Rice (Oryza sativa L.) OsNADP-ME4 gene responds to adversity stresses  

Limin Chen1 , Daisuke Tsugama2 , Tetsuo Takano2 , Shenkui Liu1
1. Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field (SAVER), Ministry of Education, Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University, Harbin, 150040
2. Asian Natural Environmental Science Center (ANESC), The University of Tokyo, Nishi-tokyo-shi, Tokyo, Japan
Author    Correspondence author
Cell Biology and Biophysics, 2015, Vol. 4, No. 1   doi: 10.5376/cbb.2015.04.0001
Received: 30 May, 2015    Accepted: 31 May, 2015    Published: 01 Jun., 2015
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This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Chen et al., 2015, Rice (Oryza sativa L.) OsNADP-ME4 gene responds to adversity stresses, Cell Biology and Biophysics, Vol.4, No.1 1-7 (doi: 10.5376/cbb.2015.04.0001)


NADP-malic enzyme (NADP-ME, EC1.1.1.40) is involved in many metabolic pathways in plants, and has been recently reported in plant defense responses to a variety of environmental stress factors. In this study, an analysis of rice OsNADP-ME4 (NCBI: NM_001050754) gene was performed, the gene open reading frame (ORF) was 1756 bp, encoding 585 amino acids, and predicted location was in cytoplasmic. OsNADP-ME4 had a identity of 79% and 90% with AtNADP-ME1 and ZmcytME respectively, and a close genetic relationship with them in the evolutionary tree. 12 hours after different concentrations of NaCl, PEG6000,and NaHCO3 stresses with 14-days rice seedlings, the transcription of the OsNADP-ME4 gene increased in response to almost all the treatments in the roots, while decreased in the leaves, especially at 30 mM NaHCO3 stress, the transcription increased to 3 times in roots. Over-expressing OsNADP-ME4 gene enhanced the tolerance to salt and drought stresses of transgenic Arabidopsis.

