Characterization of Plant Latex and Other Exudates
In addition to latex, plants produce several types of exudates such as gums, mucilage,
essential oil, oleoresins, resins, and phloem-like saps [1]. The differentiation of exudates is mainly associated with species specificity,
anatomical origin, morphological appearance, or their chemical composition. Nonetheless,
plant exudates are generally classified based on their chemical composition ([Fig. 1]).
Fig. 1 Plant exudatesʼ classification, their main chemical components and their producing
structures. Pictures were taken by one of the authors, Luis Francisco Salomé Abarca.
Gums are defined as solids usually produced from stem barks or roots. They flow out
naturally upon mechanical damage or at an infection point, readily solidifying on
contact with air [2]. The main constituents of gums are polysaccharides, which are polymers of arabinose,
galactose, mannose, and glucuronic acid [1]. They are thus hydrophilic, being either water-soluble or capable of absorbing water
and swelling up to form a gel, but they are insoluble in oils or organic solvents.
Mucilages are water-soluble acidic or neutral polysaccharides that yield mixtures
of simple sugars (e.g., galactose, arabinose, xylose, rhamnose, and galacturonic acid)
or uronic acid [2], [12], [13] by hydrolysis. The localization of mucilages is very specific, being produced intracellularly
and having some of their constituents related to cell wall components [14]. Differently to gums, mucilages are strongly related to seed survival and also act
as water storage in plants [14].
Oleoresins are referred to as resins or, more specifically, soft resins. They are
exudates with a high content of volatile compounds. The physical state of a resin
varies from solid to semi-solid, being composed of lipophilic terpenes such as mono-,
sesqui-, and diterpenes. While most plants can produce resins, only a few families
such as Fabaceae, Burseraceae, and Pinaceae are known for their high resin production
[1].
Essential oils are mainly composed of organic volatile compounds, which confer their
characteristic properties such as odor and density and low melting points [15]. Unlike other types of plant exudates, their vapor pressure under atmospheric conditions
is so high that they diffuse through the air [16], [17]. These volatile compounds belong to various chemical classes such as alcohols, ethers
or oxides, aldehydes, ketones, esters, amines, amides, phenols, heterocycles, and
mainly monoterpenes [16].
Extra fascicular phloem is a particular type of sap localized in a special network
of sieve tubes outside of the vascular bundles. It is found in a very limited number
of families such as Curcurbitaceae [18], [19]. They contain abundant amino acids and a wide range of low levels of small molecules
together with some proteins [20]. The biological functions of the phloem are likely associated with their proteins
such as instant wound signaling and plugging of the sieve elements to avoid the loss
of nutrients. They have additionally been found to play a direct role as anti-insect
agents against generalist insect-herbivores [21].
The definition of latex has varied throughout time according to the review paper by
Hunter (1994) [22]. Polhamus (1962) stated that “no one has demonstrated why a plant makes rubber and
it does not appear to be a food reserve. Strong evidence indicates that rubber is
an end-product that is not reused in the metabolism of the plant” [23]. Five years later Metcalfe (1967) stated, “The term latex is used loosely by plant
anatomists for fluids with a milky appearance due to the suspension of many small
particles in a liquid dispersion medium with a very different refractive index” [24]. Further, he added, “According to Esau (1965) the dispersed particles are commonly
hydrocarbons of the terpene type which include essential oils, balsams, and rubber.”
Moreover, “The fact that latex is restricted to a small number of plant families which
have little taxonomic relationship suggests that the capacity to produce latex has
been evolved more than once” [25]. Even after all these concepts and hypothesis, Webster and Baulk will (1989) concluded
that “as none of the above hypotheses has been proven, the function of latex and rubber
in the plant remains unknown” [26].
Nowadays, there are more definitions of latex, based on their anatomical origin, storage,
or ecological roles. Particularly, the ecological approach explains possible functions
of latex in plants. For example, Konno (2011) stated that “plant latex is a sap, typically
a white sap, that is stored in the tissue called a laticifer and that is exuded from
a point of damage in plant tissues immediately after insect herbivory” [5]. Currently, latex can be defined in a general manner, as an exudate that is kept
in extremely elongated cells extended along the plant. Such canal structures are called
laticifers. Anatomically, latexes are originated from vacuoles because a large part
of the laticifer cells are occupied by vacuoles [27].
A number of phytochemical studies investigating latex biochemistry have been published,
covering complex aqueous mixtures of rubber to specialized metabolites such as alkaloids,
cardenolides, phenolics, starch, furanocoumarins, sugars, and non-protein amino acids
[4], [28], [29]. However, most previous works focused on polymeric ingredients while specialized
metabolites were usually neither properly identified nor quantified. If any, research
was done on a limited number of species (e.g., on the latex of Taraxacum officinale L., Asteraceae) [6].
