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Plant Physiol. 2011 Nov;157(3):1342-62. doi: 10.1104/pp.111.183129. Epub 2011 Sep 9.

In-depth temporal transcriptome profiling reveals a crucial developmental switch with roles for RNA processing and organelle metabolism that are essential for germination in Arabidopsis.

Narsai R, Law SR, Carrie C, Xu L, Whelan J.

Source

Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, Western Australia 6009, Australia. reena@cyllene.uwa.edu.au

Abstract

Germination represents a rapid transition from dormancy to a high level of metabolic activity. In-depth transcriptomic profiling at 10 time points in Arabidopsis (Arabidopsis thaliana), including fresh seed, ripened seed, during stratification, germination, and postgermination per se, revealed specific temporal expression patterns that to our knowledge have not previously been identified. Over 10,000 transcripts were differentially expressed during cold stratification, with subequal numbers up-regulated as down-regulated, revealing an active period in preparing seeds for germination, where transcription and RNA degradation both play important roles in regulating the molecular sequence of events. A previously unidentified transient expression pattern was observed for a group of genes, whereby a significant rise in expression was observed at the end of stratification and significantly lower expression was observed 6 h later. These genes were further defined as germination specific, as they were most highly expressed at this time in germination, in comparison with all developmental tissues in the AtGenExpress data set. Functional analysis of these genes using genetic inactivation revealed that they displayed a significant enrichment for embryo-defective or -arrested phenotype. This group was enriched in genes encoding mitochondrial and nuclear RNA-processing proteins, including more than 45% of all pentatricopeptide domain-containing proteins expressed during germination. The presence of mitochondrial DNA replication factors and RNA-processing functions in this germination-specific subset represents the earliest events in organelle biogenesis, preceding any changes associated with energy metabolism. Green fluorescent protein analysis also confirmed organellar localization for 65 proteins, largely showing germination-specific expression. These results suggest that mitochondrial biogenesis involves a two-step process to produce energetically active organelles: an initial phase at the end of stratification involving mitochondrial DNA synthesis and RNA processing, and a later phase for building the better-known energetic functions. This also suggests that signals with a mitochondrial origin and retrograde signals may be crucial for successful germination.

PMID:: 21908688; [PubMed - indexed for MEDLINE]
PMCID:: PMC3252162

Free PMC Article

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Figure 3.

Identification of GS gene expression. A, Publicly available microarrays carried out on wild-type tissues in the AtGenExpress developmental set (Schmid et al., 2005; E-AFMX-9) were normalized together with the arrays in this study. Hierarchical clustering of the relative expression levels are for 775 unique genes identified as showing the highest expression during germination, with expression levels less than 50% of these levels in all other tissues. B, Examination of over/underrepresented GO categories for these transiently expressed genes revealed significant overrepresentation of genes encoding proteins targeted to the mitochondria, nucleus, and ribosomes, corresponding with the observed overrepresentation of DNA- and RNA-binding functions. The overrepresentation of these functions, specifically during germination, implies that these genes are likely to encode the regulatory proteins required specifically for germination progression and later development. Over/underrepresented functional categories were identified by z-score analysis. Statistical significance is represented by a false-color heat map (up, orange; down, green) where a z-score of 1.96 represents a P value of 0.05. C, The distribution of genes into functional subcategories, categorized as follows: i, mitochondrial; ii, nuclear. D, i, The percentage of PPR-encoding genes in each cluster is displayed as a pie chart showing the percentage of genes in each cluster in the genome. ii, Expression levels during germination without (no stratification) and including stratification for the 75 PPR genes identified as possessing GS expression in this study. iii, Expression of the rice genes orthologous to the 75 Arabidopsis genes shown across germination under aerobic and anaerobic conditions in rice.

In-Depth Temporal Transcriptome Profiling Reveals a Crucial Developmental Switch with Roles for RNA Processing and Organelle Metabolism That Are Essential for Germination in Arabidopsis1[W][OA]

Plant Physiol. 2011 November;157(3):1342-1362.

Figure 5.

GFP analysis confirms the localization of proteins encoded by genes expressed during germination. A subset of 65 genes predicted to encode organelle-targeted proteins were analyzed to determine subcellular localization. Only those genes most highly expressed during germination (i.e. showing low expression in dry seed and postgermination [48 h SL]) were selected for analysis. A, Examples of fluorescence images for proteins targeted to the mitochondria, plastids, and peroxisomes. The images for every protein identified as targeting to an organelle can be found in Supplemental Figure S5. Fusion proteins containing GFP and the first 100 amino acids (AA) of the protein of interest (or the last 100 amino acids in the case of the peroxisomal predicted proteins) were constructed and transiently transformed into cell culture using biolistic transformation. Organelle targeting was verified using AOX-RFP as a mitochondrial control, SSU-RFP as a plastid control, and RFP-SRL as a control for peroxisomal targeting. B, A heat map showing expression levels during germination for the 65 genes analyzed by GFP targeting. The Arabidopsis Gene Identifier for each gene, brief gene symbol/description, and localization determined by GFP are shown next to the heat map. Predicted localizations are indicated in parentheses (M, mitochondria; Pl, plastids; Per, peroxisomes) next to the experimentally determined localization. Note that genes that were defined as most highly expressed during germination in Figure 3a are indicated by carets. Known phenotypes when the gene is knocked out/silenced are as follows: defective in flower (F), stress (St), embryo (ED), growth (G), and seedling (SL). Reference for phenotypes are as follows: ¹ Boavida et al. (2009); ² Kwak et al. (2011); ³ Ding et al. (2006); ⁴ Keren et al. (2009); ⁵ Hilson et al. (2004); ⁶ Hammani et al. (2011); ⁷ Meinke et al. (2008); ⁸ Kuromori et al. (2006); ⁹ Chen et al. (2010); ¹⁰ Saiga et al. (2008); ¹¹ Osakabe et al. (2005). AGI numbers in gray indicate those annotated as involved in RNA binding/processing.

In-Depth Temporal Transcriptome Profiling Reveals a Crucial Developmental Switch with Roles for RNA Processing and Organelle Metabolism That Are Essential for Germination in Arabidopsis1[W][OA]

Plant Physiol. 2011 November;157(3):1342-1362.