Effects of low temperature on photoinhibition and singlet oxygen production in four natural accessions of Arabidopsis.

MAIN CONCLUSIONS: Low temperature decreases PSII damage in vivo, confirming earlier in vitro results. Susceptibility to photoinhibition differs among Arabidopsis accessions and moderately decreases after 2-week cold-treatment. Flavonols may alleviate photoinhibition. The rate of light-induced inactivation of photosystem II (PSII) at 22 and 4 °C was measured from natural accessions of Arabidopsis thaliana (Rschew, Tenela, Columbia-0, Coimbra) grown under optimal conditions (21 °C), and at 4 °C from plants shifted to 4 °C for 2 weeks. Measurements were done in the absence and presence of lincomycin (to block repair). PSII activity was assayed with the chlorophyll a fluorescence parameter Fv/Fm and with light-saturated rate of oxygen evolution using a quinone acceptor. When grown at 21 °C, Rschew was the most tolerant to photoinhibition and Coimbra the least. Damage to PSII, judged from fitting the decrease in oxygen evolution or Fv/Fm to a first-order equation, proceeded more slowly or equally at 4 than at 22 °C. The 2-week cold-treatment decreased photoinhibition at 4 °C consistently in Columbia-0 and Coimbra, whereas in Rschew and Tenela the results depended on the method used to assay photoinhibition. The rate of singlet oxygen production by isolated thylakoid membranes, measured with histidine, stayed the same or slightly decreased with decreasing temperature. On the other hand, measurements of singlet oxygen from leaves with Singlet Oxygen Sensor Green suggest that in vivo more singlet oxygen is produced at 4 °C. Under high light, the PSII electron acceptor QA was more reduced at 4 than at 22 °C. Singlet oxygen production, in vitro or in vivo, did not decrease due to the cold-treatment. Epidermal flavonols increased during the cold-treatment and, in Columbia-0 and Coimbra, the amount correlated with photoinhibition tolerance.

Low temperature induced modulation of photosynthetic induction in non-acclimated and cold-acclimated Arabidopsis thaliana: chlorophyll a fluorescence and gas-exchange measurements.

Cold acclimation modifies the photosynthetic machinery and enables plants to survive at sub-zero temperatures, whereas in warm habitats, many species suffer even at non-freezing temperatures. We have measured chlorophyll a fluorescence (ChlF) and CO2 assimilation to investigate the effects of cold acclimation, and of low temperatures, on a cold-sensitive Arabidopsis thaliana accession C24. Upon excitation with low intensity (40 µmol photons m- 2 s- 1) ~ 620 nm light, slow (minute range) ChlF transients, at ~ 22 °C, showed two waves in the SMT phase (S, semi steady-state; M, maximum; T, terminal steady-state), whereas CO2 assimilation showed a linear increase with time. Low-temperature treatment (down to - 1.5 °C) strongly modulated the SMT phase and stimulated a peak in the CO2 assimilation induction curve. We show that the SMT phase, at ~ 22 °C, was abolished when measured under high actinic irradiance, or when 3-(3, 4-dichlorophenyl)-1, 1- dimethylurea (DCMU, an inhibitor of electron flow) or methyl viologen (MV, a Photosystem I (PSI) electron acceptor) was added to the system. Our data suggest that stimulation of the SMT wave, at low temperatures, has multiple reasons, which may include changes in both photochemical and biochemical reactions leading to modulations in non-photochemical quenching (NPQ) of the excited state of Chl, "state transitions," as well as changes in the rate of cyclic electron flow through PSI. Further, we suggest that cold acclimation, in accession C24, promotes "state transition" and protects photosystems by preventing high excitation pressure during low-temperature exposure.

Mass spectrometry-based metabolomic fingerprinting for screening cold tolerance in Arabidopsis thaliana accessions.

