The role of GDP-l-galactose phosphorylase in the control of ascorbate biosynthesis.

The enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization. All transgenic lines expressing functional SW protein green fluorescent protein fusions driven by their endogenous promoters showed a high accumulation of the fusion proteins, with the exception of those lines expressing GDP-l-galactose phosphorylase (GGP) protein, which had very low abundance. Transient expression of individual or combinations of SW pathway enzymes in N. benthamiana only increased ascorbate concentration if GGP was included. Although we did not detect direct interaction between the different enzymes of the pathway using yeast-two hybrid analysis, consecutive SW enzymes, as well as the first and last enzymes (GMP and l-GalDH) associated in coimmunoprecipitation studies. This association was supported by gel filtration chromatography, showing the presence of SW proteins in high-molecular weight fractions. Finally, metabolic control analysis incorporating known kinetic characteristics showed that previously reported feedback repression at the GGP step, combined with its relatively low abundance, confers a high-flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.

Hydrogen peroxide metabolism and functions in plants.

Contents Summary 1197 I. Introduction 1198 II. Measurement and imaging of H2 O2 1198 III. H2 O2 and O2·- toxicity 1199 IV. Production of H2 O2 : enzymes and subcellular locations 1200 V. H2 O2 transport 1205 VI. Control of H2 O2 concentration: how and where? 1205 VII. Metabolic functions of H2 O2 1207 VIII. H2 O2 signalling 1207 IX. Where next? 1209 Acknowledgements 1209 References 1209 SUMMARY: Hydrogen peroxide (H2 O2 ) is produced, via superoxide and superoxide dismutase, by electron transport in chloroplasts and mitochondria, plasma membrane NADPH oxidases, peroxisomal oxidases, type III peroxidases and other apoplastic oxidases. Intracellular transport is facilitated by aquaporins and H2 O2 is removed by catalase, peroxiredoxin, glutathione peroxidase-like enzymes and ascorbate peroxidase, all of which have cell compartment-specific isoforms. Apoplastic H2 O2 influences cell expansion, development and defence by its involvement in type III peroxidase-mediated polymer cross-linking, lignification and, possibly, cell expansion via H2 O2 -derived hydroxyl radicals. Excess H2 O2 triggers chloroplast and peroxisome autophagy and programmed cell death. The role of H2 O2 in signalling, for example during acclimation to stress and pathogen defence, has received much attention, but the signal transduction mechanisms are poorly defined. H2 O2 oxidizes specific cysteine residues of target proteins to the sulfenic acid form and, similar to other organisms, this modification could initiate thiol-based redox relays and modify target enzymes, receptor kinases and transcription factors. Quantification of the sources and sinks of H2 O2 is being improved by the spatial and temporal resolution of genetically encoded H2 O2 sensors, such as HyPer and roGFP2-Orp1. These H2 O2 sensors, combined with the detection of specific proteins modified by H2 O2 , will allow a deeper understanding of its signalling roles.

Plant genetics. Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome.

Oilseed rape (Brassica napus L.) was formed ~7500 years ago by hybridization between B. rapa and B. oleracea, followed by chromosome doubling, a process known as allopolyploidy. Together with more ancient polyploidizations, this conferred an aggregate 72× genome multiplication since the origin of angiosperms and high gene content. We examined the B. napus genome and the consequences of its recent duplication. The constituent An and Cn subgenomes are engaged in subtle structural, functional, and epigenetic cross-talk, with abundant homeologous exchanges. Incipient gene loss and expression divergence have begun. Selection in B. napus oilseed types has accelerated the loss of glucosinolate genes, while preserving expansion of oil biosynthesis genes. These processes provide insights into allopolyploid evolution and its relationship with crop domestication and improvement.

Urinary inositol phosphoglycan-P type: near patient test to detect preeclampsia prior to clinical onset of the disease. A study on 416 pregnant Mauritian women.

Preeclampsia and eclampsia account for major pregnancy complications in Mauritius, an emerging country (maternal mortality rate of 60 per 100,000 deliveries). This prospective longitudinal study was carried out in the main regional hospital in the north of the island, to measure inositol phosphoglycan-P type (IPG-P) in the urine of pregnant women (using an ELISA-based assay). Women had approximately 10 prenatal visits per pregnancy and a complete follow-up in this same referral centre after the first trimester of pregnancy. Urine samples were collected every 1-4 weeks in all women. In a cohort of 416 patients, preeclampsia (PE) was diagnosed in 34 women. In established PE (hypertension and proteinuria), the assay as a diagnostic test showed a positive likelihood ratio of 18.73, a negligible negative likelihood ratio with area under the curve (AUC) of 0.99, sensitivity of 96.7%, specificity of 94.8% and remained negative in control women (n=312), women with gestational hypertension (without proteinuria (n=56), and gestational diabetic mothers (n=14). Moreover, as a predictive screening test two weeks before the diagnosis of PE, the assay showed sensitivity of 84.2% and specificity of 83.6%. Detection of urinary inositol phosphoglycan-P type in pregnant women can be a useful confirmatory marker of PE, as well as a predictive marker, two weeks before the onset of the disease.

Prevalence of gene expression additivity in genetically stable wheat allohexaploids.

The reprogramming of gene expression appears as the major trend in synthetic and natural allopolyploids where expression of an important proportion of genes was shown to deviate from that of the parents or the average of the parents. In this study, we analyzed gene expression changes in previously reported, highly stable synthetic wheat allohexaploids that combine the D genome of Aegilops tauschii and the AB genome extracted from the natural hexaploid wheat Triticum aestivum. A comprehensive genome-wide analysis of transcriptional changes using the Affymetrix GeneChip Wheat Genome Array was conducted. Prevalence of gene expression additivity was observed where expression does not deviate from the average of the parents for 99.3% of 34,820 expressed transcripts. Moreover, nearly similar expression was observed (for 99.5% of genes) when comparing these synthetic and natural wheat allohexaploids. Such near-complete additivity has never been reported for other allopolyploids and, more interestingly, for other synthetic wheat allohexaploids that differ from the ones studied here by having the natural tetraploid Triticum turgidum as the AB genome progenitor. Our study gave insights into the dynamics of additive gene expression in the highly stable wheat allohexaploids.

Genome-wide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa.

In Eukaryotes, LIM proteins act as developmental regulators in basic cellular processes such as regulating the transcription or organizing the cytoskeleton. The LIM domain protein family in plants has mainly been studied in sunflower and tobacco plants, where several of its members exhibit a specific pattern of expression in pollen. In this paper, we finely characterized in poplar six transcripts encoding these proteins. In Populus trichocarpa genome, the 12 LIM gene models identified all appear to be duplicated genes. In addition, we describe several new LIM domain proteins deduced from Arabidopsis and rice genomes, raising the number of LIM gene models to six for both species. Plant LIM genes have a core structure of four introns with highly conserved coding regions. We also identified new LIM domain proteins in several other species, and a phylogenetic analysis of plant LIM proteins reveals that they have undergone one or several duplication events during the evolution. We gathered several LIM protein members within new monophyletic groups. We propose to classify the plant LIM proteins into four groups: alphaLIM1, betaLIM1, gammaLIM2, and deltaLIM2, subdivided according to their specificity to a taxonomic class and/or to their tissue-specific expression. Our investigation of the structure of the LIM domain proteins revealed that they contain many conserved motifs potentially involved in their function.