Alfalfa (Medicago sativa L.) is a perennial and highly outcrossing forage legume crop grown predominantly for hay, silage, and pasture. It is the most widely cultivated forage legume in the United States with approximately 16 million hectares planted (1). The high nutritional value of alfalfa with about 15–22% crude protein and an abundance of vitamins and minerals makes it well suited for animal and livestock feed. Alfalfa also brings long-term ecological benefits to society by improving soil fertility through its symbiotic association with the soil bacterium Sinorhizobium meliloti for atmospheric nitrogen fixation, which augments the nitrogen content in the soil for future crops (2–4). The perennial nature of alfalfa along with its deep root system (up to 15 m) helps to prevent soil erosion. However, genetic improvement in terms of forage yield has been relatively stagnant in alfalfa (1).
In fact, the high out-crossing nature, genomic complexity, severe inbreeding depression upon selfing and self-incompatibility complicates alfalfa breeding. Although the multi-purpose use of alfalfa is increasing in demand, production is hindered by changing environmental conditions leading to abiotic stresses like heat, drought, and salinity. In the context of stagnant genetic improvement, cultivation of stress-resilient alfalfa germplasm will help to improve production in response to climate change. However, identification of stress-resilient germplasm requires identification of stress-responsive genes, which, in alfalfa, are very few due to incomplete genomic information and limited expression profile data.
The sessile nature of plants inevitably exposes them to adverse environmental conditions such as abiotic stress. Plants have developed diverse mechanisms to cope with these abiotic stresses. One mechanism is the synthesis of proteins, metabolites, and other compounds to aid in survival through abiotic stress, which are often controlled by transcription factors (TFs). Transcription factors play a critical role in responses to environmental stresses via binding to cis-regulatory elements in promoters to regulate downstream gene expression. In plants, approximately 7% of the genome codes for transcriptional regulators, which bind promoter elements of downstream genes through their conserved sequence specific DNA-binding domain (5). Among the 64 families (6) of transcription factors identified in the plant kingdom, the bZIP (basic leucine zipper) family is one of the largest and most diverse (6–8).
The basic leucine zipper (bZIP) family is distinguished by its highly conserved bZIP domain composed of 60–80 amino acids (9). Structurally, the bZIP domain is divided into two functionally distinct regions: a basic region and a leucine zipper motif (9). The basic region is composed of an invariant motif (N-x7-R/K-x9) of 18 amino acids residues that facilitates sequence-specific DNA binding, while the leucine zipper contains several heptad repeats of leucine or other bulky hydrophobic amino acids such as isoleucine, valine, phenylalanine, or methionine, for dimerization specificity (7, 10). Molecular studies of bZIP genes in Arabidopsis thaliana show that they are involved in the regulation of diverse biological processes including pathogen defense, light and stress signaling, seed maturation and flower development (10). Additional information on the bZIP transcription factor family has provided evidence of their role in response to biotic and abiotic stresses in a diversity of plant species (10, 11).
The availability of whole genome sequences for plants allows the identification or prediction of bZIP TF family members at the genome-wide level. The number of bZIP TFs identified in different plant and crop species varies from 78 (AtbZIPs) in Arabidopsis thaliana (8), 89 (OsbZIPs) in Oryza sativa subs. japonica (7), 125 (ZmbZIPs) in Zea mays (11), 131 (GmbZIPs) in Glycine max (12), 92 (SbbZIPs) in Sorghum bicolor (13), 55 (VvbZIPs) in Vitis vinifera (14), 64 (CsbZIPs) in Cucumis sativus (15) and 247 (BnbZIPs) in Brassica napus (16). The bZIP transcription factors play crucial roles in developmental processes and environmental tolerance in response to multiple stresses. They are involved in the regulation of seed development (17, 18), cell elongation (19, 20), vascular development (20), flower development (21–24), somatic embryogenesis (25), as well as in nitrogen/carbon and energy metabolism (26–28).
In addition to functions in plant growth and development, bZIPs also play an important role in responses to abiotic and biotic stresses. Several bZIPs from A. thaliana (AtbZIP17, AtbZIP24, AtbZIP12), rice (OsbZIP12, OsbZIP72, OsABF1) and soybean (GmbZIP44, GmbZIP62, GmbZIP78) were found to positively regulate salt stress adaptation in plants either directly or indirectly (12, 29–34). Several bZIPs from rice (OsbZIP52/RISBZ5, OsbZIP16, OsbZIP23, OsbZIP45, AREB1, AREB2, ABF3) were also found to be involved in drought tolerance (35–38). OsbZIP52/RISBZ5 negatively regulates cold stress responses (36) while OsbZIP72 was a positive regulator of ABA responses (33). Similarly, overexpression of GmbZIP44, GmbZIP62 and GmbZIP78 reduced ABA sensitivity (12). Interestingly, group D or so-called TGA bZIPs plant a role in systemic acquired resistance (SAR) and pathogen resistance (39, 40). However, there is little published information about the bZIP transcription factor family in cultivated alfalfa and its role in stress resistance.
With the availability of a chromosome-level genome assembly in alfalfa (41) we conducted a genome-wide search to identify and characterize the alfalfa bZIP transcription factors. Since bZIP transcription factors were identified to play significant roles in the regulation of abiotic stress tolerance (10, 11), we speculated various bZIP transcription factors would be differentially expressed throughout distinct developmental stages and abiotic stresses in alfalfa as well. The present study identifies several bZIPs from a proteomic database in tetraploid alfalfa (Medicago sativa). We also analyzed differential gene expression from transcriptome sequences during ABA, drought, salt, and cold stress conditions. This study will facilitate functional analysis of the bZIP transcription factor family in alfalfa. The identification of functions of alfalfa bZIP transcription factors during abiotic stress conditions will further help breeding efforts for improved stress tolerance.