Differential and reciprocal regulation of ethylene pathway genes regulates petal abscission in fragrant and non-fragrant roses

Accepted Manuscript Title: Differential and reciprocal regulation of ethylene pathway genes regulates petal abscission in fragrant and non-fragrant roses Authors: Priya Singh, Amar Pal Singh, Aniruddha P. Sane PII: DOI: Reference:

S0168-9452(18)31172-5 https://doi.org/10.1016/j.plantsci.2018.12.013 PSL 10022

To appear in:

Plant Science

Received date: Revised date: Accepted date:

25 September 2018 12 December 2018 14 December 2018

Please cite this article as: Singh P, Singh AP, Sane AP, Differential and reciprocal regulation of ethylene pathway genes regulates petal abscission in fragrant and nonfragrant roses, Plant Science (2018), https://doi.org/10.1016/j.plantsci.2018.12.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Differential and reciprocal regulation of ethylene pathway genes regulates petal abscission in fragrant and non-fragrant roses Priya Singh1,2, Amar Pal Singh1,3 and Aniruddha P. Sane1,2 * 1

Plant Gene Expression Lab, CSIR-National Botanical Research Institute (CSIR), Lucknow-

226001, India, 2Academy of Scientific and Innovative Research (AcSIR), Ghaziabad-201002,

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India and 3Current address: National Institute for Plant Genome Research, New Delhi, India *Author for correspondence

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Email: [email protected]; [email protected]

Highlights

Abstract

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Rose ethylene pathway genes identified by transcriptomic sequencing Up-regulation of ethylene pathway in ethylene-induced abscission in R. bourboniana Delayed and differential upregulation of ethylene pathway during natural abscission Suppression of ethylene pathway by ethylene during abscission in R. hybrida Reciprocal regulation of ethylene pathway governs differential abscission in roses

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The fragrant rose, Rosa bourboniana, is highly sensitive to ethylene and shows rapid petal abscission (within 16-18 h) while the non-fragrant hybrid rose, R. hybrida, shows delayed abscission (50-52 h) due to reduced ethylene sensitivity. To understand the molecular basis

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governing these differences, all components of the ethylene pathway (biosynthesis/ receptor/signalling) were studied for expression during abscission. Transcript accumulation of most ethylene biosynthesis genes (ACS/ACO families) increased rapidly in petal abscission zones of R. bourboniana within 4-8 h of ethylene treatment. The expression of most receptor and signalling genes encoding CTRs, EIN2 and EIN3/EIL homologues also followed similar kinetics. Under natural field conditions where abscission takes longer, there was a temporal delay in transcript accumulation of most ethylene pathway genes while some

biosynthesis genes (showing reduced ethylene sensitivity) were more strongly up-regulated by abscission cues. In contrast, in R. hybrida where even ethylene-induced abscission is considerably delayed, transcript accumulation of most ethylene biosynthesis and signalling genes was, surprisingly, reduced by ethylene and showed an opposite regulation compared to R. bourboniana. The results suggest that differential and reciprocal regulation of ethylene pathway is one of the major reasons for differences in petal abscission and vase-life between

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Rosa bourboniana and R. hybrida.

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Key words: ethylene signalling; petal; senescence; cell separation; flower; vase-life

1. Introduction

Abscission is the process of natural shedding of plant parts such as leaves, buds, flowers,

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floral parts, fruits, seeds etc. in response to developmental or environmental cues. The

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process is associated with a series of changes at the molecular and biochemical level within

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the abscission zone (AZ) that separates the organ from the parent plant [1]. It culminates with the secretion of several wall modifying proteins and enzymes that lead to cell wall

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disassembly and the separation of the organ [2-5]. The plant hormone ethylene plays a particularly important role in acceleration of the abscission process [6]. Abscission zone cells

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of most tissues are therefore sensitive to ethylene although their sensitivity differs between different abscising tissues [7]. For instance, AZ cells of flowers and petals of many dicots are

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extremely sensitive to ethylene and can abscise within 1-2 hours of ethylene treatment in a dose-dependent manner compared to leaf abscission tissues [8-11]. The role of ethylene in

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abscission has been demonstrated in several plants [6] including Arabidopsis [12], tomato [13], citrus [14, 15], cotton [16], apple [17] and ornamentals like Pelargonium [10], tulips [18], rose [19-24] etc. Mutants of the ethylene pathway show delayed abscission [12, 25] while inhibitors of ethylene perception like silver nitrate and 1-methyl cyclopropene delay

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organ abscission providing further evidence of the role of ethylene in organ abscission [19, 26, 27]. The ability of ethylene to specifically affect cells of the abscission zone necessitates a complex regulation of the pathway genes governing its biosynthesis, perception and signalling within this zone. The components and function of the ethylene pathway machinery have been well elucidated in Arabidopsis and a few other plants like tomato and rice [28-33].

Most components of the pathway such as ACC synthase, ACC oxidase, ethylene receptors and transcription factors of the EIN3 and ERF family exist as members of a multi-gene family that show overlapping as well as unique functions in different tissues and processes [34-39]. Their tissue- and stage-specific regulation by developmental and environmental cues and by hormones other than ethylene, provides evidence for the complexity of signalling that is necessary to fine tune responses during development. The identity and role of individual members of the ethylene pathway in different processes in plants other than Arabidopsis is

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still a challenge.

Roses are amongst the most popular ornamentals worldwide. The fragrant varieties of roses,

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important in the perfume industry and as garden flowers, show rapid petal abscission within

35-42 h of anthesis in the field [19, 20]. Petal abscission in these flowers is highly sensitive to ethylene and can be delayed considerably by the ethylene perception inhibitor 1-methyl cyclopropene (1-MCP) [19]. In contrast, almost all hybrid rose varieties (largely non-

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fragrant) do not undergo petal abscission but show petal fading/wilting and shrivel on the

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plant several days after anthesis [24]. They show relatively reduced sensitivity to exogenous

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ethylene [40-42]. The presence of these two distinct types of flowers makes rose an interesting system for the study of petal abscission, flower life and ethylene sensitivity. In this

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work, we have identified and characterized several new components of the ethylene biosynthesis, perception and signalling machinery through a transcriptomic analysis of rose

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petal AZ cDNA. Our studies show that differences in the expression of the ethylene pathway genes during the progression of abscission underlie the differences in abscission between the

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fragrant R. bourboniana and the non-fragrant R. hybrida.

