bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Inositol 1,3,4-Trisphosphate 5-Kinase and Inositol 1,3,4,5-Tetrakisphosphate 6-Kinase in Immature Soybean Seeds
Authors: Phillippy Brian Q.
Published: July 2002

In extracts of immature soybean (Glycine max [L.] Merr.) seeds inositol tetrakisphosphate was formed from [3H]inositol 1,3,4-trisphosphate but not from [3H]inositol 1,4,5-trisphosphate. Inositol 1,3,4-trisphosphate kinase was purified to a specific activity of 3.55 min−1 mg−1 by polyethylenimine clarification and anion-exchange chromatography. The partially purified enzyme converted [3H]inositol 1,3,4-trisphosphate to inositol 1,3,4,5-tetrakisphosphate as the major product and inositol 1,3,4,6- and/or 1,2,3,4-tetrakisphosphate as the minor product. Subsequent experiments revealed a separate inositol 1,3,4,5-tetrakisphosphate 6-kinase activity, which could link these enzymes to inositol hexakisphosphate synthesis via the previously reported inositol 1,3,4,5,6-pentakisphosphate 2-kinase. The apparent Km values for inositol 1,3,4-trisphosphate kinase were 200 ± 0 nm for inositol 1,3,4-trisphosphate and 171 ± 4 μm for ATP, and the reaction was not reversible. The kinetics were such that no activity could be detected using unlabeled inositol 1,3,4-trisphosphate and [γ-32P]ATP, which suggested that other kinases may have been observed when less purified fractions were incubated with radiolabeled ATP. Inositol 1,3,4-trisphosphate kinase was nonspecifically inhibited more than 80% by various inositol polyphosphates at a concentration of 100 μm.
Evidence for a Cytoskeleton-Associated Binding Site Involved in Prolamine mRNA Localization to the Protein Bodies in Rice Endosperm Tissue1
Authors: Muench Douglas G. , Wu Yujia , Coughlan Sean J. , Okita Thomas W.
Published: July 2002

Previous studies have demonstrated that the mRNAs encoding the prolamine and glutelin storage proteins are localized to morphologically distinct membranes of the endoplasmic reticulum (ER) complex in developing rice (Oryza sativa L.) endosperm cells. To gain insight about this mRNA localization process, we investigated the association of prolamine polysomes on the ER that delimit the prolamine protein bodies (PBs). The bulk of the prolamine polysomes were resistant to extraction by 1% Triton X-100 either alone or together with puromycin, which suggests that these translation complexes are anchored to the PB surface through a second binding site in addition to the well-characterized ribosome-binding site of the ER-localized protein translocation complex. Suppression of translation initiation shows that these polysomes are bound through the mRNA, as shown by the simultaneous increase in the amounts of ribosome-free prolamine mRNAs and decrease in prolamine polysome content associated with the membrane-stripped PB fraction. The prolamine polysome-binding activity is likely to be associated with the cytoskeleton, based on the association of actin and tubulin with the prolamine polysomes and PBs after sucrose-density centrifugation.
Differential Expression of a Novel Gene in Response to Coronatine, Methyl Jasmonate, and Wounding in the Coi1Mutant of Arabidopsis1
Authors: Benedetti Celso E. , Costa Cinthia L. , Turcinelli Silvia R. , Arruda Paulo
Published: July 2002

Coronatine is a phytotoxin produced by some plant-pathogenic bacteria. It has been shown that coronatine mimics the action of methyl jasmonate (MeJA) in plants. MeJA is a plant-signaling molecule involved in stress responses such as wounding and pathogen attack. In Arabidopsis thaliana, MeJA is essential for pollen grain development. The coi1 (forcoronatine-insensitive) mutant of Arabidopsis, which is insensitive to coronatine and MeJA, produces sterile male flowers and shows an altered response to wounding. When the differential display technique was used, a message that was rapidly induced by coronatine in wild-type plants but not incoi1 was identified and the corresponding cDNA was cloned. The coronatine-induced gene ATHCOR1 (forA.thalianacoronatine-induced) is expressed in seedlings, mature leaves, flowers, and siliques but was not detected in roots. The expression of this gene was dramatically reduced in coi1plants, indicating that COI1 affects its expression.ATHCOR1 was rapidly induced by MeJA and wounding in wild-type plants. The sequence of ATHCOR1 shows no strong homology to known proteins. However, the predicted polypeptide contains a conserved amino acid sequence present in several bacterial, animal, and plant hydrolases and includes a potential ATP/GTP-binding-site motif (P-loop).
Contribution of Malic Enzyme, Pyruvate Kinase, Phosphoenolpyruvate Carboxylase, and the Krebs Cycle to Respiration and Biosynthesis and to Intracellular pH Regulation during Hypoxia in Maize Root Tips Observed by Nuclear Magnetic Resonance Imaging and Gas Chromatography-Mass Spectrometry1
Authors: Edwards Shaune , Nguyen Bich-Ty , Do Binh , Roberts Justin K.M.
Published: July 2002

