bluuebunny/crossref_metadata_2025_split huggingface dataset:
Starch Biosynthesis and Intermediary Metabolism in Maize Kernels. Quantitative Analysis of Metabolite Flux by Nuclear Magnetic Resonance
Authors: Glawischnig Erich , Gierl Alfons , Tomas Adriana , Bacher Adelbert , Eisenreich Wolfgang 
Published: December 2002

Abstract
The seeds of cereals represent an important sink for metabolites during the accumulation of storage products, and seeds are an essential component of human and animal nutrition. Understanding the metabolic interconversions (networks) underpinning storage product formation could provide the foundation for effective metabolic engineering of these primary nutritional sources. In this paper, we describe the use of retrobiosynthetic nuclear magnetic resonance analysis to establish the metabolic history of the glucose (Glc) units of starch in maize (Zea mays) kernels. Maize kernel cultures were grown with [U-13C6]Glc, [U-13C12]sucrose, or [1,2-13C2]acetate as supplements. After 19 d, starch was hydrolyzed, and the isotopomer composition of the resulting Glc was determined by quantitative nuclear magnetic resonance analysis. [1,2-13C2]Acetate was not incorporated into starch. [U-13C6]Glc or [U-13C12]sucrose gave similar labeling patterns of polysaccharide Glc units, which were dominated by [1,2,3-13C3]- and [4,5,6-13C3]-isotopomers, whereas the [U-13C6]-, [3,4,5,6-13C4]-, [1,2-13C2]-, [5,6-13C2], [3-13C1], and [4-13C1]-isotopomers were present at lower levels. These isotopomer compositions indicate that there is extensive recycling of Glc before its incorporation into starch, via the enzymes of glycolytic, glucogenic, and pentose phosphate pathways. The relatively high abundance of the [5,6-13C2]-isotopomer can be explained by the joint operation of glycolysis/glucogenesis and the pentose phosphate pathway.
Starch Biosynthesis during Pollen Maturation Is Associated with Altered Patterns of Gene Expression in Maize
Authors: Datta Rupali , Chamusco Karen C. , Chourey Prem S. 
Published: December 2002

Abstract
Starch biosynthesis during pollen maturation is not well understood in terms of genes/proteins and intracellular controls that regulate it in developing pollen. We have studied two specific developmental stages: “early,” characterized by the lack of starch, before or during pollen mitosis I; and “late,” an actively starch-filling post-pollen mitosis I phase in S-type cytoplasmic male-sterile (S-CMS) and two related male-fertile genotypes. The male-fertile starch-positive, but not the CMS starch-deficient, genotypes showed changes in the expression patterns of a large number of genes during this metabolic transition. In addition to a battery of housekeeping genes of carbohydrate metabolism, we observed changes in hexose transporter, plasma membrane H+-ATPase, ZmMADS1, and 14-3-3 proteins. Reduction or deficiency in 14-3-3 protein levels in all three major cellular sites (amyloplasts [starch], mitochondria, and cytosol) in male-sterile relative to male-fertile genotypes are of potential interest because of interorganellar communication in this CMS system. Further, the levels of hexose sugars were significantly reduced in male-sterile as compared with male-fertile tissues, not only at “early” and “late” stages but also at an earlier point during meiosis. Collectively, these data suggest that combined effects of both reduced sugars and their reduced flux in starch biosynthesis along with a strong possibility for altered redox passage may lead to the observed temporal changes in gene expressions, and ultimately pollen sterility.
Humic Acids Isolated from Earthworm Compost Enhance Root Elongation, Lateral Root Emergence, and Plasma Membrane H+-ATPase Activity in Maize Roots
Authors: Canellas Luciano Pasqualoto , Olivares Fabio Lopes , Okorokova-Façanha Anna L. , Façanha Arnoldo Rocha 
Published: December 2002

