A PCC analysis was performed between the contents of the most abundant sugars, Suc, Glu, Fru, Sor, and G6P , and the expression levels of the 5727 DEGs throughout S2 and S4-II stages, in pairwise PCC comparisons . The combination of defined GO terms and known sugar metabolism-related genes resulted in 28 sugar metabolism-associated genes that were significantly correlated with sugar contents in at least one pairwise PCC comparison . In parallel, WGCNA was performed . Fifteen WGCNA modules labeled by different colors were identified . The majority of the 5727 DEGs were grouped into the ‘darkred’ module , followed by the ‘darkorange’ and ‘blue’ modules that contained 20.9% and 13.8% of the total DEGs, respectively . The other 12 modules comprised 0.5–5.7% of total DEGs . The transcriptome analysis identified 271 sugar metabolism related genes; the majority of these were also grouped into the ‘darkred’ module , followed by the ‘darkorange’ and ‘blue’ modules that contained 21.4% and 16.2% of the total sugar metabolism-related genes, respectively . The other 12 modules included 0–7% of the total genes . Approximately half of the 28 sugar metabolism-related genes identified through PCC analysis were grouped in the ‘blue’ and ‘darkred’ modules, stackable flower pots while the ‘darkorange2’, ‘lightpink3’, and ‘coral1’ each contained 14.3% of the genes .

Modules ‘lightpink4’, ‘yellow4’, and ‘darkseagreen3’ each included 3.6% of the total highly correlated genes . Furthermore, the ‘darkorange2’ module showed the highest number of edges between the genes in the subnetwork  , followed by the ‘blue’ and ‘darkred’ modules, with 270 000 and 126 000 edges, respectively. Since genes showing the highest number of edges were considered as hub genes , we further determined the number of edges of each of the 271 sugar metabolism-related genes , within each module . Genes within each module were ranked from largest to smallest based on their number of edges . By combining results obtained from PCC analysis and WGCNA , we were able to identify 11 key sugar metabolism-associated genes significantly correlated with sugar contents and showing the highest number of edges within their respective modules . Among these 11 genes, 5 were related to Suc metabolism , while the other 6 genes were related to Gal , Tre , Gol and Raf metabolism .A comprehensive validation of the sugar metabolism-associated genes and their related metabolites was carried out using qRT–PCR and enzymatic assays of Santa Rosa and Sweet Miriam fruits and leaves during pit hardening and throughout three ripening stages . Given the significant increase in Sor content in Sweet Miriam fruits , genes associated with Sor metabolism were also included in our analysis.In Santa Rosa and Sweet Miriam cultivars, Suc contents gradually increased in fruits and decreased in leaves throughout development .

Suc contents in Santa Rosa fruits were higher than those in Sweet Miriam fruits during all analyzed stages . In leaves, except at the pit hardening stage, Santa Rosa displayed higher Suc contents than Sweet Miriam . SPS activity was higher in Santa Rosa than Sweet Miriam fruits and leaves and remained constant throughout ripening in Santa Rosa, while SPS activity decreased in Sweet Miriam fruits and leaves . SPS transcript levels in Santa Rosa and Sweet Miriam fruits and leaves displayed similar patterns to the SPS activities . Santa Rosa fruits and leaves displayed higher SPS activities and SPS expression levels with respect to Sweet Miriam . In general, Glu and Fru contents of fruits and leaves from both cultivars decreased towards the fully ripe stage, with Sweet Miriam showing a steeper decrease . Glu and Fru contents of fruits and leaves were higher in Santa Rosa throughout the ripening stages . Suc catabolism can be mediated by the action of SuSy and invertases . Although SuSy could also facilitate the reverse reaction, in fruits and leaves SuSy mediates the breakdown of Suc into UDP-glucose and Fru . Both SuSy expression and activity decreased in Santa Rosa fruits towards the fully ripe stage, while in Sweet Miriam fruits its activity increased from S2 to S4-II stages . In Santa Rosa leaves, SuSy activity increased towards S4-II and remained constant in Sweet Miriam . Three types of invertase activities , cleaving Suc to Glu and Fru , were detected in fruits and leaves from both cultivars. In fruits, CytINV and VINV activities were higher than that of CWINV . In general, all fruit invertase activities were high at the S2 stage and decreased towards the fully ripe stage .

