Florigen repression complexes involving rice CENTRORADIALIS2 regulate grain size.

Grain size is one of the crucial factors determining grain yield. However, the genetic and molecular mechanisms of florigen repression complexes (FRCs) underlying grain size in rice (Oryza sativa L.) have not been reported. Here, we report that the rice CENTRORADIALIS (CEN) family member OsCEN2 (also known as Rice TFL1/CEN homolog, RCN1), a phosphatidylethanolamine-binding protein (PEBP) family protein, negatively controls grain size in rice. Overexpression of OsCEN2 led to small grains, and knockout of OsCEN2 resulted in large, heavy grains. OsCEN2 influenced grain size by restricting cell expansion in the spikelet hull and seed filling. In in vivo and in vitro experiments, OsCEN2 physically interacted with a G-box factor 14-3-3 homolog, GF14f, which negatively regulates grain size. Bimolecular fluorescence complementation and yeast two-hybrid assays revealed that GF14f directly interacts with the basic leucine zipper (bZIP) transcription factor, OsFD2. Plants overexpressing OsFD2 produced smaller and lighter grains than wild-type plants. We found that OsFD2 also influences grain size by controlling cell expansion and division in the spikelet hull. Our results reveal the molecular mechanisms of the OsCEN2-GF14f-OsFD2 regulatory module in controlling grain size. Additionally, our study provides insight into the functions of the FRC in rice and suggests a strategy for improving seed size and weight.

A cytosolic pentatricopeptide repeat protein is essential for tapetal plastid development by regulating OsGLK1 transcript levels in rice.

Most plant pentatricopeptide repeat (PPR) proteins localize to and function inside plastids and mitochondria. However, the function of PPRs that only localize to the cytoplasm remains unknown. Here, we demonstrated that the rice (Oryza sativa) PPR protein CYTOPLASM-LOCALIZED PPR1 (OsCPPR1) contributes to pollen development and localizes to the cytoplasm. Knocking down OsCPPR1 led to abnormal plastid development in tapetal cells, prolonged tapetal programmed cell death (PCD) and tapetum degradation, and significantly reduced pollen fertility. Transcriptome analysis revealed that the transcript level of OsGOLDEN-LIKE1 (OsGLK1), which encodes a transcription factor that regulates plastid development and maintenance, was significantly higher in the OsCPPR1 knockdown plants compared to wild-type plants. We further determined that OsCPPR1 downregulates OsGLK1 transcription by directly binding to the single-stranded regions of OsGLK1 mRNAs. Overexpression of OsGLK1 resulted in abnormal tapetum and plastid development, similar to that seen in OsCPPR1 knockdown plants, and suppression of OsGLK1 partially restored pollen fertility in the OsCPPR1 knockdown plants. We therefore conclude that OsCPPR1 suppresses OsGLK1 in the regulation of plastid development and PCD in the tapetum. Our work revealed novel functions for a cytosolic PPR, demonstrating the diverse roles of PPRs in plants and identifying a new regulatory mechanism for regulating pollen development in rice.

Rice CENTRORADIALIS 2 regulates seed germination and salt tolerance via ABA-mediated pathway.

KEY MESSAGE: A FT/TFL1 subfamily gene, rice CENTRORADIALIS 2, also known as RCN1, regulates seed germination and increase salt tolerance via ABA-mediated pathway. The ABA synthesis and metabolism related genes were changed relative expression levels. Seed germination is a complex biological process that is affected by many factors. Although a number of germination-related genes have been reported, the molecular mechanism of germination regulation has not yet been fully elucidated. Here, we reported that the rice OsCEN2 gene can negatively regulate seed germination. The germination speed of OsCEN2-RNAi seeds was significantly faster while that of OsCEN2-overexpression (OE) seeds was slower than that of the wild type (WT). The results of qRT-PCR showed that the OsCEN2 expression was increased in the early stage of seed germination. Exogenous application of abscisic acid (ABA) on seeds and seedlings showed that OsCEN2-OE seeds and seedlings were highly sensitive to ABA during germination and post-germination growth, respectively. The determination of endogenous ABA content in seeds also showed that the ABA content of OsCEN2-RNAi seeds was lower, while that of OsCEN2-OE seeds was higher. Moreover, the transgenic plants changed salt tolerance because of the altered ABA level. In addition, differences were also observed in the expression of genes related to ABA synthesis and metabolism in the seeds of OsCEN2-transgenic lines. This study reveals that OsCEN2 regulates the germination speed by affecting the content of ABA during seed germination and provides a theoretical basis for research on rice direct seeding.

