The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 Is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis.

In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood.

Overexpression of ALDH10A8 and ALDH10A9 Genes Provides Insight into Their Role in Glycine Betaine Synthesis and Affects Primary Metabolism in Arabidopsis thaliana.

Betaine aldehyde dehydrogenases oxidize betaine aldehyde to glycine betaine in species that accumulate glycine betaine as a compatible solute under stress conditions. In contrast, the physiological function of betaine aldehyde dehydrogenase genes is at present unclear in species that do not accumulate glycine betaine, such as Arabidopsis thaliana. To address this question, we overexpressed the Arabidopsis ALDH10A8 and ALDH10A9 genes, which were identified to code for betaine aldehyde dehydrogenases, in wild-type A.

Genes of primary sulfate assimilation are part of the glucosinolate biosynthetic network in Arabidopsis thaliana.

Glucosinolates are plant secondary metabolites involved in responses to biotic stress. The final step of their synthesis is the transfer of a sulfo group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) onto a desulfo precursor. Thus, glucosinolate synthesis is linked to sulfate assimilation.

The plastidic bile acid transporter 5 is required for the biosynthesis of methionine-derived glucosinolates in Arabidopsis thaliana.

Aliphatic glucosinolate biosynthesis is highly compartmentalized, requiring import of 2-keto acids or amino acids into chloroplasts for side chain elongation and export of the resulting compounds into the cytosol for conversion into glucosinolate. Aliphatic glucosinolate biosynthesis in Arabidopsis thaliana is regulated by three R2R3-MYB transcription factors, the major player being High Aliphatic Glucosinolate 1 (HAG1/MYB28). Here, we show that BAT5, which belongs to the putative bile acid transporter family, is the only member of this family that is transactivated by HAG1/MYB28, HAG2/MYB76, and HAG3/MYB29.

HAG2/MYB76 and HAG3/MYB29 exert a specific and coordinated control on the regulation of aliphatic glucosinolate biosynthesis in Arabidopsis thaliana.

In a previous transactivation screen, two Arabidopsis thaliana R2R3-MYB transcription factors, HAG2/MYB76 and HAG3/MYB29, along with the already characterized HAG1/MYB28, were identified as putative regulators of aliphatic glucosinolate biosynthesis. Molecular and biochemical characterization of HAG2/MYB76 and HAG3/MYB29 functions was performed using transformants with increased or repressed transcript levels. Real-time PCR assays, cotransformation assays and measurements of glucosinolate contents were used to assess the impact of both MYB factors on the steady-state level of glucosinolate biosynthetic genes and accumulation of aliphatic glucosinolates.

The R2R3-MYB transcription factor HAG1/MYB28 is a regulator of methionine-derived glucosinolate biosynthesis in Arabidopsis thaliana.

Methionine-derived glucosinolates belong to a class of plant secondary metabolites that serve as chemoprotective compounds in plant biotic defense reactions and also exhibit strong anticancerogenic properties beneficial to human health. In a screen for the trans-activation potential of various transcription factors toward glucosinolate biosynthetic genes, we could identify the HAG1 (HIGH ALIPHATIC GLUCOSINOLATE 1, also referred to as MYB28) gene as a positive regulator of aliphatic methionine-derived glucosinolates. The content of aliphatic glucosinolates as well as transcript levels of aliphatic glucosinolate biosynthetic genes were elevated in gain-of-function mutants and decreased in HAG1 RNAi knock-down mutants.

The transcription factor HIG1/MYB51 regulates indolic glucosinolate biosynthesis in Arabidopsis thaliana.

Glucosinolates are a class of plant secondary metabolites that serve as antiherbivore compounds in plant defence. A previously identified Arabidopsis thaliana activation-tagged line, displaying altered levels of secondary metabolites, was shown here to be affected in the content of indolic and aliphatic glucosinolates. The observed chemotype was caused by activation of the R2R3-MYB transcription factor gene HIG1 (HIGH INDOLIC GLUCOSINOLATE 1, also referred to as MYB51).