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2. Materials and Methods

2.1 Plant material and ethylene treatments Rose flowers of the ethylene sensitive, fragrant variety (Rosa bourboniana var. Gruss an Teplitz) and the less sensitive, non-fragrant variety (Rosa hybrida var. Opening Night) were

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chosen for the study. Flowers of the same developmental stage were collected early in the morning (prior to sunrise; marked as time point 0), as described previously [19, 20]. Flowers were treated with 0.5 µl L-1 ethylene in a sealed glass desiccator for 18 hours for R. bourboniana (time of abscission 16–18 h) and 52 h for R. hybrida (time of abscission 50–52 h) as described [19]. The desiccators were placed under fluorescent light of the same photoperiod (about 125 µmoles of light for 12 h, temp between 25-27 degrees) to mimic field

conditions to the extent possible. Petal abscission zones (2 mm2 at the base of the petal in contact with the thalamus) were collected at 0 h (ethylene untreated), 4 h, 8 h and 12 h during ethylene treatment for R. bourboniana and further at 24 h, 36 h and 48 h for R. hybrida and frozen immediately in liquid nitrogen. Sampling was performed in three separate pools (each pool containing at least 50 flowers) for each stage. For natural abscission studies (time of abscission 35–42 h), R. bourboniana flowers were tagged prior to sunrise at the stage of opening of the outermost whorl (as in case of ethylene treated flowers) and petal AZs

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collected at 0, 4, 8, 12, 24 and 36 h without ethylene treatment and processed as above.

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2.2 RNA extraction and preparation of cDNA

Total RNA from was isolated and processed for cDNA as described [19] and used for the expression analysis of ethylene biosynthesis and signalling genes by real-time polymerase

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chain reaction (PCR).

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2.3 Real-time RT–PCR

The real time reaction was run in technical triplicates for each of the three biological samples

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and the data analyzed was the mean of triplicates in real time reaction. Reactions were run on

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an ABI Step One Plus real time PCR machine (Applied Biosystems Inc, USA) with Fast SYBR Green Master Mix (Applied Biosystems) as a fluorescent indicator dye in all real time

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reactions (Applied Biosystem). The reactions were set up in a final volume of 20 µl as follows - step 1, 50C, 2 min, step 2, 95C 10 min, step 3 (95C 15sec, 60C 1 min), x 40 cycles. The relative mRNA levels of the individual genes was normalized with respect to validated

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internal control gene ACTIN [21] and PP2A [43]. Relative gene expression was analyzed by

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the 2-∆CT method [44].

2.4 De-novo assembly of transcriptome and identification of ethylene biosynthesis and signaling genes in rose petal AZs.

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Total RNA from ethylene-untreated (0 h) and 0.5 μl/L ethylene-treated (8 h) petal and petal AZs of R. bourboniana along with 0.5 l/l ethylene-treated (8 h) petal AZs of R. hybrida was isolated in triplicates. DNase-treated RNA from respective tissues (in triplicates) was reverse transcribed and sequenced on an Illumina platform along with the SFF files obtained from 454 pyrosequencing platform. Paired-end de novo transcriptome assembly was performed using Illumina HiSeq (Quality score >Q30, Scigenome, Kochi, India). The Illumina fastq reads were trimmed using Trimmomatic tool to remove adapter sequences before performing

assembly. Simultaneously, Fastq files were generated from SFF file format of 454 data using sff_extract tool and tag sequences were removed from 454 data using tag cleaner along with trimming. Assembly of trimmed Illumina and 454 reads were then performed using Trinity. Further, trimmed reads were aligned using Bowtie program to get the assembled transcriptome. The assembled transcripts (>100 000 transcripts) were first compared with Rosaceae and later with NCBI, UniProt and other databases for annotation at an e-value of E-

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value <= 10-5 and similarity score >= 40%. The assembled transcript sequences were analysed for putative ethylene biosynthesis (ACOs,

ACSs), perception (ETRs) and signaling (CTRs, EIN2, EILs) gene sequences in the NCBI

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database against all species. Six new ethylene biosynthesis (five ACOs, one ACS) and seven

new signalling genes (two CTRs, three ethylene receptors and two signalling genes) were identified from the Illumina transcriptome. A total of 16 genes of the ethylene pathway (one

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ACO, five ACSs, two CTRs, five ETRs, three EIN3s) extracted from NCBI were also included in the study. Of the 29 genes, complete ORFs could be obtained for 22 genes through

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Illumina sequencing including three complete sequences (ACS1, CTR1 and EIN3-3) of NCBI.

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With the recent availability of the Rosa chinensis sequence [45, 46] the full length sequences

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of the remaining genes have also been obtained (supplementary table S1). Gene-specific primers (supplementary table S2) were designed for all genes (following

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nucleotide sequence alignment of paralogues) for validation such that at least one primer was from the unique 3’/5’UTR region for those genes where full length sequences were available.

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2.5 Statistical analysis. Statistical assistance was performed using Duncan’s multiple range tests (P <0.05) by using SPSS software. Standard error of the mean was calculated and

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represented as vertical bars in the figures. 3. Results

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3.1 Isolation of several new components in the ethylene signal pathway in rose Previous studies have shown differences in petal abscission between the fragrant rose, R. bourboniana, and the hybrid non-fragrant rose, R. hybrida [19-23]. The fragrant roses are ethylene sensitive and abscise naturally within 35-42 h while exogenous ethylene treatment (0.5 µl/L) accelerates abscission to just 16-18 h in a dose dependent manner. In contrast, R. hybrida does not abscise naturally but undergoes senescence. It does show abscission after 48-52 h if treated with 0.5 µl/L exogenous ethylene [21]. This was indicative of differences in

the ethylene biosynthesis pathway as well as signalling between the two. In order to get an insight into how these were regulated during petal abscission, an analysis of all the ethylene biosynthesis and signalling genes was first carried out. Prior to this work, a total of 16 genes of the ethylene pathway in rose were available in the database. These included five ACC synthases, one ACC oxidase, five ethylene receptor, two CTRs and three EIN3 homologues [47-54]. We have renamed the three EIN3 homologues EIN3-1, EIN3-2, EIN3-3 as EIL1, EIL2, EIL3 in this manuscript. Of the above genes, full length ORFs for only three genes

