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Summary The LRK10‐like receptor kinases (LRK10L‐RLKs) are ubiquitously present in higher plants, but knowledge of their expression and function is still limited. Here, we report expression and functional analysis of TtdLRK10L‐1, a typical LRK10L‐RLK in durum wheat (Triticum turgidum L. ssp. durum). The introns of TtdLRK10L‐1 contained multiple kinds of predicted cis‐elements. To investigate the potential effect of these cis‐elements on TtdLRK10L‐1 expression and function, two types of transgenic wheat lines were prepared, which expressed a GFP‐tagged TtdLRK10L‐1 protein (TtdLRK10L‐1:GFP) from the cDNA or genomic DNA (gDNA) sequence of TtdLRK10L‐1 under the native promoter. TtdLRK10L‐1:GFP expression was up‐regulated by the powdery mildew pathogen Blumeria graminis f. sp. tritici (Bgt) in both types of transgenic plants, with the scale of the elevation being much stronger in the gDNA lines. Both types of transgenic plants exhibited enhanced resistance to Bgt infection relative to wild type control. Notably, the Bgt defence activated in the gDNA lines was significantly stronger than that in the cDNA lines. Further analysis revealed that a putative MYB transcription factor binding site (MYB‐BS, CAGTTA) located in TtdLRK10L‐1 intron I was critical for the efficient expression and function of TtdLRK10L‐1 in Bgt defence. This MYB‐BS could also increase the activity of a superpromoter widely used in ectopic gene expression studies in plants. Together, our results deepen the understanding of the expression and functional characteristics of LRK10L‐RLKs. TtdLRK10L‐1 is likely useful for further dissecting the molecular processes underlying wheat defence against Bgt and for developing Bgt resistant wheat crops.

DNA methylation plays key roles in transposable element (TE) silencing and gene expression regulation. DNA methylation occurs at CG, CHG and CHH sequence contexts in plants. However, the synergistic and redundant roles of CG and non-CG methylation are poorly understood. By introducing CRISPR/Cas9-induced met1 mutation into the ddcc (drm1 drm2 cmt2 cmt3) mutant, we attempted to knock out all five DNA methyltransferases in Arabidopsis and then investigate the synergistic and redundant roles of CG and non-CG DNA methylation. We found that the homozygous ddcc met1 quintuple mutants are embryonically lethal, although met1 and ddcc mutants only display some developmental abnormalities. Unexpectedly, the ddcc met1 quintuple mutations only reduce transmission through the male gametophytes. The ddcc met1+/- mutants show apparent size divergence, which is not associated with difference in DNA methylation patterns, but associated with the difference in the levels of DNA damage. Finally, we show that a group of TEs are specifically activated in the ddcc met1+/- mutants. This work reveals that CG and non-CG DNA methylation synergistically and redundantly regulate plant reproductive development, vegetative development and TE silencing in Arabidopsis. Our findings provide insights into the roles of DNA methylation in plant development.

Trichomes are specialized epidermal cells that act as barriers against biotic and abiotic stresses. Although the formation of trichomes on hairy organs is well studied, the molecular mechanisms of trichome inhibition on smooth organs are still largely unknown. Here, we demonstrate that the CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors inhibit the formation of trichomes on cotyledons in Arabidopsis (Arabidopsis thaliana). The tcp2/3/4/5/10/13/17 septuple mutant produces cotyledons with ectopic trichomes on the adaxial sides. The expression patterns of TCP genes are developmentally regulated during cotyledon development. TCP proteins directly interact with GLABRA3 (GL3), a key component of the MYB transcription factor/basic helix–loop–helix domain protein/WD40-repeat proteins (MYB–bHLH–WD40, MBW) complex essential for trichome formation, to interfere with the transactivation activity of the MBW complex in cotyledons. TCPs also disrupt the MBW complex–R3 MYB negative feedback loop by directly promoting the expression of R3 MYB genes, which enhance the repression of the MBW complex. Our findings reveal a molecular framework in which TCPs suppress trichome formation on adaxial sides of cotyledons by repressing the activity of the MBW complex at the protein level and the transcripts of R3 MYB genes at the transcriptional level.

