Ultrastructural distribution of a MAP kinase and transcripts in quiescent and cycling plant cells and pollen grains

Mitogen-activated protein kinases (MAPKs) are components of a kinase module that plays a central role in the transduction of diverse extracellular sti muli, including mitogens, specific differentiation and developmental signal s and stress treatments. This shows that reversible protein phosphorylation cascades play a pivotal role in signal transduction in animal cells and ye ast, particularly the entry into mitosis of arrested cells. Homologues of M APKs have been found and cloned in various plant species, but there have be en no data about their in situ localization at the subcellular level and th eir expression in plant cells so far. In the present paper we report the fi rst data on the ultrastructural in situ localization of MAPK and their mRNA s in various plant cells. Proliferating and quiescent meristematic plant cells were studied to evalua te whether changes in MAPK presence, distribution and expression accompany the entry into proliferation of dormant cells. Moreover, MAPK localization was analyzed in vacuolate microspores, Polyclonal antibodies against the de duced MAPK from the tobacco Ntf6 clone were able to recognize homologue epi topes by immunocytochemical techniques in the cell types studied. The patte rn of protein distribution is similar in ail the cases studied: it is local ized in the cytoplasm and in the nucleus, mainly in the interchromatin regi on. The quantitative study of the density showed that MAPK labelling is mor e abundant in cycling than in quiescent cells, also suggesting that, in pla nts, MAPK pathways might play a role in cell proliferation, RNA probes for conserved regions of the catalytic domain of plant MAPK homologue genes wer e used to study MAPK expression in those plant cells. In situ hybridization (ISH) showed the presence of MAPK transcripts in the three plant cell type s studied, but levels were very low in quiescent cells compared to those in cycling cells, The quantification of labelling density of ISH signals stro ngly suggests a higher level of MAPK expression in proliferating cells, but also some basal messenger presence and/or expression in the quiescent ones , Immunogold and ISH results show the presence and distribution of MAPK pro teins and mRNAs in vacuolate microspores, This represents a very dynamic st age during pollen development in which the cell nucleus is being prepared f or an asymmetrical mitotic division, giving rise to both the generative and the vegetative nuclei of the bicellular pollen grain. Taken together, the data indicate a role played by MAPK in the re-entry into proliferation in p lant cells.