Transcription Factors ZjMYB39 and ZjMYB4 Regulate Farnesyl Diphosphate Synthase- and Squalene Synthase-Mediated Triterpenoid Biosynthesis in Jujube.

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.

Methyl Jasmonate- and Salicylic Acid-Induced Transcription Factor ZjWRKY18 Regulates Triterpenoid Accumulation and Salt Stress Tolerance in Jujube.

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.

Identification of the key genes contributing to the LOX-HPL volatile aldehyde biosynthesis pathway in jujube fruit.

Jujube (Ziziphus jujuba Mill.) is a traditional popular fruit widely grown in China. The volatiles in jujube determine its unique flavor and the high fruit quality required by consumers. However, the biosynthesis of volatiles in jujube were remain unknown. By using gas chromatography-mass spectrometry, there were 46 volatile compounds were identified and determined from three representative jujube fruit types at six developmental stages, including the dry-used (Z. jujuba cv. 'Junzao'), the fresh-used (Z. jujuba cv. 'Dongzao'), and wild jujube (Z. jujuba var. spinosa Hu. cv. 'Qingjiansuanzao'). The aldehydes were identified as major volatile contributors to flavor, of which (E)-2-hexenal was the primary volatile in jujube fruit. Then LOX and HPL gene family were identified in jujube, which were involved in aldehyde biosynthesis through the lipoxygenase-hydroperoxide lyase (LOX-HPL) pathway. Gene expression analysis suggested that ZjLOX3, ZjLOX4, and ZjHPL1 were highly correlated with the accumulation of (E)-2-hexenal, and their proteins were localized to the nucleus and cytoplasm. Transient over-expression of ZjLOX3, ZjLOX4, and ZjHPL1 in jujube fruit significantly enhanced the accumulation of (E)-2-hexenal. Our study provides valuable information on the major volatiles and their biosynthesis in different types of jujube fruit. These results will help determine flavor improvements for future breeding.

Metabolite and Gene Expression Analysis Underlying Temporal and Spatial Accumulation of Pentacyclic Triterpenoids in Jujube.

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.

The apple columnar gene candidate MdCoL and the AP2/ERF factor MdDREB2 positively regulate ABA biosynthesis by activating the expression of MdNCED6/9.

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 produces the columnar trait is unclear. Here, we show that MdCoL influences abscisic acid (ABA) biosynthesis through its interactions with the MdDREB2 transcription factor. Expression analyses and transgenic tobacco studies have 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 has showed that MdCoL interacts with MdDREB2. Transient expression and electrophoretic mobility shift assays have 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 has been 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.

Optimization of internals for Selective Catalytic Reduction (SCR) for NO removal.

This work tried to identify the relationship between the internals of selective catalytic reduction (SCR) system and mixing performance for controlling ammonia (NH(3)) slip. In the SCR flow section, arranging the flow-guided internals can improve the uniformity of velocity distribution but is unfavorable for the uniformity of NH(3) concentration distribution. The ammonia injection grids (AIG) with four kinds of nozzle diameters (i.e., 1.0 mm, 1.5 mm, 2.0 mm, and mixed diameters) were investigated, and it was found that the AIG with mixed nozzle diameters in which A3, A4, B3, and B4 nozzles' diameters are 1.0 mm and other nozzles' diameters are 1.5 mm is the most favorable for the uniformity of NH(3) concentration distribution. In the SCR reactor section, the appropriate space length between two catalyst layers, which serves as gas mixing in order to prevent maldistribution of gas concentrations into the second catalyst layer, under the investigated conditions is about 100, 1000, and 12 mm for honeycomb-like cordierite catalyst, plate-type catalysts with parallel channel arrangement, and with cross channel arrangement, respectively. Therefore, the cross channel arrangement is superior to the parallel channel arrangement in saving the SCR reactor volume.