Capsaicinoids: Pungency beyond

Capsicum Emmanuel Rezende Naves &

Lucas De Ávila Silva & Ronan Sulpice &
Wagner L. Araújo & Adriano
Nunes-Nesi & Lázaro E.P. Peres &
Agustin Zsögön
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Opinion

Capsaicinoids: Pungency beyond Capsicum

Emmanuel Rezende Naves,1 Lucas de Ávila Silva,1 Ronan Sulpice,2 Wagner L. Araújo,1,3
Adriano Nunes-Nesi,1 Lázaro E.P. Peres,4 and Agustin Zsögön1,*

Capsaicinoids are metabolites responsible for the appealing pungency of Highlights

Capsicum (chili pepper) species. The completion of the Capsicum annuum Capsaicinoids are secondary metabo-
lites that confer Capsicum (‘chili’ pep-
genome has sparked new interest into the development of biotechnological
pers) fruits their appealing pungency
applications involving the manipulation of pungency levels. Pungent dishes are (‘heat’) and have multiple proven
already part of the traditional cuisine in many countries, and numerous health health benefits and industrial
applications.
benefits and industrial applications are associated to capsaicinoids. This raises
the question of how to successfully produce more capsaicinoids, whose Five Capsicum species have been
biosynthesis is strongly influenced by genotype–environment interactions in domesticated. However, even in fruits
of the same species, pungency is not
fruits of Capsicum. In this Opinion article we propose that activating the consistent owing to the strong influ-
capsaicinoid biosynthetic pathway in a more amenable species such as tomato ence of the environment on capsaici-
noid levels. Furthermore, multiple
could be the next step in the fascinating story of pungent crops.
agronomic issues are prevalent in chili
pepper cultivation, such as wilting in
The Nutritional Value of Capsaicinoids hot days, flower drop, and virus
susceptibility.
The momentous voyage of Christopher Columbus in search of a new route to India was driven
by the European demand for spices – mainly black pepper (Piper nigrum L.), which at the time Capsicum belongs to the Solanaceae
fetched prices similar to gold [1,2]. Instead, Columbus arrived in Central America and brought family, and recent completion of its full
back a species that would quickly become ‘the world’s most popular condiment’: chili peppers genome sequence shows that inactive
genes for the biosynthetic pathway of
of the Capsicum genus [3]. The genus comprises more than 30 species, with only five capsaicinoids are also present in
domesticated crops, namely the closely related C. annuum, C. chinense, and C. frutescens, tomato. We propose that the use of
as well as C. baccatum and C. pubescens [4], which display a broad variety of fruit size, shape, state-of-the-art genome engineering
color, and pungency (see Glossary; Figure 1). The appealing pungency (‘heat’ or ‘bite’; Box 1) techniques to activate capsaicinoid
biosynthesis in tomato is a suitable
of hot chilies is produced by the accumulation of a group of alkaloids, called capsaicinoids, option to exploit these valuable sec-
whose major representatives are capsaicin and dihydrocapsaicin [5]. These secondary metab- ondary compounds.
olites are produced through a complex series of reactions involving the convergence of the
phenylpropanoid and branched-chain fatty acid pathways (Figure 2) [6].
1
Departamento de Biologia Vegetal,
Universidade Federal de Viçosa,
In the context of the debate on food security, nutritional and nutraceutical properties are quickly 36570-900, Viçosa, MG, Brazil
gaining importance alongside crop yield as breeding targets [7,8]. Plants represent a funda- 2
Plant Systems Biology Laboratory,
mental reservoir of chemical compounds beneficial to human health, mainly derived from their Plant and AgriBiosciences Research
Centre (PABC) and Ryan Institute,
extraordinarily rich secondary metabolism [9,10]. However, breeding efforts mostly focused National University of Ireland Galway,
on attaining high yields have resulted in a serious nutritional decline of grains, fruits, and Galway H91 TK33, Ireland
3
vegetables [11]. Finding sustainable pathways to boost the production of vitamins and sec- Max-Planck Partner Group at the
Departamento de Biologia Vegetal,
ondary metabolites such as polyphenols and carotenoids in crops is thus a top priority in the Universidade Federal de Viçosa,
plant research agenda [12,13]. Capsicum fruits contain high levels of antioxidants such as 36570-900, Viçosa, MG, Brazil
4
vitamins A, C, and flavonoids [14]. Hot peppers also produce variable amounts of capsaici- Departamento de Ciências
Biológicas, Escola Superior de
noids, which have demonstrated anti-inflammatory [15], antioxidant [16], antitumoral [17], and Agricultura ‘Luiz de Queiroz’,
weight-loss properties [18]. All of these effects suggest that a regular intake of capsaicinoids is Universidade de São Paulo, 13418-
beneficial for health and that, therefore, hot peppers can be considered to be functional foods 900 Piracicaba, SP, Brazil

[19]. Furthermore, topical capsaicin applications are an effective pain-management treatment

with low risk for adverse drug–drug interactions [20]. Remarkably, only Capsicum fruits contain *Correspondence:
these appealing compounds. We next examine their physiological roles in plants. agustin.zsogon@ufv.br (A. Zsögön).

Trends in Plant Science, Month Year, Vol. xx, No. yy https://doi.org/10.1016/j.tplants.2018.11.001 1

© 2018 Elsevier Ltd. All rights reserved.
 TRPLSC 1754 No. of Pages 12

(A) (B) (C) (D) Glossary

Biofactory: a biological system used
to produce useful amounts of
chemical compounds. Many
organisms are ‘customized’ (through
selection or genetic manipulation) as
production platforms for the
manufacture of biopharmaceuticals
such as enzymes, recombinant
(E) (F) (G) (H) proteins (in E. coli or yeast), vaccines,
and antibodies (generally in plants).
Capsinoids: compounds that are
structurally similar to capsaicinoids,
but that lack the pungent taste. The
main capsinoids found in chili
peppers are capsiate,
dihydrocapsiate, and
nordihydrocapsiate. Many of the
health benefits described for
(I) (J) (K) (L) (M) capsaicinoids have also been
attributed to capsinoids. Thus,
manipulation of the capsinoid
pathway is also an appealing
prospect given that a segment of the
population cannot tolerate the
pungent flavor.
Cisgenesis: a form of genetic
engineering whereby a crop plant is
genetically modified using DNA
fragments contained in the species
Figure 1. Variation in Plant and Fruit Morphology in the Capsicum genus. Accessions of Capsicum annuum itself or in closely related, interfertile
(A–M) and Capsicum chinense (N). Scale bar, 10 cm. species.
Directed deterrence hypothesis
(DDH): first proposed by Cipollini
The How and Why of Pungency in Capsicum and Levey in 1997 [25], the
Pungency and Seed Dispersal hypothesis states that plants
The development of nutritious and highly caloric fruits to enlist efficient agents of seed dispersal produce secondary metabolites that
selectively preclude foraging by
presented plants with the conundrum of avoiding seed predators [21]. How a fruiting plant can specific animals (by being toxic or
repel inefficient dispersal agents and favor more effective ones is a long-standing question in unpalatable), but not by others that
angiosperm ecology [22]. One possible solution is the production of secondary metabolites, can then act as efficient vectors for
seed dispersal.
which are thus abundant in fleshy fruits [23,24]. The directed deterrence hypothesis (DDH)
Exaptation: a shift in the function of
posits that fruit secondary metabolites can selectively repel seed predators without deterring a trait during evolution. In the case of
seed dispersers [25,26]. There is mounting evidence that secondary metabolites have indeed capsaicinoids, it is possible that their
been instrumental in shifting plant–animal relationships from predation to mutualism [27,28]. original function was as protective
agents for the plants against attack
Experiments with captive animals showed that capsaicinoids in hot chili fruits were irritant to
by pathogens, and subsequently
mammals but not to birds. The basis for this selective toxicity of capsaicinoids was eventually they acquired a role as deterrents of
demonstrated at the molecular level. The vanilloid receptor 1 (VR1), a cation channel on sensory fruit-eating mammals.
Functional foods: whole, enhanced,
or fortified foods containing
ingredients that aid specific body
Box 1. The Measure of Chili Pungency functions for health benefits (beyond
The most widely used measure of chili pepper pungency is the ‘Scoville scale’, after its inventor, Wilbur Scoville, who in basic nutrition).
1912 created the Scoville organoleptic test [116]. Volunteers test serial dilutions of pepper extract, and the degree of Knock-in: targeted insertion of a
dilution is translated into Scoville heat units (SHU). The scale remains popular although it is unreliable and subjective; in DNA sequence that is integrated into
modern times has been superseded by the use of HPLC [117]. Pungency units are thus defined as parts of capsaicin per a specific genomic locus.
million parts of pepper fruit dry mass. Pungency units can be converted to SHU by multiplying by 16 (e.g., pure capsaicin Mutualism: a cooperative
is 16M SHU, pepper spray is 5M SHU). Some of the hottest chili pepper varieties can exceed 1M SHU, and have fanciful relationship between two organisms
names such as Scorpion, Carolina Reaper, or Dragon’s Breath. whereby both benefit from the
interaction.

