The transcriptomic landscapes of rice cultivars with diverse root system architectures grown in upland field conditions.

Root system architecture affects plant drought resistance and other key agronomic traits such as lodging. However, although phenotypic and genomic variation has been extensively analyzed, few field studies have integrated phenotypic and transcriptomic information, particularly for below-ground traits such as root system architecture. Here, we report the phenotypic and transcriptomic landscape of 61 rice (Oryza sativa) accessions with highly diverse below-ground traits grown in an upland field. We found that four principal components explained the phenotypic variation and that accessions could be classified into four subpopulations (indica, aus, japonica and admixed) based on their tiller numbers and crown root diameters. Transcriptome analysis revealed that differentially expressed genes associated with specific subpopulations were enriched with stress response-related genes, suggesting that subpopulations have distinct stress response mechanisms. Root growth was negatively correlated with auxin-inducible genes, suggesting an association between auxin signaling and upland field conditions. A negative correlation between crown root diameter and stress response-related genes suggested that thicker crown root diameter is associated with resistance to mild drought stress. Finally, co-expression network analysis implemented with DNA affinity purification followed by sequencing analysis identified phytohormone signaling networks and key transcription factors negatively regulating crown root diameter. Our datasets provide a useful resource for understanding the genomic and transcriptomic basis of phenotypic variation under upland field conditions.

Development of Neutral pH-Responsive Microgels by Tuning Cross-Linking Conditions.

Polymer microgels that respond in a range of neutral pH can be useful for the development of molecular imaging tools and drug-delivery carriers. Here, we describe a simple approach in developing microgels that undergo volume phase transitions and substantial nuclear magnetic resonance (NMR) relaxometric changes within a narrow pH range of 6.4 to 7.4. The pH-responsive microgels were synthesized using methacrylic acid and a series of ethylene glycol dimethacrylate cross-linkers with repeating units of ethylene glycol that range from one to four. NMR relaxometry demonstrated that the transverse relaxation time (T2) of a suspension containing microgels that were cross-linked with diethylene glycol dimethacrylate sharply decreases at the pH where volume phase transition occurs. The polymer microgels cross-linked with 40 and 45 mol% of diethylene glycol dimethacrylate caused about 50% T2 reduction with decreasing pH from 6.8 to 6.4. These results demonstrated that responses of microgels to a range of neutral pH can be easily tuned by using appropriate cross-linkers with certain cross-linking degree. This approach can be useful in developing highly sensitive molecular sensors for magnetic resonance imaging (MRI) of tissue pH values.

Backhoe-assisted monolith method for plant root phenotyping under upland conditions.

Root system architecture (RSA) is one of the most important traits determining water and nutrient availability for plants. Modification of RSA is known to be a useful approach for improving root performance of crops. However, for conducting root phenotyping, there are few alternatives for the rapid collection of root samples from a constant soil volume. In this report, we propose a rapid root-sampling method, which uses a steel cylinder known as round monolith and backhoes to reduce the physical effort. The monolith was set on the ground surrounding individual rice plants and vertically driven back by a backhoe. Soil samples with 20 cm width and 25 cm depth were excavated by the monolith, from which root samples were then isolated. This backhoe-assisted monolith method requires at most five minutes to collect root samples from one plant. Using this method, we quantified the root traits of three rice lines, reported to form different types of root system such as shallow-, intermediate-, and deep-roots, using a root image analysis software. The data obtained through this method, which showed the same trend as previously reported, clearly demonstrated that this method is useful for quantitative evaluation of roots in the soil.

Development of defective and persistent Sendai virus vector: a unique gene delivery/expression system ideal for cell reprogramming.

The ectopic expression of transcription factors can reprogram differentiated tissue cells into induced pluripotent stem cells. However, this is a slow and inefficient process, depending on the simultaneous delivery of multiple genes encoding essential reprogramming factors and on their sustained expression in target cells. Moreover, once cell reprogramming is accomplished, these exogenous reprogramming factors should be replaced with their endogenous counterparts for establishing autoregulated pluripotency. Complete and designed removal of the exogenous genes from the reprogrammed cells would be an ideal option for satisfying this latter requisite as well as for minimizing the risk of malignant cell transformation. However, no single gene delivery/expression system has ever been equipped with these contradictory characteristics. Here we report the development of a novel replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic variant virus, which fulfills all of these requirements for cell reprogramming. The SeVdp vector could accommodate up to four exogenous genes, deliver them efficiently into various mammalian cells (including primary tissue cells and human hematopoietic stem cells) and express them stably in the cytoplasm at a prefixed balance. Furthermore, interfering with viral transcription/replication using siRNA could erase the genomic RNA of SeVdp vector from the target cells quickly and thoroughly. A SeVdp vector installed with Oct4/Sox2/Klf4/c-Myc could reprogram mouse primary fibroblasts quite efficiently; ∼1% of the cells were reprogrammed to Nanog-positive induced pluripotent stem cells without chromosomal gene integration. Thus, this SeVdp vector has potential as a tool for advanced cell reprogramming and for stem cell research.

