Pathway and control of sucrose import into initiating cotton fibre cells

This paper originates from a presentation at the International Conference on Assimilate Transport and Partitioning, Newcastle, NSW, August 1999 Our aim is to unravel the mechanisms controlling fibre cell initiation from the epidermis of cotton (Gossypium hirsutum L.) ovules. We compared the development of fibres and trichomes in wild type cotton and a fibreless seed (fls) mutant, and determined the cellular pathway of sucrose transport into fibre initials on the day of anthesis. Although fibre initiation is inhibited in the fls mutant, leading to the fibreless phenotype, trichome development in other parts of the plant is normal. Confocal imaging analysis revealed that the fluorescent molecule, 5(6)-carboxyfluorescein, which is transported symplastically, moved readily from the integument phloem into initiating fibres. Plasmolysis studies showed that the fibre initials and adjacent non-initiating ovule epidermal cells have similar osmotic potential. Immunolocalisation analysis showed the absence of sucrose transporter proteins in the initiating fibre, but their abundance in the transfer cell precursors at the innermost integument. These results (i) demonstrate that fibre cell initiation is controlled by unique mechanism(s) that differ from that for normal trichome development; (ii) show a symplastic pathway of sucrose import into initiating fibres and strengthen the current opinion that sucrose synthase is likely to be the key enzyme mobilising sucrose into initiating fibres; and (iii) suggest that the initial protrusion of the fibre cells above the ovule surface is largely achieved by increased cell wall extensibility rather than higher turgor as is commonly thought.

Download Full-text

Overexpression of arginine decarboxylase in transgenic plants

Biochemical Journal ◽

10.1042/bj3250331 ◽

1997 ◽

Vol 325 (2) ◽

pp. 331-337 ◽

Cited By ~ 46

Author(s):

Daniel BURTIN ◽

Anthony J. MICHAEL

Keyword(s):

Transgenic Plants ◽

Arginine Decarboxylase ◽

Polyamine Metabolism ◽

Morphological Development ◽

Polyamine Biosynthesis ◽

Adenosylmethionine Decarboxylase ◽

Two Generations ◽

Key Enzyme ◽

Polyamine Conjugate ◽

And Control

The activity of arginine decarboxylase (ADC), a key enzyme in plant polyamine biosynthesis, was manipulated in two generations of transgenic tobacco plants. Second-generation transgenic plants overexpressing an oat ADC cDNA contained high levels of oat ADC transcript relative to tobacco ADC, possessed elevated ADC enzyme activity and accumulated 10–20-fold more agmatine, the direct product of ADC. In the presence of high levels of the precursor agmatine, no increase in the levels of the polyamines putrescine, spermidine and spermine was detected in the transgenic plants. Similarly, the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were unchanged. No diversion of polyamine metabolism into the hydroxycinnamic acid–polyamine conjugate pool or into the tobacco alkaloid nicotine was detected. Activity of the catabolic enzyme diamine oxidase was the same in transgenic and control plants. The elevated ADC activity and agmatine production were subjected to a metabolic/physical block preventing increased, i.e. deregulated, polyamine accumulation. Overaccumulation of agmatine in the transgenic plants did not affect morphological development.

Download Full-text

Abstract 500: High Basal Ca 2+ / Calmodulin Kinase II Activity Modulates Spontaneous Sarcoplasmic Reticulum Ca 2+ Cycling That Drives Normal Automaticity in Sinoatrial Nodal Cells

Circulation ◽

10.1161/circ.116.suppl_16.ii_86-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Yue Li ◽

Syevda G. Sirenko ◽

Tatiana M. Vinogradova ◽

Alexey E. Lyashkov ◽

Weizhong Zhu ◽

...

Keyword(s):

