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Chlorophyll fluorescence measurements have been widely applied as non-destructive methods to study the photosynthetic efficiency of plants, under control or stress conditions. Compared to most protocols of pulse amplitude modulation (PAM) fluorometry, laser induced chlorophyll fluorescence (LIF) has the advantages of not requiring pulses to be delivered at close range, allowing the remote analysis of a great number of individual plants in a short period of time. Such analyses are extremely useful, for instance, when doing large phenotyping screens of Arabidopsis thaliana mutants or ecotypes. Water deficit is a major abiotic stress compromising plant growth and productivity. Arabidopsis has been adopted as the main model organism in plant sciences and it has been widely used in plant stress studies. However, reports on the applications of LIF techniques to this model plant are scarce. Here we report the use of LIF to investigate changes in chlorophyll a (Chl a ) fluorescence signature under progressive drought of potted Arabidopsis plants (Slow Stress) and under fast dehydration of detached leaves (Fast Stress). Results show that the two dehydration methods cause distinct modifications on the red/far-red Chl a fluorescence ratio ( F 690 / F 730 ) and on the wavelengths of Chl a fluorescence maxima. These differences are likely related to distinct changes in water content, photosynthetic pigments, anthocyanins, fatty acid composition and other metabolic adaptations, which are differently regulated in Slow and Fast Stress. Of particular interest are Chl a emission fluorescence changes, which take place under progressive drought, before a substantial decrease in leaf water content. Additionally, no differences were found on LIF emission spectra between fully expanded and young leaves. However, the choice of leaf surface influenced fluorescence emission, with the abaxial surface displaying lower fluorescence and higher F 690 / F 730 ratios. Results suggest that LIF is a fast and non-destructive tool suitable for high-throughput phenotyping of Arabidopsis under water deficit.

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.

Phenotypic screening after transformation experiments aiming to identify lines with the enhanced/desired trait is still a time consuming process for most agricultural crops, especially when dealing with complex physiological responses such as water deficit. In this study we evaluated the suitability of non-destructive leaf gas-exchange analysis and imaging-PAM chlorophyll a fluorescence to select transgenic lines of Medicago truncatula expressing the Trehalose-6-Phosphate Synthase 1 (AtTPS1) from Arabidopsis thaliana with altered response to water deficit (WD) and WD recovery (WDR) in the early stages of the transformation process (T0). Primary transformants (T0) with different expression levels of a constitutive AtTPS1 construct were used. Additionally, we evaluated if the expression of the transgene could be correlated with the phenotype assessed. Among tested techniques and parameters measured, the net carbon assimilation (A) from gas-exchange analysis was the best parameter to early detect lines with WD and WDR improved performance, at the earliest stages of the transformation process. With this multidisciplinary approach, we selected 3 transgenic lines TPS7, TPS10 and TPS16 for further studies, which have higher or intermediate expression levels of the transgene and improved response to WD and WDR. This work will contribute to speed-up the identification of elite lines with confidence within a large number of individuals, thus reducing time, cost and labor associated with this plant improvement strategy.

Photosynthesis and lipid allocation were investigated in Rubisco small subunit mutants of the microalga Chlamydomonas reinhardtii. Comparative analyses were undertaken with cells grown photoheterotrophically under sulphur-replete or sulphur-depleted conditions. The Y67A Rubisco mutant, which has previously demonstrated a pronounced reduction in Rubisco levels and higher hydrogen production rates than the wild type, also shows the following divergences in photosynthetic phenotype and lipid allocation: (i) low Fv/Fm (maximum photochemical efficiency), (ii) low effective quantum yield of photosystem II (ΦPSII), (iii) low effectiveness at protection against high light intensities, (iv) a higher level of total lipids per pigment and (v) changes in the relative proportions of different fatty acids, with a marked decrease in unsaturated fatty acids (FAs). The most abundant thylakoid membrane lipid, monogalactosyldiacylglycerol, decreased in amount, while the neutral lipid/polar lipid ratio increased in the mutant. The low amount and activity of the mutated Rubisco Y67A enzyme seems to have an adverse effect on photosynthesis and causes changes in carbon allocation in terms of membrane fatty acid composition and storage lipid accumulation. Our results suggest that Rubisco mutants of Chlamydomonas might be useful in biodiesel production.

