Starch is the major storage carbohydrate in higher plants and of considerable importance for the human diet and for numerous technical applications. In addition, starch can be accumulated transiently in chloroplasts as a temporary deposit of carbohydrates during ongoing photosynthesis. This transitory starch has to be mobilized during the subsequent dark period. Mutants defective in starch mobilization are characterized by high starch contents in leaves after prolonged periods of darkness and therefore are termed starch excess (sex) mutants. Here we describe the molecular characterization of the Arabidopsis sex1 mutant that has been proposed to be defective in the export of glucose resulting from hydrolytic starch breakdown. The mutated gene in sex1 was cloned using a map-based cloning approach. By complementation of the mutant, immunological analysis, and analysis of starch phosphorylation, we show that sex1 is defective in the Arabidopsis homolog of the R1 protein and not in the hexose transporter. We propose that the SEX1 protein (R1) functions as an overall regulator of starch mobilization by controlling the phosphate content of starch.

Massively parallel sequencing of DNA by pyrosequencing technology offers much higher throughput and lower cost than conventional Sanger sequencing. Although extensively used already for sequencing of genomes, relatively few applications of massively parallel pyrosequencing to transcriptome analysis have been reported. To test the ability of this technology to provide unbiased representation of transcripts, we analyzed mRNA from Arabidopsis (Arabidopsis thaliana) seedlings. Two sequencing runs yielded 541,852 expressed sequence tags (ESTs) after quality control. Mapping of the ESTs to the Arabidopsis genome and to The Arabidopsis Information Resource 7.0 cDNA models indicated: (1) massively parallel pyrosequencing detected transcription of 17,449 gene loci providing very deep coverage of the transcriptome. Performing a second sequencing run only increased the number of genes identified by 10%, but increased the overall sequence coverage by 50%. (2) Mapping of the ESTs to their predicted full-length transcripts indicated that all regions of the transcript were well represented regardless of transcript length or expression level. Furthermore, short, medium, and long transcripts were equally represented. (3) Over 16,000 of the ESTs that mapped to the genome were not represented in the existing dbEST database. In some cases, the ESTs provide the first experimental evidence for transcripts derived from predicted genes, and, for at least 60 locations in the genome, pyrosequencing identified likely protein-coding sequences that are not now annotated as genes. Together, the results indicate massively parallel pyrosequencing provides novel information helpful to improve the annotation of the Arabidopsis genome. Furthermore, the unbiased representation of transcripts will be particularly useful for gene discovery and gene expression analysis of nonmodel plants with less complete genomic information.For approximately 30 years, sequencing of DNA by the dideoxy terminator strategy introduced by Sanger (1977) has provided the basis for almost all available information about nucleotide sequences. Pyrosequencing is an alternative technology that detects the pyrophosphate released during DNA polymerase-catalyzed incorporation of nucleotides. The pyrophosphate liberated with each nucleotide addition can generate light in a reaction coupled to ATP sulfurylase and luciferase.

Knowledge on short‐term and long‐term availability of nitrogen (N) after application of organic fertilizers (e.g., farmyard manure, slurry, sewage sludge, composts) provides an important basis to optimize fertilizer use with benefits for the farmer and the environment. Nitrogen from many organic fertilizers often shows little effect on crop growth in the year of application, because of the slow‐release characteristics of organically bound N. Furthermore, N immobilization after application can occur, leading to an enrichment of the soil N pool. However, this process finally increases the long‐term efficiency of organic fertilizers. Short‐term N release from organic fertilizers, measured as mineral‐fertilizer equivalents (MFE), varies greatly from 0% (some composts) to nearly 100% (urine). The most important indicators to be used for predicting the short‐term availability of N are total and NH$ _4^+ $‐N contents, C : N ratio (especially of the decomposable organic fraction), and stability of the organic substances. Processing steps before organic fertilizers are applied in the field particularly can influence N availability. Composting reduces mineral‐N content and increases the stability of the organic matter, whereas anaerobic fermentation increases NH$ _4^+ $‐N content as well as the stability of organic matter, but decreases the C : N ratio remarkably, resulting in a product with a high content of directly available N. Nevertheless, long‐term effects of organic fertilizers rather slowly releasing N have to be considered to enable optimization of fertilizer use. After long‐term application of organic fertilizers, the overall N‐use efficiency is adequate to a MFE in the range of 40%–70%.

C 4 photosynthesis involves alterations to the biochemistry, cell biology, and development of leaves. Together, these modifications increase the efficiency of photosynthesis, and despite the apparent complexity of the pathway, it has evolved at least 45 times independently within the angiosperms. To provide insight into the extent to which gene expression is altered between C 3 and C 4 leaves, and to identify candidates associated with the C 4 pathway, we used massively parallel mRNA sequencing of closely related C 3 (Cleome spinosa) and C 4 (Cleome gynandra) species. Gene annotation was facilitated by the phylogenetic proximity of Cleome and Arabidopsis (Arabidopsis thaliana). Up to 603 transcripts differ in abundance between these C 3 and C 4 leaves. These include 17 transcription factors, putative transport proteins, as well as genes that in Arabidopsis are implicated in chloroplast movement and expansion, plasmodesmatal connectivity, and cell wall modification. These are all characteristics known to alter in a C 4 leaf but that previously had remained undefined at the molecular level. We also document large shifts in overall transcription profiles for selected functional classes. Our approach defines the extent to which transcript abundance in these C 3 and C 4 leaves differs, provides a blueprint for the NAD-malic enzyme C 4 pathway operating in a dicotyledon, and furthermore identifies potential regulators. We anticipate that comparative transcriptomics of closely related species will provide deep insight into the evolution of other complex traits.

