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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.

BACKGROUND: NAC domain transcription factors initiate secondary cell wall biosynthesis in Arabidopsis fibres and vessels by activating numerous transcriptional regulators and biosynthetic genes. NAC family member SND2 is an indirect target of a principal regulator of fibre secondary cell wall formation, SND1. A previous study showed that overexpression of SND2 produced a fibre cell-specific increase in secondary cell wall thickness in Arabidopsis stems, and that the protein was able to transactivate the cellulose synthase8 (CesA8) promoter. However, the full repertoire of genes regulated by SND2 is unknown, and the effect of its overexpression on cell wall chemistry remains unexplored. RESULTS: We overexpressed SND2 in Arabidopsis and analyzed homozygous lines with regards to stem chemistry, biomass and fibre secondary cell wall thickness. A line showing upregulation of CesA8 was selected for transcriptome-wide gene expression profiling. We found evidence for upregulation of biosynthetic genes associated with cellulose, xylan, mannan and lignin polymerization in this line, in agreement with significant co-expression of these genes with native SND2 transcripts according to public microarray repositories. Only minor alterations in cell wall chemistry were detected. Transcription factor MYB103, in addition to SND1, was upregulated in SND2-overexpressing plants, and we detected upregulation of genes encoding components of a signal transduction machinery recently proposed to initiate secondary cell wall formation. Several homozygous T4 and hemizygous T1 transgenic lines with pronounced SND2 overexpression levels revealed a negative impact on fibre wall deposition, which may be indirectly attributable to excessive overexpression rather than co-suppression. Conversely, overexpression of SND2 in Eucalyptus stems led to increased fibre cross-sectional cell area. CONCLUSIONS: This study supports a function for SND2 in the regulation of cellulose and hemicellulose biosynthetic

Eucalyptus tree species, commonly referred to as eucalypts, are among the most planted hardwoods in the world (Doughty 2000). They are generally long-lived, evergreen species belonging to the predominantly southern-hemisphere, angiosperm family Myrtaceae (Ladiges et al. 2003). They are native to Australia and islands to its north (Potts and Pederick 2000; Ladiges et al. 2003), where they occur naturally from sea level to the alpine tree line, from high rainfall to semiarid zones, and from the tropics to latitudes as high as 43° south (Williams and Woinarski 1997). Eucalypts are the dominant or codominant species of virtually all vegetation types in Australia except rainforest, the vegetation of the central arid zone, and higher montane regions (Wiltshire 2004). They are generally sclerophyllous and adapted to low nutrient soils (Eldridge et al. 1993; Florence 1996; Specht 1996) and fire (Pryor 1976;Ashton 2000; Burrows 2002). The eucalypt lineage is old, possibly extending back to the Late Cretaceous -ca. 70 million years ago (Hill et al. 1999; Ladiges et al. 2003; Crisp et al. 2004). Their ancestors were likely to have been widely dispersed on the supercontinent of Gondwana, as there are macrofossils ascribed to eucalypts of Eocene (55 to 34 Mya) age from northeastern Australia (Rozefelds 1996) and possibly Patagonia (Hill et al. 1999) and of Miocene (27 to 10 Mya) age from New Zealand (Pole et al. 1993) and Australia (Hill et al. 1999). The tectonic isolation of Australia (ca. 32 Mya) led to cooler, drier, and more seasonal climates and consequently a transition from a rainforest-dominated flora to Australia's unique sclerophyll flora (Hill et al. 1999; Ladiges et al. 2003; Crisp et al. 2004; Hill 2004). There is little doubt that the current dominance of the Australian continent by eucalypts is relatively recent and linked with the onset of severe aridity during the Late Miocene (10 to 7 Mya) and the present climatic system of extreme wet-dry glacial cycles that

