Nitric oxide is required for, and promotes auxin-mediated activation of, cell division and embryogenic cell formation but does not influence cell cycle progression in alfalfa cell cultures

• Published in: The Plant journal : for cell and molecular biology 2005-09, Vol.43 (6), p.849-860
• Main Authors: Otvos, K, Pasternak, T.P, Miskolczi, P, Domoki, M, Dorjgotov, D, Szucs, A, Bottka, S, Dudits, D, Feher, A
• Format: Article
• Language: English
• Subjects: 2,4-D; 2,4-Dichlorophenoxyacetic Acid - pharmacology; 2,4‐dichlorophenoxyacetic acid; Alfalfa; auxins; Biological and medical sciences; bromodeoxyuridine; bromodeoxyuridine (BrdU) incorporation; cell biology; Cell culture; Cell cycle; Cell Cycle - physiology; cell division; Cell kinetics; Cell physiology; cell suspension culture; Cells, Cultured; cultured cells; cyclin-dependent protein kinases; cyclins; embryo (plant); forage crops; Fundamental and applied biological sciences. Psychology; gene expression regulation; Herbicides - pharmacology; Hormones; Indoleacetic Acids - physiology; L-N(G)-monomethyl-L-arginine; Leaves; Medicago sativa; Medicago sativa - cytology; Medicago sativa - drug effects; Medicago sativa - embryology; Medicago sativa - physiology; molecular sequence data; Nitric oxide; Nitric Oxide - physiology; nitrogen monoxide; nucleotide sequences; omega-N-Methylarginine - pharmacology; plant biochemistry; Plant growth; plant physiology; Plant physiology and development; plant proteins; protein kinases; protoplast; protoplasts; Seeds - cytology; Seeds - physiology; Signal transduction; sodium nitroprusside; somatic embryogenesis
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313X.2005.02494.x

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.