Ammonia is a morphogen in Dictyostelium and is known to arise from the catabolism of proteins and RNA. However, we show that extracellular adenosine deamination catalysed by ‘adenosine deaminase-related growth factor’ (ADGF) is a major source of ammonia and demonstrate a direct role of ammonia in tip organizer development. The tip formed during early development in Dictyostelium functions analogously to the embryonic organizer of higher vertebrates. Dictyostelium strains carrying mutations in the gene adgf fail to establish an organizer, and this could be reversed by exposing the mutants to volatile ammonia. Interestingly, Klebsiella pneumoniae physically separated from the Dictyostelium adgf mutants in a partitioned dish also rescues the mound arrest phenotype. Both the substrate, adenosine and the product, ammonia, regulate adgf expression, and ADGF acts downstream of the histidine kinase DhkD in regulating tip formation. Thus, the consecutive transformation of extracellular cAMP to adenosine, and adenosine to ammonia is integral steps during Dictyostelium development. Remarkably, in higher vertebrates, adgf expression is elevated during gastrulation and thus adenosine deamination may be a conserved process driving organizer development in different organisms.

In the model plant Arabidopsis thaliana , parental age is known to affect somatic mutation rates in their immediate progeny and here we show that this age dependent effect persists across successive generations. Using a set of detector lines carrying the mutated uidA gene, we examined if a particular parental age maintained across five consecutive generations affected the rates of base substitution (BSR), intrachromosomal recombination (ICR), frameshift mutation (FS), and transposition. The frequency of functional GUS reversions were assessed in seedlings as a function of identical/different parental ages across generations. In the context of a fixed parental age, BSR/ICR rates were unaffected in the first three generations, then dropped significantly in the 4th and increased in most instances in the 5th generation (e.g. BSR (F1 38 = 0.9, F2 38 = 1.14, F3 38 = 1.02, F4 38 = 0.5, F5 38 = 0.76)). On the other hand, with advancing parental ages, BSR/ICR rates remained high in the first two/three generations, with a striking resemblance in the pattern of mutation rates (BSR (F1 38 = 0.9, F1 43 = 0.53, F1 48 = 0.79, F1 53 = 0.83 and F2 38 = 1.14, F2 43 = 0.57, F2 48 = 0.64, F2 53 = 0.94). We adopted a novel approach of identifying and tagging flowers pollinated on a particular day, thereby avoiding biases due to potential emasculation induced stress responses. Our results suggest a time component in counting the number of generations a plant has passed through self-fertilization at a particular age in determining the somatic mutation rates.

Many cells can pause their growth cycle, a topic much enriched by studies of the stationary phase (SP) of model microorganisms. Although several kinases are implicated in SP onset, whether protein kinase C has a role remains unknown. We show that Dictyostelium discoideum cells lacking pkcA entered SP at a reduced cell density, but only in shaking conditions. Precocious SP entry occurs because levels of extracellular polyphosphate (polyP) reach the threshold needed to induce the SP onset at a lower cell density than seen in wild-type cells; adding exopolyphosphatase to pkcA- cells reverses the effect and mimics wild-type growth. PkcA-mediated regulation of polyP depended on inositol hexakisphosphate kinase and phospholipase D. PkcA- mutants also had higher F-actin levels, higher rates of exocytosis and lower pinocytosis rates. Postlysosomes were smaller and present in fewer pkcA- cells compared to the wild type. Overall, the results suggest that a reduced PkcA level triggers SP primarily because cells do not acquire or retain nutrients as efficiently, thus mimicking, or amplifying, the conditions of actual starvation. This article has an associated First Person interview with the first author of the paper.

