Protein storage vacuole acidification as a control of storage protein mobilization in soybeans

Soybean protease C1 (EC 3.4.21.25), the subtilisin-like serine protease that initiates the proteolysis of seed storage proteins in germinating soybean [Glycine max (L.) Merrill], was localized to the protein storage vacuoles of parenchyma cells in the cotyledons by immunoelectron microscopy. This was demonstrated not only in germination and early seedling growth as expected, but also in two stages of protein storage vacuole development during seed maturation. Thus, the plant places the proteolytic enzyme in the same compartment as the storage proteins, but is still able to accumulate those protein reserves. Since soybean protease C1 activity requires acidic conditions for activity, the hypothesis that the pH condition in the protein storage vacuole would support protease C1 activity in germination, but not in seed maturation, was tested. As hypothesized, acridine orange accumulation in the protein storage vacuole of storage parenchyma cells was detected by fluorescence confocal microscopy in seedlings before the onset of mobilization of reserve proteins as noted by SDS-PAGE. Accumulation of the dye was reversed by inclusion of the weak base methylamine to dissipate the pH gradient across the vacuolar membrane. Also as hypothesized, acridine orange did not accumulate in the protein storage vacuole of those parenchyma cells during seed maturation. These results were obtained using cells separated by pectolyase treatment and also using cotyledon slices.     

In situ localization of storage protein mRNAs in developing meristems of Brassica napus embryos

Probes derived from cDNA clones of napin and cruciferin, the major storage proteins of Brassica napus, and in situ hybridization techniques were used to examine changes in the spatial and temporal distribution of storage protein messages during the course of embryogeny, with a special emphasis on the developing apical meristems. Napin mRNAs begin to accumulate in the cortex of the axis during late heart stage, in the outer faces of the cotyledons during torpedo stage and in the inner faces of the cotyledons during cotyledon stage. Cruciferin mRNAs accumulate in a similar pattern but approximately 5 days later. Cells in the apical regions where root and shoot meristems develop do not accumulate storage protein messages during early stages of embryogeny. In the upper axis, the boundary between these apical cells and immediately adjacent cells that accumulate napin and cruciferin mRNAs is particularly distinct. Our analysis indicates that this boundary is not related to differences in tissue or cell type, but appears instead to be coincident with the site of a particular set of early cell divisions. A major change in the mRNA accumulation patterns occurs halfway through embryogeny, as the embryos enter maturation stage and start drying down. Final maturation of the shoot apical meristem is associated with the development of leaf primordia and the accumulation of napin mRNAs in the meristem, associated leaf primordia and vascular tissue. Cruciferin mRNAs accumulate only in certain zones of the shoot apical meristem and on the flanks of leaf primordia. Neither type of mRNA accumulates in the root apical meristem at any stage.     

From embryo sac to oil and protein bodies: embryo development in the model legume Medicago truncatula

? The cell and developmental biology of zygotic embryogenesis in the model legume Medicago truncatula has received little attention. We studied M. truncatula embryogenesis from embryo sac until cotyledon maturation, including oil and protein body biogenesis. ? We characterized embryo development using light and electron microscopy, measurement of protein and lipid fatty acid accumulation and by profiling the expression of key seed storage genes. ? Embryo sac development in M. truncatula is of the Polygonum type. A distinctive multicellular hypophysis and suspensor develops before the globular stage and by the early cotyledon stage, the procambium connects the developing apical meristems. In the storage parenchyma of cotyledons, ovoid oil bodies surround protein bodies and the plasma membrane. Four major lipid fatty acids accumulate as cotyledons develop, paralleling the expression of OLEOSIN and the storage protein genes, VICILIN and LEGUMIN. ? Zygotic embryogenesis in M. truncatula features the development of a distinctive multicellular hypophysis and an endopolyploid suspensor with basal transfer cell. A clear procambial connection between the apical meristems is evident and there is a characteristic arrangement of oil bodies in the cotyledons and radicle. Our data help link embryogenesis to the genetic regulation of oil and protein body biogenesis in legume seed.     

