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. 2007 Apr;143(4):1669-79.
doi: 10.1104/pp.107.096057. Epub 2007 Feb 23.

Gene expression profiling reveals defined functions of the ATP-binding cassette transporter COMATOSE late in phase II of germination

Esther Carrera  1 , Tara Holman, Anne Medhurst, Wendy Peer, Heike Schmuths, Steven Footitt, Frederica L Theodoulou, Michael J Holdsworth
Affiliations

Gene expression profiling reveals defined functions of the ATP-binding cassette transporter COMATOSE late in phase II of germination

Esther Carrera et al. Plant Physiol. 2007 Apr.

Abstract

Phase II of germination represents a key developmental stage of plant growth during which imbibed seeds either enter stage III of germination, completing the germination process via radicle protrusion, or remain dormant. In this study, we analyzed the influence of the peroxisomal ATP-binding cassette transporter COMATOSE (CTS) on the postimbibition seed transcriptome of Arabidopsis (Arabidopsis thaliana) and also investigated interactions between gibberellin (GA) and CTS function. A novel method for analysis of transcriptome datasets allowed visualization of developmental signatures of seeds, showing that cts-1 retains the capacity to after ripen, indicating a germination block late in phase II. Expression of the key GA biosynthetic genes GA3ox1 and 2 was greatly reduced in cts seeds and genetic analysis suggested that CTS was epistatic to RGL2, a germination-repressing DELLA protein that is degraded by GA. Comparative analysis of seed transcriptome datasets indicated that specific cohorts of genes were influenced by GA and CTS. CTS function was required for expression of the flavonoid biosynthetic pathway. Confocal imaging demonstrated the exclusive accumulation of flavonoids in the epidermis of wild-type seeds. In contrast, flavonoids were absent from cts and kat2-1 mutant seeds, but accumulated following the application of sucrose, indicating an essential role for beta-oxidation in inducing flavonoid biosynthetic genes. These results demonstrate that CTS functions very late in phase II of germination and that its function is required for the expression of specific gene sets related to an important biochemical pathway associated with seedling establishment and survival.

