Bernard Epel | Tel Aviv University (original) (raw)

Papers by Bernard Epel

Research paper thumbnail of Plasmodesmal Gate Keepers for Intercellular Communication

[Plant Signaling & Behavior 2:5, 404-407, 2007

Abstract Plasmodesmata (Pd), coaxial membranous channels that connect adjacent plant cells, are n... more Abstract
Plasmodesmata (Pd), coaxial membranous channels that connect adjacent plant cells, are not static, but show a dynamic nature and can be opened or closed. These controlled changes in Pd conductivity regulate plant symplasmic permeability and play a role both in development and defense processes. One of the mechanisms shown to produce these
changes is the deposition and hydrolysis of callose by b‑1‑3‑synthase and glucanase, respectively. Recently we have identified the first b‑1,3‑glucanase Arabidopsis enzyme that is associated to the macromolecular Pd complex, termed AtBG_pap. When fused to
GFP, this previously identified GPI‑anchored protein localizes to the ER and the plasma membrane where it appears in a punctuate pattern that colocalizes with callose present around Pd. In T‑DNA insertion mutants that do not transcribe AtBG_pap, GFP cell‑to‑cell movement between epidermal cells is reduced and callose levels around Pd are elevated.
In this addenda we review the plant developmental processes of symplasmic regulation that have been shown to include callose deposition and b‑1,3‑glucanase activity, and suggest a role for AtBG_pap in these processes. Additionally, based on the ability of viral movement proteins (MPs) to interact with ankyrin repeat proteins, and together with
our recent findings showing the involvement of viral particles in callose degradation, we also purpose a new model for the ability of viruses to overcome Pd‑callose deposition, and mediate their cell‑to‑cell movement.

Research paper thumbnail of The Mechanismby Whichan Asymmetricdistributionof Plant Growthhormoneis Atfained

Zea mays (sweetcorn) seedlingsattain an asymmetricdistributionof the growth hormoneindole-3-aceti... more Zea mays (sweetcorn) seedlingsattain an asymmetricdistributionof the growth hormoneindole-3-aceticacid (IAA)within 3 minutesfollowinga gravitystimulus. Both freeand esterifiedIAA (that is total IAA) accumulateto a greaterextentin the lower halfof the mesocotylcortex of a honzontallyplacedseedlingthanmtheupper half. Thus, changesin the ratio of free IAAto ester IAAcannot accountfor the asymmetncdistribution. Our studiesdemonstratethereis no de novo synthesisof IAA in youngseedlings. Weconcludethat asymmetric IAAdistributionis attainedby a gravity-induced,potential-regulated gating of the movementof IAA from kernel to shootand from stele to cortex. As a workingtheory, whichwe call the PotentialGatingTheory,we proposethat perturbationof the plant’sbioelectricfield, inducedby gravity, causesopeningand closingof transport channelsin the plasmodesmata connectingthe vascularstele to the surroundingcortical tissues. This results in asymmetricgrowthhormonedistributionwhichresults in the asym...

Research paper thumbnail of Cytology of the (1-3)-β-Glucan (Callose) in Plasmodesmata and Sieve Plate Pores

Chemistry, Biochemistry, and Biology of 1-3 Beta Glucans and Related Polysaccharides, 2009

Publisher Summary This chapter discusses the cytology of the (1-3)-β-glucan (callose) in plasmode... more Publisher Summary This chapter discusses the cytology of the (1-3)-β-glucan (callose) in plasmodesmata and sieve plate pores. Plasmodesmata (Pd) are co-axial membranous channels that cross walls of adjacent plant cells, linking the cytoplasm, plasma membranes and endoplasmic reticulum (ER) of cells and allowing direct cytoplasmic cell-to-cell communication of both small molecules and macromolecules (proteins and RNA). Transport through Pd mediates many processes in plants, among them information transfer for coordination of development, movement of photosynthesis products from mature to developing and storage tissues, responses to pathogen infection and systemic gene silencing. A basic structure of simple primary Pd consists of two coaxial membrane tubes. The inner membrane along the Pd axis, termed the desmotubule, is continuous with and connects the ER of the bordering cells; the outer coaxial membrane is continuous with and connects plasma membranes of adjacent cells. Between the membranes is a sleeve that interconnects the cytoplasm of the neighboring cells. Within the cytoplasmic sleeve are particles whose identity is still unknown, but have been interpreted to be cytoskeletal proteins.

Research paper thumbnail of Isolation and Characterization of Plasmodesmata Components by Association with Tobacco Mosaic Virus Movement Proteins Fused with the Green Fluorescent Protein from Aequorea victoria

The coordination and regulation of growth and development in multicellular organisms is dependent... more The coordination and regulation of growth and development in multicellular organisms is dependent, in part, on the controlled short and long-distance transport of signaling molecule: In plants, symplastic communication is provided by trans-wall co-axial membranous tunnels termed plasmodesmata (Pd). Plant viruses spread cell-to-cell by altering Pd. This movement scenario necessitates a targeting mechanism that delivers the virus to a Pd and a transport mechanism to move the virion or viral nucleic acid through the Pd channel. The identity of host proteins with which MP interacts, the mechanism of the targeting of the MP to the Pd and biochemical information on how Pd are alter are questions which have been dealt with during this BARD project. The research objectives of the two labs were to continue their biochemical, cellular and molecular studies of Pd composition and function by employing infectious modified clones of TMV in which MP is fused with GFP. We examined Pd composition, a...

