RESUMEN
The effects of changes in the chlorophyll (chl) content on the kinetics of the OJIP fluorescence transient were studied using two different approaches. An extensive chl loss (up to 5-fold decrease) occurs in leaves suffering from either an Mg(2+) or SO(4)(2-) deficiency. The effects of these treatments on the chl a/b ratio, which is related to antenna size, were very limited. This observation was confirmed by the identical light intensity dependencies of the K, J and I-steps of the fluorescence rise for three of the four treatments and by the absence of changes in the F(685 nm)/F(695 nm)-ratio of fluorescence emission spectra measured at 77K. Under these conditions, the F(0) and F(M)-values were essentially insensitive to the chl content. A second experimental approach consisted of the treatment of wheat leaves with specifically designed antisense oligodeoxynucleotides that interfered with the translation of mRNA of the genes coding for chl a/b binding proteins. This way, leaves with a wide range of chl a/b ratios were created. Under these conditions, an inverse proportional relationship between the F(M) values and the chl a/b ratio was observed. A strong effect of the chl a/b ratio on the fluorescence intensity was also observed for barley Chlorina f2 plants that lack chl b. The data suggest that the chl a/b ratio (antenna size) is a more important determinant of the maximum fluorescence intensity than the chl content of the leaf.
Asunto(s)
Clorofila/metabolismo , Hojas de la Planta/metabolismo , Beta vulgaris/efectos de los fármacos , Beta vulgaris/metabolismo , Beta vulgaris/efectos de la radiación , Clorofila A , Fluorescencia , Hordeum/efectos de los fármacos , Hordeum/metabolismo , Hordeum/efectos de la radiación , Magnesio/metabolismo , Oligonucleótidos Antisentido/farmacología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/efectos de la radiación , Proteínas de Plantas/metabolismo , Sulfatos/metabolismo , Triticum/efectos de los fármacos , Triticum/metabolismo , Triticum/efectos de la radiaciónRESUMEN
KEY MESSAGE : Wheat genes differentially expressed in the egg cell before and after fertilization were identified. The data support zygotic gene activation before the first cell division in wheat. To have an insight into fertilization-induced gene expression, cDNA libraries have been prepared from isolated wheat egg cells and one-celled zygotes. Two-hundred and twenty-six egg cell and 253 zygote-expressed EST sequences were determined. Most of the represented transcripts were detected in the wheat egg cell or zygote transcriptome at the first time. Expression analysis of fourteen of the identified genes and three controls was carried out by real-time quantitative PCR. The preferential expression of all investigated genes in the female gametophyte-derived samples (egg cells, zygotes, two-celled proembryos, and basal ovule parts with synergids) in comparison to the anthers, and the leaves were verified. Three genes with putative signaling/regulatory functions were expressed at a low level in the egg cell but exhibited increased (2-to-33-fold) relative expression in the zygote and the proembryo. Genes with high EST abundance in cDNA libraries exhibited strong expression in the egg cell and the zygote, while the ones coding for unknown or hypothetical proteins exhibited differential expression patterns with preferential transcript accumulation in egg cells and/or zygotes. The obtained data support the activation of the zygotic genome before the first cell division in wheat.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/genética , Óvulo Vegetal/genética , Triticum/genética , Etiquetas de Secuencia Expresada , Fertilización , Expresión Génica , Perfilación de la Expresión Génica , Biblioteca de Genes , Genoma de Planta/genética , Especificidad de Órganos , Reacción en Cadena en Tiempo Real de la Polimerasa , Semillas/genética , CigotoRESUMEN
Selective inhibition of gene expression by antisense oligodeoxynucleotides (ODNs) is widely applied in gene function analyses; however, experiments with ODNs in plants are scarce. In this work, we extend the use of ODNs in different plant species, optimizing the uptake, stability, and efficiency of ODNs with a combination of molecular biological and biophysical techniques to transiently inhibit the gene expression of different chloroplast proteins. We targeted the nucleus-encoded phytoene desaturase (pds) gene, encoding a key enzyme in carotenoid biosynthesis, the chlorophyll a/b-binding (cab) protein genes, and the chloroplast-encoded psbA gene, encoding the D1 protein. For pds and psbA, the in vivo stability of ODNs was increased by phosphorothioate modifications. After infiltration of ODNs into juvenile tobacco (Nicotiana benthamiana) leaves, we detected a 25% to 35% reduction in mRNA level and an approximately 5% decrease in both carotenoid content and the variable fluorescence of photosystem II. In detached etiolated wheat (Triticum aestivum) leaves, after 8 h of greening, the mRNA level, carotenoid content, and variable fluorescence were inhibited up to 75%, 25%, and 20%, respectively. Regarding cab, ODN treatments of etiolated wheat leaves resulted in an up to 59% decrease in the amount of chlorophyll b, a 41% decrease of the maximum chlorophyll fluorescence intensity, the cab mRNA level was reduced to 66%, and the protein level was suppressed up to 85% compared with the control. The psbA mRNA and protein levels in Arabidopsis (Arabidopsis thaliana) leaves were inhibited by up to 85% and 72%, respectively. To exploit the potential of ODNs for photosynthetic genes, we propose molecular design combined with fast, noninvasive techniques to test their functional effects.
