OsTKPR1 proteins with a single amino acid substitution fail the synthesis of a specific sporopollenin precursor and cause abnormal exine and pollen development in rice.

Fatty acid derivatives are key components of rice pollen exine. The synthesis of aliphatic sporopollenin precursors are initiated in the plastids of the tapetal cells, followed by multiple-step reactions conducted in the endoplasmic reticulum (ER). However, the relative contribution of different precursors to the precise structure of sporopollenin remains largely elusive, let alone the underlying mechanism. Here, we report that two complete male sterile mutants ostkpr1-3 (Tetraketide α-pyrone reductase 1-3, with OsTKPR1P124S substitution) and ostkpr1-4 (with truncated OsTKPR1stop) are defective in pollen exine, Ubisch body and anther cuticle development where ostkpr1-4 display severer phenotypes. Remarkably, OsTKPR1 could produce reduced hydroxylated tetraketide α-pyrone and reduced tetraketide α-pyrone, whereas OsTKPR1P124S fails to produce the latter. Pairwise interaction assays show that mutated OsTKPR1P124S is able to integrate into a recently characterized metabolon, thus its altered catalytic activity is not due to dis-integrity of the metabolon. In short, we find that reduced tetraketide α-pyrone is a key sporopollenin precursor required for normal exine formation, and the conserved 124th proline of OsTKPR1 is essential for the reduction activity. Therefore, this study provided new insights into the sporopollenin precursor constitution critical for exine formation.

OsTKPR2 is part of a sporopollenin-producing metabolon required for exine formation in rice.

The sporopollenin polymer is a major component of the pollen exine. Fatty acid derivatives synthesized in the tapetum are among the precursors of sporopollenin. Progress has been made to understand sporopollenin metabolism in rice; however, the underlying molecular mechanisms remain elusive. We found that OsTKPR2 and OsTKPR1 share a similar expression pattern, and their coding proteins have a similar subcellular localization and enzyme activities towards reduced tetraketide α-pyrone and hydroxylated tetraketide α-pyrone. Unexpectedly, OsTKPR1pro:OsTKPR2-eGFP could not rescue the phenotype of ostkpr1-4. Three independent ostkpr2 mutant lines generated by CRISPR/Cas9 displayed reduced male fertility to various extents which were correlated with the severity of gene disruptions. Notably, the anther cuticle, Ubisch bodies, and pollen development were affected in the ostkpr2-1 mutant, where a thinner pollen exine was noticed. OsTKPR1 and OsTKPR2 were integrated into a metabolon including OsACOS12 and OsPKS2, which resulted in a significant increased enzymatic efficiency when both OsTKPR1 and OsTKPR2 were present, indicating the mutual dependence of OsTKPR2 and OsTKPR1 for their full biochemical activities. Thus, our results demonstrated that OsTKPR2 is required for anther and pollen development where an OsTKPR2-containing metabolon is functional during rice sporopollenin synthesis. Furthermore, the cooperation and possible functional divergence between OsTKPR2 and OsTKPR1 is also discussed.

One-step synthesis of nanosilver embedding laser-induced graphene for H2O2 sensor.

During the novel coronavirus pandemic, hydrogen peroxide (H2O2) played an important role as a disinfectant. However, high concentrations of H2O2 can also cause damage to the skin and eyes. Therefore, the quantitative and qualitative detection of H2O2 is an important research direction. In this work, we report a one-step laser-induced synthesis of graphene doped with Ag NPs composites. It directly trims screen printed electrodes (SPE). Firstly, we did the timekeeping current method (CA) test on H2O2 using a conventional platinum sheet as the counter electrode, and obtained linear ranges of 1-110 µM and 110-800 µM with a sensitivity of 118.7 and 96.3 µAmM-1cm-2 and a low detection limit of (LOD) 0.24 µM and 0.31 µM. On this basis we have also achieved a good result in CA testing using Screen printed carbon electrodes (SPCE), laying the foundation for portable testing. The sensor has excellent interference immunity and high selectivity.