OsNADP-ME4; Abiotic stress; Gene expression; Transgenic Arabidopsis

NADP-ME is an oxidation decarboxylase existing widely in plants, animals and microorganisms. It catalyzes the oxidative decarboxylation of L-malate to pyruvate, CO2 and NADPH in the presence of a bivalent cation Mg2+ or Mn2+ (Drincovich, Casati and Andreo, 2001; Chang and Tong, 2003). NADP-MEs in plant exist in the forms of isoenzymes encoded by a small gene family and locate in plastid and cytosol (Edwards and Andreo, 1992). In plants, NADP-MEs participate in the photosynthesis and lignin biosynthesis (Lai, Wang and Nelson, 2002; Pinto, Casati, Hsu, Ku and Edwards, 1999), maintain cell osmotic pressure, stable cytoplasmic pH (Martinoia and Rentsch, 1994), and provide carbon skeletons and reducing power for lipid biosynthesis (Veronica, Mariel, Carlos and Marı´a, 2009).
In recent years, NADP-ME in plant defense response to environmental stress factors such as salt, drought, low temperature, and oxidative stress attracted more and more attention of the researchers.A research indicated that the expression of NADP-ME gene in Aloe vera L. was induced by high salt, dehydration, and exogenous ABA (Sun, Shen, Wan and Liu, 2003). In hexaploid wheat, the activity of the NADP-ME enzyme was increased by 20% PEG6000, 4, 200 mM NaCl, abscisic acid and salicylic acid, but the transcription of TaNADP-ME1 gene and TaNADP-ME2 was both down-regulated at almost all the stresses, predicted the existence of other NADP-ME forms probably (Fu, Zhang, Liu, Hu, Xu, 2011). In Populus trichocarpa, the expressions of five NADP-ME genes were obviously regulated by NaCl, PEG and mannitol stresses (Cheng and Yang, 2011). The activity of NADP-ME enzyme from rice increased under NaCl and NaHCO3, over-expression of NADP-ME2 and NADP-ME3 gene in Arabidopsis showed a resistance to a variety of environmental stress factors (Liu, Cheng, Zhang, Guan, Nishiuchi, Hase and Takano, 2007; Cheng and Long, 2007; Li, Zhang, Takano and Liu, 2012).11 days after drought stress, NADP-ME activity in the tobacco leaves increased 3.9 times, the amount of NADP-ME protein and transcription of mRNA for chloroplastic NADP-ME isoform were increased and mRNA for cytosolic isoform was decreased (Veronika, Lucia, Jana, Radomira and Helena, 2014).NADP-ME enzyme activity in Arabidopsis thaliana was increased when treated with pathogens and O3, the expression quantity of AtNADP-ME2 gene increased obviously (Lars, Martina, Timo, Alexandra, Mariel, Maria, Andreas and Veronica, 2012; Li, Mhamdi, Clement and Graham Noctor, 2013). Thus, NADP-ME in plant is closely associated with adversity stress, but there are different expression patterns between genes, suggesting the different functions of isozymes and the complexity in the division and cooperation process.
The rice NADP-ME family is composed of four members, one plastidic and three cytosolic isoforms, as the cytosolic ones (NADP-ME2 and NADP-ME3) are involved in plant defense with adversity stresses, so in this study, another gene expressing rice NADP-ME named OsNADP-ME4 (NCBI: NM_001050754) was examined under NaCl, NaHCO3, and PEG6000. Over-expressing OsNADP-ME4 gene Arabidopsis plants were obtained, and transgenic Arabidopsis seedlings were analyzed with salt and drought tolerance.
1 Materials and methods
1.1 Sequence analysis and phylogenetic tree
Nucleic acid and amino acid sequences of OsNADP-ME4 (NCBI: NM_001050754) were analysed according to NCBI (http://www.ncbi.nlm.nih.gov/) and ExPASy (http://www.expasy.org/proteomics) databases. Complete amino acid sequences of different plant sources were obtained from NCBI database, the Homology alignment ofmultiple sequences was done with BioEdit software and phylogenetic tree was constructed with MEGA3.1 software.
1.2 Plant growth and stress treatments
Rice (Oryza sativa L., cultivar: Nipponbare) seeds were grown in 1/2MS liquid medium for 14 days in a greenhouse with12h illumination at 25, then treated with 75mMNaCl, 100mMNaCl, 125mMNaCl, 10mMNaHCO3, 30mMNaHCO3, 50mMNaHCO3, 5% PEG6000, 10% PEG6000, 15%PEG6000 for 12h respectively andcontrol was performed without treatment. The leaf and root were harvested and conserved in -80 for RNA extraction.
1.3 Quantitative real time PCR
Rice total RNA was extracted with Trizol reagent,the trace DNA in RNA was digested with DNAase, and the first-strand cDNA was synthesized according to the Prime Script RT reagent Kit (TaKaRa, Janpan) instruction.Real-time fluorescent quantitative PCR was done in the ABI 7500 (Applied Biosystems Inc., USA) PCR instrument using SYBRGreen Master mix(TaKaRa, Janpan). The pairs of special primers were as follows: OsNADP-ME4-F (5′-CAGCTTCATTTGGCTACAGC-3′) and OsNADP-ME4-R (5′-GGGATAAAATGCTACTGCCG-3),and actin-F (5′-TCTCAGCACATTCCAGCAGG-3′) and actin-R (5′- CAGCCTTGGCAATCCACATC-3′). The PCR reaction system was 20 μL containing 10.0μL of 20×SybrGreenmix, 0.5μL of each primer (10μmol·L-1), 1.0μL of cDNA, 8.0μL of ddH2O. Amplification conditions were as follow: Pre-degeneration for 3min at 94, 40 cycles of 30s at 95, 30 s at 58and 30s at 72, then 5 min at 72.
1.4 Identification of Northern blot
The T0 generation seeds of Arabidopsis for genetic transformation were harvested, and grown in 1/2 MS solid medium with 40 mg/L kanamycin for screening, then resistant seedling were transplanted tovegetative soil at normal conditions for development .The leaves of these transgenic plants were collectedrespectively, and RNA was extracted using Trizol reagent, 10 μg of total RNA were separated on a denaturing formaldehyde 1.2 % (w/v) agarose gel, and blotted onto a nylon membrane. RNA hybridization was performed using DIG-labelled OsNADP-ME4 special DNA probe, and signal detection with CDP-Star reagent using ImageMaster VDS-CL system. The specific primers used forNorthern blot were Primer-F (5′-AGGATTTACTGAAGGACGAG-3′) and Primer-R (5′-ATAGTGCGAGGATGATTGGT -3′).
1.5 Adversity stress treatments
Transgenic Arabidopsis T3 generation seedlings were grown in 1/2 MS solid medium for 5 days in a culture room at 25 with a light cycle of 8h light and 16h dark, then transferred into 1/2 MS solid medium containing 100 mM NaCl, 125 mM NaCl, 150 mM NaCl, 225 mM Mannitol ,250 mM Mannitol and 275 mM Mannitol for 10 days, and took 1/2 MS medium without any stress as control at the same time. Three independent biological replicates were performed. The growth and development of transgenic and WT seedlings were observed, the length of roots and fresh weight were measured.
2 Results and Discussion
2.1Sequence analysis ofOsNADP-ME4 gene

