Drought Stress Responses in Arabica Coffee Genotypes: Physiological and Metabolic Insights.

Understanding the impact of drought stress on Arabica coffee physiology and metabolism is essential in the pursuit of developing drought-resistant varieties. In this study, we explored the physiological and metabolite changes in coffee genotypes exhibiting varying degrees of tolerance to drought-namely, the relatively tolerant Ca74110 and Ca74112, and the sensitive Ca754 and CaJ-19 genotypes-under well-watered conditions and during terminal drought stress periods at two time points (0 and 60 days following the onset of stress). The metabolite profiling uncovered significant associations between the growth and the physiological characteristics of coffee genotypes with distinct drought tolerance behaviors. Initially, no marked differences were observed among the genotypes or treatments. However, at the 60-day post-drought onset time point, notably higher shoot growth, biomass, CO2 assimilation, pigments, and various physiological parameters were evident, particularly in the relatively tolerant genotypes. The metabolite profiling revealed elevations in glucose, maltose, amino acids, and organic acids, and decreases in other metabolites. These alterations were more pronounced in the drought-tolerant genotypes, indicating a correlation between enhanced compatible solutes and energy-associated metabolites crucial for drought tolerance mechanisms. This research introduces GC-MS-based metabolome profiling to the study of Ethiopian coffee, shedding light on its intricate responses to drought stress and paving the way for the potential development of drought-resistant coffee seedlings in intensified agro-ecological zones.

Characterization of the Metallothionein Gene Family in Avena sativa L. and the Gene Expression during Seed Germination and Heavy Metal Stress.

Metallothioneins (MTs) are a family of small proteins rich in cysteine residues. The sulfhydryl group of metallothioneins can bind to metal ions, maintaining metal homeostasis and protecting the cells from damage caused by toxic heavy metals. Moreover, MTs can function as reactive oxygen species scavengers since cysteine thiols undergo reversible and irreversible oxidation. Here, we identified 21 metallothionein genes (AsMTs) in the oat (Avena sativa L.) genome, which were divided into four types depending on the amino acid sequences of putative proteins encoded by identified genes. Analysis of promoter sequences showed that MTs might respond to a variety of stimuli, including biotic and abiotic stresses and phytohormones. The results of qRT-PCR showed that all four types of AsMTs are differentially expressed during the first 48 hours of seed germination. Moreover, stress induced by the application of zinc, cadmium, and a mixture of zinc and cadmium affects the expression of oat MTs variously depending on the MT type, indicating that AsMT1-4 fulfil different roles in plant cells.

Application Potential of Trichoderma in the Degradation of Phenolic Acid-Modified Chitosan.

The aim of the study was to determine the potential use of fungi of the genus Trichoderma for the degradation of phenolic acid-modified chitosan in compost. At the same time, the enzymatic activity in the compost was checked after the application of a preparation containing a suspension of the fungi Trichoderma (spores concentration 105/mL). The Trichoderma strains were characterized by high lipase and aminopeptidase activity, chitinase, and ß-1,3-glucanases. T. atroviride TN1 and T. citrinoviride TN3 metabolized the modified chitosan films best. Biodegradation of modified chitosan films by native microorganisms in the compost was significantly less effective than after the application of a formulation composed of Trichoderma TN1 and TN3. Bioaugmentation with a Trichoderma preparation had a significant effect on the activity of all enzymes in the compost. The highest oxygen consumption in the presence of chitosan with tannic acid film was found after the application of the consortium of these strains (861 mg O2/kg after 21 days of incubation). Similarly, chitosan with gallic acid and chitosan with ferulic acid were found after the application of the consortium of these strains (849 mgO2/kg and 725 mg O2/kg after 21 days of incubation). The use of the Trichoderma consortium significantly increased the chitinase activity. The application of Trichoderma also offers many possibilities in sustainable agriculture. Trichoderma can not only degrade chitosan films, but also protect plants against fungal pathogens by synthesizing chitinases and ß-1,3 glucanases with antifungal properties.

