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First published online November 22, 2006; 10.1104/pp.106.089425 Plant Physiology 143:410-424 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Aminocyclopropane Carboxylic Acid Synthase Is a Regulated Step in Ethylene-Dependent Induced Conifer Defense. Full-Length cDNA Cloning of a Multigene Family, Differential Constitutive, and Wound- and Insect-Induced Expression, and Cellular and Subcellular Localization in Spruce and Douglas Fir1,2,[W],[OA]Michael Smith Laboratories (S.G.R., J.W.H., S.J., J.B.) and Departments of Botany and Forest Sciences (J.B.), University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4; and School of Biological Sciences, Washington State University, Pullman, Washington 99164–4236 (J.W.H., V.R.F.)
In conifer stems, formation of chemical defenses against insects or pathogens involves specialized anatomical structures of the phloem and xylem. Oleoresin terpenoids are formed in resin duct epithelial cells and phenolics accumulate in polyphenolic parenchyma cells. Ethylene signaling has been implicated in the induction of these chemical defenses. Recently, we reported the cloning of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) from spruce (Picea spp.) and Douglas fir (Pseudotsuga menziesii). ACO protein was constitutively expressed in Douglas fir and only weakly induced upon wounding. We now cloned seven full-length and one near full-length cDNA representing four distinct 1-aminocyclopropane-1-carboxylic acid synthases (ACS; ACS1, ACS2, ACS3, and ACS4) from spruce and Douglas fir. Cloning of ACS has not previously been reported for any gymnosperm. Using gene-specific, quantitative real-time polymerase chain reaction, we measured constitutive expression for the four ACS genes and the single-copy ACO gene in various tissues of Sitka spruce (Picea sitchensis) and in white spruce (Picea glauca) somatic embryos. ACO and ACS4 were ubiquitously expressed at high levels; ACS1 was predominantly expressed in developing embryos and ACS2 and ACS3 were expressed only at very low levels. Insect attack or mechanical wounding caused strong induction of ACS2 and ACS3 in Sitka spruce bark, a moderate increase in ACO transcripts, but had no effect on ACS1 and ACS4. ACS protein was also strongly induced following mechanical wounding in Douglas fir and was highly abundant in resin duct epithelial cells and polyphenolic parenchyma cells. These results suggest that ACS, but not ACO, is a regulated step in ethylene-induced conifer defense.
Conifers in the pine family (Pinaceae) represent the most economically important forest trees and are fundamental species to many ecosystems across the northern Hemisphere. Because of their extensive geographic range and often very long life spans of up to several hundred years, conifers are exposed to a wide variety of potential herbivores, such as insects and mammals, as well as to free-living or insect-associated fungal pathogens. In general, conifers demonstrate resistance or tolerance to most herbivores, although some specialized conifer insect pests, such as certain bark beetles and weevils, cause substantial damage and mortality to individual trees and to large conifer forests (Alfaro et al., 2002  ; Raffa et al., 2005; Seybold et al., 2006).
As a resistance strategy against pathogens and herbivores, conifers have evolved a variety of anatomical and chemical defense systems (Franceschi et al., 2005
Despite the importance of induced terpenoid and phenolic defenses in conifer resistance, relatively little is known about insect- or pathogen-induced signaling events that lead to activation of resin duct and PP cells or their de novo formation. In a series of studies using exogenously applied ethylene (Hudgins and Franceschi, 2004
Ethylene is well known for regulating many processes in plant biology, such as cell differentiation, growth, development, reproduction, and response to environmental stress (for review, see Bleecker and Kende, 2000
A substantial amount of information exists on ACS in various angiosperm species. However, to our knowledge, cloning and characterization of ACS has not been reported for a gymnosperm. ACS is a cytosolic enzyme that requires pyridoxal phosphate as a cofactor (Bleecker and Kende, 2000 Here, we describe the FL-cDNA or near FL-cDNA cloning and sequence characterization of eight ACSs from white spruce, interior spruce (Picea glauca x engelmannii), and Douglas fir, representing four distinct ACS or ACS-like genes in these conifers. We provide detailed quantitative and gene-specific expression analyses of ACS and ACO transcripts showing differential patterns of spatial and temporal expression in constitutive as well as wound- and insect-induced Sitka spruce tissues. Using Douglas fir, we demonstrate wound-induced ACS protein expression and show cellular and subcellular localization of ACS in the cytosol of specialized epithelial cells of cortical resin ducts, PP cells, and ray parenchyma cells of wound-induced bark tissue. Our results suggest that ACS is a regulated step in ethylene signaling in induced cellular and chemical defense in conifers.
