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:A number of taxa utilize C4 photosynthesis, to limit the impact of photorespiration upon photosynthetic performance. In order to achieve a local elevation of CO2 concentration, maize plants possess two photosynthetic cell types. Rubisco accumulation is restricted to bundle sheath (BS) cells that surround the leaf veins. Carbon fixation occurs initially in adjacent mesophyll (ME) cells. C4 compounds are transported into the BS cells where they are subsequently decarboxylated, releasing CO2. Although the major components of the C4 pathway have been well characterized, less is known about further metabolic partitioning in the maize leaf. Microarray hybridizations have been performed in order to further investigate metabolic differences between BS and ME cell types. BS strands and ME protoplasts were isolated from the leaves of 10 day old maize seedlings by mechanical disruption and enzymatic digestion respectively. To control for differences arising from these different protocols, total leaf (TO) and total leaf stress (ST) samples were also isolated. The ST sample was subjected to the same treatments as the ME sample, with the omission of cell-wall degrading enzymes. Leaves for the TO sample were harvested as for the BS strand sample. An interwoven loop design was used to compare the four treatment groups. A biological group consisted of a growth of plants from which pooled individuals were taken for the four treatments. Six biological replicates (groups) were used. Labeling was performed using the Genisphere Array 900-MPX kit according to the manufacturer's protocol. Post hybridization washes were performed according to the recommendations of the Maize Oligo Array Project. Scan settings were used for detection of moderate to high expression signals (gain ~ 60%. power 90%). Following hybridization with TO cDNA, ~1/3 of features provided signal above twice background and below saturation. A number of taxa utilize C4 photosynthesis, to limit the impact of photorespiration upon photosynthetic performance. In order to achieve a local elevation of CO2 concentration, maize plants possess two photosynthetic cell types. Rubisco accumulation is restricted to bundle sheath (BS) cells that surround the leaf veins. Carbon fixation occurs initially in adjacent mesophyll (ME) cells. C4 compounds are transported into the BS cells where they are subsequently decarboxylated, releasing CO2. Although the major components of the C4 pathway have been well characterized, less is known about further metabolic partitioning in the maize leaf. Microarray hybridizations have been performed in order to further investigate metabolic differences between BS and ME cell types. BS strands and ME protoplasts were isolated from the leaves of 10 day old maize seedlings by mechanical disruption and enzymatic digestion respectively. To control for differences arising from these different protocols, total leaf (TO) and total leaf stress (ST) samples were also isolated. The ST sample was subjected to the same treatments as the ME sample, with the omission of cell-wall degrading enzymes. Leaves for the TO sample were harvested as for the BS strand sample. An interwoven loop design was used to compare the four treatment groups. A biological group consisted of a growth of plants from which pooled individuals were taken for the four treatments. Six biological replicates (groups) were used. Labeling was performed using the Genisphere Array 900-MPX kit according to the manufacturer's protocol. Post hybridization washes were performed according to the recommendations of the Maize Oligo Array Project. Scan settings were used for detection of moderate to high expression signals (gain ~ 60%. power 90%). Following hybridization with TO cDNA, ~1/3 of features provided signal above twice background and below saturation. Keywords: Gene expression profiling of bundle sheath and mesophyll cell types

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.

Dataset Information

Developmental Dynamics of Kranz Cell Transcriptional Specificity in Maize Leaf Reveals Early Onset of C4-related Processes

Publications

Developmental dynamics of Kranz cell transcriptional specificity in maize leaf reveals early onset of C4-related processes.

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