A loline alkaloid is a member of the 1-aminopyrrolizidines (often referred to as lolines), which are bioactive

natural product A natural product is a natural compound or substance produced by a living organism—that is, found in nature. In the broadest sense, natural products include any substance produced by life. Natural products can also be prepared by chemical s ...

s with several distinct biological and chemical features. The lolines are

insecticidal Insecticides are pesticides used to kill insects. They include ovicides and larvicides used against insect eggs and larvae, respectively. The major use of insecticides is in agriculture, but they are also used in home and garden settings, in ...

and insect-deterrent compounds that are produced in

grasses Poaceae ( ), also called Gramineae ( ), is a large and nearly ubiquitous family of monocotyledonous flowering plants commonly known as grasses. It includes the cereal grasses, bamboos, the grasses of natural grassland and species cultivated in ...

infected by

endophytic An endophyte is an endosymbiont, often a bacterium or fungus, that lives within a plant for at least part of its life cycle without causing apparent disease. Endophytes are ubiquitous and have been found in all species of plants studied to date; ...

fungal A fungus (: fungi , , , or ; or funguses) is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms. These organisms are classified as one of the tradit ...

symbionts Symbiosis (Ancient Greek : living with, companionship < : together; and ''bíōsis'': living) is any type of a close and long-term

Epichloë ''Epichloë'' is a genus of ascomycete fungi forming an endophytic symbiosis with grasses. Grass choke disease is a symptom in grasses induced by some ''Epichloë'' species, which form spore-bearing mats (stromata) on tillers and suppress the de ...

'' (

anamorph In mycology, the terms teleomorph, anamorph, and holomorph apply to portions of the life cycles of fungi in the phyla Ascomycota and Basidiomycota: *Teleomorph: the sexual reproductive stage (morph), typically a fruiting body. *Anamorph: an ase ...

species: '' Neotyphodium''). Lolines increase resistance of endophyte-infected grasses to insect

herbivore A herbivore is an animal anatomically and physiologically evolved to feed on plants, especially upon vascular tissues such as foliage, fruits or seeds, as the main component of its diet. These more broadly also encompass animals that eat ...

s, and may also protect the infected plants from environmental stresses such as

drought A drought is a period of drier-than-normal conditions.Douville, H., K. Raghavan, J. Renwick, R.P. Allan, P.A. Arias, M. Barlow, R. Cerezo-Mota, A. Cherchi, T.Y. Gan, J. Gergis, D. Jiang, A. Khan, W. Pokam Mba, D. Rosenfeld, J. Tierney, ...

and spatial competition. They are

alkaloids Alkaloids are a broad class of naturally occurring organic compounds that contain at least one nitrogen atom. Some synthetic compounds of similar structure may also be termed alkaloids. Alkaloids are produced by a large variety of organisms i ...

, organic compounds containing basic nitrogen atoms. The basic

chemical structure A chemical structure of a molecule is a spatial arrangement of its atoms and their chemical bonds. Its determination includes a chemist's specifying the molecular geometry and, when feasible and necessary, the electronic structure of the target m ...

of the lolines comprises a saturated

pyrrolizidine Pyrrolizidine is a heterocyclic organic compound. Formally, it is a saturated derivative of pyrrolizine. Pyrrolizidine forms the central chemical structure of a variety of alkaloids known collectively as pyrrolizidine alkaloids. It is one of fi ...

ring, a

primary amine In chemistry, amines (, ) are organic compounds that contain carbon-nitrogen bonds. Amines are formed when one or more hydrogen atoms in ammonia are replaced by alkyl or aryl groups. The nitrogen atom in an amine possesses a lone pair of elec ...

at the C-1 carbon, and an internal

ether In organic chemistry, ethers are a class of compounds that contain an ether group, a single oxygen atom bonded to two separate carbon atoms, each part of an organyl group (e.g., alkyl or aryl). They have the general formula , where R and R� ...

bridge—a hallmark feature of the lolines, which is uncommon in organic compounds—joining two distant ring (C-2 and C-7) carbons (see Fig. 1). Different

substituent In organic chemistry, a substituent is one or a group of atoms that replaces (one or more) atoms, thereby becoming a moiety in the resultant (new) molecule. The suffix ''-yl'' is used when naming organic compounds that contain a single bond r ...

s at the C-1 amine, such as

methyl In organic chemistry, a methyl group is an alkyl derived from methane, containing one carbon atom bonded to three hydrogen atoms, having chemical formula (whereas normal methane has the formula ). In formulas, the group is often abbreviated as ...

