Different regulatory modules of two mango ERS1 promoters modulate specific gene expression in response to phytohormones in transgenic model plants

Plant Science 289 (2019) 110269

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Different regulatory modules of two mango ERS1 promoters modulate specific gene expression in response to phytohormones in transgenic model plants

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Patrick Winterhagen , Michael H. Hagemann, Jens N. Wünsche University of Hohenheim, Institute of Crop Science, Section Crop Physiology of Specialty Crops, Stuttgart, Germany

A R T I C LE I N FO

A B S T R A C T

Keywords: Mangifera indica (mango) ethylene receptor MiERS1 Transcription factor binding sites (TFBS) Regulatory promoter module Ethylene Abscisic acid Auxin

Ethylene is a key element of plant physiology, thus ethylene research is important for both, fundamental research and agriculture. Previous work on ethylene receptors focused on expression level and protein interaction, but knowledge on regulation of gene transcription is scarce. Promoters of mango ethylene receptor genes (pMiERS1a, pMiERS1b) were analysed particularly regarding responsiveness to hormones. The promoter sequences reveal some variation and they were characterized by identifying functional regulatory candidate modules via truncated-promoter approach. Based on ectopic gene expression studies in transgenic Arabidopsis and Nicotiana it is demonstrated that both promoters are positively responsive to ethylene. For pMiERS1a the AHBP/DOFF1 module is linked to ethylene responsiveness, while for pMiERS1b it is the module MYBL/OPAQ1. A negative gene regulation in response to abscisic acid (ABA) is linked to MYBL/DOFF2. A positive response to indole-3-acetic acid (IAA) was found for GTBX/MYCL1, containing the motifs IBOX/IDDF/TEFB, which are present in this combination only in pMiERS1b, but not in pMiERS1a. Conclusively, the general response of the ethylene receptor genes is conserved, but similar regulation can be linked to different modules. Further, a minor variation in a transcription factor binding site (TFBS) motif within an overall conserved module type can lead to a different expression.

1. Introduction Ethylene signaling is an important and conserved mechanism in higher plants that plays a key role in biotic and abiotic stress responses as well as in developmental processes, like fruit ripening, organ abscission and senescence [1,2]. As described for Arabidopsis, different ethylene receptors of two subfamilies participate in the signal transduction pathway and the receptors of subfamily I (ERS1, ETR1) are considered essential, as their function cannot be completely replaced by receptors of subfamily II (ERS2, ETR2, EIN4) [3,4]. Although ethylene signaling and the involved receptors appear to be highly conserved in plants, some diversity is found regarding number and variability of ethylene receptor isoforms in different plant species. The model plant Arabidopsis hosts five receptors, while for many crop species a higher number of receptors and an increased variability is described, like for example tomato (six receptors), apple (nine receptors), coffee (ETR1 splicing variants), or peach (ETR1 splicing variants) [5–8]. The mango ethylene receptors MiETR1 and MiERS1 were previously identified and their expression was described for fruit ripening and organ abscission

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[9–12]. Besides those full-length MiERS1 sequences, novel short forms of MiERS1 were discovered - and also for MiETR1 different isoforms were distinguished - indicating that ethylene receptors are quite variable and can be present in multiple copies in mango [13,14]. The increased number of receptors, identification of additional or novel isoforms and the intraspecific variability of receptors suggests a more complex ethylene signaling mechanism in horticultural crop plants, especially compared to the model plant Arabidopsis. It is well established and was reviewed previously that ethylene causes a feedback response and is also interwoven with other phytohormones’ signaling pathways depending on plant development, tissue and physiological status [1,15,16]. Hence, the interaction of different hormone pathways – including auxin and abscisic acid – and the influence to each other adds another level of complexity on signaling [17,18]. This suggests that ethylene itself and other hormones are involved in the regulation of ethylene key genes, like ethylene receptors. Although signal transduction and the transcriptional regulation of the receptors seems to be a key in ethylene dependent responses and feedback mechanisms, to our knowledge, no systematic promoter

Corresponding author. E-mail address: [email protected] (P. Winterhagen).

https://doi.org/10.1016/j.plantsci.2019.110269 Received 10 July 2019; Received in revised form 10 September 2019; Accepted 11 September 2019 Available online 13 September 2019 0168-9452/ © 2019 Published by Elsevier B.V.

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Fig. 1. Schematic overview of MiERS1 promoter regions and constructs used for expression studies. Constructs with reporter genes under control of MiERS1 promoter sequences and organisation of the pMiERS1 regions according to the ModelInspector analysis output. Open boxes indicate the genes’ coding regions, grey boxes represent promoter sequences in constructs, solid lines with black boxes display gDNA and assigned regulatory modules. The promoter constructs and the size of modules displayed schematically on the sequences is not on scale. TFBS and modules on top of the DNA strand define positive orientation, below the DNA strand means negative orientation. Numbers indicate the size or position in base pairs [bp] on the gDNA upstream of MiERS1 start codon ATG.

to inner position): (I) 5´-CTA AAG GCT TCT TTG GTA TCG-3´, (II) 5´GCC AGT CTC TTG AAG ACT C-3´, (III) 5´-CTT TAC AGC ATT ACC CAC AAT G-3´. The protocol of Li et al. [22] was modified and a three-step semi-nested PCR was performed using the same degenerate primers in combination with reverse primers specific for MiERS1. Subsequently, the pattern and size of the obtained amplification products were evaluated by gel electrophoresis to determine candidate fragments. The fragments of interest were cloned into the vector pGEM T-Easy (Promega) and transformed into E. coli TOP10 cells (Life Technologies). After cultivation of bacteria under selection, plasmids were extracted and sent for sequencing to GATC Biotech (Germany).

analysis of ethylene receptor genes has been performed yet. In order to characterise a promotor sequence one possibility is to search for known transcription factor binding sites (TFBS). However, these typically very short sequences are likely to occur by chance and are excessively present in most DNA-sequences. Therefore, statistical analysis of known TFBS and common features of TFBS families have led to the development of nucleotide weight matrix patterns and matrix families [19–21]. These patterns extend the conserved core of a TFBS by considering the sequence neighbourhood, which is also used for scoring TFBS prediction. Additionally, it has been shown that gene regulation can depend on higher order TFBS organisation. These so-called promoter modules contain two or more TFBS in combination which are located in a certain distance to each other. Bioinformatic tools typically used to identify TFBS and modules depend on databases of empirical data sets verified by experimental promotor studies. Certainly, prediction of TFBS or modules is based on a set of assumptions and this may not be accurate in all cases, but facilitates promotor characterization. In this study, two MiERS1 genes were discriminated by their promoter sequences upstream of the start codon and the question emerged, if expression of related isoforms is regulated differently. To answer this question, TFBS modules were bioinformatically predicted in the putative promoter sequences and subsequently functionally tested by a truncated-promoter approach in transgenic Arabidopsis thaliana and Nicotiana benthamiana. This work presents the detailed analysis of ethylene receptor promoter sequences and the role of the identified TFBS and regulatory modules. It is demonstrated that the two highly related genes are specifically regulated by different TFBS motifs or modules in response to various phytohormones.

