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Title: Decline in RNA integrity of dry-stored soybean seeds correlates with loss of germination potential.

Author
item Fleming, Margaret
item Richards, Christopher
item Walters, Christina

Submitted to: Journal of Experimental Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/30/2017
Publication Date: 4/12/2017
Citation: Fleming, M.B., Richards, C.M., Walters, C.T. 2017. Decline in RNA integrity of dry-stored soybean seeds correlates with loss of germination potential. Journal of Experimental Botany. 68: 2219-2230. doi: 10.1093/jxb/erx100.

Interpretive Summary: Seeds deteriorate during storage in a process called aging and the rate that aging occurs is often unpredictable. Monitoring seed viability to detect the progress of aging is one of the most expensive aspects of seed banking and viability monitoring can unnecessarily deplete seed samples. Our goal is to develop tools that detect lost viability of stored seeds without having to use large quantities of seeds in a germination assay. This research reports on a promising new marker of seed deterioration that involves assessments of RNA integrity. RNA molecules are more labile than DNA, and show signs of fragmentation contemporaneously with loss of seed viability (unlike DNA, which stays intact long after the seed has died). Using a unique collection of soybean seed cohorts stored for up to 30 years, we were able to demonstrate a chemical signature of deterioration. This research may lead to improved assessments of seed quality as well as more economical seed banking procedures.

Technical Abstract: This study investigates the relationship between germination ability and damage to RNA in soybean seeds (cv “Williams ‘82”) stored dry at 5 oC for 1 – 27 years. Total germination of 14 age cohorts harvested between 2015 and 1989 ranged from 100 to 3%. Germination decline followed classic seed viability kinetics, with symptomatic seed aging beginning after 17 years of storage. RNA integrity was assessed by electrophoresis of total RNA, followed by calculation of the RNA Integrity Number (RIN, Agilent Bioanalyzer software), which evaluates the distribution of RNA fragment sizes. Analysis of RNA extracted from cotyledons, embryonic axes, plumules and seed coats across the range of age cohorts showed a consistent pattern of RNA degradation: older seeds had over-representation of small RNAs compared to younger seeds, which had nearly a 2:1 ratio of 25S and 18S rRNAs. RIN values for cotyledons and embryonic axes from the same seed were correlated. Decline in RIN followed a similar trend as reduced germination, with a pronounced decrease in RIN after 17 years of storage. This led to a high correlation between the mean RIN of cotyledon RNA and total germination % (R2 = 0.91, p < 0.0001). Despite this relationship, germinable and non-germinable seeds within cohorts could not be distinguished unless the RIN was less than 3.5, indicative of high deterioration. Our work demonstrates that seed RNA incurs damage over time, detectable as a change in the size distribution of fragments. Under the experimental conditions used here, RIN appears to be an excellent surrogate for germination tests used to monitor viability of stored seeds.