- thaliana lines carrying a loss-of-function mutation in BRI1 gene, bri1– 5, that exhibits a dwarf phenotype and its three activation-tag suppressor lines that were able to partially revert the bri1–5 mutant phenotype to a WS2 phenotype, namely bri1–5/bri1–1D , bri1–5/brs1–1D, and bri1–5/bak1–1D. - From the three investigated bri1–5 suppressors, bri1–5/bak1–1D was the most effective suppressor at the transcriptional level. - All three bri1–5 suppressors showed altered expression of the genes in the abscisic acid (ABA signaling) pathway, indicating that ABA likely contributes to the partial recovery of the wild-type phenotype in these bri1–5 suppressors. - For example, the function of BZR1 has been unveiled by using the null allele of BRI, bri1–116. - The weak mutant of bri1, bri1–5, can be rescued by overexpression of BAK1 and BRI Suppressor 1 (BRS1) [3, 12]. - Moreover, three of the five overexpressed BRS1’s homo- logs amongst which ECS1 (Extra Carpels and Seeds 1) can also partially suppress the phenotype of the bri1–5 mutant observed in leaves. - bri1–5/bak1–1D , bri1–5/brs1–1D and bri1–5/bri1–1D partially reconstitute bri1–5 gene expression. - bak1–1D and bri1–5/brs-1D were obtained from [12].. - An additional bri1–5/bri1–1D mutant was generated in. - Se- quencing the BRI1 flanking region from the suppressor bri1–5/bri1–1D showed that the activation tag was inserted 534 bp downstream of the BRI1 gene (Supple- mentary Fig. - Phenotypically, all bri1–5 suppressors (bri1–5/bak1–1D, bri1–5/brs1–1D, and bri1–5/bri1–1D) lines displayed larger seedlings than the bri1–5 mutant, but still signifi- cantly smaller than the WS2 (Fig. - Of all suppressor mutants, the bri1–5/bak1–1D line best approximated the growth phenotype of the WS2, and its larger seedling seemed to be mainly the effect of its larger root length and to a lower extent of its larger hypocotyl length (both of which were significantly larger than the bri1–5 mu- tant). - The contribution of the epidermal cell length in recovering the bri1–5 is marginal in the bri1–5/bak1–. - 1D (line with the largest seeding) but seems much more pronounced in the bri1–5/brs1–1D (Fig. - This indicates that in the bri1–5/brs1–. - 1D mechanisms other than those in the bri1–5/bak1–1D line play a role in alleviating the bri1–5 phenotype.. - To gain insight into which pathways in each of the studied lines were responsible for recovering the bri1–5 growth phenotype to wild-type level, we performed gene expression analysis. - All suppressor lines, together with the wild-type (WS2) and the bri1–5 background were sampled at a 7-day seedling stage. - S4 (A-C) show that of all suppressor lines, bri1–5/bak1–1D could restore the largest number of genes that were affected in expression in bri1–5 (about two-thirds of the genes that were differentially expressed in bri1–5 were no longer differentially expressed in bri1–5/bak1–1D). - 3 Differentially expressed genes (DEGs relative to WS2) being compared between bri1–5 and its three suppressors. - Group A (restored genes, 270 genes): genes differentially expressed in the bri1–5 mutant but no longer in at least two of the suppressor lines. - Group C (genes that were not restored, 371 genes): Genes that are aberrantly expressed in bri1–5 and at least two of the suppressor lines. - Group D (333 genes), E (167), and F (94 genes) contain genes that are exclusively differentially expressed in respectively the bri1–5/brs1–1D , bri1–5/bri1–1D, and bri1–5/bak1–1D suppressor lines.. - Root (A), hypocotyl (B), and epidermal cell length (C) of WS2, bri1–5 , bri1–5/brs1–1D , bri1–5/bak1–1D, and bri1–5/bri1–1D , measured 7 days after germination. - bri1–5/bak1–1D seems to also phenotypically best com- pensate for the bri1–5 mutation.. - 5/brs1–1D and bri1–5/bri1–1D lines share the largest fraction of similarly affected genes. - S4 panel D-F which shows that from all pairwise comparisons between suppressor lines, the level of differential expression relative to the mutant bri1–5 is most correlated between the suppressor lines bri1–5/. - brs1–1D and bri1–5/bri1–1D (i.e. - Al- though this results in lower correlation values than when directly comparing the expression values of the mutant lines, it better reflects the consistency between mutant lines in restoring genes affected in the bri1–5 mutant.. - To confirm the extent to which the different suppres- sor strains molecularly restore the defects in the bri1–5 mutation, we compiled a list of marker genes representa- tive of downstream pathways affected by BR signaling (Additional file 2). - Of those marker genes, only those that were significantly affected in the bri1–5 line were retained in order to identify the mutant line that best suppresses the bri1–5 mutation (96 marker genes). - S5 show how the expression of these genes is, as compared to WS2 affected in the bri1–5 mutant and how some of those genes got restored in the suppressor mutants. - bak1–1D restored the bri1–5 affected marker genes to the largest extent, and that molecularly the bri1–5/brs1–. - 1D and bri1–5/bri1–1D mutant tend to behave more