Expression patterns and subcellular localization of a 52 kDa sucrose-binding protein homologue of Vicia faba (VfSBPL) suggest different functions during development

A cDNA coding for a 54 kDa signal sequence containing protein has been isolated from a faba bean cotyledonary library and characterized. The deduced protein is designated Vicia faba SBP-like protein (VfSBPL) since it shares 58% homology to a 62 kDa soybean (Glycine max) protein (GmSBP) which has been described as a sucrose-binding and sucrose-transporting protein (SBP). VfSBPL as well as GmSBP are outgroup members of the large vicilin storage protein family. We were unable to measure any sucrose transport activity in mutant yeast cells expressing VfSBPL. During seed maturation in late (stage VII) cotyledons mRNA was localized by in situ hybridization in the storage parenchyma cells. At the subcellular level, immunolocalization studies proved VfSBPL accumulation in storage protein vacuoles. However, mRNA localization in stage VI cotyledons during the pre-storage/storage transition phase was untypical for a storage protein in that, in addition to storage parenchyma cell labelling, strong labelling was found over seed coat vascular strands and the embryo epidermal transfer cell layer reminiscent of sucrose transporter localization. The VfSBPL gene is composed of 6 exons and 5 introns with introns located at the same sites as in a Vicia faba 50 kDa vicilin storage protein gene. The time pattern of expression as revealed by northern blotting and the GUS accumulation pattern caused by a VfSBPL-promoter/GUS construct in transgenic tobacco seeds was similar to a seed protein gene with increasing expression during seed maturation. Our data suggest different functions of VfSBPL during seed development.     

莲种子萌发和幼苗生长时期营养物质的代谢变化

莲子叶细胞中储存了丰富的营养物质, 主要为蛋白质、淀粉和淀粉质体DNA。这些贮藏物质为种子萌发和幼苗的生长提供必需的能量和养料。通过组织化学和显微镜观察, 研究莲从种子萌发到植株生长至具有4个节时, 子叶中贮藏物质消耗的全过程。在此过程中, 子叶中的贮藏物质不断降解,营养物质发生转运。蛋白体首先发生降解, 其大量降解主要发生在幼苗三叶期。淀粉质体降解时会聚 集成团, 之后体积逐渐减小, 最后完全降解。种子萌发后65天是子叶贮藏物质消耗末期, 淀粉质体DNA的含量比萌发后20天的三叶期明显减少。细胞壁的形态结构发生多种形式的变化, 细胞壁发生的这些变化与子叶细胞间物质的运输有关。含多糖的球形颗粒通过维管束在子叶中运输。     

莲种子萌发和幼苗生长时期营养物质的代谢变化

莲子叶细胞中储存了丰富的营养物质,主要为蛋白质、淀粉和淀粉质体DNA.这些贮藏物质为种子萌发和幼苗的生长提供必需的能量和养料.通过组织化学和显微镜观察,研究莲从种子萌发到植株生长至具有4个节时,子叶中贮藏物质消耗的全过程.在此过程中,子叶中的贮藏物质不断降解,营养物质发生转运.蛋白体首先发生降解,其大量降解主要发生在幼苗三叶期.淀粉质体降解时会聚集成团,之后体积逐渐减小,最后完全降解.种子萌发后65天是子叶贮藏物质消耗末期,淀粉质体DNA的含量比萌发后20天的三叶期明显减少.细胞壁的形态结构发生多种形式的变化,细胞壁发生的这些变化与子叶细胞间物质的运输有关.含多糖的球形颗粒通过维管束在子叶中运输.     

The Formation of Polyploid Cells in Ripening Cotyledons of Pisum sativum L. in Relation to Ribosome and Protein Synthesis

The present paper deals with the formation of polyploid nucleiand the synthesis of RNA and protein in the parenchyma cellsof developing cotyledons of Pisum sativum L. During seed formationthese cells synthesize large amounts of reserve proteins andstarch, which later on are used up by the embryo during seedgermination. The changes of the amount of DNA per cell in the ripening cotyledontissue have been estimated by Feulgen histophotometry. The amountsof DNA, RNA, and protein in the whole cotyledons have been estimatedby chemical methods. In this way it was possible to correlatethe fluctuations of the amount of DNA, RNA, and protein withchanges at the cellular level. During a preparatory phase, preceding the phase of real cellexpansion and intensive accumulation of seed globulins and starch,the storage cells attain a high level of polyploidy: nucleiwith up to a 64 C DNA content are formed. The results indicate a correlation between the high degree ofpolyploidy in the parenchyma cells of the cotyledon and thehigh rate of RNA and protein synthesis (seed globulins) in thisstorage organ (gene dosage effect).     

