BUSCADOR RECOLECTA

1 figure., Fruit ripening is an important process that affects fruit quality. A QTL in melon, ETHQV6.3, involved in climacteric ripening regulation, has been found to be encoded by CmNAC-NOR, a homologue of the tomato NOR gene. To further investigate CmNAC-NOR function, we obtained two CRISPR/Cas9-mediated mutants (nor-3 and nor-1) in the climacteric Védrantais background. nor-3, containing a 3-bp deletion altering the NAC domain A, resulted in ~8 days delay in ripening without affecting fruit quality. In contrast, the 1-bp deletion in nor-1 resulted in a fully disrupted NAC domain, which completely blocked climacteric ripening. The nor-1 fruits did not produce ethylene, no abscission layer was formed and there was no external color change. Additionally, volatile components were dramatically altered, seeds were not well developed and flesh firmness was also altered. There was a delay in fruit ripening with the nor-1 allele in heterozygosis of ~20 days. Our results provide new information regarding the function of CmNAC-NOR in melon fruit ripening, suggesting that it is a potential target for modulating shelf life in commercial climacteric melon varieties., Peer reviewed

Supplementary Table S1: Databases and corresponding search strings for data retrieval., Peer reviewed

1 table. -- Supplemental Table 4. Primers used in the qPCR analyses performed in this study., In plants salt and water stress result in an induction of respiration and accumulation of stress-related metabolites (SRMs) with osmoregulation and osmoprotection functions that benefit photosynthesis. The synthesis of SRMs may depend on an active respiratory metabolism, which can be restricted under stress by the inhibition of the cytochrome oxidase pathway (COP), thus causing an increase in the reduction level of the ubiquinone pool. However, the activity of the alternative oxidase pathway (AOP) is thought to prevent this from occurring while at the same time, dissipates excess of reducing power from the chloroplast and thereby improves photosynthetic performance. The present research is based on the hypothesis that the accumulation of SRMs under osmotic stress will be affected by changes in folial AOP activity. To test this, the oxygen isotope-fractionation technique was used to study the in vivo respiratory activities of COP and AOP in leaves of wild-type Arabidopsis thaliana plants and of aox1a mutants under sudden acute stress conditions induced by mannitol and salt treatments. Levels of leaf primary metabolites and transcripts of respiratory-related proteins were also determined in parallel to photosynthetic analyses. The lack of in vivo AOP response in the aox1a mutants coincided with a lower leaf relative water content and a decreased accumulation of crucial osmoregulators. Additionally, levels of oxidative stress-related metabolites and transcripts encoding alternative respiratory components were increased. Coordinated changes in metabolite levels, respiratory activities and photosynthetic performance highlight the contribution of the AOP in providing flexibility to carbon metabolism for the accumulation of SRMs., Peer reviewed

Supplemental Table 3. Absolute metabolite levels in leaves of WT and AOX1a plants under control conditions and after 1 day of severe (300 mM) NaCl and Mannitol treatments as measured by GC-MS (see material and methods for details). Data is presented as means ± SE for 4 to 6 biological replicates. Bold numbers denote significant differences (P < 0.05) to the control condition in each genotype separately. Asterisks denote significant differences (P < 0.05) between WT and aox1a plants in each experimental condition. †Denotes metabolites detected only in one replicate in WT at control conditions. ‘n.d.’ denotes cases for not detected metabolites., In plants salt and water stress result in an induction of respiration and accumulation of stress-related metabolites (SRMs) with osmoregulation and osmoprotection functions that benefit photosynthesis. The synthesis of SRMs may depend on an active respiratory metabolism, which can be restricted under stress by the inhibition of the cytochrome oxidase pathway (COP), thus causing an increase in the reduction level of the ubiquinone pool. However, the activity of the alternative oxidase pathway (AOP) is thought to prevent this from occurring while at the same time, dissipates excess of reducing power from the chloroplast and thereby improves photosynthetic performance. The present research is based on the hypothesis that the accumulation of SRMs under osmotic stress will be affected by changes in folial AOP activity. To test this, the oxygen isotope-fractionation technique was used to study the in vivo respiratory activities of COP and AOP in leaves of wild-type Arabidopsis thaliana plants and of aox1a mutants under sudden acute stress conditions induced by mannitol and salt treatments. Levels of leaf primary metabolites and transcripts of respiratory-related proteins were also determined in parallel to photosynthetic analyses. The lack of in vivo AOP response in the aox1a mutants coincided with a lower leaf relative water content and a decreased accumulation of crucial osmoregulators. Additionally, levels of oxidative stress-related metabolites and transcripts encoding alternative respiratory components were increased. Coordinated changes in metabolite levels, respiratory activities and photosynthetic performance highlight the contribution of the AOP in providing flexibility to carbon metabolism for the accumulation of SRMs., Peer reviewed

