Efficient extraction of pectic polysaccharides from thinned unripe kiwifruits by deep eutectic solvent-based methods: Chemical structures and bioactivities.

To promote the potentially industrial applications of thinned unripe kiwifruits, two deep eutectic solvent-based methods, including deep eutectic solvent-assisted extraction (DAE) and microwave-assisted deep eutectic solvent extraction (MDE), were optimized for the extraction of polysaccharides from thinned unripe kiwifruits (YKP). Results showed that the yields of YKP-D prepared by DAE and YKP-DM prepared by MDE were extremely higher than YKP-H prepared by hot water extraction. Furthermore, YKP-H, YKP-D, and YKP-DM were mainly composed of pectic polysaccharides, including homogalacturonan (HG) and rhamnogalacturonan I (RG I) domains. Besides, both YKP-D and YKP-DM exhibited stronger antioxidant, anti-glycosylation, and immunomodulatory effects than those of YKP-H, and their higher contents of uronic acids and bound polyphenols as well as lower molecular weights could partially contribute to their bioactivities. Overall, these results revealed that the developed MDE method could be utilized as a promising method for highly efficient extraction of YKP with superior beneficial effects.

Pseudomonas syringae Type III Secretion Protein HrpP Manipulates Plant Immunity To Promote Infection.

The bacterial plant pathogen Pseudomonas syringae deploys a type III secretion system (T3SS) to deliver effector proteins into plant cells to facilitate infection, for which many effectors have been characterized for their interactions. However, few T3SS Hrp (hypersensitive response and pathogenicity) proteins from the T3SS secretion apparatus have been studied for their direct interactions with plants. Here, we show that the P. syringae pv. tomato DC3000 T3SS protein HrpP induces host cell death, suppresses pattern-triggered immunity (PTI), and restores the effector translocation ability of the hrpP mutant. The hrpP-transgenic Arabidopsis lines exhibited decreased PTI responses to flg22 and elf18 and enhanced disease susceptibility to P. syringae pv. tomato DC3000. Transcriptome analysis reveals that HrpP sensing activates salicylic acid (SA) signaling while suppressing jasmonic acid (JA) signaling, which correlates with increased SA accumulation and decreased JA biosynthesis. Both yeast two-hybrid and bimolecular fluorescence complementation assays show that HrpP interacts with mitogen-activated protein kinase kinase 2 (MKK2) on the plant membrane and in the nucleus. The HrpP truncation HrpP1-119, rather than HrpP1-101, retains the ability to interact with MKK2 and suppress PTI in plants. In contrast, HrpP1-101 continues to cause cell death and electrolyte leakage. MKK2 silencing compromises SA signaling but has no effect on cell death caused by HrpP. Overall, our work highlights that the P. syringae T3SS protein HrpP facilitates effector translocation and manipulates plant immunity to facilitate bacterial infection. IMPORTANCE The T3SS is required for the virulence of many Gram-negative bacterial pathogens of plants and animals. This study focuses on the sensing and function of the T3SS protein HrpP during plant interactions. Our findings show that HrpP and its N-terminal truncation HrpP1-119 can interact with MKK2, promote effector translocation, and manipulate plant immunity to facilitate bacterial infection, highlighting the P. syringae T3SS component involved in the fine-tuning of plant immunity.

De Novo Assembly of a Sarcocarp Transcriptome Set Identifies AaMYB1 as a Regulator of Anthocyanin Biosynthesis in Actinidia arguta var. purpurea.