The embryonic organizer is essential to determine one or more developmental polarities during chordate early development1,2. Functionally similar organizers also occur in more ancient animals3, and even in some protozoans such as Dictyostelium, in which the tip of the multicellular mound acts as an organizer4, establishing the main developmental axis, and regulating the size of the fruiting body5. However, our understanding of how the Dictyostelium organizer arises, and functions, is limited. Here we show that monoamine oxidase A (maoA), which degrades serotonin, confers the fate of an organizer to the Dictyostelium tip. Conversely, once a tip has formed, serotonin contributes to tip dominance. It inhibits further tip formation, and thus ensures the mound retains the size specified during an earlier developmental stage. Reducing the expression of maoA through RNA interference or by adding MAO specific inhibitors suppresses tip formation. Conversely, adding human MAOA enzyme, or an antagonist or antibodies against serotonin, restores tip formation in maoA knockdowns. Overexpression of maoA or adding a serotonin antagonist to the wildtype induces multiple tips from a single mound in a dose dependent manner. Using an array of genetic and molecular techniques, we show that serotonin’s inhibition of cAMP signalling and cell-cell adhesion is the basis of its regulation of tip formation. Our study demonstrates that serotonin, recently appreciated for its developmental roles in widespread phyla6, also has a novel and ancient role in the formation and function of an organizer.

Crossing over, the exchange of DNA between the chromosomes during meiosis, contributes significantly to genetic variation. The rate of crossovers (CO) varies depending upon the taxon, population, age, external conditions, and also, sometimes, between the sexes, a phenomenon called heterochiasmy. In the model plant Arabidopsis thaliana , the male rate of crossovers (mCO) is typically nearly double the female rate (fCO). With increasing parental age, it has been reported that the disparity decreases, because fCO rises while mCO remains stable. That finding, however, is based on chromosome-based averaging, and it is unclear whether all parts of the genome respond similarly. We addressed this point by examining how the level of heterochiasmy responded to parental age in eight genomic intervals distributed across the five chromosomes of Arabidopsis . Unlike the previous work, in each of the eight intervals, the level of heterochiasmy did not change with age, that is, the ratio mCO:fCO remained stable. As expected, though, amongst the intervals, the levels of heterochiasmy at any of the four ages examined, did vary. We propose that while the levels of heterochiasmy in Arabidopis might decrease with age on a chromosomal basis, as reported earlier, this is not true for all locations within each chromosome. This has practical implications for plant breeding research, a major aim of which is identifying ways to induce local increases in CO rates.

Crossing over, the exchange of DNA between the chromosomes during meiosis, contributes significantly to genetic variation. The rate of crossovers (CO) varies depending upon the taxon, population, age, external conditions, and also, sometimes, between the sexes, a phenomenon called heterochiasmy. In the model plant Arabidopsis thaliana, the male rate of all crossover events (mCO) is typically nearly double the female rate (fCO). A previous, PCR-based genotyping study has reported that the disparity decreases with increasing parental age, because fCO rises while mCO remains stable. We revisited this topic using a fluorescent tagged lines approach to examine how heterochiasmy responded to parental age in eight genomic intervals distributed across the organism's five chromosomes. We determined recombination frequency for, on average, more than 2000 seeds, for each interval, for each of four age groups, to estimate sex-specific CO rates. mCO did not change with age, as reported previously, but, here, fCO did not rise, and thus the levels of heterochiasmy were unchanged. We can see no methodological reason to doubt that our results reflect the underlying biology of the accessions we studied. The lack of response to age could perhaps be due to previously reported variation in CO rate among accessions of Arabidopsis.

