RESUMEN
Species distributions are closely associated with moisture availability, but the underlying mechanisms remain unresolved. Drought relations are especially important for plants such as C4 grasses that dominate seasonally dry ecosystems. Here, we test the hypothesis that C4 grass species sampled across global precipitation gradients show variation in survival under drought that can be explained by their traits.Our experiment subjected 18 C4 grass species to a lethal drought under controlled environmental conditions. The number of days until death was measured, along with root traits, senescence, and aspects of hydraulic function.We identified two strategies: Drought-avoiding species that stayed green as the water potential declined and drought-tolerating species that senesced more quickly but could extend survival via drought-tolerant meristems.Plants that stay-green for longer occupied drier habitats and had the longest survival under drought, facilitated by narrow root diameter and isohydric stomatal behavior. Plants that senesced quickly had thicker roots, an anisohydric strategy, and occupied wetter habitats.Global distributions of C4 grasses can be predicted by variation in rates of senescence, meristem survival, root traits, and stomatal strategy, showing the value of these traits for understanding plant distributions in relation to climate.
RESUMEN
Plant populations persist under recurrent fire via resprouting from surviving tissues (resprouters) or seedling recruitment (seeders). Woody species are inherently slow maturing, meaning that seeders are confined to infrequent fire regimes. However, for grasses, which mature faster, the relationships between persistence strategy and fire regime remain unknown. Globally, we analysed associations between fire regimes experienced by hundreds of grass species and their persistence strategy, within a phylogenetic context. We also tested whether persistence strategies are associated with morphological and physiological traits. Resprouters were associated with less frequent fire than seeders. Whilst modal fire frequencies were similar (fire return interval of 4-6 yr), seeders were restricted to regions with more frequent fire than resprouters, suggesting that greater competition with long-lived resprouters restricts seeder recruitment and survival when fire is rare. Resprouting was associated with lower leaf N, higher C:N ratios and the presence of belowground buds, but was unrelated to photosynthetic pathway. Differences between the life histories of grasses and woody species led to a contrasting prevalence of seeders and resprouters in relation to fire frequency. Rapid sexual maturation in grasses means that seeder distributions, relative to fire regime, are determined by competitive ability and recruitment, rather than time to reproductive maturity.
Asunto(s)
Incendios , Poaceae , Ecosistema , Filogenia , Hojas de la Planta , PlantasRESUMEN
DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5'-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via 'phosphate steering', basic residues energetically steer an inverted ss 5'-flap through a gateway over FEN1's active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA)n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5'-flap specificity and catalysis, preventing genomic instability.
Asunto(s)
ADN/genética , Endonucleasas de ADN Solapado/metabolismo , Inestabilidad Genómica , Fosfatos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , ADN/química , ADN/metabolismo , Reparación del ADN , Replicación del ADN , Endonucleasas de ADN Solapado/química , Endonucleasas de ADN Solapado/genética , Humanos , Mutación , Fosfatos/química , Alineación de Secuencia , Especificidad por SustratoRESUMEN
[This corrects the article DOI: 10.1038/ncomms15855.].
RESUMEN
The prototypical 5'-nuclease, flap endonuclease-1 (FEN1), catalyzes the essential removal of single-stranded flaps during DNA replication and repair. FEN1 hydrolyzes a specific phosphodiester bond one nucleotide into double-stranded DNA. This specificity arises from double nucleotide unpairing that places the scissile phosphate diester on active site divalent metal ions. Also related to FEN1 specificity is the helical arch, through which 5'-flaps, but not continuous DNAs, can thread. The arch contains basic residues (Lys-93 and Arg-100 in human FEN1 (hFEN1)) that are conserved by all 5'-nucleases and a cap region only present in enzymes that process DNAs with 5' termini. Proline mutations (L97P, L111P, L130P) were introduced into the hFEN1 helical arch. Each mutation was severely detrimental to reaction. However, all proteins were at least as stable as wild-type (WT) hFEN1 and bound substrate with comparable affinity. Moreover, all mutants produced complexes with 5'-biotinylated substrate that, when captured with streptavidin, were resistant to challenge with competitor DNA. Removal of both conserved basic residues (K93A/R100A) was no more detrimental to reaction than the single mutation R100A, but much less severe than L97P. The ability of protein-Ca(2+) to rearrange 2-aminopurine-containing substrates was monitored by low energy CD. Although L97P and K93A/R100A retained the ability to unpair substrates, the cap mutants L111P and L130P did not. Taken together, these data challenge current assumptions related to 5'-nuclease family mechanism. Conserved basic amino acids are not required for double nucleotide unpairing and appear to act cooperatively, whereas the helical cap plays an unexpected role in hFEN1-substrate rearrangement.
