Structure of two maize phytase genes and their spatio-temporal expression during seedling development.

Up to 80% of Zea mays L. grain phosphorus is stored in the form of phytin in the embryo. Our objective is to determine the control of phytin mobilization during germination and seedling growth. A maize phytase cDNA, phy S11, has been previously characterized (Maugenest et al., Biochem J 322: 511-517, 1997). In the present work, phy S11 was used to screen a maize genomic library and two distinct genes, PHYT I and PHYT II, were isolated and sequenced. The transcribed sequences of these two genes presented a strong homology whereas the untranscribed upstream and downstream sequences appeared very different. Northern blot analysis and in situ hybridization showed a high accumulation of phytase mRNA at the early steps of germination in the coleorhiza, radicle cortex and coleoptile parenchyma. Phytase expression was also detected at a lower extent in the scutellum. In adult plants, northern blot analyses revealed low but significant levels of phytase mRNA in the roots. In situ hybridizations on root cross-sections localized phytase mRNA in rhizodermis, endodermis and pericycle layers. Immunolocalization analysis showed phytase accumulation at the same sites as its mRNA. A RT-PCR approach was used in an attempt to discriminate between the transcripts from each gene in the different situations. These experiments indicate that both genes are expressed during germination, whereas only PHYT I is expressed in adult roots. This suggests that signals responsible for phytase gene expression in roots are different from those responsible for gene expression during germination.

Cloning and characterization of a cDNA encoding a maize seedling phytase.

During germination, maize seedlings express a phytase able to hydrolyse the large amount of phytin stored in the dry seed. Previous studies allowed purification and characterization of this enzyme as a homodimer of 38 kDa subunits [Laboure, Gagnon and Lescure, Biochem. J. (1993) 295, 413-419]. In the present work, an antibody against the purified maize phytase has been used to screen a maize seedling cDNA expression library. Several positive clones containing an insert of about 1400 bp were isolated. The nucleotide sequence of the insert of one of these clones has been established. This cDNA, called phy S11, was 1335 bp long and contained an open reading frame of 387 amino acids. The sequence of N-terminal residues (23 amino acids) of the purified phytase has been established. These residues are found at positions 19-41 of the amino acid sequence encoded by phy S11. This confirms that this cDNA codes for the maize phytase. The deduced amino acid sequence appears to be very different from those of published Aspergillus niger phytases; however, an homologous region of 33 amino acids was detected. This region of the fungal sequence contains the RHGxRxP consensus motif found in various high molecular mass acid phosphatases and believed to be the acceptor site for phosphate. Expression of the phy S11 cDNA in Escherichia coli allowed the production of the phytase subunit and its assembly to give a protein of the same size as the native phytase. The time course of phy S11 mRNA accumulation during germination showed that no transcript was present in dry seeds. The mRNA accumulated during the first day of germination, to reach a maximum after 2 days (radicle protrusion), and then decreased in young seedlings. Genomic Southern blot analyses suggest the existence of at least two genes and genetic mapping reveals two loci separated by 1 cM on chromosome 3 of maize. The cloning of this first cDNA coding for a plant phytase, will allow the isolation of the corresponding genes and the study of their regulation during germination.

Purification and characterization of a phytase (myo-inositol-hexakisphosphate phosphohydrolase) accumulated in maize (Zea mays) seedlings during germination.

Phytase (myo-inositol-hexakisphosphate phosphohydrolase, EC 3.1.3.8) has been purified from 5-7-day-old maize (Zea mays) seedlings, using a four-step purification procedure. The native protein has a molecular mass of about 76 kDa and is built up from two 38 kDa subunits. The pH and temperature optima of the purified enzyme were respectively 4.8 and 55 degrees C. The apparent Km for phytate was estimated to be 117 microM. Like other acidic phytases, the maize seedling enzyme exhibited a broad affinity for various phosphorylated substrates and especially for penta- and tri-phosphate esters of myo-inositol. The amino acid composition of the h.p.l.c.-purified protein indicated a high hydrophobicity (44% non-polar amino acids). Rabbit antibodies were produced in response to maize seedling phytase. Western-blot analyses clearly demonstrate that the increase of phytase activity observed during the first 7 days of germination corresponded to an accumulation of the protein in maize seedlings. Phytase accumulated essentially in the shoots (mesocotyl plus coleoptiles.

Ferritin gene transcription is regulated by iron in soybean cell cultures.

