GsLTP, a Non-Specific Lipid Transfer Protein from Glycine soja, Enhances Drought and Salt Stress Tolerance in Transgenic Tobacco

GsLTP, a Non-Specific Lipid Transfer Protein from Glycine soja, Enhances Drought and Salt Stress Tolerance in Transgenic Tobacco | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article GsLTP , a Non-Specific Lipid Transfer Protein from Glycine soja , Enhances Drought and Salt Stress Tolerance in Transgenic Tobacco Jinping Fan, Jingshuang Liu, Lei Cao, Jiayue Wang, Qingxue Meng, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-52965/v3 This work is licensed under a CC BY 4.0 License Status: Posted Version 3 posted You are reading this latest preprint version Show more versions Abstract Background: Lipid transfer proteins (LTPs) mainly exist in plant cell walls where they make an important impact in the transport of diverse lipophilic compounds. They have an important effect on the stress tolerance of plants by mediating plant responses to environmental stimuli and cell signal transduction pathways. Although the functions of several LTPs in different plant species have been identified, little is known about the biochemical and enzymatic activities of LTP family members in soybeans. Results: Herein, GsLTP was identified from Glycine soja using soybean gene microarray expression and screened in a salt stress expressed sequence tag (EST) library. The 369 bp open reading frame (ORF) encodes a protein of 122 amino acids. The cDNA sequence shares similarity with LTP genes in other plants such as soybean (93%) and Vigna (72%). The function of the gene was characterized in transgenic tobacco. Various physiological characteristics under different environmental stresses were investigated. We discovered that overexpression of GsLTP enhanced tolerance to drought and salt stresses. Conclusions: Our results indicate that GsLTP plays an important role in multiple abiotic signalling pathways, and provide a theoretical basis and practical gene resource for crop breeding. Biotechnology and Bioengineering Functional analysis gene cloning Glycine soja lipid transfer protein Southern blotting RT-PCR crop breeding Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Abiotic stresses, including low temperature, drought and salinity, can significantly reduce crop yield and quality [1]. Therefore, improving resistance to such adverse environments is extremely important for agriculture. New genes associated with stress resistance have been identified, and transgenic plants carrying these genes exhibit greater tolerance to drought stress [2], salt stress [3], cold stress [4], heavy metals [5], paraquat and wounding stress [1]. Non-specific lipid transfer proteins (nsLTPs) are widely present in the plant kingdom and are involved in the binding and transport of various lipids. All known plant nsLTP precursors include an N-terminal signal peptide, indicating that nsLTPs are secreted [6]. Mature nsLTPs are small proteins characterized by an eight-cysteine motif (8CM) with the consensus sequence C-Xn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C [7,8]. The eight cysteines are part of the four disulfide bonds that stabilize the three-dimensional structure of the hydrophobic cavity. This allows different lipids and hydrophobic compounds to bind [8-10]. Based on sequence similarity, ten types of nsLTPs are divided from Arabidopsis , rice, Solanaceae and other plants [11]. Lipids play an important role in stress tolerance by mediating plant responses to environmental stimuli and cell signal transduction pathways, resulting in changes in energy storage and maintaining basic cellular functions. In these processes, lipid transformation proteins (LTPs) act as lipid transporters. Non-specific lipid transfer proteins (nsLTPs) can bind or transfer various hydrophobic molecules in vitro , such as fatty acids, fatty acyl-coenzyme A, phospholipids, glycolipids and cutin monomers [6]. GsLTP belongs to the nsLTP family members which play important roles in numerous physiological processes, such as cutin and wax biosynthesis, abiotic and biotic stresses, seed development and germination, sexual reproduction, cell wall growth, nodulation, calmodulin (CaM) binding and responses to plant allergens [9,12,13]. Functions of LTPs under multiple abiotic stresses have been studied in various plants including sugarcane [14], Triticum turgidum [15], Solarium tuberosum [16] and Setaria italica [17]. NsLTPs from sugarcane respond to abiotic stresses and the signalling molecules salicylic acid and methyl jasmonate [14]. The wheat protein TdLTP4 promotes tolerance to abiotic and biotic stresses in Arabidopsis thaliana [15]. Transgenic potato lines over-expressing StnsLTP1 display enhanced cell membrane integrity under stress [16]. SiLTP enhances salt and drought stress tolerance in foxtail millet and may be partially upregulated by SiARDP [17]. Overexpression of a pepper nsLTP gene ( CALTP1 ) in transgenic Arabidopsis increased the resistance of Arabidopsis against infection by Pseudomonas syringae pv. tomato and Botrytis cinerea . The tolerance of Arabidopsis to NaCl and drought stress at all vegetative growth stages was improved [18]. Plant nsLTPs are involved in a variety of physiological functions, such as sebum transport, cutin synthesis, cell wall extension, pollen development, pollen tube growth and guidance, stigma and pollen adhesion, plant signalling, biological stresses, abiotic stresses and seed maturation [15,19,20]. Evidence suggests that nsLTPs are expressed in a wide range of plant cells. They are involved in various physiological and biochemical processes, including cuticle synthesis and embryo development, adaption to a variety of stress environments, and resistance to microorganisms. However, the exact biological functions of these proteins have not been elucidated. At low temperatures and under drought and NaCl treatment, high expression of nsLTPs has been detected in plant stems of tomato [21], barley [22] and sunflower [23]. These results indicate that nsLTPs help plants adapt to adverse environmental conditions. Glycine soja ( Glycine soja Sieb. et Zucc ), grown widely in Jilin Province in Northeast China, has a high tolerance to environmental challenges [24]. It is a model species for molecular mechanisms of low temperature and salt stresses. The ancestor of cultivated soybean is a highly adaptable plant species that grows well in desert conditions [8,25,26]. It is also an excellent genetic germplasm for mining abiotic resistance genes for agricultural crop breeding [27]. In one of our earlier studies in which Glycine soja was grown under 150mM NaCl and salinity responsive ESTs were identified. The expression of GsLTP was found to be induced to high salinity condition. In this study, we investigated the functions of GsLTP by analyzing the physiological responses of transgenic tobacco plants which were subjected to low temperature (4°C), salinity (300 mM NaCl) or drought (without water for 2, 4, 6, 8, 10, 12 or 14 days) treatment. Our results showed that GsLTP enhanced the salt tolerance and drought resistance of plants by regulating the synthesis of protective compounds. Thus, this study enhanced our understanding of the functions of LTP genes and the mechanisms by which plants tolerate multiple stresses. Moreover, it provides a theoretical basis and practical genetic resources for crop breeding. Results T he identification of full-length GsLTP The full-length sequence information of GsLTP was derived from extending the 254 bp partial EST to 689 bp by Phrap program. The extended GsLTP gene shares high similarity with genes in the LTP family, indicating its association with fatty acid transport in soybean. Characterisation of GsLTP gene and protein sequences After sequencing, multiple sequence alignment was performed with known LTP sequences. This comparison revealed high homology and the expected splice sites without nucleotide mutations. BLASTx analysis revealed that GsLTP is 369 bp in length, and encodes a predicted polypeptide of 122 amino acids. The molecular weight of the protein is ~13 kDa and the predicted isoelectric point (pI) is 8.74. Amino acid sequence analysis with the NCBI BLASTp program revealed similarity with LTP proteins in a various plants, further indicating membership of the LTP gene family. Therefore, the gene was named GsLTP and submitted to GenBank (accession number FJ825765) after confirming its sequence. Identification of transgenic tobacco lines To identify transgenic tobacco lines, total DNA was isolated and the inserted fragment was verified by PCR (Figure 1). The pBI121- GsLTP construct was successfully transferred into tobacco line #1, #3, #5, #6, #8, #10, #11, #12, #14, #15, #16, #17, #18, #19, #20, #21, #23, #24, #25, #26, #27 and #28. Total DNA was isolated from positive seedlings and digested with Bam HI for Southern blotting (Figure 2) and RT-PCR (Figure 3). Southern blot hybridization results showed that three transgenic tobacco lines yielded positive electrophoretic bands (lanes 4, 5 and 7), indicating that the GsLTP gene was successfully transferred into the plant genome. Conversely, tobacco lines without a hybridization signal indicated no gene integration. The signal in lanes 4 and 7 implied single-copy insertion into the genome. However, lane 5 has two hybridization bands, indicating the insertion of two gene copies. As expected, the positive control displayed one band, validating the appropriateness of the experimental procedure (Fig. 2). RT-PCR was also used to investigate gene transcription in plants. The successfully amplified fragments in lanes 3, 6, 8, 10, 12, 14 and 16 confirmed these independent lines harbor the target gene (Fig. 3). After reliable identification, two transgenic lines (#85 and #96) were chosen for phenotype experiments analysis, and transgenic line #96 were chosen for the chlorophyll, MDA and proline content of plants as well as conductivity of the leaves analysis. Overexpression of GsLTP enhances drought and salt stress tolerance in transgenic tobacco but not improves low temperature resistance Chlorophyll content, MDA and conductivity are three critical indexes of cell damage induced by the stresses. The results in Tables 1 and 2 reveal no significant difference between transgenic plants and non-transgenic plants regarding chlorophyll content under low temperature treatment ( p = 0.2248, a = 0.05). However, significant differences in chlorophyll content were observed under drought ( p = 0.0077, a = 0.05) and salinity ( p = 0.0101, a = 0.05) treatments. The chlorophyll content of transgenic seedlings was also significantly higher than that of control seedlings under drought treatment. We, therefore, concluded that transgenic seedlings displayed higher tolerance to drought conditions than control seedlings. In addition, the MDA content of transgenic seedlings in each treatment was significantly higher than that of control seedlings ( p <0.0001, a = 0.05). This suggests that transgenic seedlings were more resistant to osmotic stress than control seedlings. Under all treatments, there was no significant difference in the conductivity between transgenic and control seedlings. Thus, transgenic seedlings did not demonstrate higher tolerance to osmotic stress than control seedlings under any of the treatments. Proline (Pro) is one of the common canonical amino acids which are critical to plants stress tolerance. There was a significant difference between transgenic and control plants in terms of Pro content ( p <0.0001, a = 0.05) under low temperature and salinity treatments, but the difference was less significant ( p = 0.0413, a = 0.05) under drought treatment. Because the Pro content of transgenic seedlings was much higher than that of control seedlings, transgenic seedlings exhibited better resistance to osmotic stress. Tobacco seedlings harboring the GsLTP gene displayed greater resistance than control seedlings based on membrane permeability and enzymatic activity. Based on chlorophyll content, the GsLTP gene significantly enhanced tolerance to drought but did not enhance tolerance to low temperature stress or salinity stress. Based on the MDA and Pro content, the LTP gene significantly enhanced resistance to low temperature, drought and salinity. The accumulation of these osmotic protective substances in transgenic seedlings in response to stress is an important biochemical indicator for measuring tolerance to low temperature, drought and salinity. Our results clearly showed that resistance of transgenic tobacco seedlings to drought and salinity was improved (Fig. 4). Compared with control seedlings, all physiological parameters tested were enhanced to various degrees in transgenic tobacco seedlings. Thus, GsLTP improved the tolerance of tobacco plants to drought and salinity stresses. Table 1. Variance analysis of chlorophyll content, MDA content, Conductance and Pro content under different treatment. Pr>F Source Chlorophyll content MDA content Conductance Pro content Low temperature Treat 0.2248 <0.0001 0.2317 <0.0001 (4 ℃ ) Data <0.0001 <0.0001 0.0303 <0.0001 Repeat 0.4631 0.9256 0.6446 0.8066 Drought treatment Treat 0.0077 <0.0001 0.7990 0.0413 Data 0.0001 0.0005 0.0076 <0.0001 Repeat 0.5453 0.6738 0.8940 0.3755 Salt treatment Treat 0.0101 <0.0001 0.0795 <0.0001 Data <0.0001 <0.0001 0.0041 <0.0001 Repeat 0.3382 0.8806 0.7632 0.8378 Table 2. T-tests (LSD) for chlorophy content, MDA content, Conductance and Pro content in different treatment. T-tests (LSD) Treat Low temperature (4°C) Drought treatment Salt treatment Chlorophyll content control 0.71847a 0.70110b 0.65486a LTP 0.73573a 0.89190a 0.94614a MDA content control 0.0129333a 0.0110952a 0.0123810a LTP 0.0078667b 0.0080000b 0.0071905b Conductance control 81.727a 73.631a 80.064a LTP 86.061a 76.024a 86.879a Pro conten t control 0.045333b 0.036000b 0.049238b LTP 0.077000a 0.052667a 0.082952a Note ： control ： wild type plant; LTP: Seedlings transformed with LTP gene Discussion LTPs are an important class of proteins in plants [6]. In the past decade, some progresses have been made in analyzing the expression profiles of LTPs in response to plant stresses [18,28,29,30]. It has been discovered that the expression levels of LTPs were induced to low temperature, drought and salinity [31-33]. Although these prior studies indicated that LTPs may participate in the physiological regulation of plant stress response, very limited studies have demonstrated the protection role of this LTPs in plants under stress growth conditions. In the present study, a 369 full-length sequence of the GsLTP gene that encodes a predicted polypeptide of 122 amino acids was obtained. Multiple tobacco plants harboring the GsLTP gene have been produced. The physiological analysis on these transgenic plants clearly showed that overexpression of GsLTP can exert a protection function to tobacco plants under drought and salinity growth conditions. Under abiotic stress, e.g., drought and salinity, growth conditions, cell membrane stabilities may be decreased and permeability is increased. These alterations may lead to the release of cytoplasmic components to cell wall and degradation of chlorophyll. For example, the exosmosis of inorganic ions and soluble sugars in the cell leads to the increase of relative conductivity [34]. The higher concentration of chlorophyll and low conductivity in the transgenic plants indicated that overexpression of GsLTP in tobacco protected the cell membrane and prevent chlorophyll degradation. Under abiotic stress condition, the destruction of the cell membrane is also related to the enhancement of the peroxidation of the cell membrane [34]. The lower accumulation of MDA, a main product of membrane lipid peroxidation, further indicated the less deleterious modification on the cell membrane in the transgenic plants. Pro is normally accumulated under abiotic stress and plays a positive role in maintaining water balance and cell membrane integrity in plants [35]. After the application of stress, the more significant accumulation of Pro in the transgenic tobacco indicated that the overexpression of GsLTP promote the biosynthesis of Pro. This might be another factor that contributed to the improved resistance to drought and salinity in the transgenic plants. Future more in-depth transcriptomic analysis, e.g., RNA Seq, on these transgenic plants will elucidate the molecular mechanisms imposing the resistance to drought and salinity. The growth and yield of soybean are negatively affected by adverse growing environment such as drought and salinity. The combination of transgenic technology and traditional breeding is the most effective way to rapidly improve the resilience of soybean. In this study, only physiological indexes of transgenic tobacco were measured. In future studies, the LTP gene can be transferred into soybean and the related physiological parameters will be measured. Other components discovered from a more detailed transcriptomic analysis can also be used to improve abiotic stress resistance in soybean. Transformation strategies harboring LTP and other genes with known abiotic stress resistance ability may lead to more significant improvement in soybean. Conclusion We isolated the GsLTP from G. Soja and transformed it into tobacco. Transgenic plants were assessed for stress resistance by analyzing physiological parameters and biochemical indicators following low temperature, drought and salinity stress treatments. GsLTP improved tolerance to drought and salinity to different degrees. However, resistance to low temperature was not enhanced. In summary, the genetic transformation of GsLTP into plants can improve resistance to environmental stresses. Methods Plant materials and growing conditions The