Gymnosperms represent an ancient lineage that diverged from early spermatophytes during the Devonian. The long fossil records and low diversity in living species prove their complex evolutionary history, which included ancient radiations and massive extinctions. Due to their ultra-large genome size, the whole-genome assembly of gymnosperms has only generated in the past 10 years and is now being further expanded into more taxonomic representations. Here, we provide an overview of the publicly available gymnosperm genome resources and discuss their assembly quality and recent findings in large genome architectures. In particular, we describe the genomic features most related to changes affecting the whole genome. We also highlight new realizations relative to repetitive sequence dynamics, paleopolyploidy, and long introns. Based on the results of relevant genomic studies of gymnosperms, we suggest additional efforts should be made toward exploring the genomes of medium-sized (5–15 gigabases) species. Lastly, more comparative analyses among high-quality assemblies are needed to understand the genomic shifts and the early species diversification of seed plants. Arabidopsis thaliana was first sequenced, the number of assembled genomes of seed plants has reached a considerable number (>800) thanks to the fast innovation of sequencing technologies [Ginkgo, conifers, and gnetophytes , the first draft genome of a gymnosperm species [Picea abies) conducted a comparative analysis of the genome architectures of seed plants [Ginkgo, gnetophytes, cupressophytes (Conifer II), and cycads have been produced and refined with an improved assembly quality [Over the past 20 years, since nologies , 2. Amonnologies , and lownologies , 5. Extates Fig. . Due to tes Fig. . A miles number >00 thanks species ; a prepu species . Notably number >00 thanksd plants . Two setd plants . Based od plants . The subd plants  and the d plants , 17. Witd plants . The unr quality , 23–28.  quality , 26, 29.Here, we summarize the progress made in the whole-genome assembly of gymnosperms and describe the considerably varied genomic features observed in different lineages, focusing on the early genome divergence patterns of gymnosperms. We also discuss the concerns relative to inferred paleopolyploid events and provide insights for future research directions. Additionally, we review the current knowledge on the effect of genomic changes on the diversification of gymnosperms and suggest that more efforts should be focused on medium-sized genomes. Finally, to understand the function of long introns, we recommend further examinations with reverse-genetic tools, which can enhance our understanding of plant genome evolution and adaptation.Thus far, compared with flowering plants, the quantities and qualities of the assembled genomes of gymnosperms are relatively lower, with an average BUSCO value of 56.92% computed from 15 decoded species Fig. . These lGinkgo draft showed that the contig N50 had remarkably grown from 48 kb to 1.58 Mb in length [Sequoiadendron giganteum, 8.1 Gb) and the coast redwood , were successively decoded with conspicuously enhanced contiguity [Taxus, were released almost simultaneously, reflecting the great interest in the gymnosperm genomes [Taxus chinensis, 2.89 Mb in Taxus yunnanensis, and 8.60 Mb in Taxus wallichiana) and the coverage of the core Embryophyta gene library [Cycas panzhihuaensis showed outstanding assembled quality, with a contig N50 length of 12 Mb [Thanks to the advanced sequencing technologies of the PacBio RSII and Oxford Nanopore platforms, there has recently been a dramatic increase in the high-quality assembly of these gigantic genomes Fig. . For insn length , 26; alsntiguity . Additio genomes , 39, 40. library   [Ginkgo genome [T. wallichiana (10.9 Gb), a species belonging to the cupressophytes [Gnetum montanum (4.13 Gb) and Welwitschia mirabilis (6.86 Gb), showed an elevated frequency of the recombination-based removal of retroelements [Larix). Furthermore, the low occurrence rate of the solo-LTRs in Pinaceae was mostly inferred from either fragmental assembly [AtBRCC36A [Comparative genomic studies revealed that angiosperm genomes are considerably flexible and dynamic in terms of the rate of DNA sequence integration and elimination . Apart folo-LTRs , 15. Thi glauca) , 52. Howo genome . Likewissophytes . Moreoveelements , 27. Henassembly , 52 or tassembly . Mobile assembly , 54. Theassembly  and AtBRtBRCC36A ) involveKs) between each 5′-LTR and 3′-LTR flanking sequences, which are calculated based on appropriate mutation rates (per base per year) [–9 is normally adopted, assuming that gymnosperms evolved at a slower pace than angiosperms. Thus, the various ages estimated by different studies of the LTR outbreaks of the same gymnosperm could be partially explained by the different neutral mutation rates assigned  . The int. mairei , 40). It aridity . The hig aridity  are both aridity , 62.Arabidopsis suggested that the Dnmt1-type defense enzyme methyltransferase 1, the plant-specific chromomethylase 3, and the chromatin remodeler decrease in DNA methylation 1 are altogether involved in the DNA methylation of cytosines at CpG and non-CpG loci [The subsequent ancient insertions and the unusual recent burst of LTRs raise an intriguing question regarding the differences in TE surveillance between gymnosperms and angiosperms since the genome size is generally smaller in the latter. The necessity of TE silencing has been widely acknowledged, and the epigenetic control of DNA sequences is considered the vital nuclear defense system of plant genomes to the destructive potential of TEs . ApproacCpG loci .P. tabuliformis  and W. mirabilis  [P. abies [P. abies [P. abies and Pinus sylvestris), implying potential distinctive genome silencing mechanisms [RNA-directed DNA methylation (RdDM) is an epigenetic pathway that evolved to guide the modeling of DNA condensation and TE silencing . This cofor CHG) , 27, conP. abies . FurtherP. abies , 74. In P. abies . The actP. abies . The oscP. abies , 27, comP. abies , 72. Howchanisms .A fundamental shift in repeats’ dynamic has been observed in giant genomes, as indicated by the changes in repeats’ abundance and the curvilinear relationship between genome size and repeats’ proportion among 101 seed plant species  . In partThe extant gymnosperms have painted quite a different picture of the rarity of ancient polyploidizations known as whole-genome duplications (WGDs), which are often found with high frequency in flowering plants , 78 Fig. These eKs distribution of all paralogous pairs has been vital for distinguishing WGD-derived and small-scale duplication-derived paralogues [Ks values of syntenic gene pairs among P. tabuliformis, Sequoiadendron giganteum, and Ginkgo biloba [ζ-) and predicted that a lineage-specific WGD occurred in Welwitschia—the latter prediction was validated in a recent Welwitschia genome investigation [Ks values were accordingly assigned to ζ- . The variable Ks values could be attributed to the heterogeneous mutation rate and different versions of phylogenetic analysis by maximum likelihood used. Whereas we fully recognize the salience of the study both for its data sampling and analytical refinement, it still might be vulnerable to the contested phylogenetic relationships remaining in gymnosperms (the placements of Ginkgo and gnetophytes) [-WGD were preferentially retained over the duplicates derived from the gymnosperm-WGD in all the species surveyed. In addition, a Ks peak (∼0.8) that was recently observed in the Cycas genome was similar to the Ks peak of Ginkgo [ζ-WGD but not of the ω-WGD, rendering the placement of the Cycas + Ginkgo WGD highly controversial [Combining syntenic analysis with the ralogues , 85. Howralogues , 78. Heuralogues , 86, 87.ralogues  first pro biloba . Furthertigation . Anothertigation . Howevertigation , 26, 40 ophytes) . The conophytes) , 20. Altf Ginkgo , suggestf Ginkgo . This anf Ginkgo . Howeveroversial   than conifer-specific tandem duplicates (87 pairs) were preserved in the Pinaceae genomes. A highly conserved genome macrostructure was found between spruce and pine, which diverged at least 120 Ma ago [S. sempervirens) implied that a very slow diploidization process followed WGD and found the persistence of multisomic inheritance in this hexaploidy species (2n = 66). These findings may contribute to explaining why there are so few polyploid species in modern gymnosperms [Sequoia would preclude the emergence of any evolutionary advantages in polyploidy lineages. Hence, Scott et al. [Fizroya cupressoides [Gnetum ula to 35.28 Gb in Pinus ayacahuite) exhibited across gymnosperms [W. mirabilis is the only gymnosperm species known to have a family-specific WGD that occurred ∼86 Ma ago while showing an extremely low level of intrachromosomal syntenic relationships compared to angiosperms) [Given the considerable number of predicted ancient WGDs, based at least on the increased signals of gene duplication (restricted to the WGD-derives) , 20, then shifts  likely sn shifts ). Besiden shifts . One rep0 Ma ago . The lar0 Ma ago . Interspnosperms . Normallnosperms , 93. In essoides . Aside fessoides . If so, nosperms   . The unuosperms) . To concosperms) , 100 andosperms) , which eThe presence of astonishingly long genes has been extensively reported in many gymnosperms from distinct lineages , 15, 23 P. glauca and P. taeda with data from BAC clones and genome scaffolds indicated a relatively conserved signature in the long introns [P. abies, P. sylvestris, and G. montanum. They suggested that an early intron expansion might have already occurred in the MRCAs of all conifers, which would explain the identical trend in the increased length of orthologous introns. However, this point of view was changed by subsequent comparisons conducted within more species of early diverged seed plants [Ginkgo and P. taeda with the accumulation of LTR-RTs . By contrast, a high proportion of long interspersed nuclear elements (LINEs) was found in orthologous long introns between G. montanum and Amborella trichopoda , and boichopoda . This red plants . Howeverion rate , 104 andion rate , 106). Inosperms .Ginkgo or Welwitschia, considering their lower effective population size compared to conifers since the loosening of natural selection often allows the fixation of potentially deleterious mutations in the genome [P. tabuliformis, similar to the situation observed in P. glauca, Oryza sativa, and A. thaliana [Physcomitrium patens [Caenorhabditis elegans, and Homo sapiens [Exploring the biological relevance of long introns could be insightful for addressing a fundamental scientific inquiry: “Why are some genomes really big and others quite compact?” Unfortunately, this matter has been poorly addressed in gymnosperms  except fe genome . In addithaliana , 108. Hom patens , Caenorh sapiens , where c sapiens , nucleos sapiens ). Nevert sapiens .Ginkgo. The hypothetical ω-WGD is still highly contested and needs to be reconsidered by future studies. The sporadic and long-awaited releases of genome drafts inevitably limit the conclusions of species-specific cases. Despite the low level of cladogenesis and the rarity of polyploids, the fundamental shift of genomic dynamics and the potential signature of the slow process of diploidization probably offer new insights into the complex evolution of the genome architectures of gymnosperms. Additionally, the dominant model of recent allopolyploidy speciation in Ephedra [Juniperus [In this review, while appreciating the advances in our knowledge of the genome evolution of gymnosperms, we demonstrated that some essential characteristics, such as repeat dynamics, ancient WGD inference, and the biological relevance of long introns, are far from understood. The state of “genome paralysis” may be confined to Pinaceae rather than all conifers or gymnosperms since a high frequency of TE removal does exist in cupressophytes, gnetophytes, and  Ephedra , as welluniperus , contrasuniperus . With reuniperus , 118. Couniperus , 11, 75.uniperus . Moreoveuniperus , 119. FiBAC ; BUSCO ; CMT3 (chromomethylase 3); DDM1 (Decrease in DNA Methylation 1); GCE (gene conversion event); Hi-C (highthroughput chromosome conformation capture ); Ks (the synonymous substitutions per synonymous site); LINEs (long interspersed nuclear elements); LTR-RTs ; Ma (million years); MET1 (Dnmt1-type defence enzyme methyltransferase); MRCA (the most recent common ancestors); RdDM (RNA-directed DNA methylation); TEs ((retro)transposable elements); UR ; WGDs (whole-genome duplications); WGS (whole-genome shotgun sequencing).The authors declare no competing interests.This work was supported by the Scientific Research Program of Sino-Africa Joint Research Center (grant no. SAJC202105) and the National Natural Science Foundation of China grants (grant no. 31 870 206).T.W. and Q.F.W. designed the outline of the manuscript. T.W. and Y.B.G. wrote the manuscript. T.W., C.D., and Q.F.W. polished the article. Z.M.L. and Y.D.Z. worked on the revisitation of the genomic data. T.W. and Y.B.G. are joint first authors.giac078_Authors_Response_To_Reviewer_CommentsClick here for additional data file.giac078_GIGA-D-22-00123_Original_SubmissionClick here for additional data file.giac078_GIGA-D-22-00123_Revision_1Click here for additional data file.giac078_GIGA-D-22-00123_Revision_2Click here for additional data file.giac078_Response_to_Reviewer_Comments_Original_SubmissionClick here for additional data file.giac078_Reviewer_1_Report_Original_SubmissionSteven L. Salzberg, Ph.D. -- 1/15/2022 ReviewedClick here for additional data file.giac078_Reviewer_1_Report_Revision_1Steven L. Salzberg, Ph.D. -- 6/21/2022 ReviewedClick here for additional data file.giac078_Reviewer_2_Report_Original_SubmissionDavid Neale -- 6/3/2022 ReviewedClick here for additional data file.


