Molecular mechanisms of desiccation tolerance in the resurrection glacial relic Haberlea rhodopensis

Abstract

Haberlea rhodopensis is a resurrection plant with remarkable tolerance to desiccation. Haberlea exposed to drought stress, desiccation, and subsequent rehydration showed no signs of damage or severe oxidative stress compared to untreated control plants. Transcriptome analysis by next-generation sequencing revealed a drought-induced reprogramming, which redirected resources from growth towards cell protection. Repression of photosynthetic and growth-related genes during water deficiency was concomitant with induction of transcription factors (members of the NAC, NF-YA, MADS box, HSF, GRAS, and WRKY families) presumably acting as master switches of the genetic reprogramming, as well as with an upregulation of genes related to sugar metabolism, signaling, and genes encoding early light-inducible (ELIP), late embryogenesis abundant (LEA), and heat shock (HSP) proteins. At the same time, genes encoding other LEA, HSP, and stress protective proteins were constitutively expressed at high levels even in unstressed controls. Genes normally involved in tolerance to salinity, chilling, and pathogens were also highly induced, suggesting a possible cross-tolerance against a number of abiotic and biotic stress factors. A notable percentage of the genes highly regulated in dehydration and subsequent rehydration were novel, with no sequence homology to genes from other plant genomes. Additionally, an extensive antioxidant gene network was identified with several gene families possessing a greater number of antioxidant genes than most other species with sequenced genomes. Two of the transcripts most abundant during all conditions encoded catalases and five more catalases were induced in water-deficient samples. Using the pharmacological inhibitor 3-aminotriazole (AT) to compromise catalase activity resulted in increased sensitivity to desiccation. Metabolome analysis by GC or LC-MS revealed accumulation of sucrose, verbascose, spermidine, and γ-aminobutyric acid during drought, as well as particular secondary metabolites accumulating during rehydration. This observation, together with the complex antioxidant system and the constitutive expression of stress protective genes suggests that both constitutive and inducible mechanisms contribute to the extreme desiccation tolerance of H. rhodopensis.

Fig. 1

Haberlea rhodopensis under normal conditions (a), drought stress (b), desiccation (c), and after rehydration (d). Drought stress (b) was achieved by withholding the water supply for 4 days until RWC reached 50 %. Desiccated plant (c) obtained after 20 days without water had 5 % RWC; rehydrated plant (d) pictured 4 days after resuming water supply had the same RWC as control plants (a)

Fig. 2

Haberlea rhodopensis is a genuine resurrection plant. Well-watered, fully hydrated unstressed control plants were subjected to drought stress, desiccation, and subsequent rehydration. a Relative water content (RWC, %); b conductivity (mS), a measure of electrolyte leakage and cell damage; c malondialdehyde (MDA) level, a measure of lipid peroxidation and oxidative stress; d chlorophyll fluorescence (Fv/Fm). Data are means from three biological replicates ± SD

Fig. 3

Top species distribution of Haberlea rhodopensis genes obtained by BLAST analysis (Blast2Go annotation tool). Of the transcripts, 44 % are most similar to transcripts from V. vinifera, R. communis, and P. trichocarpa. 41 % of the transcripts did not match any species and may be a source for gene discovery

Fig. 4

Haberlea rhodopensis with inhibited catalase activity displays increased sensitivity to desiccation. Well-watered controls, drought-stressed, desiccated, and rehydrated plants were sprayed either with water (white columns) or the catalase inhibitor 3-aminotriazole (AT, gray columns), and electrolyte leakage (a measure for cell damage) expressed as conductivity (mS) was determined. Data are means of three biological replicates ± SD

Fig. 5

Relative abundance of metabolites in H. rhodopensis during dehydration and following rehydration. The relative metabolite levels are normalized by dry weight of the samples and depicted on a metabolic map using the VANTED software [38]. The map is drawn according to the metabolic network in A. thaliana. The white, pale red, red, and gray bars represent the four conditions: unstressed controls (RWC 80 %), drought stress (4 days without watering, RWC 50 %), severe desiccation (20 days without watering, RWC 5 %), and rehydration (4 days after re-watering to desiccated plants, RWC as in unstressed controls). The values are the mean ± SEM of 3–6 biological replicates. A star indicates the value showed significant difference from that of the unstressed controls in the t test by VANTED

Fig. 6

A unifying model based on the physiological, transcriptomic, and metabolome data representing the collective actions of different mechanisms that contribute to the establishment of desiccation tolerance and other drought-related biological effects in Haberlea rhodopensis