Asunto(s)
Arabidopsis/genética , Proteínas de Cloroplastos/genética , Regulación de la Expresión Génica de las Plantas/genética , Nicotiana/genética , Oligodesoxirribonucleótidos/genética , Triticum/genética , Arabidopsis/metabolismo , Transporte Biológico , Carotenoides/metabolismo , Núcleo Celular/metabolismo , Proteínas de Unión a Clorofila/genética , Proteínas de Unión a Clorofila/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Técnicas de Silenciamiento del Gen , Oligodesoxirribonucleótidos/síntesis química , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/genética , Oxidorreductasas/genética , Oligonucleótidos Fosforotioatos/síntesis química , Oligonucleótidos Fosforotioatos/genética , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ARN Mensajero/genética , ARN de Planta/genética , Nicotiana/metabolismo , Triticum/metabolismoRESUMEN
During mitosis, cells detach, and the cell-matrix interactions become restricted. At the completion of cytokinesis, the two daughter cells are still connected transiently by an intercellular bridge (ICB), which is subjected to abscission, as the terminal step of cytokinesis. Cell adhesion to the matrix is mediated by syndecan-4 (SDC4) transmembrane heparan sulfate proteoglycan. Our present work demonstrated that SDC4 promotes cytokinesis in a phosphorylation-dependent manner in MCF-7 breast adenocarcinoma cells. The serine179-phosphorylation and the ectodomain shedding of SDC4 changed periodically in a cell cycle-dependent way reaching the maximum at G2/M phases. On the contrary, the phospho-resistant Ser179Ala mutant abrogated the shedding. The phosphorylated full-length and shed remnants enriched along the mitotic spindles, and subsequently in the ICBs, however, proper membrane insertion was necessary for midbody localization. Expression of phosphomimicking Ser179Glu SDC4 resulted in incomplete abscission, whereas expression of the phospho-resistant SDC4 led to giant, multinucleated cells.
Asunto(s)
Citocinesis/fisiología , Sindecano-4/fisiología , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Sustitución de Aminoácidos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular , Línea Celular Tumoral , Femenino , Células Gigantes/metabolismo , Células Gigantes/patología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Fluorescente , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina/química , Huso Acromático/metabolismo , Sindecano-4/química , Sindecano-4/genéticaRESUMEN
Based on the in silico analysis of the representation of expressed sequence tags (ESTs) in wheat grain-related cDNA libraries, a specific 15k oligonucleotide microarray has been developed in order to monitor environmental stress-dependent gene expression changes in the wheat caryopses. Using this array, the effect of water withdrawal, with and without additional heat stress, has been investigated during the first five days of kernel development on two wheat cultivars differing in their drought sensitivity. Water shortage affected (more than twofold change) the expression of only 0.5% of the investigated genes. A parallel heat treatment increased the ratio of responding genes to 5-7% because of the temperature stress and/or the increased water deficit because of enhanced evaporation. It could be established that the two cultivars, differing in their long-term adaptation capabilities to drought, responded to the short and direct stress treatments on the same way. In response to the combined drought and heat treatment, the coordinately altered expression of genes coding for storage proteins, enzymes involved in sugar/starch metabolism, histone proteins, heat shock proteins, proteases, tonoplast aquaporins as well as several transcription factors has been observed. These gene expression changes were in agreement with histological data that demonstrated the accelerated development of the embryo as well as the endosperm.
