Histone H2B Monoubiquitination Regulates Salt Stress-Induced Microtubule Depolymerization In Arabidopsis

Plants usually suffer from various biotic and abiotic stresses during their growth and development.Salt stress is one of the most important environmental factors limiting the productivity of agricultural crops.Plants have evolved a variety of regulatory mechanisms which are essential for the adaptation to environmental stresses.Histone H2B monoubiquitination(H2Bub1)is being recognized as a regulatory event that controls a range of cellular processes and response to biotic stress.However,knowledge concerning molecular regulatory mechanisms of H2Bubl in response to abiotic stress in plants remains limited.In this study,we used wild type and H2Bub1 loss-of-function mutants hub1-4,hub2-2,hub1-4 hub2-2 to elucidate the role of H2Bub1 in response to salt stress.H2Bub1 loss-of-function mutants showed salt-sensitive phenotypes,including discolored and bleached of the leaves,the lower rate of seedling survival and the shorter root length.Interestingly,both HUB1 and HUB2 were induced by treatment with NaCl of different times or different concentrations.The results suggest H2Bub1 loss-of-function mutants are hypersensitive to salt,HUB1(Histone monoubiquitination 1)and HUB2 are positive regulators in response to salt stress.To further illustrate the intracellular mechanisms,we used confocal laser scanning microscope(CLSM)to observe microtubules(MTs)dynamics of plant cell after NaCl treatment.The results showed that a weaker depolymerization of cortical MTs was observed in H2Bub1 mutants compared with wild type.These data indicate that H2Bub1 modulates MT depolymerization in response to salt stress.To further determine the mechanism of H2Bub1 modulating MT depolymerization in response to salt stress.The relative expression levels of tyrosine phosphatase(PTP)genes in H2Bub1 mutants were confirmed by real-time PCR.The transcript levels of AtPTP1(Protein Tyrosine Phosphatase 1),AtDsPTP1(Dual-Specificity Protein Tyrosine Phosphatase 1),AtMKPl(MAP Kinase Phosphatase 1),AtMKP2(MAP Kinase Phosphatase 2),At1BR5(Indole-3-Butyric Acid Response 5)and AtPHSl(Propyzamide Hypersensitive 1)were induced strongly in the wild type;whereas transcript levels of the five genes increased slightly in H2Bub1 mutants.The results suggest that H2Bub1 is a positive regulator of expression of PTP genes during salt stress.The 35S:PTP1/hub1-4 and 35S:MKP1/hub1-4 lines(which were generated by transforming hubl-4 mutant with a vector expressing the 35S:PTP1 and 35S:MKP1)largely restored the salt-sensitive and MT depolymerization phenotypes of hubl-4 mutant.These data indicate that PTP1 and MKP1 play important roles to regulate MT depolymerization in response to salt stress.Yeast two-hybrid assay and bimolecular fluorescence complementation(BiFC)assay showed that PTP1 and MKP1 physically interacted with MPK3(Mitogen-Activated Protein Kinase 3)and MPK6.Genetic and biochemical analyses revealed that PTP1 and MKP1 play important roles in regulating the activity of MPK3 and MPK6.Subsequently,we validated the transcript levels,protein levels and activation of MPK3 and MPK6 in wild type and H2Bub1 mutants seedlings during salt stress.The results displayed H2Bub1 affected MPK3 and MPK6 activity during salt stress.MKK5DD(a constitutively constitutively active MKK5 kinase mutant,that leads to long-lasting activation of MPK3 and MPK6)was used to determine salt-sensitive phenotypes.The MKK5DD seedlings had increased salt tolerance.And more massive depolymerization of cortical MTs was induced in MKK5DD compared with wild type.These data indicate that the activation of MPK3 and MPK6 modulates MT depolymerization in response to salt stress,MPK3 and MPK6 are required to mediate the response to salt stress.To determine whether tyrosine phosphorylation affected MT depolymerization of wild type and H2Bubl mutants in salt stress response.The results demonstrated that protein tyrosine kinase(PTK)inhibitor GN caused a significant inhibition of MT depolymerization and decreased salt tolerance especially in the wild type,while protein tyrosine phosphatase(PTP)inhibitor PAO led to significant stimulation of MT depolymerization and increased salt tolerance especially in H2Bubl mutants in salt stress response.Further,these data verify that H2Bubl modulating MT depolymerization is required to tyrosine phosphorylation in salt stress response.Microtubule-associated proteins(MAPs)are the main microtubule structural and function proteins,One well-studied MAPs is MAP65-1(Microtubule-Associated Protein 65-1).Here,we determined the role of MAP65-1 during salt stress.The result displayed that MAP65-1 was involved in response to salt stress and affected MT depolymerization,what's more,it also regulated MT reorganization.A luciferase complementation imaging(LCI)assay showed that MAP65-1 interacted with MPK3 and MPK6.In summary,H2Bub1 regulates salt stress-induced MT depolymerization,and the PTP-MPK3/6 signalling module is responsible for integrating signalling pathways that regulate MT stability,which is critical for plant salt stress tolerance.These results will shed new light on our understanting of the molecular regulatory mechanisms in responses to salt stress in plant.Our results contribute in filling the lack of H2Bubl in response to abiotic stress in plant.