Fruit quality depends on a series of biochemical events that modify appearance, flavour and texture throughout fruit development and ripening. Cell wall polysaccharide remodelling largely contributes to the elaboration of fleshy fruit texture. Although several genes and enzymes involved in cell wall polysaccharide biosynthesis and modifications are known, their coordinated activity in these processes is yet to be discovered.
Combined transcriptomic and biochemical analyses allowed the identification of putative enzymes and related annotated members of gene families involved in cell wall polysaccharide composition and structural changes during apple fruit growth and ripening. The early development genes were mainly related to cell wall biosynthesis and degradation with a particular target on hemicelluloses. Fine structural evolutions of galactoglucomannan were strongly correlated with mannan synthase, glucanase (GH9) and β-galactosidase gene expression. In contrast, fewer genes related to pectin metabolism and cell expansion (expansin genes) were observed in ripening fruit combined with expected changes in cell wall polysaccharide composition.
Hemicelluloses undergo major structural changes particularly during early fruit development. The high number of early expressed β-galactosidase genes questions their function on galactosylated structures during fruit development and storage. Their activity and cell wall substrate remains to be identified. Moreover, new insights into the potential role of peroxidases and transporters, along with cell wall metabolism open the way to further studies on concomitant mechanisms involved in cell wall assembly/disassembly during fruit development and storage.
Apple (Malus domestica) fruit development involves a series of biochemical events determinant for qualitative traits, such as appearance, flavour and texture . Fruit growth involves cell divisions and cell expansion resulting from a dynamic interplay between cell turgor pressure, cell wall biosynthesis and remodelling . Apple ripening involves starch conversion to simple sugars, skin colour changes, ethylene production, a respiration burst and flesh softening . Reduction in tissue firmness combines a decrease in cell turgor pressure as well as cell wall polysaccharide remodelling and metabolism [4–6].
Cell walls largely contribute to fruit textural characteristics. In apple, like other fleshy fruit, they are made of pectin, hemicellulose and cellulose, together with some structural proteins . Apple cell wall polysaccharide composition and structure varies with genetics, developmental stages and growth conditions [7, 8]. The relative content of the major cell wall sugars represented by galacturonic acid attributed to pectin, and glucose from cellulose and hemicelluloses increase during apple ripening [9, 10]. Galactose and arabinose content decreases during fruit expansion and further declines during ripening [10–13]. This is due in part to β-galactosidases and α-arabinofuranosidases degradation of the galactan and arabinan side chains of the pectic rhamnogalacturonan I (RGI) [6, 14, 15]. Methyl ester substitutions of the homogalacturonan structural domain of pectins (HG) are partly removed by the action of pectin methylesterases (PME) during apple development [16, 17].
This metabolism of pectin increases cell wall porosity, decreases cell adhesion and affects fruit texture [6, 18]. The loss of RGI galactan and arabinan side chains was associated with softening , whereas high content of galactan side chains was associated with firmness . A high arabinofuranosidase activity related to MdAF3 gene expression was reported in mealy apples . Pectin HG structure and its methyl esterification are also important for apple texture. Down regulation of the MdPG1 gene coding a polygalacturonase maintains fruit firmness during ripening . In contrast local action of PME (MdPME2) was associated with mealiness development .
Unlike pectin, the overall apple hemicellulose composition and molecular weight are not significantly affected during fruit development and ripening . However, their structure and interactions with cellulose are likely remodelled, as observed in the changes of activities and gene expression levels of endo-1,4-β-D-glucanase, xyloglucan endotransglycosylase/hydrolase (XTH) and expansin which are involved in cutting, cutting and pasting and breaking hydrogen bonds between xyloglucan and cellulose [14, 17, 23–27].
In addition to cell wall chemistry and macromolecular interactions, apple texture elaboration involves other complex mechanisms related to tissue organization [28–31] and cellular water partition [8, 20, 29, 32].
As the whole fruit development is involved in texture elaboration , we investigated the parallel evolutions of cell wall chemical composition and structure with that of cell wall related gene expression during fruit development and cold storage. The transcriptomic analysis focused on genes annotated for cell wall polysaccharide biosynthesis, remodelling and degrading proteins as well as for structural proteins. Because turgor pressure is involved in fruit development and texture, genes annotated for transporters were also analysed. Gene expression results and correlation analyses between biochemical and transcriptomic profiles highlighted new candidate genes and provided new insights into possible coordinated activities involved in cell wall biosynthesis and metabolism during apple development and storage.
The global sugar composition of cell wall prepared as an alcohol insoluble material (AIM) was analysed at each developmental and storage stage (Table 1). As expected, apple fruits accumulated starch during the developmental phases reaching 47.1 % of the AIM dry weight at 110DAF. Starch content decreased at harvest and during the cold storage period. The cell wall polysaccharides after deduction of starch glucose content in AIM sugars (non-starch polysaccharides, NSP) were mainly glucose, uronic acids (UA), arabinose and galactose in decreasing order of proportion. The total amount of these 4 main sugars reached 85 to 88 % of NSP depending on developmental and storage stages. Galactose content decreased constantly from 18.7 to 7.2 % of NSP while uronic acids content increased slightly from 22.4 to 29 % of NSP when fruits reached late development stages. Smaller amounts of xylose, mannose, and traces of rhamnose and fucose were also detected. Xylose and fucose contents increased slightly at ripening stages while mannose contents decreased. Acetyl ester content also decreased during the ripening stages from 1.5 % at 60DAF to 1.2 % of NSP at 2 M. In contrast, methyl ester content did not show any significant change.
A structural profiling approach by enzymatic digestion coupled with MALDI-TOF MS analysis of the degradation products was used to follow modifications of hemicellulose fine structure. As apple hemicelluloses include xyloglucan (XyG), galactoclucomannan (GgM) and glucuronoarabinoxylan (GAX) [33–35], mannanase, xylanase and glucanase degradations were done sequentially. The order of the enzymatic treatments was chosen to maximise oligosaccharides release.
To facilitate enzymatic treatments, AIM was first washed with water (water soluble fraction, WS, Table 2) and then partially depectinated by pectin lyase and rhamnogalacturonase (pectinase-soluble fraction, PS). Uronic acids (UA) and neutral sugars (NS) contents were analysed after each treatment. In the WS fraction, UA content increased continuously from 0.5 % at 60DAF to 1.3 % of NSP at 2 M (Table 2). In contrast, NS content decreased from 1.3 % at 60DAF to 0.9 % of NSP after 2 months of cold storage (2 M). This decrease affected the mannose, galactose and glucose content, but not that of arabinose, which was the major sugar of this fraction (Table 3A). As expected, the subsequent pectinase treatment (PS) had a drastic effect and removed 15.2 to 25.3 % of NSP depending on the fruit stages (Table 2). The amount of NS content decreased from 20.4 to 10.9 % of NSP from 60DAF to 2 M (Table 2), including the majority of the released rhamnose, arabinose, mannose and galactose (Table 3B). The difference between 60DAF and 2 M was mainly due to the decrease in galactose content in the PS fraction (Table 3A). No significant change was observed in UA content between 60DAF and 2 M (Table 2) but the majority of UA was released with this treatment (Table 3B).
Endo-β-mannanase treatment on the remaining extracts allowed access to mannan-rich hemicelluloses. The treatment released 2.8 to 3.3 % of NSP at 60DAF and 110DAF and significantly less from harvest to 2 M with only 1.3%NSP (Table 2). No UA was detectable at early stages of development and only traces afterwards (Table 2). At 60DAF, hydrolysis products were mainly composed of glucose, arabinose, mannose and galactose, each representing 0.3 to 0.4 % of NSP (Table 3A) and respectively only 1.2, 1.8, 6.2 and 1.3 % of their respective initial content in NSP, as most of them was already removed in the PS fraction (Table 3B). The lower NS content of the mannanase fraction at 2 M was mainly due to the decline of galactose and glucose content with only 0.1 % NSP remaining for each.
Galactoglucomannan fine structures recovered in the mannanase hydrolysates were assessed qualitatively by MALDI-TOF MS spectra analysis (Fig. 1a). As expected, MS spectra showed a series of more or less acetyl-esterified hexo-oligosaccharides with degrees of polymerization from 4 to 8 attributed to mannans/glucomannans/galactoglucomannans fragments. Major fragments in the mean spectrum were attributed to Hex4a1 (4 hexose residues substituted by 1 acetyl group, see legend of Fig. 1 for nomenclature, m/z 731), Hex4a2 (m/z 773), Hex5a1 (m/z 893) Hex5a2 (m/z 935), and Hex6a1 (m/z 1055) oligomers. An ion with mass corresponding to hexose and pentose containing structures, Hex3a1 and Pen4, respectively, was observed at mz 569 (Pen4: 4 pentose residues). Minor structures identified were Hex4 (m/z 689), Hex5 (m/z 851), Hex5a2 (m/z 935), Hex7a1 (m/z 1217), Hex7a2 (m/z 1259), Hex8a1 (m/z 1379) and Hex8a2 (m/z 1421). The spectra also revealed the presence of minor pento-oligosaccharides: Pen3U1 (3 pentose residues substituted by 1 uronic acid, m/z 613), Pen3U1a1 (m/z 655), Pen4a1 (m/z 611), Pen4a2 (m/z 653), Pen4U1m1 (m/z 759), Pen4U1m1a1 (m/z 801) and Pen5a1 (m/z 743) arising from the minor contamination of the commercial mannanase by xylanase. Principal components analysis (PCA) of annotated oligosaccharides ion intensity was done to provide a synthetic view of sample variations as well as of the variables contributing to these variations. This analysis revealed a clear change in fine structure of mannose-containing hemicelluloses during fruit development particularly during the early phases (Fig. 1b, c). PCA of MS spectra showed that the acetylated oligomers Hex7a1 and Hex8a1 differentiated the fruits at 60DAF.
Treatment of the endo-β-mannanase residues by endo-β-xylanase was performed to analyse xylose-containing hemicelluloses. This treatment released a small amount of neutral sugars, only 1 % of NSP at 60DAF, which decreased during cold storage to 0.5 % of NSP at 2 M (Table 2). No acidic sugar was detected in this fraction (Table 2). Arabinose, galactose and glucose were the main neutral sugars detected at 60DAF with 0.1 to 0.2 % of the initial NSP (Table 3A). After 2 months cold storage, more arabinose (0.9%NSP), galactose (0.3%NSP) and glucose (0.2%NSP) were detected (Table 3A). They represented 6.2, 3.6 and 0.6 % of their respective initial content in NSP (Table 3B). Rhamnose and xylose contents increased in 2 M samples with respectively 0.1 and 0.5 % of the initial NSP, representing 5.4 and 6.0 % of their initial content in NSP. In contradiction with the global NS measurements by colorimetry (Table 2), the GC method showed an increase in neutral sugars released by the treatment after 2 M when compared to 60DAF (Table 3A). Due to the overall low amounts of the xylose-containing oligosaccharides in the hydrolysis products (Table 3B), xylanase hydrolysates were not further analysed.
Endo-β-glucanase was applied on xylanase residues as the last enzymatic treatment to access xyloglucan structures. The treatment solubilized from 13.9 %NSP at 60DAF to a maximum of 15.7 %NSP at harvest stage and decreased to 13.9 % NSP at 2 M (Table 2). Only a very small amount of UA (0.2 % NSP) was released from the harvest stage and thereafter. As expected, the main soluble sugar was glucose with respectively 5.1 and 5.7 % NSP at 60DAF and 2 M (Table 3A) and representing 16.4 % at 60 DAF and 15.8 % at 2 M of the initial content in NSP. With no remarkable differences between 60DAF and 2 M stages, smaller amounts of the other sugars were also solubilized. Most of the released fucose, glucose and xylose were found in the glucanase hydrolysis products (Table 3B).
In consistence with the high content of glucose in the hydrolysates (Table 3), xyloglucan oligosaccharides (XyGOs) were identified by MALDI-TOF MS analysis (Fig. 1b). The mean spectrum revealed the presence of major acetyl-esterified XyGOs: XXFGa1 (m/z 1435) and XLFGa1 (m/z 1597) together with other structures attributed according to their respective mass to XXG (m/z 791), XLG (m/z 953), GFG (m/z 967), XFG (m/z 1099), XLXG (m/z 1247), XLXGa1 (m/z 1289), XXFG (m/z 1393), XLGa1 (m/z 1451), XXFGa2 (m/z 1477) and XLFGa2 (m/z 1639). Minor fragments were also detected as hexo or pento-oligosaccharides and attributed to Hex4 (m/z 689), Hex4a1 (m/z 731), Hex4a2 (m/z 773), Hex5 (m/z 851), Hex5a1 (m/z 893), Hex5a2 (m/z 935), Hex6a1 (m/z 1055), Hex7a1 (m/z 1217) and Hex8a1 (m/z 1379), Pen3U1 (m/z 613), Pen3U1a1 (m/z 655), Pen4a1 (m/z 611), Pen4U1m1 (m/z 759), Pen4U1m1a1 (m/z 801), Pen5a1 (m/z 743) and Pen5U1m1a1 (m/z 933). These fragments reflected the activity of the commercial glucananase on glucomannan as well as the presence of minor contaminating xylanolytic activities. If no significant change in global NS composition was observed, a clear change of the oligosaccharide fine structures occurred during fruit development, particularly between the early developmental phases (60DAF and 110DAF) and the matures stages (H, 1 M and 2 M). While most of the XyGOs oligomers, and particularly XXG, GFG, XLXG, XXFGa2, XLFGa2, were representative of mature stages, the hexo and pento-polysaccharides distinguished the spectra of fruits in early development. Particularly, Hex6a1, Hex5a1, Hex7a1, Hex4a1, Hex4a2, Hex5a2, Pen5U1m1a1, Hex8a1, Hex4, Pen4U1m1a1, Hex5, Pen4U1m1 and Pen3U1a1 were representative of the early stages (Fig. 1b).
In order to identify genes potentially involved in the structural modifications of cell wall polysaccharides, transcriptome analyses were performed on the same samples used for cell wall biochemical analyses. Transcriptomic profiling performed with the AryANE_v1 microarray revealed that 42 % of the tested transcripts were expressed at one or more developmental stages for at least one of the 8 genotypes analysed. Differentially expressed transcripts between subsequent developmental stages were identified with significant P-values for t-tests (P-value <0.01; Fig. 2). The highest numbers of differentially expressed transcripts were observed between 110DAF and harvest, and harvest and 1 M storage. To study the changes between apple development and fruit ripening, the transcriptome at 60DAF was compared with the transcriptome at 2 M. A total of 23,001 differentially expressed transcripts were identified. Subsequent hierarchical clustering analysis on expression profiles led to the selection of 5150 transcripts displaying similar expression profiles for the 8 genotypes grown in both orchards when considering the 16 time series (Additional file 1). Microarray data were validated by RT-qPCR experiments on a subset of differentially expressed genes, using cDNA from 60DAF and 2 M apple fruits. A similar difference between gene expression levels was observed with both techniques (Pearson correlation = 0.82, P-value < 0.01) (Additional file 2).
In AryANE_v1 microarray, sense (S) and antisense (AS) probes were designed for each annotated apple coding DNA sequence (CDS), and 26 % of the differentially expressed probes corresponded to AS transcripts. Celton et al.  demonstrated that these AS transcripts were likely to be involved in small interfering RNA (siRNA) dependent negative regulation of the coding mRNAs. This study only considered genes with differentially expressed sense transcripts.
Sense transcripts with higher expression during early fruit development (60DAF and 110DAF: cluster A) or during fruit ripening and cold storage (harvest, 1 M, 2 M: cluster B) were selected for further analyses, and represented respectively 6.2 and 10.5 % of the selected differentially expressed transcripts. Based on genes annotations, they were classified into functional categories. 0.5 % had annotations related to cell wall biosynthesis and/or remodelling, or solutes flux changes (Additional file 3). In order to refine the selection, deduced protein sequence from these genes were analysed for subcellular targeting and protein domain annotation. The potential cellular locations of 96.5 % of the proteins corresponding to these genes were analysed with the ProtAnnDB tool  (Additional file 3). In concordance with a function on cell wall modifications, 66 proteins with a signal peptide for endoplasmic reticulum (ER) targeting were potential candidates to be exported to the apoplast. For 21 predicted transporters, 2 had surprisingly no predicted transmembrane domain. 5 of the 6 peroxidases had a predicted signal peptide for ER targeting and 4 were predicted to belong to Class III peroxidase superfamily when analysed through the PeroxiBase tool. Among the enzymes and proteins related to cell wall modification, 15 % could not be allocated to a coherent subcellular compartment. This was probably due to the prediction models used and/or to potentially truncated protein sequences which were deduced from the apple genome sequence and annotation . In addition, some annotations were different between ProtAnnDB and CAZy databases. For example, several proteins were identified as pectin lyase-like with ProtAnnDB, but were grouped as glycoside hydrolases 28 (GH28) (MDP0000147794; MDP0000175027; MDP0000251956; MDP0000270685; MDP0000665344; MDP0000818931 and MDP0000249285) or carbohydrate esterases 8 (CE8) (MDP0000177299; MDP0000212502; MDP0000251256; MDP0000252508; MDP0000287234) in CAZy database. Such discordances probably resulted from the markedly different methods and criteria used for protein annotation.
