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Article

Identification of Picea mongolica LEA Gene Family Implicates PmLEA25 in Drought Resistance

by
Yaping Wang
1,
Jiaqi Li
2 and
Yu’e Bai
1,*
1
College of Forestry, Inner Mongolia Agricultural University, Huhhot 010018, China
2
Inner Mongolia Autonomous Region Forestry and Grassland Seedling Station, Huhhot 010052, China
*
Author to whom correspondence should be addressed.
Forests 2024, 15(11), 1988; https://doi.org/10.3390/f15111988
Submission received: 1 October 2024 / Revised: 6 November 2024 / Accepted: 8 November 2024 / Published: 11 November 2024
(This article belongs to the Special Issue Abiotic and Biotic Stress Responses in Trees Species)
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Abstract

:
Picea mongolica is a rare and valuable tree species in China, having high tolerance for drought, cold, and sand burial. The late embryogenesis abundant protein (LEA protein) is a crucial transcription factor that plays a key role in both plant embryonic development and stress response. LEA genes have, however, not yet been reported in P. mongolica. In this study, through the analysis of genome data from Picea abies and transcriptome data from P. mongolica, a total of 49 PmLEAs were discovered and categorized into eight subfamilies based on their Pfam domain and phylogenetic relationship. RNA-Seq research revealed that 37 PmLEAs were differentially expressed at various stages of embryonic development. Using qRT-PCR, we found that most PmLEAs responded strongly to drought stress, with genes in the same subfamily exhibiting identical expression patterns. In particular, PmLEA25 is the most highly induced by drought treatment. Furthermore, we heterologously transformed PmLEA25 into Arabidopsis. The overexpression of PmLEA25 remarkably increased the germination rate, root length, and antioxidant capacity in Arabidopsis under drought treatment, compared with WT. The results serve as a point of reference for gaining a deeper comprehension of the function of PmLEA25 in the molecular process of stress resistance in P. mongolica. Additionally, they offer significant genetic materials for the purpose of breeding stress-resistant spruce species.

1. Introduction

Picea mongolica, an evergreen tree in the family Pinaceae, is a rare and endangered tree species in China, as well as an outstanding windbreak and sand-fixing tree in northern China. It grows naturally in the Hunsandak sands of Inner Mongolia and is highly resistant to drought, cold, and sand burial. The wood is mostly utilized for papermaking, furniture, and musical instrument manufacturing, while the bark is used to extract turpentine [1]. Spruce forests, as a natural protective barrier, contribute significantly to local agricultural production and have a vital impact on the ecological protection of the capital city of Beijing, which is only 300 km away, as well as the ecological environment of northern China. As a result, many scholars have been drawn to study its distribution [2], seedling [3], physiological biochemistry [4], somatic embryogenesis [5], and endophytic diversity [6], but its molecular biology is rarely studied, particularly the limited research on resistance gene mining and the resistance improvement research process.
Transcription factors (TFs) regulate gene expression in a variety of important plant developmental processes, including cell morphogenesis, signal transduction, and stress responses [7]. LEA proteins are a type of protein that plays an important function in plant stress responses [8]. With low concentrations of cysteine (Cys), tryptophan (Trp), and hydrophilic amino acid residues like glycine (Gly) and lysine (Lys), the majority of LEA proteins are extremely hydrophilic [9]. Plant LEA proteins can be categorized into distinct subfamilies using various classification criteria, such as conserved motifs and evolutionary relationships of LEA proteins [10]. Dure et al. [11] classified LEA proteins into three subfamilies based on the comparison of their amino acid sequences: the first subfamily includes proteins derived from the Em gene, the second comprises proteins from the RAB and dehydrin genes, and the third encompasses proteins from other LEA genes. As more LEA proteins are identified, the classification criteria for LEA proteins are evolving. Currently, LEA protein families are typically categorized into eight subfamilies based on their original sequence: LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, LEA_6/PvLEA18, DHN, SMP, and AtM [10].
LEA proteins were initially identified in cotton (Gossypium spp.) and accumulate in considerable amounts during late embryonic development [12]. In recent years, the continuous release of plant genome and transcriptome data have shown the presence of 51, 34, 32, 27, 88, and 23 LEAs in Arabidopsis thaliana [13], Oryza sativa [14], Zea mays [15], Solanum lycopersicum [16], Populus tremula [17], and Pinus tabulaeformis [18], respectively. The role of LEA protein in biological processes like drought resistance, salt tolerance, and low-temperature tolerance in plants has been shown by a number of studies. Drought, cold, and salt stress may all considerably boost the expression of 12 LEAs in Arabidopsis [19,20], with AtLEA14 overexpression greatly increasing salt resistance [21]. SiLEA4 and SiLEA5 can enhance tomato resilience to low temperatures and dehydration via modulating antioxidants [22,23]. In soybean, the heterologous expression of GmLEA2-1 in Arabidopsis enhanced drought and salt tolerance, whereas GmLEA4 interacted with GmCaM1 to significantly improve Arabidopsis seed vigor under conditions of an elevated temperature and humidity stress [24,25]. The MsLEA3-1 gene, located in the nucleus, exhibits high expression levels when induced by salt and ABA, and its overexpression in tobacco significantly enhances salt tolerance [26]. The LEA4-4 gene augments antioxidant enzyme activity in transgenic Arabidopsis and is involved in the ABA signaling system, providing tolerance to salt and drought stress in transgenic Arabidopsis [27]. In alfalfa, MsABF2 modulates gene expression by binding to the promoter region of MsLEA-D34, thereby augmenting the plant’s resilience to drought and salinity stress [28]. MsDIUP1 improves the plant’s salt tolerance by regulating stress signaling, antioxidant defense, ion homeostasis, osmotic regulation, and photosynthesis [29]. Additionally, poplar PtrDHN-3 transgenic yeast and Arabidopsis thaliana improved their salt tolerance [30].
In previous studies, the LEA gene family has been identified and studied across various species, but no reports have been published on P. mongolica. In our study, we identified all PmLEA gene family members in P. mongolica and investigated their evolutionary relationships and expression patterns at various stages of embryonic development and during drought stress. Simultaneously, PmLEA25 was evaluated for its role in drought stress, enhancing understanding of the LEA family and providing essential genetic resources for future breeding of stress-resistant plants.