Water deficit, when rapidly imposed on three C4 grasses of the different metabolic subtypes, Paspalum dilatatum Poiret (NADP-malic enzyme), Cynodon dactylon (L.) Pers (NAD-malic enzyme) and Zoysia japonica Steudel (phosphoenolpyruvate carboxykinase), caused decreases in photosynthetic rates, in the quantum yield of PS II and photochemical quenching, and in the activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC). The results provide evidence for non-stomatal limitations of photosynthesis differing in nature between the three species.

The photosynthetic performance of Cistus crispus, Centaurea africana and Thymus villosus, three shrubs of the Mediterranean-type maquis vegetation of Central Portugal, were studied between July and December 2003 by means of in vivo chlorophyll a fluorescence measurements. In C. africana total leaf decay occurred between August and October, in T. villosus no clear pattern of variation of the basal fluorescence (Fo) was observed, and in C. crispus an increase of this parameter was observed in late autumn. In all species, the limitation to photosynthesis by photoinhibition and the contribution of non-reversible processes to total photoinhibition were more pronounced in the summer than in the autumn. However, a pronounced decrease of the effective quantum yield of photosystem 2 (ΦPS2) was found in late autumn, probably due to a decrease of the carboxylation efficiency of the Calvin-Benson cycle, which was also supported by a parallel decrease of the photochemical quenching coefficient (qP).

Two stress imposing systems were used: a rapid stress developed by allowing excised leaves to loose water by transpiration, and a slow stress developed by withholding watering of potted plants. Carboxylating enzymes reacted differently on both types of stress. Rapid stress increased ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activation, but both activities (initial and total) showed little variation with stress. Under slow stress the activation did not change, although both activities decreased much under stress. Phosphoenolpyruvate carboxylase (PEPC) showed a deep decrease of activity under rapid stress, nevertheless, a certain recovery was found under extreme stress. On the other hand, under slow stress the activity of PEPC showed a linear increase with decreasing relative water content. The ratio between physiological and maximal activity increased slightly under both types of stress. The activity of malic enzyme did not change under rapid stress, and decreased linearly under slow stress.

Microphytobenthos are frequently subjected to light intensities higher than those required to saturate photosynthesis, which consequently can cause photoinhibition. Photosystem II (PSII) protein D1 (the main target of photoinhibition) and xanthophyll cycle pigments were quantified in epipelic benthic diatom assemblages under high irradiance, in the presence of inhibitors and promoters of photoprotection mechanisms. Levels of D1 protein were significantly lower under high irradiance (1 h, 1500 µmol photons m–2 s–1) than under low light (80 µmol photons m–2 s–1), corresponding to a photoinhibition of 22 to 29%. Photoinhibition increased to 44 and 80% in the presence of lincomycin (inhibitor of chloroplast-protein synthesis) and dithiothreitol (inhibitor of the xanthophyll cycle), respectively. High light treatment had no significant effect on D1 protein concentrations in the presence of added glutathione and ascorbate, scavengers of reactive oxygen species (ROS). In contrast, the ROS promoter methylviologen increased photoinhibition to 63%. Under light stress, the functional stability of PSII reaction centres of the studied epipelic benthic diatoms was more dependent on xanthophyll cycle activation than on D1 protein recycling mechanisms, and our results substantiate the role of antioxidants in photoprotection via ROS scavenging. published

The aim of the present work was to develop a method for the remote assessment of the impact of fire and drought stress on Mediterranean forest species such as the cork oak (Quercus suber) and maritime pine (Pinus pinaster). The proposed method is based on laser induced fluorescence (LIF): chlorophyll fluorescence is remotely excited by frequency-doubled YAG:Nd laser radiation pulses and collected and analyzed using a telescope and a gated high sensitivity spectrometer. The plant health criterion used is based on the I 685/I 740 ratio value, calculated from the fluorescence spectra. The method was benchmarked by comparing the results achieved with those obtained by conventional, continuous excitation fluorometric method and water loss gravimetric measurements. The results obtained with both methods show a strong correlation between them and with the weight-loss measurements, showing that the proposed method is suitable for fire and drought impact assessment on these two species.

