Cytochemical study of diaminobenzidine oxidation by isolated Vicia chloroplasts under illumination.

Imaizumi, Machiko; Hiraoka, Toshisuke

doi:10.1267/ahc.15.208

Cited by 2 publications

(1 citation statement)

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“…Numerous authors (7,15,17,18,21,22,25) have used the photooxidation of DAB as a sensitive probe to localize PSI. This reaction is insensitive to inhibitors of PSII activity (15,17) and is completely dependent upon light (17).…”

Section: Resultsmentioning

confidence: 99%

Immunocytochemical and Cytochemical Localization of Photosystems I and II

Vaughn¹,

Vierling²,

Duke³

et al. 1983

Plant Physiol.

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Cytochemical and immunocytochemical methods were used to localize photosystems I and II in barley (Hordeum vulgare L. cv Himalaya) chloroplasts. PSI activity, monitored by diaminobenzidine oxidation, was associated with the lumen side of the thylakoids of both grana and stroma lamellae. The P700 chlorophyll a protein, the reaction center of PSI, was localized on thin sections of barley chloroplasts using monospecific antibodies to this protein and the peroxidase-antiperoxidase procedure. Results obtained by immunocytochemistry were similar to those of the diaminobenzidine oxidation: both grana and stroma lamellae contained immunocytochemically reactive material. Both the grana and stroma lamellae were also labeled when isolated thylakoids were reacted with the P700 chlorophyll a protein antiserum and then processed by the peroxidase-antiperoxidase procedure. PSII activity was localized cytochemically by monitoring the photoreduction of thiocarbamyl nitroblue tetrazolium, a reaction sensitive to the PSII inhibitor, DCMU. PSII reactions occurred primarily on the grana lamellae, with weaker reactions on the stroma lamellae.Despite the many ultrastructural and biochemical studies of thylakoid membranes of the higher plant chloroplast (1)(2)(3)(4)(5)16), the precise locations of PSI and II in these membranes have not been firmly established. Current models of thylakoid organization (4, 16) suggests that both PSI and II are Cytochemistry. Small leaf segments (1-mm2 sections taken approximately 5-10 mm from the tip of the primary leaf) were fixed in 2% (w/v) paraformaldehyde in 0.10 M phosphate buffer (pH 7.2) for 1 h at 0 to 4°C in the dark and were washed 3 times over 2 h in 5% (w/v) RNase-free sucrose in 0.10 M phosphate buffer (pH 7.2). The material was then pre-incubated for 1 h at 0 to 4°C in the dark in the appropriate reaction mixture (1 mg/ ml DAB in phosphate buffer or 1 mg/ml TCNBT in 5% dimethyl sulfoxide in phosphate buffer) to allow for uptake of the reagent. The specimens were then transferred to a fresh reaction medium and incubated under 400 ME.m2 s-' (PAR) for 1 h at 22°C. Control specimens were incubated in the dark, in the absence of the staining reagent, or with the addition of 10-6 M DCMU. After incubation, the specimens were washed in two changes of phosphate-buffered sucrose (15 min each, in the dark) to reduce thylakoid swelling and postfixed in 1% (w/v) OS04 in 0.10 M cacodylate buffer (pH 7.2) for 1 h at 0 to 4°C. Specimens were washed twice in cold (0-4°C) distilled H20, dehydrated in acetone (to 70% acetone at 0-4°C), and embedded with Ladd's Ultralow viscosity resin (Ladd Inc., Burlington, VT). Gold-silver sections were observed without poststaining with a Hitachi HU-11 C electron microscope. Further discussion of these two cytochemical techniques can be found in Reference 21.

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