The role of alanine and aspartate aminotransferases in C4 photosynthesis.

Alanine and aspartate are essential transfer metabolites for C4 species of the NAD-malic enzyme and phosphoenolpyruvate carboxykinase subtype. To some degree both amino acids are also part of the metabolite shuttle in NADP-malic enzyme plants. In comparison with C3 species, the majority of C4 species are therefore characterised by enhanced expression and activity of alanine and aspartate aminotransferases (AT) in the photosynthetically active tissue. Both enzymes exist in multiple copies and have been found in different subcellular compartments. We tested whether different C4 species show preferential recruitment of enzymes from specific lineages and subcellular compartments. Phylogenetic analysis of alanine and aspartate ATs from a variety of monocot and eudicot C4 species and their C3 relatives was combined with subcellular prediction tools and analysis of the subsequent transcript amounts in mature leaves. Recruitment of aspartate AT from a specific subcellular compartment was strongly connected to the biochemical subtype. Deviation from the main model was however observed in Gynandropsis gynandra. The configuration of alanine AT generally differed in monocot and eudicot species. C4 monocots recruited an alanine AT from a specific cytosolic branch, but eudicots use alanine AT copies from a mitochondrial branch. Generally, plants display high plasticity in the setup of the C4 pathway. Beside the common models for the different C4 subtypes, individual solutions were found for plant groups or lineages.

The transcriptional landscape of polyploid wheat.

The coordinated expression of highly related homoeologous genes in polyploid species underlies the phenotypes of many of the world's major crops. Here we combine extensive gene expression datasets to produce a comprehensive, genome-wide analysis of homoeolog expression patterns in hexaploid bread wheat. Bias in homoeolog expression varies between tissues, with ~30% of wheat homoeologs showing nonbalanced expression. We found expression asymmetries along wheat chromosomes, with homoeologs showing the largest inter-tissue, inter-cultivar, and coding sequence variation, most often located in high-recombination distal ends of chromosomes. These transcriptionally dynamic genes potentially represent the first steps toward neo- or subfunctionalization of wheat homoeologs. Coexpression networks reveal extensive coordination of homoeologs throughout development and, alongside a detailed expression atlas, provide a framework to target candidate genes underpinning agronomic traits in wheat.

Comparative proteomics of chloroplasts envelopes from bundle sheath and mesophyll chloroplasts reveals novel membrane proteins with a possible role in c4-related metabolite fluxes and development.

As the world population grows, our need for food increases drastically. Limited amounts of arable land lead to a competition between food and fuel crops, while changes in the global climate may impact future crop yields. Thus, a second "green revolution" will need a better understanding of the processes essential for plant growth and development. One approach toward the solution of this problem is to better understand regulatory and transport processes in C4 plants. C4 plants display an up to 10-fold higher apparent CO2 assimilation and higher yields while maintaining high water use efficiency. This requires differential regulation of mesophyll (M) and bundle sheath (BS) chloroplast development as well as higher metabolic fluxes of photosynthetic intermediates between cells and particularly across chloroplast envelopes. While previous analyses of overall chloroplast membranes have yielded significant insight, our comparative proteomics approach using enriched BS and M chloroplast envelopes of Zea mays allowed us to identify 37 proteins of unknown function that have not been seen in these earlier studies. We identified 280 proteins, 84% of which are known/predicted to be present in chloroplasts. Seventy-four percent have a known or predicted membrane association. Twenty-one membrane proteins were 2-15 times more abundant in BS cells, while 36 of the proteins were more abundant in M chloroplast envelopes. These proteins could represent additional candidates of proteins essential for development or metabolite transport processes in C4 plants. RT-PCR confirmed differential expression of 13 candidate genes. Chloroplast association for seven proteins was confirmed using YFP/GFP labeling. Gene expression of four putative transporters was examined throughout the leaf and during the greening of leaves. Genes for a PIC-like protein and an ER-AP-like protein show an early transient increase in gene expression during the transition to light. In addition, PIC gene expression is increased in the immature part of the leaf and was lower in the fully developed parts of the leaf, suggesting a need for/incorporation of the protein during chloroplast development.

The dicotyledonous NAD malic enzyme C4 plant Cleome gynandra displays age-dependent plasticity of C4 decarboxylation biochemistry.

