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Chloroplasts are ancient organelles responsible for photosynthesis and various biosynthetic functions essential to most life on Earth. Many of these functions require tightly controlled regulatory processes to maintain homeostasis at the protein level. One such regulatory mechanism is the ubiquitin-proteasome system whose fundamental role is increasingly emerging in chloroplasts. In particular, the role of E3 ubiquitin ligases as determinants in the ubiquitination and degradation of specific intra-chloroplast proteins. Here, we highlight recent advances in understanding the roles of plant E3 ubiquitin ligases SP1, COP1, PUB4, CHIP, and TT3.1 as well as the ubiquitin-dependent segregase CDC48 in chloroplast function.

Strigolactones (SLs) are a unique and novel class of phytohormones that regulate numerous processes of growth and development in plants. Besides their endogenous functions as hormones, SLs are exuded by plant roots to stimulate critical interactions with symbiotic fungi but can also be exploited by parasitic plants to trigger their seed germination. In the past decade, since their discovery as phytohormones, rapid progress has been made in understanding the SL biosynthesis and signaling pathway. Of particular interest are the diversification of natural SLs and their exact mode of perception, selectivity, and hydrolysis by their dedicated receptors in plants. Here we provide an overview of the emerging field of SL perception with a focus on the diversity of canonical, non-canonical, and synthetic SL probes. Moreover, this review offers useful structural insights into SL perception, the precise molecular adaptations that define receptor-ligand specificities, and the mechanisms of SL hydrolysis and its attenuation by downstream signaling components.

Key Points Monomeric ubiquitin is relatively stable; however, it appears to be degraded by the proteasome following its own ubiquitylation, which is mediated by the thyroid receptor-interacting protein 12 (TRIP12) ligase. Ubiquitin is also degraded through two other mechanisms: along with the target substrate as part of the polyubiquitin chain attached to it, and along with a peptide attached, either linearly or in an isopeptide bond, to its carboxy-terminal Gly residue. Ubiquitin-protein ligases (E3s) are largely responsible for conferring substrate specificity to the ubiquitin–proteasome system (UPS). An increasing number of these ligases are being shown to be subject to self-ubiquitylation (also known as auto-ubiquitylation), ubiquitylation by heterologous ligases, or both. In some cases, both self-ubiquitylation and ubiquitylation by heterologous ligases lead to degradation of the protein. In other cases, self-ubiquitylation can regulate the cellular function of the ligase, whereas ubiquitylation by a heterologous E3 results in degradation of the target ligase. Other components of the UPS, including ubiquitin-conjugating enzymes (E2s) and deubiquitylating enzymes, are also subject to ubiquitylation. Components of the ubiquitin system are also subject to modification by other ubiquitin-like protein modifiers. The 26S proteasome is a stable, long-lived complex and is probably degraded through microautophagy. As part of the response to some specific cellular signals, such as oxidative stress, starvation, and stimulation of the NMDA ( N -methyl- D -aspartate) receptor, it is disassembled into its two subcomplexes, the 19S regulatory particle (RP) and the 20S catalytic (or core) particle (CP). The RP is probably disassembled into its individual subunits, which are degraded by the proteasome following ubiquitylation. Caspase-mediated cleavage of specific 19S subunits has also been shown to regulate proteasomal activity under certain conditions. The effect of disassembly of the 26S proteasome on the 20S complex has remained unclear: in some cases it was shown to inhibit its activity, to avoid damage of uncontrolled degradation, whereas in others cases it has been shown to stimulate activity and to efficiently remove — apparently in a ubiquitin-independent manner — excess damaged proteins. Ubiquitylation regulates essentially all of the intracellular processes in eukaryotes by modifying numerous cellular proteins in a spatially and temporally controlled manner. Many components of the ubiquitin–proteasome system are themselves modified by ubiquitylation; this regulates their activity or targets them for degradation. Ubiquitylation (also known as ubiquitination) regulates essentially all of the intracellular processes in eukaryotes through highly specific modification of numerous cellular proteins, which is often tightly regulated in a spatial and temporal manner. Although most often associated with proteasomal degradation, ubiquitylation frequently serves non-proteolytic functions. In light of its central roles in cellular regulation, it has not been surprising to find that many of the components of the ubiquitin system itself are regulated by ubiquitylation. This observation has broad implications for pathophysiology.

