Histochemical studies on the distribution and accumulation of trans-polyisoprene in the rubber-producing plant Eucommia ulmoides.

Eucommia ulmoides rubber (EUR) is a high-quality natural rubber resource, which can be extracted from different organs of the Eucommia ulmoides tree. In this study, EUR was isolated from the leaves, barks, and pericarps, and the structural characteristics and physicochemical properties of EUR were systematically determined. The accumulation and distribution of EUR in different tissues were assessed through in situ observations combined with cellular and subcellular scales. The preliminary analyses indicated that the variations in the physicochemical properties of EUR across different tissues were associated with its accumulation microstructure. Further analyses by SEM and TEM showed that the initial cell differentiation and fusion resulted in the formation of tubular structures without any nucleus. A limited number of rubber particles were generated within the cytoplasm, concurrent with aggregation and fusion. Eventually, rubber particles filled the entire cytoplasm, and organelles disappeared to form highly aggregated filamentous structures. In addition, the number and area of EUR-containing cells were closely related to the organization sizes of barks and leaves. This study provided valuable insights into Eucommia ulmoides histology and the rubber industry.

The leucine-rich repeat receptor-like kinase OsERL plays a critical role in anther lobe formation in rice.

In Arabidopsis, ERECTA (ER) subfamily of leucine-rich repeat (LRR) receptor kinases (LRR-RKs) play important roles in cell division and cell elongation. However, the functions of OsER genes in rice are still very much unknown. In this study, sixty-seven TILLING and four gene-edited mutants were identified for one of the three OsERs, OsERL, and used for functional analyses. Results showed that mutations in OsERL led to striking defects in anther development. Compete male sterility and reduced numbers of anther lobes, more severe than knockout mutants, were observed in mutants with amino acid substitutions in the kinase domain. Among alleles with amino acid changes in LRRs, only one mutation in the 16th LRR showed evident phenotype, suggesting a role of the LRR in ligand sensing. OsERL is expressed in shoot apcies, internodes and anthers, and within the anther OsERL is expressed in sporophytic and tapetal cells. Cell biological analyses revealed that mutations in OsERL led to defected periclinal division in archesporial cells in anthers, suggesting a critical role of OsERL in rice anther development.

Defective mitochondrial function by mutation in THICK ALEURONE 1 encoding a mitochondrion-targeted single-stranded DNA-binding protein leads to increased aleurone cell layers and improved nutrition in rice.

Cereal endosperm comprises an outer aleurone and an inner starchy endosperm. Although these two tissues have the same developmental origin, they differ in morphology, cell fate, and storage product accumulation, with the mechanism largely unknown. Here, we report the identification and characterization of rice thick aleurone 1 (ta1) mutant that shows an increased number of aleurone cell layers and increased contents of nutritional factors including proteins, lipids, vitamins, dietary fibers, and micronutrients. We identified that the TA1 gene, which is expressed in embryo, aleurone, and subaleurone in caryopses, encodes a mitochondrion-targeted protein with single-stranded DNA-binding activity named OsmtSSB1. Cytological analyses revealed that the increased aleurone cell layers in ta1 originate from a developmental switch of subaleurone toward aleurone instead of starchy endosperm in the wild type. We found that TA1/OsmtSSB1 interacts with mitochondrial DNA recombinase RECA3 and DNA helicase TWINKLE, and downregulation of RECA3 or TWINKLE also leads to ta1-like phenotypes. We further showed that mutation in TA1/OsmtSSB1 causes elevated illegitimate recombinations in the mitochondrial genome, altered mitochondrial morphology, and compromised energy supply, suggesting that the OsmtSSB1-mediated mitochondrial function plays a critical role in subaleurone cell-fate determination in rice.

Efficient CELI endonuclease production in Nicotiana benthamiana through transient expression and applications in detections of mutation and gene editing events.

