A "White" Anthocyanin-less Pomegranate (Punica granatum L.) Caused by an Insertion in the Coding Region of the Leucoanthocyanidin Dioxygenase (LDOX; ANS) Gene

Abstract

Color is an important determinant of pomegranate fruit quality and commercial value. To understand the genetic factors controlling color in pomegranate, chemical, molecular and genetic characterization of a "white" pomegranate was performed. This unique accession is lacking the typical pomegranate color rendered by anthocyanins in all tissues of the plant, including flowers, fruit (skin and arils) and leaves. Steady-state gene-expression analysis indicated that none of the analyzed "white" pomegranate tissues are able to synthesize mRNA corresponding to the PgLDOX gene (leucoanthocyanidin dioxygenase, also called ANS, anthocyanidin synthase), which is one of the central structural genes in the anthocyanin-biosynthesis pathway. HPLC analysis revealed that none of the "white" pomegranate tissues accumulate anthocyanins, whereas other flavonoids, corresponding to biochemical reactions upstream of LDOX, were present. Molecular analysis of the "white" pomegranate revealed the presence of an insertion and an SNP within the coding region of PgLDOX. It was found that the SNP does not change amino acid sequence and is not fully linked with the "white" phenotype in all pomegranate accessions from the collection. On the other hand, genotyping of pomegranate accessions from the collection and segregating populations for the "white" phenotype demonstrated its complete linkage with the insertion, inherited as a recessive single-gene trait. Taken together, the results indicate that the insertion in PgLDOX is responsible for the "white" anthocyanin-less phenotype. These data provide the first direct molecular, genetic and chemical evidence for the effect of a natural modification in the LDOX gene on color accumulation in a fruit-bearing woody perennial deciduous tree. This modification can be further utilized to elucidate the physiological role of anthocyanins in protecting the tree organs from harmful environmental conditions, such as temperature and UV radiation.

Fig 1. Schematic representation of the flavonoid-biosynthesis pathway leading to the production of anthocyanins and proanthocyanidins.

Enzyme name abbreviations are as follows: PAL, phenylalanine ammonia-lyase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3'H, flavonoid 3'-hydroxylase; F3'5'H, flavonoid 3',5'-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol reductase; LDOX, leucoanthocyanidin oxidase; ANS, anthocyanidin synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; UFGT, UDP glucose:flavonoid 3-O-glucosyltransferase.

Fig 2. HPLC chromatograms at 520 nm of methanolic extracts of different tissues of cv. Wonderful (right) and the "white" pomegranate (left).

Chromatogram peak identities were based on the UV/Vis spectrum and retention times: (1) delphinidin 3,5-diglucoside; (2) cyanidin 3,5-diglucoside; (3) pelargonidin 3,5-diglucoside; (4) delphinidin 3-glucoside; (5) cyanidin 3-glucoside; (6) pelargonidin 3-glucoside.

Fig 3. Expression patterns of the studied anthocyanin biosynthesis structural and regulatory genes in different tissues of the "white" pomegranate.

The expression patterns of the structural and regulatory genes that are predicted to be involved in anthocyanin biosynthesis in pomegranate, are represented. The analysis was done on skin and arils of the "white" pomegranate, during fruit development [from flowers (stage 1) to fully mature fruit (stage 12)] and on young leaves. W, young leaves of the "white" pomegranate; R, young leaves of the red cv. Wonderful. Semi-quantitative RT-PCR analysis was performed as described in Materials and methods. Samples were normalized to 18S and 28S ribosomal RNA (rRNA) as the reference gene for constitutive expression. PCR products were separated on a 1% agarose gel.

Fig 4. Expression of PgLDOX in red and “White” pomegranate during fruit developmental stages.

Quantitative RT-PCR analysis was performed on samples from red (left) and “white” (right) pomegranate fruit skin during different developmental stages ranging from flower (stage 1) to fully mature fruit (stage 12). The relative expression is expressed as the fold increases relative to stage 3. Error bars are SE for three replicate reactions. The various fruit developmental stage of the red and “white” pomegranate are displayed in the bottom of the figure.

Fig 5. Comparison of the content of flavonoid and proanthocyanidin precursors in the skin of mature fruit of the "white" pomegranate and the red cv. Wonderful.

Fig 6. Comparison of genomic sequences of PgLDOX in the "white" and colored pomegranate accessions.

Schematic representation of PgLDOX genomic structure from the "white" and colored pomegranates. The white boxes indicate exons and the gray boxes indicate translated regions. The insertion in PgLDOX from the "white" pomegranate is shown as a triangle and located between positions 90–91 downstream of the ATG initiation codon. The SNP is located 1,008 bp downstream of ATG and does not change the amino acid sequence. [*] Represents the site of the short sequence from the insertion that was amplified and sequenced.

Fig 7. PCR amplification for the absence of the insertion within the PgLDOX gene in the F2 progeny.

White numbers indicate progeny with "white" phenotype and red numbers indicate progeny with colored phenotype. A scheme of the location of the primers (F2 and R2) with relevance to the insert is in the bottom. PCR products were separated in a 1% agarose gel.

Fig 8. Co-dominance primer markers that distinguish between heterozygote and homozygote alleles for the insert in the red F2 progeny.

White numbers indicate progeny with "white" phenotype and red numbers indicate progeny with colored phenotype. The red lettering C/C indicates homozygocity for the SNP marker (1,008 bp downstream to the ATG, shown also in S3 Table). A scheme of the location of the F18 and R7 primers with relevance to the insert is in the bottom (F18 primer is based on the 18bp that were sequencing from the insert, using the AFLP method). PCR products were separated in a 1% agarose gel.