scholarly journals How Cysteine Protease Gene PtCP5 Affects Seed Germination by Mobilizing Storage Proteins in Populus trichocarpa

2021 ◽  
Vol 22 (23) ◽  
pp. 12637
Author(s):  
Xiatong Liu ◽  
Lijie Mo ◽  
Xiaorui Guo ◽  
Qiang Zhang ◽  
Hui Li ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Seed Germination ◽  
Cysteine Protease ◽  
Storage Proteins ◽  
Populus Trichocarpa ◽  
Cysteine Proteases ◽  
Protein Accumulation ◽  
Wild Type ◽  
Protein Storage Vacuoles ◽  
In The Wild

In higher plants, seed storage proteins are deposited in protein storage vacuoles (PSVs) and degraded by protease, especially cysteine proteases, as a source of nitrogen for seed germination. In this study, a cathepsin B-like cysteine protease PtCP5, which is important for seed germination and pollen development, was first cloned in Populus trichocarpa. The GUS staining of the ProPtCP5-GUS reporter line showed that PtCP5 is expressed in the roots, stems, leaves, flowers, siliques and seeds of Arabidopsis. We reveal that PtCP5 is present in plasma membrane and co-localizes with the plasma membrane marker REM1.3. Both seed germination and early seedling development are slower in OX-PtCP5 transgenic Arabidopsis when compared with the wild-type. Further analysis revealed that, when stained with toluidine blue, the observed storage protein accumulation was lower in OX-PtCP5 than in the wild-type. Our results also show that the number of abnormal pollen grains is higher and the germination rate of pollen is lower in OX-PtCP5 than in the wild-type. These results indicate that PtCP5 is an important factor in mobilizing storage proteins and that the proper expression of PtCP5 is necessary for both pollen and seed maturation and germination. This study sheds further light on the biological functions of cysteine proteases and provides further reference for seed development research on woody plants.

Cleavage of Arpin by Calpain as a Potential Regulation Mechanism of the Arp2/3 Complex and Cell Motility

2018 ◽  
Author(s):  
Renee Potashner
Keyword(s):  
Plasma Membrane ◽  
Cell Motility ◽  
Cancer Metastasis ◽  
Competitive Inhibition ◽  
Cell Movement ◽  
Personal Communication ◽  
Cysteine Proteases ◽  
Regulation Mechanism ◽  
Wild Type ◽  
In The Wild

Cell movement is mediated by cycles of actin polymerisation. A novel protein, Arpin, was discovered that has been suggested to decrease cell motility through competitive inhibition of WASP family proteins, the activators of the complex driving actin polymerisation. In preliminary studies, Arpin was found to inhibit the Arp2/3 complex (Gautreau and Blanchoin, personal communication). Arpin contains sequence homology to the Arp2/3-binding site of WASP proteins. Calpains, a family of intracellular calcium-dependent cysteine proteases, can be found near the plasma membrane and the concentration of calcium ion required for activation is decreased when calpain is bound to the plasma membrane in the presence of phospholipids (Leloup et al. 2010). The common localization of calpain and Arpin, along with the known contributions calpain has in regulating other cell motility proteins, makes calpain a likely candidate for Arpin regulation. By transfecting calpain wild-type (pz/pz), knockdown (p/p) and lentivirus rescue mouse embryonic fibroblasts with a plasmid containing the Arpin gene (c15orf38), I hope to show the presence of differential cleavage of Arpin by calpain in the wild-type cells compared to the calpain knock-down cells through Western blot analysis. Understanding how Arpin is regulated will provide a basis for further research in cell motility, which has contributions to cancer metastasis and other diseases.  

