Elsevier

Plant Physiology and Biochemistry

Volume 82, September 2014, Pages 95-104
Plant Physiology and Biochemistry

Research article
Overexpression of tomato chloroplast-targeted DnaJ protein enhances tolerance to drought stress and resistance to Pseudomonas solanacearum in transgenic tobacco

https://doi.org/10.1016/j.plaphy.2014.05.011Get rights and content

Highlights

  • LeCDJ2 is a novel tomato chloroplast-targeted DnaJ gene.

  • LeCDJ2 expression was triggered by drought, bacteria stresses and salicylic acid.

  • Overexpression of LeCDJ2 in tobacco enhanced the tolerance to drought stress.

  • Overexpression of LeCDJ2 enhanced the resistance to P. solanacearum in tobacco.

Abstract

DnaJ proteins as co-chaperones have critical functions in biotic and abiotic stress responses, but their biological functions remain largely uninvestigated. This study investigates the function of a tomato (Lycopersicon esculentum) chloroplast-targeted DnaJ protein (LeCDJ2) using transgenic tobacco. Quantitative real-time polymerase chain reaction analysis showed that LeCDJ2 expression was triggered by salicylic acid (SA), drought and pathogen attack. Ectopic expression of LeCDJ2 in transgenic tobacco reduced the accumulation of superoxide anion radical (O2) and hydrogen peroxide (H2O2) under drought stress. Compared with Vec plants, the maximum photochemical efficiency of photosystem II (PSII) (Fv/Fm), net photosynthetic rate (Pn), and content of D1 protein were relatively higher in transgenic plants. The transgenic plants showes better growth, higher chlorophyll content, lower malondialdehyde (MDA) accumulation and relative electrolyte leakage (REL) under drought stress. In addition, overexpression of LeCDJ2 improved the resistance to the pathogen Pseudomonas solanacearum in transgenic tobacco. These results indicate that overexpression of a tomato chloroplast-targeted DnaJ gene enhances tolerance to drought stress and resistance to P. solanacearum in transgenic tobacco.

Introduction

Several environmental stresses exist in the life cycle of plants, including abiotic and biotic stresses, such as light, drought, salinity, extreme temperatures and pathogen infections, which severely restrict growth rates and crop yields. Among the environmental stresses, drought stress is one of the most adverse factors of plant growth and productivity. Drought stress can cause stomata closedown and induce reduction in the contents and enzyme activities in the Calvin cycle (Reddy et al., 2004), which reduces the utilization of absorbed light energy for CO2 assimilation (Payton et al., 2001). Delayed consumption of excess light energy decreases photosynthetic efficiency and results in photoinhibition and photooxidative damage to the photosynthetic reaction center (Pastenes et al., 2005). D1 protein, an important photosystem II (PSII) reaction center complex subunit, will also be damaged. D1 protein degradation, synthesis, and reassembly into the PSII complex contribute to PSII damage repair (Nishiyama et al., 2006, Kato and Sakamoto, 2009, Zhou et al., 2013). However, reactive oxygen species (ROS) whose production is increased during drought stress significantly inhibits de novo D1 protein synthesis (Murata et al., 2007). ROS production also leads to chlorophyll loss and lipid peroxidation (Leshem et al., 1988).

DnaJ proteins or J proteins, also known as HSP40s (heat-shock protein 40), are a family of conserved co-chaperones for HSP70s (heat-shock protein 70) (Qiu et al., 2006). J domain is highly conserved in DnaJ proteins, which contain a 75 amino acid consensus sequence (Hennessy et al., 2005). Through this domain, DnaJ proteins can interact with the ATPase domain of Hsp70 and hydrolyze ATP to ADP, facilitating client capture (Kampinga and Craig, 2010). DnaJ was originally characterized in Escherichia coli as a 41 kDa HSP (Georgopoulos et al., 1980). A large number of DnaJ proteins have been found ubiquitously in all living creatures and characterized from a variety of different organisms (Craig et al., 2006, Zhou et al., 2012). Members of the J-protein family function as molecular chaperones, alone or in association with HSP70 partners, and are involved in various essential cellular processes, including protein folding, degradation and refolding (Hennessy et al., 2005, Craig et al., 2006).

DnaJ proteins localize in different subcellular compartments, such as in the cytosol, mitochondria, chloroplasts, endoplasmic reticulum, cytoskeleton, nucleus, plasma membrane and vacuole (Rajan and D'Silva, 2009). These proteins are also involved in various stress responses (Yang et al., 2010, Zhou et al., 2012), chloroplast movement (Suetsugu et al., 2005), and developmental processes (Park et al., 2011, Shen et al., 2011). In pathogen responses, a J domain virulence effector of Pseudomonas syringae remodels host chloroplasts and suppresses defenses (Jelenska et al., 2007). Silencing NtMPIP1, a tobacco DnaJ-like protein, significantly inhibits the spread of TMV (Shimizu et al., 2009). Overexpression of a soybean nuclear localized type III DnaJ domain-containing HSP40 indicates its functions in cell death and disease resistance (Liu and Whitham, 2013).

Many studies are carried out on DnaJ proteins, but their functions in vegetables such as tomato are rarely reported, especially the chloroplast-targeted DnaJ proteins. Therefore, we isolated and characterized a Lycopersicon esculentum chloroplast DnaJ protein 2 (LeCDJ2) from tomato to investigate its functions. Overexpression of LeCDJ2 enhanced tolerance to drought stress and resistance to Pseudomonas solanacearum in transgenic tobacco.

Section snippets

Plant growth and treatments

WT tomato cultivar (Solanum lycopersium cv. Zhongshu 6), WT tobacco plants (Nicotiana tabacum cv NC 89), T2 transgenic tobacco plants, and empty vector control plants (Vec plants) were grown in quartz sands with a photoperiod of 16/8 h (day/night), a temperature of 25/20 °C (day/night), and a photon flux density (PFD) of 200 μmol m−2s−1. The plants were irrigated with Hoagland nutrient solution once a week.

Six week-old WT tomato plants were subjected to various stress treatments at 25 °C with a

Subcellular localization of LeCDJ2

To determine the subcellular localization of LeCDJ2 protein, the 35 S:GFP and 35 S:LeCDJ2-GFP fusion proteins were transiently expressed in Arabidopsis protoplasts from the leaf tissue (Fig. 1). In the protoplasts transfected with p35 S:LeCDJ2-GFP, GFP fluorescence was distinctly confined to the chloroplasts and co-localized with the red autofluorescence of the chloroplasts. However, the green fluorescence derived from the control p35 S:GFP protein was detected in the cytoplasm surrounding the

Discussion

J proteins reportedly participate in various biological processes and stress responses (Rajan and D'Silva, 2009). They are involved in optimizing photosynthetic reactions (Chen et al., 2010) and facilitating thermotolerance (Zhou et al., 2012). However, their physiological functions under drought stress are still not clearly elucidated. In this study, LeCDJ2 was not only triggered by PEG and pathogen attack but also by the signal molecule SA (Fig. 2). Both germination rate, seedling growth (

Acknowledgments

We thank Associate Professor Lixing Zhang of the Institute of Botany, Chinese Academy of Sciences, for the D1 protein antibody. This research was supported by the State Key Basic Research and Development Plan of China (Grant No. 2009CB118505) and the Natural Science Foundation of China (Grant Nos. 31171474 and 31371553).

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