Biochemistry -- Faculty Publications Copyright (c) 2013 University of Nebraska - Lincoln All rights reserved. http://digitalcommons.unl.edu/biochemfacpub Recent documents in Biochemistry -- Faculty Publications en-us Fri, 22 Feb 2013 08:11:45 PST 3600 Probing Structural Selectivity of Synthetic Heparin Binding to Stabilin Protein Receptors http://digitalcommons.unl.edu/biochemfacpub/109 http://digitalcommons.unl.edu/biochemfacpub/109 Fri, 16 Nov 2012 12:23:39 PST As one of the most widely used drugs worldwide, heparin is an essential anticoagulant required for surgery, dialysis, treatment of thrombosis, cancer, and general circulatory management. Stabilin- 2 is a scavenger clearance receptor with high expression in the sinusoidal endothelium of liver. It is believed that Stabilin- 2 is the primary receptor for the clearance of unfractionated and low molecular weight heparins in the liver. Here, we identify the modifications and length of the heparin polymer that are required for binding and endocytosis by both human Stabilin receptors: Stabilin-2 and its homolog Stabilin-1 (also found in liver endothelium). Using enzymatically synthesized 35S-labeled heparan sulfate oligomers, we identified that sulfation of the 3-OH position of N-sulfated glucosamine (GlcNS) is the most beneficial modification for binding and endocytosis via both Stabilin receptors. In addition, our data suggest that a decasaccharide is the minimal size for binding to the Stabilin receptors. These findings define the physical parameters of the heparin structure required for efficient clearance from blood circulation. These results will also aid in the design of synthetic heparins with desired clearance rates.

]]> Elizabeth H. Pempe et al. Genome-wide ribosome profiling reveals complex translational regulation in response to oxidative stress http://digitalcommons.unl.edu/biochemfacpub/108 http://digitalcommons.unl.edu/biochemfacpub/108 Tue, 16 Oct 2012 12:45:35 PDT Information on unique and coordinated regulation of transcription and translation in response to stress is central to the understanding of cellular homeostasis. Here we used ribosome profiling coupled with next-generation sequencing to examine the interplay between transcription and translation under conditions of hydrogen peroxide treatment in Saccharomyces cerevisiae. Hydrogen peroxide treatment led to a massive and rapid increase in ribosome occupancy of short upstream ORFs, including those with non-AUG translational starts, and of the N-terminal regions of ORFs that preceded the transcriptional response. In addition, this treatment induced the synthesis of N-terminally extended proteins and elevated stop codon read-through and frameshift events. It also increased ribosome occupancy at the beginning of ORFs and potentially the duration of the elongation step. We identified proteins whose synthesis was regulated rapidly by hydrogen peroxide posttranscriptionally; however, for the majority of genes increased protein synthesis followed transcriptional regulation. These data define the landscape of genome-wide regulation of translation in response to hydrogen peroxide and suggest that potentiation (coregulation of the transcript level and translation) is a feature of oxidative stress.

]]> Maxim V. Gerashchenko et al. Detection of Mercuric Ions in Water by ELISA with a Mercury- Specific Antibody http://digitalcommons.unl.edu/biochemfacpub/107 http://digitalcommons.unl.edu/biochemfacpub/107 Tue, 18 Sep 2012 14:32:48 PDT An immunoassay that detects mercuric ions in water at concentrations of 0.5 ppb and above is described. The assay utilizes a monoclonal antibody that binds specifically to mercuric ions immobilized in wells of microtiter plates. Within the range of 0.5-10 ppb mercury, the absorbance in the enzyme-linked immunosorbent assay (ELISA) is linear to the log of the mercuric ion concentration. The quantitation of mercury by ELISA correlates closely with results from cold-vapor atomic absorption. Other divalent metal cations do not interfere with the assay, although there is interference in the presence of 1 mM chloride ions. The optimum pH for mercury detection is 7.0, although 2 ppb mercury can be detected over a wide pH range. The assay is as sensitive as coldvapor atomic absorption for mercury detection and can be performed with only 100 μl of sample.

