Characterization of soybean vegetative storage proteins and genes

Summary Soybean vegetative storage proteins (VSPs) were purified and characterized. Anion exchange HPLC resolved partially purified VSPs into fractions containing 27-kD/27-kD and 29-kD/29-kD homodimers and 27-kD/29-kD heterodimers. Reversed-phase HPLC resolved partially purified VSPs into three fractions. One fraction contained only 27-kD VSP and the other two contained 29-kD VSP. The two 29-kD VSP fractions differed with respect to their cyanogen bromide cleavage patterns, an observation that indicated the 29-kD VSPs were heterogeneous. Genomic clones that contained 29-kD VSP genes were also isolated and characterized. One genomic clone contained a complete 29-kD VSP gene and was sequenced. The coding region in the clone contained two introns whose borders had regulatory sequences typical of other eukaryotic genes. Putative polyadenlyation signals were present in the 3-flanking region of the gene, while putative TATA, CAAT, and enhancer core sequences were found in the 5-flanking regions. A second genomic clone that was studied contained the 5 regions of two partial 29-kD VSP genes in an inverted linkage. Genomic DNA gel blots showed that the two genes were organized in the same arrangement in the soybean genome.Cooperative research between USDA/Agricultural Research Service and the Indiana Agricultural Experiment Station. Journal Paper No. 12,192 from the Indiana Agricultural Experiment Station     

Specialized cellular arrangements in legume leaves in relation to assimilate transport and compartmentation: comparison of the paraveinal mesophyll

Leaves of eight species of Leguminosae-Papilionoideae were examined for the presence of a highly specialized cell layer called the paraveinal mesophyli (PVM). Three species, Glycine max (L.) Merr, Psophocarpus tetragonolobus D.C. and Vigna radiata L., contained PVM; five (Medicago sativa L., Phaseolus vulgaris L., Pisum sativum L., Vicia faba L., Vigna unguiculata L.) did not. The PVM of G. max and P. tetragonolobus was anatomically identical and consisted of large, interconnected, multiarmed cells forming a network, one cell thick, spanning the region between vascular bundles and abutting the bundle sheath at the level of the phloem. The PVM of V. radiata differed in that elaborate extensions of individual bundle-sheath cells comprised the entire intervascular network. The PVM cells of all three species were large, contained a dense, thin peripheral layer of cytoplasm, and a large central vacuole. The cytoplasm contained few small chloroplasts and few microbodies, but was enriched in rough endoplasmic reticulum. Plasmodesmata were common in crosswalls between adjacent PVM cells and between PVM cells and other cell types abutting them. Vacuolar material was present in all three species, but was variable in appearance. That of G. max was present in large amounts, semifibrillar and finely dispersed. That of P. tetragonolobus was also present in large amounts but primarily as large aggregates, although some fibrillar material was also present. Vigna radiata had small amounts of vacuolar material evenly distributed between small aggregates and dispersed fibrils. Removal of flowers or young pods resulted in further increase of the vacuolar material in G. max PVM and increase of the fibrillar material in P. tetragonolobus, but had no appreciable affect on the vacuolar material in V. radiata. Histochemical staining indicated the vacuolar material in G. max and P. tetragonolobus was proteinaceous.Abbreviations PTA phosphotungstic acid - PVM paraveinal mesophyll Contribution No. 3215 from the Central Research and Development Department, E.I. du Pont de Nemours & Co     

Ringing induces the accumulation of vegetative storage proteins in poplar bark

Poplar branches were ringed in late spring to determine whether the interruption of the phloem flow could induce the accumulation of vegetative storage proteins (VSPs) in the bark of adult trees. Eight days after ringing, an increased deposition of starch as well as a premature rise in the soluble-protein level occurred in the bark tissues located 1 cm above the ring. Changes in the SDS-PAGE pattern of bark proteins were characterized by the accumulation of three polypeptides (32, 36 and 38 kDa), which exhibited the same molecular weight as VSPs described in poplar bark during winter, cross-reacted to antibodies raised against a poplar VSP, and bound to several lectins in the same way as poplar bark VSPs. These results indicate that during the vegetative period, ringing induces the accumulation of VSPs in the bark of poplar.     