seeds of G.soja G50109 were obtained from the Jilin Academy of Agricultural Sciences (Changchun, China). To remove waxiness and promote germination, treat seeds with sulfuric acid (98%) for 10 minutes, wash them in sterile water 5 times, and store them in darkness for at least 2 days. After that, wild soybean seedlings were transferred to soil and incubated for 19 days at 24-26°C under a 16 hours light / 8 hours dark cycle condition. Leaves and roots were collected and stored at -80°C after being snap-frozen in liquid nitrogen. Wild-type (WT) Nicotiana tabacum Longjiang 911, from Mudanjiang Normal University, China, was used for transformation. Tobacco seeds were soaked for 3-5 h in water, sterilized with 70% ethanol for 30 s, treated with 0.1% mercuric chloride for 3 min, and rinsed with sterile water five times. Finally, all seeds were placed on M 0 medium (1/8 MS + 0.8% agar, pH 5.8). When young leaves reached 2-3 cm, edges were removed, leaves were cut into discs (7 mm diameter) and placed with adaxial sides facing downwards on M 1 medium (MS + 1.0 mg L6-BA + 0.1 mg LNAA + 0.75% agar, pH 5.8) for pre-culturing for 2 to 3 days. Leaf disc incisions began to swell and callus tissue started growing. WT and transgenic lines were treated with 0 mM or 300 mM NaCl, or subjected to drought stress, to analyze gene function. Isolation and sequence analysis of GsLTP Gene expression profiles were obtained with the soybean gene expression microarray. Salt stress expressed sequence tag (EST) library was used to determine gene expression during early osmotic stress. The GsLTP gene is highly similar to clone pTE-6424 in the NCBI database, as revealed by electronic extension using Phrap program (http://www.phrap.org). A BLAST analysis was carried out (http://www.ncbi.nlm.nih.gov/blast/) and the full-length sequence was obtained. The full sequence of the ORF was translated from the nucleic acid sequence by DNAMAN software (Version 4.0, Lynnon Biosoft, Quebec, Canada). The full-length cDNA of GsLTP was obtained by homologous cloning. Total RNA from G. soja G50109 was extracted from soybean leaves using a GoldScript cDNA Synthesis Kit (Invitrogen) and converted into cDNA through reverse transcription. The full- length cDNA of GsLTP was amplified using gene-specific primers 5’-AGATGGCAGAGACATTCC-3’ and 5’-TGCACCTTTCAATAC-3’. The reaction contained 2.5 μl of 10× PCR buffer (Mg 2+ -free), 3 μl of 25 mM Mg 2+ , 2 μl of 2.5 mM each dNTP, 1 μl of DNA template (DNA content <1 ng), and 0.2 μl of Taq polymerase (5 U/µl) or Pyrobest TM DNA polymerase. The PCR products were purified and cloned into the pGEM-T vector, and the Pyrobest TM DNA polymerase was used for PCR identification and DNA sequencing. Sequence alignment was performed using DNAMAN. Sequence similarity was verified by BLASTP ( http://blast.ncbi.nlm.nih.gov/Blast.cgi) . The vector was transformed to Escherichia coli DH5α and cultured on Luria Bertani (LB) solid medium containing ampicillin (100 mg/L). Construction of the pBI121- GsLTP expression vector The PCR-amplified LTP gene was cloned into the plant expression vector pBI121, and the insert was verified using forward (5’-TAGGATCC( Bam HI)TAAGTCTTTGTTCATTTGAGTAG -3’) and reverse (5’-TACGAGCTC( Sac I)ATATATAGAATTCTGCGACT-3’) primers. PCR was performed as described above, and a 447 bp fragment was identified and purified as described above. Finally, the target plant expression vector harboring the LTP gene was transformed into Agrobacterium tumefaciens LBA4404 . Tobacco transformation The A. tumefaciens LBA4404 strain containing the pBI121- GsLTP vector was transformed into tobacco leaf disks following pre-incubation for 2-3 days. After co-culture for 3-4 days, leaf disks were degermed for 2-3 h with a liquid medium containing antibiotics that prohibit Agrobacterium growth. These leaf discs were dried on filter paper and then placed on shoot regeneration media for further selection. Five to six weeks later, small shoots of 3-4 cm in size were transferred to rooting medium. Approximate 14 days later, most seedlings developed a proliferate root system. These putative transgenic plantlets were then moved into a humid culture room without sealing for 1 week. Thereafter, seedlings were planted in flower pots (grass peat: sandy soil = 2:1) and grown in an artificial climatic cabinet (26°C, 126 μmol m -2 s -1 , 16 h day -1 illumination, 85-90% relative humidity). Molecular biological identification Genomic DNA was extracted from selected transgenic and wild type plants, and integration of the GsLTP gene was confirmed by PCR and Southern blotting using a probe consisting of a 375 bp fragment of the GsLTP gene generated from total DNA in plants digested with Bam HI. GsLTP mRNA transcription was determined by RT-PCR. The above experiments were carried out on the T 3 generation of all lines. A total of 180 seedlings were confirmed to be transgenic and used in further stress tolerance tests. The material did not been deposited in a publicly available herbarium. Analysis of stress tolerance and GsLTP function Three groups of seedlings at a similar developmental stage (30 transgenic seedlings and 30 untransformed control seedlings per group) were subjected to low temperature (4°C), salinity (300 mM NaCl) and drought (without water for 2, 4, 6, 8, 10, 12 or 14 days). Chlorophyll content [36], malondialdehyde (MDA) content (TBA assay) [37,38], proline (Pro) content (ninhydrin assay) [39], leaf membrane permeability (relative conductivity assay) [40] and morphology were investigated for each treatment. Physiological indices and gene functions were statistically analyzed using SAS6.12. Abbreviations LTPs: Lipid transfer proteins ORF: open reading frame EST: expressed sequence tag nsLTPs: Non-specific lipid transfer proteins MDA: malondialdehyde Pro: proline ABA: abscisic acid Declarations Ethics approval and consent to participate: Not applicable Consent for publication: Not applicable Availability of data and materials: All data generated or analyzed during this study are included in this published article. Competing interests: The authors declare that they have no competing interests. Funding: This work was supported by National Key Research and Development Projects, grant number 2016YFC0500306-02, Heilongjiang Natural Science Fund Project Plan Mission Statement, grant number C2017030 and Heilongjiang Province Education Project Fund, grant number 12531014. The funders had no role in study design, data collection, interpretation, or decision-making in relation to submission for publication. Authors ’ contributions: DC and JF conceived and designed research. 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Jia X, Xiaoguang D, Jun Y, Beeching JR, Peng Z: Enhanced reactive oxygen species scavenging by overproduction of superoxide dismutase and catalase delays postharvest physiological deterioration of cassava storage roots . Plant Physiology 2013, 161 (3):1517-1528. Zhang T, Mo J, Zhou K, Chang Y, Liu Z: Overexpression of Brassica campestris BcICE1 gene increases abiotic stress tolerance in tobacco . Plant Physiology and Biochemistry . Ben RW, Ben-Saad R, Meynard D, Verdeil JL, Azaza J, Zouari N, Fki L, Guiderdoni E, Al-Doss A, Hassairi A: Ectopic Expression of Aeluropus littoralis Plasma Membrane Protein Gene AlTMP1 Confers Abiotic Stress Tolerance in Transgenic Tobacco by Improving Water Status and Cation Homeostasis . International Journal of Molecular Sciences 2017, 18 (4):692. 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University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jingshuang","middleName":"","lastName":"Liu","suffix":""},{"id":7116847,"identity":"d1c74ab0-26b5-460b-946e-8378fbb5f566","order_by":2,"name":"Lei Cao","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Cao","suffix":""},{"id":7116848,"identity":"eb5454fb-b272-4ba3-8f51-3c95414ed24e","order_by":3,"name":"Jiayue Wang","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiayue","middleName":"","lastName":"Wang","suffix":""},{"id":7116849,"identity":"69d886f9-4271-4302-a6cd-5ba4f9cdbb89","order_by":4,"name":"Qingxue Meng","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qingxue","middleName":"","lastName":"Meng","suffix":""},{"id":7116850,"identity":"9462741a-b404-432f-a36f-c0900576992e","order_by":5,"name":"Daidi Che","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYDCCA4wNEgkMBxj4oXzGBqK1SDYQr4WBQQJEGhwgVgvf8cONNx7U3JEzvt188DMPg43shgPMzx7g0yJ5JrHZIuHYM2OzO8eSpXkY0ow3HGAzN8CnxeBAYptEYsPhxG03cgyAWg4nbjjAwyaBV8v5h2At9Ztn5H/+zcPwnwgtNyC2JBhI5LABbTlAWIvkjYcgvxw2nHEjzcxyjkGy8czDbGZ4tfCdT39480fNYXn+GcmPb7ypsJPtO978DK8WdHcCMTMJ6kfBKBgFo2AUYAcASzlRwOonWukAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-0477-9026","institution":"Northeast Agricultural University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Daidi","middleName":"","lastName":"Che","suffix":""}],"badges":[],"createdAt":"2020-08-03 12:07:29","currentVersionCode":3,"declarations":"","doi":"10.21203/rs.3.rs-52965/v3","doiUrl":"https://doi.org/10.21203/rs.3.rs-52965/v3","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":4794655,"identity":"0a68b001-b1cb-4824-a904-0df727a1ded1","added_by":"auto","created_at":"2021-01-07 21:36:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":316982,"visible":true,"origin":"","legend":"PCR identification of transgenic tobacco plants M: DL 2000 marker. +: positive control. -: negative control. 