bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of Homogentisate Phytyltransferase and Other Tocopherol Pathway Enzymes in the Regulation of Tocopherol Synthesis during Abiotic Stress
Authors: Collakova Eva , DellaPenna Dean 
Published: October 2003

AbstractTocopherols are amphipathic antioxidants synthesized exclusively by photosynthetic organisms. Tocopherol levels change significantly during plant growth and development and in response to stress, likely as a consequence of the altered expression of pathway-related genes. Homogentisate phytyltransferase (HPT) is a key enzyme limiting tocopherol biosynthesis in unstressed Arabidopsis leaves (E. Collakova, D. DellaPenna [2003] Plant Physiol 131: 632–642). Wild-type and transgenic Arabidopsis plants constitutively overexpressing HPT (35S::HPT1) were subjected to a combination of abiotic stresses for up to 15 d and tocopherol levels, composition, and expression of several tocopherol pathway-related genes were determined. Abiotic stress resulted in an 18- and 8-fold increase in total tocopherol content in wild-type and 35S::HPT1 leaves, respectively, with tocopherol levels in 35S::HPT1 being 2- to 4-fold higher than wild type at all experimental time points. Increased total tocopherol levels correlated with elevated HPT mRNA levels and HPT specific activity in 35S::HPT1 and wild-type leaves, suggesting that HPT activity limits total tocopherol synthesis during abiotic stress. In addition, substrate availability and expression of pathway enzymes before HPT also contribute to increased tocopherol synthesis during stress. The accumulation of high levels of β-, γ-, and δ-tocopherols in stressed tissues suggested that the methylation of phytylquinol and tocopherol intermediates limit α-tocopherol synthesis. Overexpression of γ-tocopherol methyltransferase in the 35S::HPT1 background resulted in nearly complete conversion of γ- and δ-tocopherols to α- and β-tocopherols, respectively, indicating that γ-tocopherol methyltransferase activity limits α-tocopherol synthesis in stressed leaves.
Cis-acting Elements and DNA-Binding Proteins Involved in CO2-Responsive Transcriptional Activation of Cah1 Encoding a Periplasmic Carbonic Anhydrase in Chlamydomonas reinhardtii
Authors: Kucho Ken-ichi , Yoshioka Satoshi , Taniguchi Fumiya , Ohyama Kanji , Fukuzawa Hideya 
Published: October 2003

Abstract
Expression of Cah1, encoding a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii Dangeard, is activated when cells are exposed to low-CO2 conditions (0.04% [v/v]) in light. By using an arylsulfatase reporter gene, a regulatory region essential for the transcriptional activation of Cah1 was delimited to a 63-bp fragment between –293 and –231 relative to the transcription start site. Linker-scan analysis of the 63-bp region identified two enhancer elements, EE-1 (AGATTTTCACCGGTTGGAAGGAGGT) and EE-2 (CGACTTACGAA). Gel mobility shift assays indicated that nuclear extracts purified from cells grown under low-CO2 conditions in light contained DNA-binding proteins specifically interacting with EE-1 and EE-2. Gel mobility shift assays using mutant oligonucleotide probes revealed that the protein binding to EE-1 preferentially recognized a 9-bp sequence stretch (AGATTTTCA) of EE-1, containing a conserved sequence motif named EEC, GANTTNC, which is also present in EE-2. The EE-1- and EE-2-binding proteins interacted with the EECs contained in both of the two enhancer elements in vitro. Four EECs in the 5′-upstream region from –651 to –231 of Cah1 played a central role in the transcriptional activation of Cah1 under low-CO2 conditions. These EEC-binding proteins were present even in cells grown under high-CO2 conditions (5% [v/v]) or in the dark when Cah1 is not activated. On the basis of these results, the relationship between the transcriptional regulation of Cah1 and protein-binding to the enhancer elements in the 5′-upstream region of Cah1 is discussed.
A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta
Authors: Curtis Mark D. , Grossniklaus Ueli 
Published: October 2003