In vivo pyruvate synthesis by malic enzyme (ME) and pyruvate kinase and in vivo malate synthesis by phosphoenolpyruvate carboxylase and the Krebs cycle were measured by 13C incorporation from [1-13C]glucose into glucose-6-phosphate, alanine, glutamate, aspartate, and malate. These metabolites were isolated from maize (Zea mays L.) root tips under aerobic and hypoxic conditions. 13C-Nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry were used to discern the positional isotopic distribution within each metabolite. This information was applied to a simple precursor-product model that enabled calculation of specific metabolic fluxes. In respiring root tips, ME was found to contribute only approximately 3% of the pyruvate synthesized, whereas pyruvate kinase contributed the balance. The activity of ME increased greater than 6-fold early in hypoxia, and then declined coincident with depletion of cytosolic malate and aspartate. We found that in respiring root tips, anaplerotic phosphoenolpyruvate carboxylase activity was high relative to ME, and therefore did not limit synthesis of pyruvate by ME. The significance of in vivo pyruvate synthesis by ME is discussed with respect to malate and pyruvate utilization by isolated mitochondria and intracellular pH regulation under hypoxia.
Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga1
Authors: Bauer Claudia S. , Plieth Christoph , Bethmann Birgit , Popescu Ondina , Hansen Ulf-Peter , Simonis Wilhelm , Schönknecht Gerald
Published: July 2002

The divalent cation Sr2+ induced repetitive transient spikes of the cytosolic Ca2+ activity [Ca2+]cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green algaEremosphaera viridis. [Ca2+]cymeasurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr2+-induced [Ca2+]cyoscillations. Sr2+ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca2+]cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca2+-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr2+-induced repetitive [Ca2+]cy spikes, whereas the compartmentalization of Sr2+ was not influenced. A repetitive Ca2+ release and Ca2+ re-uptake by the ER probably generated repetitive [Ca2+]cyspikes in E. viridis in the presence of Sr2+. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr2+-induced Ca2+ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca2+ channel. The blockage of Sr2+-induced repetitive [Ca2+]cy spikes by La3+ or Gd3+ indicated the necessity of a certain influx of divalent cations for sustained [Ca2+]cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca2+]cy oscillations in E. viridis.
Implications of a Developmental-Stage-Dependent Thylakoid-Bound Protease in the Stabilization of the Light-Harvesting Pigment-Protein Complex Serving Photosystem II during Thylakoid Biogenesis in Red Kidney Bean1
Authors: Tziveleka Leto-Aikaterini , Argyroudi-Akoyunoglou Joan H.
Published: July 2002

Intact etioplasts of bean (Phaseolus vulgaris) plants exhibit proteolytic activity against the exogenously added apoprotein of the light-harvesting pigment-protein complex serving photosystem II (LHCII) that increases as etiolation is prolonged. The activity increases in the membrane fraction but not in the stroma, where it remains low and constant and is mainly directed against LHCII and protochlorophyllide oxidoreductase. The thylakoid proteolytic activity, which is low in etioplasts of 6-d-old etiolated plants, increases in plants pretreated with a pulse of light or exposed to intermittent-light (ImL) cycles, but decreases during prolonged exposure to continuous light, coincident with chlorophyll (Chl) accumulation. To distinguish between the control of Chl and/or development on proteolytic activity, we used plants exposed to ImL cycles of varying dark-phase durations. In ImL plants exposed to an equal number of ImL cycles with short or long dark intervals (i.e. equal Chl accumulation but different developmental stage) proteolytic activity increased with the duration of the dark phase. In plants exposed to ImL for equal durations to such light-dark cycles (i.e. different Chl accumulation but same developmental stage) the proteolytic activity was similar. These results suggest that the protease, which is free to act under limited Chl accumulation, is dependent on the developmental stage of the chloroplast, and give a clue as to why plants in ImL with short dark intervals contain LHCII, whereas those with long dark intervals possess only photosystem-unit cores and lack LHCII.
Phosphorylated Nitrate Reductase and 14-3-3 Proteins
Authors: Athwal Gurdeep S. , Huber Joan L. , Huber Steven C.
Published: July 2002

The inactivation of phosphorylated nitrate reductase (NR) by the binding of 14-3-3 proteins is one of a very few unambiguous biological functions for 14-3-3 proteins. We report here that serine and threonine residues at the +6 to +8 positions, relative to the known regulatory binding site involving serine-543, are important in the interaction with GF14ω, a recombinant plant 14-3-3. Also shown is that an increase in ionic strength with KCl or inorganic phosphate, known physical effectors of NR activity, directly disrupts the binding of protein and peptide ligands to 14-3-3 proteins. Increased ionic strength attributable to KCl caused a change in conformation of GF14ω, resulting in reduced surface hydrophobicity, as visualized with a fluorescent probe. Similarly, it is shown that the 5′ isomer of AMP was specifically able to disrupt the inactive phosphorylated NR:14-3-3 complex. Using the 5′-AMP fluorescent analog trinitrophenyl-AMP, we show that there is a probable AMP-binding site on GF14ω.
Acid-Growth Response and α-Expansins in Suspension Cultures of Bright Yellow 2 Tobacco
Authors: Link Bruce M. , Cosgrove Daniel J.
Published: July 2002

The possibility that Bright Yellow 2 (BY2) tobacco (Nicotiana tabacum L.) suspension-cultured cells possess an expansin-mediated acid-growth mechanism was examined by multiple approaches. BY2 cells grew three times faster upon treatment with fusicoccin, which induces an acidification of the cell wall. Exogenous expansins likewise stimulated BY2 cell growth 3-fold. Protein extracted from BY2 cell walls possessed the expansin-like ability to induce extension of isolated walls. In western-blot analysis of BY2 wall protein, one band of 29 kD was recognized by anti-expansin antibody. Six different classes of α-expansin mRNA were identified in a BY2 cDNA library. Northern-blot analysis indicated moderate to low abundance of multiple α-expansin mRNAs in BY2 cells. From these results we conclude that BY2 suspension-cultured cells have the necessary components for expansin-mediated cell wall enlargement.
The Role of Cytosolic Potassium and pH in the Growth of Barley Roots
Authors: Walker David J. , Black Colin R. , Miller Anthony J.
Published: July 2002

