Background The soybean (Glycine max) cotyledon is a specialized tissue whose main function is to serve as a nutrient reserve that supplies the needs of the young plant throughout seedling development. During this process the cotyledons experience a functional transition to a mainly photosynthetic tissue. To identify at the genetic level the specific active elements that participate in the natural transition of the cotyledon from storage to photosynthetic activity, we studied the transcript abundance profile at different time points using a new soybean oligonucleotide chip containing 19,200 probes (70-mer long). Results After normalization and statistical analysis we determined that 3,594 genes presented a statistically significant altered expression in relation to the imbibed seed in at least one of the time points defined for the study. Detailed analysis of this data identified individual, specific elements of the glyoxylate pathway that play a fundamental role during the functional transition of the cotyledon from nutrient storage to photosynthesis. The dynamics between glyoxysomes and peroxisomes is evident during these series of events. We also identified several other genes whose products could participate co-ordinately throughout the functional transition and the associated mechanisms of control and regulation and we described multiple unknown genetic elements that by association have the potential to make a major contribution to this biological process. Conclusions We demonstrate that the global transcript profile of the soybean cotyledon during seedling development is extremely active, highly regulated and dynamic. We defined the expression profiles of individual gene family members, enzymatic isoforms and protein subunits and classified them accordingly to their involvement in different functional activities relevant to seedling development and the cotyledonary functional transition in soybean, especially the ones associated with the glyoxylate cycle. Our data suggests that in the soybean cotyledon a very complex and synchronized system of control and regulation of several metabolic pathways is essential to carry out the necessary functions during this developmental process. Keywords: Time Course
Overall design
Glycine max Williams dry seeds were planted per small pot containing pre-wetted Universal Mix SB300 soil in the green house. Two days after planting, Cotyledons from 10 imbibed seeds were collected and pooled, washed several times with distilled water, frozen in liquid nitrogen and freeze dried for 48 hours. Total RNA was extracted from the cotyledonary tissue and four different preparations from the same tissue were pooled and quantified. The imbibed cotyledon tissue (Stage 1) was used as the reference time point to compare a total of 6 more subsequent cotyledonary developmental stages (Stages 2-7) during the process of germination and emergence. A second set of seeds was planted at a different time and cotyledons from each stage were collected in a similar manner to obtain data from a biological replicate. A total of 20 µg of the reference Stage 1 RNA was used to be labeled by reverse transcription with Cy3-dUTP and compared to 20 µg of each of stages 2-7 labeled by reverse transcription with Cy5-dUTP following the protocol by Hedge et al., 2000. Two 70-mer Oligo chips containing 19,200 features were hybridized to compare differential transcript abundance between each stage and the reference. The dyes were swapped in two experimental replicates to avoid potential dye bias for a total of 4 Oligo chips per biological replicate. Since two biological replicates were set up, a total of 8 chips were used to compare transcript abundance per germination stage defined. The corresponding GPR files containing raw data were uploaded into GeneSpring 7.2 (Silicon Genetics, CA), transformed for dye swap and normalized by using the Per spot and Per chip intensity dependent (Lowess) normalization tool. Time was defined as the only parameter to be taken into account for analysis and 6 different conditions were defined: one for each time point. A Welch analysis of variance (ANOVA) of these genes in 48 samples with a P-value cutoff 0.05, a false discovery rate (FDR) of 0.05 and between 0.1 and 100 fold expression change in at least 1 out of 6 conditions, defined a gene list of 3,594 genes with statistical significant data. K-means clustering analysis on this list grouped genes with similar transcript abundance profiles during germination and emergence in 12 clusters using standard correlation as similarity measure
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