Tingyun Kuang

Proteomic analysis of the cold stress response in the moss, Physcomitrella patens.

Cold stress has adverse effects on plant growth and development. Plants respond and acclimate to cold stress through various biochemical and physiological processes, thereby acquiring stress tolerance. To better understand the basis for tolerance, we carried out a proteomic study in the model moss, Physcomitrella patens, characterizing gametophore proteins with 2‐DE and mass spectroscopy. Following exposure to 0°C for up to 3 days, out of the more than 1000 protein spots reproducibly resolved, only 45 changed in abundance by at least 1.5‐fold. Of these, 35 were identified by tryptic digestion and mass spectroscopy. Photosynthetic proteins decreased, whereas many catabolic proteins increased. In addition, cold stress up‐regulated a variety of signaling, cytoskeleton, and defense proteins and few proteins in these classes were down‐regulated. Up‐regulated proteins include the 14‐3‐3‐like protein, actin, HSP70s, lipoxygenases, and cytochrome P450 proteins. These results point to pathways that are important for the mechanism of cold stress response in P. patens and by extension to the entire plant kingdom.

Coordination of carbon fixation and nitrogen metabolism in Salicornia europaea under salinity: Comparative proteomic analysis on chloroplast proteins

Halophyte, like Salicornia europaea, could make full use of marginal saline land for carbon fixation. How the photosynthesis of S. europaea is regulated under high salinity implicates a significant aspect to exploit this pioneer plant in future. Measurement of photosynthesis parameters demonstrated the reduction of photosynthesis for the 0 and 800 mM NaCl treated plants are more likely due to non‐stomatal limitation, which might be caused by changes in the enzymes associated with photosynthesis. Different salinity induced ultrastructure changes other than photosynthetic apparatus damage, suggesting the photosynthesis of S. europaea might be affected via biochemical regulation. Comparative proteomics analysis of chloroplast proteins by 2‐D gel electrophoresis reproducibly detected 90 differentially expressed proteins, among which 66 proteins were identified by nanoLC MS/MS. Further study of thylakoid membrane proteins by Blue‐Native PAGE proved the increase in abundance of light reaction proteins under salinity. Analysis of gene expression patterns of 12 selected proteins provides evidence for the correlations between transcription and proteomics data. Based on our results, a putative model of photosynthesis regulatory network figured out proper coordination of carbon fixation and nitrogen metabolism in chloroplast of S. europaea under salinity, which provided subcellular level insight into salt tolerance mechanism in S. europaea.

An efficient method for the extraction of chloroplast proteins compatible for 2-DE and MS analysis

Comparative proteomic analysis of chloroplast by 2‐DE has received significant attention in recent years. However, the complication of membrane systems in chloroplast made it challenging to elucidate entire chloroplast proteome by 2‐DE. Here, we developed an efficient method for extracting chloroplast proteins, and produced excellent 2‐DE profiles from both Arabidopsis thaliana and Salicornia europaea. Comparison of this method with another two protocols for the extraction of A. thaliana chloroplast proteins showed that our method obtained higher protein yields and produced more protein spots on both pH 3–10 and 4–7 2‐DE gels. Moreover, this method recovered more proteins in the basic and high Mr regions, thereby offering the best extraction of chloroplast proteins. Identification of 15 specific chloroplast‐targeted proteins on our gels by MALDI‐TOF MS revealed that this method was compatible with MS, and recovered more chloroplast membrane proteins than the commonly used methods. This protocol is expected to have a wide application in future chloroplast proteomic analysis.

Inhibitory effect of Salicornia europaea on the marine alga Skeletonema costatum

Exploiting the negative biochemical interference between plants and algal species has been suggested as a method to control harmful algal blooms. In this work, we investigated the inhibitory effect of the salt marsh halophyte Salicornia europaea against the marine alga Skeletonema costatum. S. europaea suppressed the growth of S. costatum in a nutrient-sufficient co-culture system, indicating that the inhibition of algal growth was because of the phytotoxic effect of S. europaea, rather than nutrient competition. We tested aqueous and organic extracts from S. europaea roots against S. costatum. The organic extracts inhibited growth and affected the cell size and chlorophyll a content of S. costatum in a dose-dependent manner. Among the three tested organic extracts, the methanol extract had the greatest effects on S. costatum, followed by butanol extract, and then the chloroform extract. Two flavonoids, rutin and quercetin-3-β-D-glucoside, were identified in the methanol extract by high performance liquid chromatography. The concentration of rutin was much higher than that of quercetin-3-β-D-glucoside. In an algal bioassay, rutin inhibited the growth of S. costatum and the inhibitory effect increased with increasing rutin concentration and with decreasing initial algal density. Therefore, we concluded that S. europaea negatively affects the growth of S. costatum, and that rutin, a metabolite of S. europaea, may play a role in this inhibitory effect.

New structure model of oxygen-evolving center and mechanism for oxygen evolution in photosynthesis

So far, many important questions and problems concerning the structure and mechanism of photosynthetic oxygen evolution are still unsolved. On the basis of recent achievements in this field, a new structure model is proposed whereby two H2O molecules bind asymmetrically to two manganese ions (Mn1II and Mn4III) at the open end of “C” shaped cluster and keep rather large distance. Two histidine residues coordinate to the other two manganese ions in higher oxidation state (Mn2IV and Mn3IV) through their nitrogen atoms of the imidazole. CI bound as terminal ligand to Mn4III is connected to Ca, and the latter is needed to maintain the special configuration of two Mn2O2 units by bridged-oxo and bridged-carboxylate ligands. The whole structure of oxygen evolution center is asymmetry. A new mechanism for oxygen evolution invokes predictions of asymmetric oxidation of two H2O molecules, dynamic structural changes of oxygen evolving center and indirect proton transport, etc. Only in S2 state, could Mn1IV = O, intermediate with high oxidation potential be formed. The S2→ S3 process occurs with significant structural changes, as well as intramolecular and intermolecular hydrogen transfer. The S3 state corresponds to intermediate of Mn1IV-O … H … O-Mn4IV. During S3 → [S4] → S0, the 0-0 bond is formed only in S4 state. The change of nucleophilic interaction between Cl and manganese ions different oxidation states has consequence for the significant structural changes in H2O oxidation process.