In an earlier paper we showed that in fully developed barley (Hordeum vulgare L.) root epidermal cells a decrease in cytosolic K+ was associated with an acidification of the cytosol (D.J. Walker, R.A. Leigh, A.J. Miller [1996] Proc Natl Acad Sci USA 93: 10510–10514). To show that these changes in cytosolic ion concentrations contributed to the decreased growth of K+-starved roots, we first measured whether similar changes occurred in cells of the growing zone. Triple-barreled ion-selective microelectrodes were used to measure cytosolic K+ activity and pH in cells 0.5 to 1.0 mm from the root tip. In plants growing from 7 to 21 d after germination under K+-replete conditions, the mean values did not change significantly, with values ranging from 80 to 84 mm for K+ and 7.3 to 7.4 for pH. However, in K+-starved plants (external [K+], 2 μm), the mean cytosolic K+ activity and pH had declined to 44 mm and 7.0, respectively, after 14 d. For whole roots, sap osmolality was always lower in K+-starved than in K+-replete plants, whereas elongation rate and dry matter accumulation were significantly decreased after 14 and 16 d of K+ starvation. The rate of protein synthesis in root tips did not change for K+-replete plants but declined significantly with age in K+-starved plants. Butyrate treatment decreased cytosolic pH and diminished the rate of protein synthesis in K+-replete roots. Procaine treatment of K+-starved roots gave an alkalinization of the cytosol and increased protein synthesis rate. These results show that changes in both cytosolic pH and K+ can be significant factors in inhibiting protein synthesis and root growth during K+deficiency.
Ozone Sensitivity in Hybrid Poplar Is Correlated with a Lack of Defense-Gene Activation
Authors: Riehl Koch Jennifer , Scherzer Amy J. , Eshita Steven M. , Davis Keith R.
Published: July 2002

Ozone is a major gaseous pollutant thought to contribute to forest decline. Although the physiological and morphological responses of forest trees to ozone have been well characterized, little is known about the molecular basis for these responses. Our studies compared the response to ozone of ozone-sensitive and ozone-tolerant clones of hybrid poplar (Populus maximowizii × Populus trichocarpa) at the physiological and molecular levels. Gas-exchange analyses demonstrated clear differences between the ozone-sensitive clone 388 and the ozone-tolerant clone 245. Although ozone induced a decrease in photosynthetic rate and stomatal conductance in both clones, the magnitude of the decrease in stomatal conductance was significantly greater in the ozone-tolerant clone. RNA-blot analysis established that ozone-induced mRNA levels for phenylalanine ammonia-lyase, O-methyltransferase, a pathogenesis-related protein, and a wound-inducible gene were significantly higher in the ozone-tolerant than in the ozone-sensitive plants. Wound- and pathogen-induced levels of these mRNAs were also higher in the ozone-tolerant compared with the ozone-sensitive plants. The different physiological and molecular responses to ozone exposure exhibited by clones 245 and 388 suggest that ozone tolerance involves the activation of salicylic-acid- and jasmonic-acid-mediated signaling pathways, which may be important in triggering defense responses against oxidative stress.
Plastome Engineering of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Tobacco to Form a Sunflower Large Subunit and Tobacco Small Subunit Hybrid1
Authors: Kanevski Ivan , Maliga Pal , Rhoades Daniel F. , Gutteridge Steven
Published: July 2002

Targeted gene replacement in plastids was used to explore whether the rbcL gene that codes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, the key enzyme of photosynthetic CO2 fixation, might be replaced with altered forms of the gene. Tobacco (Nicotiana tabacum) plants were transformed with plastid DNA that contained the rbcL gene from either sunflower (Helianthus annuus) or the cyanobacteriumSynechococcus PCC6301, along with a selectable marker. Three stable lines of transformants were regenerated that had alteredrbcL genes. Those containing therbcL gene for cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase produced mRNA but no large subunit protein or enzyme activity. Those tobacco plants expressing the sunflower large subunit synthesized a catalytically active hybrid form of the enzyme composed of sunflower large subunits and tobacco small subunits. A third line expressed a chimeric sunflower/tobacco large subunit arising from homologous recombination within the rbcL gene that had properties similar to the hybrid enzyme. This study demonstrated the feasibility of using a binary system in which different forms of the rbcL gene are constructed in a bacterial host and then introduced into a vector for homologous recombination in transformed chloroplasts to produce an active, chimeric enzyme in vivo.
Pvlea-18, a Member of a New Late-Embryogenesis-Abundant Protein Family That Accumulates during Water Stress and in the Growing Regions of Well-Irrigated Bean Seedlings1
Authors: Colmenero-Flores José M. , Moreno Liz P. , Smith Claudia E. , Covarrubias Alejandra A.
Published: July 2002

Pvlea-18 is a novel stress gene whose transcript is present in the dry embryo and the endosperm from bean (Phaseolus vulgaris) seeds. It accumulates in vegetative tissues in response to water deficit and abscisic acid application (J.M. Colmenero-Flores, F. Campos, A. Garciarrubio, A.A. Covarrubias [1997] Plant Mol Biol 35: 393–405). We show that the Pvlea-18 gene encodes a 14-kD protein that accumulates during late embryogenesis. Related proteins have been detected in both monocots and dicots, indicating that PvLEA-18 is a member of a new family of LEA (LateEmbryogenesis Abundant) proteins. We also show that the PvLEA-18 transcript and protein accumulate not only in different organs of the bean seedlings during water stress but also in well-irrigated seedlings. This accumulation occurs in seedling regions with more negative values of water and osmotic potentials, such as the growing region of the hypocotyl. This phenomenon has not previously been described for LEA proteins. Immunohistochemical localization showed that the PvLEA-18 protein is present in the nucleus and cytoplasm of all cell types, with a higher accumulation in the epidermis and vascular cylinder tissues, particularly in protoxylem cells and root meristematic tissues. We found a similar localization but a higher abundance in water-stressed seedlings.
Accumulation of Small Heat-Shock Protein Homologs in the Endoplasmic Reticulum of Cortical Parenchyma Cells in Mulberry in Association with Seasonal Cold Acclimation1
Authors: Ukaji Norifumi , Kuwabara Chikako , Takezawa Daisuke , Arakawa Keita , Yoshida Shizuo , Fujikawa Seizo
Published: July 2002

