MdCoL, which encodes a putative 2OG-Fe(II) oxygenase, is a strong candidate gene for control of the columnar growth phenotype in apple. However, the mechanism by which MdCoL could produce the columnar trait is unclear. Here, we show that MdCoL influences ABA biosynthesis through its interactions with the MdDREB2 transcription factor. Expression analyses and transgenic tobacco studies confirmed that MdCoL is likely a candidate for control of the columnar phenotype. Furthermore, the ABA level in columnar apple trees is significantly higher than that in standard apple trees. A protein interaction experiment showed that MdCoL interacts with MdDREB2. Transient expression and EMSA assays demonstrated that MdDREB2 binds directly to the DRE motif in the MdNCED6 and MdNCED9 (MdNCED6/9) gene promoters, thereby activating the transcription of these ABA biosynthesis genes. In addition, a higher ABA content was detected following co-overexpression of MdCoL–MdDREB2 when compared with the overexpression of MdCoL or MdDREB2 alone. Taken together, our results indicate that an interaction between MdCoL and MdDREB2 promotes the expression of MdNCED6/9 and increases ABA levels, a phenomenon that may underlie the columnar growth phenotype in apple.

Jujube (Ziziphus jujuba Mill.) has attracted increasing attention because of its fruits’ high nutritional and medicinal value, which produce pentacyclic triterpenoids with valuable pharmacological activities beneficial to human health. However, the dynamic accumulation and metabolism pathway of triterpenoids remain unknown in jujube. Here, we performed metabolite assays of triterpenoids and expression analysis of genes involved in the corresponding metabolic processes on cultivated jujube (Z. jujuba cv. Junzao) and one type of wild jujube (Z. jujuba var. spinosa cv. Qingjiansuanzao). Our results showed that the triterpenoids accumulate predominantly in young leaves, annual stems, buds, and white-mature and beginning red stage fruit. Besides, the total triterpenoid content, ceanothic acid, oleanonic acid, and 3-ketoursolic acid were higher in ‘Qingjiansuanzao’ than in ‘Junzao’. Moreover, we found 23 genes involved in terpenoids metabolism were expressed in all organs, and the ZjSQS1, ZjCYP450/1, ZjCYP450/3, ZjOSC1, ZjFPS, and ZjAACT2 gene expression patterns were consistent with metabolites accumulation during fruit development. In addition, 100 μM MeJA induced ZjSQS1, ZjFPS, and ZjHMGR3 expression in leaves and enhanced triterpenoids accumulation. These findings will help understand the unique metabolism of terpenoids and will benefit further utilization and breeding of jujube as both edible fruit and functional food.

Triterpenoids are important, pharmacologically active substances in jujube (Ziziphus jujuba Mill.), and play an important role in the plant’s resistance to abiotic stress. However, regulation of their biosynthesis, and the underlying mechanism of their balance with stress resistance, remain poorly understood. In this study, we screened and functionally characterized the ZjWRKY18 transcription factor, which is associated with triterpenoid accumulation. The transcription factor is induced by methyl jasmonate and salicylic acid, and its activity was observed by gene overexpression and silencing experiments, combined with analyses of transcripts and metabolites. ZjWRKY18 gene silencing decreased the transcription of triterpenoid synthesis pathway genes and the corresponding triterpenoid content. Overexpression of the gene promoted the biosynthesis of jujube triterpenoids, as well as triterpenoids in tobacco and Arabidopsis thaliana. In addition, ZjWRKY18 binds to W-box sequences to activate promoters of 3-hydroxy-3-methyl glutaryl coenzyme A reductase and farnesyl pyrophosphate synthase, suggesting that ZjWRKY18 positively regulates the triterpenoid synthesis pathway. Overexpression of ZjWRKY18 also increased tolerance to salt stress in tobacco and Arabidopsis thaliana. These results highlight the potential use of ZjWRKY18 to improve triterpenoid biosynthesis and salt stress tolerance in plants, and provide a strong basis for metabolic engineering to improve the content of triterpenoids and breeding of jujube varieties that are resistant to stress.

No abstract

Jujube (Ziziphus jujuba Mill.) is rich in valuable bioactive triterpenoids. However, the regulatory mechanism underlying triterpenoid biosynthesis in jujube remains poorly studied. Here, we characterized the triterpenoid content in wild jujube and cultivated jujube. The triterpenoid content was higher in wild jujube than in cultivated jujube, triterpenoids were most abundant in young leaves, buds, and later stages of development. The transcriptome analysis and correlation analysis showed that differentially expressed genes (DEGs) were enriched in the terpenoid synthesis pathways, and triterpenoids content was strongly correlated with farnesyl diphosphate synthase (ZjFPS), squalene synthase (ZjSQS), and transcription factors ZjMYB39 and ZjMYB4 expression. Gene overexpression and silencing analysis indicated that ZjFPS and ZjSQS were key genes in triterpenoid biosynthesis and transcription factors ZjMYB39 and ZjMYB4 regulated triterpenoid biosynthesis. Subcellular localization experiments showed that ZjFPS and ZjSQS were localized to the nucleus and endoplasmic reticulum and ZjMYB39 and ZjMYB4 were localized to the nucleus. Yeast one-hybrid, glucuronidase activity, and dual-luciferase activity assays suggested that ZjMYB39 and ZjMYB4 regulate triterpenoid biosynthesis by directly binding and activating the promoters of ZjFPS and ZjSQS. These findings provide insights into the underlying regulatory network of triterpenoids metabolism in jujube and lay theoretical and practical foundation for molecular breeding.