2. Materials and Methods

2.1. Plant Materials and Drought Stress Treatment

Viable seeds of P. mongolica from one single tree were soaked in water overnight before being sown in sandy soil under conditions of 25 °C, 65% relative humidity, and a 16 h light/8 h dark cycle. The one-year-old potted seedlings exhibiting constant growth and vitality were subjected to 20% PEG 6000 drought stress. Following treatment durations of 0, 6, 12, and 24 h, the plants were rapidly frozen in liquid nitrogen and subsequently stored at −80 °C in a refrigerator.
The Arabidopsis wild-type (WT) seeds utilized in this investigation were derived from the Columbia (Col-0) ecotype. The seeds first underwent sterilization with 75% ethanol for 5 min, followed by three washes with sterile water, and subsequently underwent sterilization with 5% NaClO for 10 min, concluding with 5 to 6 washes with sterile water. For drought treatment, wild-type and transgenic Arabidopsis seeds were placed in a 1/2 MS medium supplemented with 200/300 mM Mannitol. Three-week-old potted seedlings were subjected to drought treatment using a 20% PEG 6000 solution for one week prior to phenotypic and physiological investigation. The cultivation parameters for A. thaliana were established at 16 h of illumination and 8 h of darkness, 75% humidity, 22 °C, and 5000 Lux.

2.2. Identification of PmLEAs and Sequence Analysis

The amino acid sequences of 51 LEAs from the Arabidopsis genome (https://www.arabidopsis.org/, accessed on 6 January 2024) were used as probes to identify LEA proteins in P. mongolica. To identify LEA transcription factor candidate genes, a local BlastP search of P. mongolica transcriptome data (PRJNA649217) was performed using BioEdit 7.2.0 software [31], with both BLASTP and HMM alignment, using default parameters and E values less than 0.001. The conserved domain of the potential LEA protein was then evaluated using SMART, and only LEA sequences with complete conserved domains were retained. The LEAs were further aligned using the Picea abies genome (https://plantgenie.org/, accessed on 10 January 2024), and the members of the LEAs were eventually identified and named after Arabidopsis-comparable genes. Furthermore, the sequence length, molecular weight (MW), isoelectric point (PI), and GRAVY of LEA proteins were ascertained utilizing ExPASy (https://www.expasy.org/, accessed on 16 January 2024). Additionally, we used the MEME (https://meme-suite.org/meme/, accessed on 23 January 2024) web platform to examine protein conserved motifs in all LEA proteins from P. mongolica and Arabidopsis, and 20 motifs were selected for prediction.

2.3. Phylogenetic and Evolutionary Tree Construction

Multiple sequence alignments of all LEA proteins in P. mongolica and Arabidopsis were performed using Cluustal X2.1 software. The phylogenetic and evolutionary tree was generated using MEGA X with the neighborhood connection (NJ) method, utilizing a bootstrap option set to 1000 iterations while retaining default settings for all parameters.

2.4. Expression Analysis of PmLEAs

The LEA gene family was analyzed by hierarchical clustering and an expression heat map by TBtools-II v2.003 software based on FPKM in the transcriptome database (PRJNA649217) during the different developmental stages of somatic embryos of P. mongolica. The Plant Total RNA Extraction Kit (TAKARA) was used to extract total RNA from the various samples, and its integrity and concentration were determined using 1.0% agarose gel electrophoresis and Nanodrop 2000. The synthesized cDNA products were stored at −20 °C and diluted five times before use as templates. The qRT-PCR primers were designed using Primer3 software (Table S1). The qRT-PCR was carried out using iCycler iQ equipment (Bio-Rad, Hercules, CA, USA) using SYBR qPCR Master Mix (Vazyme, Nanjing, China) according to the manufacturer’s instructions. EF1α (MA_505653g0010) was utilized as the reference gene. Relative gene expression levels were estimated using the 2−ΔΔCt technique. A significant difference analysis and histograms were obtained using GraphPad Prism 10 software. Each treatment included three biological replicates and three technical replicates per sample.

