Exogenous Influences on Plant Secondary Metabolite Levels

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Exogenous inﬂuences on plant secondary metabolite levels

a b a

Daniel Petinatti Pavarini , Saulo Petinatti Pavarini , Michael Niehues ,

a,∗

Norberto Peporine Lopes

Núcleo de Pesquisas em Produtos Naturais e Sintéticos, Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto,

Universidade de São Paulo, ZIP code 14040-903, Ribeirão Preto, SP, Brazil

Faculdade de Veterinária, Departamento de Patologia e Clínica Veterinária Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, ZIP code

91540-000, Porto Alegre, RS, Brazil

a r t i c l e i n f o a b s t r a c t

Plant secondary metabolites are a group of naturally occurring compound classes biosyn-

thesized by differing biochemical pathways whose plant content and regulation is strongly

susceptible to environmental inﬂuences and to potential herbal predators. Such abiotic and

biotic factors might be speciﬁcally induced by means of various mechanisms, which create

Keywords:

variation in the accumulation or biogenesis of secondary metabolites. Hence the dynamic

Natural products

aspect of bioactive compound synthesis and accumulation enables plants to communicate

Plant secondary metabolites

and react in order to overcome imminent threats. This contribution aims to review the

Variation levels

most important mechanisms of various abiotic and biotic interactions, such as pathogenic

Biotic and abiotic factors

Livestock microorganisms and herbivory, by which plants respond to exogenous inﬂuences, and will

also report on time-scale variable inﬂuences on secondary metabolite proﬁles. Transmis-

sion of signals in plants commonly occurs by ‘semiochemicals’, which are comprised of

terpenes, phenylpropanoids, benzenoids and other volatile compounds. Due to the impor-

tant functions of volatile terpenes in communication processes of living organisms, as well

as its emission susceptibility relative to exogenous inﬂuences, we also present different

scenarios of concentration and emission variations. Toxic effects of plants vary depending

on the level and type of secondary metabolites. In farming and cattle raising scenarios, the

toxicity of plant secondary metabolites and respective concentration shifts may have severe

consequences on livestock production and health, culminating in adverse effects on crop

yields and/or their human consumers, or have an adverse economic impact. From a wider

perspective, herbal medicines, agrochemicals or other natural products are also associated

with variability in plant metabolite levels, which can impact the safety and reliable efﬁ-

cacy of these products. We also present typical examples of toxic plants which inﬂuence

livestock production using Brazilian examples of toxicity of sapogenins and alkaloids on

1. Introduction

Plant secondary metabolism has been investigated for decades as a dynamic phenomenon (Schultz, 2002) with ﬁxed and

predictable results. What guided us to such a paradigm? Possibly it was cases of plants in which compounds of pleasant

ଝ

This paper is part of the special issue entitled: Plant Bioactive Compounds in Ruminant Agriculture – Impacts and Opportunities, Guest Edited by A.Z.M.

Salem and S. López, and Editor for Animal Feed Science and Technology, P.H. Robinson.

∗

Corresponding author. Tel.: +55 16 36024707; fax: +55 16 36024243.

E-mail address: [email protected] (N.P. Lopes).

6 D.P. Pavarini et al. / Animal Feed Science and Technology 176 (2012) 5–16

Fig. 1. Chemical structures of bioactive compounds from the plant secondary metabolism. Taxol structure has its terpenic carbon skeleton moiety

highlighted in bold.

odors, and therefore easily noticeable, display an accumulation of major compounds, such as eugenol (Fig. 1) in cloves

(Syzygium aromaticum) or nutmeg (Myristica fragrans) and 1,8-cineole (Fig. 1) in Eucalyptus spp. However in recent decades

of plant metabolism research, the boundaries of science were pushed toward consideration of the chemical (Reddy and

