A CYP78As-small grain4-coat protein complex â ¡ pathway promotes grain size in rice.

CYP78A, a cytochrome P450 subfamily that includes rice (Oryza sativa L.) BIG GRAIN2 (BG2, CYP78A13) and Arabidopsis thaliana KLUH (KLU, CYP78A5), generate an unknown mobile growth signal (referred to as a CYP78A-derived signal) that increases grain (seed) size. However, the mechanism by which the CYP78A pathway increases grain size remains elusive. Here, we characterized a rice small grain mutant, small grain4 (smg4), with smaller grains than its wild type due to restricted cell expansion and cell proliferation in spikelet hulls. SMG4 encodes a multidrug and toxic compound extrusion (MATE) transporter. Loss of function of SMG4 causes smaller grains while overexpressing SMG4 results in larger grains. SMG4 is mainly localized to endoplasmic reticulum (ER) exit sites (ERESs) and partially localized to the ER and Golgi. Biochemically, SMG4 interacts with coat protein complex Ⅱ (COPⅡ) components (Sar1, Sec23, and Sec24) and CYP78As (BG2, GRAIN LENGTH 3.2 [GL3.2], and BG2-LIKE 1 [BG2L1]). Genetically, SMG4 acts, at least in part, in a common pathway with Sar1 and CYP78As to regulate grain size. In summary, our findings reveal a CYP78As-SMG4-COPⅡ regulatory pathway for grain size in rice, thus providing new insights into the molecular and genetic regulatory mechanism of grain size.

FLOURY ENDOSPERM24, a heat shock protein 101 (HSP101), is required for starch biosynthesis and endosperm development in rice.

Endosperm is the main storage organ in cereal grain and determines grain yield and quality. The molecular mechanisms of heat shock proteins in regulating starch biosynthesis and endosperm development remain obscure. Here, we report a rice floury endosperm mutant flo24 that develops abnormal starch grains in the central starchy endosperm cells. Map-based cloning and complementation test showed that FLO24 encodes a heat shock protein HSP101, which is localized in plastids. The mutated protein FLO24T296I dramatically lost its ability to hydrolyze ATP and to rescue the thermotolerance defects of the yeast hsp104 mutant. The flo24 mutant develops more severe floury endosperm when grown under high-temperature conditions than normal conditions. And the FLO24 protein was dramatically induced at high temperature. FLO24 physically interacts with several key enzymes required for starch biosynthesis, including AGPL1, AGPL3 and PHO1. Combined biochemical and genetic evidence suggests that FLO24 acts cooperatively with HSP70cp-2 to regulate starch biosynthesis and endosperm development in rice. Our results reveal that FLO24 acts as an important regulator of endosperm development, which might function in maintaining the activities of enzymes involved in starch biosynthesis in rice.

Young Leaf White Stripe encodes a P-type PPR protein required for chloroplast development.

Pentatricopeptide repeat (PPR) proteins function in post-transcriptional regulation of organellar gene expression. Although several PPR proteins are known to function in chloroplast development in rice (Oryza sativa), the detailed molecular functions of many PPR proteins remain unclear. Here, we characterized a rice young leaf white stripe (ylws) mutant, which has defective chloroplast development during early seedling growth. Map-based cloning revealed that YLWS encodes a novel P-type chloroplast-targeted PPR protein with 11 PPR motifs. Further expression analyses showed that many nuclear- and plastid-encoded genes in the ylws mutant were significantly changed at the RNA and protein levels. The ylws mutant was impaired in chloroplast ribosome biogenesis and chloroplast development under low-temperature conditions. The ylws mutation causes defects in the splicing of atpF, ndhA, rpl2, and rps12, and editing of ndhA, ndhB, and rps14 transcripts. YLWS directly binds to specific sites in the atpF, ndhA, and rpl2 pre-mRNAs. Our results suggest that YLWS participates in chloroplast RNA group II intron splicing and plays an important role in chloroplast development during early leaf development.

Decreased grain size1, a C3HC4-type RING protein, influences grain size in rice (Oryza sativa L.).

KEY MESSAGE: We reported that DGS1 plays a positive role in regulating grain size in rice and was regulated by OsBZR1. Grain size is an important agronomic trait that contributes to grain yield. However, the underlying molecular mechanisms that determine final grain size are still largely unknown. We isolated a rice mutant showing reduced grain size in a 60Co-irradiated variety Nanjing 35 population. We named the mutant decreased grain size1 (dgs1). Map-based cloning and subsequent transgenic CRISPR and complementation assays indicated that a mutation had occurred in LOC_Os03g49900 and that the DGS1 allele regulated grain size. DGS1 encodes a protein with a 7-transmembrane domain and C3HC4 type RING domain. It was widely expressed, especially in young tissues. DGS1 is a membrane-located protein. OsBZR1 (BRASSINAZOLE-RESISTANT1), a core transcription activator of BR signaling, also plays a positive role in grain size. We provided preliminary evidence that OsBZR1 can bind to the DGS1 promoter to activate expression of DGS1.