Research paper thumbnail of Date of Submission of the report : Nov 6 , 2005 BARD Project Number : Project Title : Mechanisms of intra-and intercellular targeting and movement of Tobacco mosaic virus

To cause disease, plant viruses must replicate and spread locally and systemically within the hos... more To cause disease, plant viruses must replicate and spread locally and systemically within the host. Cell-to-cell virus spread is mediated by virus-encoded movement proteins (MPs), which modify the structure and function of plasmodesmata (Pd), trans-wall co-axial membranous tunnels that interconnect the cytoplasm of neighboring cells. Tobacco mosaic virus (TMV) employ a single MP for cellcell spread and for which CP is not required. The PIs, Beachy (USA) and Epel (Israel) and co-workers, developed new tools and approaches for study of the mechanism of spread of TMV that lead to a partial identification and molecular characterization of the cellular machinery involved in the trafficking process. Original research objectives: Based on our data and those of others, we proposed a working model of plant viral spread. Our model stated that MP an integral ER membrane protein with its C-terminus exposed to the cytoplasm (Reichel and Beachy, 1998), alters the Pd SEL, causes the Pd cytoplasmic...

Research paper thumbnail of Studies on the Mechanism and Regulation of Plant Cell-Cell Communication: Effects of Hormones and Light on the Symplast Continuum

Research paper thumbnail of BARD Report -Mechanisms of intra- and intercellular targeting and movement of tobacco mosaic virus

BARD Report 2005, 2005

To cause disease, plant viruses must replicate and spread locally and systemically within the hos... more To cause disease, plant viruses must replicate and spread locally and systemically within the host. Cell-to-cell virus spread is mediated by virus-encoded movement proteins (MPs), which modify the structure and function of plasmodesmata (Pd), trans-wall co-axial membranous tunnels that interconnect the cytoplasm of neighboring cells. Tobacco mosaic virus (TMV) employ a single MP for cell- cell spread and for which CP is not required. The PIs, Beachy (USA) and Epel (Israel) and co-workers, developed new tools and approaches for study of the mechanism of spread of TMV that lead to a partial identification and molecular characterization of the cellular machinery involved in the trafficking process. Original research objectives: Based on our data and those of others, we proposed a working model of plant viral spread. Our model stated that MPᵀᴹⱽ, an integral ER membrane protein with its C-terminus exposed to the cytoplasm (Reichel and Beachy, 1998), alters the Pd SEL, causes the Pd cytop...

Research paper thumbnail of Glycosylphosphatidylinositol (GPI) modification serves as a primary plasmodesmal targeting signal

Plant Physiology, 2016

Plasmodesmata (Pd) are membranous channels that serve as a major conduit for cell-to-cell communi... more Plasmodesmata (Pd) are membranous channels that serve as a major conduit for cell-to-cell communication in plants. The Pdassociated b-1,3-glucanase (BG_pap) and CALLOSE BINDING PROTEIN1 (PDCB1) were identified as key regulators of Pd conductivity. Both are predicted glycosylphosphatidylinositol-anchored proteins (GPI-APs) carrying a conserved GPI modification signal. However, the subcellular targeting mechanism of these proteins is unknown, particularly in the context of other GPI-APs not associated with Pd. Here, we conducted a comparative analysis of the subcellular targeting of the two Pd-resident and two unrelated non-Pd GPI-APs in Arabidopsis (Arabidopsis thaliana). We show that GPI modification is necessary and sufficient for delivering both BG_pap and PDCB1 to Pd. Moreover, the GPI modification signal from both Pd-and non-Pd GPI-APs is able to target a reporter protein to Pd, likely to plasma membrane microdomains enriched at Pd. As such, the GPI modification serves as a primary Pd sorting signal in plant cells. Interestingly, the ectodomain, a region that carries the functional domain in GPI-APs, in Pd-resident proteins further enhances Pd accumulation. However, in non-Pd GPI-APs, the ectodomain overrides the Pd targeting function of the GPI signal and determines a specific GPI-dependent non-Pd localization of these proteins at the plasma membrane and cell wall. Domainswap analysis showed that the non-Pd localization is also dominant over the Pd-enhancing function mediated by a Pd ectodomain. In conclusion, our results indicate that segregation between Pd-and non-Pd GPI-APs occurs prior to Pd targeting, providing, to our knowledge, the first evidence of the mechanism of GPI-AP sorting in plants.

Research paper thumbnail of Differences in the Physical Properties of Native and Partially Degraded Phytochrome as Probed by Their Differential Sensitivity to Permanganate Oxidation

PLANT PHYSIOLOGY, 1983

The differential sensitivities to permanganate oxidation of the red and far-red forms of native p... more The differential sensitivities to permanganate oxidation of the red and far-red forms of native phytochrome from Avena sativa L. cv Mulaga (isolated as Pfr from red-irradiated tissue) and of partially degraded phytochrome (isolated as Pr from nonirradiated tissue) were determined. The far-red absorbing form of partially degraded phytochrome was 5 times more sensitive than its red-absorbing form, while both the far-red and red forms of native phytochrome exhibited identical sensitivity. The present data obtained with partially degraded phytochrome are in apparent agreement with the data and model of Hahn, Kang, and Song (1980 Biochem Biophys Res Commun 97: 1317-1323). Their model suggests that the chromophore of the red-absorbing form of phytochrome is buried in a hydrophobic crevice in the protein, while that of the far-red form is exposed. The data obtained with native phytochrome, however, are at variance with their model. Our data obtained with native phytochrome suggests that the chromophore of the red and the far-red absorbing forms of native phytochrome both are in a relatively protected environment and that only following partial proteolytic degradation of the phytochrome does the chromophore of its far-red form become relatively more exposed. The protective influence of the labile peptide could either be direct, because of its close physical proximity to the chromophore, or indirect, resulting in an alteration in chromophore-protein interaction.