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namely ACS1, CTR1, EIN3-3 were available [51, 53, 54] while the rest were partial sequences. Following an Illumina transcriptome analysis of abscission zone tissue, a total of

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12 ethylene biosynthesis genes including 6 ACOs (1-6), and 6 ACSs (1-6) were identified in

ethylene treated petal AZs of R. bourboniana and R. hybrida. These included ACS1-ACS5 and ACO1 known previously and an additional ACS6 and ACO2-ACO6 obtained from the present study. In addition, full length sequences were obtained for ACS2, ACS5 and ACO1

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known previously. Similarly genes encoding four CTRs, seven ethylene receptors and five

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signalling genes were identified in both varieties from the transcriptome study. Apart from

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CTR1 and EIN3-3, for which full length ORFs were already available, these included CTR2, ETR1, ETR2, ETR3, ETR4, ETR5, EIN3-1 and EIN3-2 known previously as partial genes. In

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addition, CTR2, CTR3, ETR2, ETR3, ETR5, ETR7, ETR8, EIN2, EIN3-1 and EIN3-2 were identified as complete ORFs in the present study. Thus a total of 29 genes were identified

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(supplementary table S1) and chosen for study. A phylogenetic analysis of the full length protein sequences of the ACS/ACO/ETR/CTR/EIN2/EIL families with corresponding

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members from Arabidopsis and tomato is shown in supplementary fig S1 (A-F). Expression studies with all identified genes were carried out under ethylene-untreated (natural abscission

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zones) and ethylene-treated conditions in a time-dependent manner. 3.2 Expression of ethylene biosynthesis genes Studies on ethylene treated rose flowers revealed expression of four of the six ACS genes in

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AZ cDNA while two, RbACS2 and RbACS4, failed to amplify in the samples and were not considered. Of the four, the most prominently expressed gene (in terms of relative transcript level) was RbACS1 (fig. 1). Its expression shot up almost ~60 fold within 4 h of ethylene treatment and remained high up to 12 h in R. bourboniana. A considerable increase was also seen for RbACS5 (about 3 folds) and RbACS6 (about 5 folds). For RbACS5 increase was transient and only seen up to 8 h after which levels decreased to less than 5% at 12 h. In

contrast, RbACS3 transcripts were rapidly depleted to 60% of control within 4 h of treatment and to less than 10% by 12 h. Expression patterns changed under natural field conditions where abscission is delayed (fig. 1). The rapid ethylene induced increase seen in RbACS1 was not observed under field conditions. Instead, expression showed a cyclic pattern, increasing at 12 h, followed by a decrease at 24 h and again an increase at 36 h. Nevertheless, the scale of increase was only 4-

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5 fold compared to the 60 fold seen upon ethylene treatment. On the other hand, both RbACS5 and RbACS6 showed a much higher increase during natural abscission with expression going up 5 folds for RbACS5 and 20 folds for RbACS6 from 12 h onwards.

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RbACS3 remained suppressed during natural abscission although the extent of suppression was lesser compared to ethylene treatment.

In R. hybrida, which shows a considerable delay in ethylene-induced abscission, the

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expression of ACS1 began increasing from 8 h (fig. 1) but reached peak levels only at 24 h.

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Moreover, the scale of expression was much reduced (about 6-8 folds) compared to that in R.

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bourboniana. ACS3 was not affected much by ethylene except at a later time point while ACS5 was strongly suppressed by ethylene although the level of suppression was reduced

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compared to that in R. bourboniana Interestingly, the expression of ACS6 (which also increased only after 12 h) was much higher in R. hybrida upon ethylene treatment compared

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to that in R. bourboniana, going up to 40 folds by 48 h. Of the six ACC oxidase genes studied, ACO2, ACO3 and ACO4 were most prominent in

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transcript abundance compared to ACO1, ACO5 and ACO6 (which was least abundant). All ACO genes (except RbACO3) showed an ethylene induced accumulation of transcripts in R.

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bourboniana. Of these, the transcript abundance of RbACO1, RbACO2, RbACO4 and RbACO5 increased substantially by 4-10 folds in petal abscission zones of R. bourboniana within 8 h of ethylene treatment (fig. 2). The increase was transient in most cases and observed at 4-8 h but decreased at 12 h prior to abscission. Unlike these, RbACO6 transcript

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levels were suppressed 4-8 h after ethylene treatment although they increased at 12 h. The expression of RbACO3 showed a continuous decrease following ethylene treatment to about 50% by 12 h. Under natural abscission, the expression profiles for most genes like RbACO1, RbACO2, RbACO4 and RbACO5 were largely similar to that observed after ethylene treatment although the increase in transcript levels was delayed and seen at 12-24 h (fig. 2). Interestingly, the increase in transcript levels for RbACO1, RbACO2, RbACO4 and RbACO5

was much higher than that seen in ethylene treated flowers. Surprisingly RbACO3, which was suppressed by ethylene treatment, also increased during the course of natural abscission by almost 2.5 folds at 12 h. RbACO6 transcript levels showed a reduction during the course of natural abscission. Compared to R. bourboniana, the response of ACO genes in R. hybrida to ethylene was very different. Expression of all ACOs, except ACO6, was strongly suppressed in R. hybrida post-

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ethylene treatment to just 10-50% of the 0 h samples (fig. 2). The expression of ACO6, however, increased two folds in R. hybrida after ethylene treatment by 12 h and up to 8 folds

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by 48 h. 3.3 Expression of ethylene receptor genes

The transcriptome data revealed seven ethylene receptor genes that were active in petal

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abscission zones. Of these, ETR2, ETR5 and ETR6 were low transcript abundance genes. The transcript abundance of RbETR1, RbETR2, RbETR3, RbETR7 and RbETR8, increased within

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4-8 h of ethylene treatment (fig. 3). Transcript levels remained high throughout the

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progression of abscission for RbETR1 and RbETR3 but decreased to basal levels for ETR2,

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ETR7 and ETR8 by 12 h. For RbETR1 the increase was almost 300 fold while for RbETR7 it was about 50 fold. In contrast, RbETR5 and RbETR6 showed a decrease in transcript

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accumulation to just 5-10% of basal levels in response to ethylene. Interestingly, under conditions of natural abscission only RbETR1 showed an increase during

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abscission (fig. 3). RbETR2, RbETR3 and RbETR8 did not show much of a change except for a decrease at early time points while steady state levels of RbETR5 were strongly reduced

