Dynamic feedback of the photosystem II reaction centre on photoprotection in plants.

Photosystem II of higher plants is protected against light damage by thermal dissipation of excess excitation energy, a process that can be monitored through non-photochemical quenching of chlorophyll fluorescence. When the light intensity is lowered, non-photochemical quenching largely disappears on a time scale ranging from tens of seconds to many minutes. With the use of picosecond fluorescence spectroscopy, we demonstrate that one of the underlying mechanisms is only functional when the reaction centre of photosystem II is closed, that is when electron transfer is blocked and the risk of photodamage is high. This is accompanied by the appearance of a long-wavelength fluorescence band. As soon as the reaction centre reopens, this quenching, together with the long-wavelength fluorescence, disappears instantaneously. This allows plants to maintain a high level of photosynthetic efficiency even in dangerous high-light conditions.

Structure of the chloroplast ribosome with chl-RRF and hibernation-promoting factor.

Oxygenic photosynthesis produces oxygen and builds a variety of organic compounds, changing the chemistry of the air, the sea and fuelling the food chain on our planet. The photochemical reactions underpinning this process in plants take place in the chloroplast. Chloroplasts evolved ~1.2 billion years ago from an engulfed primordial diazotrophic cyanobacterium, and chlororibosomes are responsible for synthesis of the core proteins driving photochemical reactions. Chlororibosomal activity is spatiotemporally coupled to the synthesis and incorporation of functionally essential co-factors, implying the presence of chloroplast-specific regulatory mechanisms and structural adaptation of the chlororibosome1,2. Despite recent structural information3-6, some of these aspects remained elusive. To provide new insights into the structural specialities and evolution, we report a comprehensive analysis of the 2.9-3.1 Å resolution electron cryo-microscopy structure of the spinach chlororibosome in complex with its recycling factor and hibernation-promoting factor. The model reveals a prominent channel extending from the exit tunnel to the chlororibosome exterior, structural re-arrangements that lead to increased surface area for translocon binding, and experimental evidence for parallel and convergent evolution of chloro- and mitoribosomes.

FASTmiR: an RNA-based sensor for in vitro quantification and live-cell localization of small RNAs.

Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a variety of important regulatory roles in many eukaryotes. Their small size has made it challenging to study them directly in live cells. Here we describe an RNA-based fluorescent sensor for small RNA detection both in vitro and in vivo, adaptable for any small RNA. It utilizes an sxRNA switch for detection of miRNA-mRNA interactions combined with a fluorophore-binding sequence 'Spinach', a GFP-like RNA aptamer for which the RNA-fluorophore complex exhibits strong and consistent fluorescence under an excitation wavelength. Two example sensors, FASTmiR171 and FASTmiR122, can rapidly detect and quantify the levels of miR171 and miR122 in vitro. The sensors can determine relative levels of miRNAs in total RNA extracts with sensitivity similar to small RNA sequencing and northern blots. FASTmiR sensors were also used to estimate the copy number range of miRNAs in total RNA extracts. To localize and analyze the spatial distribution of small RNAs in live, single cells, tandem copies of FASTmiR122 were expressed in different cell lines. FASTmiR122 was able to quantitatively detect the differences in miR122 levels in Huh7 and HEK293T cells demonstrating its potential for tracking miRNA expression and localization in vivo.

Enhancement of photoassembly of the functionally active water-oxidizing complex in Mn-depleted photosystem II membranes upon transition to anaerobic conditions.

It has been shown earlier (Khorobrykh and Klimov, 2015) that molecular oxygen is directly involved in the general mechanism of the donor side photoinhibition of photosystem II (PSII) membranes. In the present work the effect of oxygen on photoassembly ("photoactivation") of the functionally active inorganic core of the water-oxidizing complex (WOC) in Mn-depleted PSII preparations (apo-WOC-PSII) in the presence of exogenous Mn(2+), Ca(2+) as well as ferricyanide was investigated. It was revealed that the efficiency of the photoassembly of the WOC was considerably increased upon removal of oxygen from the medium during photoactivation procedure using the enzymatic oxygen trap or argon flow. The lowering of O2 concentration from 250µM to 75µM, 10µM and near 0µM results in 29%, 71% and 92%, respectively, stimulation of the rate of O2 evolution measured after the photoactivation. The increase in the intensity of light used during the photoactivation was accompanied by a decrease of both the efficiency of photoassembly of the WOC and the stimulation effect of removal of O2 (that may be due to the enhancement of the processes leading to the photodamage to PSII). It is concluded that the enhancement in photoactivation of oxygen-evolving activity of apo-WOC-PSII induced by oxygen removal from the medium is due to the suppression of the donor side photoinhibition of PSII in which molecular oxygen can be involved.

