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Article

Identification and Functional Prediction of Salt/Alkali-Responsive lncRNAs during Alfalfa Germination

by
Yajiao Liu
1,†,
Lei Xu
2,†,
Tiejun Zhang
1,
Bilig Sod
2,
Yanchao Xu
2,
Mingna Li
2,
Junmei Kang
2,
Qingchuan Yang
2,
Xiao Li
1,* and
Ruicai Long
2,*
1
School of Grassland Science, Beijing Forestry University, Beijing 100083, China
2
Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2024, 14(6), 930; https://doi.org/10.3390/agriculture14060930
Submission received: 23 April 2024 / Revised: 8 June 2024 / Accepted: 11 June 2024 / Published: 13 June 2024
(This article belongs to the Special Issue Breeding for Abiotic Stress Resistance in Crops: Biotechnology and Bioinformatics)
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Abstract

:
Long non-coding RNAs (lncRNAs) are pivotal regulators of the abiotic stress responses in plants, yet their specific involvement in salt/alkali stress during alfalfa germination remains incompletely understood. Here, we subjected Zhongmu No.1 alfalfa (Medicago sativa L.) seeds to salt stress (20 mM NaCl and 20 mM Na2SO4 solutions) or alkali stress (5 mM NaHCO3 and 5 mM Na2CO3 solutions) treatments for 3 days, followed by total RNA extraction and RNA-seq analysis to delineate stress-responsive alfalfa lncRNAs. We identified 17,473 novel alfalfa lncRNAs, among which 101 and 123 were differentially expressed lncRNAs (DElncRNAs) under salt and alkali stress, respectively, compared to the control. Furthermore, we predicted 16 and 237 differentially expressed target genes regulated by DElncRNAs through cis/trans-regulatory mechanisms under salt or alkali stress, respectively. A functional enrichment analysis of DElncRNA target genes indicated that lncRNAs were implicated in the fatty acid metabolism pathway under salt stress, while they played a significant role in the phenylpropanoid and flavonoid biosynthesis pathway under alkali stress. Notably, lncRNAs were found to participate in the plant hormone signal transduction pathway, a common regulatory mechanism in both salt and alkali stress responses. These findings contribute to a deeper understanding of the mechanisms underlying alfalfa’s response to salt and alkali stresses.

1. Introduction

Plant growth and development are significantly impacted by salt stress and alkali stress. During plant growth and development, an excessive accumulation of Na+ in the soil can lead to Na+ stress on plant, and CO32−/HCO3− can cause an increase in soil pH, a decrease in soil permeability, and ultimately lead to a decrease in biomass and even death [1,2]. Soil salinity impacts over 8000 billion m2 of arable land globally [3,4]. In China, there are approximately 335.1 billion m2 of saline–alkaline land [5].
As sessile organisms, plants have evolved complex mechanisms to overcome diverse abiotic stresses, such as salt stress and alkali stress. Studies have demonstrated that lncRNA is one kind of key regulators involved in these mechanisms. LncRNAs are a class of RNAs with transcript lengths exceeding 200 bp that lack the ability to encode protein products [6]. Normally, lncRNAs are shorter in length than mRNAs, with a low transcriptional level, limited conservation, and fewer exons [7]. However, in some cases, lncRNAs are similar to mRNAs: they are transcribed, undergo polyadenylation, carry a 5′ cap structure, and could be alternatively spliced [8]. In addition to the canonical three RNA polymerases (Pol I, Pol II, Pol III), Pol IV and Pol V are responsible for lncRNAs’ transcription [9]. Importantly, lncRNAs can regulate gene expression at the pre-transcription, middle-transcription, or post-transcription stage with regulatory diversity [10]. LncRNAs can be separated into intronic lncRNAs, intergenic lncRNAs (lincRNAs), natural antisense lncRNAs (lncNATs), and sense lncRNAs based on their genomic location and position relative to adjacent or overlapping protein-coding genes [9].
Currently, many lncRNAs which are responsive to salt/alkali stress have been identified. For instance, Npc536 is a natural antisense transcripts lncRNA induced under salt stress in Arabidopsis thaliana [11]. DROUGHT INDUCED lncRNA (DRIR) is induced by salt and drought stresses, and the overexpression of DRIR substantially improves plant resistance to drought and salt stresses [12]. The lncRNA973 in cotton (Gossypium hirsutum L.) is responsive to salt stress, and lncRNA973 gene-silenced plants exhibit attenuated salt stress tolerance [13]. The lncRNA354 is a lincRNA, and the silencing of lncRNA354 can improve the salt tolerance of cotton [14].
LncRNAs participate in various biological pathways under environmental stresses, mainly through regulating the expression of their target genes. For instance, lncRNAs in sesame (Sesamum indicum L.) could be involved in its salt stress response by regulating the expression of the genes in several important pathways, such as flavonoid biosynthesis and monoterpene biosynthesis [15]. Under a combination of salinity and excess boron stress, the identified lncRNAs in maize (Zea mays L.) can regulate the expression of genes involved in nicotinamide metabolism process [16]. Under drought stress, the lncRNAs in Chinese prickly ash (Zanthoxylum bungeanum Maxim.) leaves can activate the JA, ABA, auxin, ethylene and GA signaling pathways [17].
Alfalfa is an important forage with optimal qualities such as a high nutritional content, good palatability, a strong regeneration ability, and resistance to multiple stresses [18,19,20,21,22,23]. Currently, most research investigating the salt or alkali tolerance mechanisms of alfalfa is conducted at the physiological and molecular levels during the seedling growth stage. However, the regulatory mechanism of lncRNAs in response to salt/alkali stress during the germination stage of alfalfa is still vague.
Therefore, the main objectives of this study are to identify the lncRNAs responsive to salt and alkali stresses during the germination stage of alfalfa and to elucidate their potential regulatory roles in stress tolerance.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Zhongmu No.1 alfalfa (Medicago sativa L.) was bred by us and used in this study; it is a synthetic variety widely cultivated in China, known for its high yield and salt–alkali stress tolerance. This variety was bred through multiple rounds of selection and hybridization in the salt–alkali soil of Cangzhou, Hebei province. Zhongmu No.1 exhibits a high-yield performance in medium to mild salt–alkali soil.
In prior investigations, we subjected alfalfa seeds to varying concentrations of salt and alkali treatments, ranging from 5 to 50 mM of salt (NaCl and Na2SO4 mixtures) and from 5 to 15 mM of alkali (NaHCO3 and Na2CO3 mixtures). Our findings revealed that, among these gradients, treatments with 20 mM of salt and 5 mM of alkali exhibited notable inhibitory effects on seed germination and seedling growth [24,25]. These concentrations were selected to balance the induction of measurable stress responses while maintaining seed viability, enabling a clearer delineation of the molecular mechanisms activated under stress conditions.
Seeds of Zhongmu No.1 were disinfected with 75% alcohol for 10 min and then washed with ddH2O twice. Seeds were germinated in plastic Petri dishes (diameter: 12 cm) containing two sheets of filter paper moistened with salt solutions (20 mM NaCl and 20 mM Na2SO4), alkali solutions (5 mM NaHCO3 and 5 mM Na2CO3), or ddH2O for 3 days at a temperature of 23 °C, with a 16 h light/8 h dark photoperiod, and a relative humidity of 80%. The seeds germinated in ddH2O were used as a control group to establish a reliable baseline for comparison.
The germination rate, fresh weight, and root length of each treatment group (salt, alkali, and control) were measured 3 days after germination. Measurements were conducted across three biological replicates, with each replicate involving 50 seedlings. Statistical analysis was performed using a one-way analysis of variance (ANOVA) [26]. Before conducting the ANOVA, assumptions of the normality and homogeneity of these variances were tested using the Shapiro–Wilk test and Levene’s test, respectively. Since these assumptions were met, an ANOVA was deemed appropriate for the analysis. For multiple comparisons, Dunnett’s test was applied to determine the significance of the differences between the control and treatment groups. Differences compared to the control (NC group) were considered significant at p < 0.05 (*) and highly significant at p < 0.01 (**) for both the salt stress (SS) and alkali stress (AS) groups.

