Kiwifruit Monodehydroascorbate Reductase 3 Gene Negatively Regulates the Accumulation of Ascorbic Acid in Fruit of Transgenic Tomato Plants.

Ascorbic acid is a potent antioxidant and a crucial nutrient for plants and animals. The accumulation of ascorbic acid in plants is controlled by its biosynthesis, recycling, and degradation. Monodehydroascorbate reductase is deeply involved in the ascorbic acid cycle; however, the mechanism of monodehydroascorbate reductase genes in regulating kiwifruit ascorbic acid accumulation remains unclear. Here, we identified seven monodehydroascorbate reductase genes in the genome of kiwifruit (Actinidia eriantha) and they were designated as AeMDHAR1 to AeMDHAR7, following their genome identifiers. We found that the relative expression level of AeMDHAR3 in fruit continued to decline during development. The over-expression of kiwifruit AeMDHAR3 in tomato plants improved monodehydroascorbate reductase activity, and, unexpectedly, ascorbic acid content decreased significantly in the fruit of the transgenic tomato lines. Ascorbate peroxidase activity also increased significantly in the transgenic lines. In addition, a total of 1781 differentially expressed genes were identified via transcriptomic analysis. Three kinds of ontologies were identified, and 106 KEGG pathways were significantly enriched for these differently expressed genes. Expression verification via quantitative real-time PCR analysis confirmed the reliability of the RNA-seq data. Furthermore, APX3, belonging to the ascorbate and aldarate metabolism pathway, was identified as a key candidate gene that may be primarily responsible for the decrease in ascorbic acid concentration in transgenic tomato fruits. The present study provides novel evidence to support the feedback regulation of ascorbic acid accumulation in the fruit of kiwifruit.

Three metabolic pathways are responsible for the accumulation and maintenance of high AsA content in kiwifruit (Actinidia eriantha).

BACKGROUND: Actinidia eriantha is a precious material to study the metabolism and regulation of ascorbic acid (AsA) because of its high AsA content. Although the pathway of AsA biosynthesis in kiwifruit has been identified, the mechanism of AsA metabolism and regulation is still unclear. The purpose of this experiment is to reveal the AsA metabolic characteristics of A. eriantha 'Ganmi 6' from the molecular level, and lay a theoretical foundation for the research on the genetic improvement of kiwifruit quality. RESULTS: We found that AsA reached the accumulation peak at S7 (110 DAF) during the process of fruit growth and development. The activity of GalDH, GalLDH, MDHAR and DHAR in fruit was similar to AsA accumulation trend, and both of them were significantly positively correlated with AsA content. It was speculated that GalDH and GalLDH were key enzymes in AsA biosynthesis, while MDHAR and DHAR were key enzymes in AsA regeneration cycle, which together regulated AsA accumulation in fruit. Also, we identified 98,656 unigenes with an average length of 932 bp from the transcriptome libraries using RNA-seq technology after data assembly. There were 50,184 (50.87%) unigenes annotations in four databases. Two thousand nine hundred forty-nine unigenes were enriched into the biosynthesis pathway of secondary metabolites, among which 133 unigenes involved in the AsA and aldehyde metabolism pathways, and 23 candidate genes related to AsA biosynthesis, cycling and degradation were screened out. CONCLUSIONS: Considering gene expression levels and changes of physiological traits and related enzyme activity, we concluded that the accumulation of AsA depends mainly on the L-galactose pathway, and the D-galacturonic acid pathway and AsA recycling pathway as the secondary pathways, which co-maintain the high AsA content in fruit of A. eriantha.

Genome-wide identification and comprehensive analysis of NAC family genes involved in fruit development in kiwifruit (Actinidia).

BACKGROUND: NAC transcription factors (TFs) are plant-specific proteins encoded by a large gene family. They play important roles in diverse biological processes, such as plant growth and development, leaf senescence, and responses to biotic or abiotic stresses. Functions of a number of NAC TFs have been identified mainly in model plants. However, very few studies on NAC TFs have been conducted in the fruit tree of kiwifruit. RESULTS: Genome-wide NAC genes were identified and their phylogeny, genomic structure, chromosomal location, synteny relationships, protein properties and conserved motifs were analyzed. In addition, the fruit developmental process was evaluated in a new kiwifruit cultivar of Actinidia eriantha 'Ganlu 1'. And expressions for all those NAC genes were analyzed by quantitative real-time PCR method in fruits of 'Ganlu 1' during its developmental process. Our research identified 142 NAC TFs which could be phylogenetically divided into 23 protein subfamilies. The genomic structures of those NAC genes indicated that their exons were between one and ten. Analysis of chromosomal locations suggested that 116 out of 142 NACs distributed on all the 29 kiwifruit chromosomes. In addition, genome-wide gene expression analysis showed that expressions of 125 out of 142 NAC genes could be detected in fruit samples. CONCLUSION: Our comprehensive study provides novel information on NAC genes and expression patterns in kiwifruit fruit. This research would be helpful for future functional identification of NAC genes involved in kiwifruit fruit development.

Predicting asymptomatic neurosyphilis using peripheral blood indicators.

