Evolutionary characterization and transcript profiling of β-tubulin genes in flax (Linum usitatissimum L.) during plant development

Abstract

Background: Microtubules, polymerized from alpha and beta-tubulin monomers, play a fundamental role in plant morphogenesis, determining the cell division plane, the direction of cell expansion and the deposition of cell wall material. During polarized pollen tube elongation, microtubules serve as tracks for vesicular transport and deposition of proteins/lipids at the tip membrane. Such functions are controlled by cortical microtubule arrays. Aim of this study was to first characterize the flax β-tubulin family by sequence and phylogenetic analysis and to investigate differential expression of β-tubulin genes possibly related to fibre elongation and to flower development.

Results: We report the cloning and characterization of the complete flax β-tubulin gene family: exon-intron organization, duplicated gene comparison, phylogenetic analysis and expression pattern during stem and hypocotyl elongation and during flower development. Sequence analysis of the fourteen expressed β-tubulin genes revealed that the recent whole genome duplication of the flax genome was followed by massive retention of duplicated tubulin genes. Expression analysis showed that β-tubulin mRNA profiles gradually changed along with phloem fibre development in both the stem and hypocotyl. In flowers, changes in relative tubulin transcript levels took place at anthesis in anthers, but not in carpels.

Conclusions: Phylogenetic analysis supports the origin of extant plant β-tubulin genes from four ancestral genes pre-dating angiosperm separation. Expression analysis suggests that particular tubulin subpopulations are more suitable to sustain different microtubule functions such as cell elongation, cell wall thickening or pollen tube growth. Tubulin genes possibly related to different microtubule functions were identified as candidate for more detailed studies.

Keywords: Bast fibres; Duplicated genes; Expression analysis; Flax; Gene family; Phylogenetic analysis; Pollen tube; β-tubulin.

Fig. 1

cDNA ends amplification of flax β-tubulin genes. (a) Cloning scheme with indication of primers and expected amplicons. (b) Test of the primer pair MREI/Rex1 on flax genomic DNA (lane 1) and Fex1/Rex1 control amplification (lane 2). (c) Agarose gel of 5′ and 3’ cDNA ends amplification

Fig. 2

Exon/intron structure of flax β-tubulin genes. cDNA sequences were aligned to their putative genomic counterparts, listed in Table 1. Only a short sequence corresponding to LusTub7c was found in the NCBI database (dotted line indicates the missing part)

Fig. 3

CE-TBP profiling of flax β-tubulin introns. (a) first intron. (b) Second intron. Insets: magnification of the short size amplicon peaks

Fig. 4

Similarity tree of plant β-tubulins. Maximum likelihood analysis was performed on the complete β-tubulin sets of flax, aspen, cotton, Arabidopsis, Medicago, barley, rice and maize. Accession numbers are listed in Additional file 2. The β-tubulin of the green alga C. reinhardtii was used as an outgroup. Close circles indicate flax tubulins. Open circles indicate the nodes of the four tubulin classes. Grey background indicates monocot tubulin clades

Fig. 5

N-terminal and C-terminal sequence alignment of flax β-tubulins. Aminoacid sequences were aligned with Clustal W. Background color code: Yellow: identical; green: block of similar; light blue: conserved; white: non-similar. B: beta-sheet; H: alpha helix. (a) Amino-terminal region, from residues 1 to 103. (b) Carboxy-terminal region, from residue 350 to 452. CTT: C-terminal tail

Fig. 6

Relative differential expression of flax β-tubulin genes in stem sections and hypocotyls. Relative transcript levels of the 14 flax β-tubulin genes, normalized to the geometric mean of two reference genes, as evaluated by real-time qPCR in stem sections (a and c) and hypocotyls (b and d). Expression levels, grouped by sample and relative to the avarage expression of each target, are shown in panels a and b. In panels c and d, transcript amounts are grouped by sample and normalized to a control sample: the apex in panel c and the DAS 3 sample in panel d. A Log2 scaling was used for the y axis

Fig. 7

Relative differential expression of flax β-tubulin genes in reproductive organs. Flax reproductive organs were collected the day before anthesis (a) and the day of flowering (b) and observed under a stereomicroscope. Scale bar: 1 mm. c, relative transcript level of the 14 flax β-tubulin genes in stamens and carpels collected the day before anthesis (pA) and the day of anthesis (a) were measured by RT-qPCR and normalized to the the geometric mean of two reference genes

Fig. 8

Clustergram of differential β-tubulin gene expression in stem sections and hypocotyls. Color ranges from green to red, through black, according to the manitude of relative gene expression, as shown in the scale bar. Targets are clustered according to their similarity in the expression pattern