Lcsain3, A Novel Gene From Sheepgrass, Regulates Arabidopsis Seed Germination And Seedling Growth Under Salt Stress

Sheepgrass is a perennial native grass species with an aggressive and vigorous rhizome system, and it can tolerate high levels of salt stress. Many salt stress-responsive genes have been identied in sheepgrass. Here, we identied and characterized a novel salt-induced gene, LcSAIN3 (Leymus chinensis salt-induced 3), from sheepgrass. Expression analysis conrmed that LcSAIN3 is induced by PEG, ABA and salt stress. Subcellular localization analysis indicated that the LcSAIN3 protein is mainly localized in the chloroplasts. The heterologous of LcSAIN3 in Arabidopsis increases seed germination under various stress conditions. More importantly, the seedling survival, plant height and weight of the transgenic plants are higher than those of the WT plants under salt stress. The overexpression of LcSAIN3 causes a relatively high accumulation of free proline; enhances SOD activity; and leads to the upregulated expression of several stress-responsive genes, such as AtRAB26, AtRD29B, AtSOS1 and AtP5CS1. Our results suggest that LcSAIN3 may be a useful gene for the molecular breeding to improve plants salt stress tolerance.


Introduction
Soil salinization is a severe problem that affects plant growth, development and productivity worldwide (Munns and Tester 2008). The effects of salt on plants lead to osmotic, ion toxicity and oxidative stress (Deinlein et al. 2014;Isayenkov and Maathuis 2019;Munns and Tester 2008). Changes in physiological, biochemical, cellular and molecular processes in plants under salt stress have been investigated by many researchers (Deinlein et al. 2014;Ma et al. 2020;Reddy et al. 2011;Xiong et al. 2002;, while genetic sources for salt tolerance development in crops were also studied (Isayenkov 2019).
Extensive numbers of transcription factor-encoding genes in response to salt stress have been identi ed, including DREB, bZIP, NAC, and MYB family genes (Bo et al. 2020;Hu et al. 2008;Liang et al. 2017;Wang et al. 2017;Yang et al. 2012), and overexpressing these genes can enhance salt stress tolerance in transgenic plants (Deinlein et al. 2014;Hao et al. 2011;He et al. 2012;Peng et al. 2011;. Previous studies suggest that the transcription factors can activate many stress-induced genes, such as LEA genes (RD26, RD29A, RD29B, and RAB18) and proline biosynthesis genes (P5CS), and the LEA proteins are mainly involved in protection to desiccation by acting as cellular dewatering protectants under stress conditions . Proline plays an important role in osmoregulation, and can be also used as an active oxygen scavenger to stabilize protein and membrane structure under pressure (Deinlein et al. 2014;Szabados and Savoure 2010).Molecular regulatory networks related to salt stress are complex and have not been fully explored (Xu et al. 2019); thus, mining key and novel salt tolerancerelated genes is required for developing breeding strategies to enhance salt stress tolerance in crops.
Sheepgrass (Leymus chinensis (Trin.) Tzvel) is a perennial gramineous plant species belonging to the Leymus, Triticeae, and Poaceae classi cation groups and is widely distributed on the eastern Eurasian steppe (Lu et al. 2019). This species can survive when the soil moisture content is less than 6% in the dry season, and grows well in environments of 600 mmol/L NaCl and 175 mmol/L Na 2 CO 3 Gao et al. 2016;Nevo and Chen 2010). Many stress-induced genes have been identi ed and characterized in sheepgrass using transcriptome sequencing, including LcDREB2, LcDREB3a, LcDREB21, LcMYB1, LcWRKY5, LcP5CSs, and LcSAMDCs Liu et al. 2017;Ma et al. 2014;Peng et al. 2011). In addition, several novel genes were discovered; LcSAIN1 and LcSAIN2 genes have been identi ed to improve the greening rate of cotyledons, root elongation, plant height, and survival under salt stress in the transgenic plants ; and ectopic expression of LcFIN1 and LcFIN2 signi cantly increased freezing stress tolerance in transgenic Arabidopsis and rice (Gao et al. 2016;.
In this study, we characterized a novel gene, LcSAIN3 from sheepgrass, and the expression of LcSAIN3 was induced by salt stress. We found that the LcSAIN3 gene can improve salt tolerance in Arabidopsis, and thus propose that LcSAIN3 plays an important role in regulating salinity tolerance.