Cortical parenchyma cells of mulberry (Morus bombycis Koidz.) trees acquire extremely high freezing tolerance in winter as a result of seasonal cold acclimation. The amount of total proteins in endoplasmic reticulum (ER)-enriched fractions isolated from these cells increased in parallel with the process of cold acclimation. Protein compositions in the ER-enriched fraction also changed seasonally, with a prominent accumulation of 20-kD (WAP20) and 27-kD (WAP27) proteins in winter. The N-terminal amino acid sequence of WAP20 exhibited homology to ER-localized small heat-shock proteins (smHSPs), whereas that of WAP27 did not exhibit homology to any known proteins. Like other smHSPs, WAP20 formed a complex of high molecular mass in native-polyacrylamide gel electrophoresis. Furthermore, not only WAP20 but also 21-kD proteins reacted with antibodies against WAP20. Fractionation of the crude microsomes by isopycnic sucrose-gradient centrifugation revealed that both WAP27 and WAP20 were distributed on a density corresponding to the fractions with higher activity of ER marker enzyme, suggesting localization of these proteins in the ER. When ER-enriched fractions were treated with trypsin in the absence of detergent, WAP20 and WAP27 were undigested, suggesting localization of these proteins inside the ER vesicle. The accumulation of a large quantity of smHSPs in the ER in winter as a result of seasonal cold acclimation indicates that these proteins may play a significant role in the acquisition of freezing tolerance in cortical parenchyma cells of mulberry trees.
A Maize Glycine-Rich Protein Is Synthesized in the Lateral Root Cap and Accumulates in the Mucilage1
Authors: Matsuyama Takashi , Satoh Hidetaka , Yamada Yasuyuki , Hashimoto Takashi
Published: July 2002

The root cap functions in the perception of gravity, the protection of the root apical meristem, and facilitation of the passage of roots through the soil, but the genes involved in these functions are poorly understood. Here we report the isolation of a root-specific gene from the cap of maize (Zea mays L.) primary root by cDNA subtraction and differential screening. The gene zmGRP4(Z.maysglycinerich protein 4) encodes a member of the glycine-rich proteins with a putative signal peptide at the amino terminus. The deduced molecular mass of mature zmGRP4 is 14.4 kD. In situ-hybridization analysis has shown zmGRP4 to be strongly expressed in the lateral root cap and weakly expressed in the root epidermis. A polyclonal antibody raised against recombinant zmGRP4 detected a protein of 36 kD in the insoluble protein fraction extracted from the root tip and the root proper, indicating posttranslational modification(s) of zmGRP4. Immunohistochemical analysis showed the accumulation of zmGRP4 in the mucilage that covers the root tip. These results indicate that lateral root-cap cells secrete modified zmGRP4 into the mucilage to which the protein may contribute to its characteristic physical properties.
Sucrose Synthase in Legume Nodules Is Essential for Nitrogen Fixation1
Authors: Gordon Anthony J. , Minchin Frank R. , James Caron L. , Komina Olga
Published: July 2002

The role of sucrose synthase (SS) in the fixation of N was examined in the rug4 mutant of pea (Pisum sativum L.) plants in which SS activity was severely reduced. When dependent on nodules for their N supply, the mutant plants were not viable and appeared to be incapable of effective N fixation, although nodule formation was essentially normal. In fact, N and C resources invested in nodules were much greater in mutant plants than in the wild-type (WT) plants. Low SS activity in nodules (present at only 10% of WT levels) resulted in lower amounts of total soluble protein and leghemoglobin and lower activities of several enzymes compared with WT nodules. Alkaline invertase activity was not increased to compensate for reduced SS activity. Leghemoglobin was present at less than 20% of WT values, so O2 flux may have been compromised. The two components of nitrogenase were present at normal levels in mutant nodules. However, only a trace of nitrogenase activity was detected in intact plants and none was found in isolated bacteroids. The results are discussed in relation to the role of SS in the provision of C substrates for N fixation and in the development of functional nodules.
Antisense Repression of Hexokinase 1 Leads to an Overaccumulation of Starch in Leaves of Transgenic Potato Plants But Not to Significant Changes in Tuber Carbohydrate Metabolism
Authors: Veramendi Jon , Roessner Ute , Renz Andreas , Willmitzer Lothar , Trethewey Richard N.
Published: July 2002

