Protective Strategies of Haberlea rhodopensis for Acquisition of Freezing Tolerance: Interaction between Dehydration and Low Temperature

Abstract

Resurrection plants are able to deal with complete dehydration of their leaves and then recover normal metabolic activity after rehydration. Only a few resurrection species are exposed to freezing temperatures in their natural environments, making them interesting models to study the key metabolic adjustments of freezing tolerances. Here, we investigate the effect of cold and freezing temperatures on physiological and biochemical changes in the leaves of Haberlea rhodopensis under natural and controlled environmental conditions. Our data shows that leaf water content affects its thermodynamical properties during vitrification under low temperatures. The changes in membrane lipid composition, accumulation of sugars, and synthesis of stress-induced proteins were significantly activated during the adaptation of H. rhodopensis to both cold and freezing temperatures. In particular, the freezing tolerance of H. rhodopensis relies on a sucrose/hexoses ratio in favor of hexoses during cold acclimation, while there is a shift in favor of sucrose upon exposure to freezing temperatures, especially evident when leaf desiccation is relevant. This pattern was paralleled by an elevated ratio of unsaturated/saturated fatty acids and significant quantitative and compositional changes in stress-induced proteins, namely dehydrins and early light-induced proteins (ELIPs). Taken together, our data indicate that common responses of H. rhodopensis plants to low temperature and desiccation involve the accumulation of sugars and upregulation of dehydrins/ELIP protein expression. Further studies on the molecular mechanisms underlying freezing tolerance (genes and genetic regulatory mechanisms) may help breeders to improve the resistance of crop plants.

Keywords: carbohydrates; desiccation; fatty acids; freezing tolerance; protective proteins; resurrection plants.

Figure 1

Dynamic mechanical thermal analysis (DMTA) scans of Haberlea rhodopensis leaves (dark grey line) at full turgor (100% RWC, (left)) or partially dehydrated (55% RWC, (right)). The green line highlights the glass transition temperature (Tg), which is identified as a peak in the Tan δ (upper panels). The Red line represents a putative winter temperature in H. rhodopensis natural environment. One representative curve from two independent biological replicates is shown for each hydration state.

Figure 2

A differential scanning calorimetry (DSC) scan of a hydrated and of a partially dehydrated (RWC = 55%) leaf of Haberlea rhodopensis. The scan was recorded at a cooling rate of 0.05 °C min⁻¹. One representative scan is shown for each hydration level (blue: hydrated samples, red: dehydrated samples). The average ice nucleation temperature was −5.81 ± 0.30 and −6.62 ± 0.06 °C for leaves at 100% and at 55% of RWC, respectively. Water content was 4.1 ± 0.2 g H₂O g⁻¹ DW and 2.8 ± 0.1 g H₂O g⁻¹ DW for leaves at 100% and 55% of RWC, respectively (average ± SE, n = 7).

Figure 3

Changes in electrolyte leakage (EL, % of total) during cold acclimation of Haberlea rhodopensis (CA, period: 7–28 November), after short-term exposure to freezing stress (FS, −10 °C, 30 November), freezing-induced desiccation (FS + D, period: 1 December–30 January) and after recovery of plants in early spring (R; period: 22 March) under natural ex situ environmental conditions. Percentages at the bottom of the columns show the relative water content (RWC) of leaves at each time point. Data represent the mean of n = 6. The same letters within a graph indicate no significant differences assessed by Fisher’s LSD test (p ≤ 0.05) after performing ANOVA.

Figure 4

Changes in leaf malondialdehyde (MDA) content (nmol g⁻¹ DW) of Haberlea rhodopensis after exposure to low temperatures during cold acclimation (CA, period: 7–28 November), freezing stress (FS, −10 °C, 30 November), freezing-induced desiccation (FS + D, period: 8 December–30 January), and after recovery of plants in early spring (R; period: 22 March) under ex situ environmental conditions. The percentages at the bottom of the columns show the relative water content (RWC) of leaves at each time point. Data represent the mean of n = 9. The same letters within a graph indicate no significant differences assessed by Fisher’s LSD test (p ≤ 0.05) after performing ANOVA.