2.5. Characterization of Cis-Elements in Promoter Region of PmLEA25

PlantCARE suggested that the PmLEA25 genes’ promoter region (2.0 kb upstream of genomic DNA sequences) contains putative cis-elements.

2.6. Construction of PmLEA25 Overexpression Vector and Genetic Transformation of Arabidopsis thaliana

The coding sequence (CDS) of the PmLEA25 gene was cloned from P. mongolica cDNA, and the cloning primers are specified in Table S1. The CDS was inserted into the pDONR222 vector using Gateway technology. The resulting pDONR222-PmLEA25 plasmid was freeze-thawed into Agrobacterium tumefaciens GV3101 before being heterologously transformed into Arabidopsis using the floral dip method [32].

2.7. Screening of Positive Plants

Genomic DNA was extracted from WT and positive Arabidopsis thaliana leaves. PCR was conducted using primers pMDC32-F and PmLEA25-R (Table S1) to verify the effective integration of the target gene into the genome, based on the size of the positive plasmid band. Samples with the correct band size were further analyzed for PmLEA25 expression levels in Arabidopsis, using ACT3 as the internal reference gene.

2.8. Phenotypic Observation and Physiological Assays

The germination rates of normal and stress-treated WT and OE-PmLEA25 Arabidopsis seedlings were measured over one week, and root length was assessed after 14 d. The growth phenotypes of Arabidopsis potted seedlings were assessed under normal and drought stress treatments for 7 d, comparing WT and OE-PmLEA25. Standard methods were used to determine the activity of antioxidant enzymes, including malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as per established protocols (jiubang Biotechnology Co., Ltd., Quanzhou, China).

3. Results

3.1. Identification and Phylogenetic Study of LEA Gene Family in P. mongolica

A total of 49 PmLEAs were identified in the P. abies entire genome and the P. mongolica transcriptome database using HMM Search. The 49 PmLEAs were systematically designated according to their resemblance to Arabidopsis homologs, representing the inaugural instance of systematic nomenclature (Table 1). The physicochemical study revealed considerable heterogeneity in the amino acid sequence lengths of the 49 PmLEAs (Table 1). The longest sequence, PmLEA31, consisted of 698 amino acid residues, while the shortest sequence, PmLEA1.2b, consisted of only 60 amino acid residues. The theoretical molecular weights of these sequences varied from 6.7194 kDa to 22.872 kDa. The theoretical isoelectric points (pIs) of PmLEAs ranged from 4.07 kDa to 9.82 kDa, with 22 (45%) having a pI greater than 7 and 27 (55%) having a pI less than 7. Furthermore, the majority of the PmLEAs encode hydrophilic proteins; 46 of them have a GRAVY value less than zero. The most hydrophilic are from the LEA_1 subfamily, with average GRAVY values of −1.472 and −1.461, respectively. In contrast, three genes have GRAVY values greater than zero, and these are entirely from the LEA_2 and DHN subfamilies.
A phylogenetic tree was generated utilizing the protein sequences of 49 PmLEAs and 51 AtLEAs. The tree demonstrated that all LEAs grouped together based on the class of Pfam structural domains (Figure 1), confirming the alignment of the P. mongolica subfamily classification with the Arabidopsis LEA gene subfamily. The analysis of Pfam structural domains indicated their widespread occurrence in the majority of PmLEAs, with numerous instances exhibiting multiple LEA structural domains. The 49 PmLEAs were categorized into eight subfamilies according to their structural domains (Figure 1): 5 members of PmLEA_1, 1 member of PmLEA_2, 12 members of PmLEA_3, 16 members of PmLEA_4, 2 members of PmLEA_5, 6 members of DHN, 5 members of ATM, and 2 members of SMP. Notably, no genes from the PmLEA_6 subfamily were found in P. mongolica.

3.2. Protein Conserved Motif Analysis of LEAs in P. mongolica

The study revealed the discovery of 20 conserved motifs (Figure 2), each with an average length ranging from 14 to 50 amino acids. The results showed that all subfamilies had unique conserved patterns in their genes, except for PmLEA19, PmLEA22, PmLEA37, and PmLEA4.3. There is considerable variation in the number of motifs present in PmLEAs. Among them, PmLEA31 stands out with the highest number of motifs, totaling 10, surpassing all other PmLEA protein sequences. Motif 2 is present in all LEA_3 subfamily proteins, whereas Motif 3 is observed in most LEA_4 subfamily proteins. Generally, the content of motifs varies greatly within subfamilies, and most members within a subfamily display similar motifs. This implies that genes belonging to the same subfamily carry out comparable functions. However, the conserved patterns of different PmLEAs within specific subfamilies also differ. For example, the dehydrin subfamily contains PmLEA20 and PmLEA24, which have a higher prevalence of Motif 9. This suggests that PmLEA proteins belonging to the same subfamily may have different functions. Furthermore, they contain K, S, and Y segments (Table S2).

