TY - CONF TI - Artemisia annua L. Transformation with different Agrobacterium rhizogenesis and large scale culture of hairy roots for Artemisinin (Qinghaosu) production AU - Xie, Deyu AU - Ye, Hechun AU - Li, Guofeng AU - Guo, Zhongchen A2 - Larkin, P.J. C2 - 1998/// C3 - Agricultural biotechnology : laboratory, field and market : proceedings of the 4th Asia-Pacific Conference on Agricultural Biotechnology, 13-16 July 1998, Darwin, Australia DA - 1998/// SP - 134–136 PB - Under the Counter Publishing ER - TY - JOUR TI - Identification of sucrose synthase as an actin-binding protein AU - Winter, H. AU - L. Huber, J. AU - Huber, S.C. T2 - FEBS Letters AB - Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo. DA - 1998/// PY - 1998/// DO - 10.1016/S0014-5793(98)00659-0 VL - 430 IS - 3 SP - 205-208 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0032479338&partnerID=MN8TOARS KW - actin-binding protein KW - actin cytoskeleton KW - aldolase KW - sucrose synthase KW - phalloidin ER - TY - JOUR TI - Microbial physiology applied to process optimisation: Lactic acid bacteria T2 - Advances in Bioprocess Engineering Volume Ii DA - 1998/// PY - 1998/// DO - 10.1007/978-94-017-0643-8_6 UR - https://publons.com/publon/31684361/ ER - TY - JOUR TI - Fragile cells of Lactobacillus casei suitable for plasmid DNA isolation T2 - Biotechnology Techniques DA - 1998/// PY - 1998/// UR - https://publons.com/publon/31684369/ ER - TY - JOUR TI - Chemostat production of plantaricin C by Lactobacillus plantarum LL441 T2 - Applied and Environmental Microbiology DA - 1998/// PY - 1998/// UR - https://publons.com/wos-op/publon/28290537/ ER - TY - JOUR TI - Chemostat production of plantaricin C by Lactobacillus plantarum LL441 T2 - Applied and Environmental Microbiology DA - 1998/// PY - 1998/// VL - 64 IS - 9 SP - 3512-3514 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-3543035172&partnerID=MN8TOARS ER - TY - JOUR TI - Intragenic Recombination and Diversifying Selection Contribute to the Evolution of Downy Mildew Resistance at the RPP8 Locus of Arabidopsis AU - McDowell, J.M. AU - Dhandaydham, M. AU - Long, T.A. AU - Aarts, M.G.M. AU - Goff, S. AU - Holub, E.B. AU - Dangl, J.L. T2 - The Plant Cell AB - Pathogen resistance (R) genes of the NBS-LRR class (for nucleotide binding site and leucine-rich repeat) are found in many plant species and confer resistance to a diverse spectrum of pathogens. Little is known about the mechanisms that drive NBS-LRR gene evolution in the host-pathogen arms race. We cloned the RPP8 gene (for resistance to Peronospora parasitica) and compared the structure of alleles at this locus in resistant Landsberg erecta (Ler-0) and susceptible Columbia (Col-0) accessions. RPP8-Ler encodes an NBS-LRR protein with a putative N-terminal leucine zipper and is more closely related to previously cloned R genes that confer resistance to bacterial pathogens than it is to other known RPP genes. The RPP8 haplotype in Ler-0 contains the functional RPP8-Ler gene and a nonfunctional homolog, RPH8A. In contrast, the rpp8 locus in Col-0 contains a single chimeric gene, which was likely derived from unequal crossing over between RPP8-Ler and RPH8A ancestors within a Ler-like haplotype. Sequence divergence among RPP8 family members has been accelerated by positive selection on the putative ligand binding region in the LRRs. These observations indicate that NBS-LRR molecular evolution is driven by the same mechanisms that promote rapid sequence diversification among other genes involved in non-self-recognition. DA - 1998/11// PY - 1998/11// DO - 10.1105/tpc.10.11.1861 VL - 10 IS - 11 SP - 1861-1874 UR - http://dx.doi.org/10.1105/tpc.10.11.1861 ER - TY - JOUR TI - Nuclear events in ethylene signaling: A transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1 AU - Solano, R. AU - Stepanova, A. AU - Chao, Q. AU - Ecker, J.R. T2 - Genes and Development AB - Response to the gaseous plant hormone ethylene in Arabidopsis requires the EIN3/EIL family of nuclear proteins. The biochemical function(s) of EIN3/EIL proteins, however, has remained unknown. In this study, we show that EIN3 and EILs comprise a family of novel sequence-specific DNA-binding proteins that regulate gene expression by binding directly to a primary ethylene response element (PERE) related to the tomato E4-element. Moreover, we identified an immediate target of EIN3, ETHYLENE-RESPONSE-FACTOR1 (ERF1), which contains this element in its promoter. EIN3 is necessary and sufficient for ERF1 expression, and, like EIN3-overexpression in transgenic plants, constitutive expression of ERF1 results in the activation of a variety of ethylene response genes and phenotypes. Evidence is also provided that ERF1 acts downstream of EIN3 and all other components of the ethylene signaling pathway. The results demonstrate that the nuclear proteins EIN3 and ERF1 act sequentially in a cascade of transcriptional regulation initiated by ethylene gas. DA - 1998/// PY - 1998/// DO - 10.1101/gad.12.23.3703 VL - 12 IS - 23 SP - 3703-3714 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0032417391&partnerID=MN8TOARS KW - plants KW - Arabidopsis KW - EIN3 KW - ERF1 KW - transcription ER - TY - CHAP TI - Plant P-Hydroxyphenylpyruvate Dioxygenase: A Target for New Bleaching Herbicides AU - Garcia, I. AU - Rodgers, M. AU - Pépin, R. AU - Hsieh, Tzung-Fu AU - Matringe, M. T2 - Photosynthesis: Mechanisms and Effects PY - 1998/// DO - 10.1007/978-94-011-3953-3_900 SP - 3861-3864 OP - PB - Springer Netherlands SN - 9780792355472 9789401139533 UR - http://dx.doi.org/10.1007/978-94-011-3953-3_900 DB - Crossref ER - TY - JOUR TI - Fragile cells of Lactobacillus casei suitable for plasmid DNA isolation. AU - Bruno-Bárcena, J.M. AU - Azcárate-Peril, M.A. AU - Ragout, A. AU - Font De Valdez, G. AU - Raya, R. AU - Siñeriz, F. T2 - Biotechnology techniques. DA - 1998/2// PY - 1998/2// DO - 10.1023/a:1017139713930 VL - 2 IS - 2 SP - 97-99 UR - http://europepmc.org/abstract/AGR/IND20904300 ER - TY - JOUR TI - Chemostat production of plantaricin C by Lactobacillus plantarum LL441. T2 - Applied and environmental microbiology DA - 1998/9// PY - 1998/9// VL - 9 UR - http://europepmc.org/abstract/med/9726907 ER - TY - JOUR TI - Spatial Variability in In Situ Aerobic Respiration and Denitrification Rates in a Petroleum-Contaminated Aquifer AU - Schroth, M. H. AU - Istok, J. D. AU - Conner, G. T. AU - Hyman, M. R. AU - Haggerty, R. AU - O'Reilly, K. T. T2 - Groundwater AB - Abstract An extensive series of single‐well, push‐pull tests was performed to quantify horizontal and vertical spatial variability in aerobic respiration and denitrification rates in a petroleum‐contaminated aquifer. The results indicated rapid consumption of injected O 2 or NO 3 − in shallow and deep test intervals across a large portion of the site. Computed first‐order rate coefficients for aerobic respiration ranged from 0.15 to 1.69 h −1 in the shallow test interval, and from 0.08 to 0.83 h −1 in the deep test interval. The largest aerobic respiration rates occurred on the upgradient edge of the contaminant plume where concentrations of petroleum hydrocarbons and dissolved O 2 were relatively high. Computed first‐order rate coefficients for denitrification ranged from 0.09 to 0.42 h −1 in the shallow test interval, and from 0.11 to 0.28 h −1 in the deep test interval. The largest denitrification rates occurred on the downgradient edge of the plume where hydrocarbon concentrations were relatively high but dissolved oxygen concentrations were small. The rates reported here represent maximal rates of aerobic respiration and denitrification, as supported by high concentrations of electron acceptors in the injected test solutions. Production of dissolved CO 2 during aerobic respiration and denitrification tests provided evidence that O 2 and NO 3 − consumption was largely due to microbial activity. Additional evidence for microbial NO 3 − consumption was provided by reduced rates of NO 3 − consumption when dissolved O 2 was injected with NO 3 − , and by increased N 2 O production when C 2 H 2 was injected with NO 3 − . DA - 1998/// PY - 1998/// DO - 10.1111/j.1745-6584.1998.tb02099.x VL - 36 IS - 6 SP - 924-937 LA - en SN - 1745-6584 UR - https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1745-6584.1998.tb02099.x DB - Wiley Online Library Y2 - 2019/2/1/ ER - TY - CHAP TI - rbcL sequence divergence and phylogenetic relationships of Cornaceae sensu lato AU - Xiang, Q.-Y. AU - Soltis, D.E. T2 - Sino-Japanese Flora - Its Characteristics and Diversification A2 - Boufford, D.E. A2 - Ohba, H. PY - 1998/// SP - 123–139 PB - The University of Tokyo Press ER - TY - JOUR TI - Molecular evidence for origins of polyploid Saxifraga (Saxifragaceae): the narrow arctic endemic S. svalbardensis and its widespread allies AU - Brochmann, C. AU - Xiang, Q.Y. AU - Brunsfeld, S.J. AU - Soltis, D.E. AU - Soltis, P.S. T2 - American Journal of Botany AB - The recently described polyploid Saxifraga svalbardensis is endemic to the arctic archipelago of Svalbard. We investigated relationships among four closely related species of Saxifraga in Svalbard and tested three previously proposed hypotheses for the origin of S. svalbardensis : (1) differentiation from the morphologically and chromosomally variable polyploid S. cernua; (2) hybridization between the diploid S. hyperborea and S. cernua; and (3) hybridization between the tetraploid S. rivularis and S. cernua . Fifteen populations were analyzed using random amplified polymorphic DNAs (RAPDs) and nucleotide sequences of the chloroplast gene matK and the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). RAPD and matK data suggest that S. svalbardensis has originated from a hybrid with S. rivularis as the maternal parent and S. cernua as the paternal parent, possibly a single time, whereas ITS data could not be used to discriminate among the hypotheses. The data also suggest that the diploid S. hyperborea is a progenitor of the tetraploid S. rivularis . The four populations examined of S. svalbardensis were virtually identical for RAPD and ITS markers, whereas S. cernua showed high levels of variation, suggesting that the latter polyploid either has formed recurrently or has undergone considerable differentiation since its origin. DA - 1998/// PY - 1998/// DO - 10.2307/2446562 VL - 85 SP - 135–143 KW - Arctic KW - sequences KW - matK nucleotide sequences KW - polyploid evolution KW - RAPDs KW - Saxifraga cernua KW - Saxifraga svalbardensis KW - Saxifragaceae ER - TY - JOUR TI - The eastern Asian and eastern and western North American floristic disjunction: Congruent phylogenetic patterns in seven diverse genera AU - Xiang, QY AU - Soltis, DE AU - Soltis, PS T2 - MOLECULAR PHYLOGENETICS AND EVOLUTION AB - One of the most remarkable examples of intercontinental disjunction of the North Temperate Flora involves eastern Asia and eastern and western North America. Although there has been considerable interest in this phytogeographic pattern for over 150 years (e.g., Gray, 1859; Li, 1952; Graham, 1972; Boufford and Spongberg, 1983; Wu, 1983; Tiffney, 1985a, 1985b), relationships among taxa displaying the disjunction remain obscure. Understanding phylogenetic relationships is, however, a prerequisite for historical biogeographic analyses of this distributional pattern. To understand better the relationships of taxa displaying this intercontinental disjunction, phylogenetic analyses were conducted using a variety of DNA data sets for species of four genera (Cornus, Boykinia, Tiarella, and Trautvetteria) that occur in eastern Asia, eastern North America, and western North America. An area cladogram was constructed for each of the four genera, all of which show a similar pattern of relationship: the eastern Asian species are sister to all North American species. An identical phylogenetic pattern is also found in three other taxa exhibiting this disjunction (Aralia sect. Aralia, Calycanthus, and Adiantum pedatum). The congruent phylogenetic pattern found in these seven diverse genera raises the possibility of a common origin of the eastern Asia, eastern and western North America disjunction. The data are in agreement with the long-standing hypothesis that this well-known floristic disjunction represents the fragmentation of a once continuous Mixed Mesophytic forest community and suggest that the disjunction may have involved only two major vicariance events: an initial split between Eurasia and North America, followed by the isolation of floras between eastern and western North America. However, congruence between phylogenies and geographic distributions does not necessarily indicate an identical phytogeographic history. Taxa exhibiting the same phylogenetic pattern may have originated at different geological times. Analysis of divergence times using the molecular clock indicates that species of Cornus, Boykinia, and Calycanthus may have diverged at different geological times, suggesting that the floristic disjunction involving eastern Asia and North America may not be simple; it may have involved multiple historical events at very different geological times in different genera. DA - 1998/10// PY - 1998/10// DO - 10.1006/mpev.1998.0524 VL - 10 IS - 2 SP - 178-190 SN - 1095-9513 