bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Characterization of a β-D-Xylosidase and an Endo-Xylanase from Wheat Flour
Authors: Cleemput G. , Hessing M. , van Oort M. , Deconynck M. , Delcour J. A.
Published: October 2016

A β-D-xylosidase and an endo-xylanase were purified from European wheat (Triticum aestivum) flour. The β-D-xylosidase had a molecular weight of approximately 64,000 and an isoelectric point of 5.5. It hydrolyzed p-nitrophenyl-β-D-xylopyranoside and xylo-oligosaccharides and released D-xylose units from wheat arabinoxylan and oat spelts xylan. An endo-xylanase with a molecular weight of approximately 55,000 was also obtained and it consisted of a number of isoforms with isoelectric points between 4.0 and 5.0. The action of the isolated endo-xylanase depended on the degree of substitution of the polysaccharide. Unbranched polymers were preferentially hydrolyzed. Since xylo-oligosaccharides were not hydrolyzed, the enzyme appeared to need at least five or more consecutive unsubstituted xylose units. Finally, an α--L-arabinofuranosidase that hydrolyzed p-nitrophenyl-α--L-arabinofuranoside was partially purified.
Differential Ammonia-Elicited Changes of Cytosolic pH in Root Hair Cells of Rice and Maize as Monitored by 2[prime],7[prime]-bis-(2-Carboxyethyl)-5 (and -6)-Carboxyfluorescein-Fluorescence Ratio
Authors: Kosegarten H. , Grolig F. , Wieneke J. , Wilson G. , Hoffmann B.
Published: October 2016

Intact hair cells of young rice (Oryza sativa L.) and maize roots (Zea mays L.), grown without external nitrogen, were specifically loaded with 2[prime],7[prime]-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein acetoxymethyl ester to monitor fluorescence ratio cytosolic pH changes in response to external ammonia (NH4+/NH3) application. In neutral media, cytosolic pH of root hairs was 7.15 [plus or minus] 0.13 (O. sativa) and 7.08 [plus or minus] 0.11 (Z. mays). Application of 2 mM ammonia at external pH 7.0 caused a transient cytosolic alkalization (7.5 [plus or minus] 0.15 in rice; 7.23 [plus or minus] 0.13 in maize). Alkalization increased with an increase of external pH; no pH changes occurred at external pH 5.0. The influx of 13N-labeled ammonia in both plant species did not differ between external pH 5.0 and 7.0 but increased significantly with higher pH. Pretreatment with 1 mM 1-methionine sulfoximine significantly reduced the ammonia-elicited pH increase in rice but not in maize. Application of 2 mM methylammonia only caused a cytosolic pH increase at high external pH; the increase in both species compared with the ammonia-elicited alkalization in 1-methionine sulfoximine-treated roots. The differential effects indicate that cytosolic alkalization derived from (a) NH3 protonation after passive permeation of the plasma membrane and, particularly in rice, (b) additional proton consumption via the glutamine synthetase/glutamate synthase cycle.
Reduction of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by Antisense RNA in the C4 Plant Flaveria bidentis Leads to Reduced Assimilation Rates and Increased Carbon Isotope Discrimination
Authors: von Caemmerer S. , Millgate A. , Farquhar G. D. , Furbank R. T.
Published: October 2016

Transgenic Flaveria bidentis (a C4 species) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were used to examine the relationship between the CO2 assimilation rate, Rubisco content, and carbon isotope discrimination. Reduction in the amount of Rubisco in the transgenic plants resulted in reduced CO2 assimilation rates and increased carbon isotope discrimination of leaf dry matter. The H2O exchange was similar in transgenic and wild-type plants, resulting in higher ratios of intercellular to ambient CO2 partial pressures. Carbon isotope discrimination was measured concurrently with CO2 and H2O exchange on leaves of the control plants and T1 progeny with a 40% reduction in Rubisco. From the theory of carbon isotope discrimination in the C4 species, we conclude that the reduction in the Rubisco content in the transgenic plants has led to an increase in bundle-sheath CO2 concentration and CO2 leakage from the bundle sheath; however, some down-regulation of the C4 cycle also occurred.
Al Partitioning Patterns and Root Growth as Related to Al Sensitivity and Al Tolerance in Wheat
Authors: Samuels T. D. , Kucukakyuz K. , Rincon-Zachary M.
Published: October 2016

Studies of Al partitioning and accumulation and of the effect of Al on the growth of intact wheat (Triticum aestivum L.) roots of cultivars that show differential Al sensitivity were conducted. The effects of various Al concentrations on root growth and Al accumulation in the tissue were followed for 24 h. At low external Al concentrations, Al accumulation in the root tips was low and root growth was either unaffected or stimulated. Calculations based on regression analysis of growth and Al accumulation in the root tips predicted that 50% root growth inhibition in the Al-tolerant cv Atlas 66 would be attained when the Al concentrations were 105 [mu]M in the nutrient solution and 376.7 [mu]g Al g-1 dry weight in the tissue. In contrast, in the Al-sensitive cv Tam 105, 50% root growth inhibition would be attained when the Al concentrations were 11 [mu]M in the nutrient solution and 546.2 [mu]g Al g-1 dry weight in the tissue. The data support the hypotheses that differential Al sensitivity correlates with differential Al accumulation in the growing root tissue, and that mechanisms of Al tolerance may be based on strategies to exclude Al from the root meristems.
Molecular Genetic Evidence of the Ability of Alternative Oxidase to Support Respiratory Carbon Metabolism
Authors: Vanlerberghe G. C. , Vanlerberghe A. E. , McIntosh L.
Published: October 2016

