Ricardo Cruz de Carvalho

Abstract Background and Aims The aquatic moss Fontinalis antipyretica requires a slow rate of dehydration to survive a desiccation event. The present work examined whether differences in the dehydration rate resulted in corresponding differences in the production of reactive oxygen species (ROS) and therefore in the amount of cell damage.

During desiccation, plant cells are subjected to very low water potentials. Osmoregulation through increase of soluble materials (e.g. soluble sugars, compatible inorganic ions) is a response to the decreasing turgor pressure in the cells. In bryophytes, sucrose acts as an osmolyte and also stabilizing membranes and proteins through vitrification. We used psychrometric measurements in the aquatic bryophyte Fontinalis antipyretica Hedw. to construct pressure–volume isotherms and determine the water relations parameters under fast and slow dehydration rates. Sucrose was also quantified. The starting hypothesis was that a slow dehydration rate would increase sucrose concentration, thereby decreasing the osmotic potential at turgor loss point, and would also increase cell wall elasticity, postponing turgor loss and allowing time for induction of molecular and structural acclimation mechanisms. In fact, we found that slowly dehydrated samples presented more elastic cell walls, allowing cells to shrink and maintain turgor, helping to better preserve their metabolic functions and therefore to induce desiccation tolerance (DT). On the other hand, in fast dehydrated samples the osmotic potential at turgor loss point decreased, indicating the activity of osmoregulation processes, possibly connected to the increase observed in sucrose content. Upon rehydration, fast dried samples lost 50% of the sucrose through leakage due to cell membrane rupture, while slow dehydrated leaves maintained their sucrose content constant. DT appears to be achieved through slow dehydration, meaning that a high sucrose content alone does not contribute to DT establishment. Moreover, in natural conditions external water can be maintained at very high values due to the life form of F. antipyretica, which grows in long and compact floating stems in streams, allowing a slow dehydration rate required for induction of other DT mechanisms.

"† Background and Aims The aquatic moss Fontinalis antipyretica requires a slow rate of dehydration to survive a desiccation event. The present work examined whether differences in the dehydration rate resulted in corresponding differences in the production of reactive oxygen species (ROS) and therefore in the amount of cell damage.

† Methods Intracellular ROS production by the aquatic moss was assessed with confocal laser microscopy and the ROS-specific chemical probe 2,7-dichlorodihydrofluorescein diacetate. The production of hydrogen peroxide was also quantified and its cellular location was assessed.

† Key Results The rehydration of slowly dried cells was associated with lower ROS production, thereby reducing the amount of cellular damage and increasing cell survival. A high oxygen consumption burst accompanied the
initial stages of rehydration, perhaps due to the burst of ROS production.

†Conclusions A slow dehydration rate may induce cell protection mechanisms that serve to limit ROS production and reduce the oxidative burst, decreasing the number of damaged and dead cells due upon rehydration."

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.

"Photosynthesis, chlorophyll fluorescence, and
leaf water parameters were measured in six Portuguese
maize (Zea mays L.) cultivars during and following a
period of drought stress. The leaf relative water content
(RWC) responded differently among cultivars but except
for cultivar PB369, recovered close to initial values after
watering was restored. Photosynthetic rate and stomatal
conductance decreased with drought but more slowly in
cultivars PB269 and PB260 than in cultivars AD3R, PB64,
PB304 and PB369. Water use efficiency (WUE) decreased
during the water stress treatment although with cultivar
PB260 the decrease was marked only when the RWC fell
below 40%. Recovery of WUE was seen with all cultivars
except PB369. The maximum quantum efficiency of photosystem
II, the photochemical quenching coefficient, the
electron transport rate in PSII and the estimated functional
plastoquinone pool tended to decrease with drought, while
the non-photochemical quenching coefficient increased.
The parameters estimated from chlorophyll fluorescence
did not recover in PB369, during re-watering. The results
show that PB260 and PB269 were the most tolerant and
PB369 was the least tolerant cultivars to water stress. The variation found among the cultivars tested suggests the
existence of valuable genetic resources for crop improvement
in relation to drought tolerance."

Fontinalis antipyretica has increasingly been used as an environmental pollution biomonitor of stream water quality. This bryophyte goes through periodical desiccation especially in Mediterranean intermittent streams in the driest season. Although there is some information concerning the physiological effects of desiccation on terrestrial bryophytes, only few information is available on the aquatic ones. Thereby, the present study aimed to understand the effect of desiccation on the primary energy metabolism and oxidative stress of the aquatic moss Fontinalis antipyretica both during the desiccation process and recovery. Samples were dehydrated at different rates using controlled atmospheres with different relative humidity (RH) and rapidly rehydrated by immersion on water. Measurements were made before stress and immediately after rehydration. The results showed that desiccation stress induced membrane damage decreases of Gross Photosynthesis and of maximum photochemical efficiency of PSII (Fv/Fm). The data will be interpreted in terms of the effect of RWC and RWL. In this experiment a strong respiratory burst (circa 1200 µmol O2 g-1 DW h-1) was observed during rehydration which correlated better with RWL than with RWC. KCN, sodium azide and SHAM showed an inhibitory effect on oxygen consumption (about 60%, 40% and 25%, respectively) on unstressed samples. However, only KCN was able to totally suppress this respiratory burst in desiccated samples but not by SHAM or Na3N.
To elucidate this respiratory burst an approach in terms of oxidative stress involving production of nitric oxide (NO), reactive oxygen species (ROS) and hydrogen peroxide (H2O2) was investigated. Diaminobenzidine (DAB) staining showed an increased production in H2O2 on F. antipyretica tips upon immediate rehydration and also after 15 minutes rehydration. Quantification of H2O2 production showed an increase with decreasing RWC, but over a period of 25 minutes of rehydration, this rate slowly decreased. However this production is not enough to significantly explain the respiratory burst observed in stress conditions. In order to localize in the aquatic moss where the ROS and NO production was occurring, fluorescent probes and epifluorescence and confocal microscopy were used in the first moments after rehydration. Using 2,7-dichlorofluorescin diacetate (DCFH2-DA), a probe used for intracellular ROS detection, a generalized oxidation inside the cells was observed particularly in the chloroplasts. Some NO production was observed in the tissues applying the NO probe, 2,3-diamino naphthalene (DAN).
In what concerns the recovery, photosynthesis and respiration slowly recover 5 days after the end of desiccation, but recovery was slower when the mosses reached further lower RWC or when the desiccation rate was further higher. Photosynthesis and respiration did not fully achieved pre-desiccation values even after 5 days recovery from desiccation stress. Neverthelss, chlorophyll a fluorescence parameters recover almost completely, with the exception of NPQ values which remain lower than pre-desiccation ones.
These results show that the membrane integrity and the photosynthetic processes depend more on the amount than on the rate of water loss, although higher RWL exacerbate the stress effects. On the contrary, the non-specific respiratory burst is more dependent on the desiccation rate. This burst may be due to generalized oxidative processes and thereby could be the reason why the rate of desiccation seems to be crucial for recovery and survival. On the other hand, the rate of water loss also played an important role implying that a faster desiccation rate would not allow time for the activation of protective mechanisms against desiccation. The reactive species that are produced in stress conditions as a result of miscellaneous reactions can also have an important role in cell signaling and cell-to-cell communication for desiccation protection measures.