Currently, there is no scientific evidence of the metabolic role of latex in plants.
Instead, a large amount of evidence has been accumulated proposing latex as a natural
defense barrier against herbivores. In the strict sense, a plant defense is any feature
that improves the fitness of plants under herbivory pressure [30]. In the case of latex, it is used by plants as a complementary chemical and mechanical
defensive system [31] ([Fig. 2]). However, only a few studies have linked the variation of latex in planta to plant fitness [32].
Fig. 2 A model of latex coagulation process. Four main steps are represented: (1) equilibrium
state, (2) laticifer rupture, (3) plug formation, and (4) plug polymerization.
The composition of latexes
Laticifers are specialized latex-producing cells with a characteristic anatomy and
distinct cytoplasm [33]. The cytoplasm of the cells is a colloidal suspension of small particles in an aqueous
sap of unspecified composition, but normally with different refractive index from
its particles [34], [35]. They contain latexes that are generally milky or white but may sometimes be colored
(yellow, orange, red, brown) or even colorless [36].
The mature laticifer has not yet been reported to contain chloroplasts [37] so it is believed to have no functional plasmodesmatal connections with its neighboring
tissues. Thus, laticifer cells are expected to obtain their energy from the apoplast.
Hence, its electrophysiological behavior might display an electrogenic sequelae of
active transport. Specifically, the loading of laticifers is thought to be related
to the symplastic transport of photoassimilates and other minerals, from the phloem
to the contiguous associated parenchyma. This process is followed by their entrance
to the apoplast, and further transport into the laticifer by an H+/sugar symport system [36], [38] to be transformed into latex.
Lutoids and Frey-Wyssling complexes
The ultracentrifugation of latex at 59 000 g yielded 3 major fractions [39]. The bottom layer consisted in a solid pellet corresponding to around 20% of the
latex volume, a rubber-clear aqueous supernatant (C-serum) mid-layer, and an upper
layer consisting of rubber particles that floated to the top [39]. The bottom fraction was characterized by the presence of a special type of plastids,
called lutoids [40]. Electron microscopy shows them as spherical particles ranging in size from 0.5
to 3.0 µm. Lutoids are highly osmosensitive [41]. Lutoids accumulate chemicals from the cytoplasmic environment. Some of these, specifically
pyrophosphate (Pi), citrate, and Mg2+ are about 10 times more concentrated than in the cytoplasm. They also contain many
amino acids, acid hydrolases, peroxidases, lysozyme, and α-mannosidase [42]. Lutoidic membranes were reported to have a low or undetectable content of phosphatidic
acid and no nitrogen phospholipids [43], while both types of compounds were detected in the membrane of rubber particles.
It was hypothesized that the large quantity of negative charges carried by the phosphatidic
acid favors the colloidal stability of the main chemical components present in latex
[42], [43].
Similar sensitive particles to lutoids can be found as a thin vivid yellow layer conform
of specialized plastids. These layers are form by double membrane vesicles that contain
carotenes and polyphenol oxidases (PPOs) [44]. These modified plastids, so-called Frey-Wyssling complexes, contribute to the coagulation
of latex under open-air conditions because of their o-diphenoloxidase content [41]. This activity is related to the oxidation of o-diphenols to o-quinones [45]. These transformed metabolites can react with a big variety of substances in the
cytosol such as proteins or oligosaccharides to form brown polymers. These phenomena
are well documented in T. officinale latex L. (Asteraceae) [46].
Rubber
Rubber is a biopolymer with exceptional properties such as high elasticity, resilience,
and efficient heat dispersion [47]. This polymer is synthesized from cis-isopentenyl diphosphate units, poly-(cis-1,4-isoprene ([Fig. 3 A]) [48], [49], [50]. These polymers in rubbery latexes are present in the form of globules surrounded
by a phospholipoprotean membrane [41]. According to Agrawal and Konno (2009), the stickiness of latex is attributed mainly
to factors related to rubber: (a) elasticity of cis-polyisoprene, (b) coagulation of rubber particles, and (c) adhesiveness of rubber particles to the surfaces of insects. Therefore, the primary
role of rubber in latex that is generally accepted is to confer self-stickiness [4]. In addition to the stickiness, the white color of latexes is also due to the rubber
particles [51].
Fig. 3 Chemical structures of typical terpenoids and steroids found in latexes. A cis-1,4-polyisoprene, B lactucopicrin oxalate, C phytol, D phytoecdysones, E cardenolides (5β,14β-androstane-3β,14-diols), F digitoxin, and G ouabain.
Proteins and enzymes
A variety of proteins and enzymes have been isolated and identified from plant latexes.
