Comparative biochemical and morphological changes in imbibed cotton seed hypocotyls and radicles in situ and in vitro – Protein breakdown and elongation growth

1995 ◽  
Vol 5 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Eugene L. Vigil ◽  
Tung K. Fang
Keyword(s):  
Matrix Protein ◽  
Storage Proteins ◽  
Morphological Changes ◽  
Fold Increase ◽  
Hypocotyl Length ◽  
Cortical Cells ◽  
Protein Storage Vacuoles ◽  
Sds Page ◽  
Cotton Seeds

AbstractAxes, hypocotyls and radicles excised from dry cotton seeds (Gossypium hirsutum L. cv. M-8, a double haploid) were imbibed for 24 h and compared with axial segments (excised sections of embryos below the cotyledons) of imbibed, intact seeds. Radicles of excised axes had a 7.4-fold increase in length compared with only 5.2- and 5.7-fold increases, respectively, in radicles of intact seeds and in those isolated when dry. Change in hypocotyl length was not as extensive. EM data for hypocotyl and radicle cortical cells from dry and imbibed seeds revealed a major reduction in matrix protein in protein storage vacuoles along with significant organelle development at 24 h from the start of imbibition. This occurred in parallel with a reduction in salt-extracted proteins and an increase in 2% SDS-extractable proteins. SDS-PAGE of protein from low (0.2 M NaCI) and high (1.0 M NaCI) salt extracts showed a reduction in amount of the major storage proteins (53 and 48 kDa), these bands being almost totally absent in gels of protein extracts from imbibed radicles and significantly reduced in hypocotyls, within 24 h from the start of imbibition. These results indicate that initial elongation of hypocotyls and radicles in intact seeds or of excised axes, after 24 h imbibition, involves breakdown of storage proteins in these axial parts to supply nutrients for growth, with very limited contribution from the cotyledons.

Comparative cytological and biochemical analysis of protein storage vacuoles from cotyledons and radicles of cotton seeds

1996 ◽  
Vol 6 (1) ◽  
pp. 31-37 ◽  
Author(s):  
E.L. Vigil ◽  
A.L. Fleming ◽  
T. Fang ◽  
N. Chaney ◽  
W. P. Wergin
Keyword(s):  
Mineral Composition ◽  
Storage Proteins ◽  
Radicle Growth ◽  
Protein Storage ◽  
Protein Storage Vacuoles ◽  
Sds Page ◽  
Lower Molecular Mass ◽  
Elemental Analyses ◽  
Cotton Seeds ◽  
Protein Complement

AbstractProtein storage vacuoles (PSVs) from radicles and cotyledons of dry cotton seeds were isolated by differential centrifugation following homogenization in glycerol. Protein complement analysis of isolated PSVs with one dimensional SDS-PAGE gels revealed similar major storage proteins, viz. 53 and 48 kDa, with differences in lower molecular mass proteins. Radicle PSVs have apparently more 35-kDa and less 22-kDa storage protein than do cotyledon PSVs. The mineral composition of whole radicles, cotyledons and isolated PSVs from radicles and cotyledons was determined by atomic absorption spectroscopy and colorimetric elemental analyses. The concentration of calcium (Ca), magnesium (Mg), potassium (K) and phosphate (P) was lower in isolated PSVs from radicles than from cotyledons, resulting in a marked difference in the Mg/Ca and (Mg+Ca)/K ratios in PSVs from these two sources. Analysis of radicle and cotyledon tissue from dry seeds for mineral distribution with EDX and scanning electron microscopy revealed major concentrations of Mg, K and P in PSVs. These observations indicate that PSVs in radicles are similar in protein and mineral composition to PSVs in cotyledons. PSVs in radicles have the potential function as storage organelles to provide minerals and nutrients for radicle growth during imbibition and germination.

