Project description:To investigate the developmental gradient of the third maize leaf, the light exposed area of the leaf (corresponding to 18cm of leaf) and 2cm shaded by the sheath were sampled in ten slices. Four replicates were collected, immediately shock frozen in liquid nitrogen and subsequently cut into 2cm slices. At least 10 plants were pooled for each biological replicate. We have systematically analyzed a developmental gradient of the third maize leaf from the point of emergence into the light to the tip in ten continuous leaf slices to study organ development and physiological and biochemical functions. Transcriptome analysis, oxygen sensitivity of photosynthesis, delta-13C values, and photosynthetic rate measurements showed that the maize leaf undergoes a sink to source transition without an intermediate phase of C3 photosynthesis or operation of a photorespiratory carbon pump. Metabolome and transcriptome analysis, chlorophyll and protein measurements, as well as dry weight determination showed continuous gradients for all analyzed items. The absence of binary on-off switches and regulons pointed to a morphogradient along the leaf as the determining factor of developmental stage. Analysis of transcription factors for differential expression along the leaf gradient defined a list of putative regulators orchestrating the sink-to-source transition and establishment of C4 photosynthesis. Finally, transcriptome and metabolome analysis, as well as enzyme activity measurements, and absolute quantification of selected metabolites revised the current model of maize C4 photosynthesis. All datasets are included within the publication to serve as a resource for maize leaf systems biology. For the transcriptional analysis, the goal of the study was to (i) identify whether the leaf contains binary switches for genes involved in photosynthesis, (ii)characterize the patterns of gene expression in the leaf, (iii) provide independent validation of maize leaf expression experiments published in Li et al. (2011) and (iv) determine transcripts co-expressed with key transcripts of C4 photosynthesis. To this end, changed transcripts were determined by ANOVA and characterized by K-means and hierachical clustering.

Project description:To investigate the developmental gradient of the third maize leaf, the light exposed area of the leaf (corresponding to 18cm of leaf) and 2cm shaded by the sheath were sampled in ten slices. Four replicates were collected, immediately shock frozen in liquid nitrogen and subsequently cut into 2cm slices. At least 10 plants were pooled for each biological replicate. We have systematically analyzed a developmental gradient of the third maize leaf from the point of emergence into the light to the tip in ten continuous leaf slices to study organ development and physiological and biochemical functions. Transcriptome analysis, oxygen sensitivity of photosynthesis, delta-13C values, and photosynthetic rate measurements showed that the maize leaf undergoes a sink to source transition without an intermediate phase of C3 photosynthesis or operation of a photorespiratory carbon pump. Metabolome and transcriptome analysis, chlorophyll and protein measurements, as well as dry weight determination showed continuous gradients for all analyzed items. The absence of binary on-off switches and regulons pointed to a morphogradient along the leaf as the determining factor of developmental stage. Analysis of transcription factors for differential expression along the leaf gradient defined a list of putative regulators orchestrating the sink-to-source transition and establishment of C4 photosynthesis. Finally, transcriptome and metabolome analysis, as well as enzyme activity measurements, and absolute quantification of selected metabolites revised the current model of maize C4 photosynthesis. All datasets are included within the publication to serve as a resource for maize leaf systems biology. For the transcriptional analysis, the goal of the study was to (i) identify whether the leaf contains binary switches for genes involved in photosynthesis, (ii)characterize the patterns of gene expression in the leaf, (iii) provide independent validation of maize leaf expression experiments published in Li et al. (2011) and (iv) determine transcripts co-expressed with key transcripts of C4 photosynthesis. To this end, changed transcripts were determined by ANOVA and characterized by K-means and hierachical clustering. Four replicates were collected for each of the ten consecutive leaf slices resulting in 40 one color arrays. Slice 1 represents the tip of the leaf, slice 10 the lowermost slice which is shaded by the sheath with all slices in between consecutively numbered.