The following are available online at https://www.mdpi.com/article/10.3390/cancers14020449/s1. Supplementary Figures. Figure S1: Distribution of pathological CSF samples (without healthy ones) among each phase of study and the different groups according to the incidence of the pathology, depending on the infiltration (CSF +/− LM) and the primary tumor (hematologic and solid tumor). Figure S2: Quality control images of the planar protein microarrays generated. Figure S3: A quantile normalization in planar protein microarrays. Figure S4: Coomasie gels which indicate protein distribution across samples. Figure S5: Venn diagrams of total identified proteins with LC-MS/MS. Figure S6: Plots showing the functional proteins using the Reactome for different conditions. Figure S7: Differential protein profiles within CSF + LM according to primary tumor (Lymphoma) by protein microarrays. Figure S8: Differential protein profiles within CSF + LM according to primary tumor (Leukemia) by protein microarrays. Figure S9: Differential protein profiles within CSF + LM according to primary tumor (Lymphoma) by affinity proteomics. Figure S10: Differential protein profiles within CSF + LM according to primary tumor (Leukemia) by affinity proteomics. Figure S11: Summary of the multipronged proteomics characterization among the different phases of study. Supplementary Tables. Table S1: Table of clinical-biological characteristics from the whole CSF samples used in the study. Table S2: Antibodies list used in Planar Protein Microarrays. Table S3: Antibodies list used in Beads Suspension Microarrays. Table S4: Protein identification with LC-MS/MS among the different strategies and their emPAI quantification. Table S5: Boxplots of the protein identified in validation and confirmation phases, respectively, comparing the different groups of study. Table S6: Intensity data results from Planar Protein Microarrays. Table S7: Intensity data results from Beads Suspension Microarrays. Table S8: ROC analysis list of potential biomarker panel on CSF +/− LM and the different comparisons by protein arrays and affinity proteomics., Peer reviewed

1 table. -- Supplemental Table 2. Relative metabolite levels in leaves of WT and aox1a plants under control conditions and after 1 day of severe (300 mM) NaCl and Mannitol treatments as measured by GC-MS (see material and methods for details). Data is presented as means ± SE for 4 to 6 biological replicates normalized to the mean level of the WT plants under control conditions. Bold numbers denote significant differences (P < 0.05) to the control condition in each genotype separately. Asterisks denote significant differences (P < 0.05) between WT and aox1a plants in each experimental condition. †Denotes metabolites detected only in one replicate in WT at control conditions. ‘n.d.’ denotes cases for not detected metabolites., In plants salt and water stress result in an induction of respiration and accumulation of stress-related metabolites (SRMs) with osmoregulation and osmoprotection functions that benefit photosynthesis. The synthesis of SRMs may depend on an active respiratory metabolism, which can be restricted under stress by the inhibition of the cytochrome oxidase pathway (COP), thus causing an increase in the reduction level of the ubiquinone pool. However, the activity of the alternative oxidase pathway (AOP) is thought to prevent this from occurring while at the same time, dissipates excess of reducing power from the chloroplast and thereby improves photosynthetic performance. The present research is based on the hypothesis that the accumulation of SRMs under osmotic stress will be affected by changes in folial AOP activity. To test this, the oxygen isotope-fractionation technique was used to study the in vivo respiratory activities of COP and AOP in leaves of wild-type Arabidopsis thaliana plants and of aox1a mutants under sudden acute stress conditions induced by mannitol and salt treatments. Levels of leaf primary metabolites and transcripts of respiratory-related proteins were also determined in parallel to photosynthetic analyses. The lack of in vivo AOP response in the aox1a mutants coincided with a lower leaf relative water content and a decreased accumulation of crucial osmoregulators. Additionally, levels of oxidative stress-related metabolites and transcripts encoding alternative respiratory components were increased. Coordinated changes in metabolite levels, respiratory activities and photosynthetic performance highlight the contribution of the AOP in providing flexibility to carbon metabolism for the accumulation of SRMs., Peer reviewed

