A loline alkaloid is a member of the 1-aminopyrrolizidines (often referred to as lolines), which are bioactive with several distinct biological and chemical features. The lolines are Insecticide and insect-deterrent compounds that are produced in Pooideae infected by Endophyte Fungus Symbiosis of the genus Epichloë (anamorph species: Neotyphodium). Lolines increase resistance of endophyte-infected grasses to insect , and may also protect the infected plants from environmental stresses such as drought and spatial competition. They are alkaloids, organic compounds containing basic nitrogen atoms. The basic chemical structure of the lolines comprises a saturated pyrrolizidine ring, a primary amine at the C-1 carbon, and an internal 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 at the C-1 amine, such as methyl, formyl, and acetyl groups, yield loline species that have variable bioactivity against insects. Besides endophyte–grass symbionts, loline alkaloids have also been identified in some Eudicot; namely, Adenocarpus species (family Fabaceae) and Argyreia mollis (family Convolvulaceae).
Discovery
A member of the loline alkaloids was first isolated from the grass
Lolium temulentum 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 medium,
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,
Coleoptera,
Hymenoptera,
Lepidoptera, and
Blattodea, such as the bird cherry-oat aphid (genus
Rhopalosiphum), large milkweed bug (
Oncopeltus fasciatus), and American cockroach (
Periplaneta americana).
(1991). 047161324X, John Wiley & Sons. 047161324X
LC
50 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 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
Neurotoxin 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, 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.
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,
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 , 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
Inflorescence 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—which commonly signals predation by insects—decreases
Gene expression 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
mycelium growth for fungal reproduction (
stromata) is regulated by the expression of loline genes.
Biosynthesis
The lolines are structurally 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
.
However, feeding studies with carbon isotope–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 is assembled in several biosynthetic steps from the amino acid precursors,
Proline and
Homoserine.
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 catalysis 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
Oxidation of the
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.
