RESUMO
Exposure to abiotic stresses accelerates leaf senescence in most crop plant species, thereby reducing photosynthesis and other assimilatory processes. In some cases, genotypes with delayed leaf senescence (i.e. 'stay-green') show stress resistance, particularly in cases of water deficit, and this has led to the proposal that senescence delay improves crop performance under some abiotic stresses. In this review, we summarize the evidence for increased resistance to abiotic stress, mostly water deficit, in genotypes with delayed senescence, and specifically focus on the physiological mechanisms and agronomic conditions under which the stay-green trait may ameliorate grain yield under stress.
Assuntos
Produtos Agrícolas , Senescência Vegetal , Estresse Fisiológico , Produtos Agrícolas/fisiologia , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/genética , Senescência Vegetal/fisiologia , Folhas de Planta/fisiologiaRESUMO
Leaf senescence is characterized by massive degradation of chloroplast proteins, yet the protease(s) involved is(are) not completely known. Increased expression and/or activities of serine, cysteine, aspartic, and metalloproteases were detected in senescing leaves, but these studies have not provided information on the identities of the proteases responsible for chloroplast protein breakdown. Silencing some senescence-associated proteases has delayed progression of senescence symptoms, yet it is still unclear if these proteases are directly involved in chloroplast protein breakdown. At least four cellular pathways involved in the traffic of chloroplast proteins for degradation outside the chloroplast have been described (i.e., "Rubisco-containing bodies," "senescence-associated vacuoles," "ATI1-plastid associated bodies," and "CV-containing vesicles"), which differ in their dependence on the autophagic machinery, and the identity of the proteins transported and/or degraded. Finding out the proteases involved in, for example, the degradation of Rubisco, may require piling up mutations in several senescence-associated proteases. Alternatively, targeting a proteinaceous protein inhibitor to chloroplasts may allow the inhibitor to reach "Rubisco-containing bodies," "senescence-associated vacuoles," "ATI1-plastid associated bodies," and "CV-containing vesicles" in essentially the way as chloroplast-targeted fluorescent proteins re-localize to these vesicular structures. This might help to reduce proteolytic activity, thereby reducing or slowing down plastid protein degradation during senescence.
RESUMO
The apoplast, i.e. the cellular compartment external to the plasma membrane, undergoes important changes during senescence. Apoplastic fluid volume increases quite significantly in senescing leaves, thereby diluting its contents. Its pH elevates by about 0.8 units, similar to the apoplast alkalization in response to abiotic stresses. The levels of 159 proteins decrease, whereas 24 proteins increase in relative abundance in the apoplast of senescing leaves. Around half of the apoplastic proteins of non-senescent leaves contain a N-terminal signal peptide for secretion, while all the identified senescence-associated apoplastic proteins contain the signal peptide. Several of the apoplastic proteins that accumulate during senescence also accumulate in stress responses, suggesting that the apoplast may constitute a compartment where developmental and stress-related programs overlap. Other senescence-related apoplastic proteins are involved in cell wall modifications, proteolysis, carbohydrate, ROS and amino acid metabolism, signaling, lipid transport, etc. The most abundant senescence-associated apoplastic proteins, PR2 and PR5 (e.g. pathogenesis related proteins PR2 and PR5) are related to leaf aging rather than to the chloroplast degradation program, as their levels increase only in leaves undergoing developmental senescence, but not in dark-induced senescent leaves. Changes in the apoplastic space may be relevant for signaling and molecular trafficking underlying senescence.
