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. 2015 Jul;66(13):3753-64.
doi: 10.1093/jxb/erv168. Epub 2015 Apr 28.

Mannans and endo-β-mannanases (MAN) in Brachypodium distachyon: expression profiling and possible role of the BdMAN genes during coleorhiza-limited seed germination

Virginia González-Calle  1 , Cristina Barrero-Sicilia  1 , Pilar Carbonero  1 , Raquel Iglesias-Fernández  2
Affiliations

Mannans and endo-β-mannanases (MAN) in Brachypodium distachyon: expression profiling and possible role of the BdMAN genes during coleorhiza-limited seed germination

Virginia González-Calle et al. J Exp Bot. 2015 Jul.

Abstract

Immunolocalization of mannans in the seeds of Brachypodium distachyon reveals the presence of these polysaccharides in the root embryo and in the coleorhiza in the early stages of germination (12h), decreasing thereafter to the point of being hardly detected at 27h. Concurrently, the activity of endo-β-mannanases (MANs; EC 3.2.1.78) that catalyse the hydrolysis of β-1,4 bonds in mannan polymers, increases as germination progresses. The MAN gene family is represented by six members in the Brachypodium genome, and their expression has been explored in different organs and especially in germinating seeds. Transcripts of BdMAN2, BdMAN4 and BdMAN6 accumulate in embryos, with a maximum at 24-30h, and are detected in the coleorhiza and in the root by in situ hybridization analyses, before root protrusion (germination sensu stricto). BdMAN4 is not only present in the embryo root and coleorhiza, but is abundant in the de-embryonated (endosperm) imbibed seeds, while BdMAN2 and BdMAN6 are faintly expressed in endosperm during post-germination (36-42h). BdMAN4 and BdMAN6 transcripts are detected in the aleurone layer. These data indicate that BdMAN2, BdMAN4 and BdMAN6 are important for germination sensu stricto and that BdMAN4 and BdMAN6 may also influence reserve mobilization. Whether the coleorhiza in monocots and the micropylar endosperm in eudicots have similar functions, is discussed.

Keywords: BdMAN gene family; Brachypodium distachyon; MAN gene expression; coleorhiza; endo-β-mannanases; germination; mRNA in situ hybridization.; mannan immunolocalization.

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Figures

Fig. 1.
Fig. 1.
(A) Longitudinal sections of the different phases of Brachypodium distachyon germination sensu stricto, stained with toluidine blue. C, coleorhiza; Co, coleoptile; E, endosperm; Sc, scutellum; Sh, shoot; R, root. Scale bar, 200 μm. (B) Endo-β-mannanase activity (white bars) in embryo and endosperm (de-embryonated seed) upon B. distachyon seed germination (0–24h). One unit of MAN activity is defined as the amount of enzyme that releases 1 nmol of reducing sugar per minute and per mg of protein. Percentage germination evaluated as coleorhiza emergence (CE; open circles) and root emergence (RE; close circles) are represented. In the inset, the time needed for 50% of CE (t50CE) and RE (t50RE) is indicated. Data are means ± standard error (SE) of three technical replicates of three biological samples.
Fig. 2.
Fig. 2.
Mannan polymer immunolocalization at the root tip and the coleorhiza in longitudinal sections of Brachypodium germinating embryos at (A–C) 12h and at (D–F) 27h. (G–I) DIC images of D, E and F. (C, F, I) Close-up of the coleorhizae. C, coleorhiza; R, root. Scale bars: (A, D, G), 50 μm; (B, E, H), 25 μm; (C, F, I), 10 μm.
Fig. 3.
Fig. 3.
(A) Phylogenetic dendrogram with deduced protein sequence of the mannanase gene families form Brachypodium distachyon, Oryza sativa and Arabidopsis thaliana; bootstrapping values are indicated in the branches. (B) Schematic distribution of conserved motifs among the deduced protein sequences in the phylogenetic tree (A), identified by means of the MEME analysis. Asterisks indicate those motifs important for enzymatic activity. Motifs in grey share >85% of similar amino acid residues. This figure is available in colour at JXB online.
Fig. 4.
Fig. 4.
(A) Different stages of Brachypodium distachyon seed development (4, 6, 8, 10, 12 d after pollination; dap). (B) Expression of the BdMAN1, BdMAN2 and BdMAN6 genes by RT-qPCR during seed development. Data are means ± standard error (SE) of three technical replicates of three biological samples.
Fig. 5.
Fig. 5.
Transcript accumulation of BdMAN2, BdMAN4 and BdMAN6 in (A) embryos, and in (B) endosperms (de-embryonated seeds) upon B. distachyon seed germination. Data are means ± standard error (SE) of three technical replicates of three biological samples.
Fig. 6.
Fig. 6.
In situ mRNA hybridization analysis of BdMAN2, BdMAN4 and BdMAN6 in 27h germinating Brachypodium seeds. (A–D) BdMAN2, (E–H) BdMAN4, (I–L) BdMAN6. (A, E, I) Longitudinal sections of germinating embryos. (B, F, J) Close-up of the coleorhiza and the root tip. (C, G, K) Close-up of the endosperm and the aleurone. (D, H, L) Control sense probes. Al, aleurone layer; C, coleorhiza; Co, coleoptile; Cp, calyptra; E, endosperm; Sc, scutellum; Sh, shoot; R, root. The black arrow indicates the localization of transcripts. Scale bar, 50 μm.
Fig. 7.
Fig. 7.
Polysaccharide and protein mobilization upon B. distachyon seed germination. Bright field microscopy of longitudinal seed sections stained with PAS-Naphthol Blue Black. (A, D, G, J) Longitudinal sections from dry and water-imbibed seeds at 27h and 36h. (B, E, H, K) Close-up of the coleorhiza in A, D, G, J and (C, F, I, L) close-up of the endosperm in A, D, G, J, respectively. Proteins stain in blue and polysaccharide-rich cell walls in pink. C, coleorhiza; Co, coleoptile; E, endosperm; M, mesocotile; Sc, scutellum; Sh, shoot; R, root. Scale bar: 50 μm.
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