Abiotic Stress Resistance Analysis of Lilium pumilum Overexpressing the LpMT2 Gene

Plant metallothioneins (MT) are cysteine-rich proteins present in plants that can improve a plant’s salt tolerance. Therefore, a greater understanding of the MT gene in lily (Lilium pumilum), Liliaceae, is an important factor in the development and cultivation of improved salt-tolerant varieties and enriching plant resources for saline soils. A type 2 MT gene (GenBank access number: MH319787, designated as LpMT2) was isolated from L. pumilum leaves. The response mechanism to stress was then investigated, which provided the basis for molecular breeding of L. pumilum for stress tolerance. The LpMT2 gene amino acid sequence is highly homologous to that of type 2 MT protein. Quantitative real-time PCR (qPCR) determined that different plant tissues expressed the LpMT2 gene differently and these expressions were dependent on the specific stress. Transgenic plants with LpMT2 gene exhibited significantly increased resistance to salt and oxidative stress compared with untransgenic plants. The LpMT2 transgenic plants had better growth, greater chlorophyll and proline content, less malondialdehyde (MDA) content and cell membrane permeability, greater superoxide dismutase (SOD) activity, less Na content, greater K content and Na efflux, and less K efflux. These results determined that the transformed LpMT2 gene in L. pumilium plays an important role in enhancing the plant’s salt tolerance and antioxidant capacity.


Northeast China Salinity and Lily (Lilium pumilum)
Northeast China is one of the most severe saline areas in China and one of the three largest saline soils in the world with an area of 3.84 km 2 (Yao et al., 2006), accounting for 3.1% of the total area of the northeast China region (Y. H. Wang & S. X. Wang, 1994). Only a few salt tolerant plants can survive in this saline area (Jin et al., 2017). L. pumilum can grow well in saline soils, but there is limited research on its salt tolerant genes. The Lilium genus, in the family Liliaceae, was established by Linnaeus in 1753 (Takhtajian, 1986), and there are about 115 species identified worldwide (Fu, 2002). L. pumilum is a perennial herb of Lilium and distributed widely in north China. L. pumilum research mainly focuses on plant resources (Wu et al., 2006), hybrid breeding (Yang, 2016), the flowering biological characteristics (Fukai & Goi, 2001), tissue culture , and reproduction (Chojnowski, 1996). It is also reported that Lilium has resistance to drought and salt stress (Yue, 2012). However, research on the salt tolerance and cloning salt-tolerant genes in Lilium is limited. In Phase #1 of this research the objective was to screen a salt-tolerant Metallothionein (MT) gene from L. pumilum by qPCR under salt stress and investigate the gene function. A transcript fragment was amplified by PCR from the cDNA with the forward primer (5'-ATGTCTTGCTGTGGTGGAAA-3') and reverse primer (5'-TTAGCACTTGCATGGGTTG-3') based on the transcriptome contract sequencing results of L. pumilum. Primers were designed using Primer Premier 5.0 software (Premier Biosoft, Canada). The PCR product was ligated into plasmid pMD18-T vector (Takara Bio, Japan) and sequenced. The sequences were identified using DNAMAN 6.0 software (Lynnon Biosoft, USA). The new gene was designated as LpMT2.

The qPCR Analyses of LpMT2 Expression in Different Organs of L. pumulum and Under Different Stress
RNAs were extracted from the roots (three years old), bulbs (three years old), young leaves (eight week old), mature leaves (three years old), flowers (three years old) and seeds (harvested from three-year old plants) of L. pumulum using RNeasy Plant Mini Kit. First-strand cDNA was synthesized by reverse transcribing 500 ng of total RNA with using PrimeScriptTM RT reagent Kit. The primers for qPCR were the same as primers for gene cloning primers. Subsequent qPCR analyses were conducted using SYBR green (TaKaRa Bio, Japan). Each amplification in a 96-well plate was performed in a 20 μL final volume containing 2.0 μL of 2× diluted cDNA template; 0.5 μL of each specific primer pair at 10 μM; 10 μL of 2× SYBR Premix Ex Taq

Construction of Plant Expression Vectors and L. pumilum Transformation
The coding region of the LpMT2 gene was amplified from pMD18-T-LpMT2 with the BamHI forward primer 5'-GGATCCATGTCTTGCTGTGGTGGAAA-3' and XhoI reverse primer 5'-C'TCGAGTTAGCACTTGCATGG GTTG-3'. The PCR product was ligated into plasmid pMD18-T vector. The plasmids were digested with BamHI and XhoI and then ligated into the BamHI and XhoI sites of pBI121 binary vector plasmids (We altered the vector). The plasmid DNA of pBI121-LpMT2 was transformed into the L. pumilum bulbs by Agrobacterium tumefaciens (strain EHA105 Takara, Tokyo, Japan) mediated transformation (Ishida et al., 1996;Zhao et al., 2002). The bulbs were germinated on MS + 0.5 mg·mL -1 BA + 0.05 mg·mL -1 NAA plates containing 50 mg·L -1 kanamycin (kana) to select kana-resistance plants, and the kana-resistance plants were transferred to fresh MS medium at 25 °C under 2000 Lux irradiation with a 16 h light/8 h dark photoperiod. Finally transgenic plants were identified using Northern blot (Jin et al., 2017). RNAs were extracted from eight-week old transgenic L. pumilum seedlings.