Small grain and semi-dwarf 3, a WRKY transcription factor, negatively regulates plant height and grain size by stabilizing SLR1 expression in rice.

KEY MESSAGE: OsWRKY36 represses plant height and grain size by inhibiting gibberellin signaling. Plant height and grain size are important agronomic traits affecting yield in cereals, including rice. Gibberellins (GAs) are plant hormones that promote plant growth and developmental processions such as stem elongation and grain size. WRKYs are transcription factors that regulate stress tolerance and plant development including height and grain size. However, the relationship between GA signaling and WRKY genes is still poorly understood. Here, we characterized a small grain and semi-dwarf 3 (sgsd3) mutant in rice cv. Hwayoung (WT). A T-DNA insertion in the 5'-UTR of OsWRKY36 induced overexpression of OsWRKY36 in the sgsd3 mutant, likely leading to the mutant phenotype. This was confirmed by the finding that overexpression of OsWRKY36 caused a similar small grain and semi-dwarf phenotype to the sgsd3 mutant whereas knock down and knock out caused larger grain phenotypes. The sgsd3 mutant was also hyposensitive to GA and accumulated higher mRNA and protein levels of SLR1 (a GA signaling DELLA-like inhibitor) compared with the WT. Further assays showed that OsWRKY36 enhanced SLR1 transcription by directly binding to its promoter. In addition, we found that OsWRKY36 can protect SLR1 from GA-mediated degradation. We thus identified a new GA signaling repressor OsWRKY36 that represses GA signaling through stabilizing the expression of SLR1.

Characterization of Angle Accuracy and Precision of 3-Degree-of-Freedom Absolute Encoder Based on NanoGPS OxyO Technology.

An absolute encoder based on vision system nanoGPS OxyO was developed by HORIBA France. This encoder provides three types of position information, namely, two inplane co-ordinates and inplane angular orientation. This paper focuses on the characterization of its angular performance. To this aim, the nanoGPS OxyO system was compared with the national angle standard of the National Metrology Institute of Italy (INRIM) that had evaluated accuracy of about 0.1 µrad. The effect of image size and illumination conditions on angular measurements was studied. Precision better than 10 µrad and accuracy better than 63 µrad over 2π rotation were demonstrated. Moreover, the application of nanoGPS OxyO to the characterization of rotation bearing is presented. Small deviations from pure rotational behavior were evidenced that would have not been possible using laser interferometers. As a consequence of its accuracy and versatility, the nanoGPS OxyO encoder is expected to be useful for laboratory experiments and quality-control tasks.

WSL5, a pentatricopeptide repeat protein, is essential for chloroplast biogenesis in rice under cold stress.

Chloroplasts play an essential role in plant growth and development, and cold conditions affect chloroplast development. Although many genes or regulators involved in chloroplast biogenesis and development have been isolated and characterized, many other components affecting chloroplast biogenesis under cold conditions have not been characterized. Here, we report the functional characterization of a white stripe leaf 5 (wsl5) mutant in rice. The mutant develops white-striped leaves during early leaf development and is albinic when planted under cold stress. Genetic and molecular analysis revealed that WSL5 encodes a novel chloroplast-targeted pentatricopeptide repeat protein. RNA sequencing analysis showed that expression of nuclear-encoded photosynthetic genes in the mutant was significantly repressed, and expression of many chloroplast-encoded genes was also significantly changed. Notably, the wsl5 mutation causes defects in editing of rpl2 and atpA, and splicing of rpl2 and rps12. wsl5 was impaired in chloroplast ribosome biogenesis under cold stress. We propose that the WSL5 allele is required for normal chloroplast development in maintaining retrograde signaling from plastids to the nucleus under cold stress.

Targeted next-generation sequencing on Hirschsprung disease: a pilot study exploits DNA pooling.