cDNA of rice OsNADP-ME4 (NCBI: NM_001050754) had been cloned and transferred into Arabidopsis in the previous study in our lab. According to NCBI database (http://www.ncbi.nlm.nih.gov/), the full length cDNA is 2055 bp, the ORF contains 1758 bp from 112 site ATG to 1869 site TGA, encoding a putative 585 aminoacid protein. The amino acid sequence of OsNADP-ME4 is analysed by ExPASy tools (http://www.expasy.org/proteomics), and the predicted molecular weight of the protein is about 64.27 kDa, the theoretical pIis6.50,and the protein is an unstable hydrophilicity protein with instability index (II) of 43.37, aliphatic index of 95.90 and Grand average of hydropathicity of -0.127. There is no transmembrane region or signal peptide in the sequence, and predicted positioning of the protein is within the cytoplasm.

A phylogenetic analysis based on the alignment of OsNADP-ME4 protein with NADP-MEs full-length amino acid sequences of 7 kinds of plants was done with Bioedit, and a phylogenetic tree was constructed using MEGA3.1 by N-J method (Figure 1). Results show that OsNADP-ME4 has a high identity of 75%~90% with other species. Within rice NADP-ME family, OsNADP-ME4 has 76% identity with NADP-ME1, 78% with NADP-ME2 and 77% with NADP-ME3, they are in different branches of the phylogenetic tree. However, OsNADP-ME4 has a close genetic relationship with AtNADP-ME1 and ZmcytME, and the identities are 79% and 90% respectively. Other studies also indicate that AtNADP-ME1, ZmcytME and OsNADP-ME4have a divergence from the other NADP-MEs of their families and are classified to a new group compared with previous three branch system, containing both monocot and dicot NADP-MEs and presenting a different evolutionary course (Chi, Yang, Wu and Zhang, 2004; Mariel, Marcos, Maria, Carlos, Ulf-Ingo and Veronica, 2005; Fu, Zhang, Hu, Shao and Xu, 2009). In addition, AtNADP-ME1 was detected expressing in roots by semi-quantitative RT-PCR, and the promoter driving GUS gene expressed during the late stages of embryogenesis and in the root tip during germination and some second roots in the mature plant (Mariel, Marcos, Maria, Carlos, Ulf-Ingo and Veronica, 2005), ZmcytME only expressed in embryo and emerging roots (Enrique, Maurino, Alvarez and Andreo, 2008). So we consider that OsNADP-ME4 gene may probably have a close relationship with metabolic activity in the root. Whether there is a same characterization with OsNADP-ME4 of this Group, is required a further study.

