Molecular phylogeography and species distribution modelling evidence of 'oceanic' adaptation for Actinidia eriantha with a refugium along the oceanic-continental gradient in a biodiversity hotspot.

BACKGROUND: Refugia is considered to be critical for maintaining biodiversity; while discerning the type and pattern of refugia is pivotal for our understanding of evolutionary processes in the context of conservation. Interglacial and glacial refugia have been studied throughout subtropical China. However, studies on refugia along the oceanic-continental gradient have largely been ignored. We used a liana Actinidia eriantha, which occurs across the eastern moist evergreen broad-leaved forests of subtropical China, as a case study to test hypotheses of refugia along the oceanic-continental gradient and 'oceanic' adaptation. RESULTS: The phylogeographic pattern of A. eriantha was explored using a combination of three cpDNA markers and 38 nuclear microsatellite loci, Species distribution modelling and dispersal corridors analysis. Our data showed intermediate levels of genetic diversity [haplotype diversity (hT) = 0.498; unbiased expected heterozygosity (UHE) = 0.510] both at the species and population level. Microsatellite loci revealed five clusters largely corresponding to geographic regions. Coalescent time of cpDNA lineages was dated to the middle Pliocene (ca. 4.03 Ma). Both geographic distance and climate difference have important roles for intraspecific divergence of the species. The Zhejiang-Fujian Hilly Region was demonstrated to be a refugium along the oceanic-continental gradient of the species and fit the 'refugia in refugia' pattern. Species distribution modelling analysis indicated that Precipitation of Coldest Quarter (importance of 44%), Temperature Seasonality (29%) and Mean Temperature of Wettest Quarter (25%) contributed the most to model development. By checking the isolines in the three climate layers, we found that A. eriantha prefer higher precipitation during the coldest quarter, lower seasonal temperature difference and lower mean temperature during the wettest quarter, which correspond to 'oceanic' adaptation. Actinidia eriantha expanded to its western distribution range along the dispersal corridor repeatedly during the glacial periods. CONCLUSIONS: Overall, our results provide integrated evidence demonstrating that the Zhejiang-Fujian Hilly Region is a refugium along the oceanic-continental gradient of Actinidia eriantha in subtropical China and that speciation is attributed to 'oceanic' adaptation. This study gives a deeper understanding of the refugia in subtropical China and will contribute to the conservation and utilization of kiwifruit wild resources in the context of climate change.

Pseudomonas syringae pv. actinidiae Effector HopAU1 Interacts with Calcium-Sensing Receptor to Activate Plant Immunity.

Kiwifruit canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is a destructive pathogen that globally threatens the kiwifruit industry. Understanding the molecular mechanism of plant-pathogen interaction can accelerate applying resistance breeding and controlling plant diseases. All known effectors secreted by pathogens play an important role in plant-pathogen interaction. However, the effectors in Psa and their function mechanism remain largely unclear. Here, we successfully identified a T3SS effector HopAU1 which had no virulence contribution to Psa, but could, however, induce cell death and activate a series of immune responses by agroinfiltration in Nicotiana benthamiana, including elevated transcripts of immune-related genes, accumulation of reactive oxygen species (ROS), and callose deposition. We found that HopAU1 interacted with a calcium sensing receptor in N. benthamiana (NbCaS) as well as its close homologue in kiwifruit (AcCaS). More importantly, silencing CaS by RNAi in N. benthamiana greatly attenuated HopAU1-triggered cell death, suggesting CaS is a crucial component for HopAU1 detection. Further researches showed that overexpression of NbCaS in N. benthamiana significantly enhanced plant resistance against Sclerotinia sclerotiorum and Phytophthora capsici, indicating that CaS serves as a promising resistance-related gene for disease resistance breeding. We concluded that HopAU1 is an immune elicitor that targets CaS to trigger plant immunity.

Defence-related pathways, phytohormones and primary metabolism are key players in kiwifruit plant tolerance to Pseudomonas syringae pv. actinidiae.

