Arabidopsis STAY-GREEN2 Is a Negative Regulator of Chlorophyll Degradation during Leaf Senescence

Chlorophyll （Chl） degradation causes leaf yellowing during senescence or under stress conditions. For Chl breakdown, STAY-GREEN1 （SGR1） interacts with Chl catabolic enzymes （CCEs） and light-harvesting complex II （LHCII） at the thylakoid membrane, possibly to allow metabolic channeling of potentially phototoxic Chl breakdown intermediates. Among these Chl catabolic components, SGR1 acts as a key regulator of leaf yellowing. In addition to SGR1 （At4g22920）, the Arabidopsis thaliana genome contains an additional homolog, SGR2 （At4g11910）, whose biological function remains elusive. Under senescence-inducing conditions, SGR2 expression is highly up-regulated, similarly to SGR1 expression. Here we show that SGR2 function counteracts SGR1 activity in leaf Chl degradation; SGR2-overexpressing plants stayed green and the sgr2-1 knockout mutant exhibited early leaf yellowing under age-, dark-, and stress-induced senescence conditions. Like SGR1, SGR2 interacted with LHCII but, in contrast to SGR1, SGR2 interactions with CCEs were very limited. Furthermore, SGR1 and SGR2 formed homo- or heterodimers, strongly suggesting a role for SGR2 in negatively regulat- ing Chl degradation by possibly interfering with the proposed CCE-recruiting function of SGR1. Our data indicate an antagonistic evolution of the functions of SGR1 and SGR2 in Arabidopsis to balance Chl catabolism in chloroplasts with the dismantling and remobilizing of other cellular components in senescing leaf cells.     

Molecular Evolution of VEF-Domain-Containing PcG Genes in Plants

Arabidopsis VERNALIZATION2 （VRN2）, EMBRYONIC FLOWER2 （EMF2）, and FERTILIZATION-INDEPENDENT SEED2 （FIS2） are involved in vernalization-mediated flowering, vegetative development, and seed development, respectively. Together with Arabidopsis VEF-L36, they share a VEF domain that is conserved in plants and animals. To investigate the evolution of VEF-domain-containing genes （VEF genes）, we analyzed sequences related to VEF genes across land plants. To date, 24 full-length sequences from 11 angiosperm families and 54 partial sequences from another nine families were identified. The majority of the full-length sequences identified share greatest sequence similarity with and possess the same major domain structure as Arabidopsis EMF2. EMF2-1ike sequences are not only widespread among angiosperms, but are also found in genomic sequences of gymnosperms, lycophyte, and moss. No FIS2- or VEF-L36-1ike sequences were recovered from plants other than Arabidopsis, including from rice and poplar for which whole genomes have been sequenced. Phylogenetic analysis of the full-length sequences showed a high degree of amino acid sequence conservation in EMF2 homologs of closely related taxa. VRN2 homologs are recovered as a clade nested within the larger EMF2 clade. FIS2 and VEF-L36 are recovered in the VRN2 clade. VRN2 clade may have evolved from an EMF2 duplication event that occurred in the rosids prior to the divergence of the eurosid I and eurosid II lineages. We propose that dynamic changes in genome evolution contribute to the generation of the family of VEF-domain-containing genes, Phylogenetic analysis of the VEF domain alone showed that VEF sequences continue to evolve following EM F2NRN2 divergence in accordance with species relationship. Existence of EMF2-1ike sequences in animals and across land plants suggests that a prototype form of EMF2 was present prior to the divergence of the plant and animal lineages. A proposed sequence of events, based on domain organization and occurrence of intermediate seque     

A Histone H3 Lysine-27 Methyltransferase Complex Represses Lateral Root Formation in Arabidopsis thaliana

Root branching or lateral root formation is crucial to maximize a root system acquiring nutrients and water from soil. A lateral root （LR） arises from asymmetric cell division of founder cells （FCs） in a pre-branch site of the primary root, and FC establishment is essential for lateral root formation. FCs are known to be specified from xylem pole pericycle cells, but the molecular genetic mechanisms underlying FC establishment are unclear. Here, we report that, in Arabidopsis thaliana, a PRC2 （for Polycomb repressive complex 2） histone H3 lysine-27 （H3K27） methyltransferase complex, functions to inhibit FC establishment during LR initiation. We found that functional loss of the PRC2 subunits EMF2 （for EMBRYONIC FLOWER 2） or CLF （for CURLY LEAF） leads to a great increase in the number of LRs formed in the primary root. The CLF H3K27 methyltransferase binds to chromatin of the auxin efflux carrier gene PIN FORMED 1 （PIN1）, deposits the repres- sive mark H3K27me3 to repress its expression, and functions to down-regulate auxin maxima in root tissues and inhibit FC establishment. Our findings collectively suggest that EMF2-CLF PRC2 acts to down-regulate root auxin maxima and show that this complex represses LR formation in Arabidopsis.     

