Transcription factors BBX11 and HY5 interdependently regulate the molecular and metabolic responses to UV-B.

UV-B radiation acts as a developmental cue and a stress factor for plants, depending on dose. Activation of the transcription factor ELONGATED HYPOCOTYL 5 (HY5) in a UV RESISTANCE LOCUS 8 (UVR8)-dependent manner leads to the induction of a broad set of genes under UV-B. However, the underlying molecular mechanisms regulating this process are less understood. Here, we use molecular, biochemical, genetic, and metabolomic tools to identify the B-BOX transcription factor B-BOX PROTEIN 11 (BBX11) as a component of the molecular response to UV-B in Arabidopsis (Arabidopsis thaliana). BBX11 expression is induced by UV-B in a dose-dependent manner. Under low UV-B, BBX11 regulates hypocotyl growth suppression, whereas it protects plants exposed to high UV-B radiation by promoting the accumulation of photo-protective phenolics and antioxidants, and inducing DNA repair genes. Our genetic studies indicate that BBX11 regulates hypocotyl elongation under UV-B partially dependent on HY5. Overexpression of BBX11 can partially rescue the high UV-B sensitivity of hy5, suggesting that HY5-mediated UV-B stress tolerance is partially dependent on BBX11. HY5 regulates the UV-B-mediated induction of BBX11 by directly binding to its promoter. BBX11 reciprocally regulates the mRNA and protein levels of HY5. We report here the role of a BBX11-HY5 feedback loop in regulating photomorphogenesis and stress tolerance under UV-B.

Insights into sustainable resource and energy recovery from leachate towards emission mitigation for environmental management: A critical approach.

The exponential generation of municipal solid waste (MSW) and landfill disposal without any treatment has increased the continuous generation of landfill leachate. Improper MSW and leachate management are contributing to environmental degradation and water and soil pollution, which must be treated. Numerous works have been conducted on leachate treatments for energy and resource recovery. This review presents a comprehensive study of leachate management in which different treatment methods are discussed to analyze the suitability of processes that can be employed to treat leachate efficiently. Further, the characteristics of leachate are examined as properties of leachate may be varied depending upon the region. Still, several challenges related to leachate management and its treatments are discussed in this study. An integrated system could be a better option for treating leachate because it contains large amounts of organic and inorganic compounds. Proper leachate management would help to recover energy and value-added products (metals).

Reprint of Organic waste conversion through anaerobic digestion: A critical insight into the metabolic pathways and microbial interactions.

Anaerobic digestion is a promising method for energy recovery through conversion of organic waste to biogas and other industrial valuables. However, to tap the full potential of anaerobic digestion, deciphering the microbial metabolic pathway activities and their underlying bioenergetics is required. In addition, the behavior of organisms in consortia along with the analytical abilities to kinetically measure their metabolic interactions will allow rational optimization of the process. This review aims to explore the metabolic bottlenecks of the microbial communities adopting latest advances of profiling and 13C tracer-based analysis using state of the art analytical platforms (GC, GC-MS, LC-MS, NMR). The review summarizes the phases of anaerobic digestion, the role of microbial communities, key process parameters of significance, syntrophic microbial interactions and the bottlenecks that are critical for optimal bioenergetics and enhanced production of valuables. Considerations into the designing of efficient synthetic microbial communities as well as the latest advances in capturing their metabolic cross talk will be highlighted. The review further explores how the presence of additives and inhibiting factors affect the metabolic pathways. The critical insight into the reaction mechanism covered in this review may be helpful to optimize and upgrade the anaerobic digestion system.

Organic waste conversion through anaerobic digestion: A critical insight into the metabolic pathways and microbial interactions.

