bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamics of Changing Intercellular CO2 Concentration (ci) during Drought and Determination of Minimum Functional ci
Authors: Brodribb T.
Published: October 2016

Nine conifer species with narrow (<5 mm), single-veined leaves were selected for the purpose of examining changes in intercellular CO2 concentration (ci) during drought. Due to the leaf morphology of the study plants, the confounding effects of nonhomogenous photosynthesis common to most reticulate-veined angiosperms were largely avoided, giving a clear picture of ci dynamics under increasing drought. A characteristic biphasic response was observed in all species, with an initial stomatal control phase resulting in a substantial reduction in ci as stomatal conductance (gs) decreased. As gs reached low levels, a strong nonstomatal limitation phase was observed, causing ci to increase as gs approached a minimum. This nonstomatal phase was linked to a concomitant rapid decrease in the fluorescence parameter quantum efficiency, indicating the onset of nonreversible photoinhibition. The ratio of internal to atmospheric CO2 concentration (ci/ca) decreased from values of between 0.68 and 0.57 in undroughted plants to a minimum, (ci/ca)min, which was well defined in each species, ranging from 0.10 in Actinostrobus acuminatus to 0.36 in Acmopyle pancheri. A high correlation was found to exist between (ci/ca)min and leaf water potential measured at (ci/ca)min. Species developing high maximum intrinsic water use efficiencies (low [ci/ca]min), such as A. acuminatus, did so at lower leaf water potentials (-4.5 MPa) than more mesic species (-1.75 MPa for A. pancheri). It is concluded that in the absence of patchy stomatal closure, (ci/ca)min gives a good representation of the drought tolerance of foliage.
Calorimetric Properties of Dehydrating Pollen (Analysis of a Desiccation-Tolerant and an Intolerant Species)
Authors: Buitink J. , Walters-Vertucci C. , Hoekstra F. A. , Leprince O.
Published: October 2016

The physical state of water in the desiccation-tolerant pollen of Typha latifolia L. and the desiccation-sensitive pollen of Zea mays L. was studied using differential scanning calorimetry in an attempt to further unravel the complex mechanisms of desiccation tolerance. Melting transitions of water were not observed at water content (wc) values less than 0.21 (T. latifolia) and 0.26 (Z. mays) g H2O/g dry weight. At moisture levels at which melting transitions were not observable, water properties could be characterized by changes in heat capacity. Three hydration regions could be distinguished with the defining wc values changing as a function of temperature. Shifts in baseline power resembling second-order transitions were observed in both species and were interpreted as glass-to-liquid transitions, the glass-transition temperatures being dependent on wc. Irrespective of the extent of desiccation tolerance, both pollens exhibited similar state diagrams. The viability of maize pollen at room temperature decreased gradually with the removal of the unfrozen water fraction. In maize, viability was completely lost before grains were sufficiently dried to enter into a glassy state. Apparently, the glassy state per se cannot provide desiccation tolerance. From the existing data, we conclude that, although no major differences in the physical behavior of water could be distinguished between desiccation-tolerant and -intolerant pollens, the physiological response to the loss of water varies between the two pollen types.
Characterization of Membrane Properties in Desiccation-Tolerant and -Intolerant Carrot Somatic Embryos
Authors: Tetteroo FAA. , de Bruijn A. Y. , Henselmans RNM. , Wolkers W. F. , van Aelst A. C. , Hoekstra F. A.
Published: October 2016

In previous studies, we have shown that carrot (Daucus carota L.) somatic embryos acquire complete desiccation tolerance when they are treated with abscisic acid during culture and subsequently dried slowly. With this manipulable system at hand, we have assessed damage associated with desiccation intolerance. Fast drying caused loss of viability, and all K+ and carbohydrates leached from the somatic embryos within 5 min of imbibition. The phospholipid content decreased by about 20%, and the free fatty acid content increased, which was not observed after slow drying. However, the extent of acyl chain unsaturation was unaltered, irrespective of the drying rate. These results indicate that, during rapid drying, irreversible changes occur in the membranes that are associated with extensive leakage and loss of germinability. The status of membranes after 2 h of imbibition was analyzed in a freeze-fracture study and by Fourier transform infrared spectroscopy. Rapidly dried somatic embryos had clusters of intramembraneous particles in their plasma membranes, and the transition temperature of isolated membranes was above room temperature. Membrane proteins were irreversibly aggregated in an extended β-sheet conformation and had a reduced proportion of α--helical structures. In contrast, the slowly dried somatic embryos had irregularly distributed, but non-clustered, intramembraneous particles, the transition temperature was below room temperature, and the membrane proteins were not aggregated in a β-sheet conformation. We suggest that desiccation sensitivity of rapidly dried carrot somatic embryos is indirectly caused by an irreversible phase separation in the membranes due to de-esterification of phospholipids and accumulation of free fatty acids.
Chloroplast Distribution in Arabidopsis thaliana (L.) Depends on Light Conditions during Growth
Authors: Trojan A. , Gabrys H.
Published: October 2016

Chloroplasts of Arabidopsis thaliana move in response to blue light. Sensitivity to light and the range of fluence rates to which the chloroplasts respond were found to be comparable to those of other higher plants studied. We investigated typical chloroplast distributions in Arabidopsis grown under three different light conditions:standard-light conditions, similar to natural light intensities; weak-light intensities, close to the compensation point of photosynthesis; and strong-light intensities, close to the saturation of the light-response curve of photosynthesis. We observed a striking difference in chloroplast arrangement in darkness between plants grown under weak- and strong-light conditions. There was a slight difference after weak-light pretreatment, and the arrangements of chloroplasts after strong-light pretreatment in both plant groups were very similar. These results support the ecological significance of chloroplast movements.
The N-1-Naphthylphthalamic Acid-Binding Protein Is an Integral Membrane Protein
Authors: Bernasconi P. , Patel B. C. , Reagan J. D. , Subramanian M. V.
Published: October 2016

