Plant Physiol, 2005 Jan, 137(1), 328 - 40 Epub 2004 Dec 23. B1-phytoprostanes trigger plant defense and detoxification responses; Loeffler C et al.; Phytoprostanes are prostaglandin/jasmonate-like products of nonenzymatic lipid peroxidation that not only occur ubiquitously in healthy plants but also increase in response to oxidative stress . In this work, we show that the two naturally occurring B(1)-phytoprostanes (PPB(1)) regioisomers I and II (each comprising two enantiomers) are short-lived stress metabolites that display a broad spectrum of biological activities . Gene expression analysis of Arabidopsis (Arabidopsis thaliana) cell cultures treated with PPB(1)-I or -II revealed that both regioisomers triggered a massive detoxification and defense response . Interestingly, expression of several glutathione S-transferases, glycosyl transferases, and putative ATP-binding cassette transporters was found to be increased by one or both PPB(1) regioisomers, and hence, may enhance the plant's capacity to inactivate and sequester reactive products of lipid peroxidation . Moreover, pretreatment of tobacco (Nicotiana tabacum) suspension cells with PPB(1) considerably prevented cell death caused by severe CuSO(4) poisoning . Several Arabidopsis genes induced by PPB(1), such as those coding for adenylylsulfate reductase, tryptophan synthase beta-chain, and PAD3 pointed to an activation of the camalexin biosynthesis pathway that indeed led to the accumulation of camalexin in PPB(1) treated leaves of Arabidopsis . Stimulation of secondary metabolism appears to be a common plant reaction in response to PPB(1) . In three different plant species, PPB(1)-II induced a concentration dependent accumulation of phytoalexins that was comparable to that induced by methyl jasmonate . PPB(1)-I was much weaker active or almost inactive . No differences were found between the enantiomers of each regioisomer . Thus, results suggest that PPB(1) represent stress signals that improve plants capacity to cope better with a variety of stresses. Biotechnol Bioeng . 2004 Dec 22; {Epub ahead of print} Effect of nitric oxide on catharanthine production and growth of Catharanthus roseus suspension cells; Xu M et al.; Sodium nitroprusside (SNP) was used as the donor of nitric oxide (NO) to investigate its effect on catharanthine synthesis and the growth of Catharanthus roseus suspension cells . The results showed that SNP at high concentrations (10.0 and 20.0 mmol/L) stimulated catharanthine formation of C . roseus cells, but inhibited growth of the cells . Low concentrations of SNP (0.1 and 0.5 mmol/L) enhanced the growth of C . roseus cells, but had no effect on catharanthine synthesis . The maximum total catharanthine production was achieved by the addition of 0.5 and 10.0 mmol/L SNP to the cultures at day 0 and day 10, respectively, being about threefold of the control . NO-induced catharanthine production of C . roseus cells was strongly suppressed by jasmonic acid (JA) biosynthesis inhibitor ibuprofen (IBU) and nordihydroguaiaretic (NDGA) . The result suggests that the stimulatory role of NO on catharanthine production is partially JA-dependent . (c) 2004 Wiley Periodicals, Inc. Plant Cell Physiol, 2004 Nov, 45(11), 1715 - 9 Ectopic expression of the NtSET1 histone methyltransferase inhibits cell expansion, and affects cell division and differentiation in tobacco plants; Shen WH et al.; The tobacco NtSET1 gene encodes a member of the SUV39H family of histone methyltransferases . Ectopic expression of NtSET1 causes an increase in methylated histone H3 lysine 9 and abnormal chromosome segregation in tobacco suspension cells, and inhibits tobacco plant growth . Here we show that the inhibition of plant growth was caused by reduced cell expansion as well as by abnormal cell division and differentiation . We found that deletion of the C-terminally located catalytic domain of the protein abolished the ectopic effects of NtSET1 on plant growth . Our results indicate that histone H3 lysine 9 methylation is a critical mark of epigenetic control for plant development. Nucleosides Nucleotides Nucleic Acids, 2004 Oct, 23(8-9), 1451 - 4 Online fluorescent method to assess BCRP/ABCG2 activity in suspension cells; Hooijberg JH et al.; An online method was developed to monitor BCRP mediated efflux of fluorescent substrates in suspension cells . To this end, a 2-compartment system consisting of a transwell cup and a cuvette was used . In this system we were able to observe differences in efflux kinetics between BCRP overexpressing RPMI 8226/MR cells and parental myeloid RPMI 8226(s) cells using only 50,000 cells per experiment . 8226/MR cells displayed a larger cellular efflux rate of the BCRP substrate Hoechst 33342, as compared to the wildtype cells . This difference in efflux rate was completely decreased in the presence of the BCRP inhibitor Ko143. Plant Cell Rep, 2004 Nov, 23(6), 371 - 6 Epub 2004 Nov. Efficient plant regeneration from suspension cells of Allium cepa L; Zhang W et al.; Plant regeneration from calli of three cultivars of Allium cepa (Senshuki, O.Pki and Shojovaka) was investigated . Callus was induced on four variations of BDS medium containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP) . The regeneration frequency of calli of cvs . Senshuki and O.Pki subcultured on solid MS medium supplemented with BAP ranged from 50% to 80%; this frequency decreased to less than 30% after subculture in the dark in liquid BDS medium . By repeating the dark/light transitions of the culture protocol and by selecting for green cell clusters, we were able to increase the regeneration frequency to more than 80% in all three cultivars . These cell clusters maintained a high regeneration capacity in subsequent subcultures in the absence of light for 2 months . Most (97%) of the regenerated plantlets had a normal diploid karyotype (2 n=16) that was identical to that of the mother plants, although 3% of the regenerated plants of cv . Shojovaka had a tetraploid karyotype. J Biomed Mater Res A, 2005 Jan 1, 72A(1), 1 - 9 Adhesion of human U937 macrophages to phosphorylcholine-coated surfaces; Gong YK et al.; A new type of amphiphilic phosphorylcholine (PC) polymer was used in this work to develop a cell culture surface that allows the attachment of U937 macrophages . The PC polymer was a random copolymer of N-isopropylacrylamide (45%), N-(phosphorylcholine)-N'-(ethylenedioxy-bis(ethyl)) acrylamide (41%), and the hydrophobic monomer N-(n-octadecyl) acrylamide (14%) . Polypropylene (PP) films (1 cm2) were coated with the polymer solution by immersion . U937 macrophage suspensions were applied on PC polymer-coated surfaces and incubated for up to 72 h at 37 degrees C . While U937 cells did not adhere to PP, ammonia, nitrogen, or oxygen plasma-treated surfaces, they attached rapidly on PC-coated surfaces (< 1 h), proliferated, and stayed attached to the modified surface for at least 72 h, suggesting that unique features of the PC polymer, and the U937 macrophages, are responsible for the attachment of these cells . We compared the effect of Co2+ and Cr3+ ions on the expression of bone-resorbing cytokines (TNF-alpha, IL-6, IL-1beta) in U937 macrophages cultured on PC-coated surfaces to the response of U937 macrophages in suspension . Cytokine gene expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR) . Addition of Co2+ and Cr3+ ions led to a significant increased expression of TNF-alpha in both cultured and suspension cells . On the other hand, Co2+ and Cr3+ ions had a weak stimulatory effect or no effect on IL-1beta and IL-6, respectively, in both cultured and suspension cells . In conclusion, the use of PC polymer-modified surfaces might offer promising new opportunities for the culture of human U937 cells and may also point to the mechanism by which macrophages interact with lipid bilayers of biological membranes. Apoptosis, 2004 Nov, 9(6), 833 - 41 Acidic-rich region of amyloid precursor protein induces glial cell apoptosis; Sun KH et al.; Amyloid precursor protein (APP) has several caspase cleavage sites in its C-terminal cytoplasmic domain and N-terminal extracellular domain . Caspase cleavages of APP at its cytosolic tail may result in releasing the domain and inducing cell death . During apoptosis, the N-terminal domain may also be processed at amino acids 197 and 219 by caspases leading to unmasking of an acidic-rich region (AR) . In this study, AR-exposing APP was shown to inhibit cell growth after transfection into RBA-1 astrocytes and BV-2 microglial cells . The recombinant AR from residue 220 to 288 of APP (APP220-288) was produced and its biological activities were analyzed . APP220-288 induced morphological changes, cell death, and DNA fragmentation in BV-2 and RBA-1 cells . However, AR was determined to have no apparent effects in suspension cells, erythroleukemia K562 cells, and Jurkat T cells . The cytotoxicity was depending on negative charge cluster and the apoptotic activity of AR was attributed to the inhibition of cell adhesion . In BV-2 microglial cells, AR significantly stimulated Fas expression, although expressions of the pro-inflammatory cytokine genes were not detected . APP220-288 also induced nitric oxide synthase (iNOS) expression and nitric oxide (NO) production . These findings indicate that the acidic-rich domain of APP may have apoptotic activity due to inhibition of cell adhesion and induction of iNOS and Fas expressions . Moreover, unmasking the apoptosis-induced AR may activate and exacerbate glial cells which in turn lead to further progression of the death program. Planta . 2004 Oct 6; {Epub ahead of print} Catalase and alternative oxidase cooperatively regulate programmed cell death induced by beta-glucan elicitor in potato suspension cultures; Mizuno M et al.; In potato ( Solanum tuberosum L.) suspension cells, the expression of the gene encoding alternative oxidase (AOX) and H(2)O(2) accumulation were induced by treatment with beta-glucan elicitor . The inhibition of catalase activity enhanced both AOX mRNA expression and the production of H(2)O(2), whereas the ascorbate peroxidase inhibitor did not have any effect on these responses . Simultaneous inhibition of catalase and AOX activities in elicited cells dramatically increased H(2)O(2) accumulation, leading to the disruption of mitochondrial membrane potential (DeltaPsi(m)) and programmed cell death (PCD) . The results demonstrate, for the first time, that not only AOX but also catalase plays a central role in the suppression of mitochondrial DeltaPsi(m) breakdown and PCD induced by beta-glucan elicitor. Appl Microbiol Biotechnol . 2004 Oct 5; {Epub ahead of print} Elicitor-induced nitric oxide burst is essential for triggering catharanthine synthesis in Catharanthus roseus suspension cells; Xu M et al.; Elicitor prepared from the cell walls of Penicillium citrinum induced multiple responses in Catharanthus roseus suspension cells, including rapid generation of nitric oxide (NO), sequentially followed by enhancement of catharanthine production by C . roseus cells . Elicitor-induced catharanthine biosynthesis was blocked by NO-specific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and nitric oxide synthase (NOS) inhibitor S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU) . PBITU also strongly inhibited elicitor-induced NO generation by C . roseus suspension cells . The inhibiting effect of PBITU on elicitor-induced catharanthine production was reversed by external application of NO via the NO-donor sodium nitroprusside . The results strongly suggested that NO, generated by NOS or NOS-like enzymes in C . roseus suspension cells when treated with the fungal elicitor, was essential for triggering catharanthine synthesis. Plant Physiol Biochem, 2004 Jul-Aug, 42(7-8), 617 - 22 Characterization of DNA end-binding activities in higher plants; Yan KH et al.; DNA double-strand-breaks (DSB) are the most severe lesion in cells exposing to ionizing radiation and many other stress environments . Repair of DNA DSB is therefore critical to cellular survival . In this work, we observed the double-stranded DNA end-binding (DEB) like activities in rice (Oryza sativa L . cv . TN5) suspension cells and hypocotyls from etiolated mung bean (Vigna radiata L . TN5) seedlings . Higher plant DEB-like protein binds primarily to linearized double-stranded DNA ends . Competition of unlabeled probe was examined in double-stranded DEB assay of cell extracts from rice and mung bean . DEB-like activities of higher plants did not depend on sequence and types of double-stranded DNA ends . Distinct electrophoretic mobility shift patterns and binding features further indicate that DEB-like factors from various sources might not share identical structure and function, and probably belong to different types of DEB proteins from higher plants . Our evidence suggests that DEB proteins are certainly ubiquitous in all organisms probably for repairing and processing double-stranded DNA breaks from formidable lethal lesion . FEBS Lett, 2004 Aug 27, 573(1-3), 51 - 4 Phosphate or phosphite addition promotes the proteolytic turnover of phosphate-starvation inducible tomato purple acid phosphatase isozymes; Bozzo GG et al.; Within 48 h of the addition of 2.5 mM phosphate (HPO42-, Pi) or phosphite (H2PO3-, Phi) to 8-day-old Pi-starved (-Pi) tomato suspension cells: (i) secreted and intracellular purple acid phosphatase (PAP) activities decreased by about 12- and 6-fold, respectively and (ii) immunoreactive PAP polypeptides either disappeared (secreted PAPs) or were substantially reduced (intracellular PAP) . The degradation of both secreted PAP isozymes was correlated with the de novo synthesis of two extracellular serine proteases having M(r)s