MicroProteins in the Regulation of Flowering Time

Plants are, as sessile organisms, highly dependent on their ability to adapt to an ever changing environment. In order to do so they developed a remarkable variety of regulatory networks which enable them to perceive, integrate and adapt towards their surroundings on a molecular level. Transcription factors as regulators of gene activity are an essential component of those networks. They often interact via specific interaction domains and form high order complexes with further proteins. MicroProteins, small proteins with a single protein-protein interaction domain, function as negative regulators of protein complex formation by sequestering their target proteins in a non-functional state. Their mode of function enables them to modulate the activity transcription factors in regulatory networks and in the past years several examples for processes in which microProteins play important roles have been described. In order to learn more about the role of microProteins in development, we performed a computational screen to identify proteins with microProtein characteristics in the model plant Arabidopsis thaliana. Among the identified proteins where two small B-Box proteins – subsequently named miP1a and miP1b- which we further characterized. Overexpression of miP1a/b in Arabidopsis causes late flowering under inductive long day conditions whereas artificial reduction of their expression causes plants to flower slightly earlier. Both microProteins are able to interact with the flower promoting B-Box protein CONSTANS and we characterized miP1a/b as negative regulators of CONSTANS activity. Analysis of the temporal and spatial expression of miP1a and miP1b revealed a diurnal pattern of expression with high mRNA levels in the night period and a vascular expression of miP1a and miP1b. Finally, we characterized the interaction of miP1a/b with the transcriptional co-repressor TOPLESS and showed, that the two microProteins are able to engage CONSTANS and TOPLESS in a trimeric complex. Our findings point towards a novel role for CONSTANS, not only as a flowering promoting factor, but also as a repressor of the floral transition in the presence of miP1a and miP1b, were it becomes engaged together with TOPLESS in a trimeric complex.Pflanzen sind als sessile Organismen darauf angewiesen, sich Veränderungen in ihrer Umwelt anpassen zu können. Um dies zu bewerkstelligen habe sie in ihrer Evolution bemerkenswert vielseitige molekulare Netzwerke entwickelt, die es ihnen erlauben Veränderungen in ihrer Umgebung wahrzunehmen und sich diesen anzupassen. Transkripitionsfaktoren sind Proteine, welche die Aktivität von Genen regulieren und sind ein wesentlicher Bestandteil dieser molekularen Netzwerke. Oft interagieren sie mit anderen Proteinen anhand von spezifischen Interaktionsdomänen und bilden größere Proteinkomplexe. MikroProteine, eine Klasse von kleinen Proteinen die lediglich aus einer solche Interaktionsdomäne bestehen, interagieren spezifisch mit anderen Proteinen und verhindern so die Bildung eines funktionellen Komplexes. Diese Funktionsweise ermöglicht es ihnen die Aktivität von Transkriptionsfaktoren in regulatorischen Netzwerken zu beeinflussen. Mehrere Beispiele für solche durch MikroProteine regulierten Prozesse wurden in den letzten Jahren beschrieben. Um die Rolle von MikroProteinen in Entwicklungsprozessen besser zu verstehen, haben wir in der Modellpflanze Arabidopsis thaliana systematisch nach Proteinen mit den Eigenschaften von MikroProtein gesucht. Unter anderem fanden wir dabei zwei kleine B-Box Proteine, im Folgenden miP1a und miP1b genannt, die wir genauer untersucht haben. Die Überexpression der beiden Proteine bewirkt spätes Blühen unter normalerweise blühinduzierenden Wachstumsbedingungen wohingegen die künstliche Reduktion ihrer Expression leicht früheres Blühen bewirkt. Die beiden Proteine sind in der Lage mit CONSTANS, einem anderen B-Box Protein welches maßgeblich an der Blühinduktion beteiligt ist, zu interagieren. Wir konnten zeigen, dass miP1a und miP1b CONSTANS in seiner Aktivität inhibieren. Die beiden MikroProteine weisen eine zyklische Expression im Verlauf des Tages mit einem Expressionsmaximum während der Nacht auf. Vornehmlich sind sie in der Vaskulatur der oberirdischen Pflanzenteile exprimiert. Zusätzlich konnten wir zeigen, dass miP1a und miP1b mit TOPLESS, einem transkriptionellem Ko-Repressor interagieren und CONSTANS mit diesem zusammen in einem trimeren Komplex binden. Diese Entdeckung beschriebt eine neue Funktion von CONSTANS, dass somit nicht nur als Blühinduzierender Faktor sondern auch als reprimierender Faktor fungieren kann, wenn es durch miP1a und miP1b in einem Komplex zusammen mit TOPLESS eingebunden wird

