DELLA Methylglyoxalation-modulated Flowering Under Salt Stress

As a common abiotic stress,salt stress has an important impact on plant growth and development.It is known that salt stress can stabilize DELLA proteins,key proteins in the gibberellin signaling pathway,leading to late flowering in Arabidopsis.However,how salt stress regulates the stability of DELLA proteins remains unknown.Additionally,methylglyoxal(MG),an intrinsic by-product of glycolysis,plays a role in plants responses to various external stress with unknown mechanisms.Here,we focused on RGA,one of MG modified DELLA proteins in the process.We found that salt stress promoted MG accumulation and stabilized RGA protein by methylglyoxalation,leading to late flowering in Arabidopsis.Our results are as follows :1.Salt stress promotes MG accumulation and causes late flowering in Arabidopsis.When Arabidopsis plants were treated with NaCl or MG,MG content was increased and the flowering was delayed.Further,we overexpressed GLYI2 and GLYII4 to degrade MG,and found that the transgenic lines flower early than the wild type when treated with NaCl or MG.Consistently,the treated transgenic plants lines had lower MG than the treated wild-type plant.2.MG delays flowering by stabilizing RGA proteinThen,we examined the stability of RGA,one of DELLA proteins,by using RGA::RGA-GFP with flourescent microscopic observation and western blot analyses,and found that both treatment can significantly increase RGA stability.Further,when all DELLA genes were inactivated in the della mutant,either NaCl or MG can not repress the early flowering of the mutant.3.MG improves the stability of RGA protein by inhibiting RGA ubiquitinationWe expressed and purified the RGA and SLY1(a E3 ligase)from E.coli,and carried out in vitro ubiquitination experiments with RGA.We found that MG treatment significantly inhibited RGA ubiquitination.4.MG modifies RGA protein in vitro and in vivoWe treated RGA with different MG concentrations,and examined whether RGA can be modified by using Anti-MMP,an antibody to recognize MG-modified proteins.Our results indicated that more RGA protein was modified with increasing MG concentration.Further,our pull down experiments with pER8::RGA-Myc-His-Flag and35S::RGA-Myc-His-Flag also verified MG modification of RGA protein.5.Detection of MG-modified amino acids by mass spectrometryIt is known that MG modifies arginine and lysine.Thus we assayed MG-modified amino acids using MG-treated RGA and pull down enriched proteins with Anti-Flag from MG-treated pER8::RGA-Myc-His-Flag and 35S::RGA-Myc-His-Flag,we found that five arginine and three lysine residues of RGA protein can be modified by MG.