Characterization of DREB family genes in Lotus japonicus and LjDREB2B overexpression increased drought tolerance in transgenic Arabidopsis

Background Drought stress affects plant growth and development. DREB proteins play important roles in modulating plant growth, development, and stress responses, particularly under drought stress. To study the function of DREB transcription factors (TFs), we screened key DREB-regulating TFs for drought in Lotus japonicus. Results Forty-two DREB TFs were identified, and phylogenetic analysis of proteins from L. japonicus classified them into five subfamilies (A1, A2, A4, A5, A6). The gene motif composition of the proteins is conserved within the same subfamily. Based on the cis-acting regulatory element analysis, we identified many growth-, hormone-, and stress-responsive elements within the promoter regions of DREB. We further analyzed the expression pattern of four genes in the A2 subfamily in response to drought stress. We found that the expression of most of the LjDREB A2 subfamily genes, especially LjDREB2B, was induced by drought stress. We further generated LjDREB2B overexpression transgenic Arabidopsis plants. Under drought stress, the growth of wild-type (WT) and overexpressing LjDREB2B (OE) Arabidopsis lines was inhibited; however, OE plants showed better growth. The malondialdehyde content of LjDREB2B overexpressing lines was lower than that of the WT plants, whereas the proline content and antioxidant enzyme activities in the OE lines were significantly higher than those in the WT plants. Furthermore, after drought stress, the expression levels of AtP5CS1, AtP5CS2, AtRD29A, and AtRD29B in the OE lines were significantly higher than those in the WT plants. Conclusions Our results facilitate further functional analysis of L. japonicus DREB. LjDREB2B overexpression improves drought tolerance in transgenic Arabidopsis. These results indicate that DREB holds great potential for the genetic improvement of drought tolerance in L. japonicus. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-024-05225-y.


Background
Drought influences plant growth and development.This seriously affects the efficiency of agricultural production, and ecological and environmental security.Plants form a complex transcriptional regulatory network that resists drought stress [1].Transcription factors (TFs) play an important role in drought stress response.They specifically bind to cis-acting elements in promoter regions and synergistically regulate the expression of downstream genes [2].Dehydration-responsive element binding (DREB) family TFs are a subfamily of the APETALA2/ Ethylene-Responsive Element Binding Factor (AP2/ ERF) which regulates the response to biotic and abiotic stress [3,4].Recent studies demonstrated that DREB TFs respond to drought, salt, cold, heat, and other abiotic stressors in plants [5,6].
DREB genes play crucial roles in plant hormone signaling pathways for protection against pathogens and abiotic stress [7].The response of plants to stress is controlled by multiple genes in the signal transduction network, and DREB participates in multiple signal transduction pathways [8].Adversity can induce the expression of DREB genes and regulate the expression of target genes in response to stress [9,10].DREB specifically binds to the dehydration response element/C-repeat (DRE/CRT) and cis-acting elements (G/ACCGAC) in the promoter regions of stress resistance genes [11].DREB interacts with ABA-responsive element-binding proteins (AREB) and heat shock transcription factors (HSF) in response to adversity and regulates stress resistance [12].
The DREB subfamily can be divided into six subgroups, namely A1-A6, in Arabidopsis [13].The DREB A1 subfamily members are sensitive to cold stress and regulate the expression of cold stress-related genes [14].The expression of DREB 2 A is induced by drought and overexpression of DREB A2 significantly improved drought stress tolerance in transgenic Arabidopsis [15].Arabidopsis AtTINY belongs to the DREB A3 family Overexpression of AtTINY improves cold resistance by upregulating cold stress-related genes such as COR6.6,COR15A, and COR78 [16].SlDREBA4 of tomato DREB A4 family is involved in regulating heat resistance [17].GmDREB2 was classified into the A5 subgroup; GmDREB2 binds to the DRE element and overexpression of GmDREB2 improves drought resistance under drought stress [18].The DREB A6 gene CiDREB6 improves the heat tolerance of plants by regulating the expression of several heat shock protein (Hsp) genes [19].The mechanism by which DREB participates in stress regulation is complex and different species have different stress resistance mechanisms.
The DREB family of genes plays an important role in the drought stress [20].The sequences and expression patterns of DREB genes have been identified and analyzed from model plants to higher plants [21,22].For example, using comprehensive genome-wide screening, 20 AcoDREB genes in pineapple (Ananas comosus) were identified, some of the AcoDREB genes responded to drought stress [23].The expression of FvDREB8 in subgroup A2 is rapidly induced under drought stress [24].DREB TFs RAP2.4 activates cuticular wax biosynthesis in transgenic Arabidopsis leaves under drought stress [25].Overexpression of OsDREB improved drought resistance in rice [26].Overexpression of StDREB2 enhanced drought stress tolerance in cotton [27].Overexpression of DcDREB1A enhanced drought tolerance in transgenic Arabidopsis and modulated lignin levels by regulating lignin-biosynthesis-related genes [28].These studies show that the DREB family genes play an important role in plant resistance to drought stress.
Lotus japonicus is an important leguminous forage that is used as a protein feed source, biological nitrogen fixation resource, and in ecological conservation.Drought significantly affects the growth and geographical distribution of L. japonicus.Therefore, it is important to breed L. japonicus cultivars with enhanced drought resistance.Expression of Medicago truncatula MtDREB2A is induced by drought, salt, and heat stress, and overexpression of MtDREB2A results in significant dwarfism in transgenic M. truncatula plants [29].Mizoi found that GmDREB2A was induced by drought, heat, and lowtemperature stress and improved drought resistance in transgenic Arabidopsis [30].The DREB family gene in the legumes has been identified and studied, while the DREB in L. japonicus has not been studied.In the present study, we identified 42 DREB TFs in L. japonicus.To provide insights into the evolution and function of DREB genes, we analyzed their phylogeny, physicochemical properties, structure, classification, and promoter cis-acting elements.We found that LjDRE2B expression was strongly induced by drought stress.Further investigation showed that overexpression of LjDRE2B increased the drought resistance of transgenic Arabidopsis plants.

