Publications

Additional file 5: Table S4. RNAseq profiling of dcl234 in comparision to Col-0 and GO annotation of UP and Down regulated DEGs.

In Arabidopsis thaliana, the calcium binding protein Salt Overly Sensitive3 (SOS3) interacts with and activates the protein kinase SOS2, which in turn activates the plasma membrane Na+/H+ antiporter SOS1 to bring about sodium ion homeostasis and salt tolerance. Constitutively active alleles of SOS2 can be constructed in vitro by changing Thr168 to Asp in the activation loop of the kinase catalytic domain and/or by removing the autoinhibitory FISL motif from the C-terminal regulatory domain. We expressed various activated forms of SOS2 in Saccharomyces cerevisiae (yeast) and in A. thaliana and evaluated the salt tolerance of the transgenic organisms. Experiments in which the activated SOS2 alleles were coexpressed with SOS1 in S. cerevisiae showed that the kinase activity of SOS2 is partially sufficient for SOS1 activation in vivo, and higher kinase activity leads to greater SOS1 activation. Coexpression of SOS3 with SOS2 forms that retained the FISL motif resulted in more dramatic increases in salt tolerance. In planta assays showed that the Thr168-to-Asp–activated mutant SOS2 partially rescued the salt hypersensitivity in sos2 and sos3 mutant plants. By contrast, SOS2 lacking only the FISL domain suppressed the sos2 but not the sos3 mutation, whereas truncated forms in which the C terminus had been removed could not restore the growth of either sos2 or sos3 plants. Expression of some of the activated SOS2 proteins in wild-type A. thaliana conferred increased salt tolerance. These studies demonstrate that the protein kinase activity of SOS2 is partially sufficient for activation of SOS1 and for salt tolerance in vivo and in planta and that the kinase activity of SOS2 is limiting for plant salt tolerance. The results also reveal an essential in planta role for the SOS2 C-terminal regulatory domain in salt tolerance.

While multiple chromatin-based epigenetic pathways are well characterized, their genome-wide coordination and hierarchical interplay remain poorly understood. We previously identified the BAH-PHD-CPL2 complex, where AIPP3 binds H3K27me3 via its BAH domain and, in concert with PHD2 and PHD3 (PHD2/3), recruits CPL2 to repress transcription by dephosphorylating RNA Polymerase II. Here, we show that CPL2 and PHD2/3 form a distinct module (CPL2-PHD2/3) that safeguards CHH DNA methylation and silences CHH-methylated sites at transposable elements (TEs) through engagement of multiple DNA methylation pathways. The CPL2-PHD2/3 module physically associates with SUVH4 and SUVH5 (SUVH4/5) to preserve SUVH4/5-CMT2-mediated CHH methylation at a subset of H3K9me2-marked regions. Chromatin enrichment of PHD3 likewise enables the module to sustain RNA-directed DNA methylation (RdDM)-mediated CHH DNA methylation at sites bearing H3K9me2, H3K27me3, or neither. Thus, CPL2-PHD2/3 emerges as a previously underappreciated, multifunctional regulator of plant DNA methylation networks. Unexpectedly, its gene repression function is uncoupled from DNA methylation. CPL2-PHD2/3 represses Polycomb-marked genes by interacting with LHP1 or recognizing hypomethylated H3K4. While it cooperates with LHP1 at a subset of targets, it also independently silences many more genes that LHP1 alone cannot repress, both sets of genes being critical for proper development. This dual, yet largely LHP1-insufficient, repression mode singles out CPL2-PHD2/3 as the essential executor of Polycomb silencing. Together, our findings establish CPL2-PHD2/3 as a chromatin-responsive integrator that spans DNA methylation and Polycomb-associated repression, providing a unifying mechanism for epigenetic control of both TEs and genes across diverse chromatin landscapes.

Purpose The purpose of this paper is to develop a dual peg-in-hole insertion strategy. Dual peg-in-hole insertion is the most common task in manufacturing. Most of the previous work develop the insertion strategy in a two- or three-dimensional space, in which they suppose the initial yaw angle is zero and only concern the roll and pitch angles. However, in some case, the yaw angle could not be ignored due to the pose uncertainty of the peg on the gripper. Therefore, there is a need to design the insertion strategy in a higher-dimensional configuration space. Design/methodology/approach In this paper, the authors handle the insertion problem by converting it into several sub-problems based on the attractive region formed by the constraints. The existence of the attractive region in the high-dimensional configuration space is first discussed. Then, the construction of the high-dimensional attractive region with its sub-attractive region in the low-dimensional space is proposed. Therefore, the robotic insertion strategy can be designed in the subspace to eliminate some uncertainties between the dual pegs and dual holes. Findings Dual peg-in-hole insertion is realized without using of force sensors. The proposed strategy is also used to demonstrate the precision dual peg-in-hole insertion, where the clearance between the dual-peg and dual-hole is about 0.02 mm. Practical implications The sensor-less insertion strategy will not increase the cost of the assembly system and also can be used in the dual peg-in-hole insertion. Originality/value The theoretical and experimental analyses for dual peg-in-hole insertion are proposed without using of force sensor.

