Anthocyanins are distributed ubiquitously to terrestrial plants and chalcone isomerase (CHI) catalyzes the stereospecific isomerization of chalcones – a committed step in the anthocyanin biosynthesis pathway. In this study, one gene encoding CHI was isolated from Ophiorrhiza japonica and designated as OjCHI. Multiple sequence alignments and phylogenetic analysis revealed that OjCHI had the conserved CHI active site residues and was classified into type I CHI group. In order to better understand the mechanisms of anthocyanin synthesis in O. japonica, integrative analysis between metabolites and OjCHI expression was conducted. The results showed OjCHI expression matched the accumulation patterns of anthocyanins not only in different tissues but also during the flower developmental stages, suggesting the potential roles of OjCHI in the biosynthesis of anthocyanin. Then biochemical analysis indicated that recombinant OjCHI protein exhibited a typical type I CHI activity which catalyzed the production of naringenin from naringenin chalcone. Moreover, expressing OjCHI in Arabidopsis tt5 mutant restored the anthocyanins and flavonols phenotype of hypocotyl, cotyledon and seed coat, indicating its function as a chalcone isomerase in vivo. In summary, our findings reveal the in vitro as well as in vivo functions of OjCHI and provide a resource to understand the mechanism of anthocyanin biosynthesis in O. japonica.

Rosa roxburghii Tratt (Rosaceae) has a fruit that is flavorful, economically valuable, and highly nutritious, providing numerous health benefits. Myeloblastosis (MYB) proteins play key roles in the development and fruit quality of R. roxburghii. However, there is little available genomic and transcriptomic information for R. roxburghii. Here, a normalized cDNA library was constructed from five tissues, including the stem, leaf, flower, young fruit, and mature fruit, using the Illumina HiSeq 3000 platform. De novo assembly was performed, and 470.66 million clean reads were obtained. In total, 63,727 unigenes, with an average GC content of 42.08%, were discovered, 60,406 of which were annotated. In addition, 9,354 unigenes were assigned to Gene Ontology categories, and 20,202 unigenes were assigned to 25 Eukaryotic Ortholog Groups. Additionally, 19,508 unigenes were classified into 140 pathways of the Kyoto Encyclopedia of Genes and Genomes database. Based on the transcriptome, 163 unigenes associated with MYBs were detected. Among these genes, 75 genes were significantly expressed in the various tissues, including 10 R1 MYB, 42 R2R3 MYB, one R1R2R3 MYB, three R4 MYB and 19 atypical MYB-like proteins. The expression levels of the 12 MYB genes randomly selected for quantitative real-time PCR analysis corroborated the RNA sequencing results. A total of 37,545 microsatellites were detected, with an average expressed sequence tag–simple sequence repeat frequency of 0.59 (37,545/63,727). This transcriptome data improves our understanding of the role of MYB in R. roxburghii and will be valuable for identifying genes of interest.

Dihydroflavonol 4-reductase (DFR), a key regulatory enzyme, participated in the biosynthesis of anthocyanins, proanthocyanidins and other flavonoids that essential for plant survival and human health. However, the role of this enzyme in Ophiorrhiza japonica is still unknown. Here, three putative DFR-like genes were firstly isolated from O. japonica. Phylogenetic analysis indicated that OjDFR1 was classified into DFR subgroup, while the rest two were clustered into other NADPH-dependent reductases. Then, functions of the three genes were further characterized. Expression analysis showed that OjDFR1 transcripts had strong correlations with the accumulation pattern of anthocyanin during the flower developmental, whereas other two were not, this suggested the potential roles of OjDFR1 in anthocyanin biosynthesis. Subsequently, all three clones were functionally expressed in Escherichia coli, but confirming that only OjDFR1 encode active DFR proteins that catalyzed the reduction of dihydroflavonols to leucoanthocyanidin. Consistant with the biochemical assay results, overexpressing OjDFR1 in Arabidopsis tt3-1 mutant successfully restored the deficiency of anthocyanin and proanthocyanidin, hinting its function as DFR in planta. Additionally, heterologous expression of OjDFR1 in transgenic tobacco contributed to darker flower color via up-regulating the expressions of endogenous NtANS and NtUFGT, which suggested that OjDFR1 was involved in flower color development. In summary, this study validates the functions of OjDFR1 and expands our understanding of anthocyanin biosynthesis in O. japonica.

The current research was aimed to study the effects of different land use types (LUT) and soil depth (SD) on soil enzyme activity, metal content, and soil fungi in the karst area. Soil samples with depths of 0–20 cm and 20–40 cm were collected from different land types, including grassland, forest, Zanthoxylum planispinum land, Hylocereus spp. land and Zea mays land. The metal content and enzyme activity of the samples were determined, and the soil fungi were sequenced. The results showed that LUT had a significant effect on the contents of soil K, Mg, Fe, Cu and Cr; LUT and SD significantly affected the activities of invertase, urease, alkaline phosphatase and catalase. In addition, Shannon and Chao1 index of soil fungal community was affected by different land use types and soil depths. Ascomycota, Basidiomycota and Mortierellomycota were the dominant phyla at 0–20 cm and 20–40 cm soil depths in five different land types. Land use led to significant changes in soil fungal structure, while soil depth had no significant effect on soil fungal structure, probably because the small-scale environmental changes in karst areas were not the dominant factor in changing the structure of fungal communities. Additionally, metal element content and enzyme activity were related to different soil fungal communities. In conclusion, soil mineral elements content, enzyme activity, and soil fungal community in the karst area were strongly affected by land use types and soil depths. This study provides a theoretical basis for rational land use and ecological restoration in karst areas.