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AOBPreview originally published online on June 30, 2008
Annals of Botany 2008 102(3):317-330; doi:10.1093/aob/mcn110
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© The Author 2008. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

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

Melissa J. Roach and Michael K. Deyholos*

Department of Biological Sciences, University of Alberta, Edmonton, Canada T6G 2E9

* For correspondence. E-mail deyholos{at}ualberta.ca

Received: 24 November 2007    Returned for revision: 23 April 2008    Accepted: 6 June 2008    Published electronically: 30 June 2008


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY INFORMATION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

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 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.

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 β-galactosidase activity suggests a role for this enzyme in remodelling of flax bast fibre cell walls during secondary cell wall deposition.

Key words: Bast, fibre, flax, hypocotyl, Linum usitatissimum, phloem, microarray, galactosidase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY INFORMATION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Phloem fibres are produced in both the stem and hypocotyl of flax (Linum usitatissimum). These fibres originate within the primary phloem from protophloem precursor cells (Esau, 1943). In flax stems, phloem fibres (also known as bast fibres) are found in bundles of 12–40 cells located in the primary phloem poles of the stele, directly opposite the protoxylem (Ageeva et al., 2005). In flax hypocotyls, the bast fibres are also arranged in bundles opposite the protoxylem, but the bundles contain fewer fibre cells than in the stem. Mature phloem fibres in both stems and hypocotyls are characterized by two distinct anatomical traits: an extremely long length and a thickened, gelatinous secondary cell wall (Gorshkova and Morvan, 2006). Since hypocotyl and stem phloem fibres share these anatomical traits, there is expected to be some similarity in the mechanisms controlling development of these fibres.

In stems, the development of bast fibres is separated into two non-overlapping phases. First, the fibre undergoes extensive elongation by coordinate and intrusive growth (Ageeva et al., 2005). After elongation is completed, the fibre begins to deposit a thick secondary cell wall consisting of 70–75 % cellulose, 15 % hemicellulose and 15 % pectin, with trace amounts of lignin, containing a large proportion of H-units (Gorshkova et al., 1996, 2000; Mooney et al., 2001; Day et al., 2005b). In flax stems, primary phloem fibres differentiate in a gradient along the length of the stem, resulting in a spatial separation of the phases of differentiation (Gorshkova et al., 2003).

Hypocotyls are a commonly used model for studying cell elongation in species such as Arabidopsis thaliana (Le et al., 2005), Ricinus communis (Eisenbarth and Weig, 2005), Cucumis sativus (Takahashi et al., 2006) and Cucurbita moschata (Taguchi et al., 1999), since post-germinative growth of the hypocotyl is mainly due to elongation of the hypocotyl cells, rather than cell division. Hypocotyls have also been used previously to study other physiological processes such as vascular development (Zhao et al., 2005), photomorphogenesis (Achard et al., 2007), hormone interactions (Smalle et al., 1997; Collett et al., 2000) and immunolocalization of cell wall polymers (Andeme-Onzighi et al., 2000).

In contrast to the stem, the size and patterning of the hypocotyl is established during embryogenesis, and subsequent development is generally constrained to a finite number of cells (Gendreau et al., 1997). During embryogenesis, the cells of the hypocotyl are specified but not fully differentiated. As the hypocotyl elongates, differentiation continues until most cells have attained their terminal identity. Although the majority of primary growth is achieved through cell elongation rather than division, some cell division does occur in the hypocotyl, including within a vascular cambium, which contributes to the overall girth of the hypocotyl (Zhao et al., 2005).

During previous microarray analysis of transcripts enriched during stages of fibre development in the flax stem (Roach and Deyholos, 2007), β-galactosidases (EC 3·2·1·23) were found enriched specifically at the transition zone (i.e. snap point) from fibre elongation to secondary cell wall deposition (Gorshkova et al., 2003). β-Galactosidases degrade terminal, non-reducing ends of β-D-galactosides, and appear to have specific roles in cell wall development, either through degrading, re-cycling or remodelling pectins (Martin et al., 2005), xyloglucan oligosaccharides (de Alcantara et al., 2006; Iglesias et al., 2006) or arabinogalactan proteins (Hirano et al., 1994; Kotake et al., 2005). In flax, the transition from fibre cell elongation to cell wall deposition is marked by the fibre cell-specific occurrence of a β-1,4-galactan epitope (Gorshkova et al., 2004) which is believed to be involved in either orienting cellulose microfibrils early in wall deposition or remodelling and cross-linking the cell wall later in development (Gorshkova and Morvan, 2006). Developmentally regulated deposition of β-1,4-galactans has also been reported in flax roots (Vicre et al., 1998), pea cotyledons (McCartney et al., 2000), potato tubers (Bush et al., 2001) Asclepias laticifers (Serpe et al., 2001), and arabidopsis roots (McCartney et al., 2003). Hypocotyls were used to further extend the hypothesis that β-galactosidases are involved in the transition from elongation to cell wall deposition in flax phloem fibres.

