Affinity Purification Protocol Starting with a Small Molecule as Bait.

Protein-metabolite interactions (PMIs) are fundamental for several biological processes. Even though PMI studies have increased in recent years, our knowledge is still limited. The screening of PMIs using small molecules as bait will broaden our ability to uncover novel PMIs, setting the basis for establishing their biological relevance. Here, we describe a protocol that allows the identification of multiple protein partners for one ligand. This protocol describes a straightforward methodology that can be adapted to a wide variety of organisms.

LEAfing through literature: late embryogenesis abundant proteins coming of age-achievements and perspectives.

To deal with increasingly severe periods of dehydration related to global climate change, it becomes increasingly important to understand the complex strategies many organisms have developed to cope with dehydration and desiccation. While it is undisputed that late embryogenesis abundant (LEA) proteins play a key role in the tolerance of plants and many anhydrobiotic organisms to water limitation, the molecular mechanisms are not well understood. In this review, we summarize current knowledge of the physiological roles of LEA proteins and discuss their potential molecular functions. As these are ultimately linked to conformational changes in the presence of binding partners, post-translational modifications, or water deprivation, we provide a detailed summary of current knowledge on the structure-function relationship of LEA proteins, including their disordered state in solution, coil to helix transitions, self-assembly, and their recently discovered ability to undergo liquid-liquid phase separation. We point out the promising potential of LEA proteins in biotechnological and agronomic applications, and summarize recent advances. We identify the most relevant open questions and discuss major challenges in establishing a solid understanding of how these intriguing molecules accomplish their tasks as cellular sentinels at the limits of surviving water scarcity.

Plant Stress Granules: Trends and Beyond.

Stress granules (SGs) are dynamic membrane-less condensates transiently assembled through liquid-liquid phase separation (LLPS) in response to stress. SGs display a biphasic architecture constituted of core and shell phases. The core is a conserved SG fraction fundamental for its assembly and consists primarily of proteins with intrinsically disordered regions and RNA-binding domains, along with translational-related proteins. The shell fraction contains specific SG components that differ among species, cell type, and developmental stage and might include metabolic enzymes, receptors, transcription factors, untranslated mRNAs, and small molecules. SGs assembly positively correlates with stalled translation associated with stress responses playing a pivotal role during the adaptive cellular response, post-stress recovery, signaling, and metabolic rewire. After stress, SG disassembly releases mRNA and proteins to the cytoplasm to reactivate translation and reassume cell growth and development. However, under severe stress conditions or aberrant cellular behavior, SG dynamics are severely disturbed, affecting cellular homeostasis and leading to cell death in the most critical cases. The majority of research on SGs has focused on yeast and mammals as model organism. Nevertheless, the study of plant SGs has attracted attention in the last few years. Genetics studies and adapted techniques from other non-plant models, such as affinity capture coupled with multi-omics analyses, have enriched our understanding of SG composition in plants. Despite these efforts, the investigation of plant SGs is still an emerging field in plant biology research. In this review, we compile and discuss the accumulated progress of plant SGs regarding their composition, organization, dynamics, regulation, and their relation to other cytoplasmic foci. Lastly, we will explore the possible connections among the most exciting findings of SGs from mammalian, yeast, and plants, which might help provide a complete view of the biology of plant SGs in the future.

PEST sequences from a cactus dehydrin regulate its proteolytic degradation.

Dehydrins (DHNs) are intrinsically disordered proteins expressed under cellular dehydration-related stresses. In this study, we identified potential proteolytic PEST sequences located at the central and C-terminal regions from the Opuntia streptacantha OpsDHN1 protein. In order to evaluate these PEST sequences as proteolytic tags, we generated a translational fusion with the GUS reporter protein and OpsDHN1 coding sequence. We found a GUS degradation effect in tobacco agro-infiltrated leaves and Arabidopsis transgenic lines that expressed the fusion GUS::OpsDHN1 full-length. Also, two additional translational fusions between OpsDHN1 protein fragments that include the central (GUS::PEST-1) or the C-terminal (GUS::PEST-2) PEST sequences were able to decrease the GUS activity, with PEST-2 showing the greatest reduction in GUS activity. GUS signal was abated when the OpsDHN1 fragment that includes both PEST sequences (GUS::PEST-1-2) were fused to GUS. Treatment with the MG132 proteasome inhibitor attenuated the PEST-mediated GUS degradation. Point mutations of phosphorylatable residues in PEST sequences reestablished GUS signal, hence these sequences are important during protein degradation. Finally, in silico analysis identified potential PEST sequences in other plant DHNs. This is the first study reporting presence of PEST motifs in dehydrins.

In vivo evidence for homo- and heterodimeric interactions of Arabidopsis thaliana dehydrins AtCOR47, AtERD10, and AtRAB18.

