Characterization of two carnation petal prolyl 4 hydroxylases

Abstract

Prolyl 4-hydroxylases (P4Hs) catalyze the proline hydroxylation, a major post-translational modification, of hydroxyproline-rich glycoproteins. Two carnation petal P4H cDNAs, (Dianthus caryophyllus prolyl 4-hydroxylase) DcP4H1 and DcP4H2, were identified and characterized at the gene expression and biochemical level in order to investigate their role in flower senescence. Both mRNAs showed similar patterns of expression with stable transcript abundance during senescence progression and differential tissue-specific expression with DcP4H1 and DcP4H2 strongly expressed in ovaries and stems, respectively. Recombinant DcP4H1 and DcP4H2 proteins were produced and their catalytic properties were determined. Pyridine 2,4-dicarboxylate (PDCA) was identified as a potent inhibitor of the in vitro enzyme activity of both P4Hs and used to determine whether inhibition of proline hydroxylation in petals is involved in senescence progression of cut carnation flowers. PDCA suppressed the climacteric ethylene production indicating a strong correlation between the inhibition of DcP4H1 and DcP4H2 activity in vitro by PDCA and the suppression of climacteric ethylene production in cut carnation flowers.

Promotion of root elongation by pyridinecarboxylic acids known as novel cut flower care agents

“…However, the mechanism of these diverse effects of PCA and PDCA analogs remain unknown. Previously, 2,4-PDCA was shown to inhibit the activity of prolyl-4-hydroxylase by acting as a structural analog of 2-oxoglutarate, the co-substrate of the enzyme (Vlad et al 2010), and that of ACC oxidase by acting as a possible structural analog of ascorbate, the co-substrate of the enzyme (Vlad et al 2010, Satoh et al 2014. Moreover, aconitase has been reported to be inactivated markedly by 2,6-PDCA, but not by 2,3-, 2,4-, and 2,5-PDCA (Murakami et al 2007).…”

“…Regarding the action mechanism of 2,4-PDCA for retarding senescence of carnation flowers, Vlad et al (2010) hypothesized that 2,4-PDCA inhibits ACC oxidase by competing with ascorbate, a cos u b s t r a t e o f t h e e n z y m e a c t i o n . T h e n , Fragkostefanakis et al (2013) showed that 2,4-PDCA inhibited the in vitro activity of ACC oxidase prepared from tomato pericarp tissues.…”

Pyridinedicarboxylic Acids Prolong the Vase Life of Cut Flowers of Spray-type ‘Light Pink Barbara’ Carnation by Accelerating Flower Opening in Addition to an Already-known Action of Retarding Senescence

“…Satoh et al (2014) hypothesized on the association of GA with the promoting action of 2,4-PDCA on flower (bud) opening in cut spray-type carnation flowers. This hypothesis arose from the notion that 2,4-PDCA is a structural analog of 2-oxoglutarate (2-OxoGA) and, by competing with 2-OxoGA, 2,4-PDCA probably inhibits 2-OxoGA-dependent dioxygenases (Vlad et al, 2010;Flower Senescence and P4Hs, from HP of Laboratory of Horticultural Genetics and Biotechnology, Mediterranean Agronomic Institute of Chania, Greece, http:// www.maich.gr/en/research/horticultural_genetics_and_ biotechnology, September 13, 2014), which include those responsible for GA biosynthesis and metabolism, such as GA 3β-dioxygenase (GA 3β-hydroxylase), GA-44 dioxygenase, and GA 2β-dioxygenase (GA 2β-hydroxylase) (Hedden and Kamiya, 1997;Lange et al, 1994a, b;MacMillan, 1984, 1986).…”

“…Treatment with these inhibitors prolongs the vase life of cut carnation flowers as well as other ethylene-sensitive ornamental flowers. Vlad et al (2010) reported that 2,4-pyridinedicarboxylic acid (2,4-PDCA) inhibited ethylene production in detached flowers of 'White Sim' carnation, which belongs to the standard category of carnation flowers, and delayed senescence of the flowers. The latter action was interesting practically; however, flower senescence was delayed in only 40%, but not in the remaining 60%, after treatment with the chemical.…”

2,4-Pyridinedicarboxylic Acid Prolongs the Vase Life of Cut Flowers of Spray Carnations

“…Such OxoGAdependent dioxygenases include proline-4-hydroxylase (Kivirikko and Myllyharju, 1998;Kivirikko and Pihlajaniemi, 1998;Vlad et al, 2010) and enzymes involved in gibberellin biosythesis and metabolism, such as gibberellin 3b-dioxygenase (gibberellin 3b-hydroxylase), gibberellin-44 dioxygenase, gibberellin 2b-dioxygenase (gibberellin 2b-hydroxylase) (Hedden and Kamiya, 1997;Lange et al, 1994a, b;MacMillan, 1984, 1986).…”