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Analysis of LEA protein family members in Lepidium apetalum seeds and the expression of LaLEA1 in seedlings in response to abiotic stresses

To study the biological function of late embryogenesis abundant (LEA) proteins in Lepidium apetalum Willd., genes encoding LEA family proteins were identified from the seed transcriptome. The sequence most closely related to germination at a low temperature was selected and gene expressions in response to low temperature stress further studied. The results show that 27 LEA genes were expressed in seeds germinating at the low temperature: 3 genes were upregulated, 20 were downregulated, and 4 were not significantly different from controls. The most prominent of the upregulated genes, LaLEA1, contained an open reading frame of 624 bp and encoded 208 amino acids. The protein was rich in hydrophilic amino acids including threonine, alanine, glutamine, and lysine. It is predicted that the secondary structure contains α-helices and irregular curls. Real-time quantitative PCR results show that under low temperature stress, the expression of LaLEA1 was first downregulated and then upregulated rapidly, reaching its highest expression at 12 h, then the expression of LaLEA1 was reduced slightly but maintained higher than that of the non-stress group. As the expression of LaLEA1 was significantly altered in response to low temperature stress, we investigated the expression of LaLEA1 also in response to other abiotic stresses, i.e., salinity and drought. L. apetalum seedlings wilted in the early stage following NaCl or osmotic (polyethylene glycol) stresses, but recovered quickly, showing a strong tolerance. Real-time quantitative PCR results show that LaLEA1 was rapidly upregulated following salt and osmotic stresses, and its expression profile was closely related to NaCl or PEG concentrations. Expression was up to 7.9-fold higher than that of the control after 6 h of salt stress. These results suggest that L. apetalum seedlings responded quickly to salt stress. The response to osmotic stress was slightly slower; expression of LaLEA1 was 6.0-fold higher than that of the control after 12 h. Thus, LaLEA1 played an important role in abiotic stress tolerance. These results provide a basis for further analysis of the role of the LEA genes in the stress resistance of L. apetalum. Additional key words: gene expression, low temperature, osmotic stress, salinity. Submitted 27 September 2019, last revision 15 December 2019, accepted 19 December 2019. Abbreviations: LEA late embryogenesis abundant; PEG polyethylene glycol. Acknowledgments: This work was supported by grants from the “13th Five-Year” Plan for Key Discipline Biology, Xinjiang Normal University (17SDKD0201), the National Natural Science Foundation of China (31660079 and 31460041), and the Open projects of the Beijing Key Laboratory of Resistance Genes Resource and Molecular Development (2017GD03), Research and Innovation Project for Graduate Students in Xinjiang Uygur Autonomous Region. The first two authors contributed equally to this work. * Corresponding author; e-mail: 954843437@qq.com Introduction Lepidium apetalum Willd. (family Brassicaceae), which has medicinal and nutritional value, is distributed worldwide from 400 to 2 000 m above sea level. This plant has the ability to adapt to a widely differing environments producing different ecotypes. For example, the ecotype in northern Xinjiang (China) has been reported to grow at 0 to 5 °C in some cases (Mao and Zhang, 1994). The climate in the mountainous areas of northern Xinjiang is very dry with some snow in winter. In early spring, ephemeral plants germinate and grow rapidly in the cold soil, which is soaked with melted snow, completing their life cycles before the soil dries up. This harsh environment makes L. apetalum living in northern Xinjiang, China unique in its ability to tolerate cold early spring weather, and to germinate and grow under such conditions (Yang et al. 2015). In a study of seed germination characteristics, it was found that the low-temperature germination of L. apetalum proceeds as follows: at temperatures of 0 to


Introduction