Table S2. Summary of 466 hyper DMRs and 2300 hypo DMRs in Fa3 compared to Fa1. (XLSX 283 kb)

We report the identification and characterization of an Arabidopsis mutant, hos10-1 (for high expression of osmotically responsive genes), in which the expression of RD29A and other stress-responsive genes is activated to higher levels or more rapidly activated than in wild-type by low temperature, exogenous abscisic acid (ABA), or salt stress (NaCl). The hos10-1 plants are extremely sensitive to freezing temperatures, completely unable to acclimate to the cold, and are hypersensitive to NaCl. Induction of NCED3 (the gene that encodes the rate-limiting enzyme in ABA biosynthesis) by polyethylene glycol-mediated dehydration and ABA accumulation are reduced by this mutation. Detached shoots from the mutant plants display an increased transpiration rate compared with wild-type plants. The hos10-1 plants exhibit several developmental alterations, such as reduced size, early flowering, and reduced fertility. The HOS10 gene encodes a putative R2R3-type MYB transcription factor that is localized to the nucleus. Together, these results indicate that HOS10 is an important coordinating factor for responses to abiotic stress and for growth and development.

Epigenetic alterations and metabolic dysfunction are two hallmarks of aging. However, the mechanism of how their interaction regulates aging, particularly in mammals, remains largely unknown. Here we show ELOVL fatty acid elongase 2 (Elovl2), a gene whose epigenetic alterations are most highly correlated with age prediction, contributes to aging by regulating lipid metabolism. Impaired Elovl2 function disturbs lipid synthesis with increased endoplasmic reticulum stress and mitochondrial dysfunction, leading to key accelerated aging phenotypes. Restoration of mitochondrial activity can rescue age-related macular degeneration (AMD) phenotypes induced by Elovl2 deficiency in human retinal pigmental epithelial (RPE) cells. We revealed an epigenetic-metabolism axis contributing to aging and potentially to antiaging therapy.