Post-translational processing of 7S and 11S components of soybean storage proteins

The synthesis and processing of the major storage proteins in soybean cotyledons was studied both in vivo and in vitro. The and subunits of 7S as well as the 11S proteins are synthesized as higher molecular weight-precursors on membrane-bound polysomes. The initial translation products of the 7S are proteolytically cleaved during translation suggesting the removal of a signal peptide as evidenced by the presence of 2 and 2 peptides immunoreactive with 7S antibody in the in vitro chain completion products of the membrane-bound polysomes. This is followed or accompanied by cotranslational glycosylation, which increases their size equivalent to that of initially-synthesized precursors. In vivo pulse-labelled 7S and products are of slightly higher molecular weights than the immunoprecipitable chain-completion products, indicating further post-translational modifications. A slow post-translational processing during a period of 1.5 to 16 h yields the final 7S and glycoproteins.Acidic and basic subunits of the 11S protein appear to be synthesized from common large molecular weight (60K-59K) precursors. Antibodies to the 11S acidic component recognize both acidic and basic domains in the precursor while those raised against basic subunits appear to be specific for that region only. The processing of the 11S precursor is also very slow and occurs post-translationally. This slow rate of processing, coupled with a temporal difference in the synthesis of 7S and 11S components, suggests a highly coordinated mechanism for synthesis and packaging of these proteins into protein bodies during seed development.     

Vacuole biogenesis and protein transport to the plant vacuole: A comparison with the yeast vacuole and the mammalian lysosome

Summary The vacuole is often termed the lytic compartment of the plant cell. The yeast cell also possesses a vacuole containing acid hydrolases. In animal cells these enzymes are localized in the lysosome. Recent research suggests that there is good reason to regard these organelles as homologous in terms of protein transport. Although sorting motifs for the recognition of vacuolar proteins within the endomembrane system differ between the three organelles, there is an underlying similarity in targeting determinants in the cytoplasmic tails of Golgi-based receptors. In all three cases these determinants appear to interact with adaptins of clathrin-coated vesicles which ferry their cargo first of all to an endosomal compartment. The situation in sorting and targeting of plant vacuolar proteins is complicated by the fact that storage and lytic vacuoles may exist together in the same cell. The origin of these two types of vacuole is also a matter of some uncertanity.Abbrevations AP assembly protein - ALP alkaline phosphatase - ARF adenosine diphosphate ribosylation factor - BiP immunoglobulin binding protein - CCV clathrin coated vesicle - CPY carboxypeptidase-Y - DPAP dipeptidyl aminopeptidase - ER endoplasmic reticulum - GApp Golgi apparatus - LAMPs lysosomal associated membrane protein(s) - LAP lysosomal acid phosphatase - LIMPs lysosomal integral membrane protein(s) - MPRs mannosyl 6-phosphate receptors - MVB multivesicular bodies - NSF N-ethylmaleimide sensitive fusion (protein) - PAT phosphinotricine acetyltransferase - PB protein body - PHA phytohemagglutinin - PM plasma membrane - PSV protein storage vacuole - SNAPs soluble NSF attachment protein(s) - SNAREs SNAP receptor(s) - TGN trans Golgi network - TIP tonoplast integral protein - VPS vacuolar protein sorting - ZIO zinc iodide/osmium     

The riddle of the plant vacuolar sorting receptors

Proteins synthesized on membrane-bound ribosomes are sorted at the Golgi apparatus level for delivery to various cellular destinations: the plasma membrane or the extracellular space, and the lytic vacuole or lysosome. Sorting involves the assembly of vesicles, which preferentially package soluble proteins with a common destination. The selection of proteins for a particular vesicle type involves the recognition of proteins by specific receptors, such as the vacuolar sorting receptors for vacuolar targeting. Most eukaryotic organisms have one or two receptors to target proteins to the lytic vacuole. Surprisingly, plants have several members of the same family, seven in Arabidopsis thaliana. Why do plants have so many proteins to sort soluble proteins to their respective destinations? The presence of at least two types of vacuoles, lytic and storage, seems to be a partial answer. In this review we analyze the last experimental evidence supporting the presence of different subfamilies of plant vacuolar sorting receptors.     