Lepidium apetalum Willd. (family Brassicaceae), which has medicinal and nutritional value, is distributed worldwide from 400 to 2 000 m above sea level. This plant has the ability to adapt to a widely differing environments producing different ecotypes. For example, the ecotype in northern Xinjiang (China) has been reported to grow at 0 to 5 °C in some cases (Mao and Zhang, 1994). The climate in the mountainous areas of northern Xinjiang is very dry with some snow in winter. In early spring, ephemeral plants germinate and grow rapidly in the cold soil, which is soaked with melted snow, completing their life cycles before the soil dries up. This harsh environment makes L. apetalum living in northern Xinjiang, China unique in its ability to tolerate cold early spring weather, and to germinate and grow under such conditions (Yang et al. 2015). In a study of seed germination characteristics, it was found that the low-temperature germination of 5 °C, the initial germination of seeds can occur, but there is a stagnation period. Interestingly, brief exposure (50 min) to a higher temperature (e.g. 25 °C) allows the seeds to circumvent the stagnation period. When the germinated seeds are again placed in a low-temperature environment, they can tolerate the low temperature, and continue to germinate and grow (Zhao et al. 2010). Considering the seed response to temperature, the transcriptome of seeds under stagnant, low-temperature conditions was compared to that of seeds resuming germination following a short, high-temperature treatment; differentially expressed genes were identified (Shah et al. 2018, Young et al. 2018. In addition to low-temperature damage, the plants also experience drought and salt stress during seed germination and seedling growth. Considering the harsh growth environment of L. apetalum, late embryogenesis abundant (LEA) proteins have recently been investigated; LEA family proteins play an important role in abiotic stress tolerance in plants, including cold, drought, and salt stresses (Xiang et al. 2018).
LEA proteins are a class of small hydrophilic proteins that were first isolated from cotton embryos by Capdevila and Dure (1981). These proteins are widespread throughout eukaryotic organisms, including plants (Liang et al. 2016, Lin et al. 2017, He et al. 2018. According to homologous and conserved domains, the LEA protein family was divided into the groups D-19, D-11, D-7, D-113, D-29, D-34, and D-73, as well as several additional groups. Their common characteristic is high content of glycine (Gly), lysine (Lys), arginine (Arg), glutamic acid (Glu), and threonine (Thr) (Arroyo et al. 2000). LEA proteins are highly hydrophilic and exhibit thermal stability. LEA proteins have been reported to be involved in regulating the embryonic development (Galau et al. 1987, Ingram and Bartels 1996, Oliveira et al. 2010. There is also evidence that plant LEA proteins play a role in plant tolerance to abiotic stresses (Cai et al. 2006, Wang et al. 2008, Jiang et al. 2017. LEA proteins are thought to regulate plant stress resistance by stabilizing proteins, thus protecting against denaturation (Liu, 2014, He and Hu 2015, reactive oxygen species , Li et al. 2017a, or by stabilizing the cell membrane (Mouillon et al. 2006, Alan andWise 2007). However, LEA family proteins differ significantly among different plants, and the mechanism underlying the regulation of plant development and stress resistance by LEA proteins is not fully understood.
The seedlings of L. apetalum also exhibited strong tolerance to low temperature (Meng et al. 2008, Zhao et al. 2010, Yang et al. 2015, 2017b, Yuan et al. 2018. The transcriptomes of the two different groups of seeds were sequenced ) and many highly expressed LEA protein family genes were identified. However, whether the expressions of these genes were related to tolerance of the seed germinated at low temperature, or to the other abiotic stresses, has not yet been determined. In this study, expressions of LEA genes in the two groups were analyzed, before and after lowtemperature germination. Expression of the LaLEA1 gene under salinity and drought was also investigated. These results provide an experimental basis to better understand the function of LEA genes and the adaptation mechanisms of L. apetalum seedlings.

Materials and methods
Plant, treatments, and related data sources: Lepidium apetalum Willd. ripe seeds were collected from Liyushan mountain, Urumqi, Xinjiang in northern China. After drying at room temperature, the seeds were stored at 4 °C. Seeds were treated with 98 % (m/v) sulfuric acid to remove the seed coat, rinsed with distilled water several times, placed on wet filter paper, and left at a temperature of 24 -26 °C, a 16-h·photoperiod, an irradiance of 400 to 650 µmol m -2 s -1 , and a relative humidity of 60 ± 5 % to germinate for 1 week. Seven-day-old seedlings of uniform size were selected to clone the cDNA of the LaLEA1 gene, and to verify the response of the LaLEA1 gene to abiotic stress. LEA protein information of the seeds of L. apetalum was obtained from our previous transcriptome sequencing results. Gene sequence information for other species was obtained from the NCBI database.
