## 4 Materials and Methods
### Gene Expression Analysis by Real Time PCR  
Quantitative real-time PCR was carried out on a LightCycler 480 instrument (Roche, Madrid, Spain), using the LightCycler 480 SYBRGreen I Master kit (Roche, Madrid, Spain). Reaction mix and conditions followed the manufacturer's instructions with some modifications. The PCR mix contained 1 \(\upmu\)L of diluted cDNA, 5 \(\upmu\)L of SYBR Green I Master Mix, 1 \(\upmu\)L of 3 \(\upmu\)M primer F, and 1 \(\upmu\)L of 3 \(\upmu\)M primer R, with a final volume of 10 \(\upmu\)L. The primers (PSF purified, Isogen, Utrecht, Netherlands) used for the amplification of each gene are described by [PERSON] et al. [19]. The cycling protocol, for all genes, consisted of 10 min at 95 \({}^{\circ}\)C for pre-incubation, then 40 cycles of 10 s at 95 \({}^{\circ}\)C for denaturation, 10 s at 59 \({}^{\circ}\)C for annealing, and 10 s at 72 \({}^{\circ}\)C for extension. Fluorescent intensity data was acquired during the extension time with the LightCycler 480 Software release 1.5.0, version 1.5.0.39 (Roche, Madrid, Spain) and were transformed into mRNA levels by using specific standard curves for all analyzed genes. Amplification efficiency (E) and correlation coefficient (\(r^{2}\)) of each primer were calculated using the standard curve method and the formula E = 10\({}^{(-1/\mathrm{slope})}\)[68].  
The specificity of the PCR reaction was assessed by the presence of a single peak in the dissociation curve performed after the amplification steps followed by the sequencing of the amplicon. In this experiment _ACTIN, ELONGATION FACTOR 1 (EF1)_, and \(\beta\)-_TUBULIN_ (\(\beta\)-_TUB_) were tested as potential housekeeping genes based on previously published primer sequences of _Citrus_ genes using BestKeeper software [69]. The _ACTIN_ gene was the best housekeeping gene for our analysis, having the lowest SD [\(\pm\)CP], therefore the normalization of the gene expression levels was done against it. Expression levels were calculated using the Relative Expression Software Tool (REST, [70]) and relative to the expression value obtained for each gene in the flavedo of immature-green Valencia Late fruits (collected in June), which was arbitrarily given the expression value of 1. Results are the mean of at least three replicates.  
Search for Putative Transcription Factor Binding Sites in the Promoter Region of Genes Involved in AsA Metabolism  
The DNA sequences of the promoter regions (600 bp upstream of the start codon ATG) were obtained for the 23 genes involved in AsA metabolism analyzed in this work. Sequences were downloaded from the _Citrus sinensis_ (sweet orange) genome databases Huazhong Agricultural University ([[http://citrus.hzau.edu.cn/orange](http://citrus.hzau.edu.cn/orange)]([http://citrus.hzau.edu.cn/orange](http://citrus.hzau.edu.cn/orange)), accessed on 14 April 2021; [36]) and Phytozome (JGI Phytozome v12; [37]), and are available in Table S5. Subsequently, the promoter regions were used to carry out a screening analysis in the MatInspector library of matrices for plant transcription binding sites version 9.4 (Genomatix software suite v3.7, Munich, Germany, [71; 72]) Only the matrices with a \(p\)-value equal or lower than 0.05 were considered in the present work. The \(p\)-value represents the probability to obtain an equal or greater number of sequences with a match in a randomly drawn sample of the same size as the input sequence set, hence the lower this probability, the higher is the importance of the observed TFBS.