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Germany↗
Biomedical subjects
U Wobus
Publications and source records attributed to U Wobus.
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Academies and Institutes↗
Sugar levels altered by ectopic expression of a yeast-derived invertase affect cellular differentiation of developing cotyledons of Vicia narbonensis L.
In order to change the sugar status during seed development a yeast-derived invertase gene was expressed in cotyledons of Vicia narbonensis. As a result, sucrose decreased whereas hexoses accumulated. We analysed cell structure and cellular differentiation in cotyledons expressing the yeast-invertase. Transgenic cells contained large and long-persisting vacuoles apparently serving as storage compartments for hexoses and clusters of storage-protein aggregates. In the wild-type, large vacuoles did not persist but were replaced by smaller protein bodies. During maturation and desiccation, the transgenic cells showed plasmolysis and vesiculation of the endo-membrane system. Immunogold-labelling revealed that the storage proteins vicilin and legumin were present within the cytoplasm and the extraprotoplasmic space and were attached to membranes of the endoplasmic reticulum and the nuclei. Protein storage vacuoles in mature seeds appeared heterogeneous and only partially filled. The data suggest that sugars control the subcellular organisation of the vacuolar system. Transcript levels encoding a tonoplast intrinsic protein, a marker for membranes of protein storage vacuoles, remained unchanged whereas mRNA levels of a hexose and a sucrose transporter increased. Generally, transgenic seeds appeared to be physiologically younger than wild-type seeds of the same age. The data underline the important role of sugars in legume seed development.
Cell Differentiation↗
Sucrose transport into barley seeds: molecular characterization of two transporters and implications for seed development and starch accumulation.
In order to understand sucrose transport in developing seeds of cereals at the molecular level, we cloned from a caryopses library two cDNAs encoding sucrose transporters, designated HvSUT1 and HvSUT2. Sucrose uptake activity was confirmed by heterologous expression in yeast. Both transporter genes are expressed in maternal as well as filial tissues. In a series of in situ hybridizations we analysed the cell type-specific expression in developing seeds. HvSUT1 is preferentially expressed in caryopses in the cells of the nucellar projection and the endospermal transfer layer, which represent the sites of sucrose exchange between the maternal and the filial generation and are characterized by transfer cell formation. HvSUT2 is expressed in all sink and source tissues analysed and may have a general housekeeping role. The rapid induction of HvSUT1 gene expression in caryopses at approximately 5-6 days after fertilization coincides with increasing levels of sucrose as well as sucrose synthase mRNA and activity, and occurs immediately before the onset of rapid starch accumulation within the endosperm. Starch biosynthesis requires sucrose to be imported into the endosperm, as direct precursor for starch synthesis and to promote storage-associated processes. We discuss the possible role of HvSUT1 as a control element for the endospermal sucrose concentration.
Base Sequence↗
Antisense-inhibition of ADP-glucose pyrophosphorylase in developing seeds of Vicia narbonensis moderately decreases starch but increases protein content and affects seed maturation.
The small subunit of a Vicia faba ADP-glucose pyrophosphorylase (AGP) cDNA was expressed in antisense orientation in Vicia narbonensis under the control of the seed-specific legumin B4 promoter. From several independent transgenic lines both ADP-glucose pyrophosphorylase AGP-mRNA and AGP enzyme activity were reduced by up to 95% in the cotyledons during the mid- to late-maturation phase. Starch was moderately decreased and sucrose was increased. In two of three lines, transcripts encoding the large subunit of AGP and the storage protein vicilin were increased, whereas legumin B-mRNA was decreased. Transcripts of other storage-associated genes were not altered. The cotyledons contained more protein and total nitrogen. Despite the reduction in starch, total carbon was not decreased and dry weight was unchanged. Compared to the wild type, transgenic seeds contained more water and accumulated dry weight during a longer period, and therefore had a prolonged seed-filling period. Transgenic cotyledon cells of comparable age to the wild type were more highly vacuolated and contained smaller starch grains, indicating a delay in cellular differentiation. We conclude that a specific alteration in carbon metabolism can have pleiotropic effects on water and nitrogen content and induces temporal changes in seed development.
Cotyledon↗
Phosphoenolpyruvate carboxylase in developing seeds of Vicia faba L.: gene expression and metabolic regulation.