Rapid and low-cost methods of detecting mutations and polymorphisms are crucial for genotyping applications including mutagenesis and gene editing. S1 family endonucleases such as T7E1, EndoV and CELI can potentially be used in enzymatic mismatch detection. Among them, CELI has been shown to be effective in detecting mutations in Targeting Induced Local Lesions IN Genomes (TILLING). However, current method of CELI purification from celery is laborious, and challenging for many non-biochemical laboratories, and the presence of post-translational modifications hinders efficient production of the enzyme in E. coli. Here, we report an efficient system for bulk production of enzymatically active CELI endonuclease through transient expression in a model plant Nicotiana benthamiana. We also optimized the reaction buffer, by additions of Mn2+ and DTT, with enhanced mismatch cleavage activity. Using the new CELI production and reaction system, we were able to routinely detect mismatches in 1/32 mixed mutant and wildtype DNA samples. We believe the newly established system has many applications in characterization of mutations occurred in natural variations, mutagenized populations and gene editing.

The Copy Number Variation of OsMTD1 Regulates Rice Plant Architecture.

Copy number variation (CNV) may have phenotypic effects by altering the expression level of the gene(s) or regulatory element(s) contained. It is believed that CNVs play pivotal roles in controlling plant architecture and other traits in plant. However, the effects of CNV contributing to special traits remain largely unknown. Here we report a CNV involved in rice architecture by modulating tiller number and leaf angle. In the genome of Oryza sativa ssp. japonica cv. Nipponbare, we found a locus Loc_Os08g34249 is derived from a 13,002-bp tandem duplication in the nearby region of OsMTD1, a gene regulating tillering in rice. Further survey of 230 rice cultivars showed that the duplication occurred in only 13 japonica rice cultivars. Phenotypic investigation indicated that this CNV region may contribute to tiller number. Moreover, we revealed that OsMTD1 not only influences rice tiller number and leaf angle, but also represses pri-miR156f transcription in the CNV region. Intriguingly, this CNV performs function through both the dosage and position effects on OsMTD1 and pri-miR156f. Thus, our work identified a CNV and revealed a molecular regulatory basis for its effects on plant architecture, implying this CNV may possess importance and application potential in molecular breeding in rice.

An efficient TILLING platform for cultivated tobacco.

Targeting-induced local lesions in genomes (TILLING) is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants. Although TILLING has been developed for many diploid plants, the technology has been used in very few polyploid species due to their genomic complexity. Here, we established an efficient capillary electrophoresis-based TILLING platform for allotetraploid cultivated tobacco (Nicotiana tabacum L.) using an ethyl methanesulfonate (EMS)-mutagenized population of 1,536 individuals. We optimized the procedures for endonuclease preparation, leaf tissue sampling, DNA extraction, normalization, pooling, PCR amplification, heteroduplex formation, and capillary electrophoresis. In a test screen using seven target genes with eight PCR fragments, we obtained 118 mutants. The mutation density was estimated to be approximately one mutation per 106 kb on average. Phenotypic analyses showed that mutations in two heavy metal transporter genes, HMA2S and HMA4T, led to reduced accumulation of cadmium and zinc, which was confirmed independently using CRISPR/Cas9 to generate knockout mutants. Our results demonstrate that this powerful TILLING platform (available at http://www.croptilling.org) can be used in tobacco to facilitate functional genomics applications.

PINOID Is Required for Formation of the Stigma and Style in Rice.

The stigma is the entry point for sexual reproduction in plants, but the mechanisms underlying stigma development are largely unknown. Here, we disrupted putative auxin biosynthetic and signaling genes to evaluate their roles in rice (Oryza sativa) development. Disruption of the rice PINOID (OsPID) gene completely eliminated the development of stigmas, and overexpression of OsPID led to overproliferation of stigmas, suggesting that OsPID is a key determinant for stigma development. Interestingly, ospid mutants did not display defects in flower initiation, nor did they develop any pin-like inflorescences, a characteristic phenotype observed in pid mutants in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). We constructed double mutants of OsPID and its closest homolog, OsPIDb, yet the double mutants still did not develop any pin-like inflorescences, indicating that either ospid is compensated by additional homologous genes or OsPID has different functions in rice compared with PID in other organisms. We then knocked out one of the NAKED PINS IN YUC MUTANTS (NPY) genes, which cause the formation of pin-like inflorescences in Arabidopsis when compromised, in the ospid background. The ospid osnpy2 double mutants developed pin-like inflorescences, which were phenotypically similar to pid mutants in Arabidopsis and maize, demonstrating that the roles of OsPID in inflorescence development are likely masked by redundant partners. This work identified a key determinant for stigma development in rice and revealed a complex picture of the PID gene in rice development. Furthermore, the stigma-less ospid mutants are potentially useful in producing hybrid rice.