The C(4) photosynthetic pathway enriches carbon dioxide in the vicinity of Rubisco, thereby enabling plants to assimilate carbon more efficiently. Three canonical subtypes of C(4) exist, named after their main decarboxylating enzymes: NAD-dependent malic enzyme type, NADP-dependent malic enzyme type and phosphoenolpyruvate carboxykinase type. Cleome gynandra is known to perform NAD-ME type C(4) photosynthesis. To further assess the mode of C(4) in C. gynandra and its manifestation in leaves of different age, total enzyme activities of eight C(4) -related enzymes and the relative abundance of 31 metabolites were measured. C. spinosa was used as a C(3) control. C. gynandra was confirmed as an NAD-ME type C(4) plant in mid-aged leaves, whereas a mixed NAD-ME and PEPCK type was observed in older leaves. Young leaves showed a C(3) -C(4) intermediate state with respect to enzyme activities and metabolite abundances. Comparative transcriptome analysis of mid-aged leaves of C. gynandra and C. spinosa showed that the transcript of only one aspartate aminotransferase (AspAT) isoform is highly abundant in C. gynandra. However, the canonical model of the NAD-ME pathway requires two AspATs, a mitochondrial and a cytosolic isoform. Surprisingly, our results indicate the existence of only one highly abundant AspAT isoform. Using GFP-fusion, this isozyme was localised exclusively to mitochondria. We propose a revised model of NAD-ME type C(4) photosynthesis in C. gynandra, in which both AspAT catalysed reactions take place in mitochondria and PEPCK catalyses an alternative decarboxylating pathway.

What can next generation sequencing do for you? Next generation sequencing as a valuable tool in plant research.

Next generation sequencing (NGS) technologies have opened fascinating opportunities for the analysis of plants with and without a sequenced genome on a genomic scale. During the last few years, NGS methods have become widely available and cost effective. They can be applied to a wide variety of biological questions, from the sequencing of complete eukaryotic genomes and transcriptomes, to the genome-scale analysis of DNA-protein interactions. In this review, we focus on the use of NGS for plant transcriptomics, including gene discovery, transcript quantification and marker discovery for non-model plants, as well as transcript annotation and quantification, small RNA discovery and antisense transcription analysis for model plants. We discuss the experimental design for analysis of plants with and without a sequenced genome, including considerations on sampling, RNA preparation, sequencing platforms and bioinformatics tools for data analysis. NGS technologies offer exciting new opportunities for the plant sciences, especially for work on plants without a sequenced genome, since large sequence resources can be generated at moderate cost.

The freshwater biodiversity crisis.

PIP: This article concerns the threat on freshwater ecosystems, which harbor a disproportionate amount of the world's biodiversity. In many parts of the world, freshwater ecosystems are already degraded from a range of human activities, including water extraction, pollution and physical alteration. The data that showed a biodiversity crisis in ecosystems included species loss and breakdown of the ecological processes and resources. Furthermore, several case studies were cited to illustrate the status of freshwater diversity. Numerous reasons for freshwater biodiversity loss were mentioned, which included pollution from pesticides and agricultural and mine run-off, and physical alteration through channelization and impoundments that affected the hydrology and benthic habitat. Despite the successful establishment of institutions to conserve water birds and wetland habitats, there was a lower priority for conservation of freshwater biodiversity in terms of species and habitats. This bias has had important and serious implications for allocation of resources to increase the knowledge and understanding of freshwater ecosystems, as well as for the adequacy of impact assessments for development projects affecting them.^ieng

Transcription unit mapping in bacteriophage T7. I. In vivo transcription by Escherichia coli RNA polymerase.

Premature termination of transcription at UV lesions in DNA permits a study of the sequential order of transcription by E. coli RNA polymerase of the genes in the early region of T7. This analysis is extended to the transcription by E. coli polymerase of the late region of T7 by deleting the early terminator. The results demonstrate the existence of a single large transcription unit spanning the early region, with a promotor located at the left end of the T7 genome. Furthermore, there are no initiation sites for E. coli RNA polymerase in the late region. When E. coli polymerase transcribes the late region, it does so exclusively by initiation at the early promotor.

Transcription unit mapping in bacteriophage T7. II. Proportionality of number of gene copies, mRNA, and gene product.

