Characteristics of Salvia miltiorrhiza methylome and the regulatory mechanism of DNA methylation in tanshinone biosynthesis

Salvia miltiorrhiza is a model medicinal plant with significant economic and medicinal value.Its roots produce a group of diterpenoid lipophilic bioactive components,termed tanshinones.Biosynthesis and regulation of tanshinones has attracted widespread interest.However,the methylome of S.miltiorrhiza has not been analysed and the regulatory mechanism of DNA methylation in tanshinone production is largely unknown.Here we report single-base resolution DNA methylomes from roots and leaves.Comparative analysis revealed differential methylation patterns for CG,CHG,and CHH contexts and the association between DNA methylation and the expression of genes and small RNAs.Lowly methylated genes always had higher expression levels and 24-nucleotide sRNAs could be key players in the RdDM pathway in S.miltiorrhiza.DNA methylation variation analysis showed that CHH methylation contributed mostly to the difference.Go enrichment analysis showed that diterpenoid biosynthetic process was significantly enriched for genes with downstream overlapping with hypoCHHDMR in July_root when comparing with those in March_root.Tanshinone biosynthesis-related enzyme genes,such as DXS2,CMK,IDI1,HMGR2,DXR,MDS,CYP76AH1,2OGD25,and CYP71D373,were less CHH methylated in gene promoters or downstream regions in roots collected in July than those collected in March.Consistently,gene expression was up-regulated in S.miltiorrhiza roots collected in July compared with March and the treatment of DNA methylation inhibitor 5-azacytidine significantly promoted tanshinone production.It suggests that DNA methylation plays a significant regulatory role in tanshinone biosynthesis in S.miltiorrhiza through changing the levels of CHH methylation in promoters or downstreams of key enzyme genes.

Genome sequence and evolution of Betula platyphylla

Betula L.(birch)is a pioneer hardwood tree species with ecological,economic,and evolutionary importance in the Northern Hemisphere.We sequenced the Betula platyphylla genome and assembled the sequences into 14 chromosomes.The Betula genome lacks evidence of recent whole-genome duplication and has the same paleoploidy level as Vitis vinifera and Prunus mume.Phylogenetic analysis of lignin pathway genes coupled with tissue-specific expression patterns provided clues for understanding the formation of higher ratios of syringyl to guaiacyl lignin observed in Betula species.Our transcriptome analysis of leaf tissues under a time-series cold stress experiment revealed the presence of the MEKK1–MKK2–MPK4 cascade and six additional mitogen-activated protein kinases that can be linked to a gene regulatory network involving many transcription factors and cold tolerance genes.Our genomic and transcriptome analyses provide insight into the structures,features,and evolution of the B.platyphylla genome.The chromosome-level genome and gene resources of B.platyphylla obtained in this study will facilitate the identification of important and essential genes governing important traits of trees and genetic improvement of B.platyphylla.

Current status and trends in forest genomics

Forests are not only the most predominant of the Earth's terrestrial ecosystems,but are also the core supply for essential products for human use.However,global climate change and ongoing population explosion severely threatens the health of the forest ecosystem and aggravtes the deforestation and forest degradation.Forest genomics has great potential of increasing forest productivity and adaptation to the changing climate.In the last two decades,the field of forest genomics has advanced quickly owing to the advent of multiple high-throughput sequencing technologies,single cell RNA-seq,clustered regularly interspaced short palindromic repeats(CRISPR)-mediated genome editing,and spatial transcriptomes,as well as bioinformatics analysis technologies,which have led to the generation of multidimensional,multilayered,and spatiotemporal gene expression data.These technologies,together with basic technologies routinely used in plant biotechnology,enable us to tackle many important or unique issues in forest biology,and provide a panoramic view and an integrative elucidation of molecular regulatory mechanisms underlying phenotypic changes and variations.In this review,we recapitulated the advancement and current status of 12 research branches of forest genomics,and then provided future research directions and focuses for each area.Evidently,a shift from simple biotechnology-based research to advanced and integrative genomics research,and a setup for investigation and interpretation of many spatiotemporal development and differentiation issues in forest genomics have just begun to emerge.

