JXB Advance Access published online on January 13, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erl246
RESEARCH PAPER |
Microarray gene expression profiling of developmental transitions in Sitka spruce (Picea sitchensis) apical shoots
1Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4
2Department of Botany, University of British Columbia, Vancouver, BC, Canada V6T 1Z4
3Department of Forest Sciences, University of British Columbia, Vancouver, BC, Canada V6T 1Z4
* To whom correspondence should be addressed. E-mail: cdouglas{at}interchange.ubc.ca
Received 17 July 2006; Revised 20 September 2006 Accepted 16 October 2006
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Abstract |
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The apical shoot drives the yearly new stem growth of conifer trees, is the primary site for the establishment of chemical and physical defences, and is important in establishing subsequent perennial growth. This organ presents an interesting developmental system, with growth and development progressing from a meristematic tip through development of a primary vascular system, to a base with fully differentiated and lignified secondary xylem on the inside and bark tissue with constitutive defence structures such as resin, polyphenolic phloem parenchyma cells, and sclereids on the outside. A spruce (Picea spp.) microarray containing approximately 16.7K unique cDNAs was used to study transcript profiles that characterize the developmental transition in apical shoots of Sitka spruce (Picea sitchensis) from their vegetative tips to their woody bases. Along with genes involved in cell-wall modification and lignin biosynthesis, a number of differentially regulated genes encoding protein kinases and transcription factors with base-preferred expression patterns were identified, which could play roles in the formation of woody tissues inside the apical shoot, as well as in regulating other developmental transitions associated with organ maturation. Preferential expression of known conifer defence genes, genes encoding defence-related proteins, and genes encoding regulatory proteins was observed at the apical shoot tip and in the green bark tissues at the apical shoot base, suggesting a commitment to constitutive defence in the apical shoot that is co-ordinated with rapid development of secondary xylem.
Key words: Conifer ESTs, lipid transfer protein, resin duct, secondary cell wall, terpenoid secondary metabolism, white pine weevil (Pissodes strobi), xylem
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Introduction |
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Sitka spruce (Picea sitchensis) is native to the west coast of North America, and Sitka spruce trees originating from coastal British Columbia (Canada) are an important timber species in planted forests in Scotland and other parts of Europe. Compared with other conifers, Sitka spruce has a fast growth rate, it can attain very large sizes, and its fibre and wood are of very high quality (Burns, 1990; Petersen et al., 1997). However, planting of Sitka spruce in Canada is now severely restricted by white pine weevil (Pissodes strobi) infestations (King et al., 1997).
In the last few years, a large-scale conifer genomics programme has been developed that has facilitated studies on the defence and resistance mechanisms of Sitka spruce against white pine weevil (Miller et al., 2005; Ralph et al., 2006b) as well as studies on secondary xylem development in conifers. The shoot apical leader is an important organ for the study of both processes.
A key feature of conifer defence is the development of cellular and anatomical structures for chemical defence in the apical shoot. These anatomical structures include primary resin ducts filled with terpenoid oleoresin that remain active in the bark and xylem for long periods of time, phloem polyphenolic parenchyma (PP) cells which accumulate vacuolar phenolics, and lignified sclereids (Franceschi et al., 2005). While much is known about the genes and enzymes of terpenoid biosynthesis in conifer defence (Keeling and Bohlmann, 2006), as well as their inducible expression (Miller et al., 2005), relatively little is known about their levels of constitutive expression relative to the early development of resin ducts in the apical shoot. Also, very little is known about expression profiles of genes potentially associated with the early development of PP cells or sclereids in the spruce apical shoot.
Wood formation is a characteristic feature of tree growth and development that requires the development of a vascular cambium and subsequent formation of specialized xylogenic tissue at the apex of woody plants that drives secondary growth (Mellerowicz et al., 2001; Plomion et al., 2001; Savidge, 2001). As cells differentiate during secondary growth, and make the transition from cambial derivatives to mature tracheary elements, a progression of cell differentiation processing towards the inside of the stem is established. This progression, which involves cell elongation, formation of a cellulose-rich secondary cell wall, lignin deposition, and programmed cell death, is under tight developmental control. Transcription factors and other regulatory proteins are therefore likely to play critical roles in modulating the expression of appropriate suites of genes required for cell expansion, secondary wall formation, and lignin deposition (Anterola et al., 2002; Newman et al., 2004; Schrader et al., 2004; Ehlting et al., 2005).
Many recent studies, using genomic tools, have addressed the genetic regulation of tree growth, wood formation, and fibre quality. For instance, gene expression profiles have been described for different stages of developing secondary xylem in poplar (Hertzberg et al., 2001; Schrader et al., 2004), heartwood development in black locust (JM Yang et al., 2003, 2004), differentiating secondary xylem and compression wood formation in pine (SH Yang et al., 2004), in vitro differentiation of xylem vessel elements in Zinnia (Demura et al., 2002), secondary xylem development in Eucalyptus (Paux et al., 2004), and interfascicular fibre formation in Arabidopsis (Oh et al., 2003; Ehlting et al., 2005). Genes involved in the synthesis of lignin and cellulose, the main components of secondary cell walls, have been characterized in the genome of Arabidopsis, and in trees such as poplar, aspen, and pine (Anterola et al., 2002; Doblin et al., 2002; Boerjan et al., 2003; Gardiner et al., 2003; Joshi, 2003; Raes et al., 2003; Djerbi et al., 2004). Nevertheless, the contributions of most genes preferentially expressed in woody tissues to the control of wood formation and wood quality remain undefined. In particular, the transition from primary to secondary growth at the apex of trees has not been studied extensively.
The apical shoot of conifer trees drives the new annual stem growth, is the primary site for the establishment of long-lasting chemical and anatomical defences, and lays the foundation for subsequent perennial and secondary stem growth. This organ presents an interesting developmental system, with growth and development progressing from a meristematic tip that controls primary growth, through development of the primary vascular system, to a woody base with fully differentiated and lignified secondary xylem on the inside and a complex bark tissue with constitutive resin ducts, polyphenolic phloem parenchyma cells, and scereids on the outside. As far as is known, the patterns of gene expression associated with development of conifer apical shoots have not been described in the literature. Using a newly developed 16.7K element spruce cDNA microarray (S Ralph, J Bohlmann, unpublished data), the transcripts associated with developmental transition from primary to secondary growth and the parallel development of constitutive defence systems in apical shoots of Sitka spruce, from their green shoot tips to their woody bases, were profiled. This work revealed sets of structural and regulatory genes, which are likely to be involved in these processes, and apparent conservation of many genes with those in angiosperm systems.