Increased sulfur amino acids in soybean plants overexpressing the maize 15 kDa zein protein

Summary Four transgenic soybean [Glycine max (L.) Merrill] lines were generated containing the maize 15 kDa zein protein gene using somatic embryogenic protocols. The zein gene was inserted behind the β-phaseolin promoter for seed-specific expression. All four lines represent separate transformation events as they were generated in different experiments at different locations. Two of the transformation events produced multiple plants, and these produced identical Southern hybridization patterns (UKY/Z1, UKY/Z2 and UKY/Z3 from the first; and OSU/Z4, OSU/Z8 and OSU/Z10 from the second). Molecular characterization revealed that multiple copies of the zein gene were present in all of the transgenic lines. Immunoblot analysis confirmed the accumulation of the zein protein product in the seeds of the UKY/Z1, UKY/Z2, UKY/Z3, OSU/Z4, OSU/Z8 and OSU/Z10 transgenic lines. However, there was no accumulation of zein protein in the UGA/Z1 line and Northern analysis confirmed that the zein transgene was silenced in this line. It was not possible to analyze the zein expression in the seeds of the UKY/Z4 line, as it was sterile. Amino acid analysis of the UKY and OSU lines confirmed that there was a 12–20% increase in methionine, and 15–35% increase in cysteine content in these lines compared to the control. There were no consistent changes in the content of the other amino acids in the transgenic lines. These results suggest that while the increase in methionine content in these lines is modest, it is possible to increase the methionine content without adversely affecting the protein composition of soybean.     

大豆microRNA基因GmMIR160A负调控植物叶片衰老进程

:叶片衰老是受内外多种因子影响的遗传发育进程.生长素、细胞分裂素和乙烯等多种植物激素是调 控叶片衰老的重要内部因子,它们通过长或短距离运输形成叶片组织内特定的区域分布和浓度梯度,从而直 接或间接参与植物叶片衰老过程.分子遗传学表明,细胞分裂素和乙烯分别是叶片衰老的抑制子和正调节 子,而生长素如何参与叶片衰老的分子机制目前还不清晰.植物体内成熟小分子RNA 由小RNA 基因转录 并通过特定酶加工形成的２１~２３bp的双链RNA分子.这些小分子通过不完全配对方式抑制其靶基因转录 和/或表达,参与植物生长发育多个过程,然而这类小RNA 分子如何调控植物叶片衰老发育过程目前则还鲜 有报告.大豆是重要的油料作物,具有典型的单次结实性衰老特征.研究大豆叶片衰老具有重要的科学意义 和深远的应用价值.该文采用实时荧光定量PCR(qPCR)技术分析大豆(Glycinemax)microRNA基因Gm- MIR１６０A 的表达模式,发现大豆第一复叶中GmMIR１６０A 表达受外源生长素和黑暗处理的诱导,暗示该基 因是生长素快速响应的叶片衰老相关基因.为进一步探究GmMIR１６０A 在大豆叶片发育中的功能,构建了 肾上腺皮质激素(Glucocorticoid,GR)类似物地塞米松(Dexamethasone,DEX)诱导表达GmMIR１６０A 双元表 达载体并通过农杆菌介导的子叶节方法转化野生型大豆.通过抗性筛选和基因组PCR 鉴定并结合表型分 析,共获得了４株诱导表达的稳定遗传转基因植株(株系OXＧ３、OXＧ５、OXＧ７和OXＧ８).GmMIR１６０A 过表达 植株根、茎、叶、花和果实在形态学上与野生型相比无显著差异,但叶片的叶绿素含量增加、最大光量子效率 (Fv/Fm)增强.进一步分子分析发现,转基因大豆叶片中GmARFs 和衰老标记基因(GmCYSP１)表达明显下 降,表明大豆Gma-miR１６０通过抑制靶基因GmARFs 的表达来负调控植物叶片的衰老进程.该文揭示了生 长素通过小分子RNA调控叶片发育一条新途径,为研究植物激素调控植物叶片衰老提供了新的思路.     

