Lc-transgenic tobacco lines exhibited more intense pigmentation in floral parts
More than 10 Lc-transgenic plants were cultivated and screened, among which, the lines Lc2, Lc4, Lc6, and Lc10 were used for the following assay. For SR1, at the vegetative stage, the leaves and stems were green, while at the bloom stage, the calyxes were green, the petals and filaments were pale red, and the anthers as well as the stamens exhibited light green. For Lc-transgenic lines, green leaves and stems were similar to those of SR1, while the flowers appeared light red. The colour intensity was consistent with the TAC in the different floral organs. The TAC in young and mature leaves at the seedling stage as well as the anthers and the pistils at floral stage was very low, and the TAC was greatly increased in calyxes, in petals, and in filaments. Flowers of all Lc-transgenic lines displayed dark red (Fig. 1A). As compared to the counterparts in SR1, the TAC in calyxes, in petals, and in filaments of the Lc-transgenic lines was increased by 18, 51 and 132-folds, respectively. Though the TAC in anthers was low, it was still much higher than that of SR1 (Fig. 1B).
Cyanidin is responsible for the intense pigmentation in Lc-transgenic tobacco lines
To clarify the major component of anthocyanins responsible for intense pigmentation in Lc-transgenic flowers, three most common anthocyanins, Cya (cyanidin), Pel (pelargonidin), and Del (delphinidin) were further quantified with HPLC. The HPLC extraction and analysis system of the three anthocyanidins were established. The retention times of Cya, Del and Pel standard sample were 32.148, 37.960 and 41.213 min, respectively (Fig. 1C). Only Cya was identified in Lc-transgenic tobacco petals. Moreover, the Cya concentrations of three Lc-transgenic lines were at least 38-fold higher than that of SR1 (Fig. 1D), which demonstrated that the increased cyanidin was responsible for the intense pigmentation in the petals.
Boosted expression of key anthocyanin-biosynthetic genes in Lc-transgenic lines
Intense pigmentation was detected in the floral parts, especially in petals and in filaments of Lc-transgenic plants, compared to SR1 plants, which suggested that ectopic expression of Lc was responsible for the anthocyanin increases. Northern blot displayed that Lc expressed comparatively weak in calyxes, strong in petals and filaments (Fig. 2), which agreed with the differential TAC increases in calyx, petals and filaments in Lc-transgenic lines (Fig. 1).
To elucidate how ectopic expression of Lc enhanced anthocyanin biosynthesis in the different parts of Lc-transgenic flowers, six key anthocyanin biosynthetic genes (NtPAL, Nt4CL, NtCHS, NtDFR, NtANS and NtGST) were analyzed with Northern blot. NtPAL was highly expressed in the petals, filaments and pistils and its expression pattern were similar between SR1 and Lc-transgenic lines. Nt4CL expressed weakly in the calyxes and anthers, strongly in the petals, filaments and pistils in both SR1 and Lc-transgenic lines. NtCHS expressed exclusively in petals of SR1, while it was greatly upregulated in petals and filaments in Lc-transgenic lines. NtDFR expressed highly in the petals, filaments and anthers, low in the calyx, and absent in the pistils of SR1, while it expressed significantly higher in the petals and filaments of Lc-transgenic lines. Similar expression profile was detected for both NtANS and NtCHS, with an exclusive expression in petals in SR1, and a significantly upregulated expression in the petals and filaments in Lc-transgenic lines. NtGST was ubiquitously expressed in the five floral parts of SR1 and Lc-transgenic lines (Fig. 2).
The anthocyanin biosynthesis pathway was enhanced in petals of Lc-transgenic plants
To further discriminate the molecular mechanism of the TAC increase in the Lc-transgenic lines, we analyzed the expression of genes involved in the anthocyanin biosynthetic pathway in the petals with multiplex RT-PCR. Lc was only detected in the petals of three Lc-transgenic lines (Fig. 3L), the expression of NtPAL, NtCHS, NtCHI, NtF3′H, NtDFR, NtANS increased by 47, 47, 70, 170, 139 and 124 % in Lc-transgenic line as compared to those in SR1 (Fig. 3A, D, E, G–I). Nt4CL and NtF3H expressed similarly between SR1 and Lc-transgenic lines (Fig. 3C, F). Additionally, the flavonol synthase gene NtFLS expression was much lower in the two Lc-transgenic lines than that in SR1 (Fig. 3J).
For the anthocyanin regulatory gene, Lc was only detected in Lc-transgenic lines (Fig. 3L) not in SR1. The expression of NtAN2, the partner of Lc-like bHLH proteins in regulating the anthocyanin biosynthesis pathway was not changed in the Lc-transgenic lines (Fig. 3K), which suggested that ectopic expression of Lc rather than NtAN2 is responsible for the up-regulation of anthocyanin biosynthesis genes as well as the anthocyanin accumulation in the Lc-transgenic lines.
Taken together, the four key genes NtCHS, NtF3’H, NtDFR and NtANS were greatly upregulated in petals and filaments of Lc-transgenic lines, which resulted in enhanced anthocyanin biosynthesis and more intense pigmented calyx, petals and filaments in the Lc-transgenic lines.