A total of 114 cell wall related genes were selected, 82 % were expressed during the early developmental phases and the remaining 18 % were expressed during later developmental stages and storage. According to their expression pattern, early expressed genes were grouped in cluster A while the later expressed genes were in cluster B (Table 4; Additional file 3). Cell wall genes from cluster A included mainly genes potentially involved in pectin and cellulose/hemicellulose metabolism, respectively 20 and 26 genes. Several genes potentially coding expansins, galactosyltransferases, glycoproteins and many β-galactosidases were also identified in this cluster, as well as peroxidases and transporters. In contrast, few cell wall related genes were identified in cluster B. Those identified were mainly involved in pectin degradation, and very few genes were involved in cellulose/hemicelluloses metabolism including genes coding expansins (Table 4; Additional file 3).
Gene expression networks were realized within each cluster (Additional file 4). Gene correlation in cluster A yielded one large network composed of 70 genes (r > 0.7). A subset of genes in the network showed strong correlations (P < 0.05, r > 0.9) and was centred on a gene annotated for a glycoside hydrolase belonging to family 9 (GH9) grouping mainly glucanases (MDP0000131397). This subset was composed of GH9, β-glucosidase (MDP0000140817), β-galactosidase (MDP0000899966), XTH (MDP0000378203), FLA (MDP0000525641), AGP (MDP0000893240), peroxidase (MDP0000221335) and sugar transporter (MDP0000318992). Another subset containing CSLA (MDP0000717000), FLA (MDP0000658332) and β-galactosidase (MDP0000310582) also significantly correlated (P < 0.05, r > 0.9). Two small correlation networks were drawn for genes in cluster B (Additional file 4). Two gene networks showed significant correlations (P < 0.05, r > 0.7). One showed correlations between genes encoding pectin-degrading enzymes such as PG and pectin esterases (MDP0000249285; MDP0000251256; MDP0000252508; MDP0000287234) and genes encoding transporters (MDP0000216376; MDP000219430; MDP0000266249; MDP0000403872). The other network showed strong correlations (P < 0.05, r > 0.8) between genes encoding expansin like A (MDP0000906812), expansin like B (MDP0000214811; MDP0000292477), β-galactosidase (MDP0000416548) and peroxidase (MDP0000142485).
Transcriptomic profiles were tentatively correlated with the cell wall monosaccharides contents and the oligosaccharides enzymatically released from hemicelluloses in order to reveal concomitant events (Additional file 5). Total monosaccharide contents in AIM (as %NSP) were considered, except for arabinose and rhamnose whose content did not change (Table 1). Oligosaccharides relative contents in glucanase digests were also considered as markers of hemicellulose structural changes (Fig. 1b).
Glucose and uronic acids contents were the least correlated with the selected gene expression levels. Expression profiles of cluster A genes expressed during early developmental stages were positively correlated with galactose and mannose contents, as well as oligosaccharides content attributed to mannans (Hex4a1, Hex4a2, Hex5a1, Hex5a2, Hex6a1, Hex7a1 and Hex8a1) and xylans (Pen5U1m1a1, Pen4U1m1 and Pen4U1m1a1). They were also negatively correlated with fucose and xylose content as well as with oligosaccharides attributed to xyloglucans (XXG, XLG, GFG, XFG, XLXG, XXFG, XXFGa2, XLFGa2). The opposite correlations were observed with expression profiles from genes belonging to cluster B, showing higher expression at mature stages.
Strong correlations were observed between expression profiles of β-galactosidases and galactose content (Fig. 3a), between expression profiles of expansins and the structure XXG of xyloglucans (Fig. 3b), between expression profiles of cellulose synthase like-A and glycoside hydrolase family 9 and the structure Hex6a1 of mannans during apple development and ripening (Fig. 3c and d, respectively).
Modifications in the chemical composition of apple cell wall polysaccharides during fruit development and ripening have already been described [6, 9, 10, 22, 30] as well as enzymes and genes expression involved in ripening [14, 16, 17, 23, 26, 27, 39, 40]. However a more detailed view of genes potentially involved in cell wall polysaccharide chemical composition, structure, water flux during apple development and storage provides insights into the mechanisms affecting texture characteristics and highlights novel candidate genes involved in these processes.
Biochemical cell wall analyses were done to assess the changes in polysaccharide composition and particularly that of hemicelluloses fine structure during apple fruit development. For the latter analyses, pectin was partially removed by water washes and pectinolytic enzymes as it was reported to mask hemicelluloses . Our results showed that compared with previous studies of XGos profiles (Fig. 1), pectin in apple parenchyma cell wall did not have a major impact on hemicellulose accessibility to enzymes [7, 33, 42]. Furthermore, the changes observed by MALDI-TOF MS in the relative proportion of GgM oligomers in the glucanase hydrolysate, followed the decrease in mannose content in the cell wall of the fruit in development. Although these observations pointed to some degree of representativeness of the cell wall hemicelluloses enzymatic profiling, the hydrolyzates composition likely reflected readily accessible structures and not those in strong interaction. Additionally, the endogenous modifications of polysaccharides structure and access during fruit development possibly affected the enzymatic hydrolyzates composition.
Transcriptomic analysis provided access to genes encoding specific proteins and enzymes related to cell wall construction and remodelling during apple development, ripening and cold storage. Genes were selected according to their annotations from different databases but their respective biochemical activities remain to be characterized. Genome-wide expression analysis of apple fruit development has already revealed the coordination between gene expressions with specific fruit developmental stages from floral bud to ripe fruit [1, 43, 44]. Genes expressed during early fruit development are mainly involved in cell proliferation and expansion [43, 44]. Recently, this approach revealed that the down regulation of MdPME2, an early-expressed pectin methylesterase-coding gene during fruit development was linked to the apparition of mealiness, during fruit cold storage . Several other functional categories have also been reported, such as solute transport and cell wall metabolism .
The present analysis confirmed that the number of transcripts detected was similar from early apple development to harvest stage and that it was not affected up to 2 months after cold storage . However, remarkably more cell wall-related genes were specific to early developmental stages than to ripening and storage phases. This could be explained by the fact that analyses were carried out on distinct genotypes with different fruit texture evolution after harvest (Additional file 6). This difference in gene expression profiles highlights the plasticity of the genome with different expression time-frames and/or other genetic/environmental factors affecting markedly metabolic pathways during the ripening process. Indeed, variations in transcript profiles already observed between different apple genotypes support a genetic dependent regulation of fruit growth and ripening [43, 46].
During early apple development, genes involved in pectin metabolism were co-expressed with genes involved in hemicellulose metabolism and their remodelling by XTH (Table 4, Additional files 3 and 4). Concomitant expression of genes involved in biosynthetic and degradation functions is in line with observations that hydrolytic mechanisms are required to achieve a proper cell wall polysaccharide synthesis and organ development [47–49].
Changes in cell wall composition were observed as expected during fruit development and ripening, such as a decrease in galactose content [10, 11, 30, 50]. The increase in xylose, fucose and uronic acids likely resulted from both the cell wall enrichment in XyG and pectin depletion in neutral side-chains. The presence of uronic acids in the water washes of apple AIM at the ripening stage supports the hypothesis that HG depolymerization by PG contributes to the decrease of fruit firmness [14, 20, 51]. The removal of methyl esters facilitates PG action [3, 52] but also favours cell adhesion and the rigidity of pectin network . In peach and tomato, pectin methyl-esterification decreased during development and ripening concomitantly with the increased activity of PME [52, 54, 55]. In apple, PME activity was reported to decrease during ripening [14, 17]. In this study, 2 pectin esterases coding gene (MDP0000177299, MDP0000212502) showed a decreased expression during apple development and ripening. However, as previously reported [10, 19, 21], no global significant variation was observed in methyl ester content of pectin during apple ripening. It was suggested that these enzymes could have a very local activity, at tricellular junctions, whose effect could not be evaluated at the whole fruit level . Acetyl-esterification is also a common feature of apple pectin  and may be the target for the ill-defined function of these esterases. The latter may contribute to the significant decrease in the global cell wall acetyl esterification observed between 110 DAF and 2 M.
The specific expression of a GAUT coding gene (MDP0000609623) was also noticed during the ripening phases, as well as a significant increase of UA content. This suggests that new pectin could be incorporated into cell wall even after the developmental stages. This is in accordance with the observation that early ripening tomato has still cell wall synthetic capacities while being disassembled [57, 58].
The semi-quantitative variations of hemicellulose structural domains observed during fruit development (Fig. 1) were in accordance with observations made on other plant organs or fruits [59, 60]. This variation suggests different roles for different XyG fine structures in relation with cell wall expansion and extensibility mechanical properties by yet unclear mechanisms . In several fruits, including apple, distinct xyloglucan transglycosylase/hydrolases (XTH)-coding genes are expressed in young and mature fruits and are likely to contribute to XyG structure [23, 62, 63]. Particularly found expressed in the early development phases in this study, XTH gene expression was highly and positively correlated (P-value < 0.01) with the expression of glycoside hydrolase GH9 (MDP0000131397), β-glucosidase (MDP0000140817), β-galactosidase (MDP0000899966), glycoproteins (FLA: MDP0000525641, AGP: MDP0000893240), peroxidase (MDP0000221335) and sugar transporter (MDP0000318992) (Additional file 4). It points out the key role of XyG in the cell wall dynamic together with other cell wall events including remodelling of cell wall polysaccharides by hydrolases, oxidative reactions and likely cell turgor regulation. Indeed, xyloglucans have been shown to be involved in cell wall mechanics, acting on the stability of microtubule cytoskeleton and the cellulose microfibrils biosynthesis and organization . The concomitant expression of FLA genes (MDP0000525641, MDP0000658332) with XyG remodelling suggests that the proposed cell adhesion and plant mechanical implications of FLA proteins [65, 66] associate specific xyloglucan structures. Genes potentially coding a dual α-L-arabinofuranosidase/α -xylosidase (MDP0000208161) and α-L-fucosidase (MDP0000166406) were also highly expressed during early apple development. These genes should be further characterized as they may be involved in developmental regulation of XyG structure . In addition, the xylosidase/arabinofuranosidase may also be implied in the remodelling of arabinan side chains of pectin, glucuronoarabinoxylan and/or AGP structures.
The present study revealed that galactoglucomannan (GgM), a minor component of apple hemicellulose, undergoes fine structural changes during early fruit development. It co-occurred with the high expression of one CSLA gene (MDP0000673496) (Additional file 5), whose Arabidopsis homolog was shown to encode mannan synthase  and has a potential key role on cell expansion . In particular, this CSLA gene expression was positively correlated with the detection of the Hex6a1 structure in glucanase digest (Fig. 3c) and with the expression of the glycoside hydrolase GH9 gene (MDP0000131397) (Fig. 3d). The strong correlation of one CSLA (MDP0000717000) gene expression with the β-galactosidase expression profile (MDP0000310582) (P-value < 0.01) (Additional file 4) suggests that β-galactosidases may also be involved in the control of GgM synthesis and deposition in the wall. The function of GgM in primary walls remains unclear. Their potential interactions with cellulose  make them a candidate to control microfibrils aggregation and thus cell wall expansion . This is consistent with the existence, at least in tomato, of mannan-degrading enzymes with transglycosylase activity (MTH) similar to XTH activity .
The large amount of mannose and glucose released by the combined action of pectin lyase and rhamnogalacturonase (PS; Table 3B) suggests that GgM could be associated with pectin. Such an association would also be affected by ripening as the amount of glucose decreased in 2 M samples when compared with 60DAF. The specific timing of GgM biosynthesis in early fruit development and metabolism as fruit enlarges points to a specific role most likely during and/or just after cell division with an implication in cell-cell adhesion as in tomato  and/or in the first rapid firmness decline observed during early fruit development .
Xylanase profiling did not show fine structural modifications of the minor glucuronoarabinoxylan (GAX) content in apple. This was likely due to the partial hydrolysis of the GAX by the xylanase contaminating the mannanase used in the previous hydrolysis step but also probably due to hindrance of the binding/active sites by xylan substitutions. Indeed, the xylanase activity contained in the following glucanase was able to release some more xylan oligomers. Overall, the mannanase- and glucanase-released xylo-oligomers revealed that GAX was particularly present at 60DAF and 110DAF, with different fine structures. There is no information on fine structure modifications of xylan-based polysaccharides in other fruits. As for GgM, the function of GAX in apple remains to be established. In Arabidopsis, xylan can be linked to pectin through AGP  and in tomato glucuronoxylan (GUX) are partly linked to GgM . The identification of arabinose, galactose and rhamnose in the products of mannanase and xylanase hydrolysis supports the proposed connections between GAX and the AGP/pectin RGI complex. As with XTH and MTH, xylanase with hydrolase but also transglycosylase activity exists in fruit , opening the way for xylan remodelling mechanisms. Since tomato GUX were located in cell wall lining the intercellular spaces , it would be of interest to search for apple GAX in a similar location and test their involvement in the formation of large intercellular spaces in fruit flesh during cell expansion.
Expansins are also important proteins for loosening XyG-cellulose interactions during cell expansion [77, 78]. They have been found in developing fruit, such as tomato , pear , strawberry  and apple . Our results showed that 4 expansin (EXPA) or expansin-like (EXLA) genes (MDP0000257797, MDP0000259640, MDP0000785413, and MDP0000568045) were specifically more expressed during early fruit development (Table 4; Additional file 3). Their deduced protein sequence had various level of similarity with expansin domains from others fleshy fruit [79–81, 83] (Additional file 7). In contrast, the protein sequence from the 3 expansin-like genes (EXL) (MDP0000214811, MDP0000292477, MDP0000906812) identified during fruit ripening was very different, suggesting different biochemical characteristics and biological functions (Additional file 7). Their gene expression profiles were correlated with oligosaccharides XXG relative content (Fig. 3b; Additional file 5), suggesting a target preference for this fine structure, or their involvement in cell wall integration of new XXG structures. Expansin genes (MDP0000214811; MDP0000292477; MDP0000906812) expression was also correlated with that of one β-galactosidase (MDP0000416548). This observation suggests that galactosylation of XyG structure may be involved in the recognition of XyG/cellulose complex by expansin.
β-galactosidases are common highly active enzymes during apple development and particularly during the ripening stage [6, 19, 25, 39, 40]. In the present study, 11 genes coding β-galactosidases were expressed during early apple development and had glycoside hydrolase 35 domain (GH35). They are similar to those identified in tomato and Japanese pear, but different from the Arabidopsis AtBGAL10 acting on xyloglucans  (Additional file 7). In particular, 7 of these apple β-galactosidases were very similar to PpGAL5, PpGAL6, and PpGAL7 whose expression was at their highest in expanding fruit but decreased drastically upon the onset of ripening . These enzymes can potentially target several cell wall structures, such as AGP, pectic RGI galactan side-chains, xyloglucan or galactoglucomannans. The positive correlation of the 11 apple β-galactosidases expression pattern with galactose content (Fig. 3a, Additional file 5) suggests that RGI galactan side chains hydrolysis might not be their target unless these enzymes act also as transglycosylases as suggested by Franková and Fry . In such a case, these enzymes would have more complex functions in the remodelling of cell wall polysaccharides. They might control pectin and hemicellulose polysaccharides interactions with cellulose through the modulation of their side chains structure [87–89], or regulate remodelling enzymes such as XTH/XET by modifying XyG galactosylation [90–92]. In any case, these glycosidases appear central in the remodelling of cell wall along with cell expansion.
Three other β-galactosidase genes were observed preferentially expressed during apple ripening (Fig. 3a). These β-galactosidases gene expression profiles were weakly and negatively correlated with galactose content (Additional file 5). Two of them (MD0000416548 and MD0000127542) corresponded to the up regulated β-galactosidase genes (Mdβ-GAL1 and Mdβ-GAL2) during apple storage in controlled or regular atmosphere at 1 °C . Protein analysis showed that they were highly similar to the ripening specific pear proteins PpGAL4 and PpGAL1, respectively (Additional file 7), which suggests that they may play similar roles in apple and pear ripening [40, 94, 95] in pruning of RGI galactan side chains [12, 96]. Ripening-specific β-galactosidase gene expression has also been reported in tomato  and down-regulation of one of them, TBG4, resulted in reduced fruit softening .