Trichoderma viride Colonizes the Roots of Brassica napus L., Alters the Expression of Stress-Responsive Genes, and Increases the Yield of Canola under Field Conditions during Drought.

In this work, we present the results of the inoculation of canola seeds (Brassica napus L.) with Trichoderma viride strains that promote the growth of plants. Seven morphologically different strains of T. viride (TvI-VII) were shown to be capable of synthesizing auxins and exhibited cellulolytic and pectinolytic activities. To gain a deeper insight into the molecular mechanisms underlying canola-T. viride interactions, we analyzed the canola stress genes metallothioneins (BnMT1-3) and stringent response genes (BnRSH1-3 and BnCRSH). We demonstrated the presence of cis-regulatory elements responsive to fungal elicitors in the promoter regions of B. napus MT and RSH genes and observed changes in the levels of the transcripts of the above-mentioned genes in response to root colonization by the tested fungal strains. Of the seven tested strains, under laboratory conditions, T. viride VII stimulated the formation of roots and the growth of canola seedlings to the greatest extent. An experiment conducted under field conditions during drought showed that the inoculation of canola seeds with a suspension of T. viride VII spores increased yield by 16.7%. There was also a positive effect of the fungus on the height and branching of the plants, the number of siliques, and the mass of a thousand seeds. We suggest that the T. viride strain TvVII can be used in modern sustainable agriculture as a bioinoculant and seed coating to protect B. napus from drought.

New seed coating containing Trichoderma viride with anti-pathogenic properties.

Background: To ensure food security in the face of climate change and the growing world population, multi-pronged measures should be taken. One promising approach uses plant growth-promoting fungi (PGPF), such as Trichoderma, to reduce the usage of agrochemicals and increase plant yield, stress tolerance, and nutritional value. However, large-scale applications of PGPF have been hampered by several constraints, and, consequently, usage on a large scale is still limited. Seed coating, a process that consists of covering seeds with low quantities of exogenous materials, is gaining attention as an efficient and feasible delivery system for PGPF. Methods: We have designed a new seed coating composed of chitin, methylcellulose, and Trichoderma viride spores and assessed its effect on canola (Brassica napus L.) growth and development. For this purpose, we analyzed the antifungal activity of T. viride against common canola pathogenic fungi (Botrytis cinerea, Fusarium culmorum, and Colletotrichum sp.). Moreover, the effect of seed coating on germination ratio and seedling growth was evaluated. To verify the effect of seed coating on plant metabolism, we determined superoxide dismutase (SOD) activity and expression of the stress-related RSH (RelA/SpoT homologs). Results: Our results showed that the T. viride strains used for seed coating significantly restricted the growth of all three pathogens, especially F. culmorum, for which the growth was inhibited by over 40%. Additionally, the new seed coating did not negatively affect the ability of the seeds to complete germination, increased seedling growth, and did not induce the plant stress response. To summarize, we have successfully developed a cost-effective and environmentally responsible seed coating, which will also be easy to exploit on an industrial scale.

Changes in physio-biochemical parameters and expression of metallothioneins in Avena sativa L. in response to drought.

Drought is one of the major threats to food security. Among several mechanisms involved in plant stress tolerance, one protein family-the plant metallothioneins (MTs)-shows great promise for enhancing drought resistance. Plant metallothioneins in oat (Avena sativa L.) have not yet been deeply analysed, and the literature lacks a comprehensive study of the whole family of plant MTs in response to drought. In this study, we showed that the number and nature of cis-elements linked with stress response in promoters of AsMTs1-3 differed depending on the MT type. Drought stress in oat plants caused an increase in the expression of AsMT2 and AsMT3 and a decrease in the expression of AsMT1 compared to well-watered plants. Moreover, the low values of relative water content, water use efficiency, net photosynthesis (PN), transpiration (E), stomatal conductance (gs), chlorophyll a, and carotenoid were accompanied by high levels of electrolyte leakage, internal CO2 concentration (Ci) and abscisic acid content, and high activity of antioxidants enzymes in plants under drought stress. The present study puts forward the idea that AsMTs are crucial for oat response to drought stress not only by regulating antioxidant activity but also by changing the plant water regime and photosynthesis. Our results support the hypothesis that structural differences among types of plant MTs reflect their diversified physiological roles.

