Pineapple stem-derived bromelain based priming improves pepper seed protein reserve mobilization and germination

Pepper fruit has a great agronomic, nutritional and commercial value given that it is rich in antioxidant compounds such as carotenes, and vitamins C and E. However, pepper seeds are slow to germinate and emergence is often non-uniform and incomplete. Seed priming and invigoration treatments have been explored for a number of pepper varieties but success has been variable and generally limited. The present report describes the effects of pineapple stem – derived protease (stem bromelain) based priming on pepper seed germination in relation to reserve mobilization (specically, proteins and aminoacids). Germination capacity in bromelain treated seeds was signicantly higher on days 7, 14 and 21 than in unsoaked (control) and deionized water-soaked seeds but comparable across the treatments and the control on day 28. Germination rate was signicantly highest in bromelain treated seeds. Light microscopy revealed an abundance of protein bodies in the endosperm of the seeds investigated at before imbibition and when observed over a period of 96 h, these bodies were progressively degraded, with the rate of this degradation being fastest in bromelain treated seeds. Quantitative measurements of protein levels (and free amino content) conrmed this observation. The results motivate the use of bromelain extracts for priming pepper seeds based on their proteolytic activity, since germination is dependent on the availability of crude protein and essential amino acids.


Introduction
Pepper (Capsicum annuum L.) fruit has a great agronomic, nutritional and commercial importance based on its high levels of antioxidant compounds such as carotenes, and vitamins C and E (Chávez-Mendoza et al. 2015). According to the Food and Agriculture Organization (FAO), world´s production of pepper reached 4 164 594 t in 2018 (FAOSTAT 2018). However, seedling establishment in this cash crop is often not maximized owing to the low speed and uniformity in the germination exhibited by pepper seeds (Demir et al. 2018).
As in other vegetable species (Delian et al. 2017;Anwar et al. 2020), efforts have been made to ameliorate these undesirable seed traits in pepper seeds (Bradford et al. 1990;Andreoli and Khan 1999).
For example, Dutta et al. (2015) reported that K 2 SO 4 (1%) priming exerted a positive in uence via increasing pepper seed germination percentage by 31.29% compared to non primed seeds. Siri et al. (2013) reported that seeds primed in a polyethylene glycol (PEG 6000) solution with the osmotic potential of -1.5 MPa for 6 days improved germination compared to control seeds. The disadvantages of using these chemical substances as priming agents is that the cost of osmotics such as PEG is high, and they also present obstacles to their commercialization due to their relative complexity. In addition, they must be removed from the seeds before they dry out. Studies on the priming of peppers seeds with plant extracts are rare (Barchenger and Bosland 2016), and to the best of our knowledge, bromelain extracts from pineapple stems has never been used to prime pepper seeds.
Proteases play an important role in mobilizing protein reserves in seeds (Zhao et al. 2018). Cysteine proteases are the most abundant group of proteases responsible for the degradation and mobilization of reserve proteins to promote seed germination (Grudkowska and Zagdańska 2004). Bromelain is a cysteine protease (Heinicke and Gortner 1957) with several applications in elds such as medicine and biotechnology (Mohri and Matsushita 1984;Carvajal et al. 2010;Zhao et al. 2013;Kwatra 2019). In 1997, the Bioplant Centre patented a simple and e cient new technology to obtain bromelain from pineapple stems (harvest remains) (Hernández et al. 1997). Reference to the literature indicated only one study, published in 1994, where the effect of exogenous bromelain application on germination was assessed.
This study, which was conducted on the seeds of Rosa multi ora Thunb (Kuska 1994), used reagent grade bromelain from stem of pineapple and increased seed germination percentage three times compared to unprimed seeds. However, studies on bromelains potential mechanism(s) of action in terms of its effects on the biochemical processes that in uence germination were not conducted. This motivated the present report which describes the use of pineapple stem -derived bromelain for pepper seed priming. Seed germination capacity and rate were related to levels of proteins and aminoacids to identify bromelains potential mechanism of action in this species. Light microscopy was also used to assess levels of protein degradation during imbibition.