To adopt an efficient approach of identifying rare variants possibly related to Hirschsprung disease (HSCR), a pilot study was set up to evaluate the performance of a newly designed protocol for next generation targeted resquencing. In total, 20 Chinese HSCR patients and 20 Chinese sex-matched individuals with no HSCR were included, for which coding sequences (CDS) of 62 genes known to be in signaling pathways relevant to enteric nervous system development were selected for capture and sequencing. Blood DNAs from eight pools of five cases or controls were enriched by PCR-based RainDance technology (RDT) and then sequenced on a 454 FLX platform. As technical validation, five patients from case Pool-3 were also independently enriched by RDT, indexed with barcode and sequenced with sufficient coverage. Assessment for CDS single nucleotide variants showed DNA pooling performed well (specificity/sensitivity at 98.4%/83.7%) at the common variant level; but relatively worse (specificity/sensitivity at 65.5%/61.3%) at the rare variant level. Further Sanger sequencing only validated five out of 12 rare damaging variants likely involved in HSCR. Hence more improvement at variant detection and sequencing technology is needed to realize the potential of DNA pooling for large-scale resequencing projects.

Single YVO4:Eu nanoparticle emission spectra using direct Eu3+ ion excitation with a sum-frequency 465-nm solid-state laser.

We report emission spectrum measurements on single YxEu(1-x)VO4 nanoparticles. The inhomogeneous widths of the emission peaks are identical for single nanoparticles and for ensembles of nanoparticles, while being broader than those of the bulk material. This indicates that individual nanoparticles are identical in terms of the distribution of different local Eu3+ sites due to crystalline defects and confirms their usability as identical, single-particle oxidant biosensors. Moreover, we report a 465 nm solid-state laser based on sum-frequency mixing that provides a compact, efficient solution for direct Eu3+ excitation of these nanoparticles. Both these two aspects should broaden the scope of Eu-doped nanoparticle applications.

Fresh Human Umbilical Cord Arteries as a Potential Source for Small-Diameter Vascular Grafts.

The demand for small-diameter vascular grafts has been globally increased but still lacks optimal solutions in this category. This study evaluated the feasibility of utilizing human pretreated fresh and nondecellularized umbilical cord arteries (hUCAs) as vascular grafts without needing any immunosuppression process. A mixed lymphocyte reaction assay revealed that hUCAs did not induce lymphocyte proliferation or cytokine production. To assess the in vivo inflammatory response, hUCAs were buried in fatty tissue under the skin of the abdominal wall in the left and right iliac fossas of rats. The average sizes of the implanted hUCAs remained consistent at 30 days post implantation. To evaluate xenogeneic transplantation, hUCAs were grafted to the abdominal aorta below the kidney of Wister rats. Remarkably, all rats exhibited positive revascularization and perfusion, maintaining blood pressure values of around 110/70 mmHg. Doppler ultrasound consistently indicated good circulation, with the three separate echogenic layers corresponding to the three arterial wall layers throughout the assessment period. Grafted rats exhibited normal motor behavior, accompanied by positive responses to thermal and pain stimulation. Blood biochemical values and whole blood cell counts showed no significant differences between pre and post-transplantation. Histological analysis of the grafts revealed no calcification or thrombosis, and a mild chronic inflammatory response was presented. In conclusion, hUCAs maintained their structural and functional properties after transplantation in rats without immunosuppression. This highlights their potential as a source for allogeneic, readily accessible, small-diameter vascular grafts.

Different regions and environments have critical roles on yield, main quality and industrialization of an industrial purple-fleshed sweetpotato (Ipomoea batatas L. (Lam.)) "Xuzishu8".

Purple-fleshed sweetpotato (PFSP) (Ipomoea batatas (L.) Lam), whose flesh is purple to dark purple, is a special variety type of sweetpotato, which has the characteristics of food crop, industrial crop and medicinal crop. The storage root (SR) of PFSP is rich in anthocyanins, starch, protein, soluble sugar, mineral elements, polyphenol, dietary fiber and so on, which has balanced and comprehensive nutritional value. And in recent years, its unique nutritional elements are increasingly known for their health functions. At present, there is no article on the characteristics and quality analysis of industrial xz8 variety. To explore the influence of different environments on the processing quality of xz8, we selected nine regions (Xuzhou, Jiawang, Pizhou, Xinyi, Peixian, Sihong, Yanchen, Xiangyang and Tianshui) to measure its yield and quality changes. The data demonstrated that xz8 has a very consistent high yield performance. In Tianshui, the anthocyanins, protein and minerals contents were significantly higher and yield also above average. Moreover, the variety with the lowest starch content exhibited the best taste. On the basis of the above results, it suggested that quite practicable to promote xz8 cultivation and suitable for processing in these areas. Thus, our present findings improve our understanding of xz8 variety and provide the basis for the industrial production of PFSP with strong prospects for success.

