Induced somatic sector analysis of cellulose synthase (CesA) promoter regions in woody stem tissues
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The increasing focus on plantation forestry as a renewable source of cellulosic biomass has emphasized the need for tools to study the unique biology of woody genera such as Eucalyptus, Populus and Pinus. The domestication of these woody crops is hampered by long generation times, and breeders are now looking to molecular approaches such as marker-assisted breeding and genetic modification to accelerate tree improvement. Much of what is known about genes involved in the growth and development of plants has come from studies of herbaceous models such as Arabidopsis and rice. However, transferring this information to woody plants often proves difficult, especially for genes expressed in woody stems. Here we report the use of induced somatic sector analysis (ISSA) for characterization of promoter expression patterns directly in the stems of Populus and Eucalyptus trees. As a case study, we used previously characterized primary and secondary cell wall-related cellulose synthase (CesA) promoters cloned from Eucalyptus grandis. We show that ISSA can be used to elucidate the phloem and xylem expression patterns of the CesA genes in Eucalyptus and Populus stems and also show that the staining patterns differ in Eucalyptus and Populus stems. These findings show that ISSA is an efficient approach to investigate promoter function in the developmental context of woody plant tissues and raise questions about the suitability of heterologous promoters for genetic manipulation in plant species.
KeywordsCAMV35S promoter Eucalyptus GUS reporter gene Populus Secondary cell wall Wood formation
Average of transformed sectors per cm2
Induced somatic sector analysis
Plantation tree species such as those from the genera Populus and Eucalyptus are receiving worldwide attention for their capacity to produce cellulosic biomass which can be used for pulp and, potentially, biofuel production (Hinchee et al. 2011). Unlike first generation biofuel crops such as sugarcane and maize, forest trees are less likely to directly compete with food production and have a greater biomass production capacity (Rathmann et al. 2010), although the processing of lignin-rich woody biomass to liberate cell wall biopolymers remains a challenge (Mansfield 2009). Furthermore, the genetic improvement of forest trees is hindered by long generation times and late expression of mature traits. Tree breeders attempting to enhance properties such as wood quality and cellulose deposition will benefit from the application of molecular approaches such as marker-assisted breeding (MAB) and genetic modification (Grattapaglia et al. 2009; Seguin 2011). These molecular approaches are now also benefiting from the application of next-generation genomics technologies, which can be used to study the genetics of wood formation as a system and to rapidly identify candidate genes for further functional analysis (Mizrachi et al. 2012).
Cellulose is deposited in plant cell walls by large, membrane-bound, protein complexes composed of several different cellulose synthase (CESA) proteins (Kimura et al. 1999), the identity of which depends on the type of cell wall being laid down (Song et al. 2010). In Arabidopsis and other plant species, three CesA genes (CesA4, 7 and 8) have been associated with secondary cell wall deposition, while a different set of CesA genes were found to be involved in primary cell wall formation (Turner and Somerville 1997; Taylor et al. 2000, 2003; Hamann et al. 2004; Samuga and Joshi 2004; Ranik and Myburg 2006). During primary cell wall formation in Arabidopsis two CesA genes, AtCesA1 and 3, are essential for cell development with knock-out mutants being lethal (Arioli et al. 1998; Scheible et al. 2001). Five other CesA genes (AtCesA2, 4, 5, 6 and 9) have been linked to primary cell wall formation in Arabidopsis, but these are functionally redundant when mutated and appear to be involved in tissue-specific primary cell wall formation (Beeckman et al. 2002; Desprez et al. 2002; Stork et al. 2010; Carroll and Specht 2011).
While there are many similarities in cellulose biosynthesis across plant genera (Popper et al. 2011), there are also a number of species-specific features. The CesA gene family has ten members in Arabidopsis (Richmond and Somerville 2000), while Populus has 18 expressed CesA genes (Djerbi et al. 2005; Suzuki et al. 2006; Kumar et al. 2009). A phylogenetic analysis of the PopulusCesA gene family revealed that the 18 CesA genes grouped with the ten Arabidopsis orthologs in all of the primary and secondary cell wall-related clades and that Populus has two or more paralogs of some Arabidopsis genes (Kumar et al. 2009). In particular, it was noted that Populus has duplicated genes for the secondary cell wall-associated AtCesA7 and AtCesA8 genes. In each case, one of the two Populus paralogs (PtiCesA7-A or PtiCesA8-B) was more highly expressed in xylem, suggesting differential regulation of the paralogs and possible loss of regulation of the lower expressed paralog (Suzuki et al. 2006). Similarly, the primary cell wall-associated AtCesA3 gene has four close orthologs in Populus, and each of these has a different expression pattern (Suzuki et al. 2006). The differentiated expression patterns of the duplicated CesA genes in Populus suggest that the Populus paralogs may be undergoing subfunctionalization.
