A Crucial Role of the RGS Domain in Trans- Golgi Network Export of AtRGS1 in the Protein Secretory Pathway

The secretory pathway is responsible for the transport of newly synthesized transmembrane proteins from the endoplasmic reticulum to their destinations via the Golgi/trans-Golgi network （TGN）, Cargo proteins at each sta- tion are actively sorted by specific sorting signals on the cargo and the corresponding coat complexes. Here, we used the Arabidopsis regulator of G-protein signaling （AtRGS1）, which contains an N-terminal potentially sensing glucose seven-transmembrane domain and a C-terminal RGS domain, as a model to uncover sorting motifs required for its cell surface expression. Expression of wild-type and truncated or mutated AtRGS1 fluorescent fusion proteins identified two cysteine residues in the extracellular N-terminus that are essential for endoplasmic reticulum exit and/or correct folding of AtRGS1. The linker between the seven-transmembrane and RGS domains contains an endoplasmic reticulum export signal, whereas the C-terminus is dispensable for the plasma membrane expression of AtRGS1. Interestingly, deletion of the RGS domain results in Golgi/TGN localization of the truncated AtRGS1. Further analysis using site-directed mutagen- esis showed that a tyrosine-based motif embedded in the RGS domain is essential for Golgi/TGN export of AtRGS1. These results reveal a new role for the RGS domain in regulating AtRGS1 trafficking from the Golgi/TGN to the plasma membrane and explain the interaction between the seven-transmembrane and RGS domains.     

Import Determinants of Organelle-Specific and Dual Targeting Peptides of Mitochondria and Chloroplasts in Arabidopsis thaliana

Most of the mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins carrying an N-terminal targeting peptide （TP） directing them specifically to a correct organelle. However, there is a group of proteins that are dually targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide （dTP）. Here, we have investigated pattern properties of import determinants of organelle-specific TPs and dTPs combining mathematical multivariate data analysis （MVDA） with in vitro organellar import studies. We have used large datasets of mitochondrial and chloroplastic proteins found in organellar proteomes as well as manually selected data sets of experimentally confirmed organelle-specific TPs and dTPs from Arabidopsis thaliana. Two classes of organelle-specific TPs could be distinguished by MVDA and potential patterns or periodicity in the amino acid sequence contributing to the separation were revealed, dTPs were found to have intermediate sequence features between the organelle-specific TPs. Interestingly, introducing positively charged residues to the dTPs showed clustering towards the mitochondrial TPs in silico and resulted in inhibition of chloroplast, but not mitochondrial import in in vitro organellar import studies. These findings suggest that positive charges in the N-terminal region of TPs may function as an ＇avoidance signal＇ for the chloroplast import.     

Methods for Analysis of Protein Glutathionylation and their Application to Photosynthetic Organisms

Protein S-glutathionylation, the reversible formation of a mixed-disulfide between glutathione and protein thiols, is involved in protection of protein cysteines from irreversible oxidation, but also in protein redox regulation. Recent studies have implicated S-glutathionylation as a cellular response to oxidative/nitrosative stress, likely playing an important role in signaling. Considering the potential importance of glutathionylation, a number of methods have been developed for identifying proteins undergoing glutathionylation. These methods, ranging from analysis of purified proteins in vitro to large-scale proteomic analyses in vivo, allowed identification of nearly 200 targets in mammals. By contrast, the number of known glutathionylated proteins is more limited in photosynthetic organisms, although they are severely exposed to oxidative stress. The aim of this review is to detail the methods available for identification and analysis of glutathionylated proteins in vivo and in vitro. The advantages and drawbacks of each technique will be discussed as well as their application to photosynthetic organisms. Furthermore, an overview of known glutathionylated proteins in photosynthetic organisms is provided and the physiological importance of this post-translational modification is discussed.     

Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica

Phosphoribulokinase （PRK）, a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction （Calvin） cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc （sea slug） Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclearencoded proteins that are directed to complex （i.e. four membrane-bound） algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V/itorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months.     

