The cytokinin receptor OHK4/OsHK4 regulates inflorescence architecture in rice via an IDEAL PLANT ARCHITECTURE1/WEALTHY FARMER'S PANICLE-mediated positive feedback circuit.

Inflorescence architecture is important for rice (Oryza sativa) grain yield. The phytohormone cytokinin (CK) has been shown to regulate rice inflorescence development; however, the underlying mechanism mediated by CK perception is still unclear. Employing a forward genetic approach, we isolated an inactive variant of the CK receptor OHK4/OsHK4 gene named panicle length1, which shows decreased panicle size due to reduced inflorescence meristem (IM) activity. A 2-amino acid deletion in the long α-helix stalk of the sensory module of OHK4 impairs the homodimerization and ligand-binding capacity of the receptor, even though the residues do not touch the ligand-binding domain or the dimerization interface. This deletion impairs CK signaling that occurs through the type-B response regulator OsRR21, which acts downstream of OHK4 in controlling inflorescence size. Meanwhile, we found that IDEAL PLANT ARCHITECTURE1(IPA1)/WEALTHY FARMER'S PANICLE (WFP), encoding a positive regulator of IM development, acts downstream of CK signaling and is directly activated by OsRR21. Additionally, we revealed that IPA1/WFP directly binds to the OHK4 promoter and upregulates its expression through interactions with 2 TCP transcription factors, forming a positive feedback circuit. Altogether, we identified the OHK4-OsRR21-IPA1 regulatory module, providing important insights into the role of CK signaling in regulating rice inflorescence architecture.

Rice transcription factor OsNAC2 maintains the homeostasis of immune responses to bacterial blight.

Rice (Oryza sativa) bacterial blight, caused by Xanthomonas oryzae pv. Oryzae (Xoo), threatens plant growth and yield. However, the molecular mechanisms underlying rice immunity against Xoo remain elusive. Here, we identified a NAC (NAM-ATAF-CUC) transcription factor OsNAC2 as a negative regulator in the resistance to bacterial blight disease in rice. Constitutive overexpression of OsNAC2 inhibited the expression of salicylic acid (SA) biosynthesis-related genes (i.e. isochorismate synthase 1 (OsICS1), phenylalanine ammonia lyase 3 (OsPAL3), etc.) with adverse impacts on the pathogenesis-related proteins (PRs) responses and compromised blight resistance. Moreover, OsNAC2 interacted with APETALA2/ethylene-responsive element binding protein (AP2/EREBP) transcription factor OsEREBP1 and possibly threatened its protein stability, destroying the favorable interaction of OsEREBP1-Xa21-binding protein OsXb22a in the cytoplasm during Xoo-induced infection. On the contrary, downregulation of OsNAC2 resulted in enhanced resistance to bacterial blight in rice without any growth or yield penalties. Our results demonstrated that OsNAC2 inhibits SA signaling and stably interacted with OsEREBP1 to impair disease resistance. This OsNAC2-OsEREBP1-based homeostatic mechanism provided insights into the competition between rice and bacterial pathogens, and it will be useful to improve the disease resistance of important crops through breeding.

Chatter-suppressing ruling method based on double-layer elastic support.

A cross-hinge spring is the preferred support for a ruling tool because of its excellent flexibility. However, there are high precision requirements for the tool installation, which make the installation and adjustments difficult. There also is poor robustness against interference, which readily results in tool chatter. These issues affect the quality of the grating. This paper proposes an elastic ruling tool carrier with a double-layer parallel-spring mechanism, establishes a torque model of the spring, and analyzes its force state. In a simulation, the spring deformation and frequency modes of the two ruling tool carriers are compared and the overhang length of the parallel-spring mechanism is optimized. In addition, the performance of the optimized ruling tool carrier is analyzed in a grating ruling experiment to verify the carrier's effectiveness. The results show that compared to the cross-hinge elastic support, the deformation of the parallel-spring mechanism by a ruling force in the X direction is on the same order of magnitude. However, the deformation in the Y direction is reduced by a factor of 270, and the deformation in the Z direction is reduced by a factor of 32. The torque of the proposed tool carrier is slightly higher (12.8%) in the Z direction but lower by a factor of 2.5 in the X direction and by a factor of 60 in the Y direction. The overall stiffness of the proposed tool carrier is improved and the first-order frequency of the proposed structure is higher by a factor of 2.8. The proposed tool carrier thus better suppresses chatter, effectively reducing the effect of the ruling tool installation error on the grating quality. The flutter suppression ruling method can provide a technical basis for further research on high-precision grating ruling manufacturing technology.