Is There Excitation Energy Transfer between Different Layers of Stacked Photosystem-II-Containing Thylakoid Membranes?

We have compared picosecond fluorescence decay kinetics for stacked and unstacked photosystem II membranes in order to evaluate the efficiency of excitation energy transfer between the neighboring layers. The measured kinetics were analyzed in terms of a recently developed fluctuating antenna model that provides information about the dimensionality of the studied system. Independently of the stacking state, all preparations exhibited virtually the same value of the apparent dimensionality, d = 1.6. Thus, we conclude that membrane stacking does not affect the efficiency of the delivery of excitation energy toward the reaction centers but ensures a more compact organization of the thylakoid membranes within the chloroplast and separation of photosystems I and II.

CARBONIC ANHYDRASE ACTIVITY OF INTEGRAL-FUNCTIONAL COMPLEXES OF THYLAKOID MEMBRANES OF SPINACH CHLOROPLASTS.

Isolated thylakoid membranes were disrupted by treatment with nonionic detergents digitonin or dodecyl maltoside. Solubilized polypeptide complexes were separated by native gel charge shift electrophoresis. The position of ATP-synthase complex and its isolated catalytic part (CF1) within gel was determined using the color reaction for ATPase activity. Due to the presence of cytochromes, the red band in unstained gels corresponded to the cytochrome b6f complex. Localization of the cytochrome b6f complex, ATP synthase and coupling CF1 in the native gel was confirmed by their subunit composition determined after SDS-electrophoretic analysis. Carbonic anhydrase (CA) activity in polypeptide zones of PS II, cytochrome b6f complex, and ATP-synthase CF1 was identified in native gels using indicator bromothymol blue. CA activity of isolated CF1 in solution was determined by infrared gas analysis as the rate of bicarbonate dehydration. The water-soluble acetazolamide, an inhibitor of CA, unlike lipophilic ethoxyzolamide inhibited CA activity of CF1 Thus, it was shown for the first time that ATP-synthase has a component which is capable of catalyzing the interconversion of forms of carbonic acid associated with proton exchange. The data obtained suggest the presence of multiple forms of carbonic anhydrase in the thylakoid membranes of spinach chloroplasts and confirm their involvement in the proton transfer to the ATP synthase.

Spinach (Spinacia oleracea L.) modulates its proteome differentially in response to salinity, cadmium and their combination stress.

Cadmium (Cd) contamination and salinity are common stressors in agricultural soils all over the globe. Sensitivity and modulation of plant proteome lead to proper signal execution and adaptation to abiotic stress via molecular responses, which strengthen plant defence system. A comparative proteomic study, employing 2DE-MALDI TOF/TOF MS, of Spinacia oleracea plants exposed to cadmium (50 µg CdCl2 g(-1) soil), salinity (10 mg NaCl g(-1) soil) and their combination (NaCl + Cd) was conducted to understand the minimum common adaptation to multiple stress. Analysis of 2D gel maps showed significant increase and decrease in relative abundance of 14 and 39 proteins by Cd; 11 and 46 by salinity and 22 and 37 by combined stress of Cd and salinity, respectively. Peptide mass fingerprinting (PMF) helped in the identification of maturase K and PPD4 with increased relative abundance under all stresses; whereas salinity stress and combination stress silenced the presence of one protein (polycomb protein EZ2) and two proteins (cellulose synthase-like protein and ubiquitin conjugation factor E4), respectively. The identified proteins were functionally associated with signal transduction (15%), protein synthesis (16%), stress response and defence (33%), photosynthesis (13%), plant growth/cell division (9%), energy generation (4%), transport (4%), secondary metabolism (3%), and cell death (3%); clearly indicating the importance and necessity of keeping a higher ratio of defence and disease-responsive proteins. The results suggest that plant may increase the abundance of defence proteins and may also lower the abundance of catabolic proteins. Proteins with altered ratios of abundance belonged to different functional categories, suggesting that plants have differential mechanisms to respond to Cd, salinity, and their combined stress, but with unique sets of proteins.