Iron-regulated ferritin synthesis in animals is dominated by translational control of stored mRNA; iron-induced transcription of ferritin genes, when it occurs, changes the subunit composition of ferritin mRNA and protein and is coupled to translational control. Ferritins in plants and animals have evolved from a common progenitor, based on the similarity of protein sequence; however, sequence divergence occurs in the C termini; structure prediction suggests that plant ferritin has the E-helix, which, in horse ferritin, forms a large channel at the tetrameric interface. In contemporary plants, a transit peptide is encoded by ferritin mRNA to target the protein to plastids. Iron-regulated synthesis of ferritin in plants and animals appears to be very different since the 50- to 60-fold increases of ferritin protein, previously observed to be induced by iron in cultured soybean cells, is accompanied by an equivalent accumulation of hybridizable ferritin mRNA and by increased transcription of ferritin genes. Ferritin mRNA from iron-induced cells and the constitutive ferritin mRNA from soybean hypocotyls are identical. The iron-induced protein is translocated normally to plastids. Differences in animal ferritin structure coincide with the various iron storage functions (reserve for iron proteins and detoxification). In contrast, the constancy of structure of soybean ferritin, iron-induced and constitutive, coupled with the potential for vacuolar storage of excess iron in plants suggest that rapid synthesis of ferritin from a stored ferritin mRNA may not be needed in plants for detoxification of iron.

Purification and characterization of an iron-induced ferritin from soybean (Glycine max) cell suspensions.

Ferric citrate induces ferritin synthesis and accumulation in soybean (Glycine max) cell suspension cultures [Proudhon, Briat & Lescure (1989) Plant Physiol. 90, 586-590]. This iron-induced ferritin has been purified from cells grown for 72 h in the presence of either 100 microM- or 500 microM-ferric citrate. It has a molecular mass of about 600 kDa and is built up from a 28 kDa subunit which is recognized by antibodies raised against pea (Pisum sativum) seed ferritin and it has the same N-terminal sequence as this latter, except for residue number 3, which is alanine in pea seed ferritin instead of valine in iron-induced soybean cell ferritin. It contains an average of 1800 atoms of iron per molecule whatever the ferric citrate concentration used to induce its synthesis. It is shown that the presence of 100 microM- or 500 microM-ferric citrate in the culture medium leads respectively to an 11- and 28-fold increase in the total intracellular iron concentration and to a 30- and 60-fold increase in the ferritin concentration. However, the percentage of iron stored in the mineral core of ferritin remains constant whatever the ferric citrate concentration used and represents only 5-6% of cellular iron.

Iron induction of ferritin synthesis in soybean cell suspensions.

In animal cells specialized for iron storage, iron-induced accumulation of ferritin is known to result from a shift of stored mRNA from the ribonucleoprotein fraction to polysomes. Previous reports with bean leaves suggested that in plants iron induction of ferritin synthesis would result from a regulation at the transcriptional level (F van der Mark, F Bienfait, H van der Ende [1983] Biochem Biophys Res Commun 115:463-469). Soybean (Glycine max, cv Mandarin) cell suspension cultures have been used here to support these findings. Ferritin induction is obtained by addition of Fe-citrate to the culture medium. A good correlation is found between cellular iron content and the amount of ferritin accumulation. This protein accumulation corresponds to an increase of in vitro translatable ferritin mRNA. Addition of 4 micrograms actinomycin D per milliliter to the cultures inhibits completely in vivo RNA synthesis, whereas protein synthesis was poorly affected, at least for 24 hours. During the same time, this concentration of actinomycin D strongly inhibits the iron-induced synthesis of ferritin. These results show that in soybean cell cultures, the mechanism of regulation of ferritin synthesis in response to iron does not result from recruitment of preexisting mRNA. They confirm that in plant systems, ferritin synthesis results from increased transcription of the corresponding genes.

Evidence for a translation-mediated attenuation of a spinach chloroplast rDNA operon.

The presence of potential hairpin structures H1, H2, H3 in the leader region of a spinach rDNA operon led us to postulate that this operon is regulated by premature termination. The mechanism would be controlled by the presence or absence of ribosomes translating a leader peptide. In vitro synchronized transcription by E. coli RNA polymerase shows that pauses do occur in the leader region. By their sizes, the transient transcripts could correspond to pauses on H1 and H2 as predicted by the model in the absence of ribosomes. The complete leader sequence (pKOPH) and the leader sequence with the hairpin structures deleted (pKOP) have been used to the GalK gene in the pK01 plasmid. The resulting plasmids have been used to transform a GalK- E. coli strain. Measurements of GalK expression show that the promoter region of spinach chloroplast rDNA is neither subjected to the growth rate nor to the stringent control. However, under growth conditions leading to an excess of free ribosomes, the expression of GalK gene appears systematically to be reduced in pKOPH when compared with that of pKOP. These results are consistent with a role of the leader region in a translation-mediated attenuation of the chloroplast rDNA expression.

Purification and characterization of ferritins from maize, pea, and soya bean seeds. Distribution in various pea organs.

Ferritins from maize, pea, and soya bean seeds were purified. They contain two polypeptides of 28 and 26.5 kDa. The molecular weight of native pea seed ferritin has been estimated to be 540,000. Pea and maize seed ferritins were compared by reverse phase high performance liquid chromatography, amino acid composition, and two-dimensional gel electrophoresis. They are very similar, although four isoforms of the 28-kDa polypeptide from the pea were observed in contrast to a unique polypeptide in maize. No isoforms of the 26.5-kDa polypeptide were detected. Rabbit antibodies were produced in response to pea seed ferritin. It was shown by Western blot analysis that ferritins of the three plants analyzed share immunological determinants. However, horse spleen ferritin was not recognized by the phytoferritin antibodies. Antibodies were also used to demonstrate that ferritins are not uniformly distributed in different pea organs from 30-day-old iron-unloaded plants. The protein was more abundant in flowers than in fruits and roots, and was not detected in leaves.

