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

E Balus'ka et al.: Mitogen-activated protein kinase SIMK in alfalfa 265
in Fig. 1 K, L), SIMK was seen to have re-entered the
nuclei (Fig. 1 K), but a clear association with the young
cell wall was also apparent (Fig. 1 K). The association
of SIMK with young cell walls was consistently found
to extend well into the G1 phase, when cells had clearly
finished cytokinesis, as can be seen by inspecting the
cells in Fig. 1 A, E, and I.
Salt-stress-associated changes in intracellular
localization of SIMK
A concentration of 200 mM NaC1 induces the activation
of SIMK in suspension-cultured cells, leaves, and
roots (Munnik et al. 1999). Root tip cells of alfalfa
seedlings that were treated at this salt concentration
for t h showed little changes in the intracellular localization
of SIMK. In interphase cells (Fig. 1M, N),
SIMK was found to be still predominantly nuclear.
However, some cells also showed an association of
SIMK with preprophase bands (Fig. 1M). Whereas
prophase and metaphase cells showed a SIMK staining
pattern that was similar to nonstressed cells (data
not shown), some salt-treated root tip cells in telophase
(Fig. 1 O, P) revealed colocalization of SIMK
with phragmoplasts (Fig. 1 O). In these cells, SIMK
labeling of the newly forming cell walls was distinctly
absent.
Tissue-specific expression of SIMK influenced
by salt stress
When root tips were analyzed for SIMK expression at
a lower magnification, it became apparent that SIMK
was abundantly present in most tissues of the root
tip (Fig. 2 A). An exception to this rule were the quiescent
center and the statocytes at the very root apex
(Fig. 2A) showing much lower amounts of SIMK.
Interestingly, root tips of salt-stressed seedlings
showed much higher SIMK protein amounts (Fig.
2B). In the transition zone of unstressed roots, SIMK
showed considerably higher expression levels in the
epidermis than in the adjacent cortex (Fig. 2C), and
salt stress exacerbated this difference even further
(Fig. 2 D). When cells of the elongation region were
analyzed after salt stress, a large percentage of epidermal
cells was observed to have undergone plasmolysis
(Fig. 2E). In these cells, the protoplast
retained most of SIMK protein, but some SIMK
protein was also found to be associated with the
plasma membrane and the cell wall (Fig. 2 E).
Discussion
In animals, yeasts, and plants, specific MAP kinase
pathways are involved in mediating responses to
hyperosmotic stress. The family of mammalian MAP
kinases including the SAPK(stress-activated protein
kinase)/JNK(Jun N-terminal kinase)/p38 is activated
by hyperosmotic as well as various other types of stress
(Waskiewicz and Cooper 1995). In Saccharomyces
cerevisiae, the HOG1 MAP kinase pathway is exclusively
used for mediating hyperosmotic stress (Brewster
et al. 1993). In alfalfa plants, the SIMK pathway is
involved in signaling hyperosmotic stress (Munnik
et al. 1999).
Activation of MAP kinases often involves the
nuclear import of the MAP kinase from the cytoplasm
after activation. Studies on the mammalian ERK1/
ERK2 kinases showed that the phosphorylation of the
MAP kinase by the upstream MAP kinase kinase was
an essential step to induce the nuclear translocation
(Chen et al. 1992, Lenormand et al. 1993). Recent data
revealed that MAP kinases can shuttle between cytoplasmic
and nuclear compartments, and that the time
spent in any one compartment can be influenced by a
number of parameters that may differ in a systemspecific
way. In mammalian cells, MAPKKs may act as
cytoplasmic anchors for inactive MAPKs (Fukuda
et al. 1997). Phosphorylation of MAPK is thought to
induce dissociation from the MAPKK, thereby allowing
nuclear import of the MAPK. Other evidence
suggests that phosphorylation-induced dimerization
may also contribute to nuclear import of MAPKs
(Khokhlatchev et al. 1998). Investigations of the Schizosaccharomyces
pornbe Spcl/Styl stress-signaling
MAP kinase pathway revealed that the nuclear target
of the Spcl/Styl kinase, the transcription factor Atfl,
plays an active role in retaining the MAPK in the
nucleus (Gaits et al. 1998). Finally, studies on the
HOG1 kinase in budding yeast added more complexity
by showing that besides regulation of entry and
anchoring of the MAPK in the nucleus, nuclear export
of the MAPK also contributes to the overall time of
MAPK nuclear residence (Ferrigno et al. 1998).
To study the intracellular localization of SIMK, indirect
immunofluorescence microscopy was performed
on root tip sections of seedlings that were either
untreated or stressed with 200 mM NaC1 for different
times. Our analysis in nonstressed roots revealed a cell
cycle phase-dependent localization of SIMK, showing
constitutive nuclear staining during interphase. The