USDA-ARS, National Laboratory for Genetic Resource Preservation, 1111 S. Mason Street, Fort Collins, CO 80521, USA.
J Exp Bot. 2017 Apr 1;68(9):2219-2230. doi: 10.1093/jxb/erx100.
This study investigates the relationship between germination ability and damage to RNA in soybean seeds (cv 'Williams 82') stored dry at 5 °C 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 in dry seeds by electrophoresis of total RNA, followed by calculation of the RNA integrity number (RIN, Agilent Bioanalyzer software), which evaluates RNA fragment size distributions. Analysis of RNA extracted from cotyledons, embryonic axes, plumules, and seed coats across the range of age cohorts showed consistent RNA degradation: older seeds had over-representation of small RNAs compared with 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 tracked 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 the total germination percentage (R2=0.91, P<0.0001). Despite this relationship, germinable and non-germinable seeds within cohorts could not be distinguished unless the RIN was <3.5, indicating substantial deterioration. Our work demonstrates that seed RNA incurs damage over time, observable in fragment size distributions. Under the experimental conditions used here, RIN appears to be a promising surrogate for germination tests used to monitor viability of stored seeds.
本研究调查了在 5°C 下干燥储存 1-27 年的大豆种子（品种 'Williams 82'）的发芽能力与 RNA 损伤之间的关系。在 2015 年至 1989 年收获的 14 个年龄组中，总发芽率从 100%到 3%不等。发芽率下降遵循经典的种子活力动力学，在储存 17 年后开始出现有症状的种子老化。通过总 RNA 的电泳评估干燥种子中的 RNA 完整性，然后计算 RNA 完整性数（RIN，Agilent Bioanalyzer 软件），该值评估 RNA 片段大小分布。对来自不同年龄组的子叶、胚胎轴、胚芽和种皮中提取的 RNA 进行分析表明，RNA 降解一致：与年轻种子相比，年老种子的小 RNA 含量过高，年轻种子的 25S 和 18S rRNA 比例接近 2:1。来自同一种子的子叶和胚胎轴提取的 RNA 的 RIN 值相关。RIN 值的下降与发芽率的降低相关，在储存 17 年后 RIN 值显著降低。这导致子叶 RNA 的平均 RIN 值与总发芽率之间存在高度相关性（R2=0.91，P<0.0001）。尽管存在这种关系，但在 RIN<3.5 的情况下，无法区分群体内的可发芽和不可发芽种子，表明种子严重恶化。我们的工作表明，种子 RNA 会随着时间的推移而受损，可在片段大小分布中观察到。在本研究中使用的实验条件下，RIN 似乎是监测储存种子活力的发芽试验的一个很有前途的替代指标。