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Materials and methods |
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Plant material
Seedlings of Sitka spruce [Picea sitchensis (Bong.) Carriere] clone FB3-425 were derived from somatic embryo cultures and were generously provided by Dr David Ellis (CellFor, Victoria, BC, Canada). Maintenance of seedlings and growth conditions were as previously described (Miller et al., 2005; Ralph et al., 2006b). Two-year-old Sitka spruce trees were grown outdoors in 2 gallon pots containing a peat:fine bark:pumice mix (2:1:1), balanced to a target pH of 5.5–6.5 with limestone and dolomite. During the summer months the trees were watered daily and received a 15–15–15 Cal-Mag fertilizer at 120 ppm (Scotts, Marysville, OH, USA) every other day. During the winter months, the trees were watered very infrequently, and received no fertilizer. For the third year of growth, the trees were transferred to 6 gallon pots.
In early September 2003, before the onset of dormancy, the apical shoot representing the current year's growth on 2-year-old trees showed an obvious progression between primary vascular growth at the tip and secondary xylem growth at the base. Similar-sized apical shoots (13–18 cm in length) were harvested from three trees and hand-sectioned at 2 cm intervals for histological characterization of the developmental stages along the apical shoot. The extent of lignification was visualized in hand sections by UV autofluorescence using a fluorescent microscope (DMR, Leica, Wetzlar, Germany) with excitation filters at 340–380 nm and emission at 450+ nm. Images were processed using the Openlab software (version 4.0.2; Improvision, MA, USA). Tissues were then collected from 20 trees for cDNA microarray profiling. As shown in Fig. 1A, the very top 0.5 cm of the apical shoot was discarded, and a 2 cm section just below it was excised, its needles were removed, and the stem section was immediately frozen in liquid nitrogen. The collected sections were pooled. Likewise, a 3 cm section was collected from the base of the apical shoot, c. 3 cm above the junction with the previous year's growth. Needles were removed, bark was peeled off, and the remaining woody stem containing secondary xylem was immediately frozen in liquid nitrogen.
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In September 2004, apical shoot tissues were collected in a similar manner from the same clonal group of trees (now 3 years old) (Fig. 1B, C). Tip and base tissues were collected from 10 trees as before (Fig. 1B). Additional 2–3-cm-long segments were taken from another 10 trees at similar positions at the tip and base of the apical shoot, as well as at two approximately central positions separated by 1–2 cm, depending on the overall length of the particular apical shoot. These intermediate segments were harvested and frozen in liquid nitrogen without separating bark and xylem (Fig. 1C). Small (0.5 cm) segments were also collected into fixative (4% formaldehyde; Canemco, Ontario, Canada) in 50 mM PIPES buffer (pH 7.2). After passage through a dehydration series in ethanol (30%, 50%, 70%, 80%, 95%, and three times 100%), these were embedded in LR White (Canemco) in a progression series with 100% ethanol (1:3, 1:1, 3:1, and 100% resin), under vacuum to remove bubbles. The embedded blocks were trimmed, and sectioned into 2 µm sections using a Leica microtome (model Ultracut T). Sections were visualized under light microscopy, using a Zeiss model AxioPlan 2 microscope, and UV autofluorescence observed using a mercury UV source. Micrographs were captured directly into the Northern Eclipse program (EmPix Imaging Inc., Ontario, Canada).
RNA isolation
RNA was isolated from frozen tissues following the method of Chang et al. (1993) with modifications. Approximately 1 g of tissue was ground to a powder in liquid N2, and extracted with 14 ml extraction buffer (100 mM TRIS-HCl, 25 mM EDTA, 2 M NaCl, 2% CTAB, 2% PVP, 0.5 µg ml–1 spermidine, 2% ß-mercaptoethanol, pH 8, pre-warmed to 65 °C), vortex mixed, and an equal volume of chloroform:isoamyl alcohol (24:1 v/v) was added and the samples centrifuged at 3000 g (Sorvall RT-7 tabletop) for 20 min. The upper phase was re-extracted with 15 ml of chloroform: isoamyl alcohol and centrifuged. RNA was precipitated from the combined aqueous phases overnight at 4 °C by adding a one-quarter volume 10 M LiCl. RNA was pelleted by centrifugation for 30 min at 3000 g as above and resuspended in 1–2 ml SSTE buffer (1 M NaCl, 0.5% SDS, 10 mM TRIS-HCl, 1 mM EDTA, pH 8). RNA samples were extracted with chloroform:isoamyl alcohol, and RNA precipitated by the addition of one-tenth volume 5 M NaCl and 2 volumes EtOH at –20 °C overnight. After pelleting by centrifugation at 4 °C, RNA was rinsed with 70% EtOH, and resuspended in DEPC-treated water. RNA concentration was determined spectrophotometrically (Pharmacia UltroSpec 3000), and an aliquot visualized by agarose gel electrophoresis. RNA quality was assessed by first-strand cDNA synthesis using MMLV reverse transcriptase (Gibco-BRL, Gaithersburg, USA) with the addition of [P32]dGTP, as described (Ralph et al., 2006b).
Microarray hybridizations
The 16.7K spruce microarray contains 16 700 cDNA elements in addition to negative controls, and spots corresponding to positive spikes and orientation markers. This array shares many features with a previously described spruce 9.7K cDNA microarray (Ralph et al., 2006a) and will be fully described elsewhere (S Ralph and J Bohlmann, unpublished data). First strand synthesis reactions incorporating a dendrimer-trapping oligonucleotide were conducted as described in the Dendrimer 350 kit (Genisphere) with minor modifications as described by Ralph et al. (2006b). Microarray pre-treatments, hybridizations, and washes were carried out according to the Genisphere kit instructions with modifications as described by Ralph et al. (2006b). For these experiments, the hybridization mix was prepared using the Dendrimer 350 kit SDS-based hybridization buffer, complemented with LNA blocker included in the kit (3 µl per reaction), salmon sperm DNA (2 µg per reaction, Sigma), Cy5-labelled GFP (orientation marker), and the cDNA samples for a total volume of 45 µl. Hybridizations were carried out at 60 °C for 16 h, and the second dendrimer hybridizations were carried out at 60 °C for 3 h and processed as described by Ralph et al. (2006b). For comparisons between apical shoot tip and base tissue collected in 2003, hybridizations were repeated 10 times with pooled tissue, with an equal number of dye swaps. For the 2004 tip versus base biological replicate, six hybridizations with dye swaps were performed using pooled tissue. For comparison of apical shoot tip to base in the 2004 samples without removal of the bark (Fig. 1C), six hybridizations with dye swaps were carried out. Hybridized slides were scanned on a Scan Array Express (Perkin Elmer, Foster City, CA, USA). For slides from tissues harvested in 2003, laser settings were at 90%, the Cy3 channel was scanned for most slides at a photomultiplier tube (PMT) setting of 69, and the Cy5 at a setting of 80. For slides from tissues harvested in 2004, settings were Cy3 PMT 68 and Cy5, 78. Scan intensities were comparable between each set of slides for a given hybridization.