N-1-Naphthylphthalmic acid (NPA)-binding protein is a plasmalemma (PM) protein involved in the control of cellular auxin efflux. We re-evaluated the spatial relationship of this protein with the PM of zucchini (Cucurbita pepo L.) hypocotyls. First, Triton X-114 partitioning indicated that the NPA-binding protein was more hydrophobic than most PM proteins. Second, the NPA-binding activity was found to be resistant to proteolytic digestion in membranes. Maximum concentrations of binding sites for NPA were virtually identical in untreated and proteinase K-treated PMs: 19.2 and 20.6 pmol [3H]NPA bound/mg protein, respectively. The insensitivity of the NPA-binding protein was not due to its presence inside tightly sealed vesicles or due to lack of protease activity in the conditions tested. This protein could be made sensitive to proteolytic degradation upon solubilization by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in the presence of sodium molybdate. Proteinase K treatment decreased the concentration of binding sites to 0.84 pmol [3H]NPA bound/mg protein from 9.2 for untreated, solubilized PM. Third, this activity could not be solubilized by chaotropic agents or sodium carbonate treatment of intact PM. This study indicates that the NPA-binding protein may be an integral membrane protein and contradicts previously reported findings that suggested that this protein was peripheral to the PM.
Alterations in Structural Polysaccharides during Liquefaction of Tomato Locule Tissue
Authors: Cheng G. W. , Huber D. J.
Published: October 2016

The locule tissue of tomato (Lycopersicon esculentum, Mill.) undergoes extensive liquefaction during ripening. In this study, the solubility, molecular mass, and glycosyl composition of locule pectic and alkali-soluble polysaccharides were examined with the aim of identifying features contributing to the unique properties of this tissue. Ethanol-insoluble solids were prepared from de-seeded locule tissue from tomato fruit at the immature green (IMG), mature green, and breaker stages of development. Ethanol-insoluble pectins were extracted sequentially in H2O, 50 mM trans-1,2-diaminocyclohexane-N,N,N[prime],N[prime]-tetraacetic acid, 50 mM Na2CO3, and 4 M KOH. At the IMG stage, nearly 85% of the locule pectins were solubilized by water, trans-1,2-diaminocyclohexane-N,N,N[prime],N[prime]-tetraacetic acid, and Na2CO3 solutions. Solubility increased only slightly with further locule development. The noncovalently associated polymers were of high molecular mass throughout liquefaction. Polymers extracted in mild alkali were of considerably lower molecular mass. Locule pectins in IMG fruit were heavily glycosylated with galactose, arabinose, and xylose. All pectin classes exhibited similar deglycosylation trends during liquefaction. Locule hemicelluloses were rich in glucose, xylose, and arabinose. These polymers collectively showed molecular mass downshifts with minimal compositional changes during liquefaction. The KOH-soluble material also included xylose-rich acidic polymers not matching the neutral sugar profile of the noncovalently associated pectic polymers.
Volatile Semiochemicals Released from Undamaged Cotton Leaves (A Systemic Response of Living Plants to Caterpillar Damage)
Authors: Rose USR. , Manukian A. , Heath R. R. , Tumlinson J. H.
Published: October 2016

Cotton plants (Gossypium hirsutum L.), attacked by herbivorous insects release volatile semiochemicals (chemical signals) that attract natural enemies of the herbivores to the damaged plants. We found chemical evidence that volatiles are released not only at the damaged site but from the entire cotton plant. The release of volatiles was detected from upper, undamaged leaves after 2 to 3 d of continuous larval damage on lower leaves of the same plant. Compounds released systemically were (Z)-3-hexenyl acetate, (E)-β-ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene, (E)-β-farnesene, (E,E)-α--farnesene, and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene. All systemically released compounds are known to be induced by caterpillar damage and are not released in significant amounts by undamaged plants. Other compounds, specifically indole, isomeric hexenyl butyrates, and 2-methylbutyrates, known to be released by cotton in response to caterpillar damage, were not released systemically. However, when upper, undamaged leaves of a caterpillar-damaged plant were damaged with a razor blade, they released isomeric hexenyl butyrates, 2-methylbutyrates, and large amounts of constitutive compounds in addition to the previously detected induced compounds. Control plants, damaged with a razor blade in the same way, did not release isomeric hexenyl butyrates or 2-methylbutyrates and released significantly smaller amounts of constitutive compounds. Indole was not released systemically, even after artificial damage.
Differential Expression of Lipoxygenase Isoenzymes in Embryos of Germinating Barley
Authors: Holtman W. L. , van Duijn G. , Sedee NJA. , Douma A. C.
Published: October 2016