cDNA Cloning of ACS Genes from Spruce and Douglas Fir
To obtain initial sequence information for ACS genes from conifers, a TBLASTN search of the spruce expressed sequence tag (EST) and FL-cDNA databases of the Treenomix project (Ralph et al., 2006b
ACS cDNA sequences from Douglas fir were obtained by reverse transcription (RT)-PCR using sets of primers both within the UTRs and at the ORF termini based upon PgeACS2, PgeACS3a, and PgACS4 transcripts with two tissue sources as templates; wounded bark (including phloem and cambium) and green shoot tips. These three primer combinations produced distinct PCR products in both tissue sources, which, upon further purification and sequencing of PCR products from the bark RNA template, combined with 5' RACE to extend a truncated PmACS2 cDNA, provided unique cDNAs of 1,734 (PmACS2), 1,907 (PmACS3), and 1,687 bp (PmACS4; Table I). PmACS2 possesses 5' and 3' UTRs of 146 and 208 bp, respectively, in length, and when translated encodes a 460-amino acid protein with a predicted pI value of 5.79 and molecular mass of 52.0 kD. PmACS4 possesses 5' and 3' UTRs of 61 and 36 bp, respectively, in length, and when translated encodes a 530-amino acid protein with a predicted pI value of 7.49 and molecular mass of 58.9 kD. Pairwise sequence alignments of the deduced amino acid sequences indicate that the putative starting Met and stop codon of PmACS2 overlaps with PgeACS2, as does PmACS4 and PgACS4, and stop codons are present upstream within the 5' UTRs of both Douglas fir proteins, suggesting that PmACS2 and PmACS4 are FL-cDNAs. The spruce and Douglas fir ACS2 proteins share 84.1% amino acid identity (Table II) and 85.1% nucleotide identity between the overlapping transcript regions. PgACS4 and PmACS4 share 85.3% amino acid identity (Table II) and 93.3% nucleotide identity between the overlapping transcript regions. PmACS3 encodes a predicted protein of 399 amino acids, which, based on pairwise sequence alignment with PgeACS3a/PgeACS3b, likely represents a truncated ACS protein due to a frameshift. The putative starting Met of PmACS3 overlaps with that of PgeACS3a/PgeACS3b; however, the C terminus of PmACS3 is approximately 130 amino acids shorter than the complete ORF in spruce. Multiple sets of primers were also designed based on the spruce PgACS1 sequence in an attempt to obtain a related transcript from Douglas fir by RT-PCR, but we were unable to obtain even a partial transcript using bark or green shoot tip tissue as templates. In retrospect, this was likely due to the fact we did not analyze Douglas fir embryogenic tissue because PgACS1 is primarily expressed in spruce embryos (see below).
