Citrus is one of the world's most important fruit crops, contributing essential nutrients, such as vitamin C and minerals, to the human diet. It is characterized by two important traits: first, its major edible part is composed of juice sacs, a unique structure among fruit, and second, relatively high levels of citric acid are accumulated in the vacuole of the juice sac cell. Although the major routes of primary metabolism are generally the same in citrus fruit and other plant systems, the fruit's unique structural features challenge our understanding of carbon flow into the fruit and its movement through all of its parts. In fact, acid metabolism and accumulation have only been summarized in a few reviews. Here we present a comprehensive view of sugar, acid and amino acid metabolism and their connections within the fruit, all in relation to the fruit's unique structure.

Many fruit trees undergo cycles of heavy fruit load (ON-Crop) in one year, followed by low fruit load (OFF-Crop) the following year, a phenomenon known as alternate bearing (AB). The mechanism by which fruit load affects flowering induction during the following year (return bloom) is still unclear. Although not proven, it is commonly accepted that the fruit or an organ which senses fruit presence generates an inhibitory signal that moves into the bud and inhibits apical meristem transition. Indeed, fruit removal from ON-Crop trees (de-fruiting) induces return bloom. Identification of regulatory or metabolic processes modified in the bud in association with altered fruit load might shed light on the nature of the AB signalling process. The bud transcriptome of de-fruited citrus trees was compared with those of ON- and OFF-Crop trees. Fruit removal resulted in relatively rapid changes in global gene expression, including induction of photosynthetic genes and proteins. Altered regulatory mechanisms included abscisic acid (ABA) metabolism and auxin polar transport. Genes of ABA biosynthesis were induced; however, hormone analyses showed that the ABA level was reduced in OFF-Crop buds and in buds shortly following fruit removal. Additionally, genes associated with Ca(2+)-dependent auxin polar transport were remarkably induced in buds of OFF-Crop and de-fruited trees. Hormone analyses showed that auxin levels were reduced in these buds as compared with ON-Crop buds. In view of the auxin transport autoinhibition theory, the possibility that auxin distribution plays a role in determining bud fate is discussed.

The enzyme 1-amino-cyclopropane-1-carboxylic acid synthase (ACS) participates in the ethylene biosynthesis pathways and it is tightly regulated transcriptionally and post-translationally. Notwithstanding its major role in climacteric fruit ripening, the transcriptional regulation of ACS during ripening is not fully understood. We studied fruit ripening in two Japanese plum cultivars, the climacteric Santa Rosa (SR) and its non-climacteric bud sport mutant, Sweet Miriam (SM). As the two cultivars show considerable difference in ACS expression, they provide a good system for the study of the transcriptional regulation of the gene. To investigate the differential transcriptional regulation of ACS1 genes in the SR and SM, their promoter region, which showed only minor sequence differences, was isolated and used to identify the binding of transcription factors interacting with specific ACS1 cis-acting elements. Three transcription factors (TFs), abscisic acid-insensitive 5 (ABI5), GLABRA 2 (GL2), and TCP2, showed specific binding to the ACS1 promoter. Synthetic DNA fragments containing multiple cis-acting elements of these TFs fused to β-glucuronidase (GUS), showed the ABI5 binding site mediated ethylene and abscisic acid (ABA) responses of the promoter. While TCP2 and GL2 showed constant and similar expression levels in SM and SR fruit during ripening, ABI5 expression in SM fruits was lower than in SR fruits during advanced fruit ripening states. Overall, the work demonstrates the complex transcriptional regulation of ACS1.

We investigated sugar metabolism in leaves and fruits of two Japanese plum (Prunus salicina Lindl.) cultivars, the climacteric Santa Rosa and its bud sport mutant the non-climacteric Sweet Miriam, during development on the tree. We previously characterized differences between the two cultivars. Here, we identified key sugar metabolic pathways. Pearson coefficient correlations of metabolomics and transcriptomic data and weighted gene co-expression network analysis (WGCNA) of RNA sequencing (RNA-Seq) data allowed the identification of 11 key sugar metabolism-associated genes: sucrose synthase, sucrose phosphate synthase, cytosolic invertase, vacuolar invertase, invertase inhibitor, α-galactosidase, β-galactosidase, galactokinase, trehalase, galactinol synthase, and raffinose synthase. These pathways were further assessed and validated through the biochemical characterization of the gene products and with metabolite analysis. Our results demonstrated the reprogramming of sugar metabolism in both leaves and fruits in the non-climacteric plum, which displayed a shift towards increased sorbitol synthesis. Climacteric and non-climacteric fruits showed differences in their UDP-galactose metabolism towards the production of galactose and raffinose, respectively. The higher content of galactinol, myo-inositol, raffinose, and trehalose in the non-climacteric fruits could improve the ability of the fruits to cope with the oxidative processes associated with fruit ripening. Overall, our results support a relationship between sugar metabolism, ethylene, and ripening behavior.

We recently identified a Citrus gene encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor that contained a sequence complementary to miR156. Genes of the SPL family are known to play a role in flowering regulation and phase transition. In Citrus, the mRNA levels of the gene were significantly altered by fruit load in buds; under heavy fruit load (ON-Crop trees), known to suppress next year flowering, the mRNA levels were down-regulated, while fruit removal (de-fruiting), inducing next-year flowering, resulted in its up-regulation. In the current work, we set on to study the function of the gene. We showed that the Citrus SPL was able promote flowering independently of photoperiod in Arabidopsis, while miR156 repressed its flowering-promoting activity. In order to find out if fruit load affected the expression of additional genes of the SPL family, we identified and classified all SPL members in the Citrus genome, and studied their seasonal expression patterns in buds and leaves, and in response to de-fruiting. Results showed that two additional SPL-like genes and miR172, known to be induced by SPLs in Arabidopsis, were altered by fruit load. The relationships between these factors in relation to the fruit-load effect on Citrus flowering are discussed.