2.2. Bioinformatic sequence analysis The putative MiERS1 promoter sequences (pMiERS1) were analyzed with tools from the Genomatix Software Suite: for prediction of transcription factor binding sites (TFBS) the MatInspector tool was applied, while more complex promoter modules of higher order were identified by the ModelInspector tool [20]. Following settings for the ModelInspector analysis were chosen: library Plant_Modules Version 5.5, Matrix Family Library Version 8.4, both strands were searched, threshold for number of elements: 100%. To identify common conserved TFBS and modules of higher order, sequences containing the ERS1 promoter region of Arabidopsis (Genbank Sequence ID: CP002685.1, At_chr2 clone T20B5 map CIC11C08_AC002409.3) and grapevine (Genoscope: Vv_chr7_scaff42) were compared with the isolated pMiERS1 sequences. 2.3. Infiltration for transient expression in Nicotiana and transformation of Arabidopsis

2. Material and methods 2.1. Promoter sequence isolation and cloning

Based on the bioinformatic characterization, constructs for functional analysis were made for both MiERS1 promoter sequences. Fulllength promoters (pMiERS1a1180 and pMiERS1b-1396), as well as truncated promoter versions were designed with 5′ unidirectional deletions to stepwise remove predicted modules (pMiERS1a: ΔpMiERS11067, ΔpMiERS1-954, ΔpMiERS1-506, ΔpMiERS1-455 and for pMiERS1b: ΔpMiERS1-1180, ΔpMiERS1-506, ΔpMiERS1-455) as displayed in Fig. 1. The shortest construct ΔpMiERS1455 represents both isoforms, because this part of sequence is conserved. Gateway cloning technology compatible PCR products of the promoter sequences were

Genomic DNA (gDNA) from mango (Mangifera indica cv. Hôi, tree age: 12 years) was extracted from randomly sampled fully developed mature and healthy leaves using the Master Pure Plant Leaf DNA Purification kit (Epicentre) following manufacturer´s recommendations. Sequences upstream of the mango ERS1 (MiERS1) gene containing the putative promoter region was obtained by thermal asymmetric interlaced (TAIL) PCR using previously described degenerate primers [22,23] and MiERS1 specific reverse primers (order from outer 2

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produced and inserted into the entry vector pDONR221 for subsequent transfer into the binary vector pKGWFS7 [24] following the manufacturer´s guidelines (invitrogen life technologies). Agrobacteria ATHV were transformed with the constructs via electroporation and cultivated for subsequent plant transformation. For transient expression assays, leaves of ten weeks old Nicotiana benthamiana were infiltrated with agrobacteria by standard procedure using a needleless syringe. Inhibition of gene silencing was ensured by coinfiltration with pBAR-p19BS containing the sequence of the TBSV silencing suppressor p19 [25,26]. For transformation of Arabidopsis thaliana Col-0 the floral-dip method was applied and seedlings were grown under kanamycin selection [27]. Transgenic Arabidopsis were further cultivated to obtain at least two independent lines of the T3 generation for each construct.

variance (ANOVA) of both experiments were checked by examining the residual plots (R packages “hist()”, “qqnorm()”). For the GUS-enzyme activity testing in N. benthamiana, the ANOVA was done with the R package “lmer()” for linear models with interaction effects in order to account for the effect of infiltration, since more than one infiltration has been applied to a single leaf. The results presented are the estimated means (estimates) of enzyme activity. For analysis of GUS activity in Arabidopsis, ANOVA was conducted with the R package “lm()” for simple linear models. These experiments have been conducted on different dates, thus the ‘day of experiment’ has been included in the statistical model, since there is a significant date-dependent effect on GUS staining results. The presented results (estimates) account for this effect.

2.4. Plant cultivation and hormone treatments

3. Results

N. benthamiana were cultivated in pots with soil under 16/8 light/ dark cycle and 24 °C for infiltration experiments. Subsequent hormone treatments were performed three days post-infiltration. Arabidopsis seedlings were germinated and cultivated on silicone sand and ¼ MS medium for one week under 16/8 light/dark cycle and 24 °C, then hormone treatments were conducted. Plants were treated by spray application with 10 μM indole acetic acid (IAA) or 10 μM abscisic acid (ABA) combined with 0.02% netting agent Etalfix (Syngenta). For ethylene treatment, plants were sealed in a gas tight experimental chamber and exposed to an atmosphere of 10 ppm ethylene. Control plants were not exposed to hormones. Experiments were repeated at least twice. Plant samples (N. benthamiana: 13 mm leaf discs of infiltrated area, Arabidopsis: whole seedlings) were collected 6 h after hormone treatment and stored at 80 °C until further analysis.

