Comprehensive assessment of drought resistance and recovery in kiwifruit genotypes using multivariate analysis.

As global climate change persists, ongoing warming exposes plants, including kiwifruit, to repeated cycles of drought stress and rewatering, necessitating the identification of drought-resistant genotypes for breeding purposes. To better understand the physiological mechanisms underlying drought resistance and recovery in kiwifruit, moderate (40-45% field capacity) and severe (25-30% field capacity) drought stresses were applied, followed by rewatering (80-85% field capacity) to eight kiwifruit rootstocks in this study. We then conducted a multivariate analysis of 20 indices for the assessment of drought resistance and recovery capabilities. Additionally, we identified four principal components, each playing a vital role in coping with diverse water conditions. Three optimal indicator groups were pinpointed, enhancing precision in kiwifruit drought resistance and recovery assessment and simplifying the evaluation system. Finally, MX-1 and HW were identified as representative rootstocks for future research on kiwifruit's responses to moderate and severe drought stresses. This study not only enhances our understanding of the response mechanisms of kiwifruit rootstocks to progressive drought stress and recovery but also provides theoretical guidance for reliable screening of drought-adaptive kiwifruit genotypes.

Rapid and Visual Detection of Actinidia Chlorotic Ringspot-Associated Virus Using One-Step Reverse-Transcription Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay.

Actinidia chlorotic ringspot-associated virus (AcCRaV) occurs widely in major kiwifruit producing areas of China and is often accompanied by coinfecting viruses, affecting the growth, yield, and quality of kiwifruit. Therefore, a rapid and sensitive detection method is crucial for diagnosing and developing effective AcCRaV management strategies. In this study, a one-step reverse-transcription recombinase polymerase amplification combined with a lateral flow dipstick (RT-RPA-LFD) assay was developed for rapid detection of AcCRaV. Specific primers and a probe were designed based on the conserved region of the coat protein gene sequence of AcCRaV. The one-step RT-RPA reaction can be performed at 35 and 40°C within 10 to 30 min, and the amplification results can be read directly on the LFD within 5 min. The detection limit of the one-step RT-RPA-LFD assay was 10-8 ng (about 20 viral copies), which was equal with one-step RT-qPCR and 100 times more sensitive than one-step RT-PCR. Moreover, the one-step RT-RPA-LFD assay was successfully applied to detect AcCRaV from crude extracts, and the entire detection process can be completed within 40 min. These results indicate that the RT-RPA-LFD assay is a simple, rapid, and sensitive strategy that can be used for accurate diagnosis of AcCRaV-infected kiwifruit plants in the field. To our knowledge, this is the first study applying the one-step RT-RPA-LFD assay to detect a kiwifruit virus.

Inactivation of Salmonella using ultrasound in combination with Litsea cubeba essential oil nanoemulsion and its bactericidal application on cherry tomatoes.

The presence of Salmonella in nature poses a significant and unacceptable threat to the human public health domain. In this study, the antibacterial effect and mechanism of ultrasound (US) combined with Litsea cubeba essential oil nanoemulsion (LEON) on Salmonella. LEON + US treatment has a significant bactericidal effect on Salmonella. Reactive oxygen species (ROS), malondialdehyde (MDA) detection, N-phenyl-l-naphthylamine (NPN) uptake and nucleic acid release assays showed that LEON + US exacerbated cell membrane lipid peroxidation and increased the permeability of the cell membrane. The results of field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM) showed that LEON + US treatment was able to alter cell morphology. It can be observed by flow cytometry (FCM) that LEON + US treatment can cause cell apoptosis. In addition, bacterial counts of cherry tomatoes treated with LEON (0.08 µL/mL) + US (345 W/cm2) for 9 min were reduced by 6.50 ± 0.20 log CFU/mL. This study demonstrates that LEON + US treatment can be an effective way to improve the safety of fruits and vegetables in the food industry.