Of the numerous identified proteins, cysteine proteases, serine proteases, chitinases,
lectins, protease inhibitors, and oxidases are the most reported in literature [52]. These proteins play many physiological roles such as signal transduction and oxidative
defense [53], [54]. Due to the variety of proteins, latexes were assumed to be a promising reservoir
of defense proteins [5], providing a strong evidence of their defensive role against the plantʼs natural
enemies. Intriguingly, some of the proteins were found to possess not only direct
toxicity for the enemies but were also capable of triggering a mechanical defense
against herbivores (e.g., coagulation process to form an initial barrier) [55], [56], [57].
In the latex of Chelidonium majus L. (Papaveraceae) cystains (thiol protease inhibitors) were determined to be involved
in stress adaptation mechanisms and defense plant responses. Moreover, these enzymes
could also confer antimicrobial and antiviral properties to this latex [58]. Moreover, in some plant species, the functions of these proteins were related to
specialized metabolites such as alkaloids [59].
Besides, the construction of a comprehensive dataset containing 1208 nonredundant
latex proteins from diverse latex producing species and its in silico predictions showed latex proteins to be related to a broad range of internal molecular
processes and also to external environmental changes. Further, the function similarity
between latex and phloem proteins suggested a functional conservation in plant vascular
proteins [60]. Also, latex proteins have been reviewed and liked to a new type of constitutive
immediate response against viral agents [61].
Lectins are natural specific carbohydrate-binding proteins and similarly to these
exert a protective function against a wide range of pests [62], [63], including homopteran, lepidopteran, and coleopteran insects [64], [65], [66]. A review of literature on this activity reveals different possible mechanisms,
one of the most important being their stability in the digestive system of herbivores
[63]. They also displayed a significant inhibitory activity of protein synthesis as exemplified
with an in vitro test with 2 lectins isolated from the latex of Hura crepitans L. (Euphorbiaceae) [67]. Another report showed them to be effective against sucking insects because of their
specific residues that show mannose-binding to cell walls [64]. They are also able to act indirectly against herbivores by enhancing latex coagulation
[57] unlike other types of proteins that can only act as a direct chemical defense.
Another relevant type of latex proteins are protein inhibitors since they can confer
anti-nutritive properties to the latex (e.g., protease inhibition to prevent protein
digestion) [68]. Trypsin and serine protease inhibitors were found to be highly induced in the latex
of Ficus carica L. (Moraceae) [69] and Carica papaya L. (Caricaceae) upon herbivore and pathogenic microbesʼ attacks [70]. This type of molecule can counteract degradative enzymes produced by some microorganisms
during pathogenic interactions or by other specialized insects. The complexity of
the enzymatic biochemical system in the latex of Euphorbia species has been discussed previously by Pintus et al. [52].
Specialized metabolites in latex
In general, plants have 2 types of defense systems, primary and secondary. Primary
defenses are the first line of defense against herbivore attacks and involve morphologically
protective structures on the surface, such as hairs, trichomes, thorns, spines, or
even thicker leaves. Secondary defenses are considered to be mediated by plant chemicals,
especially by specialized metabolites (e.g., phenolics, terpenoids, and alkaloids).
The metabolites induced during plant defense can directly kill or retard the development
of the herbivores and phytopathogenic microorganisms [71]. The main defensive role of plant exudates is very likely as a barrier: acting firstly
mechanically rather than chemically because of their inherent stickiness or sliminess.
These exudates are able to tangle some herbivores by reducing their movements or disabling
their mouthparts and sense organs [72]. They can also seal wounds, preventing the entrance of possible pathogens [73]. On the other hand, a specific type of exudates such as latex, phloem-like sap,
and resins can exert a secondary type of chemical defense due to their wide range
of specialized metabolites as well as functional proteins that are frequently in much
higher concentrations than in other organs or tissues of the plant, such as leaves,
stems, or roots [4]. It has been found that many of these compounds provide strong resistance to herbivores
via toxicity or anti-feeding effects [5].
In many latexes, the local concentrations of defensive substances increase at the
point of damage to levels estimated to be between 20 and 2000 times those in leaves
[74], [75], [76]. In this sense, latex is similar to an inducible defense system despite being pre-formed
in the plant. However, there is a great advantage in latex-borne defense, since the
local increase of the level of specialized metabolites occurs within a few seconds
after herbivory, which is much faster than inducible defense systems [5]. The advantage of the latex response is even greater considering the mobility that
allows for the immediate transport of defense substances to the precise point of damage
by herbivores or pathogenic infections [5]. It is thus a perfect example of the complementation of primary and secondary defenses.