Trehalose stimulation of photoinduced electron transfer and oxygen photoconsumption in Mn-depleted photosystem 2 membrane fragments.

It is known that the removal of manganese from the water-oxidizing complex (WOC) of photosystem 2 (PS2) leads to activation of oxygen photoconsumption (OPC) [Khorobrykh et al., 2002; Yanykin et al., 2010] that is accompanied by the formation of organic hydroperoxides on the electron-donor side of PS2 [Khorobrykh et al., 2011]. In the present work the effect of trehalose on the OPC in Mn-depleted PS2 preparations (apo-WOC-PS2) was investigated. A more than two-fold increase of the OPC is revealed upon the addition of 1M trehalose. Drastic (30%-70%) inhibition of the OPC upon the addition of either electron acceptor or electron donor indicates that the trehalose-induced activation of the OPC occurs on both donor and acceptor sides of PS2. A two-fold increase in the rate of superoxide-anion radical photoproduction on the electron-acceptor side of PS2 was also shown. Applying the "variable" chlorophyll fluorescence (ΔF) it was shown that the addition of trehalose induces: (i) a significant increase in the ability of exogenous Mn(2+) to donate electrons to the reaction center of PS2, (ii) slowing down the photoaccumulation of the primary quinone electron acceptor of PS2 (QA(-)) under aerobic conditions, (iii) acceleration of the reoxidation of QA(-) by QB (and by QB(-)) as well as the replacement of QB(2-) by a fully oxidized plastoquinone, and (iv) restoration of the electron transfer between the quinone electron carriers in the so-called "closed reaction centers of PS2" (their content in the apo-WOC-PS2 is 41%). It is suggested that the trehalose-induced increase in efficiency of the O2 interaction with the electron-donor and electron-acceptor sides of apo-WOC-PS2 is due to structural changes leading to both a decrease in the proportion of the "closed PS2 reaction centers" and an increase in the electron transfer rate in PS2.

Characterization of the structural changes and photochemical activity of photosystem I under Al(3+) effect.

The photochemical activity of photosystem I (PSI) as affected by Al(3+) was investigated in thylakoid membranes and PSI submembrane fractions isolated from spinach. Biophysical and biochemical techniques such as oxygen uptake, light induced absorbance changes at 820nm, chlorophyll fluorescence emission, SDS-polyacrylamide gel electrophoresis, and FTIR spectroscopy have been used to analyze the sites and action modes of this cation on the PSI complex. Our results showed that Al(3+) above 3mM induces changes in the redox state of P700 reflected by an increase of P700 photooxidation phase and a delay of the slower rate of P700 re-reduction which reveals that Al(3+) exerted an inhibitory action at the donor side of PSI especially at plastocyanin (PC). Furthermore, results of P700 photooxidation monitored in the presence of DCMU with or without MV suggested that the same range of Al(3+) concentrations impairs the photochemical reaction centers (RC) of PSI, as shown by the decline in the amount of active population of P700, and disrupts the charge separation between P700 and the primary electron acceptor A0 leading to the inhibition of electron transfer at the acceptor side of PSI. These inhibitory actions were also accompanied by an impairment of the energy transfer from light harvesting complex (LHCI) to RC of PSI, following the disconnection of LHCI antenna as illustrated by an enhancement of chlorophyll fluorescence emission spectra at low temperature (77K). The above results coincided with FTIR measurements that indicated a conformational change of the protein secondary structures in PSI complex where 25% of α-helix was converted into ß-sheet, ß-antiparallel and turn structures. These structural changes in PSI complex proteins are closely related with the alteration photochemical activity of PSI including the inhibition of the electron transport through both acceptor and donor sides of PSI.

Frequency of Verticillium Species in Commercial Spinach Fields and Transmission of V. dahliae from Spinach to Subsequent Lettuce Crops.