2.2. RNA Extraction, Library Preparation, and Sequencing

Three days after germination, seedlings from each treatment group (salt, alkali, and control) were separately collected, quick-frozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted using RNAiso Plus (Takara, Beijing, China) reagent following the manufacturer’s protocol. Library preparation, RNA sequencing and analysis were conducted by Biomarker Technologies (Beijing, China). An epicentre Ribo-ZeroTM kit (Madison, WI, USA) was used to remove the rRNA from each sample. After quality control of the constructed cDNA library, the libraries were pooled according to the target offline data volume, and the cDNA library was sequenced using an Illumina Hi-Seq platform (San Diego, CA, USA). Sequencing data quality control was performed on the original sequences, which included the removal of reads containing adaptors and low-quality reads (including those with a ratio of N greater than 10% and those with quality values of Q ≤ 10 with their base proportions exceeding 50% of the total reads). Using Hisat v2.2.1 [27], clean reads were mapped to the reference genome of the cultivated alfalfa “Zhongmu No. 1” (https://figshare.com/articles/dataset/Medicago_sativa_genome_and_annotation_files/12623960, accessed on 10 December 2022) [28]. StringTie v2.0.4 software was used to assemble and appropriately quantify the mapped data. Fragments Per Kilobase of transcript per Million fragments mapped (FPKM) was used as a criterion for the transcript or gene expression levels [29]. Three biological replicates were conducted for each treatment group and labeled as “NC-1, NC-2, NC-3”, “SS-1, SS-2, SS-3”, and “AS-1, AS-2, AS-3”, respectively. NC, SS, and AS represent the non-specific control, salt stress, and alkali stress, respectively. A total of 150 seedlings were collected for one biological replicate of each treatment group.

2.3. LncRNA Identification

Assembled transcripts possessing the classification codes “i”, “x”, “u”, “o”, and “e” were selected as members of the potential lncRNA pool [30]. Transcripts with length < 200 bp, number of exons < 2, or FPKM < 0.1 were excluded from the potential lncRNA pool [31]. The coding ability of the remaining potential lncRNAs was evaluated using the Coding Potential Calculator (CPC2) [32], Coding-Non-Coding Index (CNCI) [33], Coding Potential Assessment Tool (CPAT) [34], and the Protein Family database (Pfam) [35]. The lncRNAs with a predicted coding ability were excluded from the potential lncRNA pool. Ultimately, the intersecting transcripts from the above four analyses were identified as lncRNAs and utilized for subsequent analyses. According to their position relationship with protein-coding genes, the identified lncRNAs were classified as either lincRNAs, antisense-lncRNAs, intronic-lncRNAs, or sense-lncRNAs.

