Chloroplast biogenesis. Cell-free transfer of envelope monogalactosylglycerides to thylakoids.

An ATP- and temperature-dependent transfer of monogalactosylglycerides from the chloroplast envelope to the chloroplast thylakoids was reconstituted in a cell-free system prepared from isolated chloroplasts of garden pea (Pisum sativum) or spinach (Spinacia oleracea). Isolated envelope membranes, in which the label was present exclusively in monogalactosylglycerides, were prepared radiolabeled in vitro with [14C]galactose from UDP-[14C]galactose to label galactolipids as the donor. ATP-dependent transfer of radioactivity from donor to unlabeled acceptor thylakoids, immobilized on nitrocellulose strips, was observed. In some experiments linear transfer for longer than 30 min of incubation was facilitated by the addition of stroma proteins but in other experiments stroma was without effect or inhibitory suggesting no absolute requirements for a soluble protein carrier. Transfer was donor specific. No membrane fraction tested (plasma membrane, tonoplast, endoplasmic reticulum, nuclei, Golgi apparatus, mitochondria or thylakoids) (isolated from tissue radiolabeled in vivo with [14C]acetate) other than chloroplast envelopes demonstrated any significant ability to transfer labeled membrane lipids to immobilized thylakoids. Acceptor specificity, while not absolute, showed a 3-10-fold greater ATP-dependent transfer of labeled galactolipids from chloroplast envelopes to immobilized thylakoids than to other leaf membranes. The results provide independent confirmation of the potential for transfer of galactolipids between chloroplast envelopes and thylakoids suggested previously from ultrastructural studies and of the known location of thylakoid galactolipid biosynthetic activities in the chloroplast envelope.

The two spectroscopically different short wavelength protochlorophyllide forms in pea epicotyls are both monomeric.

The spectral properties of the protochlorophyllide forms in the epicotyls of dark-grown pea seedlings have been studied in a temperature range, from 10 to 293 K with conventional fluorescence emission and excitation spectroscopy as well as by fluorescence line narrowing (FLN) at cryogenic temperatures. The conventional fluorescence techniques at lower temperatures revealed separate bands at 628, 634-636, 644 and 655 nm. At room temperature (293 K) the 628 and 634-636 nm emission bands strongly overlapped and the band shape was almost independent of the excitation wavelength. Under FLN conditions, vibronically resolved fluorescence spectra could be measured for the 628 and 634-636 nm bands. The high resolution of this technique excluded the excitonic nature of respective excited states and made it possible to determine the pure electronic (0,0) range of the spectra of the two components. Thus it was concluded that the 628 and 634-636 nm (0,0) emission bands originate from two monomeric forms of protochlorophyllide and the spectral difference is interpreted as a consequence of environmental effects of the surrounding matrix. On the basis of earlier results and the data presented here, a model is discussed in which the 636 nm form is considered as an enzyme-bound protochlorophyllide and the 628 nm form as a protochlorophyllide pool from which the substrate is replaced when the epicotyl is illuminated with continuous light.

Leaf Developmental Age Controls Expression of Genes Encoding Enzymes of Chlorophyll and Heme Biosynthesis in Pea (Pisum sativum L.).

The effects of leaf developmental age on the expression of three nuclear gene families in pea (Pisum sativum L.) coding for enzymes of chlorophyll and heme biosynthesis have been examined. The steady-state levels of mRNAs encoding aminolevulinic acid (ALA) dehydratase, porphobilinogen (PBG) deaminase, and NADPH:protochlorophyllide reductase were measured by RNA gel blot and quantitative slot-blot analyses in the foliar leaves of embryos that had imbibed for 12 to 18 h and leaves of developing seedlings grown either in total darkness or under continuous white light for up to 14 d after imbibition. Both ALA dehydratase and PBG deaminase mRNAs were detectable in embryonic leaves, whereas mRNA encoding the NADPH:protochlorophyllide reductase was not observed at this early developmental stage. All three gene products were found to increase to approximately the same extent in the primary leaves of pea seedlings during the first 6 to 8 d after imbibition (postgermination) regardless of whether the plants were grown in darkness or under continuous white-light illumination. In the leaves of dark-grown seedlings, the highest levels of message accumulation were observed at approximately 8 to 10 d postgermination, and, thereafter, a steady decline in mRNA levels was observed. In the leaves of light-grown seedlings, steady-state levels of mRNA encoding the three chlorophyll biosynthetic enzymes were inversely correlated with leaf age, with youngest, rapidly expanding leaves containing the highest message levels. A corresponding increase in the three enzyme protein levels was also found during the early stages of development in the light or darkness; however, maximal accumulation of protein was delayed relative to peak levels of mRNA accumulation. We also found that although protochlorophyllide was detectable in the leaves immediately after imbibition, the time course of accumulation of the phototransformable form of the molecule coincided with NADPH:protochlorophyllide reductase expression. In studies in which dark-grown seedlings of various ages were subsequently transferred to light for 24 and 48 h, the effect of light on changes in steady-state mRNA levels was found to be more pronounced at later developmental stages. These results suggest that the expression of these three genes and likely those genes encoding other chlorophyll biosynthetic pathway enzymes are under the control of a common regulatory mechanism. Furthermore, it appears that not light, but rather as yet unidentified endogenous factors, are the primary regulatory factors controlling gene expression early in leaf development.

