Clathrin-mediated endocytosis is the major mechanism for eukaryotic plasma membrane-based proteome turn-over. In plants, clathrin-mediated endocytosis is essential for physiology and development, but ...the identification and organization of the machinery operating this process remains largely obscure. Here, we identified an eight-core-component protein complex, the TPLATE complex, essential for plant growth via its role as major adaptor module for clathrin-mediated endocytosis. This complex consists of evolutionarily unique proteins that associate closely with core endocytic elements. The TPLATE complex is recruited as dynamic foci at the plasma membrane preceding recruitment of adaptor protein complex 2, clathrin, and dynamin-related proteins. Reduced function of different complex components severely impaired internalization of assorted endocytic cargoes, demonstrating its pivotal role in clathrin-mediated endocytosis. Taken together, the TPLATE complex is an early endocytic module representing a unique evolutionary plant adaptation of the canonical eukaryotic pathway for clathrin-mediated endocytosis.
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•A multimeric protein complex is essential for plant clathrin-mediated endocytosis•The TPLATE complex is essential and functions in concert with the AP2 complex•Impaired TPLATE complex function abates internalization of several cargo molecules•The TPLATE complex represents an evolutionarily unique strategy to internalize cargo
The characterization of TPLATE, a plant-specific complex involved in clathrin-mediated endocytosis, highlights an evolutionary adaptation of an essential endocytic pathway in plants.
The target of rapamycin (TOR) kinase is a conserved regulatory hub that translates environmental and nutritional information into permissive or restrictive growth decisions. Despite the increased ...appreciation of the essential role of the TOR complex in plants, no large-scale phosphoproteomics or interactomics studies have been performed to map TOR signalling events in plants. To fill this gap, we combined a systematic phosphoproteomics screen with a targeted protein complex analysis in the model plant Arabidopsis thaliana. Integration of the phosphoproteome and protein complex data on the one hand shows that both methods reveal complementary subspaces of the plant TOR signalling network, enabling proteome-wide discovery of both upstream and downstream network components. On the other hand, the overlap between both data sets reveals a set of candidate direct TOR substrates. The integrated network embeds both evolutionarily-conserved and plant-specific TOR signalling components, uncovering an intriguing complex interplay with protein synthesis. Overall, the network provides a rich data set to start addressing fundamental questions about how TOR controls key processes in plants, such as autophagy, auxin signalling, chloroplast development, lipid metabolism, nucleotide biosynthesis, protein translation or senescence.
Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein ...interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (∼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; ∼5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.
In addition to the nucleus, mitochondria and chloroplasts in plant cells also contain genomes. Efficient DNA repair pathways are crucial in these organelles to fix damage resulting from endogenous ...and exogenous factors. Plant organellar genomes are complex compared with their animal counterparts, and although several plant-specific mediators of organelle DNA repair have been reported, many regulators remain to be identified. Here, we show that a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein, SWIB5, is capable of associating with mitochondrial DNA (mtDNA) in Arabidopsis thaliana. Gain- and loss-of-function mutants provided evidence for a role of SWIB5 in influencing mtDNA architecture and homologous recombination at specific intermediate-sized repeats both under normal and genotoxic conditions. SWIB5 interacts with other mitochondrial SWIB proteins. Gene expression and mutant phenotypic analysis of SWIB5 and SWIB family members suggests a link between organellar genome maintenance and cell proliferation. Taken together, our work presents a protein family that influences mtDNA architecture and homologous recombination in plants and suggests a link between organelle functioning and plant development.
The quiescent center (QC) plays an essential role during root development by creating a microenvironment that preserves the stem cell fate of its surrounding cells. Despite being surrounded by highly ...mitotic active cells, QC cells self-renew at a low proliferation rate. Here, we identified the ERF115 transcription factor as a rate-limiting factor of QC cell division, acting as a transcriptional activator of the phytosulfokine PSK5 peptide hormone. ERF115 marks QC cell division but is restrained through proteolysis by the APC/C CCS52A2 ubiquitin ligase, whereas QC proliferation is driven by brassinosteroid-dependent ERF115 expression. Together, these two antagonistic mechanisms delimit ERF115 activity, which is called upon when surrounding stem cells are damaged, revealing a cell cycle regulatory mechanism accounting for stem cell niche longevity.
