Analysis of the nuclear proteome of the resurrection plant Xerophyta viscosa in response to dehydration stress using iTRAQ with 2DLC and tandem mass spectrometry

Xerophyta viscosa Baker (family Velloziaceae) is a desiccation tolerant plant which survives extremes of dehydration down to 5% relative water content (RWC) and resumes full physiological activity within 80h of rehydration. The nuclear proteome of Xerophyta viscosa and its response to dehydration at 35% RWC as compared to fully hydrated plants was analysed using iTRAQ together with 2DLC and ESI-MS/MS. RWC at 35% is unique for desiccation tolerant species as it represents a distinct phase of the dehydration process where induction of late protection mechanisms are initiated. We reproducibly identified 122 proteins with confidence≥95% (ρ<0.05). In response to dehydration, 65% of the identified proteins had the same protein abundance as the hydrated, 22% were shown to be more abundant while 9.8% were less abundant. Classification of the nuclear proteins according to GO annotation showed that most proteins were part of cellular processes (77.43%) and had binding activity (85.47%) respectively. Ontological classification according to Interpro and Pfam databases categorized most nuclear proteins as part of gene regulation (21%) while the functions of the mapped proteins using MapMan showed involvement in protein synthesis (22%), degradation (9%), DNA structure (8%) and regulation (8%).     

Protein dynamics in thylakoids of the desiccation-tolerant plant Boea hygroscopica during dehydration and rehydration

Plants of Boea hygroscopica F. Muell were dehydrated to 9% relative water content (RWC) by withholding water for 26 d, and afterward the plants were rehydrated. Leaves were taken from control plants after 7, 12, and 26 d from the beginning of dehydration, and after 6 and 48 h from rehydration. The RWC decreased by 80% during dehydration, but the leaves regained RWC with rehydration. Dehydrated plants showed lesser amounts of proteins, lipids, and chlorophyll, all of which increased following rewatering. The lipid-to-protein ratio, which decreased during dehydration, returned to control level after 48 h of rehydration. Thylakoid lipids were more unsaturated when RWC reached the value of 9%. EPR measurements of spin-labeled proteins showed the presence of three different groups of proteins with different mobility in thylakoid membranes. The rotational correlation time of groups 1 and 2 increased with dehydration and decreased upon rehydration, whereas group 3 showed little changes. Desiccation did not cause thylakoid swelling or breakage, but the membrane system assemblage showed changes in thylakoid stacking. After 48 h of rehydration the membrane system recovered completely the organization of the fully hydrated state, showing several well-defined and regularly distributed grana.     

Protection mechanisms against excess light in the resurrection plants Craterostigma wilmsii and Xerophyta viscosa

Mechanisms of avoidance and protection against light damage were studied in the resurrection plants Craterostigma wilmsii and Xerophyta viscosa.In C. wilmsii, a combination of both physical and chemical changes appeared to afford protection against free radical damage. During dehydration leaves curled inwards, and the abaxial surface became exposed to light. The tissue became purple/brown in colour, this coinciding with a three-fold increase in anthocyanin content and a 30% decline in chlorophyll content. Thus light-chlorophyll interactions are progressively reduced as chlorophyll became masked by anthocyanins in abaxial layers and shaded in the adaxial layers. Ascorbate peroxidase (AP) activity increased during this process but declined when the leaf was desiccated (5% RWC). During rehydration leaves uncurled and the potential for normal light-chlorophyll interaction was possible before full hydration had occurred. Superoxide dismutase (SOD) and glutathione reductase (GR) activities increased markedly during this stage, possibly affording free radical protection until full hydration and metabolic recovery had occurred.In contrast, the leaves of X. viscosa did not curl, but light-chlorophyll interactions were minimised by the loss of chlorophyll and dismantling of thylakoid membranes. During dehydration, free radical protection was afforded by a four-fold increase in anthocyanin content and increased activities of AP, GR and SOD. These declined during rehydration. It is suggested that potential free radical damage may be avoided by the persistence of anthocyanins during the period of thylakoid membrane re-assembly and full chlorophyll restitution which only occurred once the leaves were fully rehydrated.     

