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Organic fertilizers and microbial biofertilizers are now widely recognized to effectively complement traditional mineral fertilizers for plant growth. The present study shows that bio-organic fertilizers can be enhanced by the addition of functional plant-growth-promoting rhizobacteria (PGPR) that provide additional benefits to plants. We hypothesized that not all beneficial soil bacteria are functional in different farm soils and plant varieties; hence, the most effective PGPR that are suitable to each farm’s individual cropping conditions were selected. Five different field soils and their respective crops were tested for compatibility with six microbial biofertilizers (including three new bacterial strains) to supplement a commercially available bio-organic fertilizer. In pot trials with lucerne plants, four out of the six microbial treatments led to significant (p < 0.05) growth promotion benefits (up to 79.8% more leaves and dry weight) compared to mock-treated or bio-organic fertilizer-only-treated control plants. A trial with industrial hemp demonstrated that compatibility with PGPR occurs in a cultivar-specific manner, leading to growth promotion ranging from −3.4% to 68.9%, with each cultivar displaying a preference for a different PGPR. Finally, pot trials with Rhodes grass and two different soils demonstrated high yield increases compared to control plants, with Bacillus amyloliquefaciens 33YE being most effective for one soil and Bacillus velezensis UQ9000N/Pseudomonas lini SMX2 for the other soil. Yield advantages reduced after several cuts of grass, but a repeat biofertilizer treatment at 69 days after the initial treatment restored high yield advantages, with the same PGPR again being most effective. These results demonstrate the importance of customization of microbial inoculants to identify the most compatible PGPR–cultivar–soil interaction. The customization of microbial biofertilizers to soils and plant cultivars, combined with complementary fertilizer applications, can potentially lead to more reliable and more sustainable agricultural practices.

The increase in global population and industrial development has led to a significant release of organic and inorganic pollutants into water streams, threatening human health and ecosystems. Microalgae, encompassing eukaryotic protists and prokaryotic cyanobacteria, have emerged as a sustainable and cost-effective solution for removing these pollutants and mitigating carbon emissions. Various microalgae species, such as C. vulgaris, P. tricornutum, N. oceanica, A. platensis, and C. reinhardtii, have demonstrated their ability to eliminate heavy metals, salinity, plastics, and pesticides. Synthetic biology holds the potential to enhance microalgae-based technologies by broadening the scope of treatment targets and improving pollutant removal rates. This review provides an overview of the recent advances in the synthetic biology of microalgae, focusing on genetic engineering tools to facilitate the removal of inorganic (heavy metals and salinity) and organic (pesticides and plastics) compounds. The development of these tools is crucial for enhancing pollutant removal mechanisms through gene expression manipulation, DNA introduction into cells, and the generation of mutants with altered phenotypes. Additionally, the review discusses the principles of synthetic biology tools, emphasizing the significance of genetic engineering in targeting specific metabolic pathways and creating phenotypic changes. It also explores the use of precise engineering tools, such as CRISPR/Cas9 and TALENs, to adapt genetic engineering to various microalgae species. The review concludes that there is much potential for synthetic biology based approaches for pollutant removal using microalgae, but there is a need for expansion of the tools involved, including the development of universal cloning toolkits for the efficient and rapid assembly of mutants and transgenic expression strains, and the need for adaptation of genetic engineering tools to a wider range of microalgae species.

Tomer Snir,1 Ilya Kirgner,2 Nachum Nesher,3 Yaacov Abramov,3 Marina Kolodii,3 Sharbel Azzam,3 Michael Peer3 1Medical Students Elective Program, University of Milan, Milan, Italy; 2Blood Bank, Ichilov Medical Center, Tel Aviv, Israel; 3Department of Thoracic Surgery, Ichilov Medical Center, Tel Aviv, IsraelCorrespondence: Michael Peer, Tel +972-3-6972021, Fax +972-3 - 6973235, Email michaelpe@tlvmc.gov.ilBackground: Blood transfusion rates during surgery and hospitalization for thoracic surgery vary from 16% to 55%. The religious beliefs of Jehovah’s Witnesses (JW) permit medical and surgical procedures but exclude the use of blood and blood products. Performing major pulmonary resection without the possibility of compensating for blood loss is a daunting challenge that few surgeons are willing to meet.Methods: The clinical and surgical data on two JW patients who underwent major pulmonary resections for non-small cell lung carcinoma (NSCLC) in Tel Aviv Medical Center between the years 2019 and 2020 were retrieved from the departmental databases and analyzed for the requirement and consumption of blood products. The patients were a 70-year-old female and a 49-year-old man that have underwent a completion right upper lobe lobectomy and a left lower lobe lobectomy, respectively.Results: None required blood transfusions and the surgeries were successful, demonstrating that it is possible to successfully perform “bloodless” major pulmonary resection while respecting the limitations set by the patient’s religious beliefs.Conclusion: This concept paves the way for the consideration of major pulmonary resection for patients who are currently denied such procedures due to religious restrictions or to their being high-risk surgical candidates, when resources are limited or lacking.Keywords: bloodless thoracic surgery, lobectomy, Jehovah’s Witnesses

Rhizosphere bacteria can provide multiple benefits to plants, including increased nutrient supply, pathogen/disease control, and abiotic stress tolerance, but results from pot trials do not always translate to field conditions. This study tested whether rhizosphere biocontrol bacteria can also provide plant growth promotion and how benefits can be provided at a commercial farm. Commercial lettuce seeds and plants were treated with rhizosphere biocontrol bacteria Bacillus velezensis UQ9000N, B. amyloliquefaciens 33YE, Brevibacillus laterosporus 4YE, and Pseudomonas azotoformans UQ4510An. 33YE increased the head diameter, plant height, and fresh weight of the Green Moon cultivar, while 33YE, UQ4510An, and UQ9000N increased the fresh and dry weight of Liston, a more heat-tolerant cultivar, via a single seed treatment or repeat root treatments under nursery and field conditions across different inoculation schedules and growth stages. Significant growth promotion was also demonstrated when inoculating field plants after transplanting (in particular for 33YE). Applications of these microbial biostimulants to lettuce seeds or plantlets potentially enable earlier transplanting and earlier harvests. Repeat inoculations using irrigation water and long-lasting formulations may further advance the benefits of these biostimulants as microbial biofertilisers for plant growth promotions in the field.