Screening for LEA protein family genes: The seeds which had been deposited at 4 °C for 9 d were used as the sample of germination at low temperature. After that some seeds were exposed to 25 °C for 55 min. Sequencing libraries were constructed for the both seed groups (Zhou et al. , 2018. High throughput sequencing using Illumina HiSeqTM2000 was then performed. Using LEA as the key word, LEA gene related sequences were obtained from the transcriptome data of L. apetalum seeds. Each sequence screened from the transcriptome database was analyzed in the NCBI database for BLAST analysis (https:// www. ncbi.nlm.nih.gov). Only those sequences that contain the conservative domain of LEA and are highly homologous (consistency greater than 85 %) with the sequences annotated as LEA in NCBI can be identified as members of the LEA-related family. Following removal of redundant sequences, cDNA sequences of LEA family members of L. apetalum were determined. Functional determination of LEA protein family genes was carried out using the nonredundant (Nr) protein and eukaryotic ortholog groups (KOG) databases, as well as gene ontology (GO) analysis.
Extraction of total RNA from seedlings of Lepidium apetalum: TRIzol reagent was used to extract total RNA from ??-d-old seedlings. RNA purity and quality were detected by agarose gel electrophoresis and a UV spectrophotometer. The cDNA synthesis was carried out using a reverse transcription kit (RevertAid First Strand cDNA synthesis kit) according to the manufacturer's instructions. The cDNAs were stored at -80 °C until further use.

Differential expression analysis of LEA protein family genes before and after low-temperature germination:
Based on the fragments per kilobase of transcript per million fragments mapped (FPKM) method, differential expression analysis of LEA protein coding genes in the transcriptome was performed. Genes that were up-or down-regulated more than 0.5-1.5 times were considered differentially expressed. Target genes of L. apetalum seeds with significant differences in expression before versus after germination at low temperature were identified.
Cloning and sequence analysis of the fulllength cDNA sequence of LaLEA1: Based on the transcriptome sequencing results of the seeds, specific primers for the amplification of LaLEA1 (LaLEA1-2F, 5'-CGGGGTACCAACAAAAATGGCGTCTCACC-3' and LaLEA1-2R, 5'-ACGCGTCGACGCTCACTTCCTC TGATA-3') were designed. Using the cDNA of L. apetalum seedlings as a template, the full-length sequence of the LaLEA1 gene was amplified (commissioned by the Beijing PMAD company for sequencing). DNAMAN software was used to compare these sequences with the transcriptome sequences. The NCBI database was used to analyze sequence homology. The properties of the protein were analyzed by ProtParam and the secondary structure was predicted using ExPASy. SWISS-MODEL was used to predict the tertiary structure of the proteins. By using MEGA 6 software to align amino acid sequences, and based on the maximum likelihood method, the phylogenetic tree was constructed through 500 bootstrap tests.

Statistical analysis:
The Student's t-test was used for analysis of statistical significance following a Gaussian distribution. Results are expressed as means ± standard errors (SEs), n = 3. Different letters (a-e) indicate significant differences (P < 0.05).

Results
Based on the transcriptome sequencing results of L. apetalum seeds, 35 sequences containing the key word LEA were identified. Following removal of redundant sequences, 27 LEA gene sequences were identified. The phylogenetic tree was constructed using 27 LEA protein enconding gene sequences, which showed that all of them belong to the LEA protein family (Fig. 1 Suppl.). The longest and shortest sequences were 882 and 164 bp, respectively. Of the 27 LEA sequences, seven encoded complete open reading frames.
Lepidium apetalum late embryogenesis abundant (LEA) protein family unigene distribution based on the Nr database and GO analysis indicated that the identified LEA family genes were highly homologous to known LEA genes of five species of Brassicaceae, 63 % were homologous with those of Arabidopsis thaliana (Fig. 1).