2.4. Differentially Expressed lncRNAs and mRNAs Analyses and qPCR Validation

Differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) were identified based on their expression levels in the stress group vs. control group using a Fold Change ≥ 2 and FDR < 0.05. The hierarchical clustering of the selected DElncRNAs or DEmRNAs was performed using Heatmap Illustrator in TBtools v1.8 [36]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database and clusterProfiler were used for pathway enrichment analysis and enrichment analysis of the biological process, molecular function, and cellular fraction of the DElncRNAs’ targets, respectively [37,38]. The clustering analysis of the DElncRNAs and DEmRNAs was conducted using the Short Time-series Expression Miner (STEM, http://www.sb.cs.cmu.edu/stem/, accessed on 5 June 2023).
Total RNA from each group was reverse transcribed into cDNA using a PrimeScript™ RT reagent Kit with a gDNA Eraser (Perfect Real Time) (Genesand, Beijing, China) following the manufacturer’s instructions. Quantitative Real-Time PCR (qPCR) analyses were conducted using a CFX96 Real-Time PCR Detection System (Bio-rad, Hercules, CA, USA) with a total reaction volume of 20 μL, containing 10 μL 2× Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), 0.4 μL of each 10 μM primer, 2 μL of cDNA template, and 7.2 μL of ddH2O. The qPCR procedure consisted of a two-step reaction: step 1 involved a denaturation at 95 °C for 30 s; step 2 involved 40 cycles of denaturation at 95 °C for 5 s, followed by annealing and extension at 60 °C for 30 s. The 2−ΔΔCt method was used to calculate the relative quantitative gene expression level [39]. Three biological replicates were conducted for each measurement. All primers used in the qPCR analyses can be found in Table S1.

2.5. LncRNA Target Gene Predication

Cis-regulation refers to the transcription activation or expression regulation of lncRNA to adjacent mRNAs. Therefore, mRNAs within 100 kb upstream or downstream of the lncRNA were predicted to be cis-target genes of the lncRNA according to their positional relationship. The trans-regulation of lncRNA refers to its regulation of distal mRNA. Therefore, trans-targeted genes were predicted by analyzing the co-expression relationship between the lncRNA and mRNA expression levels in samples. Using a Pearson correlation coefficient method, the genes with an absolute correlation value greater than 0.9 (|ρ| > 0.9) and a significance p value < 0.01 were predicted to be the trans-targeted genes of a particular lncRNA.

3. Results

3.1. Stress Response of Alfalfa during Germination

We observed the growth status of alfalfa seeds after 3 days of exposure to different treatments and statistically analyzed their germination rates, fresh weights, and root lengths (Figure 1). The germination rate in each treatment group was over 90%, with no distinct difference between the SS and NC groups but a significant difference between the AS and NC groups (Figure 1B). The average fresh weight and root length of the control group were 11.54 mg and 11.82 mm, respectively. However, the average fresh weight and root length were 9.87 mg and 7.47 mm, respectively, under salt stress. And the average fresh weight and root length were 10.02 mg and 7.01 mm, respectively, under alkali stress. The fresh weights and root lengths of the two treatment groups were significantly different compared to the control group (Figure 1C,D).

3.2. Alfalfa lncRNAs’ Identification and Characterization

To explore the salt/alkali-stress-responsive lncRNAs in alfalfa, we conducted strand-specific RNA sequencing using 3-day-old seedlings germinated under different stress treatments. A total of 205.70 Gb of clean data were generated from nine test samples (three biological repeats for each treatment), of which 81.09% to 83.89% mapped to the alfalfa reference genome.
In total, 17,473 novel lncRNAs were identified (Figure 2A), which were classified as lincRNAs (81.5%), antisense-lncRNAs (6.8%), intronic-lncRNAs (6.9%), or sense-lncRNAs (4.8%) (Figure 2B). Most lncRNAs (96%) and mRNAs (80%) can be mapped onto eight chromosomes of the reference genome, and the assembled lncRNAs and mRNAs were evenly distributed across the eight chromosomes (Figure 2C). The ORF (open reading frame) length corresponding to the lncRNAs was mainly concentrated around 100 bp, while the ORF length corresponding to mRNAs was primarily concentrated around 100–300 bp (Figure 2D), indicating the low coding potential of the identified lncRNAs. Additionally, the expression levels of the lncRNAs were lower than those of the mRNAs (Figure 2E). Although the longest identified lncRNA was 13,028 bp and the identified lncRNAs had up to 14 exons, the length of most lncRNAs was around 1000 bp (69.93%) and they had 2 exons (68.60%) (Figure 2F,G).

3.3. DElncRNAs’ Analysis and qPCR Validation

A total of 257 DElncRNAs were identified, of which 68, 84, and 59 DElncRNAs were uniquely identified in the NC vs. SS, NC vs. AS, and SS vs. AS groups, respectively (Figure 3A). Under salt stress, the expression levels of 24 lncRNAs were up-regulated, and those of 77 lncRNAs were down-regulated. Under alkali stress, the expression levels of 44 lncRNAs were up-regulated, and those of 79 lncRNAs were down-regulated (Figure 3B). There were more DElncRNAs identified under alkali stress compared to salt stress. To validate the RNA-seq results, eight lncRNAs were randomly selected for qPCR, and the results were, overall, consistent with the sequencing data (Figure 3C).

