A comparative structural analysis of direct and indirect shoot ...

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Protoplasma (2000) 212:262-267 PROTOPLASMA 9 Springer-Verlag 2000 Printed in Austria Salt stress induces changes in amounts and localization of the mitogen-activated protein kinase SIMK in alfalfa roots Frantigek Balugka 1,2, Miroslav Ovecka 2, and Heribert Hirt 3'* Institute of Botany, University of Bonn, Bonn, ; Institute of Botany, Slovak Academy of Sciences, Bratislava, and 3 Institute of Microbiology and Genetics, University of Vienna, Vienna Biocenter, Vienna Received October 1, 1999 Accepted December 22, 1999 Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday Summary. SIMK is an alfalfa mitogen-activated protein kinase (MAPK) that is activated by salt stress and shows a nuclear localization in suspension-cultured cells. We investigated the localization of SIMK in alfalfa (Medicago sati a) roots. Although SIMK was expressed in most tissues of the root apex, cells of the quiescent center and statocytes showed much lower SIMK protein amounts. In cells of the elongation zone, SIMK was present in much higher amounts in epidermal than in cortex cells. In dividing cells of the root tip, SIMK revealed a cell cycle phase-dependent localization, being predominantly nuclear in interphase but associating with the cell plate and the newly formed cell wall in telophase and early G~ phase. In dividing cells, salt stress resulted in an association of part of the SIMK with the preprophase band. Generally, salt stress resulted in much higher amounts of SIMK in dividing cells of the root apex and epidermal cells of the elongation zone. These data demonstrate that amounts and subcellular localization of SIMK in roots is highly regulated and sensitive to environmental stress. Keywords: Mitogen-activated protein kinase; Salt stress; Osmotic stress; Medicago sati a. Introduction Hyperosmotic-stress signaling in yeasts, mammals, and plants is mediated through highly conserved MAPK cascades (Brewster et al. 1993, Galcheva-Gargova et al. 1994, Han et al. 1994, Munnik et al. 1999). MAP kinase pathways are found in all eukaryotes, including * Correspondence and reprints: Institute of Microbiology and Genetics, University of Vienna, Vienna Biocenter, Dr.-Bohr-Gasse 9, A-1030 Vienna, Austria. E-mail: hehi@gem.univie.ac.at plants, and are involved in transducing a variety of extracellular signals including growth factors, hormones, as well as biotic and abiotic stresses (Jonak et al. 1999, Waskiewicz and Cooper 1995). MAPK cascades are usually composed of three protein kinases that upon activation undergo sequential phosphorylation (Robinson and Cobb 1997). By phosphorylation of conserved threonine and tyrosine residues, a MAPK becomes activated by a specific MAPK kinase (MAPKK). A MAPKK kinase (MAPKKK) activates MAPKK through phosphorylation of conserved threonine and/or serine residues. MAPK pathways may integrate a variety of upstream signals through interaction with other kinases or G proteins (Robinson and Cobb 1997). The latter factors often directly serve as coupling agent between a plasma-membranelocated receptor protein that senses an extracellular stimulus and a cytoplasmic MAPK module. At the downstream end of the module, activation of the cytoplasmic MAPK module often induces the translocation of the MAPK into the nucleus, where the kinase activates certain sets of genes through phosphorylation of specific transcription factors (Treisman 1996). In other cases, a given MAPK may translocate to other sites in the cytoplasm to phosphorylate specific enzymes (protein kinases, phosphatases, lipases, etc.) or cytoskeletal components (Cohen 1997, Robinson and Cobb 1997). By tight regulation of MAPK localization and through expression of certain signaling components and substrates in particular cells, tissues, or organs, particular MAPK pathways can mediate
E Balus"ka et al.: Mitogen-activated protein kinase SIMK in alfalfa 263 signaling of a multitude of extracellular stimuli and bring about a large variety of specific responses. We have investigated whether hyperosmotic stress in plants is also mediated by MAP kinase pathways. We have found that SIMK (stress-inducible MAP kinase) (originally named MsK7; Jonak et al. 1993) is activated by hyperosmotic stress in alfalfa cells (Munnik et al. 1999). In suspension-cultured cells, SIMK was found to be a constitutively nuclear protein and was not undergoing changes in its intracellular location upon salt stress. In this report, we have investigated the presence and localization of SIMK in alfalfa roots. Our results indicate that the tissuespecific pattern and subcellular localization of SIMK is influenced by both cell cycle cues and environmental conditions. Material and methods Plant material and sample preparation Seeds of Medicago sati a L. cv. Europa were placed on moist filter paper in petri dishes and germinated for 3 days in cultivating chambers in darkness at 25 ~ For salt stress treatment roots of 3-dayold seedlings were incubated in 0 or 200 mM NaC1. 