The experimental approach was designed with two main objectives. The first was to determine the degree of similarity in gene expression patterns during phloem fibre development in hypocotyl as compared with stem. This helps to define the extent to which hypocotyls can be used as a general model for flax phloem fibre development. The second objective was to identify genes associated with the transition from fibre cell elongation to secondary cell wall deposition. This approach will further our knowledge of flax bast fibre development, as well as provide insight into mechanisms that control the temporal and spatial coordination of cell elongation and cell wall deposition.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 SUPPLEMENTARY INFORMATION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Plant material
Seeds of Linum usitatissimum L. flax variety Norlin were donated by Gordon Rowland (Crop Development Center, Saskatoon). This variety of flax (i.e. linseed) is used primarily for its seed oil and was selected for this study because of local interest in developing dual-purpose crops. Plants were grown in Metromix 360 (Scotts, Marysville, OH, USA) in controlled environment chambers at 24 °C with 50 % humidity, and a light intensity of 200 µE supplied by high output fluorescent bulbs (CRI of 85, colour temperature of 3500 K) on a 16/8 h light/dark cycle.

Hypocotyl measurements and staging
Developmental staging of hypocotyls was done by daily measurement of hypocotyl length and fibre cell wall thickness. Fibre cell wall thickness was measured by hand sectioning through the centre of the hypocotyl. Sections were stained with toluidine blue, rinsed in water, and viewed under a light microscope. ImageJ software (Research Services Branch, NIH, Bethesda, MD, USA) was used to measure cell wall thickness. Wall thicknesses were measured in at least five individual fibres of four independent plants.

Microarray analysis
Construction of a 9600 element microarray from anonymous cDNA clones was described previously (Roach and Deyholos, 2007). The microarray slide is a cDNA microarray representing transcripts from a flax stem-peel cDNA library. A three-way comparison was conducted using hypocotyls harvested at 7 d (elongation phase), 9 d (onset of secondary cell wall deposition) and 15 d (late cell wall deposition).

Hypocotyls were dissected from seedlings 4 h after the start of the 16-h light cycle, and were immediately frozen in liquid N2. For each time-point used in the microarray analysis, hypocotyls from ten plants were pooled and total RNA was extracted using an RNeasy Plant kit (Qiagen). cDNA was synthesized using SuperscriptII (Invitrogen), primed with RT poly(A)-capture oligomers from the 3D Array900 microarray labelling kit (Genisphere, Hatfield, PA, USA). Hybridizations were performed as previously described (Roach and Deyholos 2007). Dye-related labelling efficiency bias was avoided by performing dye-flip hybridizations for each sample comparison. Three individual biological replicates of each comparison (7 d/9 d, 9 d/15 d and 7 d/15 d) were conducted for a total of 18 hybridizations.

Microarray and EST sequence analysis
Microarray spot intensities were extracted using Spotfinder v3·0, and the intensities were normalized through an implementation of the Loess algorithm in MIDAS v2·19 (Saeed et al., 2003). Significance Analysis of Microarrays (SAM) was applied to find spots for which the fold change (fc) differed significantly between one or more stem segments in a multiclass test, using a false discovery rate (fdr) of ≤5 % (Tusher et al., 2001). From among these significant spots, clones were identified which showed a ≥2-fold signal intensity between two or more hypocotyl developmental stages, and sequenced the corresponding clones using M13 reverse primers at the Plant Biology Institute (Saskatoon, Canada) on an ABI capillary sequencer (Applied Biosystems, Foster City, CA, USA). The chromatograms were processed with the phrep, phrap and consed software packages, using default parameters, except for phrap-minmatch 35 -minscore 50 during contig assembly (Ewing et al., 1998; Gordon et al., 1998). The raw DNA sequence for each EST was deposited in GenBank, under accessions EX720038 [GenBank] -EX720492. The individual sequences, which had been trimmed by phred to remove low-quality reads and the vector sequence were aligned, sequences to GenBank's non-redundant protein databases were finished using BLASTx, and this information was used to assign tentative annotations (Altschul et al., 1997). EST sequences that did not align to any GenBank protein sequence with an e-value of <10–10 were further processed using ESTScan2, to predict potential open reading frames (Iseli et al., 1999).

Quantitative real-time PCR (qRT-PCR)
Three biologically independent replicates of qRT-PCR were conducted on nine genes. Each replicate used aliquots of the same RNA samples used for microarrays. RNA was extracted and reverse transcription was performed as described above, using 2·5 µg of total RNA pretreated with DNase I (Ambion, DNA-free), and primed with oligo(dT)12–18 (Invitrogen). Real-time PCR was performed in an Applied Biosystems 7500 Fast Real-Time PCR System. For quantitative PCR reactions, 2·5 µL of a one-sixteenth dilution of the reverse-transcription reaction was used in a total volume of 10 µL with 0·4 µM of each forward and reverse gene-specific primer, 0·2 mM dNTPs, 0·25x SYBR Green, 1x ROX and 0·075 U Platinum Taq (Invitrogen). Threshold cycles (CT) were determined using 7500 Fast Software. CT values were normalized using elongation factor 1-{alpha} (EF1{alpha}) as an endogenous control. {Delta}{Delta}CT values were generated using the 7-d sample as a reference and log2 ratios calculated to show relative expression values between 7-d, 9-d and 15-d hypocotyls (Livak and Schmittgen, 2001).