3.4. DEmRNAs’ Analysis and Clustering of DEmRNAs and DElncRNAs

A total of 245 DEmRNAs were identified, with 40, 111, and 38 DEmRNAs being identified in the NC vs. SS, NC vs. AS and SS vs. AS groups, respectively (Figure 4A). Under salt stress, 51 DEmRNAs were down-regulated, while, under alkali stress, 86 DEmRNAs were down-regulated (Figure 4B). Three replicates of each group were clustered together. The most significant changes in gene expression levels were observed in the AS vs. NC group (Figure 4C).
Transcription factors are essential for plant responses to external stresses. Thirteen transcription factors were identified from all DEmRNA. Broadly speaking, these 13 transcription factors can be divided into two groups: those up-regulated under salt/alkali stress and those down-regulated under stress (Figure 4D). All DElncRNAs and DEmRNAs were separated into six groups depending on their expression patterns (Figure 4E). The DElncRNAs and DEmRNAs up-regulated under salt/alkali stress can be further divided into three groups: those with the highest expression level under salt stress, those with the highest expression level under alkali stress, and those with relatively the same expression levels under salt stress and alkali stress. The DElncRNAs and DEmRNAs down-regulated under salt/alkali stress can be further divided into two groups: those with the lowest expression under salt stress and those with relatively the same expression levels under salt stress and alkali stress. Another group was the DElncRNAs and DEmRNAs down-regulated under alkali stress but up-regulated under salt stress. Remarkably, the “down-regulated under salt stress and alkali stress” group had the most DElncRNAs and DEmRNAs, whereas the “down-regulation under alkali stress, up-regulation under salt stress” group had the fewest DElncRNAs and DEmRNAs (Figure 4E).

3.5. Cis- and Trans-Target Genes of DElncRNAs

A total of 21 DElncRNAs were predicted to regulate 16 DEmRNAs in cis and 257 DElncRNAs were predicted to regulate 237 DEmRNAs in trans (Figure 5A). Among them, the lncRNAs MSTRG.42963.2 and MSTRG.14821.2 regulate their respective target genes, MsG0680034981.01 and MsG0380012011.01, which are located 38,891 bp and 47,045 bp downstream of their respective lncRNAs (Figure 5B). MSTRG.16487.3 is on the positive chain of chromosome 3 and predicted to regulate its trans-target gene MsG0680030362.01, which is on the negative chain of chromosome 6 (Figure 5C). In addition, MSTRG.15329.3 is on the negative chain of chromosome 3 and predicted to regulate the trans-target gene MsG0380013406.01, which is on the positive chain of chromosome 3 (Figure 5C).

3.6. Functional Prediction of DElncRNA Target Genes during Alfalfa Germination under Salt/Alkali Stress

All mRNAs potentially targeted by DElncRNAs in salt/alkali stress responses were subjected to a KEGG analysis. Under salt/alkali stress, the top 20 pathways with the most significant enrichment were further analyzed. Under salt stress, the potential target genes of DElncRNAs were primarily involved in the alpha-linolenic acid metabolism (ko00592), arginine and proline metabolism (ko00330), pentose and glucuronate interconversions (ko00040), amino sugar and nucleotide sugar metabolism (ko00520), spliceosome (ko03040), and plant hormone signal transduction (ko04075) pathways (Figure 6A). The potential target genes of alkali-responsive DElncRNAs were primarily implicated in the arginine and proline metabolism (ko00330), phenylalanine metabolism (ko00360), phenylpropanoid biosynthesis (ko00940), plant hormone signal transduction (ko04075), and ABC transporters (ko02010) pathways (Figure 6B). The Gene Ontology (GO) enrichment analysis of the potential target genes of DElncRNAs showed that these genes were associated with 20 biological processes, 18 cell components, and 15 molecular functions under salt/alkali stress (Figures S1 and S2).
Under salt-stress conditions, 31 potential target genes of the DElncRNAs were enriched in the fatty acid metabolism pathway. Four of these genes were selected for further analysis. MsG0480023715.01 and MsG0480023716.01, which encode a 3-oxyacyl [acyl carrier protein] synthase, an enzyme regulating the carbon chain elongation of long-chain fatty acids, were both regulated by MSTRG.38508.5 and MSTRG.6911.4. MsG0680033143.01 and MsG0180001034.01 were both regulated by three different DElncRNAs. Both of these genes encode the stearoyl-acyl carrier protein desaturase, which governs the ratio of saturated to unsaturated fatty acids in plants. The expression levels of the above genes were all down-regulated under salt-stress conditions, which could result in decrease in the content of fatty acids and the ratio of saturated fatty acids to unsaturated fatty acids during alfalfa germination (Figure 7A).
Under alkali-stress conditions, 179 potential target genes of the DElncRNAs were enriched in the phenylpropanoid biosynthesis pathway, and 42 target genes were enriched in the flavonoid biosynthesis pathway. In both pathways, many genes promoting phenylpropanoid and flavonoid biosynthesis were found to be down-regulated. For example, MsG0880045643.01, MsG0380014506.01, and MsG0880045644.01 were potential DElncRNA target genes. They all encode naringenin, which is a flavonoid compound with free radical scavenging and antioxidant activity. MSTRG.34461.1 regulated MsG0380015574.01, a flavonol synthetase encoding gene which catalyzes the synthesis of flavonoids. The above genes were all down-regulated under alkali-stress conditions. Four lncRNAs regulated MsG0180006194.01, which encodes a chalcone flavanone isomerase, which catalyzes the initiation of flavonoid biosynthesis. It was down-regulated under alkali-stress conditions. MsG0580024209.01, MsG0880047488.01, and MsG0480021654.01 were potentially regulated by MSTRG.29651.1, MSTRG.11680.1, and MSTRG.17290.1, respectively. The above three genes encode 4-coumarate-CoA ligase, which catalyzes the formation of 4-coumaric acid and its derivatives in the flavonoid synthesis pathway. Phenylalanine ammonia lyase (PAL) is a key rate-limiting enzyme in the metabolism of phenylpropanoid in plants. MSTRG.15926.11, MSTRG.34568.3, and MSTRG.9730.16 regulated their potential target genes MsG0180005188.01, MsG0180005189.01, and MsG0780040945.01, respectively. They are all PAL coding genes. The down-regulation of these target genes could result in a reduction of the flavonoid content in plants under alkali stress (Figure 7A).
Under salt- or alkali-stress conditions, 107 common potential target genes were involved in the plant hormone signaling transduction pathway. For example, MSTRG.27989.1 potentially regulated MsG0880041991.01 and MsG0880042916.01, while MSTRG.58791.4 potentially regulated MsG0880045184.01 and MsG0880045185.01. The above four genes all encode MYC transcription factors, core regulators in the JA signal transduction pathway. MSTRG.39008.1 potentially regulated MsG0680034609.01, MsG0880047288.01, MsG0880042776.01, and MsG0280008436.01, all of which encode JASMONATE-ZIM-DOMAIN (JAZ) proteins. These MYC- and JAZ-coding genes participating in the JA signal transduction pathway were down-regulated under salt/alkali-stress conditions (Figure 7A).

