UBP12 and UBP13 negatively regulate the activity of the ubiquitin-dependent peptidases DA1, DAR1 and DAR2

Protein ubiquitination is a very diverse post-translational modification leading to protein degradation or delocalization, or altering protein activity. In Arabidopsis thaliana, two E3 ligases, BIG BROTHER (BB) and DA2, activate the latent peptidases DA1, DAR1 and DAR2 by mono-ubiquitination at multiple sites. Subsequently, these activated peptidases destabilize various positive regulators of growth. Here, we show that two ubiquitin-specific proteases, UBP12 and UBP13, deubiquitinate DA1, DAR1 and DAR2, hence reducing their peptidase activity. Overexpression of UBP12 or UBP13 strongly decreased leaf size and cell area, and resulted in lower ploidy levels. Mutants in which UBP12 and UBP13 were downregulated produced smaller leaves that contained fewer and smaller cells. Remarkably, neither UBP12 nor UBP13 were found to be cleavage substrates of the activated DA1. Our results therefore suggest that UBP12 and UBP13 work upstream of DA1, DAR1 and DAR2 to restrict their protease activity and hence fine-tune plant growth and development

Transferring an optimized TAP-toolbox for the isolation of protein complexes to a portfolio of rice tissues

Proteins are the cell's functional entities. Rather than operating independently, they interact with other proteins. Capturing in vivo protein complexes is therefore crucial to gain understanding of the function of a protein in a cellular context. Affinity purification coupled to mass spectrometry has proven to yield a wealth of information about protein complex constitutions for a broad range of organisms. For Oryza sativa, the technique has been initiated in callus and shoots, but has not been optimized ever since. We translated an optimized tandem affinity purification (TAP) approach from Arabidopsis thaliana toward Oryza sativa, and demonstrate its applicability in a variety of rice tissues. A list of non-specific and false positive interactors is presented, based on re-occurrence over more than 170 independent experiments, to filter bona fide interactors. We demonstrate the sensitivity of our approach by isolating the complexes for the rice ANAPHASE PROMOTING COMPLEX SUBUNIT 10 (APC10) and CYCLIN-DEPENDENT KINASE D (CDKD) proteins from the proliferation zone of the emerging fourth leaf. Next to APC10 and CDKD, we tested several additional baits in the different rice tissues and reproducibly retrieved at least one interactor for 81.4 % of the baits screened for in callus tissue and T1 seedlings. By transferring an optimized TAP tag combined with state-of-the-art mass spectrometry, our TAP protocol enables the discovery of interactors for low abundance proteins in rice and opens the possibility to capture complex dynamics by comparing tissues at different stages of a developing rice organ

Recent trends in plant protein complex analysis in a developmental context

Because virtually all proteins interact with other proteins, studying protein-protein interactions (PPIs) is fundamental in understanding protein function. This is especially true when studying specific developmental processes, in which proteins often make developmental stage-or tissue specific interactions. However, studying these specific PPIs in planta can be challenging. One of the most widely adopted methods to study PPIs in planta is affinity purification coupled to mass spectrometry (AP/MS). Recent developments in the field of mass spectrometry have boosted applications of AP/MS in a developmental context. This review covers two main advancements in the field of affinity purification to study plant developmental processes: increasing the developmental resolution of the harvested tissues and moving from affinity purification to affinity enrichment. Furthermore, we discuss some new affinity purification approaches that have recently emerged and could have a profound impact on the future of protein interactome analysis in plants

The transcriptional repressor complex FRS7-FRS12 regulates flowering time and growth in Arabidopsis

Most living organisms developed systems to efficiently time environmental changes. The plant-clock acts in coordination with external signals to generate output responses determining seasonal growth and flowering time. Here, we show that two Arabidopsis thaliana transcription factors, FAR1 RELATED SEQUENCE 7 (FRS7) and FRS12, act as negative regulators of these processes. These proteins accumulate particularly in short-day conditions and interact to form a complex. Loss-of-function of FRS7 and FRS12 results in early flowering plants with overly elongated hypocotyls mainly in short days. We demonstrate by molecular analysis that FRS7 and FRS12 affect these developmental processes in part by binding to the promoters and repressing the expression of GIGANTEA and PHYTOCHROME INTERACTING FACTOR 4 as well as several of their downstream signalling targets. Our data reveal a molecular machinery that controls the photoperiodic regulation of flowering and growth and offer insight into how plants adapt to seasonal changes