, formyl, and

acetyl In organic chemistry, an acetyl group is a functional group denoted by the chemical formula and the structure . It is sometimes represented by the symbol Ac (not to be confused with the element actinium). In IUPAC nomenclature, an acetyl grou ...

groups, yield loline species that have variable

bioactivity In pharmacology, biological activity or pharmacological activity describes the beneficial or adverse effects of a drug on living matter. When a drug is a complex chemical mixture, this activity is exerted by the substance's active ingredient or ...

against insects. Besides endophyte–grass symbionts, loline alkaloids have also been identified in some other plant species; namely, ''

Adenocarpus ''Adenocarpus'' is a genus of flowering plants in the family Fabaceae. It belongs to the subfamily Faboideae. The plants are broom-like shrubs with bright yellow flowers. The genus is native to the Mediterranean Basin and sub-Saharan Africa, b ...

'' species (family

Fabaceae Fabaceae () or Leguminosae,International Code of Nomen ...

) and ''Argyreia mollis'' (family

Convolvulaceae Convolvulaceae (), commonly called the bindweed, bindweeds or morning glory, morning glories, is a Family (biology), family of about 60 genera and more than 1,650 species. These species are primarily herbaceous vines, but also include trees, sh ...

Discovery

A member of the loline alkaloids was first isolated from the grass ''

Lolium temulentum ''Lolium temulentum'', typically known as darnel, poison darnel, darnel ryegrass or cockle, is an annual plant of the genus '' Lolium'' within the family Poaceae. The plant stem can grow up to one meter tall, with inflorescence in the ears and pu ...

'' and its elemental composition determined in 1892. It was initially named ''temuline'' and later renamed ''norloline''. (Reviewed by Schardl et al. (2007).) Studies in the 1950s and 1960s by Russian researchers established the name ''loline'' and identified the characteristic 2,7 ether bridge in its molecular structure. Since then the analytical methods for purification and analysis of the lolines have been refined and several different loline species have been identified in many ''

Lolium ''Lolium'' is a genus of tufted grasses in the bluegrass subfamily (Pooideae). It is often called ryegrass, but this term is sometimes used to refer to grasses in other genera. They are characterized by bunch-like growth habits. ''Lolium'' ...

'' and related grasses infected by the ''Epichloë/Neotyphodium'' (epichloae) endophytes. Lolines are absent in grass plants that do not harbor the epichloae endophytes, and not all epichloae produce the lolines. Because of the very intimate association of plant and endophyte and difficulties to reproduce the symbiotic conditions ''in vitro'', it was long unknown if the fungus was the producer of the lolines, or if they were synthesized by the plant in response to endophyte infection. In 2001, it was demonstrated that the endophyte ''Neotyphodium uncinatum'' produces lolines in some chemically defined

growth media A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss ''Physcomitrella patens''. Differe ...

, which suggests that the endophyte is also the producer of the lolines in the grass plant. The lolines have also been reported from some plants in several

plant families Plants are the eukaryotes that form the kingdom Plantae; they are predominantly photosynthetic. This means that they obtain their energy from sunlight, using chloroplasts derived from endosymbiosis with cyanobacteria to produce sugars fro ...

, suggesting a more widespread occurrence of these compounds in nature.

Mechanism of action

Lolines are insecticidal and deterrent to a broad range of insects, including species in the

Hemiptera Hemiptera (; ) is an order of insects, commonly called true bugs, comprising more than 80,000 species within groups such as the cicadas, aphids, planthoppers, leafhoppers, assassin bugs, bed bugs, and shield bugs. They range in size from ...

Coleoptera Beetles are insects that form the Taxonomic rank, order Coleoptera (), in the superorder Holometabola. Their front pair of wings are hardened into wing-cases, elytra, distinguishing them from most other insects. The Coleoptera, with about 40 ...