Gypsophila paniculata, belonging to the Caryophyllaceae of the Caryophyllales, is one of the most famous worldwide cut flowers. It is commonly used as dried flowers, whereas the underlying mechanism of flower senescence has not yet been addressed. Here, we present a chromosome-scale genome assembly for G. paniculata with a total size of 749.58 Mb. Whole-genome duplication signatures unveil two major duplication events in its evolutionary history: an ancient one occurring before the divergence of Caryophyllaceae and a more recent one shared with Dianthus caryophyllus. The integrative analyses combining genomic and transcriptomic data reveal the mechanisms regulating floral development and ethylene response of G. paniculata. The reduction of AGAMOUS expression probably caused by sequence polymorphism and the mutation in miR172 binding site of PETALOSA are associated with the double flower formation in G. paniculata. The low expression of ETHYLENE RESPONSE SENSOR (ERS) and the reduction of downstream ETHYLENE RESPONSE FACTOR (ERF) gene copy number collectively lead to the ethylene insensitivity of G. paniculata, affecting flower senescence and making it capable of making dried flowers. This study provides a cornerstone for understanding the underlying principles governing floral development and flower senescence, which could accelerate the molecular breeding of the Caryophyllaceae species. Gypsophila belongs to the Caryophyllaceae family, and comprises ~150 species of the annual, biennial, and perennial plants which mainly originated from temperate Asia and Europe [G. paniculata (2n = 34) is a perennial herbaceous shrub, the only species used as cut flowers in the genus Gypsophila [G. paniculata is commonly used as fresh or dried filler in flower arrangements and bouquets. Due to its ornamental value, G. paniculata is listed in the top ten best-selling cut flowers in the global floricultural market [G. paniculata contain various bioactive compounds with potential medicinal values, such as flavonoids, triterpene saponins, sterols, and volatiles, which increase the utilization and economic value of this ornamental plant [The genus niculata n = 34 isG. paniculata, and several commercial varieties were released, such as ‘Million Stars’ and ‘Huixing 1’ [Flower type is one of the most critical ornamental traits and thetop interest of many floricultural species . Among those diverse flower types, the double flower is considered to be the key and precious one. Thereby, breeding efforts have been invested in the creation and improvement of desirable double flower varieties . The samixing 1’ . Howeverixing 1’ . As a coixing 1’ .Arabidopsis which also fits other species [. The loss-of-function of AGAMOUS (AG) results in the elevated expression of A-class genes in the stamens, turning them into petals [APETALA2/PETALOSA (AP2/PET) due to the defects of miR172 binding reduces the AG expression in the stamens, leading to the double flower phenotype likewise [G. paniculata [In the past decades, the genetic and molecular networks regulating flower development have been investigated in roses, carnations, and gerberas –11. An A species , 13. Theo petals . Similarlikewise , 10. Altniculata .Limonium and Gypsophila [RhHB1, RhMYB108, RhWRKY33, and DcWRKY75 affect petal senescence through positive or negative regulation of phytohormone synthesis and signal transduction [G. paniculata were highly sensitive to ethylene, and the endogenous ethylene content instantaneously increased in flower initiating senescent process [G. paniculata flowers is limited.Flowers that remain open and colored long after senescence initiation are normally used as dried flowers, such as species in psophila . This phpsophila , 18. In psophila . Moreovesduction –23. Prev process , 25. How process , 27. To G. paniculata was presented. The genome was sequenced by a combination of long-read sequencing and Hi-C scaffolding technologies. In total, a 749.58 Mb genome was assembled and anchored to 17 pseudo-chromosomes. The molecular basis of double flower formation and regulation in G. paniculata was revealed through gene family and transcriptome analyses generated from diverse floral developmental stages, together with a forward genetic ethyl methane sulfonate (EMS) mutant screen. Meanwhile, we provide new insights into the influence of ethylene on flower senescence with a plausible explanation of the formation of dried flowers in G. paniculata. This chromosome-scale reference genome and the genetic mechanisms governing floral development in G. paniculata offer valuable resources for developing consumer-oriented selective breeding of Gypsophila.In this study, a chromosome-level genome assembly of G. paniculata was conducted from a wild-type plant with pink single flowers. The genome was sequenced using Illumina HiSeq, Nanopore sequencing, and Hi-C technique. Approximately 82.85 Gb of Nanopore long reads were generated, covering 102.92-fold of the 805.00 Mb genome sequence , consisting of retroelement (71.33%), DNA Transposon (23.14%), and tandem repeats (9.13%). Interestingly, we found a remarkable feature in the distribution of genes in the assembly of  central , which i central . TherefoGypsophila, we first clustered the orthologous genes derived from the intergenomic and intragenomic analysis of G. paniculata, Dianthus caryophyllus, Spinacia oleracea, Beta vulgaris, Fagopyrum tataricum, Arabidopsis thaliana, Vitis vinifera, Oryza sativa, Ananas comosus, and Nymphaea colorata. Then single-copy orthologs were adopted for the construction of the phylogenetic tree. Synonymous substitution is thought to occur at a relatively stable rate during the evolution process of orthologous genes, allowing them to be used as molecular clocks due to their absence from selection [G. paniculata , AGAMOUS-LIKE 16 (AGL16), APETALA 1 (AP1), FLOWERING LOCUS C (FLC), AGAMOUS/SEEDSTICK (AG/STK), SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), SHORT VEGETATIVE PHASE (SVP), AGAMOUS-LIKE 15 (AGL15), ARABIDOPSIS NITRATE REGULATED 1 (ANR1), AGAMOUS-LIKE 12 (AGL12), OsMADS32, APETALA 3/PISTILLATA (AP3/PI), ARABIDOPSIS BSISTER (BS), and *MIKC. TM8 and AGL32 were found neither in the G. paniculata genome nor the transcriptome. In addition, no significant expansion of gene number in the 11 groups of Type II MADS-box genes was detected in G. paniculata, except for OsMADS32, FLC, and AGL12.Through the blast of identified MADS-box genes from ox genes . In the G. paniculata were further investigated, and the homologous genes for the ABCE model were identified  is largely decreased in two double flower cultivars compared with wild type, while the expression levels of A-, B-, and E-class genes increased to a first approximation. We then further investigated the expression pattern of ABCE model genes in different floral organs  at the fully open stage  were examined in different floral organs. All the expression levels of the C-class genes were reduced significantly in petal of double flower cultivars, while the expression levels of B-class genes PI and AP3b increased dramatically in the same organs, compared with those in wild type. The relative expression levels of A-class genes were quite low in floral organs except for AP1a and PETa. Although slight differences were displayed between double flower cultivars and wild type, no clear pattern was summarized. The E-class genes SEPa and SEPb showed different expression patterns as well. SEPa expressed more or less in all the samples, whereas the expression levels were significantly high in floral organs of double flowers rather than wild type.To investigate the organ determination and development of double flowers, the floral development process was divided into three stages in wild type and double flower cultivars (‘YX1’ and ‘YX4’), namely flower bud stage, flower semi-open stage, and flower fully open stage . ‘YX1’ al stages . The resen stage . We did G. paniculata were isolated, namely dfm1, dfm2, and dfm3, through EMS induced mutation using single flower wild type seeds. The three double flower mutants displayed unequal variation in flower type and color compared with wild-type flowers  of AP2/PET is also responsible for the double flower formation in flower plants [PETa miR172 binding site (G to C) was detected in ‘YX1’ and ‘YX4’, but not in dfm lines. Lastly, random crosses between wild type and commercial double flower cultivars were performed, and several double flower progenies were isolated in the seeds harvested from wild type, indicating the dominance of the double flower phenotype  like genes, are highly expressed at the S3 stage in ‘YX1’ compared with other two plants, suggesting that the senescent process is accelerated in cultivar ‘YX1’ compared with wild type and ‘YX4’. Likely, Gpan01g01319 encodes a purple acid phosphatase, which regulates phosphate during leaf senescence and is upregulated during leaf senescence [Gpan01g01319 is all upregulated during flower development in wild type and two cultivars, indicating that it is possibly involved in petal senescence as well. Moreover, Gpan01g00371, Gpan01g01321, and Gpan01g01319 also show hub roles in the gene networks in the magenta module, together with the unknown gene Gpan01g01389 (Gpan07g00598 (gibberellin receptor) and Gpan05g00038 , are highly expressed at stage S3 in wild type rather than that in the two cultivars. The accumulation of peroxidase helps to eliminate reactive oxygen species in plants and thus delays senescence [G. paniculata naturally harboured the ability of anti-senescence and this ability was partially lost during the process of artificial breeding.In the magenta module, 406 genes are highly expressed at the flower fully open stage (S3) in two cultivars. Based on the gene expression pattern and annotation, we obtained four genes that might be associated with flower development and senescence , ETHYLENE INSENSITIVE 3-LIKE , and ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR (Gpan02g00593 and Gpan08g01162). Most of the ethylene-related genes show lower expression levels in “YX1” and “YX4”, suggesting that the two cultivars might be insensitive to the ethylene, resulting in the delayed flower senescence. Three unknown genes, Gpan02g00074, Gpan01g00708, and Gpan01g00461, show the hub regulation roles in the gene network of the turquoise module, indicating that they may have functions in the regulation of flower senescence in the two cultivars  in the two cultivars. A lot of genes related to ethylene signal transduction and response were discovered and clustered in the turquoise module , includiultivars . The RNANyctophila colorata), monocots , and eudicots . Eleven core gene families were identified via comparative genome analysis, namely S-ADENOSYL METHIONINE (SAM), 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE (ACS), 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID OXIDASE (ACO), ERS, RESPONSIVE TO ANTAGONIST 1 (RAN1), REVERSION TO ETHYLENE SENSITIVITY 1 (RTE1), CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1), ETHYLENE INSENSITIVE (EIN), EIN3-BINDING F-BOX PROTEIN (EBF), EIL, and ETHYLENE RESPONSE FACTOR (ERF) . A huge gene number shrinkage is identified in ERF family, in which only 34 genes are detected in G. paniculata, while there are 205 and 118 members identified in banana and Arabidopsis, respectively, implying that there is a dramatical reduction of the ERF gene number in G. paniculata.Next, the key genes involved in the pathways