Missing pieces in protein deposition and mobilization inside legume seed storage vacuoles: calcium and magnesium ions

2012 ◽  
Vol 22 (4) ◽  
pp. 249-258 ◽  
Author(s):  
Cláudia N. Santos ◽  
Marta M. Alves ◽  
Isabel T. Bento ◽  
Ricardo B. Ferreira
Keyword(s):  
Alkaline Earth ◽  
Storage Proteins ◽  
Lupinus Albus ◽  
Physiological Role ◽  
Experimental Conditions ◽  
Protein Storage Vacuoles ◽  
Aggregation And Disaggregation ◽  
Alkaline Earth Cations

AbstractDuring the maturation of dicotyledonous seeds, organic carbon, nitrogen and sulphur are stored in protein storage vacuoles (PSVs) as storage globulins. Several studies point to the coexistence of storage proteins with proteases responsible for their degradation inside PSVs. Different mechanisms have been proposed to explain why there is no proteolysis during this period. Protein aggregation to form large supramolecular structures resistant to proteolytic attack could be the reason. However, during germination, and particularly following its completion, the globulin aggregates must undergo disintegration to allow protease attack for protein reserve mobilization. Based on the well-described concentration-dependent ability of Ca2+ and Mg2+ to promote in vitro aggregation and disaggregation of globulins, we explored a possible role for these alkaline earth cations in globulin packaging and mobilization. Ca2+ and Mg2+ measurements in purified PSVs [6.37 μmol and 43.9 μmol g− 1 dry weight (DW) of cotyledons, respectively] showed the presence of these two alkaline earth cations within this compartment. To our knowledge, this is the first time that Ca2+ and Mg2+ have been quantified in purified PSVs from Lupinus albus seeds. Considering the importance of these two alkaline earth cations inside PSVs, which represent 14.6% and 60.7% of the total seed Mg2+and Ca2+, respectively, globulin aggregation and disaggregation profiles were assayed using experimental conditions closer to those that are physiologically present (proportion of Ca2+ and Mg2+, and acidic pH). Based on: (1) the high in vivo abundance of Ca2+ and Mg2+ inside PSVs; and (2) globulin aggregation and disaggregation profiles, together with structural and physiological evidence already reported in the literature, an important physiological role for Ca2+ and Mg2+ in globulin packaging and mobilization inside PSVs is suggested.

scholarly journals Dimethylsulfoxide exposure modulates HL-60 cell rolling interactions

2012 ◽  
Vol 32 (4) ◽  
pp. 375-382 ◽  
Author(s):  
David J. Gee ◽  
L. Kate Wright ◽  
Jonathan Zimmermann ◽  
Kayla Cole ◽  
Karen Soule ◽  
...  
Keyword(s):  
Shear Stress ◽  
Cell Surface ◽  
Morphological Changes ◽  
Flow Cytometric Analysis ◽  
Fold Increase ◽  
Flow Chamber ◽  
Granulocytic Differentiation ◽  
Rolling Velocity

Human leukaemic HL-60 cells are widely used for studying interactions involving adhesion molecules [e.g. P-selectin and PSGL-1 (P-selectin glycoprotein ligand-1)] since their rolling behaviour has been shown to mimic the dynamics of leucocyte rolling in vitro. HL-60 cells are neutrophilic promyelocytes that can undergo granulocytic differentiation upon exposure to compounds such as DMSO (dimethylsulfoxide). Using a parallel plate flow chamber functionalized with recombinant P-selectin–Fc chimaera, undifferentiated and DMSO-induced (48, 72 and 96 h) HL-60 cells were assayed for rolling behaviour. We found that depending on P-selectin incubation concentration, undifferentiated cells incurred up to a 6-fold increase in rolling velocity while subjected to an approximately 10-fold increase in biologically relevant shear stress. HL-60 cells exposed to DMSO for up to 72 h incurred up to a 3-fold increase in rolling velocity over the same shear stress range. Significantly, cells exposed for up to 96 h incurred up to a 9-fold decrease in rolling velocity, compared with undifferentiated HL-60 cells. Although cell surface and nuclear morphological changes were evident upon exposure to DMSO, flow cytometric analysis revealed that PSGL-1 expression was unchanged, irrespective of treatment duration. The results suggest that DMSO-treated HL-60 cells may be problematic as a substitute for neutrophils for trafficking studies during advanced stages of the LAC (leucocyte adhesion cascade). We suggest that remodelling of the cell surface during differentiation may affect rolling behaviour and that DMSO-treated HL-60 cells would behave differently from the normal leucocytes during inflammatory response in vivo.

scholarly journals Structural insights into how vacuolar sorting receptors recognize the sorting determinants of seed storage proteins

2022 ◽  
Vol 119 (1) ◽  
pp. e2111281119
Author(s):  
Hsi-En Tsao ◽  
Shu Nga Lui ◽  
Anthony Hiu-Fung Lo ◽  
Shuai Chen ◽  
Hiu Yan Wong ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Storage Proteins ◽  
Salt Bridges ◽  
Protein Storage ◽  
Protein Storage Vacuoles ◽  
C Terminus ◽  
Vacuolar Sorting ◽  
Structural Insights ◽  
Cargo Binding