A phosphoinositide hub connects CLE peptide signaling and polar auxin efflux regulation

Auxin efflux through plasma-membrane-integral PIN-FORMED (PIN) carriers is essential for plant tissue organization and tightly regulated. For instance, a molecular rheostat critically controls PIN-mediated auxin transport in developing protophloem sieve elements of Arabidopsis roots. Plasma-membrane-association of the rheostat proteins, BREVIS RADIX (BRX) and PROTEIN KINASE ASSOCIATED WITH BRX (PAX), is reinforced by interaction with PHOSPHATIDYLINOSITOL-4-PHOSPHATE-5-KINASE (PIP5K). Genetic evidence suggests that BRX dampens autocrine signaling of CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 45 (CLE45) peptide via its receptor BARELY ANY MERISTEM 3 (BAM3). How excess CLE45-BAM3 signaling interferes with protophloem development and whether it does so directly or indirectly remains unclear. Here we show that rheostat polarity is independent of PIN polarity, but interdependent with PIP5K. Catalytically inactive PIP5K confers rheostat polarity without reinforcing its localization, revealing a possible PIP5K scaffolding function. Moreover, PIP5K and PAX cooperatively control local PIN abundance. We further find that CLE45-BAM3 signaling branches via RLCK-VII/PBS1-LIKE (PBL) cytoplasmic kinases to destabilize rheostat localization. Our data thus reveal antagonism between CLE45-BAM3-PBL signaling and PIP5K that converges on auxin efflux regulation through dynamic control of PAX polarity. Because second-site bam3 mutation suppresses root as well as shoot phenotypes of pip5k mutants, CLE peptide signaling likely modulates phosphoinositide-dependent processes in various developmental contexts

MicroProtein-mediated recruitment of CONSTANS into a TOPLESS trimeric complex represses flowering in Arabidopsis

MicroProteins are short, single domain proteins that act by sequestering larger, multi-domain proteins into non-functional complexes. MicroProteins have been identified in plants and animals, where they are mostly involved in the regulation of developmental processes. Here we show that two Arabidopsis thaliana microProteins, miP1a and miP1b, physically interact with CONSTANS (CO) a potent regulator of flowering time. The miP1a/b-type microProteins evolved in dicotyledonous plants and have an additional carboxy-terminal PF(V/L)FL motif. This motif enables miP1a/b microProteins to interact with TOPLESS/TOPLESS-RELATED (TPL/TPR) proteins. Interaction of CO with miP1a/b/TPL causes late flowering due to a failure in the induction of FLOWERING LOCUS T (FT) expression under inductive long day conditions. Both miP1a and miP1b are expressed in vascular tissue, where CO and FT are active. Genetically, miP1a/b act upstream of CO thus our findings unravel a novel layer of flowering time regulation via microProtein-inhibition

Preliminary tests of dosimetric quality and projected therapeutic outcomes of multi-phase 4D radiotherapy with proton and carbon ion beams