Image processing and data analysis
Scanned images were processed using Imagene (v. 5.0) extraction software (BioDiscovery, Marina Del Rey, USA) as described by Ralph et al. (2006b). For all analyses, the median pixel intensities for each spot were used. Further analyses were performed excluding control elements using customized scripts for R and Bioconductor (R Development Core Team, www.r-project.org). For background correction, the mean of the lowest 10% of spot foreground intensities from a particular subgrid was taken as the background for that subgrid, and subtracted from the foreground intensity of each spot in the same subgrid. Each experiment consisted of comparing apical shoot base with apical shoot tip. A paired t-test on the background corrected, normalized set of intensity differences for each unique gene or cDNA in each experiment was performed. Normalization for each array independently was achieved by passing the two sets of intensities from the two channels of the arrays to the variance stabilizing transformation (Huber et al., 2002) function in the Bioconductor package of the same name. The difference between signal intensities derived from shoot base and shoot tip was calculated and fold-change differentials were calculated by exponentiating the mean of the differences (Huber et al., 2002). Statistical robustness of differential intensities from each gene being assessed was based on comparison of the paired t-statistics obtained from the set of differences shoot base–shoot tip to the tabulated values of t-statistics (i.e. parametric P-values) and by choosing limiting values for fold-difference (>2-fold difference with a P-value <0.01 in the 2003A experiment).
Validation of differentially expressed genes by real-time (RT)-PCR
Appropriate reference genes for quantitative RT-PCR, WS00912_N13 (hypothetical protein) and WS0109_C05 (peroxisomal targeting signal receptor), were identified by screening the microarray data for cDNAs whose signals across all arrays remained apparently unchanged (fold-difference ratios between 0.99 and 1.01), and also displayed very low standard deviations for technical replicates. Presence of these transcripts was confirmed by RT-PCR to be consistently stable across the samples profiled (Table 1). Details of quantitative RT-PCR analysis of spruce transcripts have been described previously (Ralph et al., 2006b). In brief, total RNA (18 µg) from the 2004B and 2004C samples was treated with DNase I (Invitrogen, Carlsbad, CA, USA). Absence of DNA in the treated RNA (10 ng) was confirmed by PCR using primers for WS00912_N13. Next, RNA (three reactions of 4 µg each per sample) was reverse transcribed and cDNA (10 ng) was analysed by PCR in a total volume of 20 µl, in the presence of 10 µl DyNAmo SYBR Green Mastermix (FinnZymes, Finland) and 0.3 µM each of a forward and a reverse primer (see Supplementary Table S5 at JXB online). Primers of 20–24 nucleotides in length were designed to amplify a gene-specific 160–200 bp fragment of the target cDNA (usually in the 3'-untranslated region) with a Tm of 60 °C. Reactions were carried out in triplicate in an MJR Opticon2 RT-PCR machine with an initial step of 15 min at 95 °C, followed by 40 cycles of 10 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C. Each cycle was followed by a data-acquisition step. After the last cycle and a final 10 min extension at 72 °C, a melting curve was measured from 65 °C to 95 °C, with readings every 0.2 °C and holding for 1 s. Data analysis of the results was carried out by first adjusting threshold cycles manually, and subtracting the baseline fluorescence from cycles 3–9, and then determining individual efficiency values for each well using the LinReg program (Raemakers et al., 2003). The 
CT values between woody stem and tip were determined for each gene and for the two references, and fold change in expression determined from the CT ratio between them (Pfaffl, 2001). When comparing expression along the apical shoot without removing bark, the fold-difference ratios were compared with the level of expression at the tip. In this case, the apical shoot was separated into four approximately equal segments along its length, usually with 1.5–2 cm between them, covering the apical shoot from the tip to the base.
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Phylogenetic analysis
Twelve spruce full-length cDNAs for putative lipid transfer proteins (LTPs) represented on the array were identified by the presence of seven characteristic conserved Cys residues. Alignment of their deduced amino acid sequences to an Arabidopsis LTP gene (At3g13870) was carried out using DIALIGN (http://www.infobiogen.fr/services/analyseq/cgi-bin/dialign2_in.pl). The aligned sequences were edited manually, and a section covering a highly divergent N-terminal portion of the proteins was excluded from the analysis, so that the alignment covered a conserved region spanning most of the protein (c. 97 amino acids), starting at a conserved Glu residue five amino acids upstream of the first conserved Cys residue that forms part of the characteristic signature of the LTP family (Arondel et al., 2000). A phylogeny was reconstructed using the PHYML maximum likelihood method (PHYML, http://www.lirmm.fr/
guindon/phyml/body.html) based on a default NJ tree, JTT amino acid evolution model, and generation of 100 bootstraps. |
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Anatomy of Sitka spruce apical shoots
In order to define appropriate developmental stages to be compared by gene expression profiling, spruce apical shoots were first hand-sectioned at 1 cm intervals from the top to the base, and different segments examined by light and fluorescence microscopy (Fig. 2). Sections from apical shoots harvested in 2003 and 2004 showed a rapid progression from individual vascular bundles at the top of the apical shoot, visualized by UV autofluorescence, to secondary xylem and the beginnings of a woody stem at the base (Fig. 2B, C). Micrographs of thin sections revealed additional details of the anatomy, including the presence of constitutive, axial resin ducts and PP cells in the bark (Fig. 2D, E). In the xylem, the transition to secondary growth was already detected in sections at about 2 cm below the top segment (data not shown). Extensive lignification of secondary cell walls visualized by UV autofluorescence was in the lower sections of the leader (Fig. 2C).
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Expression profiling of spruce apical shoot sections
Gene expression profiling was carried out on the tip and base apical shoot sections in three separate experiments (2003A, 2004B, and 2004C; Fig. 1). In each experiment, a direct comparison was made of the gene expression profiles in the apical shoot tip, which included all tissues, and the woody stem at the base of the apical shoot by co-hybridizing Cy3- and Cy5-labelled probes derived from RNA samples from both tissues. Experiments 2003A and 2004B were identical except that the trees being sampled were 1 year older in 2004B, and in these experiments, samples from the woody base were enriched for secondary xylem by removal for the green bark tissue. In the 2004C experiment, the same material was used as in 2004B, but the bark was not removed at the base of the apical shoot, so that gene expression data could be obtained for the apical shoot base containing all tissues (secondary xylem and bark). Functional annotation of array elements was assigned according to BLAST searches against TAIR (The Arabidopsis Information Resource; www.arabidopsis.org) Arabidopsis peptide set, with a BLASTX expect value (E) <1e–05 threshold, as described by Ralph et al. (2006b).
