A comparison of prolamellar bodies from wheat, Scots pine and Jeffrey pine. Pigment spectra and properties of protochlorophyllide oxidoreductase

Inner etioplast membrane fractions were isolated from wheat ( Triticum aestivum L. cv. Starkell), Scots pine ( Pinus sylvestris L.) and Jeffrey pine ( Pinus jeffreyi Murr), in order to investigate whether cotyledons of dark-grown conifers have protochlorophyllide associated to protochlorophyllide oxidoreductase (EC 1.6.99.–) in the pro-lamellar body in the same way as angiosperms. Protochlorophyllide was found to be present in dark-grown seedlings of Scots pine and Jeffrey pine to the same extent as in dark-grown wheat, 10–15.8 nmol (g fresh weight)⁻¹. Fluorescence emission spectra at 77 K showed accumulation of protochlorophyllide with emission maximum at 657 nm in the prolamellar body fractions of the three species studied. Also the light- and NADPH-dependent activity of protochlorophyllide oxidoreductase was consistently localized in the prolamellar body fractions. The three prolamellar body fractions were dominated by the same polypeptide. Its molecular weight was estimated to be 38 000 by sodium dodecylsulphate polyacrylamide gel electrophoresis.     

The regular ultrastructure of isolated prolamellar bodies depends on the presence of membrane-bound NADPH-protochlorophyllide oxidoreductase

Light-induced alterations of isolated prolamellar bodies (PLBs) were studied in flash-irradiated suspensions of a PLB-enriched fraction and a mixed membrane fraction isolated from dark-grown seedlings of wheat (Triticum aestivum L. cv. Walde). The mixed membrane fraction consisted of PLB fragments and membrane vesicles originating from the prothylakoids. Ultrastructural and spectral properties, as well as pigment and protein composition of non-irradiated and of flash-irradiated suspensions were studied. The addition of 0.3 mM NADPH prevented spectral shifts towards shorter wavelengths in irradiated as well as in non-irradiated PLB-fractions. as measured by fluorescence emission at – 196°C. In non-irradiated PLB-fractions the amount of phototransformable protochlorophyllide (PChlide) as compared to nonphototransformable PChlide decreased when NADPH was not added. The emission maximum due to chlorophyll(ide) shifted from 696 nm to 680 um in the flashirradiated fractions where no NADPH was added. The amount of chlorophyllous pigments, as well as the amount of NADPH-protochlorophyllide oxidoreductase, decreased during the experimental period of 4 h in the suspensions without added NADPH. especially in the irradiated ones. The ultrastructure of the pelletable material in the different suspensions was analyzed by transmission and scanning electron microscopy. The non-irradiated PLBs appeared as cottonball-like structures in the scanning electron microscope. Without NADPH added more PLBs with an irregular tubular appearance were seen. After irradiation and storage for 1 h in darkness the surface was covered with vesicles. These vesicles were still present after 4 h. In the presence of NADPH no vesicle-formation occurred and the regular network of the PLBs was preserved also after an irradiation which caused transformation of PChlide to chlorophyllide. Thus, the regular structure seems to depend on an ample supply of NADPH. which in turn may be necessary to stabilize the pigment-protein complex in the lipid moiety of the PLB membranes. The formation of vesicles may thus be caused by a loss of this pigment-protein complex in suspensions with a low level of NADPH. The possible significance of an NADPH-dependence in vivo is discussed.     

Plastid‐associated polyamines: their role in differentiation,structure, functioning,stress response and senescence

Polyamines are low‐molecular weight biogenic amines. They are a specific group of cell growth and development regulators. In the past decade biochemical, molecular and genetic studies have contributed much to a better understanding of the biological role of polyamines in the plant cell. Substantial evidence has also been added to our understanding of the role of polyamines in plastid development. In developing chloroplasts, polyamines serve as a nitrogen source for protein and chlorophyll synthesis. In chloroplast structure, thylakoid proteins linked to polyamines belong mainly to antenna proteins of light‐harvesting chlorophyll a/b–protein complexes. The fact that LHCII oligomeric forms are much more intensely labelled by polyamines, in comparison to monomeric forms, suggests that polyamines participate in oligomer stabilisation. In plastid metabolism, polyamines modulate effectiveness of photosynthesis. The role of polyamines in mature chloroplasts is also related to the photo‐adaptation of the photosynthetic apparatus to low and high light intensity and its response to environmental stress. The occurrence of polyamines and enzymes participating in their metabolism at every stage of plastid development indicates that polyamines play a role in plastid differentiation, structure, functioning and senescence.     