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during abscission. No expression was detected for RbETR6 and RbETR7. In R. hybrida, treatment with ethylene failed to induce ETR1, ETR2, ETR7 and ETR8 in the way seen in R. bourboniana (fig. 3). Only ETR3 showed an increase in transcript accumulation as seen in R. bourboniana. In fact, ETR1 and ETR7 which were most strongly

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up-regulated in R. bourboniana by ethylene, showed a reduction in transcript level to less than 10% of the basal level especially at the 36-48 h time points. The difference between the endogenous levels of R. bourboniana and R. hybrida following ethylene treatment was 100500 fold (being high in R. bourboniana). In contrast, ETR5 which showed greatly reduced transcript accumulation upon ethylene treatment in R. bourboniana, displayed a strong

increase with transcript levels going up 100 fold or more for ETR5. ETR2, ETR6 and ETR8 were largely unaffected by ethylene except for a slight increase just prior to abscission. 3.4 Expression of ethylene signalling genes Unlike Arabidopsis, which has a single CTR1 gene (that negatively regulates the ethylene signal pathway), at least four CTR1 homologues, designated as CTR1-CTR4, could be identified from rose petal abscission zones. The transcript abundance of at least three, namely

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CTR1-CTR3, increased within 4 h of exogenous ethylene treatment in R. bourboniana (fig. 4). The increase ranged from 2-10 folds for all. CTR4 also showed an increase in transcript

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accumulation but only at the 8 h stage unlike the other three which increased from 4-12 h.

CTR1 was the most abundant of the four as far as transcript levels were concerned while CTR4 was the least abundant.

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Under conditions of natural abscission too, three of the CTRs, CTR2, CTR3 and CTR4 showed an increase in transcript accumulation, although the increase was delayed compared

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to that of ethylene treated samples. Transcript levels rose from the 12 h stage in CTR2 and

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CTR3 and remained high up to 36 h while they increased just prior to abscission at 36 h for

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CTR4 which remained largely suppressed during natural abscission. Surprisingly, RbCTR1, which was up-regulated during ethylene-induced abscission, showed a substantial decrease in

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transcript accumulation to just 20% of its levels during natural abscission (fig. 4). Unlike in R. bourboniana, the strong ethylene responsiveness of CTR1, CTR2 and CTR3 could not be observed R. hybrida. In fact, expression of CTR2 and CTR3 was strongly reduced in R.

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hybrida. In contrast, RbCTR4 which was only slightly affected by ethylene in R. bourboniana and was actually down-regulated by 12 h, was up-regulated by about 2-3 fold in R. hybrida in

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response to ethylene.

EIN2, one of the key positive regulators of ethylene signalling in Arabidopsis, was identified as single gene in rose. Expression of RbEIN2 was rapidly up-regulated by ethylene within 4 h

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of ethylene treatment in Rosa bourboniana and remained 3-5 folds higher during the progression of abscission (fig. 5). Its expression was also up-regulated during natural abscission with transcripts increasing from 12 h onwards. In contrast to Rosa bourboniana, ethylene did not elicit an induction of EIN2 in R. hybrida. Transcript levels remained as high as at the 0 h stage with very little change or a reduction.

EIN3/EILs are central in transducing the ethylene signal to downstream targets and activating ethylene responses. Of the four EIN3-like genes identified in rose, namely RbEIL1-RbEIL4, RbEIL1 was most abundant while RbEIL4 was least abundant. Transcript accumulation of RbEIL2, RbEIL3 and RbEIL4 increased several fold upon treatment with ethylene in R. bourboniana with a 2-6 fold increase from 4-8 h of ethylene treatment (fig. 5). Of these, RbEIL2 was most responsive to ethylene with transcripts increasing within 4 h of ethylene exposure. RbEIL1 showed only a slight increase upon ethylene treatment. Under natural

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abscission, the expression of all EIL genes was enhanced at 8-12 h although it was preceded

by a decrease at 4 h and after 24 h. Interestingly, the expression of EIL1 and EIL4 showed a

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more prominent increase during natural abscission compared to ethylene-treated flowers with

transcript levels increasing to 4-7 folds. The increase occurred after a temporal delay compared to ethylene treated flowers and in most cases was seen at the 12 h stage. Unlike R. bourboniana, transcript levels of the EIL genes showed a considerable decrease in R. hybrida

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upon ethylene treatment with levels going down to 75% of the basal levels (EIL2) or further

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down to 50% (EIL3). Transcript levels of EIL1 were also reduced although it showed a

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transient increase at the early time point of 12 h. EIL4 did not undergo much of a change.

4. Discussion

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In rose, the flower life in fragrant varieties like R. bourboniana is considerably shorter (ending a day or two post-anthesis) due to rapid petal abscission [19, 21-24]. In contrast, most

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R. hybrida varieties (used mainly as cut flowers and non-fragrant) stay for several days postanthesis showing only petal fading and wilting instead of petal abscission [24, 40-42]. The

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differences in flower life between these species are closely associated with differences in ethylene sensitivity, its responses and the speed of abscission [21-23, 42]. In fragrant roses, abscission can be substantially delayed by 1-MCP indicating the role of ethylene in the process [19]. We hypothesized that differential regulation of the ethylene pathway

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components in petal abscission zones may, in large part, be responsible for early petal abscission in R. bourboniana and delayed senescence in R. hybrida flowers. Ethylene has been identified as an important hormone in different abscission processes [6] in various plants and the regulation of some components of either the ethylene biosynthesis or signalling genes has been studied in plants like citrus, tomato, apple and peach during abscission of leaves, fruits, fruitlets etc [17, 37, 55, 56]. Yet, the complex coordinated regulation of the

entire pathway in abscission remains undeciphered not just in rose but in most plants due to lack of sequence information of all pathway components and difficulties of studying the minute abscission zone tissues. Firstly, through the current study, we have established the components of the ethylene pathway in R. bourboniana and R. hybrida through transcriptomic sequencing. Our data show a much larger number of the genes than that previously reported with 6 ACSs, 6 ACOs, 7

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ETRs, 4 CTRs, one EIN2 and 4 EILs (supplementary table S1) and matches with the data that was recently released for R. chinensis [45, 46]. Secondly, we show that all components of the pathway, namely those pertaining to ethylene biosynthesis (ACS, ACO family), its perception

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(ETR family) and signalling (CTRs, EIN2, EIL families), are expressed and differentially

regulated during ethylene-induced abscission not only between R. bourboniana and R. hybrida but also between ethylene-induced and natural abscission within R. bourboniana.