In Arabidopsis, vacuolar sorting receptor isoform 1 (VSR1) sorts 12S globulins to the protein storage vacuoles during seed development. Vacuolar sorting is mediated by specific protein–protein interactions between VSR1 and the vacuolar sorting determinant located at the C terminus (ctVSD) on the cargo proteins. Here, we determined the crystal structure of the protease-associated domain of VSR1 (VSR1-PA) in complex with the C-terminal pentapeptide (468RVAAA472) of cruciferin 1, an isoform of 12S globulins. The 468RVA470 motif forms a parallel β-sheet with the switch III residues (127TMD129) of VSR1-PA, and the 471AA472 motif docks to a cradle formed by the cargo-binding loop (95RGDCYF100), making a hydrophobic interaction with Tyr99. The C-terminal carboxyl group of the ctVSD is recognized by forming salt bridges with Arg95. The C-terminal sequences of cruciferin 1 and vicilin-like storage protein 22 were sufficient to redirect the secretory red fluorescent protein (spRFP) to the vacuoles in Arabidopsis protoplasts. Adding a proline residue to the C terminus of the ctVSD and R95M substitution of VSR1 disrupted receptor–cargo interactions in vitro and led to increased secretion of spRFP in Arabidopsis protoplasts. How VSR1-PA recognizes ctVSDs of other storage proteins was modeled. The last three residues of ctVSD prefer hydrophobic residues because they form a hydrophobic cluster with Tyr99 of VSR1-PA. Due to charge–charge interactions, conserved acidic residues, Asp129 and Glu132, around the cargo-binding site should prefer basic residues over acidic ones in the ctVSD. The structural insights gained may be useful in targeting recombinant proteins to the protein storage vacuoles in seeds.

Protease activities and elongation growth of excised cotton seed axes during the first 24 hours of imbibition

1995 ◽  
Vol 5 (4) ◽  
pp. 201-207 ◽  
Author(s):  
Eugene L. Vigil ◽  
Tung K. Fang
Keyword(s):  
De Novo ◽  
Storage Proteins ◽  
Dry Weight ◽  
Major Axis ◽  
Elongation Growth ◽  
Lag Phase ◽  
Sds Page ◽  
Axis Elongation ◽  
Triphasic Pattern

AbstractExcised axes from cotton (Gossypium hirsutum L. cv. M-8, a double haploid) were imbibed in vitro for 3,6,9,12,18 and 24 h. Data for length, fresh and dry weight and percentage water showed that excised axes undergo a triphasic pattern of growth: an initial burst, a short lag phase and then protracted rapid growth. Analysis of protein content with SDS-PAGE, and of activities of amino-, carboxy- and endopeptidases provided data indicating a direct correlation between major axis elongation growth between 12 and 24 h and enzymatic breakdown of storage proteins (especially those of 48 kDa) by carboxy- and endopeptidase. Data for axes imbibed at 30°C and 0–5°C for 12, 18 and 24 h indicated that a reduction in length and percentage moisture in the cold occurred in parallel with reductions in carboxy- and endopeptidase activities but not in ami-nopeptidase activity. The tentative conclusion is that carboxy- and endopeptidase are probably synthesized de novo in the axis during imbibition and are important in initial breakdown of storage proteins for axial elongation growth.

scholarly journals Wetting of phase-separated droplets on plant vacuole membranes leads to a competition between tonoplast budding and nanotube formation

2021 ◽  
Vol 118 (36) ◽  
pp. e2024109118
Author(s):  
Halim Kusumaatmaja ◽  
Alexander I. May ◽  
Mistianne Feeney ◽  
Joseph F. McKenna ◽  
Noboru Mizushima ◽  
...  
Keyword(s):  
Storage Proteins ◽  
Spontaneous Curvature ◽  
Liquid Droplets ◽  
Protein Storage Vacuoles ◽  
Plant Vacuole ◽  
Dicotyledonous Plants ◽  
Membrane Budding ◽  
Membrane Wetting

