MusaDREB1G‐Like Protein Modulates Cold and Drought Tolerance in Musa x paradisica

Summary statement

MusaDREB1G acts as an abiotic stress responsive transcription factor that modulates drought or cold stress tolerance in Musa x paradisica through multifaceted mechanisms. This study paves the way for engineering stress‐resistant banana crops using MusaDREB1G.

1.

The unfavorable environmental conditions (high temperature, drought, salinity, etc.) that exist at different points during the lifecycle of a crop plant negatively impact yield. Under such a scenario, development of stress‐resilient crops has assumed widespread significance. Modern gene‐based approaches have played a crucial role in identifying and characterizing key regulators of abiotic stress tolerance, which have been utilized to improve the genotype of susceptible crops. Though the dehydration‐responsive element binding (DREB) transcription factors have been demonstrated to be crucial for stress tolerance in model plants, their role in economically important crops largely remains unclear. In the present study, the role of MusaDREB1G in mitigating drought or cold stress was investigated in banana, a crop vital for global agriculture and food security.

Sequence/phylogenetic analysis showed the MusaDREB1G protein to harbor a classical AP2/ERF domain and this protein very closely resembled the DREB1G protein of Oryza sativa (Supporting Information S1: Figure S1). Quantitative RT‐PCR of banana plant (Musa cv Karibale Monthan) subjected to abiotic stressors (cold, drought or salinity) or exposure to abscisic acid (ABA) showed strong induction of MusaDREB1G under cold as well as on exposure to ABA (Figure 1Ai,ii, Supporting Information S1: Figure S2). To understand its regulation/expression patterns, the 5′‐regulatory region of MusaDREB1G was ligated upstream of the GUS reporter gene and the corresponding construct (in pCAMBIA‐1301) was transformed into tobacco leaf disks (Supporting Information S1: Figure S3). After exposure to various stresses, tobacco lines harboring Pro _MusaDREB1G ‐GUS were analyzed by GUS‐staining and fluorescent β‐galactosidase assay (MUG). Activation of Pro _MusaDREB1G ‐GUS in tobacco was distinctly noticeable after drought, salinity or cold (Figure 1Aiii, Supporting Information S1: Figure S3). Interestingly, Pro _MusaDREB1G ‐GUS was primarily active in the vascular region under the standard conditions of growth, whereas on imposition of abiotic stresses, expression of Pro _MusaDREB1G ‐GUS was also strongly activated in the nonvascular tissues (Figure 1Aiii).

Figure 1.

(A) Expression profiling of the transcription factor MusaDREB1G under (i) cold stress & (ii) 100 μM ABA. (iii) Expression of GUS under Pro _MusaDREB1G after 6 h of drought or 24 h of cold stress. (B) (i) Levels of the MusaDREB1G transcript in banana lines overexpressing the MusaDREB1G gene. (ii) Phenotype of MusaDREB1G‐overexpressing banana lines during hardening in green house. (iii) In vitro stress recovery assay. Gain in the fresh weight of banana lines during recovery from osmotic stress (10% PEG 6000) or cold stress (exposure to 4°C). (C) (i) EMSA of MusaDREB1G protein and ds‐oligonucleotides (D1G3, D1G6, D1G7) that contain DRE‐elements or mutated, D1G3 (M1), D1G6 (M2) & D1G7 (M3), oligonucleotides. Protein concentration 1X:2 µg, 2X:4 µg, 3X: 6 µg) (ii) Sequence of the different ds‐oligonucleotides with MusaDREB1G binding sites (underlined). (iii) Fluorometric GUS activity (transactivation assay) in banana embryogenic cells transformed with either reporter only (control) or co‐transformed with reporter and effector constructs (P _CaMV35s ::MusaDREB1G + P _SPX /P _{HG_Nat} /P _CYP450 ::GUS).

Transgenic lines overexpressing MusaDREB1G were developed using Agrobacterium‐mediated transformation (Supporting Information S1: Figure S4). The randomly selected transgenic lines, which showed significant up regulation in the MusaDREB1G transcript, displayed a dwarf‐growth phenotype (Figure 1Bi,ii). Leaf disk and in vitro stress recovery assays to assess the stress tolerance of banana lines revealed the presence of significantly higher chlorophyll content in the transgenic banana lines than the control lines at the end of osmotic or cold shock (Supporting Information S1: Figure S5). However, sensitivity of these transgenic banana lines to salinity stress was akin to that of the control plants. The transgenic banana lines showed higher fresh weight and better root growth during recovery from drought or cold (Figure 1Biii, Supporting Information S1: Figure S6). Thus, overexpression of MusaDREB1G improved drought/cold tolerance, but not salinity tolerance, in banana lines.

