Integrated transcriptomic and proteomic analysis reveals the complex molecular mechanisms underlying stone cell formation in Korla pear.

Korla pear (Pyrus sinkiangensis Yü) is a landrace selected from a hybrid pear species in the Xinjiang Autonomous Region in China. In recent years, pericarp roughening has been one of the major factors that adversely affects fruit quality. Compared with regular fruits, rough-skin fruits have a greater stone cell content. Stone cells compose sclerenchyma tissue that is formed by secondary thickening of parenchyma cell walls. In this work, we determined the main components of stone cells by isolating them from the pulp of rough-skin fruits at the ripening stage. Stone cell staining and apoptosis detection were then performed on fruit samples that were collected at three different developmental stages (20, 50 and 80 days after flowering (DAF)) representing the prime, late and stationary stages of stone cell differentiation, respectively. The same batches of samples were used for parallel transcriptomic and proteomic analysis to identify candidate genes and proteins that are related to SCW biogenesis in Korla pear fruits. The results showed that stone cells are mainly composed of cellulose (52%), hemicellulose (23%), lignin (20%) and a small amount of polysaccharides (3%). The periods of stone cell differentiation and cell apoptosis were synchronous and primarily occurred from 0 to 50 DAF. The stone cell components increased abundantly at 20 DAF but then decreased gradually. A total of 24,268 differentially expressed genes (DEGs) and 1011 differentially accumulated proteins (DAPs) were identified from the transcriptomic and proteomic data, respectively. We screened the DEGs and DAPs that were enriched in SCW-related pathways, including those associated with lignin biosynthesis (94 DEGs and 31 DAPs), cellulose and xylan biosynthesis (46 DEGs and 18 DAPs), S-adenosylmethionine (SAM) metabolic processes (10 DEGs and 3 DAPs), apoplastic ROS production (16 DEGs and 2 DAPs), and cell death (14 DEGs and 6 DAPs). Among the identified DEGs and DAPs, 63 significantly changed at both the transcript and protein levels during the experimental periods. In addition, the majority of these identified genes and proteins were expressed the most at the prime stage of stone cell differentiation, but their levels gradually decreased at the later stages.

Benzoate-CoA ligase contributes to the biosynthesis of biphenyl phytoalexins in elicitor-treated pear cell cultures.

KEY MESSAGE: Benzoate-Coenzyme A ligase enzyme activity catalyzing the conversion of free benzoic acid to benzoyl-CoA was detected and biochemically characterized in the elicitor-treated pear cell cultures. Asian pear (Pyrus pyrifolia) is an economically and nutritionally important fruit-bearing tree of the subtribe Malinae. Upon pathogen attack, pears produce unique benzoate-derived biphenyl phytoalexins. The upstream biosynthesis of the biphenyl in Malinae is still incomplete. Previously, protein preparations from yeast extract-treated pear cultures were able to convert L-phenylalanine to cinnamic acid catalyzed by the activity of the phenylalanine ammonia lyase. The same extract was able to perform a C2 side-chain cleavage of cinnamic acid to benzaldehyde followed by oxidation of the latter to benzoic acid owing to the molecularly-undefined benzaldehyde synthase and benzaldehyde dehydrogenase activities, respectively. The biosynthesis of biphenyls starts with benzoate-Coenzyme A ligase (BZL), which converts benzoic acid to benzoyl-CoA. Subsequently, the previously-defined biphenyl synthase uses benzoyl-CoA to form the biphenyls. The current study reports the first time detection and characterization of BZL activity in elicitor-treated pear cell cultures. The preferred substrate was benzoic acid (Km = 62 ± 4 µM). Magnesium or manganese was prerequisite for the activity, which was enhanced by ~ 70% in the presence of potassium. Maximum BZL activity was observed 18 h post elicitation, which is in agreement with the coordinate induction reported for the enzymes in the same pathway. The induced BZL activity preceded the accumulation of biphenyls supporting its involvement in their biosynthesis.

