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.

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.

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.

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.

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LEA proteins are involved in plant stress tolerance. In Arabidopsis, the LEA_4 Pfam group is the biggest group with the majority of its members being expressed in dry seeds. To assess subcellular localization in vivo, we investigated 11 seed-expressed LEA_4 proteins in embryos dissected from dry seeds expressing LEA_4 fusion proteins under its native promoters with the Venus fluorescent protein (proLEA_4::LEA_4:Venus). LEA_4 proteins were shown to be localized in the endoplasmic reticulum, nucleus, mitochondria, and plastids. LEA9, in addition to the nucleus, was also found in cytoplasmic condensates in dry seeds dependent on cellular hydration level. Most investigated LEA_4 proteins were detected in 4-d-old seedlings. In addition, we assessed bioinformatic tools for predicting subcellular localization and promoter motifs of 11 seed-expressed LEA_4 proteins. Ratiometric bimolecular fluorescence complementation assays showed that LEA7, LEA29, and LEA48 form homodimers while heterodimers were formed between LEA7-LEA29 and LEA42-LEA48 in tobacco leaves. Interestingly, LEA48 homodimers and LEA42-LEA48 heterodimers formed droplets structures with liquid-like behavior. These structures, along with LEA9 cytoplasmic condensates, may have been formed through liquid-liquid phase separation. These findings suggest possible important roles of LLPS for LEA protein functions.

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.

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 recap the 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, posttranslational modifications or water deprivation, we give a detailed summary of the 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 (LLPS). 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.