Cytotype distribution and chloroplast phylogeography of the Actinidia chinensis complex.

BACKGROUND: Plant phylogeographic studies of species in subtropical China have mainly focused on rare and endangered species, whereas few studies have been conducted on taxa with relatively wide distribution, especially polyploid species. We investigated the cytotype and haplotype distribution pattern of the Actinidia chinensis complex, a widespread geographically woody liana with variable ploidy in subtropical China comprising two varieties, with three chloroplast fragments DNA (ndhF-rpl132, rps16-trnQ and trnE-trnT). Macroevolutionary, microevolutionary and niche modeling tools were also combined to disentangle the origin and the demographic history of the species or cytotypes. RESULTS: The ploidy levels of 3338 individuals from 128 populations sampled throughout the species distribution range were estimated with flow cytometry. The widespread cytotypes were diploids followed by tetraploids and hexaploids, whereas triploids and octoploids occurred in a few populations. Thirty-one chloroplast haplotypes were detected. The genetic diversity and genetic structure were found to be high between varieties (or ploidy races) chinensis and deliciosa. Our results revealed that these two varieties inhabit significantly different climatic niche spaces. Ecological niche models (ENMs) indicate that all varieties' ranges contracted during the Last Inter Glacial (LIG), and expanded eastward or northward during the Last Glacial Maximum (LGM). CONCLUSIONS: Pliocene and Plio-Pleistocene climatic fluctuations and vicariance appear to have played key roles in shaping current population structure and historical demography in the A. chinensis complex. The polyploidization process also appears to have played an important role in the historical demography of the complex through improving their adaptability to environmental changes.

Protuberances are organized distinct regions of long-term callus: histological and transcriptomic analyses in kiwifruit.

KEY MESSAGE: Macroscopic, ultrastructural, and molecular features-like a ball shape, the presence of starch granules, and the up-regulation of genes involved in carbohydrate metabolism and secondary metabolite biosynthesis-distinguish PT regions within a callus. The modification of the mass of pluripotent cells into de novo shoot bud regeneration is highly relevant to developmental biology and for agriculture and biotechnology. This study deals with protuberances (PT), structures that appear during the organogenic long-term culturing of callus (OC) in kiwifruit. These ball-shaped regions of callus might be considered the first morphological sign of the subsequent shoot bud development. Sections of PT show the regular arrangement of some cells, especially on the surface, in contrast to the regions of OC beyond the PT. The cells of OC possess chloroplasts; however, starch granules were observed only in PTs' plastids. Transcriptomic data revealed unique gene expression for each kind of sample: OC, PT, and PT with visible shoot buds (PT-SH). Higher expression of the gene involved in lipid (glycerol-3-phosphate acyltransferase 5 [GPAT5]), carbohydrate (granule-bound starch synthase 1 [GBSS1]), and secondary metabolite (beta-glucosidase 45 [BGL45]) pathways were detected in PT and could be proposed as the markers of these structures. The up-regulation of the regulatory associated protein of TOR (RAPTOR1) was found in PT-SH. The highest expression of the actinidain gene in leaves from two-year-old regenerated plants suggests that the synthesis of this protein takes place in fully developed organs. The findings indicate that PT and PT-SH are specific structures within OC but have more features in common with callus tissue than with organs.

Extracellular matrix and wall composition are diverse in the organogenic and non-organogenic calli of Actinidia arguta.