Comparative Transcriptomic Analysis Provides Insight into the Domestication and Improvement of Pear (P. pyrifolia) Fruit.

Knowledge of the genetic changes that occurred during the domestication and improvement of perennial trees at the RNA level is limited. Here, we used RNA sequencing analysis to compare representative sets of wild, landrace, and improved accessions of pear (Pyrus pyrifolia) to gain insight into the genetic changes associated with domestication and improvement. A close population relationship and similar nucleotide diversity was observed between the wild and landrace groups, whereas the improved group had substantially reduced nucleotide diversity. A total of 11.13 Mb of genome sequence was identified as bearing the signature of selective sweeps that occurred during pear domestication, whereas a distinct and smaller set of genomic regions (4.04 Mb) was identified as being associated with subsequent improvement efforts. The expression diversity of selected genes exhibited a 20.89% reduction from the wild group to the landrace group, but a 23.13% recovery was observed from the landrace to the improved group, showing a distinctly different pattern with variation of sequence diversity. Module-trait association analysis identified 16 distinct coexpression modules, six of which were highly associated with important fruit traits. The candidate trait-linked differentially expressed genes associated with stone cell formation, fruit size, and sugar content were identified in the selected regions, and many of these could also be mapped to the previously reported quantitative trait loci. Thus, our study reveals the specific pattern of domestication and improvement of perennial trees at the transcriptome level, and provides valuable genetic sources of fruit traits that could contribute to pear breeding and improvement.

More for less: Improving the biomass yield of a pear cell suspension culture by design of experiments.

Plant cell suspension cultures are widely used for the production of recombinant proteins and secondary metabolites. One of the most important steps during process development is the optimization of yields by testing different cultivation parameters, including the components of the growth medium. However, we have shown that the biomass yield of a cell suspension culture derived from the pear cultivar Pyrus communis cv. Champagner Bratbirne can be significantly improved solely by varying the temperature, inoculum density, illumination, and incubation time. In contrast to medium optimization, these simple physical factors are easily controlled and varied, thereby reducing the effort required. Using an experimental design approach, we improved the biomass yield from 146 g fresh weight (FW)/L to 407 g FW/L in only 5 weeks, simultaneously reducing the costs of goods sold per kg biomass from € 125 to € 45. Our simple approach therefore offers a rapid, efficient and economical process for the optimization of plant cell suspension cultures.

The stiffening of the cell walls observed during physiological softening of pears.

MAIN CONCLUSION: The Young's modulus of the primary cell walls of pears decreases linearly during the pre-harvest on-tree maturation and increases during postharvest storage, and does not correlate with firmness of fruit. The determination of mechanical properties of cell walls is indispensable for understanding the mechanism of physiological softening and deterioration of quality of fruits during postharvest storage. The Young's modulus of the primary cell walls from pear fruit (Pyrus communis L., cultivars 'Conference' and 'Xenia') during pre-harvest maturation and postharvest storage in an ambient atmosphere at 2 °C followed by shelf life was studied using atomic force microscopy (AFM). The results were related to the firmness of fruits, galacturonic acid content in water, chelator, sodium carbonate and insoluble pectin fractions, polygalacturonase and pectin methylesterase activities. The Young's modulus of the primary cell walls decreased linearly during the last month of pre-harvest maturation from 3.2 ± 1.8 to 1.1 ± 0.7 MPa for 'Conference' and from 1.9 ± 1.2 to 0.2 ± 0.1 MPa for 'Xenia' which correlated with linear firmness decrease. During postharvest storage the cell wall Young's modulus increased while firmness continued to decrease. Correlation analysis for the entire period of the experiment showed a lack of straightforward relation between the Young's modulus of primary cell walls and fruit firmness. The Young's modulus of cell walls correlated negatively either with galacturonic acid content in sodium carbonate soluble pectin ('Conference') or with insoluble pectin fractions ('Xenia') and positively with polygalacturonase activity. It was therefore evidenced that covalently linked pectins play the key role for the stiffness of fruit cell walls. Based on the obtained results, the model explaining the fruit transition from firm and crispy to soft and mealy was proposed.

