Introduction

Droughts have devastating impacts on crop production and food security. In this respect, developing crops with increased drought tolerance is a major focus for plant breeders and researchers. Whilst most plants can withstand mild drought for short periods of time, loss of water content below 40% results in extensive damage and ultimately death (Höfler & Rottenburg, 1941). A small group of plants (< 0.2% of the total flora) termed ‘resurrection plants’ are unique in that they can survive long periods of time desiccated with water content < 10% and recover within hours upon hydration (Oliver et al., 2005, 2020). Whilst desiccation tolerance is observed in ferns, mosses, pollen and orthodox seeds, desiccation tolerance within angiosperm vegetative tissues is a rare phenomenon (Gaff & Oliver, 2013). Consequently, resurrection plants are valuable models to study the molecular mechanisms involved in desiccation tolerance and a comprehensive knowledge of the fundamental mechanisms involved is crucial. The Balkan endemic Haberlea rhodopensis can survive unusually long periods of desiccation for ≤ 2 yr and resume normal growth within hours of hydration (Gechev et al., 2013).

Several studies have investigated the physiological, cellular and molecular mechanisms involved in establishing desiccation tolerance. Specialized mechanisms minimize damage and maintain cellular integrity during desiccation and rapidly mobilize cellular function and repair mechanisms upon rehydration (Oliver et al., 2020). Among the core protective mechanisms are the accumulation of late embryogenesis abundant (LEA) proteins, small heat shock proteins (sHSPs) and early light-induced protein (ELIP) (reviewed by Gechev et al., 2021), and osmolytes such as proline (Forlani et al., 2019), proposed to protect proteins from dehydration and aggregation. Sucrose and oligosaccharides accumulate and act as osmoprotectants to stabilize membranes (Martinelli, 2008; Djilianov et al., 2011; Moyankova et al., 2014).

Nonenzymatic and enzymatic antioxidant systems also are activated to establish desiccation tolerance. Located within the energy-producing organelles, mitochondria and chloroplasts, antioxidant molecules such as glutathione, ascorbate, tocopherols and polyphenols have been observed to increase during desiccation (Djilianov et al., 2011; Moyankova et al., 2014; Georgieva et al., 2017). Scavengers such as superoxide dismutase, ascorbate peroxidase, catalase and glutathione reductase also accumulate during desiccation (Gechev et al., 2013), probably to prevent reactive oxygen species (ROS) damage.

The specific role of chloroplasts (and photosynthesis) has been determined during the desiccation of several resurrecting species (Koonjul et al., 2000; Dinakar et al., 2012; Mladenov et al., 2015; Georgieva et al., 2020; Nadal et al., 2021). Inhibition of photosynthesis is a central response, observed in both desiccation-tolerant and desiccation-sensitive plants affected by drought (Challabathula et al., 2018). Desiccation of sensitive plants leads to irreparable damage of the photosynthetic membranes, however, in desiccation-tolerant resurrecting plants, the photosynthetic apparatus is deactivated during desiccation, followed by complete recovery upon rehydration. Two mechanisms have been described for this process; (i) poikilochlorophyllous plants degrade chlorophyll (Chl) and the photosynthetic apparatus in a regulated manner requiring de novo synthesis during rehydration (Tuba et al., 1998) whilst (ii) homoiochlorophyllous plants preserve Chl and thylakoid membranes and instead initiate active protection mechanisms. The homoiochlorophyllous H. rhodopesis maintains chloroplast morphology, preserving the integrity of the thylakoid membrane, photosystems I (PSI) and II (PSII) and a high Chl content during desiccation, and initiate protective mechanisms during desiccation to prevent damage and maintain the integrity of the photosynthetic apparatus (Georgieva et al., 2020). Molecular responses protecting photosynthetic machinery include the upregulation of genes encoding early light-inducible proteins (ELIP), LEA, antioxidant enzymes and cell-wall modification enzymes (Gechev et al., 2013; Liu et al., 2018).