Seeds of dicotyledonous plants store proteins in dedicated membrane-bounded organelles called protein storage vacuoles (PSVs). Formed during seed development through morphological and functional reconfiguration of lytic vacuoles in embryos [M. Feeney et al., Plant Physiol. 177, 241–254 (2018)], PSVs undergo division during the later stages of seed maturation. Here, we study the biophysical mechanism of PSV morphogenesis in vivo, discovering that micrometer-sized liquid droplets containing storage proteins form within the vacuolar lumen through phase separation and wet the tonoplast (vacuolar membrane). We identify distinct tonoplast shapes that arise in response to membrane wetting by droplets and derive a simple theoretical model that conceptualizes these geometries. Conditions of low membrane spontaneous curvature and moderate contact angle (i.e., wettability) favor droplet-induced membrane budding, thereby likely serving to generate multiple, physically separated PSVs in seeds. In contrast, high membrane spontaneous curvature and strong wettability promote an intricate and previously unreported membrane nanotube network that forms at the droplet interface, allowing molecule exchange between droplets and the vacuolar interior. Furthermore, our model predicts that with decreasing wettability, this nanotube structure transitions to a regime with bud and nanotube coexistence, which we confirmed in vitro. As such, we identify intracellular wetting [J. Agudo-Canalejo et al., Nature 591, 142–146 (2021)] as the mechanism underlying PSV morphogenesis and provide evidence suggesting that interconvertible membrane wetting morphologies play a role in the organization of liquid phases in cells.

scholarly journals RNAi-Mutants of Sorghum bicolor (L.) Moench with Improved Digestibility of Seed Storage Proteins

2021 ◽  
Author(s):  
Lev A. Elkonin ◽  
Valery M. Panin ◽  
Odissey A. Kenzhegulov ◽  
Saule Kh. Sarsenova
Keyword(s):  
Rna Interference ◽  
Storage Proteins ◽  
Marker Gene ◽  
Seed Storage ◽  
Seed Storage Proteins ◽  
Genetic Construct ◽  
Rnai Silencing ◽  
Sds Page ◽  
Developing Area

Modification of the composition of grain storage proteins is an intensively developing area of plant biotechnology, which is of particular importance for sorghum – high-yielding drought tolerant crop. Compared to other cereals, the majority of sorghum cultivars and hybrids are characterized by reduced nutritional value that is caused by a low content of essential amino acids in the seed storage proteins (kafirins), and resistance of kafirins to protease digestion. RNA interference (RNAi) by suppressing synthesis of individual kafirin subclasses may be an effective approach to solve this problem. In this chapter, we review published reports on RNAi silencing of the kafirin-encoding genes. In addition, we present new experimental data on phenotypic effects of RNAi-silencing of γ-KAFIRIN-1 gene in sorghum cv. Avans. To obtain RNAi mutants with γ-KAFIRIN-1 gene silencing we used Agrobacterium-mediated genetic transformation. Transgenic kernels had modified endosperm type with reduced vitreous layer and significantly improved in vitro protein digestibility (93% vs. 57%, according to the densitometry of SDS-PAGE patterns). SDS-PAGE of transgenic kernels showed lowered level of kafirins and appearance of globulin proteins, which were not observed in the original cultivar. For the first time, the cases of instability of inserted genetic construct were identified: elimination of ubi1-intron that is a constituent part of the genetic construct for RNAi silencing, or nos-promotor governing expression of the marker gene (bar) (in the RNAi mutants of cv. Zheltozernoe 10). The research findings presented in this chapter provide strong evidence that RNA interference can be used for improvement of the nutritional properties of sorghum grain.

Platelet shape change and protein phosphorylation induced by ADP and thrombin are not sensitive to short periods of microgravity

1993 ◽  
Vol 104 (3) ◽  
pp. 805-810
Author(s):  
D.A. Schmitt ◽  
P. Ohlmann ◽  
C. Gachet ◽  
J.P. Cazenave
Keyword(s):  
Protein Phosphorylation ◽  
Normal Gravity ◽  
Phorbol Esters ◽  
Morphological Changes ◽  
Shape Change ◽  
Animal Cells ◽  
Kinase C ◽  
Sds Page ◽  
Platelet Shape

Recent experiments have shown that the stimulation of animal cells in vitro by direct protein kinase C (PKC) activators is significantly reduced under microgravity (micro g). Platelets undergo protein phosphorylation and morphological changes a few seconds after stimulation by agonists such as phorbol esters which activate PKC. Therefore, taking advantage of parabolic plane flight to obtain short periods of microgravity, we studied phosphorylation of myosin light chain (20K), specific PKC-dependent phosphorylation of a 40,000 M(r) protein, pleckstrin (40K) and platelet shape change. SDS-PAGE analysis and electron microscopy were performed on platelets subjected to 20 seconds microgravity as compared to normal gravity (1 g) conditions. These investigations showed that neither Ca(2+)-calmodulin-mediated activation nor the PKC-dependent pathways are inhibited during short periods of microgravity.