Bacterial biocontrol agent/s (BCAs) against plant diseases are eco-friendly and sustainable options for profitable agricultural crop production. Specific beneficial strains of Bacillus subtilis are effective in controlling many fungal diseases including Alternaria blight caused by a notorious pathogen “Alternaria solani”. In the present study, the biocontrol attributes of a newfangled strain of B. subtilis (BS-01) have been investigated and its bioactive compounds were also identified against A. solani. The volatile organic compounds (VOCs) produced by BS-01 in organic solvents viz., n-hexane, dichloromethane, and ethyl acetate were extracted and their antifungal efficacy has evaluated against A. solani. Also, the preventive and curative biocontrol method to reduce the fungal load of A. solani was estimated by both foliar and seed applications on infected tomato (Solanum lycopersicum) plants as determined by quantitative PCR assays. Growth chamber bioassay revealed that both foliar and seed application of BS-01 on tomato plants previously or subsequently infected by A. solani significantly reduced the pathogen load on inoculated tomato foliage. Results showed that antifungal bioassays with various concentrations (10-100 mg mL-1) of extracted metabolites produced by BS-01 in ethyl acetate fraction showed the highest inhibition in fungal biomass (extracellular metabolites: 69-98% and intracellular metabolites: 48-85%) followed by n-hexane (extracellular metabolites: 63-88% and intracellular metabolites: 35-62%) and dichloromethane (extracellular metabolites: 41-74% and intracellular metabolites: 42-70%), respectively. The extracted volatile compounds of BS-01 were identified via GC-MS analysis and were found in great proportions in the organic fractions as major potent antifungal constituents including triphenylphosphine oxide; pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl); pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(phenylmethyl); n-hexadecanoic acid; n-tridecan-1-ol; octadecane; octadecanoic acid; eicosane and dodecyl acrylate. Separate or mixture of these bioactive VOCs had the potential to mitigate the tomato early blight disease severity in the field that would act as a sustainable plant protection strategy to generate profitable tomato production.

Abstract Background Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that facilitates the adaptation of cancer cells to hypoxic conditions and may be prognostic of breast cancer recurrence. We evaluated the association of HIF-1α expression with breast cancer recurrence, and its association with timing of breast cancer recurrence. Methods In this population-based case-control study, we included women diagnosed with stage I–III breast cancer between 1985 and 2001, aged 35–69 years, registered in the Danish Breast Cancer Group. We identified 541 cases of breast cancer recurrence among women with estrogen receptor (ER)-positive disease who were treated with tamoxifen for at least 1 year (ER+ TAM+). We also enrolled 300 breast cancer recurrence cases among women with ER-negative disease, not treated with tamoxifen, who survived at least 1 year (ER−/TAM−). Controls were recurrence-free breast cancer patients at the time of case diagnosis, matched to recurrence cases on ER/TAM status, date of surgery, menopausal status, cancer stage, and county of residence. Expression of HIF-1α was measured by immunohistochemistry on tissue microarrays. We fitted logistic regression models to compute odds ratios (ORs) and 95% confidence intervals (CIs) associating HIF-1α expression with recurrence, and with timing of recurrence. Results HIF-1α expression was observed in 23% of cases and 20% of controls in the ER+/TAM+ stratum, and in 47% of cases and 48% of controls in the ER−/TAM− stratum. We observed a near-null association between HIF-1α expression in both ER/TAM groups (ER+/TAM+ OR = 1.21, 95%CI 0.88, 1.67 and ER−/TAM− OR = 0.97, 95%CI 0.68, 1.39). HIF-1α expression was not associated with time to recurrence among women in the ER+/TAM+ stratum, but was associated with early recurrence among women in the ER−/TAM− stratum. Conclusion In this study, HIF-1α expression was not associated with breast cancer recurrence overall but may be associated with early recurrence among women diagnosed with ER− breast cancer.

In recent decades, marine microorganisms have become known for their ability to produce a wide variety of secondary bioactive metabolites. Several compounds have been isolated from marine microorganisms for the development of novel bioactives for the food and pharmaceutical industries. In this study, a number of microalgae were evaluated for their antimicrobial activity against gram-positive and gram-negative bacteria, including food and plant pathogens, using various extraction techniques and antimicrobial assays. Disc diffusion and spot-on-lawn assays were conducted to confirm the antimicrobial activity. To measure the potency of the extracts, minimum inhibition concentrations (MIultCs) were measured. Three microalgae, namely Isochrysis galbana, Scenedesmus sp. NT8c, and Chlorella sp. FN1, showed strong inhibitory activity preferentially against gram-positive bacteria. These microalgal species were then selected for further purification and analysis, leading to compound identification. By using a mixture of different chromatography techniques gas chromatography–mass spectrometry (GC–MS) and high-performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS), we were able to separate and identify the dominant compounds that are responsible for the inhibitory activity. Additionally, nuclear magnetic resonance (NMR) was used to confirm the presence of these compounds. The dominant compounds that were identified and purified in the extracts are linoleic acid, oleic acid, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). These compounds are the potential candidates that inhibit the growth of gram-positive bacteria. This indicates the potential use of microalgae and their antimicrobial compounds as biocontrol agents against food and plant pathogens.

Breast cancer heterogeneity in histology and molecular subtype influences metabolic and proliferative activity and hence the acid load on cancer cells. We hypothesized that acid-base transporters and intracellular pH (pHi) dynamics contribute inter-individual variability in breast cancer aggressiveness and prognosis. We show that Na+,HCO3– cotransport and Na+/H+ exchange dominate cellular net acid extrusion in human breast carcinomas. Na+/H+ exchange elevates pHi preferentially in estrogen receptor-negative breast carcinomas, whereas Na+,HCO3– cotransport raises pHi more in invasive lobular than ductal breast carcinomas and in higher malignancy grade breast cancer. HER2-positive breast carcinomas have elevated protein expression of Na+/H+ exchanger NHE1/SLC9A1 and Na+,HCO3– cotransporter NBCn1/SLC4A7. Increased dependency on Na+,HCO3– cotransport associates with severe breast cancer: enlarged CO2/HCO3–-dependent rises in pHi predict accelerated cell proliferation, whereas enhanced CO2/HCO3–-dependent net acid extrusion, elevated NBCn1 protein expression, and reduced NHE1 protein expression predict lymph node metastasis. Accordingly, we observe reduced survival for patients suffering from luminal A or basal-like/triple-negative breast cancer with high SLC4A7 and/or low SLC9A1 mRNA expression. We conclude that the molecular mechanisms of acid-base regulation depend on clinicopathological characteristics of breast cancer patients. NBCn1 expression and dependency on Na+,HCO3– cotransport for pHi regulation, measured in biopsies of human primary breast carcinomas, independently predict proliferative activity, lymph node metastasis, and patient survival.