We performed maximum likelihood analysis using 28 ACS protein sequences from spruce, Douglas fir, Arabidopsis, and tomato to analyze the evolutionary relationships between conifer (gymnosperm) and angiosperm ACS genes. Multiple protein sequence alignments were performed using ClustalW and then manually adjusted to define a conserved sequence of about 430 amino acids. Using the neighbor-joining algorithm, we generated a phylogenetic tree (Fig. 2
), which shows that six of the eight conifer ACS proteins form a distinct cluster, suggesting that ACS genes from gymnosperms and angiosperms have diverged significantly over time since separation of these major land plant lineages. Amino acid identity among the five full-length conifer proteins in this group (i.e. excluding PmACS3) ranges from 56.7% to 98.5%, and from 44.7% to 62.7% between these conifer ACS proteins and Arabidopsis ACS proteins (i.e. excluding AtACS10 and AtACS12; Table II). The remaining two conifer ACS proteins, PgACS4 and PmACS4, group more closely with AtACS10 and AtACS12 (Fig. 2), both of which have been demonstrated to complement E. coli aminotransferase mutants (Yamagami et al., 2003
Constitutive Transcript Profiles of Spruce ACS and ACO Assessed by Real-Time PCR The existence of a multigene ACS family in species of spruce and Douglas fir may suggest different roles of individual ACSs in constitutive or induced processes. In an attempt to illustrate possible differences in spatial patterns of RNA expression, the relative constitutive abundance of the four different spruce ACS transcripts (ACS1–ACS4) was quantified using real-time PCR in total RNA isolated from different stem tissues (cortex, phloem, cambium, and xylem), young lateral shoot tips, and root tissues from Sitka spruce. We also measured ACS expression in developing somatic embryos of white spruce. Real-time PCR expression data were normalized to housekeeping gene control levels (i.e. eukaryotic translation initiation factor [TIF]5A). Gene-specific ACS primers were designed based on FL-cDNA sequences for PgACS1, PgeACS2, PgeACS3a/b, and PgACS4. ACS1 transcript expression was most dominant in developing somatic embryos (Fig. 3A ; please note different scale for ACS1 in embryos), with the next most abundant tissue expression in roots at 33.0-fold lower levels than embryos. ACS1 was only marginally detectable in shoots, cortex, phloem, and cambium (about 4 or 5 orders of magnitude lower than TIF5A), with no detectable transcript expression in xylem (Fig. 3A). ACS2 was expressed at moderate to low levels (about 2–4 orders of magnitude lower than TIF5A) in all tissues examined, with highest expression in roots and cortex, and no detectable transcript in xylem. ACS3 showed a pattern of tissue expression similar to that of ACS2, but was present at 5.6- to 30.0-fold lower levels in each tissue (about 4 or 5 orders of magnitude lower than TIF5A), except for developing embryos where ACS2 was only 2.2-fold more abundant (Fig. 3A). ACS3 expression was highest in roots and cortex, with no detectable transcripts in xylem. In contrast to the spatial patterns of transcript abundance for other spruce ACS genes, ACS4 was ubiquitously expressed at high levels (about 1 or 2 orders of magnitude lower than TIF5A) in all tissues, with greatest transcript abundance in the cortex and roots and lowest in developing somatic embryos.
To complement monitoring the spatial distribution of the four spruce ACS transcripts, we also examined transcript levels for ACO, which is the final step in the ethylene biosynthesis pathway and appears to be represented by a single gene in spruce (Hudgins et al., 2006 ). ACO was ubiquitously expressed at high levels (about 1 or 2 orders of magnitude lower than TIF5A), with the transcript 4.7- to 21.7-fold more abundant in shoots than any other tissues (Fig. 3A).
Having established the spatial patterns of expression for the four ACS and one ACO transcript in spruce, we next examined these genes for a possible role in the wound- and insect-induced defense response of Sitka spruce by assessing their temporal patterns of expression in response to treatment. In brief, 2-year-old Sitka spruce saplings were subjected on their stems to either feeding by adult white pine weevils or mechanical wounding using a needle. Transcript profiles were monitored in bark tissues using real-time PCR 2, 6, and 48 h after the onset of treatment and compared to untreated control tissues. Data are presented as transcript abundance normalized to TIF5A levels (Fig. 3B) and as fold induction relative to untreated control tissues (Table III ). In general, three of the five transcripts examined (i.e. ACS2, ACS3, and ACO) showed statistically significant increases in abundance following mechanical wounding and/or weevil feeding. All three transcript species were rapidly induced and had reached peak levels at 2 h (ACS2 and ACS3) or 6 h (ACO), following mechanical wounding. Both ACS2 and ACS3 transcripts were expressed at relatively low levels in control bark, with increases of 155.9-fold and 174.2-fold, respectively, 2 h after mechanical wounding (Fig. 3B; Table III). The levels of both transcript species diminished over time, but were still 8.1-fold (ACS2) and 3.7-fold (ACS3) more abundant than in control tissues 48 h after wounding. In contrast to the temporal pattern of rapid induction following mechanical wounding, induction by weevil feeding was slower and of lower magnitude, with peak induction at 48 h for ACS2 (48.9-fold) and ACS3 (27.9-fold; Fig. 3B; Table III). The differing temporal response to insect herbivory and mechanical wounding treatments likely reflects the very slow, but continuous, feeding by stem-boring weevils compared with a single artificial wounding event.
