Molecular Genetic Analysis of Citric Acid Accumulation in Citrus Fruit
Budget
Duration
$300,000
3 years
Sadka, A.
ARO, Min. Ag.
Roose, M.L.
UC, Riverside
Erner, Y.
ARO, Min. Ag.
Achievements Fruit juice pH, titratable acidity or citric acid content was measured in 6 populations derived from an acidless pummelo (pummelo 2240) [Citrus maxima (Burm.) Merrill]. The acidless trait in pummelo 2240 is controlled by a single gene called acitric. Using bulked segregant analysis, three random amplified polymorphic DNA (RAPD) markers were identified as linked to acitric (Paper #1). RAPD marker OpZ20410, which mapped 1.2 cM from acitric, was cloned, sequenced and a sequence characterized amplified region (SCAR) marker (SCZ20) was developed. The SCZ20-410 marker allele that is linked to the acitric allele occurs only in pummelo 2240 and other pummelos, and therefore, this SCAR marker should be useful as a dominant or codominant marker for introgressing acitric into mandarins and other citrus species. Using the cloned OpZ20410 band as a hybridization probe revealed a codominant RFLP marker called RFZ20 that mapped 1.2 cM from acitric. Progeny homozygous for the acitric allele (genotype acac) had citric acid content in juice below 10 mm, the minimum detection level by HPLC. The citric acid content of fruit juice and marker OpZ2041O were studied in 82 progeny of a cross between Chandler pummelo (genotype Acac) and Poncirus trifoliata (genotype AcAc). Progeny predicted to be heterozygous for acitric (genotype Acac) by the marker had juice with about 30% less citric acid than individuals predicted to be homozygous AcAc. The significance of this result is that it indicates that the same gene (or at least chromosome segment) of pummelo 2240 that blocks citric acid accumulation when homozygous will also reduce acidity when heterozygous. Markers OpZ20410, SCZ20, and RFZ20 were highly polymorphic among 59 citrus accessions and would allow citrus breeders to select seedling progeny heterozygous for acitric in nearly all crosses between hybrids of 2240 pummelo and other citrus genotypes. assisted transfer of acitric into other genetic backgrounds should be possible.
The molecular physiology of acid accumulation (Israel)
1. Cloning of the genes for organic acid metabolism from juice sac cells. The cDNAs for a most of the genes for organic acid metabolism (citrate synthase, cytosolic aconitase, NADP and NAD isocitrate dehydrogenase (ICDH), fumarase, malate dehydrogenase, malic enzyme) were cloned from lemon pulp (partially unpublished). This, in addition to other findings, indicates that citrate, among other organic acids, is synthesized in the fruit, and is not translocated from other plant organs.
2.The activity and expression of citrate synthase in acidless lime, acid lemon and in 'Minneola' fruits and tissue cultures treated with arsenite. The mitochondrial citrate synthase was compared between acidless lime and sour lemon (Paper #5) . Its mRNA level in sour lemon increased 3-4 fold early in fruit development, in association with acid accumulation. This increase was followed by an increase in the activity of citrate synthase in the mitochondria, as expected from a biosynthetic
enzyme. Similar results were also obtained with sweet lime. Considering these results it seems that the mitochondrial citrate synthase plays a role in citrate accumulation. However, its action cannot explain the difference between low- and high-acid varieties. The activity of citrate synthase of 'Minneola' tangelo fruit was transiently inhibited by sodium arsenite, a treatment that reduced acid content in citrus fruit (paper #3). The recovery in the enzymatic activity could be explained by induction in the transcript level, detected in the fruit and in tissue cultures following arsenite treatment.
3. Aconitase activity and expression in the fruit. It has long been hypothesized that a metabolic block in the mitochondrial aconitase (which catabolizes citrate) plays a major role in citrate accumulation. Indeed, it has been shown that the activity of this enzyme in sour lemon is greatly reduced early in fruit development, while no reduction was detected during the development of acidless fruits (Paper #2). These results provide a reasonable scenario for acid accumulation: the reduction in the mitochondrial aconitase creates a local increase in the citric acid level; the additional acid is removed from the mitochondria to the cytosol, and stored in the vacuole. When the acid level declines toward fruit maturation, citrate is actively removed or leaks from the vacuole. It was expected that aconitase activity would be re-induced at later stages of fruit development. Indeed, it was found that a cytosolic aconitase activity is re-induced in the pulp towards maturation. cDNA for a cytosolic aconitase was cloned in the lab. The putative amino acid sequence of this clone showed homology to a group of RNA binding proteins, Iron Regulated Proteins (IRP), which also possesses aconitase activity. The involvement of IRP-like protein in citrate catabolism raises an interesting possibility in regards to the involvement of iron homeostasis in citric acid accumulation. It is thought that iron limitation inhibits acid decline. This possibility is currently being investigated in the lab, and preliminary results strongly support it.
4. The activity and expression of NADP-isocitrate dehydrogenase in lemon fruit. Following the catabolism of citric acid to isocitric acid, the later is converted into a-ketoglutarate by isocitrate dehydrogenase (ICDH). The expression and the enzymatic activity of the cytosolic form of this enzyme were examined (Paper #4). Both of them were greatly induced toward fruit maturity. The induction of this enzyme, as well as the cytosolic aconitase, towards fruit maturation suggests that some of the catabolism of citric acid occurs in the cytosol.
5. Fruit total acidity (pH) and citric acid accumulation. Previous work showed that the accumulation of citric acid in the vacuole of the juice sac cells was accompanied by a massive influx of protons generated by a tonoplastic H+ATPase. This proton influx reduces the vacuolar pH to about 2.5, and probably provides a driving force for citrate uptake into the vacuole, where it acts as a buffer. A detailed analysis of citric acid level and total acidity (representing the vacuolar pH) during 'Minneola' fruit development was performed (Paper #3). Differences between the patterns of total acidity and citric acid could be detected, indicating that these two mechanisms are indeed independent, although most likely tightly co-regulated.