The enzyme hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT) is involved in the production of methoxylated monolignols that are precursors to guaiacyl and syringyl lignin in angiosperm species. We identified and cloned a putative HCT gene from Pinus radiata, a coniferous gymnosperm that does not produce syringyl lignin. This gene was up-regulated during tracheary element (TE) formation in P. radiata cell cultures and showed 72.6% identity to the amino acid sequence of the Nicotiana tabacum HCT isolated earlier. RNAi-mediated silencing of the putative HCT gene had a strong impact on lignin content, monolignol composition, and interunit linkage distribution. AcBr assays revealed an up to 42% reduction in lignin content in TEs. Pyrolysis-GC/MS, thioacidolysis, and NMR detected substantial changes in lignin composition. Most notable was the rise of p-hydroxyphenyl units released by thioacidolysis, which increased from trace amounts in WT controls to up to 31% in transgenics. Two-dimensional 13C-1H correlative NMR confirmed the increase in p-hydroxyphenyl units in the transgenics and revealed structural differences, including an increase in resinols, a reduction in dibenzodioxocins, and the presence of glycerol end groups. The observed modifications in silenced transgenics validate the targeted gene as being associated with lignin biosynthesis in P. radiata and thus likely to encode HCT. This enzyme therefore represents the metabolic entry point leading to the biosynthesis of methoxylated phenylpropanoids in angiosperm species and coniferous gymnosperms such as P. radiata.

https://www.pnas.org/content/pnas/104/28/11856.full.pdf

Exploring lignification in conifers by silencing hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase in Pinus radiata

At the outset the author states that: ‘This book is about how to grow Pinus radiata (radiata pine) forest plantations.’ It certainly covers a very wide range of topics – from the ecology of radiata pine within its natural range to the complexities of current research into tree breeding, silviculture and wood utilisation. The book also reviews the social, economic and environmental aspects of radiata pine plantations and includes a chapter on the use of radiata pine on farms.

Sustainable management of Pinus radiata plantations.

Forest tree biotechnology

Physical methods for genetic plant transformation.

In angiosperms, the formation of the flower meristem is controlled by partially redundant flower meristem identity genes of which FLORICAULA (FLO)/LEAFY (LFY ) plays a central role. It is not known whether formation of reproductive organs of pre-angiosperm species is similarly regulated. Recently, a FLO/LFY-like cDNA, NEEDLY (NLY ), has been cloned in a conifer species Pinus radiata (D. Don). Here we report cloning of a different P
inus
radiata F
LO/L
FY-l ike cDNA, PRFLL. PRFLL had two large regions of high similarity to angiosperm FLO/LFY orthologues: amino acids 61–126 and 247–406 (50% and 81% identity, and 75% and 88% similarity, respectively, to LFY) and shorter regions of local similarity. Overall identity was 53% to LFY and 61% to NLY. Phylogenetic analysis of deduced protein sequences including partial LFY-like sequences from Pseudotsuga menziesii indicated that conifer proteins constituted a separate clade that could be divided into two groups represented by NLY and PRFLL. In contrast to angiosperms, both conifers had two paralogous proteins resembling LFY. Northern hybridisation analysis revealed expression of PRFLL in vegetative buds of juvenile, adolescent and mature trees. The transcript was not detected in vascular cambium, roots or secondary needles. To follow PRFLL expression during the early stages of cone development we analysed a temporal series of buds containing cone primordia, and developing cones, using Northern hybridisation and confocal microscopy in parallel. PRFLL mRNA was detected in buds from dominant and subordinate branches, in which cone and shoot primordia develop, and in developing male cones but not in developing female cones. Expression was particularly high in buds containing axillary primordia prior to their differentiation as male cone primordia. This is consistent with PRFLL being involved in determination of the male cone primordium identity.

PRFLL – a Pinus radiata homologue of FLORICAULA and LEAFY is expressed in buds containing vegetative shoot and undifferentiated male cone primordia

A biolistic particle delivery system was used to genetically transform embryogenic tissue of Pinus radiata. The introduced DNA contained a uidA reporter gene under the control of either the tandem CaMV 35S or the artificial Emu promoter, and the npt II selectable marker controlled by the CaMV 35S promoter. The average number of stable, geneticin-resistant lines recovered was 0.5 per 200 mg fresh weight bombarded tissue. Expression of the uidA reporter gene was detected histochemically and fluorimetrically in transformed embryogenic tissue and in derived mature somatic embryos and regenerated plants. The integration of uidA and npt II genes into the Pinus radiata genome was demonstrated using PCR amplification of the inserts and Southern hybridisation analysis. The expression of both genes in transformed tissue was confirmed by Northern hybridisation analysis. More than 150 transgenic Pinus radiata plants were produced from 20 independent transformation experiments with four different embryogenic clones.

Stable transformation and regeneration of transgenic plants of Pinus radiata D. Don

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Stable transformation and regeneration of transgenic plants of Pinus radiata D. Don