In plants, the formation of isopentenyl diphosphate and dimethylallyl diphosphate, the central intermediates in the biosynthesis of isoprenoids, is compartmentalized: the mevalonate (MVA) pathway, which is localized to the cytosol, is responsible for the synthesis of sterols, certain sesquiterpenes, and the side chain of ubiquinone; in contrast, the recently discovered MVA-independent pathway, which operates in plastids, is involved in providing the precursors for monoterpenes, certain sesquiterpenes, diterpenes, carotenoids, and the side chains of chlorophylls and plastoquinone. Specific inhibitors of the MVA pathway (lovastatin) and the MVA-independent pathway (fosmidomycin) were used to perturb biosynthetic flux in Arabidopsis thaliana seedlings. The interaction between both pathways was studied at the transcriptional level by using GeneChip (Affymetrix) microarrays and at the metabolite level by assaying chlorophylls, carotenoids, and sterols. Treatment of seedlings with lovastatin resulted in a transient decrease in sterol levels and a transient increase in carotenoid as well as chlorophyll levels. After the initial drop, sterol amounts in lovastatin-treated seedlings recovered to levels above controls. As a response to fosmidomycin treatment, a transient increase in sterol levels was observed, whereas chlorophyll and carotenoid amounts decreased dramatically when compared with controls. At 96 h after fosmidomycin addition, the levels of all metabolites assayed (sterols, chlorophylls, and carotenoids) were substantially lower than in controls. Interestingly, these inhibitor-mediated changes were not reflected in altered gene expression levels of the genes involved in sterol, chlorophyll, and carotenoid metabolism. The lack of correlation between gene expression patterns and the accumulation of isoprenoid metabolites indicates that posttranscriptional processes may play an important role in regulating flux through isoprenoid metabolic pathways.1-deoxy-D-xylulose 5-phosphate ͉ fosmidomycin ͉ lovastatin ͉ mevalonate T he isoprenoids, which constitute the most diverse group of natural products, serve numerous biochemical functions in plants. They play important roles as quinones in electron transport chains, as components of membranes (sterols), in subcellular targeting and regulation (prenylation of proteins), as photosynthetic pigments (carotenoids, side chain of chlorophyll), as hormones (gibberellins, brassinosteroids, abscisic acid, cytokinins), and as plant defense compounds as well as attractants for pollinators (monoterpenes, sesquiterpenes, and diterpenes) (1). Isoprenoids are synthesized ubiquitously among prokaryotes and eukaryotes through condensation of the five-carbon intermediates isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (2). In higher plants, two distinct biosynthetic routes to IPP and DMAPP exist (Fig. 1). The cytosolic pathway, which starts from acetyl-CoA and proceeds through the intermediate mevalonate (MVA), provides the precursors for sterols and ubiquinone (3). ...

SummaryThe Arabidopsis PR-1 gene is one of a suite of genes induced co-ordinately during the onset of systemic acquired resistance (SAR), a plant defense pathway triggered by pathogen infection or exogenous application of chemicals such as salicylic acid (SA) and 2,6-dichloroisonicotinic acid (INA). We have characterized cis-acting regulatory elements in the PR-1 promoter involved in INA induction using deletion analysis, linker-scanning mutagenesis, and in vivo footprinting. Compared to promoter fragments of 815 bp or longer (which show greater than 10-fold inducibility after INA treatment), induction of a 698 bp long promoter fragment is reduced by half and promoter fragments of 621 bp or shorter have lost all inducibility. Additionally, two 10-bp linker-scanning mutations centered at 640 bp and 610 bp upstream from the transcription initiation site are each sufficient to abolish chemical inducibility of a GUS reporter fusion. The -640 linker-scanning mutation encompasses a region highly homologous to recognition sites for transcription factors of the basic leucine zipper class, while the -610 linker-scanning mutation contains a sequence similar to a consensus recognition site for the transcription factor NF-κB. Furthermore, several inducible in vivo footprints located at or nearby these motifs demonstrate significant and highly reproducible changes in DNA accessibility following SAR induction. This in vivo signature of protein-DNA interactions after INA induction is tightly correlated with the functionally important regions of the promoter identified by mutation analysis.

