Changes in photosynthetic rate and stress volatile emissions through desiccation‐rehydration cycles in desiccation‐tolerant epiphytic filmy ferns (Hymenophyllaceae)

Exposure to recurrent desiccation cycles carries a risk of accumulation of reactive oxygen species that can impair leaf physiological activity upon rehydration, but changes in filmy fern stress status through desiccation and rewatering cycles have been poorly studied. We studied foliage photosynthetic rate and volatile marker compounds characterizing cell wall modifications (methanol) and stress development (lipoxygenase [LOX] pathway volatiles and methanol) through desiccation–rewatering cycles in lower‐canopy species Hymenoglossum cruentum and Hymenophyllum caudiculatum, lower‐ to upper‐canopy species Hymenophyllum plicatum and upper‐canopy species Hymenophyllum dentatum sampled from a common environment and hypothesized that lower canopy species respond more strongly to desiccation and rewatering. In all species, rates of photosynthesis and LOX volatile emission decreased with progression of desiccation, but LOX emission decreased with a slower rate than photosynthesis. Rewatering first led to an emission burst of LOX volatiles followed by methanol, indicating that the oxidative burst was elicited in the symplast and further propagated to cell walls. Changes in LOX emissions were more pronounced in the upper‐canopy species that had a greater photosynthetic activity and likely a greater rate of production of photooxidants. We conclude that rewatering induces the most severe stress in filmy ferns, especially in the upper canopy species.     

Physiological consequences of desiccation in the aquatic bryophyte <Emphasis Type="Italic">Fontinalis antipyretica</Emphasis>

The moss Fontinalis antipyretica, an aquatic bryophyte previously described as desiccation-intolerant, is known to survive intermittent desiccation events in Mediterranean rivers. To better understand the mechanisms of desiccation tolerance in this species and to reconcile the apparently conflicting evidence between desiccation tolerance classifications and field observations, gross photosynthesis and chlorophyll a fluorescence were measured in field-desiccated bryophyte tips and in bryophyte tips subjected in the laboratory to slow, fast, and very fast drying followed by either a short (30 min) or prolonged (5 days) recovery. Our results show, for the first time, that the metabolic response of F. antipyretica to desiccation, both under field and laboratory conditions, is consistent with a desiccation-tolerance pattern; however, drying must proceed slowly for the bryophyte to regain its pre-desiccation state following rehydration. In addition, the extent of dehydration was found to influence metabolism whereas the drying rate determined the degree of recovery. Photosystem II (PSII) regulation and structural maintenance may be part of the induced desiccation tolerance mechanism allowing this moss to recover from slow drying. The decrease in the photochemical quenching coefficient (qP) immediately following rehydration may serve to alleviate the effects of excess energy on photosystem I (PSI), while low-level non-photochemical quenching (NPQ) would allow an energy shift enabling recovery subsequent to extended periods of desiccation. The findings were confirmed in field-desiccated samples, whose behavior was similar to that of samples slowly dried in the laboratory.     

Dehydration rate and time of desiccation affect recovery of the lichenic algae <Emphasis Type="Italic">Trebouxia erici</Emphasis>: alternative and classical protective mechanisms

The mechanisms involved in desiccation tolerance of lichens and their photobionts are still poorly understood. To better understand these mechanisms we have studied dehydration rate and desiccation time in Trebouxia, the most abundant chlorophytic photobiont in lichen. Our findings indicate that the drying rate has a profound effect on the recovery of photosynthetic activity of algae after rehydration, greater than the effects of desiccation duration. The basal fluorescence (F′_o) values in desiccated algae were significantly higher after rapid dehydration, than after slow dehydration, suggesting higher levels of light energy dissipation in slow-dried algae. Higher values of PSII electron transport were recovered after rehydration of slow-dried Trebouxia erici compared to rapid-dried algae. The main component of non-photochemical quenching after slow dehydration was energy dependent (q _E), whereas after fast dehydration it was photoinhibition (q _I). Although q _E seems to play a role during desiccation recovery, no significant variations were detected in the xanthophyll cycle components. Desiccation did not affect PSI functionality. Classical antioxidant activities like superoxide dismutase or peroxidase decreased during desiccation and early recovery. Dehydrins were detected in the lichen-forming algae T. erici and were constitutively expressed. There is probably a minimal period required to develop strategies which will facilitate transition to the desiccated state in this algae. In this process, the xanthophyll cycle and classical antioxidant mechanisms play a very limited role, if any. However, our results indicate that there is an alternative mechanism of light energy dissipation during desiccation, where activation is dependent on a sufficiently slow dehydration rate.     