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Figures

Figure 1.
Figure 1.
Germination characteristics of wild-type and cts-1 mutant seeds. A, Germination potential of freshly harvested or S (AR) wild-type and cts-1 mutant seeds used for microarray analysis. Germination was assessed at 1 d (24 h) and 7 d on WA media. Values shown are averages (+sd) of triplicate experiments using seeds derived from different plants. B, Frequency of testa (black circles, dotted line) and endosperm (white circles, solid line) rupture of AR Ler wild-type seeds. Values shown are average of triplicate experiments using seeds derived from different plants. Error bars indicate sd.
Figure 2.
Figure 2.
PCA applied to seed transcriptome datasets. Black circles indicate Ler wild-type D or AR seeds; white circles cts-1 mutant F or S seeds.
Figure 3.
Figure 3.
Analysis of transcriptome profiles using the GO molecular function or TAGGIT approaches. A, Ontological classification of genes in the Ler D > Ler AR or Ler AR > Ler D sets using GO molecular function. The proportional representation of the total gene set is shown in each case; genes of unknown function using this methodology are colored white. B, Flow diagram illustrating the workflow used to reannotate gene lists using the TAGGIT macro. C, Classification of genes in the Ler D > Ler AR or Ler AR > Ler D sets using the TAGGIT macro. The proportional representation of the total gene set is shown in each case, TAIR6 annotated genes of no known function are shown in gray, TAGGIT unannotated genes in white, and TAGGIT annotated genes in black, expanded by the side to show proportional representation of individual classes. Datasets derived from Supplemental Table S1. In each case, > indicates the up-regulated gene set in the comparison.
Figure 4.
Figure 4.
Comparisons of developmental signatures of differentially expressed transcriptomes. A, Comparison of genes differentially expressed in Ler AR and D 24-h imbibed seed samples. B, Comparison of genes differentially expressed in accession Cvi AR and D samples (Cadman et al., 2006). Dataset obtained from NASCarrays. C, Comparison of genes differentially expressed from seeds imbibed for 0 to 24 h (Nakabayashi et al. 2005). According to the design of the reported experiments, early time points were compared to unimbibed (dry) seeds, later time points to 0-h imbibed seeds. D, Comparison of genes differentially expressed in cts-1 S and F 24-h imbibed seed samples. E, Comparison of genes differentially expressed in cts-1 S and Ler AR 24-h imbibed seed samples. Each graph shows gene-set name and the proportional representation of the genes identified using the TAGGIT workflow (see Table I for total numbers of differentially expressed genes; Fig. 3 for key to gene groups). Percentage of genes identified using the TAGGIT workflow in each comparison are given in corresponding Supplemental figures. In each case, > indicates the up-regulated gene set in the comparison.
Figure 5.
Figure 5.
Gene expression profiles during 48 h following the initiation of imbibition for Ler and cts-1 seeds. Quantitative RT-PCR analysis was carried out using RNA obtained from Ler D and AR and cts-1 S and F seeds imbibed on WA over 48 h. AGI codes and names of encoded proteins are indicated for each transcript. In each case, the expression characteristics of genes in transcriptome datasets at 24-h imbibition are given as titles above graphs. A, Expression of CTS. B, Transcripts showing no change in expression in cts-1 S or F seeds. C, Transcripts differentially regulated in cts-1 S or F seeds. D, Transcripts showing highly induced expression in Ler AR seeds only.
Figure 6.
Figure 6.
Germination potential of cts-1 rgl2-1 seeds and expression of GA biosynthesis genes in wild-type and cts-1 seeds. A, Germination potential after 7 d is shown for freshly harvested and S/AR seed lots assayed on WA media either with or without exogenous PAC (10 μm). Data represent mean and sd. B, Quantitative RT-PCR analysis of genes encoding AtGA3ox1 and 2 in Ler and cts-1 seeds. RNA was obtained from Ler D and AR and cts-1 S and F seeds imbibed on WA over 48 h. AGI numbers and function of encoded proteins are indicated for each transcript.
Figure 7.
Figure 7.
Comparison of transcriptomes up-regulated by GA4 treatment of ga1-3 seeds and up-regulated in Ler in comparison to cts-1. A, Venn diagram resulting from comparison of GA4 up-regulated gene set (229 genes) with the Ler AR > cts-1 S gene set (465 genes, 279 not on the Affymetrix 8K Genechip). Transcriptome dataset for GA4 treatment of ga1-3 seeds (GA up-regulated) was obtained from Ogawa et al. (2003). Differential gene expression is represented by Venn diagrams. Original developmental signatures and total numbers of genes from these gene sets are shown next to the Venn diagram. Numbers of genes represented in both gene sets are shown within the intersections of the two sets. B, Representations of the developmental signatures of genes from the different Venn diagram sets. Specific functional groups are highlighted with white (increased representation in the CTS set) or black (increased representation in the GA set) arrows. Numbers next to arrows indicate absolute numbers of genes in each functional category of each set. Percentage of genes identified using the TAGGIT workflow in each comparison are given in Supplemental figures. Colors correspond to functional categories shown in Figure 3.
Figure 8.
Figure 8.
Expression of genes of the flavonoid biosynthesis pathway in wild-type and cts-1 seeds. A, Schematic representation of flavonoid biosynthesis (redrawn from Peer et al., 2001). Enzymes are shown with associated position in the Ler AR > cts-1 S seed gene set and fold differential up-regulation in AR seeds (see Supplemental Table S1). 4CL, 4-Coumarate-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; FLS, flavonol synthase; TT, transparent testa; PAP2, PRODUCTION OF ANTHOCYANIN PIGMENT 2; EGL3, ENHANCER OF GLABRA 3. B, Quantitative RT-PCR analysis of gene expression in wild-type Ler and cts-1 S seeds imbibed on WA.
Figure 9.
Figure 9.
Visualization of flavonoid accumulation in 24-h imbibed radicles of mutant and wild-type seeds. Orange fluorescence corresponds to quercetin-DPBA, green fluorescence to kaempferol-DPBA, and yellow fluorescence to colocalization of flavonoids. Respective wild-type seedlings are shown next to mutants (Ler, cts-1; Col-BM, cts-3, 5; Ws4, kat2-1). A, Bar represents 100 μm. B, Closeup of epidermis of Ler and cts-1. C, Visualization of flavonoids in Ler and cts-1 seed radicles incubated in the presence of 0.5% Suc. D, Quantitative RT-PCR analysis of flavonoid-associated gene expression in cts-1 seeds imbibed 24 h on WA in the presence of Suc (germination after 7 d: +Suc 11%, −Suc 0%).
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