RESUMO
Plant senescence is accompanied by a marked increase in proteolytic activities, and cysteine proteases (Cys-protease) represent the prevailing class among the responsible proteases. Cys-proteases predominantly locate to lytic compartments, i.e., to the central vacuole (CV) and to senescence-associated vacuoles (SAVs), the latter being specific to the photosynthetic cells of senescing leaves. Cellular fractionation of vacuolar compartments may facilitate Cys-proteases purification and their concentration for further analysis. Active Cys-proteases may be analyzed by different, albeit complementary approaches: (1) in vivo examination of proteolytic activity by fluorescence microscopy using specific substrates which become fluorescent upon cleavage by Cys-proteases, (2) protease labeling with specific probes that react irreversibly with the active enzymes, and (3) zymography, whereby protease activities are detected in polyacrylamide gels copolymerized with a substrate for proteases. Here we describe the three methods mentioned above for detection of active Cys-proteases and a cellular fractionation technique to isolate SAVs.
Assuntos
Envelhecimento , Cisteína Proteases/metabolismo , Fenômenos Fisiológicos Vegetais , Vacúolos/enzimologia , Ativação Enzimática , Proteínas de Plantas/metabolismo , Coloração e RotulagemRESUMO
PURPOSE: To present a case of a complicated posterior melanocytoma that was successfully treated with intravitreal bevacizumab. CASE REPORT: A 50-year-old Caucasian man was referred with sudden-onset metamorphopsia and decreased vision in his right eye over the course of the last 2 months. His best-corrected visual acuity was 20/80 and poorer than Jaeger 14 in the right eye, and 20/20 and Jaeger 1 in his left eye. In the right fundus, there was a melanocytic lesion occupying the inferotemporal quadrant of the optic disk, extending to the adjacent choroid inferiorly; optic nerve edema, superotemporal retinal vein dilatation, and subretinal fluid under the macula and nasal half of the posterior pole were observed, and a subretinal choroidal neovascularization complex was observed adjacent to the superotemporal margin of the optic disk, confirmed by fluorescein angiography, surrounded by a dense subretinal hemorrhage. Optical coherence tomography showed retinal edema and detachment of neurosensory retina. The patient was treated with three consecutive doses on a monthly basis of intravitreal 1.25 mg/0.05 mL bevacizumab. Visual acuity recovered rapidly, and at 4 months after treatment, it was 20/20 and Jaeger 1, with complete resolution of macular edema and subretinal fluid and hemorrhage. After 3 years of follow-up, best-corrected visual acuity remained stable, macular area was normal, and there was no evident optic nerve edema, retinal vein caliber and aspect were normal, and there was no significant change of the tumor. Fluorescein angiography only evidenced late staining of choroidal neovascularization scar, and optical coherence tomography showed a normal macular anatomy. CONCLUSION: Intravitreal bevacizumab was effective in the treatment of choroidal neovascularization, optic nerve edema, venous dilatation, and local capillary telangiectasia, complicating an optic disk melanocytoma.
RESUMO
Senescence involves increased expression of proteases, which may participate in nitrogen recycling or cellular signalling. 2D zymograms detected two protein species with increased proteolytic activity in senescing leaves of Arabidopsis thaliana. A proteomic analysis revealed that both protein species correspond to a subtilisin protease encoded by At3g14067, termed Senescence-Associated Subtilisin Protease (SASP). SASP mRNA levels and enzyme activity increase during leaf senescence in leaves senescing during both the vegetative or the reproductive phase of the plant life cycle, but this increase is more pronounced in reproductive plants. SASP is expressed in all above-ground organs, but not in roots. Putative AtSASP orthologues were identified in dicot and monocot crop species. A phylogenetic analysis shows AtSASP and its putative orthologues clustering in one discrete group of subtilisin proteases in which no other Arabidospsis subtilisin protease is present. Phenotypic analysis of two knockout lines for SASP showed that mutant plants develop more inflorescence branches during reproductive development. Both AtSASP and its putative rice orthologue (OsSASP) were constitutively expressed in sasp-1 to complement the mutant phenotype. At maturity, sasp-1 plants produced 25% more inflorescence branches and siliques than either the wild-type or the rescued lines. These differences were mostly due to an increased number of second and third order branches. The increased number of siliques was compensated for by a small decrease (5.0%) in seed size. SASP downregulates branching and silique production during monocarpic senescence, and its function is at least partially conserved between Arabidopsis and rice.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Sementes/crescimento & desenvolvimento , Sementes/genética , Subtilisinas/genética , Subtilisinas/metabolismo , Sequência de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Regulação da Expressão Gênica no Desenvolvimento , Filogenia , Proteômica , Sementes/enzimologia , Alinhamento de Sequência , Subtilisinas/químicaRESUMO
Degradation of chloroplasts and chloroplast components is a distinctive feature of leaf senescence. In spite of its importance in the nutrient economy of plants, knowledge about the mechanism(s) involved in the breakdown of chloroplast proteins is incomplete. A novel class of vacuoles, "senescence-associated vacuoles" (SAVs), characterized by intense proteolytic activity appear during senescence in chloroplast-containing cells of leaves. Since SAVs contain some chloroplast proteins, they are candidate organelles to participate in chloroplast breakdown. In this review we discuss the characteristics of SAVs, and their possible involvement in the degradation of Rubisco, the most abundant chloroplast protein. Finally, SAVs are compared with other extra-plastidial protein degradation pathways operating in senescing leaves.