The availability of rapid and reliable tools for monitoring of plants' cold tolerance is a prerequisite for research aimed at breeding of cold-tolerant crop plants. Therefore, we have tested the capacity of metabolomics-based methods employing ultra-high-performance liquid chromatography (UHPLC)-mass spectrometry and direct analysis in real time-mass spectrometry for high-throughput screening of cold tolerance in eight differentially cold-tolerant accessions of Arabidopsis thaliana. Metabolomic fingerprinting of leaf tissues was performed in methanolic extracts for (1) 6-week-old non-acclimated (NAC) plants grown at room temperature, (2) NAC plants cold-acclimated (ACC) at 4 °C for 2 weeks, and (3) cold-acclimated plants given sub-zero-temperature treatments by slow cooling at -4 °C for 8 h. The generated chromatograms and mass spectra were processed with the use of multivariate statistical analysis employing principal component analysis (PCA) and linear discriminant analysis. The PCA of metabolomic fingerprints classified the investigated A. thaliana accessions into three categories with low, intermediate, and high cold tolerance for both the cold-acclimated and the sub-zero-temperature-treated plants. This indicates the potential application of metabolomics-based fingerprinting for measuring cold tolerance in the cold-acclimated state, i.e., without treating plants at freezing temperatures that is required by currently available methods. Furthermore, we employed UHPLC coupled to the quadrupole-time-of-flight mass spectrometry to identify characteristic metabolites in ACC state and found the abundance of gluconapin and flavon-3-ol glycosides, respectively, in the cold-sensitive and the cold-tolerant accessions.

Evidence for a role of raffinose in stabilizing photosystem II during freeze-thaw cycles.

A role of non-reducing sugars like sucrose and raffinose in the protection of plant cells against damage during freezing has been proposed for many species, but reports on physiological effects are conflicting. Non-aqueous fractionation of mesophyll cell compartments in Arabidopsis thaliana was used to show that sucrose and raffinose accumulate in plastids during low temperatures, pointing to a physiological role in protecting the photosynthetic apparatus. Comparing a previously described raffinose synthase (RS) mutant of A. thaliana with its corresponding wild type, accession Col-0, revealed that a lack of raffinose has no effect on electrolyte leakage from leaf cells after freeze-thaw cycles, supporting that raffinose is not essential for protecting the plasma membrane. However, in situ chlorophyll fluorescence showed that maximum quantum yield of PS II photochemistry (F (v)/F (m)) and other fluorescence parameters of cold acclimated leaves subjected to freeze-thaw cycles were significantly lower in the raffinose synthase mutant than in the corresponding wild type, indicating that raffinose is involved in stabilizing PS II of cold acclimated leaf cells against damage during freezing.

Towards discrimination of plant species by machine vision: advanced statistical analysis of chlorophyll fluorescence transients.

Automatic discrimination of plant species is required for precision farming and for advanced environmental protection. For this task, reflected sunlight has already been tested whereas fluorescence emission has been only scarcely considered. Here, we investigated the discriminative potential of chlorophyll fluorescence imaging in a case study using three closely related plant species of the family Lamiaceae. We compared discriminative potential of eight classifiers and four feature selection methods to identify the fluorescence parameters that can yield the highest contrast between the species. Three plant species: Ocimum basilicum, Origanum majorana and Origanum vulgare were grown separately as well as in pots where all three species were mixed. First, eight statistical classifiers were applied and tested in simulated species discrimination. The performance of the Quadratic Discriminant Classifier was found to be the most efficient. This classifier was further applied in combination with four different methods of feature selection. The Sequential Forward Floating Selection was found as the most efficient method for selecting the best performing subset of fluorescence images. The ability of the combinatorial statistical techniques for discriminating the species was also compared to the resolving power of conventional fluorescence parameters and found to be more efficient.

Degradation of chlorophyll and synthesis of flavonols during autumn senescence-the story told by individual leaves.