Guerrero, 2004), ecological (Baldwin et al., 2006; Baldwin and Schultz, 1983) and evolutionary (Conrath et al., 2006) roles of

secondary metabolites. Evolved mechanisms of plant defense such as tri-trophic herbivory protection (Degenhardt, 2008)

by release of volatile compounds, and also the jasmonate induced elicitation of cellular responses against predators (Liechti

and Farmer, 2002) were reported and are now under further investigation. Considered in a larger perspective, these roles

are of tremendous importance for safe and reliable use of plants, be it for drug discovery and agrochemical research and

development or in animal feed science and technology. Recent reviews dealing with variations in secondary metabolites in

medicinal plants showed the inﬂuences of abiotic factors and reinforced the importance of chemical control for safe use of

such plants in phytomedicine (Gobbo-Neto and Lopes, 2007; Ramakrishna and Ravishankar, 2011; Gouvea et al., 2012).

Considering all the evidence raised by the joint efforts of phytochemistry and plant biology it can be assumed that

secondary metabolites are a way which plants communicate or respond to external stimuli (Bouwmeester et al., 2007;

Maffei, 2010; Rasmann and Turlings, 2008; Frost et al., 2008). Such bioactive compounds accumulate in plant tissues through

biochemistry mechanisms encrypted by other mechanisms of regulation. More speciﬁcally, we might conclude that each

plant species has evolved up to a singular set of mechanisms for the biosynthetic regulation of secondary metabolites, from

prior generations, which successfully expressed these strategies.

This review therefore aims to review key factors inﬂuencing biosynthesis and accumulation of secondary metabolites

in plant tissues. By reviewing a number of reports on common biotic and abiotic factors which inﬂuence plant secondary

compounds, we report on the prevailing views of regulatory mechanisms of plant secondary metabolism. Despite the inte-

grative aspects of plant physiology and cell biochemistry, we categorize these traits in order to describe effects on each more

D.P. Pavarini et al. / Animal Feed Science and Technology 176 (2012) 5–16 7

comprehensively. A brief discussion of main cases of plant toxicity effects occurring with livestock in Brazil, as well as their

chemical proﬁles and variability, is presented.

1.1. Bioactive compounds: general aspects

The bioactivity of secondary metabolites has been investigated since ancient Greece and Dioscorides’ De Materia Medica

(Dioscorides, 2000) in vivo experimentation with plant and poison types, until today as demonstrated by the exploitation by

the pharmaceutical industry of the prominent natural anticancer drug Taxol by (Fig. 1; Clardy and Walsh, 2004). However,

despite progress achieved from biodiversity conservation (Mendehall, 2011; Gibson et al., 2011; Bayon and Jenkins, 2010),

habitats hosting plants which accumulate bioactive secondary metabolites still remain poorly understood and so endanger

the survival of such species (Aguilar-Støen and Moe, 2007).

A large number of plant secondary metabolites studies demonstrate or search for bioactive and protective compounds,

such as elicitors and phytoalexins. An example of a prominent compound in this research area is capsidiol (Fig. 1), which

is biosynthesized in tobacco leaves from its terpenic precursor, aristolochene (Fig. 1; Takahashi et al., 2005). Mediated by

the activity of various isoforms of terpene synthase, the prenyl phosphate units are cyclized to create the aristolochene

carbon skeleton type in Nicotiana tabacum (Maldonado-Bonilla et al., 2008). This bicyclic unit of alkene is considered the

most important biosynthetic step in biogenesis of capsidiol. This biosynthetic pathway, speciﬁcally the terpene synthase

transcripts level, has been shown to be inﬂuenced by Manduca sexta (Sphingidae) infestation of shoots in Nicotiana attenuata

leaves (Bohlmann et al., 2002).

This type of direct contact between plants and others species which induces changes in secondary metabolite biosynthesis

may be considered to be a biotic effect on biosynthesis and accumulation of compounds. In cases of environmental ﬁtness due

to this responsiveness, initial plant populations can represent, in an evolutionary time scale, the starting point of successful

acquisition as a primary defense mechanism (Wink, 2003).