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Differences in regulation of individual genes in each group of the family seem to be at least partly associated with abscission outcome. By and large, the ethylene pathway genes

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themselves strongly respond to ethylene in the ethylene-sensitive Rosa bourboniana while

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their response to ethylene is muted or even opposite in Rosa hybrida. Genes encoding CTRs,

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EIN2 and EILs which are not transcriptionally activated in Arabidopsis undergo a transcriptional change in rose in response developmental cues and in response to ethylene

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indicating differences in regulation of the ethylene pathway between different plants. Abscission in R. bourboniana, induced by 0.5 μl/L ethylene, is associated with a strong

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activation of most genes of the ethylene pathway within 4-8 h. This activation is seen for all ethylene pathway components involved in biosynthesis, perception and signalling except ACS3, ACO3, ETR5 and ETR6 which are suppressed. It is particularly high (within 4 h) in

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ACS1, ETR1, ETR3 and to a lesser extent in ETR2 and EIL2. The auto-activation of the ethylene pathway genes by ethylene may be a means for autocatalytic production of ethylene as seen in climacteric fruits during ripening [57, 58]. It may not only be necessary for

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abscission but also for ethylene-induced flower opening that is seen in roses. In fact, ethylene treatment of roses does increase ethylene levels in petals during flower opening [50]. Unlike ethylene-induced abscission, the course of natural field abscission is slow, taking more than twice as long for abscission. Accordingly, most components of the ethylene pathway in R. bourboniana, although up-regulated during natural abscission, show a temporal delay, being activated between 12 h and continuing up to 24 h or 36 h compared to 4-8 h in ethyleneinduced abscission. Strangely, natural abscission proceeded with a decrease in transcription

of most genes at the 4 h and 8 h time points – an observation that is difficult to explain as of now and calls for further studies. The delay in expression of ethylene pathway genes during field abscission would delay ethylene production and downstream ethylene signalling processes under field conditions thereby delaying abscission as expected. Nevertheless there are clear qualitative differences between ethylene-induced abscission and field abscission that cannot be explained merely by a delay in the biosynthesis of ethylene. For instance, there is a difference in the scale of regulation of the specific components such as ACS5, ACS6, ACO3-

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ACO5, CTR2, CTR3 and EIL4, all of which show a greater up-regulation during natural

abscission as compared to ethylene-induced abscission. It is interesting to note that some of

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these genes do not undergo much of an induction by ethylene. On the other hand, genes encoding ETRs do not seem to undergo much of a transcriptional change during natural

abscission, unlike their regulation in ethylene-induced abscission. In all probability, the genes up-regulated during natural abscission could be the primary genes that respond to abscission

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cues under field conditions. Their up-regulation may initiate ethylene biosynthesis and

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thereafter activate other primarily ethylene-responsive members of the pathway such as

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ACS1, ACO1, EIN2 and EIL2 for autocatalytic production of ethylene and further activation of its signalling. Since ETRs suppress ethylene signalling, these may not respond to

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abscission cues (when initial ethylene biosynthesis and pathway activation is needed) but only respond to ethylene when its levels increase so as to suppress and prevent the ethylene

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signal from going out of control. The ethylene-induced activation of downstream components like EIN2/EILs then amplifies the ethylene signal and accelerates abscission as shown in a

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hypothetical model (Fig 6). The results suggest that the regulation of ethylene pathway genes in R. bourboniana, is under a complex control exerted by developmental cues during natural abscission as well as ethylene. The abscission-specific developmental cues may fine regulate

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the expression of target genes of ethylene pathway to initiate ethylene biosynthesis. This is particularly apparent in the case of ACO3 and CTR1. ACO3 is strongly up-regulated during natural abscission but actually suppressed upon ethylene treatment while CTR1 (a negative

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regulator) is activated during ethylene-induced abscission but strongly down-regulated during natural abscission. With the availability of the R. chinensis sequence, identifying the changes occurring at the promoters in these genes may become easier. While the expression of most ethylene pathway genes showed a temporal delay during natural abscission (with few exceptions), the expression pattern in R. hybrida was quite different from R. bourboniana and largely reciprocal nature in nature. Thus, while many of

the genes in R. bourboniana are up-regulated by ethylene there is a corresponding decrease in their transcript levels in R. hybrida by ethylene and vice versa. These differences are most prominently seen in the regulation of genes encoding ACC oxidases where all genes (except ACO3) are strongly up-regulated in R. bourboniana while transcripts of all genes, except ACO6, are reduced in R. hybrida in response to ethylene. The ethylene induced suppression of the ethylene pathway in R. hybrida could affect autocatalytic ethylene production. This might, in part, explain the inability of the R. hybrida flowers to abscise in the field. The

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reciprocal regulation between R. bourboniana and R. hybrida is also observed in genes encoding ethylene receptors like ETR1, ETR5, ETR6, ETR7 and ETR8. Of these ETR5 and

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ETR6 are strongly reduced in R. bourboniana but up-regulated in R. hybrida several folds.

Signalling genes encoding CTR2, CTR3, EIN2, EIL2 and EIL3 are also reciprocally regulated being suppressed in R. hybrida. On the other hand, a few genes such as ACO3, ETR2, ETR3, CTR4, EIL1 and EIL4 show a regulation similar to that of R. bourboniana.