Characterization of Protein Synthetic Changes in a Desiccation-Tolerant Fern, Polypodium virginianum. Comparison of the Effects of Drying, Rehydration and Abscisic Acid

The desiccation-tolerant fern, Polypodium virginianum, was ableto lose more than 60% of its fresh weight during periods ofprolonged water stress and fully recover during periods of wateravailability. Rehydration from the air-dry state occurred rapidly(within 24 h) with a concurrent initiation of protein synthesis.A low-molecular-weight doublet was synthesized during waterstress and was stable for at least 24 h after rehydration. Theseproteins were expressed also when the fully hydrated frondswere subjected to exogenous ABA even though drying resultedin a 10-fold reduction in ABA content. The large subunit ofRubisco was synthesized during drying, but despite temporaryaccumulation, the de novo synthesized component was degradedduring rehydration. During rehydration from the air-dry state,unique rehydration-specific polypeptides were produced. Thesepolypeptides were synthesized at different water contents andtimes of rehydration, and were transiently expressed. This transientexpression may mean that they are only necessary during theearly stages of rehydration when the rapid initiation of physiologicaland repair processes is essential. The response of this fernto desiccation has features which are similar to those reportedfor desiccation-tolerant mosses, and others which are reminiscentof desiccation-tolerant angiosperms. Key words: Desiccation-tolerance, fern, Polypodium virginianum, protein synthesis, abscisic acid     

Increased activities of superoxide dismutase and catalase are not the mechanism of desiccation tolerance induced by hardening in the moss Atrichum androgynum

Abstract

The effects of treatments that increase desiccation tolerance were tested on the activity of the enzymes superoxide dismutase (SOD) and catalase (CAT) in the moss Atrichum androgynum subjected to a drying/wetting cycle. Hardening by both abscisic acid (ABA) pretreatment and partial dehydration significantly increased the rate of recovery of photosynthesis during rehydration following desiccation. Hardening treatments had little effect on SOD activity. In non-hardened plants, SOD activity increased three-fold during desiccation for 32 h at 52% rh, but hardened material tended to display smaller increases in activity. During rehydration, SOD activities rapidly declined to their initial values in all treatments. Hardening by partial dehydration, but not ABA, reduced CAT activity. After desiccation for 32 h, material from all treatments displayed about half the initial CAT activity, and activity did not change during subsequent rehydration. Results show that, while the induction of SOD appears to play a role in desiccation tolerance, a similar induction occurred in both hardened and non-hardened mosses. Induction of greater activities of enzymes that scavenge reactive oxygen species is not responsible for the added tolerance induced by hardening treatments.     

Vertical distribution of Hymenophyllaceae species among host tree microhabitats in a temperate rain forest in Southern Chile

Question: Are differences in microhabitat preferences of co‐occurring epiphytic Hymenophyllaceae species (filmy ferns) correlated with differences in ecophysiological responses to light availability and humidity in the host tree? Location: The Andean foothills in south‐central Chile. Methods: We evaluated the distribution pattern of nine filmy fern species in microhabitats that differ in light availability and humidity in four host tree species. A DCA was developed to assess Hymenophyllaceae species microhabitat preference in terms of canopy openness (CO) and relative humidity. We assessed whether differences in chlorophyll content, maximum photochemical efficiency (Fv/Fm), photosynthetic capacity (A_max), evapotranspiration (E) and instantaneous water use efficiency (WUE) are consistent with any pattern. Results: CO and relative humidity differed significantly with height in the host trees. While CO increased with height in a host tree, relative humidity decreased. DCA analysis showed that filmy fern species distribution within and among trees was mainly explained by the relative humidity of the microhabitat. Chlorophyll content, chlorophyll a/b ratio, A_max and E differed significantly among filmy fern species. A_max and E were correlated with axis 1 scores from the DCA analysis. Conclusions: The vertical distribution and abundance of filmy fern species in Chilean temperate rain forest seems to be closely related to the different microhabitats offered by host trees. This pattern may reflect interspecific differences in ecophysiological traits related both to light availability and humidity. Our results suggest that humidity is the main environmental factor driving functional responses and habitat preferences of these filmy fern species.     