Materials And Methods
Plant materials, growth conditions and stress treatment Sheepgrass variety Zhongke No. 1 (released from Institute of Botany, the Chinese Academy of Sciences) seeds were grown in a mixture of peat moss and vermiculite (2:1, v/v) in an incubator at 28 °C/16 °C under a 16 h light/8 h dark photoperiod. For analysis of speci c expression in different tissues, the roots, stems, leaves, and seeds were collected from two-year-old sheepgrass plants under normal condition. For salt stress analyses, 4-week-old sheepgrass seedlings were immersed in the solution of 400 mM NaCl.
Seedlings were treated with 100 mM abscisic acid (ABA) and 20% PEG6000 for drought stress treatment, respectively. A total of 40 plants were sampled at 0, 1, 3, 5, 12, and 24 h after various stresses treatments, and three replicates of each sample were collected, immediately frozen in liquid nitrogen and stored at -80 °C Li et al. 2019).
Arabidopsis thaliana (ecotype Columbia (Col-0)) and Tobacco (Nicotiana benthamiana) seeds were grown in a greenhouse under a 16 h light/8 h dark photoperiod with an average temperature of 23 °C.
Cloning and sequence analysis of the LcSAIN3 gene In our previous study, a number of candidate salinity-induced transcripts were identi ed using transcriptome analyses of sheepgrass seedlings subjected to salinity stress or not . Among them, one transcript designated LcSAIN3 was encoded by an unknown functional gene and was signi cantly induced by salt stress treatment. To obtain the full-length cDNA of LcSAIN3, 4-week-old sheepgrass seedlings under 400 mM NaCl stress for 12 h were harvested.
Total RNA was isolated using a TRIzol kit (Invitrogen, Carlsbad, CA, USA), and rst-strand cDNA synthesis was performed with a SMART RACE cDNA Ampli cation Kit (Clontech, Palo Alto, CA, USA) according to the manufacturers' instructions. Full-length LcSAIN3 cDNA was ampli ed using the primers 5'-GTAGCCCGTGAGGAAGTT − 3' and 5'-CACTAGAAGGGCCCCGAA − 3', and the cDNA of 5' RACE was used as a template. The ampli cation conditions were as follows: 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min; and a nal extension at 72 °C for 10 min. All of the PCR products were cloned into a pMD19-T vector and sequenced at Sangon Biotech (Shanghai Co., Ltd., China). The LcSAIN3 sequences were analyzed using the BLAST program of the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/), and subcellular localization was predicted using the Plant-mPLoc program (Chou and Shen 2010).

RNA extraction and qRT-PCR analysis
Total RNA from Arabidopsis and sheepgrass seedlings was extracted using a TRIzol kit (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized, and qRT-PCR was conducted following the manufacturer's instructions using a PrimeScript™ PCR Kit (TaKaRa, Dalian, China). The cDNA template was ampli ed with a qRT-PCR system (Roche Light Cycler 480 II, Switzerland), and the data were quanti ed using the comparative 2 −ΔΔCT method after the PCR program was run (Livak and Schmittgen 2001). The primer sequences for qRT-PCR are listed in Table S1. LcActin and AtActin2 were used as internal controls for assessing the expression levels in sheepgrass and Arabidopsis, respectively.

Subcellular localization of LcSAIN3
The open reading frame (ORF) of the LcSAIN3 was recombined into a pMDC45 vector (containing GFP) using the Gateway cloning while the construct was introduced into Agrobacterium tumefaciens (EHA105) cells. The intact leaves of 4-week-old wild-type tobacco (N. benthamiana) plants were injected with A. tumefaciens strain EHA105 harboring pMDC45 and pMDC45-LcSAIN3 (35S::GFP-LcSAIN3) and the in ltration was performed with a 2 ml syringe without niddle. The transgene-derived expression was monitored 2 to 3 d after in ltration by confocal microscopy on a Leica TCS SP5 microscope (Leica Microsystems, Wetzlar, Germany) ). Fluorophores were excited using an argon laser at 488 nm (GFP) and 382 bright-eld images were collected using the transmitted light detector.