ER - TY - JOUR TI - Phylogenetic relationships of cornaceae and close relatives inferred from matK and rbcL sequences AU - Xiang, QY AU - Soltis, DE AU - Soltis, PS T2 - AMERICAN JOURNAL OF BOTANY AB - Phylogenetic relationships were inferred using nucleotide sequences of the chloroplast gene matK for members of Cornales, a well‐supported monophyletic group comprising Cornaceae and close relatives. The shortest trees resulting from this analysis were highly concordant with those based on previous phylogenetic analysis of rbcL sequences. Analysis of a combined matK and rbcL sequence data set (a total of 2652 bp [base pairs]) provided greater resolution of relationships and higher internal support for clades compared to the individual data sets. Four major clades (most inclusive monophyletic groups) of Cornales are indicated by both sets of genes: (1) Cornus‐Alangium, (2) nyssoids ( Nyssa‐Davidia‐Camptotheca )‐mastixioids ( Mastixia, Diplopanax ), (3) Curtisia, and (4) Hydrangeaceae‐Loasaceae. The combined evidence indicates that clades 2 and 3 are sisters, with clade 4 sister to the remainder of Cornales. These relationships are also supported by other lines of evidence, including synapomorphies in fruit and pollen morphology and gynoecial vasculature. Comparisons of matK and rbcL sequences based on one of the most parsimonious rbcL‐matK trees indicate that matK has a much higher A‐T content (66.9% in matK vs. 55.8% in rbcL ) and a lower transition:transversion ratio (1.23 in matK vs. 2.21 in rbcL ). The total number of nucleotide substitutions per site for matK is 2.1 times that of rbcL in Cornales. These findings are similar to recent comparisons of matK and rbcL in other dicots. Variable sites of matK are almost evenly distributed among the three codon positions (1.0:1.0:1.3), whereas variable sites of rbcL are mostly at the third position (1.8:1.0:7.5). Among‐lineages rates of nucleotide substitutions in rbcL are basically homogeneous throughout Cornales, but are more heterogeneous in matK . DA - 1998/2// PY - 1998/2// DO - 10.2307/2446317 VL - 85 IS - 2 SP - 285-297 SN - 1537-2197 KW - Cornales KW - matK KW - molecular evolution KW - molecular phylogeny KW - rbcL ER - TY - JOUR TI - Origin and biogeography of Aesculus L. (Hippocastanaceae): a molecular phylogenetic perspective AU - Xiang, Qiu-Yun AU - Crawford, D. J. AU - Wolfe, A. D. AU - Tang, Y.-C. AU - DePamphilis, C. W. T2 - Evolution AB - Sequences of chloroplast gene matK and internal transcribed spacers of nuclear ribosomal RNA genes were used for phylogenetic analyses of Aesculus, a genus currently distributed in eastern Asia, eastern and western North America, and southeastern Europe. Phylogenetic relationships inferred from these molecular data are highly correlated with the geographic distributions of species. The identified lineages closely correspond to the five sections previously recognized on the basis of morphology. Ancestral character-state reconstruction, a molecular clock, and fossil evidence were used to infer the origin and biogeographic history of the genus within a phylogenetic framework. Based on the molecular phylogenetic reconstruction of the genus, sequence divergence, and paleontological evidence, we infer that the genus originated during the transition from the Cretaceous to the Tertiary (~65 M.Y.B.P.) at a high latitude in eastern Asia and spread into North America and Europe as an element of the "boreotropical flora"; the current disjunct distribution of the genus resulted from geological and climatic changes during the Tertiary. DA - 1998/// PY - 1998/// DO - 10.1111/j.1558-5646.1998.tb01828.x VL - 52 IS - 1998 SP - 988–997 ER - TY - JOUR TI - Assessing hybridization in natural populations of Penstemon (Scrophulariaceae) using hypervariable intersimple sequence repeat (ISSR) bands AU - Wolfe, AD AU - Xiang, QY AU - Kephart, , SR T2 - MOLECULAR ECOLOGY AB - Inferences regarding hybridization rely on genetic markers to differentiate parental taxa from one another. Intersimple sequence repeat (ISSR) markers are based on single‐primer PCR reactions where the primer sequence is derived from di‐ and trinucleotide repeats. These markers have successfully been used to assay genetic variability among cultivated plants, but have not yet been tested in natural populations. We used genetic markers generated from eight ISSR primers to examine patterns of hybridization and purported examples of hybrid speciation in Penstemon (Scrophulariaceae) in a hybrid complex involving P. centranthifolius , P. grinnellii , P. spectabilis and P. clevelandii . This hybrid complex has previously been studied using three molecular data sets (allozymes, and restriction‐site variation of nuclear rDNA and chloroplast DNA). These studies revealed patterns of introgression involving P. centranthifolius , but were unsuccessful in determining whether gene flow occurs among the other species, and support for hypotheses of diploid hybrid speciation was also lacking. In this study, we were able to fingerprint each DNA accession sampled with one to three ISSR primers and most accessions could be identified with a single primer. We found population‐ and species‐specific markers for each taxon surveyed. Our results: (i) do not support the hybrid origin of P. spectabilis ; (ii) do support the hypothesis that P. clevelandii is a diploid hybrid species derived from P. centranthifolius and P. spectabilis ; and (iii) demonstrate that pollen‐mediated gene flow via hummingbird vectors is prevalent in the hybrid complex. DA - 1998/9// PY - 1998/9// DO - 10.1046/j.1365-294x.1998.00425.x VL - 7 IS - 9 SP - 1107-1125 SN - 1365-294X KW - diploid hybrid speciation KW - genetic marker KW - introgression KW - ISSR KW - Penstemon KW - pollen-mediated gene flow ER - TY - CHAP TI - The dependence of Fed-1 light regulation on translation AU - Petracek, M. E. AU - Dickey, L. F. AU - Hansen, E. R. AU - Sowinski, D. A. AU - Nguyen, T. AU - Allen, G. C. AU - Thompson, G. F. T2 - A look beyond transcription: Mechanisms determining mRNAstability and translation in plants A2 - J. Bailey-Serres, A2 - Gallie, D. R. CN - QK981.4 .L66 1998 PY - 1998/// SP - 96-101 PB - Washington, DC: American Society of Plant Physiologists ER - TY - JOUR TI - Active oxygen in fungal pathogenesis of plants: the role of cercosporin in Cercospora diseases AU - Daub, M. E. AU - Ehrenshaft, M. AU - Jenns, A. E. AU - Chung, K. R. T2 - Recent Advances in Phytochemistry DA - 1998/// PY - 1998/// VL - 32 IS - 1998 SP - 31-56 ER - TY - CHAP TI - Inositol lipid signaling: what can we learn from plants? AU - Heilmann, I. AU - Perera, I. Y. AU - Stevenson, J. M. AU - Ransom, W. D. AU - Gross, W. AU - Boss, W. F. T2 - Advances in lipids research A2 - J. Sanchez, E. Cerda-Olmedo A2 - Martinez-Force, E. PY - 1998/// SP - 394-397 PB - Sevilla, Spain: University of Sevilla Press ER - TY - JOUR TI - Characterization of montane cedar-hardwood woodlands in the Piedmont and Blue Ridge provinces of North Carolina AU - Small, C. J. AU - Wentworth, T. R. T2 - Castanea DA - 1998/// PY - 1998/// VL - 63 IS - 3 SP - 241-261 ER - TY - JOUR TI - Growth dynamics and cytoskeleton organization during stem maturation and gravity-induced stem bending in Zea mays L. AU - Collings, DA AU - Winter, H AU - Wyatt, SE AU - Allen, NS T2 - PLANTA DA - 1998/12// PY - 1998/12// DO - 10.1007/s004250050480 VL - 207 IS - 2 SP - 246-258 SN - 1432-2048 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0032435367&partnerID=MN8TOARS KW - actin KW - cell elongation KW - gravitropism KW - microtubule KW - pulvinus KW - Zea (gravitropism) ER - TY - JOUR TI - A mutation in a purported regulatory gene affects control of sterol uptake in Saccharomyces cerevisiae AU - Crowley, J. H. AU - Leak, F. W. AU - Shianna, K. V. AU - Tove, S. AU - Parks, L. W. T2 - Journal of Bacteriology DA - 1998/// PY - 1998/// VL - 180 IS - 16 SP - 4177-4183 ER - TY - JOUR TI - Relative toxicity of the maize endosperm ribosome-inactivating protein to insects AU - Dowd, PF AU - Mehta, AD AU - Boston, RS T2 - JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY AB - The relative toxicity of proenzyme and protease-activated forms of maize seed ribosome-inactivating protein (b-32) to several insect species was determined. Only the protease-activated form had significant toxicity to any caterpillars when fed in diets at 1 mg/g of diet. Activity ranged from 70% mortality to cabbage looper (Trichoplusia ni) to no effect to Indian meal moth (Plodia interpunctella). Neither form of the protein showed activity against larvae of the Freeman sap beetle, (Carpophilus freemani). However, the proenzyme and protease-activated forms were approximately equally deterrent in choice assays to other sap beetles and maize weevils (Sitophilus zeamais), with relative feeding rates reduced by up to 6-fold. Because this protein can naturally occur at the 1 mg/g endosperm range in the endosperm of Opaque-2 (normal) plants versus 2 orders of magnitude lower in opaque-2 mutants, it is likely that this RIP plays a natural defensive role against insects. However, some insects appear to have adapted to this protein. Keywords: Host plant resistance; corn; Zea; Carpophilus; Helicoverpa; Spodoptera; Ostrinia; Sitophilus; Plodia; b-32 DA - 1998/9// PY - 1998/9// DO - 10.1021/jf980334w VL - 46 IS - 9 SP - 3775-3779 SN - 0021-8561 KW - host plant resistance KW - corn KW - Zea KW - Carpophilus KW - Helicoverpa KW - Spodoptera KW - Ostrinia KW - Sitophilus KW - Plodia KW - b-32 ER - TY - PAT TI - Plant nuclear scaffold attachment region and method for increasing gene expression in transgenic cells C2 - 1998/// DA - 1998/// PY - 1998/// ER - TY - JOUR TI - Phosphoglycerylethanolamine posttranslational modification of plant eukaryotic elongation factor 1 alpha AU - Ransom, WD AU - Lao, PC AU - Gage, DA AU - Boss, WF T2 - PLANT PHYSIOLOGY AB - Abstract Eukaryotic elongation factor 1α (eEF-1A) is a multifunctional protein. There are three known posttranslational modifications of eEF-1A that could potentially affect its function. Except for phosphorylation, the other posttranslational modifications have not been demonstrated in plants. Using matrix-assisted laser desorption/ionization-mass spectrometry and peptide mass mapping, we show that carrot (Daucus carotaL.) eEF-1A contains a phosphoglycerylethanolamine (PGE) posttranslational modification. eEF-1A was the only protein labeled with [14C]ethanolamine in carrot cells and was the predominant ethanolamine-labeled protein in Arabidopsis seedlings and tobacco (Nicotiana tabacum L.) cell cultures. In vivo-labeling studies using [3H]glycerol, [32P]Pi, [14C]myristic acid, and [14C]linoleic acid indicated that the entire phospholipid phosphatidylethanolamine is covalently attached to the protein. The PGE lipid modification did not affect the partitioning of eEF-1A in Triton X-114 or its actin-binding activity in in vitro assays. Our in vitro data indicate that this newly characterized posttranslational modification alone does not affect the function of eEF-1A. Therefore, the PGE lipid modification may work in combination with other posttranslational modifications to affect the distribution and the function of eEF-1A within the cell. DA - 1998/7// PY - 1998/7// DO - 10.1104/pp.117.3.949 VL - 117 IS - 3 SP - 949-960 SN - 0032-0889 ER - TY - PAT TI - Method of increasing expression for foreign genes in plant cells C2 - 1998/// DA - 1998/// PY - 1998/// ER - TY - JOUR TI - Ferredoxin-1 mRNA is destabilized by changes in photosynthetic electron transport AU - Petracek, ME AU - Dickey, LF AU - Nguyen, TT AU - Gatz, C AU - Sowinski, DA AU - Allen, GC AU - Thompson, WF T2 - PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA AB - In transgenic tobacco, pea Ferredoxin-1 ( Fed-1 ) mRNA accumulates rapidly in response to photosynthesis even when the transgene is driven by a constitutive promoter. To investigate the role of photosynthesis on Fed-1 mRNA stability, we used the tetracycline repressible Top10 promoter system to specifically shut off transcription of the Fed-1 transgene. The Fed-1 mRNA has a half-life of approximately 2.4 hr in the light and a half-life of only 1.2 hr in the dark or in the presence of the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). These data indicate that cessation of photosynthesis, either by darkness or DCMU results in a destabilization of the Fed-1 mRNA. Furthermore, the Fed-1 mRNA half-life is reduced immediately upon transfer to darkness, suggesting that Fed-1 mRNA destabilization is a primary response to photosynthesis rather than a secondary response to long-term dark adaptation. Finally, the two different methods for efficient tetracycline delivery reported here generally should be useful for half-life measurements of other mRNAs in whole plants. DA - 1998/7/21/ PY - 1998/7/21/ DO - 10.1073/pnas.95.15.9009 VL - 95 IS - 15 SP - 9009-9013 SN - 0027-8424 ER - TY - JOUR TI - A calcium-dependent ergosterol mutant of Saccharomyces cerevisiae AU - Crowley, JH AU - Tove, S AU - Parks, LW T2 - CURRENT GENETICS DA - 1998/8// PY - 1998/8// DO - 10.1007/s002940050371 VL - 34 IS - 2 SP - 93-99 SN - 1432-0983 KW - ergosterol KW - antifungal KW - calcium KW - fenpropimorph KW - azole KW - ERG24 gene ER - TY - JOUR TI - Tomato