DataSheet_1_Changes in Medicago truncatula seed proteome along the rehydration–dehydration cycle highlight new players in the genotoxic stress response.pdf

IntroductionSeveral molecular aspects underlying the seed response to priming and the resulting vigor profile are still poorly understood. Mechanisms involved in genome maintenance deserve attention since the balance between stimulation of germination and DNA damage accumulation versus active repair is a key determinant for designing successful seed priming protocols.MethodsChanges in the Medicago truncatula seed proteome were investigated in this study, using discovery mass spectrometry and label-free quantification, along the rehydration-dehydration cycle of a standard vigorization treatment (hydropriming plus dry-back), and during post-priming imbibition.Resuts and discussionFrom 2056 to 2190 proteins were detected in each pairwise comparison, among which six were differentially accumulated and 36 were detected only in one condition. The following proteins were selected for further investigation: MtDRP2B (DYNAMIN-RELATED PROTEIN), MtTRXm4 (THIOREDOXIN m4), and MtASPG1 (ASPARTIC PROTEASE IN GUARD CELL 1) showing changes in seeds under dehydration stress; MtITPA (INOSINE TRIPHOSPHATE PYROPHOSPHORYLASE), MtABA2 (ABSCISIC ACID DEFICIENT 2), MtRS2Z32 (SERINE/ARGININE-RICH SPLICING FACTOR RS2Z32), and MtAQR (RNA HELICASE AQUARIUS) that were differentially regulated during post-priming imbibition. Changes in the corresponding transcript levels were assessed by qRT-PCR. In animal cells, ITPA hydrolyses 2’-deoxyinosine triphosphate and other inosine nucleotides, preventing genotoxic damage. A proof of concept was performed by imbibing primed and control M. truncatula seeds in presence/absence of 20 mM 2’-deoxyinosine (dI). Results from comet assay highlighted the ability of primed seeds to cope with dI-induced genotoxic damage. The seed repair response was assessed by monitoring the expression profiles of MtAAG (ALKYL-ADENINE DNA GLYCOSILASE) and MtEndoV (ENDONUCLEASE V) genes that participate in the repair of the mismatched I:T pair in BER (base excision repair) and AER (alternative excision repair) pathways, respectively.

种子对预浸和由此产生的活力轮廓背后的若干分子层面仍处于理解不足的状态。涉及基因组维护的机制值得关注，因为激发萌发与DNA损伤积累之间的平衡，以及主动修复的激活，是设计成功种子预浸方案的至关重要因素。
方法在本研究中，通过发现质谱和免标记定量分析，研究了在标准活力化处理（水预浸加干燥回潮）的再水化-脱水循环期间以及预浸后吸水阶段，黄芪草（Medicago truncatula）种子蛋白质组的变化。
结果与讨论在每个成对比较中检测到2056至2190种蛋白质，其中6种在特定条件下积累不同，36种仅在一种条件下检测到。以下蛋白质被选为进一步研究：在脱水应激下表现出变化的MtDRP2B（DYNAMIN-RELATED PROTEIN）、MtTRXm4（THIOREDOXIN m4）和MtASPG1（ASPARTIC PROTEASE IN GUARD CELL 1）；在预浸后吸水阶段差异调节的MtITPA（INOSINE TRIPHOSPHATE PYROPHOSPHORYLASE）、MtABA2（ABSCISIC ACID DEFICIENT 2）、MtRS2Z32（SERINE/ARGININE-RICH SPLICING FACTOR RS2Z32）和MtAQR（RNA HELICASE AQUARIUS）。通过qRT-PCR评估了相应转录水平的改变。在动物细胞中，ITPA水解2'-脱氧肌苷三磷酸和其他肌苷核苷，防止致毒损伤。通过将预浸和对照黄芪草种子在含有/不含有20 mM 2'-脱氧肌苷（dI）的条件下吸水，进行了一个概念验证实验。彗星实验的结果突出了预浸种子应对dI诱导的致毒损伤的能力。通过监测参与BER（碱基切除修复）和AER（替代切除修复）途径中不匹配I:T对修复的MtAAG（ALKYL-ADENINE DNA GLYCOSILASE）和MtEndoV（ENDONUCLEASE V）基因的表达谱，评估了种子修复反应。