. https://doi.org/10.1186/1471-2164-12-178 PMID: 21473768

33. Kalluri UC, DiFazio SP, Brunner AM, Tuskan GA. Genome-wide analysis of Aux/IAA and ARF gene families in Populus trichocarpa. BMC Plant Biol. 2007; 7: 59. https://doi.org/10.1186/1471-2229-7-59 PMID: 17986329

34. Wu J, Wang F, Cheng L, Kong F, Peng Z, Liu S, et al. Identification, isolation and expression analysis of auxin response factor (ARF) genes in Solanum lycopersicum. Plant Cell Rep. 2011; 30: 2059. https://doi.org/10.1007/s00299-011-1113-z PMID: 21735233

35. Tomato Genome Consortium. The tomato genome sequence provides insights into fleshy fruit evolution. Nature. 2012; 485: 635. https://doi.org/10.1038/nature11119 PMID: 22660326

36. Mun J-H, Yu H-J, Shin JY, Oh M, Hwang H-J, Chung H. Auxin response factor gene family in Brassica rapa: genomic organization, divergence, expression, and evolution. Mol Genet Genomics. 2012; 287: 765–784. https://doi.org/10.1007/s00438-012-0718-4 PMID: 22915303

37. Hu W, Zuo J, Hou X, Yan Y, Wei Y, Liu J, et al. The auxin response factor gene family in banana: genome-wide identification and expression analyses during development, ripening, and abiotic stress. Front Plant Sci. 2015; 6.

38. Sunkar R, Zhu J-K. Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell. 2004; 16: 2001–2019. https://doi.org/10.1105/tpc.104.022830 PMID: 15258262

39. Zhang J, Jia W, Yang J, Ismail AM. Role of ABA in integrating plant responses to drought and salt stresses. Field Crops Res. 2006; 97: 111–119.

40. Mallory AC, Bartel DP, Bartel B. MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell. 2005; 17: 1360–1375. https://doi.org/10.1105/tpc.105.031716 PMID: 15829600

41. Liu N, Wu S, Van Houten J, Wang Y, Ding B, Fei Z, et al. Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato. J Exp Bot. 2014; 65: 2507–2520. https://doi.org/10.1093/jxb/eru141 PMID: 24723401
42. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. cell. 2004; 116: 281-297.
PMID: 14744438

43. Curaba J, Singh MB, Bhalla PL. miRNAs in the crosstalk between phytohormone signalling pathways. J Exp Bot. 2014; 65: 1425-1438. https://doi.org/10.1093/jxb/eru002 PMID: 24523503

44. SOGO [Internet]. [cited 6 Dec 2017]. Available: https://sogo.dna-affrc.go.jp/cgi-bin/sogo.cgi?lang=en

45. Zouine M, Maza E, Djari A, Lauvernier M, Frasse P, Smouni A, et al. TomExpress, a unified tomato RNA-Seq platform for visualization of expression data, clustering and correlation networks. Plant J. 2017; 92: 727-735. https://doi.org/10.1111/tpj.13711 PMID: 28873253

46. Wang H, Jones B, Li Z, Frasse P, Delalande C, Regad F, et al. The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis. Plant Cell. 2005; 17: 2676-2692. https://doi.org/10.1105/tpc.105.033415 PMID: 16126837

47. Broughton W, Dilworth M. Control of leghaemoglobin synthesis in snake beans. Biochem J. 1971; 125: 1075-1080. PMID: 5144223

48. Kirch H-H, Berkel J van, Glaczinski H, Salamini F, Gebhardt C. Structural organization, expression and promoter activity of a cold-stress-inducible gene of potato (Solanum tuberosum L.). Plant Mol Biol. 33: 897-909. https://doi.org/10.1023/A:1005759925962 PMID: 9106513

49. Hsieh T-H, Lee J, Charng Y, Chan M-T. Tomato plants ectopically expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress. Plant Physiol. 2002; 130: 618-626. https://doi.org/10.1104/pp.006783 PMID: 12376629

50. Nie X, Singh RP, Tai GC. Molecular characterization and expression analysis of 1-aminocyclopropane-1-carboxylate oxidase homologs from potato under abiotic and biotic stresses. Genome. 2002; 45: 905-913. PMID: 12416623

51. Abe H, Yamaguchi-Shinozaki K, Urao T, Iwasaki T, Hosokawa D, Shinozaki K. Role of Arabidopsis MYC and MYB homologs in drought-and abscisic acid-regulated gene expression. Plant Cell. 1997; 9: 1859-1868. https://doi.org/10.1105/tpc.9.10.1859 PMID: 9368419

52. Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell. 2003; 15: 63-78. https://doi.org/10.1105/tpc.006130 PMID: 12509522

53. Dang HQ, Tran NQ, Tuteja R, Tuteja N. Promoter of a salinity and cold stress-induced MCM6 DNA helicase from pea. Plant Signal Behav. 2011; 6: 1006-1008. https://doi.org/10.4161/psb.6.7.15502 PMID: 21691155

