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Type-II metacaspases are conserved cysteine proteases found in eukaryotes with oxygenic photosynthesis, including green plants and some algae, such as Chlamydomonas and Volvox. Genetic and biochemical studies showed that some members in this protease family could be involved in oxidative stress-induced cell death in higher plants, but their regulatory mechanisms remain unclear. Biochemically, two distinct classes of type-II metacaspases are exemplified by AtMC4 and AtMC9 from Arabidopsis, with AtMC4 activation dependent on calcium under neutral pH, whereas AtMC9 is active only under mildly acidic pH, regardless of the availability of calcium. Here, we constructed all six possible combinations between the p20, linker, and p10 domains from AtMC4 and AtMC9. Our results show that calcium stimulation of AtMC4 activity is associated with essential amino acids located in its p20 domain. In contrast, the acidic pH optimum trait is lost from AtMC9 if one or two of its domains are replaced by that from AtMC4, suggesting that multiple interactions between domains in AtMC9 may be responsible for this property. Consistent with this, we found conserved 'signature' residues in each of the three domains that distinguish AtMC4- and AtMC9-like classes of metacaspases. Tracing the origin of the AtMC9 class, we found evidence for its appearance between lycophytes and gymnosperms, coincident with the evolution of more complex root archetypes in terrestrial plants. Our work suggests that the distinctive properties of the AtMC9-like protease could be associated with special cellular physiology in the roots of gymnosperms and angiosperms that are distinct from lycophytes., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Duckweeds are the fastest growing angiosperms and have the potential to become a new generation of sustainable crops. Although a seed plant, Spirodela polyrhiza clones rarely flower and multiply mainly through vegetative propagation. Whole-genome sequencing using different approaches and clones yielded two reference maps. One for clone 9509, supported in its assembly by optical mapping of single DNA molecules, and one for clone 7498, supported by cytogenetic assignment of 96 fingerprinted bacterial artificial chromosomes (BACs) to its 20 chromosomes. However, these maps differ in the composition of several individual chromosome models. We validated both maps further to resolve these differences and addressed whether they could be due to chromosome rearrangements in different clones. For this purpose, we applied sequential multicolor fluorescence in situ hybridization (mcFISH) to seven S. polyrhiza clones, using 106 BACs that were mapped onto the 39 pseudomolecules for clone 7498. Furthermore we integrated high-depth Oxford Nanopore (ON) sequence data for clone 9509 to validate and revise the previously assembled chromosome models. We found no major structural rearrangements between these seven clones, identified seven chimeric pseudomolecules and Illumina assembly errors in the previous maps, respectively. A new S. polyrhiza genome map with high contiguity was produced with the ON sequence data and genome-wide synteny analysis supported the occurrence of two Whole Genome Duplication events during its evolution. This work generated a high confidence genome map for S. polyrhiza at the chromosome scale, and illustrates the complementarity of independent approaches to produce whole-genome assemblies in the absence of a genetic map., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Spirodela polyrhiza is a fast-growing aquatic monocot with highly reduced morphology, genome size and number of protein-coding genes. Considering these biological features of Spirodela and its basal position in the monocot lineage, understanding its genome architecture could shed light on plant adaptation and genome evolution. Like many draft genomes, however, the 158-Mb Spirodela genome sequence has not been resolved to chromosomes, and important genome characteristics have not been defined. Here we deployed rapid genome-wide physical maps combined with high-coverage short-read sequencing to resolve the 20 chromosomes of Spirodela and to empirically delineate its genome features. Our data revealed a dramatic reduction in the number of the rDNA repeat units in Spirodela to fewer than 100, which is even fewer than that reported for yeast. Consistent with its unique phylogenetic position, small RNA sequencing revealed 29 Spirodela-specific microRNA, with only two being shared with Elaeis guineensis (oil palm) and Musa balbisiana (banana). Combining DNA methylation data and small RNA sequencing enabled the accurate prediction of 20.5% long terminal repeats (LTRs) that doubled the previous estimate, and revealed a high Solo:Intact LTR ratio of 8.2. Interestingly, we found that Spirodela has the lowest global DNA methylation levels (9%) of any plant species tested. Taken together our results reveal a genome that has undergone reduction, likely through eliminating non-essential protein coding genes, rDNA and LTRs. In addition to delineating the genome features of this unique plant, the methodologies described and large-scale genome resources from this work will enable future evolutionary and functional studies of this basal monocot family., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