4. Discussion

Abiotic stresses can alter plant growth and development. The inhibition of alfalfa seed germination and the growth of seedlings were observed under salt/alkali stress treatments (Figure 1A). In the presence of either type of abiotic stress, the average fresh weight and root length of seeds germinated for three days were both significantly inhibited compared to the control group (Figure 1C,D), which is consistent with the findings of Wang et al. [40].
The numbers of identified lncRNAs could vary among different species. In the present study, a total of 17,473 lncRNAs were identified in 3-day-old alfalfa seedlings under salt/alkali stress (Figure 2A). This number was similar to that of the lncRNAs identified in three alfalfa varieties (11,677) under salt and drought stresses [41]. However, it was 7-fold higher than that found in Medicago truncatula (2448) under salt stress [42], and less than the number identified in two wheat varieties (19,000) under alkali stress [43].
Subsequently, the general characteristics of the lncRNAs in alfalfa seedlings were uncovered. Of the four lncRNA types, lincRNAs were the most numerous (Figure 2B), and chromosome 6 had the largest number of lncRNAs (Figure 2C). This finding was inconsistent with that of Medicago truncatula. Medicago truncatula lncRNAs were divided into six types, with the largest number being antisense lncRNAs, while chromosome 4 had the highest number of lncRNAs [42]. The lncRNAs identified in alfalfa can be divided into eight categories, with lincRNAs being the most numerous [41]. The inconsistency of the lncRNA types in alfalfa may be caused by genomic differences between varieties and classification tools. The ORF length of the lncRNAs was approximately 200 bp (Figure 2D), and lncRNAs had lower expression levels compared to mRNAs (Figure 2E), which was in agreement with findings reported in rice [44]. Many lncRNAs were shorter than 1000 bp in length and contained two exons (Figure 2F,G), similar to the findings reported for Zea mays and Medicago truncatula [16,42,45]. However, these data contradict what has been reported in wheat, as more than half of the lncRNAs in wheat contain only one exon [43].
In our study, 101 DElncRNAs were identified under salt stress and 123 DElncRNAs were identified under alkali stress, with 79 DElncRNAs being common to both (Figure 3A). These results suggest that lncRNAs play a role in the salt/alkali stress response through key pathways such as plant hormone signaling and metabolic pathways like alpha-linolenic acid and phenylpropanoid biosynthesis. This observation resonates with prior transcriptional analyses of alfalfa seedlings under salt stress, which emphasized lipid metabolism and transcription factors such as MYB, WRKY, NAC, and bHLH, albeit with a broader emphasis on protein kinase activity and structural molecule function [46]. In the response to salt stress in Medicago truncatula, a whole-transcriptome RNA sequencing analysis revealed the significant roles of transcription factors (e.g., MYB, NAC), metabolic pathways, and secondary metabolite biosynthesis [41]. Likewise, transcriptional studies of alfalfa under saline–alkaline stress confirmed the involvement of protein kinases and transcription factors, emphasizing the role oxidative stress markers and flavonoid metabolism under different stress durations [47]. Transcriptomic analyses of two alfalfa cultivars differing in salinity tolerance (GN5 and GN3) revealed significantly higher expression levels of mRNAs and lncRNAs related to osmotic and ionic stress [48]. A comparative transcriptome analysis of alfalfa leaves under salt stress at different pH values further extends these findings by revealing an increasing number of differentially expressed genes (DEGs) and KEGG pathways with rising pH levels, highlighting pathways like plant hormone signal transduction, glutathione metabolism, MAPK signaling, and photosynthesis [40]. Another alfalfa alkali stress study at the seedling stage highlighted the importance of pathways related to catalytic activities, electron carrier activities, and signal transduction. Additionally, the study also involved metabolomic analyses, which indicated that phenylpropanoid and flavonoid biosynthesis were significantly increased under alkali stress [49]. All studies noted enriched pathways like the photosynthesis pathway and plant hormone signal transduction, underscoring common stress responses. Our unique focus on specific biosynthetic pathways’ down-regulation complements the broader molecular insights from previous studies, offering a comprehensive view of alfalfa’s stress response mechanisms.
