Molecular Mechanism For The Altered Traits Of The Red Flesh Bud Sport Of 'Anliu' Sweet Orange

Bud mutations arise often in citrus. The selection of bud mutants is one of the most important breeding channels in citrus. However, the molecular basis of bud mutation has rarely been studied.In this study we describe a novel, pleiotropic sweet orange (Citrus sinensis [L.] Osbeck) mutant, 'Hong Anliu'. This mutation causes carotenoid accumulation, high sugar and low acid in the fruits. Gas chromatography analysis revealed that high sugar and low acid in the fruit were caused by the accumulation of sucrose and the deficiency of citric acid. The dominant carotenoid accumulated in albedo, segment membranes and juice sacs is lycopene, which can reach levels that are a thousand-fold higher than those in comparable wild-type fruits. This mutation does not affect carotenoid composition of leaves. Carotenoid concentration and biosynthetic gene expression of albedo, segment membranes and juice sacs were dramatically altered by the mutation. Lycopene accumulation in the juice sacs was regulated by coordinate expression of carotenoid biosynthetic genes. However, in albedo and segment membranes, the expression of downstream carotenogenic genes seems to be feedback induced by lycopene accumulation. This implies that there must be at least two modes regulating lycopene accumulation in 'Hong Anliu' fruit. Taken together, these results suggest that massive lycopene might be synthesized in the juice sacs and then transported to the segment membrane and the albedo, which leads to lycopene accumulation there.Organic acid concentration and biosynthetic gene expression of albedo, segment membranes and juice sacs were dramatically altered by the mutation. In juice sacs of 'Hong anliu', the concerntration of citric acid was among 1/4 and 1/11 of 'Anliu'. The expression of both upstream (CS) and downstream (Acon, IDH, MDH) genes of 'Hong Anliu'in the citric acid metabolism were higher than those of 'Anliu'. Moreover, the enzymes activities of downstream genes in citric acid metabolism were higher in 'Hong anliu'than those of 'Anliu'. Thus, we speculate that, the citric acid deficiency in the juice sacs of 'Hong anliu' was caused by the up-regulation of the downstream genes.Sugar concentration and metablic gene expression of albedo, segment membranes and juice sacs were dramatically altered by the mutation. The concerntration of sucrose in the juice sacs of 'Hong anliu' was significangly higher than that of 'Anliu'. The expression of sucrose cleavage genes (Ivr and SPS) and sucrose synthetic gene (SuS) in the juice sacs of 'Hong anliu' were higher than those of 'Anliu'. We speculate that the sucrose accumulation in the juice of 'Hong anliu' may be caused by the up-regulation of SuS. However, the sucrose accumulation in the juice of 'Hong anliu' could also be synthesized in the leaves and transported to the juice sacs.To identify potential important or novel genes involved in a spontaneous sweet orange (C. sinensis [L.] Osbeck) bud mutation causing lycopene accumulation, low citric acid, and high sucrose in fruit, suppression subtractive hybridization (SSH) and microarray were performed to decipher this bud mutation during fruit development.After sequencing of the differentially expressed clones, a total of 267 non-redundant transcripts were obtained and 182 (68.2%) of them shared homology (E-value≤1×10^-10) with known gene products. Few genes were consecutively up- or down-regulated (fold change≥2) in the bud mutation during fruit development. SOTA Algorithm analysis results showed that 95.1% of the differentially expressed genes were extensively coordinated with the initiation of lycopene accumulation. Cellular metabolic-, primary metabolic-, localization-, macromolecular metabolic-related transcripts were among the most regulated genes. These genes were involved in many metabolic pathways such as pyruvate metabolism, starch and sucrose metabolism, citrate cycle, glycolysis, and carotenoid biosynthesis pathways. Moreover, 13 genes which were differentially regulated at 170DAF shared homology with previously described signal transduction or transcription factors.This is the first global approach addressing a simultaneous evaluation of transcription changes to bud mutation, and these differentially expressed genes constitute relevant candidates for bud mutation in citrus fruits.