Photosynthetic activity of homoiochlorophyllous desiccation tolerant plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis> during dehydration and rehydration

The functional state of the photosynthetic apparatus of flowering homoiochlorophyllous desiccation tolerant plant Haberlea rhodopensis during dehydration and subsequent rehydration was investigated in order to characterize some of the mechanisms by which resurrection plants survive drought stress. The changes in the CO₂ assimilation rate, chlorophyll fluorescence parameters, thermoluminescence, fluorescence imaging and electrophoretic characteristics of the chloroplast proteins were measured in control, moderately dehydrated (50% water content), desiccated (5% water content) and rehydrated plants. During the first phase of desiccation the net CO₂ assimilation decline was influenced by stomatal closure. Further lowering of net CO₂ assimilation was caused by both the decrease in stomatal conductance and in the photochemical activity of photosystem II. Severe dehydration caused inhibition of quantum yield of PSII electron transport, disappearance of thermoluminescence B band and mainly charge recombination related to S₂Q_A⁻ takes place. The blue and green fluorescence emission in desiccated leaves strongly increased. It could be suggested that unchanged chlorophyll content and amounts of chlorophyll–proteins, reversible modifications in PSII electron transport and enhanced probability for non-radiative energy dissipation as well as increased polyphenolic synthesis during desiccation of Haberlea contribute to drought resistance and fast recovery after rehydration.     

Desiccation and rehydration dynamics in the epiphytic resurrection fern Pleopeltis polypodioides

The epiphytic resurrection—or desiccation-tolerant (DT)—fern Pleopeltis polypodioides can survive extreme desiccation and recover physiological activity within hours of rehydration. Yet, how epiphytic DT ferns coordinate between deterioration and recovery of their hydraulic and photosynthetic systems remains poorly understood. We examined the functional status of the leaf vascular system, chlorophyll fluorescence, and photosynthetic rate during desiccation and rehydration of P. polypodioides. Xylem tracheids in the stipe embolized within 3–4 h during dehydration. When the leaf and rhizome received water, tracheids refilled after ∼24 h, which occurred along with dramatic structural changes in the stele. Photosynthetic rate and chlorophyll fluorescence recovered to predesiccation values within 12 h of rehydration, regardless of whether fronds were connected to their rhizome. Our data show that the epiphytic DT fern P. polypodioides can utilize foliar water uptake to rehydrate the leaf mesophyll and recover photosynthesis despite a broken hydraulic connection to the rhizome.     

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.     

Dehydration-mediated activation of the xanthophyll cycle in darkness: is it related to desiccation tolerance?

The development of desiccation tolerance by vegetative tissues was an important step in the plants’ conquest of land. To counteract the oxidative stress generated under these conditions the xanthophyll cycle plays a key role. Recent reports have shown that desiccation itself induces de-epoxidation of xanthophyll cycle pigments, even in darkness. The aim of the present work was to study whether this trait is a common response of all desiccation-tolerant plants. The xanthophyll cycle activity and the maximal photochemical efficiency of PS II (F _v/F _m) as well as β-carotene and α-tocopherol contents were compared during slow and rapid desiccation and subsequent rehydration in six species pairs (with one desiccation-sensitive and one desiccation-tolerant species each) belonging to different taxa. Xanthophyll cycle pigments were de-epoxidised in darkness concomitantly with a decrease in F _v/F _m during slow dehydration in all the desiccation-tolerant species and in most of the desiccation-sensitive ones. De-epoxidation was reverted in darkness by re-watering in parallel with the recovery of the initial F _v/F _m. The stability of the β-carotene pool confirmed that its hydroxylation did not contribute to zeaxanthin formation. The α-tocopherol content of most of the species did not change during dehydration. Because it is a common mechanism present in all the desiccation-tolerant taxa and in some desiccation-sensitive species, and considering its role in antioxidant processes and in excess energy dissipation, the induction of the de-epoxidation of xanthophyll cycle pigments upon dehydration in the dark could be understood as a desiccation tolerance-related response maintained from the ancestral clades in the initial steps of land occupation by plants.     