Unlike ACS2 and ACS3, ACO transcripts were present at relatively high levels in untreated control bark and showed only a moderate (3.9-fold), although equally rapid, increase in transcript abundance following mechanical wounding that also diminished with time (Fig. 3B; Table III). The response of ACO to weevil feeding was less pronounced, with slightly reduced transcript abundance after 2 and 6 h of weevil feeding and only a small increase (2.4-fold) after 48 h. The two remaining transcript species examined, ACS1 and ACS4, did not follow the above pattern of induction following wounding or insect damage. ACS1 was expressed at marginally detectable constitutive levels in bark from untreated control trees, with no consistent pattern of altered transcript levels following either treatment (Fig. 3B; Table III). ACS4 was the most constitutively abundant of the ACS and ACO transcripts examined in control bark tissue and demonstrated a weak, and not always statistically significant, down-regulation in response to both treatments at all time points (Fig. 3B; Table III).
In previous work (Hudgins and Franceschi, 2004 ACS antibodies showed strong signals with a single band on western blots of protein extracts from wound-induced stem bark, but did not yield protein signals with extracts from nonwounded control tissue (Fig. 4 ). The molecular mass of the protein detected was approximately 55 kD on SDS-PAGE, consistent with the predicted molecular mass of conifer ACS proteins (Table I). ACS protein was detected at the first time point of the induced samples at 10 h following wounding, and protein levels remained increased relative to controls over the entire time course until 96 h after wounding (Fig. 4). Next, we tested the specific localization of ACS in wound-induced bark tissues. Immunocytochemistry using ACS antibody showed the most extensive presence of ACS in two different cell types associated with terpenoid and phenolic chemical defenses in wounded bark tissues, namely, epithelial cells that line the inner surface of activated cortical resin ducts (Fig. 5A ) and active PP cells that are characterized by their large and densely stained vacuoles (Fig. 5C), respectively. As a negative control, the nonimmune serum did not generate any reproducible labeling of any cell type (Fig. 5B). In wound-induced bark, ACS labeling was also detected in ray parenchyma cells (Fig. 5, C and D). These cells connect the xylem and outer bark via the cambial zone. Labeling was also detected within the cambial zone itself and was found highly abundant in a ray initial captured in mitosis with nearly complete separation of the duplicated set of chromosomes (Fig. 5D). In control sections of noninduced bark tissues, ACS protein was not detected above the background signal in any cell type (data not shown), confirming the results of western-blot analysis.
High-resolution subcellular localization detected ACS protein in the cytoplasm of PP cells (Fig. 5E), but not in the membranes, cell walls, vacuoles, or other organelles. In the lower magnification images shown in Figure 5, C and D, the antibody also appears to label some nuclear regions of ray parenchyma cells; however, this may be a result of vacuole compression of cytoplasm around the nuclear domains.
Conifer trees are able to survive hundreds of years and often dominate large ecosystems due to their apparently highly successful defense systems, which protects them against most herbivores and pathogens. Most prominent among the chemical defenses of conifers in the pine family are the constitutive and inducible terpenoid oleoresins (Martin and Bohlmann, 2005 ; Keeling and Bohlmann, 2006) that are formed and accumulate in specialized anatomical structures, such as resin ducts, resin blisters, or resin cells in stems, roots, and needles. Another highly abundant, although less well characterized, chemical defense system of conifers are phenolics in bark (Franceschi et al., 2005). Both terpenoid and phenolic chemical defenses are inducible in response to wounding, insect feeding or oviposition, or fungal inoculation. The induced biosynthesis and accumulation of terpenoids and phenolics can be associated with activation of existing resin ducts and PP cells, respectively, or in the case of terpenoid accumulation, with the de novo formation of TDs (Franceschi et al., 2005; Martin and Bohlmann, 2005; Keeling and Bohlmann, 2006).