Jasmonates as the jasmonic acid or methyl jasmonate (Fig. 1), plus a huge diversity of derivatives once determined as

phytoalexins, are now considered as the low-weight molecules responsible for the gene response induced by an herbivore

injury of leaf tissue (Avanci et al., 2010; Fonseca et al., 2009; Wasternack and Parthier, 1997).

A new class of Phytohormones, the strigolactones, was once classiﬁed as secondary metabolites. Strigolactones (Fig. 1)

are biosynthesized by the carotenoid biosynthesis pathway and can suppress lateral shoot branching. Orobanchyl acetate

(Gomez-Roldan et al., 2008), for example, was shown to regulate root development (Koltai, 2011) and promote seed germi-

nation (Kohlen et al., 2011).

Homalanthus nutans (Euphorbiaceae) is commonly used by Samoan healers for hepatitis treatment and contains the diter-

pene prostratin (Fig. 1), reported to have anti-HIV potential, is a compound with intra-plant quantitative variability (Johnson

et al., 2008) which may be related to ontogenesis under- and/or over-expression of diterpene synthase, so exemplifying a

biotic effect on variability of bioactive compound biogenesis.

2. Effects governing plant metabolism variability

Within a wide variety of factors which regulate biogenesis of secondary metabolites in plants, two distinct plant stimuli

can be determined, being biotic and abiotic effects. Considering the rhythms in which living organisms are under, we include

discussion of the ultradian and circadian time-scales once they are well documented and assumed to be of high inﬂuence

on secondary metabolism of volatile compounds and phenolic derivatives.

2.1. Abiotic Effects

With reference to previous reports on abiotic factors inﬂuencing the content of secondary metabolites in plants (Gobbo-

Neto and Lopes, 2007; Ramakrishna and Ravishankar, 2011; Gouvea et al., 2012) or more speciﬁcally in Populus species (Chen

et al., 2009), our review mainly considers and summarizes (Table 1) recent articles which reported abiotic interactions on

a wide group of plant species. Moreover, a selected number of prominent cases presenting secondary metabolite level

variability are discussed in detail. Abiotic factors overviewed in Table 1 demonstrate speciﬁc interactions with determine

plant species inﬂuences on secondary metabolite accumulation and biosynthesis related gene expression.

Abiotic effects include all physical effects governing habitat, such as light, or UV–vis radiation, water availability (which

leads to drought inﬂuence), temperature and soil composition. Phenolic compounds are a chemical deﬁnition which com-

prises a wide variety of carbon skeletons and a diversity of structures (Goldstein and Brown, 1990; Winkel-Shirley, 2001).

Several phenolics have their biosynthesis and bioactivity affected by UV. Coumarins from vegetables of the Umbeliferae

family, now re-named Apiaceae, are biosynthesized and accumulated in a non-toxic form in plant tissue. Nevertheless, the

stressed leaves of celery (Apium graveolens), when exposed to UV radiation, can damage eukaryotic DNA (Taiz and Zeiger,

2006). Coumarins, such as psoralen, bergapten, and xanthotoxin (Fig. 2) which are responsible for this effect have a sea-

sonal rhythm in a nontoxic concentration on A. graveolens petioles, where these compounds have lower levels depending

on the plant age, and leaves (Trumble et al., 1992). Consistent with acquisition of this evolutionary ﬁtness, umbeliferones,

which are coumarin-type metabolites from Ruta graveolensis, are known to be biosynthesized through a pathway involving

a UV-inducible gene, which itself enhances umbeliferone levels in leaves of the R. graveolensis (Vialart et al., 2012).

8 D.P. Pavarini et al. / Animal Feed Science and Technology 176 (2012) 5–16

Table 1

Recent reports of shifts on secondary metabolites levels and also of transcripts and proteins related to biosynthetic pathways of each metabolite. At the

left column, table displays each abiotic factor inﬂuencing responses of secondary metabolism.