In leaves, similar levels of CWINV, CytINV, and VINV activities were detected . In Santa Rosa leaves, CWINV and VINV remained unchanged during leaf development while CytINV decreased . In Sweet Miriam leaves, CWINV, CytINV, and VINV activities increased during ripening. Since invertase activities are regulated through post-translational suppression via the action of invertase inhibitors , we assessed INVINH transcript levels in both fruits and leaves . In fruits, INVINH transcript levels were similar in Santa Rosa and Sweet Miriam and increased towards the fully ripe stage . In Santa Rosa leaves, INVINH transcript levels were very low as compared with Sweet Miriam . Sweet Miriam leaves displayed 10-fold higher INVINH transcripts during early and mature developmental stages, but decreased to levels similar to Santa Rosa leaves towards the fully ripe stage .Similar to Suc , Sor contents increased in fruits and decreased in leaves of both cultivars during development . Notably, Sor contents of Sweet Miriam fruits and leaves were much higher than those in Santa Rosa at all stages . Sor synthesis is mediated by the activity of S6PDH , which reduces G6P to sorbitol-6-phosphate . S6PDH activity increased towards the fully ripe stage in Sweet Miriam, while it remained unchanged in Santa Rosa fruits . The high Sor contents and increased S6PDH activity were well correlated with the elevated S6PDH transcripts in Sweet Miriam fruits . In fruits from both cultivars, G6P contents increased throughout the ripening-related stages , while Santa Rosa leaves displayed a reduction towards the S4-II stage and Sweet Miriam showed increased G6P contents . Hexokinase activity, that phosphorylatesGlu to G6P , decreased in Santa Rosa fruits throughout the four assayed stages, while it remained unchanged in Sweet Miriam fruits . In leaves, enzymatic activity and mRNA levels were constant throughout the four assayed stages in both cultivars . Degradation of Sor is mediated by the action of NAD+- SDH into Fru using NAD+ as a cofactor, and through SOX into Glu . In fruits from both cultivars, NAD+-SDH and SOX activities decreased towards the fully ripe stage and increased only in Santa Rosa leaves . Nevertheless, NAD+-SDH and SOX activities were 2- to 4-fold higher in Santa Rosa fruits and leaves as compared with Sweet Miriam, and this was paralleled by lower NAD+ levels in fruits and leaves of Santa Rosa .Gal contents in fruits and leaves decreased towards the fully ripe stage in both cultivars yet Gal contents in Santa Rosa were higher than those of Sweet Miriam . Gal synthesis via α-GAL results from the hydrolysis of Raf to yield free Gal and Suc . In fruits and leaves from both cultivars, α-GAL mRNA levels decreased towards the fully ripe stage, tower garden except for Sweet Miriam fruits where these remained unchanged . Additionally, the cleavage of galactosyl residues from cell wall polysaccharides via β- GAL could also contribute to the increase in the free Gal pool . α-GAL and β-GAL transcripts were higher in Santa Rosa fruits and leaves than those in Sweet Miriam and correlated well with the higher Santa Rosa Gal contents . Gal can also be phosphorylated by GalK into Gal 1-P which is converted to UDP-galactose possibly by a pyrophosphorylase . GalK mRNA levels increased in Santa Rosa fruits and decreased in Sweet Miriam fruits throughout development, and were higher in Santa Rosa fruits . In leaves, GalK transcripts remained unchanged in both cultivars, with Sweet Miriam displaying higher transcripts levels . On the other hand, UDP-Gal together with Ino are used as substrates by GolS for the synthesis of Gol . During development and ripening, Gol contents remained constant in Santa Rosa fruits and leaves while Gol increased in Sweet Miriam fruits, but not in leaves . GolS transcripts in Sweet Miriam fruits and leaves, paralleled the increased Gol contents observed in this cultivar . Gol, together with Suc, form Raf through the action of RS , releasing Ino . RS displayed increasing mRNA levels towards the fully ripe stage in fruits from Santa Rosa and Sweet Miriam and only in leaves from Sweet Miriam, while Santa Rosa remained unchanged . Accordingly, Raf contents increased in Sweet Miriam but not in Santa Rosa fruits and Raf contents in Sweet Miriam leaves were higher than those in Santa Rosa .

