Distinct roles for Arabidopsis SUMO protease ESD4 and its closest homolog ELS1

SUMO conjugation affects a broad range of processes in Arabidopsis thaliana, including flower initiation, pathogen defense, and responses to cold, drought and salt stress. We investigated two sequence-related SUMO-specific proteases that are both widely expressed and show that they differ significantly in their properties. The closest homolog of SUMO protease ESD4, ESD4-LIKE SUMO PROTEASE 1 (ELS1, alternatively called AtULP1a) has SUMO-specific proteolytic activity, but is functionally distinct from ESD4, as shown by intracellular localization, mutant phenotype and heterologous expression in yeast mutants. Furthermore, we show that the growth defects caused by loss of ESD4 function are not due to increased synthesis of the stress signal salicylic acid, as was previously shown for a SUMO ligase, indicating that impairment of the SUMO system affects plant growth in different ways. Our results demonstrate that two A. thaliana SUMO proteases showing close sequence similarity have distinct in vivo functions

Arabidopsis thaliana SPF1 and SPF2 are nuclear-located ULP2-like SUMO proteases that act downstream of SIZ1 in plant development

Post-translational modifiers such as the small ubiquitin-like modifier (SUMO) peptide act as fast and reversible protein regulators. Functional characterization of the sumoylation machinery has determined the key regulatory role that SUMO plays in plant development. Unlike components of the SUMO conjugation pathway, SUMO proteases (ULPs) are encoded by a relatively large gene family and are potential sources of specificity within the pathway. This study reports a thorough comparative genomics and phylogenetic characterization of plant ULPs, revealing the presence of one ULP1-like and three ULP2-like SUMO protease subgroups within plant genomes. As representatives of an under-studied subgroup, Arabidopsis SPF1 and SPF2 were subjected to functional characterization. Loss-of-function mutants implicated both proteins with vegetative growth, flowering time, and seed size and yield. Mutants constitutively accumulated SUMO conjugates, and yeast complementation assays associated these proteins with the function of ScUlp2 but not ScUlp1. Fluorescence imaging placed both proteins in the plant cell nucleoplasm. Transcriptomics analysis indicated strong regulatory involvement in secondary metabolism, cell wall remodelling, and nitrate assimilation. Furthermore, developmental defects of the spf1-1 spf2-2 (spf1/2) double-mutant opposed those of the major E3 ligase siz1 mutant and, most significantly, developmental and transcriptomic characterization of the siz1 spf1/2 triple-mutant placed SIZ1 as epistatic to SPF1 and SPF2.We thank Mark Hochstrasser (Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA) for kindly providing the ulp1-ts yeast mutant strain. This research was funded by FEDER (through COMPETE), and by Fundacao para a Ciencia e Tecnologia (FCT), within the scope of project SUMOdulator (FCOMP-01-0124-FEDER-028459 and PTDC/BIA-PLA/3850/2012). PHC was supported by FCT (SFRH/BD/44484/2008). HA and FF were supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000007 and Norte-01-0145-FEDER-000008, respectively). The work was supported by FEDER through the COMPETE 2020-Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT, within the framework of projects 'Rede de Investigacao em Biodiversidade e Biologia Evolutiva' (POCI-01-0145-FEDER-006821) and 'Institute for Research and Innovation in Health Sciences' (POCI-01-0145-FEDER-007274). This research was also supported by a grant from the Spanish Ministerio de Ciencia y Tecnologia (AGL2016-75819-C2-1-R) and FEDER (PCQ, AGC, ERB)

The α-helix of the second chromodomain of the 43 kDa subunit of the chloroplast signal recognition particle facilitates binding to the 54 kDa subunit

AbstractChloroplasts of higher plants contain a unique signal recognition particle (cpSRP) that consists of two proteins, cpSRP54 and cpSRP43. CpSRP43 is composed of a four ankyrin repeat domain and three functionally distinct chromodomains (CDs). In this report we confirm previously published data that the second chromodomain (CD2) provides the primary binding site for cpSRP54. However, quantitative binding analysis demonstrates that cpSRP54 binds to CD2 significantly less efficiently than it binds to full-length cpSRP43. Further analysis of the binding interface of cpSRP by mutagenesis studies and a pepscan approach demonstrates that the C-terminal α-helix of CD2 facilitates binding to cpSRP54

Beeinflusst der Vorwissensstand die Interaktionsdynamik und den Lernerfolg im digitalen problemorientierten Lernen? Eine Pilotstudie

