Article Figures & Data
Figures
- Figure 1.
Developmental profiling of mitochondria, plastids, and peroxisomes in an Arabidopsis embryo/seedling over a germination time course. A, Seeds from a stably transformed line of Arabidopsis plants expressing mitochondria-targeted GFP, plastid-targeted RFP, and peroxisome-targeted CFP were examined at a number of time points during seed germination. i, The cotyledon of an embryo dissected from a dry seed. Peroxisomes (cyan) and proplastids (red) are visible in high numbers. ii, The cotyledon of an embryo dissected from a seed after 48 h of stratification (48 h S; prior to transfer to continuous light). Peroxisomes (cyan) and proplastids (red) are visible in high numbers; mitochondria (green) can begin to be identified, although these are not as distinct as in later time points. iii, The cotyledon of an embryo dissected at 12 h in continuous light (12 h SL). All three organelles are clearly visible. iv, The cotyledon of an embryo dissected at 48 h in continuous light (48 h SL). All three organelles are more clearly visible. The plastids (red) have quadrupled in size and are colocalizing with the peroxisomes. Bars = 15 μm. B, Total protein was extracted from Arabidopsis seeds collected during the germination time course and separated by SDS-PAGE (30 μg). Separated proteins were transferred to a polyvinylidene difluoride membrane and subjected to western-blot analysis. Following quantitation of band intensities using ImageQuant TL software (GE Healthcare), values were normalized to the highest level of intensity over the time course and graphed.
- Figure 2.
Concordance/discordance in transcript and protein abundance data during germination. Transcript and protein abundance data were normalized to maximum and visualized as a heat map in order of functional category. Pearson’s correlation coefficient was calculated for each transcript/protein abundance pair. Correlation coefficients greater than 0.625 were considered statistically significant (P < 0.05) and are indicated by the gray line. Annotation and localization details for each gene are shown in Supplemental Table S1, in accordance with the displayed order.
- Figure 3.
Distribution of overrepresented motifs (left panel) and transcription factor (TF)-binding sites (right panel) and in the promoters of mitochondrial genes (center panel). All transcripts for genes encoding mitochondrial proteins called present at a minimum of one time point were normalized to the highest expression value of each gene and hierarchically clustered. Cluster 3 of the mitochondrial set was observed to be significantly enriched compared with the clustered distribution of the genome, indicated with an asterisk. On the left, putative regulator-binding motifs calculated to be overrepresented (by z-score analysis; P < 0.05) are indicated for clusters 1 and 3 (i.e. where transcripts were observed to increase during the time course of the experiment). On the right are listed known transcription factor-binding sites enriched in clusters 1 (pink) and 3 (green), as determined by Pscan (Zambelli et al., 2009), with the expression of the profile of the transcription factors indicated according to cluster (e.g. the binding site for PIF3 is enriched in genes present in clusters 1 and 3, and the expression of PIF3 is assigned to cluster 1 by hierarchical clustering).
- Figure 4.
Transcript abundance profiles of genes encoding mitochondrial proteins during germination. Transcript abundance was normalized to the maximum expression level during the time course, classified into nontrivial BINS, and displayed on a custom pathway image. A method of color-coding was utilized to distinguish subtle variations between the profiles. Three shades of green were used to denote transcript abundance peaking at 48 h S/1 h SL (light green), 6 h SL (medium green), and 12 h SL (dark green). Two shades of red were used to denote transcript abundance increasing steadily, with maximal abundance in the final time point (dark red) and transcript abundance increasing steadily until 24 h SL, followed by a small decrease/stabilization in abundance at 48 h SL (light red). For a number of transcripts, one or more proteins were quantitated. Purple asterisks have been used to indicate when transcript and protein abundances peak at the same time point.
- Figure 5.
Coordination of nucleus- and organelle-encoded components of the mitochondrial electron transport chain. Expression values derived from microarray analysis of nucleus-encoded components of the mitochondrial electron transport chain were profiled in parallel to corresponding mitochondria-encoded components quantitated by qRT-PCR. For each gene displayed, gene names, annotations, and abundance levels are detailed in Supplemental Table S3.
- Figure 6.
Profiling mitochondria-targeted PPRs and their known organelle-encoded transcript targets. A, Hierarchical clustering of the relative expression levels of 261 genes encoding PPR proteins targeted to the mitochondria. Of the four clusters identified, cluster 3, which is characterized by transient expression, was significantly (P < 0.001) enriched (62%) when compared with the total genome (15%). B, PPR proteins that have been characterized previously in the literature were normalized alongside qRT-PCR data showing expression of their known mitochondria-encoded RNA targets.
- Figure 7.
Phenotypes observed in a transiently expressed set of genes encoding mitochondrial proteins during seed germination. Of the 239 transiently expressed genes (Supplemental Fig. S1B; cluster 3), one in three (78 genes) were observed to have a previously published phenotype when silenced or knocked out (Supplemental Table S5). Of these, 37% presented an embryo-arrested/lethal phenotype, which is three times greater than the expected number of embryo-arrested/lethal phenotypes for the whole genome. Other categories are as follows: leaf shape/development/amino acid content/fatty acid content/polysaccharide (AA/FA/PS) content, whole plant affected, developmental, flower development/timing/siliques, reduced/altered hypocotyl and/or root growth, and seed pigment/yield/amino acid content/fatty acid content (for details, see Supplemental Table S5). WT, Wild type.
- Figure 8.
A model for mitochondrial biogenesis during germination. Mitochondria in dry seeds lack cristae structures (Stasis). The earliest events that occur in mitochondrial biogenesis occur at the end of stratification and the first hour of transfer to light and are associated with RNA transcription and translation, leading to an increase in the transcript abundance of mitochondria-encoded genes. The next stage, which occurs at 24 h after transfer to light, is the increase in the transcript abundance of genes encoding various metabolic components. For a number of transcripts, one or more proteins were quantitated. The stages at which the lowest/highest corresponding protein abundances are observed are indicated with black/green asterisks, respectively.
Additional Files
Supplemental Data
Supplemental Figures, Methods, and Tables
Files in this Data Supplement:
- Supplemental Data - Supplemental Figure 1
- Supplemental Data - Supplemental Figure 2
- Supplemental Data - Supplemental Figure 3
- Supplemental Data - Supplemental Figure 4
- Supplemental Data - Supplemental Figure 5
- Supplemental Data - Supplemental Figure 6
- Supplemental Data - Supplemental Methods
- Supplemental Data - Supplemental Tables I-VI
