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

Natural product

A natural product is a chemical compound or substance produced by a living organism - found in nature that usually has a pharmacological or biological activity for use in pharmaceutical drug discovery and drug design...

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

Insecticide

An insecticide is a pesticide used against insects. They include ovicides and larvicides used against the eggs and larvae of insects respectively. Insecticides are used in agriculture, medicine, industry and the household. The use of insecticides is believed to be one of the major factors behind...

 and insect-deterrent compounds that are produced in grasses

Pooideae

The Pooideae is a subfamily of the true grass family Poaceae. It includes some major cereals such as wheat, barley, oat, rye and many lawn and pasture grasses.- List :...

 infected by endophytic

Endophyte

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

 fungal

Fungus

A fungus is a member of a large group of eukaryotic organisms that includes microorganisms such as yeasts and molds , as well as the more familiar mushrooms. These organisms are classified as a kingdom, Fungi, which is separate from plants, animals, and bacteria...

 symbionts

Symbiosis

Symbiosis is close and often long-term interaction between different biological species. In 1877 Bennett used the word symbiosis to describe the mutualistic relationship in lichens...

 of the genus Epichloë

Epichloë

Epichloë species and their close relatives, the Neotyphodium species, are systemic and constitutive symbionts of cool-season grasses , and belong to the fungal family Clavicipitaceae...

(anamorphic species: Neotyphodium

Neotyphodium

Neotyphodium is a form genus containing species of endophytic fungi. These endophytes are asexual, seed-borne symbionts of cool-season grasses, and grow intercellularly throughout the aerial tissues of their hosts, including shoot apical meristems, leaf sheaths and blades, inflorescences, seeds and...

). Lolines increase resistance of endophyte-infected grasses to insect herbivore

Herbivore

Herbivores are organisms that are anatomically and physiologically adapted to eat plant-based foods. Herbivory is a form of consumption in which an organism principally eats autotrophs such as plants, algae and photosynthesizing bacteria. More generally, organisms that feed on autotrophs in...

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

Drought

A drought is an extended period of months or years when a region notes a deficiency in its water supply. Generally, this occurs when a region receives consistently below average precipitation. It can have a substantial impact on the ecosystem and agriculture of the affected region...

 and spatial competition. They are alkaloids, organic compounds containing basic nitrogen atoms. The basic chemical structure

Molecular geometry

Molecular geometry or molecular structure is the three-dimensional arrangement of the atoms that constitute a molecule. It determines several properties of a substance including its reactivity, polarity, phase of matter, color, magnetism, and biological activity.- Molecular geometry determination...

 of the lolines comprises a saturated pyrrolizidine

Pyrrolizidine

Pyrrolizidine alkaloids are a group of naturally occurring alkaloids based on the structure of pyrrolizidine. Pyrrolizidine alkaloids are produced by plants as a defense mechanism against insect herbivores. More than 660 PAs and PA N-oxides have been identified in over 6,000 plants, and about...

 ring, a primary amine at the C-1 carbon, and an internal ether

Ether

Ethers are a class of organic compounds that contain an ether group — an oxygen atom connected to two alkyl or aryl groups — of general formula R–O–R'. A typical example is the solvent and anesthetic diethyl ether, commonly referred to simply as "ether"...

 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

Substituent

In organic chemistry and biochemistry, a substituent is an atom or group of atoms substituted in place of a hydrogen atom on the parent chain of a hydrocarbon...

s at the C-1 amine, such as methyl, formyl, and acetyl

Acetyl

In organic chemistry, acetyl is a functional group, the acyl with chemical formula COCH3. It is sometimes represented by the symbol Ac . The acetyl group contains a methyl group single-bonded to a carbonyl...

 groups, yield loline species that have variable bioactivity against insects. Besides endophyte–grass symbiota, loline alkaloids have also been identified in some other plant species; namely, Adenocarpus

Adenocarpus

Adenocarpus is a genus of flowering plants in the legume family, Fabaceae. It belongs to the sub family Faboideae....

species (family Fabaceae

Fabaceae

The Fabaceae or Leguminosae, commonly known as the legume, pea, or bean family, is a large and economically important family of flowering plants. The group is the third largest land plant family, behind only the Orchidaceae and Asteraceae, with 730 genera and over 19,400 species...

) and Argyreia mollis (family Convolvulaceae

Convolvulaceae

Convolvulaceae, known commonly as the bindweed or morning glory family, are a group of about 60 genera and more than 1,650 species of mostly herbaceous vines, but also trees, shrubs and herbs.- Description :...

).

Discovery

A member of the loline alkaloids was first isolated from the grass Lolium temulentum

Lolium temulentum

Lolium temulentum, typically known as darnel, poison darnel or cockle, is an annual plant that forms part of the Poaceae family and part of the Lolium genus. The plant stem can grow up to one meter tall, with inflorescence in the ears and purple grain...

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 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

Growth medium

A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens.There are different types of media for growing different types of cells....

, 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, 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

Hemiptera is an order of insects most often known as the true bugs , comprising around 50,000–80,000 species of cicadas, aphids, planthoppers, leafhoppers, shield bugs, and others...