Research paper thumbnail of Tobacco mosaic virus (TMV) Replicase and Movement Protein Function Synergistically in Facilitating TMV Spread by Lateral Diffusion in the Plasmodesmal Desmotubule of Nicotiana benthamiana

Molecular Plant-Microbe Interactions, 2008

Virus spread through plasmodesmata (Pd) is mediated by virus-encoded movement proteins (MPs) that... more Virus spread through plasmodesmata (Pd) is mediated by virus-encoded movement proteins (MPs) that modify Pd structure and function. The MP of Tobacco mosaic virus (TMVMP) is an endoplasmic reticulum (ER) integral membrane protein that binds viral RNA (vRNA), forming a vRNA:MP:ER complex. It has been hypothesized that TMVMP causes Pd to dilate, thus potentiating a cytoskeletal mediated sliding of the vRNA:MP:ER complex through Pd; in the absence of MP, by contrast, the ER cannot move through Pd. An alternate model proposes that cell-to-cell spread takes place by diffusion of the MP:vRNA complex in the ER membranes which traverse Pd. To test these models, we measured the effect of TMVMP and replicase expression on cell-to-cell spread of several green fluorescent protein-fused probes: a soluble cytoplasmic protein, two ER lumen proteins, and two ER membrane-bound proteins. Our data support the diffusion model in which a complex that includes ER-embedded MP, vRNA, and other components d...

Research paper thumbnail of Characterization of Cytochromes in Lithotrophically and Organotrophically Grown Cells of Thiobacillus A2

Journal of General Microbiology (1982), 128, 2371-2378., 1982

The dominant cytochrome in thiosulphate-grown Thiobacillus A2 was found to be of the c-type with... more The dominant cytochrome in thiosulphate-grown Thiobacillus A2 was found to be of the c-type
with a reduced or-band at 548 nm (c548). This c548 component did not constitute an integral part
of the membrane carrier system. It did, however, appear to be part of a large complex not tightly
bound to membranes. Reconstitution experiments showed that cytochromes of the membrane
‘abc’ system could be reduced by the c548 component and vice versa. The reduction of membrane
cytochromes of either lithotrophic or organotrophic origin by thiosulphate electrons was
achieved, but it required the presence of a soluble fraction containing cytochrome c548. Evidence tending to rule out a reductive cleavage as the first step of the thiosulphate oxidation
pathway in ThiobaciIZus A2 was obtained by following the reduction of partially purified c548
particles under various conditions.

Research paper thumbnail of Studies on the Longitudinal and Lateral Transport of IAA in the Shoots of Etiolated Corn Seedlings

Journal of Plant Physiology, 1992

The auxin, indole-3-acetic acid, and the symplastic probe, carboxyfluorescein diacetate, were app... more The auxin, indole-3-acetic acid, and the symplastic probe, carboxyfluorescein diacetate, were applied to the cut mesocotyl base or coleoptile apex of etiolated Zea mays seedlings and their transport measured and tissue distribution determined. The longitudinal transport of indole-3-acetate was strongly basipolar, while that of carboxyfluorescein was essentially apolar. The longitudinal transport of IAA, like carboxyfluorescein, was mainly in the stele. IAA exhibited a much higher lateral mobility from stele to cortex than did carboxyfluorescein. Based on the calculation of moles probe/kgfw, IAA is 4 times more concentrated in the stele than in the cortex while CF is 24 times higher in concentration in the stele than in the cortex. The structure of the node and the mesocotyl regions just below the node, regions of maximum growth, were examined and plasmodesmatal structure and frequency in these regions determined. The plasmodesmatal frequency, about 3 per 11m 2 , between all cell types of the mesocotyl was found to be about 5-8 fold higher than that found for the root. Hypotheses of lateral auxin transport are discussed.

Research paper thumbnail of Circadian changes in membrane properties of human red blood cells in vitro, as measured by a membrane probe

Research paper thumbnail of A Spectroscopic Analysis of a High Fluorescent Mutant of Chlamydomonas Reinhardi

Biophysical Journal, 1972

Research paper thumbnail of Changing Patterns of Localization of the Tobacco Mosaic Virus Movement Protein and Replicase to the Endoplasmic Reticulum and Microtubules during Infection

The Plant Cell, 1998

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green... more Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.