Autumn senescence of deciduous trees is characterized by chlorophyll degradation and flavonoid synthesis. In the present study, chlorophyll and flavonol contents were measured every morning and evening during the whole autumn with a non-destructive method from individual leaves of Sorbus aucuparia, Acer platanoides, Betula pendula and Prunus padus. In most of the studied trees, the chlorophyll content of each individual leaf remained constant until a phase of rapid degradation commenced. The fast phase lasted only ~1 week and ended with abscission. In S. aucuparia, contrary to the other species, the chlorophyll content of leaflets slowly but steadily decreased during the whole autumn, but rapid chlorophyll degradation commenced only prior to leaflet abscission also in this species. An increase in flavonols commonly accompanied the rapid degradation of chlorophyll. The results may suggest that each individual tree leaf retains its photosynthetic activity, reflected by a high chlorophyll content, until a rapid phase of chlorophyll degradation and flavonoid synthesis begins. Therefore, in studies of autumn senescence, leaves whose chlorophyll content is decreasing and leaves with summertime chlorophyll content (i.e. the leaves that have not yet started to degrade chlorophyll) should be treated separately.

Distinct growth and physiological responses of Arabidopsis thaliana natural accessions to drought stress and their detection using spectral reflectance and thermal imaging.

Reduced growth and stomatal closure are the two main responses of plants to drought stress. The extent to which these processes are connected and whether different genotypes prefer one over the other remains unclear. To understand the genotype-specific interconnections of these two processes and evaluate potential utilisation of this knowledge for drought tolerance phenotyping, six natural accessions of Arabidopsis thaliana (L.) Heynh. were exposed to drought stress for 10 days. Projected leaf area of rosette, light-saturated CO2 assimilation rate (Amax), relative water content (RWC), leaf temperature (thermal imaging), and spectral reflectance were measured through the course of induced drought stress. Three types of acclimation were identified: (i) growth not affected but Amax significantly reduced, (ii) both growth and Amax significantly reduced, and (iii) growth significantly reduced but only small decrease in Amax. Within the last type, the smallest decline in RWC was evident. These results show that a substantial reduction in leaf area may cause a decline in transpiration that enables maintenance of both RWC and physiological processes. Both non-invasive thermal imaging and spectral reflectance measurements proved reliable tools for tracking drought-induced changes in Amax and RWC across all accessions tested and thus are effective tools for phenotyping stress tolerance.

Chlorophyll a fluorescence, under half of the adaptive growth-irradiance, for high-throughput sensing of leaf-water deficit in Arabidopsis thaliana accessions.

BACKGROUND: Non-invasive and high-throughput monitoring of drought in plants from its initiation to visible symptoms is essential to quest drought tolerant varieties. Among the existing methods, chlorophyll a fluorescence (ChlF) imaging has the potential to probe systematic changes in photosynthetic reactions; however, prerequisite of dark-adaptation limits its use for high-throughput screening. RESULTS: To improve the throughput monitoring of plants, we have exploited their light-adaptive strategy, and investigated possibilities of measuring ChlF transients under low ambient irradiance. We found that the ChlF transients and associated parameters of two contrasting Arabidopsis thaliana accessions, Rsch and Co, give almost similar information, when measured either after ~20 min dark-adaptation or in the presence of half of the adaptive growth-irradiance. The fluorescence parameters, effective quantum yield of PSII photochemistry (ΦPSII) and fluorescence decrease ratio (RFD) resulting from this approach enabled us to differentiate accessions that is often not possible by well-established dark-adapted fluorescence parameter maximum quantum efficiency of PSII photochemistry (FV/FM). Further, we screened ChlF transients in rosettes of well-watered and drought-stressed six A. thaliana accessions, under half of the adaptive growth-irradiance, without any prior dark-adaptation. Relative water content (RWC) in leaves was also assayed and compared to the ChlF parameters. As expected, the RWC was significantly different in drought-stressed from that in well-watered plants in all the six investigated accessions on day-10 of induced drought; the maximum reduction in the RWC was obtained for Rsch (16%), whereas the minimum reduction was for Co (~7%). Drought induced changes were reflected in several features of ChlF transients; combinatorial images obtained from pattern recognition algorithms, trained on pixels of image sequence, improved the contrast among drought-stressed accessions, and the derived images were well-correlated with their RWC. CONCLUSIONS: We demonstrate here that ChlF transients and associated parameters measured even in the presence of low ambient irradiance preserved its features comparable to that of measured after dark-adaptation and discriminated the accessions having differential geographical origin; further, in combination with combinatorial image analysis tools, these data may be readily employed for early sensing and mapping effects of drought on plant's physiology via easy and fully non-invasive means.