Natural polyamines and synthetic analogs modify the growth and the morphology of Pyrus communis pollen tubes affecting ROS levels and causing cell death.

Polyamines (PAs) are small molecules necessary for pollen maturation and tube growth. Their role is often controversial, since they may act as pro-survival factors as well as factors promoting Programmed Cell Death (PCD). The aim of the present work was to evaluate the effect of exogenous PAs on the apical growth of pear (Pyrus communis) pollen tube and to understand if PAs and reactive oxygen species (ROS) are interconnected in the process of tip-growth. In the present study besides natural PAs, also aryl-substituted spermine and methoctramine (Met 6-8-6) analogs were tested. Among the natural PAs, Spm showed strongest effects on tube growth. Spm entered through the pollen tube tip, then diffused in the sub-apical region that underwent drastic morphological changes, showing enlarged tip. Analogs were mostly less efficient than natural PAs but BD23, an asymmetric synthetic PAs bearing a pyridine ring, showed similar effects. These effects were related to the ability of PAs to cause the decrease of ROS level in the apical zone, leading to cell death, counteracted by the caspase-3 inhibitor Ac-DEVD-CHO (DEVD). In conclusions, ROS are essential for pollen germination and a strict correlation between ROS regulation and PA concentration is reported. Moreover, an imbalance between ROS and PAs can be detrimental thereby driving pollen toward cell death.

Development of flower buds in the Japanese pear (Pyrus pyrifolia) from late autumn to early spring.

We periodically investigated the lateral flower bud morphology of 1-year shoots of 'Kosui' pears (Pyrus pyrifolia Nakai) in terms of dormancy progression, using magnetic resonance imaging. The size of flower buds did not change significantly during endodormancy, but rapid enlargement took place at the end of the ecodormancy stage. To gain insight into the physiological status during this period, we analyzed gene expression related to cell cycle-, cell expansion- and water channel-related genes, namely cyclin (CYC), expansin (EXPA), tonoplast intrinsic proteins (TIP) and plasma membrane intrinsic proteins (PIP). Constant but low expression of pear cyclin genes (PpCYCD3s) was observed in the transition phase from endodormancy to ecodormancy. The expression levels of PpCYCD3s were consistent with few changes in flower bud size, but up-regulated before the sprouting stage. In contrast, the expression of pear expansin and water channel-related genes (PpEXPA2, PpPIP2A, PpPIP2B, PpIδTIP1A and PpIδTIP1B) were low until onset of the rapid enlargement stage of flower buds. However, expression of these genes rapidly increased during sprouting along with a gradual increase of free water content in the floral primordia of buds. Taken together, these results suggest that flower bud size tends to stay constant until the endodormancy phase transition. Rapid enlargement of flower buds observed in March is partly due to the enhancement of the cell cycle. Then, sprouting takes place concomitant with the increase in cell expansion and free water movement.

Apoplastic calmodulin promotes self-incompatibility pollen tube growth by enhancing calcium influx and reactive oxygen species concentration in Pyrus pyrifolia.

Calmodulin (CaM) has been associated with various physiological and developmental processes in plants, including pollen tube growth. In this study, we showed that CaM regulated the pear pollen tube growth in a concentration-dependent bi-phasic response. Using a whole-cell patch-clamp configuration, we showed that apoplastic CaM induced a hyperpolarization-activated calcium ion (Ca²âº) current, and anti-CaM largely inhibited this type of Ca²âº current. Moreover, upon anti-CaM treatment, the reactive oxygen species (ROS) concentration decreased and actin filaments depolymerized in the pollen tube. Interestingly, CaM could partially rescue the inhibition of self-incompatible pear pollen tube growth. This phenotype could be mediated by CaM-enhanced pollen plasma membrane Ca²âº current, tip-localized ROS concentration and stabilized actin filaments. These data indicated that Ca²âº, ROS and actin filaments were involved with CaM in regulating pollen tube growth and provide a potential way for overcoming pear self-incompatibility.