Modulation of frequency and height of cytosolic calcium spikes by plasma membrane anion channels in guard cells

2021 ◽  
Author(s):  
Md Tahjib-Ul-Arif ◽  
Shintaro Munemasa ◽  
Toshiyuki Nakamura ◽  
Yoshimasa Nakamura ◽  
Yoshiyuki Murata
Keyword(s):  
Plasma Membrane ◽  
Stomatal Closure ◽  
Anion Channel ◽  
Guard Cells ◽  
Cytosolic Calcium ◽  
Peak Height ◽  
Extracellular Calcium ◽  
Wild Type ◽  
Anion Channels ◽  
In The Wild

Abstract Cytosolic calcium ([Ca2+]cyt) elevation activates plasma membrane anion channels in guard cells, which is required for stomatal closure. However, involvement of the anion channels in the [Ca2+]cyt elevation remains unclear. We investigated the involvement using Arabidopsis thaliana anion channel mutants, slac1-4 slah3-3 and slac1-4 almt12-1. Extracellular calcium induced stomatal closure in the wild-type plants but not in the anion channel mutant plants whereas extracellular calcium induced [Ca2+]cyt elevation both in the wild-type guard cells and in the mutant guard cells. The peak height and the number of the [Ca2+]cyt spike were lower and larger in the slac1-4 slah3-3 than in the wild-type and the height and the number in the slac1-4 almt12-1 were much lower and much larger than in the wild-type. These results suggest that the anion channels are involved in the regulation of [Ca2+]cyt elevation in guard cells.

scholarly journals Identification and expression analysis of a novel phytocystatin in developing and germinating seeds of triticale (×Triticosecale Wittm.)

2015 ◽  
Vol 84 (1) ◽  
pp. 139-142 ◽  
Author(s):  
Joanna Simińska ◽  
Wiesław Bielawski
Keyword(s):  
Seed Germination ◽  
Seed Development ◽  
Storage Protein ◽  
Cdna Sequence ◽  
Storage Proteins ◽  
Cysteine Proteinase ◽  
Protein Accumulation ◽  
Protein Levels ◽  
Germinating Seeds ◽  
Cysteine Proteinase Activity

In this paper the complete cDNA sequence of a newly identified triticale phytocystatin, TrcC-7, was analyzed. Because <em>TrcC-7</em> transcripts were present in seeds, we hypothesized that it may regulate storage protein accumulation and degradation. Therefore, changes in mRNA and protein levels during the entire period of seed development and germination were examined. Expression of <em>TrcC-7</em> increased during development and decreased at the end of maturation and subsequently increased during seed germination. Based on these results, TrcC-7 likely regulates cysteine proteinase activity during the accumulation and mobilization of storage proteins.

scholarly journals Exomer Is Part of a Hub Where Polarized Secretion and Ionic Stress Connect

2021 ◽  
Vol 12 ◽  
Author(s):  
Sandra Moro ◽  
Esteban Moscoso-Romero ◽  
Abhishek Poddar ◽  
Jose M. Mulet ◽  
Pilar Perez ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transient Receptor Potential ◽  
Ion Homeostasis ◽  
Wild Type ◽  
Plasma Membrane Atpase ◽  
General Role ◽  
Membrane Hyperpolarization ◽  
Polarized Secretion ◽  
In The Wild ◽  
Golgi Network

Plasma membrane and membranous organelles contribute to the physiology of the Eukaryotic cell by participating in vesicle trafficking and the maintenance of ion homeostasis. Exomer is a protein complex that facilitates vesicle transport from the trans-Golgi network to the plasma membrane, and its absence leads to the retention of a set of selected cargoes in this organelle. However, this retention does not explain all phenotypes observed in exomer mutants. The Schizosaccharomyces pombe exomer is composed of Cfr1 and Bch1, and cfr1Δ and bch1Δ were sensitive to high concentrations of potassium salts but not sorbitol, which showed sensitivity to ionic but not osmotic stress. Additionally, the activity of the plasma membrane ATPase was higher in exomer mutants than in the wild-type, pointing to membrane hyperpolarization, which caused an increase in intracellular K+ content and mild sensitivity to Na+, Ca2+, and the aminoglycoside antibiotic hygromycin B. Moreover, in response to K+ shock, the intracellular Ca2+ level of cfr1Δ cells increased significantly more than in the wild-type, likely due to the larger Ca2+ spikes in the mutant. Microscopy analyses showed a defective endosomal morphology in the mutants. This was accompanied by an increase in the intracellular pools of the K+ exporting P-type ATPase Cta3 and the plasma membrane Transient Receptor Potential (TRP)-like Ca2+ channel Pkd2, which were partially diverted from the trans-Golgi network to the prevacuolar endosome. Despite this, most Cta3 and Pkd2 were delivered to the plasma membrane at the cell growing sites, showing that their transport from the trans-Golgi network to the cell surface occurred in the absence of exomer. Nevertheless, shortly after gene expression in the presence of KCl, the polarized distribution of Cta3 and Pkd2 in the plasma membrane was disturbed in the mutants. Finally, the use of fluorescent probes suggested that the distribution and dynamics of association of some lipids to the plasma membrane in the presence of KCl were altered in the mutants. Thus, exomer participation in the response to K+ stress was multifaceted. These results supported the notion that exomer plays a general role in protein sorting at the trans-Golgi network and in polarized secretion, which is not always related to a function as a selective cargo adaptor.

