Einat Sadot - Academia.edu (original) (raw)

Papers by Einat Sadot

Journal of Integrative Plant Biology, Oct 14, 2014

Plant organelles are highly motile, with speed values of 3-7 µm/s in cells of land plants and abo... more Plant organelles are highly motile, with speed values of 3-7 µm/s in cells of land plants and about 20-60 µm/s in characean algal cells. This movement is believed to be important for rapid distribution of materials around the cell, for the plant&amp;amp;amp;#39;s ability to respond to environmental biotic and abiotic signals and for proper growth. The main machinery that propels motility of organelles within plant cells is based on the actin cytoskeleton and its motor proteins the myosins. Most plants express multiple members of two main classes: myosin VIII and myosin XI. While myosin VIII has been characterized as a slow motor protein, myosins from class XI were found to be the fastest motor proteins known in all kingdoms. Paradoxically, while it was found that myosins from class XI regulate most organelle movement, it is not quite clear how or even if these motor proteins attach to the organelles whose movement they regulate.

The study has been focused on proline-rich proteins from the HyPRP family. Three proline-rich pro... more The study has been focused on proline-rich proteins from the HyPRP family. Three proline-rich proteins have been characterized with the CWLP as the main objective. We showed that this unique protein is assembled in the plasma membrane (PM) and forms a continuum between the cell wall (CW) and cytosol via the PM. While spanning the PM, it is arranged in lipid rafts as CWLP-aquaporin complexes that recruit PP2A-β”, as a part of PP2A enzyme, close to the aquaporin moiety where it dephosphorylates two crucial Ser residues and induces closure of the aquaporin water channels. The closure of water channels renders cells more tolerant to plasmolysis and plants to dehydration. This unique effect was observed not only in Arabidopsis, but also in potato plants over expressing the CWLP, suggesting a possible usage in crop plants as a valve that reduces loss of water or/and elevates cold resistance. The CWLP is a member of the HyPRP protein family that all possess structurally similar 8CM domain, predicted to localize to PM lipid rafts. In this study, two additional highly homologous HyPRP proteins were also studied. The GPRP showed the same localization and it’s over expression increased tolerance to lack of water. However, the third one, PRP940, despite sharing high homology in the 8CM domain, is completely different and is assembled in parallel to cortical microtubules in the cell. Moreover, our data suggest that this protein is not involved in rendering plants resistant to lack of water. We suggest implying CWLP as a tool for better regulation of water maintenance in crop plants.

Plant cell monographs, 2019

Actin and myosin (i.e., the actomyosin system) play pivotal roles in plants, including organelle ... more Actin and myosin (i.e., the actomyosin system) play pivotal roles in plants, including organelle movement, cytoplasmic streaming, cell expansion, responses to microbes, cell signaling, and cell division. Among the plant biological processes attributed to actin and myosin function, understanding their precise role in cell division has been one of the more challenging problems to address. The difficulties in linking actomyosin function to cell division come in large part from inconsistent actin labeling in the cell division apparatus to the mild cell division phenotypes of actomyosin mutants. While the latter can be explained by functional redundancy, the presence of actin and myosin in the mitotic spindle has been somewhat controversial. Nonetheless, genetically encoded live actin and myosin probes have confirmed some classic microscopy results reported decades ago while also uncovering unique structures associated with the plant cell division machinery. In this chapter, we discuss how early microscopic work and recent live cell imaging data are beginning to provide a more unified view on how the actomyosin system facilitates cell division in plants.