Potato (Solanum tuberosum L.) plants transformed with sense and antisense constructs of a cDNA encoding the potato hexokinase 1 (StHK1) exhibited altered enzyme activities and expression of StHK1 mRNA. Measurements of the maximum catalytic activity of hexokinase revealed a 22-fold variation in leaves (from 22% of the wild-type activity in antisense transformants to 485% activity in sense transformants) and a 7-fold variation in developing tubers (from 32% of the wild-type activity in antisense transformants to 222% activity in sense transformants). Despite the wide range of hexokinase activities, no change was found in the fresh weight yield, starch, sugar, or metabolite levels of transgenic tubers. However, there was a 3-fold increase in the starch content of leaves from the antisense transformants after the dark period. Starch accumulation at the end of the night period was correlated with a 2-fold increase of glucose and a decrease of sucrose content. These results provide strong support for the hypothesis that glucose is a primary product of transitory starch degradation and is the sugar that is exported to the cytosol at night to support sucrose biosynthesis.
The Generation of Active Oxygen Species Differs in Tobacco and Grapevine Mesophyll Protoplasts
Authors: Papadakis Anastasia K. , Roubelakis-Angelakis Kalliopi A.
Published: July 2002

Our previous results have shown that oxidative stress may reduce the regeneration potential of protoplasts, but only protoplasts that are able to supply extracellularly H2O2 can actually divide (C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1993] Physiol Plant 87: 263–270; C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1994] Plant Physiol 1105: 1375–1383; A. de Marco, K.A. Roubelakis-Angelakis [1996a] Plant Physiol 110: 137–145; A. de Marco, K.A. Roubelakis-Angelakis [1996b] J Plant Physiol 149: 109–114). In the present study we have attempted to break down the oxidative burst response into the individual active oxygen species (AOS) superoxide (O2  ·−) and H2O2, and into individual AOS-generating systems during the isolation of regenerating tobacco (Nicotiana tabacum L.) and non-regenerating grape (Vitis vinifera L.) mesophyll protoplasts. Wounding leaf tissue or applying purified cellulase did not elicit AOS production. However, the application of non-purified cellulase during maceration induced a burst of O2  ·− and H2O2accumulation in tobacco leaf, while in grape significantly lower levels of both AOS accumulated. AOS were also generated when protoplasts isolated with purified cellulase were treated with non-purified cellulase. The response was rapid: after 5 min, AOS began to accumulate in the culture medium, with significant quantitative differences between the two species. In tobacco protoplasts and plasma membrane vesicles, two different AOS synthase activities were revealed, one that showed specificity to NADPH and sensitivity to diphenyleneiodonium (DPI) and was responsible for O2  ·−production, and a second NAD(P)H activity that was sensitive to KCN and NaN3, contributing to the production of both AOS. The first activity probably corresponds to a mammalian-like NADPH oxidase and the second to a NAD(P)H oxidase-peroxidase. In grape, only one AOS-generating activity was detected, which corresponded to a NAD(P)H oxidase-peroxidase responsible for the generation of both AOS.
Arabidopsis Alcohol Dehydrogenase Expression in Both Shoots and Roots Is Conditioned by Root Growth Environment
Authors: Chung Hwa-Jee , Ferl Robert J.
Published: July 2002

It is widely accepted that the Arabidopsis Adh (alcohol dehydrogenase) gene is constitutively expressed at low levels in the roots of young plants grown on agar media, and that the expression level is greatly induced by anoxic or hypoxic stresses. We questioned whether the agar medium itself created an anaerobic environment for the roots upon their growing into the gel. β-Glucuronidase (GUS) expression driven by theAdh promoter was examined by growing transgenic Arabidopsis plants in different growing systems. Whereas roots grown on horizontal-positioned plates showed high Adh/GUS expression levels, roots from vertical-positioned plates had no Adh/GUS expression. Additional results indicate that growth on vertical plates closely mimics the Adh/GUS expression observed for soil-grown seedlings, and that growth on horizontal plates results in induction of high Adh/GUS expression that is consistent with hypoxic or anoxic conditions within the agar of the root zone. Adh/GUS expression in the shoot apex is also highly induced by root penetration of the agar medium. This induction of Adh/GUS in shoot apex and roots is due, at least in part, to mechanisms involving Ca2+ signal transduction.
Signaling Events Leading to Crassulacean Acid Metabolism Induction in the Common Ice Plant
Authors: Taybi Tahar , Cushman John C.
Published: July 2002

A rapid, semiquantitative reverse transcriptase-polymerase chain reaction assay was developed to investigate signal transduction events involved in the induction of Crassulacean acid metabolism (CAM) in detached common ice plant (Mesembryanthemum crystallinum) leaves. Transcript abundance of Ppc1, a gene encoding the CAM-specific isoform of phosphoenolpyruvate carboxylase, increased rapidly in response to osmotic stress (dehydration and mannitol), ionic stress (NaCl), and exogenous abscisic acid treatment, but failed to accumulate in response to exogenous cytokinin or methyl jasmonate. Stress-induced accumulation of Ppc1, GapC1, and Mdh1transcripts was inhibited by pretreating leaves with the calcium chelator ethyleneglycol-bis(aminoethyl ether)-N,N′-tetraacetic acid, suggesting that extracellular calcium participates in signaling events leading to CAM induction. Treatment of unstressed detached leaves with ionomycin, a Ca2+ ionophore, and thapsigargin, a Ca2+-ATPase inhibitor, enhanced Ppc1 transcript accumulation, indicating that elevations in cytosolic [Ca2+] are likely to participate in signaling CAM induction. Inhibitors of Ca2+- or calmodulin-dependent protein kinases (N-[6-aminohexyl]-5-chloro-1-napthalenesulfonamide, Lavendustin C) and protein phosphatase 1 and 2A (okadaic acid) activity suppressedPpc1 transcript accumulation in response to ionic and osmotic stresses, as well as abscisic acid treatment. These results suggest that both protein phosphorylation and dephosphorylation events participate in signaling during CAM induction. In contrast, pretreatment with cyclosporin A or ascomycin, inhibitors of protein phosphatase 2B activity, stimulated Ppc1 gene expression either directly or indirectly through promoting water loss.
Light Induction of Cell Type Differentiation and Cell-Type-Specific Gene Expression in Cotyledons of a C4Plant,Flaveria trinervia
Authors: Shu Guoping , Pontieri Vincenza , Dengler Nancy G. , Mets Laurens J.
Published: July 2002