Distribution of transglutaminase in pear pollen tubes in relation to cytoskeleton and membrane dynamics.

Transglutaminases (TGases) are ubiquitous enzymes that take part in a variety of cellular functions. In the pollen tube, cytoplasmic TGases are likely to be involved in the incorporation of primary amines at selected peptide-bound glutamine residues of cytosolic proteins (including actin and tubulin), while cell wall-associated TGases are believed to regulate pollen tube growth. Using immunological probes, we identified TGases associated with different subcellular compartments (cytosol, membranes, and cell walls). Binding of cytosolic TGase to actin filaments was shown to be Ca(2+) dependent. The membrane TGase is likely associated with both Golgi-derived structures and the plasma membrane, suggesting a Golgi-based exocytotic delivery of TGase. Association of TGase with the plasma membrane was also confirmed by immunogold transmission electron microscopy. Immunolocalization of TGase indicated that the enzyme was present in the growing region of pollen tubes and that the enzyme colocalizes with cell wall markers. Bidimensional electrophoresis indicated that different TGase isoforms were present in distinct subcellular compartments, suggesting either different roles or different regulatory mechanisms of enzyme activity. The application of specific inhibitors showed that the distribution of TGase in different subcellular compartments was regulated by both membrane dynamics and cytoskeleton integrity, suggesting that delivery of TGase to the cell wall requires the transport of membranes along cytoskeleton filaments. Taken together, these data indicate that a cytoplasmic TGase interacts with the cytoskeleton, while a different TGase isoform, probably delivered via a membrane/cytoskeleton-based transport system, is secreted in the cell wall of pear (Pyrus communis) pollen tubes, where it might play a role in the regulation of apical growth.

Fast loading ester fluorescent Ca2+ and pH indicators into pollen of Pyrus pyrifolia.

Loading of Ca(2+)-sensitive fluorescent probes into plant cells is an essential step to measure activities of free Ca(2+) ions in cytoplasm with a fluorescent imaging technique. Fluo-3 is one of the most suitable Ca(2+) indicators for CLSM. We loaded pollen with fluo-3/AM at three different temperatures. Fluo-3/AM was successfully loaded into pollen at both low (4°C) and high (37°C) temperatures. However, high loading temperature was best suited for pollen, because germination rate of pollen and growth of pollen tubes were relatively little impaired and loading time was shortened. Moreover, Ca(2+) distribution increased in the three apertures of pollen after hydration and showed a Ca(2+) gradient, similar to the tip of growing pollen tubes. The same protocol can be used with the AM-forms of other fluorescent dyes for effective labeling. When loading BCECF-AM into pollen at high temperature, the pollen did not show a pH gradient after hydration. Ca(2+) activities and fluxes had the same periodicity as pollen germination, but pH did not show the same phase and mostly lagged behind. However, the clear zone was alkaline when pollen tube growth was slowed or stopped and turned acidic when growth recovered. It is likely that apical pH(i) regulated pollen tube growth.

S-RNase disrupts tip-localized reactive oxygen species and induces nuclear DNA degradation in incompatible pollen tubes of Pyrus pyrifolia.