KEY MESSAGE: Differences in the composition and the structural organisation of the extracellular matrix correlate with the morphogenic competence of the callus tissue that originated from the isolated endosperm of kiwifruit. The chemical composition and structural organisation of the extracellular matrix, including the cell wall and the layer on its surface, may correspond with the morphogenic competence of a tissue. In the presented study, this relationship was found in the callus tissue that had been differentiated from the isolated endosperm of the kiwiberry, Actinidia arguta. The experimental system was based on callus samples of exactly the same age that had originated from an isolated endosperm but were cultured under controlled conditions promoting either an organogenic or a non-organogenic pathway. The analyses which were performed using bright field, fluorescence and scanning electron microscopy techniques showed significant differences between the two types of calli. The organogenic tissue was compact and the outer walls of the peripheral cells were covered with granular structures. The non-organogenic tissue was composed of loosely attached cells, which were connected via a net-like structure. The extracellular matrices from both the non- and organogenic tissues were abundant in pectic homogalacturonan and extensins (LM19, LM20, JIM11, JIM12 and JIM20 epitopes), but the epitopes that are characteristic for rhamnogalacturonan I (LM5 and LM6), hemicellulose (LM25) and the arabinogalactan protein (LM2) were detected only in the non-organogenic callus. Moreover, we report the epitopes, which presence is characteristic for the Actinidia endosperm (LM21 and LM25, heteromannan and xyloglucan) and for the endosperm-derived cells that undergo dedifferentiation (loss of LM21 and LM25; appearance or increase in the content of LM5, LM6, LM19, JIM11, JIM12, JIM20, JIM8 and JIM16 epitopes).

Genetic and cytological analyses reveal the recombination landscape of a partially differentiated plant sex chromosome in kiwifruit.

BACKGROUND: Angiosperm sex chromosomes, where present, are generally recently evolved. The key step in initiating the development of sex chromosomes from autosomes is the establishment of a sex-determining locus within a region of non-recombination. To better understand early sex chromosome evolution, it is important to determine the process by which recombination is suppressed around the sex determining genes. We have used the dioecious angiosperm kiwifruit Actinidia chinensis var. chinensis, which has an active-Y sex chromosome system, to study recombination rates around the sex locus, to better understand key events in the development of sex chromosomes. RESULTS: We have confirmed the sex-determining region (SDR) in A. chinensis var. chinensis, using a combination of high density genetic mapping and fluorescent in situ hybridisation (FISH) of Bacterial Artificial Chromosomes (BACs) linked to the sex markers onto pachytene chromosomes. The SDR is a subtelomeric non-recombining region adjacent to the nucleolar organiser region (NOR). A region of restricted recombination of around 6 Mbp in size in both male and female maps spans the SDR and covers around a third of chromosome 25. CONCLUSIONS: As recombination is suppressed over a similar region between X chromosomes and between and X and Y chromosomes, we propose that recombination is suppressed in this region because of the proximity of the NOR and the centromere, with both the NOR and centromere suppressing recombination, and this predates suppressed recombination due to differences between X and Y chromosomes. Such regions of suppressed recombination in the genome provide an opportunity for the evolution of sex chromosomes, if a sex-determining locus develops there or translocates into this region.

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.

Studies on the Infection, Colonization, and Movement of Pseudomonas syringae pv. actinidiae in Kiwifruit Tissues Using a GFPuv-Labeled Strain.

Kiwifruit bacterial canker, an economically important disease caused by Pseudomonas syringae pv. actinidiae (Psa), has caused severe losses in all major areas of kiwifruit cultivation. Using a GFPuv-labeled strain of Psa, we monitored the invasion, colonization, and movement of the pathogen in kiwifruit twigs, leaves and veins. The pathogen can invade twigs through both wounds and natural openings; the highest number of Psa is obtained in cut tissues. We determined that, following spray inoculation, Psa-GFPuv could infect leaves and cause lesions in the presence and absence of wounds. Light and transmission electron microscopic observations showed that bacterial cells colonize both phloem and xylem vessels. Bacterial infection resulted in marked alterations of host tissues including the disintegration of organelles and degeneration of protoplasts and cell walls. Furthermore, low temperature was conducive to colonization and movement of Psa-GFPuv in kiwifruit tissues. Indeed, the pathogen migrated faster at 4°C than at 16°C or 25°C in twigs. However, the optimum temperature for colonization and movement of Psa in leaf veins was 16°C. Our results, revealing a better understanding of the Psa infection process, might contribute to develop more efficacious disease management strategies.

High-temperature inhibition of biosynthesis and transportation of anthocyanins results in the poor red coloration in red-fleshed Actinidia chinensis.

