Fe homeostasis in plant cells: does nicotianamine play multiple roles in the regulation of cytoplasmic Fe concentration?

The cellular and intracellular localization of the non-proteogenic amino acid nicotianamine (NA) in leaves and root elongation zones was immunochemically investigated in pea (Pisum sativum L.) and tomato (Lycopersicon esculentum Mill.) plants grown under various iron regimes and in three mutants defective in the regulation of iron uptake. Strongest immunostaining was observed in the over-accumulating pea mutants brz and dgl, and in iron-loaded wild-type plants. Fe concentration and NA level paralleled staining intensity, indicating that NA synthesis is induced by high iron availability. While label was mainly present in the cytoplasm under normal (10 microM) Fe supply and under Fe deprivation, most of the labeling was present in the vacuole in iron-loaded plants. This pattern resembled the distribution of NA in Fe over-accumulating mutants, indicating the possible importance of vacuolar sequestration in the detoxification of excess Fe. Based on the dependence of the cellular distribution of NA on the iron nutritional status of the plant, a possible role of NA in buffering free Fe in root and leaf cells was inferred. We show here for the first time that the NA concentration is increased in response to iron overload, indicating that, besides other classes of intracellular metal-binding ligands, NA may play an essential role in iron tolerance.

Immunomodulation of phytohormones and functional proteins in plant cells.

Recombinant antibodies expressed ectopically in plant cells recognize their corresponding antigens and can therefore bind specifically to phytohormones and proteins in vivo. The generation of antibody-antigen complexes interferes with the functions of the targets and affects the phenotype of transgenic plants. Recombinant antibodies can accumulate in different cell compartments and organs of transgenic plants at different stages of development. High levels of expression of specific, high-affinity antibodies are required for immunomodulation. Here, we discuss several models and examples of the antibody-mediated modulation of phytohormone and protein functions in terms of their potential for plant research.

Immunomodulation of ABA function affects early events in somatic embryo development.

Immunomodulation of abscisic acid (ABA) function during somatic embryogenesis of Nicotiana plumbaginifolia has been used to demonstrate for the first time the effect of this phytohormone on early embryonic events. A homozygous transgenic line constitutively expressing an anti-abscisic acid (ABA) single chain fragment variable antibody in the endoplasmic reticulum was established. Development of somatic embryos from the transgenic line and the wild type was compared. The ABA biosynthesis mutants aba1 and aba2 and wild type cultures treated with the ABA biosynthesis inhibitor fluridone were also used for the comparative investigations. The development of embryonic structures was disturbed in the early stages of all cultures in which ABA function was blocked or which were ABA-deficient. After ABA complementation of the in vitro cell cultures normal somatic embryo development was restored.

Molecular characterization of cDNAs encoding G protein alpha and beta subunits and study of their temporal and spatial expression patterns in Nicotiana plumbaginifolia Viv.

We have isolated cDNA sequences encoding alpha and beta subunits of potential G proteins from a cDNA library prepared from somatic embryos of Nicotiana plumbaginifolia Viv. at early developmental stages. The predicted NPGPA1 and NPGPB1 gene products are 75-98% identical to the known respective plant alpha and beta subunits. Southern hybridizations indicate that NPGPA1 is probably a single-copy gene, whereas at least two copies of NPGPB1 exist in the N. plumbaginifolia genome. Northern analyses reveal that both NPGPA1 and NPGPB1 mRNA are expressed in all embryogenic stages and plant tissues examined and their expression is obviously regulated by the plant hormone auxin. Immunohistological localization of NPGPalpha1 and NPGPbeta1 preferentially on plasma and endoplasmic reticulum membranes and their immunochemical detection exclusively in microsomal cell fractions implicate membrane association of both proteins. The temporal and spatial expression patterns of NPGPA1 and NPGPB1 show conformity as well as differences. This could account for not only cooperative, but also individual activities of both subunits during embryogenesis and plant development.

Evolutionary conservation of kinetochore protein sequences in plants.

The evolutionary conservation of structural/functional kinetochore proteins has been studied on isolated nuclei and pro-/metaphase chromosomes of mono- and dicot plants. The cross-reactivities of antibodies against human CENPC, CENPE and CENPF, and against maize CENPCa with the centromeric regions of mitotic chromosomes of Vicia faba and/or Hordeum vulgare are shown. Putative homologs of the kinetochore protein SKP1 (suppressor of kinetochore protein 1p of yeast) were found in both species and of CBF5p (centromere binding factor 5 of yeast) in barley. Antibodies against synthetic peptides derived from partial sequences encoding these proteins were produced and recognized the centromeric regions on mitotic chromosomes as detected by indirect immunofluorescence.

