HSI2/VAL1 Silences AGL15 to Regulate the Developmental Transition from Seed Maturation to Vegetative Growth in Arabidopsis.

Gene expression during seed development in Arabidopsis thaliana is controlled by transcription factors including LEAFY COTYLEDON1 (LEC1) and LEC2, ABA INSENSITIVE3 (ABI3), FUSCA3 (FUS3), known as LAFL proteins, and AGAMOUS-LIKE15 (AGL15). The transition from seed maturation to germination and seedling growth requires the transcriptional silencing of these seed maturation-specific factors leading to downregulation of structural genes including those that encode seed storage proteins, oleosins, and dehydrins. During seed germination and vegetative growth, B3-domain protein HSI2/VAL1 is required for the transcriptional silencing of LAFL genes. Here, we report chromatin immunoprecipitation analysis indicating that HSI2/VAL1 binds to the upstream sequences of the AGL15 gene but not at LEC1, ABI3, FUS3, or LEC2 loci. Functional analysis indicates that the HSI2/VAL1 B3 domain interacts with two RY elements upstream of the AGL15 coding region and at least one of them is required for HSI2/VAL1-dependent AGL15 repression. Expression analysis of the major seed maturation regulatory genes LEC1, ABI3, FUS3, and LEC2 in different genetic backgrounds demonstrates that HSI2/VAL1 is epistatic to AGL15 and represses the seed maturation regulatory program through downregulation of AGL15 by deposition of H3K27me3 at this locus. This hypothesis is further supported by results that show that HSI2/VAL1 physically interacts with the Polycomb Repressive Complex 2 component protein MSI1, which is also enriched at the AGL15 locus.

HSI2/VAL1 and HSL1/VAL2 function redundantly to repress DOG1 expression in Arabidopsis seeds and seedlings.

DELAY OF GERMINATION1 (DOG1) is a primary regulator of seed dormancy. Accumulation of DOG1 in seeds leads to deep dormancy and delayed germination in Arabidopsis. B3 domain-containing transcriptional repressors HSI2/VAL1 and HSL1/VAL2 silence seed dormancy and enable the subsequent germination and seedling growth. However, the roles of HSI2 and HSL1 in regulation of DOG1 expression and seed dormancy remain elusive. Seed dormancy was analysed by measurement of maximum germination percentage of freshly harvested Arabidopsis seeds. In vivo protein-protein interaction analysis, ChIP-qPCR and EMSA were performed and suggested that HSI2 and HSL1 can form dimers to directly regulate DOG1. HSI2 and HSL1 dimers interact with RY elements at DOG1 promoter. Both B3 and PHD-like domains are required for enrichment of HSI2 and HSL1 at the DOG1 promoter. HSI2 and HSL1 recruit components of polycomb-group proteins, including CURLY LEAF (CLF) and LIKE HETERCHROMATIN PROTEIN 1 (LHP1), for consequent deposition of H3K27me3 marks, leading to repression of DOG1 expression. Our findings suggest that HSI2- and HSL1-dependent histone methylation plays critical roles in regulation of seed dormancy during seed germination and early seedling growth.

Genome-Wide Identification of Medicago Peptides Involved in Macronutrient Responses and Nodulation.

Growing evidence indicates that small, secreted peptides (SSPs) play critical roles in legume growth and development, yet the annotation of SSP-coding genes is far from complete. Systematic reannotation of the Medicago truncatula genome identified 1,970 homologs of established SSP gene families and an additional 2,455 genes that are potentially novel SSPs, previously unreported in the literature. The expression patterns of known and putative SSP genes based on 144 RNA sequencing data sets covering various stages of macronutrient deficiencies and symbiotic interactions with rhizobia and mycorrhiza were investigated. Focusing on those known or suspected to act via receptor-mediated signaling, 240 nutrient-responsive and 365 nodulation-responsive Signaling-SSPs were identified, greatly expanding the number of SSP gene families potentially involved in acclimation to nutrient deficiencies and nodulation. Synthetic peptide applications were shown to alter root growth and nodulation phenotypes, revealing additional regulators of legume nutrient acquisition. Our results constitute a powerful resource enabling further investigations of specific SSP functions via peptide treatment and reverse genetics.