Abstract
Earthworms (Eisenia foetida) produce humic substances that can influence plant growth by mechanisms that are not yet clear. In this work, we investigated the effects of humic acids (HAs) isolated from cattle manure earthworm compost on the earliest stages of lateral root development and on the plasma membrane H+-ATPase activity. These HAs enhance the root growth of maize (Zea mays) seedlings in conjunction with a marked proliferation of sites of lateral root emergence. They also stimulate the plasma membrane H+-ATPase activity, apparently associated with an ability to promote expression of this enzyme. In addition, structural analysis reveals the presence of exchangeable auxin groups in the macrostructure of the earthworm compost HA. These results may shed light on the hormonal activity that has been postulated for these humic substances.
Molecular Characterization of a Novel Gene Family Encoding ACT Domain Repeat Proteins in Arabidopsis
Authors: Hsieh Ming-Hsiun , Goodman Howard M. 
Published: December 2002

Abstract
In bacteria, the regulatory ACT domains serve as amino acid-binding sites in some feedback-regulated amino acid metabolic enzymes. We have identified a novel type of ACT domain-containing protein family in Arabidopsis whose members contain ACT domain repeats (the “ACR” protein family). There are at least eight ACR genes located on each of the five chromosomes in the Arabidopsis genome. Gene structure comparisons indicate that the ACR gene family may have arisen by gene duplications. Northern-blot analysis indicates that each member of the ACR gene family has a distinct expression pattern in various organs from 6-week-old Arabidopsis. Moreover, analyses of an ACR3 promoter-β-glucuronidase (GUS) fusion in transgenic Arabidopsis revealed that the GUS activity formed a gradient in the developing leaves and sepals, whereas low or no GUS activity was detected in the basal regions. In 2-week-old Arabidopsis seedlings grown in tissue culture, the expression of the ACR gene family is differentially regulated by plant hormones, salt stress, cold stress, and light/dark treatment. The steady-state levels of ACR8 mRNA are dramatically increased by treatment with abscisic acid or salt. Levels of ACR3 and ACR4 mRNA are increased by treatment with benzyladenine. The amino acid sequences of Arabidopsis ACR proteins are most similar in the ACT domains to the bacterial sensor protein GlnD. The ACR proteins may function as novel regulatory or sensor proteins in plants.
Independent Control of Gibberellin Biosynthesis and Flowering Time by the Circadian Clock in Arabidopsis
Authors: Blázquez Miguel A. , Trénor Marta , Weigel Detlef 
Published: December 2002

Abstract
Flowering of the facultative long-day plant Arabidopsis is controlled by several endogenous and environmental factors, among them gibberellins (GAs) and day length. The promotion of flowering by long days involves an endogenous clock that interacts with light cues provided by the environment. Light, and specifically photoperiod, is also known to regulate the biosynthesis of GAs, but the effects of GAs and photoperiod on flowering are at least partially separable. Here, we have used a short-period mutant,toc1, to investigate the role of the circadian clock in the control of flowering time by GAs and photoperiod. We show thattoc1 affects expression of several floral regulators and a GA biosynthetic gene, but that these effects are independent.
Forms of Zinc Accumulated in the HyperaccumulatorArabidopsis halleri
Authors: Sarret Géraldine , Saumitou-Laprade Pierre , Bert Valérie , Proux Olivier , Hazemann Jean-Louis , Traverse Agnès , Marcus Matthew A. , Manceau Alain 
Published: December 2002

