Cytological Mechanism Of Heat Stress-impacted Male Meiosis In Arabidopsis Thaliana

Meiosis is an important stage in the process of sexual reproduction of higher plants,which can affect the fertility of plants.Homologous recombination leads to the recombination of alleles,which provides a basis for the development of biodiversity.Male meiosis is sensitive to the changes of ambient temperature.Under the condition of heat stress at 28℃,Arabidopsis can still be fertile,and the frequency of homologous recombination increases.However,the effect of heat stress beyond the fertile range on Arabidopsis meiosis and its cytological mechanism are still unclear.In order to explore the effect of heat stress beyond the fertile range on Arabidopsis meiosis and its cytological mechanism,the following studies have been performed:(1)In order to explore the effect of heat stress on the reproductive development of Arabidopsis,the following experiments were carried out.When Arabidopsis thaliana was treated at heat stress(36-38℃),it was found that its seed pod became shorter.Alexander staining was used to examine the fertility of pollen grains in the anthers.It was found that some pollen grains in anthers lost viability dand produced abnormal pollen grains under heat stress.The above results show that the severe heat stress compromised the fertility of Arabidopsis.(2)Orcein stain was used to examine the meiotic products after heat stress,and it was found that it contained a high proportion of abnormal tetrads.Chromosome spreads method was used to further explore the behavior changes of chromosomes during meiosis.It was found that chromosomes showed univalent after heat stress(5 bivalents were produced at control temperature),indicating that heat stress destroyed the process of homologous recombination and crossover formation.(3)Fluorescence in Situ Hybridization(FISH)combined with centromere probe was used to explore the changes of chromosome behaviors after heat stress.The results showed that heat stress caused defects in the process of homologous pairing,synapsis and/or meiotic recombination.The formation of univalents leaded to the unbalanced separation of homologous chromosomes after the first meiotic division.The callose cell wall of pollen mother cell was examined by Aniline blue staining.It was found that the structure of cell wall was disordered in the tetrad stage.(4)Immunofluorescence localization technique was used to detect the behavior of microtubules during meiosis.After heat stress,the spindle and phragmoplast structures are changed.After heat stress,during the metaphase of meiotic first division,the α-tubulin signal was punctate.In the late stage of the first meiotic division,chromosomes are unevenly separated.The spindle structure in the metaphase of meiotic second division is incomplete.At the end of the second meiotic division,chromosomes are unevenly separated to produce aneuploid microspores.The above experimental results showed that the heat stress destroyed the organization of microtubule cytoskeleton,resulting in unequal chromosome separation and cytoplasmic division defects.(5)In order to explore the effect of heat stress on meiotic recombination,we first examined DSB formation by detecting γH2A.X foci.After heat stress,the signal of γH2A.X in Col-0 decreased.However,in spo11-1-1 mutant the signal of γH2A.X before and after heat stress were always at a low level.The fluorescence quantitative experiment of DMC1 proteins verified this conclusion.(6)To elucidate the impact of heat stress on synaptonemal complex(SC),the axial protein ASY1 was checked by immunofluorescence technique.In the control plants,the ASY1 signal was punctate at leptotene,and was linear at zygotene.After heat stress treatment,the signal of ASY1 showed dotted configuration.The above experimental results showed that the heat stress affects the stability of lateral element of SC.(7)We next examined central element protein ZYP1 of synaptonemal complex.In wildtype Col-0,ZYP1 was linear at pachytene.After heat stress,the structure of ZYP1 was destroyed.It hence concluded that heat stress impaired the formation of synaptonemal complex.(8)The formation and distribution of DSB and SC are regulated by DNA methylation.In order to explore whether heat stress affects DSB and CO formation via an impact on DNA methylation,5-methylcytosine(5m C)was examined on prophase I chromosomes.Under the control condition,5m C signals were mainly distributed at heterochromatin regions.After heat stress treatment,the 5m C landscape was changed.In this paper,Arabidopsis thaliana was used as experimental material to explore the cytological changes during meiosis of plants after heat stress,and rigorous experiments were carried out with cytological and molecular biological test methods.The experimental method is relatively novel and can objectively reflect the facts.It is of certain significance to explore the changes in the cellular mechanism of plants in response to environmental changes.