The NOP-1 peptide derived from the central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers

The plant hormone ethylene was identified as important triggering factor and primary regulator of flower senescence in many species. Consequently, application of chemical inhibitors of ethylene biosynthesis and action is used to extend the longevity of ethylene-sensitive flowers. Here, we show that the peptide NOP-1, a biological derived from the nuclear localization signal of ethylene regulator EIN2 tightly binds to the ethylene receptor of carnation plants - a model to study flower senescence. When applied on cut flowers the peptide biological delays petal senescence similar to previously identified and currently used chemical inhibitors, but offers significant advances to these chemicals in biodegradability, sustainability and ecotoxicity. Our bioinformatic analysis of a wide range of ethylene receptors indicates complete sequence conservation of the anticipated NOP-1 binding site in flower species supporting a widespread use of the peptide on flowering ornamentals to delay senescence and decay in cut flowers. We anticipate our innovative approach to extend flower longevity by a new class of biomolecules such as peptides, peptide analogues and peptide mimetics will significantly advance our technological capability to delay flower senescence and expand vase-life of cut flowers in a sustainable and environmentally friendly manner.

Flower senescence is a tightly regulated developmental process that plays a crucial role in the overall reproductive strategy of many plants. Initial work on this developmental process was motivated primarily by the commercial interest in increasing the life time of cut flowers which according to import statistics reported to United Nations (UN) show an overall world trade value of around 4 billion USD. Europe (66.7%), the US (19.3%) and Japan (10.7%) form the three most important floriculture consumption regions in this market. Globally, both, traders and customers, demand for cut flowers with a long vase-life no matter whether those have already experienced long-distance transport from their cultivation areas in Latin America or Eastern Africa. Accordingly, chemicals or processes that decelerate or delay flower senescence are of broad social and economic interest.
Identification of the plant hormone ethylene as primary regulator of carnation senescence 1 and the drastic extension of life of petals after treatment of cut flowers by inhibitors of ethylene biosynthesis 2 or ethylene action inhibitors resulted in a range of commercial treatments to extend the vase life of cut flowers 3 . Small molecule inhibitors amino-ethoxyvinyl glycine (AVG) and amino-oxyacetic acid (AOA) which interfere with ethylene biosynthesis have been shown effective in blocking ethylene production that accompanies senescence. Consequently, a number of commercial products based on these chemicals have been introduced to the market to delay floral senescence and abscission [4][5][6][7][8] . However, these inhibitors are ineffective in preventing the effects of exogenous ethylene on flower senescence during transport and storage. Hence, more commercial interest was received for inhibitors blocking ethylene perception such as silver ions 3 and the cyclopropene derivative 1-MCP 9 . Silver salts which were shown to decrease the number of active ethylene binding sites and alter signal output of the receptors 10 have been an industry standard for preventing ethylene action in ornamentals for decades. But nowadays the use of the heavy metal pollutant is banned in many countries due to serious concerns about its potential as a groundwater pollutant. In recent years, the cyclic olefine 1-MCP has been widely adopted in the ornamental plant industry as a non-toxic alternative to silver salts, although it does not control senescence for as long as silver ions when applied in a single treatment. However, repetitive treatments with the gaseous inhibitor resulted in a marked increase in vase life and efficiently blocked floral senescence of cut flower 11 . Hence, 1-MCP today is widely used as ethylene action inhibitor at a wide range of ornamental plants 12 which due to the gaseous character of the inhibitor are treated in enclosed, gas-tight areas. The inhibitor was shown to be highly efficient at very small concentrations 12 , although treatment depends on temperature achieving complete inhibition at 20 °C, but almost no inhibition at 0 °C 13 . In addition to chemical treatments, transgenic approaches targeting ethylene biosynthesis or ethylene signaling were applied to extend flower vase life 14,15 . Overall, each method comes with its own chances and drawbacks 16 . Hence, a sustainable and easy-to-use method to improve longevity of cut flower has not been found yet. The different strategies developed in the past to delay floral senescence have been used on a wide range of ornamental plants. However, among ethylene-sensitive flowers carnations are probably the most studied model system 13,17,18 as they are highly sensitive and rapidly respond to the plant hormone by clear physiological and morphological changes which can be studied even on individual petals 19 .
Much of the current knowledge on ethylene perception and transduction has been established by physiological, biochemical and genetic studies in the small crucifer weed Arabidopsis, and crops such as tomato or rice. These studies clarified that ethylene is perceived by a family of receptor proteins (ETRs), which form homo-and heterodimers at the ER membrane [20][21][22][23] . Although the exact output of the receptors is still obscure, genetic and biochemical studies established that in the absence of ethylene, a receptor associated kinase (CTR1) phosphorylates a central regulator of the signaling cascade (EIN2). Conversely, in the presence of ethylene the CTR1 kinase is inactivated leading to dephosphorylation of EIN2 24 . In turn, the EIN2 C-terminal domain (aa 462-1294) containing a highly conserved nuclear localization signal 25,26 , is processed by a so far unknown protease and translocated to the nucleus 24,26,27 where it activates a variety of ethylene response genes and phenotypes. Recent studies in our lab propose an efficient way to interfere with ethylene signaling based on a yet unknown function of the nuclear localization signal (NLS) in EIN2. Peptides mimicking this NLS motif were shown to affect ethylene responses in Arabidopsis and also function as potent inhibitor of tomato fruit ripening 28,29 .

