The large vascular meristem of poplar trees with its highly organized secondary xylem enables the boundaries between different developmental zones to be easily distinguished. This property of wood-forming tissues allowed us to determine a unique tissuespecific transcript profile for a well defined developmental gradient. RNA was prepared from different developmental stages of xylogenesis for DNA microarray analysis by using a hybrid aspen unigene set consisting of 2,995 expressed sequence tags. The analysis revealed that the genes encoding lignin and cellulose biosynthetic enzymes, as well as a number of transcription factors and other potential regulators of xylogenesis, are under strict developmental stage-specific transcriptional regulation.T ranscript profiling has the potential to reveal transcriptional hierarchy during development for thousands of genes, as well as providing expression data for many genes of unknown function (1, 2). This is especially true when expression patterns can be obtained for well defined tissues at specific developmental stages. However, this is technically demanding and so far there are no reports demonstrating tissue-specific analysis on cell types from a single developmental sequence. We have studied the developing secondary xylem of poplar, which is highly organized with easily recognized and distinct boundaries between the different developmental stages. Wood formation is initiated in the vascular cambium. Cambial derivatives develop into xylem cells through the processes of division, expansion, secondary wall formation, lignification, and finally, programmed cell death. The large physical size of the vascular meristem in trees offers a unique possibility to obtain samples from defined developmental stages by tangential cryo sectioning (3). To determine the steady-state mRNA levels at specific stages during the ontogeny of wood formation in Populus tremula ϫ Populus tremuloides (hybrid aspen) we sampled 30-m-thick sections through the wood development region and subsequently analyzed the samples by using a spotted cDNA-microarray (4) consisting of 2,995 unique ESTs from hybrid aspen. Our study provides a unique global examination of gene expression patterns that encompasses a developmental gradient within a multicellular organism. Materials and MethodsThe Unigene set was selected from the expressed sequence tags (ESTs) presented in ref. 5, using cluster analysis. ESTs were transformed into Escherichia coli by using TSS competent cells (6), plasmids were prepared by using 96-well Multiscreen FB plates (Millipore), inserts were PCR amplified by using vectorspecific primers, and PCR products were purified on Multiscreen PCR filter plates (Millipore) and spotted in duplicate onto CMT GAPS slides (Corning) by using the GMS 417 Arrayer (Affymetrix, Santa Clara, CA) as described (7). All PCR products were checked on ethidium bromide-stained agarose gels. Nine clones giving double PCR bands were excluded from the analysis.A subset of 2,085 of the 2,995 PCR products in the Unigene set...

The non-specific lipid transfer proteins (LTPs) constitute a large protein family found in all land plants. They are small proteins characterized by a tunnel-like hydrophobic cavity, which makes them suitable for binding and transporting various lipids. The LTPs are abundantly expressed in most tissues. In general, they are synthesized with an N-terminal signal peptide that localizes the protein to spaces exterior to the plasma membrane. The in vivo functions of LTPs are still disputed, although evidence has accumulated for a role in the synthesis of lipid barrier polymers, such as cuticular waxes, suberin, and sporopollenin. There are also reports suggesting that LTPs are involved in signaling during pathogen attacks. LTPs are considered as key proteins for the plant’s survival and colonization of land. In this review, we aim to present an overview of the current status of LTP research and also to discuss potential future applications of these proteins. We update the knowledge on 3D structures and lipid binding and review the most recent data from functional investigations, such as from knockout or overexpressing experiments. We also propose and argument for a novel system for the classification and naming of the LTPs.

Differentiation of xylem cells in dicotyledonous plants involves expansion of the radial primary cell walls and intrusive tip growth of cambial derivative cells prior to the deposition of a thick secondary wall essential for xylem function. Expansins are cell wall-residing proteins that have an ability to plasticize the cellulose-hemicellulose network of primary walls. We found expansin activity in proteins extracted from the cambial region of mature stems in a model tree species hybrid aspen (Populus tremula × Populus tremuloides Michx). We identified three α-expansin genes (PttEXP1, PttEXP2, and PttEXP8) and one β-expansin gene (PttEXPB1) in a cambial region expressed sequence tag library, among which PttEXP1 was most abundantly represented. Northern-blot analyses in aspen vegetative organs and tissues showed that PttEXP1 was specifically expressed in mature stems exhibiting secondary growth, where it was present in the cambium and in the radial expansion zone. By contrast, PttEXP2 was mostly expressed in developing leaves. In situ reverse transcription-PCR provided evidence for accumulation of mRNA of PttEXP1 along with ribosomal rRNA at the tips of intrusively growing xylem fibers, suggesting that PttEXP1 protein has a role in intrusive tip growth. An examination of tension wood and leaf cDNA libraries identified another expansin, PttEXP5, very similar to PttEXP1, as the major expansin in developing tension wood, while PttEXP3 was the major expansin expressed in developing leaves. Comparative analysis of expansins expressed in woody stems in aspen, Arabidopsis, and pine showed that the most abundantly expressed expansins share sequence similarities, belonging to the subfamily A of α-expansins and having two conserved motifs at the beginning and end of the mature protein, RIPVG and KNFRV, respectively. This conservation suggests that these genes may share a specialized, not yet identified function.