Abiotic factor Plant Effect Reports

Lower temperature Artemisia spp. Higher levels of artemisin Wallaart et al. (2000), Brown

(2010)

Lower temperature Nicotiana tabacum Higher levels of anthocyanins Huang et al. (2012)

Lower temperature; UV-B irridiation Malus sp. Higher levels of anthocyanins Ban et al. (2007), Ubi et al.

(2006), Lin-Wang et al. (2011)

Longer light exposure Panax quinquefolius Higher levels of ginsenosides Fournier et al. (2003)

Altitude Leontodon autumnalis Shifting ﬂavonoids contents Grass et al. (2006), Zidorn and

Stuppner (2001)

Higher altitudes Matricaria chamomilla Higher ﬂavonoids and phenolic Ganzera et al. (2008)

acids ﬂowers contents

Higher altitudes and lower temperatures Arnica montana Phenolics derivatives Albert et al. (2009)

Continuous solar radiation Vaccinium myrtillus Flavonoid biosynthesis Jaakola et al. (2004)

pathway activation

Shifting latitudes Several (review paper) Intragenus shifts of ﬂavonoid Jaakola and Hohtola (2010)

contents

Enriched carbon dioxide atmosphere Quercus spp.; Galactia elliottii Higher tannin levels Stiling and Cornelissen (2007) (meta-analysis)

Exposure to red light ( = 600–700 nm) Ocimun basilicum Rosmarinic acid accumulation Shiga et al. (2009)

Drought stress Quercus ilex Lower monoterpene emissions Lavoir et al. (2009)

High conditions of Cu and Mn nutrition Eugenia uniﬂora Lower both tanins and Santos et al. (2011)

ﬂavonoids contents

Temperature and light variations Populus × canescens Shifts on transcript levels of Mayrhofer et al. (2005),

terpene biosynthesis-related Sharkey et al. (2008), Tholl

genes (2006)

Exposure to UV Catharanthus roseus (cell Enhanced production of Ramani and Chelliah (2007),

culture) catharanthine Jenkins (2009)

Light effects also have crucial importance on pre-harvest of Camellia sp. (Tea) shoots (Ashihara et al., 2008). Synthesis of

phenolic derivatives (i.e., ﬂavanols and catechin types (Fig. 2)) is upregulated when shoots are overexposed to light which,

in leaves results in accumulation of phenolic compounds (Anan and Nakagawa, 1974; Ashihara et al., 2008). Hence, light

exclusion of shoots just before harvest enhances purine alkaloid levels such as the caffeine (Fig. 2) proportion in leaves, in

consequence yielding higher tea quality (Ashihara et al., 2008).

Flavonoids confer UV protection to plant tissues, and their accumulation due to UV-exposure is also well documented.

Cistus sp. can have the contents of ﬂavonoids in its leaf exudates enhanced by UV radiation induction, or by drought, and

even by a positive synergic regulation of both factors on synthesis (Chaves et al., 1997). Cistus sp. seasonal accumulation of

secondary metabolites in leaf exudates is a convenient model to comprehend the multi-task trait of secondary metabolite

accumulation and biosynthesis. Resveratrol (Fig. 2), a stilbene derivative produced in Vitis vinifera, is considered the major

Fig. 2. Chemical structures of bioactive compounds from plant secondary metabolism as inﬂuenced by abiotic effects.

D.P. Pavarini et al. / Animal Feed Science and Technology 176 (2012) 5–16 9

Fig. 3. Chemical structures of bioactive compounds from plants secondary metabolism as inﬂuenced by biotic factors.

factor in the wine orthomolecular trait (Pezzuto, 2008; Quiroga et al., 2012), and has been reported to respond to UV stimulus

(Adrian et al., 2000; Quiroga et al., 2012).

Water stress also causes physiological adaptations of plant organisms based on an exogenous factor, like any stress,

from pathogen infestation even to seasonality of climate conditions. Among the earlier physiological responses, such as

leaf area decrease and abscission induction, the osmotic shifts due to water loss can induce purine alkaloids biosynthesis

(Godoy-Hernandez and Loyola-Vargas, 1991; Ashihara et al., 2008; Frischknecht et al., 1987).

