Physiology and proteomics research on the leaves of ancient Platycladus orientalis (L.) during winter Sheng Zhang a , Lingling Zhang b , Yongyu Chai c , Fei Wang a,c , Yiming Li a , Li Su a , Zhong Zhao a, ⁎ a Key Laboratory of Environment and Ecology in Western China of Ministry of Education, College of Forestry, Northwest A&F University, Yangling, Shaanxi 712100, PR China b College of Forestry, Northwest A&F University, Yangling, Shaanxi 712100, PR China c Northwest Institute of Forest Inventory, Planning and Design, State Forestry Administration, Xi' an, Shaanxi 710048, PR China abstract article info Article history: Received 20 April 2015 Received in revised form 16 June 2015 Accepted 26 June 2015 Available online 6 July 2015 Keywords: Low-temperature stress Enzyme activity Ancient trees Leaf proteomics Ancient trees have an important value in humanities and history, and also have an important scientific value in the investigation of the decline and senescence mechanisms. Thus, we conducted an environmental stress study using ancient trees. To evaluate age-dependent changes in physiology and the leaf proteome, we assessed the low-temperature stress responses of 20 ± 5-, 500 ± 100- and 1200 ± 200-year-old Platycladus orientalis (L.) samples obtained outdoors during winter. Several physiological parameters were evaluated. Leaf proteomes were obtained using two-dimensional electrophoresis gels, and 77 protein spots were identified successfully using MALDI TOF/TOF MS/MS. The majority of the identified protein species were classified into functional categories including defense/stress-related, energy and carbohydrate metabolism, photosynthesis, and hormone-related functions. A general reduction in the abundance of protein species was observed as the age of the studied trees increased; reduction in photosynthesis and defense/stress-related categories were particular- ly apparent in the leaves of ancient trees. However, the number of protein species with functions in energy and carbohydrate metabolism increased with age. An increase in the abundance of lipid metabolism and hormone- related protein species was a primary characteristic of the leaves of ancient trees under low-temperature stress during winter. These results improve our understanding of the biochemical mechanisms of stress responses in ancient trees. Biological significance: Low temperature is the most common meteorological challenge in the study area. For evergreen plants, low-temperature stress has a great impact on the leaves of ancient P. orientalis. Thus, we conducted an environmental stress study using ancient trees. Recently, various studies were carried out in ancient trees. However, no information is available on the molecular mechanisms of defense to low- temperature stress in ancient trees. Therefore, this original study comprises the following differential proteomic analysis of ancient P. orientalis: (1) age-dependent changes in the physiology and leaf proteome are evaluated under low-temperature stress to (2) understand the differences in metabolic responses between ancient and adult trees under low-temperature stress during winter. This analysis will provide an understanding of the complex physiological changes that occur in ancient trees. The results suggest that certain identified proteins can be used as markers of low-temperature stress in ancient P. orientalis. © 2015 Elsevier B.V. All rights reserved. 1. Introduction In nature, temperature acts as a selective pressure for plant growth and limits the geographical distribution of many species [1]. Subzero temperature stress is a common and constant environ- mental stress that trees face during their life cycles, particularly trees growing in mountainous areas with higher altitudes during winter and early spring. For trees adapted to low temperatures, low (even subzero) temperatures induce gene expression and metabolic changes that allow the plant to tolerate and resist low-temperature stress. The leaves of evergreen plants have evolved a process known as cold acclimation to permit survival when exposed to low tempera- tures during winter. The accumulation of soluble sugars [2] and defense-related protein species and changes in gene expression associ- ated with tolerance to cellular dehydration [3] are some of the promi- nent alterations observed during cold acclimation. Compared with the low-temperature stress response in Platycladus orientalis, the differ- ences in the responses of adult and ancient trees to temperature stress remain elusive. Moreover, whether adult and ancient trees differ in their responses to stress remains unknown. Previous studies have shown that age-dependent decreases in biomass production commence Journal of Proteomics 126 (2015) 263–278 ⁎ Corresponding author. E-mail addresses: zhaozhlunwen2010@126.com, zhaozh@nwsuaf.edu.cn (Z. Zhao). http://dx.doi.org/10.1016/j.jprot.2015.06.019 1874-3919/© 2015 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Journal of Proteomics journal homepage: www.elsevier.com/locate/jprot