Anaerobic digestion is a promising method for energy recovery through conversion of organic waste to biogas and other industrial valuables. However, to tap the full potential of anaerobic digestion, deciphering the microbial metabolic pathway activities and their underlying bioenergetics is required. In addition, the behavior of organisms in consortia along with the analytical abilities to kinetically measure their metabolic interactions will allow rational optimization of the process. This review aims to explore the metabolic bottlenecks of the microbial communities adopting latest advances of profiling and 13C tracer-based analysis using state of the art analytical platforms (GC, GC-MS, LC-MS, NMR). The review summarizes the phases of anaerobic digestion, the role of microbial communities, key process parameters of significance, syntrophic microbial interactions and the bottlenecks that are critical for optimal bioenergetics and enhanced production of valuables. Considerations into the designing of efficient synthetic microbial communities as well as the latest advances in capturing their metabolic cross talk will be highlighted. The review further explores how the presence of additives and inhibiting factors affect the metabolic pathways. The critical insight into the reaction mechanism covered in this review may be helpful to optimize and upgrade the anaerobic digestion system.

The unique bryophyte-specific repeat-containing protein SHORT-LEAF regulates gametophore development in moss.

Convergent evolution of shoot development across plant lineages has prompted numerous comparative genetic studies. Though functional conservation of gene networks governing flowering plant shoot development has been explored in bryophyte gametophore development, the role of bryophyte-specific genes remains unknown. Previously, we have reported Tnt1 insertional mutants of moss defective in gametophore development. Here, we report a mutant (short-leaf; shlf) having two-fold shorter leaves, reduced apical dominance, and low plasmodesmata frequency. UHPLC-MS/MS-based auxin quantification and analysis of soybean (Glycine max) auxin-responsive promoter (GH3:GUS) lines exhibited a striking differential auxin distribution pattern in the mutant gametophore. Whole-genome sequencing and functional characterization of candidate genes revealed that a novel bryophyte-specific gene (SHORT-LEAF; SHLF) is responsible for the shlf phenotype. SHLF represents a unique family of near-perfect tandem direct repeat (TDR)-containing proteins conserved only among mosses and liverworts, as evident from our phylogenetic analysis. Cross-complementation with a Marchantia homolog partially recovered the shlf phenotype, indicating possible functional specialization. The distinctive structure (longest known TDRs), absence of any known conserved domain, localization in the endoplasmic reticulum, and proteolytic cleavage pattern of SHLF imply its function in bryophyte-specific cellular mechanisms. This makes SHLF a potential candidate to study gametophore development and evolutionary adaptations of early land plants.

Mutants lacking global regulators, fis and arcA, in Escherichia coli enhanced growth fitness under acetate metabolism by pathway reprogramming.

Global regulatory transcription factors play a significant role in controlling microbial metabolism under genetic and environmental perturbations. A system-level effect of carbon sources such as acetate on microbial metabolism under disrupted global regulators has not been well established. Acetate is one of the major substrates available in various nutrient niches such as the mammalian gut and a keto diet. A substantial amount of acetate gets secreted in aerobic metabolism. Therefore, investigating the study on acetate metabolism is highly significant. It is known that the global regulators fis and arcA regulate acetate uptake genes in E. coli under glucose conditions. This study deciphered the growth and flux distribution of E. coli transcription regulatory knockouts Δfis, ΔarcA and double deletion mutant, ΔarcAΔfis under acetate using 13C-metabolic flux analysis (MFA), which has not been investigated before. We observed that the mutants exhibited an expeditious growth rate (~ 1.2-1.6-fold) with a proportionate increase in acetate uptake rates compared to the wild type. 13C-MFA displayed the distinct metabolic reprogramming of intracellular fluxes via the TCA cycle, anaplerotic pathway and gluconeogenesis, which conferred an advantage of a faster growth rate with better carbon usage in all the mutants. This resulted in higher metabolic fluxes through the TCA cycle (~ 18-90%), lower gluconeogenesis (~ 15-35%) and higher CO2 and ATP production with the proportional increase in growth rate. The study reveals a novel insight by stating the sub-optimality of the wild-type strain grown under acetate substrate aerobically. These mutant strains efficiently oxidize acetate, thus acting as potential candidates for the biosynthesis of isoprenoids, biofuels, vitamins and various pharmaceutical products.Key Points• Mutants exhibited a better balance between energy and precursor synthesis than WT.• Leveraged in the unravelling of regulatory control under various nutrient shifts.• Metabolic readjustment resulted in optimal biomass requirement and faster growth.