1-28: putative transgenic tobacco plants. 21: #85.26: #96","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-52965/v3/18fa7e615b82cef3b97cead8.png"},{"id":4794556,"identity":"93589ae2-dc02-4449-a20b-f76064cebfc6","added_by":"auto","created_at":"2021-01-07 21:33:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":242575,"visible":true,"origin":"","legend":"Southern blot analysis of transgenic tobacco plants M: DL 2000 marker. 1: positive control. 2:negative control (without hybridization signal). 3-9: putative transgenic plants. 4: #85.5: #96","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-52965/v3/191f7fffb5dfdc2c550b985a.png"},{"id":4794687,"identity":"b2f0cd05-7b63-4b9c-bd41-60d05d203d14","added_by":"auto","created_at":"2021-01-07 21:39:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":210913,"visible":true,"origin":"","legend":"RT-PCR identification of transgenic tobacco plants M: DL2000 marker. 1: control I: PCR product of wild-type tobacco RNA. 2: control II: PCR product of wild-type tobacco cDNA. 3: positive control: PCR product of pBI-GsLTP plasmid. 4: negative control: PCR product of water. 5, 7, 9, 11, 13, 15: PCR product results of all identified putative transgenic tobacco plants RNA. 6, 8, 10, 12, 14, 16: PCR product results of 6: #85.7, 8: #96","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-52965/v3/42993ed63a7b63c2b20bac60.png"},{"id":4794793,"identity":"ca336c2e-3d29-4044-9593-89779e3f8c10","added_by":"auto","created_at":"2021-01-07 21:42:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1056551,"visible":true,"origin":"","legend":"Phenotype experiments analysis of WT and plants under drought and 300mM NaCl treatment GsLTP#85 and GsLTP#96: transgenic tobacco plants","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-52965/v3/6f038f00756cf623e74a623a.png"},{"id":15670547,"identity":"3ffc6386-6048-4c8f-8f71-77e3820c198c","added_by":"auto","created_at":"2021-11-18 14:00:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3156857,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-52965/v3/d3a3ecb0-9fde-41e3-8ada-22351afdd1e0.pdf"},{"id":4794658,"identity":"79da7d23-a604-4e1e-8513-fa1ee343b690","added_by":"auto","created_at":"2021-01-07 21:36:31","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":319021,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-52965/v3/4144e518baf64d1dd86984f1.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cem\u003eGsLTP\u003c/em\u003e, a Non-Specific Lipid Transfer Protein from \u003cem\u003eGlycine soja\u003c/em\u003e, Enhances Drought and Salt Stress Tolerance in Transgenic Tobacco\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eAbiotic stresses, including low temperature, drought and salinity, can significantly reduce crop yield and quality [1]. Therefore, improving resistance to such adverse environments is extremely important for agriculture. New genes associated with stress resistance have been identified, and transgenic plants carrying these genes exhibit greater tolerance to drought stress [2], salt stress [3], cold stress [4], heavy metals [5], paraquat and wounding stress [1].\u003c/p\u003e\n\u003cp\u003eNon-specific lipid transfer proteins (nsLTPs) are widely present in the plant kingdom and are involved in the binding and transport of various lipids. All known plant nsLTP precursors include an N-terminal signal peptide, indicating that nsLTPs are secreted [6]. Mature nsLTPs are small proteins characterized by an eight-cysteine motif (8CM) with the consensus sequence C-Xn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C [7,8]. The eight cysteines are part of the four disulfide bonds that stabilize the three-dimensional structure of the hydrophobic cavity. This allows different lipids and hydrophobic compounds to bind [8-10]. Based on sequence similarity, ten types of nsLTPs are divided from \u003cem\u003eArabidopsis\u003c/em\u003e, rice, \u003cem\u003eSolanaceae \u003c/em\u003eand other plants [11].\u003c/p\u003e\n\u003cp\u003eLipids play an important role in stress tolerance by mediating plant responses to environmental stimuli and cell signal transduction pathways, resulting in changes in energy storage and maintaining basic cellular functions. In these processes, lipid transformation proteins (LTPs) act as lipid transporters. Non-specific lipid transfer proteins (nsLTPs) can bind or transfer various hydrophobic molecules \u003cem\u003ein vitro\u003c/em\u003e, such as fatty acids, fatty acyl-coenzyme A, phospholipids, glycolipids and cutin monomers [6]. \u003cem\u003eGsLTP\u003c/em\u003e belongs to the nsLTP family members which play important roles in numerous physiological processes, such as cutin and wax biosynthesis, abiotic and biotic stresses, seed development and germination, sexual reproduction, cell wall growth, nodulation, calmodulin (CaM) binding and responses to plant allergens [9,12,13].\u003c/p\u003e\n\u003cp\u003eFunctions of LTPs under multiple abiotic stresses have been studied in various plants including sugarcane [14], \u003cem\u003eTriticum turgidum\u003c/em\u003e [15], \u003cem\u003eSolarium tuberosum\u003c/em\u003e [16] and \u003ca href=\"http://apps.webofknowledge.com/full_record.do?product=UA\u0026amp;search_mode=GeneralSearch\u0026amp;qid=3\u0026amp;SID=6ChNwJejytFKoIOw9ci\u0026amp;page=1\u0026amp;doc=5\"\u003e\u003cem\u003eSetaria italica\u003c/em\u003e\u003c/a\u003e [17]. NsLTPs from sugarcane respond to abiotic stresses and the signalling molecules salicylic acid and methyl jasmonate [14]. The wheat protein \u003cem\u003eTdLTP4\u003c/em\u003e promotes tolerance to abiotic and biotic stresses in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e [15]. Transgenic potato lines over-expressing \u003cem\u003eStnsLTP1\u003c/em\u003e display enhanced cell membrane integrity under stress [16]. \u003cem\u003eSiLTP\u003c/em\u003e enhances salt and drought stress tolerance in foxtail millet and may be partially upregulated by \u003cem\u003eSiARDP\u003c/em\u003e [17]. Overexpression of a pepper nsLTP gene (\u003cem\u003eCALTP1\u003c/em\u003e) in transgenic \u003cem\u003eArabidopsis \u003c/em\u003eincreased the resistance of \u003cem\u003eArabidopsis\u003c/em\u003e against infection by \u003cem\u003ePseudomonas syringae \u003c/em\u003epv. tomato and \u003cem\u003eBotrytis cinerea\u003c/em\u003e. The tolerance of \u003cem\u003eArabidopsis\u003c/em\u003e to NaCl and drought stress at all vegetative growth stages was improved [18].\u003c/p\u003e\n\u003cp\u003ePlant nsLTPs are involved in a variety of physiological functions, such as sebum transport, cutin synthesis, cell wall extension, pollen development, pollen tube growth and guidance, stigma and pollen adhesion, plant signalling, biological stresses, abiotic stresses and seed maturation [15,19,20]. Evidence suggests that nsLTPs are expressed in a wide range of plant cells. They are involved in various physiological and biochemical processes, including cuticle synthesis and embryo development, adaption to a variety of stress environments, and resistance to microorganisms. However, the exact biological functions of these proteins have not been elucidated. At low temperatures and under drought and NaCl treatment, high expression of nsLTPs has been detected in plant stems of tomato [21], barley [22] and sunflower [23]. These results indicate that nsLTPs help plants adapt to adverse environmental conditions.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGlycine soja \u003c/em\u003e(\u003cem\u003eGlycine soja Sieb. et Zucc\u003c/em\u003e), grown widely in Jilin Province in Northeast China, has a high tolerance to environmental challenges [24]. It is a model species for molecular mechanisms of low temperature and salt stresses. The ancestor of cultivated soybean is a highly adaptable plant species that grows well in desert conditions [8,25,26]. It is also an excellent genetic germplasm for mining abiotic resistance genes for agricultural crop breeding [27]. In one of our earlier studies in which \u003cem\u003eGlycine soja \u003c/em\u003ewas grown under 150mM NaCl and salinity responsive ESTs were identified. The expression of \u003cem\u003eGsLTP\u003c/em\u003e was found to be induced to high salinity condition. In this study, we investigated the functions of \u003cem\u003eGsLTP\u003c/em\u003e by analyzing the physiological responses of transgenic tobacco plants which were subjected to low temperature (4\u0026deg;C), salinity (300 mM NaCl) or drought (without water for 2, 4, 6, 8, 10, 12 or 14 days) treatment. Our results showed that \u003cem\u003eGsLTP \u003c/em\u003eenhanced the salt tolerance and drought resistance of plants by regulating the synthesis of protective compounds. Thus, this study enhanced our understanding of the functions of\u003cem\u003e LTP\u003c/em\u003e genes and the mechanisms by which plants tolerate multiple stresses. Moreover, it provides a theoretical basis and practical genetic resources for crop breeding.