The kiwifruit (Actinidia arguta var. purpurea) produces oval shaped fruits containing a slightly green or mauve outer exocarp and a purple-flesh endocarp with rows of tiny black seeds. The flesh color of the fruit results from a range of anthocyanin compounds, and is an important trait for kiwifruit consumers. To elucidate the molecular mechanisms involved in anthocyanin biosynthesis of the sarcocarp during A. arguta fruit development, de novo assembly and transcriptomic profile analyses were performed. Based on significant Gene Ontology (GO) biological terms, differentially expressed genes were identified in flavonoid biosynthetic and metabolic processes, pigment biosynthesis, carbohydrate metabolic processes, and amino acid metabolic processes. The genes closely related to anthocyanin biosynthesis, such as phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and anthocyanidin synthase (ANS), displayed significant up-regulation during fruit development according to the transcriptomic data, which was further confirmed by qRT-PCR. Meanwhile, a series of transcription factor genes were identified among the DEGs. Through a correlation analysis. AaMYB1 was found to be significantly correlated with key genes of anthocyanin biosynthesis, especially with CHS. Through a transient expression assay, AaMYB1 induced anthocyanin accumulation in tobacco leaves. These data provide an important basis for exploring the related mechanisms of sarcocarp anthocyanin biosynthesis in A. arguta. This study will provide a strong foundation for functional studies on A. arguta and will facilitate improved breeding of A. arguta fruit.

Ultrasound-Assisted Extraction, Identification, and Quantification of Antioxidants from 'Jinfeng' Kiwifruit.

Kiwifruit (Actinidia chinensis) is a nutrient-dense fruit abundant in vitamin C and phenolic compounds, and it exhibits strong antioxidant capacity. However, the antioxidants in 'Jinfeng' kiwifruit have seldom been extracted and analyzed, and the conditions for the extraction of kiwifruit antioxidants by ultrasound-assisted extraction (UAE) have seldom been investigated. In this study, response surface methodology (RSM) was used to optimize UAE conditions to extract antioxidants from 'Jinfeng' kiwifruit. In addition, the antioxidant capacity, contents of total phenolics and total flavonoids, ascorbic acid, and the profiles of antioxidants were also analyzed. The results showed that the optimal UAE conditions included 68% ethanol, liquid/solid ratio at 20 mL/g, extraction time at 30 min, extraction temperature at 42 °C, and ultrasonic power at 420 W. Under these conditions, the ABTS value of kiwifruit was 70.38 ± 1.38 µM TE/g DW, which was 18.5% higher than that of the extract obtained by conventional solvent extraction. The total phenolic and flavonoid contents were 15.50 ± 0.08 mg GAE/g DW and 5.10 ± 0.09 mg CE/g DW, respectively. Moreover, 20 compounds were tentatively identified by UPLC-MS/MS, and the content of main compounds, such as procyanidin B2, neochlorogenic acid, and epicatechin, were determined by HPLC-DAD. This research revealed the profiles of antioxidant phytochemicals in 'Jinfeng' kiwifruit, which can be a good dietary source of natural antioxidants with potential health functions.

The comparative studies of complete chloroplast genomes in Actinidia (Actinidiaceae): novel insights into heterogenous variation, clpP gene annotation and phylogenetic relationships.

The genus Actinidia, also called kiwifruit, is characterized with abundant balanced nutritional metabolites, including exceptionally high vitamin C content. However, the traditional classification could not fully reflect the actual Actinidia species' relationships, which need further revision through more accurate approaches. Compared to the nuclear genome, the chloroplast genome has simple heredity characteristics, conserved genome structure and small size, suitable for deciphering complicated species' phylogenetic relationships. Here, the genome-wide comprehensive comparative analyses were performed over 29 independent chloroplast genomes' sequences derived from 25 Actinidia taxa. The average genome size is 156,673.38 bp, with an average 37.20% GC content. The long repeat sequences rather than SSRs (simple sequence repeats) in Actinidia were revealed to be the causal agent leading to the chloroplast genome size expansion. The clpP gene sequences with exon merge and intron deletion were annotated in all the 29 chloroplast genomes tested, which has been previously reported to be lost in Actinidia species. Comprehensive sequence analyses indicated the distinct variation at the clpP gene locus was Actinidiaceae-specific, emerging after the Actinidiaceae-other Ericales species divergence. Four highly divergent sequences (i.e., rps16 ~ trnQ-UUG, rps4 ~ trnT-UGU, petA ~ psbJ, and rps12 ~ psbB) evolved in the LSC (large single-copy) and SSC (small single-copy) regions embodying rps12 ~ psbB (including clpP gene and its up/downstream noncoding sequence) were identified as variation hot spots in Actinidia species. Based on either LSC region alone, combined sequences of LSC and SSC or the whole chloroplast genome sequences, three identical phylogenetic trees of the 25 Actinidia taxa with relatively improved resolution were reconstructed, consistently supporting the reticulate evolutionary lineage in Actinidia. Our findings could help to better understand the evolution characteristics of chloroplast genomes and phylogenetic relationships among Actinidia species.