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.

Mutations in the DNA demethylase OsROS1 result in a thickened aleurone and improved nutritional value in rice grains.

The rice endosperm, consisting of an outer single-cell layer aleurone and an inner starchy endosperm, is an important staple food for humans. While starchy endosperm stores mainly starch, the aleurone is rich in an array of proteins, vitamins, and minerals. To improve the nutritional value of rice, we screened for mutants with thickened aleurones using a half-seed assay and identified thick aleurone 2-1 (ta2-1), in which the aleurone has 4.8 ± 2.2 cell layers on average. Except for starch, the contents of all measured nutritional factors, including lipids, proteins, vitamins, minerals, and dietary fibers, were increased in ta2-1 grains. Map-based cloning showed that TA2 encodes the DNA demethylase OsROS1. A point mutation in the 14th intron of OsROS1 led to alternative splicing that generated an extra transcript, mOsROS1, with a 21-nt insertion from the intron. Genetic analyses showed that the ta2-1 phenotype is inherited with an unusual gametophytic maternal effect, which is caused not by imprinted gene expression but rather by the presence of the mOsROS1 transcript. Five additional ta2 alleles with the increased aleurone cell layer and different inheritance patterns were identified by TILLING. Genome-wide bisulfite sequencing revealed general increases in CG and CHG methylations in ta2-1 endosperms, along with hypermethylation and reduced expression in two putative aleurone differentiation-related transcription factors. This study thus suggests that OsROS1-mediated DNA demethylation restricts the number of aleurone cell layers in rice and provides a way to improve the nutrition of rice.

OsNAC2 integrates auxin and cytokinin pathways to modulate rice root development.

The rice root system is important for growth. The crosstalk between auxin and cytokinin mediates root initiation and elongation. However, it remains unclear how the transcriptional network upstream of the auxin and cytokinin signalling pathways determines root development. Here, we observed that the knockdown of OsNAC2, which encodes a NAC transcription factor, increased the primary root length and the number of crown roots. OsNAC2 predominantly expressed in primary root tips, crown roots and lateral root primordia, implying it influences root development. Molecular analyses revealed that the expressions of auxin- and cytokinin-responsive genes were affected in OsNAC2-overexpressing (OsNAC2-OX; ON7 and ON11), RNA interference (OsNAC2-RNAi; RNAi25 and RNAi31) and CRISPR/Cas9 plants. Additionally, OsNAC2 can directly bind to the promoters of IAA inactivation-related genes (GH3.6 and GH3.8), an IAA signalling-related gene (OsARF25), and a cytokinin oxidase gene (OsCKX4). Furthermore, genetic analysis of ON11/osgh3.6 and RNAi31/osckx4 homozygote confirmed that OsCKX4 and OsGH3.6 functioned downstream of OsNAC2. The mRNA levels of CROWN ROOTLESS (CRL) genes and cyclin-dependent protein kinase (CDK) genes increased in OsNAC2-RNAi and OsNAC2-cas9 lines while reduced in OsNAC2-OX lines. Thus, we describe that OsNAC2 functions as an upstream integrator of auxin and cytokinin signals that affect CRL and CDK production to regulate cell division during root development. This novel auxin-OsNAC2-cytokinin model should provide a new insight into the understanding of NAC TFs and crosstalk of auxin and cytokinin pathway, and can be potentially applied in agriculture to enhance rice yields by genetic approaches.

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.

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.

OsCIPK7 point-mutation leads to conformation and kinase-activity change for sensing cold response.

Calcineurin B-like interacting protein kinases (CIPKs) play important roles via environmental stress. However, less is known how to sense the stress in molecular structure conformation level. Here, an OsCIPK7 mutant via TILLING procedure with a point mutation in the kinase domain showed increased chilling tolerance, which could be potentially used in the molecular breeding. We found that this point mutation of OsCIPK7 led to a conformational change in the activation loop of the kinase domain, subsequently with an increase of protein kinase activity, thus conferred an increased tolerance to chilling stress.

Abscisic acid negatively modulates plant defence against rice black-streaked dwarf virus infection by suppressing the jasmonate pathway and regulating reactive oxygen species levels in rice.

