DamID-seq: A Genome-Wide DNA Methylation Method that Captures Both Transient and Stable TF-DNA Interactions in Plant Cells.

Capturing the dynamic and transient interactions of a transcription factor (TF) with its genome-wide targets whose regulation leads to plants' adaptation to their changing environment is a major technical challenge. This is a widespread problem with biochemical methods such as chromatin immunoprecipitation-sequencing (ChIP-seq) which are biased towards capturing stable TF-target gene interactions. Herein, we describe how DNA adenine methyltransferase identification and sequencing (DamID-seq) can be used to capture both transient and stable TF-target interactions by DNA methylation. The DamID technique uses a TF protein fused to a DNA adenine methyltransferase (Dam) from E. coli. When expressed in a plant cell, the Dam-TF fusion protein will methylate adenine (A) bases near the sites of TF-DNA interactions. In this way, DamID results in a permanent, stable DNA methylation mark on TF-target gene promoters, even if the target gene is only transiently "touched" by the Dam-TF fusion protein. Here we provide a step-by-step protocol to perform DamID-seq experiments in isolated plant cells for any Dam-TF fusion protein of interest. We also provide information that will enable researchers to analyze DamID-seq data to identify TF-binding sites in the genome. Our protocol includes instructions for vector cloning of the Dam-TF fusion proteins, plant cell protoplast transfections, DamID preps, library preparation, and sequencing data analysis. The protocol outlined in this chapter is performed in Arabidopsis thaliana, however, the DamID-seq workflow developed in this guide is broadly applicable to other plants and organisms.

Soybean photosynthesis and crop yield are improved by accelerating recovery from photoprotection.

Crop leaves in full sunlight dissipate damaging excess absorbed light energy as heat. This protective dissipation continues after the leaf transitions to shade, reducing crop photosynthesis. A bioengineered acceleration of this adjustment increased photosynthetic efficiency and biomass in tobacco in the field. But could that also translate to increased yield in a food crop? Here we bioengineered the same change into soybean. In replicated field trials, photosynthetic efficiency in fluctuating light was higher and seed yield in five independent transformation events increased by up to 33%. Despite increased seed quantity, seed protein and oil content were unaltered. This validates increasing photosynthetic efficiency as a much needed strategy toward sustainably increasing crop yield in support of future global food security.

Into the Shadows and Back into Sunlight: Photosynthesis in Fluctuating Light.

Photosynthesis is an important remaining opportunity for further improvement in the genetic yield potential of our major crops. Measurement, analysis, and improvement of leaf CO2 assimilation (A) have focused largely on photosynthetic rates under light-saturated steady-state conditions. However, in modern crop canopies of several leaf layers, light is rarely constant, and the majority of leaves experience marked light fluctuations throughout the day. It takes several minutes for photosynthesis to regain efficiency in both sun-shade and shade-sun transitions, costing a calculated 10-40% of potential crop CO2 assimilation. Transgenic manipulations to accelerate the adjustment in sun-shade transitions have already shown a substantial productivity increase in field trials. Here, we explore means to further accelerate these adjustments and minimize these losses through transgenic manipulation, gene editing, and exploitation of natural variation. Measurement andanalysis of photosynthesis in sun-shade and shade-sun transitions are explained. Factors limiting speeds of adjustment and how they could be modified to effect improved efficiency are reviewed, specifically nonphotochemical quenching (NPQ), Rubisco activation, and stomatal responses.

Transient genome-wide interactions of the master transcription factor NLP7 initiate a rapid nitrogen-response cascade.