Eucalyptus tree species, commonly referred to as eucalypts, are among the most planted hardwoods in the world (Doughty 2000). They are generally long-lived, evergreen species belonging to the predominantly southern-hemisphere, angiosperm family Myrtaceae (Ladiges et al. 2003). They are native to Australia and islands to its north (Potts and Pederick 2000; Ladiges et al. 2003), where they occur naturally from sea level to the alpine tree line, from high rainfall to semiarid zones, and from the tropics to latitudes as high as 43° south (Williams and Woinarski 1997). Eucalypts are the dominant or codominant species of virtually all vegetation types in Australia except rainforest, the vegetation of the central arid zone, and higher montane regions (Wiltshire 2004). They are generally sclerophyllous and adapted to low nutrient soils (Eldridge et al. 1993; Florence 1996; Specht 1996) and fire (Pryor 1976;Ashton 2000; Burrows 2002). The eucalypt lineage is old, possibly extending back to the Late Cretaceous -ca. 70 million years ago (Hill et al. 1999; Ladiges et al. 2003; Crisp et al. 2004). Their ancestors were likely to have been widely dispersed on the supercontinent of Gondwana, as there are macrofossils ascribed to eucalypts of Eocene (55 to 34 Mya) age from northeastern Australia (Rozefelds 1996) and possibly Patagonia (Hill et al. 1999) and of Miocene (27 to 10 Mya) age from New Zealand (Pole et al. 1993) and Australia (Hill et al. 1999). The tectonic isolation of Australia (ca. 32 Mya) led to cooler, drier, and more seasonal climates and consequently a transition from a rainforest-dominated flora to Australia's unique sclerophyll flora (Hill et al. 1999; Ladiges et al. 2003; Crisp et al. 2004; Hill 2004). There is little doubt that the current dominance of the Australian continent by eucalypts is relatively recent and linked with the onset of severe aridity during the Late Miocene (10 to 7 Mya) and the present climatic system of extreme wet-dry glacial cycles that

Eucalyptus tree species, commonly referred to as eucalypts, are among the most planted hardwoods in the world (Doughty 2000). They are generally long-lived, evergreen species belonging to the predominantly southern-hemisphere, angiosperm family Myrtaceae (Ladiges et al. 2003). They are native to Australia and islands to its north (Potts and Pederick 2000; Ladiges et al. 2003), where they occur naturally from sea level to the alpine tree line, from high rainfall to semiarid zones, and from the tropics to latitudes as high as 43° south (Williams and Woinarski 1997). Eucalypts are the dominant or codominant species of virtually all vegetation types in Australia except rainforest, the vegetation of the central arid zone, and higher montane regions (Wiltshire 2004). They are generally sclerophyllous and adapted to low nutrient soils (Eldridge et al. 1993; Florence 1996; Specht 1996) and fire (Pryor 1976;Ashton 2000; Burrows 2002). The eucalypt lineage is old, possibly extending back to the Late Cretaceous -ca. 70 million years ago (Hill et al. 1999; Ladiges et al. 2003; Crisp et al. 2004). Their ancestors were likely to have been widely dispersed on the supercontinent of Gondwana, as there are macrofossils ascribed to eucalypts of Eocene (55 to 34 Mya) age from northeastern Australia (Rozefelds 1996) and possibly Patagonia (Hill et al. 1999) and of Miocene (27 to 10 Mya) age from New Zealand (Pole et al. 1993) and Australia (Hill et al. 1999). The tectonic isolation of Australia (ca. 32 Mya) led to cooler, drier, and more seasonal climates and consequently a transition from a rainforest-dominated flora to Australia's unique sclerophyll flora (Hill et al. 1999; Ladiges et al. 2003; Crisp et al. 2004; Hill 2004). There is little doubt that the current dominance of the Australian continent by eucalypts is relatively recent and linked with the onset of severe aridity during the Late Miocene (10 to 7 Mya) and the present climatic system of extreme wet-dry glacial cycles that