Construct creation and plant genetic transformation
The ORF of LcSAIN3 was inserted into a pSN1301 vector under the control of the Cauli ower mosaic virus (CaMV) 35S promoter via the BamHI and KpnI sites, after which the construct pSN1301-LcSAIN3 plasmid was transformed into Arabidopsis using the oral dip method (Clough and Bent 1998). T1 transgenic Arabidopsis seeds were subsequently sterilized in 30% (v/v) bleach for 15 min, rinsed ve times with sterile water, and selected on Murashige and Skoog (MS) agar supplemented with 30 µg ml − 1 hygromycin, and the seedlings were con rmed by PCR analysis using the gene-speci c primers.

Stress treatment for the transgenic plants
For the seed germination of transgenic Arabidopsis plants under stress treatments, T3 homozygous seeds of three transgenic plants (line 5, line 6 and line 8) were incubated at 4 °C for 2 d to break dormancy, after which they were germinated on half-strength MS media (sucrose concentration 15 g l − 1 , agar concentration 5 g l − 1 and pH 5.8) supplemented with different concentrations of ABA (1 and 2 µM), mannitol (200 and 300 mM), and NaCl (100, 125, 150, and 200 mM). The germination rate was scored daily for 7 d by observing radical protrusion, and at least 120 seeds from each transgenic line were evaluated. To test the salt tolerance of transgenic Arabidopsis, 3-week-old plants were treated with 200 mM NaCl for 3 weeks at 3-day intervals ).
Measurement of proline and (superoxide dismutase) SOD Proline was measured as previously described (Shan et al. 2007), and total superoxide dismutase (SOD) activity was measured using nitro blue tetrazolium (NBT) reduction as previously described (Durak et al. 1993;Li et al. 2019).

Statistical analysis
The data concerning Arabidopsis seed germination and seedling growth parameters, proline content and SOD activity were subjected to ANOVA using the SPSS 21.0 program (IBM, Chicago).

Isolation and expression analysis of LcSAIN3
Our previous studies identi ed many stress-induced genes using transcriptome sequencing techniques ). Among those transcripts, the full length of stress-induced gene designated LcSAIN3 was obtained by the RACE technique while its function was unknown.. The LcSAIN3 (GenBank ID: MN901606) gene is 847 bp long and encodes a protein comprising 198 amino acids, which has high homology (72%) with a wheat cDNA clone, WT004_K04 (GenBank ID: AK331493), but rice does not have an endogenous homolog of LcSAIN3. Furthermore, the amino acid sequence of LcSAIN3 shows 52% homology with predicted protein product of Triticum turgidum subsp. Durum, suggesting that LcSAIN3 is a novel protein with unknown function.
The expression of LcSAIN3 transcripts under control conditions was highly expressed in the stems, but less expressed in the leaves, seeds, and roots (Fig. 1a). To investigate the effects of stress conditions on the expression of LcSAIN3, sheepgrass seedlings were exposed to various abiotic stresses. QRT-PCR was performed using the total RNA extracted from 4-week-old sheepgrass plants subjected to stress treatments at different time intervals. As shown in Fig. 1, the transcript levels of LcSAIN3 were signi cantly increased beginning at 3 h and reached highest at 5 h after NaCl treatment (Fig. 1b). Similar to the treatment with salt stress, treatment with PEG and ABA also led to a signi cant increase in expression levels at 5 h (Fig. 1c). These results indicate that salt, ABA and PEG treatments signi cantly induce the expression of LcSAIN3 in sheepgrass seedlings.

Subcellular localization of LcSAIN3
The LcSAIN3 protein was predicted by the Plant-mPLoc program (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) being a chloroplast protein. To determine the actual subcellular localization of LcSAIN3 in vivo, the ORF sequence was inserted into a pMDC45 vector fused to a GFP reporter gene under the control of the CaMV 35S promoter, after which the construct was in ltrated into tobacco (Nicotiana tabacum) leaf cells. As shown in Fig. 2 and Figure S1, the uorescent signals from the 35S::GFP-LcSAIN3 fusion protein and auto uorescent signals of chloroplasts were merged together, and demonstrating that LcSAIN3 is a chloroplast-localized protein.