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1's primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.

Abstract Sustainable alternatives to fossil fuels are urgently needed to avoid severe climate impacts and further environmental degradation. Microalgae are one of the most productive crops globally and do not need to compete for arable land or freshwater resources. Hence, they may become a promising, more sustainable cultivation alternative for the large‐scale production of biofuels provided that substantial reductions are achieved in their production costs. In this study, we identify the most suitable areas globally for siting microalgal farms for biodiesel production that maximize profitability and minimize direct competition with food production and direct impacts on biodiversity, based on a spatially explicit multiple‐criteria decision analysis. We further explore the relationships between microalgal production, agricultural value, and biodiversity, and propose several solutions for siting microalgal production farms, based on current and future targets in energy production using integer linear programming. If using seawater for microalgal cultivation, biodiesel production could reach 5.85 × 1011 L/year based on top suitable lands (i.e., between 13% and 16% of total transport energy demands in 2030) without directly competing with food production and areas of high biodiversity value. These areas are particularly abundant in the dry coasts of North and East Africa, the Middle East, and western South America. This is the first global analysis that incorporates economic and environmental feasibility for microalgal production sites. Our results can guide the selection of best locations for biofuel production using microalgae while minimizing conflicts with food production and biodiversity conservation.

For efficient downstream processing, harvesting remains as one of the challenges in producing Nannochloropsis biomass, a microalga with high-value omega-3 oils. Flocculation is an effective, low-energy, low-cost method to harvest microalgae. Chitosan has been shown to be an effective food-grade flocculant; however, commercial chitosan is sourced from crustaceans, which has disadvantages including concerns over heavy-metal contamination. Thus, this study tests the flocculation potential of mushroom chitosan. Our results indicate a 13% yield of chitosan from mushroom. The identity of the prepared chitosan was confirmed by Fourier-transform infrared (FTIR) spectroscopy. Furthermore, results show that mushroom chitosan can be an alternative flocculant with >95% flocculation efficiency when tested in 100-mL jar and 200-L vertical column photobioreactor. Applications in a 2000-L raceway pond demonstrated that thorough mixing of mushroom chitosan with the algal culture is required to achieve efficient flocculation. With proper mixing, mushroom chitosan can be used to produce food-grade Nannochloropsis biomass suitable for the production of vegan omega-3 oils as a fish oil alternative.

Mediator subunits play key roles in numerous physiological pathways and developmental processes in plants. Arabidopsis Mediator subunits, MED18 and MED25, have previously been shown to modulate disease resistance against fungal and bacterial pathogens through their role in jasmonic acid (JA) signaling. In this study, Arabidopsis mutant plants of the two Mediator subunits, med18 and med25, were tested against three ssRNA viruses and one dsDNA virus belonging to four different families: Turnip mosaic virus (TuMV), Cauliflower mosaic virus (CaMV), Alternanthera mosaic virus (AltMV), and Cucumber mosaic virus (CMV). Although both subunits are utilized in JA signaling, they occupy different positions (Head and Tail domain, respectively) in the Mediator complex and their absence affected virus infection differently. Arabidopsis med18 plants displayed increased resistance to RNA viral infection and a trend against the DNA virus, while med25 mutants displayed increased susceptibility to all viruses tested at 2 and 14 days post inoculations. Defense marker gene expression profiling of mock- and virus-inoculated plants showed that med18 and med25 mutants exhibited an upregulated SA pathway upon virus infection at 2 dpi for all viruses tested. JA signaling was also suppressed in med18 plants after virus infection, independent of which virus infected the plants. The upregulation of SA signaling and suppression of JA signaling in med18 may have led to more targeted oxidative burst and programmed cell death to control viruses. However, the susceptibility exhibited by med25 mutants suggests that other factors, such as a weakened RNAi pathway, might play a role in the observed susceptibility. We conclude that MED18 and MED25 have clear and opposite effects on accumulation of plant viruses. MED18 is required for normal virus infection, while MED25 is important for defense against virus infection. Results from this study provide a better understanding of the role of Mediator subunits during plant-virus interactions, viral disease progression and strategies to develop virus resistant plants.

We hypothesized that environmental microbiomes contain a wide range of bacteria that produce yet uncharacterized antimicrobial compounds (AMCs) that can potentially be used to control pathogens. Over 600 bacterial strains were isolated from soil and food compost samples, and 68 biocontrol bacteria with antimicrobial activity were chosen for further studies based on inhibition assays against a wide range of food and plant pathogens. For further characterization of the bioactive compounds, a new method was established that used living pathogens in a liquid culture to stimulate bacteria to produce high amounts of AMCs in bacterial supernatants. A peptide gel electrophoresis microbial inhibition assay was used to concurrently achieve size separation of the antimicrobial peptides. Fifteen potential bioactive peptides were then further characterized by tandem MS, revealing cold-shock proteins and 50S ribosomal proteins. To identify non-peptidic AMCs, bacterial supernatants were analyzed by HPLC followed by GC/MS. Among the 14 identified bioactive compounds, 3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione and 2-acetyl-3-methyl-octahydropyrrolo[1,2-a]piperazine-1,4-dione were identified as new AMCs. Our work suggests that antimicrobial compound production in microbes is enhanced when faced with a threat from other microorganisms, and that this approach can rapidly lead to the development of new antimicrobials with the potential for upscaling.