In plants, the role of anthocyanins trafficking in response to high temperature has been rarely studied, and therefore poorly understood. Red-fleshed kiwifruit has stimulated the world kiwifruit industry owing to its appealing color. However, fruit in warmer climates have been found to have poor flesh coloration, and the factors responsible for this response remain elusive. Partial correlation and regression analysis confirmed that accumulative temperatures above 25 °C (T25) was one of the dominant factors inhibiting anthocyanin accumulation in red-fleshed Actinidia chinensis, 'Hongyang'. Expression of structural genes, AcMRP and AcMYB1 in inner pericarp sampled from the two high altitudes (low temperature area), was notably higher than the low altitude (high temperature area) during fruit coloration. AcMYB1 and structural genes coordinate expression supported the MYB-bHLH (basic helix-loop-helix)-WD40 regulatory complex mediated downregulation of anthocyanin biosynthesis induced by high temperatures in kiwifruit. Moreover, cytological observations using the light and transmission electronic microscopy showed that there were a series of anthocyanic vacuolar inclusion (AVI)-like structures involved in their vacuolization process and dissolution of the pigmented bodies inside cells of fruit inner pericarp. Anthocyanin transport was inhibited by high temperature via retardation of vacuolization or reduction in AIV-like structure formation. Our findings strongly suggested that complex multimechanisms influenced the effects of high temperature on red-fleshed kiwifruit coloration.

Ozone-induced kiwifruit ripening delay is mediated by ethylene biosynthesis inhibition and cell wall dismantling regulation.

Ozone treatments are used to preserve quality during cold storage of commercially important fruits due to its ethylene oxidizing capacity and its antimicrobial attributes. To address whether or not ozone also modulates ripening by directly affecting fruit physiology, kiwifruit (Actinidia deliciosa cv. 'Hayward') were stored in very low ethylene atmosphere at 0°C (95% RH) in air (control) or in the presence of ozone (0.3µLL(-1)) for 2 or 4 months and subsequently ripened at 20°C (90% RH) for up to 8d. Ozone-treated kiwifruit showed a significant delay of ripening during maintenance at 20°C, accompanied by a marked decrease in ethylene biosynthesis due to inhibited AdACS1 and AdACO1 expression and reduced ACC synthase (ACS) and ACC oxidase (ACO) enzyme activity. Furthermore, ozone-treated fruit exhibited a marked reduction in flesh softening and cell wall disassembly. This effect was associated with reduced cell wall swelling and pectin and neutral sugar solubilization and was correlated with the inhibition of cell wall degrading enzymes activity, such as polygalacturonase (PG) and endo-1,4-ß-glucanase/1,4-ß-glucosidase (EGase/glu). Conclusively, the present study indicated that ozone may exert major residual effects in fruit ripening physiology and suggested that ethylene biosynthesis and cell walls turnover are specifically targeted by ozone.

Tapetum and middle layer control male fertility in Actinidia deliciosa.

BACKGROUND AND AIMS: Dioecism characterizes many crop species of economic value, including kiwifruit (Actinidia deliciosa). Kiwifruit male sterility occurs at the microspore stage. The cell walls of the microspores and the pollen of the male-sterile and male-fertile flowers, respectively, differ in glucose and galactose levels. In numerous plants, pollen formation involves normal functioning and degeneration timing of the tapetum, with calcium and carbohydrates provided by the tapetum essential for male fertility. The aim of this study was to determine whether the anther wall controls male fertility in kiwifruit, providing calcium and carbohydrates to the microspores. METHODS: The events occurring in the anther wall and microspores of male-fertile and male-sterile anthers were investigated by analyses of light microscopy, epifluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL assay) and transmission electron microscopy coupled with electron spectroscopy. The possibility that male sterility was related to anther tissue malfunctioning with regard to calcium/glucose/galactose provision to the microspores was also investigated by in vitro anther culture. KEY RESULTS: Both tapetum and the middle layer showed secretory activity and both degenerated by programmed cell death (PCD), but PCD was later in male-sterile than in male-fertile anthers. Calcium accumulated in cell walls of the middle layer and tapetum and in the exine of microspores and pollen, reaching higher levels in anther wall tissues and dead microspores of male-sterile anthers. A specific supply of glucose and calcium induced normal pollen formation in in vitro-cultured anthers of the male-sterile genotype. CONCLUSIONS: The results show that male sterility in kiwifruit is induced by anther wall tissues through prolonged secretory activity caused by a delay in PCD, in the middle layer in particular. In vitro culture results support the sporophytic control of male fertility in kiwifruit and open the way to applications to overcome dioecism and optimize kiwifruit production.