3.3. Transcript Pattern of PmLEAs During Somatic Embryo Development by RNA-Seq

Based on the heatmap analysis (Figure 3), we found that out of the 49 PmLEAs, 37 were expressed in six different stages. Most of these genes exhibited high levels of transcripts during the early (Megagametophy) and late (cotyledon embryos and mature embryos) stages of somatic embryo development. Notably, PmLEA1.2a/11/38/41, PmLEA14/11/18/26, and PmLEA7/9/17/22 displayed exceptionally high transcript abundance in Megagametophy, cotyledon embryos, and mature embryos, respectively. This indicates that these genes are essential for somatic embryo beginning and subsequent developmental events. Furthermore, it is important to mention that two genes (PmLEA1.2b/24) exhibited high expression levels in cleavage polyembryony, two genes (PmLEA21/29) showed high expression levels in dominant embryos, and two genes (PmLEA1.2c/19) displayed high expression levels in pre-cotyledon embryos. These genes exhibited significant expression levels in pre-cotyledon embryos. In summary, a total of five genes (PmLEA5/10/13/31/43) were consistently up-regulated, while three genes (PmLEA11/38/41) were consistently down-regulated during somatic embryo development. The aforementioned findings indicate that various PmLEAs may have distinct roles in the process of somatic embryo development in P. mongolica.

3.4. Expression Analysis of PmLEAs Under Drought Stress

To further examine the response of the PmLEAs to abiotic stress, we analyzed the expression profiles of 49 PmLEAs under drought conditions (Figure 4). The findings showed that, with extended stress treatment duration, most PmLEAs show significant differential expression under drought stress conditions. Specifically, 11 genes (PmLEA8/14/18/20/25/30/31/34/35/40/41) were up-regulated by over 5-fold under drought treatment. In contrast, PmLEA22 was continuously down-regulated during drought therapy. Additionally, we observed that PmLEA8 showed a more than 7-fold increase in expression after 6 h of drought stress. Furthermore, we noted that the expression of PmLEA25/35/41 increased by more than 18-fold after 6 h of drought stress, the expression of PmLEA20/25 increased by more than 70-fold after 12 h of drought stress, and the expression of PmLEA25/41 increased by more than 20-fold after 24 h of drought stress. In general, under drought stress, most PmLEAs reached peak expression at 24 h. The results indicate that the PmLEA gene family may have various functions in the context of abiotic stress development. Notably, PmLEA25 exhibited up-regulation by over 80-fold during drought stress, suggesting its possible role as a candidate gene in the control of abiotic stress responses in P. mongolica.
There were multiple regulatory elements associated with stress and hormone responses within the promoter of PmLEA25 (Figure S1). The results revealed an abundance of regulatory elements associated with stress and hormone responses within the promoter of PmLEA25. These include STRE elements related to stress response, anaerobic induction-regulating elements such as ARE, damage and pathogen response elements like the W box, and MeJA-responsive cis-regulatory elements. Additionally, motifs such as the TGACG motif associated with the jasmonic acid signaling pathway and TGA element linked to salicylic acid response were identified (Table S3). These cis-elements collectively indicate the significant involvement of this gene in stress response, potentially subject to complex regulatory networks involving jasmonic acid and salicylic acid signaling pathways.

3.5. Overexpression of PmLEA25 Enhances Drought Resistance in Transgenic Arabidopsis

Using the primers pMDC32-F and PmLEA25-R, the DNA from Arabidopsis leaves was extracted and confirmed by PCR (Figure 5B). In positive plants, gel electrophoresis showed a single band of the anticipated size (500 bp), indicating that the PmLEA25 gene had been integrated into the Arabidopsis genome. Additionally, using the RT-PCR analysis, three stable overexpressing PmLEA25 lines were chosen for treatments against drought stress (Figure 5C).
Under drought conditions, the germination rate of Arabidopsis thaliana diminished as Mannitol concentration increased, and the germination rate of WT Arabidopsis was markedly lower than that of the overexpressed PmLEA25 lines. Under 200 mM Mannitol stress conditions, the germination rates of the L2, L3, and L4 strains began to be approximately twice that of the WT after the first day. On day 4, the germination rate of the WT was only 30.23%, while the germination rates of the L2, L3, and L4 lines were 77.85%, 80.60%, and 81.34%, respectively. Moreover, compared to the WT, the overexpressed PmLEA25 lines exhibited a faster growth rate. Under 300 mM Mannitol stress, similar to the trend observed with 200 mM Mannitol, the germination rates of the L2, L3, and L4 lines continuously surpassed those of the WT plants beginning on day 3. On day 6, the peak values were seen, with germination rates of 72.36%, 73.06%, and 68.41% for the L2, L3, and L4 lines, respectively, whereas the WT exhibited a germination rate of merely 42.65% (Figure 6).
In the normal 1/2 MS medium, there was no notable variation in the primary roots and lateral roots of the L2, L3, and L4 lines compared with the WT. In the 1/2 MS medium supplemented with 200 mM Mannitol, the cotyledons of all plants were shrunk and curled to a certain extent, and the primary root lengths of the L2, L3, and L4 lines were significantly greater than those of the WT. In the 1/2 MS medium with 300 mM Mannitol, the primary root lengths of the L2, L3, and L4 lines were much greater than that of the WT, and the number of lateral roots also exhibited a significant increase (Figure 7).
Through subjecting 20-day-old WT and overexpressed PmLEA25 plants to one week of drought stress treatments, we observed chlorosis and wilting in leaves of both WT and overexpressed PmLEA25 lines. However, the WT exhibited more severe wilting symptoms (Figure 8A). Abiotic stress enhances the generation of reactive oxygen species (ROS), resulting in oxidative damage to the cellular membrane. Antioxidant enzymes are crucial for ROS scavenging and plant defense mechanisms. To further investigate the correlation between the enhanced drought tolerance of overexpressed PmLEA25 lines and antioxidant enzyme activity, we measured the activity of antioxidant enzymes in plants grown subjected to identical drought stress conditions (Figure 8B). The findings indicated that in Arabidopsis, the overexpressed PmLEA25 lines displayed markedly reduced malondialdehyde (MDA) levels relative to the WT, although the activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) demonstrated an inverse pattern (Figure 6B). The above results indicate that the expression of the PmLEA25 reduces membrane damage in plants due to drought and enhances the detoxification of active oxygen free radicals, thereby increasing its drought resistance.