The predicted functions of the products encoded by the 27 LEA genes of L. apetalum were obtained by GO analysis. In total, 21 LEA genes were annotated to the biological process category, 10 to the molecular function category, and 15 to the cellular component category (Fig. 2). Among the 27 LEA genes, only 8 were annotated to the three categories. It is speculated that these 8 genes may have multiple functions in plant development and stress resistance (Fig. 2 Suppl.). Three genes were not annotated to terms in the GO database.
Based on the conserved sites, the plant LEA protein sequences were grouped into 26 functional components using the KOG database. In this study, 5 LEA protein family gene sequences isolated from the seeds of L. apetalum were functionally annotated in KOG. Only one sequence was reported, which was annotated as O (post-translational modification, protein turnover, chaperones); the remaining four sequences were annotated as S (function unknown) ( Table 1). The other 22 sequences were not annotated in the KOG database.
LEA proteins generally contain a highly conserved motif rich in alkaline and hydrophilic amino acids, and their structures are primarily composed of α-helices. LEA proteins are highly hydrophilic, and when subjected to abiotic stresses such as drought, salinity, and high temperature, are able to maintain function of membrane.
The 27 LEA protein family genes expressed during seed germination belonged to the groups D-7, D-113, D-73, and LEA_2 based on analysis of conserved domains. The hydrophilic features of these proteins resulted in their high thermal stability (Table 1 Suppl.).
The transcriptional data of LEA protein family genes of L. apetalum seeds were analyzed before and after germination at low temperature (Fig. 2). We conducted RT-qPCR to validate the RNA-seq data and to analyze the gene expression changes of randomly selected genes. Although the expressions of selected genes were different between RNA-seq and RT-qPCR, the trend of expressions were the same (Fig. 3 Suppl.). Three upregulated and twenty downregulated genes were identified, the  remaining four genes showed no significant difference based on transcriptional data (Fig. 4 Suppl.). The c37384_ g1 sequence was selected for further analysis based on the relationship between its expression and abiotic stress. This sequence was named LaLEA1 in this study. Via cloning and amplification of LaLEA1 from L. apetalum by RT-qPCR, one band of the expected size was obtained. Text to sequence analysis showed that the gene contained a complete open reading frame of 624 bp and encoded 208 amino acids (Fig. 5 Suppl.). Bioinformatic analysis showed that the molecular mass of the protein encoded by this gene was 22294.12. The protein was rich in hydrophilic amino acids. The threonine content was 17.9 %, glutamine content was 12.1 %, and lysine content was 11.6 %, whereas the highest content of hydrophobic amino acids was content alanine (15.9 %). Thus, the protein was highly hydrophilic. The tertiary structure of the protein was predicted to be dominated by α-helices, covering 72.95 % of the peptide chain. Irregular curls accounted for 17.39 % of the peptide (Fig. 6A Suppl.). The secondary structure suggests the protein is folded into an ellipsoid (Fig. 6B Suppl.). Side chains of polar amino acid residues were observed on the surface of the protein; thus, the protein is highly hydrophilic and able to bind water.
BLAST analysis revealed 10 species with LEA amino acid sequences close to LaLEA1, as identified in GenBank. A phylogenetic tree of the LEA proteins was constructed. LaLEA1 was found in one branch along with LEA76 of Camelina sativa and LEA29 of Capsella rubella, indicating that the conservation of LEA protein was tightly coupled to species (Fig. 3).
Homology analysis of the amino acid sequence of LaLEA1 and the LEA of 10 plant species revealed that the proximal C-and N-terminal sequences of the proteins were conserved, but the amino acid sequence in the middle (amino acids 30 -180) varied significantly and the overall homology was relatively low (Fig. 7 Suppl.).
