Publications

AtHKT1 Facilitates Na+ Homeostasis and K+ Nutrition in Planta

Abstract Genetic and physiological data establish that Arabidopsis AtHKT1 facilitates Na+ homeostasis in planta and by this function modulates K+ nutrient status. Mutations that disrupt AtHKT1 function suppress NaCl sensitivity of sos1-1 and sos2-2, as well as of sos3-1 seedlings grown in vitro and plants grown in controlled environmental conditions. hkt1 suppression of sos3-1 NaCl sensitivity is linked to higher Na+ content in the shoot and lower content of the ion in the root, reducing the Na+ imbalance between these organs that is caused by sos3-1. AtHKT1 transgene expression, driven by its innate promoter, increases NaCl but not LiCl or KCl sensitivity of wild-type (Col-0 gl1) or of sos3-1 seedlings. NaCl sensitivity induced by AtHKT1 transgene expression is linked to a lower K+ to Na+ ratio in the root. However, hkt1 mutations increase NaCl sensitivity of both seedlings in vitro and plants grown in controlled environmental conditions, which is correlated with a lower K+ to Na+ ratio in the shoot. These results establish that AtHKT1 is a focal determinant of Na+ homeostasis in planta, as either positive or negative modulation of its function disturbs ion status that is manifested as salt sensitivity. K+-deficient growth of sos1-1, sos2-2, and sos3-1 seedlings is suppressed completely by hkt1-1. AtHKT1 transgene expression exacerbates K+ deficiency of sos3-1 or wild-type seedlings. Together, these results indicate that AtHKT1 controls Na+ homeostasis in planta and through this function regulates K+ nutrient status.

Dissecting the Subnuclear Localization Patterns of Argonaute Proteins and Other Components of the RNA-Directed DNA Methylation Pathway in Plants

Emerging roles of RNA processing factors in regulating long non-coding RNAs

Long non-coding RNAs (lncRNAs) can be important regulators of various biological processes such as RNA-directed DNA methylation (RdDM). In the RdDM pathway, recruitment of the DNA methylation complex is mediated through complementary pairing between scaffold RNAs and Argonaute-associated siRNAs. Scaffold RNAs are chromatin-associated lncRNAs transcribed by RNA polymerase Pol V or Pol II, while siRNAs originate from Pol IV- or Pol II-dependent production of lncRNAs. In contrast to the vast literature on co-transcriptional and post-transcriptional processing of mRNAs, information is limited for lncRNA regulation that enables their production and function. Recently Arabidopsis RRP6L1, a plant paralog of the conserved nuclear RNA surveillance protein Rrp6, was shown to mediate RdDM through retention of lncRNAs in the chromatin, thereby revealing that accumulation of functional lncRNAs requires more than simply RNA polymerases. By focusing on the canonical RdDM pathway, here we summarize recent evidence that indicate co-transcriptional and/or post-transcriptional regulation of lncRNAs, and highlight the emerging theme of lncRNA regulation by RNA processing factors.

Before and beyond ABA: upstream sensing and internal signals that determine ABA accumulation and response under abiotic stress

Sensing and signalling events that detect abiotic stress-induced changes in plant water status and initiate downstream stress responses such as ABA (abscisic acid) accumulation and osmoregulation remain uncharacterized in plants. Although conclusive results are lacking, recent results from plants, and analogies to signalling in other organisms, suggest possible mechanisms for sensing altered water status and initial transduction of that signal. Internal signals that act downstream of ABA and modulate stress responses to reflect the type and severity of the stress and the metabolic status of the plant are also not well understood. Two specific types of signalling, sugar sensing and reactive oxygen signalling, are likely to be modulators of ABA response under stress. For both upstream sensing and signalling of plant water status as well as downstream modulation of ABA response, present results suggest several genetic strategies with high potential to increase our understanding of the molecular basis by which plants sense and respond to altered water status.

