@article{ma_shatil-cohen_ben-dor_wigoda_perera_im_diminshtein_yu_boss_moshelion_et al._2014, title={Do phosphoinositides regulate membrane water permeability of tobacco protoplasts by enhancing the aquaporin pathway?}, volume={241}, ISSN={0032-0935 1432-2048}, url={http://dx.doi.org/10.1007/s00425-014-2216-x}, DOI={10.1007/s00425-014-2216-x}, abstractNote={Enhancing the membrane content of PtdInsP 2 , the already-recognized protein-regulating lipid, increased the osmotic water permeability of tobacco protoplasts, apparently by increasing the abundance of active aquaporins in their membranes. While phosphoinositides are implicated in cell volume changes and are known to regulate some ion channels, their modulation of aquaporins activity has not yet been reported for any organism. To examine this, we compared the osmotic water permeability (P f) of protoplasts isolated from tobacco (Nicotiana tabacum) cultured cells (NT1) with different (genetically lowered or elevated relative to controls) levels of inositol trisphosphate (InsP3) and phosphatidyl inositol [4,5] bisphosphate (PtdInsP2). To achieve this, the cells were transformed with, respectively, the human InsP3 5-phosphatase ('Ptase cells') or human phosphatidylinositol (4) phosphate 5-kinase ('PIPK cells'). The mean P f of the PIPK cells was several-fold higher relative to that of controls and Ptase cells. Three results favor aquaporins over the membrane matrix as underlying this excessive P f: (1) transient expression of the maize aquaporin ZmPIP2;4 in the PIPK cells increased P f by 12-30 μm s(-1), while in the controls only by 3-4 μm s(-1). (2) Cytosol acidification-known to inhibit aquaporins-lowered the P f in the PIPK cells down to control levels. (3) The transcript of at least one aquaporin was elevated in the PIPK cells. Together, the three results demonstrate the differences between the PIPK cells and their controls, and suggest a hitherto unobserved regulation of aquaporins by phosphoinositides, which could occur through direct interaction or indirect phosphoinositides-dependent cellular effects.}, number={3}, journal={Planta}, publisher={Springer Science and Business Media LLC}, author={Ma, Xiaohong and Shatil-Cohen, Arava and Ben-Dor, Shifra and Wigoda, Noa and Perera, Imara Y. and Im, Yang Ju and Diminshtein, Sofia and Yu, Ling and Boss, Wendy F. and Moshelion, Menachem and et al.}, year={2014}, month={Dec}, pages={741–755} } @article{ischebeck_werner_krishnamoorthy_lerche_meijón_stenzel_löfke_wiessner_im_perera_et al._2013, title={Phosphatidylinositol 4,5-Bisphosphate Influences PIN Polarization by Controlling Clathrin-Mediated Membrane Trafficking in Arabidopsis}, volume={25}, ISSN={1040-4651 1532-298X}, url={http://dx.doi.org/10.1105/tpc.113.116582}, DOI={10.1105/tpc.113.116582}, abstractNote={AbstractThe functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P2 levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)–green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A–induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P2 in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P2 in the control of clathrin dynamics and in auxin distribution in Arabidopsis.}, number={12}, journal={The Plant Cell}, publisher={American Society of Plant Biologists (ASPB)}, author={Ischebeck, Till and Werner, Stephanie and Krishnamoorthy, Praveen and Lerche, Jennifer and Meijón, Mónica and Stenzel, Irene and Löfke, Christian and Wiessner, Theresa and Im, Yang Ju and Perera, Imara Y. and et al.}, year={2013}, month={Dec}, pages={4894–4911} } @article{dieck_wood_brglez_rojas-pierce_boss_2012, title={Increasing phosphatidylinositol (4,5) bisphosphate biosynthesis affects plant nuclear lipids and nuclear functions}, volume={57}, ISSN={["1873-2690"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84861821133&partnerID=MN8TOARS}, DOI={10.1016/j.plaphy.2012.05.011}, abstractNote={In order to characterize the effects of increasing phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P(2)) on nuclear function, we expressed the human phosphatidylinositol (4)-phosphate 5-kinase (HsPIP5K) 1α in Nicotiana tabacum (NT) cells. The HsPIP5K-expressing (HK) cells had altered nuclear lipids and nuclear functions. HK cell nuclei had 2-fold increased PIP5K activity and increased steady state PtdIns(4,5)P(2). HK nuclear lipid classes showed significant changes compared to NT (wild type) nuclear lipid classes including increased phosphatidylserine (PtdSer) and phosphatidylcholine (PtdCho) and decreased lysolipids. Lipids isolated from protoplast plasma membranes (PM) were also analyzed and compared with nuclear lipids. The lipid profiles revealed similarities and differences in the plasma membrane and nuclei from the NT and transgenic HK cell lines. A notable characteristic of nuclear lipids from both cell types is that PtdIns accounts for a higher mol% of total lipids compared to that of the protoplast PM lipids. The lipid molecular species composition of each lipid class was also analyzed for nuclei and protoplast PM samples. To determine whether expression of HsPIP5K1α affected plant nuclear functions, we compared DNA replication, histone 3 lysine 9 acetylation (H3K9ac) and phosphorylation of the retinoblastoma protein (pRb) in NT and HK cells. The HK cells had a measurable decrease in DNA replication, histone H3K9 acetylation and pRB phosphorylation.}, journal={PLANT PHYSIOLOGY AND BIOCHEMISTRY}, publisher={Elsevier BV}, author={Dieck, Catherine B. and Wood, Austin and Brglez, Irena and Rojas-Pierce, Marcela and Boss, Wendy F.}, year={2012}, month={Aug}, pages={32–44} } @article{boss_im_2012, title={Phosphoinositide Signaling}, volume={63}, ISSN={["1545-2123"]}, DOI={10.1146/annurev-arplant-042110-103840}, abstractNote={ All things flow and change…even in the stillest matter there is unseen flux and movement. Attributed to Heraclitus (530–470 BC) From The Story of Philosophy by Will Durant ( 28 ) Heraclitus, a Greek philosopher, was thinking on a much larger scale than molecular signaling; however, his visionary comments are an important reminder for those studying signaling today. Even in unstimulated cells, signaling pathways are in constant metabolic flux and provide basal signals that travel throughout the organism. In addition, negatively charged phospholipids, such as the polyphosphorylated inositol phospholipids, provide a circuit board of on/off switches for attracting or repelling proteins that define the membranes of the cell. This template of charged phospholipids is sensitive to discrete changes and metabolic fluxes—e.g., in pH and cations—which contribute to the oscillating signals in the cell. The inherent complexities of a constantly fluctuating system make understanding how plants integrate and process signals challenging. In this review we discuss one aspect of lipid signaling: the inositol family of negatively charged phospholipids and their functions as molecular sensors and regulators of metabolic flux in plants. }, journal={ANNUAL REVIEW OF PLANT BIOLOGY, VOL 63}, author={Boss, Wendy F. and Im, Yang Ju}, year={2012}, pages={409–429} } @article{boss_sederoff_im_moran_grunden_perera_2010, title={Basal Signaling Regulates Plant Growth and Development}, volume={154}, ISSN={["0032-0889"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-77957739073&partnerID=MN8TOARS}, DOI={10.1104/pp.110.161232}, abstractNote={The term signal transduction refers to the classical paradigm where an external stimulus is sensed and initiates an increase in second messengers. Each second messenger transmits and amplifies the signal by activating a subset of downstream pathways. This complex network of interwoven downstream}, number={2}, journal={PLANT PHYSIOLOGY}, author={Boss, Wendy F. and Sederoff, Heike Winter and Im, Yang Ju and Moran, Nava and Grunden, Amy M. and Perera, Imara Y.