Cytokinin stimulates polyribosome loading of nuclear-encoded mRNAs for the plastid ATP synthase in etioplasts of Lupinus luteus: the complex accumulates in the inner-envelope membrane with the CF(1) moiety located towards the stromal space

Three of the nine subunits of the plastid ATP synthase, including the subunit of the CF(1) moiety (gene AtpC), are encoded in the nucleus. Application of cytokinin to etiolated lupine seedlings induces polyribosome association of their mRNAs. This appears to be specific as no such regulation was observed for messages for three ribosomal proteins. Cytokinin-mediated polyribosome loading was also observed for the spinach AtpC message in etiolated transgenic tobacco seedlings. Analysis of various spinach AtpC mRNA derivatives uncovered that the 5' untranslated region (5' UTR) of this message is sufficient to direct polyribosome loading, and that sequences at the 3' end of the AtpC 5' UTR, including an UC-rich motif, are crucial for this regulation. The increase in polyribosome loading of the AtpC message correlated with an increased synthesis of the polypeptide. The subunit, together with the ATP synthase complex, accumulates in the inner-envelope membrane with the CF(1) moiety located towards the stromal space of the etioplast. These results suggest that cytokinin promotes accumulation of the ATP synthase in the inner-envelope membrane of lupine etioplasts by stimulating the translation efficiency of their nuclear-encoded messages.     

Chloroplast structure: from chlorophyll granules to supra-molecular architecture of thylakoid membranes

This review provides a brief historical account of how microscopical studies of chloroplasts have contributed to our current knowledge of the structural and functional organization of thylakoid membranes. It starts by tracing the origins of the terms plastid, grana, stroma and chloroplasts to light microscopic studies of 19th century German botanists, and then describes how different types of electron microscopical techniques have added to this field. The most notable contributions of thin section electron microscopy include the elucidation of the 3-D organization of thylakoid membranes, the discovery of prolamellar bodies in etioplasts, and the structural changes in thylakoid architecture that accompany the light-dependent transformation of etioplasts into chloroplasts. Attention is then focused on the roles that freeze-fracture and freeze-etch electron microscopy and immuno electron microscopy have played in defining the extent to which the functional complexes of thylakoids are non-randomly distributed between appressed, grana and non-appressed stroma thylakoids. Studies reporting on how this lateral differentiation can be altered experimentally, and how the spatial organization of functional complexes is affected by alterations in the light environment of plants are also included in this discussion. Finally, the review points to the possible uses of electron microscope tomography techniques in future structural studies of thylakoid membranes. This revised version was published online in August 2006 with corrections to the Cover Date.     

ADP/ATP and protein phosphorylation dependence of phototransformable protochlorophyllide in isolated etioplast membranes

The effects of modulated ADP/ATP and NADPH/NADP⁺ ratios, and of protein kinase inhibitors, on the in vitro reformation of phototransformable protochlorophyllide, i.e. the aggregated ternary complexes between NADPH, protochlorophyllide, and NADPH-protochlorophyllide oxidoreductase (POR, EC 1.3.1.33), in etioplast membranes isolated from dark-grown wheat (Triticum aestivum) were investigated. Low temperature fluorescence emission spectra (–196 °C) were used to determine the state of the pigments. The presence of spectral intermediates of protochlorophyllide and the reformation of phototransformable protochlorophyllide were reduced at high ATP, but favoured by high ADP. Increased ADP level partly prevented the chlorophyllide blue-shift. The protein kinase inhibitor K252a prevented reformation of phototransformable protochlorophyllide without showing any effect on the chlorophyllide blue-shift. Addition of NADPH did not overcome the inhibition. The results indicate that protein phosphorylation plays a role in the conversion of the non-phototransformable protochlorophyllide to POR-associated phototransformable protochlorophyllide. The possible presence of a plastid ADP-dependent kinase, the activity of which favours the formation of PLBs, is discussed. Reversible protein phosphorylation is suggested as a regulatory mechanism in the prolamellar body formation and its light-dependent dispersal by affecting the membrane association of POR. By the presence of a high concentration of phototransformable protochlorophyllide, prolamellar bodies can act as light sensors for plastid development. The modulation of plastid protein kinase and protein phosphatase activities by the NADPH/NADP⁺ ratio is suggested. This revised version was published online in June 2006 with corrections to the Cover Date.     

Defective etioplasts observed in variegation mutants may reveal the light-independent regulation of white/yellow sectors of Arabidopsis leaves

Leaf variegation resulting from nuclear gene mutations has been used as a model system to elucidate the molecular mechanisms of chloroplast development. Since most variegation genes also function in photosynthesis, it remains unknown whether their roles in photosynthesis and chloroplast development are distinct. Here, using the variegation mutant thylakoid formation1 (thf1) we show that variegation formation is light independent. It was found that slow and uneven chloroplast development in thf1 can be attributed to defects in etioplast development in darkness. Ultrastructural analysis showed the coexistence of plastids with or without prolamellar bodies (PLB) in cells of thf1, but not of WT. Although THF1 mutation leads to significant decreases in the levels of Pchlide and Pchllide oxidoreductase (POR) expression, genetic and 5-aminolevulinic acid (ALA)-feeding analysis did not reveal Pchlide or POR to be critical factors for etioplast formation in thf1. Northern blot analysis showed that plastid gene expression is dramatically reduced in thf1 compared with that in WT, particularly in the dark. Our results also indicate that chlorophyll biosynthesis and expression of plastidic genes are coordinately suppressed in thf1. Based on these results, we propose a model to explain leaf variegation formation from the plastid development perspective.     