of 137 and 121 kDa . In vitro proteolysis of purified secreted tomato PAP isozymes occurred following their 24 h incubation with culture filtrate from Pi-resupplied cells . The results indicate that Pi or Phi addition to -Pi tomato cells induces serine proteases that degrade Pi-starvation inducible extracellular proteins. Plant Physiol, 2004 Aug, 135(4), 2330 - 47 Epub 2004 Aug 13. Transcriptome profiling of the response of Arabidopsis suspension culture cells to Suc starvation; Contento AL et al.; Upon encountering nutrient stress conditions, plant cells undergo extensive metabolic changes and induce nutrient recycling pathways for their continued survival . The role of nutrient mobilization in the response of Arabidopsis suspension cells to Suc starvation was examined . Vacuolar autophagy was induced within 24 h of starvation, with increased expression of vacuolar proteases that are likely to be required for degradation of cytoplasmic components delivered to the vacuole, and thus for nutrient recycling . After 48 h of starvation, culture viability began to decrease, and substantial cell death was evident by 72 h . To provide further insight into the pathways required for survival during Suc deficit, transcriptional profiling during Suc starvation was performed using the ATH1 GeneChip array containing 22,810 probe sets . A significant increase in transcript levels was observed for 343 genes within 48 h of starvation, indicating a response to nutrient stress that utilizes the recycling of cellular components and nutrient scavenging for maintaining cell function, the protection of the cell from death through activation of various defense and stress response pathways, and regulation of these processes by specific protein kinases and transcription factors . These physiological and molecular data support a model in which plant cells initiate a coordinated response of nutrient mobilization at the onset of Suc depletion that is able to maintain cell viability for up to 48 h . After this point, genes potentially involved in cell death increase in expression, whereas those functioning in translation and replication decrease, leading to a decrease in culture viability and activation of cell death programs. Plant Cell Rep . 2004 Jul 28; {Epub ahead of print} Efficient plant regeneration from suspension cells of Allium cepa L; Zhang W et al.; Plant regeneration from calli of three cultivars of Allium cepa (Senshuki, O.Pki and Shojovaka) was investigated . Callus was induced on four variations of BDS medium containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP) . The regeneration frequency of calli of cvs . Senshuki and O.Pki subcultured on solid MS medium supplemented with BAP ranged from 50% to 80%; this frequency decreased to less than 30% after subculture in the dark in liquid BDS medium . By repeating the dark/light transitions of the culture protocol and by selecting for green cell clusters, we were able to increase the regeneration frequency to more than 80% in all three cultivars . These cell clusters maintained a high regeneration capacity in subsequent subcultures in the absence of light for 2 months . Most (97%) of the regenerated plantlets had a normal diploid karyotype (2 n=16) that was identical to that of the mother plants, although 3% of the regenerated plants of cv . Shojovaka had a tetraploid karyotype. Plant Mol Biol, 2004 Feb, 54(3), 311 - 23 Purification, cloning and functional expression of hydroxyphenylpyruvate reductase involved in rosmarinic acid biosynthesis in cell cultures of Coleus blumei; Kim KH et al.; Hydroxyphenylpyruvate reductase (HPPR) is an enzyme involved in the biosynthesis of rosmarinic acid in Lamiaceae reducing hydroxyphenylpyruvates in dependence of NAD(P)H to the corresponding hydroxyphenyllactates . The HPPR protein was purified from suspension cells of Coleus blumei accumulating high levels of rosmarinic acid by ammonium sulfate precipitation, anion exchange chromatography, hydroxylapatite chromatography, chromatography on 2',5'-ADP-Sepharose 4B and SDS-polyacrylamide gel electrophoresis . The protein was tryptically digested and the peptides sequenced . Sequence information was used to isolate a full-length cDNA-clone for HPPR (EMBL accession number AJ507733) by RT-PCR, screening of a C . blumei cDNA-library and 5'-RACE-PCR . The open reading frame of the HPPR-cDNA consists of 939 nucleotides encoding a protein of 313 amino acid residues . The sequence showed that HPPR belongs to the family of D-isomer-specific 2-hydroxyacid dehydrogenases . The HPPR-cDNA was heterologously expressed in Escherichia coli and the protein was shown to catalyse the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvate to 4-hydroxyphenyllactate and 3,4-dihydroxyphenylpyruvate to 3,4-dihydroxyphenyllactate. Plant Cell Rep, 2004 Oct, 23(4), 256 - 62 Epub 2004 Jul 24. Expression of a salt-induced protein (SALT) in suspension-cultured cells and leaves of rice following exposure to fungal elicitor and phytohormones; Kim ST et al.; Phytohormones are essential signal compounds in the regulation of stress-related and defense-related genes . However, there is no clear evidence for any effect of these signal molecules and biotic elicitors on the regulation of the SALT gene in suspension-cultured rice cells . We characterized the expression of a SALT gene following treatment with fungal elicitor, phytohormones, cycloheximide, and inhibitors of protein kinase/phosphatases . SALT expression was up-regulated following treatment with a fungal elicitor, jasmonic acid (JA), abscisic acid (ABA), and NaCl . However, salicylic acid (SA) alone or in combination with one of the other elicitors not only strongly inhibited SALT gene expression but also exhibited an antagonistic effect in suspension cells and leaves . Cycloheximide inhibited SALT accumulation in suspension cells and in leaves, but the inhibitors of protein kinase/phosphatase did not . Immunolocalization revealed that SALT protein was present in xylem parenchyma cells of vascular bundles in the major and minor leaf veins. J Gen Virol, 2004 Aug, 85(Pt 8), 2459 - 69 Nucleo-cytoplasmic shuttling of the beet necrotic yellow vein virus RNA-3-encoded p25 protein; Vetter G et al.; The protein p25 encoded by beet necrotic yellow vein virus (BNYVV) RNA-3 is involved in symptom expression of infected plants . Confocal microscopy analysis of wild-type and mutated p25 fused to GFP and transiently expressed in BY-2 tobacco suspension cells identified a nuclear localization signal (NLS) in the N-terminal part of the protein . Functionality of the NLS was confirmed by pull-down assays using rice and pepper importin-alpha . Furthermore, it was demonstrated that p25 contains a nuclear export sequence sensitive to leptomycin B . The nuclear export signal (NES) was characterized by mutagenesis . A GFP-p25 fusion protein expressed during a BNYVV infection of Chenopodium quinoa leaves had the same subcellular localization as observed during transient expression in BY-2 cells . The symptom phenotype induced by expression of GFP-p25 during infection was similar to that induced by wild-type virus . Studies with mutated derivatives of GFP-p25 revealed that symptom phenotype was altered when the subcellular localization of GFP-p25 was modified. Cells Tissues Organs, 2004, 177(1), 37 - 46 Establishment and characterization of a human gastric carcinoma cell line TMC-1; Shyu RY et al.; Established cancer cell lines are useful in the study of various cancers . We established a human gastric carcinoma cell line TMC-1 derived from the lymph node of a moderately differentiated adenocarcinoma of the stomach . TMC-1 cells grew in vitro as a mixture of attached and suspension cells, and exhibited spindle or ovoid morphology . They had a population doubling time of 15 h, a plating efficiency of 61%, formed colonies in semisolid agar, secreted the tumor marker CA 19-9, and were tumorigenic in athymic nude mice . The cells expressed E-cadherin and beta-catenin . The karyotypic analysis demonstrated hyperdiploid features with a modal chromosome of 53 . The cell had the deletion at chromosome 18q and gains at chromosome 2p13-25, 5p15, 5q21-35, 7, 8q24, 9q, 11, 12p, 14q24-32 and 20 . Analysis by fluorescence in situ hybridization showed the deletion at 7qtel and duplication at 7q11.2 at the rearranged chromosome 7 . Growth of TMC-1 cells was inhibited by 27-32% by interferon-alpha (2,000 U/ml) and by interferon-gamma with an IC50 of 125 U/ml . The cell line is tumorigenic in vivo, and its growth is moderately inhibited by interferon-alpha and interferon-gamma . It can be used to develop new modalities of human gastric cancer treatment . Plant Physiol, 2004 Jul, 135(3), 1367 - 77 Epub 2004 Jul 02. Signal peptide-dependent targeting of a rice alpha-amylase and cargo proteins to plastids and extracellular compartments of plant cells; Chen MH et al.; alpha-Amylases are important enzymes for starch degradation in plants . However, it has been a long-running debate as to whether alpha-amylases are localized in plastids where starch is stored . To study the subcellular localization of alpha-amylases in plant cells, a rice (Oryza sativa) alpha-amylase, alphaAmy3, with or without its own signal peptide (SP) was expressed in transgenic tobacco (Nicotiana tabacum) and analyzed . Loss-of-function analyses revealed that SP was required for targeting of alphaAmy3 to chloroplasts and/or amyloplasts and cell walls and/or extracellular compartments of leaves and suspension cells . SP was also required for in vitro transcribed and/or translated alphaAmy3 to be cotranslationally imported and processed in canine microsomes . alphaAmy3, present in chloroplasts of transgenic tobacco leaves, was processed to a product with Mr similar to alphaAmy3 minus its SP . Amino acid sequence analysis revealed that the SP of chloroplast localized alphaAmy3 was cleaved at a site only one amino acid preceding the predicted cleavage site . Function of the alphaAmy3 SP was further studied by gain-of-function analyses . beta-Glucuronidase (GUS) and green fluorescence protein fused with or without the alphaAmy3 SP was expressed in transgenic tobacco or rice . The alphaAmy3 SP directed translocation of GUS and green fluorescence protein to chloroplasts and/or amyloplasts and cell walls in tobacco leaves and rice suspension cells . The SP of another rice alpha-amylase, alphaAmy8, similarly directed the dual localizations of GUS in transgenic tobacco leaves . This study is the first evidence of SP-dependent dual translocations of proteins to plastids and extracellular compartments, which provides new insights into the role of SP in protein targeting and the pathways of SP-dependent protein translocation in plants. Protoplasma, 2004 Jun, 223(2-4), 93 - 102 Epub 2004 Jun 22. Plasmodesmata in Arabidopsis thaliana suspension cells; Bayer E et al.; A current challenge in plant biology is to identify the structural and functional components of plasmodesmata (PDs) . The use of plant tissue as a source material for plasmodesmal characterisation has had limited success, so we have explored the frequency and features of PDs occurring in suspension cell cultures of Arabidopsis thaliana . This material has the advantages of homogeneity, quantity, and ease of disruption . Using light and electron microscopy and immunostaining for callose and calreticulin, we showed that suspension cells laid down abundant PDs in division walls, and that vestiges of these structures were retained as half PDs even when the cell-to-cell contacts were disrupted during culture growth . Although callose was a reliable marker for PD distribution, which was deposited in an organised collar around the neck of PDs, it was not abundant in unstressed cells . Calreticulin and the chemical stain 3,3'-dihexyloxacarbocyanine iodide also provided useful markers when monitoring PDs in cell wall preparations by light microscopy . Purified cell walls were shown to be virtually free of contamination from cytoplasmic components, except for the presence of small amounts of cortical endoplasmic reticulum attached to PDs . Hence, clean cell walls from A . thaliana suspension cells provide a valuable resource for a proteomic approach to the analysis of plasmodesmal components. Plant Physiol Biochem, 2004 May, 42(5), 429 - 35 Ergosterol elicits oxidative burst in tobacco cells via phospholipase A2 and protein kinase C signal pathway; Kasparovsky T et al.; Ergosterol, a typical fungal sterol, induced in tobacco (Nicotiana tabacum L . cv . Xanthi) suspension cells the synthesis of reactive oxygen species and alkalization of the external medium that are dependent on the mobilization of calcium from internal stores . We used specific inhibitors to elucidate the signal pathway triggered by ergosterol compared with cryptogein, a proteinaceous elicitor of Phytophthora cryptogea . Herbimycin A and genistein, inhibitors of tyrosine protein kinases, had no effect on the oxidative burst and pH changes induced by both elicitors . Similarly, H-89, an inhibitor of protein kinase A, had no effect on the induction of these defense reactions . However, the response to both elicitors was completely blocked by NPC-15437, a specific inhibitor of animal protein kinase C (PKC) . The responses induced by cryptogein but not those induced by ergosterol were inhibited by U73122 and neomycin, inhibitors of phospholipase C (PLC) . On the other hand, the activity of phospholipase A2 (PLA2) measured using a fluorogenic substrate was stimulated by ergosterol and not by cholesterol and cryptogein . A specific inhibitor of PLA2, arachidonic acid trifluoromethyl ketone (AACOCF3), inhibited the pathway stimulated by ergosterol but not that induced by cryptogein . These results suggest that the cryptogein-induced signal pathway leading to the oxidative burst and DeltapH changes includes PLC and PKC, whereas this response induced by ergosterol includes PLA2 and PKC . Plant Cell Rep, 2004 Aug, 23(1-2), 1 - 8 Epub 2004 May 25. Cryopreservation of embryogenic suspension cultures of Cyclamen persicum mill; Winkelmann T et al.; We have developed a simple protocol for the cryopreservation of embryogenic suspension cultures of Cyclamen persicum . Embryogenic suspension cultures in the linear growth phase 7-10 days after subculture were used for cryopreservation . Of the different cryoprotectants tested during a 2-day pre-culture, 0.6 M sucrose resulted in the highest re-growth rates of 75% . An additional pre-treatment with 0.6 M sucrose and 10% DMSO (dimethylsulfoxide) for 1 h also positively affected re-growth . Microscopic studies on viability revealed that only few small embryogenic cells survived cryopreservation, while vacuolated single cells died . Experiments in which the duration of the pre-culture period--i.e . the length of time the embryogenic suspension cells were exposed to 0.6 M sucrose--was varied showed that 2-4 days was the most optimal exposure time to 0.6 M sucrose . Callus re-grown after cryopreservation showed growth rates similar to that of unfrozen callus and regenerated even higher numbers of somatic embryos than unfrozen callus. FEBS Lett, 2004 May 21, 566(1-3), 307 - 10 Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells; Unnamalai N et al.; We have used cationic oligopeptide polyarginine-12mer (POA) to deliver double-stranded RNA (dsRNA), prepared in vitro, to tobacco (Nicotiana tabacum) suspension cells . POA interacts electrostatically with dsRNA to form a complex . When dsRNA for the GUS or NPTII gene was delivered into cells carrying the same genes, the corresponding mRNA was degraded . Using RNase protection assay we were able to detect 21-bp small interfering RNA in dsRNA/POA-treated cells . These results demonstrate that POA can be used to deliver dsRNA to induce post-transcriptional gene silencing in plant cells. Plant Physiol, 2004 May, 135(1), 231 - 43 Plasma membrane depolarization induced by abscisic acid in Arabidopsis suspension cells involves reduction of proton pumping in addition to anion channel activation, which are both Ca2+ dependent; Brault M et al.; In Arabidopsis suspension cells a rapid plasma membrane depolarization is triggered by abscisic acid (ABA) . Activation of anion channels was shown to be a component leading to this ABA-induced plasma membrane depolarization . Using experiments employing combined voltage clamping, continuous measurement of extracellular pH, we examined whether plasma membrane H(+)-ATPases could also be involved in the depolarization . We found that ABA causes simultaneously cell depolarization and medium alkalinization, the second effect being abolished when ABA is added in the presence of H+ pump inhibitors . Inhibition of the proton pump by ABA is thus a second component leading to the plasma membrane depolarization . The ABA-induced depolarization is therefore the result of two different processes: activation of anion channels and inhibition of H(+)-ATPases . These two processes are independent because impairing one did not suppress the depolarization . Both processes are however dependent on the {Ca2+}cyt increase induced by ABA since increase in {Ca(2+)}(cyt) enhanced anion channels and impaired H(+)-ATPases. Plant Physiol, 2004 May, 135(1), 507 - 15 Epub 2004 May 07. Characterization of GaWRKY1, a cotton transcription factor that regulates the sesquiterpene synthase gene (+)-delta-cadinene synthase-A; Xu YH et al.; The cotton (+)-delta-cadinene synthase (CAD1), a sesquiterpene cyclase, catalyzes a branch-point step leading to biosynthesis of sesquiterpene phytoalexins, including gossypol . CAD1-A is a member of CAD1 gene family, and its promoter contains a W-box palindrome with two reversely oriented TGAC repeats, which are the proposed binding sites of WRKY transcription factors . We isolated several WRKY cDNAs from Gossypium arboreum . One of them, GaWRKY1, encodes a protein containing a single WRKY domain and a putative N-terminal Leu zipper . Similar to genes encoding enzymes of cotton sesquiterpene pathway, GaWRKY1 was down-regulated in a glandless cotton cultivar that contained much less gossypol . GaWRKY1 showed a temporal and spatial pattern of expression comparable to that of CAD1-A in various aerial organs examined, including sepal, stigma, anther, and developing seeds . In suspension cells, expression of both GaWRKY1 and CAD1-A genes and biosynthesis of sesquiterpene aldehydes were strongly induced by a fungal elicitor preparation and methyl jasmonate . GaWRKY1 interacted with the 3x W-box derived from CAD1-A promoter in yeast (Saccharomyces cerevisiae) one-hybrid system and in vitro . Furthermore, in transgenic Arabidopsis plants, overexpression of GaWRKY1 highly activated the CAD1-A promoter, and transient assay in tobacco (Nicotiana tabacum) leaves demonstrated that W-box was required for this activation . These results suggest that GaWRKY1 participates in regulation of sesquiterpene biosynthesis in cotton, and CAD1-A is a target gene of this transcription factor. Plant Physiol . 2004 Apr 30; {Epub ahead of print} Plasma Membrane Depolarization Induced by Abscisic Acid in Arabidopsis Suspension Cells Involves Reduction of Proton Pumping in Addition to Anion Channel Activation, Which Are Both Ca2+ Dependent; Brault M et al.; In Arabidopsis suspension cells a rapid plasma membrane depolarization is triggered by abscisic acid (ABA) . Activation of anion channels was shown to be a component leading to this ABA-induced plasma membrane depolarization . Using experiments employing combined voltage clamping, continuous measurement of extracellular pH, we examined whether plasma membrane H(+)-ATPases could also be involved in the depolarization . We found that ABA causes simultaneously cell depolarization and medium alkalinization, the second effect being abolished when ABA is added in the presence of H(+) pump inhibitors . Inhibition of the proton pump by ABA is thus a second component leading to the plasma membrane depolarization . The ABA-induced depolarization is therefore the result of two different processes: activation of anion channels and inhibition of H(+)-ATPases . These two processes are independent because impairing one did not suppress the depolarization . Both processes are however dependent on the {Ca(2+)}cyt increase induced by ABA since increase in {Ca(2+)}cyt enhanced anion channels and impaired H(+)-ATPases. In Vitro Cell Dev Biol Anim, 2003 Nov-Dec, 39(10), 420 - 3 Differential expression of mammalian or viral promoter-driven gene in adherent versus suspension cells; Feng G et al.; Although expression vectors using viral and mammalian promoters constitutively express genes of interest in adherent cells, few studies have examined whether the function of these vectors in suspended cells, such as in over-agar or soft agar assay (an in vitro cell transformation assay), is as robust as when they are in adherent cells . The selection of appropriate expression vector to optimally express genes in suspended cells would be useful in determining whether these genes play a critical role in maintaining colony formation or cell transformation . To compare promoter-driven expression vector function in adherent versus suspension cells, we performed transient transfection assays using viral (simian virus 40 {SV40} and cytomegalovirus {CMV}) and mammalian (beta-actin) promoters fused to luciferase or beta-galactosidase reporter gene . Over-agar assay was used to suspend cells on top of agar, which allowed cell retrieval and analysis . We found that beta-actin and SV40 promoters exhibited suppressed gene expression of 70 and 56%, respectively, in cells suspended on agar compared with those attached on plates . The suppressed response by the exogenous beta-actin promoter in suspension was consistent with the response of the endogenous beta-actin promoter activity because the steady-state level of beta-actin messenger ribonucleic acid in suspended cells was significantly reduced by 50% relative to that expressed in attached cells . In contrast to SV40 promoter, CMV promoter activity was not decreased in cells suspended in over-agar when compared with adherent cells . These studies show that regardless of mammalian or viral vectors, one cannot assume that all expression vectors behave similarly in both suspension and adherent state. Plant Cell Rep, 2004 Jul, 22(12), 903 - 9 Epub 2004 Apr 07. Transgenic Russian wildrye (Psathyrostachys juncea) plants obtained by biolistic transformation of embryogenic suspension cells; Wang ZY et al.; Russian wildrye (Psathyrostachys juncea (Fisch.) Nevski) is a cool-season forage species well adapted to semi-arid climates . We are interested in developing biotechnological methods to improve this monocot forage species . Single genotype-derived embryogenic suspension cultures were established from the Russian wildrye cultivar Bozoisky-Select, and were used as target cells for biolistic transformation . A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker, and a chimeric beta-glucuronidase (gusA) gene was co-transformed with hph . Resistant calli were obtained from 29% of the bombarded dishes after selection with 200 mg/l hygromycin . Plants were regenerated from 45% of the hygromycin resistant calli . Thirty-six transgenic Russian wildrye plants were recovered after microprojectile bombardment of suspension cells and subsequent hygromycin selection . The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples . When a second gene (gusA) was co-transformed with hph, a reasonably high co-transformation frequency of 78% was observed . Transgenic expression of gusA was confirmed by GUS staining of shoot and leaf tissues . Fertile transgenic plants were obtained after two winters of vernalization under field conditions . This is the first report on the generation of transgenic plants in Russian wildrye. Biosci Biotechnol Biochem, 2004 Mar, 68(3), 705 - 13 Sequential Development of Cysteine Proteinase Activities and Gene Expression during Somatic Embryogenesis in Carrot; Mitsuhashi W et al.; Three bands of proteinase activity (Rf values of 0.5, 0.6, and 0.7) were detected on activity-stained gels after native gel electrophoresis of carrot (Daucus carota L . cv US-Harumakigosun) suspension cells . After the induction of somatic embryogenesis, one activity band (0.7 band) rapidly disappeared; the 0.6 band was absent at the heart-shaped embryo stage . However, the intensity of the 0.5 band increased during embryogenesis . An additional band (0.25 band) appeared after the torpedo-shaped stage . Three bands (0.25, 0.5, and 0.6) were also detected in zygotic seeds . Two activity bands (0.5 and 0.6) were classified as cysteine proteinases based on sensitivities to N-Ethylmaleimide (NEM) or L-3-trans-Carboxyoxirane-2-Carbonyl-L-Leucyl-Agmatine (E-64) . To find candidate genes for the cysteine proteinases, we cloned seven cDNAs encoding putative cysteine proteinases from suspension cells and developing somatic embryos . The expression patterns of the seven genes were categorized into three types (Type A, mRNAs increase concomitantly with somatic embryogenesis; Type B, mRNAs decrease quickly in organized cells; Type C, no significant change in transcript level during somatic embryogenesis). Plant Cell Rep, 2004 Jun, 22(11), 848 - 58 Epub 2004 Mar 26. Methylmalonate-semialdehyde dehydrogenase is induced in auxin-stimulated and zinc-stimulated root formation in rice; Oguchi K et al.; Proteins induced in rice by auxin and zinc were determined by proteome analysis . Cultured suspension cells of rice were treated with 2,4-dichlorophenoxyacetic acid and ZnSO4 and then proteins were separated by two-dimensional polyacrylamide gel electrophoresis; seven proteins were found to be induced by auxin and zinc . Of these seven, methylmalonate-semialdehyde dehydrogenase (MMSDH) was elevated by treatment with auxin alone . MMSDH was detected in cultured suspension cells, root and leaf sheath, but not in leaf blades . MMSDH responded to auxin and gibberellin, but did not respond to brassinolide and cytokinin . Furthermore, the amount of MMSDH in slr1, a constitutive gibberellin response mutant, was 2-fold that of wild type . MMSDH mRNA and protein were stimulated in root formation induced by auxin and/or zinc over a 4-week period . These results suggest that MMSDH may be necessary for root formation in rice induced by auxin and/or zinc . Cell Res, 2004 Feb, 14(1), 27 - 33 Alpha-picolinic acid, a fungal toxin and mammal apoptosis-inducing agent, elicits hypersensitive-like response and enhances disease resistance in rice; Zhang HK et al.; Alpha-picolinic acid (PA), a metabolite of tryptophan and an inducer of apoptosis in the animal cell, has been reported to be a toxin produced by some of plant fungal pathogens and used in screening for disease resistant mutants . Here, we report that PA is an efficient apoptosis agent triggering cell death of hypersensitive-like response in planta . Confirmed by Fluorescence Activated Cell Sorter (FACS), rice suspension cells and leaves exhibited programmed cell death induced by PA . The PA-induced cell death was associated with the accumulation of reactive oxygen species that could be blocked by diphenylene iodonium chloride, indicating that the generation of reactive oxygen species was NADPH-oxidase dependent . We also demonstrated the induction of rice defense-related genes and subsequent resistant enhancement by PA against the rice blast fungus Magnaporthe grisea . Hence, it was