}, year={2010}, month={Oct}, pages={439–443} } @article{haidar_boss_2009, title={Blue light induced changes in inositol 1,4,5-trisphosphate in Cuscuta campestris seedlings}, volume={49}, ISSN={["1365-3180"]}, DOI={10.1111/j.1365-3180.2009.00732.x}, abstractNote={SummaryPrevious studies revealed that blue light stimulates and red light inhibits prehaustoria development in young seedlings of the parasitic weed Cuscuta campestris (field dodder). This study showed a positive correlation between blue light induced increases in inositol 1,4,5‐trisphosphate (IP3) and blue light‐mediation of prehaustoria development, prior to host attachment. Blue light induced a significant increase in the level of IP3, with a peak at about 30 min. Thereafter, the level of IP3 declined to the resting value after 2 h of blue light. Irradiation with 10 min red light pulse applied directly at the end of each 0.05, 0.1, 0.15, 0.2, 0.3, 1, 2 and 4 h blue light significantly reduced IP3, while high levels of IP3 were observed after 10 min far‐red pulse. The G‐protein inhibitor pertussis toxin inhibited prehaustoria developed under blue light, suggesting that receptor‐coupled G‐proteins are likely to be involved in prehaustoria development. These results are the first in vivo demonstration of a possible role for IP3 as a second messenger in the blue light signal transduction process in prehaustoria development in Cuscuta.}, number={6}, journal={WEED RESEARCH}, author={Haidar, M. A. and Boss, W. F.}, year={2009}, month={Dec}, pages={628–633} } @article{im_ji_lee_killens_grunden_boss_2009, title={Expression of Pyrococcus furiosus Superoxide Reductase in Arabidopsis Enhances Heat Tolerance}, volume={151}, ISSN={["1532-2548"]}, DOI={10.1104/pp.109.145409}, abstractNote={Abstract Plants produce reactive oxygen species (ROS) in response to environmental stresses sending signaling cues, which, if uncontrolled, result in cell death. Like other aerobic organisms, plants have ROS-scavenging enzymes, such as superoxide dismutase (SOD), which removes superoxide anion radical (O2  −) and prevents the production and buildup of toxic free radicals. However, increasing the expression of cytosolic SODs is complex, and increasing their production in vivo has proven to be challenging. To avoid problems with endogenous regulation of gene expression, we expressed a gene from the archaeal hyperthermophile Pyrococcus furiosus that reduces O2  −. P. furiosus uses superoxide reductase (SOR) rather than SOD to remove superoxide. SOR is a thermostable enzyme that reduces O2  − in a one-electron reduction without producing oxygen. We show that P. furiosus SOR can be produced as a functional enzyme in planta and that plants producing SOR have enhanced tolerance to heat, light, and chemically induced ROS. Stress tolerance in the SOR-producing plants correlates positively with a delayed increase in ROS-sensitive transcripts and a decrease in ascorbate peroxidase activity. The SOR plants provide a good model system to study the impact of cytosolic ROS on downstream signaling in plant growth and development. Furthermore, this work demonstrates that this synthetic approach for reducing cytosolic ROS holds promise as a means for improving stress tolerance in crop plants.}, number={2}, journal={PLANT PHYSIOLOGY}, author={Im, Yang Ju and Ji, Mikyoung and Lee, Alice and Killens, Rushyannah and Grunden, Amy M. and Boss, Wendy F.}, year={2009}, month={Oct}, pages={893–904} } @article{khodakovskaya_sword_wu_perera_boss_brown_sederoff_2010, title={Increasing inositol (1,4,5)-trisphosphate metabolism affects drought tolerance, carbohydrate metabolism and phosphate-sensitive biomass increases in tomato}, volume={8}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-73949085190&partnerID=MN8TOARS}, DOI={10.1111/j.1467-7652.2009.00472.x}, abstractNote={SummaryInositol‐(1,4,5)‐trisphosphate (InsP3) is a second messenger in plants that increases in response to many stimuli. The metabolic consequences of this signalling pathway are not known. We reduced the basal level of InsP3 in tomato (Solanum lycopersicum cv. Micro‐Tom) by expressing the human type I inositol polyphosphate 5‐phosphatase (InsP 5‐ptase) gene. Transgenic lines producing InsP 5‐ptase protein had between 15% and 30% of the basal InsP3 level of control plants. This increased hydrolysis of InsP3 caused dramatic increases in drought tolerance, vegetative biomass and lycopene and hexose concentrations in the fruits. Transcript profiling of root, leaf and fruit tissues identified a small group of genes, including a cell‐wall invertase inhibitor gene, that were differentially regulated in all tissues of the InsP 5‐ptase expressing plants. Significant differences were found in the amounts of carbohydrates and organic phosphate in these plants. Plants with increased hydrolysis of InsP3 in the cytosol also showed increased net CO2‐fixation and sucrose export into sink tissue and storage of hexoses in the source leaves. The increase in biomass was dependent on the supply of inorganic phosphate in the nutrient medium. Uptake and storage of phosphate was increased in the transgene expressing lines. This suggests that in tomato, increased flux through the inositol phosphate pathway uncoupled phosphate sensing from phosphate metabolism. Altering the second messenger, InsP3, revealed multiple coordinated changes in development and metabolism in tomato that have potential for crop improvement.}, number={2}, journal={Plant Biotechnology Journal}, author={Khodakovskaya, M. and Sword, C. and Wu, Q. and Perera, I. Y. and Boss, W. F. and Brown, C. S. and Sederoff, Heike}, year={2010}, pages={170–183} } @article{ma_shor_diminshtein_yu_im_perera_lomax_boss_moran_2009, title={Phosphatidylinositol (4,5)Bisphosphate Inhibits K+-Efflux Channel Activity in NT1 Tobacco Cultured Cells}, volume={149}, ISSN={["1532-2548"]}, DOI={10.1104/pp.108.129007}, abstractNote={Abstract In the animal world, the regulation of ion channels by phosphoinositides (PIs) has been investigated extensively, demonstrating a wide range of channels controlled by phosphatidylinositol (4,5)bisphosphate (PtdInsP2). To understand PI regulation of plant ion channels, we examined the in planta effect of PtdInsP2 on the K+-efflux channel of tobacco (Nicotiana tabacum), NtORK (outward-rectifying K channel). We applied a patch clamp in the whole-cell configuration (with fixed “cytosolic” Ca2+ concentration and pH) to protoplasts isolated from cultured tobacco cells with genetically manipulated plasma membrane levels of PtdInsP2 and cellular inositol (1,4,5)trisphosphate: “Low PIs” had depressed levels of these PIs, and “High PIs” had elevated levels relative to controls. In all of these cells, K channel activity, reflected in the net, steady-state outward K+ currents (IK), was inversely related to the plasma membrane PtdInsP2 level. Consistent with this, short-term manipulations decreasing PtdInsP2 levels in the High PIs, such as pretreatment with the phytohormone abscisic acid (25 μ  m) or neutralizing the bath solution from pH 5.6 to pH 7, increased IK (i.e. NtORK activity). Moreover, increasing PtdInsP2 levels in controls or in abscisic acid-treated high-PI cells, using the specific PI-phospholipase C inhibitor U73122 (2.5–4 μ  m), decreased NtORK activity. In all cases, IK decreases stemmed largely from decreased maximum attainable NtORK channel conductance and partly from shifted voltage dependence of channel gating to more positive potentials, making it more difficult to activate the channels. These results are consistent with NtORK inhibition by the negatively charged PtdInsP2 in the internal plasma membrane leaflet. Such effects are likely to underlie PI signaling in intact plant cells.