Storage globulins pass through the Golgi apparatus and multivesicular bodies in the absence of dense vesicle formation during early stages of cotyledon development in mung bean

During seed development and maturation, large amounts of storage proteins are synthesized and deposited in protein storage vacuoles (PSVs). Multiple mechanisms have been proposed to be responsible for transporting storage proteins to PSVs in developing seeds. In this study, a specific antibody was raised against the mung bean (Vigna radiata) seed storage protein 8S globulin and its deposition was followed via immunogold electron microscopy in developing mung bean cotyledons. It is demonstrated that non-aggregated 8S globulins are present in multivesicular bodies (MVBs) in early stages of cotyledon development where neither dense vesicles (DVs) nor a PSV were recognizable. However, at later stages of cotyledon development, condensed globulins were visible in both DVs and distinct MVBs with a novel form of partitioning, with the internal vesicles being pushed to one sector of this organelle. These distinct MVBs were no longer sensitive to wortmannin. This study thus indicates a possible role for MVBs in transporting storage proteins to PSVs during the early stage of seed development prior to the involvement of DVs. In addition, wortmannin treatment is shown to induce DVs to form aggregates and to fuse with the plasma membrane.     

Endoplasmic Reticulum of Mung Bean Cotyledons: ACCUMULATION DURING SEED MATURATION AND CATABOLISM DURING SEEDLING GROWTH

Homogenates of mung bean cotyledons were subjected to equilibrium density centrifugation on linear sucrose gradients and the positions of the various organelles determined by assay of marker enzymes. Measurement of phospholipid distribution on such gradients showed that the major peak of phospholipid at a density of 1.11 to 1.13 grams per cubic centimeter coincided with the position of the endoplasmic reticulum (ER), confirming ultrastructural evidence that storage parenchyma cells are rich in ER. Germination and seedling growth were accompanied by a rapid decline in ER-associated phospholipid but a marked increase in the ER marker enzyme NADH cytochrome c reductase. Similar experiments with developing seeds indicated that the amount of ER-associated phospholipid increases during cotyledon expansion reaching a maximum during seed maturation. There was no subsequent decline during seed desiccation, instead ER-associated phospholipid levels were maintained in the dry seed until germination when catabolism was initiated 12 to 24 hours after the start of imbibition. This timing indicates that the observed ER breakdown is not an expression of the overall senescence of the cotyledons, but may represent the dismantling of the extensive rough ER used for reserve protein synthesis during cotyledon development.     

A Rapid Increase in the Level of Binding Protein (BiP) is Accompanied by Synthesis and Degradation of Storage Proteins in Pumpkin Cotyledons

The binding protein (BiP) has been implicated in cotranslationalfolding of nascent polypeptides, and in the recognition anddisposal of aberrant polypeptides. To elucidate the involvementof BiP in the biosynthesis of vacuolar proteins, we have characterizedthe protein in pumpkin cotyledons during seed maturation andseedling growth. Isolated microsomes from maturing pumpkin cotyledonscontained a significant amount of BiP, protein-disulfide isomeraseand calreticulin. We have purified a 70-kDa protein; sequencesof the N-terminus and internal fragments of this protein exhibiteda high identity to the sequence of soybean BiP. Immunoblot analysiswith specific antibodies raised against the purified BiP showedthat the amount of BiP in a cotyledon increased markedly atthe middle stages and then decreased. The increase was accompaniedby the synthesis of storage proteins and the development ofthe endoplasmic reticulum in the cotyledons at the middle stageof seed maturation. Most of these storage proteins degradeddramatically between 2 and 5 days after seed germination, andthe degradation was also accompanied by a rapid increase inthe level of BiP. Subcellular fractionation of the 4-day-oldcotyledons showed a high accumulation of BiP in the endoplasmicreticulum. It is possible that BiP might be involved in thesynthesis of seed storage proteins during maturation and inthe synthesis of hydrolytic enzymes responsible for the degradationof the storage proteins during seed germination. (Received September 18, 1996; Accepted January 8, 1997)     

Seed-specific overexpression of a potato sucrose transporter increases sucrose uptake and growth rates of developing pea cotyledons