Figure 1 Phylogenetic tree of plant NADP-ME. The Phylogenetic tree was constructed by neiighbor-joining method with the amino acid sequence using MEGA3.1. Statistical significance of each branch of the tree was evaluated by bootstrap analysis by 100 iterations of bootstrap samplings. The following sequences were included: Vitis vinifera: VvME1 (NM_001281213), VvME2 (U67426); Lycopersicon esculentum: LeME1 (AF001269), LeME2 (AF001270); Aloe arborescens: AME1 (BAA74735), AME2 (AB005808);Zea mays: ZmchlME1 (J05130), ZmchlME2 (U39958), ZmcytME (AJ224847); Arabidopsis thaliana: AtNADP-ME1 (NC_003071), AtNADP-ME2 (NM_121205), AtNADP-ME3 (NM_122489), AtNADP-ME4 (NC_003070); Oryza sativa. L: OsNADP-ME1 (NM_001048792), OsNADP-ME2 (AB053295), OsNADP-ME3 (NM_001061367), OsNADP-ME4 (NM_001050754); Triticum aestivum. L: TaNADP-ME1 (EU170134), TaNADP-ME2 (EU082065)

2.2 Expression pattern of
OsNADP-ME4 gene under various abiotic stresses
In CAM plant Aloe vera L, C4 plant maize (Maurino, Mariana and Andreo, 2001), C3 monocot rice, wheat, dioct Arabidopsis, tobacco and woody plant Populus trichocarpa, NADP-MEs were reported involved in plant defense response to abiotic stresses including salt, drought, low temperature, ABA and oxidative stresses as described in introduction. This kind of function of NADP-MEs may be widespread in plants, and the roles between the family members are varied. To further investigate whether OsNADP-ME4 gene responses to adversity, stress treatments were applied to 14-days rice seedlings, the expression of OsNADP-ME4 gene was analysed by quantitative real-time PCR (Figure 2). After treating rice seedlings with different concentrations of NaCl, PEG6000 and NaHCO3 for 12 h, the abundance of OsNADP-ME4 transcripts relative to the expression in control showed a similar trend that reducing in rice leaves and increasing in roots at almost all the stresses. There existed small differences between each stress. For the NaCl assay, while low concentration induced the detection of OsNADP-ME4 transcripts reduced in leaves and a trace of increase in roots of control, 125 mM NaCl induced a equivalent level in leaves and a high degree in roots (Figure 2 A, B). At PEG6000 stress, the decrease in leaves was obvious when the increase in roots was inconspicuous (Figure 2 C, D). NaHCO3 may be a special sensitizing material to OsNADP-ME4 gene, as the transcript reduced to a very low stage in leaves and performed a substantial increase in roots, especially at 30 mM NaHCO3the transcriptional level in roots was three times of the control (Figure 2 E, F). NaCl, PEG6000 and NaHCO3 can regulate the expression of OsNADP-ME4 gene as with the cytosolic NADP-ME2 (Liu, Cheng, Zhang, Guan, Nishiuchi, Hase and Takano, 2007) and NADP-ME3 (Li, Zhang, Takano and Liu, 2012). Rice NADP-ME gene family contains four members, apart from the cytosolic ones, the plastid NADP-ME was also involved in plant defense to stress factors (Fushimi, Umeda, Shimazaki, Kato, Toriyama and Uchimiya, 1994), but the induced expression levels were varied between four genes (Chi, Yang, Wu and Zhang, 2004). The expression profiles of the four genes were different in tissues of rice root, leaf and panicle and in the leaves of different developmental stage (for 5-days, 10-days, 15-days and 25-days seeding), and in particular, OscytME3 (OsNADP-ME4) expressed at very low levels in all the tissues and in the leaves with development (Chi, Yang, Wu and Zhang, 2004). AlthoughOsNADP-ME4 gene expressed slightly in rice, but had an active expression when composed with NaCl, PEG6000 and NaHCO3, these suggested that the gene was closely related to plant defense response to adversity stresses.

