Regulation of etioplast pigment-protein complexes, inner membrane architecture, and protochlorophyllide a chemical heterogeneity by light-dependent NADPH:protochlorophyllide oxidoreductases A and B.

The etioplast of dark-grown angiosperms is characterized by the prolamellar body (PLB) inner membrane, the absence of chlorophyll, and the accumulation of divinyl and monovinyl derivatives of protochlorophyll(ide) a [Pchl(ide) a]. Either of two structurally related, but differentially expressed light-dependent NADPH:Pchlide oxidoreductases (PORs), PORA and PORB, can assemble the PLB and form dark-stable ternary complexes containing enzymatically photoactive Pchlide-F655. Here we have examined in detail whether these polypeptides play redundant roles in etioplast differentiation by manipulating the total POR content and the PORA-to-PORB ratio of etiolated Arabidopsis seedlings using antisense and overexpression approaches. POR content correlates closely with PLB formation, the amounts, spectroscopic properties, and photoreduction kinetics of photoactive Pchlide, the ratio of photoactive Pchlide-F655 to non-photoactive Pchl(ide)-F632, and the ratio of divinyl- to monovinyl-Pchl(ide). This last result defines POR as the first endogenous protein factor demonstrated to influence the chemical heterogeneity of Pchl(ide) in angiosperms. It is intriguing that excitation energy transfer between different spectroscopic forms of Pchl(ide) in etiolated cotyledons remains largely independent of POR content. We therefore propose that the PLB contains a minimal structural unit with defined pigment stoichiometries, within which a small amount of non-photoactive Pchl(ide) transfers excitation energy to a large excess of photoactive Pchlide-F655. In addition, our data suggests that POR may bind not only stoichiometric amounts of photoactive Pchlide, but also substoichiometric amounts of non-photoactive Pchl(ide). We conclude that the typical characteristics of etioplasts are closely related to total POR content, but not obviously to the specific presence of PORA or PORB.

Etioplast differentiation in arabidopsis: both PORA and PORB restore the prolamellar body and photoactive protochlorophyllide-F655 to the cop1 photomorphogenic mutant.

The etioplast plastid type of dark-grown angiosperms is defined by the accumulation of the chlorophyll (Chl) precursor protochlorophyllide (Pchlide) and the presence of the paracrystalline prolamellar body (PLB) membrane. Both features correlate with the presence of NADPH:Pchlide oxidoreductase (POR), a light-dependent enzyme that reduces photoactive Pchlide-F655 to chlorophyllide and plays a key role in chloroplast differentiation during greening. Two differentially expressed and regulated POR enzymes, PORA and PORB, have recently been discovered in angiosperms. To investigate the hypothesis that etioplast differentiation requires PORA, we have constitutively overexpressed PORA and PORB in the Arabidopsis wild type and in the constitutive photomorphogenic cop1-18 (previously det340) mutant, which is deficient in the PLB and Pchlide-F655. In both genetic backgrounds, POR overexpression increased PLB size, the ratio of Pchlide-F655 to nonphotoactive Pchl[ide]-F632, and the amount of Pchlide-F655. Dramatically, restoration of either PORA or PORB to the cop1 mutant led to the formation of etioplasts containing an extensive PLB and large amounts of photoactive Pchlide-F655.

Overexpression of light-dependent PORA or PORB in plants depleted of endogenous POR by far-red light enhances seedling survival in white light and protects against photooxidative damage.

