Seed aging has become a topic of renewed interest but its mechanism remains poorly understood. Our recent analysis of stored mRNA degradation in aged Arabidopsis seeds found that the stored mRNA degradation rates (estimated as the frequency of breakdown per nucleotide per day or β value) were constant over aging time under stable conditions. However, little is known about the generality of this finding to other plant species. We expanded the analysis to aged seeds of wheat (Triticum aestivum) and canola (Brassica napus). It was found that wheat and canola seeds required much longer periods than Arabidopsis seeds to lose seed germination ability completely under the same aging conditions. As what had been observed for Arabidopsis, stored mRNA degradation (∆Ct value in qPCR) in wheat and canola seeds correlated linearly and tightly with seed aging time or mRNA fragment size, while the quality of total RNA showed little change during seed aging. The generated β values reflecting the rate of stored mRNA degradation in wheat or canola seeds were similar for different stored mRNAs assayed and constant over seed aging time. The overall β values for aged seeds of wheat and canola showed non-significant differences from that of Arabidopsis when aged under the same conditions. These results are significant, allowing for better understanding of controlled seed aging for different species at the molecular level and for exploring the potential of stored mRNAs as seed aging biomarkers.

The ubiquitin-conjugating enzyme (E2) is required for protein ubiquitination. Arabidopsis has 37 E2s grouped into 14 subfamilies and the functions for many of them are unknown. We utilized genetic and biochemical methods to study the roles of Arabidopsis UBC4, UBC5, and UBC6 of the E2 subfamily IV. The Arabidopsis ubc4/5/6 triple mutant plants had higher levels of glucose, sucrose, and starch than the control plants, as well as a higher protein level of a key gluconeogenic enzyme, cytosolic fructose 1,6-bisphosphatase 1 (cyFBP). In an in vitro assay, the proteasome inhibitor MG132 inhibited the degradation of recombinant cyFBP whereas ATP promoted cyFBP degradation. In the quadruple mutant ubc4/5/6 cyfbp, the sugar levels returned to normal, suggesting that the increased sugar levels in the ubc4/5/6 mutant were due to an increased cyFBPase level. In addition, the ubc4/5/6 mutant plants showed early leaf senescence at late stages of plant development as well as accelerated leaf senescence using detached leaves. Further, the leaf senescence phenotype remained in the quadruple ubc4/5/6 cyfbp mutant. Our results suggest that UBC4/5/6 have two lines of important functions, in sugar metabolism through regulating the cyFBP protein level and in leaf senescence likely through a cyFBP-independent mechanism.