The Importance of Microorganisms for Sustainable Agriculture-A Review.

In the face of climate change, progressive degradation of the environment, including agricultural land negatively affecting plant growth and development, endangers plant productivity. Seeking efficient and sustainable agricultural techniques to replace agricultural chemicals is one of the most important challenges nowadays. The use of plant growth-promoting microorganisms is among the most promising approaches; however, molecular mechanisms underneath plant-microbe interactions are still poorly understood. In this review, we summarized the knowledge on plant-microbe interactions, highlighting the role of microbial and plant proteins and metabolites in the formation of symbiotic relationships. This review covers rhizosphere and phyllosphere microbiomes, the role of root exudates in plant-microorganism interactions, the functioning of the plant's immune system during the plant-microorganism interactions. We also emphasized the possible role of the stringent response and the evolutionarily conserved mechanism during the established interaction between plants and microorganisms. As a case study, we discussed fungi belonging to the genus Trichoderma. Our review aims to summarize the existing knowledge about plant-microorganism interactions and to highlight molecular pathways that need further investigation.

Birch Tar Introduced into Polylactide and Its Influence on the Barrier, Thermal, Functional and Biological Properties of the Film Obtained by Industrial Extrusion.

The aim of the study was to evaluate possibility of producing a polylactide film with birch tar by the industrial extrusion method and whether the addition of 10% birch tar can ensure adequate biocidal properties of PLA against pathogenic microorganisms (E. coli, S. aureus, P. aeruginosa, A. tumefaciens, X. campestris, P. brassicacearum, P. corrugate and P. syringae) and fungi (A. niger, A. flavus and A. versicolor) while ensuring beneficial functional properties, such as water vapor, nitrogen, oxygen and carbon dioxide permeability, which are of considerable importance in the packaging industry. The main test methods used were ISO 22196, ISO 846, ISO 2556, ASTM F 1927 and ASTM F 2476-20. The obtained results prove the possibility of extruding polymer films with a biocidal additive, i.e., birch tar, and obtaining favorable properties that qualify the produced film for applications in the packaging industry.

Biodegradability of Novel Polylactide and Polycaprolactone Materials with Bacteriostatic Properties Due to Embedded Birch Tar in Different Environments.

The microbial biodegradation of new PLA and PCL materials containing birch tar (1-10% v/v) was investigated. Product of dry distillation of birch bark (Betula pendula Roth) was added to polymeric materials to obtain films with antimicrobial properties. The subject of the study was the course of enzymatic degradation of a biodegradable polymer with antibacterial properties. The results show that the type of the material, tar concentration, and the environment influenced the hydrolytic activity of potential biofilm degraders. In the presence of PCL films, the enzyme activities were higher (except for α-D-glucosidase) compared to PLA films. The highest concentration of birch tar (10% v/v) decreased the activity of hydrolases produced by microorganisms to the most significant extent; however, SEM analysis showed the presence of a biofilm even on plastics with the highest tar content. Based on the results of the biological oxygen demand (BOD), the new materials can be classified as biodegradable but, the biodegradation process was less efficient when compared to plastics without the addition of birch tar.

In Silico Study of the RSH (RelA/SpoT Homologs) Gene Family and Expression Analysis in Response to PGPR Bacteria and Salinity in Brassica napus.