Argonaute (AGO) family proteins are conserved key components of small RNA-induced silencing pathways. In the RNA-directed DNA methylation (RdDM) pathway in Arabidopsis, AGO6 is generally considered to be redundant with AGO4. In this report, our comprehensive, genomewide analyses of AGO4- and AGO6-dependent DNA methylation revealed that redundancy is unexpectedly negligible in the genetic interactions between AGO4 and AGO6. Immunofluorescence revealed that AGO4 and AGO6 differ in their subnuclear co-localization with RNA polymerases required for RdDM. Pol II and AGO6 are absent from perinucleolar foci, where Pol V and AGO4 are co-localized. In the nucleoplasm, AGO4 displays a strong co-localization with Pol II, whereas AGO6 co-localizes with Pol V. These patterns suggest that RdDM is mediated by distinct, spatially regulated combinations of AGO proteins and RNA polymerases. Consistently, Pol II physically interacts with AGO4 but not AGO6, and the levels of Pol V-dependent scaffold RNAs and Pol V chromatin occupancy are strongly correlated with AGO6 but not AGO4. Our results suggest that AGO4 and AGO6 mainly act sequentially in mediating small RNA-directed DNA methylation.

Rrp6-mediated nuclear RNA surveillance tunes eukaryotic transcriptomes through non-coding RNA degradation and mRNA quality control, including exosomal RNA decay and transcript retention triggered by defective RNA processing. It is unclear whether Rrp6 can positively regulate non-coding RNAs and whether RNA retention occurs in normal cells. Here we report that AtRRP6L1, an Arabidopsis Rrp6-like protein, controls RNA-directed DNA methylation through positive regulation of non-coding RNAs. Discovered in a forward genetic screen, AtRRP6L1 mutations decrease DNA methylation independently of exosomal RNA degradation. Accumulation of Pol V-transcribed scaffold RNAs requires AtRRP6L1 that binds to RNAs in vitro and in vivo. AtRRP6L1 helps retain Pol V-transcribed RNAs in chromatin to enable their scaffold function. In addition, AtRRP6L1 is required for genome-wide Pol IV-dependent siRNA production that may involve retention of Pol IV transcripts. Our results suggest that AtRRP6L1 functions in epigenetic regulation by helping with the retention of non-coding RNAs in normal cells.

Retraction of: The EMBO Journal DOI 10.15252/embj.201694284 | Published online 28.07.2016 The authors have requested retraction of the paper noting: ‘We found a mistake for the materials used …

REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylation enzyme that was previously identified during a genetic screen for the silencing of both RD29A-LUC and 35S-NPTII transgenes on a T-DNA construct. Here we performed a genetic screen to identify additional mutants in which the 35S-NPTII transgene is silenced. We identified several alleles of ros1 and of the following components of the RNA-directed DNA methylation (RdDM) pathway: NRPD1 (the largest subunit of polymerase IV), RDR2, NRPE1 (the largest subunit of polymerase V), NRPD2, AGO4, and DMS3. Our results show that the silencing of 35S-NPTII in the RdDM pathway mutants is due to the reduced expression of ROS1 in the mutants. We also identified a putative histone acetyltransferase (ROS4) from the genetic screen. The acetyltransferase contains a PHD-finger domain that binds to unmethylated histone H3K4. The mutation in ROS4 led to reduction of H3K18 and H3K23 acetylation levels. We show that the silencing of 35S-NPTII and some transposable element genes was released by the ddm1 mutation but that this also required ROS4. Our study identifies a unique antisilencing factor, and reveals that the RdDM pathway has an antisilencing function due to its role in maintaining ROS1 expression.