During the storage phase, cotyledons of developing pea seeds are nourished by nutrients released to the seed apoplasm by their maternal seed coats. Sucrose is transported into pea cotyledons by sucrose/H+ symport mediated by PsSUT1 and possibly other sucrose symporters. PsSUT1 is principally localised to plasma membranes of cotyledon epidermal and subepidermal transfer cells abutting the seed coat. We tested the hypothesis that endogenous sucrose/H+ symporter(s) regulate sucrose import into developing pea cotyledons. This was done by supplementing their transport activity with a potato sucrose symporter (StSUT1), selectively expressed in cotyledon storage parenchyma cells under control of a vicilin promoter. In segregating transgenic lines, enhanced [(14)C]sucrose influx into cotyledons above wild-type levels was found to be dependent on StSUT1 expression. The transgene significantly increased (approximately 2-fold) transport activity of cotyledon storage parenchyma tissues where it was selectively expressed. In contrast, sucrose influx into whole cotyledons through the endogenous epidermal transfer cell pathway was increased by only 23% in cotyledons expressing the transgene. A similar response was found for rates of biomass gain by intact cotyledons and by excised cotyledons cultured on a sucrose medium. These observations demonstrate that transport activities of sucrose symporters influence cotyledon growth rates. The attenuated effect of StSUT1 overexpression on sucrose and dry matter fluxes by whole cotyledons is consistent with a large proportion of sucrose being taken up at the cotyledonary surface. This indicates that the cellular location of sucrose transporter activity plays a key role in determining rates of sucrose import into cotyledons.     

切除上胚轴对绿豆子叶衰老逆转及其核酸和蛋白质代谢的效应

在绿豆子叶衰老达到不归点(萌发后5～6d)前切除上胚轴可使开始衰老的子叶中核酸和蛋白质含量回升,衰老短期逆转。衰老不归点后的子叶中核酸和蛋白质变化的主要特征是:丧失了较多的核主带DNA、25S、18S rRNA以及大部分可溶性蛋白质组分,一种小分子DNA 组分完全消失。不归点前切除上胚轴可使上述核酸和蛋白质组分明显增加,表明子叶衰老的逆转可能与这些重要功能物质的回升有关。在切除上胚轴的茎顶涂抹IAA,能阻止子叶核酸和蛋白质回升,也消除了切除上胚轴对子叶裹老的逆转作用。     

Sequence of a plant cDNA from Vicia faba encoding a novel Ran-related GTP-binding protein

A clone obtained from a broad bean (Vicia faba) developing cotyledon cDNA library contained the complete coding sequence of a polypeptide with very high homology to the small GTP-binding proteins Ran from human cells and Spi1 from yeast. These proteins belong to the ras superfamily of proteins involved in different basic cellular processes. The Ran/Spi1 proteins interact with a protein bound to DNA (RCC1) and are thought to function in the regulation of the cell cycle. The amino acid sequence of the obtained plant Ran-homologue, designated Vfa-ran, is 74% and 76% identical to Ran and Spi1, respectively. The five functional, conserved domains of ras-related proteins are present in the Vfa-ran sequence. However, as in Ran/Spi1 the C-terminus of Vfa-ran is very acidic and lacks the Cys motif for isoprenylation.Northern blotting revealed a corresponding mRNA expression in broad bean roots, leaves, and cotyledons with the highest level in roots.     

DNA changes involving repeated sequences in senescing soybean (Glycine max) cotyledon nuclei

Age-related DNA changes in soybean ( Glycine max L. cv. Ransom) cotyledon nuclei were investigated by Feulgen cytophotometry and cloned DNA probes. The amount of nuclear DNA in 17-day-old senescing cotyledons was 23% lower than that of 5-day-old young cotyledons. In order to understand the nature of senescence-related DNA loss, fragments of repetitive DNA from young cotyledons were cloned into Escherichia coli HB101 cells by DNA recombination. The cloned DNA probed changes in specific repeated nucleotide sequences in senescing cotyledons. The colony hybridization between cloned DNA and [³²P]-labeled total DNA from 5- and 17-day cotyledons indicated loss of specific repeated sequences. The selected sequences of repeated DNA further showed a loss in their copy numbers. The study suggested that some repetitive DNA sequences were degraded selectively in the genome of senescing cotyledons.     

