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Abstract PremiseSeed longevity is critical for successful genebanking, but it is hard to detect or predict. We examined survival of genebanked seeds from species native to the United States to estimate longevity. We tested whether RNA integrity (RIN) can be used to detect aging and predict mortality. MethodsDry seeds from >100 species were stored for 28 ± 7 yr at −18°C. A recently harvested sample (cohort) from the same population provides a zero‐time reference. Germination and RIN were assessed and differences between cohorts were used to distinguish short‐lived seeds from long‐lived seeds. ResultsNo differences in germination or RIN were detected between cohorts in about one‐fourth of the species. Viability and/or RIN was lower in the stored cohort than in the recently harvested cohort in most species, and the size of the difference was used to infer aging rates. Differences in germination and RIN were correlated among the 100 samples tested; moderate correlation coefficients indicate that additional factors are involved in seed aging and its detection. ConclusionsOverall, longevity in the genebank appears to be similar for seeds from wild and domesticated species. We identified species that appeared to produce quite long‐lived and short‐lived seeds. Seeds from wild species tend to germinate slowly and asynchronously, and this confounds comparisons across storage times; deterioration is detected mostly after severe mortality. By contrast, RIN values decline before viability loss is detected and appear to be unaffected by wild seed traits. RIN tests during early storage can help predict seed longevity. 

Abstract PremiseGenebanks must maintain viable seeds for decades. Seeds that germinate are clearly alive, but some seeds, often from wild populations, do not germinate because they are dormant, empty, aged, or damaged (D.E.A.D.). This work evaluates the effects of D.E.A.D. factors on genebanked seeds using a unique dataset to improve genebanking practices and standards for ex situ conservation of seed collections. MethodsSeeds from over 100 species were recently collected from the same populations as seeds that were genebanked decades ago. Germination proportion and speed were measured after applying various temperature, chemical, or seed coat abrasion treatments. Viability was further tested using vital staining of samples with a low germination proportion. Proportions of dormant, empty, aged, and damaged seeds were compared between seed cohorts. ResultsGermination proportion and speed varied among samples, and cues to stimulate germination of dormant seeds were identified for individual species, leading to a positive correlation between viability metrics of germination and vital staining. Empty seeds primarily contribute to low germination in this study. Aging, indicated by lower and slower germination, was evident in several of the stored samples, compared to those that had been recently harvested. DiscussionThis unique approach demonstrates the feasibility of genebanking seeds from diverse endangered plant species using freezer storage. Genebanking methods that are more relevant for crop seeds need to be modified when applied to seeds from wild populations because the sample sizes tend to be small and the seeds tend to germinate slowly and asynchronously. 

Abstract Ubiquitin-binding shuttle proteins are important components of stress-induced biomolecular condensates in cells. Yeast Dsk2 scaffolds proteasome-containing condensates via multivalent interactions with proteasomes and polyubiquitinated substrates under stress conditions. Here, we identify the chaperone-binding STI1 domain as the main driver of Dsk2 self-association and phase separation. Using nuclear magnetic resonance (NMR) spectroscopy and computational simulations, we find that the STI1 domain interacts with three transient amphipathic helices within the intrinsically disordered regions of Dsk2. Removal of either the STI1 domain or these helices significantly reduces Dsk2’s propensity to form condensates. In vivo, perturbing STI1-helix interactions, specifically removal of the transient helices, reduces the formation of azide stress-induced Dsk2/proteasome condensates, in line with our in vitro results. Modeling of Dsk2 STI1-helix interactions reveals a binding mode reminiscent of chaperone STI1/DP2 domains interacting with client helices. Our findings support a model whereby STI1-helix interactions important for Dsk2 condensate formation can be replaced by STI1-client interactions for downstream chaperone or other protein quality control outcomes. 