The fold differential transcript abundance [‘fold change’ (FC)] values for all array elements for all three experiments, arranged according to preferred expression at the apical shoot base or tip, are shown in Supplementary Tables S3 and S4 at JXB online. Overall, the microarray data were comparable between the two years (experiments 2003A and 2004B), as shown by the year-to-year similarity of FC ratios and trends for hybridization to most of the cDNA elements (Tables 2–4; see Supplementary Tables S1–S4 at JXB online). For the 2003A data set, a total of 3522 genes were differentially expressed over 2-fold between the two apical shoot segments (P-value <0.01), and 2652 such genes were observed in 2004B. Of these two gene sets, 930 (35% of the 2004B set) were differentially expressed over 2-fold (P <0.01) in both years of the replicated experiment (2003A and 2004B). A higher percentage was found when only the most highly differentially expressed genes were considered. For example, of the 343 genes in experiment 2004B that had an FC value of 5-fold or greater, 311 (90%) displayed an FC value >2 in the replicate experiment, 2003A.
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Interestingly, by contrast to the 2652 differentially expressed genes detected in the 2004B experiment that excluded bark from the apical shoot base, in experiment 2004C, where bark tissue was included (Fig. 1C), the number of genes differentially expressed between tip and base was reduced to only 610, due at least partially to the exclusion of genes encoding defence-related proteins expressed both at the shoot tip and in the bark tissue of the apical shoot base. Many trends in differential expression of genes involved in wood formation in common with experiments 2003A and 2004B were still apparent, especially for the most highly differentially expressed genes in the 2003A and 2004B datasets.
Patterns of differential gene regulation were generally comparable between experiments 2003A and 2004B and the specificity of expression patterns within gene families was also reproducible. For instance, there are 39 cDNAs from the xyloglucan endo-glycosyl transferase (XET) gene family represented on the array, and they group into 27 different apparent gene family members, based on >90% sequence identity. Only two cDNAs (WS00813_I07 and WS0087_M10) representing two distinct gene family members showed strong preferential expression in the leader stem base in the 2003A experiment (FC >5; see Supplementary Table S1 at JXB online) and this specificity in XET gene expression was found again in the subsequent year (2004B), and in the bark-included experiment (experiment 2004C; see Supplementary Table S1 at JXB online). Likewise, 15 cDNAs on the array were annotated as encoding members of the arabinogalactan family of proteins (AGP), and these fall into six groups, based on at least 90% sequence identity at the nucleotide level (see Supplementary Table S2 at JXB online). Of these, a single group containing three cDNAs very similar in sequence showed marked preferential expression in the xylem-enriched apical shoot base in both the 2003A and 2004B experiments, with a similar trend in the 2004C experiment in which bark was included in apical shoot base samples (see Supplementary Table S2 at JXB online). These data show that reproducible differential expression of genes, and of individual members of multi-gene families, can be readily detected when comparing the bases of apical shoots with the tips.
Representative cDNAs corresponding to genes and gene families of particular biological interest (e.g. cell wall and lignin formation, regulation, defence) were selected from the genes found to be consistently differentially expressed in experiments 2003A and 2004B and are presented in Tables 2–4. Also included in these selections are some genes that, while differentially expressed by at least 2-fold with P-values of 
0.01 in the 2003A experiment, met slightly lower but still stringent criteria for differential expression in the less replicated 2004B experiment (P-value for differential expression generally 0.05). All other differentially expressed array elements are found in the supplementary data (see Supplementary Tables S3 and S4 at JXB online).
Secondary cell wall synthesis, carbohydrate metabolism, and lignin biosynthesis
Genes encoding enzymes involved in secondary cell wall synthesis, carbohydrate metabolism, and lignin biosynthesis were strongly differentially expressed in secondary xylem-enriched samples at the base of apical shoots (experiments 2003A and 2004B; Table 2). These include genes encoding cellulose synthase subunits, sucrose synthase, XET, fasciclin-like AGP, and family 8 glucosyl transferase. Also preferentially expressed at the xylem-enriched apical shoot bases were genes which collectively represent almost every step in lignin biosynthesis: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate CoA ligase (4CL), p-coumarate 3-hydroxylase (C3H), hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase (HCT), cinnamoyl CoA reductase (CCR), caffeoyl-CoA O-methyltransferase (CCoOMT), caffeic acid O-methyltransferase (COMT), and cinnamyl alcohol dehydrogenase (CAD), as well as members of the shikimate pathway that provides precursors for lignin and other phenolic pathways. Other genes whose functions could be related to cellular processes involved in secondary xylem differentiation, secondary wall formation, and lignin deposition, such as proteases, laccases, and aquaporins were also more highly expressed in secondary xylem-enriched tissue at the stem base (experiments 2003A and 2004B; Table 2). This group also includes a number of apparent spruce orthologues of Arabidopsis genes that have been shown to be co-expressed during secondary cell wall formation in Arabidopsis, such as the fasciclin-like AGP gene mentioned above, a germin-like protein, a chitinase-like protein (family 19), and a COBRA-like 4 protein. Additional differentially expressed genes representative of other functional categories are also shown in Table 2.
Regulatory proteins and transcription factors at the shoot base
Transcripts with consistent up-regulation in the xylem-enriched woody stem base (experiments 2003A and 2004B) included genes encoding potential signalling proteins and transcription factors (Table 3). Of 136 cDNAs for transcription factors on the array (annotated by similarity to Arabidopsis; http://arabidopsis.med.ohio-state.edu/AtTFDB; see Supplementary Tables S3 and S4 at JXB online), eight were differentially expressed (Table 3). A Myb gene family member (WS00712_A21) with marked similarity to AtMyb43 showed >10-fold higher expression in the xylem-enriched apical shoot base, which is the highest differential expression ratio observed for any transcription factor on the array. Another Myb gene family member (WS00917_H19) highly up-regulated at the shoot base is most closely related to AtMyb123 (TT2), a regulator of proanthocyanidin accumulation in Arabidopsis (Nesi et al., 2001). Other transcription factors preferentially expressed in the xylem-enriched apical shoot base showed similarity to two different members of the Arabidopsis HD-Zip family (WS0092-N10/ATHB-15; WS00915_B21/HAT14), as well as to members of the WRKY, GRAS, and squamosa promoter binding protein-like families (Table 3). Transcripts hybridizing with array element WS0083_O11, whose sequence is similar to the Arabidopsis auxin response factor (ARF) genes ARF4 and ARF5, were also preferentially expressed at the xylem-enriched base.
