Fig. 3.

Effect of LaCHS8 RNAi silencing on flavonoid accumulation and lateral rootlet density. Reduced expression of LaCHS8 was achieved by subcloning an mRNA fragment of LaCHS8 into an RNAi vector containing DsRED as a reporter and subsequent transformation of white lupin roots using A. rhizogenes. In parallel, roots transformed with a human myosin gene were used as a control to exclude effects due to the formation of hairy roots. Hairy roots were incubated in DPBA to show flavonoid accumulation. (A–D) Green fluorescence of flavonoid–DPBA was detected by confocal laser scanning microscopy (scale bar = 1.0 mm). Images of (A) flavonoid fluorescence in root apices of MYO RNAi (MYOi) hairy roots, (B) root apices of LaCHS8 RNAi (LaCHS8i) hairy roots, (C) lateral rootlet emergence zones of MYOi hairy roots and (D) lateral rootlet emergence zones of LaCHS8i hairy roots. (E) qRT–PCR confirmed the reduction of the LaCHS8 transcript in the corresponding RNAi roots relative to the control. (F) Fluorescence intensity (arbitrary fluorescence units) in different transgenic hairy root segments: root apexes (RA) and juvenile cluster roots (JCR). (G) Lateral rootlet density of LaCHS8i and MYOi hairy roots. Asterisks in (E–G) indicate significant differences compared with the control (MYOi) by Student’s t-test: *P < 0.05; **P < 0.01. Significant difference was based on four biological replicates. The bars on the graphs are standard errors.