Hymenoptera Hymenoptera is a large order of insects, comprising the sawflies, wasps, bees, and ants. Over 150,000 living species of Hymenoptera have been described, in addition to over 2,000 extinct ones. Many of the species are parasitic. Females typi ...

Lepidoptera Lepidoptera ( ) or lepidopterans is an order (biology), order of winged insects which includes butterflies and moths. About 180,000 species of the Lepidoptera have been described, representing 10% of the total described species of living organ ...

, and

Blattodea Blattodea is an order (biology), order of insects that contains cockroaches and termites. Formerly, termites were considered a separate order, Isoptera, but genetics, genetic and molecular evidence suggests they evolved from within the cockroach ...

, such as the bird cherry-oat aphid (genus ''

Rhopalosiphum ''Rhopalosiphum'' is a genus of aphid of the family Aphididae that includes 17 species worldwide. Apart from sucking the phloem of host plants and thereby being treated in agriculture as pests, some species are vectors for viral plant pathogen ...

''),

large milkweed bug ''Oncopeltus fasciatus'', known as the large milkweed bug, is a medium-sized hemipteran (true bug) of the family Lygaeidae.Attisano, A. (2013) Oosorption and migratory strategy of the milkweed bug,''Oncopeltus fasciatus. Animal Behaviour 86(3):65 ...

(''Oncopeltus fasciatus''), and

American cockroach The American cockroach (''Periplaneta americana'') is the largest species of common cockroach, and often considered a pest. In certain regions of the U.S. it is colloquially known as the waterbug, though it is not a true waterbug since it is not ...

(''Periplaneta americana''). LC₅₀ values of ''N''-formylloline or ''N''-acetylloline from grass seed extracts are 1-20 μg/ml for aphids and milkweed bugs and impair insect development and

fecundity Fecundity is defined in two ways; in human demography, it is the potential for reproduction of a recorded population as opposed to a sole organism, while in population biology, it is considered similar to fertility, the capability to produc ...

and cause avoidance of loline-containing grass tissues. However, results of feeding tests with grass extracts are occasionally difficult to interpret due to the presence of other endophyte alkaloids in these extracts, and the exact mechanisms of the insecticidal actions of the lolines are unknown. The lolines may be

neurotoxic Neurotoxicity is a form of toxicity in which a biological, chemical, or physical agent produces an adverse effect on the structure or function of the central and/or peripheral nervous system. It occurs when exposure to a substance – specifical ...

to the insects, and differences in the chemical groups at the C-1-amine result in different levels of insect toxicity; for example, ''N''-formylloline (see Fig. 2), which occurs in higher concentrations in endophyte-infected grass plants, has greater insect toxicity than some other lolines, which occur at lower concentrations in the grass plant. N-formylloline

Loline bioactivities show some unexpected variability with variation in their concentration in grass tissues. For example, the tall fescue endophyte, '' N. coenophialum'', has been associated with enhanced resistance to the migratory root-endoparasitic nematode, '' Pratylenchus scribneri''. At low concentrations, ''N''-formylloline serves as a chemoattractant for ''P. scribneri'', but acts as a repellant at higher concentrations. However,

ergot alkaloid Ergoline is a core structure in many alkaloids and their synthetic derivatives. Ergoline alkaloids were first characterized in ergot. Some of these are implicated in the condition of ergotism, which can take a convulsive form or a gangrenous for ...

s also have repellent and immobilizing effects on ''P. scribneri'', and an endophyte of perennial ryegrass lacking lolines, and genetically engineered to produce no ergot alkaloids, exhibits resistance to this nematode. Therefore, the relative importance of the loline and ergot alkaloids to nematode resistance remains unclear. Many epichloae endophytes—including '' N. coenophialum'' symbiotic with '' Lolium arundinaceum'' (syn. ''Festuca arundinacea'', tall fescue)—also produce ergot alkaloids that are toxic to mammalian herbivores. The ergot alkaloids occur at relatively low concentrations in the plant and are often difficult to detect analytically. By contrast, the lolines frequently accumulate to very high levels in grass tissues, and were, therefore, initially associated also with toxicity to

mammal A mammal () is a vertebrate animal of the Class (biology), class Mammalia (). Mammals are characterised by the presence of milk-producing mammary glands for feeding their young, a broad neocortex region of the brain, fur or hair, and three ...