]]> Dwane E. Wylie et al. Activation of the Carbon Concentrating Mechanism by CO2 Deprivation Coincides with Massive Transcriptional Restructuring in <i>Chlamydomonas reinhardtii</i> http://digitalcommons.unl.edu/biochemfacpub/106 http://digitalcommons.unl.edu/biochemfacpub/106 Tue, 03 Jul 2012 09:39:33 PDT A CO2-concentrating mechanism (CCM) is essential for the growth of most eukaryotic algae under ambient (392 ppm) and very low (<100 ppm) CO2 concentrations. In this study, we used replicated deep mRNA sequencing and regulatory network reconstruction to capture a remarkable scope of changes in gene expression that occurs when Chlamydomonas reinhardtii cells are shifted from high to very low levels of CO2 (£100 ppm). CCM induction 30 to 180 min post-CO2 deprivation coincides with statistically significant changes in the expression of an astonishing 38% (5884) of the 15,501 nonoverlapping C. reinhardtii genes. Of these genes, 1088 genes were induced and 3828 genes were downregulated by a log2 factor of 2. The latter indicate a global reduction in photosynthesis, protein synthesis, and energy-related biochemical pathways. The magnitude of transcriptional rearrangement and its major patterns are robust as analyzed by three different statistical methods. De novo DNA motif discovery revealed new putative binding sites for Myeloid oncogene family transcription factors potentially involved in activating low CO2–induced genes. The (CA)n repeat (9 ≤ n ≤ 25) is present in 29% of upregulated genes but almost absent from promoters of downregulated genes. These discoveries open many avenues for new research.

Includes supplemental materials.

]]> Andrew J. Brueggeman et al. Homologous recombination in <i>Nannochloropsis</i>: A powerful tool in an industrially relevant alga http://digitalcommons.unl.edu/biochemfacpub/105 http://digitalcommons.unl.edu/biochemfacpub/105 Wed, 23 May 2012 08:35:55 PDT The availability of facile methods for targeted gene knockout and gene replacement based on homologous recombination in bacteria and yeast systems has driven rapid progress in understanding many of the complex metabolic and regulatory networks in prokaryotic and eukaryotic cells. The lack of such tools in other organisms is a major impediment to progress both in fundamental research and in research that is directed toward practical economic and societal outcomes. For example, the current goal to replace fossil fuels with renewable energy sources produced by plants and algae is hampered in both the short term and the long term by our limited knowledge of their metabolic systems and how they can be modified to create organisms that produce more and better energy-rich molecules. Thus, the report in PNAS by Kilian et al. (1) of efficient and reliable genetic transformation of the commercially important alga, Nannochloropsis, via homologous recombination is a significant step forward.

]]> Donald P. Weeks Targeted insertion of cysteine by decoding UGA codons with mammalian selenocysteine machinery http://digitalcommons.unl.edu/biochemfacpub/104 http://digitalcommons.unl.edu/biochemfacpub/104 Wed, 23 May 2012 08:32:42 PDT Cysteine (Cys) is inserted into proteins in response to UGC and UGU codons. Herein, we show that supplementation of mammalian cells with thiophosphate led to targeted insertion of Cys at the UGA codon of thioredoxin reductase 1 (TR1). This Cys was synthesized by selenocysteine (Sec) synthase on tRNA[Ser]Sec and its insertion was dependent on the Sec insertion sequence element in the 3′ UTR of TR1 mRNA. The substrate for this reaction, thiophosphate, was synthesized by selenophosphate synthetase 2 from ATP and sulfide and reacted with phosphoseryl-tRNA[Ser]Sec to generate Cys-tRNA[Ser]Sec. Cys was inserted in vivo at UGA codons in natural mammalian TRs, and this process was regulated by dietary selenium and availability of thiophosphate. Cys occurred at 10% of the Sec levels in liver TR1 of mice maintained on a diet with normal amounts of selenium and at 50% in liver TR1 of mice maintained on a selenium deficient diet. These data reveal a novel Sec machinerybased mechanism for biosynthesis and insertion of Cys into protein at UGA codons and suggest new biological functions for thiophosphate and sulfide in mammals.