Transcriptome analysis revealed that the LaLEA1 gene was highly expressed during the low-temperature germination. We further investigated whether this gene was involved in the response to short-term low temperature stress. For this purpose, 7-d-old seedlings of L. apetalum were exposed to 0 °C. After 2 h, the expression of LaLEA1 began to increase, reaching its highest level at 8 -12 h. Expression began to decrease after 24 h and, after 48 h expression was stable but still higher than that prior the treatment, suggesting that the LaLEA1 protein might play a role in the response to low temperature stress.
In contrast to exposure to low temperature, the seedlings under salt stress firstly wilted and then gradually recovered. The wilting rate increased with increased NaCl concentration. Following treatments with 50 -300 mM NaCl, all of the seedlings recovered completely at 48 h (Fig. 5A). To investigate whether salt stress influence the expression of LaLEA1, seedlings were treated with 150 and 300 mM NaCl. In the early stage of salt stress, the expression of LaLEA1 increased rapidly (Fig. 5B). Following treatment with 300 mM NaCl for 6 h, the seedling was highest and the expression of LaLEA1 was also the highest, up to 7.9-fold that of untreated seedlings. The rate of seedling wilting was also high at 6 h following treatment with 150 mM NaCl; however, the highest expression of LaLEA1 was detected at 12 h. These results suggested that salt stress induced LaLEA1 expression, and the expression was positively correlated with the NaCl concentration. With prolonged salt stress, the expression of LaLEA1 following treatment with both 150 and 300 mM NaCl decreased. However, a relatively high expression was maintained. These results suggest that LaLEA1 may play an important role in salt tolerance of L. apetalum seedlings.
Drought conditions were simulated by treatment with different concentrations of PEG 6000. Similar to salt stress, drought treatment can cause temporary wilting and with prolongation of the processing time, wilting was restored. However, unlike salt stress, the wilting was not reversible in all cases; high concentrations of PEG 6000 can cause permanent wilting. Wilting was completely reversed within 48 h with lower concentrations (10 and 15 % of PEG 6000), whereas it was only partially reversed at 25 and 30 % PEG 6000. Treatment of seedlings with 20 % PEG 6000 caused wilting that was almost completely reversible. Therefore, seedlings treated with 20 and 30 % PEG 6000 were chosen to investigate the relationship between LaLEA1 expression and osmotic stress. Our results showed that, with an increase in the wilting rate, LaLEA1 expression rapidly increased in the early stage of stress. With the prolongation of osmotic stress, the expression of LaLEA1 decreased gradually. However, a higher expression was maintained compared to that in control. Following treatment with 30 % PEG 6000, the wilting rate and LaLEA1 expression were highest at 24 h; the expression of LaLEA1 was up to 6.5-fold higher than that in control. Following treatment with 20 % PEG 6000, the highest LaLEA1 expression occurred at 12 h, whereas the highest wilting rate was observed at 24 h. These results suggest that the increase in LaLEA1 expression was related to the severity of the osmotic stress and that the LaLEA1 gene may play an important role in drought tolerance of L. apetalum seedlings. These results are similar to those obtained under low temperature and salt stress.

Discussion
The number of LEA family genes differs among plants. It has been reported that there are 36 LEA genes in Glycine max (Li et al. 2011), 27 in Ricinus communis (Zou et al. 2013), 23 in Medicago truncatula (Liu et al. 2015), 29 in Solanum tuberosum (Safa et al. 2015), 27 in Solanum lycopersicum (Cao and Li 2015), and 87 in Poplar sp. . Here, 27 genes encoding LEA family proteins were identified from the transcriptome of L. apetalum seeds during germination at low-temperature. It is possible that some LEA protein coding genes were not expressed in the germinated seeds of L. apetalum, and thus the number of LEA protein family genes in this plant may be greater than 27. More experimental evidence is needed to determine the number of LEA protein family coding genes in L. apetalum.
