Studies of stored mRNAs during seed aging in Arabidopsis thaliana, Brassica napus and wheat (Triticum aestivum)
How plant seeds age remains poorly understood and effective tools for monitoring seed aging are lacking. Dry seeds contain various stored mRNAs which are believed to be required for protein synthesis during early stages of seed germination. We reasoned that seed stored mRNAs would undergo degradation during seed aging, based on previous research and the propensity of mRNAs to degrade. I performed RT-PCR, qPCR, and MinION nanopore sequencing analyses to study the changes in stored mRNAs of Arabidopsis seeds, and the hypothesis was approved by the following results. First, all stored mRNAs analyzed were gradually degraded in naturally and acceleratedly aged seeds. The difference in the threshold cycle (Ct) number of qPCR analysis between aged and control seeds (△Ct value) was highly correlated with the mRNA fragment size and seed aging time. Second, mathematical equations were derived for estimating the relative amount of undamaged stored mRNAs and frequency of the breakdown at the one nucleotide level for individual mRNAs. Third, results indicated that stored mRNAs were broken down randomly. The frequency of breaks per nucleotide per day, named as a β value, remained fairly constant over the aging time under the same aging conditions, but increased greatly with temperature. Fourth, RNA-seq analysis using MinION nanopore sequencing revealed a genome-wide trend of decreasing stored mRNA lengths in aged seeds. However, the changes were not as profound as observed with the qPCR analysis. Fifth, the method based the △Ct value reflecting stored mRNA change was found to be more precise than three existing methods for seed aging assessment. The findings in Arabidopsis were extended with similar observations in wheat and canola seeds. The △Ct value also highly correlated with the mRNA fragment size and seed aging time for these two crop seeds. Interestingly, the β values for the three plant species were generally similar suggesting comparable rates of stored mRNA degradation under the same conditions. These observations have raised interesting questions on the traditionally proposed mechanisms for seed aging and differences in seed aging among plants. Further, the methods developed here should be useful for studying stored mRNAs and seed aging.
Arabidopsis thaliana, real-time quantitative PCR (qPCR), seed aging, seed germination, stored mRNA, long-lived mRNA
Doctor of Philosophy (Ph.D.)