The <i>Arabidopsis HOS1</i> gene negatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo–cytoplasmic partitioning

Low temperature is one of the most important environmental stimuli that control gene transcription programs and development in plants. In Arabidopsis thaliana , the HOS1 locus is a key negative regulator of low temperature-responsive gene transcription. The recessive hos1 mutation causes enhanced induction of the CBF transcription factors by low temperature as well as of their downstream cold-responsive genes. The hos1 mutant plants flower early, and this correlates with a low level of Flowering Locus C gene expression. The HOS1 gene was isolated through positional cloning. HOS1 encodes a novel protein with a RING finger motif near the amino terminus. HOS1 is ubiquitously expressed in all plant tissues. HOS1–GFP translational fusion studies reveal that HOS1 protein resides in the cytoplasm at normal growth temperatures. However, in response to low temperature treatments, HOS1 accumulates in the nucleus. Ectopic expression of HOS1 in wild-type plants causes cosuppression of HOS1 expression and mimics the hos1 mutant phenotypes.

Root twisting drives halotropism via stress-induced microtubule reorientation

A miRNA Involved in Phosphate-Starvation Response in Arabidopsis

A Histone Acetyltransferase Regulates Active DNA Demethylation in <i>Arabidopsis</i>

Active DNA demethylation is an important part of epigenetic regulation in plants and animals. How active DNA demethylation is regulated and its relationship with histone modification patterns are unclear. Here, we report the discovery of IDM1, a regulator of DNA demethylation in Arabidopsis. IDM1 is required for preventing DNA hypermethylation of highly homologous multicopy genes and other repetitive sequences that are normally targeted for active DNA demethylation by Repressor of Silencing 1 and related 5-methylcytosine DNA glycosylases. IDM1 binds methylated DNA at chromatin sites lacking histone H3K4 di- or trimethylation and acetylates H3 to create a chromatin environment permissible for 5-methylcytosine DNA glycosylases to function. Our study reveals how some genes are indicated by multiple epigenetic marks for active DNA demethylation and protection from silencing.

Enrichment of vitronectin‐ and fibronectin‐like proteins in NaCl‐adapted plant cells and evidence for their involvement in plasma membrane–cell wall adhesion

Summary Cells of tobacco adapted to grow in high concentrations of NaCl develop tight zones of adhesion between the plasma membrane and cell wall, revealed by concave plasmolysis in osmotic solutions. Unadapted cells exhibit mostly convex plasmolysis and exhibit little or no adhesive character. Wall‐less protoplasts isolated from the adapted cells retain the complementary adhesive character and adhere tightly to each other, whereas protoplasts from unadapted cells do not. The hexapeptide gly‐ arg‐gly‐asp ‐ser‐pro, in which the arg‐gly‐asp represents the integrin‐binding domain of several animal extracellular matrix proteins, specifically blocks adhesion of the protoplasts. A control hexapeptide, gly‐arg‐gly‐glu‐ser‐pro, is ineffective in blocking adhesion. Tobacco proteins immunologically related to human vitronectin were found in cell walls and membranes of unadapted and NaCl‐adapted cells, but the total extractable vitronectin‐like protein was enriched in the adapted cells. Tobacco proteins immunologically related to human fibronectin were found in membranes and cell walls of NaCl‐adapted cells but not in those from unadapted cells. Our observations indicate that plant cells possess cell‐matrix adhesion complexes similar to animal cells, and these adhesion complexes accumulate in growth‐limited cells adapted to saline stress.

Genetic Analysis of Salt Tolerance in Arabidopsis: Evidence for a Critical Role of Potassium Nutrition

A RAF-SnRK2 kinase cascade mediates early osmotic stress signaling in higher plants

Abstract Osmoregulation is important for plant growth, development and response to environmental changes. SNF1-related protein kinase 2s (SnRK2s) are quickly activated by osmotic stress and are central components in osmotic stress and abscisic acid (ABA) signaling pathways; however, the upstream components required for SnRK2 activation and early osmotic stress signaling are still unknown. Here, we report a critical role for B2, B3 and B4 subfamilies of Raf-like kinases (RAFs) in early osmotic stress as well as ABA signaling in Arabidopsis thaliana . B2, B3 and B4 RAFs are quickly activated by osmotic stress and are required for phosphorylation and activation of SnRK2s. Analyses of high-order mutants of RAFs reveal critical roles of the RAFs in osmotic stress tolerance and ABA responses as well as in growth and development. Our findings uncover a kinase cascade mediating osmoregulation in higher plants.

