The RNA-mediated silencing of one of the Pin genes in allohexaploid wheat simultaneously decreases the expression of the other, and increases grain hardness

Abstract

The RNAi-mediated silencing of Pina and Pinb, the two genes responsible for the grain texture of allohexaploid wheat, was induced and analysed in two wheat cultivars, Kontesa and Torka. A characterization of the two genes in non-transgenic plants revealed that Pinb carries a point mutation, designated Pinb-D1c in both cultivars. This mutation does not influence transcript abundance or protein content. Two silencing cassettes of the hpRNA type were constructed and used for stable transformation via Agrobacterium. In total, 43 transgenic lines representing the two cultivars were obtained, transformed with the silencing cassettes for Pina or for Pinb or co-transformed with both cassettes. The relative transcript levels of the two genes in the same progeny plant were found to be similar, independent of the silencing cassette used. The reduction in the Pina and Pinb transcript levels in the segregating T(1) progeny of Kontesa and Torka transformed with one of the silencing cassettes exceeded 80%. Co-transformation with the silencing cassettes for both genes resulted in a reduction of over 91% of Pina and Pinb transcripts in some segregating T(1) progeny of Kontesa. The silencing was transmitted to the T(4) kernel generation of the T(3) lines. A significant reduction or lack of both puroindoline proteins in the silenced lines correlated with an essential increase in grain hardness. The discussion covers some new insights into the function of the Pin genes, including the simultaneous silencing of both, independent of the siRNA signal.

Changes in the starch-protein interface depending on common wheat grain hardness revealed using atomic force microscopy

“…RNAi-mediated silencing of one of the PIN genes was shown to decrease the expression of the others and led to a significant increase of grain hardness [27].…”

Sina and Sinb genes in triticale do not determine grain hardness contrary to their orthologs Pina and Pinb in wheat

“…As previously documented, the RNA-mediated silencing of one of the Pin genes in wheat simultaneously decreased the expression of the other, which resulted in a significant increase of grain hardness [22]. This is the first report on the RNAi silencing of their orthologs, Sin genes in triticale, with the aim of explaining their function.…”

“…Each ortholog contained several long, exceeding 21 nt 100% homologous fragments. This feature allowed us to apply the same, hpRNA type of silencing cassette, which has already been successfully used for silencing of Pin genes in wheat [22] to silence Sin genes in triticale. (Pina) contained the whole coding sequence of Pina-D1a in sense and antisense orientation; (Pinb) contained the respective Pinb-D1a sequences, both 447 bp long.…”

“…Specific mutation or RNAi silencing of both Pin genes decreased the level of puroindolines and increased grain hardness up to the level of hard T. turgidum var. durum cultivar [22]. The function of their orthologs, Sin genes in triticale, is expected to be similar; however, a few papers on the subject have reported contradictory data [13,14,23].…”

“…The technology is especially useful in the case of polyploid cereals, such as wheat [22,24-27] or triticale, for which all homologous genes and their copies might be silenced. Application of RNAi technology is possible with the well-established genetic transformation method.…”