The Recruitment of AMP-activated Protein Kinase to Glycogen Is Regulated by Autophosphorylation

The mammalian AMP-activated protein kinase (AMPK) is an obligatory αβγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the β-subunit (AMPKβ) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or β1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKβ is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins.     

Unraveling Hidden Components of the Chloroplast Envelope Proteome: Opportunities and Limits of Better MS Sensitivity

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Highlights

• •Identification of previously undetected chloroplast envelope proteins.
• •Up to date manual annotation of genuine (or shared) envelope components.
• •New hypotheses for localizations, functions, interactions among cell compartments.
• •A new resource of significant value to the broader plant science community.

     

Methylation of chloroplast DNA does not affect viability and maternal inheritance in tobacco and may provide a strategy towards transgene containment

We report the integration of a type II restriction-methylase, mFokI, into the tobacco chloroplast genome and we demonstrate that the introduced enzyme effectively directs the methylation of its target sequence in vivo and does not affect maternal inheritance. We further report the transformation of tobacco with an E. coli dcm methylase targeted to plastids and we demonstrate efficient cytosine methylation of the plastid genome. Both adenosine methylation of FokI sites and cytosine methylation of dcm sites appeared phenotypically neutral. The ability to tolerate such plastid genome methylation is a pre-requisite for a proposed plant transgene containment system. In such a system, a chloroplast located, maternally inherited restriction methylase would provide protection from a nuclear-encoded, plastid targeted restriction endonuclease. As plastids are not paternally inherited in most crop species, pollen from such plants would carry the endonuclease transgene but not the corresponding methylase; the consequence of this should be containment of all nuclear transgenes, as pollination will only be viable in crosses to the appropriate transplastomic maternal background.     

The control of chloroplast number in wheat mesophyll cells

Chloroplast number per cell and mesophyll cell plan area were determined in populations of separated cells from the primary leaves of different wheat species representing three levels of ploidy. Mean chloroplast number per cell increases with ploidy level as mean cell size increases. But in addition the analysis of individual cells clearly shows that cells of a similar size but from species of different ploidies have similar numbers of chloroplasts. We conclude that the number of chloroplasts within a cell is closely correlated (P<0.001) with the size of the cell and this relationship is consistent for species of different ploidies over a wide range of cell sizes. These results are discussed in relation to the hypothesis that chloroplast number in leaf mesophyll cells is determined by the size of the cell.     

Genetic control of plastid carotenoids and transformation in the skin of Cucurbita pepo L. fruit

Summary The influence of allelic state of gene B on skin pigmentation in two cultivars of Cucurbita pepo L. has been studied. Total carotenoids were lower at early stages of fruit development in cultivar (cv.) Early Prolific (EP) BB YY fruit skin, than in EP B ⁺ B ⁺ YY fruit skin, but no differences were observed in total skin carotenoids twenty days after anthesis. Total carotenoids were lower in cv. Fordhook Zucchini (FZ) BB yy fruit skin, than in FZ B ⁺ B ⁺ yy fruit skin at all developmental stages from anthesis to maturity. Both green and yellow tissues contained typical foliar carotenoids. The carotenoids from yellow fruit skin of both EP genotypes and of FZ BB were characterized by a low carotene: xanthophyll ratio, with a high proportion of the xanthophylls esterified to fatty acids. The xanthophylls of the yellow tissues were esterified with 120, 140, 160 fatty acids. The carotenoids from the green fruit skin of FZ B ⁺ B ⁺ had a higher percentage of carotenes (primarily -carotene) and a lower percentage of esterified xanthophylls. Spectral shapes of carotenoid fractions from all yellow tissues were similar and distinguishable from those of green FZ B ⁺ B ⁺ tissue. The results of these studies are discussed in terms of the genetic control of plastid transformation in Cucurbita pepo L.New Jersey Agricultural Experiment Station No. D99201 (NE-9) 32-83, supported by state funds and funds from the Rutgers University Research Council     

CRM1-dependent nuclear export and dimerization with hMSH5 contribute to the regulation of hMSH4 subcellular localization

MSH4 and MSH5 are members of the MutS homolog family, a conserved group of proteins involved in DNA mismatch correction and homologous recombination. Although several studies have provided compelling evidences suggesting that MSH4 and MSH5 could act together in early and late stages of meiotic recombination, their precise roles are poorly understood and recent findings suggest that the human MSH4 protein may also exert a cytoplasmic function. Here we show that MSH4 is present in the cytoplasm and the nucleus of both testicular cells and transfected somatic cells. Confocal studies on transfected cells provide the first evidence that the subcellular localization of MSH4 is regulated, at least in part, by an active nuclear export pathway dependent on the exportin CRM1. We used deletion mapping and mutagenesis to define two functional nuclear export sequences within the C-terminal part of hMSH4 that mediate nuclear export through the CRM1 pathway. Our results suggest that CRM1 is also involved in MSH5 nuclear export. In addition, we demonstrate that dimerization of MSH4 and MSH5 facilitates their nuclear localization suggesting that dimerization may regulate the intracellular trafficking of these proteins. Our findings suggest that nucleocytoplasmic traffic may constitute a regulatory mechanism for MSH4 and MSH5 functions.     