Expression of lipoxygenase was studied in barley (Hordeum distichum L.) embryos during germination. Total lipoxygenase activity was high in quiescent grains, dropped during the 1st d of germination, and subsequently increased to a level similar to that in quiescent grains. The contribution of two isoenzymes, lipoxygenases 1 (LOX-1) and 2 (LOX-2), was studied at the activity, protein, and mRNA levels. Activity ratios of the two isoforms were determined via the ratio of 9- and 13-hydroperoxides, which are formed from linoleic acid. Isoenzyme protein levels were determined using specific monoclonal antibodies. mRNA levels were studied using the specific cDNA probes LoxA and LoxC, which correspond to LOX-1 and LOX-2, respectively. The major difference in temporal expression of LOX-1 and LOX-2 was observed in quiescent grains. At this stage, LOX-1 contributed almost exclusively to total lipoxygenase activity. LOX-2 activity rapidly increased until d 2 of germination. From this time point onward, LOX-1 and LOX-2 showed similar patterns at both activity and protein levels. The tissue distribution of the two isoenzymes in the germinating embryo was closely similar, with the highest expression levels in leaves and roots. The levels of LOX-1 and LOX-2 may be regulated mainly pretranslationally, as suggested by the similarity of the protein and mRNA patterns corresponding to the two isoforms.
Growth Inhibition in Suspension-Cultured Rice Cells under Phosphate Deprivation Is Mediated through Putrescine Accumulation
Authors: Shih C. Y. , Kao C. H.
Published: October 2016

The effects of phosphate deprivation on the growth and polyamine levels of suspension-cultured rice (Oryza sativa) cells were investigated. When rice suspension cells were deprived of phosphate, cell growth was markedly inhibited. Phosphate deprivation resulted in a higher putrescine level and lower spermidine and spermine levels in rice suspension cells. The growth of rice cells cultured in the absence of phosphate did not recover as a result of spermidine and spermine addition. D-Arginine and α--methylornithine, inhibitors of putrescine biosynthesis, caused a reduced level of putrescine in rice suspension cells cultured under phosphate deprivation. The growth of rice cells cultured in the absence of phosphate was completely recovered after the addition of D-arginine but not α--methylornithine. Our results indicate that putrescine accumulation is a factor causing growth inhibition of suspension-cultured rice cells under phosphate deprivation.
Biosynthesis of Caffeine in Leaves of Coffee
Authors: Ashihara H. , Monteiro A. M. , Gillies F. M. , Crozier A.
Published: October 2016

The levels of endogenous caffeine and theobromine were much higher in buds and young leaves of Coffea arabica L. cv Kent than in fully developed leaves. Biosynthesis of caffeine from 14C-labeled adenine, guanine, xanthosine, and theobromine was observed, whereas other studies (H. Ashihara, A.M. Monteiro, T. Moritz, F.M. Gillies, A. Crozier [1996] Planta 198: 334–339) have indicated that there is no detectable incorporation of label into caffeine when theophylline and xanthine are used as substrates for in vivo feeds with leaves of C. arabica. The capacity for caffeine biosynthesis, especially from guanine and xanthosine, was reduced markedly in both fully developed mature and aged leaves. Data obtained in pulse-chase experiments with young leaves indicate the operation of an AMP -> IMP -> xanthosine 5[prime]-monophosphate (or GMP -> guanosine) -> xanthosine -> 7-methylxanthosine -> 7-methylxanthine -> theobromine -> caffeine pathway. The data obtained provide strong evidence against proposals by G.M. Nazario and C.J. Lovatt ([1993] Plant Physiol 103: 1203–1210) concerning the independence of caffeine and theobromine biosynthesis pathways and the role of xanthine as a key intermediate in caffeine biosynthesis.
Analysis of Cytokinin Metabolism in ipt Transgenic Tobacco by Liquid Chromatography-Tandem Mass Spectrometry
Authors: Redig P. , Schmulling T. , Van Onckelen H.
Published: October 2016

The endogenous levels of the major, naturally occurring cytokinins in Pisum sativum ribulose-1,5-bisphosphate carboxylase small subunit promoter-isopentenyl transferase gene (Pssu-ipt)-transformed tobacco (Nicotiana tabacum L.) callus were quantified using electrospray-liquid chromatography-tandem mass spectrometry during a 6-week subcultivation period. An ipt gene was expressed under control of a tetracycline-inducible promoter for a more detailed study of cytokinin accumulation and metabolism. Activation of the ipt in both expression systems resulted in the production of mainly zeatin-type cytokinins. No accumulation of isopentenyladenine or isopentenyladenosine was observed. In Pssu-ipt-transformed calli, as well as in the tetracycline-inducible ipt leaves, metabolic inactivation occurred through O-glucoside conjugation. No significant elevation of cytokinin N-glucosides levels was observed. Side-chain reduction to dihydrozeatin-type cytokinins was observed in both systems. The levels of the endogenous cytokinins varied in time and were subject to homeostatic regulatory mechanisms. Feeding experiments of ipt transgenic callus with [3H]isopentenyladenine and [3H]isopentenyladenosine mainly led to labeled adenine-like compounds, which are degradation products from cytokininoxidase activity. Incorporation of radioactivity in zeatin riboside was observed, although to a much lesser extent.
Role of Jasmonates in the Elicitor- and Wound-Inducible Expression of Defense Genes in Parsley and Transgenic Tobacco
Authors: Ellard-Ivey M. , Douglas C. J.
Published: October 2016

Jasmonates have been proposed to be signaling intermediates in the wound and/or elicitor-activated expression of plant defense genes. We used parsley (Petroselinum crispum) cell cultures and transgenic tobacco (Nicotiana tabacum) plants expressing 4CL1-GUS gene fusions to investigate the potential role played by jasmonates in mediating the wound and/or elicitor activation of phenylpropanoid and other defense-related genes. Jasmonates and α--linolenic acid strongly induced the expression of 4CL in a dose-dependent manner in parsley cells; methyl jasmonate also activated the coordinate expression of other phenylpropanoid genes and the accumulation of furanocoumarin phytoalexins. However, the response of the cells to optimal methyl jasmonate concentrations was distinct quantitatively and qualitatively from the response of elicitor-treated cells. In transgenic tobacco wound-inducible tobacco 4CL genes and a 4CL1 promoter-GUS transgene were responsive to jasmonates and α--linolenic acid in a dose-dependent manner. Pre-treatment of parsley cells or tobacco leaves with a lipoxygenase inhibitor reduced their responsiveness to the elicitor and to wounding. These results show that the elicitor response in parsley cells can be partially mimicked by jasmonate treatment, which supports a role for jasmonates in mediating wound-induced expression of 4CL and other phenylpropanoid genes.
Correlation of Rapid Cell Death with Metabolic Changes in Fungus-Infected, Cultured Parsley Cells
Authors: Naton B. , Hahlbrock K. , Schmelzer E.
Published: October 2016

