Clostridioides difficile spores are the infective form for this endospore-forming organism. The vegetative cells are intolerant to oxygen and poor competitors with a healthy gut microbiota. Therefore, in order for C. difficile to establish infection, the spores have to germinate in an environment that supports vegetative growth. To initiate germination, C. difficile uses Csp-type germinant receptors that consist of the CspC and CspA pseudoproteases as the bile acid and cogerminant receptors, respectively. CspB is a subtilisin-like protease that cleaves the inhibitory propeptide from the pro-SleC cortex lytic enzyme, thereby activating it and initiating cortex degradation. Though several locations have been proposed for where these proteins reside within the spore (i.e., spore coat, outer spore membrane, cortex, and inner spore membrane), these have been based, mostly, on hypotheses or prior data in Clostridium perfringens. In this study, we visualized the germination and outgrowth process using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and used immunogold labeling to visualize key germination regulators. These analyses localize these key regulators to the spore cortex region for the first time. IMPORTANCE Germination by C. difficile spores is the first step in the establishment of potentially life-threatening C. difficile infection (CDI). A deeper understanding of the mechanism by which spores germinate may provide insight for how to either prevent spore germination into a disease-causing vegetative form or trigger germination prematurely when the spore is either in the outside environment or in a host environment that does not support the establishment of colonization/disease.

For soilborne pathogens, germination of the resting or dormant propagule that enables persistence within the soil environment is a key point in pathogenesis. Spongospora subterranea is an obligate soilborne protozoan that infects the roots and tubers of potato causing root and powdery scab disease for which there are currently no effective controls. A better understanding of the molecular basis of resting spore germination of S. subterranea could be important for development of novel disease interventions. However, as an obligate biotroph and soil dwelling organism, the application of new omics techniques for the study of the pre-infection process in S. subterranea has been problematic. Here, RNA sequencing was used to analyse the reprogramming of S. subterranea resting spores during the transition to zoospores in an in-vitro model. More than 63 million mean high-quality reads per sample were generated from the resting and germinating spores. By using a combination of reference-based and de novo transcriptome assembly, 6,664 unigenes were identified. The identified unigenes were subsequently annotated based on known proteins using BLAST search. Of 5,448 annotated genes, 570 genes were identified to be differentially expressed during the germination of S. subterranea resting spores, with most of the significant genes belonging to transcription and translation, amino acids biosynthesis, transport, energy metabolic processes, fatty acid metabolism, stress response and DNA repair. The datasets generated in this study provide a basic knowledge of the physiological processes associated with spore germination and will facilitate functional predictions of novel genes in S. subterranea and other plasmodiophorids. We introduce several candidate genes related to the germination of an obligate biotrophic soilborne pathogen which could be applied to the development of antimicrobial agents for soil inoculum management.

An obvious relationship between germination sensu stricto and seedling development during post-germination has been considered, but not explained concerning vigor. Taking this into account, we used measurements of water dynamics in germinating seeds and seedling development to clarify that relationship. The biological model was soybean seeds, since it is the most relevant 'true seed' produced around world. Our findings suggest that the way energy is used (acceleration) and not its input (velocity) is the main aspect relating seed germination and seedling development, especially when considering vigor. However, velocity and acceleration can be complementary in analyses of seed physiology. Other measurements proposed here also have potential uses for testing vigor in seed lots, such as seedling vigor index and biological activity in the lot. Therefore, water dynamics in germinating seeds can be an interesting way for testing seed lots, because it is an easier, faster and cheaper method in relation to other non-destructive procedures.

Seed germination, a complex, physiological-morphogenetic process, is a critical stage in the life cycle of plants. Biological changes in germinating seeds have not been investigated in poplar, a model woody plant.