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(A) (B) Predation: an interaction between

Calyx Mitochondrium Plasd two organisms in which one (the
Mesocarp
Pyruvate Shikimate predator) kills and feeds off the other
pathway (the prey).
L-valine
Seeds Pungency: technical term for food
L-valine commonly referred to as ‘spicy’ or
‘hot’.
Isobutyryl-CoA Chorismate
Secondary metabolism: pathways
and chemical products of
Placenta Isobutyryl- metabolism with specific functions
Epicarp CoA 8-methyl-6-
Phenylalanine that are generally associated with
nonenoic acid
plant–environment interactions,
including stress responses. Humans
8-methyl-6-
use a myriad of secondary
noneoyl-CoA 8-methyl-6- Phenylalanine metabolites for a variety of purposes,
Cucle-bound

Endocarp nonenoic acid

for instance alkaloids (nicotine,
blister

caffeine, morphine), terpenes

(artemisin, limonene, pinene) and
Capsaicin flavonoids (hesperidin, rutin,
Blisters Vanillylamine Feruloyl-CoA quercetin).
Cytosol Synteny: conservation in the relative
position of genetic loci within a
chromosome among different
species. A powerful tool in
Figure 2. Capsaicinoid Biosynthetic Pathway. (A) Cross-section of chili pepper fruit. (B) Schematic representations of comparative genomics that is used
the subcellular locations of the main steps involved in capsaicinoid biosynthesis. The convergence of two pathways is to infer gene function and
required – the phenylpropanoid pathway, which leads formation of vanillylamine, and the branched chain fatty acid phylogenetic relationships between
pathway, which forms the 8-methyl-6-nonenoyl-CoA; the two final compounds are condensed by capsaicinoid synthase species.
to form capsaicin. Abbreviations: ACS, acyl-CoA synthetase; AMT, aminotransferase; BCAT, branched-chain amino acid
aminotransferase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl-CoA ligase; COMT, caffeoyl-CoA 3-O-methyltrans-
ferase; CS, capsaicinoid synthase; FatA, acyl-ACP thioesterase; HCHL, hydroxycinnamoyl-CoA hydratase lyase; PAL,
phenylalanine ammonia-lyase. Redrawn from Mazourek et al. [6] and Kim et al. [95].

nerve endings which responds to noxious thermal stimuli, is activated by capsaicin in mice [29].
Cloning and functional characterization of the chicken VR1 ortholog showed that, in birds, it fails
to be activated by capsaicin owing to differences in the amino acid sequence at key positions
that affect ligand binding [30].

The evolutionary emergence of pungency is considered to be a derived trait within the Capsicum
genus, and is not found in species of the Andean clade, the most ancient group of Capsicum
species [31]. However, it is not known for certain why pungency has been fixed in some species of
the genus and not in others. It could be the result of an ecological trade-off which allows the
biosynthetic pathway to remain active [32]. Pungency precludes fruit consumption by small
mammals, to avoid loss of seed viability in their digestive tract, and instead favors consumption
by birds, which can provide seed dispersion over greater distances [33]. Ironically, the compounds
produced to deter mammal consumption were instrumental in the formation of a reciprocal
relationship with mammals that helped Capsicum to spread to all continents: humans [34]. Seeds
of pungent fruits are also less prone to infection by the fungal pathogen Fusarium, and thus
pungent individuals propagate their offspring more efficiently than non-pungent ones [35]. It is
possible that this was the original function of capsaicinoids and that the selective deterrence of
mammals was an exaptation occurring later during the evolution of Capsicum species [36].

Challenges of Producing Capsicum Fruits with Stable Pungency Levels

Growth and Cultivation of Capsicum Species
Unlike some of its domesticated counterparts in the Solanaceae family (tomato, Solanum
lycopersicum; potato, S. tuberosum; tobacco, Nicotiana tabacum), Capsicum species are

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notoriously labor-intensive and difficult to cultivate crops. Pungent varieties are predominantly
cultivated in the open field, and are thus vulnerable to environmental conditions detrimental to
fruit yield. High air temperature, high CO2 concentration, and excess precipitation can all retard
growth and increase the incidence of disease [37]. Seed germination can be very slow and
uneven depending on species, cultivar, and post-harvest handling [38,39]. Seed viability is
affected by the maturity of fruits, and can be reduced considerably if they are harvested either
too early or too late [40].

Soil-borne diseases and nematodes are prevalent in Capsicum, and grafting onto disease-
resistant rootstocks is a common, although expensive and time-consuming, cultural practice
[41]. Capsicum yield is poorly responsive to N and K supply because most of the effect is
translated into vegetative instead of reproductive growth [42,43]. Fruit set can be erratic
owing to flower drop induced by excessive N fertilization, high wind, and lack of pollination
[44]. Capsicum species are self-pollinating, but their floral structure readily allows cross-
pollination (mostly by insects) [45], making the production of pure seed cumbersome and
costly [46]. Even if all these agronomic shortcomings can be resolved satisfactorily, the highly
variable nature of capsaicinoid biosynthesis, which is also under strong environmental
control, represents one further hurdle towards the production of consistent levels of pun-
gency in fruits [47].

Capsicum Breeding and Biotechnology

Varieties with more consistent pungency levels could be produced by either breeding or
biotechnological manipulation. Classical breeding programs have contributed to the creation
of elite Capsicum varieties, although numerous interspecific crossing barriers are present within
the genus that prevent the full exploitation of favorable traits [48]. The alternative is to
manipulate traits of interest by Agrobacterium-mediated genetic transformation [49]. Capsicum
species, however, are notoriously recalcitrant to transformation by Agrobacterium [50]. In vitro
regeneration capacity is not an obstacle, and protocols can be adjusted for each particular
genotype of interest [51]. The limiting step appears to be the introduction of exogenous DNA
into the plant. Multiple factors influence the process of DNA transfer and integration into the
plant genome, many of which are not fully understood [52]. Currently available Capsicum
transformation protocols have low efficiency, poor reproducibility, and high genotype-depen-
dence [53,54]. Thus, biotechnological manipulation of capsaicinoids in Capsicum is not a
suitable option in the toolbox of the breeder.