}, number={2}, journal={PLANT PHYSIOLOGY}, author={Ma, Xiaohong and Shor, Oded and Diminshtein, Sofia and Yu, Ling and Im, Yang Ju and Perera, Imara and Lomax, Aaron and Boss, Wendy F. and Moran, Nava}, year={2009}, month={Feb}, pages={1127–1140} } @misc{perera_hung_moore_stevenson-paulik_boss_2008, title={Transgenic Arabidopsis Plants Expressing the Type 1 Inositol 5-Phosphatase Exhibit Increased Drought Tolerance and Altered Abscisic Acid Signaling}, volume={20}, ISSN={["1040-4651"]}, DOI={10.1105/tpc.108.061374}, abstractNote={AbstractThe phosphoinositide pathway and inositol-1,4,5-trisphosphate (InsP3) are implicated in plant responses to stress. To determine the downstream consequences of altered InsP3-mediated signaling, we generated transgenic Arabidopsis thaliana plants expressing the mammalian type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), which specifically hydrolyzes soluble inositol phosphates and terminates the signal. Rapid transient Ca2+ responses to a cold or salt stimulus were reduced by ∼30% in these transgenic plants. Drought stress studies revealed, surprisingly, that the InsP 5-ptase plants lost less water and exhibited increased drought tolerance. The onset of the drought stress was delayed in the transgenic plants, and abscisic acid (ABA) levels increased less than in the wild-type plants. Stomatal bioassays showed that transgenic guard cells were less responsive to the inhibition of opening by ABA but showed an increased sensitivity to ABA-induced closure. Transcript profiling revealed that the drought-inducible ABA-independent transcription factor DREB2A and a subset of DREB2A-regulated genes were basally upregulated in the InsP 5-ptase plants, suggesting that InsP3 is a negative regulator of these DREB2A-regulated genes. These results indicate that the drought tolerance of the InsP 5-ptase plants is mediated in part via a DREB2A-dependent pathway and that constitutive dampening of the InsP3 signal reveals unanticipated interconnections between signaling pathways.}, number={10}, journal={PLANT CELL}, author={Perera, Imara Y. and Hung, Chiu-Yueh and Moore, Candace D. and Stevenson-Paulik, Jill and Boss, Wendy F.}, year={2008}, month={Oct}, pages={2876–2893} } @article{davis_im_dubin_tomer_boss_2007, title={Arabidopsis phosphatidylinositol phosphate kinase 1 binds F-actin and recruits phosphatidylinositol 4-kinase beta 1 to the actin cytoskeleton (Retracted article. See vol. 284, pg. 16060, 2009)}, volume={282}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M611728200}, abstractNote={The actin cytoskeleton can be influenced by phospholipids and lipid-modifying enzymes. In animals the phosphatidylinositol phosphate kinases (PIPKs) are associated with the cytoskeleton through a scaffold of proteins; however, in plants such an interaction was not clear. Our approach was to determine which of the plant PIPKs interact with actin and determine whether the PIPK-actin interaction is direct. Our results indicate that AtPIPK1 interacts directly with actin and that the binding is mediated through a predicted linker region in the lipid kinase. AtPIPK1 also recruits AtPI4Kbeta1 to the cytoskeleton. Recruitment of AtPI4Kbeta1 to F-actin was dependent on the C-terminal catalytic domain of phosphatidylinositol-4-phosphate 5-kinase but did not require the presence of the N-terminal 251 amino acids, which includes 7 putative membrane occupation and recognition nexus motifs. In vivo studies confirm the interaction of plant lipid kinases with the cytoskeleton and suggest a role for actin in targeting PIPKs to the membrane.}, number={19}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Davis, Amanda J. and Im, Yang Ju and Dubin, Joshua S. and Tomer, Kenneth B. and Boss, Wendy F.}, year={2007}, month={May}, pages={14121–14131} } @article{galvao_kota_soderblom_goshe_boss_2008, title={Characterization of a new family of protein kinases from Arabidopsis containing phosphoinositide 3/4-kinase and ubiquitin-like domains}, volume={409}, ISSN={["1470-8728"]}, DOI={10.1042/bj20070959}, abstractNote={At least two of the genes predicted to encode type II PI4K (phosphoinositide 4-kinase) in Arabidopsis thaliana (thale cress), namely AtPI4Kγ4 and AtPI4Kγ7, encode enzymes with catalytic properties similar to those of members of the PIKK (phosphoinositide kinase-related kinase) family. AtPI4Kγ4 and AtPI4Kγ7 undergo autophosphorylation and phosphorylate serine/threonine residues of protein substrates, but have no detectable lipid kinase activity. AtPI4Kγ4 and AtPI4Kγ7 are members of a subset of five putative AtPI4Ks that contain N-terminal UBL (ubiquitin-like) domains. In vitro analysis of AtPI4Kγ4 indicates that it interacts directly with, and phosphorylates, two proteins involved in the ubiquitin–proteasome system, namely UFD1 (ubiquitin fusion degradation 1) and RPN10 (regulatory particle non-ATPase 10). On the basis of the present results, we propose that AtPI4Kγ4 and AtPI4Kγ7 should be designated UbDKγ4 and UbDKγ7 (ubiquitin-like domain kinases γ4 and γ7). These UBL-domain-containing AtPI4Ks correspond to a new PIKK subfamily of protein kinases. Furthermore, UFD1 and RPN10 phosphorylation represents an additional mechanism by which their function can be regulated.}, journal={BIOCHEMICAL JOURNAL}, author={Galvao, Rafaelo M. and Kota, Uma and Soderblom, Erik J. and Goshe, Michael B. and Boss, Wendy F.}, year={2008}, month={Jan}, pages={117–127} } @article{im_perera_brglez_davis_stevenson-paulik_phillippy_johannes_allen_boss_2007, title={Increasing plasma membrane phosphatidylinositol(4,5)bisphosphate biosynthesis increases phosphoinositide metabolism in Nicotiana tabacum}, volume={19}, ISSN={["1532-298X"]}, DOI={10.1105/tpc.107.051367}, abstractNote={AbstractA genetic approach was used to increase phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2] biosynthesis and test the hypothesis that PtdInsP kinase (PIPK) is flux limiting in the plant phosphoinositide (PI) pathway. Expressing human PIPKIα in tobacco (Nicotiana tabacum) cells increased plasma membrane PtdIns(4,5)P2 100-fold. In vivo studies revealed that the rate of 32Pi incorporation into whole-cell PtdIns(4,5)P2 increased >12-fold, and the ratio of [3H]PtdInsP2 to [3H]PtdInsP increased 6-fold, but PtdInsP levels did not decrease, indicating that PtdInsP biosynthesis was not limiting. Both [3H]inositol trisphosphate and [3H]inositol hexakisphosphate increased 3-and 1.5-fold, respectively, in the transgenic lines after 18 h of labeling. The inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] binding assay showed that total cellular Ins(1,4,5)P3/g fresh weight was >40-fold higher in transgenic tobacco lines; however, even with this high steady state level of Ins(1,4,5)P3, the pathway was not saturated. Stimulating transgenic cells with hyperosmotic stress led to another 2-fold increase, suggesting that the transgenic cells were in a constant state of PI stimulation. Furthermore, expressing Hs PIPKIα increased sugar use and oxygen uptake. Our results demonstrate that PIPK is flux limiting and that this high rate of PI metabolism increased the energy demands in these cells.}, number={5}, journal={PLANT CELL}, author={Im, Yang Ju and Perera, Imara Y. and Brglez, Irena and Davis, Amanda J. and Stevenson-Paulik, Jill and Phillippy, Brian Q. and Johannes, Eva and Allen, Nina S. and Boss, Wendy F.