Sergio González-Pérez

Early Transcriptional Defense Responses in Arabidopsis Cell Suspension Culture under High-Light Conditions

The early transcriptional defense responses and reactive oxygen species (ROS) production in Arabidopsis (Arabidopsis thaliana) cell suspension culture (ACSC), containing functional chloroplasts, were examined at high light (HL). The transcriptional analysis revealed that most of the ROS markers identified among the 449 transcripts with significant differential expression were transcripts specifically up-regulated by singlet oxygen (1O2). On the contrary, minimal correlation was established with transcripts specifically up-regulated by superoxide radical or hydrogen peroxide. The transcriptional analysis was supported by fluorescence microscopy experiments. The incubation of ACSC with the 1O2 sensor green reagent and 2′,7′-dichlorofluorescein diacetate showed that the 30-min-HL-treated cultures emitted fluorescence that corresponded with the production of 1O2 but not of hydrogen peroxide. Furthermore, the in vivo photodamage of the D1 protein of photosystem II indicated that the photogeneration of 1O2 took place within the photosystem II reaction center. Functional enrichment analyses identified transcripts that are key components of the ROS signaling transduction pathway in plants as well as others encoding transcription factors that regulate both ROS scavenging and water deficit stress. A meta-analysis examining the transcriptional profiles of mutants and hormone treatments in Arabidopsis showed a high correlation between ACSC at HL and the fluorescent mutant family of Arabidopsis, a producer of 1O2 in plastids. Intriguingly, a high correlation was also observed with ABA deficient1 and more axillary growth4, two mutants with defects in the biosynthesis pathways of two key (apo)carotenoid-derived plant hormones (i.e. abscisic acid and strigolactones, respectively). ACSC has proven to be a valuable system for studying early transcriptional responses to HL stress.

Programmed cell death activated by Rose Bengal in Arabidopsis thaliana cell suspension cultures requires functional chloroplasts

Summary Arabidopsis cell suspension cultures are used as a biological tool to better understand the key role of functional chloroplasts in singlet oxygen-mediated programmed cell death in plants.

Raman Spectroscopy Adds Complementary Detail to the High-Resolution X-Ray Crystal Structure of Photosynthetic Psbp from Spinacia Oleracea.

Raman microscopy permits structural analysis of protein crystals in situ in hanging drops, allowing for comparison with Raman measurements in solution. Nevertheless, the two methods sometimes reveal subtle differences in structure that are often ascribed to the water layer surrounding the protein. The novel method of drop-coating deposition Raman spectropscopy (DCDR) exploits an intermediate phase that, although nominally “dry,” has been shown to preserve protein structural features present in solution. The potential of this new approach to bridge the structural gap between proteins in solution and in crystals is explored here with extrinsic protein PsbP of photosystem II from Spinacia oleracea. In the high-resolution (1.98 Å) x-ray crystal structure of PsbP reported here, several segments of the protein chain are present but unresolved. Analysis of the three kinds of Raman spectra of PsbP suggests that most of the subtle differences can indeed be attributed to the water envelope, which is shown here to have a similar Raman intensity in glassy and crystal states. Using molecular dynamics simulations cross-validated by Raman solution data, two unresolved segments of the PsbP crystal structure were modeled as loops, and the amino terminus was inferred to contain an additional beta segment. The complete PsbP structure was compared with that of the PsbP-like protein CyanoP, which plays a more peripheral role in photosystem II function. The comparison suggests possible interaction surfaces of PsbP with higher-plant photosystem II. This work provides the first complete structural picture of this key protein, and it represents the first systematic comparison of Raman data from solution, glassy, and crystalline states of a protein.

Peroxynitrite inhibits electron transport on the acceptor side of higher plant photosystem II

Peroxynitrite is a strong oxidant that has been proposed to form in chloroplasts. The interaction between peroxynitrite and photosystem II (PSII) has been investigated to determine whether this oxidant could be a hazard for PSII. Peroxynitrite is shown to inhibit oxygen evolution in PSII membranes in a dose-dependent manner. Analyses by PAM fluorimetry and EPR spectroscopy have demonstrated that the inhibition target of peroxynitrite is on the PSII acceptor side. In the presence of the herbicide DCMU, the chlorophyll (Chl) a fluorescence induction curve is inhibited by peroxynitrite, but the slow phase of the Chl a fluorescence decay does not change. EPR studies demonstrate that the Signal II(slow) and Signal II(fast) of peroxynitrite-treated Tris-washed PSII membranes are induced at room temperature, implying that the redox active tyrosines Y(Z) and Y(D) of PSII are not significantly nitrated. A featureless EPR signal with a g value of approximately 2.0043+/-0.0003 and a line width of 10+/-1G is induced under continuous illumination in the presence of peroxynitrite. This new EPR signal corresponds with the semireduced plastoquinone Q(A) in the absence of magnetic interaction with the non-heme Fe2+. We conclude that peroxynitrite impairs PSII electron transport in the Q(A)Fe2+ niche.