Arabidopsis exocyst subunit SEC3A has been reported to participate in embryo development. Here we report that SEC3A is involved during pollen germination. A T-DNA insertion in SEC3A leads to an absolute, male-specific transmission defect that can be complemented by the expression of SEC3A coding sequence from the LAT52 promoter or SEC3A genomic DNA. No obvious abnormalities in the microgametogenesis are observed in the sec3a/SEC3A mutant, however, in vitro and in vivo pollen germination are defective. Further studies reveal that the callose, pectin, and cellulose are apparently not deposited at the germination site during pollen germination. SEC3A is expressed ubiquitously, including in pollen grains and pollen tubes. Notably, SEC3A-GFP fusion proteins are specifically recruited to the future pollen germination site. This particular localization pattern is independent of phosphatidylinositol 4,5-bisphosphate (PI-4,5P2), although SEC3-HIS fusion proteins are able to bind to several phosphoinositols in vitro. These results suggest that SEC3A plays an important role in the establishment of the polar site for pollen germination.

Seed germination is inherently related to seed metabolism, which changes throughout its maturation, desiccation and germination processes. The metabolite content of a seed and its ability to germinate are determined by underlying genetic architecture and environmental effects during development.

Clostridioides difficile infections begin when its metabolically dormant spores germinate in response to sensing bile acid germinants alongside amino acid and divalent cation co-germinants in the small intestine. While bile acid germinants are essential for C. difficile spore germination, it is currently unclear whether both co-germinant signals are required. One model proposes that divalent cations, particularly Ca2+, are essential for inducing germination, while another proposes that either co-germinant class can induce germination. The former model is based on the finding that spores defective in releasing large stores of internal Ca2+ in the form of calcium dipicolinic acid (CaDPA) cannot germinate when germination is induced with bile acid germinant and amino acid co-germinant alone. However, since the reduced optical density of CaDPA-less spores makes it difficult to accurately measure their germination, we developed a novel automated, time-lapse microscopy-based germination assay to analyze CaDPA mutant germination at the single-spore level. Using this assay, we found that CaDPA mutant spores germinate in the presence of amino acid co-germinant and bile acid germinant. Higher levels of amino acid co-germinants are nevertheless required to induce CaDPA mutant spores to germinate relative to WT spores because CaDPA released by WT spores during germination can function in a feedforward loop to potentiate the germination of other spores within the population. Collectively, these data indicate that Ca2+ is not essential for inducing C. difficile spore germination because amino acid and Ca2+ co-germinant signals are sensed by parallel signaling pathways. IMPORTANCE Clostridioides difficile spore germination is essential for this major nosocomial pathogen to initiate infection. C. difficile spores germinate in response to sensing bile acid germinant signals alongside co-germinant signals. There are two classes of co-germinant signals: Ca2+ and amino acids. Prior work suggested that Ca2+ is essential for C. difficile spore germination based on bulk population analyses of germinating CaDPA mutant spores. Since these assays rely on optical density to measure spore germination and the optical density of CaDPA mutant spores is reduced relative to WT spores, this bulk assay is limited in its capacity to analyze germination. To overcome this limitation, we developed an automated image analysis pipeline to monitor C. difficile spore germination using time-lapse microscopy. With this analysis pipeline, we demonstrate that, although Ca2+ is dispensable for inducing C. difficile spore germination, CaDPA can function in a feedforward loop to potentiate the germination of neighboring spores.

Seed germination, the foundation of plant propagation, involves a series of changes at the molecular level. Poplar is a model woody plant, but the molecular events occurring during seed germination in this species are unclear.

Red light promotes germination after activating phytochrome phyB, which destabilizes the germination repressor PIF1. Early upon seed imbibition, canopy light, unfavorable for photosynthesis, represses germination by stabilizing PIF1 after inactivating phyB. Paradoxically, later upon imbibition, canopy light stimulates germination after activating phytochrome phyA. phyA-mediated germination is poorly understood and, intriguingly, is inefficient, compared to phyB-mediated germination, raising the question of its physiological significance. A genetic screen identified polyamine uptake transporter 2 (put2) mutants that overaccumulate polyamines, a class of antioxidant polycations implicated in numerous cellular functions, which we found promote phyA-mediated germination. In WT seeds, our data suggest that canopy light represses polyamines accumulation through PIF1 while red light promotes polyamines accumulation. We show that canopy light also downregulates PIF1 levels, through phyA; however, PIF1 reaccumulates rapidly, which limits phyA-mediated germination. High polyamines levels in decaying seeds bypass PIF1 repression of germination and stimulate phyA-mediated germination, suggesting an adaptive mechanism promoting survival when viability is compromised.

