SENSITIVE TO FREEZING5: The Multiple Functions of AVP1 in Plant Freezing Tolerance

Frost-induced crop losses significantly decrease yield. Therefore, identifying genes involved in freezing tolerance is of agricultural importance. Arabidopsis thaliana SFR5 was identified in 1996 as one such gene, due to its mutant’s sensitivity to freezing temperatures. More recently, next generation sequencing has allowed several SFR5 candidate genes to be identified, including AVP1. Arabidopsis vacuolar proton- pumping pyrophosphatase (AVP1) is primarily known for its proton-pumping activity, utilising the energy derived from the hydrolysis of pyrophosphate to transport protons across the tonoplast. In doing so, it impacts the cytoplasmic PPi content. However, the enzyme has also been shown to influence sucrose phloem loading. In sieve element– companion cells, AVP1 is localised to the plasma membrane, and carries out the reverse function to maintain the pyrophosphate supply required for transporting sucrose into the phloem. One or multiple of these three activities may contribute to plant freezing tolerance. In the present study, the potential role of AVP1 in plant freezing tolerance was explored. An increase in freezing sensitivity was observed in the known avp1 mutants fugu5-1 and fugu5-3, and in another independent mutant generated using gene editing. This was observed both in non-acclimated and cold acclimated plants, indicating that it is not, or not solely, related to the pathways activated during the acclimation process, indicating a role in basal freezing tolerance. Partial complementation to restore specific functions or expression in particular subcellular locations was utilised to investigate the mechanism by which AVP1 contributes to freezing tolerance, and implicated the enzyme’s pyrophosphatase activity. Expression of the cytosolic inorganic pyrophosphatase1 (IPP1) gene from budding yeast in an avp1 mutant background restored freezing tolerance to WT levels, suggesting that it is AVP1’s role in maintaining phosphate homeostasis that influences freezing tolerance. Understanding the mechanism by which AVP1 contributes to freezing tolerance would facilitate more targeted manipulation of AVP1 in efforts to improve crop freezing tolerance.