Trolox, a Water-Soluble Analogue of α-Tocopherol, Photoprotects the Surface-Exposed Regions of the Photosystem II Reaction Center in Vitro. Is This Physiologically Relevant?

Can Trolox, a water-soluble analogue of α-tocopherol and a scavenger of singlet oxygen ((1)O(2)), provide photoprotection, under high irradiance, to the isolated photosystem II (PSII) reaction center (RC)? To answer the question, we studied the endogenous production of (1)O(2) in preparations of the five-chlorophyll PSII RC (RC5) containing only one β-carotene molecule. The temporal profile of (1)O(2) emission at 1270 nm photogenerated by RC5 in D(2)O followed the expected biexponential behavior, with a rise time, unaffected by Trolox, of 13 ± 1 μs and decay times of 54 ± 2 μs (without Trolox) and 38 ± 2 μs (in the presence of 25 μM Trolox). The ratio between the total (k(t)) and chemical (k(r)) bimolecular rate constants for the scavenging of (1)O(2) by Trolox in aqueous buffer was calculated to be ~1.3, with a k(t) of (2.4 ± 0.2) × 10(8) M(-1) s(-1) and a k(r) of (1.8 ± 0.2) × 10(8) M(-1) s(-1), indicating that most of the (1)O(2) photosensitized by methylene blue chemically reacts with Trolox in the assay buffer. The photoinduced oxygen consumption in the oxygen electrode, when RC5 and Trolox were mixed, revealed that Trolox was a better (1)O(2) scavenger than histidine and furfuryl alcohol at low concentrations (i.e., <1 mM). After its incorporation into detergent micelles in unbuffered solutions, Trolox was able to photoprotect the surface-exposed regions of the D1-D2 heterodimer, but not the RC5 pigments, which were oxidized, together with the membrane region of the protein matrix of the PSII RC, by (1)O(2). These results are discussed and compared with those of studies dealing with the physiological role of tocopherol molecules as a (1)O(2) scavenger in thylakoid membranes of photosynthetic organisms.

Does singlet oxygen activate cell death in Arabidopsis cell suspension cultures?: analysis of the early transcriptional defense responses to high light stress.

Can Arabidopsis cell suspension cultures (ACSC) provide a useful working model to investigate genetically-controlled defense responses with signaling cascades starting in chloroplasts? In order to provide a convincing answer, we analyzed the early transcriptional profile of Arabidopsis cells at high light (HL). The results showed that ACSC respond to HL in a manner that resembles the singlet oxygen (1O2)-mediated defense responses described for the conditional fluorescent (flu) mutant of Arabidopsis thaliana. The flu mutant is characterized by the accumulation of free protochlorophyllide (Pchlide) in plastids when put into darkness and the subsequent production of 1O2 when the light is on. In ACSC, 1O2 is produced in chloroplasts at HL when excess excitation energy flows into photosystem II (PSII). Other reactive oxygen species are also produced in ACSC at HL, but to a lesser extent. When the HL stress ceases, ACSC recovers the initial rate of oxygen evolution and cell growth continues. We can conclude that chloroplasts of ACSC are both photosynthetically active and capable of initiating 1O2-mediated signaling cascades that activate a broad range of genetically-controlled defense responses. The upregulation of transcripts associated with the biosynthesis and signaling pathways of OPDA (12-oxophytodienoic acid) and ethylene (ET) suggests that the activated defense responses at HL are governed by these two hormones. In contrast to the flu mutant, the 1O2-mediated defense responses were independent of the upregulation of EDS1 (enhanced disease susceptibility) required for the accumulation of salicylic acid (SA) and genetically-controlled cell death. Interestingly, a high correlation in transcriptional expression was also observed between ACSC at HL, and the aba1 and max4 mutants of Arabidopsis, characterized by defects in the biosynthesis pathways of abscisic acid (ABA) and strigolactones, respectively.