Abstract Background Several frequently used prescription drugs may affect bleeding risk. We investigated use of aspirin, nonsteroidal anti-inflammatory drugs (NSAIDs), selective serotonin reuptake inhibitors (SSRIs), and statins and risk of postoperative red blood cell transfusion in breast cancer patients. Methods Using Danish population-based registries, we identified a cohort of women who underwent surgery for primary breast cancer (n = 22,238) during 2005–2012 and ascertained their use of aspirin, NSAIDs, SSRIs, and statins. For each drug, patients were categorized as users if they filled ≥1 prescription in the 60 days prior to surgery. We calculated the 14-day risk of red blood cell transfusion and relative risks (RRs) with 95% confidence intervals (CIs), comparing users with nonusers for each drug and adjusting for age, cancer stage, and Charlson Comorbidity Index score. Results In our cohort, 1385 (6.2%) women were aspirin users, 1794 (8.0%) were NSAID users, 1110 (4.9%) were SSRI users, and 2053 (9.1%) were statin users. The overall risk of red blood cell transfusion was 1.3%. The 14-day risk of RBC transfusion was 3.5% among aspirin users versus 1.1% among aspirin nonusers (adjusted RR = 1.9, 95% CI: 1.4–2.7), and 1.8% among SSRI users versus 1.2% among SSRI nonusers (adjusted RR = 1.2, 95% CI: 0.7–1.9). Red blood cell transfusion risk was increased among NSAID users, but not in a sensitivity analysis with a 30-day exposure window. Red blood cell transfusion risk was not increased among SSRI and statin users. Conclusions Primary breast cancer surgery confers a low risk of RBC transfusion. Still, use of aspirin and possibly NSAIDs, but not SSRIs and statins, is associated with increased red blood cell transfusion. This increased risk is not explained by differences in age, stage, or comorbidity level.

Despite being established as a sustainable feedstock for biofuel production with tremendous potential, the microalgae biofuel industry still struggles to make large-scale production economically viable. An overriding aspect in microalgae oil production is strain selection, as it affects nearly all stages of production. This chapter presents the key traits that microalgae should possess for successful lipid production, as well as suitable isolation and selection strategies. It highlights the various metabolic engineering methods that are currently available for the biological improvement of microalgae strains, comparing GM vs non-GM approaches.

Abstract Food‐grade rather than synthetic or chemical flocculants are needed for microalgae harvesting by settling, if used for food products. Chitosan is effective in harvesting freshwater microalgae, but it is expensive and typically not suitable for marine microalgae like Nannochloropsis. To minimize costs for food‐grade flocculation, a number of potentially important parameters are considered, including chitosan solubility and optimized chitosan‐mediated flocculation of Nannochloropsis sp. BR2 by a five‐factor central composite design experiment. Results show that an optical density (440 nm) of 2 (0.23 g dry weight L−1), initial pH of 6, final pH of 10, and 22 ppm chitosan with a viscosity of 1808 cP provide optimum flocculation efficiency, which is predicted to be in the range of 97.01% to 99.93%. These predictions are verified on 4.5 and 8 L Nannochloropsis sp. BR2 cultures.

Biofuel production is a key strategy for reducing CO _2 emissions globally and is expected to increase substantially in the coming decades, particularly in tropical developing countries. The adoption of sustainable biofuel production technologies that do not place large demands on agricultural or forested lands, has the potential to make a substantial contribution to decreasing greenhouse gas emissions while reducing biodiversity losses and degradation of native ecosystems resulting from high demand for land. With their high productivity per unit area and ability to grow on non-arable lands, microalgal biofuel production systems could become a major sustainable alternative to biofuel production from food crops (first-generation biofuels). However, the potential impacts of microalgal biofuels on food production, biodiversity, and carbon storage, compared to other biofuel production alternatives, are largely unknown. In the present study, the most suitable areas for siting microalgae production farms to fulfill 30% of future transport energy demands were determined within four Neotropical countries with high population densities and high importance for agricultural expansion and biodiversity conservation globally (Colombia, Ecuador, Panama, and Venezuela). These results were contrasted with the best areas for siting oil palm and sugarcane crops to fulfill the same target in future transport energy demands. Microalgal production systems offer the most sustainable alternative for future biofuel production within the Neotropics. Meeting 30% of future transport energy demands with microalgal biofuels reduced land area requirements by at least 52% compared to oil palm and sugarcane. Furthermore, microalgal biofuel production reduced direct competition with agricultural lands, biodiverse areas, and carbon-rich systems within countries, with little overlap with the biodiverse and carbon-rich rainforests. This study can guide decision making towards the identification and adoption of more sustainable biofuel production alternatives in the Neotropics, helping in avoiding unnecessary environmental impacts from biofuel expansion in the region.

Bacterial isolates obtained from the rhizosphere of Arabidopsis and a plantless compost potting mix was screened for anti-oomycete activity against Phytophthora capsici, Phytophthora citricola, Phytophthora palmivora, and Phytophthora cinnamomi. Three out of 48 isolates exhibited more than 65% inhibition against all tested Phytophthora species and were selected for further studies. These strains, named UQ154, UQ156, and UQ202, are closely related to Bacillus amyloliquefaciens, Bacillus velezensis, and Acinetobacter sp., respectively, based on 16S rDNA sequence analysis. The isolates were evaluated for their ability to fix nitrogen, solubilize phosphate, as well as for siderophore, indoleacetic acid, cell wall degrading enzymes and biofilm production. Their plant growth promoting activities were evaluated by measuring their effect on the germination percentage, root and shoot length, and seedling vigor of lettuce plants. All of these traits were significantly enhanced in plants grown from seeds inoculated with the isolates compared with control plants. Moreover, bacteria-inoculated P. capsici-infected chili plants exhibited improved productivity based on CO2 assimilation rates. Both real-time quantitative PCR and disease severity index revealed significant decreases in pathogen load in infected chili root tissues when plants were previously inoculated with the isolates. Biocontrol activity may result from the secretion of diketopiperazines as identified by Gas chromatography-mass spectrometry analysis of bacterial cultures’ extracts. Collectively, this work demonstrates the potential of bacterial isolates to control Phytophthora infection and promote plant growth. They can, therefore be considered as candidate microbial biofertilizers and biopesticides.

One of the most exciting scientific advances in recent decades has been the realization that the diverse and immensely active microbial communities are not only ‘passengers’ with plants, but instead play an important role in plant growth, development and resistance to biotic and abiotic stresses. A picture is emerging where plant roots act as ‘gatekeepers’ to screen soil bacteria from the rhizosphere and rhizoplane. This typically results in root endophytic microbiome dominated by Proteobacteria, Actinobacteria and to a lesser extent Bacteroidetes and Firmicutes, but Acidobacteria and Gemmatimonadetes being almost depleted. A synthesis of available data suggest that motility, plant cell-wall degradation ability and reactive oxygen species scavenging seem to be crucial traits for successful endophytic colonization and establishment of bacteria. Recent studies provide solid evidence that these bacteria serve host functions such as improving of plant nutrients through acquisition of nutrients from soil and nitrogen fixation in leaves. Additionally, some endophytes can engage ‘priming’ plants which elicit a faster and stronger plant defense once pathogens attack. Due to these plant growth-promoting effects, endophytic bacteria are being widely explored for their use in the improvement of crop performance. Updating the insights into the mechanism of endophytic bacterial colonization and interactions with plants is an important step in potentially manipulating endophytic bacteria/microbiome for viable strategies to improve agricultural production.

