Ascorbic acid, also referred to as vitamin C (Vc), is an important nutrient found in fruits and vegetables that promotes produce quality and human health. Rosa roxburghii is an underutilized natural fruit that contains very high levels of Vc. However, the Vc content of R. roxburghii varies considerably during plant development and ripening. To better understand the molecular mechanisms that underlie fluctuations in Vc content of R. roxburghii fruit at different developmental stages, we performed transcriptomic and metabolomic analyses and identified two significant gene networks/modules and 168 transcription factors directly involved in Vc synthesis. Promoter analysis of two core genes involved in Vc synthesis, RrGGP and RrGalUR, revealed the presence of a retroviral long terminal repeat (LTR) insert in the RrGalUR promoter. Using yeast one-hybrid and dual-luciferase assays, we demonstrated that the transcription factors RrHY5H and RrZIP9 bind to the promoter of RrGGP to promote its expression. RrZIP6 and RrWRKY4 bind to the LTR in the RrGalUR promoter to promote its expression. Our results reveal a molecular mechanism that controls Vc synthesis and accumulation in R. roxburghii fruit.

Plants with crassulacean acid metabolism (CAM) generally utilize water 20%–80% more efficiently than non-CAM plants. The whole genomes of several CAM plants have been sequenced or are being sequenced. For effective genome characterization and genome editing of CAM plants, an efficient transformation system is essential. In this study, we developed an Agrobacterium-mediated transformation protocol for Kalanchoe laxiflora, an obligate CAM plant,by optimizing several factors affecting the transformation efficiency. Agrobacterium strains AGL1, C58, EHA105, and GV3101 were all suitable for K. laxiflora transformation. Fifty-nine percent of the leaf explants yielded kanamycin-resistant and GUS-positive shoots. Polymerase chain reaction and quantitative real-time PCR (qRT-PCR) using gusA-, gusPlus-, nptⅡ- and hpt-specific primers confirmed that the transgenes were integrated into K. laxiflora genome and expressed. This efficient transformation system will allow effective functional characterization of genes through over- or down-expression, knockout, or genome editing.