The reasons underlying the differential tolerance of Actinidia spp. to the pandemic pathogen Pseudomonas syringae pv. actinidiae (Psa) have not yet been elucidated. We hypothesized that differential plant-defence strategies linked to transcriptome regulation, phytohormones and primary metabolism might be key and that Actinidia chinensis susceptibility results from an inefficient activation of defensive mechanisms and metabolic impairments shortly following infection. Here, 48 h postinoculation bacterial density was 10-fold higher in A. chinensis var. deliciosa than in Actinidia arguta, accompanied by significant increases in glutamine, ornithine, jasmonic acid (JA) and salicylic acid (SA) (up to 3.2-fold). Actinidia arguta showed decreased abscisic acid (ABA) (0.7-fold), no changes in primary metabolites, and 20 defence-related genes that were only differentially expressed in this species. These include GLOX1, FOX1, SN2 and RBOHA, which may contribute to its higher tolerance. Results suggest that A. chinensis' higher susceptibility to Psa is due to an inefficient activation of plant defences, with the involvement of ABA, JA and SA, leading to impairments in primary metabolism, particularly the ammonia assimilation cycle. A schematic overview on the interaction between Psa and genotypes with distinct tolerance is provided, highlighting the key transcriptomic and metabolomic aspects contributing to the different plant phenotypes after infection.

AvNAC030, a NAC Domain Transcription Factor, Enhances Salt Stress Tolerance in Kiwifruit.

Kiwifruit (Actinidia chinensis Planch) is suitable for neutral acid soil. However, soil salinization is increasing in kiwifruit production areas, which has adverse effects on the growth and development of plants, leading to declining yields and quality. Therefore, analyzing the salt tolerance regulation mechanism can provide a theoretical basis for the industrial application and germplasm improvement of kiwifruit. We identified 120 NAC members and divided them into 13 subfamilies according to phylogenetic analysis. Subsequently, we conducted a comprehensive and systematic analysis based on the conserved motifs, key amino acid residues in the NAC domain, expression patterns, and protein interaction network predictions and screened the candidate gene AvNAC030. In order to study its function, we adopted the method of heterologous expression in Arabidopsis. Compared with the control, the overexpression plants had higher osmotic adjustment ability and improved antioxidant defense mechanism. These results suggest that AvNAC030 plays a positive role in the salt tolerance regulation mechanism in kiwifruit.

Full-length transcriptome profiling reveals insight into the cold response of two kiwifruit genotypes (A. arguta) with contrasting freezing tolerances.

BACKGROUND: Kiwifruit (Actinidia Lindl.) is considered an important fruit species worldwide. Due to its temperate origin, this species is highly vulnerable to freezing injury while under low-temperature stress. To obtain further knowledge of the mechanism underlying freezing tolerance, we carried out a hybrid transcriptome analysis of two A. arguta (Actinidi arguta) genotypes, KL and RB, whose freezing tolerance is high and low, respectively. Both genotypes were subjected to - 25 °C for 0 h, 1 h, and 4 h. RESULTS: SMRT (single-molecule real-time) RNA-seq data were assembled using the de novo method, producing 24,306 unigenes with an N50 value of 1834 bp. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs showed that they were involved in the 'starch and sucrose metabolism', the 'mitogen-activated protein kinase (MAPK) signaling pathway', the 'phosphatidylinositol signaling system', the 'inositol phosphate metabolism', and the 'plant hormone signal transduction'. In particular, for 'starch and sucrose metabolism', we identified 3 key genes involved in cellulose degradation, trehalose synthesis, and starch degradation processes. Moreover, the activities of beta-GC (beta-glucosidase), TPS (trehalose-6-phosphate synthase), and BAM (beta-amylase), encoded by the abovementioned 3 key genes, were enhanced by cold stress. Three transcription factors (TFs) belonging to the AP2/ERF, bHLH (basic helix-loop-helix), and MYB families were involved in the low-temperature response. Furthermore, weighted gene coexpression network analysis (WGCNA) indicated that beta-GC, TPS5, and BAM3.1 were the key genes involved in the cold response and were highly coexpressed together with the CBF3, MYC2, and MYB44 genes. CONCLUSIONS: Cold stress led various changes in kiwifruit, the 'phosphatidylinositol signaling system', 'inositol phosphate metabolism', 'MAPK signaling pathway', 'plant hormone signal transduction', and 'starch and sucrose metabolism' processes were significantly affected by low temperature. Moreover, starch and sucrose metabolism may be the key pathway for tolerant kiwifruit to resist low temperature damages. These results increase our understanding of the complex mechanisms involved in the freezing tolerance of kiwifruit under cold stress and reveal a series of candidate genes for use in breeding new cultivars with enhanced freezing tolerance.