Cutin plays a role in differentiation of endosperm-derived callus of kiwifruit.

Cutin fluorescence, after auramine O treatment, was detected on the surface of organogenic areas (protuberances) of endosperm derived callus induced on Murashige and Skoog medium with thidiazuron (0.5 mg l(-1)) in darkness. Electron micrographs of the protuberances revealed cuticle, visible as a dark-staining layer, and amorphous waxes on the cell wall. In some cases the cells of the epidermis-like layer and shoot buds at early stages of development showed thick and characteristically wavy cutin. This waviness corresponds with the wrinkled appearance of the cell wall as observed by scanning electron microscopy. The role of multivesicular bodies in cutin production and transfer to the plasma membrane is discussed.

Artificial neural networks as an alternative to the traditional statistical methodology in plant research.

In this work, we compared the unique artificial neural networks (ANNs) technology with the usual statistical analysis to establish its utility as an alternative methodology in plant research. For this purpose, we selected a simple in vitro proliferation experiment with the aim of evaluating the effects of light intensity and sucrose concentration on the success of the explant proliferation and finally, of optimizing the process taking into account any influencing factors. After data analysis, the traditional statistical procedure and ANNs technology both indicated that low light treatments and high sucrose concentrations are required for the highest kiwifruit microshoot proliferation under experimental conditions. However, this particular ANNs software is able to model and optimize the process to estimate the best conditions and does not need an extremely specialized background. The potential of the ANNs approach for analyzing plant biology processes, in this case, plant tissue culture data, is discussed.

The cell wall of kiwifruit pollen tubes is a target for chromium toxicity: alterations to morphology, callose pattern and arabinogalactan protein distribution.

Trivalent chromium has previously been found to effectively inhibit kiwifruit pollen tube emergence and elongation in vitro. In the present study, a photometric measure of increases in tube wall production during germination showed that 25 and 50 mum CrCl(3) treatment induced a substantial reduction in levels of polysaccharides in walls over those in controls. Moreover, chromium-treated kiwifruit pollen tubes had irregular and indented cell walls. Callose, the major tube wall polysaccharide, was deposited in an anomalous punctuate pattern. Arabinogalactan proteins (AGPs), which are integral in maintaining correct tube growth and shape in kiwifruit pollen, were found to be strongly altered in their distribution after CrCl(3) treatment compared to control tube walls. Transmission electron microscopy-immunogold analysis using four monoclonal antibodies (JIM8, JIM13, JIM14 and MAC207) revealed discontinuous AGP distribution within the treated tube walls. Such clearly discernable alterations in the molecular and morphological architecture of pollen tube walls may be detrimental in vivo for the male gametophyte to accomplish its vital role in the fertilisation process.

Changes in the cell-wall polysaccharides of outer pericarp tissues of kiwifruit during development.

