Aine L. Plant

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

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 (—)-pinene synthase. The recombinant (—)-pinene synthase catalyzes the formation of (—)-α-pinene and (—)-β-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 (—)-pinene synthase gene expression is an important element of the direct defense system deployed in Sitka spruce after insect attack.

Clone history shapes Populus drought responses

Just as animal monozygotic twins can experience different environmental conditions by being reared apart, individual genetically identical trees of the genus Populus can also be exposed to contrasting environmental conditions by being grown in different locations. As such, clonally propagated Populus trees provide an opportunity to interrogate the impact of individual environmental history on current response to environmental stimuli. To test the hypothesis that current responses to an environmental stimulus, drought, are contingent on environmental history, the transcriptome- level drought responses of three economically important hybrid genotypes—DN34 (Populus deltoides × Populus nigra), Walker [P. deltoides var. occidentalis × (Populus laurifolia × P. nigra)], and Okanese [Walker × (P. laurifolia × P. nigra)]—derived from two different locations were compared. Strikingly, differences in transcript abundance patterns in response to drought were based on differences in geographic origin of clones for two of the three genotypes. This observation was most pronounced for the genotypes with the longest time since establishment and last common propagation. Differences in genome-wide DNA methylation paralleled the transcriptome level trends, whereby the clones with the most divergent transcriptomes and clone history had the most marked differences in the extent of total DNA methylation, suggesting an epigenomic basis for the clone history-dependent transcriptome divergence. The data provide insights into the interplay between genotype and environment in the ecologically and economically important Populus genus, with implications for the industrial application of Populus trees and the evolution and persistence of these important tree species and their associated hybrids.

Wound-Induced Terpene Synthase Gene Expression in Sitka Spruce That Exhibit Resistance or Susceptibility to Attack by the White Pine Weevil

We analyzed the expression pattern of various terpene synthase (TPS) genes in response to a wounding injury applied to the apical leader of Sitka spruce (Picea sitchensis Bong. Carr.) genotypes known to be resistant (R) or susceptible (S) to white pine weevil (Pissodes strobi Peck.) attack. The purpose was to test if differences in constitutive or wound-induced TPS expression can be associated with established weevil resistance. All wounding treatments were conducted on 9-year-old R and S trees growing under natural field conditions within the range of variation for weevil R and S genotypes. Representative cDNAs of the monoterpene synthase (mono-TPS), sesquiterpene synthase (sesqui-TPS), and diterpene synthase (di-TPS) classes were isolated from Sitka spruce to assess TPS transcript levels. Based on amino acid sequence similarity, the cDNAs resemble Norway spruce (Picea abies) (−)-linalool synthase (mono-TPS; PsTPS-Linl) and levopimaradiene/abietadiene synthase (di-TPS; PsTPS-LASl), and grand fir (Abies grandis) δ-selinene synthase (sesqui-TPS; PsTPS-Sell). One other mono-TPS was functionally identified as (−)-limonene synthase (PsTPS-Lim). No significant difference in constitutive expression levels for these TPSs was detected between R and S trees. However, over a postwounding period of 16 d, only R trees exhibited significant transcript accumulation for the mono- and sesqui-TPS tested. Both R and S trees exhibited a significant accumulation of PsTPS-LASl transcripts. An assessment of traumatic resin duct formation in wounded leaders showed that both R and S trees responded by forming traumatic resin ducts; however, the magnitude of this response was significantly greater in R trees. Collectively, our data imply that the induced resinosis response is an important aspect of defense in weevil R Sitka spruce trees growing under natural conditions.

Characterization of salt-induced changes in gene expression in tomato (Lycopersicon esculentum) roots and the role played by abscisic acid

Examination of tomato (Lycopersicon esculentum Mill) root mRNA profiles by differential display-polymerase chain reaction (DD-PCR) revealed that a salt treatment induced, promoted or repressed the expression of a number of genes. The majority of the observed changes were indicative of a rapid and transient salt-induced alteration in gene expression. Twenty partial cDNAs corresponding primarily to salt-induced or up-regulated mRNAs were subsequently cloned and sequenced. The role of abscisic acid (ABA) in regulating salt-responsive gene expression in roots was explored. The DD-PCR data indicate that the majority of the salt-induced changes in the root mRNA profile occurred in an ABA-independent manner. The expression of genes corresponding to six cDNAs was shown unequivocally to be responsive to a salt treatment by RNA blot hybridization. Just two of these were responsive to exogenous ABA and, in salt-treated roots of the ABA-deficient mutant flacca, all were expressed to a level comparable to that in the wild-type. The identity of two of the salt-responsive partial cDNAs is known. The deduced amino acid sequence of one was similar to that of laccases that polymerize a variety of substrates to form resilient structures within the cell wall. One other shared amino acid sequence similarity with the C-terminus of a tobacco pathogen-induced oxygenase (PIOX). It is possible that the PIOX is involved in generating signaling molecules that mediate a general stress response.