The structurally related light-dependent protochlorophyllide (Pchlide) oxidoreductases PORA and PORB mediate the only light-requiring step in chlorophyll (Chl) biosynthesis in higher plants. Correlative evidence suggests that some in vivo functions of PORA and PORB may be unique, including a postulated photoprotective role for PORA. For example, wild-type Arabidopsis thaliana seedlings grown in non-photooxidative far-red light (cFR) resemble those grown in white light (WL), but they are yellow and do not green normally thereafter in WL. This defect is accompanied by the absence of detectable PORA and reduced levels of PORB expression. Here, direct evidence is provided that the presence of POR, either as PORA or PORB, can confer photoprotection in plants. In contrast to the wild-type, the plastids of transgenic PORA- or PORB-overexpressing Arabidopsis seedlings grown in cFR possess extensive prolamellar bodies. Upon a subsequent shift to WL, POR-overexpressing seedlings develop thylakoid membranes, accumulate large amounts of Chl and are viable at fluence rates lethal to the wild-type. Intriguingly, the plastid membrane architectures of greening transgenic seedlings seem to depend on whether PORA or PORB has been overproduced. POR-overexpressing seedlings shifted from cFR to WL of fluence rates from 20 to 500 muE m-2 sec-1 accumulate substantially higher amounts of Chl than does the wild-type. Furthermore, the WL fluence rate that permits maximal Chl accumulation increases from 8 muE m-2 sec-1 in the wild-type to 125 muE m-2 sec-1 in transgenic seedlings. POR overexpression during growth in cFR also correlates with a fourfold decrease in the steady-state content of Pchlide, a potentially lethal photosensitizer.

Genetic polymorphism of AHSG, FXIIIB, HP and PLG serum proteins in six Jewish groups in Israel.

The allelic distribution of the polymorphic serum proteins AHSG, PLG, FXIIIB and HP was studied in six Jewish groups who migrated to Israel from the Middle East, North Africa, Rumania, Bulgaria, Central and Eastern Europe. The observed AHSG and PLG allele frequencies in these Jewish groups were more or less similar to the observed distributions in non-Jewish populations from their respective areas of origin, while FXIIIB and HP frequencies were similar to those in European populations. Therefore, no uniform pattern of genetic relationships between the Jewish groups was observed. A genetic distance analysis including comparative data from Europe and the Middle East reflected differences between the Jewish groups according to their areas of origin.

Chlorophyll Synthesis in a Deetiolated (det340) Mutant of Arabidopsis without NADPH-Protochlorophyllide (PChlide) Oxidoreductase (POR) A and Photoactive PChlide-F655.

Chlorophyll (Chl) synthesis in Arabidopsis is controlled by two light-dependent NADPH-protochlorophyllide (PChlide) oxidoreductases (PORs), one (POR A) that is active transiently in etiolated seedlings at the beginning of illumination and another (POR B) that also operates in green plants. The function of these two enzymes during the light-induced greening of dark-grown seedlings has been studied in the wild type and a deetiolated (det340) mutant of Arabidopsis. One of the consequences of the det mutation is that POR A is constitutively down-regulated, and therefore, synthesis of the POR A enzyme is shut off. When grown in the dark, the det340 mutant lacks POR A and the photoactive PChlide-F655 species but maintains the second PChlide reductase, POR B. Previously, photoactive PChlide-F655 has often been considered to be the only PChlide form that leads to Chl formation. Despite its deficiency in POR A and photoactive PChlide-F655, the det340 mutant is able to green when placed in the light. Chl accumulation, however, proceeds abnormally. At the beginning of illumination, seedlings of det340 mutants are extremely susceptible to photooxidative damage and accumulate Chl only at extremely low light intensities. They form core complexes of photosystems I and II but are almost completely devoid of light-harvesting structures. The results of this study demonstrate that in addition to the route of Chl synthesis that has been studied extensively in illuminated dark-grown wild-type plants, a second branch of Chl synthesis exists that is driven by POR B and does not require POR A.

Substrate-dependent transport of the NADPH:protochlorophyllide oxidoreductase into isolated plastids.