These results emphasize the complexity of the β-galactosidase family which merits further studies to assess if it could be split into two groups, one principally acting on the regulation of cell wall polysaccharides and glycoproteins galactosylation, and another one mainly pruning pectin RGI side chains.
Peroxidases participate in a range of physiological processes , such as cell wall degradation by cleaving polysaccharides bonds through the generation of reactive oxygen species (ROS) [100–104]. Genes encoding peroxidases were found mostly expressed during early apple development. Some of them had expression profiles correlated with those from proteins and enzymes involved in biosynthesis, remodelling and degradation of cell wall polysaccharides. These results support a role for these enzymes in cell development as reported for Arabidospis cell root elongation  but their mechanism of action remains unclear. Indeed, beside polysaccharide degradation, certain ROS can inversely contribute to cell wall cross-linking and therefore limiting cell expansion . Results from this study suggest that the regulation of apoplastic ROS production is important for cell wall biosynthesis and modifications during apple development.
During fruit growth and ripening, changes of internal turgor pressure lead to modifications of tissue mechanical properties [4, 8, 107]. Transporters have a major role in the regulation of turgor pressure by acting on apoplastic/cell solutes concentration and pH that affect osmotic pressure [2, 108, 109]. As previously reported [43, 44], genes encoding ions and sugar transporters were found highly expressed during early developmental phases (Table 4; Additional file 3). Their expression was highly correlated with genes involved in polysaccharides biosynthesis and degradation. Four genes encoding ion and sugar channels/transporters were preferentially expressed during apple ripening (Table 4; Additional file 3) and correlated with genes related to pectin degradation (Additional file 4). These early and late expressed transporter genes require further studies with regard to their implication in the regulation of turgor pressure during fruit expansion and ripening. During ripening, apple softening is known to involve a decrease in cell turgor pressure . It would be of interest to assess the role of the hexose/sugar transporter genes expressed during late phases on the fate of the phloem downloaded sugars in the apoplast  with regard to the change in water compartmentalization observed in ripening apples . Regulation of osmolytes concentration in ripening apples may contribute to changes in cell turgor pressure and loss of fruit firmness as proposed for grape berries . This mechanism concomitant with cell wall polysaccharide remodelling and degradation may also participate in the elaboration of different apple textures, such as those perceived as mealiness and meltiness.
In addition to known cell wall pectin changes during apple fruit development and cold storage, this study revealed changes in hemicellulose fine structure particularly during early fruit development. The associated characterization of the transcriptome highlighted genes potentially involved in hemicellulose metabolism and the changes observed. At the ripening stage, the low number of genes identified was more specific of pectin metabolism. This study also pointed to β-galactosidases whose roles during fruit growth and ripening remain to be characterized in regard to the targeted substrates and molecular function. Correlations observed between expression profiles from ion and sugar transporters, peroxidases and cell wall-related genes open the way to further studies on the interplay between cell wall assembly/disassembly mechanisms and cell turgor regulation during fruit development.
Eight hybrids (H074, H097, I016, I062, I095, V034, V083 and W029) from the 'HIVW' segregate population resulting from the cross between X3259 and X3263 done in our laboratory at INRA Angers [112, 113] were grown on 2 experimental plots (PH and P12) connected to the INRA laboratory at Bois l'Abbé domain (INRA, Angers). Fruits were collected from the middle and outside parts of the tree canopy at 60 and 110 days after flowering (DAF), and optimum maturity (Harvest, H). Skin colour and starch index (7–8) were used to evaluate fruit maturity at harvest . Fruit collected at optimum maturity were also stored in a cold room at 1 °C for 1 month (1 M) and 2 months (2 M) before sampling. For RNA extraction, outer cortex from 5 fruits was sampled and frozen in liquid nitrogen immediately after collection or after 24 h at room temperature for 1 M and 2 M samples. For biochemical analyses, the cortex tissue of one fruit from each genotype and each plot was sampled and frozen in liquid nitrogen immediately after collection or after 24 h at room temperature for 1 M and 2 M samples (three to five fruits frozen per genotype and per plot). Fruits were sampled in 2012 except 60 DAF and 110 DAF fruits from PH plot harvested in 2013. Fruit softening from harvest to 2 M was checked using an automated penetrometer (TA.XT.-PLUS, Stable Micro system) equipped with a 4 mm diameter convex probes as described by Galvez-Lopez et al.  (Additional file 6).
Total RNAs were extracted using a CTAB extraction buffer from 3 g of frozen fruit flesh tissue ground in liquid nitrogen, as described in Nobile et al.,  and Rienth et al., . mRNAs were amplified, labelled and co-hybridized according to Celton et al.,  as following: aRNAs were produced with Message AmpII aRNA amplification kit (Ambion) from 200 ng of total RNA. Then, 5 μg of each aRNAs were retrotranscribed and labelled with either Cyanine-3 or Cyanine-5 fluorescent dye (Interchim, Montluçon, France). Labelled samples were combined as 30 pmol for each dye and co-hybridized to the Nimblegen microarray AryANE v1.0 containing 135,000 60-mers oligonucleotide probes as described in Celton et al., . Deva software (Nimblegen) was used to extract pair-data files from the scanned images, obtained using the MS200 microarray scanner (Roche Nimblegen).
Genotypes were associated in 4 pairs (I062/V083, V034/W029, H097/I095, I016/H074) for competitive hybridizations at each time point (60 DAF, 110 DAF, H, 1 M and 2 M). Two independent biological repeats were performed with fruits from PH or P12 plots and technical replicates with dye swap were included for a total of 38 arrays.
All statistical analyses were performed as described in Celton et al.,  using R software . Briefly, for each intensity comparison data were normalized with the Lowess method between microarrays. Normalized intensity values were then subtracted from the background to provide an estimation of the transcript expression levels. A second normalization by the quantiles method was then performed on expression values from all comparisons using the normalize.quantiles function from the R package preprocessCore  (Bioconductor project). Differential expression analyses between the different time points were carried out using the lmFit function and the Bayes moderated t test using the R package LIMMA  from the Bioconductor project. Genes were considered differentially expressed if the t-test P-values of the samples were below 1 % between 60 DAF and 2 M. To determine number probes expressed, only signals from the most specific probes belonging to classes 1–3 were considered for subsequent analysis (96,120 probes) .
The microarray data have been submitted to the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE64079.
cDNA synthesis and qPCR were performed on total-RNA samples used for the microarray experiment as described in Segonne-Mikol et al., . Primers were designed for short and specific amplification of the microarray probe region from the selected sequences with Primer 3 plus software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/) (Additional file 2). Amplicons were sequenced once for each genotype to verify primer specificity. For each run, single product amplification was confirmed by melting curve analysis. The amplification efficiency was tested for each primer pairs using a dilution curve method over a 6 points dilution series (from 1.10−1 to 1.10−6) on a pool of cDNAs containing all genotypes and developmental stages included in the study. Primers pairs selected for further analysis have efficiency above 90 %. RT-qPCR was carried out for 7 genes at 2 different time points.
Based on the microarray results, three reference genes with similar expression level in all samples were selected to calculate a normalization factor: MDP0000645828 (GAPDH), MDP0000146514 and MDP0000207727 (respectively annotated as Protein prenylyltransferase superfamily protein and DC1 domain-containing protein). Relative expression level was calculated using the formula ∆Ct = (Cttag-Ctref) derived from the 2−ΔΔC T method, where Ct is the threshold cycle, tag is the target gene, and ref is the reference gene  (Additional file 3).
Apple genes annotation based on sequence similarity with Arabidopsis thaliana were retrieve from the Genome Database for Rosaceae (GDR, https://www.rosaceae.org/). Mapman gene ontology was used for functional classification and enrichment Wilcoxon test (P-value < 0.05) .
Hierarchical clustering of differentially expressed genes was performed with Genesis software (http://www.aics.riken.jp/labs/cbrt/)  using the average linkage hierarchical clustering method as agglomeration rule, and the distance was the similarity between gene expression. Correlation between expression profiles from selected genes were carried out using the cor.test function and the Pearson's test using the R Stats package . The expression correlation between two genes was selected if r >0.7 and P-value <0.01. The gene networks were generated using Cytoscape software .
Proteins annotations and predictions for sub-cellular localization were retrieve from the ProtAnnDB database (http://www.polebio.scsv.ups-tlse.fr/ProtAnnDB/) . Potential peroxidases sequences were also analyzed through PeroxiBase database (http://peroxibase.toulouse.inra.fr/index.php) . Proteins sequences multiple alignment were performed using Kalign tool (http://msa.sbc.su.se/cgi-bin/msa.cgi) with default parameters . All protein sequences were retrieve from GenBank (http://www.ncbi.nlm.nih.gov/genbank/).
Pectin lyase (PL) [EC 4.2.2.10] was purified from Peclyve (Lyven, France) . Rhamnogalacturonase (RG) [EC 3.2.1.171] was from Aspergillus aculeatus (Novozyme, Denmark). Endo-1,4-β-mannanase [EC 3.2.1.78] was from Aspergillus niger (E-BMANN Megazyme, Ireland). Endo-1,4-β-xylanase [EC 3.2.1.8] was from Trichoderma viride (E-XYTR1 Megazyme) and endo-β-glucanase [EC 3.2.1.6] was from Trichoderma sp. (E-CELTR Megazyme) and contained low activity towards xylan and glucomannan.
A pool of the 8 genotypes at each date was realized by picking randomly one fruit per genotype, per date for each orchard. Each sample was cut and lyophilized. Each dried sample was reduced to a fine powder with a mortar and a pestle. Cell walls were prepared as alcohol insoluble material (AIM) using an automated solvent extractor (ASE™ 350, Thermo Scientific™ Dionex™) using 80 % ethanol at 100 °C during 15 min until the ethanol extract was free of soluble sugars. The AIM was dried at 40 °C under vacuum over P2O5. The AIM obtained was reduced in powder using a benchtop homogenizer (FastPrep, MP Biomedicals, USA) at a speed of 6.5 m.s−1 for 20 s.
Cell wall neutral sugars content were identified and quantified from 5 mg AIM by sulfuric acid hydrolysis according to Hoebler et al., . AIM was dispersed in 13 M sulfuric acid during 30 min at 30 °C under agitation and then hydrolyzed in 1 M sulfuric acid (2 h at 100 °C). Sugars were reduced to alditols with NaBH4 (100 mg mL−1, NH3 3.7 N) during 1 h at 40 °C under agitation. Then alditols were acetylated using acetic anhydride and imidazole during 20 min at room temperature . Alditol acetates recovered in dichloromethane were analysed by GC (TRACE GC ULTRA, Thermo Scientific, USA) using TG-225MS column (Thermo Scientific, USA). Standard solution of sugars and inositol as an internal standard was used for calibration. Uronic acids content was quantified using the metahydroxydiphenyl colorimetric method [94, 97] with galacturonic acid as a standard.
To quantify starch content in cell wall material, AIM (10 mg) was incubated overnight at room temperature in 200 μL of MOPS (50 mM, pH7) followed by 5 min at 120 °C. Commercial thermostable α-amylase from Bacillus licheniformis (Megazyme, 3000 U mL−1) was added and the sample was incubated further for 6 min at 100 °C. After cooling, the sample was adjusted to pH 4.5 by addition of 400 μL of acetate buffer (200 mM, pH 4.5). It was further incubated for 30 min at 50 °C with commercial amyloglucosidase from Aspergillus niger (Megazyme, 3300 U mL−1). Glucose released was quantified in HPAEC-PAD using a CarboPac® PA1 column (4 mm × 250 mm, Thermo Scientific, USA), thermostated at 25 °C. An isocratic elution of 500 mM of NaOH was used at a flow rate 1 mL min−1. Rhamnose was used as an internal standard for calibration.
Methyl and acetyl esters was quantified from 5 mg AIM as described in Levigne et al., . Briefly, samples were saponified for 1 h at 4 °C in NaOH 1 N and CuSO4 1 mg mL−1. After centrifugation (7400 g, 4 °C for 10 min), the supernatant was filtered (Alltech Maxi-Clean IC-H, Grace, USA) and analysed by HPLC with a C18 column (4 mm × 250 mm, 5 μm, Interchim, France), thermostated at 15 °C. An isocratic elution with 4 mM H2SO4 was used at a flow rate 1.0 mL min−1. Elution was monitored by differential refractometer (2414 refractive index detector, Waters, USA). Standard solution containing methanol, acetic acid and isopropanol as an internal standard was used for calibration. The degree of methylesterification (DM) of pectin was calculated as the number of moles of methanol per 100 moles of galacturonic acid (GalA).
Pectin was partially removed from 5 mg AIM by deionized water (1 mL) at 40 °C under gentle agitation for 15 min followed by boiling for 10 min and centrifugation (15,300 g, 20 °C for 10 min). The supernatant was removed and the AIM pellet was suspended in deionized water (1 mL) and digested by a combination of pectin lyase and rhamnogalacturonase (0.12 U each) at 40 °C under gentle agitation for 3 h. After centrifugation (as above), the pellet was washed 3 times with 1 mL deionized water under agitation. Then sequential enzymatic digestion was applied on the pellet with endo-1,4-β-mannanase (10 U), endo-1,4-β-xylanase (25 U) and endo-β-glucanase (10 U) at 40 °C under gentle agitation for 17 h  (Additional file 8). The three water washes between enzymatic degradations were discarded. All supernatants were boiled 10 min prior to filtration (0.45 μm filter; Millex-Hv, PVDF, Millipore, France) and analysis by MALDI-TOF MS. Three replicates were realized for biological sample.
The degradation products were analysed by mass spectrometry MALDI. Each supernatant was combined with the ionic liquid matrix N,N-dimethylaniline/2,5-dihydroxybenzoic (DMA/DHB)  and dried on the MALDI polished steel plate. Three replicates were spotted per enzyme hydrolysate. MALDI-TOF MS analysis was performed in the positive mode on an Autoflex III (Bruker Daltonics, Germany) combined with a Smartbeam laser (355 nm, 1000 Hz). The instrument was externally calibrated using galactomannan oligomers (DP 3-9) of known mass. Spectra were recorded in the mass range m/z 500-3000. Ion masses and intensities were normalized on m/z 1085 (XXXG oligosaccharide), 731 (Hex4a1 oligosaccharide) and 655 (Pen3U1a1 oligosaccharide) for glucanase, mannanase and xylanase hydrolyzates, respectively. The nomenclature of xyloglucan was from Fry et al.,  extended to acetyl esters groups noted as a, followed by the number of ester groups in the oligosaccharide. In the oligosaccharides, Hex refers to hexose, Pen refers to pentose, U refers to uronic acid (UA), and m to methyl esters. For each letter, the following figure corresponds to the number of residues or substituents in the oligosaccharide.
Principal component analyses were performed using the PCA function using the R Factominer package . Analysis of variances of chemical data was performed using the anova function on R Stats package  and data were considered significantly different if p < 0.0001.
The correlation analysis between biochemical and transcriptomic data sets was performed using cor function and the Pearson's test (P-value <0.01) from the R Stats package . Expression data were grouped in three random pools and averaged (Pool1: V034, I062 and V083, Pool2: W029, I095 and H097, Pool3: H074 and I016), to match the three replicates available for biochemical data (Additional file 5).
The datasets supporting the conclusions of this article are included within the article and its additional files. The microarray dataset supporting the conclusions of this article is available in the Gene Expression Omnibus repository, under the accession number GSE64079 (http://www.ncbi.nlm.nih.gov/geo/).
The authors would like to thank Sylvain Hanteville, Maryline Bruneau and Jacqueline Vigouroux for their help in orchard and lab analyses, Jean-Marc Celton for his help in transcriptomic analyses, the INRA-Horticultural experimental unit (UEH0449) for the apple trees maintenance, the ANAN platform of the SFR QuaSaV for microarray facilities access, the BIBS (INRA Nantes) platform for biochemical and MS analyses and Thomas Baldwin for English proofreading.
This work was supported by the Institut National de la Recherche Agronomique and by the Region Pays de la Loire (program AI FRUIT).
The data sets supporting the results of this article are included within the article and its additional files. The microarray data have been submitted to the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE64079. The fruit material is available from INRA-IRHS Angers.
ED, SLG, JPR, MO, EB and ML conceived and designed the research. ED carried out all experiments with assistance of MCG for transcriptomic experiment and SLG for biochemical analyses. ED, SLG, MO, JPR, EB and ML wrote the paper. All authors read and approved the final manuscript.
R Development Core Team: R: A language and environment for statistical computing. 2013. https://www.r-project.org/.


