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High throughput confocal imaging poses challenges in the computational image analysis of complex subcellular structures such as the microtubule cytoskeleton. Here, we developed CellArchitect, an automated image analysis tool that quantifies changes to subcellular patterns illustrated by microtubule markers in plants. We screened microtubule-targeted herbicides and demonstrate that high throughput confocal imaging with integrated image analysis by CellArchitect can distinguish effects induced by the known herbicides indaziflam and trifluralin. The same platform was used to examine six other compounds with herbicidal activity, and at least three different effects induced by these compounds were profiled. We further show that CellArchitect can detect subcellular patterns tagged by actin and endoplasmic reticulum markers. Thus, the platform developed here can be used to automate image analysis of complex subcellular patterns for purposes such as herbicide discovery and mode of action characterisation. The capacity to use this tool to quantitatively characterise cellular responses lends itself to application across many areas of biology.

On April 6, 1865, Friedrich Engelhorn founded the company "Badische Anilin- & Sodafabrik" in Mannheim, Germany. This Essay, commemorating the 150th anniversary of BASF, introduces several outstanding examples of innovation from the history of BASF and highlights how chemical and technical competencies developed in the past still play an essential role in current projects.

In response to changing market dynamics, the discovery of new herbicides has declined significantly over the past few decades and has only seen a modest upsurge in recent years. Nevertheless, the few introductions have proven to be interesting and have brought useful innovation to the market. In addition, herbicide-tolerant or herbicide-resistant crop technologies have allowed the use of existing nonselective herbicides to be extended into crops. An increasing and now major challenge is being posed by the inexorable increase in biotypes of weeds that are resistant to herbicides. This problem is now at a level that threatens future agricultural productivity and needs to be better understood. If herbicides are to remain sustainable, then it is a must that we adopt diversity in crop rotation and herbicide use as well as increase the use of nonchemical measures to control weeds. Nevertheless, despite the difficulties posed by resistant weeds and increased regulatory hurdles, new screening tools promise to provide an upsurge of potential herbicide leads. Our industry urgently needs to supply agriculture with new, effective resistance-breaking herbicides along with strategies to sustain their utility.

Intron sequences from monocotyledonous and dicotyledonous origin were used to abolish marker gene expression in prokaryotes (Escherichia coli and Agrobacterium tumefaciens) but permit expression in selected eukaryotic systems using the eukaryotic specific splicing mechanism. A 1014 bp maize Shrunken-1 (Sh 1) intron 1 flanked by exon1 and exon2 sequences was cloned into the N-terminal of the NPT II-coding region. Transient gene expression analysis revealed that the modified neomycin phosphotransferase II (NPT II) gene, driven by the cauliflower mosaic virus (CaMV) 35S promoter, is expressed in barley protoplasts, but poorly expressed in tobacco protoplasts. In dicotyledonous cells AU-rich sequences are known to be important for efficient splicing and therefore an attempt was made to improve expression of the NPT II gene, containing the Sh 1 intron 1, in tobacco by increasing the AU content from 57% to 69%. Reverse transcriptase PCR analysis of RNA from transiently expressed NPT II transcripts from tobacco protoplasts revealed that despite the increase in AU-content, NPT II was still poorly expressed. Cryptic splice sites were identified as one possible cause for missplicing of the Sh1 intron 1 in dicots and poor levels of expression. Alternatively, cloning of the 198 bp intron 2 of the potato STLS 1 gene (81% AU) into the N-terminal part of the NPT II-coding region resulted in proper expression of NPT II in tobacco as well as in barley protoplasts and abolished marker gene expression in prokaryotes. The successful insertion of an intron into a selectable marker gene which completely abolishes gene expression in prokaryotes, without affecting expression of chimeric genes in monocotyledonous and dicotyledonous plant cells provides a suitable system to reduce the number of false-positives in transgenic plant production.

