Action of ethylene , 1-methylcyclopropene and silver thiosulfate in two developmental stages of potted ornamental pepper

The species of Capsicum genus have great genetic variability with enormous potential for marketing as ornamental potted plants. The exposure at ethylene induces various deleterious responses during plants life cycle, but there are few studies on how the ethylene affects early developmental stages of these ornamental plants. Thus, this study aimed to evaluate the effects of the application of ethylene and ethylene inhibitors, 1-methylcyclopropene (1-MCP) and silver thiosulphate (STS) in two early development stages (seedling or initial flowering) of potted ornamental peppers of the cultivars ‘Calypso’ and ‘MG 302’. Cultivar ‘MG 302’ showed intermediate sensitivity to ethylene, while the cultivar ‘Calypso’ showed complete abscission of leaves when exposed to 10 μl of ethylene for 48 hours in both development stages. Plants treated with 1-MCP showed similar durability to control for both varieties and developmental stages, and the treatment with 1-MCP + ethylene was beneficial only for ‘MG 302’. STS was the most efficient substance in delaying the ethylene, wherein at stage 1 there was no leaf abscission and at stage 2 had much reduced leaf abscission occurred, regardless the cultivar. Treatment with STS + ethylene was efficient only at stage 1 for ‘Calypso’ and at stage 2 for ‘MG 302’. According to the data, cultivars showed different levels of sensitivity to ethylene, ‘Calypso’ is very sensitive and ‘MG 302’ showed intermediate sensitivity. Regardless the cultivar, STS treatment significantly blocked the ethylene action in both development stages and the use of 1-MCP was less efficient.


INTRODUCTION
Capsicum annuum species is considered the most cultivated and greater variability within the genus Capsicum (CARVALHO and BIANCHETTI, 2008).When evaluating the ethylene sensitivity within plant families, not all families have been found to have the same level of sensitivity between species or even among cultivars (MACNISH et al., 2011), because multiple receptors might be involved in contrasting responses in different tissues, as well as different developmental stages or different environmental stresses (IQBAL et al., 2017).Ethylene is a developmental regulator that operates in manifold physiological processes in all tissues throughout the plant life cycle ( VAN DE POEL et al., 2015), regulates growth and senescence, depending on its concentration, timing of application and plant species (KHAN et al., 2008).
The use of ethylene inhibitors such as 1-methylcyclopropene (1-MCP) and silver thiosulfate (STS) help to control of ethylene action on ornamentals plants (SEGATTO et al., 2013).1-MCP, is the most useful compound for this purpose among cyclopropenes, is nontoxic, stable at room temperature, active at relatively low concentrations providing protection for a long time (FINGER and BARBOSA, 2006).In addition, ion silver is a potent inhibitor of ethylene action by binding to ethylene receptors (KUMAR et al., 2009).Silver has better effect when applied as silver thiosulfate (STS) -[Ag(S 2 O 3 ) 2 -3 ], because has more mobile and less phytotoxic than silver nitrate (AgNO 3 ) ( VAN ALTVORST and BOVY, 1995).The use of inhibitors treatments affect at different levels the ethylene signaling pathway, reveals collectively an intricate network of interactions as exemplified by numerous studies of senescence in flowers reviewed in Ferrante et al. (2015).
During plant life, the transition from vegetative to reproductive stages and senescence is largely influenced by ethylene and its interplay with other plant hormones.These studies should be extended to different organs and development stages to deeply understand the intricate network affecting relevant agronomic traits such as yield, longevity and morphological appearance (IQBAL et al., 2017).
Currently there are underway studies about postproduction of ornamental potted peppers (LIMA et al., 2017;FINGER et al., 2015;SEGATTO et al., 2013), but little is known how ethylene affects early developmental stages, in which the plants can also be marketed.Besides, the production of seedlings can be considered as one of the most important stages in the cultivation of vegetables (SIPIONI et al., 2016) and can affect post-production.In general, exposure to ethylene during greenhouse production, shipping, and retailing can reduce the shelf life and garden performance of ornamental plants (JONES and LING, 2012).
This study aimed to evaluate the effects of the application of ethylene and ethylene inhibitors, 1-methylcyclopropene (1-MCP) and silver thiosulphate (STS) in two developmental stages of potted ornamental peppers of the cultivars 'Calypso' and 'MG 302'.