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003cstrong\u003ehe identification of\u003c/strong\u003e\u003cstrong\u003e full-length\u003cem\u003e GsLTP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe full-length sequence information of \u003cem\u003eGsLTP\u003c/em\u003e was derived from extending the 254 bp partial EST to 689 bp by Phrap program. The extended \u003cem\u003eGsLTP\u003c/em\u003e gene shares high similarity with genes in the \u003cem\u003eLTP\u003c/em\u003e family, indicating its association with fatty acid transport in soybean.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterisation of \u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eGsLTP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e gene and protein sequences\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter sequencing, multiple sequence alignment was performed with known \u003cem\u003eLTP\u003c/em\u003e sequences. This comparison revealed high homology and the expected splice sites without nucleotide mutations. BLASTx analysis revealed that \u003cem\u003eGsLTP \u003c/em\u003eis 369 bp in length, and encodes a predicted polypeptide of 122 amino acids. The molecular weight of the protein is ~13 kDa and the predicted isoelectric point (pI) is 8.74. Amino acid sequence analysis with the NCBI BLASTp program revealed similarity with \u003cem\u003eLTP\u003c/em\u003e proteins in a various plants, further indicating membership of the \u003cem\u003eLTP\u003c/em\u003e gene family. Therefore, the gene was named \u003cem\u003eGsLTP\u003c/em\u003e and submitted to GenBank (accession number FJ825765) after confirming its sequence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of transgenic tobacco lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify transgenic tobacco lines, total DNA was isolated and the inserted fragment was verified by PCR (Figure 1). The pBI121-\u003cem\u003eGsLTP\u003c/em\u003e construct was successfully transferred into tobacco line #1, #3, #5, #6, #8, #10, #11, #12, #14, #15, #16, #17, #18, #19, #20, #21, #23, #24, #25, #26, #27 and #28. Total DNA was isolated from positive seedlings and digested with \u003cem\u003eBam\u003c/em\u003eHI for Southern blotting (Figure 2) and RT-PCR (Figure 3). Southern blot hybridization results showed that three transgenic tobacco lines yielded positive electrophoretic bands (lanes 4, 5 and 7), indicating that the \u003cem\u003eGsLTP\u003c/em\u003e gene was successfully transferred into the plant genome. Conversely, tobacco lines without a hybridization signal indicated no gene integration. The signal in lanes 4 and 7 implied single-copy insertion into the genome. However, lane 5 has two hybridization bands, indicating the insertion of two gene copies. As expected, the positive control displayed one band, validating the appropriateness of the experimental procedure (Fig. 2). RT-PCR was also used to investigate gene transcription in plants. The successfully amplified fragments in lanes 3, 6, 8, 10, 12, 14 and 16 confirmed these independent lines harbor the target gene (Fig. 3). After reliable identification, two transgenic lines (#85 and #96) were chosen for phenotype experiments analysis, and transgenic line #96 were chosen for the chlorophyll, MDA and proline content of plants as well as conductivity of the leaves analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverexpression of \u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eGsLTP \u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eenhances drought and salt stress tolerance in transgenic tobacco but not improves low temperature resistance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChlorophyll content, MDA and conductivity are three critical indexes of cell damage induced by the stresses. The results in Tables 1 and 2 reveal no significant difference between transgenic plants and non-transgenic plants regarding chlorophyll content under low temperature treatment (\u003cem\u003ep\u003c/em\u003e = 0.2248, a = 0.05). However, significant differences in chlorophyll content were observed under drought (\u003cem\u003ep\u003c/em\u003e = 0.0077, a = 0.05) and salinity (\u003cem\u003ep\u003c/em\u003e = 0.0101, a = 0.05) treatments. The chlorophyll content of transgenic seedlings was also significantly higher than that of control seedlings under drought treatment. We, therefore, concluded that transgenic seedlings displayed higher tolerance to drought conditions than control seedlings. In addition, the MDA content of transgenic seedlings in each treatment was significantly higher than that of control seedlings (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, a = 0.05). This suggests that transgenic seedlings were more resistant to osmotic stress than control seedlings. Under all treatments, there was no significant difference in the conductivity between transgenic and control seedlings. Thus, transgenic seedlings did not demonstrate higher tolerance to osmotic stress than control seedlings under any of the treatments.\u003c/p\u003e\n\u003cp\u003eProline (Pro) is one of the common canonical amino acids which are critical to plants stress tolerance. There was a significant difference between transgenic and control plants in terms of Pro content (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, a = 0.05) under low temperature and salinity treatments, but the difference was less significant (\u003cem\u003ep\u003c/em\u003e = 0.0413, a = 0.05) under drought treatment. Because the Pro content of transgenic seedlings was much higher than that of control seedlings, transgenic seedlings exhibited better resistance to osmotic stress.\u003c/p\u003e\n\u003cp\u003eTobacco seedlings harboring the \u003cem\u003eGsLTP\u003c/em\u003e gene displayed greater resistance than control seedlings based on membrane permeability and enzymatic activity. Based on chlorophyll content, the \u003cem\u003eGsLTP\u003c/em\u003e gene significantly enhanced tolerance to drought but did not enhance tolerance to low temperature stress or salinity stress. Based on the MDA and Pro content, the \u003cem\u003eLTP\u003c/em\u003e gene significantly enhanced resistance to low temperature, drought and salinity. The accumulation of these osmotic protective substances in transgenic seedlings in response to stress is an important biochemical indicator for measuring tolerance to low temperature, drought and salinity. Our results clearly showed that resistance of transgenic tobacco seedlings to drought and salinity was improved (Fig. 4). Compared with control seedlings, all physiological parameters tested were enhanced to various degrees in transgenic tobacco seedlings. Thus, \u003cem\u003eGsLTP \u003c/em\u003eimproved the tolerance of tobacco plants to drought and salinity stresses.\u003c/p\u003e\u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:justify;text-indent:0in;line-height:200%;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;line-height:200%;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eTable 1.\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:15px;line-height:200%;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eVariance analysis of\u003c/span\u003e\u003cspan style='font-size:15px;line-height:200%;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026nbsp;chlorophyll content, MDA content, Conductance and Pro content\u003c/span\u003e\u003cspan style='font-size:15px;line-height:200%;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026nbsp;under different treatment.