Autophagy, Dec 19, 2020

The caspase-like vacuolar processing enzyme (VPE) is a key factor in programmed cell death (PCD) ... more The caspase-like vacuolar processing enzyme (VPE) is a key factor in programmed cell death (PCD) associated with plant stress responses. Growth medium lacking a carbon source and dark conditions caused punctate labeling of 35S::VPE1-GFP (StVPE1-GFP) in potato leaves. Under conditions of carbon starvation, VPE activity and PCD symptoms strongly increased in BY-2 cells, but to a much lesser extent in VPE-RNAi BY-2 cells. During extended exposure to carbon starvation, VPE expression and activity levels peaked, with a gradual increase in BY-2 cell death. Histological analysis of StVPE1-GFP in BY-2 cells showed that carbon starvation induces its translocation from the endoplasmic reticulum to the central vacuole through tonoplast engulfment. Exposure of BY-2 culture to the macroautophagy/autophagy inhibitor concanamycin A led to, along with an accumulation of autophagic bodies, accumulation of StVPE1-GFP in the cell vacuole. This accumulation did not occur in the presence of 3-methyladenine, an inhibitor of early-stage autophagy. BY-2 cells constitutively expressing RFP-StATG8IL, an autophagosome marker, showed colocalization with the StVPE1-GFP protein in the cytoplasm and vacuole. RNAi silencing of the core autophagy component ATG4 in BY-2 cells reduced VPE activity and cell death. These results are the first to suggest that VPE translocates to the cell vacuole through the autophagy pathway, leading to PCD.

Methods in molecular biology, 2023

Protoplasma, Apr 1, 2007

Narcissus tazetta is one of the major geophyte crops worldwide, but little is known about its cel... more Narcissus tazetta is one of the major geophyte crops worldwide, but little is known about its cell biology. The narcissus storage organ was studied by monitoring scale cell biology during the growth stage and dormancy, and it was found that amyloplasts gradually increased in size and reached a maximum at dormancy. In parallel, microtubules changed their organisation: during the growth phase (February to March) they were oblique; during April and May, microtubules formed a network with round "holes"; by late June and the beginning of July, when dormancy started, they were organised in parallel arrays. The holes formed in the microtubule array corresponded to amyloplasts. A closer look showed that during a short time window, while the plants were preparing for dormancy, the microtubules surrounded the amyloplasts. In vitro reconfirmation of this phenomenon was obtained when fluorescent bovine brain microtubules enwrapped isolated amyloplasts that had been purified between April and July but not those purified between January and March. Interestingly, protease treatment of amyloplasts did not completely prevent binding of microtubules, which suggests the existence of a protease-resistant factor that docks microtubules to the outer membrane of amyloplasts.

BMC Genomics, Jun 25, 2014

Background: The change from juvenile to mature phase in woody plants is often accompanied by a gr... more Background: The change from juvenile to mature phase in woody plants is often accompanied by a gradual loss of rooting ability, as well as by reduced microRNA (miR) 156 and increased miR172 expression. Results: We characterized the population of miRNAs of Eucalyptus grandis and compared the gradual reduction in miR156 and increase in miR172 expression during development to the loss of rooting ability. Forty known and eight novel miRNAs were discovered and their predicted targets are listed. The expression pattern of nine miRNAs was determined during adventitious root formation in juvenile and mature cuttings. While the expression levels of miR156 and miR172 were inverse in juvenile and mature tissues, no mutual relationship was found between high miR156 expression and rooting ability, or high miR172 expression and loss of rooting ability. This is shown both in E. grandis and in E. brachyphylla, in which explants that underwent rejuvenation in tissue culture conditions were also examined. Conclusions: It is suggested that in these Eucalyptus species, there is no correlation between the switch of miR156 with miR172 expression in the stems and the loss of rooting ability.

Israel Journal of Plant Sciences, Feb 25, 2020

In the original proposal we planned to focus on two proteins related to the actin cytoskeleton: T... more In the original proposal we planned to focus on two proteins related to the actin cytoskeleton: TCH2, a touch-induced calmodulin-like protein which was found by us to interact with the IQ domain of myosin VIII, ATM1; and ERD10, a dehydrin which was found to associate with actin filaments. As reported previously, no other dehydrins were found to interact with actin filaments. In addition so far we were unsuccessful in confirming the interaction of TCH2 with myosin VIII using other methods. In addition, no other myosin light chain candidates were found in a yeast two hybrid survey. Nevertheless we have made a significant progress in our studies of the role of myosins in plant cells. Plant myosins have been implicated in various cellular activities, such as cytoplasmic streaming (1, 2), plasmodesmata function (3-5), organelle movement (6-10), cytokinesis (4, 11, 12), endocytosis (4, 5, 13-15) and targeted RNA transport (16). Plant myosins belong to two main groups of unconventional m...

Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, my... more Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck regions to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins, CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a member of the myosin VIII class. Here, usingin vivoandin vitroassays we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among ten CML isoforms tested forin plantabinding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals usingin plantasplit-luciferase protein-interaction assays.In vitro,recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. Subcellular localization analysis indicated that CaM, CML13, and...

Clonal propagation of plants by induction of adventitious roots (ARs) from stem cuttings is a req... more Clonal propagation of plants by induction of adventitious roots (ARs) from stem cuttings is a requisite step in breeding programs. Nevertheless, a major barrier exists for propagating valuable plants that naturally have low capacity to form ARs. Due to the central role of auxin in organogenesis, indole-3-butyric acid (IBA) is often utilized, yet many recalcitrant plants do not form ARs in response to such treatment. We describe the synthesis and screening of a focused library of synthetic auxin conjugates inEucalyptus grandiscuttings, highlighting 4-chlorophenoxyacetic acid-L-tryptophan-OMe as a competent enhancer of adventitious rooting in a number of recalcitrant woody plants. Comprehensive metabolic and functional analyses revealed that this activity is engendered by prolonged auxin signaling due to initial fast uptake and slow release and clearance of the free auxin 4-chlorophenoxyacetic acid. This work highlights the utility of a slow-release strategy for bioactive compounds an...

Frontiers in Plant Science

Argania spinosa trees have attracted attention in recent years due to their high resistance to ex... more Argania spinosa trees have attracted attention in recent years due to their high resistance to extreme climate conditions. Initial domestication activities practiced in Morocco. Here we report on selection and vegetative propagation of A. spinosa trees grown in Israel. Trees yielding relatively high amounts of fruit were propagated by rooting of stem cuttings. High variability in rooting ability was found among the 30 clones selected. In-depth comparison of a difficult-to-root (ARS7) and easy-to-root (ARS1) clone revealed that the rooted cuttings of ARS7 have a lower survival rate than those of ARS1. In addition, histological analysis of the adventitious root primordia showed many abnormal fused primordia in ARS7. Hormone profiling revealed that while ARS1 accumulates more cytokinin, ARS7 accumulates more auxin, suggesting different auxin-to-cytokinin ratios underlying the different rooting capabilities. The hypothesized relationship between rooting and grafting abilities was addres...

The C-type hybrid-proline-rich protein (HyPRP) AtCWLP and its homolog AtPRP940 are referred as ce... more The C-type hybrid-proline-rich protein (HyPRP) AtCWLP and its homolog AtPRP940 are referred as cell wall (CW)-plasma-membrane (PM) linker proteins, but little is known about their functions. Here we show that N-terminal proline-rich domains of CWLP and PRP940, containing glycosylated hydroxyproline residues, contact the CW, while their C-terminal 8CM domains function as PM-scaffolds. Both proteins are detected in PM nanodomains (PM-ND) and show co-localization and co-immunoprecipitation with aquaporins PIP2;1 and PIP2;7. Inhibition of actin polymerization by latrunculin B promotes CWLP-endosome appearance, while blocking the actomyosin-based transport by a truncated form of myosin XI-K relaxes lateral boundaries of CWLP-PIP2;1 PD-NDs. Mass spectrometry data indicate that CWLP co-purifies with dynamins implicated in fission of endocytic PD-ND invaginations. Lack of co-localization and co-immunoprecipitation with aquaporin-binding flotillin (FLOT2) indicates that CWLP and PRP940 mark ...

The original objectives of the approved proposal were: 1. To construct a YFP fused Arabidopsis cD... more The original objectives of the approved proposal were: 1. To construct a YFP fused Arabidopsis cDNA library in a mammalian expression vector. 2. To infect the library into a host fibroblast cell line and to screen for new cytoskeletal associated proteins using an automated microscope. 3. Isolate the new genes. 4. Characterize their role in plants. The project was approved as a feasibility study to allow proof of concept that would entail building the YFP library and picking up a couple of positive clones using the fluorescent screen. We report here on the construction of the YFP library, the development of the automatic microscope, the establishment of the screen and the isolation of positive clones that are plant cDNAs encoding cytoskeleton associated proteins. The rational underling a screen of plant library in fibroblasts is based on the high conservation of the cytoskeleton building blocks, actin and tubulin, between the two kingdoms (80-90% homology at the level of amino acids ...