Results and Discussion
To clarify whether NOP-1 also has the potential to serve as an inhibitor of senescence in cut flowers, we applied the peptide on cut carnations (Dianthus caryophyllus) and analyzed the effect on floral senescence in this model ethylene response system 9,13,17,30 . In analogy to treatment with the chemical senescence inhibitor STS containing silver ions as active ingredient which target plant ethylene receptors 31 the peptide was directly supplied to the vase water. Senescence was monitored visually throughout the experiment and quantified by changes observed in petal size and/or color (Fig. 1A,B). While controls with no additions to vase water show a clear senescence phenotype of petal inrolling and petal fading after 6 days (Fig. 1A, upper panel), the addition of 1 mM silver salt delays the appearance of these morphological manifestations by several days and petals show almost no signs of senescence after 15 days (Fig. 1A, middle panel). Similarly, addition of NOP-1 peptide at 0.5 mM concentration clearly delays the onset of petal senescence and significantly increases flower longevity, although the delay is shorter than observed with the potent silver salt inhibitor (Fig. 1A, lower panel). To further substantiate the effect of the peptide biological on floral senescence observed with the carnation model system, we used roses as another species of highly ethylene sensitive ornamental plants. Similar to carnations untreated roses showed obvious signs of senescence such as wilting and petal bending by day 6 ( Fig. 2A, upper panel). The untreated controls completely ended their vase life on day 9 due to these morphological changes. In contrast, cut roses treated with the silver salt inhibitor ( Fig. 2A, middle panel) and flowers treated with the NOP-1 peptide ( Fig. 2A, lower panel) showed a prolonged vase life of about 6-8 days relative to buffer controls. While the silver salt chemical inhibitor was noticeably more efficient in extending the vase life of cut carnations than the peptide biological ( Fig. 1B), the difference on flower longevity between both treatments is less pronounced for cut roses (Fig. 2B). Nevertheless, on the entire trial period, NOP-1 was less effective than the silver treatment similar to what is seen for carnation. Noteworthy, no beneficial effect on flower longevity was observed by multiple treatments with the NOP-1 peptide in contrast to studies with the gaseous ethylene antagonist 1-MCP which significantly delayed flower senescence when applied on a daily basis 11 . Hence, further testing remains to be done to uncover the full potential of the peptide biological in order to identify optimum treatment conditions (concentration, duration, temperature) and strategies for efficient application.
To demonstrate uptake of the peptide from the vase water and vascular transport to petals fluorescently labeled NOP-1 was used. To this end, dansylated NOP-1 was added directly to the vase water of cut carnation flowers at 1 mM concentration. Confocal microscopy and spectroscopic analysis of mock and NOP-1 treated flowers clearly demonstrate significant accumulation of the peptide in petal cells (Fig. 3). Consequently, this also verifies efficient transport of the NOP-1 peptide across the vascular system. No fluorescence was detected in mock controls. Moreover, we noted that there is no indication of cytotoxic effects on carnation cells by NOP-1 or dansylated NOP-1.
To elucidate the mode of action of NOP-1 on flower senescence and to determine whether the peptide biological works by the same molecular mechanism as determined for NOP-1 controlled ripening delay, we initiated interaction studies of the carnation ethylene receptor DcETR1, the central regulator of the ethylene signaling cascade EIN2 and the NOP-1 peptide. To this end, DcETR1 was cloned in E. coli, expressed and purified according to protocols established in our lab 32 for ethylene receptors from Arabidopsis thaliana, Physcomitrella patens, tomato and apple ( Supplementary Fig. S1). Functional folding of the recombinant purified proteins was demonstrated by CD spectroscopy (Supplementary Fig. S2) and purified DcETR1 was labeled with fluorescent dye AlexaFluor488. To determine binding affinity and dissociation constants of the purified recombinant proteins and the NOP-1 peptide we used microscale thermophoresis. In these experiments we observed tight binding of the DcETR1 receptor to the EIN2 ethylene central regulator (Fig. 4A the interaction of the corresponding proteins from Arabidopsis and tomato 29,33 . These data indicate a high affinity and central protein protein interaction (PPI) common in ethylene signaling among plant species. Consequently, targeting this interaction by small-molecule modulators has the potential to shut down ethylene signaling and related plant responses. Therefore, we tested binding of the ripening delaying peptide NOP-1 identified in previous studies to target the ETR1-EIN2 complex with our purified carnation receptor DcETR1. Our data on the carnation protein confirm binding of the peptide biological at similar affinities (Fig. 4B, K D = 3.49 µM ± 0.55 µM) as earlier observed for Arabidopsis and tomato receptors 29,34 . The precise interaction site of NOP-1 at the Arabidopsis ETR1 receptor was recently resolved in our lab by molecular and computational studies 34 . In these studies, we uncovered binding of the peptide biological at the GAF domain of the receptor. Based on the resolved binding site and binding mode we proposed that the bound peptide arrests intra-and intermolecular downstream signaling in the receptor resulting in the observed ripening inhibition. To verify that the NOP-1 binding site identified in the Arabidopspis ETR1 receptor is also present in ethylene receptors of cut flowers and ornamental plants, we compared the corresponding sequence (aa 143-174 in Arabidopsis) in 17 flowering ornamentals widely used as cut flowers. Remarkably, all tested varieties show an identical protein sequence (Fig. 5). The extreme conservation found in this part of the receptor (see also Supplementary Fig. S3) further emphasizes a potential critical role of this element in downstream signal transfer. Moreover, the exact match of this patch in the receptors of different flower species supports a general usage of NOP-1 as a senescence inhibitor in a variety of cut flowers without the need to genetically engineer ornamental plants in ethylene biosynthesis or perception. Compared to current approaches to expand the vase-life of cut flowers, NOP-1 offers a number of advantages, such as application as aqueous solution, ultimate biodegradability, sustainability and low ecotoxicity due to the lack of adverse effects to the environment. In contrast, silver salts although they offer long lasting senescence protection have various drawbacks. As a heavy metal they are highly toxic and harmful to the environment, persist in soil and groundwater for long periods and pollute drinking water 35 . 1-MCP, another senescence inhibitor used to extend longevity of cut flowers, lacks most of these disadvantages, is easy to dispose and safe, but as a gas is active by fumigation only complicating its usage on cut flowers. Hence, the peptide biological NOP-1 combines the strengths of current senescence inhibitors and thereby offers an alternative and sustainable approach to prolong the vase-life of cut flowers. To further improve the effect on the vase-life of cut flowers, additional studies on more flower species and variants of the NOP-1 biological (peptide length, stability, modifications) are needed. Nevertheless, our present work highlights a novel way to address flower longevity and presents future opportunities to control senescence delay in cut flowers.    rose (Rosa hybrida), graded for marketable quality, were obtained from a local flower producer. Upon arrival in the laboratory, the cut flower stems were re-cut to a length of approximately 23 cm. Flowers were treated with NOP-1 by adding the peptide dissolved in 50 mM Tris/Acetic acid pH 8 at a final concentration of 0.5 mM directly to the vase reservoir. Cut carnations placed in 50 mM Tris/Acetic acid pH 8 were used as mock control. Flowers with 1 mM of senescence inhibitor AgNO 3 added to the buffer served as positive control. Six flowers were used for each treatment to evaluate vase life throughout the experiment. Progression of senescence was recorded photographically for a period of 15 days. Images were acquired using fixed camera setting and set-up to ensure high data quality and reliability. Changes in petal size and color were quantified using ImageJ (32 bit v1.51t.). Relative discoloration of petals was quantified by drawing multiple lines across each flower (petals only) and calculating the average values for red (R), green (G) and blue (B). Data shown represent the values for blue as these showed the most significant difference between fresh and senescent flowers. Relative size of flowers was quantified using a macro that specifically recognizes the flower (petals + sepals) but not the flower stem with flower size in pixel² as the output. All Data are normalized to the maximum.

