Importance of the flavonoid pathway in plant growth promotion
In the ineffective rhizobacteria-plant partnerships, flavonoids showed considerably higher accumulation while they were not significantly changed or significantly reduced in effective and highly effective partnerships, respectively. Considering our results and other reports on the negative association between flavonoids and auxin transport, auxin distribution and turnover (Brown et al. 2001; Buer et al.2013; Yin et al. 2014; Kuhn et al. 2017), we further investigated the effects of Pbg on growth of 12 isogenic homozygous Arabidopsis transparent testa (tt )-mutants (Appelhagen et al. 2014) disrupted in biosynthetic genes of different branches of the flavonoid pathway (Figure 4a ). First, the inherent variation in shoot biomass of each of the 12 mutant lines and wild type (WT) Col-0 was assessed in absence of Pbg . Second, the effect of Pbg on growth of these 12 mutants and Col-0 was assessed by calculating the percent change in shoot biomass relative to the biomass of the respective non-treated mutant line or WT. Both measures were subjected to analysis of variance (ANOVA) by considering the ‘genotype’ as a factor and root biomass and percent change in shoot biomass as dependent variables.
In absence of Pbg , we observed significant variation in shoot biomass among the tt- mutants and between several of thett- mutants and WT Col-0 (Figures 4b and 4c and Supplementary material, Table S5 ). Mutants disrupted in the initial steps of the flavonoid pathway (tt- 4 and tt- 6) showed similar shoot biomass as WT Col-0 and higher shoot biomass than many of the mutants with genes disrupted in the downstream steps of the pathway, especially mutants tds 4, aha 10, and tt 10 that are associated with the biosynthesis of flavan-3-ols (PA) (Figure 4c ).
Root tip inoculation with Pbg led to significant changes in shoot fresh biomass between the 12 mutants (Figure 4d and Supplementary material, Table S6 ). Interestingly, Pbghad the strongest growth-promoting effect on mutants affected in the downstream part of the flavonoid pathway, particularly on mutants in the proanthocyanidin branch (tt 10-8, aha 10-6, tt 12-2,tds -4, tt 15-5 and tt 18-5) (Figure 4d ). Thett- 7 mutant that accumulates kaempferol-derived flavonols showed inherently the lowest shoot biomass accumulation (Figure 4d ) and root inoculation of this mutant with Pbg had no significant growth-promoting effect as compared to the WT (Figure 4d ). Collectively, these results indicate that the proanthocyanidin (PA) branch plays a key role in Pbg -mediated growth promotion.