Tentoxin does not cause chlorosis in greening mung bean leaves by inhibiting photophosphorylation

Effects of the fungal toxin, tentotoxin, on development and chlorophyll accumulation of plastids of primary leaves of mung bean [ Vigna radiata (L.) Wilczek cv. Berken] were studied using spectrophotometric, electrophoretic, and microscopic procedures. In etioplasts of control tissues both prolamellar bodies and prothylakoids occurred, whereas small vesicles were associated with structurally distinct prolamellar bodies in tentoxin-affected etioplasts. As determined by in vivo spectrophotometry, tentoxin-affected etioplasts had 25% less phototransformable protochlorophyll(ide) and 35% less non-phototransformable protochlorophyll(ide) than had control etioplasts after 5 days of dark seedling growth. Tentoxin had no effect on the rate of the Shibita shift. Protochlorophyll(ide) resynthesis in the dark immediately after protochlorophyll(ide) phototransformation was five to six times slower in tentoxintreated than in control tissues. Effects on chlorophyll(ide) content were observed within 30 min of the beginning of continuous white light exposure. In vivo measurement of cytochrome f redox activity revealed that this cytochrome was linked to light-driven electron flow in control tissues within 20 min of the beginning of continuous white light, whereas in the tentoxin-treated tissues there was no linkage (despite the presence of cytochrome f ) at any time. Coupling factor 1 was present and had potential ATPase activity in both control and tentoxin-affected plastids. There was about sixteen times more chlorophyll in control than in tentoxin-treated tissues in continuous as well as in intermittent (2 min light/118 min dark) light. These data are consistent with the view that tentoxin disrupts normal etioplast and chloroplast development through a mechanism unrelated to photophosphorylation.     

Involvement of photobleaching and inhibition of protochlorophyll(ide) accumulation in tentoxin effects on greening of mung bean seedlings

Several types of evidence indicate that tentoxin-caused reduction of chlorophyll accumulation in greening primary leaves of mung bean [ Vigna radiata (L.) Wilczek cv. Berken] is due to both photobleaching and decreased protochlorophyll(ide) synthesis. Greening was greater under dim (2.5 μmol m_-2 s_-1) far-red or white light than under bright (180 to 200 μmol m_-2 s_-1) white light in tentoxin-treated tissues, whereas there was a positive correlation between fluence rate and greening in control tissues. Under continuous white light (100 μmol m_-2 s_-1) chorophyll(ide) accumulation was slower in tentoxin-treated than in control tissues. This was caused by greater photobleaching of newly formed chlorophyll(ide), as well as by decreased protochlorophyll(ide) synthesis. Photobleaching did not affect protochlorophyll(ide) synthesis in control or tentoxin-treated tissues. Chlorophyll(ide) was less stable in tentoxin-treated than in control tissues during a 24 h period of darkness. Plastids of tentoxin-treated tissues had all of the chlorophyll-proteins of control plants. Etioplasts of tentoxin-treated plants contained normal galactolipid contents, but galactolipids in these plants were greatly reduced in white light. Reduced chlorophyll accumulation caused by tentoxin is apparently the result of both photodestruction and of reduced synthesis of chlorophyll.     

Isoelectric focusing of pigment-protein complexes solubilized from non-irradiated and irradiated prolamellar bodies

Preparative isoelectric focusing was employed to compare the association of protochlorophyllide and chlorophyllide with the enzyme NADPH-protochlorophyllide oxidoreductase (PCR; EC 1.3.1.33). Photoactive protochlorophyllide-PCR complexes were solubilized with 1-O- n -octyl-β- d -glucopyranoside from non-irradiated prolamellar bodies of wheat ( Triticum aestivum ). Also, chlorophyllide-PCR complexes were solubilized from prolamellar bodies irradiated under conditions either preventing or favouring a spectral shift of chlorophyllide to shorter wavelengths. Independently of the treatment prior to the solubilization, the pigments and the PCR focused together at pHs of 4 to 5. The results indicate that protochlorophyllide-PCR complexes are conformationally similar to chlorophyllide-PCR complexes. The results support the hypothesis that the spectral shift, referred to as the Shibata shift, reflects a breaking-up of large chlorophyllide-PCR aggregates to smaller chlorophyllide-PCR units, rather than a dissociation of the chlorophyllide from the enzyme protein.