Expression of a yeast-derived invertase in developing cotyledons of Vicia narbonensis alters the carbohydrate state and affects storage functions.

In plants the carbohydrate state provides signals to adjust metabolism to specific physiological conditions. Storage-active sink organs like seeds often contain high levels of sucrose. In order to change the sugar status during seed development a yeast-derived invertase gene was expressed in Vicia narbonensis under control of the LeguminB4 promoter. A signal sequence targeted the invertase to the apoplast in maturing embryos. In the cotyledons, sucrose was decreased whereas hexoses strongly accumulated. There was a major reduction of starch whereas proteins were less affected. Vacuoles of cotyledon cells were enlarged and dry seeds wrinkled. Transcripts and enzyme activity of sucrose synthase, the small and large subunit of ADP-glucose pyrophosphorylase as well as vicilin were downregulated. Sucrose phosphate synthase and legumin-mRNAs were not affected. Analysing single seeds with different sucrose levels revealed a positive correlation of sucrose concentration to mRNA levels of sucrose synthase and most pronounced to ADP-glucose pyrophosphorylase-mRNA levels as well as to starch content. Glucose on the other hand did not show any correlation. After feeding 14C-sucrose in vitro, the invertase-expressing cotyledons partitioned less carbon into starch compared to the wild-type. In the transgenic cotyledons, a relatively higher amount was directed into proteins compared to starch. We conclude that starch accumulation in developing cotyledons could be a function of sucrose concentration. Our results are consistent with a possible sucrose-mediated induction of storage-associated differentiation indicated by upregulation of specific genes of the starch synthesis pathway.

Calreticulin expression in plant cells: developmental regulation, tissue specificity and intracellular distribution.

The tissue-specific expression pattern and the intracellular distribution of the Ca(2+)-binding protein calreticulin at the mRNA and protein levels have been studied during somatic and zygotic embryogenesis of Nicotiana plumbaginifolia Viv. A full-length cDNA sequence encoding calreticulin was isolated from a lembda Zap cDNA library from early developmental stages of somatic embryogenesis. The deduced amino acid sequence of the calreticulin from N. plumbaginifolia shows high homology to the corresponding proteins of tobacco (98.2% identity), maize (80%) and barley (76.5%), and more than 55% homology to animal calreticulins, and the sequence motifs with established functions found in calreticulins of other species were quite conserved. Northern experiments revealed a developmental regulation of the calreticulin transcript with a maximum during the early stages of somatic embryogenesis and an auxin dependence during in-vitro cell culture. alpha-Naphthaleneacetic acid stimulated calreticulin expression whereas 2,4-dichlorophenoxyacetic acid reduced it. Immunohistological analysis of calreticulin distribution in the ovaries during zygotic embryogenesis showed that calreticulin biosynthesis started tissue specifically, with a high abundance in the endothelium of the integument in the ovules, followed by calreticulin accumulation in the embryo proper and in the associated endosperm at the late globular stage of embryogenesis. Using immunogold labeling, calreticulin was intracellularly localized with a high abundance to the Golgi compartment and to patches on the surface of dividing protoplasts. Smaller amounts were found in the endoplasmic reticulum and plasma membranes. The functional role of calreticulin in posttranslational processing and translocation processes, apart from its postulated function in cellular Ca2+ homeostasis, is discussed.

Evidence for secretion of vacuolar alpha-mannosidase, class I chitinase, and class I beta-1,3-glucanase in suspension cultures of tobacco cells.

We have investigated the possibility that vacuolar proteins can be secreted into the medium of cultured cells of Nicotiana tabacum L. Time-course and balance-sheet experiments showed that a large fraction, up to ca. 19%, of vacuolar alpha-mannosidase (EC 3.2.1.24) and vacuolar class I chitinase (EC 3.2.1.14) in suspension cultures accumulated in the medium within one week after subculturing. This effect was most pronounced in media containing 2,4-dichlorophenoxyacetic acid (2,4-D). Under comparable conditions only a small fraction, 1.8-5.1% of the total protein and ca. 1% of malate dehydrogenase (EC 1.1.1.37), which is localized primarily in the mitochondria and cytoplasm, accumulated in the medium. Pulse-chase experiments showed that newly synthesized vacuolar class I isoforms of chitinase and beta-1,3-glucanase (EC 3.2.1.39) were released into the medium. Post-translational processing, but not the release of these proteins, was delayed by the secretion inhibitor brefeldin A. Only forms of the proteins present in the vacuole, i.e. mature chitinase and pro-beta-1,3-glucanase and mature beta-1,3-glucanase, were chased into the medium of tobacco cell-suspension cultures. Our results provide strong evidence that vacuolar alpha-mannosidase, chitinase and beta-1,3-glucanase can be secreted into the medium. They also suggest that secretion of chitinase and beta-1,3-glucanase might be via a novel pathway in which the proteins pass through the vacuolar compartment.