concluded that the PA-stimulated defense response likely involves the onset of the hypersensitive response in rice, which also provides a simple eliciting tool for studying apoptosis in the plant cell. Cell Res, 2004 Feb, 14(1), 8 - 15 Arabidopsis RAV1 is down-regulated by brassinosteroid and may act as a negative regulator during plant development; Hu YX et al.; RAV1 is a novel DNA-binding protein with two distinct DNA-binding domains unique in higher plants, but its role in plant growth and development remains unknown . Using cDNA array, we found that transcription of RAV1 is down-regulated by epibrassinolide (epiBL) in Arabidopsis suspension cells . RNA gel blot analysis revealed that epiBL-regulated RAV1 transcription involves neither protein phosphorylation/dephosphorylation nor newly synthesized protein, and does not require the functional BRI1, suggesting that this regulation might be through a new BR signaling pathway . Overexpressing RAV1 in Arabidopsis results in a retardation of lateral root and rosette leaf development, and the underexpression causes an earlier flowering phenotype, implying that RAV1 may function as a negative regulatory component of growth and development. Int J Biochem Cell Biol, 2004 May, 36(5), 814 - 25 Methotrexate differentially affects growth of suspension and adherent cells; Kimura E et al.; The effects of low concentrations of methotrexate (MTX) on the growth of suspension (FM3A, 2B4 and THP-1) and adherent (NIH3T3 and V79) cells were compared . The concentration of methotrexate to cause the inhibition of cell growth was lower in suspension cells than in adherent cells . The IC(50) for FM3A, 2B4, THP-1, NIH3T3 and V79 cells were 3.5, 5, 9, 30 and 50 nM, respectively . The inhibition of cell growth was reversed completely by tetrahydrofolate and was fully or significantly reversed by adenosine and thymidine, suggesting that the effects of low concentrations of methotrexate result from the inhibition of biosynthesis of purines and pyrimidines . In suspension cells but not in adherent cells there was a decrease in the levels of S-adenosylmethionine and polyamines after methotrexate treatment . Growth of suspension but not adherent cells was significantly recovered by treatment with S-adenosylmethionine . However, treatment with spermidine did not reverse the effects of methotrexate in any of the cell lines . The preferential inhibitory effect of methotrexate in suspension cells versus adherent cells was due mainly to a more rapid uptake of methotrexate . This may be relevant to the in vivo effects of low doses of methotrexate, which have immunosuppressive and anti-inflammatory effects, because lymphocytes are suspension cells. J Pineal Res, 2004 Mar, 36(2), 126 - 31 Attenuation of cold-induced apoptosis by exogenous melatonin in carrot suspension cells: the possible involvement of polyamines; Lei XY et al.; Pretreatment with 43 nM (10 ng/mL) to 86 nM melatonin for 5 days significantly attenuated cold-induced apoptosis in carrot suspension cells (Daucus carota L.) as evidenced by the TUNEL procedure, DNA fragmentation and the morphological changes revealed by electronic microscopy observations . The antiapoptotic effect of melatonin was initially thought to be a result of its antioxidant actions . In our study, however, reactive oxygen species (ROS) generation remained unaffected by melatonin treatment, suggesting that melatonin plays its protective role not related to its direct ROS scavenger . At the same time, notable increases in putrescine and spermidine levels were observed in melatonin-treated cells, which may be responsible for the alleviation of the cold-induced apoptosis . The possible involvement of polyamines in the antiapoptotic effect of melatonin was further confirmed by the inhibitory effect of exogenous polyamines on apoptosis as displayed by the DNA laddering assay. Plant J, 2004 Mar, 37(5), 654 - 67 Phosphorylation and 14-3-3 binding of Arabidopsis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Kulma A et al.; Fructose 2,6-bisphosphate (fru-2,6-P2) is a signalling metabolite that regulates photosynthetic carbon partitioning in plants . The content of fru-2,6-P2 in Arabidopsis leaves varied in response to photosynthetic activity with an abrupt decrease at the start of the photoperiod, gradual increase through the day, and modest decrease at the start of the dark period . In Arabidopsis suspension cells, fru-2,6-P2 content increased in response to an unknown signal upon transfer to fresh culture medium . This increase was blocked by either 2-deoxyglucose or the protein phosphatase inhibitor, calyculin A, and the effects of calyculin A were counteracted by the general protein kinase inhibitor K252a . The changes in fru-2,6-P2 at the start of dark period in leaves and in the cell experiments generally paralleled changes in nitrate reductase (NR) activity . NR is inhibited by protein phosphorylation and binding to 14-3-3 proteins, raising the question of whether fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase protein from Arabidopsis thaliana (AtF2KP), which both generates and hydrolyses fru-2,6-P2, is also regulated by phosphorylation and 14-3-3s . Consistent with this hypothesis, AtF2KP and NR from Arabidopsis cell extracts bound to a 14-3-3 column, and were eluted specifically by a synthetic 14-3-3-binding phosphopeptide (ARAApSAPA) . 14-3-3s co-precipitated with recombinant glutathione S-transferase (GST)-AtF2KP that had been incubated with Arabidopsis cell extracts in the presence of Mg-ATP . 14-3-3s bound directly to GST-AtF2KP that had been phosphorylated on Ser220 (SLSASGpSFR) and Ser303 (RLVKSLpSASSF) by recombinant Arabidopsis calcium-dependent protein kinase isoform 3 (CPK3), or on Ser303 by rat liver mammalian AMP-activated protein kinase (AMPK; homologue of plant SNF-1 related protein kinases (SnRKs)) or an Arabidopsis cell extract . We have failed to find any direct effect of 14-3-3s on the F2KP activity in vitro to date . Nevertheless, our findings indicate the possibility that 14-3-3 binding to SnRK1-phosphorylated sites on NR and F2KP may regulate both nitrate assimilation and sucrose/starch partitioning in leaves. FEBS Lett, 2004 Jan 30, 558(1-3), 85 - 8 Histidine-containing phosphotransfer domain extinction by RNA interference turns off a cytokinin signalling circuitry in Catharanthus roseus suspension cells; Papon N et al.; We previously reported that cytokinins (CK) induce the fast and specific transcription of CrRR1, a gene encoding a type A response regulator in Catharanthus roseus cell cultures . Here, we characterized the CrHPt1 gene that encodes a histidine-containing phosphotransfer domain . CrHPt1 was silenced through RNA interference (RNAi) to test its possible implication in the CK signalling pathway . In transgenic lines stably transformed with an intron-spliced construct, the degradation of CrHPt1 transcripts abolishes the CK inductive effect on CrRR1 transcription . These result give a new in vivo functional argument for the crucial role of HPt proteins in the CK signalling pathway leading to the expression of the genes encoding type A response regulators . They also show that RNAi is a powerful strategy to turn off the CK signalling circuitry. Cell Motil Cytoskeleton, 2004 Apr, 57(4), 246 - 58 Microtubules become more dynamic but not shorter during preprophase band formation: a possible "search-and-capture" mechanism for microtubule translocation; Vos JW et al.; The dynamic behavior of the microtubule cytoskeleton plays a crucial role in cellular organization, but the physical mechanisms underlying microtubule (re)organization in plant cells are poorly understood . We investigated microtubule dynamics in tobacco BY-2 suspension cells during interphase and during the formation of the preprophase band (PPB), the cytoskeletal structure that defines the site of cytokinesis . Here we show that after 2 h of microtubule accumulation in the PPB and concurrent disappearance elsewhere in the cortex, the PPB is completed and starts to breakdown exponentially already 20 min before the onset of prometaphase . During formation of the PPB, the dynamic instability, i.e., the stochastic alternating between growing and shrinking phases, of the cortical microtubules outside the PPB increases significantly, but the microtubules do not become shorter . Based on this, as well as on the cross-linking of microtubules in the PPB and the lack of evidence for motor involvement, we propose a "search-and-capture" mechanism for PPB formation, in which the regulation of dynamic instability causes the cortical microtubules to become more dynamic and possibly longer, while the microtubule cross-linking activity of the developing PPB preferentially stabilizes these "searching" microtubules . Thus, microtubules gradually disappear from the cortex outside the PPB and aggregate to the forming PPB . Cytometry A, 2004 Feb, 57(2), 100 - 7 Comparison of flow cytometry and laser scanning cytometry for the analysis of CD34+ hematopoietic stem cells; Oswald J et al.; BACKGROUND: Characterization of hematopoietic stem cells (HSCs) by laser scanning cytometry (LSC) was compared with conventional flow cytometry (FCM) . The method was evaluated for application in the development of advanced cell culture substrates that were supposed to support the ex vivo expansion of HSC . For this purpose, adherent HSCs were grown in culture on thin polymer films coated with reconstituted collagen I fibrils and subsequently analyzed by LSC . METHODS: CD34+ HSCs were isolated from cord blood by immunomagnetic separation and cultivated on polymer films coated with reconstituted collagen I fibrils . Cell surface antigens (CD34, CD29) were stained with antibodies, and nuclei were labeled with a DNA stain (TO-PRO-3 iodide) that does not interfere with the fluorochromes of the antibodies . Fluorescence intensity of the adherent cells was measured by means of LSC . Before and after in vitro expansion for time periods of up to 7 days, suspension cells were analyzed with LSC and FCM . RESULTS: LSC-based analysis enabled reliable quantification of CD34+ cells with bright antigen expression before cell culture . At this stage, LSC and FCM data for CD34 expression at given HSC samples largely coincided . After in vitro expansion, LSC data deviated from FCM data for cells with dim CD34 antigen expression, whereas the fluorescence intensity of the CD29 antigen remained comparable . The deviation between LSC and FCM data for CD34dim was attributed to the better resolution of weak fluorescence by FCM . Based on the preceding evaluation of the method, LSC analysis could be applied to characterize HSCs cultivated on collagen I-coated polymer films without detachment of the cells from the substrate . CONCLUSIONS: LSC-based analysis allows for the automated evaluation of adherent HSCs . Although resolution of weakly expressed antigens can be achieved more precisely with FCM, the method provides a valuable tool to study interactions of HSCs with bioartificial substrates . J Zhejiang Univ Sci, 2004 Feb, 5(2), 137 - 43 Programmed cell death features in apple suspension cells under low oxygen culture; Xu CJ et al.; Suspension-cultured apple fruit cells (Malus pumila Mill . cv . Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions . Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342) . DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding . About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability . Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen . The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit. Biosci Biotechnol Biochem, 2003 Nov, 67(11), 2438 - 47 Gibberellin is essentially required for carrot (Daucus carota L.) somatic embryogenesis: dynamic regulation of gibberellin 3-oxidase gene expressions; Mitsuhashi W et al.; A GA biosynthesis inhibitor, uniconazole, caused many shrunken embryos when it was supplied to cultured carrot (Daucus carota L.) cells at the induction of somatic embryos . The abnormality was prevented by exogenous GA(1) or GA(4) . To analyze the status of GA biosynthesis during somatic embryogenesis, expression patterns of newly isolated genes encoding GA biosynthetic enzymes, two GA 20-oxidases, three GA 3-oxidases, and two GA 2-oxidases were observed by using a semi-quantitative reverse-transcription-polymerase chain reaction with gene-specific primers . Transcript levels of GA 20-oxidases and GA 2-oxidases did not change greatly during development of the somatic embryo . On the other hand, drastic changes were found in three GA 3-oxidase genes . Strikingly, expression of a GA 3-oxidase gene, DcGA3ox2, was elevated once in somatic embryogenesis, but not in the non-induced suspension cells . The enzymatic functions of these gene products were also confirmed using recombinant proteins expressed in Escherichia coli . Our results indicate that GA biosynthesis is required for carrot somatic embryogenesis. Mol Genet Genomics, 2004 Jan, 270(6), 485 - 96 Epub 2003 Nov 21. Proteome analysis of rice tissues by two-dimensional electrophoresis: an approach to the investigation of gibberellin regulated proteins; Tanaka N et al.; Protein databases constructed using high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) were used to explore the proteome expressed in various rice tissues . Proteins from leaf sheath, root, and cultured suspension cells were systematically analyzed using 2D-PAGE, mass spectrometry and Edman sequencing, followed by database searching . In all, 79 of the 431 spots detected by 2D-PAGE in the leaf sheath, 73 of the 508 spots in the root and 140 of the 962 spots in the cultured suspension cells could be identified . Protein lists were constructed for each tissue and used to