}, year={2007}, month={May}, pages={1603–1616} } @article{im_davis_perera_johannes_allen_boss_2007, title={The N-terminal membrane occupation and recognition nexus domain of Arabidopsis phosphatidylinositol phosphate kinase 1 regulates enzyme activity}, volume={282}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M611342200}, abstractNote={The type I B family of phosphatidylinositol phosphate kinases (PIPKs) contain a characteristic region of Membrane Occupation and Recognition Nexus (MORN) motifs at the N terminus. These MORN motifs are not found in PIPKs from other eukaryotes. To understand the impact of the additional N-terminal domain on protein function and subcellular distribution, we expressed truncated and full-length versions of AtPIPK1, one member of this family of PIPKs, in Escherichia coli and in tobacco cells grown in suspension culture. Deletion of the N-terminal MORN domain (amino acids 1–251) of AtPIPK1 increased the specific activity of the remaining C-terminal peptide (ΔMORN) >4-fold and eliminated activation by phosphatidic acid (PtdOH). PtdOH activation could also be eliminated by mutating Pro396 to Ala (P396A) in the predicted linker region between the MORN and the kinase homology domains. AtPIPK1 is product-activated and the MORN domain binds PtdIns(4,5)P2. Adding back the MORN peptide to ΔMORN or to the PtdOH-activated full-length protein increased activity ∼2-fold. Furthermore, expressing the MORN domain in vivo increased the plasma membrane PtdInsP kinase activity. When cells were exposed to hyperosmotic stress, the MORN peptide redistributed from the plasma membrane to a lower phase or endomembrane fraction. In addition, endogenous PtdInsP kinase activity increased in the endomembrane fraction of hyperosmotically stressed cells. We conclude that the MORN peptide can regulate both the function and distribution of the enzyme in a manner that is sensitive to the lipid environment.}, number={8}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Im, Yang Ju and Davis, Amanda J. and Perera, Imara Y. and Johannes, Eva and Allen, Nina S. and Boss, Wendy F.}, year={2007}, month={Feb}, pages={5443–5452} } @article{perera_hung_brady_muday_boss_2006, title={A universal role for inositol 1,4,5-trisphosphate-mediated signaling in plant gravitropism}, volume={140}, ISSN={["1532-2548"]}, DOI={10.1104/pp.105.075119}, abstractNote={Abstract Inositol 1,4,5-trisphosphate (InsP3) has been implicated in the early signaling events of plants linking gravity sensing to the initiation of the gravitropic response. However, at present, the contribution of the phosphoinositide signaling pathway in plant gravitropism is not well understood. To delineate the role of InsP3 in plant gravitropism, we generated Arabidopsis (Arabidopsis thaliana) plants constitutively expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme that specifically hydrolyzes InsP3. The transgenic plants show no significant differences in growth and life cycle compared to wild-type plants, although basal InsP3 levels are reduced by greater than 90% compared to wild-type plants. With gravistimulation, InsP3 levels in inflorescence stems of transgenic plants show no detectable change, whereas in wild-type plant inflorescences, InsP3 levels increase approximately 3-fold within the first 5 to 15 min of gravistimulation, preceding visible bending. Furthermore, gravitropic bending of the roots, hypocotyls, and inflorescence stems of the InsP 5-ptase transgenic plants is reduced by approximately 30% compared with the wild type. Additionally, the cold memory response of the transgenic plants is attenuated, indicating that InsP3 contributes to gravisignaling in the cold. The transgenic roots were shown to have altered calcium sensitivity in controlling gravitropic response, a reduction in basipetal indole-3-acetic acid transport, and a delay in the asymmetric auxin-induced β-glucuronidase expression with gravistimulation as compared to the controls. The compromised gravitropic response in all the major axes of growth in the transgenic Arabidopsis plants reveals a universal role for InsP3 in the gravity signal transduction cascade of plants.}, number={2}, journal={PLANT PHYSIOLOGY}, author={Perera, IY and Hung, CY and Brady, S and Muday, GK and Boss, WF}, year={2006}, month={Feb}, pages={746–760} } @article{perera_davis_galanopilou_im_boss_2005, title={Characterization and comparative analysis of Arabidopsis phosphatidylinositol phosphate 5-kinase 10 reveals differences in Arabidopsis and human phosphatidylinositol phosphate kinases}, volume={579}, ISSN={["1873-3468"]}, DOI={10.1016/j.febslet.2005.05.018}, abstractNote={ Arabidopsis phosphatidylinositol phosphate (PtdInsP) kinase 10 (AtPIPK10; At4g01190) is shown to be a functional enzyme of the subfamily A, type I AtPtdInsP kinases. It is biochemically distinct from AtPIPK1 (At1g21980), the only other previously characterized AtPtdInsP kinase which is of the B subfamily. AtPIPK10 has the same K m, but a 10‐fold lower V max than AtPIPK1 and it is insensitive to phosphatidic acid. AtPIPK10 transcript is most abundant in inflorescence stalks and flowers, whereas AtPIPK1 transcript is present in all tissues. Comparative analysis of recombinant AtPIPK10 and AtPIPK1 with recombinant HsPIPKIα reveals that the Arabidopsis enzymes have roughly 200‐ and 20‐fold lower V max/K m, respectively. These data reveal one explanation for the longstanding mystery of the relatively low phosphatidylinositol‐(4,5)‐bisphosphate:phosphatidylinositol‐4‐phosphate ratio in terrestrial plants.}, number={16}, journal={FEBS LETTERS}, author={Perera, IY and Davis, AJ and Galanopilou, D and Im, YJ and Boss, WF}, year={2005}, month={Jun}, pages={3427–3432} } @article{im_ji_lee_boss_grunden_2005, title={Production of a thermostable archaeal superoxide reductase in plant cells}, volume={579}, ISSN={["1873-3468"]}, DOI={10.1016/j.febslet.2005.09.015}, abstractNote={ Pyrococcus furiosus superoxide reductase (SOR) is a thermostable archaeal enzyme that reduces superoxide without producing oxygen. When produced as a fusion protein with the green fluorescent protein in plant cells, P. furiosus SOR is located in the cytosol and nucleus. The recombinant SOR enzyme retains its function and heat stability when assayed in vitro. Importantly, expressing SOR in plant cells enhances their survival at high temperature indicating that it functions in vivo. The archaeal SOR provides a novel mechanism to reduce superoxide and demonstrates the potential for using archaeal genes to alter eukaryotic metabolism.}, number={25}, journal={FEBS LETTERS}, author={Im, YJ and Ji, MK and Lee, AM and Boss, WF and Grunden, AM}, year={2005}, month={Oct}, pages={5521–5526} } @article{davis_perera_boss_2004, title={Cyclodextrins enhance recombinant phosphatidylinositol phosphate kinase activity}, volume={45}, ISSN={["1539-7262"]}, DOI={10.1194/jlr.D400005-JLR200}, abstractNote={Inositol lipid kinases have been studied extensively in both plant and animal systems. However, major limitations for in vitro studies of recombinant lipid kinases are the low specific activity and instability of the purified proteins. Our goal was to determine if cyclodextrins would provide an effective substrate delivery system and enhance the specific activity of lipid kinases. For these studies, we have used recombinant Arabidopsis thaliana phosphatidylinositol phosphate kinase 1 (At PIPK1). At PIPK1 was produced as a fusion protein with glutathione-S-transferase and purified on glutathione-Sepharose beads. A comparison of lipid kinase activity using substrate prepared in α-, β-, or γ-cyclodextrin indicated that β-cyclodextrin was most effective and enhanced lipid kinase activity 6-fold compared with substrate prepared in Triton X-100-mixed micelles. We have optimized reaction conditions and shown that product can be recovered from the cyclodextrin-treated recombinant protein, which reveals a potential method for automating the assay for pharmacological screening.