Base editors, presently including cytidine base editors (CBEs) and adenine base editors (ABEs), enable precise base alterations in the genome without inducing DNA double-stranded breaks (DSBs). Base editors are valuable tools for precision plant molecular breeding since many agronomic traits are controlled by variations in one or few DNA bases. The early developed CBE and ABE systems, consisting of the rat cytidine deaminase APOBEC1 (rAPOBEC1) or activation-induced cytidine deaminase (AID) PmCDA1, and the evolved tRNA adenine deaminase TadA, respectively, have been applied to many plant species. To improve the base editing efficiency, more effective cytidine deaminases such as the human APOBEC3A have been tested (Zong et al., 2018). On the other hand, for expanding the base editing scope in plants, several SpCas9 and SaCas9 variants such as VQR-Cas9, VRER-Cas9 and SaKKH-Cas9 that recognize PAMs other than the canonical NGG motif were introduced into the CBE and ABE toolbox (Hua et al., 2018a; Qin et al., 2018). However, relative to the widely used CRISPR/Cas gene editing technologies for inducing DSBs and subsequent repair-caused mutations, the efficiency of base editing is still low. In addition, base editors reported thus far are constrained by recognition of only a few kinds of PAM sequences. We have previously reported the initial adoption of CBEs and ABEs in rice (Hua et al., 2018b; Lu and Zhu, 2017). In our CBE system, we fused rAPOBEC1 to the N-terminus of SpCas9 nickase (Cas9n, D10A) using the unstructured 16-residue peptide XTEN as linker. A traditional nuclear localization signal, SV40 NLS peptide, was added to the C-terminus of the Cas9n. Two agronomically important genes of rice, NRT1.1B and SLR1, were selected for editing by this CBE system. However, the base substitution efficiencies were low, with only 2.7% for NRT1.1B and 13.3% for SLR1, respectively (Lu and Zhu, 2017). In our ABE system, we synthesized wild-type ecTadA and its mutant form ecTadA*7.10 and linked them together using a 32-amino acid (aa) linker; the resulting recombinant protein was fused to the N-terminus of the SpCas9 or SaCas9 nickase with the same linker. Testing at different targets showed that the base substitution efficiencies ranged from 5% to 60%, with most of the target sites having efficiencies lower than 30% as reported by other groups (Hua et al., 2018b). Recently, Koblan et al. (2018) found that the expression levels of base editors are major bottlenecks for base editing efficiency. They improved BE4 and ABE7.10 base editors by adopting bipartite nuclear localization signals (bpNLS), optimizing codon usage and ancestral reconstruction of the deaminase component. The resulting BE4max, AncBE4max and ABEmax editors showed increased editing efficiencies in a variety of settings, especially under suboptimal conditions or at sites previously edited with low efficiencies (Koblan et al., 2018). To improve the base editing efficiency in plants, we directly adopted the above GenScript codon-optimized nucleotide sequences of bpNLS-Anc689 APOBEC-32 aa Linker and bpNLS-adenine deaminase of ABE7.10-32 aa Linker into our previous CBE and ABE editors, resulting in Anc689BE4max and ABEmax, respectively (Figure 1a,b). To directly compare the performance of Anc689BE4max with our previous CBE, the NRT1.1B and SLR1 were selected for editing using the previously tested sgRNA. As shown in Figure 1c, 72.4% of the transgenic rice lines harboured the target C to T replacement at NRT1.1B target site, and 76.2% of these lines (55.2% of total transgenic lines) are homozygous (Figure 1d). Most of the regenerated plantlets transformed with Anc689BE4max-sgRNASLR1 displayed an obvious dwarf phenotype (Figure 1e,f). Genotyping and sequencing results showed that 82.8% of the transgenic lines converted C to T at their target site, and 72.5% of the transgenic lines were homozygous or biallelic C to T substitutions (Figure 1g,h). The sequencing results are also consistent with the phenotype of each plantlet (Figure 1f,i). Compared with the efficiency of 2.7% for NRT1.1B and 13.3% for SLR1 from our previous CBE (Lu and Zhu, 2017), the Anc689BE4max showed much higher base editing efficiencies. We also noticed that the deamination window ranged from the 4th to 15th target bases, but the substitutions were concentrated at the 4th to 7th bases, which is similar to our previously reported CBE (Lu and Zhu, 2017). To further evaluate the efficiency of Anc689BE4max, we designed an sgRNA (ALS-sg1) for modifying the acetolactate synthase gene (ALS) in rice. It is known that a mutated form of ALS, ALSS627N (G1880 to A in Nipponbare DNA sequence), confers tolerance to imidazolinone herbicides (Piao et al., 2018) (Figure 1j). Similar to the results from the base editing of NRT1.1B and SLR1, 71.4% of the transgenic lines contained C to T substitution at their target site in ALS, and most of them were homozygous or biallelic (Figure 1k,l). Our result also shows that although the target C ranging from 4th to 10th of the protospacer could be replaced by T, the substitution preferentially occurred within the window from the 5th to 7th base. Only two of the edited lines contained the intended G1880 to A conversion, since this target base is located outside of the 'hot spot' of the deamination window (Figure 1k). Recently, Nishimasu et al. (2018) reported that a rationally engineered SpCas9 variant, SpCas9-NG, containing the R1335A/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R seven amino acid alteration, can recognize relaxed NG PAMs in human cells. During the preparation of our manuscript, Endo et al. (2018) reported that the nickase of this variant (D10A) fused to cytidine deaminases such as PmCDA1 and rAPOBEC1 could mediate C to T conversion at sites bearing NG PAMs in rice calli, but the nSpCas9NG-APOBEC1 base editor showed a low activity at most of the tested target sites. To expand the access range of base editors, we adopted the SpCas9NG nickase into our Anc689BE4max and ABEmax to replace the SpCas9 nickase, resulting in Anc689BE4max-nCas9NG and ABEmax-nCas9NG system, respectively (Figure 1a,b). Testing of the Anc689BE4max-nCas9NG system at SLR1 using the same sgRNA with GGG PAM showed an editing efficiency of 40.9%, lower than that of Anc689BE4max-nCas9 (Figure 1s). To facilitate the G1880 to A substitution in ALS, we designed another sgRNA, ALS-sg2, harbouring the AGC PAM, for editing by the Anc689BE4max-nCas9NG system (Figure 1j). 57.1% of the transgenic lines showed the intended G1880 to A replacement, although the combined base substitution efficiency was lower than that of Anc689BE4max-nCas9 with ALS-sg1 (Figure 1m,s). The mutants homozygous for the G1880 to A substitution were tolerant to imidazolinone herbicide, whereas wild-type plants were not (Figure 1n,o). Taking advantage of the nCas9NG-derived base editor that can recognize relaxed PAMs, we further applied it to modify the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. Previous work showed that a single base transition of C317-T within OsEPSPS (C530 in Nipponbare genome), changing proline-106 to leucine (P106L), led to resistance to another herbicide, glyphosate, in the EPSPS-deficient Escherichia coli strain AB2829 (Zhou et al., 2006). Here, we designed an sgRNA harbouring the TGA PAM (EPSPS-sg1) for editing by our Anc689BE4max-nCas9NG system, since there are no suitable NGG PAMs near the protospacer (Figure 1p). We successfully obtained five plantlets containing the targeted C530 to T replacement from 29 transgenic lines (Figure 1p,q,s). The glyphosate tolerance will be tested in the T1 generation since there was no homozygous mutant in the T0 plants. To evaluate the activity of our ABEmax editor, we designed a third sgRNA (ALS-sg3) to edit the ALS gene by ABEmax and our previous ABE side by side. The results showed that ABEmax doubled the editing efficiency of ABE (Figure 1r,t). We further tested ABEmax at the ALS-sg1 target site, and the results showed that 48.3% of the transgenic lines harboured A to G substitution (Figure 1t). The general editing efficiencies of ABEmax seem lower than those of Anc689BE4max (Figure 1s,t). We further evaluated the ABEmax-nCas9NG system with non-canonical NGG PAMs. Testing at the EPSPS-sg2 target site harbouring the AGT PAM showed an editing efficiency of 41.2%. However, testing at another target site (ALS-sg4) bearing the CGT PAM showed the editing efficiency lower than 10% (Figure 1t). In summary, our upgraded base editors not only show substantially increased editing efficiencies, but also have expanded editing scopes compared to previously reported CBEs and ABEs. These improved base editors are more powerful tools for molecular breeding of crops, although more plant species and more target sites need to be tested in the future. This work was supported by the Chinese Academy of Sciences. The authors declare no conflicts of interest with respect to this work.

