ZjHXK5 and ZjHXK6 negatively regulate the sugar metabolism of Ziziphus jujuba Mill.

Hexokinase (HXK) plays a crucial role in plants, catalyzing the phosphorylation of hexose substances, which is one of the key steps in sugar metabolism and energy production. While HXK genes have been well-studied in model plants, the evolutionary and functional characteristics of HXK gene family in jujube is unknow. In this study, the HXK gene family members were identified by bioinformatics methods, the key members regulating glucose metabolism were identified by transcriptome data, and finally the function of the key genes was verified by instantaneous and stable genetic transformation. Our results showed that seven HXK genes were identified in the jujube genome, all of which were predict located in the chloroplast and contain Hexokinase-1 (PF00349) and Hexokinase-2 (PF03727) conserved domains. Most of HXK proteins were transmembrane protein with stable, lipid-soluble, hydrophilic. The secondary structure of ZjHXK proteins main α-helix, and contains two distinct tertiary structure. All ZjHXK genes contain nine exons and eight introns. Predictions of cis-regulatory elements indicate that the promoter region of ZjHXK contains a large number of MeJA responsive elements. Finally, combined with the analysis of the relationship between the expression and glucose metabolism, found that ZjHXK5 and ZjHXK6 may the key genes regulating sugar metabolism. Transient overexpression of ZjHXK5 and ZjHXK6 on jujube, or allogeneic overexpression of ZjHXK5 and ZjHXK6 on tomato would significantly reduce the content of total sugar and various sugar components. Transient silencing of ZjHXK5 and ZjHXK6 genes results in a significant increase in sucrose and total sugar content. Interestingly, the expression of ZjHXK5 and ZjHXK6 were also affected by methyl jasmonate.

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.

L-Ascorbic acid metabolism and regulation in fruit crops.

L-Ascorbic acid (AsA) is more commonly known as vitamin C and is an indispensable compound for human health. As a major antioxidant, AsA not only maintains redox balance and resists biological and abiotic stress but also regulates plant growth, induces flowering, and delays senescence through complex signal transduction networks. However, AsA content varies greatly in horticultural crops, especially in fruit crops. The AsA content of the highest species is approximately 1,800 times higher than that of the lowest species. There have been significant advancements in the understanding of AsA accumulation in the past 20 years. The most noteworthy accomplishment was the identification of the critical rate-limiting genes for the 2 major AsA synthesis pathways (L-galactose pathway and D-galacturonic acid pathway) in fruit crops. The rate-limiting genes of the former are GMP, GME, GGP, and GPP, and the rate-limiting gene of the latter is GalUR. Moreover, APX, MDHAR, and DHAR are also regarded as key genes in degradation and regeneration pathways. Interestingly, some of these key genes are sensitive to environmental factors, such as GGP being induced by light. The efficiency of enhancing AsA content is high by editing upstream open reading frames (uORF) of the key genes and constructing multi-gene expression vectors. In summary, the AsA metabolism has been well understood in fruit crops, but the transport mechanism of AsA and the synergistic improvement of AsA and other traits is less known, which will be the focus of AsA research in fruit crops.

Genome-wide identification and expression profiling analysis of sucrose synthase (SUS) and sucrose phosphate synthase (SPS) genes family in Actinidia chinensis and A. eriantha.

Sucrose synthase (SUS) is a common sugar-base transfer enzyme in plants, and sucrose phosphate synthase (SPS) is one of the major enzymes in higher plants that regulates sucrose synthesis. However, information of the SPS and SUS gene families in Actinidia, as well as their evolutionary and functional properties, is limited. According to the SPS and SUS proteins conserved domain of Arabidopsis thaliana, we found 6 SPS genes and 6 SUS genes from A. chinensis (cultivar: 'Hongyang'), and 3 SPS genes and 6 SUS genes from A. eriantha (cultivar: 'White'). The novel CDC50 conserved domains were discovered on AcSUS2, and all members of the gene family contain similar distinctive conserved domains. The majority of SUS and SPS proteins were hydrophilic, lipid-soluble enzymes that were expected to be found in the cytoplasm. The tertiary structure of SPS and SUS protein indicated that there were many tertiary structures in SPS, and there were windmill-type and spider-type tertiary structures in SUS. The phylogenetic tree was created using the neighbor-joining method, and members of the SPS and SUS gene families are grouped into three subgroups. Genes with comparable intron counts, conserved motifs, and phosphorylation sites were clustered together first. SPS and SUS were formed through replication among their own family members. AcSPS1, AcSPS2, AcSPS4, AcSPS5, AcSUS5, AcSUS6, AeSPS3, AeSUS3 and AeSUS4 were the important genes in regulating the synthesis and accumulation of sucrose for Actinidia during the fruit growth stages.

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.