Figure 2 Regulation of OsNADP-ME4 transcripts by stress treatments. A/C/E: Detections of OsNADP-ME4 transcripts in rice leaves under NaCl, PEG6000 and NaHCO3 stress. B/D/F: Expressions of OsNADP-ME4 gene in rice roots respone toabiotic stresses. Each experiment was repeated three times

NADP-ME4 transcripts in rice leaves under NaCl, PEG6000 and NaHCO3 stress. B/D/F: Expressions of OsNADP-ME4 gene in rice roots respone toabiotic stresses. Each experiment was repeated three times.
2.3 Over-expression of OsNADP-ME4 enhanced the resistance to salt and drought stresses Arabidopsis
Quantitative RT-PCR analysis revealed that OsNADP-ME4 gene was induced by drought and salt, it may have a potential role in salt and drought resistance. So the ORF of this gene was cloned and 35s: OsNADP-ME4 genewas transformed into Arabidopsis, then T0 generation seeds had been screened by 40 mg/L kana. The green seedlings after screening grown at normal condition for 30 days were used for molecular identification by Northern blot, among them, four transgenic lines (4#, 5#, 6# and 12#) expressed rice OsNADP-ME4 transcripts, and 4#, 5# and 12# were chosen for further study (Figure 3).

Figure 3 Identification of NADP-ME4 transgenic lines by Northern blot. WT is wild type plant, and #3, #4, #5, #6, #9, #12 are different transgenic plant strains

The three T3 generation independent transgenic plants (4#, 5# and 12#) and WT seeds harvested at the same period were grown in 1/2 MS solid medium for 5 days, then transferred to medium with NaCl and mannitol stresses. In 1/2 MS medium without stresses, there were no obvious differences in phenotypic, root length and fresh weight at germination stage (Figure 4). When distributed to different levels of NaCl, transgenic plants showed better growth state than the wild-type,
with the increase of concentration, transgenic seedlings showed a significant tolerance with respect to the wild-type of the same concentration. Salt stress seriously inhibited root growth, especially at 150 mM NaCl, the root elongation of WT was stopped at a very short root period, but transgenic ones were not seriously influenced (Figure 4 A-C). While cultivated in mannitol, the growth was inhibited with all the plants withleaf withering, root system undeveloped (Figure 4 D), and root length and fresh weight was significantly decreased compared with the control group (Figure 4 E, F). At each concentration, over-expression lines were growing better than WT, these ones were more resistant to drought stress (Figure 4 D-F).

Figure 4 Resistance analysis of transgenic plants under adversity stresses. A: Grouth of WT and transgenic seedings on 1/2 MS solid medium with different concentrations of NaCl. B/C: Root length and fresh weight of WT and transgenic plants were measured after NaCl stresses. D: WT and transgenic seedings grown with mannitol stresses. E/F: Root length and fresh weight of transgenic seedings under mannitol stresses

Salt and drought stresses
often cause osmosis stress, producing large amounts of reactive oxygen species (ROS), destroy the structures of plasma membrane, and affect the photosynthetic rate in plant,NADP-ME catalyzes malic acid metabolism producing reducing substances NADPH, may provide reducing power to ROS metabolism, and NADPH is also required for biosynthesis of pigments and defensive substance such as lignin and flavonoids (Pinto, Casati, Hsu, Ku and Edwards, 1999; Wu, Lu, Wang and Li, 2008; LIU, SHAO, CHU and ZHANG, 2010). OsNADP-ME4 showed a low level of constitutive pattern of expression in rice (Chi, Yang, Wu and Zhang, 2004), but it could improve plant abiotic stress tolerance to a certain extent, preliminary speculated that the catalytic synthesis of a variety of products were participated in promoting resilience material and the repair process probably, and this needed more studies.
In conclusion, OsNADP-ME4 had high homology of 75%~90% with other plant NADP-MEs, OsNADP-ME4 gene could be regulated by different concentrations of NaCl, PEG6000, NaHCO3, and the expressions were increased in roots, decreased in leaves. Over-expressing OsNADP-ME4 gene Arabidopsis grew better than wild type under NaCl and mannitol stresses, the expression of OsNADP-ME4 improved the tolerance to salt and drought resistance of transgenic Arabidopsis.
This work was supported by specific fund for forest scientific research in the public welfare (201404220) and Program for Changjiang Scholars and Innovative Research Team in University of China (PCSIRT) (IRT13053).
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