Phylogenetic analysis of the DREB gene family
A phylogenetic tree was constructed using the fulllength DREB protein sequences of Lotus japonicus and Arabidopsis and was divided into six groups, A1 to A6 (Fig. 1).Lotus japonicus and M. truncatula were located on branches close together and exhibited a close kinship relationship.Glycine max had more family members, which is closely related to their chromosome numbers (Fig. S1A).The proportions of subgroups among different species were similar (Fig. S1B).The number of DREB genes in group A4 was the highest, ranging from 27.27 to 38.64%, whereas group A3 had the lowest, ranging from 1.41 to 2.27%.

Physicochemical properties analysis of the DREB gene family
According to the sequence analysis of the six species, amino acid lengths ranged from 83 to 627 amino acids (aa), with molecular weights varying from 9408.36 Da to 60,208.07Da (Table S1).Lotus japonicus showed the minimum variation in length (83-526 aa), whereas Trifolium pratense (106-627 aa) showed the maximum variation in length.The isoelectric points (pI) ranged from 3.8867 to 12.1988, indicating members from different physiological environments.More than 80.07% of the DREB family proteins from the six species were weakly acidic because their pI was < 7. The grand average of hydropathicity (GRAVY) values was negative and ranged from − 1.186 to -0.279, thus implying their hydrophilic nature.The aliphatic indices ranged from 48.19 to 80.73.

Conservative domain and motif composition analysis
Multiple sequence alignment showed that DREB A4 and A5 have a close homologous evolutionary relationship (Fig. 2A).The conserved protein motifs obtained using the MEME program is shown (Fig. 2B).The top 10 enriched motifs were identified (Fig. S2).We compiled Major MEME motif sequences (Table S2).Noteworthily, we observed that motif 4 was present only in Group A1, suggesting that Group A1 genes evolved from the same gene.The structural characteristics of the conserved domain are shown in Fig. 2C; all family members demonstrated a common AP2 domain.The advanced structures of plant proteins are related to their biological functions and activities.