Rapid identification of causative insertions underlying Medicago truncatula Tnt1 mutants defective in symbiotic nitrogen fixation from a forward genetic screen by whole genome sequencing.

BACKGROUND: In the model legume Medicago truncatula, the near saturation genome-wide Tnt1 insertion mutant population in ecotype R108 is a valuable tool in functional genomics studies. Forward genetic screens have identified many Tnt1 mutants defective in nodule development and symbiotic nitrogen fixation (SNF). However, progress toward identifying the causative mutations of these symbiotic mutants has been slow because of the high copy number of Tnt1 insertions in some mutant plants and inefficient recovery of flanking sequence tags (FSTs) by thermal asymmetric interlaced PCR (TAIL-PCR) and other techniques. RESULTS: Two Tnt1 symbiotic mutants, NF11217 and NF10547, with defects in nodulation and SNF were isolated during a forward genetic screen. Both TAIL-PCR and whole genome sequencing (WGS) approaches were used in attempts to find the relevant mutant genes in NF11217 and NF10547. Illumina paired-end WGS generated ~16 Gb of sequence data from a 500 bp insert library for each mutant, yielding ~40X genome coverage. Bioinformatics analysis of the sequence data identified 97 and 65 high confidence independent Tnt1 insertion loci in NF11217 and NF10547, respectively. In comparison to TAIL-PCR, WGS recovered more Tnt1 insertions. From the WGS data, we found Tnt1 insertions in the exons of the previously described PHOSPHOLIPASE C (PLC)-like and NODULE INCEPTION (NIN) genes in NF11217 and NF10547 mutants, respectively. Co-segregation analyses confirmed that the symbiotic phenotypes of NF11217 and NF10547 are tightly linked to the Tnt1 insertions in PLC-like and NIN genes, respectively. CONCLUSIONS: In this work, we demonstrate that WGS is an efficient approach for identification of causative genes underlying SNF defective phenotypes in M. truncatula Tnt1 insertion mutants obtained via forward genetic screens.

High-resolution hydrodynamic chromatographic separation of large DNA using narrow, bare open capillaries: a rapid and economical alternative technology to pulsed-field gel electrophoresis?

A high-resolution, rapid, and economical hydrodynamic chromatographic (HDC) method for large DNA separations in free solution was developed using narrow (5 µm diameter), bare open capillaries. Size-based separation was achieved in a chromatographic format with larger DNA molecules being eluting faster than smaller ones. Lambda DNA Mono Cut Mix was baseline-separated with the percentage resolutions generally less than 9.0% for all DNA fragments (1.5 to 48.5 kbp) tested in this work. High efficiencies were achieved for large DNA from this chromatographic technique, and the number of theoretical plates reached 3.6 × 10(5) plates for the longest (48.5 kbp) and 3.7 × 10(5) plates for the shortest (1.5 kbp) fragments. HDC parameters and performances were also discussed. The method was further applied for fractionating large DNA fragments from real-world samples (SacII digested Arabidopsis plant bacterial artificial chromosome (BAC) DNA and PmeI digested Rice BAC DNA) to demonstrate its feasibility for BAC DNA finger printing. Rapid separation of PmeI digested Rice BAC DNA covering from 0.44 to 119.041 kbp was achieved in less than 26 min. All DNA fragments of these samples were baseline separated in narrow bare open capillaries, while the smallest fragment (0.44 kbp) was missing in pulsed-field gel electrophoresis (PFGE) separation mode. It is demonstrated that narrow bare open capillary chromatography can realize a rapid separation for a wide size range of DNA mixtures that contain both small and large DNA fragments in a single run.

HSI2/VAL1 PHD-like domain promotes H3K27 trimethylation to repress the expression of seed maturation genes and complex transgenes in Arabidopsis seedlings.