OsAGO2 controls ROS production and the initiation of tapetal PCD by epigenetically regulating OsHXK1 expression in rice anthers.

Proteins of the ARGONAUTE (AGO) family function in the epigenetic regulation of gene expression. Although the rice (Oryza sativa) genome encodes 19 predicted AGO proteins, few of their functions have thus far been characterized. Here, we show that the AGO protein OsAGO2 regulates anther development in rice. OsAGO2 was highly expressed in anthers. Knockdown of OsAGO2 led to the overaccumulation of reactive oxygen species (ROS) and abnormal anther development, causing premature initiation of tapetal programmed cell death (PCD) and pollen abortion. The expression level of Hexokinase 1 (OsHXK1) increased significantly, and the methylation levels of its promoter decreased, in plants with knocked-down OsAGO2 expression. Overexpression of OsHXK1 also resulted in the overaccumulation of ROS, premature initiation of PCD, and pollen abortion. Moreover, knockdown of OsHXK1 restored pollen fertility in OsAGO2 knockdown plants. Chromatin immunoprecipitation assays demonstrated that OsAGO2 binds directly to the OsHXK1 promoter region, suggesting that OsHXK1 is a target gene of OsAGO2. These results indicate that OsHXK1 controls the appropriate production of ROS and the proper timing of tapetal PCD and is directly regulated by OsAGO2 through epigenetic regulation.

Hexokinase gene OsHXK1 positively regulates leaf senescence in rice.

BACKGROUND: Leaf senescence is a highly complex and meticulous regulatory process, and the disruption of any factor involved in leaf senescence might lead to premature or delayed leaf senescence and thus result in reduced or increased crop yields. Despite sincere efforts by scientists, there remain many unsolved problems related to the regulatory factors and molecular mechanisms of leaf senescence. RESULTS: This study successfully revealed that OsHXK1 was highly expressed in senescent leaves of rice. The upregulation of OsHXK1 led to premature senescence of rice leaves, a decreased level of chlorophyll, and damage to the chloroplast structure. The overexpression of OsHXK1 resulted in increases in glucose and ROS levels and produced programmed cell death (PCD) signals earlier at the booting stage. Further analysis showed that expression level of the respiratory burst oxidase homolog (RBOH) genes and OsGLO1 were increased in OsHXK1-overexpressing plants at the booting stage. CONCLUSIONS: Overall, the outcomes of this study suggested that OsHXK1 could act as a positive regulator of rice leaf senescence by mediating glucose accumulation and inducing an increase in ROS.

Initiation and Execution of Programmed Cell Death and Regulation of Reactive Oxygen Species in Plants.

Programmed cell death (PCD) plays crucial roles in plant development and defence response. Reactive oxygen species (ROS) are produced during normal plant growth, and high ROS concentrations can change the antioxidant status of cells, leading to spontaneous cell death. In addition, ROS function as signalling molecules to improve plant stress tolerance, and they induce PCD under different conditions. This review describes the mechanisms underlying plant PCD, the key functions of mitochondria and chloroplasts in PCD, and the relationship between mitochondria and chloroplasts during PCD. Additionally, the review discusses the factors that regulate PCD. Most importantly, in this review, we summarise the sites of production of ROS and discuss the roles of ROS that not only trigger multiple signalling pathways leading to PCD but also participate in the execution of PCD, highlighting the importance of ROS in PCD.

