RESUMO
Pseudoroegneria is a small genus of the Triticeae tribe; its St genome is present in over half of allopolyploid Triticeae species. The high molecular weight (HMW) subunits of glutenin (GS) encoded by the St genome are not well described. In this paper, we report the characterization of fourteen alleles of HMW-GS genes from the two species Pd. spicata and Pd. strigosa. Analysis shows that all fourteen sequences possess a typical primary structure shared by other known HMW-GS, but with some unique modifications. All fourteen Glu-St1 alleles are significantly smaller than normal Glu-1 genes due to fewer repeat motifs in a repetitive region with no indication of large deletion in other conserved regions. Thus, the small size is a common feature of HMW-GS encoded by Glu-St1 loci of Pseudoroegneria species. Sequence analysis indicated that all fourteen Glu-St1 alleles were intermediate type between x- and y-type, which represent an intermediate stage in the evolutionary divergence of x- and y-type subunits.
Assuntos
DNA de Plantas/genética , Glutens/genética , Proteínas de Plantas/genética , Poaceae/genética , Alelos , Sequência de Aminoácidos , DNA de Plantas/química , DNA de Plantas/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Glutens/metabolismo , Dados de Sequência Molecular , Peso Molecular , Filogenia , Proteínas de Plantas/metabolismo , Poaceae/classificação , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Plants have a profound capacity to regenerate organs from differentiated somatic tissues, based on which propagating plants in vitro was made possible. Beside its use in biotechnology, in vitro shoot regeneration is also an important system to study de novo organogenesis. Phytohormones and transcription factor WUSCHEL (WUS) play critical roles in this process but whether and how epigenetic modifications are involved is unknown. Here, we report that epigenetic marks of DNA methylation and histone modifications regulate de novo shoot regeneration of Arabidopsis through modulating WUS expression and auxin signaling. First, functional loss of key epigenetic genes-including METHYLTRANSFERASE1 (MET1) encoding for DNA methyltransferase, KRYPTONITE (KYP) for the histone 3 lysine 9 (H3K9) methyltransferase, JMJ14 for the histone 3 lysine 4 (H3K4) demethylase, and HAC1 for the histone acetyltransferase-resulted in altered WUS expression and developmental rates of regenerated shoots in vitro. Second, we showed that regulatory regions of WUS were developmentally regulated by both DNA methylation and histone modifications through bisulfite sequencing and chromatin immunoprecipitation. Third, DNA methylation in the regulatory regions of WUS was lost in the met1 mutant, thus leading to increased WUS expression and its localization. Fourth, we did a genome-wide transcriptional analysis and found out that some of differentially expressed genes between wild type and met1 were involved in signal transduction of the phytohormone auxin. We verified that the increased expression of AUXIN RESPONSE FACTOR3 (ARF3) in met1 indeed was due to DNA demethylation, suggesting DNA methylation regulates de novo shoot regeneration by modulating auxin signaling. We propose that DNA methylation and histone modifications regulate de novo shoot regeneration by modulating WUS expression and auxin signaling. The study demonstrates that, although molecular components involved in organogenesis are divergently evolved in plants and animals, epigenetic modifications play an evolutionarily convergent role in this process.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Ácidos Indolacéticos/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Proteínas de Homeodomínio/metabolismo , Mutação , Transdução de SinaisRESUMO
Protein arginine methylation plays important roles in diverse biological processes, but its role in regulating shoot regeneration remains elusive. In this study, we characterized the function of the protein arginine methyltransferase AtPRMT5 during de novo shoot regeneration in Arabidopsis. AtPRMT5 encodes a type II protein arginine methyltransferase that methylates proteins, including histones and RNA splicing factors. The frequency of shoot regeneration and the number of shoots per callus were decreased in the atprmt5 mutant compared with those in the wild type. Chromatin immunoprecipitation analysis revealed that AtPRMT5 targets KIP-RELATED PROTEINs (KRPs), which encode the cyclin-dependent kinase inhibitors that repress the cell cycle. During shoot regeneration, the KRP transcript level increased in the atprmt5 mutant, which resulted from reduced histone H4R3 methylation in the KRP promoter. Overexpression of KRP significantly reduced the frequency of shoot regeneration and shoot number per callus. Furthermore, abnormal pre-mRNA splicing in the gene RELATED TO KPC1 (RKP), which encodes an ubiquitin E3 ligase, was detected in the atprmt5 mutant. RKP functions in regulating KRP protein degradation, and mutation in RKP inhibited shoot regeneration. Thus, AtPRMT5 regulated shoot regeneration through histone modification-mediated KRP transcription and RKP pre-mRNA splicing. Our findings provide new insights into the function of protein arginine methylation in de novo shoot regeneration.