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

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

Dosimetric Validation of a System to Treat Moving Tumors Using Scanned Ion Beams That Are Synchronized With Anatomical Motion

PURPOSE: The purpose of this study was to validate the dosimetric performance of scanned ion beam deliveries with motion-synchronization to heterogenous targets. METHODS: A 4D library of treatment plans, comprised of up to 10 3D sub-plans, was created with robust and conventional 4D optimization methods. Each sub-plan corresponded to one phase of periodic target motion. The plan libraries were delivered to a test phantom, comprising plastic slabs, dosimeters, and heterogenous phantoms. This phantom emulated range changes that occur when treating moving tumors. Similar treatment plans, but without motion synchronization, were also delivered to a test phantom with a stationary target and to a moving target; these were used to assess how the target motion degrades the quality of dose distributions and the extent to which motion synchronization can improve dosimetric quality. The accuracy of calculated dose distributions was verified by comparison with corresponding measurements. Comparisons utilized the gamma index analysis method. Plan quality was assessed based on conformity, dose coverage, overdose, and homogeneity values, each extracted from calculated dose distributions. RESULTS: High pass rates for the gamma index analysis confirmed that the methods used to calculate and reconstruct dose distributions were sufficiently accurate for the purposes of this study. Calculated and reconstructed dose distributions revealed that the motion-synchronized and static deliveries exhibited similar quality in terms of dose coverage, overdose, and homogeneity for all deliveries considered. Motion-synchronization substantially improved conformity in deliveries with moving targets. Importantly, measurements at multiple locations within the target also confirmed that the motion-synchronized delivery system satisfactorily compensated for changes in beam range caused by the phantom motion. Specifically, the overall planning and delivery approach achieved the desired dose distribution by avoiding range undershoots and overshoots caused by tumor motion. CONCLUSIONS: We validated a dose delivery system that synchronizes the movement of the ion beam to that of a moving target in a test phantom. Measured and calculated dose distributions revealed that this system satisfactorily compensated for target motion in the presence of beam range changes due to target motion. The implication of this finding is that the prototype system is suitable for additional preclinical research studies, such as irregular anatomic motion

Dosimetric Validation of a System to Treat Moving Tumors Using Scanned Ion Beams That Are Synchronized With Anatomical Motion

Purpose: The purpose of this study was to validate the dosimetric performance of scanned ion beam deliveries with motion-synchronization to heterogenous targets. Methods: A 4D library of treatment plans, comprised of up to 10 3D sub-plans, was created with robust and conventional 4D optimization methods. Each sub-plan corresponded to one phase of periodic target motion. The plan libraries were delivered to a test phantom, comprising plastic slabs, dosimeters, and heterogenous phantoms. This phantom emulated range changes that occur when treating moving tumors. Similar treatment plans, but without motion synchronization, were also delivered to a test phantom with a stationary target and to a moving target; these were used to assess how the target motion degrades the quality of dose distributions and the extent to which motion synchronization can improve dosimetric quality. The accuracy of calculated dose distributions was verified by comparison with corresponding measurements. Comparisons utilized the gamma index analysis method. Plan quality was assessed based on conformity, dose coverage, overdose, and homogeneity values, each extracted from calculated dose distributions. Results: High pass rates for the gamma index analysis confirmed that the methods used to calculate and reconstruct dose distributions were sufficiently accurate for the purposes of this study. Calculated and reconstructed dose distributions revealed that the motion-synchronized and static deliveries exhibited similar quality in terms of dose coverage, overdose, and homogeneity for all deliveries considered. Motion-synchronization substantially improved conformity in deliveries with moving targets. Importantly, measurements at multiple locations within the target also confirmed that the motion-synchronized delivery system satisfactorily compensated for changes in beam range caused by the phantom motion. Specifically, the overall planning and delivery approach achieved the desired dose distribution by avoiding range undershoots and overshoots caused by tumor motion. Conclusions: We validated a dose delivery system that synchronizes the movement of the ion beam to that of a moving target in a test phantom. Measured and calculated dose distributions revealed that this system satisfactorily compensated for target motion in the presence of beam range changes due to target motion. The implication of this finding is that the prototype system is suitable for additional preclinical research studies, such as irregular anatomic motion