The effect of cane vigour on the kiwifruit (Actinidia chinensis) and kiwiberry (Actinidia arguta) quality.

Kiwifruit has not been studied as much as other well-known fruits especially when it comes to studies about plant vigour and training systems. The aim of the study was to determine the importance of cane vigour of Actinidia chinensis var. deliciosa 'Hayward' and Actinidia arguta 'Issai' in order to develop the proper pruning technique that results in the best fruit quality. In addition, the effect of storage parameters such as weight, firmness and quality of the fruit was also studied. The study showed that the fruit size and weight are lower in low vigour canes in A. arguta, in contrast to A. chinensis, where the fruit size and weight are smaller on high-vigorous canes. For A. arguta, it is recommended to choose high-vigour canes as the optimal fruit wood during pruning. In this way, the fruits will ripen more evenly. The other possibility is to perform the harvest two to three times per season to achieve a more uniform fruit quality. In the case of A. chinensis the fruit are less variable between different cane vigour, so harvesting can be done in a single picking. In A. chinensis the less vigorous canes tend to show a slightly better fruit quality.

Genome-wide analysis of the bZIP gene family and the role of AchnABF1 from postharvest kiwifruit (Actinidia chinensis cv. Hongyang) in osmotic and freezing stress adaptations.

Chilling injury (CI) is a barrier to the refrigeration of kiwifruit, resulting in decreased fruit quality and increased nutrient loss during storage. Understanding the molecular basis underlying the cold response and its regulation in refrigerated kiwifruit is therefore highly important. Basic (region) leucine zipper (bZIP) transcription factors (TFs) have been widely studied for their roles in abiotic stress resistance in various species. In this study, we identified 81 bZIP family proteins in kiwifruit and classified them into 11 groups. Further transcriptome analysis revealed that the expression of members of the AREB/ABF family was strongly induced by low temperature and abscisic acid (ABA). Ectopic expression of AchnABF1 enhanced plant cold tolerance by upregulating the expression of several key genes associated with ABA-dependent and ABA-independent pathways in Arabidopsis thaliana. Reactive oxygen species (ROS) metabolism was suggested to be involved in the AchnABF1-mediated osmotic stress response. For instance, enhanced ROS-scavenging ability was observed in transgenic plants with enhanced activity of catalase (CAT) and peroxidase (POD), which resulted in decreased in situ O2.- and H2O2 accumulation, ion leakage, and malondialdehyde (MDA) content under various abiotic stresses. In addition, AchnABF1 also participated in the osmotic stress response during both the germination and postgermination stages. We concluded that AchnABF1 may play an important role in kiwifruit during refrigeration.

Orchard layout and plant traits influence fruit yield more strongly than pollinator behaviour and density in a dioecious crop.

Mutualistic plant-pollinator interactions are critical for the functioning of both non-managed and agricultural systems. Mathematical models of plant-pollinator interactions can help understand key determinants in pollination success. However, most previous models have not addressed pollinator behavior and plant biology combined. Information generated from such a model can inform optimal design of crop orchards and effective utilization of managed pollinators like western honey bees (Apis mellifera), and help generate hypotheses about the effects of management practices and cultivar selection. We expect that the number of honey bees per flower and male to female flower ratio will influence fruit yield. To test the relative importance of these effects, both singly and simultaneously, we utilized a delay differential equation model combined with Latin hypercube sampling for sensitivity analysis. Empirical data obtained from historical records and collected in kiwifruit (Actinidia chinensis) orchards in New Zealand were used to parameterize the model. We found that, at realistic bee densities, the optimal orchard had 65-75% female flowers, and the most benefit was gained from the first 6-8 bees/1000 flowers, with diminishing returns thereafter. While bee density significantly impacted fruit production, plant-based parameters-flower density and male:female flower ratio-were the most influential. The predictive model provides strategies for improving crop management, such as choosing cultivars which have their peak bloom on the same day, increasing the number of flowers with approximately 70% female flowers in the orchard, and placing enough hives to maintain more than 6 bees per 1000 flowers to optimize yield.