Plant latexes have been found to display a broad chemical diversity that, as mentioned
before, is distinctive from other organs or exudates of the plant. Their chemical
uniqueness is not limited to a specific group of metabolites but includes a wide range
of specialized metabolites such as terpenes, phenolics, alkaloids, and cardenolides
[4], [28], [29]. Furthermore, some multitask and biologically active proteins including cysteine
and serine proteases, and several other enzymes have been also found in these exudates
[52].
Terpenoids and steroids
One of the most abundant metabolites in latexes is a polymeric terpenoid derived from
isoprene units (C5). The most important C5 compound in latex is rubber (cis-1,4-polyisoprene) ([Fig. 3 A]). This compound is found in many plant species (i.e., 300 genera and 8 plant families)
[22], [51], [52].
Apart from rubber, smaller terpenoids have also been found in latex. The latex of
Lactuca sativa L., Asteraceae, contains several sesquiterpene lactones. In particular, the concentration
of lactucopicrin oxalate ([Fig. 3 B]) in latex was found to be 1000 times higher than in leaves. Together with these
sesquiterpene lactones, lettucenin A was reported to be induced in latex by fungal
or bacterial challenge [77]. The involvement of the induced compounds in plant defenses was partially supported
by some antifungal activity against Cladosporium herbarum
[4], [78]. Some Euphorbiaceae latexes (e.g., Euphorbia biglandulosa Desf., Euphorbiaceae) contain diterpenes, phorbols, and their derivatives in higher
concentrations than in other organs [78]. These compounds exhibit a high toxicity against many insects and mammals [79]. Further, phytol ([Fig. 3 C]), which is found in chlorophyll as a side chain, is supposed to help some compounds
to adhere to cell membranes [80]. Steroids, particularly phytoecdysones ([Fig. 3 D]) have some defensive roles against insects, disrupting molting and other developmental
and physiological processes with lethal consequences [81].
Euphorbia is a genus that is well-known to contain triterpenoids as the major components of
their latexes [82]. For example, the latexes of Euphorbia officinarum L. (Euphorbiaceae) and Euphorbia peplus L. (Euphorbiaceae) were found to have several steroids and triterpenes including
obtusifoloiol, cycloartenol and 24-methylenecycloartenol, lupeol, lupeol acetate,
lanosterol, and 24-methylenelanosterol, among others [83]. The biological roles of the steroids and the triterpenoids are still unclear, although
they are always present in high levels in rubbery latexes. As an assumed role, these
compounds could form part of the laticifer or the lutoidic membrane or even could
be freely suspended in the C-serum. This could imply that the latex steroids and terpenoids
might attach to the cell membranes of herbivores or pathogens, consequently facilitating
the transportation of non-lipophilic molecules through cell membranes of insects or
microorganisms. They could be intercalated in the cell membranes, forming channels
that allow the entrance of small toxic hydrophilic molecules such as phenolics or
others [84].
Another specific type of steroids, cardenolides, a type of cardiac glycoside that
has a 5- or 6-membered lactone ring in β position at C17, are also well-known metabolites in latexes. The cardenolides are
able to inhibit Na+/K+-ATPases, which are essential for maintaining the electric potential in most animal
cells [5], [85]. Cardiac glycosides have 3 characteristic molecular features: sugar, steroid, and
lactone moieties. Lactone rings and sugar moieties are not likely to be essential
parts for their biological activity [86], [87]. However, these lactone and sugar moieties could also be involved in the interaction
with the Na+⁄K+-ATPase inhibition process [88]. Studies on structure-activity relationships of cardenolide toxicity in animals
revealed their steroid nucleus 5β,14β-androstane-3β,14-diol ([Fig. 3 E]) as the structure required for receptor recognition [87]. Further, the differences in the hydrophilicity of cardenolides is associated with
the degree of absorption by animal tissues, which consequently resulted in potential
toxicity. Usually, lipophilic cardenolides like digitoxin ([Fig. 3 F]) are rapidly and almost completely absorbed but polar ones such as ouabain ([Fig. 3 G]) are slowly absorbed in the intestine [86]. In the case of herbivory, cardenolides can also avert insect egg-laying and herbivory
and disrupt their development, especially for generalist insects [89], [90], [91], [92], [93], [94].
The latexes of many Apocynaceae plants, another main latex-bearing family, contain
high levels of cardenolides (up to 30% considering the latex dried mass) [74], [85]. For example, the latex of Antiaris toxicaria Lesch. (Moraceae) in tropical Southeast Asia have cardenolides (toxicariosides).
Interestingly the cardenolides in the latex are very different from those of the leaves
[95], [96]. Although the unique pattern of cardenolides in the latex might result from the
specific selection for defense compounds to be located in latex as a defensive trait,
more studies are required to prove these roles.
