Plant tissues subjected to short or prolonged freezing to a fixed sub-freezing temperature are expected to undergo similar freeze-desiccation but former causes substantially less injury than the latter. To gain metabolic insight into this differential response, metabolome changes in spinach (Spinacia oleracea L.) leaves were determined following short-term (0.5 and 3.0 h) vs. prolonged freezing (5.5 and 10.5 h) at -4.5°C resulting in reversible or irreversible injury, respectively. LD50 , the freezing duration causing 50% injury, was estimated to be ~3.1 h, and defined as the threshold beyond which tissues were irreversibly injured. From 39 identified metabolites, 19 were selected and clustered into 3 groups: (1) signaling-related (salicylic acid, aliphatic and aromatic amino acids), (2) injury-related (GABA, lactic acid, maltose, fatty acids, policosanols, TCA intermediates), and (3) recovery-related (ascorbic acid, α-tocopherol). Initial accumulation of salicylic acid during short-term freezing followed by a decline may be involved in triggering tolerance mechanisms in moderately injured tissues, while its resurgence during prolonged freezing may signal programmed cell death. GABA accumulated with increasing freezing duration, possibly to serve as a 'pH-stat' against cytoplasmic acidification resulting from lactic acid accumulation. Mitochondria seems to be more sensitive to prolonged freezing than chloroplast since TCA intermediates decreased after LD50 while salicylic acid and maltose, produced in chloroplast, accumulate even at 10.5-h freezing. Fatty acids and policosanols accumulation with increasing freezing duration indicates greater injury to membrane lipids and epicuticular waxes. Ascorbic acid and α-tocopherol accumulated after short-term freezing, supposedly facilitating recovery while their levels decreased in irreversibly injured tissues. This article is protected by copyright. All rights reserved.

中文翻译：

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短期和长期冷冻对菠菜叶冻融损伤的不同影响：通过代谢物分析的机制洞察

植物组织在固定的亚冰点温度下短期或长期冷冻，预计会经历类似的冷冻干燥，但前者造成的伤害远小于后者。为了获得对这种差异反应的代谢洞察，在 -4.5°C 下短期（0.5 和 3.0 小时）与长期冷冻（5.5 和 10.5 小时）后确定菠菜（Spinacia oleracea L.）叶的代谢组变化，导致可逆性或不可逆转的伤害，分别。LD50 是造成 50% 损伤的冷冻持续时间，估计约为 3.1 小时，并定义为组织不可逆损伤的阈值。从 39 种已鉴定的代谢物中，选择了 19 种并将其分为 3 组：（1）信号相关（水杨酸、脂肪族和芳香族氨基酸），（2）损伤相关（GABA、乳酸、麦芽糖、脂肪酸，多廿烷醇、TCA 中间体）和 (3) 恢复相关（抗坏血酸、α-生育酚）。短期冷冻期间水杨酸的初始积累随后下降可能与触发中度损伤组织的耐受机制有关，而长期冷冻期间水杨酸的重新出现可能表明程序性细胞死亡。GABA随着冷冻时间的增加而积累，可能作为“pH-stat”对抗由乳酸积累引起的细胞质酸化。线粒体似乎比叶绿体对长时间冷冻更敏感，因为 TCA 中间体在 LD50 后减少，而叶绿体中产生的水杨酸和麦芽糖即使在冷冻 10.5 小时时也会积累。随着冷冻持续时间的增加，脂肪酸和多廿烷醇的积累表明对膜脂和表皮蜡的更大伤害。抗坏血酸和 α-生育酚在短期冷冻后会积累，据说有助于恢复，而它们在不可逆损伤组织中的水平下降。本文受版权保护。版权所有。