Jasmonic acid (JA) is an essential hormone in plant development and defense responses in Arabidopsis thaliana. Exogenous treatment with JA has recently been shown to alter root exudate profiles and the composition of root-associated bacterial communities. However, it is currently unknown whether disruptions of the JA in the rhizosphere affect root exudation profiles and the relative abundance of bacteria and archaea in the rhizosphere. In the present study, two Arabidopsis mutants that are disrupted in different branches of the jasmonate pathway, namely myc2 and med25, were cultivated in nutrient solution and soil to profile root exudates and bacterial and archaeal communities, respectively. Compared with the wild type, both mutants showed distinct exudation patterns, including lower amounts of asparagine, ornithine, and tryptophan, as well as distinct bacterial and archaeal community composition, as illustrated by an increased abundance of Streptomyces, Bacillus, and Lysinibacillus taxa in the med25 rhizosphere and an Enterobacteriaceae population in myc2. Alternatively, the Clostridiales population was less abundant in the rhizosphere of both mutants. Similarities between plant genotypes were highly correlated, as determined by operational taxonomic units in the rhizosphere and metabolites in root exudates. This strongly suggests that root exudates play a major role in modulating changes in microbial community composition upon plant defense responses.

Studies investigating the action of small RNAs on computationally predicted target genes require some form of experimental validation. Classical molecular methods of validating microRNA action on target genes are laborious, while approaches that tag predicted target sequences to qualitative reporter genes encounter technical limitations. The aim of this study was to address the challenge of experimentally validating large numbers of computationally predicted microRNA-target transcript interactions using an optimized, quantitative, cost-effective, and scalable approach. The presented method combines transient expression via agroinfiltration of Nicotiana benthamiana leaves with a quantitative dual luciferase reporter system, where firefly luciferase is used to report the microRNA-target sequence interaction and Renilla luciferase is used as an internal standard to normalize expression between replicates. We report the appropriate concentration of N. benthamiana leaf extracts and dilution factor to apply in order to avoid inhibition of firefly LUC activity. Furthermore, the optimal ratio of microRNA precursor expression construct to reporter construct and duration of the incubation period post-agroinfiltration were determined. The optimized dual luciferase assay provides an efficient, repeatable and scalable method to validate and quantify microRNA action on predicted target sequences. The optimized assay was used to validate five predicted targets of rice microRNA miR529b, with as few as six technical replicates. The assay can be extended to assess other small RNA-target sequence interactions, including assessing the functionality of an artificial miRNA or an RNAi construct on a targeted sequence.

The conserved protein complex known as Mediator conveys transcriptional signals by acting as an intermediary between transcription factors and RNA polymerase II. As a result, Mediator subunits play multiple roles in regulating developmental as well as abiotic and biotic stress pathways. In this report we identify the head domain subunits MEDIATOR18 and MEDIATOR20 as important susceptibility factors for Fusarium oxysporum infection in Arabidopsis thaliana. Mutants of MED18 and MED20 display down-regulation of genes associated with jasmonate signaling and biosynthesis while up-regulation of salicylic acid associated pathogenesis related genes and reactive oxygen producing and scavenging genes. We propose that MED18 and MED20 form a sub-domain within Mediator that controls the balance of salicylic acid and jasmonate associated defense pathways.

Mediator is a multiprotein transcriptional co-regulator complex composed of four modules; Head, Middle, Tail, and Kinase. It conveys signals from promoter-bound transcriptional regulators to RNA polymerase II and thus plays an essential role in eukaryotic gene regulation. We describe subunit localization and activities of Mediator in Arabidopsis through metabolome and transcriptome analyses from a set of Mediator mutants. Functional metabolomic analysis based on the metabolite profiles of Mediator mutants using multivariate statistical analysis and heat-map visualization shows that different subunit mutants display distinct metabolite profiles, which cluster according to the reported localization of the corresponding subunits in yeast. Based on these results, we suggest localization of previously unassigned plant Mediator subunits to specific modules. We also describe novel roles for individual subunits in development, and demonstrate changes in gene expression patterns and specific metabolite levels in med18 and med25, which can explain their phenotypes. We find that med18 displays levels of phytoalexins normally found in wild type plants only after exposure to pathogens. Our results indicate that different Mediator subunits are involved in specific signaling pathways that control developmental processes and tolerance to pathogen infections.

With the depletion of global fish stocks, caused by high demand and effective fishing techniques, alternative sources for long chain omega-3 fatty acids are required for human nutrition and aquaculture feeds. Recent research has focused on land-based cultivation of microalgae, the primary producers of omega-3 fatty acids in the marine food web. The effect of salinity on fatty acids and related gene expression was studied in the model marine microalga, Tetraselmis sp. M8. Correlations were found for specific fatty acid biosynthesis and gene expression according to salinity and the growth phase. Low salinity was found to increase the conversion of C18:4 stearidonic acid (SDA) to C20:4 eicosatetraenoic acid (ETA), correlating with increased transcript abundance of the Δ-6-elongase-encoding gene in salinities of 5 and 10 ppt compared to higher salinity levels. The expression of the gene encoding β-ketoacyl-coenzyme was also found to increase at lower salinities during the nutrient deprivation phase (Day 4), but decreased with further nutrient stress. Nutrient deprivation also triggered fatty acids synthesis at all salinities, and C20:5 eicosapentaenoic acid (EPA) increased relative to total fatty acids, with nutrient starvation achieving a maximum of 7% EPA at Day 6 at a salinity of 40 ppt.

Reactive oxygen species (ROS) are highly controlled signaling species that are involved in regulating gene expression in response to different environmental cues. The production of heat shock proteins (HSPs) is a key strategy that plants use to defend themselves against diverse stresses, including oxidative stress. In this study, expression patterns of the Arabidopsis HSP17.4CI gene, a cytosolic class I small HSP, were systematically profiled under different abiotic, biotic and oxidative stresses. Our data show that HSP17.4CI was early and highly induced by heat, cold, salt, drought and high-light. HSP17.4CI also showed high expression levels in Arabidopsis plants infected with the biotrophic pathogen Pseudomonas syringae, but not in response to the necrotrophic pathogens Alternaria brassicicola and Fusarium oxysporum. Oxidative stress treatments including H2O2 and the herbicide methyl viologen led to induction of HSP17.4CI. The plant hormones abscisic acid (ABA) and salicylic acid (SA) induced the expression of HSP17.4CI, whereas methyl jasmonate (MJ) did not affect the expression level of this gene. Furthermore, we found enhanced expression of HSP17.4CI in catalase mutant plants, which are deficient in catalase 2 activity and accumulate intracellular H2O2. Taken together, data presented here suggest that HSP17.4CI expression is regulated by various signals that connect biotic and abiotic stresses with ROS and can be used as a molecular marker for oxidative stress.