ian herbivores. Specifically, the lolines were thought to be responsible for toxic symptoms called ''fescue toxicosis'' displayed by

livestock Livestock are the Domestication, domesticated animals that are raised in an Agriculture, agricultural setting to provide labour and produce diversified products for consumption such as meat, Egg as food, eggs, milk, fur, leather, and wool. The t ...

grazing on grasses infected by ''N. coenophialum''. However, subsequently it was demonstrated that only the endophyte-produced ergot alkaloids are responsible for the symptoms of ''fescue toxicosis'' (or ''summer syndrome''), and not the lolines which, even at high doses, have only very small physiological effects on mammalians feeders. Another group of alkaloids, the senecio-type alkaloids, are produced by various plants and like the lolines, the ''senecio alkaloids'' possess a pyrrolizidine ring structure. Unlike the lolines, however, the senecio alkaloids exhibit strong

hepatotoxicity Hepatotoxicity (from ''hepatic toxicity'') implies chemical-driven liver damage. Drug-induced liver injury (DILI) is a cause of acute and chronic liver disease caused specifically by medications and the most common reason for a drug to be withdr ...

, owing to a

double bond In chemistry, a double bond is a covalent bond between two atoms involving four bonding electrons as opposed to two in a single bond. Double bonds occur most commonly between two carbon atoms, for example in alkenes. Many double bonds exist betw ...

between C-1 and C-2 in their ring structure. This double bond is absent in the lolines, explaining the lack of hepatotoxicity of this group of compounds. The lolines have been suggested to inhibit seed germination or growth of other plants (

allelopathy Allelopathy is a biological phenomenon by which an organism produces one or more biochemicals that influence the germination, growth, survival, and reproduction of other organisms. These biochemicals are known as allelochemicals and can have ben ...

), and to increase resistance of infected grasses against

, but such effects have not been substantiated under more natural conditions of cultivation or in habitats.

Production and distribution in the grass plant

Lolines are produced by several grass–endophyte symbioses involving epichloae species, often along with other bioactive metabolites including ergot alkaloids and

indole Indole is an organic compound with the formula . Indole is classified as an aromatic heterocycle. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered pyrrole ring. Indoles are derivatives of indole ...

diterpenoid Diterpenes are a class of terpenes composed of four isoprene units, often with the molecular formula C20H32. They are biosynthesized by plants, animals and fungi via the HMG-CoA reductase pathway, with geranylgeranyl pyrophosphate being a primar ...

s, and the unusual pyrrolopyrazine alkaloid, peramine, which is not found in other biological communities or organisms. The lolines are produced at levels, however, that can exceed 10 mg/g grass tissue (ranging from 2–20,000 μg/g), exceeding the concentrations of the other endophyte alkaloids by >1000-fold. Lolines produced in the grasses ''

Lolium pratense ''Lolium pratense'', meadow fescue is a perennial species of grass, which is often used as an ornamental in gardens, and is also an important forage crop. It grows in meadows, roadsides, old pastures, and riversides on moist, rich soils, especial ...

'' (syn. ''Festuca pratensis'', meadow fescue) and tall fescue infected by ''N. uncinatum'' and ''N. coenophialum'' (see Fig. 3), respectively, exhibit variable concentrations in grass tissues. Higher loline concentrations (100–1000 μg/g) are present in the seeds and in younger leaf tissues, and the lolines display seasonal changes in concentration levels throughout the plant. The periodical appearance of tissues with high loline concentrations, such as flowering stems and seeds, contributes to this seasonal variation. Loline concentrations often increase in grass tissues regrown after defoliation and clipping of plants, suggesting an inducible defense response mechanism, involving both symbiotic partners. However, this increase appears to be due to higher loline levels in younger leaves compared to older leaves, but loline increases resembling inducible plant defenses have also been reported. Variation of loline concentration with the developmental stage of specific grass tissues suggests regulation of ''in planta'' loline distributions, providing greater protection of newly grown or embryonic tissues against attacks by insects. Surprisingly, exogenous application of the plant signaling compound,

methyl jasmonate Methyl jasmonate (abbreviated MeJA) is a volatile organic compound used in plant defense and many diverse developmental pathways such as seed germination, root growth, flowering, fruit ripening, and senescence. Methyl jasmonate is derived from ja ...