of ethylene biosynthesis and signaling transduction were investigated through genomic data of eight species, including early angiosperm (OR (ERF) . Among tytoplasm , have thC. salicifolius, A. thaliana, R. chinensis, and G. paniculata to infer the narrowed mechanism of the ERF gene family in G. paniculata .The expression profiles of the above gene families in different flower development stages and tissues were conducted using ultivars . The ethG. paniculata to ethylene is due to the abnormal function of ethylene receptors, we investigated the expression level of ERS under short exposure  to exogenous ethylene using the double flowers of two cultivars (‘YX1’ and ‘YX4’). Eight ETR genes identified in the genome of G. paniculata were detected, which could be divided into two groups based on the phylogenetic analysis, namely ERS1 and ERS2  showed decreasing after 4 h ethylene treatment in cultivar ‘YX4’. This is also consistent with the observed senescent process that ‘YX1’ ages faster than ‘YX4’. These results prove that ethylene receptors of G. paniculata do not respond to exogenous ethylene in commercial cultivars and the ethylene sensitivity varies in different varieties, which may be caused by artificial selection during plant breeding. Our data collectively suggest that the low expression level of ERS genes and the reduced number of downstream ERF genes might be related to ethylene insensitivity in G. paniculata due to non-ethylene response, resulting in the petal reservation during the process of flower senescence.To support the hypothesis that the insensitivity of and ERS2  and c. TG. paniculata, the only species used as cut flowers in the genus Gypsophila, is an important cut flower for global floricultural markets. In this study, we have sequenced the genome of G. paniculata wild type, which is the first genome of the Gypsophila genus. On the premise of a pseudochromosome genome assembly and a relatively complete annotation, the WGT events are well illustrated in different ways, such as genome collinearity and orthologs analysis, which are shared by G. paniculata and D. caryphyllus and may play an essential role in the evolution of Caryophyllaceae.G. paniculata provides a better understanding of floral development, which is the key ornamental trait in its breeding process [G. paniculata, together with the comparative transcriptome data analyses. In the expression profile of MADS-box genes in G. paniculata, the expression of AG genes showed a significant decrease in double flower cultivars, whereas the expression of AP2/PET genes showed a sharp increase in the whole flower at different stages. In EMS mutants and double flower cultivars, variety mutations were identified in AG genes, which is a core gene playing an essential role in double flower formation in many flowering plants [AGa and AGb, the AG subfamily, are widely expressed in stamens and carpels in most flower plants, such as Nymphaea colorata, D. caryphyllus, and Eustoma grandiflorum, and the mutation or reduced expression in petal of AG will cause the formation of double flower [AP2/PET were responsible for the double flower formation in Arabidopsis, petunia, rose, peach, and Dianthus [PETa from two double flower cultivars, but no mutations in EMS mutants, which had fewer petals compared to the cultivar. This could be that the disrupted AG have a cumulative effect with overaccumulation of PET on double flower formation. In all, the revealing of the floral development regulation in G. paniculata provides the genetic basis for molecular breeding using genetic engineering technology, such as genome editing.The genome of  process . The appg plants –38. For e flower –41. MoreDianthus . In presG. paniculata flowers. Our results suggested that low expression of ethylene receptors and the massive loss of ethylene response factors were related to ethylene insensitivity during flower senescence. This phenomenon has also been observed in carnation flowers, which are highly sensitive to ethylene but petals do not fall naturally [ETR1, ERS1, and ERS2 genes were classified in one subfamily via phylogenetic analysis. These three genes were expressed constitutively or at undetectable levels in different floral tissues during flower senescence in carnation reported by previous independent research [ERF genes, were largely lost in G. paniculata genome , as well as in carnation genome (41 ERFs of carnation vs 106 ERFs of rose) [G. paniculata during flower senescence. Moreover, similar results have also been achieved in geranium and Delphinium, demonstrating that the effect of ethylene on flower senescence may not be carried out by regulating the expression of ethylene receptor genes [ERF genes.Dried flowers and dyed flowers are important forms of cut aturally , 43. Sixresearch . We founof rose) . These ror genes –48. ThusG. paniculata, the first reference genome of the Gypsophila genus. This G. paniculata genome will be a significant contribution to this ornamental plant research community. The whole-genome duplication signatures demonstrate two major duplication events in the evolutionary history of G. paniculata. Through this high-quality chromosome-level genome, we identified and analysed the MADS-box genes to illustrate the floral development regulation of G. paniculata. Notably, with the sequence polymorphism analyses of EMS double-flowered mutants and commercial cultivars, the disruption of AG and/or the misregulation of miR172-mediated PET expression are found to be associated with the double flower formation. We also found that the transcriptional regulation and the reduction in the number of ethylene-related genes lead to the insensitivity of G. paniculata to ethylene, satisfying the characteristics of dried flowers. This G. paniculata genome will also serve as a great resource to improve ornamental traits through molecular breeding in the future due to the fact that G. paniculata is increasingly consumed and demanded for new varieties in the global floricultural market.In summary, we have presented a high-quality genome of G. paniculata. RNA for transcriptome sequencing was extracted from several organs and tissues of wild type, ‘YX-1’ and ‘YX-4’, including root, shoot, leaf, flower bud, semi-open flower, and fully open flower.Total DNA for genome sequencing was extracted from fresh young leaves of a single wild type plant of After the samples were qualified, the genomic DNA was randomly cut off with Megaruptor. Large DNA fragments were enriched and purified by magnetic beads. BluePippin automatic nucleic acid recycling instrument is used to cut and recover large fragments and repair the damage of fragmented DNA. After purification, the terminal repair was carried out at both ends of the DNA fragment. After purification, the joints in the LSK108 connection kit were used for connection reaction. The Qiagen kit was used to extract high-quality DNA, and a Nanopore library with insertion fragments larger than 20 KB was constructed. The Oxford Nanopore Technology  sequencer was applied to conduct single-molecule real-time sequencing of DNA, after which the original reads were filtered to remove low-quality data and adapter sequences.https://github.com/gmarcais/Jellyfish) program was used for K-mer (k = 17) distribution analysis of G. paniculata to estimate the genome size. For the low error rate of next-generation sequencing, it is commonly used in K-mer distribution analysis, whose correctness is closely related to the accuracy of the sequencing result. At the same time, we also performed the genome size determination by a flow cytometer  using leaf samples. The nuclei of the samples were stained with DNA fluorochrome PI  and were measured through flow cytometer with at least 10 000 nuclei. The coefficient of variation (CV) of both sample and reference for every measurement was controlled within 5%. The genome size was determined by comparing with a standard reference genome of Oryza sativa L. japonica. cv. Nipponbare (genome size is 420 Mb) based on the method of Arumuganathan and Earle [The Jellyfish (nd Earle .https://github.com/broadinstitute/pilon). The Hi-C sequencing result was adopted for the construction of pseudo chromosome level scaffolds. BWA (0.7.17-r1188) [We applied MECAT process  to assem7-r1188)  was appl7-r1188)  for prelWe aligned the NGS reads to the assembly using bwa-mem2 (v2.2.1) (bwa-mem2 mem -t 40 -o Gpan.sam Gpan_WG.chromosome.fasta.gz R1.fastq.gz R2.fastq.gz) . SamtoolS. oleracea, B. vulgaris, F. tataricum, A. thaliana, V. vinifera, O. sativa, A. comosus, and Nymphaea colorata were used as homologous reference sequences to predict the gene structure in the genome. Combined with the transcriptome data, the maker (http://www.yandell-lab.org/software/maker.html) was applied to annotate the genome. After the structural annotation, we applied BUSCO to judge the quality of the annotation (python run_BUSCO.py -i Gpan.pep -o Gpan.prot.busco -l ~/data/busco/eudicotyledons_odb10/ -m proteins -c 1). Then we used eggnog-mapper [http://pfam.xfam.org) to annotate the genome functionally. MCScan, a collinearity-analysing tool contained in JCVI toolset [The protein sequences of g-mapper   and RepeatModler (RepeatModeler -pa 20 -engine wublast -database dbname) were used for the searching and detection of transposable elements and Tandem Repeat Finder for tandem repeat sequences.G. paniculata, we analysed the gene families with the genome of G. paniculata. The protein sequences of G. paniculata, N. coloratacolorata, Amborella trichopoda, V. vinifera, Solanum lycopersicum, A. thaliana, O. sativa, A. comosus, and Sorghum bicolor were selected to construct a species phylogenetic tree. We clustered the datasets of proteins of chosen species into orthologs and paralogs at first, then filtered the genes of single-copy orthologs for the alignment and construction of species phylogenetic tree. We chose Orthofinder, a commonly adopted gene-family-clustering tool, as the main tool of the following analysis (orthofinder -t 20 -a 20 -f July11 -S diamond -A mafft). It is an integrated workflow combined with mainstream bioinformatic tools, such as DIAMOND and IQTREE, which can separate and cluster protein sequences from different genomes into paralogous gene groups and orthologous gene groups.To study the evolutional status of B. vulgaris and S. oleracea and of O. sativa and A. comosus from the TimeTree database, whose divergence records are usually supported by synthetical research. The calibration time is then used as a correction to our analysis in further construction of divergence time. To estimate the divergence time, single-copy orthologous genes are in need to analyse the fourfold degenerate site. After the single-copy ortholog orthologous genes and basic phylogenetic tree were obtained, the longest coding sequences (CDS) of each single-copy orthologous gene was extracted according to the orthofinder results. Clustalo (v1.2.4) [Before further analysis, we get the calibrated divergence time of (v1.2.4)  was used(v1.2.4) , to cons(v1.2.4)  workflowG. paniculata, B. vulgaris, C. arabica, and D. caryophyllus were used for synteny and collinearity detection within themselves to shed light on polyploidization events. PAML was chosen for the computing of Ks value between gene pairs of synteny gene blocks.The longest protein sequences of each gene within the genome of G. paniculata, N. colorata, A. trichopoda, V. vinifera, S. lycopersicum, A. thaliana, O. sativa, A. comosus, and