With the cytochrome pathway inhibited, AOX was able to support considerable growth of cultured tobacco (Nicotiana tabacum cv Petit Havana SR1) cells but the efficiency of carbon utilization decreased dramatically. Antisense cells with decreased AOX protein did not grow, whereas sense cells with elevated AOX protein had higher growth and respiration rates than the wild type. In antisense cells a large accumulation of pyruvate resulted in aerobic ethanolic fermentation.
Expression of an Aspartate Kinase Homoserine Dehydrogenase Gene Is Subject to Specific Spatial and Temporal Regulation in Vegetative Tissues, Flowers, and Developing Seeds
Authors: Zhu-Shimoni J. X. , Lev-Yadun S. , Matthews B. , Galili G.
Published: October 2016

Although the regulation of amino acid synthesis has been studied extensively at the biochemical level, it is still not known how genes encoding amino acid biosynthesis enzymes are regulated during plant development. In the present report, we have used the β-glucuronidase (GUS) reporter gene to study the regulation of expression of an Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase (AK/HSD) gene in transgenic tobacco plants. The polypeptide encoded by the AK/HSD gene comprises two linked key enzymes in the biosynthesis of aspartate-family amino acids. AK/HSD-GUS gene expression was highly stimulated in apical and lateral meristems, lateral buds, young leaves, trichomes, vascular and cortical tissues of growing stems, tapetum and other tissues of anthers, pollen grains, various parts of the developing gynoecium, developing seeds, and, in some transgenic plants, also in stem and leaf epidermal trichomes. AK/HSD-GUS gene expression gradually dimished upon maturation of leaves, stems, floral tissues, and embryos. GUS expression was relatively low in roots. During seed development, expression of the AK/HSD gene in the embryo was coordinated with the initiation and onset of storage protein synthesis, whereas in the endosperm it was coordinated with the onset of seed desiccation. Upon germination, AK/HSD-GUS gene expression in the hypocotyl and the cotyledons was significantly affected by light. The expression pattern of the A. thaliana AK/HSD-GUS reporter gene positively correlated with the levels of aspartate-family amino acids and was also very similar to the expression pattern of the endogenous tobacco AK/HSD mRNA as determined by in situ hybridization.
Abscisic Acid Activates a 48-Kilodalton Protein Kinase in Guard Cell Protoplasts
Authors: Mori I. C. , Muto S.
Published: October 2016

A 49- and a 46-kD Ca2+-independent protein kinase and a 53-kD Ca2+-dependent protein kinase were detected in Vicia faba guard cell protoplasts (GCPs) by an in-gel protein kinase assay using myelin basic protein as a substrate. A 48-kD protein kinase designated as abscisic acid (ABA)-responsive protein kinase (ABR kinase) appeared when GCPs were treated with ABA. The activation of ABR kinase was suppressed by the protein kinase inhibitor staurosporine, indicating that a putative activator protein kinase phosphorylates and activates ABR kinase. The treatment of GCPs with 1,2-bis(o-aminophenoxy)ethan-N,N,N′,N′-tetraacetic acid, a calcium chelator, suppressed the activation of ABR kinase, suggesting that an influx of extracellular Ca2+ is required for the activation. Staurosporine and K-252a inhibited both the activity of ABR kinase and the stomatal closure induced by ABA treatment of V. faba epidermal peels. These results suggest that ABR kinase and its activator kinase may consist of a protein kinase cascade in a signal transduction pathway linking ABA perception to stomatal closure. The mobility of the 53-kD Ca2+-dependent protein kinase in sodium dodecyl sulfate-polyacrylamide gel was shifted upon Ca2+ binding to the enzyme, thus exhibiting the characteristics of a Ca2+-dependent or calmodulin-like domain protein kinase. This kinase may be the activator of ABR kinase.
The cdc2Ms Kinase Is Differently Regulated in the Cytoplasm and in the Nucleus
Authors: Bogre L. , Zwerger K. , Meskiene I. , Binarova P. , Csizmadia V. , Planck C. , Wagner E. , Hirt H. , Heberle-Bors E.
Published: October 2016

To study a cyclin-dependent kinase (CDK) from alfalfa (Medicago sativa L.), an antibody was raised against the C-terminal 16 amino acids of the protein cdc2aMs. The cdc2Ms protein was immunopurified with this antibody and its histone kinase activity was measured. The cdc2Ms kinase is activated at the G1/S transition when phosphate-starved cells from the G0 phase re-enter the cell cycle and remain active as cells transit the S, G2, and M phases, indicating that the same CDK regulates all of these phases in alfalfa. In contrast, when cdc2Ms kinase was purified by binding to p13suc1, it was active only in the G2 and M phases. In immunoblots the C-terminal antibody detected an equal amount of the cdc2Ms protein in the cytoplasm and in the nucleus. By indirect immunofluorescence, however, the cytoplasmic form of cdc2Ms could not be found in the S phase of the cells, indicating that the epitope for the cdc2 antibody is not accessible. Binding of putative inhibitor proteins to cdc2 was shown by inactivation of purified plant CDK when cell extracts were added. Furthermore, purified CDK inhibitors, such as the mouse p27kip1 and the yeast p40sic1, blocked the purified plant CDK activity.
Proteinase Activity during Tracheary Element Differentiation in Zinnia Mesophyll Cultures
Authors: Beers E. P. , Freeman T. B.
Published: October 2016