]]> Xue-Ming Xu et al. Differential transcription of cytochrome P450s and glutathione S transferases in DDT-susceptible and -resistant <i>Drosophila melanogaster</i> strains in response to DDT and oxidative stress http://digitalcommons.unl.edu/biochemfacpub/103 http://digitalcommons.unl.edu/biochemfacpub/103 Wed, 23 May 2012 08:14:06 PDT Metabolic DDT resistance in Drosophila melanogaster has previously been associated with constitutive over-transcription of cytochrome P450s. Increased P450 activity has also been associated with increased oxidative stress. In contrast, over-transcription of glutathione S transferases (GSTs) has been associated with resistance to oxidative stress. However, little is known in regards to the impact of xenobiotics on induction of P450s and GSTs and if there exist differences in inducibility between the pesticide susceptible and resistant strains. Thus, we investigated the transcriptional expression of GSTs and P450s in DDT resistant (Wisconsin) and susceptible (Canton-S) Drosophila strains in response to exposure to DDT and the oxidative stressor H2O2. Wisconsin constitutively over-transcribed P450s, constitutively under-transcribed 27% of its total GSTs, and was more susceptible to H2O2 than Canton-S. DDT exposure induced GST expression only in the Wisconsin strain and not in the Canton-S strain. These results suggest that there are potentially more differences between pesticide susceptible and resistant strains than just constitutive expression of P450s; there may also exist, at least in some strains, differences in their patterns of inducibility of P450s and GSTs. Within the context of the Wisconsin strain, these differences may be contributing to the fly lines increased susceptibility to oxidative stress.

]]> Lijie Sun et al. Functional Characterization of a Higher Plant Sphingolipid Δ4-Desaturase: Defining the Role of Sphingosine and Sphingosine-1-Phosphate in Arabidopsis http://digitalcommons.unl.edu/biochemfacpub/102 http://digitalcommons.unl.edu/biochemfacpub/102 Wed, 30 Nov 2011 08:49:23 PST The role of Δ4-unsaturated sphingolipid long-chain bases such as sphingosine was investigated in Arabidopsis (Arabidopsis thaliana). Identification and functional characterization of the sole Arabidopsis ortholog of the sphingolipid Δ4-desaturase was achieved by heterologous expression in Pichia pastoris. A P. pastoris mutant disrupted in the endogenous sphingolipid Δ4-desaturase gene was unable to synthesize glucosylceramides. Synthesis of glucosylceramides was restored by the expression of Arabidopsis gene At4g04930, and these sphingolipids were shown to contain Δ4-unsaturated long-chain bases, confirming that this open reading frame encodes the sphingolipid Δ4-desaturase. At4g04930 has a very restricted expression pattern, transcripts only being detected in pollen and floral tissues. Arabidopsis insertion mutants disrupted in the sphingolipid Δ4-desaturase At4g04930 were isolated and found to be phenotypically normal. Sphingolipidomic profiling of a T-DNA insertion mutant indicated the absence of Δ4-unsaturated sphingolipids in floral tissue, also resulting in the reduced accumulation of glucosylceramides. No difference in the response to drought or water loss was observed between wild-type plants and insertion mutants disrupted in the sphingolipid Δ4-desaturase At4g04930, nor was any difference observed in stomatal closure after treatment with abscisic acid. No differences in pollen viability between wild-type plants and insertion mutants were detected. Based on these observations, it seems unlikely that Δ4-unsaturated sphingolipids and their metabolites such as sphingosine-1-phosphate play a significant role in Arabidopsis growth and development. However, Δ4-unsaturated ceramides may play a previously unrecognized role in the channeling of substrates for the synthesis of glucosylceramides.