The Dynamics of Capsaicinoid Accumulation in Fruits

Capsaicinoid levels are highly dynamic during fruit development. Their levels appear to be
influenced by the ontogenetic trajectory of the fruit. Capsaicinoids begin to accumulate from the
early stages (10 days after pollination, DAP) of fruit development, peak at around 40 DAP, and
then decrease sharply [55–58]. The late decrease in capsaicinoid content appears to result
from high peroxidase activity which oxidizes capsaicinoids in the presence of H2O2 [59,60].
Therefore, the final amount of capsaicinoids in the fruit depends on a complex balance between
the factors that stimulate their synthesis and degradation. Capsaicinoid levels are thus highly
plastic and are most likely determined by a complex set of ecological and physiological trade-
offs with strong input from the environment.

Light
Light stimulates capsaicinoid formation in fruits harvested immature and kept under artificial
lighting [61]. Moreover, ripe pepper fruits maintained under a fluorescent light contained twice
the capsaicin content of peppers stored in darkness [62]. Keeping fruits constantly illuminated

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at moderate irradiance (150–350 mmol m
2 s
1) was found to be more inductive of capsai-
cinoid biosynthesis than maintaining plants at high luminous intensities in the greenhouse
during the summer season [63]. This suggests that photoperiod is seemingly more important to
capsaicin accumulation than light intensity per se. Light has a positive influence on the
expression of the capsaicin synthase gene (CS, discussed further below), which harbors
light-responsive motifs in its promoter region [64], KAS (keto-acyl ACP synthase) and AMT
(aminotransferase) [65], and a negative effect through the induction of peroxidases that
degrade capsaicin [60]. It is hitherto unknown how this balance is controlled and fine-tuned.

Temperature
Pungency level is highly variable in response to air temperature between cultivars and species
[66], and therefore there is no consensus regarding the temperature effects on capsaicinoid
synthesis and accumulation. For some varieties, higher temperature favors the accumulation of
total capsaicinoids, while in others the accumulation of capsaicinoids is affected negatively. It
has been hypothesized that respiration might play an important role in temperature responses
by altering the consumption of photoassimilates and carbon partitioning between plant growth,
lignin, and capsaicinoid synthesis [67]. Furthermore, high night-time temperatures can have a
greater impact on capsaicin contents than daylight temperatures [63]. The CS gene promoter
region contains heat stress consensus elements (HSEs) which could possibly stimulate the
synthesis of capsaicinoids in response to high temperature [64].

Water
In general, water stress reduces fruit yield and increases capsaicinoid accumulation in hot
pepper, albeit in a strongly variety-dependent manner [68]. Water stress decreases fruit size
compared to well-watered plants, but affect less the formation of placental tissue, leading to a
higher proportion of placental tissue per fruit [69]. This is consistent with a higher production of
capsaicinoids because the placenta is their site of synthesis and accumulation. This effect,
however, is also cultivar-dependent [70]. Drought increases the activity of enzymes necessary
for capsaicin biosynthesis (PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxy-
lase; and CS) and reduces capsaicin degradation by peroxidases [69,71]. By contrast, the
proportion of pungent plants in wild Capsicum populations native from Bolivia increases under
higher moisture [32]. In this case, pungency could be beneficial because capsaicinoids protect
the fruit from pathogens, especially from fungi that thrive under high-moisture conditions. Thus,
pungency is increased by both water shortage and excess, but for different ecological and
physiological reasons.

Mineral Nutrition
Mineral nutrition influences the metabolism of capsaicinoids. In this sense, N and K are the main
players. N availability directly affects capsaicin accumulation because the synthesis of a single
capsaicin molecule involves three amino acids – phenylalanine, valine, and leucine – and the still
unidentified amino donor required for the formation of vanillylamine (Figure 2). The capsaicinoid
content in Capsicum fruits has been associated positively with available N in the soil [42,43].
Similarly, high nitrate nutrition enhanced capsaicin levels in placenta from Capsicum fruits
cultured in vitro [72] or harvested from hydroponic plants [73]. C. annuum fruits from plants
grown under different concentrations of K did not show alterations in capsaicinoid accumula-
tion [42]. By contrast, even though K does not participate in capsaicinoid metabolism, an
increase in K application resulted in a significant decrease of capsaicin levels and leaf N content
in C. chinense [43]; thus, the level of K levels might indirectly affect capsaicin accumulation
through its effects on fruit development [74].

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Environmental factors have complex and sometimes contrasting effects on pungency levels
(Figure 3). Genotypes, either species or cultivars, are highly variable in their response to specific
environmental conditions. Analysis of 49 accessions of C. chinense, collected in different
regions of Brazil, revealed wide variation in metabolic parameters [75]. Similar results were
found for 32 accessions of Capsicum with respect to productivity, fruit pigments and mor-
phology, capsaicinoids, capsinoids, and vitamins C and E [24]. The relative scarcity of data in
this area makes it difficult to draw definitive conclusions. Studies harnessing the abundant
natural variation in the genus, associated with high-throughput genotyping, may shed more
light on this interesting topic [76–78].

Activating Pungency Biosynthesis in Tomato Fruits: Towards ‘Hot’ Tomatoes

The Capsicum and tomato clades split at least 19 Mya ago [79]. The genomes of Capsicum and
tomato are well preserved, with a basic chromosome number of x = 12 in both species and
major conserved syntenic segments between them [80]. Capsicum fruits are generally
considered non-climacteric; however, the physiological and morphological events during
fruit-set are similar to those in tomato [81,82]. Yields of hot pepper seldom exceed 3 tons
per hectare on the field in about 4–5 months of culture [83]. For tomato, on the other hand, it is
not uncommon to reach 110 t/ha on the field over a 120 day cropping cycle [84]. In addition to
being one of the main horticultural crops in the world, tomato is a well-established model
species [85] that is highly amenable to biotechnological manipulation [86], and there are
miniature model cultivars, such as Micro-Tom, that can be grown quickly and in large numbers
in reduced space [87]. Thus, engineering tomato into a capsaicinoid biofactory is a concep-
tually promising approach for sustainable production of these secondary compounds [88,89].

Capsaicinoids consist of at least 23 different chemical species [90], of which capsaicin (trans-8-
methyl-N-vanillyl-6-nonenamide) and dihydrocapsaicin (8-methyl-N-vanillylnonanamide)
together account for 90% of the total fruit capsaicinoid content [56]. Capsaicinoids are

Water
High moisture Drought stress

Selecon pressure Capsaicin synthesis enzymes

Secondary metabolites

Irradiance
Temperature
CS expression
CS expression
CS enzyme acvity
CS expression
CS enzyme acvity
Secondary metabolites Capsaicinoid precursors
Fruit development
Wounding
Mineral nutrion ( N and K)

Figure 3. Some Factors Directly or Indirectly Influencing Pungency Levels in Capsicum. The expression of the
gene (CS) encoding capsaicinoid synthase is directly affected by irradiance, temperature, and wounding. Higher
temperatures and wounding also increase this enzyme activity. The supply of N directly influences the capsaicin pathway
because this element is directly related to the production of the precursor molecules of capsaicin. K influences devel-
opment of the fruit, and thus indirectly influences the size of the placenta by allowing the production of larger fruits. The
scheme does not apply to all varieties in all situations but presents the standard behavior in most cases.

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synthesized in the fruit placenta through the integration of two different biochemical pathways
that involve multiple intracellular compartments (Figure 2). Using phenylalanine as a substrate,
the phenylpropanoid pathway provides vanillylamine, whereas the branched-chain fatty acid
pathway produces 8-methyl nonenoic acid from valine. The enzyme CS, also known as PUN1,
combines both compounds to produce capsaicin and, by condensing vanillylamine with
different fatty acid substrates, other capsaicinoids [91,92]. The intracellular location and
molecular mechanism of this last step is not yet clear, although it appears to be located in
the cytosol. Tomato has an active phenylpropanoid pathway in the fruit peel that can be
upregulated by overexpression of specific transcription factors [93]. Although valine is found in
the tomato fruit, the intermediary compounds in the branched-chain fatty acid pathway have
not hitherto been described, either because of technical limitations or because their levels are
below the level of detection with currently available methods [94].