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There are at least 81 cDNAs in the array with annotations related to protein kinases, and several of these were differentially expressed in the xylem-enriched apical shoot base (see Supplementary Tables S3 and S4 at JXB online). For both the shoot tip and woody base, elements annotated as receptor kinases were among the most prominent differentially expressed kinase-related genes, and six of these showed consistent, preferential expression at the xylem-enriched shoot base (Table 3). These include putative leucine-rich repeat (LRR) receptor-like kinases (RLKs) (WS00812_M21, WS0039_K14, WS00813_I21), which are similar to Arabidopsis RLKs of unknown functions (Table 3). WS00812_M21 is most closely related to the CLV1 family of LRR–RLKs, and shares a stretch of c. 90 highly conserved amino acids with the Arabidopsis CLV1-like gene, At3g24240. Preferential expression of other genes with putative kinase and phosphatase functions in the xylem-enriched tissue at the shoot base suggests an active role for protein phosphorylation associated with secondary xylem development (Table 3). Intriguingly, one of the genes (WS0031_P03) most highly specific to the xylem-enriched apical shoot base, resembles the Arabidopsis Ca-dependent protein kinase CDPK4 (At5g24430) and a Ca-CaM-dependent protein kinase from maize (MCK2). Another highly apical shoot base-specific gene (WS0039_K18) is similar to the Arabidopsis Ca2+-binding protein, RD20 (At2g33380).
Effect of xylem enrichment on analysis of apical shoot base differential gene expression
In experiments 2003A and 2004B (Fig. 1), bark tissue was removed from the base of the apical shoot in order to enrich the sample for xylem tissue. Consequently, it was possible to detect transcripts that were preferentially present in xylem compared with green apical shoot tips, but whose signals would otherwise have been diluted by the presence of transcripts associated with green bark tissue. A number of highly differentially expressed genes in the xylem-enriched tissue at the base of the apical shoot, such as cellulose synthase, XET, aquaporins, and laccases also retained this specificity even when the bark and phloem were included in the leader base samples in experiment 2004C (Tables 2, 3), although the FC ratios were correspondingly lower. Other genes with lower FC values for base versus tip in experiments 2004A and 2004B had non-significant FC values when bark was retained in experiment 2004C. Overall, most of the differentially regulated genes with highest expression at the apical shoot base (Tables 2, 3) showed an expression pattern that was strikingly enhanced in the xylem-enriched base samples. These include genes represented by cDNAs without significant sequence similarity to Arabidopsis genes (Table 2), suggesting that they may play conifer-specific roles in secondary xylem differentiation and wood formation.
Genes preferentially expressed in apical shoot tips
A large number of differentially expressed transcripts in experiments 2003A and 2004B showed preferential expression in the green apical shoot tip relative to the xylem-enriched base. Prevalent within this subset were genes generally associated with defence responses against pests or pathogens, responses to stress, and known conifer defence genes of terpenoid (oleoresin) and phenolic secondary metabolism. Representative examples of defence genes predominantly expressed in the green shoot tip relative to xylem-enriched shoot base are shown in Table 4. When gene expression was examined in tips versus intact bases including green bark tissues (experiment 2004C; Fig. 2) about 25% of these defence- and stress-related genes retained significant differential expression in tips compared with the full apical shoot base (experiment 2004C; Table 4), supporting a model according to which the apical shoot tip commits significantly enhanced gene expression to the early formation of a constitutive defence barrier. Spruce genes annotated as PR-proteins showed particularly high ratios (Table 4; see Supplementary Table S4 at JXB online). In addition, of the 34 chitinase elements represented on the array, most showed tip-specific transcript hybridizations (see Supplementary Table S4 at JXB online) similar to element WS0108_K13 (Table 4). Similarly, most of the highly tip-specific ß-1,3 glucanase transcripts detected on the array also showed patterns of preferential expression in apical shoot tip relative to the intact apical shoot base in experiment 2004C (Table 4). A group of protease inhibitor (PI) transcripts were also preferentially detected in the apical shoot tip relative to the xylem-enriched shoot base (e.g. element WS0064_D18; Table 4; see Supplementary Table S4 at JXB online). Many of these (15 cDNA array elements) share similarity to the protein encoded by the Arabidopsis PI II gene, At2g02120, a protease inhibitor II containing a gamma-thionin domain. However, unlike the ß-1,3 glucanase and chitinase gene family members, these spruce PI genes appeared to be expressed at similar levels in the tips and the intact bases of leader stems in experiment 2004C (Table 4), suggesting that they may contribute to constitutive defence both in the apical shoot tip and the young apical shoot bark.
Genes for terpenoid and phenolic secondary metabolism and constitutive defence
Several genes of the methylerythritol phosphate (MEP) pathway leading to the formation of the isopentenyl diphosphate and dimethylallyl diphosphate precursors for mono- and diterpenoid oleoresin defence, as well as other terpenoids, were preferentially expressed in the green apical shoot tips when compared with the xylem-enriched apical shoot base. Genes corresponding to cDNAs for 1-deoxy-D-xylulose-5-phosphate (DXR, At5g62790) and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MECPS, At1g63970) showed the highest shoot tip-preferred expression in this group of enzymes (Table 4). Similarly, spruce terpene synthase genes, specifically involved in spruce oleoresin defence and represented on the array by several previously characterized cDNAs (Martin et al., 2004), showed higher expression in apical shoot tips relative to the xylem-enriched base (5–20-fold), corresponding with the phloem and shoot tip location of developing constitutive resin ducts. When the apical shoot tip was compared with the intact apical shoot base, including bark and phloem tissues (experiment 2004C; Fig. 2), genes encoding enzymes in the terpenoid pathway were similarly expressed in both sections. These results support association of constitutive terpenoid biosynthesis with constitutive resin ducts that are initiated very early in the development of the green apical shoot tip and remain active in mature phloem.
In phenolic secondary metabolism, several genes encoding proteins related to Arabidopsis flavonoid biosynthetic enzymes were also strongly differentially expressed in apical shoot tips relative to xylem-enriched bases, as were other phenylpropanoid pathway-related genes (Table 4). Most of these genes remained abundantly expressed in bark tissues at the apical shoot base (see data on experiment 2004C; Table 4). At least 28 cDNAs on the array have been annotated as dirigent or dirigent-like proteins (Ralph et al., 2006a), and almost all of these elements showed preferential expression in the apical shoot tip relative to the xylem-enriched leader base in experiments 2003A and 2004B (see Table 4 for examples; see Supplementary Table S4 at JXB online). However, some were preferentially expressed in xylem-enriched tissue at the leader base (experiments 2003A and 2004B; Table 2), in particular those with similarity to At1g64160 (DIR5).
Genes encoding putative regulatory proteins with preferential shoot tip expression
Several genes with potential regulatory functions were differentially expressed in the apical shoot tip relative to the xylem-enriched base (experiments 2003A and 2004B; Table 4). For example, elements WS0011_C05 and WS0091_B15 encode homeodomain and bHLH transcription factors related to the Arabidopsis genes ANL and EGL3, respectively, which are regulators of anthocyanin biosynthesis in Arabidopsis (Kubo et al., 1999; Broun, 2005). At least four WRKY transcription factor family members showed differential expression in tip samples. Of these, element WS0082_I17 retained a strong tip preferred expression even in experiment 2004C (Table 4). Several genes potentially involved in protein kinase signalling also showed differential expression in the apical shoot tip relative to the xylem-enriched base and, among these, two RLKs (IS0014_A12, WS00913_F03; Table 4) appeared strongly tip-preferred.