ABA Increases the Desiccation Tolerance of Photosynthesis in the Afromontane Understorey Moss Atrichum androgynum

The effect of pretreatment with abscisic acid (ABA) on the physiologyof the moss Atrichum androgynum during a desiccation–rehydrationcycle was examined. During rehydration following desiccationfor 16 h, net CO₂fixation recovered much more slowly than photosystemII (PSII) activity, conditions conducive to the formation ofreactive oxygen species (ROS) in the photosynthetic apparatus.Pretreatment with ABA increased the rate of recovery of photosynthesisand PSII activity, and also doubled non-photochemical quenching(NPQ). Increased NPQ activity will reduce ROS formation, andmay explain in part how ABA hardens the moss to desiccation.In ABA-pretreated, but not untreated mosses, desiccation significantlyincreased the concentration of soluble sugars. Sugar accumulationmay promote vitrification of the cytoplasm and protect membranesduring desiccation. Starch concentrations in freshly collectedA. androgynum were only approx. 40 mg g^-1dry mass; they roseslightly during desiccation but were only slightly affectedby ABA pretreatment. ABA did not reduce chlorophyll breakdownduring desiccation. Copyright 2001 Annals of Botany Company Moss, desiccation, abscisic acid, photosynthesis, chlorophyll fluorescence     

ABA treatment increases both the desiccation tolerance of photosynthesis,and nonphotochemical quenching in the moss Atrichum undulatum

Pulse amplitude modulation fluorescence was used to investigate whether abscisic acid (ABA) pretreatment increases the desiccation tolerance of photosynthesis in the moss Atrichum undulatum. In unstressed plants, ABA pretreatment decreased the F _V/F _m ratio, largely as a result of an increase in F _o. This indicated a reduction in energy transfer between LHCII and PSII, possibly hardening the moss to subsequent stress by reducing the production of the reactive oxygen species near PSII. During desiccation, F ₀, F _m, F _v/F _m, PSII, and NPQ and F ₀ quenching declined in ABA-treated and nontreated mosses. However, during rehydration, F ₀, F _m, F _v/F _m, and PSII recovered faster in ABA-treated plants, suggesting that ABA improved the tolerance of photosystem II to desiccation. NPQ increased upon rehydration in mosses from both treatments, but much more rapidly in ABA-treated plants; during the first hour of rehydration, NPQ was two-fold greater in plants treated with ABA. F ₀quenching followed a similar pattern, indicating that ABA treatment stimulated zeaxanthin-based quenching. The implications of these results for the mechanisms of ABA-induced desiccation tolerance in A. undulatum are discussed.     

Desiccation tolerance of Hymenophyllacea filmy ferns is mediated by constitutive and non-inducible cellular mechanisms

The Hymenophyllaceae is a primitive family within the Filicopsidae. One of the most exceptional features of this family of ferns is the presence of fronds with one or just a few cell layers (hence their name of filmy ferns), and the absence of stomata. Hymenophyllum caudiculatum and Hymenophyllum dentatum are able to lose more than 82% of their fully hydrated water content, to remain dry for extended periods of time (days or weeks), and to survive and remain viable following rehydration. The aim of this work was to understand whether the adaptive strategy of the Hymenophyllaceae for desiccation tolerance is constitutive or inducible. A proteomic approach was adopted in combination with physiological parameters to assess whether there were changes in the protein content during dehydration and following rehydration. Detached fronds were used to monitor the rates of photosynthesis in desiccation experiments, sugar accumulation, and high-resolution 2-DE to analyze proteome variation during a desiccation–rehydration cycle. The analyzed proteome exhibited little variation (3–4%) between hydrated and desiccated states, while variation was greater between the desiccated and rehydrated states (8.7–10%). Eighty-two discrete proteins were analyzed by MS/MS, and 65 were identified. About 21% of the analyzed proteins (17) were mixtures of two or more different polypeptides. Of the identified proteins, more than a half (33 spots, 55%) had functions related to energy-photosynthesis. The second largest category with known function (five spots, 8%) was related to cell rescue, defense, and virulence. More than one in every four proteins analyzed belonged to a group of hypothetical proteins (18 spots, 28%). The results suggest that the Hymenophyllaceae represent an example of a change in adaptive strategy from a typical vascular to the poikilohydric homoiochlorophyllous adaptation, which they share with the bryophytes that grow in profusion in the same habitats. The speed at which desiccation takes place therefore precludes the induction of protective systems, suggesting a constitutive mechanism of cellular protection.     