InFlaveria trinervia (Asteraceae) seedlings, light-induced signals are required for differentiation of cotyledon bundle sheath cells and mesophyll cells and for cell-type-specific expression of Rubisco small subunit genes (bundle sheath cell specific) and the genes that encode pyruvate orthophosphate dikinase and phosphoenolpyruvate carboxylase (mesophyll cell specific). Both cell type differentiation and cell-type-specific gene expression were complete by d 7 in light-grown seedlings, but were arrested beyond d 4 in dark-grown seedlings. Our results contrast with those found for another C4 dicot, Amaranthus hypochondriacus, in which light was not required for either process. The differences between the two C4 dicot species in cotyledon cell differentiation may arise from differences in embryonic and post-embryonic cotyledon development. Our results illustrate that a common C4 photosynthetic mechanism can be established through different developmental pathways in different species, and provide evidence for independent evolutionary origins of C4 photosynthetic mechanisms within dicotyledonous plants.
Distinguishing between Luminal and Localized Proton Buffering Pools in Thylakoid Membranes
Authors: Ewy Robert G. , Dilley Richard A.
Published: July 2002

The dual gradient energy coupling hypothesis posits that chloroplast thylakoid membranes are energized for ATP formation by either a delocalized or a localized proton gradient geometry. Localized energy coupling is characterized by sequestered domains with a buffering capacity of approximately 150 nmol H+ mg−1chlorophyll (Chl). A total of 30 to 40 nmol mg−1 Chl of the total sequestered domain buffering capacity is contributed by lysines with anomolously low pKas, which can be covalently derivatized with acetic anhydride. We report that in thylakoid membranes treated with acetic anhydride, luminal acidification by a photosystem I (duraquinol [DQH2] to methyl viologen [MV]) proton pumping partial reaction was nearly completely inhibited, as measured by three separate assays, yet surprisingly, H+ accumulation still occurred to the significant level of more than 100 nmol H+ mg Chl−1, presumably into the sequestered domains. The treatment did not increase the observed rate constant of dark H+ efflux, nor was electron transport significantly inhibited. These data provide support for the existence of a sequestered proton translocating pathway linking the redox reaction H+ ion sources with the CF0H+ channel. The sequestered, low-pKa Lys groups appear to have a role in the H+ diffusion process and chemically modifying them blocks the putative H+ relay system.
Direct Measurement of Aluminum Uptake and Distribution in Single Cells of Chara corallina
Authors: Taylor Gregory J. , McDonald-Stephens Julie L. , Hunter Douglas B. , Bertsch Paul M. , Elmore David , Rengel Zdenko , Reid Robert J.
Published: July 2002

Quantitative information on the uptake and distribution of Al at the cellular level is required to understand mechanisms of Al toxicity, but direct measurement of uptake across the plasma membrane has remained elusive. We measured rates of Al transport across membranes in single cells of Chara corallina using the rare26Al isotope, an emerging technology (accelerator mass spectrometry), and a surgical technique for isolating subcellular compartments. Accumulation of Al in the cell wall dominated total uptake (71–318 μg m−2 min−1), although transport across the plasma membrane was detectable (71–540 ng m−2 min−1) within 30 min of exposure. Transport across the tonoplast was initially negligible, but accelerated to rates approximating uptake across the plasma membrane. The avacuolate protoplasm showed signs of saturation after 60 min, but continued movement across the plasma membrane was supported by sequestration in the vacuole. Saturation of all compartments was observed after 12 to 24 h. Accumulation of Al in the cell wall reflected variation in {Al3+} induced by changes in Al supply or complexing ligands, but was unaffected by pH. In contrast, transport across the plasma membrane peaked at pH 4.3 and increased when {Al3+} was reduced by complexing ligands. Cold temperature (4°C) reduced accumulation in the cell wall and protoplasm, whereas 2,4-dinitrophenol andm-chlorocarbonylcyanidephenyl hydrazone increased membrane transport by 12- to 13-fold. Our data suggest that the cell wall is the major site of Al accumulation. Nonetheless, membrane transport occurs within minutes of exposure and is supported by subsequent sequestration in the vacuole. The rapid delivery of Al to the protoplasm suggests that intracellular lesions may be possible.
Involvement of Brassinosteroids in the Gravitropic Response of Primary Root of Maize
Authors: Kim Seong-Ki , Chang Soo Chul , Lee Eun Joo , Chung Woo-Sook , Kim Young-Soo , Hwang Seongbin , Lee June Seung
Published: July 2002

Exogenously applied brassinolide (BL, 10−9–10−5  m) increased gravitropic curvature in maize (Zea mays) primary roots. The BL-enhanced gravitropic curvature was clearly promoted in the presence of indole-3-acetic acid (IAA, 10−10–10−8  m), indicating that BL is interactive with IAA during the gravitropic response. The interactive effect between BL and IAA was completely diminished by treatment of p-chlorophenoxy isobutric acid, an auxin action antagonist. The activation of the gravitropic response by BL in the absence and in the presence of IAA was nullified by application of 2,3,5-triiodobenzoic acid, a polar auxin transport inhibitor. The data indicate that brassinosteroids (BRs) might be involved in auxin-mediated processes for the gravitropic response. Gas chromotography-selected ion-monitoring analysis revealed that maize primary roots contained approximately 0.3 ng g−1 fresh weight castasterone as an endogenous BR. Exogenously applied castasterone also increased the gravitropic response of maize roots in an IAA-dependent manner. This study provides the first evidence, to our knowledge, for occurrence and gravitropic activity of BRs in plant roots.
Identification of a CYP84 Family of Cytochrome P450-Dependent Mono-Oxygenase Genes in Brassica napus and Perturbation of Their Expression for Engineering Sinapine Reduction in the Seeds
Authors: Nair Ramesh B. , Joy Richard W. , Kurylo Eugen , Shi Xiaohong , Schnaider Joan , Datla Raju S.S. , Keller Wilf A. , Selvaraj Gopalan
Published: July 2002

