Tatsuo Oku

Inhibition of Blue Light-Dependent H+ Pumping by Abscisic Acid in Vicia Guard-Cell Protoplasts

Blue-light (BL)-dependent H+ pumping in guard-cell protoplasts (GCPs) from Vicia faba was inhibited by 65% in the presence of abscisic acid (ABA). The inhibition increased with the time after application of ABA and was concentration dependent with a saturating concentration of 1 [mu]M at pH 6.2. The inhibition was nearly independent of the pH of the medium in the range 5.4 to 7.2 when ABA was applied at 10 [mu]M, whereas it was dependent on pH when the ABA concentration was decreased. The protonated form of ABA was saturating at 40 nM in inhibiting BL-dependent H+ pumping under various experimental conditions, whereas the dissociated form at 500 nM had no inhibitory effect on the pumping, suggesting that the protonated form of ABA is the form active in inhibiting the pumping. Fusicoccin (10 [mu]M), an activator of plasma membrane H+-ATPase, induced H+ pumping from GCPs, and the rate of H+ pumping was decreased to 70% by ABA. In contrast, ABA did not inhibit H+ pumping in isolated microsome vesicles from GCPs. These results suggest that the inhibition of BL-dependent H+ pumping by ABA in GCPs may be due to indirect inactivation of plasma membrane H+-ATPase and/or inhibition of the BL-signaling pathway. The pump inhibition by ABA causes membrane depolarization and can be an initial step to induce stomatal closure and reduces the transpirational water loss under drought stress in the daytime.

Light-dependent development of thermoluminescence, delayed emission and fluorescence variation in dark-grown spruce leaves.

Thermoluminescence profiles of spruce leaves grown under various light or dark conditions were measured after excitation at a low temperature (-70 to -20 degrees C) by 1-min illumination with red light, and the following results were obtained. Mature spruce leaves showed five thermoluminescence bands at -30, -5, +20, +40 (or +35) and +70 degrees C (denoted as Zv, A, B1, B2 and C bands, respectively), but dark-grown spruce leaves with a similar chlorophyll content showed only two bands, at -30 and +70 degrees C (the Zv and C bands) and were devoid of the three other bands (the A, B1, and B2 bands). On exposure of the dark-grown leaves to continuous red light, the A, B1and B2 bands were rapidly developed, and the development was accompanied by enhancement of delayed emission, fluorescence variation and the Hill activity (photoreduction of 2,6-dichlorophenolindophenol with water as electron donor). It was demonstrated that the dark-grown spruce leaves are devoid of the water-splitting system in Photosystem II, and that the latent water-splitting activity is rapidly photoactivated by exposure of the leaves to continuous red light. These results on the gymnosperm spruce leaves, in which greening proceeds in complete darkness, being independent of the development of the water-splitting system in light, were discussed in relation to previous observations on angiosperm leaves, in which both greening and the activity generation proceed in the light.

Properties of Proton Pumping in Response to Blue Light and Fusicoccin in Guard Cell Protoplasts Isolated from Adaxial Epidermis of Vicia Leaves

Guard cell protoplasts (GCPs) were isolated from the adaxial epidermis of Vicia leaves. The properties of isolated adaxial GCPs (ad GCPs) were compared with those of abaxial GCPs (ab GCPs) with respect to H+-pumping activity. A saturating pulse of blue light (200 [mu]mol m-2 s-1, 30 s) induced H+ pumping in both ad GCPs and ab GCPs under red light. The maximum rate of blue-light-dependent H+ pumping was slightly higher in ad GCPs than in ab GCPs, but the magnitude of H+ pumping in ad GCPs was 68% of that in ab GCPs. H+ pumping was responsive to the second pulse, and the rate and magnitude of the pumping increased with the time between two pulses. The periods required to achieve 50% of the maximum rate were 12 and 22 min for ad GCPs and ab GCPs, respectively. The rates of blue-light-dependent H+ pumping were saturable, with half-saturation at 630 [mu]mol m-2 (21 [mu]mol m-2 s-1, 30 s) for ad GCPs and 105 [mu]mol m-2 (3.5 [mu]mol m-2 s-1, 30s) for ab GCPs. In contrast, fusicoccin, an activator of the plasma membrane H+- ATPase, induced H+ pumping with a slightly higher rate in ad GCPs than in ab GCPs. Both types of protoplast swelled similarly in response to fusicoccin. These results suggest that ad GCPs have almost the same activity for H+ pumping as ab GCPs, whereas ad GCPs require a larger number of photons to activate the H+ pump than ab GCPs.

Multiple-flash development of thermoluminescence bands in dark-grown spruce leaves

Spruce leaves greened in darkness were devoid of three of the five thermoluminescence bands found for mature leaves. These bands were developed rapidly by exposure of the dark-grown leaves to continuous light. The development of these bands was studied by illumination with repetitive flashes at varied intervals. Flashes at intervals of 1 s were the most effective in inducing these bands. Those at shorter or longer intervals were less effective. It was deduced from these data that the development of these bands is a multiquantum process which involves at least two photo-events with a dark reaction between them.