Transcriptome and Metabolomics Integrated Analysis Reveals MdMYB94 Associated with Esters Biosynthesis in Apple (Malus × domestica).

Volatile esters are major aromas contributing to the organoleptic quality of apple fruit. However, the molecular mechanisms underlying the regulation of volatile ester biosynthesis in apple remain elusive. This study investigated the volatile profiles and transcriptomes of 'Qinguan' (QG) apple fruit during development and/or postharvest storage. Although the constitution of volatiles varied widely between the peel and flesh, the volatile profiles of the peel and flesh of ripening QG fruit were dominated by volatile esters. WGCNA results suggested that 19 genes belonging to ester biosynthesis pathways and 11 hub transcription factor genes potentially participated in the biosynthesis and regulation of esters. To figure out key regulators of ester biosynthesis, correlation network analysis, dual-luciferase assays, and yeast one-hybrid assay were conducted and suggested that MdMYB94 trans-activated the MdAAT2 promoter and participated in the regulation of ester biosynthesis. This study provides a framework for understanding ester biosynthesis and regulation in apple.

The Pollen Donor Affects Seed Development, Taste, and Flavor Quality in 'Hayward' Kiwifruit.

To investigate how different species or ploidy level of pollen donors affects the fruit quality of kiwifruit, flowers of 'Hayward' kiwifruit (a hexaploid Actinidia deliciosa cultivar, 6x) were hand-pollinated with pollen from ten different male donors. Kiwifruit plants pollinated with four distant species-M7 (2x, A. kolomikta), M8 (4x, A. arguta), M9 (4x, A. melanandra), and M10 (2x, A. eriantha)-had a low fruit-setting rate and therefore were not investigated further. Of the other six treatments, kiwifruit plants pollinated with M4 (4x, A. chinensis), M5 (6x, A. deliciosa) M6 (6x, A. deliciosa) had a larger fruit size and weight than those pollinated with M1 (2x, A. chinensis) and M2 (2x, A. chinensis). However, pollination with M1 (2x) and M2 (2x) resulted in seedless fruits, having few small and aborted seeds. Notably, these seedless fruits had higher fructose, glucose, and total sugar and lower citric acid content. This resulted in a higher sugar to acid ratio compared to fruits from plants pollinated with M3 (4x, A. chinensis), M4 (4x), M5 (6x), and M6 (6x). Most volatile compounds increased in the M1 (2x)- and M2 (2x)-pollinated fruit. A combination of principal component analysis (PCA), electronic tongue, and electronic nose suggested that the different pollen donors significantly affected the kiwifruit's overall taste and volatiles. Specifically, two diploid donors had the most positive contribution. This was in agreement with the findings from the sensory evaluation. In conclusion, the present study showed that the pollen donor affected the seed development, taste, and flavor quality of 'Hayward' kiwifruit. This provides useful information for improving the fruit quality and breeding of seedless kiwifruit.

Interaction of AcMADS68 with transcription factors regulates anthocyanin biosynthesis in red-fleshed kiwifruit.

In red-fleshed kiwifruit, anthocyanin pigmentation is a crucial commercial trait. The MYB-bHLH-WD40 (MBW) complex and other transcription factors regulate its accumulation. Herein, a new SEP gene, AcMADS68, was identified as a regulatory candidate for anthocyanin biosynthesis in the kiwifruit by transcriptome data and bioinformatic analyses. AcMADS68 alone could not induce the accumulation of anthocyanin both in Actinidia arguta fruit and tobacco leaves. However, in combination with AcMYBF110, AcMYB123, and AcbHLH1, AcMADS68 co-overexpression increased anthocyanin biosynthesis, whereas its silencing reduced anthocyanin accumulation. The results of the dual-luciferase reporter, firefly luciferase complementation, yeast two-hybrid and co-immunoprecipitation assays showed that AcMADS68 could interact with both AcMYBF110 and AcMYB123 but not with AcbHLH1, thereby co-regulating anthocyanin biosynthesis by promoting the activation of the target genes, including AcANS, AcF3GT1, and AcGST1. Moreover, AcMADS68 also could activate the promoter of AcbHLH1 surported by dual-luciferase reporter and yeast one-hybrid assays, thereby further amplifying the regulation signals from the MBW complex, thus resulting in enhanced anthocyanin accumulation in the kiwifruit. These findings may facilitate better elucidation of various regulatory mechanisms underlying anthocyanin accumulation and contribute to the quality enhancement of red-fleshed kiwifruit.