Over the last few years, it has been shown that two phytohormones, the octadecanoid compound MeJA and ethylene, induce similar chemical and cellular defense responses in conifers as do wounding, insect attack, or fungal inoculation (e.g. Martin et al., 2002
We identified five different ACS cDNA sequences by mining spruce EST and FL-cDNA databases, which, at more than 200,000 sequences, are second only to loblolly pine (Pinus taeda) as the largest gymnosperm sequence resource (Ralph et al., 2006b
Phylogenetic comparison of the spruce and Douglas fir ACS protein sequences with angiosperm ACS proteins demonstrates that conifers ACS1, ACS2, and ACS3 are relatively closely related to the majority of previously known and functionally characterized angiosperm ACSs (Fig. 2). Within this larger group of gymnosperm and angiosperm proteins, the conifer (gymnosperm) ACS forms a distinct cluster. This topology of the ACS tree suggests that angiosperm and gymnosperm ACSs share a common ancestor and multiple ACS forms evolved by gene duplication and sequence divergence in both lineages after the separation of angiosperms and gymnosperms. Like Arabidopsis, both spruce and Douglas fir also have more distantly related ACS-like proteins (i.e. PgACS4 and PmACS4). The Arabidopsis AtACS10 and AtACS12 proteins have been tentatively classified as aminotransferases (Yamagami et al., 2003
Unlike the apparently single-copy conifer ACO gene (Hudgins et al., 2006
To test whether all four conifer ACS genes are associated with induced defense, we examined transcript abundance in response to both mechanical wounding and weevil feeding over a time course of 2 to 48 h in Sitka spruce bark tissues (Fig. 3B). ACS1 was expressed at very low levels in control bark tissue, with only minor fluctuations in abundance following mechanical wounding or insect feeding. A similar pattern was seen with ACS4, which was abundantly expressed in both treated and control bark. In contrast, ACS2 and ACS3 were rapidly (2 h) and strongly (>150-fold) induced following mechanical wounding, with a similar, although slower, response to weevil feeding. Interestingly, both of these transcripts were present at low levels in untreated control bark, as well as all other tissues examined for constitutive expression, suggesting that ACS2 and ACS3 genes likely function in the regulation of induced ethylene signaling in conifer defense (Hudgins and Franceschi, 2004
Western-blot analysis confirmed strong induction of ACS at the protein level in Douglas fir bark following wounding (Fig. 4), consistent with ACS transcript accumulation in Sitka spruce and also with previous findings of wound-induced ethylene evolution in this system (Hudgins and Franceschi, 2004
In conclusion, rapid accumulation of transcripts and proteins for enzymes involved in ethylene biosynthesis following wounding or insect feeding in conifer bark tissues supports the importance of this phytohormone in signaling inducible defense responses in conifer bark. The previously demonstrated high constitutive expression of ACO protein in conifer bark and its weak inducibility following wounding (Hudgins and Franceschi, 2004
Plant and Insect Materials and Treatments
Three-year-old Douglas fir (Pseudotsuga menziesii) saplings were from a commercial nursery (Lawyer Nursery). Growth conditions and details of treatment and tissue harvest of Douglas fir for ACS protein expression and immunolocalization were exactly as previously described (Hudgins et al., 2006
To monitor spruce ACS transcript expression in response to wounding or insect attack, Sitka spruce (Picea sitchensis) trees (clone FB3-425; derived from somatic embryogenesis [CellFor]) were grown outside at the UBC greenhouse as described by Ralph et al. (2006a)