The world’s food supply is nearing a crisis in meeting the demands of an ever-growing global population, and an increase in both yield and nutrient value of major crops is vitally necessary to meet the increased population demand. Nutrients play an important role in plant metabolism as well as growth and development, and nutrient deficiency results in retarded plant growth and leads to reduced crop yield. A variety of cellular processes govern crop plant nutrient absorption from the soil. Among these, nutrient membrane transporters play an important role in the acquisition of nutrients from soil and transport of these nutrients to their target sites. In addition, as excess nutrient delivery has toxic effects on plant growth, these membrane transporters also play a significant role in the removal of excess nutrients in the crop plant. The key function provided by membrane transporters is the ability to supply the crop plant with an adequate level of tolerance against environmental stresses, such as soil acidity, alkalinity, salinity, drought, and pathogen attack. Membrane transporter genes have been utilized for the improvement of crop plants, with enhanced nutrient uptake leading to increased crop yield by providing tolerance against different biotic and abiotic stresses. Further understanding of the basic mechanisms of nutrient transport in crop plants could facilitate the advanced design of engineered plant crops to achieve increased yield and improve nutrient quality through the use of genetic technologies as well as molecular breeding. This review is focused on nutrient toxicity and tolerance mechanisms in crop plants to aid in understanding and addressing the anticipated global food demand.

Summary Creating true‐breeding lines is a critical step in plant breeding. Novel, completely homozygous true‐breeding lines can be generated by doubled haploid technology in single generation. Haploid induction through modification of the centromere‐specific histone 3 variant (CENH3), including chimeric proteins, expression of non‐native CENH3 and single amino acid substitutions, has been shown to induce, on outcrossing to wild type, haploid progeny possessing only the genome of the wild‐type parent, in Arabidopsis thaliana. Here, we report the characterization of 31 additional EMS‐inducible amino acid substitutions in CENH3 for their ability to complement a knockout in the endogenous CENH3 gene and induce haploid progeny when pollinated by the wild type. We also tested the effect of double amino acid changes, which might be generated through a second round of EMS mutagenesis. Finally, we report on the effects of CRISPR/Cas9‐mediated in‐frame deletions in the αN helix of the CENH3 histone fold domain. Remarkably, we found that complete deletion of the αN helix, which is conserved throughout angiosperms, results in plants which exhibit normal growth and fertility while acting as excellent haploid inducers when pollinated by wild‐type pollen. Both of these technologies, CRISPR mutagenesis and EMS mutagenesis, represent non‐transgenic approaches to the generation of haploid inducers.

Strigolactones (SLs) are a class of plant hormones that regulate numerous processes of growth and development. SL perception and signal activation involves interaction between F-box E3 ubiquitin ligase D3/MAX2 and DWARF14 (D14) α/β-hydrolase in a SL-dependent manner and targeting of D53/SMXL6/7/8 transcriptional repressors (SMXLs) for proteasome-mediated degradation. D3/MAX2 has been shown to exist in multiple conformational states in which the C-terminal helix (CTH) undergoes a closed-to-open dynamics and regulates D14 binding and SL perception. Despite the multiple modes of D3–D14 interactions found in vitro, the residues that regulate the conformational switch of D3/MAX2 CTH in targeting D53/SMXLs and the subsequent effect on SL signalling remain unclear. Here we elucidate the functional dynamics of ASK1–D3/MAX2 in SL signalling by leveraging conformational switch mutants in vitro and in plants. We report the crystal structure of a dislodged CTH of the ASK1–D3 mutant and demonstrate that disruptions in CTH plasticity via either CRISPR–Cas9 genome editing or expression of point mutation mutants result in impairment of SL signalling. We show that the conformational switch in ASK1–D3/MAX2 CTH directly regulates ubiquitin-mediated protein degradation. A dislodged conformation involved in D53/SMXLs SL-dependent recruitment and ubiquitination and an engaged conformation are required for the release of polyubiquitinated D53/SMXLs and subsequently D14 for proteasomal degradation. Finally, we uncovered an organic acid metabolite that can directly trigger the D3/MAX2 CTH conformational switch. Our findings unravel a new regulatory function of a SKP1–CUL1–F-box ubiquitin ligase in plant signalling.This study elucidates the functional dynamics of the ubiquitin ligase SCF D3/MAX2 as a key element in strigolactone signalling. The switch between D3/MAX2 conformational states regulates substrate targeting and can be elicited by a primary metabolite.

