Barley grain maturation and germination: metabolic pathway and regulatory network commonalities and differences highlighted by new MapMan/PageMan profiling tools

Abstract

Plant seeds prepare for germination already during seed maturation. We performed a detailed transcriptome analysis of barley (Hordeum vulgare) grain maturation, desiccation, and germination in two tissue fractions (starchy endosperm/aleurone and embryo/scutellum) using the Affymetrix Barley1 GeneChip. To aid data evaluation, Arabidopsis thaliana MapMan and PageMan tools were adapted to barley. The analyses allow a number of conclusions: (1) Cluster analysis revealed a smooth transition in transcription programs between late seed maturation and germination within embryo tissues, but not in the endosperm/aleurone. (2) More than 12,000 transcripts are stored in the embryo of dry barley grains, many of which are presumably activated during germination. (3) Transcriptional activation of storage reserve mobilization events occurs at an early stage of germination, well before radicle protrusion. (4) Key genes of gibberellin (GA) biosynthesis are already active during grain maturation at a time when abscisic acid peaks suggesting the formation of an endogenous store of GA in the aleurone. This GA probably acts later during germination in addition to newly synthesized GA. (5) Beside the well-known role of GA in gene activation during germination spatiotemporal expression data and cis-element searches in homologous rice promoters confirm an equally important gene-activating role of abscisic acid during this developmental period. The respective regulatory webs are linked to auxin and ethylene controlled networks. In summary, new bioinformatics PageMan and MapMan tools developed in barley have been successfully used to investigate in detail the transcriptome relationships between seed maturation and germination in an important crop plant.

Figure 1.

Key stages of grain development and germination used for transcriptome analysis. A, Grain appearance. B, Hierarchical cluster dendrogram of normalized transcript abundances from 32 experiments including biological replicates based on complete distance linkage. Two tissue fractions (E/A, brown; Em, blue) were analyzed during grain development (DAF; dark coloring) and germination (HAI; light coloring). Pericarp (P; green) was analyzed during grain development only.

Figure 2.

PageMan display of coordinated changes of gene categories activated during barley seed development and germination. A, Storage product accumulation and its mobilization. B, Selected major gene categories of primary and secondary metabolism pathways. Affymetrix Barley1 GeneChip normalized gene expression data collected from E/A- and Em-fractions during seed development (4, 8, 16, and 25 DAF) and seed germination (0, 24, 48, and 72 HAI) were subjected to overrepresentation analysis of functional categories using PageMan. Fisher's exact test was used to test whether significantly more genes in a given category at a given developmental point were up-regulated when normalized to their average expression. (Color scale is: red, significant enrichment of up-regulated genes; blue, significant depletion of up-regulated genes). In the display, the overrepresented MapMan functional categories are given by collapsing nonsignificant categories. The complete analysis and its display are provided in Supplemental Figure S2.

Figure 3.

MapMan Metabolism overview maps showing differences in transcript levels between late seed maturation (25 DAF) and seed germination (48 HAI) in two genetically distinct tissues. A, E/A-fraction. B, Em-fraction. Log₂ ratios for average transcript abundance based on two independent replicates of Affymetrix Barley1 GeneChip normalized gene expression data of E/A 25 DAF versus E/A 48 HAI and Em 25 DAF versus Em 48 HAI were calculated. The resulting file was loaded into the MapMan Image Annotator module to generate the metabolism overview map. On the logarithmic color scale ranging from −3 to 3, dark blue represents at least 6-fold higher gene expression during seed maturation in comparison to seed germination, and red represents 6-fold higher gene expression during seed germination in comparison to late seed maturation. Color saturates at an 8-fold or higher change. Significant coregulation is indicated with arrow marks (blue, 25 DAF seed maturation; red, 48 HAI seed germination). The complete set of genes, derived functional categories, normalized expression values, and calculated ratios are given in Supplemental Tables S3 and S4.

Figure 4.

