Impact of differential DNA methylation on transgene expression in cotton (Gossypium hirsutum L.) events generated by targeted sequence insertion.

Targeted Genome Optimization (TGO) using site-specific nucleases to introduce a DNA double-strand break (DSB) at a specific target locus has broadened the options available to breeders for generation and combination of multiple traits. The use of targeted DNA cleavage in combination with homologous recombination (HR)-mediated repair, enabled the precise targeted insertion of additional trait genes (2mepsps, hppd, axmi115) at a pre-existing transgenic locus in cotton. Here we describe the expression and epigenome analyses of cotton Targeted Sequence Insertion (TSI) events over generations. In a subset of events, we observed variability in the level of transgene (hppd, axmi115) expression between independent but genetically identical TSI events. Transgene expression could also be differential within single events and variable over generations. This expression variability and silencing occurred independently of the transgene sequence and could be attributed to DNA methylation that was further linked to different DNA methylation mechanisms. The trigger(s) of transgene DNA methylation remains elusive but we hypothesize that targeted DSB induction and repair could be a potential trigger for DNA methylation.

The Cyclin-Dependent Kinase Inhibitor KRP6 Induces Mitosis and Impairs Cytokinesis in Giant Cells Induced by Plant-Parasitic Nematodes in Arabidopsis.

In Arabidopsis thaliana, seven cyclin-dependent kinase (CDK) inhibitors have been identified, designated interactors of CDKs or Kip-related proteins (KRPs). Here, the function of KRP6 was investigated during cell cycle progression in roots infected by plant-parasitic root-knot nematodes. Contrary to expectations, analysis of Meloidogyne incognita-induced galls of KRP6-overexpressing lines revealed a role for this particular KRP as an activator of the mitotic cell cycle. In accordance, KRP6-overexpressing suspension cultures displayed accelerated entry into mitosis, but delayed mitotic progression. Likewise, phenotypic analysis of cultured cells and nematode-induced giant cells revealed a failure in mitotic exit, with the appearance of multinucleated cells as a consequence. Strong KRP6 expression upon nematode infection and the phenotypic resemblance between KRP6 overexpression cell cultures and root-knot morphology point toward the involvement of KRP6 in the multinucleate and acytokinetic state of giant cells. Along these lines, the parasite might have evolved to manipulate plant KRP6 transcription to the benefit of gall establishment.

ANGUSTIFOLIA3 binds to SWI/SNF chromatin remodeling complexes to regulate transcription during Arabidopsis leaf development.

The transcriptional coactivator ANGUSTIFOLIA3 (AN3) stimulates cell proliferation during Arabidopsis thaliana leaf development, but the molecular mechanism is largely unknown. Here, we show that inducible nuclear localization of AN3 during initial leaf growth results in differential expression of important transcriptional regulators, including GROWTH REGULATING FACTORs (GRFs). Chromatin purification further revealed the presence of AN3 at the loci of GRF5, GRF6, CYTOKININ RESPONSE FACTOR2, CONSTANS-LIKE5 (COL5), HECATE1 (HEC1), and ARABIDOPSIS RESPONSE REGULATOR4 (ARR4). Tandem affinity purification of protein complexes using AN3 as bait identified plant SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin remodeling complexes formed around the ATPases BRAHMA (BRM) or SPLAYED. Moreover, SWI/SNF ASSOCIATED PROTEIN 73B (SWP73B) is recruited by AN3 to the promoters of GRF5, GRF3, COL5, and ARR4, and both SWP73B and BRM occupy the HEC1 promoter. Furthermore, we show that AN3 and BRM genetically interact. The data indicate that AN3 associates with chromatin remodelers to regulate transcription. In addition, modification of SWI3C expression levels increases leaf size, underlining the importance of chromatin dynamics for growth regulation. Our results place the SWI/SNF-AN3 module as a major player at the transition from cell proliferation to cell differentiation in a developing leaf.