Project description:FOXO transcription factors control cellular formation of reactive oxygen species (ROS), which critically contribute to cell survival and cell death in neuroblastoma. Here, we report that C10orf10, also named M-bM-^@M-^\Decidual Protein induced by Progesterone (DEPP)M-bM-^@M-^], is a direct transcriptional target of FOXO3 in human neuroblastoma. As FOXO3-mediated apoptosis involves a biphasic ROS accumulation, we analyzed cellular ROS levels in DEPP-knockdown cells by live-cell imaging. Knockdown of DEPP prevented the primary and secondary ROS accumulation during FOXO3 activation and attenuates FOXO3-induced apoptosis, whereas its overexpression raises cellular ROS levels and sensitizes to cell death. In neuronal cells, cellular steady state ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that enables protein import into peroxisomes, we analyzed the effect of DEPP on peroxisomal function by measuring the catalase enzyme activity. Catalase activity was reduced by conditional DEPP overexpression and significantly increased in DEPP-knockdown cells. Using live cell imaging and fluorescent peroxisomal and mitochondrial probes we demonstrate that DEPP localizes to peroxisomes and mitochondria in neuroblastoma cells. The combined data indicate that DEPP reduces peroxisomal activity and thereby impairs the cellular ROS detoxification capacity and contributes to death sensitization. SH-EP, NB15 neuroblastoma cells and CCRF-CEM-C7H2 acute lymphoblastic leukemia cells were infected with the retrovirus plasmid pLIB-FOXO3(A3)-Ertm-iresNeo. Gene expression measures of samples with activated FOXO3 transcription factor (3h OHT treated) have been compared to untreated samples (0h time point). To rule out gene regulations by estrogen samples treated for 3 hours with tamoxifen have been compared to the untreated samples. Only genes that were more than two-fold regulated in the first, but not in the second comparison were defined to be FOXO3 regulated.

Project description:Ascorbate (vitamin C) is an important antioxidant in leaves with multiple functions in photosynthesis and its levels are tightly regulated by light quality. The majority of work to date has focussed on regulation of ascorbate levels via biosynthesis. However, the pathway of ascorbate degradation, which is localised in the apoplast may additionally play a significant role. The degradative pathway begins with ascorbate oxidation by the enzyme ascorbate oxidase (AO) and hence we have examined the impact of manipulation of ascorbate oxidase activity on the content of ascorbate and the acclimation of photosynthesis to changing light levels. Leaf ascorbate content was highly light dependent in Nicotiana tobaccum being double in high compared with low light conditions. In contrast ascorbate oxidase activity was not altered by changing light levels. Transgenic tobacco lines with reduced or enhanced AO activity exhibited no morphological phenotype and photosynthesis under high light was similarly impaired irrespective of the measured AO activity. However further investigation between AO activity and leaf acclimation to changing light intensity revealed a suite of metabolic and transcriptome changes indicating a role for the apoplastic ascorbate pool in chloroplast to nucleus communication. In all genotypes, transcripts associated with ascorbate synthesis and recycling were less abundant following transition from high to low light. However transcripts associated with glutathione recycling, light harvesting, reaction centre function and the Calvin cycle were all increased in abundance. Following the transition from high to low light increases in leaf threonate, an ascorbate degradation product, were proportional to AO activity. A number of chloroplast synthesised amino acids were significantly increased both during high light and following transfer back to LL for 12 h. Similarly, several primary carbohydrates and TCA intermediates were significantly enhanced following high light treatment. In addition to light effects, AO activity had a significant impact on ï¢-alanine, aspartate and threonine. Intriguingly, a large number of fatty acids were reduced in antisense AO and wild-type plants immediately after high light stress however the content of these compounds were significantly increased in overexpressing sense lines. We conclude that AO mediated turnover plays an important role in adjusting cellular ascorbate content to photosynthetic need in a fluctuating light environment and that light mediated signals rapidly adjust that the apoplastic ascorbate pool.

Dataset Information

Pexophagy suppresses ROS-induced damage in leaf cells under high-intensity light

Publications

Pexophagy suppresses ROS-induced damage in leaf cells under high-intensity light.

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