Modification of Cellulose Fibre via Hormone Genes

Hormones are involved in regulation of many features during plant development like shoot growth and flower formation. Genetically transferring hormone or hormone-like genes for changing shoot growth may be an attractive starting point when aiming for modifying phenotypic and wood anatomical features in trees (Table 6.1). Classically, modifications of shoot growth resulting in so-called dwarf or semi-dwarf mutants are very often caused by mutations in genes encoding proteins that regulate synthesis and/or signalling of gibberellin, a major plant hormone (for reviews: Hedden 1999; Olszewski et al. 2002; Sun and Gubler 2004; Fleet and Sun 2005). The gene mainly responsible for internode length is gibberellin acid20 (GA20) -oxidase, as identified in dwarf mutants of rice (Spielmeyer et al. 2002), potato (Carrera et al. 2000) or aspen (Eriksson and Moritz 2002). Other dwarf mutants like the brachytic2 (br2) in maize or the dwarf3 (dw3) in sorghum are defective of a protein responsible for auxin transport (Multani et al. 2003).

Induction of dwarfism has been achieved in tobacco by over-expressing gibberellin acid2 (GA2) -oxidase gene from Arabidopsis (Biemelt et al. 2004). In poplar, the same gene was found to be over-expressed in a dwarf trans-genic hybrid poplar line obtained in a screening of several independently activation-tagged transgenic poplar lines in tissue culture, greenhouse, and field environments (Busov et al. 2003). Experiments on over-expression of the Arabidopsis GA3-oxidase gene in transgenic aspen clearly show that ectopic expression of this gene alone cannot increase the flux towards bioac-tive GAs; thus the 20-oxidation is the limiting step (Israelsson et al. 2004).

When the GA20-oxidase gene is over-expressed in transgenic poplar, elongated shoot growth (Fig. 6.1a) with increased biomass production was observed (Huang et al. 1998; Eriksson et al. 2000; Biemelt et al. 2004). These characteristics were positively correlated with rate of photosynthesis (Biemelt et al. 2004), early flowering and decreased seed dormancy (Huang et al. 1998), and small leaves (Fladung, unpublished). Detailed investigations on gene expression alterations in wood-forming tissue of GA20-oxidase over-expressing trees showed that highest transcript changes occurred in genes generally involved in the early stages of xylogenesis, e.g. cell division, early and late expansion (Israelsson et al. 2003).

Transgenic plants over-expressing GA20-oxidase reveal a faster growth (Fig. 6.1b) with increased biomass production (Eriksson et al. 2000). The xylem of these transgenic trees form fibres with up to an 8% increased length compared to untransformed wildtype. Over-expression of active GAs in plant tissues on the other hand also lead to pleiotropic or other deleterious effects. In sorghum, expression analysis of a GA20-oxidase gene in embryos revealed a possible role of this gene in the hormonal control of germination (Perez-Flores et al. 2003). In poplar, GA20-oxidase transgenic plants reveal a poor rooting capacity causing problems during the transfer of transgenic plants into soil (Eriksson et al. 2000). GA20-oxidase gene was also identified as a candidate gene involved in seed dormancy and vernalization processes (Oka et al. 2001).

Transfer of the cytokinin biosynthesis gene ipt into poplar resulted in transgenic plants with severe effects on plant phenotype (von Schwartzenberg et al. 1994). ipt-transgenic poplar revealed reduced apical dominance, a dwarfed appearance and inability to rooting. When

Table 6.1. Overexpression of hormone and hormone-like genes and their effects in transgenic trees

Hormone and hormone-like genes

Tree species

Overall effects

Effects on wood features

References

Gibberellins

GA2O oxidase

GA2 oxidase GA3 oxidase

Cytokinins Ipt

Auxins

IaaH, IaaM

IaaL

Rol genes

Populus trémula x P. tremuloides

Populus tremula x P. alba

Populus tremula x P tremuloides

Populus trémula x P. alba

Populus tremula x P. tremuloides

Populus tremula x P. tremuloides

Betula pendula

Populus tremula

Elongated internodes and biomass improvement

Dwarf plants

Nearly unaltered -growth pattern

Bud formation in the absence of exogenous cytokinins; shoots unable to root

Reduced growth rate

Pending leaves

Dwarf plants, smaller leaves, altered growth, precocious flushing

Longer cellulose fibres

Altered wood anatomical traits like vessel size and density

Bushy growth habit, smaller leaves

Breaking of stem -apical dominance, higher cumulative stem length

Shortened xylem fibres

Eriksson et al. (2000); Fladung, unpublished

Busov et al.