Selection for Improved Energy Use Efficiency and Drought Tolerance in Canola Results in Distinct Transcriptome and Epigenome Changes.

To increase both the yield potential and stability of crops, integrated breeding strategies are used that have mostly a direct genetic basis, but the utility of epigenetics to improve complex traits is unclear. A better understanding of the status of the epigenome and its contribution to agronomic performance would help in developing approaches to incorporate the epigenetic component of complex traits into breeding programs. Starting from isogenic canola (Brassica napus) lines, epilines were generated by selecting, repeatedly for three generations, for increased energy use efficiency and drought tolerance. These epilines had an enhanced energy use efficiency, drought tolerance, and nitrogen use efficiency. Transcriptome analysis of the epilines and a line selected for its energy use efficiency solely revealed common differentially expressed genes related to the onset of stress tolerance-regulating signaling events. Genes related to responses to salt, osmotic, abscisic acid, and drought treatments were specifically differentially expressed in the drought-tolerant epilines. The status of the epigenome, scored as differential trimethylation of lysine-4 of histone 3, further supported the phenotype by targeting drought-responsive genes and facilitating the transcription of the differentially expressed genes. From these results, we conclude that the canola epigenome can be shaped by selection to increase energy use efficiency and stress tolerance. Hence, these findings warrant the further development of strategies to incorporate epigenetics into breeding.

A generic tool for transcription factor target gene discovery in Arabidopsis cell suspension cultures based on tandem chromatin affinity purification.

Genome-wide identification of transcription factor (TF) binding sites is pivotal to our understanding of gene expression regulation. Although much progress has been made in the determination of potential binding regions of proteins by chromatin immunoprecipitation, this method has some inherent limitations regarding DNA enrichment efficiency and antibody necessity. Here, we report an alternative strategy for assaying in vivo TF-DNA binding in Arabidopsis (Arabidopsis thaliana) cells by tandem chromatin affinity purification (TChAP). Evaluation of TChAP using the E2Fa TF and comparison with traditional chromatin immunoprecipitation and single chromatin affinity purification illustrates the suitability of TChAP and provides a resource for exploring the E2Fa transcriptional network. Integration with transcriptome, cis-regulatory element, functional enrichment, and coexpression network analyses demonstrates the quality of the E2Fa TChAP sequencing data and validates the identification of new direct E2Fa targets. TChAP enhances both TF target mapping throughput, by circumventing issues related to antibody availability, and output, by improving DNA enrichment efficiency.

Arabidopsis ULTRAVIOLET-B-INSENSITIVE4 maintains cell division activity by temporal inhibition of the anaphase-promoting complex/cyclosome.

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin ligase that regulates progression through the cell cycle by marking key cell division proteins for destruction. To ensure correct cell cycle progression, accurate timing of APC/C activity is important, which is obtained through its association with both activating and inhibitory subunits. However, although the APC/C is highly conserved among eukaryotes, no APC/C inhibitors are known in plants. Recently, we have identified ULTRAVIOLET-B-INSENSITIVE4 (UVI4) as a plant-specific component of the APC/C. Here, we demonstrate that UVI4 uses conserved APC/C interaction motifs to counteract the activity of the CELL CYCLE SWITCH52 A1 (CCS52A1) activator subunit, inhibiting the turnover of the A-type cyclin CYCA2;3. UVI4 is expressed in an S phase-dependent fashion, likely through the action of E2F transcription factors. Correspondingly, uvi4 mutant plants failed to accumulate CYCA2;3 during the S phase and prematurely exited the cell cycle, triggering the onset of the endocycle. We conclude that UVI4 regulates the temporal inactivation of APC/C during DNA replication, allowing CYCA2;3 to accumulate above the level required for entering mitosis, and thereby regulates the meristem size and plant growth rate.

Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana.