Pseudomonas syringae pv. actinidiae: Ecology, Infection Dynamics and Disease Epidemiology.

Since 2008, the kiwifruit industry has been devastated by a pandemic outbreak of Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial canker. This disease has become the most significant limiting factor in kiwifruit production. Psa colonizes different organs of the host plant, causing a specific symptomatology on each of them. In addition, the systemic invasion of the plant may quickly lead to plant death. Despite the massive risk that this disease poses to the kiwifruit industry, studies focusing on Psa ecology have been sporadic, and a comprehensive description of the disease epidemiology is still missing. Optimal environmental conditions for infection, dispersal and survival in the environment, or the mechanisms of penetration and colonization of host tissues have not been fully elucidated yet. The present work aims to provide a synthesis of the current knowledge, and a deeper understanding of the epidemiology of kiwifruit bacterial canker based on new experimental data. The pathogen may survive in the environment or overwinter in dormant tissues and be dispersed by wind or rain. Psa was observed in association with several plant structures (stomata, trichomes, lenticels) and wounds, which could represent entry points for apoplast infection. Environmental conditions also affect the bacterial colonization, with lower optimum values of temperature and humidity for epiphytic than for endophytic growth, and disease incidence requiring a combination of mild temperature and leaf wetness. By providing information on Psa ecology, these data sets may contribute to plan efficient control strategies for kiwifruit bacterial canker.

Genome-wide analysis of coding and non-coding RNA reveals a conserved miR164-NAC regulatory pathway for fruit ripening.

Kiwifruit (Actinidia spp.) is a climacteric fruit with high sensitivity to ethylene, influenced by multiple ethylene-responsive structural genes and transcription factors. However, the roles of other post-transcriptional regulators (e.g. miRNAs) necessary for ripening remain elusive. High-throughput sequencing sRNAome, degradome and transcriptome methods were used to identify further contributors to ripening control in the kiwifruit (A. deliciosa cv 'Hayward'). Two NAM/ATAF/CUC domain transcription factors (AdNAC6 and AdNAC7), both predicted targets for miR164, showed significant upregulation by exogenous ethylene. Gene expression analysis and luciferase reporter assays indicated that Ade-miR164 and one of its precursor miRNAs (Ade-MIR164b) were repressed by ethylene treatment and negatively correlated with AdNAC6/7 expression. Subsequent analysis indicated that both AdNAC6 and AdNAC7 proteins are transcriptional activators and physically bind the promoters of AdACS1 (1-aminocyclopropane-1-carboxylate synthase), AdACO1 (1-aminocyclopropane-1-carboxylic acid oxidase), AdMAN1 (endo-ß-mannanase) and AaTPS1 (terpene synthase). Moreover, subcellular analysis indicated that the location of the AdNAC6/7 proteins was influenced by Ade-miR164. Multiple omics-based approaches revealed a novel regulatory link for fruit ripening that involved ethylene-miR164-NAC. The regulatory pathway for miR164-NAC is present in various fruit (e.g. Rosaceae fruit, citrus, grape), with implications for fruit ripening regulation.

Expression analysis of ethylene synthesis and signalling genes in kiwifruit stigmatic arms and their involvement in programmed cell death processes.