spotted wilt virus resistance in chrysanthemum expressing the viral nucleocapsid gene AU - Sherman, JM AU - Moyer, JW AU - Daub, ME T2 - PLANT DISEASE AB - Three tomato spotted wilt virus (TSWV) nucleocapsid (N) gene constructs were employed for Agrobacterium-mediated transformation of chrysanthemum (Dendranthema grandiflora) cv. Polaris. These constructs contained either a full-length N gene (pTSWVN+), a full-length N gene encoding a truncated N protein (pTSWVNt), or an antisense version of the full-length N gene (pTSWVN-), all derived from a dahlia isolate of TSWV (TSWV-D). Initial resistance screens were conducted on cuttings made from 152 pTSWVN+, 37 pTSWVNt, and 47 pTSWVN- transformed plants employing a highly virulent, heterologous strain of TSWV (TSWV-GB) isolated from chrysanthemum and vectored by thrips. This screening served to eliminate the majority of TSWV-susceptible transgenic lines. More rigorous resistance tests with three rounds of mechanical inoculation with TSWV-GB identified one pTSWVNt and two pTSWVN- transformed lines that exhibited a total lack of systemic symptoms and no virus accumulation. Six other lines, including some pTSWVN+, exhibited a lack of one or more of the destructive necrotic TSWV symptoms (stem canker and apical bud death) and a delay in symptom expression. Both sense and antisense constructs, therefore, were found to be effective at yielding TSWV resistance in chrysanthemum. Molecular analysis revealed that the highly TSWV-resistant pTSWVNt line had no detectable levels of N protein. All three resistant lines had low levels of N gene transcript and at least three transgene insertion sites within their genomes, although susceptible lines often had a similar number of insertion sites. The generation of Polaris lines resistant to TSWV transmitted either mechanically or by thrips represents the first time a major ornamental crop has been genetically engineered for disease resistance. DA - 1998/4// PY - 1998/4// DO - 10.1094/PDIS.1998.82.4.407 VL - 82 IS - 4 SP - 407-414 SN - 0191-2917 KW - floral crop biotechnology ER - TY - CONF TI - Polarization modulation DIC microscopy: an improvement for video microscopy AU - Allen, N. S. AU - Moxley, D. AU - Collings, D. AU - Holzwarth, G. C2 - 1998/// C3 - Proceedings of the Electron Microscopy Society of America, 1998 CN - QH212.E4 E4 DA - 1998/// SP - 130-131 ER - TY - JOUR TI - Light regulation of Fed-1 mRNA requires an element in the 5' untranslated region and correlates with differential polyribosome association AU - Dickey, L. F. AU - Petracek, M. E. AU - Nguyen, T. T. AU - Hansen, E. R. AU - Thompson, William T2 - Plant Cell DA - 1998/// PY - 1998/// DO - 10.2307/3870603 VL - 10 IS - 3 SP - 475–484 ER - TY - JOUR TI - Effects of microbial utilization of phenolic acids and their phenolic acid breakdown products on allelopathic interactions AU - Blum, U T2 - JOURNAL OF CHEMICAL ECOLOGY DA - 1998/4// PY - 1998/4// DO - 10.1023/A:1022394203540 VL - 24 IS - 4 SP - 685-708 SN - 1573-1561 KW - allelopathy KW - phenolic acids KW - sorption KW - microbial utilization KW - soil extractions KW - ferulic acid KW - p-coumaric acid KW - p-hydroxybenzoic acid KW - vanillic acid ER - TY - PAT TI - DNA encoding Bacillus lichenformis PWD-1 keratinase AU - Shih, J. C. H. AU - Lin, X. AU - Miller, E. S. C2 - 1998/// DA - 1998/// PY - 1998/// ER - TY - JOUR TI - Conserved sequence and structural motifs contribute to the DNA binding and cleavage activities of a geminivirus replication protein AU - Orozco, BM AU - Hanley-Bowdoin, L T2 - JOURNAL OF BIOLOGICAL CHEMISTRY AB - Tomato golden mosaic virus (TGMV), a member of the geminivirus family, has a single-stranded DNA genome that replicates through a rolling circle mechanism in nuclei of infected plant cells. TGMV encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its host. AL1 is a multifunctional protein that binds double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and forms oligomers. Earlier experiments showed that the region of TGMV AL1 necessary for DNA binding maps to the N-terminal 181 amino acids of the protein and overlaps the DNA cleavage (amino acids 1–120) and oligomerization (amino acids 134–181) domains. In this study, we generated a series of site-directed mutations in conserved sequence and structural motifs in the overlapping DNA binding and cleavage domains and analyzed their impact on AL1 function in vivo and in vitro. Only two of the fifteen mutant proteins were capable of supporting viral DNA synthesis in tobacco protoplasts. In vitroexperiments demonstrated that a pair of predicted α-helices with highly conserved charged residues are essential for DNA binding and cleavage. Three sequence motifs conserved among geminivirus AL1 proteins and initiator proteins from other rolling circle systems are also required for both activities. We used truncated AL1 proteins fused to a heterologous dimerization domain to show that the DNA binding domain is located between amino acids 1 and 130 and that binding is dependent on protein dimerization. In contrast, AL1 monomers were sufficient for DNA cleavage and ligation. Together, these results established that the conserved motifs in the AL1 N terminus contribute to DNA binding and cleavage with both activities displaying nearly identical amino acid requirements. However, DNA binding was readily distinguished from cleavage and ligation by its dependence on AL1/AL1 interactions. Tomato golden mosaic virus (TGMV), a member of the geminivirus family, has a single-stranded DNA genome that replicates through a rolling circle mechanism in nuclei of infected plant cells. TGMV encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its host. AL1 is a multifunctional protein that binds double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and forms oligomers. Earlier experiments showed that the region of TGMV AL1 necessary for DNA binding maps to the N-terminal 181 amino acids of the protein and overlaps the DNA cleavage (amino acids 1–120) and oligomerization (amino acids 134–181) domains. In this study, we generated a series of site-directed mutations in conserved sequence and structural motifs in the overlapping DNA binding and cleavage domains and analyzed their impact on AL1 function in vivo and in vitro. Only two of the fifteen mutant proteins were capable of supporting viral DNA synthesis in tobacco protoplasts. In vitroexperiments demonstrated that a pair of predicted α-helices with highly conserved charged residues are essential for DNA binding and cleavage. Three sequence motifs conserved among geminivirus AL1 proteins and initiator proteins from other rolling circle systems are also required for both activities. We used truncated AL1 proteins fused to a heterologous dimerization domain to show that the DNA binding domain is located between amino acids 1 and 130 and that binding is dependent on protein dimerization. In contrast, AL1 monomers were sufficient for DNA cleavage and ligation. Together, these results established that the conserved motifs in the AL1 N terminus contribute to DNA binding and cleavage with both activities displaying nearly identical amino acid requirements. However, DNA binding was readily distinguished from cleavage and ligation by its dependence on AL1/AL1 interactions. tomato golden mosaic virus intergenic region rolling circle replication bean golden mosaic virus tomato yellow leaf curl virus glutathione S-transferase nucleotide(s). Tomato golden mosaic virus (TGMV)1 is a member of the geminivirus family of plant-infecting viruses characterized by twin icosahedral particles and small, single-stranded DNA genomes (reviewed in Refs. 1Timmermans M.C.P. Das O.P. Messing J. Annu. Rev. Plant Physiol. 1994; 45: 79-112Crossref Scopus (103) Google Scholar and 2Hanley-Bowdoin, L., Settlage, S. B., Orozco, B. M., Nagar, S., and Robertson, D. (1998) Crit. Rev. Plant Sci., in pressGoogle Scholar). The single-stranded DNA is converted to a double-stranded form in the nucleus of infected cells and then serves as a template for rolling circle replication (RCR; Refs. 3Saunders K. Lucy A. Stanley J. Nucleic Acids Res. 1991; 19: 2325-2330Crossref PubMed Scopus (167) Google Scholar, 4Stenger D.C. Revington G.N. Stevenson M.C. Bisaro D.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8029-8033Crossref PubMed Scopus (289) Google Scholar, 5Heyraud F. Matzeit V. Kammann M. Schaefer S. Schell J. Gronenborn B. EMBO J. 1993; 12: 4445-4452Crossref PubMed Scopus (90) Google Scholar) and viral gene transcription (6Sunter G. Gardiner W.E. Bisaro D.M. Virology. 1989; 170: 243-250Crossref PubMed Scopus (53) Google Scholar, 7Sunter G. Bisaro D.M. Virology. 1989; 173: 647-655Crossref PubMed Scopus (47) Google Scholar). Geminiviruses encode only a few proteins for these processes and depend on host DNA and RNA polymerases as well as their accessory factors. These characteristics make geminiviruses excellent model systems for studying plant DNA replication and transcription mechanisms. The TGMV genome consists of two circular DNA molecules, designated as A and B (8Hamilton W.D.O. Bisaro D.M. Coutts R.H.A. Buck K.W. Nucleic Acids Res. 1983; 11: 7387-7396Crossref PubMed Scopus (92) Google Scholar). Both components have a conserved 5′ intergenic region (IR) that separates divergent open reading frames (9Bisaro D.M. Hamilton W.D.O. Coutts R.H.A. Buck K.W. Nucleic Acids Res. 1982; 10: 4913-4922Crossref PubMed Scopus (52) Google Scholar). The IR includes the plus-strand origin of replication (10Orozco B.M. Gladfelter H.J. Settlage S.B. Eagle P.A. Gentry R. Hanley-Bowdoin L. Virology. 1998; 242: 346-356Crossref PubMed Scopus (68) Google Scholar) and the promoters for leftward and rightward transcription (6Sunter G. Gardiner W.E. Bisaro D.M. Virology. 1989; 170: 243-250Crossref PubMed Scopus (53) Google Scholar, 7Sunter G. Bisaro D.M. Virology. 1989; 173: 647-655Crossref PubMed Scopus (47) Google Scholar). A directly repeated sequence in the TGMV IR is required for recognition of the plus-strand origin and negative regulation of the overlapping promoter for leftward transcription (11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar, 12Eagle P.A. Orozco B.M. Hanley-Bowdoin L. Plant Cell. 1994; 6: 1157-1170PubMed Google Scholar). Related motifs are found in the genomes of most dicot-infecting geminiviruses (13Arguello-Astorga G.R. Guevara-Gonzalez R.G. Herrera-Estrella L.R. Rivera-Bustamante R.F. Virology. 1994; 203: 90-100Crossref PubMed Scopus (278) Google Scholar), and their roles in virus-specific replication have been confirmed for bean golden mosaic virus (BGMV) and beet curly top virus (14Fontes E.P.B. Gladfelter H.J. Schaffer R.L. Petty I.T.D. Hanley-Bowdoin L. Plant Cell. 1994; 6: 405-416Crossref PubMed Scopus (171) Google Scholar, 15Choi I.R. Stenger D.C. Virology. 1995; 206: 904-912Crossref PubMed Scopus (64) Google Scholar, 16Gladfelter H.J. Eagle P.A. Fontes E.P.B. Batts L.A. Hanley-Bowdoin L. Virology. 1997; 239: 186-197Crossref PubMed Scopus (47) Google Scholar). The IR also contains a hairpin with a 9-base pair loop sequence conserved among all geminiviruses that is cleaved during initiation and termination of RCR (5Heyraud F. Matzeit V. Kammann M. Schaefer S. Schell J. Gronenborn B. EMBO J. 1993; 12: 4445-4452Crossref PubMed Scopus (90) Google Scholar, 17Laufs J. Traut W. Heyraud F. Matzeit V. Rogers S.G. Schell J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3879-3883Crossref PubMed Scopus (260) Google Scholar, 18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). Genetic experiments established that the hairpin structure is essential for TGMV replication (18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). TGMV encodes two proteins, AL1 and AL3, that are required for efficient viral replication. AL1 is necessary for replication, whereas AL3 enhances viral DNA accumulation by an unknown mechanism (19Elmer J.S. Brand L. Sunter G. Gardiner W.E. Bisaro D.M. Rogers S.G. Nucleic Acids Res. 1988; 16: 7043-7060Crossref PubMed Scopus (212) Google Scholar, 20Sunter G. Hartitz M.D. Hormuzdi S.G. Brough C.L. Bisaro D.M. Virology. 1990; 179: 69-77Crossref PubMed Scopus (167) Google Scholar). AL1 is a multifunctional protein that confers virus-specific recognition to its cognate plus-strand origin (15Choi I.R. Stenger D.C. Virology. 1995; 206: 904-912Crossref PubMed Scopus (64) Google Scholar, 16Gladfelter H.J. Eagle P.A. Fontes E.P.B. Batts L.A. Hanley-Bowdoin L. Virology. 1997; 239: 186-197Crossref PubMed Scopus (47) Google Scholar, 21Jupin I. Hericourt F. Benz B. Gronenborn B. FEBS Lett. 1995; 362: 116-120Crossref PubMed Scopus (74) Google Scholar) and initiates RCR (17Laufs J. Traut W. Heyraud F. Matzeit V. Rogers S.G. Schell J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3879-3883Crossref PubMed Scopus (260) Google Scholar, 18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar,22Heyraud-Nitschke F. Schumacher S. Laufs J. Schaefer S. Schell J. Gronenborn B. Nucleic Acids Res. 1995; 23: 910-916Crossref PubMed Scopus (143) Google Scholar). It also actively represses its own transcription in a virus-specific manner (12Eagle P.A. Orozco B.M. Hanley-Bowdoin L. Plant Cell. 1994; 6: 1157-1170PubMed Google Scholar, 16Gladfelter H.J. Eagle P.A. Fontes E.P.B. Batts L.A. Hanley-Bowdoin L. Virology. 