Changes in pectin, hemicelluloses and cellulose in the cell walls of outer pericarp tissues of kiwifruit (Actinidia deliciosa cv. Hayward) were determined during development. An extensive amylase digestion was employed to remove possible contaminating starch before and after fractionation of wall polysaccharides. An initial treatment of crude cell walls with alpha-amylase and iso-amylase or DMSO, was found to be insufficient removing the contaminating starch from wall polysaccharides. After EDTA and alkaline extraction, the pectic and hemicellulose fractions were again treated with the combination of alpha-amylase and iso-amylase. The amounts of predominant pectic sugars Gal, Rha and Ara, unaffected by the first and second amylase digestion, decreased markedly during the early fruit enlargement (8-12 weeks after anthesis, WAA), then increased during 16-20 WAA, and finally declined during fruit maturity (20-25 WAA). The molecular-mass of pectic polysaccharides decreased during fruit enlargement (8-16 WAA), and then changed little during fruit maturity. The higher molecular-mass components of hemicelluloses in HC-I and HC-II fractions detected at the early stage of fruit enlargement (8-12 WAA) were degraded at the late stage of fruit enlargement (16 WAA), but then remained stable at the much lower molecular-mass till fruit maturity. The amount of Xyl in the HC-II fraction decreased during the early fruit enlargement and fruit maturity, an observation that was consistent with xyloglucan (XG) content. The gel permeation profiles of XG showed a slight increase in higher molecular-mass components during 8-12 WAA, but thereafter there was no significant down-shift of molecular-mass until harvest time. The cellulose fraction increased steadily during fruit enlargement through maturity, but the XG contents in HC-I and HC-II fractions remained at a low level during these stages. Methylation analysis of HC-I and HC-II fractions confirmed the low level of XG in the hemicellulosic fractions. It was suggested that pectin in the outer pericarp of kiwifruit was degraded at the early stage of fruit enlargement, but XG remains constant during fruit enlargement and maturation.

Morphological anomalies in pollen tubes of Actinidia deliciosa (kiwi) exposed to 50 Hz magnetic field.

The role of the pollen grain, with respect to the reproductive process of higher plants, is to deliver the spermatic cells to the embryo sac for egg fertilisation. Delivery occurs through the pollen tube, a self produced organ that is generated when the pollen grain reaches the stigma surface. The effect of magnetic fields on pollen tube growth was reported in a recent publication by Germanà et al. Pollen tube growth is an interesting candidate for the detailed study of the effects of electromagnetic fields on cytoplasmic structures and organelles. In this research Actinidia deliciosa (kiwifruit) pollen grains were germinated in the presence of an alternating magnetic field (50 Hz). Our results, although of preliminary nature, show that pollen tube growth is affected by magnetic fields. The analysis of the observed anomalies in the pollen tube appear to be the result of changes in the ionic charges within the pollen tube cytoplasm.

Programmed cell death induces male sterility in Actinidia deliciosa female flowers.

The importance of programmed cell death (PCD) during the life cycle of plants is well established, although the underlying molecular mechanisms are still poorly defined. An emerging system for the study of PCD during development in plants is that of sex organ abortion. In this work we investigate the degeneration of microspores in the anthers of Actinidia deliciosa female flowers. The kiwifruit, A. deliciosa, is a dioecious species native to China. Pollen development in female flowers is equivalent to pollen development in the male flowers, until the microspores are released from the tetrads. At this time the first differences appear, and include the condensation and shrinkage of the cytoplasm, blebbing of the plasma membrane and of the nuclear envelope, and condensation of chromatin. However, at the time these events are occurring, all other cellular organelles, including mitochondria, have their structures well preserved. Fragmentation of DNA was detected in situ by the TUNEL procedure, which involves the end labeling of the DNA fragments by terminal deoxynucleotidyl transferase with UTP conjugated to a detectable marker. This assay confirmed the morphological characterization of PCD in this system.

A one-step organelle capture: gynogenetic kiwifruits with paternal chloroplasts.

Androgenesis, the development of a haploid embryo from a male nucleus, has been shown to result in the instantaneous uncoupling of the transmission of the organelle and nuclear genomes (with the nuclear genome originating from the male parent only and the organelle genomes from the female parent). We report, for the first time, uncoupling resulting from gynogenesis, in Actinidia deliciosa (kiwifruit), a plant species known for its paternal mode of chloroplast inheritance. After pollen irradiation, transmission of nuclear genes from the pollen parent to the progeny was inhibited, but transmission of the chloroplast genome was not. This demonstrates that plastids can be discharged from the pollen tube into the egg with little or no concomitant transmission of paternal nuclear genes. Such events of opposite inheritance of the organelle and nuclear genomes must be very rare in nature and are unlikely to endanger the long-term stability of the association between the different genomes of the cell. However, they could lead to incongruences between organelle gene trees and species trees and may constitute an alternative to the hybridization/introgression scenario commonly invoked to account for such incongruences.