Divergent cytosine DNA methylation patterns in single-cell, soybean root hairs.

Chromatin modifications, such as cytosine methylation of DNA, play a significant role in mediating gene expression in plants, which affects growth, development, and cell differentiation. As root hairs are single-cell extensions of the root epidermis and the primary organs for water uptake and nutrients, we sought to use root hairs as a single-cell model system to measure the impact of environmental stress. We measured changes in cytosine DNA methylation in single-cell root hairs as compared with multicellular stripped roots, as well as in response to heat stress. Differentially methylated regions (DMRs) in each methylation context showed very distinct methylation patterns between cell types and in response to heat stress. Intriguingly, at normal temperature, root hairs were more hypermethylated than were stripped roots. However, in response to heat stress, both root hairs and stripped roots showed hypomethylation in each context, especially in the CHH context. Moreover, expression analysis of mRNA from similar tissues and treatments identified some associations between DMRs, genes and transposons. Taken together, the data indicate that changes in DNA methylation are directly or indirectly associated with expression of genes and transposons within the context of either specific tissues/cells or stress (heat).

Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds.

Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes to our knowledge the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits.

Effect of lipo-chitooligosaccharide on early growth of C4 grass seedlings.

Although lipo-chitooligosaccharides (LCOs) are important signal molecules for plant-symbiont interactions, a number of reports suggest that LCOs can directly impact plant growth and development, separate from any role in plant symbioses. In order to investigate this more closely, maize and Setaria seedlings were treated with LCO and their growth was evaluated. The data indicate that LCO treatment significantly enhanced root growth. RNA-seq transcriptomic analysis of LCO-treated maize roots identified a number of genes whose expression was significantly affected by the treatment. Among these genes, some LCO-up-regulated genes are likely involved in root growth promotion. Interestingly, some stress-related genes were down-regulated after LCO treatment, which might indicate reallocation of resources from defense responses to plant growth. The promoter activity of several LCO-up-regulated genes using a ß-glucuronidase reporter system was further analysed. The results showed that the promoters were activated by LCO treatment. The data indicate that LCO can directly impact maize root growth and gene expression.

P2X7 receptor activation induces inflammatory responses in salivary gland epithelium.

Inflammation of the salivary gland is a well-documented aspect of salivary gland dysfunction that occurs in Sjogren's syndrome (SS), an autoimmune disease, and in γ-radiation-induced injury during treatment of head and neck cancers. Extracellular nucleotides have gained recognition as key modulators of inflammation through activation of cell surface ionotropic and metabotropic receptors, although the contribution of extracellular nucleotides to salivary gland inflammation is not well understood. In vitro studies using submandibular gland (SMG) cell aggregates isolated from wild-type C57BL/6 mice indicate that treatment with ATP or the high affinity P2X7R agonist 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) induces membrane blebbing and enhances caspase activity, responses that were absent in SMG cell aggregates isolated from mice lacking the P2X7R (P2X7R(-/-)). Additional studies with SMG cell aggregates indicate that activation of the P2X7R with ATP or BzATP stimulates the cleavage and release of α-fodrin, a cytoskeletal protein thought to act as an autoantigen in the development of SS. In vivo administration of BzATP to ligated SMG excretory ducts enhances immune cell infiltration into the gland and initiates apoptosis of salivary epithelial cells in wild-type, but not P2X7R(-/-), mice. These findings indicate that activation of the P2X7R contributes to salivary gland inflammation in vivo, suggesting that the P2X7R may represent a novel target for the treatment of salivary gland dysfunction.

Generation of Soybean (Glycine max) Transient Transgenic Roots.

Legumes-because of their nitrogen-fixing capacity-have both ecological and agronomic importance, and are also the major plant protein source for animal consumption. The model legume species are Lotus japonicus, Medicago truncatula, and soybean (Glycine max). These species have sequenced genomes and are amenable to genetic manipulation, as well as to various functional genomic and cell biology approaches. Plant transformation mediated by Agrobacterium is one of the most powerful methods in plant biotechnology. Using the traditional Agrobacterium tumefaciens method, stable transgenic plants take 6 to 12 months to create, depending on species. Besides being time consuming, this approach is often quite laborious. Hence, there is a need for more rapid methods to create transgenic tissues. In the case of roots, this can be done using hairy root transformation mediated by Agrobacterium rhizogenes. This protocol describes a method to generate transgenic soybean roots in as little as 3 weeks. © 2016 by John Wiley & Sons, Inc.

Soybean Roots Grown under Heat Stress Show Global Changes in Their Transcriptional and Proteomic Profiles.

Heat stress is likely to be a key factor in the negative impact of climate change on crop production. Heat stress significantly influences the functions of roots, which provide support, water, and nutrients to other plant organs. Likewise, roots play an important role in the establishment of symbiotic associations with different microorganisms. Despite the physiological relevance of roots, few studies have examined their response to heat stress. In this study, we performed genome-wide transcriptomic and proteomic analyses on isolated root hairs, which are a single, epidermal cell type, and compared their response to stripped roots. On average, we identified 1849 and 3091 genes differentially regulated in root hairs and stripped roots, respectively, in response to heat stress. Our gene regulatory module analysis identified 10 key modules that might control the majority of the transcriptional response to heat stress. We also conducted proteomic analysis on membrane fractions isolated from root hairs and compared these responses to stripped roots. These experiments identified a variety of proteins whose expression changed within 3 h of application of heat stress. Most of these proteins were predicted to play a significant role in thermo-tolerance, as well as in chromatin remodeling and post-transcriptional regulation. The data presented represent an in-depth analysis of the heat stress response of a single cell type in soybean.