Evaluation of eleven kiwifruit genotypes for bicarbonate tolerance and characterization of two tolerance-contrasting genotypes.

Screening bicarbonate-tolerant genotypes is an environmentally-friendly and long-term effective strategy to cope with bicarbonate-induced chlorosis in fruit crops grown on calcareous soils. We investigated eleven genotypes from four kiwifruit species (Actinidia chinensis, A. macrosperma, A. polygama, and A. valvata) for differences in bicarbonate tolerance. We also characterized the physiological and molecular differences in two contrasting genotypes of this group. In the first experiment, bicarbonate-treated plantlets were irrigated with 3.0 g L-1 CaCO3 and 5.04 g L-1 NaHCO3 in peat and perlite medium culture. Based on principal component analysis, weight-based membership function method and cluster analysis, the tested genotypes were classified into three groups: (1) tolerant, including YX, Av-1, Acd, Ap, Av-2, and QM; (2) moderately tolerant, including Av-3, Am, Av-4, and HWD; and (3) sensitive, including only QH. In the second experiment, QH (bicarbonate-sensitive) and YX (bicarbonate-tolerant) were grown in sand culture with 4.0 g L-1 CaCO3 and 0.84 g L-1 or 1.26 g L-1 NaHCO3. Compared with QH, YX showed a better ability to take up iron (Fe) by roots and to transport Fe from roots to shoots in the bicarbonate treatments, probably due to a better capacity to protect from oxidative damage and to excrete protons, and a differential expression of genes associated with Fe uptake and translocation, including HA8, IRT1, YSL3 and NRAMP3. The results can facilitate identifying potential resources for bicarbonate tolerance and breeding new rootstocks, and contribute to the elucidation of the bicarbonate tolerance mechanisms in the genus Actinidia.

Integration of morphological, physiological and multi-omics analysis reveals a comprehensive mechanism for cuticular wax during development of greasiness in postharvest apples.

Skin greasiness is a common postharvest disorder of apple (Malus × domestica). However, the molecular mechanism of skin greasiness is unclear. In this study, fruits of 'Golden Delicious' (GD), 'Granny Smith', and 'Fuji' with distinct characteristics of greasiness were used for greasiness scoring, wax morphology, wax metabolite, and RNA-seq analyses. Additionally, GD fruit were treated with 1-methylcyclopropene (1-MCP), which repressed greasiness. A partial least squares discriminant analysis (PLS-DA) revealed that wax esters were the critical wax fraction for skin greasiness. Among these wax esters, liquid linoleate esters of short-chain alcohols (C4-C6) and farnesol showed increased contents with increasing greasiness. Their concentrations were significantly correlated with greasiness score. To identify the genes encoding key enzymes for the synthesis of liquid linoleate esters, a weighted gene co-expression network analysis was conducted. MdDCR1, encoding an acyltransferase (defective in cuticular ridges, DCR), was selected as a candidate gene. MdDCR1 was significantly upregulated in greasy skin, and significantly suppressed by 1-MCP treatment. MdDCR1 silencing suppressed the accumulation of liquid linoleate esters of short-chain alcohols, including butyl linoleate, pentyl linoleate, and hexyl linoleate, in GD skin. These results provide insights into the molecular mechanisms of cuticular wax metabolism related to skin greasiness in apple. Our results show that transcriptional regulation of MdDCR1, encoding an acyltransferase that catalyzes the biosynthesis of liquid linoleate esters of short-chain alcohols (C4-C6) via an independent side branch of the C18:2 CoA pathway, regulates the formation of greasiness.