RESUMO
Breakdown of leaf proteins, particularly chloroplast proteins, is a massive process in senescing leaves. In spite of its importance in internal N recycling, the mechanism(s) and the enzymes involved are largely unknown. Senescence-associated vacuoles (SAVs) are small, acidic vacuoles with high cysteine peptidase activity. Chloroplast-targeted proteins re-localize to SAVs during senescence, suggesting that SAVs might be involved in chloroplast protein degradation. SAVs were undetectable in mature, non-senescent tobacco leaves. Their abundance, visualized either with the acidotropic marker Lysotracker Red or by green fluorescent protein (GFP) fluorescence in a line expressing the senescence-associated cysteine protease SAG12 fused to GFP, increased during senescence induction in darkness, and peaked after 2-4 d, when chloroplast dismantling was most intense. Increased abundance of SAVs correlated with higher levels of SAG12 mRNA. Activity labelling with a biotinylated derivative of the cysteine protease inhibitor E-64 was used to detect active cysteine proteases. The two apparently most abundant cysteine proteases of senescing leaves, of 40kDa and 33kDa were detected in isolated SAVs. Rubisco degradation in isolated SAVs was completely blocked by E-64. Treatment of leaf disks with E-64 in vivo substantially reduced degradation of Rubisco and leaf proteins. Overall, these results indicate that SAVs contain most of the cysteine protease activity of senescing cells, and that SAV cysteine proteases are at least partly responsible for the degradation of stromal proteins of the chloroplast.
Assuntos
Senescência Celular , Cloroplastos/enzimologia , Cisteína Proteases/metabolismo , Nicotiana/enzimologia , Folhas de Planta/enzimologia , Proteínas de Plantas/metabolismo , Vacúolos/enzimologia , Senescência Celular/efeitos dos fármacos , Senescência Celular/efeitos da radiação , Cloroplastos/efeitos dos fármacos , Cloroplastos/genética , Cloroplastos/efeitos da radiação , Cisteína Proteases/genética , Inibidores de Cisteína Proteinase/farmacologia , Escuridão , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/efeitos da radiação , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteólise/efeitos dos fármacos , Proteólise/efeitos da radiação , Nicotiana/efeitos dos fármacos , Nicotiana/genética , Nicotiana/efeitos da radiação , Vacúolos/efeitos dos fármacos , Vacúolos/genética , Vacúolos/efeitos da radiaçãoRESUMO
Cellular proteins are extensively degraded during leaf senescence, and this correlates with an up-regulation of protease gene expression, particularly cysteine proteases. The objectives of this work were (i) to detect cysteine proteases associated with senescence of wheat leaves under different conditions and (ii) to find out their subcellular location. Activity labelling of cysteine proteases with the biotinylated inhibitor DCG-04 detected five bands at 27, 36, 39, 42, and 46 kDa in leaves of wheat senescing under continuous darkness. In-gel activity assays showed that these proteases are only active in an acid milieu (pH 4), and their activity increased several-fold in senescing leaves. Fractionation experiments showed that the senescence-associated cysteine proteases of 36, 39, 42, and 46 kDa localize to a vacuolar-enriched fraction. The vacuolar cysteine proteases of 36, 39, and 42 kDa increased in activity in attached flag leaves senescing naturally during post-anthesis, and in attached leaves of plants subjected to a period of water deficit. Thus, the activity of these vacuolar cysteine proteases is associated with developmental (post-anthesis) senescence and with senescence induced by stress factors (i.e. protracted darkness or drought). This suggests that vacuoles are involved in senescence-associated cellular degradation, and that different senescence-inducing factors may converge on a single degradation pathway.