Telomerase, particularly its main subunit, the reverse transcriptase, TERT, prevents DNA erosion during eukaryotic chromosomal replication, but also has poorly understood non-canonical functions. Here, in the model social amoeba Dictyostelium discoideum, we show that the protein encoded by tert has telomerase-like motifs, and regulates, non-canonically, important developmental processes. Expression levels of wild-type (WT) tert were biphasic, peaking at 8 and 12 h post-starvation, aligning with developmental events, such as the initiation of streaming (~7 h) and mound formation (~10 h). In tert KO mutants, however, aggregation was delayed until 16 h. Large, irregular streams formed, then broke up, forming small mounds. The mound-size defect was not induced when a KO mutant of countin (a master size-regulating gene) was treated with TERT inhibitors, but anti-countin antibodies did rescue size in the tert KO. Although, conditioned medium (CM) from countin mutants failed to rescue size in the tert KO, tert KO CM rescued the countin KO phenotype. These and additional observations indicate that TERT acts upstream of smlA/countin: (i) the observed expression levels of smlA and countin, being respectively lower and higher (than WT) in the tert KO; (ii) the levels of known size-regulation intermediates, glucose (low) and adenosine (high), in the tert mutant, and the size defect's rescue by supplemented glucose or the adenosine-antagonist, caffeine; (iii) the induction of the size defect in the WT by tert KO CM and TERT inhibitors. The tert KO's other defects (delayed aggregation, irregular streaming) were associated with changes to cAMP-regulated processes (e.g. chemotaxis, cAMP pulsing) and their regulatory factors (e.g. cAMP; acaA, carA expression). Overexpression of WT tert in the tert KO rescued these defects (and size), and restored a single cAMP signaling centre. Our results indicate that TERT acts in novel, non-canonical and upstream ways, regulating key developmental events in Dictyostelium.

Apomixis or asexual reproduction through seeds occurs in about 400 species of flowering plants producing genetically uniform progeny. During apomixis, meiosis is bypassed and embryos develop by parthenogenesis. However, the endosperm could form either autonomously without fertilization or sexually, depending on the plant species. Most probably, a heterochronic expression of sexually expressed genes is one of the reason that causes apomixis. A better understanding of the genetic components regulating apomixis is important for developmental and evolutionary studies and also for engineering apomixis traits into crop plants that may realize a possibility to propagate hybrid vigor in a range of subsequent generations.

Closely related to the model plant Arabidopsis thaliana, the genus Boechera is known to contain both sexual and apomictic species or accessions. Boechera retrofracta is a diploid sexually reproducing species and is thought to be an ancestral parent species of apomictic species. Here we report the de novo assembly of the B. retrofracta genome using short Illumina and Roche reads from 1 paired-end and 3 mate pair libraries. The distribution of 23-mers from the paired end library has indicated a low level of heterozygosity and the presence of detectable duplications and triplications. The genome size was estimated to be equal 227 Mb. N50 of the assembled scaffolds was 2.3 Mb. Using a hybrid approach that combines homology-based and de novo methods 27,048 protein-coding genes were predicted. Also repeats, transfer RNA (tRNA) and ribosomal RNA (rRNA) genes were annotated. Finally, genes of B. retrofracta and 6 other Brassicaceae species were used for phylogenetic tree reconstruction. In addition, we explored the histidine exonuclease APOLLO locus, related to apomixis in Boechera, and proposed model of its evolution through the series of duplications. An assembled genome of B. retrofracta will help in the challenging assembly of the highly heterozygous genomes of hybrid apomictic species.

Plants exposed to environmental stresses draw upon many genetic and epigenetic strategies, with the former sometimes modulated by the latter. This can help the plant, and its immediate progeny, at least, to better endure the stress. Some evidence has led to proposals that (epi) genetic changes can be both selective and sustainably heritable, while other evidence suggests that changes are effectively stochastic, and important only because they induce genetic variation. One type of stress with an arguably high level of stochasticity in its effects is temperature stress. Studies of how heat and cold affect the rates of meiotic recombination (MR) and somatic mutations (SMs, which are potentially heritable in plants) report increases, decreases, or no effect. Collectively, they do not point to any consistent patterns. Some of this variability, however, might arise from the stress being applied for such an extended time, typically days or weeks. Here, we adopted a targeted approach by (1) limiting exposure to one hour; and (2) timing it to coincide with (a) gamete, and early gametophyte, development, a period of high stress sensitivity; and (b) a late stage of vegetative development.

Little is known on the effect of parental reproductive age on the somatic mutation rates in flowering plants. With a set of Arabidopsis mutation detector lines, we examined the effect of parental reproductive age on the spectrum of mutations induced and their rates in the subsequent progeny. We scored rates of point and frameshift mutations, as well as homologous recombination (HR) and transposition events based on functional GUS reversion of a mutated or truncated uid A gene. We observed that T-C transition rates increased with the parental reproductive age in the offspring. Similarly, frameshift mutation (G10) and transposition rates rose in the progeny as a consequence of increased parental age with a pronounced effect of maternal age. However, the HR frequency decreased with the age of parents. Our study shows that parental reproductive age alters the somatic mutation rates in progeny of Arabidopsis plants.