Dynamic reprogramming of gene regulatory networks (GRNs) enables organisms to rapidly respond to environmental perturbation. However, the underlying transient interactions between transcription factors (TFs) and genome-wide targets typically elude biochemical detection. Here, we capture both stable and transient TF-target interactions genome-wide within minutes after controlled TF nuclear import using time-series chromatin immunoprecipitation (ChIP-seq) and/or DNA adenine methyltransferase identification (DamID-seq). The transient TF-target interactions captured uncover the early mode-of-action of NIN-LIKE PROTEIN 7 (NLP7), a master regulator of the nitrogen signaling pathway in plants. These transient NLP7 targets captured in root cells using temporal TF perturbation account for 50% of NLP7-regulated genes not detectably bound by NLP7 in planta. Rapid and transient NLP7 binding activates early nitrogen response TFs, which we validate to amplify the NLP7-initiated transcriptional cascade. Our approaches to capture transient TF-target interactions genome-wide can be applied to validate dynamic GRN models for any pathway or organism of interest.

Photosystem II Subunit S overexpression increases the efficiency of water use in a field-grown crop.

Insufficient water availability for crop production is a mounting barrier to achieving the 70% increase in food production that will be needed by 2050. One solution is to develop crops that require less water per unit mass of production. Water vapor transpires from leaves through stomata, which also facilitate the influx of CO2 during photosynthetic assimilation. Here, we hypothesize that Photosystem II Subunit S (PsbS) expression affects a chloroplast-derived signal for stomatal opening in response to light, which can be used to improve water-use efficiency. Transgenic tobacco plants with a range of PsbS expression, from undetectable to 3.7 times wild-type are generated. Plants with increased PsbS expression show less stomatal opening in response to light, resulting in a 25% reduction in water loss per CO2 assimilated under field conditions. Since the role of PsbS is universal across higher plants, this manipulation should be effective across all crops.

Engineering the lutein epoxide cycle into Arabidopsis thaliana.

Although sunlight provides the energy necessary for plants to survive and grow, light can also damage reaction centers of photosystem II (PSII) and reduce photochemical efficiency. To prevent damage, plants possess photoprotective mechanisms that dissipate excess excitation. A subset of these mechanisms is collectively referred to as NPQ, or nonphotochemical quenching of chlorophyll a fluorescence. The regulation of NPQ is intrinsically linked to the cycling of xanthophylls that affects the kinetics and extent of the photoprotective response. The violaxanthin cycle (VAZ cycle) and the lutein epoxide cycle (LxL cycle) are two xanthophyll cycles found in vascular plants. The VAZ cycle has been studied extensively, owing in large part to its presence in model plant species where mutants are available to aid in its characterization. In contrast, the LxL cycle is not found in model plants, and its role in photosynthetic processes has been more difficult to define. To address this challenge, we introduced the LxL cycle into Arabidopsis thaliana and functionally isolated it from the VAZ cycle. Using these plant lines, we showed an increase in dark-acclimated PSII efficiency associated with Lx accumulation and demonstrated that violaxanthin deepoxidase is responsible for the light-driven deepoxidation of Lx. Conversion of Lx to L was reversible during periods of low light and occurred considerably faster than rates previously described in nonmodel species. Finally, we present clear evidence of the LxL cycle's role in modulating a rapid component of NPQ that is necessary to prevent photoinhibition in excess light.

Dissecting and modeling zeaxanthin- and lutein-dependent nonphotochemical quenching in Arabidopsis thaliana.

Photosynthetic organisms use various photoprotective mechanisms to dissipate excess photoexcitation as heat in a process called nonphotochemical quenching (NPQ). Regulation of NPQ allows for a rapid response to changes in light intensity and in vascular plants, is primarily triggered by a pH gradient across the thylakoid membrane (∆pH). The response is mediated by the PsbS protein and various xanthophylls. Time-correlated single-photon counting (TCSPC) measurements were performed on Arabidopsis thaliana to quantify the dependence of the response of NPQ to changes in light intensity on the presence and accumulation of zeaxanthin and lutein. Measurements were performed on WT and mutant plants deficient in one or both of the xanthophylls as well as a transgenic line that accumulates lutein via an engineered lutein epoxide cycle. Changes in the response of NPQ to light acclimation in WT and mutant plants were observed between two successive light acclimation cycles, suggesting that the character of the rapid and reversible response of NPQ in fully dark-acclimated plants is substantially different from in conditions plants are likely to experience caused by changes in light intensity during daylight. Mathematical models of the response of zeaxanthin- and lutein-dependent reversible NPQ were constructed that accurately describe the observed differences between the light acclimation periods. Finally, the WT response of NPQ was reconstructed from isolated components present in mutant plants with a single common scaling factor, which enabled deconvolution of the relative contributions of zeaxanthin- and lutein-dependent NPQ.