Dynamic changes in ANGUSTIFOLIA3 complex composition reveal a growth regulatory mechanism in the maize leaf

Most molecular processes during plant development occur with a particular spatio-temporal specificity. Thus far, it has remained technically challenging to capture dynamic protein-protein interactions within a growing organ, where the interplay between cell division and cell expansion is instrumental. Here, we combined high-resolution sampling of the growing maize (Zea mays) leaf with tandem affinity purification followed by mass spectrometry. Our results indicate that the growth-regulating SWI/SNF chromatin remodeling complex associated with ANGUSTIFOLIA3 (AN3) was conserved within growing organs and between dicots and monocots. Moreover, we were able to demonstrate the dynamics of the AN3-interacting proteins within the growing leaf, since copurified GROWTH-REGULATING FACTORs (GRFs) varied throughout the growing leaf. Indeed, GRF1, GRF6, GRF7, GRF12, GRF15, and GRF17 were significantly enriched in the division zone of the growing leaf, while GRF4 and GRF10 levels were comparable between division zone and expansion zone in the growing leaf. These dynamics were also reflected at the mRNA and protein levels, indicating tight developmental regulation of the AN3-associated chromatin remodeling complex. In addition, the phenotypes of maize plants overexpressing miRNA396a-resistant GRF1 support a model proposing that distinct associations of the chromatin remodeling complex with specific GRFs tightly regulate the transition between cell division and cell expansion. Together, our data demonstrate that advancing from static to dynamic protein-protein interaction analysis in a growing organ adds insights in how developmental switches are regulated

The SBT6.1 subtilase processes the GOLVEN1 peptide controlling cell elongation

Maturation of GLV signaling peptides requires two SBT6 subtilases. SBT6 proteolytic activity is further regulated by the Serpin1 inhibitor, implying a complex network that controls cell elongation in Arabidopsis.The GOLVEN (GLV) gene family encode small secreted peptides involved in important plant developmental programs. Little is known about the factors required for the production of the mature bioactive GLV peptides. Through a genetic suppressor screen in Arabidopsis thaliana, two related subtilase genes, AtSBT6.1 and AtSBT6.2, were identified that are necessary for GLV1 activity. Root and hypocotyl GLV1 overexpression phenotypes were suppressed by mutations in either of the subtilase genes. Synthetic GLV-derived peptides were cleaved in vitro by the affinity-purified SBT6.1 catalytic enzyme, confirming that the GLV1 precursor is a direct subtilase substrate, and the elimination of the in vitro subtilase recognition sites through alanine substitution suppressed the GLV1 gain-of-function phenotype in vivo. Furthermore, the protease inhibitor Serpin1 bound to SBT6.1 and inhibited the cleavage of GLV1 precursors by the protease. GLV1 and its homolog GLV2 were expressed in the outer cell layers of the hypocotyl, preferentially in regions of rapid cell elongation. In agreement with the SBT6 role in GLV precursor processing, both null mutants for sbt6.1 and sbt6.2 and the Serpin1 overexpression plants had shorter hypocotyls. The biosynthesis of the GLV signaling peptides required subtilase activity and might be regulated by specific protease inhibitors. The data fit with a model in which the GLV1 signaling pathway participates in the regulation of hypocotyl cell elongation, is controlled by SBT6 subtilases, and is modulated locally by the Serpin1 protease inhibitor

Unraveling the MAX2 protein network in Arabidopsis thaliana : identification of the protein phosphatase PAPP5 as a novel MAX2 interactor