Correct targeting of a vacuolar tobacco chitinase in Saccharomyces cerevisiae--post-translational modifications are dependent on the host strain.

The chitinase gene FB7-1 of Nicotiana tabacum cv. samsun line 5 was expressed in the two Saccharomyces cerevisiae strains, INVSC2 and H4, under the control of the GAL1 promoter from S. cerevisiae and a multicopy plasmid vector. Both yeast strains express the plant gene as enzymatic active proteins. In transformants of the strain INVSC2, 94% of the total plant chitinase is contained inside the cells, probably within the vacuole which has been confirmed by subcellular fractionation as well as immunohistochemical experiments. This retention inside the cells is due to the C-terminally located 7 amino acids long vacuolar targeting peptide of the prochitinase. When this sequence was removed, chitinase was transported into the culture medium. Pulse-chase experiments revealed that during translation in transformants of both yeast strains one chitinase polypeptide can be immunoadsorbed with specific antibodies. In the case of INVSC2-transformants, newly formed chitinase is modified in a 60 min chase to slightly increase its molecular mass, whereas in H4-transformants the molecular mass constantly remained 32 kDa. By Western blot analysis two chitinase corresponding polypeptides of 32 and 37 kDa were accumulated in the culture medium of both transformants carrying the chitinase gene without the vacuolar targeting sequence. The larger one was very likely O-glycosylated. Whereas, both polypepitdes were also detected in cell extracts of the H4-transformant, only the smaller one was found in the INVSC2-transformant. The plant chitinase passed through the endoplasmic reticulum on its way to the vacuole. The N-terminal signal peptide responsible for the uptake into the endoplasmic reticulum is cleaved correctly. However, cleavage of the vacuolar targeting peptide located at the C-terminus, to give the mature chitinase is obviously influenced by the genetic background of the host strain. In INVSC2-transformants chitinase accumulates in its mature form whereas both the polypeptides of H4-transformants retain their vacuolar targeting peptide. Our results demonstrate that in the case of plant class I chitinase, the plant sorting signal is recognized in yeast cells but post-translational modifications are influenced by the host strain.

Molecular-cytogenetic characterization of a higher plant centromere/kinetochore complex.

The centromeric region of a telocentric field bean chromosome that resulted from centric fission of the metacentric satellite chromosome was microdissected. The DNA of this region was amplified and biotinylated by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR)/linker-adapter PCR. After fluorescence in situ hybridization (FISH) the entire chromosome complement of Vicia faba was labelled by these probes except for the nucleolus organizing region (NOR) and the interstitial heterochromatin, the chromosomes of V. sativa and V. narbonensis were only slightly labelled by the same probes. Dense uniform labelling was also observed when a probe amplified from a clearly delimited microdissected centromeric region of a mutant of Tradescantia paludosa was hybridized to T. paludosa chromosomes. Even after six cycles of subtractive hybridization between DNA fragments amplified from centromeric and acentric regions no sequences specifically located at the field bean centromeres were found among the remaining DNA. A mouse antiserum was produced which detected nuclear proteins of 33 kDa and 68 kDa; these were predominantly located at V. faba kinetochores during mitotic metaphase. DNA amplified from the chromatin fraction adsorbed by this serum out of the sonicated total mitotic chromatin also did not cause specific labelling of primary constrictions. From these results we conclude: (1) either centromere-specific DNA sequences are not very conserved among higher plants and are - at least in species with large genomes - intermingled with complex dispersed repetitive sequences that prevent the purification of the former, or (2) (some of) the dispersed repeats themselves specify the primary constrictions by stereophysical parameters rather than by their base sequence.

Subcellular Localization and Characterization of Excessive Iron in the Nicotianamine-less Tomato Mutant chloronerva.