investigate the effects of gibberellin (GA) treatment . In the leaf sheath, root and cultured suspension cells, 8, 21, and 14 of the identified proteins, respectively, were regulated by GA . These proteins included polypeptides involved in general metabolism, energy production, transcriptional regulation and signal transduction in the leaf sheath; in metabolism and defense in the root; and in metabolism, energy production, cell growth, defense and signal transduction in the cultured suspension cells . These results indicate that the proteome databases assembled in these studies will be useful for the rapid assessment of changes in protein content in specific tissues, and that proteins regulated by GA may play a significant role in tissue growth. J Biomol Screen, 2002 Dec, 7(6), 515 - 25 A homogeneous enzyme fragment complementation cyclic AMP screen for GPCR agonists; Golla R et al.; In the new high-throughput screening (HTS) campaign, receptor functional assays, 3',5'-cyclic adenosine monophosphate (cAMP), intracellular {Ca(2)+}(i), phosphatidylinositol turnover, and reporter-based assays are being used as primary screens as they are now developed as homogeneous and automation-friendly assays . FlashPlate assay and scintillation proximity assay using radiolabeled cAMP have been used for measuring cAMP . A nonradioactive homogeneous HTS assay using HitHunter trade mark enzyme fragment complementation (EFC) technology was evaluated for measuring cAMP in adherent and suspension cells overexpressing a Galpha(s)-coupled receptor . In the EFC-cAMP assay, the beta-galactosidase (beta-gal) donor fragment-cAMP (ED-cAMP) conjugate complements with the beta-gal enzyme acceptor (EA) fragment to form an active beta-gal enzyme . Binding of ED-cAMP conjugate to the anti-cAMP antibody prevents its complementation with the EA fragment to form an active enzyme . Cyclic AMP in the samples compete with ED-cAMP to bind to the anti-cAMP antibody, thus increasing the free ED-cAMP that can complement with the EA fragment to form an active enzyme that is assayed with a luminescent substrate . Thus, this assay results in a positive signal unlike other technologies, wherein the signal is completed by cAMP in the sample . Glucagon-like peptide (GLP)-1 binds to GLP-1 receptor (with a Kd of 0.2 nM) signals through Galpha(s) to activate adenylate cyclase, which results in an increase of intracellular cAMP (EC(50) of 0.3 nM) . GLP-1 stimulation of cAMP levels measured by the EFC method was similar in both adherent and suspension cell formats (EC(50)~0.3 nM) at different cell numbers . The assay was further validated with forskolin, exendin, and several active GLP-1 peptide analogues . The stimulation of cAMP by GLP-1 and forskolin was effectively inhibited by the adenylate cyclase inhibitors MDL-12330A and SQ-22536, confirming that the increased cAMP is through the AC pathway . The assay tolerates dimethyl sulfoxide (DMSO) up to 10%, and tartrazine does not interfere with the assay with the adherent cells up to 1 mM and affects minimally up to 10 microM in suspension cells . The assay is very robust, with a Z' value of 0.7 to 0.8 . The assay was validated with several plates of low molecular weight nonpeptide compounds and peptide agonists with different potencies . The suspension cell protocol is a robust homogeneous assay that involves fewer steps than the adherent cell protocol and is suitable for HTS . The cAMP assay using EFC technology is advantageous in that it has a greater dynamic range of detection; is nonradioactive, very sensitive, robust; has minimal interference from DMSO and colored compounds; and is amenable for automation . An added advantage of this assay is that the cAMP is measured as a positive signal, thereby reducing the incidence of false positives. Biochem J, 2004 Jan 15, 377(Pt 2), 419 - 28 Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures; Bozzo GG et al.; An intracellular acid phosphatase (IAP) from P(i)-starved (-P(i)) tomato ( Lycopersicon esculentum ) suspension cells has been purified to homogeneity . IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (lambda(max)=546 nm) and was insensitive to L-tartrate . PAGE, periodic acid-Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (alpha-subunit) and 57 kDa (beta-subunit) polypeptides . However, the nine N-terminal amino acids of the alpha- and beta-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs . Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P(i)-deprivation was correlated with similar increases in the amount of antigenic IAP alpha- and beta-subunits . IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg(2+), but was potently inhibited by Zn(2+), Cu(2+), Fe(3+), molybdate, vanadate, fluoride and P(i) . Although IAP demonstrated broad substrate selectivity, its specificity constant ( V (max)/ K (m)) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate . IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg(2+) or molybdate . This IAP is proposed to scavenge P(i) from intracellular phosphate esters in -P(i) tomato . A possible secondary IAP role in the metabolism of reactive oxygen species is discussed . IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of -P(i) tomato cells {Bozzo, Raghothama and Plaxton (2002) Eur . J . Biochem . 269, 6278-6286}. J Exp Bot, 2003 Nov, 54(392), 2587 - 8 Epub 2003 Sep 09. CrMYC1, a Catharanthus roseus elicitor- and jasmonate-responsive bHLH transcription factor that binds the G-box element of the strictosidine synthase gene promoter; Chatel G et al.; A cDNA encoding a bHLH transcription factor was isolated by the yeast one-hybrid system from a Catharanthus roseus cDNA library using the G-box element of the Strictosidine synthase gene promoter as bait . The corresponding protein (named CrMYC1) was shown to bind specifically to the G-box in yeast . In C . roseus suspension cells CrMYC1 mRNA levels are induced by fungal elicitor and jasmonate suggesting that CrMYC1 may be involved in the regulation of gene expression in response to these signals. Plant Cell, 2003 Sep, 15(9), 2058 - 75 Involvement of the secretory pathway and the cytoskeleton in intracellular targeting and tubule assembly of Grapevine fanleaf virus movement protein in tobacco BY-2 cells; Laporte C et al.; Grapevine fanleaf virus (GFLV) is one of a large class of plant viruses whose cell-to-cell transport involves the passage of virions through tubules composed of virus-encoded movement protein (MP) . The tubules are embedded within modified plasmodesmata, but the mechanism of targeting of MP to these sites is unknown . To study intracellular GFLV MP trafficking, a green fluorescent protein-MP fusion (GFP:MP) was expressed in transgenic tobacco BY-2 suspension cells under the control of an inducible promoter . We show that GFP:MP is targeted preferentially to calreticulin-labeled foci within the youngest cross walls, where it assembles into tubules . During cell division, GFP:MP colocalizes in the cell plate with KNOLLE, a cytokinesis-specific syntaxin, and both proteins are linked physically, as shown by coimmunoprecipitation of the two proteins from the same microsomal fraction . In addition, treatment with various drugs has revealed that a functional secretory pathway, but not the cytoskeleton, is required for tubule formation . However, correct GFP:MP targeting to calreticulin-labeled foci seems to be cytoskeleton dependent . Finally, biochemical analyses have revealed that at least a fraction of the MP behaves as an intrinsic membrane protein . These findings support a model in which GFP:MP would be transported to specific sites via Golgi-derived vesicles along two different pathways: a microtubule-dependent pathway in normal cells and a microfilament-dependent default pathway when microtubules are depolymerized. Planta, 2003 Dec, 218(2), 204 - 16 Epub 2003 Aug 21. The association of peroxisomes with the developing cell plate in dividing onion root cells depends on actin microfilaments and myosin; Collings DA et al.; We have investigated changes in the distribution of peroxisomes through the cell cycle in onion ( Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek ( Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein . During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane . Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate . However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules . Peroxisome aggregation depends on actin microfilaments and myosin . Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments . Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation . We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the beta-oxidation pathway . Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants. Planta, 2003 Dec, 218(2), 233 - 9 Epub 2003 Aug 14. Phosphite accelerates programmed cell death in phosphate-starved oilseed rape (Brassica napus) suspension cell cultures; Singh VK et al.; Phosphite (H(2)PO(3)(-), Phi) prevents the acclimation of plants and yeast to orthophosphate (Pi, HPO(4)(2-)) deprivation by specifically obstructing the derepression of genes encoding proteins characteristic of their Pi-starvation response . In this study, we report that prolonged (i.e., 3-4 weeks) culture of Brassica napus L . suspension cells in Pi-deficient (-Pi) media leads to programmed cell death (PCD) . However, when the B . napus cells were subcultured into -Pi media containing 2 mM Phi, they initiated PCD within 5 days, with 95% cell death observed by day 9 . Dying cells exhibited several morphological and biochemical features characteristic of PCD, including protoplast shrinkage, chromatin condensation, and fragmentation of nuclear DNA . Immunoblotting indicated that B . napus cells undergoing PCD upregulated a 30-kDa cysteine endoprotease that is induced during PCD in the inner integument cells of developing B . napus seeds . It is concluded that PCD in B . napus suspension cells is triggered by extended Pi starvation, and that Phi treatment greatly accelerates this process . Our results also infer that the adaptive value of acclimating at the molecular level to Pi-stress is to extend the viability of -Pi B . napus cell cultures by about 3 weeks. Plant Cell Rep, 2003 Aug, 21(12), 1199 - 206 Epub 2003 May 15. A promoter derived from taro bacilliform badnavirus drives strong expression in transgenic banana and tobacco plants; Yang IC et al.; Taro bacilliform virus (TaBV) is a pararetrovirus of the genus Badnavirus which infects the monocotyledonous plant, taro ( Colocasia esculenta) . A region of the TaBV genome spanning nucleotides 6,281 to 12 (T1200), including the 3' end of open reading frame 3 (ORF 3) and the intergenic region to the end of the tRNA(met)-binding site, was tested for promoter activity along with four different 5' deletion fragments (T600, T500, T250 and T100) . In transient assays, only the T1200, T600, T500 fragments were shown to have promoter activity in taro leaf, banana suspension cells and tobacco callus . When these three promoters were evaluated in stably transformed, in vitro-grown transgenic banana and tobacco plants, all were found to drive near-constitutive expression of either the green fluorescent protein or beta-glucuronidase (GUS) reporter gene in the stem (or pseudostem), leaves and roots, with strongest expression observed in the vascular tissue . In transgenic banana leaves, the T600 promoter directed four-fold greater GUS activity than that of the T1200, T500 and the maize polyubiquitin-1 promoters . In transgenic tobacco leaves, the levels of GUS expression directed by the three promoters was between four- and ten-fold lower than that of the double Cauliflower mosaic virus 35S promoter . These results indicate that the TaBV-derived promoters may be useful for the high-level constitutive expression of transgenes in either monocotyledonous or dicotyledonous species. World J Gastroenterol, 2003 Jul, 9(7), 1594 - 7 Effects of carbon dioxide and nitrogen on adhesive growth and expressions of E-cadherin and VEGF of human colon cancer cell CCL-228; Cai KL et al.; AIM: To study the effects of carbon dioxide on the metastatic capability of cancer cells, and to compare them with that of nitrogen . METHODS: The colon cancer cell CCL-228 was treated with 100 % carbon dioxide or nitrogen at different time points and then cultured under normal condition . Twelve hours after the treatment, the survival rates of suspension cells and the expressions of e-cadherin and VEGF were examined . RESULTS: After 60 min of carbon dioxide and longer time of nitrogen treatment, the suspended cells increased and the expression of e-cadherin decreased while the expression of VEGF was enhanced significantly . And the effects of nitrogen were similar to, but weaker than, those of carbon dioxide . CONCLUSION: Carbon dioxide may improve the metastatic capability of cancer cells and its effects are significantly stronger than that of nitrogen . A sequential use of carbon dioxide and nitrogen in pneumoperitoneum may take the advantage of both gases. Biocell, 2003 Apr, 27(1), 47 - 55 Inhibition of focal adhesion kinase by antisense oligonucleotides enhances the sensitivity of breast cancer cells to camptothecins; Satoh TH et al.; This study shows a strong association between cell attachment to substratum and activation of beta 1-integrin-signaling with resistance to the camptothecin derivative topotecan (TPT) in breast cancer cells . We propose a mechanistic-driven approach to sensitize the cells to camptothecins . ZR-75-1 anchorage-dependent breast cancer cell line, its derivative 9D3S suspension cells (9D3S-S), and 9D3S cells attached to fibronectin-coated plates (9D3S-A) were treated with TPT (1 microM) or CPT-11 (40 microM) for 48 h . Programmed