The F-box protein MORE AXILLARY GROWTH 2 (MAX2) is a central component in the signaling cascade of strigolactones (SLs) as well as of the smoke-derived karrikins (KARs) and the so far unknown endogenous KAI2 ligand (KL). The two groups of molecules are involved in overlapping and unique developmental processes, and signal-specific outcomes are attributed to perception by the paralogous α/β-hydrolases DWARF14 (D14) for SL and KARRIKIN INSENSITIVE 2/HYPOSENSITIVE TO LIGHT (KAI2/HTL) for KAR/KL. In addition, depending on which receptor is activated, specific members of the SUPPRESSOR OF MAX2 1 (SMAX1)-LIKE (SMXL) family control KAR/KL and SL responses. As proteins that function in the same signal transduction pathway often occur in large protein complexes, we aimed at discovering new players of the MAX2, D14, and KAI2 protein network by tandem affinity purification in Arabidopsis cell cultures. When using MAX2 as a bait, various proteins were copurified, among which were general components of the Skp1-Cullin-F-box complex and members of the CONSTITUTIVE PHOTOMORPHOGENIC 9 signalosome. Here, we report the identification of a novel interactor of MAX2, a type 5 serine/threonine protein phosphatase, designated PHYTOCHROME-ASSOCIATED PROTEIN PHOSPHATASE 5 (PAPP5). Quantitative affinity purification pointed at PAPP5 as being more present in KAI2 rather than in D14 protein complexes. In agreement, mutant analysis suggests that PAPP5 modulates KAR/KL-dependent seed germination under suboptimal conditions and seedling development. In addition, a phosphopeptide enrichment experiment revealed that PAPP5 might dephosphorylate MAX2 in vivo independently of the synthetic SL analog, rac-GR24. Together, by analyzing the protein complexes to which MAX2, D14, and KAI2 belong, we revealed a new MAX2 interactor, PAPP5, that might act through dephosphorylation of MAX2 to control mainly KAR/KL-related phenotypes and, hence, provide another link with the light pathway

Évaluations par Cartes Conceptuelles à trous et apprentissage par les pairs

Cet article décrit un nouveau dispositif d’évaluation des acquis d’apprentissage basé sur des cartes conceptuelles « à trous » (CCàT) permettant également l’apprentissage par les pairs en grands auditoires durant les tests et une correction automatisée par des formulaires QCM.L’intérêt du dispositif est de garantir une évaluation qualitative (à haut niveau taxonomique) des apprentissages tout en facilitant la conception et la correction de ces évaluations par l’enseignant, même pour de grands groupes d’étudiants (>500) et en favorisant la coopération entre étudiants en amont et pendant les évaluations

GSyellow, a multifaceted tag for functional protein analysis in monocot and dicot plants

The ability to tag proteins has boosted the emergence of generic molecular methods for protein functional analysis. Fluorescent protein tags are used to visualize protein localization, and affinity tags enable the mapping of molecular interactions by, for example, tandem affinity purification or chromatin immunoprecipitation. To apply these widely used molecular techniques on a single transgenic plant line, we developed a multifunctional tandem affinity purification tag, named GS(yellow), which combines the streptavidin-binding peptide tag with citrine yellow fluorescent protein. We demonstrated the versatility of the GS(yellow) tag in the dicot Arabidopsis (Arabidopsis thaliana) using a set of benchmark proteins. For proof of concept in monocots, we assessed the localization and dynamic interaction profile of the leaf growth regulator ANGUSTIFOLIA3 (AN3), fused to the GS(yellow) tag, along the growth zone of the maize (Zea mays) leaf. To further explore the function of ZmAN3, we mapped its DNA-binding landscape in the growth zone of the maize leaf through chromatin immunoprecipitation sequencing. Comparison with AN3 target genes mapped in the developing maize tassel or in Arabidopsis cell cultures revealed strong conservation of AN3 target genes between different maize tissues and across monocots and dicots, respectively. In conclusion, the GS(yellow) tag offers a powerful molecular tool for distinct types of protein functional analyses in dicots and monocots. As this approach involves transforming a single construct, it is likely to accelerate both basic and translational plant research

Functional characterization of the Arabidopsis transcription factor bZIP29 reveals its role in leaf and root development

Plant bZIP group I transcription factors have been reported mainly for their role during vascular development and osmosensory responses. Interestingly, bZIP29 has been identified in a cell cycle interactome, indicating additional functions of bZIP29 in plant development. Here, bZIP29 was functionally characterized to study its role during plant development. It is not present in vascular tissue but is specifically expressed in proliferative tissues. Genome-wide mapping of bZIP29 target genes confirmed its role in stress and osmosensory responses, but also identified specific binding to several core cell cycle genes and to genes involved in cell wall organization. bZIP29 protein complex analyses validated interaction with other bZIP group I members and provided insight into regulatory mechanisms acting on bZIP dimers. In agreement with bZIP29 expression in proliferative tissues and with its binding to promoters of cell cycle regulators, dominant-negative repression of bZIP29 altered the cell number in leaves and in the root meristem. A transcriptome analysis on the root meristem, however, indicated that bZIP29 might regulate cell number through control of cell wall organization. Finally, ectopic dominant-negative repression of bZIP29 and redundant factors led to a seedling-lethal phenotype, pointing to essential roles for bZIP group I factors early in plant development