similarly in restoring the same marker genes.. - S6, S7 and S8 and Table S2 show a number of pathways that are differ- entially expressed in both bri1–5 and all of the suppres- sor lines. - These represent the pathways that are responsible for the aberrant growth phenotype in the bri1–5 mutant and that could not entirely be restored or compensated for in the suppressor lines. - We assumed that if the suppressor strains alleviate the phenotype of the bri1–5 mutant, they could do so be- cause they either restore the pathways disrupted in the bri1–5 mutant to wild-type levels or they induce genes that compensate for the bri1–5 affected pathways. - Pro- cesses that are aberrantly expressed in the bri1–5 mu- tant, but not in any of the suppressor lines, represent pathways that are restored to WS2 levels in all of the suppressors. - S7, Table S2) are largely down-regulated in the bri1–5 mu- tant, restored to normal in bri1–5/bri1–1D and bri1–5/. - bak1–1D and up-regulated as compared to WS2 levels in the bri1–5/brs1–1D suppressor, indicating that some overcompensation is needed for this pathway in the bri1–5/brs1–1D background in order to restore the bri1–5 phenotype. - S8 and Table S2) seems to have been affected by all suppressors, but at least not to a significant level in the bri1–5 mutant. - a pathway that needs to be triggered in the suppressor strains in order to re- store the bri1–5 affected pathways and phenotype.. - the suppressor lines can restore the bri1–5 phenotype to WS2 levels. - the genes with altered ex- pression in the bri1–5 mutant, but restored to WS2 level in at least 2 suppressors), ii) genes that were compensa- tory in most of the suppressors (genes of group B i.e. - the genes not differentially expressed in the bri1–5 mutant, but differentially expressed in at least two suppressor strains) and iii) genes altered in the bri1–5 mutant that most likely were not restored in the suppressors (genes of group C i.e. - the genes, differentially expressed in the bri1–5 mutant and at least two of the suppressor strains). - This indicates that these are the pathways that contribute to alleviating bri1–5 signaling deficiency in the suppressor strains.. - genes: these were are up-regulated in at least two bri1–5 suppressor lines compared to wild-type, but were not af- fected in the bri1–5 mutant (HAI1 and HAB1 being sig- nificantly up-regulated in all suppressor mutants. - In addition, HAI2 was affected in the bri1–5 line, but could not be restored in at least two suppressors (non-restored gene), and HAB2 was identified as a connector node.. - This indicates that ABA signaling has indeed been affected in the suppressor strains to compensate for the bri1–5 sig- naling deficiency. - Genes were only selected as representative for downstream BR signaling if the up/down regulation of their expression was confirmed by at least 5 independent references and also affected in the bri1–5 line of our study (compared to WS2). - Columns bri1–5 , bri1–5/bri1–1D , bri1–5/brs1–1D , bri1–5/bak1–1 indicate whether the genes were found to be up or down-regulated compared to WS2 according to our expression data. - However, they appear to become up-regulated in suppressors, indicat- ing that further compensatory repression of the ABA sig- naling is required in order to restore the bri1–5 phenotype.. - expressed in the bri1–5 lines and all of the suppressors.. - CYP708A3, another BR biosynthesis gene was found to be differentially expressed in all suppressors and the bri1–5 mutant (Fig. - 6 Comparing the mean expression values of BR-biosynthesis genes in the bri1–5 mutant and suppressor lines. - For each line (WS2, bri1–5, and suppressors) the average log2 expression values of gene expression across replicates are given for the indicated BRs biosynthesis genes. - The main BR-biosynthesis genes are affected in the bri1–5 mutant and all suppressors. - However, the plots show that BR-biosynthesis genes are less affected in the line ( bri1–5/bak1–1D) that best suppresses the bri1–5 phenotype . - overexpression of the BR signaling and biosynthesis genes in the bri1–5 and suppressor mutants. - 6, the best suppressor of bri1–5, bri1–5/bak1-D, shows the lowest expression change of the biosynthesis genes). - bri1–5”, “GO_only_bri1–1D”, “GO_only_bak1–1D”,. - Iron ion homeostasis, ferroxidase, and glutathione transferase activity are identified as compensatory mechanisms unique to the bri1–5/brs1–1D suppressor Unlike for BRI1 and BAK1, much less is known about the role of BRS1 in BR signaling. - Therefore we had a closer look at genes of group D which are exclusively differentially expressed in bri1–5/brs1–1D mutant and hence comprise compensatory pathways specific for bri1–5/brs1–1D. - These re- sults showed how in the bri1–5/brs1–1D mutant the expression of the glutathione transferase was not only restored as compared to the other suppressor lines, but even overcompensated as compared to WS2 levels (Fig. - We also found that several members of the CCAAT-binding factor complex (CBC) (NFYA2, NFYA3, NFYA6, NFYA10) were uniquely up-regulated in