The Effect of 1-MCP on the Expression of Carotenoid, Chlorophyll Degradation, and Ethylene Response Factors in 'Qihong' Kiwifruit.

The development of yellow color is an important aspect of fruit quality in yellow fleshed kiwifruit during fruit ripening, and it has a large influence on consumer preference. The yellow color is determined by carotenoid accumulation and chlorophyll degradation and is likely affected by ethylene production. This study investigates the expression of carotenoid, chlorophyll degradation, and ethylene response factors in 'Qihong' fruit, which had reached the near ripening stage (firmness ≈ 20 N) and were either left untreated (controls) or treated with 0.5 µL L-1 of 1-MCP for 12 h. Both the accumulation of ß-carotene (not lutein) and degradation of chlorophyll a and b increased in response to the 1-MCP treatment, resulting in more yellow colored flesh in the 1-MCP treated fruit with higher carotenoid and lower chlorophyll contents. 1-MCP up-regulated AcLCY-ß, AcSGR1, and AcPAO2, but reduced the expression of AcCCD1. These four genes were correlated with the concentrations of ß-carotene and the chlorophylls. The expression of three ethylene response factors, including Acc29730, Acc25620, and Acc23763 were delayed and down-regulated in 1-MCP treated fruit, showing the highest correlation with the expression of AcLCY-ß, AcSGR1, AcPAO2, and AcCCD1. Dual-Luciferase assays showed that 1-MCP treatment not only eliminated the inhibition of Acc23763 on the promoters of both AcPAO2 and AcLCY-ß, but also reduced the activation of Acc29730 and Acc25620 on the AcCCD1 promoter. Our findings indicate that Acc29730, Acc25620, and Acc23763 may play an important role in the response to 1-MCP treatment during the fruit eating ripe stage, which likely altered the promoter activities of carotenoid and chlorophyll-related genes (AcPAO2, AcLCY-ß and AcCCD1) to regulate their transcripts, resulting in more yellow color in the fruit flesh of 'Qihong'.

Transcriptional Regulation of Anthocyanin Synthesis by MYB-bHLH-WDR Complexes in Kiwifruit (Actinidia chinensis).

The anthocyanin synthetic pathway is regulated centrally by an MYB-bHLH-WD40 (MBW) complex. Anthocyanin pigmentation is an important fruit quality trait in red-fleshed kiwifruit; however, the underlying regulatory mechanisms involving the MBW complex are not well understood. In this study, one R2R3MYB (AcMYBF110 expressed in fruit characteristically), one bHLH (AcbHLH1), two upstream regulators of AcbHLH1 (AcbHLH4 and AcbHLH5), and one WDR (AcWDR1) are characterized as being involved in the regulation of anthocyanin synthesis in kiwifruit. AcMYBF110 plays an important role in the regulation of anthocyanin accumulation by specifically activating the promoters of several anthocyanin pathway genes including AcCHS, AcF3'H, AcANS, AcUFGT3a, AcUFGT6b, and AcGST1. Coexpression of AcbHLH1, AcbHLH4, or AcbHLH5 together with AcMYBF110 induces much greater anthocyanin accumulation in both tobacco leaves and in Actinidia arguta fruit compared with AcMYBF110 alone. Moreover, this activation is further enhanced by adding AcWDR1. We found that both AcMYBF110 and AcWDR1 interact with all three AcbHLH factors, while AcMYBF110 also interacts with AcWDR1 to form three different MBW complexes that have different regulatory roles in anthocyanin accumulation of kiwifruit. The AcMYBF110-AcbHLH1-AcWDR1 complex directly targets the promoters of anthocyanin synthetic genes. Other features of the regulatory pathways identified include promotion of AcMYBF110, AcbHLH1,and AcWDR1 activities by this MBW complex, providing for both reinforcement and feedback regulation, whereas the AcMYBF110-AcbHLH4/5-AcWDR1 complex is indirectly involved in the regulation of anthocyanin synthesis by activating the promoters of AcbHLH1 and AcWDR1 to amplify the regulation signals of the first MBW complex.

