Identification of long-lived and stable mRNAs in the aged seeds of wheat

Seed germination relies on preserving mRNA integrity in dry seeds. However, the quality of mRNA in aged wheat seeds is not well understood. Here, we investigated 20 wheat varieties for seed longevity using controlled deterioration treatment (CDT) and identified that Chinese Spring seeds exhibit moderate longevity. We observed correlations between seed viability and RNA integrity in the aleurone and embryo cells after aging-treatment. Nanopore sequencing of whole seeds from natural aging treatment (NAT) and CDT for 25 days identified 3,083 full-length transcripts. We performed RNA-seq transcriptome profiling to classify the tissue origin of these transcripts under eight aging treatments, revealing the presence of 2,064 overlapping long-lived mRNAs (LLRs) in the seed embryo and 2,130 in the aleurone layers. These LLRs corresponded to genes with detectable transcription levels and at least one full-length transcript in their coding sequence. Notably, degradation percentages of these mRNAs varied among seeds of different wheat varieties with similar ages. We predicted 21 most stable LLRs with high GC% content and short coding sequence length, among which only one LLR was seed-specifically expressed and belonged to the late-embryogenesis-abundant (LEA) protein family. RT-PCR confirmed the expression of the seven LLR fragments in the aleurone layer and embryo of Chinese Spring seeds. We found three of the most stable LLRs (LLR13, LLR15, and LLR20) identified in Chinese Spring were more stable in high longevity varieties than in short longevity varieties after aging, indicating their potential roles in seed longevity and germination.