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However, even in these genes, the up-regulation seen within 4-8 h in R. bourboniana is only

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seen in R. hybrida much later and is of a lesser scale. Interestingly, the reciprocal regulation

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was not seen in genes of the ACS family but only in components downstream to ACS. As evident from these results, both ethylene-mediated activation and degradation of transcripts in

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R. bourboniana was dampened and delayed in R. hybrida. Previous studies have described the reduced ethylene sensitivity of different varieties of hybrid roses. In these, symptoms of

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petal fading and wilting are observed several days post-ethylene treatment even in the relatively sensitive varieties like Bronze [42]. In some respects, our data on expression

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patterns of ethylene pathway in R. hybrida petal abscission zones are different from the expression patterns reported previously [50, 51, 53] in that they show suppression instead of activation by ethylene. These might reflect differences in the tissue chosen for study namely,

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abscission zone in the present case compared to petals in the previous study [50, 51, 53] in addition to varietal differences. The reciprocal change in the transcriptional regulation of ethylene pathway genes between

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the two roses is rather interesting. One possible explanation for these changes could be differences in promoters of the ethylene pathway genes between R. bourboniana and R. hybrida. However, given the similarity in sequences between R. bourboniana, R. hybrida and R. chinensis, invoking a large scale change in promoter sequences of all genes appears a bit far-fetched, although it cannot be ruled out. A more likely explanation is the presence of a factor that regulates ethylene sensitivity and responses in a manner similar to tomato fruits. In

tomato fruits, the sensitivity to ethylene is stage-dependent and the basal ethylene levels generated by system I ethylene, maintained by ACS1a and ACS6, is auto-inhibitory and suppresses the ethylene pathway [30, 31, 58]. In contrast, system II ethylene generated by ACS2, ACS4, ACO1 and ACO4 is autocatalytic in nature and activated by ethylene [57, 58]. One could envisage the operation of such a system in rose, wherein there persists an autoinhibitory system I ethylene in R. hybrida and an auto-catalytic system II ethylene in R. bourboniana. This could explain the distinct gene regulatory patterns seen in these plants in

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response to ethylene. Another possibility that is not mutually exclusive is the specific ethylene-dependent degradation of transcripts of certain ethylene pathway genes by an

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ethylene responsive RNase in R. hybrida. In Arabidopsis, AtEIN5/XRN4 encodes an RNase

that specifically targets transcripts of AtEBF1 and AtEBF2 for degradation in response to ethylene but not in its absence [59]. It is likely that the activation of a similar RNase in R. hybrida could lead to reduction in steady state levels of many ethylene pathway genes. It

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should be noted that the rose cysteine protease gene, RbCP1, shows differential regulation in

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response to ethylene, being activated by ethylene in most tissues but showing an ethylene-

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induced decrease in transcript levels in thalamus [20]. The decrease was attributed to suppression of ethylene related processes to aid fruit development. Thus, there do exist within

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tissues means to either activate or suppress the expression of the same gene by ethylene. In this context, it is worth noting that there appears to be a tight association between increased

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ethylene sensitivity, fragrance and abscission as seen in R. bourboniana and its absence in R. hybrida. If the fragrance (or the lack of it) provides an evolutionary advantage to the flowers

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and is indeed also associated with ethylene sensitivity, then the sensitivity to abscission may only be a by-product of these sensitivities. Although ethylene is the prime regulator of abscission, especially in petals, one cannot exclude the role of other hormones like abscisic

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acid, auxin etc. Fine regulation as exerted by these at certain checkpoints like ACS and EIN2 in different developmental processes in Arabidopsis may also be important. Finally translational regulation of every gene and the post-translational changes of various

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components also govern these processes [60] and are also likely to regulate abscission. Interestingly, ethylene plays a dual role in rose. It is necessary for flower opening as well as for petal abscission [23, 51, 53]. The increase in ethylene sensitivity activates both these processes in R. bourboniana while reduced sensitivity to ethylene delays both these processes in R. hybrida. Compared to complete flowering opening within 12 hours of ethylene treatment in R. bourboniana, flowers of R. hybrida take at least 36 hours for opening with the

same treatment (data not shown). They remain largely unopened at 12 h unlike R. bourboniana flowers. The activation of ethylene pathway in both (R. bourboniana and R. hybrida) may lead to flower opening in both, albeit at different times, through cell wall associated changes by XTHs in the abscission zone that allow angular movement of petals from a vertical position (in a closed bud) to a horizontal position (when the flower is fully open) [23]. This ethylene may, as a consequence, activate flower abscission in R.

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bourboniana while suppressing abscission in R. hybrida. In conclusion, our studies show how differences between ethylene-induced and natural abscission in the fragrant Rosa bourboniana are associated with temporal and specific

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differences in expression of the individual components of the ethylene pathway. The studies

also show how differences in ethylene sensitivity and rapidity of petal abscission, that characterize the fragrant and non-fragrant rose varieties of Rosa bourboniana and Rosa

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hybrida respectively, are associated with differential and reciprocal regulation of the ethylene pathway genes. The studies shed new light on how ethylene signalling within the abscission

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zone may differentially affect the abscission outcome and thereby vase life in roses.

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Supplementary material Supplementary Tables:

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Table S1. Accession numbers of the ethylene pathway genes identified in the study

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Table S2: List of oligonucleotide primers specific for genes used in this study. Supplementary figure S1. Phylogenetic analysis of various members of the ACS, ACO, ETR,

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CTR, EIN2 and EIN3/EIL families of rose with those from Arabidopsis and tomato.

Acknowledgements

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We thank the Council of Scientific and Industrial Research (Govt of India) for Senior Research Fellowships to PS and APS and for financial support to APS (under BSC0107) for the work. We are grateful to Mr Ram Awadh for taking care of the rose plants.

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Figure legends

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Figure 1: Real-time PCR analysis of transcript accumulation of RbACSs in petal AZs of

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0.5 µl L-1 ethylene-treated flowers of R. bourboniana and R. hybrida and in flowers of R.

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bourboniana undergoing natural abscission (NAZ). Total RNA from petal AZs was isolated from control, ethylene-untreated flowers (0 h) as well as from petal AZs of flowers

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treated with 0.5 µl L-1 ethylene (4hE, 8hE… etc). Reactions were run in triplicate from three separate pools of AZ RNA samples (each pool containing at least 50 flowers). Rose beta-

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ACTIN and PP2A was used as an internal control for normalization. Error bars represent ± SE of three biological replicates. Expression values were analyzed by one-way ANOVA and

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compared using Duncan’s Multiple Range Test (DMRT). Values on the bar carrying different letters are significantly different.

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Figure 2: Real-time PCR analysis of transcript accumulation of RbACOs in petal AZs of 0.5 µl L-1 ethylene-treated flowers of R. bourboniana and R. hybrida and in flowers of R. bourboniana undergoing natural abscission (NAZ). Analysis was carried out as described

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in Fig 1.

Figure 3: Real-time PCR analysis of transcript accumulation of RbETRs in petal AZs of 0.5 µl L-1 ethylene-treated flowers of R. bourboniana and R. hybrida and in flowers of R. bourboniana undergoing natural abscission (NAZ). Analysis was carried out as described in (Fig. 1).