Improving photosynthesis and crop productivity by accelerating recovery from photoprotection.

Crop leaves in full sunlight dissipate damaging excess absorbed light energy as heat. When sunlit leaves are shaded by clouds or other leaves, this protective dissipation continues for many minutes and reduces photosynthesis. Calculations have shown that this could cost field crops up to 20% of their potential yield. Here, we describe the bioengineering of an accelerated response to natural shading events in Nicotiana (tobacco), resulting in increased leaf carbon dioxide uptake and plant dry matter productivity by about 15% in fluctuating light. Because the photoprotective mechanism that has been altered is common to all flowering plants and crops, the findings provide proof of concept for a route to obtaining a sustainable increase in productivity for food crops and a much-needed yield jump.

Evolution of an atypical de-epoxidase for photoprotection in the green lineage.

Plants, algae and cyanobacteria need to regulate photosynthetic light harvesting in response to the constantly changing light environment. Rapid adjustments are required to maintain fitness because of a trade-off between efficient solar energy conversion and photoprotection. The xanthophyll cycle, in which the carotenoid pigment violaxanthin is reversibly converted into zeaxanthin, is ubiquitous among green algae and plants and is necessary for the regulation of light harvesting, protection from oxidative stress and adaptation to different light conditions(1,2). Violaxanthin de-epoxidase (VDE) is the key enzyme responsible for zeaxanthin synthesis from violaxanthin under excess light. Here we show that the Chlorophycean VDE (CVDE) gene from the model green alga Chlamydomonas reinhardtii encodes an atypical VDE. This protein is not homologous to the VDE found in plants and is instead related to a lycopene cyclase from photosynthetic bacteria(3). Unlike the plant-type VDE that is located in the thylakoid lumen, the Chlamydomonas CVDE protein is located on the stromal side of the thylakoid membrane. Phylogenetic analysis suggests that CVDE evolved from an ancient de-epoxidase that was present in the common ancestor of green algae and plants, providing evidence of unexpected diversity in photoprotection in the green lineage.

Transient expression in Nicotiana benthamiana for rapid functional analysis of genes involved in non-photochemical quenching and carotenoid biosynthesis.

Plants must switch rapidly between light harvesting and photoprotection in response to environmental fluctuations in light intensity. This switch can lead to losses in absorbed energy usage, as photoprotective energy dissipation mechanisms can take minutes to hours to fully relax. One possible way to improve photosynthesis is to engineer these energy dissipation mechanisms (measured as non-photochemical quenching of chlorophyll a fluorescence, NPQ) to induce and relax more quickly, resulting in smaller losses under dynamic light conditions. Previous studies aimed at understanding the enzymes involved in the regulation of NPQ have relied primarily on labor-intensive and time-consuming generation of stable transgenic lines and mutant populations - approaches limited to organisms amenable to genetic manipulation and mapping. To enable rapid functional testing of NPQ-related genes from diverse organisms, we performed Agrobacterium tumefaciens-mediated transient expression assays in Nicotiana benthamiana to test if NPQ kinetics could be modified in fully expanded leaves. By expressing Arabidopsis thaliana genes known to be involved in NPQ, we confirmed the viability of this method for studying dynamic photosynthetic processes. Subsequently, we used naturally occurring variation in photosystem II subunit S, a modulator of NPQ in plants, to explore how differences in amino acid sequence affect NPQ capacity and kinetics. Finally, we functionally characterized four predicted carotenoid biosynthesis genes from the marine algae Nannochloropsis oceanica and Thalassiosira pseudonana and examined the effect of their expression on NPQ in N. benthamiana. This method offers a powerful alternative to traditional gene characterization methods by providing a fast and easy platform for assessing gene function in planta.