—which commonly signals predation by insects—decreases

expression Expression may refer to: Linguistics * Expression (linguistics), a word, phrase, or sentence * Expression (mathematics), Symbolic description of a mathematical object * Fixed expression, a form of words with a specific meaning * Idiom, a type of ...

of the genes for the lolines. The factors that control loline production vary also among endophyte-infected grass tissues: whereas plant-supplied amino acids that are loline precursors limit accumulation of lolines in many grass tissues, their production in tissues that bear external

mycelial Mycelium (: mycelia) is a root-like structure of a fungus consisting of a mass of branching, thread-like hyphae. Its normal form is that of branched, slender, entangled, anastomosing, hyaline threads. Fungal colonies composed of mycelium are fou ...

growth for fungal reproduction (

stromata The ''Stromata'' (), a mistake for ''Stromateis'' (Στρωματεῖς, "Patchwork," i.e., ''Miscellanies''), attributed to Clement of Alexandria (c. 150 – c. 215), is the third of a trilogy of works regarding the Christian life. The oldest ...

) is regulated by the expression of loline genes.

Biosynthesis

The lolines are

structurally A structure is an arrangement and organization of interrelated elements in a material object or system, or the object or system so organized. Material structures include man-made objects such as buildings and machines and natural objects such as ...

similar to pyrrolizidine alkaloids produced by many plants, notably the necine ring containing a

tertiary amine In chemistry, amines (, ) are organic compounds that contain carbon-nitrogen bonds. Amines are formed when one or more hydrogen atoms in ammonia are replaced by alkyl or aryl groups. The nitrogen atom in an amine possesses a lone pair of elec ...

. This led to the early hypothesis that the biosynthesis of the lolines is similar to that of the plant pyrrolizidines, which are synthesized from

polyamine A polyamine is an organic compound having two or more amino groups. Alkyl polyamines occur naturally, but some are synthetic. Alkylpolyamines are colorless, hygroscopic, and water soluble. Near neutral pH, they exist as the ammonium derivatives. ...

s. However, feeding studies with

carbon isotope Carbon (6C) has 14 known isotopes, from to as well as , of which only and are stable. The longest-lived radioisotope is , with a half-life of years. This is also the only carbon radioisotope found in nature, as trace quantities are formed ...

–labeled amino acids or related molecules in pure cultures of the loline-producing fungus ''N. uncinatum'' recently demonstrated that the loline alkaloid pathway is fundamentally different from that of the plant pyrrolizidines. The basic loline

is assembled in several biosynthetic steps from the amino acid

precursor Precursor or Precursors may refer to: *Precursor (religion), a forerunner, predecessor ** The Precursor, John the Baptist Science and technology * Precursor (bird), hypothesized genus of fossil birds that was composed of fossilized parts of unre ...

s, L-proline and L-homoserine. In the proposed first step in loline biosynthesis, these two amino acids are coupled in a

condensation reaction In organic chemistry, a condensation reaction is a type of chemical reaction in which two molecules are combined to form a single molecule, usually with the loss of a small molecule such as water. If water is lost, the reaction is also known as a ...

linking the γ-carbon in homoserine to the

secondary amine In chemistry, amines (, ) are organic compounds that contain carbon-nitrogen bonds. Amines are formed when one or more hydrogen atoms in ammonia are replaced by alkyl or aryl groups. The nitrogen atom in an amine possesses a lone pair of elec ...

in proline in a PLP–type enzyme–catalyzed reaction to form the loline intermediate, ''N''-(3-amino-3-carboxy)propylproline (NACPP). Further steps in loline biosynthesis are thought to proceed with sequential PLP-enzyme-catalyzed and

oxidative Redox ( , , reduction–oxidation or oxidation–reduction) is a type of chemical reaction in which the oxidation states of the reactants change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is ...