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Home Energy Costa Rica Is Running 100% on Renewable Electricity
Wind power in the country
Costa Rica Is Running 100% on Renewable Electricity
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Costa Rica continues its quest to become the most sustainable and renewable energy country on the planet and has now powered the entire country for more than 75 days (as of the March 24th ) using only renewable energy. The country hasn't needed fossil fuel for its energy supply for all of 2015 because of its commitment to hydropower and other renewable sources.
Costa Rica with a population of approximately 5 million also utilizes renewable energies from solar, geothermal and wind power.
Costa Rica relies heavily the power generated by water flow and river power, but in recent years Costa Rica has invested $1 billion into the abundant geothermal power available throughout the country. While geothermal energy contributed only 10 % of Costa Rica's power last year, this number is expect to increase quickly as more geothermal system come on line.
Costa Rica has set ambitious goals in its commitment to protect its environment such as its quest to be entirely carbon neutral by 2021. Costa Rica now tops the world's list of nations producing much of their power from renewable energy sources.
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redpajama_set_name: RedPajamaCommonCrawl


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The technical design of sources of polarized light used earlier was not adequate for producing materially larger light-emitting surfaces.
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#The Wild Silence. A Review of Raynor Winn's sequel to #The Salt Path
First of all a big shout out to Son of Rune (the elder) for the amazing transformation of the design of this blog. Thank you.
The Wild Silence is Raynor Winn's second book which covers the time from her and her husband Moth's return home (where is home?) to the writing of the book and trying in middle age to establish a new life for themselves. When Winn wrote her first book The Salt Path, she thought she was writing a personal account of a personal event. But this memoire of homelessness, with its unassuming displays of courage and endurance touched a universal nerve. The Salt Path became a runaway best seller in 2018. Being infinitely well qualified to write on these issues, Raynor Winn and her husband Moth were made homeless – aged in their 'fifties. Moth also received a terminal diagnosis more or less in the same week. It seemed that the only way was up
After Winn and her husband complete the massive undertaking of walking the 650 mile South West Coast Path (in the UK) finding a new normal isn't easy. The Wild Silence moves both backward and forward in time – backward as Winn remembers her rural childhood and the meeting of Moth; how she overcame her mother's disapproval of the man she chose for her life partner. Then forward to the beginnings of writing The Salt Path and to a proposed tenancy of an old farm and apple orchard in great need of care and renewal. It is hard to avoid the analogy between watching new green shoots of a return to life for an arid and over farmed corner of land, and a parallel return for the protagonists who have to decide who they can ever trust again, if anyone.
Raynor Winn writes so beautifully that sometimes her books seem less of a story and more of an ode – to a vanishing landscape, to a lost childhood of (relative) rural peace when there were still meadows and insects, but mostly to Winn's life's consuming passion, her husband Moth and the mountains, both literal and metaphorical, that they have climbed together. But there is not much that is wistful and certainly no sentiment in these pages. Winn is too practical, too strong and far sighted. Rather than wasting time mourning what has been lost, Raynor Winn sits down with a notebook and pen and plans what the heck she can do about it.
W.H. Davies, poet and author of Autobiography of a Supertramp once wrote:
What is this life if, full of care,
We have no time to stand and stare.
No time to stand beneath the boughs
And stare as long as sheep or cows.
No time to see, when woods we pass,
Where squirrels hide their nuts in grass.
No time to see, in broad day light,
Streams full of stars, like skies at night.
No time to turn at beauty's glance,
And watch her feet, how they can dance.
No time to wait till her mouth can
Enrich that smile her eyes began.
A poor life this if, full of care,
'Leisure' From: Songs of Joy and Others (1911)
Davies was born in Newport, South Wales (my hometown) and knew real poverty. As the title of his autobiography suggests, he became a drifter. His poetic gifts, whether or not related to his peripatetic lifestyle, were born out of his deep understanding of the human connection to the natural world. To me, Raynor Winn's writing is also redolent of those connections. Here she describes a walking holiday they once took in Scotland:
"Days later, among the rocks at the summit of Ben More, the highest point on Mull, the island spread beneath us in an undulation of hills and glistening lochs. On every side, a sheet of lush green draped over an ancient volcanic land, falling softly to the sea. And there in an upswell of air, lifting without moving wing or feather, the huge, terrifying, magnificent shape of a golden eagle. Glowing rust in the afternoon sun…"
However, while from the safety of historical perspective, we all wallow in the romance of the supertramp, the reality is somewhat different. Society's current ethos on these matters of wandering and penury – unless you are Simon Armitage (with whom Moth was confused at one stage on their walk and asked for a recitation) – can be summed up by 'if you don't have a job and a house you must be a criminal, or mad, or both.'
But Raynor and Moth are just themselves, having to learn to avoid using the 'H' word while walking. So instead they find a story they can tell, about selling up the house and retraining to teach, which people could accept. Better to be thought of as a bit eccentric than homeless.
In her first book Winn wrote:
" If you ask someone to describe a homeless person, the majority will give you a description of a rough sleeper, unrolling a mat and bedding down in a street, perhaps with a dog, invariably begging for money for drugs or alcohol. A stereotype that evokes a range of emotions from the feet that pass them as they sleep in doorways, from mildly uncomfortable to aggressively violent."
Homeless folk are associated by others in society with potential trouble, mental health issues, drugs. Often local Councils deal with anyone they consider 'suspicious' by compassionately engaging the police to employ the nineteenth century Vagrancy Act to move them on, or arrest them. But anyone can be homeless. Jobs are becoming a rarity and the cost of property continues to spiral. Safety nets are getting threadbare and more full of holes and at the time when Winn and Moth were walking and at the time she began these books the pandemic hadn't even started. Criminalising poverty is not going to make it go away. Post pandemic, these issues are only going to require even more urgent action, and funding. Achieving this level of awareness is one of Winn's aims having experienced the sharp end of homelessness herself. With these lyrical books she has found the perfect medium for her voice.
2 responses to "#The Wild Silence. A Review of Raynor Winn's sequel to #The Salt Path"
Graham Black says:
Your book was recommended to me by two young hikers on the coast path at Portleven following in your footsteps l told them that I had read Mark Wallington book many years ago now but still remember how l enjoyed it. Do you still live in Polruan? I am a member of the Plymouth Ramblers
Ah. I only wrote the review, not the book sadly. You are lucky. It must be wonderful to live close to that beautiful coast and be able to enjoy regular walks there.