BACKGROUND: The novel mutant allele hsi2-4 was isolated in a genetic screen to identify Arabidopsis mutants with constitutively elevated expression of a glutathione S-transferase F8::luciferase (GSTF8::LUC) reporter gene in Arabidopsis. The hsi2-4 mutant harbors a point mutation that affects the plant homeodomain (PHD)-like domain in HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE2 (HSI2)/VIVIPAROUS1/ABI3-LIKE1 (VAL1). In hsi2-4 seedlings, expression of this LUC transgene and certain endogenous seed-maturation genes is constitutively enhanced. The parental reporter line (WT LUC ) that was used for mutagenesis harbors two independent transgene loci, Kan R and Kan S . Both loci express luciferase whereas only the Kan R locus confers resistance to kanamycin. RESULTS: Here we show that both transgene loci harbor multiple tandem insertions at single sites. Luciferase expression from these sites is regulated by the HSI2 PHD-like domain, which is required for the deposition of repressive histone methylation marks (H3K27me3) at both Kan R and Kan S loci. Expression of LUC and Neomycin Phosphotransferase II transgenes is associated with dynamic changes in H3K27me3 levels, and the activation marks H3K4me3 and H3K36me3 but does not appear to involve repressive H3K9me2 marks, DNA methylation or histone deacetylation. However, hsi2-2 and hsi2-4 mutants are partially resistant to growth inhibition associated with exposure to the DNA methylation inhibitor 5-aza-2'-deoxycytidine. HSI2 is also required for the repression of a subset of regulatory and structural seed maturation genes in vegetative tissues and H3K27me3 marks associated with most of these genes are also HSI2-dependent. CONCLUSIONS: These data implicate HSI2 PHD-like domain in the regulation of gene expression involving histone modifications and DNA methylation-mediated epigenetic mechanisms.

A modified aeroponic system for growing small-seeded legumes and other plants to study root systems.

BACKGROUND: Various growth systems are available for studying plant root growth and plant-microbe interactions including hydroponics and aeroponics. Although some of these systems work well with Arabidopsis thaliana and smaller cereal model plants, they may not scale up as well for use with hundreds of plants at a time from a larger plant species. The aim of this study is to present step-by-step instructions for fabricating an aeroponic system, also called a "caisson," that has been in use in several legume research labs studying the development of symbiotic nitrogen fixing nodules, but for which detailed directions are not currently available. The aeroponic system is reusable and is adaptable for many other types of investigations besides root nodulation. RESULTS: An aeroponic system that is affordable and reusable was adapted from a design invented by French engineer René Odorico. It consists of two main components: a modified trash can with a lid of holes and a commercially available industrial humidifier that is waterproofed with silicon sealant. The humidifier generates a mist in which plant roots grow, suspended from holes in trash can lid. Results from use of the aeroponic system have been available in the scientific community for decades; it has a record as a workhorse in the lab. CONCLUSIONS: Aeroponic systems present a convenient way for researchers to grow plants for studying root systems and plant-microbe interactions in root systems. They are particularly attractive for phenotyping roots and following the progress of nodule development in legumes. Advantages include the ability to precisely control the growth medium in which the plants grow and easy observations of roots during growth. In this system, mechanical shear potentially killing microbes found in some other types of aeroponic devices is not an issue. Disadvantages of aeroponic systems include the likelihood of altered root physiology compared to root growth on soil and other solid substrates and the need to have separate aeroponic systems for comparing plant responses to different microbial strains.

A novel HSI2 mutation in Arabidopsis affects the PHD-like domain and leads to derepression of seed-specific gene expression.

Two related B3 domain transcriptional repressors, HSI2 (HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE2)/VAL1 (VP1/ABI3-LIKE1) and HSL1 (HSI2-LIKE1)/VAL2, function redundantly to repress key transcriptional regulators of seed maturation genes in Arabidopsis thaliana seedlings. Using a forward genetic screen designed to isolate trans-acting mutants that affected expression of a transgene containing the glutathione S-transferase F8 promoter::luciferase (GSTF8::LUC) reporter, we identified a novel HSI2 mutant allele, hsi2-4, that exhibits constitutively elevated luciferase expression while expression of the endogenous GSTF8 transcript remains unchanged. The hsi2-4 lesion was found to be a missense mutation that results in the substitution of a conserved cysteine within the plant homeodomain-like (PHD) motif of HSI2. Microarray analysis of hsi2-4 and hsi2-4 hsl1 mutants indicated that the HSI2 PHD-like domain functions non-redundantly to repress a subset of seed maturation genes, including those that encode AGL15 (AGAMOUS-LIKE15), FUSCA3 (FUS3), cruciferins, cupin family proteins, late-embryogenesis abundant protein, oleosins, 2S albumins and other seed-specific proteins in Arabidopsis seedlings. Many genes that are responsive to this mutation in the HSI2 PHD-like domain are enriched in histone H3 trimethylation on lysine 27 residues (H3K27me3), a repressive epigenetic mark. Chromatin immunoprecipitation analysis showed that sequences of the GSTF8::LUC transgene are enriched in H3K27me3 in a HSI2 PHD domain-dependent manner. These results indicate that the transcriptional repression activity of the HSI2 PHD domain could be mediated, at least in part, by its participation in the deposition of H3K27me3 on the chromatin of specific target genes.