Direct and Bicarbonate-Induced Iron Deficiency Differently Affect Iron Translocation in Kiwifruit Roots.

Bicarbonate-induced iron (Fe) deficiency (+Bic) is frequently observed in kiwifruit orchards, but more research attention has been paid to direct Fe deficiency (-Fe) in plants, including kiwifruit. Here we compared the differences of kiwifruit plants between -Fe and +Bic in: (1) the traits of 57Fe uptake and translocation within plants, (2) Fe forms in roots, and (3) some acidic ions and metabolites in roots. The concentration of 57Fe derived from nutrient solution (57Fedfs) in roots was less reduced in +Bic than -Fe treatment, despite similar decrease in shoots of both treatments. +Bic treatment increased 57Fedfs distribution in fine roots but decreased it in new leaves and stem, thereby displaying the inhibition of 57Fedfs translocation from roots to shoots and from fine roots to xylem of coarse roots. Moreover, +Bic imposition induced the accumulation of water-soluble Fe and apoplastic Fe in roots. However, the opposite was observed in -Fe-treated plants. Additionally, the cell wall Fe and hemicellulose Fe in roots were less reduced by +Bic than -Fe treatment. +Bic treatment also triggered the reduction in H+ extrusion and the accumulation of NH4+, succinic acid, and some amino acids in roots. These results suggest that, contrary to -Fe, +Bic treatment inhibits Fe translocation to shoots by accumulating water-soluble and apoplastic Fe and slowing down the release of hemicellulose Fe in the cell wall in kiwifruit roots, which may be related to the decreased H+ extrusion and the imbalance between C and N metabolisms.

Cryopreservation of shoot tips, evaluations of vegetative growth, and assessments of genetic and epigenetic changes in cryo-derived plants of Actinidia spp.

A droplet-vitrification protocol was described for cryopreservation of shoot tips of kiwifruit 'Yuxiang' (Actinidia chinensis var. deliciosa). No significant differences were found in root formation and shoot growth between the in vitro-derived shoots (the control) and cryo-derived ones when cultured in vitro. No significant differences were detected in survival and vegetative growth between the in vitro-derived plants (the control) and cryo-derived ones after re-establishment in greenhouse conditions. Inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) did not detect any polymorphic bands in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-establishment in greenhouse conditions. These data indicate rooting ability, vegetative growth and genetic stability are maintained in the cryo-derived kiwifruit plants recovered from the droplet-vitrification cryopreservation. Methylation sensitive amplification polymorphism (MSAP) detected 12.8% and 1.6% DNA methylation in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-established in greenhouse conditions, respectively. This droplet-vitrification was applied to five cultivars and three rootstocks belonging to A. chinensis var. deliciosa, A. chinensis var. chinensis, A. macrosperma, A. polygama and A. valvata. The highest (68.3%) and lowest (22.5%) shoot regrowth were obtained in A. macrosperma and A. chinensis var. chinensis 'Jinmi', respectively, with an average of 46.4% shoot regrowth obtained across the eight genotypes. The droplet-vitrification protocol described here can be considered the most applicable cryopreservation method so far reported for the genus Actinidia. Results reported here provide theoretical and technical supports for setting up cryo-banks of genetic resources of Actinidia spp.

The Manufacturing Process of Kiwifruit Fruit Powder with High Dietary Fiber and Its Laxative Effect.