Kiwifruit (Actinidia chinensis var. deliciosa (A. Chev) A. Chev.) is a widely cultivated crop due to the nutritional value of its fruits. Its commercialization is related to the fruit size, which is directly linked with the number of seeds and, consequently, with pollination. In this dioecious species pollination is dependent on a short effective pollination period which is related to a Programmed Cell Death (PCD) process. At the same time, this PCD process allows the growth of many pollen tubes. Several studies suggest that ethylene can play an important role in PCD in a number of systems. In this report, we determined the full sequence of the AcACS gene, encoding the enzyme that catalyses a rate-limiting step of the ethylene synthesis. Next, we monitored the expression pattern of this gene as well as of other genes involved in ethylene synthesis (ACO2-5) and signalling (AdERS1a, AdERS1b, AdETR1, AdETR2, AdETR3, AdCTR1, AdCTR2, AdEIL1) in pollinated and non-pollinated stigmatic arms of kiwifruit female flowers. The relative expression patterns observed for AcACS, ACOs and ethylene perception and signalling genes (AdERS1, AdETR1, AdCTR1 and AdEIL1) showed that they are expressed before anthesis. After anthesis, expression of the studied genes was detected earlier in pollinated than in non-pollinated stigmatic arms, as it was previously determined for PCD hallmarks. In addition, the expression pattern of the studied genes showed a clear relationship with the PCD hallmarks described in a previous report in the secretory tissue both in non-pollinated stigmatic arms (related to the short EPP in this species) and in pollinated ones (related to the growth of many pollen tubes during progamic phase). Overall, these results suggest an involvement of ethylene with PCD contributing to the high reproductive success of this species.

Downhill seed dispersal by temperate mammals: a potential threat to plant escape from global warming.

Vertical seed dispersal, i.e. seed dispersal towards a higher or lower altitude, is considered a critical process for plant escape from climate change. However, studies exploring vertical seed dispersal are scarce, and thus, its direction, frequency, and mechanisms are little known. In the temperate zone, evaluating vertical seed dispersal of animal-dispersed plants fruiting in autumn and/or winter is essential considering the dominance of such plants in temperate forests. We hypothesized that their seeds are dispersed towards lower altitudes because of the downhill movement of frugivorous animals following the autumn-to-winter phenology of their food plants which proceeds from the mountain tops to the foot in the temperate zone. We evaluated the vertical seed dispersal of the autumn-fruiting wild kiwi, Actinidia arguta, which is dispersed by temperate mammals. We collected dispersed seeds from mammal faeces in the Kanto Mountains of central Japan and estimated the distance of vertical seed dispersal using the oxygen isotope ratios of the dispersed seeds. We found the intensive downhill seed dispersal of wild kiwi by all seed dispersers, except the raccoon dog (bear: mean -393.1 m; marten: -245.3 m; macaque: -98.5 m; and raccoon dog: +4.5 m). Mammals with larger home ranges dispersed seeds longer towards the foot of the mountains. Furthermore, we found that seeds produced at higher altitudes were dispersed a greater distance towards the foot of the mountains. Altitudinal gradients in autumn-to-winter plant phenology and other mountain characteristics, i.e. larger surface areas and more attractive human crops at lower altitudes compared to higher altitudes, were considered drivers of downhill seed dispersal via animal movement. Strong downhill seed dispersal by mammals suggests that populations of autumn-to-winter fruiting plants dispersed by animals may not be able to sufficiently escape from current global warming in the temperate zone.

Positive Regulation of the Transcription of AchnKCS by a bZIP Transcription Factor in Response to ABA-Stimulated Suberization of Kiwifruit.

Wound-induced suberization is an essentially protective healing process for wounded fruit to reduce water loss and microbial infection. It has been demonstrated that abscisic acid (ABA) could promote wound suberization, but the molecular mechanism of ABA regulation remains little known. In this study, the transcript level of Achn030011 (designated as AchnKCS), coding a ß-ketoacyl-coenzyme A synthase (KCS) involved in suberin biosynthesis, was found to be significantly upregulated by ABA in wounded kiwifruit. A bZIP transcription factor (Achn270881), a possible downstream transcription factor in the ABA signaling pathway, was screened and designated as AchnbZIP12 according to its homology with related Arabidopsis transcription factors. A yeast one-hybrid assay demonstrated that AchnbZIP12 could interact with the AchnKCS promoter. Furthermore, significant trans-activation of AchnbZIP12 on AchnKCS was verified. The transcript level of AchnbZIP12 was also upregulated upon treatment with ABA. These results imply that AchnbZIP12 acts as a positive regulator in ABA-mediated AchnKCS transcription during wound suberization of kiwifruit.