AbstractThe current challenge, now that two plant genomes have been sequenced, is to assign a function to the increasing number of predicted genes. In Arabidopsis, approximately 55% of genes can be assigned a putative function, however, less than 8% of these have been assigned a function by direct experimental evidence. To identify these functions, many genes will have to undergo comprehensive analyses, which will include the production of chimeric transgenes for constitutive or inducible ectopic expression, for antisense or dominant negative expression, for subcellular localization studies, for promoter analysis, and for gene complementation studies. The production of such transgenes is often hampered by laborious conventional cloning technology that relies on restriction digestion and ligation. With the aim of providing tools for high throughput gene analysis, we have produced a Gateway-compatible Agrobacterium sp. binary vector system that facilitates fast and reliable DNA cloning. This collection of vectors is freely available, for noncommercial purposes, and can be used for the ectopic expression of genes either constitutively or inducibly. The vectors can be used for the expression of protein fusions to the Aequorea victoria green fluorescent protein and to the β-glucuronidase protein so that the subcellular localization of a protein can be identified. They can also be used to generate promoter-reporter constructs and to facilitate efficient cloning of genomic DNA fragments for complementation experiments. All vectors were derived from pCambia T-DNA cloning vectors, with the exception of a chemically inducible vector, for Agrobacterium sp.-mediated transformation of a wide range of plant species.
The Sulfolipids 2′-O-Acyl-Sulfoquinovosyldiacylglycerol and Sulfoquinovosyldiacylglycerol Are Absent from aChlamydomonas reinhardtiiMutant Deleted inSQD1
Authors: Riekhof Wayne R. , Ruckle Michael E. , Lydic Todd A. , Sears Barbara B. , Benning Christoph 
Published: October 2003

AbstractThe biosynthesis of thylakoid lipids in eukaryotic photosynthetic organisms often involves enzymes in the endoplasmic reticulum (ER) and the chloroplast envelopes. Two pathways of thylakoid lipid biosynthesis, the ER and the plastid pathways, are present in parallel in many species, including Arabidopsis, but in other plants, e.g. grasses, only the ER pathway is active. The unicellular alga Chlamydomonas reinhardtii diverges from plants like Arabidopsis in a different way because its membranes do not contain phosphatidylcholine, and most thylakoid lipids are derived from the plastid pathway. Here, we describe an acylated derivative of sulfolipid, 2′-O-acyl-sulfoquinovosyldiacylglycerol (ASQD), which is present in C. reinhardtii. Although the fatty acids of sulfoquinovosyldiacylglycerol (SQDG) were mostly saturated, ASQD molecular species carried predominantly unsaturated fatty acids. Moreover, directly attached to the head group of ASQD was preferentially an 18-carbon fatty acid with four double bonds. High-throughput robotic screening led to the isolation of a plasmid disruption mutant of C. reinhardtii, designated Δsqd1, which lacks ASQD as well as SQDG. In this mutant, the SQD1 ortholog was completely deleted and replaced by plasmid sequences. It is proposed that ASQD arises from the sugar nucleotide pathway of sulfolipid biosynthesis by acylation of the 2′-hydroxyl of the sulfoquinovosyl head group. At the physiological level, the mutant showed increased sensitivity to a diuron herbicide and reduced growth under phosphate limitation, suggesting a role for SQDG and/or ASQD in photosynthesis as conducted by C. reinhardtii, particularly under phosphate-limited conditions.
Proteomics of
Chlamydomonas reinhardtii
Light-Harvesting Proteins
Authors: Stauber Einar J. , Fink Andreas , Markert Christine , Kruse Olaf , Johanningmeier Udo , Hippler Michael 
Published: October 2003

ABSTRACT

With the recent development of techniques for analyzing transmembrane thylakoid proteins by two-dimensional gel electrophoresis, systematic approaches for proteomic analyses of membrane proteins became feasible. In this study, we established detailed two-dimensional protein maps of
Chlamydomonas reinhardtii
light-harvesting proteins (Lhca and Lhcb) by extensive tandem mass spectrometric analysis. We predicted eight distinct Lhcb proteins. Although the major Lhcb proteins were highly similar, we identified peptides which were unique for specific
lhcbm
gene products. Interestingly,
lhcbm6
gene products were resolved as multiple spots with different masses and isoelectric points. Gene tagging experiments confirmed the presence of differentially N-terminally processed Lhcbm6 proteins. The mass spectrometric data also revealed differentially N-terminally processed forms of Lhcbm3 and phosphorylation of a threonine residue in the N terminus. The N-terminal processing of Lhcbm3 leads to the removal of the phosphorylation site, indicating a potential novel regulatory mechanism. At least nine different
lhca
-related gene products were predicted by comparison of the mass spectrometric data against
Chlamydomonas
expressed sequence tag and genomic databases, demonstrating the extensive variability of the
C. reinhardtii
Lhca antenna system. Out of these nine, three were identified for the first time at the protein level. This proteomic study demonstrates the complexity of the light-harvesting proteins at the protein level in
C. reinhardtii
and will be an important basis of future functional studies addressing this diversity.
Introduction to chromosome dynamics in mitosis
Authors: Watrin Erwan , Legagneux Vincent 
Published: October 2003