A robust method of extraction and GC-MS analysis of Monophenols exhibited UV-B mediated accumulation in Arabidopsis.

Studies on specialized metabolites like phenolics are of immense interest owing to their significance to agriculture, nutrition and health. In plants, phenolics accumulate and exhibit spatial and temporal regulations in response to growth conditions. Robust methodologies aimed at efficient extraction of plant phenolics, their qualitative and quantitative analysis is desired. We optimized the analytical and experimental bottlenecks that captured free, ester, glycoside and wall-bound phenolics after acid or alkali treatments of the tissue extracts and subsequent GC-MS analysis. Higher recovery of phenolics from the methanolic extracts was achieved through (a) Ultrasonication assisted extraction along with Methyl tert-butyl ether (MTBE) enrichment (b) nitrogen gas drying and (c) their derivatization using MSTFA for GC-MS analysis. The optimized protocol was tested on Arabidopsis rosette exposed to UV-B radiation (280-315 nm) which triggered enhanced levels of 11 monophenols and might be attributed to photoprotection and other physiological roles. Interestingly, coumaric acid (308 m/z) and caffeic acid (396 m/z) levels were enhanced by 12-14 folds under UV-B. Other phenolics such as cinnamic acid (220 m/z), hydroxybenzoic acid (282 m/z), vanillic acid (312 m/z, gallic acid (458 m/z), ferulic acid (338 m/z), benzoic acid (194 m/z), sinapinic acid (368 m/z) and protocatechuic acid (370 m/z) also showed elevated levels by about 1 to 4 folds. The protocol also comprehensively captured the variations in the levels of ester, glycoside and wall-bounded phenolics with high reproducibility and sensitivity. The robust method of extraction and GC-MS analysis can readily be adopted for studying phenolics in plant systems. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01150-2.

Allosteric inhibition of MTHFR prevents futile SAM cycling and maintains nucleotide pools in one-carbon metabolism.

Methylenetetrahydrofolate reductase (MTHFR) links the folate cycle to the methionine cycle in one-carbon metabolism. The enzyme is known to be allosterically inhibited by SAM for decades, but the importance of this regulatory control to one-carbon metabolism has never been adequately understood. To shed light on this issue, we exchanged selected amino acid residues in a highly conserved stretch within the regulatory region of yeast MTHFR to create a series of feedback-insensitive, deregulated mutants. These were exploited to investigate the impact of defective allosteric regulation on one-carbon metabolism. We observed a strong growth defect in the presence of methionine. Biochemical and metabolite analysis revealed that both the folate and methionine cycles were affected in these mutants, as was the transsulfuration pathway, leading also to a disruption in redox homeostasis. The major consequences, however, appeared to be in the depletion of nucleotides. 13C isotope labeling and metabolic studies revealed that the deregulated MTHFR cells undergo continuous transmethylation of homocysteine by methyltetrahydrofolate (CH3THF) to form methionine. This reaction also drives SAM formation and further depletes ATP reserves. SAM was then cycled back to methionine, leading to futile cycles of SAM synthesis and recycling and explaining the necessity for MTHFR to be regulated by SAM. The study has yielded valuable new insights into the regulation of one-carbon metabolism, and the mutants appear as powerful new tools to further dissect out the intersection of one-carbon metabolism with various pathways both in yeasts and in humans.

ELONGATED HYPOCOTYL5 Negatively Regulates DECREASE WAX BIOSYNTHESIS to Increase Survival during UV-B Stress.