Abortive and productive elongation catalysed by purified spinach chloroplast RNA polymerase.

Experimental conditions are reported under which purified spinach chloroplast RNA polymerase catalyses the abortive elongation reaction on a synthetic poly[d(A-T)] template. The reaction only occurs under very stringent conditions and absolutely requires Mn2+ as the metal activator. No reaction can be detected in the presence of Mg2+. Furthermore, the rate of abortive elongation with the chloroplast enzyme is extremely sensitive to the presence of added salts, such as KCl or (NH4)2SO4, in the reaction assays. In the combined presence of Mn2+ and Mg2+, a marked inhibition of abortive elongation is associated with an activation of productive elongation and an increased length of RNA chains. Thus, whereas Mn2+ is more active than Mg2+ for phosphodiester bond formation, it appears that Mg2+ favors the stabilization of the ternary transcription complexes. These results are compared with those obtained under similar conditions for wheat germ RNA polymerase II and Escherichia coli RNA polymerase.

Relations between the plastid gene dosage and the levels of 16S rRNA and rbcL gene transcripts during amyloplast to chloroplast change in mixotrophic spinach cell suspensions.

Spinach cell suspension cultures maintained in photomixotrophic conditions exhibit plastids which undergo cyclic morphological transformations along a growth cycle. Ultrastructural studies show that the green chloroplasts present at the initial stage differentiate into amyloplasts during the subsequent log phase and then return to chloroplasts in stationary phase. The changes of the levels of plastid DNA (pt DNA) per cell have been determined along the growth cycle, as a percentage of total DNA by hybridization of definite amounts of total DNA to a radioactive probe of cloned pt DNA. The number of pt DNA copies have been estimated to 1125 per cell at the maximum of amyloplast development and to 5940 copies per cell at the maximum of chloroplast differentiation. Hybridizations of defined amounts of total cellular RNA to labelled probes of the 16S rDNA and of the rbcL gene allowed estimations of the variations of the corresponding cellular RNA pools. These variations are well correlated with the changes of the ptDNA cellular levels. These results show that the ptDNA gene dosage plays a central role in the regulation of the plastid transcript levels in this system.

Transcription of the chloroplast DNA: a review.

The transcription systems of chloroplasts and bacteria share different properties. The genetic material of chloroplasts is organized in the same way as bacterial nucleoids. The regulatory DNA sequences for transcription have a strong homology with their E. coli counterparts and some regulatory mechanisms could be conserved. The RNA polymerase subunits and some transcription factors also share similarities with prokaryotes. However, the chloroplast core-enzyme seems to be synthesized in the cytoplasm from nuclear encoded messages.

In vitro transcription initiation of the spinach chloroplast 16S rRNA gene at two tandem promoters.

Two potential prokaryotic promoters, P1 and P2, are characterized 164 and 114 bp upstream of the spinach chloroplast 16S rRNA gene. The strengths of these promoters, calculated according to an homology score established for E. coli RNA-polymerase, are identical. Experiments performed with a Taq I-DNA fragment, containing 16 bp of the 16S rDNA and 243 bp upstream of the gene, give evidence that in vitro, E. coli RNA-polymerase starts transcription at these two promoters. These results are based on both the size of the transcripts and their nucleotide sequences. A possible regulation by differential control of these dual promoters is suggested. S1 mapping with RNAs extracted either from green or from etiolated spinach plants, indicates that, at these two steps of plastid development, transcription in vivo starts at P1. Surprisingly only P2 appears to be conserved in the homologous sequences reported for maize, mustard and Spirodela.

Site of synthesis of spinach chloroplast ribosomal proteins and formation of incomplete ribosomal particles in isolated chloroplasts.

Chloroplast ribosomal proteins from spinach have been prepared in the presence of a protease inhibitor and some modifications have been introduced to the previous characterization of the 50S subunits (Mache et al., MGG, 177, 333, 1980): 33 ribosomal proteins are detected instead of 34. No change has been observed for the 30S subunits.Using a light-driven system of protein synthesis it is shown that up to ten ribosomal proteins of the 30S and eight proteins of the 50S subunits are made in the chloroplast.Newly synthesized ribosomal subunits have been analysed on CsCl gradients after sedimentation at equilibrium, allowing the separation of fully assembled subunits from incomplete ribosomal particles. Most of the newly made 50S subunits are fully assembled (ρ=1.634). A small amount of incomplete 50S particles (ρ=1.686) is detectable. Newly made 30S subunits (ρ=1.598) and incomplete 30S particles (ρ=1.691) are also observed. The ribosomal proteins of the incomplete 30S have been determined. They contain eight or nine of the 30S-proteins, seven of which are synthesized within the chloroplast. It is suggested that incomplete ribosomal particles resulted from a step in the assembly of ribosomal subunits.