Eucalyptus tree species, commonly referred to as eucalypts, are among the most planted hardwoods in the world (Doughty 2000). They are generally long-lived, evergreen species belonging to the predominantly southern-hemisphere, angiosperm family Myrtaceae (Ladiges et al. 2003). They are native to Australia and islands to its north (Potts and Pederick 2000; Ladiges et al. 2003), where they occur naturally from sea level to the alpine tree line, from high rainfall to semiarid zones, and from the tropics to latitudes as high as 43° south (Williams and Woinarski 1997). Eucalypts are the dominant or codominant species of virtually all vegetation types in Australia except rainforest, the vegetation of the central arid zone, and higher montane regions (Wiltshire 2004). They are generally sclerophyllous and adapted to low nutrient soils (Eldridge et al. 1993; Florence 1996; Specht 1996) and fire (Pryor 1976;Ashton 2000; Burrows 2002). The eucalypt lineage is old, possibly extending back to the Late Cretaceous -ca. 70 million years ago (Hill et al. 1999; Ladiges et al. 2003; Crisp et al. 2004). Their ancestors were likely to have been widely dispersed on the supercontinent of Gondwana, as there are macrofossils ascribed to eucalypts of Eocene (55 to 34 Mya) age from northeastern Australia (Rozefelds 1996) and possibly Patagonia (Hill et al. 1999) and of Miocene (27 to 10 Mya) age from New Zealand (Pole et al. 1993) and Australia (Hill et al. 1999). The tectonic isolation of Australia (ca. 32 Mya) led to cooler, drier, and more seasonal climates and consequently a transition from a rainforest-dominated flora to Australia's unique sclerophyll flora (Hill et al. 1999; Ladiges et al. 2003; Crisp et al. 2004; Hill 2004). There is little doubt that the current dominance of the Australian continent by eucalypts is relatively recent and linked with the onset of severe aridity during the Late Miocene (10 to 7 Mya) and the present climatic system of extreme wet-dry glacial cycles that

Eucalyptus tree species, commonly referred to as eucalypts, are among the most planted hardwoods in the world (Doughty 2000). They are generally long-lived, evergreen species belonging to the predominantly southern-hemisphere, angiosperm family Myrtaceae (Ladiges et al. 2003). They are native to Australia and islands to its north (Potts and Pederick 2000; Ladiges et al. 2003), where they occur naturally from sea level to the alpine tree line, from high rainfall to semiarid zones, and from the tropics to latitudes as high as 43° south (Williams and Woinarski 1997). Eucalypts are the dominant or codominant species of virtually all vegetation types in Australia except rainforest, the vegetation of the central arid zone, and higher montane regions (Wiltshire 2004). They are generally sclerophyllous and adapted to low nutrient soils (Eldridge et al. 1993; Florence 1996; Specht 1996) and fire (Pryor 1976;Ashton 2000; Burrows 2002). The eucalypt lineage is old, possibly extending back to the Late Cretaceous -ca. 70 million years ago (Hill et al. 1999; Ladiges et al. 2003; Crisp et al. 2004). Their ancestors were likely to have been widely dispersed on the supercontinent of Gondwana, as there are macrofossils ascribed to eucalypts of Eocene (55 to 34 Mya) age from northeastern Australia (Rozefelds 1996) and possibly Patagonia (Hill et al. 1999) and of Miocene (27 to 10 Mya) age from New Zealand (Pole et al. 1993) and Australia (Hill et al. 1999). The tectonic isolation of Australia (ca. 32 Mya) led to cooler, drier, and more seasonal climates and consequently a transition from a rainforest-dominated flora to Australia's unique sclerophyll flora (Hill et al. 1999; Ladiges et al. 2003; Crisp et al. 2004; Hill 2004). There is little doubt that the current dominance of the Australian continent by eucalypts is relatively recent and linked with the onset of severe aridity during the Late Miocene (10 to 7 Mya) and the present climatic system of extreme wet-dry glacial cycles that