1997; 239: 186-197Crossref PubMed Scopus (47) Google Scholar, 23Sunter G. Hartitz M.D. Bisaro D.M. Virology. 1993; 195: 275-280Crossref PubMed Scopus (100) Google Scholar, 24Eagle P.A. Hanley-Bowdoin L. J. Virol. 1997; 71: 6947-6955Crossref PubMed Google Scholar) and induces the expression of a host DNA synthesis protein, proliferating cell nuclear antigen, in nondividing plant cells (25Nagar S. Pedersen T.J. Carrick K. Hanley-Bowdoin L. Robertson D. Plant Cell. 1995; 7: 705-719Crossref PubMed Scopus (156) Google Scholar). Several biochemical activities have been described for AL1 in vitro. TGMV AL1 binds to double-stranded DNA at the conserved repeated motif in the plus-strand origin (11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar, 26Fontes E.P.B. Luckow V.A. Hanley-Bowdoin L. Plant Cell. 1992; 4: 597-608Crossref PubMed Scopus (136) Google Scholar) and cleaves single-stranded DNA in the invariant sequence of the hairpin loop (18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). Analysis of the C1 protein of tomato yellow leaf curl virus (TYLCV), a TGMV AL1 homologue, revealed that it covalently attaches to the 5′ end of the cleaved DNA by a phosphotyrosyl bond and catalyzes ligation of the cleavage products (27Laufs J. Schumacher S. Geisler N. Jupin I. Gronenborn B. FEBS Lett. 1995; 377: 258-262Crossref PubMed Scopus (67) Google Scholar). ATPase activity has also been demonstrated for the AL1/C1 proteins from TYLCV and TGMV (28Desbiez C. David C. Mettouchi A. Laufs J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5640-5644Crossref PubMed Scopus (106) Google Scholar, 29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). TGMV AL1 is involved in several protein interactions. It forms large multimeric complexes (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), binds AL3 (30Settlage S.B. Miller B. Hanley-Bowdoin L. J. Virol. 1996; 70: 6790-6795Crossref PubMed Google Scholar), and interacts with a maize homologue of the animal cell cycle regulatory protein, retinoblastoma (31Ach R.A. Durfee T. Miller A.B. Taranto P. Hanley-Bowdoin L. Zambriski P.C. Gruissem W. Mol. Cell. Biol. 1997; 17: 5077-5086Crossref PubMed Scopus (202) Google Scholar). The C1 protein from wheat dwarf virus also interacts with retinoblastoma proteins from human and maize (32Xie Q. Suarezlopez P. Gutierrez C. EMBO J. 1995; 14: 4073-4082Crossref PubMed Scopus (156) Google Scholar, 33Collin S. Fernandez-Lobato M. Gooding P.S. Mullineaux P.M. Fenoll C. Virology. 1996; 219: 324-329Crossref PubMed Scopus (66) Google Scholar, 34Xie Q. Sanzburgos P. Hannon G.J. Gutierrez C. EMBO J. 1996; 15: 4900-4908Crossref PubMed Scopus (195) Google Scholar). In an earlier study, we used truncated proteins produced in a baculovirus expression system to map the regions of TGMV AL1 that are responsible for DNA cleavage, DNA binding and oligomerization (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). These experiments showed that the DNA cleavage domain is located in the first 120 amino acids of AL1 whereas the oligomerization domain maps to the protein center between amino acids 121 and 181. DNA binding requires a larger region that fully overlaps the DNA cleavage and oligomerization domains. Based on these experiments, we proposed that additional amino acids between position 121 and 181 are necessary for AL1/DNA binding and/or that AL1 complex formation is required for DNA binding. In this study, we identified key sequence and structural motifs in the DNA binding and cleavage domains. A series of site-directed mutations in the AL1 N terminus were analyzed for their impact on function in vivo and in vitro. These studies focused on three amino acid motifs, which are conserved among all geminivirus AL1/C1 proteins and many initiator proteins from other RCR systems (35Koonin E.V. Ilyina T.V. J. Gen. Virol. 1992; 73: 2763-2766Crossref PubMed Scopus (132) Google Scholar, 36Ilyina T.V. Koonin E.V. Nucleic Acids Res. 1992; 20: 3279-3285Crossref PubMed Scopus (508) Google Scholar), and on a predicted helix-loop-helix motif in the N termini of AL1/C1 proteins of dicot-infecting geminiviruses (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). We also used a heterologous protein interaction domain to assess the importance of oligomerization for AL1/DNA binding and coupled DNA cleavage/ligation. The plasmid pNSB148, which contains the AL1 coding sequence in a pUC118 background, was used as the template for site-directed mutagenesis (37Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 482-492Google Scholar). The oligonucleotide primers and resulting clones are listed in Table I. The sequences of fragments containing the mutations and used for subsequent cloning were verified by DNA sequence analysis. Plant expression cassettes with the mutant AL1 coding sequences were generated by subcloning NdeI/SalI fragments (AL1 amino acids 1–120) from the mutant clones into the same sites in a wild type AL1 plant expression cassette pMON1549 (11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). In pMON1549, AL1 expression is under the control of the cauliflower mosaic virus 35 S promoter with a duplicated enhancer (38Kay R. Chan A. Daly M. McPherson J. Science. 1987; 236: 1299-1302Crossref PubMed Scopus (736) Google Scholar) and the 3′ end from the pea E9 rbcS gene (39Coruzzi G. Broglie R. Edwards C. Chua N.-H. EMBO J. 1984; 3: 1671-1679Crossref PubMed Scopus (208) Google Scholar). Baculovirus expression vectors coding for AL1 proteins fused to a glutathione S-transferase tag (GST-AL1) were generated by digesting the mutant plant expression cassettes with NdeI and BamHI and repairing the ends with Escherichia coli DNA polymerase I (Klenow). The fragments, which included complete AL1 coding regions, were inserted into the SmaI site of pNSB314. The pNSB314 vector contains the GST coding sequence, followed by a glycine linker, a thrombin cleavage site, and a multiple cloning site for generating in-frame fusion proteins (18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). The mutant GST-AL1 expression cassettes are listed in Table I.Table IAL1 mutationsMotifMutationOligonucleotideBaculovirus vectorPlant expressionHelix 1H1–1GAGAAAGTGATgCggCcgcGGACAAGGAGCACpNSB627pNSB655H1–2GTAATTGAGAAAGTacTTCTTCTTTGGACpNSB434Helix 2H2–1CTTCATGAAGCgCTgcaCAGATTgcTATGAATTTTTTGTTAATCGGpNSB628pNSB656H2–2CTCTGCAGATcTTTccGAATTTTTTGTTAATCGGpNSB629pNSB657H2–3CATCTTCATGtcGCTCTtcGCAGAcTTTTATGAATTTTTTGTTAATCGGpNSB630pNSB658Motif 1M1–1GCACTGAGGAgcgGccgcAgcATAATTTTTGGCATpNSB649pNSB652M1–2CACCTCGAGGATAcGTAAGAgcATAATTTTTGGCATTpNSB685pNSB681M1–3GACATGAGGAgcgGTAAGAAAATAATTTTTGGGGCATTpNSB686pNSB682Motif 2M2–1GAATAAGCACGgcggccgcAGGTTGCCCATCpNSB650pNSB653Motif 3M3–1GAGTATCTCCGgCTTTaTCGATGgcCGTCTTGACGTCGpNSB651pNSB654M3–2CTTTGTCGATcgcCGTCTTGACGTCGpNSB687pNSB683M3–3GAGTATCTCCGgCTTTaTCGATGTACGpNSB688pNSB684M3–4TACAAGAGTAgCTCCGgCTTTcgCGATGTACGTCTTGACpNSB741pNSB747M3–5CTCCGTCTTTaTCGATGaACGTCTTGACpNSB781pNSB779M3–6GAGTATCTCCGTCTgcaTCGATGTACGpNSB782pNSB780 Open table in a new tab Wild type and mutant GST-AL1 fusion proteins were expressed in Spodoptera frugiperda (Sf9) cells using a Tn7-based baculovirus expression system (40Luckow V.A. Lee S.C. Barry G.F. Olins P.O. J. Virol. 1993; 67: 4566-4579Crossref PubMed Google Scholar) and purified from Sf9 cell cultures by glutathione affinity chromatography according to published protocols (18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). Purified proteins were visualized by electrophoresis in 16% polyacrylamide-SDS gels and staining with Coomassie Brilliant Blue dye. Interactions between authentic AL1 and mutant GST-AL1 proteins were assayed by copurification on glutathione-Sepharose, followed by immunoblot analysis using AL1 polyclonal antisera (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 30Settlage S.B. Miller B. Hanley-Bowdoin L. J. Virol. 1996; 70: 6790-6795Crossref PubMed Google Scholar). DNA gel shift assays were performed as described previously (18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). An 83-base pair EcoRI fragment containing the AL1 DNA binding motif (TGMV A positions 28–84) was isolated from pNSB378 and 3′ end-labeled using Klenow and [α-32P]dATP. The radiolabeled DNA was incubated with purified GST-AL1 fusion proteins for 1 h at room temperature at the concentrations indicated in the figure legends. The bound and free probe were resolved on 1% agarose gels, dried onto DE-81 paper, and analyzed by autoradiography. For DNA cleavage and ligation, oligonucleotides corresponding to sequences in the hairpin of the TGMV (+)-strand origin were 5′ end-labeled using polynucleotide kinase and [γ-32P]ATP. The oligonucleotides CR13 (5′-GTTTAATATTACCGGATGGCCGC) and CR33 (5′-GCGGCCATCCGTTTAATATT) were used for assays with full-length mutant and wild type AL1 proteins. For assays with truncated AL1 proteins, only one oligonucleotide, CR13 or CR34 (5′-GCGGCCATCCGTTTAATATTACCGGATGG) was radiolabeled and the cold oligonucleotide was titrated into the reactions as described in the figure legend. Approximately 5000 cpm of each labeled DNA was incubated with ∼100 ng of purified GST-AL1 fusion protein in 10 μl of cleavage buffer (25 mm Tris-HCl, pH 7.5, 75 mmNaCl, 5 mm MgCl2, 2.5 mm EDTA, 2.5 mm dithiothreitol, and 250 ng of poly(dI-dC)) at 37 °C for 30 min. The reactions were terminated by adding 6 μl of gel loading buffer (95% formamide, 20 mm EDTA, 0.05% bromphenol blue) and heating to 90 °C for 2 min. The reaction products were resolved on 15% polyacrylamide denaturing gels and analyzed by autoradiography. Protoplasts were isolated fromNicotiana tabacum NT-1 suspension cells, electroporated, and cultured according to published methods (11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). The transfections contained 15 μg each of replicon DNA containing a partial tandem copy of TGMV B (pTG1.4B described in Ref. 14Fontes E.P.B. Gladfelter H.J. Schaffer R.L. Petty I.T.D. Hanley-Bowdoin L. Plant Cell. 1994; 6: 405-416Crossref PubMed Scopus (171) Google Scholar), wild type or mutant AL1 expression cassette, and an AL3 plant expression cassette (pNSB46 described in Ref. 11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). Total DNA was extracted 3 days after transfection and analyzed for double-stranded viral DNA accumulation by DNA gel blot hybridization (11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). Two key steps in initiation of RCR are origin recognition and generation of a free 3′-OH for priming plus-strand DNA synthesis. TGMV AL1 mediates both of these processes by binding double-stranded DNA in a sequence-specific manner and by cleaving at a unique site in the origin. Earlier studies established that the AL1 N terminus is necessary for both activities (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). This region of AL1 contains several conserved sequence and structural motifs (Fig. 1), including a highly predicted pair of α-helices between amino acids 25 and 52 (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The sequences of both helices are strongly conserved among dicot-infecting geminiviruses, and the second helix is amphipathic in character. This conservation suggested that the predicted α-helices may be necessary for AL1 function. To test this hypothesis, we generated four site-directed mutants of TGMV AL1 that are modified in either helix 1 or helix 2 (Fig. 1 B) and compared their activities to the wild type protein in vivo and in vitro (Fig. 1 B). The helix mutants were first tested for the ability to direct replication of TGMV B DNA in tobacco protoplasts with wild type TGMV AL3 also supplied in trans. Helix 1-mutant 2 (H1–2), which contains a S25V change and resembles the AL1 protein of the closely related geminivirus BGMV, supported wild type levels of TGMV B replication (Fig. 2 A, cf. lanes 1 and 2). In contrast, H1–1, which has alanine substitutions at three conserved charged residues in helix 1, failed to support viral DNA replication (Fig. 2 A, lane 3). The mutations in helix 2 either converted three conserved charged residues to alanine (H2–1), included a I45G change (H2–2), or altered the helix to resemble BGMV AL1 (H2–3). All helix 2 mutations abolished transient replication (Fig. 2 A, lanes 3–5). The mutant phenotypes established that, in general, the amino acid sequences of the predicted helices are essential for AL1 activity in vivo. The negative effect of the I45G replacement in H2–2, which should disrupt helix 2, suggested that the structure is also required for AL1 function. The AL1 helix mutants defective in replication assays were expressed as GST-AL1 fusion proteins in insect cells, purified by glutathione affinity chromatography, and examined for various AL1 activitiesin vitro. H1–2 was not included in these experiments because of its wild type replication phenotype. The mutant proteins were tested for DNA binding activity in gel shift assays using a radiolabeled double-stranded DNA probe that includes the TGMV AL1 DNA binding site. None of the helix mutants bound DNA (Fig. 2 B, lanes 3–6), even though wild type AL1 efficiently shifted the probe in a parallel reaction (lane 2). The mutant AL1 proteins were also tested for DNA cleavage activity using a radiolabeled, single-stranded oligonucleotide corresponding to the loop and right side of the hairpin in the plus-strand origin. Wild type GST-AL1 cleaved the 23-nt substrate to give a 10-nt radiolabeled product (Fig. 2 C, lane 2). Mutants H1–1 (Fig. 2 C, lane 3) and H2–3 (lane 6) displayed severely attenuated DNA cleavage activity, whereas H2–1 (lane 4) and H2–2 (lane 5) had no detectable activity. These results demonstrated that the predicted helices 1 and 2 are required both for DNA binding and cleavage by TGMV AL1. Helices 1 and 2 are outside of the AL1 interaction domain, and their mutation should have no effect on oligomerization if the proteins are properly expressed and folded. To verify that the failure of the AL1 helix mutants to bind and cleave DNA was not due to global misfolding, the proteins were assayed for their abilities to form oligomers with authentic AL1. Wild type GST-AL1 (Fig. 3 A, lane 1) and GST fusions of H1–1 (lane 2), H2–1 (lane 3), H2–2 (lane 4), and H2–3 (lane 5) were coexpressed with authentic AL1 in insect cells, as determined by immunoblotting of total protein extracts. Like wild type GST-AL1 (Fig. 3 A, lane 6), all of the mutant proteins copurified with authentic AL1 on glutathione-Sepharose (lanes 7–10), indicating that the helix mutations did not impair AL1/AL1 interactions and, instead, specifically affected the DNA binding and cleavage activities of AL1. The N terminus of AL1 also includes three conserved sequence motifs that are found in many RCR initiator proteins (Fig. 1; Refs. 35Koonin E.V. Ilyina T.V. J. Gen. Virol. 