Hydrogen peroxide induces the activation of the phospholipase C-γ1 survival pathway in PC12 cells： protective role in apoptosis

It has been reported that phospholipase C-γ1 （PLC-γ1） plays an important protective role in hydrogen peroxide （H2O2）-induced pheochromocytoma （PC） 12 cells death. However, most studies have used high doses of H2O2 and the downstream targets of PLC-γ1 activation remain to be identified. The present study was designed to examine the roles of PLC-γ1 signaling pathway in the apoptosis of PC12 cells induced by low dose of H2O2, as well as the downstream factors involved in this pathway. Low-dose treatment of H2O2 resulted in PLC-γ1 tyrosine phosphorylation in a time-dependent manner and H2O2 killed the PC12 cells by inducing necrosis. In contrast, pretreatment of PCI2 cells with U73122, a specific inhibitor of PLC, markedly increased the percentage of dead cells. The mode of cell death was converted to apoptosis as determined by Hoechst/PI nuclear staining and fluorescence microscopy. Western blot analysis demonstrated that the expression of Bcl-2 protein and the activation of pro-caspase-3 were not significantly affected by low dose of H2O2 alone. However, after pretreatment with U73122, Bcl-2 protein expression was dramatically decreased and the activation of pro-caspase-3 was significantly increased. We concluded that PLC-γ1 plays an important protective role in H2O2-induced PC12 cells death. Bcl-2 and caspase-3 probably participate in the signaling pathway as downstream factors.     

GNOM-LIKE 2, Encoding an Adenosine Diphosphate-Ribosylation Factor-Guanine Nucleotide Exchange Factor Protein Homologous to GNOM and GNL1, is Essential for Pollen Germination in Arabidopsis

In flowering plants, male gametes are delivered to female gametophytes by pollen tubes. Although it is important for sexual plant reproduction, little is known about the genetic mechanism that controls pollen germination and pollen tube growth. Here we report the identification and characterization of two novel mutants, gnom-like 2-1 （gnl2-1） and gn12-2 in Arabidopsis thaliana, in which the pollen grains failed to germinate in vitro and in vivo. GNL2 encodes a protein homologous to the adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factors, GNOM and GNL1 that are involved in endosomal recycling and endoplasmic reticulum-Golgi vesicular trafficking. It was prolifically expressed in pollen grains and pollen tubes. The results of the present study suggest that GNL2 plays an important role in pollen germination.     

Camouflage Patterning in Maize Leaves Results from a Defect in Porphobilinogen Deaminase

Maize leaves are produced from polarized cell divisions that result in clonal cell lineages arrayed along the long axis of the leaf. We utilized this stereotypical division pattern to identify a collection of mutants that form chloroplast pigmentation sectors that violate the clonal cell lineages. Here, we describe the camouflage1 （cfl） mutant, which develops nonclonal, yellow-green sectors in its leaves. We cloned the cfl gene by transposon tagging and determined that it encodes porphobilinogen deaminase （PBGD）, an enzyme that functions early in chlorophyll and heme biosynthesis. While PBGD has been characterized biochemically, no viable mutations in this gene have been reported in plants. To investigate the in vivo function of PBGD, we characterized the cfl mutant. Histological analyses revealed that cfl yellow sectors display the novel phenotype of bundle sheath cell-specific death. Light-shift experiments determined that constant light suppressed cfl sector formation, a dark/light transition is required to induce yellow sectors, and that sectors form only during a limited time of leaf development. Biochemical experiments determined that of 1 mutant leaves have decreased PBGD activity and increased levels of the enzyme substrate in both green and yellow regions. Furthermore, the cfl yellow regions displayed a reduction in catalase activity. A threshold model is hypothesized to explain the cfl variegation and incorporates photosynthetic cell differentiation, reactive oxygen species scavenging, and PBGD function.     

Alb4 of Arabidopsis Promotes Assembly and Stabilization of a Non Chlorophyll-Binding Photosynthetic Complex,the CF1CF0-ATP Synthase

All members of the YidC/Oxal/Alb3 protein family are evolutionarily conserved and appear to function in membrane protein integration and protein complex stabilization. Here, we report on a second thylakoidal isoform of Alb3, named Alb4. Analysis of Arabidopsis knockout mutant lines shows that AIb4 is required in assembly and/or stability of the CF1CF0-ATP synthase （ATPase）. alb4 mutant lines not only have reduced steady-state levels of ATPase subunits, but also their assembly into high-molecular-mass complexes is altered, leading to a reduction of ATP synthesis in the mutants. Moreover, we show that Alb4 but not AIb3 physically interacts with the subunits CF1β and CF0ll. Summarizing, the data indicate that AIb4 functions to stabilize or promote assembly of CF1 during its attachment to the membrane-embedded CF0 part.     