Recent development in zebrafish model for bioactivity and safety evaluation of natural products.

The zebrafish is a species of freshwater fish, popular in aquariums and laboratories. Several advantageous features have facilitated zebrafish to be extensively utilized as a valuable vertebrate model in the lab. It has been well-recognized that natural products possess multiple health benefits for humans. With the increasing demand for natural products in the development of functional foods, nutraceuticals, and natural cosmetics, the zebrafish has emerged as an unprecedented tool for rapidly and economically screening and identifying safe and effective substances from natural products. This review first summarized the key factors for the management of zebrafish in the laboratory, followed by highlighting the current progress on the establishment and applications of zebrafish models in the bioactivity evaluation of natural products. In addition, the zebrafish models used for assessing the potential toxicity or health risks of natural products were involved as well. Overall, this review indicates that zebrafish are promising animal models for the bioactivity and safety evaluation of natural products, and zebrafish models can accelerate the discovery of novel natural products with potential health functions.

Phenolic Content, Main Flavonoids, and Antioxidant Capacity of Instant Sweet Tea (Lithocarpus litseifolius [Hance] Chun) Prepared with Different Raw Materials and Drying Methods.

This study aims to investigate the effects of raw materials and drying methods on the phytochemical and antioxidant capacities of instant sweet tea powder. Four raw materials of sweet tea leave powders (STUT) were extracted and dried with two methods (freeze-drying and spray-drying). The antioxidant capacity, total phenolic content (TPC), total flavonoid content (TFC), and phlorizin and trilobatin contents of obtained instant sweet tea powders were compared. In addition, the single-factor experiments coupled with response surface methodology were used to study the influences of solvent-to-sample ratio, extraction temperature, extraction time, and their interactions on instant sweet tea yield. Results showed that the optimal conditions for extraction were the solvent-to-sample ratio of 19:1 mL/g, extraction temperature of 88 °C, and extraction time of 30 min. The TPC, TFC, antioxidant capacities, and phloridzin and trilobatin contents of instant sweet teas were higher than those of STUT, and the TPC and TFC of freeze-dried instant sweet teas were higher than those of spray-dried instant sweet teas. Significant correlations were found among TPC, TFC, and antioxidant capacities (p < 0.01). The freeze-dried instant sweet tea produced by young leaves (prepared by oven-drying) showed the highest TPC, TFC, and antioxidant capacities, compared with other raw materials and drying methods.

Comparison of fruit morphology and nutrition metabolism in different cultivars of kiwifruit across developmental stages.

Kiwifruit (Actinidia) is becoming increasingly popular worldwide due to its favorable flavour and high vitamin C content. However, quality parameters vary among cultivars. To determine the differences in quality and metabolic parameters of kiwifruit, we monitored the growth processes of 'Kuilv' (Actinidia arguta), 'Hongyang' (Actinidia chinensis) and 'Hayward' (Actinidia deliciosa). We found that 'Kuilv' required the shortest time for fruit development, while 'Hayward' needed the longest time to mature. The fruit size of 'Hayward' was the largest and that of 'Kuilv' was the smallest. Furthermore, 'Hongyang' showed a double-S shape of dry matter accumulation, whereas 'Kuilv' and 'Hayward' showed a linear or single-S shape pattern of dry matter accumulation during development. The three cultivars demonstrated the same trend for total soluble solids accumulation, which did not rise rapidly until 90-120 days after anthesis. However, the accumulation of organic acids and soluble sugars varied among the cultivars. During later fruit development, the content of glucose, fructose and quinic acid in 'Kuilv' fruit was far lower than that in 'Hongyang' and 'Hayward'. On the contrary, 'Kuilv' had the highest sucrose content among the three cultivars. At maturity, the antioxidative enzymatic systems were significantly different among the three kiwifruit cultivars. 'Hongyang' showed higher activities of superoxide dismutase than the other cultivars, while the catalase content of 'Hayward' was significantly higher than that of 'Hongyang' and 'Kuilv'. These results provided knowledge that could be implemented for the marketing, handling and post-harvest technologies of the different kiwifruit cultivars.