Comparison of Different Label-Free Raman Spectroscopy Approaches for the Discrimination of Clinical MRSA and MSSA Isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) is classified as one of the priority pathogens that threaten human health. Resistance detection with conventional microbiological methods takes several days, forcing physicians to administer empirical antimicrobial treatment that is not always appropriate. A need exists for a rapid, accurate, and cost-effective method that allows targeted antimicrobial therapy in limited time. In this pilot study, we investigate the efficacy of three different label-free Raman spectroscopic approaches to differentiate methicillin-resistant and -susceptible clinical isolates of S. aureus (MSSA). Single-cell analysis using 532 nm excitation was shown to be the most suitable approach since it captures information on the overall biochemical composition of the bacteria, predicting 87.5% of the strains correctly. UV resonance Raman microspectroscopy provided a balanced accuracy of 62.5% and was not sensitive enough in discriminating MRSA from MSSA. Excitation of 785 nm directly on the petri dish provided a balanced accuracy of 87.5%. However, the difference between the strains was derived from the dominant staphyloxanthin bands in the MRSA, a cell component not associated with the presence of methicillin resistance. This is the first step toward the development of label-free Raman spectroscopy for the discrimination of MRSA and MSSA using single-cell analysis with 532 nm excitation. IMPORTANCE Label-free Raman spectra capture the high chemical complexity of bacterial cells. Many different Raman approaches have been developed using different excitation wavelength and cell analysis methods. This study highlights the major importance of selecting the most suitable Raman approach, capable of providing spectral features that can be associated with the cell mechanism under investigation. It is shown that the approach of choice for differentiating MRSA from MSSA should be single-cell analysis with 532 nm excitation since it captures the difference in the overall biochemical composition. These results should be taken into consideration in future studies aiming for the development of label-free Raman spectroscopy as a clinical analytical tool for antimicrobial resistance determination.

Fiber Probe-Based Raman Spectroscopic Identification of Pathogenic Infection Microorganisms on Agar Plates.

Rapid identification of microorganisms is clinically meaningful, and it helps to decelerate the spread of drug resistance and improve patient treatment. In this study, we present a rapid fiber probe-based Raman technique with an excitation wavelength of 785 nm, which is applied to classify and identify nine different species of microorganisms. The cost-effective fiber probe compresses the dimension of the system and provides a more reliable and stable database. All microorganisms were simply cultivated on Luria-Bertani (LB) agar, and Raman spectra were obtained directly from the microbial colonies with the fiber probe within 30 s. The classification model consists of principal component analysis (PCA) in combination with linear discriminant analysis (LDA) and was examined by applying leave-one-batch-out cross-validation (LOBOCV). This model achieved an accuracy of 98.9%. In addition, the validation and identification processes based on independent replicates achieved accuracies of 99.8% and 100%, respectively. The results demonstrated that fiber probe Raman spectroscopy in combination with chemometric analysis allowed a rapid classification and identification of microorganisms only with a normal culture. Therefore, it is promising especially for medical applications and could moreover be helpful to investigate and identify microorganisms rapidly in further studies.

In Vitro Fiber-Probe-Based Identification of Pathogens in Biofilms by Raman Spectroscopy.

Biofilms are the preferred habitat of microorganisms on living and artificial surfaces. Biofilm-related infections, such as infections of medical implants, are difficult to treat, and due to a reduced cultivability of the included bacteria, difficult to diagnose. Therefore, it is highly important to rapidly identify and investigate biofilms on implant surfaces, e.g., during surgery. In this study, we present fiber-probe-based Raman spectroscopy with an excitation wavelength of 785 nm, which was applied to investigate six different pathogen species involved in biofilm-related infections. Biofilms were cultivated in a drip flow reactor, which can model a biofilm growth environment. The signals collected from a fiber probe allowed us to collect Raman spectra not only from the embedded bacterial and yeast cells but also the surrounding extracellular polymeric substance matrix. This information was used in a classification model. The model consists of a principal component analysis in combination with linear discriminant analysis and was examined by applying a leave-one-batch-out cross-validation. This model achieved a classification accuracy of 93.8%. In addition, the identification accuracy increased up to 97.5% when clinical strains were used for identification. A fiber-probe-based Raman spectroscopy method combined with a chemometric analysis might therefore serve as a fast, accurate, and portable strategy for the species identification of biofilm-related infections, e.g., during surgical procedures.