]]> Louise V. Michaelson et al. Functional Characterization of the Arabidopsis β-Ketoacyl-Coenzyme A Reductase Candidates of the Fatty Acid Elongase http://digitalcommons.unl.edu/biochemfacpub/101 http://digitalcommons.unl.edu/biochemfacpub/101 Wed, 30 Nov 2011 08:39:30 PST In plants, very-long-chain fatty acids (VLCFAs; >18 carbon) are precursors of sphingolipids, triacylglycerols, cuticular waxes, and suberin. VLCFAs are synthesized by a multiprotein membrane-bound fatty acid elongation system that catalyzes four successive enzymatic reactions: condensation, reduction, dehydration, and a second reduction. A bioinformatics survey of the Arabidopsis (Arabidopsis thaliana) genome has revealed two sequences homologous to YBR159w encoding a Saccharomyces cerevisiae β-ketoacyl reductase (KCR), which catalyzes the first reduction during VLCFA elongation. Expression analyses showed that both AtKCR1 and AtKCR2 genes were transcribed in siliques, flowers, inflorescence stems, leaves, as well as developing embryos, but only AtKCR1 transcript was detected in roots. Fluorescent protein-tagged AtKCR1 and AtKCR2 were localized to the endoplasmic reticulum, the site of fatty acid elongation. Complementation of the yeast ybr159Δ mutant demonstrated that the two KCR proteins are divergent and that only AtKCR1 can restore heterologous elongase activity similar to the native yeast KCR gene. Analyses of insertional mutants in AtKCR1 and AtKCR2 revealed that loss of AtKCR1 function results in embryo lethality, which cannot be rescued by AtKCR2 expression using the AtKCR1 promoter. In contrast, a disruption of the AtKCR2 gene had no obvious phenotypic effect. Taken together, these results indicate that only AtKCR1 is a functional KCR isoform involved in microsomal fatty acid elongation. To investigate the roles of AtKCR1 in postembryonic development, transgenic lines expressing RNA interference and overexpression constructs targeted against AtKCR1 were generated. Morphological and biochemical characterization of these lines confirmed that suppressed KCR activity results in a reduction of cuticular wax load and affects VLCFA composition of sphingolipids, seed triacylglycerols, and root glycerolipids, demonstrating in planta that KCR is involved in elongation reactions supplying VLCFA for all these diverse classes of lipids.

]]> Frédéric Beaudoin et al. Very-Long-Chain Fatty Acids Are Involved in Polar Auxin Transport and Developmental Patterning in <i>Arabidopsis</i> http://digitalcommons.unl.edu/biochemfacpub/100 http://digitalcommons.unl.edu/biochemfacpub/100 Wed, 30 Nov 2011 08:25:53 PST Very-long-chain fatty acids (VLCFAs) are essential for many aspects of plant development and necessary for the synthesis of seed storage triacylglycerols, epicuticular waxes, and sphingolipids. Identification of the acetyl-CoA carboxylase PASTICCINO3 and the 3-hydroxy acyl-CoA dehydratase PASTICCINO2 revealed that VLCFAs are important for cell proliferation and tissue patterning. Here, we show that the immunophilin PASTICCINO1 (PAS1) is also required for VLCFA synthesis. Impairment of PAS1 function results in reduction of VLCFA levels that particularly affects the composition of sphingolipids, known to be important for cell polarity in animals. Moreover, PAS1 associates with several enzymes of the VLCFA elongase complex in the endoplasmic reticulum. The pas1 mutants are deficient in lateral root formation and are characterized by an abnormal patterning of the embryo apex, which leads to defective cotyledon organogenesis. Our data indicate that in both tissues, defective organogenesis is associated with the mistargeting of the auxin efflux carrier PIN FORMED1 in specific cells, resulting in local alteration of polar auxin distribution. Furthermore, we show that exogenous VLCFAs rescue lateral root organogenesis and polar auxin distribution, indicating their direct involvement in these processes. Based on these data, we propose that PAS1 acts as a molecular scaffold for the fatty acid elongase complex in the endoplasmic reticulum and that the resulting VLCFAs are required for polar auxin transport and tissue patterning during plant development.