Cis-regulatory elements in the promoters of DREB in L. japonicus
The 2000 bp upstream sequence of LjDREB gene was extracted, and cis-regulatory elements in the promoter were obtained.In total, 42 cis-acting elements were identified and classified them into three basic categories: hormone response, stress response, and plant growth metabolic cycle (Fig. 3A, B).ABREs for abscisic acid (ABA) responsiveness are the most common elements of the DREB family.The others were associated with plant hormones, including the CGTCA-and TGACG-motif for MeJA-responsive elements, the TATC-box and P-box for gibberellin-responsive elements, and the TGA-element for auxin-responsive elements.Anaerobic induction (ARE), low-temperature responsiveness (LTR), and binding sites involved in drought inducibility (MBS and MYB) Fig. 1 Phylogenetic tree of DREB proteins from Lotus japonicus and Arabidopsis thaliana.The tree was divided into six clades, which are marked by different colors and named as A1, A2, A3, A4, A5, and A6.The tree was constructed using the neighbor-joining algorithm with 1000 bootstraps based on the amino acid sequence of DREB proteins were also found.The number of cis-acting elements was also determined (Fig. 3B).We further analyzed the gene promoters of the DREB A2 group; the cis-elements were classified into hormone-related elements (Fig. 3C) and stress-related elements (Fig. 3D).Overall, the hormone and stress response genes were more abundant than the plant growth metabolic cycle genes.These results suggest that DREB genes likely play a crucial role in abiotic stress responses.

Gene expression response patterns under drought stress
To further investigate the function of DREB A2 in the drought stress response, we identified the expression patterns of DREB A2 family genes using qRT-PCR.The results showed that the expression of three genes in this family of four genes rapidly increased, reaching the highest level at 12 h of drought stress, and then decreased (Fig. 4A).The expression level of the Lj0g3v0359549.1 gene was the highest, reaching 7.8-fold after 12 h of drought stress (Fig. 4B).Lj0g3v0359549.1 may play an important role in drought stress.Furthermore, Lj0g3v0359549.1 was orthologous to AtDREB2B through sequence comparison (Fig. S3).Therefore, we named Lj0g3v0359549.1 as LjDREB2B.

Interaction network of DREB proteins
To obtain further insight into LjDREB2B, an interaction network was constructed using the STRING software (Fig. S4A).LjDREB2B was predicted to interact with the Delta-1-pyrroline-5-carboxylate synthase (P5CS) protein, which plays a critical role in plant drought responses.We also predicted that LjDREB2B interacted with Dehydration-respective protein A (RD29A), Dehydration-respective protein 22 (RD22), Heat stress transcription factor (HSFA3), and Alpha-neurotoxin homolog 7 (NHX7) which are all involved in responses to abiotic stresses.In addition, we predicted the interaction proteins of the A2 family members (Fig. S4B-D).Functional enrichment analysis was based on Gene Ontology in the network of LjDREB2B proteins that responded to salt, temperature, and abiotic stress, and water deprivation (Table S4).LjDREB2B proteins may play an important role in stress response.These findings are consistent with the mechanisms of LjDREB2B in drought response regulation.

Overexpression of LjDREB2B improves drought tolerance in transgenic Arabidopsis
To further investigate the function of LjDREB2B under drought stress, we generated 35 S::LjDREB2B overexpression transgenic Arabidopsis (OE) lines and selected 2 single insertion lines as drought stress (Fig. S5, Table S5).Under drought treatment, the growth of both the WT and OE lines was inhibited, although the transgenic plants showed better growth (Fig. 5A).The malondialdehyde (MDA) and proline levels of WT plants were higher and lower than those of LjDREB2B overexpression lines, respectively (Fig. 5B C).H 2 O 2 and O 2 − contents of WT plants were also higher than those of LjDREB2B overexpression lines (Fig. 5D and E).Plants have the capacity to scavenge excessive ROS by promoting an enzymatic antioxidant defense system that includes SOD, POD, and CAT.We measured the activities of SOD, POD, and CAT, and the results indicated that the activities of these enzymes rapidly increased after 7 days of drought stress (Fig. 5F-H).The enzyme activities in the OE lines were significantly higher than those in the WT lines.Furthermore, the expression levels of AtP5CS1, AtP5CS2, AtRD29A, and AtRD29B in the OE lines were significantly higher than in the WT plants after drought stress (Fig. 6A-D).Therefore, these results indicated that overexpression of LjDREB2B increased the drought tolerance of transgenic Arabidopsis plants.

