Fig 1.
Tripogon loliiformis leaf tissues are alive at desiccation.
T. loliiformis plants were grown from seed harvested from a single source and grown in the glasshouse for two months, dehydrated to desiccation and rehydrated with the addition of water. Left Panels—fully hydrated (Top), dehydrating and desiccated (Middle) and 48h after rehydration (Bottom). Right Panels—Leaf tissue was harvested from hydrated and dehydrating plants at 60, 40 and <10% RWC and stained with Evans Blue. As a control, a hydrated leaf was stained after immersion in boiling water for 5 minutes (samples as labelled).
Fig 2.
T. loliiformis suppresses PCD pathway during dehydration and desiccation.
Hydrated, dehydrating, desiccated and rehydrated T. loliiformis leaf tissues were harvested and subjected to TUNEL assay and propidium iodide counter staining for the detection of DNA fragmentation and Apoptotic-like cell death. Hydrated leaves treated with DNase were used as the positive control.
Fig 3.
Tripogon loliiformis employs autophagy as a pro-survival mechanism during desiccation.
Dehydrating (60, 40%RWC) and completely desiccated (<10% RWC) T. loliiformis leaves were harvested and subjected to Immunoblotting using an ATG8 antibody. ATG8 proteins were made visible by immunoblot analysis with a polyclonal ATG8 antibody following separation by Urea-SDS PAGE*. Approximately 30 μg of total protein as calculated by Bradford assay was loaded for each sample (Ponceau staining of PVDF membrane post-transfer). Protein extracts were subjected to phospholipase D treatment to verify ATG8 lipidation. Signal intensities of the ATG8-PE band were calculated using ImageJ software and were normalised using the Ponceau stained membrane post-transfer for normalisation. Each bar represents the mean ± SEM of triplicate values from a representative experiment. RWC = relative water content, U = undigested, D = digested.
Fig 4.
Exogenous application of trehalose triggers autophagy in T.loliiformis.
Hydrated leaves from three month old glasshouse grown T. loliiformis plants were harvested, divided into 1 cm segments, vacuum infiltrated with 1 and 5 mM trehalose solution containing 1 μM concamycin A prepared in ½ MS basal salts and incubated for 24 hrs. Autophagosomes were visualised by Transmission Electron Microscopy. Top Panel–(A) Concanamycin control, (B) 1 mM trehalose, (C) 1 mM trehalose, higher magnification, (D) 5 mM trehalose, (E) 5 mM trehalose, higher magnification, (F) Tunicamycin control, Bottom panel—The sucrose and trehalose contents of hydrated, dehydrating and desiccated leaves were assessed by GCMS analysis.
Fig 5.
Autophagy-mediated desiccation tolerance in Tripogon loliiformis.
Tripogon loliiformis uses a multi-pronged strategy to survive desiccation stress. I) rapid shutdown of photosynthesis triggers caloric deficiency and the onset of autophagy pathways during the early stages of dehydration. ii) autophagic removal of damaged, unfolded and redundant organelles and proteins as well as ROS mitigates stress to delay the onset of apoptosis. The efficacy of autophagy pathways is bolstered by the accumulation of trehalose throughout dehydration. iii) Nutrient recycling as a product of autophagy mediated breakdown of proteins and organelles suspends “aging” senescence pathways.