Lipid transfer proteins
Among the most prevalent transcripts showing particularly high FC ratios between the apical shoot tip and the xylem-enriched shoot base were those hybridizing with cDNA array elements annotated as lipid transfer proteins (LTPs). There are at least 35 LTP-like cDNAs on the spruce array that show similarity to this gene family in Arabidopsis, and these fall into 10 groups. Within a group, each LTP shares over 90% EST nucleotide sequence similarity with other members. A large fraction of the LTP array elements showed very high differential expression in tips relative to the xylem-enriched tissue at the shoot base (10–30x FC, experiments 2003A and 2004B; Table 4), and most of these share the greatest similarity with Arabidopsis LTP4 or LTP3 genes. However, they did not retain a significant differential expression in tips relative to the intact leader base including the green bark tissue (Table 4; experiment 2004C). On the other hand, a small number of LTP genes were highly differentially expressed in the xylem-enriched leader base (e.g. cDNAs WS0033_C20 and WS0044_K03; Table 2). Sequence alignment and phylogenetic reconstruction of 12 spruce full-length LTP sequences revealed that LTP genes showing preferential expression in xylem-enriched leader base samples grouped into a distinct phylogenetic clade (Fig. 4).
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Validation of candidate gene expression by RT-PCR
Candidate genes for expression validation by quantitative RT-PCR were chosen from those genes encoding putative signalling proteins and transcription factors with apparently preferential expression either at the apical shoot tip or base. The XET gene whose expression was specific to xylem-enriched tissue at the apical shoot base was also included in this analysis. The corresponding cDNAs were fully sequenced, which allowed the design of sequence-specific PCR primer pairs. RT-PCR assays performed on the same 2004 RNA samples validated microarray results for all the selected cDNAs, not only in specificity of expression, but also in the magnitude (Table 1). Expression of a subset of this group of genes was also monitored along the length of the spruce leader by RT-PCR. For this analysis, four segments were taken from the tip to the base of the leader, without removing the bark (Fig. 1C). Expression relative to the tip changed gradually along the leader for some genes (Fig. 3), while for others there was a sharp transition in expression between the tip and the segment immediately below it, where secondary growth was already evident.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsSupplementary dataReferences |
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Apical shoots of Sitka spruce provide a useful system to study gene expression associated with early events of stem development in conifers. In the present study, it was possible to identify genes that displayed consistent patterns of differential expression over the course of apical shoot development, including those involved in secondary xylem formation and the developmental transition to the woody apical shoot base, and in constitutive defence in the shoot tip and young bark tissues. Also, differentially expressed regulatory proteins were identified that provide new candidates for control with secondary xylem development and constitutive defence structures and secondary metabolite production.
Secondary cell wall development
The spruce apical shoot begins to form secondary xylem immediately below the apical shoot tip, characterized by extensive development of lignified secondary cell walls (Fig. 2). Prominent among the cell secondary cell wall-related genes preferentially expressed in the xylem tissue present at the apical shoot base (Table 2) were members of the cellulose synthase (CesA) superfamily (Doblin et al., 2002). In Arabidopsis, distinct CesA subunits are associated with secondary and primary cell wall formation (Doblin et al., 2002; Zhong et al., 2003). The two spruce CesA genes (WS0073_D24, WS00812_N07) that stood out in their differential expression in the xylem-enriched apical shoot base are very similar to the loblolly pine (Pinus taeda) CesA3 gene, proposed to be the pine orthologue of the Arabidopsis secondary cell wall-associated CesA7 gene (Nairn and Haselkorn, 2005). This is consistent with the suggestion that distinct CesA isoforms with functions in primary and secondary wall biosynthesis evolved both in conifers and angiosperms (Nairn and Haselkorn, 2005). As well, recent studies have identified, in silico, Arabidopsis genes whose expression is highly correlated with CesA gene expression during secondary wall formation (Brown et al., 2005; Ehlting et al., 2005; Persson et al., 2005). It is interesting that a set of genes represented on the spruce cDNA microarray with similarity to these Arabidopsis genes is also up-regulated in the xylem-enriched tissue, together with the CesA genes. These spruce genes are annotated as encoding COBRA-like 4 (COBL4), germin-like, chitinase-like 2 (CTL2), fasciclin-like arabinogalactan (FLA12), and KORRIGAN (KOR) proteins. This correspondence could suggest that the spruce proteins play similar roles in cellulose deposition during secondary wall formation, either as part of the cellulose synthase complex, or in subsequent modification of cellulose microfibrils, and that this process is highly conserved in angiosperms and gymnosperms. Two genes with homology to sucrose synthase that appear to be up-regulated in the apical shoot base may correspond to spruce orthologues of a sucrose synthase that delivers the UDP-Glc substrate to the CesA complex (Haigler et al., 2001).
The present results also showed differential expression among the members of the arabinogalactan-proteins (AGP) gene family (see Supplementary Table S2 at JXB online). The spruce AGP gene (WS0038_G03) preferentially expressed in the xylem-enriched apical shoot base is nearly identical to an AGP abundant in loblolly pine xylem (p14A9) (Loopstra and Sederoff, 1995; Yang et al., 2005), and the similarity of this gene to Arabidopsis FLA12 reinforces the suggestion that FLA12-like AGPs play an important role in cellulose deposition during secondary xylogenesis. However, two other Sitka spruce AGP genes, with similarities to Arabidopsis FLA1 and FLA9, were more preferentially expressed in the leader tip (Tables 2, 4; see Supplementary Table S2 at JXB online). Similarly, of the several xyloglucan endoglycosyl transferase (XET) genes represented on the microarray, one (WS0087_M10) showed marked preferential expression at the apical shoot base (Tables 1, 2; see Supplementary Table S1 at JXB online; Fig. 3). The sequence of the corresponding cDNA is very similar to that of a loblolly pine cDNA isolated from a salt-treated root cDNA library (TC69813, pine gene index, TIGR: www.tigr.org/tigrscripts/tgi/T_index.cgi?species=pinus), and is similar (65% amino acid sequence similarity) to a cotton XET gene highly expressed in elongating fibres (Ji et al., 2003). This result is consistent with previous studies showing differential expression of XET genes in cambial and expanding xylem tissues (Vander Mijnsbrugge et al., 2000; Hertzberg et al., 2001; Mellerowicz et al., 2001; Paux et al., 2004; Schrader et al., 2004; SH Yang et al., 2004), suggesting that the woody stem-specific spruce XET is involved in cell expansion during secondary xylem formation.