Among several mechanisms involved in the plant stress response, synthesis of guanosine tetra and pentaphosphates (alarmones), homologous to the bacterial stringent response, is of crucial importance. Plant alarmones affect, among others, photosynthetic activity, metabolite accumulation, and nutrient remobilization, and thus regulate plant growth and development. The plant RSH (RelA/SpoT homolog) genes, that encode synthetases and/or hydrolases of alarmones, have been characterized in a limited number of plant species, e.g., Arabidopsis thaliana, Oryza sativa, and Ipomoea nil. Here, we used dry-to-wet laboratory research approaches to characterize RSH family genes in the polyploid plant Brassica napus. There are 12 RSH genes in the genome of rapeseed that belong to four types of RSH genes: 6 RSH1, 2 RSH2, 3 RSH3, and 1 CRSH. BnRSH genes contain 13-24 introns in RSH1, 2-6 introns in RSH2, 1-6 introns in RSH3, and 2-3 introns in the CRSH genes. In the promoter regions of the RSH genes, we showed the presence of regulatory elements of the response to light, plant hormones, plant development, and abiotic and biotic stresses. The wet-lab analysis showed that expression of BnRSH genes is generally not significantly affected by salt stress, but that the presence of PGPR bacteria, mostly of Serratia sp., increased the expression of BnRSH significantly. The obtained results show that BnRSH genes are differently affected by biotic and abiotic factors, which indicates their different functions in plants.

Potential of Serratia plymuthica IV-11-34 strain for biodegradation of polylactide and poly(ethylene terephthalate).

Serratia plymuthica strain IV-11-34 belongs to the plant growth promoting bacteria (PGPR). In the sequenced genome of S. plymuthica IV-11-34, we have identified the genes involved in biodegradation and metabolisms of xenobiotics. The potential of S. plymuthica IV-11-34 for the degradation of biodegradable aliphatic polyester polylactide (PLA) and resistant to biodegradation - poly(ethylene terephthalate) (PET) was assessed by biochemical oxygen consumption (BOD) and carbon dioxide methods. After seven days of growth, the bacteria strain showed more than 80% and 60% increase in respiratory activity in the presence of PLA and PET, respectively. We assume that during biodegradation, S. plymuthica IV-11-34 colonise the surface of PLA and PET, since the formation of a biofilm on the surface of polymers was shown by the LIVE/DEAD method. We have demonstrated for the relA gene, which is an alarmone synthetase, a 1.2-fold increase in expression in the presence of PLA, and a 4-fold decrease in expression in the presence of PET for the spoT gene, which is a hydrolase of alarmones. Research has shown that the bacterium has the ability to biodegrade PLA and PET, and the first stage of this process involves bacterial stringent response genes responsible for survival under extreme conditions.

Comparative Study of Structural Changes of Polylactide and Poly(ethylene terephthalate) in the Presence of Trichoderma viride.

Plastic pollution is one of the crucial global challenges nowadays, and biodegradation is a promising approach to manage plastic waste in an environment-friendly and cost-effective way. In this study we identified the strain of fungus Trichoderma viride GZ1, which was characterized by particularly high pectinolytic activity. Using differential scanning calorimetry, Fourier-transform infrared spectroscopy techniques, and viscosity measurements we showed that three-month incubation of polylactide and polyethylene terephthalate in the presence of the fungus lead to significant changes of the surface of polylactide. Further, to gain insight into molecular mechanisms underneath the biodegradation process, western blot hybridization was used to show that in the presence of poly(ethylene terephthalate) (PET) in laboratory conditions the fungus produced hydrophobin proteins. The mycelium adhered to the plastic surface, which was confirmed by scanning electron microscopy, possibly due to the presence of hydrophobins. Further, using atomic force microscopy we demonstrated for the first time the formation of hydrophobin film on the surface of aliphatic polylactide (PLA) and PET by T. viride GZ1. This is the first stage of research that will be continued under environmental conditions, potentially leading to a practical application.

Combined use of Bacillus strains and Miscanthus for accelerating biodegradation of poly(lactic acid) and poly(ethylene terephthalate).

BACKGROUND: The aim of this study was to verify whether the presence of Bacillus strains and of miscanthus influence biodegradation and formed of biofilm of poly(lactic acid) (PLA) and poly(ethylene terephthalate) (PET). METHODS: The experiment conducted in compost soil showed that strains Bacillus subtilis and Bacillus cereus isolated from heavy metal contaminated environment have biochemical activity and accelerate biodegradation of both plastic materials. RESULTS: For PLA film it was found that the carbonyl index dropped by over 15% in the presence of B. subtilis, while the film tensile strength decreased by 35% and the oxygen to carbon O/C ratio was higher by 3% in the presence of B. cereus, and the presence of miscanthus resulted in a loss of weight. For PET film, a decrease in the carbonyl index by 16% was observed following inoculation with B. cereus. The metabolic activity of this strain contributed to the reduction of the film's tensile strength by 17% and to the increase in the permeability to O2 and CO2. The most intense degradation of PET film was observed in the presence of bacteria and plants. B. subtilis strain combined with miscanthus plantings may be a promising method for accelerating PLA and PET degradation in compost soil.

