Protection of thylakoids against combined light and drought by a lumenal substance in the resurrection plant Haberlea rhodopensis

Abstract

Background and aims: Haberlea rhodopensis is a perennial, herbaceous, saxicolous, poikilohydric flowering plant that is able to survive desiccation to air-dried state under irradiance below 30 micromol m-2 s-1. However, desiccation at irradiance of 350 micromol m-2 s-1 induced irreversible changes in the photosynthetic apparatus, and mature leaves did not recover after rehydration. The aim here was to establish the causes and mechanisms of irreversible damage of the photosynthetic apparatus due to dehydration at high irradiance, and to elucidate the mechanisms determining recovery.

Methods: Changes in chloroplast structure, CO2 assimilation, chlorophyll fluorescence parameters, fluorescence imaging and the polypeptide patterns during desiccation of Haberlea under medium (100 micromol m-2 s-1; ML) irradiance were compared with those under low (30 micromol m-2 s-1; LL) irradiance.

Key results: Well-watered plants (control) at 100 micromol m-2 s-1 were not damaged. Plants desiccated at LL or ML had similar rates of water loss. Dehydration at ML decreased the quantum efficiency of photosystem II photochemistry, and particularly the CO2 assimilation rate, more rapidly than at LL. Dehydration induced accumulation of stress proteins in leaves under both LL and ML. Photosynthetic activity and polypeptide composition were completely restored in LL plants after 1 week of rehydration, but changes persisted under ML conditions. Electron microscopy of structural changes in the chloroplast showed that the thylakoid lumen is filled with an electron-dense substance (dense luminal substance, DLS), while the thylakoid membranes are lightly stained. Upon dehydration and rehydration the DLS thinned and disappeared, the time course largely depending on the illumination: whereas DLS persisted during desiccation and started to disappear during late recovery under LL, it disappeared from the onset of dehydration and later was completely lost under ML.

Conclusions: Accumulation of DLS (possibly phenolics) in the thylakoid lumen is demonstrated and is proposed as a mechanism protecting the thylakoid membranes of H. rhodopensis during desiccation and recovery under LL. Disappearance of DLS during desiccation in ML could leave the thylakoid membranes without protection, allowing oxidative damage during dehydration and the initial rehydration, thus preventing recovery of photosynthesis.

Fig. 1

Changes in relative water content (RWC) of Haberlea rhodopensis leaves after 2 d (stage D1; RWC about 70 %), 4 d (stage D2; RWC about 25 %) and 7 d of dehydration (stage D3; RWC about 6 %) as well as after 1 d (stage R1) and 7 d (stage R7) of rehydration under low (LL, 30 µmol m⁻² s⁻¹) or medium (ML, 100 µmol m⁻² s⁻¹) irradiance. Control plants, kept either at LL or at ML (C, C7), were regularly watered throughout the experiment. Values are mean of six replicates with standard error.

Fig. 2

Effect of dehydration and rehydration of Haberlea rhodopensis plants on the maximum quantum efficiency of PSII (F_v/F_m), the actual quantum yield of PSII electron transport in the light-adapted state (ΦPSII) and the proportion of thermal energy dissipation in the antenna (1 – F_v′/F_m′) under low (LL) or medium (ML) light conditions. The water status of the leaves was as described in Fig. 1. Values are mean of six replicates with standard error.

Fig. 3

Changes in the blue/red fluorescence ratio (F₄₄₀/F₆₉₀) during dehydration and after rehydration of Haberlea rhodopensis under low (LL) or medium (ML) light conditions. The water status of the leaves was as described in Fig. 1. Values are mean of six replicates with standard error.

Fig. 4

Changes in net CO₂ assimilation (P_N), transpiration and stomatal conductance rates in leaves of Haberlea rhodopensis during dehydration and after rehydration under low (LL) or medium (ML) light conditions. The water status of the leaves was as described in Fig. 1. Values are mean of six replicates with standard error.

Fig. 5

Polypeptide distribution in thylakoids isolated from plants treated at (A) low light (LL) and (B) medium light (ML) conditions. C, control; D1, D2, D3, different stages of drying; R7, rehydrated for 1 week. Inset: gel patterns of thylakoids of differently treated plants. Regions including the apoproteins of the main chlorophyll–protein complexes are marked as PSI, PSII and LHC; LMW, proteins with molecular mass <20 kDa. Differences within the corresponding groups either at LL or at ML are not statistically significant. St, SIGMA LMW protein standards (kDa): bovine serum albumin (66), ovalbumin (45), glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle (36), carbonic anhydrase (29), trypsinogen from bovine pancreas (24), soybean trypsin inhibitor (20·1), α-lactalbumin, bovine milk (14·2).

Fig. 6

Changes in total leaf proteins in plants dried under low light (LL) (A) and medium light (ML) (B) conditions. C, control; St, standards (see legend to Fig. 5). Polypeptide patterns of the different lanes/treatments were compared after normalization (equal amounts of total stained protein). Polypeptides present in higher amounts are marked with arrowheads. Band heights of marked polypeptides were 1·5–2 times greater on the densitograms in D3 stage than those of the corresponding controls.

Fig. 7

Parts of chloroplasts in leaves dried under low light conditions. (A) Control (to D3 stage), (B) D3 stage, (C, D) R7 stage, (E) control (to R7 stage). dl, dense thylakoid lumen; s, starch grain; tl, transparent thylakoid lumen. Scale bars = 0·5 µm.

Fig. 8

Parts of chloroplasts in leaves dried under medium light conditions. (A) Control (to D3 stage), (B) D1 stage, (C) D3 stage, (D) R7 stage, (E) control (to R7 stage). dl, dense thylakoid lumen; lt, lumen in transition; pg, plastoglobule; s, starch grain; tl, transparent thylakoid lumen. Scale bars = 0·5 µm.