Plant mitochondria play an essential role in energy production, are a major nexus of carbon and nitrogen metabolism, and play critical roles linked to photosynthesis and in responses to oxidative and environmental stresses. Unlike photosynthesis, mitochondrial function during desiccation has not been well-studied in resurrection plants. Tuba et al. (1997) reported that mitochondrial respiration rates correlated to tissue water content in Xerophyta humilis (Tuba et al., 1997). Likewise, respiration rates declined only after drying to 40% relative water content (RWC) in the homoiochlorophyllous species Craterostigma wilmsii and Myrothamnus flabellifolius, whilst in the poikilochlorophyllous monocot Xerophyta humilis, respiration rates declined when RWC decreased to 20% and ceased in all three species at ≤ 10% (Farrant, 2000). Recently, a comprehensive transcriptomic, proteomic and metabolic study on Craterostigma plantagineum has provided some insight into the role of mitochondria in desiccation tolerance (Xu et al., 2021). RNA-Seq analysis data showed a high abundance of transcripts encoding mitochondrial protein import components TIM17 and TIM23, suggesting an upregulation of mitochondrial biogenesis during desiccation, in addition to an accumulation of tricarboxylic acid cycle (TCA) intermediates and oxidative phosphorylation (OXPHOS) machinery (Xu et al., 2021). These findings suggest mitochondrial biogenesis and activity may be maintained during desiccation, to minimize ROS damage and provide an energy advantage upon rehydration.

Here we investigated mitochondrial activity during the dehydration and subsequent rehydration of detached H. rhodopensis leaves with an emphasis on the role of mitochondria in desiccation tolerance. We found that mitochondrial respiration is established almost immediately upon rehydration and before the reactivation of photosynthesis. Transcript and protein abundance of proteins involved in mitochondrial biogenesis are high in desiccated leaves and remain constant during rehydration, along with fully assembled oxidative phosphorylation machinery. Furthermore, the alternative respiratory components and mitochondrial stress-responsive components were observed to be most abundant in desiccated tissues suggesting that specific mitochondrial mechanisms play a role in maintaining organelle integrity during desiccation and allow for rapid activation of function.

中文翻译：

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Haberlea rhodopensis 复活过程中的线粒体活性和生物发生

介绍

干旱对作物生产和粮食安全具有破坏性影响。在这方面，开发具有更高耐旱性的作物是植物育种者和研究人员的主要关注点。虽然大多数植物可以在短时间内承受轻度干旱，但水分含量低于 40% 的损失会导致广泛的损害并最终死亡 (Höfler & Rottenburg,  1941 )。一小群植物（< 总植物群的 0.2%）被称为“复活植物”，它们的独特之处在于它们可以在含水量 < 10% 的干燥环境中存活很长时间，并在水合后数小时内恢复（Oliver等人，  2005 年） , 2020). 虽然在蕨类植物、苔藓、花粉和正统种子中观察到干燥耐受性，但被子植物营养组织内的干燥耐受性是一种罕见现象 (Gaff & Oliver,  2013 )。因此，复活植物是研究干燥耐受性分子机制的宝贵模型，全面了解所涉及的基本机制至关重要。巴尔干地区特有的Haberlea rhodopensis可以在异常长时间的干燥中存活 ≤ 2 年，并在水合后数小时内恢复正常生长（Gechev等人，  2013 年）。

几项研究调查了建立干燥耐受性所涉及的生理、细胞和分子机制。专门的机制可最大限度地减少干燥过程中的损伤并保持细胞完整性，并在补液时迅速调动细胞功能和修复机制（Oliver等人，  2020 年）。核心保护机制包括晚期胚胎发生丰富 (LEA) 蛋白、小热休克蛋白 (sHSP) 和早期光诱导蛋白 (ELIP)（由 Gechev等人综述，2021 年）以及脯氨酸等渗透物的积累（ Forlani等人，  2019 年), 建议保护蛋白质免于脱水和聚集。蔗糖和低聚糖积累并充当渗透保护剂以稳定细胞膜（Martinelli，  2008 年；Djilianov等人，  2011 年；Moyankova等人，2014 年）。