Several studies have indicated that fatty acids are involved in multiple abiotic stress responses. For example, the tomato (Solanum lycopersicum L.) endoplasmic reticulum omega-3 fatty acid desaturase gene (LeFAD3) was induced at low temperatures (4 °C) and inhibited at high temperatures (40 °C). LeFAD3 overexpression in tomato increased the levels of linolenic acid in both the leaves and roots, while a corresponding decrease in the level of linoleic acid was observed, which led to resistance to low temperatures [50]. In tobacco (Nicotiana tabacum L.), the overexpression of FAD3 or FAD8 genes enhanced drought and salt resistance by increasing the α-linolenic acid content [51]. STEAROYL-ACYL CARRIER PROTEIN ∆9-DESATURASE6 (SAD6) is also a fatty acid desaturase-coding gene whose expression is inhibited under hypoxia. Under hypoxic drought stress, SAD6, together with FAD3, increased the level of unsaturated fat in the crown of Arabidopsis thaliana [52]. In our study, MsG0180001034.01 and MsG0680033143.01 were predicted to be SAD6-coding genes in alfalfa. The expression of these two genes did not significantly change under alkali stress but was significantly down-regulated under salt stress, which could result in ratio changes of saturated fatty acids to unsaturated fatty acids in the plant cell membrane, affecting the normal biological function of the cells and in response to salt stress (Figure 7).
Li et al. found that differential proteins in alfalfa under a mixture of Na2CO3 and NaHCO3 alkali stress were enriched in their phenylpropanoid biosynthesis and flavonoid biosynthesis metabolic pathways [53]. Flavonoids are a class of polyphenolic secondary metabolites widely present in plants. They participate in plants’ resistance to abiotic stresses through their antioxidant activity and free radical scavenging activity. Flavonoids can be roughly grouped into eight subclasses, including flavones, flavonols, flavanones, flavanols, flavanonols, isoflavones, chalcones, and anthocyanins [54]. Chalcone isomerase (CHI) is a key enzyme in flavonoid biosynthesis. A gene encoding chalcone isomerase 2 (OsCHI2) in rice was up-regulated under abiotic stress. Under abiotic stress conditions, stress-induced OsCHI2 expression enhanced the transcript levels of the structural genes related to the flavonoids biosynthesis pathway and the accumulation of metabolites, thereby enhancing the tolerance of rice to drought, cold, and salinity [55]. In the present study, the expression level of MsG0180006194.01, a chalcone-flavonone isomerase 2 encoding gene in alfalfa which converts chalcone to flavanone, was down-regulated under alkali stress. Naringenin is a kind of flavanone. In common beans (Phaseolus vulgaris L.), 0.1~0.4 mM of flavonoid naringenin can relieve the growth inhibition caused by salt/PEG and regulate these plants’ antioxidant capacity [56]. MsG0880045643.01, MsG0380014506.01, and MsG0880045644.01 were annotated as genes encoding naringenin in alfalfa. The expression levels of the above three genes were all down-regulated under alkali stress. Studies have demonstrated that the PAL gene family of alfalfa (MePAL) is responsive to various stresses [57]. The up-regulation of MePAL expression in cassava leaves under low-temperature conditions resulted in increased plant flavonoid synthesis and improved resistance to oxidative damage [58]. The nucleotide sequence of MsG0780040945.01 was 98.81% consistent with MePAL, while its expression level was down-regulated under alkali stress. The down-regulation of all the above genes could lead to decreased flavonoid compounds and thereby decreased oxidative damage resistance in alfalfa under alkali stress (Figure 7).
Plant hormones are signaling compounds that regulate key aspects of plants’ growth, development, and environmental stress responses. In this study, a large number of DElncRNAs’ potential target genes were enriched in the plant hormone signal transduction pathway under salt/alkali stress (Figure 6). JA is one of the most common plant hormones; it has roles in biotic and abiotic stress responses, as well as in plant growth and development [59]. In Arabidopsis thaliana, the transcriptional programming induced by JA is mainly conducted by the MYC2 transcription factor. The JAZ proteins, key repressors involved the JA pathway, inhibit jasmonate signaling and plant defense [60,61]. In our study, the differentially expressed potential target genes MsG0880042916.01, MsG0880045184.01, and MsG0880045185.01 were annotated as MYC2 transcription factor-coding genes. MsG0680034609.01, MsG0880047288.01, MsG0880042776.01, and MsG0280008436.01 were annotated as JAZ-coding genes in alfalfa (Figure 7). It seems contradictory that both MYC-coding genes and JAZ-coding genes were down-regulated under salt/alkali conditions. However, studies have shown that there was also a negative feedback regulation of MYC transcription factors. In Liu et al.’s study, they found that MTBs, which were activated by MYC2, could in turn impair the activation effect of MYC2 to terminate JA signaling [62]. In our study, we collected samples after a 3-day salt/alkali treatment. It could be possible that the expression levels of MYC transcription factor-coding genes were up-regulated in the early stage when exposed to salt/alkali stress and then were down-regulated after 3 days of exposure due to a negative feedback circuit.
Overall, we constructed a network model of salt/alkali-stress-responsive lncRNAs and their potential target genes during alfalfa germination (Figure 7). However, the functions of these lncRNAs have not been sufficiently characterized and further research is required.