4. Discussion

P. mongolica, one of Inner Mongolia’s three main sand management tree species, is widely distributed in the Hunsandak Desert and has excellent cold and drought tolerance, making it an important resource for resistance gene mining. LEA proteins assist plants in responding to various abiotic stress, such as salt stress, drought stress, high-temperature stress, and low-temperature stress [33,34,35]. LEA-related genes have recently been discovered in a range of plant species [15,17,36,37,38,39]; however, they have not been detected in P. mongolica.
Research showed that various plants differ in the number of members and distribution of subfamilies within the LEA gene family. For instance, Jin et al. [40] identified 48 CsLEAs in the tea tree, which were divided into the LEA_1 to LEA_5, DHN, and SMP subfamilies, but lacked the LEA_6 and ATM subfamilies. Similarly, Yu et al. [41] identified 26 CkLEAs in Caragana korshinskii, classified into eight subfamilies but lacking the LEA_5 subfamily. In soybean, 36 GmLEAs were identified, which can be divided into eight subfamilies, among which the ATM subfamily is missing [42]. In poplar, a total of 87 LEAs were identified, divided into eight subfamilies, with the LEA_2 subfamily including the largest number of members [43]. In our study, we discovered 49 PmLEAs in P. mongolica (Table 1). The LEA_4 subfamily comprises the highest number of members, while the LEA_6 subfamily is absent (Figure 1). Members of the LEA gene family have been found in several species, each exhibiting a characteristic structural domain, but the distribution of members across different subfamilies shows significant variation, frequently accompanied by common subfamily deletions. In addition, in the tea tree, due to different identification methods and database ranges, 33 CsLEAs were identified by Wang et al. [44] while 48 CsLEAs were identified by Jin et al. [40]. Therefore, we hypothesize that there are more than 49 PmLEAs in P. mongolica, and there may be other members that have not been identified. The abundance of members in the LEA_4 subfamily suggests that it has evolved more rapidly and may have more complex physiological functions (Figure 1). Additionally, based on the physicochemical property analysis, most of the PmLEAs are small, hydrophilic molecules with highly conserved motifs within the same subfamily but widely varying motifs among subfamilies, such as the K, S, and Y fragments in all DHN subfamily members (Figure 2, Table S2), consistent with the findings of Jin et al. [40], Yu et al. [41], and Liang et al. [36], implying subfamily-specific functional roles for LEAs.
The mRNA of LEAs remains at elevated levels in desiccation-tolerant embryos, whereas the transcripts of storage proteins are entirely degraded during the final stages of embryonic development [45]. Our research findings indicate that most PmLEAs exhibit high levels of transcripts during the late phases of somatic embryonic development, especially notable in mature embryos (Figure 3). Similar results have been reported in wheat [46], and soybeans [47], indicating that during the development of somatic embryos in P. mongolica, PmLEAs may have different functions at various stages. LEAs are abundantly expressed in response to stressors such as drought, low temperature, high temperature, and high salinity [48]. In Caragana korshinskii, 19 CkLEAs were up-regulated over 2-fold in response to drought and salt stress and were influenced by stresses including ABA, high temperature, and low temperature [41]. In wheat, 46 TaLEAs exhibited up-regulation in expression due to low-temperature stress, whereas 177 TaLEAs were induced under drought stress, with 50 TaLEAs up-regulated during drought stress and 25 consistently up-regulated under both high-salt and low-temperature stress [49]. In the tea tree, 23 CsLEAs were up-regulated by low-temperature stress, 44 by high-temperature stress, 33 by drought stress, and 28 by ABA stress, of which 14 responded to all stresses [40]. Following drought treatment in P. mongolica, it was demonstrated that the expression of numerous LEA genes was up-regulated due to drought stress, with PmLEA25 being the most notable (Figure 4), suggesting that PmLEA25 plays a crucial role in the response to drought stress. The promoter was further predicted and analyzed to contain elements responsive to ARE, MEJA, and STRE (Figure S1), which were speculated to play a protective role in relevant adversities and may augment the resistance of P. mongolica to diverse abiotic adversities.