scholarly journals Transcriptional Response of Leptospira interrogans to Iron Limitation and Characterization of a PerR Homolog

2010 ◽  
Vol 78 (11) ◽  
pp. 4850-4859 ◽  
Author(s):  
Miranda Lo ◽  
Gerald L. Murray ◽  
Chen Ai Khoo ◽  
David A. Haake ◽  
Richard L. Zuerner ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Iron Homeostasis ◽  
Storage Proteins ◽  
Iron Acquisition ◽  
Bacterial Species ◽  
Transcriptional Response ◽  
Oxidative Stress Response ◽  
Wild Type ◽  
In The Wild

ABSTRACT Leptospirosis is a globally significant zoonosis caused by Leptospira spp. Iron is essential for growth of most bacterial species. Since iron availability is low in the host, pathogens have evolved complex iron acquisition mechanisms to survive and establish infection. In many bacteria, expression of iron uptake and storage proteins is regulated by Fur. L. interrogans encodes four predicted Fur homologs; we have constructed a mutation in one of these, la1857. We conducted microarray analysis to identify iron-responsive genes and to study the effects of la1857 mutation on gene expression. Under iron-limiting conditions, 43 genes were upregulated and 49 genes were downregulated in the wild type. Genes encoding proteins with predicted involvement in inorganic ion transport and metabolism (including TonB-dependent proteins and outer membrane transport proteins) were overrepresented in the upregulated list, while 54% of differentially expressed genes had no known function. There were 16 upregulated genes of unknown function which are absent from the saprophyte L. biflexa and which therefore may encode virulence-associated factors. Expression of iron-responsive genes was not significantly affected by mutagenesis of la1857, indicating that LA1857 is not a global regulator of iron homeostasis. Upregulation of heme biosynthetic genes and a putative catalase in the mutant suggested that LA1857 is more similar to PerR, a regulator of the oxidative stress response. Indeed, the la1857 mutant was more resistant to peroxide stress than the wild type. Our results provide insights into the role of iron in leptospiral metabolism and regulation of the oxidative stress response, including genes likely to be important for virulence.

scholarly journals Phosphorylation of UT-A1 on serine 486 correlates with membrane accumulation and urea transport activity in both rat IMCDs and cultured cells

2010 ◽  
Vol 298 (4) ◽  
pp. F935-F940 ◽  
Author(s):  
Janet D. Klein ◽  
Mitsi A. Blount ◽  
Otto Fröhlich ◽  
Chad E. Denson ◽  
Xiaoxiao Tan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Cell Lines ◽  
Specific Antibody ◽  
Cultured Cells ◽  
Apical Plasma Membrane ◽  
Wild Type ◽  
Amino Acid Peptide ◽  
In The Wild