Asunto(s)
Calcio/química , ADN/química , Endonucleasas de ADN Solapado/química , Sustitución de Aminoácidos , Calcio/metabolismo , ADN/genética , ADN/metabolismo , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Mutación Missense , Prolina , Estructura Secundaria de ProteínaRESUMEN
The structure- and strand-specific phosphodiesterase flap endonuclease-1 (FEN1), the prototypical 5'-nuclease, catalyzes the essential removal of 5'-single-stranded flaps during replication and repair. FEN1 achieves this by selectively catalyzing hydrolysis one nucleotide into the duplex region of substrates, always targeting the 5'-strand. This specificity is proposed to arise by unpairing the 5'-end of duplex to permit the scissile phosphate diester to contact catalytic divalent metal ions. Providing the first direct evidence for this, we detected changes induced by human FEN1 (hFEN1) in the low-energy CD spectra and fluorescence lifetimes of 2-aminopurine in substrates and products that were indicative of unpairing. Divalent metal ions were essential for unpairing. However, although 5'-nuclease superfamily-conserved active-site residues K93 and R100 were required to produce unpaired product, they were not necessary to unpair substrates. Nevertheless, a unique arrangement of protein residues around the unpaired DNA was detected only with wild-type protein, suggesting a cooperative assembly of active-site residues that may be triggered by unpaired DNA. The general principles of FEN1 strand and reaction-site selection, which depend on the ability of juxtaposed divalent metal ions to unpair the end of duplex DNA, may also apply more widely to other structure- and strand-specific nucleases.
Asunto(s)
ADN/metabolismo , Endonucleasas de ADN Solapado/química , 2-Aminopurina/química , Dominio Catalítico , ADN/química , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Humanos , Modelos Moleculares , Mutación , Conformación de Ácido NucleicoRESUMEN
OBJECTIVE: Insulin-like growth factor-I (IGF-I) and insulin are important vasoactive peptides but little is known about their effects in hypertension. DESIGN: We compared the responses of stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rat aortae to IGF-I and insulin. METHODS: Aortae were removed from WKY and SHRSP, cut into 2-3 mm rings, and contractile responses to phenylephrine and endothelin-1 studied in organ chambers in the presence of vehicle, IGF-I (0.1 micromol/l) or insulin (0.1 micromol/l). In addition, the effects of nitric oxide synthase (NOS) inhibition, phosphatidylinositol 3-kinase (PI3-kinase) inhibition and superoxide scavenging on these responses were investigated. RESULTS: Incubation with IGF-I and insulin caused attenuation of phenylephrine-induced and endothelin-1-induced vasoconstriction in arteries from normotensive but not hypertensive animals. In the arteries from WKY rats, co-incubation with either wortmannin or LY294002, inhibitors of PI3-kinase, attenuated the effect of IGF-I. The vasorelaxant effect of IGF-I was also abolished by removal of the endothelium or addition of the NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME). Co-incubation with tiron, a superoxide scavenger, suggested that the attenuation of IGF-I vasodilation in SHRSP arteries was not due to excess superoxide production. CONCLUSION: In WKY, IGF-I/insulin attenuate phenylephrine-mediated constrictions via PI3-kinase/nitric oxide pathways. In contrast, in SHRSP these pathways are dysfunctional and IGF-I has little effect on vascular responses.
Asunto(s)
Aorta Torácica/efectos de los fármacos , Hipertensión/fisiopatología , Factor I del Crecimiento Similar a la Insulina/farmacología , Insulina/farmacología , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Androstadienos/farmacología , Animales , Cromonas/farmacología , Endotelina-1/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Morfolinas/farmacología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Fenilefrina/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Especificidad de la Especie , Vasoconstrictores/farmacología , WortmaninaRESUMEN
OBJECTIVE: To determine the effects of the angiotensin II receptor antagonist irbesartan, the calcium-channel blocker amlodipine, and hydrochlorothiazide/hydralazine on superoxide, NAD(P)H oxidase and nitric oxide bioavailability in spontaneously hypertensive stroke-prone rats (SHRSP). METHODS: Drugs or vehicle were administered for 8 weeks to SHRSP and blood pressure was measured weekly by tail-cuff plethysmography. After 8 weeks, superoxide levels in carotid arteries and aortas were measured by lucigenin chemiluminescence and p22phox expression quantified by immunohistochemistry. In vitro the effects of exposure to drugs and vehicle for 30 min and 4 h on superoxide levels and nitric oxide bioavailability were examined. The latter was expressed as the increase in contractile responses of carotid arteries to phenylephrine in the presence of the nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester(l-NAME). RESULTS: In vivo irbesartan, amlodipine and hydrochlorothiazide/hydralazine produced similar falls in blood pressure, from 162 +/- 4 to 125 +/- 5, 132 +/- 4 and 131 +/- 6 mmHg, respectively, but irbesartan caused a greater reduction in superoxide and p22phox; superoxide levels in carotid arteries being 3.1 +/- 0.3, 1.1 +/- 0.2, 1.9 +/- 0.3 and 2.0 +/- 0.3 nmoles/mg per min, respectively. In vitro 4 h exposure to irbesartan decreased superoxide levels in the aorta from 2.08 +/- 0.68 to 1.48 +/- 0.62 nmoles/mg per min and increased nitric oxide bioavailability in carotid arteries. Neither 30 min incubation with irbesartan nor 4 h with amlodipine or hydrochlorothiazide/hydralazine altered superoxide levels. CONCLUSIONS: These studies support the hypothesis that AT1 receptor blockade has beneficial effects on superoxide production and nitric oxide bioavailability above that of other classes of antihypertensive agents. Reduced expression of components of the NAD(P)H oxidase may contribute to these effects.