Induction of secreted acid phosphatase (APase) is a universal response of higher plants to phosphate (Pi) limitation. These enzymes are thought to scavenge Pi from organophosphate compounds in the rhizosphere and thus to increase Pi availability to plants when Pi is deficient. The tight association of secreted APase with the root surface may make plants more efficient in the utilization of soil Pi around root tissues, which is present in organophosphate forms. To date, however, no systematic molecular, biochemical, and functional studies have been reported for any of the Pi starvation-induced APases that are associated with the root surface after secretion. In this work, using genetic and molecular approaches, we identified Arabidopsis (Arabidopsis thaliana) Purple Acid Phosphatase10 (AtPAP10) as a Pi starvation-induced APase that is predominantly associated with the root surface. The AtPAP10 protein has phosphatase activity against a variety of substrates. Expression of AtPAP10 is specifically induced by Pi limitation at both transcriptional and posttranscriptional levels. Functional analyses of multiple atpap10 mutant alleles and overexpressing lines indicated that AtPAP10 plays an important role in plant tolerance to Pi limitation. Genetic manipulation of AtPAP10 expression may provide an effective means for engineering new crops with increased tolerance to Pi deprivation.

In Arabidopsis, an RNA-directed DNA methylation pathway (RdDM) is responsible for de novo establishment of DNA methylation and contributes to transcriptional gene silencing. Recently, the microrchidia (MORC)-type ATPases were shown to play essential roles in enforcing transcriptional gene silencing of a subset of genes and transposons by regulating the formation of higher-order chromatin architecture. However, how MORC proteins cooperate with the RdDM pathway components to regulate gene expression remains largely unclear. In this study, SUVH9 and MORC6 were identified from a screening of suppressors of idm1, which is a mutant defective in active DNA demethylation. SUVH9 and MORC6 are required for silencing of two reporter genes and some endogenous genes without enhancing DNA methylation levels. SUVH9, one of SU(VAR)3-9 homologs involved in RdDM, directly interacts with MORC6 and its two close homologs, MORC1 and MORC2. Similar to MORC6, SUVH9 and its homolog SUVH2 are required for heterochromatin condensation and formation of 3D chromatin architecture at SDC and Solo-LTR loci. We propose that SUVH2 and SUVH9 bind to the methylated DNA and facilitate the recruitment of a chromatin-remodeling complex to the target loci in association with MORC proteins.

Phosphomannomutase (PMM) catalyzes the interconversion of mannose-6-phosphate and mannose-1-phosphate. However, systematic molecular and functional investigations on PMM from higher plants have hitherto not been reported. In this work, PMM cDNAs were isolated from Arabidopsis, Nicotiana benthamiana, soybean, tomato, rice and wheat. Amino acid sequence comparisons indicated that plant PMM proteins exhibited significant identity to their fungal and mammalian orthologs. In line with the similarity in primary structure, plant PMM complemented the sec53-6 temperature sensitive mutant of Saccharomyces cerevisiae. Histidine-tagged Arabidopsis PMM (AtPMM) purified from Escherichia coli converted mannose-1-phosphate into mannose-6-phosphate and glucose-1-phosphate into glucose-6-phosphate, with the former reaction being more efficient than the latter one. In Arabidopsis and N. benthamiana, PMM was constitutively expressed in both vegetative and reproductive organs. Reducing the PMM expression level through virus-induced gene silencing caused a substantial decrease in ascorbic acid (AsA) content in N. benthamiana leaves. Conversely, raising the PMM expression level in N. benthamiana using viral-vector-mediated ectopic expression led to a 20-50% increase in AsA content. Consistent with this finding, transgenic expression of an AtPMM-GFP fusion protein in Arabidopsis also increased AsA content by 25-33%. Collectively, this study improves our understanding on the molecular and functional properties of plant PMM and provides genetic evidence on the involvement of PMM in the biosynthesis of AsA in Arabidopsis and N. benthamiana plants.