AdRAP2.3, a Novel Ethylene Response Factor VII from Actinidia deliciosa, Enhances Waterlogging Resistance in Transgenic Tobacco through Improving Expression Levels of PDC and ADH Genes.

APETALA2/ethylene-responsive factor superfamily (AP2/ERF) is a transcription factor involved in abiotic stresses, for instance, cold, drought, and low oxygen. In this study, a novel ethylene-responsive transcription factor named AdRAP2.3 was isolated from Actinidia deliciosa 'Jinkui'. AdRAP2.3 transcription levels in other reproductive organs except for the pistil were higher than those in the vegetative organs (root, stem, and leaf) in kiwi fruit. Plant hormones (Salicylic acid (SA), Methyl-jasmonate acid (MeJA), 1-Aminocyclopropanecarboxylic Acid (ACC), Abscisic acid (ABA)), abiotic stresses (waterlogging, heat, 4 °C and NaCl) and biotic stress (Pseudomonas Syringae pv. Actinidiae, Psa) could induce the expression of AdRAP2.3 gene in kiwi fruit. Overexpression of the AdRAP2.3 gene conferred waterlogging stress tolerance in transgenic tobacco plants. When completely submerged, the survival rate, fresh weight, and dry weight of transgenic tobacco lines were significantly higher than those of wile type (WT). Upon the roots being submerged, transgenic tobacco lines grew aerial roots earlier. Overexpression of AdRAP2.3 in transgenic tobacco improved the pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) enzyme activities, and improved the expression levels of waterlogging mark genes NtPDC, NtADH, NtHB1, NtHB2, NtPCO1, and NtPCO2 in roots under waterlogging treatment. Overall, these results demonstrated that AdRAP2.3 might play an important role in resistance to waterlogging through regulation of PDC and ADH genes in kiwi fruit.

Ozone-induced inhibition of kiwifruit ripening is amplified by 1-methylcyclopropene and reversed by exogenous ethylene.

BACKGROUND: Understanding the mechanisms involved in climacteric fruit ripening is key to improve fruit harvest quality and postharvest performance. Kiwifruit (Actinidia deliciosa cv. 'Hayward') ripening involves a series of metabolic changes regulated by ethylene. Although 1-methylcyclopropene (1-MCP, inhibitor of ethylene action) or ozone (O3) exposure suppresses ethylene-related kiwifruit ripening, how these molecules interact during ripening is unknown. RESULTS: Harvested 'Hayward' kiwifruits were treated with 1-MCP and exposed to ethylene-free cold storage (0 °C, RH 95%) with ambient atmosphere (control) or atmosphere enriched with O3 (0.3 µL L- 1) for up to 6 months. Their subsequent ripening performance at 20 °C (90% RH) was characterized. Treatment with either 1-MCP or O3 inhibited endogenous ethylene biosynthesis and delayed fruit ripening at 20 °C. 1-MCP and O3 in combination severely inhibited kiwifruit ripening, significantly extending fruit storage potential. To characterize ethylene sensitivity of kiwifruit following 1-MCP and O3 treatments, fruit were exposed to exogenous ethylene (100 µL L- 1, 24 h) upon transfer to 20 °C following 4 and 6 months of cold storage. Exogenous ethylene treatment restored ethylene biosynthesis in fruit previously exposed in an O3-enriched atmosphere. Comparative proteomics analysis showed separate kiwifruit ripening responses, unraveled common 1-MCP- and O3-dependent metabolic pathways and identified specific proteins associated with these different ripening behaviors. Protein components that were differentially expressed following exogenous ethylene exposure after 1-MCP or O3 treatment were identified and their protein-protein interaction networks were determined. The expression of several kiwifruit ripening related genes, such as 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1), ethylene receptor (ETR1), lipoxygenase (LOX1), geranylgeranyl diphosphate synthase (GGP1), and expansin (EXP2), was strongly affected by O3, 1-MCP, their combination, and exogenously applied ethylene. CONCLUSIONS: Our findings suggest that the combination of 1-MCP and O3 functions as a robust repressive modulator of kiwifruit ripening and provide new insight into the metabolic events underlying ethylene-induced and ethylene-independent ripening outcomes.