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Portrait of Frank P. Walsh
Portrait of Frank P. Walsh, progressive lawyer and labor advocate. Taken around 1915.
Photograph of Frank P. Walsh and Family
Photograph of Frank P. Walsh, his wife Katherine, and three of their children. Taken between 1918 and 1921.
Photograph of Frank P. Walsh
Photograph of Frank P. Walsh, progressive lawyer and labor advocate. Taken between 1911-1920.
Portrait of Frank P. Walsh, progressive lawyer and labor advocate. Taken between 1910 and 1925.
Maurice M. Milligan
Head and shoulders autographed portrait of Maurice M. Milligan, U.S. attorney who prosecuted Tom Pendergast.
Ralph T. Harding, Lillie Knight, and Clarence Click
Photograph of Ralph T. Harding, Lillie Knight, and Clarence Click (left to right) in the court room. The caption on the back of the image reads, "Watch your credit, International Chicago, 8817, 12-8-33, Charged with Kidnaping Mary McElroy: Clarence Click (right), discussing his case with Miss Lillie Knight, young woman attorney, who pleaded with a Kansas City jury not to send click to the gallows; at left is chief counsel for defense, Ralph T. Harding. Click is charged with kidnaping Miss Mary McElroy, daughter of City Manager H. F.
Trial of Walter H. McGee
Photograph of Ralph T. Harding, Walter McGee, John V. Hill, and Joseph B. Keenan (left to right) at the trial of Walter McGee. The caption on the back of the image reads, "STATE ASKS DEATH PENALTY FOR KIDNAPER, Photo shows the courtroom of Judge Allen C. Southern at Kansas City, Mo. During the trial of Walter McGee, who is charged with being the ringleader of the gang that kidnaped Mary McElroy, daughter of the City Manager of Kansas City, and held her till a ransom of $30,000 was paid left to right: Ralph T.
George McGee, Lillie Knight, and Ralph T. Harding
Photograph of George McGee, Lillie Knight, and Ralph T. Harding (left to right) with R. H. Moore removed from the picture to the right. The caption on the back of the image reads, "Like in the case of his brother, Walter, the state has asked the death penalty for George McGee, youthful accused member of the gang which kidnaped Miss Mary McElroy, daughter of City Manager H. F. McElroy of Kansas City. Walter McGee was the first kidnaper ever given the death penalty in this country." George McGee is shown in this picture conferring with his three attorneys.
Louise Byers, Tiera Farrow, Anna Campbell, Sue Mandell, et al.
Snapshot of Louise Byers with friends from Kansas City School of Law (Tiera Farrow, Anna Campbell, and Sue Mandell included), ca. 1919. The back of the photograph includes the following text: "Brother, These are the girls who were out when the Juniors had their party at our house. Susan Mandell, Glady Asel, Anna Mae Campbell, Mrs. Rogers, Emma Chaquette, Miss Farrow, Elsie Asel, Esther Johnson, Clara Austin, & me [Louise Byers] at the bottom." Byers and Farrow were co-founders of the Women's Bar Association of Kansas City.