BACKGROUND: The high misdiagnosis rate of asymptomatic neurosyphilis (ANS) has long challenged infectious disease clinicians. We aim to develop a model for diagnosing ANS in asymptomatic syphilis (AS) patients without CSF indicators. RESULTS: 277 AS patients with HIV-negative and underwent lumbar puncture were enrolled in this horizontal study.The area under the curve for predicting ANS by CSF leukocytes and protein was 0.643 and 0.675 [95% CI, 0.583-0.699VS.0.616-0.729]. Through LRM, the AUC increased to 0.806 [95% CI, 0.732-0.832], and the Youden's index was 0.430. If the score is ≤ 0.159, ANS can be excluded with a predictive value of 92.9%; we can identify ANS while the score is over 0.819, with a predictive value of 91.7% and a specificity of 99.25%. This study showed that the LRM can diagnose ANS in AS patients effectively. CONCLUSION: Given a large number of misdiagnosis ANS patients and CSF results' insufficiency, the model is more practical. Our research will help clinicians track suspected syphilis, especially those who cannot accept the CSF test.

Prolonged On-Vine vs. Cold of Actinidiaeriantha: Differences in Fruit Quality and Aroma Substances during Soft Ripening Stage.

In order to find an efficient, economical and feasible method for soft ripening storage of kiwifruit, two softening methods (on-vine, cold) were utilized for the 'Ganlv-2' kiwifruit (Actinidia. eriantha) cultivar. A comprehensive evaluation was conducted on the quality changes in 'Ganlv-2' under different methods after fruit ripening by principal component analysis and mathematical modeling. Compared to kiwifruit under cold softening, kiwifruit treated with on-vine soft ripening had slightly greater sugar-acid ratios and flesh firmness and higher contents of dry matter, soluble solids, and soluble sugar. The titratable acid content was slightly lower in the on-vine group than in the cold group. The sensory evaluation results manifested little difference in fruit flavor between the two groups. However, at the end of the trial, the overripe taste of the on-vine group was lighter and the taste was sweeter than those of the cold group. More aromatic substances were emitted from the kiwifruit in the on-vine group. According to the mathematic model, there was no significant difference in fruit quality and flavor between the on-vine and traditional cold groups. The fruit in the on-vine group had a stronger flavor and lighter overripe flavor when they reached the edible state. This paper provided a novel storage method of A. eriantha, it can reduce the cost of traditional cold storage and reduce the pressure on centralized harvesting, and the feasibility of this method was verified from the fruit quality.

Metabolome and Transcriptome Reveal Novel Formation Mechanism of Early Mature Trait in Kiwifruit (Actinidia eriantha).

Kiwifruit (Actinidia eriantha) is a peculiar berry resource in China, and the maturation period is generally late. Fortunately, we found an early mature A. eriantha germplasm. In order to explore the formation mechanism of its early mature trait, we determined the main carbohydrate and endogenous hormone content of the fruit, and used off-target metabolomics and transcriptomics to identify key regulatory metabolites and genes. We found that early mature germplasm had faster starch conversion rate and higher sucrose, glucose, and fructose content when harvested, while with lower auxin (IAA), abscisic acid (ABA), and zeatin (ZR) content. Through the non-targeted metabolome, 19 and 20 metabolites closely related to fruit maturity and early maturity were identified, respectively. At the same time, weighted correlation network analysis (WGCNA) showed that these metabolites were regulated by 73 and 99 genes, respectively, especially genes related to sugar metabolism were mostly. Based on above, the formation of early mature trait of A. eriantha was mainly due to the sucrose decomposition rate was reduced and the soluble solid content (SSC) accumulated at low levels of endogenous hormones, so as to reach the harvest standard earlier than the late mature germplasm. Finally, ten single nucleotide polymorphism (SNP) loci were developed which can be used for the identification of early mature trait of A. eriantha.

Differences of sucrose accumulation concentration and related genes expression between two sucrose accumulation types of Actinidia eriantha.

According to the investigation of wild Actinidia eriantha in Jiangxi province of China, we found that soluble solids content of fruit was lower than edible standard (14%). However, we found a high-sugar type A. eriantha line (code was 'MM24', test material) during investigative process at Nancheng county (E 116° 48', N 26° 23', 845 m). We sheared its scions to asexual reproduction in Fengxin County (rootstock was A. deliciosa 'Miliang 1' with 7 years old) and at the same time DUS (Distinctness, Uniformity and Stability) test was also carried out. There were uncontested differences between the two comparative genotypes according to the results of polyacrylamide gel electrophoresis, it can be judged as a new cultivar. In addition, there was great similarity on most important morphological and quality characteristics. While, there was difference on SSC, DM and TS between the two materials on ripen fruit, these indicators were much higher on test material than on control. The sugar degree assessment showed that the sugar degree of test material was strong and retention time was long. Further, no sucrose was found before DAF 135 d in test material and sucrose were significantly higher than in control only at DAF 165 d and DAF 175 d. The qRT-PCR results of sucrose-related genes showed that the relative expression levels of AcSPS1, AcSPS3, AcSPS5 and AcSUS5 genes were consistent with the sucrose accumulation trend, which was probably the main genes for the difference in sugar degree.