L-Ascorbic acid (AsA) is more commonly known as vitamin C, and an indispensable compound for human health. As a major antioxidant, AsA can not only maintain redox balance, resist biological and abiotic stress, but also regulate plant growth, induce flowering and delay senescence through complex signal transduction networks. However, AsA content varied greatly in horticultural crops, especially in fruit crops. The AsA content of the highest specie is about 10,000 times higher than that of the lowest specie. There have been significant advancements in the understanding of AsA accumulation in the recent twenty years. The most noteworthy accomplishment was the identification of the critical rate-limiting genes for the two major AsA synthesis pathway (L-galactose pathway and D-galacturonic acid) in fruit crops. The rate-limiting genes of the former were GMP, GME, GGP and GPP, while the rate-limiting genes of the latter was GalUR. Moreover, APX, MDHAR and DHAR were also regarded as key genes in degradation and regeneration pathways. Interestingly, some of these key genes were sensitive to environmental factors, such as GGP induced by light. The efficiency of enhancing AsA content was high by editing 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.

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.

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.

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-wideNACgenes 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 ofActinidia eriantha‘Ganlu 1’. And expressions for all thoseNACgenes 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 thoseNACgenes indicated that their exons were between one and ten. Analysis of chromosomal locations suggested that 116 out of 142NACs distributed on all the 29 kiwifruit chromosomes. In addition, genome-wide gene expression analysis showed that expressions of 125 out of 142NACgenes could be detected in fruit samples.Conclusion: Our comprehensive study provides novel information onNACgenes and expression patterns in kiwifruit fruit. This research would be helpful for future functional identification ofNACgenes involved in kiwifruit fruit development.

The widespread ascorbic acid (AsA) plays a vital role in plant development and abiotic stress tolerance, but AsA concentration varies greatly among different plants. GDP-D-mannose epimerase (GME), which catalyzes GDP-D-mannose to GDP-L-galactose or GDP-L-gulose, is a key enzyme in plant AsA biosynthesis pathway. Functions and expression patterns of GME have been well studied in previous works, however, little information is known about the evolutionary patterns of the gene. In this study, GME gene structure, corresponding conserved protein motifs and evolutionary relationships were systematically analyzed. A total of 111 GME gene sequences were retrieved from 59 plant genomes, which representing almost all the major lineages of Viridiplantae: dicotyledons, monocotyledons, gymnosperms, pteridophytes, bryophytes, and chlorophytes. Results showed that homologs of GME were widely present in Viridiplantae. GME gene structures were conservative in higher plants, while varied greatly in the basal subgroups of the phylogeny including lycophytes, bryophytes, and chlorophytes, suggesting GME gene structure might have undergone severe differentiation at lower plant and then gradually fixed as plant evolution. The basic motifs of GME were strongly conserved throughout Viridiplantae, suggesting the conserved function of the protein. Molecular evolution analysis showed that strong purifying selection was the predominant force in the evolution of GME. A few branches and sites under episodic diversifying selection were identified and most of the branches located in the subgroup of chlorphytes, indicating episodic diversifying selection at a few branches and sites may play a role in the evolution of GME and diversifying selection may have occurred at the early stage of Viridiplantae. Our results provide novel insights into functional conservation and the evolution of GME.