Vegetation Mapping of the Mond Protected Area of Bushehr Province （South-west Iran）

Arid regions of the world occupy up to 35% of the earth＇s surface, the basis of various definitions of climatic conditions, vegetation types or potential for food production. Due to their high ecological value, monitoring of arid regions is necessary and modern vegetation studies can help in the conservation and management of these areas. The use of remote sensing for mapping of desert vegetation is difficult due to mixing of the spectral reflectance of bright desert soils with the weak spectral response of sparse vegetation. We studied the vegetation types in the semiarid to arid region of Mond Protected Area, south-west Iran, based on unsupervised classification of the Spot XS bands and then produced updated maps. Sixteen map units covering 12 vegetation types were recognized in the area based on both field works and satellite mapping. Halocnemum strobilaceum and Suaeda fruticosa vegetation types were the dominant types and Ephedra foliata, Salicornia europaea-Suaeda heterophylla vegetation types were the smallest. Vegetation coverage decreased sharply with the increase in salinity towards the coastal areas of the Persian Gulf. The highest vegetation coverage belonged to the riparian vegetation along the Mond River, which represents the northern boundary of the protected area. The location of vegetation types was studied on the separate soil and habitat diversity maps of the study area, which helped in final refinements of the vegetation map produced.     

Oxygation Enhances Growth,Gas Exchange and Salt Tolerance of Vegetable Soybean and Cotton in a Saline Vertisol

Impacts of salinity become severe when the soil is deficient in oxygen. OxygaUon （using aerated water for subsurface drip irrigation of crop） could minimize the impact of salinity on plants under oxygen-limiting soil environments. Pot experiments were conducted to evaluate the effects of oxygation （12% air volume/volume of water） on vegetable soybean （moderately salt tolerant） and cotton （salt tolerant） in a salinized vertisol at 2, 8, 14, 20 dS/m ECe. In vegetable soybean, oxygation increased above ground biomass yield and water use efficiency （WUE） by 13% and 22%, respectively, compared with the control. Higher yield with oxygation was accompanied by greater plant height and stem diameter and reduced specific leaf area and leaf Na^＋ and CI^- concentrations. In cotton, oxygation increased lint yield and WUE by 18% and 16%, respectively, compared with the control, and was accompanied by greater canopy light interception, plant height and stem diameter. Oxygation also led to a greater rate of photosynthesis, higher relative water content in the leaf, reduced crop water stress index and lower leaf water potential. It did not, however, affect leaf Na^＋ or CI^- concentration. Oxygation invariably increased, whereas salinity reduced the K^＋： Na^＋ ratio in the leaves of both species. Oxygation improved yield and WUE performance of salt tolerant and moderately tolerant crops under saline soil environments, and this may have a significant impact for irrigated agriculture where saline soils pose constraints to crop production.     

Arabidopsis Protein Phosphatase 2C ABI1 Interacts with Type I ACC Synthases and Is Involved in the Regulation of Ozone-Induced Ethylene Biosynthesis

Ethylene plays a crucial role in various biological processes and therefore its biosynthesis is strictly regu- lated by multiple mechanisms. Posttranslational regulation, which is pivotal in controlling ethylene biosynthesis, impacts 1-aminocyclopropane 1-carboxylate synthase （ACS） protein stability via the complex interplay of specific factors. Here, we show that the Arabidopsis thaliana protein phosphatase type 2C, ABI1, a negative regulator of abscisic acid signaling, is involved in the regulation of ethylene biosynthesis under oxidative stress conditions. We found that ABI1 interacts with ACS6 and dephosphorylates its C-terminal fragment, a target of the stress-responsive mitogen-activated protein kinase, MPK6. In addition, ABI1 controls MPK6 activity directly and by this means also affects the ACS6 phosphorylation level. Consistently with this, ozone-induced ethylene production was significantly higher in an ABI1 knockout strain （abiltd） than in wild-type plants. Importantly, an increase in stress-induced ethylene production in the abiltd mutant was compen- sated by a higher ascorbate redox state and elevated antioxidant activities. Overall, the results of this study provide evi- dence that ABI1 restricts ethylene synthesis by affecting the activity of ACS6. The ABI1 contribution to stress phenotype underpins its role in the interplay between the abscisic acid （ABA） and ethylene signaling pathways.     