Photosynthetic limitations and volatile and non‐volatile isoprenoids in the poikilochlorophyllous resurrection plant Xerophyta humilis during dehydration and rehydration

We investigated the photosynthetic limitations occurring during dehydration and rehydration of Xerophyta humilis, a poikilochlorophyllous resurrection plant, and whether volatile and non‐volatile isoprenoids might be involved in desiccation tolerance. Photosynthesis declined rapidly after dehydration below 85% relative water content (RWC). Raising intercellular CO₂ concentrations during desiccation suggest that the main photosynthetic limitation was photochemical, affecting energy‐dependent RuBP regeneration. Imaging fluorescence confirmed that both the number of photosystem II (PSII) functional reaction centres and their efficiency were impaired under progressive dehydration, and revealed the occurrence of heterogeneous photosynthesis during desiccation, being the basal leaf area more resistant to the stress. Full recovery in photosynthetic parameters occurred on rehydration, confirming that photosynthetic limitations were fully reversible and that no permanent damage occurred. During desiccation, zeaxanthin and lutein increased only when photosynthesis had ceased, implying that these isoprenoids do not directly scavenge reactive oxygen species, but rather protect photosynthetic membranes from damage and consequent denaturation. X. humilis was found to emit isoprene, a volatile isoprenoid that acts as a membrane strengthener in plants. Isoprene emission was stimulated by drought and peaked at 80% RWC. We surmise that isoprene and non‐volatile isoprenoids cooperate in reducing membrane damage in X. humilis, isoprene being effective when desiccation is moderate while non‐volatile isoprenoids operate when water deficit is more extreme.     

Changes in leaf hexokinase activity and metabolite levels in response to drying in the desiccation-tolerant species Sporobolus stapfianus and Xerophyta viscosa.

The phosphorylation of glucose and fructose is an important step in regulating the supply of hexose sugars for biosynthesis and metabolism. Changes in leaf hexokinase (EC 2.7.1.1) activity and in vivo metabolite levels were examined during drying in desiccation-tolerant Sporobolus stapfianus and Xerophyta viscosa. Leaf hexokinase activity was significantly induced from 85% to 29% relative water content (RWC) in S. stapfianus and from 89% to 55% RWC in X. viscosa. The increase in hexokinase corresponded to the region of sucrose accumulation in both species, with the highest activity levels coinciding with region of net glucose and fructose removal. The decline of hexose sugars and accumulation of sucrose in both plant species was not associated with a decline in acid and neutral invertase. The increase in hexokinase activity may be important to ensure that the phosphorylation and incorporation of glucose and fructose into metabolism exceeded production from potential hydrolytic activity. Total cellular glucose-6-phosphate (Glc-6-P) and fructose-6-phosphate (Fru-6-P) levels were held constant throughout dehydration. In contrast to hexokinase, fructokinase activity was unchanged during dehydration. Hexokinase activity was not fully induced in leaves of S. stapfianus dried detached from the plant, suggesting that the increase in hexokinase may be associated with the acquisition of desiccation-tolerance.     

The use of aeroponics to investigate antioxidant activity in the roots of <Emphasis Type="Italic">Xerophyta viscosa</Emphasis>

In order to ultimately understand the whole plant mechanism of attaining desiccation tolerance, we undertook to investigate the root tissues of the resurrection plant Xerophyta viscosa, as previous work has only been conducted on the leaf tissues of resurrection plants. An aeroponic plant growth system was designed and optimised to observe the root’s response to desiccation without the restrictions of a soil medium, allowing easy access to roots. Successful culture of both X.viscosa and the control, Zea mays, was achieved and dehydration stress was implemented through reduction of nutrient solution spraying of the roots. After drying to the air dry state (achieved after 7 days for roots and 10 days for shoots), rehydration was achieved by resumption of root spraying. X.viscosa plants survived desiccation and recovered but Z. mays did not. The activity of the antioxidant enzymes superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase and quantities of ascorbate and glutathione were determined during root desiccation. There was an initial decline in activity in all enzymes upon drying to 80% RWC, but activity thereafter remained constant, at rates indicative of potential metabolic activity, to the air-dry state. This data suggests that these enzymes are not denatured by desiccation of the root tissue. Ascorbate and glutathione content remained constant at concentrations of 70 and 100 μM, respectively during drying. Thus root tissues appear to retain antioxidant potential during drying, for use in recovery upon rehydration, as has been reported for leaf tissues of this and other resurrection plants.     