S. bicolor to analyse the differences of MADS-box genes between them. HMMsearch [−8. The abnormal sequences that are too long or too short were manually deleted, and the longest transcript was chosen when multiple transcripts coexist. Different groups of MADS-box genes were separated to be aligned by MAFFT [We used the peptide sequences of MMsearch  was applby MAFFT  and MUSCby MAFFT  to constG. paniculata, 21, 737 genes  were selected to do weight correlation network analysis (WGCA) using R package [In order to search the hub genes involved in the flower senescence of  package . An adjaAG, AP2, and PET genes were isolated from the genome dataset of G. paniculata. The primers for gene cloning were designed by Primer Premier 5  with QuantiFast® SYBR® Green PCR Kit . RT-qPCR reactions were performed as described previously [ACTIN was used as a reference gene for RT-qPCR reactions. The gene-specific primers for qPCR of the target genes were shown in Severn eviously . ACTIN wF.L. acknowledges funding from National Natural Science Foundation of China (31960608), Yunnan Fundamental Research Projects (202101AT070147), and High-level Talent Introduction Program of Yunnan Province – Industrial Talent Special Project (YNQR-CYRC-2020-004). C.Y. thanks the Green Food Brand—Build a Special Project (Floriculture) supported by Science and Technology (530000210000000013742). J.W. is funded by China Agriculture Research System of MOF and MARA (CARS-23-G56). L.Z. thanks the Fundamental Research Funds for the Central Universities (2021QNA6008). We want to thank the support from Yuxi Yunxing Biological Technology Co., Ltd. .F.L. managed the project. F.L, L.Z., and J.W. conceived and designed the study. F.L., Y.G., C.J., and X.W. wrote the manuscript. F.L., J.R., C.Y., and J.W. collected the plant materials for sequencing and did the cross breeding. F.L., Y.C., and H.G. conducted the EMS mutagenesis experiment, cloned the genes, and analysed the sequence. Y.G., H.Q., and L.Z. assembled and annotated the genome, performed gene family clustering, and comparative phylogenomics. F.L., C.J., X.W., F.Z., and L.Z. conducted transcriptome sequencing and analysis. F.L., C.J., X.W., X.L., S.F., and F.Z. conducted the ethylene analysis. All authors reviewed and approved the manuscript.https://www.cngb.org/) under project number CNP0003304. The genome assembly sequences and gene annotations are available at https://ftp.cngb.org/pub/CNSA/data4/CNP0003304/CNS0579838/CNA0050895/.All data are publicly available in the China National GeneBank (The authors declare no competing interests.Horticulture Research  online.Web_Material_uhac176Click here for additional data file.


Bacillus species, is active against a broad range of microorganisms. Because of its simple structure and excellent mode of action, i.e., through the inhibition of glucosamine 6-phosphate synthase, it has drawn the attention of researchers. In addition, it acts as a pleiotropic signaling molecule that affects different cellular activities. However, all Bacillus species are not capable of producing bacilysin. The biosynthesis of bacilysin by Bacillus species is not uniform throughout the population; specificity and heterogeneity at both the strain and species levels has been observed. This review discusses how bacilysin is biosynthesized by Bacillus species, the regulators of its biosynthesis, its importance in the host, and the abiotic factors affecting bacilysin production.Microbes produce a diverse range of secondary metabolites in response to various environmental factors and interspecies competition. This enables them to become superior in a particular environment. Bacilysin, a dipeptide antibiotic produced by  Antimicrobial peptides (AMPs) are short amino acid sequences produced by both unicellular and multicellular organisms to protect a host from pathogenic microbes, such as bacteria, viruses, fungi, and parasites . NumerouBacillus species are well known for producing a variety of AMPs, such as bacilysin, bacitracin, surfactin, fengycin, amylolysin, plantazolicin, bacillomycin D, lactosporin, and thuricin [Bacillus brevis. Thus, this peptide may be involved in gene regulation during sporulation [B. brevis when the cells were grown under low-nitrogen concentration in the culture medium [B. subtilis PY79 was obtained by N-Methyl-N′-nitro-N–nitrosoguanidine mutagenesis, which showed higher sensitivity to heat, chemicals, and lysozymes, as well as lower dipicolinic acid content than the wild-type strain [B. subtilis PY79 since this effect was observed upon supplementation of bacterial culture with this compound before reaching the mid-log phase of growth [thuricin . AMPs arrulation . A slighrulation . Sporulae medium . In conte medium . A bacile strain ,14. The f growth .12H18N2O5 and a molecular mass of 270.28 g/mol [Bacillus, and it causes cell lysis in bacteria and fungi [N-terminus and L-anticapsin (a non-proteinogenic amino acid) at the C-terminus. The antimicrobial activity of bacilysin mainly due to the presence of L-anticapsin at the C-terminus [B. subtilis in 1946 and was originally named bacillin [Staphylococcus aureus and Escherichia coli [B. subtilis A14; however, it was not extractable using organic solvents [B. subtilis A14 at a very low yield [B. pumilus B-180 was shown to exhibit chemical and physical similarities to bacilysin [B. subtilis A14 was described as a heat-tolerant antibacterial compound that is stable between pH 1.4 and 12.0 for four hours at 20 °C and is insoluble in organic solvents [Bacilysin is a dipeptide antibiotic compound with the molecular formula C28 g/mol . It is pnd fungi . This coterminus . Bacilysow yield . When baow yield . In 1973acilysin . In 1975acilysin . Bacilysbac operon , which is responsible for bacilysin production, has been examined in molecular studies to understand bacilysin biosynthesis and molecular regulation in B. subtilis [B. subtilis A14 and B. pumilus B-180. The researchers concluded that bacilysin is produced by both strains at the beginning of the death phase [Bacilysin is an antimicrobial compound that has attracted the attention of researchers owing to its simple structure and high antimicrobial activity against a broad range of microorganisms, including bacteria, fungi, and algae ,17. The subtilis ,25. Anotth phase ,27. bac operon, its molecular regulation, and the surrounding environment that influences bacilysin biosynthesis.This review aims to explain the mode of action of bacilysin, its role in the producer organisms, its biosynthesis by the N-acetylglucosamine synthesis [Studies on the antimicrobial activity of bacilysin revealed that it inhibits glucosamine 6-phosphate (GlcN6P) synthase, which helps in the synthesis of GlcN6P from fructose-6-phosphate and glutamine, which is an essential component of the peptidoglycan of the bacterial cell wall . As a reynthesis . Intake ynthesis .E. coli, was studied using bacilysin. Bacilysin can be hydrolyzed by an intracellular peptidase of C. albicans and anticapsin is released into the cell [The inhibition of the enzyme GlcN6P synthase, which was heterologously expressed in the cell . Moleculthe cell . Many stthe cell . Therefothe cell . B. amyloliquefaciens FZB42 exhibited strong antibacterial activity against the cyanobacterium Microcystis aeruginosa , with a killing efficiency of 98.78% [aro gene cluster, and the sfp-mutant strain CH03, unable to synthesize lipopeptides (LPs) or polyketides (PKs), was able to inhibit the growth of M. aeruginosa. This indicated the production of another antibacterial compound by B. amyloliquefaciens FZB42, which inhibited the growth of M. aeruginosa. Further studies showed that bacilysin produced by B. amyloliquefaciens FZB42 caused cell lysis and changed the membranes of many cell organelles of M. aeruginosa. Bacilysin has also been associated with the inhibition of harmful algae such as Aphanizomenon flos-aquae, Nostoc sp., and Anabaena sp. [S. aureus are associated with bacilysin-uptake, whereas E. coli has both di- and oligopeptide transport systems [S. aureus NCTC 6571 compete to receive bacilysin [Bacilysin synthesized by f 98.78% . Biosyntaena sp. . Bacilys systems . Severalacilysin .B. amyloliquefaciens GSB272 transformed with plasmid pSB767 containing bacABCDE produced over ten times more bacilysin than the non-recombinant laboratory strain B. subtilis 168 [B. amyloliquefaciens has a unique genetic backup for the production of bacilysin. This may be related to the presence of genes that positively regulate bacilysin biosynthesis [B. amyloliquefaciens FZB42, also known as B. velezensis [Erwinia amylovora, a causative agent of fire blight disease [E. amylovora growth [sfp plays a role in the production of lipopeptides ; although mutants of this gene are unable to synthesize non-ribosomal LPs and PKs, the growth of E. amylovora is still suppressed. These results suggest that B. amyloliquefaciens has an antagonistic effect in suppressing E. amylovora growth [E. amylovora growth, indicating that the additional inhibitory effect is due to the synthesis of bacilysin, and bacilysin biosynthesis is not dependent on sfp [B. velezensis FZB42 efficiently antagonizes Phytophthora sojae, which causes soybean root- rot disease. FZB42 mutants deficient in lipopeptides (bacillomycin D and fengycin) and PKs  did not impair antagonism against P. sojae. However, mutants deficient in bacilysin gene clusters completely lost their antagonistic effect against the pathogen, indicating the antifungal activity of this dipeptide antibiotic against the pathogen. Electron microscopy showed that bacilysin damaged the hyphal structures and loosened the cellular contents [B. velezensis exhibited biocontrol potential against rice blight and leaf streak pathogens (Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola) [ilis 168 . These dynthesis ,36,37. Glezensis , showed  disease ,40. A mua growth . Moreovea growth . A doublt on sfp . Bacilyscontents . In a siS. aureus when ethylenediaminetetraacetic acid (EDTA) inhibits the hydrolysis of bacilysin [S. aureus strains have evolved altered cell surface receptors. Bacilysin-sensitive S. aureus strains utilize L-alanine produced by the hydrolysis of bacilysin within the cell, whereas resistant strains do not utilize it [B. subtilis 168 in synthetic medium containing sucrose and glutamate was also inhibited by certain growth conditions, such as growth supplements and temperatures above 30 °C [B. subtilis against E. coli and S. aureus in yeast extract glucose agar medium was higher than that of yeast extract agar medium [E. coli activity of B. subtilis [The amount of bacilysin production depends on the nutritional composition of the growth medium and the growth conditions. Its GlcN6P synthase inhibitory activity decreases significantly in crude extracts of acilysin . Moreoveilize it . Bacilysve 30 °C ,43. The ve 30 °C . Antibacr medium . Moreovesubtilis .Bacillus species. In addition to B. subtilis A14, many Bacillus species have been reported to synthesize bacilysin [Bacillus species such as B. amyloliquefaciens, B. velezensis, B. licheniformis, B. pumilus, and B. subtilis revealed that the bacilysin gene cluster is present in all species, except B. licheniformis [Bacillus species, including B. velezensis HNA3, B. velezensis FZB42, B. amyloliquefaciens DSM7, and B. subtilis 168 [B. coagulans, B. licheniformis ATCC 9789, and B. megaterium PV361 upon transformation with plasmid pSB660 containing