\u003c/span\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\n \u003ctable style=\"width: 5.0e+2pt;border: none;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 99.4pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;line-height:107%;font-family:\"Calibri\",sans-serif;'\u003ePr\u0026gt;F\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;line-height:107%;font-family:\"Calibri\",sans-serif;'\u003eSource\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.9pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan 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style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;line-height:107%;font-family:\"Calibri\",sans-serif;'\u003eConductance\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:\"Times New Roman\",serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;line-height:107%;font-family:\"Calibri\",sans-serif;'\u003ePro content\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 99.4pt;border: none;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eLow temperature\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65.5pt;border: none;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eTreat\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.9pt;border: none;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.2248\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.15pt;border: none;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026lt;0.0001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border: none;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n 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style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e(4\u003c/span\u003e\u003cspan style='font-size:15px;font-family:\"Cambria Math\",serif;color:windowtext;'\u003e℃\u003c/span\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65.5pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eData\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.0303\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026lt;0.0001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 99.4pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65.5pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eRepeat\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.9pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.4631\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.15pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.9256\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.6446\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.8066\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 99.4pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eDrought treatment\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65.5pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eTreat\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.9pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp 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style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.7990\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.0413\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 99.4pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65.5pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eData\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.9pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.0001\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.15pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.0005\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.0076\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026lt;0.0001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 99.4pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65.5pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eRepeat\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.9pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.5453\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.15pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.6738\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.8940\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.3755\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 99.4pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eSalt treatment\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65.5pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eTreat\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.9pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.0101\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.15pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026lt;0.0001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.0795\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026lt;0.0001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 99.4pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65.5pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eData\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.9pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026lt;0.0001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.15pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026lt;0.0001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.0041\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65pt;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026lt;0.0001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 99.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eRepeat\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.3382\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73.15pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.8806\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.7632\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.2pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.8378\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:justify;text-indent:0in;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:justify;text-indent:0in;line-height:200%;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;line-height:200%;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eTable 2.\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;line-height:200%;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:15px;line-height:200%;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eT-tests (LSD) for chlorophy content, MDA content, Conductance and Pro content in different treatment.\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\n \u003ctable style=\"width: 5.0e+2pt;border: none;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:124.35pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:40.7pt;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eT-tests (LSD)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:45.4pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:40.7pt;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eTreat\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:106.35pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:40.7pt;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eLow temperature (4\u0026deg;C)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:106.3pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:40.7pt;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eDrought treatment\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:114.0pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:40.7pt;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eSalt treatment\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n 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0.25in;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eLTP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 0.25in;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.077000a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 0.25in;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.052667a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 0.25in;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:center;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-size:15px;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003e0.082952a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style='margin-top:12.0pt;margin-right:0in;margin-bottom:12.0pt;margin-left:0in;text-align:left;line-height:200%;font-size:12px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:22.0pt;'\u003e\u003cspan style='font-size:15px;line-height:200%;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003eNote\u003c/span\u003e\u003cspan style=\"font-size:15px;line-height:200%;font-family:SimSun;color:windowtext;\"\u003e：\u003c/span\u003e\u003cspan style='font-size:15px;line-height:200%;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003econtrol\u003c/span\u003e\u003cspan style=\"font-size:15px;line-height:200%;font-family:SimSun;color:windowtext;\"\u003e：\u003c/span\u003e\u003cspan style='font-size:15px;line-height:200%;font-family:\"Calibri\",sans-serif;color:windowtext;'\u003ewild type plant; LTP: Seedlings transformed with \u003cem\u003eLTP\u003c/em\u003e gene\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u003cspan style='font-size:15px;line-height:107%;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLTPs are an important class of proteins in plants [6]. In the past decade, some progresses have been made in analyzing the expression profiles of LTPs in response to plant stresses [18,28,29,30]. It has been discovered that the expression levels of LTPs were induced to low temperature, drought and salinity [31-33]. Although these prior studies indicated that LTPs may participate in the physiological regulation of plant stress response, very limited studies have demonstrated the protection role of this LTPs in plants under stress growth conditions. In the present study, a 369 full-length sequence of the\u003cem\u003e GsLTP\u003c/em\u003e gene that encodes a predicted polypeptide of 122 amino acids was obtained. Multiple tobacco plants harboring the \u003cem\u003eGsLTP\u003c/em\u003e gene have been produced. The physiological analysis on these transgenic plants clearly showed that overexpression of \u003cem\u003eGsLTP\u003c/em\u003e can exert a protection function to tobacco plants under drought and salinity growth conditions.