AbstractThe chemical forms of zinc (Zn) in the Zn-tolerant and hyperaccumulator Arabidopsis halleri and in the non-tolerant and nonaccumulator Arabidopsis lyrata subsp. petraea were determined at the molecular level by combining chemical analyses, extended x-ray absorption spectroscopy (EXAFS), synchrotron-based x-ray microfluorescence, and μEXAFS. Plants were grown in hydroponics with various Zn concentrations, and A. halleri specimens growing naturally in a contaminated site were also collected. Zn speciation in A. halleri was independent of the origin of the plants (contaminated or non-contaminated) and Zn exposure. In aerial parts, Zn was predominantly octahedrally coordinated and complexed to malate. A secondary organic species was identified in the bases of the trichomes, which contained elevated Zn concentrations, and in which Zn was tetrahedrally coordinated and complexed to carboxyl and/or hydroxyl functional groups. This species was detected thanks to the good resolution and sensitivity of synchrotron-based x-ray microfluorescence and μEXAFS. In the roots of A. halleri grown in hydroponics, Zn phosphate was the only species detected, and is believed to result from chemical precipitation on the root surface. In the roots of A. halleri grown on the contaminated soil, Zn was distributed in Zn malate, Zn citrate, and Zn phosphate. Zn phosphate was present in both the roots and aerial part of A. lyrata subsp. petraea. This study illustrates the complementarity of bulk and spatially resolved techniques, allowing the identification of: (a) the predominant chemical forms of the metal, and (b) the minor forms present in particular cells, both types of information being essential for a better understanding of the bioaccumulation processes.
Cloning, Functional Expression, and Subcellular Localization of Multiple NADPH-Cytochrome P450 Reductases from Hybrid Poplar
Authors: Ro Dae-Kyun , Ehlting Jürgen , Douglas Carl J. 
Published: December 2002

Abstract
NADPH:cytochrome P450 reductase (CPR) provides reducing equivalents to diverse cytochrome P450 monooxygenases. We isolated cDNAs for three CPR genes (CPR1,CPR2, and CPR3) from hybrid poplar (Populus trichocarpa × Populus deltoides). Deduced CPR2 and CPR3 amino acid sequences were 91% identical, but encoded isoforms divergent from CPR1 (72% identity). CPR1 and CPR2 were co-expressed together with the P450 enzyme cinnamate-4-hydroxylase (C4H) in yeast (Saccharomyces cerevisiae). Microsomes isolated from strains expressing CPR1/C4H or CPR2/C4H enhanced C4H activities approximately 10-fold relative to the C4H-only control strain, and catalyzed NADPH-dependent cytochrome c reduction. The divergent CPR isoforms (CPR1 and CPR2/3) contained entirely different N-terminal sequences, which are conserved in other plant CPRs and are diagnostic for two distinct classes of CPRs within the angiosperms. C-terminal green fluorescent protein fusions to CPR1 and CPR2 were constructed and expressed in both yeast and Arabidopsis. The fusion proteins expressed in yeast retained the ability to support C4H activity and, thus, were catalytically active. Both CPR::green fluorescent protein fusion proteins were strictly localized to the endoplasmic reticulum in transgenic Arabidopsis. The lack of localization of either isoform to chloroplasts, where P450s are known to be present, suggests that an alternative P450 reduction system may be operative in this organelle. Transcripts for the three poplar CPR genes were present ubiquitously in all tissues examined, but CPR2 showed highest expression in young leaf tissue.
Expression and Biochemical Properties of a Ferredoxin-Dependent Heme Oxygenase Required for Phytochrome Chromophore Synthesis
Authors: Muramoto Takuya , Tsurui Noriyuki , Terry Matthew J. , Yokota Akiho , Kohchi Takayuki 
Published: December 2002

