SummaryPlant biology is currently experiencing a growing demand for easy and reliable mRNA and protein localisation techniques. Here, we present novel whole mount in situ hybridisation and immunolocalisation protocols, suitable to localise mRNAs and proteins in Arabidopsis seedlings. We demonstrate that these methods can be used in different organs of Arabidopsis seedlings as well as in other plant species. In order to achieve better reproducibility and higher throughput, we modi®ed these protocols for automation to be performed by a liquid handling robot. In addition, we show that other procedures such as reporter enzyme assays and tissue clearing can be similarly automated. We present examples of application of our protocols including mRNA localisation and proteins and epitope tag (co)localisations which demonstrate that these methods provide reliable and versatile tools for expression, localisation and anatomical studies in plants.

Proteins similar to the bacterial Dnaj protein have been implicated as molecular chaperones in different compartments of eukaryots. A plant equivalent is now described in tissues of dark-grown cucumber seedlings. Using a cucumber Dnaj protein produced by expression in bacteria, we raised polyclonal antibodies against the protein and used them for localization studies. In etiolated cucumber seedlings, both cotyledons and hypocotyledons were found to contain Dnaj proteins. Cell fractionation of etiolated cotyledons showed that Dnaj proteins were detectable mainly in the postnuclear cell fraction after sedimentation at lOOOOXg, and in the microsomes. Following subfractionation by sucrose density gradient centrifugation and analysis by immunoblotting, a 53-kDa protein was attributed to the glyoxysomal fraction and an 80-kDa protein to the mitochondria1 fraction. The glyoxysomal Dnaj protein behaved as a membrane-bound form. Upon heat shock, a slight increase in the content of the glyoxysomal Dnaj protein was found. When glyoxysomes were treated with protease and subsequently isolated by gradient centrifugation, virtually all immunologically detectable Dnaj protein was removed. Administration of radiolabelled mevalonic acid to cotyledons and isolation of glyoxysomes yielded labelled Dnaj protein which remained membrane bound during the purification of glyoxysomal membranes by floatation in a density gradient.The knowledge about molecular chaperones participating in the unidirectional intracellular transport of proteins had great implications in formulating concepts of organelle biosynthesis [l-31. However, new concepts and approaches are required, particularly for improving our understanding of the biosynthesis of peroxisomes. Despite the elegant work with in-vivo-expressed chimeric proteins and the characterization of peroxisomal-targeting sequences [4], the studies needed to understand the import machinery were only partly successful [5-81. Hence, genetic approaches have been applied [9, 101 to overcome inadequacies in known methodologies. Also promising are investigations of peroxisomal membrane components which are responsible for the specific binding of cytosolic precursor proteins destined for the peroxisomal matrix. It is anticipated that the peroxisomal location signal itself or a potential cytosolic receptor, a peroxisomal location signal-binding protein, may be involved in selecting the precursor protein from the cytosolic pool.Cells programmed for increased peroxisome or glyoxysome biosynthesis are assumed to promote substantial production of components of the peroxisomal-import machinery. To detect components of this apparatus, we began with a cDNA library representing the stage just prior to the stage of maximal fatty acid degradation. This metabolic process is preceded by a stage of extensive synthesis and import ofCorrespondence to H. Kindl,

We have isolated, cloned and characterized a cDNA from Zea mays L., denoted ZmAPI, coding for an anionic peroxidase. The open reading frame of ZmAP1 starting 72 residues from the 5' end of the cDNA predicts a 37778 dalton protein of 356 amino acid residues. The protein has high similarity to other peroxidases and contains two peroxidase motifs that carry two highly conserved histidines in the active center. We expressed recombinant ZmAPl protein in E. coli as a fusion with maltose-binding protein. The fusion protein was biochemically active after addition of hemin to the apoprotein. The maize peroxidase ZmAPl has a pH optimum at pH 4.0 and a K,, of 0.2 mM for the substrate 2,2'-azino-bis-(3-ethyl-benzothiazolin-6-sulfonic acid) at this pH. In maize seedlings the ZmAPl gene is expressed predominantly in roots, the mesocotyl, the coleoptile and to a lower extent in the node, whereas no expression in the primary leaf was found. In situ hybridization shows that the expression of ZinAPl in the young maize root is confined to the epidermis, hypodermis and the pericycle.

The major auxin-binding protein (ZmERabp1) from maize (Zea mays L.) has been structurally characterized. We determined the position of a disulfide bridge in ZmERabp1 by mass-spectrometric analysis. We show that Cys2 and Cys61 are covalently linked and that residue Cys155 bears the free sulfhydryl group. By making use of electrospray mass spectrometry, the molecular mass of ZmERabp1 was determined to be 20 243 Da comprising a sugar moiety of 1865 Da, corresponding to a high mannose-type glycan structure. Due to the high homology among all characterized ABPs, the information on the disulfide bonds will be important for functional analysis of recombinantly expressed ABP1. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.