DNA cytosine methylation dynamics and functional roles in horticultural crops

Methylation of cytosine is a conserved epigenetic modification that maintains the dynamic balance of methylation in plants under the regulation of methyltransferases and demethylases.In recent years,the study of DNA methylation in regulating the growth and development of plants and animals has become a key area of research.This review describes the regulatory mechanisms of DNA cytosine methylation in plants.It summarizes studies on epigenetic modifications of DNA methylation in fruit ripening,development,senescence,plant height,organ size,and under biotic and abiotic stresses in horticultural crops.The review provides a theoretical basis for understanding the mechanisms of DNA methylation and their relevance to breeding,genetic improvement,research,innovation,and exploitation of new cultivars of horticultural crops.

Breeding exceptionally fragrant soybeans for soy milk with strong aroma

Soybean(Glycine max(L.)Merr.)is a major source of vegetable protein and oil in human diet and animal nutrition.Soybean seeds have been extensively used in various food products and snacks.Taste quality,particularly the aroma,affects cooking and eating,and ultimately influences consumer preference.Soy milk is particularly popular in China and has been gaining popularity in many other countries in the world.

Knockout of miR396 genes increases seed size and yield in soybean

Yield improvement has long been an important task for soybean breeding in the world in order to meet the increasing demand for food and animal feed.mi R396 genes have been shown to negatively regulate grain size in rice,but whether mi R396 family members may function in a similar manner in soybean is unknown.Here,we generated eight soybean mutants harboring different combinations of homozygous mutations in the six soybean mi R396genes through genome editing with clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease(Cas)12SF01 in the elite soybean cultivar Zhonghuang 302(ZH302).Four triple mutants(mir396aci,mir396acd,mir396adf,and mir396cdf),two quadruple mutants(mir396-abcd and mir396acfi),and two quintuple mutants(mir396abcdf and mir396bcdfi)were characterized.We found that plants of all the mir396 mutants produced larger seeds compared to ZH302 plants.Field tests showed that mir396adf and mir396cdf plants have significantly increased yield in growth zones with relatively high latitude which are suited for ZH302 and moderately increased yield in lower latitude.In contrast,mir396abcdf and mir396bcdfiplants have increased plant height and decreased yield in growth zones with relatively high latitude due to lodging issues,but they are suited for low latitude growth zones with increased yield without lodging problems.Taken together,our study demonstrated that loss-of-function of mi R396 genes leads to significantly enlarged seed size and increased yield in soybean,providing valuable germplasms for breeding high-yield soybean.

A group of SUVH methyl-DNA binding proteins regulate expression of the DNA demethylase ROS_1 in Arabidopsis

DNA methylation is typically regarded as a repressive epigenetic marker for gene expression.Genome-wide DNA methylation patterns in plants are dynamically regulated by the opposing activities of DNA methylation and demethylation reactions. In Arabidopsis, a DNA methylation monitoring sequence(MEMS) in the promoter of the DNA demethylase gene ROS_1 functions as a methylstat that senses these opposing activities and regulates genome DNA methylation levels by adjusting ROS_1 expression. How DNA methylation in the MEMS region promotes ROS_1 expression is not known. Here, we show that several Su(var)3-9 homologs(SUVHs) can sense DNA methylation levels at the MEMS region and function redundantly to promote ROS_1 expression. The SUVHs bind to the MEMS region, and the extent of binding is correlated with the methylation level of the MEMS.Mutations in the SUVHs lead to decreased ROS_1 expression, causing DNA hypermethylation at more than 1,000 genomic regions. Thus, the SUVHs function to mediate the activation of gene transcription by DNA methylation.

Expanding the scope of CRISPR/Cas9-mediated genome editing in plants using an xCas9 and Cas9-NG hybrid^FA

The widely used Streptococcus pyogenes Cas9(SpCas9)requires NGG as a protospacer adjacent motif(PAM)for genome editing.Although Sp Cas9 is a powerful genome-editing tool,its use has been limited on the targetable genomic locus lacking NGG PAM.The Sp Cas9 variants xCas9 and Cas9-NG have been developed to recognize NG,GAA,and GAT PAMs in human cel s.Here,we show that xCas9 cannot recognize NG PAMs in tomato,and Cas9-NG can recognize some of our tested NG PAMs in the tomato and Arabidopsis genomes.In addition,we engineered BrSp Cas9(XNG-Cas9)based on mutations from both xCas9 and Cas9-NG,and found that XNG-Cas9 can efficiently mutagenize endogenous target sites with NG,GAG,GAA,and GAT PAMs in the tomato or Arabidopsis genomes.The PAM compatibility of XNG-Cas9 is the broadest reported to date among Cas9 s(SpCas9 and Cas9-NG)active in plant.

A novel protein complex that regulates active DNA demethylation in Arabidopsis

Active DNA demethylation is critical for altering DNA methylation patterns and regulating gene expression.The 5-methylcytosine DNA glycosylase/lyase ROS1 initiates a base-excision repair pathway for active DNA demethylation and is required for the prevention of DNA hypermethylation at 1000 s of genomic regions in Arabidopsis.How ROS1 is regulated and targeted to specific genomic regions is not well understood.Here,we report the discovery of an Arabidopsis protein complex that contains ROS1,regulates ROS1 gene expression,and likely targets the ROS1 protein to specific genomic regions.ROS1 physically interacts with a WD40 domain protein(RWD40),which in turn interacts with a methyl-DNA binding protein(RMB1)as well as with a zinc finger and homeobox domain protein(RHD1).RMB1 binds to DNA that is methylated in any sequence context,and this binding is necessary for its function in vivo.Loss-of-function mutations in RWD40,RMB1,or RHD1 cause DNA hypermethylation at several tested genomic regions independently of the known ROS1 regulator IDM1.Because the hypermethylated genomic regions include the DNA methylation monitoring sequence in the ROS1 promoter,plants mutated in RWD40,RMB1,or RHD1 show increased ROS1 expression.Importantly,ROS1 binding to the ROS1 promoter requires RWD40,RMB1,and RHD1,suggesting that this complex dictates ROS1 targeting to this locus.Our results demonstrate that ROS1 forms a protein complex with RWD40,RMB1,and RHD1,and that this novel complex regulates active DNA demethylation at several endogenous loci in Arabidopsis.