2.2. Biotic effects

Biotic effects include more sophisticated interactions with plant biochemistry and plant physiology (Briskin, 2000). In

a larger sense it can be assumed that biotic effects are related either to plant interactions with microorganisms or plant

physiological aspects, as phenology and ontogeny.

Fungus infested V. vinifera varieties as, for instance, in Spanish fruits of V. vinifera by Botrytis spp. (Laquitaine et al., 2006)

can either act as a crop disease or as a wine making technique to create higher value products (Choquer et al., 2007).

Once phenology is accepted to be the way plant physiology operates relative to time of the year or, more speciﬁcally,

how phenophases are distributed among seasons, this rhythm can explain what sometimes is misunderstood as the seasonal

variability of plant metabolisms. It is commonly accepted that temperate climate occurring species function under easily

detected points, such as autumn and spring, of environmental temperature and photoperiod. Phenology describes all ultra-

dian, or even seasonal, features of a living organism such as ﬂowering in angiosperms or senescence in temperate climate

occurring gymnosperms, while ontogeny determines physiological differences within a lifetime of a living organism.

Key enzymes of the polyacetylene compounds (i.e., fatty acid derivatives) synthesis in Helianthus annus are encoded by

the gene Crep1 (Minto and Blacklock, 2008), which desaturates micro molecules, and is over-expressed during H. annus

ﬂowering and accumulated in a scale 10 times higher in ﬂowers than in seeds.

Ecological interactions are common to several living organisms. Agrobacterium rhizogenes is a pathogenic soil bacterium

which promotes formation of hairy roots and multi-branched proliferative adventitious roots, by insertion of transfer DNA

contained in the bacterial plasmid into the plant genome at the infection surface of a damaged root (Zid and Orihara, 2005).

Ambrosia maritime is a weed often infected with A. rhizogenes and its hairy roots, if in contact with methyl jasmonate, are

able to have genes elicited (i.e., genes are elicited when in contact with an elicitor which is deﬁned by Angelova et al. (2006)

as a “compound stimulating any type of plant defense” while Mueller et al. (1993) state that elicitors are responsible for

“formation of low and high molecular weight defense compounds in plant cell”). Genes encoding enzymes of synthesis of

fatty acid derivatives, the polyacetylenes, are highlighted since their transcription in roots is established by symbiosis (Zid

and Orihara, 2005). Nine-fold higher levels of pentayneene, the precursor of thiarubrine and derivatives, were detected in

hairy roots while no infected cells remained with normal polyacetylene activity. Azadirachtin-A (Fig. 3), a highly oxidized

tetranortriterpenoid, is biosynthesized at higher levels in hairy roots of Azadiracta indica under the same interaction with A.

rizogenes (Satdive et al., 2007).

10 D.P. Pavarini et al. / Animal Feed Science and Technology 176 (2012) 5–16

OH O

O O O

HO P HO O OH P P OH O O OH

OH HO OH OH OH

methyl-erythritol-phosphate mevalo nic acid isopentenyl diphosphate

Phenylpropen e isoprene

Fig. 4. Chemical structures of key molecules involved in volatile compound synthesis.

Herbivory is an exogenous factor which triggers an intrinsic defense mechanism which can include several known sec-

ondary metabolites as deterrents, as shown for the caterpillar wound induced storage of secoguaianolide type (Fig. 3)

sesquiterpene lactones in Vernonieae species such as Viguiera robusta (Ambrosio et al., 2008). One of the most studied

classes of herbivory inducible secondary metabolites are volatile compounds which act in tritroﬁc ecological interaction

(i.e., a plant can release such compounds immediately after predator attack to attract predators of its own predators).

