RNAi-based functional elucidation of PtrPRP, a gene encoding a hybrid proline rich protein, in cold tolerance of Poncirus trifoliata.

Ting Peng,Mao-Mao Jia,Ji-Hong Liu

doi:10.3389/fpls.2015.00808

Abstract

Hybrid proline-rich proteins (HyPRPs) have been suggested to play important roles in various plant development and stress response. In this study, we report the cloning and functional analysis of PtrPRP, a HyPRP-encoding gene of Poncirus trifoliata. PtrPRP contains 176 amino acids, among which 21% are proline residues, and has an 8-cysteine motif (8 CM) domain at the C terminal, a signal peptide and a proline-rich region at the N terminal. PtrPRP is constitutively expressed in root, stem and leaf, with the highest expression levels in leaf. It was progressively induced by cold, but transiently upregulated by salt and ABA. Transgenic P. trifoliata plants with knock-down PtrPRP by RNA interference (RNAi) were generated to investigate the role of PtrPRP in cold tolerance. When challenged by low temperature, the PtrPRP-RNAi plants displayed more sensitive performance compared with wild type (WT), as shown by higher electrolyte leakage and malondialdehyde content. In addition, the RNAi lines accumulated more reactive oxygen species (ROS) and lower levels of proline relative to WT. These results suggested that PtrPRP might be positively involved in cold tolerance by maintaining membrane integrity and ROS homeostasis.

Highlights

Plants are frequently challenged with a variety of abiotic stresses, among which cold, at either freezing, or chilling regimes, constitutes an important factor leading to adverse impacts on plant growth, development and yield potential
Isolation of PtrPRP from P. trifoliata In a previous work, an EST annotated as prolinerich proteins (PRPs) was fished out by SSH-based screening of a cold-treated cDNA library of P. trifoliata (Peng et al, 2012)
5 -Rapid amplification of cDNA ends (RACEs) and 3 -RACE PCR were carried out using primers designed base on the original EST, leading to amplification of two fragments of 554 and 802 bp, respectively, which were shown to be homologous to known PRP genes by Blastn against NCBI

Summary

Introduction

Plants are frequently challenged with a variety of abiotic stresses, among which cold, at either freezing, or chilling regimes, constitutes an important factor leading to adverse impacts on plant growth, development and yield potential. Chilling and freezing stresses lead to physiological or structural alterations, such as elevation of reactive oxygen species (ROS), imbalance of osmotic pressure, and formation of ice crystals. Accumulating evidences show that extensive reprogramming of a cohort of cold-responsive genes is an elegant strategy for the plants to adapt to the harsh environments (Stockinger et al, 1997; Zarka et al, 2003; Nakashima and Yamaguchi-Shinozaki, 2006; Shi et al, 2015) These genes are generally classified into two major categories, functional proteins, and regulatory proteins, which play either direct protective or regulatory roles in stress tolerance (Yamaguchi-Shinozaki and Shinozaki, 2005). It is worth mentioning that functional characterization of a stress-responsive gene is required before it can be efficiently manipulated

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