Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci Overexpression of Lsi1 in cold-sensitive rice mediates transcriptional regulatory networks and enhances resistance to chilling stress Changxun Fang a,c , Pengli Zhang a,c , Xin Jian a,c , Weisi Chen a,c , Hongmei Lin a,c , Yingzhe Li a,c , Wenxiong Lin a,b,c, ⁎ a Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 35002, PR China b Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, PR China c Key Laboratory of Crop Ecology and Molecular Physiology (Fujian Agriculture and Forestry University), Fujian Province University, Fuzhou 35002, PR China ARTICLE INFO Keywords: Chilling stress Rice Silicon OsWRKY53 Reactive oxygen species ABSTRACT Frequent cold spells in late spring can damage early rice seedlings. However, overexpression of the silicon- uptake gene Lsi1 (Lsi1-OX) in cold-sensitive rice (Oryza sativa L., accession: Dular) notably enhances its chilling resistance. In this study, we found that continual chilling led to chlorophyll and RNA degradation in wild-type Dular leaves, whereas leaves from a Lsi1-OX line exhibited no obvious changes. A comparison of the global mRNA expression between the two rice lines showed that genes encoding photosynthesis-antenna proteins were downregulated and those encoding the proteasome were upregulated in the wild-type organism. Moreover, the differential responses of the two rice lines to chilling stress were found to correlate with the transcription factor OsWRKY53, which was predicted target of the respective microRNA (miRNA) novel-m0586-5p. In addition, miRNAs that targeted genes involved in the process of reactive oxygen species (ROS) metabolism were differ- entially expressed in the two rice lines after chilling stress, when comparative analysis of the outcomes of RNA sequencing on the two rice lines. Our results suggest that when overexpressed Lsi1 in cold-sensitive rice, it possibility regulates the transcription factor OsWRKY53 in addition to the genes involved in the ROS metabo- lism, thus mediating resistance to chilling stress. 1. Introduction In China, cold spells in late spring (usually in April) can seriously damage rice (Oryza sativa L.) seed germination and seedling growth, leading to an incidence of 30%–50% rotting seedlings per year over the past 30 years and reductions in rice yields of 3–5 billion kilograms per year [1]. Since chilling stress adversely affect rice growth and pro- ductivity, it is especially important to develop chilling tolerant rice. Silicon is known to support crop plant defense mechanisms. Exogenous Si increased antioxidant defense activity and reduced lipid peroxidation and membrane permeability on wheat and cucumber, which contributed to alleviate freezing stress and enhance plant growth under freezing stress [2,3]. Rice is a typical silicophilic plant [4,5]. During its entire growth period, a rice crop with a yield of 1000 kg would uptake 130 kg of Si from the soil, or an amount twice as much that of N, P, and K combined [6]. In rice, silicon strengthens the plant cell wall and activates defenses against biotic and abiotic stresses, including salinity, heavy metal toxicity, drought stress, radiation, high- temperature stress, chilling stress, and ultraviolet light [for reviews,7]. In this organism, silicon absorption is controlled by a member of the Nod26-like major intrinsic protein (NIP) subfamily, which is encoded by Low silicon gene 1 (Lsi1). This gene, which belongs to the aquaporin family, was identified from a mutant with low silicon absorption ability and is constitutively expressed in plant roots [8]. The transcript level of Lsi1 in addition with silicon content in the sensitive rice cultivar Dular was lower than that of the resistant accession Lemont [9], which sug- gests that Lsi1 acts as a positive role in regulation of rice resistance. Conformably, it was documented that overexpression of Lsi1 in rice significantly increases the silicon content and enhances resistance to ultraviolet light, cadmium stress, and chilling stress [9–12]. Nowadays, an increasing number of reports have addressed the regulatory biological activities of miRNAs in plant growth development and stress responses [13–15]. The small (21–24 nucleotides; nt) non- coding RNAs play a vital regulatory role in gene expression [16–19], http://dx.doi.org/10.1016/j.plantsci.2017.06.002 Received 19 February 2017; Received in revised form 21 May 2017; Accepted 2 June 2017 ⁎ Corresponding author at: Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 35002, PR China. E-mail address: lwx@fafu.edu.cn (W. Lin). Plant Science 262 (2017) 115–126 Available online 13 June 2017 0168-9452/ © 2017 Elsevier B.V. All rights reserved. MARK