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Biochemical and molecular characterization of Avena indolines and their role in kernel texture

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Abstract

Among cereals, Avena sativa is characterized by an extremely soft endosperm texture, which leads to some negative agronomic and technological traits. On the basis of the well-known softening effect of puroindolines in wheat kernel texture, in this study, indolines and their encoding genes are investigated in Avena species at different ploidy levels. Three novel 14 kDa proteins, showing a central hydrophobic domain with four tryptophan residues and here named vromindoline (VIN)-1,2 and 3, were identified. Each VIN protein in diploid oat species was found to be synthesized by a single Vin gene whereas, in hexaploid A. sativa, three Vin-1, three Vin-2 and two Vin-3 genes coding for VIN-1, VIN-2 and VIN-3, respectively, were described and assigned to the A, C or D genomes based on similarity to their counterparts in diploid species. Expression of oat vromindoline transgenes in the extra-hard durum wheat led to accumulation of vromindolines in the endosperm and caused an approximate 50 % reduction of grain hardness, suggesting a central role for vromindolines in causing the extra-soft texture of oat grain. Further, hexaploid oats showed three orthologous genes coding for avenoindolines A and B, with five or three tryptophan residues, respectively, but very low amounts of avenoindolines were found in mature kernels. The present results identify a novel protein family affecting cereal kernel texture and would further elucidate the phylogenetic evolution of Avena genus.

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Author notes

  1. Michela Janni

    Present address: CNR-Istituto di Genetica Vegetale, Via Amendola 165/A, Bari, Italy

Authors and Affiliations

  1. Consiglio per la Ricerca e la Sperimentazione in Agricoltura (CRA-QCE), Via Cassia, 176, 00191, Rome, Italy

    Laura Gazza, Federica Taddei, Salvatore Conti, Gloria Gazzelloni & Norberto E. Pogna

  2. Department of Chemical Sciences, University of Catania, Via Andrea Doria 6, Catania, Italy

    Vera Muccilli

  3. Department DAFNE, University “La Tuscia”, Via C. De Lellis snc, Viterbo, Italy

    Michela Janni & Renato D’Ovidio

  4. Consiglio per la Ricerca e la Sperimentazione in Agricoltura (CRA-MAC), Via Stezzano 24, Bergamo, Italy

    Michela Alfieri & Rita Redaelli

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  1. Laura Gazza
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Correspondence to Laura Gazza.

Electronic supplementary material

Online Resource 1 DNA construct obtained by insertion of the coding region of Vin-D2a (JQ518369) or Vin-A3a (JQ518372) between the Dx5Prom promoter and the Dx5T terminator of gene Glu-D1-1a at the SalI and Xba sites of the pRDPT5 plasmid.

Online Resource 2 (A) Two-dimensional A-PAGE x SDS-PAGE fractionation of starch-bound proteins from oat cv. Primula and (B) immunostaining with the polyclonal antiserum specific to protein 3B3.

Online Resource 3 Agarose gel fractionation of cDNA amplicons obtained with primers specific to (A) Vin-2 (B) Vin-3 and (C) Aina in immature kernels of oat cv. Donata. Immature kernels were harvested 7, 14, 21, 28 and 35 days post anthesis (DPA). Tub = amplicons of oat β-tubulin.

Online Resource 4 (A) A-PAGE fractionation of starch-bound proteins extracted from immature kernels harvested 7, 14, 21, 28 and 35 DPA from oat cv. Donata and (B) immunostaining with the polyclonal antiserum specific to VIN-2 (3B3). Mat: mature kernels (> 45 DPA). VIN = vromindolines.

Online Resource 6 Starch-bound proteins fractionated by A-PAGE and immunostained with the anti-VIN-3 (anti-3B3T) antibody. Kernels from: (1,2) T1 plants homozygous for Vin-D2a and Vin-A3a; (3) null T1 plant; (4,5) T1 plants homozygous for Vin-D2a and Vin-A3a; (6) A. sativa cv. Donata and (7) durum wheat cv. Svevo.

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Gazza, L., Taddei, F., Conti, S. et al. Biochemical and molecular characterization of Avena indolines and their role in kernel texture. Mol Genet Genomics 290, 39–54 (2015). https://doi.org/10.1007/s00438-014-0894-5

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