As an early spring ephemeral plant, the germination of L. apetalum seeds is an interesting process. Based on the transcriptome data, expression of the LaLEA1 gene was high in L. apetalum seeds, which were able to germinate at low temperature, as well as being higher than that of seeds that could not germinate significantly at low temperature. It has been suggested that the LaLEA1 gene plays an important role in the tolerance of seed germination to low temperature. It has also been shown that the amino acid sequence of the protein encoded by LaLEA1 was highly homologous to that of LEA76, of Camelina sativa and Brassica campestris. In terms of structure classification, LaLEA1 and LEA76 belong to the third group of LEA Fig. 4. Lepidium apetalum late embryogenesis abundant 1 (LaLEA1) expression in L. apetalum seedlings in response to short-term low temperature stress (0 °C). Means ± SEs, n = 3. Different letters indicate significant differences (P < 0.05; the Student's t-test). proteins. Members of this group of LEA proteins have been reported to bind to hydrophobic surfaces to form dimers, thereby performing an ionic chelating function, when plants are dehydrated. Dure and Galau (1976) reported that LEA76 has 13 repetitive motives, but did not specify the function of LEA76 for plants in KOG annotations. The secondary structure of the LaLEA1 protein is primarily composed of α-helices, and dimers are formed by hydrophobic interactions. The side chains of hydrophilic amino acids are distributed on the surface of the molecule, rendering the protein more bound water. Because of its hydrophilic properties, LaLEA1 can bind several water molecules. When plant cells are exposed to low temperature stress, the LEA proteins enhance plant tolerance to that stress by lowering the freezing point of bound water. Under salt and osmotic stress, the hydrophilic LEA proteins maintain cell water content via their strong water binding capacity, reducing the amount of damage caused by loss of water. This result is consistent with the characteristics of L. apetalum seedlings, which are tolerant to low temperature, high salinity, and drought. We believe that the expression of LaLEA1 is closely related to plant tolerance to abiotic stresses, such as low temperature, salinity, and drought.
The RT qPCR indicated that the expression of LaLEA1 was sensitive to low temperature stress, such as 0 °C. In the early stage of stress (within 1 h), the expression decreased significantly, and then increased rapidly; expression was highest at 8 -12 h. This suggests that the change in gene expression is not a direct response to low temperature stress, but rather to a later step in signal transduction (Fig. 7). One possible explanation is that the expression of LaLEA1 at the early stage of cold stress increased sharply in responce to sudden hypothermia. Over time, the seedlings gradually adapted to the low temperature; while the expression of LaLEA1 decreased slowly, it still remained at a relatively high level. This latter phenomenon suggests that LaLEA1 may play a role in the regulation of seedling growth under sustained low temperature.
Following salt or osmotic stress, L. apetalum seedlings wilt rapidly and the greater the NaCl or PEG concentration the higher the rate of wilting. However, when the stress was present for more than 48 h, most of the seedlings returned to normal, i.e., the wilting rate decreased significantly. Interestingly, the change in LaLEA1 gene expression was closely linked to the change in seedling wilting rate. That Fig. 6. Wilting rate of Lepidium apetalum seedlings. A -Wilting the seedlings treated with different concentrations of PEG 6000 for different stress duration. Slightly yellow and curly leaves were thought to have wilted. B -Quantitative fluorescenceanalysis of L. apetalum late embryogenesis abundant 1 (LaLEA1) expression in L. apetalum seedlings treated with PEG 6000 for different duration (48+Rew is the seedlings recovery at 0 mM NaCl for 12, 24, and 48 h after 48-h salt stress). Fig. 7. Lepidium apetalum late embryogenesis abundant 1 (LaLEA1) expression in L. apetalum seedlings in response to abiotic stresses for 0, 1, 12, 24, and 48 h, CK -7-d-old control seedling, 48+Rew -seedlings recovered at 0 mM NaCl for 12, 24, and 48 h after 48-h salt stress. is, seedlings wilted rapidly when under both drought or salt stresses, and the expression of LaLEA1 also increased rapidly in a short time; the wilting rate and expression of LaLEA1 was highest at similar times. Then, the seedlings gradually returned to normal; expression of LaLEA1 decreased but remained higher than that under non-stress conditions.
These results suggest that LaLEA1 expression might play an important role in the tolerance of L. apetalum seedlings to low temperature, salinity, and osmotic stress although additional experimental evidence is needed to determine whether LaLEA1 increases the ability of plants to retain water.