Increasing Freezing Tolerance: Kinase Regulation of ICE1

A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

A genotype‐independent and highly efficient <i>Agrobacterium</i> ‐mediated soybean genetic transformation system

A stable and efficient transformation system is crucial for functional genomics and trait improvement in soybean. This study developed a tissue culture based genetic transformation system incorporating dual selection (Spectinomycin and RUBY). This system significantly enhances transformation efficiency, shortens the transformation cycle, and demonstrates broad genotype independence, providing a powerful tool for soybean research and breeding.

Additional file 3 of 3D organization of regulatory elements for transcriptional regulation in Arabidopsis

Additional file 3: Table S2. Chromatin interactions validated by DNA fluorescence in situ hybridization.

Gene targeting in <i>Arabidopsis</i> via an all‐in‐one strategy that uses a translational enhancer to aid Cas9 expression

Gene targeting, that is precise sequence knockin or replacement, is the ultimate goal of precision genome engineering. Although homologous recombination (HR)-mediated gene targeting is widely used in many organisms, including mammals (Thomas and Capecchi, 1987) (Chandrasegaran and Carroll, 2016), the low frequencies of HR in higher plants limit its utilization for gene targeting (Fauser et al., 2012). We recently described a sequential transformation approach for efficient (5.3%–9.1%) HR-mediated gene targeting in Arabidopsis. Briefly, a construct encoding an HR donor and a single-guide RNA (sgRNA) is transformed into parental transgenic plants that stably express the Cas9 nuclease in egg cells and the early embryo (Miki et al., 2018). Given that the sequential approach requires genetically modified plants already expressing Cas9, which limits its use in diverse genetic backgrounds, we aimed to establish an efficient all-in-one method for gene targeting in the T1 generation of Arabidopsis. Previously, we attempted an all-in-one strategy targeting the GL2 locus. We transformed Arabidopsis with an 'all-in-one' construct that contained: DD45pro::Cas9 (which drives Cas9 expression in egg cells and the early embryo), an HR donor sequence and GL2 sgRNA. Because we had previously found that the DD45pro::Cas9 system in Arabidopsis led to an elevated mutation frequency in T2 populations compared with T1 (Mao et al., 2016), we screened T2 seedlings in bulk (Miki et al., 2018). However, we did not identify any heritable gene targeting events at the GL2 locus after screening 28 T2 pools (Miki et al., 2018). Since we had observed heritable gene targeting of the ROS1 locus upon sequential transformation, we next tested whether we could use an all-in-one strategy to generate a heritable GFP knockin at this locus. We designed an all-in-one construct that contained DD45pro::Cas9, as well as the ROS1 sgRNA and HR donor from our previous report (Miki et al., 2018; Figure 1a). Given that the DD45 promoter drives Cas9 expression in the egg/early embryo, we hypothesized that gene targeting could be established in the T1 generation. We transformed this construct into the nontransgenic Col-0 accession, selected for T1 transformants that were resistant to hygromycin, and performed PCR-based genotyping to detect gene targeting events. We used two strategies for PCR genotyping: one primer set amplifies only the knockin allele ('GT-specific') whereas the other primer set amplifies both the endogenous and the knockin alleles ('external'; Figure 1a). We screened a large number of T1 transformants, given the potentially low efficiency of gene targeting (Wolter et al., 2018), and obtained two heterozygous gene targeting-positive T1 plants from 293 independent T1 transformants (Figure 1b). The GT-specific primer set gave rise to some false positive signals that were not detected by the external primer set (Figure 1b). All plants that were positive for both genotyping strategies could be confirmed by Southern blotting (Figure 1b), and all knockins were inherited in T2 plants (Figure 1c). The gene targeting efficiency of the all-in-one strategy at the ROS1 locus was 0.68% (Figure 1d), almost 10-fold lower than our previously reported sequential transformation strategy (Miki et al., 2018). Transcriptional and translational enhancer sequences have been reported to improve Cas9 expression and, in turn, the efficiency of CRISPR/Cas9-mediated targeted mutagenesis in plants (Gasparis et al., 2018; Kusano et al., 2018). We therefore investigated whether the omega translational enhancer from tobacco mosaic virus (TMV) would improve gene targeting by enhancing Cas9 translation (Gallie and Kado, 1989; Mitsuhara et al., 1996; Figure 1a). We obtained three heterozygous gene targeting-positive T1 plants from 125 independent T1 transformants of the DD45pro::omega enhancer-Cas9 all-in-one (Figure 1e), which were heritable to the T2 generation (Figure 1f). The gene targeting efficiency of the DD45pro::omega enhancer-Cas9 all-in-one strategy was 2.4%, which is at least three times higher than without omega enhancer (Figure 1d). In addition, a previous all-in-one strategy was shown to successfully target the ALS locus in Arabidopsis. This strategy used a chimeric EC1.2/DD45 enhancer with the EC1.1 promoter to drive Cas9 expression in egg cells (Wolter et al., 2018). Therefore, we also generated an all-in-one construct that drives omega translational enhancer-Cas9 expression from a chimeric EC1.2/DD45 enhancer with the EC1.1 promoter (Figure 1a). We screened 190 independent T1 transformants of this all-in-one construct, but all the tested plants were negative (Figure 1d). These data suggest that the EC1.2/DD45 enhancer and EC1.1 promoter are not as efficient for gene targeting compared with the DD45 promoter in our system. Another strategy that has been used to improve gene targeting efficiency in planta is to excise the donor fragment from the T-DNA by using sequence-specific nucleases (SSNs) (Wolter et al., 2018). To determine if excision of the HR donor improves the efficiency of our DD45-promoter-based all-in-one gene targeting system, we designed a construct in which the HR donor sequence was flanked by two recognition sites for the same sgRNA as endogenous ROS1 (Figure 1a). We screened 123 independent T1 transformants, but did not obtain any positive lines with gene targeting events (Figure 1d). These data suggest that excising the HR donor template from the T-DNA does not increase but rather reduces the efficiency of the DD45 promoter-based all-in-one strategy. Our sequential transformation gene targeting strategy did not affect cytosine DNA methylation at the homology arms of homozygous ROS1-GFP T4 plants (Miki et al., 2018). In this study, we did not observe substantial changes in cytosine methylation in either the 5′ or 3′ homology arm regions in two heterozygous DD45pro::Cas9 all-in-one ROS1-GFP T1 GT plants (data not shown), confirming that gene targeting does not affect DNA methylation at the target locus (Miki et al., 2018). To examine whether any new DNA methylation is established de novo at the knockin sequence, we analyzed homozygous T2 DD45pro::Cas9 all-in-one ROS1-GFP GT plants by cytosine methylation sensitive restriction enzymes-based Southern blotting (Figure 1a). All cytosine contexts, CG, CHG and CHH, lacked methylation (Figure 1g), suggesting that de novo methylation did not occur. In summary, our data show that heritable ROS1-GFP GT-positive plants can be obtained by an all-in-one strategy, though at a much lower frequency than with the sequential approach. Further, placing the omega translational enhancer upstream of Cas9 improves the efficiency of gene targeting by the all-in-one strategy. Although our data further suggest that egg cell- and early embryo-specific expression of Cas9 contributes to heritable gene targeting, our all-in-one strategy using a chimeric EC1.2/DD45 enhancer with EC1.1 promoter to drive Cas9 expression in egg cells did not support efficient gene targeting. Previous experiments used the same chimeric promoter to drive Staphylococcus aureus Cas9 (Wolter et al., 2018), whereas we used Streptococcus pyogenes Cas9, which might have contributed to the lower efficiency. Although sequential transformation gives rise to higher gene targeting frequencies, our results suggest that the DD45 promoter-based all-in-one strategy could be optimized to achieve gene targeting, so that gene targeting may be carried out at any genetic background or natural accession of Arabidopsis, or other plant species that can be transformed by floral dipping. We propose that high Cas9 protein levels in the egg cells/early embryos generate efficiently DNA double stranded breaks that promote HR-mediated gene targeting. This work was supported by the Chinese Academy of Sciences to J.K.Z. We would like to thank Life Science Editors for editorial assistance. D.M. and J.K.Z. designed the research; F.N.P., W.X.Z., W.J.Z. and D.M. performed the experiments; D.M. and J.K.Z. supervised the project; D.M., F.N.P., W.X.Z. and J.K.Z. wrote the paper. Correspondence and requests for materials should be addressed to D.M. or J.K.Z. The authors declare no competing financial interests.