Chlorophyll fluorescence emission can screen cold tolerance of cold acclimated Arabidopsis thaliana accessions.

BACKGROUND: An easy and non-invasive method for measuring plant cold tolerance is highly valuable to instigate research targeting breeding of cold tolerant crops. Traditional methods are labor intensive, time-consuming and thereby of limited value for large scale screening. Here, we have tested the capacity of chlorophyll a fluorescence (ChlF) imaging based methods for the first time on intact whole plants and employed advanced statistical classifiers and feature selection rules for finding combinations of images able to discriminate cold tolerant and cold sensitive plants. RESULTS: ChlF emission from intact whole plant rosettes of nine Arabidopsis thaliana accessions was measured for (1) non-acclimated (NAC, six week old plants grown at room temperature), (2) cold acclimated (AC, NAC plants acclimated at 4°C for two weeks), and (3) sub-zero temperature (ST) treated (STT, AC plants treated at -4°C for 8 h in dark) states. Cold acclimation broadened the slow phase of ChlF transients in cold sensitive (Co, C24, Can and Cvi) A. thaliana accessions. Similar broadening in the slow phase of ChlF transients was observed in cold tolerant (Col, Rsch, and Te) plants following ST treatments. ChlF parameters: maximum quantum yield of PSII photochemistry (FV/FM) and fluorescence decrease ratio (RFD) well categorized the cold sensitive and tolerant plants when measured in STT state. We trained a range of statistical classifiers with the sequence of captured ChlF images and selected a high performing quadratic discriminant classifier (QDC) in combination with sequential forward floating selection (SFFS) feature selection methods and found that linear combination of three images showed a reasonable contrast between cold sensitive and tolerant A. thaliana accessions for AC as well as for STT states. CONCLUSIONS: ChlF transients measured for an intact whole plant is important for understanding the impact of cold acclimation on photosynthetic processes. Combinatorial imaging combined with statistical classifiers and feature selection methods worked well for the screening of cold tolerance without exposing plants to sub-zero temperatures. This opens up new possibilities for high-throughput monitoring of whole plants cold tolerance via easy and fully non-invasive means.

Chlorophyll fluorescence emission as a reporter on cold tolerance in Arabidopsis thaliana accessions.

Non-invasive, high-throughput screening methods are valuable tools in breeding for abiotic stress tolerance in plants. Optical signals such as chlorophyll fluorescence emission can be instrumental in developing new screening techniques. In order to examine the potential of chlorophyll fluorescence to reveal plant tolerance to low temperatures, we used a collection of nine Arabidopsis thaliana accessions and compared their fluorescence features with cold tolerance quantified by the well established electrolyte leakage method on detached leaves. We found that, during progressive cooling, the minimal chlorophyll fluorescence emission rose strongly and that this rise was highly dependent on the cold tolerance of the accessions. Maximum quantum yield of PSII photochemistry and steady state fluorescence normalized to minimal fluorescence were also highly correlated to the cold tolerance measured by the electrolyte leakage method. In order to further increase the capacity of the fluorescence detection to reveal the low temperature tolerance, we applied combinatorial imaging that employs plant classification based on multiple fluorescence features. We found that this method, by including the resolving power of several fluorescence features, can be well employed to detect cold tolerance already at mild sub-zero temperatures. Therefore, there is no need to freeze the screened plants to the largely damaging temperatures of around -15°C. This, together with the method's easy applicability, represents a major advantage of the fluorescence technique over the conventional electrolyte leakage method.