Cell proliferation is the main driving force for plant growth. Although genome sequence analysis revealed a high number of cell cycle genes in plants, little is known about the molecular complexes steering cell division. In a targeted proteomics approach, we mapped the core complex machinery at the heart of the Arabidopsis thaliana cell cycle control. Besides a central regulatory network of core complexes, we distinguished a peripheral network that links the core machinery to up- and downstream pathways. Over 100 new candidate cell cycle proteins were predicted and an in-depth biological interpretation demonstrated the hypothesis-generating power of the interaction data. The data set provided a comprehensive view on heterodimeric cyclin-dependent kinase (CDK)-cyclin complexes in plants. For the first time, inhibitory proteins of plant-specific B-type CDKs were discovered and the anaphase-promoting complex was characterized and extended. Important conclusions were that mitotic A- and B-type cyclins form complexes with the plant-specific B-type CDKs and not with CDKA;1, and that D-type cyclins and S-phase-specific A-type cyclins seem to be associated exclusively with CDKA;1. Furthermore, we could show that plants have evolved a combinatorial toolkit consisting of at least 92 different CDK-cyclin complex variants, which strongly underscores the functional diversification among the large family of cyclins and reflects the pivotal role of cell cycle regulation in the developmental plasticity of plants.

An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes.

Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (∼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; ∼5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.

Arabidopsis WEE1 kinase controls cell cycle arrest in response to activation of the DNA integrity checkpoint.

Upon the incidence of DNA stress, the ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) signaling kinases activate a transient cell cycle arrest that allows cells to repair DNA before proceeding into mitosis. Although the ATM-ATR pathway is highly conserved over species, the mechanisms by which plant cells stop their cell cycle in response to the loss of genome integrity are unclear. We demonstrate that the cell cycle regulatory WEE1 kinase gene of Arabidopsis thaliana is transcriptionally activated upon the cessation of DNA replication or DNA damage in an ATR- or ATM-dependent manner, respectively. In accordance with a role for WEE1 in DNA stress signaling, WEE1-deficient plants showed no obvious cell division or endoreduplication phenotype when grown under nonstress conditions but were hypersensitive to agents that impair DNA replication. Induced WEE1 expression inhibited plant growth by arresting dividing cells in the G2-phase of the cell cycle. We conclude that the plant WEE1 gene is not rate-limiting for cycle progression under normal growth conditions but is a critical target of the ATR-ATM signaling cascades that inhibit the cell cycle upon activation of the DNA integrity checkpoints, coupling mitosis to DNA repair in cells that suffer DNA damage.

Novel plant-specific cyclin-dependent kinase inhibitors induced by biotic and abiotic stresses.

The EL2 gene of rice (Oryza sativa), previously classified as early response gene against the potent biotic elicitor N-acetylchitoheptaose and encoding a short polypeptide with unknown function, was identified as a novel cell cycle regulatory gene related to the recently reported SIAMESE (SIM) gene of Arabidopsis thaliana. Iterative two-hybrid screens, in vitro pull-down assays, and fluorescence resonance energy transfer analyses showed that Orysa; EL2 binds the cyclin-dependent kinase (CDK) CDKA1;1 and D-type cyclins. No interaction was observed with the plant-specific B-type CDKs. The amino acid motif ELERFL was identified to be essential for cyclin, but not for CDK binding. Orysa;EL2 impaired the ability of Orysa; CYCD5;3 to complement a budding yeast (Saccharomyces cerevisiae) triple CLN mutant, whereas recombinant protein inhibited CDK activity in vitro. Moreover, Orysa;EL2 was able to rescue the multicellular trichome phenotype of sim mutants of Arabidopsis, unequivocally demonstrating that Orysa;EL2 operates as a cell cycle inhibitor. Orysa;EL2 mRNA levels were induced by cold, drought, and propionic acid. Our data suggest that Orysa;EL2 encodes a new type of plant CDK inhibitor that links cell cycle progression with biotic and abiotic stress responses.

The cyclin-dependent kinase inhibitor KRP2 controls the onset of the endoreduplication cycle during Arabidopsis leaf development through inhibition of mitotic CDKA;1 kinase complexes.