cell death (PCD), as shown by poly(ADP-ribose) polymerase (PARP), pro-caspase-3 and pro-caspase-9 cleavage, was observed in 9D3S-S cells but not in ZR-75-1 or 9D3S-A cells . Because p125 focal adhesion kinase (FAK) is a transducer in the beta 1-integrin signaling pathway, it is essential to cell adhesion and it is overexpressed in metastatic breast cancer, we hypothesized that attenuation of FAK might enhance the sensitivity of breast cancer cells to camptothecins . Moreover, inhibition of FAK gene expression by a phosphorothioated antisense oligodeoxynucleotide targeting the portion of the gene encoding amino acids 262-268, increased the sensitivity of ZR-75-1, MDA-MB-231 and MCF7 breast cancer cells to treatment with TPT or CPT-11. J Exp Bot, 2003 Jul, 54(388), 1793 - 5 Differential expression of two type-A response regulators in plants and cell cultures of Catharanthus roseus (L.) G . Don; Papon N et al.; Two full-length cDNAs named CrRR2 and CrRR3 have been isolated by PCR from a C . roseus cDNA library . The first one encodes a 154 amino acid putative protein with a high percentage of identity with the Arabidopsis thaliana response regulators ARR16 and ARR17, and is not expressed in C . roseus organs and cell cultures . The second one encodes a 188 amino acid ORF sharing the highest homologies with the A . thaliana ARR8 and ARR9 response regulators . Its expression is root-specific and the transcripts are transiently up-regulated after trans-zeatin treatment in C . roseus suspension cells . CrRR3 protein might be involved in the cytokinin-enhanced alkaloid production in C . roseus cell cultures. J Biol Chem, 2003 Sep 12, 278(37), 35732 - 42 Epub 2003 Jun 16. Equilibrative nucleoside transporters of Arabidopsis thaliana . cDNA cloning, expression pattern, and analysis of transport activities; Li G et al.; Equilibrative nucleoside transporters (ENTs) occur in diverse organisms . In the model plant Arabidopsis thaliana, eight potential ENTs (AtENTs) have been predicted by genome sequencing . We here report the cloning of the cDNAs for AtENTs 2, 3, 4, 6, 7, and 8 . Conceptual translation of the cDNAs of AtENTs 2, 3, 4, 6, 7, and 8 yielded polypeptides possessing strong similarities to ENTs characterized previously . Eleven putative transmembrane domains were identified in each of the six AtENTs . In suspension cells, the transcription of AtENTs 1, 3, 4, 6, and 8 was increased by two treatments (nitrogen deprivation, application of 5-fluorouracil and methotrexate) that inhibited the de novo pathway of nucleotide synthesis, indicating that multiple members of the Arabidopsis ENT family may function in the salvage pathway of nucleotide synthesis . Except for AtENT1, the transcription of the remaining six AtENTs showed varying degrees of organ specificity . However, all seven AtENTs were expressed in the leaf and flower . In plant, insect, and yeast cells, ectopically expressed AtENT3 was targeted to the plasma membrane . AtENT3 expressed in yeast cells transported adenosine and uridine with high affinity . Furthermore, the activities of AtENT3 appear not to require a transmembrane proton gradient because protonophores did not abolish adenosine or uridine transport . In competition experiments, the transport of {3H}adenosine by AtENT3 was most significantly inhibited by a number of different purine and pyrimidine nucleosides and 2'-deoxynucleosides, although certain nucleobases and nucleotides were also found to have some inhibitory effect . This indicates that AtENT3 may possess broad substrate specificity . Adenosine and uridine transport by AtENT3, although partly sensitive to the vasodilator drugs dilazep and dipyridamole, was resistant to the nucleoside analogue nitrobenzylmercaptopurine ribonucleoside . We conclude that AtENT3 represents the first ei type ENT characterized from higher plants . The potential functions of ENTs in the biology of A . thaliana are discussed. Protoplasma, 2003 Mar, 220(3-4), 111 - 8 Development and disintegration of phragmoplasts in living cultured cells of a GFP::TUA6 transgenic Arabidopsis thaliana plant; Ueda K et al.; Cultured suspension cells of Arabidopsis thaliana that stably express a green-fluorescent protein-alpha-tubulin 6 fusion protein were used to follow the development and disintegration of phragmoplasts . The development and disintegration of phragmoplasts in the living cultured cells could be successively observed by detecting the green-fluorescent protein fluorescence of the microtubules . In the early telophase spindle, where two kinetochore groups and two daughter chromosome groups had completely separated from one another, fluorescence appeared in the interzone between the two chromosome groups . The fluorescent region was gradually condensed at the previous equator and increased in fluorescence intensity, and finally it formed the initial phragmoplast . The initial phragmoplast moved from the cell center towards the cell periphery, and it lost fluorescence at its center and became double rings in shape . The expansion orientation of the phragmoplast was not always the same as that of the future new cell wall before it came in contact with the cell wall . The phragmoplast did not usually come in contact with the cell wall simultaneously with its entire length . A portion of the phragmoplast which was earlier in contact with the cell wall disappeared earlier than other portions of the phragmoplast . The duration of contact between any portions of the phragmoplast and the plasma membrane of the cell wall was 15-30 min . The fluorescence intensity of the cytoplasm did not seem to be elevated by the disintegration of the strongly fluorescent phragmoplast. J Biol Chem, 2003 Jun 13, 278(24), 21395 - 407 Epub 2003 Mar 19. Expression of the telomeric repeat binding factor gene NgTRF1 is closely coordinated with the cell division program in tobacco BY-2 suspension culture cells; Yang SW et al.; Telomeres are vital for preserving chromosome integrity during cell division . Several genes encoding potential telomere-binding proteins have recently been identified in higher plants, but nothing is known about their function or regulation during cell division . In this study, we have isolated and characterized a cDNA clone, pNgTRF1, encoding a putative double-stranded telomeric repeat binding factor of Nicotiana glutinosa, a diploid tobacco plant . The predicted protein sequence of NgTRF1 (Mr = 75,000) contains a single Myb-like domain with significant homology to a corresponding motif in human TRF1/Pin2 and TRF2 . Gel retardation assays revealed that bacterially expressed full-length NgTRF1 was able to form a specific complex only with probes containing three or more contiguous telomeric TTTAGGG repeats . The Myb-like domain of NgTRF1 is essential, but not sufficient, to bind the telomeric repeat sequence . The glutamine-rich extreme C-terminal region, which does not exist in animal proteins, was additionally required to form a specific telomere-protein complex . The dissociation constant (Kd) of the Myb motif plus the glutamine-rich domain of NgTRF1 to the two-telomeric repeat sequence was evaluated to be 4.5 +/- 0.2 x 10-9 m, which is comparable to that of the Myb domain of human TRF1 . Expression analysis showed that NgTRF1 gene activity was inversely correlated with the cell division capacity of tobacco root cells and during the 9-day culture period of BY-2 suspension cells, while telomerase activity was positively correlated with cell division . In synchronized BY-2 cells, NgTRF1 was selectively expressed in G1 phase, whereas telomerase activity peaked in S phase . These findings suggest that telomerase activity and NgTRF1 expression are differentially regulated in an opposing fashion during growth and cell division in tobacco plants . The possible physiological functions of NgTRF1 in tobacco cells are also discussed. J Cell Physiol, 2003 Apr, 195(1), 108 - 18 Lipid factor (bVLF) from bovine vitreous body evokes in EGFR-T17 cells a Ca2+-dependent K+ current associated with inositol 1,4,5-trisphosphate-independent Ca2+ mobilization; Camina JP et al.; Bovine vitreous lipid factor (bVLF) is a complex phospholipid isolated from bovine vitreous body with strong Ca(2+)-mobilizing activity . In this study, the effects of bVLF on membrane potential were investigated in EGFR-T17 fibroblasts with the whole-cell patch clamp technique on monolayer cells, as well as with the fluorescent dye bis-oxonol as membrane potential-sensitive probe on monolayer and suspension cells . bVLF induced a transient hyperpolarization characterized by an initial peak and subsequent return to resting membrane potential levels within 1-2 min . The increase of {Ca(2+)}(i) was concomitant with an outward current responsible for the hyperpolarizing response . Results with: (a) high {K(+)}(o) media; (b) the monovalent cation ionophore gramicidin; and (c) substitution of K(+) with Cs(+) in the intracellular solution were consistent with the involvement of K(+) channels . The bVLF-induced hyperpolarization was blocked by the K(+) channel blockers, quinine and tetraethylamonium chloride, and partially affected by 4-aminopyridine . The calcium ionophore ionomycin caused a similar hyperpolarization as bVLF . When intracellular calcium was buffered by adding BAPTA to the pipette solution, bVLF-activated outward current was prevented . Moreover, the hyperpolarization response was strongly reduced at low doses (3 nM) of specific Ca(2+)-activated K(+) channel blockers, charybdotoxin and iberiotoxin . Based on these observations we conclude that bVLF hyperpolarizes the cells via the activation of a Ca(2+)-dependent K(+) current . In addition, it was observed that bVLF did not have a significant effect on intercellular communication measured by a single patch-electrode technique . Thus, membrane potential changes appeared to belong to the earliest cellular responses triggered by bVLF, and are closely associated with phosphatidic acid-dependent {Ca(2+)}(i) mobilization . Biochim Biophys Acta, 2003 Feb 20, 1625(3), 261 - 8 Identification and characterization of two new members of the GRAS gene family in rice responsive to N-acetylchitooligosaccharide elicitor; Day RB et al.; We identified two new members of the GRAS gene family from rice, CIGR1 and CIGR2, which are rapidly induced upon N-acetylchitooligosaccharide elicitor perception . The predicated proteins encoded by CIGR1 and CIGR2 possess significant sequence similarity with previously identified members of the GRAS family, such as Arabidopsis SCARECROW, GAI, RGA, tomato Lateral suppressor, and rice SLR1, all of which have VHIID regions, likely to play a role in cellular signaling . Fusions of CIGR1 and CIGR2 with Green Fluorescent Protein were detected exclusively in the nuclei of onion epidermal cells . The expression of CIGR1 and CIGR2 was dependent on the structure of N-acetylchitooligosaccharides, which parallels the structural specificity for chitin binding to the plasma membrane-localized chitin-binding protein, and independent of de novo protein synthesis . Co-cultivation of rice cells with rice blast fungus strongly induced the expression of CIGR1 and CIGR2, whereas inoculation of suspension cells with phytopathogenic bacteria did not . We hypothesize that CIGR1 and CIGR2 act as transcriptional regulators in the early events of the elicitor-induced defense response in rice. Zhong Yao Cai, 2000 Jan, 23(1), 1 - 4 {Effects of phytohormones on growth and content of depsides in Salvia miltiorrhiza suspension cells}; Huang L et al.; This paper deals with the effects of 2,4-D, BA and GA3 on the growth and content of two depsides (rosmarinic acid and lithospermic acid B) in suspension cells of Salvia miltiorrhiza . The results showed cell growth and rosmarinic acid content reached the maximum on the 12th day and lithospermic acid B content on the 16th day after incubating cells in the subculture medium MS + 2, 4-D 1 mg/L + KT 0.1 mg/L . This cell line was a growth-product-associated . With the same concentration (1 mg/L), 2, 4-D stimulated the cell growth but prohibited the formation of lithospermic acid B; GA3 inhabited the cell growth but stimulated the formation of two depsides; The effects of BA is between 2, 4-D and GA3 . The concentration optimum of phytohormones tested displayed 3 mg/L for BA and 1 mg/L for GA3 . For the optimum time of adding BA and GA3 was in med-term (the 8th day) and early term (at the beginning) of culture period respectively . The synergiatic function of BA and GA3 on the depside formation was also showed that adding GA3 (1 mg/L) was favour for the formation of lithospermic acid B of the suspension cell cultured in the medium containing BA 3 mg/L . The suitable time of adding GA3 was the 6the day after incubating cells in the medium of MS + BA 3 mg/L. Plant Cell Physiol, 2003 Jan, 44(1), 93 - 5 The plastid clpP gene may not be essential for plant cell viability; Cahoon AB et al.; The plastid gene clpP is widely regarded as essential for chloroplast function and general plant cell survival . In this note we provide evidence that certain lines of non-photosynthetic maize (Zea mays) Black Mexican Sweet (BMS) suspension cells do not carry clpP in their plastid genomes . We also discuss several incidences in the literature where clpP is either missing or not expressed in other non-green cell lines and plants . We conclude that clpP is not required for general plant cell survival but instead may only be essential for the development and/or function of plastids with active gene expression. Shi Yan Sheng Wu Xue Bao, 2000 Mar, 33(1), 85 - 8 {Factors influence on transformation by particle bombardment in Indica rice}; Tao LZ et al.; Four factors influence on transformation of indica