The key regulatory enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR), is a nuclear-encoded plastid protein. Its post-translational transport into plastids is determined by its substrate. The precursor of POR (pPOR) is taken up and processed to mature size by plastids only in the presence of protochlorophyllide (Pchlide). In etioplasts, the endogenous level of Pchlide saturates the demands for pPOR translocation. During the light-induced transformation of etioplasts into chloroplasts, the Pchlide concentration declined drastically, and isolated chloroplasts rapidly lost the ability to import the precursor enzyme. The chloroplasts' import capacity for the pPOR, however, was restored when their intraplastidic level of Pchlide was raised by incubating the organelles in the dark with delta-aminolevulinic acid, a common precursor of tetrapyrroles. Additional evidence for the involvement of intraplastidic Pchlide in regulating the transport of pPOR into plastids was provided by experiments in which barley seedlings were grown under light/dark cycles. The intraplastidic Pchlide concentration in these plants underwent a diurnal fluctuation, with a minimum at the end of the day and a maximum at the end of the night period. Chloroplasts isolated at the end of the night translocated pPOR, whereas those isolated at the end of the day did not. Our results imply that the Pchlide-dependent transport of the pPOR into plastids might be part of a novel regulatory circuit by which greening plants fine tune both the enzyme and pigment levels, thereby avoiding the wasteful degradation of the imported pPOR as well as photodestruction of free Pchlide.

Isolation and classification of chlorophyll-deficient xantha mutants of Arabidopsis thaliana.

Mutant lines of Arabidopsis thaliana that are either blocked at various steps of the biosynthetic pathway of chlorophyll (Chl) or that are disturbed in one of the subsequent steps leading to the assembly of an active photosynthetic membrane were isolated by screening for Chl-deficient xantha (xan) mutants. Only mutants that segregated in a 3:1 ratio, that contained the same carotenoid spectrum as etiolated wild-type seedlings and less than 2% of the Chl of wild-type control seedlings, and whose Chl content was not affected by the addition of sucrose to the growth medium were selected for a more detailed analysis. As a final test for the classification of the selected mutants, light-grown xan mutants were vacuum-infiltrated and incubated with the common precursor of tetrapyrroles, delta-aminolevulinic acid (ALA), in the dark. Two major groups of mutants could be distinguished. Some of the mutants were blocked at various steps of the Chl pathway between ALA and protochlorophyllide (Pchlide) and did not accumulate the latter in the dark. The other mutants accumulated Pchlide in the dark regardless of whether exogenous ALA was added. This latter group could be subdivided into mutants with a biochemical lesion in a recently discovered second light-dependent Pchlide reduction step that occurs in green plants and mutants that have blocks in the assembly of Chl protein complexes. In the present work a total of seven different loci could be defined genetically in Arabidopsis that affect the synthesis of Chl and its integration into the growing photosynthetic membrane.

Biochemical, immunochemical, and ultrastructural studies of protein storage in poplar(Populus × canadensis 'robusta') wood.

The seasonal changes in protein content have been followed in the wood of Populus × canadensis Moench 'robusta', both biochemically and electronmicroscopically at the cellular level. In the storage-parenchyma cells of the twig wood, 4-6 µg · mg(-1) DW protein were deposited in the fall, parallel to the yellowing of leaves, and mobilized completely again during the outgrowth of buds in the spring. Environmental impacts on the leaves, e.g. a fungal attack and mechanical injury by a hurricane, were found to affect protein deposition in the wood considerably. Accumulation of protein bodies in the fall and their disappearance from the cells in the spring proceeded parallel to the changes in protein content measured biochemically, proving that these organelles are the main sites of protein storage in the wood parenchyma cells. Using immunogold labelling and an anti-32-kDa poplar storage-protein antibody the protein bodies were shown to be the exclusive sites of storage of a 32-kDa polypeptide. Transient changes in protein content were also observed during fall and winter. Because these changes coincided with changes in protein-body structure and with changes in the population of vesicles and-or tubular membrane cisternae of the cells, an exchange of nitrogen compounds from the storage pool into the structural protein of membranes possibly takes place during these periods. The structural events observed during proteolysis in spring are very similar to those found in seeds. The possible roles of small cytoplasmic vesicles found within protein bodies during proteolysis and of multimembraneous vacuolar compartments during membrane retrieval are discussed.