Comparison of the Capsicum genome to that of tomato has revealed the presence of all the
necessary genes for capsaicinoid production in the tomato genome [82,95]. Genes of the
capsaicinoid biosynthetic pathway have been analyzed in both species to better understand
the absence of pungency in tomato. In sweet peppers, loss of pungency can occur through a
variety of different mutations that affect gene expression in the pathway [96,97]. Pungency,
therefore, appears to be under transcriptional control, as borne out by the higher expression
levels of capsaicinoid biosynthesis genes in the placental septum of pungent cultivars [58,98].
Compared to chili peppers, in tomato some genes have lower levels of expression (PAL; C4H;
ACL, acyl carrier protein, and AMT, aminotransferase); some have lower expression levels with
temporally restricted expression (COMT, caffeoyl-CoA 3-O-methyltransferase; and FaTA, acyl-
ACP thioesterase), in addition to KAS; and two genes are not expressed at all (BCAT,
branched-chain amino acid aminotransferase, and CS) [95].

Two genome-engineering strategies could be used in tandem to activate capsaicinoid biosyn-

thesis in the tomato (Figure 4, Key Figure). One is the use of transcriptional activator-like
effectors (TALEs), a suite of proteins secreted by pathogenic Xanthomonas spp. bacteria when
they infect plant hosts [99]. Tools for robust transcriptional activation of endogenous genes
exploiting customized TALEs have recently been developed that allowed multiplex activation of
up to four genes in plants [100]. Rapid assembly of the TALE genes into a single T-DNA vector is
possible using a MultiSite Gateway recombination system [101,102] and would allow simul-
taneous upregulation of the expression of PAL, KAS, COMT, and FaTA. Interestingly, recent
work has shown that a CaMYB31 transcription factor of the R2R3–MYB family regulates
positively the transcription of multiple key capsaicinoid biosynthesis genes (C4H, COMT, Kas,
and AMT) [65]. It remains to be determined whether the transcript levels achieved will be
sufficient for the capsaicinoid pathway to be functional because expression could either be to
low, and thus insufficient to activate the capsaicinoid pathway, or too high, which could trigger
gene silencing [100].

The second strategy is the use of genome engineering for targeted replacement of promoters.
The feasibility of this method has been demonstrated in tomato using a constitutive 35S
promoter inserted upstream of the ANT1 gene, which encodes an R2R3–MYB transcription
factor regulating anthocyanin biosynthesis [103]. Promoter regions of the inactive genes in the
capsaicinoid pathway (BCAT, CS), could be replaced with endogenous tomato fruit-specific
promoters, such as E8 [104], to produce cisgenic plants with transcriptionally active genes
[105,106]. It remains to be determined, however, if the products of these genes are fully
functional, biochemically active, and catalyze the appropriate reactions.

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Key Figure
How To Make a Hot Tomato

Hot Sweet
Tomato
pepper pepper
PAL PAL PAL
BCAT BCAT BCAT
C4H C4H C4H Increased Promoter
transcripon replacement
KAS KAS KAS
ACL ACL ACL
Genome PAL
engineering BCAT
COMT COMT COMT KAS
FaTA FaTA FaTA CS
COMT
AMT AMT AMT
FaTA
CS CS CS

Zero Intermediate High

Hot tomato
Transcript level

Figure 4. Transcriptional profile of genes related to the metabolism of pungency in hot pepper, sweet pepper, and tomato
(adapted from [14]). The relative values are the highest measured over all the course of fruit development. Abbreviations:
PAL, phenylalanine ammonia-lyase; BCAT, branched-chain amino acid aminotransferase; C4H, cinnamate 4-hydroxylase;
COMT, caffeoyl-CoA 3-O-methyltransferase; AMT, aminotransferase; FatA, acyl-ACP thioesterase; CS, capsaicinoid
synthase; KAS, keto-acyl ACP synthase; ACL, acyl carrier protein. Pungency in tomato could be achieved increasing the
expression of PAL, KAS, COMT, and FaTA, and by activating the expression of BCAT and CS using state-of-the-art
genome engineering techniques.

Technical Challenges of Engineering Capsaicin Biosynthesis in Tomato

The most frequently described type of genome modification up to date is the creation of loss-of-
function alleles using TALENs or CRISPR/Cas9 [107]. Three recently published studies have
used the latter to perform genome editing of wild relatives of the tomato by multiplex editing of a
suite of genes to improve agronomic performance [108–110]. While this type of modification
occurs through the pathway of non-homologous end-joining for DNA repair in somatic cells,
more precise modifications such as base substitutions or targeted insertions require the use of
homology-directed repair. This involves further technical challenges because sequence-spe-
cific nucleases induce double-strand breaks in a specific sequence while a DNA donor template
is provided for homologous recombination repair to insert the desired alteration. Homology-
directed repair is not the preferred type of repair in plant cells, but efficient methods to create
knock-ins are still forthcoming, although successful integration of promoters [103] or larger
DNA sequences, including entire genes [111,112], has been reported.

The creation of novel ‘expression alleles’, namely gene variants with differential transcription
compared to the original allele, can be achieved by two alternative approaches. The first is the
use of TALE or CRISPR/Cas9 transcriptional activators, which have been successfully applied
in N. benthamiana, Arabidopsis thaliana, and tomato [100,113]. The second, described above,
is the targeted insertion of a promoter sequence upstream of the gene of interest [114]. This
requires a priori knowledge of the spatial and temporal expression profiles produced by specific

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 TRPLSC 1754 No. of Pages 12

Box 2. Production of Other Valuable Metabolites in Tomato Outstanding Questions

Successful implementation of the approach proposed here would represent an important proof-of-concept that could Given that other species of Solana-
be extended to the production of other valuable metabolites in tomato in the future. Other pathways that could ceae, such as tomato, harbor the bio-
potentially be manipulated using this approach include production of the food and cosmetic pigment bixin (annatto). synthetic pathway for capsaicinoids,
Bixin is the second most important natural color additive used in industry and it is produced by a single tropical plant what is the evolutionary explanation
species, Bixa orellana [118]. Three B. orellana genes encode the enzymes necessary to convert lycopene into bixin: for the restriction of pungency to spe-
carotenoid cleavage dioxygenase 1 (CCD1) or lycopene cleavage dioxygenase (LCD), bixin aldehyde dehydrogenase cies of the Capsicum genus?
(BADH), and norbixin methyltransferase (BMT). Because tomato has a high fruit lycopene content and also harbors
orthologs of those genes, their manipulation might allow bixin production in a species that is cultivated worldwide with
Could capsaicinoid biosynthesis be
improved agronomic practices. Alternatively, editing of the tomato lycopene b-cyclase gene (SlCyc-b) could improve
activated specifically in the tomato fruit
b-carotene (provitamin A) accumulation at expense of lycopene (and vice versa) [109]. b-Carotene is a powerful
using state-of-the-art genome-engi-
antioxidant that is readily available in large amounts in sweet potato (Ipomoea batatas), winter squash (Cucurbita pepo),
neering platforms?
and carrot (Daucus carota). However, 45% of the population carry a genetic mutation that reduces their ability to convert
b-carotene into the more active retinol (vitamin A) [119,120]. Tomatoes with massively increased levels of b-carotene
(with subsequent cleavage into vitamin A) would therefore represent a valuable addition to their diet. Would the novel genomically engi-
neered ‘hot tomatoes’ yield relevant
and stable amounts of capsaicinoids
under different environmental condi-
tions and cultural practices?
promoter sequences and also knowledge of the desired expression pattern. Some further
challenges to be addressed include the expression levels that can be achieved, in particular the
coordination of expression between genes, both at the spatial (tissue specificity) and temporal
(developmental stage) levels, as well as the subcellular localization of the resulting gene
products. These hurdles, however, are not insurmountable if a systems-biology approach
is employed, integrating knowledge from multiple research areas [115].