The zinnia (Zinnia elegans) mesophyll cell culture tracheary element (TE) system was used to study proteinases active during developmentally programmed cell death. Substrate-impregnated gels and single-cell assays revealed high levels of proteinase activity in differentiating TEs compared with undifferentiated cultured cells and expanding leaves. Three proteinases (145, 28, and 24 kD) were exclusive to differentiating TEs. A fourth proteinase (59 kD), although detected in extracts from all tissues examined, was most active in differentiating TEs. The 28- and 24-kD proteinases were inhibited by thiol proteinase inhibitors, leupeptin, and N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine (E-64). The 145- and 59-kD proteinases were inhibited by the serine proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF). Extracts from the TE cultures contained sodium dodecyl sulfate-stimulated proteolytic activity not detected in control cultures. Sodium dodecyl sulfate-stimulated proteolysis was inhibited by leupeptin or E-64, but not by PMSF. Other tissues, sucrose-starved cells and cotyledons, that contain high levels of proteolytic activity did not contain TE-specific proteinases, but did contain higher levels of E-64-sensitive activities migrating as 36- to 31-kD enzymes and as a PMSF-sensitive 66-kD proteinase.
Growth, Water Relations, and Accumulation of Organic and Inorganic Solutes in Roots of Maize Seedlings during Salt Stress
Authors: Rodriguez H. G. , Roberts JKM. , Jordan W. R. , Drew M. C.
Published: October 2016

Seedlings of maize (Zea mays L. cv Pioneer 3906), hydroponically grown in the dark, were exposed to NaCl either gradually (salt acclimation) or in one step (salt shock). In the salt-acclimation treatment, root extension was indistinguishable from that of unsalinized controls for at least 6 d at concentrations up to 100 mM NaCl. By contrast, salt shock rapidly inhibited extension, followed by a gradual recovery, so that by 24 h extension rates were the same as for controls, even at 150 mM NaCl. Salt shock caused a rapid decrease in root water and solute potentials for the apical zones, and the estimated turgor potential showed only a small decline; similar but more gradual changes occurred with salt acclimation. The 5-bar decrease in root solute potential with salt shock (150 mM NaCl) during the initial 10 min of exposure could not be accounted for by dehydration, indicating that substantial osmotic adjustment occurred rapidly. Changes in concentration of inorganic solutes (Na+, K+, and Cl-) and organic solutes (proline, sucrose, fructose, and glucose) were measured during salt shock. The contribution of these solutes to changes in root solute potential with salinization was estimated.
Thermostability and Photostability of Photosystem II in Leaves of the Chlorina-f2 Barley Mutant Deficient in Light-Harvesting Chlorophyll a/b Protein Complexes
Authors: Havaux M. , Tardy F.
Published: October 2016

The chlorophyll-b-less chlorina-f2 barley mutant is deficient in the major as well as some minor light-harvesting chlorophyll-protein complexes of photosystem II (LHCII). Although the LHCII deficiency had relatively minor repercussions on the leaf photosynthetic performances, the responses of photosystem II (PSII) to elevated temperatures and to bright light were markedly modified. The chlorina-f2 mutation noticeably reduced the thermostability of PSII, with thermal denaturation of PSII starting at about 35[deg]C and 38.5[deg]C in chlorina-f2 and in the wild type, respectively. The increased susceptibility of PSII to heat stress in chlorina-f2 leaves was due to the weakness of its electron donor side, with moderate heat stress causing detachment of the 33-kD extrinsic PSII protein from the oxygen-evolving complex. Prolonged dark adaptation of chlorina-f2 leaves was also observed to inhibit the PSII donor side. However, weak illumination slowly reversed the dark-induced inhibition of PSII in chlorina-f2 and cancelled the difference in PSII thermostability observed between chlorina-f2 and wild-type leaves. The mutant was more sensitive to photoinhibition than the wild type, with strong light stress impairing the PSII donor side in chlorina-f2 but not in the wild type. This difference was not observed in anaerobiosis or in the presence of 3-(3,4-dichlorophenyl)- 1,1-dimethylurea, diuron. The acceptor side of PSII was only slightly affected by the mutation and/or the aforementioned stress conditions. Taken together, our results indicate that LHCII stabilize the PSII complexes and maintain the water-oxidizing system in a functional state under varying environmental conditions.
Leaf Respiration in Light and Darkness (A Comparison of Slow- and Fast-Growing Poa Species)
Authors: Atkin O. K. , Westbeek MHM. , Cambridge M. L. , Lambers H. , Pons T. L.
Published: October 2016

We investigated whether leaf dark respiration (nonphotorespiratory mitochondrial CO2 release) is inhibited by light in several Poa species, and whether differences in light inhibition between the species are related to differences in the rate of leaf net photosynthesis. Four lowland (Poa annua L., Poa compressa L., Poa pratensis L., and Poa trivialis L.), one subalpine (Poa alpina L.), and two alpine (Poa costiniana Vick. and Poa fawcettiae Vick.) Poa species differing in whole plant relative growth rates were grown under identical controlled conditions. Nonphotorespiratory mitochondrial CO2 release in the light (Rd) was estimated according to the Laisk method. Photosynthesis was measured at ambient CO2 partial pressure (35 Pa) and 500 [mu]mol photons m-2 s-1. The rate of photosynthesis per unit leaf mass was positively correlated with the relative growth rate, with the slow-growing alpine Poa species exhibiting the lowest photosynthetic rates. Rates of both Rd and respiration in darkness were also substantially lower in the alpine species. Nonphotorespiratory CO2 release in darkness was higher than Rd in all species. However, despite some variation between the species in the level of light inhibition of respiration, no relationship was observed between the level of inhibition and the rate of photosynthesis. Similarly, the level of inhibition was not correlated with the relative growth rate. Our results support the suggestion that rates of leaf respiration in the light are closely associated with rates in darkness.
Diurnal Fluctuations in Ethylene Formation in Chenopodium rubrum
Authors: Machackova I. , Chauvaux N. , Dewitte W. , van Onckelen H.
Published: October 2016