Special secretory cells in the soybean seed coat

Summary The seed coat of soybean (Glycine max L. Merr.) is of physiological interest for synthesis and transport of amino acids and photosynthates during embryo development. A transmission and scanning electron microscopic study to elucidate the structure of the seed coat disclosed a specialized convex area (antipit) appressed to a concave pit in the center of the abaxial surface of the cotyledon. The antipit, which lies on the inner surface of the seed coat at a medial point in the anterior to posterior direction of the seed, contained specialized secretory cells bounded by loose multi-layered cell walls. These cells were rectangular in the developing seed, varied in length, and contributed directly to the convex morphology of the antipit seen on the ventral surface of the seed coat. At maturity these cells assumed the shape of a cone, extending from the aleurone layer in a perpendicular array. The aleurone and cone cells contained numerous Golgi apparatus, laminated rough endoplasmic reticulum, secretory vesicles, and amyloplasts. Secretory vesicles arose directly from tubules of fenestrated trans cisternae of the Golgi apparatus. Mitochondria were clustered with the amyloplasts; stacks of lamellar cisternae of rough endoplasmic reticulum were associated with the nucleus and Golgi apparatus. The cellular contents, the interconnections by plasmodesmata, and the close physical association with the cotyledon suggested that the aleurone and cone cells may be involved in symplastic transport of nutrients for use by the developing embryo.This paper is dedicated to the memory of my parents, Joseph and Theresa Yaklich, who by their example taught me the value of work and the enjoyment of simple things.     

Genetic improvement of the somatic embryogenesis and regeneration in soybean and transformation of the improved breeding lines

Somatic embryos of soybean [Glycine max (L.) Merrill] have been used to generate transgenic plants by particle bombardment. The induction and proliferation of somatic embryos from immature cotyledons are dependent on the genotype of the cultivar. Whereas somatic embryogenesis and plant regeneration are inefficient in most cultivars, they are efficient in the cultivar Jack. We previously established a breeding line, QF2, by the integration of null mutations of each subunit of the major seed storage proteins glycinin and β-conglycinin, but the embryogenic response of this line is insufficient to allow efficient transformation. We have now backcrossed QF2 to cultivar Jack in order to combine the null traits with competence for somatic embryogenesis. The backcrossed breeding lines selected on the basis of the absence of the major storage proteins exhibited an improved capacity for the induction and proliferation of somatic embryos compared with that of QF2. The induced somatic embryogenic tissue of these breeding lines was successfully used for the production of transgenic plants by particle bombardment. These results also indicate that somatic embryogenesis in soybean is genetically controlled and inherited in a manner independent of the null traits of the major seed storage proteins.     

Molecular and structural analysis of electrophoretic variants of soybean seed storage proteins

Soybean (Glycine max L.) storage proteins are composed mainly of two major components, beta-conglycinin and glycinin. Electrophoretic variants of the beta subunit of beta-conglycinin and the A3 polypeptide of glycinin were detected on SDS-PAGE, and designated them as beta* and A3*, respectively. beta* and A3* exhibited higher and lower mobilities, respectively, than the common beta subunit and A3 polypeptide. The N-terminal nine and 10 amino acid sequences of beta* and A3* were completely identical to the previously reported sequences of the beta subunit and the A3 polypeptide, respectively. Analysis using concanavalin A-horseradish peroxidase and treatment with N-glycosidase indicated that glycans were not responsible for the difference in electrophoretic mobility of beta* or A3*. Furthermore, five clones of beta* or beta and three clones of A3*, respectively, were sequenced but we could not detect deletions and insertions except for a single or a few amino acid substitutions as compared with the common beta subunit and A3 polypeptide. These results indicate that a single or a few amino acid substitution affects the electrophoretic mobilities of beta* and A3*.     