Concluding Remarks and Future Perspectives

Capsaicinoids are the secondary metabolites that are responsible for the appealing pungency
of hot peppers, a central element in the culinary activities of many countries. Their multiple
health benefits make capsaicinoids a valuable nutraceutical product, which is likely to
increase in demand in the coming years. Given the notorious challenges of producing stable
yields of Capsicum with consistent levels of pungency in the fruit, we are here proposing, and
working towards, an alternative approach by harnessing recent advances in genome engi-
neering. The completion of the Capsicum genome, and the discovery that the tomato
contains the full suite of genes necessary for pungency, paves the way for the production
of ‘hot tomatoes’ as capsaicinoid biofactories through targeted genome manipulation. The
convergence of basic knowledge with the rapidly growing repertoire of genome-manipulation
tools should pave the way for engineering this and other more imaginative biosynthetic
pathways (Box 2) using the tomato fruit as a chassis [88]. Our approach could provide not
only new basic knowledge to help to elucidate the capsaicinoid biosynthetic pathway but also
to understand its evolution and divergence in different Solanaceae species. Furthermore, it
could allow the development of new functional foods appealing to a wide range of consumers
(also see Outstanding Questions).

Acknowledgments
We gratefully acknowledge Eugenio Butelli (John Innes Centre, UK), the editor, and two anonymous reviewers for critical
reading and valuable suggestions on the manuscript. We thank Prof. Fábio DaMatta for valuable support on this project.
This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant 402511/
2016-6), the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG; grants APQ-01078-15 and CRA-
RED-00053-16) and the Max Planck Society. R.S. is recipient of a Visiting Professorship from the CNPq (grant
PVPE401090/2014-0), and the CNPq has granted research fellowships to A.N-N., L.E.P.P., and W.L.A., and a scholarship
to L.d.A.S. The Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) provided a scholarship to E.R.N.
We thank Acácio Rodrigues Salvador and Dr Jorge Condori Apfata for help with figures.