The genes involved in the 2,3-butanediol pathway coding for ct-acetolactate decarboxylase, a-acetolactate synthase (ae-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiela terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budAl gene, coding for ot-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrgena genes budB and budC, coding for at-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long cx-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic a-ALS enzymes encoded by the genes ilvB, ilvG, and ilvl. The K. terrigena a-ALS is also shown to complement an anabolic ce-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.

We have identified a gene, denoted PttMAP20, which is strongly up-regulated during secondary cell wall synthesis and tightly coregulated with the secondary wall-associated CESA genes in hybrid aspen (Populus tremula × tremuloides). Immunolocalization studies with affinity-purified antibodies specific for PttMAP20 revealed that the protein is found in all cell types in developing xylem and that it is most abundant in cells forming secondary cell walls. This PttMAP20 protein sequence contains a highly conserved TPX2 domain first identified in a microtubule-associated protein (MAP) in Xenopus laevis. Overexpression of PttMAP20 in Arabidopsis (Arabidopsis  thaliana) leads to helical twisting of epidermal cells, frequently associated with MAPs. In addition, a PttMAP20-yellow fluorescent protein fusion protein expressed in tobacco (Nicotiana tabacum) leaves localizes to microtubules in leaf epidermal pavement cells. Recombinant PttMAP20 expressed in Escherichia coli also binds specifically to in vitro-assembled, taxol-stabilized bovine microtubules. Finally, the herbicide 2,6-dichlorobenzonitrile, which inhibits cellulose synthesis in plants, was found to bind specifically to PttMAP20. Together with the known function of cortical microtubules in orienting cellulose microfibrils, these observations suggest that PttMAP20 has a role in cellulose biosynthesis.

The non-specific lipid transfer proteins (nsLTP) are unique to land plants. The nsLTPs are characterized by a compact structure with a central hydrophobic cavity and can be classified to different types based on sequence similarity, intron position or spacing between the cysteine residues. The type G nsLTPs (LTPGs) have a GPI-anchor in the C-terminal region which attaches the protein to the exterior side of the plasma membrane. The function of these proteins, which are encoded by large gene families, has not been systematically investigated so far. In this study we have explored microarray data to investigate the expression pattern of the LTPGs in Arabidopsis and rice. We identified that the LTPG genes in each plant can be arranged in three expression modules with significant coexpression within the modules. According to expression patterns and module sizes, the Arabidopsis module AtI is functionally equivalent to the rice module OsI, AtII corresponds to OsII and AtIII is functionally comparable to OsIII. Starting from modules AtI, AtII and AtIII we generated extended networks with Arabidopsis genes coexpressed with the modules. Gene ontology analyses of the obtained networks suggest roles for LTPGs in the synthesis or deposition of cuticular waxes, suberin and sporopollenin. The AtI-module is primarily involved with cuticular wax, the AtII-module with suberin and the AtIII-module with sporopollenin. Further transcript analysis revealed that several transcript forms exist for several of the LTPG genes in both Arabidopsis and rice. The data suggests that the GPI-anchor attachment and localization of LTPGs may be controlled to some extent by alternative splicing.