Gossypium can biosynthesize bioactive compounds as gossypol (Fig. 3), which is accumulated in wounded Gossypium spp.

leaves (Gershenzon and Dudareva, 2007; Opitz et al., 2008). At another level of interaction, much of a bilateral responsive-

ness, aliphatic terpenes such as farnesene derivatives are biosynthesized de novo by Gossypium leaves within a determined

time-frame after attack (Paré and Tumlinson, 1997).

2.3. Time-scale variability

Wild type secondary metabolite proﬁles are, most commonly, singular to each population of plants. This idiosyncratic

feature is time dependent, and plant secondary metabolite proﬁles and contents can be related to conditions such as season,

time of day, tissue age and temperature.

Vernonieae species (Asteraceae) that are endemic to central Brazil display an accumulation of sesquiterpene lactones

and ﬂavonoids (Keles et al., 2010). Lychnophora spp. (Gobbo-Neto et al., 2010) and Eremanthus spp. (Sakamoto et al., 2005;

Lopes et al., 1991) are examples of plants from this Compositae which display different metabolite proﬁles and bioactivity.

Also of a peculiar way of accumulation are the volatile terpenes from Virola surinamensis (Lopes et al., 1997), which respond

to the seasonal cycle of the Amazon forest by shifting terpene accumulation levels. For example, during wet winter time

total monoterpene levels are lower at night.

Aspects of synergistic effects due to UV radiation, humidity and hot temperatures can explain seasonal trends of plant

secondary metabolite accumulation. Saponins as the medicagenic acid are, for example, accumulated in older leaves of

Medicago sativa, as well as in summer cuts (Pecetti et al., 2006; Szakiel et al., 2011).

3. Volatiles

Volatile compounds as semi-chemical agents are not an exclusive aspect of plant communication. Several pheromones

occur in vertebrates and invertebrates, and are fundamental to eusocial insect breeding as well as being important to

mammals (Wyatt, 2009). We present the cases in which these compounds are reported to have shifting levels of accumulation

and emission at, and by, determined tissues of organisms in the plant kingdom. Despite the occurrence of amino acids,

benzenoids, aliphatic aldehydes and alkene type volatiles (Dudareva et al., 2006), this section focuses on two types of

secondary metabolite classes, being phenylpropanoids and terpenes. Usually biosynthesis and compound storage occurs in

specialized plant structures (Gang et al., 2001; Besser et al., 2009; Franceschi et al., 2005; Gobbo-Neto et al., 2008; Wang et al.,

1997; Maffei, 2010), which is important to consider when searching for important shifting levels of bioactive compounds

from plants considered for use in animal technology and, even more importantly, to avoid side effects of usage, as often

occurs in livestock (Radi et al., 2004; Wilson et al., 2001)

3.1. Terpenes

The major parts of all ∼35,000 terpenoids that are known (Mcmurry, 2009) are reported as secondary metabo-

lites. All types of terpenes (alkenes) and terpenoids (oxygenated alkene derivatives), from the smallest monoterpenes

to carotenoids are formed by oligomerization of isopentenyl diphosphate units (C5H9P207; Fig. 4). Isopentenyl diphos-

phate units can be produced by mevalonic acid (Qureshi and Porter, 1981; Newman and Chappell, 1999; Fig. 4) or by the

D.P. Pavarini et al. / Animal Feed Science and Technology 176 (2012) 5–16 11

Fig. 5. Chemical structures of the saponin protodioscine and the sapogenol B carbon skeleton (Pecetti et al., 2006) reported to be accumulated in Brachiaria

spp.

methyl-erythritol-phosphate (Fig. 4) pathway (Tholl, 2006), as well as by an intermediate pathway. Examples of volatile

terpene emissions patterns will be discussed and temporal rhythms are a major aspect of the discussion. However examples

of how abiotic factors, such as temperature, can impact emission patterns of volatile terpenes will be presented.

Wild type Arabidopsis thaliana is known to have a diurnal pattern of terpene emissions from its aerial parts (Aharoni et al.,

2003). Such a pattern does not occur for volatile entities such as aldehydes and linear chained hydrocarbons or non-terpenic

metabolites.