Understanding how the distinct cell types of the shoot apical meristem (SAM) withstand ultraviolet radiation (UVR) stress can improve cultivation of plants in high-UVR environments. Here, we show that UV-B irradiation selectively kills epidermal and niche cells in the shoot apex. Plants harboring a mutation in DECREASE WAX BIOSYNTHESIS (DEWAX) are tolerant to UV-B. Our data show that DEWAX negatively regulates genes involved in anthocyanin biosynthesis. ELONGATED HYPOCOTYL5 (HY5) binds to the DEWAX promoter elements and represses its expression to promote the anthocyanin biosynthesis. The HY5-DEWAX regulatory network regulates anthocyanin content in Arabidopsis (Arabidopsis thaliana) and influences the survivability of plants under UV-B irradiation stress. Our cell sorting-based study of the epidermal cell layer transcriptome confirms that core UV-B stress signaling pathway genes are conserved and upregulated in response to UV-B irradiation of the SAM. Furthermore, we show that UV-B induces genes involved in shoot development and organ patterning. We propose that the HY5-DEWAX regulatory relationship is conserved; however, changes in the expression levels of these genes can determine anthocyanin content in planta and, hence, fitness under UV-B irradiation stress.

Overexpression of improved EPSPS gene results in field level glyphosate tolerance and higher grain yield in rice.

Glyphosate is a popular, systemic, broad-spectrum herbicide used in modern agriculture. Being a structural analog of phosphoenolpyruvate (PEP), it inhibits 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) which is responsible for the biosynthesis of aromatic amino acids and various aromatic secondary metabolites. Taking a lead from glyphosate-resistant weeds, two mutant variants of the rice EPSPS gene were developed by amino acid substitution (T173I + P177S; TIPS-OsEPSPS and G172A + T173I + P177S; GATIPS-OsEPSPS). These mutated EPSPS genes were overexpressed in rice under the control of either native EPSPS or constitutive promoters (maize ubiquitin [ZmUbi] promoter). The overexpression of TIPS-OsEPSPS under the control of the ZmUbi promoter resulted in higher tolerance to glyphosate (up to threefold of the recommended dose) without affecting the fitness and related agronomic traits of plants in both controlled and field conditions. Furthermore, such rice lines produced 17%-19% more grains compared to the wild type (WT) in the absence of glyphosate application and the phenylalanine and tryptophan contents in the transgenic seeds were found to be significantly higher in comparison with WT seeds. Our results also revealed that the native promoter guided expression of modified EPSPS genes did not significantly improve the glyphosate tolerance. The present study describing the introduction of a crop-specific TIPS mutation in class I aroA gene of rice and its overexpression have potential to substantially improve the yield and field level glyphosate tolerance in rice. This is the first report to observe that the EPSPS has role to play in improving grain yield of rice.

The B-Box-Containing MicroProtein miP1a/BBX31 Regulates Photomorphogenesis and UV-B Protection.

The bZIP transcription factor ELONGATED HYPOCOTYL5 (HY5) represents a major hub in the light-signaling cascade both under visible and UV-B light. The mode of transcriptional regulation of HY5, especially under UV-B light, is not well characterized. B-BOX (BBX) transcription factors regulate HY5 transcription and also posttranscriptionally modulate HY5 to control photomorphogenesis under white light. Here, we identify BBX31 as a key signaling intermediate in visible and UV-B light signal transduction in Arabidopsis (Arabidopsis thaliana). BBX31 expression is induced by UV-B radiation in a fluence-dependent manner. HY5 directly binds to the promoter of BBX31 and regulates its transcript levels. Loss- and gain-of-function mutants of BBX31 indicate that it acts as a negative regulator of photomorphogenesis under white light but is a positive regulator of UV-B signaling. Genetic interaction studies suggest that BBX31 regulates photomorphogenesis independent of HY5 We found no evidence for a direct BBX31-HY5 interaction, and they primarily regulate different sets of genes in white light. Under high doses of UV-B radiation, BBX31 promotes the accumulation of UV-protective flavonoids and phenolic compounds. It enhances tolerance to UV-B radiation by regulating genes involved in photoprotection and DNA repair in a HY5-dependent manner. Under UV-B radiation, overexpression of BBX31 enhances HY5 transcriptional levels in a UV RESISTANCE LOCUS8-dependent manner, suggesting that BBX31 might regulate HY5 transcription.

A Nuclear Magnetic Resonance (NMR) Platform for Real-Time Metabolic Monitoring of Bioprocesses.

