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DNA methylation is a conserved epigenetic modification that is important for gene regulation and genome stability. Aberrant patterns of DNA methylation can lead to plant developmental abnormalities. A specific DNA methylation state is an outcome of dynamic regulation by de novo methylation, maintenance of methylation and active demethylation, which are catalysed by various enzymes that are targeted by distinct regulatory pathways. In this Review, we discuss DNA methylation in plants, including methylating and demethylating enzymes and regulatory factors, and the coordination of methylation and demethylation activities by a so-called methylstat mechanism; the functions of DNA methylation in regulating transposon silencing, gene expression and chromosome interactions; the roles of DNA methylation in plant development; and the involvement of DNA methylation in plant responses to biotic and abiotic stress conditions.

Plants cannot move, so they must endure abiotic stresses such as drought, salinity and extreme temperatures. These stressors greatly limit the distribution of plants, alter their growth and development, and reduce crop productivity. Recent progress in our understanding of the molecular mechanisms underlying the responses of plants to abiotic stresses emphasizes their multilevel nature; multiple processes are involved, including sensing, signalling, transcription, transcript processing, translation and post-translational protein modifications. This improved knowledge can be used to boost crop productivity and agricultural sustainability through genetic, chemical and microbial approaches.In this Review, Zhang et al. summarize our current understanding of the molecular mechanisms underlying the responses of plants to abiotic stresses, and how this knowledge can be used to improve crop resilience through genetic, chemical and microbial approaches.

Summary Base editing is a novel genome editing strategy that enables irreversible base conversion at target loci without the need for double stranded break induction or homology‐directed repair. Here, we developed new adenine and cytosine base editors with engineered SpCas9 and SaCas9 variants that substantially expand the targetable sites in the rice genome. These new base editors can edit endogenous genes in the rice genome with various efficiencies. Moreover, we show that adenine and cytosine base editing can be simultaneously executed in rice. The new base editors described here will be useful in rice functional genomics research and will advance precision molecular breeding in crops.

Homologous recombination-based gene targeting is a powerful tool for precise genome modification and has been widely used in organisms ranging from yeast to higher organisms such as Drosophila and mouse. However, gene targeting in higher plants, including the most widely used model plant Arabidopsis thaliana , remains challenging. Here we report a sequential transformation method for gene targeting in Arabidopsis . We find that parental lines expressing the bacterial endonuclease Cas9 from the egg cell- and early embryo-specific DD45 gene promoter can improve the frequency of single-guide RNA-targeted gene knock-ins and sequence replacements via homologous recombination at several endogenous sites in the Arabidopsis genome. These heritable gene targeting can be identified by regular PCR. Our approach enables routine and fine manipulation of the Arabidopsis genome. Efficient gene targeting in higher plants remains challenging. Here, the authors develop a sequential transformation method for CRISPR/Cas9-mediated gene targeting in Arabidopsis and demonstrate its functionality at five genomic sites in two endogenous loci.

Previous studies on base editing in dicots showed that improvement of nCas9 expression level could significantly increase the editing efficiency (Kang et al., 2018). [...]we sought to optimize pCXPE01 to improve editing efficiency by increasing nCas9‐MMLV expression level. According to the sequencing results, we detected desired edits at two genes, ALS2 and PDS1. [...]no plant phenotype results were reported in these studies. [...]our prime‐edited T0 tomato plants were chimeras and did not display any obvious phenotypes (Figure 1e, g and h). [...]for both monocots and dicots, assessment of the utility of prime editing awaits future analysis of large populations of edited lines and their off‐springs.

A major problem facing humanity is that our numbers are growing but the availability of land and fresh water for agriculture is not. This problem is being exacerbated by climate change-induced increases in drought, and other abiotic stresses. Stress-resistant crops are needed to ensure yield stability under stress conditions and to minimize the environmental impacts of crop production. Evolution has created thousands of species of naturally stress-resistant plants (NSRPs), some of which have already been subjected to human domestication and are considered minor crops. Broader cultivation of these minor crops will diversify plant agriculture and the human diet, and will therefore help improve global food security and human health. More research should be directed toward understanding and utilizing NSRPs. Technologies are now available that will enable researchers to rapidly improve the genetics of NSRPs, with the goal of increasing NSRP productivity while retaining NSRP stress resistance and nutritional value. There are multiple strategies to fortify crop nutrition and support global food security and sustainable agriculture. Here the authors propose to increase the diversity of crops by devoting more efforts to studying minor crops that are naturally stress resistant.