Overexpression of LcSAIN3 regulates seed germination under salt stress
To con rm the function of LcSAIN3 in response to salt stress, the LcSAIN3 gene was transferred to Arabidopsis. Three lines homozygous (line 5, line 6, and line 8) with higher expression levels of LcSAIN3 were selected for further analysis, and con rmed by qRT-PCR ( Figure S2). The salt tolerance of the WT and transgenic LcSAIN3 plants was then tested at seed germination stage. WT and transgenic seeds were germinated on MS media supplemented with different concentrations of NaCl (100, 125, 150, 175, and 200 mM) after 2 d of strati cation, and no obvious differences were detected between the WT and transgenic plants on the MS media. However, the germination rates of the LcSAIN3-overexpressing lines were signi cantly higher than those of the WT plants in the presence of NaCl (Fig. 3a). As shown in Fig. 3b, the three transgenic lines (L5, L6, and L8) showed signi cantly higher germination rates (92%, 86%, and 81%, respectively) than did the WT plants under 150 mM NaCl (~ 55%) (Fig. 3b). Thus, LcSAIN3 overexpression in Arabidopsis reduces sensitivity to salt stress at seed germination stage.

LcSAIN3 overexpression in Arabidopsis enhanced seed germination under ABA and osmotic stress
Our previous qRT-PCR analysis showed that the expression of the LcSAIN3 gene was signi cantly induced by ABA and drought stress. To determine whether LcSAIN3 also increases tolerance to ABA, the tolerance of transgenic lines and WT was examined. Under 2 µM ABA treatment, the germination rate of WT seeds was only 57%, while the germination rates of the three LcSAIN3 overexpression lines, i.e. L5, L6 and L8 seeds were 79%, 87% and 75% respectively ( Fig. 4a and 4b). When LcSAIN3 overexpression lines and WT were grown on MS plates containing 200 and 300 mM mannitol, the transgenic plants germinated, while the germination of the WT plants was completely inhibited (Fig. 4c and 4d). This implies that LcSAIN3 reduces the sensitivity to ABA and osmotic stresses at seed germination stage.

LcSAIN3 overexpression enhances tolerance to salt stress in Arabidopsis seedlings
To determine whether enhanced salt tolerance is present in adult seedlings, 3-week-old Arabidopsis plants were treated with 200 mM NaCl for 2 weeks at 3-day intervals . Under salt stress treatment, the germination of the transgenic lines and WT were all inhibited, but the sensitivity of LcSAIN3 overexpression lines to salt stress was reduced.. For example, most of the WT seedlings were bleached and wilted after 3-week salt treatment; in contrast, most seedlings of the three LcSAIN3 overexpression lines survived and had both green and yellow leaves. Three transgenic lines had signi cantly higher survival rate (93, 87, and 98%) compared to that of the WT plants (~ 30%) (Fig. 5a, b). Furthermore, plant height and weight were signi cantly greater for the transgenic plants than for the WT plants (Fig. 5c, d).
These results indicate that LcSAIN3 confers salt tolerance to Arabidopsis.

LcSAIN3 regulates proline accumulation and SOD activity in response to salt stress
To further explore the possible mechanisms that may be responsible for the improved tolerance of the transgenic plants to salt stress relative to the WT plants, proline content and SOD (a major antioxidant enzyme) activity were measured. Proline content in both transgenic plants and control plants increased under salt stress conditions, but the increased levels were signi cantly higher in the transgenic plants than in the control plants (Fig. 6a). Further, the SOD activity was also signi cantly higher in the transgenic plants than in the wild-type plants under salt stress (P < 0.01) (Fig. 6b). Taken together, our results indicate that LcSAIN3 overexpression in Arabidopsis increases the proline content and SOD activities under salt stress.

LcSAIN3 overexpression alters the expression of salt-related genes in Arabidopsis plants
The expression levels of several known salt stress-responsive marker genes were compared between the transgenic Arabidopsis lines and WT plants using qRT-PCR under salt stress condition. The functional genes RAB26 and RD29B exhibited increased expression levels in the transgenic plants compared with the WT plants under salt stress (Fig. 7). Furthermore, SOS1 and P5CS1 also exhibited increased expression levels in the transgenic lines (Fig. 7). Altogether, these data suggest that LcSAIN3 confers salt stress tolerance to plants by upregulating the expression of salt stress-responsive genes.

