Jorge Marques da Silva

NONDESTRUCTIVE TRACING OF MIGRATORY RHYTHMS OF INTERTIDAL BENTHIC MICROALGAE USING IN VIVO CHLOROPHYLL A FLUORESCENCE1,2

In vivo chlorophyll (Chl) a fluorescence was measured in undisturbed intertidal sediments with the purpose of tracing the vertical migratory rhythms of benthic microalgae. A pulse amplitude fluorometer, an instrument which does not require physical contact with the sample, was used, thus allowing successive measurements to be taken on the same sample without causing any type of disturbance to the sediment structure. The basis of the method is the possibility to detect changes in the Chl a concentration near the sediment surface caused by the vertical movement of the microalgae. This requires the verification of two conditions: the possibility to follow changes in the sediment Chl a content from fluorescence intensity, and a sediment photic depth smaller than the vertical distances covered by the moving microalgae. Both conditions were experimentally verified in intertidal muddy sediments of the Tagus estuary, Portugal. In vivo fluorescence was shown to vary linearly with the sediment Chl a content, and the sediment photic depth was estimated to reach 0.27 mm, a value clearly smaller than the reported depths for microalgal migrations. Sediment samples kept under in situ conditions exhibited large hourly Variations (over 400%) in the Chl a fluorescence intensity, which were closely synchronized with the daytime periods of emersion. The rhythmic fluctuations in Chl a fluorescence were confirmed further to represent microalgal migration by (1) its endogenous nature (fluorescence continued to follow diurnal and tidal cycles after removal of environmental stimuli), (2) its dependence on the vertical distribution of the microalgal population within the sediment (vertically homogenized samples failed to display fluorescence variations), and (3) the lack of significant temperature and light effects on the fluorescence emission under in situ conditions (tested in three species representative of the main groups found in the studied microphytobenthic communities—the diatom Phaeodactylum tricornutum (Böhlin), the cyanobacterium Spirulina maxima (Setch. et Gard.), and the euglenophyte Euglena granulata (Klebs) Lemm.). The results obtained indicate that, in spite of the potential concurrent effects of factors other than the Chl a concentration on the fluorescence intensity, in vivo Chl a fluorescence can be used to trace nondestructively the migratory behavior of benthic microalgae.

Contributions of soluble carbohydrates to the osmotic adjustment in the C4 grass Setaria sphacelata: A comparison between rapidly and slowly imposed water stress

Photosynthetic carbohydrate content in Setaria sphacelata var. splendida under rapidly and slowly induced water deficit and its contribution to osmotic adjustment were studied. In short-term stress experiments, a decrease in the total content of sucrose (Su) and starch (St) was observed in leaf discs submitted to stress. An increase in the ratio between free hexoses and sucrose was found in stressed leaves, but no significant differences were found in the amount of free hexoses nor in the ratio between soluble and insoluble sugars. In long-term stress experiments, a higher amount of soluble sugars and a lower amount of starch were found in stressed leaves, when compared to the control. The ratios of free hexoses to sucrose and of soluble to insoluble sugars were also higher in stressed leaves. The contribution of the accumulation of soluble sugars to osmotic adjustment was absent in rapidly stressed leaves and was of minor importance in slowly stressed leaves.

Photosynthetic responses of three C4 grasses of different metabolic subtypes to water deficit

C4 plants are considered to be less sensitive to drought than C3 plants because of their CO2 concentrating mechanism. The C4 grasses, Paspalum dilatatum Poiret (NADP-ME), Cynodon dactylon (L.) Pers (NAD-ME) and Zoysia japonica Steudel (PEPCK) were compared in their response to water deficit imposed by the addition of polyethylene glycol to the nutrient solution in which they were grown. The effects of drought on leaf relative water content (RWC), net photosynthesis, stomatal conductance, carboxylating enzyme activities and chlorophyll a fluorescence were investigated. In C. dactylon the RWC was more sensitive, but the photosynthetic activity was less sensitive, to water deficit than in P. dilatatum and Z. japonica. The decrease of photosynthesis in P. dilatatum under water deficit was not closely related to the activities of the carboxylating enzymes or to chlorophyll a fluorescence. However, decreased activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase, in addition to decreased stomatal conductance, may have contributed to the decrease of photosynthesis with drought in C. dactylon and Z. japonica. The different responses to water deficit are discussed in relation to the natural habitats of C4 grasses.

The impact of dehydration rate on the production and cellular location of reactive oxygen species in an aquatic moss

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.

Abiotic Stress Responses in Plants: Unraveling the Complexity of Genes and Networks to Survive

Plants are often subjected to unfavorable environmental conditions – abiotic factors, causing abiotic stresses that play a major role in determining productivity of crop yields [1] but also the differential distribution of the plants species across different types of environment [2]. Some examples of abiotic stresses that a plant may face include decreased water availability, extreme temperatures (heating or freezing), decreased availability of soil nutrients and/or excess of toxic ions, excess of light and increased hardness of drying soil that hamper roots growth [3]. The ability of plants to adapt and/or acclimate to different environments is directly or indirectly related with the plasticity and resilience of photosynthesis, in combination with other processes, determining plant growth and development, namely reproduction [4]. A remarkable feature of plant adaptation to abiotic stresses is the activation of multiple responses involving complex gene interactions and crosstalk with many molecular pathways [5, 6].

