File S1 - Analysis of Papaya Cell Wall-Related Genes during Fruit Ripening Indicates a Central Role of Polygalacturonases during Pulp Softening

These are the legends for Supporting Tables / Figures presented in File S1. Table S1. Cell wall-related genes from ripe papaya and mature A. thaliana plant. Table S2. Similarity percentage of amino acid from papaya and other plants PGs. Table S3. Nucleotide sequences used in PCR reactions. Table S4. Nucleotide sequences used in qPCR. Table S5. Calibration curves for relative gene expression. Table S6. Calibration curves for absolute gene expression. Figure S1. Up-regulation of cell wall-related genes during papaya ripening. Real-time PCR (qPCR) was used to determine the absolute quantitation of the mRNA levels of various genes during papaya ripening. The quantification is represented by the column height. The error bars on each column indicate the SD from four technical replicates from samplings I and II. The different letters represent samples that were significantly different from those collected on other days post-harvest (within the same gene) as determined by one-way ANOVA and Tukey's test (αn = 4). Figure B shows the threshold cycle values (Ct) for the two genes used as internal controls (actin gene – cpACT and elongation factor 1-alpha gene - cp_EF1). Figure S2. Genomic and mRNA organization of different PGs from papaya fruit. Grey boxes represent coding regions (exons), while black lines represent non-coding regions (introns). White boxes represent the mRNA sequences concatenated from the above compared exons. Figure S3. Unrooted phylogram encompassing PGs from papaya, Arabidopsis and several other plant organisms. A phylogenetic tree was calculated using the neighbor-joining method based on the ClustalW alignment of the deduced amino acid sequences. The putative signal peptide from all of the proteins was removed from the sequence. The following proteins and their corresponding GenBank IDs were used: A. thaliana 1, 2 and 3 (NP_191544, NP_191310, NP_187454), P. persica 1 and 2 (AAC64184, CAA54448), P. communis 1 and 2 (CAH18935, BAC22688), D. carota (BAC87792), S. lycopersicum 1 and 2 (ABW38780; AC28947), C. melo 1 and 2 (AAC26510; AAC26512), V. vinifera 1 and 2 (XP_002263164, XP_002282759), B. napus (CAA65072), G. Max 1 and 2 (ABC70314, AAL30418), D. kaki (ACJ06506), R. communis 1, 2 and 3 (XP_002529090, XP_002529616, XP_002517823), A. deliciosa (P35336), O. europaea (ACA49228), P. trichocarpa 1, 2 and 3 (XP_002331621, XP_002322711, XP_002313308) and C. papaya 1, 2, 3 and 4 (FJ007644, GQ479791, GQ479794 e GQ479795). The values for the branch lengths are based on the scale bar; there are 0.1 residue substitutions per site. Letters A and B show the two distinct clades resulting from the phylogenetic analyses. Figure S4. Recombinant proteins sequencing. Figures show the query sequences of cpPG1 (A) protein, and the correspondent peptides that were visualized in protein sequencing. Residues highlighted by gray boxes and black letters are from expression vector; residues highlighted by black boxes and white letters are those obtained in protein sequencing. The histidine tags are underlined and asterisks indicate the end of protein. Figure S5. Expression of recombinant proteins from papaya pulp. The figures show representative gels from heterologous expression of cpPG1 (A) gene. Figure A. Proteins profiles from bacteria carrying pET45-cpPG1 before IPTG induction (PG), after 16 hs growth non-induced (PG-NI) and after 16 hs growth IPTG-induced (PG-I); Ni-purified recombinant proteins can be seen after properly elution (E 1 and 2). Proteins markers are highlighted with weight numbers. Figure B. The figure displays the hybridization of antiserum for cpPG1 recombinant protein. The arrow indicates the molecular weight for commercially available protein markers. The quantities of proteins are also indicated. Figure S6. Protein alignment of four distinct papaya PGs. The corresponding proteins from cpPG1 (PG1 - FJ007644), cpPG2 (PG2 - GQ479791), cpPG3 (PG3 - GQ479794) and cpPG4 (PG4 - GQ479795) genes were aligned using ClustalW and viewed with BOXSHADE program and then manually edited. Gaps were introduced to optimize alignment. Identic amino acids are highlighted by black boxes and white letters, similar amino acids are highlighted by grey boxes and white letters and different amino acids are highlighted by white boxes and black letters. The conserved domain of glycosil hydrolases from family 28 (PS00502) is indicated by a dotted-line box. The probable catalytic residues are indicated by asterisks, being possible to observe the cpPG2 protein has a Glu346 instead of a Lys346 as the other three polygalacturonases have. (ZIP)