Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation

Abstract

SummaryEucalyptus are of tremendous economic importance being the most planted hardwoods worldwide for pulp and paper, timber and bioenergy. The recent release of the Eucalyptus grandis genome sequence pointed out many new candidate genes potentially involved in secondary growth, wood formation or lineage-specific biosynthetic pathways. Their functional characterization is, however, hindered by the tedious, time-consuming and inefficient transformation systems available hitherto for eucalypts. To overcome this limitation, we developed a fast, reliable and efficient protocol to obtain and easily detect co-transformed E. grandis hairy roots using fluorescent markers, with an average efficiency of 62%. We set up conditions both to cultivate excised roots in vitro and to harden composite plants and verified that hairy root morphology and vascular system anatomy were similar to wild-type ones. We further demonstrated that co-transformed hairy roots are suitable for medium-throughput functional studies enabling, for instance, protein subcellular localization, gene expression patterns through RT-qPCR and promoter expression, as well as the modulation of endogenous gene expression. Down-regulation of the Eucalyptus cinnamoyl-CoA reductase1 (EgCCR1) gene, encoding a key enzyme in lignin biosynthesis, led to transgenic roots with reduced lignin levels and thinner cell walls. This gene was used as a proof of concept to demonstrate that the function of genes involved in secondary cell wall biosynthesis and wood formation can be elucidated in transgenic hairy roots using histochemical, transcriptomic and biochemical approaches. The method described here is timely because it will accelerate gene mining of the genome for both basic research and industry purposes.

A systems biology view of wood formation in Eucalyptus grandis trees submitted to different potassium and water regimes

“…The DNA fragments were then cloned into Gateway‐adapted binary vectors pGWAY‐0 and pGWAY‐1, for overexpression and promoter activity analyses respectively and transformed into Eucalyptus hairy roots as described by Plasencia et al . (). Eucalyptus composite plants harboring transgenic hairy roots were grown in OIL DRI substrate US‐Special Substrate (Type III/R; Damolin, Fur, Denmark) in 200 ml pots for a period of 5 months (16 h photoperiod, 25/22°C).…”

The Eucalyptus linker histone variant EgH1.3 cooperates with the transcription factor EgMYB1 to control lignin biosynthesis during wood formation

“…RNA was extracted from hairy roots as described by Plasencia et al . (). Once extracted, the RNA was digested with TurboDNase (Ambion) to remove residual DNA.…”

“…Then, EgH1.3 cDNA was inserted into the gateway region by LR cloning. For transactivation assays we also used, as reporter constructs, the binary vectors harbouring the EgCAD2 and EgCCR1 promoters fused to the gene encoding b-glucuronidase (GUS) (Plasencia et al, 2015).…”

“…Eucalyptus grandis hairy root transformation was performed as described by Plasencia et al (2015). Briefly, E. grandis seeds were germinated in vitro and their hypocotyls were infected with a needle swabbed with A. rhizogenes by stabbing.…”

“…RNA was extracted from stems using a protocol for seeds and siliques (Oñate-S anchez & Vicente-Carbajosa, 2008). RNA was extracted from hairy roots as described by Plasencia et al (2015). Once extracted, the RNA was digested with TurboDNase (Ambion) to remove residual DNA.…”