Verticillium wilt caused by V. dahliae is a devastating disease of lettuce in California (CA). The disease is currently restricted to a small geographic area in central coastal CA, even though cropping patterns in other coastal lettuce production regions in the state are similar. Infested spinach seed has been implicated in the introduction of V. dahliae into lettuce fields but direct evidence linking this inoculum to wilt epidemics in lettuce is lacking. In this study, 100 commercial spinach fields in four coastal CA counties were surveyed to evaluate the frequency of Verticillium species recovered from spinach seedlings and the area under spinach production in each county was assessed. Regardless of the county, V. isaacii was the most frequently isolated species from spinach followed by V. dahliae and, less frequently, V. klebahnii. The frequency of recovery of Verticillium species was unrelated to the occurrence of Verticillium wilt on lettuce in the four counties but was related to the area under spinach production in individual counties. The transmission of V. dahliae from infested spinach seeds to lettuce was investigated in microplots. Verticillium wilt developed on lettuce following two or three plantings of Verticillium-infested spinach, in independent experiments. The pathogen recovered from the infected lettuce from microplots was confirmed as V. dahliae by polymerase chain reaction assays. In a greenhouse study, transmission of a green fluorescence protein-tagged mutant strain of V. dahliae from spinach to lettuce roots was demonstrated, after two cycles of incorporation of infected spinach residue into the soil. This study presents conclusive evidence that V. dahliae introduced via spinach seed can cause Verticillium wilt in lettuce.

Molecular cloning and characterization of pathogenesis-related protein family 10 gene from spinach (SoPR10).

PR10 genes encode small, intracellular proteins that respond to biotic and abiotic stresses. In this study, a cDNA clone (designated as SoPR10, GenBank Accession No. KC142174) encoding a PR10 protein from spinach (Spinacia oleracea L.) was isolated and characterized. SoPR10 encoded a 161-amino acid polypeptide with a predicted molecular mass of 19.76 kDa and a pI of 4.61. Real-time quantitative analysis indicated that SoPR10 was constitutively expressed in root and shoot. The abundance of SoPR10 in salt-resistant cultivar (Chaoji) was generally greater than in salt-sensitive cultivar (Daye) under 160 mM L(-1) NO3(-) treatment for 0.5, 3, and 6 h. The expression of SoPR10 was also induced by other abiotic stresses including polyethylene glycol, NaCl, salicylic acid, and H2O2. Our results indicated that SoPR10 might play important roles under nitrate stress and other abiotic stresses.

Heat- and light-induced detachment of the light-harvesting antenna complexes of photosystem I in isolated stroma thylakoid membranes.

The multisubunit pigment-protein complex of photosystem I (PSI) consists of a core and peripheral light-harvesting antenna (LHCI). PSI is thought to be a rather rigid system and very little is known about its structural and functional flexibility. Recent data, however, suggest LHCI detachment from the PSI supercomplex upon heat and light treatments. Furthermore, it was suggested that the splitting off of LHCI acts as a safety valve for PSI core upon photoinhibition (Alboresi et al., 2009). In this work we analyzed the heat- and light-induced reorganizations in isolated PSI vesicles (stroma membrane vesicles enriched in PSI). Using differential scanning calorimetry we revealed a stepwise disassembly of PSI supercomplex above 50°C. Circular dichroism, sucrose gradient centrifugation and 77K fluorescence experiments identified the sequence of events of PSI destabilization: 3min heating at 60°C or 40min white light illumination at 25°C resulted in pronounced Lhca1/4 detachment from the PSI supercomplex, which is then followed by the degradation of Lhca2/3. The similarity of the main structural effects due to heat and light treatments supports the notion that thermo-optic mechanism, structural changes induced by ultrafast local thermal transients, which has earlier been shown to be responsible for structural changes in the antenna system of photosystem II, can also regulate the assembly and functioning of PSI antenna.

Quality control of Photosystem II: the molecular basis for the action of FtsH protease and the dynamics of the thylakoid membranes.

The reaction center-binding D1 protein of Photosystem II is damaged by excessive light, which leads to photoinhibition of Photosystem II. The damaged D1 protein is removed immediately by specific proteases, and a metalloprotease FtsH located in the thylakoid membranes is involved in the proteolytic process. According to recent studies on the distribution and organization of the protein complexes/supercomplexes in the thylakoid membranes, the grana of higher plant chloroplasts are crowded with Photosystem II complexes and light-harvesting complexes. For the repair of the photodamaged D1 protein, the majority of the active hexameric FtsH proteases should be localized in close proximity to the Photosystem II complexes. The unstacking of the grana may increase the area of the grana margin and facilitate easier access of the FtsH proteases to the damaged D1 protein. These results suggest that the structural changes of the thylakoid membranes by light stress increase the mobility of the membrane proteins and support the quality control of Photosystem II.