Asunto(s)
Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Semillas/genética , Triticum/genética , Adaptación Fisiológica/genética , Análisis por Conglomerados , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Calor , Proteínas de Plantas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semillas/anatomía & histología , Semillas/crecimiento & desarrollo , Especificidad de la Especie , Estrés Fisiológico , Triticum/anatomía & histología , Triticum/crecimiento & desarrollo , Agua/metabolismo , Agua/farmacologíaRESUMEN
The ability of different synthetic cell penetrating peptides, as Antennapedia (wild and Phe(6) mutated penetratins), flock house virus, and integrin peptides to form complexes with a 25mer antisense oligonucleotide was compared and their conformation was determined by circular dichroism spectroscopy. The efficiency for oligonucleotide delivery into cells was measured using peptides labeled with a coumarin derivative showing blue fluorescence and the fluorescein-labeled antisense oligonucleotide showing green fluorescence. Fluorescence due to the excitation energy transfer confirmed the interaction of the antisense oligonucleotide and cell-penetrating peptides. The most efficient oligonucleotide delivery was found for penetratins. Comparison of the two types of penetratins shows that the wild-type penetratin proved to be more efficient than mutated penetratin. The paper also emphasizes that the attachment of a fluorescent label may have an effect on the conformation and flexibility of cell-penetrating peptides that must be taken into consideration when evaluating biological experiments.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/farmacocinética , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Oligonucleótidos Antisentido/farmacocinética , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/genética , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/síntesis química , Proteínas Portadoras/genética , Línea Celular Transformada , Péptidos de Penetración Celular , Dicroismo Circular , Regulación hacia Abajo , Portadores de Fármacos/síntesis química , Técnicas de Transferencia de Gen , Ratones , Datos de Secuencia Molecular , Mutación , Células 3T3 NIH , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/genética , Conformación Proteica , Transporte de Proteínas , Proteína EWS de Unión a ARNRESUMEN
Because of the relative impermeability of the blood-brain barrier (BBB), many drugs are unable to reach the CNS in therapeutically relevant concentration. One method to deliver drugs to the CNS is the osmotic opening of the BBB using mannitol. Hyperosmotic mannitol induces a strong phosphorylation on tyrosine residues in a broad spectrum of proteins in cerebral endothelial cells, the principal components of the BBB. Previously, we have shown that among targets of tyrosine phosphorylation are beta-catenin, extracellular signal-regulated kinase 1/2 and the non-receptor tyrosine kinase Src. The aim of this study was to identify new signalling pathways activated by hypertonicity in cerebral endothelial cells. Using an antibody array and immunoprecipitation we identified the receptor tyrosine kinase Axl to become tyrosine phosphorylated in response to hyperosmotic mannitol. Besides activation, Axl was also cleaved in response to osmotic stress. Degradation of Axl proved to be metalloproteinase- and proteasome-dependent and resulted in 50-55 kDa C-terminal products which remained phosphorylated even after degradation. Specific knockdown of Axl increased the rate of apoptosis in hyperosmotic mannitol-treated cells; therefore, we assume that activation of Axl may be a protective mechanism against hypertonicity-induced apoptosis. Our results identify Axl as an important element of osmotic stress-induced signalling.
Asunto(s)
Barrera Hematoencefálica/enzimología , Arterias Cerebrales/enzimología , Células Endoteliales/enzimología , Proteínas Oncogénicas/metabolismo , Presión Osmótica/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Barrera Hematoencefálica/efectos de los fármacos , Línea Celular , Arterias Cerebrales/citología , Arterias Cerebrales/efectos de los fármacos , Regulación hacia Abajo/fisiología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Humanos , Soluciones Hipertónicas/farmacología , Manitol/farmacología , Metaloproteasas/metabolismo , Proteínas Oncogénicas/genética , Fosforilación/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Proteínas Tirosina Quinasas Receptoras/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tirosina Quinasa del Receptor AxlRESUMEN
It has been reported that muscles of myoD-/- mice present a lower potential to regenerate, but there are no reports on the effect of acute interference with myoD expression limited in space and time to only a particular regenerating muscle. Here we relied on antisense inhibition of this factor. Four different oligos were tested. The suppression of regeneration indices (the expression of desmin, the formation of myotubes and the initiation of endplates) was the most pronounced, with the oligomer targeting a region encompassing the translation start site of myoD. A mixed backbone phosphorothioate-phosphate diester oligo (200 microl at 20 microM) was still detectable in the muscles 1 h after its administration and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the level of the targeted 5' end of the myoD mRNA was selectively decreased. The level of myoD protein was also lowered. Four hours after the antisense treatment, when the oligos were no longer detectable, the myoD mRNA level was restored and 24 h later it exceeded controls together with that of myf-5 and myogenin. After 4 weeks, the antisense-treated soleus muscles were similar to the control-treated and the untreated regenerated soleus with respect to fiber types and motor endplates, however, they contained smaller fibers which reflected the asynchronity of regeneration. This shows that successfully targeted simple antisense oligonucleotides can be used as selective tools for inhibition of individual factors in studying the process of muscle regeneration.