To analyze the role of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) during seed development, two cDNA clones encoding two isoforms of PEPCase were isolated from a seed-specific library of Vicia faba. The two sequences (VfPEPCase1 and VfPEPCase2) have a sequence identity of 82 and 89% on the nucleotide and amino acid levels. The VfPEPCase1 mRNA was found to be predominantly expressed in roots and developing cotyledons whereas the VfPEPCase2 mRNA was more abundant in green and maternal tissues. In the cotyledons, PEPCase mRNAs accumulated from early to mid cotyledon stage and decreased thereafter. The PEPCase activity increased continuously during cotyledon development. The enzyme was strongly activated by glucose-6-phosphate, but not by glucose, fructose or sucrose. Asparagine was weakly activating whereas malate, aspartate and glutamate were inhibitory. The inhibitors became less effective with increasing pH. Aspartate was a much stronger inhibitor of cotyledonary PEPCase than glutamate at both pH 7.0 and 7.5. The sensitivity of PEPCase to malate inhibition decreased from early to mid cotyledon stage at a time when storage proteins are synthesized. This indicates activation on the protein level, possibly by protein phosphorylation. Nitrogen starvation in the presence of hexoses but not sucrose decreased mRNA levels of VfPEPCase1 and enzyme activity, indicating control on the mRNA level by both carbon and nitrogen. It is concluded that in developing cotyledons PEPCase is probably important for the synthesis of organic acids to provide carbon skeletons for amino acid synthesis.
Base Sequence↗
Seed maturation: genetic programmes and control signals.
Seed maturation is mainly governed by a few genes best studied in maize and Arabidopsis. The isolation of the LEC1 and FUS3 genes, besides the previously known VP1/AB/3 genes, and their identification as transcriptional regulators provides the first direct hints as to their molecular mode of action. With the identification of new effector genes, the investigation of the role of hormones with new methods such as immunomodulation and the increasingly recognised role of metabolites like sugars as important modulators of seed development, we increasingly understand the complexity and structure of the regulatory network underlying seed maturation.
Abscisic Acid↗
Homodimers and heterodimers of Pho1-type phosphorylase isoforms in Solanum tuberosum L. as revealed by sequence-specific antibodies.
Higher plants possess two types of glucan phosphorylase (EC 2.4.1.1). One isozyme type, designated as Pho1, is located in the plastid whereas the other type, Pho2, is restricted to the cytosol. For Solanum tuberosum L. two Pho1 type phosphorylases have been sequenced [Nakano, K. & Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236; Sonnewald, U., Basner, A., Greve, B. & Steup, M. (1995) Plant Mol. Biol. 27, 567-576]. Both proteins (referred to as Pho1a and Pho1b, respectively) are highly similar (81-84% amino acid identity over most parts of the two sequences) with the exception of the N-terminal transit peptide and the large insertion located between the N- and the C-terminal domains. In this communication antibodies that bind specifically to either Pho1a or Pho1b were used to study both isoforms at the protein level. The antibodies were applied to both potato tuber and leaf extracts following either denaturing or non-denaturing electrophoresis. Pho1a but not Pho1b was immunochemically detectable in tuber extracts whereas leaf extracts contained both the Pho1a and Pho1b protein. During denaturing electrophoresis the two antigens comigrated. When the leaf Pho1 isoforms were separated by affinity electrophoresis three bands of activity were resolved; all of them were recognized by the anti-Pho1a antibodies, but only two of these reacted with the anti-Pho1b antibodies. The isoform binding exclusively to the anti-Pho1a antibodies comigrated with the Pho1 isozyme from potato tubers. Immunoprecipitation experiments performed with anti-Pho1a antibodies removed the entire Pho1 phosphorylase activity from both tuber and leaf extracts. Addition of anti-Pho1b antibodies to tuber extracts did not affect the enzyme pattern, whereas in leaf extracts one isoform remained unchanged but the two other bands were strongly retarded. This indicates that the Pho1a protein is present in all three forms and Pho1b is associated with Pho1a. Association of Pho1a and Pho1b was further demonstrated by cross-linking experiments using bis(sulfosuccinimidyl)suberate as linker. Immunoprecipitation experiments were also performed using extracts of transformed Escherichia coli cells that expressed either Pho1a or Pho1b or both simultaneously. Under these conditions a homodimeric Pho1b phosphorylase was observed that had a lower electrophoretic mobility than the heterodimer from leaves. In leaves of transgenic potato plants antisense inhibition of the Pho1a gene affected the formation of (Pho1a)2 more strongly than that of the heterodimer. Thus, in leaves, Pho1a exists both as a homodimer, (Pho1a)2 and as heterodimer, (Pho1a-Pho1b); a part of it appears to be covalently modified. Pho1b, in the homodimeric form, is often below the limit of detection. In tubers the homodimer, (Pho1a)2, is the only detectable Pho1-type enzyme. To our knowledge this is the first report on a heterodimeric structure of plant phosphorylase.
