**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.

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

### **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.*

*Padrón Peppers, Some Are Hot, and Some Are Not DOI: http://dx.doi.org/10.5772/intechopen.110435*

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**).

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,


### **Table 1.**

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

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**).

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,

### **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.*

*Padrón Peppers, Some Are Hot, and Some Are Not DOI: http://dx.doi.org/10.5772/intechopen.110435*

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**).

### **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.

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].

### **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).*

#### **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).*

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.