Phenolics
Many types of phenolics, usually synthesized in the shikimate pathway, are ubiquitous
in plants (e.g., tannins, lignans, coumarins, anthraquinones, phenylpropanoids, and
flavonoids). It is thus expected that these diverse phenolics are involved in many
biological and physiological processes of plants in general [97]. While the ecological significance of phenolics is undoubted, their presence in
latexes has not been studied extensively. One study reported the presence of gallic
acid, naphthoic acid, quercetin, chlorogenic acid, and rutin in the latex of Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. (Euphorbiaceae) [98] while higher levels of hexadecyl, octadecyl, and eicosyl esters of p-coumaric acids ([Fig. 4 A]) were found in the latex of sweet potato, Ipomoea batatas (L.) Lam. (Convolvulaceae), as compared to those of other organs. Similar results
were observed in fresh vine latex and root latex that showed a 3% and 10% higher content
of p-coumarate esters than leaves and roots, respectively [99]. Interestingly, the concentration of this type of coumarins (e.g., C16, C18, C20
(Z)-coumarates) is inversely correlated to the palatability for weevils. Thus, (Z)-coumarate esters are assumed to participate in the defense of sweet potato against
insect herbivores [99]. The latter would support the role of latex as a defense strategy.
Fig. 4 Chemical structures of typical phenolics and alkaloids found in latexes. A p-coumarate esters, B 4-hydroxyphenyl acid derivative of inositol, C chelidonine, D sanguinarine, and E coptisine.
Oxidative activation may explain much of the variation observed at the individual
and ecosystem level responses to phenolics [97]. Phenolic inositol esters ([Fig. 4 B]) were found to be 1 of the 3 major predominant metabolites classes in leaves, roots,
and especially latex of 3 genotypes of dandelion (T. officinale agg. L., Asteraceae), and again in this case, the metabolites in latex were strongly
repellent against generalist insects as compared to those of leaves and roots [6].
Based on these results, it can be assumed that a certain phenolic compound plays a
specific role in latex. This is also supported by the fact that their oxidative activation
is perfectly matched with the hypoxic conditions in the laticifer, therefore preventing
pre-activation and self-plant intoxication. Furthermore, it is known that many fungi
produce oxidases as detoxification mechanisms against plants, but in this case this
fungal strategy could in fact activate and potentiate the biological activity of phenolic
compounds.
Alkaloids
Alkaloids are the most bioactive group of specialized metabolites found in nature.
Even if many plant organs contain these compounds, especially high concentrations
are found in young organs [100]. Their main function in plants is to work as defensive compounds against herbivores,
especially mammals, because of their wide range of toxic effects and deterrence capability
[101], [102], [103]. Some alkaloids disrupt the nervous system, especially chemical transmitters, while
others can affect membrane transport, protein synthesis, and miscellaneous enzyme
activities [70], [104]. Many types of alkaloids are found in plant exudates. They are irregularly distributed
and highly concentrated amongst angiosperm families, including Apocynaceae, Papaveraceae,
and Moraceae. For example, isoquinoline alkaloids such as chelidonine ([Fig. 4 C]), sanguinarine ([Fig. 4 D]), and coptisine ([Fig. 4 E]) can reach almost 20% of the fresh latex mass in C. majus L. (Papaveraceae) [105]. Sanguinarine is especially toxic to insects and vertebrates because of its capability
to affect neurotransmission, several neuroreceptors, and DNA synthesis [4], [106].
In general, most alkaloids have some degree of toxicity and appear to be used by plants
in defense against herbivores and pathogenic microorganisms. Therefore, latexes could
constitute an important site for the production or storage of these compounds. In
fact, some enzymes involved in the first stages of alkaloidal production are contained
in parenchymal cells proximate to laticifer cells [107] and those involved in their final biosynthetic phases are contained in the laticifer
cytoplasm [108]. Alkaloids in latexes are believed to have many biological roles, but to date there
are not yet many reports to support such allegations. One of the few studies explored
a possible mechanism of sugar-mimicking alkaloids. These imino-sugars are powerful
inhibitors of glycosidases and other essential enzymes related to sugar metabolism
in insects [109]. They were found in the latexes of mulberry (Morus spp., Moraceae) at levels as high as 2.5% of the latex fresh mass and 18% of the
dry mass [76]. Additionally, these alkaloids were found to be related to poor development of insects
by growth disruption because of the inhibition of sucrose uptake and trehalose usage
in several tissues [110].