非酶促和酶促抗氧化系统也被激活以建立干燥耐受性。谷胱甘肽、抗坏血酸、生育酚和多酚等抗氧化分子位于产生能量的细胞器、线粒体和叶绿体中，已被观察到在干燥过程中会增加（Djilianov等人，  2011 年；Moyankova等人，  2014 年；Georgieva等人，  2017 年）。超氧化物歧化酶、抗坏血酸过氧化物酶、过氧化氢酶和谷胱甘肽还原酶等清除剂也在干燥过程中积累（Gechev等人，  2013 年），可能是为了防止活性氧 (ROS) 损伤。

叶绿体（和光合作用）的具体作用已在几个复活物种的干燥过程中确定（Koonjul等人，  2000 年；Dinakar等人，  2012 年；Mladenov等人，2015 年；Georgieva等人，  2020 年；Nadal等人等人，  2021 年）。光合作用的抑制是一种主要反应，在受干旱影响的耐干燥和敏感植物中观察到（Challabathula等人，  2018 年）). 敏感植物的干燥会导致光合膜无法修复的损坏，然而，在耐干燥的复活植物中，光合装置在干燥过程中会失活，然后在补水后完全恢复。已经为这个过程描述了两种机制；(i) 杂叶绿素植物以一种受控方式降解叶绿素 (Chl) 和光合装置，需要在再水化过程中从头合成（Tuba等人，  1998），而 (ii) 同源叶绿素植物保留 Chl 和类囊体膜，并启动主动保护机制。同型叶绿素H. rhodopesis维持叶绿体形态，保持类囊体膜、光系统 I (PSI) 和 II (PSII) 的完整性以及干燥期间的高叶绿素含量，并在干燥期间启动保护机制以防止损坏并保持光合装置的完整性（Georgieva等人）等人，2020 年）。保护光合机制的分子反应包括上调编码早期光诱导蛋白 (ELIP)、LEA、抗氧化酶和细胞壁修饰酶的基因（Gechev等人，  2013 年；Liu等人，2018 年）。

植物线粒体在能量生产中起着至关重要的作用，是碳和氮代谢的主要联系，并在与光合作用以及对氧化和环境胁迫的反应中发挥关键作用。与光合作用不同，在复活植物中，干燥过程中的线粒体功能尚未得到充分研究。图巴等人。( 1997 ) 报道线粒体呼吸速率与旱生植物中的组织含水量相关(Tuba et al .,  1997 )。同样，只有在同型叶绿素物种Craterostigma wilmsii和Myrothamnus flabellifolius干燥至 40% 相对含水量 (RWC) 后，呼吸率才会下降，而在杂叶绿素单子叶植物旱生植物中，当 RWC 降至 20% 时呼吸速率下降，所有三个物种在 ≤ 10% 时呼吸速率均停止（Farrant，  2000）。最近，一项针对车前草的转录组学、蛋白质组学和代谢学的综合研究为线粒体在干燥耐受性中的作用提供了一些见解（Xu et al .,  2021）。RNA-Seq 分析数据显示大量转录本编码线粒体蛋白导入成分TIM17和TIM23，这表明除了三羧酸循环 (TCA) 中间体和氧化磷酸化 (OXPHOS) 机制的积累外，干燥过程中线粒体生物发生的上调 (Xu et al .,  2021 )。这些发现表明线粒体的生物发生和活性可以在干燥过程中保持，以最大限度地减少 ROS 损伤并在补液时提供能量优势。

在这里，我们研究了分离的H. rhodopensis脱水和随后再水化过程中的线粒体活性强调线粒体在耐干燥中的作用。我们发现线粒体呼吸作用几乎是在补水后和光合作用重新激活之前立即建立的。参与线粒体生物发生的蛋白质的转录本和蛋白质丰度在干燥的叶子中很高，并且在再水化过程中保持不变，以及完全组装的氧化磷酸化机制。此外，观察到替代呼吸成分和线粒体应激反应成分在干燥组织中最为丰富，这表明特定的线粒体机制在干燥过程中维持细胞器完整性并允许快速激活功能方面发挥作用。