Identifying and fixing in-plane positioning and stability issues on a microscope using machine-readable patterned position scales.

Investigations of the in-plane positioning capabilities of microscopes using machine-readable encoded patterned scales are presented. The scales have patterns that contain absolute position information, and adequate software accurately determines the in-plane position from the scale images captured by the microscope camera. This makes in-plane positioning experiments simple and fast. The scales and software used in this study are commercially available. We investigated different microscopy systems and found that positioning performance is a system issue that is not determined solely by stage performance. In some cases, our experiments revealed software or hardware glitches that limited the positioning performance, which we easily fixed. We have also shown that it is possible to investigate vibrations using this approach and quantify their impact on image blurring. This is, for example, useful for experimentally determining the settling time after a stage movement.

WEAK SEED DORMANCY 1, an aminotransferase protein, regulates seed dormancy in rice through the GA and ABA pathways.

Seed dormancy is a critical trait that enhances plant survival by preventing seed germination at the wrong time or under unsuitable conditions. Lack of seed dormancy in rice can lead to pre-harvest sprouting on mother plants leading to reduced yield and seed quality. Although some genes have been identified, knowledge of regulation of seed dormancy is limited. Here, we characterized a weak seed dormancy mutant named weak seed dormancy 1 (wsd1) that showed a higher seed germination percentage than the wild-type following the harvest ripeness. We cloned the WSD1 encoding an aminotransferase protein using a MutMap approach. WSD1 was stably expressed after imbibition and its protein was localized in the endoplasm reticulum. A widely targeted metabolomics assay and amino acid analysis showed that WSD1 had a role in regulating homeostasis of amino acids. PAC treatment and RNA-seq analysis showed that WSD1 regulates seed dormancy by involvement in the GA biosynthesis pathway. GA1 content and expression of GA biosynthesis-related genes were increased in the wsd1 mutant compared with the wild-type. The wsd1 mutant had reduced sensitivity to ABA. Our overall results indicated that WSD1 regulates seed dormancy by balancing the ABA and GA pathways.

Identification of QTLs for Heat Tolerance at the Flowering Stage Using Chromosome Segment Substitution Lines in Rice.

High temperature is a major stress in rice production. Although considerable progress has been made in investigating heat tolerance (HT) in rice, the genetic basis of HT at the heading stage remains largely unknown. In this study, a novel set of chromosome segment substitution lines (CSSLs) consisting of 113 lines derived from a heat-resistant indica variety N22 and a heat-sensitive indica variety 9311 was developed and used for the analysis of the genetic basis of HT. The heat sensitivity index (HSI) calculated based on seed-setting rates under natural and high-temperature environments was used to evaluate the influence of HT at the rice heading stage. In total, five quantitative trait loci (QTLs) associated with HT were detected based on seed-setting rate (SSR) evaluation; these were named qSSR6-1, qSSR7-1, qSSR8-1, qSSR9-1 and qSSR11-1 located on chromosomes 6, 7, 8, 9 and 11, respectively. Heat-tolerant alleles of the QTLs were all derived from N22. Among them, qSSR9-1 overlapped with QTLs identified previously, while the remaining QTLs were found novel. In particular, qSSR7-1 explained a high phenotypic variation of 26.35% with a LOD score of 10.75, thus deserved to be further validated. These findings will increase our understanding of the genetic mechanism underlying HT and facilitate the breeding of heat-tolerant rice varieties.

Comparative Transcriptome Profiling Reveals the Genes Involved in Storage Root Expansion in Sweetpotato (Ipomoea batatas (L.) Lam.).

Sweetpotato (Ipomoea batatas (L.) Lam.) is recognized as one of the most important root crops in the world by the Food and Agriculture Organization of the United Nations. The yield of sweetpotato is closely correlated with the rate of storage root (SR) formation and expansion. At present, most of the studies on sweetpotato SR expansion are focused on the physiological mechanism. To explore the SR expansion mechanism of sweetpotato, we performed transcriptome sequencing of SR harvested at 60, 90, 120, and 150 days after planting (DAP) to analyze two sweetpotato lines, Xuzishu 8 and its crossing progenies named Xu 18-192, which were selected from an F1 segregation population of Xuzishu 8 and Meiguohong, in which SR expansion was delayed significantly. A total of 57,043 genes were produced using transcriptome sequencing, of which 1312 were differentially expressed genes (DEGs) in four SR growth periods of the sweetpotato lines. The combination of the KEGG and trend analysis revealed several key candidate genes involved in SR expansion. The SBEI gene involved in starch metabolism, and transcription factors ARF6, NF-YB3 and NF-YB10 were all significantly up-regulated during SR expansion. The data from this study provide insights into the complex mechanisms of SR formation and expansion in sweetpotato and identify new candidate genes for increasing the yield of sweetpotato.