bri1–1D/brs1–1D (Fig. - In addition, iron ion homeostasis/ferroxidase activity was also found to be down-regulated, specifically in the bri1–5/brs1–1D. - Accordingly, we also found that the main inhibitor of H + -ATPase trans- porters, CBC1, was significantly down-regulated in bri1–5/brs1–1D (fold change − 1.7, adj p-value 8.36e- 06), but not in the other suppressors. - S9) might be essential to compensate for the more acidic environ- ment in the bri1–5/brs1–1D and would be required for maintaining redox homeostasis. - In addition, we hypothesize that the observed acidification could gen- erate a cellular environment that improves BRI1-BRs binding or BRI1-BAK1 dimerization and hence con- tributes to restore the bri1–5 mutant phenotype.. - Our analysis identified ABA signaling as a mainly com- pensatory pathway, and hormone signaling pathways re- lated to ethylene, auxin, and ROS, as pathways involved in partially restoring the bri1–5 expression phenotype to WS2 levels. - In the absence of BRs (or BR signaling deficiency like bri1–5), SnRK2.3 can mimic the presence of ABA in triggering ABA signaling, once it is phosphorylated by BIN2 [25]. - 7 GO enrichment for differentially expressed genes (DEGs) exclusively in bri1 – 5/brs1 – 1D compared to WS2. - Links between BR signaling and auxin we observed are also supported by literature and in line with the ob- served phenotype of the bri1–5 mutant. - As expected, fatty acid metabol- ism (subnetwork 3) and developmental processes (sub- network 4) have been partially restored by bri1–5 suppressor lines, supporting the impact of BR signaling on the development, and the composition of fatty acids.. - This is in line with our results: oligopeptide transport (subnet- work 5) is affected in the bri1–5 mutant and is partially restored by suppressor lines.. - Our results also show that the level of negative feedback depends on the degree to which the suppressor could compensate for the phenotypic differ- ence between the bri1–5 and WS2. - bri1 – 5) the activated SnRK2 by BIN2 mimics the presence of ABA, activates ABI5, and finally regulates stress response genes (Fig. - bes1-D) described in the literature or why treatment with exogenous BR give rises to a phenotypic response that is worse than the one observed in the bri1 – 5 mutant (shorter root) [47]. - Our analysis of the genes/pathways that are uniquely in- volved in the bri1–5/brs1–1D suppressor line to com- pensate for the bri1–5 mutant showed that BRS1 seems involved in the acidification of the apoplast environment.. - We hypothesize that this acidification could contribute to an improved BRI1-BRs binding or BRI1-BAK1 dimerization and hence restoration of the bri1–5 mutant phenotype. - This is confirmed by the phenotypic analysis which shows that indeed the bri1–5/brs1–1D line at least partially restores the epidermal cell length (Fig. - The bri1–5/brs1–1D suppressor line also induces glutathione transferase activity which is necessary for redox homeostasis. - Our results suggest that ABA signaling plays a significant role in alleviating the bri1–5 dwarf phenotype. - The fact that also other phytohormone signaling pathways are restored to the wild-type expression level in all bri1–5 suppressors con- firms the crosstalk between BR and other phytohormone signalings. - bri1–1, bri1–5/brs1–1, and bri1–5/bak1–1 or by study- ing the effect of suppressors not only in the BRI1 as a loss-of-function background but also using double or triple mutants of multiple genes of the BRI1 gene family and/or BRI1-like genes [52] could be interesting to fur- ther elucidate the mechanism of BRI signaling. - The two activation tagging suppressors of bri1–5, bri1–. - 5/bak1–1D, and bri1–5/brs1–1D were obtained from our previous study [12]. - An additional activation tagging suppressor line bri1–5/bri1–1D was generated in this study as previously described [3]. - Wild-type (WS2), the loss-of-function BR mutant (bri1–5), and its three sup- pressor mutants (bri1–5/brs1–1D, bri1–5/bak1–1D, bri1–5/bri1–1D) were grown at 22 °C in a long-day con- dition (16 h of light and 8 h of dark) in a greenhouse for 7 days. - The intersection of the genes that were differentially expressed in the bri1–5 mutant line with the list of. - genes that were significantly differentially expressed in all three bri1 – 5 suppressors, but not in bri1 – 5 mutant. - The core of group C (non-restored genes) consists of 149 genes that are affected in bri1 – 5 mutant but not restored by any suppressors. - The T-DNA insertion site for bri1–1D (A), and microscopic images of 7-day old hypocotyl cells for WS2 (B), bri1–5 (C. - bri1–5/bak1–1D ( D. - bri1–5/bri1–1D (E. - bri1–5/brs1–1D (F). - Comparing genome-wide expression impact between bri1–5 suppressor lines. - Heatmap of expression of the marker genes that up/down regulation of their expression was confirmed by at least 5 independent references and also affected in the bri1–5 line of our study. - Panel A: bri1–5, Panel B : bri1–5/bri1–1D , Panel C: bri1–5/brs1–1D , Panel D: bri1–5/bak1–1D
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