Vasopressin is the primary hormone regulating urine-concentrating ability. Vasopressin phosphorylates the UT-A1 urea transporter in rat inner medullary collecting ducts (IMCDs). To assess the effect of UT-A1 phosphorylation at S486, we developed a phospho-specific antibody to S486-UT-A1 using an 11 amino acid peptide antigen starting from amino acid 482 that bracketed S486 in roughly the center of the sequence. We also developed two stably transfected mIMCD3 cell lines: one expressing wild-type UT-A1 and one expressing a mutated form of UT-A1, S486A/S499A, that is unresponsive to protein kinase A. Forskolin stimulates urea flux in the wild-type UT-A1-mIMCD3 cells but not in the S486A/S499A-UT-A1-mIMCD3 cells. The phospho-S486-UT-A1 antibody identified UT-A1 protein in the wild-type UT-A1-mIMCD3 cells but not in the S486A/S499A-UT-A1-mIMCD3 cells. In rat IMCDs, forskolin increased the abundance of phospho-S486-UT-A1 (measured using the phospho-S486 antibody) and of total UT-A1 phosphorylation (measured by 32P incorporation). Forskolin also increased the plasma membrane accumulation of phospho-S486-UT-A1 in rat IMCD suspensions, as measured by biotinylation. In rats treated with vasopressin in vivo, the majority of the phospho-S486-UT-A1 appears in the apical plasma membrane. In summary, we developed stably transfected mIMCD3 cell lines expressing UT-A1 and an S486-UT-A1 phospho-specific antibody. We confirmed that vasopressin increases UT-A1 accumulation in the apical plasma membrane and showed that vasopressin phosphorylates UT-A1 at S486 in rat IMCDs and that the S486-phospho-UT-A1 form is primarily detected in the apical plasma membrane.

scholarly journals Role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1

2000 ◽  
Vol 350 (3) ◽  
pp. 677-683 ◽  
Author(s):  
Ana M. PAJOR ◽  
Esther S. KAHN ◽  
Rama GANGULA
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Xenopus Oocytes ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Conserved Residues ◽  
In The Wild ◽  
Cationic Amino Acids

The role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1 was investigated by site-directed mutagenesis and subsequent expression of mutant transporters in Xenopus oocytes. Of the ten residues chosen for mutagenesis, eight (Lys-34, Lys-107, Arg-108, Lys-333, Lys-390, Arg-368, Lys-414 and Arg-541) were found to be non-essential for function or targeting. Only two conserved residues, Lys-84 (at the cytoplasmic end of helix 3) and Arg-349 (at the extracellular end of helix 7), were found to be important for transport. Both mutant transporters were expressed at the plasma membrane. The mutation of Lys-84 to Ala resulted in an increased Km for succinate of 1.8mM, compared with 0.3mM in the wild-type NaDC-1. The R349A mutant had Na+ and citrate kinetics that were similar to those of the wild type. However, succinate handling in the R349A mutant was altered, with evidence of inhibition at high succinate concentrations. In conclusion, charge neutralization of Lys-84 and Arg-349 in NaDC-1 affects succinate handling, suggesting that these residues might have roles in substrate binding.

Calcium release from Synechocystis cells induced by depolarization of the plasma membrane: MscL as an outward Ca2+ channel

Microbiology ◽  
2003 ◽  
Vol 149 (5) ◽  
pp. 1147-1153 ◽  
Author(s):  
Lyudmila V. Nazarenko ◽  
Igor M. Andreev ◽  
Alexander A. Lyukevich ◽  
Tatiana V. Pisareva ◽  
Dmitry A. Los
Keyword(s):  
Plasma Membrane ◽  
Calcium Release ◽  
Channel Blocker ◽  
Membrane Depolarization ◽  
Wild Type ◽  
Exact Function ◽  
Intracellular Ca ◽  
In The Wild ◽  
Mechanosensitive Ion Channel ◽  
Mutant Cells

Cells of the cyanobacterium Synechocystis sp. PCC 6803 are equipped with a mechanosensitive ion channel MscL that is located in their plasma membrane. However, the exact function of the channel in this freshwater cyanobacterium is unknown. This study shows that cells of Synechocystis are capable of releasing Ca2+ in response to depolarization of the plasma membrane by the K+ ionophore valinomycin in the presence of K+ or by tetraphenylphosphonium (TPP+). A fluorescent dye, diS-C3-(5), sensitive to membrane potential and the metallochromic Ca2+ indicator arsenazo III were used to follow the plasma membrane depolarization and the Ca2+ release, respectively. The Ca2+ release from wild-type cells was temperature-dependent and it was strongly inhibited by the Ca2+ channel blocker verapamil and by the mechanosensitive channel blocker amiloride. In MscL-deficient cells, Ca2+ release was about 50 % of that from the wild-type cells. The mutant cells had lost temperature sensitivity of Ca2+ release completely. However, verapamil and amiloride inhibited Ca2+ release from these cells in same manner as in the wild-type cells. This suggests the existence of additional Ca2+ transporters in Synechocystis, probably of a mechanosensitive nature. Evidence for the putative presence of intracellular Ca2+ stores in the cells was obtained by following the increase in fluorescence intensity of the Ca2+ indicator chlortetracycline. These results suggest that the MscL of Synechocystis might operate as a verapamil/amiloride-sensitive outward Ca2+ channel that is involved in the plasma-membrane depolarization-induced Ca2+ release from the cells under temperature stress conditions.