WSL9 Encodes an HNH Endonuclease Domain-Containing Protein that Is Essential for Early Chloroplast Development in Rice.

BACKGROUND: The plant chloroplast is essential for photosynthesis and other cellular processes, but an understanding of the biological mechanisms of plant chloroplast development are incomplete. RESULTS: A new temperature-sensitive white stripe leaf 9(wsl9) rice mutant is described. The mutant develops white stripes during early leaf development, but becomes green after the three-leaf stage under field conditions. The wsl9 mutant was albinic when grown at low temperature. Gene mapping of the WSL9 locus, together with complementation tests indicated that WSL9 encodes a novel protein with an HNH domain. WSL9 was expressed in various tissues. Under low temperature, the wsl9 mutation caused defects in splicing of rpl2, but increased the editing efficiency of rpoB. Expression levels of plastid genome-encoded genes, which are transcribed by plastid-coded RNA polymerase (PEP), chloroplast development genes and photosynthesis-related genes were altered in the wsl9 mutant. CONCLUSION: WSL9 encodes an HNH endonuclease domain-containing protein that is essential for early chloroplast development. Our study provides opportunities for further research on regulatory mechanisms of chloroplast development in rice.

Rice OsBT1 regulates seed dormancy through the glycometabolism pathway.

Seed dormancy and germination in rice (Oryza sativa L.) are complex and important agronomic traits that involve a number of physiological processes and energy. A mutant named h470 selected from a60Co-radiated indica cultivar N22 population had weakened dormancy that was insensitive to Gibberellin (GA) and Abscisic acid (ABA). The levels of GA4 and ABA were higher in h470 than in wild-type (WT) plants. The gene controlling seed dormancy in h470 was cloned by mut-map and transgenesis and confirmed to encode an ADP-glucose transporter protein. A 1 bp deletion in Os02g0202400 (OsBT1) caused the weaker seed dormancy in h470. Metabolomics analyses showed that most sugar components were higher in h470 seeds than the wild type. The mutation in h470 affected glycometabolism.

WRKY Transcription Factor OsWRKY29 Represses Seed Dormancy in Rice by Weakening Abscisic Acid Response.

For efficient plant reproduction, seed dormancy delays seed germination until the environment is suitable for the next generation growth and development. The phytohormone abscisic acid (ABA) plays important role in the induction and maintenance of seed dormancy. Previous studies have identified that WRKY transcription factors can regulate ABA signaling pathway. Here, we identified an Oswrky29 mutant with enhanced dormancy in a screen of T-DNA insertion population. OsWRKY29 is a member of WRKY transcription factor family which located in the nuclear. The genetic analyses showed that both knockout and RNAi lines of OsWRKY29 had enhanced seed dormancy whereas its overexpression lines displayed reduced seed dormancy. When treated with ABA, OsWRKY29 knockout and RNAi lines showed greater sensitivity than its overexpression lines. In addition, the expression levels of ABA positive response factors OsVP1 and OsABF1 were higher in the OsWRKY29 mutants but were lower in its overexpression lines. Further assays showed that OsWRKY29 could bind to the promoters of OsABF1 and OsVP1 to inhibit their expression. In summary, we identified a new ABA signaling repressor OsWRKY29 that represses seed dormancy by directly downregulating the expression of OsABF1 and OsVP1.

Regulation of the ROS response dynamics and organization to PDGF motile stimuli revealed by single nanoparticle imaging.

Although reactive oxygen species (ROS) are better known for their harmful effects, more recently, H2O2, one of the ROS, was also found to act as a secondary messenger. However, details of spatiotemporal organization of specific signaling pathways that H2O2 is involved in are currently missing. Here, we use single nanoparticle imaging to measure the local H2O2 concentration and reveal regulation of the ROS response dynamics and organization to platelet-derived growth factor (PDGF) signaling. We demonstrate that H2O2 production is controlled by PDGFR kinase activity and EGFR transactivation, requires a persistent stimulation, and is regulated by membrane receptor diffusion. This temporal filtering is impaired in cancer cells, which may determine their pathological migration. H2O2 subcellular mapping reveals that an external PDGF gradient induces an amplification-free asymmetric H2O2 concentration profile. These results support a general model for the control of signal transduction based only on membrane receptor diffusion and second messenger degradation.