3.1. Bioinformatic analysis of ERS1 promoter regions Two different sequence fragments upstream of MiERS1 genes with the size of 2197 bp (includes pMiERS1a) (Genbank ID KU886218) and 2466 bp (includes pMiERS1b) (Genbank ID KU886217) were isolated by TAIL-PCR. The fragments containing the putative promoter sequences to regulate MiERS1 were analyzed with Genomatix Software Suite and screened for putative transcription factor binding sites (TFBS) and regulatory modules of higher order. Further, BLAST analysis at NCBI revealed that the 5′end of each DNA fragment also contains the 3′part of an ATP-dependent RNA helicase (ADRH) isoform, which was described as MiADRH7-1 and MiADRH7-2 because they are most similar to ADRH7 from cocoa [33]. Hence, each pMiERS1 region is framed by an adjacent 3′-ADRH gene end and the MiERS1 coding region. The schematic organization of the pMiERS1 regions including putative regulatory modules are displayed in Fig. 1. Overall, binding sites for transcription factors from 60 families were found and several are present in multiple motifs. TFBS predicted by the MatInspector analysis tool, their (core) sequences and their positions on the promoter sequences are listed in supporting information S2-Table 1. Both pMiERS1 sequences were compared and TFBS/motifs were assigned to the modules according to the output of the ModelInspector analysis tool (Fig. 1, Table 1, S2-Table 1). From the start codon on going upstream, the modules MYBL/DOFF2, DOFF/OPAQ3, GBOX/ABRE1, and GTBX/ MYCL1 are predicted for both pMiERS1 sequences. Although conserved, some of those modules reveal variability concerning the presence and position of certain TFBS. The DOFF/OPAQ3 module is conserved in both pMiERS1 sequences, but an extra P$DOFF site in pMiERS1b is responsible for the prediction of an additional overlapping MYBL/ OPAQ1 module in this region. The GBOX/ABRE1 module is conserved in both pMiERS1 isoforms. However, the sequence of the overlapping GTBX/MYCL1 module is variable: pMiERS1a contains P$AHBP/P $L1BX/P$MYBL sites, while pMiERS1b contains P$IBOX/P$IDDF/P $TEFB. In addition, and besides the mentioned MYBL/OPAQ1 module, pMiERS1b contains further upstream a GBOX/GBOX1 module. For pMiERS1a the additional modules AHBP/DOFF1 and MYCL/MYBL1 are predicted. To investigate, if the predicted regulatory modules are conserved also in other plants, genomic ERS1 promoter sequences of Arabidopsis (pAtERS1) and grapevine (pVvERS1) were analysed and compared. One module has been identified in pAtERS1 and 6 modules are present in pVvERS1. However, only two of the predicted modules present in pMiERS1 were found in the corresponding ERS1-promotors in Arabidopsis or grapevine. The module MYBL/DOFF2 closely located to the MiERS1 start codon was found in the pAtERS1 sequence, but the module is located there in distance from the ERS1 start codon (−2154 bp to −2013 bp). Because this predicted module is in great distance to AtERS1 and since it is part of the adjacent gene bZIP17 (NM_129659.3), it might not belong to the pAtERS1 regulatory system. Like pMiERS1, the upstream region of VvERS1 contains a GTBX/MYCL1 module. This

2.5. Quantitation of GUS enzyme activity in N. benthamiana extracts N. benthamiana leaf samples were homogenized in extraction buffer using a micropestle. Supernatant was collected and extracts were used for protein quantitation and glucuronidase (GUS) assay according to Aich et al. [28] and Toth et al. [29] with minor modifications. Plant extracts were incubated with 1 mM 4-nitrophenyl β-D-glucuronide (pNPG, Merck-Sigma) for GUS assay. Absorbance of samples and standards (p-nitrophenol, pNP, Merck-Sigma) was determined in duplicates by spectrophotometry. The detailed protocol can be found in supporting information S1-MM. 2.6. GUS staining for localisation and quantitation of enzyme activity in Arabidopsis Histological analysis of GUS reporter gene activity is based on protocols from Jefferson et al. [30]. For GUS staining 0.1 mM substrate X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid cyclohexyl ammonium salt) was used. Samples were rinsed in ethanol series and mounted on slides for brightfield microscopy (Zeiss Stemi SV 11) and imaging. Specific staining was analyzed and quantified in situ with ImageJ software [31] following the recommendations of Beziat et al. [32] with some modifications. For normalization, from each image background brightness was determined and subtracted from the signal. GUS staining intensity was quantified and evaluated for different plant organs. The detailed protocol can be found in supporting information S1-MM. 2.7. Data analysis and statistics The effects of the hormone treatments on GUS-enzyme activity in N. benthamiana or Arabidopsis plantlets was evaluated by pairwise comparison of the means at a probability level of p ≤ 0.05 and Tukey's honest significant difference (HSD) (R version 3.3.2). The Model assumptions (normality and variance homogeneity) for the analysis of 3

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Table 1 Predicted modules according to ModelInspector analysis, their orientation and position on the pMiERS1 sequences. Sequence isoform

Module

Strand

Position from [bp]

Position to [bp]

pMiERS1a pMiERS1a pMiERS1a pMiERS1a pMiERS1a pMiERS1a pMiERS1b pMiERS1b pMiERS1b pMiERS1b pMiERS1b pMiERS1b

MYCL/MYBL1 AHBP/DOFF1 GTBX/MYCL1 GBOX/ABRE1 DOFF/OPAQ3 MYBL/DOFF2 GBOX/GBOX1 GTBX/MYCL1 GBOX/ABRE1 MYBL/OPAQ1 DOFF/OPAQ3 MYBL/DOFF2

+ + + – – – – + – + – –

−1123 −1041 −681 −538 −462 −286 −1285 −679 −536 −494 −459 −285

−1103 −979 −540 −572 −497 −427 −1387 −538 −570 −454 −495 −426

Transgenic Arabidopsis seedlings from various independent lines containing constructs with full length or stepwise truncated promoter versions of pMiERS1a or pMiERS1b were grown for one week and basic GUS-expression was analyzed (Fig. 3). The GUS expression was evaluated by imaging and average pixel brightness values indicating GUS staining intensity for following tissues: cotyledons (COT), shoot apical meristem (SAM), hypocotyl (HYP), root (ROOT), and root apical meristem (RAM). The average values for whole seedlings (avg. plant) were calculated based on the brightness values acquired for the specific tissues. Transgenic lines with the full size promoter constructs pMiERS1a1180 or pMiERS1b1396 reveal an overall similar GUS staining pattern. Distinct GUS expression is found in COT and SAM, while weaker expression is detected in HYP, ROOT, and RAM. The first truncation at the 5′-end of both promoters (ΔpMiERS1a-1067 and ΔpMiERS1b-1180) causes still a high GUS expression in most tissues, but further truncations generally result in a reduced GUS expression. Throughout the different transgenic lines HYP has the weakest GUS expression, while the other tissues COT, SAM, ROOT, and RAM regularly show distinct GUS staining. However, staining intensity appears to be linked to construct length and presence of particular modules. Considering the GUS staining pattern in more detail, Arabidopsis containing the first truncated construct version ΔpMiERS1a-1067 display a similar pattern as plants with the full promoter construct pMiERS1a-1180. In transgenic lines with the further truncated construct ΔpMiERS1a-954 a significantly reduced GUS activity is found for COT and SAM. In plants containing the ΔpMiERS1a-506 construct, occasionally faint GUS staining is found in RAM (33%) but mostly no GUS activity at all is detectable in the seedlings. Plants with the minimal promoter construct ΔpMiERS1-455 reveal weak but more consistent GUS activity, mostly confined to ROOT or RAM. However, some individuals show faint GUS staining also in other tissues. Regarding pMiERS1b, transgenic plants containing the truncated construct ΔpMiERS1b-1180 show an overall similar GUS staining pattern compared to plants with the corresponding full size promoter construct pMiERS1b-1396; although a more intense staining was found in COT. In contrast, plants with the ΔpMiERS1b-506 construct reveal a severely reduced or even no GUS expression (only 22% show some faint GUS expression confined to ROOT or RAM) leading to a low average staining value.

