Padrón Peppers, Some Are Hot, and Some Are Not

Abstract

Some peppers are pungent due to the presence of their secondary metabolite contents—capsaicinoids. The ability to synthesize capsaicinoids is a genetic trait, but the control of their accumulation in the fruit is more complex than just biosynthesis. Besides biosynthesis, other metabolic pathways, such as oxidation and conjugation, are also involved in capsaicinoid homeostasis. Moreover, all these pathways are modulated by different factors, namely plant hormones, transcription factors, ontogeny, and the environment, including both abiotic and biotic agents. In the present chapter, the present knowledge about the control of capsaicin metabolism in pepper is reviewed. Based on the literature and our own experience, there is a correlation between pungency and lignification. We have a clue about the reason: capsaicinoid and lignin metabolic pathways are related, and their biosynthesis predate from the same intermediate compounds. Finally, this chapter mainly focuses on the cultivar Padrón, a pungent variety used in our experiments because of its economic and cultural value.

1. Introduction

The title of this chapter is a translation of a local saying in Galicia (Northwest of Spain) that literally states in the Galician language, “Os pementos de Padrón, uns pican e outros non”. The saying refers to the typical local dish consisting of green, immature fruits of pepper fried in oil and salted. When a dish of these peppers is served, you find that most fruits are sweet or mild, but sometimes you eat one that is extremely hot by chance. Padrón pepper production is of great economic relevance not only in Galicia but also in other parts of Spain, and even it is produced in other countries like Marocco (Figure 1).

Figure 1.

A). Commercial bag of PDO “Pemento de Herbón” with the PDO label. B). Three different labels of Padrón commercial brands.

These pepper fruits belong to a local landrace called “Padrón,” which is characterized by the presence of capsaicinoids (the pungent substances in peppers), but in low amounts when fruits are immature [1]. The origin of the landrace can be traced back from the seeds that were taken in the 17th Century from Tabasco (Mexico) to the monastery of Herbón (a place in the municipality of Padrón) by monks of the Franciscan order [2]. The monks transferred seeds to the local farmers. The landrace arose from local breeding and established the tradition of restricting seed transfer within the local community. Interestingly, Padrón seeds are always part of women’s dowry.

The quality of the fruit, not only the heat but also other organoleptic traits, is considered of great importance by consumers. To guarantee this quality, some pepper growers in Padrón and other closer municipalities promoted the creation by the European Union of the Protected Designation of Origin (PDO) Pemento de Herbón, established in 2010.

On the other hand, the popularity of these peppers has led to the release of numerous commercial products, which include these peppers as an ingredient (paprika, jam, sauces, candies, chocolates, liquors, etc.) or items like t-shirts, socks, etc. as a part of Padrón-pepper passion (Figure 2).

Figure 2.

Commercial products include Padrón peppers as an ingredient or as a motto. A) Paprika powder made with PDO Herbón peppers. B) PDO Herbón pepper jam. C) Hot candies. D) PDO Herbón chocolates. E) Padrón pepper sauce mimicking tabasco. F) Padrón pepper liquor. G) t-shirt with a “Galician roulette” pun regarding the Galician saying. H) Socks. I) Key chain.

Padron pepper consumers are divided into those who like such a pungency Russian roulette and those who prefer only sweet or hot fruits. Therefore, finding technical solutions to control the heat level is essential. Furthermore, it is not only a local issue: many other pepper landraces and cultivars are hot or sweet, and this trait has to be preserved when pepper lines are bred for, e.g., plant resistance to diseases, pests, and abiotic stress. Consumers expect a specific level of pungency (heat) in a particular cultivar’s fruit, which has to be bear in mind to achieve success in sales. Moreover, even a pepper with the pungency (heat) trait can show a modulation of the level of capsaicinoids due to several factors [3].

This chapter reviews the present knowledge about pepper pungency and its control.

2. To be or not to be hot: capsaicinoid presence as a genetic trait

Some peppers are pungent, and others are not. Pepper pungency is a trait conferred by the presence of the functional allele of Pun1 gene [4]. It is considered to be a dominant trait [5]. Still, the recent report of hot pepper hybrids derived from the crossing of two non-pungent parents [6] makes such a heterosis effect on capsaicinoid accumulation deserve further studies in the future.