Effects of a regenerating matrix on the survival of birds in tropical forest fragments

Background: Vast areas of lowland neotropical forest have regenerated after initially being cleared for agricultural purposes. The ecological value of regenerating second growth to forest-dwelling birds may largely depend on the age of the forest, associated vegetative structure, and when it is capable of sustaining avian demographics similar to those found in pristine forest.Methods: To determine the influence of second growth age on bird demography, we estimated the annual survival of six central Amazonian bird species residing in pristine forest, a single 100 and a single 10 ha forest fragment, taking into consideration age of the surrounding matrix(i.e. regenerating forest adjacent to each fragment) as an explanatory variable.Results: Study species exhibited three responses: arboreal, flocking and ant-following insectivores(Willisornis poecilinotus, Thamnomanes ardesiacus and Pithys albifrons) showed declines in survival associated with fragmentation followed by an increase in survival after 5 years of matrix regeneration. Conversely, Percnostola rufifrons, a gap-specialist, showed elevated survival in response to fragmentation followed by a decline after 5 years of regeneration. Lastly, facultative flocking and frugivore species(Glyphorynchus spirurus and Dixiphia pipra, respectively) showed no response to adjacent clearing and subsequent regeneration.Conclusions: Our results in association with previous studies confirm that the value of regenerating forest surrounding habitat patches is dependent on two factors: ecological guild of the species in question and second growth age. Given the rapid increase in survival following succession, we suggest that the ecological value of young tropical forest should not be based solely on a contemporary snapshot, but rather, on the future value of mature second growth as well.

The diverse roles of cytokinins in regulating leaf development

Leaves provide energy for plants,and consequently for animals,through photosynthesis.Despite their important functions,plant leaf developmental processes and their underlying mechanisms have not been well characterized.Here,we provide a holistic description of leaf developmental processes that is centered on cytokinins and their signaling functions.Cytokinins maintain the growth potential(pluripotency)of shoot apical meristems,which provide stem cells for the generation of leaf primordia during the initial stage of leaf formation;cytokinins and auxins,as well as their interaction,determine the phyllotaxis pattern.The activities of cytokinins in various regions of the leaf,especially at the margins,collectively determine the final leaf morphology(e.g.,simple or compound).The area of a leaf is generally determined by the number and size of the cells in the leaf.Cytokinins promote cell division and increase cell expansion during the proliferation and expansion stages of leaf cell development,respectively.During leaf senescence,cytokinins reduce sugar accumulation,increase chlorophyll synthesis,and prolong the leaf photosynthetic period.We also briefly describe the roles of other hormones,including auxin and ethylene,during the whole leaf developmental process.In this study,we review the regulatory roles of cytokinins in various leaf developmental stages,with a focus on cytokinin metabolism and signal transduction processes,in order to shed light on the molecular mechanisms underlying leaf development.

Breeding polyploid Populus:progress and perspective

Populus is a genus of 25−30 species of deciduous flowering plants in the family Salicaceae,which are primarily planted in short-rotation planations for producing timber,pulpwood,wooden products as well as bioenergy feedstock;they are also widely planted in agricultural fields and along roadsides as shelter forest belts for windbreak,decoration,and reduction of pollutants and noise.Moreover,their fast-growth and good adaptation to marginal lands enable them to provide some critical ecosystem services at various phytoremediation sites for land restoration and reclaimation.Thanks to their important roles,breeding for fast growing poplar trees has been one of the most important objectives for nearly a century.One of the most demonstrated,documented achievements in this aspect is polyploid breeding,especially triploid breeding.This paper critically reviews the various techniques used in inducing triploid plants,including natural 2n formation,artificial induction of 2n male and female gemmates through chemical or physical treatments,trait characterization of the triploid and tetraploid breeding populations,unveiling the molecular mechanisms underpinning the significantly improved traits,and identification and selection of the best triploid progenies.This review also recapitulated the challenges and strategies facing the future of triploid breeding in Populus,including amelioration of 2n gamete induction techniques and efficiency,selection of the best parents and identification of the best progrenies,utilization of the huge amount of genomic,transcriptomic,proteomic,metabolomic,and other omics data for selecting parents for improving target traits.