Dehydrins (DHNs) are intrinsically disordered proteins that play central roles in plant abiotic stress responses; however, how they work remains unclear. Herein, we report the in planta subcellular localization of Arabidopsis thaliana DHNs AtCOR47, AtERD10, and AtRAB18 through GFP translational fusions. To explore the dimerization ability of the Arabidopsis acidic DHNs AtCOR47 and AtERD10, we conducted an in planta DHN binding assay using the Bimolecular Fluorescence Complementation (BiFC) technique. Our analyses revealed homodimeric interactions for AtCOR47 and AtERD10; interestingly, heterodimeric associations also occurred with these DHNs, and these interactions were observed in the cytosol of tobacco cells. Furthermore, we evaluated whether Arabidopsis basic DHNs, such as AtRAB18, could also interact with itself and/or with AtCOR47 and AtERD10 in the BiFC system. Our data revealed homodimeric RAB18 complexes in the nucleus and cytosol, while heterodimeric associations between AtRAB18 and acidic DHNs occurred only in the cytosol. Finally, we demonstrated the presence of heterodimeric complexes among Arabidopsis AtCOR47, AtERD10, and AtRAB18 DHNs with their acidic ortholog the OpsDHN1 from Opuntia streptacantha; these heterodimeric interactions showed different subcellular distributions. Our results guide DHN research toward a new scenario where DHN/DHN oligomerization could be explored as a part of their molecular mechanism.

Characterization of maize spermine synthase 1 (ZmSPMS1): Evidence for dimerization and intracellular location.

Polyamines are ubiquitous positively charged metabolites that play an important role in wide fundamental cellular processes; because of their importance, the homeostasis of these amines is tightly regulated. Spermine synthase catalyzes the formation of polyamine spermine, which is necessary for growth and development in higher eukaryotes. Previously, we reported a stress inducible spermine synthase 1 (ZmSPMS1) gene from maize. The ZmSPMS1 enzyme differs from their dicot orthologous by a C-terminal extension, which contains a degradation PEST sequence involved in its turnover. Herein, we demonstrate that ZmSPMS1 protein interacts with itself in split yeast two-hybrid (Y2H) assays. A Bimolecular Fluorescence Complementation (BiFC) assay revealed that ZmSPMS1 homodimer has a cytoplasmic localization. In order to gain a better understanding about ZmSPMS1 interaction, two deletion constructs of ZmSPMS1 protein were obtained. The ΔN-ZmSPMS1 version, where the first 74 N-terminal amino acids were eliminated, showed reduced capability of dimer formation, whereas the ΔC-ZmSPMS1 version, lacking the last 40 C-terminal residues, dramatically abated the ZmSPMS1-ZmSPMS1 protein interaction. Recombinant protein expression in Escherichia coli of ZmSPMS1 derived versions revealed that deletion of its N-terminal domain affected the spermine biosynthesis, whereas C-terminal ZmSPMS1 truncated version fail to generate this polyamine. These data suggest that N- and C-terminal domains of ZmSPMS1 play a role in a functional homodimer.

Nuclear localization of the dehydrin OpsDHN1 is determined by histidine-rich motif.

The cactus OpsDHN1 dehydrin belongs to a large family of disordered and highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins, which accumulate during the late stages of embryogenesis and in response to abiotic stresses. Herein, we present the in vivo OpsDHN1 subcellular localization by N-terminal GFP translational fusion; our results revealed a cytoplasmic and nuclear localization of the GFP::OpsDHN1 protein in Nicotiana benthamiana epidermal cells. In addition, dimer assembly of OpsDHN1 in planta using a Bimolecular Fluorescence Complementation (BiFC) approach was demonstrated. In order to understand the in vivo role of the histidine-rich motif, the OpsDHN1-ΔHis version was produced and assayed for its subcellular localization and dimer capability by GFP fusion and BiFC assays, respectively. We found that deletion of the OpsDHN1 histidine-rich motif restricted its localization to cytoplasm, but did not affect dimer formation. In addition, the deletion of the S-segment in the OpsDHN1 protein affected its nuclear localization. Our data suggest that the deletion of histidine-rich motif and S-segment show similar effects, preventing OpsDHN1 from getting into the nucleus. Based on these results, the histidine-rich motif is proposed as a targeting element for OpsDHN1 nuclear localization.

A dehydrin-dehydrin interaction: the case of SK3 from Opuntia streptacantha.

Dehydrins belongs to a large group of highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins. It is well known that dehydrins are intrinsically disordered plant proteins that accumulate during the late stages of embryogenesis and in response to abiotic stresses; however, the molecular mechanisms by which their functions are carried out are still unclear. We have previously reported that transgenic Arabidopsis plants overexpressing an Opuntia streptacantha SK3 dehydrin (OpsDHN1) show enhanced tolerance to freezing stress. Herein, we show using a split-ubiquitin yeast two-hybrid system that OpsDHN1 dimerizes. We found that the deletion of regions containing K-segments and the histidine-rich region in the OpsDHN1 protein affects dimer formation. Not surprisingly, in silico protein sequence analysis suggests that OpsDHN1 is an intrinsically disordered protein, an observation that was confirmed by circular dichroism and gel filtration of the recombinantly expressed protein. The addition of zinc triggered the association of recombinantly expressed OpsDHN1 protein, likely through its histidine-rich motif. These data brings new insights about the molecular mechanism of the OpsDHN1 SK3-dehydrin.