Trends in Plant Science, Month Year, Vol. xx, No. yy 9

 TRPLSC 1754 No. of Pages 12
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Prisoner at the bar—you are trifling with the court.... You have not
answered the charge.
Have I not!—well then—I prepare to answer it now. I swear that I
loved them that I have buried there—there!—loved them with a love
passing all that I ever heard of, or read of. I swear too that I
nourished and comforted and ministered to the dear creatures, who,
ye are told, have come out of the earth to destroy me—even me—
me, their husband, their lover, and the father of their children! I swear
too—but why continue the terrible outrage? Let my accusers appear!
Let them walk up, if they will, out of their graves!—their graves are
before me. I am not afraid—I shall not be afraid—so long as they
wear the blessed shape, or the blessed features of them that have
disappeared from their bridal chamber, with a——
He was interrupted by great noises and shrieks that were enough to
raise the dead—noises from every part of the grave-yard—shrieks
from people afar off in the wood, shrieks from the multitude on the
outside of the house—and shrieks from the sea-shore; and
immediately certain of the accusers fell down as if they saw
something approach; and several that were on the outside of the
meeting-house came rushing in with a fearful outcry, saying that a
shed which had been built up over a part of the burial-ground was
crowded with strange faces, and with awful shapes, and that among
them were the two dead wives of the prisoner.
There they go—there they go! screamed other voices outside the
door; and immediately the cry was repeated by the accusers who
were within the house—all shrieking together. “Here they come!—
here they come!—here they come!”—And Judith Hubbard looking up
and uncovering her face, about which her cloak had been gathered
in the first hurry of her distraction, declared that the last wife of
Burroughs, on whom her eyes were fixed at the time, was then
actually standing before him and looking him in the face, “O, with
such a look—so calm, so piteous and so terrible!”
After the uproar had abated in some degree, the judges who were
huddled together, as far as they could possibly get from the crowd
below, ordered up three more of the witnesses, and were about to
speak to them, when Burroughs happening to turn that way also,
they cried out as if they were stabbed with a knife, and fell upon the
floor at their whole length and were speechless.
Whereupon the chief judge, turning toward him, asked him what
hindered these poor people from giving their testimony.
I do not know said Burroughs, who began to give way himself now,
with a convulsion of the heart, before the tremendous array of
testimony and weight of delusion; to fear that of a truth preternatural
shapes were about him, and that the witnesses were over-
persuaded by irresistible power, though he knew himself to be no
party in the exercise of such power. I do not know, said he: I am
utterly confounded by their behaviour. It may be the devil.
Ah—and why is the devil so loath to have testimony borne against
you?
“Which query,” says a writer who was there at the time, and saw the
look of triumph which appeared in the faces of the whole bench, “did
cast Burroughs into very great confusion.”
And well it might, for he was weighed to the earth, and he knew that
whatever he said, and whatever he did; and whether he spoke with
promptitude or with hesitation; whether he showed or did not show a
sign of dismay, everything would be, and was regarded by the
judges, and the jury, and the people, as further corroboration of his
turpitude.
Here the trial ended. Here the minds of the jury were made up; and
although he grew collected at last, and arose and spoke in a way
that made everybody about him weep and very bitterly too, for what
they called the overthrow of a mind of great wisdom and beauty and
power; and although he gave up to the judges a written argument of
amazing ingenuity and vigor which is yet preserved in the records of
that people, wherein he mocked at their faith in witchcraft, and
foretold the grief and the shame, the trouble and the reproach that
were to follow to them that were so busy in the work of death; yet—
yet—so impressed were the twelve, by the scene that had occurred
before their faces, that they found him guilty; and as if the judges
were afraid of a rescue from the powers of the air, they gave
judgment of death upon him before they left the bench, and contrary
to their established practice, ordered him to be executed on the
morrow.
On the morrow? said he, with a firm steady eye and a clear tone,
though his lip quivered as he spoke. Will ye afford me no time to
prepare?
We would not that the body and soul both perish; and we therefore
urge you to be diligent in the work brother, very diligent for the little
time that is now left to make your calling and election sure. Be ready
for the afternoon of the morrow.
Hitherto the prisoner at the bar had shown little or no emotion;
hitherto he had argued and looked as if he did not believe the jury
nor the judges capable of doing what they had now done, nor the
multitude that knew him, capable of enduring it. Hitherto he had been
as it were a spectator of the terrible farce, with no concern for the
issue; but now ... now ... all eyes were rivetted upon him with fear, all
thoughts with alarm; for though he stood up as before, and made no
sort of reply to the judges, and bore the wracking of the heavy irons
with which they were preparing to load him, as if he neither felt nor
saw them; yet was there a something in his look which made the
officers of the court unsheathe their swords, and lift up their axes,
and the people who were occupied about him, keep as far out of his
reach as they possibly could.
Yet he neither moved nor spoke, till he saw the women crowding up
to a part of the house where he had seen Elizabeth Dyer, and stoop
as if she that had been kneeling there a few moments before, lay
very low, and lift her up as if she had no life in her, and carry her
away, guarded by men with pikes, and with swords and with huge
firelocks. Then he was moved—and his chains were felt for the first
time, and he would have called out for a breath of air—prayed for a
drop of water to save a life more precious by far than his—but before
he could open his mouth so as to make himself heard, he saw
Rachel Dyer pressing up to the bar of death, and heard the judges
call out to the high-sheriff and his man to guard the door, and look to
the prisoner.
He will get away if you turn your head, Mr. sheriff, said one of the
judges.
That he will, added a witness, that he will! if you don’t look sharp, as
sure as my name is Peter P.
Watch and pray—watch and pray—added another.
Burroughs looked up to the bench with surprise, then at the people,
who were watching every motion of his body as if they expected him
to tear away the ponderous fetters and walk forth as free as the wind
of the desert, and then at the blacksmith who stood near with his
hammer uplifted in the air; and then his chest heaved and his chains
shook, and the people hurried away from his path, and tumbled over
each other in their eagerness to escape, and the chief-judge cried
out again to the officer to look to the door and be prepared for a
rescue.
Let me be tried now! I entreat thee, said Rachel Dyer, throwing up
her locked-hands before Judge Winthrop, and speaking as if she
was about to plead not for death but for life. Let me be tried now, I
beseech thee.
Now.—
Yea—now!—before the maiden is brought back to life. O let her be at
peace, ye men of power, till I have a—have a—
She gathered herself up now with a strong effort, and spoke with
deliberate firmness....
——Till I have gone through the work which is appointed for me by
the twelve that I see there—
Be it so.—I say, Mr. high-sheriff!
Well, Mr. judge Winthrop?
This way, this way; you’ll be so good as to remove a—a—a—
(Looking at Burroughs who stood leaning against the wall)—you are
to be a—a—(in a whisper of authority)—you are to be careful of what
you do—a very hard case, very—very—
Yes judge—
Well, well—well, well—why don’t ye do as I bid you?
What am I to do?
What are you to do ... remove the prisoner—poor soul.
Which prisoner?
Why that are ... poh poh, poh—(pointing to Burroughs.)
Where to?
Where to Sir?—Take him away; away with him—pretty chap you are
to be sure, not to know where to take a man to, after its all over with
him—poh, poh, poh.
I say, Mr. Judge, none o’ that now—
Take the man away Sir. Do as you are bid.
Who—me—cried Burroughs, waking up from his fit of apathy and
looking about on every side.
Away with him.
Judges—judges—hear me. Let me remain, I pray you, cried he,
setting his back to the wall and lifting his loaded arms high up in the
air—suffer me to stay here till the jury have said whether or no this
heroic woman is worthy of death—I do beseech you!
Take him away, I tell you—what are ye afraid of?
Judges—men—I would that ye would have mercy, not on me, but on
the people about me. I would that ye would suffer me to tarry here—
in fetters—till the jury have given their verdict on Rachel Dyer. Suffer
me to do so, I beseech you, and I will go away then, I swear to you,
whithersoever it may please you, like a lamb to the slaughter. I swear
this to you before God!—but, so help me God, I will not be carried
away alive before. I will not stir, nor be stirred while I have power to
lift my arms, or to do what you now see me do——
As he spoke, he lifted up his arms in the air—up—up, as high as he
could reach, standing on tip-toe the while; and brought them down
with such force, loaded as they were, that he doubled the iron guard
which kept him in the box, and shattered the heavy door from the top
to the bottom.
—Behold—shorn though I be of my youth, betrayed though I have
been, while I forgot where I was, I do not lack power. Now bid your
people tear me away if you have courage! For lo, my feet are upon
the foundations of your strength ... and by Jehovah—the God of the
strong man of other days!—I’ll not be dragged off till I know the fate
of the giantess before you.
We shall see—cut him down officer—cut him down!
Very well. Come thou near enough to cut me down, officer, and I’ll
undertake for thee.
Judges—how little ye know of that man’s power—why not suffer him
to stay? cried Rachel Dyer. Why will ye provoke it? On your heads
be the issue, if ye drive your ministers to the toil! on yours their
blood, if they approach him!
The sheriff hung back—and the judges, after consulting together,
told Burroughs he might stay, and ordered the trial of the women to
