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.

Luminescence method for detection of aflatoxin B1 using ATP-releasing nucleotides.

Determination of aflatoxin B1 (AFB1) is still a big issue in food safety. In this paper, we developed a luminescence AFB1 detection method combined with ATP-releasing nucleotides (ARNs) and AFB1 aptamer. Firstly, using a new coupling method, we synthesized two ARNs (dTP4A and dGP4A) in a yield of 67% and 58%, respectively. The newly prepared ARNs show a much lower background. Then, we developed a new isothermal polymerase amplification method. In this method, two DNA hairpins were used to substitute the circle DNA template in rolling circle amplification. Using this amplification method and combined with AFB1 aptamer, a new AFB1 detection method is developed. A detection limit as low as 0.3 pM is achieved. This method is simple and efficient, and will have a great potential to be used for food safety and public health.

Differences among the Anthocyanin Accumulation Patterns and Related Gene Expression Levels in Red Pears.

Differences in coloration exist among red pear cultivars. Here, we selected six red pear cultivars with different genetic backgrounds to elucidate the characteristics of fruit pigmentation. We detected anthocyanin contents and the expression levels of anthocyanin synthesis-related genes in these cultivars at different stages of fruit development. The anthocyanin contents of all six cultivars showed a rise-drop tendency. Principal component and hierarchical cluster analyses were used to distinguish the types of cultivars and the genes crucial to each anthocyanin accumulation pattern. The six cultivars were divided into three groups. Red Zaosu were clustered into one group, Red Sichou and Starkrimson into another group, and Palacer, Red Bartlett, and 5 Hao clustered into a third group. The expression levels of F3H, UFGT2, MYB10, and bHLH3 were similar among the differential coloration patterns of the six cultivars, suggesting a critical and coordinated mechanism for anthocyanin synthesis. Anthocyanin transporters (GST) and light-responsive genes, such as COP1, PIF3.1, and PIF3.2 played limited roles in the regulation of anthocyanin accumulation. This study provides novel insights into the regulation of anthocyanins synthesis and accumulation in red pears.

PbCOP1.1 Contributes to the Negative Regulation of Anthocyanin Biosynthesis in Pear.

The synthesis of anthocyanin in pear (Pyrus bretschneideri) fruit is regulated by light. However, little is known about the molecular mechanisms of pear fruit coloring mediated by upstream light-signaling regulators. Here, the photoresponse factors CONSTITUTIVE PHOTOMORPHOGENIC (COP) 1.1 and 1.2 were cloned from 'Red Zaosu' peel to study their functions in pear fruit coloring. The overexpression vectors pBI121-PbCOP1.1 and pBI121-PbCOP1.2 were constructed to analyze their effects on anthocyanin synthesis in pear fruit. A protein sequence alignment and phylogenetic tree analysis revealed that PbCOP1 proteins are highly homologous with those of other species. An analysis of tissue differential expression showed that the greatest expression levels of PbCOP1s occurred in the leaves. Their expression levels increased in the leaves during development, when the leaves changed from red to green. The overexpression of PbCOP1s in the peel resulted in reduced anthocyanin synthesis at the injection sites. A quantitative PCR analysis of the injection sites showed that PbCOP1.1 significantly inhibited the expression of the anthocyanin synthesis-related genes CHI, DFR, UFGT2, bHLH3, HY5 and GST. Based on the above results, we hypothesize that PbCOP1.1 is an anthocyanin synthetic inhibitory factor of pear coloration.

2,4-D-induced parthenocarpy in pear is mediated by enhancement of GA4 biosynthesis.

Parthenocarpy, the productions of seedless fruit without pollination or fertilization, is a potentially desirable trait in many commercially grown fruits, especially in pear, which is self-incompatible. Phytohormones play important roles in fruit set, a process crucial for parthenocarpy. In this study, 2,4-dichlorophenoxyacetic acid (2,4-D), an artificially synthesized plant growth regulator with functions similar to auxin, was found to induce parthenocarpy in pear. Histological observations revealed that 2,4-D promoted cell division and expansion, which increased cortex thickness, but the effect was weakened by paclobutrazol (PAC), a gibberellin (GA) biosynthesis inhibitor. Phenotypic differences in pear may therefore be due to different GA contents. Hormone testing indicated that 2,4-D mainly induced the production of bioactive GA4 , rather than GA3. Three key oxidase genes function in the GA biosynthetic pathway: GA20ox, GA3ox and GA2ox. In a pear group treated with only 2,4-D, PbGA20ox2-like and PbGA3ox-1 were significantly upregulated. When treated with 2,4-D supplemented with PAC, however, expression levels of these genes were significantly downregulated. Additionally, PbGA2ox1-like and PbGA2ox2-like expression levels were significantly downregulated in pear treated with either 2,4-D only or 2,4-D supplemented with PAC. We thus hypothesize that 2,4-D can induce parthenocarpy by enhancing GA4 biosynthesis.