Exit from the mitotic cell cycle and initiation of cell differentiation frequently coincides with the onset of endoreduplication, a modified cell cycle during which DNA continues to be duplicated in the absence of mitosis. Although the mitotic cell cycle and the endoreduplication cycle share much of the same machinery, the regulatory mechanisms controlling the transition between both cycles remain poorly understood. We show that the A-type cyclin-dependent kinase CDKA;1 and its specific inhibitor, the Kip-related protein, KRP2 regulate the mitosis-to-endocycle transition during Arabidopsis thaliana leaf development. Constitutive overexpression of KRP2 slightly above its endogenous level only inhibited the mitotic cell cycle-specific CDKA;1 kinase complexes, whereas the endoreduplication cycle-specific CDKA;1 complexes were unaffected, resulting in an increase in the DNA ploidy level. An identical effect on the endoreduplication cycle could be observed by overexpressing KRP2 exclusively in mitotically dividing cells. In agreement with a role for KRP2 as activator of the mitosis-to-endocycle transition, KRP2 protein levels were more abundant in endoreduplicating than in mitotically dividing tissues. We illustrate that KRP2 protein abundance is regulated posttranscriptionally through CDK phosphorylation and proteasomal degradation. KRP2 phosphorylation by the mitotic cell cycle-specific CDKB1;1 kinase suggests a mechanism in which CDKB1;1 controls the level of CDKA;1 activity through regulating KRP2 protein abundance. In accordance with this model, KRP2 protein levels increased in plants with reduced CDKB1;1 activity. Moreover, the proposed model allowed a dynamical simulation of the in vivo observations, validating the sufficiency of the regulatory interactions between CDKA;1, KRP2, and CDKB1;1 in fine-tuning the mitosis-to-endocycle transition.

Conditional, recombinase-mediated expression of genes in plant cell cultures.

In plant cells, overexpression of critical genes can be hampered by deleterious effects on development that results in a counterselection of transgenic cells harboring the gene of interest. Inducible expression systems have been reported, but many of them show unwanted leaky expression. To circumvent this potential problem, a novel inducible system was developed based on two previously characterized systems: the CRE-loxP site-specific recombination system of bacteriophage P1 and the subcellular targeting of proteins by a mammalian glucocorticoid receptor (GR). By fusing the receptor domain of the rat GR to the carboxyl terminus of the CRE recombinase, a double-lock conditional transcriptional induction system was created that is highly useful to overexpress genes whose expression may block transgenic regeneration. Furthermore, because the designed vector utilizes the GATEWAY recombination technology, cloning was restriction- and ligation-free, thus rendering the vector suitable for high-throughput research. The system was tested in Nicotiana tabacum bright yellow-2 (BY-2) cells and its efficiency was demonstrated for the controlled overexpression of the gus reporter gene and a mutant allele of the A-type cyclin-dependent kinase (CDKA), which is known to be a potent inhibitor of the cell cycle.

B1-type cyclin-dependent kinases are essential for the formation of stomatal complexes in Arabidopsis thaliana.

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle. In yeasts, only one CDK is sufficient to drive cells through the cell cycle, whereas higher eukaryotes developed a family of related CDKs. Curiously, plants contain a unique class of CDKs (B-type CDKs), whose function is still unclear. We show that the CDKB1;1 gene of Arabidopsis (Arabidopsis thaliana) is highly expressed in guard cells and stomatal precursor cells of cotyledons, suggesting a prominent role for B-type CDKs in stomatal development. In accordance, transgenic Arabidopsis plants with reduced B-type CDK activity had a decreased stomatal index because of an early block of meristemoid division and inhibition of satellite meristemoid formation. Many aberrant stomatal cells were observed, all of them blocked in the G2 phase of the cell cycle. Although division of stomatal precursors was inhibited, cells still acquired stomatal identity, illustrating that stomatal cell differentiation is independent of cellular and nuclear division.