Ethylene formation was studied in 5- to 6-d-old Chenopodium rubrum seedlings under the following light regimes: continuous light (CL), continuous darkness (CD), and alternating light/darkness (12 h of each). No significant regular oscillations in ethylene formation were found in either the CL or CD groups. In the light/dark regime, pronounced diurnal fluctuations in ethylene formation were observed. Activity of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase was transiently increased on transfer from light to dark and vice versa. In CL, ACC oxidase activity did not change significantly, whereas in CD, it decreased continuously after the initial increase. The in vivo levels of ACC and N-malonyl-ACC (MACC) were constant for the first few hours of darkness, then decreased dramatically, but increased again in the light. In constant darkness, the level of ACC displayed endogenous rhythm, with minimum values at h 12 and 44, and a maximum value at h 32 to 36. The level of MACC in both shoots and roots decreased in the CD group until h 12, and then remained constant until h 30 before decreasing continuously. We conclude that the photoperiodic regime affects both ACC and MACC levels, as well as the conversion of ACC to ethylene. Correlation of the described changes in ethylene formation to photoperiodic flower induction is discussed.
Reduction of Uroporphyrinogen Decarboxylase by Antisense RNA Expression Affects Activities of Other Enzymes Involved in Tetrapyrrole Biosynthesis and Leads to Light-Dependent Necrosis
Authors: Mock H. P. , Grimm B.
Published: October 2016

We introduced a full-length cDNA sequence encoding tobacco (Nicotiana tabacum) uroporphyrinogen III decarboxylase (UROD; EC 4.1.1.37) in reverse orientation under the control of a cauliflower mosaic virus 35S promoter derivative into the tobacco genome to study the effects of deregulated UROD expression on tetrapyrrole biosynthesis. Transformants with reduced UROD activity were characterized by stunted plant growth and necrotic leaf lesions. Antisense RNA expression caused reduced UROD protein levels and reduced activity to 45% of wild type, which was correlated with the accumulation of uroporphyrin(ogen) and with the intensity of necrotic damage. Chlorophyll levels were only slightly reduced (up to 15%), indicating that the plants sustained cellular damage from accumulating photosensitive porphyrins rather than from chlorophyll deficiency. A 16-h light/8-h dark regime at high-light intensity stimulates the formation of leaf necrosis compared with a low-light or a 6-h high-light treatment. Transgenic plants grown at high light also showed inactivation of 5-aminolevulinate dehydratase and porphobilinogen deaminase, whereas the activity of coproporphyrinogen oxidase and the 5-aminolevulinate synthesizing capacity were not altered. We conclude that photooxidation of accumulating uroporphyrin(ogen) leads to the generation of oxygen species, which destabilizes other enzymes in the porphyrin metabolic pathway. This porphyrin-induced necrosis resembles the induction of cell death observed during pathogenesis and air pollution.
P Metabolism in the Bean-Rhizobium tropici Symbiosis
Authors: Al-Niemi T. S. , Kahn M. L. , McDermott T. R.
Published: October 2016

Nodulated legumes require more P than legumes growing on mineral nitrogen, but little is known about the basis for the higher P requirement. Experiments were conducted to determine how Rhizobium tropici responds to P limitation and to understand how P is partitioned between the symbionts under conditions of adequate or limiting P. Free-living R. tropici responds to P stress by increasing P transport capacity and inducing both an acid and an alkaline phosphatase. This P-stress response occurs when the medium P concentration decreases below 1 [mu]M. Both P-stress-inducible phosphatases are found in bacteroids taken from plants growing with adequate P, suggesting that P levels in the symbiosome space is low enough to induce the expression of these enzymes. Bacteroid alkaline phosphatase-specific activity was highest during vegetative growth of the bean plant, but decreased approximately 75% during the host reproductive stages. In hydroponic experiments 32P-tracer studies showed that in vivo rates of P accumulation were significantly higher in bacteroids from P-limited plants compared with those from plants that had been supplied with adequate P. In contrast, label accumulation in leaves was greatest in plants grown with adequate P.
Relationship of Endo-β-D-Mannanase Activity and Cell Wall Hydrolysis in Tomato Endosperm to Germination Rates
Authors: Dahal P. , Nevins D. J. , Bradford K. J.
Published: October 2016

The endosperm tissue enclosing the radicle tip (endosperm cap) governs radicle emergence in tomato (Lycopersicon esculentum Mill.) seeds. Weakening of the endosperm cap has been attributed to hydrolysis of its mannan-rich cell walls by endo-β-D-mannanase. To test this hypothesis, we measured mannanase activity in tomato endosperm caps from seeds allowed to imbibe under conditions of varying germination rates. Over a range of suboptimal temperatures, mannanase activity prior to radicle emergence increased in accordance with accumulated thermal time. Reduced water potential delayed or prevented radicle emergence but enhanced mannanase activity in the endosperm caps. Abscisic acid did not prevent the initial increase in mannanase activity, although radicle emergence was markedly delayed. Sugar composition and percent mannose (Man) content of endosperm cap cell walls did not change prior to radicle emergence under any condition. Man, glucose, and other sugars were released into the incubation solution by endosperm caps isolated from intact seeds during imbibition. Pregerminative release of Man was suppressed and the release of glucose was enhanced when seeds were incubated in osmoticum or abscisic acid; the opposite occurred in the presence of gibberellin. Thus, whereas sugar release patterns were sensitive to environmental and hormonal factors affecting germination, neither assayable endo-β-D-mannanase activity nor changes in cell wall sugar composition of endosperm caps correlated well with tomato seed germination rates under all conditions.
Characterization of a Light-Controlled Anion Channel in the Plasma Membrane of Mesophyll Cells of Pea
Authors: Elzenga JTM. , Van Volkenburgh E.
Published: October 2016