Soil microbial communities play an important role in plant health and soil quality. Researchers have developed a wide range of methods for studying the structure, diversity, and activity of microbes to better understand soil biology and plant-microbe interactions. Functional microbiological analyses of the rhizosphere have given new insights into the role of microbial communities in plant nutrition and plant protection against diseases. In this review, we present the most commonly used traditional as well as new culture-independent molecular methods to assess the diversity and function of soil microbial communities. Furthermore, we discuss advantages and disadvantages of these techniques and provide a perspective on emerging technologies for soil microbial community profiling.

In the wake of intensive fossil fuel usage and CO2 accumulation in the environment, research is targeted towards sustainable alternate bioenergy that can suffice the growing need for fuel and also that leaves a minimal carbon footprint. Oil production from microalgae can potentially be carried out more efficiently, leaving a smaller footprint and without competing for arable land or biodiverse landscapes. However, current algae cultivation systems and lipid induction processes must be significantly improved and are threatened by contamination with other algae or algal grazers. To address this issue, we have developed an efficient two-stage cultivation system using the marine microalga Tetraselmis sp. M8. This hybrid system combines exponential biomass production in positive pressure air lift-driven bioreactors with a separate synchronized high-lipid induction phase in nutrient deplete open raceway ponds. A comparison to either bioreactor or open raceway pond cultivation system suggests that this process potentially leads to significantly higher productivity of algal lipids. Nutrients are only added to the closed bioreactors while open raceway ponds have turnovers of only a few days, thus reducing the issue of microalgal grazers.

Current technologies have changed biology into a data-intensive field and significantly increased our understanding of signal transduction pathways in plants. However, global defense signaling networks in plants have not been established yet. Considering the apparent intricate nature of signaling mechanisms in plants (due to their sessile nature), studying the points at which different signaling pathways converge, rather than the branches, represents a good start to unravel global plant signaling networks. In this regard, growing evidence shows that the generation of reactive oxygen species (ROS) is one of the most common plant responses to different stresses, representing a point at which various signaling pathways come together. In this review, the complex nature of plant stress signaling networks will be discussed. An emphasis on different signaling players with a specific attention to ROS as the primary source of the signaling battery in plants will be presented. The interactions between ROS and other signaling components, e.g. calcium, redox homeostasis, membranes, G-proteins, MAPKs, plant hormones and transcription factors will be assessed. A better understanding of the vital roles ROS are playing in plant signaling would help innovate new strategies to improve plant productivity under the circumstances of the increasing severity of environmental conditions and the high demand of food and energy worldwide

Biodiesel production from microalgae is being widely developed at different scales as a potential source of renewable energy with both economic and environmental benefits. Although many microalgae species have been identified and isolated for lipid production, there is currently no consensus as to which species provide the highest productivity. Different species are expected to function best at different aquatic, geographical and climatic conditions. In addition, other value-added products are now being considered for commercial production which necessitates the selection of the most capable algae strains suitable for multiple-product algae biorefineries. Here we present and review practical issues of several simple and robust methods for microalgae isolation and selection for traits that maybe most relevant for commercial biodiesel production. A combination of conventional and modern techniques is likely to be the most efficient route from isolation to large-scale cultivation.

Oil-accumulating microalgae have the potential to enable large-scale biodiesel production without competing for arable land or biodiverse natural landscapes. High lipid productivity of dominant, fast-growing algae is a major prerequisite for commercial production of microalgal oil-derived biodiesel. However, under optimal growth conditions, large amounts of algal biomass are produced, but with relatively low lipid contents, while species with high lipid contents are typically slow growing. Major advances in this area can be made through the induction of lipid biosynthesis, e.g., by environmental stresses. Lipids, in the form of triacylglycerides typically provide a storage function in the cell that enables microalgae to endure adverse environmental conditions. Essentially algal biomass and triacylglycerides compete for photosynthetic assimilate and a reprogramming of physiological pathways is required to stimulate lipid biosynthesis. There has been a wide range of studies carried out to identify and develop efficient lipid induction techniques in microalgae such as nutrients stress (e.g., nitrogen and/or phosphorus starvation), osmotic stress, radiation, pH, temperature, heavy metals and other chemicals. In addition, several genetic strategies for increased triacylglycerides production and inducibility are currently being developed. In this review, we discuss the potential of lipid induction techniques in microalgae and also their application at commercial scale for the production of biodiesel.

Fusarium oxysporum is a root-infecting fungal pathogen that causes wilt disease on a broad range of plant species, including the model plant Arabidopsis thaliana. Currently, very little is known about the molecular or physiological processes that are activated in the host during infection and the roles these processes play in resistance and susceptibility to F. oxysporum. In this study, we analyzed global gene expression profiles of F. oxysporum-infected Arabidopsis plants. Genes involved in jasmonate biosynthesis as well as jasmonate-dependent defense were coordinately induced by F. oxysporum. Similarly, tryptophan pathway genes, including those involved in both indole-glucosinolate and auxin biosynthesis, were upregulated in both the leaves and the roots of inoculated plants. Analysis of plants expressing the DR5:GUS construct suggested that root auxin homeostasis was altered during F. oxysporum infection. However, Arabidopsis mutants with altered auxin and tryptophan-derived metabolites such as indole-glucosinolates and camalexin did not show an altered resistance to this pathogen. In contrast, several auxin-signaling mutants were more resistant to F. oxysporum. Chemical or genetic alteration of polar auxin transport also conferred increased pathogen resistance. Our results suggest that, similarly to many other pathogenic and nonpathogenic or beneficial soil organisms, F. oxysporum requires components of auxin signaling and transport to colonize the plant more effectively. Potential mechanisms of auxin signaling and transport-mediated F. oxysporum susceptibility are discussed.