PmLEA25 proteins are classified as members of the DHN subfamily, while dehydrin proteins are classified as members of the second set of family proteins, also referred to as LEB proteins or water stress proteins (WSPs) [50]. Dehydrins, which are proteins found in plants, have significant functions in the way plants react to various abiotic stressors [51,52]. Various stress conditions lead to the accumulation of different types of dehydrins. The overexpression of the wheat LEA gene DHN-5 in Arabidopsis dramatically enhances the plant’s ability to withstand osmotic stress [53]. The introduction of the barley LEA gene HVA7 into rice resulted in a substantial improvement in the tolerance of transgenic rice lines to drought and salt treatments [54]. An increased expression of the wheat LEA gene (WCOR410) in strawberry plants can greatly enhance the ability of leaves to withstand cold temperatures [55]. Furthermore, studies have verified that DHNS can greatly enhance the ability of pepper, peanut, and cotton plants to withstand or respond to abiotic stress [56,57,58]. The overexpression of PmLEA25 enhanced drought tolerance, with transgenic plants showing better growth and less damage than wild-type plants under stress conditions (Figure 6 and Figure 7), suggesting that PmLEA25 has a positive regulatory role in abiotic stress tolerance.
In response to abiotic stress, plants generate significant quantities of ROS and membrane lipid peroxidation products, such as MDA, which compromise cell membrane stability and cause cellular injury [59]. To mitigate this damage, plants synthesize antioxidant enzymes to neutralize ROS, as well as osmoregulatory compounds, such as proline, to maintain osmotic balance within the cell membrane [60]. The LEA protein has the ability to directly decrease the amounts of intracellular ROS and strengthen the plant’s antioxidant system, hence diminishing oxidation [35]. The DHN gene HbDHN2 was discovered in Hevea brasiliensis, and its overexpression in Arabidopsis greatly significantly enhanced tolerance to salt and drought. Under drought and salt stress, the transgenic lines exhibited longer root systems, reduced electrolyte leakage, and enhanced SOD, APX, and CAT activity, as well as elevated proline levels compared to the WT [61]. Furthermore, overexpressing the lemon (Leguminosae) group II LEA gene, CkLEA2-3, in Arabidopsis resulted in overexpression lines that accumulated less malondialdehyde and maintained higher SOD activity and GSH content under drought stress, thereby improving drought tolerance [41]. After converting the alfalfa MfLEA3 gene into tobacco, the transgenic lines collected less ROS and had considerably greater SOD, CAT, and APX activity after a low temperature, drought, and strong light irradiation [62]. Under drought stress, we discovered that the overexpression of PmLEA25 in Arabidopsis plants efficiently enhanced SOD, POD, and CAT enzyme activities; reduced MDA production; and improved antioxidant capacity (Figure 8). To summarize, the activation of the PmLEA25 gene in plants positively regulates the plants’ ability to withstand stress. This study offers significant stress resistance gene resources for P. mongolica and serves as a reference for further investigation into the plant’s stress resistance mechanism.
Drought, salinity, and cold temperatures can induce water deficits in plants, leading to the accumulation of various protective proteins, with LEA proteins being the most prevalent [63]. The LEA gene family has a crucial role in drought-tolerant plants like P. mongolica, particularly in contexts of escalating drought and desertification. Enhancing the expression of this gene will be essential for the growth and survival of P. mongolica. Our research indicates that the overexpression of the PmLEA25 gene can markedly enhance the drought resistance of transgenic Arabidopsis plants. While the precise molecular mechanisms underlying this enhancement warrant further investigation, the findings suggest a potential avenue for improving drought tolerance in P. mongolica. The lengthy and unstable traditional breeding cycle allows for the utilization of the LEA gene to enhance the drought tolerance of P. mongolica by advanced molecular breeding, hence augmenting its adaptability and survival rate in desertified regions. This not only mitigates ecological issues arising from drought but also establishes a scientific foundation for desertification prevention and ecological restoration.

5. Conclusions

In summary, this study offers a comprehensive and systematic analysis of the LEA family in P. mongolica. We found 49 PmLEA genes, categorized into eight subfamilies. The transcriptional profiling and gene expression pattern analysis revealed that several PmLEA genes have unique functions during somatic embryo development or drought stress treatment, with the PmLEA25 gene exhibiting considerable up-regulation under drought conditions. Subsequent functional verification demonstrated that Arabidopsis plants overexpressing the PmLEA25 gene enhanced their drought resistance by modulating antioxidant enzyme activity. Consequently, PmLEA25 may serve as a candidate gene for drought resistance and offer theoretical support for the molecular breeding of P. mongolica and other plant species. Moreover, our findings enhance the comprehension of the function of LEA under drought stress and establish a basis for further investigation into the molecular mechanisms of drought resistance in P. mongolica.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f15111988/s1, Figure S1. Analysis of cis-elements in the promoter of PmLEA25 gene. Table S1. Primer sequences used in this study. Table S2. Conserved motifs in different subfamilies of the LEA gene of Picea mongolica. Table S3. PmLEA25 promoter cis-acting element.

