Effects of inside-out heat-shock via microwave on the fruit softening and quality of persimmon during postharvest storage.

Rapid postharvest softening largely limits the shelf-life of persimmon (Diospyros kaki L.) fruit. Microwave is a new environmental-friendly inside-out heat-shock approach, whose effect on the fruit softening and quality has not yet been investigated. The current study applied two kinds of microwave treatments (low-power long-time, LPLT, or high-power short-time, HPST) to persimmon fruit with comparison to the hot water (HW) treatments. The results showed both microwave treatments maintained firmness, facilitated the deastringency, and increased soluble solid contents (SSC) and sugar-acid ratio of persimmon fruit. The microwave treatments reduced the cellulose and pectin degradation, and inhibited the cellulase activity, resulting in a significantly higher firmness than HW treatment and control after 2 and 4 days of storage. Moreover, application of HPST treatment down-regulated gene expression of DkPG1, DkPE2 and DkEGase1 compared with untreated fruits. These results indicated that microwave treatment is a promising soft-delaying method for the preservation of persimmon fruit.

MBW complexes impinge on anthocyanidin reductase gene regulation for proanthocyanidin biosynthesis in persimmon fruit.

MBW protein complexes containing MYB, bHLH and WD40 repeat factors are known transcriptional regulators of secondary metabolites production such as proanthocyanidins and anthocyanins, and developmental processes such as trichome formation in many plant species. DkMYB2 and DkMYB4 (MYB-type), DkMYC1 (bHLH-type) and DkWDR1 (WD40-type) factors have been proposed by different authors to take part of persimmon MBW complexes for proanthocyanidin accumulation in immature fruit, leading to its characteristic astringent flavour with important agronomical and ecological effects. We have confirmed the nuclear localization of these proteins and their mutual physical interaction by bimolecular fluorescence complementation analysis. In addition, transient expression of DkMYB2, DkMYB4 and DkMYC1 cooperatively increase the expression of a persimmon anthocyanidin reductase gene (ANR), involved in the biosynthesis of cis-flavan-3-ols, the structural units of proanthocyanidin compounds. Collectively, these data support the presence of MBW complexes in persimmon fruit and suggest their coordinated participation in ANR regulation for proanthocyanidin production.

DkWRKY interacts with pyruvate kinase gene DkPK1 and promotes natural deastringency in C-PCNA persimmon.

PAs, also known as condensed tannins, cause the astringency sensation in the persimmon fruit. The astringency of Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) can be naturally removed on the tree, but the regulatory mechanisms of deastringency remain to be elucidated. In our previous research, DkPK1 was shown to be involved in the natural loss of astringency of C-PCNA persimmon fruit. In the present study, yeast one-hybrid (Y1H) library screening using the DkPK1 promoter as baits identified two DkWRKY transcription factor genes (DkWRKY3 and -15). The transcript levels of both DkWRKY3 and -15 exhibited a positive correlation with the decrease in soluble proanthocyanidin (PA) content during the last developmental stage in C-PCNA persimmon. Multiple sequence analysis and subcellular localization confirmed that DkWRKY3 and -15 belonging to the group II and I families, respectively, were both located in the nucleus. Dual-luciferase and Y1H assays demonstrated that DkWRKY3 and -15 can transactivate the DkPK1 promoters. The combination of DkWRKY3 and -15 most likely produced an additive activation effect compared to a single activator on DkPK1, although the two transcriptional activators were not capable of interacting. Notably, DkWRKY3 and -15 showed ubiquitous expression in various organs and abundant upregulation in seeds. Furthermore, transient overexpression of both DkWRKY3 and -15 in persimmon leaves led to a significant decrease in the content of soluble PAs but a significant increase in the expression levels of the acetaldehyde metabolism-related DkPK, DkPDC and DkADH genes. Thus, we suggest that DkWRKY3 and -15 are the upstream regulators of DkPK1 and positively regulate the natural deastringency in C-PCNA persimmon.

Gene networks orchestrated by MeGI: a single-factor mechanism underlying sex determination in persimmon.

