Isolation and Functional Characterization of a Salt-Responsive Calmodulin-Like Gene MpCML40 from Semi-Mangrove Millettia pinnata

Abstract

Salt stress is a major increasing threat to global agriculture. Pongamia (Millettia pinnata), a semi-mangrove, is a good model to study the molecular mechanism of plant adaptation to the saline environment. Calcium signaling pathways play critical roles in the model plants such as Arabidopsis in responding to salt stress, but little is known about their function in Pongamia. Here, we have isolated and characterized a salt-responsive MpCML40, a calmodulin-like (CML) gene from Pongamia. MpCML40 protein has 140 amino acids and is homologous with Arabidopsis AtCML40. MpCML40 contains four EF-hand motifs and a bipartite NLS (Nuclear Localization Signal) and localizes both at the plasma membrane and in the nucleus. MpCML40 was highly induced after salt treatment, especially in Pongamia roots. Heterologous expression of MpCML40 in yeast cells improved their salt tolerance. The 35S::MpCML40 transgenic Arabidopsis highly enhanced seed germination rate and root length under salt and osmotic stresses. The transgenic plants had a higher level of proline and a lower level of MDA (malondialdehyde) under normal and stress conditions, which suggested that heterologous expression of MpCML40 contributed to proline accumulation to improve salt tolerance and protect plants from the ROS (reactive oxygen species) destructive effects. Furthermore, we did not observe any measurable discrepancies in the development and growth between the transgenic plants and wild-type plants under normal growth conditions. Our results suggest that MpCML40 is an important positive regulator in response to salt stress and of potential application in producing salt-tolerant crops.

Characteristics of SlCML39, a Tomato Calmodulin-like Gene, and Its Negative Role in High Temperature Tolerance of Arabidopsis thaliana during Germination and Seedling Growth

“…CML20 negatively modulates ABA signals in guard cells and drought tolerance [19]. MpCML40 overexpression strongly increases Arabidopsis salt tolerance [20]. In all, these studies suggest that CMLs likely interpret Ca 2+ signals during development and stress responses.…”

“…SlCML39 shares a high degree of similarity to Os-CML19 (56.74%), AtCML37 (56.03%), AtCML38 (54.48%), AtCML39 (54.031%), and AtCML41 (41.14%). Based on the InterPro protein families database (EF hand domain IPR002048) and Zhang et al's [20] analysis, the conservative functional domains identified in these CMLs are Ca 2+ -binding EF hands. S1.…”

A C2H2-Type Zinc-Finger Protein from Millettia pinnata, MpZFP1, Enhances Salt Tolerance in Transgenic Arabidopsis

“…Transcriptome profiles of leaf and/or root tissues of Pongamia have been conducted to address the issue [ 7 , 8 ], while the detailed functional study of salt stress responsive gene is scarce. To date, only two salt-tolerant Pongamia genes, MpCHI and MpCML40 , have been cloned and characterized [ 9 , 10 ]. The heterologous expression of MpCHI in yeast ( Saccharomyces cerevisiae ), which encodes the chalcone isomerase involved in flavonoid biosynthesis, could enhance the salt tolerance capacity of recipient cells [ 9 ].…”

“…Recently, we characterized a salt-induced Calmodulin-like gene, MpCML40 , that enabled transgenic yeast and Arabidopsis to become salt-tolerant. The transgenic MpCML40 Arabidopsis did not show any growth retardation, which differed from most genetically modified stress-tolerant plants [ 10 ].…”

Molecular Aspects of Plant Salinity Stress and Tolerance

“…Previously, calmodulin-like proteins (CMLs) were found to be involved in salinity stress tolerance in Arabidopsis. Zhang et al isolated a CML gene, MpCML40 , from Pongamia, and found that its heterologous expression could improve salinity tolerance in yeast cells and enhance the rate of seed germination and the length of roots when exposed to salt and osmotic stresses in Arabidopsis [ 6 ]. The authors suggested that MpCML40 contributed to the proline accumulation for reducing the damage caused by reactive oxygen species.…”