MicroRNA-mediated control of floral homeotic functions in Antirrhinum: characterization of the FISTULATA gene

The identity of Antirrhinum reproductive organs is controlled by two class C- MADS box genes, PLENA (PLE) and FARINELLI (FAR), which are expressed in the third and the fourth whorl of the flower. The fistulata mutant (fis) in Antirrhinum and blind (bl) in Petunia show partial homeotic conversion of petals to stamenoid structures, which is caused by ectopic expression of PLE/FAR and pMADS3/FBP6, the Petunia C-genes, respectively. FIS and BL thus control the C- expression domain in the wild type. FISTULATA was cloned by a map-based strategy combined with a candidate gene approach and show that both genes encode a microRNA corresponding to miR169. The miR169 target site is present in the 3´UTR of members of the NF-YA transcription factor family that bind a cis-acting CCAAT motif present in the second intron of the PLE/FAR and pMADS3 genes. This suggests a miRNA-mediated and NF-YA-dependent control of the C- function. We cloned six members of the Antirrhinum NF-YA gene family (AmYAs), four of which contain the miRNA recognition site. Data derived from in vitro infitration experiments indicate miRFIS-dependent post-transcriptional regulation of AmYAs. Expression studies in wild type and fis mutant flowers showed that miRFIS and the AmYAs are spatially co-expressed in the flower suggesting that by fine tuning expression of the NF-YAs, miRFIS indirectly regulates the level of C-gene expression. In addition, genetic evidence is presented demonstrating the functional relevance of the unexpected miRFIS function in the centre of the flower. Based on these results a dynamic model is proposed in which miRFIS maintains the boundaries of the C- domain by preventing formation of a radially extending molecular gradient from the centre of the flower (where C-products are activated) toward peripheral regions of the meristem. Experimental approaches are suggested to solve open questions

Enhanced AGAMOUS expression in the centre of the Arabidopsis flower causes ectopic expression over its outer expression boundaries

Spatial regulation of C-function genes controlling reproductive organ identity in the centre of the flower can be achieved by adjusting the level of their expression within the genuine central expression domain in Antirrhinum and Petunia. Loss of this control in mutants is revealed by enhanced C-gene expression in the centre and by lateral expansion of the C-domain. In order to test whether the level of central C-gene expression and hence the principle of ‘regulation by tuning’ also applies to spatial regulation of the C-function gene AGAMOUS (AG) in Arabidopsis, we generated transgenic plants with enhanced central AG expression by using stem cell-specific CLAVATA3 (CLV3) regulatory sequences to drive transcription of the AG cDNA. The youngest terminal flowers on inflorescences of CLV3::AG plants displayed homeotic features in their outer whorls indicating ectopic AG expression. Dependence of the homeotic feature on the age of the plant is attributed to the known overall weakening of repressive mechanisms controlling AG. Monitoring AG with an AG-I::GUS reporter construct suggests ectopic AG expression in CLV3::AG flowers when AG in the inflorescence is still repressed, although in terminating inflorescence meristems, AG expression expands to all tissues. Supported by genetic tests, we conclude that upon enhanced central AG expression, the C-domain laterally expands necessitating tuning of the expression level of C-function genes in the wild type. The tuning mechanism in C-gene regulation in Arabidopsis is discussed as a late security switch that ensures wild-type C-domain control when other repressive mechanism starts to fade and fail

Robotic simple prostatectomy vs HOLEP, a 'multi single-center' experiences comparison

Introduction: The aim of this study was to compare peri-operative and mid-term outcomes of patients who underwent robot-assisted simple prostatectomy (RASP) vs holmium laser enucleation of the prostate (HOLEP). RASP and HOLEP are the treatments of choice for men with symptomatic benign prostatic obstruction (BPO) and a prostate ≥80 g, achieving comparable short and mid-term efficacy. No randomized controlled studies have proved the superiority of one technique over the other. Material and methods: The prospectively maintained databases of the participating institutions were queried for patients with a prostate volume (PV) ≥80 g, who underwent surgery for BPO between 2011 and 2021. The study population was divided into two subgroups based on surgical approach. Demographics, baseline characteristics, and 12 months outcomes were compared between groups: χ2 and Student t-tests were used for categorical and continuous variables, respectively. The Trifecta composite outcome (post-operative Q-max >15 ml/sec, International Prostate Symptom Score (IPSS) <8 and absence of complications) was used to define surgical quality and the two groups were compared accordingly. Logistic regression analyses investigated predictors of Trifecta achievement. Results: We included 97 patients with comparable pre-operative features (all p >0.30): 43 underwent RASP, 54 HOLEP. Median PV was 102 g (IQR 89-120) and Q-max was 7.2 ml/s (IQR 5.4-9.0). The Trifecta rate was 43% overall, higher in the RASP subgroup (56% vs 33%; p = 0.02). The endoscopic approach was its only independent predictor (OR 0.5; 95% CI 0.28-0.88; p = 0.016). Conclusions: At univariable regression analysis, surgical approach was the only independent predictor of Trifecta achievement, which was significantly higher in the RASP group compared to HOLEP

Conservation versus divergence in LEAFY and APETALA functions between Arabidopsis thaliana and Cardamine hirsuta