Asunto(s)
Proteínas Musculares/genética , Músculo Esquelético/fisiología , Proteína MioD/antagonistas & inhibidores , Miogenina/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Regeneración/genética , Proteínas Represoras , Transactivadores , Animales , Secuencia de Bases , Bromodesoxiuridina/metabolismo , Proteínas de Unión al ADN/genética , Desmina/metabolismo , Expresión Génica , Proteína 1 Inhibidora de la Diferenciación , Proteína 2 Inhibidora de la Diferenciación , Masculino , Músculo Esquelético/efectos de los fármacos , Proteína MioD/genética , Proteína MioD/fisiología , Factor 5 Regulador Miogénico , Cadenas Pesadas de Miosina/metabolismo , Oligodesoxirribonucleótidos Antisentido/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Regeneración/efectos de los fármacos , Factores de Transcripción/genéticaRESUMEN
SET/I(2)(PP2A), a member of the family of nucleosome assembly proteins (NAPs), has been previously described as a multifunctional protein inhibiting protein phosphatase 2A (PP2A)-mediated histone H3((pSer10)) dephosphorylation during the heat shock response in animal cells. In the present work we demonstrate that its plant orthologs, designated as NAP-related proteins (NRPs), have a similar in vitro biochemical activity and interact with PP2A and histone H3((pSer10))in vivo. Although heat shock gene promoters were found to be associated with histone H3((pSer10))-marked chromatin following a high temperature treatment, heat shock gene expression was not affected in NRP-deficient mutant Arabidopsis thaliana (L.) plantlets. These observations indicate that NRPs are potential regulators of histone dephosphorylation in plants, but they are dispensable for gene expression reorganization in response to heat shock.
Asunto(s)
Arabidopsis/enzimología , Proteínas de Choque Térmico/genética , Medicago sativa/enzimología , Nucleosomas/metabolismo , Proteína Fosfatasa 2/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Anticuerpos , Arabidopsis/genética , Ensamble y Desensamble de Cromatina , Inhibidores Enzimáticos , Expresión Génica , Histonas/genética , Histonas/metabolismo , Calor , Medicago sativa/genética , Datos de Secuencia Molecular , Mutación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas/genética , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Conejos , Alineación de SecuenciaRESUMEN
The receptor-like cytoplasmic protein kinases (RLCKs) are plant-specific proteins encoded by almost 200 genes in the Arabidopsis genome. Despite of their high number, the available information on the potential function of RLCKs is very limited. In this report, the sequence analysis and the gene expression pattern of 14 members of one of the Arabidopsis RLCK families (RLCK class VI) are described. Sequence comparison indicated that gene duplication played a significant role in the formation of the kinase family and that several members carry an N-terminal "universal stress protein" (UspA) domain. In order to gain insight into the potential function of the RLCK VI kinases, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to determine the relative transcript levels in the various organs of the Arabidopsis plant as well as under a series of abiotic stress/hormone treatments in seedlings. The obtained data revealed the differentially regulated expression of the genes in agreement with a high variability of sequence elements in their promoters. The divergent expression patterns indicate that the encoded kinase proteins may be involved in a wide variety of signal transduction pathways related to plant development and stress responses. The significance of gene duplication and expression divergence in the extension of the Arabidopsis RLCK VI family during evolution is discussed.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Proteínas Quinasas/genética , Ácido Abscísico/farmacología , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Secuencia de Bases , Frío , Desastres , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Cloruro de Sodio/farmacologíaRESUMEN
We present a method that offers the possibility to directly apply and measure torque on particles in an optical trap. It can be used to rotationally manipulate biopolymers attached to appropriate particles. A flat object is trapped and oriented in the focus of a linearly polarized laser light. The direction and power of the orientational trap are controlled by the polarization state of the light. As a demonstration of the capabilities of the method, we examined the torsional stiffness of dsDNA (lambda-DNA) in its linear torsional regime by directly measuring the torque generated by the molecule.
Asunto(s)
ADN/química , Óptica y Fotónica , Torque , Factores de TiempoRESUMEN
Octanoyl and palmitoyl groups were coupled to the N-terminus of an analog of the SV40 nuclear localization signal peptide, SV126-133(Ser128), to study the effect of the fatty acid chain length on the complex formation with a single-stranded antisense oligodeoxynucleotide (ODN) and on the cellular uptake of the complex. The strongest binding affinity was observed for the palmitoylated peptide, indicating the better accessibility of the positively charged lysyl and arginyl side-chains to the phosphate groups due to the turn structures stabilized by the palmitoyl group. On increase of the peptide to ODN molar ratio (rM), gradual unstacking of the bases was observed, the maximal rate being reached at rM=10. At rM>10 restacking of the nucleotide bases was detected and the ODN was completely encapsulated in a liposome-like structure made up of palmitoylated peptides. Cell translocation experiments revealed a highly efficient cell transport of the ODN by palmitoylated SV40 peptide at rM>10.