Genome-Wide Association Studies Provide Insights into the Genetic Determination of Flower and Leaf Traits of Actinidia eriantha.

Kiwifruit (Actinidia eriantha) is a dioecious vine, and the pollen of its male cultivars has a direct effect on the quality of its fruits. In this study, to facilitate molecular breeding and gene identification, we performed genome-wide association studies (GWAS) on 11 traits of flower and leaf. A total of 946,337 highly consistent SNP markers were obtained in the whole genome. Phylogenetic tree analysis and population structure analysis showed that the 143 germplasms can be divided into two groups. The linkage disequilibrium analysis showed that A. eriantha have a relatively fast attenuation rate, and that the average attenuation distance of LD was 0.1-0.3 Kb. The MLM (QK) model was determined as best for correlation analysis, and eight and three SNPs associated with flower- and leaf-related traits were identified, respectively, at 0.01 significance level. However, SNP markers associated with stamen number per flower, pollen viability, total chlorophyll content, and total flavonoid content were not identified at the 0.01 significant level, although it is worth noting that one, one, five, and two SNPs were identified to be associated with these traits at the 0.05 significant level. This study provides insights into the complex flower- and leaf-related biology, and identifies genes controlling important traits in A. eriantha through GWAS, which extends the genetic resources and basis for facilitating molecular breeding in kiwifruits.

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.

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.

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.

Identification and expression profiling analysis of ascorbate peroxidase gene family in Actinidia chinensis (Hongyang).

Ascorbate peroxidase (APX) is one of the important antioxidant enzymes in the active oxygen metabolism pathway of plants and animals, especially it is the key enzyme to clear H2O2 in chloroplast and the main enzyme of vitamin C metabolism. However, knowledge about APX gene family members and their evolutionary and functional characteristics in kiwifruit is limited. In this study, we identified 13 members of the APX gene family in the kiwifruit (cultivar: Hongyang) genome according the APX proteins conserved domain of Arabidopsis thaliana. Phylogenetic analysis by maximum likelihood split these 13 genes into four groups. The APX gene family members were distributed on nine chromosomes (Nos. 4, 5, 11, 13, 20, 21, 23, 25, 28). Most of the encoded hydrophilic and lipid-soluble enzymes were predicted to be located in the cytoplasm, nucleus and chloroplast. Among them, AcAPX4, AcAPX5, AcAPX8, AcAPX12 were transmembrane proteins, and AcAPX8 and AcAPX12 had the same transmembrane domain. The gene structure analysis showed that AcAPXs were composed of 4-22 introns, except that AcAPX10 was intron-free. Multiple expectation maximization for motif elicitation program (MEME) analyzed 13 APX protein sequences of Actinidia chinensis and identified 10 conserved motifs ranging in length from 15 to 50 amino acid residues. Additionally, the predicted secondary structures of the main motifs consisted of α-helix and random coils. The gene expression of fruits in different growth stages and bagging treatment were determined by qRT-PCR. The results showed that 8 AcAPXs had the highest expression levels during the color turning period and only the gene expression of AcAPX3 was consistent with the ascorbic acid content; five AcAPXs were consistent with the ascorbic acid content after bagging. Our data provided evolutionary and functional information of AcAPX gene family members and revealed the gene expression of different members in different growth stages and bagging treatments These results may be useful for future studies of the structures and functions of AcAPX family members.

Characterization of Organic Acid Metabolism and Expression of Related Genes During Fruit Development of Actinidia eriantha 'Ganmi 6'.

Studies on organic acid metabolism have been mainly concentrated on the fruit, whereas, few have focused on the mechanism of high organic acids content in the fruit of Actinidia eriantha. Fruits of 'Ganmi 6' harvested at eleven developmental periods were used as materials. The components and content of organic acids were determined by high-performance liquid chromatography (HPLC) system, the activities of the related enzyme were detected, and gene expression levels were measured by quantitative real-time PCR (qRT-PCR). Components of ascorbic acid (AsA) and eight kinds of organic acids were detected. These results showed that quinic acid and citric acid were the main organic acids in the fruit of 'Ganmi 6'. Correlation analysis showed that NADP-Quinate dehydrogenase (NADP-QDH), NADP-Shikimate dehydrogenase (NADP-SDH), and Cyt-Aconitase (Cyt-Aco) may be involved in regulating organic acids biosynthesis. Meanwhile, the SDH gene may play an important role in regulating the accumulation of citric acid. In this study, the activities of NADP-SDH, Mit-Aconitase (Mit-Aco), and NAD-Isocitrate dehydrogenase (NAD-IDH) were regulated by their corresponding genes at the transcriptional level. The activity of Citrate synthase (CS) may be affected by post-translational modification. Our results provided new insight into the characteristics of organic acid metabolism in the fruit of A. eriantha.