In leaf mesophyll cells of pea (Pisum sativum) light induces a transient depolarization that is at least partly due to an increased plasma membrane conductance for anions. Several channel types were identified in the plasma membrane of protoplasts from mesophyll cells using the patch-clamp technique. One of these was an anion channel with a single-channel conductance of 32 picasiemens in symmetrical 100/100 KCl solutions. In asymmetrical solutions the reversal potential indicates a high selectivity for Cl- over K+ at high cytoplasmic Cl-. At negative membrane voltages the channel openings were interrupted by very short closures. In the open channel conductance several substrates were identified. At a cytoplasmic negative logarithm of Ca concentration higher than 6.3, no channel openings were observed. When the protoplast was illuminated in the cell-attached configuration, at least one channel type had a higher opening probability. This channel can tentatively be identified as the above-described anion channel based on conductance and the characteristic short closures at negative membrane potentials. This light activation of the 32-picasiemen anion channel is a strong indication that this channel conducts the light-induced depolarizing current. Because channel activity is strongly Ca2+-dependent, a role of cytoplasmic Ca2+ concentration changes in the light activation of the conductance is discussed.
Glutathione-Mediated Regulation of ATP Sulfurylase Activity, SO42- Uptake, and Oxidative Stress Response in Intact Canola Roots
Authors: Lappartient A. G. , Touraine B.
Published: October 2016

The dual role of glutathione as a transducer of S status (A.G. Lappartient and B. Touraine [1996] Plant Physiol 111: 147–157) and as an antioxidant was examined by comparing the effects of S deprivation, glutathione feeding, and H2O2 (oxidative stress) on SO42- uptake and ATP sulfurylase activity in roots of intact canola (Brassica napus L.). ATP sulfurylase activity increased and SO42- uptake rate severely decreased in roots exposed to 10 mM H2O2, whereas both increased in S-starved plants. In split-root experiments, an oxidative stress response was induced in roots remote from H2O2 exposure, as revealed by changes in the reduced glutathione (GSH) level and the GSH/oxidized glutathione (GSSG) ratio, but there was only a small decrease in SO42- uptake rate and no effect on ATP sulfurylase activity. Feeding plants with GSH increased GSH, but did not affect the GSH/GSSG ratio, and both ATP sulfurylase activity and SO42- uptake were inhibited. The responses of the H2O2-scavenging enzymes ascorbate peroxidase and glutathione reductase to S starvation, GSH treatment, and H2O2 treatment were not to glutathione-mediated S demand regulatory process. We conclude that the regulation of ATP sulfurylase activity and SO42- uptake by S demand is related to GSH rather than to the GSH/GSSG ratio, and is distinct from the oxidative stress response.
Asymmetric Distribution of Acetylcholinesterase in Gravistimulated Maize Seedlings
Authors: Momonoki Y. S.
Published: October 2016

Acetylcholinesterase (AChE) activity has previously been studied by this laboratory and shown to occur at the interface between the stele and cortex of the mesocotyl of maize (Zea mays L.) seedlings. In this work we studied the distribution of AChE activity in 5-d-old maize seedlings following a gravity stimulus. After the stimulus, we found an asymmetric distribution of the enzyme in the coleoptile, the coleoptile node, and the mesocotyl of the stimulated seedlings using both histochemical and colorimetric methods for measuring the hydrolysis of acetylthiocholine. The hydrolytic capability of the esterase was greater on the lower side of the horizontally placed seedlings. Using the histochemical method, we localized the hydrolytic capability in the cortical cells around the vascular stele of the tissues. The hydrolytic activity was inhibited 80 to 90% by neostigmine, an inhibitor of AChE. When neostigmine was applied to the corn kernel, the gravity response of the seedling was inhibited and no enzyme-positive spots appeared in the gravity-stimulated seedlings. We believe these results indicate a role for AChE in the gravity response of maize seedlings.
Influence of Environmental Stress on Biomass Partitioning in Transgenic Tobacco Plants Expressing the Movement Protein of Tobacco Mosaic Virus
Authors: Balachandran S. , Hull R. J. , Martins R. A. , Vaadia Y. , Lucas W. J.
Published: October 2016

The influence of various environmental factors on biomass partitioning between shoots and roots in transgenic tobacco (Nicotiana tabacum) plants expressing the movement protein (MP) of tobacco mosaic virus (TMV) was investigated. TMV-MP-expressing transgenic plants exhibited a root-to-shoot ratio that was approximately 40% below that of transgenic vector control plants. When transgenic plants expressing the TMV-MP were subjected to water-stress conditions, the root-to-shoot ratio was increased to a value comparable to that of control plants subjected to the same water-stress treatment. Although the root-to-shoot ratio was increased by N or P deficiencies, the TMV-MP-induced alteration in biomass partitioning was not overcome. Surprisingly, under K+-deficient growth conditions, both TMV-MP-expressing and control plants exhibited reduced root-to-shoot ratios when compared with plants grown in the presence of sufficient K+. Furthermore, plant growth under K+-deficient conditions did not alleviate the influence of the TMV-MP over resource allocation to the roots. These results are discussed in terms of possible mechanisms by which stress signals could cause an alteration in biomass partitioning between shoots and roots in control and transgenic tobacco plants expressing the TMV-MP.
Metabolic Control of Avocado Fruit Growth (Isoprenoid Growth Regulators and the Reaction Catalyzed by 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase)
Authors: Cowan A. K. , Moore-Gordon C. S. , Bertling I. , Wolstenholme B. N.
Published: October 2016