}, number={9}, journal={JOURNAL OF LIPID RESEARCH}, author={Davis, AJ and Perera, IY and Boss, WF}, year={2004}, month={Sep}, pages={1783–1789} } @article{akesson_persson_love_boss_widell_sommarin_2005, title={Overexpression of the Ca2+-binding protein calreticulin in the endoplasmic reticulum improves growth of tobacco cell suspensions (Nicotiana tabacum) in high-Ca2+ medium}, volume={123}, ISSN={["1399-3054"]}, DOI={10.1111/j.1399-3054.2004.00434.x}, abstractNote={ Calreticulin (CRT) is a eukaryotic, highly conserved, Ca2+‐binding protein predominantly located in the endoplasmic reticulum (ER) lumen. In addition to being involved in the regulation of cellular Ca2+, calreticulin is a key quality control element during protein folding in the ER lumen. Tobacco (Nicotiana tabacum L.) suspension cells overexpressing a maize CRT (CRT1a) were used here to examine the properties of CRT in growing plant cells with respect to stress exposure. The endogenous CRT gene was induced rapidly after subculturing of the cells to new medium. In accordance, the CRT protein levels increased, peaking at days 3–4. At day 5, when the CRT transcript levels had levelled off, a further increase in endogenous CRT expression was obtained when the cells were treated with excess Ca2+ or the N‐linked glycosylation inhibitor tunicamycin. Whereas the response to Ca2+ occurred within 30 min, the induction by tunicamycin took several hours to be established. Transforming tobacco cells with maize CRT1a, under a constitutive mannopine synthase promoter, resulted in a stable level of expressed CRT1a during the growth cycle compared with endogenous CRT. The CRTs showed differences in attached glycans, but both contained the high mannose‐rich‐type glycans characteristic of ER proteins. In agreement with an ER location, both tobacco CRT and the transgene product CRT1a codistributed with the ER marker NADH cytochrome c reductase after density gradient centrifugation of microsomal fractions from tobacco cells. Increased production of CRT, as was obtained in the transgenic tobacco cell lines, made cells more tolerant than wild‐type cells to high Ca2+ during growth. These data suggest that overexpression of CRT1a in plant cells results in a more efficient calcium buffering capacity in the ER. }, number={1}, journal={PHYSIOLOGIA PLANTARUM}, author={Akesson, A and Persson, S and Love, J and Boss, WF and Widell, S and Sommarin, M}, year={2005}, month={Jan}, pages={92–99} } @article{kimbrough_salinas-mondragon_boss_brown_sederoff_2004, title={The fast and transient transcriptional network of gravity and mechanical stimulation in the Arabidopsis root Apex}, volume={136}, ISSN={["1532-2548"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-16544389872&partnerID=MN8TOARS}, DOI={10.1104/pp.104.044594}, abstractNote={Abstract Plant root growth is affected by both gravity and mechanical stimulation (Massa GD, Gilroy S [2003] Plant J 33: 435–445). A coordinated response to both stimuli requires specific and common elements. To delineate the transcriptional response mechanisms, we carried out whole-genome microarray analysis of Arabidopsis root apices after gravity stimulation (reorientation) and mechanical stimulation and monitored transcript levels of 22,744 genes in a time course during the first hour after either stimulus. Rapid, transient changes in the relative abundance of specific transcripts occurred in response to gravity or mechanical stimulation, and these transcript level changes reveal clusters of coordinated events. Transcriptional regulation occurs in the root apices within less than 2 min after either stimulus. We identified genes responding specifically to each stimulus as well as transcripts regulated in both signal transduction pathways. Several unknown genes were specifically induced only during gravitropic stimulation (gravity induced genes). We also analyzed the network of transcriptional regulation during the early stages of gravitropism and mechanical stimulation.}, number={1}, journal={PLANT PHYSIOLOGY}, author={Kimbrough, JM and Salinas-Mondragon, R and Boss, WE and Brown, CS and Sederoff, HW}, year={2004}, month={Sep}, pages={2790–2805} } @misc{wyatt_tsou_robertson_boss_2004, title={Transgenic plants with increased calcium stores}, volume={6,753,462}, number={2004 June 22}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Wyatt, S. and Tsou, P.-L. and Robertson, D. and Boss, W.}, year={2004} } @article{stevenson-paulik_love_boss_2003, title={Differential regulation of two Arabidopsis type III phosphatidylinositol 4-kinase isoforms. A regulatory role for the pleckstrin homology domain}, volume={132}, ISSN={["0032-0889"]}, DOI={10.1104/pp.103.021758}, abstractNote={Abstract Here, we compare the regulation and localization of the Arabidopsis type III phosphatidylinositol (PtdIns) 4-kinases, AtPI4Kα1 and AtPI4Kβ1, in Spodoptera frugiperda (Sf9) insect cells. We also explore the role of the pleckstrin homology (PH) domain in regulating AtPI4Kα1. Recombinant kinase activity was found to be differentially sensitive to PtdIns-4-phosphate (PtdIns4P), the product of the reaction. The specific activity of AtPI4Kα1 was inhibited 70% by 0.5 mm PtdIns4P. The effect of PtdIns4P was not simply due to charge because AtPI4Kα1 activity was stimulated approximately 50% by equal concentrations of the other negatively charged lipids, PtdIns3P, phosphatidic acid, and phosphatidyl-serine. Furthermore, inhibition of AtPI4Kα1 by PtdIns4P could be alleviated by adding recombinant AtPI4Kα1 PH domain, which selectively binds to PtdIns4P (Stevenson et al., 1998). In contrast, the specific activity of AtPI4Kβ1, which does not have a PH domain, was stimulated 2-fold by PtdIns4P but not other negatively charged lipids. Visualization of green fluorescent protein fusion proteins in insect cells revealed that AtPI4Kα1 was associated primarily with membranes in the perinuclear region, whereas AtPI4Kβ1 was in the cytosol and associated with small vesicles throughout the cytoplasm. Expression of AtPI4Kα1 without the PH domain in the insect cells compromised PtdIns 4-kinase activity and caused mislocalization of the kinase. The green fluorescent protein-PH domain alone was associated with intracellular membranes and the plasma membrane. In vitro, the PH domain appeared to be necessary for association of AtPI4Kα1 with fine actin filaments. These studies support the idea that the Arabidopsis type III PtdIns 4-kinases are responsible for distinct phosphoinositide pools.}, number={2}, journal={PLANT PHYSIOLOGY}, author={Stevenson-Paulik, J and Love, J and Boss, WF}, year={2003}, month={Jun}, pages={1053–1064} } @article{persson_rosenquist_svensson_galvao_boss_sommarin_2003, title={Phylogenetic analyses and expression studies reveal two distinct groups of calreticulin isoforms in higher plants}, volume={133}, ISSN={["0032-0889"]}, DOI={10.1104/pp.103.024943}, abstractNote={Abstract Calreticulin (CRT) is a multifunctional protein mainly localized to the endoplasmic reticulum in eukaryotic cells. Here, we present the first analysis, to our knowledge, of evolutionary diversity and expression profiling among different plant CRT isoforms. Phylogenetic studies and expression analysis show that higher plants contain two distinct groups of CRTs: a CRT1/CRT2 group and a CRT3 group. To corroborate the existence of these isoform groups, we cloned a putative CRT3 ortholog from Brassica rapa. The CRT3 gene appears to be most closely related to the ancestral CRT gene in higher plants. Distinct tissue-dependent expression patterns and stress-related regulation were observed for the isoform groups. Furthermore, analysis of posttranslational modifications revealed differences in the glycosylation status among members within the CRT1/CRT2 isoform group. Based on evolutionary relationship, a new nomenclature for plant CRTs is suggested. The presence of two distinct CRT isoform groups, with distinct expression patterns and posttranslational modifications, supports functional specificity among plant CRTs and could account for the multiple functional roles assigned to CRTs.