The Pun1 gene (also known as AT3) has been demonstrated to be involved in capsaicinoid biosynthesis. Firstly, there is a correlation between its expression with capsaicinoid accumulation and the lack of the pungent trait in pepper lines with mutations in this gene [4, 7]. Moreover, in the last years, several authors have demonstrated that virus silencing (VIGS) of the gene in pepper leads to a decrease in capsaicinoid accumulation [4, 8, 9, 10]. It should be noted that the pepper plant is recalcitrant to Agrobacterium transformation [3, 10]; therefore, the usual functional approach by genetic transformation of the plant has not been possible so far.

Another approach was to demonstrate that the protein encoded by Pun1/AT3 is the so-called capsaicin synthase. Ogawa et al. [9] expressed Pun1 protein in Escherichia coli, purified it, and obtained antibodies anti-Pun1, which inhibited capsaicinoid formation in an assay using pepper protoplasts. However, they did not assay the enzymatic activity of purified Pun1 itself. Recently, Muratovska et al. [11] expressed Pun1/AT3 and another aminotransferase gene (CaAT) in Saccharomyces cerevisiae and proved that both lines of transformed yeasts were able to synthesize the capsaicinoid nonivamide. Finally, Milde et al. [12] not only purified Pun1 expressed in E. coli but also demonstrated the ability of the purified protein to catalyze the capsaicin synthase reaction, that is, the transformation of trans 8-methyl-6-nonenoyl-CoA and vanilloylamine into capsaicin. Thus, Pun1 is a bonafide capsaicin synthase. However, the possibility that other pepper proteins, such as CaAT, could also be involved in the biocatalysis of this reaction cannot be excluded [11].

3. Being hot, but how much? Regulation of capsaicinoid levels

The presence of capsaicin synthase (Pun1/AT3 or other protein) is enough to allow the biosynthesis of capsaicinoids in a pepper line because their precursors are synthesized from common plant pathways. However, there are other pathways involved in capsaicin metabolism. Moreover, all those pathways are targets of fine-tune modulation in response to variations of different endogenous and exogenous factors. Hence, it is worthwhile to look at the present knowledge of regulation.

3.1 Capsaicinoid metabolic pathways

The capsaicin synthase reaction is the final step of the pathway, converting vanillylamine and 8-methyl-6-nonenoyl-Coa into capsaicin. The two substrates of the last reaction are synthesized in two different pathways. Thus, the aromatic/phenolic moiety comes from phenylalanine, and the aliphatic moiety from valine (Figure 3). More detailed information on this pathway can be found in [5, 13]. Branches of this pathway lead to the biosynthesis of capsinoids and capsaicinoids (details in [13]), non-pungent analogs of capsaicinoids that are usually synthesized in pepper lines with low content of the latter because of mutations in Pun1. Capsaicinoid biosynthesis shares precursors with other pathways. Thus, phenolic compounds are required to synthesize lignins, some phytoanticipins, flavonoids, etc. Therefore, there is competition among the different pathways that predate the same precursors.

Figure 3.

A general overview of the pathways involved in capsaicinoid metabolism: biosynthesis, oxidation, and conjugation. Original figure created by the authors based on the cited literature.

Oxidases such as peroxidases and polyphenol oxidases can degrade capsaicinoids in pepper (Figure 3). Peroxidases can catalyze capsaicin oxidation in vitro, and their expression is correlated with capsaicin decrease at the end of fruit development [5]. The main products of such oxidation are capsaicin dimers, such as 5,5′-dicapsaicin [5], which are present in natural sources such as pepper cell cultures and fruits [5, 14]. Immunoinhibition assays of capsaicin oxidation by peroxidases [14] supported the idea that other types of enzymes, such as polyphenol oxidases, could also be partially responsible for capsaicin oxidation [5].

Another metabolic fate of capsaicinoids is conjugation with other molecules (Figure 3). Thus, glucosides and glucopiranosides of capsaicin have been found in pepper fruits [5, 15]. However, we cannot exclude that other conjugated forms of capsaicinoids, e.g., with other sugars or amino acids, could be present in peppers.