An enzyme-free and label-free visual sensing strategy for the detection of thrombin using a plasmonic nanoplatform.

An enzyme-free and label-free visual sensing strategy was developed for sensitively detecting thrombin using a plasmonic nanoplatform. Both the thrombin-triggered catalytic hairpin assembly (CHA) amplification reaction and G-quadruplex/hemin DNAzyme-controlled plasmonic signal readout were engineered on an electrospun nanofibrous membrane. Owing to its large specific surface area and porous structure, the nanofibrous membrane enhanced the loading capacity of B-H2 and the interface interaction efficiency. This plasmonic nanoplatform was used to perform the sensitive and naked-eye detection of thrombin as low as 1.0 pM in human serum samples. This visual strategy can discriminate thrombin from other co-existing proteins very well. Moreover, the visual sensing platform exhibited excellent reusability and long-term stability. The proposed enzyme-free and label-free plasmonic nanoplatform is low-cost, easy to operate and highly sensitive, and has potential applications in the point-of-care detection of protein biomarkers.

Mean irradiance profile of a Gaussian beam under random jitter.

The mean irradiance profile has been widely applied in performance analysis and atmospheric channel research for free space optical communication; however, the modeling method and a closed-form expression of the mean irradiance profile under random jitter have rarely been discussed. To the best of our knowledge, this work presents a modeling method and a closed-form expression for a mean irradiance profile under random jitter through the ensemble average principle for the first time. We find that, with an increase in random jitter variance, the mean irradiance profile varied from an approximately Gaussian profile to a flat-topped profile to a hollow profile. These findings were verified using multilayer random phase screen simulations. The model of the mean irradiance profile under random jitter developed in this study will improve the accuracy of performance analysis and atmospheric channel research.

Overexpression of miR164b-resistant OsNAC2 improves plant architecture and grain yield in rice.

Plant architecture is a major target of rice (Oryza sativa) breeding and selection, but the underlying regulatory networks remain unclear. Here, we overexpressed an OsNAC2 mutant (OErN) that cannot be cleaved by the miRNA miR164b. OErN plants had better plant architecture and longer panicles, and produced more grains. The parental line averaged 12.2 primary and 31.5 secondary branches in the main panicles; two OErN lines averaged 15.0 and 15.2 primary, and 41.5 and 44.3 secondary branches. In large-scale field trials, OErN plants produced at least 58.62% more total grain (by weight) compared with the parental line. They also had more large and small vascular bundles in the stem internodes and leaves. Overexpression of miR164b or down-regulation of OsNAC2 led to decreased panicle length and grain yield in the main panicle. The OErN plants showed significant up-regulation of the grain number and plant architecture-related genes IPA1 and DEP1. A survey of >3000 rice varieties found no natural mutations in the miR164b-binding site of OsNAC2. OErN increased yield in Nipponbare and the commonly grown Yangyujing 3 cultivars. In summary, we identified an efficient new strategy to increase rice yield substantially and improve plant architecture through overexpression of OsmiR164b-resistant OsNAC2.

Principle and performance analysis of coherent tracking sensor based on local oscillator beam nutation.

By integrating coherent tracking sensor functionality into a coherent communication receiver, a dual-function receiver with coherent boresight error sensing is developed for tracking in coherent free-space optical communication systems. The sensor principles are analyzed according to optical interference theory, and the boresight error detection algorithm and small signal linear model are derived. Analysis of local-oscillator beam nutation on system performance shows that the best nutation half-angle is 0.5-1 µrad, the noise equivalent angle is less than 0.02 µrad, and the communication sensitivity degradation is 0.2-0.6 dB. This technology opens new avenues for free-space optical communication system design.

Advancements in Rice Leaf Development Research.