5. Conclusions

A total of 17,473 lncRNAs were identified during alfalfa seed germination under salt/alkali stress. Of these, 101 DElncRNAs were identified under salt stress, and 123 DElncRNAs were identified under alkali stress. The KEGG analysis of the DElncRNAs’ potential target genes indicated that fatty acid metabolism was involved in the response to salt stress. At the same time, the DElncRNAs’ potential target genes were enriched in the phenylpropanoid and flavonoid biosynthesis pathways under alkali stress. The DElncRNAs’ potential target genes were enriched in the plant hormone signal transduction pathway under both salt stress and alkali stress. The genes involved in these pathways, such as MsG0180001034.01, MsG0780040945.01, and MsG0880042916.01, could serve as crucial candidate genes in the response to salt stress or alkali stress during alfalfa germination, while their functions still require further verification.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agriculture14060930/s1, Figure S1: GO enrichment analysis of target genes of DElncRNAs under salt stress; Figure S2: GO enrichment analysis of target genes of DElncRNAs under alkali stress; Table S1: Primer sequences used for qPCR of actin and some lncRNAs.

Author Contributions

Y.L., L.X., X.L. and R.L. conceived and designed the experiments; Y.L., L.X. and B.S. conducted the experiments; Y.L., L.X., T.Z., X.L. and M.L. performed the bioinformatics analyses; Y.L., L.X. and X.L. wrote the first draft; Y.L., L.X., X.L. and Y.X. prepared the figures and table; J.K., Q.Y., X.L. and R.L. revised and refined the manuscripts. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 32071865), the China Postdoctoral Science Foundation (Grant No. 2023M740283), the China Agriculture Research System of MOF and MARA (Grant No. CARS-34),the Agricultural Biological Breeding Project (Grant No. 2022ZD04011) and Breeding and Industrialization Demonstration of New Highquality Alfalfa Varieties (Grant No. 2022JBGS0020).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Upon reasonable request, the corresponding author will provide the data that support the conclusions of this study. The RNA sequencing raw data have been deposited in the Genome Sequence Archive at the National Genomics Data Center, China National Center for Bioinformation (GSA: CRA012302), and are publicly accessible at https://ngdc.cncb.ac.cn/gsa (accessed on 25 August 2023).

Acknowledgments

We are grateful to all colleagues in the laboratory for providing us with their valuable experimental and technical guidance. We are very grateful to the editors and reviewers for their critical evaluation of the manuscript and for providing constructive suggestions for its improvement.

Conflicts of Interest

The authors declare that they have no known competing interests that could have appeared to influence the work reported in this paper.