may belong to the regulatory region of the grapevine ERS1 gene, although it is located in a greater distance (position −1429 bp to −1289 bp) from the start codon when compared to the mango sequences. Other modules predicted for the pVvERS1 sequence - but not for mango or Arabidopsis - are: MYCL/GCCF1 (−1782 bp to −1800 bp), MYCL/ GCCF1 (−1306 bp to −1288 bp), SUCB/SURE1 (−1034 bp to −1058 bp), MYBL/MYBS1 (−845 bp to −880 bp), and SUCB/SURE1 (−816 bp to −841 bp). 3.2. pMiERS1 constructs and basic expression of the GUS-reporter Stepwise truncated versions were designed for both pMiERS1 fulllength promoter sequences and transformed into Arabidopsis to investigate the influence of predicted modules on GUS-reporter gene expression. With the full promoter constructs (pMiERS1-1396 or pMiERS1-1180) and the truncated promoter constructs (ΔpMiERS11180, ΔpMiERS1-1067, ΔpMiERS1-954, ΔpMiERS1-506, and ΔpMiERS1-455) the role of individual modules of both promoters pMiERS1a and pMiERS1b was analyzed (Fig. 1). However, the ΔpMiERS1b-1180, ΔpMiERS1a-954 and the ΔpMiERS1b-506 constructs contain merged target modules that could not be dissected for analysis because of their overlapping sequences. Hence, the regulatory function of the modules GTBX/MYCL1 and GBOX/ABRE1 in ΔpMiERS1b-1180 and ΔpMiERS1a-954 could not be analyzed separately. The target modules investigated with ΔpMiERS1-506 constructs are variable, as both pMiERS1 isoforms contain the DOFF/OPAQ3 module, but the overlapping MYBL/OPAQ1 module is only predicted for pMiERS1b. Therefore, the role of such overlapping modules was evaluated by comparing gene expression regarding their presence or absence in the particular combination. Transgenic Arabidopsis containing the full promoter constructs pMiERS1a-1180 or pMiERS1b1396 were analyzed after staining to identify the basic GUS expression pattern in different plant organs (Fig. 2). The independent transgenic lines have a similar basic GUS expression pattern and it is obvious that enzyme activity is not evenly distributed in the plant but confined to specific tissues. GUS activity can be found for both promoter isoforms in vascular tissue and leaf veins (Fig. 2a, b, e, f, i, j, m, n). Faint GUS staining can also be detected to some extent in the leaf blade (Fig. 2e, f, m, n). Intense and spatially confined GUS staining is located at the axilla from cauline leaves, from flowers and from siliques. Further, strong GUS activity is evident at the base of flowers (receptacle) and also regularly at the apex of the pistil (stigma) (Fig. 2c, g, k, o). Developing siliques resemble the GUS expression pattern of the flowers at their base and apex (Fig. 2d, h, l, p). For comparison the expression pattern of ERS1 in various plant organs from Arabidopsis Col-0 is displayed (Fig. 2q), based on eFP Browser data [34]. Arabidopsis ERS1 expression is slightly elevated in stamen of open flowers and cauline leaves, however, the display does not indicate organ structures in detail like axilla or leaf veins. No GUS staining was detectable in wild type (WT) Col-0 control plants (data not shown).

3.3. Hormone-induced GUS expression under the control of pMiERS1 constructs Leaves from N. benthamiana plants were infiltrated with agrobacteria to transfer pMiERS1-constructs, three days post-infiltration they were treated with hormones for 6 h. Infiltrated control samples without hormone treatment were compared to samples from leaves exposed to hormones after infiltration. GUS activity in extracts from leaf discs was determined by analyzing the accumulation of the end product pNP in μM/total protein content (Fig. 4a, b). A significantly higher GUS activity can be found only after ethylene treatment for 4

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Fig. 2. Basic GUS expression under control of full length MiERS1 promoters in transgenic Arabidopsis. GUS staining is displayed for different tissues of plants from independent lines containing the full length constructs pMiERS1a-1180 (a to h) and pMiERS1b-1396 (i to p). For comparison the basic expression pattern of Arabidopsis ERS1 is displayed according to the output of the eFP Browser online tool [34] (q). Online source information: visualized data refers to Gene Expression Map of Arabidopsis Development [50], 245098_at was used as the probe set identifier for At2g40940 (ERS1). The color code indicates the gene expression response.

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Fig. 3. Basic GUS expression in transgenic Arabidopsis seedlings containing full length or truncated MiERS1 promoter constructs. Detection of GUS expression is shown for seedlings containing stepwise truncated promoter of pMiERS1a and pMiERS1b. GUS staining for each construct is displayed exemplarily by microscopic images of representative individual plantlets. The color intensity and the values indicate average GUS expression in the different plant tissues based on pixel brightness analysis (in arbitrary units). For each construct at least two independent transgenic lines were analyzed and the number of evaluated individual seedlings per construct is: ΔpMiERS1-455: n = 32, ΔpMiERS1a-506: n = 9, ΔpMiERS1a954: n = 28, ΔpMiERS1a-1067: n = 18, pMiERS1a-1180: n = 14; ΔpMiERS1b-506: n = 32, ΔpMiERS1b-1180: n = 21, pMiERS1b-1396: n = 18. Plant tissues evaluated: COT, cotyledons; SAM, shoot apical meristem; HYP, hypocotyl; ROOT, root; RAM, root apical meristem; avg. plant, average over all tissues. The promoters and size or position of modules are displayed schematically and not on scale.