A simplified method for the analysis of transcription factor-promoter interactions that allows high-throughput data generation.

Transient expression systems are intensively used to study the transactivation potential of transcription factors and to confirm target promoters. Here we present a novel system based on the high-efficiency transformation of cultured Arabidopsis thaliana cells by agrobacteria. To demonstrate the potential of this system, we compared it with a commonly used protoplast transfection assay, and studied the regulation of phenylpropanoid biosynthetic pathway genes by various transcription factors.

Identification and expression analysis of twelve members of the nucleobase-ascorbate transporter (NAT) gene family in Arabidopsis thaliana.

By screening genome databases, 12 genes encoding membrane proteins homologous to nucleobase-ascorbate transporters (NATs) were identified in Arabidopsis thaliana. A similar number of genes was found in the rice genome. The plant NAT proteins split into five clades (I-V) based on protein multisequence alignments.

Forward genetic screening of insertional mutants.

Insertional mutagenesis has contributed to the success of forward genetics in Arabidopsis thaliana. The availability of large collections of lines mutagenized by either transposon or T-DNA insertions, in combination with the systematic sequencing of insertion/genome junctions, enables the identification of mutations for any given gene in the nuclear genome of this plant species. Protocols for the identification and confirmation of mutations by forward genetics, the isolation of insertion/genome junctions, and the stabilization of transposon-induced mutations are provided, together with an overview of available insertional mutant collections and of their characteristics.

Differential gene expressions in arbuscular mycorrhizal-colonized tomato grown under heavy metal stress.

When tomato was grown in either "Breinigerberg" soil, which has a high content of Zn and of other heavy metals or in non-polluted soil enriched with up to 1 mM CdCl2, plants colonized with the arbuscular mycorrhizal fungus (AMF) Glomus intraradices grew distinctly better than non-mycorrhizal controls. An analysis of differential mRNA transcript formations was performed on several plant genes coding for products potentially involved in heavy metal tolerance. Northern blot analyses indicated that the mRNA from either roots or leaves was not differentially expressed in the case of LePCS1 (coding for phytochelatin synthase), Lemt1, Lemt3 and Lemt4 (for metallothioneins) or LeNramp2 (for a broad range heavy metal transporter) in both mycorrhizal and non-mycorrhizal plants, grown either with or without heavy metals.

The Arabidopsis plastidic glucose 6-phosphate/phosphate translocator GPT1 is essential for pollen maturation and embryo sac development.

Plastids of nongreen tissues can import carbon in the form of glucose 6-phosphate via the glucose 6-phosphate/phosphate translocator (GPT). The Arabidopsis thaliana genome contains two homologous GPT genes, AtGPT1 and AtGPT2. Both proteins show glucose 6-phosphate translocator activity after reconstitution in liposomes, and each of them can rescue the low-starch leaf phenotype of the pgi1 mutant (which lacks plastid phosphoglucoisomerase), indicating that the two proteins are also functional in planta.

PHOSPHATE TRANSLOCATORS IN PLASTIDS.

During photosynthesis, energy from solar radiation is used to convert atmospheric carbon dioxide into intermediates that are used within and outside the chloroplast for a multitude of metabolic pathways. The daily fixed carbon is exported from the chloroplasts as triose phosphates and 3-phosphoglycerate. In contrast, nongreen plastids rely on the import of carbon, mainly hexose phosphates.

Characterization of two functional phosphoenolpyruvate/phosphate translocator (PPT) genes in Arabidopsis--AtPPT1 may be involved in the provision of signals for correct mesophyll development.

The Arabidopsis thaliana chlorophyll a/b-binding protein underexpressed 1 (cue1) mutant shows a reticulate leaf phenotype and is defective in a plastidic phosphoenolpyruvate (PEP)/phosphate translocator (AtPPT1). A functional AtPPT1 providing plastids with PEP for the shikimate pathway is therefore essential for correct leaf development. The Arabidopsis genome contains a second PPT gene, AtPPT2.