If you or a loved one is looking at pursuing a head injury compensation claim in the Wirral area, it would be of great benefit to enlist an expert head injury solicitor on the Wirral, ready to provide trusted legal advice and possible representation of your claim. Hampson Hughes Solicitors provide a team of expert head injury solicitors to our clients on the Wirral, through our serious injury team who possess the experience and expertise to offer expert legal guidance for claims of this type.
How can I know if I'm eligible to claim?
Individuals can suffer a range of head injury types, from small cuts and bruises right through to a Traumatic Brain Injury (TBI). At present, over 150,000 people a year are admitted to UK hospitals in relation to a head injury. NHS guidelines make it clear that while it is difficult to predict and protect against specific head injuries, there are several key provisions that can be made in order to reduce possible injury risk.
In cases where such risks were not properly protected against, resulting in a person incurring a head injury due to negligence from another party, a victim should explore a head injury compensation claim. When you've made the step of finding an expert head injury solicitor in the Wirral area, you can begin a claim related to your accident and injury, whether it happened at work, a sports field, on the road or even in your home.
Hampson Hughes Solicitors offer a wealth of experience in this compensation claim type, allowing us to offer a team of serious injury solicitors to represent claims expertly for clients on the Wirral. In any case where we take on a client's representation, we work hard to display that not only was a head injury suffered due to the incident in question, but that the situation is one where legal accountability should fall on a specific defendant.
Our team will also ensure the any head injury client we represent receives an expert medical assessment on the severity of any head injury they suffered. This assessment not only provides helpful medical advice to the head injury compensation claim, it can also support and inform a compensation assessment, ensuring any injured party receives fair compensation relating to the full scope of support and/or ongoing care they are in need of.
Are there other head injury claim types?
While it is true that the majority of head injury cases are handled by our serious injury solicitors, in cases where there is a brain injury caused by negligent actions from a medical professional – for example misdiagnosis, or surgical error – then this type of injury is classed as an Acquired Brain Injury (ABI). In a claim of this type – usually made against a medical professional or facility – you receive expert legal representation from our Clinical Negligence team.
What if a victim shares partial responsibility?
In this type of situation, most people assume that if the actions of a victim contributed to their suffering of a head injury, it will end their chances of claiming compensation. Often, this is not so, as even in cases where a claimant is deemed to share a measure of responsibility, there could still be the award of a compensation amount. An exact figure is determined in correlation to the level of specific blame that is apportioned, known as a 'split-liability' agreement.
Examples of such a situation could be where a worker is offered protective headgear but they were found to not be utilising the item when the incident took place. A split-liability agreement gives recognition that while the head injury claimant may not have been liable for the event where their head injury happened, they could have taken one or several steps that would have reduced the risk of injury and/or mitigated levels of harm suffered.
The compensation awarded for each type of head injury will vary, with the value depending on impact, whether short-term or life-changing. For example, a facial injury such as breaking a nose or cheekbone brings one level of compensation, while a serious brain injury, often bringing long-term harm, brings higher compensation amounts.
In the event that you or a loved one has suffered a head injury and are looking into beginning a legal claim, you can get in touch with our Serious Injury or Clinical Negligence legal teams today. They are available by email, web enquiry form, or telephone on 0151 236 1222. Hampson Hughes Solicitors offers the expertise of our head injury solicitors to clients around the Wirral and our team can chat over our legal services with you in more depth.