Separating male and female sex organs is one of the main strategies used to maintain genetic diversity within a species. However, the genetic determinants and their regulatory mechanisms have been identified in only a few species. In dioecious persimmons, the homeodomain transcription factor, MeGI, which is the target of a Y chromosome-encoded small-RNA, OGI, can determine floral sexuality. The basic features of this system are conserved in the monoecious hexaploid Oriental persimmon, in which an additional epigenetic regulation of MeGI determines floral sexuality. The downstream regulatory pathways of MeGI remain uncharacterized. In this study, we examined transcriptomic data for male and female flowers from monoecious persimmon cultivars to unveil the gene networks orchestrated by MeGI. A network visualization and cistrome assessment suggested that class-1 KNOTTED-like homeobox (KNOX)/ovate family protein (OFP)/growth regulating factors (GRFs) and short vegetative phase (SVP) genes mediate the differences in gynoecium and androecium development between male and female flowers, respectively. The expression of these genes is directly controlled by MeGI. The gene networks also suggested that some cytokinin, auxin, and gibberellin signaling genes function cooperatively in the KNOX/OFP/GRF pathway during gynoecium differentiation. Meanwhile, SVP may repress PI expression in developing androecia. Overall, our results suggest that MeGI evolved the ability to promote gynoecium development and suppress androecium development by regulating KNOX/OFP/GRF and SVP expression levels, respectively. These insights may help to clarify the molecular mechanism underlying the production of unisexual flowers, while also elucidating the physiological background enabling a single-factor system to establish dioecy in plants.

Hormonal control of parthenocarpic fruit set in 'Rojo Brillante' persimmon (Diospyros kaki Thunb.).

In persimmon (Diospyros kaki Thunb.), one to three waves of fruit abscission can occur. The parthenocarpic cv. Rojo Brillante may abscise close to 50% of flowers, which implies a major economic losses. In order to study this process, 700 flowers were labelled, 600 had the lobes of the calyx removed at three stages to promote abscission. Half of them were also treated with gibberellic acid (GA3; 10 mg l-1) to counteract the effect, and 100 were used as control. In the second year, GA3 (25 mg l-1) was applied to whole trees. Calyx lobe removal (CLR) reduced fruit growth rates and advanced and increased fruitlet abscission, whereas GA3 counteracted this effect. Furthermore, when GA3 was applied to the whole tree, fruit set was increased. The time-course of fruit abscission paralleled a decreased in hormonal and carbohydrate contents. Control fruit showed a peak of gibberellin (GA1 and GA4) and IAA concentration at anthesis. Hexose concentrations remained almost constant from flower bud to fruit set, whereas that of sucrose diminished with time. A peak in ethylene production occurred at anthesis, which increased when CLR was performed prior to or at anthesis, but not when performed at fruit set, when ethylene was markedly smaller. GA3 also counteracted it. Accordingly, we suggest that fruit set depends on the induction of gibberellin (GA) and IAA responses in the persimmon, and since there is no shortage of hormones or carbohydrates at anthesis, ethylene production at anthesis seems the most plausible cause of the physiological fruitlet abscission.

Review on the significance of chlorine for crop yield and quality.

The chloride concentration in the plant determines yield and quality formation for two reasons. First, chlorine is a mineral nutrient and deficiencies thereof induce metabolic problems that interfere with growth. However, due to low requirement of most crops, deficiency of chloride hardly appears in the field. Second, excess of chloride, an event that occurs under chloride-salinity, results in severe physiological dysfunctions impairing both quality and yield formation. The chloride ion can effect quality of plant-based products by conferring a salty taste that decreases market appeal of e.g. fruit juices and beverages. However, most of the quality impairments are based on physiological dysfunctions that arise under conditions of chloride-toxicity: Shelf life of persimmon is shortened due to an autocatalytic ethylene production in fruit tissues. High concentrations of chloride in the soil can increase phyto-availability of the heavy metal cadmium, accumulating in wheat grains above dietary intake thresholds. When crops are cultivated on soils that are moderately salinized by chloride, nitrate fertilization might be a strategy to suppress uptake of chloride by means of an antagonistic anion-anion uptake competition. Overall, knowledge about proteins that catalyse chloride-efflux out of the roots or that restrict xylem loading is needed to engineer more resistant crops.

Co-occurring woody species have diverse hydraulic strategies and mortality rates during an extreme drought.