Physical and Chemical Implications in Latex Transportation and Coagulation
Latex transportation
Latex is immediately mobilized to the site of damage after either mechanical or herbivore
damage. The latex of Cryptostegia grandiflora Roxb. ex R. Br. (Apocynaceae) was reported to be transported within the plant over
a distance of more than 70 cm to a damaged point [111]. The non-punctured laticifer is turgid probably due to osmotic water uptake, which
could contribute to the out flow of latex whenever the laticifer is broken by any
type of tissue disruption. Qualitative observations on Euphorbia species showed that the recovery of the normal laticifer oozing pressure takes around
1 min [112]. However, the process of emptying and refilling of single laticifers has not been
accurately measured yet [112].
Another fact that supports the role of latex in defense instances is that the amount
of the exuded latex can increase in response to insect herbivory. For example, herbivory
by the larvae of the monarch butterfly (Danaus plexippus) on 2 Asclepias species resulted in an almost 2-fold increment in the amount of exuded latex [4], [113], [114]. However, the laticifer-borne system is very susceptible to damage, especially when
ducts in the proximal areas are destroyed. In such cases, the leaf area that is distant
to the point of destruction has no supply of latex from the main reservoir in the
proximal parts and also loses pressure to exude latex [5].
Chemical coagulation: a physiological role of latex in plants
The coagulation process of latex has been extensively studied due to its practical
importance as it is the main limiting factor in rubber extraction for its utilization
on industry. In the case of H. brasiliensis (Willd. ex A. Juss.) Müll. Arg. (Euphorbiaceae), one of the most well studied plants
for rubber extraction, Gidrol et al. (1994) proposed that hevein, a lectin-like protein, might be involved in the coagulation
of latex [56]. Later on, a new anticoagulating factor in the C-serum of H. brasiliensis (Willd. ex A. Juss.) Müll. Arg. (Euphorbiaceae) latex was discovered [57]. This factor, called hevea latex lectin (HLL), reinforced the previous model of
latex coagulation. The HLL binding affinity to the soluble C-serumʼs HLL binding protein
was at least 6-fold higher than the rubber particle binding protein to small rubber
particles, providing a plausible explanation for the colloidal stability of latex
under normal conditions [57].
In addition to these studies, a recent report on the latex coagulation mechanism of
H. brasiliensis (Willd. ex A. Juss.) Müll. Arg. (Euphorbiaceae) showed chitinase and glucanase in
primary and secondary lutoids to be playing a crucial role in rubber particle aggregation
in the plant [115]. Under normal conditions, chitinase does not interact with glucanase. However, upon
wounding or any mechanical damage, fresh latex flows out and the turgor pressure from
laticifers suddenly decreases. This pressure decrease results in bursting of the lutoids.
Consequently, the membrane-embedded hevamine interacts with membrane-localized glucanase
facilitating the rubber particle aggregation and subsequent rubber latex coagulation.
Finally, latex vessels become plugged with rubber coagulates, which stops the latex
flow [46], [115]. Apart from the role of proteins on rubber particle aggregation connected to latex
coagulation, the effects of other chemical factors such as enzymes and reactive oxygen
species have been investigated [116], [117]. The activity of enzymes such as peroxidase, catalase, and PPO have been well characterized
in T. officinale L. (Asteraceae), Taraxacum kok-saghyz L. E. Rodin (Asteraceae), and other latex-bearing plants [116]. Especially in Taraxacum species, PPO activity is one of the main factors controlling the coagulation mechanism.
The rate of latex coagulation was found to be strongly correlated with the level of
laticifer-specific PPO activity in Ficus elastica Roxb. ex. Hornem (Moraceae) and some Asteraceae plants, but not in H. brasiliensis (Willd. ex A. Juss.) Müll. Arg. (Euphorbiaceae). These findings show that the coagulation
chemical processes are species dependent. However, it is important to bear in mind
that coagulation processes do not involve only chemical changes but can also involve
physical factors [118]. Physical differences such as rubber particle size, temperature, and water evaporation
can also have a significant influence on the coagulation process. Larger particle
sizes provide a larger surface area for rubber particle contact during rubber aggregation,
leading most likely to different coagulation speed amongst species [118]. Besides, water evaporation speed at the latex plug contributes to the reduction
of the latex coagulum volume, also increasing the rubber particles collision frequency
and, consequently, the coagulation speed [118].
The speed of water evaporation is affected by environmental factors such as temperature
and humidity. For example, high temperature combined with low humidity can produce
faster water evaporation and faster coagulation. In general, different latex coagulation
mechanisms seem to occur not only among different plant families but also among species
belonging to the same plant family [118]. However, if one would ignore differences in specific coagulation factors and instead
would focus on chemical families, it could be possible to find a general principle
in latex coagulation process in rubbery latexes at least. This common trend always
involves the rubber and protein fractions, plus the main environmental factors, temperature
and humidity, and their physicochemical consequences such as divalent cation stoichiometric
ratios and pH changes.