低温胁迫期间水稻光合膜色素与蛋白水平的变化

对４℃和１１℃两种低温胁迫过程中水稻类囊体膜色素与蛋白组成的变化进行了比较研究。结果表明：４℃低温不仅使类囊体膜中的光合色素（叶绿素、类胡萝卜素）含量降低,而且还引起膜蛋白组成的深刻变化,表现在大部分原有膜蛋白组分的含量在低温下明显降低,同时在低温处理的第３天诱导出一条３２．５ＫＤ的新蛋白带。与４℃处理相比,１１℃低温处理只引起了光合色素含量的降低,而对类囊体膜蛋白组成的影响不大,另外发现,两种低     

Photosystem II photochemical efficiency,zeaxanthin and antioxidant contents in the poikilohydric Ramonda serbica during dehydration and rehydration

Changes in photochemical efficiency, non-radiative energy dissipation (NRD), de-epoxidation state of xanthophyll cycle components (DPS) and contents of the antioxidants ascorbic acid and glutathione were studied in leaves of the poikilohydric Ramonda serbica Panc. (Gesneriaceae) during cycles of dehydration and subsequent rehydration. In drying leaves, the intrinsic efficiency of PS II photochemistry and the photon yield of PS II electron transport showed strong progressive decreases. Simultaneously, the fraction of excitation energy dissipated as heat in the PS II antenna increased markedly. The energy-dependent component of non-photochemical quenching (NPQ) showed an increase in dehydrating leaves down to relative water contents (RWC) values near 30%. Further decreases in RWC below these values caused a decrease in NPQ. Accordingly, DPS showed a similar behaviour, with a sharp increase and a subsequent decrease at very low RWC, although the maximum DPS was reached at slightly lower RWC than that for the maximum NPQ. The pools of reduced ascorbate and glutathione increased strongly when the RWC values fell below 40% and remained high in fully dehydrated leaves. When plants were re-watered photosynthetic efficiency, NRD, DPS and antioxidant contents recovered their initial control values. However, during rehydration, the zeaxanthin content showed a transient increase, as did NPQ, indicating an increasing demand for non-radiative dissipation. On the other hand, the contents of reduced ascorbate and reduced glutathione decreased but were still relatively high in the initial phase of rehydration, when the rate of photosynthetic electron transport, proton pumping and NRD were still relatively low. These results indicate that several photoprotective mechanisms are operating in R. serbica. Protection from photo-oxidation and photoinhibition appears to be achieved by co-ordinated contributions by ascorbate, glutathione and zeaxanthin-mediated NPQ. This variety of photoprotective mechanisms may be essential for conferring desiccation-tolerance.This revised version was published online in October 2005 with corrections to the Cover Date.     

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

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 µmol m⁻² s⁻¹. However, desiccation at irradiance of 350 µmol 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 µmol m⁻² s⁻¹; ML) irradiance were compared with those under low (30 µmol m⁻² s⁻¹; LL) irradiance.

Key Results

Well-watered plants (control) at 100 µmol 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.Key words: Haberlea rhodopensis, resurrection plant, electron microscopy, blue–green fluorescence, chlorophyll fluorescence     

Impacts of Boron Application on Maize Plants Growing under Flooded and Unflooded Conditions

Plants of maize (Zea mays L.) were waterlogged for 7 d and irrigated (root application) or sprayed (foliar application) with 0, 1, 3 and 6 mg dm-3 of boron. The stability of leaf membranes was assessed by determining leakage of electrolytes from leaf discs exposed to heat (51 °C) and dehydration (40 % PEG). Leaf membranes were more stable to heat than to dehydration. The membranes of waterlogged plants were more injured than those of control plants. Waterlogging reduced contents of dry mass, chlorophyll, soluble proteins, total free amino acids and soluble sugars and leaf relative water content (RWC). Application of boron increased the stability of leaf membranes, chlorophyll, soluble sugars, soluble proteins, amino acids contents, leaf RWC and dry mass accumulation. Foliar boron application was more effective. Application of boron alleviated the deleterious effect of waterlogging. This revised version was published online in July 2006 with corrections to the Cover Date.     