From 2011 to 2013, Texas experienced its worst drought in recorded history. This event provided a unique natural experiment to assess species-specific responses to extreme drought and mortality of four co-occurring woody species: Quercus fusiformis, Diospyros texana, Prosopis glandulosa, and Juniperus ashei. We examined hypothesized mechanisms that could promote these species' diverse mortality patterns using postdrought measurements on surviving trees coupled to retrospective process modelling. The species exhibited a wide range of gas exchange responses, hydraulic strategies, and mortality rates. Multiple proposed indices of mortality mechanisms were inconsistent with the observed mortality patterns across species, including measures of the degree of iso/anisohydry, photosynthesis, carbohydrate depletion, and hydraulic safety margins. Large losses of spring and summer whole-tree conductance (driven by belowground losses of conductance) and shallower rooting depths were associated with species that exhibited greater mortality. Based on this retrospective analysis, we suggest that species more vulnerable to drought were more likely to have succumbed to hydraulic failure belowground.

DkXTH8, a novel xyloglucan endotransglucosylase/hydrolase in persimmon, alters cell wall structure and promotes leaf senescence and fruit postharvest softening.

Fruit softening is mainly associated with cell wall structural modifications, and members of the xyloglucan endotransglucosylase/hydrolase (XTH) family are key enzymes involved in cleaving and re-joining xyloglucan in the cell wall. In this work, we isolated a new XTH gene, DkXTH8, from persimmon fruit. Transcriptional profiling revealed that DkXTH8 peaked during dramatic fruit softening, and expression of DkXTH8 was stimulated by propylene and abscisic acid but suppressed by gibberellic acid and 1-MCP. Transient expression assays in onion epidermal cells indicated direct localization of DkXTH8 to the cell wall via its signal peptide. When expressed in vitro, the recombinant DkXTH8 protein exhibited strict xyloglucan endotransglycosylase activity, whereas no xyloglucan endohydrolase activity was observed. Furthermore, overexpression of DkXTH8 resulted in increased leaf senescence coupled with higher electrolyte leakage in Arabidopsis and faster fruit ripening and softening rates in tomato. Most importantly, transgenic plants overexpressing DkXTH8 displayed more irregular and twisted cells due to cell wall restructuring, resulting in wider interstitial spaces with less compact cells. We suggest that DkXTH8 expression causes cells to be easily destroyed, increases membrane permeability and cell peroxidation, and accelerates leaf senescence and fruit softening in transgenic plants.

Processes Driving the Adaptive Radiation of a Tropical Tree (Diospyros, Ebenaceae) in New Caledonia, a Biodiversity Hotspot.

Due to its special geological history, the New Caledonian Archipelago is a mosaic of soil types, and in combination with climatic conditions this results in a heterogeneous environment across relatively small distances. A group of over 20 endemic species of Diospyros (Ebenaceae) has rapidly and recently radiated on the archipelago after a single long-distance dispersal event. Most of the Diospyros species in the radiating group are morphologically and ecologically well differentiated, but they exhibit low levels of DNA variability. To investigate the processes that shaped the diversification of this group we employed restriction site associated DNA sequencing (RADseq). Over 8400 filtered SNPs generally confirm species delimitations and produce a well-supported phylogenetic tree. Our analyses document local introgression, but only a limited potential for gene flow over longer distances. The phylogenetic relationships point to an early regional clustering among populations and species, indicating that allopatric speciation with respect to macrohabitat (i.e., climatic conditions) may have had a role in the initial differentiation within the group. A later, more rapid radiation involved divergence with respect to microhabitat (i.e., soil preference). Several sister species in the group show a parallel divergence in edaphic preference. Searches for genomic regions that are systematically differentiated in this replicated phenotypic divergence pointed to loci potentially involved in ion binding and cellular transport. These loci appear meaningful in the context of adaptations to soil types that differ in heavy-metal and mineral content. Identical nucleotide changes affected only two of these loci, indicating that introgression may have played a limited role in their evolution. Our results suggest that both allopatric diversification and (parapatric) ecological divergence shaped successive rounds of speciation in the Diospyros radiation on New Caledonia.

Plant genetics. A Y-chromosome-encoded small RNA acts as a sex determinant in persimmons.

In plants, multiple lineages have evolved sex chromosomes independently, providing a powerful comparative framework, but few specific determinants controlling the expression of a specific sex have been identified. We investigated sex determinants in the Caucasian persimmon, Diospyros lotus, a dioecious plant with heterogametic males (XY). Male-specific short nucleotide sequences were used to define a male-determining region. A combination of transcriptomics and evolutionary approaches detected a Y-specific sex-determinant candidate, OGI, that displays male-specific conservation among Diospyros species. OGI encodes a small RNA targeting the autosomal MeGI gene, a homeodomain transcription factor regulating anther fertility in a dosage-dependent fashion. This identification of a feminizing gene suppressed by a Y-chromosome-encoded small RNA contributes to our understanding of the evolution of sex chromosome systems in higher plants.