MATERIAL AND METHODS
Seeds from peppers of the cultivars Calypso and MG 302 (Capsicum annuum) were sown in flats with 36 single cells and transplanted to 760 mL plastic pots when the plantlets had four to five true leaves.Flats and pots containing one plantlet each were filled with commercial substrate containing pine bark, vermiculite, superphosphate, potassium nitrate and were fertilized at every week with 0.1 g (flats) and 10 g (pots) of NPK 10-10-10.Plants were grown in a greenhouse for about 35 days (seedling -stage 1) or about 50 days (initial flowering -stage 2).
Each variety was analyzed at two early developmental stages: seedlings and initial flowering.After the grown period in greenhouse, the pots were transferred to a room at 25 ± 1 o C, illuminated for 12 hours period with white fluorescent light (OSRAM, L40W/10S, USA) with intensity of 8-10 μmol s -1 m -2 during the experimental period (SEGATTO et al., 2013).
Pots were placed in a 90 L sealed chambers and were treated with 10 μL L -1 ethylene for 48 hours; fumigation with 1 μL L -1 1-MCP for 6 hours and fumigation with 1 μL L -1 1-MCP for 6 hours followed by 10 μL L -1 ethylene for 48 hours according to the methodology described by Segatto et al. (2013).Plants were sprayed until runoff with 5 mL of 2 mmol L -1 STS and sprayed with 2 mmol L -1 STS followed by treatment with 10 μL L -1 ethylene for 48 hours according to the methodology described by Lima et al. (2017).The control plants were kept on the laboratory bench.Plants of all treatments were kept at 25 ± 1 °C under white fluorescent light with intensity of 8-10 μmol s -1 m -2 , 60-65% relative humidity and watered when needed (FINGER et al., 2015).
Leaf abscission was determined at every three days by counting the total leaves during the whole period of shelf life, expressed in percentage compared to the initial leaf counting.The end of longevity occurred when there is 50% of leaf abscission and/or 50% yellowing of leaves (FINGER et al., 2015).
Degree of green color and flavonoids of the leaves was evaluated in three leaves in each plant: base, center and top, the average of determinations were considered.The chlorophyll content of leaves was determined by the chlorophyll index -Chl, the flavonoids index -Flav and nitrogen balance index -NBI were also determined, giving the reason CHL/FLAV, nitrogen deficiency and protein rate marker.All these variables were evaluated at every three days with the help of a leaf-clip portable meter Dualex® 4, design to measure chlorophyll, epidermal flavonoids and nitrogen balance index (FORCE-A, Université Paris Sud, 91893 ORSAY CEDEX, France).This instrument is used for non-destructive assessment of plant status, especially in regards to the simultaneous assessment of Chl and Flav on the same leaf spot, and was equipped with 6 mm diameter sensor in the measuring area and silicon photodiode detector (CEROVIC et al., 2012).
Pots were arranged in complete randomized block design with five replicates of one pot each.The average of the parameters analyzed were submitted to the standard V. 23, N o .3, 2017, p. 240-249 error of the average (n=5) and SigmaPlot software was used for data analysis and graph design.

