DNA methylation-mediated modulation of rapid desiccation tolerance acquisition and dehydration stress memory in the resurrection plant Boea hygrometrica

Pre-exposure of plants to various abiotic conditions confers improved tolerance to subsequent stress. Mild drought acclimation induces acquired rapid desiccation tolerance (RDT) in the resurrection plant Boea hygrometrica, but the mechanisms underlying the priming and memory processes remain unclear. In this study, we demonstrated that drought acclimation-induced RDT can be maintained for at least four weeks but was completely erased after 18 weeks based on a combination of the phenotypic and physiological parameters. Global transcriptome analysis identified several RDT-specific rapid dehydration-responsive genes related to cytokinin and phospholipid biosynthesis, nitrogen and carbon metabolism, and epidermal morphogenesis, most of which were pre-induced by drought acclimation. Comparison of whole-genome DNA methylation revealed dehydration stress-responsive hypomethylation in the CG, CHG, and CHH contexts and acclimation-induced hypermethylation in the CHH context of the B. hygrometrica genome, consistent with the transcriptional changes in methylation pathway genes. As expected, the global promoter and gene body methylation levels were negatively correlated with gene expression levels in both acclimated and dehydrated plants but showed no association with transcriptional divergence during the procedure. Nevertheless, the promoter methylation variations in the CG and CHG contexts were significantly associated with the differential expression of genes required for fundamental genetic processes of DNA conformation, RNA splicing, translation, and post-translational protein modification during acclimation, growth, and rapid dehydration stress response. It was also associated with the dehydration stress-induced upregulation of memory genes, including pre-mRNA-splicing factor 38A, vacuolar amino acid transporter 1-like, and UDP-sugar pyrophosphorylase, which may contribute directly or indirectly to the improvement of dehydration tolerance in B. hygrometrica plants. Altogether, our findings demonstrate the potential implications of DNA methylation in dehydration stress memory and, therefore, provide a molecular basis for enhanced dehydration tolerance in plants induced by drought acclimation.

Drought tolerance and acclimation in Pinus ponderosa seedlings: the influence of nitrogen form

Drought is a limiting factor to forest regeneration and restoration, which is likely to increase in intensity and duration under future climates. Nitrogen (N) nutrition is related to drought-resistance mechanisms in trees. However, the influence of chemical N form (inorganic and organic N) on physiological traits related to drought resistance has been sparsely studied in conifer seedlings. We investigated the effect of N forms on morpho-physiological traits of Pinus ponderosa Dougl. ex Laws. seedlings and subsequent influences in drought tolerance and acclimation. One-year-old seedlings were fertilized during 10 weeks at 9 mM N with different N forms [either NH4+, NO3− or organic N (amino acids mixture)] in their second year of growth. After fertilization, we measured traits associated with intrinsic drought tolerance (shoot water relations, osmotic regulation, photosynthesis and cell membrane stability). Seedlings were then subjected to an 8-week drought period at varying drought intensities to evaluate plant acclimation mechanisms. We demonstrated that P. ponderosa seedlings could efficiently use amino acids as a primary N source, showing similar performance to those grown with inorganic N forms. Nitrogen form influenced mainly drought-acclimation mechanisms rather than intrinsic drought tolerance. Osmotic potential at saturation (Ψπsat) was marginally affected by N form, and a significant relationship between proline concentration in needles and Ψπsat was found. During acclimation, seedlings fertilized with organic N minimized needle senescence, retained more nutrients in the oldest needles, had maximum increments in proline concentration and hastened the development of water-use efficiency mechanisms compared with those fertilized with inorganic N sources. Our results suggest an improved physiological drought acclimation of organic N-fertilized seedlings.

Highly ABA-Induced 1 (HAI1)-Interacting protein HIN1 and drought acclimation-enhanced splicing efficiency at intron retention sites

The Highly ABA-Induced 1 (HAI1) protein phosphatase is a central component of drought-related signaling. A screen for HAI1-interacting proteins identified HAI1-Interactor 1 (HIN1), a nuclear protein of unknown function which could be dephosphorylated by HAI1 in vitro. HIN1 colocalization and interaction with serine-arginine rich (SR) splicing factors and appearance of nuclear speckle-localized HIN1 during low water potential (ψw) stress suggested a pre-mRNA splicing-related function. RNA sequencing of Arabidopsis Col-0 wild type identified more than 500 introns where moderate severity low ψw altered intron retention (IR) frequency. Surprisingly, nearly 90% of these had increased splicing efficiency (decreased IR) during stress. For one-third of these introns, ectopic HIN1 expression (35S:HIN1) in unstressed plants mimicked the increased splicing efficiency seen in stress-treated wild type. HIN1 bound to a GAA-repeat, Exonic Splicing Enhancer-like RNA motif enriched in flanking sequence around HIN1-regulated introns. Genes with stress and HIN1-affected splicing efficiency were enriched for abiotic stress and signaling-related functions. The 35S:HIN1 plants had enhanced growth maintenance during low ψw, while hin1 mutants had reduced growth, further indicating the role of HIN1 in drought response. HIN1 is annotated as an MYB/SANT domain protein but has limited homology to other MYB/SANT proteins and is not related to known yeast or metazoan RNA-binding proteins or splicing regulators. Together these data identify HIN1 as a plant-specific RNA-binding protein, show a specific effect of drought acclimation to promote splicing efficiency of IR-prone introns, and also discover HAI1–HIN1 interaction and dephosphorylation that connects stress signaling to splicing regulation.