The moss Physcomitrella patens is an important model organism for evo-devo studies. Here, we determined the genome-wide chromatin landscape of five important histone three (H3) modifications (H3K4me3, H3K27me3, H3K27Ac, H3K9Ac and H3K9me2) and describe the changes to these histone marks in two contrasted situations, developmental transition and abiotic (drought) stress. Integrative analysis of these histone H3 modifications revealed their preferential association into 15 chromatin states (CS) in genic regions of the P. patens genome. Synergistic relationships that influence expression levels were revealed for the three activating marks H3K4me3, H3K27Ac and H3K9Ac, while an antagonistic relationship was found between CS containing the H3K27me3 and H3K27Ac marks, suggesting that H3K27 is a key indexing residue regarding transcriptional output. Concerning the alteration of histone marks in response to developmental transition (juvenile to adult) and drought stress, the three activating marks H3K4me3, H3K27Ac and H3K9Ac show significant changes in both situations. However, changes to H3K27me3 are central only for genes differentially expressed during development. Interestingly, genes induced during drought stress show significant histone mark toggling during developmental transition. This situation suggests that drought induced adult (gametophore expressed) genes are primed to respond to this stress during the juvenile to adult transition., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

Genome-wide analyses of epigenomic and transcriptomic profiles provide extensive resources for discovering epigenetic regulatory mechanisms. However, the construction of functionally relevant hypotheses from correlative patterns and the rigorous testing of these hypotheses may be challenging. We combined bioinformatics-driven hypothesis building with mutant analyses to identify potential epigenetic mechanisms using the model plant Arabidopsis thaliana. Genome-wide maps of nine histone modifications produced by ChIP-seq were used together with a strand-specific RNA-seq dataset to profile the epigenome and transcriptome of Arabidopsis. Combinatorial chromatin patterns were described by 42 major chromatin states with selected states validated using the re-ChIP assay. The functional relevance of chromatin modifications was analyzed using the ANchored CORrelative Pattern (ANCORP) method and a newly developed state-specific effects analysis (SSEA) method, which interrogates individual chromatin marks in the context of combinatorial chromatin states. Based on results from these approaches, we propose the hypothesis that cytosine methylation (5mC) and histone methylation H3K36me may synergistically repress production of natural antisense transcripts (NATs) in the context of actively expressed genes. Mutant analyses supported this proposed model at a significant proportion of the tested loci. We further identified polymerase-associated factor as a potential repressor for NAT abundance. Although the majority of tested NATs were found to localize to the nucleus, we also found evidence for cytoplasmically partitioned NATs. The significance of the subcellular localization of NATs and their biological functions remain to be defined., (© 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.)