Inter-specific differentiation can affect regulatory sequences in promoters and produce discordant results when different orthologs are used in transgene constructs. Fei et al. (2006) found that a promoter construct which increased glutamine synthase expression in both Lotusjaponicus and Sesbaniarostrata, did not produce a corresponding increase in expression in Pisumsativum. In an extensive study on mammalian and Drosophila cell lines eight supposedly constitutive promoters were tested and most promoters showed variation in reporter gene expression between both cell line and species (Qin et al. 2010). Even the highly utilized CAMV 35S promoter has been shown to have differential expression across different species (Benfey and Chua 1990; Zhang et al. 2003). This evidence suggests that for some promoters it may be desirable to perform functional analysis in the native genetic backgrounds; however, this may not be practical in species that are recalcitrant to genetic transformation.
Induced somatic sector analysis (ISSA), first proposed by Spokevicius et al. (2005) and developed further by Van Beveren et al. (2006), uses a novel in planta transformation method, which has been successfully applied in the analysis of transgenes in woody stem tissues of Pinus, Populus and Eucalyptus (Hussey et al. 2011). In this method, Agrobacterium carrying the promoter and transgene of interest is applied to the exposed cambium on the stem of a living tree. The gene construct is transferred by Agrobacterium into actively dividing cambial, xylem, phloem and ray initial cells, creating a number of transformants in this small section (~1 cm2) of the tree stem (Van Beveren et al. 2006). When the cambium is resealed and the stem is allowed to grow for a few months where the transformed cells divide and multiply within the stem, producing somatic sectors of transformed cells. This area of transformed cells can then be analysed for transgene (e.g. β-glucuronidase) expression and changes in cell wall morphology by comparing transformed sectors with adjacent non-transformed stem cells. ISSA has great potential for functional genetic studies, as it allows for the analysis of transgenes and promoters directly in the stem tissues of the tree and, for wood-specific constructs, may give a more accurate picture of the native functions or expression patterns of transgenes in woody tissues (Spokevicius et al. 2007).
Here, we used ISSA to study the expression patterns of six previously characterized promoters of EucalyptusgrandisCesA genes (Creux et al. 2008) in woody stem tissues of Eucalyptus and Populus trees. The first objective of this study was to investigate the suitability of ISSA for the analysis of promoter function in various woody stem tissues. Second, we assessed whether ISSA could be used to compare reporter gene expression patterns in Populus and Eucalyptus stems. To our knowledge, this is the first study to directly compare the expression patterns of promoter::reporter gene constructs in woody tissues of Populus, the model tree genus for molecular studies, and Eucalyptus, a globally important fibre crop.
Materials and methods
Three-month-old ramets of five Eucalyptus camaldulensis × globulus and six Eucalyptus camaldulensis × grandis clones were purchased from a specialist forestry nursery (Narromine Transplants, Narromine, NSW, Australia), potted in premium potting mix and maintained in a greenhouse for another 4 months. A single Populus alba (L.) ‘pyramidalis’ clone, growing at the University of Melbourne Creswick Campus (Vic. Australia), was used to generate plant material through rooted cuttings. Dormant stems were sourced and established in cutting beds following treatment with a commercial rooting hormone powder (Yates Striking Powder, Homebush, NSW), transplanted into premium potting mix after 6 weeks and maintained in the greenhouse for 3 months until required. Greenhouse temperatures were maintained between 14 and 17 °C at night and between 21 and 25 °C during the day. A 16-h photoperiod was kept through supplementary lighting. Supplementary lighting was supplied by six 1000 W Metal Halide globes in a glasshouse chamber of approximately 16 m2. All plants were watered regularly with tap water (as required, depending on season) and fertilised with a slow release formulation (Osmocote Exact Mini, Scotts-Sierra Horticultural Products, Marysville, OH, USA) every 3 months.
Promoter isolation, vector and Agrobacterium preparation
Arabidopsis thaliana and Populus trichocarpaCesA orthologs and the corresponding Eucalyptus grandis CesA genes included in this study
Primary cell wall-associated CesA genes
Secondary cell wall-associated CesA genes
Two CAMV 35S promoter vectors were used as a positive control. The first, 35S-F, was the pCAMBIA1305.1 vector (http://www.cambia.org/ verified 5/5/10) and the second, 35S-G, was based on the same pCAMBIA1305.1 vector backbone, but with a Gateway recombinase cassette in the multi-cloning region. An empty (promoter-less) pMDC162 vector was also used as negative control.