Multicellular development in Dictyostelium discoideum involves tightly regulated signaling events controlling the entry into development, initiation of aggregation and chemotaxis, and cellular differentiation. Here we show that PkcA, a Dictyostelium discoideum Protein Kinase C-orthologue, is involved in quorum sensing and the initiation of development, as well as cAMP sensing during chemotaxis. Additionally, by epistasis analysis we provide evidence that PkcA and PldB (a Phospholipase D-orthologue) functionally interact to regulate aggregation, differentiation, and cell-cell adhesion during development. Finally, we show that PkcA acts as a positive regulator of intracellular PLD-activity during development. Taken together, our results suggest that PkcA act through PldB, by regulating PLD-activity, in order to control events during development.

A number of organisms possess several isoforms of protein kinase C but little is known about the significance of any specific isoform during embryogenesis and development. To address this we characterized a PKC ortholog (PkcA; DDB_G0288147) in Dictyostelium discoideum. pkcA expression switches from prestalk in mound to prespore in slug, indicating a dynamic expression pattern. Mutants lacking the catalytic domain of PkcA (pkcA−) did not exhibit tip dominance. A striking phenotype of pkcAˉ was the formation of an aggregate with a central hollow, and aggregates later fragmented to form small mounds, each becoming a fruiting body. Optical density wave patterns of cAMP in the late aggregates showed several cAMP wave generation centers. We attribute these defects in pkcA− to impaired cAMP signaling, altered cell motility and decreased expression of the cell adhesion molecules – CadA and CsaA. pkcA− slugs showed ectopic expression of ecmA in the prespore region. Further, the use of a PKC-specific inhibitor, GF109203X that inhibits the activity of catalytic domain phenocopied pkcA−.

In humans, it is well known that the parental reproductive age has a strong influence on mutations transmitted to their progeny. Meiotic nondisjunction is known to increase in older mothers, and base substitutions tend to go up with paternal reproductive age. Hence, it is clear that the germinal mutation rates are a function of both maternal and paternal ages in humans. In contrast, it is unknown whether the parental reproductive age has an effect on somatic mutation rates in the progeny, because these are rare and difficult to detect. To address this question, we took advantage of the plant model system Arabidopsis (Arabidopsis thaliana), where mutation detector lines allow for an easy quantitation of somatic mutations, to test the effect of parental age on somatic mutation rates in the progeny. Although we found no significant effect of parental age on base substitutions, we found that frameshift mutations and transposition events increased in the progeny of older parents, an effect that is stronger through the maternal line. In contrast, intrachromosomal recombination events in the progeny decrease with the age of the parents in a parent-of-origin-dependent manner. Our results clearly show that parental reproductive age affects somatic mutation rates in the progeny and, thus, that some form of age-dependent information, which affects the frequency of double-strand breaks and possibly other processes involved in maintaining genome integrity, is transmitted through the gametes.

Agrobacterium infection, which is widely used to generate transgenic plants, is often accompanied by T-DNA-linked mutations and transpositions in flowering plants. It is not known if Agrobacterium infection also affects the rates of point mutations, somatic homologous recombinations (SHR) and frame-shift mutations (FSM). We examined the effects of Agrobacterium infection on five types of somatic mutations using a set of mutation detector lines of Arabidopsis thaliana. To verify the effect of secreted factors, we exposed the plants to different Agrobacterium strains, including wild type (Ach5), its derivatives lacking vir genes, oncogenes or T-DNA, and the heat-killed form for 48 h post-infection; also, for a smaller set of strains, we examined the rates of three types of mutations at multiple time-points. The mutation detector lines carried a non-functional β-glucuronidase gene (GUS) and a reversion of mutated GUS to its functional form resulted in blue spots. Based on the number of blue spots visible in plants grown for a further two weeks, we estimated the mutation frequencies.