The phenotype of the Arabidopsis cue1 mutant is not simply caused by a general restriction of the shikimate pathway.

The Arabidopsis thalianachlorophyll a/b binding protein underexpressed (cue1) mutant, which has been isolated in a screen for chlorophyll a/b binding protein (CAB) underexpressors, exhibits a reticulate leaf phenotype combined with delayed chloroplast development and aberrant shape of the palisade parenchyma cells. The affected gene in cue1 is a phosphoenolpyruvate (PEP)/phosphate translocator (PPT) of the plastid inner envelope membrane. The proposed function of the PPT in C3-plants is the import of PEP into the stroma as one of the substrates for the shikimate pathway, from which aromatic amino acids and a variety of secondary plant products derive.

Analysis of the plastidic phosphate translocator gene family in Arabidopsis and identification of new phosphate translocator-homologous transporters, classified by their putative substrate-binding site.

Analysis of the Arabidopsis genome revealed the complete set of plastidic phosphate translocator (pPT) genes. The Arabidopsis genome contains 16 pPT genes: single copies of genes coding for the triose phosphate/phosphate translocator and the xylulose phosphate/phosphate translocator, and two genes coding for each the phosphoenolpyruvate/phosphate translocator and the glucose-6-phosphate/phosphate translocator. A relatively high number of truncated phosphoenolpyruvate/phosphate translocator genes (six) and glucose-6-phosphate/phosphate translocator genes (four) could be detected with almost conserved intron/exon structures as compared with the functional genes.

An Arabidopsis thaliana knock-out mutant of the chloroplast triose phosphate/phosphate translocator is severely compromised only when starch synthesis, but not starch mobilisation is abolished.

The Arabidopsis thaliana tpt-1 mutant which is defective in the chloroplast triose phosphate/phosphate translocator (TPT) was isolated by reverse genetics. It contains a T-DNA insertion 24 bp upstream of the start ATG of the TPT gene. The mutant lacks TPT transcripts and triose phosphate (TP)-specific transport activities are reduced to below 5% of the wild type.

The arbuscular mycorrhizal fungus Glomus geosporum in European saline, sodic and gypsum soils.

Plants of saline and sodic soils of the Hungarian steppe and of gypsum rock in the German Harz mountains, thus soils of high ionic strength and electric conductivity, were examined for their colonization by arbuscular mycorrhizal fungi (AMF). Roots of several plants of the saline and sodic soils such as Artemisia maritima, Aster tripolium or Plantago maritima are strongly colonized and show typical AMF structures (arbuscules, vesicles) whereas others like the members of the Chenopodiaceae, Salicornia europaea, Suaeda maritima or Camphorosma annua, are not. The vegetation of the gypsum rock is totally different, but several plants are also strongly colonized there.

The evolution of gymnosperms redrawn by phytochrome genes: the Gnetatae appear at the base of the gymnosperms.

Gymnosperms possess two to four phytochrome types which apparently are the result of successive gene duplications in the genomes of their common ancestors. Phytochromes are nuclear-encoded proteins whose genes, contrary to chloroplast, mitochondrion, and rRNA genes, have hitherto rarely been used to examine gymnosperm phylogenies. Since the individual phytochrome gene types implied phylogenies that were not completely congruent to one another, conflicting branching orders were sorted by the number of gene lineages present in a taxon.

Overexpression of C(4)-cycle enzymes in transgenic C(3) plants: a biotechnological approach to improve C(3)-photosynthesis.