Identification,expression, and characterization of the highly conserved D-xylose isomerase in animals

D-xylose is a necessary sugar for animals. The xylanase from a mollusk, Ampullaria crossean, was previously reported by our laboratory. This xylanase can degrade the xylan into D-xylose. But there is still a gap in our knowledge on its metabolic pathway. The question is how does the xylose enter the pentose pathway？ With the help of genomic databases and bioinformatic tools, we found that some animals, such as bacteria, have a highly conserved D-xylose isomerase （EC 5.3.1.5）. The xyiose isomerase from a sea squirt, Ciona intestinali, was heterogeneously expressed in Escherichia coli and purified to confirm its function. The recombinant enzyme had good thermal stability in the presence of Mg^2＋. At the optimum temperature and optimum pH environment, its specific activity on D-xylose was 0.331 μmol/mg/min. This enzyme exists broadly in many animals, but it disappeared in the genome of Amphibia-like Xenopus laevis. Its sequence was highly conserved. The xylose isomerases from animals are very interesting proteins for the study of evolution.     

The Chloroplast Kinase Network： New Insights from Large-Scale Phosphoproteome Profiling

Protein phosphorylation is one of the most important posttranslational modifications in eukaryotic cells and affects almost all basic cellular processes. The chloroplast as plant-specific cell organelle with important metabolic functions is integrated into the cellular signaling and phosphorylation network. Recent large-scale chloroplast phosphoproteome analyses in Arabidopsis have provided new information about phosphorylation targets and expanded the list of chloroplast metabolic and regulatory functions that are potentially controlled by protein phosphorylation. Phosphorylated peptides identified from chloroplast proteins provide new insights into phosphorylation motifs, protein kinase activities, and substrate utilization. Phosphorylation sites in protein kinases can reveal chloroplast phosphorylation cascades that may network different functions by integrating signaling chains. Our review provides a meta-analysis of currently available chloroplast phosphoproteome information and discusses biological insights from large-scale chloroplast phosphoprotein profiling as well as technological constraints of kinase network analysis.     

The Response Difference of Mitochondria in Recalcitrant Antiaris toxicaria Axes and Orthodox Zea mays Embryos to Dehydration Injury

Long-term preservation of recalcitrant seeds is very difficult because the physiological basis on their desiccation sensitivity is poorly understood. Survival of Antiaris toxicaria axes rapidly decreased and that of immature maize embryos very slowly decreased with dehydration. To understand their different responses to dehydration, we examined the changes in mitochondria activity during dehydration. Although activities of cytochrome （Cyt） c oxidase and malate dehydrogenase of the A. toxicaria axis and maize embryo mitochondria decreased with dehydration, the parameters of maize embryo mitochondria were much higher than those of A. toxicaria, showing that the damage was more severe for the A. toxicaria axis mitochondria than for those of maize embryo. The state I and III respiration of the A. toxicaria axis mitochondria were higher than those of maize embryo, the former rapidly decreased, and the latter slowly decreased with dehydration. The proportion of Cyt c pathway to state III respiration for the A. toxicaria axis mitochondria was low and rapidly decreased with dehydration, and the proportion of alternative oxidase pathway was high and slightly increased with dehydration. In contrast, the proportion of Cyt c pathway for maize embryo mitochondria was high, and that of alternative oxidase pathway was low. Both pathways decreased slowly with dehydration.     

