Data_Sheet_1_Distinct Gene Expression Patterns of Two Heat Shock Protein 70 Members During Development, Diapause, and Temperature Stress in the Freshwater Crustacean Daphnia magna.docx

Dormancy is a lifecycle delay that allows organisms to escape suboptimal environmental conditions. As a genetically programmed type of dormancy, diapause is usually accompanied by metabolic depression and enhanced tolerance toward adverse environmental factors. However, the drivers and regulators that steer an organism’s development into a state of suspended animation to survive environmental stress have not been fully uncovered. Heat shock proteins 70 (HSP70s), which are often produced in response to various types of stress, have been suggested to play a role in diapause. Considering the diversity of the Hsp70 family, different family members may have different functions during diapause. In the present study, we demonstrate the expression of two hsp70 genes (A and B together with protein localization of B) throughout continuous and diapause interrupted development of Daphnia magna. Before and after diapause, the expression of Dmhsp70-A is low. Only shortly before diapause and during diapause, Dmhsp70-A is significantly upregulated and may therefore be involved in diapause preparation and maintenance. In contrast, Dmhsp70-B is expressed only in developing embryos but not in diapausing embryos. During continuous development, the protein of this Hsp70 family member is localized in the cytosol. When we expose both embryo types to heat stress, expression of both hsp70 genes increases only in developing embryos, and the protein of family member B is translocated to the nucleus. In this stress formation, this protein provides effective protection of nucleoplasmic DNA. As we also see this localization in diapausing embryos, it seems that Daphnia embryo types share a common subcellular strategy when facing dormancy or heat shock, i.e., they protect their DNA by HSP70B nuclear translocation. Our study underlines the distinctive roles that different Hsp70 family members play throughout continuous and diapause interrupted development.

滞育（dormancy）是一种可使生物体逃离不适宜环境条件的生命周期延迟机制。作为一类由遗传程序调控的滞育（diapause）类型，滞育通常伴随代谢抑制以及对不良环境因子的耐受性增强。然而，驱动生物体发育进入停滞状态以抵御环境胁迫的调控因子与作用机制，目前尚未被完全阐明。热休克蛋白70（HSP70s）通常可响应多种胁迫并被诱导表达，已有研究提示其在滞育过程中发挥功能。鉴于Hsp70家族的多样性，不同家族成员在滞育过程中可能承担不同的生物学功能。在本研究中，我们针对大型溞（Daphnia magna）的连续发育与滞育中断发育过程，分析了两个hsp70基因（A与B）的表达模式，并解析了B蛋白的亚细胞定位情况。在滞育前后，Dmhsp70-A的表达量均处于较低水平；仅在滞育前短期以及滞育期间，Dmhsp70-A的表达量才会显著上调，因此该基因可能参与滞育的准备与维持过程。与之相反，Dmhsp70-B仅在发育中的胚胎中表达，而在滞育胚胎中无表达。在连续发育过程中，该Hsp70家族成员的蛋白定位于细胞质中。当对两种胚胎均施加热胁迫时，仅在发育中的胚胎中，两个hsp70基因的表达量均出现上调，且家族成员B的蛋白会易位至细胞核内。在此胁迫响应过程中，该蛋白可有效保护核质DNA。我们在滞育胚胎中同样观察到了该定位现象，这表明两种溞胚胎在面临滞育或热胁迫时，共享一套保守的亚细胞调控策略：即通过HSP70B的核易位来保护其DNA。本研究明确了不同Hsp70家族成员在连续发育与滞育中断发育过程中所发挥的独特功能。