## 4 Discussion
in tobacco leaves, it was observed that in higher P treatment there was a reduction in the transcript levels of NtCHS (challenge synthase), NtCHI (chalcone isomerase), NtF3H (flavanone 3-hydroxylase), NtF30H (flavanone 30-hydroxylase) and NtFLS (flavonol synthase), while NtFLS transcript levels were increased in both juvenile and mature leaves in the lower P treatment. Pi (inorganic P) deficiency led to a lack of bHLH transcription factor and negatively affected the key gene for anthocyanin biosynthesis (NtDFR) [56]. In Arabidopsis, a mutation in the regulator of the flavonoid biosynthesis AtMYB12, which encodes the R2R3-MYB TF and influences the expression of CHS, CHI, F3H and FLS, causes remarkable downregulation of these genes. In maize, the P1 regulator, which encodes the R2R3-MYB TF, induces the expression of these genes in a similar way except for the F3H and genes that are only found in the anthocyanin branch of the phenylpropanol pathway [57; 58]. The bHLH transcription factor and F3H are identified among the genes occupying pivotal positions in the phenylpropanol pathway. The expression of the bHLH gene exhibited a significant reduction, particularly in the S3 maturity stage of P0. In general, the results suggest that the maturity stage played a central role in the downregulation of bHLH, with a similar expression pattern observed in the variations of F3H expression. Plant responses to P alterations often involve changes in gene expression at the post-transcriptional level. In Arabidopsis, rice and soybean for example, the miRNA339 is induced by P stress and regulates phosphate homeostasis by suppressing the activity of the PHO\({}_{2}\) enzyme; additionally, P stresses can activate genes due to alterations in transcription factor expression, such as TF of the bHLH families [59]. Alterations in F3H expression in common bean seeds affected the concentration of dihydroquercetin-related compounds and proanthocyanidins [60]. In tobacco leaves, P treatments in both low and high concentrations affected the regulation of the transcripts involved in the flavonoid pathway, which is a branch of the phenylpropanol. KEGG pathway analysis indicated an increase in the upregulated genes in P2 treatment in comparison with P0, while the downregulated genes showcased a decrease only in P2 treatment. On the other hand, the upregulated genes in the flavonoid biosynthesis pathway exhibited a one-gene increase in P2, whereas the downregulated genes rose in both P1 and P2 treatments. The alterations observed in enriched DEGs as a result of changes in P levels do not align with the selected genes within the pathway, except for F3H and FLS. Consequently, the results of the KEGG analysis may be associated with other genes expressed in the broader phenylpropanol pathway. In a comparative transcriptomic study conducted on apple seedlings under different P treatments, DEG analysis revealed significant changes in flavonoids and anthocyanin concentration in the treatments, while the KEGG enrichment analysis showed that the flavonoid and phenylpropanol biosynthesis pathways were highly enhanced in different P treatments [61]. In tobacco leaves, P treatments of both low and high concentrations, affected the regulation of the transcripts involved in the flavonoid pathway, which is a branch of the phenylpropanol [56]. ExcessP (P2) increased the DFR expression, but the seed maturity stage negatively affected the expression since in every P treatment there was a remarkable reduction in the expression in S3 compared to S2. The downregulation of the genes of the phenylpropanol pathway and the consistent even lower expression in most cases in P1 and P2 treatments suggest that excess P is a limiting factor for proanthocyanidin (tannin) formation. Ongoing research aims to determine if the transcriptional downregulation of phenylpropanol gene expression correlates with lower seed tannin accumulation.