To study in detail the hypersensitive reaction, one of the major defense responses of plants against microbial infection, we used a model system of reduced complexity with cultured parsley (Petroselinum crispum) cells infected with the phytopathogenic fungus Phytophthora infestans. Experimental conditions were established to maintain maximal viability of the cultured cells during co-cultivation with fungal germlings, and a large proportion of the infected parsley cells responded to fungal infection with rapid cell death, thereby exhibiting major features of the hypersensitive reaction in whole-plant-pathogen interactions. Rapid cell death clearly correlated with termination of further growth and development of the fungal pathogen. Thus, the system fulfilled important prerequisites for investigating cell-death-related metabolic changes in individual infected cells. Using cytochemical methods, we monitored the increase of mitochondrial activity in single infected cells and the intracellular accumulation of reactive oxygen species prior to the occurrence of rapid cell death. We obtained strong correlative evidence for the involvement of these intracellularly accumulating reactive oxygen species in membrane damage and in the resulting abrupt collapse of the cell.
Tissue-Specific and Light-Mediated Expression of the C4 Photosynthetic NAD-Dependent Malic Enzyme of Amaranth Mitochondria
Authors: Long J. J. , Berry J. O.
Published: October 2016

In the C4 dicotyledonous grain plant amaranth (Amaranthus hypochondriacus), a mitochondrial NAD-dependent malic enzyme (NAD-ME; EC 1.1.1.39) serves a specialized and essential role in photosynthetic carbon fixation. In this study we have examined specialized photosynthetic gene expression patterns for the NAD-ME α- subunit. We show here that the α- subunit gene is preferentially expressed in leaves and cotyledons (the most photosynthetically active tissues), and this expression is specific to the bundle-sheath cells of these tissues from the earliest stages of development. Synthesis of the α- subunit polypeptide and accumulation of its corresponding mRNA are strongly light-dependent, but this regulation is also influenced by seedling development. In addition, light-dependent accumulation of the α- subunit mRNA is regulated at transcriptional as well as posttranscriptional levels. Our findings demonstrate that the NAD-ME of amaranth has acquired numerous complex tissue-specific and light-mediated regulation patterns that define its specialized function as a key enzyme in the C4 photosynthetic pathway.
The Effects of Exogenous Auxins on Endogenous Indole-3-Acetic Acid Metabolism (The Implications for Carrot Somatic Embryogenesis)
Authors: Ribnicky D. M. , Ilic N. , Cohen J. D. , Cooke T. J.
Published: October 2016

The effect of auxin application on auxin metabolism was investigated in excised hypocotyl cultures of carrot (Daucus carota). Concentrations of both free and conjugated indole-3-acetic acid (IAA), [2H4]IAA, 2,4-dichlorophenoxyacetic acid, and naphthaleneacetic acid (NAA) were measured by mass spectroscopy using stable-isotope-labeled internal standards. [13C1]NAA was synthesized for this purpose, thus extending the range of auxins that can be assayed by stable-isotope techniques. 2,4-Dichlorophenoxyacetic acid promoted callus proliferation of the excised hypocotyls, accumulated as the free form in large quantities, and had minor effects on endogenous IAA concentrations. NAA promoted callus proliferation and the resulting callus became organogenic, producing both roots and shoots. NAA was found mostly in the conjugated form and had minor effects on endogenous IAA concentrations. [2H4]IAA had no visible effect on the growth pattern of cultured hypocotyls, possibly because it was rapidly metabolized to form inactive conjugates or possibly because it mediated a decrease in endogenous IAA concentrations by an apparent feedback mechanism. The presence of exogenous auxins did not affect tryptophan labeling of either the endogenous tryptophan or IAA pools. This suggested that exogenous auxins did not alter the IAA biosynthetic pathway, but that synthetic auxins did appear to be necessary to induce callus proliferation, which was essential for excised hypocotyls to gain the competence to form somatic embryos.
Stimulation of Symbiotic N2 Fixation in Trifolium repens L. under Elevated Atmospheric pCO2 in a Grassland Ecosystem
Authors: Zanetti S. , Hartwig U. A. , Luscher A. , Hebeisen T. , Frehner M. , Fischer B. U. , Hendrey G. R. , Blum H. , Nosberger J.
Published: October 2016

Symbiotic N2 fixation is one of the main processes that introduces N into terrestrial ecosystems. As such, it may be crucial for the sequestration of the extra C available in a world of continuously increasing atmospheric CO2 partial pressure (pCO2). The effect of elevated pCO2 (60 Pa) on symbiotic N2 fixation (15N-isotope dilution method) was investigated using Free-Air-CO2-Enrichment technology over a period of 3 years. Trifolium repens was cultivated either alone or together with Lolium perenne (a nonfixing reference crop) in mixed swards. Two different N fertilization levels and defoliation frequencies were applied. The total N yield increased consistently and the percentage of plant N derived from symbiotic N2 fixation increased significantly in T. repens under elevated pCO2. All additionally assimilated N was derived from symbiotic N2 fixation, not from the soil. In the mixtures exposed to elevated pCO2, an increased amount of symbiotically fixed N (+7.8, 8.2, and 6.2 g m-2 a-1 in 1993, 1994, and 1995, respectively) was introduced into the system. Increased N2 fixation is a competitive advantage for T. repens in mixed swards with pasture grasses and may be a crucial factor in maintaining the C:N ratio in the ecosystem as a whole.
Identification and Characterization of an Inducible NAD(P)H Dehydrogenase from Red Beetroot Mitochondria
Authors: Menz R. I. , Day D. A.
Published: October 2016