A systems biology approach identifies a regulator,BplERF1,of cold tolerance in Betula platyphylla

Cold is an abiotic stress that can greatly affect the growth and survival of plants.Here,we reported that an AP2/ERF family gene,BplERF1,isolated from Betula platyphylla played a contributing role in cold stress tolerance.Overexpression of BplERF1 in B.platyphylla transgenic lines enhanced cold stress tolerance by increasing the scavenging capability and reducing H_(2)O_(2) and malondialdehyde(MDA)content in transgenic plants.Construction of BplERF-mediated multilayered hierarchical gene regulatory network(ML-hGRN),using Top-down GGM algorithm and the transcriptomic data of BplERF1 overexpression lines,led to the identification of five candidate target genes of BplERF1 which include MPK20,ERF9,WRKY53,WRKY70,and GIA1.All of them were then verified to be the true target genes of BplERF1 by chromatin-immunoprecipitation PCR(ChIP-PCR)assay.Our results indicate that BplERF1 is a positive regulator of cold tolerance and is capable of exerting regulation on the expression of cold signaling and regulatory genes,causing mitigation of reactive oxygen species.

HB-PLS:A statistical method for identifying biological process or pathway regulators by integrating Huber loss and Berhu penalty with partial least squares regression

Gene expression data features high dimensionality,multicollinearity,and non-Gaussian distribution noise,posing hurdles for identification of true regulatory genes controlling a biological process or pathway.In this study,we integrated the Huber loss function and the Berhu penalty(HB)into partial least squares(PLS)framework to deal with the high dimension and multicollinearity property of gene expression data,and developed a new method called HB-PLS regression to model the relationships between regulatory genes and pathway genes.To solve the Huber-Berhu optimization problem,an accelerated proximal gradient descent algorithm with at least 10 times faster than the general convex optimization solver(CVX),was developed.Application of HB-PLS to recognize pathway regulators of lignin biosynthesis and photosynthesis in Arabidopsis thaliana led to the identification of many known positive pathway regulators that had previously been experimentally validated.As compared to sparse partial least squares(SPLS)regression,an efficient method for variable selection and dimension reduction in handling multicollinearity,HB-PLS has higher efficacy in identifying more positive known regulators,a much higher but slightly less sensitivity/(1-specificity)in ranking the true positive known regulators to the top of the output regulatory gene lists for the two aforementioned pathways.In addition,each method could identify some unique regulators that cannot be identified by the other methods.Our results showed that the overall performance of HB-PLS slightly exceeds that of SPLS but both methods are instrumental for identifying real pathway regulators from high-throughput gene expression data,suggesting that integration of statistics,machine leaning and convex optimization can result in a method with high efficacy and is worth further exploration.

MicroRNA319-mediated gene regulatory network impacts leaf development and morphogenesis in poplar

MicroRNA319(miR319)has been implicated in leaf development in a number of plant species.Here we study the roles of miR319a and its regulated network in leaf development in poplars.Over-expression of miR319a in Populus alba×Populus glandulosa caused dwarf statures,narrow leaf blades and serrated leaf margins.The vascular bundles and bundle sheaths in transgenic leaves had more layers of cells than those in the leaves of control plants,indicating enhanced lignification in these cells.Among the 93 putative targets of miR319a predicted with the psRNATarget tool,only three genes,TCP(TEOSINTE BRANCHED1,CYCLOIDEA,and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR),were differentially expressed in the leaves of MIR319a-over-expression transgenic lines.With the RNA-seq data sets from multiple MIR319a over-expression transgenic lines,we built a three-layered gene regulatory network mediated by miR319a using Top-down graphic Gaussian model(GGM)algorithm that is capable of capturing causal relationships from transcriptomic data.The results support that miR319a primarily regulates the lignin biosynthesis,leaf development and differentiation as well as photosynthesis via miR319-MEE35/TCP4,miR319-TCP2 and miR319-TCP2-1 regulatory modules.