3.2 Hormone and transcriptional regulation

3.2.1 Plant hormones

A previous review [5] summarized some evidence of plant hormone regulation of capsaicin metabolism, pointing to the role of ethylene, jasmonates, and salicylic acid, based on data from exogenous application of the hormones to cell cultures and plants. Since then, several pieces of information have been published, but the most interesting are those that have used new approaches. Thus, several transcription factors involved in capsaicin biosynthesis (see Section 3.2.2) are responsive to plant hormones such as ethylene (CcERF2, [16]) or jasmonates (CaMYB108, [17]). The evidence of the ethylene regulation of the capsaicinoid biosynthesis is clear: CcERF2 silencing resulted in decreased capsaicin accumulation, and the treatment of peppers with inhibitors of ethylene perception (1-methylcyclopropene) and biosynthesis (piperazine) also leads to a reduction of the pungent compounds in the fruit [16]. In the case of jasmonates, silencing a jasmonate-responsive transcription factor leads to a decrease in capsaicin and dihydrocapsaicin accumulation [17].

Moreover, the expression of a gene encoding an enzyme involved in jasmonate biosynthesis (2-oxophytoeienoic acid reductase) in pepper fruit is correlated with the stage of development when capsaicin accumulates [18]. However, more studies are needed before we can fully determine the hormone regulation of capsaicin pathways. For instance, limited studies have addressed the regulation of capsaicinoid oxidation or conjugation.

3.2.2 Transcriptional regulation

As in the case of hormones, gene silencing has been used in the last years to test the involvement of several transcription factors (TFs) in the biosynthesis of capsaicinoids. Most of them belong to the MYB type, but also, an AP2/ERF TF has been successfully proven to regulate the expression of capsaicin biosynthesis genes and capsaicinoid accumulation in the fruit (Table 1).

Capsicum species	Gene	Type of TF	References
Capsicum chinense	CcMYB31*	MYB	[18]
Capsicum annuum	CaMYB31	MYB	[19]
C. annuum	CaMYB37	MYB	[20]
C. annuum	CaMYB48	MYB	[21]
C. annuum	CaMYB108	MYB	[17]
C. chinense	CcERF2	AP2 superfamily	[16]

Table 1.

Transcription factors (TFs) proved to regulate capsaicin biosynthesis in pepper by gene silencing.

*

In this case, overexpression of the gene in pepper was also used.

There are limited studies on the TF regulation of genes involved in capsaicinoid oxidation or conjugation. Other studies are based on the correlation between their expression and the expression of capsaicin biosynthesis genes or by computer-based analysis of their promoters. However, their involvement still has to be confirmed by gene silencing or other methods that provide equivalent information.

3.3 Organ and plant development

There are many reports of the trend of the capsaicinoid content in pepper fruit development: it increases continuously during most of the course of development, and just at the end of the process, it decreases [3]. In most cases, only the genes or proteins involved in the biosynthesis pathway were studied. Furthermore, usually, the study of competition among different capsaicinoid pathways was not addressed. However, Estrada et al. [1] demonstrated a negative correlation between lignin deposition and capsaicinoid accumulation, pointing to such competition (Figure 4).

Figure 4.

Competition among different biosynthetic and catabolic pathways affects capsaicinoid levels in pepper fruit. A) Trends in the levels of capsaicinoids, phenolics, lignin, and peroxidases in Padrón pepper fruit (based on data from [1]). B) Overview of the involved pathways. Original figures created by the authors based on the cited literature.

Intriguingly, peroxidases and polyphenol oxidases (e.g., laccases) can affect capsaicinoid accumulation in at least two different ways: participating in the oxidation of these compounds or driving the flow of phenolic compounds to the lignin biosynthesis pathway by the catalysis of the last reaction of the pathway (Figure 4). In any case, this competition is underexplored, and conjugation has also been an oversight, even though it may be linked to the transport in vegetative organs.