Research paper thumbnail of Relationship Between Stimuli, IAA and Growth

Plant Growth Substances 1988, 1990

“Ohne Wuchsstoff kein Wachstum”: without auxin no growth [e.g. 32]. A particularly valuable test ... more “Ohne Wuchsstoff kein Wachstum”: without auxin no growth [e.g. 32]. A particularly valuable test of this dictum was made by Dolk [e.g. 32] who showed that double decapitation of the A vena coleoptile stopped growth. Growth could then be partially restored by means of applied auxin. As beautiful as this experiment is, it is important to test the conclusion by determining if there is a quantitative relationship between endogenous IAA and growth. The relationship between numbers of coleoptile tips placed on a receiver block and growth induced by applying the receiver block to shoot stumps does not establish this relationship, since the tip supplies both free IAA and IAA derived from the seed auxin precursor [2, 23] presumably by hydrolysis of bound IAA [28]. Thus, we wish to make a quantitative test of the dictum by determining the relationship between growth rate and amount of free IAA. We wish further to determine the effect of various environmental stimuli on the amount of IAA in the tissue. In that manner we can determine the extent to which the transduction of environmental stimuli is accomplished by varying the amount of free IAA in the experimental system. Lastly, we wish to understand how the plant can so precisely regulate its IAA levels so as to have more IAA on one side than on the other of a tropically stimulated stem. Lastly, we wish to understand tropic curvatures in terms of IAA metabolism and transport.

Research paper thumbnail of A plasmodesmata-associated beta-1,3-glucanase in Arabidopsis

The Plant journal : for cell and molecular biology, 2007

Plasmodesmal conductivity is regulated in part by callose turnover, which is hypothesized to be d... more Plasmodesmal conductivity is regulated in part by callose turnover, which is hypothesized to be determined by beta-1,3-glucan synthase versus glucanase activities. A proteomic analysis of an Arabidopsis thaliana plasmodesmata (Pd)-rich fraction identified a beta-1,3-glucanase as present in this fraction. The protein encoded by the putative plasmodesmal associated protein (ppap) gene, termed AtBG_ppap, had previously been found to be a post-translationally modified glycosylphosphatidylinositol (GPI) lipid-anchored protein. When fused to green fluorescent protein (GFP) and expressed in tobacco (Nicotiana tabacum) or Nicotiana benthamiana epidermal cells, this protein displays fluorescence patterns in the endoplasmic reticulum (ER) membrane system, along the cell periphery and in a punctate pattern that co-localizes with aniline blue-stained callose present around the Pd. Plasma membrane localization was verified by co-localization of AtBG_ppap:GFP together with a plasma membrane marke...

Research paper thumbnail of Inhibition of Respiration in Prototheca zopfii by Light

PLANT PHYSIOLOGY, 1970

Irradiation of cells of Prototheca zopfii with blue light inhibited the respiratory capacity of t... more Irradiation of cells of Prototheca zopfii with blue light inhibited the respiratory capacity of the cells. The inhibition of respiration was correlated with a photodestruction of cytochrome c(551), cytochrome b(559), and cytochrome a3. Cytochrome c(549), cytochrome b(555), and cytochrome b(564) were unaffected by the irradiation treatment. The a-band of reduced cytochrome a was shifted from 599 to 603 nm by irradiation, an effect similar to that observed when methanol was added to nonirradiated cells. The presence of oxygen was required during irradiation for both photoinhibition of respiration and photodestruction of the cytochromes. Cytochrome a3 was protected against photodestruction by cyanide. Photodestruction of these same cytochromes also occurred when washed mitochondria of P. zopfii were irradiated. Blue and near-ultraviolet radiation are inhibitory or lethal to a wide variety of prokaryotic and eukaryotic microorganisms and to a number of tissues of higher plants and animals. Among the prokaryotes visible radiation and near-ultraviolet radiation have been reported to kill or inhibit the growth of the heterotropic bacteria Streptococcus salivarius (4), Escherichia coli (17, 19, 20), Bacterium prodigiosum (38), Pseudomonas abruginosa (20), and Haemophilus infiuenzae (18), and a number of carotenoidless mutants of such normally carotenoid-containing bacteria as Sarcina lutea (23), Mycobacterium sp. (43), Halobacterium salinarium (9), and Myxococcus xanthus (5, 6) and the chemoatotrophic bacteria Nitrosomonas europaea (1, 36) and Nitrobacter winogradskyi and the denitrifying bacterium Micrococcus

Research paper thumbnail of The mechanism of the oxidation of ascorbate and Mn2+ by chloroplasts: The role of the radical superoxide

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1973

Research paper thumbnail of A Kinetic Analysis of Phytochrome Controlled Mesocotyl Growth in Zea mays Seedlings

Plant Physiology, 1987

ABSTRACr Mesocotyl elongtio in 4 day old etdolated s s imm tely following 3 hours of white light ... more ABSTRACr Mesocotyl elongtio in 4 day old etdolated s s imm tely following 3 hours of white light (3 h W) is reversibly controlled by phytochrome. Time-lapse video measurements were made of the 5 millimeter zone just below the coleoptile which is the main growth region of the mesocotyl. The growth kinetics were determined for five contguous 1 millimeter zon subti the coleoptile node for n ted seedlings, for seedlng given 3 h W, and 3 h W followed by tmin farred (FR) or red subsequent to the far-red (FI/R) irdtio. Each zoe in ted s lis exhibits exp eltion kinetics duing the early stages of elogtio. Tis finig suggests that duing elonption, a growth limitig fctr is also exponentially i sing. Folowing 3 h W differences in the kinetic responses were found for each zone. In all zones, the inhibitory effect following the 3 h W is totally FR reversible. The effect of FR is reversed by R. The upper zone exhibits the fastest response and is the most plastic in its growth response. The three upper zones all exhibit spontaneous and sharp recoveries with time. It is suggested that the control by phytochrome is not inductive but rather continuous, the controlling factor being either the level of the far redabsorbing form of phytochrome (Pfr) or the ratio Pfr to total phytochrome.