The effect of isoprenoid growth regulators on avocado (Persea americana Mill. cv Hass) fruit growth and mesocarp 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity was investigated during the course of fruit ontogeny. Both normal and small-fruit phenotypes were used to probe the interaction between the end products of isoprenoid biosynthesis and the activity of HMGR in the metabolic control of avocado fruit growth. Kinetic analysis of the changes in both cell number and size revealed that growth was limited by cell number in phenotypically small fruit. In small fruit a 70% reduction in microsomal HMGR activity was associated with an increased mesocarp abscisic acid (ABA) concentration. Application of mevastatin, a competitive inhibitor of HMGR, reduced the growth of normal fruit and increased mesocarp ABA concentration. These effects were reversed by co-treatment of fruit with mevalonic acid lactone, isopentenyladenine, or N-(2-chloro-4-pyridyl)-N-phenylurea, but were not significantly affected by either gibberellic acid or stigmasterol. However, stigmasterol appeared to partially restore fruit growth when co-injected with mevastatin in either phase II or III of fruit growth. In vivo application of ABA reduced fruit growth and mesocarp HMGR activity and accelerated fruit abscission, effects that were reversed by co-treatment with isopentenyladenine. Together, these observations indicate that ABA accumulation down-regulates mesocarp HMGR activity and fruit growth, and that in situ cytokinin biosynthesis modulates these effects during phase I of fruit ontogeny, whereas both cytokinins and sterols seem to perform this function during the later phases.
Characterization of the Cell Wall Microdomain Surrounding Plasmodesmata in Apple Fruit
Authors: Roy S. , Watada A. E. , Wergin W. P.
Published: October 2016

In fleshy fruits ripening is generally associated with a loss in tissue firmness resulting from depolymerization of wall components and separation of adjacent cells. In the regions of the wall that contain plasmodesmata, the usual sequences of ripening events, i.e. depolymerization of the middle lamellae and splitting of the walls, are not observed. In the present study we attempted to characterize in apple (Malus domestica Borkh.) fruit the structural microdomain of the cell wall that surrounds the plasmodesmata by in muro visualization of the cell wall components. Anionic sites of galacturonic acids were labeled with cationic gold. Low-esterified homogalacturonans were labeled with the monoclonal antibody JIM 5. In addition, a polyclonal antibody directed toward [beta](1->3)-glucopyranose was used to target callose in situ. The results indicated that the plasmodesmata-wall complexes were surrounded by a pectic microdomain. This domain was composed of low-esterified homogalacturonans that were not involved in calcium cross-bridging but were probably surrounded by a cationic environment. These structural features may result in the prevention of normal cell wall separation in regions containing plasmodesmata. However, observations by low-temperature scanning electron microscopy suggested that splitting of these walls ruptured the plasmodesmata and ultimately resulted in the spatial separation of adjacent cells.
Regulation of the Accumulation and Reduction of Nitrate by Nitrogen and Carbon Metabolites in Maize Seedlings
Authors: Sivasankar S. , Rothstein S. , Oaks A.
Published: October 2016

The accumulation and reduction of nitrate in the presence of the nitrogen metabolites asparagine (Asn) and glutamine (Gln) and the carbon metabolite sucrose (Suc) were examined in maize (Zea mays L.) seedlings in an attempt to separate their effects on the nitrate uptake system and the nitrate reduction system. After 8 h of exposure to nitrate in the presence of 1 mM Asn, tissue nitrate accumulation was reduced at 250 [mu]M external nitrate, but not at 5 mM Asn. The induction of nitrate reductase (NR) activity was reduced at both external nitrate concentrations. In the presence of 1 mM Gln or 1% Suc, tissue nitrate concentration was not significantly altered, but the induction of root NR activity was reduced or enhanced, respectively. The induction of root nitrite reductase (NiR) activity was also reduced in the presence of Asn or Gln and enhanced in the presence of Suc. Transcript levels of NR and NiR in roots were reduced in the presence of the amides and enhanced in the presence of Suc. When Suc was present in combination with either amide, there was complete relief from the inhibition of NiR transcription observed in the presence of amide alone. In the case of NR, however, this relief from inhibition was negligible. The inhibition of the induction of NR and NiR activities in the presence of Gln and Asn is a direct effect and is not the result of altered nitrate uptake in the presence of these metabolites.
ATP Hydrolysis Activity and Polymerization State of Ribulose-1,5-Bisphosphate Carboxylase Oxygenase Activase (Do the Effects of Mg2+, K+, and Activase Concentrations Indicate a Functional Similarity to Actin?)
Authors: Lilley R. M. , Portis Jr A. R.
Published: October 2016