, Homoptera

Homoptera

Homoptera is a deprecated suborder of order Hemiptera; recent morphological studies and DNA analysis strongly suggests that the order is paraphyletic. It was therefore split into the suborders Sternorrhyncha, Auchenorrhyncha, and Coleorrhyncha....

, Coleoptera, Hymenoptera

Hymenoptera

Hymenoptera is one of the largest orders of insects, comprising the sawflies, wasps, bees and ants. There are over 130,000 recognized species, with many more remaining to be described. The name refers to the heavy wings of the insects, and is derived from the Ancient Greek ὑμήν : membrane and...

, Lepidoptera

Lepidoptera

Lepidoptera is a large order of insects that includes moths and butterflies . It is one of the most widespread and widely recognizable insect orders in the world, encompassing moths and the three superfamilies of butterflies, skipper butterflies, and moth-butterflies...

, and Blattaria, such as the bird cherry-oat aphid (genus Rhopalosiphum

Rhopalosiphum

Rhopalosiphum is a genus of aphid of the family Aphididae that includes 16 species worldwide. In addition to direct behavior as pests on host plants, some species are vectors for viral pathogens.-Species:Species include:...

), large milkweed bug (Oncopeltus fasciatus), and American cockroach

American cockroach

The American cockroach , also known as the waterbug, or misidentified as the palmetto bug , is the largest species of common cockroach, and often considered a pest. None of the Periplaneta species are endemic to the Americas; despite the name, P...

 (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

Fecundity, derived from the word fecund, generally refers to the ability to reproduce. In demography, fecundity is the potential reproductive capacity of an individual or population. In biology, the definition is more equivalent to fertility, or the actual reproductive rate of an organism or...

 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

Neurotoxin

A neurotoxin is a toxin that acts specifically on nerve cells , usually by interacting with membrane proteins such as ion channels. Some sources are more general, and define the effect of neurotoxins as occurring at nerve tissue...

 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.
Loline bioactivities show some unexpected variability with variation in their concentration in grass tissues. For example, the tall fescue endophyte, N. coenophialum

Neotyphodium coenophialum

Neotyphodium coenophialum is a systemic and seed-transmissible symbiont of Schedonorus arundinaceus , a grass endemic to Eurasia and North Africa, but widely naturalized in North America, Australia and New Zealand / Aotearoa...

, has been associated with enhanced resistance to the migratory root-endoparasitic nematode, Pratylenchus scribneri

Pratylenchus scribneri

Pratylenchus scribneri is a plant pathogenic nematode.- External links :*...

. Interestingly, at low concentrations, N-formylloline serves as a chemoattractant for P. scribneri, but acts as a repellant at higher concentrations. However, ergot alkaloids 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

Neotyphodium coenophialum

Neotyphodium coenophialum is a systemic and seed-transmissible symbiont of Schedonorus arundinaceus , a grass endemic to Eurasia and North Africa, but widely naturalized in North America, Australia and New Zealand / Aotearoa...

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

Mammal

Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...

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

Livestock

Livestock refers to one or more domesticated animals raised in an agricultural setting to produce commodities such as food, fiber and labor. The term "livestock" as used in this article does not include poultry or farmed fish; however the inclusion of these, especially poultry, within the meaning...

 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

Hepatotoxicity implies chemical-driven liver damage.The liver plays a central role in transforming and clearing chemicals and is susceptible to the toxicity from these agents. Certain medicinal agents, when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure...

, owing to a double bond

Covalent bond

A covalent bond is a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms. The stable balance of attractive and repulsive forces between atoms when they share electrons is known as covalent bonding....

 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

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

), and to increase resistance of infected grasses against drought

Drought

A drought is an extended period of months or years when a region notes a deficiency in its water supply. Generally, this occurs when a region receives consistently below average precipitation. It can have a substantial impact on the ecosystem and agriculture of the affected region...

, 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

Indole is an aromatic heterocyclic organic compound. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring. Indole is a popular component of fragrances and the precursor to many pharmaceuticals. Compounds that contain an...

 diterpenoids, 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 (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

Inflorescence

An inflorescence is a group or cluster of flowers arranged on a stem that is composed of a main branch or a complicated arrangement of branches. Strictly, it is the part of the shoot of seed plants where flowers are formed and which is accordingly modified...

 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

Plant embryogenesis

Plant embryogenesis is the process that produces a plant embryo from a fertilised ovule by asymmetric cell division and the differentiation of undifferentiated cells into tissues and organs. It occurs during seed development, when the single-celled zygote undergoes a programmed pattern of cell...

 tissues against attacks by insects. Surprisingly, exogenous application of the plant signaling compound, methyl jasmonate—which commonly signals predation by insects—decreases expression

Gene expression

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...

 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

thumb|right|Fungal myceliaMycelium is the vegetative part of a fungus, consisting of a mass of branching, thread-like hyphae. The mass of hyphae is sometimes called shiro, especially within the fairy ring fungi. Fungal colonies composed of mycelia are found in soil and on or within many other...

 growth for fungal reproduction (stromata

Stromata

The Stromata is the third in Clement of Alexandria's trilogy of works on the Christian life. Clement titled this work Stromateis, "patchwork," because it dealt with such a variety of matters...