The effect of bafilomycin A1 and protease inhibitors on the degradation and recycling of a Class 5-mutant LDLR

The low-density lipoprotein receptor （LDLR） mediates cholesterol homeostasis through endocytosis of lipoprotein particles, particularly low-density lipoproteins （LDLs）. Normally, the lipoprotein particles are released in the endosomes and the receptors recycle to the cell surface. Familial hypercholesterolemia （FH） is an autosomal dominant disease caused by mutations in the gene encoding the LDLR. These mutations are divided into five functional classes where Class 5 mutations encode receptors that suffer from ligand-induced degradation and recycling deficiency. The aim of this study was to investigate whether it is possible to prevent the fast ligand-induced degradation of Class 5-mutant LDLR and to restore its ability to recycle to the cell surface. E387K is a naturally occurring Class 5 mutation found in FH patients, and in the present study, we used Chinese hamster ovary cells transfected with an E387K-mutant LDLR. Abrogation of endosomal acidification by adding bafdomycin A1 or addition of the irreversible serine protease inhibitors, 4-（2-aminoethyl）-benzenesulfonyl fluoride （AEBSF） and 3,4-dichloroisocoumarin （DCI）, prevented the degradation of the E387K-mutant LDLR. However, the undegraded receptor did not recycle to the cell surface in the presence of LDL. Unexpectedly, AEBSF caused aggregation of early endosome antigen-1positive endosomes and the intracellular trapped LDLR co-localized with these aggregated early endosomes.     

Arabidopsis Profilin Isoforms,PRF1 and PRF2 Show Distinctive Binding Activities and Subcellular Distributions

Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether these isoforms are functionally different. In the present study, two profilin isoforms from Arabidopsis, PRF1 and PRF2 were fused with green fuorescent protein （GFP） tag and expressed in Escherichia coil and A. thaliana in order to compare their biochemical properties in vitro and their cellular distributions in vivo. Biochemical analysis revealed that fusion proteins of GFP-PRF1 and GFP-PRF2 can bind to poly-L-proline and G-actin showing remarkable differences. GFP-PRF1 has much higher affinities for both poly-L-proline and G-actin compared with GFP-PRF2. Observations of living cells in stable transgenic A. thaliana lines revealed that 35S：：GFP-PRF1 formed a filamentous network, while 35S：：GFP-PRF2 formed polygonal meshes. Results from the treatment with latrunculin A and a subsequent recovery experiment indicated that filamentous alignment of GFP-PRF1 was likely associated with actin filaments. However, GFP-PRF2 localized to polygonal meshes resembling the endoplasmic reticulum. Our results provide evidence that Arabidopsis profllin isoforms PRF1 and PRF2 have different biochemical affinities for poly-L-proline and G-actin, and show distinctive Iocalizations in living cells. These data suggest that PRF1 and PRF2 are functionally different isoforms.     

Protective effects of DIDS against ethanol-induced gastric mucosal injury in rats

The compound 4,4＇-diisothiocyanostilbene-2,2＇-disulfonic acid （DIDS） is an efficient anion exchanger inhibitor that can block the activities of anion exchanger 2 （AE2）, which plays an indispensable role in gastric acid secretion. DIDS also has potent anti-oxidative and antiapoptosis activities. This study aimed to investigate the effect of DIDS on ethanol-induced mucosal damage in rats and to evaluate the underlying mechanisms that mediate the action of the compound. The rats received 1 ml of absolute ethanol or saline orally. DIDS [50 mg/kg intravenous （i.v.）] was given 5 min before ethanol administration. Gastric lesions were evaluated macroscopically, microscopically, and electron microscopically at 60 min after ethanol challenge. Gastric myeloperoxidase （MPO） activity, malonyldialdehyde （MDA） level, prostaglandin E2 （PGE2） synthesis, and cyclooxygenase-2 （COX-2） expression were assessed. For the evaluation of the effect of DIDS on gastric acid secretion, histamine-stimulatory gastric acid secretion was examined with or without pretreatment of DIDS （50 mg/kg; i.v.）. Ethanol-induced gastric lesions were characterized by increasing gastric MDA level, MPO activity, and COX-2 expression, and decreasing PGE2 synthesis. It was found that DIDS significantly reduced the extent of gastric mucosal damage and reversed tissue MDA level and MPO activity. DIDS further enhanced the expression of COX-2 and reversed the decrease of PGE2. Our results suggested that DIDS is beneficial in rat model of gastric injury through mechanisms that involve inhibiting inflammatory cell infiltration and lipid peroxidation and up-regulating the COX-2/PGE2 pathway.