1992; 73: 2763-2766Crossref PubMed Scopus (132) Google Scholar and 36Ilyina T.V. Koonin E.V. Nucleic Acids Res. 1992; 20: 3279-3285Crossref PubMed Scopus (508) Google Scholar). Laufset al. (27Laufs J. Schumacher S. Geisler N. Jupin I. Gronenborn B. FEBS Lett. 1995; 377: 258-262Crossref PubMed Scopus (67) Google Scholar) showed that motif 3 corresponds to the endonucleolytic active site, but the roles of motifs 1 and 2 in RCR have not been investigated. We specifically modified these motifs in TGMV AL1 and analyzed the impact of the mutations on protein function (Fig. 1 B). In M1–1, all four motif I residues (F16LTY19) were replaced with alanine. In M1–2 and M1–3, individual aromatic residues were altered to give F16A and Y19A, respectively. The motif 2 mutant contained alanine substitutions for the core HLH sequence. Transient replication assays revealed that none of the motif 1 or 2 mutants (Fig. 4 A, lanes 2–5) supported TGMV B amplification in tobacco protoplasts, establishing that both motifs are essential for AL1 activity in vivo. To gain insight into the biochemical basis of the mutant replication phenotypes, we purified GST-AL1 fusion proteins corresponding to the motif 1 and 2 mutants from insect cells and tested them for DNA binding and cleavage in vitro. All of the mutant proteins failed to bind double-stranded DNA (Fig. 4 B, lanes 2–5). Similarly, none of the mutants had detectable single-stranded DNA cleavage activity (Fig. 4 C, lanes 2–5). In parallel assays, wild type GST-AL1 (lane 1) efficiently bound (Fig. 4 B) and cleaved (Fig. 4 C) the respective DNA probes. Protein interaction experiments (Fig. 3 B) established that M1–1 (lanes 3 and 8) and M2–1 (lanes 4 and 9) can form oligomers with wild type AL1 and, thus, are not globally misfolded. Together, these results showed that motifs 1 and 2 of TGMV AL1 are required for both DNA binding and cleavage during initiation of RCR. Motif 3 includes several conserved amino acids that may contribute to AL1 function. We generated six TGMV AL1 mutants that modified one or more residues in motif 3 and analyzed their activities in vivo and in vitro. In M3–1, alanines were substituted for the catalytic Tyr-103 and conserved Asp-107 residues. As expected, M3–1 did not support TGMV B replication in tobacco protoplasts (Fig. 5 A, lane 2) and was defective for DNA cleavage (Fig. 5 C, lane 2). Gel shift assays with M3–1 revealed that double-stranded DNA binding was also attenuated over a range of protein concentrations (Fig. 5 B, lanes 5–7), even though wild type GST-AL1 readily formed protein/DNA complexes at the same concentrations (lanes 2–4). In some experiments, low levels of DNA binding were observed with the M3–1 protein (data not shown). Protein interaction experiments showing that M3–1 can oligomerize with wild type AL1 (Fig. 3 B, lanes 5 and 10) indicated that the motif 3 mutant is not globally misfolded. The DNA binding defect of M3–1 was unexpected because of the direct involvement of motif 3 in catalysis of DNA cleavage. To better characterize the role of motif 3 and specifically Tyr-103 in DNA binding, we generated two different mutations at this position. In M3–2, Tyr-103 was changed to alanine, while M3–5 contained a phenylalanine substitution. Like M3–1, neither M3–2 or M3–5 supported viral DNA replication (Fig. 5 A, lanes 3 and 6) or cleaved DNA (Fig. 5 C, lanes 3 and 6), confirming that Tyr-103 is necessary for these processes. However, only M3–2 was impaired for DNA binding over a range of protein concentrations (Fig. 5 B, lanes 5–7), whereas M3–5 displayed wild type DNA binding properties (lanes 17–19). These results demonstrated that Tyr-103 contributes to both the DNA bind DA - 1998/9/18/ PY - 1998/9/18/ DO - 10.1074/jbc.273.38.24448 VL - 273 IS - 38 SP - 24448-24456 SN - 0021-9258 ER - TY - JOUR TI - A flexible, multipurpose method for recording vegetation composition and structure AU - Peet, R. K AU - Wentworth, T. R. AU - White, P. S. T2 - Castanea DA - 1998/// PY - 1998/// VL - 63 IS - 3 SP - 262-274 ER - TY - JOUR TI - The wound response in tomato involves rapid growth and electrical responses, systemically up-regulated transcription of proteinase inhibitor and calmodulin and down-regulated translation AU - Stankovic, B AU - Davies, E T2 - PLANT AND CELL PHYSIOLOGY AB - Localized heat wounding of tomato plants triggered rapid changes in growth and in electrical activity. The growth alterations were manifested as a transient increase in growth (petiole elongation), followed by a massive, long-lasting growth reduction. The electrical potential changes consisted of a wave of depolarization and re-polarization, i.e., a variation potential (VP). The tissue deformation apparently resulted from a pressure surge rapidly transmitted through the xylem, and preceded the changes in electrical potential. Externally-applied pressure mimicked flame wounding by triggering an electrical response resembling a VP. Our findings suggest that the VP results from a pressure surge in the xylem causing change in activity of mechanosensitive ion channels or pumps in adjacent living cells. The ensuing ion fluxes evoke plasma membrane depolarization, monitored extracellularly as a VP. Wounding also evoked a systemic decrease in polysomes, as well as a decrease in their protein synthesizing capacity in vitro. Very little of the newly-synthesized proteinase inhibitor (pin) and calmodulin (cal) mRNA was recruited into polysomes during the first hour following wounding. Since the VP appearance in distant tissue preceded the systemic molecular responses, the VP might be the long-distance signal up-regulating transcription of proteinase inhibitors and calmodulin, and down-regulating translation. DA - 1998/3// PY - 1998/3// DO - 10.1093/oxfordjournals.pcp.a029367 VL - 39 IS - 3 SP - 268-274 SN - 0032-0781 KW - calmodulin KW - electrophysiology KW - Lycopersicon KW - proteinase inhibitor KW - translation ER - TY - JOUR TI - Multiple cis elements contribute to geminivirus origin function AU - Orozco, BM AU - Gladfelter, HJ AU - Settlage, SB AU - Eagle, PA AU - Gentry, RN AU - Hanley-Bowdoin, L T2 - VIROLOGY AB - The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules which are dissimilar in sequence except for a highly conserved 200-bp common region that includes the origin for rolling circle replication. To better characterize the plus-strand origin, we analyzed the capacities of various TGMV common region sequences to support episomal replication in tobacco protoplasts when the viral replication proteins AL1 and AL3 were supplied in trans. These experiments demonstrated that the minimal origin is located in 89-bp common region fragment that includes the known AL1 binding motif and a hairpin structure containing the DNA cleavage site. Analyses of mutant origin sequences identified two additional cis elements--one that is required for origin activity and a second that greatly enhances replication. In contrast, a conserved partial copy of the AL1 binding site did not contribute to origin function. Mutational analysis of the functional AL1 binding site showed that both spacing and sequence of this motif are important for replication in vivo and AL1/DNA binding in vitro. Spacing changes between the AL1 binding site and hairpin also negatively impacted TGMV origin function in a position-dependent manner. Together, these results demonstrated that the organization of TGMV plus-strand origin is complex, involving multiple cis elements that are likely to interact with each other during initiation of replication. DA - 1998/3/15/ PY - 1998/3/15/ DO - 10.1006/viro.1997.9013 VL - 242 IS - 2 SP - 346-356 SN - 0042-6822 ER - TY - JOUR TI - Improvement of poult performance following Bordetella avium challenge by administration of a novel oxy-halogen formulation AU - Pardue, SL AU - Luginbuhl, GH T2 - AVIAN DISEASES AB - The ability of a novel oxy-halogen formulation (OHF) to alter the development of bordetellosis (turkey coryza) in large white turkey poults was assessed. Bordetella avium (BA)-infected (1-day-of-age) and noninfected control poults received 0, 0.008%, or 0.016% of an OHF continuously in the drinking water. At 4, 7, 10, 14, and 17 days of age, reisolation of BA from infected poults was attempted. Infected poults receiving 0.016% OHF exhibited significantly lower cumulative BA reisolation rates (90%) when compared with infected poults receiving 0 (96.7%) or 0.008% OHF (100%). At 7, 14, and 17 days of age, infected poults in the OHF-treated groups were significantly heavier than those BA-challenged poults receiving control water. Feed utilization was significantly improved from hatch to 7 days of age in BA-infected poults receiving OHF when compared with infected poults receiving control water. Clinical symptoms were severe only in untreated, infected poults and were mild or absent in all others. Damage to the tracheal epithelium, as measured by scanning electron microscopy, paralleled the clinical signs. Tracheal epithelial damage was virtually eliminated by OHF administration in infected poults. These results suggest that OHF treatment ameliorates many of the symptoms frequently associated with bordetellosis in young turkeys. DA - 1998/// PY - 1998/// DO - 10.2307/1592586 VL - 42 IS - 1 SP - 140-145 SN - 1938-4351 KW - Bordetella avium KW - turkey coryza KW - oxy-halogen KW - growth suppression KW - tracheal epithelium ER - TY - JOUR TI - Action potentials and variation potentials in sunflower: An analysis of their relationships and distinguishing characteristics AU - Stankovic, B AU - Witters, DL AU - Zawadzki, T AU - Davies, E T2 - PHYSIOLOGIA PLANTARUM AB - Sunflower plants ( Helianthus annuus L.) were given an electrical stimulus to the stem or a heat (flame)‐wound to a single leaf or a cotyledon. The resulting electrical activity was monitored with extracellular electrodes. An electrical stimulus applied to the stem frequently evoked an action potential (AP), but never a variation potential (VP). In contrast, a heat‐wound applied to a leaf virtually always elicited a VP, which was often accompanied by one or more superimposed spikes (putative APs). The kinetic parameters of the AP and the VP were investigated. The AP appears to propagate without decrement in velocity or magnitude, whereas the VP parameters decrease significantly with distance. The heat stimulus triggered rapid alterations in stem elongation/contraction, which preceded changes in electrical potential, indicating the transmission of a hydraulic signal. Light‐off and light‐on stimuli evoked negative‐ and positive‐going changes in extracellular electrical potential, respectively, corresponding to de‐ and hyper‐polarization of the plasma membrane. Membrane depolarization (extracellularly manifested as a VP) evoked by both the light‐off and heat‐wounding stimuli was able to trigger one or more APs. We interpret these results to suggest that APs are “genuine” electrical signals involving voltage‐gated ion channels or pumps, which can be evoked directly by electrical stimulation or indirectly by changes in membrane potential occurring during the VP or after the light‐off stimulus. In contrast, VPs appear to be a local (non‐transmissible) electrical consequence of the passage of a rapidly transmitted hydraulic signal in the xylem, presumably acting on mechanosensitive ion channels or pumps in adjacent living cells. DA - 1998/5// PY - 1998/5// DO - 10.1034/j.1399-3054.1998.1030107.x VL - 103 IS - 1 SP - 51-58 SN - 1399-3054 KW - action potential KW - Helianthus annuus KW - hydraulic signal KW - sunflower KW - variation potential KW - wounding ER - TY - JOUR TI - A regeneration and Agrobacterium-mediated transformation system for genetically