Sarcoplasmic Reticulum ◽

Surface Membrane ◽

Phosphorylation Site ◽

Major Effect ◽

Physiological Solution ◽

Confocal Imaging ◽

Ventricular Cells ◽

Calmodulin Kinase ◽

Recovery From Inactivation ◽

And Control

The crucial dependence of normal automaticity of sinoatrial nodal cells (SANC) on CaMKII signaling has previously been linked to an effect to facilitate recovery from inactivation of L-type Ca 2+ channels. More recently, however, it has been discovered that spontaneous, rhythmic, local Ca 2+ releases (LCR’s) from sarcoplasmic reticulum (SR) activate Na + /Ca 2+ exchanger current, imparting an exponential increase to the later part of the spontaneous depolarization that brings the surface membrane to threshold to fire an action potential (AP). Here, in single isolated intact SANC, using the phosphorylation site-specific polyclonal antibody, we show (Fig. A ) that, basal state phospholamban (PLB) phosphorylation at Thr-17, a CaMKII phosphorylation site, is over 3 times greater in SANC than in ventricular cells (VC). The CaMKII inhibitor, KN-93, but not its inactive analog, KN-92, markedly inhibits (by 80%) PLB Thr-17 phosphorylation (Fig. B ). Confocal imaging of saponin permeablized SANC, superfused in physiological solution with 150nM free Ca 2+ and 0.5mM EGTA, showed that KN-93 reduced the LCR frequency by 80% (from 5.6 ± 1.5 to 1.1 ± 0.3/1s × 100 μm) and size by 50% (from 4.0 ± 0.3 to 2.0 ± 0.3 μm). Thus, in addition to an effect on L-type Ca 2+ channels, the high basal CaMKII activation (indexed by PLB phosphorylation at Thr-17) has a major effect to determine the characteristics of spontaneous LCR’s that initiate AP’s and control normal automaticity of SANC.

Download Full-text

Source physiology and assimilate transport: the interaction of sucrose metabolism, starch storage and phloem export in source leaves and the effects on sugar status in phloem

Functional Plant Biology ◽

10.1071/pp99127 ◽

2000 ◽

Vol 27 (6) ◽

pp. 497 ◽

Cited By ~ 13

Author(s):

Ewald Komor

Keyword(s):

Genetic Manipulation ◽

Sieve Tube ◽

Phloem Loading ◽

Assimilate Transport ◽

Vascular Bundles ◽

Ambient Conditions ◽

Sucrose Transport ◽

Carbon Export ◽

Export Rate ◽

Long Day

Phloem loading of sucrose is decisive for the speed of mass flow, because sucrose is the dominant solutein the sieve tube sap of nearly all plant species. The export rate of carbon is linearly correlated to the concentration of sucrose in green leaves. Saturation of export was not observed, because surplus of assimilates is converted to starch, a process which is regulated by the sucrose level in the cytosol. Consequently, an increase of sucrose synthesis by overexpression of SPS did not enhance carbon export (at least under normal ambient conditions). Saturation of sucrose export could be observed only in experimental systems, where sucrose was fed directly to the phloem (e.g. in Ricinus seedling) or where constraints on transport activity were imposed by genetic manipulation either on the transporters (e.g. in sucrose transporter antisense plants) or on the path of sucrose (e.g. in plants trans ormed with TMV movement protein, or by incubation in salts). The balance between carbon storage and carbon export is subject to adaptation to meet growth requirements under special circumstances. For example, in a starch-deficient mutant, the day time export rate is nearly doubled compared to wild type plants. Furthermore, plants under short day illumination greatly accelerated starch storage compared to plants under long day illumination (a modulation which persists even a few days after a shift to long day conditions). Plants with a higher assimilation rate due to elevated ambient CO2 increase the nightly carbon export rate, whereas the export rate in day time rate appeared to work at its upper limit. The overall efficiency of sucrose export and incorporation into biomass is ca 0.65, which is close to the theoretical value of 0.75. Sucrose transport along the phloem strands is modulated according to the input at the source, but the individual phloem strands show also partial coordination with respect to sucrose concentrations (as revealed by NMR-imaging), especially obvious after physical interruption of some vascular bundles.

Download Full-text

Confocal imaging analysis of ATP-induced Ca2+ response in individual endothelial cells of the artery in situ

AJP Cell Physiology ◽

10.1152/ajpcell.1997.272.6.c1980 ◽

1997 ◽

Vol 272 (6) ◽

pp. C1980-C1987 ◽

Cited By ~ 60

Author(s):

H. Ohata ◽

Y. Ujike ◽

K. Momose

Keyword(s):

Endothelial Cells ◽

Laser Scanning ◽

Cultured Cells ◽

Laser Scanning Confocal Microscopy ◽

Confocal Imaging ◽

Transient Increase ◽

Similar Response ◽

Imaging Analysis ◽

Acetoxymethyl Ester

The mechanisms for mobilization of intracellular free Ca2+ have been studied in various types of isolated and cultured cells, but little is known about Ca2+ mobilization in individual cells in situ. We tried to establish imaging analysis of intracellular free Ca2+ concentration ([Ca2+]i) in individual cells loaded with the acetoxymethyl ester of fluo 3 in situ, using laser scanning confocal microscopy. The method permitted us to distinguish signals from endothelial and smooth muscle cells of guinea pig artery. Addition of ATP to the artery caused a transient increase in endothelial [Ca2+]i. It was concluded that the response was induced via P2Y purinoceptors, because adenosine 5'-O-(2-thiodiphosphate), but not UTP, caused a similar response independent of extracellular Ca2+. The percentage of cells that responded to ATP (1-10 microM) and the peak amplitude of the transient increase in [Ca2+]i were dose dependently increased. Using rapid xy-scanning and line-scanning modes, we confirmed that 10 microM ATP induced Ca2+ waves, at a rate of 10-30 microns/s, after a lag time of approximately 3 s. These results show that [Ca2+]i waves within endothelial cells are physiologically induced by ATP via P2Y purinoceptor, but not P2U purinoceptor, in aortic strips in situ. The method should be of use in the study of vascular physiology and pathophysiology.

