Retrospective and perspective of plant epigenetics in China

Epigenetics refers to the study of heritable changes in gene function that do not involve changes in the DNA sequence. Such effects on cellular and physiological phenotypic traits may result from external or environmental factors or be part of normal developmental program. In eukaryotes, DNA wraps on a histone octamer(two copies of H2A, H2B, H3 and H4) to form nucleosome, the fundamental unit of chromatin. The structure of chromatin is subjected to a dynamic regulation through multiple epigenetic mechanisms, including DNA methylation, histone posttranslational modifications(PTMs), chromatin remodeling and noncoding RNAs. As conserved regulatory mechanisms in gene expression, epigenetic mechanisms participate in almost all the important biological processes ranging from basal development to environmental response. Importantly, all of the major epigenetic mechanisms in mammalians also occur in plants. Plant studies have provided numerous important contributions to the epigenetic research. For example, gene imprinting, a mechanism of parental allele-specific gene expression, was firstly observed in maize; evidence of paramutation, an epigenetic phenomenon that one allele acts in a single locus to induce a heritable change in the other allele, was firstly reported in maize and tomato.Moreover, some unique epigenetic mechanisms have been evolved in plants. For example, the 24-nt siRNA-involved RNA-directed DNA methylation(RdDM) pathway is plant-specific because of the involvements of two plant-specific DNA-dependent RNA polymerases, Pol IV and Pol V. A thorough study of epigenetic mechanisms is of great significance to improve crop agronomic traits and environmental adaptability. In this review, we make a brief summary of important progress achieved in plant epigenetics field in China over the past several decades and give a brief outlook on future research prospects.We focus our review on DNA methylation and histone PTMs, the two most important aspects of epigenetic mechanisms.

EXPLICIT-Kinase:A gene expression predictor for dissecting the functions of the Arabidopsis kinome

Protein kinases regulate virtually all cellular processes,but it remains challenging to determine the functions of all protein kinases,collectively called the“kinome”,in any species.We developed a computational approach called EXPLICIT-Kinase to predict the functions of the Arabidopsis kinome.Because the activities of many kinases can be regulated transcriptionally,their gene expression patterns provide clues to their functions.A universal gene expression predictor for Arabidopsis was constructed to predict the expression of 30,172 nonkinase genes based on the expression of 994 kinases.The model reconstituted highly accurate transcriptomes for diverse Arabidopsis samples.It identified the significant kinases as predictor kinases for predicting the expression of Arabidopsis genes and pathways.Strikingly,these predictor kinases were often regulators of related pathways,as exemplified by those involved in cytokinesis,tissue development,and stress responses.Comparative analyses revealed that portions of these predictor kinases are shared and conserved between Arabidopsis and maize.As an example,we identified a conserved predictor kinase,RAF6,from a stomatal movement module.We verified that RAF6 regulates stomatal closure.It can directly interact with SLAC1,a key anion channel for stomatal closure,and modulate its channel activity.Our approach enables a systematic dissection of the functions of the Arabidopsis kinome.

Modern phenomics to empower holistic crop science, agronomy, and breeding research

Crop phenomics enables the collection of diverse plant traits for a large number of samples along different time scales,representing a greater data collection throughput compared with traditional measurements.Most modern crop phenomics use different sensors to collect reflective,emitted,and fluorescence signals,etc.,from plant organs at different spatial and temporal resolutions.Such multi-modal,high-dimensional data not only accelerates basic research on crop physiology,genetics,and whole plant systems modeling,but also supports the optimization of field agronomic practices,internal environments of plant factories,and ultimately crop breeding.Major challenges and opportunities facing the current crop phenomics research community include developing community consensus or standards for data collection,management,sharing,and processing,developing capabilities to measure physiological parameters,and enabling farmers and breeders to effectively use phenomics in the field to directly support agricultural production.

Nucleocytoplasmic Trafficking of the Arabidopsis WD40 Repeat Protein XIW1 Regulates ABI5 Stability and Abscisic Acid Responses

WD40 repeat-containing proteins(WD40 proteins)serve as versatile scaffolds for protein-protein interac-tions,modulating a variety of cellular processes such as plant stress and hormone responses.Here we report the identification of a WD40 protein,XIW1(for XPO1-interacting WD40 protein 1),which positively regulates the abscisic acid(ABA)response in Arabidopsis.XIW1 is located in the cytoplasm and nucleus.We found that it interacts with the nuclear transport receptor XPO1 and is exported by XPO1 from the nucleus.Mutation of XIW1 reduces the induction of ABA-responsive genes and the accumulation of ABA Insensitive 5(ABI5),causing mutant plants with ABA-insensitive phenotypes during seed germination and seedling growth,and decreased drought stress resistance.ABA treatment upregulates the expression of XIW1,and both ABA and abiotic stresses promote XIW1 accumulation in the nucleus,where it interacts with ABI5.Loss of XIW1 function results in rapid proteasomal degradation of ABI5.Taken together,these findings suggest that XIW1 is a nucleocytoplasmic shuttling protein and plays a positive role in ABA responses by interacting with and maintaining the stability of ABI5 in the nucleus.