Abscisic acid (ABA) plays a multifaceted role in plant immunity and can either increase resistance or increase susceptibility to some bacterial and fungal pathogens depending on the pathosystem. ABA is also known to mediate plant defence to some viruses. In this study, the relationship between the ABA pathway and rice black-streaked dwarf virus (RBSDV) was investigated in rice. The expression of ABA pathway genes was significantly reduced upon RBSDV infection. Application of exogenous hormones and various ABA pathway mutants revealed that the ABA pathway plays a negative role in rice defence against RBSDV. Exogenous hormone treatment and virus inoculation showed that ABA inhibits the jasmonate-mediated resistance to RBSDV. ABA treatment also suppressed accumulation of reactive oxygen species by inducing the expression of superoxidase dismutases and catalases. Thus, ABA modulates the rice-RBSDV interaction by suppressing the jasmonate pathway and regulating reactive oxygen species levels. This is the first example of ABA increasing susceptibility to a plant virus.

Design and analysis of a worm gear turntable off-axis assembly method in a three-grating monochromator.

To solve the problem where the actual grating aperture decreases with an increasing scanning angle during the scanning of a three-grating monochromator, we propose an off-axis assembly method for the worm gear turntable that makes it possible to suppress this aperture reduction. We simulated and compared the traditional assembly method with the off-axis assembly method in the three-grating monochromator. Results show that the actual grating aperture can be improved by the off-axis assembly method. In fact, for any one of the three gratings, when the monochromator outputs the longest wavelength in the corresponding wavelength band, the actual grating aperture increases by 45.93%. Over the entire monochromator output band, the actual grating aperture increased by an average of 32.56% and can thus improve the monochromator's output energy. Improvement of the actual grating aperture can also reduce the stray light intensity in the monochromator and improve its output signal-to-noise ratio.

Correcting groove error in gratings ruled on a 500-mm ruling engine using interferometric control.

Groove error is one of the most important factors affecting grating quality and spectral performance. To reduce groove error, we propose a new ruling-tool carriage system based on aerostatic guideways. We design a new blank carriage system with double piezoelectric actuators. We also propose a completely closed-loop servo-control system with a new optical measurement system that can control the position of the diamond relative to the blank. To evaluate our proposed methods, we produced several gratings, including an echelle grating with 79 grooves/mm, a grating with 768 grooves/mm, and a high-density grating with 6000 grooves/mm. The results show that our methods effectively reduce groove error in ruled gratings.

[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.

[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.

[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.

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.

Efficient separation and production of high-quality rubber, lignin nanoparticles and fermentable sugars from Eucommia ulmoides pericarp via deep eutectic solvent pretreatment.

The natural barriers of lignocellulose hinder the separation of Eucommia ulmoides rubber (EUR) from Eucommia ulmoides pericarp (EUP), whereas traditional separation methods normally lead to the waste of Eucommia ulmoides lignocellulose resource and environmental pollution. In this study, an acidic deep eutectic solvent composed of lactic acid and ZnCl2 was developed as a pretreatment medium to reduce the separation barriers of EUR while producing lignin nanoparticles and fermentable sugars. Results showed that DES pretreatment could accelerate the extraction efficiency (91.0 %) and purity (>99 %) of EUR and maintain its chemical structure compared to the traditional alkaline and mechanical methods. Meanwhile, the regenerated nano-lignin showed excellent antioxidant activity (IC50 = 46.3 µg/mL) comparable to commercial antioxidant BHA. Besides, the enzymatic hydrolysis efficiency of EUP with DES pretreatment was significantly enhanced about 9 times than the control groups. Overall, the acidic DES pretreatment could be considered a promising pretreatment method for separation of high-quality EUR and valorization of lignocellulosic components.

Roxadustat: Do we know all the answers?

Anemia is a common complication of chronic kidney disease (CKD), and its prevalence rises as the disease progresses. Intravenous or subcutaneous erythropoiesis-stimulating agents (ESAs) are advised to treat CKD-associated anemia, since shortage of erythropoietin (EPO) and iron are the main cause of anemia. However, ESA resistance and safety have spurred a lot of interest in the development of alternate anemia therapies. Roxadustat, an orally administered hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) that increases erythropoiesis and may modulate iron metabolism, was recently licensed in China, Chile, South Korea, Japan and the European Union for the treatment of CKD-related anemia. Despite this, clinical trials have shown a number of adverse effects, including cardiovascular disease, hyperkalemia, and infections. In addition, of concern is roxadustat's possible effects on other organs and systems. In this review, based on clinical evidence, we discuss the potentially detrimental effects of roxadustat to the known biology on systems other than kidney, and the need for long-term follow-up in order for roxadustat to be approved in more countries in the future.