ONPG assay of β-galactosidase activity
Crude protein extracts were extracted by grinding hypocotyl samples under liquid nitrogen in 0·05 M sodium phosphate buffer, pH 7·2. Homogenate was centrifuged at 4 °C for 30 min at 14 000 g. The collected supernatant was used as the crude protein extract. Extracts were quantified using 2D Quant Kit (GE Life Sciences). The ONPG assay of β-galactosidase activity was adapted from Dopico et al. (1989) and Sambrook and Russell (2001). Crude protein extracts were diluted in 0·05 M sodium citrate buffer pH 7 containing 0·1 M MgCl2, 4·5 M β-mercaptoethanol and 6 mM ONPG. Solutions were incubated for 30 min at 37 °C. The reaction was stopped by adding 500 µL 1 M Na2CO3. Absorbance of free p-nitrophenol was measured at 420 nm. β-Galactosidase activity for each sample was extrapolated from a standard curve and boiled protein samples were used as negative controls.

X-gal enzyme activity staining
X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) was used as a stain to detect β-galactosidase activity. Hypocotyls from 7 d, 9 d and 15 d were sectioned using a vibratome, fixed in 90 % cold acetone for 10 min, then rinsed in 50 mM sodium phosphate buffer pH 7·2. Sections were then immersed in staining solution (50 mM sodium phosphate buffer pH 7·2, 0·5 mM K3Fe(CN)6, 0·5 mM K4Fe(CN)6) containing 1 mM X-gal. Sections were stained for 2 h at 37 °C, then cleared in 70 % ethanol for 24 h, and then transferred to 95 % ethanol. Sections were viewed under a light microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 SUPPLEMENTARY INFORMATION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Hypocotyl developmental staging
The first objective was to define stages of phloem fibre development in hypocotyls. Hypocotyls were observed every 2 d during germination and early seedling growth, first by measuring the overall elongation rate of the hypocotyl. Then the internal anatomy of tissues in the stem, including the extent of fibre cell wall development, was studied. Rapid elongation of the hypocotyl occurred between 3 d and 9 d (Fig. 1), with an average maximum hypocotyl length of 25·6 mm attained by 9 d. Within 7-d hypocotyls, elongated cells were visible in longitudinal sections of phloem tissues (data not shown) and, although secondary walls were not detectable within phloem fibres, the secondary walls of xylem tissues were thick and well developed (Fig. 2A). The first evidence of cambial activity was visible at 9 d (Fig. 2B), which coincided with the cessation of hypocotyl elongation (Fig. 1). This phase also marked the onset of secondary cell wall thickening in fibres (Fig. 2B). A second increase in deposition of secondary cell wall occurred just prior to 15 d (Fig. 1). At this stage, the tightly and loosely packed layers (Gorshkova et al., 2003) of the fibre secondary cell wall were visible (Fig. 2C). Thus, three time-points (7 d, 9 d and 15 d) were chosen to represent stages of fibre elongation (7 d), completion of fibre elongation/onset of wall deposition (9 d) and late secondary wall thickening (15 d). These three time-points were used for further analysis by microarrays. The 7-d, 9-d and 15-d time-points therefore define processes in hypocotyl fibre development that are generally analogous to those that occur in the three regions of the stem (TOP, MID, BOT, respectively) that had been defined previously in Roach and Deyholos (2007), although fibre elongation in hypocotyls does not require the extensive intrusive growth that occurs in stems.


Figure 1
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  		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.

 


Figure 2
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  		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.

 
Microarray analysis of hypocotyl fibre stages
A unique characteristic of vascular tissues in hypocotyls is that they have a region known as the transition zone. The transition zone is where the vascular tissues undergo rearrangement from an exarch protostele arrangement in the root to an endarch eustele organization in the shoot (Esau, 1977). The rearrangement of the vasculature down the length of the hypocotyl makes it very difficult to peel ‘fibre-rich’ outer layers, as previously done in stems (Day et al., 2005a; De Pauw et al., 2007; Roach and Deyholos, 2007). For this reason, the entire hypocotyl (20–25 mm in length) was used for the microarray analyses, as opposed to peels or dissected fibre layers. While the advantage of this method is that fibres would be accurately represented within each stage as they existed in vivo, the disadvantage of this is that other tissues and cell types would be present and the differences in developmental programmes of these cells would be reflected in the analysis. However, as described above (Fig. 2), the development of xylem tissues continued through all of the time-points analysed. Therefore, it was expected that a substantial portion of the differentially expressed transcripts detected would be reflective of phloem-specific developmental events.

In this manuscript, clones that constituted an apparently unique contig or singleton will be referred to as probesets (ps). In total, 1568 probesets were found to have microarray hybridization signal intensities that differed significantly between one or more stem segments (fdr ≤ 5 %; see Table in Supplementary Information, available online). Of these statistically significant probesets, 660 were enriched by at least 2-fold in comparisons of one or more stem segments. The microarray analysis showed that 7-d and 9-d stages were both more similar to each other than to the 15-d samples (Table in Supplementary Information). Only 37 (37/660, 5·6 %) transcripts were significantly differentially expressed with an fc greater than 2 in the 7 d/9 d comparison (Table in Supplementary information). In contrast, by these same criteria (fdr ≤ 5 %, fc ≥ 2), the 7 d/15 d and 9 d/15 d comparisons had 523 (523/660, 79 %) and 434 (434/660, 65 %) differentially expressed transcripts, respectively. Of these, 180 (180/660, 27 %) transcripts were commonly enriched (fdr ≤ 5 %, fc ≥ 2) in both 7 d and 9 d when compared with 15 d. Conversely, only 15 (15/660, 2·2 %) transcripts were enriched in both 9 d and 15 d compared with 7 d. If the fold-change cut-off was lowered to 1·5, the number of differentially expressed probesets increased from 37 to 194 in the comparison between 7 d and 9 d. It was interesting, however, that the 9-d expression pattern did overlap with both the 7-d and 15-d samples, consistent with the idea that the 9-d stage was a transition point for development.