We present a Nuclear Magnetic Resonance (NMR) compatible platform for the automated real-time monitoring of biochemical reactions using a flow shuttling configuration. This platform requires a working sample volume of ∼11 mL and it can circulate samples with a flow rate of 28 mL/min., which makes it suitable to be used for real-time monitoring of biochemical reactions. Another advantage of the proposed low-cost platform is the high spectral resolution. As a proof of concept, we acquire 1H NMR spectra of waste orange peel, bioprocessed using Trichoderma reesei fungus, and demonstrate the real-time measurement capability of the platform. The measurement is performed over more than 60 h, with a spectrum acquired every 7 min, such that over 510 data points are collected without user intervention. The designed system offers high resolution, automation, low user intervention, and, therefore, time-efficient measurement per sample.

Light signaling and UV-B-mediated plant growth regulation.

Light plays an important role in plants' growth and development throughout their life cycle. Plants alter their morphological features in response to light cues of varying intensity and quality. Dedicated photoreceptors help plants to perceive light signals of different wavelengths. Activated photoreceptors stimulate the downstream signaling cascades that lead to extensive gene expression changes responsible for physiological and developmental responses. Proteins such as ELONGATED HYPOCOTYL5 (HY5) and CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) act as important factors which modulate light-regulated gene expression, especially during seedling development. These factors function as central regulatory intermediates not only in red, far-red, and blue light pathways but also in the UV-B signaling pathway. UV-B radiation makes up only a minor fraction of sunlight, yet it imparts many positive and negative effects on plant growth. Studies on UV-B perception, signaling, and response in plants has considerably surged in recent times. Plants have developed different strategies to use UV-B as a developmental cue as well as to withstand high doses of UV-B radiation. Plants' responses to UV-B are an integration of its cross-talks with both environmental factors and phytohormones. This review outlines the current developments in light signaling with a major focus on UV-B-mediated plant growth regulation.

Nitric oxide accelerates germination via the regulation of respiration in chickpea.

Seed germination is crucial for the plant life cycle. We investigated the role of nitric oxide (NO) in two chickpea varieties that differ in germination capacity: Kabuli, which has a low rate of germination and germinates slowly, and Desi, which shows improved germination properties. Desi produced more NO than Kabuli and had lower respiratory rates. As a result of the high respiration rates, Kabuli had higher levels of reactive oxygen species (ROS). Treatment with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) reduced respiration in Kabuli and decreased ROS levels, resulting in accelerated germination rates. These findings suggest that NO plays a key role in the germination of Kabuli. SNAP increased the levels of transcripts encoding enzymes involved in carbohydrate metabolism and the cell cycle. Moreover, the levels of amino acids and organic acids were increased in Kabuli as a result of SNAP treatment. 1H-nuclear magnetic resonance analysis revealed that Kabuli has a higher capacity for glucose oxidation than Desi. An observed SNAP-induced increase in 13C incorporation into soluble alanine may result from enhanced oxidation of exogenous [13C]glucose via glycolysis and the pentose phosphate pathway. A homozygous hybrid that originated from a recombinant inbred line population of a cross between Desi and Kabuli germinated faster and had increased NO levels and a reduced accumulation of ROS compared with Kabuli. Taken together, these findings demonstrate the importance of NO in chickpea germination via the control of respiration and ROS accumulation.

Genome-scale metabolic model led engineering of Nothapodytes nimmoniana plant cells for high camptothecin production.

