Abstract
Plant growth is regulated by internal and external cues using a limited number of genes coded in the genome. Comparative transcriptome analysis often provides an indication of which particular sets of genes are co-regulated in different physiological events. We have recently reported that protein synthesis-related genes were highly overrepresented in the genes upregulated during germination in Arabidopsis. In particular, these included ∼100 ribosomal protein (RP) genes. The promoters of these genes had a higher frequency of two cis elements, designated as Up1 and Up2. Up1 is almost identical to the target cis element of TCP transcription factors. We also found that a similar set of RP genes is upregulated during decapitation-induced axillary shoot outgrowth. Thus, Up1-mediated activation of RP genes appears to be a common mechanism for growth induction in axillary shoots and imbibed seed. Moreover, co-regulation of a large number of RP genes suggests that these genes are useful markers to monitor growth states for microarray analyses.
Key words: Arabidopsis, axillary shoot, cis element, co-regulation, growth activation, microarray analysis, ribosomal protein genes, seed, TCP transcription factors, genome-wide gene expression analysis
Identification of Unidentified Roles of Transcription Factors by Gene Expression Profiling
The adjustment of growth rate to the environment is a fundamental aspect of the life cycle of many organisms. Genetic analysis has contributed greatly to the discovery of the functions of genes involved in the regulation of plant growth and development. However, it is often difficult to explain gene function in relation to mutant phenotype. Microarray-based expression analysis provides a highly sensitive method in molecular phenotyping of a mutant by revealing expression patterns of 20–40 K genes. Although transcriptional regulation is not necessarily the limiting regulatory mechanism of every biological event, genome-wide expression analyses lead us more frequently to an unexpected link between the visible phenotype and the corresponding mechanisms. Moreover, current advances in the technology for the isolation of specific cell types through cell sorting or laser microdissection enable us to analyze gene expression patterns in a particular cell type.1,2 We are interested in whether genome-wide expression patterns reveal traits of their regulatory mechanism, which can be seen as enriched cis elements in their promoters.3–5 To test this, we carried out gene expression profiling based on the enriched cis elements in the promoters of genes, whose mRNA levels were altered during Arabidopsis seed germination.3–4
We firstly analyzed the enriched cis elements in the promoters of the genes for stored mRNA in dry seed and found that abscisic acid-responsive element (ABRE)-like sequences were overrepresented in their promoters.3 These contain ACGT core sequences that are the typical target sequence for bZIP transcription factors including ABSCISIC ACID-INSENSITIVE5 (ABI5), an essential regulator for seed ABA responses.6,7 The enrichment of these ABRE-like sequences disappeared in the transcriptomes of imbibed seed, which correlated with the reduction in endogenous ABA levels. Moreover, this enrichment was severely diminished in the promoters of highly expressed genes in the abi5 mutant dry seeds.3 Based on these analyses, we concluded that the enrichment of these elements reflected a prominent role of ABI5 in determining the transcriptomes of stored mRNA. We also found that these ABREs highly co-localized with elements similar to RY repeats and the coupling element (CE1), the target cis elements for ABI3 and ABI4 transcription factors, respectively.3 These pilot experiments suggest that a collection of promoter sequences from co-regulated genes contains useful information to identify the unidentified regulatory elements.
In order to search for unidentified cis elements regulating seed germination, we carried out in silico promoter analysis on the upregulated genes after imbibition.4 As a result, two putative cis elements, Up1 and Up2, were found to be overrepresented significantly in the 500-base upstream regions of the upregulated genes. The synthetic promoter harboring the tandem repeats of Up1 induced the reporter gene expression during germination, suggesting that Up1 is a functional cis element responsible for germination-associated gene expression. Up1 resembled the site II motif, which is the target sequence for TCP transcription factors.5,8–10 Microarray expression data suggested at least 12 AtTCP genes were expressed in imbibed seed. The AtTCP14, which showed the highest transcript levels in imbibed seed, were chosen for functional analysis by reverse genetics. The attcp14 mutants showed delayed and ABA-hypersensitive germination. Furthermore, the AtTCP14 mRNA was predominantly localized in the vascular tissue of the embryo, suggesting that AtTCP14 is the positive regulator of embryonic growth potential. Notably, regardless of the significant contribution of Up1-mediated gene expression to the germination-associated transcriptome, the phenotypes of the attcp14 mutants were subtle. This might be attributed to the redundant function of other AtTCP genes. The role of AtTCP14 cannot be elucidated without a prediction of its involvement by gene expression profiling.