Functional categorization of differentially expressed transcripts
Because the microarray was printed from a library of 9600 anonymous (i.e. unsequenced) cDNA clones, 615 of the 660 significant differentially expressed (fdr < 5 %, fc > 2) probesets were sequenced and were deposited in GenBank under the accession numbers EX720038 [GenBank] –EX720492. Of the sequenced clones, 43 did not align to any known proteins in GenBank, but did contain open reading frames ranging from 56 aa to 256 aa long, indicating that these sequences likely represented novel, previously undescribed proteins. Some of these transcripts of undescribed proteins appear to be highly abundant; for example, probeset (ps 54) is represented by nine different clones in the fibre-rich stem peels that were used to make the cDNA library. These proteins of unknown function are an intriguing target for future studies, but will not be discussed here any further.

The 378 probesets that that aligned to previously described sequences in Genbank (BLASTx e-values of ≤10–10) were categorized based on their putative functions and their expression pattern, for each of the six microarray slide comparisons (Fig. 3). The largest functional category corresponded to photosynthetic-related transcripts, which were most enriched in the 7 d/15 d and 9 d/15 d comparisons. Transporters were also enriched in the 7-d and 9-d samples compared with the 15-d samples. Secondary metabolism-associated transcripts were enriched later in hypocotyl development, with all but one transcript related to secondary metabolism being more abundant in the 15-d sample than either the 7-d or 9-d samples. Because of their predominance on the microarray and inferred biological roles in the development of hypocotyls and fibres, the expression of selected transcripts will be described in more detail in the following paragraphs.


Figure 3
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  		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).

 
Photosynthesis
The largest functional category represented in the present results was photosynthesis-related transcripts. The majority of photosynthesis-related transcripts were enriched in the 7-d sample when compared with the 15-d sample, and some of these were also significantly differently expressed in the 9-d sample when compared with the 15-d sample. The most common transcripts within the photosynthesis category encoded chlorophyll a/b-binding proteins. There were 62 separate probesets for chlorophyll a/b-binding proteins enriched in the 7-d hypocotyls (see Supplementary Information, available online). Based on available sequence information, each of these 62 probesets appeared to represent a unique transcript, but further sequencing is likely to produce a more accurate estimate of the number of genes represented here. Chlorophyll a/b-binding proteins are expected to comprise a large gene family in flax, as there are 30 members of this gene family in arabidopsis (Jansson, 1999). Other photosynthetic-related transcripts that were enriched in 7-d or 9-d samples corresponded to photosystem I or II reaction centre subunits, Rubisco and chlorophyll biosynthesis (uroporphyrin III-C-methyltransferase, ps 8475; EC 2·1·1·107). Previous microarray analysis of photomorphogenesis in arabidopsis showed the importance and co-regulation of transcripts involved in organization of pigment–protein complexes in the thylakoid membranes during photomorphogenesis (Ghassemian et al., 2006).

Transporters
Probesets corresponding to transporter-related proteins were most highly enriched in the 7-d and 9-d samples compared with the 15-d samples (Table 1). Probesets that were similarly enriched in the 7-d and 9-d samples corresponded to aquaporins. Five distinct probesets (ps 111, 214, 4929, 6675 and 7812) corresponded to different types of aquaporins including two tonoplast-intrinsic proteins and a plasma membrane intrinsic protein. Also similarly enriched in the 7-d and 9-d samples were transcripts for ion, amino acid and sugar transporters. The putative ion transporters included: a homologue of CCC1, a cation:chloride cotransporter from arabidopsis (ps 6030); a potassium channel protein (ps 8727); and a bile acid:sodium symporter (ps 946). In other plant species, transcripts with similarity to bile transporters have been proposed to act as brassinosteroid transporters (Rzewuski and Sauter, 2002). Only one amino acid transporter (ps 4287) and one hexose transporter (ps 8710) were enriched in transcript abundance in the 7-d and 9-d samples as compared with 15-d samples. Fewer transporters were enriched in later (15 d) in hypocotyl development, as compared with earlier stages. These included a mannitol transporter (ps 6186), a MATE-efflux antiporter (ps 4698) and an UDP-N-acetylglucosamine transporter (ps 435).