In contrast, the treatment with ABA causes a significant downregulation of GUS expression in plants with ΔpMiERS1b-1180 and ΔpMiERS1b506 constructs. For comparison, Arabidopsis ERS1 expression in response to hormone treatments is visualized according to the eFP Browser online source [34] (Fig. 4e). ERS1 is downregulated in seedlings after ABA treatment, while treatment with IAA or ACC (1-aminocyclopropane carboxylic acid, to induce endogenous ethylene synthesis) leads to increased ERS1 expression. To further dissect the regulation of GUS under the control of the different constructs, the expression after hormone treatment was determined for various plant organs in transgenic Arabidopsis seedlings. For pMiERS1a it is found that in plants with pMiERS1a-1180 and ΔpMiERS1a1067 constructs the GUS expression is significantly upregulated in response to ethylene in ROOT and RAM, and in COT, SAM and ROOT, respectively (Fig. 5a, b). The shorter constructs do not respond to the ethylene treatment. After treatment with ABA the GUS expression is significantly downregulated in plants with the constructs pMiERS1a-1180, ΔpMiERS1a-954 and ΔpMiERS1-455 (Fig. 5a, c, e) and a non-significant trend is found for ΔpMiERS1a506 (Fig. 5d). A significant downregulation is visible only in COT, SAM and ROOT for the full promoter construct. Plants with the truncated constructs show a consistent downregulation of GUS in all tissues. After auxin treatment a

those leaves infiltrated with the constructs pMiERS1b-1396 or ΔpMiERS1b-1180, compared to controls not treated with hormones. Shorter constructs versions of pMiERS1b and none of the pMiERS1a constructs show a significant response to ethylene. The treatments with auxin (IAA) or abscisic acid (ABA) after infiltration do not result in significantly altered GUS activity in comparison to controls. Independently, hormone-induced GUS expression was determined in transgenic Arabidopsis seedlings by histological GUS staining and image analysis (Fig. 4c, d). Compared to the infiltration experiments, transgenic Arabidopsis lines show an overall similar pattern regarding the response to hormones; but these results reveal more details and significant differences between the treatments. A significant upregulation in response to ethylene is found with the constructs pMiERS1a-1180 and ΔpMiERS1a-1067, and after treatment with ABA a significant downregulation is determined in plant lines with ΔpMiERS1a-954, pMiERS1a-1180, and ΔpMiERS1-455 (Fig. 4c). Furthermore, plants with the minimal promoter construct ΔpMiERS1-455 show a significant downregulation in response to the IAA treatment (Fig. 4c, d). Transgenic lines with the constructs pMiERS1b-1396 and ΔpMiERS1b-1180 significantly upregulate GUS expression after ethylene treatment (Fig. 4d). Also in response to the IAA treatment, a significant upregulation of GUS activity is found for plants containing ΔpMiERS1b-1180. 6

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Fig. 4. GUS expression under the control of pMiERS1a and pMiERS1b constructs after treatment with hormones. Agrobacteria-infiltration experiments on N. benthamiana and subsequent GUS enzyme assay with leaf disc (n ≥ 6) extracts for pMiERS1a (a) and pMiERS1b (b) constructs. GUS expression in transgenic Arabidopsis seedlings with pMiERS1a (c) and pMiERS1b (d) constructs determined by histological GUS staining and image analysis. For each construct at least two independent transgenic lines were analyzed. Estimates and the standard errors are shown. The colors identify the treatments. Significant differences of treatment effects are indicated by different letters (HSD, p ≤ 0.05). The corresponding promoter schemes on top of the graphs refer to the presence of modules in constructs. For comparison the Arabidopsis ERS1 expression in response to hormone treatments is displayed according to the output of the eFP Browser online tool [34] (e). Online source information: seven day old WT Col-0 seedlings were treated with hormones for three hours, RNA was hybridized to ATH1 geneChip, 245098_at was used as the probe set identifier for At2g40940 (ERS1). The color code indicates the gene expression response.

to auxin, an indistinct pattern is visible. A significant upregulation of GUS expression is detected in RAM for plants with the constructs pMiERS1b-1396, ΔpMiERS1b-1180 and ΔpMiERS1b506 (Fig. 6a–c). Further, GUS expression is increased in COT, SAM and HYP in plants harbouring ΔpMiERS1b-1180 (Fig. 6b). However, GUS expression is downregulated in ROOT of plants with ΔpMiERS1b-506. The response of plants with the minimal promoter construct is already described under Fig. 5.

significant downregulation is identified only in ROOT for plants with ΔpMiERS1a-1067, as well as in HYP and ROOT for plants with the construct ΔpMiERS1-455 (Fig. 5b, e). The response to hormone treatments on the tissue level for plants with pMiERS1b-constructs is displayed in Fig. 6. Similarly, the longer promoter constructs (pMiERS1b-1396, ΔpMiERS1b-1180) are responsive to ethylene. Distinct GUS upregulation is found for both construct versions in COT, ROOT and RAM, and for ΔpMiERS1b1180 also for SAM (Fig. 6a, b). Plants containing the shorter construct ΔpMiERS1b-506 reveal significantly increased GUS expression after ethylene treatment in SAM, HYP and ROOT (Fig. 6c). All pMiERS1a constructs are responsive to ABA and downregulate GUS expression in various tissues. Generally, a significant downregulation (and in one case a non-significant trend) is detected in SAM and HYP. Furthermore, in plant lines with ΔpMiERS1b-1180, ΔpMiERS1b-506 and ΔpMiERS1455 GUS is also downregulated in COT (Fig. 6b–d). However, plants with the full promoter construct pMiERS1b1396 reveal an increased GUS expression in RAM (Fig. 6a). Regarding the response

4. Discussion 4.1. Evaluation of predicted modules by basic GUS reporter expression pattern The isolated sequences are similarly organized: the putative MiERS1 promoter regions are framed by the MiERS1 open reading frame and a helicase gene, which was previously described [33]. The in silico analysis reveals that both promoters share highly conserved regions, but 7

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Fig. 5. GUS expression under the control of pMiERS1a constructs in various tissues of transgenic Arabidopsis seedlings after treatment with hormones. For each construct at least two independent lines were analyzed. Estimates and the standard errors are shown. The colors identify the treatments. Significant differences of treatment effects are indicated by different letters (HSD, p ≤ 0.05). The corresponding promoter scheme on the right of the graphs refers to the presence of modules in constructs. COT, cotyledons; SAM, shoot apical meristem; HYP, hypocotyle; ROOT, root; RAM, root apical meristem.

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Fig. 6. GUS expression under the control of pMiERS1b constructs in various tissues of transgenic Arabidopsis seedlings after treatment with hormones. For each construct at least two independent transgenic lines were analyzed. Estimates and the standard errors are shown. The colors identify the treatments. Significant differences of treatment effects are indicated by different letters (HSD, p ≤ 0.05). The corresponding promoter scheme on the right of the graphs refers to the presence of modules in constructs. COT, cotyledones; SAM, shoot apical meristem; HYP, hypocotyle; ROOT, root; RAM, root apical meristem.