Facile method for spectroscopic examination of radical ions of hydrophilic carotenoids

Hydrophilic carotenoids, unusual members of an intrinsically hydrophobic family, and their radical ions are important reactants. An all-optical method for generating singly charged radical ions of a hydrophilic carotenoid (Car) is described. It relies on photolyzing an aqueous mixture of Car and a photoionizable auxiliary solute (A), and making conditions conducive to the capture, by Car, of the hydrated electron (e(aq)(-)) or the positive hole in A(*)(+) or both. When A is Trolox (TOH), only e(aq)(-) can be captured, since TOH (*)(+) deprotonates too rapidly to be a hole donor; when A is Trolox methyl ether (TOMe), both Car(*)(-) and Car(*)(+) are formed, since TOMe (+) lives long enough to transfer its positive hole to Car; formation of Car(*)(-) is prevented under aerobic conditions.

Singlet oxygen triggers chloroplast rupture and cell death in the zeaxanthin epoxidase defective mutant aba1 of Arabidopsis thaliana under high light stress

The two Arabidopsis thaliana mutants, aba1 and max4, were previously identified as sharing a number of co-regulated genes with both the flu mutant and Arabidopsis cell suspension cultures exposed to high light (HL). On this basis, we investigated whether aba1 and max4 were generating high amounts of singlet oxygen (1O2) and activating 1O2-mediated cell death. Thylakoids of aba1 produced twice as much 1O2 as thylakoids of max4 and wild type (WT) plants when illuminated with strong red light. 1O2 was measured using the spin probe 2,2,6,6-tetramethyl-4-piperidone hydrochloride. 77-K chlorophyll fluorescence emission spectra of thylakoids revealed lower aggregation of the light harvesting complex II in aba1. This was rationalized as a loss of connectivity between photosystem II (PSII) units and as the main cause for the high yield of 1O2 generation in aba1. Up-regulation of the 1O2 responsive gene AAA-ATPase was only observed with statistical significant in aba1 under HL. Two early jasmonate (JA)-responsive genes, JAZ1 and JAZ5, encoding for two repressor proteins involved in the negative feedback regulation of JA signalling, were not up-regulated to the WT plant levels. Chloroplast aggregation followed by chloroplast rupture and eventual cell death was observed by confocal imaging of the fluorescence emission of leaf cells of transgenic aba1 plants expressing the chimeric fusion protein SSU-GFP. Cell death was not associated with direct 1O2 cytotoxicity in aba1, but rather with a delayed stress response. In contrast, max4 did not show evidence of 1O2-mediated cell death. In conclusion, aba1 may serve as an alternative model to other 1O2-overproducing mutants of Arabidopsis for investigating 1O2-mediated cell death.

12 – Sunflower Proteins

Publisher Summary Sunflower seeds are mainly used for their oil, but as with other oilseeds, the meal left behind after oil extraction is a valuable product because of its high protein content. Sunflower seeds are attractive due to their high protein content and extensive availability. Compared to other vegetable protein sources, sunflower seeds contain low or no anti-nutritional factors and, except for lysine present at low concentration, their amino acid composition conforms to the Food and Agriculture Organization outline of human requirements. Sunflower protein consists largely of albumins and globulins and, consequently, has a high intrinsic solubility. Nowadays, the main market outlet for sunflower protein relies on animal feed. This chapter discusses sunflower proteins, principally seed proteins, including composition, structure, stability, and biochemical and molecular characterization.

Femtosecond Laser Disruption of Filamentous Cyanobacteria Unveils Dissimilar Cellular Stability Between Heterocysts and Vegetative Cells

Filamentous cyanobacteria develop heterocysts in response to deprivation for combined nitrogen under aerobic conditions. The most prominent structural change in heterocysts is the biosynthesis of an envelope that restricts gas permeability, providing an appropriate micro‐oxic environment for N2 fixation inside. The additional thickness of the differentiated cells, when compared to vegetative cells, makes filamentous cyanobacteria an attractive biological system to investigate cellular response against femtosecond laser processing. By irradiating the cyanobacterial filaments with 120 fs, 795 nm, 1 kHz pulses focused through a 100× microscope objective with a numerical aperture of 0.85, we have determined that the pulse energy threshold for an apparent disruption of the cell wall of vegetative cells is 13 ± 4 nJ per pulse. A further increase in the pulse energy to 43 ± 13 nJ causes the complete removal of vegetative cells. In contrast, the pulse energy threshold has to be augmented about three‐fold for heterocyst envelope disruption or two‐fold for complete removal of heterocysts. We propose that the singular cross‐linked structure of the glycolipid multilayer of the envelope, required to restrict gas permeability, accounts for the remarked difference in the ablation energy threshold between vegetative cells and heterocysts.