Regulation of developmental senescence is conserved between Arabidopsis and Brassica napus

SAG12 is a developmentally controlled, senescence-specific gene from Arabidopsis which encodes a cysteine protease. Using SAG12 as a probe, we isolated two SAG12 homologues (BnSAG12–1 and BnSAG12–2) from Brassica napus. Structural comparisons and expression studies indicate that these two genes are orthologues of SAG12. The expression patterns of BnSAG12–1 and BnSAG12–2 in Arabidopsis demonstrate that the senescence-specific regulation of this class of cysteine proteases is conserved across these species. Gel-shift assays using the essential promoter regions of SAG12, BnSAG12–1, and BnSAG12–2 show that the extent of binding of a senescence-specific, DNA-binding protein from Arabidopsis is proportional to the expression levels of these genes in Arabidopsis. Therefore, the expression levels of these genes may reflect the affinities of the senescence-specific DNA-binding protein for the promoter element.     

Human sensitive to apoptosis gene protein inhibits peroxynitrite-induced DNA damage

Sensitive to apoptosis gene (SAG) protein, a novel zinc RING finger protein, which is redox responsive and protects mammalian cells from apoptosis, is a metal chelator and a potential reactive oxygen species scavenger, but its antioxidant properties have not been completely defined. The present study was undertaken to test the hypothesis that human SAG protects from DNA damage induced by peroxynitrite, a potent physiological inorganic toxin. The present study has shown that SAG significantly inhibits single strand breaks in supercoiled plasmid DNA induced by synthesized peroxynitrite (ONOO(-)) and 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a generator of peroxynitrite through the reaction between nitric oxide and superoxide anion. The formation of 8-hydroxy-2(')-deoxyguanosine in calf thymus DNA by peroxynitrite and SIN-1 was also significantly inhibited by SAG. The protective effect on peroxynitrite-mediated DNA damage was completely abolished by the reaction of SAG with N-ethylmaleimide, a chemical modification agent for the sulfhydryl group of proteins. These observations suggested that the sulfhydryl group of cysteines in SAG could react directly with peroxynitrite to prevent DNA damage.     

Delay of leaf senescence in <Emphasis Type="Italic">Medicago sativa</Emphasis> transformed with the <Emphasis Type="Italic">ipt</Emphasis> gene controlled by the senescence-specific promoter SAG12

We report the successfull delay of leaf senescence in Medicago sativa. A highly regenerable clone of alfalfa was transformed with the construct SAG12-IPT, an approach that has already proved efficient in other crops. Several independent transformants were obtained as determined by Southern analysis and all the transformants expressed the transgene as measured by RT-PCR. In vitro and in vivo analyses showed that SAG12-IPT plants exhibited a stay-green phenotype that has the potential to greatly improve the quantity and quality of alfalfa forage.     

Membrane interactions of the hydrophobic segment of diacylglycerol kinase epsilon

Diacylglycerol kinase epsilon (DGKε) is unique among mammalian DGK isoforms in having a segment of hydrophobic amino acids as a putative membrane anchor. To model the conformation, and stoichiometry of this segment in membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of DGKε of sequence KKKKLILWTLCSVLLPVFITFWKKKKK-NH₂. Flanking Lys residues mimic the natural setting of this peptide in DGKε, while facilitating peptide synthesis and characterization. Circular dichroism and fluorescence spectroscopic analysis demonstrated that the peptide has increased helical content and significant blue shifts in the presence of anionic - but not zwitterionic - bilayer membranes. When labeled with fluorophores that can undergo fluorescence resonance energy transfer, the peptide was found to dimerize - a result also observed from migration rates on SDS-PAGE gels under both reducing and non-reducing disulfide bridge conditions. The peptide was shown to preferentially interact with cholesterol in lipid films comprised of homogeneous mixtures of cholesterol and phosphatidylcholine, yet the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. The peptide was also able to inhibit the activity of DGKε protein in vitro. Our overall findings suggest that the peptide ultimately cannot leave the bulk water for attachment/insertion into the outer leaflet of an erythrocyte-like bilayer, yet its core sequence is sufficiently hydrophobic to insert into membrane core regions when membrane attachment is promoted by electrostatic attraction to anionic lipid head groups of the inner leaflet of an erythrocyte-like bilayer.     

转PSAG12-ipt基因水稻叶片中叶绿体超微结构的变化

研究了转PSAG12-ipt基因水稻和对照植株发育过程中叶片中的叶绿体结构的变化。发现水稻发育到乳熟期,转基因植株叶片中的叶绿体与对照植株开始出现明显的差别。对照叶绿体中嗜锇体体积增大,数目增多,大部分基粒的类囊体膜膨胀、裂解,片层结构解体。而转基因植株叶片中的叶绿体结构变化不大,嗜锇体相对有所增加,但体积较小,大部分基粒类囊体片层结构仍然排列整齐,少数类囊体垛叠化丧失。     

Exploring the uncultured microeukaryote majority in the oceans: reevaluation of ribogroups within stramenopiles