Pear (Pyrus pyrifolia L.) has an S-RNase-based gametophytic self-incompatibility (SI) mechanism, and S-RNase has also been implicated in the rejection of self-pollen and genetically identical pollen. However, RNA degradation might be only the beginning of the SI response, not the end. Recent in vitro studies suggest that S-RNase triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube of Pyrus pyrifolia, and it seems that a relationship exists between self S-RNase, actin depolymerization and DNA degradation. To further uncover the SI response in pear, the relationship between self S-RNase and tip-localized reactive oxygen species (ROS) was evaluated. Our results show that S-RNase specifically disrupted tip-localized ROS of incompatible pollen tubes via arrest of ROS formation in mitochondria and cell walls. The mitochondrial ROS disruption was related to mitochondrial alteration, whereas cell wall ROS disruption was related to a decrease in NADPH. Tip-localized ROS disruption not only decreased the Ca(2+) current and depolymerized the actin cytoskeleton, but it also induced nuclear DNA degradation. These results indicate that tip-localized ROS disruption occurs in Pyrus pyrifolia SI. Importantly, we demonstrated nuclear DNA degradation in the incompatible pollen tube after pollination in vivo. This result validates our in vitro system in vivo.

Pollen density on the stigma affects endogenous gibberellin metabolism, seed and fruit set, and fruit quality in Pyrus pyrifolia.

To clarify the relationship between pollen density and gametophytic competition in Pyrus pyrifolia, gametophytic performance, gibberellin metabolism, fruit set, and fruit quality were investigated by modifying P. pyrifolia pollen grain number and density with Lycopodium spores. Higher levels of pollen density improved seed viability, fruit set, and fruit quality. Treatments with the highest pollen density showed a significantly increased fruit growth rate and larger fruit at harvest. High pollen density increased germination rate and gave a faster pollen tube growth, both in vivo and in vitro. Endogenous gibberellin (GA) concentrations increased in pollen tubes soon after germination and the concentration of two growth-active GAs, GA(3), and GA(4), was positively correlated to final fruit size, cell numbers in the mesocarp, and pollen tube growth rate. These two GAs appear to be biosynthesized de novo in pollen tube and are the main pollen-derived bioactive GAs found after pollen germination. GA(1) levels in the pollen tube appear to be related to a pollen-style interaction that occurred after the pollen grains landed on the stigma.

Microscale mechanisms of gas exchange in fruit tissue.

* Gas-filled intercellular spaces are considered the predominant pathways for gas transport through bulky plant organs such as fruit. Here, we introduce a methodology that combines a geometrical model of the tissue microstructure with mathematical equations to describe gas exchange mechanisms involved in fruit respiration. * Pear (Pyrus communis) was chosen as a model system. The two-dimensional microstructure of cortex tissue was modelled based on light microscopy images. The transport of O(2) and CO(2) in the intercellular space, cell wall network and cytoplasm was modelled using diffusion laws, irreversible thermodynamics and enzyme kinetics. * In silico analysis showed that O(2) transport mainly occurred through intercellular spaces and less through the intracellular liquid, while CO(2) was transported at equal rates in both phases. Simulations indicated that biological variation of the apparent diffusivity appears to be caused by the random distribution of cells and intercellular spaces in tissue. Temperature does not affect modelled gas exchange properties; it rather acts on the respiration metabolism. * This modelling approach provides, for the first time, detailed information about gas exchange mechanisms at the microscopic scale in bulky plant organs, such as fruit, and can be used to study conditions of anoxia.

Characterisation of secreted polysaccharides and (glyco)proteins from suspension cultures of Pyrus communis.

High molecular weight material recovered from the culture filtrate of cell suspension cultured Pyrus communis was composed of 81% carbohydrate, 13% protein and 5% inorganic material. This material was separated into three fractions (one neutral (Fraction A) and two acidic (Fractions B and C)), by anion-exchange chromatography on DEAE-Sepharose CL-6B using a gradient of imidazole-HCl at pH 7.0. The monosaccharide and linkage composition of each fraction was determined after carboxyl reduction of uronic acid residues. From the combined results of the carbohydrate analyses, we conclude that the high molecular weight extracellular material consists of three major and two minor polysaccharides: a (fucogalacto)xyloglucan (36%) in the unbound neutral Fraction A; a type II arabinogalactan (as an arabinogalactan-protein, 29%) and an acidic (glucurono)arabinoxylan (2%) in Fraction B; and a galacturonan (33%) and a trace of heteromannan in Fraction C. The main amino acids in the proteins were Glx, Thr, Ser, Hyp/Pro and Gly. Further separation of Fraction B by solvent partition, SDS-PAGE and analysis by LC-MS/MS identified the major proteins as two chitanases, two thaumatin-like proteins, a beta-1,3-glucanase, an extracellular dermal glycoprotein and a pathogenesis-related protein.