bacA-bacBCDEF, which indicates that these genes are not solely responsible for bacilysin production [bacABCD genes or that of the vector pSB679 containing bacAB genes enabled bacilysin biosynthesis in B. pumilus ATCC12140 and B. amyloliquefaciens GSB272 [The mechanism of bacilysin biosynthesis differs among various strains of acilysin . In siliniformis . In a siilis 168 . Bacilysoduction . In conts GSB272 . B. subtilis showed wide variation in protein expression in the presence and absence of the bac operon. Both gel- and gel-free proteomics analysis were performed to observe differences in protein expression. Based on these findings, it was concluded that bacilysin acts as a pleiotropic signaling molecule that affects different cellular activities [scoC in B. pumilus BA06 and B. subtilis genomes, which causes transcriptomic and phenotypical changes [scoC gene in B. pumilus BA06 increased total extracellular protease activity and reduced cell motility, as flagella formation was affected. Transcriptome analysis showed that more than a thousand genes were altered during multiple growth stages at the transcription level, including many protease genes, particularly the aprE gene [B. subtilis, ScoC was reported to regulate at least 560 genes [B. pumilus also downregulated the aprN gene encoding a neutral protease in the scoC mutant, indicating that ScoC plays a strain-specific role [B. subtilis [Bacillus species differs at both the strain and species levels [Bacillus species.tivities . Similar changes ,47. MutaprE gene . In B. sfic role . AbrB, Dsubtilis . Products levels . Presentbac operon, which plays a key role in the conversion of prephenate to bacilysin. Some regulatory genes also regulate the expression of the bac operon, which is discussed in the latter part of the article. This complex and unique genetic setup of B. subtilis, B. amyloliquefaciens, and B. pumilus enables them to produce bacilysin more efficiently than any other Bacillus strain. The bacilysin biosynthesis pathway starts from prephenate with the help of genes of bac operon  isomer into the (Z) isomer and vice versa [2HPP, converting it to 3E-ex-H2HPP or 2-oxo-3- propanoic acid, which is the precursor of L-anticapsin [bacB was unable to produce L-anticapsin or bacilysin, indicating that the end product of bacB leads to the production of L-anticapsin and bacilysin [The enzyme encoded by ubstrate ,51. The ubstrate . The gence versa ; althougce versa . The encticapsin . A mutanacilysin .bacG gene of B. subtilis is an integral component of the bacilysin biosynthesis gene cluster. The gene was cloned and expressed and the product of the gene was purified and characterized similarly to bacA or bacB [bacG. Therefore, the substrate for bacG is epoxy-3E-H2HPP, which is converted to epoxy-4S-H4HPP by the end of this reaction [The  or bacB . A previreaction ,56. bacF. BacF protein is a fold-type I pyridoxal 5-phosphate (PLP)-dependent stereospecific transaminase [4HPP as its substrate, converting it to l-dihydroanticapsin, which is the precursor of L-anticapsin [Another essential component of the bacilysin biosynthetic gene cluster is saminase . This enticapsin ,56.bacC encodes a dehydrogenase or reductase that oxidizes the C4-hydroxyl of L-dihydroanticapsin, which takes place immediately after the cyclohexenol double bond epoxidation. Mutants with bacC deficiency were unable to synthesize L-anticapsin or bacilysin, suggesting that it is an essential element for bacilysin production. Computational analysis of BacC proved that it is a member of the NAD+-dependent oxidoreductase family [e family . bacD gene of the bac operon encodes an amino acid ligase. It was previously investigated and determined to be an unorganized dipeptide ligase [The e ligase . BacC  [B. subtilis. At the beginning of this procedure, cell-derived pheromones ComX and CSF  accumulate outside the cells [B. subtilis [B. subtilis through QS [Bacteria produce extracellular signaling molecules at high cell densities that are involved in drastic changes in gene expression through a mechanism known as quorum sensing (QS). QS is the bacterial response or communication at high cell concentrations that allows them to control specific processes through gene regulation. QS governs antibiotic production, sporulation, and competence development in all  ComQXPA . Two QS se (Opp) . ComX anhe cells ,58,59,60he cells . ComX, Che cells . RapC ishe cells . RapC ishe cells . CSF is he cells ,64. CSF,he cells . Activatsubtilis . Bacilysrough QS ,37.phrA, phrC, and comA deletion mutants of B. subtilis PY79 were constructed, and the results showed that these genes depend on Opp for proper function [phrC resulted in a bacilysin-negative phenotype in B. subtilis PY79 [B. subtilis PY79. In addition, an insertion mutation in comA also caused the same result, where 43 out of 50 transformed cells were phenotypically bacilysin-negative [phrA did not reveal any relationship with bacilysin production [Opp is significantly involved in bacilysin production via the QS pathway and handles sporulation, competence development, and the initiation of surfactin production . Opp impfunction . An inselis PY79 . Out of negative . In contoduction . This rebac operon. The signaling pathway of Phr peptides is also coupled with ComX. Together with these data, it can be hypothesized that Phr peptides may be involved in the expression of the bac operon [bacA-lacZ fusion was expressed in a mutant lacking phrC, phrF, and phrK, a huge variation in the expression of the bac operon was found [phrC results in complete cessation of the bac operon [bac operon is possible in some strains without ComX. Therefore, it can be concluded that ComX-mediated signaling is strain specific, whereas PhrC is species specific. The addition of PhrC could compensate for ComX-mediated signaling in deficient strains of B. subtilis [A group of Phr peptides , induces sporulation and competence development ,68. The c operon . When thas found . Most sic operon . In contsubtilis .srfABCD operon of B. subtilis encodes surfactin, a non-ribosomally synthesized LPs known to act against several pathogenic microbes, including L. monocytogenes, Enterococcus faecalis, S. aureus, Pseudomonas aeruginosa, E. coli, Fusarium oxysporum, F. moniliforme, F. solani, Trichoderma atroviride, and T. reesei [B. subtilis PY79 [srfA mutant isolates were investigated; they could express bacA-lacZ, but the expression of the bac operon was not observed [srfA [B. subtilis. B. subtilis that lacked spo0H and/or spo0A (repressor of the abrB gene) could not produce bacilysin, whereas blocking AbrB significantly increased bacilysin production in the mutant strain [bac promoter and positively and indirectly regulates its expression and enhances the expression of the bac operon by suppressing the abrB gene. When Spo0A directly binds to the bac promoter, AbrB cannot bind to the promoter; thus Spo0A indirectly regulates the expression of the bac operon positively by inhibiting AbrB from binding to the bac operon [B. subtilis strains that could not produce bacilysin were suppressed by an abrB mutation in spo0A-blocked mutants. All these reports suggest that gene transcription for bacilysin biosynthesis is negatively controlled by AbrB and is relieved by Spo0A [The . reesei ,73,74. Slis PY79 . To veriobserved . AbrB ised [srfA , and hast strain . Spo0A dc operon . B. subtby Spo0A . bac operon. A decrease in GTP level results in improved expression of the bac operon [B. subtilis, the addition of decoyinine (an inhibitor of GMP synthetase) enhanced the expression of the bac operon, resulting in a 2.5 fold increase in bacilysin biosynthesis [B. subtilis, encoding peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, along with the stationary phase and virulence [bac operon was increased in the mutant strains of Bacillus spp. lacking codY gene, suggesting that its product negatively regulates transcription of these genes [bac operon; however, both can bind to the promoter region of the bac operon. As a result, they act mutually to bind to the bac operon and do not interfere with each other’s activity [Intracellular GTP levels are directly related to the c operon . In wildynthesis . CodY isirulence ,77. Intese genes . Anotheractivity .hpr) negatively regulates protease synthesis and sporulation in B. subtilis [B. subtilis strains revealed that they all have scoC mutation. The expression of the bac operon was higher in mutants lacking scoC, and it has been identified that ScoC directly binds to the promoter of the bac operon and, with AbrB and CodY, negatively regulates the transcription of the bac operon [bac promoter and negatively regulate its transcription, while ComA and Spo0A positively regulate the transcription of the bac operon.ScoC  is essential for bacilysin biosynthesis [B. subtilis PY79 in which nucleotides 196–314 of the lutR gene was deleted resulted in a bacilysin-negative phenotype. In contrast, mutations in the lacR gene located downstream of the lutR gene did not affect bacilysin biosynthesis. These results indicate that LacR does not influence bacilysin biosynthesis, whereas LutR is involved in bacilysin biosynthesis [The expression of the unctions ,81. Simiynthesis . GntR, aynthesis . A mutanynthesis . All genBacillus species and how bacilysin biosynthesis is regulated at the molecular level. The review also reveals how bacilysin production and the expression of several extracellular proteases are controlled in Bacillus species by investigating studies on the involvement of various transcriptional regulators or pleiotropic signaling molecules. However, the control mechanism of bacilysin biosynthesis in Bacillus species and its function are still unclear. For example, it is well known that bacilysin inhibits GlcN6P synthase; however, which proteins are involved in its trafficking inside the cell, or which amino acids are involved in the interaction between GlcN6P synthase and anticapsin-GlcN6P complex formation is unknown. Moreover, bacilysin extraction from its producers in its original form is very challenging. Thus, more research is needed to ensure that bacilysin can be extracted efficiently without losing its biological function. In addition, Bacillus species that produce high amounts of bacilysin can be genetically manipulated to enhance bacilysin production. In the future, this approach might be a potential method for producing long-lasting biocontrol agents using Bacillus spp. for sustainable agriculture.The information presented above demonstrates how bacilysin is synthesized in