Growth parameters and overall transformation efficiency for Eucalyptus and Populus plants
Average height at inoculation
91.7 cm (SE = 5.0 cm)
168.9 cm (SE = 6.7 cm)
Average height at harvest
205.8 cm (SE = 6.9 cm)
247.7 cm (SE = 9.186 cm)
Average diameter at inoculation (at stem height = 10 cm)
6.2 mm (SE = 0.09 mm)
7.5 mm (SE = 0.17 mm)
Average diameter at harvest (at stem height = 10 cm)
11.4 mm (SE = 0.22 mm)
11.2 mm (SE = 0.33 mm)
Average total radial growth of cambial window xylogenic tissue (from wound site)
2.55 mm (SE = 0.03 mm)
1.67 mm (SE = 0.069 mm)
Average total radial expansion rate
0.022 mm/day (SE = 0.001 mm/day)
0.013 mm/day (SE = 0.001 mm/day)
Total number of sectors counted
Inoculation and harvest of transformed tissues for Induced Somatic Sector Analysis
During the start of the growing season (early summer) 40 P. alba ‘pyramidalis’, 20 E. camaldulensis × globulus (four ramets of each clone) and 24 E. camaldulensis × grandis (four ramets of each clone) potted plants were selected on the basis of good form and growth for experimentation (Table 2). Along the stem of each tree, 11 approximately 1 cm2 cambial windows were opened using the in vivo stem ISSA method described in Van Beveren et al. (2006). Each cambial window was inoculated with 5 μL of Agrobacterium suspension containing one of the 11 promoter constructs under investigation (one promoter per window) and subsequently sealed using parafilm. Due to lower sector numbers for the EgCesA1 and EgCesA2 promoters (see “Results”) a further 30 windows were produced for each promoter in both species (Eucalyptus and Populus) during the following year using the same clonal material. Plant height and stem diameter (measured at a height of 10 cm from the trunk base) were recorded. Plants were fertilised after inoculation and maintained in the greenhouse until harvest.
At harvest, plant height and stem diameter (measured at a height of 10 cm from the trunk base) were again recorded and stem sections harbouring cambial windows were excised from the main stem and placed in 10 ml Falcon tubes for transport. Un-inoculated stem tissue (outside the window area) was removed and the remaining cambial tissue was cut transversely into 1 mm half-discs and placed back into the 10 mL tubes for GUS assays. Cambial discs were washed twice with 0.1 M NaPO4 buffer (pH 7) prior to the addition of 5 mL (approx) of GUS solution (0.1 M NaPO4 buffer pH 7, 0.5 % v/v Triton X-100 (Sigma), 10 mM EDTA (Sigma), 0.5 mM potassium ferricyanide (III) (Sigma), 0.5 mM potassium hexacyanoferrate (II) trihydrate (Sigma), 0.5 mM X-Gluc). Cambial discs were incubated in a water bath in GUS solution at 55 °C for 10 min prior to being placed upright in the dark on a rotary shaker (150 rpm) at 37 °C overnight. The GUS solution was then replaced with 70 % ethanol and samples were stored at 4 °C until assessment (Spokevicius et al. 2005; Alwen et al. 1992).
Assessment of GUS staining patterns
Cambial sectors were further analysed to gain insight into the temporal and spatial activity of the promoters under investigation, referred to here as ‘cambial sector ratio’. For this analysis, cambial sectors were assessed for presence or absence of GUS staining in three distinct regions defined as X1, X2, and P (Fig. 1b). The X2 region extended outward from the initial wound parenchyma cells up to the end of the mature xylem. Staining observed in this region was indicative of promoter activity in ray cells which extend radially through the stem. Most xylem fibres and vessels in the X2 region have already undergone programmed cell death (PCD) and one would therefore only expect X2 GUS expression and staining in ray cells which have not undergone PCD. The X1 region was characterized by staining in developing xylem cells close to and including the cambial zone (but no staining on the phloem side). Staining observed in the X1 region was indicative of promoter activity in differentiating xylem cells (before the onset of PCD). The P region comprised all phloem tissues and GUS staining in this region was indicative of promoter activity in phloem tissue in general. It is important to again note here that the final staining pattern observed (combination of P, X1 and X2) was determined by the cell type initially transformed and the specificity of the promoter tested.
