Synchrony between flower opening and petal-color change from red to blue in morning glory, Ipomoea tricolor cv. Heavenly Blue

Abstract

Petal color change in morning glory Ipomoea tricolor cv. Heavenly Blue, from red to blue, during the flower-opening period is due to an unusual increase in vacuolar pH (pHv) from 6.6 to 7.7 in colored epidermal cells. We clarified that this pHv increase is involved in tonoplast-localized Na+/H+ exchanger (NHX). However, the mechanism of pHv increase and the physiological role of NHX1 in petal cells have remained obscure. In this study, synchrony of petal-color change from red to blue, pHv increase, K+ accumulation, and cell expansion growth during flower-opening period were examined with special reference to ItNHX1. We concluded that ItNHX1 exchanges K+, but not Na+, with H+ to accumulate an ionic osmoticum in the vacuole, which is then followed by cell expansion growth. This function may lead to full opening of petals with a characteristic blue color.

Fig. 1

Petal color change and expansion growth of Ipomoea tricolor cv. Heavenly Blue. (A) Whole flower growth. The right photos are half-cut buds. Bar: 2.0 cm. (B) Transverse section of petals. Bar: 50 μm. (C) Structure of heavenly blue anthocyanin (HBA) (D) Cryo-SEM appearance of a fully-opened petal at 0 h. Bar: 100 μm. (E) Cell volume increase of adaxial colored epidermis during the last 24 h. (n = 9, mean ± SD). ***: significant differences (P < 0.001) between −24 h; §: significant difference (P < 0.05) between −12 h; § § §: significant difference (P < 0.001) between −12 h.

Fig. 2

The change in ion content per colored protoplast depending on flower-opening period. (n = 2, mean ± SE). (A) Change in cation contents. (B) Change in anion contents. (C) Change of K⁺ concentration in colored cells calculated as average K⁺ content/average cell volume (n = 2, mean ± SE). § indicates the data of one experiment.

Fig. 3

Cellular features of strange chimera petals. (A) Chimera flower petals. Bar: 2.0 cm. (B) A transverse section of the border region of a chimera petal. Bar: 50 μm. (C) The cell volume of red and blue parts in chimera-colored petals at 0 h (n = 10, mean ± SD). ***: significant difference (P < 0.001) between blue cells. (D) The pHv of red and blue cells. (blue cell, n = 28, mean ± SD; red cell, n = 5, mean ± SD). ***: significant difference (P < 0.001) between blue cells. (E) HPLC chromatograms of the extracts from blue and red parts of the chimera petals (detection: 530 nm). (F) The difference in the ion contents of blue and red chimera petal cells at 0 h. (n = 3, mean ± SD). *: significant difference (P < 0.05) between blue chimera petal cells.

Fig. 4

The change in the color and ion contents of red protoplasts (−7.5 h) incubated with indicated salts. (n = 3, mean ± SD). ** and * indicate significant differences, P < 0.01 and P < 0.05, respectively, between the control. (A) Control cells and their color change by indicated salt. Bar: 50 μm. (B) Change in cation contents of protoplasts after treatment with 50 mM alkali salts. (C) Change in anion contents of protoplasts after treatment with 50 mM alkali salts.

Fig. 5

Cell color and ion contents of protoplast prepared from the red parts of chimera petals with the KCl treatment. (A) The protoplasts incubated with 50 mM KCl for 3 h. Bar: 50 μm. (B) The cation and anion contents of the salt-treated protoplasts. (n = 3, mean ± SD). **: a significant difference (P < 0.01) between the control.

Fig. 6

Expressions of ItNHX1, ItNHX1 protein and other membrane proteins in petals. (A) Timecourse of the relative expression level of ItNHX1/γ-sub in normal petals. (n = 3, mean ± SD). * and ** (P < 0.05) and * and *** (P < 0.001) indicate significant differences. (B) Comparison of the relative expression levels of ItNHX1/γ-sub in the blue and red parts of chimera petals (0 h). (C) Western blot analysis of crude membrane fractions prepared from chimera petals (0 h). The expression of ItNHX1 in red cells: 14%, V-AT-Pase: 94% and V-PPase: 81%. The protein level of PM-AT-Pase was lower (56%) in the red cells, but that of Bip did not show any difference (99%).

Fig. 7

A schematic model of expansive growth with blue coloration of morning glory petals maintained by a coordinated work of ItNHX1 and proton pumps. At −24 h tonoplast-localized proton pumps worked to maintain acidic vacuole and HBA already transported into the vacuole, thus the cell color was red. Approaching 0 h, up-regulated V-ATPase and V-PPase work to establish the proton motive force (pmf, Δp) and may energize tonoplast around +30 mV (vacuolar side is positive). Using this pmf, ItNHX1 exchanges K⁺, which may be transported into cytosol by certain channels, with H⁺ until the difference between pHv and pHc (ΔpH) is greater than 0.5. Thus, K⁺ content in the vacuole increases, which is then followed by rising pHv and cell expansion growth. As K⁺ accumulates, HBA dissociates to form HBA anion (HBA−) which produces blue color. At the same time, PM-ATPase pumps out protons, which may maintain the cytosolic pH around 7.2 and affect cell wall loosening.