Molecular Mechanisms In Regulating UV-B Tolerance Of OsPRFs In Rice

UV-B(Ultraviolet B)light,as an intrinsic part of sunlight,markedly affects and regulates plant growth and development.However,excessive ultraviolet radiation causes severe biological effects on plants and other organisms at many levels,including altering DNA,proteins,and lipids,modifying the activities of some enzymes.Besides these,a high level of UV-B enhances the production of reactive oxygen species(ROS),which activates stress signaling pathways.The ozone layer can effectively protect organisms from the damage of ultraviolet radiation.Ultraviolet radiation reached the surface of the earth increased constantly with the change of the environment,especially the destruction of the ozone layer,and UV-B a challenging threat constraining global agricultural production.Rice is one of the three major cereal crops in the world,therefore it is particularly important to explore the molecular mechanism of rice to UV-B stress.The cytoskeleton had shown its pivotal role in plant growth and development,as well as light signaling transduction pathway mediated by microfilament.As an actin-binding protein,profilin participates in the regulation of actin polymerization.However,the biological nature of OsPRFs is less-investigated at present,which motivated us to study systematically of all the rice profilin proteins.In this work,we characterized the profilin genes in rice genome and explored the effect of them on the cytoskeleton.We also examined the physiological process they participate in by creating prfs mutants and conducting abiotic treatments.More importantly,we clarified the molecular mechanism of profilin in regulating of UV-B tolerance by using techniques and methods related to crop physiology,cell biology,biochemistry and molecular biology.The main research results are as follows:1.Cloning and characterization of OsPRFs in rice genomeThree genes encoding profilin protein in rice were identified:OsPRF1,LOC＿Os10g17660;OsPRF2,LOC＿Os10g17680 and OsPRF3,LOC＿Os06g05880,by using RGAP,NCBI,RAP-DB,Uni Prot and other databases as well as SMART,MEGA5,Ex PASy.Rice profilin proteins and the other monocots profilin proteins have a high degree of homology in generally and has similar biochemical and physical properties.It predicted that there are five sites have high affinity to monomeric actin and eleven sites have high affinity to poly-proline proteins in the three rice profilins.The three profilins are divided into vegetative and reproductive types.2.Expression pattern and subcellular localization of profilinThe three profilins present completely different expression patterns.Generally,the transcription of OsPRF3 is the highest and OsPRF1 is the lowest,according to the q RT-PCR analysis.OsPRF1 and OsPRF2 are mainly expressed in the reproductive organs and belong to the reproductive type,while OsPRF3 has a higher expression level in each tissue and belongs to the vegetative type.Meanwhile,GUS staining confirmed the above conclusions.Moreover,we used PRF-CFP fusion protein to assess the subcellular localization of OsPRFs in rice protoplasts and found that,similar to the OsPRF1 and OsPRF2,OsPRF3 was also suggested to localize in cytoplasm and nucleus.However,it is notable that the cyan fluorescent signal representing of OsPRF3 is not evenly distributed in the nucleus and cytoplasm and the OsPRF3signal is dominantly observed in the nucleus.3.Response of OsPRFs to abiotic stresses and hormonesPlant CARE analysis showed that there are diverse cis-elements associated with light and a few hormone response elements in OsPRF1 and OsPRF2 promoter.However,the light response elements in OsPRF3 promoter region are less,while the hormone response elements are predominant.Under UV-B stimulation,both OsPRF1 and OsPRF2 expression are repressed quickly within 30mins,while OsPRF3 gene was up-regulated greatly upon salt treatment.In addition,OsPRF2 can also be inhibited by hormones SA and GR-24.prf1,prf2 and prf1prf2 were obtained by artificial small RNA interference method.However,prf1,prf2 and prf1prf2 lack obvious developmental defects and only prf1prf2 shows slightly dwarf phenotype.The prf3 generated by the CRISPR/Cas9 method,which inserted an additional“T”nucleotide at the first exon and presumably to produce a premature stop codon.Moreover,prf1prf2 showed high tolerance to UV-B stress and low tolerance to heat stress,and prf3 decreased its resistance to salt treatment.4.Effect of OsPRFs on plant cytoskeletonThe three rice profilin proteins purified in vitro can bind rabbit muscle actin monomer,and the affinity of OsPRF1 and OsPRF2 is slightly higher than that of OsPRF3.OsPRF1-GFP-HIS,OsPRF2-GFP-HIS and OsPRF3-GFP-HIS can promote actin polymerization,but the polymerization ability of OsPRF3 is slightly weaker than that of OsPRF1 and OsPRF2.Compared with wild-type,the aggregation state and arrangement of microfilaments changed in the root cells of prf1,prf2,prf1prf2 and prf3.In the cells of prf1,prf2 and prf3,the filaments present local aggregation,with high curvature and wavy curve.Moreover,prf1prf2 showed similar or even more severe microfilament polymerization and organization defects than prf1,prf2 and prf3,such as short,thick,discontinuous and dispersive actin filaments throughout the whole cell,and the bundle and linear filament bundles are missing.In addition,OsPRF1/2 and OsPRF3 can also bind to formin protein(DRT1),and the binding site is in the FH1 domain of DRT1.5.Functional analysis of OsPRFs in UV-B stressAmong NIP,prf1,prf2 and prf1prf2,prf1prf2 is the most tolerant to UV-B stress and has better photosynthetic performance than NIP,prf1 and prf2 in the presence of UV-B stress.In addition,the content of ROS in prf1prf2 cells is the lowest,meanwhile the activity of CAT and SOD is the highest,followed by prf1 and prf2.On the contrary,OsPRF1-OE is more sensitive to UV-B stress than the wild-type,and the sensitivity is proportional to the expression level.The level of ROS in OsPRF1-OE cells is higher than that in the wild-type.The expression level of UVR8-1 is significantly reduced by UV-B treatment,but it is no induced in prf1prf2 background.In addition,Y2H and LCI experiments show that OsPRF1interacted with UVR8-1,and the interaction mainly occurred in the nucleus.Moreover,UV-B enhanced the transition efficiency of OsPRF1 from the cytoplasm to nucleus,thereby greatly improved their interaction in the nucleus.UV-B stress response genes are affected in prf1prf2.OsFLS1,OsCHS1,OsCHS2 and OsCPDP,positively regulatory genes in UV-B tolerance,are up-regulated.Consequently,prf1prf2 show the UV-B resistance,while OsPRF1-OE possess the opposite effect.More importantly,uvr8-1 knockout mutant is less tolerant to UV-B,and the expression level of OsPRF1 in the mutant is increased,and not inhibited by UV-B.6.Funcational analysis of OsPRFs on chloroplast movement in riceTo explore whether OsPRFs involves in microfilament-mediated chloroplast relocation,we established the observation system for chloroplast movement in rice.The rice seedlings cultured for 10 days are treated with darkness,50μmol/m~2/s(weak light)and 300μmol/m~2/s(strong light)light,and the leaf sheath is used to detect chloroplast movement and the distribution of chloroplast in the cell can be clearly observed by optical microscope or confocal laser scanning microscope.Using this system,it was found that the chloroplast movement in prf1prf2 was abnormal,indicating that the cytoskeleton regulated by OsPRF1 and OsPRF2participated in the light-induced chloroplast movement.