Rice leaf morphology is a pivotal component of the ideal plant architecture, significantly impacting rice yield. The process of leaf development unfolds through three distinct stages: the initiation of leaf primordia, the establishment and maintenance of polarity, and leaf expansion. Genes regulating leaf morphology encompass transcription factors, hormones, and miRNAs. An in-depth synthesis and categorization of genes associated with leaf development, particularly those successfully cloned, hold paramount importance in unraveling the complexity of rice leaf development. Furthermore, it provides valuable insights into the potential for molecular-level manipulation of rice leaf types. This comprehensive review consolidates the stages of rice leaf development, the genes involved, molecular regulatory pathways, and the influence of plant hormones. Its objective is to establish a foundational understanding of the creation of ideal rice leaf forms and their practical application in molecular breeding.

Involvement of the Metallothionein gene OsMT2b in Drought and Cadmium Ions Stress in Rice.

Abiotic stress is one of the major factors restricting the production of rice (Oryza sativa L.). Developing rice varieties with dual abiotic stress tolerance is essential to ensure sustained rice production, which is necessary to illustrate the regulation mechanisms underlying dual stress tolerance. At present, only a few genes that regulate dual abiotic stress tolerance have been reported. In this study, we determined that the expression of OsMT2b was induced by both drought and Cd2+ stress. After stress treatment, OsMT2b-overexpression lines exhibited enhanced drought tolerance and better physiological performance in terms of relative water content and electrolyte leakage compared with wild-type (WT). Further analysis indicated that ROS levels were lower in OsMT2b-overexpression lines than in WT following stress treatment, suggesting that OsMT2b-overexpression lines had a stronger ability to scavenge ROS under stress. Reverse transcription-quantitative PCR (RT-qPCR) results demonstrated that under drought stress, OsMT2b influenced the expression of genes involved in ROS scavenging to enhance drought tolerance in rice. In addition, OsMT2b-overexpression plants displayed increased tolerance to Cd2+ stress, and physiological assessment results were consistent with the observed phenotypic improvements. Thus, enhancing ROS scavenging ability through OsMT2b overexpression is a novel strategy to boost rice tolerance to both drought and Cd2+ stress, offering a promising approach for developing rice germplasm with enhanced resistance to the abiotic stressors.

A rapid on-site visualization platform based on RPA coupled with CRISPR-Cas12a for the detection of genetically modified papaya 'Huanong No.1'.

The Papaya ringspot virus (PRSV)-resistant genetically modified (GM) papaya 'Huanong No.1' has been certified as safe for consumption and widely planted in China for about 18 years. To protect consumers' rights and facilitate government supervision and monitoring, it is necessary to establish a simple, rapid, and specific detection method for 'Huanong No.1'. Herein, we developed a platform based on recombinase polymerase amplification (RPA) coupled with CRISPR-Cas12a for the detection of 'Huanong No.1'. The RPA-CRISPR-Cas12a platform was found to have high specificity, with amplification signals only present in 'Huanong No.1'. Additionally, the platform was highly sensitive, with a limit of detection (LOD) of approximately 20 copies. The detection process was fast and could be completed in less than 1 h. This novel platform enables the rapid on-site visualization detection of 'Huanong No.1', eliminating dependence on laboratory conditions and specialized instruments, and can serve as a technical reference for the rapid detection of other GM plants.

Analysis of the role of the rice metallothionein gene OsMT2b in grain size regulation.

Seed size is one of the three main characteristics determining rice yield. Clarification of the mechanisms regulating seed size in rice has implications for improving rice yield. Although several genes have been reported to regulate seed size, most of the reports are fragmentary. The role of metallothioneins (MTs) in regulating seed size remains unknown. Here, we found that OsMT2b was expressed in both spikelets and developing seeds. OsMT2b-overexpression lines had large and heavy seeds, and RNAi (RNA interference) lines had small and light seeds. Scanning electron microscopy (SEM) observations revealed that OsMT2b regulated spikelet hull size by affecting cell expansion in the outer epidermis. Histological analysis indicated that OsMT2b affected the number of cells in the cross-section of spikelet hulls, which affected seed size. The fresh weight of seeds was consistently higher in OsMT2b-overexpression lines than in seeds of the wild-type (WT) and RNAi lines from 6 DAP (days after pollination) until maturity, indicating that OsMT2b affected seed filling. Reverse transcription-quantitative PCR (RT-qPCR) analyses revealed that OsMT2b regulates the expression of reactive oxygen species scavenging-related genes involved in seed size regulation. In conclusion, our results indicated that OsMT2b positively regulates seed size, which provides a novel approach for regulating seed size with genetic engineering technology.