The transition from seed to seedling represents a critical developmental step in the life cycle of higher plants, dramatically affecting plant ontogenesis and stress tolerance. The release from dormancy to acquiring germination ability is defined by a balance of phytohormones, with the substantial contribution of abscisic acid (ABA), which inhibits germination. We studied the embryonic axis of Pisum sativum L. before and after radicle protrusion. Our previous work compared RNA sequencing-based transcriptomics in the embryonic axis isolated before and after radicle protrusion. The current study aims to analyze ABA-dependent gene regulation during the transition of the embryonic axis from the germination to post-germination stages. First, we determined the levels of abscisates (ABA, phaseic acid, dihydrophaseic acid, and neo-phaseic acid) using ultra-high-performance liquid chromatography-tandem mass spectrometry. Second, we made a detailed annotation of ABA-associated genes using RNA sequencing-based transcriptome profiling. Finally, we analyzed the DNA methylation patterns in the promoters of the PsABI3, PsABI4, and PsABI5 genes. We showed that changes in the abscisate profile are characterized by the accumulation of ABA catabolites, and the ABA-related gene profile is accompanied by the upregulation of genes controlling seedling development and the downregulation of genes controlling water deprivation. The expression of ABI3, ABI4, and ABI5, which encode crucial transcription factors during late maturation, was downregulated by more than 20-fold, and their promoters exhibited high levels of methylation already at the late germination stage. Thus, although ABA remains important, other regulators seems to be involved in the transition from seed to seedling.

Miscanthus is a leading second generation bio-energy crop. It is mostly rhizome propagated; however, the increasing use of seed is resulting in a greater need to investigate germination. Miscanthus seed are small, germination is often poor and carried out without sterilisation; therefore, automated methods applied to germination detection must be able to cope with, for example, thresholding of small objects, low germination frequency and the presence or absence of mould.

Rhodospirillum centenum is a Gram-negative alphaproteobacterium that is capable of differentiating into dormant cysts that are metabolically inactive and desiccation resistant. Like spores synthesized by many Gram-positive species, dormant R. centenum cysts germinate in response to an environmental signal, indicating that conditions favor survival and proliferation. Factors that induce germination are called germinants and are often both niche and species specific. In this study, we have identified photosynthesis as a niche-specific germinant for R. centenum cyst germination. Specifically, excitation of wild-type cysts suspended in a nutrient-free buffer with far-red light at >750 nm results in rapid germination. This is in stark contrast to mutant strains deficient in photosynthesis that fail to germinate upon exposure to far-red light under all assayed conditions. We also show that photosynthesis-induced germination occurs in a carbon- and nitrogen-free buffer even in strains that are deficient in carbon or nitrogen fixation. These results demonstrate that photosynthesis not only is necessary for germination but is itself sufficient for the germination of R. centenum cysts.IMPORTANCE Environmental cues that signal Gram-positive spores to germinate (termed germinants) have been identified for several Bacillus and Clostridium species. These studies showed that germinants are niche and species specific. For example, Clostridium difficile spores sense bile salts as a germinant as their presence informs these cells of an intestinal environment. Bacillus fastidiosus spores use uric acid as a germinant that is present in soil and poultry litter as this species inhabits poultry litter. It is evident from these studies that dormant cells sample their environment to assess whether conditions are advantageous for the propagation and survival of vegetative cells. To date, a limited number of germinants have been defined for only a few Gram-positive spore-forming species. Beyond that group, there is scant information on what cues signal dormant cells to exit dormancy. In our study, we show that the versatile Gram-negative photosynthetic bacterium Rhodospirillum centenum uses light-driven photosynthesis, and not the availability of nutrients, to trigger the germination of dormant cysts. This use of light-driven photosynthesis as a germinant is surprising as this species is also capable of growing under dark conditions using exogenous carbon sources for energy. Consequently, photosynthetic growth appears to be the preferred growth mechanism by this species.