Active DNA demethylation plays crucial roles in the regulation of gene expression in both plants and animals. In Arabidopsis thaliana, active DNA demethylation is initiated by the ROS1 subfamily of 5-methylcytosine-specific DNA glycosylases via a base excision repair mechanism. Recently, IDM1 and IDM2 were shown to be required for the recruitment of ROS1 to some of its target loci. However, the mechanism(s) by which IDM1 is targeted to specific genomic loci remains to be determined. Affinity purification of IDM1- and IDM2- associating proteins demonstrated that IDM1 and IDM2 copurify together with two novel components, methyl-CpG-binding domain protein 7 (MBD7) and IDM2-like protein 1 (IDL1). IDL1 encodes an α-crystallin domain protein that shows high sequence similarity with IDM2. MBD7 interacts with IDM2 and IDL1 in vitro and in vivo and they form a protein complex associating with IDM1 in vivo. MBD7 directly binds to the target loci and is required for the H3K18 and H3K23 acetylation in planta. MBD7 dysfunction causes DNA hypermethylation and silencing of reporter genes and a subset of endogenous genes. Our results suggest that a histone acetyltransferase complex functions in active DNA demethylation and in suppression of gene silencing at some loci in Arabidopsis., Author Summary DNA cytosine methylation (5-methylcytosine, 5-meC) plays important roles in genome organization, genomic imprinting, transposon silencing and gene expression. DNA methylation patterns are dynamically controlled by methylation and demethylation reactions during development and reproduction in eukaryotes. In Arabidopsis thaliana, ROS1, a 5-methylcytosine DNA glycosylase, is responsible for active DNA demethylation via a base excision repair pathway. Our previous work showed that a histone acetyltransferase, IDM1, is an important regulator of active DNA demethylation. Here we identified two components, IDL1 and MBD7, as additional novel regulators of active DNA demethylation. Our data suggest that a histone acetyltransferase complex, composed of MBD7, IDL1, IDM2 and IDM1, is required for the recruitment of ROS1 to specific loci for active DNA demethylation. Our findings significantly expand our understanding of the regulatory mechanisms underlying active DNA demethylation in plants.

An elevated growth temperature often inhibits plant defense responses and renders plants more susceptible to pathogens. However, the molecular mechanisms underlying this modulation are unknown. To genetically dissect this regulation, we isolated mutants that retain disease resistance at a higher growth temperature in Arabidopsis. One such heat-stable mutant results from a point mutation in SNC1, a NB-LRR encoding gene similar to disease resistance (R) genes. Similar mutations introduced into a tobacco R gene, N, confer defense responses at elevated temperature. Thus R genes or R-like genes involved in recognition of pathogen effectors are likely the causal temperature-sensitive component in defense responses. This is further supported by snc1 intragenic suppressors that regained temperature sensitivity in defense responses. In addition, the SNC1 and N proteins had a reduction of nuclear accumulation at elevated temperature, which likely contributes to the inhibition of defense responses. These findings identify a plant temperature sensitive component in disease resistance and provide a potential means to generate plants adapting to a broader temperature range., Author Summary It has been known that temperature modulates plant immune responses, but the molecular mechanisms underlying this modulation are unknown. Our study describes a novel finding that the NB-LRR type of R or R-like protein is the temperature-sensitive component of plant defense responses. R or R-like proteins have ‘receptor’ like functions involved in specific recognition of pathogens. Through genetic screens and targeted mutagenesis, we found that alterations in the R-like gene SNC1 and the R gene N can change temperature sensitivity of defense responses. Further, an elevated temperature reduced the nuclear accumulation of SNC1, which likely contributes to the inhibition of disease resistance at high temperatures. Our study indicates that NB-LRR proteins mediate temperature sensitivity in plant immune responses.

High molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe a more detailed characterization of the HMW glutenin subunits from Aegilops searsii, which is diploid and contains the S(s) genome related to the S genome of Aegilops speltoides and the A, B and D genomes of hexaploid wheat. SDS-PAGE experiments revealed two subunits (one x and one y) for each of the nine Ae. searsii accessions analyzed, indicating that the HMW glutenin subunit gene locus of Ae. searsii is similar to the Glu-1 locus found in wheat in containing both x and y genes. The primary structure of the four molecularly cloned subunits (from two Ae. searsii accessions) was highly similar to that of the previously reported x and y subunits. However, in one accession (IG49077), the last 159 residues of the x subunit (1S(s)x49077), which contained the sequence element GHCPTSPQQ, were identical to those of the y subunit (1S(s)y49077) from the same accession. Consequently, 1S(s)x49077 contains an extra cysteine residue located at the C-terminal part of its repetitive domain, which is novel compared to the x-type subunits reported so far. Based on this and previous studies, the structure and expression of the Glu-1 locus in Ae. searsii is discussed. A hypothesis on the genetic mechanism generating the coding sequence for the novel 1S(s)x49077 subunit is presented.

Purple acid phosphatases (PAPs) are metallo-phosphoesterases. Their expression and function have not been systematically investigated in higher plants. In this work, we compared the transcript levels of 28 Arabidopsis PAP (AtPAP) genes in five Arabidopsis organs. The 28 members, although differed in their expression patterns in vegetative organs, were all transcribed in flower. Furthermore, the transcription of seven members (AtPAPs 6, 11, 14, 19, 23, 24 and 25) occurred predominantly in the flower. To begin dissecting the role of AtPAP genes in flower development, further expression and functional analyses were conducted using AtPAP23. Histochemical staining of transgenic plants expressing AtPAP23 promoter-beta-glucuronidase (GUS) gene construct revealed that AtPAP23 transcription was strong in flower apical meristems, but became restricted to petals and anther filaments in fully developed flower. A GST (glutathione S-transferase) fusion protein of AtPAP23 (GST:AtPAP23) was expressed in bacterial cells, and was found to contain significant amounts of Fe and Mn (whereas the control GST protein contained none). In biochemical tests, GST:AtPAP23 showed typical acid phosphatase activities. The fusion protein was also highly active on phosphoserine, but not phosphotyrosine. Despite its highly specific expression pattern and the demonstrated biochemical function of its protein product, the RNAi (RNA interference), T-DNA knock-out and overexpression lines of AtPAP23 were indistinguishable from wild type plants in the development of flower (or other organs). Interestingly, the Fe and Mn contents were found significantly increased in AtPAP23 overexpression lines, which may offer a new direction for further functional studies of AtPAPs in Arabidopsis.

Background Phosphomannomutase (PMM) is an essential enzyme in eukaryotes. However, little is known about PMM gene and function in crop plants. Here, we report molecular evolutionary and biochemical analysis of PMM genes in bread wheat and related Triticeae species. Results Two sets of homoeologous PMM genes (TaPMM-1 and 2) were found in bread wheat, and two corresponding PMM genes were identified in the diploid progenitors of bread wheat and many other diploid Triticeae species. The duplication event yielding PMM-1 and 2 occurred before the radiation of diploid Triticeae genomes. The PMM gene family in wheat and relatives may evolve largely under purifying selection. Among the six TaPMM genes, the transcript levels of PMM-1 members were comparatively high and their recombinant proteins were all enzymatically active. However, PMM-2 homoeologs exhibited lower transcript levels, two of which were also inactive. TaPMM-A1, B1 and D1 were probably the main active isozymes in bread wheat tissues. The three isozymes differed from their counterparts in barley and Brachypodium distachyon in being more tolerant to elevated test temperatures. Conclusion Our work identified the genes encoding PMM isozymes in bread wheat and relatives, uncovered a unique PMM duplication event in diverse Triticeae species, and revealed the main active PMM isozymes in bread wheat tissues. The knowledge obtained here improves the understanding of PMM evolution in eukaryotic organisms, and may facilitate further investigations of PMM function in the temperature adaptability of bread wheat.