Discussion
Plant development is a complex process that occurs in various environments.DREB are an important class of TFs involved in plant development and stress responses.There are 56 DREB members divided into six subfamilies in Arabidopsis thaliana [31].A total of 73 DREB members have been identified in the soybean genome [32].In  S1).Forty-two DREB proteins were identified in L. japonicus (Fig. 1).They were distributed on branches A1-A6 of the phylogenetic tree, with the exception of A3.These results indicate that DREB family genes are widespread and evolutionarily diverse in plants.Previous studies have shown that DREB family genes have a variety of motifs and structures in plants and participate in various functions [33].Su performed a genome-wide identification and expression analysis of DREB family genes in cotton, which indicated that the DREB family gene domains were highly conserved [34].In the present study, the DREB family genes had different conserved motifs in L. japonicus (Fig. 2B and C).Some differences were observed in motif distribution among the different subfamilies, but most of the motifs were shared on the same branch.All DREB genes contain motif 1, which belongs to the conserved AP2 domain.Our results are consistent with those of previous studies [35].
Under stress, some cis-acting elements of gene promoters, such as hormone-and stress-responsive elements, recognize upstream signals and regulate downstream gene expression at the transcriptional level, thereby enabling plants to resist stress.DREB TFs are sensitive to plant hormones and play key roles in plant hormone signaling pathways [36].Filyushins reported that AsaDREB1 promoters contain seven hormones and nine stressresponsive cis-regulatory elements [37].Nakashima showed that most DREB genes have cis-acting AAGAA and ABRE that are responsive to ABA [38].In the present study, the promoter elements of the DREB genes were highly diversified in L. japonicus (Fig. 3A and B).The most common elements were ABRE and AAGAA (Fig. 3A and B).Almost all family gene promoters have MYB/MYC and the drought-related cis-acting elements MBS, STRE, and ARE [37].We further analyzed the gene promoters of the DREB A2 group; the cis-elements were classified into hormone-and stress-related elements (Fig. 3C, D).Therefore, DREB A2 genes may be involved in different adversity pathways and play an important role in plant drought stress.
Plant DREB TFs play important roles in transcriptional activation under abiotic stress.DREB A2 participates in drought resistance regulation in plants [39].The expression of DREB A2 TFs was induced by drought stress [40,41].DREB2-type proteins are particularly important in plant responses to drought [42].Our results show that the expression of these three DREB A2 genes was rapidly induced by drought stress (Fig. 4A).The expression level of LjDREB2B was the highest and reached 7.8-fold after 12 h of drought stress (Fig. 4B).DREB genes participate in hormone signaling pathways and regulate proteins interacting with downstream genes to improve plant stress tolerance [43].We further predicted LjDREB2B might interact with P5CS, RD29, and RD22 (Fig. S4), although protein-protein interactions need to be further investigated.Therefore, our results indicated that LjDREB2B may play an important role in drought stress.
To investigate the role of LjDREB2B in drought stress, we overexpressed LjDREB2B in Arabidopsis.Under drought stress, the growth of the transgenic lines was significantly better than that of the WT lines (Fig. 5A).Previous studies have shown that DREB overexpression increases drought resistance [44,45].For instance, overexpression of BrDREB2B in transgenic Arabidopsis enhances tolerance to salt, heat, and drought stress [31].Overexpression of Phyllostachys edulis PeDREB28 improved plant salt and cold tolerance in Arabidopsis and bamboo [46].Overexpression of Syntrichia caninervis ScDREB10 enhanced transgenic Arabidopsis plant stress tolerance by regulating phenylpropanoid biosynthesis and starch metabolism [47].Eremosparton songoricum DREB2-type transcription factor EsDREB2B enhances tolerance to multiple abiotic stresses in transgenic tobacco [48].MaDREB1F overexpression increases banana resistance to cold and drought stress by regulating soluble sugar and proline levels and activating antioxidant systems [49].Overexpression of VuDREB2A improves drought and heat stress in cowpea by enhancing osmotic adjustment, antioxidant defense, and photosynthetic efficiency [50].In the present study, the MDA and ROS contents of the WT plants was higher than those of LjDREB2B overexpression lines (Fig. 5B-E).Furthermore, the proline content and antioxidant enzyme activities of the WT plants were lower than those of LjDREB2B overexpression lines (Fig. 5C-H).These results showed that the overexpression of LjDREB2B increased drought tolerance by enhancing the enzymatic antioxidant defense system in transgenic Arabidopsis.Previous studies have indicated that DREB family TFs regulate the expression of downstream stress-related genes, including HsfA3, RD29A, RD29B, COR15a, kin1, kin2, and erd10 [51,52].The overexpression of DREB1A upregulates expression of RD29A and COR15A and improves drought stress tolerance in Arabidopsis plants [15,53].Overexpression of Ammopiptanthus nanus AnDREB5.1 increased osmotic and cold stress tolerances by enhanced antioxidant enzyme activity and expression of stress-tolerant related genes in transgenic tobacco [54].Overexpression of Cerasus humilis ChDREB2C increased salt tolerance by enhancing scavenging capacity of ROS and expression of AtSOS1, AtNHX1 and AtRD29B in transgenic plant [55].Our results indicated that overexpression LjDREB2B increased the expression levels of AtP5CS1, AtP5CS2, AtRD29A, and AtRD29B (Fig. 6).These results suggest that the overexpression of LjDREB2B might increase drought resistance in Arabidopsis plants by upregulating the expression of genes related to stress.Therefore, the DREB gene family may provide high-quality plant resources and drought tolerance for molecular breeding.