In this regard, here we propose a general model for latex coagulation involving 4
general steps. The first step is an “equilibrium state.” In these conditions, the
laticifers are in normal physiological conditions and not disrupted by any mechanical
rupture. All latex components such as lutoids, Frey-Wyssling complexes, rubber particles,
specialized metabolites, and coagulant and anticoagulant proteins are suspended in
the C-serum at neutral pH. The negatively charged rubber particles help to stabilize
the latex colloidal suspension by negative-negative charge repulsion [49], [119], [120], [121]. In the second step, “laticifer rupture,” the strong decrease of pressure plus the
entrance of water by apoplastic transport through the vascular bundles lead to the
lysis of the lutoids [36]. After the cells burst, Ca2+ and Mg2+ ions are released into the C-serum [121]. Then an electrostatic interaction between the positive charges of the released
ions and the negative charges of the rubber particles start the rubber aggregation
process. After this and depending on the bursting lutoid index (number of broken lutoids),
the concentration of coagulant proteins will surpass the concentration of anticoagulant
proteins, as occurs in H. brasiliensis (Willd. ex A. Juss.) Müll. Arg. (Euphorbiaceae) [56], [115]. This phenomenon reinforces the process of rubber aggregation. The presence of exposed
lutoid membranes attached with positively charged proteins additionally serves as
an agglutination factor. These membrane complexes are very specific for rubber particle
recognition in rubber aggregation, working as a bridge for rubber particle interconnections.
After this, all of the agglutinated particles and the secondary metabolites start
to move carried by the out flow to the rupture point, in a manner comparable to Brownian
diffusion. Because of this, all of the lutoidic components will be concentrated at
one single point lowering the pH at the damage point. This acidifying effect rises
the coagulation speed [122].
In a third step, the “plug formation,” the latex mass starts to lose water because
of the environmental temperature and humidity. The resulting volume reduction increases
the concentration of solutes and the collision frequency among the rubber aggregates,
which could remove the phospholipidic membrane covering the rubber particles and activate
the rubber elongation factor [123]. The activation of this processes starts the coalescence among free-membrane rubber
structures to produce a bigger rubber polymer [118]. At this point the rubber polymerization has reached an irreversible state. Finally,
in the fourth step, “plug polymerization,” the latex plug is completely dry and the
plug has been transformed into a polymer composed of rubber, ions, residues of lutoid
membranes, and protein complexes ([Fig. 2]). This dried plug is able to completely seal the injury, avoiding the loss of water
and other metabolites. It can also obstruct the entrance of pathogenic microorganisms
to the plant vascular system. In addition to this, in this polymeric cover there is
a mixture of extremely high concentrated specialized metabolites and proteins that
confer additional chemical protection against herbivores via toxicity or deterrence
([Fig. 2]).
Endophytes-plant interactions shapes chemical profile of plants and their exudates
Endophytes are defined as any microorganism that intra- or extracellularly colonizes
healthy plant tissues without causing apparent pathologic symptoms [124]. They have been isolated from scale primordia to diverse plant organs such as roots
or even resin ducts [125], [126], [127], [128]. During plant-microorganism interactions, plants limit the development of endophytic
populations, obliging these endophytes to evolve several biochemical strategies to
gradually adapt to their new living environments [129]. This adaptation involves circumventing several physical and chemical barriers that
must be overcome to establish a successful association [130]. In this context, the balanced antagonism hypothesis was initially proposed to address
how an endophyte avoids the activation of the host immune system [131], [132]. This hypothesis explains how microorganisms become resistant to toxic host metabolites
and how they increase their population density without causing visible manifestations
of infection [133], [134].
Taking into account the fact that endophytes reside within plants and are continuously
interacting with their hosts, it is conceivable that plants have a substantial influence
on the metabolic processes of endophytes and vice versa [135]. As examples, plant homoserine and asparagine act as host signals to activate the
expression of a lethal gene in virulent strains of Nectria haematococca, which is only expressed in planta
[10], and a camptothecin-producing endophyte, Fusarium solani, has been isolated from Camptotheca acuminate
[11]. This endophyte was able to produce the precursors of camptothecin locally. Therefore,
this type of plant-microorganism interaction raises the possibility of various recognized
“plant metabolites” to actually be the metabolic products of their endophytes [136].
Endophytic microorganisms inhabiting latex?
As previously mentioned, most of the plant exudates contain a large spectrum of active
specialized metabolites and biomolecules. In this context, it is unlikely that they
could act as possible niche for microorganisms. However, as usually happens in nature,
nothing is 100% certain. Consider, for example, that the role of herbivores in the
transmission of bacteria that can later behave as endophytes has already been proven
[137]. In that case even if not discussed by the authors, it is possible that the phloem
could be as disperser of the bacterial community to all of the organs of the plant.