Cytosol originated sesquiterpenes are biosynthesized by hypothetical regulation of phosphate prenylated precursors

in cytosol rather than plastids (Dudareva et al., 2005). Such a regulation hypothesis may explain how infradian shifts of

sesquiterpene emission levels can occur in Arabidopsis leaves, thereby indicating a more complex mechanism which goes

beyond simple temperature regulation.

Ultradian rhythms of volatiles emission by ﬂowers of Rosa hybrida are periodic within one daylight time frame. However its

ability of keeping this rhythm, even with no light/dark period remains the same (Helsper et al., 1998). An apparently abiotic

factor of secondary metabolite production variability is, in this case, hiding the internal clock regulation of biosynthesis.

Greenhouse monitoring of sesquiterpene emission levels does conﬁrm this circadian rhythm (Helsper et al., 1998).

However there are a number of articles pointing out abiotic effects on volatile emissions by aerial parts of plants, and most

link diurnal patterns of emissions to temperature shifts within day. Temperature has an important role on terpene emissions

since these volatile compounds would only leave storage structures as would trichomes (Besser et al., 2009), and bark tissue

on gymnosperms (Ghirardo et al., 2010) into the atmosphere when they are exposed to higher temperatures, since energy

is needed to reach a suitable vapor pressure. Temperature and ultraviolet visible radiation (UV–vis) are connected to the

regulatory processes of gene encoding of key enzymes of isoprene (Fig. 4) derivatives (Mayrhofer et al., 2005). Considering

reports of abiotic effects controlling terpene emissions by Citrus (Hansen and Seufert, 2003) as well as in Pinus (Wang et al.,

2011) and other northern hemisphere endemic species (Ibrahim et al., 2010), we conclude that understanding the dynamics

behind those shifts is of great interest for many ﬁelds.

4. Shifting levels of secondary metabolites from toxic plants: impacts on Brazilian livestock

Within the diversity of Brazilian ﬂora, 113 species are known to be toxic to livestock, where a ‘toxic plant’ is any plant able

to cause injury or death after consumption (Riet-Correa et al., 2007, 1993). Despite the lack of information throughout Brazil,

12 D.P. Pavarini et al. / Animal Feed Science and Technology 176 (2012) 5–16

Fig. 6. Chemical structures of pyrrolizidine alkaloids from Senecio spp. and Crotalaria spp. reported to have toxic effects on livestock and plant tissue levels

variations.

researches in the south of Brazil, particularly Rio Grande do Sul, and Santa Catarina, have estimated annual losses at US$22

million since 2000, or 14% of livestock deaths. Extrapolating that ﬁgure to all of Brazil suggests that annual losses during

this period were US$160–224 million (Riet-Correa and Medeiros, 2001). Understanding how saponins and pyrrolizidine

alkaloids are produced, and how often they occur in plant tissues, are crucial to diminishing this economic impact. These

two major causes of toxicity in livestock are discussed below.

4.1. Saponins from Brachiaria spp.

Of the Cerrado biome area in central Brazil used for livestock breeding since 2000, 85% of the area was occupied by

Brachiaria species for animal feed (Macedo, 2005). Despite its high nutritional value, its phototoxicity due to saponins effects

(Brum et al., 2007, 2009; Santos, 2008) is a cause of losses related to plant secondary metabolite accumulation (Riet-Correa

et al., 2011).

Among a diversity of saponins reported to occur in Brachiaria spp., sapogenol B type aglycone, pentacyclic derivatives

and protodioscine (Fig. 5), are probably the most common (Brum et al., 2007, 2009; Santos, 2008). An “age dependent” shift

on protodioscine levels occurs in Brachiaria leaves. During the aging of leaf tissue, protodioscine is found in higher levels in

young leaves (Riet-Correa et al., 2011; Santos, 2008).