IPA1 functions as a downstream transcription factor repressed by D53 in strigolactone signaling in rice.

Strigolactones (SLs), a group of carotenoid derived terpenoid lactones, are root-to-shoot phytohormones suppressing shoot branching by inhibiting the outgrowth of axillary buds. DWARF 53 (D53), the key repressor of the SL signaling pathway, is speculated to regulate the downstream transcriptional network of the SL response. However, no downstream transcription factor targeted by D53 has yet been reported. Here we report that Ideal Plant Architecture 1 (IPA1), a key regulator of the plant architecture in rice, functions as a direct downstream component of D53 in regulating tiller number and SL-induced gene expression. We showed that D53 interacts with IPA1 in vivo and in vitro and suppresses the transcriptional activation activity of IPA1. We further showed that IPA1 could directly bind to the D53 promoter and plays a critical role in the feedback regulation of SL-induced D53 expression. These findings reveal that IPA1 is likely one of the long-speculated transcription factors that act with D53 to mediate the SL-regulated tiller development in rice.

[Study on the Design of Prism Hyperspectral Imaging System Based on Off-Axis Two-Mirror Littrow Configuration].

In order to meet the requirements of high spectral resolution and high image quality on the hyperspectral imaging system, and to meet the new demands of miniaturization, light weight, and high optical efficiency in practical applications, a prism known as hyperspectral imaging system based on Littrow configuration is designed. The use of off-axis two-mirror Littrow configuration is to reduce the size of the optical system and provide a collimated beam for the plane prisms. To avoid the optical path interference, the macro programming optimization is applied. The application of two correct lens and aspheric mirrors can correct the spectral smile and the keystone of the hyperspectral imaging system. It is indicated that the distortion is less than 2.1 µm and the spectral bend is less than 1.3 µm, both are controlled within 18% pixel. The analytical results indicate that the MTF in the visible-near infrared(VNIR) spectral region from 400 to 1 080 nm is above 0.9 while spectrum resolution is about 1.6～5.0 nm, the spectral transmittance more than 51.5%. The results show that the system has high transmittance and image quality within the whole spectral range.

[Algorithm for Background Removal in Spectral Image of Echelle Spectrometer].

Echelle spectrometer gets full spectrum by transient direct reading because of the characteristic of cross-dispersion. The two-dimension spectra received by flat-plane detector needs to be reduced to one-dimension spectra so that the effective wavelength can be detected. Because of huge original data and few effective data, background removal plays an important role of decreasing the amount of data and improving data processing speed. The two-dimension spectrum of echelle spectrometer is analyzed and a suitable background removal algorithms is came up. The edge detection method is applied to diffuse spot detection. Selecting appropriate operator to convolute original image to get edge image and calculating global threshold to segment edge image which can be used to map original image to get the background removed image. Two-dimensional spectral images based on different elements at different integration time are used to judge the effect of different background removal algorithm and different operator are analyzed to figure out their effect of speed and accuracy for algorithm. Experimental result shows that the algorithm came up by this letter is better for image background removal than the others. The background removed image can be used in spectrum reductionand the speed of data processing is notable promoted.

[An Effective Wavelength Detection Method Based on Echelle Spectra Reduction].

Echelle spectrometer with high dispersion, high resolution, wide spectral coverage, full spectrum transient direct-reading and many other advantages, is one of the representative of the advanced spectrometer. In the commercialization trend of echelle spectrometer, the method of two-dimension spectra image processing is becoming more and more important. Currently, centroid extraction algorithm often be used first to detect the centroid position of effective facula and then combined with echelle spectrum reduction method to detect the effective wavelength, but this method is more difficult to achieve the desired requirements. To improve the speed, accuracy and the ability of imaging error correction during detecting the effective wavelength, an effective wavelength detection method based on spectra reduction is coming up. At the beginning, the two-dimension spectra will be converted to a one-dimension image using echelle spectra reduction method instead of finding centroid of effective facula. And then by setting appropriate threshold the one-dimension image is easy to be dealing with than the two-dimension spectra image and all of the pixel points stand for effective wavelength can be detected at one time. Based on this new idea, the speed and accuracy of image processing have been improved, at the same time a range of imaging errors can be compensated. Using the echelle spectrograph make a test applying this algorithm for data processing to check whether this method is fit for the spectra image processing or not. Choosing a standard mercury lamp as a light source during the test because the standard mercury lamp have a number of known characteristic lines which can be used to examine the accuracy of wavelength detection. According to experimental result, this method not only increase operation speed but improve accuracy of wavelength detection, also the imaging error lower than 0.05 mm (two pixel) can be corrected, and the wavelength accuracy would up to 0.02 nm which can satisfy the requirements of echelle spectrograph for image processing.