Amino Acid Sequence↗
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.
Carbohydrate Metabolism↗
A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds.
To analyze sugar transport processes during seed development of fava bean, we cloned cDNAs encoding one sucrose and one hexose transporter, designated VfSUT1 and VfSTP1, respectively. sugar uptake activity was confirmed after heterologous expression in yeast. Gene expression was studied in relation to seed development. Transcripts were detected in both vegetative and seed tissues. In the embryo, VfSUT1 and VfSTP1 mRNAs were detected only in epidermal cells, but in a different temporal and spatial pattern. VfSTP1 mRNA accumulates during the midcotyledon stage in epidermal cells covering the mitotically active parenchyma, whereas the VfSUT1 transcript was specific to outer epidermal cells showing transfer cell morphology and covering the storage parenchyma. Transfer cells developed at the contact area of the cotyledonary epidermis and the seed coat, starting first at the early cotyledon stage and subsequently spreading to the abaxial region at the late cotyledon stage. Feeding high concentrations of sugars suppressed both VfSUT1 expression and transfer cell differentiation in vitro, suggesting a control by carbohydrate availability.
Carbohydrate Metabolism↗
Cloning and characterization of full-length cDNA encoding sucrose phosphate synthase from faba bean.
A 3419-bp cDNA clone encoding sucrose phosphate synthase was isolated from a seed coat-specific cDNA library of Vicia faba L. and conceptually translated into a protein of 1059 amino acid residues. The corresponding 3.4-kb mRNA was found in both source and sink tissues. Southern hybridization data are consistent with a single SPS gene.
Cloning, Molecular↗
Glucan phosphorylases in Vicia faba L.: cloning, structural analysis and expression patterns of cytosolic and plastidic forms in relation to starch.
We have isolated and characterised cDNA sequences from a Vicia faba cotyledonary library encoding a plastidic isoform (VfPho1) and a cytosolic isoform (VfPho2) of an alpha-1,4-glucan phosphorylase (EC 2.4.1.1; Commission on Plant Gene Nomenclature 1994). The Pho1 isoform is characterized by the presence of a plastidial transit peptide and an 81-residue stretch of additional amino acids in the middle of the polypeptide which are not found in the Pho2 isoform. We define the position of this so-called insertion sequence differently from previous authors. The Pho1 transcripts were found predominantly in the early seed coat and in cotyledons, and accumulated until the late desiccation phase, whereas Pho2 transcripts were about equally abundant in all investigated tissues. Activity patterns of both enzymes in cotyledons roughly followed mRNA accumulation patterns, with the exception of the late desiccation phases when mRNAs were degraded but enzyme activities remained at high level, even in long-stored seeds. The distinct Pho1 and Pho2 gene expression patterns in seed coats coincided with the transient accumulation pattern of starch. Similarly, in-situ hybridisation revealed a relationship between Pho1 gene expression and starch granule formation in developing cotyledons. Expression data and enzyme activity patterns were associated with starch formation during seed development, and could simply reflect a continuous accumulation of enzyme protein, ensuring immediate participation in starch degradation during germination.
Amino Acid Sequence↗
Sucrose metabolism during cotyledon development of Vicia faba L. is controlled by the concerted action of both sucrose-phosphate synthase and sucrose synthase: expression patterns, metabolic regulation and implications for seed development.
The roles of sucrose-phosphate synthase (Sps) and sucrose synthase (Sus) in developing embryos of Vicia faba have been characterized. In the cotyledons the expression of both Sps and Sus is initiated in cells differentiating into storage tissue. This stage is characterized by a switch in the carbohydrate state from a high to a low hexoses to sucrose ratio. The carbohydrate state was found earlier to be controlled by seed coat-associated invertase. During cotyledon development the Sps-enzyme undergoes a cycle of deactivation and reactivation: the activated state is associated with the prestorage phase, desiccation and germination and the deactivated state with the storage phase. Sus activity is associated with the storage phase. Sps and Sus are differentially influenced by free sugars. Feeding hexoses to storage phase cotyledons increases levels of Sps-mRNA but not Sus-mRNA, Sps activity and Sps activation state and impairs storage functions evidenced by an increased sucrose to starch ratio and a downregulation of storage protein legumin B-mRNA. Sus enzyme activity is inhibited by free hexoses in vitro. It is proposed that the changing carbohydrate state during cotyledon development controls the ratio of Sps to Sus. Sps may have some significance for the initiation of the storage process possibly decreasing hexoses and/or increasing sucrose. The relevance of the changing carbohydrate state with respect to development and storage processes is discussed.