Figure 4: Real-time PCR analysis of transcript accumulation of RbCTRs in petal AZs of 0.5 µl L-1 ethylene-treated flowers of R. bourboniana and R. hybrida and in flowers of R. bourboniana undergoing natural abscission (NAZ). Analysis was carried out as described in (Fig. 1). Figure 5: Real-time PCR analysis of transcript accumulation of RbEIN2 and RbEILs in petal AZs of 0.5 µl L-1 ethylene-treated flowers of R. bourboniana and R. hybrida and in

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flowers of R. bourboniana undergoing natural abscission (NAZ). Analysis was carried out as described in (Fig. 1).

Figure 6: A hypothetical model for the sequence of events that accelerate abscission in

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R. bourboniana and suppress it in R. hybrida.

Abscission cues activate ethylene biosynthesis genes (ACS/ACOs) that are responsive to abscission cues in R. bourboniana, leading to biosynthesis of ethylene. This then activates the

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ethylene responsive members of biosynthesis to a greater increase the ethylene levels (shown in larger letters). This ethylene in turn, simultaneously activates downstream ethylene

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responsive components of signalling (EIN2/EILs) and the negative regulators (ETRs/CTRs

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which suppress and control the ethylene signal). The amplified ethylene signal activates

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abscission within a few hours. In R. hybrida, most genes are not responsive to ethylene and are actually suppressed by it. Hence the amount of ethylene produced is low (as shown by the

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reduced letter size). Moreover, this reduced ethylene primarily activates many suppressors of ethylene pathway and suppresses activators of ethylene signal leading to a reduced signal that

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does not activate abscission but instead leads to delayed senescence after several days.

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Time points (h)

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Figure 1

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Time points (h)

Figure 3

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Time points (h)

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Figure 4

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Abscission cues

R. hybrida

ACS1, 5, 6 ACO1, 2, 3, 4, 5 EIL4

ACS1, ACS6, ACO6

Ethylene

Ethylene

ACS1, 5 ACO1, 2, 4, 5, 6 ETR3, 5, 6, CTR4

ETR1, 7, CTR1, 2, 3 EIN2, EIL2, 3

Ethylene ETR1, 2, 7, 8, CTR1, 2, 3

EIL1, 4

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EIN2, EIL2, 3, 4

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Figure 6

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Delayed senescence (in days)

Petal abscission (in hours)

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R. bourboniana

Rosa hybrida

Gene (accession no.)

Gene (NCBI accession no.)

RbACS1 (MH276988) RbACS2 (MH276989) RbACS3 (AY525067.1) RbACS4 (AY525068.1) RbACS5 (MH276990) RbACS6 (MH276991) RbACO1 (MH276992) RbACO2 (MH276993) RbACO3 (MH276994) RbACO4 (MH276995) RbACO5 (MH276996) RbACO6 (MH276997) RbETR1 (AF394914.1) RbETR2 (MH277012) RbETR3 (MH277010) RbETR4 (AF159172.1) RbETR5 (MH277013) RbETR6 (MH277014) RbETR7 (MH277011) RbETR8 (MH277009) RbCTR1 (MH277000) RbCTR2 (MH277001) RbCTR3 (MH277002) RbCTR4 (MH277003) RbEIL1 (MH277006) RbEIL2 (MH277007) RbEIL3 (MH277008) RbEIL4 (MH277005) RbEIN2 (MH277004) RbACT (MH277016) RbPP2A (MH277015)

RhACS1 (AY378152.1) RhACS2 (AY803737.1) RhACS3 (AY525067.1) RhACS4 (AY525068.1) RhACS5 (AY061946.1)

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RhACO1 (AF441282.1)

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RhETR1 (AF394914.1) RhETR2 (AF127220.1) RhETR3 (AY953392.1) RhETR4 (AF159172.1) RhETR5 (AF441283.1)

RhCTR1 (AY032953.1) RhCTR2 (AY029067.1)

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30* 31*

Rosa bourboniana

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Serial no.

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Supplementary table S1: Accession numbers of the ethylene pathway genes identified in the study

RhEIN3-1 (AF443783.1) RhEIN3-2 (AY919867.1) RhEIN3-3 (KC484653.1)

RhACT (KF985187.1) RhPP2A (JN399224)

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* genes used for normalization

Supplementary Table S2: List of oligonucleotide primers specific for genes used in this

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study Gene

Forward primers(5’-3’)

Reverse primers(5’3’)

RbACS1

GAATGTAGGTAAGCTCAATG TTACT GTCACCTCTTGTTCGAGCCA CTTAG AAGCATTCAGAAGATTCCAA CCG

TGAACGAACATTTCAACTGTG

RbACS2 RbACS3

GACTAACTTCTTAGGTGGAAT ACCT TGCGGTTAGGACTATCCTCTG

Amplicon length (bp) 169 190 168

RbETR2 RbETR3 RbETR4 RbETR5 RbETR6 RbETR7 RbETR8 RbCTR1 RbCTR2 RbCTR3 RbCTR4 RbEIL1

GTACATTCAGACTTTCCTG ATCTCATTGGATACAAACCA GC GATGTCGGATGAGCTTCGG CCCACACCTCAACCTAGTCA TGC CGCGTCCTCCTAACCTCTCT ATC TAGACGAGGTCATCACTAAT C GAACGAGAGGTACTTCTGC CCAG CAGAGTGAGCAAGACAGAA CT CTCGTAACAATGTGCCTGAA CC CCTGTGAGTTCGTTCAGTGT C TGGTTCTAAAGTCTTGGTAG TAG CTCCAACATCTGCTCCTT

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RbEIL2 RbEIL3 RbEIL4

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RbEIN2

RbACT (beta actin) RbPP2A

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CTTGGATTTGCCAATACC GCTGCGGATGGGAGGAAATC GTGCTGTAAACACCTGAAGG G GCAAATGATAAAATCATATAA ACCGCA GTTGAGCTTACGGTTGTTACA