[Nanomechanical properties of vertebral trabeculae in ovariectomized rats].

OBJECTIVE: To study the nanomechanical properties of the vertebral trabeculae of ovariectomized rat using nanoindentation. METHODS: Twenty 10-month-old SD rats were randomly divided into 2 equal groups: ovariectomized (OVX) group and Sham operation (SHAM) group. Fifteen weeks post-operationally dual energy X-ray absorptiometry was used to measure the bone mineral density (BMD) of the total body and of the sixth lumbar vertebra. Then the rats were killed. The BMD values of the sixth lumbar vertebrae were measured by DXA. Bone histomorphometry was performed on the proximal metaphysis of the right tibia. Three of the sixth lumbar vertebrae were randomly selected from each group and embedded in methyl methacrylate. Each vertebra was cut into two parts along the transverse direction in the middle point of longitudinal axis so as to expose the trabeculae on the cross section. The lower part was polished, trabeculae were randomly selected from 4 places, and 5 points from each place were randomly selected to undergo nanoindentation so as to measure the nanomechanical properties. RESULTS: Compared with the SHAM rats, the BMD of the sixth lumbar vertebra of the OVX rats was reduced significantly (P < 0.05). The histomorphometry of the tibia showed an increase in trabecular separation and a decrease in trabecular bone area fraction (both P < 0.05); the trabecular number and thickness decreased in these 2 groups, however, without significant difference between them. Nanoindentation tests showed that the values of hardness and elastic modulus of the trabeculae of the OVX rats were 0.91 GPa +/- 0.13 GPa and 21.01 GPa +/- 2.48 GPa respectively, not significantly different from those of the SHAM rats, 0.90 GPa +/- 0.09 GPa and 22.03 GPa +/- 2.44 GPa respectively. CONCLUSION: A novel technique, nanoindentation is able to directly measure the nanomechanical properties of trabeculae. Estrogen deficiency after ovariectomy induces significant osteoporotic change, but has no significant influence on the trabecular nanomechanical properties.

[Digital speckle correlation method: a technique to evaluate the tensile property of articular cartilage].

OBJECTIVE: To establish a new technique for evaluation of the tensile property of articular cartilage using digital speckle correlation method (DSCM). METHODS: Three specimens of whole layer articular cartilage of the size of 10 mm x 4 mm were prepared from the cartilage of head of femur replaced from a 52-year-old male suffering from fracture of neck of femur (old control), the amputated tibia plateau of a 16-year-old female suffering from osteosarcoma (young control), and the cartilage of head of femur of a 19-year-old female suffering from spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDT-PA) and then underwent DSCM in U and V fields. RESULTS: The specimens of the 2 controls showed a smooth surface of cartilage and homologous deformation while the specimen of the SEDT-PA patient showed a rough surface of cartilage and deformation with increased undulation. Under a changed loading of 3.3 N the values of average strain of cartilage were 3800, 8800, and 9500 micro epsilon, and the values of tensile elastic modularity were 227.23, 89.59, and 127.25 MPa respectively for the old control, young control, and SEDT-PA patient. The numbers of pixel in U field after 20 pixels were moved in the X direction were significantly different between the old control subject and the SEDT-PA patient (0.101 +/- 0.022 vs 0.220 +/- 0.053, P = 0.023). The numbers of pixel in V field after 20 pixels were moved in the Y direction were significantly different among the old control subject and the SEDT-PA patient. Differences were also significant among the three kinds of cartilage (0.055 +/- 0.018, 0.196 +/- 0.057 vs 0.658 +/- 0.144, both P < 0.05). CONCLUSION: DSCM is a reliable technique to measure the tensile property of articular cartilage, especially for evaluation of small specimens. SEDT-PA is characterized by a dramatic decrease of tensile property, causing destruction and loss of the articular cartilage.