ABSTRACT
Biosynthesis of D-allulose has been achieved using ketose 3-epimerases (KEases), but its application is limited by poor catalytic performance. In this study, we redesigned a genetically encoded biosensor based on a D-allulose-responsive transcriptional regulator for real-time monitoring of D-allulose. An ultrahigh-throughput droplet-based microfluidic screening platform was further constructed by coupling with this D-allulose-detecting biosensor for the directed evolution of the KEases. Structural analysis of Sinorhizobium fredii D-allulose 3-epimerase (SfDAE) revealed that a highly flexible helix/loop region exposes or occludes the catalytic center as an essential lid conformation regulating substrate recognition. We reprogrammed SfDAE using structure-guided rational design and directed evolution, in which a mutant M3-2 was identified with 17-fold enhanced catalytic efficiency. Our research offers a paradigm for the design and optimization of a biosensor-based microdroplet screening platform.
Subject(s)
Fructose , Racemases and Epimerases , Fructose/chemistryABSTRACT
PI3K and its product PI3P are both involved in plant development and stress responses. In this study, the down-regulation of PI3K activity accelerated leaf senescence induced by methyl jasmonate (MeJA) and suppressed the activation of vacuolar H(+)-ATPase (V-ATPase). Yeast two-hybrid analyses indicated that PI3K bound to the V-ATPase B subunit (VHA-B). Analysis of bimolecular fluorescence complementation in tobacco guard cells showed that PI3K interacted with VHA-B2 in the tonoplasts. Through the use of pharmacological and genetic tools, we found that PI3K and V-ATPase promoted vacuolar acidification and stomatal closure during leaf senescence. Vacuolar acidification was suppressed by the PIKfyve inhibitor in 35S:AtVPS34-YFP Arabidopsis during MeJA-induced leaf senescence, but the decrease was lower than that in YFP-labeled Arabidopsis. These results suggest that PI3K promotes V-ATPase activation and consequently induces vacuolar acidification and stomatal closure, thereby delaying MeJA-induced leaf senescence.
Subject(s)
Arabidopsis Proteins/metabolism , Phosphatidylinositol 3-Kinase/metabolism , Plant Leaves/physiology , Protein Subunits/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , Vacuoles/metabolism , Acetates/pharmacology , Acids/chemistry , Arabidopsis Proteins/genetics , Base Sequence , Blotting, Western , Cyclopentanes/pharmacology , Hydrogen-Ion Concentration , Microscopy, Confocal , Microscopy, Electron, Scanning , Mutation , Oxylipins/pharmacology , Phosphatidylinositol 3-Kinase/genetics , Plant Growth Regulators/pharmacology , Plant Leaves/drug effects , Plant Leaves/genetics , Plant Stomata/drug effects , Plant Stomata/genetics , Plant Stomata/physiology , Plants, Genetically Modified , Protein Binding , Protein Subunits/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Two-Hybrid System Techniques , Vacuolar Proton-Translocating ATPases/genetics , Vacuoles/chemistryABSTRACT
In plants, extensive efforts have been devoted to understanding the crosstalk between salicylic acid (SA) and jasmonic acid (JA) signaling in pathogen defenses, but this crosstalk has scarcely been addressed during senescence. In this study, the effect of SA application on methyl jasmonate (MeJA)-induced leaf senescence was assessed. We found that low concentrations of SA (1-50 µM) played a delayed role against the senescence promoted by MeJA. Furthermore, low concentrations of SA enhanced plant antioxidant defenses and restricted reactive oxygen species (ROS) accumulation in MeJA-treated leaves. When applied simultaneously with MeJA, low concentrations of SA triggered a nitric oxide (NO) burst, and the elevated NO levels were linked to the nitric oxide associated 1 (NOA1)-dependent pathway via nitric oxide synthase (NOS) activity. The ability of SA to up-regulate plant antioxidant defenses, reduce ROS accumulation, and suppress leaf senescence was lost in NO-deficient Atnoa1 plants. In a converse manner, exogenous addition of NO donors increased the plant antioxidant capacity and lowered the ROS levels in MeJA-treated leaves. Taken together, the results indicate that SA at low concentrations counteracts MeJA-induced leaf senescence through NOA1-dependent NO signaling and strengthening of the antioxidant defense.
Subject(s)
Acetates/metabolism , Cyclopentanes/metabolism , Nitric Oxide Synthase/metabolism , Oxylipins/metabolism , Plant Growth Regulators/physiology , Plant Leaves/physiology , Salicylic Acid/metabolism , Aging/physiology , Arabidopsis/metabolism , Arabidopsis/physiology , Chlorophyll/metabolism , Lipid Peroxidation , Nitric Oxide Synthase/physiology , Real-Time Polymerase Chain Reaction , Up-RegulationABSTRACT
Crosstalk between salicylic acid (SA) and jasmonic acid (JA) signaling plays an important role in regulation of plant senescence. Our previous work found that SA could delay methyl jasmonate (MeJA)-induced leaf senescence in a concentration-dependent manner. Here, the effect of low concentration of SA (LCSA) application on MeJA-induced leaf senescence was further assessed. High-throughput sequencing (RNA-Seq) results showed that LCSA did not have dominant effects on the genetic regulatory pathways of basal metabolism like nitrogen metabolism, photosynthesis and glycolysis. The ClusterONE was applied to identify discrete gene modules based on protein-protein interaction (PPI) network. Interestingly, an autophagy-related (ATG) module was identified in the differentially expressed genes (DEGs) that exclusively induced by MeJA together with LCSA. RT-qPCR confirmed that the expression of most of the determined ATG genes were upregulated by LCSA. Remarkably, in contrast to wild type (Col-0), LCSA cannot alleviate the leaf yellowing phenotype in autophagy defective mutants (atg5-1 and atg7-2) upon MeJA treatment. Confocal results showed that LCSA increased the number of autophagic bodies accumulated in the vacuole during MeJA-induced leaf senescence. Collectively, our work revealed up-regulation of autophagy by LCSA as a key regulator to alleviate MeJA-induced leaf senescence.
Subject(s)
Aging/genetics , Autophagy/genetics , Plant Leaves/genetics , Salicylic Acid/metabolism , Acetates/pharmacology , Autophagy/drug effects , Autophagy-Related Protein 5/genetics , Autophagy-Related Protein 7/genetics , Cyclopentanes/metabolism , Cyclopentanes/pharmacology , Gene Expression Regulation, Plant/drug effects , Oxylipins/metabolism , Oxylipins/pharmacology , Plant Leaves/growth & development , RNA-Seq , Signal Transduction/drug effectsABSTRACT
PURPOSE: To compare tumor size by mammography and sonography and align with pathological results in primary breast cancer cases. MATERIALS AND METHODS: We retrospectively reviewed 95 primary breast cancer patients who underwent mammography and sonography from January 2011 to June 2012. The largest tumor diameter was chosen as sizing reference for each imaging modality. The measurements of mammography and sonography were considered concordant if they were within the measurement of pathological results±0.5 cm. Pearson's correlation coefficient was calculated for imaging results. RESULTS: The range of the maximum diameter was 0.6 cm-10.5 cm and mean value was 3.81±2.04 cm by pathological results, 0.7 cm-12.4 cm and 3.99±2.19 cm by mammography, and 0.9 cm-11.0 cm and 3.63±2.01 cm by sonography, respectively. Sonography (R: 0.754), underestimated tumor size, but had a better correlation with pathological tumor size compared to mammography (R: 0.676), which overestimated tumor size. CONCLUSIONS: Sonography is superior to mammography in assessment of primary breast cancer.