Camptothecin (CPT) is a vital monoterpene indole alkaloid used in anti-cancer therapeutics. It is primarily derived from Camptotheca acuminata and Nothapodytes nimmoniana plants that are indigenous to Southeast Asia. Plants have intricate metabolic networks and use them to produce secondary metabolites such as CPT, which is a prerequisite for rational metabolic engineering design to optimize their production. By reconstructing metabolic models, we can predict plant metabolic behavior, facilitating the selection of suitable approaches and saving time, cost, and energy, over traditional hit and trial experimental approaches. In this study, we reconstructed a genome-scale metabolic model for N. nimmoniana (NothaGEM iSM1809) and curated it using experimentally obtained biochemical data. We also used in silico tools to identify and rank suitable enzyme targets for overexpression and knockout to maximize camptothecin production. The predicted over-expression targets encompass enzymes involved in the camptothecin biosynthesis pathway, including strictosidine synthase and geraniol 10-hydroxylase, as well as targets related to plant metabolism, such as amino acid biosynthesis and the tricarboxylic acid cycle. The top-ranked knockout targets included reactions responsible for the formation of folates and serine, as well as the conversion of acetyl CoA and oxaloacetate to malate and citrate. One of the top-ranked overexpression targets, strictosidine synthase, was chosen to generate metabolically engineered cell lines of N. nimmoniana using Agrobacterium tumefaciens-mediated transformation. The transformed cell line showed a 5-fold increase in camptothecin production, with a yield of up to 5 µg g-1.

Phytochemical rich Himalayan Rhododendron arboreum petals inhibit SARS-CoV-2 infection in vitro.

Phytochemicals with potential to competitively bind to the host receptors or inhibit SARS-CoV-2 replication, may prove to be useful as adjunct therapeutics for COVID-19. We profiled and investigated the phytochemicals of Rhododendron arboreum petals sourced from Himalayan flora, undertook in vitro studies and found it as a promising candidate against SARS-CoV-2. The phytochemicals were reported in various scientific investigations to act against a range of virus in vitro and in vivo, which prompted us to test against SARS-CoV-2. In vitro assays of R. arboreum petals hot aqueous extract confirmed dose dependent reduction in SARS-CoV-2 viral load in infected Vero E6 cells (80% inhibition at 1 mg/ml; IC50 = 173 µg/ml) and phytochemicals profiled were subjected to molecular docking studies against SARS CoV-2 target proteins. The molecules 5-O-Feruloyl-quinic acid, 3-Caffeoyl-quinic acid, 5-O-Coumaroyl-D-quinic acid, Epicatechin and Catechin showed promising binding affinity with SARS-CoV-2 Main protease (MPro; PDB ID: 6LU7; responsible for viral replication) and Human Angiotensin Converting Enzyme-2 (ACE2; PDB ID: 1R4L; mediate viral entry in the host). Molecular dynamics (MD) simulation of 5-O-Feruloyl-quinic acid, an abundant molecule in the extract complexed with the target proteins showed stable interactions. Taken together, the phytochemical profiling, in silico analysis and in vitro anti-viral assay revealed that the petals extract act upon MPro and may be inhibiting SARS-CoV-2 replication. This is the first report highlighting R. arboreum petals as a reservoir of antiviral phytochemicals with potential anti-SARS-CoV-2 activity using an in vitro system.

Utilization of agricultural residues for energy and resource recovery towards a sustainable environment.

Fungal pre-treatment using Pleurotus ostreatus (PO) was carried out on individual and combinations of agro-waste wheat straw (WS), rice straw (RS), and pearl millet straw (PMS) with the addition of biochar (5%,7.5% and 10%) to reduce the pre-treatment duration. Further remaining substrate known as spent mushroom substrate (SMS) was used in anaerobic digestor (AD) for estimation enhanced biomethane yield. Equal ratios of RS + WS, WS + PMS, PMS + RS, and RS + PMS + WS and biochar addition were taken for enhancing pre-treatment, PO growth and AD process. The extent of pre-treatment was recorded with the maximum lignin removal of 40.4% for RS + PMS + WS as compared to untreated counterparts and 0.5%, 2.2%, and 3.3% times more lignin removal from individual PMS, RS, and WS respectively. Addition of biochar to the substrates reduced the total pre-treatment duration by days as compared to the non-biochar substrates. Biological efficiency (BE) used for the analysis of mushroom growth varied from 51-92%. Further, the average bio-methane yield was 187 ml/gVS for SMS of PMS + WS + RS with 10% biochar indicating an increment of 83.33% from untreated SMS of PMS + WS + RS. This, higher biomethane yield was 9.35%, 22.22% and 57.14% times higher than individual SMS of PMS, RS, and WS respectively. The current study shows that biochar not only enhances the bio-methane yield but also reduces the biological pre-treatment duration and removes the dependency on one lignocellulosic biomass for energy (bio-methane) and food (mushroom) production.