Phytochrome controlled, long-day photoperiod-inducible protein in rice leaves

A new protein in the leaves of NK58S and NK58 (Oryza sativa L. subsp.japonica), which can be induced by 10 d-long-day photoperiod (14 h lightid) and cannot be induced by 10 d-short-day photoperiod (10 h light/d), has been found by two-dimensional gel electrophoresis. The protein, whose molecular weight and isoelectric point are 36 ku and pH 5.2 respectively, is found to be controlled by phytochrome as shown by the experiment of red light induction-far red light reversion.The existence of this protein in both NK58S and NK58 reflects that some of the responses of NK58S and NK58 might be similar in response to long-day photoperiod, a mild stress.

Theoretical study on primary reaction of photosynthetic bacteria.

Theoretical calculation was carried out on the primary electron donor P870 of photosynthetic bacteria. The results show that: (i) the bimolecular structure of the primary electron donor is more advantageous in energy than monomolecular structure; (ii) the initial configuration of primary electron donor is no longer stable and changes to the configuration with lower energy and chemical reactivity after the charge separation. In the P870, such structural change is completed through the rotation of C3 acetyl, so the oxygen atom of acetyl interacts with the magnesium atom of another bacterio-chlorophyll molecule, and the total energy and chemical reactivity are reduced evidently. It is suggested that the structural change of the primary electron donor is important in preventing the occurrence of charge recombination during the primary reaction and maintaining the high efficiency of the conversion of sun-light to chemical energy. A new mechanism of primary reaction has been proposed, which can give reasonable explanations to the results of kinetic and site mutation studies.

Erratum to High-light-induced superoxide anion radical formation in cytochrome b 6 f complex from spinach as detected by EPR spectroscopy

The generation of superoxide anion radical (O2 ·−) in the cytochrome b 6 f complex (Cyt b 6 f) of spinach under high-light illumination was studied using electron paramagnetic resonance spectroscopy. The generation of O2 ·− was lost in the absence of molecular oxygen. It was also suppressed in the presence of NaN3 and could be scavenged by extraneous antioxidants such as ascorbate, β-carotene, and glutathione. The results also indicate that O2 ·−, which is produced under high-light illumination of the Cyt b 6 f from spinach, might be generated from a reaction involing 1O2, and the Rieske Fe-S protein could serve as the electron donor in the O2 ·− production. The mechanism of photoprotection of the Cyt b 6 f complex by antioxidants is discussed.

Two-dimensional crystallization and preliminary structure analysis of LHC-II from cucumber and spinach

Large and well-ordered two-dimensional (2D) crystals of the light-harvesting chlorophyll a/b protein complexes (LHC-II) from cucumber and spinach chloroplasts were produced by the so-called batch method. The twodimensional structures of these crystals were examined at about 1.5 nm resolution by electron microscopy and image processing. The projection maps showed that there were similar, but not identical, structure features between two different LHC-II complexes. A cmparison between 2D crystal formations of the two different LHC-II complexes was done and some factors affecting 2D crystallization of the membrane proteins were analyzed. The relations of the structures of the LHC-II complexes to their polypeptide components and Chl a/b ratio were also discussed.Large and well-ordered two-dimensional (2D) crystals of the light-harvesting chlorophyll a/b protein complexes (LHC-II) from cucumber and spinach chloroplasts were produced by the so-called batch method. The twodimensional structures of these crystals were examined at about 1.5 nm resolution by electron microscopy and image processing. The projection maps showed that there were similar, but not identical, structure features between two different LHC-II complexes. A cmparison between 2D crystal formations of the two different LHC-II complexes was done and some factors affecting 2D crystallization of the membrane proteins were analyzed. The relations of the structures of the LHC-II complexes to their polypeptide components and Chl a/b ratio were also discussed.

The peroxidase activity of cytochrome b6f complex from spinach chloroplasts

The cytochrome b6f (Cyt b6f) complex, which functions as a plastoquinol-plastocyanin oxidoreductase and mediates the linear electron flow between photosystem II (PSII) and photosystem I (PSI) and the cyclic electron flow around PSI, was isolated from spinach (Spinacia oleracea L.) chloroplasts using n-octyl-β-D-glucopyranoside (β-OG). The preparation was also able to catalyze the peroxidase-like reaction in the presence of hydrogen peroxide (H2O2) and guaiacol. The optimal conditions for peroxidase activity of the preparation included: pH 3.6, ionic strength 0.1, and temperature 35°C. The apparent Michaelis constant (Km) values for H2O2 and guaiacol were 50 mM and 2 mM, respectively. The bimolecular rate constant (kobs) was about 26 M−1 s−1 and the turnover number (Kcat) was about 60 min−1 (20 mM guaiacol, 100 mM sodium phosphate, pH 3.6, 25°C, [H2O2]<100mM). These parameters were similar to those of several other heme-containing proteins, such as myoglobin and Cyt c.