Abstract Background and AimsRecent studies on desiccation-tolerant Australian rainforest seeds demonstrated that some were short-lived in storage. We sought to understand structural changes of storage lipids that might occur during storage at −20 °C that could contribute to a short lifespan. MethodsWe used differential scanning calorimetry (DSC) to examine exothermic and endothermic transitions during freezing and thawing in dry seed samples of 23 species. Seed samples and extracted triacylglycerols (TAGs) were cooled to −150 °C and rewarmed to 50 °C at 10 °C min−1; slower and faster rates of cooling/warming were used for a subset of species to examine lipid crystallization and melting kinetics. Thermograms were analysed for temperature and enthalpy of observed peaks, and these were compared with expected values to detect anomalies. Extracted lipids were further analysed using gas chromatography to characterize fatty acid composition. The thermal profiles of six species were used to design experiments comparing the impact of storage at −20 °C with storage at temperatures outside the range of thermal transitions. Key ResultsThermal activity was detected in 22 species within the narrow temperature range of −30 and −10 °C; activity at broader temperature ranges was also detected depending on species, cooling protocol and fatty acid composition. A profound interaction between DSC parameters and time at low temperature, as well as fatty acid composition, suggested that TAG crystallization rates contribute to low-temperature sensitivity. We confirmed that damage from TAG crystallization could be avoided by storing seeds at temperatures above TAG crystallization and melting events; storage at cryogenic temperatures improved survival over storage at −20 °C but requires further optimization to maintain pre-storage germination potential. ConclusionsWe conclude that the crystallization and melting of TAGs during storage may negatively impact seed longevity. Seed thermal profiles and rate of TAG crystallization may serve as predictive tools for sensitivity to storage at −20 °C. 

Abstract Multidomain proteins consist of folded domains connected by intrinsically disordered regions. The flexibility afforded by the disordered regions coupled to the structure and surface chemistry of folded regions allows for unique structural and functional features in these proteins. Yet how intramolecular interactions between disordered regions and folded domains affect multidomain protein structure and function remain poorly understood. Here we use a range of biophysical and computational approaches to measure the intramolecular interactions between the folded domains and disordered regions of ubiquilins (UBQLNs) - essential components of protein quality control that shuttle poly-ubiquitinated client proteins to proteasomal degradation or autophagy. Starting with the yeast UBQLN homolog Dsk2, we find that interactions between two folded domains located at the opposite ends of UBQLN bring about a closed conformation. The prevalence of this closed conformation, however, is modulated by intramolecular interactions involving the disordered regions and folded STI1 domain at the center of the protein. Simulations and analysis of UBQLN homologs across multiple eukaryotic lineages reveals that these disordered:folded domain interactions exist in some UBQLN homologs but are absent in others, indicating possible fundamental differences in function among proteins with the same multidomain architecture. 

Abstract Desiccation tolerance (DT), the ability to survive near‐complete cellular dehydration, is widespread in diaspores but rare in the vegetative tissues of land plants. The patchy and punctuated phylogenetic distribution of vegetative desiccation tolerance (VDT) suggests that the trait is both ancient and recurrent, yet the evolutionary trajectories remain unresolved. Here, we synthesize evidence across land plants to propose a framework for the evolution of VDT in embryophytes. We build on the current understanding of VDT as an ancestral trait, present in the gametophyte of early land plants. The transition to sporophyte dominance and resulting homiohydry in vascular plants coincides with the widespread loss of VDT, likely driven by relaxed selection for VDT, coupled with new structural constraints and anatomical innovations that facilitated water acquisition, transport, and retention. The core molecular modules of DT were retained in the diaspores of most land plants, where they served as evolutionary refugia for the essential building blocks of the trait. Some species later reestablished VDT by co‐opting deeply conserved diaspore modules and evolving key anatomical innovations to support them. We argue that such reestablishments of VDT are dependent on both anatomical predispositions as well as exposure to key selective pressures and ecological filters. We conclude that VDT is not a simple presence–absence trait, but rather a modular system, subject to anatomical constraints and contingent on the ecological context. Ultimately, we suggest that VDT serves as an elegant example of how complex traits emerge, persist, and shift across time. 