Research paper thumbnail of Plasmodesmal Gate Keepers for Intercellular Communication

[Plant Signaling & Behavior 2:5, 404-407, 2007

Abstract Plasmodesmata (Pd), coaxial membranous channels that connect adjacent plant cells, are n... more Abstract
Plasmodesmata (Pd), coaxial membranous channels that connect adjacent plant cells, are not static, but show a dynamic nature and can be opened or closed. These controlled changes in Pd conductivity regulate plant symplasmic permeability and play a role both in development and defense processes. One of the mechanisms shown to produce these
changes is the deposition and hydrolysis of callose by b‑1‑3‑synthase and glucanase, respectively. Recently we have identified the first b‑1,3‑glucanase Arabidopsis enzyme that is associated to the macromolecular Pd complex, termed AtBG_pap. When fused to
GFP, this previously identified GPI‑anchored protein localizes to the ER and the plasma membrane where it appears in a punctuate pattern that colocalizes with callose present around Pd. In T‑DNA insertion mutants that do not transcribe AtBG_pap, GFP cell‑to‑cell movement between epidermal cells is reduced and callose levels around Pd are elevated.
In this addenda we review the plant developmental processes of symplasmic regulation that have been shown to include callose deposition and b‑1,3‑glucanase activity, and suggest a role for AtBG_pap in these processes. Additionally, based on the ability of viral movement proteins (MPs) to interact with ankyrin repeat proteins, and together with
our recent findings showing the involvement of viral particles in callose degradation, we also purpose a new model for the ability of viruses to overcome Pd‑callose deposition, and mediate their cell‑to‑cell movement.

Research paper thumbnail of The Mechanismby Whichan Asymmetricdistributionof Plant Growthhormoneis Atfained

Zea mays (sweetcorn) seedlingsattain an asymmetricdistributionof the growth hormoneindole-3-aceti... more Zea mays (sweetcorn) seedlingsattain an asymmetricdistributionof the growth hormoneindole-3-aceticacid (IAA)within 3 minutesfollowinga gravitystimulus. Both freeand esterifiedIAA (that is total IAA) accumulateto a greaterextentin the lower halfof the mesocotylcortex of a honzontallyplacedseedlingthanmtheupper half. Thus, changesin the ratio of free IAAto ester IAAcannot accountfor the asymmetncdistribution. Our studiesdemonstratethereis no de novo synthesisof IAA in youngseedlings. Weconcludethat asymmetric IAAdistributionis attainedby a gravity-induced,potential-regulated gating of the movementof IAA from kernel to shootand from stele to cortex. As a workingtheory, whichwe call the PotentialGatingTheory,we proposethat perturbationof the plant’sbioelectricfield, inducedby gravity, causesopeningand closingof transport channelsin the plasmodesmata connectingthe vascularstele to the surroundingcortical tissues. This results in asymmetricgrowthhormonedistributionwhichresults in the asym...

Research paper thumbnail of Cytology of the (1-3)-β-Glucan (Callose) in Plasmodesmata and Sieve Plate Pores

Chemistry, Biochemistry, and Biology of 1-3 Beta Glucans and Related Polysaccharides, 2009

Publisher Summary This chapter discusses the cytology of the (1-3)-β-glucan (callose) in plasmode... more Publisher Summary This chapter discusses the cytology of the (1-3)-β-glucan (callose) in plasmodesmata and sieve plate pores. Plasmodesmata (Pd) are co-axial membranous channels that cross walls of adjacent plant cells, linking the cytoplasm, plasma membranes and endoplasmic reticulum (ER) of cells and allowing direct cytoplasmic cell-to-cell communication of both small molecules and macromolecules (proteins and RNA). Transport through Pd mediates many processes in plants, among them information transfer for coordination of development, movement of photosynthesis products from mature to developing and storage tissues, responses to pathogen infection and systemic gene silencing. A basic structure of simple primary Pd consists of two coaxial membrane tubes. The inner membrane along the Pd axis, termed the desmotubule, is continuous with and connects the ER of the bordering cells; the outer coaxial membrane is continuous with and connects plasma membranes of adjacent cells. Between the membranes is a sleeve that interconnects the cytoplasm of the neighboring cells. Within the cytoplasmic sleeve are particles whose identity is still unknown, but have been interpreted to be cytoskeletal proteins.

Research paper thumbnail of Isolation and Characterization of Plasmodesmata Components by Association with Tobacco Mosaic Virus Movement Proteins Fused with the Green Fluorescent Protein from Aequorea victoria

The coordination and regulation of growth and development in multicellular organisms is dependent... more The coordination and regulation of growth and development in multicellular organisms is dependent, in part, on the controlled short and long-distance transport of signaling molecule: In plants, symplastic communication is provided by trans-wall co-axial membranous tunnels termed plasmodesmata (Pd). Plant viruses spread cell-to-cell by altering Pd. This movement scenario necessitates a targeting mechanism that delivers the virus to a Pd and a transport mechanism to move the virion or viral nucleic acid through the Pd channel. The identity of host proteins with which MP interacts, the mechanism of the targeting of the MP to the Pd and biochemical information on how Pd are alter are questions which have been dealt with during this BARD project. The research objectives of the two labs were to continue their biochemical, cellular and molecular studies of Pd composition and function by employing infectious modified clones of TMV in which MP is fused with GFP. We examined Pd composition, a...

