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

Abstract

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.

Fig. 1.

Hypocotyl elongation and phloem fibre thickening in developing flax seedlings. Hypocotyl length was measured. Then sections were made to visualize the internal tissues of the hypocotyl and measure fibre cell wall thickness. Each data point is the average of 20 individual measurements. Error bars show the standard error, n = 20.

Fig. 2.

Fibre development in flax hypocotyls. Sections were stained with toluidine blue. (A) and (D) 7-d hypocotyls; (B) and (E) 9-d hypocotyls; (C) and (F) 15-d hypocotyls. Abbreviations: x, xylem; s, sieve element of phloem; f, phloem fibre with distinct secondary wall. Scale bars = 100 µm.

Fig. 3.

Functional categorization of significantly (fc > 2, fdr < 5 %) differentially expressed transcripts in all six comparisons. The figure does not include probe sets that could not be classified into the nine categories represented. Since microarrays were done in a three-way comparison (7 d/9 d, 9 d/15 d, 7 d/15 d), each comparison will have two categories of differentially expressed transcripts. For example, the 7 d/9 d comparison will have transcripts which are expressed more in 7 d compared with 9 d (7 > 9) and also will have transcripts which are expressed more in 9 d compared with 7 d (9 > 7).

Fig. 4.

Correlation of microarray signal intensities in pair-wise comparisons of hypocotyl and stem developmental stages. The correlation co-efficient (R²) for each comparison was calculated using mean signal intensities for each probeset in each of the 7-d, 9-d and 15-d microarray analyses reported here and corresponding probesets from each of the three previously defined stem developmental stages (TOP, MID, BOT; Roach and Deyholos, 2007).

Fig. 5.

β-Galactosidase activity in hypocotyl protein extracts assayed by ONPG. Each datum point represents the average of three independent biological replicates. Values that were significantly different (α = 0·05) in an ANOVA are labelled with different letters. Error bars are the standard error of the mean.

Fig. 6.

In situ detection of β-galactosidase enzyme activity. X-gal staining of fixed tissue sections was used to detect the activity of β-galactosidase in flax hypocotyls. Arrows show phloem fibres with thickened walls. The scale bar in each panel is equivalent to 100 µm. (A), (D) and (G) 7-d hypocotyls; (B), (E) and (H) 9-d hypocotyls; (C), (F) and (I) 15-d hypocotyls; (A), (B), (C), (G), (H) and (I) transverse sections; (D), (E) and (F) longitudinal sections.