The Identification of the Banana Endogenous Reference Gene MaSPS1 and the Construction of Qualitative and Quantitative PCR Detection Methods.

Endogenous reference genes play a crucial role in the qualitative and quantitative PCR detection of genetically modified crops. Currently, there are no systematic studies on the banana endogenous reference gene. In this study, the MaSPS1 gene was identified as a candidate gene through bioinformatics analysis. The conservation of this gene in different genotypes of banana was tested using PCR, and its specificity in various crops and fruits was also examined. Southern blot analysis showed that there is only one copy of MaSPS1 in banana. The limit of detection (LOD) test showed that the LOD of the conventional PCR method is approximately 20 copies. The real-time fluorescence quantitative PCR (qPCR) method also exhibited high specificity, with a LOD of approximately 10 copies. The standard curve of the qPCR method met the quantitative requirements, with a limit of quantification (LOQ) of 1.14 × 10-2 ng-about 20 copies. Also, the qPCR method demonstrated good repeatability and stability. Hence, the above results indicate that the detection method established in this study has strong specificity, a low detection limit, and good stability. It provides a reliable qualitative and quantitative detection system for banana.

Methylesterification of cell-wall pectin controls the diurnal flower-opening times in rice.

Flowers are the core reproductive organ of plants, and flowering is essential for cross-pollination. Diurnal flower-opening time is thus a key trait influencing reproductive isolation, hybrid breeding, and thermostability in plants. However, the molecular mechanisms controlling this trait remain unknown. Here, we report that rice Diurnal Flower Opening Time 1 (DFOT1) modulates pectin methylesterase (PME) activity to regulate pectin methylesterification levels of the lodicule cell walls, which affect lodicule swelling to control diurnal flower-opening time. DFOT1 is specifically expressed in the lodicules, and its expression gradually increases with the approach to flowering but decreases with flowering. Importantly, a knockout of DFOT1 showed earlier diurnal flower opening. We demonstrate that DFOT1 interacts directly with multiple PMEs to promote their activity. Knockout of PME40 also resulted in early diurnal flower opening, whereas overexpression of PME42 delayed diurnal flower opening. Lower PME activity was observed to be associated with higher levels of pectin methylesterification and the softening of cell walls in lodicules, which contribute to the absorption of water by lodicules and cause them to swell, thus promoting early diurnal flower opening. Higher PME activity had the opposite effect. Collectively, our work uncovers a molecular mechanism underlying the regulation of diurnal flower-opening time in rice, which would help reduce the costs of hybrid breeding and improve the heat tolerance of flowering plants by avoiding higher temperatures at anthesis.

One-step fast and label-free imaging array for multiplexed detection of trace avian influenza viruses.

Rapid and low-cost diagnosis of multiple infectious diseases is of great significance especially in densely populated or resource-constrained settings. Herein, we developed a one-step fast and label-free imaging array for multiplexed detection of trace avian influenza virus (AIV) DNA biomarkers. By designing a series of specific and efficient catalytic hairpin assembly (CHA) amplification reactions and utilizing thioflavin T, a specific G-quadruplex fluorescence probe, three subtypes of AIV DNA biomarkers (H1N1, H7N9 and H5N1) were simultaneously and quickly detected within only 20 min, which just needed a small reagent volume of 50 µL and a smartphone instead of a spectrometer. With the combination of fluorescence imaging output and grey-level analysis, the array sensor can be on-site with the limit of detection of 136 pM, 141 pM and 129 pM for H1N1, H7N9 and H5N1, respectively. The imaging array also displayed good mismatch discrimination, excellent anti-interference, and real sample application. In view of its advantages of fast detection, low cost and multiplexed analysis, the imaging array is expected to have potential applications for early infectious disease diagnosis in resource-constrained settings.