Kiwifruit is rich in vitamins, minerals, dietary fiber and other functional components, and it has long been used as a functional food to treat intestinal ailments such as constipation. The current research made full use of the kiwifruit, the juice was prepared by microencapsulation, and the dietary fiber in kiwifruit pomace was modified by enzymatic hydrolysis and grinding, then, the two were mixed to obtain an ultra-micro kiwifruit powder (UKP). In addition, the laxative effect of the UKP was verified by a diphenoxylate induced constipated mice model. The results demonstrated that compared with the raw samples, the retention rate of vitamin C, lutein and catechin in UKP were 83.3%, 81.9% and 88.3%, respectively, thus effectively avoiding the loss of functional components during the processing of kiwifruit. Moreover, α-amylase, protease and the ball milling process effectively reduced the size of dietary fiber in kiwifruit pomace, and its water-holding capacity (WHC), oil-holding capacity (OHC) and swelling capacity (SWC) were enhanced by 1.26, 1.65 and 1.10 times, respectively. Furthermore, to analyze the laxative effect of the UKP, a constipation mice model was established by diphenoxylate treatment (5 mg·kg-1, i.g.) for the last week, with or without UKP supplementation (2.4 g·kg-1 B.W. per day) for 4 weeks. The results demonstrated that UKP significantly increased feces condition (fecal output and dejecta moisture content, gut transit (the intestinal propulsion rates) and substance P (SP) levels in portal vein plasma, and it decreased the whole gut transit time and mucinogen granules secreted by goblet cell in constipated mice.

Molecular cloning and functional characterization of AcGST1, an anthocyanin-related glutathione S-transferase gene in kiwifruit (Actinidia chinensis).

KEY MESSAGE: AcGST1, an anthocyanin-related GST, may functions as a carrier to transport anthocyanins from ER to tonoplast in kiwifruit. It was positively regulated by AcMYBF110 through directly binding to its promoter. Anthocyanins are synthesized in the cytoplasmic surface of the endoplasmic reticulum but accumulate predominantly in the vacuole. Previous studies in model and ornamental plants have suggested that a member of the glutathione S-transferase (GST) gene family is involved in sequestration of anthocyanins into the vacuole. However, little is known about anthocyanin-related GST protein in kiwifruit. Here, four putative AcGSTs were identified from the genome of the red-fleshed Actinidia chinensis cv 'Hongyang'. Expression analyses reveal only the expression of AcGST1 was highly consistent with anthocyanin accumulation. Molecular complementation of Arabidopsis tt19 demonstrates AcGST1 can complement the anthocyanin-less phenotype of tt19. Transient expression in Actinidia arguta fruits further confirms that AcGST1 is functional in anthocyanin accumulation in kiwifruit. In vitro assays show the recombinant AcGST1 increases the water solubility of cyanidin-3-O-galactoside (C3Gal) and cyanidin-3-O-xylo-galactoside (C3XG). We further show that AcGST1 protein is localized not only in the ER but also on the tonoplast, indicating AcGST1 (like AtTT19) may functions as a carrier protein to transport anthocyanins to the tonoplast in kiwifruit. Moreover, the promoter of AcGST1 can be activated by AcMYBF110, based on results from transient dual-luciferase assays and yeast one-hybrid assays. EMSAs show that AcMYBF110 binds directly to CAGTTG and CCGTTG motifs in the AcGST1 promoter. These results indicate that AcMYBF110 plays an important role in transcriptional regulation of AcGST1 and, therefore, in controlling accumulation of anthocyanins in kiwifruit.

Difference of resistance to postharvest blue mold between Hongyang and Qihong kiwifruits.

