Isolation And Functional Analysis Of LEA Protein Genes,ZmDHN13, ZmLEA3and ZmLEA5C In Zea Mays

Environmental stresses such as drought, high salinity and disease induce changes in enzyme activities and gene expression in crop plants, leading to considerable reduction in their growth and productivity. In response to various stresses, plants produce a series of proteins to protect cell metabolism. The synthesis of hydrophilic proteins is a major part of the plant response to stress conditions. LEA proteins were first identified in cotton seeds at1981, which are accumulated during the late stages of seed development and are associated with the acquisition of desiccation tolerance in maturing seeds. Late embryogenesis abundant （LEA） proteins are one of the major hydrophilic proteins, which can reduce the damage caused by adverse conditions. According to amino acid sequences and conserved motifs, LEA proteins are categorized into seven distinctive groups.In the present study, three novel maize （Zea mays L.） LEA genes, ZmDHN13（group2, dehydirn）, ZmLEA3（group3A） and ZmLEA5C （group5C） were identified and later characterized its functions. We analyzed the sequence characteristic, expression analysis, physiological and biochemical functions of the three genes. The main results are as follows:1. The ZmDHN13（NP₀01150115.1） cDNA has an open reading frame of324bp that encodes a protein of107amino acid residues. According to the analysis of the conserved domains, ZmDHN13belongs to KS-dehydrin. The ZmLEA3cDNA has an open reading frame （ORF） of546bp that encodes a protein of182amino acids. According to the analysis of the conserved domains, the ZmLEA3protein is classified as LEA3A group. ZmLEA5C （NM₀01155750.1） was isolated from maize. The ZmLEA5C cDNA has an open reading frame （ORF） of624bp that encodes a protein of207amino acids. Motif search analysis using Blastp （NCBI） revealed that the LEA protein contains a "LEA₂" motif （PF03168,1.16e-11）, which was classified as group5C （D-95family）.2. ZmDHN13gene was constitutively expressed in maize, but the transcript accumulation of ZmDHN13could be induced by high osmosis, low temperature, H2O2and signal molecular. ZmLEA3transcript accumulation could be regulated by various environmental stresses and signal molecular. Organ-specific expression studies showed that the transcription of ZmDHN13, ZmLEA5C and ZmLEA5C was higher in seeds than that in roots, stems and leaves3. The proteins ZmDHN13mainly accumulated in the nucleus, which depended on the phosphorylation. Futher study indicated that the S-segment and the NLS-segment were essential for its targeting to the nucleus. The ZmLEA3proteins specifically accumulated in the cytosol and nucleus. The ZmLEA5C proteins mainly accumulated in the chloroplast.4. ZmDHN13and ZmLEA3could bind metal ions, which could reduce the concentration of the free metal ions in plants. These free ions are required to produce ROS via the Haber-Weiss reactions.5. ZmDHN13, ZmLEA3and ZmLEA5C proteins protected lactate dehydrogenase （LDH） activity from damage caused by environmental stresses.6. The proteins ZmDHN13and ZmDLEA5C could be phosphorylated by the Casein kinase Ⅱ （CKII）. The phosphorylation of ZmDHN13could influence the location and the physiological and biochemical functions. The S-segment and the NLS-segment played important roles in the phosphorylation and the nuclear location. The mechanism and function of phosphorylated of ZmLEA5C was not clear.7. ZmDHN13had the DNase activity, which could be inhibited by EDTA. The proteins ZmDHN13△S and ZmDHN13△NLS also possessed the DNase activity, but the activity was weaker than that in ZmDHN13. Further studies indicated that the activity of the deleted protein ZmDHN13△K was significantly inhibited. Thus, we speculated that the K-segment could be the reaction center of DNA cleavage activity and that the NLS-segment represents a non-specific DNA-binding site, the role of S-segment was not clear.8. Overexpression of ZmDHN13enhanced transgenic tobacco tolerance to Cu2+ions and oxidative stresses. The three conserved segments of ZmDHN13exhibited a cooperative effect in response to environmental stresses in vivo. Overexpression of ZmLEA3in transgenic tobacco（Nicotonia tobaccum） and yeast （GS115） conferred tolerance to osmotic and oxidative stresses. Overexpression of ZmLEA5C in transgenic tobacco（Nicotonia tobaccum） and yeast （GS115） confered tolerance to osmotic and low temperature stresses.9. Tobacco’s defense response to Pst DC3000（Pseudomonas syringae pv. Tomato DC3000） was conducted primarily through the SA signaling pathway. ZmLEA3and ZmLEA5C might influence the plant defense response to Pst DC3000.