Author Contributions

Y.W. and J.L. devised the experiment. Y.W. and Y.B. conducted the experiment. Y.W. and J.L. analyzed bioinformatics, phenotypic, and physiological data. Y.W. wrote and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Natural Science Foundation of China (32360402) and the Natural Science Foundation of Inner Mongolia Autonomous Region (No. 2022MS03037).

Data Availability Statement

The raw RNA-Seq data used in this study have been deposited in the Sequence Read Archive (SRA) database of the National Center for Biotechnology Information (NCBI) with the access number PRJNA649217, “https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA649217 (accessed on 10 November 2024)”.

Conflicts of Interest

The authors affirm that there are no conflicts of interest regarding the publication of this paper.

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Figure 1. Phylogenetic relationship between 49 LEAs of P. mongolica and 51 LEAs of A. thaliana. Red circles and blue squares represent P. mongolica and A. thaliana.
Figure 1. Phylogenetic relationship between 49 LEAs of P. mongolica and 51 LEAs of A. thaliana. Red circles and blue squares represent P. mongolica and A. thaliana.
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Figure 2. Analysis of conserved motifs of 49 LEAs in P. mongolica and 51 LEAs in Arabidopsis.
Figure 2. Analysis of conserved motifs of 49 LEAs in P. mongolica and 51 LEAs in Arabidopsis.
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Figure 3. Transcription expression profiles of PmLEAs during embryonic development. SDYS1: Megagametophy; SDYS2: cleavage polyembryony; SDYS3: dominant embryos; SDYS4: pre-cotyledon embryos; SDYS5: cotyledon embryos; SDYS6: mature embryos. Red indicates high expression, and green indicates low expression.
Figure 3. Transcription expression profiles of PmLEAs during embryonic development. SDYS1: Megagametophy; SDYS2: cleavage polyembryony; SDYS3: dominant embryos; SDYS4: pre-cotyledon embryos; SDYS5: cotyledon embryos; SDYS6: mature embryos. Red indicates high expression, and green indicates low expression.
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Figure 4. Expression analysis of 49 PmLEAs identified from P. mongolica under drought stress. Red indicates high expression, and green indicates low expression.
Figure 4. Expression analysis of 49 PmLEAs identified from P. mongolica under drought stress. Red indicates high expression, and green indicates low expression.
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Figure 5. Screening and identification of PmLEA25 transgenic Arabidopsis. (A) Hygromycin screening of transgenic plants. (B) PCR results of PmLEA25. M: 2000 bp DNA Marker. +: Positive control. -: Negative control. 1–10: Different transgenic lines. (C) Expression levels of PmLEA25 in WT and three overexpressing PmLEA25 lines analyzed by RT-PCR. **** p value < 0.0001.
Figure 5. Screening and identification of PmLEA25 transgenic Arabidopsis. (A) Hygromycin screening of transgenic plants. (B) PCR results of PmLEA25. M: 2000 bp DNA Marker. +: Positive control. -: Negative control. 1–10: Different transgenic lines. (C) Expression levels of PmLEA25 in WT and three overexpressing PmLEA25 lines analyzed by RT-PCR. **** p value < 0.0001.
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Figure 6. Effects of drought stress on the seed germination rate of WT and overexpressed PmLEA25 lines. (A) Germination phenotypes of WT and overexpressed PmLEA25 lines at varying doses of Mannitol. (B) Germination rates of WT and overexpressed PmLEA25 lines under varying amounts of Mannitol.
Figure 6. Effects of drought stress on the seed germination rate of WT and overexpressed PmLEA25 lines. (A) Germination phenotypes of WT and overexpressed PmLEA25 lines at varying doses of Mannitol. (B) Germination rates of WT and overexpressed PmLEA25 lines under varying amounts of Mannitol.
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Figure 7. Effects of drought stress on the root length of WT and overexpressed PmLEA25 lines. (A) Root phenotypes of WT and overexpressed PmLEA25 lines at varying amounts of Mannitol. (B) Root length statistics of WT and overexpressed PmLEA25 lines under varying amounts of Mannitol (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