Enzymatic degradation and biofilm formation during biodegradation of polylactide and polycaprolactone polymers in various environments.

The present article presents the results of research on the susceptibility of polylactide, poly(ɛ-caprolactone) and mixtures to biodegradation in conditions imitating natural extracts of compost, activated sludge, sea and river water, determined by the biochemical oxygen consumption by microorganisms and susceptibility to enzymatic degradation with the use of enzyme solutions of fungal microbial origin. Analyzes of both types of degradation were carried out over a period of seven days and in four environments: compost, activated sludge, river and sea water, and four enzymatic solutions containing proteinase K, protease, esterase, and lipase. The amount of oxygen consumed by microorganisms in the presence of the tested films was determined, as well as the weight loss determined after the samples were incubated in enzymatic solutions. Images of the surface of individual samples, taken by fluorescence microscopy and scanning electron microscopy, confirm the formation of bacterial biofilm and the results of biochemical oxygen consumption by microorganisms, or weight loss. It was shown that the compost and activated sludge extract as well as the enzymes proteinase K from Engyodontium album (synonym Tritirachium album) and protease from Bacillus licheniformis had the greatest impact on the biodegradation of the tested materials.

The Role of Birch Tar in Changing the Physicochemical and Biocidal Properties of Polylactide-Based Films.

The objective of this study was to produce bactericidal polymer films containing birch tar (BT). The produced polymer films contain PLA, plasticiser PEG (5% wt.) and birch tar (1, 5 and 10% wt.). Compared to plasticised PLA, films with BT were characterised by reduced elongation at break and reduced water vapour permeability, which was the lowest in the case of film with 10% wt. BT content. Changes in the morphology of the produced materials were observed by performing scanning electron microscopy (SEM) and atomic force microscopy (AFM) analysis; the addition of BT caused the surface of the film to be non-uniform and to contain recesses. FTIR analysis of plasticised PLA/BT films showed that the addition of birch tar did not change the crystallinity of the obtained materials. According to ISO 22196: 2011, the PLA film with 10% wt. BT content showed the highest antibacterial effect against the plant pathogens A. tumefaciens, X. campestris, P. brassicacearum, P. corrugata, P. syringae. It was found that the introduction of birch tar to plasticised PLA leads to a material with biocidal effect and favourable physicochemical and structural properties, which classifies this material for agricultural and horticultural applications.

Identification and characterization of the Ipomoea nil RelA/SpoT Homologs (InRSHs) and potential directions of their transcriptional regulation.

Although the stringent response has been known for more than half a century and has been well studied in bacteria, only the research of the past 19 years revealed that the homologous mechanism is conserved in plants. The plant RelA/SpoT Homolog (RSH) genes have been identified and characterized in a limited number of plant species, whereas products of their catalytic activities, (p)ppGpp (alarmones), have been shown to accumulate mainly in chloroplasts. Here, we identified full-length sequences of the Ipomoea nil RSH genes (InRSH1, InRSH2 and InCRSH), determined their copy number in the I. nil genome as well as the structural conservancy between InRSHs and their Arabidopsis and rice orthologs. We showed that InRSHs are differentially expressed in I. nil organ tissues and that only InRSH2 is upregulated in response to salt, osmotic and drought stress. Our results of the E. coli relA/spoT mutant complementation test suggest that InRSH1 is likely a (p)ppGpp hydrolase, InCRSH - synthetase and InRSH2 shows both activities. Finally, we referred our results to the recently published I. nil genomic and proteomic data and uncovered the complexity of the I. nil RSH family as well as potential ways of the InRSH transcriptional regulation.