]]> Françoise Roudier et al. Sphingolipid Long-Chain Base Hydroxylation Is Important for Growth and Regulation of Sphingolipid Content and Composition in <i>Arabidopsis</i> http://digitalcommons.unl.edu/biochemfacpub/99 http://digitalcommons.unl.edu/biochemfacpub/99 Wed, 30 Nov 2011 08:03:50 PST Sphingolipids are structural components of endomembranes and function through their metabolites as bioactive regulators of cellular processes such as programmed cell death. A characteristic feature of plant sphingolipids is their high content of trihydroxy long-chain bases (LCBs) that are produced by the LCB C-4 hydroxylase. To determine the functional significance of trihydroxy LCBs in plants, T-DNA double mutants and RNA interference suppression lines were generated for the two Arabidopsis thaliana LCB C-4 hydroxylase genes Sphingoid Base Hydroxylase1 (SBH1) and SBH2. These plants displayed reductions in growth that were dependent on the content of trihydroxy LCBs in sphingolipids. Double sbh1 sbh2 mutants, which completely lacked trihydroxy LCBs, were severely dwarfed, did not progress from vegetative to reproductive growth, and had enhanced expression of programmed cell death associated–genes. Furthermore, the total content of sphingolipids on a dry weight basis increased as the relative amounts of trihydroxy LCBs decreased. In trihydroxy LCB–null mutants, sphingolipid content was ~2.5-fold higher than that in wild-type plants. Increases in sphingolipid content resulted from the accumulation of molecular species with C16 fatty acids rather than with very-long-chain fatty acids, which are more commonly enriched in plant sphingolipids, and were accompanied by decreases in amounts of C16-containing species of chloroplast lipids. Overall, these results indicate that trihydroxy LCB synthesis plays a central role in maintaining growth and mediating the total content and fatty acid composition of sphingolipids in plants.

]]> Ming Chen et al. An Inositolphosphorylceramide Synthase Is Involved in Regulation of Plant Programmed Cell Death Associated with Defense in <i>Arabidopsis</i> http://digitalcommons.unl.edu/biochemfacpub/98 http://digitalcommons.unl.edu/biochemfacpub/98 Wed, 30 Nov 2011 07:56:55 PST The Arabidopsis thaliana resistance gene RPW8 triggers the hypersensitive response (HR) to restrict powdery mildew infection via the salicylic acid–dependent signaling pathway. To further understand how RPW8 signaling is regulated, we have conducted a genetic screen to identify mutations enhancing RPW8-mediated HR-like cell death (designated erh). Here, we report the isolation and characterization of the Arabidopsis erh1 mutant, in which the At2g37940 locus is knocked out by a T-DNA insertion. Loss of function of ERH1 results in salicylic acid accumulation, enhanced transcription of RPW8 and RPW8-dependent spontaneous HR-like cell death in leaf tissues, and reduction in plant stature. Sequence analysis suggests that ERH1 may encode the long-sought Arabidopsis functional homolog of yeast and protozoan inositolphosphorylceramide synthase (IPCS), which converts ceramide to inositolphosphorylceramide. Indeed, ERH1 is able to rescue the yeast aur1 mutant, which lacks the IPCS, and the erh1 mutant plants display reduced (~53% of wild type) levels of leaf IPCS activity, indicating that ERH1 encodes a plant IPCS. Consistent with its biochemical function, the erh1 mutation causes ceramide accumulation in plants expressing RPW8. These data reinforce the concept that sphingolipid metabolism (specifically, ceramide accumulation) plays an important role in modulating plant programmed cell death associated with defense.

]]> Wenming Wang et al. Kinetic properties of bifunctional 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase from spinach leaves http://digitalcommons.unl.edu/biochemfacpub/97 http://digitalcommons.unl.edu/biochemfacpub/97 Mon, 14 Nov 2011 12:57:54 PST A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Spinacia oleracea leaf library and used to ex­press a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. The soluble protein had a molecular mass of 320 kDa, estimated by gel filtration chromatography, and a subunit size of 90.8 kDa. The purified pro­tein had activity of both 6-phosphofructo-2-kinase (specific activity 10.4–15.9 nmol∙min–1∙mg protein–1) and fructose-2,6- bisphosphatase (specific activity 1.65–1.75 nmol∙min–1∙mg protein–1). The 6-phosphofructo-2-kinase activity was activated by inorganic phosphate, and in­hibited by 3-carbon phosphorylated metabolites and pyrophosphate. In the presence of phosphate, 3-phosphoglycerate was a mixed in­hibitor with respect to both fructose 6-phosphate and ATP. Fructose-2,6-bisphosphatase activity was sensitive to product inhibition; in­hibition by inorganic phosphate was uncompetitive, whereas inhibition by fructose 6-phosphate was mixed. These kinetic properties support the view that the level of fructose 2,6-bisphosphate in leaves is determined by the relative concentrations of hexose phosphates, three-carbon phosphate esters and inorganic phosphate in the cytosol through reciprocal modulation of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities of the bifunctional enzyme.