Exogenous NADH oxidation of mitochondria isolated from red beetroots (Beta vulgaris L.) increased dramatically upon slicing and aging the tissue. Anion-exchange chromatography of soluble fractions derived by sonication from fresh and aged beetroot mitochondria yielded three NADH dehydrogenase activity peaks. The third peak from aged beetroot mitochondria was separated into two activities by blue-affinity chromatography. One of these (the unbound peak) readily oxidized dihydrolipoamide, whereas the other (the bound peak) did not. The latter was an NAD(P)H dehydrogenase with high quinone and ferricyanide reductase activity and was absent from fresh beet mitochondria. Further affinity chromatography of the NAD(P)H dehydrogenase indicated enrichment of a 58-kD polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We propose that this 58-kD protein is the inducible, external NADH dehydrogenase.
Coarse and Fine Control and Annual Changes of Sucrose-Phosphate Synthase in Norway Spruce Needles
Authors: Loewe A. , Einig W. , Hampp R.
Published: October 2016

Annual changes of activity of sucrose-phosphate synthase (SPS) from spruce (Picea abies [L.] Karst.) needles were studied with respect to three regulatory levels: metabolic fine control, covalent modification (phosphorylation), and protein amount. Glucose-6-phosphate served as an allosteric activator of spruce SPS by shifting the Michaelis constant for the substrate fructose-6-phosphate from 4.2 to 0.59 mM, whereas inorganic phosphate competitively inhibited this activation. The affinity for the other substrate, UDP-glucose, was unaffected. Incubation of the crude extract with ATP resulted in a time- and concentration-dependent decrease of the maximal velocity of SPS. This inactivation was sensitive to staurosporine, a potent protein kinase inhibitor, indicating the participation of a protein kinase. Probing SPS protein with heterologous antibodies showed that the subunit of spruce SPS is an approximately 139-kD protein and that changes in the extractable activity during the course of a year were correlated with the amount of SPS protein. High SPS activities in winter were paralleled by increased levels of the activator glucose-6-phosphate and the substrate fructose-6-phosphate, indicating a high capacity for sucrose synthesis that may be necessary to maintain photosynthetic CO2 fixation in cold-hardened spruce needles.
Antioxidant Response to NaCl Stress in a Control and an NaCl-Tolerant Cotton Cell Line Grown in the Presence of Paraquat, Buthionine Sulfoximine, and Exogenous Glutathione
Authors: Gossett D. R. , Banks S. W. , Millhollon E. P. , Lucas M. C.
Published: October 2016

A cotton (Gossypium hirsutum L.) control and NaCl-tolerant cell line (cv Coker 312) were grown on media with or without NaCl in the presence or absence of paraquat, buthionine sulfoximine, and oxidized glutathione. On medium with 150 mM NaCl the NaCl-tolerant cell line exhibited no reduction in growth, whereas a 96% reduction was observed in the control line. The NaCl-tolerant cell line that was grown on 150 mM NaCl exhibited significantly greater catalase (341%), peroxidase (319%), glutathione reductase (287%), ascorbate peroxidase (450%), [gamma]-glutamylcysteine synthetase (224%), and glutathione S-transferase (500%) activities than the intolerant control. The NaCl-tolerant cell line had a significantly lower dehydroascorbic acid/ascorbic acid ratio. Paraquat reduced growth by 20 and 53.7%, respectively, in the NaCl-tolerant and control cell line. The NaCl-tolerant cell line also showed a slight tolerance to buthionine sulfoximine. In the buthionine sulfoximine experiments reduced glutathione restored growth in both cell lines, whereas oxidized glutathione restored growth only in the NaCl-tolerant cell line. These data indicate that the NaCl-tolerant cell line exhibited a cross-tolerance to a variety of stress variables and had a more active ascorbate-glutathione cycle.
Arginase, Arginine Decarboxylase, Ornithine Decarboxylase, and Polyamines in Tomato Ovaries (Changes in Unpollinated Ovaries and Parthenocarpic Fruits Induced by Auxin or Gibberellin)
Authors: Alabadi D. , Aguero M. S. , Perez-Amador M. A. , Carbonell J.
Published: October 2016

Arginase (EC 3.5.3.1) activity has been found in the ovaries and Young fruits of tomato (Lycopersicon esculentum Mill. cv Rutgers).Changes in arginase, arginine decarboxylase (EC 4.1.1.19), and ornithine decarboxylase activity (EC 4.1.1.17) and levels of free and conjugated putrescine, spermidine, and spermine were determined in unpollinated ovaries and in parthenocarpic fruits during the early stages of development induced by 2,4-dichlorophenoxyacetic acid (2,4-D) or gibberellic acid (GA3). Levels of arginase, free spermine, and conjugates of the three polyamines were constant in unpollinated ovaries and characteristic of a presenescent step. A marked decrease in arginase activity, free spermine, and polyamine conjugates was associated with the initiation of fruit growth due to cell division, and when cell expansion was initiated, the absence of arginase indicated a redirection of nitrogen metabolism to the synthesis of arginine. A transient increase in arginine decarboxylase and ornithine decarboxylase was also observed in 2,4-D-induced fruits. In general, 2,4-D treatments produced faster changes than GA3, and without treatment, unpollinated ovaries developed only slightly and senescence was hardly visible. Sensitivity to 2,4-D and GA3 treatment remained for at least 2 weeks postanthesis.
3,7-Dichloroquinolinecarboxylic Acid Inhibits Cell-Wall Biosynthesis in Maize Roots
Authors: Koo S. J. , Neal J. C. , DiTomaso J. M.
Published: October 2016