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  		TABLE 1. Transcript abundance and tissue bias of probe sets in microarray comparisons of 7-d, 9-d and 15-d hypocotyls

 
Secondary metabolism and one-carbon metabolism
Most of the transcripts sequenced that were putatively related to secondary metabolism or one-carbon metabolism were enriched in the 15-d hypocotyls (Table 1). There was, however, one transcript related to secondary metabolism that was enriched early in hypocotyl development. Mevalonate diphosphate decarboxylase (ps 8743; EC 4·1·1·33) is an enzyme in the mevalonate pathway, which is upstream of many different secondary metabolic pathways in plants, including synthesis of terpenoids, carotenoids, steroids, ABA, cytokinin and gibberellin. It is interesting that transcripts putatively encoding this single, upstream component of so multiple metabolitic pathways is enriched relatively early in hypocotyl development, and that subsequently in the 15-d hypocotyls, transcripts putatively encoding a much more diverse range of downstream pathways accumulate. Secondary metabolitic pathways represented by transcripts enriched in 15-d hypocotyls included terpenoid synthesis (geranylgeranyl transferase ps 8427, EC 2·5·1·41), the flavonoid synthesis (dihydroflavonol reductase ps 5334; EC 1·1·1·219; and anthocyanin permease ps 1129, No EC) and representatives of both the tetrahydrofolate, and S-adenosylmethionine pathways for one-carbon metabolism. The S-adenosylmethionine cycle was represented by five transcripts (ps 2951, 3790, 4227, 4488 and 8386) up-regulated in 15-d hypocotyls, and tetrahydrofolate one-carbon metabolism only had one transcript represented (phosphoribosylaminoimidazolcarboxamide formyltransferase ps 5370; EC 2·1·2·3). The large representation of one-carbon metabolism transcripts in relation to cell-wall deposition has been reported previously (Oh et al., 2003; Prassinos et al., 2005; Andersson-Gunneras et al., 2006; De Pauw et al., 2007). One-carbon metabolism is involved in many plant processes including methylation of cell wall structural components such as lignin precursors (Ye et al., 1994) or pectin side chains (Goubet et al., 1998). Together, these data indicate that maturation of the hypocotyl involves an increased diversity in the types of metabolic pathways acting in the hypocotyl.

Protein and amino acid metabolism
Transcripts related to protein and amino acid metabolism were expressed throughout all three stages of hypocotyl development (Table 1). Throughout all three stages, different probesets related to ribosomal proteins increased in transcript abundance. In 7-d and 9-d samples there was an enrichment of transcripts putatively encoding DnaJ proteins (eight probesets), believed to be involved in protein folding. 15-d samples had an enrichment of other types of folding proteins, including heat shock proteins and chaperonins (ps 4914, 4962, 7822 and 7940). The 15-d sample also showed specific enrichment of transcripts related to amino acid metabolism. These included three transcripts corresponding to ketol-acid reductoisomerases (ps 4341, 7683 and 7685; EC 1·1·1·86), which are involved in the biosynthesis of the amino acids valine, leucine and isoleucine. Serine-related transcripts enriched in 15-d hypocotyls were serine hydroxymethyltransferase (ps 7104; EC 2·1·2·1) and serine carboxypeptidase (ps 8808; EC 3·4·16·1). Other amino acid-related transcripts corresponded to aromatic amino acid biosynthesis (3-deoxy-7-phosphoheptulonate synthase, ps 588; EC 2·5·1·54), two glutamine amidotransferases (ps 4582 and 4595; EC 4·1·3·27) and a transcript related to glutathione synthesis (gamma-glutamylcysteine synthetase, ps 6716; EC 6·3·2·2). Overall, at least several different pathways of amino acid biosynthesis were represented among transcripts enriched at later stages of hypocotyl development.

Signalling and gene regulation
Transcripts related to signalling and transcriptional regulation of gene expression differed in abundance at all stages of fibre development observed (Table 1). This functional category included hormone-related genes, calcium-signalling genes, signal kinases and putative transcription factors. Protein kinases (ps 280, 2967, 8092, 8443) were also up-regulated early in hypocotyl development as they were more present in the 7-d and 9-d samples compared with 15-d hypocotyls. Although in general, very few transcripts were differentially expressed between the 7-d and 9-d samples, one transcript that was specifically enriched in 7-d hypocotyls was homologous to a GA-stimulated transcript (GAST1-like) transcript from arabidopsis. Two other hormone-related transcripts were enriched at 15 d compared with 7 d and 9 d, respectively. These were another putative gibberellin-regulated transcript, GASA5-like protein from spruce (ps 138) and a transcript related to ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate oxidase (ps 7199; EC 1·14·17·4). Transcripts related to calcium signalling were also enriched in 15-d hypocotyls, as evidenced by the up-regulation of two calcium-binding proteins, calmodulin (ps 3749) and calnexin (ps 7182).

Transcription factors also demonstrated specific patterns of transcript expression at each stage of hypocotyl development. Hypocotyls of 7-d seedlings were specifically enriched in transcripts putatively encoding two zinc finger proteins (ps 5707 and 5961) one RAU1 transcription factor (ps 979) and a LIM transcription factor (ps7041), while 9-d hypocotyls were specifically enriched in transcripts with similarity to an arabidopsis switching protein (AtSWI3C; ps 7333). In both 7-d and 9-d hypocotyls, enrichment of transcripts for another zinc finger protein (ps 6228) and a CONSTANS1-like protein (ps 6762) was detected. Transcription factors that were up-regulated in 15-d hypocotyls included a CONSTANS-interacting protein (ps 2398) and a NAC domain protein (ps 5323). Because transcription factors control specific developmental programmes, these genes are interesting targets for future characterization through forward and reverse genetics.