Evidence for a general light-dependent negative control of NADPH-protochlorophyllide oxidoreductase in angiosperms.

The effect of light on NADPH-protochlorophyllide oxidoreductase and its mRNA has been studied in five different species of dicotyledonous plants, bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), tomato (Lycopersicon esculentum Mill.), sunflower (Helianthus annuns L.) and mustard (Sinapis alba L.), and in two monocotyledonous plant species, maize (Zea mays L.) and barley (Hordeum vulgare L.). In all these species, illumination of etiolated seedlings led to a rapid decline of both the activity and the content of the enzyme protein. These results indicate that there may be a general light-dependent regulation of the enzyme common to higher plants.

Protein bodies in ray cells of Populus x canadensis Moench 'robusta'.

Light- and electron-microscopical investigations revealed distinct intravacuolar protein aggregates of 0.3-0.8 µm in diameter in ray cells of poplar during the dormant season. In semi-thin sections, these bodies showed positive protein staining and enzymatic digestibility with pepsin, indicating their proteinaceous nature. Morphometric measurements showed such protein bodies in 7-13% of the area of the ray-cell lumen. This amount corresponded with the protein content of the wood determined biochemically, e.g. 2.0-5.0 µg·mg(-1) dry weight. Polyacrylamide gel electrophoresis of the total protein fraction extracted from wood showed prominent polypeptide species with an apparent molecular weight of 30-32 kilodaltons. The results indicate considerable protein storage in ray cells, especially in the form of protein-storage vacuoles.

Light-induced changes in the distribution of the 36000-Mr polypeptide of NADPH-protochlorophyllide oxidoreductase within different cellular compartments of barley (Hordeum vulgare L.) : II. Localization by immunogold labelling in ultrathin sections.

The cellular distribution of the 36000-Mr polypeptide of NADPH-protochlorophyllide oxidoreductase has been determined in ultrathin sections of barley leaves by the method of immunogold labelling. In leaves of etiolated seedlings a large portion of the immunoreactive protein was localized within the prolamellar body. However, approximately one third of the total immunoreactive protein was present outside the plastid in the area of the plasmalemma. During illumination of etiolated seedlings the two polypeptide populations were differentially affected by light. While the concentration of the plastid-localized immunoreactive protein rapidly decreased and was hardly detectable after 16 h of continuous white-light treatment, the concentration of the extraplastidic polypeptide did not decline significantly during this illumination period. A similar distribution pattern of the immunoreactive polypeptide was also found in maize and rye. The chlorophyll-deficient barley mutant xantha-l(81) contained the immunoreactive 36000-Mr polypeptide, even though the prolamellar body was not detectable in etioplasts of this mutant. All of the immunoreactive polypeptide was localized outside the plastid in the area of the plasmalemma. Despite the apparent absence of the enzyme protein from the plastid, dark-grown mutant plants contained the same relative concentration of mRNA activity for the NADPH-protochlorophyllide oxidoreductase, which declined rapidly during illumination, as in wild-type plants. The antigenic properties and the apparent molecular weight of the plastid-localized NADPH-protochlorophyllide oxidoreductase and the 36000-Mr immunoreactive polypeptide outside the plastid were so similar as to indicate that the two proteins may be of common origin.

Recovery of Viable Staphylococcus aureus in Corn and Green Beans Stored at Ambient Temperature.

Staphylococcus aureus was recovered from post-process inoculated commercially canned green beans after periods of up to 6 weeks of storage and from inoculated commercially canned corn after up to 26 weeks of storage under various conditions. Variance in duration of viability of the culture was apparently due to intrinsic factors of the food and presence of inoculated competing organisms. Drying of the inoculated corn had no effect on viability of the test culture, but drying shortened its length of viability in green beans.