Free solution hydrodynamic separation of DNA fragments from 75 to 106,000 base pairs in a single run.

Gel electrophoresis is commonly used to separate DNA, but narrow capillaries or microchannels desired for high throughput efficient separations are difficult to fill with gels. We report here that a narrow capillary can be used to hydrodynamically separate a wide size range of DNA fragments in a single run without the need for gels, wall coatings, or an electric field. We also demonstrate that attractive separation is possible in a few minutes and that the separated DNA can be collected into individual fractions that remain viable for amplification via the polymerase chain reaction.

Bare nanocapillary for DNA separation and genotyping analysis in gel-free solutions without application of external electric field.

In this work, we demonstrate DNA separation and genotyping analysis in gel-free solutions using a nanocapillary under pressure-driven conditions without application of an external electric field. The nanocapillary is a approximately 50-cm-long and 500-nm-radius bare fused-silica capillary. After a DNA sample is injected, the analytes are eluted out in a chromatographic separation format. The elution order of DNA molecules follows strictly with their sizes, with the longer DNA being eluted out faster than the shorter ones. High resolutions are obtained for both short (a few bases) and long (tens of thousands of base pairs) DNA fragments. Effects of key experimental parameters, such as eluent composition and elution pressure, on separation efficiency and resolution are investigated. We also apply this technique for DNA separations of real-world genotyping samples to demonstrate its feasibility in biological applications. PCR products (without any purification) amplified from Arabidopsis plant genomic DNA crude preparations are directly injected into the nanocapillary, and PCR-amplified DNA fragments are well resolved, allowing for unambiguous identification of samples from heterozygous and homozygous individuals. Since the capillaries used to conduct the separations are uncoated, column lifetime is virtually unlimited. The only material that is consumed in these assays is the eluent, and hence, the operation cost is low.

Keel petal incision: a simple and efficient method for genetic crossing in Medicago truncatula.

BACKGROUND: Genetic crossing is an essential tool in both forward and reverse genetic approaches to understand the biological functions of genes. For Medicago truncatula (barrel medic) various crossing techniques have been used which differ in the methods used to dissect the female parent's unopened flower bud to remove immature anthers for prevention of self-pollination. Previously described methods including front, side or back incision methods may damage the flower bud, impeding successful fertilization and/or seed development because they may allow pollen to dislodge and floral organs to desiccate after crossing, all of which diminish the success rates of crossing. RESULTS: We report the keel petal incision method for genetic crossing in M. truncatula ecotype R108 and demonstrate successful crosses with two other M. truncatula ecotypes, A17 and A20. In the method presented here, an incision is made along the central line of the keel petal from the bottom 1/3rd of the female parent's flower bud to its distal end. This allows easy removal of anthers from the flower bud and access for cross-pollination. After pollination, the stigma and the deposited pollen from the male donor are covered by the keel petal, wing petals and standard petal, forming a natural pouch. The pouch prevents dislodging of deposited pollen from the stigma and protects the internal floral organs from drying out, without using cling-film or water-containing chambers to maintain a humid environment. The keel petal incision method showed an approximate 80% success rate in the M. truncatula R108 ecotype and also in other ecotypes including Jemalong A17 and A20. CONCLUSIONS: Our keel petal incision protocol shows marked improvement over existing methods with respect to the ease of crossing and the percentage of successful crosses. Developed for the M. truncatula R108 ecotype, the protocol has been demonstrated with A17 and A20 ecotypes and is expected to work with other ecotypes. Investigators of varying experience have achieved genetic crosses in M. truncatula using this method.