Cell wall/polysaccharide related
Because, in relation to phloem fibres, the developmental series represented the stages that distinguished primary cell from secondary wall deposition in fibre, it was expected that probesets related to cell wall and polysaccharide metabolism would be identified in all three comparisons (Fig. 3). Within the cell wall and polysaccharide category, most of the probesets were enriched in the 15-d section compared with both the 7-d and 9-d sections (Table 1). Transcripts for two sucrose synthases were enriched in the 15-d hypocotyl (ps 2833 and 5428; EC 2·4·1·13). The largest group of 15-d enriched cell wall-related transcripts corresponded to glycosyl hydrolases, which are likely to be involved in the deposition or remodelling of secondary walls. Within the glycosyl hydrolases, transcripts of eight distinct chitinases (ps 26, 65, 118, 220, 5881, 7887, 8749 and 8825; EC 3·2·1·14) and three glucanases (ps 6, 28 and 3726; EC 3·2·1.–) were detected as enriched in15-d hypocotyls.

One of the glucanases (ps 3726; EC 3·2·1·4) that was enriched among 15-d transcripts had homology to cotton fibre KORRIGAN, which is an endo-β-1,4-glucanase required for proper cellulose deposition (Sato et al., 2001). A second KORRIGAN transcript was identified as differentially expressed, but this KORRIGAN was significantly up-regulated in 7-d and 9-d hypocotyls, and not in 15-d hypocotyls (ps 4893). This leads to speculation that two distinct KOR transcripts had been detected: one KOR that is related to primary wall cellulose deposition, and a second KOR potentially involved specifically in secondary wall deposition. Different forms of KOR proteins have been described previously (Molhøj et al., 2001) and although KOR function in cellulose biosynthesis was originally described in relation to primary wall CesA proteins (Sato et al., 2001), the requirement of KOR in secondary wall deposition has also been recently described in cotton and arabidopsis (Peng et al., 2002; Szyjanowicz et al., 2004).

The 7-d and 9-d hypocotyls had a specific enrichment of cell wall transcripts with putative functions in cell wall loosening. Three transcripts related to xyloglucan remodelling were up-regulated in these early stages of growth compared with the 15-d ones. Two xyloglucan-endotransglycosylase/hydrolases (ps 4309, 8742; EC 2·4·1·207) and a xyloglucan galactosyltransferase (ps 4781; EC 2·4·1.–) were detected. Also in this expression group were glucanases with putative roles in cell wall loosening, including an {alpha}-galactosidase (ps 4378; EC 3·2·1·22) and a β-1,3-glucanase (ps 9041; EC 3·2·1·6).

Even though detectable secondary wall deposition began at 9 d, there were very few transcripts that increased in abundance in both the 9-d and 15-d samples. Only two probesets fell into this category, and they were both fasciclin-like arabinogalactan proteins (ps 152 and 224). This corresponds with previous data (Roach and Deyholos, 2007) which showed that fasciclin-like arabinogalactan proteins were specifically up-regulated at the snap point of flax stems, where fibre development transitions from cell elongation to cell-wall deposition.

Only two cell wall-related probesets were differentially expressed between 7 d and 9 d, one was more enriched in the 7-d hypocotyl and the other was enriched in 9-d hypocotyls. The 7-d enriched probeset corresponded to a UDP:glucose glucosyltransferase (ps 6015; EC 2·4·1·35). This enzyme diverts UDP:glucose from cellulose synthase and builds short glucosides. The 9-d enriched probeset corresponded to a β-galactosidase (ps 9; EC 3·2·1·23). β-Galactosidases can catabolize galactans, and have been reported to act on the side chains of RG-1 pectins (Martin et al., 2005). This β-galactosidase is enriched in both the 9-d and 15-d samples compared with the 7-d samples, indicating that accumulation of this transcript is induced at the onset of secondary wall deposition and remains at elevated abundance in the later stages of wall deposition.

qRT-PCR
To confirm that the RNA expression patterns identified by microarray could be reproduced using other techniques, nine transcripts were selected as targets for qRT-PCR analysis. These nine cDNAs were selected to represent a variety of functional categories and expression patterns. In almost all cases, the temporal pattern of transcript enrichment was the same in both types of analyses (Table 2), although in quantitative terms, the microarray data tended to underestimate the differences in expression between samples when compared with qRT-PCR. The exception the sugar transporter (ps 6186), for which the relative differences measured between samples in the microarray data were larger than for qRT-PCR. The only major inconsistency was the UDP-N-acetylglucosamine transporter (ps 435), which, according to qRT-PCR, showed very little difference in expression in all stages, whereas the microarray data showed it to have greater expression in the 15-d sample. This discrepancy could be due to cross hybridization on the microarray slide which is not detected when using qRT-PCR which uses a more specific, primer-based amplification technique to quantify transcript abundance. Further knowledge of other UDP-N-acetylglucosamine transporter sequences in flax would help determine whether this was indeed the case.