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likely is relevant for downregulation of GUS. An inhibitory function is further supported, as a further truncation to the minimal promoter results in increased expression. Seedlings with such minimal promoter construct, containing only the MYBL/DOFF2 module, show clearly detectable GUS in most tissues. Hence, the inhibition is released and the minimal promoter is able to express the GUS reporter.

different regulatory modules also occur (Fig. 1). The promoter pMiERS1a contains the additional modules AHBP/DOFF1 and MYCL/ MYBL1, while the promoter pMiERS1b has the extra modules MYBL/ OPAQ1 and GBOX/GBOX1. Further, the common GTBX/MYCL1 module is present in both promoters, but this module is variable regarding TFBS motif combination. Comparing MiERS1 promoters with ERS1 promoter regions of other plant species (Arabidopsis and grapevine) reveals, that identified modules are different considering presence and position. Although ethylene receptors are highly conserved (S3-Fig. 1) and ethylene signaling is a common and important response pathway in plants [1,35], the identified variability of promoters suggests a different regulation of MiERS1. However, both full length mango promoters reveal a similar reporter expression pattern in adult transgenic Arabidopsis (Fig. 2). Intense GUS staining was found in leaf veins, cauline leaf axils and flower axils, as well as in flower and fruit, which indicates a tissue specific basic promoter activity. The Arabidopsis ERS1 expression pattern overall appears similar according to the eFP Browser [34]. However, a specific ERS1 expression - as found for pMiERS1:GUS - is not indicated for organ axilla or flower and fruit tissues. Comparing the ectopic reporter expression in transgenic Arabidopsis with the endogenous MiERS1 expression pattern in mango [14], it is found that the expression is quite similar in some organs. However, it should be kept in mind that Arabidopsis is a weed while mango is a tree, consequently, findings for one or another might not be fully comparable between those species. In Winterhagen et al. [14] it is shown that pre-mature mango flowers have a higher MiERS1 expression level than the mature male or perfect flowers. In stamen from mango flowers only a very low MiERS1 expression was found. Also in mango leaves and leaf peduncles MiERS1 was detected. The reporter expression in transgenic Arabidopsis overall mirrors this expression pattern. Although the target gene is expressed in fruit organs, a different expression is found in mango fruit and fruit pedicel compared to Arabidopsis fruit. However, mango produces a single stone fruit while Arabidopsis has a seed pod, so the species specific fruit physiology and developmental characteristics may limit a direct comparability. Transgenic Arabidopsis seedlings reveal high GUS activity in cotyledons and shoot apical meristem, but less activity in hypocotyl, root and the root apical meristem (Fig. 3), however, no data for MiERS1 expression in mango seedlings are available for comparison. Overall, both MiERS1 promoters show a similar and to some extend conserved organ specific expression pattern, although distinct differences are found in the composition of regulatory modules. Truncated promoter constructs were designed (Fig. 1) and the basic expression pattern of the GUS reporter was investigated in transgenic Arabidopsis seedlings (Fig. 3). The removal of the most distant predicted modules (pMiERS1a: MYCL/MYBL1, pMiERS1b: GBOX/GBOX1) did not significantly change the expression pattern. But for pMiERS1a, further truncation by removing the AHBP/DOFF1 module leads to significant reduction of GUS expression. Hence, this module seems to be responsible for a high basic expression level in most tissues. The overlapping modules GTBX/MYCL1 and GBOX/ABRE1 are present in both promoter isoforms, but the overall GUS expression is higher in pMiERS1b. Removing those modules results in a generally reduced expression in all tissues for both promoter isoforms, but particularly for pMiERS1b. This suggests that the GTBX/MYCL1 and/or the GBOX/ ABRE1 module of pMiERS1b contain a key motif for a high basic expression level in various organs. Interestingly, these modules in pMiERS1a appear not be able to keep the high expression level. The reason might be the variation of motifs within the GTBX/MYCL1 modules. It is concluded, that the motif combination IBOX/IDDF/TEFB present in pMiERS1b leads to a higher basic expression level. The motif combination AHBP/L1BX/MYBL of pMiERS1a obviously is not able to keep up such expression level. Further, in both promoters the module DOFF/OPAQ3 is present, but pMiERS1b contains additionally the overlapping module MYBL/OPAQ1. As both promoters cause a similarly reduced expression level, the conserved DOFF/OPAQ3 module

4.2. GUS expression under the control of pMiERS1 constructs after hormone treatment To investigate the response to phytohormones, agrobacteria-mediated infiltration with subsequent hormone application (ABA, IAA, ethylene) was performed on N. benthamiana and GUS activity was determined by enzyme assay (Fig. 4). The two longest constructs of pMiERS1b show significantly increased enzyme activity after ethylene treatment. But no additional significant response was found for any construct of pMiERS1a. Nevertheless, a trend can be seen: an increasing GUS activity after treatment is found also here for the longer constructs. After treatments with ABA or IAA, no response on GUS activity can be found for any construct when compared to controls. Independently, in transgenic Arabidopsis seedlings the GUS expression after hormone treatment was evaluated by tissue staining (Fig. 4). Considering methods, GUS quantification on histological samples of Arabidopsis reveals distinct responses, while the enzyme assays on N. benthamiana appears to be less sensitive or mask treatment effects (high standard error). In transgenic Arabidopsis a significant upregulation of GUS is found after ethylene treatment for the two longest constructs of both pMiERS1 isoforms. Furthermore, seedlings with the construct ΔpMiERS1b-506 also show significant upregulation of GUS after ethylene treatment. This suggests that both MiERS1 promoter isoforms are responsive to ethylene, and therefore, both MiERS1 receptors might be involved in positive ethylene feedback mechanisms. It was demonstrated for mango that MiERS1 is upregulated in fruit and pedicel after ethephon treatment [9,11], hence, an activation of the mango promoter in the transgenic model could be expected after ethylene treatment. As displayed in the eFP Browser [34], ERS1 is upregulated after ACC application (Fig. 4e), which is the precursor for endogenous ethylene synthesis. The positive ethylene feedback signaling and the participating pathway components in Arabidopsis have been reviewed earlier [35,36]. Based on the presented results, it is concluded that for pMiERS1a the presence of the AHBP/DOFF1 module is crucial for a positive response to ethylene. Adequately, such a module was described to be activated by wounding or methyl jasmonate treatments, which are linked to ethylene responses [37]. For pMiERS1b the module MYBL/ OPAQ1 in the ΔpMiERS1-506 construct appears to be linked to ethylene responses. This module is not present in the corresponding construct of pMiERS1a, which shows no response to ethylene. The conserved module DOFF/OPAQ3 at the same position may not be relevant for ethylene responses. The promoter analysis suggests that different regulatory modules control ethylene-induced gene transcription in related pMiERS1 isoforms. For most constructs the treatment with ABA leads to significant downregulation of GUS. The analysis reveals that already the shortest construct leads to a severe downregulation of GUS in response to ABA. It is concluded, that the MYBL/DOFF2 module has an ABA-dependent inhibitory function. The negative response is quite stable and can also be found in seedlings with longer constructs, suggesting that the inhibitory effect of ABA is linked to the MYBL/DOFF2 module and dominates other positively acting modules leading to consistent downregulation of the gene expression. However, the degree of the downregulation appears to be fine-tuned by other positively acting modules. According to the eFP Browser [34], ERS1 expression is also downregulated in Arabidopsis after ABA treatment. This indicates, that a negative response to ABA is conserved. Earlier studies on Arabidopsis and other plants describe an antagonistic cross-talk between ethylene and ABA signaling [38–40]. However, the interaction between ABA and 10