Plasmonic nanoplatform for point-of-care testing trace HCV core protein.

Early diagnosis of hepatitis C virus (HCV) infection is still urgently desired as there is a global healthy burden and no vaccine available. In this work, a plasmonic nanoplatform was engineered with catalytic hairpin assembly (CHA) amplification reaction specifically of HCV core protein (HCVcp), G-quadruplex/hemin DNAzyme and nanofibrous membrane together. HCVcp was detected in whole serum at the ultralow concentration of 1.0 × 10-4 pg/mL with naked eye. By testing serum samples from 30 donors with different viral loads, detection sensitivity of the plasmonic nanoplatform turned out to be much better than that of the commercial ELISA kit. In addition, the plasmonic nanoplatform exhibited high specificity, excellent reusability and long-term stability. Naked-eye detection based on the plasmonic nanoplatform is expected to have potential applications in point-of-care testing (POCT) and early diagnosis of hepatitis C and other infectious diseases.

One-step sensitive thrombin detection based on a nanofibrous sensing platform.

Convenient and time-saving one-step strategies for detecting ultralow concentrations of protein biomarkers play key roles in rapid disease diagnosis. In this study, we report a one-step detection method based on a nanofibrous sensing platform via the combination of proximity-induced DNA strand displacement (PiDSD), catalytic hairpin assembly (CHA) amplification and thioflavin T (ThT) binding. The interface behaviors on the nanofibrous membrane were studied to promote interface reaction kinetics and thermodynamics. Thrombin was used as a model biomarker, and the nanofibrous sensing platform achieved a limit of detection as low as 1.0 pM, a wide linear range of 50 pM to 5 nM, excellent specificity and good long-term stability. Compared with previous one-step thrombin detection methods, our one-step detection method is label-free, convenient and much more sensitive; it has potential applications for protein detection in point-to-care testing (POCT) and early diagnosis.

Overexpression of a microRNA-targeted NAC transcription factor improves drought and salt tolerance in Rice via ABA-mediated pathways.

BACKGROUND: The NAC (NAM, AFAT, and CUC) transcription factors play critical roles in rice (Oryza sativa) development and stress regulation. Overexpressing a microRNA (miR164b)-resistant OsNAC2 mutant gene, which generates transcripts that cannot be targeted by miR164b, improves rice plant architecture and yield; however, the performance of these mOsNAC2-overexpressing lines, named ZUOErN3 and ZUOErN4, under abiotic stress conditions such as drought have not yet been fully characterized. RESULTS: In this study, we showed that the germination of ZUOErN3 and ZUOErN4 seeds was delayed in comparison with the wild-type (WT) seeds, although the final germination rates of all lines were over 95%. The quantification of the endogenous ABA levels revealed that the germinating mOsNAC2-overexpressing seeds had elevated ABA levels, which resulted in their slower germination. The mOsNAC2-overexpressing plants were significantly more drought tolerance than the WT plants, with the survival rate increasing from 11.2% in the WT to nearly 70% in ZUOErN3 and ZUOErN4 plants after a drought treatment. Salt (NaCl) tolerance was also increased in the ZUOErN3 and ZUOErN4 plants due to significantly increased ABA levels. A reverse transcription quantitative PCR (RT-qPCR) analysis showed a significant increase in the expression of the ABA biosynthesis genes OsNCED1 and OsNCED3 in the mOsNAC2-overexpressing lines, and the expression levels of the stress-responsive genes OsP5CS1, OsLEA3, and OsRab16 were significantly increased in these plants. Moreover, OsNAC2 directly interacted with the promoters of OsLEA3 and OsNCED3 in yeast one-hybrid assays. CONCLUSIONS: Taken together, our results show that OsNAC2 plays a positive regulatory role in drought and salt tolerance in rice through ABA-mediated pathways.