International audienceA conserved genetic toolkit underlies the development of diverse floral forms among angiosperms. However, the degree of conservation vs divergence in the configuration of these gene regulatory networks is less clear. We addressed this question in a parallel genetic study between the closely related species Arabidopsis thaliana and Cardamine hirsuta. We identified leafy (lfy) and apetala1 (ap1) alleles in a mutant screen for floral regulators in C. hirsuta. C. hirsuta lfy mutants showed a complete homeotic conversion of flowers to leafy shoots, mimicking lfy ap1 double mutants in A. thaliana. Through genetic and molecular experiments, we showed that AP1 activation is fully dependent on LFY in C. hirsuta, by contrast to A. thaliana. Additionally, we found that LFY influences heteroblasty in C. hirsuta, such that loss or gain of LFY function affects its progression. Overexpression of UNUSUAL FLORAL ORGANS also alters C. hirsuta leaf shape in an LFY-dependent manner. We found that LFY and AP1 are conserved floral regulators that act nonredundantly in C. hirsuta, such that LFY has more obvious roles in floral and leaf development in C. hirsuta than in A. thaliana

Pan-European study of genotypes and phenotypes in the Arabidopsis relative Cardamine hirsuta reveals how adaptation, demography, and development shape diversity patterns

We study natural DNA polymorphisms and associated phenotypes in the Arabidopsis relative Cardamine hirsuta. We observed strong genetic differentiation among several ancestry groups and broader distribution of Iberian relict strains in European C. hirsuta compared to Arabidopsis. We found synchronization between vegetative and reproductive development and a pervasive role for heterochronic pathways in shaping C. hirsuta natural variation. A single, fast-cycling ChFRIGIDA allele evolved adaptively allowing range expansion from glacial refugia, unlike Arabidopsis where multiple FRIGIDA haplotypes were involved. The Azores islands, where Arabidopsis is scarce, are a hotspot for C. hirsuta diversity. We identified a quantitative trait locus (QTL) in the heterochronic SPL9 transcription factor as a determinant of an Azorean morphotype. This QTL shows evidence for positive selection, and its distribution mirrors a climate gradient that broadly shaped the Azorean flora. Overall, we establish a framework to explore how the interplay of adaptation, demography, and development shaped diversity patterns of 2 related plant species

Pulsatile Urea Excretion in Gulf Toadfish: Neuroendocrine Control and Potential Involvement in Chemical Communication

Gulf toadfish (Opsanus beta) are well studied for their ability to produce and excrete urea as an alternate form of nitrogenous waste. Urea is only produced by a few adult teleost species in substantial quantities because its production is energetically costly compared to ammonia. Toadfish uniquely excrete urea in distinct pulses and the process is controlled physiologically by the stress hormone cortisol and the neurotransmitter serotonin (5-HT). Decades of research have ruled out several explanations for the adaptive advantage of this pulsatile urea excretion and the remaining hypothesis is that signals are release via urea pulses for chemical communication. However, the use of pheromones for communication is not well understood in marine teleosts as most studies focus on chemical signals released in freshwater fish and crustacean urine. Therefore, the main objectives of this dissertation are to further characterize the neuroendocrine control of pulsatile urea excretion and determine whether toadfish utilize urea pulses across the gill to releases signals that communicate reproductive and social status. Using physiological and behavioral experimental approaches, the present studies determined that the 5-HT that triggers urea pulses is likely coming from circulation rather from local release within the gill because plasma 5-HT decreases over the course of a urea pulse and then increases after the pulse. Additionally, we confirmed that pulsatile urea excretion is not under nervous control through the branchial branches of the glossopharyngeal and vagus cranial nerves. These nerves are also not responsible for local release of 5-HT from gill storage sites as nerve-sectioning did not affect abundance of gill 5-HT and its metabolite. Ours was the first study to determine that plasma 5-HT fluctuates seasonally in teleosts. We suggest that higher circulating 5-HT measured during breeding season, that was strongly correlated with toadfish gonadosomatic index (GSI), may be responsible for consistently elevated pulsatile urea excretion during breeding season. Toadfish respond with a reduced pulse latency, a greater pulse frequency, and an increase in swimming activity when presented with chemical cues of a conspecific of the opposite sex during breeding season, suggesting that they may be releasing chemical signals via urea pulses to communicate sex and reproductive status. However, perhaps more convincingly, when olfaction was experimentally blocked in pairs of male toadfish, pulsatile urea excretion was inhibited in anosmic males compared to controls and behavior in subordinate anosmic males was altered. These changes in behavior and urea excretion in response to anosmia suggest that the transfer of chemical signals plays a role in establishing social hierarchies. Additionally, peaks in agonistic behavior occurred around the time of urea pulses in the majority of toadfish pairs, suggesting coordination of urea pulses and behavior. This dissertation significantly advanced our understanding of pulsatile urea excretion by providing physiological and behavioral evidence that it is involved in the communication of reproductive and social status. Furthermore, because it has already been found that circulating 5-HT plays a role in teleost reproduction and aggressive behavior, the newly determined importance of circulating 5-HT in the control of pulsatile urea excretion and its seasonality provides an essential link between communication of reproductive and social status and pulsatile urea excretion.</p

Maria Albri

Video interview with Maria Albri as part of the Italian Cinema Audiences projec