Regulation and Levels of the Thylakoid K+/H+ Antiporter KEA3 Shape the Dynamic Response of Photosynthesis in Fluctuating Light.

Crop canopies create environments of highly fluctuating light intensities. In such environments, photoprotective mechanisms and their relaxation kinetics have been hypothesized to limit photosynthetic efficiency and therefore crop yield potential. Here, we show that overexpression of the Arabidopsis thylakoid K+/H+ antiporter KEA3 accelerates the relaxation of photoprotective energy-dependent quenching after transitions from high to low light in Arabidopsis and tobacco. This, in turn, enhances PSII quantum efficiency in both organisms, supporting that in wild-type plants, residual light energy quenching following a high to low light transition represents a limitation to photosynthetic efficiency in fluctuating light. This finding underscores the potential of accelerating quenching relaxation as a building block for improving photosynthetic efficiency in the field. Additionally, by overexpressing natural KEA3 variants with modification to the C-terminus, we show that KEA3 activity is regulated by a mechanism involving its lumen-localized C-terminus, which lowers KEA3 activity in high light. This regulatory mechanism fine-tunes the balance between photoprotective energy dissipation in high light and maximum quantum yield in low light, likely to be critical for efficient photosynthesis in fluctuating light conditions.

An evaluation of new and established methods to determine T-DNA copy number and homozygosity in transgenic plants.

Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL-)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T-DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL-PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T1 progeny from 26 T0 plants showed that at least 19% of the lines carried multiple T-DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T-DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided.

Lineage-specific chromatin signatures reveal a regulator of lipid metabolism in microalgae.

Alga-derived lipids represent an attractive potential source of biofuels. However, lipid accumulation in algae is a stress response tightly coupled to growth arrest, thereby imposing a major limitation on productivity. To identify transcriptional regulators of lipid accumulation, we performed an integrative chromatin signature and transcriptomic analysis to decipher the regulation of lipid biosynthesis in the alga Chlamydomonas reinhardtii. Genome-wide histone modification profiling revealed remarkable differences in functional chromatin states between the algae and higher eukaryotes and uncovered regulatory components at the core of lipid accumulation pathways. We identified the transcription factor, PSR1, as a pivotal switch that triggers cytosolic lipid accumulation. Dissection of the PSR1-induced lipid profiles corroborates its role in coordinating multiple lipid-inducing stress responses. The comprehensive maps of functional chromatin signatures in a major clade of eukaryotic life and the discovery of a transcriptional regulator of algal lipid metabolism will facilitate targeted engineering strategies to mediate high lipid production in microalgae.

Major transcriptome reprogramming underlies floral mimicry induced by the rust fungus Puccinia monoica in Boechera stricta.

Pucciniamonoica is a spectacular plant parasitic rust fungus that triggers the formation of flower-like structures (pseudoflowers) in its Brassicaceae host plant Boecherastricta. Pseudoflowers mimic in shape, color, nectar and scent co-occurring and unrelated flowers such as buttercups. They act to attract insects thereby aiding spore dispersal and sexual reproduction of the rust fungus. Although much ecological research has been performed on P. monoica-induced pseudoflowers, this system has yet to be investigated at the molecular or genomic level. To date, the molecular alterations underlying the development of pseudoflowers and the genes involved have not been described. To address this, we performed gene expression profiling to reveal 256 plant biological processes that are significantly altered in pseudoflowers. Among these biological processes, plant genes involved in cell fate specification, regulation of transcription, reproduction, floral organ development, anthocyanin (major floral pigments) and terpenoid biosynthesis (major floral volatile compounds) were down-regulated in pseudoflowers. In contrast, plant genes involved in shoot, cotyledon and leaf development, carbohydrate transport, wax biosynthesis, cutin transport and L-phenylalanine metabolism (pathway that results in phenylethanol and phenylacetaldehyde volatile production) were up-regulated. These findings point to an extensive reprogramming of host genes by the rust pathogen to induce floral mimicry. We also highlight 31 differentially regulated plant genes that are enriched in the biological processes mentioned above, and are potentially involved in the formation of pseudoflowers. This work illustrates the complex perturbations induced by rust pathogens in their host plants, and provides a starting point for understanding the molecular mechanisms of pathogen-induced floral mimicry.