The importance of the C-terminal region and Cys residues for the membrane association of the NADPH:protochlorophyllide oxidoreductase in pea.

In vitro chloroplast import reactions and thylakoid association reactions have been performed with a series of C-terminal deletions and Cys-to-Ser substitution mutants of the pea NADPH:protochlorophyllide oxidoreductase (POR; EC 1.6.99). C-terminal deletions of the precursor POR (Delta362-400, Delta338-400, Delta315-400 and Delta300-400) were efficiently translocated across the chloroplast envelope. However, except the Delta396-400 mutant, no C-terminal deletion mutants or Cys-to-Ser substitution (Cys119, Cys281 and Cys309) mutants resisted post-treatment with thermolysin after the thylakoid association reactions. This suggests that these mutants were unable to properly associate to the thylakoids due to changes of the protein conformation of POR.

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 degrees 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.

Tissue specific protochlorophyll(ide) forms in dark-forced shoots of grapevine (Vitis viniferaL.).

Cuttings of grapevine (Vitis vinifera L. cv. Chardonnay) were dark-forced at least three weeks. Pigment contents, 77 K fluorescence emission, excitation spectra of the leaves, petioles, stems, transmission electron micrographs of the etioplasts from leaves, the chlorenchyma tissues of the stems were analysed. The dark-grown leaves, stems contained 8 to 10, 3 to 5 mug/g fresh weight protochlorophyllide, its esters, respectively. HPLC analysis showed that the molar ratio of the unesterified, esterified pigments was 7:3 in the shoot developed in darkness. The dark-forced leaves contained carotenoids identified as: neoxanthin, violaxanthin, antheraxanthin, lutein, beta-carotene. Detailed analyses of the fluorescence spectra proved that all tissues of the dark-forced shoots had protochlorophyllide or protochlorophyll forms with emission maxima at 628, 636, 644, 655, 669 nm. The 628, 636 nm emitting forms were present in all parts of the dark-forced shoot, but dominated in the stems, which may indicate an organ specificity of the etioplast development. Variations in the distribution of the pigment forms were even found in the different tissues of the stem. The subepidermal layers were more abundant in the 655 nm form than the parenchyma cells of the inner part of the cortex, the pith. In the latter cells, the plastid differentiation stopped in intermediary stages between proplastids, etioplasts. The plastids in the subepidermal layers had developed prolamellar body structures, which were similar to those of etiolated leaves. The results highlight the importance of organ-, tissue specificity of plastid differentiation for chlorophyll biosynthesis, greening of different plant organs.

Carotenoid dependence of the protochlorophyllide to chlorophyllide phototransformation in dark-grown wheat seedlings.

The influence of carotenoids on partial protochlorophyllide (Pchlide) photoreduction and the successive formation of long-wavelength chlorophyllide (Chlide) forms was studied by low-temperature fluorescence spectroscopy (77 K). Wheat leaves with a decreased content of carotenoids obtained from norflurazon-treated seedlings (10 and 100 micromol l(-1)) were compared with leaves containing normal amounts of these pigments. Partial photoreduction of Pchlide was achieved by irradiation of the leaves with one light flash in combination with a number of neutral gray and/or red Perspex filters. There were significant differences between the fluorescence emission spectra (the position and height of the peaks) of dark-grown normal and carotenoid-deficient leaves irradiated with non-saturating white light of increasing intensity. The long-wavelength Chlide forms appeared first in the leaves nearly devoid of carotenoids (treated with 100 micromol l(-1) norflurazon), then in the leaves with carotenoid deficiency (treated with 10 micromol l(-1) norflurazon), and finally in normal leaves. After irradiation with non-saturating light of the same intensity, the ratio Chlide/Pchlide(657) was always the highest in the leaves nearly deficient of carotenoids, medium in the leaves with carotenoid deficiency and lowest in the normal leaves. Similarly to white light, red light of low intensity induced faster formation of long-wavelength Chlide species in the leaves with carotenoid deficiency in comparison to the normal leaves. We propose that, in leaves with reduced carotenoid content, a greater number of Pchlide molecules transform to Chlide per light flash than in normal leaves. The results are discussed in relation to the involvement of carotenoids in competitive absorption and light screening, as well as to their influence on Pchlide-Chlide interactions.