}, number={3}, journal={PLANT PHYSIOLOGY}, author={Persson, S and Rosenquist, M and Svensson, K and Galvao, R and Boss, WF and Sommarin, M}, year={2003}, month={Nov}, pages={1385–1396} } @article{perera_love_heilmann_thompson_boss_2002, title={Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase}, volume={129}, ISSN={["1532-2548"]}, DOI={10.1104/pp.003426}, abstractNote={Abstract To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP3) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP3. The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP3 compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP3 in both wild-type and transgenic cells. However, even with stimulation, InsP3 levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP3 signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP2), the lipid precursor of InsP3, was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP2 metabolism showed that the activity of the PtdInsP2-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of 32P into PtdInsP2 in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP2 synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP2 synthesis as a regulatory step in this system.}, number={4}, journal={PLANT PHYSIOLOGY}, author={Perera, IY and Love, J and Heilmann, I and Thompson, WF and Boss, WF}, year={2002}, month={Aug}, pages={1795–1806} } @article{persson_love_tsou_robertson_thompson_boss_2002, title={When a day makes a difference. Interpreting data from endoplasmic reticulum-targeted green fluorescent protein fusions in cells grown in suspension culture}, volume={128}, ISSN={1532-2548 0032-0889}, url={http://dx.doi.org/10.1104/pp.010840}, DOI={10.1104/pp.010840}, abstractNote={The stability of the self-contained structure of green fluorescent protein (GFP) has made it the most widely utilized fluorescent marker for gene expression and subcellular localization studies ([Chalfie et al., 1994][1]; [Tsien, 1998][2]; [De Giorgi et al., 1999][3]; [Haseloff et al., 1999][4]).}, number={2}, journal={Plant Physiology}, publisher={Oxford University Press (OUP)}, author={Persson, S. and Love, J. and Tsou, P. L. and Robertson, D. and Thompson, W. F. and Boss, W. F.}, year={2002}, pages={341–344} } @article{perera_hilmann_chang_boss_kaufman_2001, title={A role for inositol 1,4,5-trisphosphate in gravitropic signaling and the retention of cold-perceived gravistimulation of oat shoot pulvini}, volume={125}, ISSN={["1532-2548"]}, DOI={10.1104/pp.125.3.1499}, abstractNote={Abstract Plants sense positional changes relative to the gravity vector. To date, the signaling processes by which the perception of a gravistimulus is linked to the initiation of differential growth are poorly defined. We have investigated the role of inositol 1,4,5-trisphosphate (InsP3) in the gravitropic response of oat (Avena sativa) shoot pulvini. Within 15 s of gravistimulation, InsP3 levels increased 3-fold over vertical controls in upper and lower pulvinus halves and fluctuated in both pulvinus halves over the first minutes. Between 10 and 30 min of gravistimulation, InsP3 levels in the lower pulvinus half increased 3-fold over the upper. Changes in InsP3 were confined to the pulvinus and were not detected in internodal tissue, highlighting the importance of the pulvinus for both graviperception and response. Inhibition of phospholipase C blocked the long-term increase in InsP3, and reduced gravitropic bending by 65%. Short-term changes in InsP3 were unimpaired by the inhibitor. Gravitropic bending of oat plants is inhibited at 4°C; however, the plants retain the information of a positional change and respond at room temperature. Both short- and long-term changes in InsP3 were present at 4°C. We propose a role for InsP3 in the establishment of tissue polarity during the gravitropic response of oat pulvini. InsP3 may be involved in the retention of cold-perceived gravistimulation by providing positional information in the pulvini prior to the redistribution of auxin.}, number={3}, journal={PLANT PHYSIOLOGY}, author={Perera, IK and Hilmann, I and Chang, SC and Boss, WF and Kaufman, PB}, year={2001}, month={Mar}, pages={1499–1507} } @inproceedings{boss_perera_love_heilmann_2001, title={Altering phosphoinositide metabolism by expressing human type I inositol polyphosphate 5 ' phosphatase in tobacco cells}, volume={12}, number={2001 Nov}, booktitle={Molecular Biology of the Cell}, author={Boss, W. F. and Perera, I. Y. and Love, J. and Heilmann, I.}, year={2001}, pages={820} } @article{shank_su_brglez_boss_dewey_boston_2001, title={Induction of lipid metabolic enzymes during the endoplasmic reticulum stress response in plants}, volume={126}, ISSN={["0032-0889"]}, DOI={10.1104/pp.126.1.267}, abstractNote={AbstractThe endoplasmic reticulum (ER) stress response is a signal transduction pathway activated by the perturbation of normal ER metabolism. We used the maize (Zea mays)floury-2 (fl2) mutant and soybean (Glycine max) suspension cultures treated with tunicamycin (Tm) to investigate the ER stress response as it relates to phospholipid metabolism in plants. Four key phospholipid biosynthetic enzymes, including DG kinase and phosphatidylinositol (PI) 4-phosphate 5-kinase were up-regulated in the fl2 mutant, specifically in protein body fractions where the mutation has its greatest effect. The third up-regulated enzyme, choline-phosphate cytidylyltransferase, was regulated by fl2 gene dosage and developmental signals. Elevated accumulation of the fourth enzyme, PI 4-kinase, was observed in the fl2 endosperm and soybean cells treated with Tm. The activation of these phospholipid biosynthetic enzymes was accompanied by alterations in membrane lipid synthesis and accumulation. The fl2 mutant exhibited increased PI content in protein body membranes at 18 d after pollination and more than 3-fold higher triacylglycerol accumulation in the endosperm by 36 d after pollination. Incorporation of radiolabeled acetate into phospholipids in soybean culture cells increased by about 30% with Tm treatment. The coordinated regulation of ER stress related proteins and multiple components of phospholipid biosynthesis is consistent with signaling through a common pathway. We postulate that the plant ER stress response has an important role in general plant metabolism, and more specifically in integrating the synthesis of protein and lipid reserves to allow proper seed formation.