Global analysis of Arabidopsis/downy mildew interactions reveals prevalence of incomplete resistance and rapid evolution of pathogen recognition.

Interactions between Arabidopsis thaliana and its native obligate oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) represent a model system to study evolution of natural variation in a host/pathogen interaction. Both Arabidopsis and Hpa genomes are sequenced and collections of different sub-species are available. We analyzed ∼400 interactions between different Arabidopsis accessions and five strains of Hpa. We examined the pathogen's overall ability to reproduce on a given host, and performed detailed cytological staining to assay for pathogen growth and hypersensitive cell death response in the host. We demonstrate that intermediate levels of resistance are prevalent among Arabidopsis populations and correlate strongly with host developmental stage. In addition to looking at plant responses to challenge by whole pathogen inoculations, we investigated the Arabidopsis resistance attributed to recognition of the individual Hpa effectors, ATR1 and ATR13. Our results suggest that recognition of these effectors is evolutionarily dynamic and does not form a single clade in overall Arabidopsis phylogeny for either effector. Furthermore, we show that the ultimate outcome of the interactions can be modified by the pathogen, despite a defined gene-for-gene resistance in the host. These data indicate that the outcome of disease and disease resistance depends on genome-for-genome interactions between the host and its pathogen, rather than single gene pairs as thought previously.

Structural elucidation and functional characterization of the Hyaloperonospora arabidopsidis effector protein ATR13.

The oomycete Hyaloperonospora arabidopsidis (Hpa) is the causal agent of downy mildew on the model plant Arabidopsis thaliana and has been adapted as a model system to investigate pathogen virulence strategies and plant disease resistance mechanisms. Recognition of Hpa infection occurs when plant resistance proteins (R-genes) detect the presence or activity of pathogen-derived protein effectors delivered to the plant host. This study examines the Hpa effector ATR13 Emco5 and its recognition by RPP13-Nd, the cognate R-gene that triggers programmed cell death (HR) in the presence of recognized ATR13 variants. Herein, we use NMR to solve the backbone structure of ATR13 Emco5, revealing both a helical domain and a disordered internal loop. Additionally, we use site-directed and random mutagenesis to identify several amino acid residues involved in the recognition response conferred by RPP13-Nd. Using our structure as a scaffold, we map these residues to one of two surface-exposed patches of residues under diversifying selection. Exploring possible roles of the disordered region within the ATR13 structure, we perform domain swapping experiments and identify a peptide sequence involved in nucleolar localization. We conclude that ATR13 is a highly dynamic protein with no clear structural homologues that contains two surface-exposed patches of polymorphism, only one of which is involved in RPP13-Nd recognition specificity.

Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation.

Peripheral blood neutrophils form highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs), that have been implicated in innate immune response to bacterial infection. Neutrophils express high levels of peptidylarginine deiminase 4 (PAD4), which catalyzes histone citrullination. However, whether PAD4 or histone citrullination plays a role in chromatin structure in neutrophils is unclear. In this study, we show that the hypercitrullination of histones by PAD4 mediates chromatin decondensation. Histone hypercitrullination is detected on highly decondensed chromatin in HL-60 granulocytes and blood neutrophils. The inhibition of PAD4 decreases histone hypercitrullination and the formation of NET-like structures, whereas PAD4 treatment of HL-60 cells facilitates these processes. The loss of heterochromatin and multilobular nuclear structures is detected in HL-60 granulocytes after PAD4 activation. Importantly, citrullination of biochemically defined avian nucleosome arrays inhibits their compaction by the linker histone H5 to form higher order chromatin structures. Together, these results suggest that histone hypercitrullination has important functions in chromatin decondensation in granulocytes/neutrophils.

Recognition of the Hyaloperonospora parasitica effector ATR13 triggers resistance against oomycete, bacterial, and viral pathogens.