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The expression of the jellyfish green fluorescent protein (GFP) in plants was analyzed by transient expression in protoplasts from Nicotiana tabacum, Arabidopsis thaliana, Hordeum vulgare, and Zea mays. Expression of GFP was only observed with a mutated cDNA, from which a recently described cryptic splice site had been removed. However, detectable levels of green fluorescence were only emitted from a small number of protoplasts. Therefore, other mutations in the GFP cDNA leading to single-amino acid exchanges in the chromophore region, which had been previously studied in Escherichia coli, were tested in order to improve the sensitivity of this marker protein. Of the mutations tested so far, the exchange of GFP amino acid tyrosine 66 to histidine (Y66H) led to detection of blue fluorescence in plant protoplasts, while the exchange of amino acid serine 65 to cysteine (S65C) and threonine (S65T) increased the intensity of green fluorescence drastically, thereby significantly raising the detection level for GFP. For GFP S65C, the detectable number of green fluorescing tobacco (BY-2) protoplasts was raised up to 19-fold, while the fluorimetricly determined fluorescence was raised by at least 2 orders of magnitude.

The isolation and analysis of several cDNAs and of one genomic clone encoding ST-LS1, a single-copy gene fromSolanum tuberosum with leaf/stem-specific, light-inducible expression, is described. The structure of the gene was determined by sequencing several overlapping partial cDNA clones as well as the genomic clone and by determining the transcription start site by RNA protection experiments. A “tagged” derivative of the gene, obtained by exon modification, was reintroduced into potato and into tobacco shoots usingAgrobacterium/Ti-plasmid vector systems. The modified gene was expressed in both tobacco and potato shoots giving rise to an RNA of approximately 1,200 nucleotides which exceeded the length of the RNA made from the endogenous gene by the expected size of the “tag” (470 nucleotides). The level of expression of the modified gene varied substantially between independent transformants. A high proportion of the transformants (20%–40%) synthesized as much RNA from the added gene as from the resident gene. Expression of the transferred gene was light induced. Qualitatively and quantitatively the expression of the introduced gene was similar in a homologous (potato) and in a heterologous, but related, cellular background (tobacco).

Today there are no longer any basic problems involved in isolating and characterizing plant genes. Our knowledge of the structure and function of the genes so far sequenced provides a beginning understanding of gene regulation in plants. In connection with the recently developed methods of gene transfer to plant cells, the most important application is the production of plants with new characteristics through genetic engineering. Since the genes can be transferred from any kind of plant—indeed even from any organism—completely new possibilities are opened up for plant breeding.

A cDNA library established from polyA+RNA from tubers of potato (Solanum tuberosum) was analysed for sequences specifically transcribed in tubers. Screening more than 8000 cDNA clones in a differential hybridization assay using RNA from leaves, roots, stems and tubers resulted in four different classes of cDNA clones. These could be categorized further into two groups according to the size distribution of mRNAs to which they hybridize. Thus the first group comprising cDNA clones pcT700, pcT800 and pcT1500 were in each case complementary to a single RNA species. Hybrid select/in vitro translation data indicate that one of these clones, i.e. pcT1500, is derived from an RNA encoding the major storage protein of potato tubes, i.e. a 40 kd species (called “patatin”).

A cDNA library established from polyA+ RNA of leaves from potato (Solanum tuberosum) was analysed for sequences specifically present in leaves. Three cDNA-clones, one of which is the small subunit of Ribulose-1,5-bis-phosphate carboxylase (ss Rubisco), were obtained via differential screening using RNA from leaves, roots and tubers. Northern and RNA dot blot analysis demonstrate that the expression of these clones is higher by at least a factor of 100–500 in leaves as compared to roots and tubers. Genomic blot analysis shows that all three cDNA clones are encoded by small, multigene families.

Biologie de la cellule vegetale: techniques de cultures cellulaires, utilisation de protoplastes, selection de mutants in vitro, importance de la biologie de la cellule pour le genie genetique. Transfert d'ADN: systeme utilisant Agrobacterium tumefaciens, transformation directe de cellules avec du DNA isole. Expression genique chez les plantes. Applications du genie genetique: obtention de plantes resistantes, modifications de la composition des substances vegetales, manipulation des processus physiologiques, fixation de l'azote. Problemes et risques

Synthesis of (+)-Aspicilin Using Building Blocks from Renewable Resources The 18-membered lichen macrolide (+)-aspicilin (1) is easily built up from compounds 14, 16, 3, and 24 following the C3 + C7 + C6 + C2 = C18 pattern (see Schemes). The synthesis requires 15 steps and gives 1 in 13% overall yield from D-mannose (2). The latter compound provides the stereogenic centers C(4), C(5), and C(6). The stereogenic center C(17) is supplied by building block 14 (from (−)-(S)-ethyl lactate).