The nodule autoregulation receptor kinase (GmNARK) of soybean (Glycine max) is essential for the systemic auto-regulation of nodulation. Based on quantitative reverse-transcriptase polymerase chain reaction, GmNARK is expressed to varying levels throughout the plant; the transcript was detected at high levels in mature leaves and roots but to a lesser extent in young leaves, shoot tips, and nodules. The transcript level was not significantly affected by Bradyrhizobium japonicum during the first week following inoculation. In addition, the activities of the promoters of GmNARK and Lotus japonicus HAR1, driving a β-glucuronidase (GUSPlus) reporter gene, were examined in stably transformed L. japonicus and transgenic hairy roots of soybean. Histochemical GUS activity in L. japonicus plants carrying either a 1.7-kb GmNARKpr∷GUS or 2.0-kb LjHAR1pr∷GUS construct was clearly localized to living cells within vascular bundles, especially phloem cells in leaves, stems, roots, and nodules. Phloem-specific expression also was detected in soybean hairy roots carrying these constructs. Our study suggests that regulatory elements required for the transcription of these orthologous genes are conserved. Moreover, rapid amplification of 5′ cDNA ends (5′ rapid amplification of cDNA ends) revealed two major transcripts of GmNARK potentially originating from two TATA boxes. Further analysis of the GmNARK promoter has confirmed that these two TATA boxes are functional. Deletion analysis also located a region controlling phloem-specific expression to a DNA sequence between 908 bp and 1.7 kb upstream of the translation start site of GmNARK.

Microalgal biomass can be used for biodiesel, feed and food production. Collection and identification of local microalgal strains in the Northern Territory – Australia was conducted to identify strains with high protein and lipid contents as potential feedstock for animal feed and biodiesel production, respectively. A total of 36 strains were isolated from 13 samples collected from a variety of freshwater locations, such as dams, ponds and streams and subsequently classified by 18S rDNA sequencing. All of the strains were green microalgae and predominantly belong to Chlorella sp., Scenedesmus sp., Desmodesmus sp., Chlamydomonas sp., Pseudomuriella sp., Tetraedron caudatum, Graesiella emersonii and Mychonastes timauensis. Among the fastest growing strains, Scenedesmus sp. NT1d possessed the highest content of protein; reaching up to 33% of its dry weight. In terms of lipid production, Chlorella sp. NT8a and Scenedesmus dimorphus NT8e produced the highest triglyceride contents of 116.9 µg mL-1 culture and 99.13 µg mL-1, respectively, as measured by gas chromatography-mass spectroscopy (GC-MS) of fatty acid methyl esters (FAMEs). These strains may present suitable candidates for biodiesel production after further optimization of culturing conditions, while their protein-rich biomass could be used for animal feed.

Microalgal lipid recovery for biodiesel production is currently considered suboptimal, but pre-treatment of algal biomass, the use of solvent mixtures and the positioning of transesterification can lead to increased yields. Here, the effect of various reportedly successful pre-treatments and solvent mixtures were directly compared to each other and combined with direct and indirect transesterification methods using the oleaginous microalga Tetraselmis sp. M8. Microwave and thermal pre-treatments were applied and the total lipid and fatty acid methyl ester (FAME) recoveries were investigated. The application of pre-treatments increased FAME recovery through indirect transesterification when a Soxhlet system was used but they had no significant effect for direct transesterification. Gravimetric analyses of total lipids revealed that lipid recovery was highest when utilizing the chloroform-based Bligh and Dyer extraction method; however FAME yield was the highest when applying a Soxhlet system utilizing a solvent mixture of hexane-ethanol (3:1). Total lipid recovery did not necessarily correlate with the recovery of FAMEs. The highest FAME recovery was achieved from thermal or microwave pre-treated biomass followed by indirect transesterification through Soxhlet extraction. FAME recovery could be more than doubled (increase of up to 171%) under these conditions. We conclude that a simple thermal pre-treatment (80°C for 10 min) in combination with solvent mixture extraction through indirect transesterification may present a cost-effective and scalable option for large-scale lipid extraction from microalgae.

In vitro evolution imitates the natural evolution of genes and has been very successfully applied to the modification of coding sequences, but it has not yet been applied to promoter sequences. We propose an alternative method for functional promoter analysis by applying an in vitro evolution scheme consisting of rounds of error-prone PCR, followed by DNA shuffling and selection of mutant promoter activities. We modified the activity in embryogenic sugarcane cells of the promoter region of the “Goldfinger” isolate of banana streak virus and obtained mutant promoter sequences that showed an average mutation rate of 2.5% after applying one round of error-prone PCR and DNA shuffling. Selection and sequencing of promoter sequences with decreased or unaltered activity allowed us to rapidly map the position of one cis-acting element that influenced promoter activity in embryogenic sugarcane cells and to discover neutral mutations that did not affect promoter function. The “selective-shotgun” approach of this promoter analysis method immediately after the promoter boundaries have been defined by 5′ deletion analysis dramatically reduces the labor associated with traditional “linker-scanning” deletion analysis to reveal the position of functional promoter domains. Furthermore, this method allows the entire promoter to be investigated at once, rather than selected domains or nucleotides, increasing the prospect of identifying interacting promoter regions.

Plants in natural and agricultural environments are continuously exposed to a plethora of diverse microorganisms resulting in microbial colonization of roots and the rhizosphere. This process is believed to be accompanied by an intricate network of ongoing simultaneous interactions. In this study, we examined Arabidopsis thaliana roots and shoots in the presence or absence of whole microbial communities extracted from compost soil. The results show a clear growth promoting effect on Arabidopsis shoots in the presence of soil microbes compared to plants grown in microbe-free soil under otherwise identical conditions. Element analyses showed that iron uptake was facilitated by these mixed microbial communities which also led to transcriptional downregulation of genes required for iron transport. In addition, soil microbial communities suppressed the expression of marker genes involved in nitrogen uptake, oxidative stress/redox signaling, cell wall modification and salicylic acid (SA)-mediated plant defense while upregulating jasmonate (JA) signaling and photosynthesis. Multi-species analyses such as simultaneous transcriptiptional profiling of plants and their interacting microorganisms (metatransciptomics) coupled to metagenomics may further increase our understanding of the intricate networks underlying plant-microbe interactions in their diverse environments.