Roles of DEMETER in regulating DNA methylation in vegetative tissues and pathogen resistance

DNA methylation is an epigenetic mark important for genome stability and gene expression.In Arabidopsis thaliana,the 5-methylcytosine DNA glycosylase/demethylase DEMETER(DME)controls active DNA demethylation during the reproductive stage;however,the lethality of loss-of-function dme mutations has made it difficult to assess DME function in vegetative tissues.Here,we edited DME using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 and created three weak dme mutants that produced a few viable seeds.We also performed central cell-specific complementation in a strong dme mutant and combined this line with mutations in the other three Arabidopsis demethylase genes to generate the dme ros1 dml2 dml3(drdd)quadruple mutant.A DNA methylome analysis showed that DME is required for DNA demethylation at hundreds of genomic regions in vegetative tissues.A transcriptome analysis of the drdd mutant revealed that DME and the other three demethylases are important for plant responses to biotic and abiotic stresses in vegetative tissues.Despite the limited role of DME in regulating DNA methylation in vegetative tissues,the dme mutants showed increased susceptibility to bacterial and fungal pathogens.Our study highlights the important functions of DME in vegetative tissues and provides valuable genetic tools for future investigations of DNA demethylation in plants.

Genome-wide distribution and functions of the AAE complex in epigenetic regulation in Arabidopsis

Heterochromatin is widespread in eukaryotic genomes and has diverse impacts depending on its genomic context.Previous studies have shown that a protein complex,the ASI1-AIPP1-EDM2(AAE)complex,participates in polyadenylation regulation of several intronic heterochromatin-containing genes.However,the genome-wide functions of AAE are still unknown.Here,we show that the ASI1 and EDM2 mostly target the common genomic regions on a genome-wide level and preferentially interacts with genetic heterochromatin.Polyadenylation(poly(A)sequencing reveals that AAE complex has a substantial influence on poly(A)site usage of heterochromatin-containing genes,includingnotonlyintronicheterochromatincontaining genes but also the genes showing overlap with heterochromatin.Intriguingly,AAE is also involved in the alternative splicing regulation of a number of heterochromatin-overlapping genes,such as the disease resistance gene RPP4.We provided evidence that genic heterochromatin is indispensable for the recruitment of AAE in polyadenylation and splicing regulation.In addition to conferring RNA processing regulation at genic heterochromatin-containing genes,AAE also targets some transposable elements(TEs)outside of genes(including TEs sandwiched by genes and island TEs)for epigenetic silencing.Our results reveal new functions of AAE in RNA processing and epigenetic silencing,and thus representimportantadvancesinepigenetic regulation.

EXPORTIN 1A prevents transgene silencing in Arabidopsis by modulating nucleo-cytoplasmic partitioning of HDA6

In eukaryotic cells,transport of macromolecules across the nuclear envelope is an essential process that ensures rapid exchange of cellular components,including protein and RNA molecules.Chromatin regulators involved in epigenetic control are among the molecules exported across the nuclear envelope,but the significance of this nucleo-cytoplasmic trafficking is not well understood.Here,we use a forward screen to isolate XPO1 A(a nuclear export receptor in Arabidopsis)as an anti-silencing factor that protects transgenes from transcriptional silencing.Loss-of-function of XPO1 A leads to locus-specific DNA hypermethylation at transgene promoters and some endogenous loci.Wefound that XPO1 A directly interacts with histone deacetylase HDA6 in vivo and that the xpo1 a mutation causes increased nuclear retention of HDA6 protein and results in reduced histone acetylation and enhanced transgene silencing.Our results reveal a new mechanism of epigenetic regulation through the modulation of XPO1 A-dependent nucleo-cytoplasm partitioning of a chromatin regulator.

Large-scale identification of expression quantitative trait loci in Arabidopsis reveals novel candidate regulators of immune responses and other processes

The extensive phenotypic diversity within natural populations of Arabidopsis is associated with differences in gene expression.Transcript levels can be considered as in-heritable quantitative traits,and used to map expression quantitative trait loci(eQTL)in genome-wide association studies(GWASs).In order to identify putative genetic de-terminants for variations in gene expression,we used pub-licly available genomic and transcript variation data from 665 Arabidopsis accessions and applied the single nucleotide polymorphism-set(Sequence)Kernel Association Test(SKAT)method for the identification of eQTL.Moreover,we used the penalized orthogonal-components regression(POCRE)method to increase the power of statistical tests.Then,gene annotations were used as test units to identify genes that are associated with natural variations in transcript accumulation,which correspond to candidate regulators,some of which may have a broad impact on gene ex-pression.Besides increasing the chances to identify real as-sociations,the analysis using POCRE and SKAT significantly reduced the computational cost required to analyze large datasets.As a proof of concept,we used this approach to identify eQTL that represent novel candidate regulators of immune responses.The versatility of this approach allows its application to any process that is subjected to natural var-iation among Arabidopsis accessions.