Among opportunistically pathogenic filamentous fungi of the Aspergillus genus, Aspergillus fumigatus stands out as a drastically more prevalent cause of infection than others. Utilizing the zebrafish embryo model, we applied a combination of non-invasive real-time imaging and genetic approaches to compare the infectious development of A. fumigatus with that of the less pathogenic A. niger. We found that both species evoke similar immune cell migratory responses, but A. fumigatus is more efficiently phagocytized than A. niger. Though efficiently phagocytized, A. fumigatus conidia retains the ability to germinate and form hyphae from inside macrophages leading to serious infection even at relatively low infectious burdens. By contrast, A. niger appears to rely on extracellular germination, and rapid hyphal growth to establish infection. Despite these differences in the mechanism of infection between the species, galactofuranose mutant strains of both A. fumigatus and A. niger display attenuated pathogenesis. However, deficiency in this cell wall component has a stronger impact on A. niger, which is dependent on rapid extracellular hyphal growth. In conclusion, we uncover differences in the interaction of the two fungal species with innate immune cells, noticeable from very early stages of infection, which drive a divergence in their route to establishing infections.

The germination of Clostridium difficile spores is an important stage of the C. difficile life cycle. In other endospore-forming bacteria, the composition of the medium in which the spores are generated influences the abundance of germination-specific proteins, thereby influencing the sensitivity of the spores towards germinants. In C. difficile media composition on the spores has only been reported to influence the number of spores produced. One of the measures of spore germination is the analysis of the release of DPA from the spore core. To detect DPA release in real time, terbium chloride is often added to the germination conditions because Tb3+ complexes with the released DPA and this can be detected using fluorescence measurements. Although C. difficile spores germinate in response to TA and glycine, recently calcium was identified as an enhancer for spore germination. Here, we find that germination by spores prepared in peptone rich media, such as 70:30, is positively influenced by terbium. We hypothesize that, in these assays, Tb3+ functions similarly to calcium. Although the mechanism(s) causing increased sensitivity of the C. difficile spores that are prepared in peptone rich media to terbium is still unknown, we suggest that the TbCl3 concentration used in the analysis of C. difficile DPA release be carefully titrated so as not to misinterpret future findings.

An Arabidopsis gene trap line (GT606), which disrupted the AtCSP1 gene, exhibited an early germination phenotype that was affected by stratification treatment. Comparative analysis of GUS expression in seeds at the early germination stage, with or without stratification, demonstrated that AtCSP1 expression was affected by cold temperature. Evaluation of germination assays with varying concentrations of ABA or NaCl revealed a reduced sensitivity of the atcsp1 mutant to both ABA and NaCl. Taken together, these data support the hypothesis that AtCSP1 affects early stages of seed germination subsequent to stratification treatment of seeds.

Spore germination in Saccharomyces cerevisiae is a process in which a quiescent cell begins to divide. During germination, the cell undergoes dramatic changes in cell wall and membrane composition, as well as in gene expression. To understand germination in greater detail, we screened the S. cerevisiae deletion set for germination mutants. Our results identified two genes, TRF4 and ERG6, that are required for normal germination on solid media. TRF4 is a member of the TRAMP complex that, together with the exosome, degrades RNA polymerase II transcripts. ERG6 encodes a key step in ergosterol biosynthesis. Taken together, these results demonstrate the complex nature of germination and two genes important in the process.

Spore germination plays an essential role in the pathogenesis of Clostridium perfringens-associated food poisoning. Germination is initiated when bacterial spores sense various stimuli, including chemicals and enzymes. A previous study showed that dipicolinic acid (DPA) chelated with calcium (Ca-DPA) significantly stimulated spore germination in C. perfringens. However, whether Ca2+ or DPA alone can induce germination is unknown. Therefore, we aimed to evaluate the possible roles of Ca2+ and other divalent cations present in the spore core, such as Mn2+ and Mg2+, in C. perfringens spore germination. Our study demonstrated that (i) Ca-DPA, but not DPA alone, induced C. perfringens spore germination, suggesting that Ca2+ might play a signaling role; (ii) all tested calcium salts induced spore germination, indicating that Ca2+ is critical for germination; (iii) the spore-specific divalent cations Mn2+ and Mg2+, but not Zn2+, induced spore germination, suggesting that spore core-specific divalent cations are involved in C. perfringens spore germination; and (iv) endogenous Ca2+ and Mg2+ are not required for induction of C. perfringens spore germination, whereas exogenous and partly endogenous Mn2+ are required. Collectively, our results suggest that exogenous spore core-specific divalent cation signals are more important than endogenous signals for the induction of spore germination.