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  		TABLE 2. Comparison of log2 ratios obtained by qRT-PCR and microarray analysis

 
Comparison of hypocotyl fibre development to stem fibre development
Part of the motivation behind this study was to determine whether the stages identified in hypocotyl fibre development were analogous to the stem fibre developmental stages that had previously been analysed by microarrays (Roach and Deyholos, 2007), and to thereby also provide additional context for interpreting biochemical analyses of fibre development in flax hypocotyls (Alexandre et al., 1997; His et al., 1997; Janeau et al., 1997; Quentin et al., 1997; Andeme-Onzighi et al., 2000; Douchiche et al., 2007). Microarray data from 7-d, 9-d and 15-d hypocotyls were therefore compared with each of the microarray expression patterns reported for the TOP, MID and BOT segments of flax stems. In both hypocotyls and stems, the elongation phase (7 d in hypocotyl, TOP in stem) was most different from late secondary wall deposition (15 d in hypocotyl, BOT in stem) (Fig. 4). The major difference between the analyses was that in stems, the transition stage (snap point, MID) was most similar to the late secondary wall deposition (BOT) stage, whereas in hypocotyls the transition stage (9 d) was most similar to the elongation (7 d) stage. Together these data suggest that transition of fibres from elongation to secondary wall development is accompanied by an overlap of gene expression from both the elongation and wall deposition stages.


Figure 4
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  		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).

 
β-Galactosidase activity in fibre development
In the current study and in previously described stem microarrays (Roach and Deyholos, 2007), the sample representing the transition from fibre elongation to secondary wall deposition (i.e. MID stem segment or the 9-d hypocotyl sample) had the smallest number of stage-specific transcripts. In both studies, however, β-galactosidase was identified as being one of the transcripts whose expression is correlated with the onset of secondary wall deposition in phloem fibres. β-Galactosidase proteins had also been identified previously as being enriched in individual fibres from the MID stem segment (Hotte and Deyholos, 2008). Based on these data and research by Gorshkova et al. (2004), which reported the remodelling of a β-1,4-galactan at the transition in flax fibre development, it was inferred that this specific β-galactosidase had an important function during cell wall deposition in flax bast fibres. Because the qRT-PCR results confirmed the stage-specific expression pattern of this β-galactosidase transcript (Table 2), this gene was chosen as a candidate for further characterization.

ONPG assay of β-galactosidase activity
To determine whether the increase in β-galactosidase transcript observed actually corresponded to an increase in β-galactosidase enzyme activity, crude protein was extracted from hypocotyls and β-galactosidase activity measured using an in vitro assay system. Although this method would detect all active β-galactosidases present in the hypocotyl tissue, it was assumed that an increase in expression of the secondary wall-associated β-galactosidase would be still be detectable above this background. The data showed that β-galactosidase activity increased after 7 d, and continued to increase until at least 15 d (Fig. 5). By 15 d, the amount of β-galactosidase activity had more than tripled the original activity level in 7-d hypocotyls. This showed that an increase in β-galactosidase activity is temporally correlated with cell wall deposition in hypocotyls.


Figure 5
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  		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 ({alpha} = 0·05) in an ANOVA are labelled with different letters. Error bars are the standard error of the mean.

 
In situ detection of β-galactosidase activity
To identify the spatial distribution of β-galactosidase activity, an in situ enzyme assay was conducted on fixed hypocotyls. The chromogenic substrate X-gal was used to stain locations of β-galactosidase activity in developing hypocotyls (Fig. 6). In 7-d sections, there was no detectable staining after 2-h incubation with X-gal. In transverse sections, fibres were not easily distinguished, as no secondary wall thickening could be detected (Fig. 6A, D and G). Also, in this stage the vascular cambium had not yet developed. In 9-d sections, staining was still not yet detected after 2-h incubation, although the cambium had developed and a thin secondary wall could be seen around a few fibre cells (Fig. 6B and H). In 15-d fibres after 2 h staining, blue stain could be detected in the cytoplasm of fibre cells with thick secondary cell walls (Fig. 6C, F and I). Longitudinal sections showed that staining was mostly associated with cytoplasm of the fibre cells (Fig. 6F). Less staining was detected in the fibre wall itself compared with the fibre cytoplasm. Further research into the localization and structure of wall polymers, localization of β-galactosidase protein, and transgenic analysis in flax will help decipher whether this protein has an integral role in the secondary wall development of flax bast fibres.


Figure 6
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  		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.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
SUPPLEMENTARY INFORMATION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
To better understand the diversity of processes by which cells acquire secondary walls, and to enhance the utility of flax hypocotyls as a model system for phloem fibre development, anatomical, transcriptomic and enzymatic analyses of tissues dissected from flax seedlings were conducted (Figs 1 and 2). It was observed that phloem fibre development was closely synchronized with hypocotyl elongation. Both the hypocotyl and its phloem fibres elongated until approx. 9 d, after which point hypocotyl elongation ceased and secondary wall deposition in phloem fibres began to be detectable, with the bulk of the secondary wall accumulating rapidly before 15 d. In contrast, secondary wall deposition in xylem tissues was evident throughout seedling development, including at time-points that preceded the rapid deposition of secondary walls in phloem fibres (e.g. 7 d, Fig. 2A). Microarrays were therefore used to compare transcript abundance in 7-d, 9-d and 15-d hypocotyls, with the assumption that some of the differentially expressed transcripts would be associated specifically with developing phloem fibres. It is noted that because phloem fibre cells were not individually dissected in this study, some of the transcripts identified may be associated with other developmentally regulated processes that differed between the three time-points examined. Nevertheless, transcript expression patterns in 7-d, 9-d and 15-d hypocotyls was generally correlated (Fig. 4) with previously described expression patterns in serial segments (TOP, MID, BOT, respectively) of developing stems, supporting the further use of hypocotyls as models for genetic and biochemical analyses of phloem fibre development (Deyholos and Roach, 2007).