Stimulation of testicular function during the nonbreeding season in the woodchuck (Marmota monax).

Treatment of male woodchucks with a series of s.c. injections of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) induced testicular growth, sperm production and marked increase in serum testosterone levels in the fall, approximately 4 mo before the expected spontaneous onset of testicular activity. These results suggest that it may be possible to induce and/or maintain reproductive competence in this species outside of its normally brief breeding season.

The use of LOTUS 1-2-3 in statistics.

This paper describes a convenient way of performing statistical tests in biology. The recent development of powerful spreadsheet programs for microcomputers has made it possible to easily apply various statistical significance tests on biological data. Presently the following tests have been implemented in the LOTUS 1-2-3 framework: Student's t-test, chi-square test, analysis of variance (single classification random ANOVA), Student-Neumann-Kuels test, correlation analysis and analysis of linear regression (single and multilevel design). The most important advantages gained by using 1-2-3 instead of the commercial statistical software packages are the simplicity of entering data, the possibility of asking "what-if?" questions, the simple, but useful graphical presentation of data and the ease of actual building of the tests.

A radioimmunoassay program for Lotus 1-2-3.

Spreadsheet programs have become very popular as convenient ways of performing mathematical operations, as well as entering and organizing data. This paper describes how Lotus 1-2-3 can be used to calculate the results of a radioimmunoassay, a widely used technique in biomedical laboratories.

Improved sperm counts in mink males (Mustela vison) treated with clomiphene citrate.

A group of 12 sterile, azoospermic mink males were treated with clomiphene citrate (10 mg/kg/day) for 10 days during the mating season; 50% of the males showed improved sperm counts already after 2 days of treatment and the maximum effect was seen 4-6 days after the start of the treatment. The other half of the group did not respond to the treatment and their sperm counts remained near zero during the whole experiment.

Oat leaf base: tissue with an efficient regeneration capacity.

An efficient short term regeneration system using seedling derived oat (Avena sativa) leaf tissue has been developed. Callus derived from the leaf base showed a higher response of plant regeneration than callus initiated from mesocotyls and more mature parts of the leaves. A correlation between the nuclear DNA content of the donor material, as analysed with flow cytometry, and its ability to form callus was observed. Somatic embryogenesis was histologically recognised from callus derived from tissue close to the apical meristem. Plant regeneration media with various concentrations of auxin were tested. Callus from three different cultivars had a similar regeneration potential with an optimal regeneration frequency of 60%. About 2 months after inoculation regenerated plantlets could be moved to a greenhouse for cultivation.

Testicular aspiration biopsy in evaluation of fertility of mink (Mustela vison).

A 19-gauge needle biopsy was taken of the testis of mink in late January. When scores from 1 to 10 were given according to the developmental stage and number of spermatogenic cells, males scoring 8-10 returned significantly better breeding results than did males having scores less than 7. The biopsy did not affect libido or induce other disturbances of fertility. Fine-needle aspiration biopsy of the testis is possibly the most convenient and accurate infertility assay in mink breeding.

Adherence and toxicity of Yersinia enterocolitica 0:3 and 0:9 containing virulence-associated plasmids for various cultured cells.