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treatment. However, seedlings with the full promoter construct show consistent downregulation only in SAM and HYP, but GUS expression is upregulated in RAM. This suggests that the GBOX/GBOX1 module regulates gene expression in response to ABA differently and tissue specific. This is different in comparison to the pMiERS1a full promoter, which generally downregulates GUS expression in response to ABA. Our results are supported by earlier studies that describe the role of GBOX core elements on gene expression due to environmental or developmental factors and in response to ABA [47,48]. Interestingly, it was demonstrated for Arabidopsis by Bakshi et al. [49], that ABA affects the expression of the ethylene receptors ETR1, ETR2 and EIN4, while the expression level of the receptors ERS1 and ERS2 was not influenced by ABA. Therefore, the response of pMiERS1 constructs to ABA suggests that MiERS1 genes are differently regulated than Arabidopsis ERS1. The GUS expression pattern after IAA treatment is inconsistent for various tissues and constructs. For the minimal promoter construct a downregulation is found in HYP and ROOT. However, this inhibitory effect is lost for most other constructs of the isoform pMiERS1a and a downregulation is detected in ROOT only for ΔpMiERS1-1067. A different expression pattern on tissue level is found for pMiERS1b constructs. While for ΔpMiERS1b-506 the gene expression is downregulated in ROOT, at the same time it is upregulated in RAM. Seedlings containing ΔpMiERS1b-1180 show significantly upregulated GUS expression in SAM, HYP, ROOT and RAM. This suggests that the module GTBX/MYCL1 (motif combination: IBOX/IDDF/TEFB) in pMiERS1b positively responds to IAA, but it appears to be not very tissue specific. The expression pattern under the control of the pMiERS1b full promoter is different again: GUS is upregulated after IAA treatment solely in RAM, cancelling upregulation in the other tissues found for ΔpMiERS1b-1180. Comparing the GUS expression pattern for both promoter isoforms suggests, that they are differently responsive to IAA. While isoform pMiERS1a does not reveal a clear response pattern, pMiERS1b contains a GTBX/MYCL1 module that is responsive to IAA; and depending on the truncation of the construct GUS is up- or downregulated. However, to our knowledge this module has not yet been linked to auxin signaling in previous publications.

ethylene, and the distinct response depends on the organ or the developmental stage of the plant [41,42]. Transgenic seedlings treated with IAA did not reveal a consistent pattern and GUS expression appears to be quite erratic. A significant downregulation is found for seedlings with the shortest construct and the construct ΔpMiERS1-1067 of pMiERS1a. In contrast, GUS expression was significantly increased in seedlings with the construct ΔpMiERS1-1180 of pMiERS1b. At this point, there is no consistency or trend and it is difficult to interpret the pattern, or even link modules to a specific auxin response. However, cross-talk between ethylene and auxin signaling is described and it was indicated that auxin acts downstream of ethylene signaling [17, Stepanova, 2009 #1673]. Interestingly, according to the eFP Browser [34], Arabidopsis ERS1 is responsive to auxin, while GUS under control of pMiERS1 is not. This suggests that MiERS1 genes are regulated differently in response to auxin. However, auxin-ethylene interaction appears quite variable and dependent on the species: e.g. the ACC synthase genes CitACS1 and CitACS2 are upregulated by IAA in an ethylene independent manner in melon, indicating that auxin also can act upstream of ethylene signaling [43,44]. Furthermore, polar auxin transport and ethylene signaling are tightly connected in fruit abscission processes in apple or mango. Auxin export from the developing embryo is involved in setting the sink status of the fruit, and a reduced auxin transport could activate the abscission zone by increasing ethylene susceptibility [11,16]. 4.3. Tissue specific GUS expression after hormone treatment Tissue specific GUS staining in transgenic seedlings was investigated by histology and image analysis (Figs. 5, 6). Seedlings with the two longest constructs of pMiERS1a show significantly increased GUS expression after ethylene treatment in some, but not in all tissues. In agreement with this finding, a AHBP/DOFF1 module was previously described as tissue specific [45]. Shorter constructs without the AHBP/ DOFF1 module are not responsive to ethylene, therefore, it is concluded that upregulation of GUS is based on this module. When the MCYL/ MYBL1 module is present (full promoter), the expression level in the shoot is reduced, but enhanced in root tissues. Hence, this module may also be involved in tissue specific transcriptional regulation. Regarding pMiERS1b the response to ethylene is different. Elevated GUS expression is found in several tissues for ΔpMiERS1b-506, and the two longest constructs promote further GUS upregulation - besides for HYP. This indicates that the MYBL/OPAQ1 module is relevant for initial ethylene response and transcriptional regulation is not very tissue specific. With the presence of the GTBX/MYCL1 module (motif combination: IBOX/ IDDF/TEFB), GUS expression was further upregulated and also significant in root tissues, but also here not for HYP. GTBX/MYCL modules are described to be responsive to various factors, including ethylenerelated processes like etiolation or senescence [46]. Both full promoters cause ethylene induced GUS expression in ROOT and RAM, but only isoform pMiERS1b leads to upregulation of GUS also in COT and SAM. Finally, not all plant organs of the seedlings show the same response to ethylene and results indicate that the modules cause distinct differences on responsiveness and tissue specificity. For the whole seedlings it was presented earlier (Fig. 4) that ABA negatively controls expression with the shortest construct containing the MYBL/DOFF2 module. On the level of plant organs this is further strengthened as GUS expression is significantly downregulated in all tissues after ABA treatment. This leads to the conclusion that MYBL/ DOFF2 is responsive to ABA and does not act in tissue specific manner. However, this effect appears to be counter-regulated by AHBP/DOFF1 in pMiERS1a, where a response to ABA is mitigated. But significantly decreased GUS expression is found again with the full promoter containing MYCL/MYBL1. This module appears to be functional and may dominate AHBP/DOFF1 leading again to downregulation of GUS in COT, SAM and ROOT. Overall, a similar pattern is found for pMiERS1b constructs, where GUS expression is also downregulated after ABA