The fungal phytotoxin alternariol 9-methyl ether and some of its synthetic analogues inhibit the photosynthetic electron transport chain.

Alternariol and monomethylalternariol are natural phytotoxins produced by some fungal strains, such as Nimbya and Alternaria. These substances confer virulence to phytopathogens, yet no information is available concerning their mode of action. Here we show that in the micromolar range alternariol 9-methyl ether is able to inhibit the electron transport chain (IC50 = 29.1 ± 6.5 µM) in isolated spinach chloroplasts. Since its effectiveness is limited by poor solubility in water, several alternariol analogues were synthesized using different aromatic aldehydes. The synthesized 6H-benzo[c]cromen-6-ones, 5H-chromene[4,3-b]pyridin-5-one, and 5H-chromene[4,3-c]pyridin-5-one also showed inhibitory properties, and three 6H-benzo[c]cromen-6-ones were more effective (IC50 = 12.8-22.8 µM) than the lead compound. Their addition to the culture medium of a cyanobacterial model strain was found to inhibit algal growth, with a relative effectiveness that was consistent with their activity in vitro. In contrast, the growth of a nonphotosynthetic plant cell culture was poorly affected. These compounds may represent a novel lead for the development of new active principles targeting photosynthesis.

Kinetics of structural reorganizations in multilamellar photosynthetic membranes monitored by small-angle neutron scattering.

We demonstrate the power of time-resolved small-angle neutron scattering experiments for the investigation of the structure and structural reorganizations of multilamellar photosynthetic membranes. In addition to briefly summarizing our results on thylakoid membranes isolated from higher plants and in unicellular organisms, we discuss the advantages and technical and methodological limitations of time-resolved SANS. We present a detailed and more systematical investigation of the kinetics of light-induced structural reorganizations in isolated spinach thylakoid membranes, which show how changes in the repeat distance and in the long-range order of the multilamellar membranes can be followed with a time resolution of seconds. We also present data from comparative measurements performed on thylakoid membranes isolated from tobacco.

Changes in the energy transfer pathways within photosystem II antenna induced by xanthophyll cycle activity.

Energy transfer pathways between photosystem II (PSII) antenna complexes in intact thylakoid membranes have been studied using low-temperature (77 K) excitation fluorescence spectroscopy. The focus of this study was to see whether de-epoxidation of violaxanthin into zeaxanthin causes any alterations in the energetic couplings between the core antenna complexes CP43 and CP47 and the peripheral light-harvesting antenna (LHCII). It was discovered that the appearance of zeaxanthin caused characteristic alterations in the PSII excitation fluorescence spectra in the Soret-band region. While in the dark violaxanthin was found to be largely uncoupled from any emitting chlorophylls, its intensive de-epoxidation resulted in the appearance of two additional bands at 509 and 492 nm. The former was attributed to weak coupling of zeaxanthin to emitters in the CP43 and LHCII complexes and the latter to efficient coupling of violaxanthin of the CP29 complex to emitters in the CP43, CP47, and LHCII complexes. The role of CP29-bound violaxanthin is discussed in light of both its efficient energetic coupling and strong physical binding to this complex. The finding that zeaxanthin is energetically coupled to chlorophyll a emitters of the PSII antenna is discussed with respect to its suggested role as a quencher involved in photoprotective energy dissipation, or non-photochemical quenching (NPQ), in the photosynthetic membrane.

The endophytic lifestyle of Escherichia coli O157:H7: quantification and internal localization in roots.