Relationship of cyst nematode gene frequencies to soybean resistance

Summary Soybean (S, Glycine max (L.) Merr.) lines with relatively few cysts of soybean cyst nematode (CN, Heterodera glycines Ichinohe) populations are usually called CN-resistant. The phenotype of number of cysts per plant is of the CN-S (Cyst Nematode-Soybean) association and determined by the interactions of genes for avirulence-resistance. The acronym alins was proposed for these alleles for incompatibility, with xalin representing the interaction X of one microsymbiont malin with its host h-alin. These alins are dominant in the gene-for-gene model but may be mostly recessive with CN-S. Definitive genetic studies have been hindered by the heterogeneity of sexually reproducing CN populations and lack of the appropriate genetic models. Loegering's abstract interorganismal genetic model was modified so that one model represented all four possible interactions of dominant-recessive alins for an incompatible phenotype. This involved redefining the Boolean algebra symbol 1 to represent both the alins AND their frequencies. The model was used to derive the relationship: {ie893-01} where the expectation E of cysts (of any CN-S combination, as proportion of number of cysts on a check cultivar) is proportional to the product of CN genotypic frequencies expressed as functions of m-alin frequencies. Each m-alin is at a different locus, i.e., {ie893-02}. The number of terms multiplied for each CN-S is equal to the number of alins in the S line (or F₂ plant). There are too many unknowns in the equation to solve for any of them. The relationship does explain the continuous distributions of phenotypes that were nearly always observed. Basic genetic principles were used to concurrently derive the models and to obtain discontinuous distributions of numbers of cyst phenotypes in segregating generations due to one recessive alin in a CN-susceptible soybean line.Contribution from the Missouri Agricultural Experiment Station, Journal Series No. 9739     

Microcallus formation from leaf mesophyll protoplasts in the genus Actinidia Lindl

Summary Microcallus (more than 60 cells) formation was obtained from leaf mesophyll protoplasts of 6 species and varieties in the genus Actinidia Lindl. (kiwifruit). The best results were achieved by using liquid over agarose culture for A. arguta var. arguta, liquid and agarose disc type culture for A. arguta var. purpurea, agarose disc type culture for A. arguta cv. Issaï and A. deliciosa and liquid agarose bead type- and disc type culture for A. kolomikta and A. polygama. Several factors influencing purification, browning, survival and sustained division of the protoplasts are briefly discussed.Abbreviations BAP benzylaminopurine - CPW cell and protoplast washing solution - 2,4-D dichlorophenoxyacetic acid - GA3 gibberellic acid - IAA indole-3-acetic acid - MES 2-(N-morpholino) ethanesulfonic acid - NAA 1-naphthaleneacetic acid - PVP polyvinylpyrrolidone - BT agarose bead type culture - DT agarose disc type culture     

The fine structure of aleurone cells in the soybean seed coat

Summary The morphology and fine structure of aleurone cells of soybean [Glycine max (L.) Merr.] seed coats were analyzed with transmission electron microscopy for the period of rapid seed fill up to physiological maturity. Thin sections and freeze-fracture replicas were prepared for each stage. The aleurone is a tissue lining the embryo sac and consists of a single layer of cells attached to the aerenchyma of the seed coat proper. During seed fill, aleurone cells contained numerous Golgi-derived vesicles in the basal region of the cytoplasm that were either free or attached to the plasma membrane along the lateral and basal regions of the cell wall. Correspondingly, the Golgi apparatus were well developed with individual dictyosomes having 5 to 8, highly fenestrated stacked cisternae. The degree of fenestration along the periphery of each cisterna increased from the cis to trans region. Rough endoplasmic reticulum (RER) was also abundant, often consisting of up to 30, stacked swollen cisternae which occupied large regions of cytoplasm. Plasmodesmata which connected adjacent aleurone cells was not observed along the dorsal walls of aleurone cells that faced aerenchyma. At physiological maturity, dictyosome cisternae were less fenestrated and had fewer associated secretory vesicles. Stacked lamellae of RER were absent, being replaced by short tubular cisternae and small vesicles. At physiological maturity, the aleurone cells had thick walls, and contained numerous lipid bodies in apposition to the plasma membrane. The cytoplasm appeared densely stained in thin-sections and contained protein bodies and amyloplasts with large starch grains. We conclude that during the period of rapid seed fill aleurone cells produce, package, transport and secrete vesicular contents toward the embryo, that is followed at physiological maturity by the storage of lipid, protein and starch in the same cells. The embryo is the most likely destination for secretory products during the period of rapid seed fill. The fate of the stored food reserves in aleurone cells at physiological maturity may be analogous to that of aleurone tissue of grasses, being utilized during imbibition for processes important to germination.     