Statistical analysis of ISSA results
Details of the statistical analysis are outlined in the Results section. Confidence intervals (95 %) were calculated for growth data using Minitab (Minitab Inc., State College, PA, USA) to compare growth rates. Chi-squared tests were performed using Minitab to compare the frequency of GUS expressing sectors observed in the X1, X2 and P regions (at α = 0.05). Promoter constructs for which fewer than ten sectors were observed were excluded from the statistical analysis, as was the case for the EgCesA2 and EgCesA4 in Populus stems. However, the majority of sectors observed for these promoters were cambial sectors and data for these promoters were included in the graphs, but should be validated in future experiments.
The number of transformed sectors varied dependent on species and promoter
Average number of transformation events per cm−2 of tissue (ATS−2) observed for the different promoter constructs
Sector type and frequency differed between promoter constructs
Cambial sectors were the most abundant and varied among species and promoter constructs
Cambial sectors were highly abundant in Populus and Eucalyptus stems (Fig. 2a, b) and these sectors were further classified into expression patterns (Fig. 2c, d). Five different cambial expression patterns were observed in the two species which included X2 + X1 + P, X2 + X1, X1 + P, X1-only and P-only (Fig. 1b). The X2 + X1 + P sector type was most likely produced by the transformation of a cambial initial, which subsequently gave rise to a ray sector extending into the P, X1 and X2 region followed by promoter activity in all three regions (Fig. 1c). The X2 + X1 sector type could be the result of transformation of a ray initial on the xylem side, or xylem-specific promoter activity in a cambial sector giving rise to ray cells. The X1 + P sector type was most likely the result of transformation of a cambial initial differentiating into phloem (P) and xylem (X1), but terminating at the zone of PCD (X1/X2 border), and subsequent promoter activity in phloem and xylem cells. X1-only and P-only sectors could be produced by the transformation of a cambial initial followed by xylem or phloem promoter activity, or the transformation of a xylem or phloem mother cell, respectively, followed by promoter activity in the resulting xylem or phloem sector. No X2-only or X2 + P staining patterns were detected in either species.
In the case of the two 35S promoter constructs (F and G) all cambial sector types (Fig. 1b) were found in Eucalyptus stems (Fig. 2d), but three (X2 + X1 + P, X1 + P and P-only) accounted for the majority of cambial staining patterns observed in Populus stems (Fig. 2c). In both species, the X2 + X1 + P sector type was the highest frequency cambial staining pattern observed for the 35S promoter constructs (approx 40 % in Eucalyptus and 65 % in Populus stems). For the CesA promoter constructs, all cambial staining patterns (Fig. 1b) were identified, but with distinct differences in the frequencies of staining patterns between Populus and Eucalyptus stems.
In Eucalyptus stems, cambial sector X1-only was the most frequent type observed for the promoters of secondary cell wall-related genes EgCesA1, 2, 3 and AtCesA8, whereas for the promoters of the primary cell wall-related genes EgCesA 4, 5 and 7 much higher frequencies of the X1 + P cambial sector types in addition to X1-only were observed (Fig. 2d). In Populus stems, the secondary cell wall-related CesA promoters also showed a high frequency of X1-only staining patterns; however, in Populus these promoters also displayed a higher frequency of the X1 + P type sectors. The primary cell wall-related CesA promoters produced a high proportion of X1 + P sectors in the Populus stems, similar to the pattern observed in Eucalyptus, but there was also a number of X2 + X1 + P and P only sectors present (Fig. 2c).
Some CesA promoters showed similar activity to the 35S promoter
Comparison of β-glucuronidase (GUS) expression frequencies observed for the CesA promoter constructs and for the CAMV35S promoter constructs in Eucalyptus and Populus stem tissues
Patterns of individual CesA promoter activity between tree species
The inter-specific comparison of β-glucuronidase (GUS) expression patterns observed in Populus and Eucalyptus cambial tissues for the EgCesA and CAMV35S promoters
ISSA provides a rapid and efficient approach to evaluate promoter expression in woody stems
Gene and promoter testing in tree genera such as Eucalyptus, Populus or Pinus require time-consuming and laborious manipulation through tissue culture and greenhouse studies. In this study we investigated the use of ISSA (Spokevicius et al. 2005; Van Beveren et al. 2006) as an approach for rapid functional genetic analysis of promoter expression patterns in developing woody tissues based on large numbers of independent transgenic events. We demonstrate the suitability of ISSA for promoter expression analysis of six Eucalyptus cellulose synthase (CesA) genes in the stems of Eucalyptus and Populus trees. We show that in the Eucalyptus genetic background the EgCesA promoters produced distinct staining patterns, which were consistent with the primary and secondary cell wall-associated expression patterns previously demonstrated for these genes (Samuga and Joshi 2004; Ranik and Myburg 2006), whereas in the heterologous Populus genetic background the staining patterns of the two groups of EucalyptusCesA genes were less distinct.