RESULTS AND DISCUSSION
Regardless the stage of development, treatment with 10 µL L -1 ethylene for 48 hours induced total abscission of leaves for 'Calypso' (Figures 1A, 1C Wonder 300', and 'Habanero Big Sun' and concluded that seedling ethylene sensitivity is lower than in the mature plant (EDELMAN and JONES, 2014).
Ethylene can trigger the senescence process, especially in the sensitive species, in any part of the plant and at any stage of leaf development.The exposure to ethylene induces premature senescence symptoms such as leaf yellowing, abscission, or desiccation/necrosis. Leaf senescence is activated at mature stage of leaf development when leaves are fully expanded, in the process three different stages can be identified: initiation, organization of degradation, and cell death processes (IQBAL et al., 2017).
1-MCP treatment was more efficient to the ethylene control, but showed high rate of accumulated leaf abscission, about 43% and 69% at sixth evaluation day (Figures 1A, 1C, 2C and 2D) and about 39% and 57% at nine evaluation day (Figures 1B, 1D, 3C and 3D) in stages 1 and 2 for 'Calypso' and 'MG 302', respectively.Treatment with 1-MCP + Ethylene was not efficient on 'Calypso', because showed complete leaf abscission after treatment on stage 1, and in the stage 2 showed 96% leaf abscission at 6 rd evaluation day (Figures 1A, 1C, 2E and 2F); but was efficient on 'MG 302', having 54% and 70% accumulated leaf abscission at nine evaluation day for development stages 1 and 2, respectively (Figures 1B, 1D, 3E and 3F).Similarly to this research, Lima et al. (2017) concluded that pretreatment with 1-MCP (with or without ethylene) was more beneficial in 'MG 302' at marketing stage.
If applied in mature plants, Finger et al. (2015) concluded that 1-MCP treatment was effective in partially preventing the sudden fall of leaves in presence of exogenous ethylene in post-production of 'Calypso'.According to Phebe and Ong (2010), the effectiveness of 1-MCP varies with species and variety, development stages, and exposure temperature, concentration and duration.Moreover, 1-MCP may have contrary effects, either reducing or enhancing physiological disorders (ESTABLÉS-ORTIZ et al., 2016).Different reports suggest that the production of new ethylene receptors may be responsible for the loss of efficacy of 1-MCP in blocking the perception of ethylene (PRANGE et al., 2005).Furthermore, 1-MCP may be metabolized in plants (HUBER et al., 2010) or ethylene and 1-MCP may bind with different affinities to different ethylene receptors (COOLS et al., 2011).
Plants treated with STS showed improved durability when compared with other treatments, at stage 1 there was no leaf abscission up to the thirty-six evaluation day in both varieties (Figures 1A,1B,2G and 3G), while in stage 2 there were 44% and 17% accumulated foliar abscission at twenty-fourth evaluation day in 'Calypso ' and 'MG 302',respectively (Figures 1C,1D,2H and 3H).At stage 1, treatment with STS + Ethylene was efficient in 'Calypso', because at the thirty-six evaluation day there was no leaf abscission, but induced phytotoxicity in 'MG 302' with expressive leaf wilt on third day (Figures 1A,1B,2I and 3I), already at stage 2, the treatment was efficient in 'MG 302' showing 29% leaf abscission at twenty-fourth day, but caused phytotoxicity in 'Calypso' (Figures 1C,1D,2J and 3J).According Moura et al. (2010) the performance of the STS depends on the cultivar, stage of development and concentration used.Similarly, Lima et al. (2017) observed that STS is more effective in inhibits ethylene action in same evaluated cultivars at marketing stage.
During evaluations, there was complete flowers abscission and the plants have not reached subsequent development stages, there was only leaf emergence at the seedling stage, this fact is probably due to low irradiance (8-10 μmol photons s -1 m -2 ) the interior of the room, which reduced the photosynthetic process and by the increase of ethylene synthesis (SEGATTO et al., 2013).Buildup of ethylene in the atmosphere occurs mainly during transport and retail sales, which effects are increased by stresses (SÁ et al., 2008), which can promote or inhibit its development, altering the phenology and productive potential (TOMAZETTI et al., 2015).These responses to ethylene are influenced by environmental factors including light (ZHONG et al., 2012).
The decreases observed may be related to the fact of ethylene synthesis and sensitivity are enhanced during certain stages of plant development, as well as by a number of biotic and abiotic stresses, which stimulates chlorophyll destruction and yellowing, abscission and senescence of leaves (SALVEIT, 1999), because acts by converting chlorophyll a and b in chlorophyllide and phytol, resulting in loss of green color usually at concentration varying from 1 to 10μL L -1 (FERRANTE and FRANCINI, 2006).The same authors mention that ethylene increases activity of chlorophyllase and oxidases responsible for chlorophyll degradation by regulating up their gene expression, in the presence or absence of light.

CONCLUSIONS
Regardless of the development stages, varieties showed different levels of sensitivity to ethylene, 'Calypso' is very sensitive and 'MG 302' showed intermediate sensitivity, indicating that ethylene sensitivity at the early developmental stages correlated with ethylene sensitivity in marketing stage.Treatment with STS significantly blocked the ethylene in both development stages studied and that the use of 1-MCP was less efficient, probably due to the concentration of 1-MCP was used caused no effect in blocking the ethylene, the exposure time was inadequate or there was accelerated production of new ethylene receptor sites.
, 2A and 2B), while the 'MG 302' showed intermediate response to ethylene with about 88% abscission at third evaluation day for stage 1 and 44% abscission after treatment in stage 2 (Figures 1B, 1D, 3A and 3B).As previously shown by Segatto et al. (2013) and Lima et al. (2017) 'Calypso' and 'MG 302' in the post-production stage have different levels of sensitivity to ethylene, in which the 'Calypso' is highly sensitive and 'MG 302' showed intermediate response to treatment.On other hand, Edelman and Jones evaluated three accessions of Capsicum annum including 'Yellow Bell', 'California

Figure 2 .
Figure 2. Appearance of the ornamental pepper 'Calypso' on development stage 1 (seedlings) and development stage 2 (initial flowering).A and B -treatment with ethylene compared to control; C and D -treatment with 1-MCP compared to control; E and F -treatment with 1-MCP + Ethylene compared to control; G and H -treatment with STS compared to control and I and J -treatment with STS + Ethylene compared to control.

Figure 3 .
Figure 3. Appearance of the ornamental pepper 'MG 302' on development stage 1 (seedlings) and development stage 2 (initial flowering).A and B -treatment with ethylene compared to control; C and D -treatment with 1-MCP compared to control; E and F -treatment with 1-MCP + Ethylene compared to control; G and H -treatment with STS compared to control and I and J -treatment with STS + Ethylene compared to control.