decarboxylation Decarboxylation is a chemical reaction that removes a carboxyl group and releases carbon dioxide (CO2). Usually, decarboxylation refers to a reaction of carboxylic acids, removing a carbon atom from a carbon chain. The reverse process, which is ...

s of the

carboxy group In organic chemistry, a carboxylic acid is an organic acid that contains a carboxyl group () attached to an R-group. The general formula of a carboxylic acid is often written as or , sometimes as with R referring to an organyl group (e.g. ...

s in the homoserine and proline moieties, respectively,

cyclization A cyclic compound (or ring compound) is a term for a compound in the field of chemistry in which one or more series of atoms in the compound is connected to form a ring. Rings may vary in size from three to many atoms, and include examples where ...

to form the core loline ring structure, and oxidation of the C-2 and C-7 carbons to give the oxygen bridge spanning the two pyrrolizidine rings. Genetic studies agree with the biosynthetic routes established in the precursor-feeding experiments. AFLP-based studies using crosses between strains of the endophyte, ''Epichloë festucae'', that differ in the capacity to produce lolines, show that loline production and protection of the grass, ''Lolium giganteum'', from feeding by the

aphid Aphids are small sap-sucking insects in the Taxonomic rank, family Aphididae. Common names include greenfly and blackfly, although individuals within a species can vary widely in color. The group includes the fluffy white Eriosomatinae, woolly ...

, ''Rhopalosiphum padi'', segregate in a

Mendelian Mendelian inheritance (also known as Mendelism) is a type of biological inheritance following the principles originally proposed by Gregor Mendel in 1865 and 1866, re-discovered in 1900 by Hugo de Vries and Carl Correns, and later popularized ...

fashion. The presence of a single

locus Locus (plural loci) is Latin for "place". It may refer to: Mathematics and science * Locus (mathematics), the set of points satisfying a particular condition, often forming a curve * Root locus analysis, a diagram visualizing the position of r ...

for loline production was later confirmed by the finding that loline-producing epichloae endophytes contain a

gene cluster A gene cluster is a group of two or more genes found within an organism's DNA that encode similar peptide, polypeptides or proteins which collectively share a generalized function and are often located within a few thousand base pairs of each othe ...

(''LOL'' cluster) of at least eleven genes. The ''LOL'' genes are greatly and coordinately

upregulated In biochemistry, in the biological context of organisms' regulation of gene expression and production of gene products, downregulation is the process by which a cell decreases the production and quantities of its cellular components, such as RNA ...

during loline alkaloid production, and experimental genetic tests involving manipulation of selected ''LOL'' genes by

RNA interference RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression. Historically, RNAi was known by ...

and

gene knockout Gene knockouts (also known as gene deletion or gene inactivation) are a widely used genetic engineering technique that involves the gene targeting, targeted removal or inactivation of a specific gene within an organism's genome. This can be done t ...

have directly confirmed the involvement of two of the ''LOL'' genes in loline biosynthesis. These tests and similarities in the peptide sequences of the proteins encoded by these genes to known

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

s indicate that one gene, termed ''lolC'', is likely required for the first step in loline biosynthesis (condensation of L-proline and L-homoserine for NACPP formation), and another gene, ''LolP'' —likely encoding a

cytochrome P450 monooxygenase Cytochromes P450 (P450s or CYPs) are a superfamily of enzymes containing heme as a cofactor that mostly, but not exclusively, function as monooxygenases. However, they are not omnipresent; for example, they have not been found in ''Escherichi ...

—, for

oxygenation Oxygenation may refer to: * Oxygenation (environmental), a measurement of dissolved oxygen concentration in soil or water * Oxygen saturation (medicine), The percent of hemoglobin saturated by oxygen, usually in arterial blood. * Water oxygenatio ...

of one methyl group on the C-1 amine of ''N''-methylloline, which gives the most abundant loline in many grass–endophyte symbionts, ''N''-formylloline.

References

{{Reflist, colwidth=30em Pyrrolizidine alkaloids Mycotoxins Insecticides