PuHox52 promotes coordinated uptake of nitrate,phosphate, and iron under nitrogen deficiency in Populus ussuriensis

It is of great importance to better understand how trees regulate nitrogen(N) uptake under N deficiency conditions which severely challenge afforestation practices, yet the underlying molecular mechanisms have not been well elucidated. Here,we functionally characterized PuHox52, a Populus ussuriensis HD-ZIP transcription factor, whose overexpression greatly enhanced nutrient uptake and plant growth under N deficiency. We first conducted an RNA sequencing experiment to obtain root transcriptome using PuHox52-overexpression lines of P. ussuriensis under low N treatment. We then performed multiple genetic and phenotypic analyses to identify key target genes of PuHox52 and validated how they acted against N deficiency under PuHox52 regulation.PuHox52 was specifically induced in roots by N deficiency, and overexpression of PuHox52promoted N uptake, plant growth, and root development. We demonstrated that several nitrate-responsive genes(PuNRT1.1, PuNRT2.4,PuCLC-b, PuNIA2, PuNIR1, and PuNLP1),phosphate-responsive genes(PuPHL1A and PuPHL1B), and an iron transporter gene(PuIRT1) were substantiated to be direct targets of PuHox52. Among them, PuNRT1.1, PuPHL1A/B, and PuIRT1 were upregulated to relatively higher levels during PuHox52-mediated responses against N deficiency in PuHox52-overexpression lines compared to WT. Our study revealed a novel regulatory mechanism underlying root adaption to N deficiency where PuHox52 modulated a coordinated uptake of nitrate, phosphate, and iron through 'PuHox52-PuNRT1.1', 'PuHox52-PuPHL1A/PuPHL1B', and'PuHox52-PuIRT1' regulatory relationships in poplar roots.

Regulation of regeneration in Arabidopsis thaliana

We employed several algorithms with high efficacy to analyze the public transcriptomic data,aiming to identify key transcription factors(TFs)that regulate regeneration in Arabidopsis thaliana.Initially,we utilized CollaborativeNet,also known as TF-Cluster,to construct a collaborative network of all TFs,which was subsequently decomposed into many subnetworks using the Triple-Link and Compound Spring Embedder(CoSE)algorithms.Functional analysis of these subnetworks led to the identification of nine subnetworks closely associated with regeneration.We further applied principal component analysis and gene ontology(GO)enrichment analysis to reduce the subnetworks from nine to three,namely subnetworks 1,12,and 17.Searching for TF-binding sites in the promoters of the co-expressed and co-regulated(CCGs)genes of all TFs in these three subnetworks and Triple-Gene Mutual Interaction analysis of TFs in these three subnetworks with the CCGs involved in regeneration enabled us to rank the TFs in each subnetwork.Finally,six potential candidate TFs-WOx9A,LEC2,PGA37,WIP5,PEI1,and AIL1 from subnetwork 1-were identified,and their roles in somatic embryogenesis(GO:0010262)and regeneration(GO:0031099)were discussed,so were the TFs in Subnetwork 12 and 17 associated with regeneration.The TFs identified were also assessed using the CIS-BP database and Expression Atlas.Our analyses suggest some novel TFs that may have regulatory roles in regeneration and embryogenesis and provide valuable data and insights into the regulatory mechanisms related to regeneration.The tools and the procedures used here are instrumental for analyzing high-throughput transcriptomic data and advancing our understanding of the regulation of various biological processes of interest.