Isodon is also phylogenetically and taxonomically recalcitrant largely ascribed to its recent rapid radiation in the Hengduan Mountains. Previous molecular phylogenetic studies using limited loci have only successfully resolved the backbone topology of the genus, but the interspecific relationships suffered from low resolution, especially within the largest clade (Clade IV) which comprises over 80% species. In this study, we attempted to further elucidate the phylogenetic relationships within Isodon especially Clade IV using plastome sequences with a broad taxon sampling of ca. 80% species of the genus. To reduce systematic errors, twelve different plastome data sets  were employed to reconstruct phylogeny using maximum likelihood and Bayesian inference. Our results revealed largely congruent topologies of the 12 data sets and recovered major lineages of Isodon consistent with previous studies, but several incongruences are also found among these data sets and among single plastid loci. Most of the shallow nodes within Clade IV were resolved with high support but extremely short branch lengths in plastid trees, and showed tremendous conflicts with the nrDNA tree, morphology and geographic distribution. These incongruences may largely result from stochasticity  and hybridization and plastid capture. Therefore, the uniparental-inherited plastome sequences are insufficient to disentangle relationships within a genus which has undergone recent rapid diversification. Our findings highlight a need for additional data from nuclear genome to resolve the relationships within Clade IV and more focused studies to assess the influences of multiple processes in the evolutionary history of Isodon. Nevertheless, the morphology of the shape and surface sculpture/indumentum of nutlets is of systematic importance that they can distinguish the four major clades of Isodon.As one of the largest genera of Lamiaceae and of great medicinal importance,  Isodon (Schrad. ex Benth.) Spach  consists of approximately 100 species, mostly occurring in subtropical to tropical Asia, with two endemic species disjunctly distributed in tropical Africa  calyces, strongly two-lipped (4/1) corollas, and free filaments inserted at the base of the corolla tubes  H.W. Li (7 spp.), sect. Amethystoides (Benth.) H.W. Li (7 spp.), sect. Isodon (91 spp.), and sect. Melissoides (Benth.) H.W. Li (6 spp.) – based on the types of inflorescence, and morphology of fruiting calyx  and corolla tube . Ten series were further delimited within the largest section (sect. Isodon) by l Africa . It is ml Africa . As a mela tubes . In a woIsodon  have long been used as traditional folk medicine in China and Japan and the genus is abundant in diterpenoids with diverse structural scaffolds and important pharmaceutical functions ] sequences, representing the most comprehensive taxonomic sampling of the genus to date. We aimed to answer the following questions: (a) whether plastome-scale data can further resolve the shallow-level relationships within Isodon based on a comprehensive taxa sampling? (b) if there are any conflicts among different plastome data sets, among single plastid genes, and between the plastid and nrDNA data sets? What might be the causes of these incongruences?In this study, we carried out the phylogenetic analyses of 86 taxa of Isodon in East Asia and Africa were sampled as ingroups, representing all recognized sections and series of A total of 99 accessions of 86 taxa (including 80 species and six varieties) from the major distribution areas of ® Ultra IITMDNA Library Prep Kit for Illumina®). Sequencing with 2 × 150 bp paired-end reads was conducted to generate approximately 2 Gb data for each accession using an Illumina HiSeq 2000 platform  at BGI Genomics .Total genomic DNA was either extracted from silica-gel dried leaves using a modified CTAB method  or from PCR mixtures and procedures for the amplification of ITS  and ETS followed those described in de novo assembling of clean paired-end reads was carried out using the GetOrganelle Toolkit (Isodon amethystoides (Benth.) H. Hara  Britton  were generated after removing the 19 loci , NCR , and CR + NCR (the concatenated CR and NCR) data sets. The script “concatenate_fasta.py” developed by For the nuclear sequences, the nrITS and ETS regions were directly concatenated to construct the nrDNA data set.Phylogenetic reconstruction was carried out using maximum likelihood (ML) and Bayesian inference (BI) analyses for each of the 12 plastome data sets and the nrDNA data set. The best partitioning schemes and substitution models for the 12 plastome data sets were estimated by PartitionFinder2 v.2.1.1  with the1 Analysis of 1,000 rapid bootstrap replicates (-x) was followed by a search for the best-scoring ML tree in a single program run (-f a), both phases using the GTRGAMMA model for nucleotide data and other parameters being the default settings.Partitioned ML analysis was implemented using RAxML-HPC2 v.8.2.12  on XSEDEPartitioned BI analysis was performed with MrBayes v.3.2.7a  on XSEDEGene trees for each of the 207 loci extracted from plastome sequences were also reconstructed using RAxML-HPC2 for 1,000 rapid bootstrap replicates and a search for the best-scoring tree.2 Concordance among single gene trees was also analyzed using PhyParts by mapping the RAxML trees inferred from the 207 loci against the CR + NCR-GB tree, which is the best resolved and most concordant with the remaining 11 concatenated trees. All 207 single gene trees were rooted and only bipartitions with at least 70% BS were considered informative. Finally, incongruence between the CR + NCR-GB tree and the nrDNA tree was visualized using the “tanglegram” function in Dendroscope v.3.8.2  region and a small single copy (SSC) region separated by two inverted repeat (IRa and IRb) regions (I. yuennanensis (Hand.-Mazz.) H. Hara] to 152,824 bp [I. ternifolius (D. Don) Kudô] (I. yuennanensis) to 83,640 bp (I. ternifolius), the SSC regions varied between 17,532 bp [I. megathyrsus (Diels) H. Hara] and 17,729 bp [I. lophanthoides var. graciliflorus (Benth.) H. Hara], and the IR regions ranged from 25,671 bp [I. phyllopodus (Diels) Kudô] to 25,759 bp [I. rugosus  Codd]. The overall GC content of the Isodon plastomes was similar and ranged from 37.5% in I. ramosissimus (Hook. f.) Codd to 37.7% in I. pharicus (Prain) Murata and I. adenanthus (Diels) Kudô  Kudô] . The LSCls) Kudô . Plastomonserved .The sequence characteristics of all 12 plastome data sets and the nrDNA data set were summarized in R2 values lower than 0.90 (three coding and 16 non-coding loci), the resolution of the CR-R2 tree was largely consistent with that of CR tree, but were slightly poorer in the NCR-R2 tree and CR + NCR-R2 tree compared with the NCR tree and CR + NCR tree, respectively. Likewise, the CR-slope tree was also consistent with the CR tree after removing eleven coding loci with slope values lower than 0.85. In contrast, with nearly half (61 out of 127) of the non-coding loci excluded, the NCR-slope tree had the lowest resolution among the 12 concatenated trees. Interestingly, the support value of the sister clade of Isodon formed by ten outgroup species was improved by ambiguously aligned regions and saturated loci being removed.All 12 plastome data sets yielded almost identical tree topologies at deep nodes and exhibited largely congruent relationships at shallow nodes with some differences or conflicts . For theIsodon was shown to be monophyletic (BS = 100%/PP = 1.00) and four major clades (Clades I–IV) were recovered within the genus. As the first diverging clade, Clade I (BS = 100%/PP = 1.00) consisted of 11 taxa which can be further divided into two strongly supported subclades. The first subclade comprised I. lophanthoides (Buch.-Ham. ex D. Don) H. Hara var. lophanthoides, I. lophanthoides var. graciliflorus, I. atroruber R.A. Clement, I. villosus Y.P. Chen and H. Peng, and I. scrophularioides  Murata, and the other subclade included I. oreophilus (Diels) A.J. Paton and Ryding, I. flavidus (Hand.-Mazz.) H. Hara. I. phyllopodus, and I. yuennanensis. The next split lineage was composed of the two African endemic species I. ramosissimus and I. schimperi (Vatke) J.K. Morton . Clade III consists of the two individuals of I. ternifolius (BS = 100%/PP = 1.00), which was further sister to the largest Clade IV (BS = 100%/PP = 1.00). Despite a few shallow nodes were collapsed into polytomies or weakly supported, most of the nodes within Clade IV were resolved with robust support. However, the internal branch lengths within Clade IV were extremely short. The single gene trees were largely unresolved, with some of the loci exhibiting conflicts with the concatenated data set  consisting of four major lineages: Clade I (BS = 96%/PP = 1.00), Clade II (BS = 100%/PP = 1.00), Clade III (BS = 100%/PP = 1.00), and Clade IV (BS = 72%/PP = 1.00). Even though the relationships within Clade IV of the nrDNA tree were almost unresolved with a large polytomy, the plastid-nuclear tanglegram demonstrated widespread discordances  with acute apex. Six different surface types of nutlets can be recognized within Isodon, including psilate, reticulate, reticulate-papillate, striate, cellular, and glandular (and pubescent) types (Isodon ternifolius (Clade III) has distinct nutlets with a striate surface, whereas most species of Clade IV possess nutlets with cellular surfaces. Nutlets with glandular or glandular and pubescent trichomes are only possessed by the remaining species of Clade IV.Nutlets of rk brown . All spet) types . Both thIsodon was aimed at resolving the phylogenetic ambiguities found by prior phylogenetic studies. Our study has increased taxonomic sampling to include all four sections and ten series, and provided additional geographic sampling to accommodate the distribution of Isodon, especially from HM. Our results recover four monophyletic clades (Clades I–IV) of Isodon species similar to Isodon with our extensive field investigation and morphological studies enables us to further discuss the relationships within each clade.This examination of infrageneric relationships for Isodon  belonging to the other subclade of Clade I . Invariable transitions between phyllotaxy such as these indicate that the current diagnosis of I. ternifolius cannot be properly applied, and must be adjusted to a more stable synapomorphic character. Our examination of nutlets  of tid tree , comparetid tree , strong tid tree . MoreoveIsodon, species of Clade IV are herbs or shrubs with a variety of leaves, inflorescences, and trichomes. It has been difficult to find out the synapomorphy for such a clade. Interestingly, herbal species are mostly distributed in eastern Asia and Japan, whereas the shrubby species, which account for a large proportion of Clade IV, are mainly accustomed to the dry valleys in HM. Two types of nutlet surface are restricted to Clade IV as revealed by our morphological study, with most species sharing the cellular nutlet ornamentation (I. tenuifolius (W.W. Sm.) Kudô] are herbs from the forest area in eastern Asia and Japan.As the most morphologically diverse group of entation . The remIsodon. Although potential sources of conflict such as selection and heteroplasmy  have been documented from many other taxa  H. Hara and I. brachythyrsus (C.Y. Wu and H.W. Li) H. Hara which receives high support (BS = 96%/PP = 1.00) in the nrDNA tree is in contrast with their significant differences in the morphology of inflorescence (spike-like panicles with bracts longer than cymes in I. interruptus vs. loose panicles with bracts shorter than cymes in I. brachythyrsus), calyx (tubular campanulate vs. broadly campanulate), and corolla , whereas I. interruptus shares all these features with I. muliensis (W.W. Sm.) Kudô  (I. gibbosus (C.Y. Wu and H.W. Li) H. Hara and I. lungshengensis (C.Y. Wu and H.W. Li) H. Hara, I. xerophilus (C.Y. Wu and H.W. Li) H. Hara and I. enanderianus (Hand.