In hypocotyls undergoing elongation (but not phloem fibre secondary wall thickening), it was found that transcripts related to photosynthesis, transport, and specific types of hormone signalling were particularly abundant (Fig. 3). Elongating hypocotyls also demonstrated enrichment of several transcripts with putative roles in the loosening and extension of primary cell walls, including enzymes acting on xyloglucan, xyloglucan endo-transglycosylase/hydrolase and xyloglucan galactosyltransferase (Pauly et al., 2001; Hernandez-Nistal et al., 2006; Jimenez et al., 2006). Also in reference to cell wall development, transcripts of a putative KORRIGAN gene were enriched in elongating hypocotyls; these transcripts show high sequence similarity to arabidopsis KOR1, which was initially identified through its correlation with hypocotyl elongation and is a required protein for cellulose synthesis (Nicol et al., 1998; Peng et al., 2002). Interestingly, the putative flax KORRIGAN transcripts that were enriched in elongating hypocotyls were distinct in sequence from a separate probe set that also encoded a KORRIGAN-like gene, but which was enriched at 15 d. This suggests that a specific KORRIGAN gene may be required for secondary wall deposition in phloem fibres. Tissue-specific and stage-specific expression of different KORRIGAN family genes has also been described in arabidopsis (Molhøj et al., 2001).

Other transcripts for which expression in hypocotyls was correlated with secondary wall thickening of phloem fibres included sucrose synthases, arabinogalactan proteins, chitinases and β-galactosidase (Tables 1 and 2). The relevance of sucrose synthases in cellulose deposition is well known (Haigler et al., 2001). On the other hand, roles for these other proteins in secondary wall development are only starting to be understood. It has been reported previously that transcripts of distinct arabinogalactan proteins were differentially enriched in specific cells and tissues of developing flax stems (Roach and Deyholos, 2007). The expression of chitinase-like proteins in phloem fibre-bearing tissues has not been characterized previously; however, the present observation is consistent with the reported co-regulation of chitinases and cellulose synthases in arabidopsis (Persson et al., 2005). Chitinase-like proteins are also preferentially expressed in cotton fibres with secondary walls (Zhang et al., 2004). Finally, β-galactosidases may be critical to the metabolism of a unique class of galactans in phloem-fibre development, as described below. The present identification of specific expression patterns for chitinases, arabinogalactan proteins and β-galactosidases within developing flax hypocotyls provides a well-defined system for further exploration of the function of these proteins.

A specific β-galactosidase is temporally and spatially correlated with phloem fibre cell-wall thickening
Presented here is the first demonstration that both β-galactosidase transcript abundance and enzyme activity increase after hypocotyl elongation is completed and continue to increase during secondary wall development, and this activity is localized to phloem fibres (Table 2, and Figs 5 and 6). This is consistent with previous studies of protein or transcript abundance in flax fibres and stems, and with analyses of a stem peel EST library (Roach and Deyholos, 2007; Hotte and Deyholos, 2008; Day et al., 2005a). Possible substrates for β-galactosidase in this context include arabinogalactan proteins (Hirano et al., 1994; Kotake et al., 2005), pectins (Martin et al., 2005), and xyloglucan or xyloglucan oligosaccharides (Edwards et al., 1988; de Alcantara et al., 2006; Iglesias et al., 2006). Differential expression of members of a β-galactosidase gene family has also been described in chick pea (Cicer arietinum; Estaban et al., 2005; Martin et al., 2005). The inferred amino acid sequence of the flax fibre-enriched β-galactosidase is more similar to the chick pea CanBGal-4 protein than to any of the other 490 plant β-galactosidases in public databases. Functional similarity of these proteins is further suggested by the observation that transcripts of CanBGal-4 are most highly expressed in non-elongating tissues, as is the flax fibre-specific β-galactosidase described here. Together, these observations support the proposal by Gorshkova and Morvan (2006) that a high molecular weight β-1,4-galactan is partially cleaved and incorporated into the developing secondary cell wall. Future studies will focus on using transgenic flax to identify whether modulating β-galactosidase function in flax affects the formation of the secondary cell wall.


   SUPPLEMENTARY INFORMATION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY INFORMATION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Supplementary information is available online at http://aob.oxfordjournals.org/ and consists of a table providing sequence, annotation and signal intensity data for all clones, probe sets and microarray experiments. Note that these data are provided both as a text file and as an Excel file.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY INFORMATION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
We thank Gordon Rowland (Crop Development Center, Saskatoon, Canada) for the generous gift of Norlin flax seeds, and Janice Cooke and Mohsen Mohammadi for useful discussions. Funding for this work was provided by Alberta Advanced Education and Technology (to M.K.D.), Natural Sciences and Engineering Research Council Canada (261583-03 to M.K.D.) and Queen Elizabeth II Scholarship (to M.J.R.).


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY INFORMATION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

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F. Paynel, A. Schaumann, M. Arkoun, O. Douchiche, and C. Morvan
Temporal regulation of cell-wall pectin methylesterase and peroxidase isoforms in cadmium-treated flax hypocotyl
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