Pyrus pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen and tubes in vitro.

Pears (Pyrus pyrifolia L.) have an S-RNase-based gametophytic self-incompatibility system, and S-RNases have also been implicated in self-pollen or genetically identical pollen rejection. Tip growth of the pollen tube is dependent on a functioning actin cytoskeleton. In this study, configurations of the actin cytoskeleton in P. pyrifolia pollen and effects of stylar S-RNases on its dynamics were investigated by fluorescence and confocal microscopy. Results show that actin filaments in normal pollen grains exist in fusiform or circular structures. When the pollen germinates, actin filaments assembled around one of the germination pores, and then actin bundles oriented axially throughout the shank of the growing tube. There was a lack of actin filaments 5-15 microm from the tube tip. When self-stylar S-RNase was added to the basal medium, pollen germination and tube growth were inhibited. The configuration of the actin cytoskeleton changed throughout the culturing time: during the first 20 min, the actin configurations in the self-pollen and tube were similar to the control; after 20 min of treatment, the actin filaments in the pollen tube gradually moved into a network running from the shank to the tip; finally, there was punctate actin present throughout the whole tube. Although the actin filaments of the self-pollen grain also disintegrated into punctate foci, the change was slower than in the tube. Furthermore, the alterations to the actin cytoskeleton occurred prior to the arrest of pollen tube growth. These results suggest that P. pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen grains and tubes.

A permeation-diffusion-reaction model of gas transport in cellular tissue of plant materials.

Gas transport in fruit tissue is governed by both diffusion and permeation. The latter phenomenon is caused by overall pressure gradients which may develop due to the large difference in O(2) and CO(2) diffusivity during controlled atmosphere storage of the fruit. A measurement set-up for tissue permeation based on unsteady-state gas exchange was developed. The gas permeability of pear tissue was determined based on an analytical gas transport model. The overall gas transport in pear tissue samples was validated using a finite element model describing simultaneous O(2), CO(2), and N(2) gas transport, taking into account O(2) consumption and CO(2) production due to respiration. The results showed that the model described the experimentally determined permeability of N(2) very well. The average experimentally determined values for permeation of skin, cortex samples, and the vascular bundle samples were (2.17+/-1.71)x10(-19) m(2), (2.35+/-1.96)x10(-19) m(2), and (4.51+/-3.12)x10(-17) m(2), respectively. The permeation-diffusion-reaction model can be applied to study gas transport in intact pears in relation to product quality.

The impact of cell division and cell enlargement on the evolution of fruit size in Pyrus pyrifolia.

BACKGROUND AND AIMS: Dramatic increases in fruit size have accompanied the domestication of Pyrus pyrifolia. To evaluate the contribution of cell division and cell enlargement in the evolution of fruit size, the following study was conducted. METHODS: Three wild Pyrus and 46 cultivated Pyrus pyrifolia cultivars were selected to examine cell number/size at time of pollination and at time of fruit harvest. The period of cell division was estimated by logarithmic curve of the increasing pattern of cell number, and its correlations with maturation period and final fruit size were analysed. KEY RESULTS: Final fruit size is directly related to the number of cells produced in the period immediately following pollination. Late-maturing cultivars are larger than earlier-maturing cultivars and this is due to an extended period of cell division. CONCLUSIONS: The evolution of fruit size in P. pyrifolia has mainly resulted from shifts in the ability of cells to divide rather than to enlarge.