Background Recently, DNA methylation was proposed to regulate fleshy fruit ripening. Fleshy fruits can be distinguished by their ripening process as climacteric fruits, such as tomatoes, or non-climacteric fruits, such as strawberries. Tomatoes undergo a global decrease in DNA methylation during ripening, due to increased expression of a DNA demethylase gene. The dynamics and biological relevance of DNA methylation during the ripening of non-climacteric fruits are unknown. Results Here, we generate single-base resolution maps of the DNA methylome in immature and ripe strawberry. We observe an overall loss of DNA methylation during strawberry fruit ripening. Thus, ripening-induced DNA hypomethylation occurs not only in climacteric fruit, but also in non-climacteric fruit. Application of a DNA methylation inhibitor causes an early ripening phenotype, suggesting that DNA hypomethylation is important for strawberry fruit ripening. The mechanisms underlying DNA hypomethylation during the ripening of tomato and strawberry are distinct. Unlike in tomatoes, DNA demethylase genes are not upregulated during the ripening of strawberries. Instead, genes involved in RNA-directed DNA methylation are downregulated during strawberry ripening. Further, ripening-induced DNA hypomethylation is associated with decreased siRNA levels, consistent with reduced RdDM activity. Therefore, we propose that a downregulation of RdDM contributes to DNA hypomethylation during strawberry ripening. Conclusions Our findings provide new insight into the DNA methylation dynamics during the ripening of non-climacteric fruit and suggest a novel function of RdDM in regulating an important process in plant development.

The widespread agricultural problem of pre-harvest sprouting (PHS) could potentially be overcome by improving seed dormancy. Here, we report that miR156, an important grain yield regulator, also controls seed dormancy in rice. We found that mutations in one MIR156 subfamily enhance seed dormancy and suppress PHS with negligible effects on shoot architecture and grain size, whereas mutations in another MIR156 subfamily modify shoot architecture and increase grain size but have minimal effects on seed dormancy. Mechanistically, mir156 mutations enhance seed dormancy by suppressing the gibberellin (GA) pathway through de-represssion of the miR156 target gene Ideal Plant Architecture 1 ( IPA1 ), which directly regulates multiple genes in the GA pathway. These results provide an effective method to suppress PHS without compromising productivity, and will facilitate breeding elite crop varieties with ideal plant architectures. Pre-harvest sprouting reduces the yield of agriculturally important crops such as rice. Here, the authors show that mutating specific members of the MIR156 gene family can suppress pre-harvest sprouting in rice without negative effects on plant architecture, suggesting a practical route to elite crop varieties.

A contribution of DNA methylation to defense against invading nucleic acids and maintenance of genome integrity is uncontested; however, our understanding of the extent of involvement of this epigenetic mark in genome-wide gene regulation and plant developmental control is incomplete. Here, we knock out all five known DNA methyltransferases in Arabidopsis , generating DNA methylation-free plants. This quintuple mutant exhibits a suite of developmental defects, unequivocally demonstrating that DNA methylation is essential for multiple aspects of plant development. We show that CG methylation and non-CG methylation are required for a plethora of biological processes, including pavement cell shape, endoreduplication, cell death, flowering, trichome morphology, vasculature and meristem development, and root cell fate determination. Moreover, we find that DNA methylation has a strong dose-dependent effect on gene expression and repression of transposable elements. Taken together, our results demonstrate that DNA methylation is dispensable for Arabidopsis survival but essential for the proper regulation of multiple biological processes. Our understanding of the extent of involvement of DNA methylation in genome-wide gene regulation and plant developmental control is incomplete. Here, the authors knock out all five known DNA methyltransferases and show the developmental and gene expression changes in the DNA methylation-free Arabidopsis plants.

Abscisic acid (ABA) is an important phytohormone regulating seed dormancy, germination, seedling growth, and plant transpiration. We report here an Arabidopsis triple mutant that is disrupted in 3 SNF1-related protein kinase subfamily 2 (SnRK2s) and nearly completely insensitive to ABA. These SnRK2s, SnRK2.2, SnRK2.3, and SnRK2.6 (also known as OST1), are activated by ABA and can phosphorylate the ABA-responsive element binding factor family of b-ZIP transcription factors, which are important for the activation of ABA-responsive genes. Although stomatal regulation of snrk2.6 and seed germination and seedling growth of the snrk2.2/2.3 double mutant are insensitive to ABA, ABA responses are still present in these mutants, and the growth and reproduction of these mutants are not very different from those of the WT. In contrast, the snrk2.2/2.3/2.6 triple mutant grows poorly and produces few seeds. The triple mutant plants lose water extremely fast when ambient humidity is not high. Even on 50 μM ABA, the triple mutant can germinate and grow, whereas the most insensitive known mutants cannot develop on 10 μM ABA. In-gel kinase assays showed that all ABA-activated protein kinase activities are eliminated in the triple mutant. Also, the expression of ABA-induced genes examined is completely blocked in the triple mutant. These results demonstrate that the protein kinases SnRK2.2, SnRK2.3, and SnRK2.6 have redundant functions, and suggest that ABA signaling is critical for plant growth and reproduction.