}, number={1}, journal={PLANT PHYSIOLOGY}, author={Shank, KJ and Su, P and Brglez, I and Boss, WF and Dewey, RE and Boston, RS}, year={2001}, month={May}, pages={267–277} } @article{heilmann_perera_gross_boss_2001, title={Plasma membrane phosphatidylinositol 4,5-bisphosphate levels decrease with time in culture}, volume={126}, ISSN={["1532-2548"]}, DOI={10.1104/pp.126.4.1507}, abstractNote={Abstract During the stationary phase of growth, after 7 to 12 d in culture, the levels of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) decreased by 75% in plasma membranes of the red alga Galdieria sulphuraria. Concomitant with the decrease in PtdInsP2 levels in plasma membranes, there was an increase in PtdInsP2 in microsomes, suggesting that the levels of plasma membrane PtdInsP2 are regulated differentially. The decline of PtdInsP2 in plasma membranes was accompanied by a 70% decrease in the specific activity of PtdInsP kinase and by reduced levels of protein cross-reacting with antisera against a conserved PtdInsP kinase domain. Upon osmotic stimulation, the loss of PtdInsP2from the plasma membrane increased from 10% in 7-d-old cells to 60% in 12-d-old cells, although the levels of inositol 1,4,5-trisphosphate (InsP3) produced in whole cells were roughly equal at both times. When cells with low plasma membrane PtdInsP2 levels were osmotically stimulated, a mild osmotic stress (12.5 mm KCl) activated PtdInsP kinase prior to InsP3 production, whereas in cells with high plasma membrane PtdInsP2, more severe stress (250 mm KCl) was required to induce an increase in PtdInsP kinase activity. The differential regulation of a plasma membrane signaling pool of PtdInsP2 is discussed with regard to the implications for understanding the responsive state of cells.}, number={4}, journal={PLANT PHYSIOLOGY}, author={Heilmann, I and Perera, IY and Gross, W and Boss, WF}, year={2001}, month={Aug}, pages={1507–1518} } @article{persson_wyatt_love_thompson_robertson_boss_2001, title={The Ca2+ status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants}, volume={126}, ISSN={["1532-2548"]}, DOI={10.1104/pp.126.3.1092}, abstractNote={Abstract To investigate the endoplasmic reticulum (ER) Ca2+ stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca2+-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca2+uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent 45Ca2+accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca2+ ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of45Ca2+ released, and a 2- to 3-fold increase in the amount of 45Ca2+ retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca2+ pool. In addition, CRTtransgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca2+-containing medium to Ca2+-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca2+ stores and thereby enhances the survival of plants grown in low Ca2+ medium.}, number={3}, journal={PLANT PHYSIOLOGY}, author={Persson, S and Wyatt, SE and Love, J and Thompson, WF and Robertson, D and Boss, WF}, year={2001}, month={Jul}, pages={1092–1104} } @article{ransom-hodgkins_brglez_wang_boss_2000, title={Calcium-regulated proteolysis of eEF1A}, volume={122}, ISSN={["0032-0889"]}, DOI={10.1104/pp.122.3.957}, abstractNote={Abstract Eukaryotic elongation factor 1α (eEF1A) can be post-translationally modified by the addition of phosphorylglycerylethanolamine (PGE). [14C]Ethanolamine was incorporated into the PGE modification, and with carrot (Daucus carota L.) suspension culture cells, eEF1A was the only protein that incorporated detectable quantities of [14C]ethanolamine (Ransom et al., 1998). When 1 mm CaCl2 was added to microsomes containing [14C]ethanolamine-labeled eEF1A ([14C]et-eEF1A), there was a 60% decrease in the amount of [14C]et-eEF1A recovered after 10 min. The loss of endogenous [14C]et-eEF1A was prevented by adding EGTA. Recombinant eEF1A, which did not contain the PGE modification, also was degraded by microsomes in a Ca2+-regulated manner, indicating that PGE modification was not necessary for proteolysis; however, it enabled us to quantify enodgenous eEF1A. By monitoring [14C]et-eEF1A, we found that treatment with phospholipase D or C, but not phospholipase A2, resulted in a decrease in [14C]et-eEF1A from carrot microsomes. The fact that there was no loss of [14C]et-eEF1A with phospholipase A2 treatment even in the presence of 1 mmCa2+ suggested that the loss of membrane lipids was not essential for eEF1A proteolysis and that lysolipids or fatty acids decreased proteolysis. At micromolar Ca2+ concentrations, proteolysis of eEF1A was pH sensitive. When 1 μmCaCl2 was added at pH 7.2, 35% of [14C]et-eEF1A was lost; while at pH 6.8, 10 μm CaCl2 was required to give a similar loss of protein. These data suggest that eEF1A may be an important downstream target for Ca2+ and lipid-mediated signal transduction cascades.}, number={3}, journal={PLANT PHYSIOLOGY}, author={Ransom-Hodgkins, WD and Brglez, I and Wang, XM and Boss, WF}, year={2000}, month={Mar}, pages={957–965} } @article{stevenson_perera_heilmann_persson_boss_2000, title={Inositol signaling and plant growth}, volume={5}, ISSN={1360-1385}, url={http://dx.doi.org/10.1016/s1360-1385(00)01652-6}, DOI={10.1016/S1360-1385(00)01652-6}, abstractNote={Living organisms have evolved to contain a wide variety of receptors and signaling pathways that are essential for their survival in a changing environment. Of these, the phosphoinositide pathway is one of the best conserved. The ability of the phosphoinositides to permeate both hydrophobic and hydrophilic environments, and their diverse functions within cells have contributed to their persistence in nature. In eukaryotes, phosphoinositides are essential metabolites as well as labile messengers that regulate cellular physiology while traveling within and between cells. The stereospecificity of the six hydroxyls on the inositol ring provides the basis for the functional diversity of the phosphorylated isomers that, in turn, generate a selective means of intracellular and intercellular communication for coordinating cell growth. Although such complexity presents a difficult challenge for bench scientists, it is ideal for the regulation of cellular functions in living organisms.}, number={6}, journal={Trends in Plant Science}, publisher={Elsevier BV}, author={Stevenson, Jill M and Perera, Imara Y and Heilmann, Ingo and Persson, Staffan and Boss, Wendy F}, year={2000}, month={Jun}, pages={252–258} } @article{heilmann_perera_gross_boss_1999, title={Changes in phosphoinositide metabolism with days in culture affect signal transduction pathways in Galdieria sulphuraria}, volume={119}, ISSN={["0032-0889"]}, DOI={10.1104/pp.119.4.1331}, abstractNote={Abstract The metabolism of phosphatidylinositol-4,5-bisphosphate (PIP2) changed during the culture period of the thermoacidophilic red alga Galdieria sulphuraria. Seven days after inoculation, the amount of PIP2 in the cells was 910 ± 100 pmol g−1fresh weight; by 12 d, PIP2 levels increased to 1200 ± 150 pmol g−1 fresh weight. In vitro assays indicated that phosphatidylinositol monophosphate (PIP) kinase specific activity increased from 75 to 230 pmol min−1mg−1 protein between d 7 and 12. When G. sulphuraria cells were osmostimulated, transient increases of up to 4-fold could be observed in inositol-1,4,5-trisphosphate (IP3) levels within 90 s, regardless of the age of the cells. In d-12 cells, the increase in IP3 was preceded by a transient increase of up to 5-fold in specific PIP kinase activity, whereas no such increase was detected after osmostimulation of d-7 cells. The increase in PIP kinase activity before IP3signaling in d-12 cells indicates that there is an additional pathway for regulation of phosphoinositide metabolism after stimulation other than an initial activation of phospholipase C. Also, the rapid activation of PIP2 biosynthesis in cells with already-high PIP2 levels suggests that the PIP2 present was not available for signal transduction. By comparing the response of the cells at d 7 and 12, we have identified two potentially distinct pools of PIP2.