Research paper thumbnail of Date of Submission of the report : Nov 6 , 2005 BARD Project Number : Project Title : Mechanisms of intra-and intercellular targeting and movement of Tobacco mosaic virus

To cause disease, plant viruses must replicate and spread locally and systemically within the hos... more To cause disease, plant viruses must replicate and spread locally and systemically within the host. Cell-to-cell virus spread is mediated by virus-encoded movement proteins (MPs), which modify the structure and function of plasmodesmata (Pd), trans-wall co-axial membranous tunnels that interconnect the cytoplasm of neighboring cells. Tobacco mosaic virus (TMV) employ a single MP for cellcell spread and for which CP is not required. The PIs, Beachy (USA) and Epel (Israel) and co-workers, developed new tools and approaches for study of the mechanism of spread of TMV that lead to a partial identification and molecular characterization of the cellular machinery involved in the trafficking process. Original research objectives: Based on our data and those of others, we proposed a working model of plant viral spread. Our model stated that MP an integral ER membrane protein with its C-terminus exposed to the cytoplasm (Reichel and Beachy, 1998), alters the Pd SEL, causes the Pd cytoplasmic...

Research paper thumbnail of Studies on the Mechanism and Regulation of Plant Cell-Cell Communication: Effects of Hormones and Light on the Symplast Continuum

Research paper thumbnail of BARD Report -Mechanisms of intra- and intercellular targeting and movement of tobacco mosaic virus

BARD Report 2005, 2005

To cause disease, plant viruses must replicate and spread locally and systemically within the hos... more To cause disease, plant viruses must replicate and spread locally and systemically within the host. Cell-to-cell virus spread is mediated by virus-encoded movement proteins (MPs), which modify the structure and function of plasmodesmata (Pd), trans-wall co-axial membranous tunnels that interconnect the cytoplasm of neighboring cells. Tobacco mosaic virus (TMV) employ a single MP for cell- cell spread and for which CP is not required. The PIs, Beachy (USA) and Epel (Israel) and co-workers, developed new tools and approaches for study of the mechanism of spread of TMV that lead to a partial identification and molecular characterization of the cellular machinery involved in the trafficking process. Original research objectives: Based on our data and those of others, we proposed a working model of plant viral spread. Our model stated that MPᵀᴹⱽ, an integral ER membrane protein with its C-terminus exposed to the cytoplasm (Reichel and Beachy, 1998), alters the Pd SEL, causes the Pd cytop...

Research paper thumbnail of Glycosylphosphatidylinositol (GPI) modification serves as a primary plasmodesmal targeting signal

Plant Physiology, 2016

Plasmodesmata (Pd) are membranous channels that serve as a major conduit for cell-to-cell communi... more Plasmodesmata (Pd) are membranous channels that serve as a major conduit for cell-to-cell communication in plants. The Pdassociated b-1,3-glucanase (BG_pap) and CALLOSE BINDING PROTEIN1 (PDCB1) were identified as key regulators of Pd conductivity. Both are predicted glycosylphosphatidylinositol-anchored proteins (GPI-APs) carrying a conserved GPI modification signal. However, the subcellular targeting mechanism of these proteins is unknown, particularly in the context of other GPI-APs not associated with Pd. Here, we conducted a comparative analysis of the subcellular targeting of the two Pd-resident and two unrelated non-Pd GPI-APs in Arabidopsis (Arabidopsis thaliana). We show that GPI modification is necessary and sufficient for delivering both BG_pap and PDCB1 to Pd. Moreover, the GPI modification signal from both Pd-and non-Pd GPI-APs is able to target a reporter protein to Pd, likely to plasma membrane microdomains enriched at Pd. As such, the GPI modification serves as a primary Pd sorting signal in plant cells. Interestingly, the ectodomain, a region that carries the functional domain in GPI-APs, in Pd-resident proteins further enhances Pd accumulation. However, in non-Pd GPI-APs, the ectodomain overrides the Pd targeting function of the GPI signal and determines a specific GPI-dependent non-Pd localization of these proteins at the plasma membrane and cell wall. Domainswap analysis showed that the non-Pd localization is also dominant over the Pd-enhancing function mediated by a Pd ectodomain. In conclusion, our results indicate that segregation between Pd-and non-Pd GPI-APs occurs prior to Pd targeting, providing, to our knowledge, the first evidence of the mechanism of GPI-AP sorting in plants.

Research paper thumbnail of Differences in the Physical Properties of Native and Partially Degraded Phytochrome as Probed by Their Differential Sensitivity to Permanganate Oxidation

PLANT PHYSIOLOGY, 1983

The differential sensitivities to permanganate oxidation of the red and far-red forms of native p... more The differential sensitivities to permanganate oxidation of the red and far-red forms of native phytochrome from Avena sativa L. cv Mulaga (isolated as Pfr from red-irradiated tissue) and of partially degraded phytochrome (isolated as Pr from nonirradiated tissue) were determined. The far-red absorbing form of partially degraded phytochrome was 5 times more sensitive than its red-absorbing form, while both the far-red and red forms of native phytochrome exhibited identical sensitivity. The present data obtained with partially degraded phytochrome are in apparent agreement with the data and model of Hahn, Kang, and Song (1980 Biochem Biophys Res Commun 97: 1317-1323). Their model suggests that the chromophore of the red-absorbing form of phytochrome is buried in a hydrophobic crevice in the protein, while that of the far-red form is exposed. The data obtained with native phytochrome, however, are at variance with their model. Our data obtained with native phytochrome suggests that the chromophore of the red and the far-red absorbing forms of native phytochrome both are in a relatively protected environment and that only following partial proteolytic degradation of the phytochrome does the chromophore of its far-red form become relatively more exposed. The protective influence of the labile peptide could either be direct, because of its close physical proximity to the chromophore, or indirect, resulting in an alteration in chromophore-protein interaction.