\u003c/p\u003e\n\u003cp\u003eUnder abiotic stress, e.g., drought and salinity, growth conditions, cell membrane stabilities may be decreased and permeability is increased. These alterations may lead to the release of cytoplasmic components to cell wall and degradation of chlorophyll. For example, the exosmosis of inorganic ions and soluble sugars in the cell leads to the increase of relative conductivity [34]. The higher concentration of chlorophyll and low conductivity in the transgenic plants indicated that overexpression of \u003cem\u003eGsLTP\u003c/em\u003e in tobacco protected the cell membrane and prevent chlorophyll degradation. Under abiotic stress condition, the destruction of the cell membrane is also related to the enhancement of the peroxidation of the cell membrane [34]. The lower accumulation of MDA, a main product of membrane lipid peroxidation, further indicated the less deleterious modification on the cell membrane in the transgenic plants. Pro is normally accumulated under abiotic stress and plays a positive role in maintaining water balance and cell membrane integrity in plants [35]. After the application of stress, the more significant accumulation of Pro in the transgenic tobacco indicated that the overexpression of \u003cem\u003eGsLTP \u003c/em\u003epromote the biosynthesis of Pro. This might be another factor that contributed to the improved resistance to drought and salinity in the transgenic plants. Future more in-depth transcriptomic analysis, e.g., RNA Seq, on these transgenic plants will elucidate the molecular mechanisms imposing the resistance to drought and salinity.\u003c/p\u003e\n\u003cp\u003eThe growth and yield of soybean are negatively affected by adverse growing environment such as drought and salinity. The combination of transgenic technology and traditional breeding is the most effective way to rapidly improve the resilience of soybean. In this study, only physiological indexes of transgenic tobacco were measured. In future studies, the \u003cem\u003eLTP \u003c/em\u003egene can be transferred into soybean and the related physiological parameters will be measured. Other components discovered from a more detailed transcriptomic analysis can also be used to improve abiotic stress resistance in soybean. Transformation strategies harboring \u003cem\u003eLTP\u003c/em\u003e and other genes with known abiotic stress resistance ability may lead to more significant improvement in soybean.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe isolated the \u003cem\u003eGsLTP\u003c/em\u003e from \u003cem\u003eG. Soja\u003c/em\u003e and transformed it into tobacco. Transgenic plants were assessed for stress resistance by analyzing physiological parameters and biochemical indicators following low temperature, drought and salinity stress treatments. \u003cem\u003eGsLTP\u003c/em\u003e improved tolerance to drought and salinity to different degrees. However, resistance to low temperature was not enhanced. In summary, the genetic transformation of \u003cem\u003eGsLTP\u003c/em\u003e into plants can improve resistance to environmental stresses.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials and growing conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe seeds of \u003cem\u003eG.soja\u003c/em\u003e G50109 were obtained from the Jilin Academy of Agricultural Sciences (Changchun, China). To remove waxiness and promote germination, treat seeds with sulfuric acid (98%) for 10 minutes, wash them in sterile water 5 times, and store them in darkness for at least 2 days. After that, wild soybean seedlings were transferred to soil and incubated for 19 days at 24-26\u0026deg;C under a 16 hours light / 8 hours dark cycle condition. Leaves and roots were collected and stored at -80\u0026deg;C after being snap-frozen in liquid nitrogen.\u003c/p\u003e\n\u003cp\u003eWild-type (WT) \u003cem\u003eNicotiana tabacum \u003c/em\u003eLongjiang 911, from Mudanjiang Normal University, China, was used for transformation. Tobacco seeds were soaked for 3-5 h in water, sterilized with 70% ethanol for 30 s, treated with 0.1% mercuric chloride for 3 min, and rinsed with sterile water five times. Finally, all seeds were placed on M\u003csub\u003e0\u003c/sub\u003e medium (1/8 MS + 0.8% agar, pH 5.8). When young leaves reached 2-3 cm, edges were removed, leaves were cut into discs (7 mm diameter) and placed with adaxial sides facing downwards on M\u003csub\u003e1\u003c/sub\u003e medium (MS + 1.0 mg L6-BA + 0.1 mg LNAA + 0.75% agar, pH 5.8) for pre-culturing for 2 to 3 days. Leaf disc incisions began to swell and callus tissue started growing. WT and transgenic lines were treated with 0 mM or 300 mM NaCl, or subjected to drought stress, to analyze gene function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and sequence analysis of \u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eGsLTP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene expression profiles were obtained with the soybean gene expression microarray. Salt stress expressed sequence tag (EST) library was used to determine gene expression during early osmotic stress. The \u003cem\u003eGsLTP\u003c/em\u003e gene is highly similar to clone pTE-6424 in the NCBI database, as revealed by electronic extension using Phrap program (http://www.phrap.org). A BLAST analysis was carried out (http://www.ncbi.nlm.nih.gov/blast/) and the full-length sequence was obtained. The full sequence of the ORF was translated from the nucleic acid sequence by DNAMAN software (Version 4.0, Lynnon Biosoft, Quebec, Canada).\u003c/p\u003e\n\u003cp\u003eThe full-length cDNA of \u003cem\u003eGsLTP\u003c/em\u003e was obtained by homologous cloning. Total RNA from \u003cem\u003eG. soja \u003c/em\u003eG50109 was extracted from soybean leaves using a GoldScript cDNA Synthesis Kit (Invitrogen) and converted into cDNA through reverse transcription. The full- length cDNA of \u003cem\u003eGsLTP\u003c/em\u003e was amplified using gene-specific primers 5\u0026rsquo;-AGATGGCAGAGACATTCC-3\u0026rsquo; and 5\u0026rsquo;-TGCACCTTTCAATAC-3\u0026rsquo;. The reaction contained 2.5 \u0026mu;l of 10\u0026times; PCR buffer (Mg\u003csup\u003e2+\u003c/sup\u003e-free), 3 \u0026mu;l of 25 mM Mg\u003csup\u003e2+\u003c/sup\u003e, 2 \u0026mu;l of 2.5 mM each dNTP, 1 \u0026mu;l of DNA template (DNA content \u0026lt;1 ng), and 0.2 \u0026mu;l of Taq polymerase (5 U/\u0026micro;l) or Pyrobest TM DNA polymerase. The PCR products were purified and cloned into the pGEM-T vector, and the Pyrobest TM DNA polymerase was used for PCR identification and DNA sequencing. Sequence alignment was performed using DNAMAN. Sequence similarity was verified by BLASTP (\u003ca href=\"http://blast.ncbi.nlm.nih.gov/Blast.cgi)\"\u003ehttp://blast.ncbi.nlm.nih.gov/Blast.cgi)\u003c/a\u003e. The vector was transformed to \u003cem\u003eEscherichia coli\u003c/em\u003e DH5\u0026alpha; and cultured on Luria Bertani (LB) solid medium containing ampicillin (100 mg/L).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of the \u003c/strong\u003e\u003cstrong\u003epBI121-\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eGsLTP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e expression vector\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PCR-amplified \u003cem\u003eLTP\u003c/em\u003e gene was cloned into the plant expression vector pBI121, and the insert was verified using forward (5\u0026rsquo;-TAGGATCC(\u003cem\u003eBam\u003c/em\u003eHI)TAAGTCTTTGTTCATTTGAGTAG -3\u0026rsquo;) and reverse (5\u0026rsquo;-TACGAGCTC(\u003cem\u003eSac\u003c/em\u003eI)ATATATAGAATTCTGCGACT-3\u0026rsquo;) primers. PCR was performed as described above, and a 447 bp fragment was identified and purified as described above. Finally, the target plant expression vector harboring the\u003cem\u003e LTP\u003c/em\u003e gene was transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e LBA4404 .