Reactive oxygen species (ROS) are produced in plant cells in response to diverse biotic and abiotic stresses as well as during normal growth and development. Although a large number of transcription factor (TF) genes are up- or down-regulated by ROS, currently very little is known about the functions of these TFs during oxidative stress. In this work, we examined the role of ERF6 (ETHYLENE RESPONSE FACTOR6), an AP2/ERF domain-containing TF, during oxidative stress responses in Arabidopsis. Mutant analyses showed that NADPH oxidase (RbohD) and calcium signaling are required for ROS-responsive expression of ERF6. erf6 insertion mutant plants showed reduced growth and increased H2O2 and anthocyanin levels. Expression analyses of selected ROS-responsive genes during oxidative stress identified several differentially expressed genes in the erf6 mutant. In particular, a number of ROS responsive genes, such as ZAT12, HSFs, WRKYs, MAPKs, RBOHs, DHAR1, APX4, and CAT1 were more strongly induced by H2O2 in erf6 plants than in wild-type. In contrast, MDAR3, CAT3, VTC2 and EX1 showed reduced expression levels in the erf6 mutant. Taken together, our results indicate that ERF6 plays an important role as a positive antioxidant regulator during plant growth and in response to biotic and abiotic stresses.

Jasmonic acid (JA) signalling plays a central role in plant defences against necrotrophic pathogens and herbivorous insects, which afflict both roots and shoots. This pathway is also activated following the interaction with beneficial microbes that may lead to induced systemic resistance. Activation of the JA signalling pathway via application of methyl jasmonate (MeJA) alters the composition of carbon containing compounds released by roots, which are implicated as key determinants of rhizosphere microbial community structure. In this study, we investigated the influence of the JA defence signalling pathway activation in Arabidopsis thaliana on the structure of associated rhizosphere bacterial communities using 16S rRNA gene amplicon pyrosequencing. Application of MeJA did not directly influence bulk soil microbial communities but significant changes in rhizosphere community composition were observed upon activation of the jasmonate signalling pathway. Our results suggest that JA signalling may mediate plant-bacteria interactions in the soil upon necrotrophic pathogen and herbivorous insect attacks.

Microalgae have been widely reported as a promising source of biofuels, mainly based on their high areal productivity of biomass and lipids as triacylglycerides and the possibility for cultivation on non-arable land. The isolation and selection of suitable strains that are robust and display high growth and lipid accumulation rates is an important prerequisite for their successful cultivation as a bioenergy source, a process that can be compared to the initial selection and domestication of agricultural crops. We developed standard protocols for the isolation and cultivation for a range of marine and brackish microalgae. By comparing growth rates and lipid productivity, we assessed the potential of subtropical coastal and brackish microalgae for the production of biodiesel and other oil-based bioproducts. This study identified Nannochloropsis sp., Dunaniella salina and new isolates of Chlorella sp. and Tetraselmis sp. as suitable candidates for a multiple-product algae crop. We conclude that subtropical coastal microalgae display a variety of fatty acid profiles that offer a wide scope for several oil-based bioproducts, including biodiesel and omega-3 fatty acids. A biorefinery approach for microalgae would make economical production more feasible but challenges remain for efficient harvesting and extraction processes for some species.

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1's primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.

Organic fertilizers and microbial biofertilizers are now widely recognized to effectively complement traditional mineral fertilizers for plant growth. The present study shows that bio-organic fertilizers can be enhanced by the addition of functional plant-growth-promoting rhizobacteria (PGPR) that provide additional benefits to plants. We hypothesized that not all beneficial soil bacteria are functional in different farm soils and plant varieties; hence, the most effective PGPR that are suitable to each farm's individual cropping conditions were selected. Five different field soils and their respective crops were tested for compatibility with six microbial biofertilizers (including three new bacterial strains) to supplement a commercially available bio-organic fertilizer. In pot trials with lucerne plants, four out of the six microbial treatments led to significant (p < 0.05) growth promotion benefits (up to 79.8% more leaves and dry weight) compared to mock-treated or bio-organic fertilizer-only-treated control plants. A trial with industrial hemp demonstrated that compatibility with PGPR occurs in a cultivar-specific manner, leading to growth promotion ranging from −3.4% to 68.9%, with each cultivar displaying a preference for a different PGPR. Finally, pot trials with Rhodes grass and two different soils demonstrated high yield increases compared to control plants, with Bacillus amyloliquefaciens 33YE being most effective for one soil and Bacillus velezensis UQ9000N/Pseudomonas lini SMX2 for the other soil. Yield advantages reduced after several cuts of grass, but a repeat biofertilizer treatment at 69 days after the initial treatment restored high yield advantages, with the same PGPR again being most effective. These results demonstrate the importance of customization of microbial inoculants to identify the most compatible PGPR–cultivar–soil interaction. The customization of microbial biofertilizers to soils and plant cultivars, combined with complementary fertilizer applications, can potentially lead to more reliable and more sustainable agricultural practices. [ABSTRACT FROM AUTHOR]

Background: Na+,HCO3–-cotransporter NBCn1/Slc4a7 accelerates murine breast carcinogenesis. Lack of specific pharmacological tools previously restricted therapeutic targeting of NBCn1 and identification of NBCn1-dependent functions in human breast cancer. Methods: We develop extracellularly-targeted anti-NBCn1 antibodies, screen for functional activity on cells, and evaluate (a) mechanisms of intracellular pH regulation in human primary breast carcinomas, (b) proliferation, cell death, and tumor growth consequences of NBCn1 in triple-negative breast cancer, and (c) association of NBCn1-mediated Na+,HCO3–-cotransport with human breast cancer metastasis. Results: We identify high-affinity (KD ≈ 0.14 nM) anti-NBCn1 antibodies that block human NBCn1-mediated Na+,HCO3–-cotransport in cells, without cross-reactivity towards human NBCe1 or murine NBCn1. These anti-NBCn1 antibodies abolish Na+,HCO3–-cotransport activity in freshly isolated primary organoids from human breast carcinomas and lower net acid extrusion effectively in primary breast cancer tissue from patients with macrometastases in axillary lymph nodes. Inhibitory anti-NBCn1 antibodies decelerate tumor growth in vivo by ~50% in a patient-derived xenograft model of triple-negative breast cancer and pH-dependently reduce colony formation, cause G2/M-phase cell cycle accumulation, and increase apoptosis of metastatic triple-negative breast cancer cells in vitro. Conclusions: Inhibitory anti-NBCn1 antibodies block net acid extrusion in human breast cancer tissue, particularly from patients with disseminated disease, and pH-dependently limit triple-negative breast cancer growth. [ABSTRACT FROM AUTHOR]