Phytopathogenic oomycetes cause some of the most devastating diseases affecting agricultural crops. Hyaloperonospora parasitica is a native oomycete pathogen of Arabidopsis and is related to other oomycete phytopathogens that include several species of Phytophthora, including the causal agent of potato late blight. Recently, four oomycete effector genes have been isolated, and several oomycete genomes have been sequenced. We have developed an efficient and genetically amenable system to test putative effector genes using the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. The H. parasitica effector protein ATR13 was delivered via P. syringae by fusing the ATR13 gene with the avrRpm1 type three secretion signal peptide, a bacterial sequence that allows transfer of proteins into the host cell through the bacterial type III secretion system. We also inserted ATR13 into the genome of the turnip mosaic virus, a single-stranded RNA virus. Our results show that delivery of ATR13 via the bacterial or viral pathogen triggers defense responses in plants containing the cognate resistance protein RPP13(Nd), which restricts proliferation of both pathogens. Hence, recognition of ATR13 by RPP13 initiates defense responses that are effective against oomycete, bacterial and viral pathogens, pointing to a common defense mechanism. We have characterized regions of the RPP13(Nd) resistance protein that are essential for effector recognition and/or downstream signaling, using transient coexpression in Nicotiana benthamiana.

Mouse cPLA2gamma, a novel oocyte and early embryo-abundant phospholipase A2 gamma-like protein, is targeted to the nuclear envelope during germinal vesicle breakdown.

This report documents the characterization of a novel mouse oocyte protein which was originally identified by microsequence analysis of a 67.8 kDa protein spot (pI 5.7) on a Coomassie-stained two-dimensional (2D) gel of murine egg proteins. Tandem mass spectroscopic analysis of the peptides obtained from the cored protein yielded sequences that appeared to match only ovary, egg, and preimplantation embryo cDNAs. We then cloned the novel gene by RACE-PCR, and analysis of the deduced cDNA sequence found that this maternal product was approximately 56% identical to human cytosolic phospholipase A2gamma (cPLA2gamma). Based on this sequence homology, we named the molecule mouse cytosolic phospholipase A2gamma (cPLA2gamma). As with human cPLA2gamma, mouse cPLA2gamma contains a lipase consensus sequence and lacks the calcium binding domain that is found in other PLA2 proteins. However, mouse cPLA2gamma is different from human cPLA2gamma in that mouse cPLA2gamma expression is restricted to the ovary and that the protein does not contain the myristoylation and prenylation lipid-anchoring motifs that are present in human cPLA2gamma. Within oocytes, mouse cPLA2gamma localizes mainly to the oocyte cortex and to the nucleoplasm. Interestingly, during germinal vesicle breakdown, mouse cPLA2gamma aggregates dynamically relocate from the oocyte cortex to the nuclear envelope, suggesting a possible role for this putative egg-restricted phospholipase A2gamma in membrane remodeling. Furthermore, mouse cPLA2gamma protein continues to be expressed in the embryo until the 4-8-cell stage of development, suggesting that mouse cPLA2gamma may function as a previously uncharacterized maternal effect gene.

Human PAD4 regulates histone arginine methylation levels via demethylimination.

Methylation of arginine (Arg) and lysine residues in histones has been correlated with epigenetic forms of gene regulation. Although histone methyltransferases are known, enzymes that demethylate histones have not been identified. Here, we demonstrate that human peptidylarginine deiminase 4 (PAD4) regulates histone Arg methylation by converting methyl-Arg to citrulline and releasing methylamine. PAD4 targets multiple sites in histones H3 and H4, including those sites methylated by coactivators CARM1 (H3 Arg17) and PRMT1 (H4 Arg3). A decrease of histone Arg methylation, with a concomitant increase of citrullination, requires PAD4 activity in human HL-60 granulocytes. Moreover, PAD4 activity is linked with the transcriptional regulation of estrogen-responsive genes in MCF-7 cells. These data suggest that PAD4 mediates gene expression by regulating Arg methylation and citrullination in histones.