The mode of action of the herbicide 3,7-dichloroquinolinecar-boxylic acid (quinclorac) was examined by measuring incorporation of [14C]glucose, [14C]acetate, [3H]thymidine, and [3H]uridine into maize (Zea mays) root cell walls, fatty acids, DNA, and RNA, respectively. Among the precursors examined, 10 [mu]M quinclorac inhibited [14C]glucose incorporation into the cell wall within 3 h. Fatty acid and DNA biosynthesis were subsequently inhibited, whereas RNA biosynthesis was unaffected. In contrast to the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile, quinclorac strongly inhibited cellulose and a hemicellulose fraction presumed to be glucuronoarabinoxylan. However, the synthesis of (1–>3),(1–>4)-β-D-glucans was only slightly inhibited. The degree of inhibition was time- and dose-dependent. By 4 h after treatment, the concentration that inhibited [14C]glucose incorporation into the cell wall, cellulose, and the sensitive hemicellulose fraction by 50% was about 15, 5, and 20 [mu]M, respectively. Concomitant with an inhibition of [14C]glucose incorporation into the cell wall, quinclorac treatment led to a marked accumulation of radioactivity in the cytosol. The increased radioactivity was found mostly in glucose and fructose. However, total levels of glucose, fructose, and uridine diphosphate-glucose were not changed greatly by quinclorac. These data suggest that quinclorac acts primarily as a cell-wall biosynthesis inhibitor in a susceptible grass by a mechanism that is different from that of 2,6-dichlorobenzonitrile.
Stomatal Guard Cells Are Totipotent
Authors: Hall R. D. , Riksen-Bruinsma T. , Weyens G. , Lefebvre M. , Dunwell J. M. , Krens F. A.
Published: October 2016

It has been successfully demonstrated, using epidermis explants of sugar beet (Beta vulgaris L.), that stomatal guard cells retain full totipotent capacity. Despite having one of the highest degrees of morphological adaptation and a unique physiological specialization, it is possible to induce a re-expression of full (embryogenic) genetic potential in these cells in situ by reversing their highly differentiated nature to produce regenerated plants via a callus stage. The importance of these findings both to stomatal research and to our understanding of cytodifferentiation in plants is discussed.
Frequencies, Timing, and Spatial Patterns of Co-Suppression of Nitrate Reductase and Nitrite Reductase in Transgenic Tobacco Plants
Authors: Palauqui J. C. , Elmayan T. , de Borne F. D. , Crete P. , Charles C. , Vaucheret H.
Published: October 2016

Frequencies, timing, and spatial patterns of co-suppression of the nitrate (Nia) and nitrite (Nii) genes were analyzed in transgenic tobacco (Nicotiana tabacum) plants carrying either Nia or Nii cDNAs under the control of the 35S promoter, or a Nii gene with its own regulatory signals (promoter, introns, and terminator) cloned downstream of two copies of the enhancer of the 35S promoter. We show that (a) the frequencies of transgenic lines affected by co- suppression are similar for the three constructs, ranging from 19 to 25%; (b) Nia and Nii co-suppression are triggered stochastically during a phenocritical period of 2 weeks between germination and flowering; (c) the timing of co-suppression (i.e. the percentage of isogenic plants affected by co-suppression reported as a function of the number of days of culture) differs from one transgenic line to another; (d) the percentage of isogenic plants affected by co-suppression is increased by growing the plants in vitro prior to their transfer to the greenhouse and to the field; and (e) at the end of the culture period, plants are either unaffected, completely co-suppressed, or variegated. Suppressed and nonsuppressed parts of these variegated plants are separated by a vertical plane through the stem in Nia co-suppression, and separated by a horizontal plane in Nii co-suppression.
Accumulation of Al in Root Mucilage of an Al-Resistant and an Al-Sensitive Cultivar of Wheat
Authors: Archambault D. J. , Zhang G. , Taylor G. J.
Published: October 2016

To estimate rates of Al accumulation within the symplasm, all apoplastic pools of Al need to be eliminated or accounted for. We have developed a revised kinetic protocol that allows us to estimate the contribution of mucilage-bound Al to total, nonexchangeable Al, and to eliminate the mucilage as an apoplastic pool of Al. By comparing the Al content of excised root tips (2 cm) of wheat (Triticum aestivum L.) with and without the removal of the mucilage (using a 10-min wash in 1 M NH4Cl), we found that Al bound to the mucilage accounted for approximately 25 to 35% of Al remaining after desorption in citric acid. The kinetics of Al uptake into mucilage were biphasic, with a rapid phase occurring in the first 30 min of uptake, followed by a linear phase occurring in the remainder of the experimental period (180 min). By adopting a step for removal of mucilage into our existing kinetic protocol, we have been able to isolate a linear phase of uptake with only a slight deviation from linearity in the first 5 min. Although we cannot unambiguously identify this phase of uptake as uptake into the symplasm, we believe this new protocol provides us with the most accurate quantitative estimate of symplastic Al yet available.
Changes in Calcium-Dependent Protein Kinase Activity during in Vitro Tuberization in Potato
Authors: MacIntosh G. C. , Ulloa R. M. , Raices M. , Tellez-Inon M. T.
Published: October 2016