-Mazz.) H.W. Li, can also be supported by their shared morphology, habitat, and distribution. Plants of I. gibbosus and I. lungshengensis are perennial herbs usually grow at moist streamside in the bordering area of Guangxi, Hunan, and Guizhou Provinces, China, while I. enanderianus and I. xerophilus are shrubs restricted to the dry valleys in southern Yunnan Province  Kudô . The minructure) , the limructure) . For exaructure) , I. gibbProvince . Howeverrophilus .I. scrophularioides likely indicates a hybrid origin, as has been shown in Isodon that will require further study  and er study . Therefoer study . Moleculom Japan . Previouom Japan . However  of which consists of the largest portion of Isodon (85% of species). The tree topology at deep nodes largely corresponds with previous molecular phylogenetic studies of Isodon, whereas we further reveal that Clade II which consisting of the two African endemic species differs from the remaining Isodon species by the reticulate-papillate nutlet surfaces. Clade III is composed of individuals of I. ternifolius with both whorled and opposite phyllotaxy, while our morphological examination demonstrates that nutlets with acute and conspicuously trigonous apex is exclusive to all individuals of I. ternifolius. Phylogenetic relationships within Clade IV are much better resolved in the plastid tree than that in previous studies, but highly incongruent with the nrDNA tree and morphology and distribution. Species of Clade IV have cellular nutlet surface type and the distinct nutlets with trichomes, the latter type is restricted to species mostly from eastern Asia and Japan. Our results also suggest that multiple processes might have been involved in the evolutionary history of Isodon and plastome sequences fail to further resolve the shallow-level relationships within the genus. Large-scale molecular markers from the nuclear genome might contribute to a better understanding of the infrageneric relationships of the rapidly radiated Isodon.PRJNA863331, and the sequence alignments and resulting tree files were deposited in figshare at https://doi.org/10.6084/m9.figshare.20170727.The original contributions presented in this study are publicly available. The raw sequence data were deposited in the NCBI BioProject database under the accession number: Y-PC, L-MG, and C-LX conceived this study. Y-PC and FZ analyzed the data. Y-PC drafted the manuscript with contributions from other authors. All authors collected the materials, read, and approved the final manuscript.


Gait velocity also improved from baseline to during RAC and post-RAC in all participants, although this improvement was not significant after correcting for multiple comparisons. The changes in cadence and gait velocity were most pronounced in PSP. In addition, our exploratory analysis showed that the cadence in the suspected TAU group (PSP+CBS) showed a significant improvement from baseline to during RAC and post-RAC . This pilot study using short-term RAC in APD patients demonstrated improvements in cadence and velocity. There is an urgent need for effective gait rehabilitation modalities for patients with APD, and rhythmic cueing can be a practical and useful intervention to improve their gait pattern.Rhythmic auditory cueing (RAC) can improve gait parameters in neurological disorders such as Parkinson's disease and stroke. However, there is a lack of research on the effects of RAC in patients with atypical parkinsonian disorders (APD). Using a smartphone metronome application, we aimed to investigate the immediate effects of RAC in patients with clinically diagnosed APD, namely Progressive Supranuclear Palsy , Corticobasal Syndrome (CBS), Multiple System Atrophy (MSA), and Dementia with Lewy Bodies (DLB). A total of 46 APD participants  walked at their preferred pace for 2 min without any rhythmic auditory cueing (RAC). Participants then walked the same path for another 2 min with RAC set at a tempo 10% faster than the baseline cadence of each participant. After a 10–15-min break, participants walked the same path for another 2 min without RAC to observe for carryover effects. Gait parameters  were collected at baseline, during RAC, and post-RAC. There was a significant improvement in cadence in all participants from baseline to during RAC and post-RAC (corrected  A large body of literature supports the use of music and rhythm-based interventions to improve gait and aspects of quality of life in patients with neurological disorders . GaiRhythmic auditory stimulus (RAS) is a Neurologic Music Therapy (NMT) technique that utilizes an auditory rhythmic cue to entrain gait to a specific rhythm . RAS, asRAS is traditionally implemented by identifying the patient's baseline cadence first and then providing rhythmic cues using a metronome and/or live music at a faster or slower frequency  in order to improve the patient's gait. Cadence, gait velocity, and stride length are the commonly used parameters to monitor changes in a patient's gait , 8. All Since each patient has a unique preferred baseline cadence, RAS is implemented in an individualized manner for each patient instead of using a fixed metronome speed for all patients , 21. ResThe effects of RAS on patients with atypical parkinsonian disorders (APD) remain largely unexplored. This study aimed to test the effects of rhythmic auditory cueing (RAC) using a metronome on patients with APD. We use the term RAC rather than RAS in this paper because the intervention was not delivered by a professional who obtained a NMT certificate, but rather by a Physical Therapist trained in delivering RAC. We hypothesized that RAC would have similar short-term therapeutic effects in APDs as for patients with PD.The classification of atypical parkinsonism includes various movement disorders, each with unique phenotypes and pathologies . The disIn summary while there are studies describing the clinical benefits of these interventions in PD, they have largely remained unexplored in atypical parkinsonian disorders (APD), which involve early gait and balance impairment and a more rapid progression to disability and death. APD currently lack therapeutic options to target gait and balance, and based on the PD literature, music-based interventions for these disorders warrant exploration.Participants were recruited for this study from the Johns Hopkins Atypical Parkinsonism Center's multidisciplinary clinic between 2015 and 2019. This study included participants who had a diagnosis of PSP , CBS 2727, MSA , or DLB Written informed consent was obtained prior to participation in the study. The study protocol was approved by the Institutional Review Board at the Johns Hopkins Medical Institutions .via a free metronome phone application , which was set to a tempo 10% faster than the baseline cadence of each participant. After a 10–15-min break, participants walked the same path for another 2 min without RAC. The physical therapist carefully counted the number of steps for participants with research assistants who timed the trials using a smart phone timer and served as a second step counter. The path walked was identical for all participants and its length, together with the number of times that length was covered by participants, was used to calculate stride length and gait velocity . Gait parameters were measured at baseline, during RAC, and post-RAC. All RAC interventions were performed by the physical therapists who were part of the study team  during Multi-Disciplinary Atypical Parkinsonism Clinic visits at the Johns Hopkins Atypical Parkinsonism Center. Each participant was exposed to the same indoor clinic environment and walked unaided  along the same walking path at baseline, during, and after RAC. Participants wore comfortable clothing and shoes and walked along a flat, well-lit unobstructed surface. Every effort was made to minimize outside noise. All of the above parameters were kept constant for all three walking trials.Participants were instructed to walk at their preferred pace for 2 min without any rhythmic auditory cues to collect baseline gait parameters . Stride length was defined as the distance between successive points of initial contact of the same foot for two consecutive steps . The parp < 0.05 for all analyses.Shapiro-Wilk tests and Levene tests were performed to test for normal distributions and equal variances, respectively, among all data groups. Non-parametric statistical tests were applied to non-normal data or data with unequal variance, all statistical analyses to ensure adequate stringency: The Wilcoxon test and the Mann-Whitney U test. Statistical significance was set to The effects of RAC on the cadence, stride length, and gait velocity for the entire cohort and each subgroup were determined using the Wilcoxon test, to assess gait parameter change between baseline (pre-RAC) vs. during RAC, pre-RAC vs. post-RAC, and during-RAC vs. post-RAC. The Mann-Whitney U test was utilized to assess differences between subgroups in each gait parameter. A priori analyses were performed using the entire subject cohort, while subgroup analyses were considered exploratory because of unequal subgroup size and lack of power to detect statistically significant differences after correcting for multiple comparisons. Statistical analysis was performed in Python (version 3.9).p-value*/] . To obtain the effect size for each comparison, Cohen's d was calculated by dividing the difference between two groups' means by their pooled standard deviation. The sample size necessary to achieve a power of 0.8 was obtained by performing power calculations using Cohen's d value for each comparison and an alpha level of 0.05 . For our subgroup analyses, we report p-values before correction due to the small and unequal numbers of participants in subgroups  and the exploratory nature of these analyses. All corrected p-values are reported in Correction for multiple comparisons was performed, setting a False Discovery Rate (FDR) by applying the Benjamini-Hochberg method [-value)]   age of 70.17 (7.15), disease duration 4.45 (2.31) years, and total MoCA scores of 20.56 (5.52) see . Of notep = 0.001 (corr. p =0.009). This improvement was retained post-RAC [94.73 (21.12)], p = 0.002 (corr. p = 0.009) (There was a significant improvement in cadence (steps/min) for all participants when comparing baseline [90.40 (19.66)] and during-RAC [94.68 (22.23)], = 0.009) .p = 0.003; baseline vs. post-RAC . In PSP, PSP-RS participants also showed significant differences between baseline (83.82 (21.58) vs. during RAC [90.03 (22.96)], p = 0.017 as well as baseline vs. post-RAC [89.15 (19.55)], p = 0.021. A trend toward improvement in cadence was seen in PSP-non-RS between baseline [96.00 (15.17)] and during RAC [102.38 (17.48)], p = 0.050 and baseline vs. post-RAC [103.44 (15.16)], p = 0.050. CBS participants also showed a significant improvement in cadence between baseline [94.00 (18.83)] and post- RAC [101.33 (21.96)], p = 0.039 and trend-level improvement from baseline to during RAC [99.72 (22.01)], p = 0.068. There was no significant cadence improvement for MSA or DLB groups ] vs. during RAC [93.98 (21.80)], B groups .p = 0.024. This improvement was also retained during retesting 15 min later post-RAC [45.69 (23.28)], p = 0.039 (A significant improvement in gait velocity (m/min) was seen in all participants between baseline [Mean (standard deviation) = 42.49 (17.19)] and with-RAC [46.05 (21.96)],  = 0.039 .p = 0.051) for baseline gait velocity and a significant difference in gait velocity for during-RAC and post-RAC . There was a significant improvement in gait velocity in PSP participants between baseline [43.70 (14.26)] vs. during RAC [49.66 (24.00)], p = 0.022. PSP-nonRS showed significant improvement in velocity between both baseline [51.97 (14.05)] vs. during-RAC [66.05 (34.22)], p = 0.018 and baseline vs. post-RAC [65.65 (31.20)], p = 0.028. MSA