Reactive oxygen species (ROS) play a crucial role in regulation of stress homeostasis in plants and elevation of ROS is known to correlate with reduced stress tolerance (Wang et al. 2024). Overexpression of MusaDREB1G led to significantly lower H₂O₂ accumulation, which corroborated very well with the remarkably higher transcripts of CAT (catalase), Trx (thioredoxin) and TrxR (Thioredoxin reductase) (Supporting Information S1: Figure S7). Phytohormones such as salicylic acid (SA), jasmonic acid (JA) and ABA are important mediators of stress signaling pathways involved in adaptation to abiotic stresses (Myers et al. 2023). ABA induces stress‐related transcription factors that govern processes such as stomatal closure, osmolyte accumulation and scavenging of ROS (Gautam and Kariyat 2025; Zheng et al. 2025). JA is known to induce antioxidant enzymes and activate transcription factors that help mitigate various stress to eventually impart tolerance (Han et al. 2025). Targeted LC‐MS analysis of banana lines revealed elevated ABA, JA, and IAA content in transgenic lines overexpressing MusaDREB1G; however, the content of SA remained unchanged (Supporting Information S1: Figure S8A–D). Moreover, ABA biosynthetic genes, ZEP (zeaxanthin epoxidase) and NCED (9‐cis‐epoxycarotenoid dioxygenase) as well as JA biosynthetic genes, PLA‐1 (phospholipase A‐1) and LOX (lipoxygenase), showed enhanced expression in the MusaDREB1G overexpressing lines (Supporting Information S1: Figure S8E–H). This indicates a possible regulation of ABA and JA biosynthetic genes by the MusaDREB1G protein, which consequently leads to improved stress tolerance in the transgenic banana lines.

Conserved domain analysis of MusaDREB1G protein sequence showed the presence of a single AP2 domain. To assess the DNA‐binding ability of MusaDREB1G, this protein with 6 additional in frame C‐terminal His‐residues (His‐tag), was overexpressed in E. coli and purified by affinity chromatography. The presence of the his‐tagged MusaDREB1G protein was verified by Western blot analysis employing the anti‐His antibody (Supporting Information S1: Figure S9A,B). The purified MusaDREB1G protein was employed for EMSA in conjunction with nine distinct ds‐oligonucleotide fragments that harbored different DRE‐elements. Out of these nine, three variations of the DRE‐elements exhibited a notable binding to MusaDREB1G (Figure 1Ci,ii). These oligos contained sequences that belonged to the AP2 superfamily i.e. AP2 family (GCACAT), RAV family (GCCGCC) or ERF family (AGCCGCC). Particularly, when these sites were mutated, MusaDREB1G failed to bind to the altered oligos, demonstrating the specificity of interaction (Figure 1Ci).

Compared to the wild‐type, RNAseq data of the MusaDREB1G‐Ox line showed elevated transcription of several stress‐related genes (Supporting Information No.3). Further, Gene ontology analysis showed these genes to regulate important biological processes, such as abiotic stress response, protein folding, etc (Supporting Information S1: Figure S10). Promoter of three highly induced genes, identified from transcriptomic analysis [SPX (Ma07_g09970), CYP450 (Ma03_g32460) and HG_NAT cat domain (Ma01_g02790)‐like genes] were employed for transactivation assays to ascertain their in vivo activation by the MusaDREB1G protein. Considerably enhanced reporter gene activity was observed with these promoters when effector (P _CaMV35S :MusaDREB1G‐nosT) and reporter constructs (P _SPX /P _CYP450 /P _{HG_NAT} ::GUS‐nosT) were co‐transformed in the banana embryogenic cells (Supporting Information S1: Figure S9C,D). Thus, MusaDREB1G does indeed activate transcription of these genes in vivo in banana (Figure 1Ciii). This evidence, along with the above‐mentioned results obtained from EMSAs, validate MusaDREB1G as a member of the AP2 superfamily of TFs.

Collectively, these findings suggest that MusaDREB1G functions as a stress‐activated transcription factor that is responsible for drought or cold stress tolerance through multifaceted mechanisms that involve gene activation, biosynthesis of stress hormones and scavenging of ROS (Supporting Information S1: Figure S11). Although the overexpression of MusaDREB1G in banana plants led to dwarfism, the transgenic banana plants showed enhanced ability to withstand drought or cold stress. Harnessing the stress‐induced expression of this DREB transcription factor in banana plants holds promise for bolstering their resilience to environmental challenges and enhancing productivity.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting figures PCE MusaDREB1G.

Supporting file M‐M.

Supporting Tables.

Supporting Information No.3_RNAseq Data of MusaDREB1G‐Ox banana plants.

Data Availability Statement

Data available in article Supporting Information.