}, number={4}, journal={PLANT PHYSIOLOGY}, author={Heilmann, I and Perera, IY and Gross, W and Boss, WF}, year={1999}, month={Apr}, pages={1331–1339} } @misc{drobak_dewey_boss_1999, title={Phosphoinositide kinases and the synthesis of polyphosphoinositides in higher plant cells}, volume={189}, ISBN={["0-12-364593-X"]}, ISSN={["0074-7696"]}, DOI={10.1016/S0074-7696(08)61386-8}, abstractNote={Phosphoinositides are a family of inositol-containing phospholipids which are present in all eukaryotic cells. Although in most cells these lipids, with the exception of phosphatidylinositol, constitute only a very minor proportion of total cellular lipids, they have received immense attention by researchers in the past 15-20 years. This is due to the discovery that these lipids, rather than just having structural functions, play key roles in a wide range of important cellular processes. Much less is known about the plant phosphoinositides than about their mammalian counterparts. However, it has been established that a functional phosphoinositide system exists in plant cells and it is becoming increasingly clear that inositol-containing lipids are likely to play many important roles throughout the life of a plant. It is not our intention to give an exhaustive overview of all aspects of the field, but rather we focus on the phosphoinositide kinases responsible for the synthesis of all phosphorylated forms of phosphatidylinositol. Also, we mention some of the aspects of current phosphoinositide research which, in our opinion, are most likely to provide a suitable starting point for further research into the role of phosphoinositides in plants.}, journal={INTERNATIONAL REVIEW OF CYTOLOGY - A SURVEY OF CELL BIOLOGY, VOL 189}, author={Drobak, BK and Dewey, RE and Boss, WF}, year={1999}, pages={95–130} } @article{perera_heilmann_boss_1999, title={Transient and sustained increases in inositol 1,4,5-hisphosphate precede the differential growth response in gravistimulated maize pulvini}, volume={96}, ISSN={["1091-6490"]}, DOI={10.1073/pnas.96.10.5838}, abstractNote={ The internodal maize pulvinus responds to gravistimulation with differential cell elongation on the lower side. As the site of both graviperception and response, the pulvinus is an ideal system to study how organisms sense changes in orientation. We observed a transient 5-fold increase in inositol 1,4,5-trisphosphate (IP 3 ) within 10 s of gravistimulation in the lower half of the pulvinus, indicating that the positional change was sensed immediately. Over the first 30 min, rapid IP 3 fluctuations were observed between the upper and lower halves. Maize plants require a presentation time of between 2 and 4 h before the cells on the lower side of the pulvinus are committed to elongation. After 2 h of gravistimulation, the lower half consistently had higher IP 3, and IP 3 levels on the lower side continued to increase up to ≈5-fold over basal levels before visible growth. As bending became visible after 8–10 h, IP 3 levels returned to basal values. Additionally, phosphatidylinositol 4-phosphate 5-kinase activity in the lower pulvinus half increased transiently within 10 min of gravistimulation, suggesting that the increased IP 3 production was accompanied by an up-regulation of phosphatidylinositol 4,5-bisphosphate biosynthesis. Neither IP 3 levels nor phosphatidylinositol 4-phosphate 5-kinase activity changed in pulvini halves from vertical control plants. Our data indicate the involvement of IP 3 and inositol phospholipids in both short- and long-term responses to gravistimulation. As a diffusible second messenger, IP 3 provides a mechanism to transmit and amplify the signal from the perceiving to the responding cells in the pulvinus, coordinating a synchronized growth response. }, number={10}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Perera, IY and Heilmann, I and Boss, WF}, year={1999}, month={May}, pages={5838–5843} } @article{stevenson_perera_boss_1998, title={A phosphatidylinositol 4-kinase pleckstrin homology domain that binds phosphatidylinositol 4-monophosphate}, volume={273}, ISSN={["0021-9258"]}, DOI={10.1074/jbc.273.35.22761}, abstractNote={Pleckstrin homology (PH) domains are found in many proteins involved in signal transduction, including the family of large molecular mass phosphatidylinositol (PI) 4-kinases. Although the exact function of these newly discovered domains is unknown, it is recognized that they may influence enzyme regulation by binding different ligands. In this study, the recombinant PI 4-kinase PH domain was explored for its ability to bind to different phospholipids. First, we isolated partial cDNAs of the >7-kilobase transcripts of PI 4-kinases from carrot (DcPI4Kα) andArabidopsis (AtPI4Kα). The deduced primary sequences were 41% identical and 68% similar to rat and human PI 4-kinases and contained the telltale lipid kinase unique domain, PH domain, and catalytic domain. Antibodies raised against the expressed lipid kinase unique, PH, and catalytic domains identified a polypeptide of 205 kDa in Arabidopsis microsomes and an F-actin-enriched fraction from carrot cells. The 205-kDa immunoaffinity-purified Arabidopsis protein had PI 4-kinase activity. We have used the expressed PH domain to characterize lipid binding properties. The recombinant PH domain selectively bound to phosphatidylinositol 4-monophosphate (PI-4-P), phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), and phosphatidic acid and did not bind to the 3-phosphoinositides. The PH domain had the highest affinity for PI-4-P, the product of the reaction. Consideration is given to the potential impact that this has on cytoskeletal organization and the PI signaling pathway in cells that have a high PI-4-P/PI-4,5-P2 ratio.}, number={35}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Stevenson, JM and Perera, IY and Boss, WF}, year={1998}, month={Aug}, pages={22761–22767} } @inbook{heilmann_perera_stevenson_ransom_gross_boss_1998, title={Inositol lipid signaling: what can we learn from plants?}, booktitle={Advances in lipids research}, publisher={Sevilla, Spain: University of Sevilla Press}, author={Heilmann, I. and Perera, I. Y. and Stevenson, J. M. and Ransom, W. D. and Gross, W. and Boss, W. F.}, editor={J. Sanchez, E. Cerda-Olmedo and Martinez-Force, E.Editors}, year={1998}, pages={394–397} } @article{ransom_lao_gage_boss_1998, title={Phosphoglycerylethanolamine posttranslational modification of plant eukaryotic elongation factor 1 alpha}, volume={117}, ISSN={["0032-0889"]}, DOI={10.1104/pp.117.3.949}, abstractNote={Abstract Eukaryotic elongation factor 1α (eEF-1A) is a multifunctional protein. There are three known posttranslational modifications of eEF-1A that could potentially affect its function. Except for phosphorylation, the other posttranslational modifications have not been demonstrated in plants. Using matrix-assisted laser desorption/ionization-mass spectrometry and peptide mass mapping, we show that carrot (Daucus carotaL.) eEF-1A contains a phosphoglycerylethanolamine (PGE) posttranslational modification. eEF-1A was the only protein labeled with [14C]ethanolamine in carrot cells and was the predominant ethanolamine-labeled protein in Arabidopsis seedlings and tobacco (Nicotiana tabacum L.) cell cultures. In vivo-labeling studies using [3H]glycerol, [32P]Pi, [14C]myristic acid, and [14C]linoleic acid indicated that the entire phospholipid phosphatidylethanolamine is covalently attached to the protein. The PGE lipid modification did not affect the partitioning of eEF-1A in Triton X-114 or its actin-binding activity in in vitro assays. Our in vitro data indicate that this newly characterized posttranslational modification alone does not affect the function of eEF-1A. Therefore, the PGE lipid modification may work in combination with other posttranslational modifications to affect the distribution and the function of eEF-1A within the cell.}, number={3}, journal={PLANT PHYSIOLOGY}, author={Ransom, WD and Lao, PC and Gage, DA and Boss, WF}, year={1998}, month={Jul}, pages={949–960} }