To understand the function of the Fe2+-complexing compound nicotianamine (NA) in the iron metabolism of plants we have localized iron and other elements in the NA-containing tomato wild type (Lycopersicon esculentum) and its NA-free mutant chloronerva by quantitative x-ray microanalysis. Comparison of element composition of the rhizodermal cell walls indicated that the wild type accumulated considerable amounts of iron and phosphorus in the cell wall, whereas in the mutant iron and phosphorus were detected in the cytoplasm and vacuoles of the rhizodermis. In mutant leaves containing high iron concentrations in the symplast, electron-dense inclusions were detected in chloroplasts and phloem. Such particles, consisting mainly of iron and phosphorus, were never found in the wild type and were very rarely detected in young chlorotic mutant leaves or after treatment of the mutant with NA. For further characterization the electron-dense inclusions in mutant leaves were isolated and compared by sodium dodecyl sulfate-gel electrophoresis and immunoblotting to ferritin from iron-loaded Phaseolus vulgaris leaves. Antibodies raised against purified Phaseolus leaf ferritin were used. Neither in mutant nor in wild type (iron loaded and control) was ferritin protein detected. These results suggest that the electron-dense inclusions in mutant leaves are not identical with ferritin. It is concluded that NA is necessary to complex ferrous iron in a soluble and available form within the cells. In the absence of NA the precipitation of excessive iron in the form of insoluble ferric phosphate compounds could protect the cells from iron overload.

Comparative biochemical, genetical and immunological studies of glucoamylase producing Arxula adeninivorans yeast strains.

Seven Arxula adeninivorans strains originating from The Netherlands (CSIR 1136, CSIR 1138 and CBS 8244T), South Africa (CSIR 1147, CSIR 1148 and CSIR 1149) and Siberia (Ls3) were compared concerning their secretory glucoamylase. Within the spectrum of secretory polypeptides obtained by SDS-polyacrylamide gel electrophoresis, the glucoamylase corresponding polypeptide, could be identified by means of specific antibodies directed against the glucoamylase of strain Ls3. The molecular masses of the glucoamylases vary from 84 to 95 kDa. After endoglycosidase F treatment of the secretory proteins, the N-linked carbohydrate content for each glucoamylase could be quantified at about 25%. Glucoamylase activity staining of secretory proteins separated under nondenaturing conditions revealed one glucoamylase active protein for the Dutch strains and two active forms for the strains originating from South Africa and Siberia. Highest activities of glucoamylase can be measured at the end of the exponential growth phase for each strain. The Dutch strain CSIR 1138 secretes the highest activity. Optimum values for temperature and pH were determined and compared. By means of pulsed field gel electrophoresis and Southern analysis, the glucoamylase corresponding gene could be localized on the second of the four chromosomes of each yeast strain.

Transient expression of a lysine-rich vicilin gene ofVicia faba in barley endosperm detected by immunological tissue printing after particle bombardment.

Using immunological tissue printing we detected transient expression of a faba bean vicilin gene with or without introns driven by the B1 hordein promoter in barley endosperm after particle bombardment. The described method generally allows the analysis of transient expression of genes without depending on reporter gene constructs and specifically suggests correct splicing of dicot introns by a monocot splicing machinery.

Tissue-specific expression of an oat 12S seed globulin gene in developing tobacco seeds: differential mRNA and protein accumulation.

We studied the expression of the oat globulin gene asglo5 in developing transgenic tobacco seeds. The asglo5 gene promoter directed transcription in the endosperm as well as in the provascular tissue, the presumptive root tip and the shoot apical meristem of the embryo as revealed by GUS reporter gene constructs and in situ hybridization. However, immunological tissue printing detected the oat protein exclusively in the tobacco endosperm, suggesting that extensive post-transcriptional regulatory processes influence the expression of the monocot transgene in the dicot host.

Location, size, and complexity of epitopes on the coat protein of beet necrotic yellow vein virus studied by means of synthetic overlapping peptides.

Five regions on the coat protein of BNYVV which had been shown previously to be involved in the formation of continuous epitopes were further analyzed by means of synthetic overlapping peptides. It was found that at least some of these regions may encompass several overlapping epitopes (or parts thereof). Four monoclonal antibodies (MAbs) which were known to be specific for the C-terminus of BNYVV coat protein (amino acids 182-188 = RTSPPGQ) were found to react with different sets of peptides which had either the sequence RTS, RTSP, RTSPP, or PPGQ in common. Two other MAbs which also had been shown previously to be specific for the C-terminus of BNYVV coat protein failed to react with overlapping decapeptides. Two epitopes which were previously located in the areas of amino acids 115-125 and 125-140 could now be located more precisely on the sequences SANVRRD (amino acids 115-121) and AESSG (amino acids 128-132), respectively. Replacement studies with alanine showed that not all amino acids within these sequences are equally important for antibody binding. On the other hand, amino acids outside these sequences may strongly influence the reactivity of epitopes. The accessibility of amino acid sequences on the particles of BNYVV is discussed.