Co-Regulation of Ribosimal Protein Genes in Growing Axillary Shoots and Germinating Seed
Our previous report indicated that Up1 induces the protein synthesis-and cell division-related gene expression during axillary shoot outgrowth.5 In the report selected for the Addendum,4 we showed that this element also regulates the protein synthesis-related genes during seed germination. Most of these genes are shared in both shoots and seeds. In particular, a large number of ribosomal protein (RP) genes are included in these protein synthesis-related genes. After seed imbibition, induction of protein synthesis is an important biological event for germination.11 Similar physiological events are observed in pea axillary buds after decapitation of the apical bud.12 Taking these matters into consideration, we proposed that Up1-mediated transcriptional regulation of RP genes is a conserved regulatory mechanism of growth activation in axillary shoots and germinating seed.
The Arabidopsis genome contains 249 RP genes corresponding to 80 distinct types of cytoplasmic ribosome (32 genes for 40S subunits and 48 genes for 60S subunits).13 Co-expression analysis on a large amount of publicly available microarray data showed that expression of RP genes is not constitutive, but is highly co-regulated.14 Consistent with such co-regulated expression, ∼70% of Arabidopsis RP genes contain both the site II motif (Up1) and telo-box (Up2) in their promoters.10 Our microarray analysis indicated that 148 genes and 50 RP genes were upregulated in germinating seed and growing axillary shoot, respectively.4–5 In general, RP gene expression tends to be more active in young tissues than in mature tissues. Published microarray data indicates that RP mRNA levels are high in the early stage of microphore development and decrease during pollen development.15 Transcript levels are high in the root tip and elongation zone.16 On the other hand, anoxia, drought stress and sucrose starvation appear not to affect their mRNA levels, but do affect the association of RP mRNA with polysomes.17–19 Co-expression of the large number of RP genes easily accounts for the biased GO functional categories in the co-regulated genes. We also note that the change in the expression patterns of RP genes appears to be tissue specific.4,5 Therefore, microarray expression analysis using tissue selected by cell sorting or laser microdissection will make these genes more sensitive markers for growth regulation.
Microarray analysis generates a massive amount of biological data, but it can be difficult to interpret. To understand these data in a systematic way, it is crucial to define groups of genes in a methodical manner. One possible way of grouping genes is by their cis elements. Grouping genes in this way often gives us biased GO functional categories (Table 1). Alternatively, co-regulation of gene expression can be grouped by functional categories. RP genes are highly co-expressed and share the common cis elements, Up1 and Up2. Thus, this set of genes fulfils both criteria. Moreover, the possible link of their expression patterns to the state of growth suggests that RP genes are useful markers to interpret plant growth status from microarray expression data.
Table 1.
Enrichment of particular functional categories on gene ontology in the gene sets containing ABRE, Up1 and Up2
| Classification | All genes | ABRE | Up1 | Up2 |
| Metabolism | 25.22% | 32.27% | 22.66% | 16.32% |
| Energy | 1.92% | 2.87% | 2.54% | 1.95% |
| Cell cycle and DNA processing | 7.66% | 7.82% | 11.66% | 12.30% |
| Transcription | 13.07% | 13.90% | 13.55% | 20.40% |
| Protein synthesis | 2.91% | 2.73% | 10.18% | 9.64% |
| Protein fate | 5.64% | 6.36% | 7.28% | 5.44% |
| Transport | 2.20% | 2.83% | 1.66% | 1.36% |
| Cellular communication | 5.90% | 4.10% | 3.02% | 4.02% |
| Stress-related | 3.87% | 3.39% | 2.01% | 1.71% |
| Cell fate and development | 2.43% | 2.97% | 1.95% | 2.72% |
| Localization | 1.48% | 1.84% | 1.78% | 1.42% |
| Others | 3.17% | 1.46% | 1.24% | 2.07% |
| Unclassified | 24.53% | 17.48% | 20.47% | 20.64% |
| Total | 100.00% | 100.00% | 100.00% | 100.00% |
Each gene set was classified into 13 categories as described in Nakabayashi et al.3 Number in each gene set is as follows; All genes: 22,749. ABRE: 2,123, Up1: 1,690, Up2: 1,691. Significant difference compared with ‘All genes’ is indicated by bold.
Abbreviations
- TCP
TEOSINTE BRANCHED1, CYCLOIDEA and PCF
- RP
ribosomal protein
Footnotes
Previously published online as a Plant Signaling & Behavior E-publication: http://www.landesbioscience.com/journals/psb/article/5577
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