Changes in some thylakoid membrane proteins and pigments upon desiccation of the resurrection plant Haberlea rhodopensis

The changes in some proteins involved in the light reactions of photosynthesis of the resurrection plant Haberlea rhodopensis were examined in connection with desiccation. Fully hydrated (control) and completely desiccated plants (relative water content (RWC) 6.5%) were used for thylakoid preparations. The chlorophyll (Chl) a to Chl b ratios of thylakoids isolated from control and desiccated leaves were very similar, which was also confirmed by measuring their absorption spectra. HPLC analysis revealed that β-carotene content was only slightly enhanced in desiccated leaves compared with the control, but the zeaxanthin level was strongly increased. Desiccation of H. rhodopensis to an air-dried state at very low light irradiance led to a little decrease in the level of D1, D2, PsbS and PsaA/B proteins in thylakoids, but a relative increase in LHC polypeptides. To further elucidate whether the composition of the protein complexes of the thylakoid membranes had changed, we performed a separation of solubilized thylakoids on sucrose density gradients. In contrast to spinach, Haberlea thylakoids appeared to be much more resistant to the same solubilization procedure, i.e. complexes were not separated completely and complexes of higher density were found. However, the fractions analyzed provided clear evidence for a move of part of the antenna complexes from PSII to PSI when plants became desiccated. This move was also confirmed by low temperature emission spectra of thylakoids.Overall, the photosynthetic proteins remained comparatively stable in dried Haberlea leaves when plants were desiccated under conditions similar to their natural habitat. Low light during desiccation was enough to induce a rise in the xanthophyll zeaxanthin and β-carotene. Together with the extensive leaf shrinkage and some leaf folding, increased zeaxanthin content and the observed shift in antenna proteins from PSII to PSI during desiccation of Haberlea contributed to the integrity of the photosynthetic apparatus, which is important for rapid recovery after rehydration.     

Environmentally modulated phosphorylation and dynamics of proteins in photosynthetic membranes

Recent advances in vectorial proteomics of protein domains exposed to the surface of photosynthetic thylakoid membranes of plants and the green alga Chlamydomonas reinhardtii allowed mapping of in vivo phosphorylation sites in integral and peripheral membrane proteins. In plants, significant changes of thylakoid protein phosphorylation are observed in response to stress, particularly in photosystem II under high light or high temperature stress. Thylakoid protein phosphorylation in the algae is much more responsive to the ambient redox and light conditions, as well as to CO(2) availability. The light-dependent multiple and differential phosphorylation of CP29 linker protein in the green algae is suggested to control photosynthetic state transitions and uncoupling of light harvesting proteins from photosystem II under high light. The similar role for regulation of the dynamic distribution of light harvesting proteins in plants is proposed for the TSP9 protein, which together with other recently discovered peripheral proteins undergoes specific environment- and redox-dependent phosphorylation at the thylakoid surface. This review focuses on the environmentally modulated reversible phosphorylation of thylakoid proteins related to their membrane dynamics and affinity towards particular photosynthetic protein complexes.     

Developmental acclimation of the thylakoid proteome to light intensity in Arabidopsis

Photosynthetic acclimation, the ability to adjust the composition of the thylakoid membrane to optimise the efficiency of electron transfer to the prevailing light conditions, is crucial to plant fitness in the field. While much is known about photosynthetic acclimation in Arabidopsis, to date there has been no study that combines both quantitative label-free proteomics and photosynthetic analysis by gas exchange, chlorophyll fluorescence and P700 absorption spectroscopy. Using these methods we investigated how the levels of 402 thylakoid proteins, including many regulatory proteins not previously quantified, varied upon long-term (weeks) acclimation of Arabidopsis to low (LL), moderate (ML) and high (HL) growth light intensity and correlated these with key photosynthetic parameters. We show that changes in the relative abundance of cytb₆f, ATP synthase, FNR2, TIC62 and PGR6 positively correlate with changes in estimated PSII electron transfer rate and CO₂ assimilation. Improved photosynthetic capacity in HL grown plants is paralleled by increased cyclic electron transport, which positively correlated with NDH, PGRL1, FNR1, FNR2 and TIC62, although not PGR5 abundance. The photoprotective acclimation strategy was also contrasting, with LL plants favouring slowly reversible non-photochemical quenching (qI), which positively correlated with LCNP, while HL plants favoured rapidly reversible quenching (qE), which positively correlated with PSBS. The long-term adjustment of thylakoid membrane grana diameter positively correlated with LHCII levels, while grana stacking negatively correlated with CURT1 and RIQ protein abundance. The data provide insights into how Arabidopsis tunes photosynthetic electron transfer and its regulation during developmental acclimation to light intensity.     