AbstractTo ensure that the genetic information, replicated in the S‐phase of the cell cycle, is correctly distributed between daughter cells at mitosis, chromatin duplication and chromosome segregation are highly regulated events. Since the early 1980's, our knowledge of the mechanisms governing these two events has greatly increased due to the use of genetic and biochemical approaches. We present here, first, an overview of the replication process, highlighting molecular aspects involved in coupling replication with chromatin dynamics in mitosis. The second part will present the current understanding of chromosome condensation and segregation during mitosis in higher eukaryotes. Finally, we will underline the links that exist between replication and mitosis.
Interaction of Calmodulin, a Sorting Nexin and Kinase-Associated Protein Phosphatase with the Brassica oleracea S Locus Receptor Kinase
Authors: Vanoosthuyse Vincent , Tichtinsky Gabrielle , Dumas Christian , Gaude Thierry , Cock J. Mark 
Published: October 2003

Abstract
Recognition of self-pollen during the self-incompatibility response in Brassica oleracea is mediated by the binding of a secreted peptide (the S locus cysteine-rich protein) to the S locus receptor kinase (SRK), a member of the plant receptor kinase (PRK) superfamily. Here, we describe the characterization of three proteins that interact with the cytosolic kinase domain of SRK. A B. oleracea homolog of Arabidopsis kinase-associated protein phosphatase was shown to interact with and dephosphorylate SRK and was itself phosphorylated by SRK. Yeast (Saccharomyces cerevisiae) two-hybrid screens identified two additional interactors, calmodulin and a sorting nexin, both of which have been implicated in receptor kinase down-regulation in animals. A calmodulin-binding site was identified in sub-domain VIa of the SRK kinase domain. The binding site is conserved and functional in several other members of the PRK family. The sorting nexin also interacted with diverse members of the PRK family, suggesting that all three of the interacting proteins described here may play a general role in signal transduction by this family of proteins.
Proteomics ofMedicago truncatulaSeed Development Establishes the Time Frame of Diverse Metabolic Processes Related to Reserve Accumulation
Authors: Gallardo Karine , Le Signor Christine , Vandekerckhove Joël , Thompson Richard D. , Burstin Judith 
Published: October 2003

AbstractWe utilized a proteomic approach to investigate seed development in Medicago truncatula, cv Jemalong, line J5 at specific stages of seed filling corresponding to the acquisition of germination capacity and protein deposition. One hundred twenty proteins differing in kinetics of appearance were subjected to matrix-assisted laser desorption ionization time of flight mass spectrometry. These analyses provided peptide mass fingerprint data that identified 84 of them. Some of these proteins had previously been shown to accumulate during seed development in legumes (e.g. legumins, vicilins, convicilins, and lipoxygenases), confirming the validity of M. truncatula as a model for analysis of legume seed filling. The study also revealed proteins presumably involved in cell division during embryogenesis (β-tubulin and annexin). Their abundance decreased before the accumulation of the major storage protein families, which itself occurs in a specific temporal order: vicilins (14 d after pollination [DAP]), legumins (16 DAP), and convicilins (18 DAP). Furthermore, the study showed an accumulation of enzymes of carbon metabolism (e.g. sucrose synthase, starch synthase) and of proteins involved in embryonic photosynthesis (e.g. chlorophyll a/b binding), which may play a role in providing cofactors for protein/lipid synthesis or for CO2 refixation during seed filling. Correlated with the reserve deposition phase was the accumulation of proteins associated with cell expansion (actin 7 and reversibly glycosylated polypeptide) and of components of the precursor accumulating vesicles, which give rise to a trypsin inhibitor on maturation. Finally, we revealed a differential accumulation of enzymes involved in methionine metabolism (S-adenosyl-methionine synthetase and S-adenosylhomo-cysteine hydrolase) and propose a role for these enzymes in the transition from a highly active to a quiescent state during seed development.
Zeaxanthin Deficiency Enhances the High Light Sensitivity of an Ascorbate-Deficient Mutant of Arabidopsis
Authors: Müller-Moulé Patricia , Havaux Michel , Niyogi Krishna K. 
Published: October 2003