SUMMARY
Plants have evolved an extensive specialized secondary metabolism. The colorful flavonoid anthocyanins, for example, not only stimulate flower pollination and seed dispersal, but also protect ...different tissues against high light, UV and oxidative stress. Their biosynthesis is highly regulated by environmental and developmental cues and induced by high sucrose levels. Expression of the biosynthetic enzymes involved is controlled by a transcriptional MBW complex, comprising (R2R3) MYB‐ and bHLH‐type transcription factors and the WD40 repeat protein TTG1. Anthocyanin biosynthesis is not only useful, but also carbon‐ and energy‐intensive and non‐vital. Consistently, the SnRK1 protein kinase, a metabolic sensor activated in carbon‐ and energy‐depleting stress conditions, represses anthocyanin biosynthesis. Here we show that Arabidopsis SnRK1 represses MBW complex activity both at the transcriptional and post‐translational level. In addition to repressing expression of the key transcription factor MYB75/PAP1, SnRK1 activity triggers MBW complex dissociation, associated with loss of target promoter binding, MYB75 protein degradation and nuclear export of TTG1. We also provide evidence for direct interaction with and phosphorylation of multiple MBW complex proteins. These results indicate that repression of expensive anthocyanin biosynthesis is an important strategy to save energy and redirect carbon flow to more essential processes for survival in metabolic stress conditions.
Significance Statement
In carbon‐ and energy‐depleting stress conditions, plants need to prioritize and redirect carbon flow from useful but expensive and non‐essential metabolites to more vital processes for metabolic homeostasis and survival. Consistently, the SnRK1 protein kinase represses activity of the MBW complex controlling anthocyanin biosynthesis gene expression, both at the transcriptional and post‐translational level.
Abstract
At meiosis, programmed meiotic DNA double-strand breaks are repaired via homologous recombination, resulting in crossovers (COs). From a large excess of DNA double-strand breaks that are ...formed, only a small proportion gets converted into COs because of active mechanisms that restrict CO formation. The Fanconi anemia (FA) complex proteins AtFANCM, MHF1 and MHF2 were previously identified in a genetic screen as anti-CO factors that function during meiosis in Arabidopsis thaliana. Here, pursuing the same screen, we identify FANCC as a new anti-CO gene. FANCC was previously only identified in mammals because of low primary sequence conservation. We show that FANCC, and its physical interaction with FANCE–FANCF, is conserved from vertebrates to plants. Further, we show that FANCC, together with its subcomplex partners FANCE and FANCF, regulates meiotic recombination. Mutations of any of these three genes partially rescues CO-defective mutants, which is particularly marked in female meiosis. Functional loss of FANCC, FANCE, or FANCF results in synthetic meiotic catastrophe with the pro-CO factor MUS81. This work reveals that FANCC is conserved outside mammals and has an anti-CO role during meiosis together with FANCE and FANCF.
Lay Summary
The Fanconi Anemia (FA) pathway is the subject of intense interest owing to the role of FA as a tumor suppressor. Three FA complex proteins, FANCM, MHF1 and MHF2, were identified as factors that suppress crossover during meiosis in the model plant Arabidopsis thaliana. Here, the authors extended these findings and identified a novel anti-crossover factor and showed that it encodes the plant FANCC homolog, which was previously thought to be vertebrate-specific. They further showed that FANCC regulates meiotic crossover together with two other FA proteins, FANCE and FANCF. This suggests that the FANCC–E–F subcomplex was already regulating DNA repair in the common ancestor of all living eukaryotes.
Graphical Abstract
Graphical Abstract
The FANCC-FANCE–FANCF complex is conserved from humans to plants, with a similar predicted structure. The complex regulates meiotic recombination and limits crossovers. Created with Biorender.com.
Multiple mechanisms limit meiotic crossovers Séguéla-Arnaud, Mathilde; Crismani, Wayne; Larchevêque, Cécile ...