CYP84 is a recently identified family of cytochrome P450-dependent mono-oxygenases defined by a putative ferulate-5-hydroxylase (F5H) from Arabidopsis. Until recently F5H has been thought to catalyze the hydroxylation of ferulate to 5-OH ferulate en route to sinapic acid. Sinapine, a sinapate-derived ester in the seeds, is antinutritional and a target for elimination in canola meal. We have isolated threeF5H-like genes (BNF5H1-3) from a cultivated Brassica napus, whose amphidiploid progenitor is considered to have arisen from a fusion of the diploidsBrassica rapa and Brassica oleracea  . Two cultivated varieties of the diploids were also found to contain BNF5H3 and additionally either BNF5H1 or BNF5H2, respectively. Whereas all three are >90% identical in their coding sequence, BNF5H1 and BNF5H2 are closer to each other than to BNF5H3. This and additional data suggest that the two groups of genes have diverged in an ancestor of the diploids. B. napus showed maximal F5Hexpression in the stems, least in the seeds, and subtle differences among the expression profiles of the three genes elsewhere. TransgenicB. napus with cauliflower mosaic virus 35S-antisenseBNF5H contained up to 40% less sinapine, from 9.0 ± 0.3 mg in the controls to 5.3 ± 0.3 mg g−1 seed. F5H from Arabidopsis and a similar enzyme from sweetgum (Liquidamber styraciflua) has recently been shown to have coniferaldehyde hydroxylase activity instead of F5H activity. Thus the supply of 5-OH coniferaldehyde or 5-OH ferulate has a bearing on sinapine accumulation in canola seeds.
Profilin Plays a Role in Cell Elongation, Cell Shape Maintenance, and Flowering in Arabidopsis
Authors: Ramachandran Srinivasan , Christensen Hans E.M. , Ishimaru Yasuko , Dong Chun-Hai , Chao-Ming Wen , Cleary Ann L. , Chua Nam-Hai
Published: July 2002

Profilin (PFN) is an ubiquitous, low-M  r, actin-binding protein involved in the organization of the cytoskeleton of eukaryotes including higher plants. PFNs are encoded by a multigene family in Arabidopsis. We have analyzed in vivo functions of Arabidopsis PFN by generating transgenic plants carrying a35S-PFN-1 or35S-antisense  PFN-1transgene. Etiolated seedlings underexpressing PFN (PFN-U) displayed an overall dwarf phenotype with short hypocotyls whose lengths were 20% to 25% that of wild type (WT) at low temperatures. Light-grown PFN-U plants were smaller in stature and flowered early. Compared with equivalent cells in WT, most cells in PFN-U hypocotyls and roots were shorter, but more isodiametric, and microscopic observations of etiolated PFN-U hypocotyls revealed a rough epidermal surface. In contrast, light-grown seedlings overexpressing PFN had longer roots and root hair although etiolated seedlings overexpressing PFN were either the same size or slightly longer than WT seedlings. Transgenic seedlings harboring aPFN-1-GUS transgene directed expression in root and root hair and in a ring of cells at the elongating zone of the root tip. As the seedlings matured PFN-1-GUS was mainly expressed in the vascular bundles of cotyledons and leaves. Our results show that Arabidopsis PFNs play a role in cell elongation, cell shape maintenance, polarized growth of root hair, and unexpectedly, in determination of flowering time.
Membrane Lipid Biosynthesis in Chlamydomonas reinhardtii. In Vitro Biosynthesis of Diacylglyceryltrimethylhomoserine
Authors: Moore Thomas S. , Du Zhirong , Chen Zhi
Published: July 2002

Diacylglyceryltrimethylhomo-Ser (DGTS) is an abundant lipid in the membranes of many algae, lower plants, and fungi. It commonly has an inverse concentration relationship with phosphatidylcholine, thus seemingly capable of replacing this phospholipid in these organisms. In some places this replacement is complete; Chlamydomonas reinhardtii is such an organism, and was used for these investigations. We have assayed headgroup incorporation to form DGTS in vitro. The precursor for both the homo-Ser moiety and the methyl groups was found to be S-adenosyl-L-Met. DGTS formation was associated with microsomal fractions and is not in plastids. By analogy with phosphatidylcholine and phosphatidylethanolamine biosynthesis in higher plants, the microsomal activity probably is associated with the endoplasmic reticulum. The pH optimum for the total reaction was between 7.5 and 8.0, and the best temperature was 30°C. The apparent K  m andV  max forS-adenosyl-L-Met in the overall reaction were 74 and 250 μm, respectively.
LEPS2, a Phosphorus Starvation-Induced Novel Acid Phosphatase from Tomato
Authors: Baldwin James C. , Karthikeyan Athikkattuvalasu S. , Raghothama Kashchandra G.
Published: July 2002