<i>Arabidopsis</i> proline-rich protein important for development and abiotic stress tolerance is involved in microRNA biogenesis

MicroRNAs (miRNAs) are important for plant development and stress responses. However, factors regulating miRNA metabolism are not completely understood. SICKLE (SIC), a proline-rich protein critical for development and abiotic stress tolerance of Arabidopsis , was identified in this study. Loss-of-function sic -1 mutant plants exhibited a serrated, sickle-like leaf margin, reduced height, delayed flowering, and abnormal inflorescence phyllotaxy, which are common characteristics of mutants involved in miRNA biogenesis. The sic-1 mutant plants accumulated lower levels of a subset of miRNAs and transacting siRNAs but higher levels of corresponding primary miRNAs than the WT. The SIC protein colocalizes with the miRNA biogenesis component HYL1 in distinct subnuclear bodies. sic-1 mutant plants also accumulated higher levels of introns from hundreds of loci. In addition, sic-1 mutant plants are hypersensitive to chilling and salt stresses. These results suggest that SIC is a unique factor required for the biogenesis of some miRNAs and degradation of some spliced introns and important for plant development and abiotic stress responses.

The ABA Receptor PYL8 Promotes Lateral Root Growth by Enhancing MYB77-Dependent Transcription of Auxin-Responsive Genes

Plants use an alternative abscisic acid signaling pathway to promote lateral root growth.

Coupling of H3K27me3 recognition with transcriptional repression through the BAH-PHD-CPL2 complex in Arabidopsis

Histone 3 Lys 27 trimethylation (H3K27me3)-mediated epigenetic silencing plays a critical role in multiple biological processes. However, the H3K27me3 recognition and transcriptional repression mechanisms are only partially understood. Here, we report a mechanism for H3K27me3 recognition and transcriptional repression. Our structural and biochemical data showed that the BAH domain protein AIPP3 and the PHD proteins AIPP2 and PAIPP2 cooperate to read H3K27me3 and unmodified H3K4 histone marks, respectively, in Arabidopsis. The BAH-PHD bivalent histone reader complex silences a substantial subset of H3K27me3-enriched loci, including a number of development and stress response-related genes such as the RNA silencing effector gene ARGONAUTE 5 (AGO5). We found that the BAH-PHD module associates with CPL2, a plant-specific Pol II carboxyl terminal domain (CTD) phosphatase, to form the BAH-PHD-CPL2 complex (BPC) for transcriptional repression. The BPC complex represses transcription through CPL2-mediated CTD dephosphorylation, thereby causing inhibition of Pol II release from the transcriptional start site. Our work reveals a mechanism coupling H3K27me3 recognition with transcriptional repression through the alteration of Pol II phosphorylation states, thereby contributing to our understanding of the mechanism of H3K27me3-dependent silencing.