Plant materials
Sweet pepper seeds cv. Lamuyo were obtained from the Ceballos Agro-Industrial Company, Ciego de Ávila, Cuba. Bromelain crude extract was obtained from pineapple cv. MD-2 stems from harvest residues.
Extraction was performed using the procedure described by Hernández et al. (1997) with 2 mmol/L sodium sulphite to stabilize the active centers of the enzyme.

Seed treatments and assessment
Seeds were soaked in bromelain crude extract with a proteolytic activity of 6.25 tU or deionized water for 3 h at 35 o C in 100 mL-Erlenmeyers without shaking in the dark. The optimal duration of pepper seed priming was previously determined in our laboratory (unpublished data). Unsoaked seeds served as a control. One total U is de ned as the amount of enzyme, in the total volume of extract used, that liberates 1 µmol of soluble TCA. One hundred pepper seeds (4 reps of 25 seeds / Erlenmeyers) were used per treatment and control. Later seeds were dried at 25°C for 48 h until reaching their initial mass (8.5 mg ± 0.61 mg per seed). For germination, all treatments were placed in Petri dishes on lter paper moistened with 10 mL of deionized water. A germination chamber (RTOP-D Series, China) was used at 30 ± 2°C for 28 days. During the rst ve days they were kept in the dark and later in a photoperiod of 16 h of light / 8 h of darkness. Germination was recorded daily and this data were used to calculate the germination rate according to Ranal and Santana (2006).

Histochemical evaluation of protein bodies in pepper seeds
A separate batch of 125 pepper seeds (5 reps of 25 seeds / Erlenmeyer) were used per treatment and control for histochemical evaluation as described above. Twenty seeds per treatment and control were randomly sampled (4 seeds / rep) at 0, 24, 48, 72 and 96 h for the histochemical analysis (Johansen 1940). Samples were xed in FAA, dehydrated in an ethanol series, and embedded in para n. Cross sections (5 µm thick) were cut with a hand rotary microtome KD-202A and collected on microscopic slides covered with a gelatin solution. Sections were stained with solutions of mercury (1%) and blue bromophenol (0.05%) according to Pearse (1985). Observations were carried out using a Carl Zeiss MicroImaging (GmbH 37081) microscope at 20x, and images captured with a Cannon (EOS 600D) digital camera.

Levels of soluble proteins and free aminoacids in pepper seeds
Another separate batch of 125 pepper seeds (5 reps of 25 seeds / Erlenmeyer) were used per treatment and control for measuring levels of soluble proteins and free aminoacids. Twenty seeds per treatment were randomly sampled (4 seeds / rep) at 0, 24, 48, 72, 96 and 120 h for protein and free aminoacids evaluation. Protein extraction was carried out with Tris-HCl 0.1 mol / L buffer, pH = 8.5-8.8, 5 mmol / L EDTA and 20 mmol / L β-mercaptoethanol (Isaacson et al. 2006). The total protein content was determined according to Bradford (1976). The amino acid extraction of each sample was carried out twice with ethanol (80%, v: v) and both supernatants were combined. Quanti cation of the total amino acid content was carried out through ninhydrin reaction according to Moore and Stein (1948). Biochemical analyzes of each sample were replicated three times.

Data analysis
The quantitative data collected were analyzed using SPSS (Version 8.0 for Windows, SPSS Inc., New York, NY). Data were subjected to t-, ANOVA and Tukey tests (p = 0.05) after normality was established (Shapiro-Wilk test).