Genes encoding enzymes involved in the synthesis of the monolignol units from phenylalanine have been well characterized in Arabidopsis (Boerjan et al., 2003; Raes et al., 2003; Ehlting et al., 2005), and many of them have also been described in pine (Anterola et al., 2002) and spruce (Ralph et al., 2006b). A set of spruce genes corresponding to cDNA microarray elements annotated as encoding lignin biosynthetic enzymes showed strongly up-regulated expression in the woody base of Sitka spruce apical shoots (Table 2; see Supplementary Table S3 at JXB online). Most of these enzymes were represented on the spruce microarray by multiple cDNAs representing putative gene family members with distinct sequences, yet for many of the putative family members only a subset were up-regulated in the woody stem base. This set, with representatives shown in Table 2, includes all enzymes known to be required for the biosynthesis of G-lignin, (PAL, C4H, 4CL, HCT, C3H, CCoOMT, CCR, CAD) and thus represents those spruce phenylpropanoid genes most likely to encode the spruce enzymes involved in developmental lignification. Interestingly, the Arabidopsis genes to which members of this group of spruce genes showed highest similarity were, in most cases, those known or inferred by expression analysis to be involved in developmental lignification in that plant as well (e.g. 4CL2, CCOMT1, CAD1, CCR1; Raes et al., 2003; Ehlting et al., 2005).
The role of conifer COMT, also known as AEOMT (Li et al., 1997), in monolignol biosynthesis is controversial (Anterola et al., 2002), since, according to current understanding in angiosperms (Boerjan et al., 2003), it is involved in S-lignin biosynthesis, which does not occur in conifers. However, of the 13 spruce cDNAs with similarity to COMT on the microarray, six displayed preferential expression in the woody apical shoot base (see Supplementary Table S3 at JXB online), and among these was WS0039_D14, the cDNA that shows the greatest similarity to the dual-function COMT (AEOMT) (Table 2) previously described in loblolly pine (Li et al., 1997). This result supports an association of AEOMT with active wood formation, while leaving unresolved the potential biosynthetic role of the bifunctional O-methylation potentially catalysed by this enzyme.
Two classes of oxidative enzymes believed to be involved in oxidative polymerization of monolignols are laccases and peroxidases (Boerjan et al., 2003). Laccases predominated among the genes showing a high expression in the woody portion of the Sitka spruce apical shoot (Table 2). Three of these showed a high degree of similarity to Arabidopsis laccases LAC4 (At2g38080), LAC12 (At5g05390), and LAC17 (At5g60020) that were found to be strongly co-expressed with cellulose synthase genes involved in secondary cell wall formation (Brown et al., 2005; Ehlting et al., 2005; Persson et al., 2005). Similarly, a spruce peroxidase with strong woody apical shoot base-preferred expression (WS0034_K23) is similar to Arabidopsis peroxidase P49 (At5g05340), which is also strongly up-regulated in concert with lignification in Arabidopsis (Ehlting et al., 2005). These results point to a conserved repertoire of oxidative enzymes involved in lignin polymerization in both conifers and angiosperms.
Several classes of proteases also show preferential expression in the woody base of Sitka spruce apical shoots, incuding subtilisin-like, Ser-, Cys-, and Asp-proteases (Table 2). These classes of proteases were shown to be up-regulated during programmed cell death during trans-differentiation of mesophyll cells into tracheary elements in Zinnia (Demura et al., 2002) and have been characterized recently in plant cell death during wood formation in poplar (Moreau et al., 2005), suggesting that they participate in this final step of spruce xylem differentiation.
Regulatory proteins in xylem development
A number of transcription factors showed consistent up-regulation at the apical shoot woody base, making them candidates for positive regulators of secondary xylem differentiation (Table 3). Given their association with wood formation and lignification in both conifers and angiosperms (Patzlaff et al., 2003; Newman et al., 2004), the two spruce Myb genes represented by spruce cDNAs WS00712_A21 and WS00917_H19, are of particular interest. Although WS00712_A21 is distinct from loblolly pine PtMYB4, reported to be a regulator of lignification (Patzlaff et al., 2003), it shows similarity to loblolly pine PtMYB1 (81% amino acid identity at the N-terminal R2R3 DNA binding domain), a putative regulator of phenylpropanoid metabolism (Patzlaff et al., 2003). Interestingly, the spruce gene sequence is moderately similar to those of the related Arabidopsis AtMyb43 and AtMyb20 genes (81% and 55% amino acid identity, respectively, in the R2R3 DNA-binding domains) whose transcription has been correlated with secondary wall formation and lignification in developing Arabidopsis interfascicular fibres (Ehlting et al., 2005). This is consistent with phylogenetic analysis, which places PtMYB1 in the same clade as AtMyb43 and AtMyb20 (Patzlaff et al., 2003). The second Myb gene WS00917_H19 shares similarity (60% amino acid identity in the R2R3 DNA-binding domain) to Arabidopsis Myb transcription factors AtMyb123 (TT2) (Nesi et al., 2001) and AtMyb75 (PAP1) (Borevitz et al., 2000), regulators of phenylpropanoid metabolism. The similarity of these two spruce genes to clades of Arabidopsis Myb genes associated with phenylpropanoid metabolism and secondary wall formation suggest potential functional conservation of Myb proteins between conifers and angiosperms.
HD-ZipIII transcription factor family members are candidates for regulators of xylogenesis in angiosperms (Baima et al., 2001; Hertzberg et al., 2001; Schrader et al., 2004; Prigge et al., 2005), and the two different spruce HD-ZipIII genes up-regulated in xylem-enriched tissue at the base (WS0092_N10 and WS00915_B21) may play analogous roles in spruce xylem differentiation. Other transcription factors showing strong woody stem-preferred expression include a member of the WRKY family (WS0083_F20) that has a high degree of amino acid sequence similarity (up to 81%) to the WRKY42 protein in Arabidopsis (At2g44745) that is involved in control of cell expansion and proanthocyandin biosynthesis in developing seeds (Johnson et al., 2002; Garcia et al., 2005), and a spruce auxin response factor (ARF) transcription factor gene (WS0083_O11) similar to Arabidopsis ARF4 and ARF5/MONOPTEROS. These results suggest potential conservation of a transcription factor function regulating cell expansion and auxin-regulated secondary xylem development in conifers and angiosperms.
The presence of multiple cDNA microarray elements related to protein signalling functions, such as LRR RLKs and protein kinases, allowed potential roles for these classes of proteins to be assessed during tissue differentiation processes in the spruce leader. Four spruce LRR RLK genes showed a pronounced up-regulation in the woody apical shoot base (Table 3) and one of these, WS00812_M21 shows sequence similarity to the Arabidopsis CLV1 gene, raising the possibility of conserved signalling pathways underlying key pathways of RLK-mediated cellular differentiation in conifers and angiosperms. In the present experiment, a putative Ca-dependent protein kinase gene, WS0031_P03, with similarity to Arabidopsis CPK4 (At5g24430) was strongly up-regulated in woody stem tissue of the apical shoot. Another gene with preferential expression in woody stem tissues of the apical shoot, WS0039_K18, encodes a protein similar to the Arabidopsis Ca2+-binding protein, RD20 (At2g33380), which has been shown to be induced by ABA upon desiccation (Takahashi et al., 2000). These observations suggest that Ca2+-signalling may also play a key role in xylem differentiation in Sitka spruce.
