Insect Attack and Wounding Induce Traumatic Resin Duct Development and Gene Expression of (—)-Pinene Synthase in Sitka Spruce

Abstract

Conifers possess inducible terpenoid defense systems. These systems are associated with the formation of traumatic resin ducts (TRD) and are underpinned by enhanced gene expression and activity of terpene synthases (TPS), enzymes responsible for oleoresin formation. We first determined that Sitka spruce (Picea sitchensis [Bong.] Carriere) had the capacity for TRD formation by mechanically wounding representative trees. We then proceeded to investigate whether the white pine weevil (Pissodes strobi Peck.), a stem-boring insect, can influence the expression of genes encoding monoterpene synthases (mono-tps) in Sitka spruce. We went on to compare this response with the effects of a simulated insect attack by drill wounding. A significant increase in mono-tps transcript level was observed in the leaders of lateral branches of weevil-attacked and mechanically wounded trees. In this study, weevils induced a more rapid enhancement of mono-tps gene expression. A full-length Sitka spruce mono-tps cDNA (PsTPS2) was isolated, expressed in Escherichia coli, and functionally identified as (Ϫ)-pinene synthase. The recombinant (Ϫ)-pinene synthase catalyzes the formation of (Ϫ)-␣-pinene and (Ϫ)-␤-pinene, both of which are known constituents of stem oleoresin in Sitka spruce and increase in abundance after weevil attack. These data suggest that increased (Ϫ)-pinene synthase gene expression is an important element of the direct defense system deployed in Sitka spruce after insect attack.Oleoresin is a complex mixture of monoterpenes, sesquiterpenes, and diterpene resin acids that provide chemical and physical protection of conifer trees against potential herbivores, stem-boring insects, and pathogens (Berryman, 1972; Bohlmann and Croteau, 1999; Phillips and Croteau, 1999; Trapp and Croteau, 2001). In Sitka spruce (Picea sitchensis [Bong.] Carrière), constitutive oleoresin is sequestered in preformed resin ducts in bark, sapwood, and needles. During the initial stages of attack by stem-boring insects, such as weevils (Curculionidae) or bark beetles (Coleopterae), this oleoresin is released and repels insects through intoxication or the formation of physical barriers. Conifers also possess inducible terpenoid defense systems. These include the formation of new traumatic resin ducts (TRD) in phloem and xylem tissue (Cheniclet, 1987; Alfaro, 1995; Nagy et al., 2000; Alfaro et al., 2002) and a hypersensitive response associated with the accumulation of terpenoids, lignin, and phenolics associated with cells surrounding an attack site (Raffa, 1991; Franceschi et al., 1998). Traumatic resinosis can be induced by a range of stimuli, including mechanical wounding, abiotic stress, insect attack, pathogen invasion, elicitor molecules derived from fungal or plant cell walls, or by treatment of trees with methyl jasmonate (MeJA; Croteau et al., 1987; Lieutier and Berryman, 1988; Nagy et al., 2000; Franceschi et al., 2002; Martin et al., 2002). Recent work with Norway spruce (Picea abies) demonstrated that differentiation of TRD in the d...

Functional Characterization of Nine Norway Spruce TPS Genes and Evolution of Gymnosperm Terpene Synthases of the TPS-d Subfamily

“…PaTPS-Myr is most similar to myrcene synthase (72% I, 80% S) from grand fir (Bohlmann et al, 1997). The third mono-TPS of this set, PaTPS-Pin, is most similar to (2)-pinene synthase (90% I, 91% S) from Sitka spruce (Byun McKay et al, 2003). The amino acid sequence of PaTPS-Lin is closest to the previously characterized terpinolene synthase (69% I, 81% S) from grand fir .…”

“…The (2)-pinene synthases of Norway spruce and grand fir both produce a greater proportion of (2)-b-pinene than (2)-a-pinene (Bohlmann et al, 1997), whereas in the closely related (2)-a-pinene synthase of Sitka spruce, (2)-a-pinene is the major product (Byun McKay et al, 2003) and in loblolly pine(2)-a-pinene is the only product Supplemental Table II). Notably, each of these still forms (2)-a-pinene as a major product and they all group closely together within the TPS-d1 cluster (Fig.…”

“…These responses include de novo formation of traumatic resin ducts in lieu of tracheids in the developing stem xylem (Franceschi et al, 2002;Martin et al, 2002), induced accumulation of terpenoid resin (Martin et al, 2002), and increased enzyme activities and elevated levels of transcripts of mono-TPS and di-TPS (Martin et al., 2002;Fäldt et al, 2003b). Mono-TPS transcripts were similarly induced in Sitka spruce (Picea sitchensis) by mechanical wounding or feeding by the spruce weevil (Pissodes strobi; Byun McKay et al, 2003). MeJA treatment also induced a rhythmic, diurnal release of volatile sesquiterpenoids and oxygenated monoterpenoids, along with methyl salicylate, from needles of Norway spruce .…”

“…Recently, a new TPS gene encoding (1)-3-carene synthase was isolated from Norway spruce (Fäldt et al, 2003b) and a (2)-apinene synthase gene was identified in Sitka spruce (Byun McKay et al, 2003). However, most spruce TPS genes, their particular biochemical functions and their contribution to chemical defense remain to be discovered and functionally characterized.…”

Induced volatiles of Medicago truncatula: molecular diversity and mechanistic aspects of a multiproduct sesquiterpene synthase from M. truncatula

“…[5] Thus, the biosynthesis of the volatiles responds in a highly complex manner to below-ground and above-ground impacts, as well as to the sequence in which microbes or herbivores infect or damage the plant. Terpenoid formation is generally assumed to be regulated at the transcript level of the TPS genes, [6] but the mode of regulation is often very complex [7,8] and needs to be studied individually with respect to the plant and the herbivore. Three putative sesquiterpenoid synthases, TPS1, TPS3 and TPS5, were therefore subcloned into the pHis8-3 expression vector and the recombinant vectors were transformed into Escherichia coli BL21-Codon Plus (DE3).…”