Assuntos
Cisteína Endopeptidases/metabolismo , Folhas de Planta/enzimologia , Triticum/enzimologia , Vacúolos/enzimologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Concentração de Íons de Hidrogênio , Reprodução/fisiologia , Fatores de Tempo , Água/metabolismoRESUMO
Vacuolar compartments associated with leaf senescence and the subcellular localization of the senescence-specific cysteine-protease SAG12 (senescence-associated gene 12) were studied using specific fluorescent markers, the expression of reporter genes, and the analysis of high-pressure frozen/freeze-substituted samples. Senescence-associated vacuoles (SAVs) with intense proteolytic activity develop in the peripheral cytoplasm of mesophyll and guard cells in Arabidopsis and soybean. The vacuolar identity of these compartments was confirmed by immunolabeling with specific antibody markers. SAVs and the central vacuole differ in their acidity and tonoplast composition: SAVs are more acidic than the central vacuole and, whereas the tonoplast of central vacuoles is highly enriched in gamma-TIP (tonoplast intrinsic protein), the tonoplast of SAVs lacks this aquaporin. The expression of a SAG12-GFP fusion protein in transgenic Arabidopsis plants shows that SAG12 localizes to SAVs. The analysis of Pro(SAG12):GUS transgenic plants indicates that SAG12 expression in senescing leaves is restricted to SAV-containing cells, for example, mesophyll and guard cells. A homozygous sag12 Arabidopsis mutant develops SAVs and does not show any visually detectable phenotypical alteration during senescence, indicating that SAG12 is not required either for SAV formation or for progression of visual symptoms of senescence. The presence of two types of vacuoles in senescing leaves could provide different lytic compartments for the dismantling of specific cellular components. The possible origin and functions of SAVs during leaf senescence are discussed.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/ultraestrutura , Glycine max/enzimologia , Glycine max/ultraestrutura , Vacúolos/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Cloroplastos , Cisteína Endopeptidases/fisiologia , Concentração de Íons de Hidrogênio , Mutação , Folhas de Planta/enzimologia , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas , Glycine max/genética , Fatores de Tempo , Vacúolos/químicaRESUMO
Photosynthesis, respiration, and other processes produce reactive oxygen species (ROS) that can cause oxidative modifications to proteins, lipids, and DNA. The production of ROS increases under stress conditions, causing oxidative damage and impairment of normal metabolism. In this work, oxidative damage to various subcellular compartments (i.e. chloroplasts, mitochondria, and peroxisomes) was studied in two cultivars of wheat differing in ascorbic acid content, and growing under good irrigation or drought. In well-watered plants, mitochondria contained 9-28-fold higher concentrations of oxidatively modified proteins than chloroplasts or peroxisomes. In general, oxidative damage to proteins was more intense in the cultivar with the lower content of ascorbic acid, particularly in the chloroplast stroma. Water stress caused a marked increase in oxidative damage to proteins, particularly in mitochondria and peroxisomes. These results indicate that mitochondria are the main target for oxidative damage to proteins under well-irrigated and drought conditions.