scholarly journals Cladophora glomerata methanolic extract promotes chondrogenic gene expression and cartilage phenotype differentiation in equine adipose-derived mesenchymal stromal stem cells affected by metabolic syndrome

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Lynda Bourebaba ◽  
Izabela Michalak ◽  
Meriem Baouche ◽  
Katarzyna Kucharczyk ◽  
Krzysztof Marycz
Keyword(s):  
Metabolic Syndrome ◽  
Stem Cells ◽  
Matrix Protein ◽  
Cultured Cells ◽  
Methanolic Extract ◽  
Therapeutic Potential ◽  
Morphological Changes ◽  
Cladophora Glomerata ◽  
Stromal Stem Cells

Abstract Background Chondrogenesis represents a highly dynamic cellular process that leads to the establishment of various types of cartilage. However, when stress-related injuries occur, a rapid and efficient regeneration of the tissues is necessary to maintain cartilage integrity. Mesenchymal stem cells (MSCs) are known to exhibit high capacity for self-renewal and pluripotency effects, and thus play a pivotal role in the repair and regeneration of damaged cartilage. On the other hand, the influence of certain pathological conditions such as metabolic disorders on MSCs can seriously impair their regenerative properties and thus reduce their therapeutic potential. Objectives In this investigation, we attempted to improve and potentiate the in vitro chondrogenic ability of adipose-derived mesenchymal stromal stem cells (ASCs) isolated from horses suffering from metabolic syndrome. Methods Cultured cells in chondrogenic-inductive medium supplemented with Cladophora glomerata methanolic extract were experimented for expression of the main genes and microRNAs involved in the differentiation process using RT-PCR, for their morphological changes through confocal and scanning electron microscopy and for their physiological homeostasis. Results The different added concentrations of C. glomerata extract to the basic chondrogenic inductive culture medium promoted the proliferation of equine metabolic syndrome ASCs (ASCsEMS) and resulted in chondrogenic phenotype differentiation and higher mRNA expression of collagen type II, aggrecan, cartilage oligomeric matrix protein, and Sox9 among others. The results reveal an obvious inhibitory effect of hypertrophy and a strong repression of miR-145-5p, miR-146-3p, and miR-34a and miR-449a largely involved in cartilage degradation. Treated cells additionally exhibited significant reduced apoptosis and oxidative stress, as well as promoted viability and mitochondrial potentiation. Conclusion Chondrogenesis in EqASCsEMS was found to be prominent after chondrogenic induction in conditions containing C. glomerata extract, suggesting that the macroalgae could be considered for the enhancement of ASC cultures and their reparative properties.

Correlation Between Cellular Functions and Morphological Changes of Human Trophoblast in Vitro

1975 ◽  
Vol 33 ◽  
pp. 600-601
Author(s):  
John C. Garancis ◽  
Robert O. Hussa ◽  
Michael T. Story ◽  
Donald Yorde ◽  
Roland A. Pattillo
Keyword(s):  
Electron Microscopy ◽  
Cellular Proliferation ◽  
Morphological Changes ◽  
Culture Fluid ◽  
Cellular Functions ◽  
Human Trophoblast ◽  
Transmission Electron ◽  
Marked Activity ◽  
Malignant Trophoblast

Human malignant trophoblast cells in continuous culture were incubated for 3 days in medium containing 1 mM N6-O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) and 1 mM theophylline. The culture fluid was replenished daily. Stimulated cultures secreted many times more chorionic gonadotropin and estrogens than did control cultures in the absence of increased cellular proliferation. Scanning electron microscopy revealed remarkable surface changes of stimulated cells. Control cells (not stimulated) were smooth or provided with varying numbers of microvilli (Fig. 1). The latter, usually, were short and thin. The surface features of stimulated cells were considerably different. There was marked increase of microvilli which appeared elongated and thick. Many cells were covered with confluent polypoid projections (Fig. 2). Transmission electron microscopy demonstrated marked activity of cytoplasmic organelles. Mitochondria were increased in number and size; some giant forms with numerous cristae were observed.

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