Capsaicinoids have been detected in vegetative organs, but eliminating the floral buds and preventing fruit formation leads to the absence of these pungent compounds in leaves and stems [22]. This suggests that capsaicinoids could be transported from fruits to other organs of plants, but so far, this was not confirmed. Moreover, exogenous feeding of capsaicin to the roots of vegetative pepper plants does not lead to capsaicin presence in aerial organs [23]. Capsaicin is a compound not soluble in water, making its transport difficult into the plant. Maybe capsaicin conjugates, more soluble than capsaicin itself, are the compounds transported throughout the plant. To our knowledge, such a possibility has not been explored so far. Indeed, there is a lack of studies regarding capsaicin conjugates in pepper plants.

The age of the plant also determines the amount of capsaicin in the fruit, and older plants usually show more pungent fruits [5]. Probably as a consequence, the Padron pepper fruits are also hotter at the end of the season, in September–October (Figure 5).

Figure 5.

Evolution of capsaicinoid levels in Padrón peppers during the commercial season. Samples were bought at Galician local markets during 2021 and 2022 (data from the Ph.D. thesis of Raquel Núñez-Fernández, in preparation).

3.4 Environmental factors and capsaicinoids

Several recent publications have reviewed the effects of several environmental abiotic factors as light, temperature, mineral nutrition, water, etc., on capsaicinoid accumulation, showing that, overall, stress causes an increase in these compounds (Figure 6A) [3, 24]. Therefore, we have focused on 1) the metabolic consequences of that effect regarding lignification and 2) biotic stress.

Figure 6.

Effects of stress on capsaicinoid levels. A) Stress causes an increase in capsaicinoids and a decrease in lignin in the fruit (based on data from [25, 26]). B) the stress-induced accumulation of capsaicinoids causes a reduction in the phenolic moieties that otherwise would be used in lignification. C) Phytophthora capsici infection causes stress in pepper plants, thus leading to increased capsaicinoid accumulation in the fruit (data from Ph.D. thesis of Raquel Núñez-Fernández, in preparation).

As we stated above, lignification competes with capsaicinoid biosynthesis during the development of the fruit. Therefore, an increase in capsaicinoid levels caused by stress should lead to a decrease in the deposition of lignin. This was exactly what we observed in previous studies regarding mineral nutrition and watering (Figure 6B) [25, 26].

Biotic stress also can modulate the amount of capsaicinoids in the fruit. However, capsaicin quantification is usually oversight in agronomic trials testing biofertilizers, biostimulants, and biological control agents (BCAs), as well as studies where a pathogen or pest is used as a challenger. However, this analysis is worthwhile because the market expects a stable pungency level [3]. Thus, Saxena et al. [27] found that pepper plants treated with Trichoderma isolates used as BCAs caused an increase in capsaicin in the fruits of plants after Colletotrichum truncatum challenge. Khan et al. [28] tested the endophyte Penicillium resedanum as a potential tool to alleviate drought stress in pepper. They reported an increase in the capsaicin levels in the fruits of the plants treated with this fungal endophyte. In our experiments, we observed that the stress in plants inoculated with Phytophthora capsici leads to increased capsaicin in the fruit Figure 6C). Therefore, in the last years, we included the analysis of capsaicinoids in greenhouse trials while testing BCAs and resistance inducers.

4. Conclusions and perspectives

Control of the capsaicin levels beyond a simple absence/presence trait is necessary. Producing pepper fruits with uniform and predictable levels of pungency is a challenge that needs further insight into the regulation of both the biosynthesis and the catabolic pathways of capsaicinoids. The knowledge of oxidation and conjugation pathways has not advanced much in the last two decades, probably because scientists paid little attention to them. On the other hand, genomic, transcriptomic, proteomic, and metabolomics studies have flourished in the last decade. Still, more molecular and physiological studies are required, particularly studies proving the function of the genes by silenced lines or mutants if the recalcitrant nature of pepper for genetic transformation is overcome. There is room for improvement in our knowledge of these physiological processes, and basic science advances will lead to applied advances. Pungent compounds can be used not only as food but also can be useful in medicine as drugs or in agriculture as fungicides or pesticides [3, 23], and all these applications need a guarantee of stable supply.

Acknowledgments

This research was funded by grant RTA2017-00019-C03-03 from Ministerio de Ciencia e Innovación.

Conflict of interest

The authors declare no conflict of interest.