]]> Jonathan E. Markham et al. Sphingolipids Containing Very-Long-Chain Fatty Acids Define a Secretory Pathway for Specific Polar Plasma Membrane Protein Targeting in <i>Arabidopsis</i> http://digitalcommons.unl.edu/biochemfacpub/96 http://digitalcommons.unl.edu/biochemfacpub/96 Mon, 31 Oct 2011 13:26:42 PDT Sphingolipids are a class of structural membrane lipids involved in membrane trafficking and cell polarity. Functional analysis of the ceramide synthase family in Arabidopsis thaliana demonstrates the existence of two activities selective for the length of the acyl chains. Very-long-acyl-chain (C > 18 carbons) but not long-chain sphingolipids are essential for plant development. Reduction of very-long-chain fatty acid sphingolipid levels leads in particular to auxin-dependent inhibition of lateral root emergence that is associated with selective aggregation of the plasma membrane auxin carriers AUX1 and PIN1 in the cytosol. Defective targeting of polar auxin carriers is characterized by specific aggregation of Rab-A2a– and Rab-A1e–labeled early endosomes along the secretory pathway. These aggregates correlate with the accumulation of membrane structures and vesicle fragmentation in the cytosol. In conclusion, sphingolipids with very long acyl chains define a trafficking pathway with specific endomembrane compartments and polar auxin transport protein cargoes.

]]> Jonathan E. Markham et al. Sphingolipids in the Root Play an Important Role in Regulating the Leaf Ionome in <i>Arabidopsis thaliana</i> http://digitalcommons.unl.edu/biochemfacpub/95 http://digitalcommons.unl.edu/biochemfacpub/95 Tue, 25 Oct 2011 14:48:10 PDT Sphingolipid synthesis is initiated by condensation of Ser with palmitoyl-CoA producing 3-ketodihydrosphinganine (3-KDS), which is reduced by a 3-KDS reductase to dihydrosphinganine. Ser palmitoyltransferase is essential for plant viability. Arabidopsis thaliana contains two genes (At3g06060/TSC10A and At5g19200/TSC10B) encoding proteins with significant similarity to the yeast 3-KDS reductase, Tsc10p. Heterologous expression in yeast of either Arabidopsis gene restored 3-KDS reductase activity to the yeast tsc10D mutant, confirming both as bona fide 3-KDS reductase genes. Consistent with sphingolipids having essential functions in plants, double mutant progeny lacking both genes were not recovered from crosses of single tsc10A and tsc10B mutants. Although the 3-KDS reductase genes are functionally redundant and ubiquitously expressed in Arabidopsis, 3-KDS reductase activity was reduced to 10% of wild-type levels in the loss-of-function tsc10a mutant, leading to an altered sphingolipid profile. This perturbation of sphingolipid biosynthesis in the Arabidopsis tsc10a mutant leads an altered leaf ionome, including increases in Na, K, and Rb and decreases in Mg, Ca, Fe, and Mo. Reciprocal grafting revealed that these changes in the leaf ionome are driven by the root and are associated with increases in root suberin and alterations in Fe homeostasis.

