Expression balances of structural genes in shikimate and flavonoid biosynthesis cause a difference in proanthocyanidin accumulation in persimmon (Diospyros kaki Thunb.) fruit

Abstract

Persimmon fruits accumulate a large amount of proanthocyanidin (PA) during development. Fruits of pollination-constant and non-astringent (PCNA) type mutants lose their ability to produce PA at an early stage of fruit development, while fruits of the normal (non-PCNA) type remain rich in PA until fully ripened. To understand the molecular mechanism for this difference, we isolated the genes involved in PA accumulation that are differentially expressed between PCNA and non-PCNA, and confirmed their correlation with PA content and composition. The expression of structural genes of the shikimate and flavonoid biosynthetic pathways and genes encoding transferases homologous to those involved in the accumulation of phenolic compounds were downregulated coincidentally only in the PCNA type. Analysis of PA composition using the phloroglucinol method suggested that the amounts of epigallocatechin and its 3-O-gallate form were remarkably low in the PCNA type. In the PCNA type, the genes encoding flavonoid 3'5' hydroxylase (F3'5'H) and anthocyanidin reductase (ANR) for epigallocatechin biosynthesis showed remarkable downregulation, despite the continuous expression level of their competitive genes, flavonoid 3' hydroxylation (F3'H) and leucoanthocyanidin reductase (LAR). We also confirmed that the relative expression levels of F3'5'H to F3'H, and ANR to LAR, were considerably higher, and the PA composition corresponded to the seasonal expression balances in both types. These results suggest that expressions of F3'5'H and ANR are important for PA accumulation in persimmon fruit. Lastly, we tested enzymatic activity of recombinant DkANR in vitro, which is thought to be an important enzyme for PA accumulation in persimmon fruits.

Gene Expression and Metabolite Profiling of Developing Highbush Blueberry Fruit Indicates Transcriptional Regulation of Flavonoid Metabolism and Activation of Abscisic Acid Metabolism

“…In apple, the PA hydroxylation pattern is the result of high F3#H gene expression and the apparent lack of the F3#5#H gene (Han et al, 2010). By contrast, persimmon fruit PAs are composed primarily of trihydroxylated subunits, corresponding to high expression of F3#5#H (Akagi et al, 2009a). Thus, the species-and tissue-specific PA subunit hydroxylation patterns may be largely dependent on the regulation of flavonoid hydroxylase genes, consistent with our results in blueberry.…”

“…The blueberry mDP range is similar to polymer lengths reported for apple fruits (3-13; Sanoner et al, 1999;Hamauzu et al, 2005) and grape seeds (3-9; Kennedy et al, 2000;Downey et al, 2003). By contrast, the skin of ripe persimmon, quince (Cydonia oblonga), and grape contains PAs with mDPs of 30 or greater (Downey et al, 2003;Hamauzu et al, 2005;Akagi et al, 2009a). Such larger PA polymers impart astringency to these fruit, while lower molecular weight PAs (less than 6) are perceived as bitter (Robichaud and Noble, 1990).…”

“…The predominance of epicatechin units in the polymers seems to parallel the substantially higher relative abundance of VcANR transcripts compared with VcLAR transcripts. ANR has been characterized as producing epicatechin-type flavan-3-ol monomers in a number of species (Xie et al, 2004;Bogs et al, 2005;Akagi et al, 2009a), while LAR is thought to make catechin-type flavan-3-ol monomers in species that make this compound (Tanner et al, 2003). It is tempting to hypothesize that the ANR activity is associated with PA synthesis in blueberry; however, the exact origin of the flavan-3-ol extension subunits in PA polymers is still in question.…”

Seasonal Abscisic Acid Signal and a Basic Leucine Zipper Transcription Factor, DkbZIP5, Regulate Proanthocyanidin Biosynthesis in Persimmon Fruit

“…PAs in persimmon fruit constitute more than 1% of its fresh weight (Taira et al, 1998). PAs are high-M r polymers with a mean degree of polymerization above 30 at the middle developmental stages, during which the accumulation of PA terminates in certain cultivars (Matsuo and Ito, 1978;Akagi et al, 2009a), which causes very strong astringency. Wild-type astringent-type (A-type) persimmon fruit accumulates PAs for a long term during its developmental stages, which ranges from the stage before full anthesis (April in Japan) to before the mature stage (August in Japan; Akagi et al, 2009a).…”

“…PAs are high-M r polymers with a mean degree of polymerization above 30 at the middle developmental stages, during which the accumulation of PA terminates in certain cultivars (Matsuo and Ito, 1978;Akagi et al, 2009a), which causes very strong astringency. Wild-type astringent-type (A-type) persimmon fruit accumulates PAs for a long term during its developmental stages, which ranges from the stage before full anthesis (April in Japan) to before the mature stage (August in Japan; Akagi et al, 2009a). In fruits with a spontaneous mutant phenotype, PA accumulation is terminated at an early fruit development stage and astringency is not expressed (Ikegami et al, 2005;Akagi et al, 2009b).…”