Exploring The Metabolic Basis Of Desiccation Tolerance In Resurrection Plants And Their Potential Use As Low Water Input Forage

Abstract

The ability to survive vegetative-tissue desiccation, defined as the near complete loss (80-95%) of protoplasmic water, is an exceedingly rare trait and occurs only in about 300 species (0.15%) of all vascular plant species. So-called "resurrection plants" that are desiccation tolerant (DT) are evolutionarily widespread across the plant kingdom from bryophytes to angiosperms. We hypothesized that these plants have likely evolved and/or retained some coping mechanisms, not present in desiccation sensitive (DS) species, that can be used to engineer crop plants with improved drought-stress tolerance.Because the metabolome (i.e., a complete set of metabolites) are the end products of metabolism and represents the ultimate phenotypes of organisms as dictated by gene expression and enzyme activity, metabolomic analysis holds a particular importance when compared to transcriptomic and proteomic approaches. To study the metabolomics of DT in resurrection plants, we used Selaginella lepidophylla as a model plant. This lycophyte belongs to a lineage that occupied the earth 400 million years ago and can recover from years of desiccation. Moreover, it has some economical importance in Mexico where it is used in the traditional medicine. We performed a large-scale sister-group metabolomic comparison with a closely-related DS lycophyte Selaginella moellendorffii as well as a metabolomic profiling at various time points during a rehydration-dehydration cycle using an integrated platform consisting of a combination of three independent approaches: ultrahigh performance liquid chromatography/tandem mass spectrometry (UHLC/MS/MS2) optimized for basic species, UHLC/MS/MS2 optimized for acidic species, and gas chromatography/mass spectrometry (GC/MS). A total of 302 metabolites were compared between the two species at two time points (100% and 50% relative water content; RWC) showing remarkable differences that reflect two different ways of adaptation to the dehydration stress. While S. moellendorffii responds to the dehydration stress through stress-induced mechanisms, S. lepidophylla is pre-equipped with defense mechanisms even under favorable conditions. In terms of abundance, S. lepidophylla exhibited greater abundance in sugar alcohols and major osmolyte, γ-glutamyl amino acids, aromatic amino acids, secondary metabolites, and polyunsaturated fatty acids, which indicates a better adaptation to maintaining cellular integrity and protecting cells from oxidative stress, mechanisms that are critical for the acquisition of the DT trait. For metabolomic profiling, 251 metabolites were identified in S. lepidophylla of which many were produced constitutively indicating preemptive adaptation to the dehydration stress. Of these, carbohydrates were the most abundant, with trehalose, sucrose and glucose representing up to 50% of the metabolite amounts on a largely constitutive basis. Although desiccation was not associated with increased energy metabolism activity, several glycolysis and tricarboxylic acid (TCA) cycle intermediates displayed increased abundance during the early stages of dehydration, indicative of readiness for the transition to the desiccated state. Unlike sugar alcohols and some glycolysis intermediates that were more abundant in the hydrated state, other metabolomic groups such as nitrogen-rich compounds and γ-glutamyl amino acids were more abundant in dehydrating and dry states, which indicates that S. lepidophylla relies upon both constitutive and rehydration/dehydration-induced metabolite production to survive desiccation. Lastly, we investigated three South African Sporobolus species: the DT species S. stapfianus, the DS species S. indicus var. pyramidalis, and the DS species S. fimbriatus for their potential use as forage grasses in arid and semi-arid regions such as Nevada. Our results showed that S. fimbriatus, although is more susceptible to drought stress, produced more biomass compared to the other species. Despite susceptibility to drought, this species requires less water than Alfalfa, common forage used in the region. For forage quality, these species contain considerable amounts of protein, tolerable amounts of fiber, and major minerals. All the minerals exceeded the minimum requirements of beef cattle, with the exception of zinc that appeared low in all the three species. Moreover, all the minerals remained below the toxicity levels for beef cattle.Findings from this study have greatly improved our understanding of the metabolisms of DT in resurrection plants and have paved the road towards domesticating low water-requiring good forage grasses in arid and semi-arid regions.