Broomcorn millet (Panicum miliaceum L.) is the most water-efficient cereal and one of the earliest domesticated plants. Here we report its high-quality, chromosome-scale genome assembly using a combination of short-read sequencing, single-molecule real-time sequencing, Hi-C, and a high-density genetic map. Phylogenetic analyses reveal two sets of homologous chromosomes that may have merged ~5.6 million years ago, both of which exhibit strong synteny with other grass species. Broomcorn millet contains 55,930 protein-coding genes and 339 microRNA genes. We find Paniceae-specific expansion in several subfamilies of the BTB (broad complex/tramtrack/bric-a-brac) subunit of ubiquitin E3 ligases, suggesting enhanced regulation of protein dynamics may have contributed to the evolution of broomcorn millet. In addition, we identify the coexistence of all three C4 subtypes of carbon fixation candidate genes. The genome sequence is a valuable resource for breeders and will provide the foundation for studying the exceptional stress tolerance as well as C4 biology.

The function of miR165/166 in plant growth and development has been extensively studied, however, its roles in abiotic stress responses remain largely unknown. Here, we report that reduction in the expression of miR165/166 conferred a drought and cold resistance phenotype and hypersensitivity to ABA during seed germination and post-germination seedling development. We further show that the ABA hypersensitive phenotype is associated with a changed transcript abundance of ABA-responsive genes and a higher expression level of ABI4, which can be directly regulated by a miR165/166 target. Additionally, we found that reduction in miR165/166 expression leads to elevated ABA levels, which occurs at least partially through the increased expression of BG1, a gene that is directly regulated by a miR165/166 target. Taken together, our results uncover a novel role for miR165/166 in the regulation of ABA and abiotic stress responses and control of ABA homeostasis.

Epigenome and cis-regulome, comprising cis-regulatory elements (CREs) and modules (CRMs), jointly define the architecture of gene regulation. However, the causal mechanisms by which epigenetic marks influence CRM function remain elusive. To address this, modular epigenome editing frameworks, exemplified by dead Cas9-coupled DNA demethylation (dCd) and DNA methylation (dCm) platforms, are developed for programmable dissection and engineering of CRM activity. The dCd system modulates methylation levels and transcriptional output at CRMs in situ or ex situ, in accordance with CRM-specific methylation responsiveness, and alters co-transcriptional RNA processing to yield predictable phenotypic outcomes in plants. These findings underscore the reliability of targeted DNA demethylation. In parallel, the dCm system reconstitutes methylation-dependent and -sensitive CRMs of diverse origins in Saccharomyces cerevisiae, a species devoid of native DNA methylation, enabling causal dissection of epigenetic regulation and revealing cross-species portability. This system further uncovers crosstalk between DNA methylation and chromatin modifications, and enables logic-gated control of endogenous genes through CRM engineering. Incorporation of optogenetic and temperature-sensitive anti-CRISPR inhibitors confers tunable, reversible regulation, proposing dCm as a foundation for input-responsive synthetic epigenome editors. Together, these frameworks provide a versatile platform to decode and reprogram cis-regulatory epigenetic logic, with broad applications in trait design and synthetic biology.