Exploring The Mechanisms Of Desiccation Tolerance In Resurrection Plants: Toward Developing Desiccation Tolerance Crops
AdvisorCushman, John C.
Biochemistry and Molecular Biology
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Desiccation tolerance (DT), which is the ability to survive and recover from the almost complete loss (80-95 %) of protoplasmic water, is extremely rare among vascular plant species. Only about 350 species (less than 0.15%) of vascular plants can survive the desiccation of vegetative tissues. Although rare, these so-called "resurrection plants" are evolutionally widespread among terrestrial organisms from bryophytes to angiosperms. The central hypothesis to be tested here is that the study of DT species will reveal novel genes and/or regulatory networks to cope with heat and drought stress that are not present in desiccation sensitive (DS) species. Understanding DT mechanisms will be a first step to strengthen the understanding of adaptive traits associated with low water demands and to engineer improved heat and drought stress tolerance in crops. Small-scale or large-scale transcriptomic analysis of resurrection plants revealed key insights into the mechanistic basis of DT. Next generation sequencing (NGS) technology has emerged recently as a tool to investigate genome-wide analysis of the transcriptome. To provide solid genomic resources, we conducted Sanger, 454, and Illumina sequencing from S. lepidophylla (DT) and Illumina sequencing from S. moellendorffii (DS), respectively. De novo and hybrid assemblies of S. lepidophylla and S. moellendorffii, respectively provided 45,959 contigs and 39,230 contigs, respectively. We detected 954 and 865 putative transcription factors (TFs) in S. lepidophylla and S. moellendorffii, respectively. Among these TF genes, basic helix-loop-helix (bHLH) TF was the most enriched in the DT S. lepidophylla. Interestingly, bHLH, ethylene insensitive3 (EIN3)-like (EIL), growth hormone-releasing factor (GRF), MADS (MINICHROMOSOME MAINTENANCE1, AGAMOUS, DEFICIENS, and SERUM RESPONSE FACTOR), NAC (NAM, ATAF, and CUC), nuclear factor Y (NF-Y), Squamosa promoter binding proteins (SBP) TF gene families were more abundant in S. lepidophylla than in S. moellendorffii.To study the transcriptome of DT in resurrection plants, a sister group comparison of two closely related Selaginella species that differed in their ability to survive desiccation of vegetative tissues. Comparative transcriptome profiling using Illumina-based next generation sequencing (NGS) revealed that Selaginella lepidophylla, a desiccation tolerant species, mounted a highly active transcriptional response to dehydration stress, whereas S. moellendorffii, a desiccation sensitive species, responded in a relatively passive manner. S. lepidophylla displayed 7,704 differentially expressed genes (DEGs) in contrast to only 348 in S. moellendorffii during a shift from 100% relative water content (RWC) to 50%. Several functional classes of genes, namely late embryogenesis abundant (LEA) proteins, heat shock proteins (HSPs), and reactive oxygen species (ROS) scavenging enzymes including 1-cysteine peroxiredoxin and glyoxalase I family enzymes showed increased or enhanced relative steady-start mRNA abundance only in the DT species following dehydration stress. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses also revealed that S. lepidophylla invokes the mRNA expression of a much wider array of biological processes and functional pathways under dehydration stress compared with those expressed in S. moellendorffii. In order to investigate the functional role of specific genetic determinants that might be associated with adaptation to desiccation tolerance, a set of late embryogenesis abundant (LEA) encoding genes from S. lepidophylla were evaluated for improving abiotic stress tolerance in stably transformed Arabidopsis thaliana. One group 4 LEA gene, SlLEA4, which showed increased steady-state mRNA accumulation in response to dehydration, was constitutively expressed A. thaliana imparted improved germination rates, increased root growth, and increased shoot biomass growth on Murashige and Skoog (MS) media containing various concentrations of sorbitol or NaCl compared with wild type (WT) or empty vector (EV) control plants. A SlLEA4::GFP fusion protein was localized to both the nucleus and the cytosol. In addition, in vitro assay revealed that SlLEA4 was capable of protecting Citrate synthase (CS) and Lactate dehydrogenase (LDH) from aggregation following freeze-thaw and drying treatments. The anti-aggregation properties of SlLEA4 indicates that this protein likely plays a critical role in tolerance to osmotic or ionic stress, by acting as a molecular shield to prevent aggregation of client proteins and the stabilization of macromolecular structures following the withdraw of bulk water from biological systems. Findings from this study will be invaluable for providing rational strategies for improved stress tolerance in crop plants.