Abstract Intrinsically disordered regions (IDRs) pervasively engage in essential molecular functions, yet they are often poorly conserved as assessed by sequence alignment. To explore the seeming paradox of how sequence variability is compatible with persistent function, we examined the functional determinants for a poorly conserved but essential IDR. We show that IDR function depends on two distinct but related properties: sequence and chemical specificity. Whereas sequence specificity operates via binding motifs and depends on the precise order and identity of residues, chemical specificity reflects the sequence-encoded chemistry of multivalent interactions across an IDR and depends on local and global chemical properties. Unexpectedly, a binding motif essential in the wild-type IDR can be removed when compensatory changes to the sequence chemistry are introduced, highlighting the orthogonality and interoperability of these properties, and expanding the sequence space compatible with function. Our results provide a general framework for the functional constraints on IDR evolution. 

Abstract Physiologically relevant drought stress is difficult to apply consistently, and the heterogeneity in experimental design, growth conditions, and sampling schemes makes it challenging to compare water deficit studies in plants. Here, we reanalyzed hundreds of drought gene expression experiments across diverse model and crop species and quantified the variability across studies. We found that drought studies are surprisingly incomparable, even when accounting for differences in genotype, environment, drought severity, and method of drying. Many studies, including most Arabidopsis (Arabidopsis thaliana) work, lack high-quality phenotypic and physiological datasets to accompany gene expression, making it challenging to assess the severity or consistency of water deficit stress events. To help address this, we developed supervised learning classifiers that can distinguish RNAseq samples that likely experienced drought stress. While not a substitute for direct measurements, these classifiers may aid in interpreting existing datasets and assessing drought severity in studies lacking physiological metadata. Together, our analyses highlight the importance of paired physiological data to quantify stress severity for reproducibility and future data analyses. 

IntroductionDry seeds do not show obvious signs of life and so testing for viability, health and life expectancy can be challenging. Usually testing seed quality involves adding water and measuring metabolic capacity or growth potential by vital staining or germination assays. Importantly, most laboratory seed tests are intended to assay immediate viability, while most genebanks need tests that predict seed performance in the distant future. All currently available assays require considerable a priori knowledge of germination conditions and seeds large enough to dissect. Germination conditions are often unknown for seeds produced from wild species and are an important criterion for seed testing. MethodsUsing standardized methods (i.e., commercially available kits) we tested the feasibility of adapting a new seed quality assay that measures RNA integrity and is promising for cultivated species, to seeds from wild species. Most of the 100 wild species we include are rare or endangered and in need of preservation through genebanking. To determine the feasibility of measuring RNA integrity in seeds from wild populations, we compared the quality of RNA extracted from seeds that were recently harvested to those of the same species that have been genebanked for 16 to 41 years, with various seed traits examined for interference with RNA extraction and characterization. ResultsWe demonstrate reliable characterization of RNA quality across a diverse group of plants, despite variation in germination requirements, seed morphology or composition. RIN (RNA Integrity Number) values were usually high across all samples and variables, attesting to both the quality of newly collected material as well as its maintenance under genebanking conditions. This study conclusively demonstrates the feasibility of reliably extracting and characterizing RNA from dry seeds collected from wild populations, regardless of a variety of seed traits and morphologies. Relationships between RNA quality and seed age and viability require further exploration. 

Abstract Abiotic stresses, including drought, salt, heat, cold, flooding, and low nitrogen, are harmful to agriculture and increasing in frequency due to climate change. Plants can experience multiple stresses within a single season, which elicit shared or overlapping responses. We searched for core stress-responsive genes in maize across stressors through meta-analysis of public RNA-seq data. Using nearly 1,900 RNA-seq samples with both set operations and random forest classification, we identified a core set of 744 stress-responsive genes across the six stressors. These are enriched in transcription factors, including the stress-responsive families AP2/ERF-ERF, NAC, bZIP, HSF, and C2C2-CO-like. Co-expression network analysis demonstrated that core transcription factors are co-expressed with stress-specific genes, supporting their role in regulating both generalized and stress-specific responses. This provides a valuable resource for understanding stress tolerance mechanisms and guiding future efforts to enhance maize resilience under climate change.