The process of photorespiration diminishes the efficiency of CO(2) assimilation and yield of C(3)-crops such as wheat, rice, soybean or potato, which are important for feeding the growing world population. Photorespiration starts with the competitive inhibition of CO(2) fixation by O(2) at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and can result in a loss of up to 50% of the CO(2) fixed in ambient air. By contrast, C(4) plants, such as maize, sugar cane and Sorghum, possess a CO(2) concentrating mechanism, by which atmospheric CO(2) is bound to C(4)-carbon compounds and shuttled from the mesophyll cells where the prefixation of bicarbonate occurs via phosphoenolpyruvate carboxylase (PEPC) into the gas-tight bundle-sheath cells, where the bound carbon is released again as CO(2) and enters the Calvin cycle.

The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants.

Plastids are the site of the reductive and the oxidative pentose phosphate pathways, which both generate pentose phosphates as intermediates. A plastidic transporter from Arabidopsis has been identified that is able to transport, in exchange with inorganic phosphate or triose phosphates, xylulose 5-phosphate (Xul-5-P) and, to a lesser extent, also ribulose 5-phosphate, but does not accept ribose 5-phosphate or hexose phosphates as substrates. Under physiological conditions, Xul-5-P would be the preferred substrate.

Single and double overexpression of C(4)-cycle genes had differential effects on the pattern of endogenous enzymes, attenuation of photorespiration and on contents of UV protectants in transgenic potato and tobacco plants.

To improve the efficiency of CO(2) fixation in C(3) photosynthesis, C(4)-cycle genes were overexpressed in potato and tobacco plants either individually or in combination. Overexpression of the phosphoenolpyruvate carboxylase (PEPC) gene (ppc) from Corynebacterium glutamicum (cppc) or from potato (stppc, deprived of the phosphorylation site) in potato resulted in a 3-6-fold induction of endogenous cytosolic NADP malic enzyme (ME) and an increase in the activities of NAD-ME (3-fold), NADP isocitrate dehydrogenase (ICDH), pyruvate kinase (PK), NADP glycerate-3-P dehydrogenase (NADP-GAPDH), and PEP phosphatase (PEPP). In double transformants overexpressing cppc and chloroplastic NADP-ME from Flaveria pringlei (fpMe1), cytosolic NADP-ME was less induced and pleiotropic effects were diminished.

ERN1, a novel ethylene-regulated nuclear protein of Arabidopsis.

Employing differential display of mRNA to investigate the transcriptionally regulated part of the ethylene response pathway in etiolated seedlings of Arabidopsis thaliana, a novel ethylene-regulated nuclear-localized protein, designated ERN1, was identified. ERN1 is one of four genes whose differential expression was confirmed by RNA blot analysis. ERN1 is represented by a single-copy gene in the Arabidopsis genome.

Plastidic metabolite transporters and their physiological functions in the inducible crassulacean acid metabolism plant Mesembryanthemum crystallinum.

The inducible crassulacean acid metabolism (CAM) plant Mesembryanthemum crystallinum accumulates malic acid during the night and converts it to starch during the day via a pathway that, because it is located in different subcellular compartments, depends on specific metabolite transport across membranes. The chloroplast glucose transporter (pGlcT) and three members of the phosphate translocator (PT) family were isolated. After induction of CAM, transcript amounts of the phosphoenolpyruvate (PEP) phosphate translocator (PPT) and the glucose-6-phosphate (Glc6P) phosphate translocator (GPT) genes were increased drastically, while triose phosphate (TP) phosphate translocator (TPT) and the pGlcT transcripts remained unchanged.

Functional analysis of the two Arabidopsis homologues of Toc34, a component of the chloroplast protein import apparatus.

Two Arabidopsis Toc34 homologues, atToc34 and atToc33, components of the chloroplast protein import machinery located in the outer envelope membrane, were recently isolated. Both proteins insert into the outer envelope, are supposed to bind GTP and to interact with Toc75 as demonstrated by in vitro import assays. We studied the expression of the two genes by RNA gel blot analysis, promoter-GUS plants and in situ hybridisations as well as immunoblot analysis.