Figure 7. Effects of drought stress on the root length of WT and overexpressed PmLEA25 lines. (A) Root phenotypes of WT and overexpressed PmLEA25 lines at varying amounts of Mannitol. (B) Root length statistics of WT and overexpressed PmLEA25 lines under varying amounts of Mannitol (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
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Figure 8. Phenotypic observation and physiological changes in WT and overexpressed PmLEA25 lines. (A) Phenotype variation in WT and overexpressed PmLEA25 lines after one-week drought treatment. (B) Antioxidant enzyme activities including MDA, SOD, POD, and CAT (*, p < 0.05; **, p < 0.01).
Figure 8. Phenotypic observation and physiological changes in WT and overexpressed PmLEA25 lines. (A) Phenotype variation in WT and overexpressed PmLEA25 lines after one-week drought treatment. (B) Antioxidant enzyme activities including MDA, SOD, POD, and CAT (*, p < 0.05; **, p < 0.01).
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Table 1. The sequence features of 49 PmLEAs in P. mongolica.
Table 1. The sequence features of 49 PmLEAs in P. mongolica.
Gene NameGene IDLength of CDS (bp)Protein
(aa)		Mw
(Da)		PIGRAVY
PmLEA1.2aMA_185382g0010252839555.365.14−1.461
PmLEA1.2bMA_68242g0010183606719.44.92−1.187
PmLEA1.2cMA_135240g0030252839499.345.14−1.261
PmLEA2MA_10436352g0020234778747.756.54−0.816
PmLEA3.3MA_17049g001031510411,318.649.45−0.641
PmLEA4.2aMA_858786g0010261869425.619.82−0.834
PmLEA4.2bMA_186086g0010264879547.778.98−1.108
PmLEA4.3aMA_235g0020237788653.656.59−1.133
PmLEA5MA_49897g002059419721,261.54.6−0.568
PmLEA6MA_33768g0010258859119.178.82−0.906
PmLEA7MA_487045g00102799210,216.449.68−0.636
PmLEA8MA_40361g0020258859247.297.99−1.048
PmLEA9MA_1400g001085528430,111.95.35−0.432
PmLEA10MA_41069g0010228758480.417.11−1.483
PmLEA11MA_121405g001045915216,652.25.470.101
PmLEA12MA_6165882g001032710811,317.45.46−1.041
PmLEA13MA_9339426g001044414715,429.688.69−1.3
PmLEA14MA_10069912g001032710811,235.265.36−0.995
PmLEA15MA_2988g002033911212,307.497.93−1.472
PmLEA16MA_183489g0030267889669.636.55−1.097
PmLEA17MA_10427927g001057619119,696.264.98−0.176
PmLEA18MA_10367411g001032710811,221.276.22−0.999
PmLEA19MA_717888g001061820522,872.864.07−0.664
PmLEA20MA_295448g00102979810,065.367.880.077
PmLEA21MA_6771381g001061520422,078.026.51−0.108
PmLEA22MA_3108g001051917218,936.526.06−0.998
PmLEA23MA_116141g0010195647691.769.46−1.188
PmLEA24MA_9957327g0010288959578.787.780.211
PmLEA25MA_10435107g001035411713,180.969.3−0.831
PmLEA26MA_722400g001035711813,335.199.3−0.892
PmLEA27MA_17597g001042914215,786.396.63−0.904
PmLEA28MA_8427244g001050416717,801.056.05−0.086
PmLEA29MA_432438g002042013915,036.169.12−0.347
PmLEA30MA_21240g00103009910,253.26.15−1.004
PmLEA31MA_111913g0010209769877,148.186.28−1.091
PmLEA32MA_590086g001032710811,317.45.46−1.041
PmLEA33MA_99592g001033010911,703.226.57−0.317
PmLEA34MA_10428902g001034511412,130.636.82−0.254
PmLEA35MA_403263g001031810511,438.776.27−0.525
PmLEA36MA_175705g0010365798798.919.86−0.644
PmLEA37MA_15415g001043814515,866.779.34−0.569
PmLEA38MA_9170359g001033010911,878.328.05−0.627
PmLEA39MA_198577g001033010911,836.389−0.483
PmLEA40MA_90896g001033311011,886.349.51−0.501
PmLEA41MA_132211g00102949710,697.916.02−0.427
PmLEA42MA_42290g001030610110,939.486.4−0.372
PmLEA43MA_326206g001047415716,742.65.59−0.693
PmLEA44MA_4752780g0010195646690.48.1−0.87
PmLEA45MA_41035g001068019020,880.299.17−1.168
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Wang, Y.; Li, J.; Bai, Y. Identification of Picea mongolica LEA Gene Family Implicates PmLEA25 in Drought Resistance. Forests 2024, 15, 1988. https://doi.org/10.3390/f15111988

AMA Style

Wang Y, Li J, Bai Y. Identification of Picea mongolica LEA Gene Family Implicates PmLEA25 in Drought Resistance. Forests. 2024; 15(11):1988. https://doi.org/10.3390/f15111988

Chicago/Turabian Style

Wang, Yaping, Jiaqi Li, and Yu’e Bai. 2024. "Identification of Picea mongolica LEA Gene Family Implicates PmLEA25 in Drought Resistance" Forests 15, no. 11: 1988. https://doi.org/10.3390/f15111988

APA Style

Wang, Y., Li, J., & Bai, Y. (2024). Identification of Picea mongolica LEA Gene Family Implicates PmLEA25 in Drought Resistance. Forests, 15(11), 1988. https://doi.org/10.3390/f15111988

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