Abstract:

The production of seeds is a vital part of agricultural development. High-quality seeds are the key to growing crops that are both high yielding and able to tolerate stress. Even under optimal storage conditions， the process of seed aging remains an unavoidable occurrence. Seed viability is affected by the balance between the production of reactive oxygen species （ROS） and antioxidant capacity， and this balance is affected by oxidative stress. It is therefore crucial to regulate ROS levels if optimal seed vigor is to be maintained. The Barley B Recombinant-Basic Pentacysteine （BBR-BPC） transcription factor family is a conserved group of transcription factors playing crucial roles in plant morphogenesis， organ development， and responses to abiotic stresses. Members of the BBR-BPC family are known to be involved in regulating the ROS balance in plants， suggesting that they may play a role in controlling seed viability. Nevertheless， it is uncertain which genes in this family are responsible for controlling the vitality of alfalfa （Medicago sativa） seeds. In this study， we focused on the BBR-BPC gene family in the alfalfa cultivar ‘Xinjiang Daye’. Their transcript profiles during the processes of swelling and germination of seeds with varying viability were determined by transcriptome and RT-qPCR analyses. These analyses highlighted the BBR-BPC family members potentially involved in the regulation of seed aging. In addition， proteins that potentially interact with members of the BBR-BPC family were predicted. Sixteen MsBBR-BPC genes were identified in the alfalfa genome and a phylogenetic analysis grouped them into eight subfamilies. The 16 MsBBR-BPC genes were distributed unevenly across 13 chromosomes. A collinearity analysis indicated that expansion within this gene family during evolution has been driven by segmental duplication. Analyses of gene transcript profiles revealed high transcript levels of MsBBR-BPC10， MsBBR-BPC13， and MsBBR-BPC16 at 24 days of seed aging， 8 days of seed aging， and 12-24 hours after imbibition， suggesting that these family members participate in the control of seed viability. A protein-protein interaction network analysis via the STRING database revealed a strong interaction between MsBBR-BPC16 and MsBBR-BPC11. In addition， MsBBR-BPC16 was predicted to interact with AGL11， PAT21， ZHD3， and GPL3 proteins. This study pinpointed three MsBBR-BPCs （MsBBR-BPC10， MsBBR-BPC13， and MsBBR-BPC16） that are involved in seed aging stress and may play a role in controlling seed viability. More research is required to clarify their roles in the control of seed viability， and to verify the protein interactions predicted here. The results of this research provide new insights into the molecular regulatory system governing alfalfa seed aging via BBR-BPC transcription factors， and provide further evidence that the ROS balance plays a role in maintaining seed viability. These findings have established a research foundation for the functional validation of candidate proteins， and for the use of their encoding genes in the genetic improvement of alfalfa seed viability.

Key words: alfalfa, BBR-BPC, gene family analysis, expression pattern, seed aging