Table_1_Changes in Medicago truncatula seed proteome along the rehydration–dehydration cycle highlight new players in the genotoxic stress response.xlsx

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.

种子对预处理反应的分子基础及其所形成的活力轮廓中，诸多方面仍鲜为人知。鉴于在萌发刺激与DNA损伤积累之间的平衡是设计成功种子预处理方案的至关重要的决定因素，因此涉及基因组维持的机制值得特别注意。在本研究中，我们采用发现质谱和免标记定量技术，调查了在标准活力恢复处理（水预处理加干燥回吸）的再水化-脱水循环过程中，以及预处理后的吸水阶段，对Medicago truncatula种子蛋白质组的变化。结果表明，在每个成对比较中检测到2056至2190种蛋白质，其中6种在不同条件下积累差异显著，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的对象：在脱水胁迫下发生变化的MtDRP2B（DYNAMIN-RELATED PROTEIN）、MtTRXm4（THIOREDOXIN m4）和MtASPG1（ASPARTIC PROTEASE IN GUARD CELL 1）；在预处理后吸水阶段中差异调节的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变化。在动物细胞中，ITPA催化2'-脱氧肌苷三磷酸和其他肌苷核苷的水解，从而预防基因毒性损伤。通过将预处理和对照的M. truncatula种子在存在/不存在20 mM 2'-脱氧肌苷（dI）的条件下吸水，进行了一种概念验证实验。彗星实验的结果突出了预处理种子应对dI诱导的基因毒性损伤的能力。通过监测参与BER（碱基外切修复）和AER（替代修复）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。