Phytohormone levels are regulated through specialized enzymes, participating not only in their biosynthesis but also in post-signaling processes for signal inactivation and cue depletion. Arabidopsis thaliana (At) carboxylesterase 15 (CXE15) and carboxylesterase 20 (CXE20) have been shown to deplete strigolactones (SLs) that coordinate various growth and developmental processes and function as signaling molecules in the rhizosphere. Here, we elucidate the X-ray crystal structures of AtCXE15 (both apo and SL intermediate bound) and AtCXE20, revealing insights into the mechanisms of SL binding and catabolism. The N-terminal regions of CXE15 and CXE20 exhibit distinct secondary structures, with CXE15 characterized by an alpha helix and CXE20 by an alpha/beta fold. These structural differences play pivotal roles in regulating variable SL hydrolysis rates. Our findings, both in vitro and in planta, indicate that a transition of the N-terminal helix domain of CXE15 between open and closed forms facilitates robust SL hydrolysis. The results not only illuminate the distinctive process of phytohormone breakdown but also uncover a molecular architecture and mode of plasticity within a specific class of carboxylesterases. Plant hormone levels are regulated by synthesis and breakdown, but the mechanism of strigolactone degradation remains unclear. This study reveals the structure of distinct carboxylesterases, which switch between open and closed conformations to facilitate strigolactone catabolism in plants.

Cryptochromes (CRYs) are evolutionarily conserved photoreceptors that mediate various light-induced responses in bacteria, plants, and animals. Plant cryptochromes govern a variety of critical growth and developmental processes including seed germination, flowering time and entrainment of the circadian clock. CRY’s photocycle involves reduction of their flavin adenine dinucleotide (FAD)-bound chromophore, which is completely oxidized in the dark and semi to fully reduced in the light signaling-active state. Despite the progress in characterizing cryptochromes, important aspects of their photochemistry, regulation, and light-induced structural changes remain to be addressed. In this study, we determine the crystal structure of the photosensory domain of Arabidopsis CRY2 in a tetrameric active state. Systematic structure-based analyses of photo-activated and inactive plant CRYs elucidate distinct structural elements and critical residues that dynamically partake in photo-induced oligomerization. Our study offers an updated model of CRYs photoactivation mechanism as well as the mode of its regulation by interacting proteins.Palayam, Ganapathy, Guercio et al. determine the crystal structure of the photosensory domain of Arabidopsis Cryptochrome-2 (CRY2) in a tetrameric active state, identifying critical residues that participate in photo-induced oligomerization of CRY2. This study offers an updated model of CRY’s photoactivation mechanism.

Plants utilize the ubiquitin proteasome system (UPS) to orchestrate numerous essential cellular processes, including the rapid responses required to cope with abiotic and biotic stresses. The 26S proteasome serves as the central catalytic component of the UPS that allows for the proteolytic degradation of ubiquitin-conjugated proteins in a highly specific manner. Despite the increasing number of studies employing cell-free degradation assays to dissect the pathways and target substrates of the UPS, the precise extraction methods of highly potent tissues remain unexplored. Here, we utilize a fluorogenic reporting assay using two extraction methods to survey proteasomal activity in different Arabidopsis thaliana tissues. This study provides new insights into the enrichment of activity and varied presence of proteasomes in specific plant tissues.

In the original publication, third author name was incorrectly published.