Stress Tolerance Analysis of the L. pumilum
Eight-week old L. pumilum untransgenic and transgenic seedlings at the same growth stage were selected for fresh MS medium + 200 mM, 250 mM, 300 mM NaCl, 9 mM, 11 mM, 13 mM H 2 O 2 , 20 mM, 50 mM, 80 mM NaHCO 3 stress treatment at 25 °C under 2000 Lux irradiation with a 16 h light/8 h dark photoperiod for 48 h to observe the extent of leaf injury. The leaf centers were determined by SPAD-502 Plus Chlorophyll Meter Model (Konica Minolta, Japan). The relative value of chlorophyll content in each leaf was measured. The free proline (Pro) content was determined by the ninhydrin method (Ma et al., 2007). Malondialdehyde (MDA) content was determined by the thiobarbituric acid method (Hodges et al., 1999). The degree of cell membrane damage was determined by conductance (Liu et al., 2008). Superoxide dismutase (SOD) was determined by its ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) (Giannopolitis & Ries, 1977).
Eight-week old untransgenic and transgenic L. pumilum seedlings were treated without (control) or with each of following solutions: 20 mM NaHCO 3 and 250 mM NaCl, respectively for 48 h. Na + and K + ion content of leaves were measured by an atomic absorption spectrophotometer (AA800, Perkin Elmer, USA) (Barragan et al., 2012). Net flux of the roots' K + and Na + were measured using Noninvasive Micro-test Technology (Zhao et al., 2017). jas.ccsenet.
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LpMT2
LpMT2   Under certain stress levels, a plant's cytochrome system will be destroyed, leading to a decrease in chlorophyll content (Li et al., 2016). The chlorophyll content of untransgenic plants' leaves had a greater decreasing trend than the transgenic L. pumilum plants under salt stress. This is an indication that LpMT2 has a significant impact on plant chlorophyll content. Proline, as an effective plant osmotic regulator, can scavenge reactive oxygen species (ROS), reduce lipid peroxidation, and prevent toxic amino acids accumulation (Li et al., 2011). When plants are subjected to environmental stress, the accumulation of proline increases significantly, which can enhance plant resistance to stress . When under stress, the proline content was greater in the transgenic plants compared to the untransgenic plants. Various adverse environments often first influence the cell membrane (Xu et al., 2007). Relative conductivity reflects the cell membrane integrity, while MDA is an indicator of membrane lipid peroxidation, which together reflects the degree of cellular damage (Verslues et al., 2006). The increase in MDA content in the early stage of stress indicates that the membrane lipid peroxidation occurred. As the treatment concentration increased, the MDA content decreased, which may be due to the excessive lipid peroxidation consumption due to respiration. The cell membrane destruction in transgenic plants was less than the untransgenic plants. SOD plays a very important role in protecting cells from oxidative damage (Giannopolitis & Ries, 1977). SOD activity decreased more in transgenic plants than that in the untransgenic plants as a result of stress. This is an indication that the LpMT2 gene transfer reduced membrane peroxidation and protected the plants. When comparing the physiological indexes between the transgenic and untransgenic L.pumilum, the transgenic resistant plants were significantly greater than the untransgenic plants.
Salt stress destroys the plant's intracellular nutritional balance, primarily due to the excessive Na + accumulation, which causes ion poisoning and other elemental deficits (Flowers et al., 2010). K + is an essential nutrient for plant growth, but because of the antagonism between Na + and K + , it is necessary to maintain a high K + content in order to improve the salt tolerance of plants (Adams et al., 1992). Under salt stress, due to the accumulation of Na + , K + uptake is inhibited and there is a decrease in plant K + . The Na + concentration in the transgenic plants was less than the untransgetic plants, while the K + concentration was greater under saline stress. These results demonstrate that transgenic plants can control Na + uptake, maintain K + content in leaves, and ensure normal growth under stress.
Salt stress weakens Na + absorption and transport, and accelerates the Na + poisoning (Guo et al., 2005). The transgenic plant Na + efflux rate under salt stress was significantly greater than the untransgenic plants, while K + efflux rate was the opposite. This is an indication that the transgenic plants can adapt to a saline environment by increasing the Na + efflux rate and reducing the K + efflux rate, therefore, the transgenic plants had greater stress resistance.
The biological MT protein has been studied for over 60 years, but its exact function is still unclear. In recent years the function of the MT gene and protein relationship to abiotic stress has been predominantly understood, but the molecular mechanism of how the MT gene regulates a plant response to stress is not clear, therefore, the investigation of plant MT continues to be important.
The over expression of LpMT2 gene improves the salt tolerance of L. pumilum, but the impact of high salt stress on plant growth and development is very complex. Halophytes can grow in a saline environment as the result of a combination of various salt-tolerant mechanisms. In addition, the function of MT in abiotic stress has been primarily understood in recent years, but the molecular mechanism of how the MT regulates a plant's response to stress is not yet clear. Further research on the physiological metabolism and cell structure of transgenic L. pumilum will be helpful to reveal the role of MT accumulation in plants and its relationship to salt tolerance in plants.

Conclusions
In this research we isolated a LpMT2 gene from L. pumilum, proved that the gene expression was different in different plant organs or under different stresses, and characterized its function in L. pumilum. This study indicates that the LpMT2 gene enhanced the transgenic plants' tolerance to salts (NaCl and NaHCO 3 ) and oxidative (H 2 O 2 ) stress. The following conclusions can be drawn from this research: 1) A 234bp long MT2 gene encoding 77 amino acids was cloned by PCR using cDNA of L. pumilum leaves as a template. QRT-PCR results showed that the LpMT2 gene has the greatest expression in flowers and the expression was significantly induced under salt and oxidative stress.
2) The plant expression vector pBI121-LpMT2 was constructed and transferred into the bulbs of L. pumilum by Agrobacterium-mediated successfully.
3) The untransgenic and transgenic L. pumilum were treated with different concentrations of NaCl, NaHCO 3 , and H 2 O 2 for 48 h. Compared with untransgenic plants, transgenic