Present-day pterygote insects have two pairs of wings, one in the mesothorax (T2), the other in the metathorax (T3), and both have diverged in structure and function in different groups. Studies in endopterygote and paraneopteran species have shown that the gene Ultrabithorax (Ubx) specifies the identity and wing structure in T3, whereas the gene apterous (ap) significantly contributes to forming modified T2 wings. We wondered whether these Ubx and ap mechanisms operate in the lineage of polyneopterans. To explore this possibility, we used the cockroach Blattella germanica (Polyneoptera and Blattodea), in which the T2 wings are sclerotized (tegmina), whereas those of the T3 are membranous. We found that Ubx determines the structure of T3 and the membranous wing, while ap significantly contributes to form the sclerotized T2 tegmina. These results along with the studies carried out on the beetle Tribolium castaneum by Tomoyasu and collaborators suggest that ap plays an important role in the sclerotization and melanization of the T2 wings in neopteran groups that have sclerotized forewings. In turn, the sclerotizing properties of ap demonstrated in beetles and cockroaches suggest that the origin of this function goes back to the emergence of Neoptera, in the mid Devonian., Peer reviewed

Primers used to measure transcript levels by qPCR in Blattella germanica tissues and to prepare the dsRNA for RNAi experiments.;Summary of the experiments carried out to study the effects of RNAi targeting Ultrabithorax (Ubx) or apterous-A (ap-A) and apterous-B (ap-B) on wing development in Blattella germanica.;Alignment of the homeodomain of Apterous proteins., Peer reviewed

1 table. -- Supplementary Table 1. Overview of the metabolite reporting list., In plants salt and water stress result in an induction of respiration and accumulation of stress-related metabolites (SRMs) with osmoregulation and osmoprotection functions that benefit photosynthesis. The synthesis of SRMs may depend on an active respiratory metabolism, which can be restricted under stress by the inhibition of the cytochrome oxidase pathway (COP), thus causing an increase in the reduction level of the ubiquinone pool. However, the activity of the alternative oxidase pathway (AOP) is thought to prevent this from occurring while at the same time, dissipates excess of reducing power from the chloroplast and thereby improves photosynthetic performance. The present research is based on the hypothesis that the accumulation of SRMs under osmotic stress will be affected by changes in folial AOP activity. To test this, the oxygen isotope-fractionation technique was used to study the in vivo respiratory activities of COP and AOP in leaves of wild-type Arabidopsis thaliana plants and of aox1a mutants under sudden acute stress conditions induced by mannitol and salt treatments. Levels of leaf primary metabolites and transcripts of respiratory-related proteins were also determined in parallel to photosynthetic analyses. The lack of in vivo AOP response in the aox1a mutants coincided with a lower leaf relative water content and a decreased accumulation of crucial osmoregulators. Additionally, levels of oxidative stress-related metabolites and transcripts encoding alternative respiratory components were increased. Coordinated changes in metabolite levels, respiratory activities and photosynthetic performance highlight the contribution of the AOP in providing flexibility to carbon metabolism for the accumulation of SRMs., Peer reviewed

1 figure., In plants salt and water stress result in an induction of respiration and accumulation of stress-related metabolites (SRMs) with osmoregulation and osmoprotection functions that benefit photosynthesis. The synthesis of SRMs may depend on an active respiratory metabolism, which can be restricted under stress by the inhibition of the cytochrome oxidase pathway (COP), thus causing an increase in the reduction level of the ubiquinone pool. However, the activity of the alternative oxidase pathway (AOP) is thought to prevent this from occurring while at the same time, dissipates excess of reducing power from the chloroplast and thereby improves photosynthetic performance. The present research is based on the hypothesis that the accumulation of SRMs under osmotic stress will be affected by changes in folial AOP activity. To test this, the oxygen isotope-fractionation technique was used to study the in vivo respiratory activities of COP and AOP in leaves of wild-type Arabidopsis thaliana plants and of aox1a mutants under sudden acute stress conditions induced by mannitol and salt treatments. Levels of leaf primary metabolites and transcripts of respiratory-related proteins were also determined in parallel to photosynthetic analyses. The lack of in vivo AOP response in the aox1a mutants coincided with a lower leaf relative water content and a decreased accumulation of crucial osmoregulators. Additionally, levels of oxidative stress-related metabolites and transcripts encoding alternative respiratory components were increased. Coordinated changes in metabolite levels, respiratory activities and photosynthetic performance highlight the contribution of the AOP in providing flexibility to carbon metabolism for the accumulation of SRMs., Peer reviewed