proceed.
 CHAPTER XX.
Already were they about to give judgment of death upon Rachel
Dyer, when two or three of her accusers, who began to fear that she
might escape, had another fit.
Why are these poor women troubled? asked a judge.
I do not know, was her unstudied reply.
But according to your belief?
I do not desire to say what my belief is. It can do no good, and it may
do harm; for who shall assure me that I do not err?
Don’t you think they are bewitched?
No.
Give us your thoughts on their behavior.
No, Ichabod, no; my thoughts can be of no worth to thee or such as
thee. If I had more proof, proof that ye would receive in law, I might
be willing to speak at large both of them and of their master——
Their master! cried a little man, with a sharp inquisitive eye, who had
not opened his mouth before. Who is their master?
If they deal in witchcraft or in the black art, Joseph Piper, thee may
know as well as I do.
Woman....are you not afraid of death?
No.........not much, though I should like to be spared for a few days
longer.
Not afraid of death!—
No—not much, I say. And why should I be afraid of death? why
should I desire to live?—what is there to attach a thing of my shape
to life, a wretched, miserable, weary....
Ah, ha—now we shall have it—she is going to confess now—she is
beginning to weep, said a judge. But he was overheard by the
woman herself, who turning to the jury with a look that awed them in
spite of their prejudice, told them to proceed.
They’ll proceed fast enough, by and by, said another judge. What
have you done to disturb the faculties of that woman there?
What woman?
Judith Hubbard.
Much. For I know her, and she knows that I know her; and we have
both known for a great while that we cannot both live. This world is
not large enough. What have I done to disturb her faculties? Much.
For that woman hath wronged me; and she cannot forgive me. She
hath pursued me and mine to death; all that are very near and dear
to me, my poor sister and my—and the beloved friend of my sister—
to death; and how would it be possible for Judith Hubbard to forgive
us?
But your apparition pursues her.
If so, I cannot help it.
But why is it your apparition?
How do I know? He that appeared in the shape of Samuel, why
should he not appear in the shape of another?
But enough—Rachel Dyer was ordered for execution also. And a
part of the charge proved against her was, that she had been spirited
away by the powers of the air, who communicated with her and
guarded her at the cost of much human life, on the night when she
fled into the deep of the wilderness in company with George
Burroughs and Mary Elizabeth Dyer; each of whom had a like body-
guard of invisible creatures, who shot with arrows of certain death on
the night of their escape.
And Mary Elizabeth Dyer was now brought up for trial; but being half
dead with fear, and very ill, so that she was reported by a jury
empannelled for the purpose, to be mute by the visitation of God,
they adjourned the court for the morrow, and gave her permission to
abide with her sister till the day after the morrow.
And so Mary Elizabeth Dyer and Rachel Dyer met again—met in the
depth of a dungeon like the grave; and Elizabeth being near the
brave Rachel once more, grew ashamed of her past weakness.
I pray thee dear sister, said Mary Elizabeth, after they had been
together for a long while without speaking a word, Rachel with her
arm about Elizabeth’s neck, and Elizabeth leaning her face upon the
shoulder of Rachel, I pray thee to forgive me.
Forgive thee ... for what pray?
Do, do forgive me, Rachel.
Why, what on earth is the matter with thee, child? Here we sit for a
whole hour in the deep darkness of the night-season, without so
much as a sob or a tear, looking death in the face with a steady
smile, and comforting our hearts, weary and sick as they are, with a
pleasant hope—the hope of seeing our beloved brother Jacob, our
dear good mother, and our pious grandmother; and now, all of a
sudden thee breaks out in this way, as if thee would not be
comforted, and as if thee had never thought of death before—
O, I’m not afraid of death sister, now—I’m prepared for death now—
I’m very willingly to die now—it isn’t that I mean.
Why now?—why do thee say so much of now? Is it only now that
thee is prepared for death?
No, no, dear sister, but some how or other I do not even desire to
live now, and yet——
And yet what?—why does thee turn away thy head? why does thee
behave so to me ... why break out into such bitter—bitter
lamentation?—what is the matter I say?—what ails thee?—
Oh dear!
Why—Elizabeth!—what am I to believe?—what has thee been doin’?
Why do thee cling to me so?—why do thee hide thy face?—
O Rachel, Rachel—do not go away,—do not abandon me—do not
cast me off!
Child—why—
No, no!—
Look me in the face, I beseech thee.
No no—I dare not—I cannot.
Dare not—cannot—
No no.—
Dare not look thy sister in the face?
Oh no—
Lift up thy head this minute, Mary Elizabeth Dyer!—let go of my neck
—let go of my neck, I say—leave clinging to me so, and let me see
thy face.
No no dear Rachel, no no, I dare not—I am afraid of thee now, for
now thee calls me Mary Elizabeth—
Afraid of me—of me—O Elizabeth, what has thee done?
Oh dear!
And what have I done to deserve this?
Thee—thee!—O nothing dear sister, nothing at all; it is I—I that have
been so very foolish and wicked after—
Wicked, say thee?
O very—very—very wicked—
But how—in what way—thee’ll frighten me to death.
Shall I—O I am very sorry—but—but—thee knows I cannot help it—
Cannot help it, Mary Elizabeth Dyer—cannot help what? Speak ...
speak ... whatever it is, I forgive thee ... we have no time to lose now;
we may never meet again. Speak out, I beseech thee. Speak out, for
the day is near, the day of sorrow——
I will, I will—cried Elizabeth, sobbing as if her heart would break, and
falling upon her knees and burying her head in the lap of her sister—
I will—I will, but—pushing aside a heap of hair from her face, and
smothering the low sweet whisper of a pure heart, as if she knew
that every throb had a voice—I will, I will, I say, but I am so afraid of
thee—putting both arms about her sister’s neck and pulling her face
down that she might whisper what she had to say—I will—I will—I’m
a goin’ to tell thee now—as soon as ever I can get my breath—nay,
nay, don’t look at me so—I cannot bear it——
Look at thee—my poor bewildered sister—how can thee tell whether
I am looking at thee or not, while thy head is there?—Get up—get
up, I say—I do not like that posture; it betokens too much fear—the
fear not of death, but of shame—too much humility, too much
lowliness, a lowliness the cause whereof I tremble to ask thee. Get
up, Elizabeth, get up, if thee do not mean to raise a grief and a
trouble in my heart which I wouldn’t have there now for the whole
world; get up, I beseech thee, Mary Elizabeth Dyer.
Elizabeth got up, and after standing for a moment or two, without
being able to utter a word, though her lips moved, fell once more
upon her sister’s neck; and laying her mouth close to her ear, while
her innocent face glowed with shame and her whole body shook with
fear, whispered—I pray thee Rachel, dear Rachel ... do ... do let me
see him for a minute or two before they put him to death.
Rachel Dyer made no reply. She could not speak—she had no voice
for speech, but gathering up the sweet girl into her bosom with a
convulsive sob, she wept for a long while upon her neck.
They were interrupted by the jailor, who came to say that George
Burroughs, the wizard, having desired much to see Rachel Dyer and
Mary Elizabeth Dyer, the confederate witches, before his and their
death, he had been permitted by the honorable and merciful judges
to do so—on condition that he should be doubly-ironed at the wrist;
wherefore he, the jailor had now come to fetch her the said Rachel to
him the said George.
I am to go too, said Elizabeth, pressing up to the side of her sister,
and clinging to her with a look of dismay.
No, no—said he, no, no, you are to stay here.
Nay, nay, sister—dear sister—do let me go with thee!
It is not for me to speak, dear, dear Elizabeth, or thee should go now
instead of me. However——
Come, come—I pity you both, but there’s no help for you now—
never cry for spilt milk—you’re not so bad as they say, I’m sure—so
make yourself easy and stay where you be, if you know when you’re
well off.
Do let me go!
Nonsense—you’re but a child however, and so I forgive you, and the
more’s the pity; must obey orders if we break owners—poh, poh,—
poh, poh, poh.
A separation like that of death followed. No hope had the two sisters
of meeting again alive. They were afraid each for the other—and
Elizabeth sat unable to speak, with her large clear eyes turned up to
the eyes of Rachel as if to implore, with a last look, a devout
consideration of a dying prayer.
If it may be, said Rachel turning her head at the door if it may be
dear maiden, it shall be. Have courage—
I have, I have!
Be prepared though; be prepared Elizabeth, my beautiful sister. We
shall not see each other again ... that is.... O I pray thee, I do pray
thee, my dear sister to be comforted.
Elizabeth got up, and staggered away to the door and fell upon her
sister’s neck and prayed her not to leave her.
I must leave thee ... I must, I must ... would thee have me forsake
George Burroughs at the point of death?
O no—no—no!
We never shall meet again I do fear—I do hope, I might say, for of
what avail is it in the extremity of our sorrow; but others may—he
and thee may Elizabeth—and who knows but after the first shock of
this thy approaching bereavement is over, thee may come to regard
this very trial with joy, though we are torn by it, as by the agony of
death now—let us pray.
The sisters now prayed together for a little time, each with her arm
about the other’s neck, interchanged a farewell kiss and parted—-
parted forever.
And Rachel was then led to the dungeons below, where she saw him
that her sister loved, and that a score of other women had loved as it
were in spite of their very natures—for they were bred up in fear of
the dark Savage. He sat with his hands locked in his lap, and
chained and rivetted with iron, his brow gathered, his teeth set, and
his keen eyes fixed upon the door.
There is yet one hope my dear friend, whispered he after they had
been together a good while without speaking a word or daring to look
at each other—one hope—laying his pinioned arm lightly upon her
shoulder, and pressing up to her side with all the affectionate
seriousness of a brother—one hope, dear Rachel—
She shut her eyes and large drops ran down her cheeks.
—One hope—and but one—