A Y-Encoded Suppressor of Feminization Arose via Lineage-Specific Duplication of a Cytokinin Response Regulator in Kiwifruit.

Dioecy, the presence of male and female flowers on distinct individuals, has evolved independently in multiple plant lineages, and the genes involved in this differential development are just starting to be uncovered in a few species. Here, we used genomic approaches to investigate this pathway in kiwifruits (genus Actinidia). Genome-wide cataloging of male-specific subsequences, combined with transcriptome analysis, led to the identification of a type-C cytokinin response regulator as a potential sex determinant gene in this genus. Functional transgenic analyses in two model systems, Arabidopsis thaliana and Nicotiana tabacum, indicated that this gene acts as a dominant suppressor of carpel development, prompting us to name it Shy Girl (SyGI). Evolutionary analyses in a panel of Actinidia species revealed that SyGI is located in the Y-specific region of the genome and probably arose from a lineage-specific gene duplication. Comparisons with the duplicated autosomal counterpart, and with orthologs from other angiosperms, suggest that the SyGI-specific duplication and subsequent evolution of cis-elements may have played a key role in the acquisition of separate sexes in this species.

Kiwifruit SVP2 controls developmental and drought-stress pathways.

KEY MESSAGE: Genome-wide targets of Actinidia chinensis SVP2 confirm roles in ABA- and dehydration-mediated growth repression and reveal a conservation in mechanism of action between SVP genes of taxonomically distant Arabidopsis and a woody perennial kiwifruit. The molecular mechanisms underlying growth and dormancy in woody perennials are largely unknown. In Arabidopsis, the MADS-box transcription factor SHORT VEGETATIVE PHASE (SVP) plays a key role in the progression from vegetative to floral development, and in woody perennials SVP-like genes are also proposed to be involved in controlling dormancy. During kiwifruit development SVP2 has a role in growth inhibition, with high-chill kiwifruit Actinidia deliciosa transgenic lines overexpressing SVP2 showing suppressed bud outgrowth. Transcriptomic analyses of these plants suggests that SVP2 mimics the well-documented abscisic acid (ABA) effect on the plant dehydration response. To corroborate the growth inhibition role of SVP2 in kiwifruit development at the molecular level, we analysed the genome-wide direct targets of SVP2 using chromatin immunoprecipitation followed by high-throughput sequencing in kiwifruit A. chinensis. SVP2 was found to bind to at least 297 target sites in the kiwifruit genome, and potentially modulates 252 genes that function in a range of biological processes, especially those involved in repressing meristem activity and ABA-mediated dehydration pathways. In addition, our ChIP-seq analysis reveals remarkable conservation in mechanism of action between SVP genes of taxonomically distant plant species.

Proteomics analysis to understand the ABA stimulation of wound suberization in kiwifruit.

