| Pollen development is crucial for plant sexual reproduction and serves as a prerequisite for successful plant reproduction.However,the molecular mechanism responsible for its regulation remains largely unexplored.Increasing evidence suggests that Armadillo(ARM)repeats play a significant role in the reproductive development of both animals and plants.Various types of ARM repeats are essential for ensuring the proper development of plant pollen.Nevertheless,the precise functionality of the majority of ARM repeats in pollen development remains elusive.Therefore,investigating the molecular mechanism by which ARM repetitive proteins regulate pollen development will not only enhance our comprehension of the plant sexual reproduction process and genetic regulation,but also yield valuable insights into the development of male sterile lines and crossbreeding strategies.These findings hold potential practical significance and application value for crop breeding and the pursuit of agricultural sustainability.Our research group previously identified a pleiotropic gene called AVB(ABNORMAL VASCULAR BUNDLES)in rice through map-based cloning(Ma et al.,2017).This gene encodes an ARM repetitive protein.However,its presence and function in dicotyledonous plants remain unreported and unknown.In this study,we conducted an analysis of the fulllength amino acid sequence of the AVB protein in rice through homologous sequence alignment and conserved domain analysis.Additionally,we identified the At AVB gene family in Arabidopsis(Arabidopsis thaliana),a model dicotyledonous plant,consisting of four members.This gene family was subsequently named EFOP(EFR3 OF PLANTS)family(Noack et al.,2022).Building upon this foundation,our study employed bioinformatics,reverse genetics,cell biology,biostatistics,biochemistry,and molecular biology to comprehensively investigate the role of the EFOP gene family in the development of the male gametophyte in Arabidopsis.Additionally,we conducted an in-depth analysis of the synergistic regulatory mechanism between EFOP3 and EFOP4 in the development of the pollen intine in Arabidopsis.The main results are as follows:1.Identification and bioinformatics analysis of the EFOP gene familyBy conducting a comparison of amino acid homology sequences and analyzing the ARM domain,we identified four members of the EFOP gene family in Arabidopsis.These members include EFOP1(At5g21080),EFOP2(At2g41830),EFOP3(At1g05960),and EFOP4(At5g26850).The analysis of cis-acting elements revealed that the EFOP promoter sequence spanning 2000 bp contains numerous important cis-acting elements associated with various essential biological processes including plant growth and development,light response,stress response,and hormone response.The analysis of physical and chemical properties indicates that EFOPs are classified as acidic,unstable,hydrophilic proteins.Conserved domain analysis showed that all EFOPs belonged to typical ARM repeat proteins.Phylogenetic analysis shows that the evolution of EFOPs underwent a complex process.EFOP1 and EFOP2 likely exhibit functional differentiation between monocotyledonous and dicotyledonous plants.Conversely,EFOP3 and EFOP4 are expected to have conserved biochemical functions across both monocotyledonous and dicotyledonous plants.2.Expression patterns and mutant constructions were examined for the EFOP gene family membersThe q RT-PCR analysis revealed the widespread expression of the EFOPs gene in various tissues and organs,including roots,stems,rosette leaves,flowers,and siliques.These findings suggest that the EFOPs gene may have a significant role in the development of diverse organs.We isolated and identified six T-DNA insertion mutants of the EFOPs gene,whose insertion disrupted the integrity of the EFOPs gene without significant phenotypic changes.Accordingly,we further generated 15 double mutants through reciprocal hybridization.Phenotypic analysis revealed that only 3 heterozygous double mutants exhibited defects in pollen development.The remaining 12 homozygous double mutants did not show any defects in vegetative or reproductive growth.As a result,subsequent research primarily focused on investigating the roles of EFOP3 and EFOP4 genes in male reproductive development.3.Functional analysis of EFOP3 and EFOP4 genes in male gametophyte developmentDefects in vegetative growth and reproductive growth were not observed in the efop3-/-and efop4-/-mutants.However,the efop3-/-efop4+/-and efop3+/-efop4-/-mutants exhibited partial pollen defects,while it was not possible to obtain efop3-/-efop4-/-homozygous double mutants through self-crossing.Further investigation revealed that the T-DNA insertion of the EFOP3 gene in the efop4-/-background and the T-DNA insertion of the EFOP4 gene in the efop3-/-background were unable to be transmitted via the male gametophyte.However,the transmission of these insertions through the female