The ATPase activity and fluoresence of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) activase were determined over a range of MgCl2, KCl, and activase concentrations. Both salts promoted ADP release from ATP and intrinsic fluorescence enhancement by adenosine 5[prime]-[[gamma]-thio] triphosphate, but Mg2+ was about 10 times more effective than K+. ATPase and fluorescence enhancement both increased from zero to saturation within the same Mg2+ and K+ concentration ranges. At saturating concentrations (5 mM Mg2+ and 22 mM K+), the specific activity of ATPase (turnover time, about 1 s) and specific intrinsic fluorescence enhancement were maximal and unaffected by activase concentration above 1 [mu]M activase; below 1 [mu]M activase, both decreased sharply. These responses are remarkably similar to the behavior of actin. Intrinsic fluorescence enhancement of Rubisco activase reflects the extent of polymerization, showing that the smaller oligomer or monomer present in low-salt and activase concentrations is inactive in ATP hydrolysis. However, quenching of 1-anilinonapthaline-8-sulfonate fluorescence revealed that ADP and adenosine 5[prime]-[[gamma]-thio] triphosphate bind equally well to activase at low- and high-salt concentrations. This is consistent with an actin-like mechanism requiring a dynamic equilibrium between monomer and oligomers for ATP hydrolysis. The specific activation rate of substrate-bound decarbamylated Rubisco decreased at activase concentrations below 1 [mu]M. This suggests that a large oligomeric form of activase, rather than a monomer, interacts with Rubisco when performing the release of bound ribulose-1,5-bisphosphate from the inactive enzyme.
Subcellular Localization of Secondary Lipid Metabolites Including Fragrance Volatiles in Carnation Petals
Authors: Hudak K. A. , Thompson J. E.
Published: October 2016

Pulse-chase labeling of carnation (Dianthus caryophyllus L. cv Improved White Sim) petals with [14C]acetate has provided evidence for a hydrophobic subcompartment of lipid-protein particles within the cytosol that resemble oil bodies, are formed by blebbing from membranes, and are enriched in lipid metabolites (including fragrance volatiles) derived from membrane fatty acids. Fractionation of the petals during pulse-chase labeling revealed that radiolabeled fatty acids appear first in microsomal membranes and subsequently in cytosolic lipid-protein particles, indicating that the particles originate from membranes. This interpretation is supported by the finding that the cytosolic lipid-protein particles contain phospholipid as well as the same fatty acids found in microsomal membranes. Radiolabeled polar lipid metabolites (methanol/water-soluble) were detectable in both in situ lipid-protein particles isolated from the cytosol and those generated in vitro from isolated radiolabeled microsomal membranes. The lipid-protein particles were also enriched in hexanal, trans-2-hexenal, 1-hexanol, 3-hexen-1-ol, and 2-hexanol, volatiles of carnation flower fragrance that are derived from membrane fatty acids through the lipoxygenase pathway. Therefore, secondary lipid metabolites, including components of fragrance, appear to be formed within membranes of petal tissue and are subsequently released from the membrane bilayers into the cytosol by blebbing of lipid-protein particles.
A Plant Chloroplast Glutamyl Proteinase
Authors: Laing W. A. , Christeller J. T.
Published: October 2016

A glutamyl proteinase was partially purified from Percoll gradient-purified spinach (Spinacia oleracea) chloroplast preparations and appeared to be predominantly localized in the chloroplast stroma. The enzyme degraded casein, but of the 11 synthetic endopeptidase substrates tested, only benzyloxycarbonyl-leucine-leucine-glutamic acid-β-napthylamide was hydrolyzed at measurable rates. In addition, the enzyme cleaved the oxidized β-chain of insulin after a glutamic acid residue. There was no evidence that native ribulose-1,5-bisphosphate carboxylase/oxygenase was cleaved by this proteinase. The apparent Km for benzyloxycarbonyl-leucine-leucine-glutamic acid-[beta]NA at the pH optimum of 8.0 was about 1 mM. Cl-ions were required for both activity and stability. Of the proteinase inhibitors covering all four classes of the endopeptidases, only 4-(2-aminoethyl)-benzenesulfonyl-fluoride HCl and L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone significantly inhibited the proteinase. The partially purified enzyme had a molecular weight of about 350,000 to 380,000, based on size-exclusion chromatography. The enzyme has both similar and distinctive properties to those of the bacterial glutamyl proteinases. To our knowledge, this is the first description of a plant glutamyl proteinase found predominantly or exclusively in the chloroplast.
Turnover of Galactans and Other Cell Wall Polysaccharides during Development of Flax Plants
Authors: Gorshkova T. A. , Chemikosova S. B. , Lozovaya V. V. , Carpita N. C.
Published: October 2016

We investigated the synthesis and turnover of cell wall polysaccharides of the flax (Linum usitatissimum L.) plant during development of the phloem fibers. One-month-old flax plants were exposed to a 40-min pulse with 14CO2 followed by 8-h, 24-h, and 1-month periods of chase with ambient CO2, and radioactivity in cell wall sugars was determined in various plant parts. The relative radioactivity of glucose in noncellulosic polysaccharides was the highest compared with all other cell wall sugars immediately after the pulse and decreased substantially during the subsequent chase. The relative radioactivities of the other cell wall sugars changed with differing rates, indicating turnover of specific polysaccharides. Notably, after 1 month of chase there was a marked decrease in the proportional mass and total radioactivity in cell wall galactose, indicating a long-term turnover of the galactans enriched in the fiber-containing tissues. The ratio of radiolabeled xylose to arabinose also increased during the chase, indicating a turnover of arabinose-containing polymers and interconversion to xylose. The pattern of label redistribution differed between organs, indicating that the cell wall turnover processes are tissue- and cell-specific.
Dissecting the Diphenylene Iodonium-Sensitive NAD(P)H:Quinone Oxidoreductase of Zucchini Plasma Membrane
Authors: Trost P. , Foscarini S. , Preger V. , Bonora P. , Vitale L. , Pupillo P.
Published: October 2016