text:
Standard atmosphere gas properties. Support for n-dim inputs, non-standard atmospheres, units, etc.

[![View Standard Atmosphere Functions on File Exchange](https://www.mathworks.com/matlabcentral/images/matlab-file-exchange.svg)](https://www.mathworks.com/matlabcentral/fileexchange/28135)

Standard atmosphere functions based on the 1976 Standard Atmosphere. Returns density, speed of sound, temperature, pressure, and viscosity for a given altitude input up to 86 km.

Functions are designed to be useful for those designing and analyzing aircraft and have the following features:
- Inputs may be scalar, vector, matrix, or n-dimensional arrays. Functions are vectorized and fast for computing conditions at a large number of points simultaneously. Especially for n-dimensional problems, it is faster than the built-in atmosisa that comes with the aerospace toolbox.
- Temperature offset option for non-standard atmospheres, e.g analyzing hot day aircraft performance.
- Absolute temperature option for non-standard atmospheres, e.g for when outside air temperature is known.
- Either SI or imperial units (and easy to set your preferred default).
- Units consistency can be enforced by using the Physical Units Toolbox, reducing errors and making code clearer.
- Returns everything needed to easily determine important parameters such as dynamic pressure, Mach number, Reynolds number, stagnation temperature, etc.
- Option for geometric instead of geopotential altitude input.
- Density altitude function allows reverse lookup of altitude based on gas properties.
- Stripped-down, troposphere-only function included for when computation speed is a priority.

References: ESDU 77022; www.pdas.com/atmos.html

redpajama_set_name: RedPajamaGithub


text:
About InvestGDA
News Copy
New land available to investors in Gdańsk – Alan Aleksandrowicz passes the word
The city-owned land neighbouring on the Port of Gdańsk waited patiently for the opening of the Still Vistula Tunnel [Martwa Wisła]. Now, InvestGDA is launching a project to commercialise the expanse of over 100 hectares in the location, plus almost 70 hectares in other locations. What will it take to find prospective lessees? What investors can we expect? Alan Aleksandrowicz, President of investGDA, answers those and other questions.
Published on: 26 March 2017
Alan Aleksandrowicz, President of the Management Board, investGDA (Gdańsk Economic Development Agency)
Jerzy Pinkas/www.gdansk.pl
Izabela Biała: At the turn of March and April, investGDA (Gdańsk Economic Development Agency) will announce a tender for the laying of infrastructure in the land it is in charge of, adjacent on the right-bank estate of the Port of Gdańsk. What is the objective the project?
Alan Aleksandrowicz: – We are launching a large-scale project aimed at commercialising the land owned by the City of Gdańsk, stretching along the port. We have called the venture the Pomeranian Investment Centre (embracing over 76 ha – ed.). The area will be provided with a new road network, plus water, sewage, and power supply systems. Our plans fit into the investment pursued by the Port of Gdańsk Authority and consisting in the building of a buffer parking lot to accommodate one thousand trucks on 10 hectares of land purchased from us. We will use the proceeds generated on the sale to complete our project.
How are the construction works intended to proceed? Where will they start?
– We have made an agreement with the Port Authority that the infrastructure we develop will reach the border of the future parking lot. Stage one of the works will consist in building roads from the Koga office building (at Andruszkiewicza St. – ed.), along Sucharskiego St., and up to the site of the planned parking lot, including a connection to Kontenerowa St. which goes to the DCT terminal. This element of the infrastructure should be ready as soon as next year.
Have any prospective investors already expressed their interest in the land, and do you expect the commenced construction works will trigger such interest?
– We are in the midst of negotiations with logistic companies whose businesses are linked to the operations of the DCT terminal. Knowing our plans, they are interested in entering into lease contracts. I cannot disclose the names of the specific investors as yet; nevertheless, in all probability they will start building their warehouses as soon as our works on the new road grid begin. The lessees have specific expectations as to the completion date of our investment project. We would like to make its outcome available to the major investors in the first six months of next year. We should obtain confirmation of whether they will actually invest here this March or April.
You say this will be the first stage into the works. When are you planning stage two?
– Things will depend on the actual developments in the process of commercialising the Pomeranian Investment Centre area. The current division into cadastral plots here is only provisional. Should it happen that after completion of the initial stage in the works, planned to bring roads and the media to the largest possible number of plots, major investors interested in leasing the entire remaining land approach us, the building of the second part of the road network may turn out redundant. I presume we will witness the same mechanism coming into play here as the one we saw in the case of our 'Maszynowa' Industry and Technology Park in Kokoszki. Once we had put the roads and media in place there, the land instantly attracted investors. We have 30 companies operating on the site today.
The Pomeranian Investment Centre marked with the blue dot (google maps)
The Pomeranian Investment Centre expanse encircled in blue (google maps)
As I understand, in the case of the Pomeranian Investment Centre one can count primarily on businesses related to port operations?
– Yes, most of all, although the location, I believe, will also be perfect for companies looking for a convenient access to national roads omitting the centre of Gdańsk. The Sucharski Route in place offers a smooth entry into the S7 road, and the Southern Ring Road into the A1 Motorway. Proximity of the Still Vistula Tunnel is also vital, as it contributes a lot to increased popularity of the port-adjacent areas.
Moreover, we would also like to use our land next to the port to set up at least two storage yards, 10 thousand square metres each. The first one will be completed as early as this year to serve the tenants of our Koga office building. Locations offering storage opportunities are currently much in demand in Gdańsk. Companies in need of storage space, operating on the verge of services and industrial production, are moving out of such areas as e.g. the Letnica district where a developer has commenced works on a residential estate. There will be no place available to them downtown either.
Did you wait for the tunnel to be opened before launching your project of the port-adjacent land?
– Yes, we did; we awaited the 'communication effect' I mentioned earlier on and that is why we first focused on commercialisation of the 'Maszynowa' project which raised high interest among investors. By the way, Goodman dealing in logistics, our first major lessee of the port-adjacent land, did not wait for the completion of the tunnel and developed the entire area of our Pomeranian Logistics Centre.
We are talking about building the roads and providing the media. What about office space? Will the Koga building erected by investGDA have its followers in the neighbourhood?
– We want to prepare for the option of erecting another office building, though at the level of the investment project design for the time being. The companies we are negotiating with are weighing the variant of investing in the construction of warehouses only against the variant of going further into building office space for rent. We do not want to spoil the market and I realise that if we enter the market with office buildings which should be erected by private investors, the industry might interpret it as taking advantage of our apparently privileged position of a municipal business entity.
Why was Koga built then?
– We built Koga to activate the area. In order to inject life there, we had to build a road, lay all media, i.e. give it a start. That same idea guided us in erecting that office building. Should, however, private investors now wish to engage in their own developments, I do not think we should follow the path any further. We will focus on hard infrastructure, possibly simple investments such as storage yards. We will not compete with commercial investors in the office space market. Indeed, sooner or later they will start looking for new locations to develop their centres. After all, the Grunwaldzka/Kołobrzeska quarters will be utilised to the full by 2020.
Is this all land investGDA has at its disposal in the neighbourhood of the Port of Gdańsk?
– There is vacant land along Ku Ujściu St. (over 50 ha – ed.), on the other side of Sucharskiego St., and it carries a high potential too. Its advantage lies in access to the railway infrastructure. PKP is pursuing a large EU-financed project in the area, consisting in redevelopment of the Northern Port reloading station. The Port of Gdańsk Authority [ZMPG] is also pursuing a similar project nearby.
We have recently established a relationship with Balticon with respect to that location. They are launching a large container storage yard with access to railway tracks, and in the future might start a container production plant on the site. As for the other land, we would like to have it contributed in kind to us by the Municipality of Gdańsk. To achieve the objective, we will also need a positive opinion from the Port of Gdańsk Authority, as the said real property lies within the estate administered by the Authority, and – consequently – consent from the Minister of Maritime Economy and Inland Waterways.
The location of the investGDA land along Ku Ujściu St., marked with the blue dot (google maps)
The InvestGDA land along Ku Ujściu St. encircled in blue (google maps)
I take it that at the moment investGDA is focusing primarily on commercialisation of the said land in the vicinity of the Port of Gdańsk. What other locations in Gdańsk do you intend to search investors for?
– We would like to focus on leasing the land in the Płonia districts (over 60 ha – ed.), near the refinery of the Lotos Group. Many companies are voicing their interest in the site even today. Benzynowa St., now in poor condition, will certainly need a revamp and readjustment to the needs of the prospective investors. For the time being, we are in the midst of the procedure of establishing a special economic zone to embrace the area, following suit of the 'Maszynowa' Park special economic zone in Kokoszki. The Gdańsk City Council approved our application last year and we hope the final decision in the matter will be taken by the Council of Ministers this year (in response to the application filed by the Pomeranian Special Economic Zone – ed.). As we see it, the investors need that incentive to resolve to invest in the Płonia districts, while I do not perceive any such need in the case of the port-adjacent land. Obviously, should a large production investment project be proposed there and should its initiator make his decision to proceed conditional on the establishment of such zone there, we will be willing to apply for it.
The location of the investGDA land in the Płonia districts, marked with the blue dot (google maps)
The InvestGDA estate in the Płonia districts, encircled in blue (google maps)
Alongside the port-neighbouring land, Płonia seem to offer the best location in Gdańsk for logistics?
– Certainly so, but it is also favourable for industry. However, we are also preparing for the development of plots on the other side of the S7, spreading over the area of 4 ha along Michałki St. on the border of the Lotos Koleje estate in Rudniki. There are already some large logistic companies, e.g. Makro and Panattoni, operating in the area. If we succeed in obtaining the land from the city and finding investors to develop it, we would like to give the said street and overhaul.
The plots at Michałki St. in the Rudniki district intended for commercialisation by investGDA, marked with the blue dot (google maps)
The plots at Michałki St. in the Rudniki district, encircled in blue (google maps)
Do the above ventures exhaust your plans for the nearest future?
– We want to activate the city-owned land in the vicinities of the airport. The decision to contribute another estate of over 4.5 ha at Azymutalna St. to investGDA was made in February. The location offers advantages to investors in production and advanced technologies. The neighbourhood of Intel and the Pomeranian Special Economic Zone (at Spadochroniarzy St.) has already been extensively developed by businesses attracted by the proximity of the airport. Our plans for the land at Azymutalna St. fit in the Airport City concept pursued by the Gdańsk Airport. The area carries a huge potential.
Let me add here that there are chances to expand the Kokoszki zone by another several hectares, though the move will exhaust the land resources available at the location.
Under the 'Invest in Pomerania' initiative, Gdańsk Economic Development Agency is also engaged as a player in the 'game' of attracting investors to the Tri-City and the land the Agency is not in direct disposal of. Can we expect any 'spectacular entries' in our city this year?
– We are in the course of negotiations, but the investors would rather not have their names disclosed yet. There will be several projects launched in Gdańsk this year, generating another one thousand or more jobs. The engaged businesses operate in such trades as: advanced services, IT, and finance, i.e. the industries we have focused on in the recent years in our project stimulation efforts.
The city-owned areas at Azymutalna St. in the vicinities of the Gdańsk Airport, marked with the blue dot (google maps)
The city-owned areas at Azymutalna St., encircled in blue (google maps)
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NEWS Informacja o wyborze najkorzystniejszej oferty – ZAPYTANIE OFERTOWE nr 2/2018/BROKER/OSLO z dnia 6 grudnia 2018 r. na świadczenie usługi organizacji i logistyki spotkań biznesowych pomorskich MŚP w ramach eksportowego wydarzenia gospodarczego w Oslo 14 January 2019
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NEWS ZAPYTANIE OFERTOWE nr 2/2018/BROKER/OSLO z dnia 6 grudnia 2018 r. Organizacja i logistyka spotkań biznesowych w Oslo. 6 December 2018
Gdansk Economic Development Agency Ltd
Headquaters:
ul. Żaglowa 11 (5th floor)
80-560 Gdańsk, Poland
office@investgda.pl
/InvestGDA
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text:
Рене Лафорг () е френски психиатър и психоаналитик, основател и първи председател на Парижкото психоаналитично общество (1926), пионер на психоанализата в Мароко.

