In this paper we report 38 transgenic lines from two genotypes with different gliadin fractions silenced by RNAi, using three different promoter combinations and three different silencing construct combinations. The lines were tested during two consecutive years period to evaluate the effect of the promoters, silencing fragments and the environment on the down regulation of wheat gliadins and the effects of that silencing on other storage proteins and quality grain constituents.
Down-regulation of α-, γ-, and ω-gliadins by RNAi is an effective approach to reduce the expression of CD-related gliadin T-cells epitopes , which could be the basis for the development of products suitable not only for CD patients but also for other gluten intolerance patients. However, as consequence of this silencing, there is a re-balance of prolamin distribution, leading to the increment of total protein content in some particular lines [15, 18], but not as a general effect. In most of the research articles where storage proteins of cereals were silenced by RNAi or mutation, the authors reported either a decrease or no variation in the total protein content [19–24] reported an overall increase of total protein in lines transformed with the ‘g’ antisense fragment, which agree with the data reported in this work. Although the total protein also increases in lines transformed with the silencing fragment ‘go’, this should be interpreted with caution, because the increase in total protein was associated with a decrease in thousand seed weight. It is known that grains that are not completely filled have higher protein content, and a higher embryo to endosperm protein ratio. Thus the increase in total protein in ‘go’ lines may come not only from non-gluten proteins (albumins and globulins) but also from a higher proportion of proteins from the embryo.
Lines transformed with silencing fragment ‘g’ showed a strong reduction of γ-gliadins, which was over-compensated by an increase of α-gliadins, ω-gliadins, HMW-GS and LMW-GS . The term over-compensation is appropriate because the reduction of γ-gliadins results in a higher content of total protein rather than retaining the level of control lines. The reduction of all gliadins, with the silencing fragment ‘o’ or with the combination ‘go’, was accompanied by an increase in HMW-GS and a reduction of LMW’GS, except in line BW2003 with silencing ‘o’ where there was also an increase of LMW-GS. Increased HMW-GS was not enough to offset the lack of gliadins, resulting in the reduction of total content of prolamins, although the total protein remained constant or even increased in the case of the lines silenced with ‘go’ fragments. Therefore, the over-compensation of total protein must be from non-gluten proteins, such as albumins and globulins, as described previously  (and as shown by the calculation of non-gluten protein presented in this paper). This compensation effect had been reported in maize and rice, where the silencing of a group of storage proteins leads to a rearrangement of other storage proteins. In maize, the reduction in 22-kDa α-zeins levels by RNAi were compensated by increases of the 19-kDa α-zeins, and vice versa . In the rice mutant line Low Glutelin Content-1 (LGC-1), the content of glutelin was reduced and the contents of other seed storage proteins, including prolamins, were increased . Such up-regulation is not specific to the LGC-1 mutant and it is thought to be a non-specific compensation for the reduction of glutelin. On the other hand, reductions of glutelins and sulfur-rich 10-kDa prolamin levels by RNAi in rice were preferentially compensated by increases of sulfur-poor and other sulfur-rich prolamins, respectively, indicating that sulfur-containing amino acids might be involved in regulating seeds storage protein composition. It could be suggested that transgenic lines, with storage proteins reduced, attempt to compensate total protein content, first with related proteins, and then with unrelated proteins, if necessary. Therefore, the protein compensation could be a selective process because it does not use any kind of protein to compensate. If the compensation is governed by the availability of amino acids, the compensation process may be selectively determined by similarities in the amino acid composition of proteins.
The data presented in this paper allow the detection of differences between the two promoters used for gliadin silencing. Although the Promoter factor explained only part of the variability, it was clear that the γ-gliadin promoter had a higher efficiency which was demonstrated with a better silencing of γ-gliadins, but the efficiency was further increased when both promoters were used in combination. Both the γ-gliadin promoter and, the combination of promoters, led to a decrease of the content of LMW-GS. The contribution to the effectiveness of the promoters that drive a silencing fragment is determined by their expression level during the target expression [27, 28]. Results reported by [29, 30] concluded that D-hordein and γ-gliadin promoters both had high expression levels in the wheat endosperm but with different expression profiles. The D-hordein promoter was expressed in later stages of grain development than the γ-gliadin promoter. The high efficiency of the γ-gliadin promoter may be due to a higher expression level and/or to a better adjustment with the target genes. However, it is clear that not only the expression level is important, but also a broad expression profile which allows the expression of target genes throughout grain development. This may be what occurred when the combination of the two complementary promoters was used, leading to greater effectiveness.
Among the gliadin fractions, the content of ω-gliadins showed the highest variability being strongly environment-dependent (the factor year explained 69% of the variance). In fact it has been reported that the ω-gliadins modify their expression in response to S deficiency (increasing their expression with low S) [31–35], and N fertilization (the higher input of N the greater accumulation of ω-gliadins) [36–38]. Moreover, the proportions of ω-gliadins increase when grain is exposed to high temperature during grain filling [36, 39]. In addition, the high environment-dependence of the ω-gliadin content also could be due to the fact that it is the group which is less efficiently silenced. Therefore, the iTarget factor has less influence on the ω-gliadin content compared with environmental factors.
The iTarget x Genotype interaction for SDSS showed that there was an increase of SDSS value in transgenic lines with iTarget ‘g’ but only for the BW2003 genotype, and a more pronounced decrease of SDSS for genotype BW2003 in comparison with genotype BW208 in iTarget lines ‘o’. Although the iTarget x Genotype interaction for SDSS in iTarget lines ‘o’ has first been analyzed in this work, the different behavior of genotypes with respect to SDSS has already been reported . The latter interaction is associated in turn with the interaction of Genotype x iTarget for LMW-GS. In fact, in the BW2003 lines with a decrease of the SDSS values, this was associated with a decrease of LMW-GS. Moreover, the NMDS ordination graph showed that the LMW-GS content and SDSS value were highly correlated. A similar association between quality parameters and LMW-GS content was also reported by , who showed a positive correlation between some mixograph parameters and the SDSS test with an individual LMW-GS peak and total LMW-GS contents.