Proceedings of the National Academy of Sciences - PNAS,
04/2015, Letnik:
112, Številka:
15
Journal Article
Recenzirano
Odprti dostop
Meiotic crossovers (COs) have two important roles, shuffling genetic information and ensuring proper chromosome segregation. Despite their importance and a large excess of precursors (i.e., DNA ...doublestrand breaks, DSBs), the number of COs is tightly regulated, typically one to three per chromosome pair. The mechanisms ensuring that most DSBs are repaired as non-COs and the evolutionary forces imposing this constraint are poorly understood. Here we identified Topoisomerase3α (TOP3α) and the RECQ4 helicases—theArabidopsisslow growth suppressor 1 (Sgs1)/Bloom syndrome protein (BLM) homologs—as major barriers to meiotic CO formation. First, the characterization of a specific TOP3α mutant allele revealed that, in addition to its role in DNA repair, this topoisomerase antagonizes CO formation. Further, we found that RECQ4A and RECQ4B constitute the strongest meiotic anti-CO activity identified to date, their concomitant depletion leading to a sixfold increase in CO frequency. In bothtop3αandrecq4abmutants, DSB number is unaffected, and extra COs arise from a normally minor pathway. Finally, both TOP3α and RECQ4A/B act independently of the previously identified anti-CO Fanconi anemia of complementation group M (FANCM) helicase. This finding shows that several parallel pathways actively limit CO formation and suggests that the RECQA/B and FANCM helicases prevent COs by processing different substrates. Despite a ninefold increase in CO frequency, chromosome segregation was unaffected. This finding supports the idea that CO number is restricted not because of mechanical constraints but likely because of the long-term costs of recombination. Furthermore, this work demonstrates how manipulating a few genes holds great promise for increasing recombination frequency in plant-breeding programs.
Plant stress responses involve numerous changes at the molecular and cellular level and are regulated by highly complex signaling pathways. Studying protein-protein interactions (PPIs) and the ...resulting networks is therefore becoming increasingly important in understanding these responses. Crucial in PPI networks are the so-called hubs or hub proteins, commonly defined as the most highly connected central proteins in scale-free PPI networks. However, despite their importance, a growing amount of confusion and controversy seems to exist regarding hub protein identification, characterization and classification. In order to highlight these inconsistencies and stimulate further clarification, this review critically analyses the current knowledge on hub proteins in the plant interactome field. We focus on current hub protein definitions, including the properties generally seen as hub-defining, and the challenges and approaches associated with hub protein identification. Furthermore, we give an overview of the most important large-scale plant PPI studies of the last decade that identified hub proteins, pointing out the lack of overlap between different studies. As such, it appears that although major advances are being made in the plant interactome field, defining hub proteins is still heavily dependent on the quality, origin and interpretation of the acquired PPI data. Nevertheless, many hub proteins seem to have a reported role in the plant stress response, including transcription factors, protein kinases and phosphatases, ubiquitin proteasome system related proteins, (co-)chaperones and redox signaling proteins. A significant number of identified plant stress hubs are however still functionally uncharacterized, making them interesting targets for future research. This review clearly shows the ongoing improvements in the plant interactome field but also calls attention to the need for a more comprehensive and precise identification of hub proteins, allowing a more efficient systems biology driven unraveling of complex processes, including those involved in stress responses.
The transcriptional coactivator ANGUSTIFOLIA3 (AN3) stimulates cell proliferation during Arabidopsis thaliana leaf development, but the molecular mechanism is largely unknown. Here, we show that ...inducible nuclear localization of AN3 during initial leaf growth results in differential expression of important transcriptional regulators, including GROWTH REGULATING FACTORS (GRFs). Chromatin purification further revealed the presence of AN3 at the loci of GRF5, GRF6, CYTOKININ RESPONSE FACTOR2, CONSTANS-UKE5 (COL5), HECATE1 (HEC1), and ARABIDOPSIS RESPONSE REGULATOR4 (ARR4). Tandem affinity purification of protein complexes using AN3 as bait identified plant SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin remodeling complexes formed around the ATPases BRAHMA (BRM) or SPLAYED. Moreover, SWI/SNF ASSOCIATED PROTEIN 73B (SWP73B) is recruited by AN3 to the promoters of GRF5, GRF3, COL5, and ARR4, and both SWP73B and BRM occupy the HEC1 promoter. Furthermore, we show that AN3 and BRM genetically interact. The data indicate that AN3 associates with chromatin remodelers to regulate transcription. In addition, modification of SWI3C expression levels increases leaf size, underlining the importance of chromatin dynamics for growth regulation. Our results place the SWI/SNF-AN3 module as a major player at the transition from cell proliferation to cell differentiation in a developing leaf.
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