Cysteine proteases such as caspases play important roles in programmed cell death (PCD) of metazoans. Plant metacaspases (MCPs), a family of cysteine proteases structurally related to caspases, have been hypothesized to be ancestors of metazoan caspases, despite their different substrate specificity. Arabidopsis thaliana contains six type II MCP genes (AtMCP2a-f). Whether and how these individual members are involved in controlling PCD in plants remains largely unknown. Here we investigated the function and regulation of AtMCP2d, the predominant and constitutively expressed member of type II MCPs, in stress-inducible PCD. Two AtMCP2d mutants (mcp2d-1 and mcp2d-3) exhibited reduced sensitivity to PCD-inducing mycotoxin fumonisin B1 as well as oxidative stress inducers, whereas AtMCP2d over-expressors were more sensitive to these agents, and exhibited accelerated cell-death progression. We found that AtMCP2d exclusively localizes to the cytosol, and its accumulation and self-processing patterns were age-dependent in leaves. Importantly, active proteolytic processing of AtMCP2d proteins dependent on its catalytic activity was observed in mature leaves during mycotoxin-induced cell death. We also found that mcp2d-1 leaves exhibited reduced cell death in response to Pseudomonas syringae carrying avirulent gene avrRpt2, and that self-processing of AtMCP2d was also detected in wild-type leaves in response to this pathogen. Furthermore, increases in processed AtMCP2d proteins were found to correlate with conditional cell-death induction in two lesion-mimic mutants (cpr22 and ssi4) that exhibit spontaneous cell-death phenotypes. Taken together, our data strongly suggest that AtMCP2d plays a positive regulatory role in biotic and abiotic stress-induced PCD., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Chromatin components can be extensively modified and dynamically regulated by a plethora of catalytic complexes. The numerous modifications may form a type of molecular pattern that defines particular local and global 'chromatin states' through extensive cross-talk. Analyses that can integrate multiple genome-wide datasets are essential to determine the interactions and biological function of chromatin modifications in various contexts. Through a combination of hierarchical clustering and pattern visualization, we categorized all annotated Arabidopsis genes into 16 chromatin state clusters using combinations of four chromatin marks (H3K4me3, H3K36me2, H3K27me3 and cytosine methylation) using publicly available data. Our results suggest that gene length may be an important factor in shaping chromatin states across transcription units. By analysis of two rare chromatin states, we found that the enrichment of H3K36me2 around the transcription start site is negatively correlated with transcriptional activities. High-resolution association analyses in the context of chromatin states have identified inter-correlations between chromatin modifications. H3K4me3 were found to be under-represented in actively transcribed regions that are modified by DNA methylation and the H3K36me2 mark, concomitant with increased nucleosome occupancy in these regions. Lastly, quantitative data from transcriptome analyses and gene ontology partitioning were integrated to determine the possible functional relevance of the corresponding chromatin states. We show that modelling the plant epigenome in terms of chromatin states and combining correlative visualization methods can be a productive approach to unravel complex relationships between epigenomic features and the functional output of the genome.

The interphase nucleus exists as a highly dynamic system, the physical properties of which have functional importance in gene regulation. Not only can gene expression be influenced by the local sequence context, but also by the architecture of the nucleus in three-dimensions (3D), and by the interactions between these levels via chromatin modifications. A challenging task is to resolve the complex interplay between sequence- and genome structure-based control mechanisms. Here, we created a collection of 277 Arabidopsis lines that allow the visual tracking of individual loci in living plants while comparing gene expression potential at these locations, via an identical reporter cassette. Our studies revealed regional gene silencing near a heterochromatin island, via DNA methylation, that is correlated with mobility constraint and nucleolar association. We also found an example of nucleolar association that does not correlate with gene suppression, suggesting that distinct mechanisms exist that can mediate interactions between chromatin and the nucleolus. These studies demonstrate the utility of this novel resource in unifying structural and functional studies towards a more comprehensive model of how global chromatin organization may coordinate gene expression over large scales.

Programmed cell death (PCD) is a common process in eukaryotes during development and in response to pathogens and stress signals. Bax inihibitor-1 (BI-1) is proposed to be a cell death suppressor that is conserved in both animals and plants, but the physiological importance of BI-1 and the impact of its loss of function in plants are still unclear. In this study, we identified and characterized two independent Arabidopsis mutants with a T-DNA insertion in the AtBI1 gene. The phenotype of atbi1-1 and atbi1-2, with a C-terminal missense mutation and a gene knockout, respectively, was indistinguishable from wild-type plants under normal growth conditions. However, these two mutants exhibit accelerated progression of cell death upon infiltration of leaf tissues with a PCD-inducing fungal toxin fumonisin B1 (FB1) and increased sensitivity to heat shock-induced cell death. Under these conditions, expression of AtBI1 mRNA was up-regulated in wild-type leaves prior to the activation of cell death, suggesting that increase of AtBI1 expression is important for basal suppression of cell death progression. Over-expression of AtBI1 transgene in the two homozygous mutant backgrounds rescued the accelerated cell death phenotypes. Together, our results provide direct genetic evidence for a role of BI-1 as an attenuator for cell death progression triggered by both biotic and abiotic types of cell death signals in Arabidopsis.