There are a number of key advantages to using ISSA for functional genetic analysis of wood formation genes and promoters. First, it allows the testing of transgenic constructs directly in native woody tissues (e.g. Pinus, Populus and Eucalyptus) in a relatively short period of time yielding measurable results within a few months (Spokevicius et al. 2005). Second, this technique requires little greenhouse space because multiple constructs or replicate transformations (up to 10 windows) can be performed on a single tree stem. Finally, a major advantage of ISSA is that each transgenic sector represents an independent transformation event, and with ten inoculation windows per tree a large number of independent events are produced, which can then be statistically analysed. For these reasons ISSA can be a useful technique to quickly screen promoter constructs for expression in woody stem tissues, to select candidate promoters for more detailed whole-plant analyses in model species such as Arabidopsis or Populus.
An important aspect to consider when analysing ISSA data is that stem tissue is comprised of different cell types at different developmental stages (Plomion et al. 2001) each of which may respond differently to transformation by Agrobacterium. Similarly, genetic background and species-specific developmental patterns may affect transformation efficiency. For example the periderm sectors, which are a result of transformed cells near the cut surface of the cambial window and have undergone rounds of division during the wounding response, were observed for most of the promoter constructs transformed into Populus stems. No such sectors were observed in Eucalyptus stems (Fig. 2) suggesting that this tissue is recalcitrant to transformation or responds differently to wounding in Eucalyptus. Another important consideration for analysing promoter regions using ISSA is the cell fate of the initially transformed cell and the cell- or tissue-specificity of the promoter construct. These two factors determine the final staining pattern observed and have to be jointly considered in the analysis of tissue- or cell type-specific promoters. We found it useful to compare the sector type frequencies obtained for the CesA promoters with those obtained for the CAMV35S promoter constructs (Fig. 3), which for the purpose of this study we assumed to be constitutively expressed in all cells derived from transformed initials. The latter is supported by the observation of a more diverse set of sector types for the CAMV35S promoter constructs including a higher frequency of wound parenchyma and tylose sectors (Fig. 2a, b), suggesting that these tissues are indeed susceptible to transformation, but that tissue-specific regulation resulted in low sector counts for these tissues when transformed with the CesA promoter constructs.
The CAMV35S control promoter was expressed in all stem tissues and exhibited similar cambial expression patterns in Eucalyptus and Populus stems
One of the aims of this study was to investigate the suitability of ISSA for assessing and comparing promoter activity in woody stems across plant species and genera. Towards this end, we first asked whether the observed sector types and staining patterns were indeed comparable among species, because it has been noted before that even constitutive promoters such as CAMV35S can show variable expression across species (Qin et al. 2010). Similar to previous results (Van Beveren et al. 2006) we found that the CAMV35S promoter was active in most sampled stem tissue types (Fig. 2a, b) and this was to be expected as the CAMV35S promoter is constitutive and will express GUS in most plant tissues (Odell et al. 1985; Jefferson et al. 1987; Benfey and Chua 1990). The comparison of CAMV35S driven GUS expression patterns in cambial derived sectors observed in Eucalyptus and Populus stems did not show any significant differences (Table 5); however, other sector types such as the tylose sectors exhibited very different frequencies presumably due to differences in the susceptibility of cell types to transformation (Fig. 2a, b). Together, these results suggest that ISSA can be used to compare promoter expression patterns across species using either sector type (Fig. 2) or cambial staining patterns (Fig. 3), provided that the inherent differences in transformation efficiency are accounted for by using a standard constitutive promoter construct such as CAMV35S.