Objective: Previous research on problem-based learning (PBL) describes that videotaped observations develop meaningful insights into cognitive processes in tutorial groups. Analysis regarding the amount of prior knowledge on learning achievement has not been investigated in medical education so far, although both are key factors of PBL success. Thus, we intended to analyse videos of digital problem-based learning (dPBL) sessions, focusing on knowledge acquisition and interaction dynamics among groups with different levels of prior knowledge to reveal any distinctions.Methods: This study employed a pilot design by dividing 60 dental students into twelve subgroups with less or more prior knowledge, determined by a pre-semester multiple choice test (MCQ). The groups engaged in videotaped dPBL cases, which were examined regarding group interactions and tutor effectiveness. The learning achievement was assessed through a post-semester MCQ, an oral and practical exam.Results: The video analysis showed that dPBL groups with less prior knowledge achieved significantly higher tutor effectiveness and group interaction utterances, but that the percentage of time in which utterances occurred was similar in both groups. Related to the MCQ results, the students with less prior knowledge learned four times more than those with profound previous abilities, but no significant difference was found in the results of the oral exam and practical exam.Conclusions: The interaction dynamics in dPBL depend on the group's amount of prior knowledge. Especially groups including participants with less prior knowledge seemed to benefit from dPBL in comparison to groups with more prior knowledge. The dPBL groups acquired knowledge in different ways during the courses but, finally, all students arrived at a similar level of knowledge.Zielsetzung: Forschungsbefunde zum problemorientierten Lernen (POL) zeigen, dass Untersuchungen von Videoaufzeichnungen tutorieller Lernsitzungen bedeutsame Einblicke in kognitive Prozesse ermöglichen. Der Einfluss von Vorwissen auf Lernerfolg unter Einbezug der Lehr-Lern-Interaktionsdynamik wurde in der medizinischen Ausbildung bisher allerdings noch nicht untersucht, obwohl die Faktoren eine Schlüsselrolle für den Erfolg von POL darstellen. Ziel der Studie ist es daher, digitale problemorientierten Lernsitzungen (dPOL) anhand von Videoaufzeichnungen zu analysieren und dabei Wissenserwerbsprozesse und die Interaktionsdynamik in den Lerngruppen in Abhängigkeit vom Vorwissensstand zu untersuchen.Methoden: In dieser Studie wurde ein Pilotdesign angewandt, bei dem 60 Zahnmedizinstudierende in zwölf Untergruppen mit geringerem oder höherem Vorwissen eingeteilt wurden. Die Erhebung des Vorwissens erfolgte durch einen Multiple-Choice-Test (MCQ) zu Beginn des Semesters. Die Gruppen bearbeiteten mit tutorieller Unterstützung dPOL-Fälle. Die Gruppeninteraktion und Tutor*innenaktivitäten wurden videographiert. Der Lernerfolg wurde am Ende des Semesters anhand eines MCQ sowie einer mündlichen und praktischen Prüfung erhoben.Ergebnisse: dPOL-Gruppen mit geringerem Vorwissen weisen eine signifikant höhere Anzahl an Aussagen in Gruppeninteraktionen und eine höhere Tutor*inneneffektivität auf. Der zeitliche Umfang der Äußerungen (Prozentsatz, die Äußerungen an der Gesamtzeit einnehmen) ist jedoch in beiden Gruppen gleich. Studierende mit geringerem Vorwissen zeigen einen höheren Lernzuwachs im MCQ. Signifikante Gruppenunterschiede in den Ergebnissen der mündlichen und praktischen Prüfung zeigen sich allerdings nicht.Schlussfolgerungen: Unterschiedliches Vorwissen der Lernenden führt zu unterschiedlichen Interaktionsdynamiken im dPOL. Im Hinblick auf Lernoutcomes profitieren insbesondere Lernende mit geringerem Vorwissen von dPOL. Die jeweiligen dPOL-Gruppen erreichten am Ende des Semesters ähnliche Lernergebnisse, die Befunde legen aber nahe, dass sich die Charakteristik des Wissenserwerbprozesses in Abhängigkeit vom Vorwissen unterscheidet

Auxin and ROP GTPase Signaling of Polar Nuclear Migration in Root Epidermal Hair Cells

Polar nuclear migration is crucial during the development of diverse eukaryotes. In plants, root hair growth requires polar nuclear migration into the outgrowing hair. However, knowledge about the dynamics and the regulatory mechanisms underlying nuclear movements in root epidermal cells remains limited. Here, we show that both auxin and Rho-of-Plant (ROP) signaling modulate polar nuclear position at the inner epidermal plasma membrane domain oriented to the cortical cells during cell elongation as well as subsequent polar nuclear movement to the outer domain into the emerging hair bulge in Arabidopsis (Arabidopsis thaliana). Auxin signaling via the nuclear AUXIN RESPONSE FACTOR7 (ARF7)/ARF19 and INDOLE ACETIC ACID7 pathway ensures correct nuclear placement toward the inner membrane domain. Moreover, precise inner nuclear placement relies on SPIKE1 Rho-GEF, SUPERCENTIPEDE1 Rho-GDI, and ACTIN7 (ACT7) function and to a lesser extent on VTI11 vacuolar SNARE activity. Strikingly, the directionality and/or velocity of outer polar nuclear migration into the hair outgrowth along actin strands also are ACT7 dependent, auxin sensitive, and regulated by ROP signaling. Thus, our findings provide a founding framework revealing auxin and ROP signaling of inner polar nuclear position with some contribution by vacuolar morphology and of actin-dependent outer polar nuclear migration in root epidermal hair cells

Auxin and ROP GTPase signaling of polar nuclear migration in root epidermal hair cells

Polar nuclear migration is crucial during the development of diverse eukaryotes. In plants, root hair growth requires polar nuclear migration into the outgrowing hair. However, knowledge about the dynamics and the regulatory mechanisms underlying nuclear movements in root epidermal cells remains limited. Here, we show that both auxin and Rho-of-Plant (ROP) signaling modulate polar nuclear position at the inner epidermal plasma membrane domain oriented to the cortical cells during cell elongation as well as subsequent polar nuclear movement to the outer domain into the emerging hair bulge in Arabidopsis (Arabidopsis thaliana). Auxin signaling via the nuclear AUXIN RESPONSE FACTOR7 (ARF7)/ARF19 and INDOLE ACETIC ACID7 pathway ensures correct nuclear placement toward the inner membrane domain. Moreover, precise inner nuclear placement relies on SPIKE1 Rho-GEF, SUPERCENTIPEDE1 Rho-GDI, and ACTIN7 (ACT7) function and to a lesser extent on VTI11 vacuolar SNARE activity. Strikingly, the directionality and/or velocity of outer polar nuclear migration into the hair outgrowth along actin strands also are ACT7 dependent, auxin sensitive, and regulated by ROP signaling. Thus, our findings provide a founding framework revealing auxin and ROP signaling of inner polar nuclear position with some contribution by vacuolar morphology and of actin-dependent outer polar nuclear migration in root epidermal hair cells