Fluorescent chromatin tagging makes possible tracking of specific loci in vivo and in situ. Loci tagged by the lac operator (lacO)/GFP-LacI/Nuclear Localization Signal (NLS) system show rapid motility and constrained chromatin dynamics in somatic nuclei of a transgenic line, designated EL702C, in Arabidopsis thaliana. The tagged loci associated with each other significantly more often than expected at random, due to homologous pairing of the lacO tandem repeat arrays. Furthermore, these arrays associated significantly more often than average euchromatic regions with heterochromatic chromocenters (CCs). We show now that the inserted lacO array in this transgenic line became strongly methylated at CG sites in the T3 generation, which can be reversed upon transfer into the mutant backgrounds of decrease in DNA methylation 1 (ddm1) and methyltransferase 1 (met1). Concomitantly, the tagged loci showed lower association frequencies as compared with the transgenics in wild-type background, which is correlated with a significant decrease in allelic and ectopic pairing of the lacO repeat arrays as visualized by fluorescence in situ hybridization. In contrast, the preferential association of the lacO arrays with heterochromatin, locus mobility in somatic nuclei and transcription of neighboring transgenes were not altered by reduced DNA methylation in ddm1 and met1 backgrounds. Our results show that repeat arrays can activate hypermethylation of the inserted locus that correlates with high frequencies of homologous pairing in somatic cells. In contrast, the preferential association of these inserted arrays with CCs in plant cells occurs through another mechanism.

Transcription factors often belong to multigene families and their individual contribution in a particular regulatory network remains difficult to assess. We show here that specific members from a family of conserved Arabidopsis bZIP transcription factors, the TGA proteins, are regulated in their protein stability by developmental stage-specific proteolysis. Using GFP fusions of three different Arabidopsis TGA factors that represent members of distinct subclasses of the TGA factor family, we demonstrate that two of these TGA proteins are specifically targeted for proteolysis in mature leaf cells. Using a supershift gel mobility assay, we found evidence for similar regulation of the cognate proteins as compared to the GFP fusion proteins expressed under the cauliflower mosaic virus (CaMV) 35S promoter. Using various inhibitors, we showed that the expression of at least one of these three TGA factors could be stabilized by inhibition of proteasome-mediated proteolysis. This study indicates that TGA transcription factors may be regulated by distinct pathways of targeted proteolysis that can serve to modulate the contribution of specific members of a multigene family in complex regulatory pathways.

One of the earliest signal transduction events that trigger the hypersensitive response (HR) of plants against pathogen attack is thought to be an alteration of proton flux across the plasma membrane (PM). However, no direct genetic evidence for the involvement of PM-localised proton channels or pumps in the induction of this response has been reported. We previously showed that expression of the bacterial proton pump bacterio-opsin (bO) in transgenic plants resulted in the spontaneous activation of the HR. Here we show that the bO protein is likely localised to the PM in transgenic tobacco plants. Furthermore, mutational analysis shows that induction of the HR by bO expression is dependent upon the capability of bO to translocate protons. Although bO functions as a light-driven proton pump in Halobacteria when assembled with retinal, we also show by mutational analysis that this chromophore binding is unnecessary for its in planta activity. Taken together, our results suggest that expression of bO in plants leads to the insertion of a passive proton channel into the PM. The activity of this channel in the PM results in spontaneous activation of cell death and HR-associated phenotypes including enhanced resistance to a broad spectrum of plant pathogens. Our work provides direct molecular evidence to support a working model in which alterations in ionic homeostasis at the level of the PM may work as one of the critical steps in the signalling pathway for the activation of the HR.

Salicylic acid (SA) is a key regulator for the induction of systemic acquired resistance (SAR), and NPR1 is a critical mediator for the biological effects of SA. Physical interactions between NPR1 and TGA factors, a conserved family of basic-leucine-zipper (bZip) proteins in plants, have suggested a role for these transcription factors in mediating SAR induction via the regulation of defense genes. To elucidate this function, we constructed a trans-dominant mutant that specifically eliminates DNA-binding activities of this class of bZip proteins in transgenic tobacco plants. Our results demonstrate that the loss of TGA DNA-binding activities is correlated with suppression of two xenobiotic-responsive genes, GNT35 and STR246, and enhanced induction of pathogenesis-related (PR) genes by SA. In addition, these TGA-suppressed plants exhibited higher levels of PR gene induction by pathogen challenge and an enhanced SAR. These results suggest that TGA transcription factors serve both negative and positive regulatory roles in mediating plant defense responses.