Terpenoid, phenolic, and other defence-related genes
The present experiment revealed genes that are preferentially expressed in non-woody tissues of the Sitka spruce apical shoot, specifically the apical shoot tip and green bark tissues at the base where anatomical structures for chemical and physical defences develop. Elevated expression of genes involved in formation of terpenoid and phenolic secondary metabolism, along with other defence-related genes such as chitinases, glucanases, and protease inhibitors in the growing apical shoot tip supports a model of an early allocation of substantial resources towards constitutive defence and protection. For example, in terpenoid defence metabolism, many of the terpene synthase gene family members on the microarray showed high expression ratios in apical shoot tips (5–20-fold) relative to the woody leader base. Similarly, almost all of the spruce DIR genes represented on the spruce microarray were preferentially expressed in apical shoot tip and green tissues (Table 4), consistent with previous results of preferential expression of many spruce DIR genes in outer stem tissues and a proposed function in conifer defence (Ralph et al., 2006a). By contrast, only two DIR genes (WS01011_J07, WS00913_G16) were found strongly differentially expressed in the woody apical shoot base, where they could be involved in lignin deposition. Many of the defence-related genes with high expression in the apical shoot tip continue to be expressed in the developing green bark tissues of the apical shoot base, suggesting that these defences remain active for a long time and are not confined to the non-woody shoot tip.
The apical shoot of Sitka spruce is the preferred site for feeding and oviposition by adult white pine weevil and the developing larvae of this insect, which is the single most destructive pest in regenerating Sitka spruce forests (King, 1997). In addition, a substantial body of research has shown that the same bark and phloem tissues of Sitka spruce apical shoots also respond strongly with a plethora of inducible defences in response to attack by adult white pine weevil (Miller et al., 2005; Ralph et al., 2006a, b). For example, in addition to the prominent up-regulation of terpenoid defences in Sitka spruce bark tissues (Miller et al., 2005) and the weevil-induced up-regulation of DIR-proteins (Ralph et al., 2006a), a number of the chitinases and glucanases identified here in the apical shoot tip and green bark (i.e. WS0017_B07; Table 4) were also induced in stem bark tissues by mechanical wounding and insect feeding (Ralph et al., 2006b). Furthermore, a number of additional genes with diverse functions (chalcone synthase, glutathione S-transferase, AP2 transcription factor, ß-glucosidase, and cytochrome b5; Table 4) with apical shoot tip-preferred expression are also induced by wounding, adult weevil feeding, or by budworm caterpillar feeding (Ralph et al., 2006b).
Differential expression of LTPs: possible roles in constitutive chemical defence and secondary cell wall formation
LTPs are small proteins with highly conserved Cys residues (Kader, 1996; Arondel et al., 2000) that are thought to transfer lipids between membranes but which appear to have other diverse roles in plant development, signalling, and response to pathogens (Ma et al., 1995; Blein et al., 2002; Horvath et al., 2002; Eklund and Edqvist, 2003; Gomes et al., 2003; Cheng et al., 2004). Most LTP genes represented on the spruce microarray showed very high relative expression at the apical shoot tip (15–30x higher than in xylem-enriched tissue at the apical shoot base). In fact, they comprised a significant portion of the genes with the highest tip-preferred expression (Table 4; see Supplementary Table S4 at JXB online). Most of these spruce LTPs show greatest similarity to Arabidopsis LTP1, LTP3, or LTP4 genes (Arondel et al., 2000). To date, mechanisms of transport and secretion of the highly lipophilic terpenoid oleoresin compounds are not known. While functions of conifer LTPs have not been characterized, some of these spruce proteins could be involved in the secretion and accumulation of terpenoid and phenolic secondary metabolites in resin ducts and PP cells, respectively, at the shoot tip. In support of a role in defence, one of the LTP genes, WS0019_O12, showed strong induction in spruce leader tips upon feeding by western budworm caterpillars (Ralph et al., 2006b).
Other spruce LTP genes were preferentially expressed in the woody apical shoot base, and all of these have sequences distinct from those with highest expression in shoot tips. Alignment and phylogenetic analysis of LTP amino acid sequences represented by full-length cDNA sequences (Ralph et al., 2006b) shows that LTPs associated primarily with the woody shoot base are located in a separate phylogenetic clade (Fig. 4). These LTPs might play roles in secondary cell wall formation, perhaps as part of the cellulose synthase complex, for which there is evidence in cotton (Ma et al., 1995; Doblin et al., 2002).
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsSupplementary dataReferences |
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The apical shoot of Sitka spruce trees is important for initiating stem growth and secondary xylem formation and is the primary target for feeding and ovipositing white pine weevil. During the growing season, resources in the apical shoot must be allocated for growth and development, constitutive defence, and induced defences when needed, requiring co-ordinated regulation of these processes. Using large-scale transcript profiling, differentially expressed genes associated with secondary xylem differentiation and constitutive defence in this developing organ have been identified. This information now serves as a basis for work on elucidating functions of specific genes or combinations of genes in spruce and other conifers. The associated sequence information available for a large spruce EST and full-length cDNA resource (Ralph et al., 2006b), together with the indications of conservation in gene function and expression patterns between conifers and angiosperms, also provide fertile ground for comparative genomic studies that address the evolution of important traits such as vascular development and metabolic specialization of secondary metabolism in the plant kingdom.
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Supplementary data |
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Supplementary data can be found at JXB online.
Table S1. Comparison of XET expression between years.
Table S2. Comparison of AGP expression between years.
Table S3. Base versus tip fold-change and P-values for all array elements on experiments 2003A, 2004B, and 2004C.
Table S4. Tip versus base fold-change and P-values for all array elements in experiments 2003A, 2004B, and 2004C.
Table S5. Quantitative RT-PCR Primer Sequences.
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Acknowledgements |
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We gratefully acknowledge financial support from Genome Canada, Genome British Columbia, and the Province of British Columbia through funding provided for the Treenomix Conifer Forest Health Project, Genome Canada Competition III (grant to JB and KR) and for the Treenomix Project, Genome Canada Competition I (grant to JB, KR, BEE, and CJD); as well as financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC; Discovery Grants to JB and CJD). We thank Claire Oddy, Hesther Yueh, and Sharon Jancsik for technical assistance with array development, David Kaplan for greenhouse support, members of the UBC Bio-imaging facility for assistance with microscopy, and Dr Jürgen Ehlting for discussions.
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