Transcriptome analysis of Chongyi wild mandarin, a wild species more cold-tolerant than Poncirus trifoliata, reveals key pathways in response to cold

Highlights

• Chongyi wild mandarin is more cold-tolerant than Poncirus trifoliata.

• BRs pathway might be dominant in cold signaling of Chongyi wild mandarin.

• Profound accumulation of osmoprotectants was cold-induced in Chongyi wild mandarin.

Abstract

Much effort has been made on the dissection of cold response in citrus, but relevant study in wild germplasm has been rare. Based on the result of the membership function value analysis with 12 physiological indexes, we reported a wild citrus germplasm, Chongyi wild mandarin (Citrus reticulata), which was more cold-tolerant than a cold-hardy rootstock, Poncirus trifoliata. To gain an overview of transcriptome changes under cold, leaves of Chongyi wild mandarin were sampled at 0, 1, 5 d after 0 °C treatment, and sequenced using an Illumina sequencing platform. Totally, 3921 and 5995 differentially expressed genes (DEGs) were identified in 1 d and 5 d, respectively. And then, RNA-seq results were further validated via real-time quantitative PCR with 17 DEGs. Many DEGs related to various cold signaling pathways were identified, especially those related to brassinosteroid cascade. Around 10 % of the total DEGs were transcription factors from 67 families, suggesting that transcriptional regulation, both C-repeat/dehydration responsive element-binding factor-dependent/independent pathways, was crucial for the cold tolerance of Chongyi wild mandarin. Our data also revealed that the transcriptional levels of many DEGs related to anti-oxidant enzymes and osmoprotectants metabolism were altered, which were consistent with the results of physiological experiments. Moreover, a number of DEGs involved in ABA, BR, GA metabolisms were isolated. Accordingly, the levels of ABA and BR were significantly increased under cold, but GAs were decreased. Taken together, this work provided a comprehensive view of physiological and transcriptional alterations in cold response of Chongyi wild mandarin.

Introduction

Low temperature, either chilling (0–15 °C) or freezing (< 0 °C), is one of the major stressors limiting plant growth, development, yield and geographic distribution (Ji et al., 2019). A period of exposure to low but non-freezing temperatures could increase the tolerance of various temperate plants to subsequent freezing temperatures, which was defined as the process of cold acclimation (Thomashow, 1999). Besides decreasing temperature, the initiation of cold acclimation also needs decreasing photoperiod and changes in light quality (Chang et al., 2021). To date, many researches have been done and tremendous progresses have been made in dissecting the mechanism of cold acclimation and cold tolerance in plants (Baier et al., 2019; Ming et al., 2020; Prerostova et al., 2021). Cold-induced changes in membrane fluidity and rearrangement of the cytoskeleton might trigger the Ca²⁺ influx, which initiated the signal perception involving receptors such as calmodulin, Ca²⁺-dependent protein kinases (CPKs) and calcineurin B-like protein-interacting protein kinase 3 (CIPK3) (Kudla et al., 2018). Mitogen-activated protein kinase (MPKs) cascades and phytohormones, like abscisic acid (ABA), brassinosteroids (BRs), played important roles in the following cold signal transduction process (Li et al., 2017; Liu et al., 2020). Subsequently, an array of transcription factors (TFs) was activated and they regulated the expression of downstream cold regulated (COR) genes (Liu et al., 2014).

C-repeat binding factors (CBFs) play important roles in the cold-responsive pathway, which is also known as the dehydration-responsive element-binding factors (DREBs) and belonged to the APETALA2/ethylene-responsive factor-type transcription factor family (Fowler and Thomashow, 2002). CBFs can regulate the expression of a subset of COR genes encoding functional proteins like low-molecular-weight compatible osmolytes and reactive oxygen species (ROS)-scavenging proteins (Peng et al., 2014; Lv et al., 2020). The expression of CBFs was regulated by some upstream transcription factors, including inducer of CBF expression 1 (ICE1), MdBBX37, MdMYB88/MdMYB124, zinc finger of Arabidopsis thaliana 12 (ZAT12), ethylene-insensitive 3 (EIN3), brassinazole-resistant 1 (BZR1) and calmodulin binding transcription activators (CAMTA) (An et al., 2020; Doherty et al., 2009; Fowler et al., 2005; Huang et al., 2015; Kim et al., 2013; Li et al., 2017; Shi et al., 2012; Xie et al., 2018). On the post-translational level, the receptor-like cytoplasmic kinase cold-responsive protein kinase 1 (CRPK1) can phosphorylate 14-3-3 proteins and facilitate their binding with CBFs, leading to the proteasome-mediated degradation of CBFs (Liu et al., 2017). In addition, some CBF-independent genes were also reported, such as high expression of osmotically responsive gene 9, WKRY6, suppressor of overexpression of constans 1 (SOC1) (Zhu et al., 2004). Most of the above-mentioned investigations were done in Arabidopsis, while the cold responsive mechanism in non-model woody plants was less explored, especially in wild germplasms.

Citrus originated in Southeast Asia and spread worldwide with human activities, which has been one of the most economically important fruit crops (Liu, 1983; Zhu et al., 2019). China is considered as one of the centers of origin for the genus Citrus L. and rich in wild citrus germplasms, such as Ichang papeda (C. ichangensis), Hongkong kumquat (Fortunella hindsii), precocious trifoliate orange (Poncirus trifoliata), C. junos Sieb. ex Tanaka (Zhang et al., 2016; Wang et al., 2018). Wild mandarins were mostly distributed in a region around the Nanling Mountains in the south of China, such as Mangshan Mountain, Daoxian and Jiangyong Counties in Hunan Province, Chongyi County in the south of Jiangxi Province and Hezhou in the Guangxi Zhuang Autonomous Region (Nie and Hu, 1987; Li et al., 2007). Since commercial cultivars are vulnerable to various biotic and abiotic stresses due to the very narrow genetic diversity, wild germplasms have been considered as the ideal materials for genetic improvement of citrus (Wu et al., 2014). Therefore, the assessment of these wild citrus species resistant to various stresses is of great significance.

In 1977, a loose-skin mandarin (Citrus reticulata) was first discovered in the valleys of Chongyi County, locally called ‘Chou Gan’ in Chinese, meaning ‘smelly tangerine’ (Luo, 1981). But its evaluation and utilization has been rare. P. trifoliata was the commonly recognized citrus species with remarkable cold hardiness after a period of acclimation (Peng et al., 2020; Wang et al., 2019). Our preliminary experiments revealed that Chongyi wild mandarin had lower electrolyte leakage (EL) and malondialdehyde (MDA) content than P. trifoliata did after cold treatment, indicating that it might be more cold-tolerant. Therefore, we validated the cold tolerance of Chongyi wild mandarin using the membership function value method and analyzed its transcriptomic alteration in response to cold stress, which may contribute to the genetic improvement of cold-tolerant citrus cultivars in future.