5. Conclusion This study shows that both pMiERS1 lead to a similar gene expression in Arabidopsis, regarding tissue specificity in mature plants or response to hormones in seedlings, when compared to endogenous AtERS1 expression in Arabidopsis. Although distinct differences were found in the promoter module composition, the overall expression response appears to be quite conserved. However, ectopic expression experiments testing sequences from other species in transgenic models should be interpreted with caution. Both full length promoters are responsive to ethylene, indicating that a positive feedback regulation of MiERS1 is well conserved. Nevertheless, the promoters reveal individual responsiveness and tissue specificity related to particular TFBS motifs and modules. The AHBP/DOFF1 module of pMiERS1a could be linked with a positive ethylene feedback, while for pMiERS1b it is the module MYBL/OPAQ1. Although the overall response to ethylene is similar for both promoter isoforms, their individual regulation depends on different regulatory modules. A specific response to ABA or IAA depends on construct and presence of particular modules. For a negative response to ABA the MYBL/DOFF2 module could be responsible, as it is present in both promoter isoforms. The GTBX/MYCL1 module containing the motifs IBOX/IDDF/TEFB, present in this combination only in pMiERS1b, is responsive to IAA in several tissues. Although both promoter isoforms contain GTBX/MYCL1 modules, a motif variation could be linked with a specific auxin response. Conclusively, although ERS1 ethylene receptors are highly conserved among plants, fine tuning of their expression is variable on the species and intraspecific level.

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Authors contribution

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PW designed and performed the experiments; PW and MHH analysed the data; PW, MHH, and JNW evaluated results, wrote and reviewed the manuscript. Declaration of Competing Interest The authors declare there is no conflict of interest. Acknowledgements The authors thank Elke Sprich for excellent technical assistance and Philippo Capezzone for statistical advice. We are grateful to Moritz Nowack for providing Arabidospis seeds and support with transformations. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors declare there is no conflict of interest. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.plantsci.2019.110269. References [1] A.N. Stepanova, J.M. Alonso, Ethylene signaling and response: where different regulatory modules meet, Curr. Opin. Plant Biol. 12 (2009) 548–555. [2] D.R. Gallie, Ethylene receptors in plants – why so much complexity? F1000Prime Rep. 7 (2015) 39. [3] A.B. Bleecker, M.A. Estelle, C. Somerville, H. Kende, Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana, Science 241 (1988) 1086–1089. [4] J. Hua, H. Sakai, S. Nourizadeh, Q.G. Chen, A.B. Bleecker, J.R. Ecker, E.M. Meyerowitz, EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis, Plant Cell 10 (1998) 1321–1332. [5] C.L. Bassett, T.S. Artlip, A.M. Callahan, Characterization of the peach homologue of the ethylene receptor, PpETR1, reveals some unusual features regarding transcript processing, Planta 215 (2002) 679–688. [6] J. Bustamante-Porras, C. Campa, V. Poncet, M. Noirot, T. Leroy, S. Hamon, A. de Kochko, Molecular characterization of an ethylene receptor gene (CcETR1) in coffee trees, its relationship with fruit development and caffeine content, Mol. Gen. Genet. 277 (2007) 701–712. [7] H.S. Ireland, F. Guillen, J. Bowen, E.J. Tacken, J. Putterill, R.J. Schaffer, J.W. Johnston, Mining the apple genome reveals a family of nine ethylene receptor genes, Postharvest Biol. Technol. 72 (2012) 42–46. [8] B.M. Kevany, D.M. Tieman, M.G. Taylor, V.D. Cin, H.J. Klee, Ethylene receptor degradation controls the timing of ripening in tomato fruit, Plant J. 51 (2007) 458–467. [9] M. Ish-Shalom, Y. Dahan, I. Maayan, V. Irihimovitch, Cloning and molecular characterization of an ethylene receptor gene, MiERS1, expressed during mango fruitlet abscission and fruit ripening, Plant Physiol. Biochem. 49 (2011) 931–936. [10] P.G. Martinez, R.L. Gomez, M.A. Gomez-Lim, Identification of an ETR1-homologue from mango fruit expressing during fruit ripening and wounding, J. Plant Physiol. 158 (2001) 101–108. [11] M.H. Hagemann, P. Winterhagen, M. Hegele, J.N. Wünsche, Ethephon induced abscission in mango: physiological fruitlet responses, Front. Plant Sci. 6 (2015) article 706. [12] M.H. Hagemann, P. Winterhagen, M. Hegele, J.N. Wünsche, Ethephon induced abscission of mango fruitlets – physiological fruit pedicel response, Acta Hortic. 1066 (2015) 109–116. [13] P. Winterhagen, J.N. Wünsche, Single nucleotide polymorphism analysis reveals heterogeneity within a seedling tree population of a polyembryonic mango cultivar, Genome 59 (2016) 319–325. [14] P. Winterhagen, M.H. Hagemann, J.N. Wünsche, Expression and interaction of the mango ethylene receptor MiETR1 and different receptor versions of MiERS1, Plant Sci. 246 (2016) 26–36. [15] R. Swarup, P. Perry, D. Hagenbeek, D. Van Der Straeten, G.T. Beemster, G. Sandberg, R. Bhalerao, K. Ljung, M.J. Bennett, Ethylene upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation, Plant Cell 19 (2007) 2186–2196. [16] A. Botton, G. Eccher, C. Forcato, A. Ferrarini, M. Begheldo, M. Zermiani, S. Moscatello, A. Battistelli, R. Velasco, B. Ruperti, A. Ramina, Signaling pathways mediating the induction of apple fruitlet abscission, Plant Physiol. 155 (2011) 185–208. [17] R. Swarup, G. Parry, N. Graham, T. Allen, M. Bennett, Auxin cross-talk: integration of signalling pathways to control plant development, Plant Mol. Biol. 49 (2002) 411–426. [18] J.J. Ross, D.E. Weston, S.E. Davidson, J.B. Reid, Plant hormone interactions: how complex are they? Physiol. Plant. 141 (2011) 299–309.

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