This study aimed to reveal the physiological mechanism of resistance to postharvest blue mold of kiwifruit. Hongyang and Qihong kiwifruits were inoculated with Penicillium expansum (P. expansum) and stored at low temperature (0 ±â€¯1 °C). The disease incidence and lesion diameter, activities of defense-related enzymes, and contents of defense-related substance of Hongyang and Qihong kiwifruits were also compared, combined with the observation of fruit pericarp structure by scanning electron microscopy. Results showed that the disease resistance of Hongyang was stronger than that of Qihong with late onset, low incidence, and small lesion diameter. And Hongyang kiwifruit showed a high biochemical resistance after inoculation with P. expansum. The epidermis structure of Hongyang kiwifruit had typical disease resistance characteristics with a dense epidermis structure, orderly cell arrangement, and less obvious microcracks. The strong biochemical resistance, dense, and complete epidermis structure of Hongyang fundamentally guarantee its strong resistance to diseases.

Phenolic compounds and antioxidant activity in red- and in green-fleshed kiwifruits.

Red-fleshed kiwifruits are receiving increasing attention because of their high phenolic contents. However, detailed information on their phenolic compounds and antioxidant capacities remains scarce. Here, six red-fleshed and six green-fleshed kiwifruits were investigated to determine their contents of phenolic compounds and their antioxidant capacities. The results showed chlorogenic acid, p-coumaric acid and ferulic acid were the main phenolic compounds found in kiwifruit. Most of red-fleshed kiwifruits contain higher amounts of total phenolics and anthocyanins, as well as higher activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). Moreover, they exhibited stronger antioxidant capacities than green-fleshed kiwifruits in ABTS, DPPH and FRAP assays. Furthermore, the reactive oxygen species (ROS) inhibition assay showed the phenolics extracted from red-fleshed kiwifruit can better protect tobacco leaves against hydrogen peroxide (H2O2)-induced oxidative damage. This is because of their abundant anthocyanins which in vitro contribute more to H2O2 scavenging than the other phenolic compounds. Based on these findings, it is fair to conclude the red-fleshed kiwifruits are promising sources of antioxidants in human nutrition.

Genome-wide identification of WRKY transcription factors in kiwifruit (Actinidia spp.) and analysis of WRKY expression in responses to biotic and abiotic stresses.

As one of the largest transcriptional factor families in plants, WRKY transcription factors play important roles in various biotic and abiotic stress responses. To date, WRKY genes in kiwifruit (Actinidia spp.) remain poorly understood. In our study, o total of 97 AcWRKY genes have been identified in the kiwifruit genome. An overview of these AcWRKY genes is analyzed, including the phylogenetic relationships, exon-intron structures, synteny and expression profiles. The 97 AcWRKY genes were divided into three groups based on the conserved WRKY domain. Synteny analysis indicated that segmental duplication events contributed to the expansion of the kiwifruit AcWRKY family. In addition, the synteny analysis between kiwifruit and Arabidopsis suggested that some of the AcWRKY genes were derived from common ancestors before the divergence of these two species. Conserved motifs outside the AcWRKY domain may reflect their functional conservation. Genome-wide segmental and tandem duplication were found, which may contribute to the expansion of AcWRKY genes. Furthermore, the analysis of selected AcWRKY genes showed a variety of expression patterns in five different organs as well as during biotic and abiotic stresses. The genome-wide identification and characterization of kiwifruit WRKY transcription factors provides insight into the evolutionary history and is a useful resource for further functional analyses of kiwifruit.

Biochemical and functional characterization of AcUFGT3a, a galactosyltransferase involved in anthocyanin biosynthesis in the red-fleshed kiwifruit (Actinidia chinensis).