Results And Discussion
The mobilization of reserves (proteins, carbohydrates and lipids) is crucial for germination e ciency and establishment of seedlings (Thompson 2018). Abud et al. (2017) pointed out that proteins constitute one of the main reserve components in Capsicum annuum L. seeds. Galão et al. (2007) observed in the seeds of Prosopis juli ora after 48 h under germination conditions, that the globular protein bodies were hydrolyzed and the protein material became more soluble. The hydrolysis of reserve proteins to their constituent amino acids is carried out by proteases, both during germination and in post-germination processes (Zhao et al. 2018).
In the present study, bromophenol blue staining developed a strong blue coloration of the cytoplasmic granules suggesting the presence of protein bodies in vacuoles of the endosperm of seeds in all treatments (Fig. 1). Numerous protein bodies were observed in pepper seed endosperm at the beginning of the imbibition period (Fig. 1A). Their number was reduced progressively during the rst 96 h of germination in all treatments, although protein degradation appeared to be markedly faster (more extensive) in bromelain primed seeds (Fig. 1B). More speci cally, during the rst 96 h of observation, higher protein disintegration (less intense coloration) was observed in bromelain-treated seeds compared with untreated (control) and hydro-primed seeds (Fig. 1B). After 48 h, a greater spacing and dispersion of the protein bodies of the seeds conditioned with crude bromelain extract was observed (Fig. 1). Quantitative analysis of protein levels con rmed this observation ( Fig. 2A). The bromelain treatment also increased levels of free aminoacids (Fig. 2B). Percentage of germination (Fig. 3) and germination rate (Fig. 4) were initially higher in bromelain-treated seeds. However, nal germination percentage was comparable across the treatments and the control.
Several authors have reported on the predominance of cysteine-proteases during the degradation processes of reserve proteins (Szewińska et al. 2016;Liu et al. 2018). Tozzi and Takaki (Tozzi and Takaki 2011) observed in seeds of Passi ora edulis the degradation of the protein bodies and the loss of these began generating spaces product of the mobilization of reserves from the sixth day of being placed in germination conditions. The biochemical analyses carried out here indicate that the content of protein reserves decreased fastest in the bromelain-treated seeds, most likely as a result of hydrolysis by the action of the proteolytic activity of bromelain (cysteine-protease) (Kwatra 2019;Mendes et al. 2019) and other proteases present in the seed.
The content of free amino acids was in agreement with these observations. The relationship observed between the content of soluble proteins and free amino acids has been described in seeds of different plants during the mobilization of reserves. In a study conducted by Satyanarayana et al. (2011) on Sterculia urens Roxb. observed a decrease in the content of soluble proteins in the cotyledons while an increase in the content of free amino acids occurred over time during the 9 d -germination period. Aragão et al. (2015) also observed a signi cant decrease in ethanol-soluble proteins and an increase in the total content of free amino acids during germination (5-7 days) of Cedrela ssilis Vellozo seeds, which suggests that amino acids could be provided by the mobilization of proteins stored in mature seeds.
The results of the present study indicate that the priming of the seeds with crude bromelain extract has a bene cial effect on the pepper seeds, increasing their initial germination percentage and rate. As alluded to above the hydrolysis of storage proteins promoting the mobilization of protein reserves and therefore the generation of amino acids. The amino acids obtained can then be used in the biosynthesis of enzymes, hormones, proteins, pyrimidines and purine bases, which contributes to the germinative development of the embryo (Aragão et al. 2015). In addition, the crude bromelain extract contains other compounds such as minerals, sugars, vitamins and amino acids that may have accelerated the development of the embryo and contributed to increasing the speed of germination, which coincides with that indicated by Mavi and Atak (2016). The results collectively motivate for the use of bromelain extracts for priming pepper seeds, since the hastening of germination through increased reserve mobilization during the early stages of germinative development can promote seedling establishment.

Declarations
Author contribution: LP, YA, LN, CC, CL, S, JCL and AP designed the research; LP, YA, LN, CC and CL conducted the experiments; LP, YA, S, JCL and AP analyzed the data; LP, YA, S, JC and AP wrote the paper; S, JCL and AP had primary responsibility for the nal content. All authors have read and approved the nal manuscript. Figure 1 In the present study, bromophenol blue staining developed a strong blue coloration of the cytoplasmic granules suggesting the presence of protein bodies in vacuoles of the endosperm of seeds in all treatments ( Fig. 1)

Figure 2
Quantitative analysis of protein levels con rmed this observation ( Fig. 2A). The bromelain treatment also increased levels of free aminoacids (Fig. 2B).

Figure 3
Percentage of germination (Fig. 3) and germination rate (Fig. 4) were initially higher in bromelain-treated seeds. However, nal germination percentage was comparable across the treatments and the control.

Figure 4
Percentage of germination (Fig. 3) and germination rate (Fig. 4) were initially higher in bromelain-treated seeds. However, nal germination percentage was comparable across the treatments and the control.