The foodborne pathogen Escherichia coli O157:H7 is increasingly associated with fresh produce (fruit and vegetables). Bacterial colonization of fresh produce plants can occur to high levels on the external tissue but bacteria have also been detected within plant tissue. However, questions remain about the extent of internalization, its molecular basis, and internal location of the bacteria. We have determined the extent of internalization of E. coli O157:H7 in live spinach and lettuce plants and used high-resolution microscopy to examine colony formation in roots and pathways to internalization. E. coli O157:H7 was found within internal tissue of both produce species. Colonization occurred within the apoplast between plant cells. Furthermore, colonies were detected inside the cell wall of epidermal and cortical cells of spinach and Nicotiana benthamiana roots. Internal colonization of epidermal cells resembled that of the phytopathogen Pectobacterium atrosepticum on potato. In contrast, only sporadic cells of the laboratory strain of E. coli K-12 were found on spinach, with no internal bacteria evident. The data extend previous findings that internal colonization of plants appears to be limited to a specific group of plant-interacting bacteria, including E. coli O157:H7, and demonstrates its ability to invade the cells of living plants.

Norovirus surrogate survival on spinach during preharvest growth.

Produce can become contaminated with human viral pathogens in the field through soil, feces, or water used for irrigation; through application of manure, biosolids, pesticides, and fertilizers; and through dust, insects, and animals. The objective of this study was to assess the survival and stability of human noroviruses and norovirus surrogates (Murine norovirus [MNV] and Tulane virus [TV]) on foliar surfaces of spinach plants in preharvest growth conditions. Spinach plants were housed in a biocontrol chamber at optimal conditions for up to 7 days and infectivity was determined by plaque assay. Virus inoculation location had the largest impact on virus survival as viruses present on adaxial leaf surfaces had lower decimal reduction time (D values) than viruses present on abaxial leaf surfaces. Under certain conditions, spinach type impacted virus survival, with greater D values observed from survival on semi-savoy spinach leaves. Additional UVA and UVB exposure to mimic sunlight affected virus survival on adaxial surfaces for both semi-savoy and smooth spinach plants for both viruses. Human GII norovirus inoculated onto semi-savoy spinach had an average D value that was not statistically significant from MNV and TV, suggesting that these surrogates may have similar survival on spinach leaves compared with human noroviruses. An understanding of the behavior of enteric viruses on spinach leaves can be used to enhance growers' guidelines and for risk assessment with certain growing conditions.

Transfer of Escherichia coli O157:H7 to spinach by house flies, Musca domestica (Diptera: Muscidae).

Filth flies are known mechanical vectors of pathogenic bacteria in hospital and restaurant settings, but their role as vectors for disseminating microbes to plants has not been demonstrated. Escherichia coli O157:H7 deposition by flies onto spinach was studied using molecular, microbiological, and microscopy techniques. Relative quantitative polymerase chain reaction studies showed that bacteria acquired by flies from contaminated cattle manure and deposited in regurgitation spots on leaves survived and multiplied. Scanning electron microscopy of the regurgitation spots of flies exposed to manure inoculated with E. coli suggested the multiplication of bacteria-like organisms within the spots. This finding implies that the bacteria were active and is consistent with a hypothesis that regurgitation spots serve as a nutrition source allowing E. coli O157:H7 to survive on the spinach phylloplane. E. coli O157:H7 persisted on fly body surfaces up to 13 days after exposure to acquisition sources, suggesting that fly cuticular surfaces are conducive to the growth of this pathogen. These results are consistent with the hypothesis of bioenhanced transmission of human pathogens by house flies and suggest that filth flies may affect the microbial safety of fresh produce.

Furoquinoline alkaloids isolated from Balfourodendron riedelianum as photosynthetic inhibitors in spinach chloroplasts.

In the search for natural inhibitors of plant growth, we investigate the mechanism of action of the natural furoquinoline alkaloids isolated from Balfourodendron riedelianum (Rutaceae): evolitrine (1), kokusaginine (2), γ-fagarine (3), skimmianine (4) and maculosidine (5) on the photosynthesis light reactions. Their effect on the electron transport chain on thylakoids was analyzed. Alkaloids 1, 2, 4 and 5 inhibited ATP synthesis, basal, phosphorylating and uncoupled electron transport acting as Hill reaction inhibitors on spinach chloroplasts. Alkaloid 3 was not active. The inhibition and interaction site of alkaloids 1, 2, 4 and 5 on the non-cyclic electron transport chain was studied by polarography and fluorescence of the chlorophyll a (Chl a). The results indicate that the target for 1 was localized on the donor and acceptor side of PS II. In addition alkaloids 2 and 5 affect the PS I electron acceptors on leaf discs.