Tre contents increased in fruits from both cultivars towards the fully ripe stage and in Sweet Miriam leaves . Catabolism of Tre is mediated by the action of TRE . TRE mRNA levels remained unchanged in fruits from both cultivars during fruit development and ripening, while it decreased in leaves .In this study, we compared biochemical and molecular characteristics of two genetically related Japanese plums . While plum fruits, in general, and the cultivar Santa Rosa, in particular, have been historically classified as climacteric, its bud sport mutant Sweet Miriam is a non-climacteric fruit . The existence of two contrasting ripening behaviors in fruits of the same genetic background offers a unique experimental system to investigate the influence of ethylene on sugar metabolism and fruit sugar composition. Although the expression of genes associated with sugar metabolism in fruits has been extensively studied in a number of climacteric and non-climacteric species , studies comparing global gene expression, enzymatic activities, and sugar contents in climacteric versus non-climacteric species/cultivars, and additionally including leaves, are scanty . Our initial study revealed altered sugar metabolism and differences in sugar contents between the fruits of Santa Rosa and Sweet Miriam cultivars . Non-climacteric fruits displayed enhanced sorbitol contents, and a link between ethylene and fruit sugar homeostasis was suggested . Here, we used transcriptomics and metabolomics to identify genes associated with the notable differences in sugar composition between the climacteric and non-climacteric fruits. Additionally, WGCNA allowed the identification of a number of sugar metabolism-associated genes that could act as putative hubs . We functionally assessed the role of these genes and demonstrated their involvement in the differences in sugar metabolism between fruits with contrasting ripening behaviors. A schematic display illustrating the overall summary of the results obtained in this work is presented in Figure 7.A number of studies suggested a possible relationship between sugar and ethylene metabolism. Suc stimulated ethylene production in tomato through increased expression of genes associated with ethylene biosynthesis and signaling , and, in strawberry, exogenous Suc accelerated fruit ripening . Our previous results showed increased Sor contents of nonclimacteric Sweet Miriam fruits, suggesting a reciprocal correlation between ethylene and Sor. This notion appeared to be supported by the high Sor contents of climacteric apple fruits treated with the inhibitor of ethylene perception, 1-methylcyclopropene  and by the decrease in the protein levels of S6PDH, catalyzing Sor synthesis, in ethylene-treated apple fruits . The gradual decrease in Sor and Suc leaf contents during development and the parallel increase in the fruits of both Santa Rosa and Sweet Miriam cultivars were in good agreement with the role of Sor and Suc as the major photo assimilates translocated from leaves to fruits in members of the Rosaceae family . In general, changes in Sor contents during fruit development and the Sor increase seen in Sweet Miriam fruits were well correlated with the high levels of G6P and higher S6PDH activity and S6PDH transcripts . Free Suc contents are determined by the balance between Suc synthesis and Suc degradation . The relatively high SPS expression levels and SPS activity in Santa Rosa leaves and fruits correlated well with the higher Suc contents . This is consistent with Hubbard et al. who reported that SPS activity within the fruit itself is an important contributor to fruit Suc contents. Supporting this notion, reports in other climacteric fruits, such as tomato , melons , jackfruit , and apples , demonstrated the importance of SPS in sucrose accumulation. With regards to Suc degradation, at least during the S2 stage, CWINV and CytINV activities were lower in Santa Rosa than in Sweet Miriam fruits, while VINV and SuSy were higher in Santa Rosa fruits .