) is regulated by the expression of loline genes.

Biosynthesis

The lolines are structurally

Molecular geometry

Molecular geometry or molecular structure is the three-dimensional arrangement of the atoms that constitute a molecule. It determines several properties of a substance including its reactivity, polarity, phase of matter, color, magnetism, and biological activity.- Molecular geometry determination...

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

Polyamine

A polyamine is an organic compound having two or more primary amino groups .This class of compounds includes several synthetic substances that are important feedstocks for the chemical industry, such as ethylene diamine , 1,3-diaminopropane , and hexamethylenediamine...

s. However, feeding studies with carbon isotope

Carbon-13

Carbon-13 is a natural, stable isotope of carbon and one of the environmental isotopes. It makes up about 1.1% of all natural carbon on Earth.- Detection by mass spectrometry :...

–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 chemical structure

Chemical structure

A chemical structure includes molecular geometry, electronic structure and crystal structure of molecules. Molecular geometry refers to the spatial arrangement of atoms in a molecule and the chemical bonds that hold the atoms together. Molecular geometry can range from the very simple, such as...

 is assembled in several biosynthetic steps from the amino acid precursor

Precursor (chemistry)

In chemistry, a precursor is a compound that participates in the chemical reaction that produces another compound. In biochemistry, the term "precursor" is used more specifically to refer to a chemical compound preceding another in a metabolic pathway....

s, L-proline

Proline

Proline is an α-amino acid, one of the twenty DNA-encoded amino acids. Its codons are CCU, CCC, CCA, and CCG. It is not an essential amino acid, which means that the human body can synthesize it. It is unique among the 20 protein-forming amino acids in that the α-amino group is secondary...

 and L-homoserine

Homoserine

Homoserine is an α-amino acid with the chemical formula HO2CCHCH2CH2OH. L-Homoserine is not one of the common amino acids encoded by DNA. It differs from the proteinogenic amino acid serine by insertion of an additional methylene group...

. In the proposed first step in loline biosynthesis, these two amino acids are coupled in a condensation reaction linking the γ-carbon in homoserine to the secondary amine in proline in a PLP–type enzyme–catalyzed

Enzyme catalysis

Enzyme catalysis is the catalysis of chemical reactions by specialized proteins known as enzymes. Catalysis of biochemical reactions in the cell is vital due to the very low reaction rates of the uncatalysed reactions....

 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 decarboxylation

Decarboxylation

Decarboxylation is a chemical reaction that releases carbon dioxide . Usually, decarboxylation refers to a reaction of carboxylic acids, removing a carbon atom from a carbon chain. The reverse process, which is the first chemical step in photosynthesis, is called carbonation, the addition of CO2 to...

s of the carboxy groups in the homoserine and proline moieties, respectively, cyclization 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

Amplified fragment length polymorphism

Amplified Fragment Length Polymorphism PCR is a PCR-based tool used in genetics research, DNA fingerprinting, and in the practice of genetic engineering. Developed in the early 1990s by Keygene, AFLP uses restriction enzymes to digest genomic DNA, followed by ligation of adaptors to the sticky...

-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

Aphid

Aphids, also known as plant lice and in Britain and the Commonwealth as greenflies, blackflies or whiteflies, are small sap sucking insects, and members of the superfamily Aphidoidea. Aphids are among the most destructive insect pests on cultivated plants in temperate regions...

, Rhopalosiphum padi, segregate in a Mendelian

Gregor Mendel

Gregor Johann Mendel was an Austrian scientist and Augustinian friar who gained posthumous fame as the founder of the new science of genetics. Mendel demonstrated that the inheritance of certain traits in pea plants follows particular patterns, now referred to as the laws of Mendelian inheritance...

 fashion. The presence of a single locus

Locus (genetics)

In the fields of genetics and genetic computation, a locus is the specific location of a gene or DNA sequence on a chromosome. A variant of the DNA sequence at a given locus is called an allele. The ordered list of loci known for a particular genome is called a genetic map...

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

Gene cluster

A gene cluster is a set of two or more genes that serve to encode for the same or similar products. Because populations from a common ancestor tend to possess the same varieties of gene clusters, they are useful for tracing back recent evolutionary history...

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

Gene expression

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...

 during loline alkaloid production, and experimental genetic tests

Reverse genetics

Reverse genetics is an approach to discovering the function of a gene by analyzing the phenotypic effects of specific gene sequences obtained by DNA sequencing. This investigative process proceeds in the opposite direction of so-called forward genetic screens of classical genetics...

 involving manipulation of selected LOL genes by RNA interference

RNA interference

RNA interference is a process within living cells that moderates the activity of their genes. Historically, it was known by other names, including co-suppression, post transcriptional gene silencing , and quelling. Only after these apparently unrelated processes were fully understood did it become...

 and gene knockout

Gene knockout

A gene knockout is a genetic technique in which one of an organism's genes is made inoperative . Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function...

 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

Enzyme

Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...

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—, for oxygenation

Redox

Redox reactions describe all chemical reactions in which atoms have their oxidation state changed....

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