Abstract
The ascorbate content of plants is usually increased in high light (HL), implying a function for ascorbate in the acclimation of plants to HL. Nevertheless, the importance of ascorbate in HL acclimation has not yet been tested directly. Here, we report on the acclimation process of an ascorbate-deficient Arabidopsis mutant to HL. The mutant vtc2 has only 10% to 30% of wild-type levels of ascorbate, and it is also slightly deficient in feedback de-excitation (qE), a photoprotective mechanism that causes the dissipation of excess light as heat. The vtc2 mutant was unable to acclimate to HL, when transferred from low light to HL. Its mature leaves bleached, and it showed an increased degree of lipid peroxidation and photoinhibition. In parallel, we tested the photosensitivity of an ascorbate-deficient xanthophyll cycle mutant, vtc2npq1, which also lacks zeaxanthin and nearly all qE. The double mutant bleached sooner and had higher degrees of lipid peroxidation and photoinhibition than the vtc2 mutant. This was in contrast to the npq1 single mutant that showed only slight deviations from the wild-type phenotype under the conditions used. These results demonstrate the antioxidant role of ascorbate in the acclimation process to HL and point to the relative importance of ascorbate in comparison with other photoprotective processes, such as specific xanthophylls or feedback de-excitation. The results also provide further support for the proposed role of zeaxanthin as an antioxidant and lipid stabilizer.
Characterization of Salt-Regulated Mannitol-1-Phosphate Dehydrogenase in the Red Alga Caloglossa continua
Authors: Iwamoto Koji , Kawanobe Hideaki , Ikawa Tomoyoshi , Shiraiwa Yoshihiro 
Published: October 2003

Abstract
Mannitol-1-phosphate (M1P) dehydrogenase (M1PDH; EC 1.1.1.17), an enzyme catalyzing the reduction of Fru-6-phosphate (F6P) to M1P in algal mannitol biosynthesis, was purified to homogeneity from a cell homogenate of the eulittoral red alga Caloglossa continua (Okamura) King et Puttock. The enzyme was a monomer with an apparent molecular mass of 53 kD, as determined by gel filtration and SDS-PAGE, and exhibited an pI of approximately 5.5. The substrate specificity was very high toward F6P and M1P for respective reductive and oxidative reactions. The enzyme was found to be a sulfhydryl-type, because its activity was inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate, and the inhibition by p-hydroxymercuribenzoate was rescued by 2-mercaptoethanol. Some unknown factors in the extract may also have inhibited the activity, because the total activity was greatly increased through the purification procedure. The optimum pH for F6P reduction was changed from 6.0 or lower to 7.2 by the addition of 200 mm NaCl. The reduction of F6P showed strong substrate inhibition above 0.5 mm. However, K m(F6P) of M1PDH was increased eight times by the addition of 200 mm NaCl, whereas V max was in a similar range with the avoidance of substrate inhibition by F6P. These results indicate that the enzyme was finely and directly regulated by the salt concentration without the requirement for gene expression. M1PDH can therefore be a key enzyme for regulating mannitol biosynthesis when the alga is stressed by a salinity change.
Transcriptional Profiling of Arabidopsis Tissues Reveals the Unique Characteristics of the Pollen Transcriptome
Authors: Becker Jörg D. , Boavida Leonor C. , Carneiro Jorge , Haury Matthias , Feijó José A. 
Published: October 2003

AbstractPollen tubes are a good model for the study of cell growth and morphogenesis because of their extreme elongation without cell division. Yet, knowledge about the genetic basis of pollen germination and tube growth is still lagging behind advances in pollen physiology and biochemistry. In an effort to reduce this gap, we have developed a new method to obtain highly purified, hydrated pollen grains of Arabidopsis through flowcytometric sorting, and we used GeneChips (Affymetrix, Santa Clara, CA; representing approximately 8,200 genes) to compare the transcriptional profile of sorted pollen with those of four vegetative tissues (seedlings, leaves, roots, and siliques). We present a new graphical tool allowing genomic scale visualization of the unique transcriptional profile of pollen. The 1,584 genes expressed in pollen showed a 90% overlap with genes expressed in these vegetative tissues, whereas one-third of the genes constitutively expressed in the vegetative tissues were not expressed in pollen. Among the 469 genes enriched in pollen, 162 were selectively expressed, and most of these had not been associated previously with pollen. Their functional classification reveals several new candidate genes, mainly in the categories of signal transduction and cell wall biosynthesis and regulation. Thus, the results presented improve our knowledge of the molecular mechanisms underlying pollen germination and tube growth and provide new directions for deciphering their genetic basis. Because pollen expresses about one-third of the number of genes expressed on average in other organs, it may constitute an ideal system to study fundamental mechanisms of cell biology and, by omission, of cell division.
Sfp1 Plays a Key Role in Yeast Ribosome Biogenesis
Authors: Fingerman Ian , Nagaraj Vijayalakshmi , Norris David , Vershon Andrew K. 
Published: October 2003