Intraspecific variation in the Populus balsamifera drought transcriptome

Drought is a major limitation to the growth and productivity of trees in the ecologically and economically important genus Populus. The ability of Populus trees to contend with drought is a function of genome responsiveness to this environmental insult, involving reconfiguration of the transcriptome to appropriately remodel growth, development and metabolism. Here we test hypotheses aimed at examining the extent of intraspecific variation in the drought transcriptome using six different Populus balsamifera L. genotypes and Affymetrix GeneChip technology. Within a given genotype there was a positive correlation between the magnitude of water-deficit induced changes in transcript abundance across the transcriptome, and the capacity of that genotype to maintain growth following water deficit. Genotypes that had more similar drought-responsive transcriptomes also had fewer genotypic differences, as determined by microarray-derived single feature polymorphism (SFP) analysis, suggesting that responses may be conserved across individuals that share a greater degree of genotypic similarity. This work highlights the fact that a core species-level response can be defined; however, the underpinning genotype-derived complexities of the drought response in Populus must be taken into consideration when defining both species- and genus-level responses.

Testing of a heterologous, wound- and insect-inducible promoter for functional genomics studies in conifer defense

Large-scale sequencing of conifer cDNAs and targeted molecular cloning have identified many putative conifer defense genes. Expression of many of these genes is induced in response to biotic stress and some may be expressed only in a few specialized tissues or cells. Proven functional genomics approaches to test these genes involve expression of proteins in Escherichia coli or yeast for biochemical characterization or constitutive over-expression in transformed plants. Plant transformation to test functions of insect-, wound- or pathogen-induced conifer defense genes would benefit from the use of an inducible expression system. We describe here the development of a heterologous, wound- and insect-inducible gene expression system for conifers using the potato proteinase inhibitor II (pinII)-promoter. Using pinII::GUS and pinII::(E)-α-bisabolene synthase expression constructs we demonstrate localized induced gene expression in white spruce seedlings (Picea glauca). Testing of these constructs in Arabidopsis thaliana and tobacco illustrates the additional potential of the pinII-promoter to be used in tests of gene function that involve cell-specific and systemic induction.

Identification of genes that contribute to drought stress tolerance in Populus

Background The cultivation of poplars (Populus spp.) is favored for forestry and reclamation purposes all over the northern hemisphere where they represent a commercially important resource. Poplars may become a component of programs to optimize carbon sequestration however; poplars are generally regarded as drought sensitive. The patterns of episodic drought over the last decade suggest that the development of drought tolerant poplar genotypes could be a useful tool to achieve sustained forest productivity [1]. Previous reports have shown that expression of hundreds of poplar genes changes in response to drought, presenting a problem in the identification of genes that are more important than others in counteracting the harmful effects of drought [2,3]. The genus Populus contains many fast growing hybrids that show varied drought tolerance according to genotype [4]. Hence, there is genetic variation among poplar hybrids that can be used to identify genes that contribute to drought stress tolerance. Despite extensive physiological and morphological descriptions of the response of Populus to drought, little work has been undertaken to explain genotype differences at the gene level. Therefore, this research has been undertaken and its major objective is to identify the genes that contribute to drought stress tolerance in poplar by correlating the physiological responses to gene expression. These genes may potentially be used as molecular markers in the drought tolerance breeding programs.

The relationship between intra-specific variation in the Populus transcriptome, stomatal development, and the metabolome in response to drought

Drought is one of the most significant factors limiting tree growth. Trees in the genus Populus are particularly noted for their drought sensitivity; therefore, understanding the mechanisms by which these economically and ecologically important forest trees respond to drought is of paramount importance. The ability of Populus trees to contend with drought is dependent on the responsiveness of the genome, and in turn, the ability of the transcriptome to appropriately remodel growth, metabolism and development. Amassing evidence indicates that different species of Populus have divergent mechanisms and adaptations to contend with drought stress; however, individuals within a given species also display divergent drought responses. In order to investigate the intra-specific variation underpinning the drought response, we examined six genotypes of P. balsamifera. Using Affymetrix Poplar GeneChips, we found a positive correlation between the magnitude of drought-induced changes in the transcriptome and the capacity of the genotype to maintain growth. Surprisingly identifiable differences at the transcriptome were observed, and similar responses were observed within the metabolome. Although common drought responses could be identified within the species, the complexities of these responses must be taken into consideration when defining species- or genus-level drought responses.