]]> Dai-Yin Chao et al. Biotin carboxyl carrier protein and carboxyltransferase subunits of the multi-subunit form of acetyl-CoA carboxylase from <i>Brassica napus</i>: cloning and analysis of expression during oilseed rape embryogenesis http://digitalcommons.unl.edu/biochemfacpub/94 http://digitalcommons.unl.edu/biochemfacpub/94 Tue, 25 Oct 2011 11:58:10 PDT In the oilseed rape Brassica napus there are two forms of acetyl- CoA carboxylase (ACCase). As in other dicotyledonous plants there is a type I ACCase, the single polypeptide 220 kDa form, and a type II multi-subunit complex analogous to that of Escherichia coli and Anabaena. This paper describes the cloning and characterization of a plant biotin carboxyl carrier protein (BCCP) from the type II ACCase complex that shows 61% identity/79% similarity with Anabaena BCCP at the amino acid level. Six classes of nuclear encoded oilseed rape BCCP cDNA were cloned, two of which contained the entire coding region. The BCCP sequences allowed the assignment of function to two previously unassigned Arabidopsis expressed sequence tag (EST) sequences. We also report the cloning of a second type II ACCase component from oilseed rape, the β-carboxyltransferase subunit (βCT), which is chloroplast-encoded. Northern analysis showed that although the relative levels of BCCP and βCT mRNA differed between different oilseed rape tissues, their temporal patterns of expression were identical during embryo development. At the protein level, expression of BCCP during embryo development was studied by Western blotting, using affinity-purified anti-biotin polyclonal sera. With this technique a 35 kDa protein thought to be BCCP was shown to reside within the chloroplast. This analysis also permitted us to view the differential expression of several unidentified biotinylated proteins during embryogenesis.

]]> Kieran M. Elborough et al. The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development http://digitalcommons.unl.edu/biochemfacpub/93 http://digitalcommons.unl.edu/biochemfacpub/93 Tue, 25 Oct 2011 11:08:26 PDT Very-long-chain fatty acids (VLCFAs) are synthesized as acyl-CoAs by the endoplasmic reticulum-localized elongase multiprotein complex. Two Arabidopsis genes are putative homologues of the recently identified yeast 3-hydroxy-acyl-CoA dehydratase (PHS1), the third enzyme of the elongase complex. We showed that Arabidopsis PASTICCINO2 (PAS2) was able to restore phs1 cytokinesis defects and sphingolipid long chain base overaccumulation. Conversely, the expression of PHS1 was able to complement the developmental defects and the accumulation of long chain bases of the pas2–1 mutant. The pas2–1 mutant was characterized by a general reduction of VLCFA pools in seed storage triacylglycerols, cuticular waxes, and complex sphingolipids. Most strikingly, the defective elongation cycle resulted in the accumulation of 3- hydroxy-acyl-CoA intermediates, indicating premature termination of fatty acid elongation and confirming the role of PAS2 in this process. We demonstrated by in vivo bimolecular fluorescence complementation that PAS2 was specifically associated in the endoplasmic reticulum with the enoyl-CoA reductase CER10, the fourth enzyme of the elongase complex. Finally, complete loss of PAS2 function is embryo lethal, and the ectopic expression of PHS1 led to enhanced levels of VLCFAs associated with severe developmental defects. Altogether these results demonstrate that the plant 3-hydroxy-acyl-CoA dehydratase PASTICCINO2 is an essential and limiting enzyme in VLCFA synthesis but also that PAS2-derived VLCFA homeostasis is required for specific developmental processes.

Includes Supplementary material.

]]> Liên Bach et al. Polyploidy creates higher diversity among <i>Cynodon</i> accessions as assessed by molecular markers http://digitalcommons.unl.edu/biochemfacpub/92 http://digitalcommons.unl.edu/biochemfacpub/92 Wed, 08 Jun 2011 10:44:30 PDT Developing a better understanding of associations among ploidy level, geographic distribution, and genetic diversity of Cynodon accessions could be beneficial to bermudagrass breeding programs, and would enhance our understanding of the evolutionary biology of this warm season grass species. This study was initiated to: (1) determine ploidy analysis of Cynodon accessions collected from Turkey, (2) investigate associations between ploidy level and diversity, (3) determine whether geographic and ploidy distribution are related to nuclear genome variation, and (4) correlate among four nuclear molecular marker systems for Cynodon accessions’ genetic analyses. One hundred and eighty-two Cynodon accessions collected in Turkey from an area south of the Taurus Mountains along the Mediterranean cost and ten known genotypes were genotyped using sequence related amplified polymorphism (SRAP), peroxidase gene polymorphism (POGP), inter-simple sequence repeat (ISSR), and random amplified polymorphic DNA (RAPD). The diploids, triploids, tetraploids, pentaploids, and hexaploids revealed by flow cytometry had a linear present band frequency of 0.36, 0.47, 0.49, 0.52, and 0.54, respectively. Regression analysis explained that quadratic relationship between ploidy level and band frequency was the most explanatory (r = 0.62, P < 0.001). The AMOVA results indicated that 91 and 94% of the total variation resided within ploidy level and provinces, respectively. The UPGMA analysis suggested that commercial bermudagrass cultivars only one-third of the available genetic variation. SRAP, POGP, ISSR, and RAPD markers differed in detecting relationships among the bermudagrass genotypes and rare alleles, suggesting more efficiency of combinatory analysis of molecular marker systems. Elucidating Cynodon accessions’ genetic structure can aid to enhance breeding programs and broaden genetic base of commercial cultivars.