Research paper thumbnail of Tobacco mosaic virus (TMV) Replicase and Movement Protein Function Synergistically in Facilitating TMV Spread by Lateral Diffusion in the Plasmodesmal Desmotubule of Nicotiana benthamiana

Molecular Plant-Microbe Interactions, 2008

Virus spread through plasmodesmata (Pd) is mediated by virus-encoded movement proteins (MPs) that... more Virus spread through plasmodesmata (Pd) is mediated by virus-encoded movement proteins (MPs) that modify Pd structure and function. The MP of Tobacco mosaic virus (TMVMP) is an endoplasmic reticulum (ER) integral membrane protein that binds viral RNA (vRNA), forming a vRNA:MP:ER complex. It has been hypothesized that TMVMP causes Pd to dilate, thus potentiating a cytoskeletal mediated sliding of the vRNA:MP:ER complex through Pd; in the absence of MP, by contrast, the ER cannot move through Pd. An alternate model proposes that cell-to-cell spread takes place by diffusion of the MP:vRNA complex in the ER membranes which traverse Pd. To test these models, we measured the effect of TMVMP and replicase expression on cell-to-cell spread of several green fluorescent protein-fused probes: a soluble cytoplasmic protein, two ER lumen proteins, and two ER membrane-bound proteins. Our data support the diffusion model in which a complex that includes ER-embedded MP, vRNA, and other components d...

Research paper thumbnail of Characterization of Cytochromes in Lithotrophically and Organotrophically Grown Cells of Thiobacillus A2

Journal of General Microbiology (1982), 128, 2371-2378., 1982

The dominant cytochrome in thiosulphate-grown Thiobacillus A2 was found to be of the c-type with... more The dominant cytochrome in thiosulphate-grown Thiobacillus A2 was found to be of the c-type
with a reduced or-band at 548 nm (c548). This c548 component did not constitute an integral part
of the membrane carrier system. It did, however, appear to be part of a large complex not tightly
bound to membranes. Reconstitution experiments showed that cytochromes of the membrane
‘abc’ system could be reduced by the c548 component and vice versa. The reduction of membrane
cytochromes of either lithotrophic or organotrophic origin by thiosulphate electrons was
achieved, but it required the presence of a soluble fraction containing cytochrome c548. Evidence tending to rule out a reductive cleavage as the first step of the thiosulphate oxidation
pathway in ThiobaciIZus A2 was obtained by following the reduction of partially purified c548
particles under various conditions.

Research paper thumbnail of Studies on the Longitudinal and Lateral Transport of IAA in the Shoots of Etiolated Corn Seedlings

Journal of Plant Physiology, 1992

The auxin, indole-3-acetic acid, and the symplastic probe, carboxyfluorescein diacetate, were app... more The auxin, indole-3-acetic acid, and the symplastic probe, carboxyfluorescein diacetate, were applied to the cut mesocotyl base or coleoptile apex of etiolated Zea mays seedlings and their transport measured and tissue distribution determined. The longitudinal transport of indole-3-acetate was strongly basipolar, while that of carboxyfluorescein was essentially apolar. The longitudinal transport of IAA, like carboxyfluorescein, was mainly in the stele. IAA exhibited a much higher lateral mobility from stele to cortex than did carboxyfluorescein. Based on the calculation of moles probe/kgfw, IAA is 4 times more concentrated in the stele than in the cortex while CF is 24 times higher in concentration in the stele than in the cortex. The structure of the node and the mesocotyl regions just below the node, regions of maximum growth, were examined and plasmodesmatal structure and frequency in these regions determined. The plasmodesmatal frequency, about 3 per 11m 2 , between all cell types of the mesocotyl was found to be about 5-8 fold higher than that found for the root. Hypotheses of lateral auxin transport are discussed.

Research paper thumbnail of Circadian changes in membrane properties of human red blood cells in vitro, as measured by a membrane probe

Research paper thumbnail of A Spectroscopic Analysis of a High Fluorescent Mutant of Chlamydomonas Reinhardi

Biophysical Journal, 1972

Research paper thumbnail of Changing Patterns of Localization of the Tobacco Mosaic Virus Movement Protein and Replicase to the Endoplasmic Reticulum and Microtubules during Infection

The Plant Cell, 1998

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green... more Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.