Much of the diversity of anthocyanin pigmentation in plant tissues is due to the action of glycosyltransferases, which attach sugar moieties to the anthocyanin aglycone. This step can increase both their solubility and stability. We investigated the pigmentation of the outer and inner pericarps of developing fruits of the red-fleshed kiwifruit Actinidia chinensis cv. 'Hongyang'. The results show that the red color of the inner pericarp is due to anthocyanin. Based on expression analyses of structural genes, AcUFGT was shown to be the key gene involved in the anthocyanin biosynthetic pathway. Expression of AcUFGT in developing fruit paralleled changes in anthocyanin concentration. Thirteen putative UFGT genes, including different transcripts, were identified in the genome of 'Hongyang'. Among these, only the expression of AcUFGT3a was found to be highly consistent with anthocyanin accumulation. Fruit infiltrated with virus-induced gene silencing showed delayed red colorations, lower anthocyanin contents and lower expressions of AcUFGT3a. At the same time, transient overexpression of AcUFGT3a in both Actinidia arguta and green apple fruit resulted in higher anthocyanin contents and deeper red coloration. In vitro biochemical assays revealed that recombinant AcUFGT3a recognized only anthocyanidins as substrate but not flavonols. Also, UDP-galactose was used preferentially as the sugar donor. These results indicate AcUFGT3a is the key enzyme regulating anthocyanin accumulation in red-fleshed kiwifruit.

Expression Differences of Pigment Structural Genes and Transcription Factors Explain Flesh Coloration in Three Contrasting Kiwifruit Cultivars.

Fruits of kiwifruit cultivars (Actinidia chinensis and A. deliciosa) generally have green or yellow flesh when ripe. A small number of genotypes have red flesh but this coloration is usually restricted to the inner pericarp. Three kiwifruit cultivars having red ('Hongyang'), or yellow ('Jinnong-2'), or green ('Hayward') flesh were investigated for their color characteristics and pigment contents during development and ripening. The results show the yellow of the 'Jinnong-2' fruit is due to the combined effects of chlorophyll degradation and of beta-carotene accumulation. The red inner pericarps of 'Hongyang' fruit are due to anthocyanin accumulation. Expression differences of the pathway genes in the inner pericarps of the three different kiwifruits suggest that stay-green (SGR) controls the degradation of chlorophylls, while lycopene beta-cyclase (LCY-ß) controls the biosynthesis of beta-carotene. The abundance of anthocyanin in the inner pericarps of the 'Hongyang' fruit is the results of high expressions of UDP flavonoid glycosyltransferases (UFGT). At the same time, expressions of anthocyanin transcription factors show that AcMYBF110 expression parallels changes in anthocyanin concentration, so seems to be a key R2R3 MYB, regulating anthocyanin biosynthesis. Further, transient color assays reveal that AcMYBF110 can autonomously induce anthocyanin accumulation in Nicotiana tabacum leaves by activating the transcription of dihydroflavonol 4-reductase (NtDFR), anthocyanidin synthase (NtANS) and NtUFGT. For basic helix-loop-helix proteins (bHLHs) and WD-repeat proteins (WD40s), expression differences show these may depend on AcMYBF110 forming a MYB-bHLH-WD40 complex to regulate anthocyanin biosynthesis, instead of it having a direct involvement.

An investigation of Chinese attitudes toward the environment: case study using the Grain for Green Project.

China is the world's most populous country and has one of the largest territories. As such, Chinese attitudes and behavior with regard to environmental issues are key factors in protecting the world's natural resources and environment. In this study, we surveyed a random sample of 5000 citizens from six Chinese provinces (Beijing, Shanghai, Hubei, Hunan, Henan, and Shaanxi) to understand their environmental attitudes, contrasts between the attitudes of citizens in different demographic groups, and their willingness to invest in environmental conservation. The results indicated that policymakers and the public increasingly recognize the key role that environmental restoration plays in protecting the overall health of the environment. In total, 91% of the interviewees believed that the environment had deteriorated severely during the past decade, compared with 44% in a 1999 survey. In addition, 78% of the interviewees supported their government's investment of more than 300 billion RMB (approximately 10% of total government revenues in 2004) in the "Grain for Green Project", which discouraged unsustainable land use by compensating farmers and herders for abandoning farming and grazing on marginal land. There was a strong correlation between environmental attitudes and net income and education levels, and other differences were based on the respondents' age, gender, job, and location. Net income and education level were the key factors that affected environmental attitudes. Based on these results, we propose that successful environmental restoration projects must include both an education component and an economic development component.