Effects of ambient UV-B radiation on soybean crops: Impact on leaf herbivory by Anticarsia gemmatalis

Replicated field experiments with large plastic filters were carried outin Buenos Aires (Argentina, 34° S) to study the impacts of current levelsofsolar UV-B radiation ( 315 ) on soybean(Glycine max L.) crops and their interactions with chewinginsects, in particular the soybean worm Anticarsiagemmatalis Hübner (Lepidoptera: Noctuidae). Solar(near-ambient)UV-B induced changes in the leaves that reduced their attractiveness toA. gemmatalis larvae in laboratory choicebioassays. When the A. gemmatalis larvae were forced toconsume leaves from field plots that received solar UV-B, they grew slightlyless rapidly and suffered more mortality than their counterparts fed withleavesfrom plots covered with polyester films that excluded the UV-B component ofsunlight. Exposure of the larvae themselves to ambient UV-B under a soybeancanopy during the feeding trials did not lower their life expectancy. At thewhole canopy level, we found that solar UV-B exclusion resulted in a two-foldincrease in the number of leaf lesions inflicted by various species of chewinginsects that naturally invaded the field plots. Leaves from canopies exposed tosolar UV-B showed significantly higher levels of soluble phenolics and lowerlevels of lignin than leaves that developed in canopies covered by polyesterfilms. No differences in specific leaf mass, leaf nitrogen or hemicellulosecontent were detected between the control and the solar-UV-B exclusiontreatments. Our results are consistent with the idea that present-day solar UV-B has an important regulatory influence on the interactions between plants and phytophagous insects.     

Actin-filament-dependent remodeling of the vacuole in cultured mesophyll protoplasts

Summary. The ability of plant cells to dedifferentiate represents an important survival strategy invoked in a range of situations from repair mechanisms following wounding to apomixis. Dedifferentiation requires that somatic cells reprogram and enter the cell division cycle. This in turn necessitates the accurate partitioning of nuclear content and organelles, such as chloroplasts, to daughter cells, thereby ensuring continuity of cellular information systems. The distribution of cytoplasm and its organelle content in mature plant cells is governed by a large, central vacuole, with connections between distant cortical and perinuclear cytoplasmic domains mediated by transvacuolar strands. Here we examined the changes to vacuolar architecture in Arabidopsis thaliana protoplasts expressing a green-fluorescent protein fusion to a δ-tonoplast-intrinsic protein (δTIP). We found that vacuolar architecture became increasingly intricate during protoplast culture with the development of numerous transvacuolar strands. The development of an intricate vacuolar architecture was an actin filament- and not microtubule-dependent process, as is the case in interphase plant cells. Furthermore, we show that myosin is required for this increased complexity of vacuolar architecture and the formation of subcortical actin filament arrays. Despite the likelihood that increased vacuolar invagination would allow better redistribution of cytoplasmic organelles, we found that repositioning of chloroplasts from cortical to perinuclear cytoplasm was not dependent on transvacuolar strands. Our findings indicate that the vacuole is a dynamic entity that develops a complex architecture before dedifferentiating plant cells enter cell division. Supplementary material to this paper is available in electronic form at Correspondence and reprints: School of Environmental and Life Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.     