Chloroplast Proteomics and the Compartmentation of Plastidial Isoprenoid Biosynthetic Pathways

Recent advances in the proteomic field have allowed high-throughput experiments to be conducted on chloroplast samples. Many proteomic investigations have focused on either whole chloroplast or sub-plastidial fractions. To date, the Plant Protein Database （PPDB, Sun et al., 2009） presents the most exhaustive chloroplast proteome available online. However, the accurate localization of many proteins that were identified in different sub-plastidial compartments remains hypothetical. Ferro et al. （2009） went a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regards to their accurate localization within the chloroplast by using a semi-quantitative proteomic approach known as spectral counting. Their proteomic strategy was based on the accurate mass and time tags （AMT） database approach and they built up AT_CHLORO, a comprehensive chloroplast proteome database with sub-plastidial localization and curated information on envelope proteins. Comparing these two extensive databases, we focus here on about 100 enzymes involved in the synthesis of chloroplast-specific isoprenoids. Well known pathways （i.e. compartmentation of the methyl erythritol phosphate biosynthetic pathway, of tetrapyrroles and chlorophyll biosynthesis and breakdown within chloroplasts） validate the spectral counting-based strategy. The same strategy was then used to identify the precise localization of the biosynthesis of carotenoids and prenylquinones within chloroplasts （i.e. in envelope membranes, stroma, and/or thylakoids） that remains unclear until now.     

The Translational Apparatus of Plastids and Its Role in Plant Development

Chloroplasts （plastids） possess a genome and their own machinery to express it. Translation in plastids occurs on bacterial-type 70S ribosomes utilizing a set of tRNAs that is entirely encoded in the plastid genome. In recent years, the components of the chloroplast translational apparatus have been intensely studied by proteomic approaches and by reverse genetics in the model systems tobacco （plastid-encoded components） and Arabidopsis （nucleus-encoded components）. This work has provided important new insights into the structure, function, and biogenesis of chloroplast ribosomes, and also has shed fresh light on the molecular mechanisms of the translation process in plastids. In addition, mutants affected in plastid translation have yielded strong genetic evidence for chloroplast genes and gene products influencing plant develop- ment at various levels, presumably via retrograde signaling pathway（s）. In this review, we describe recent progress with the functional analysis of components of the chloroplast translational machinery and discuss the currently available evidence that supports a significant impact of plastid translational activity on plant anatomy and morphology.     

Response of Chinese Wampee Axes and Maize Embryos to Dehydration at Different Rates

Survival of wampee （Clausena lansium Skeels） axes and maize （Zea mays L.） embryos decreased with rapid and slow dehydration. Damage of wampee axes by rapid dehydration was much less than by slow dehydration, and that was contrary to maize embryos. The malondialdehyde contents of wampee axes and maize embryos rapidly increased with dehydration, those of wampee axes were lower during rapid dehydration than during slow dehydration, and those of maize embryos were higher during rapid dehydration than during slow dehydration. Activities of superoxide dismutase （SOD）, ascorbate peroxidase （APX） and catalase （CAT） of wampee axes markedly increased during the early phase of dehydration, and then rapidly decreased, and those of rapidly dehydrated axes were higher than those of slow dehydrated axes when they were dehydrated to low water contents. Activities of SOD and APX of maize embryos notable decreased with dehydration. There were higher SOD activities and lower APX activities of slowly dehydrated maize embryos compared with rapidly dehydrated maize embryos. CAT activities of maize embryos markedly increased during the early phase of dehydration, and then decreased, and those of slowly dehydrated embryos were higher than those of rapidly dehydrated embryos during the late phase of dehydration.     

血管性痴呆小鼠海马神经元ERK的表达变化

目的：观察血管性痴呆（VD）小鼠海马神经元中细胞外信号调节激酶（ERK）的表达变化,探讨其在VD发病中的作用机制。方法：采用双侧颈总动脉反复缺血／再灌注法制备小鼠VD模型,设立假手术组作为对照。术后第29、30d,经跳台试验和水迷宫试验对两组小鼠进行行为学成绩测试,用免疫组化方法观察两组小鼠海马神经元中ERK的表达变化。结果：VD模型小鼠学习、记忆成绩较假手术组显著下降（P〈0．05）.模型组小鼠海马CA1区ERK1、ERK2的表达及海马CA3区p-ERK的表达较假手术组减少,均有显著性差异（P〈0．05）。结论：海马神经元内ERK的表达减少可能参与了血管性痴呆的发病机制,因此,应用能促进ERK表达的药物可能成为治疗VD的有效方法之一。