A soluble Ca2+-dependent protein kinase (CDPK) was purified to homogeneity in potato (Solanum tuberosum L.) plants. Potato CDPK was strictly dependent on Ca2+ (one-half maximal activation 0.6 [mu]M) and phosphorylated a wide diversity of substrates, in which Syntide 2 was the best phosphate acceptor (Michaelis constant = 30 [mu]M). The kinase was inhibited by Ca2+-chelating agents, phenotiazine derivatives, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (one-half maximal inhibition = 0.25 mM). Polyclonal antibodies directed against the regulatory region of the soybean CDPK recognized a 53-kD polypeptide. In an autophosphorylation assay, this same band was strongly labeled with [[gamma]-32P]ATP in the presence of Ca2+. CDPK activity was high in nontuberized plants, but increased 2.5-fold at the onset of tuber development and was reduced to one-half of its original activity when the tuber had completed formation. In the early stages of tuberization, Ca2+-dependent phosphorylation of endogenous targets (specific bands of 68, 51, and 46 kD) was observed. These polypeptides were not labeled in nontuberizing plants or in completely formed tubers, indicating that this phosphorylation is a stage-specific event. In addition, dephosphorylation of specific polypeptides was detected in tuberizing plants, suggesting the involvement of a phosphatase. Preincubation of crude extracts with phosphatase inhibitors rendered a 100% increase in CDPK activity.
Characterization of Glutathione S-Transferase Isoforms in Three Maize Inbred Lines Exhibiting Differential Sensitivity to Alachlor
Authors: Rossini L. , Jepson I. , Greenland A. J. , Gorla M. S.
Published: October 2016

Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione to several xenobiotics, including a number of important herbicides. Several GST isoforms have been identified in maize (Zea mays L.). In this study we focused on three isoforms, GST I, II, and IV, derived from homo-or heterodimerization of two subunits GST-29 and GST-27, which have been shown to be responsible for reactivity to alachlor. The expression of these isoforms was examined in three inbred lines of maize that showed tolerance, susceptibility, and intermediate resistance to alachlor (2-Cl-N-[2,6-diethylphenyl]-N-[methoxymethyl]acetamide) treatment. The different isoforms were separated by anion-exchange chromatography and subunits were quantified by western blot analysis. GST assays were performed against both 1-Cl-2,4-dinitrobenzene and alachlor. This analysis showed that the susceptible and intermediate lines exhibit impaired function in the GST-27 and GST-29 subunits, respectively. In addition, this study suggests that GST IV is the principal, detoxifying enzyme for alachlor, although GST I and II are required to achieve tolerance to high rates of the herbicide.
Solubilization, Partial Purification, and Characterization of a Binding Site for a Glycopeptide Elicitor from Microsomal Membranes of Tomato Cells
Authors: Fath A. , Boller T.
Published: October 2016

Purified glycopeptides derived from yeast invertase act as highly potent elicitors in suspension-cultured tomato (Lycopersicon esculentum [L.] Mill) cells, inducing ethylene biosynthesis and phenylalanine ammonia lyase half-maximally at concentrations of 1 to 5 nM. We previously demonstrated the presence of a high-affinity binding site that specifically recognized these glycopeptides in cells and microsomal membranes of tomato (C.W. Basse, A. Fath, T. Boller [1993] J Biol Chem 268: 14724–14731). This elicitor-binding site was solubilized in an active form from the microsomal membranes using the neutral detergents n-dodecylmaltoside and n-dodecanoylsucrose and purified 67-fold in a single step by anion-exchange chromatography. Ligand saturation studies and competition experiments with unlabeled glycopeptides and glycans demonstrated that the detergent-solubilized elicitor-binding site retained the high affinity (Kd approximately 1–4 nM) and selectivity of the membrane-bound form. The binding site was found to have a high affinity for N-linked glycans with nine mannosyl residues from fungal glycoproteins, whereas it did not recognize the typical mammalian glycans with nine mannosyl residues, demonstrating further its high selectivity.
Physiological Characterization of Root Zn2+ Absorption and Translocation to Shoots in Zn Hyperaccumulator and Nonaccumulator Species of Thlaspi
Authors: Lasat M. M. , Baker AJM. , Kochian L. V.
Published: October 2016

Radiotracer techniques were employed to characterize 65Zn2+ influx into the root symplasm and translocation to the shoot in Thlaspi caerulescens, a Zn hyperaccumulator, and Thlaspi arvense, a nonaccumulator. A protocol was developed that allowed us to quantify unidirectional 65Zn2+ influx across the root-cell plasma membrane (20 min of radioactive uptake followed by 15 min of desorption in a 100 [mu]M ZnCl2 + 5 mM CaCl2 solution). Concentration-dependent Zn2+ influx in both Thlaspi species yielded nonsaturating kinetic curves that could be resolved into linear and saturable components. The linear kinetic component was shown to be cell-wall-bound Zn2+ remaining in the root after desorption, and the saturable component was due to Zn2+ influx across the root-cell plasma membrane. This saturable component followed Michaelis-Menten kinetics, with similar apparent Michaelis constant values for T. caerulescens and T. arvense (8 and 6 [mu]M, respectively). However, the maximum initial velocity for Zn2+ influx in T. caerulescens root cells was 4.5-fold higher than for T. arvense, indicating that enhanced absorption into the root is one of the mechanisms involved in Zn hyperaccumulation. After 96 h 10-fold more 65Zn was translocated to the shoot of T. caerulescens compared with T. arvense. This indicates that transport sites other than entry into the root symplasm are also stimulated in T. caerulescens. We suggest that although increased root Zn2+ influx is a significant component, transport across the plasma membrane and tonoplast of leaf cells must also be critical sites for Zn hyperaccumulation in T. caerulescens.
Endo-β-Mannanase Activity Present in Cell Wall Extracts of Lettuce Endosperm prior to Radicle Emergence
Authors: Dutta S. , Bradford K. J. , Nevins D. J.
Published: October 2016