Selective pressure exerted by a massive decline in atmospheric CO 2 levels 55 to 40 million years ago promoted the evolution of a novel, highly efficient mode of photosynthetic carbon assimilation known as C 4 photosynthesis. C 4 species have concurrently evolved multiple times in a broad range of plant families, and this multiple and parallel evolution of the complex C 4 trait indicates a common underlying evolutionary mechanism that might be elucidated by comparative analyses of related C 3 and C 4 species. Here, we use mRNA-Seq analysis of five species within the genus Flaveria, ranging from C 3 to C 3 -C 4 intermediate to C 4 species, to quantify the differences in the transcriptomes of closely related plant species with varying degrees of C 4 -associated characteristics. Single gene analysis defines the C 4 cycle enzymes and transporters more precisely and provides new candidates for yet unknown functions as well as identifies C 4 associated pathways. Molecular evidence for a photorespiratory CO 2 pump prior to the establishment of the C 4 cycle-based CO 2 pump is provided. Cluster analysis defines the upper limit of C 4 -related gene expression changes in mature leaves of Flaveria as 3582 alterations.

In the light of social change and a transformation in the work situation, interest in the problem of burnout has grown over the past decade. There is a conspicuous discrepancy, however, between what is regarded as certain knowledge and what is published opinion. To date, there is no generally accepted definition of burnout, or binding diagnostic criteria. According to the most common description at present, burnout syndrome is characterized by exhaustion, depersonalization and reduced satisfaction in performance. Because of its aetiopathogenesis, burnout is today mainly regarded as the result of chronic stress which has not been successfully dealt with. This paper gives an overview of the current definition for burnout syndrome and states possible contemporary hypotheses for its aetiology. By examining diagnostic criteria and possible therapies, methods of prevention are discussed. There is an urgent need for further investigations to determine whether burnout syndrome is a work-related disease.

Plastids of nongreen tissues import carbon as a source of biosynthetic pathways and energy. Within plastids, carbon can be used in the biosynthesis of starch or as a substrate for the oxidative pentose phosphate pathway, for example. We have used maize endosperm to purify a plastidic glucose 6-phosphate/phosphate translocator (GPT). The corresponding cDNA was isolated from maize endosperm as well as from tissues of pea roots and potato tubers. Analysis of the primary sequences of the cDNAs revealed that the GPT proteins have a high degree of identity with each other but share only ‫ف‬ 38% identical amino acids with members of both the triose phosphate/phosphate translocator (TPT) and the phosphoenolpyruvate/phosphate translocator (PPT) families. Thus, the GPTs represent a third group of plastidic phosphate antiporters. All three classes of phosphate translocator genes show differential patterns of expression. Whereas the TPT gene is predominantly present in tissues that perform photosynthetic carbon metabolism and the PPT gene appears to be ubiquitously expressed, the expression of the GPT gene is mainly restricted to heterotrophic tissues. Expression of the coding region of the GPT in transformed yeast cells and subsequent transport experiments with the purified protein demonstrated that the GPT protein mediates a 1:1 exchange of glucose 6-phosphate mainly with inorganic phosphate and triose phosphates. Glucose 6-phosphate imported via the GPT can thus be used either for starch biosynthesis, during which process inorganic phosphate is released, or as a substrate for the oxidative pentose phosphate pathway, yielding triose phosphates. INTRODUCTIONDuring C 3 photosynthesis, energy from solar radiation is used for the formation of phosphorylated C3 sugar phosphates, triose phosphates (trioseP), and 3-phosphoglycerate (3-PGA); these products are exported from the chloroplasts into the cytosol via the trioseP/3-PGA/phosphate translocator (TPT). In the mature leaves of most plants, the exported photosynthates are then used in the formation of sucrose, which is allocated via the phloem to the heterotrophic plant organs, such as young leaves, roots, seeds, fruits, or tubers. In these sink tissues, sucrose serves as a source of carbon and energy and is first cleaved by the action of invertases or sucrose synthase; the products of these reactions are converted into hexose phosphates.Nongreen plastids of heterotrophic tissues are carbohydrate-importing organelles and, in the case of amyloplasts of storage tissues, the site of starch synthesis. Because these plastids are normally not able to generate hexose phosphates from C3 compounds due to the absence of fructose 1,6-bisphosphatase activity (Entwistle and ap Rees, 1988), they rely on the import of cytosolically generated hexose phosphates as the source of carbon for starch biosynthesis and, in addition, for the oxidative pentose phosphate pathway. The results of transport measurements with intact organelles or reconstituted tissues from different plants suggest that thi...

The Arabidopsis chlorophyll a / b binding protein ( CAB ) gene underexpressed 1 ( cue1 ) mutant underexpresses light-regulated nuclear genes encoding chloroplast-localized proteins. cue1 also exhibits mesophyll-specific chloroplast and cellular defects, resulting in reticulate leaves. Both the gene underexpression and the leaf cell morphology phenotypes are dependent on light intensity. In this study, we determine that CUE1 encodes the plastid inner envelope phosphoenolpyruvate/phosphate translocator (PPT) and define amino acid residues that are critical for translocator function. The biosynthesis of aromatics is compromised in cue1 , and the reticulate phenotype can be rescued by feeding aromatic amino acids. Determining that CUE1 encodes PPT indicates the in vivo role of the translocator in metabolic partitioning and reveals a mesophyll cell-specific requirement for the translocator in Arabidopsis leaves. The nuclear gene expression defects in cue1 suggest that a light intensity-dependent interorganellar signal is modulated through metabolites dependent on a plastid supply of phosphoenolpyruvate.