Isolation and Characterization of Maize PMP3 Genes Involved in Salt Stress Tolerance

Citation: Fu J, Zhang D-F, Liu Y-H, Ying S, Shi Y-S, et al. ( Isolation and Characterization of Maize PMP3 Genes Involved in Salt Stress Tolerance Jing Fu 0 Deng-Feng Zhang 0 Ying-Hui Liu 0 Sheng Ying 0 Yun-Su Shi 0 Yan-Chun Song 0 Yu Li 0 Tian- Yu Wang 0 Gustavo Bonaventure, Max Planck Institute for Chemical Ecology, Germany 0 1 College of Biological Sciences, China Agricultural University , Beijing , China , 2 National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), Institute of Crop Science, Chinese Academy of Agricultural Sciences , Beijing , China Plasma membrane protein 3 (PMP3), a class of small hydrophobic polypeptides with high sequence similarity, is responsible for salt, drought, cold, and abscisic acid. These small hydrophobic ploypeptides play important roles in maintenance of ion homeostasis. In this study, eight ZmPMP3 genes were cloned from maize and responsive to salt, drought, cold and abscisic acid. The eight ZmPMP3s were membrane proteins and their sequences in trans-membrane regions were highly conserved. Phylogenetic analysis showed that they were categorized into three groups. All members of group II were responsive to ABA. Functional complementation showed that with the exception of ZmPMP3-6, all were capable of maintaining membrane potential, which in turn allows for regulation of intracellular ion homeostasis. This process was independent of the presence of Ca2+. Lastly, over-expression of ZmPMP3-1 enhanced growth of transgenic Arabidopsis under salt condition. Through expression analysis of deduced downstream genes in transgenic plants, expression levels of three ion transporter genes and four important antioxidant genes in ROS scavenging system were increased significantly in transgenic plants during salt stress. This tolerance was likely achieved through diminishing oxidative stress due to the possibility of ZmPMP31's involvement in regulation of ion homeostasis, and suggests that the modulation of these conserved small hydrophobic polypeptides could be an effective way to improve salt tolerance in plants. - Funding: This work was partly supported by grants provided by the Ministry of Science and Technology of China (2011CB100100, 2009CB118401) and the China Natural Science Foundation (30730063). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Plant growth and development are affected by various abiotic stresses, such as high salinity, drought, low temperature and heavy metals. In particular, salinity is a global environmental challenge, affecting crop production over 800 million hectares, or a quarter to one third of all agricultural land on the earth [1]. Recently, physiological and genetic mechanisms of salt tolerance have been intensively investigated, and it is believed that high concentration of salts often causes ion imbalance and hyperosmotic stress to plants [2,3]. Ion homeostasis is fundamental to physiological processes of living cells. The living cells often maintain high concentration of K+ and low concentration of Na+ in the cytosol, which is important for activities of many cytosolic enzymes. However, under salt stress, Na+ accumulates extremely in cells and consequently disrupts ion homeostasis. Thus, the maintenance of Na+ and K+ homeostasis is crucial under salt stress for plants to survive. There are two kinds of mechanisms for plants to maintain ion homeostasis under salt stress. Firstly, at the organismal level, the ability of regulating Na+ uptake and transporting Na+ from roots to the shoots is critical in all plants. In saline soil, plant roots are inclined to minimize Na+ accumulation in plants. For instance, sodium influx of halophyte roots is much lower than nonhalophyte roots because the width of the Casparion band is two or three times larger in halophytes than in non-halophytes, which effectively prevent the excessive Na+ from entering into the apoplastic space [4]. When the Na+ ion enters the apoplastic space of roots, the excessive Na+ is restricted to old tissues to prevent Na+ from accumulating in reproductive and delicate organs, which can lead to irreversible damage. Secondly, at the cellular level, the maintenance of appropriate Na+ accumulation in cells is due to diffusion and active transport. Recently, some factors responsible for ion transport, such as nonselective cation channels (NSCCs), ion transporters and membrane-potential modulators, have been characterized. In plants, the NSCCs, which catalyze ion influx, can be divided into three groups according to their physical stimuli. These are respectively cyclic-nucleotide-gated NSCCs (CNGSs), amino-acidgated NSCCs (AAG-NSCCs) and reactive-oxygen-species-activated NSCCs (ROS-NSCCs) [5]. Among these NSCCs, CNGSs are perhaps the best studied. Known examples include AtCNGS3, which localizes in root epidermal and cortical cells and contributes to Na+ uptake at the initial stage of salt stress [6,7]. Other examples include AAS-NSCCs and ROS-NSCCs, which support the role of Ca2+ transporter [8]. Previous studies also revealed that there are several ion transporters that play important roles in retrieving intracellular ion homeostasis under saline conditions. As a plasma membrane Na+/H+-antiporter, SOS1 is an important tolerance determinant involved in the exclusion of sodium ions from cells [9,10]. The transcription level of SOS1 is up-regulated by salt stress but not by drought and cold stress [11]. In the presence of calcium, SOS3 activates the substrate phosphorylation activity of SOS2 [12], and then the SOS3/SOS2 complex in turn activates SOS1 probably via phosphorylation, which catalyzes sodium efflux from plant cells. Additionally, several other transporters are also involved in sodium ion transport, such as AtNHX1 and AtNHX2 [13]. Plasma membrane protein 3 (PMP3), a class of small molecular weight hydrophobic proteins in higher plants, responds to various stresses, such as low temperature, salt and dehydration. [1416]. All these proteins are highly conserved at both sequential and structural levels and contain the common conservative domain of UPF0057. The PMP3 protein (Pmp3p) was also identified in yeast (Saccharomyces cerevisiae), which functions in the maintenance of plasma membrane potential. Deletion of PMP3 increased Na+ and K+ sensitivity of yeast cells and resulted in excessive concentration of Na+ and K+ [16]. Complementation analysis in yeast revealed that plant homologues, such as RCI2 in Arabidopsis thaliana, Lti6a/b in Oryza sativa, AcPMP3-1 in sheep grass (Aneurolepidium chinense, a monocotyledonous halophyte), PutPMP3-1 and PutPMP3-2 in alkali grass (Puccinellia tenuiflora), are capable of restoring salt sensitivity of yeast mutant lacking PMP3/SNA1 [1622]. It was also demonstrated that AcPMP3 regulated cellular Na+ and K+ accumulation in the (...truncated)