The purpose of this study was to perform preliminary pre-clinical tests to compare the dosimetric quality of two approaches to treating moving tumors with ion beams: synchronously delivering the beam with the motion of a moving planning target volume (PTV) using the recently developed multi-phase 4D dose delivery (MP4D) approach, and asynchronously delivering the ion beam to a motion-encompassing internal tumor volume (ITV) combined with rescanning. We created 4D optimized treatment plans with proton and carbon ion beams for two patients who had previously received treatment for non-small cell lung cancer. For each patient, we created several treatment plans, using approaches with and without motion mitigation: MP4D, ITV with rescanning, static deliveries to a stationary PTV, and deliveries to a moving tumor without motion compensation. Two sets of plans were optimized with margins or robust uncertainty scenarios. Each treatment plan was delivered using a recently-developed motion-synchronized dose delivery system (M-DDS); dose distributions in water were compared to measurements using gamma index analysis to confirm the accuracy of the calculations. Reconstructed dose distributions on the patient CT were analyzed to assess the dosimetric quality of the deliveries (conformity, uniformity, tumor coverage, and extent of hotspots). Gamma index analysis pass rates confirmed the accuracy of dose calculations. Dose coverage was >95% for all static and MP4D treatments. The best conformity and the lowest lung doses were achieved with MP4D deliveries. Robust optimization led to higher lung doses compared to conventional optimization for ITV deliveries, but not for MP4D deliveries. We compared dosimetric quality for two approaches to treating moving tumors with ion beams. Our findings suggest that the MP4D approach, using an M-DDS, provides conformal motion mitigation, with full target coverage and lower OAR doses

A Modular System for Treating Moving Anatomical Targets With Scanned Ion Beams at Multiple Facilities: Pre-Clinical Testing for Quality and Safety of Beam Delivery

Background: Quality management and safety are integral to modern radiotherapy. New radiotherapy technologies require new consensus guidelines on quality and safety. Established analysis strategies, such as the failure modes and effects analysis (FMEA) and incident learning systems have been developed as tools to assess the safety of several types of radiation therapies. An extensive literature documents the widespread application of risk analysis methods to photon radiation therapy. Relatively little attention has been paid to performing risk analyses of nascent radiation therapy systems to treat moving tumors with scanned heavy ion beams. The purpose of this study was to apply a comprehensive safety analysis strategy to a motion-synchronized dose delivery system (M-DDS) for ion therapy. Methods: We applied a risk analysis method to new treatment planning and treatment delivery processes with scanned heavy ion beams. The processes utilize a prototype, modular dose delivery system, currently undergoing preclinical testing, that provides new capabilities for treating moving anatomy. Each step in the treatment process was listed in a process map, potential errors for each step were identified and scored using the risk probability number in an FMEA, and the possible causes of each error were described in a fault tree analysis. Solutions were identified to mitigate the risk of these errors, including permanent corrective actions, periodic quality assurance (QA) tests, and patient specific QA (PSQA) tests. Each solution was tested experimentally. Results: The analysis revealed 58 potential errors that could compromise beam delivery quality or safety. Each of the 14 binary (pass-or-fail) tests passed. Each of the nine QA and four PSQA tests were within anticipated clinical specifications. The modular M-DDS was modified accordingly, and was found to function at two centers. Conclusion: We have applied a comprehensive risk analysis strategy to the M-DDS and shown that it is a clinically viable motion mitigation strategy. The described strategy can be utilized at any ion therapy center that operates with the modular M-DDS. The approach can also be adapted for use at other facilities and can be combined with existing safety analysis systems

Environmental and Economic Performance of Yacon (Smallanthus sonchifolius) Cultivated for Fructooligosaccharide Production

As the prevalence of diabetes is predicted to rise globally in the coming decades, the demand for sugar substitutes is expected to increase significantly. In this context, natural sweeteners have been receiving particular attention, as artificial sweeteners have been associated with obesity and cardiovascular disease. One natural sweetener is yacon (Smallanthus sonchifolius) ((Poepp. and Endl.) H. Robinson), which could play a prominent role due to its high fructooligosaccharides yield. Yacon is currently only a minor crop in Europe and there is little information available on the environmental and economic impacts of its various cultivation systems. These are especially affected by nitrogen fertilization levels and genotype selection. Thus, before the crop is introduced on a larger scale, it is expedient to identify the most sustainable cultivation system. The life-cycle assessment (LCA) and life-cycle costing (LCC) analysis of yacon cultivation systems conducted in this study revealed significant differences between yacon genotypes and found that a nitrogen fertilization level of 80 kg N ha−1 significantly decreased production costs and simultaneously led to a comparatively good environmental performance. The results indicated that, for the holistic evaluation of agricultural systems, it is crucial to assess both the economic and environmental performance of new crops