Biochemical and transcriptomic analyses reveal that critical genes involved in pigment biosynthesis influence leaf color changes in a new sweet osmanthus cultivar 'Qiannan Guifei'.

Background: (Oleaceae) is one of the most important ornamental plant species in China. Many cultivars with different leaf color phenotypes and good ornamental value have recently been developed. For example, a new cultivar 'Qiannan Guifei', presents a rich variety of leaf colors, which change from red to yellow-green and ultimately to green as leaves develop, making this cultivar valuable for landscaping. However, the biochemical characteristics and molecular mechanisms underlying leaf color changes of these phenotypes have not been elucidated. It has been hypothesized that the biosynthesis of different pigments in might change during leaf coloration. Here, we analyzed transcriptional changes in genes involved in chlorophyll (Chl), flavonoid, and carotenoid metabolic pathways and identified candidate genes responsible for leaf coloration in the new cultivar 'Qiannan Guifei'.

Methods: Leaf samples were collected from 'Qiannan Guifei' plants at the red (R), yellow-green (YG) and green (G) leaf stages. We compared the different-colored leaves via leaf pigment concentrations, chloroplast ultrastructure, and transcriptomic data. We further analyzed differentially expressed genes (DEGs) involved in the Chl, flavonoid, and carotenoid metabolic pathways. In addition, we used qRT-PCR to validate expression patterns of the DEGs at the three stages.

Results: We found that, compared with those at the G stage, chloroplasts at the R and YG stages were less abundant and presented abnormal morphologies. Pigment analyses revealed that the leaves had higher flavonoid and anthocyanin levels at the R stage but lower Chl and carotenoid concentrations. Similarly, Chl and carotenoid concentrations were lower at the YG stage than at the G stage. By using transcriptomic sequencing, we further identified 61 DEGs involved in the three pigment metabolic pathways. Among these DEGs, seven structural genes (, , , , , and) involved in the flavonoid biosynthesis pathway were expressed at the highest level at the R stage, thereby increasing the biosynthesis of flavonoids, especially anthocyanins. Six putative genes, including three flavonoid-related activators and three repressors, were also highly expressed at the R stage, suggesting that they might coordinately regulate the accumulation of flavonoids, including anthocyanins. Additionally, expressions of the Chl biosynthesis-related genes , and and the carotenoid biosynthesis-related genes and were upregulated from the R stage to the G stage, which increased the accumulation of Chl and carotenoids throughout leaf development. In summary, we screened the candidate genes responsible for the leaf color changes of 'Qiannan Guifei', improved current understanding of the regulatory mechanisms underlying leaf coloration and provided potential targets for future leaf color improvement in .