Conclusions
We performed a comprehensive analysis of the evolutionary relationships and protein features of the DREB family genes.Furthermore, the motifs and domains of the proteins and promoter cis-acting elements of the DREB genes were analyzed in L. japonicus.We further analyzed the expression pattern of DREB A2 in response to drought stress and found that LjDREB2B was strongly induced by drought stress.Additionally, our results showed that overexpression of LjDREB2B could increase drought resistance in transgenic Arabidopsis plants.Therefore, our results provide a basis for further functional studies on the DREB family genes in L. japonicus and resources for prospective applications in droughtresistant breeding.

Constructs and plant transformation
The LjDREB2B gene was PCR-amplified from Lotus japonicas cDNA based on a gene sequence (Lj0g3v0359549.1),The LjDREB2B cDNA sequence was cloned into the binary pBI121vector, which carries the kanamycin resistance gene for bacterial and plant selection, and drives transgene expression using the CaMV35S promoter (Fig. S6).The construct was transformed into the Agrobacterium tumefaciens strain GV3101 by electroporation, which was then used to floral dip method in Arabidopsis thaliana plants.Two homozygous transgenic Arabidopsis lines, exhibiting high LjDREB2B expression levels, were chosen for subsequent drought stress experiment.

Evolution, domain organization, and structure analysis
Phylogenetic trees was constructed with the full-length DREB proteins of Lotus japonicus and Arabidopsis thaliana were constructed using Clustal 2.1 software using with default settings to examine the evolutionary relationships, and MEGA7.0 software was used to conduct a unrooted phylogenetic analysis by the neighbor-joining (NJ) method with 1000 bootstrap replicates [31].Conserved motifs of the DREB family of Lotus japonicus proteins were constructed using MEME Version 5.5.5 (http://meme-suite.org/tools/meme),top 10 most enriched motifs were selected [20,21].The conserved domains of the DREB family of Lotus japonicus were predicted using NCBI-CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [20,33].The distribution was drawn using a visualization tool in TBtools.

