BackgroundQuantitative real-time PCR (qRT-PCR) is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs). Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (Linum usitatissimum L).ResultsSpecific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs) and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH) as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups.qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax LuMYB1 gene (homologue of AtMYB59). LuMYB1-1 and LuMYB1-2 were highly expressed in the internal stem tissues as compared to outer stem tissues and other samples. This result was confirmed with both geNorm-designated- and NormFinder-designated-reference genes.ConclusionsThe use of 2 different statistical algorithms results in the identification of different combinations of flax HKGs for expression data normalization. Despite such differences, the use of geNorm-designated- and NormFinder-designated-reference genes enabled us to accurately compare the expression levels of a flax MYB gene in different organs and tissues. Our identification and validation of suitable flax HKGs will facilitate future developmental transcriptomic studies in this economically-important plant.

Flax (Linum usitatissimum) stems contain cells showing contrasting cell wall structure: lignified in inner stem xylem tissue and hypolignified in outer stem bast fibers. We hypothesized that stem hypolignification should be associated with extensive phenolic accumulation and used metabolomics and transcriptomics to characterize these two tissues.1 H nuclear magnetic resonance clearly distinguished inner and outer stem tissues and identified different primary and secondary metabolites, including coniferin and p-coumaryl alcohol glucoside. Ultrahigh-performance liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry aromatic profiling (lignomics) identified 81 phenolic compounds, of which 65 were identified, to our knowledge, for the first time in flax and 11 for the first time in higher plants. Both aglycone forms and glycosides of monolignols, lignin oligomers, and (neo)lignans were identified in both inner and outer stem tissues, with a preponderance of glycosides in the hypolignified outer stem, indicating the existence of a complex monolignol metabolism. The presence of coniferin-containing secondary metabolites suggested that coniferyl alcohol, in addition to being used in lignin and (neo)lignan formation, was also utilized in a third, partially uncharacterized metabolic pathway. Hypolignification of bast fibers in outer stem tissues was correlated with the low transcript abundance of monolignol biosynthetic genes, laccase genes, and certain peroxidase genes, suggesting that flax hypolignification is transcriptionally regulated. Transcripts of the key lignan genes Pinoresinol-Lariciresinol Reductase and Phenylcoumaran Benzylic Ether Reductase were also highly abundant in flax inner stem tissues. Expression profiling allowed the identification of NAC (NAM, ATAF1/2, CUC2) and MYB transcription factors that are likely involved in regulating both monolignol production and polymerization as well as (neo)lignan production.

Laccases are ubiquitous oxidases present in animals, plants, bacteria, and fungi. In plant species, they occur as large multigenic families. The involvement of peroxidases in lignification is supported by a wealth of literature data. In contrast, the role of laccases in this major plant process is less firmly established. The large number of plant laccases, which argues for a variety of functions in plant development, makes the identification of lignin-specific laccases a challenge. However, in the past decade, the development of new genetic technologies and tools has played a central role towards resolving this issue. In addition, the plant model, Arabidopsis thaliana, has recently provided novel insights about the occurrence of laccases involved in stem lignification. Information about lignin-related laccases is also available from other species, such as poplar, or other organs and tissues, such as seed coats. This review brings a short and cutting edge survey of laccases and lignification.