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTobacco transformation \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eA. tumefaciens\u003c/em\u003e LBA4404 strain containing the pBI121-\u003cem\u003eGsLTP\u003c/em\u003e vector was transformed into tobacco leaf disks following pre-incubation for 2-3 days. After co-culture for 3-4 days, leaf disks were degermed for 2-3 h with a liquid medium containing antibiotics that prohibit Agrobacterium growth. These leaf discs were dried on filter paper and then placed on shoot regeneration media for further selection. Five to six weeks later, small shoots of 3-4 cm in size were transferred to rooting medium. Approximate 14 days later, most seedlings developed a proliferate root system. These putative transgenic plantlets were then moved into a humid culture room without sealing for 1 week. Thereafter, seedlings were planted in flower pots (grass peat: sandy soil = 2:1) and grown in an artificial climatic cabinet (26\u0026deg;C, 126 \u0026mu;mol m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e, 16 h day\u003csup\u003e-1\u003c/sup\u003e illumination, 85-90% relative humidity).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular biological identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA was extracted from selected transgenic and wild type plants, and integration of the \u003cem\u003eGsLTP\u003c/em\u003e gene was confirmed by PCR and Southern blotting using a probe consisting of a 375 bp fragment of the \u003cem\u003eGsLTP\u003c/em\u003e gene generated from total DNA in plants digested with \u003cem\u003eBam\u003c/em\u003eHI. \u003cem\u003eGsLTP\u003c/em\u003e mRNA transcription was determined by RT-PCR. The above experiments were carried out on the T\u003csub\u003e3\u003c/sub\u003e generation of all lines. A total of 180 seedlings were confirmed to be transgenic and used in further stress tolerance tests. The material did not been deposited in a publicly available herbarium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of stress tolerance and \u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eGsLTP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree groups of seedlings at a similar developmental stage (30 transgenic seedlings and 30 untransformed control seedlings per group) were subjected to low temperature (4\u0026deg;C), salinity (300 mM NaCl) and drought (without water for 2, 4, 6, 8, 10, 12 or 14 days). Chlorophyll content [36], malondialdehyde (MDA) content (TBA assay) [37,38], proline (Pro) content (ninhydrin assay) [39], leaf membrane permeability (relative conductivity assay) [40] and morphology were investigated for each treatment. Physiological indices and gene functions were statistically analyzed using SAS6.12.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eLTPs: Lipid transfer proteins\u003c/p\u003e\n\u003cp\u003eORF: open reading frame\u003c/p\u003e\n\u003cp\u003eEST: expressed sequence tag\u003c/p\u003e\n\u003cp\u003ensLTPs: Non-specific lipid transfer proteins \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMDA: malondialdehyde\u003c/p\u003e\n\u003cp\u003ePro: proline\u003c/p\u003e\n\u003cp\u003eABA: abscisic acid\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication: \u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials: \u003c/strong\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests: \u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by National Key Research and Development Projects, grant number 2016YFC0500306-02, Heilongjiang Natural Science Fund Project Plan Mission Statement, grant number C2017030 and Heilongjiang Province Education Project Fund, grant number 12531014. The funders had no role in study design, data collection, interpretation, or decision-making in relation to submission for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003e contributions:\u003c/strong\u003e DC and JF conceived and designed research. JL and LC conducted experiments. JF contributed new reagents or analytical tools. JW and QM analyzed data. JF wrote the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang W, Vinocur B, Altman A: \u003cstrong\u003ePlant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance\u003c/strong\u003e. \u003cem\u003ePlanta \u003c/em\u003e2003, \u003cstrong\u003e218\u003c/strong\u003e(1):1-14.\u003c/li\u003e\n\u003cli\u003eLiu Y, Zhang L, Meng S, Liu Y, Zhao X, Pang C, Zhang H, Xu T, He Y, Qi M\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eExpression of galactinol synthase from Ammopiptanthus nanus in tomato improves tolerance to cold stress\u003c/strong\u003e. \u003cem\u003eJ Exp Bot \u003c/em\u003e2020, \u003cstrong\u003e71\u003c/strong\u003e(1):435-449.\u003c/li\u003e\n\u003cli\u003eYang Z, Sheng J, Lv K, Ren L, Zhang D: \u003cstrong\u003eY2SK2 and SK3 type dehydrins from 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In\u003cem\u003e.\u003c/em\u003e: Portland Press Limited; 1983.\u003c/li\u003e\n\u003cli\u003eDong W, Wang M, Xu F, Quan T, Peng K, Xiao L, Xia G: \u003cstrong\u003eWheat oxophytodienoate reductase gene TaOPR1 confers salinity tolerance via enhancement of abscisic acid signaling and reactive oxygen species scavenging\u003c/strong\u003e. \u003cem\u003ePlant physiology \u003c/em\u003e2013, \u003cstrong\u003e161\u003c/strong\u003e(3):1217-1228.\u003c/li\u003e\n\u003cli\u003eJia X, Xiaoguang D, Jun Y, Beeching JR, Peng Z: \u003cstrong\u003eEnhanced reactive oxygen species scavenging by overproduction of superoxide dismutase and catalase delays postharvest physiological deterioration of cassava storage roots\u003c/strong\u003e. \u003cem\u003ePlant Physiology \u003c/em\u003e2013, \u003cstrong\u003e161\u003c/strong\u003e(3):1517-1528.\u003c/li\u003e\n\u003cli\u003eZhang T, Mo J, Zhou K, Chang Y, Liu Z: \u003cstrong\u003eOverexpression of Brassica campestris BcICE1 gene increases abiotic stress tolerance in tobacco\u003c/strong\u003e. \u003cem\u003ePlant Physiology and Biochemistry\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eBen RW, Ben-Saad R, Meynard D, Verdeil JL, Azaza J, Zouari N, Fki L, Guiderdoni E, Al-Doss A, Hassairi A: \u003cstrong\u003eEctopic Expression of Aeluropus littoralis Plasma Membrane Protein Gene AlTMP1 Confers Abiotic Stress Tolerance in Transgenic Tobacco by Improving Water Status and Cation Homeostasis\u003c/strong\u003e. \u003cem\u003eInternational Journal of Molecular Sciences \u003c/em\u003e2017, \u003cstrong\u003e18\u003c/strong\u003e(4):692.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Functional analysis, gene cloning, Glycine soja, lipid transfer protein, Southern blotting, RT-PCR, crop breeding","lastPublishedDoi":"10.21203/rs.3.rs-52965/v3","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-52965/v3","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Lipid transfer proteins (LTPs) mainly exist in plant cell walls where they make an important impact in the transport of diverse lipophilic compounds. They have an important effect on the stress tolerance of plants by mediating plant responses to environmental stimuli and cell signal transduction pathways. Although the functions of several LTPs in different plant species have been identified, little is known about the biochemical and enzymatic activities of LTP family members in soybeans. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Herein, \u003cem\u003eGsLTP\u003c/em\u003e was identified from \u003cem\u003eGlycine soja \u003c/em\u003eusing soybean gene microarray expression and screened in a salt stress expressed sequence tag (EST) library. The 369 bp open reading frame (ORF) encodes a protein of 122 amino acids. The cDNA sequence shares similarity with \u003cem\u003eLTP\u003c/em\u003e genes in other plants such as soybean (93%) and Vigna (72%). The function of the gene was characterized in transgenic tobacco. Various physiological characteristics under different environmental stresses were investigated. We discovered that overexpression of \u003cem\u003eGsLTP\u003c/em\u003e enhanced tolerance to drought and salt stresses. \u003cstrong\u003eConclusions: \u003c/strong\u003eOur results indicate that \u003cem\u003eGsLTP\u003c/em\u003e plays an important role in multiple abiotic signalling pathways, and provide a theoretical basis and practical gene resource for crop breeding.\u003c/p\u003e","manuscriptTitle":"GsLTP, a Non-Specific Lipid Transfer Protein from Glycine soja, Enhances Drought and Salt Stress Tolerance in Transgenic Tobacco","msid":"","msnumber":"","nonDraftVersions":[{"code":3,"date":"2021-01-07 21:33:29","doi":"10.21203/rs.3.rs-52965/v3","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":2,"date":"2020-11-06 20:08:14","doi":"10.21203/rs.3.rs-52965/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2020-08-17 22:36:45","doi":"10.21203/rs.3.rs-52965/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d3bdfb88-c17c-41a9-b290-68a5fb103eaf","owner":[],"postedDate":"January 7th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":1783342,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2021-01-05T13:39:59+00:00","versionOfRecord":[],"versionCreatedAt":"2021-01-07 21:33:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v3","identity":"rs-52965","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-52965","identity":"rs-52965","version":["v3"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}