The basic helix–loop–helix (bHLH) family is the second largest superfamily of transcription factors that belongs to all three eukaryotic kingdoms. The key function of this superfamily is the regulation of growth and developmental mechanisms in plants. However, the bHLH gene family in Carthamus tinctorius has not yet been studied. Here, we identified 41 bHLH genes in Carthamus tinctorius that were classified into 23 subgroups. Further, we conducted a phylogenetic analysis and identified 10 conserved protein motifs found in the safflower bHLH family. We comprehensively analyzed a group of bHLH genes that could be associated with flavonoid biosynthesis in safflower by gene expression analysis, gene ontology annotation, protein interaction network prediction, subcellular localization of the candidate CtbHLH40 gene, and real-time quantitative expression analysis. This study provides genome-wide identification of the genes related to biochemical and physiological processes in safflower.

Flavonoids with significant therapeutic properties play an essential role in plant growth, development, and adaptation to various environments. The biosynthetic pathway of flavonoids has long been studied in plants; however, its regulatory mechanism in safflower largely remains unclear. Here, we carried out comprehensive genome-wide identification and functional characterization of a putative cytochrome P45081E8 gene encoding an isoflavone 2′-hydroxylase from safflower. A total of 15 CtCYP81E genes were identified from the safflower genome. Phylogenetic classification and conserved topology of CtCYP81E gene structures, protein motifs, and cis-elements elucidated crucial insights into plant growth, development, and stress responses. The diverse expression pattern of CtCYP81E genes in four different flowering stages suggested important clues into the regulation of secondary metabolites. Similarly, the variable expression of CtCYP81E8 during multiple flowering stages further highlighted a strong relationship with metabolite accumulation. Furthermore, the orchestrated link between transcriptional regulation of CtCYP81E8 and flavonoid accumulation was further validated in the yellow- and red-type safflower. The spatiotemporal expression of CtCYP81E8 under methyl jasmonate, polyethylene glycol, light, and dark conditions further highlighted its likely significance in abiotic stress adaption. Moreover, the over-expressed transgenic Arabidopsis lines showed enhanced transcript abundance in OE-13 line with approximately eight-fold increased expression. The upregulation of AtCHS, AtF3′H, and AtDFR genes and the detection of several types of flavonoids in the OE-13 transgenic line also provides crucial insights into the potential role of CtCYP81E8 during flavonoid accumulation. Together, our findings shed light on the fundamental role of CtCYP81E8 encoding a putative isoflavone 2′-hydroxylase via constitutive expression during flavonoid biosynthesis.

The rhizosphere harbors abundant plant growth-promoting rhizobacteria (PGPR) that are vital for plant health. In this study, we screened growth-promoting bacteria from tomato rhizosphere soil, verified their functions, and constructed the optimal combination of growth-promoting bacteria for promoting tomato growth. Furthermore, the effects of these bacteria on various physiological and biochemical parameters of tomato plants were evaluated. A total of 36 strains of rhizobacteria were isolated from tomato rhizosphere soil and their abilities to produce indole-3-acetic acid (IAA), solubilize phosphate and iron carriers were assessed. The bacterial strains with the highest capacities for IAA production (R62, R317), phosphate solubilization (R41, R219), and siderophore production (R25, R325) were selected to form three bacterial combinations: R62 + R219 + R317 + R325 (T1), R62 + R325 (T5), and R317 + R325 (T8). Fifteen days after inoculation, all three combinations showed a stimulatory effect on seedling growth compared to the un-inoculated control. Inoculation with T1, T5 and T8 increased the seedling vigor index by 173.7%, 204.1%, and 168.7%, respectively. Compared to the un-inoculated control, the T1 combination increased the activities of polyphenol oxidase, peroxidase, and the net photosynthetic rate by 132.7%, 18.7%, 58.5%, and upregulated the relative expression levels of the photosynthetic assimilation-related genes RbcL, RbcS, FBPase and FDA by 22.2-, 6.6-, 1.95-, and 2.0-fold, respectively. Our findings provide a potential for constructing rhizobacterial combinations of different functional groups for improving crop growth.