Complete genome sequencing of Pseudomonas syringae pv. actinidiae Biovar 3, P155, kiwifruit pathogen originating from China / Sequenciamento completo do genoma Pseudomonas syringae pv. actinidiae Biovar 3, P155, agente patogênico do kiwi, originário da China

Pseudomonas syringae pv. actinidiae is a bacterial pathogen of kiwifruit. Based on the results of the pathogenicity assay, we sequenced the strain Pseudomonas syringae (Psa3) P155 which possesses a series of virulence and resistance genes, CRISPR candidate elements, prophage related sequences, methylation modifications, genomic islands as well as one plasmid. Most importantly, the copper resistance genes copA, copB, copC, copD, and copZ as well as aminoglycoside resistance gene ksgA were identified in strain P155, which would pose a threat to kiwifruit production. The complete sequence we reported here will provide valuable information for a better understanding of the genetic structure and pathogenic characteristics of the genome of P155.

Pseudomonas syringae pv. actinidiae agente causal do cancro bacteriano do kiwi. Com base nos resultados do teste de patogenicidade, foi sequenciado um isolado de Pseudomonas syringae (Psa3) P155, que abriga a uma série de genes de virulência e resistência, elementos candidatos CRISPR, sequências relacionadas a profagos, modificações na metilação, ilhas genômicas, e também um plasmídeo. O mais importante foram os genes de resistência ao cobre, copA, copB, copC, copD e copZ, bem como, o gene de resistência aminoglicosídea ksgA identificados na estirpe P155, os quais representariam uma ameaça à produção de kiwi. A sequência completa relatada fornecerá informações valiosas para uma melhor compreensão da estrutura genética e as características patogênicas do genoma de P155.

Correlation between fruit weight and nutritional metabolism during development in CPPU-treated Actinidia chinensis 'Hongyang'.

Forchlorfenuron, N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU), is often used to promote fruit growth and improve production. The role of CPPU in kiwifruit growth has been established. However, the correlation between fruit weight and nutritional metabolism during development after CPPU treatments remains largely undetermined. Here, we surveyed the variations in weight and nutrient components of the 'Hongyang' kiwifruit (Actinidia chinensis) when CPPU was sprayed on fruit 25 days after anthesis. The CPPU application did not significantly influence the dry matter, soluble solids, starch, vitamin C or protein concentrations. However, the fresh weight, length and maximum diameter were significantly increased compared with the control. Moreover, in fruit of the same developmental stage, the fructose, glucose and soluble sugar levels increased after the CPPU treatment, compared with the control. On the contrary, citric, quinic and titratable acid concentrations decreased. However, a correlation analysis between fresh weight and the nutritional contents revealed that CPPU did not affect the concentrations of the most abundant organic acids (quinic and citric) and sugars (glucose, fructose and sucrose), compared with control fruit of the same weight. Therefore, CPPU applications enhance 'Hongyang' kiwifruit weight/size. However, there were no significant differences in the nutritional qualities of treated and untreated fruit having the same weights.

Development of Specific Markers for Identification of Biovars 1 and 2 Strains of Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae, the causal agent of canker in kiwifruit, can be divided into three biovars (biovars 1, 2, and 3). Strains belonging to biovar 1 produce phaseolotoxin and were isolated in Japan and Italy before 2008. Strains of biovar 2 produce coronatine instead of phaseolotoxin and have been isolated only in Korea. Strains belonging to biovar 3 produce neither phaseolotoxin nor coronatine and are responsible for the global outbreak of bacterial canker of kiwifruit in recent years. The biovar 3-specific primer set was developed in a previous work. In this study, two sets of PCR primers specific to strains of biovars 1 and 2, respectively, were developed based on random amplified polymorphic DNA analyses. Primers PsaJ-F and PsaJ-R produced a 481-bp region with genomic DNA of biovar 1 strains, whereas primers PsaK-F and PsaK-R amplified a 413-bp region present only in the genome of biovar 2 strains.