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Noninvasive phenotyping can quantify dynamic plant growth processes at higher temporal resolution than destructive phenotyping and can reveal phenomena that would be missed by end‐point analysis alone. Additionally, whole‐plant phenotyping can identify growth conditions that are optimal for both above‐ and below‐ground tissues. However, noninvasive, whole‐plant phenotyping approaches available today are generally expensive, complex, and non‐modular. We developed a low‐cost and versatile approach to noninvasively measure whole‐plant physiology over time by growing plants in isolated hydroponic chambers. We demonstrate the versatility of our approach by measuring whole‐plant biomass accumulation, water use, and water use efficiency every two days on unstressed and osmotically stressed sorghum accessions. We identified relationships between root zone acidification and photosynthesis on whole‐plant water use efficiency over time. Our system can be implemented using cheap, basic components, requires no specific technical expertise, and should be suitable for any non‐aquatic vascular plant species.

Desiccation tolerance evolved recurrently across diverse plant lineages to enable survival in water-limited conditions. Many resurrection plants are polyploid, and several groups have hypothesized that polyploidy contributed to the evolution of desiccation tolerance. However, due to the vast phylogenetic distance between resurrection plant lineages, the rarity of desiccation tolerance, and the prevalence of polyploidy in plants, this hypothesis has been difficult to test. Here, we surveyed natural variation in morphological, reproductive, and desiccation tolerance traits across several cytotypes of a single species to test for links between polyploidy and increased resilience. We sampled multiple natural populations of the resurrection grass Microchloa caffra across an environmental gradient ranging from mesic to xeric in South Africa. We describe two distinct ecotypes of M. caffra that occupy different extremes of the environmental gradient and exhibit consistent differences in ploidy, morphological, reproductive, and desiccation tolerance traits in both field and common growth conditions. Interestingly, plants with more polyploid genomes exhibited consistently higher recovery from desiccation, were less reproductive, and were larger than plants with smaller genomes and lower ploidy. These data indicate that selective pressures in increasingly xeric sites may play a role in maintaining and increasing desiccation tolerance and are mediated by changes in ploidy.

Tardigrades are a group of microscopic animals renowned for their ability to survive near complete desiccation. A family of proteins, unique to tardigrades, called Cytoplasmic Abundant Heat Soluble (CAHS) proteins are necessary to mediate robust desiccation tolerance in these animals. However, the mechanism(s) by which CAHS proteins help to protect tardigrades during water-loss have not been fully elucidated. Here we use thermogravimetric analysis to empirically test the proposed hypothesis that tardigrade CAHS proteins, due to their propensity to form hydrogels, help to retain water during desiccation. We find that regardless of its gelled state, both in vitro and in vivo, a model CAHS protein (CAHS D) retains no more water than common proteins and control cells in the dry state. However, we find that while CAHS D proteins do not increase the total amount of water retained in a dry system, they interact with the small amount of water that does remain. Our study indicates that desiccation tolerance mediated by CAHS D cannot be simply ascribed to water retention and instead implicates its ability to interact more tightly with residual water as a possible mechanism underlying its protective capacity. These results advance our fundamental understanding of tardigrade desiccation tolerance which could provide potential avenues for new technologies to aid in the storage of dry shelf-stable pharmaceuticals and the generation of stress tolerant crops to ensure food security in the face of global climate change.

To survive extreme drying (anhydrobiosis), many organisms, spanning every kingdom of life, accumulate intrinsically disordered proteins (IDPs). For decades, the ability of anhydrobiosis‐related IDPs to form transient amphipathic helices has been suggested to be important for promoting desiccation tolerance. However, evidence empirically supporting the necessity and/or sufficiency of helicity in mediating anhydrobiosis is lacking. Here, we demonstrate that the linker region of CAHS D, a desiccation‐related IDP from the tardigradeHypsibius exemplaris, that contains significant helical structure, is the protective portion of this protein. Perturbing the sequence composition and grammar of the linker region of CAHS D, through the insertion of helix‐breaking prolines, modulating the identity of charged residues, or replacement of hydrophobic amino acids with serine or glycine residues results in variants with different degrees of helical structure. Importantly, correlation of protective capacity and helical content in variants generated through different helix perturbing modalities does not show as strong a trend, suggesting that while helicity is important, it is not the only property that makes a protein protective during desiccation. These results provide direct evidence for the decades‐old theory that helicity of desiccation‐related IDPs is linked to their anhydrobiotic capacity.

Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins, which also function to prevent perturbations induced by drying in vitro and in heterologous systems. CAHS proteins have been shown to form gels both in vitro and in vivo, which has been speculated to be linked to their protective capacity. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of fibrillization and gelation for CAHS D similar to that of intermediate filament assembly. We show that in vitro, gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying. In vivo, we observe that CAHS D forms fibrillar networks during osmotic stress. Fibrillar networking of CAHS D improves survival of osmotically shocked cells. We observe two emergent properties associated with fibrillization; (i) prevention of cell volume change and (ii) reduction of metabolic activity during osmotic shock. We find that there is no significant correlation between maintenance of cell volume and survival, while there is a significant correlation between reduced metabolism and survival. Importantly, CAHS D's fibrillar network formation is reversible and metabolic rates return to control levels after CAHS fibers are resolved. This work provides insights into how tardigrades induce reversible biostasis through the self‐assembly of labile CAHS gels.

Water is essential for all active life processes. Despite this, there are a number of organisms that can survive prolonged desiccation. The vitrification hypothesis posits that such organisms survive desiccation by forming non-crystalline amorphous (vitrified) solids, often through the accumulation of protective disaccharides. In line with this theory, vitrification has been shown to be essential for desiccation tolerance in many organisms that survive extreme drying. However, it is known that not all vitrified materials are protective and that certain physio-chemical properties correlate with the protection in the glassy state. Furthermore, recent evidence suggests that the physio-chemical properties that correlate with protection can vary depending on the chemical nature of similarly sized protectants. While the chemistry of protectants has been probed in relation to the protective properties they induce when vitrified, the effect of protectant size on glassy properties and protection during drying has not been investigated. Here, we study the effect of the polymer size of sucrose on glassy properties associated with protection in the vitrified state. The monomer sucrose, and the polymers polysucrose 70 and polysucrose 400 (70 and 400 refer to the molecular weight of the polymers in kDa). Using these three different-sized sucrose polymers, we find that each of the glassy properties we investigated including; enzyme protection, water content, glass transition temperature, and glass former fragility, were affected by polymer size. However, only one vitrified property, glass transition temperature, correlated with protection during drying. This correlation is heavily dependent on sucrose polymer size. Increased glass transition midpoint temperature correlated positively with protection conferred by monomeric sucrose (p-value = 0.009,R²= 0.840), whereas this correlation was bi-phasic for polysucrose 70, and had an inverse relationship for polysucrose 400 (p-value = 0.120, R2 = 0.490). Our results indicate that the size of vitrifying protectants can have a profound effect on glassy properties as well as on how these properties correlate with protection in the dry state. Beyond desiccation tolerance, these findings provide insights for the development of new technologies for the stabilization of biological material in the dry state.