Download Full-text

Vacuolar chain elongation of raffinose oligosaccharides in Ajuga reptans

Functional Plant Biology ◽

10.1071/pp99165 ◽

2000 ◽

Vol 27 (9) ◽

pp. 743 ◽

Cited By ~ 3

Author(s):

Renate Braun ◽

Felix Keller

Keyword(s):

Osmotic Shock ◽

Subcellular Location ◽

Assimilate Transport ◽

Chain Elongation ◽

Raffinose Family Oligosaccharides ◽

Raffinose Oligosaccharides ◽

Ajuga Reptans ◽

Key Enzyme ◽

Autumn And Winter ◽

Long Chain

This paper originates from a presentation at the International Conference on Assimilate Transport and Partitioning, Newcastle, NSW, August 1999 Galactan : galactan galactosyltransferase (GGT) is the key enzyme responsible for the accumulation of long-chain raffinose family oligosaccharides (RFOs; α-D-galn(1,6) α-D-glc(1,2) β-D-fru) in Ajuga reptans L. leaves during autumn and winter. The exact subcellular location of GGT is not known and its elucidation was the aim of this paper. A method for the isolation of vacuoles from A. reptans mesophyll protoplasts was developed using a pH and osmotic shock to rupture the plasma membrane selectively. By comparing protoplasts with vacuoles, GGT was confirmed to be a vacuolar enzyme. By comparing vacuoles with tonoplast vesicles and cell sap fractions, GGT was further shown to reside in the cell sap and not in the tonoplast. These findings suggest the need for a tonoplast-bound mechanism for the transport of short-chain RFOs such as stachyose or raffinose into the vacuole for subsequent chain elongation.

Download Full-text

Confocal Imaging Analysis of Mitochondrial Trafficking of Individual Lipid Species in Live Cells

SSRN Electronic Journal ◽

10.2139/ssrn.3860388 ◽

2021 ◽

Author(s):

Jun Zhang ◽

Jia Nie ◽

Haoran Sun ◽

John-Paul Andersen ◽

Yuguang Shi

Keyword(s):

Confocal Imaging ◽

Live Cells ◽

Imaging Analysis ◽

Lipid Species ◽

Mitochondrial Trafficking

Download Full-text

Post-Sieve Element Transport of Sucrose in Developing Seeds

Functional Plant Biology ◽

10.1071/pp9950681 ◽

1995 ◽

Vol 22 (4) ◽

pp. 681 ◽

Cited By ~ 52

Author(s):

JW Patrick ◽

CE Offler

Keyword(s):

Plasma Membranes ◽

Grain Legumes ◽

Experimental Models ◽

Sieve Element ◽

Assimilate Transport ◽

Sucrose Transport ◽

Cell Complexes ◽

Developing Seeds ◽

Element Transport ◽

Energy Dependent

Developing seeds of cereals and grain legumes have proven to be useful experimental models to examine post-sieve element assimilate transport in sink tissues. Morphologically, these seeds offer well-defined sinks in which the processes of sucrose import plus efflux and influx plus metabolism may be examined independently. In all cases, sucrose is delivered through the phloem to the maternal seed tissues. Unloading from the sieve element-companion cell complexes is symplastic. Subsequently, sucrose moves through a symplastic route to cells responsible for sucrose efflux to the seed apoplast. The efflux cells are located at, or near, the maternal/filial interface. Sucrose is retrieved from the seed apoplast by the outermost cell layers of the filial tissues. Subsequent transfer of sucrose to the sites of storage in the filial tissues is confined principally to a symplastic route. Sucrose efflux from the maternal tissues appears to be passive in cereals and energy dependent in grain legumes, possibly through a sucrose/proton antiport system. Sucrose influx across the plasma membranes of the filial cells is energy dependent and, for grain legumes, is energy coupled through a sucrose/proton symporter. Studies on the control of post-sieve element transport of sucrose have focused largely on the membrane transport steps. The role of phytohormones as modulators of sucrose transport is uncertain in grain legumes, efflux from the maternal cells could be regulated by rates of sucrose utilisation in the filial tissues through a turgor homeostat mechanism located in the efflux cells.