Phosphate (Pi) is one of the least available plant nutrients found in the soil. A significant amount of phosphate is bound in organic forms in the rhizosphere. Phosphatases produced by plants and microbes are presumed to convert organic phosphorus into available Pi, which is absorbed by plants. In this study we describe the isolation and characterization of a novel tomato (Lycopersicon esculentum) phosphate starvation-induced gene (LePS2) representing an acid phosphatase.LePS2 is a member of a small gene family in tomato. The cDNA is 942 bp long and contains an open reading frame encoding a 269-amino acid polypeptide. The amino acid sequence of LePS2 has a significant similarity with a phosphatase from chicken. Distinct regions of the peptide also share significant identity with the members of HAD and DDDD super families of phosphohydrolases. Many plant homologs of LePS2 are found in the databases. TheLePS2 transcripts are induced rapidly in tomato plant and cell culture in the absence of Pi. However, the induction is repressible in the presence of Pi. Divided root studies indicate that internal Pi levels regulate the expression of LePS2. The enhanced expression of LePS2 is a specific response to Pi starvation, and it is not affected by starvation of other nutrients or abiotic stresses. The bacterially (Escherichia coli)expressed protein exhibits phosphatase activity against the synthetic substrate p-nitrophenyl phosphate. The pH optimum of the enzyme activity suggests that LePS2 is an acid phosphatase.
Rice Bioinformatics. Analysis of Rice Sequence Data and Leveraging the Data to Other Plant Species
Authors: Yuan Qiaoping , Quackenbush John , Sultana Razvan , Pertea Mihaela , Salzberg Steven L. , Buell C. Robin
Published: July 2002

Rice (Oryza sativa) is a model species for monocotyledonous plants, especially for members in the grass family. Several attributes such as small genome size, diploid nature, transformability, and establishment of genetic and molecular resources make it a tractable organism for plant biologists. With an estimated genome size of 430 Mb (Arumuganathan and Earle, 1991), it is feasible to obtain the complete genome sequence of rice using current technologies. An international effort has been established and is in the process of sequencing O. sativa spp.japonica var “Nipponbare” using a bacterial artificial chromosome/P1 artificial chromosome shotgun sequencing strategy. Annotation of the rice genome is performed using prediction-based and homology-based searches to identify genes. Annotation tools such as optimized gene prediction programs are being developed for rice to improve the quality of annotation. Resources are also being developed to leverage the rice genome sequence to partial genome projects such as expressed sequence tag projects, thereby maximizing the output from the rice genome project. To provide a low level of annotation for rice genomic sequences, we have aligned all rice bacterial artificial chromosome/P1 artificial chromosome sequences with The Institute of Genomic Research Gene Indices that are a set of nonredundant transcripts that are generated from nine public plant expressed sequence tag projects (rice, wheat, sorghum, maize, barley, Arabidopsis, tomato, potato, and barrel medic). In addition, we have used data from The Institute of Genomic Research Gene Indices and the Arabidopsis and Rice Genome Projects to identify putative orthologues and paralogues among these nine genomes.
Nonsense-Mediated Decay of MutantwaxymRNA in Rice
Authors: Isshiki Masayuki , Yamamoto Yoshiaki , Satoh Hikaru , Shimamoto Ko
Published: July 2002

Two rice (Oryza sativa) waxy mutations of the Japonica background were shown to contain approximately 20% of the fully spliced mRNA relative to the wild type. Sequencing analysis of the entire waxy genes of the two mutants revealed the presence of premature translation termination codons in exon 2 and exon 7. These results indicated that the lower accumulation of fully spliced RNA in the mutants was caused by nonsense-mediated decay (NMD), which is an RNA surveillance system universally found in eukaryotes. It is interesting that levels of RNA retaining intron 1 were not changed by premature nonsense codons, suggesting that splicing may be linked with NMD in plants, as previously found in mammalian cells. Measurements of the half-lives of waxy RNAs in transfected rice protoplasts indicated that the half-life of waxy RNA with a premature nonsense codon was 3.3 times shorter than that without a premature nonsense codon. Because the wild-type waxytranscripts, which are derived from the Wxbgene predominantly distributed among Japonica rice, have been shown to be less efficiently spliced and their alternative splicing has been documented, we examined whether these splicing properties influenced the efficiency of NMD. However, no effects were observed. These results established that NMD occurs in rice waxy RNA containing a premature nonsense codon.
Ectopic Expression of the Tetratricopeptide Repeat Domain of SPINDLY Causes Defects in Gibberellin Response
Authors: Tseng Tong-Seung , Swain Stephen M. , Olszewski Neil E.
Published: July 2002

The SPINDLY (SPY) protein of Arabidopsis is a negative regulator of gibberellin (GA) response. The SPY protein has 10 copies of the tetratricopeptide repeat (TPR) at the N terminus. TPR motifs function as protein-protein interaction domains. Several spyalleles are affected only in the TPR region suggesting that protein-protein interactions mediated by this domain are important for proper GA signaling. We have used a reverse genetics approach to further investigate the role of the TPR domain. The TPR domain of SPY was overexpressed in wild-type, gai, andspy plants. Expression of the TPR domain alone is not sufficient to rescue spy mutants. Expression of the TPR domain in a wild-type background produces phenotypes similar to those caused by loss-of-function spy mutants including resistance to GA biosynthesis inhibitors, short hypocotyl length, and early flowering. The dwarfing of the floral shoot internodes caused by the gai mutation was suppressed by expression of the TRP domain. Expression of the TPR domain had no effect on the abundance of endogenous SPY mRNA. The TPR domain was found to interact with SPY both in vitro and in yeast two-hybrid assays. These data indicate that the TPR domain of SPY can participate in protein-protein interactions and that these interactions are important for the proper functioning of SPY.