diverse Chrysanthemum cultivars AU - Sherman, JM AU - Moyer, JW AU - Daub, ME T2 - JOURNAL OF THE AMERICAN SOCIETY FOR HORTICULTURAL SCIENCE AB - An efficient, high-frequency regeneration and Agrobacterium -mediated transformation system was developed allowing the genetic engineering of three chrysanthemum ( Dendranthema grandiflora Tzvelev) cultivars: the formerly recalcitrant and economically important cut-flower mum `Polaris' and two potted mums, `Hekla' and `Iridon'. The regeneration protocol used leaf explants on a sequence of media with four hormone regimes. Explants were first cultured on an embryogenesis-type medium containing a high concentration of 2,4-D, which promoted callus formation. Shoot primordia were induced by culture on medium lacking 2,4-D, followed by shoot elongation on a high-cytokinin plus gibberellic acid medium. Finally, elongated shoots were rooted on a low-auxin rooting medium. Transformed plants of the three cultivars were obtained following co-culture of leaf explants with A. tumefaciens strain EHA 105 harboring the plasmid pBI121 containing genes for neomycin phosphotransferase II (NPTII) and β-glucuronidase (GUS). Stable transformation of the three cultivars was verified via GUS assays and Southern analysis. DA - 1998/3// PY - 1998/3// DO - 10.21273/jashs.123.2.189 VL - 123 IS - 2 SP - 189-194 SN - 0003-1062 KW - Dendranthema grandiflora KW - plant cell culture KW - tissue culture KW - floral crop biotechnology ER - TY - JOUR TI - Plasma membrane-associated actin in bright yellow 2 tobacco cells - Evidence for interaction with microtubules AU - Collings, DA AU - Asada, T AU - Allen, NS AU - Shibaoka, H T2 - PLANT PHYSIOLOGY AB - Abstract Plasma membrane ghosts form when plant protoplasts attached to a substrate are lysed to leave a small patch of plasma membrane. We have identified several factors, including the use of a mildly acidic actin stabilization buffer and the inclusion of glutaraldehyde in the fixative, that allow immunofluorescent visualization of extensive cortical actin arrays retained on membrane ghosts made from tobacco (Nicotiana tabacum L.) suspension-cultured cells (line Bright Yellow 2). Normal microtubule arrays were also retained using these conditions. Membrane-associated actin is random; it exhibits only limited coalignment with the microtubules, and microtubule depolymerization in whole cells before wall digestion and ghost formation has little effect on actin retention. Actin and microtubules also exhibit different sensitivities to the pH and K+ and Ca2+ concentrations of the lysis buffer. There is, however, strong evidence for interactions between actin and the microtubules at or near the plasma membrane, because both ghosts and protoplasts prepared from taxol-pretreated cells have microtubules arranged in parallel arrays and an increased amount of actin coaligned with the microtubules. These experiments suggest that the organization of the cortical actin arrays may be dependent on the localization and organization of the microtubules. DA - 1998/11// PY - 1998/11// DO - 10.1104/pp.118.3.917 VL - 118 IS - 3 SP - 917-928 SN - 1532-2548 ER - TY - JOUR TI - A phosphatidylinositol 4-kinase pleckstrin homology domain that binds phosphatidylinositol 4-monophosphate AU - Stevenson, JM AU - Perera, IY AU - Boss, WF T2 - JOURNAL OF BIOLOGICAL CHEMISTRY AB - Pleckstrin homology (PH) domains are found in many proteins involved in signal transduction, including the family of large molecular mass phosphatidylinositol (PI) 4-kinases. Although the exact function of these newly discovered domains is unknown, it is recognized that they may influence enzyme regulation by binding different ligands. In this study, the recombinant PI 4-kinase PH domain was explored for its ability to bind to different phospholipids. First, we isolated partial cDNAs of the >7-kilobase transcripts of PI 4-kinases from carrot (DcPI4Kα) andArabidopsis (AtPI4Kα). The deduced primary sequences were 41% identical and 68% similar to rat and human PI 4-kinases and contained the telltale lipid kinase unique domain, PH domain, and catalytic domain. Antibodies raised against the expressed lipid kinase unique, PH, and catalytic domains identified a polypeptide of 205 kDa in Arabidopsis microsomes and an F-actin-enriched fraction from carrot cells. The 205-kDa immunoaffinity-purified Arabidopsis protein had PI 4-kinase activity. We have used the expressed PH domain to characterize lipid binding properties. The recombinant PH domain selectively bound to phosphatidylinositol 4-monophosphate (PI-4-P), phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), and phosphatidic acid and did not bind to the 3-phosphoinositides. The PH domain had the highest affinity for PI-4-P, the product of the reaction. Consideration is given to the potential impact that this has on cytoskeletal organization and the PI signaling pathway in cells that have a high PI-4-P/PI-4,5-P2 ratio. Pleckstrin homology (PH) domains are found in many proteins involved in signal transduction, including the family of large molecular mass phosphatidylinositol (PI) 4-kinases. Although the exact function of these newly discovered domains is unknown, it is recognized that they may influence enzyme regulation by binding different ligands. In this study, the recombinant PI 4-kinase PH domain was explored for its ability to bind to different phospholipids. First, we isolated partial cDNAs of the >7-kilobase transcripts of PI 4-kinases from carrot (DcPI4Kα) andArabidopsis (AtPI4Kα). The deduced primary sequences were 41% identical and 68% similar to rat and human PI 4-kinases and contained the telltale lipid kinase unique domain, PH domain, and catalytic domain. Antibodies raised against the expressed lipid kinase unique, PH, and catalytic domains identified a polypeptide of 205 kDa in Arabidopsis microsomes and an F-actin-enriched fraction from carrot cells. The 205-kDa immunoaffinity-purified Arabidopsis protein had PI 4-kinase activity. We have used the expressed PH domain to characterize lipid binding properties. The recombinant PH domain selectively bound to phosphatidylinositol 4-monophosphate (PI-4-P), phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), and phosphatidic acid and did not bind to the 3-phosphoinositides. The PH domain had the highest affinity for PI-4-P, the product of the reaction. Consideration is given to the potential impact that this has on cytoskeletal organization and the PI signaling pathway in cells that have a high PI-4-P/PI-4,5-P2 ratio. Since the first report of changes in phosphoinositide metabolism in response to light (1Morse M.J. Crain R.C. Satter R.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7075-7078Crossref PubMed Google Scholar), there have been many studies of the metabolism of inositol phospholipids in plants (2Drøbak B.K. Biochem. J. 1992; 288: 697-712Crossref PubMed Scopus (145) Google Scholar, 3Cote G.G. Crain R.C. Bioessays. 1994; 16: 39-46Crossref Scopus (46) Google Scholar, 4Perera I.Y. Boss W.F. Briggs W.R. Heath R.L. Tobin E.M. Regulation of Plant Growth and Development by Light. American Society of Plant Physiologists, Rockville, MD1996: 114-126Google Scholar). These studies reveal two distinguishing features of phosphoinositide metabolism in higher plants: 1) [3H]PI-4-P 1The abbreviations used are: PI-4-Pphosphatidylinositol 4-monophosphatePI-45-P2, phosphatidylinositol 4,5-bisphosphatePI4Kphosphatidylinositol 4-kinasePIphosphatidylinositolPH domainpleckstrin homology domainPLCphospholipase CPI-3-Pphosphatidylinositol 3-monophosphatePI-34-P2, phosphatidylinositol 3,4-bisphosphatePAphosphatidic acidPCRpolymerase chain reaction, RACE, rapid amplification of cDNA endskbkilobaseseEF-1αelongation factor-1αPAGEpolyacrylamide gel electrophoresisNBD12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)). is 10–20-fold higher than [3H]PI-4,5-P2, and 2) changes in [3H]PI-4-P are detectable. Conversely, in the most responsive animal systems, the ratio of PI-4-P to PI-4,5-P2 is ∼1:1, and there is only a transient change in PI-4,5-P2, with little to no change in PI-4-P even though inositol 1,4,5-trisphosphate may increase 40-fold (5Cunningham E. Thomas G.M.H. Ball A. Hiles I. Cockcroft S. Curr. Biol. 1995; 5: 775-783Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). One explanation that we are exploring for these differences is that the synthesis of PI-4,5-P2 is rate-limiting in plant cells because PI-4-P is sequestered by cytoskeletal or other proteins and not readily available for phosphorylation to PI-4,5-P2 or other metabolic pathways. phosphatidylinositol 4-monophosphate 5-P2, phosphatidylinositol 4,5-bisphosphate phosphatidylinositol 4-kinase phosphatidylinositol pleckstrin homology domain phospholipase C phosphatidylinositol 3-monophosphate 4-P2, phosphatidylinositol 3,4-bisphosphate phosphatidic acid polymerase chain reaction, RACE, rapid amplification of cDNA ends kilobases elongation factor-1α polyacrylamide gel electrophoresis 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)). The first committed step in the biosynthesis of PI-4,5-P2is the phosphorylation of PI by PI 4-kinase to form PI-4-P. PI-4-P is phosphorylated by PI-4-P 5-kinase to form PI-4,5-P2. Biochemically, PI 4-kinase and PI-4-P 5-kinase activities have been studied in plant membranes (6Sommarin M. Sandelius A.S. Biochim. Biophys. Acta. 1988; 958: 268-278Crossref Scopus (82) Google Scholar, 7Sandelius A.S. Sommarin M. Morré D.J. Boss W.F. Loewus F.A. Inositol Metabolism in Plants. Wiley-Liss, New York1990: 139-161Google Scholar, 8Gross W. Yang W. Boss W.F. Biochim. Biophys. Acta. 1992; 1134: 73-80Crossref PubMed Scopus (17) Google Scholar, 9Cho M.H. Shears S.B. Boss W.F. Plant Physiol. ( Bethesda ). 1993; 103: 637-647Crossref PubMed Scopus (52) Google Scholar) and cytoskeletal (10Tan Z. Boss W.F. Plant Physiol. ( Bethesda ). 1992; 100: 2116-2120Crossref PubMed Scopus (73) Google Scholar, 11Xu P. Lloyd C.W. Staiger C.J. Drøbak B.K. Plant Cell. 1992; 4: 941-951Crossref PubMed Google Scholar) and soluble (12Okpodu C.M. Gross W. Burkhart W. Boss W.F. Plant Physiol. ( Bethesda ). 1995; 107: 491-500Crossref PubMed Scopus (18) Google Scholar) fractions. Recently, a putative PI-4-P 5-kinase was cloned fromArabidopsis (13Satterlee J.S. Sussman M.R. Plant Physiol. ( Bethesda ). 1997; 115: 864Google Scholar), but the genes encoding PI 4-kinases in plants have remained elusive. A clear distinction between two structurally different isoforms of the PI 4-kinase has emerged from the cloning and sequencing of PI 4-kinase from yeast (14Flanagan C.A. Schnieders E.A. Emerick A.W. Kunisawa R. Admon A. Thorner J. Science. 1993; 262: 1444-1448Crossref PubMed Scopus (174) Google Scholar, 15Garcia-Bustos J.F. Marini F. Stevenson I. Frei C. Hall M.N. EMBO J. 1994; 13: 2352-2361Crossref PubMed Scopus (103) Google Scholar, 16Yoshida S. Ohya Y. Goebl M. Nakano A. Anraku Y. J. Biol. Chem. 1994; 269: 1166-1171Abstract Full Text PDF PubMed Google Scholar), human (17Wong K. Cantley L.C. J. Biol. Chem. 1994; 269: 28878-28884Abstract Full Text PDF PubMed Google Scholar, 18Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 4384-4390Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), rat (19Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), and bovine (20Gehrmann T. Vereb G. Schmidt M. Klix D. Meyer H.E. Varsanyi M. Heilmeyer Jr., L.M.G. Biochim. Biophys. Acta. 1996; 1311: 53-63Crossref PubMed Scopus (31) Google Scholar, 21Balla T. Downing G.J. Jaffe H. Kim S. Zolyomi A. Catt K.J. J. Biol. Chem. 1997; 272: 18358-18366Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) tissues. The two isoforms differ in size, amino acid sequence homology, and putative location and function within the cell. The smaller type encodes a polypeptide of ∼110–125 kDa that is found in the cytosol and is associated with the Golgi apparatus (22Wong K. Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 13236-13241Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The second type encodes a larger protein of ∼200–230 kDa that is membrane-associated (19Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 22Wong K. Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 13236-13241Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). A distinctive feature of the group of higher molecular mass PI 4-kinases is that they contain putative PH domains. PH domains are poorly conserved protein modules of ∼100 amino acids in length (23Shaw G. Bioessays. 1996; 18: 35-46Crossref PubMed Scopus (256) Google Scholar, 24Lemmon M.A. Ferguson K.M. Schlessinger J. Cell. 1996; 85: 621-624Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 25Ingley E. Hemmings B.A. J. Cell. Biochem. 1994; 56: 436-443Crossref PubMed Scopus (67) Google Scholar, 26Musacchio A. Gibson T. Rice P. Thompson J. Saraste M. Trends Biochem. Sci. 1993; 18: 343-348Abstract Full Text PDF PubMed Scopus (496) Google Scholar). These motifs exist in proteins that associate with membranes during signal transduction. PH domains bind a variety of ligands ranging from other signal transduction proteins such as G-protein βγ subunits (27Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar) to polyphosphorylated inositol lipids (28Lomasney J.W. Cheng H.-F. Wang L.-P. Liu S.