Seed germination paves the way for the dormant embryo to establish itself as a new plant marking the first critical step in postembryonic plant growth and development. Germination starts with the uptake of water (imbibition), followed by induction of transcription, translation, energy metabolism, and cell division processes. Although small RNAs have been implicated in many developmental processes, their role during seed germination stages and conditions remained elusive. Here we show that seed germination conditions, like imbibition and temperature, dynamically regulate the expression of many developmentally important miRNAs and their targets. We have identified 58 miRNAs belonging to 30 different families at different seed germination conditions. Amongst these, 15 miRNAs and their targets were significantly differentially expressed in Arabidopsis seeds in dry and 12 h, 24 h and 48 h of imbibition. Interestingly, differential expression of miR390, which targets trans-acting siRNA locus (TAS3) derived transcripts, resulted in alteration of tasiR-ARF mediated regulation of expression of target AUXIN RESPONSE FACTORs (ARF2/3/4). Our results suggest that the dynamic expression of several miRNAs, their targets, and a crosstalk between miRNA and ta-siRNA pathways contribute to the regulation of seed germination in Arabidopsis thaliana.

DnaJ proteins are essential co-chaperones involved in abiotic and biotic stress responses. Arabidopsis AtDjA3 gene encodes a molecular co-chaperone of 420 amino acids, which belongs to the J-protein family. In this study, we report the functional characterization of the AtDjA3 gene using the Arabidopsis knockout line designated j3 and the 35S::AtDjA3 overexpression lines. Loss of AtDjA3 function was associated with small seed production. In fact, j3 mutant seeds showed a reduction of 24% in seed weight compared to Col-0 seeds. Expression analysis showed that the AtDjA3 gene was modulated in response to NaCl, glucose, and abscisic acid (ABA). The j3 line had increased sensitivity to NaCl and glucose treatments in the germination and cotyledon development in comparison to parental Col-0. Furthermore, the j3 mutant line exhibited higher ABA sensitivity in comparison to parental Col-0 and 35S::AtDjA3 overexpression lines. In addition, we examined the expression of ABI3 gene, which is a central regulator in ABA signaling, in j3 mutant and 35S::AtDjA3 overexpression lines. Under 5 μM ABA treatment at 24 h, j3 mutant seedlings displayed higher ABI3 expression, whereas in 35S::AtDjA3 overexpression lines, ABI3 gene expression was repressed. Taken together, these results demonstrate that the AtDjA3 gene is involved in seed development and abiotic stress tolerance.

Sorghum is a gluten-free cereal representing a staple food in many countries of Africa, where germination is traditionally used for the preparation of several sorghum-based products. This study focused on the effect of germination on total phenolic content, in vitro and ex vivo antioxidant activity, and antihypertensive action of sorghum from Togo. Total phenolic content was estimated as Folin-Ciocalteu reducing capacity, while antioxidant activities were assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric Reducing Antioxidant Power (FRAP) in vitro tests and ex vivo by the cellular antioxidant activity (CAA) assay on human erythrocytes. The antihypertensive effect of germinated and non-germinated sorghum peptides fraction was evaluated as angiotensin-converting enzyme (ACE) inhibitory activity. Despite our findings demonstrated no impact of germination on the total phenolic content, non-germinated sorghum showed significantly higher in vitro antioxidant activities than the germinated one; further, non-germinated sorghum displayed significantly higher ACE inhibition than germinated sorghum that, instead, at lower doses, exhibited better erythrocytes protection from peroxyl radicals. In conclusion, the germination process negatively impacted the in vitro antioxidant activity and the antihypertensive effect of sorghum while improved erythrocytes protection. This study evidenced better nutraceutical potential of non-germinated sorghum that, besides good antioxidant activity, represents an important source of ACE-inhibitory peptides. However, the germination process might have positively impacted the profile of bioactive compounds involved in the protection of human erythrocytes from oxidative damage.