Studies Of Nuclear Filament-containing Protein 2 In Modulating Spindle Microtubule Organization And Chromosome Segregation In Arabidopsis

In yeast and human,the kinetochore is a conserved macromolecular protein complex that assembles on centromeric（CEN）DNA and mediates the attachment of paired sister chromatids to spindle microtubules（MTs）in a bipolar manner compatible with the dissociation of the single chromatid.The kinetochore is composed of the inner layer and the outer layer.The core of the inner layer is constitutive centromere-associated network（CCAN）that loosely bound to the chromosomes;while the core of the outer layer is the network composed of KNLL subcomplex,MIS12 subcomplex,and NDC80subcomplex（KMN）,associating with spindle microtubules.The NDC80 complex composes of four subunits,including NUF2（NUCLEAR FILAMENT-CONTAINING PROTEIN 2）,SPC25（SPINDLE POLE BODY COMPONENT 25）,NDC80（NUCLEAR DIVISION CYCLE 80）/HEC1（HIGHLY EXPRESSED IN CANCER 1）,and SPC24,and acts as a bridge between kinetochore and microtubules.However,in higher plants,there are few studies on kinetochores,especially NDC80 complexes.In this study,we revealed that At NUF2 modulated the spindle microtubules organization and affected the arrangement and separation of chromosomes in the process of mitosis,and At NUF2 and At SPC25 could be bound directly to microtubules in vitro,which is great significant for the early embryo and endosperm development,as well as the post-embryo growth in Arabidopsis.The main results are as follows:1.Effects of At NUF2 on sporophyte development in ArabidopsisThree T-DNA insertion mutants in Arabidopsis thaliana,nuf2-1,nuf2-2 and nuf2-3,were identified and analyzed,finding that three mutants were inserted in the first,fourth and sixth introns of At NUF2 gene,respectively.What’s more,the offsprings of all the three mutants were wild-type（WT）and heterozygotes,and no homozygous plants.Phenotypic observation of nuf2-1/+and nuf2-3/+in vegetative growth stage showed no difference with the wild type.However,in the siliques of the mutants nuf2-1/+,nuf2-2/+and nuf2-3/+,the ratios of albino ovules were up to 24.69%,24.57%and24.89%.The segregation ratio of self-fertilized progenies about mutant nuf2-1/+was analyzed.The results showed that the genetic segregation ratio was about 2:1（heterozygous:wild type）,indicating that the T-DNA insertion caused the death of the homozygote.To determine the nuf2-1 transmission ratios of male and female gametophytes,the intercrossing results between Col and mutant nuf2-1/+showed that the separation ratios of male and female gametophytes were close to the expected ratio1:1,suggesting that the nuf2-1 transmission rates of male and female gametophytes were normal.Similar results were obtained by using Basta resistance screening for the progeny of nuf2-3/+,and cross-crossing between wild type and nuf2-3/+.Further genetic complementation experiments were proved that the phenotypes of the mutants were fully restored,suggesting that the abortion phenotypes were indeed caused by the mutation in At NUF2 gene.2.Effects of At NUF2 on early embryo and endosperm development in ArabidopsisThe ovules of heterozygous plants in the mutant nuf2-2 and nuf2-3 were observed,and it was found that some embryos of the albino ovules were stagnated at about2/4-cell embryo proper stage accompanied by rarely and expanded free endosperm nuclei,while the other embryos were stagnated at the early globular stage accompanied with relatively normal endosperm nuclei.Statistical analysis on the abnormal embryos showed that ratios of the albino ovule were 20.98%to 26.89%,similar with the seed abortion ratios.The endosperm cellularization had completed at 6 DAP（days after pollination）ovules in wild-type,while the ovules of mutant nuf2-3/+had sparse and enlarged endosperm nuclei scattered along the wall of the embryo sac and could not be cellularized.These results suggested that the mutation of At NUF2 in Arabidopsis affected early embryo and endosperm development,ultimately resulting in seed abortions.3.Kinetochore At NUF2 and At SPC25 maintained co-localization with spindle microtubulesBoth At NUF2 and At SPC25 maintained co-localization with the centromere（labeled by CENH3/HTR12 antibody）and the spindle microtubules（labeled byα-tubulin antibody）during root tip cell mitosis.The results suggested that they were conserved kinetochore proteins in Arabidopsis and may play roles in mitosis.And At NUF2,At NDC80 and At SPC25 were widely expressed in the vegetative and reproductive development organs,suggesting that they might play a similar biological role in some event.4.Obtaination of partially complementation mutant nuf2-3/+^DA plantsExpression of At NUF2 gene was driven by endosperm-specific promoter FIS2pro and embryo-specific promoter DD45pro::ABI3pro to complement the mutant nuf2-3/+,respectively,and it was found that the phenotype of the sparse and enlarged free endosperm nuclei was not complemented in nuf2-3/+^{FIS2pro::At NUF2}.In nuf2-3/+^{DD45;ABI3pro::At NUF2}(nuf2-3/+^DA),embryos could develop into the globular stage and the cotyledon stage,and the ovule abortion ratio decreased from 24.89%to 13.07%.These results indicated that At NUF2 was essential for the development of embryo and endosperm.5.At NUF2 modulates spindle microtubule formation and chromosome segregation during mitosisPhenotypic analysis of the seedlings nuf2-3/-^{DD45;ABI3pro::At NUF2}(nuf2-3/-^DA)showed that they were smaller than wild type,the root length was significantly shorter,the cell activity in the root apex meristematic area was reduced and some cells started apoptosis,and the seedlings that grew for two weeks produced only one pair of true leaves or was lost.Then,they continued to grow for about a month and eventually died.Through immunostaining withα-tubulin antibody in the meristem cells of the root tip,we found no obvious difference in the prophase and metaphase stages of the mitosis compared with wild type.In the root tip cells of WT,the umbrella spindle fibers in anaphase began to pull chromosomes to the opposite direction,while unevenly distributed spindles produced obvious chromosome behavior defects in nuf2-3/-^DA,leading to the hysteresis of chromosomes.In telophase,the spindle fibers gradually disappeared,spindle fibers accumulated at expected phragmoplast position on either side of two newly formed nuclei,eventually distributed evenly on both sides of the phragmoplast in wild-type cells.However,in the same stage,there were few spindle fibers deposition in the expected phragmoplast area in nuf2-3/-^DA.Finally,the mitotic spindle formation was disordered and the chromosome behavior was abnormal,which eventually led to the stagnation of the seedling growth.The results suggested that ATNUF2 was involved in regulating mitotic spindle formation and chromosomal behavior.6.Conservation of NDC80 complex in different speciesIt was found that At NUF2,At NDC80 and At SPC25 were conserved and contained microtubule-binding sites in different species.The interaction among At NUF2,At NDC80 and At SPC25 was confirmed by yeast two-hybrid experiments.In addition,At NUF2 and At SPC25 could be bound directly to microtubules in vitro,and At SPC25had a strong binding ability,which differed from the findings in yeast and humans,suggesting that as eukaryotes evolved,some of their biological functions occurred to change.