Staining patterns for CesA promoters in cambial derived tissues showed clear grouping of primary and secondary cell wall-related promoters
In a previous study, the expression patterns of the Arabidopsis (AtCesA8) and Eucalyptus (EgCesA1) promoters were analysed using promoter::GUS assays in Arabidopsis plants (Creux et al. 2008). This confirmed the secondary cell wall-related expression patterns of these two functional orthologs (Ranik and Myburg 2006). We included the same two promoter constructs in this study to allow comparison of the ISSA results to that obtained by whole-plant transformation in Arabidopsis. We found that the cambial staining patterns obtained in Eucalyptus stems for these two promoter constructs, as well as for the other CesA genes (Fig. 3), were consistent with the expression patterns previously observed for these genes (Taylor et al. 2003; Taylor 2008; Ko et al. 2012). The EgCesA1, 2, 3 and AtCesA8 promoters produced GUS staining mostly in developing xylem cells (X1) which actively deposit secondary cell walls before the onset of PCD (Plomion et al. 2001) and are thus expected to show high EgCesA1, 2 and 3 expression levels. Their distinct expression patterns may explain the lower net ATS−2 values (ATS−2 = 0.7 for EgCesA1 to ATS−2 = 2.0 for EgCesA3) observed for the secondary cell wall-associated EucalyptusCesA promoters (Table 3). In contrast, higher ATS−2 values were observed for the primary cell wall-related promoters (EgCesA4, 5 and 7), which reflected their expression in a wider range of cell types such as phloem (P), developing xylem (X1) and ray cells in mature xylem (X2) tissues. These results demonstrate that the ISSA approach was able to discriminate the distinct expression patterns of the EucalyptusCesA genes in woody stem tissues.
The staining patterns of the CesA promoter constructs were not as distinctive in Populus stems as was observed for the primary and secondary cell wall-associated CesA genes in Eucalyptus stems (Figs. 2c, d, 3a). In particular, the three secondary cell wall-related EucalyptusCesA promoters (EgCesA1, 2 and 3) did not predominantly produce developing xylem (X1) expression in Populus stems, but were expressed at equal frequency in phloem (P) and developing xylem (X1) tissues. This could be the result of differences between the regulatory networks of the two genera and has been reported in a number of other plant promoter studies (Zhang et al. 2003; Fei et al. 2006; Qin et al. 2010). While the transcriptional network regulating secondary cell wall deposition is thought to be largely conserved across plant species and genera (Zhong et al. 2010), there may be important differences in promoter sequence and transcription factor binding sites of these species. In well-studied models such as humans, fruit flies and yeast, cis-regulatory variation has been shown to be relatively common (Ho et al. 2009; Dowell 2010; Mu et al. 2011) and could underlie differences in reporter gene expression observed for the same promoter construct in different species, as was found in this study. Cis-element evolution within promoter sequences can give rise to subfunctionalization of duplicated gene loci in organisms such as Populus, which have undergone genome-wide or segmental duplications (Tuskan et al. 2006). Furthermore, the NAC domain transcription factor family harbouring many of the key transcription factors involved in secondary cell wall formation is highly expanded in some plant genomes and the duplicated genes may be under different evolutionary pressures (Hu et al. 2010). These differences may explain the variation observed in reporter gene expression from different genetic backgrounds. Other possible sources of variation in the reporter gene expression observed for these two species could be on an anatomical or development level, but would require further investigation to elucidate this complex issue.
In this study we show that ISSA is an efficient approach to investigate promoter expression in the stems of woody plants such as Populus and Eucalyptus. ISSA requires less time and space to test promoters in woody stems than whole-plant transformation and regeneration and provides ample independent transformation events for statistical analysis. However, it is important to include appropriate controls to interpret the ISSA staining patterns produced by transforming multiple cell types and using promoters with cell type- or developmental stage-specific expression. We found that the CesA promoter constructs produced distinct staining patterns in woody stem tissues consistent with the predicted roles of the corresponding CesA genes in primary and secondary cell wall formation. Our results suggest that, while many aspects of the secondary cell wall transcriptional network are conserved (Zhong et al. 2010), there are regulatory differences which should be considered when testing promoters in heterologous systems. ISSA should be applicable to a wider range of woody plants and various secondary cell wall-related promoters could be analysed in this manner, which will be important for elucidating the transcriptional control of woody biomass production.
We are grateful to Martin Ranik for providing the CesA-promoter::GUS constructs which made this study possible and Minique De Castro for aiding in the construction of the second CAMV35S-promoter::GUS construct. We also thank Julio Najera, Valerie Frassiant and Angelique Manuel for laboratory assistance. This work was supported through funding provided by Mondi and Sappi to the Forest Molecular Genetics (FMG) Programme, the Technology and Human Resources for Industry Programme (THRIP) and the National Research Foundation (NRF) of South Africa as well as a Linkage Grant from the Australian Research Council (LP0776563) to GB, AAM and AVS.
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