Desiccation sensitivity and tolerance in the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>: assessing limits and damage

The moss Physcomitrella patens is becoming the model of choice for functional genomic studies at the cellular level. Studies report that Physcomitrella survives moderate osmotic and salt stress, and that desiccation tolerance can be induced by exogenous ABA. Our goal was to quantify the extent of dehydration tolerance in wild type moss and to examine the nature of cellular damage caused by desiccation. We exposed Physcomitrella to humidities that generate water potentials from −4 (97% RH) to −273 MPa (13% RH) and monitored water loss until equilibrium. Water contents were measured on a dry matter basis to determine the extent of dehydration because fresh weights (FW) were found to be variable and, therefore, unreliable. We measured electrolyte leakage from rehydrating moss, assessed overall regrowth, and imaged cells to evaluate their response to drying and rehydration. Physcomitrella did not routinely survive water potentials <−13 MPa. Upon rehydration, moss dried to water contents >0.4 g g dm⁻¹ maintained levels of leakage similar to those of hydrated controls. Moss dried to lower water contents leaked extensively, suggesting that plasma membranes were damaged. Moss protonemal cells were shrunken and their walls twisted, even at −13 MPa. Moss cells rehydrated after drying to −273 MPa failed to re-expand completely, again indicating membrane damage. ABA treatment elicited tolerance of desiccation to at least −273 MPa and limited membrane damage. Results of this work will form the basis for ongoing studies on the functional genomics of desiccation tolerance at the cellular level.     

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     

Effects of dehydration rate on physiological responses and survival after rehydration in larvae of the anhydrobiotic chironomid

Strategies to combat desiccation are critical for organisms living in arid and semi-arid areas. Larvae of the Australian chironomid Paraborniella tonnoiri resist desiccation by reducing water loss. In contrast, larvae of the African species Polypedilum vanderplanki can withstand almost complete dehydration, referred to as anhydrobiosis. For successful anhydrobiosis, the dehydration rate of P. vanderplanki larvae has to be controlled. Here, we desiccated larvae by exposing them to different drying regimes, each progressing from high to low relative humidity, and examined survival after rehydration. In larvae of P. vanderplanki, reactions following desiccation can be categorized as follows: (I) no recovery at all (direct death), (II) dying by unrepairable damages after rehydration (delayed death), and (III) full recovery (successful anhydrobiosis). Initial conditions of desiccation severely affected survival following rehydration, i.e. P. vanderplanki preferred 100% relative humidity where body water content decreased slightly. In subsequent conditions, unfavorable dehydration rate, such as more than 0.7 mg water lost per day, resulted in markedly decreased survival rate of rehydrated larvae. Slow dehydration may be required for the synthesis and distribution of essential molecules for anhydrobiosis. Larvae desiccated at or above maximum tolerable rates sometimes showed temporary recovery but died soon after.     

Fungal-associated NO is involved in the regulation of oxidative stress during rehydration in lichen symbiosis

Background  

Reactive oxygen species (ROS) are normally produced in respiratory and photosynthetic electron chains and their production is enhanced during desiccation/rehydration. Nitric oxide (NO) is a ubiquitous and multifaceted molecule involved in cell signaling and abiotic stress. Lichens are poikilohydrous organisms that can survive continuous cycles of desiccation and rehydration. Although the production of ROS and NO was recently demonstrated during lichen rehydration, the functions of these compounds are unknown. The aim of this study was to analyze the role of NO during rehydration of the lichen Ramalina farinacea (L.) Ach., its isolated photobiont partner Trebouxia sp. and Asterochloris erici (Ahmadjian) Skaloud et Peksa (SAG 32.85 = UTEX 911).     

Unravelling the roles of desiccation-induced xanthophyll cycle activity in darkness: a case study in Lobaria pulmonaria

Desiccation-tolerance ability in photosynthetic organisms is largely based on a battery of photoprotective mechanisms. Xanthophyll cycle operation induced by desiccation in the absence of light has been previously proven in the desiccation-tolerant fern Ceterach officinarum. To understand the physiological function of xanthophyll cycle induction in darkness and its implication in the desiccation tolerance in more detail, we studied its triggering factors and its photochemical effects in the lichen Lobaria pulmonaria. We found that both the drying rate and the degree of desiccation play a crucial role in the violaxanthin de-epoxidase activation. De-epoxidation of violaxanthin to zeaxanthin (Z) occurs when the tissue has lost most of its water and only after slow dehydration, suggesting that a minimum period of time is required for the enzyme activity induction. Fluorescence analysis showed that Z, synthesised during tissue dehydration in the absence of light, prevents photoinhibition when rewatered tissues are illuminated. This is probably due to Z implication in both non-photochemical quenching and/or antioxidative responses.