The Tonoplast-Localized Transporter OsNRAMP2 is involved in Fe Homeostasis and affects Seed Germination in rice

2021 ◽  
Author(s):  
Yun Li ◽  
Jingjun Li ◽  
Yihong Yu ◽  
Xia Dai ◽  
Changyi Gong ◽  
...  
Keyword(s):  
Seed Germination ◽  
Natural Resistance ◽  
Fe Deficiency ◽  
Shoot Biomass ◽  
Wild Type ◽  
Crop Development ◽  
In The Wild ◽  
Macrophage Protein ◽  
Fe Homeostasis ◽  
Fe Toxicity

Abstract Vacuolar storage of iron (Fe) is important for Fe homeostasis in plants. When sufficient, the excess Fe could be stored in vacuoles for remobilization in case of Fe deficiency. Although the mechanism of Fe remobilization from vacuoles is critical for crop development under low Fe stress, the transporters that mediate vacuolar Fe translocation into the cytosol in rice remains unknown. Here, we showed that under higher Fe 2+ concentrations, the Δccc1 yeast mutant transformed with rice natural resistance-associated macrophage protein 2 (OsNRAMP2) became more sensitive to Fe toxicity. In rice protoplasts and transgenic plants expressing Pro35S: OsNRAMP2-GFP, OsNRAMP2 was localized to tonoplast. Vacuolar Fe contents in osnramp2 knockdown lines were higher than in the wild-type, while the growth of osnramp2 knockdown plants was significantly influenced by Fe deficiency. Furthermore, the germination of osnramp2 knockdown plants was arrested. Inversely, the vacuolar Fe contents of Pro35S: OsNRAMP2-GFP lines were significantly lower than in the wild-type, and overexpression of OsNRAMP2 increased shoot biomass under Fe deficiency. Taken together, we propose that OsNRAMP2 transports Fe from the vacuole to the cytosol and plays a pivotal role in seed germination.

Freeze-substituted fungal cells of Nectria haematococca: A comparison of the macroconidial walls of the adhesion-competent wild type with an adhesion-reduced mutant

1990 ◽  
Vol 48 (3) ◽  
pp. 688-689
Author(s):  
Thecan Caesar-Ton That ◽  
Lynn Epstein
Keyword(s):  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Wild Type ◽  
Nectria Haematococca ◽  
Fruit Extract ◽  
Dialysis Tubing ◽  
Tube Emergence ◽  
Contrast Microscopy ◽  
In The Wild ◽  
Freeze Damage

Nectria haematococca mating population I (anamorph, Fusarium solani) macroconidia attach to its host (squash) and non-host surfaces prior to germ tube emergence. The macroconidia become adhesive after a brief period of protein synthesis. Recently, Hickman et al. (1989) isolated N. haematococca adhesion-reduced mutants. Using freeze substitution, we compared the development of the macroconidial wall in the wild type in comparison to one of the mutants, LEI.Macroconidia were harvested at 1C, washed by centrifugation, resuspended in a dilute zucchini fruit extract and incubated from 0 - 5 h. During the incubation period, wild type macroconidia attached to uncoated dialysis tubing. Mutant macroconidia did not attach and were collected on poly-L-lysine coated dialysis tubing just prior to freezing. Conidia on the tubing were frozen in liquid propane at 191 - 193C, substituted in acetone with 2% OsO4 and 0.05% uranyl acetate, washed with acetone, and flat-embedded in Epon-Araldite. Using phase contrast microscopy at 1000X, cells without freeze damage were selected, remounted, sectioned and post-stained sequentially with 1% Ba(MnO4)2 2% uranyl acetate and Reynold’s lead citrate. At least 30 cells/treatment were examined.

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