Total RNA was isolated from all tissues, except for somatic embryos, according to the protocol of Kolosova et al. (2004)
A TBLASTN search of the Treenomix spruce ESTs and the FL-cDNA database (Ralph et al., 2006b To clone ACS cDNAs from Douglas fir, 15 µg total RNA from wounded bark and green shoot tips were treated with DNaseI (Invitrogen) according to the manufacturer's instructions, followed by RT using SuperScript II reverse transcriptase (Invitrogen) with an oligo(dT)18 primer according to the manufacturer's instructions. Efficiency of cDNA synthesis was assessed by gel electrophoresis. For PCR, primers were designed within the UTR and at the ORF termini based upon PgeACS2 (DF_WS00944_L14_F1 and DF_WS00944_L14_R2; DF_WS00944_L14_INT1 and DF_WS00944_L14_R1), PgeACS3a (WS00956_O07_F1 and WS00956_O07_R1), and PgACS4 (DF_WS00729_O03_F1 and DF_WS00729_O03_R1; DF_WS00729_O03_F2 and DF_WS00729_O03_R2; Supplemental Table S1). PCR reactions were performed using 100 ng of cDNA template, 200 µM each dNTP, 10 mM Tris-HCl (pH 9.0), 50 mM KoAC, 1.5 mM MgSO4, 1 unit of SuperTaq Plus polymerase (Ambion), and 0.5 µM of each primer in a final volume of 20 µL. The following standard thermal profile was used for all PCRs: 95°C for 2 min; 35 cycles of 95°C for 15 s; 60°C or 52°C for 30 s; and 68°C for 2 min; then 68°C for 10 min. PCR products were cloned into pCR2.1-TOPO vector (Invitrogen) and transformed into E. coli electroMAX DH10B cells (Invitrogen). Single colonies were isolated, plasmid DNA sequenced using –21M13 forward and M13 reverse primers, and additional primers designed to internal sequences (WS00956_O07_R3, DF_WS00729_O03_INT1, and DF_WS00729_O03_INT2) to yield the FL-cDNA Douglas fir PmACS4 and partial cDNAs PmACS2 and PmACS3. To obtain a FL-cDNA clone for PmACS2, the FirstChoice RLM-RACE system (Ambion) was used to generate a 5'-RACE PCR template from wounded bark as described above. Gene-specific primers for PmACS2 were DF_WS00944_L14_RACE_OUTER and DF_WS00944_L14_RACE_INNER (Supplemental Table S1). PCR products were cloned into pCR2.1-TOPO vector (Invitrogen) and transformed into E. coli electroMAX DH10B cells (Invitrogen). Single colonies were isolated and plasmid DNA sequenced using –21M13 forward and M13 reverse primers to yield the FL-cDNA PmACS2.
Predictions for pI and molecular mass were made using the entire ORF and the pI/Mw tool at Expasy (www.expasy.org/tools/pi_tool.html). Amino acid multiple sequence alignments were made with ClustalW (www.ebi.ac.uk/clustalw) and Boxshade (bioweb.pasteur.fr/seqanal/interfaces/boxshade.html) and manually adjusted to define a conserved ACS sequence of approximately 430 amino acids prior to maximum likelihood analysis using Phyml, version 2.4.1 (Guindon and Gascuel, 2003
For constitutive tissue expression profiling, 9 µg of total RNA from each tissue was treated with DnaseI (Invitrogen), divided into three aliquots of 3 µg each, and independent cDNA synthesis reactions were performed using SuperScript II reverse transcriptase (Invitrogen) with an oligo(dT)18 primer according to the manufacturer's instructions. For analysis of induced tissues, 3 µg of total RNA were pooled from each of five trees for each treatment and time point, treated with DNaseI, divided into three aliquots of 5 µg, and converted to cDNA in three independent reactions. Efficiency of each cDNA synthesis was assessed individually by gel electrophoresis. Gene-specific PCR primers were designed (Supplemental Table S1) using a stringent set of criteria, including predicted melting temperature of 64°C ± 2°C, primer lengths of 20 to 24 nucleotides, guanine-cytosine content of 40% to 60%, and PCR amplicon lengths of 110 to 280 bp. Primer specificity (single product of expected length) was confirmed by analysis on 2% agarose gel, by melting curve analysis, and for at least one PCR reaction per gene, by sequence