4.2. Pyrrolizidine alkaloids

The Senecioneae (Asteraceae) and Eupatorieae tribes are known for their secondary metabolism which expresses a notice-

able and exclusive occurrence of pyrrolizidine alkaloids (Funk et al., 2009). Several species of this genus biosynthesize and

accumulate pyrrolizidine alkaloids and the Brazilian livestock industry is affected by them. Chronic or acute toxic effects on

livestock due to pyrrolizidine alkaloid ingestion are caused by Senecio spp. (Asteraceae) and Crotalaria spp. (Leguminosae),

while an occurrence of Echium plantagineum (Boraginaceae) toxicity has been reported (Lucena et al., 2010).

Toxicity cases are, in general, caused by Senecio spp. Such cases rank among the main causes of livestock death in southern

Brazil where S. brasiliensis, S. oxyphyllus, S. heterotrichius and S. selloi are common sources of pyrrolizidine alkaloid ingestion

(Lucena et al., 2010; Tokarnia et al., 2002). Environments lacking young bushes of Senecio spp. during the winter cause

livestock to eat by these toxic plants, which biosynthesize and accumulate higher levels of pyrrolizidine alkaloids at the

winter (Karam et al., 2011). This pattern of pyrrolizidine alkaloid accumulation may be related to seasonality and phenology.

D.P. Pavarini et al. / Animal Feed Science and Technology 176 (2012) 5–16 13

Derivatives of senecionine occur in S. brasiliensis as Z-senecionine, Z-senecioﬁlina and Z-retrorsine (Krebs et al., 1996; Toma

et al., 2004; Santos-Mello et al., 2002), while S. selloi were reported to contain 18-hidroxy-jaconine, senecivernine, as well

as Z-retrorsine and Z-senecionine (Fig. 6; Krebs et al., 1996). Senecio jacobaea is suggested as an example of ontogenetic

modulation of pyrrolizidine alkaloid synthesis (Schaffner et al., 2003), and pyrrolizidine alkaloids from Senecio spp. have

high chemodiversity and can be used as chemotaxonomic marker in an infra genus scale (Trigo et al., 2003).

Toxic cases from natural exposure to Crotalaria retusa have been reported, for horses (Nobre et al., 2004, 2005) and sheep

in northeastern Brazil. Plants from Crotalaria genus accumulate monocrotaline (Fig. 6) in leaves (Rocha et al., 2009), and

related pyrrolizidine alkaloids are considered responsible for toxic effects on livestock that have eaten them. However, it

seems that there are speciﬁc tissues were these alkaloid levels are much higher, particularly seeds (Anjos et al., 2010).

5. Conclusions

The secondary metabolism of plants, and the expressed metabolite levels, may change considerably due to the inﬂuence

of several biotic and abiotic stress signals. In general, knowing these factors and the potential of plant metabolism variations

in a speciﬁc area may constitute an important economic and food safety impact. This review reveals an evident and strong

necessity to complete studies on accumulation of secondary metabolites under normal circumstances, and also under stress

conditions. Such data can provide information to improve, for instance, productivity of an agricultural area. Knowing the

factors which induce variations of plant secondary metabolite contents should further stimulate studies to deﬁne conditions

and periods where in cultivation and/or harvest can occur to achieve desirably high concentrations of bioactive compounds,

such as in the case of cultivation of medicinal plants, or to achieve desirably low concentrations of bioactive compounds, such

as in the case of cultivation of medicinal plants as human or animal foods/feeds. Identiﬁcation and understanding natural

variation in metabolite concentrations may extend the knowledge of ecologic interactions of plants with their environment

and allow alternative strategies to increase productivity of plants used in agriculture.

Acknowledgments

Authors acknowledge Prof. Dr. João Luis Callegari Lopes from NPPNS-FCFRP-USP and Prof. Dr. David Driemeier from

UFRGS (Faculdade de Veterinária, Departamento de Patologia e Clínica Veterinária) for their relevant advice on this review

subject. We are grateful to FAPESP, CNPq and CAPES for grants, ﬁnancial support, and scholarships. Authors apologize for

references not cited in this contribution due to lack of space.

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