Have a care George Burroughs. I would not have thee betray thyself
anew—there is no hope.
It is not for myself I speak. There is no hope for me. I know that—I
feel that—I am sure of it; nor, to tell you the truth, am I sorry—
Not sorry George—
No—for even as you are, so am I—weary of this world—sick and
weary of life.
Her head sunk upon his shoulder, and her breathing was that of one
who struggles with deep emotion.
No—no—it is not for myself that I speak. It is for you—
For me—
For you and for Elizabeth—
For me and for Elizabeth?—well—
And if I could bring you to do what I am persuaded you both may do
without reproach, there would be hope still for—for Elizabeth—and
for you—
For Elizabeth—and for me?—O George, George! what hope is there
now for me? What have I to do on earth, now that we are a——she
stopped with a shudder—I too am tired of life. She withdrew the
hand which till now he had been holding to his heart with a strong
terrible pressure.
Hear me, thou high-hearted, glorious woman. I have little or no hope
for thee—I confess that. I know thee too well to suppose that I could
prevail with thee; but ... but ... whatever may become of us, why not
save Elizabeth, if we may—
If we may George—but how?
Why ... draw nearer to me I pray thee; we have not much time to be
together now, and I would have thee look upon me, as one having a
right to comfort thee and to be comforted by thee—
A right ... how George?
As thy brother—
As my brother.... O, certainly——
Nay, nay ... do not forbear to lean thy head upon thy brother ... do
not, I beseech thee. What have we to do here ... what have we to do
now with that reserve which keeps the living apart ... our ashes, are
they to be hindered of communion hereafter by the unworthy law of
—ah ... sobbing ... Rachel Dyer! ... can it be that I hear you—you!
the unperturbable, the steadfast and the brave ... can it be that I hear
you sobbing at my side, as if your very heart would break....
No no....
There is to be a great change here, after we are out of the way....
Where—how?
Among the people. The accusers are going too far; they are
beginning to overstep the mark—they are flying too high.
Speak plainly, if thee would have me understand thy speech—why
do thee cleave to me so?—why so eager—why do thee speak in
parables? My heart misgives me when I hear a man like thee, at an
hour like this, weighing every word that is about to escape from his
mouth.
I deserve the rebuke. What I would say is, that the prisons of our
land are over-crowded with people of high repute. Already they have
begun to whisper the charge against our chief men. This very day
they have hinted at two or three individuals, who, a week before they
overthrew me, would have been thought altogether beyond the reach
of their audacity.
Who are they?
They speak of Matthew Paris.
The poor bewildered man ... how dare they?
And of the Governor, and of two or three more in authority; and of all
that participated in the voyage whereby he and they were made
wealthy and wise and powerful——
I thought so ... I feared as much. Poor man ... his riches are now
indeed a snare to him, his liberal heart, a mark for the arrows of
death....
Now hear me ... the accusers being about to go up to the high places
and to the seats of power, a change, there must and there will be,
and so——
And so ... why do thee stop?
Why do I stop ... did I stop?
Yea ... and thy visage too ... why does it alter?
My visage!
Yea ... thy look, thy tone of voice, the very color of thy lips.
Of a truth, Rachel?
Of a truth, George.
Why then it must be ... it is, I am sure ... on account of the ... that is
to say ... I’m afraid I do not make myself understood—
Speak out.
Well then ... may I not persuade you, my dear, dear sister ... to ... to
... in a word, Rachel....
To what pray ... persuade me to what?... Speak to me as I speak to
thee; what would thee persuade me to, George?
To ... to ... to confess ... there!
To confess what, pray?
That’s all....
George....
Nay, nay ... the fact is my dear friend, as I said before: I ... I ... if
there be a change here, it will be a speedy one.
Well—
And if—and if—a few weeks more, a few days more, it may be, and
our accusers, they who are now dealing death to us, may be brought
up in their turn to hear the words of death—in short Rachel, if you
could be persuaded just to—not to acknowledge—but just to suffer
them to believe you to be a ... to be a ... I forget what I was going to
say——
 CHAPTER XXI.
A long silence followed—a silence like that of death—at last Rachel
Dyer spoke:
George Burroughs—I understand thee now, said she, I understand it
all. Thee would have me confess that I deserve death—only that I
may live. Thee would have me acknowledge (for nothing else would
do) that I am a liar and a witch, and that I deserve to die—and all this
for what?—only that I may escape death for a few days. O George!
No, no—you mistake the matter. I would not have you confess that
you deserve death—I would only have you speak to them—God of
the faithful!—I cannot—I cannot urge this woman to betray her faith.
I understand thee, George. But if I were to do so, what should I gain
by it?
Gain by it, Rachel Dyer?
Why do thee drop my hand? why recoil at my touch now?
Gain by it! siezing both her hands with all his might, and speaking as
if he began to fear—not to hope—no, but to fear that she might be
over-persuaded—
Yea—what have I to gain by it?
Life. You escape death—a cruel ignominious death—a death, which
it is not for a woman to look at, but with horror.
Well George—
By death, you lose the opportunity of doing much good, of bringing
the wicked to justice, of aiding them that are now ready to die with
terror, of shielding the oppressed—
Well—
Well—and what more would you have? Is not this enough?
No, George.
Hear me out Rachel. Do not reprove me, do not turn away, till you
have heard me through. My duty is before me, a duty which must
and shall be done, though it break my heart. I am commanded to
argue with you, and to persuade you to live.
Commanded?—
What if you were to confess that you deserve death? What if you
were to own yourself a witch? I take your own view of the case.—I
put the query to you in a shape the least favorable to my purpose.
What if you were to do this; you would be guilty at the most but of a
—of a—
Of untruth George.
And you would save your own life by such untruth, and the lives, it
may be, of a multitude more, and the life you know, of one that is
very dear to you.
Well——
No no—do not leave me in this way! Do not go till I—I beseech you
to hear me through—
I will—it grieves me, but I will.
Which is the greater sin—to die when you have it in your power to
escape death, if you will, by a word? or to speak a word of untruth to
save your life—
George Burroughs—I pray thee—suffer me to bid thee farewell.
No no, not yet. Hear me through—hear all I have to say. By this word
of untruth, you save your own life, and perhaps many other lives.
You punish the guilty. You have leisure to repent in this world of that
very untruth—if such untruth be sinful. You have an opportunity of
showing to the world and to them that you love, that you were
innocent of that wherewith you were charged. You may root up the
error that prevails now, and overthrow the destroyer, and hereafter
obtain praise for that very untruth, whereby you hinder the shedding
of more innocent blood; praise from every quarter of the earth, praise
from every body; from the people, the preachers, the jury, the elders
—yea from the very judges for having stayed them in their headlong
career of guilt—
O George—
But if you die, and your death be sinful—and would it not be so, if
you were to die, where you might escape death?—you would have
no time to repent here, no opportunity, no leisure—you die in the
very perpetration of your guilt—
If it is guilt, I do—
And however innocent you may be of the crimes that are charged to
you, you have no opportunity of showing on this earth to them that
love you, that you are so. Yet more—the guilt of your death, if it be
not charged hereafter to you, will be charged, you may be sure, to
the wretched women that pursue you; and all who might be saved by
you, will have reason to lay their death at your door—
Well—
About life or death, you may not much care; but after death to be
regarded with scorn, or hatred or terror, by all that go by your grave,
my sister—how could you bear the idea of that? What say you—you
shudder—and yet if you die now, you must leave behind you a
character which cannot be cleared up, or which is not likely to be
cleared up on earth, however innocent you may be (as I have said
before)—the character of one, who being charged with witchcraft
was convicted of witchcraft and executed for a witchcraft. In a word
—if you live, you may live to wipe away the aspersion. If you die, it
may adhere to you and to yours—forever and ever. If you live, you
may do much good on earth, much to yourself and much to others,
much even to the few that are now thirsting for your life—you may
make lighter the load of crime which otherwise will weigh them down
—you may do this and all this, if you speak: But if you do not speak,
you are guilty of your own death, and of the deaths it may be of a
multitude, here and hereafter.
Now hear me. I do not know whether all this is done to try my truth or
my courage, but this I know—I will not leave thee in doubt
concerning either. Look at me
There——
Thee would have me confess?
I would.
Thee would have Elizabeth confess?
I would.
Do thee mean to confess.
I—I!—
Ah George—
I cannot Rachel—I dare not—I am a preacher of the word of truth.
But you may—what is there to hinder you?
Thee will not?
No.
Nor will I.
Just what I expected—give me your hand—what I have said to you, I
have been constrained to say, for it is a part of my faith Rachel, that
as we believe, so are we to be judged: and that therefore, had you
believed it to be right for you to confess and live, it would have been
right, before the Lord.—But whether you do or do not, Elizabeth may.
True—if she can be persuaded to think as thee would have her think,
she may. I shall not seek to dissuade her—but as for me, I have put
my life into the hands of our Father. I shall obey him, and trust to the
inward prompting of that which upholds me and cheers me now—
even now, George, when, but for His Holy Spirit, I should feel as I
never felt before, since I came into the world—altogether alone.
Will you advise with her, and seek to persuade her?
No.
Cruel woman!
Cruel—no no George, no no. Would that be doing as I would be
done by? Is it for me to urge a beloved sister to do what I would not
do—even to save my life?