Phytohormones Accumulation and Distribution in Shoots and Roots of Haploid,Diploid and Tetraploid Barley Seedlings Derived from Microspore Culture

Phytohormones play important roles in plant growth and development,and polyploids are thought to be an important method for plant breeding.However,the relationship between ploidy and phytohormone is still unclear.In this study,barley at three ploidy levels were produced by microspore culture.Therefore,we further analyzed the phytohormone content in the shoots and roots of the three kinds of barley materials to study the effect of ploidy on phytohormones accumulation and distribution.The results showed that Abscisic acid(ABA),gibberellin(GA),jasmonic acid(JA),auxin(IAA),salicylic acid(SA)and cytokinin(CTK)were successfully determined in shoots and roots using LC-MS(liquid chromatography mass spectrometry).By comparing the shoots of the haploid and diploid plants,it was found that the distribution trend of the six phytohormones was consistent,and another consistent trend was found in the roots of the diploid and tetraploid plants.In addition,we further analyzed the shoot/root ratio of the different phytohormones to identify the potential differences for haploid,diploid and tetraploid.Here,the relationship between ploidy and phytohormone we provided would provide new insights into understanding the new phenotypes that occur in polyploid species.

Nuclear translocation of OsMADS25 facilitated by OsNAR2.1 in reponse to nitrate signals promotes rice root growth by targeting OsMADS27 and OsARF7

Nitrate is an important nitrogen source and signaling molecule that regulates plant growth and development.Although several components of the nitrate signaling pathway have been identified,the detailed mechanisms are still unclear.Our previous results showed that OsMADS25 can regulate root development in response to nitrate signals,but the mechanism is still unknown.Here,we try to answer two key questions:how does OsMADS25 move from the cytoplasm to the nucleus,and what are the direct target genes activated by OsMADS25 to regulate root growth after it moves to the nucleus in response to nitrate?Our results demonstrated that OsMADS25 moves from the cytoplasm to the nucleus in the presence of nitrate in an OsNAR2.1-dependentmanner.Chromatin immunoprecipitation sequencing,chromatin immunoprecipitation qPCR,yeast one-hybrid,and luciferase experiments showed that OsMADS25 directly activates the expression of OsMADS27 and OsARF7,which are reported to be associated with root growth.Finally,OsMADS25-RNAi lines,the Osnar2.1 mutant,and OsMADS25-RNAi Osnar2.1 lines exhibited significantly reduced root growth compared with the wild type in response to nitrate supply,and expression of OsMADS27 and OsARF7 was significantly suppressed in these lines.Collectively,these results reveal a new mechanismby which OsMADS25 interacts with OsNAR2.1.This interaction is required for nuclear accumulation of OsMADS25,which promotes OsMADS27 and OsARF7 expression and root growth in a nitratedependent manner.

Generating homozygous mutant populations of barley microspores by ethyl methanesulfonate treatment

Induced mutations are important for genetic research and breeding.Mutations induced by physical or chemical mutagenesis are usually heterozygous during the early generations.However,mutations must be fixed prior to phenotyping or field trials,which requires additional rounds of self-pollination.Microspore culture is an effective method to produce double-haploid(DH)plants that are fixed homozygotes.In this study,we conducted ethyl methanesulfonate(EMS)-induced mutagenesis of microspore cultures of barley(Hordeum vulgare)cultivar‘Hua30’and landrace‘HTX’.The EMS concentrations were negatively correlated with the efficiency of callus induction and the frequency of mutant plant regeneration.The two genotypes showed different regeneration efficiencies.The phenotypic variation of the regenerated M1 plants and the presence of genome-wide nucleotide mutations,revealed by whole-genome sequencing,highlight the utility of EMS-induced mutagenesis of isolated microspore cultures for developing DH mutants.Genome-wide analysis of the mutation frequency in the regenerated plants revealed that a considerable proportion of mutations resulted from microspore culture(somaclonal variation)rather than EMS-induced mutagenesis.In addition to producing a population of 1972 homozygous mutant lines that are available for future field trials,this study lays the foundation for optimizing the regeneration efficiency of DH plants and the richness of mutations(mainly by fine-tuning the mutagen dosage).