Development of cellulosic secondary walls in flax fibers requires beta-galactosidase.

Bast (phloem) fibers, tension wood fibers, and other cells with gelatinous-type secondary walls are rich in crystalline cellulose. In developing bast fibers of flax (Linum usitatissimum), a galactan-enriched matrix (Gn-layer) is gradually modified into a mature cellulosic gelatinous-layer (G-layer), which ultimately comprises most of the secondary cell wall. Previous studies have correlated this maturation process with expression of a putative ß-galactosidase. Here, we demonstrate that ß-galactosidase activity is in fact necessary for the dynamic remodeling of polysaccharides that occurs during normal secondary wall development in flax fibers. We found that developing stems of transgenic (LuBGAL-RNAi) flax with reduced ß-galactosidase activity had lower concentrations of free Gal and had significant reductions in the thickness of mature cellulosic G-layers compared with controls. Conversely, Gn-layers, labeled intensively by the galactan-specific LM5 antibody, were greatly expanded in LuBGAL-RNAi transgenic plants. Gross morphology and stem anatomy, including the thickness of bast fiber walls, were otherwise unaffected by silencing of ß-galactosidase transcripts. These results demonstrate a specific requirement for ß-galactosidase in hydrolysis of galactans during formation of cellulosic G-layers. Transgenic lines with reduced ß-galactosidase activity also had biochemical and spectroscopic properties consistent with a reduction in cellulose crystallinity. We further demonstrated that the tensile strength of normal flax stems is dependent on ß-galactosidase-mediated development of the phloem fiber G-layer. Thus, the mechanical strength that typifies flax stems is dependent on a thick, cellulosic G-layer, which itself depends on ß-galactosidase activity within the precursor Gn-layer. These observations demonstrate a novel role for matrix polysaccharides in cellulose deposition; the relevance of these observations to the development of cell walls in other species is also discussed.

Microarray analysis of developing flax hypocotyls identifies novel transcripts correlated with specific stages of phloem fibre differentiation.

BACKGROUND AND AIMS: Hypocotyls are a commonly used model to study primary growth in plants, since post-germinative hypocotyls increase in size by cell elongation rather than cell division. Flax hypocotyls produce phloem fibres in bundles one to two cell layers thick, parallel to the protoxylem poles of the stele. Cell wall deposition within these cells occurs rapidly at a well-defined stage of development. The aim was to identify transcripts associated with distinct stages of hypocotyl and phloem fibre development. METHODS: Stages of flax hypocotyl development were defined by analysing hypocotyl length in relation to fibre secondary wall deposition. Selected stages of development were used in microarray analyses to identify transcripts involved in the transition from elongation to secondary cell wall deposition in fibres. Expression of specific genes was confirmed by qRT-PCR and by enzymatic assays. KEY RESULTS: Genes enriched in the elongation phase included transcripts related to cell-wall modification or primary-wall deposition. Transcripts specifically enriched at the transition between elongation and secondary wall deposition included beta-galactosidase and arabinogalactan proteins. Later stages of wall development showed an increase in secondary metabolism-related transcripts, chitinases and glycosyl hydrolases including KORRIGAN. Microarray analysis also identified groups of transcription factors enriched at one or more stages of fibre development. Subsequent analysis of a differentially expressed beta-galactosidase confirmed that the post-elongation increase in beta-galactosidase enzyme activity was localized to phloem fibres. CONCLUSIONS: Transcripts were identified associated with specific stages of hypocotyl development, in which phloem fibre cells were undergoing thickening of secondary walls. Temporal and spatial regulation of beta-galactosidase activity suggests a role for this enzyme in remodelling of flax bast fibre cell walls during secondary cell wall deposition.

Microarray analysis of flax (Linum usitatissimum L.) stems identifies transcripts enriched in fibre-bearing phloem tissues.

To better understand the molecular processes associated with the development of the unusually long (> 30 mm) and strong bast fibre cells within the phloem of flax stems, we conducted a gene discovery experiment to identify transcripts enriched in fibre-bearing tissues, with the intention that these transcripts would serve as future targets for crop improvement and research in phloem development and cell wall deposition. We produced a library of 9,600 cDNA clones from the peels of flax stems, and selected tissue-specific cDNAs for sequencing based on two series of microarray experiments. In the first microarray series, we compared transcript abundance in stem-peels and leaves, and identified stem-enriched transcripts putatively involved in the processes of polysaccharide and cell wall metabolism. In the second microarray series, we compared gene expression in three segments of the vertical stem axis, which constituted a developmental series for phloem fibres and other cell types. The expression of specific LTP and AGP transcripts was particularly well-correlated with stem segments during either the elongation phase or cell-wall thickening phase of phloem fibre development, and the phloem-specific enrichment of these transcripts was confirmed by qRT-PCR. Transcripts representing multiple, distinct chitinases, beta-galactosidases, arabinogalactan proteins (AGP), and lipid transfer proteins (LTPs) were among the interesting transcripts enriched in specific stages of the developing stem. Considered together, the results of our analyses suggest similarity between the molecular mechanisms underlying phloem fibre development and the gelatinous fibres of tension wood in trees.