Research paper thumbnail of Relationship Between Stimuli, IAA and Growth

Plant Growth Substances 1988, 1990

“Ohne Wuchsstoff kein Wachstum”: without auxin no growth [e.g. 32]. A particularly valuable test ... more “Ohne Wuchsstoff kein Wachstum”: without auxin no growth [e.g. 32]. A particularly valuable test of this dictum was made by Dolk [e.g. 32] who showed that double decapitation of the A vena coleoptile stopped growth. Growth could then be partially restored by means of applied auxin. As beautiful as this experiment is, it is important to test the conclusion by determining if there is a quantitative relationship between endogenous IAA and growth. The relationship between numbers of coleoptile tips placed on a receiver block and growth induced by applying the receiver block to shoot stumps does not establish this relationship, since the tip supplies both free IAA and IAA derived from the seed auxin precursor [2, 23] presumably by hydrolysis of bound IAA [28]. Thus, we wish to make a quantitative test of the dictum by determining the relationship between growth rate and amount of free IAA. We wish further to determine the effect of various environmental stimuli on the amount of IAA in the tissue. In that manner we can determine the extent to which the transduction of environmental stimuli is accomplished by varying the amount of free IAA in the experimental system. Lastly, we wish to understand how the plant can so precisely regulate its IAA levels so as to have more IAA on one side than on the other of a tropically stimulated stem. Lastly, we wish to understand tropic curvatures in terms of IAA metabolism and transport.

Research paper thumbnail of A plasmodesmata-associated beta-1,3-glucanase in Arabidopsis

The Plant journal : for cell and molecular biology, 2007

Plasmodesmal conductivity is regulated in part by callose turnover, which is hypothesized to be d... more Plasmodesmal conductivity is regulated in part by callose turnover, which is hypothesized to be determined by beta-1,3-glucan synthase versus glucanase activities. A proteomic analysis of an Arabidopsis thaliana plasmodesmata (Pd)-rich fraction identified a beta-1,3-glucanase as present in this fraction. The protein encoded by the putative plasmodesmal associated protein (ppap) gene, termed AtBG_ppap, had previously been found to be a post-translationally modified glycosylphosphatidylinositol (GPI) lipid-anchored protein. When fused to green fluorescent protein (GFP) and expressed in tobacco (Nicotiana tabacum) or Nicotiana benthamiana epidermal cells, this protein displays fluorescence patterns in the endoplasmic reticulum (ER) membrane system, along the cell periphery and in a punctate pattern that co-localizes with aniline blue-stained callose present around the Pd. Plasma membrane localization was verified by co-localization of AtBG_ppap:GFP together with a plasma membrane marke...

Research paper thumbnail of Inhibition of Respiration in Prototheca zopfii by Light

PLANT PHYSIOLOGY, 1970

Irradiation of cells of Prototheca zopfii with blue light inhibited the respiratory capacity of t... more Irradiation of cells of Prototheca zopfii with blue light inhibited the respiratory capacity of the cells. The inhibition of respiration was correlated with a photodestruction of cytochrome c(551), cytochrome b(559), and cytochrome a3. Cytochrome c(549), cytochrome b(555), and cytochrome b(564) were unaffected by the irradiation treatment. The a-band of reduced cytochrome a was shifted from 599 to 603 nm by irradiation, an effect similar to that observed when methanol was added to nonirradiated cells. The presence of oxygen was required during irradiation for both photoinhibition of respiration and photodestruction of the cytochromes. Cytochrome a3 was protected against photodestruction by cyanide. Photodestruction of these same cytochromes also occurred when washed mitochondria of P. zopfii were irradiated. Blue and near-ultraviolet radiation are inhibitory or lethal to a wide variety of prokaryotic and eukaryotic microorganisms and to a number of tissues of higher plants and animals. Among the prokaryotes visible radiation and near-ultraviolet radiation have been reported to kill or inhibit the growth of the heterotropic bacteria Streptococcus salivarius (4), Escherichia coli (17, 19, 20), Bacterium prodigiosum (38), Pseudomonas abruginosa (20), and Haemophilus infiuenzae (18), and a number of carotenoidless mutants of such normally carotenoid-containing bacteria as Sarcina lutea (23), Mycobacterium sp. (43), Halobacterium salinarium (9), and Myxococcus xanthus (5, 6) and the chemoatotrophic bacteria Nitrosomonas europaea (1, 36) and Nitrobacter winogradskyi and the denitrifying bacterium Micrococcus

Research paper thumbnail of The mechanism of the oxidation of ascorbate and Mn2+ by chloroplasts: The role of the radical superoxide

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1973

Research paper thumbnail of A Kinetic Analysis of Phytochrome Controlled Mesocotyl Growth in Zea mays Seedlings

Plant Physiology, 1987

ABSTRACr Mesocotyl elongtio in 4 day old etdolated s s imm tely following 3 hours of white light ... more ABSTRACr Mesocotyl elongtio in 4 day old etdolated s s imm tely following 3 hours of white light (3 h W) is reversibly controlled by phytochrome. Time-lapse video measurements were made of the 5 millimeter zone just below the coleoptile which is the main growth region of the mesocotyl. The growth kinetics were determined for five contguous 1 millimeter zon subti the coleoptile node for n ted seedlings, for seedlng given 3 h W, and 3 h W followed by tmin farred (FR) or red subsequent to the far-red (FI/R) irdtio. Each zoe in ted s lis exhibits exp eltion kinetics duing the early stages of elogtio. Tis finig suggests that duing elonption, a growth limitig fctr is also exponentially i sing. Folowing 3 h W differences in the kinetic responses were found for each zone. In all zones, the inhibitory effect following the 3 h W is totally FR reversible. The effect of FR is reversed by R. The upper zone exhibits the fastest response and is the most plastic in its growth response. The three upper zones all exhibit spontaneous and sharp recoveries with time. It is suggested that the control by phytochrome is not inductive but rather continuous, the controlling factor being either the level of the far redabsorbing form of phytochrome (Pfr) or the ratio Pfr to total phytochrome.