Effects of Chloroplast DNA Content on the Cell Proliferation and Aging in Chlamydomonas reinhardtii

Chlamydomonas is an unicellular green alga that contains one cup-shaped chloroplast with about 60 copies of cpDNA. Chloroplasts (cp) multiply in the cytoplasm of the plant cell by binary division, with multiple copies of cpDNA transmitted and maintained in successive generations. The effect of cpDNA copy number on cell proliferation and aging was investigated using a C. reinhardtii moc mutant, which has an undispersed cp-nucleoid and unequal segregation of cpDNA during cell division. When the mother cell divided into four daughters, one moc daughter cell chloroplast contained about 60 copies of cpDNA, and the chloroplasts in the three other daughter cells contained the 4–7 copies of cpDNA. In liquid medium, the number of moc cells at the period of stationary phase was about one-third that of the wild type. To observe the process of proliferation and aging in the mother cell, we used solid medium. Three out of four moc cell spores were preferentially degenerated 60 days after cell transfer. To confirm this, wild-type and moc mother cells containing four daughter cells were treated with novobiocin to inhibit cpDNA replication. Cell degeneration increased only in the moc strain following novobiocin introduction. In total, our results suggest that cells possessing smaller amounts of cpDNA degenerate and age more rapidly. Received 7 September 2000/ Accepted in revised form 14 February 2001     

Antioxidant Enzymes of Larvae of the Cabbage Looper Moth,Trzchoplusza Ni: Subcellular Distribution and Activities of Superoxide Dismutase,Catalase and Glutathione Reductase

In the mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni, the subcellular distribution of total superoxide dismutase was as follows: 3.05 units (70.0%), 0.97 units (22.3%), and 0.33 units (7.6%) mg^?1 protein in the mitochondrial, cytosolic and nuclear fractions, respectively. No superoxide dismutase activity was detected in the microsomal fraction. Catalase activity was unusually high and as follows: 283.4 units (47.3%), 150.1 units (25.1%). 142.3 units (23.8%), and 22.9 units (3.8%) mg^?1 protein in the mitochondrial, cytosolic, microsomal (containing peroxisomes), and nuclear fractions. No glutathione peroxidase activity was found, but appreciable glutathione reductase activity was detected with broad subcellular distribution as follows: 3.86 units (36.1%), 3.68 units (34.0%). 2.46 units (23.0%). and 0.70 units (6.5%) mg^?1 protein in the nuclear, mitochondrial, and cytosolic fractions, respectively. The unusually wide intracellular distribution of catalase in this phytophagous insect is apparently an evolutionary adaptation to the absence of glutathione peroxidase; hence, lack of a glutathione peroxidase-glutathione reductase role in alleviating stress from lipid peroxidation. Catalase working sequentially to superoxide dismutase, may nearly completely prevent the formation of the lipid peroxidizing OH radical from all intracellular compartments by the destruction of H₂O₂ which together with O^?₂ is a precursor of OH.     

盐胁迫下欧李叶片叶绿体结构及功能与超微弱发光激发的关系

为初步探索叶绿体及其功能与超微弱发光（Ultraweak luminescence,UWL）激发的关系,揭示UWL与植物生长生理的关系及植物中UWL产生的来源,本试验以欧李（Cerasus humilis）为材料,采用室内盆栽试验,设置不同浓度盐胁迫处理,研究盐胁迫下欧李叶片的叶绿体结构和功能（叶绿素代谢、光系统Ⅱ活性、光合性能和能量水平）及UWL的变化规律和相关性。结果表明：（1）盐胁迫降低了欧李叶片的UWL强度,且盐浓度越高,UWL强度下降程度越大;（2）盐胁迫破坏了欧李叶片叶绿体结构,并降低了其功能,具体表现为叶绿素合成主要前体物质（ALA、Mg-ProtoⅨ）含量显著降低,叶绿素降解酶叶绿素酶（Chlase）活性显著升高,导致叶绿素（Chla、Chlb、Car和Chla+b）含量显著降低;同时欧李叶片FV/Fm、FV/FO、PIABS、RC/CSm、φE0和ΨE0快速下降,光系统Ⅱ活性受到严重抑制;进一步Pn、Tr、Gs下降,Ci同时升高,光合性能显著减弱;ATP含量和EC的显著降低,导致能量水平整体下降;（3）欧李叶片UWL强度与其叶绿素代谢物质及叶绿素含量（ALA、Mg-ProtoⅨ、Chla、Chlb、Car和Chla+b）、光系统Ⅱ活性（FV/Fm、FV/FO、PIABS、RC/CSm、φE0、ΨE0）、光合性能（Pn、Tr、Gs）及能量水平（ATP、EC）等参数均呈显著或极显著性正相关关系。（4）盐浓度越高,胁迫时间越长,欧李叶片UWL强度与叶绿体功能各指标变化程度越大,且高浓度处理下的相关性整体高于低浓度处理。可见,在盐胁迫条件下,欧李叶片叶绿体结构被破坏,同时其功能受到损伤活力下降,从而导致UWL强度降低;UWL强度与叶绿体及其功能关系密切,叶绿体可能是UWL的细胞器之一;UWL强度可以用来反映欧李叶片受盐胁迫伤害的程度。