54. Guy C. Molecular responses of plants to cold shock and cold acclimation. J Mol Microbiol Biotechnol. 1999; 1: 231-242. PMID: 10943554

55. Sohn KH, Lee SC, Jung HW, Hong JK, Hwang BK. Expression and functional roles of the pepper pathogen-induced transcription factor RAV1 in bacterial disease resistance, and drought and salt stress tolerance. Plant Mol Biol. 2006; 61: 897. https://doi.org/10.1007/s11103-006-0057-0 PMID: 16927203

56. Nishiuchi T, Shinshi H, Suzuki K. Rapid and transient activation of transcription of the ERF3 Gene by Wounding in Tobacco Leaves POSSIBLE INVOLVEMENT OF NtWRKYS AND AUTOREPRESSION. J Biol Chem. 2004; 279: 55355-55361. https://doi.org/10.1074/jbc.M409674200 PMID: 15509567

57. Deb A, Kundu S. Deciphering cis-regulatory element mediated combinatorial regulation in rice under blast infected condition. PloS One. 2015; 10: e0137295. https://doi.org/10.1371/journal.pone.0137295 PMID: 26327607

58. Trivedi DK, Ansari MW, Tuteja N. Multiple abiotic stress responsive rice cyclophilin: (OsCYP-25) mediates a wide range of cellular responses. Commun Integr Biol. 2013; 6: e25260. https://doi.org/10.4161/cib.25260 PMID: 24265852

59. TomExpress Home: visualize and analyse tomato RNA-seq data [Internet]. [cited 28 Mar 2017]. Available: http://gbf.toulouse.inra.fr/tomexpress/www/welcomeTomExpress.php

60. Rosli HG, Zheng Y, Pombo MA, Zhong S, Bombarely A, Fei Z, et al. Transcriptomics-based screen for genes induced by flagellin and repressed by pathogen effectors identifies a cell wall-associated kinase involved in plant immunity. Genome Biol. 2013; 14: R139. https://doi.org/10.1186/gb-2013-14-12-r139 PMID: 24359686

61. Ulmasov T, Murfett J, Hagen G, Guilfoyle TJ. Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell. 1997; 9: 1963-1971. https://doi.org/10.1105/tpc.9.11.1963 PMID: 9401121

62. Gharbi I, Ricard B, Rolin D, Maucourt M, Andrieu M-H, Bizid E, et al. Effect of hexokinase activity on tomato root metabolism during prolonged hypoxia. Plant Cell Environ. 2007; 30: 508-517. https://doi.org/10.1111/j.1365-3040.2007.01640.x PMID: 17324236
63. Hendelman A, Buxdorf K, Stav R, Kravchik M, Arazi T. Inhibition of lamina outgrowth following Solanum lycopersicum AUXIN RESPONSE FACTOR 10 (SIARF10) derepression. Plant Mol Biol. 2012; 78: 561-576. https://doi.org/10.1007/s11103-012-9883-4 PMID: 22287097

64. Peleg Z, Blumwald E. Hormone balance and abiotic stress tolerance in crop plants. Curr Opin Plant Biol. 2011; 14: 290-295. https://doi.org/10.1016/j.pbi.2011.02.001 PMID: 21377404

65. Rahman A. Auxin: a regulator of cold stress response. Physiol Plant. 2013; 147: 28-35. https://doi.org/10.1111/j.1399-3054.2012.01617.x PMID: 22435366

66. Liu L, Guo G, Wang Z, Ji H, Mu F, Li X. Auxin in Plant Growth and Stress Responses. In: Tran L-SP, Pal S, editors. Phytohormones: A Window to Metabolism, Signaling and Biotechnological Applications. Springer New York; 2014. pp. 1-35. Available: http://link.springer.com/gate6.inist.fr/chapter/10.1007/978-1-4939-0491-4_1

67. Schmelz EA, Engelberth J, Tumlinson JH, Block A, Alborn HT. The use of vapor phase extraction in metabolic profiling of phytohormones and other metabolites. Plant J. 2004; 39: 790-808. https://doi.org/10.1111/j.1365-313X.2004.02168.x PMID: 15315639

68. Fu J, Wang S. Insights into auxin signaling in plant-pathogen interactions. Front Plant Sci. 2011; 2.

69. Du H, Liu H, Xiong L. Endogenous auxin and jasmonic acid levels are differentially modulated by abiotic stresses in rice. Front Plant Sci. 2013; 4.