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Figure 1. Characteristics of the stress responses of alfalfa during germination. (A) Growth status of alfalfa seeds after 3 days of exposure to salt/alkali stress treatments. (B) Germination rate of alfalfa seeds exposed to salt/alkali stress treatments. (C) Fresh weights of 3-day-old alfalfa seedlings exposed to salt/alkali stress treatments. (D) Root lengths of 3-day-old alfalfa seedlings exposed to salt/alkali stress treatments. NC, SS, and AS represent alfalfa treated with ddH2O, 20 mM NaCl and 20 mM Na2SO4, and 5 mM NaHCO3 and 5 mM Na2CO3, respectively. Compared with the NC group, the SS and AS groups showed significant differences when their p < 0.05 (*), while they showed extremely significant differences when their p < 0.01 (**).
Figure 1. Characteristics of the stress responses of alfalfa during germination. (A) Growth status of alfalfa seeds after 3 days of exposure to salt/alkali stress treatments. (B) Germination rate of alfalfa seeds exposed to salt/alkali stress treatments. (C) Fresh weights of 3-day-old alfalfa seedlings exposed to salt/alkali stress treatments. (D) Root lengths of 3-day-old alfalfa seedlings exposed to salt/alkali stress treatments. NC, SS, and AS represent alfalfa treated with ddH2O, 20 mM NaCl and 20 mM Na2SO4, and 5 mM NaHCO3 and 5 mM Na2CO3, respectively. Compared with the NC group, the SS and AS groups showed significant differences when their p < 0.05 (*), while they showed extremely significant differences when their p < 0.01 (**).
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Figure 2. Identification and characteristics of the lncRNAs and mRNAs in alfalfa seedlings. (A) Venn diagram illustrating the lncRNAs identified using different methods, including CNCI, CPC, Pfam, and CPAT. (B) Classification of the identified lncRNAs. (C–G) Comparison of identified lncRNAs and mRNAs, including their distribution on the chromosomes (C), ORF length distribution (D), expression levels (E), length distribution (F), and exon numbers (G).
Figure 2. Identification and characteristics of the lncRNAs and mRNAs in alfalfa seedlings. (A) Venn diagram illustrating the lncRNAs identified using different methods, including CNCI, CPC, Pfam, and CPAT. (B) Classification of the identified lncRNAs. (C–G) Comparison of identified lncRNAs and mRNAs, including their distribution on the chromosomes (C), ORF length distribution (D), expression levels (E), length distribution (F), and exon numbers (G).
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Figure 3. DElncRNAs identified under salt or alkali stress and their qPCR validation. (A) Venn diagram of DElncRNA numbers in different comparisons. NC vs. SS represents the SS compared to NC groups, and so on. (B) Histograms of numbers of up-regulated and down-regulated DElncRNAs in different comparisons. (C) Expression level of 8 lncRNAs under different growth conditions, detected by qPCR and RNA-seq. A single-factor analysis of variance was conducted between the treatment group and the control group. The results of the qPCR (relative expression level) and RNA-seq (FPKM) were analyzed using a one-way ANOVA. Compared with the NC group, the SS and AS groups showed significant differences when their p < 0.05 (*), while they showed extremely significant differences when their p < 0.01 (**).
Figure 3. DElncRNAs identified under salt or alkali stress and their qPCR validation. (A) Venn diagram of DElncRNA numbers in different comparisons. NC vs. SS represents the SS compared to NC groups, and so on. (B) Histograms of numbers of up-regulated and down-regulated DElncRNAs in different comparisons. (C) Expression level of 8 lncRNAs under different growth conditions, detected by qPCR and RNA-seq. A single-factor analysis of variance was conducted between the treatment group and the control group. The results of the qPCR (relative expression level) and RNA-seq (FPKM) were analyzed using a one-way ANOVA. Compared with the NC group, the SS and AS groups showed significant differences when their p < 0.05 (*), while they showed extremely significant differences when their p < 0.01 (**).
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Figure 4. DEmRNA analysis and the clustering of the DEmRNAs and DElncRNAs identified under salt or alkali stress. (A) Venn diagram illustrating DEmRNA numbers in the NC vs. SS, NC vs. AS, and SS vs. AS groups. (B) Numbers of up-regulated and down-regulated DEmRNAs in the NC vs. SS, NC vs. AS, and SS vs. AS groups. (C) Heatmap of DEmRNAs’ expression levels under different treatments. The expression level of the DEmRNAs (FPKM) was normalized using log2 (FPKM+1). (D) Heatmap of transcription factor expression levels under different treatments. (E) Clustering of DEmRNAs and DElncRNAs.
Figure 4. DEmRNA analysis and the clustering of the DEmRNAs and DElncRNAs identified under salt or alkali stress. (A) Venn diagram illustrating DEmRNA numbers in the NC vs. SS, NC vs. AS, and SS vs. AS groups. (B) Numbers of up-regulated and down-regulated DEmRNAs in the NC vs. SS, NC vs. AS, and SS vs. AS groups. (C) Heatmap of DEmRNAs’ expression levels under different treatments. The expression level of the DEmRNAs (FPKM) was normalized using log2 (FPKM+1). (D) Heatmap of transcription factor expression levels under different treatments. (E) Clustering of DEmRNAs and DElncRNAs.
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Figure 5. Model of DElncRNAs’ regulation of their potential target genes. (A) A brief regulatory model between DElncRNAs and DEmRNAs. (B) Examples of DElncRNAs’ cis-regulation of their target genes. (C) Examples of DElncRNAs’ trans-regulation of their target genes.
Figure 5. Model of DElncRNAs’ regulation of their potential target genes. (A) A brief regulatory model between DElncRNAs and DEmRNAs. (B) Examples of DElncRNAs’ cis-regulation of their target genes. (C) Examples of DElncRNAs’ trans-regulation of their target genes.
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Figure 6. KEGG enrichment of DElncRNAs’ potential target genes during alfalfa germination under salt stress/alkali stress. (A) KEGG enrichment of DElncRNAs’ potential target genes during alfalfa germination under salt stress. (B) KEGG enrichment of DElncRNAs’ potential target genes during alfalfa germination under alkali stress.
Figure 6. KEGG enrichment of DElncRNAs’ potential target genes during alfalfa germination under salt stress/alkali stress. (A) KEGG enrichment of DElncRNAs’ potential target genes during alfalfa germination under salt stress. (B) KEGG enrichment of DElncRNAs’ potential target genes during alfalfa germination under alkali stress.
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Figure 7. Network model of salt/alkali-responsive DElncRNAs and their potential target genes. (A) Possible regulatory pathways which lncRNAs and their potential target genes participated in under salt/alkali stress. Heatmaps represent the expression levels of lncRNAs and their potential target genes. (B) A regulatory model of the response to salt/alkali stress during alfalfa germination.
Figure 7. Network model of salt/alkali-responsive DElncRNAs and their potential target genes. (A) Possible regulatory pathways which lncRNAs and their potential target genes participated in under salt/alkali stress. Heatmaps represent the expression levels of lncRNAs and their potential target genes. (B) A regulatory model of the response to salt/alkali stress during alfalfa germination.
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MDPI and ACS Style

Liu, Y.; Xu, L.; Zhang, T.; Sod, B.; Xu, Y.; Li, M.; Kang, J.; Yang, Q.; Li, X.; Long, R. Identification and Functional Prediction of Salt/Alkali-Responsive lncRNAs during Alfalfa Germination. Agriculture 2024, 14, 930. https://doi.org/10.3390/agriculture14060930

AMA Style

Liu Y, Xu L, Zhang T, Sod B, Xu Y, Li M, Kang J, Yang Q, Li X, Long R. Identification and Functional Prediction of Salt/Alkali-Responsive lncRNAs during Alfalfa Germination. Agriculture. 2024; 14(6):930. https://doi.org/10.3390/agriculture14060930

Chicago/Turabian Style

Liu, Yajiao, Lei Xu, Tiejun Zhang, Bilig Sod, Yanchao Xu, Mingna Li, Junmei Kang, Qingchuan Yang, Xiao Li, and Ruicai Long. 2024. "Identification and Functional Prediction of Salt/Alkali-Responsive lncRNAs during Alfalfa Germination" Agriculture 14, no. 6: 930. https://doi.org/10.3390/agriculture14060930

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