Figure 5

Changes in leaf proline content (mg g⁻¹ DW) of Haberlea rhodopensis after exposure to low temperatures during cold acclimation (CA, period: 7–28 November), freezing stress (FS, −10 °C, 30 November), freezing-induced desiccation (FS + D, period: 8 December–30 January) and after recovery of plants in early spring (R; period: 22 March) under natural ex situ environmental conditions. Percentages at the bottom of the columns show the relative water content (RWC) of leaves at each time point. Data represent the mean of n = 6. The same letters within a graph indicate no significant differences assessed by Fisher’s LSD test (p ≤ 0.05) after performing ANOVA.

Figure 6

Changes in the mean concentration of carbohydrates and organic acids measured in leaves of Haberlea rhodopensis from the control under ex situ environmental conditions. Light grey bars indicate changes between control (May) and cold acclimation (CA; period 7–28 November). Dark grey bars indicate changes from CA to short-term exposure to freezing temperatures (FS; −10 °C, 30 November). Grey bars indicate changes between FS and longer-term exposure to freezing temperatures when significant desiccation occurred (FS + D; period: 5–14 December). The bars for compounds for which the trend between one phase was opposed to that observed during the precedent transition have only the grey outlines.

Figure 7

Changes in the expression of Sucrose synthase 1 (SUS1) protein in leaves of Haberlea rhodopensis after exposure to low temperatures during cold acclimation (CA, period: 7–28 November) freezing stress (FS, −10 °C, 30 November), freezing-induced desiccation (FS + D, period: 8 December–30 January) and after recovery of plants in early spring (R; period: 22 March) under natural ex situ environmental conditions. A representative Western blot is presented in the graph. 30 µg protein was applied per lane. Percentages at the bottom of the columns show the relative water content (RWC) of plants at each sampling point. The same letters within a graph indicate no significant differences assessed by Fisher’s LSD test (p ≤ 0.05) after performing ANOVA. Ponceau S staining of the membrane after blotting is presented on the right. SUS1 values are normalized according to Ponceau S staining. St: Precision Plus Dual Color Protein™ Prestained Standards (Bio-Rad, Hercules, CA, USA).

Figure 8

Representative Western blots of dehydrins (top) in leaves of Haberlea rhodopensis after exposure to low temperatures during cold acclimation (CA, period: 7–28 November), freezing stress (FS, 30 November, −10 °C), freezing-induced desiccation (FS + D, period: 8 December–30 January) and after recovery of plants in early spring (R; period: 22 March) under natural ex situ environmental conditions detected by Western blot using antibodies against the conserved K-, S- and Y-segment of the proteins. 30 µg protein was applied per lane. Ponceau S staining of the membranes after blotting is presented below the Western blots. St: Precision Plus Dual Color Protein™ Prestained Standards (Bio-Rad, Hercules, CA, USA).

Figure 9

Representative Western blots of ELIPs (left) in leaves of Haberlea rhodopensis after exposure to low temperatures during cold acclimation (CA, period: 7–28 November), freezing stress (FS, 30 November, −10 °C), freezing-induced desiccation (FS + D, period: 8 December–30 January) and after recovery of plants in early spring (R; period: 22 March) under natural ex situ environmental conditions. 30 µg protein was applied per lane. The Ponceau S staining (right) of the membrane after blotting is presented on the right. St: Precision Plus Dual Color Protein™ Prestained Standards (Bio-Rad, Hercules, CA, USA).

Figure 10

Correlation matrix for leaf relative water content (RWC), electrolyte leakage (EL), photochemical parameters (ΦPSII and Fv/Fm), and biochemical variables (sugar and fatty acids content) measured at midday in leaves of Haberlea rhodopensis when exposed from control to low temperatures. The color scale ranges from red to blue according to the maximum (−1) and minimum (+1) Pearson’s correlation coefficient, respectively. Significance correlations are indicated as: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 11

Schematic representation of the protective strategies used by H. rhodopensis to achieve freezing tolerance.