ST-LS1 is a light-inducible, single-copy gene from potato that is expressed only in photosynthetic tissues. Various sequences derived from the 5′-upstream region of this gene were fused to the coding region of the chloramphenicol acetyltransferase (CAT) gene and to the gene 7 termination region of the transfer DNA (T-DNA) from the Agrobacterium Ti plasmid pTiACH5 and transferred to tobacco using Ti-plasmid vectors. After regeneration of whole plants, tissues were assayed for the expression of the CAT gene. Sequences derived from the 5′-upstream region of the ST-LS1 gene comprising positions -334 to +11 were sufficient to confer a leaf/stem-specific as well as a light-inducible expression of the CAT gene. Destruction of chloroplasts by treatment with the herbicide norfluorazon and subsequent exposure to light drastically reduced the expression of the CAT gene indicating that this upstream sequence most likely interacts with a chloroplast-dependent signal. When sequences from position -98 to position +675 were fused to a truncated inactive fragment of the cauliflower mosaic virus 35S promoter in a head-to-head manner, the corresponding chimeric genes were again expressed in photosynthetic tissues only, indicating that these sequences have enhancer-like properties.

We have obtained transgenic tobacco plants overexpressing the enzyme glutamine synthetase (GS) by fusing an alfalfa GS gene to the cauliflower mosaic virus 35S promotor and integrating it into Nicotiana tabacum var. W38 plants by Agrobacterium tumefaciens mediated gene transfer. The amount of RNA specific to alfalfa GS was about 10 times higher in transgenic tobacco plants than in alfalfa. The alfalfa GS produced by these transgenic plants was identified by Western blotting and represented 5% of total soluble protein in the transformed plants, amounting to a 5-fold increase in specific GS activity and in a 20-fold increase in resistance to the GS inhibitor L-phosphinothricin in vitro. Tissue from GS overproducing plants showed a sevenfold lower amount of free NH3. The amino acid composition of the plant tissue was not altered significantly by GS overproduction. GS overproducing plants were fertile and grew normally. These data show that a high level of expression of a key metabolic enzyme such as glutamine synthetase does not interfere with growth and fertility of plants.

The soil bacterium Agrobaeterium tumefaciens can infect almost all dicotyledonous plants (Braun 1978, 1982). As a result of the infection, the wound tissue proliferates as a neoplastic growth, commonly referred to as a crown gall tumor. Once induced, the tumor no longer requires the presence of the bacteria to grow, and can be cultivated in vitro as an axenic culture (Braun 1943). Two main properties characterize crown galls: their ability to grow in vitro without the supplement of hormones required by normal plant cells, and their ability to synthesize a set of new metabolites termed opines, which are not present in normal cells. Opines are amino acid or sugar derivatives which can be used as a carbon and nitrogen source by the bacteria responsible for inciting the tumor (Tempe and Petit 1982).

ST-LS1, a single copy gene from potato displaying a leaf/stem specific gene expression, was tagged by an exon modification and introduced into both potato and tobacco cells using Agrobacterium vectors. After regeneration of whole plants, the expression of the tagged gene was analyzed with respect to its organ specificity and compared to the expression of the corresponding resident gene. The expression of the transferred gene in transgenic plants closely followed the expression of the resident gene. No marked influence of the plant species serving as host was observed. The level of expression of the introduced gene varied by a factor of at least 100 in independent transformants when normalized to the expression of the resident gene. Southern analysis performed on the transformed plants indicated a correlation between copy number of the introduced gene and its expression level. The activity of the tagged gene as well as of the resident gene was significantly inhibited by treatment of the transgenic plants with the herbicide norfluorazon, indicating that this gene activity is dependent on the presence of functional chloroplasts in the leaves.