MRI-based Glucose Assay Using Magnetic Nanoparticle Sensors.

Glucose sensors for NMR relaxometry and magnetic resonance imaging (MRI) can be used for the direct measurement of glucose in turbid biological specimens. Here, we proposed a magnetic glucose sensor based on superparamagnetic iron oxide (SPIO) nanoparticles conjugated to a mannopyranoside derivative and concanavalin A (ConA). The binding of mannopyranoside groups to ConA produced a nanoparticle cluster that was dissociated by competitive binding of glucose to ConA, resulting in changes in the transverse relaxation time (T2) in a glucose-dependent manner. The sensor gave rise to significant T2 changes in physiological glucose levels of 3 - 8 mM at a nanoparticle concentration of 0.5 nM. Significant T2 responses were observed within 6 min of 5 mM glucose detection. Sensor-based MRI by a benchtop 1 tesla scanner permitted a measurement of multiple samples within 8 min. These results demonstrate that the relaxometric glucose sensor could lead to high throughput direct assay of blood samples by using a compact MRI scanner for point-of-care testing.

High-throughput three-dimensional visualization of root system architecture of rice using X-ray computed tomography.

BACKGROUND: X-ray computed tomography (CT) allows us to visualize root system architecture (RSA) beneath the soil, non-destructively and in a three-dimensional (3-D) form. However, CT scanning, reconstruction processes, and root isolation from X-ray CT volumes, take considerable time. For genetic analyses, such as quantitative trait locus mapping, which require a large population size, a high-throughput RSA visualization method is required. RESULTS: We have developed a high-throughput process flow for the 3-D visualization of rice (Oryza sativa) RSA (consisting of radicle and crown roots), using X-ray CT. The process flow includes use of a uniform particle size, calcined clay to reduce the possibility of visualizing non-root segments, use of a higher tube voltage and current in the X-ray CT scanning to increase root-to-soil contrast, and use of a 3-D median filter and edge detection algorithm to isolate root segments. Using high-performance computing technology, this analysis flow requires only 10 min (33 s, if a rough image is acceptable) for CT scanning and reconstruction, and 2 min for image processing, to visualize rice RSA. This reduced time allowed us to conduct the genetic analysis associated with 3-D RSA phenotyping. In 2-week-old seedlings, 85% and 100% of radicle and crown roots were detected, when 16 cm and 20 cm diameter pots were used, respectively. The X-ray dose per scan was estimated at < 0.09 Gy, which did not impede rice growth. Using the developed process flow, we were able to follow daily RSA development, i.e., 4-D RSA development, of an upland rice variety, over 3 weeks. CONCLUSIONS: We developed a high-throughput process flow for 3-D rice RSA visualization by X-ray CT. The X-ray dose assay on plant growth has shown that this methodology could be applicable for 4-D RSA phenotyping. We named the RSA visualization method 'RSAvis3D' and are confident that it represents a potentially efficient application for 3-D RSA phenotyping of various plant species.

Podocalyxin is a glycoprotein ligand of the human pluripotent stem cell-specific probe rBC2LCN.

In comprehensive glycome analysis with a high-density lectin microarray, we have previously shown that the recombinant N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia (rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type 1 transmembrane protein, is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high-throughput antibody-overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O-glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched O-glycan comprising an H type 3 structure (Ka, 2.5 × 10(4) M(-1)) prepared from human 201B7 iPS cells, indicating that H type 3 is a most probable potential pluripotency marker. We conclude that podocalyxin is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells.

Cellular DBP and E4BP4 proteins are critical for determining the period length of the circadian oscillator.

The phenotypes of mice carrying clock gene mutations have been critical to understanding the mammalian clock function. However, behavior does not necessarily reflect cell-autonomous clock phenotypes, because of the hierarchical dominance of the central clock. We performed cell-based siRNA knockdown and cDNA overexpression and monitored rhythm using bioluminescent reporters of clock genes. We found that knockdown of DBP, D-box positive regulator, in our model led to a short-period phenotype, whereas overexpressing of DBP produced a long-period rhythm when compared to controls. Furthermore, knockdown and overexpressing of E4BP4, D-box negative regulator, led to an opposite effect of DBP. Our experiments demonstrated that D-box regulators play a crucial role in determining the period length of Per1 and Per2 promoter-driven circadian rhythms in Rat-1 fibroblasts.