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1Department of Plant Genetics, Breeding and Biotechnology, Institute of Biology, Warsaw University of Life Sciences, 02-787 Warsaw, Poland; moc.kooltuo@olzalwanna (A.W.); lp.wggs@akciceiws_aneladgam (M.Ś.); lp.wggs@retok_keram (M.D.K.); lp.wggs@iksperk_zsamot (T.K.)
3Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation, State Research Institute, 24-100 Puławy, Poland; lp.ywalup.gnui@fsa (A.S.); lp.ywalup.gnui@kyzclawokm (M.K.)
Promoter sequences were amplified by PCR (Mastercycler Nexus Gradient; Eppendorf, Hamburg, Germany) using the following program: 94 C for 5 min; 40 cycles of three-step amplification of 30 s at 94 C, 30 s at 60 C, and 45 s at 72 C; and then 5 min at 72 C. Primers were designed based on bioinformatic analysis of 1000-nt upstream regions of both genes using the open access PlantCARE database . Fragments with a high frequency of potential stress-specific motifs (SSMs) and growth- and development-specific motifs (GDSMs) were used as the templates for primer design (primer sequences are given in Table 2).
Expression patterns of the ScBx1 gene of three rye inbred lines, L318, D33, and D39 at six time points, 14, 21, 28, 42, 70, and 77 dag. The data represent mean value with standard deviation. There is no statistically significant difference between the expression level of the ScBx1 gene at subsequent time points within each of the three tested lines.
ScIgl: Starting from the first tested time point (14th dag), it was possible to detect the expression of the ScIgl gene in all three lines, albeit at a very low level. At the next time point (21st dag), its expression increased in line D39 (over 2.6-fold; being only slightly lower, with no statistically significant difference, than the expression level of ScBx1), while it remained at almost the same level in line D33 and decreased (more than 4-fold) in line L318. After the next 7 days, expression of the ScIgl gene increased in lines L318 and D33, while in D39 it fell. On the 42nd dag, the expression level of ScIgl dropped and on the 70th it increased in all tested lines. At the last time point (77th dag), the expression of ScIgl was detectable only in lines D33 and D39. Up to the 70th dag, line D39 was characterized by the highest expression level of the ScIgl gene, when line L318 showed its lowest level. Each inbred line was characterized by a unique ScIgl expression developmental pattern. The highest similarities were found for lines L318 and D33, which were found to have relatively similar expression profiles between the 21st and 70th dag. The greatest differences were observed at the second time point, when the expression level of ScIgl in line D39 was 152 and 32 times higher than those in lines L318 and D33, respectively (Figure 3).
Patterns of normalized (in respect to the empty BSMV:PDS vector) expression of ScIgl and ScBx1 genes in leaves of rye cv. Stach F1 inoculated with BSMV:ScBx1 on the 14th dpi, for methodological details related to silencing procedure see Groszyk et al. . The results represent mean value and standard deviations of three technical replicates.
Patterns of normalized (in respect to the empty BSMV:PDS vector) expression of ScIgl and ScBx1 genes in leaves of rye cv. Stach F1 inoculated with BSMV:ScBx1 on the 21st dpi; for methodological details related to silencing procedure see Groszyk et al. . The results represent mean value and standard deviations of three technical replicates.
The in silico analysis of promoter sequences of both genes was performed not only to preliminarily verify their function, but also to select fragments with high frequencies of SSMs and GDSMs for a wet experiment. In the in vivo assay, 49 proteins were found to bind to the ScBx1 and ScIgl promoter sequences selected based on bioinformatic analysis; 4 of them can be considered as stress-specific and 5 as growth- and development-specific. Among the TFs identified under stress conditions, only one (germin-like protein) was common to both genes. GLPs are encoded by a family of genes found in all plants and are associated with responses to biotic (viruses, bacteria, mycorrhizae, fungi, insects, nematodes, and parasitic plants) and abiotic stresses (salt, heat/cold, drought, nutrient, and metal), and especially with fungal pathogenesis in cereals [24,31]. The binding of this stress-specific TF to the promoters of ScBx1 and ScIgl suggests that both genes are regulated by stress. However, the binding to the promoter of ScBx1 of two other proteins (NIM1-interacting TFIIH subunit and myb-like DNA-binding domain), each of which has been proven to be associated with biotic and/or abiotic stresses [32,33,34,35], may indicate the greater roles of these genes in defense reactions. In the case of unstressed plants, one common TF (RING-H2 finger protein ATL8-like) bound to the promoter sequences of both genes. This regulatory protein plays an important role in the adaptation of plants to abiotic stresses [36,37]. In previous studies, no findings of a correlation of the TF RING-H2 finger protein ATL8-like with these processes were reported. Our results may indicate its novel function, but this needs to be confirmed through further experiments.
Similarly, as in plants inoculated with brown rust, unique TFs for the gene ScBx1, agamous-like (AGL) and EXORDIUM-like 2 (EXOL2), were identified in untreated plants. AGL, as a member of the MADS-box gene family, plays an important role in the regulation of plant growth and development ; EXOL2 has been shown to play a role in meristem function  and to suppress brassinosteroid-dependent growth [40,41]. In turn, we identified one specific gds TF as being linked only with the promoter of the second gene, namely, NAC domain-containing transcription factor, which acts as a master regulator of xylem vessel differentiation . Taking these results into account, we can conclude that both genes are developmentally regulated, but the findings for the ScBx1 gene are more robust.
The highest content of BXs was found in line D33, while the lowest one was in line D39. The same relationship was observed in the case of field-grown plants with respect to DIBOA, but not for GDIBOA; line L318 was characterized by the highest content of this BX .
In all lines, the content of BXs generally corresponded with the expression levels of the ScBx1 (at earlier stages) and ScIgl genes (at later stages). Only a few cases of deviation from this relationship, namely, a considerable increase in gene expression and a decrease in BX synthesis (and also vice versa) were observed, specifically, on the 21st dag for GDIBOA and DIBOA in lines L318 and D39, on the 28th dag for DIBOA in line D33, on the 77th dag for DIBOA in lines L318 and D39, and on the 77th dag for GDIBOA in line L318.
Our observations discussed above as well as those reported by Groszyk et al.  enable us to assert that both ScBx1 and ScIgl genes are not only regulated developmentally but are also involved in defense responses. However, the first gene is expressed at earlier stages and the second one at later stages. When the expression of ScBx1 decreases, the expression of ScIgl increases over time, although its level is low. Consequently, the ScBx1 gene provides indole to the BX biosynthesis pathway at earlier developmental stages and ScIgl at later ones (Table 7).
A.W. designed and performed the experiments analyzing ScBx1 and ScIgl expression levels in rye lines, wrote the manuscript (together with M.Ś. and M.R.-T.). M.Ś. designed and performed the experiment with virus-induced silenced plants, wrote the manuscript (together with A.W. and M.R.-T.). M.D.K. and T.K. performed in vivo analysis of promoter. A.S. and M.K. conducted biochemical analysis. L.B. performed statistical analysis. M.R.-T. performed in silico analysis of promoter, conceived and wrote the manuscript (together with A.W. and M.Ś.). All authors have read and approved the final manuscript.
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