Inactive xylem can explain differences in calibration factors for thermal dissipation probe sap flow measurements.

Thermal dissipation probes (TDPs) were calibrated in three diffuse porous fruit trees and one ornamental species in the field by comparison with heat pulse probes (nectarine and persimmon), in a greenhouse on lysimeters (apple and persimmon) and in the laboratory by pushing water through cut branches (apple, Peltophorum and nectarine). Two operational methods were used: continuous (constant thermal dissipation, CTD) and discontinuous, or transient, heating (transient thermal dissipation, TTD). Correction for the radial distribution of sap flux density was with an analytical function derived from a linear decrease in flux density with depth, as measured with a multi-depth 'Tmax' heat pulse system. When analyzed with previous calibration factors, the measured sap flow was <50% of actual value. The underestimations were consistent, and calibrations for each species in the field, greenhouse and laboratory gave approximately the same factors. Reasonable values of tree water use were obtained with the new calibration factors. Evidence is provided that even though the xylem was diffuse porous, the underestimations were caused by contact of the probes with inactive xylem along their length. The average portion of probe in contact with inactive xylem, measured in stained branches following laboratory calibrations, was 0.2-0.24. Using the measured fractions to correct temperature differentials between heated and unheated probes for CTD and TTD, based on Clearwater et al. (in Potential errors in measurement of nonuniform sap flow using heat dissipation probes. Tree Physiol 1999;19:681-687) almost completely compensated for the underestimations. Calibrations are given for each species both before and after corrections of temperature differentials, along with a multispecies calibration. These results should be an important step in reconciling many reports of different calibration factors for TDP probes.

In vitro propagation of persimmon (Diospyros kaki Thunb.).

Persimmon (Diospyros kaki Thunb.) is a temperate fruit tree species diffused in all continents. The traditional propagation method adopted by the nursery industry is based on budding/grafting scion cultivars on seedlings from D. kaki, Diospyros lotus, and Diospyros virginiana, the most important species used as rootstock, reproduced by seeds since they are not easy to root. Furthermore, most of nonastringent cultivars of persimmon are not compatible with D. lotus, a rootstock largely utilized because of its hardiness and frost resistance. The main in vitro tissue culture techniques, developed for persimmon, deal with direct regeneration (from dormant buds and root tips), and indirect regeneration through callus from dormant buds, apexes, and leaves. The bottlenecks of micropropagation are (1) the recalcitrance of many cultivars to in vitro establishment, (2) the low multiplication ratio of D. kaki compared to other fruit tree species, (3) the very low rooting ability of ex novo microcuttings both from direct and indirect regeneration, (4) the high sensitivity to transplant from in vitro to in vivo conditions. The development of reliable in vitro regeneration procedures is likely to play a key role for production of both clonal rootstocks and self-rooted cultivars. The general protocol for micropropagation of persimmon reported here is based on the establishment of winter dormant buds in vitro, shoot development, multiplication and elongation, and shoot rooting, using cytokinins (BA or zeatin) in a MS media along with an auxinic pretreatment for rooting induction.

Cryopreservation of in vitro grown shoot tips of Diospyros kaki thunb. using different methods.

The objective of this study was to compare the potential of different cryopreservation strategies for in vitro shoot tips of Diospyros kaki Thunb. The treatments consisted of three different cryopreservation methods: vitrification, droplet-vitrification and modified droplet-vitrification. The following variables were assessed: cold acclimation, sucrose concentration in the preculture medium and PVS2 treatment time. A higher average survival level was obtained using the modified droplet-vitrification method compared to the other two methods.

Expression of ethylene response genes during persimmon fruit astringency removal.

Thirteen ethylene signaling related genes were isolated and studied during ripening of non-astringent 'Yangfeng' and astringent 'Mopan' persimmon fruit. Some of these genes were characterized as ethylene responsive. Treatments, including ethylene and CO(2), had different effects on persimmon ripening, but overlapping roles in astringency removal, such as increasing the reduction in levels of soluble tannins. DkERS1, DkETR2, and DkERF8, may participate in persimmon fruit ripening and softening. The expression patterns of DkETR2, DkERF4, and DkERF5 had significant correlations with decreases in soluble tannins in 'Mopan' persimmon fruit, suggesting that these genes might be key components in persimmon fruit astringency removal and be the linkage between different treatments, while DkERF1 and DkERF6 may be specifically involved in CO(2) induced astringency removal. The possible roles of ethylene signaling genes in persimmon fruit astringency removal are discussed.