Regulation of chlorophyll and Rubisco levels in embryonic cotyledons of Phaseolus vulgaris

While deep within the maternal tissues (pods and testa), cotyledons of the bean (Phaseolus vulgaris L.) green and the plastids differentiate as chloroplasts. At the time of seed maturation the chloroplasts dedifferentiate and the green color is lost. We have used Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and chlorophyll to study chloroembryo development. Chlorophyll levels and Rubisco activity increase early in embryonic development then decline as the cotyledons enter the maturation phase. Rubisco accumulation follows a strong temporal pattern over the course of embryo development, and furthermore, occurs in total darkness. Therefore, accumulation of Rubisco during embryogenesis may occur in response to developmental signals. In embryos developed in total darkness, Rubisco accumulation was uncoupled from chlorophyll accumulation. Exposure of isolated cotyledons to abscisic acid (ABA) resulted in loss of chlorophyll and decline in Rubisco levels comparable to those seen in normal embryogenesis. This indicates that the decline in Rubisco in chloroembryos in vivo results from factors such as ABA that signal the onset of maturation. The results show that ABA not only enhances the accumulation of some proteins (e.g. storage proteins), but also depresses the accumulation of others during embryogeny.Abbreviations Rubisco ribulose-1,5-bisphosphate-carboxylase/oxygenase (EC 4.1.1.39) - LSU large subunit of Rubisco - SSU small subunit of Rubisco - ABA abscisic acid - FW fresh weight     

Deoxyribonucleic Acid and Ribonucleic Acid Synthesis during the Cell Expansion Phase of Cotyledon Development in Vicia faba L

In Vicia faba L., the tissue specific proteins, legumin and vicilin, are synthesized during the cell expansion phase of cotyledon development. During this growth period, RNA and nuclear DNA increase 8- to 10-fold. ³H-Uridine and ³H-adenosine are incorporated into ribosomal RNA, both 25S and 18S, and into transfer RNA. DNA isolated from cotyledons in the cell division phase of growth has been compared with DNA isolated from cotyledons undergoing expansion growth. Results indicate that the DNA increase involves replication of the whole genome (endoreduplication).     

Proteins of cotyledons of mature horse chestnut seeds

This is the first characterization of proteins from storage parenchyma of cotyledons of mature dormant recalcitrant horse chestnut (Aesculus hippocastanum L.) seeds and evaluation the cell protein-synthesizing capacity. It was established that the content of protein in cotyledons did not exceed 0.5% of tissue fresh weight. Soluble proteins (the proteins of the postmitochondrial supernatant or cytosol) comprised the bulk (up to 90%) of total proteins. Protein of subcellular structures (20000 g-pellet) comprised 5–7% of total protein. Cotyledon proteins were heterogenous in their charges and molecular weights of subunits. Cotyledon protein was easily extracted with a salt (1 M NaCl); they comprised 90% of water-soluble albumin-like proteins. The proportion of globulins was insignificant; it did not exceed 5%. Most water-soluble proteins (more than 80%) were tolerant to heat denaturing. Among these heat-stable proteins, two major groups of polypeptides dominated: an electrophoretically homogeneous component with a mol wt of 24–25 kD and a complex group from three to five polypeptides with mol wts in the range between 6 and 12 kD. Native heat-stable proteins had disulfide bonds. Four fractions of heat-stable proteins were obtained by ammonium sulfate fractionation; three of them were alike in their polypeptide composition and contained major components with mol wts of 24–25 and 5–12 kD. It was established that the active translational machinery functioned in the cells of storage parenchyma in cotyledons of mature dormant horse chestnut seeds. During each stage of stratification, cotyledon fragments incorporated ³⁵S-methionine into TCA-insoluble material more actively than axial organs. We discuss cotyledon protein composition, their function as a storage organ, and a possible role of heat-stable proteins.     

Influence of the axis on the enzymes of protein and amide metabolism in the cotyledons of mung bean seedlings

The growth of the mung bean (Vigna radiata) seedling is accompanied by the biosynthesis and accumulation of the endopeptidase vicilin peptidohydrolase and the catabolism of the reserve proteins in the cotyledons. If the axis is removed from the dry seeds and the cotyledons incubated on moist sand the accumulation of vicilin peptidohydrolase is reduced by 77% and the catabolism of reserve proteins slowed to 25% of the rate in intact seedlings. The cotyledons and the cotyledon exudate are rich in asparagine and this amino acid accounts for more than half of the reduced nitrogen exported from the cotyledons. Glutamine synthetase and asparagine synthetase, two key enzymes in the pathway of asparagine synthesis, are under temporal control in the cotyledons. Their activities increase 3.5- and 10-fold, respectively, then decline again. These increases in enzyme activity occur to the same extent in excised cotyledons and are prevented when the cotyledons are incubated in 5 micromolar cycloheximide. The results indicate that the axis may control certain key metabolic events in the cotyledons, such as the synthesis of vicilin peptidohydrolase, while many other anabolic activities may not depend on a growing axis.