Molecular surveys in planktonic marine systems have unveiled a large novel diversity of small protists. A large part of this diversity belongs to basal heterotrophic stramenopiles and is distributed in a set of polyphyletic ribogroups (described from rDNA sequences) collectively named as MAST (MArine STramenopiles). In the few groups investigated, MAST cells are globally distributed and abundant bacterial grazers, therefore having a putatively large impact on marine ecosystem functioning. The main aim of this study is to reevaluate the MAST ribogroups described so far and to determine whether additional groups can be found. For this purpose, we used traditional and state-of-the-art molecular tools, combining 18S rDNA sequences from publicly available clone libraries, single amplified genomes (SAGs) of planktonic protists, and a pyrosequencing survey from coastal waters and sediments. Our analysis indicated a final set of 18 MAST groups plus 5 new ribogroups within Ochrophyta (named as MOCH). The MAST ribogroups were then analyzed in more detail. Seven were typical of anoxic systems and one of oxic sediments. The rest were clearly members of oxic marine picoplankton. We characterized the genetic diversity within each MAST group and defined subclades for the more diverse (46 subclades in 8 groups). The analyses of sequences within subclades revealed further ecological specializations. Our data provide a renovated framework for phylogenetic classification of the numerous MAST ribogroups and support the notion of a tight link between phylogeny and ecological distribution. These diverse and largely uncultured protists are widespread and ecologically relevant members of marine microbial assemblages.     

An ABA-regulated and Golgi-localized protein phosphatase controls water loss during leaf senescence in Arabidopsis

It is known that a senescing leaf loses water faster than a non-senescing leaf and that ABA has an important role in promoting leaf senescence. However, questions such as why water loss is faster, how water loss is regulated, and how ABA functions in leaf senescence are not well understood. Here we report on the identification and functional analysis of a leaf senescence associated gene called SAG113. The RNA blot and GUS reporter analyses all show that SAG113 is expressed in senescing leaves and is induced by ABA in Arabidopsis. The SAG113 expression levels are significantly reduced in aba2 and abi4 mutants. A GFP fusion protein analysis revealed that SAG113 protein is localized in the Golgi apparatus. SAG113 encodes a protein phosphatase that belongs to the PP2C family and is able to functionally complement a yeast PP2C-deficient mutant TM126 (ptc1Δ). Leaf senescence is delayed in the SAG113 knockout mutant compared with that in the wild type, stomatal movement in the senescing leaves of SAG113 knockouts is more sensitive to ABA than that of the wild type, and the rate of water loss in senescing leaves of SAG113 knockouts is significantly reduced. In contrast, inducible over-expression of SAG113 results in a lower sensitivity of stomatal movement to ABA treatment, more rapid water loss, and precocious leaf senescence. No other aspects of growth and development, including seed germination, were observed. These findings suggest that SAG113, a negative regulator of ABA signal transduction, is specifically involved in the control of water loss during leaf senescence.     

Sensitive to apoptosis gene protein regulates ionizing radiation-induced apoptosis

Organisms exposed to ionizing radiation (IR) undergo increases in the production of reactive oxygen species (ROS), which are determinant components in the induction of apoptosis. Sensitive to apoptosis gene (SAG) encodes a redox-inducible and apoptosis-protective antioxidant protein. This report demonstrates that the modulation of SAG expression in cultured cells regulates IR-induced apoptosis. A protective role for SAG against IR-induced apoptosis was found in U937 cells transfected with SAG cDNA. A significant decrease in the endogenous production of ROS was also observed in SAG over-expressing cells, compared to control cells, exposed to 2 Gy γ-irradiation. These results suggest that SAG plays an important role in regulating IR-induced apoptosis, presumably by maintaining the cellular redox status. Because SAG is over-expressed in many human cancers, targeting SAG expression may have therapeutic value in cancer treatment. Transfection of the pancreatic cancer cell line PC3 with SAG small interfering RNA markedly attenuated the expression of SAG, augmenting their susceptibility to IR-induced apoptosis. The knockdown of SAG expression by RNA interference combined with radiotherapy may be a potential method for radiosensitization.     

Plant degreening: evolution and expression of tomato (Solanum lycopersicum) dephytylation enzymes

Chlorophyll is the most abundant pigment on earth and even though it is known that its high photo-excitability necessitates a tight regulation of its degradation pathway, to date there are still several steps in chlorophyll breakdown that remain obscure. In order to better understand the ‘degreening’ processes that accompany leaf senescence and fruit ripening, we characterized the enzyme-encoding genes involved in dephytylation from tomato (Solanum lycopersicum). A single pheophytinase (PPH) gene and four chlorophyllase (CLH) genes were identified in the tomato genome. A phenetic analysis revealed two groups of CLHs in eudicot species and further evolutionary analysis indicated that these enzymes are under diverse selection pressures. A comprehensive expression profile analysis also suggested functional specificity for these dephytylating enzymes. The integrated analysis allows us to propose three general roles for chlorophyll dephytylation: i) PPH, which is under high selective constraint, is responsible for chlorophyll degradation during developmentally programed physiological processes; ii) Group I CLHs, which are under relaxed selection constraint, respond to environmental and hormonal stimuli and play a role in plant adaptation plasticity; and iii) Group II CLHs, which are also under high selective constraint, are mostly involved in chlorophyll recycling.