ABSTRACT

Sfp1, an unusual zinc finger protein, was previously identified as a gene that, when overexpressed, imparted a nuclear localization defect.
sfp1
cells have a reduced size and a slow growth phenotype. In this study we show that
SFP1
plays a role in ribosome biogenesis. An
sfp1
strain is hypersensitive to drugs that inhibit translational machinery.
sfp1
strains also have defects in global translation as well as defects in rRNA processing and 60S ribosomal subunit export. Microarray analysis has previously shown that ectopically expressed
SFP1
induces the transcription of a large subset of genes involved in ribosome biogenesis. Many of these induced genes contain conserved promoter elements (RRPE and PAC). Our results show that activation of transcription from a reporter construct containing two RRPE sites flanking a single PAC element is
SFP1
dependent. However, we have been unable to detect direct binding of the protein to these elements. This suggests that regulation of genes containing RRPEs is dependent upon Sfp1 but that Sfp1 may not directly bind to these conserved promoter elements; rather, activation may occur through an indirect mechanism.
Bacterial Catalase in the Microsporidian
Nosema locustae
: Implications for Microsporidian Metabolism and Genome Evolution
Authors: Fast Naomi M. , Law Joyce S. , Williams Bryony A. P. , Keeling Patrick J. 
Published: October 2003

ABSTRACT

Microsporidia constitute a group of extremely specialized intracellular parasites that infect virtually all animals. They are highly derived, reduced fungi that lack several features typical of other eukaryotes, including canonical mitochondria, flagella, and peroxisomes. Consistent with the absence of peroxisomes in microsporidia, the recently completed genome of the microsporidian
Encephalitozoon cuniculi
lacks a gene for catalase, the major enzymatic marker for the organelle. We show, however, that the genome of the microsporidian
Nosema locustae
, in contrast to that of
E. cuniculi
, encodes a group II large-subunit catalase. Surprisingly, phylogenetic analyses indicate that the
N. locustae
catalase is not specifically related to fungal homologs, as one would expect, but is instead closely related to proteobacterial sequences. This finding indicates that the
N. locustae
catalase is derived by lateral gene transfer from a bacterium. The catalase gene is adjacent to a large region of the genome that appears to be far less compact than is typical of microsporidian genomes, a characteristic which may make this region more amenable to the insertion of foreign genes. The
N. locustae
catalase gene is expressed in spores, and the protein is detectable by Western blotting. This type of catalase is a particularly robust enzyme that has been shown to function in dormant cells, indicating that the
N. locustae
catalase may play some functional role in the spore. There is no evidence that the
N. locustae
catalase functions in a cryptic peroxisome.
Attachment of the Ubiquitin-Related Protein Urm1p to the Antioxidant Protein Ahp1p
Authors: Goehring April S. , Rivers David M. , Sprague George F. 
Published: October 2003

ABSTRACT

Urm1p is a ubiquitin-related protein that serves as a posttranslational modification of other proteins. Urm1p conjugation has been implicated in the budding process and in nutrient sensing. Here, we have identified the first in vivo target for the urmylation pathway as the antioxidant protein Ahp1p. The attachment of Urm1p to Ahp1p requires the E1 for the urmylation pathway, Uba4p. Loss of the urmylation pathway components results in sensitivity to a thiol-specific oxidant, as does loss of Ahp1p, implying that urmylation has a role in an oxidative-stress response. Moreover, treatment of cells with thiol-specific oxidants affects the abundance of Ahp1p-Urm1p conjugates. These results suggest that the conjugation of Urm1p to Ahp1p could regulate the function of Ahp1p in antioxidant stress response in
Saccharomyces cerevisiae
.