participants also demonstrated improvement in gait velocity between both baseline [34.59 (18.74)] vs. post-RAC [41.05 (18.57)], p = 0.018, as well as between during RAC [38.79 (18.43)]vs. post-RAC, p = 0.023. There was a trend of improvement between baseline vs. during-RAC, p = 0.055 . A significant difference between PSP-RS and PSP-nonRS in post-RAC, p = 0.013 was also observed.No significant improvement in stride length (cm) was seen in the assessment of the entire cohort . Howeverp = 0.039. MSA demonstrated a significant improvement in their stride length between baseline [79.78 (34.38)] and post-RAC [97.27(32.19)], p = 0.039 as well as between during RAC [91.97(33.28)] vs. post-RAC, p = 0.008. PSP-RS and CBS participants showed a decreasing trend (PSP-RS: during RAC [104.28(37.56)] vs. post-RAC [94.12 (30.70)], p = 0.057; CBS: baseline [84.87 (32.63)] vs. during-RAC [78.57 (28.81)], p = 0.050 ] and post-RAC [98.97 (27.64)],  = 0.050 .p = 0.00039 (corr. p = 0.004), and between baseline vs. post-RAC [95.74 (19.91)], p = 0.00024 (corr. p = 0.004). The TAU cohort also experienced significant improvements in gait velocity between baseline [43.11 (15.55)] vs. during RAC [47.46 (23.29)], p = 0.038. In contrast, the SYN cohort did not demonstrate significant improvements in cadence or gait velocity (in The TAU (PSP + CBS) cohort experienced significant improvements in cadence between baseline [89.38 (19.82)] vs. during RAC [95.50 (21.67)], ocity in .p = 0.023], and delta baseline vs. post-RAC between TAU and SYN [TAU: 6.35 (8.17) vs. SYN (−1.42 (8.74)], p = 0.007. When comparing changes in stride length between baseline vs. during RAC, the SYN cohort experienced an average improvement of 13.21 (22.32) cm while the TAU cohort only experienced a 2.53 (16.81) cm increase in stride length, p = 0.056. When comparing the change in stride length between baseline vs. post-RAC, the TAU cohort experienced an average shortening in stride length of −3.18 (15.16) cm, while the SYN cohort experienced an average lengthening in stride length of 11.96 (21.35) cm, p = 0.023 ] vs. SYN [−0.91 (10.41), 0.023 in .d = 0.204, and power calculation revealed that a total sample size of 199 participants would be necessary to yield a power of 0.8 to detect differences at alpha of 0.05. A total sample size of 251 participants would be necessary to yield a power of 0.8 for a comparison of change in gait velocity between baseline and during-RAC (d = 0.182) (in The comparisons between baseline and during-RAC for cadence had effect size of .182) in .This pilot study aimed to determine whether rhythmic cues using a metronome improve gait parameters  in patients with atypical parkinsonian disorders. Our data demonstrate a significant improvement in cadence and velocity in all the participants receiving RAC, an improvement which was retained 10–15 min following RAC intervention. In particular, cadence improvements across all participants remained statistically significant between baseline and during RAC as well as between baseline vs. post-RAC after correcting for multiple comparisons.Prior studies have demonstrated the effectiveness of RAS in improving gait parameters in neurological movement disorders –22, 24. Several mechanisms have been proposed for the therapeutic effects of rhythmic cues in patients with movement disorders, particularly PD. Due to impairments of the basal ganglia and loss of frontostriatal connectivity, patients with PD experience a loss of rhythm perception and timing, resulting in various gait abnormalities in PD, including freezing of gait, decreased stride length and gait velocity, and a rhythmically unstable walking pace . Externavia these mechanisms since APD and PD share overlapping affected neural pathways and clinical features, including gait slowing and freezing . It is therefore possible that differences in improvement between diagnostic subgroups may be a function of low sample size and requires validation in a future prospective study. For example, DLB, which is the smallest group (n = 4), showed less response to RAC. The degree of within-diagnostic-group variance was also high, also potentially contributing to the difference in response to RAC between subgroups. The standard deviation and standard error of the mean of DLB were larger than those of other groups, such as PSP-RS. Finally, multitasking difficulty in patients with DLB  due to more severe cognitive dysfunction than in other groups may also contribute to differences in response to RAC.Furthermore, we found different patterns of improvement in gait parameters for each diagnostic subgroup. It is worth noting that while the group that experienced the greatest improvements was PSP-RS, this was the largest subgroup  and whether it can help prevent falls are key questions for future investigation. In addition, given the consideration of each APD individual's functional needs and safety level, customizing the rhythmic cues may improve response and increase intervention safety. The decision whether the patient's gait velocity (via cadence) needs to be accelerated or decelerated can be made jointly by physical therapists, patients, and caregivers. For a functional transition into patients' daily life, a neurologic music therapist may co-facilitate rhythmic interventions with physical therapists by adding live music, advanced gait training , and pre-gait exercises through musical patterns .Several limitations of this study should be considered. Though clinical diagnostic criteria were stringently applied, autopsy confirmation of clinical diagnosis was not available for much of the cohort. A larger sample size is required to validate the efficacy of RAC in APD patients. Larger diagnostic subgroup samples would enable us to confirm differences in responses to RAC and conduct cluster analyses to determine unique characteristic of each subgroup that may aid development of more tailored RAC interventions. Furthermore, the results of this study provide guidance concerning the effect size and calculation of sample sizes required for future controlled clinical trials of RAC in patients with APD. Until that time, we emphasize that the subgroup analyses reported here should be considered exploratory. Additionally, this study investigated only the immediate effects of rhythmic cues by giving a 10% increase over baseline cadence for all participants and long-term effects of cueing were not assessed. Use of control groups  and longer follow up with repeated RAC interventions in APD are warranted, as is the comparison to natural history data. Freezing of gait was not formally assessed during the study and could have impacted our outcomes in ways we did not measure. Another limitation of the study involves the possibility of learning effects across trials, since the trials occurred in close temporal proximity. A cross-over study design would be warranted to account for learning effects as an influence on participants' gait performance. We also acknowledge that despite our best efforts minor errors in step count and distance recording could have occurred, though it is doubtful that they would impact our results. A motion capture system or electronic walk system such as GAITRiteThe present study is the first to our knowledge to demonstrate that short-term RAC may improve relevant gait parameters in patients with APD. The use of rhythmic cues and/or music in the context of an isochronous rhythmic stimulus may be an easily accessible and effective way to improve functional gait performance in patients with APD. While our study was limited by sample heterogeneity and small subgroup sample size, it paves the way for larger trials of rhythmic cueing for patients with atypical parkinsonism.The raw data supporting the conclusions of this article will be made available by the authors upon reasonable request, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board at the Johns Hopkins Medical Institutions . The patients/participants provided their written informed consent to participate in this study.AP: research project: conception, organization, and execution; statistical analysis: design, review, and critique; manuscript preparation: writing of the first draft, review, and critique. GD: statistical analysis: design, execution, review, and critique; manuscript preparation: writing of the first draft, review, and critique. KK: statistical analysis: execution, review, and critique; manuscript preparation: writing of the first draft, review, and critique. BT: statistical analysis: design, execution, review, and critique; manuscript preparation: review and critique. RP, S-KH, and AM: research project: conception, organization, and execution; manuscript preparation: review and critique. JB: research project: conception; statistical analysis: review and critique; manuscript preparation: review and critique. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Herein, we report a case of chylothorax following total thymectomy. A 46-year-old woman having an anterior mediastinal tumor underwent a thymectomy via median sternotomy. Seven days after surgery, there was no massive pleural effusion. However, on post-operative day 17, a right massive pleural effusion was detected, and it was diagnosed as chylothorax. She was successfully treated with conservative therapy. Chylothorax following thymectomy is a very rare complication. The majority of chylothorax after thoracic surgery might occur following esophagectomy, ligation of patent ductus arteriosus, aortic surgery or pulmonary resection with mediastinal lymph node dissection . ChylothHerein, we report a case of chylothorax as a complication following transsternal total thymectomy.A 46-year-old woman presented to a previous hospital with an abnormal shadow on a chest X-ray. Computed tomography (CT) revealed an anterior mediastinal mass lesion with its largest diameter of 6.6 cm . TherefoUnder general anesthesia, she underwent total thymectomy via median sternotomy. Her post-operative course was uneventful and there was no visible chyle leakage from the chest drainage tube. On post-operative day 2, the chest drainage tube was removed. The chest X-ray on post-operative day 7 revealed no massive pleural effusion , and sheOn post-operative day 17, she visited our hospital for a routine follow-up, and her chest X-ray revealed a massive right pleural effusion . TherefoFour days after fasting, we resumed the normal diet and pleural fluid changed to slightly milky again. To specifically identify the location of and reduce the chyle leakage, lipiodol lymphography was attempted in the supine position through the right inguinal lymph nodes. The lipiodol lymphography detected the suspicious location of the chyle leakage . After tChylothorax might occur due to a tear or rupture of the thoracic duct or its branches . Among aet al. reviewed 11 cases of chylothorax following thymectomy reported from 1994 to 2017 [et al. [There are only a limited number of previous reports of chylothorax as a complication following thymectomy . Zhang e to 2017 . They su to 2017 . Further to 2017 . In term to 2017 . This la to 2017  have bee [et al. . Among t [et al. , 7, but  [et al. .Conservative management would be the first choice and ideal intervention for post-operative chylothorax patients . RecentlIn conclusion, we reported a rare case of chylothorax following transsternal total thymectomy. Thoracic surgeons should recognize this rare post-operative complication.None declared.None.