Lettuce (Lactuca sativa L.) endosperm cell walls isolated prior to radicle emergence underwent autohydrolysis, the rate of which was correlated with whether radicle emergence would subsequently occur. Extraction of endosperm cell walls with 6 M LiCl suppressed autohydrolysis, and the desalted extract possessed activity that was capable of hydrolyzing purified locust bean galactomannan but not arabinogalactan, carboxymethylcellulose, glucomannan, polygalacturonic acid, tomato galactomannan, or native lettuce endosperm cell walls. Some hydrolytic activity was detected on endosperm cell walls if they were modified by partial trifluoroacetic acid hydrolysis or pretreatment with guanidinium thiocyanate. In extended incubations the cell wall enzyme extract released only large molecular mass fragments from locust bean galactomannan, indicating primarily endo-activity. Galactomannan-hydrolyzing activity in the cell wall extract increased as a function of imbibition time and was greatest just prior to radicle emergence. Thermoinhibition (imbibition at 32[deg]C) or treatment with abscisic acid at a temperature optimal for germination (25[deg]C) suppressed both germination and endosperm cell wall mannanase activity, whereas alleviation of thermoinhibition with gibberellic acid was accompanied by significant enhancement of mannanase activity. We conclude that a cell wall-bound endo-β-mannanase is expressed in lettuce endosperm prior to radicle emergence and is regulated by the same conditions that govern germination.
Influence of Gene Dosage on Carbohydrate Synthesis and Enzymatic Activities in Endosperm of Starch-Deficient Mutants of Maize
Authors: Singletary G. W. , Banisadr R. , Keeling P. L.
Published: October 2016

In cereals, starch is synthesized in endosperm cells, which have a ploidy level of three. By studying the allelic dosage of mutants affecting starch formation in maize (Zea mays L.) kernels, we determined the effect of down-regulated enzyme activity on starch accumulation and the activity of associated enzymes of carbohydrate metabolism. We found a direct relationship between the amount of starch produced in the endosperm and the gene dosage of amylose extender-1, brittle-2, shrunken1, and sugary-1 mutant alleles. Changes in starch content were found to be caused by changes in the duration as well as the rate of starch synthesis, depending on the mutant. Branching enzyme, ADP-glucose pyrophosphorylase, and sucrose synthase activities were linearly reduced in endosperm containing increasing dosages of amylose extender-1, brittle-2, and shrunken-1 alleles, respectively. De-branching enzyme activity declined only in the presence of two or three copies of sugary-1. No enzyme-dosage relationship occurred with the dull1 mutant allele. All mutants except sugary-1 displayed large increases (approximately 2- to 5-fold) in activity among various enzymes unrelated to the structural gene. This occurred in homozygous recessive genotypes, as did elevated concentrations of soluble sugars, and differed in magnitude and distribution among enzymes according to the particular mutation.
Ultraviolet B-Sensitive Rice Cultivar Deficient in Cyclobutyl Pyrimidine Dimer Repair
Authors: Hidema J. , Kumagai T. , Sutherland J. C. , Sutherland B. M.
Published: October 2016

Repair of cyclobutyl pyrimidine dimers (CPDs) in DNA is essential in most organisms to prevent biological damage by ultraviolet (UV) light. In higher plants tested thus far, UV-sensitive strains had higher initial damage levels or deficient repair of nondimer DNA lesions but normal CPD repair. This suggested that CPDs might not be important for biological lesions. The photosynthetic apparatus has also been proposed as a critical target. We have analyzed CPD induction and repair in the UV-sensitive rice (Oryza sativa L.) cultivar Norin 1 and its close relative UV-resistant Sasanishiki using alkaline agarose gel electrophoresis. Norin 1 is deficient in cyclobutyl pyrimidine dimer photoreactivation and excision; thus, UV sensitivity correlates with deficient dimer repair.
Observations of Ice Nucleation and Propagation in Plants Using Infrared Video Thermography
Authors: Wisniewski M. , Lindow S. E. , Ashworth E. N.
Published: October 2016

We evaluated the use of infrared (IR) video thermography to observe directly ice nucleation and propagation in plants. An imaging radiometer with an HgCdTe long-wave (8–12 [mu]m) detector was utilized to image the thermal response of plants during freezing. IR images were analyzed in real time and recorded on videotape. Information on the videotape was subsequently accessed and analyzed utilizing IR image analysis software. Freezing of water droplets as small as 0.5 [mu]L was clearly detectable with the radiometer. Additionally, a comparison of temperature tracking data collected by the radiometer with data collected with thermocouples showed close correspondence. Monitoring of an array of plant species under different freezing conditions revealed that ice nucleation and propagation are readily observable by thermal imaging. In many instances, the ice nucleation-active bacterium Pseudomonas syringae placed on test plants could be seen to initiate freezing of the whole plant. Apparent ice nucleation by intrinsic nucleators, despite the presence of ice nucleation-active bacteria, was also evident in some species. Floral bud tissues of peach (Prunus persica) could be seen to supercool below the temperature of stem tissues, and ice nucleation at the site of insertion of the thermocouple was frequently observed. Rates of propagation of ice in different tissues were also easily measured by thermal imaging. This study demonstrates that IR thermography is an excellent method for studying ice nucleation and propagation in plants.