RESUMO
The circadian clock coordinates the daily cyclic rhythm of numerous biological processes by regulating a large portion of the transcriptome. In animals, the circadian clock is involved in aging and senescence, and circadian disruption by mutations in clock genes frequently accelerates aging. Conversely, aging alters circadian rhythmicity, which causes age-associated physiological alterations. However, interactions between the circadian clock and aging have been rarely studied in plants. Here, we investigated potential roles for the circadian clock in the regulation of leaf senescence in plants. Members of the evening complex in Arabidopsis circadian clock, EARLY FLOWERING 3 (ELF3), EARLY FLOWERING 4 (ELF4), and LUX ARRHYTHMO (LUX), as well as the morning component PSEUDO-RESPONSE REGULATOR 9 (PRR9), affect both age-dependent and dark-induced leaf senescence. The circadian clock regulates the expression of several senescence-related transcription factors. In particular, PRR9 binds directly to the promoter of the positive aging regulator ORESARA1 (ORE1) gene to promote its expression. PRR9 also represses miR164, a posttranscriptional repressor of ORE1 Consistently, genetic analysis revealed that delayed leaf senescence of a prr9 mutant was rescued by ORE1 overexpression. Thus, PRR9, a core circadian component, is a key regulator of leaf senescence via positive regulation of ORE1 through a feed-forward pathway involving posttranscriptional regulation by miR164 and direct transcriptional regulation. Our results indicate that, in plants, the circadian clock and leaf senescence are intimately interwoven as are the clock and aging in animals.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Ritmo Circadiano/fisiologia , Fatores de Transcrição/fisiologia , Envelhecimento , Proteínas de Arabidopsis/genética , MicroRNAs/fisiologia , Folhas de Planta/fisiologia , Regiões Promotoras Genéticas , Fatores de Transcrição/genéticaRESUMO
Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS-PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min(-1) mg(-1) at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application.
Assuntos
Escherichia coli/genética , Protease de HIV/genética , HIV-1/enzimologia , Corpos de Inclusão/genética , Sequência de Aminoácidos , Sequência de Bases , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Clonagem Molecular/métodos , Eletroforese em Gel de Poliacrilamida , Infecções por HIV/virologia , Protease de HIV/química , Protease de HIV/isolamento & purificação , Protease de HIV/metabolismo , HIV-1/química , HIV-1/genética , Humanos , Dados de Sequência Molecular , Plasmídeos/genética , Redobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Ultradian rhythms have been proved to be critical for diverse biological processes. However, comprehensive understanding of the short-period rhythms remains limited. Here, we discover that leaf excision triggers a gene expression rhythm with ~3-h periodicity, named as the excision ultradian rhythm (UR), which is regulated by the plant hormone auxin. Promoter-luciferase analyses showed that the spatiotemporal patterns of the excision UR were positively associated with de novo root regeneration (DNRR), a post-embryonic developmental process. Transcriptomic analysis indicated more than 4,000 genes including DNRR-associated genes were reprogramed toward ultradian oscillation. Genetic studies showed that EXCISION ULTRADIAN RHYTHM 1 (EUR1) encoding ENHANCER OF ABSCISIC ACID CO-RECEPTOR1 (EAR1), an abscisic acid signaling regulator, was required to generate the excision ultradian rhythm and enhance root regeneration. The eur1 mutant exhibited the absence of auxin-induced excision UR generation and partial failure during rescuing root regeneration. Our results demonstrate a link between the excision UR and adventitious root formation via EAR1/EUR1, implying an additional regulatory layer in plant regeneration.