In situ localization of faba bean and oat legumin-type proteins in transgenic tobacco seeds by a highly sensitive immunological tissue print technique.

We have used a highly sensitive immunological tissue print technique to study cell- and tissue-specific expression of heterologous genes in transgenic plants. Primary polyclonal antibodies, raised against legumin of faba bean (Vicia faba L.) and 12S globulin of oat (Avena sativa L.) were used to localize these proteins in transgenic tobacco seeds in a streptavidin-alkaline phosphatase assay in combination with biotinylated secondary antibodies producing a higher sensitivity (by several amplification steps) of the assay. Both storage protein genes were found to be expressed in a specific pattern. While legumin is preferentially accumulated in certain parts of the embryo, the oat legumin-type globulin is restricted to the endosperm. The applied technique is highly sensitive with a resolution power down to the single-cell level and allows rapid screening of large numbers of samples.

Calvin cycle multienzyme complexes are bound to chloroplast thylakoid membranes of higher plants in situ.

Further evidence is provided that the Calvin cycle enzymes ribose-5-phosphate isomerase (EC 5.3.1.6), ribulose-5-phosphate kinase (Ru-5-P-K, EC 2.7.1.19), ribulose-1,5-bisphosphate carboxylase (RuP2Case, EC 4.1.1.39), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12), sedoheptulose-1,7-bisphosphatase (Sed-1,7-bPase, EC 3.1.3.37), and electron transport protein ferredoxin-NADP+ reductase (FNR, EC 1.18.1.1) are organized into stable CO2-fixing multienzyme complexes with a molecular mass of 900 kDa. Limited trypsinolysis combined with immunoblotting revealed that all of chloroplast stromal Ru-5-P-K and GAPDH is located in enzyme complexes. The Calvin cycle enzyme complexes remain intact indefinitely at lower ionic strength but dissociate into components at KCl concentrations >250 mM. Immunoelectron microscopy showed that Ru-5-P-K, GAPDH, Sed-1,7-bPase, and FNR are bound to stroma-faced thylakoid membranes in situ, whereas RuP2Case and RuP2Case activase are randomly distributed throughout chloroplasts. The results indicate that membrane-bound enzyme supercomplexes may play an important role in photosynthesis.

Dynamics of protein recovery from process filtration systems using microporous membrane filter cartridges.

The purpose of this report is to address the concern for protein concentration recovery through membrane filter cartridges at the process scale. Filtration systems consisting of pre- and/or final membrane filter cartridges were evaluated for protein concentration recovery under typical manufacturing conditions of continuous flow and high throughput volumes. Results of the study conclusively demonstrate that consideration of the protein adsorptive properties and recovery performance of cartridge filters can provide efficient bio-burden and particulate control without compromising protein yields. Understanding the dynamics of protein recovery through various prefilter and final filter cartridges can play an important role in the proper selection of a filtration system to ensure optimal life and protein yield when filtering dilute protein solutions.

Long-term cultures of barley synthesize and correctly deposit seed storage proteins.

Long-term cultures of four different cultivars of barley (Hordeum vulgare L.) have been established. Both callus and suspension cultures formed embryogenic structures at high frequency even after more than 18 months of culture. These compact proembryogenic cell clusters synthesize seed storage globulins whereas loose cell aggregates in callus culture and suspension cultures of fine dispersed consistency were free of globulins. Globulin synthesis was especially intense in compact structures of callus cultures established from suspension culture-derived protoplasts. Within the cells storage globulins are deposited in the vacuolar compartment as in zygotic embryos. The molecular data provided recommend the system for studies on factors determining seed protein gene expression and intracellular protein transport.

Immunochemical differentiation of glucose phosphate isomerase (GPI) allozymes of the mouse.

Polyclonal xenoantisera against mouse GPI-1B and GPI-1C were produced in rabbits and analyzed for their ability to recognize allozyme-specific determinants. These studies showed a high degree of serological similarity among the three allozymes of mouse glucose phosphate isomerase (GPI). However, GPI-1B and GPI-1C could be differentiated from GPI-1A as well as GPI-1A and GPI-1B from GPI-1C using quantitative solid-phase immunobinding assays. In addition, polyclonal and monoclonal alloantibodies specific for GPI-1C were produced in BALB/c (Gpi-1a/Gpi-1a) mice. As indicated by immunoblotting data, the allozyme specificity of rabbit antisera and monoclonal alloantibodies against GPI-1C is dependent on the native structure of that allozyme.