Microarray Analysis of Developing Flax Hypocotyls Identifies Novel Transcripts Correlated with Specific Stages of Phloem Fibre Differentiation

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Abstract
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. 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. 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 β-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 β-galactosidase confirmed that the post-elongation increase in β-galactosidase enzyme activity was localized to phloem fibres. 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 β-galactosidase activity suggests a role for this enzyme in remodelling of flax bast fibre cell walls during secondary cell wall deposition.