Asunto(s)
Caprilatos/química , Oligodesoxirribonucleótidos Antisentido/metabolismo , Oligopéptidos/química , Ácidos Palmíticos/química , Acilación , Animales , Transporte Biológico , Permeabilidad de la Membrana Celular , Dicroismo Circular , Portadores de Fármacos , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Ratones , Microscopía de Fuerza Atómica , Conformación Molecular , Células 3T3 NIH , Oligodesoxirribonucleótidos Antisentido/administración & dosificación , Oligodesoxirribonucleótidos Antisentido/farmacología , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Cyclin-dependent serine/threonine kinases (CDKs) have pivotal roles in regulating the eukaryotic cell cycle. Plants possess a unique class of CDKs (B-type CDKs) with preferential protein accumulation at G2/M-phases; however, their exact functions are still enigmatic. Here we describe the functional characterization of a 360-bp promoter region of the alfalfa (Medicago sativa) CDKB2;1 gene in transgenic plants and cell lines. It is shown that the activity of the analyzed promoter was characteristic for proliferating meristematic regions in planta and specific for cells in the G2/M-phases in synchronized cell cultures. Immunohistochemical analysis of transgenic root sections further confirmed the correlation of the expression of the CDKB2;1 promoter-linked reporter genes with the accumulation of the correspondent kinase. It was found that, in addition to auxin (2,4-dichlorophenoxyacetic acid) treatment, wounding could also induce both the reporter and endogenous genes in transgenic leaf explants. Furthermore, ethylene, known as a wound-response mediator, had a similar effect. The gene activation in response to wounding or ethephon was faster and occurred without the induction of cell cycle progression in contrast to the control auxin treatment. In silico analysis of this promoter indeed revealed the presence of a set of cis-elements, indicating not only cell cycle- but wound- and ethylene-dependent regulation of this CDK gene. Based on the presented data, we discuss the functional significance of the complex regulation of mitosis-specific CDK genes in plants.
Asunto(s)
Quinasas Ciclina-Dependientes/genética , Etilenos/farmacología , Medicago sativa/enzimología , Mitosis/fisiología , Proteínas de Plantas/genética , Regiones Promotoras Genéticas/fisiología , Ácido 2,4-Diclorofenoxiacético/farmacología , Secuencia de Bases , Biología Computacional , Quinasas Ciclina-Dependientes/clasificación , Quinasas Ciclina-Dependientes/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Inmunohistoquímica , Medicago sativa/efectos de los fármacos , Medicago sativa/genética , Datos de Secuencia Molecular , Proteínas de Plantas/clasificación , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Plantas Modificadas Genéticamente/anatomía & histología , Plantas Modificadas Genéticamente/fisiología , Alineación de Secuencia , Activación TranscripcionalRESUMEN
It is now well established that nitric oxide (NO) serves as a signaling molecule in plant cells. In this paper experimental data are presented which indicate that NO can stimulate the activation of cell division and embryogenic cell formation in leaf protoplast-derived cells of alfalfa in the presence of auxin. It was found that various NO-releasing compounds promoted auxin-dependent division (as shown by incorporation of bromodeoxyuridine) of leaf protoplast-derived alfalfa cells. In contrast, application of NO scavenger or NO synthesis inhibitor inhibited the same process. Both the promotion and the inhibition of cell cycle activation correlated with the amount and activity of the cognate alfalfa p34cdc2 protein Medsa;CDKA;1,2. The effect of l-NG-monomethyl-L-arginine (L-NMMA) was transient, and protoplast-derived cells spending more than 3 days in culture become insensitive to the inhibitor as far as cell cycle progression was concerned. L-NMMA had no effect on the cell cycle parameters of cycling suspension-cultured cells, but had a moderate transient inhibitory effect on cells re-entering the cell cycle following phosphate starvation. Cycling cultured cells, however, could respond to NO, as indicated by the sodium nitroprusside (SNP)- and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)-dependent accumulation of the ferritin protein. Based on these observations, it is hypothesized that L-NMMA-sensitive generation of NO is involved in the activation, but not the progression of the plant cell division cycle. In addition, SNP promoted and L-NMMA delayed the exogenous auxin [2,4-dichlorophenoxyacetic acid (2,4-D)] concentration-dependent formation of embryogenic cell clusters expressing the MsSERK1 gene; this further supports a link between auxin- and NO-dependent signaling pathways in plant cells.