Gene expression programs during Brassica oleracea seed maturation, osmopriming, and germination are indicators of progression of the germination process and the stress tolerance level

Abstract

During seed maturation and germination, major changes in physiological status, gene expression, and metabolic events take place. Using chlorophyll sorting, osmopriming, and different drying regimes, Brassica oleracea seed lots of different maturity, stress tolerance, and germination behavior were created. Through careful physiological analysis of these seed lots combined with gene expression analysis using a dedicated cDNA microarray, gene expression could be correlated to physiological processes that occurred within the seeds. In addition, gene expression was studied during early stages of seed germination, prior to radicle emergence, since very little detailed information of gene expression during this process is available. During seed maturation expression of many known seed maturation genes, such as late-embryogenesis abundant or storage-compound genes, was high. Notably, a small but distinct subgroup of the maturation genes was found to correlate to seed stress tolerance in osmoprimed and dried seeds. Expression of these genes rapidly declined during priming and/or germination in water. The majority of the genes on the microarray were up-regulated during osmopriming and during germination on water, confirming the hypothesis that during osmopriming, germination-related processes are initiated. Finally, a large group of genes was up-regulated during germination on water, but not during osmopriming. These represent genes that are specific to germination in water. Germination-related gene expression was found to be partially reversible by physiological treatments such as slow drying of osmoprimed seeds. This correlated to the ability of seeds to withstand stress.

Figure 1.

Effect of seed maturity on stress tolerance and germination performance. Seeds were sorted based on chlorophyll (CF) content and subjected to a CD test to assess stress tolerance. More mature seeds have lower chlorophyll levels. The left section shows the number of normal seedlings obtained from the various seed fractions in a standard germination test, and the right section shows the number of normal seedlings obtained when seeds are first subjected to CD for 5 d and then germinated. The very high chlorophyll fraction (very high CF; AM) and the low chlorophyll fraction (FM) were used for microarray experiments.

Figure 2.

Effect of priming and drying treatments on germination performance. Germination performance was scored based on the percentage germination (A), the percentage normal seedlings (B), and the germination speed (T₅₀; C). Prior to the germination test seeds were subjected to 0 (left), 3 (middle), or 5 (right) d of CD.

Figure 3.

EST classification of the cDNA libraries. Distribution of the cDNAs identified in the three libraries over the various gene ontology classes.

Figure 4.

Clustering the samples based on their overall gene expression programs. A, Clustering based on all genes used in the analysis (1,100 random genes). B, Clustering based on the differentially expressed genes after averaging spots representing the same gene as listed in Tables I to IV. C, Average expression profiles of gene groups obtained by hierarchical clustering. Groups 1 to 5 are described in the text and Tables I to IV and contained 10, 13, 38, 33, and 10 genes, respectively.

Figure 5.

Expression of maturation-related gene RNA blots of two maturation/stress tolerance-related genes, Em6 (AT2G40170) and RAB18 (AT5G66400). Lane 1, Bent cotyledon stage seeds; lane 2, AM seeds; lane 3, dry seeds; lane 4, FM seeds; lane 5, CD-treated seeds; lane 6, seeds after 9 months storage; lane 7, osmoprimed seeds before drying; lane 8, osmoprimed seeds after fast drying; lane 9, osmoprimed seeds after slow drying; lane 10, germinating seeds 15 h after start of imbibition on water; lane 11, germinating seeds 30 h after start of imbibition on water; and lane 12, germinating seeds 45 h after start of imbibition on water.

Figure 6.

Gene expression during priming and germination. A, RNA blots of three genes that are expressed during osmopriming and water germination: dioxygenase (AT2G25450), nucleotide translocator (AT5G13490), and Ser carboxypeptidase (AT5G08260). B, RNA blots of two genes that are preferentially expressed during water germination: expressed protein (AT3G08030) and Glu ammonia ligase (AT5G37600). Lane 1, Bent cotyledon stage seeds; lane 2, AM seeds; lane 3, dry seeds; lane 4, FM seeds; lane 5, CD-treated seeds; lane 6, seeds after 9 months storage; lane 7, osmoprimed seeds before drying; lane 8, osmoprimed seeds after fast drying; lane 9, osmoprimed seeds after slow drying; lane 10, germinating seeds 15 h after start of imbibition on water; lane 11, germinating seeds 30 h after start of imbibition on water; and lane 12, germinating seeds 45 h after start of imbibition on water.