The nonspecific lipid transfer proteins (LTPs) are small, compact proteins folded around a tunnel-like hydrophobic cavity, making them suitable for lipid binding and transport. LTPs are encoded by large gene families in all land plants, but they have not been identified in algae or any other organisms. Thus, LTPs are considered key proteins for plant survival on and colonization of land. LTPs are abundantly expressed in most plant tissues, both above and below ground. They are usually localized to extracellular spaces outside the plasma membrane. Although the in vivo functions of LTPs remain unclear, accumulating evidence suggests a role for LTPs in the transfer and deposition of monomers required for assembly of the water-proof lipid barriers-such as cutin and cuticular wax, suberin, and sporopolleninformed on many plant surfaces. Some LTPs may be involved in other processes, such as signaling during pathogen attacks. Here, we present the current status of LTP research with a focus on the role of these proteins in lipid barrier deposition and cell expansion. We suggest that LTPs facilitate extracellular transfer of barrier materials and adhesion between barriers and extracellular materials. A growing body of research may uncover the true role of LTPs in plants. Overview of plant non-specific lipid transfer proteinsThe plant non-specific lipid transfer proteins (LTPs) are abundant, secreted, soluble, cysteinerich and small proteins with a molecular size usually below 10 kDa (1, 2). In the LTPs four conserved disulfide bridges, formed by an eight-Cys motif (8CM) with the general form CXn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C, stabilize the folding of four or five α-helices into a very compact 3D-structure (3, 4 and Fig. 1). The folding of the helices results in a central hydrophobic cleft suitable for the binding of hydrophobic ligands, such as fatty acids and other lipids (Fig. 2). The compact structure renders the LTPs very insensitive to heat and denaturing agents (5, 6). LTPs are expressed in all investigated land plants, but have not been detected in any other organisms (7). LTPs are encoded by large gene families in seed plants (2,(8)(9)(10)(11). In bryophytes and ferns the gene families are significantly smaller (7,12). LTPs are classified in five major types (LTP1, LTP2, LTPc, LTPd and LTPg) and four minor types (LTPe, LTPf, LTPh, LTPj and LTPk) (7). The classification is based on the spacing between the Cys residues in the 8CM, the polypeptide sequence identity and the position of evolutionary conserved introns. The classification also reflects post-translational modifications, e.g. LTPs with a GPI-anchor belong to LTPg. LTPd and LTPg are encoded in all land plants which suggests that these were possibly the first LTP types that evolved in land plants. The most well-studied LTP types in flowering plants LTP1 and LTP2 probably evolved later since these are not found in liverworts, mosses or other non-seed plants (7). The LTPs are translated with an N-terminal signal peptide that has a potential to localize th...

Both of the Saccharomyces cerevisiae 2m circle-encoded Rep1 and Rep2 proteins are required for efficient distribution of the plasmid to daughter cells during cellular division. In this study two-hybrid and in vitro protein interaction assays demonstrate that the first 129 amino acids of Rep1 are sufficient for self-association and for interaction with Rep2. Deletion of the first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still allowed some self-association, suggesting that different but overlapping domains specify these interactions. Amino-or carboxy-terminally truncated Rep1 fusion proteins were unable to complement defective segregation of a 2m-based stability vector with rep1 deleted, supporting the idea of the requirement of Rep protein interaction for plasmid segregation but indicating a separate required function for the carboxyterminal portion of Rep1. The results of in vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both of which are capable of mediating Rep2 self-association. The amino-terminal domain interacts with Rep1, while the carboxy-terminal domain was shown by Southwestern analysis to have DNA-binding activity. The overlapping Rep1 and Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1, Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2m circle plasmid stability locus, forming a structure that mediates plasmid segregation. In this model, competition between Rep1 and Rep2 for association with Rep1 determines the formation or disassembly of the segregation complex.Most strains of the budding yeast Saccharomyces cerevisiae contain an endogenous plasmid, the 2m circle. This 6,318-bp double-stranded circular DNA plasmid is located in the nucleus at approximately 60 copies per haploid cell and replicates autonomously from, but synchronously with, the chromosomal DNA (for a review, see reference 9). The 2m circle confers no phenotype or selective advantage on the host yeast; indeed, 2m plasmid-bearing ([cir ϩ ]) cells grow 1% more slowly than isogenic plasmid-free ([cir 0 ]) cells (10). Despite this disadvantage, the 2m plasmid displays a high level of mitotic stability. This stability results from the presence of a plasmid-encoded copy number amplification system and a partition mechanism which together ensure that the rates of plasmid loss in mitosis and meiosis are very low (4,10,16,18). Partitioning of the 2m plasmid requires two proteins encoded by the plasmid genes REP1 and REP2 and a cis-acting 2m locus termed STB (16,18). The role of these three components has been examined in a variety of studies involving mainly deletion or insertion analysis of 2m-derived plasmids (17,18,23). In the absence of any one of these three components, the 2m plasmid displays a strong maternal bias in inheritance; most plasmids are retained in the mother cell (22). The 2m circle partition system overcomes this bias by an as yet unknown mechanism.The Rep1 and Rep2 proteins mediate 2m plasmid segregation, but thei...