Structural, Morphological, Optical and Electrical Characterization of Gahnite Ferroan Nano Composite Derived from Fly Ash Silica and ZnO Mixture.

The synthesis of a high value-added product, gahnite ferroan nano composite, from a mixture of fly ash silica and ZnO is a low-cost and non-expensive technique. The XRD pattern clearly reveals the synthesized product from fly ash after leaching is a product of high-purity gahnite ferroan composite. The grains are mostly cubical in shape. The optical band gap of powdered gahnite ferroan nano composite is 3.37 eV, which acts as a UV protector. However, the bulk sample shows that the 500 to 700 nm wavelength of visible light is absorbed, and UV light is allowed to pass through. So, the bulk sample acts as a band pass filter of UV light which can be used in many optical applications for conducting UV-irradiation activity. Dielectric permittivity and dielectric loss increase with a rise in temperature. The increase in the ac conductivity at higher temperatures denotes the negative temperature coefficient resistance (NTCR) behavior of the material.

Insights into the Polyhydroxybutyrate Biosynthesis in Ralstonia solanacearum Using Parallel 13C Tracers and Comparative Genome Analysis.

Bacterial accumulation of poly(3-hydroxybutyrate) [P(3HB)] is a metabolic strategy often adopted to cope with challenging surroundings. Ralstonia solanacearum, a phytopathogen, seems to be an ideal candidate with inherent ability to accumulate this biodegradable polymer of high industrial relevance. This study is focused on investigating the metabolic networks that channel glucose into P(3HB) using comparative genome analysis, 13C tracers, microscopy, gas chromatography-mass spectrometry (GC-MS), and proton nuclear magnetic resonance (1H NMR). Comparative genome annotation of 87 R. solanacearum strains confirmed the presence of a conserved P(3HB) biosynthetic pathway genes in the chromosome. Parallel 13C glucose feeding ([1-13C], [1,2-13C]) analysis mapped the glucose oxidation to 3-hydroxybutyrate (3HB), the metabolic precursor of P(3HB) via the Entner-Doudoroff pathway (ED pathway), potentially to meet the NADPH demands. Fluorescence microscopy, GC-MS, and 1H NMR analysis further confirmed the ability of R. solanacearum to accumulate P(3HB) granules. In addition, it is demonstrated that the carbon/nitrogen (C/N) ratio influences the P(3HB) yields, thereby highlighting the need to further optimize the bioprocessing parameters. This study provided key insights into the biosynthetic abilities of R. solanacearum as a promising P(3HB) producer.

Enhanced Field-Based Detection of Potato Blight in Complex Backgrounds Using Deep Learning.

Rapid and automated identification of blight disease in potato will help farmers to apply timely remedies to protect their produce. Manual detection of blight disease can be cumbersome and may require trained experts. To overcome these issues, we present an automated system using the Mask Region-based convolutional neural network (Mask R-CNN) architecture, with residual network as the backbone network for detecting blight disease patches on potato leaves in field conditions. The approach uses transfer learning, which can generate good results even with small datasets. The model was trained on a dataset of 1423 images of potato leaves obtained from fields in different geographical locations and at different times of the day. The images were manually annotated to create over 6200 labeled patches covering diseased and healthy portions of the leaf. The Mask R-CNN model was able to correctly differentiate between the diseased patch on the potato leaf and the similar-looking background soil patches, which can confound the outcome of binary classification. To improve the detection performance, the original RGB dataset was then converted to HSL, HSV, LAB, XYZ, and YCrCb color spaces. A separate model was created for each color space and tested on 417 field-based test images. This yielded 81.4% mean average precision on the LAB model and 56.9% mean average recall on the HSL model, slightly outperforming the original RGB color space model. Manual analysis of the detection performance indicates an overall precision of 98% on leaf images in a field environment containing complex backgrounds.