Background: Protein bodies (PBs) are natural endoplasmic reticulum (ER) or vacuole plantderived organelles that stably accumulate large amounts of storage proteins in seeds. The prolinerich N-terminal domain derived from the maize storage protein γ zein (Zera) is sufficient to induce PBs in non-seed tissues of Arabidopsis and tobacco. This Zera property opens up new routes for high-level accumulation of recombinant proteins by fusion of Zera with proteins of interest. In this work we extend the advantageous properties of plant seed PBs to recombinant protein production in useful non-plant eukaryotic hosts including cultured fungal, mammalian and insect cells.

The green alga Chlamydomonas reinhardtii can grow photoautotrophically utilizing CO 2 , heterotrophically utilizing acetate, and mixotrophically utilizing both carbon sources. Growth of cells in increasing concentrations of acetate plus 5% CO 2 in liquid culture progressively reduced photosynthetic CO 2 fixation and net O 2 evolution without effects on respiration, photosystem II efficiency (as measured by chlorophyll fluorescence), or growth. Using the technique of on-line oxygen isotope ratio mass spectrometry, we found that mixotrophic growth in acetate is not associated with activation of the cyanide-insensitive alternative oxidase pathway. The fraction of carbon biomass resulting from photosynthesis, determined by stable carbon isotope ratio mass spectrometry, declined dramatically (about 50%) in cells grown in acetate with saturating light and CO 2 . Under these conditions, photosynthetic CO 2 fixation and O 2 evolution were also reduced by about 50%. Some growth conditions (e.g. limiting light, high acetate, solid medium in air) virtually abolished photosynthetic carbon gain. These effects of acetate were exacerbated in mutants with slowed electron transfer through the D1 reaction center protein of photosystem II or impaired chloroplast protein synthesis. Therefore, in mixotrophically grown cells of C. reinhardtii, interpretations of the effects of environmental or genetic manipulations of photosynthesis are likely to be confounded by acetate in the medium.

Zinc finger transcription factors (TFsZF) were designed and applied to transgene and endogenous gene regulation in stably transformed plants. The target of the TFsZF is the Arabidopsis gene APETALA3 (AP3), which encodes a transcription factor that determines floral organ identity. A zinc finger protein (ZFP) was designed to specifically bind to a region upstream of AP3. AP3 transcription was induced by transformation of leaf protoplasts with a transformation vector that expressed a TFZF consisting of the ZFP fused to the tetrameric repeat of herpes simplex VP16's minimal activation domain. Histochemical staining of ␤-glucuronidase (GUS) activity in transgenic AP3::GUS reporter plants expressing GUS under control of the AP3 promoter was increased dramatically in petals when the AP3-specific TFZF activator was cointroduced. TFZF-amplified GUS expression signals were also evident in sepal tissues of these double-transgenic plants. Floral phenotype changes indicative of endogenous AP3 factor coactivation were also observed. The same AP3-specific ZFP AP3 was also fused to a human transcriptional repression domain, the mSIN3 interaction domain, and introduced into either AP3::GUS-expressing plants or wild-type Arabidopsis plants. Dramatic repression of endogenous AP3 expression in floral tissue resulted when a constitutive promoter was used to drive the expression of this TFZF. These plants were also sterile. When a floral tissue-specific promoter from APETALA1 (AP1) gene was used, floral phenotype changes were also observed, but in contrast the plants were fertile. Our results demonstrate that artificial transcriptional factors based on synthetic zinc finger proteins are capable of stable and specific regulation of endogenous genes through multiple generations in multicellular organisms.floral development ͉ APETALA3 ͉ APETALA1 I n nature, eukaryotic nuclear genes are tightly regulated at both the transcriptional and translational levels. Much of this control is achieved through DNA-binding transcription factors. The manipulation of plant traits in agricultural biotechnology would be greatly facilitated if preselected endogenous genes could be turned on or off in a controlled and selective manner. A conceptual approach to such manipulation is the engineered expression of specific native transcription factors that have evolved to control particular genes. Advances in whole-genome sequencing of Arabidopsis (1) and more recently rice (2), combined with informatics-based analysis have allowed the identification of numerous putative plant transcription factors (2, 3). However, the identification and characterization of the molecular targets of these transcription factors is still at a very early stage, and consequently it is not yet possible to use them broadly as gene-specific tools for controlled regulation of endogenous gene expression. Rational design of artificial transcription factors that target specific DNA sequences with non-native nucleotide binding domains fused to transcriptional activation or repression domain...