Discussion
Sheepgrass is an important forage grass as well as an environmentally friendly native grass species in China. It has high yield with high protein content, better palatability, strong regeneration ability, strong cold and drought resistance, as well as salt-alkali resistance Lu et al. 2019). Our previous studies demonstrated that the novel genes LcFIN1 and LcFIN2 from sheepgrass enhance tolerance to low temperature in Arabidopsis and rice, while overexpressing LcSAIN1 and LcSAIN2 could enhance the salt stress resistance of transgenic plants compared with wild-type plants (Gao et al. 2016;Li et al. 2019). The isolated salt-induced gene, LcSAIN3 from sheepgrass in present study has high homology (72%) only with a wheat cDNA clone, WT004_K04 (GenBank ID: AK331493). To further investigate the role of LcSAIN3 in the plant response to various stresses, the gene was overexpressed in Arabidopsis, as genetic transformation in sheepgrass is still very di cult (Wang et al. 2009).
We found that the expression of the LcSAIN3 gene was signi cantly induced by salinity, PEG, and ABA stresses, and heterologous of LcSAIN3 gene in Arabidopsis led to an increase in tolerance to NaCl, ABA and PEG stress at germination stage. Further, the tolerance of transgenic plants to salinity was markedly enhanced during the seedling growth stage. Moreover, our preliminary results showed LcSAIN3 is a chloroplast-localized protein (Fig. 2). Previous studies demonstrated that chloroplast proteins play a vital role in plant growth and development and participate in various abiotic stress responses ). CEST, a novel chloroplast protein, can reduce photooxidative damage and enhance tolerance to multiple environmental stresses in transgenic Arabidopsis (Yokotani et al. 2011). Heterologous expression of a chloroplast outer envelope protein from Suaeda salsa could enhance oxidative stress tolerance and induce chloroplast aggregation in transgenic Arabidopsis plants ). In addition, overexpression of chloroplast-localized rice OsRH58 is involved in the stress response and can improve seed germination and seedling growth under salt stress conditions (Nawaz and Kang 2019). Thus, novel chloroplast protein, LcSAIN3 identi ed in this study plays a positive role in the responses to salt stress and other abiotic stresses.
In plants under salt stress, the accumulation of proline has multiple protective functions, including osmotic protection and ROS scavenging (Zsigmond et al. 2012). SOD, a kind of antioxidant enzyme, also plays an important role in scavenging ROS and protects against oxidative stress under salt conditions (Xu et al. 2019).In this study, proline levels and SOD activities are signi cantly higher in the LcSAIN3 overexpression lines than in the wild type under salt stress. SOS pathway is a key regulator of Na + homeostasis, for example via SOS1 (Isayenkov and Maathuis 2019). P5CS1 is a key enzyme in the proline biosynthesis pathway, and it functions as a positive regulator in proline accumulation and plant responses to salt tolerance (Bo et al. 2019;Xu et al. 2018). Our results indicated that AtP5CS1 and AtSOS1 are expressed at much higher levels in the LcSAIN3 transgenic plants than in the WT plants under salt stress. Furthermore, in transgenic Arabidopsis plants expressing LcSAIN3, the transcription levels of the ABA-dependent genes AtRD26 and AtRD29B are higher than those in the WT plants under salt stress. The expression of these genes has been found to be induced by salinity, and they play important roles in abiotic stress. RD29B is involved in ABA-dependent signaling pathways Msanne et al. 2011). These ndings indicate that the improved tolerance of the transgenic plants under salinity stress might partly result from the enhanced proline content, SOD activity, while the salt-induced gene expression levels might result from the overexpression of LcSAIN3.
In conclusion, we characterized a novel chloroplast-localized protein, LcSAIN3, and the protein plays a positive role in regulating salt stress. Constitutive expression of LcSAIN3 in Arabidopsis accelerates seed germination and increases seedling survival when subjected to salt stress by improving proline levels and SOD activities and by regulating the expression of some stress-responsive genes.

Declarations
Author contribution statement: G.L. and X.L. planned and designed the research. X.L. and W.Y. performed the experiments. J.J., P.Z., and D.Q. made much contribution to plant material collection and experimental management. Q.L. and S.C. analyzed the data. X.L. and W.Y. wrote the manuscript. GL and L.C. edited the manuscript and gave the nal approval the manuscript. All authors read and approved the nal manuscript.