Thus, plant exudates could also have a role on the distribution of endophytic population
within individual plants.
Specifically, in the case of latexes, some trypanosomatids and other monoflagellate
microorganisms have been isolated for the first time from the laticifers of Euphorbia hirta L. (Euphorbiaceae) on the island of Mauritius [138]. Furthermore, infections in others Euphorbia species produced no evident effects
on plant growth or yield. Actually, the latex of parasitized and non-parasitized plants
was microscopically undistinguishable [139], [140]. This could reflect an equilibrium between plant host and microorganisms, avoiding
infectious interactions and maybe establishing an endophytic-plant interaction instead.
One more example that could prove the existence of endophytic microorganisms in latexes
was observed in papaya (Carica papaya L., Caricaceae) shoots. After cutting them, the shoots exude latex, containing phenolics
and pigments that represent a common problem for the initiation of plant tissue culture
from field stocks [141], [142], [143], [144], [145], [146], [147]. The bacteria appear as a cloudy dispersion mimicking a latex exudation. Eleven
strains have been isolated from these cultures, 10 Gram-negative (5X Ralstonia mannitolilytica, 2X Paenibacillus sp, 2x Sphingomonas sp., 1X Pantoea spp.) and 1 Gram-positive isolate (Lysinibacillus fusiformis) [141].
Recently, in Euphorbia latex, a study measuring communities using culture-independent
methods unexpectedly revealed complex bacterial (mean: 44 species per sample; 9 plants
analyzed) and fungal (mean: 20.9 species per sample; 22 plants analyzed) communities
contained in these latexes [148]. Many of the identified taxa are known plant endophytes but have not been found
in latex previously. All of these examples show how even if plant exudates are a hostile
environment for microbial development, some are capable of adapting to such an environment.
Other intriguing aspects that have not been considered include the following: if latexes
are chemically differentiated from their bearing tissues, is also their microbiome
differentiated from their bearing tissues? If so, is this microbial differentiation
correlated to their chemical discrimination? How do microorganisms produce their own
metabolites, are they controlled by plant signaling, do they biotransform plant metabolites,
or are they a separate symbiotic defense system? Therefore, the study of associated
microbial communities in plant exudate structures and their bearing tissues will contribute
to the understanding of plant exudates chemical diversity and selection.
Latex as a bioactive chemical resource
Historically, latexes have been used as medicinal substances by diverse cultures across
the world from the Brazilian Amazonians to Indian users [149], [150]. One of the best documented examples is the opium poppy (Papaver somniferum L., Papaveraceae). The oldest evidence of the relation between this material and
mankind was found in the Mediterranean region in northwestern Italy [151], where well conserved seeds of opium poppy–which apparently were grown for food,
oil, and medicine production or possibly for religious cult purposes–were found [151]. Nowadays, latex-bearing plants are still of great importance for the industrial
sector. H. brasiliensis (Willd. ex A. Juss.) Müll. Arg. (Euphorbiaceae) is currently the main source of natural
rubber in the world, and none of the synthetically produced rubber products have surpassed
the quality obtained from this tree [152]. Furthermore, Parthenium argentatum A. Gray (Asteraceae) and T. kok-saghyz L. E. Rodin (Asteraceae) are 2 species used for the production of hypoallergenic
and tire latex, respectively [152], [153].
In addition, the coagulation mechanisms of latex defense have been used as a bio-inspirational
source for the production of self-healing materials [154], [155], [156]. Bio-inspiration implies the conscious application of an idea or a system from nature
[156]. In this regard, the inherent protective capacity of latexes can be applied not
only in the production of healing materials, but in the understanding of the selection
of bioactive metabolites by plants [4], [5], [7]. Moreover, one should consider the fact that as usually occurs when a plant exudate
flows out of its reservoir during plant-organism interactions, the attackers are not
exposed to single compounds but to a certain combination of them [157], [158]. Thus, latexes can also provide a clue of the potentiation mechanisms (e.g., synergism
or additive effects) of plant chemical mixtures. This would help to unravel how plants
combine these specialized metabolites to achieve bioactive mixtures for their self-defense.
In this context, once unraveled, the mechanisms of plant chemical selection and their
potentiation, the bio-inspiration concept can be potentially applied to the selection
of bioactive metabolites and the design of their chemical mixtures against pathogenic
microorganisms or insect pests. Therefore, summarizing the socioeconomic impact of
the different latex-bearing plants plus their already renowned biological activities
such as antibacterials, antifungals, cytotoxicity herbivore deterrence, and insecticidal
effects further corroborates and reinforces the use of plant latex as a source of
bioactive metabolites.