Quick suberin-based healing after wounding played a protective role for plant to prevent further damage. In this study, the stimulative effect of exogenous abscisic acid (ABA) on wound suberization in postharvest kiwifruit was evaluated through suberin staining with toluidine blue O as well as the determination of suberin phenolics and aliphatics in wound tissue. Furthermore, to reveal the regulatory involvement of ABA in wound suberization, comparative quantitative proteomics and transcriptomics analyses based on iTRAQ and qRT-PCR technique were performed. In proteomics levels, a total of 95 protein species consistently showed differential abundance between ABA and control, including 29 down-regulated and 66 up-regulated protein species. The Kyoto Encyclopedia of Genes and Genomes (KEGG) with protein-protein interaction analyses revealed that ABA mainly affected the antioxidant system, phenylpropanoid metabolism and lipid metabolism associated with wound suberization. Based on the data of proteomics analysis, the differential expressions of genes encoding 11 selected protein species were confirmed by qRT-PCR analyses. GSH-Px, MDHAR, SOD, APX, POD, PAL, CCR, PPO, CYP86B1, DGGT and KCS11 were likely to be the key enzymes that involved the response of ABA to stimulate wound suberization by mediating the antioxidant system, phenylpropanoid metabolism and lipid metabolism. BIOLOGICAL SIGNIFICANCE: Kiwifruit is susceptible to physical injury causing postharvest deterioration during harvest, transportation and storage. Therefore, quick healing is important for maintaining the postharvest quality of injured fruit. This work elucidated the potential role of ABA and the proteomic mechanism of its regulation in wound suberization of postharvest kiwifruit.

Functional Characterization of Waterlogging and Heat Stresses Tolerance Gene Pyruvate decarboxylase 2 from Actinidia deliciosa.

A previous report showed that both Pyruvatedecarboxylase (PDC) genes were significantly upregulated in kiwifruit after waterlogging treatment using Illumina sequencing technology, and that the kiwifruit AdPDC1 gene was required during waterlogging, but might not be required during other environmental stresses. Here, the function of another PDC gene, named AdPDC2, was analyzed. The expression of the AdPDC2 gene was determined using qRT-PCR, and the results showed that the expression levels of AdPDC2 in the reproductive organs were much higher than those in the nutritive organs. Waterlogging, NaCl, and heat could induce the expression of AdPDC2. Overexpression of kiwifruit AdPDC2 in transgenic Arabidopsis enhanced resistance to waterlogging and heat stresses in five-week-old seedlings, but could not enhance resistance to NaCl and mannitol stresses at the seed germination stage and in early seedlings. These results suggested that the kiwifruit AdPDC2 gene may play an important role in waterlogging resistance and heat stresses in kiwifruit.

Cell separation in kiwifruit without development of a specialised detachment zone.

BACKGROUND: Unlike in abscission or dehiscence, fruit of kiwifruit Actinidia eriantha develop the ability for peel detachment when they are ripe and soft in the absence of a morphologically identifiable abscission zone. Two closely-related genotypes with contrasting detachment behaviour have been identified. The 'good-peeling' genotype has detachment with clean debonding of cells, and a peel tissue that does not tear. The 'poor-peeling' genotype has poor detachability, with cells that rupture upon debonding, and peel tissue that fragments easily. RESULTS: Structural studies indicated that peel detachability in both genotypes occurred in the outer pericarp beneath the hypodermis. Immunolabelling showed differences in methylesterification of pectin, where the interface of labelling coincided with the location of detachment in the good-peeling genotype, whereas in the poor-peeling genotype, no such interface existed. This zone of difference in methylesterification was enhanced by differential cell wall changes between the peel and outer pericarp tissue. Although both genotypes expressed two polygalacturonase genes, no enzyme activity was detected in the good-peeling genotype, suggesting limited pectin breakdown, keeping cell walls strong without tearing or fragmentation of the peel and flesh upon detachment. Differences in location and amounts of wall-stiffening galactan in the peel of the good-peeling genotype possibly contributed to this phenotype. Hemicellulose-acting transglycosylases were more active in the good-peeling genotype, suggesting an influence on peel flexibility by remodelling their substrates during development of detachability. High xyloglucanase activity in the peel of the good-peeling genotype may contribute by having a strengthening effect on the cellulose-xyloglucan network. CONCLUSIONS: In fruit of A. eriantha, peel detachability is due to the establishment of a zone of discontinuity created by differential cell wall changes in peel and outer pericarp tissues that lead to changes in mechanical properties of the peel. During ripening, the peel becomes flexible and the cells continue to adhere strongly to each other, preventing breakage, whereas the underlying outer pericarp loses cell wall strength as softening proceeds. Together these results reveal a novel and interesting mechanism for enabling cell separation.