'Rocha' pear (Pyrus communis L.) is an exclusively Portuguese certified pear cultivar commercialized worldwide. Mature unripe 'Rocha' pears were obtained from COOPVAL (Cadaval, Portugal) after 8 months at -0.5 degrees C, 94-96% RH and CA (2% O-2+0.5% CO2). Then, they were maintained in a dark room at 20+/-2 degrees C and 70% RH to simulate shelf life. For three weeks these fruit were followed along using Vis/NIR reflectance spectroscopy in the wavelength range of 400 to 950 nm, and their colour and firmness were evaluated by standard techniques. 'Rocha' pear firmness decreased significantly during shelf life, paralleled by the yellowing of the fruit skin (increase in a(star) and Hue angle). Pigments were extracted from fruit skin and assayed spectrophotometrically. Both Chla and Chlb contents decreased along ripening in shelf life, while contents of carotenoids remained constant. Vis/NIR reflectance spectra were correlated with the respective fruit skin pigments content by PLS. Prediction models were obtained for Chl (a, b, a+b), but not for carotenoids. Models were reasonably significant in regression terms [r(Chla)=0.898; r(Chlb)=0.897; r(Chla+b)=0.918], but the respective SDR (standard deviation ratio = standard deviation of the validation set/RMSEP)(2.2 (Chla), 2.3 (Chlb), 2.2 (Chla+b)) suggest that only a coarse quantitative prediction is possible for all models. Although Chla model required a higher number of latent variables [Lv(Chla)= 6; Lv(Chlb or Chla+b)= 3], similarity between RMSEC and RMSEP was lower for the other parameters [Chla: 4.6 and 4.6 g m(-2) Chlb: 3.9 and 2.3 g m(-2), Chla+b: 7.3 and 6.7 g m(-2)]. A better performance for these models has been expected, because most of the differences found in the Vis/NIR spectra in shelf life were in the Chl absorption region. However, only a coarse prediction capability was found. Thus, the data obtained suggest that changes on the background around 670 nm decrease the prediction capability of the PLS model and should be further investigated.

To study the adaptation of the C4 plant, Paspalum dilatatum, to the climate conditions of the centre of Portugal, some physiological parameters, related to photosynthesis, were determined. Measurements were made from January to July 1988. The rates of photosynthesis and transpiration increased significantly during the summer, while the chlorophyll content fluctuated from March to July. The activities of PEPC and NADP-ME were measured at 32 and 15°C. At both temperatures, there was a significant drop on the activity of PEPC, during the hottest months. The activity of NADP-ME was constant during the study.

This study aimed to test whether the chlorophyll a (Chla) fluorescence determined by a low-cost non-modulated fluorometer could provide fast, reliable and non-invasive estimators of superficial scald in 'Rocha' pear (Pyrus communis L. 'Rocha'). Fruit were harvested before the optimal maturation stage and cold stored under normal atmosphere for 7 months (NA: 0 degrees C, 90-95% RH) and 2 in controlled atmosphere (CA: 0 degrees C, 90-95% RH, 1.5 kPaO(2) + 0.5 kPa CO2) (T), or harvested at the optimal maturation stage and cold stored for 9 months under CA (C). Then, they were transferred to shelf-life conditions (22+/-2 degrees C, 70% RH) and followed for 7 d. Chla fluorescence, scald index (SI), ripening attributes, alpha-farnesene, conjugated trienols, and photosynthetic pigments were determined for each pear in both groups. Conditions chosen before shelf-life did not prevent the subsequent ripening of any fruit, but changed dramatically the superficial scald development pattern: in C fruit, the disorder developed progressively during shelf-life, whereas in T fruit, it peaked during storage. C fruit exhibited a significant negative correlation (R=-0.65; p