Uptake experiments with fluorescently labelled NOP-1. Carnations were watered with 1 mM
Dansylamide-labeled NOP-1 or tap water only, respectively. Dansylamide fluorescence was imaged after 3 days using a Zeiss LSM780 confocal microscope (Dansylamide: λ ex 405 nm, λ em 500-550 nm; auto fluorescence: λ ex 488 nm, λ em 510-650 nm). To estimate NOP-1 uptake 500 mg petals of treated and untreated carnations (triplicates) were homogenized in 500 µl water 3 days after treatment. Samples were centrifuged at 14.100 × g for 30 min. The resulting clear cell lysate was diluted 1:100 and absorbance (250 nm -400 nm) was recorded using a DU800 spectrophotometer (Beckman Coulter, Krefeld). Absorbance at 330 nm (λ ex (max) of dansylamide) was divided by the absorbance at 280 nm (proteins). Based on these data the difference between treated and untreated carnation flowers was calculated. Data shown are normalized to the maximum.

Cloning of Dianthus caryophyllus ethylene receptor ETR1 into expression vector pET16b.
According to the published sequence (

DcETR1 binding studies by Microscale Thermophoresis. Recombinant proteins DcETR1 and
AtEIN2 479-1294 were expressed, purified and labeled as described in the Supplementary Information ( Figure S1) and in 29 . For binding studies DcETR1 and NOP-1 were dissolved in 100 mM potassium phosphate buffer, 300 mM NaCl and 0.015% (w/v) FosCholine-16. DcETR1 labelled with Alexa488 was used at a concentration of 50 nM.

Data Availability
The data that support the findings of this study are available from the corresponding author upon request.