Abstract
The HY1 gene of Arabidopsis encodes a plastid heme oxygenase (AtHO1) required for the synthesis of the chromophore of the phytochrome family of plant photoreceptors. To determine the enzymatic properties of plant heme oxygenases, we have expressed theHY1 gene (without the plastid transit peptide) inEscherichia coli to produce an amino terminal fusion protein between AtHO1 and glutathione S-transferase. The fusion protein was soluble and expressed at high levels. Purified recombinant AtHO1, after glutathione S-transferase cleavage, is a hemoprotein that forms a 1:1 complex with heme. In the presence of reduced ferredoxin, AtHO1 catalyzed the formation of biliverdin IXα from heme with the concomitant production of carbon monoxide. Heme oxygenase activity could also be reconstituted using photoreduced ferredoxin generated through light irradiation of isolated thylakoid membranes, suggesting that ferredoxin may be the electron donor in vivo. In addition, AtHO1 required an iron chelator and second reductant, such as ascorbate, for full activity. These results show that the basic mechanism of heme cleavage has been conserved between plants and other organisms even though the function, subcellular localization, and cofactor requirements of heme oxygenases differ substantially.
Temperature Response of Mesophyll Conductance. Implications for the Determination of Rubisco Enzyme Kinetics and for Limitations to Photosynthesis in Vivo
Authors: Bernacchi Carl J. , Portis Archie R. , Nakano Hiromi , von Caemmerer Susanne , Long Stephen P. 
Published: December 2002

Abstract
CO2 transfer conductance from the intercellular airspaces of the leaf into the chloroplast, defined as mesophyll conductance (g m), is finite. Therefore, it will limit photosynthesis when CO2 is not saturating, as in C3 leaves in the present atmosphere. Little is known about the processes that determine the magnitude ofg m. The process dominatingg m is uncertain, though carbonic anhydrase, aquaporins, and the diffusivity of CO2 in water have all been suggested. The response ofg m to temperature (10°C–40°C) in mature leaves of tobacco (Nicotiana tabacum L. cv W38) was determined using measurements of leaf carbon dioxide and water vapor exchange, coupled with modulated chlorophyll fluorescence. These measurements revealed a temperature coefficient (Q10) of approximately 2.2 for g m, suggesting control by a protein-facilitated process because the Q10for diffusion of CO2 in water is about 1.25. Further,g m values are maximal at 35°C to 37.5°C, again suggesting a protein-facilitated process, but with a lower energy of deactivation than Rubisco. Using the temperature response of g m to calculate CO2 at Rubisco, the kinetic parameters of Rubisco were calculated in vivo from 10°C to 40°C. Using these parameters, we determined the limitation imposed on photosynthesis byg m. Despite an exponential rise with temperature, g m does not keep pace with increased capacity for CO2 uptake at the site of Rubisco. The fraction of the total limitations to CO2uptake within the leaf attributable tog m rose from 0.10 at 10°C to 0.22 at 40°C. This shows that transfer of CO2 from the intercellular air space to Rubisco is a very substantial limitation on photosynthesis, especially at high temperature.
Transcriptome Changes for Arabidopsis in Response to Salt, Osmotic, and Cold Stress,
Authors: Kreps Joel A. , Wu Yajun , Chang Hur-Song , Zhu Tong , Wang Xun , Harper Jeff F. 
Published: December 2002

Abstract
To identify genes of potential importance to cold, salt, and drought tolerance, global expression profiling was performed on Arabidopsis plants subjected to stress treatments of 4°C, 100 mm NaCl, or 200 mm mannitol, respectively. RNA samples were collected separately from leaves and roots after 3- and 27-h stress treatments. Profiling was conducted with a GeneChip microarray with probe sets for approximately 8,100 genes. Combined results from all three stresses identified 2,409 genes with a greater than 2-fold change over control. This suggests that about 30% of the transcriptome is sensitive to regulation by common stress conditions. The majority of changes were stimulus specific. At the 3-h time point, less than 5% (118 genes) of the changes were observed as shared by all three stress responses. By 27 h, the number of shared responses was reduced more than 10-fold (&lt; 0.5%), consistent with a progression toward more stimulus-specific responses. Roots and leaves displayed very different changes. For example, less than 14% of the cold-specific changes were shared between root and leaves at both 3 and 27 h. The gene with the largest induction under all three stress treatments was At5g52310 (LTI/COR78), with induction levels in roots greater than 250-fold for cold, 40-fold for mannitol, and 57-fold for NaCl. A stress response was observed for 306 (68%) of the known circadian controlled genes, supporting the hypothesis that an important function of the circadian clock is to “anticipate” predictable stresses such as cold nights. Although these results identify hundreds of potentially important transcriptome changes, the biochemical functions of many stress-regulated genes remain unknown.
Comparative Molecular and Functional Analyses of the Tobacco Cyclin-Dependent Kinase Inhibitor NtKIS1a and Its Spliced Variant NtKIS1b
Authors: Jasinski Sophie , Perennes Claudette , Bergounioux Catherine , Glab Nathalie 
Published: December 2002