The type 4 metallothionein from Brassica napus seeds folds in a metal-dependent fashion and favours zinc over other metals.

The problem of handling zinc in the cell is of great importance because zinc is an indispensable micronutrient involved in most physiological processes in all living organisms. Moreover, our understanding of mechanisms governing the discrimination between micronutrients and toxic metals on the level of individual proteins to the whole-organism level is incomplete. Metallothioneins are able to bind heavy metal ions, and roles in zinc homeostasis have been proposed. Here, we have studied the in vitro and in vivo metal-binding abilities of Brassica napus type 4 metallothionein (BnMT4) and its expression in germinating seeds in response to metal treatment. Our studies on the regulation of MT4 expression by metals at early stages of ontogenic development have revealed for the first time that the mRNA levels of BnMT4 were elevated in response to cadmium and zinc. Given this unexpected metalloregulation, and the dramatic differences in protein folding as detected by 1H NMR spectroscopy, we suggest that the BnMT4 protein may not only have a role in zinc homeostasis in early ontogenesis, but also the potential to discriminate between zinc and cadmium, perhaps via differential recognition of Cd- and Zn-complexes by cellular components involved in protein turnover.

Agents of swimmer's itch-dangerous minority in the Digenea invasion of Lymnaeidae in water bodies and the first report of Trichobilharzia regenti in Poland.

Trichobilharzia spp. have been identified as a causative agent of swimmers' itch, a skin disease provoked by contact with these digenean trematodes in water. These parasites have developed a number of strategies to invade vertebrates. Since we have little understanding of the behavior of these parasites inside the human body, the monitoring of their invasion in snail host populations is highly recommended. In our research, lymnaeid snails were collected from several Polish lakes for two vegetation seasons. The prevalence of bird schistosomes in snail host populations was significantly lower than that of other digenean species. We were the first to detect the presence of the snails emitted Trichobilharzia regenti (potentially the most dangerous nasal schistosome) in Poland. In addition, by sequencing partial rDNA genes, we confirmed the presence of the snails positive with Trichobilharzia szidati in Polish water bodies, showing that swimmer's itch is more frequent during summer months and that large snails are more often infected with bird schistosomes than small ones.

Within and beyond the stringent response-RSH and (p)ppGpp in plants.

MAIN CONCLUSION: Plant RSH proteins are able to synthetize and/or hydrolyze unusual nucleotides called (p)ppGpp or alarmones. These molecules regulate nuclear and chloroplast transcription, chloroplast translation and plant development and stress response. Homologs of bacterial RelA/SpoT proteins, designated RSH, and products of their activity, (p)ppGpp-guanosine tetra-and pentaphosphates, have been found in algae and higher plants. (p)ppGpp were first identified in bacteria as the effectors of the stringent response, a mechanism that orchestrates pleiotropic adaptations to nutritional deprivation and various stress conditions. (p)ppGpp accumulation in bacteria decreases transcription-with exception to genes that help to withstand or overcome current stressful situations, which are upregulated-and translation as well as DNA replication and eventually reduces metabolism and growth but promotes adaptive responses. In plants, RSH are nuclei-encoded and function in chloroplasts, where alarmones are produced and decrease transcription, translation, hormone, lipid and metabolites accumulation and affect photosynthetic efficiency and eventually plant growth and development. During senescence, alarmones coordinate nutrient remobilization and relocation from vegetative tissues into seeds. Despite the high conservancy of RSH protein domains among bacteria and plants as well as the bacterial origin of plant chloroplasts, in plants, unlike in bacteria, (p)ppGpp promote chloroplast DNA replication and division. Next, (p)ppGpp may also perform their functions in cytoplasm, where they would promote plant growth inhibition. Furthermore, (p)ppGpp accumulation also affects nuclear gene expression, i.a., decreases the level of Arabidopsis defense gene transcripts, and promotes plants susceptibility towards Turnip mosaic virus. In this review, we summarize recent findings that show the importance of RSH and (p)ppGpp in plant growth and development, and open an area of research aiming to understand the function of plant RSH in response to stress.