ABSTRACT
We have identified a copper P(1B)-ATPase transporter in soybean (Glycine max), named as GmHMA8, homologue to cyanobacterial PacS and Arabidopsis thaliana AtHMA8 (PAA2) transporters. A novel specific polyclonal anti-GmHMA8 antibody raised against a synthetic peptide reacted with a protein of an apparent mass of around 180-200 kDa in chloroplast and thylakoid membrane preparations isolated from soybean cell suspensions. Immunoblot analysis with this antibody also showed a band with similar apparent molecular mass in chloroplasts from Lotus corniculatus. Immunofluorescence labelling with the anti-GmHMA8 antibody and double immunofluorescence labelling with anti-GmHMA8 and anti-RuBisCo antibodies revealed the localization of the GmHMA8 transporter within the chloroplast organelle. Furthermore, the precise ultrastructural distribution of GmHMA8 within the chloroplast subcompartments was demonstrated by using electron microscopy immunogold labelling. The GmHMA8 copper transporter from soybean was localized in the thylakoid membranes showing a heterogeneous distribution in small clusters.
Subject(s)
Chloroplast Proton-Translocating ATPases/analysis , Copper/metabolism , Glycine max/enzymology , Plant Proteins/analysis , Thylakoids/enzymology , Amino Acid Sequence , Antibodies/immunology , Chloroplast Proton-Translocating ATPases/genetics , Chloroplast Proton-Translocating ATPases/immunology , DNA, Complementary/genetics , Fluorescent Antibody Technique , Ion Transport , Molecular Sequence Data , Plant Proteins/genetics , Plant Proteins/immunology , Glycine max/metabolismABSTRACT
Callose and cutin deposition were followed by staining with Aniline Blue and Nile Red and by immunolocalization using antibodies raised against callose. Along with morphogenesis induction from internodes of Humulus lupulus var. Nugget, a temporal and spatial differential deposition of callose and cutin was observed. A cutin layer showing bright yellow autofluorescence appears, surrounding cells or groups of cells committed to express morphogenic competence. This cutin layer that evolves to a randomly organized network appeared underneath a callose layer and may create a specific cellular environment with altered permeability and altered receptors providing conditions for entering the cell cycle. The incipient callose accumulation in control explants cultured on basal medium suggests the involvement of callose in the initiation of the morphogenic programme leading to nodule formation. A scanning electron microscopic study during the organogenic process showed that before shoot bud regeneration, the cutin layer increases in thickness and acquires a smooth texture. This cutin layer is specific to nodular organogenic regions and disappeared with plantlet regeneration. This layer may control permeability to water and solute transfer throughout plantlet regeneration.