-M. Fesik S.W. King K. J. Biol. Chem. 1996; 271: 25316-25326Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 29Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 30Hyvonen M. Macias M.J. Nilges M. Oschkinat H. Saraste M. Wilmanns M. EMBO J. 1995; 14: 4676-4685Crossref PubMed Scopus (309) Google Scholar, 31Harlan J.E. Hajduk P.J. Yoon H.S. Fesik S.W. Nature. 1994; 371: 168-170Crossref PubMed Scopus (687) Google Scholar)in vitro. N-terminal regions of the PH domain of phospholipase C-δ1 (PLCδ1) (28Lomasney J.W. Cheng H.-F. Wang L.-P. Liu S.-M. Fesik S.W. King K. J. Biol. Chem. 1996; 271: 25316-25326Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), β-adrenergic receptor kinase (29Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar), β-spectrin (30Hyvonen M. Macias M.J. Nilges M. Oschkinat H. Saraste M. Wilmanns M. EMBO J. 1995; 14: 4676-4685Crossref PubMed Scopus (309) Google Scholar), pleckstrin, Tsk (T-cell-specific kinase), and Ras GTPase-activating protein (31Harlan J.E. Hajduk P.J. Yoon H.S. Fesik S.W. Nature. 1994; 371: 168-170Crossref PubMed Scopus (687) Google Scholar) bind to inositol phospholipids. The affinity of PH domains for PI-4-P, PI-4,5-P2, PI-3-P, PI-3,4-P2, and PI 3,4,5-trisphosphate varies with the type of protein (32Rameh L.E. Arvidsson A. Carraway III, K.L. Couvillon A.D. Rathbun G. Crompton A. VanRenterghem B. Czech M.P. Ravichandran K.S. Burakoff S.J. Wang D.-S. Chen C.-S. Cantley L.C. J. Biol. Chem. 1997; 272: 22059-22066Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). This means that rapid cellular changes in the levels of the inositol phospholipids could affect the location and regulation of specific PH domain-containing proteins. Identifying PH domains and their ligand affinities should increase the understanding of how a protein is regulated. The PH domains of the PI 4-kinases have been described based only on primary sequence homology with other PH domains and have not been characterized biochemically. Here we show biochemical and molecular evidence for a large molecular mass PI 4-kinase in both carrot (DcPI4Kα) andArabidopsis (AtPI4Kα), and we show for the first time the affinity of a PI 4-kinase PH domain for specific phosphoinositides. The active enzyme was purified using antibodies raised against the expressed conserved domains. The molecular mass of the PI 4-kinase was estimated to be 205 kDa based on Western blot analysis of the purified protein. Western blots also indicated thatDcPI4Kα is associated with an F-actin fraction. More important, using a new technique, Fat Western blotting, we determined that the recombinant carrot PI 4-kinase PH domain binds specifically to phosphatidic acid (PA), PI-4-P, and PI-4,5-P2. The lipid binding data give new insights into potential mechanisms for regulating PI 4-kinase activity and its distribution. Total RNA was extracted from carrot cells grown in suspension culture (33Chen Q. Boss W.F. Plant Physiol. ( Bethesda ). 1990; 94: 1820-1829Crossref PubMed Scopus (39) Google Scholar) by hot borate/phenol/chloroform extraction (34Hall T.C. Ma Y. Buchbinder B.U. Pyne J.W. Sun S.M. Bliss F.A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3196-3200Crossref PubMed Scopus (164) Google Scholar, 35Perera I. Zielinski R. Plant Physiol. ( Bethesda ). 1992; 100: 812-819Crossref PubMed Scopus (19) Google Scholar). cDNA was synthesized from 5 μg of total RNA by Moloney murine leukemia virus reverse transcriptase (Promega) and primed with random hexamers (Boehringer Mannheim) as described (36Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Amplification of cDNA by the polymerase chain reaction was achieved with degenerate oligonucleotides deduced from conserved amino acid sequences of known PI 4-kinases. The conserved regions were used by Nakagawa et al. (19Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) to clone the rat PI 4-kinase. The sequences of these regions are (V/T)GDDCRQ and HIDFGF(M/V). Arabidopsis codon usage was followed to design the degenerate primers, and EcoRI sites were added to the 5′-ends of the primers to facilitate subcloning of PCR products into pBluescript (Stratagene). The sequences of the primers, using the International Union of Biochemistry code for degeneracy, were CGG AAT TCR YTG GWG AYG AYT GYC GTC AR for the sense primer and CGG AAT TCN ATR AAW CCR AAR TCD ATR TG for the antisense primer. The PCRs containing 10 μl of the cDNA synthesis mixture, 20–25 pmol of each primer, and 5 units of Taq polymerase (Promega) were amplified by 25 cycles of the following: 97 °C for 30 s, 45 °C for 1 min, and 72 °C for 1 min. The PCR products were resolved by agarose gel (1%, w/v) electrophoresis, gel-purified, digested with EcoRI, and subcloned into pBluescript. Sequence of the cDNA was determined by the dideoxy chain termination method (37Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5468Crossref PubMed Scopus (55549) Google Scholar) using the Sequenase kit (U. S. Biochemical Corp.). After determining the sequence of the PCR product, gene-specific primers were used to amplify cDNA 3′ and 5′ to the original PCR product by the rapid amplification of cDNA ends (RACE) method exactly as described previously (38Frohman M.A. Methods Enzymol. 1993; 218: 340-356Crossref PubMed Scopus (468) Google Scholar). The ArabidopsisPI 4-kinase was cloned by using the carrot 5′-RACE product as a probe to screen an Arabidopsis λYES cDNA library (a gift of Dr. Ralph Dewey, North Carolina State University). The probe was labeled with [α-32P]dCTP and random hexamers. The plated library (3 × 106 clones) was hybridized to the carrot cDNA at 55 °C for 16 h in hybridization buffer containing 6× saline/sodium phosphate/EDTA (SSPE; 1× SSPE = 10 mm NaH2PO4, 1 mm EDTA, and 149 mm NaCl) and 5× Denhardt's solution (100× Denhardt's solution = 2% (w/v) fatty acid-free bovine serum albumin, 2% (w/v) polyvinylpyrrolidone, 2% (w/v) Ficoll 400, 0.5% (w/v) SDS, and 100 μg of calf thymus DNA (Sigma)). The final wash was in 1× SSPE and 0.1% (w/v) SDS at 55 °C for 1 h. The nylon membrane was exposed to x-ray film for 24 h at −80 °C. Two hybridization-positive clones were carried through three successive screens. The larger clone was 2.5 kb and was used as a probe to screen an Arabidopsis λZap II cDNA library that had been selected for large cDNAs (39Kieber J.J. Rothenberg M. Roman G. Feldman K. Ecker J.R. Cell. 1993; 72: 427-441Abstract Full Text PDF PubMed Scopus (1525) Google Scholar). Again, two positive clones were carried through three sequential screening steps, and the largest clone (3.1 kb) was analyzed for its complete sequence by the Iowa State Sequencing Facility. Total RNA was fractionated by formaldehyde-containing agarose gel electrophoresis (40Zielinski R.E. Plant Physiol. ( Bethesda ). 1987; 84: 937-943Crossref PubMed Google Scholar). The RNA was transferred by capillary transfer and UV-cross-linked to nylon membrane and then hybridized with the Arabidopsis cDNA labeled by random priming with [α-32P]dCTP. Hybridization was at 42 °C in hybridization buffer containing 50% (v/v) formamide. The final wash of the blot was in 0.1× SSPE and 0.1% (w/v) SDS at 65 °C for 1 h. The nylon was then exposed to x-ray film for 3 days at −80 °C. A cDNA encoding the carrot PI 4-kinase PH domain was generated by PCR amplification with primers that flanked either end of the domain sequence. The sequences of the primers were CGG GAT CCC CCC TGG TTA GGC AAC ACA TT (sense) and GGA ATT CCA ACC TTG AAA ACG CAA GCT T (antisense). The primers contained BamHI (sense primer) and EcoRI (antisense primer) sites on their 5′-ends to facilitate directional subcloning into the bacterial expression vector pRSET-A (Invitrogen). The PCR product was gel-purified, digested with BamHI andEcoRI, and ligated into pRSET-A. BL21(DE3) pLys S cells were transformed with the recombinant plasmid, and expression was induced with the addition of isopropyl-β-d-thiogalactopyranoside (1 mm final concentration) to the cell culture. Bacterial cells expressing the His-tagged PH domain were lysed by sonication and solubilized in 6 m guanidine hydrochloride, and the recombinant polypeptide was purified by metal affinity chromatography using ProBond resin (Invitrogen). Because the PH domain was insoluble, purification was carried out under denaturing conditions with solutions containing 8 m urea. Column fractions were dialyzed sequentially to remove urea and to promote refolding. Tomato eEF-1α (a gift from Christine K. Shewmaker, Calgene Inc.) was expressed and purified using the same protocol. Total microsomes were prepared from carrot suspension culture cells 5 days after transfer or from whole Arabidopsis thaliana plants. Suspension culture cells were filtered by gravity and homogenized in an equal volume of buffer containing 10 mm KCl, 1 mm EDTA, 1 mm MgCl2, 50 mm Tris (pH 7.5), 95 mm LiCl, 2 mm EGTA, polyvinylpolypyrrolidone (0.1 g/g of cells), 8% (w/v) sucrose, 1 mm dithiothreitol, 2 μg/ml aprotinin, 0.1 mm phenylmethylsulfonyl fluoride, 1 mg/100 ml leupeptin, and 2 mm benzamidine. Homogenization was with an equal volume of 0.2-mm glass beads in a Virtis homogenizer in 30-s pulses for 2 min at 4 °C. Arabidopsis plants were coarsely macerated and ground in a Virtis homogenizer with an equal volume of buffer containing 3 mm EDTA, 2 mmEGTA, 30 mm Tris (pH 7.4), 250 mm sucrose, 14 mm β-mercaptoethanol, 2 mm dithiothreitol, 2 μg/ml aprotinin, 0.1 mm phenylmethylsulfonyl fluoride, 1 mg/100 ml leupeptin, and 2 mm benzamidine. The homogenate was centrifuged at 2000 × g for 5 min. The resultant supernatant was centrifuged at 40,000 × g for 60 min to obtain a microsomal fraction. Microsomes were resuspended in 30 mm Tris (pH 7.4). F-actin-rich fractions were prepared from the microsomal fraction isolated from 5-day-old carrot suspension culture cells as described previously (10Tan Z. Boss W.F. Plant Physiol. ( Bethesda ). 1992; 100: 2116-2120Crossref PubMed Scopus (73) Google Scholar). PCR was used to amplify the reading frame of the largest AtPI4Kα clone. The primers used were CGG GAT CCG TTC AGT CAC ATA TAT TAG AA (sense) and G GAA TTC TTA CTT CTC GAT GCC TTG (antisense). An internalBamHI site 45 nucleotides upstream of the region encoding the lipid kinase unique domain and the EcoRI site of the antisense primer allowed the PCR product to be digested with these two enzymes, purified, and ligated into pRSET-B. Expression and purification of the recombinant protein were exactly the same as for the recombinant PH domain described above, except that the protein was not dialyzed. Instead it was concentrated in a Centricon 10 (Amicon, Inc.), and resolved by SDS-PAGE so that ∼50 μg of recombinant protein were present in each lane of the gel. The gel was stained with 0.05% (w/v) Coomassie Brilliant Blue R-250 in water and washed copiously with water until the bands were visible. The bands containing the recombinant protein were excised from the gel and sent to Zeneca LifeScience Molecules for injection into two rabbits (662 and 663). The rabbits were given seven boosts of the recombinant protein over the course of 3 months. Sera from test bleeds and production bleeds were analyzed for cross-reactivity to the recombinant protein by immunoblotting (data not shown). Antiserum from test bleed 2 of rabbit 662 was purified for IgG on a protein A-Sepharose column (Sigma). Unspecific and His tag-generated antibodies were removed by incubating the purified IgG with an acetone precipitate of E. coli cells expressing His-tagged eEF-1α and removing the aggregates by centrifugation (41Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). A protein A-Sepharose-antibody affinity column was made by direct coupling with dimethyl pimelimidate (Sigma) as described previously (41Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Production bleed antisera from both rabbits (0.5 ml total) were pooled and added to 1 ml of protein A-Sepharose beads in 30 mm Tris (pH 7.4). After coupling the antibody to the beads, the column was washed with 20 bed volumes of 30 mm Tris (pH 8.0). The efficiency of coupling of antibody to protein A beads was analyzed by SDS-PAGE before and after the addition of dimethyl pimelimidate. Heavy chain IgG bands were present at 55 kDa before coupling, but not after. Arabidopsis microsomes (12.5 mg) were solubilized for 30 min at 4 oC in buffer used to solubilize cytoskeletal proteins (2% (v/v) Triton X-100, 100 mm Tris (pH 7.4), 10 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 150 μg/ml leupeptin, and 670 μg/ml DNase I) (42Carraway K.L. Carraway C.A.C. The Cytoskeleton: A Practical Approach. Oxford University Press, New York1992Google Scholar). Solubilized microsomes were centrifuged at 40,000 × g for 30 min. The supernatant was incubated with antibody-coupled beads in a 10 × 33-mm column resuspended in 1 bed volume of 100 mm Tris (pH 7.4) at 4 °C with shaking for 2 h. The bed was allowed to settle, and the flow-through fraction was collected. The column was washed with 10 bed volumes of 100 mm Tris (pH 7.4). In addition, to prepare the column for elution and to ensure adequate washing, the column was washed with 10 mm phosphate buffer (pH 7.0) until the flow-through fraction had a spectrophotometric absorbance reading of <0.010 at 280 nm. Bound proteins were eluted with 100 mm glycine (pH 3.0). Fractions of 1 bed volume were collected, immediately neutralized with 0.05 volume of 1m phosphate (pH 8.0), and analyzed by SDS-PAGE and immunoblotting and for PI 4-kinase activity. Each fraction eluted from the immunoaffinity column was assayed in duplicate (60 μl/assay) to determine the PI 4-kinase activity. The reaction mixture contained final concentrations of 7.5 mm MgCl2, 1 mm sodium molybdate, 0.5 mg/ml PI, 0.1% (v/v) Triton X-100, 0.9 mmATP, 30 mm Tris (pH 7.2), and 20 μCi of [γ-32P]ATP (7000 Ci/mmol) in a total volume of 100 μl. Stock PI (5 mg/ml) was solubilized in 1% (v/v) Triton X-100. The reactions were incubated at 25 °C for 2 h with intermittent shaking. The reactions were stopped with 1.5 ml of ice-cold CHCl3/MeOH (1:2) and kept at 4 °C until the lipids were extracted. Lipids were extracted as described previously (9Cho M.H. Shears S.B. Boss W.F. Plant Physiol. ( Bethesda ). 