Download Full-text

An Inducible Operon Is Involved in Inulin Utilization in Lactobacillus plantarum Strains, as Revealed by Comparative Proteogenomics and Metabolic Profiling

Applied and Environmental Microbiology ◽

10.1128/aem.02402-16 ◽

2016 ◽

Vol 83 (2) ◽

Cited By ~ 16

Author(s):

Nirunya Buntin ◽

Tipparat Hongpattarakere ◽

Jarmo Ritari ◽

François P. Douillard ◽

Lars Paulin ◽

...

Keyword(s):

Cell Wall ◽

Lactobacillus Plantarum ◽

Functional Characteristic ◽

Fermented Foods ◽

Sucrose Transport ◽

Protein Coding ◽

Content Type ◽

Genes Encoding ◽

Key Enzyme ◽

Infant Feces

ABSTRACT The draft genomes of Lactobacillus plantarum strains isolated from Asian fermented foods, infant feces, and shrimp intestines were sequenced and compared to those of well-studied strains. Among 28 strains of L. plantarum, variations in the genomic features involved in ecological adaptation were elucidated. The genome sizes ranged from approximately 3.1 to 3.5 Mb, of which about 2,932 to 3,345 protein-coding sequences (CDS) were predicted. The food-derived isolates contained a higher number of carbohydrate metabolism-associated genes than those from infant feces. This observation correlated to their phenotypic carbohydrate metabolic profile, indicating their ability to metabolize the largest range of sugars. Surprisingly, two strains (P14 and P76) isolated from fermented fish utilized inulin. β-Fructosidase, the inulin-degrading enzyme, was detected in the supernatants and cell wall extracts of both strains. No activity was observed in the cytoplasmic fraction, indicating that this key enzyme was either membrane-bound or extracellularly secreted. From genomic mining analysis, a predicted inulin operon of fosRABCDXE, which encodes β-fructosidase and many fructose transporting proteins, was found within the genomes of strains P14 and P76. Moreover, pts1BCA genes, encoding sucrose-specific IIBCA components involved in sucrose transport, were also identified. The proteomic analysis revealed the mechanism and functional characteristic of the fosRABCDXE operon involved in the inulin utilization of L. plantarum. The expression levels of the fos operon and pst genes were upregulated at mid-log phase. FosE and the LPXTG-motif cell wall anchored β-fructosidase were induced to a high abundance when inulin was present as a carbon source. IMPORTANCE Inulin is a long-chain carbohydrate that may act as a prebiotic, which provides many health benefits to the host by selectively stimulating the growth and activity of beneficial bacteria in the colon. While certain lactobacilli can catabolize inulin, this has not yet been described for Lactobacillus plantarum, and an associated putative inulin operon has not been reported in this species. By using comparative and functional genomics, we showed that two L. plantarum strains utilized inulin and identified functional inulin operons in their genomes. The proteogenomic data revealed that inulin degradation and uptake routes, which related to the fosRABCDXE operon and pstBCA genes, were widely expressed among L. plantarum strains. The present work provides a novel understanding of gene regulation and mechanisms of inulin utilization in probiotic L. plantarum generating opportunities for synbiotic product development.

Download Full-text

Abstract 330: Autophagy in Vascular Calcification

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.330 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Cynthia St. Hilaire

Keyword(s):

Vascular Calcification ◽

Premature Death ◽

Arterial Calcification ◽

In Vivo Model ◽

Ectopic Calcification ◽

Key Enzyme ◽

And Control ◽

Future Work

Vascular calcification accompanies a variety of common cardiovascular-related diseases and correlates with premature death. Vascular calcification is a highly-regulated process but the precise mechanisms inducing this pathology are not fully understood, and currently no treatment exists that halts or reverses vascular calcification. We previously discovered the rare monogenetic disease Arterial Calcification due to Deficiency of CD73 (ACDC) which presents with vessel tortuosity and extensive calcifications in the medial layer of lower-extremity arteries. To study the mechanisms underlying this disease we previously created ACDC and Control patient iPSCs, and using a teratoma assay discovered that ACDC iPSC teratomas exhibit extensive calcifications, while Control iPSC teratomas do not. Drug screening identified that rapamycin inhibited calcification in this in vivo model. Using our in vitro calcification model with ACDC fibroblasts, we again found that rapamycin inhibited calcification, as well as expression and activity of the key enzyme involved in ectopic calcification, tissue non-specific alkaline phosphatase (TNAP). To corroborate these findings in vascular cells we used coronary artery smooth muscle cells (CASMCs) in our in vitro calcification assay. We found CASMCs calcify in vitro, and that both rapamycin treatment, and importantly, specific activation of autophagy, inhibited calcification in CASMCs. This suggests autophagy as a therapeutic target for the treatment of vascular calcification. Future work will identify how autophagy prevents calcification, and whether rapamycin, which induces autophagy, can be used as a therapy for medial calcification.

Download Full-text