verification of PCR amplicons. Primers for spruce eukaryotic TIF5A (GenBank accession no. DR448953; EST IS0013_F24) served as a quantification control. PCR was performed in optical 96-well plates with a DNA Engine Opticon2 continuous fluorescence detector (MJ Research) using SYBR Green to monitor double-strand DNA synthesis. Reactions contained 7.5 µL DyNAmo HS SYBR Green quantitative PCR kit master mix (Finnzymes), 10 ng cDNA, and 0.3 µM of each primer in a final volume of 15 µL. Reactions with the cDNA template replaced by nuclease-free water or 10 ng of non-reverse-transcribed RNA were run with each primer pair as a control. The following standard thermal profile was used for all PCRs: 95°C for 15 min; 40 cycles of 95°C for 15 s; 60°C for 30 s; and 72°C for 30 s; then 72°C for 10 min. The fluorescence signal was captured at the end of each cycle and melting curve analysis was performed from 65°C to 95°C with data capture every 0.2°C during a 1-s hold. Data were analyzed using Opticon Monitor analysis software, version 2.02 (MJ Research). Quantification of each transcript in each cDNA source consisted of at least three independent (different 96-well plates) technical replicates. To generate a baseline-subtracted plot of the logarithmic increase in fluorescence signal (
Procedures for protein extractions from the 3-year-old Douglas fir saplings, western-blot analysis, microscopy, and immunocytochemistry were exactly as described in Hudgins et al. (2006) Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers EF179148 (PgACS1), EF179149 (PgeACS2), EF179150 (PgeACS3a), EF179151 (PgeACS3b), EF179152 (PgACS4), EF179153 (PmACS2), EF179154 (PmACS3), and EF179155 (PmACS4).
The following materials are available in the online version of this article.
We thank Dr. Barry Jaquish, Dr. John King, and Dr. Alvin Yanchuk from the British Columbia Ministry of Forests for access to white spruce and Sitka spruce trees, and Dr. David Ellis, CellFor Inc., for interior spruce seedlings and spruce somatic embryos; Dr. Rene I. Alfaro from the Canadian Forest Service for access to white pine weevils; Dr. Kim Rensing for technical assistance with spruce tissue sectioning, Mr. Ian Cullis for somatic embryo propagation, and Mr. David Kaplan for greenhouse support. Confocal and TEM microscopy was performed in the WSU Electron Microscopy Center. Received September 3, 2006; accepted November 17, 2006; published November 22, 2006.
1 This work was supported by the Natural Sciences and Engineering Research Council of Canada, Genome Canada, Genome British Columbia, the province of British Columbia, the Canadian Foundation for Innovation, and the British Columbia Knowledge and Development Funds (grants to J.B.). J.B. is an E.W.R. Steacie Memorial Fellow of the Natural Sciences and Engineering Research Council of Canada. 
2 This paper is dedicated to the memory of Dr. Vincent R. Franceschi.
3 These authors contributed equally to the paper. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jörg Bohlmann (bohlmann{at}interchange.ubc.ca).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.089425 * Corresponding author; e-mail bohlmann{at}interchange.ubc.ca; fax 604–822–2114.
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ABSTRACT
RESULTS
-corrected replicates (log L = –11,299). ACS nomenclature and GenBank protein accession numbers for Arabidopsis and conifer proteins are provided in 
Ct = Cttranscript – CtTIF5A. Transcript abundance was obtained from the equation (1 + E)–
0.05; **, P 
P32 dGTP incorporation. After removal of unincorporated nucleotides using gel filtration columns (Microspin S-300 HR columns; Amersham-Pharmacia Biotech), the resulting cDNA smear was resolved using a vertical 1% agarose alkaline gel and visualized using a Storm 860 phosphor imager (Amersham-Pharmacia Biotech). 
