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.

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.

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.

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.

Metallothionein from Wild Populations of the African Catfish Clarias gariepinus: From Sequence, Protein Expression and Metal Binding Properties to Transcriptional Biomarker of Metal Pollution.

Anthropogenic pollution with heavy metals is an on-going concern throughout the world, and methods to monitor release and impact of heavy metals are of high importance. With a view to probe its suitability as molecular biomarker of metal pollution, this study has determined a coding sequence for metallothionein of the African sharptooth catfish Clarias gariepinus. The gene product was recombinantly expressed in Escherichia coli in presence of Zn(II), Cd(II), or Cu, and characterised by Electrospray Ionisation Mass Spectrometry and elemental analysis. C. gariepinus MT displays typical features of fish MTs, including 20 conserved cysteines, and seven bound divalent cations (Zn(II) or Cd(II)) when saturated. Livers from wild C. gariepinus fish collected in all three seasons from four different sites on the Kafue River of Zambia were analysed for their metal contents and for MT expression levels by quantitative PCR. Significant correlations were found between Zn and Cu levels and MT expression in livers, with MT expression clearly highest at the most polluted site, Chililabombwe, which is situated in the Copperbelt region. Based on our findings, hepatic expression of MT from C. gariepinus may be further developed as a major molecular biomarker of heavy metal pollution resulting from mining activities in this region.

Cloning and biochemical characterization of indole-3-acetic acid-amino acid synthetase PsGH3 from pea.

Phytohormone conjugation is one of the mechanisms that maintains a proper hormonal homeostasis and that is necessary for the realization of physiological responses. Gretchen Hagen 3 (GH3) acyl acid amido synthetases convert indole-3-acetic acid (IAA) to IAA-amino acid conjugates by ATP-dependent reactions. IAA-aspartate (IAA-Asp) exists as a predominant amide conjugate of auxin in pea tissues and acts as an intermediate during IAA catabolism. Here we report a novel recombinant indole-3-acetic acid-amido synthetase in Pisum sativum. In silico analysis shows that amino acid sequence of PsGH3 has the highest homology to Medicago truncatula GH3.3. The recombinant His-tag-PsGH3 fusion protein has been obtained in E. coli cells and is a soluble monomeric polypeptide with molecular mass of 69.18 kDa. The PsGH3 was purified using Ni(2+)-affinity chromatography and native PAGE. Kinetic analysis indicates that the enzyme strongly prefers IAA and L-aspartate as substrates for conjugation revealing Km(ATP) = 0.49 mM, Km(L-Asp) = 2.2 mM, and Km(IAA) = 0.28 mM. Diadenosine pentaphosphate (Ap5A) competes with ATP for catalytic site and diminishes the PsGH3 affinity toward ATP approximately 1.11-fold indicating Ki = 8.5 µM. L-Tryptophan acts as an inhibitor of IAA-amido synthesizing activity by competition with L-aspartate. Inorganic pyrophosphatase (PPase) hydrolyzing pyrophosphate to two phosphate ions, potentiates IAA-Asp synthetase activity of PsGH3. Our results demonstrate that PsGH3 is a novel enzyme that is involved in auxin metabolism in pea seeds.