Photochemical and antioxidant responses in the leaves of Xerophyta viscosa Baker and Digitaria sanguinalis L. under water deficit

In this study, photochemical and antioxidant responses of the monocotyledonous resurrection plant Xerophyta viscosa Baker and the crab grass Digitaria sanguinalis L. under water deficit were investigated as a function of time. Water deficit was imposed by withholding irrigation for 21 d. Gas exchange and chlorophyll a fluorescence analyses indicated that the dehydration treatment caused photoinhibition in both species. The reduction in the photosynthesis rate in both species during water deficit probably contributed to the decline in the photochemical efficiency of PSII and electron transport rate. However, the stomatal conductance of both species did not change during treatment whereas the intercellular CO2 pressure increased after 10 d of water deficit treatment. These observations could be related to nonstomatal limitations. The increasing net transpiration rate of both species may have contributed to leaf cooling because of water limitations. Prolonged water deficit resulted in photosynthetic pigment chlorophyll (a + b) and carotenoids content loss in only D. sanguinalis. Both species especially D. sanguinalis had increased the level of anthocyanin after 15 d of treatment, possibly to prevent the damaging effect of photooxidation. The total SOD activity of D. sanguinalis was significantly different from X. viscosa during the treatment. The total peroxidase activity in D. sanguinalis was significantly higher than in X. viscosa. X. viscosa acclimated to water deficit with no ultimate apparent oxidative damage due to endogenous protective mechanisms of resurrection. In case of D. sanguinalis, water deficit induced considerable stress and possibly caused some oxidative damage, despite the upregulation of protection mechanisms.     

Resurrection Plants: The Puzzle of Surviving Extreme Vegetative Desiccation

Tolerance to near complete desiccation of vegetative organs is a widespread capability in bryophytes and is also shared by a small group of vascular plants known as resurrection plants. To date more than 300 species, belonging to pteridophytes and angiosperms, have been identified that possess this kind of desiccation-tolerance. The vegetative desiccation-tolerance of resurrection plants is an inductive process displayed only under environmental stress with or without the involvement of abscisic acid as molecular signal. The different problems associated with desiccation encountered by resurrection plants render the employment of many interacting mechanisms necessary. Preservation of cell order and correct structure of membranes and macromolecules is underpinned by the synthesis of large amounts of sugars, amino acids, and small polypeptides such as late embryogenesis abundant (LEA) proteins and dehydrins. Some of these compatible solutes, such as sucrose and LEA proteins, are also involved in cytoplasm vitrification, which occurs during the last phase of desiccation. Mechanical damage due to vacuole shrinkage in dehydrating cells is avoided by cell wall folding or by replacing the water in vacuoles with nonaqueous substances. Oxidative stress, due to enhanced production of reactive oxygen species (ROS) especially by chloroplasts, is minimized through two different strategies. The homoiochlorophyllous resurrection plants, which conserve chloroplasts with chlorophylls and thylakoids upon drying, fold leaf blades and synthesize anthocyanins, as both sunscreens and free radical scavengers, and additionally increase the activity of antioxidant systems in cells. In contrast, the chloroplasts in poikilochlorophyllous species degrade chlorophylls and thylakoid membranes yielding desiccoplasts that are devoid of any internal structures. These adaptive mechanisms preserve cells from damage by desiccation and allow them to resume vital functions once rehydrated. Even if based mainly on cell protection during drying, the vegetative desiccation-tolerance of resurrection plants also relies on systems of cell recovery and repair upon rehydration. However, most of these systems are prepared during cell dehydration.