]]> Osman Gulsen et al. Development of SRAP, SRAP-RGA, RAPD, and SCAR markers linked with a Fusarium wilt resistance gene in eggplant http://digitalcommons.unl.edu/biochemfacpub/91 http://digitalcommons.unl.edu/biochemfacpub/91 Wed, 13 Apr 2011 14:55:18 PDT Fusarium wilt (Fusarium oxysporum Schlecht. f. sp. melongenae) is a vascular disease of eggplant (Solanum melon­gena L.). The objectives of this work were (1) to confirm the monogenic inheritance of fusarium wilt resistance in eggplant, (2) to identify molecular markers linked to this resistance, and (3) to develop SCAR markers from most informative markers. We report the tagging of the gene for resistance to fusarium wilt (FOM) in eggplant using SRAP, RGA, SRAP-RGA and RAPD markers. Analysis of segregation data confirmed the monogenic inheritance of resistance. DNA from F2 and BC1 populations of eggplant segregating for fusarium wilt resis­tance was screened with 2,316 primer combinations to detect polymorphism. Three markers were linked within 2.6 cM of the gene. The codominant SRAP marker Me8/Em5 and dominant SRAP-RGA marker Em12/GLPL2 were tightly linked to each other and mapped 1.2 cM from the resistance gene, whereas RAPD marker H12 mapped 2.6 cM from the gene and on the same side as the other two markers. The SRAP marker was converted into two dominant SCAR markers that were confirmed to be linked to the resistance gene in the F2, BC1 and F2 of BC3 generations of the same cross. These markers provide a starting point for mapping the eggplant FOM re­sistance gene in eggplant and for exploring the synteny between solanaceous crops for fusarium wilt resistance genes. The SCAR markers will be useful for identifying fusarium wilt-resistant genotypes in marker-assisted se­lection breeding programs using segregating progenies of the resistant eggplant progenitor used in this study.

]]> Nedim Mutlu et al. Tissue-specific expression and developmental regulation of cytochrome b561 genes in <i>Arabidopsis thaliana</i> and <i>Raphanus sativus</i> http://digitalcommons.unl.edu/biochemfacpub/90 http://digitalcommons.unl.edu/biochemfacpub/90 Tue, 22 Mar 2011 11:56:52 PDT Ascorbate (Asc) is an essential molecule in many aspects of development and stress responses in plants and animals. Cytochromes b561 (cyts b561) are tightly coupled to Asc homeostasis. These proteins are found in mammalian tissues, where they are involved in the regeneration of Asc, serving the synthesis of catecholamine neurotransmitters, and in intestinal iron reduction. Plant genomes encode homologous membrane-associated, Asc-reducible cyts b561. The expression of these proteins in plants, however, has so far not been studied. We have now examined the expression of two Arabidopsis thaliana cyt b561-encoding genes—Artb561-1 and Artb561-2—using relative-quantitative RT-PCR and in situ hybridization (ISH) techniques. The genes show overlapping and distinct tissue- and organ-specific expression patterns. Transcripts of both genes are found in leaf epidermal cells, and expression seems to correlate with leaf maturation and cessation of cell elongation. Both genes are also expressed in the epidermal cell layer of stems and roots in the L1 layer of the shoot apex, in the vascular system of leaves, stems and roots, and in the root pericycle. In addition, Artb561-1 is expressed in the root cap, whereas Artb561-2 mRNA is found in the epidermis of lateral roots, in the root meristem, and in unfertilized ovules. These observations provide important information for the elucidation of the physiological function of cyts b561 in plants.

]]> Wim Verelst et al.