Биография
Роден е на 5 ноември 1894 година в Тан, Елзас, Германия (днес Франция). Учи медицина във Фрайбург и Берлин, където се запознава с трудовете на Фройд, с който по-късно поддържа кореспонденция. През 1919 г. Лафорг публикува статия за възможностите на психоаналитичното лечение на шизофрения, а през 1922 преминава обучителна анализа с Еугения Соколницка в клиниката за психични разстройства "Св. Ана".

Лафорг взема участие в организирането на първата конференция на психоаналитици франкофони в Швейцария (1926), който става прототип на Конгреса на психоаналитиците на франкофонията (Le Congres де Psychanalystes де ла Langue Francaise), който се провежда ежегодно.

През ноември 1926 г., с подкрепата на Мари Бонапарт, основава Парижкото психоаналитично общество (ППО) като е избран за председател, а през 1927 основава "Френски вестник за психоанализа" (La Revue Francaise де Psychanalyse). През 1945 г., е изгонен от ППО, заради сътрудничество с органите на нацистите по време на окупацията на Франция (1940 – 1944). С група от колеги лекари той емигрира в Мароко, която е под протектората на Франция и Испания, където систематично прилага методите и техниката на психоанализата в терапевтичната работа с арабско-мюсюлманското население и по този начин инициира появата на психоаналитичната практика в страната.

В творбите си, Лафорг отделя голямо внимание на изучаването на вината, чувството на неудовлетвореност и проблемите на семейните неврози.

Умира на 6 март 1962 година в Париж на 67-годишна възраст.

Библиография
 Aperçu de l'historique du mouvement psychanalytique en France (1925)
 La Psychanalyse et les névroses (en collaboration avec René Allendy, préface de Henri Claude). Payot, 1924.
 À propos de l'aperçu de l'historique du mouvement psychanalytique en France (1927)
 Relativité de la réalité
 L'Échec de Baudelaire
 Au-delà du scientisme
 Les Processus d'Auto-punition (en collaboration avec Angelo Hesnard), Denoël et Steele, Paris, 1931, 83 pp.

За него
 Alain de Mijolla, Freud et la France, 1885 – 1945, Presses Universitaires de France, 2010 (ISBN 2-13-054515-7)
 M.O. Poivet, René Laforgue. Sa place originale dans la naissance du mouvement psychanalytique français. (1978). dirigé par André Bourguignon (Université de Paris Val-de-Marne, Créteil).
 Martine Lilamand, René Laforgue, fondateur du mouvement psychanalytique français. Sa vie, son œuvre. (1980). dirigé par André Bourguignon (Université de Paris Val-de-Marne, Créteil).
 Annick Ohayon, Psychologie et psychanalyse en France. L'impossible rencontre 1919 – 1969, Ed. La Découverte, 2006, ISBN 2-7071-4779-6

Външни препратки
  Hommage a Rene Laforgue par Jean Rostand

Френски психиатри
Френски психоаналитици
Хора от Гранд Ест
Починали в Париж

redpajama_set_name: RedPajamaWikipedia


8–10 February: an opportunity to see in the e-Tax Board/e-Customs the pre-completed data on your tax return.
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text:
Ex-Indonesian militants to apologise to attack victims
A memorial to victims of the 2002 Bali bombings: Indonesia is planning reconciliation meetings between radicals and their victims
More than 100 former Indonesian militants will this week apologise to survivors of attacks and families of the dead as part of a plan to promote reconciliation and combat extremism, officials said Monday.
The meeting with victims is scheduled for Wednesday after two days of preparatory sessions that started on Monday.
The aim is to promote peace so the former radicals can take their place in society without being marginalised, said officials from Indonesia's counterterrorism agency.
All those taking part have served their prison terms but many have trouble readjusting to life outside jail.
"There are 120 former inmates who have cooperated and will take part," director of deradicalisation at the agency, Irfan Idris, told AFP.
They will include people involved in the 2002 Bali bombings that killed 202 people, mostly foreign tourists.
At meetings Monday and Tuesday they will be given lessons on Indonesia's national values such as tolerance and diversity. Seven ministers are involved in the education sessions.
The former convicts are also being briefed by officials on how to behave when they meet the 70 victims on Wednesday and get the chance to apologise.
Indonesia has the world's biggest Muslim population and has suffered a string of deadly extremist attacks this century.
The government has been trying to deradicalise militants through a programme which began more than 10 years ago.
A crackdown has also weakened the most dangerous networks. But fears have grown of a resurgence in militancy after hundreds of Indonesians flocked to the Middle East and the Philippines in recent years to join the Islamic State group or its allies.
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Single district online programs are the largest and fastest-growing segment of online and blended learning. There were an estimated 1,816,400 enrollments in distance-education courses in K-12 school districts in 2009-2010, almost all of which were online courses (Economics & Statistics, 2011).
Fueling the growth of district online learning programs are technology initiatives. School districts across the country continue to create technology enable learning environments by providing students and teachers with computing devices, often called one-to-one programs.
The expansion of technology in the hands of educators and learners has created an organic movement to online and blended learning. A natural progression to move content and leaning opportunities online once the technology infrastructure is in place to support anytime, anywhere learning. The U.S. Department of Commerce reported that as of October 2010, more than 68 percent of households used broadband internet access service (a four percent increase from 2009), and over 77 percent of households had a computer (Economics & Statistics, 2011).
Digital subscriptions, teacher sharing sites, and free web applications encourage the creation of interactive online content. As a result, districts have organically moved into developing online learning programs. Equity, engagement, and problem solving are often addressed though online learning and digital initiatives.
School administrators understand that access to a locally managed online learning program provides flexibility in scheduling, increased access to courses, professional development and the ability to be student focused. One rural public school district embarked on this journey in 2010 and has learned several lessons.


We are the UK's premier organic brewery making world class beers from the finest organic malt and hops grown on farms without chemicals, as nature intended.
Based near Inverness, the capital of the Highlands of Scotland, it is our beautiful, unspoilt, unpolluted, wild, and not a little bit wet highland home - and we love it! We have our own organic farm where we grow malting barley for brewing. We even have our own brewery house cow, called Molly, who eats the malt from the brewery mash tun and gives us 20 pints of fresh creamy milk every day.
The Black Isle is famous for the quality of its malting barley and it was here on our farm, at Allangrange, that Sir Roderick Mackenzie recorded, in the Statistical Account of 1790, that the land was first cultivated for growing barley because it was found to be of "superior quality for the brewer and distiller." Thanks Rod!
We started off brewing in a bucket in a shed, in 1998, and basically the bucket has just got bigger. Today we can brew up to 10,000 litres a day packaged in bottles, casks and kegs. We ship beer all over Scotland and the UK, and around the world from Sweden to Japan.
We live, work and brew delicious organic beer at Allangrange, translated from the Gaelic as "a fertile field of corn." We see ourselves and what we do, as a natural link between our traditional cultural heritage and the contemporary craft beer world.