188 114 171

ATGACATGGAGAAGATCTG GCATCA TGTCACTGCGTCAAAGGACA G

193 196

IP T

ATCTCATTGGATACAAACCA GC TCTCGGCAAGGGATGCACT GC TTGGGATCAATGGAGTTATC CG CACACCCTAACCCAGGTATC G TTGCCGTGCAGAAATTACG

GTTCATGGCCTCCCTGTAATC TTGG GTCCTTCGAGGTTGGCAAGC TTCCTTCCTGTACAAGTCCT

190

200

SC R

RbETR1

183

222

TGCTTTTGATGTTCGGAGAAG GA GCCCAACAAGAGGTCTAAAC CTA TACTTCATCAACAATTCATCA GCC GCACGTCCAATTCAACTGGC GCATAGATGAACTCAGGGCA TCAACT GAAGCACATGGTTAGCAAAA TC CTGAAGTGGAAGTTTAGGCA TA CAGTCCAAAATCACCTACCTT CACA GACTATCAAATCTGAACAAGG AAAG GCTTTATCAGGGAGACGCTG CC GCTTCAAATCTTACAACTTAT GC AGATCCAAATCTCAAGTCAGG A ATCAAAGGAAGGTGGCACTG G AACAAGCGATAGAGACAGAG A AATTGACTAAGCACTTTGATC C AGCCTGGATGGCAACATACA TAGC

188

GACGAATTGTCTTCTCCACCA

110

U

RbACO4 RbACO5 RbACO6

CAGCATCAGTACCCAAAGTAA T CGGTTGTTTGAAGCTAGTTG

N

RbACO2 RbACO3

165

A

RbACO1

AGCAGTGAGGACTATCCTACT

M

RbACS6

ED

RbACS5

CAACATCCAGAAGCTTCCAA CTT CACCAATGCCTCACTCGCCT CTGC GGTCCTCTTGCCACTGCACT G CTCTGAAATCAAAGATTTGG ACTGGG TCTGATCCGGATTTTCTC AGTGGAGAAGATGACGAAA G AGTGACCCTGGAGTGTTG CAAGAACGCTACTCTGTGG TCCTCGATGAGACGATCCG

PT

RbACS4

136 194 163 186 779 165 239 149 188 191 149 224 116 170 176

Supplementary figure S1

0

3 1

98 7 25

12

40

0 0

98

39

AtACS3 SlACSu2 SlACS4 AtACS6

53

34 3 81

97 26

95

60

SlACO4 RbACO3

38

AtAACO1 SlACO5 AtACO5

98

AtACO2 AtACO3

5 95

8

RbACS5 SlACS2 RbACS2 AtACS2

25

55

42

92

SlACS5 SlACS7 SlACC AtACS7 AtACS11

D

SlACO6 RbACO1 AtACO4

46

C

44

RbETR7 RbETR5

1 9

ED

0

AtETR1 SlETR3 SlETR1

4 10

PT

82

96

25

CC E

94

61

RbETR2 AtERS2 RbETR8 SlETR2 SlETR5 AtETR2 AtEIN4 SlETR6 SlETR4 RbETR3

SlTCTR2 RbCTR3 RbCTR2 AtCTR1 SlCTR3

RbCTR1

AtEIN2 RbEIN2 SlEIN2

M

1

95

AtERS1

RbACO6 RbACO2 RbACO4

SlCTR1 SlCTR4

63

E

93

SlACO1 SlACO3 SlACO2

94

SlACS3 RbACS1 AtACS12 SlACS1A SlACSu1 SlACS10 AtACS10 SlACS12 AtACS1

71

72

68

IP T

0 5

63

SC R

0

AtACS9 AtACS8 SlACS8 AtACS4 SlACS6

U

0

B

AtACS5

N

0

A

98

A

F 41

95

97

28 64 20 39

99

AtEIL1 SlEIL1 SlEIL2 AtEIL2 AtEIN3 AtEIL3 RbEIL1 SlEIL4 SlEIL3 RbEIL2 RbEIL3

A

Supplementary Figure S1: Phylogenetic analysis of the full-length RbACSs (A), RbACOs, RbCTRs, RbETRs, RbEINs amino acid sequences with Arabidopsis and tomato family members. Analysis was carried out using the MEGA5 software and the UPGMA dendrogram was constructed by the bootstrap method. The amino acid sequences of Arabidopsis and tomato were obtained from the NCBI (National Centre for Biotechnology Information) database, and accession numbers are as follows: AtACS1 (NP_191710.1), AtACS2 (NP_171655.1), AtACS3 (AT5G28360.1), AtACS4 (NP_179866.1), AtACS5 (NP_201381.1), AtACS6 (NP_192867.1), AtACS7 (NP_194350.1), AtACS8 (NP_195491.1), AtACS9 (NP_190539.1), AtACS10 (NP_564804.1), AtACS11 (NP_567330.1), AtACS12 (NP_199982.2), AtACO1 (NP_179549.1), AtACO2 (NP_176428.1), AtACO3 (NP_172665.1), AtACO4 (NP_171994.1), AtACO5 (NP_565154.1), AtETR1 (AAA70047), AtERS1 (NP_181626), AtETR2 (NP_188956), AtERS2 (AAC62209), AtEIN4 (AAD02485),

A

CC E

PT

ED

M

A

N

U

SC R

IP T

AtCTR1 (AAA32780), AtEIN2 (AAD41077.1), AtEIN3 (NP_188713), AtEIL1 (NP_180273), AtEIL2 (NP_197611) and AtEIL3 (NP_177514) in Arabidopsis; SlACS1A (NP_001266271.2), SlACS2 (NP_001234178.3), SlACS3 (NP_001234026.2), SlACS4 (NP_001233875.1), SlACS5 (NP_001234156.2), SlACS6 (AAK72433.1), SlACS7(NP_001234346.1), SlACS8 (NP_001234160.2), SlACS10 (XP_004245687.1), SlACS12 (XP_004234155.1), SlACC (XP_010313742.1), SlACSu1(XP_004252649.1), SlACSu2 (XP_004242974.1), SlACO1 (NP_001234024.2), SlACO2 (NP_001316842.1), SlACO3 (NP_001233928.1), SlACO4 (NP_001233867.2), SlACO5 (NP_001234037.1), SlACO6 (NP_001234638.1), SlETR1 (AAC02213), SlETR2 (AAC02214), SlETR3 (AAC49124), SlETR4 (AAU34076), SlETR5 (AAD31397), SlETR6 (AAL86614), SlCTR1 (AAL87456), SlTCTR2 (CAA06334), SlCTR3 (AAR89820), SlCTR4 (AAR89822), SlEIN2(NP_001234518.1),SlEIL1 (AAK58857), SlEIL2 (AAK58858), SlEIL3 (AF328786)and SlEIL4 (AB108840) in tomato.