Israelsson et al.

Schwartzenberg et al. (1994)

Tuominen et al. (1995)

Fladung and Ahuja (1996)

Piispanen et al. (2003)

Tzfira et al. (1999)

Atypical late wood Fladung et al. formation, different (1996, 1997); pyrolysis products, Nilsson et al.

lower average cellulose content, higher arabino-galactan content

Grünwald et al. (2000); Puls et al. (2003); Meier et al. (2005)

Plant height 200,00 180,00 160,00 140,00 120,00 100,00 80,00 60,00 40,00 20,00 0,00

4 weeks

8 weeks

24 weeks

Fig. 6.1(a,b). Transgenic hybrid aspen over-expressing GA20-oxidase (middle and left plant, right plant is control) show: (a) an elongated shoot growth; (b) a faster growth (E-GA20: different independent transgenic lines; Esch5 is non-transgenic control)

S Esch5 H E-GA20#1 0 E-GA20#2 ^ E-GA20#4 ^E-GA20#12 □ E-GA20#13

4 weeks

8 weeks

24 weeks

Fig. 6.1(a,b). Transgenic hybrid aspen over-expressing GA20-oxidase (middle and left plant, right plant is control) show: (a) an elongated shoot growth; (b) a faster growth (E-GA20: different independent transgenic lines; Esch5 is non-transgenic control)

Agrobacterium auxin biosynthesis genes iaaM and iaaHwere used for transformation of Populus, transgenic plants showed increased levels of auxin in leaves and roots (Tuominen et al. 1995). Growth rate of the transgenic trees was reduced, and the wood anatomical traits like vessel size and vessel density were altered. Severe phenotypic alterations were also observed in poplar transgenic to the rolC gene from Agrobacterium rhizogenes under control of the cauliflower-35S-virus promoter. Transgenic plants exhibited stunted growth with an increased number of small leaves (Fladung et al. 1996; Nilsson et al. 1996). ^olC-transgenic poplar also revealed altered hormonal levels including gibberellin, auxin and cytokinins (Fladung et al. 1997). In spring, a precocious burst from bud rest was observed in 35S-rolC transgenic trees while initiation of cambial activity was not changed (Grunwald et al. 2000). Wood of 35S-rolC poplars revealed atypical late wood formation with thin-walled fibres. Characterisation of polymers in wood by analyzing their thermal degradation products using gas chromatograph and a mass spectrometer (Py-GC/MS) showed different patterns clearly discriminating wood of 35S-rolC poplars from control (Meier et al. 2003, 2005). Determining the cellulose, hemicelluloses and lignin contents in wood of 35S-rolC transgenic poplar revealed that the average cellulose content was 9.3% lower than the one of control trees (Puls et al. 2003). Interestingly the wood of the 35S-rolC transgenic poplar had a Klason lignin content, which was 4% higher compared to the non-transformed aspen trees. The wood of the transgenic 35S-rolC transgenic poplar also excelled by a higher arabinogalactan content. This hemicellulose component is typical for not fully differentiated wood tissues.

A second difference between the primary and secondary cell wall layers to the one mentioned above is the angle of microfibrils with respect to fibre axis. In the S1 and S3 layers the microfibril angle is 50-90° but 5-30° in S2 with lower angles in early wood than in late wood (Joshi 2004). Microfibril angle changes throughout the S2 cell wall layer. Within a fibre, the microfibril angle can change as much as 8° and among fibres within the same ring it can differ as much as 21° (Joshi 2004). Many fibres must be measured to obtain an accurate and precise assessment of microfibril angle. Microfibril angle is also a function of both height and rings from the pith. Microfibril angle is important for stiffness in wood with lower angles for enhanced wood quality. Hormonal treatments can be used to investigate the signal transduction pathways involved in regulation of cellulose microfibril angle (Jackson, online 2005). However, modification of the microfibril angle via hormonal treatments or even by using hormonal genes has not been tested so far.

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