Abstract
In all eukaryotes, cell cycle progression is controlled by cyclin-dependent kinases (CDKs) whose activity is regulated at several levels including inhibition by CDK inhibitors. Here, we report a comparative molecular and functional analysis of the tobacco (Nicotiana tomentosiformis) CDK inhibitor, NtKIS1a, and its spliced variant, NtKIS1b. The C-terminal end of NtKIS1a shares strong sequence similarity with mammalian CIP/KIP inhibitors, which is not the case for NtKIS1b. Consistent with this, NtKIS1a but not NtKIS1b inhibits in vitro the kinase activity of CDK/cyclin complexes, and tobacco (Nicotiana tabacum) D-type cyclins and an A-type CDK are NtKIS1a, but not NtKIS1b, interacting partners. Although both NtKIS1a and NtKIS1b transcripts are mainly found in flowers and more precisely in stamens, NtKIS1b transcript levels are cell cycle regulated, whereas those of NtKIS1a remain constant during the cell cycle. NtKIS1a and NtKIS1b fused to fluorescent proteins are localized in the nucleus when transiently expressed in onion epidermal cells. Furthermore, there is no competition for their nuclear localization when they are simultaneously overexpressed. In vitro competition toward CDK kinase activity suggests that NtKIS1b is a strong competitor of NtKIS1a. Arabidopsis plants overexpressing NtKIS1a-green fluorescent protein (GFP) or NtKIS1b-GFP fusion proteins were obtained. In these plants, the fusion proteins are still localized in the nucleus. Interestingly, NtKIS1a-GFP-overexpressing plants display strong morphological modifications and a reduced CDK kinase activity, whereas NtKIS1b-GFP-overexpressing plants display a wild-type phenotype including a wild-type CDK kinase activity. Our results strongly suggest that the inhibition of the kinase activity is responsible for the phenotypic modifications.
Differential Regulation of RNA Levels of Gibberellin Dioxygenases by Photoperiod in Spinach
Authors: Lee Dong Ju , Zeevaart Jan A.D. 
Published: December 2002

Abstract
Previous work with spinach (Spinacia oleracea) has shown that the level of gibberellin (GA) 20-oxidase is strongly up-regulated by long days (LD). In the present work, the effect of photoperiod on expression of other GA dioxygenases was investigated and compared with that of GA 20-oxidase. Two GA 2-oxidases and one GA 3-oxidase were isolated from spinach by reverse transcription-polymerase chain reaction with degenerate primers and by 5′- and 3′-rapid amplification of cDNA ends. As determined by high-performance liquid chromatography with on-line radioactivity detection, the SoGA3ox1 gene product catalyzed 3β-hydroxylation of GA9 to GA4 and GA20 to GA1. The SoGA2ox1 and the SoGA2ox2 gene products catalyzed 2β-hydroxylation of GA9 to GA51 and GA20 to GA29. The product of GA20 metabolism by SoGA3ox1 was identified as GA1 by gas chromatography-mass spectrometry, whereas the products of GA1 and GA20 metabolism by SoGA2ox1 and SoGA2ox2 were identified as GA8 and GA29, respectively. SoGA2ox1 also metabolized GA53 to GA97. The levels ofSoGA20ox1 transcripts were greatly increased in all organs tested in LD conditions, but the levels ofSoGA3ox1 transcripts were only slightly increased in blades and petioles. A decrease in the levels of theSoGA2ox1 transcripts in young leaves and tips in LD conditions is opposite to the expression pattern of theSoGA20ox1. Expression of SoGA20ox1 in petioles and young leaves was strongly up-regulated by a supplementary 16 h of light, but the levels of SoGA3ox1 andSoGA2ox1 transcripts did not change. It is concluded that regulation and maintenance of GA1 concentration in spinach are primarily attributable to changes in expression ofSoGA20ox1.
Characterization of Arabidopsis AtAMT2, a High-Affinity Ammonium Transporter of the Plasma Membrane
Authors: Sohlenkamp Christian , Wood Craig C. , Roeb Gerhard W. , Udvardi Michael K. 
Published: December 2002