Correlations among leaf CO2-exchange rates,areas and enzyme activities among soybean cultivars

Soybean (Glycine max (L.) Merr.) genotypes varying in area per nodal unit (usually a trifoliolate) and maturity class were grown in plots at the University of Illinois experimental farm. Leaf CO₂-exchange rates per unit area (CER) were measured under sunlight on intact plants. In addition to previously reported correlations with specific leaf weight and chlorophyll, CER was positively correlated with ribulose bisphosphate carboxylase (RuBPcase) activity, specific activity, and soluble protein, and was negatively correlated with area per leaf unit. The CER: chlorophyll correlation was destroyed by high CER values in 2 chlorophyll-deficient lines. CER values for 27 of the 35 lines tested fell within the range of those for isolines of cultivar Clark varying in leaf characteristics. The CER values were highest for fully expanded leaves during rapid pod fill. These results suggested that photoperiod (maturity) genes and genes for leaf area growth interact with genes controlling photosynthetic CO₂-exchange to produce the major differences in CER values among soybean genotypes.     

Fluorescentin situ hybridization to soybean metaphase chromosomes

Repetitive DNA sequences were detected directly on somatic metaphase chromosome spreads from soybean root tips using fluorescentin situ hybridization. Methods to spread the forty small metaphase chromosomes substantially free of cellular material were developed using protoplasts. The specific DNA probe was a 1.05 kb internal fragment of a soybean gene encoding the 18S ribosomal RNA subunit. Two methods of incorporating biotin residues into the probe were compared and detection was accomplished with fluorescein-labeled avidin. The rDNA probe exhibits distinct yellow fluorescent signals on only two of the forty metaphase chromosomes that have been counterstained with propidium iodide. This result agrees with our previous analyses of soybean pachytene chromosome [27] showing that only chromosome 13 is closely associated with the nucleolus organizer region. Fluorescentin situ hybridization with the rDNA probe was detected on three of the forty-one metaphase chromosomes in plants that are trisomic for chromosome 13.     

Heterogeneity of storage proteins in maize

The extensive charge heterogeneity of maize (Zea mays L.) zeins observed in isoelectric focusing (IEF) (about 15 bands with pI's in the pH range 6–9) has been found to be independent of extraction procedures or of endosperm development. Zeins do not stain for glycoproteins and exhibit only one lipoprotein component, with pI 3, representing 3–5% of the total protein.Zeins are very resistant to in vitro deamidation, at both acidic and alkaline pH, at high temperatures, and for rather prolonged times. On the basis of the zein content in acidic and basic amino acids, and of the respective pI's exhibited in IEF (mostly in the pH range 7–8) it has been calculated that at least 90% of the glutamic and aspartic acids (52 residues out of a total of 190) are present as asparagine and glutamine.Amino acid analysis of zein fractions isolated by preparative IEF has demonstrated changes in the composition of 18 amino acid residues. However, since these changes affect only neutral and hydrophobic residues, it is concluded that the observed zein heterogeneity is partly based on in vivo deamidation of glutamine and asparagine and partly to spot mutations in some of the genes responsible for zein synthesis.Abbreviations A absorbance - Bis N,N-methylene bisacrylamide - IEF isoelectric focusing - 2-ME 2-meroaptoethanol - mol wt molecular weight - 62 opaque-2 - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - PAS periodic acid-Schiff stain - SDS sodium dodecyl sulphate - ICA trichloroacetic acid - TEMED N,N,N,N-tetramethyl ethylene diamine - Z₁ zein extracted with 55% isopropanol - Z₂ zein extracted with 55% isopropanol and 0.6% 2-ME - Z 9.6 zein of mol wt 9600 - Z 13.5 zein of mol wt 13,500 - Z 21 zein of mol wt 21,000 - Z 23 zein of mol wt 23,000     

Separation and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry

To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. Fifty-three of these protein spots were identified by searching NCBInr and SwissProt databases using the Mascot search engine. Sixty-seven spots that were not identified by MALDI-TOF-MS analysis were analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS), and 66 of these spots were identified by searching against the NCBInr, SwissProt and expressed sequence tag (EST) databases. We have identified a total of 71 unique proteins. The majority of the identified leaf proteins are involved in energy metabolism. The results indicate that 2D-PAGE, combined with MALDI-TOF-MS and LC-MS/MS, is a sensitive and powerful technique for separation and identification of soybean leaf proteins. A summary of the identified proteins and their putative functions is discussed.