rice, which were high osmotic treatment; different explant as the target tissue; pressure of rupture disk and quantity of plasmid DNA, were investigated in this experiment . High osmotic treatment of target tissue prior to and after bombardment increased 3.2-fold for Gus transient expression than control . The best treatment of high osmotic was that the target tissues were kept in the target-bed medium which contained 0.4-0.6 mol/L sorbitol and manitol each for 4 h prior to bombardment and for 16 h after bombardment . Four explants: scutellum from mature seed, young panicle, embryogenic callus and suspension cells of indica rice were tested as target explant by particle bombardment . The results of Gus transient showed that the highest expression was scutellum and for other three explants, the order from high to low was young panicle, embryogenic callus and suspension cell . Transgenic plants were obtained from all of the explants except young panicle . For the pressure of rupture disk on transformation, 1100 psi or 1300 psi of the pressure of rupture disk were best one for the transformation and higher than 1300 psi could damage the target tissue which become black and died in the following culture duration . For the quantity of plasmid DNA, the results showed that 0.83 microgram of plasmid DNA per bombardment was preferred for the transformation of indica rice. Shi Yan Sheng Wu Xue Bao, 1999 Sep, 32(3), 271 - 6 {Studies on the application of antifreeze proteins in cryopreservation of rice suspension cells}; Wang JH et al.; AFP from winter flounder was utilized in cryopreservation of plant cells . During cryopreservation of rice suspension cells by two-step method, AFP at 0.01 mg/ml damaged the cells extremely . The data obtained at relatively high concentration, however, decreased the variability of survival rate . During vitrification of rice cells, AFP at 0.2 mg/ml enhanced the viability . However, high concentration AFP (> 5 mg/ml) decreased the recovery rate . Studies indicated that the results of application of AFP in cryopreservation were closely related to the concentration of cryoprotectant . The amount of ice crystal in environment, the concentration of AFP and cryoprotectant, and the composition of plasma membrane were several key factors affecting the results of AFP application . In mechanism analysis, the authors suggested that on one hand AFP can interact with ice crystal, which inhibits ice recrystallization and prevent the cells from devitrification . On the other hand, AFP also can interact with cell membrane, resulting in the ice growth around the plasma membrane. Shi Yan Sheng Wu Xue Bao, 1999 Mar, 32(1), 88 - 92 {Involvement of anion channel in signal transduction of early defense responses elicited by harpinPss in tobacco}; Qiu JL et al.; No matter when anion channel inhibitors, DIDS (4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid) and A9C (anthracene-9-carboxylic acid) added (before, at the same time of or after harpinPss treatment), they can inhibit harpinPss-induced hypersensitive response in tobacco seedlings and release of active oxygen and extracellular alkalinization in tobacco suspension cells . DIDS and A9C also inhibit harpinPss-induced Ca2+ influx . In all these cases, DIDS is more efficient than A9C . It is postulated that anion channel positively regulates calcium channel in plasma membrane, and harpinPss may function through signal transduction mediated by anion channel and calcium channel to regulate cellular Ca2+ concentration and defense responses. Eur J Biochem, 2002 Dec, 269(24), 6278 - 86 Purification and characterization of two secreted purple acid phosphatase isozymes from phosphate-starved tomato (Lycopersicon esculentum) cell cultures; Bozzo GG et al.; Two secreted acid phosphatases (SAP1 and SAP2) were markedly up-regulated during Pi-starvation of tomato suspension cells . SAP1 and SAP2 were resolved during cation-exchange FPLC of culture media proteins from 8-day-old Pi-starved cells, and purified to homogeneity and final p-nitrophenylphosphate hydrolyzing specific activities of 246 and 940 micro mol Pi produced.min-1 mg.protein-1, respectively . SDS/PAGE, periodic acid-Schiff staining and analytical gel filtration demonstrated that SAP1 and SAP2, respectively, exist as 84 and 57 kDa glycosylated monomers . SAP1 and SAP2 are purple acid phosphatases (PAPs) as they displayed an absorption maximum at 518 and 538 nm, respectively, and were not inhibited by l-tartrate . The respective sequence of a SAP1 and SAP2 tryptic peptide was very similar to a portion of the deduced sequence of several putative Arabidopsis thaliana PAPs . CNBr peptide mapping indicated that SAP1 and SAP2 are structurally distinct . Both isozymes displayed a pH optimum of approximately pH 5.3 and were heat stable . Although they exhibited wide substrate specificities, the Vmax of SAP2 with various phosphate-esters was significantly greater than that of SAP1 . SAP1 and SAP2 were activated by up to 80% by 5 mm Mg2+, and demonstrated potent competitive inhibition by molybdate, but mixed and competitive inhibition by Pi, respectively . Interestingly, both SAPs exhibited significant peroxidase activity, which was optimal at approximately pH 8.4 and insensitive to Mg2+ or molybdate . This suggests that SAP1 and SAP2 may be multifunctional proteins that operate: (a) PAPs that scavenge Pi from extracellular phosphate-esters during Pi deprivation, or (b) alkaline peroxidases that participate in the production of extracellular reactive oxygen species during the oxidative burst associated with the defense response of plants to pathogen infection. Plant Physiol, 2002 Oct, 130(2), 999 - 1007 Activation of phospholipases C and D is an early response to a cold exposure in Arabidopsis suspension cells; Ruelland E et al.; The signaling events generated by a cold exposure are poorly known in plants . We were interested in checking the possible activation of enzymes of the phosphoinositide signaling pathway in response to a temperature drop . In Arabidopsis suspension cells labeled with (33)PO(4)(3-), a cold treatment induces a rapid increase of phosphatidic acid (PtdOH) content . This production was due to the simultaneous activation of phospholipase C (through diacylglycerol kinase activity) and phospholipase D, as monitored by the production of inositol triphosphate and of transphosphatidylation product, respectively . Moreover, inhibitors of the phosphoinositide pathway and of diacylglycerol kinase reduced PtdOH production . Enzyme activation occurred immediately after cells were transferred to low temperature . The respective contribution of both kind of phospholipases in cold-induced production of PtdOH could be estimated . We created conditions where phospholipids were labeled with (33)PO(4)(3-), but with ATP being nonradioactive . In such conditions, the apparition of radioactive PtdOH reflected PLD activity . Thus, we demonstrated that during a cold stress, phospholipase D activity accounted for 20% of PtdOH production . The analysis of composition in fatty acids of cold-produced PtdOH compared with that of different phospholipids confirmed that cold-induced PtdOH more likely derived mainly from phosphoinositides . The addition of chemical reagents modifying calcium availability inhibited the formation of PtdOH, showing that the cold-induced activation of phospholipase pathways is dependent on a calcium entry. Plant Mol Biol, 2002 Nov, 50(4-5), 735 - 42 Regulation of alternative oxidase gene expression in soybean; Djajanegara I et al.; Soybean (Glycine max cv . Stevens) suspension cells were used to investigate the expression of the alternative oxidase (Aox) multigene family . Suspension cells displayed very high rates of cyanide-insensitive respiration, but Aox3 was the only isoform detected in untreated cells . Incubation with antimycin A, citrate, salicylic acid or at low temperature (10 degrees C) specifically induced the accumulation of the Aox1 isoform . Aox2 was not observed under any conditions in the cells . Increases in Aox1 protein correlated with increases in Aox1 mRNA . Treatment of soybean cotyledons with norflurazon also induced expression of Aox1 . Reactive oxygen species (ROS) were detected upon incubation of cells with antimycin, salicylic acid or at low temperature, but not during incubation with citrate . Aox1 induction by citrate, but not by antimycin, was prevented by including the protein kinase inhibitor staurosporine in the medium . The results suggest that multiple pathways exist in soybean to regulate expression of Aox genes and that Aox1 specifically is induced by a variety of stress and metabolic conditions via at least two independent signal transduction pathways. Planta, 2002 Oct, 215(6), 914 - 23 Epub 2002 Jul 25. Nitric oxide induces transcriptional activation of the nitric oxide-tolerant alternative oxidase in Arabidopsis suspension cells; Huang X et al.; Nitric oxide (NO) is a double-edged sword - it can be either beneficial and activate defence responses in plants and animals or, together with reactive oxygen species, it can kill not only the pathogen but also the host . A prime target of NO is the cytochrome c-dependent respiration . Only plants possess alternative-pathway respiration with alternative oxidase (AOX) as a terminal electron acceptor . AOX has been suggested to be barely affected by NO . Here we show that NO affects cytochrome-dependent respiration in Arabidopsis thaliana (L.) Heynh . At the same time, treatment of Arabidopsis cell cultures with NO actually strongly induced AOX1a transcription, as determined by using a cDNA microarray and by Northern analysis . In accordance with transcript accumulation, NO treatment of suspension cells resulted in increased respiration through the alternative pathway . Addition of an AOX inhibitor to Arabidopsis cell cultures resulted in dramatically increased NO-sensitivity and cell death . In all, our data suggest that NO induces the AOX1a gene and that AOX may participate to counteract the toxicity of NO . Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00425-002-0828-z. Planta, 2002 Sep, 215(5), 880 - 4 Epub 2002 Jul 26. Regeneration of flowering plants from difficile lily protoplasts by means of a nurse culture; Horita M et al.; The regeneration of difficile lily protoplasts isolated from suspension cells of the Oriental hybrid lily ( Lilium L.) cultivars Casablanca, Siberia and Acapulco was achieved by using the nurse-culture method . The divided protoplasts grew into colonies with nurse cells that have no regeneration ability, and developed to visible calli on a medium containing picloram . Many plantlets were formed on the calli after transfer of the proliferated calli to hormone-free medium . We were able to transplant the plantlets to soil in pots without acclimatization, and the plantlets grew in a greenhouse until flowering 2 years later. Mol Plant Microbe Interact, 2002 Sep, 15(9), 932 - 8 The indolic compound hypaphorine produced by ectomycorrhizal fungus interferes with auxin action and evokes early responses in nonhost Arabidopsis thaliana; Reboutier D et al.; Signals leading to mycorrhizal differentiation are largely unknown . We have studied the sensitivity of the root system from plant model Arabidopsis thaliana to hypaphorine, the major indolic compound isolated from the basidiomycetous fungus Pisolithus tinctorius . This fungi establishes ectomycorrhizas with Eucalyptus globulus . Hypaphorine controls root hair elongation and counteracts the activity of indole-3-acetic acid on root elongation on A . thaliana, as previously reported for the host plant . In addition, we show that hypaphorine counteracts the rapid upregulation by indole-3-acetic acid and 1-naphthalenic-acetic acid of the primary auxin-responsive gene IAA1 and induces a rapid, transient membrane depolarization in root hairs and suspension cells, due to the modulation of anion and K+ currents . These early responses indicate that components necessary for symbiosis-related differentiation events are present in the nonhost plant A . thaliana and provide tools for the dissection of the hypaphorine-auxin interaction. Plant Physiol, 1994 Dec, 106(4), 1503 - 1510 Molecular Genetic Alteration of Plant Respiration (Silencing and Overexpression of Alternative Oxidase in Transgenic Tobacco); Vanlerberghe GC et al.; The alternative oxidase (AOX) of plant mitochondria is encoded by the nuclear gene Aox1 . Sense and antisense DNA constructs of Nicotiana tabacum Aox1 were introduced into tobacco, and transgenic plants with both increased and decreased levels of mitochondrial AOX protein were identified . Suspension cells derived from wild-type and transgenic plants were grown in heterotrophic batch culture . Transgenic cells with increased AOX protein had an increased capacity for cyanide-resistant, salicylhydroxamic acid-sensitive respiration compared to wild-type cells, whereas transgenic cells with decreased AOX protein had a decreased capacity for such respiration . Thus, genetic alteration of the level of AOX protein was sufficient to alter the capacity for electron transport through the alternative pathway . Under our standard growth conditions, "antisense" cells with dramatically reduced levels of AOX protein had growth and respiration rates similar to the wild type . However, whereas wild-type cells were able to grow under conditions that severely suppressed cytochrome pathway activity, antisense cells could not survive this treatment . This suggests that a critical function of AOX may be to support respiration when the cytochrome pathway is impaired . The much higher level of AOX protein in "sense" cells compared to the wild type did not appreciably alter the steady-state