Base Sequence↗
Cell-type specific, coordinate expression of two ADP-glucose pyrophosphorylase genes in relation to starch biosynthesis during seed development of Vicia faba L.
Several cDNA clones encoding two different ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27) polypeptides denoted VfAGPC and VfAGPP were isolated from a cotyledonary library of Vicia faba L. Both sequences are closely related to AGPase small-subunit sequences from other plants. Whereas mRNA levels of VfAGPP were equally high in developing cotyledons and leaves, the mRNA of VfAGPC was present in considerable amounts only in cotyledons. During development of cotyledons, both mRNAs accumulated until the beginning of the desiccation phase and disappeared afterwards. The increase of AGPase activity in cotyledons during the phase of storage-product synthesis was closely followed by the accumulation of starch. The AGPase activity in crude extracts of cotyledons was insensitive to 3-phosphoglycerate whereas the activity from leaves could be activated more than five-fold. Inorganic phosphate inhibited the enzyme from both tissues but was slightly more effective on the leaf enzyme. There was a correlation at the cellular level between the distribution of VfAGPP and VfAGPC mRNAs and the accumulation of starch, as studied by in-situ hybridisation and by histochemical staining in parallel tissue sections of developing seeds, respectively. During the early phase of seed development (12-15 days after fertilization) VfAGPase mRNA and accumulation of starch were detected transiently in the hypodermal, chlorenchymal and outer parenchymal cell layers of the seed coat but not in the embryo. At 25 days after fertilization both synthesis of VfAGPase mR-NA and biosynthesis of starch had started in parenchyma cells of the inner adaxial zone of the cotyledons. During later stages, the expression of VfAGPase and synthesis of starch extended over most of the cotyledons but were absent from peripheral cells of the abaxial zone, provascular and procalyptral cells.
Amino Acid Sequence↗
Seed coat-associated invertases of fava bean control both unloading and storage functions: cloning of cDNAs and cell type-specific expression.
We have studied the molecular physiology of photosynthate unloading and partitioning during seed development of fava bean (Vicia faba). During the prestorage phase, high levels of hexoses in the cotyledons and the apoplastic endospermal space are correlated with activity of cell wall-bound invertase in the seed coat. Three cDNAs were cloned. Sequence comparison revealed genes putatively encoding one soluble and two cell wall-bound isoforms of invertase. Expression was studied in different organs and tissues of developing seeds by RNA gel analysis, in situ hybridization, enzyme assay, and enzyme activity staining. One extracellular invertase gene is expressed during the prestorage phase in the thin-walled parenchyma of the seed coat, a region known to be the site of photoassimilate unloading. We propose a model for an invertase-mediated unloading process during early seed development and the regulation of cotyledonary sucrose metabolism. After unloading from the seed coat, sucrose is hydrolyzed by cell wall-bound invertases. Thus, invertase contributes to establish sink strength in young seeds. The resultant hexoses are loaded into the cotyledons and control carbohydrate partitioning via an influence on the sucrose synthase/sucrose-phosphate synthase pathway. The developmentally regulated degradation of the thin-walled parenchyma expressing the invertase apparently initiates the storage phase. This is characterized by a switch to a low sucrose/hexoses ratio. Feeding hexoses to storage-phase cotyledons in vitro increases the sucrose-phosphate synthase/sucrose synthase ratio and changes carbohydrate partitioning in favor of sucrose. Concomitantly, the transcript level of the major storage product legumin B is downregulated.
Base Sequence↗
The legumin gene family: a reconstructed Vicia faba legumin gene encoding a high-molecular-weight subunit is related to type B genes.
Nucleotide sequence information from a partial genomic clone, a cDNA clone, a RACE clone and a PCR fragment was combined to reconstruct the first reported complete gene sequence encoding a large legumin subunit, designated LelB3. The length difference to the well-characterized major legumin subunits is caused by an extended glutamine/glutamic acid-rich region encoded by the C-terminal part of the alpha chain. Amino acid sequence comparisons reveal that gene LelB3 is more closely related to B-type than to A-type legumin genes of Vicia faba. Gene LelB3 is a member of a small gene family as indicated by published (Pich and Schubert, Biol Zbl 112 (1993); 342-350) and limited own data.
Amino Acid Sequence↗
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.
Allergens↗