Ocal microscopy. A. Root of 5-day-old seedling. Scale bar: 50 μm. B. Lateral root of 20-day-old s... more Ocal microscopy. A. Root of 5-day-old seedling. Scale bar: 50 μm. B. Lateral root of 20-day-old seedling. Scale bar: 20 μm. C(1) and C(2). Two images of the same 20-day-old seedling root. C(1) shows the root cap, scale bar: 20 μm, and C(2) shows the upper part. Scale bar: 50 μm. A similar pattern of GFP-ATM1 localization is seen in all roots: diffuse at the root cap, then dots, then more polarized organization along the transverse sides. D. GFP-ATM1 in root hair, scale bar: 10 μm. Arrows show the direction of the root caps.<b>Copyright information:</b>Taken from "Different subcellular localizations and functions of Arabidopsis myosin VIII"http://www.biomedcentral.com/1471-2229/8/3BMC Plant Biology 2008;8():3-3.Published online 8 Jan 2008PMCID:PMC2275265.

The Cytoskeleton, 2019

Journal of Cell Science, 2002

The effect of fibroblast growth factor (FGF) on mature chondrocytes, the cells responsible for ax... more The effect of fibroblast growth factor (FGF) on mature chondrocytes, the cells responsible for axial skeletal development, is growth attenuation rather than stimulation. This singular response has been linked to signaling via FGF receptor 3 (FGFR3), partly because mutations causing chronic FGFR3 activation lead to various human disorders of bone growth. In order to study how FGF inhibits growth, we analyzed its effect on a rat chondrocyte-derived cell line. We show that the FGF-induced growth arrest occurs at the G1 phase,accompanied by profound changes in gene expression and cytoskeletal organization. Within minutes of binding, FGF induces tyrosine kinase activity in the focal substrate adhesions where it colocalizes with vinculin. Upon FGF stimulation, FGFR3 is selectively removed from the focal adhesions, which is followed by their disassembly and disruption of the organized cytoskeleton. Multiple genes are induced following FGF stimulation in chondrocytes, which has been shown b...

Israel Journal of Plant Sciences, 2020

Forest trees possess high genetic diversity and high heterozygosity which allow adaptation to cha... more Forest trees possess high genetic diversity and high heterozygosity which allow adaptation to changing environmental conditions. There is a tendency to propagate successful and unique genotypes, which are identified at their mature stage in the forests, for future improvement programs and conservation purposes. However, vegetative propagation of mature forest trees is still a challenge in many conifers. In this study, we focused on improving the rooting of cuttings of mature and old Pinus halepensis and its hybrids. We observed that storage of cuttings before rooting at 4°C for 4 weeks and prolong immersion of cuttings in a solution containing 400 mg/l of indole-3-butyric acid, 5 mg/l of the auxin conjugate 2-(2,4-dichlorophenoxy)propanoic acid-glycine methyl ester, and 0.01% of Amistar fungicide significantly improved rooting of mature cuttings. The active ingredient in Amistar is azoxystrobin, an uncoupler of respiration, which seems to directly promote rooting. Rooted cuttings of...

Current opinion in plant biology, 2014

Myosins are molecular motors that move along actin-filament tracks. Plants express two main class... more Myosins are molecular motors that move along actin-filament tracks. Plants express two main classes of myosins, myosin VIII and myosin XI. Along with their relatively conserved sequence and functions, plant myosins have acquired some unique features. Myosin VIII has the enzymatic characteristics of a tension sensor and/or a tension generator, similar to functions found in other eukaryotes. Interestingly, class XI plant myosins have gained a novel function that consists of propelling the exceptionally rapid cytoplasmic streaming. This specific class includes the fastest known translocating molecular motors, which can reach an extremely high velocity of about 60μms(-1). However, the enzymatic properties and mechanistic basis for these remarkable manifestations are not yet fully understood. Here we review recent progress in understanding the uniqueness of plant myosins, while emphasizing the unanswered questions.