70. Hannah MA, Heyer AG, Hincha DK. A global survey of gene regulation during cold acclimation in Arabidopsis thaliana. PLoS Genet. 2005; 1: e26. https://doi.org/10.1371/journal.pgen.0010026 PMID: 16121258

71. Ghanashyam C, Jain M. Role of auxin-responsive genes in biotic stress responses. Plant Signal Behav. 2009; 4: 846-848. PMID: 19847104

72. Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M, et al. A plant miRNA contributes to anti-bacterial resistance by repressing auxin signaling. Science. 2006; 312: 436-439. https://doi.org/10.1126/science.aae0382 PMID: 16627744

73. Llorente F, Muskett P, Sánchez-Vallet A, López G, Ramos B, Sánchez-Rodríguez C, et al. Repression of the auxin response pathway increases Arabidopsis susceptibility to necrotrophic fungi. Mol Plant. 2008; 1: 496-509. https://doi.org/10.1093/mp/ssn025 PMID: 19825556

74. Dowd C, Wilson IW, McFadden H. Gene expression profile changes in cotton root and hypocotyl tissues in response to infection with Fusarium oxysporum f. sp. vasinfectum. Mol Plant Microbe Interact. 2004; 17: 654-667. https://doi.org/10.1094/MPMI.2004.17.6.654 PMID: 15195948

75. Cao Y-R, Chen S-Y, Zhang J-S. Ethylene signaling regulates salt stress response. Plant Signal Behav. 2008; 3: 761-763. https://doi.org/10.4161/psb.3.10.5934 PMID: 19513226

76. Ahmad P, Prasad MNV. Abiotic stress responses in plants: metabolism, productivity and sustainability. Springer Science & Business Media; 2011.

77. Zolla G, Heimer YM, Barak S. Mild salinity stimulates a stress-induced morphogenic response in Arabidopsis thaliana roots. J Exp Bot. 2009; 61: 211-224.

78. Bhalerao RP, Eklöf J, Ljung K, Marchant A, Bennett M, Sandberg G. Shoot-derived auxin is essential for early lateral root emergence in Arabidopsis seedlings. Plant J. 2002; 29: 325-332. PMID: 11844109

79. López-Bucio J, Hernández-Abreu E, Sánchez-Calderón L, Pérez-Torres A, Rampey RA, Bartel B, et al. An auxin transport independent pathway is involved in phosphate stress-induced root architectural alterations in Arabidopsis. Identification of BIG as a mediator of auxin in pericycle cell activation. Plant Physiol. 2005; 137: 681-691. https://doi.org/10.1104/pp.104.049577 PMID: 15681664

80. He X, Mu R, Cao W, Zhang Z, Zhang J, Chen S. AtNAC2, a transcription factor downstream of ethylene and auxin signaling pathways, is involved in salt stress response and lateral root development. Plant J. 2005; 44: 903-916. https://doi.org/10.1111/j.1365-313X.2005.02575.x PMID: 16359384

81. Zhou J, Wang X, Jiao Y, Qin Y, Liu X, He K, et al. Global genome expression analysis of rice in response to drought and high-salinity stresses in shoot, flag leaf, and panicle. Plant Mol Biol. 2007; 63: 591-608. https://doi.org/10.1007/s11103-006-9111-1 PMID: 17225073

82. Okushima Y, Fukaki H, Onoda M, Theologis A, Tasaka M. ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis. Plant Cell. 2007; 19: 118-130. https://doi.org/10.1105/tpc.106.047761 PMID: 17259263

83. Goh T, Kasahara H, Mimura T, Kamiya Y, Fukaki H. Multiple AUX/IAA-ARF modules regulate lateral root formation: the role of Arabidopsis SHY2/IAA3-mediated auxin signalling. Phil Trans R Soc B. 2012; 367: 1461-1468. https://doi.org/10.1098/rstb.2011.0232 PMID: 22527388

84. Okushima Y, Mitina I, Quach HL, Theologis A. AUXIN RESPONSE FACTOR 2 (ARF2): a pleiotropic developmental regulator. Plant J. 2005; 43: 29-46. https://doi.org/10.1111/j.1365-313X.2005.02426.x PMID: 15960614
85. Chaabouni S, Jones B, Delalande C, Wang H, Li Z, Mila I, et al. Sl-IAA3, a tomato Aux/IAA at the cross-roads of auxin and ethylene signalling involved in differential growth. J Exp Bot. 2009; 60: 1349–1362. https://doi.org/10.1093/jxb/erp009 PMID: 19213814

86. Tian C, Muto H, Higuchi K, Matamura T, Tatematsu K, Koshiba T, et al. Disruption and overexpression of auxin response factor 8 gene of Arabidopsis affect hypocotyl elongation and root growth habit, indicating its possible involvement in auxin homeostasis in light condition. Plant J. 2004; 40: 333–343. https://doi.org/10.1111/j.1365-313X.2004.02220.x PMID: 15469491

87. Wang J-W, Wang L-J, Mao Y-B, Cai W-J, Xue H-W, Chen X-Y. Control of root cap formation by micro-RNA-targeted auxin response factors in Arabidopsis. Plant Cell. 2005; 17: 2204–2216. https://doi.org/10.1105/tpc.105.033076 PMID: 16006581

88. Zhao Y, Srivastava D. A developmental view of microRNA function. Trends Biochem Sci. 2007; 32: 189–197. https://doi.org/10.1016/j.tibs.2007.02.006 PMID: 17350266

89. Xia J, Zeng C, Chen Z, Zhang K, Chen X, Zhou Y, et al. Endogenous small-noncoding RNAs and their roles in chilling response and stress acclimation in Cassava. BMC Genomics. 2014; 15: 634. https://doi.org/10.1186/1471-2164-15-634 PMID: 25070534