Plasmid (47 and 44 Mdal, respectively) containing strains of Yersinia enterocolitica 0:3 and 0:9 adhered to and were toxic for HEp-2 human epithelial and Y-1 adrenal cells in vitro, At 37 degrees C, but not at room temperature, the adhesion of the bacteria lead to rounding and partial detachment of the cultured cells. UV-inactivated plasmid-positive Y. enterocolitica were neither adherent nor toxic for the cells but were readily endocytosed by HEp-2 cells. The adherence of plasmid-positive Y. enterocolitica 0:3 and 0:9 on epithelial cells may be pathogenetically important as an initial step for intestinal colonization, and possibly in Y. enterocolitica-induced diarrhoea. Plasmid-positive Y. enterocolitica also adhered to the surface of cultured human macrophages and were apparently not phagocytosed as effectively as the plasmid-negative derivatives of the same bacteria. Thus resistance to phagocytosis may form an additional plasmid-dependent virulence property of Y. enterocolitica 0:3 and 0:9.

Relationship between serum testosterone concentrations and fertility in male mink (Mustela vison).

During 7 successive months in 1982 and 1983 blood and semen samples were taken from male mink. The patterns of testosterone development in sterile and fertile males were readily distinguishable from each other. Testosterone concentrations showed a clear correlation (r = 0.73) with sperm quality of mink males. High testosterone levels (16.0-24.5 ng/ml) in early February were associated with defective sperm quality in March and low testosterone levels (2.0-13.3 ng/ml) with good sperm quality.

Reproductive capacity in male mink after long distance transportation in pregnant females.

Young American male mink born in Finland from imported pregnant females showed a clear delay (p less than 0.05) in testicular development as compared with local male mink in late February and produced semen of unsatisfactory quality during the breeding season in March. Better sperm quality (p less than 0.05) and better testicular development (p less than 0.1-0.05) was obtained in older American male mink born in Finland as compared with younger American male mink and the local breeding stock. Serum testosterone concentrations during six successive months in American males indicated a clear delay in sexual maturity and it could also be established that testosterone development reached normal levels in American males after 2 generations born in Finland. There were no differences in the serum thyroxine concentrations between American and local male mink.

Plastid microtubule-like structures in wheat are insensitive to microtubule inhibitors.

The effects of microtubule inhibitors on the spectral properties of leaves of wheat (Triticum aestivum L. cv. Walde) and on the presence of plastid microtubule-like structures (MTLS) during etioplast to chloroplast transformation were examined. Amiprophos-methyl (APM, 0.1 mM), fed to leaf sections of 7-day-old dark-grown wheat, reduced the ration of phototransformable to non-phototransformable proto-chlorophyllide (PChlide), decreased the rate of the Shibata shift, and inhibited chlorophyll accumulation and grana stacking. The spectral properties of isolated etioplasts were not affected by APM. Colchicine (10 mM), fed to leaf sections, inhibited greening but had no effect on the PChlide ratio or the Shibata shift. MTLS were still visible on electron micrographs after treatment with APM or colchicine at frequencies similar to controls. A third inhibitor, vinblastine, had no effect on the spectral properties of non-irradiated or irradiated etiolated leaves except at concentrations that produced visible tissue damage before the irradiation. The effects of APM and colchicine may reflect inhibitions of respiration and protein synthesis, respectively. It is concluded that MTLS are insensitive to microtubule inhibitors and thus are probably not composed of tubulin.

The in vitro assembly of the NADPH-protochlorophyllide oxidoreductase in pea chloroplasts.

The NADPH-protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) is the major protein in the prolamellar bodies (PLBs) of etioplasts, where it catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide during chlorophyll synthesis in higher plants. The suborganellar location in chloroplasts of light-grown plants is less clear. In vitro assays were performed to characterize the assembly process of the pchlide reductase protein in pea chloroplasts. Import reactions employing radiolabelled precursor protein of the pchlide reductase showed that the protein was efficiently imported into fully matured green chloroplasts of pea. Fractionation assays following an import reaction revealed that imported protein was targeted to the thylakoid membranes. No radiolabelled protein could be detected in the stromal or envelope compartments upon import. Assembly reactions performed in chloroplast lysates showed that maximum amount of radiolabelled protein was associated to the thylakoid membranes in a thermolysin-resistant conformation when the assays were performed in the presence of hydrolyzable ATP and NADPH, but not in the presence of NADH. Furthermore, membrane assembly was optimal at pH 7.5 and at 25 degrees C. However, further treatment of the thylakoids with NaOH after an assembly reaction removed most of the membrane-associated protein. Assembly assays performed with the mature form of the pchlide reductase, lacking the transit peptide, showed that the pre-sequence was not required for membrane assembly. These results indicate that the pchlide reductase is a peripheral protein located on the stromal side of the membrane, and that both the precursor and the mature form of the protein can act as substrates for membrane assembly.