Differential proteomics of dehydration and rehydration in bryophytes: evidence towards a common desiccation tolerance mechanism

All bryophytes evolved desiccation tolerance (DT) mechanisms during the invasion of terrestrial habitats by early land plants. Are these DT mechanisms still present in bryophytes that colonize aquatic habitats? The aquatic bryophyte Fontinalis antipyretica Hedw. was subjected to two drying regimes and alterations in protein profiles and sucrose accumulation during dehydration and rehydration were investigated. Results show that during fast dehydration, there is very little variation in protein profiles, and upon rehydration proteins are leaked. On the other hand, slow dehydration induces changes in both dehydration and rehydration protein profiles, being similar to the protein profiles displayed by the terrestrial bryophytes Physcomitrella patens (Hedw.) Bruch and Schimp. and, to what is comparable with Syntrichia ruralis (Hedw.) F. Weber and D. Mohr. During dehydration there was a reduction in proteins associated with photosynthesis and the cytoskeleton, and an associated accumulation of proteins involved in sugar metabolism and plant defence mechanisms. Upon rehydration, protein accumulation patterns return to control values for both photosynthesis and cytoskeleton whereas proteins associated with sugar metabolism and defence proteins remain high. The current results suggest that bryophytes from different ecological adaptations may share common DT mechanisms.

Response of the Diatom Phaeodactylum tricornutum to Photooxidative Stress Resulting from High Light Exposure

The response of microalgae to photooxidative stress resulting from high light exposure is a well-studied phenomenon. However, direct analyses of photosystem II (PSII) D1 protein (the main target of photoinhibition) in diatoms are scarce. In this study, the response of the diatom model species Phaeodactylum tricornutum to short-term exposure to high light was examined and the levels of D1 protein determined immunochemically. Low light (LL) acclimated cells (40 µmol photons m−2 s−1) subjected to high light (HL, 1,250 µmol photons m−2 s−1) showed rapid induction of non-photochemical quenching (NPQ) and ca. 20-fold increase in diatoxanthin (DT) concentration. This resulted from the conversion of diadinoxanthin (DD) to DT through the activation of the DD-cycle. D1 protein levels under LL decreased about 30% after 1 h of the addition of lincomycin (LINC), a chloroplast protein synthesis inhibitor, showing significant D1 degradation and repair under low irradiance. Exposure to HL lead to a 3.2-fold increase in D1 degradation rate, whereas average D1 repair rate was 1.3-x higher under HL than LL, leading to decreased levels of D1 protein under HL. There were significant effects of both HL and LINC on P. tricornutum maximum quantum yield of PSII (F v/F m), showing a reduction of active PSII reaction centres. Partial recovery of F v/F m in the dark demonstrates the photosynthetic resilience of this diatom to changes in the light regime. P. tricornutum showed high allocation of total protein to D1 and an active D1-repair cycle to limit photoinhibition.

Photosynthesis by six Portuguese maize cultivars during drought stress and recovery

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.

Water deficit and recovery response of Medicago truncatula plants expressing the ELIP-like DSP22

In this article, we present the response of Medicago truncatula Gaert. cv. Jemalong plants expressing constitutively the Dsp22 gene from Craterostigma plantagineum to water stress and rehydration. The Dsp22 gene encodes an ELIP-like protein thought to protect the chloroplast against photooxidative damage during the dehydration and rehydration. The Dsp22 transgenic homozygous M. truncatula plants showed higher amount of chlorophyll (Chl), lower Chl a/Chl b ratio and higher actual efficiency of energy conversion in photosystem 2 (ΦPSII) after rehydration, when compared to the wild type. The combined data from the Chl a fluorescence analysis, pigment quantification and biomass accumulation showed that transgenic M. truncatula plants are able to recover from water deprivation better than wild type plants.

Photosynthesis Assessment in Microphytobenthos Using Conventional and Imaging Pulse Amplitude Modulation Fluorometry

Imaging pulse amplitude modulated (Imaging‐PAM) fluorometry is a breakthrough in the study of spatial heterogeneity of photosynthetic assemblages. However, Imaging and conventional PAM uses a different technology, making comparisons between these techniques doubtful. Thereby, photosynthetic processes were comparatively assessed using conventional (Junior PAM and PAM 101) and Imaging‐PAM on intertidal microphytobenthos (MPB; mud and sand) and on cork oak leaves. Lower values of α (initial slope of the rETR, relative photosynthetic electron transport rate) vs E (incident photosynthetic active radiation) curve), ETRmax (maximum relative ETR), Ek (light saturation parameter) and Fv/Fm (maximum quantum efficiency of photosystem II of dark‐adapted samples) were obtained using the Imaging‐PAM. The level of discrepancy between conventional and Imaging‐PAM systems was dependent on the type of sample, being more pronounced for MPB muddy sediments. This may be explained by differences in the depth integration of the fluorescence signal related to the thickness of the photosynthetic layer and in the light attenuation coefficients of downwelling irradiance. An additional relevant parameter is the taxonomic composition of the MPB, as cyanobacteria present in sandy sediments rendered different results with red and blue excitation light fluorometers. These findings emphasize the caution needed when interpreting chlorophyll fluorescence data of MPB communities.