Quinone oxidoreductase activities dependent on pyridine nucleotides are associated with the plasma membrane (PM) in zucchini (Cucurbita pepo L.) hypocotyls. In the presence of NADPH, lipophilic ubiquinone homologs with up to three isoprenoid units were reduced by intact PM vesicles with a Km of 2 to 7 [mu]M. Affinities for both NADPH and NADH were similar (Km of 62 and 51 [mu]M, respectively). Two NAD(P)H:quinone oxidoreductase forms were identified. The first, labeled as peak I in gel-filtration experiments, behaves as an intrinsic membrane complex of about 300 kD, it slightly prefers NADH over NADPH, it is markedly sensitive to the inhibitor diphenylene iodonium, and it is active with lipophilic quinones. The second form (peak II) is an NADPH-preferring oxidoreductase of about 90 kD, weakly bound to the PM. Peak II is diphenylene iodonium-insensitive and resembles, in many properties, the soluble NAD(P)H:quinone oxidoreductase that is also present in the same tissue. Following purification of peak I, however, the latter gave rise to a quinone oxidoreductase of the soluble type (peak II), based on substrate and inhibitor specificities and chromatographic and electrophoretic evidence. It is proposed that a redox protein of the same class as the soluble NAD(P)H:quinone oxidoreductase (F. Sparla, G. Tedeschi, and P. Trost [1996] Plant Physiol. 112:249–258) is a component of the diphenylene iodonium-sensitive PM complex capable of reducing lipophilic quinones.
β-1,3-Glucanase Is Cryoprotective in Vitro and Is Accumulated in Leaves during Cold Acclimation
Authors: Hincha D. K. , Meins Jr F. , Schmitt J. M.
Published: October 2016

We have used isolated spinach (Spinacea oleracea L.) thylakoid membranes to investigate the possible cryoprotective properties of class I β-1,3-glucanase (1,3-β-D-glucan 3-glucanohydrolase; EC 3.2.1.39) and chitinase. Class I β-1,3-glucanase that was purified from tobacco (Nicotiana tabacum L.) protected thylakoids against freeze-thaw injury in our in vitro assays, whereas class I chitinase from tobacco had no effect under the same conditions. The β-1,3-glucanase acted by reducing the influx of solutes into the membrane vesicles during freezing and thereby reduced osmotic stress and vesicle rupture during thawing. Western blots probed with antibodies directed against tobacco class I β-1,3-glucanase showed that in spinach and cabbage (Brassica oleracea L.) leaves an isoform of 41 kD was accumulated during frost hardening under natural conditions.
Experimental Analysis of Tassel Development in the Maize Mutant Tassel Seed 6
Authors: Irish E. E.
Published: October 2016

The maize (Zea mays L.) mutation Tassel seed 6 (Ts6) disrupts both sex determination in the tassel and the pattern of branching in inflorescences. This results in the formation of supernumerary florets in tassels and ears and in the development of pistils in tassel florets where they are normally aborted. A developmental analysis indicated that extra florets in Ts6 inflorescences are most likely the result of delayed determinacy in spikelet meristems, which then initiate additional floret meristems rather than initiating floral organs as in wild type. I have used culturing experiments to assay whether delayed determinacy of Ts6 mutant tassels is reflected in an altered timing of specific determination events. Length of the tassel was used as a developmental marker. These experiments showed that although Ts6 tassels elongate much more slowly than wild type, both mutant and wild-type tassels gained the ability to form flowers with organs of normal morphology in culture at the same time. In situ hybridization patterns of expression of the maize gene Kn, which is normally expressed in shoot meristems and not in determinate lateral organs, confirmed that additional meristems, rather than lateral organs, are initiated by spikelet meristems in Ts6 tassels.
Involvement of Thylakoid Overenergization in Tentoxin-Induced Chlorosis in Nicotiana spp
Authors: Holland N. , Evron Y. , Jansen MAK. , Edelman M. , Pick U.
Published: October 2016

The purpose of this work was to clarify the mechanism of tentoxin-induced chlorosis in Nicotiana spp. seedlings. We found that chlorosis does not correlate with the inhibition of chloroplast ATP synthesis in vivo, since it occurs at tentoxin concentrations far higher than that required for the inhibition of photophosphorylation measured in the same seedlings. However, tentoxin-induced chlorosis does correlate with in vivo overenergization of thylakoids. We show that tentoxin induces overenergization in intact plants and isolated thylakoids, probably via multiple interactions with ATP synthase. Furthermore, gramicidin D, a protonophore that relieves overenergization, also relieves chlorosis. Two lines of evidence suggest that reactive oxygen species may be involved in the process of chlorosis: ascorbate, a quencher of oxygen radicals, significantly protects against chlorosis, whereas transgenic Nicotiana spp. mutants overexpressing chloroplast superoxide dismutase are partially resistant to tentoxin-induced chlorosis. It is proposed that chlorosis in developing seedlings results from overenergization of thylakoids, which leads to the generation of oxygen radicals.
Modulation of Dehydration Tolerance in Soybean Seedlings (Dehydrin Mat1 Is Induced by Dehydration but Not by Abscisic Acid)
Authors: Whitsitt M. S. , Collins R. G. , Mullet J. E.
Published: October 2016

Germinated soybean (Glycine max L. cv Williams 82) seedlings subjected to rapid dehydration begin to lose the ability to recover when the relative water content of the plant decreases below 60%. The expanded cells of the hypocotyl appear more susceptible to dehydration-induced damage than do cells in the hypocotyl zone of cell growth. Pretreatment of seedlings prior to rapid dehydration with nonlethal water deficit or exogenous abscisic acid (ABA) shifts this viability threshold to progressively lower relative water contents, indicating the acquisition of increased dehydration tolerance. Increased tolerance is associated with osmotic adjustment in the hypocotyl zone of cell growth and with increases in soybean dehydrin Mat1 mRNA levels. The accumulation of Mat1 mRNA is dehydration dependent but insensitive to ABA. Induction of Mat1 mRNA accumulation by dehydration but not by ABA makes it an unusual member of the dehydrin family.