Molecular Mechanism Of A Mitogen-Activated Protein Kinase SlMPK20 In Regulating Pollen Development In Tomato

Mitogen-activated protein kinases(MAPKs)modulate a variety of signal transduction pathways.The MAPK cascade typically consists of three consecutively acting protein kinases:MAPK,MAPK kinase(MAPKK),and MAPKK kinase(MAPKKK).MAPKs play diverse roles in plant growth and defense.However,their potential involvement in reproductive development remains largely unknown.Phylogenetic analysis revealed that MAPKs can be divided into four groups(A-D)in plants.Here,groups A,B,and C all have the ’TEY’ motif and were found in both animals and plants,while group D is specific to plant species and contain a ’ TDY’ motif.Here,we discovered a unique role of SIMPK20,a plant-specific group D MAPK in postmeiotic pollen development in tomato.SIMPK20 was preferentially expressed in stamen and reached its maximum at the binucleate microspore stage.RNAi-mediated suppression of SlMPK20 or its knockout using CRISPR/Cas9 significantly reduced or completely abolished pollen viability and seed set,respectively,without effects on maternal fertility or vegetative growth.Detailed microscopic and gene expression analyses identified the uninucleate microspore formation as the stage when SIMPK20 exerts its role in pollen development.Transcriptome analysis revealed that knock-out of SIMPK20 significantly reduced the expression of a large number of genes controlling sugar metabolism and transport as well as auxin metabolism and signaling in uninucleate anthers.Our findings on SlMPK20 provide evidence on the specific role of a TDY MAPK in plant reproductive development through controlling post-meiosis pollen development.(1)qRT-PCR analysis revealed that SIMPK20 was predominately expressed in stamens.Within the floral buds,SlMPK20 transcript level reached its maximum at binucleate microspore stage.In situ hybridization assay found that the SIMPK20 transcripts were detected strongly form tetrads to binucleate microspore stage.Strong SIMPK20 promoter GUS signals in the inflorescences,especially in anthers were observed.Furthermore,X-gluc stained transverse sections showed GUS signals from the tetrads to binucleate microspores.The SlMPK20 gene encodes a protein with an estimated molecular weight of 70.6 kDa.The SlMPK20-eYFP transgenic cells displayed fluorescence signals most strongly in the nucleus as well as in the cytoplasm,and plasma membrane.(2)To determine the role of SIMPK20,we attempted to suppress the expression of SlMPK20 in tomato by using RNA interference(RNAi)technology driven by tomato pollen specific promoter LAT52.To eliminate any contribution to pollen development by the residual SIMPK20 in the RNAi lines,we further knocked-out SIMPK20 by using the CRISPR/Cas9 system.A striking phenotype,however,did occur in the transgenic lines where a majority of pollen grains in RNAi plants and nearly all the pollen grains from the CR lines were defective.Both knockdown or knockout SlMPK20 disruppted uni-to-binuecleate microspore transition.The transgenic pollens remained normal up to the tetrad and early uninucleate stage but became largely arrested at the binuclear stage with subcellular abnormality appeared at the middle uninucleate microspore stage.TEM revealed enormous vacuolation in the cytoplasm during the middle and late uninucleate microspore stage.No visible phenotype was observed during vegetative growth and female reproductive development in the RNAi or CR transgenic lines,SIMPK20 plays a unique role in postmeiotic pollen development in tomato.(3)To identify the molecular pathways by which SIMPK20 controls postmeiotitic pollen development,we performed RNA-seq transcriptome analysis on floral buds harvested from CR-SIMPK20 and RNAi-SlMPK20 at three developmental stages:(ⅰ)tetrad stage and early uninuleate stage(the stage before microspore abortion);(ⅱ)middle and late uninuleate stage(the stage during microspore abortion)and(ⅲ)binucleate stage,named as stage 2,4 and 5,sequentially.There is a large amount of genes participated in sugar degradation and synthesis and plant hormone(auxin and JA)homeostasis and signaling were significantly downregulated.We then found that knockout of SlMPK20 increased sucrose content in anthers at both tetrad and early uninucleate stage and mid-and late uninucleate stage in CR and WT.IAA content remained unaffected at the tetrad and early uninucleate stage but was reduced in the CR at the mid-and late uninucleate stage.Knockout of SlMPK20 also reduced JA content at the mid-and late uninucleate stage in CR plants with no effect in the early stage.Apart from the major impact on expression of genes controlling carbon metabolism,our findings indicate that SlMPK20 positively contribute to post-meiotic pollent development probably in part through regulating auxin,and to a less extent,JA metabolism and signaling.(4)Protein to protein experiments revealed that SlMPK20 interacted with SlMKK4 and SIMYB32.Moreover,in vitro phosphorylation assay indicated that SlMPK20 can be phosphorylated by SlMKK4.We suggested that SlMKK4 may be updtream of SlMPK20 and SlMYB32 may acts downstream of SlMPK20.SlMYB32,a homologous transcription factor of AtMYB32 required for pollen development in Arabidopsis.Indeed,knockout of SlMYB32 did result in blockage of pollen development in tomato,the same phenotype observed in the SlMPK20 knockout line.This finding concurs with our conclusion that SlMPK20 targets SlMYB20 to exert its control over pollen development.In conclusion,we provided here molecular,cell biology and biochemical evidence that SlMPK20,a group D MAPK,positively regulate uni-to-binucleate transition during microgametogenesis,likely through interacting with SlMYB32 to modulate the expression of genes controlling sugar and auxin metabolism and signaling.