1993; 103: 637-647Crossref PubMed Scopus (52) Google Scholar). Extracted lipids were vacuum-dried, solubilized in CHCl3/MeOH (2:1), and spotted onto Whatman LK5D silica gel plates that had been completely dried in a microwave oven for 5 min after presoaking in 1% (w/v) potassium oxalate for 80 s. The lipids were separated in either a CHCl3/MeOH/NH4OH/H2O (86:76:6:16) solvent system (9Cho M.H. Shears S.B. Boss W.F. Plant Physiol. ( Bethesda ). 1993; 103: 637-647Crossref PubMed Scopus (52) Google Scholar) or a borate/pyridine-based solvent (43Walsh J.P. Caldwell K.K. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9184-9187Crossref PubMed Scopus (106) Google Scholar) and quantitated with a Bioscan System 500 Imaging Scanner. The plates were subsequently exposed to a phosphor screen for 2 days and visualized by a Storm PhosphorImager (Molecular Dynamics, Inc.). Carrot microsomes were solubilized in 1% (v/v) Triton X-100 at 4 °C overnight. To preclear unspecific antibodies and antigens, solubilized microsomes were incubated for 1 h on ice with 0.2 volume of preimmune serum from rabbit 662. 0.33 volume of protein A-Sepharose was added and incubated for 1.5 h at 4 °C with shaking. The beads were pelleted, and the supernatant was saved and used for subsequent steps. Immunoprecipitation, as described previously (41Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar), was carried out by adding to the supernatant either preimmune serum or partially purified antiserum from test bleed 2 of rabbit 662 (described above). The protein A-antibody-antigen complex was washed three times with 30 mm Tris (pH 7.2) and resuspended in SDS-PAGE sample buffer for subsequent analysis by electrophoresis and immunoblotting. Protein was separated by SDS-PAGE using 8% (w/v) polyacrylamide and transferred onto polyvinylidene difluoride membrane (44Bjerrum O.J. Schafer-Nielsen C. Dunn M.J. Analytical Electrophoresis. Verlag Chemie, Weinheim1986: 315Google Scholar). The membrane was incubated for 1 h with anti-AtPI4Kα antiserum (1:1000) at 25 °C. Cross-reactivity was detected by incubation with goat anti-rabbit IgG, F(ab′)2 conjugated to horseradish peroxidase (Pierce), and subsequent chemiluminescent detection by exposing the blot to X-Omat AR film (Eastman Kodak Co.) for the amount of time indicated. Phospholipids were from Sigma, except for PI-3-P and PI-3,4-P2, which were from Matreya, Inc., and NBD-labeled PA, NBD-labeled phosphatidylcholine, and rhodamine-labeled phosphatidylethanolamine, which were from Avanti Polar Lipids. Phospholipids were solubilized in chloroform as stock solutions of 1 mg/ml. A minimum of 10 μl containing 0.5, 1.0, or 5.0 μg of lipid were spotted onto nitrocellulose (NitroBind, MSI) at a time. The membrane and lipids were dried at 24 °C for 1 h. The nitrocellulose was incubated with 3% (w/v) fatty acid-free bovine serum albumin (isolated by cold ethanol precipitation; Sigma A-6003) in TBST (10 mm Tris (pH 8.0), 140 mm NaCl, and 0.1% (v/v) Tween 20) for 1 h and then placed in a solution containing the His-tagged fusion proteins (PH domain or eEF-1α) diluted in TBST (0.5 μg/ml) at 4 °C overnight with shaking. The nitrocellulose was washed with TBST three times for 10 min each and then incubated with T7 tag monoclonal antibody (Novagen) to the His-tagged region diluted 1:10,000 in TBST for 1 h at 24 °C. The nitrocellulose was washed three times for 10 min in TBST at 24 °C and then incubated with goat anti-mouse IgG conjugated to horseradish peroxidase (Pierce) at a titer of 1:30,000 in TBST for 1 h at 24 °C. The nitrocellulose was washed again in TBST three times for 10 min and then incubated for 5 min in a 1:1 mixture of peroxidase substrate and luminol/enhancer (Pierce) for subsequent chemiluminescent detection. The nitrocellulose was exposed to X-Omat AR film for 0.5–5 min as indicated. Using degenerate primers based on highly conserved regions of yeast PIK1 and STT4 PI 4-kinase lipid kinase domains, we amplified a cDNA of 394 nucleotides from carrot RNA. Sequence analysis of this PCR product showed that the deduced amino acid sequence was 50% identical and 64% similar to yeast STT4 and only 35% identical and 54% similar toArabidopsis PI 3-kinase (AtVPS34). 5′- and 3′-RACE were used to amplify regions of sequence beyond this initial PCR product. The 3′-RACE product was 1.1 kb in size and spanned the rest of the catalytic domain and extended through the 3′-untranslated region to the poly(A) tail. The 5′-RACE product contained 1.4 kb and had sequence homology to the lipid kinase unique domain and PH domain of the other PI 4-kinases reported in GenBankTM. The 5′-RACE product from carrot was used as a probe to screen anArabidopsis λYES cDNA library. Two positiv DA - 1998/8/28/ PY - 1998/8/28/ DO - 10.1074/jbc.273.35.22761 VL - 273 IS - 35 SP - 22761-22767 SN - 0021-9258 ER - TY - JOUR TI - SOR1, a gene required for photosensitizer and singlet oxygen resistance in Cercospora fungi, is highly conserved in divergent organisms AU - Ehrenshaft, M AU - Jenns, AE AU - Chung, KR AU - Daub, ME T2 - MOLECULAR CELL AB - Filamentous Cercospora fungi are resistant to photosensitizing compounds that generate singlet oxygen. C. nicotianae photosensitizer-sensitive mutants were restored to full resistance by transformation with SOR1 (Singlet Oxygen Resistance 1), a gene recovered from a wild-type genomic library. SOR1 null mutants generated via targeted gene replacement confirmed the requirement for SOR1 in photosensitizer resistance. SOR1 RNA is present throughout the growth cycle. Although resistance to singlet oxygen is rare in biological systems, SOR1, a gene with demonstrated activity against singlet-oxygen-generating photosensitizers, is highly conserved in organisms from widely diverse taxa. The characterization of SOR1 provides an additional phenotype to this large group of evolutionarily conserved genes. DA - 1998/3// PY - 1998/3// DO - 10.1016/S1097-2765(00)80060-X VL - 1 IS - 4 SP - 603-609 SN - 1097-2765 ER - TY - JOUR TI - Gene silencing from plant DNA carried by a Geminivirus AU - Kjemtrup, S AU - Sampson, KS AU - Peele, CG AU - Nguyen, LV AU - Conkling, MA AU - Thompson, WF AU - Robertson, D T2 - PLANT JOURNAL AB - Summary The geminivirus tomato golden mosaic virus (TGMV) replicates in nuclei and expresses genes from high copy number DNA episomes. The authors used TGMV as a vector to determine whether episomal DNA can cause silencing of homologous, chromosomal genes. Two markers were used to asses silencing: (1) the sulfur allele ( su ) of magnesium chelatase, an enzyme required for chlorophyll formation; and (2) the firefly luciferase gene ( luc ). Various portions of both marker genes were inserted into TGMV in place of the coat protein open‐reading frame and the constructs were introduced into intact plants using particle bombardment. When TGMV vectors carrying fragments of su (TGMV::su) were introduced into leaves of wild type Nicotiana benthamiana , circular, yellow spots with an area of several hundred cells formed after 3‐5 days. Systemic movement of TGMV::su subsequently produced varigated leaf and stem tissue. Fragments that caused silencing included a 786 bp 5' fragment of the 1392 bp su cDNA in sense and anti‐sense orientation, and a 403 bp 3' fragment. TGMV::su‐induced silencing was propogated through tissue culture, along with the viral episome, but was not retained through meiosis. Systemic downregulation of a constitutively expresse luciferase transgene in plants was achieved following infection with TGMV vectors carrying a 623 bp portion of luc in sense or anti‐sense orientation. These results establish that homologous DNA sequences localized in nuclear episomes can modulate the expression of active chromosomal genes. DA - 1998/4// PY - 1998/4// DO - 10.1046/j.1365-313X.1998.00101.x VL - 14 IS - 1 SP - 91-100 SN - 0960-7412 ER - TY - JOUR TI - Individual members of the light-harvesting complex II chlorophyll a/b binding protein gene family in pea (Pisum sativum) show differential responses to ultraviolet-B radiation AU - MacKerness, S. A. H. AU - Liu, L. S. AU - Thomas, B. AU - Thompson, William AU - Jordan, B. R. AU - White, M. J. T2 - Physiologia Plantarum AB - Light‐harvesting complex II chlorophyll a/b ‐binding protein (Lhcb) mRNA levels are differentially affected by ultraviolet‐B radiation (UV‐B, 280‐320 nm) at different stages of development of pea ( Pisum sativum L. cv. Feltham first) seedlings. Addition of UV‐B radiation to the light periods of diurnal cycles of white light resulted in reduction of total Lhcb mRNAs in green leaves but a transient increase in etiolated buds. The aims of this study were to determine the stage during de‐etiolation at which supplementary UV‐B began to inhibit Lhcb gene expression, and to determine whether differential regulation of individual Lhcb genes could explain the differential response to supplementary UV‐B at different developmental stages. All seven Lhcb mRNAs were shown to increase in etiolated buds transferred to a diurnal cycle with supplementary UV‐B during the light periods, but were greatly reduced in green leaves given the same treatment. Therefore, the different responses of total Lhcb mRNA levels to UV‐B radiation in green leaves and etiolated buds are not primarily due to the expression of different members of the Lhcb gene family at different developmental stages. However, the Lhcb genes could be divided into two groups based on their sensitivities to UV‐B. Transcripts from the three genes, Lhcb1 * 2 , Lhcb1 * 3 and Lhcb1 * 5 , which were undetectable in dark‐grown etiolated buds, exhibited stronger responses to supplementary UV‐B in green leaves than the four genes, Lhcb1 * 1 , Lhcb1 * 4 , Lhcb2 * 1 and Lhcb3 * 1 , which showed low levels of initial transcript accumulation in dark‐grown etiolated buds. The effect of UV‐B on Lhcb mRNA levels were, however, correlated with chlorophyll content, suggesting that the developmental stage of chloroplasts may be important in determining the responses of the Lhcb genes to supplementary UV‐B radiation in pea seedlings. DA - 1998/// PY - 1998/// DO - 10.1034/j.1399-3054.1998.1030311.x VL - 103 IS - 3 SP - 377–384 ER - TY - JOUR TI - Diploid hybrid speciation in Penstemon (Scrophulariaceae) AU - Wolfe, AD AU - Xiang, QY AU - Kephart, , SR T2 - PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA AB - Hybrid speciation has played a significant role in the evolution of angiosperms at the polyploid level. However, relatively little is known about the importance of hybrid speciation at the diploid level. Two species of Penstemon have been proposed as diploid hybrid derivatives based on morphological data, artificial crossing studies, and pollinator behavior observations: Penstemon spectabilis (derived from hybridization between Penstemon centranthifolius and Penstemon grinnellii ) and Penstemon clevelandii (derived from hybridization between P. centranthifolius and P. spectabilis ). Previous studies were inconclusive regarding the purported hybrid nature of these species because of a lack of molecular markers sufficient to differentiate the parental taxa in the hybrid complex. We developed hypervariable nuclear markers using inter-simple sequence repeat banding patterns to test these classic hypotheses of diploid hybrid speciation in Penstemon . Each species in the hybrid complex was genetically distinct, separated by 10–42 species-specific inter-simple sequence repeat markers. Our data do not support the hybrid origin of P. spectabilis but clearly support the diploid hybrid origin of P. clevelandii . Our results further suggest that the primary reason diploid hybrid speciation is so difficult to detect is the lack of molecular markers able to differentiate parental taxa from one another, particularly with recently diverged species. DA - 1998/4/28/ PY - 1998/4/28/ DO - 10.1073/pnas.95.9.5112 VL - 95 IS - 9 SP - 5112-5115 SN - 0027-8424 ER - TY - JOUR TI - Biomass of tomato seedlings exposed to an allelopathic phenolic acid and enriched atmospheric carbon dioxide AU - Shafer, , SR AU - Blum, U AU - Horton, SJ AU - Hesterberg, DL T2 - WATER AIR AND SOIL POLLUTION DA - 1998/8// PY - 1998/8// DO - 10.1023/A:1004944731826 VL - 106 IS - 1-2 SP - 123-136 SN - 0049-6979 KW - global change KW - community ecology KW - interference ER -