partitioning of electrons between the cytochrome path and the alternative pathway in vivo, suggesting that this partitioning may be subject to additional regulatory factors. Plant Physiol, 1994 Nov, 106(3), 1213 - 1216 Comparison of Dehydrin Gene Expression and Freezing Tolerance in Bromus inermis and Secale cereale Grown in Controlled Environments, Hydroponics, and the Field; Robertson AJ et al.; There have been very few reports on the expression of stress-responsive genes in field-grown material . A barley dehydrin cDNA was used to investigate the expression of dehydrin-like transcripts after low-temperature and abscisic acid-induced acclimation of bromegrass (Bromus inermis Leyss) suspension cells and of bromegrass and rye (Secale cereale) plants grown in the field and under controlled environmental conditions . Field-acclimated plants accumulated high levels of dehydrin transcripts and were very freezing tolerant . Plants grown in pots and hydroponics under controlled environments also accumulated dehydrin transcripts and showed increased freezing tolerance . Simulation of a combined drought and freezing stress in pots resulted in expression of dehydrin-like transcripts comparable to those observed in field-acclimated material. Plant Physiol, 1994 Oct, 106(2), 459 - 467 Rice Triosephosphate Isomerase Gene 5{prime} Sequence Directs {beta}-Glucuronidase Activity in Transgenic Tobacco but Requires an Intron for Expression in Rice; Xu Y et al.; In rice (Oryza sativa L.), cytosolic triosephosphate isomerase (TPI) is encoded by a single gene . TPI catalyzes a vital step in glycolysis, and RNA blots showed that the tpi gene is expressed in all vegetative tissues (root, culm, and leaves) and in rice suspension cells . No effect of light on expression was detected, but submergence of rice seedlings resulted in elevated levels of TPI mRNA in roots and culms . The 2767-bp 5{prime} upstream sequence of the tpi gene was fused translationally with the {beta}-glucuronidase (gusA) gene, and the resulting construct, TPI-GUS, was found to express constitutive, high levels of GUS activity in transgenic tobacco (Nicotiana tabacum) plants . However, the same construct yielded no GUS activity in stably transformed rice plants, and RNA blots showed that no GUS mRNA could be detected even though stable integration of functional copies of the construct was confirmed by Southern blot and genomic polymerase chain reaction analyses . Transient assays using particle bombardment yielded high levels of GUS expression from the TPI-GUS construct in tobacco leaves, but essentially no expression in rice, barley, or maize leaves . When the first intron of the tpi gene was included in the construct (TPI-int1-GUS), transient GUS activity was routinely obtained in rice leaves, revealing that the first intron of the rice tpi gene is crucial for its expression in rice . TPI-int1-GUS also directed transient GUS expression in maize and barley leaves, but little or no activity was obtained from this construct in tobacco, tomato, or soybean leaves . These results with the rice tpi promoter are in accordance with mounting evidence that differences in gene expression exist between monocots and dicots. Plant Physiol, 1994 May, 105(1), 89 - 94 Pretreatment of Parsley (Petroselinum crispum L.) Suspension Cultures with Methyl Jasmonate Enhances Elicitation of Activated Oxygen Species; Kauss H et al.; Suspension-cultured cells of parsley (Petroselinum crispum L.) were used to demonstrate an influence of jasmonic acid methyl ester (JAME) on the elicitation of activated oxygen species . Preincubation of the cell cultures for 1 d with JAME greatly enhanced the subsequent induction by an elicitor preparation from cell walls of Phytophtora megasperma f . sp . glycinea (Pmg elicitor) and by the polycation chitosan . Shorter preincubation times with JAME were less efficient, and the effect was saturated at about 5 {mu}M JAME . Treatment of the crude Pmg elicitor with trypsin abolished induction of activated oxygen species, an effect similar to that seen with elicitation of coumarin secretion . These results suggest that JAME conditioned the parsley suspension cells in a time-dependent manner to become more responsive to elicitation, reminiscent of developmental effects caused by JAME in whole plants . It is interesting that pretreatment of the parsley cultures with 2,6-dichloroisonicotinic and 5-chlorosalicylic acid only slightly enhanced the elicitation of activated oxygen species, whereas these substances greatly enhanced the elicitation of coumarin secretion . Therefore, these presumed inducers of systemic acquired resistance exhibit a specificity different from JAME. Plant Physiol, 1993 Nov, 103(3), 963 - 969 Characterization of Paraquat Transport in Protoplasts from Maize (Zea mays L.) Suspension Cells; Hart JJ et al.; Protoplasts isolated from maize (Zea mays L.) suspension cells were used to study transport of paraquat . {14C}Paraquat uptake was measured in 400-{mu}L centrifuge tubes using silicon oil centrifugation techniques . Approximately 50% of accumulation from a 100 {mu}M paraquat solution occurred in the first 10 s, and net accumulation reached a maximum after about 10 min . Membrane binding accounted for about 30% of apparent accumulation . Concentration-dependent uptake kinetics were characterized by a non-saturating curve, which was resolved into a linear and a saturable component . The Km of the saturable component was 132 {mu}M, and the Vmax was 0.512 nmol {mu}L of protoplasts-1 min-1 . In the absence of sucrose, the Vmax of the saturable component was reduced by 52%, suggesting that paraquat uptake across the plasmalemma is energy dependent . Measurement of concentration-dependent binding of paraquat to burst protoplasts showed a linear response . This suggests that the linear component from intact protoplast concentration kinetics represented paraquat binding to the plasmalemma surface . Calcium inhibited the saturable component, and this inhibition was shown by Lineweaver-Burk analysis to be noncompetitive . Putrescine, a divalent cationic polyamine with a charge distribution similar to that of paraquat, competitively inhibited paraquat uptake . These results show that paraquat transport characteristics at the plasmalemma of maize protoplasts are similar to those reported earlier for paraquat transport in roots of intact maize seedlings. Plant Physiol, 1993 Jun, 102(2), 459 - 466 Conditioning of Parsley (Petroselinum crispum L.) Suspension Cells Increases Elicitor-Induced Incorporation of Cell Wall Phenolics; Kauss H et al.; The elicitor-induced incorporation of phenylpropanoid derivatives into the cell wall and the secretion of soluble coumarin derivatives (phytoalexins) by parsley (Petroselinum crispum L.) suspension cultures can be potentiated by pretreatment of the cultures with 2,6-dichloroisonicotinic acid or derivatives of salicylic acid . To investigate this phenomenon further, the cell walls and an extracellular soluble polymer were isolated from control cells or cells treated with an elicitor from Phytophthora megasperma f . sp . glycinea . After alkaline hydrolysis, both fractions from elicited cells showed a greatly increased content of 4-coumaric, ferulic, and 4-hydroxybenzoic acid, as well as 4-hydroxybenzaldehyde and vanillin . Two minor peaks were identified as tyrosol and methoxytyrosol . The pretreatment effect is most pronounced at a low elicitor concentration . Its specificity was elaborated for coumarin secretion . When the parsley suspension cultures were preincubated for 1 d with 2,6-dichloroisonicotinic, 4- or 5-chlorosalicylic, or 3,5- dichlorosalicylic acid, the cells exhibited a greatly increased elicitor response . Pretreatment with isonicotinic, salicylic, acetylsalicylic, or 2,6-dihydroxybenzoic acid was less efficient in enhancing the response, and some other isomers were inactive . This increase in elicitor response was also observed for the above-mentioned monomeric phenolics, which were liberated from cell walls upon alkaline hydrolysis and for "lignin-like" cell wall polymers determined by the thioglycolic acid method . It was shown for 5-chlorosalicylic acid that conditioning most likely improves the signal transduction leading to the activation of genes encoding phenylalanine ammonia lyase and 4-coumarate: coenzyme A ligase . The conditioning thus sensitizes the parsley suspension cells to respond to lower elicitor concentrations . If a similar mechanism were to apply to whole plants treated with 2,6-dichloroisonicotinic acid, a known inducer of systemic acquired resistance, one can hypothesize that fungal pathogens might be recognized more readily and effectively. Plant Physiol, 1993 Feb, 101(2), 499 - 506 Characterization of Maize Acetyl-Coenzyme A Carboxylase; Egli MA et al.; Maize (Zea mays L.) leaf acetyl-CoA carboxylase (ACCase) was purified about 500-fold by ammonium sulfate fractionation and gel filtration and blue Sepharose affinity and anion-exchange chromatography . Most ACCase activity (85%) recovered from the anion-exchange column was found in a highly purified fraction (specific activity 5.5 {mu}mol acid-stable product min-1 mg-1) that consisted primarily of a single 227-kD biotinylated polypeptide . The fraction represented 29% of the original activity and was designated ACCase I . A second partially purified ACCase activity (ACCase II) eluted earlier during anion-exchange chromatography, contained a single biotinylated polypeptide of 219 kD, was poorly recognized by antiserum raised against the ACCase I polypeptide, and was less inhibited by the herbicides haloxyfop or sethoxydim than was ACCase I . ACCase I and II both utilized propionyl-CoA as substrate about 50% as effectively as acetyl-CoA, and neither utilized methylcrotonyl-CoA . Immunoprecipitation with antiserum and protein blotting of crude extracts of leaf, embryo, and endosperm tissue and suspension cells indicated that most ACCase activity in these tissues was immunologically similar and consisted of ACCase I . Only leaves contained significant amounts of the ACCase II polypeptide; however, no ACCase II polypeptide was found in isolated mesophyll chloroplasts . The ACCase I and II polypeptides appear to be subunits of distinct ACCase isoforms. Plant Physiol, 2002 Sep, 130(1), 265 - 72 Plasmalemma abscisic acid perception leads to RAB18 expression via phospholipase D activation in Arabidopsis suspension cells; Hallouin M et al.; Abscisic acid (ABA) plays a key role in the control of stomatal aperture by regulating ion channel activities and water exchanges across the plasma membrane of guard cells . Changes in cytoplasmic calcium content and activation of anion and outward-rectifying K(+) channels are among the earliest cellular responses to ABA in guard cells . In Arabidopsis suspension cells, we have demonstrated that outer plasmalemma perception of ABA triggered similar early events . Furthermore, a Ca(2+) influx and the activation of anion channels are part of the ABA-signaling pathway leading to the specific expression of RAB18 . Here, we determine whether phospholipases are involved in ABA-induced RAB18 expression . Phospholipase C is not implicated in this ABA pathway . Using a transphosphatidylation reaction, we show that ABA plasmalemma perception results in a transient stimulation of phospholipase D (PLD) activity, which is necessary for RAB18 expression . Further experiments showed that PLD activation was unlikely to be regulated by heterotrimeric G proteins . We also observed that ABA-dependent stimulation of PLD was necessary for the activation of plasma anion current . However, when ABA activation of plasma anion channels was inhibited, the ABA-dependent activation of PLD was unchanged . Thus, we conclude that in Arabidopsis suspension cells, ABA stimulation of PLD acts upstream from anion channels in the transduction pathway leading to RAB18 expression. Plant Physiol, 1996 Jul, 111(3), 721 - 724 Growth Inhibition in Suspension-Cultured Rice Cells under Phosphate Deprivation Is Mediated through Putrescine Accumulation; Shih CY et al.; The effects of phosphate deprivation on the growth and polyamine levels of suspension-cultured rice (Oryza sativa) cells were investigated . When rice suspension cells were deprived of phosphate, cell growth was markedly inhibited . Phosphate deprivation resulted in a higher putrescine level and lower spermidine and spermine levels in rice suspension cells . The growth of rice cells cultured in the absence of phosphate did not recover as a result of spermidine and spermine addition . D-Arginine and {alpha}-methylornithine, inhibitors of putrescine biosynthesis, caused a reduced level of putrescine in rice suspension cells cultured under phosphate deprivation . The growth of rice cells cultured in the absence of phosphate was completely recovered after the addition of D-arginine but not {alpha}-methylornithine . Our results indicate that putrescine accumulation is a factor causing growth inhibition of suspension-cultured rice cells under phosphate deprivation. Plant Physiol, 1996 Jun, 111(2), 589 - 595 Signals Regulating the Expression of the Nuclear Gene Encoding Alternative Oxidase of Plant Mitochondria; Vanlerberghe GC et al.; Suspension cells of tobacco (Nicotiana tabacum L . cv Bright Yellow) were used to investigate signals regulating the expression of the nuclear gene Aox1 encoding the mitochondrial alternative oxidase (AOX) protein responsible for cyanide-resistant respiration in plants . We found that an increase in the tricarboxylic acid cycle intermediate citrate (either after its exogenous supply to cells or after inhibition of aconitase by monofluoroacetate) caused a rapid and dramatic increase in the steady-state level