Activating sequence factor 1 (ASF-1) is a conserved DNA-binding activity that interacts with sequence elements containing TGACG motifs, some of which have been demonstrated to respond to exogenous application of auxin and salicylic acid. Genes encoding transcription factors with similar DNA-binding specificity to ASF-1 have been cloned from diverse plant species and these factors all contain a distinct basic-leucine-zipper (bZIP) motif. Members of this family of DNA-binding proteins, designated as TGA factors, have been shown to interact with similar DNA sequences and at least seven distinct TGA genes are present in the Arabidopsis genome. To study the roles that this family of factors may play in plant development, a trans-dominant inhibitor of ASF-1 was constructed by deleting the basic portion of the bZIP domain in the tobacco factor TGA1a. In vitro co-expression studies demonstrated that this deletion mutant, named TGA1a-D, suppresses the DNA-binding activity of wild-type tobacco TGA1a, and three different members of the Arabidopsis TGA family. In contrast, co-expression of TGA1a-D with another class of bZIP proteins, the G-Box Binding Factor family, showed no suppression of DNA-binding activity. Over-expression of TGA1a-D in transgenic tobacco significantly decreased nuclear ASF-1 relative to several other known factors, indicating that the proteins comprising ASF-1 activity in vivo are likely TGA family members. Thus, TGA1a-D may be a family-specific inhibitor for the TGA family and should facilitate the study of ASF-1 function in vivo.

A major drawback to study gene functions in plant systems is the lack of an effective gene knockout strategy. With a large number of plant genes isolated and the accelerating pace by which this collection is growing, the need for their functional analyses at the whole plant level has become increasingly urgent. Here evidence is reported for the first successful disruption of a non-selectable gene in Arabidopsis thaliana by creating a mutant of the TGA3 locus via targeted insertion of the bacterial neo gene conferring kanamycin (Km) resistance. A beta-glucuronidase (GUS) expression unit outside the region of homology was used as a screenable marker to distinguish homologous recombination events from those of ectopic insertions. PCR amplification coupled with Southern blot screening identified two putative homologous recombination events among 2580 Kmr calli. One callus line was subsequently isolated and the structure of the targeted TGA3 allele confirmed by Southern blot analyses. This study demonstrates the feasibility of targeting a non-selectable locus in Arabidopsis. Combined with future improvements in negative selection strategies and efficient transformation methodologies, gene replacement studies in plants could become a routine technique.

The genes encoding the major light-harvesting antenna chlorophyll a/b-binding protein (Cab) of higher plants are regulated by light at the transcriptional level. In addition, their expression is largely restricted to photosynthetically competent organs such as leaves. A 268 bp fragment of the Cab-1 promoter from wheat functions as a light-responsive and organ-specific enhancer in transgenic tobacco. Using DNase I footprinting, four different regions have been mapped (Cab1-A, Cab1-B, Cab1-C and Cab1-D) in this fragment that bind to protein factors in tobacco nuclear extracts. Three of these regions (A, B and C) coincide with sequences that have been found to be functionally important from previous cis-element analyses. Synthetic tetramers of these three sites interact with different proteins in gel retardation assays. In addition, cross-competition analyses demonstrate that Cab1-C is likely to interact with ASF-2, a tobacco DNA-binding activity that binds to a conserved GATA element found in many dicot Cab promoters. In transgenic tobacco, a 95 bp fragment of the Cab-1 enhancer containing the A, B and C regions can confer leaf expression when fused upstream of a truncated derivative of the cauliflower mosaic virus (CaMV) 35S promoter. However, expression observed with this enhancer fragment in the promoter context of these studies does not appear to be significantly dependent on light. Similar results were obtained with synthetic tetramers of Cab1-A, -B or -C. These data thus suggest that the wheat Cab-1 enhancer contains at least three distinct elements that contribute to leaf-specific expression in transgenic tobacco. Interaction between factors binding to these positive elements and those that bind elsewhere in the Cab-1 enhancer may be necessary for light-responsive transcriptional activation.