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.     

Enhanced alpha - amylase production by chromosomal integration of pTVA1 in industrial strain in Bacillus subtilis

Summary Strain Bacillus subtilis MS was constructed with 12–22 fold increase of -amylase production, caused by presence of multiple -amylase gene copies in the chromosome of industrial strain Bacillus subtilis CCM2722, as demonstrated by DNA hybridization. The enhanced production is a result of multiple integration of plasmid pTVA1, carrying a temperature sensitive origin of replication from pE194, and containing the -amylase gene and a modified transposon Tn917.     

Chloroplast-encoded chlB is required for light-independent protochlorophyllide reductase activity in Chlamydomonas reinhardtii.

A chloroplast-encoded gene, designated chlB, has been isolated from Chlamydomonas reinhardtii, its nucleotide sequence determined, and its role in the light-independent reduction of protochlorophyllide to chlorophyllide demonstrated by gene disruption experiments. The C. reinhardtii chlB gene is similar to open reading frame 563 (orf563) of C. moewusii, and its encoded protein is a homolog of the Rhodobacter capsulatus bchB gene product that encodes one of the polypeptide components of bacterial light-independent protochlorophyllide reduction. To determine whether the chlB gene product has a similar role in light-independent protochlorophyllide reduction in this alga, a series of plasmids were constructed in which the aadA gene conferring spectinomycin resistance was inserted at three different sites within the chlB gene. The mutated chlB genes were introduced into the Chlamydomonas chloroplast genome using particle gun-mediated transformation, and homoplasmic transformants containing the disrupted chlB genes were selected on the basis of conversion to antibiotic resistance. Individual transformed strains containing chlB disruptions were grown in the dark or light, and 17 of the 18 strains examined were found to have a "yellow-in-the-dark" phenotype and to accumulate the chlorophyll biosynthetic precursor protochlorophyllide. RNA gel blot analysis of chlB gene expression in wild-type cells indicated that the gene was transcribed at low levels in both dark- and light-grown cells. The results of these studies support the involvement of the chlB gene product in light-independent protochlorophyllide reduction, and they demonstrate that, similar to its eubacterial predecessors, this green alga requires at least three components (i.e., chlN, chlL, and chlB) for light-independent protochlorophyllide reduction.     

Purification and Characterization of Lectin from Lathyrus sativus

Lectin has been isolated and purified from Lathyrus sativus using ammonium sulphate precipitation followed by affinity chromatography. The molecular weight as determined by HPLC was found to be 42kD. The lectin is a tetramer, consisting of two types of subunits of which the heavier subunit consists of 2 polypeptides of mol wt of about 21 kD and 16 kD while the smaller subunits consists of two polypeptides of about 5kD as revealed by SDS-PAGE. The most potent sugar inhibitor of the Lathyrus lectin was found to be α-methyl D-mannoside. The N-terminal amino acid sequence was similar to that of pea lectin sequence.     

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°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.     

Euploidy in Ricinus: EUPLOIDY EFFECTS ON PHOTOSYNTHETIC ACTIVITY AND CONTENT OF CHLOROPHYLL-PROTEINS

The effects of nuclear genome duplication on the chlorophyll-protein content and photochemical activity of chloroplasts, and photosynthetic rates in leaf tissue, have been evaluated in haploid, diploid, and tetraploid individuals of the castor bean, Ricinus communis L. Analysis of this euploid series revealed that both photosystem II (2,6-dichlorophenolindophenol reduction) and photosystem I oxygen uptake (N,N,N′,N′-tetramethyl-p-phenylenediamine to methyl viologen) decrease in plastids isolated from cells with increasingly larger nuclear complement sizes. Photosynthetic O₂-evolution and ¹⁴CO₂-fixation rates in leaf tissue from haploid, diploid, and tetraploid individuals were also found to decrease with the increase in size of the nuclear genome. Six chlorophyll-protein complexes, in addition to a zone of detergent complexed free pigment, were resolved from sodium dodecyl sulfate-solubilized thylakoid membranes from cells of all three ploidy levels. In addition to the P700-chlorophyll a-protein complex and the light-harvesting chlorophyll a/b-protein complex, four minor complexes were revealed, two containing only chlorophyll a and two containing both chlorophyll a and b. The relative distribution of chlorophyll among the resolved chlorophyll-protein complexes and free pigment was found to be similar for all three ploidy levels.     

The distribution of protochlorophyllide and chlorophyll within seedlings of the lip1 mutant of Pea

The distribution of protochlorophyllide (Pchlide) and NADPH-Pchlideoxidoreductase (POR) was characterized in the epicotyls androots of wild-type pea (Pisum sativum L. cv. Alaska) and lip1,a mutant with light-independent photomorphogenesis caused bya mutation in the COP1 locus. The upper part of the dark-grownlip1 mutant epicotyls had a high Pchlide content that decreaseddownward the organ. The elevated Pchlide level in lip1 seedlingswas a result of the differentiation of more proplastids intoPchlide-containing plastids. The cortex cells in the lip1 epicotylwere filled with such plastids in contrast to the cortex cellsof wild-type seedlings. The mutant also developed Pchlide-containingplastids in the roots, indicating the suppressing effect ofthe COP1 locus on development of plastids in the correspondingtissues in dark-grown wild-type plants. The distribution ofPchlide-containing plastids in dark-grown lip1 mutant stem androot was similar to the distribution of chloroplasts in irradiatedwild-type plants. Both wild-type and lip1 epicotyls containedmostly short wavelength Pchlide fluorescing at 631 nm withonly a small shoulder at 654 nm, which was transformedto a minute amount of chlorophyllide (Chlide) by flash irradiation.In contrast, with continuous irradiation a considerable amountof Chlide was formed especially in the lip1 epicotyls. Immunoblotsindicated the presence of POR, as a 36 kDa band, in epicotylsof both dark-grown wild-type and lip1 mutant seedlings. However,lip1 stem tissue had a higher content of POR than the wild-typepea. The high content of POR was unexpected as lip1 lacked boththe 654 nm fluorescing Pchlide form and the regular PLBs.In light, a significant amount of chlorophyll was formed alsoin the roots of the lip1 seedlings. ³ Corresponding author: E-mail, mahdi.seyedi@molbio.gu.se; Fax,+46-31-773-2626.     

POR structural domains important for the enzyme activity in R. capsulatus complementation system

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes hydrogen transfer from NADPH to protochlorophyllide (PChlide) in the course of chlorophyll biosynthesis in photosynthetic organisms and is involved in the regulation of the development of photosynthetic apparatus in higher plants, algae and cyanobacteria. To approach molecular factors determining the enzyme activity in a living cell, several mutants of POR from pea (Pisum sativum) with site-directed modifications in different parts of the enzyme were generated. The mutant enzymes were expressed in a R. capsulatus mutant deficient in BChl biosynthesis, and their catalytic activity and ability to integrate in bacterial metabolism were analyzed. Our results demonstrate that in heterologous bacterial cell system, higher plant POR is integrated in the porphyrin biosynthesis network and its activity leads to the formation of photosynthetic chlorophyll-proteins (CPs). The study of POR mutants in R. capsulatus reveals several POR domains important for the association of the enzyme with other subcellular components and for its catalytic activity, including identification of putative enzyme reaction center and substrate binding site. The study also demonstrated that an unknown structural factor is important for the formation of the enzyme photoactive complex in etiolated plants. Moreover, our findings suggest that POR might be directly involved in the regulation of the metabolism of other porphyrins. This revised version was published online in June 2006 with corrections to the Cover Date.