8 mm long root tips were fixed in 3.7% formaldehyde in stabilizing buffer [SB; 50 mM piperazine-N, N9-bis(2-ethanesulfonic acid), 5 mM MgSO4, 5 mM EGTA, pH 6.9] for 1 h. After rinsing in SB and phosphatebuffered saline (PBS), root tips were dehydrated in a graded ethanol series diluted with PBS. The root tips were then infiltrated with Steedman's wax diluted in absolute ethanol in step with the proportion 1 : 1 (ethanol and wax, v/v), followed by 2 steps in pure wax. The infiltrated root tips were allowed to polymerize in pure wax at room temperature overnight. All chemicals, if not stated otherwise, were obtained from Sigma Chemical Co. (St. Louis, Mo., U.S.A.). Antibody production and specificity M23 antibody was produced against a synthetic peptide, encoding t]~e carboxyl-terminal amino acids FNPEYQQ of SIMK (Jonak et al. 1993). The antibody was found to monospecifically detect SIMK, and preincubation of the antibody with an excess of the synthetic petide completely blocked immunofluorescent labeling of SIMK in suspension-cultured cells (Munnik et al. 1999) and root sections (data not shown). Indirect immunofluorescence microscopy 7 mm thick median longitudinal sections were prepared from the fixed root tips and mounted on slides coated with glycerol-albumen (Serva, Heidelberg, Federal Republic of Germany). Sections expanded in a drop of distilled water, and they were allowed to adhere to the slides. For easy penetration of antibodies, the sections were first dewaxed in ethanol, rehydrated in an ethanol and PBS series, and kept in PBS for 30 min After a 10 rain methanol treatment at -20 ~ and a SB rinse for 30 min at room temperature, the sections were incubated with affinity-purified SIMK-specific antibody diluted 1 : 1000 with PBS for 60 rain at room temperature. After a rinse in SB the sections were incubated with secondary fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G raised in goat (Sigma) diluted 1 : 100 with PBS for 60 min at room temperature in darkness. Nuclear DNA was stained with 49,6- diamidino-2-pheuylindole (DAPI) diluted at 1 g/ml PBS for 10 rain. After rinsing with PBS, the sections were stained with 0.01% Toluidine Blue in PBS for 10 rain and mounted in antifade mountant medium containing p-phenylenediamine. Immunofluorescence observation was examined with an Axiovert 405M inverted light microscope (Zeiss, Oberkochen, Federal Republic of Germany) and Olympus BX 50 (Olympus, Tokyo, Japan) microscope equipped with epifluorescence and standard FITC exciter and barrier filters (BP 450-490 nm wavelength, LP 520 nm wavelength). Photographs were taken on Kodak T-Max films rated at 400 ASA. Results Cell cycle phase-dependent localization of SIMK SIMK encodes a MAPK that was recently identified to be activated by salt stress in suspension-cultured cells, leaves, and roots (Munnik et al. 1999). In suspension-cultured cells, salt stress was not found to alter the subcellular localization of SIMK. However, suspension-cultured cells are constantly exposed to mechanical stress (shaking), conditions that were found to activate SIMK, albeit to a much smaller extent than salt stress (Munnik et al. 1999). To study the behavior and localization of SIMK in a more natural system, roots of 3-day-old alfalfa seedlings were analyzed before and after salt stress. Figure 1 shows immunofluorescence pictures taken from longitudinal sections of nonstressed root tips. To correlate SIMK localization with the cell cycle stages, the sections were also stained with DAPI and compared. By this analysis, proliferating cells of the meristematic region revealed a predominantly nuclear localization of SIMK in interphase cells. In accordance with the cell cycle phase-dependent expression found in suspension-cultured cells (Jonak et al. 1993), G2 phase cells (Fig. 1 A) had higher amounts of SIMK protein than cells in other stages. Interestingly, in mitosis and early-Gl-phase cells, S1MK was found to accumulate in the newly formed nuclei as well as with the assembling cell plates and young cell walls (Fig. 1 A). These results prompted us to perform a more thorough analysis of the localization of SIMK during different mitotic stages. In prophase, metaphase, and anaphase, SIMK was clearly excluded from the condensing chromosomes and the spindle apparatus (Fig. 1 C-J). In telophase (lower cell in Fig. 1 K, L), SIMK started to accumulate at the periphery of the reforming nuclei (Fig. 1 K). At a slightly later stage of mitosis (upper cell
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