Abstract
AtAMT2 is an ammonium transporter that is only distantly related to the five members of the AtAMT1 family of high-affinity ammonium transporters in Arabidopsis. The short-lived radioactive ion13NH4 + was used to show that AtAMT2, expressed in yeast (Saccharomyces cerevisiae), is a high-affinity transporter with a K m for ammonium of about 20 μm. Changes in external pH between 5.0 and 7.5 had little effect on the K m for ammonium, indicating that NH4 +, not NH3, is the substrate for AtAMT2. TheAtAMT2 gene was expressed in all organs of Arabidopsis and was subject to nitrogen (N) regulation, at least in roots where expression was partially repressed by high concentrations of ammonium nitrate and derepressed in the absence of external N. Although expression of AtAMT2 in shoots responded little to changes in root N status, transcript levels in leaves declined under high CO2 conditions. Transient expression of an AtAMT2-green fluorescent protein fusion protein in Arabidopsis leaf epidermal cells indicated a plasma membrane location for the AtAMT2 protein. Thus, AtAMT2 is likely to play a significant role in moving ammonium between the apoplast and symplast of cells throughout the plant. However, a dramatic reduction in the level ofAtAMT2 transcript brought about by dsRNA interference with gene expression had no obvious effect on plant growth or development, under the conditions tested.
Nitric Oxide Improves Internal Iron Availability in Plants
Authors: Graziano Magdalena , Beligni Marı́a Verónica , Lamattina Lorenzo 
Published: December 2002

Abstract
Iron deficiency impairs chlorophyll biosynthesis and chloroplast development. In leaves, most of the iron must cross several biological membranes to reach the chloroplast. The components involved in the complex internal iron transport are largely unknown. Nitric oxide (NO), a bioactive free radical, can react with transition metals to form metal-nitrosyl complexes. Sodium nitroprusside, an NO donor, completely prevented leaf interveinal chlorosis in maize (Zea mays) plants growing with an iron concentration as low as 10 μmFe-EDTA in the nutrient solution.S-Nitroso-N-acetylpenicillamine, another NO donor, as well as gaseous NO supply in a translucent chamber were also able to revert the iron deficiency symptoms. A specific NO scavenger, 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, blocked the effect of the NO donors. The effect of NO treatment on the photosynthetic apparatus of iron-deficient plants was also studied. Electron micrographs of mesophyll cells from iron-deficient maize plants revealed plastids with few photosynthetic lamellae and rudimentary grana. In contrast, in NO-treated maize plants, mesophyll chloroplast appeared completely developed. NO treatment did not increase iron content in plant organs, when expressed in a fresh matter basis, suggesting that root iron uptake was not enhanced. NO scavengers 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and methylene blue promoted interveinal chlorosis in iron-replete maize plants (growing in 250 μm Fe-EDTA). Even though results support a role for endogenous NO in iron nutrition, experiments did not establish an essential role. NO was also able to revert the chlorotic phenotype of the iron-inefficient maize mutants yellow stripe1 and yellow stripe3, both impaired in the iron uptake mechanisms. All together, these results support a biological action of NO on the availability and/or delivery of metabolically active iron within the plant.