Resurrecting extinct interactions with extant substitutes.

There is increasing evidence that restoration ecologists should be most concerned with restoring species interactions rather than species diversity per se [1]. Rewilding with taxon substitutes, the intentional introduction of exotic species to replace the ecosystem functions of recently extinct species, is one way to reverse ecosystem dysfunction following the loss of species interactions [2]. This is highly controversial [3], in part because of a lack of rigorous scientific studies [4]. Here we present the first empirical evidence of an in situ rewilding project undertaken as a hypothesis-driven ecosystem management option. On Ile aux Aigrettes, a 25-hectare island off Mauritius, the critically endangered large-fruited endemic ebony, Diospyros egrettarum (Ebenaceae), was seed-dispersal limited after the extinction of all native large-bodied frugivores, including giant tortoises. We introduced exotic Aldabra giant tortoises, Aldabrachelys gigantea, to disperse the ebony seeds. Not only did the tortoises ingest the large fruits and disperse substantial numbers of ebony seeds, but tortoise gut passage also improved seed germination, leading to the widespread, successful establishment of new ebony seedlings. Our results demonstrate that the introduction of these exotic frugivores is aiding the recovery of ebonies. We argue for more reversible rewilding experiments to investigate whether extinct species interactions can be restored.

[Effects of climate change on photosynthesis of Diospyros kaki under different soil moisture conditions].

Taking two-year-old Diospyros kaki as test material, this paper studied the effects of high CO2 concentration (700 micromol x mol(-1)), high temperature (5 degrees C higher than the mean daily temperature); and their combination on the net photosynthesis rate (Pn), evapotranspiration (Tr), stomatal conductance (Gs), water use efficiency (WUE), chlorophyll content, Fv/Fm, and Fv/Fo under different soil moisture conditions, with the ambient air temperature and CO2 concentration (380 micromol x mol(-1)) as the control. Under all test soil moisture conditions, the combination of high CO2 concentration and high temperature decreased the Tr and Gs, but increased the WUE. This combination increased the Pn when the soil moisture content was 75%-85% and 55%-65% of field capacity, but decreased the Pn when the soil moisture content was 35%-45%. High CO2 concentration increased the Pn and WUE but decreased the Gs and Tr under all test soil moisture conditions. The effects of high temperature and its combination with high CO2 concentration on the WUE depended on soil moisture condition, with the WUE increased with increasing soil moisture content. Comparing with the control, high CO2 concentration also increased the leaf Chla, Chlb, Chl (a + b), and Car concentrations and the Fv/Fm and Fv/Fo values, relieved the water stress, and increased the stress-resistance of D. kaki.

[Changes in cell wall component metabolism and ultrastructure of postharvest persimmon fruit during softening].

The changes in cellular wall hydrolases, cellular wall components and cellular wall ultrastructure of postharvest persimmon (Diospyros kaki L. cv. Bianhua) fruit during softening were studied. Pectinesterase activity increased sharply at first and reached a peak (Fig.3A). Significant correlation was observed between the pectinesterase activity and the loss of flesh firmness (r= -0.74). Polygalacturonase activity increased slowly (Fig.3B), but there was no significant correlation between the polygalacturonase activity and the loss of flesh firmness. Beta-galactosidase activity increased sharply (Fig.3C), with a negative correlation between the b-galactosidase activity and the loss of flesh fruit firmness (r= -0.77). Cellulase activity increased markedly during ripening (Fig.3D). Significant correlation was observed between cellulase activity and the loss of flesh firmness (r= -0.90). Consistent with the increases in activity of cell wall hydrolases of cell wall constituents, fruit softening was accompanied by a progressive increase in WSP (water soluble pectin) content and a progressive decrease in protopectin and cellulose content (Fig.4). The cell wall structure was integrated when persimmon was harvested (Fig.5A). After 3 d of ripening, the middle lamella became liquefied (Fig.5B), or even the primary cell wall was dissolved in some regions (Fig.5C).