gametophyte remained unaffected.LAT52pro::EFOP3-Com and RALF4pro::EFOP4-Com were found to restore pollen fertility in efop3-/-efop4+/-and efop3+/-efop4-/-,respectively.This suggests that EFOP3 and EFOP4 function cooperatively in the development of male gametophytes.Semi-thin section and nuclear morphology analyses revealed that the concurrent loss of EFOP3 and EFOP4 gene functions resulted in pollen shrinkage at anther stage 12.This shrinkage subsequently led to rapid degradation of the trinuclear pollen nuclei,ultimately resulting in pollen abortion.Scanning electron microscope(SEM)analysis revealed that the outer surface of the shriveled pollen exhibited differences compared to normal pollen.However,no apparent defects were observed in the grid-like structure or thickness of the exine of the pollen.These findings suggest that the shriveled pollen is not a result of abnormalities in the structure of the exine.Transmission electron microscope(TEM)analysis revealed that the shriveled pollen displayed an irregular organization with uneven intine.Additionally,the intine of the shriveled pollen was completely detached from the plasma membrane,accompanied by significant cytoplasm degradation.The results of cellulose and pectin component analysis revealed significant alterations in the cellulose and pectin contents of the intine in shriveled pollens.These findings indicate that EFOP3 and EFOP4 influence the metabolism of cellulose and pectin,thereby regulating the development of the pollen intine.To analyze the expression levels of related genes in both the Col-0 and mutant,a combination of RNA-seq and q RT-PCR was employed.We found that compared with Col-0,the transcription level of cellulose synthase coding genes CESA4,CESA7 and CESA8 in efop3-/-efop4+/-mutant was significantly down-regulated,and the expression levels of pectin metabolism related genes PME13,PME21,PME28,PME45,PME49,PME67 and GATL4 were significantly down-regulated.The expression levels of PME4,PME50,PMEI1,VGD1 and GATL7 were significantly down-regulated,while the expression levels of pectin metabolismrelated genes PME8,PLL18 and PLL26 were up-regulated to varying degrees.These results suggest that loss-of-function of EFOP3 and EFOP4 affects the expression of genes involved in cellulose biosynthesis and pectin metabolism.Furthermore,the GO enrichment analysis revealed that there were 160 pathways significantly enriched with DEGs genes between the efop3-/-efop4+/-mutant and Col-0.These pathways included pectin metabolism,pectinesterase activity,and cellulose synthase activity.Out of the 568 DEGs genes,the expression levels of70 pollen-specific genes,including AGL66,ACA7,PTEN1,AGP11,and AGP40,showed significant down-regulation.The results indicated that the functional loss of EFOP3 and EFOP4 had an impact on multiple cell wall metabolic pathways and the expression of pollenspecific genes.4.Expression patterns of the EFOP3 and EFOP4 genes in pollenThe 2000 bp promoter region of EFOP3 and EFOP4 contains 28 and 44 pollen-specific expression motifs,respectively,suggesting that they possess the necessary requirements for expression in pollen.m RNA in situ hybridization and q RT-PCR results revealed a specific m RNA expression of EFOP3 and EFOP4 genes in pollen at anther stages 10–12,with the highest expression level observed in trinuclear pollen.5.The localization patterns of EFOP3 and EFOP4 proteins in pollenThe results of subcellular localization experiments revealed the specific localization of intact EFOP3 and EFOP4 proteins in the plasma membrane of mature pollen,Arabidopsis protoplasts,and Nicotiana benthamiana leaf cells.In contrast,all truncated EFOPs proteins lost their specific localization in the plasma membrane.Notably,truncated EFOPs with an intact Nterminal sequence displayed continuous plasma membrane localization signals.N-terminal cysteine point mutation analysis reveals that both EFOP3 and EFOP4 can be targeted to the plasma membrane through the three conserved cysteines in their N-terminus(Cys12,Cys16,and Cys19).There are quantitative effects of cysteines at different locations on the anchoring function of plasma membrane.N-terminal basic amino acid point mutation analysis showed that EFOP3 and EFOP4 could target the plasma membrane through their Nterminal alkaline amino acid rich regions(Arg23,Arg25,Arg27,Arg32,Lys31,Lys34,and Lys35).Furthermore,the pollen-specific complementation experiment demonstrated that any truncated EFOP3 or EFOP4 failed to rescue the pollen defects in efop3-/-efop4+/-and efop3+/-efop4-/-mutants,while only intact EFOP3 and EFOP4 were able to restore the pollen fertility of the mutants.In conclusion,the N-terminal sequence of the EFOPs protein is crucial for its continuous plasma membrane localization.Additionally,the integrity of the EFOPs protein is essential for its specific localization on the plasma membrane,a crucial aspect for pollen fertility. |