Photosynthetic properties of adaxial guard cells from Vicia leaves

Abstract Photosynthetic properties of adaxial (ad GCP) and abaxial guard-cell protoplasts (ab GCP) from Vicia faba L. were compared. The CO 2 uptake was red light-dependent, and saturated at 400 μ mol m -2 s -1 for ad GCP and ab GCP. The light dependency showed a half saturation at 55.4 for ad GCP and 22.5 μ mol m -2 s -1 for ab GCP, on a protein basis. In SDS-PAGE, ad GCP and ab GCP showed almost similar profiles of protein including a similar content of Rubisco. From the electrophoretic analysis, the enrichment of particular 21 kDa protein was found in ab GCP. Chlorophyll (Chl) fluorescence transient also showed similar phases in both cell types. The transient changes were affected by DCMU, an inhibitor of photosystem II (PS II) activity and by methyl viologen, an electron acceptor from )photosystem I) PS I. The relative height of P from GCPs of both cell types depended on the exciting light intensity. Absorption spectrum of ad GCP showed similar profiles to that of ab GCP but two-fold higher content of flavonoid was found in ad GCP. Total Chl content was somewhat higher in ab GCP than in ad GCP. Chl a/b ratios were estimated to be 3.19 for ad GCP and 2.66 for ab GCP. From the results, adaxial guard cells had high photosynthetic capacity similar to abaxial ones, and a possible explanation on the responsiveness to red light (RL) is discussed.

Stimulatory effect of calcium on photoactivation of the water oxidation system in dark-grown spruce chloroplasts

The water-oxidation system in chloroplasts from Picea abies leaves grown without light was activated and stabilized upon exposure of the isolated chloroplasts to weak light. The rate of electron transport from diphenylcarbazide to 2,6-dichloroindophenol in the photosystem II remained constant. Ca2+ added to the chloroplast suspension was incorporated into thylakoid membranes during illumination and strongly stimulated the photoactivation of latent water-oxidation system but not electron transport in photosystem II. It is concluded that the site which requires Ca2+ and is activated by light is the water-oxidation system.

Effect of polyethylene glycol on heat inactivation of the hill reaction

Abstract Protection of the Hill reaction from heat inactivation by polyethylene glycol was found in spinach chloroplasts. The protective action of the polymer is discussed in relation to chloroplast membrane structure.

The photoconversion of Chenopodium chlorophyll protein.

Abstract— Changes in the UV absorption spectrum with the photoconversion of Chenopodium chlorophyll protein, CP668 ⇆ CP743, and with the pH change of the CP668 and CP743 solutions, were measured. The change in the absorption spectrum of the apoprotein caused by the pH change was reversible, whereas that caused by the photoconversion was irreversible. The apoprotein may undergo a proton dissociation or association of the phenolic group in tyrosine residues upon pH change. A photooxidation in CP668 (loss of electron) caused by irradiation of CP668 solution may induce a change in the ionization state of some amino acid residues. The isoelectric points of CP668 and CP743 were determined to be 9.3 and 7.2, respectively.

Photoactivation and Photoinhibition of the O 2 -Evolving Complex in Dark-Grown Spruce Seedlings

Coniferous seedlings form highly-developed chloroplasts even during dark germination (1,2). The chloroplasts, however, cannot evolve oxygen since the assembly of the tetra-Mn cluster does not occur. The water-oxidizing complex in PS II membranes isolated from dark-grown spruce cotyledons can be assembled in vitro (3), as reported with NH2OH-treated PS II membranes from wheat leaves (4). This indicates that the light-induced assembly of water-oxidizing complex (photoactivation) in the dark-grown spruce cotyledons does not require de novo protein synthesis.

THE MECHANISM OF PHOTOACTIVATION OF O2 EVOLUTION IN DARK-GROWN CONIFEROUS LEAVES

Publisher Summary This chapter discusses the mechanism of photoactivation of O2 evolution in dark-grown coniferous leaves. Coniferous seedlings develop chloroplasts in darkness during germination. These chloroplasts have incomplete lamellar structure, although active photosystem-I and photosystem-II electron transport is established in their thylakoid membranes. Light activation of the O2-evolving system can also be accomplished in angiosperm leaves grown under intermittent light and in algae grown in darkness. Extraction of Mn from normal chloroplasts with Tris and hydroxylamine causes a specific inactivation of O2 evolution. Both light and Mn addition are required for the recovery of the O2-evolving activity. The activation of the latent O2-evolving system is accomplished by a multi-quantum process that involves, at least, two photoevents and a dark reaction between them. In a study discussed in the chapter, the photoactivation of the latent O2-evolving system was accompanied by a significant increase in the thiol content and oxidation of an endogenous reductant. The thiol content increased during illumination of the chloroplast suspension and saturated when the maximum photoactivation was achieved.