The effect of UV irradiation of bacteriophage T7 on in vivo early RNA synthesis has been studied by direct quantitation of the gene-specific RNA transcripts. The results show that the early region of phage T7 is transcribed from left to right as a single unit. Furthermore, gene inactivation, the UV sensitivity of synthesis of gene-specific RNA, and the UV sensitivity of synthesis of the corresponding proteins all follow pseudo first-order kinetics in multiply infected cells, demonstrating a random statistical correlation between both transcriptional sampling of gene copies and translational sampling of the resultant RNA transcripts. In addition, these simple kinetics imply an absence of positive feedback mechanisms compensating for the differential decline of individual early gene products in cells multiply infected with phage T7.

Replication of murine cytomegalovirus in reproductive tissues.

Murine cytomegalovirus (MCMV) was found in preproductive tissues of newborn mice acutely infected with this virus. Using 3H-labeled complementary RNA (cRNA) probe made from MCMV DNA, viral genetic material clearly occupied ovarian stromal cells surrounding the follicular region but not cells of the follicle or cells in the outer tunic layers. In the testes, squamous epithelial cells external to the tunica albuginea harbored viral genetic material. The immaturity of germ line cells in testes of one-week-old mice precluded identification of these cells or examination of their involvement. The presence of viral DNA in reproductive tissue during acute infection raised the possibility that the ovary and/or testes might act as a reservoir for latent virus later in life of adult mice. Hybridization kinetics analysis of DNA isolated from ovaries and testes of 5- and 6-month-old mice latently infected with MCMV suggested that the viral genome was present in both organs.

Rapid typing of herpes simplex virus isolates by deoxyribonucleic acid:deoxyribonucleic acid hybridization.

A method for typing clinical isolates of herpes simplex virus was developed. It utilizes hybridization between unlabeled deoxyribonucleic acid from infected cultures and tritium-labeled virus deoxyribonucleic acid, and it can be completed within a day using a single roller-tube culture of the clinical isolated. The data obtained are inherently quantitative, and the method yields unequivocal identification and typing. Thirty-nine coded clinical isolates were all correctly typed by this method.

Pathogenesis of murine cytomegalovirus infection: the macrophage as a permissive cell for cytomegalovirus infection, replication and latency.

Macrophages harvested from the peritoneal cavities of mice of several strains were permissive to infection with murine cytomegalovirus (MCMV). Macrophages from six mouse strains released equivalent amounts of plaque-forming virus into the culture fluids and cells from mouse strains scored similarly in numbers of infectious centres. Twenty to 50% of the infected macrophages obtained after thioglycollate activation formed infectious centres. When studied by in situ hybridization, more than 82% of infected macrophages (with or without thioglycollate activation) contained MCMV DNA. Macrophages obtained from latently infected mice were examined for their content of MCMV. Using co-cultivation assays, MCMV was frequently recovered from thioglycollate activated macrophages harvested from latently infected mice but only rarely recovered from non-activated macrophages. MCMV DNA--mouse DNA hybridization assays revealed four to seven virus genome DNA copies per 100 cells. These studies indicate that macrophages harvested from mice susceptible (BALB/cSt) or resistant (C3H) to MCMV infection replicated virus equivalently and that macrophages are a reservoir of MCMV during latent and chronic infections. Activation of macrophages may be one of the important steps leading to the exacerbation of in vivo latent infections.

Control of gene function in bacteriophage T4. I. Ribonucleic acid and deoxyribonucleic acid metabolism in T4rII-infected lambda-lysogenic hosts.

Deoxyribonucleic acid (DNA) synthesis in T4rII-infected, lambda-lysogenic strains of Escherichia coli proceeds with one-half the rate of T4 wild-infected bacteria and stops 16 min after infection at 37 C. The rates of ribonucleic acid (RNA) synthesis, however, are the same with T4rII and T4 wild. The turnover of pulse-labeled RNA is slow in K strains (half-lives 10 to 20 min) as compared with B strains (half-lives 2.5 to 6 min). Lambda-lysogeny increases the apparent messenger (m) RNA half-lives in pulse-chase experiments. The shutoff of host RNA synthesis in T4rII infected K(lambda) is incomplete. Moreover, the preferential transcription of T4 DNA ceases 13 min after infection, and transcription of host and prophage lambda DNA is resumed. The T4 RNA synthesized in rII-infected K(lambda) contains no late T4 mRNA. The early portion of the T4 genome, however, is transcribed completely. The T4-induced early modification of bacterial RNA polymerase does occur. Resumption of host DNA transcription at 13 min after infection is not associated with a reversal of the above polymerase modification. It is concluded that in lambdalysogenic bacteria T4rII infections are abortive because RNA polymerase is prevented from transcribing late T4 genes.