Temporal expression profiles of transcripts involved in storage product mobilization. Storage mobilizing transcripts reported by Chen and An (2006) to be induced in barley aleurone during seed germination by GA or ABA were chosen. By using K-mean and SOM clustering methods, three cluster groups based on temporal expression patterns were identified, two related to GA and one related to ABA. Note that cluster group 1 genes are mostly present during both grain maturation and germination in the two tissue fractions Em and E/A, i.e. they are presumably already synthesized during maturation and become reactivated during germination. On the other hand, cluster group 2 transcripts are preferentially newly synthesized during germination. The same tendency is seen in cluster group 3 transcripts regulated by ABA. Expression values are given in logarithmically scaled (base 2) signal intensities: red, high expression; yellow, moderate expression; blue, low expression. Horizontal rows represent gene expression patterns. Vertical lines represent the developmental stages indicated on top for the two tissue fractions. The predicted function of genes and functional classifications are indicated on the right. The presence of cis-elements shown to be involved in GA or ABA response in 1-kb upstream regions of homologous rice genes is given in Supplemental Table S5.

Figure 5.

MapMan transport overview maps showing differences in transcript levels between late seed maturation (25 DAF) and germination (48 HAI) in both the E/A- and the Em-fraction. In the color scale, blue represents at least 6-fold higher gene expression during seed maturation in comparison to seed germination and red represents 6-fold higher gene expression during seed germination in comparison to late seed maturation. For further details, see legend of Figure 3. For a detailed gene list, normalized expression values and calculated ratios see Supplemental Tables S3 and S4.

Figure 6.

MapMan metabolic overview maps showing differences in transcript levels of osmoprotectant, antioxidant, and stress response genes between late seed maturation (25 DAF) and germination (48 HAI) in both the E/A- and the Em-fraction. For further details, see legend to Figure 5.

Figure 7.

A, PageMan display of coordinated changes of hormone biosynthesis gene categories (for further details, see legend to Fig. 2). B, MapMan hormone overview maps showing differences in transcript levels of hormone-related genes between late seed maturation (25 DAF) and germination (24 HAI; left side) in both the E/A- and the Em-fraction and during germination between 24 and 48 HAI (right side) in the same tissue fractions. For further details, see legend to Figure 5.

Figure 8.

Transcript levels of ABA (left) and GA (right) biosynthetic and signaling pathways integrated into a hypothetical scheme related to the induction of storage mobilizing transcripts. Expression values are given in logarithmically scaled (base 2) signal intensities. The Affymetrix Contig identification and predicted function of each gene is indicated. In the lower part, the presence of GA-responsive motifs (GARE, R1-MYB, and Pyrimidine box) and ABA-responsive motifs (ABRE) in the 1-kb upstream region of rice homologous storage mobilizing genes is indicated (for predicted cis-elements see Supplemental Table S5); for detailed expression of storage mobilizing genes see Figure 4. For details about motif specific enrichment see “Materials and Methods”.

Figure 9.

A, Expression profiles of TFs showing tissue- and development-specific patterns during the period of seed development and germination. Major clusters (1–6) obtained by K-mean clustering and subclusters 6(1) and 6(6) based on the SOM method (subclusters 6[1] to 6[6]) are indicated on the left by vertical bars; the number of genes (N) represented in each individual cluster/subcluster is on the right. Log₂ expression values are plotted visualized on the color scale: red, high expression; yellow, moderate expression; blue, low expression. Horizontal rows represent gene expression patterns; the developmental stage is assigned to vertical columns on the top. B, Overrepresented functional groups predicted based on Fisher's exact test in PageMan for every individual subcluster. C, TF genes responsive to exogenous GA and ABA (Chen and An, 2006) were used to associate temporal expression profiles obtained from seed development (DAF) and germination (HAI), which fall into two major cluster groups, 6(2) and 6(5). For a detailed gene list and normalized expression values, see Supplemental Table S8.