Plant growth conditions and drought treatments
Lotus japonicus ecotype "MG20" was used in this study.The Lotus japonicus seeds were provided by Prof. Yanmin Wu's Lab (Chinese Academy of Agricultural Sciences).Arabidopsis thaliana ecotype "Columbia-0" seeds were preserved in our laboratory.We selected Lotus japonicus seeds with full grains, planted them in culture dishes covered with double-layered filter paper, and grew them in a controlled environment with a photoperiod of 16 h/8 h (light/dark) at 24 °C and 70% relative humidity in a growth chamber.The Lotus japonicus seeds were planted in pots with perlite for 30 d and then subjected to drought treatment.We removed the original culture solution and then watered the solution with 20% PEG.The leaf samples were measured at 0, 3, 6, 12, and 24 h.Each treatment included three replicates in gene expression.
For the drought stress treatments, seeds of Arabidopsis thaliana "Columbia-0" (WT) and overexpression LjDREB2B transgenic Arabidopsis thaliana were grown in MS medium for 7 d, transplanted into nutrient soil, grown for 35 d, and not watered for 15 d, and then re-watered.

Gene expression analysis
Total RNA was extracted from leaves using plant isolation kits (Cat.#B518631; Sangon Biotech, Shanghai, China).Total RNA was extracted and reverse transcribed, as previously described [58].Actin2 was used as a reference gene.Table S4 lists all the specific primer sequences.

Prediction of protein interaction network and functional annotation
The protein-protein interaction network was constructed using STRING (https://string-db.org/cgi/input.pl) [33].Functional enrichment analysis was performed using Gene Ontology.

Malondialdehyde content
Malondialdehyde content was determined using the thiobarbituric acid reaction method [59].Fresh leaf samples were powdered in liquid nitrogen and homogenized in trichloroacetic acid.The supernatant was then centrifuged, extracted, and mixed with thiobarbituric acid.The mixture was boiled and centrifuged, and the absorbance was measured at 450, 532, and 600 nm.

Proline content
The proline content was determined using the acidic ninhydrin reaction method [59].Fresh leaf samples were powdered in liquid nitrogen, homogenized in sulfosalicylic acid, and heated in boiling water.The samples were centrifuged and the supernatant was mixed with acidic ninhydrin and glacial acetic acid.After cooling, the samples were mixed with toluene, the supernatant was collected, and the absorbance was measured at 520 nm.

Antioxidant enzyme activities
The antioxidant enzyme activity was determined according to the method described by Nie et al. [60].

H 2 O 2 and O 2 − contents
Fresh leaf samples (approximately 0.2 g per sample) were powdered in liquid nitrogen.H 2 O 2 and O 2 − contents were performed as described in the instruction of Suzhou Grace Biotechnology Co.,Ltd (Suzhou, Jiangsu, China).

Statistical analysis
Statistical analyses were performed using SPSS 17.0.Mean and standard error values were calculated for variable comparisons.The experimental data were analyzed using the least significant difference (LSD) test (P < 0.05).

Fig. 2
Fig. 2 Phylogenetic relationships and conserved motifs of DREB genes from Lotus japonicus.A Phylogenetic tree of DREB proteins of Lotus japonicus.B Conserved motif of DREB proteins analyzed by MEME suite.Different colored boxes indicate different motif types.C Conserved domain of DREB proteins.Different colored boxes indicated different domains

Fig. 3
Fig. 3 Cis-acting element analysis of DREB promoters of Lotus japonicus.A Histogram of the cis-acting elements in each DREB gene promoter.Three categories of cis-acting elements in DREB.Different numbers represent different elements.B Locations of cis-acting elements in the 2 kb sequences upstream of Lotus japonicus genes.Different types of cis-acting elements are represented by different colors.C Statistical analysis of cis-acting elements of the DREB A2 subfamily gene promoters related to hormone responses.D Statistical analysis of cis-acting elements of the DREB A2 subfamily gene promoters related to stress responses.Different cis-acting elements with similar functions are shown in the same color

Fig. 4
Fig. 4 Expression levels of four DREB A2 genes under drought stress at different times.A Heatmap of four DREB A2 genes under drought stresses at different times.B Expression level of Lj0g3v0072079.1 at different times under drought stresses

Fig. 5
Fig. 5 Overexpression of LjDREB2B in transgenic Arabidopsis improved drought tolerance.A Phenotypic observation of WT and OE transgenic Arabidopsis lines under drought stress.B MDA content.C Proline content.D SOD activity.E POD activity.F CAT activity.Different letters indicate significant differences based on the LSD test (p < 0.05)