RESUMEN
Ribosome-inactivating proteins are plant cytotoxic enzymes, also present in fungi, algae and bacteria, mainly known for their ability to inhibit protein synthesis. We previously purified and structurally characterized three type 1 RIPs (PD-S1-3) from Phytolacca dioica seeds and four type 1 RIPs (PD-L1-4) from adult plant leaves. Two additional RIPs, named dioicin 1 and dioicin 2, were isolated from leaves of young plants and developing leaves of adult plants. The evidence that P. dioica synthesizes and accumulates these RIPs isoforms suggests that these proteins have been conserved during evolution. Though several aspects of P. dioica type 1 RIP characterization have been studied, some important questions remain to be answered especially with respect to Phytolaccaceae RIP evolution. One of the major problems encountered in approaching RIPs phylogeny concerns the availability of their sequences. In this study, we report the characterization of biological and structural properties of dioicin 1, including the determination of its primary structure by using a combined approach based on Edman degradation, de novo sequencing by ESI-Q-TOF-MS/MS and peptide mapping by MALDI-TOF MS. Knowledge of dioicin 1 primary structure provide us a mean to deepen Phytolaccaceae's RIPs phylogeny. We speculate that both dioicins 1 and 2 share common ancestors with PAP-II and PAP icos-II and that dioicin 1 is not closely related to other members of this clade, thus shedding lights on evolutionary relationships among type 1 RIPs from Phytolaccaceae.
Asunto(s)
Filogenia , Phytolacca/química , Proteínas de Plantas/química , Proteínas Inactivadoras de Ribosomas Tipo 1/química , Secuencia de Aminoácidos , Dicroismo Circular , Espectrometría de Masas , Datos de Secuencia Molecular , Proteínas Inactivadoras de Ribosomas Tipo 1/clasificación , Homología de Secuencia de AminoácidoRESUMEN
Saporins are type 1 ribosome-inactivating proteins (RIPs: EC 3.2.2.22) produced in various organs of Saponaria officinalis L. Two distinct saporin types, saporin-L and saporin-S isoforms, were respectively purified from the intra- and extra-cellular fractions of soapwort leaves. The saporin-L isoform was lowly identical, differed for toxicity, molecular mass and amino acid composition from saporin-S proteins forming a new monophyletic group. Genes encoding both L- and S-type isoforms were cloned from leaf-specific cDNA library; the encoded products included the N-terminal diversity observed by protein sequencing and showed compatible weights with those from mass spectra. These genes were intron-less belonging to small gene families. Reverse transcription polymerase chain reaction/quantitative reverse transcription polymerase chain reaction experiments evidenced their differential expression during leaf development, wounding and abscisic acid treatment. These results suggest that the saporin-L and -S proteins may play diversified roles during stress responses.
Asunto(s)
Ácido Abscísico/farmacología , Perfilación de la Expresión Génica , Hojas de la Planta/genética , Proteínas de Plantas/genética , Proteínas Inactivadoras de Ribosomas Tipo 1/genética , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Datos de Secuencia Molecular , Filogenia , Reguladores del Crecimiento de las Plantas/farmacología , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Inactivadoras de Ribosomas Tipo 1/clasificación , Proteínas Inactivadoras de Ribosomas Tipo 1/metabolismo , Saponaria/genética , Saponaria/crecimiento & desarrollo , Saponaria/metabolismo , Saporinas , Homología de Secuencia de Aminoácido , Estrés MecánicoRESUMEN
Ribosome-inactivating proteins (RIPs) are N-glycosidases that inhibit protein synthesis by depurinating rRNA. Despite their identification more than 25 years ago, little is known about their biological functions. Here, we report a genome-wide identification of the RIP family in rice based on the complete genome sequence analysis. Our data show that rice genome encodes at least 31 members of this family and they all belong to type 1 RIP genes. This family might have evolved in parallel to species evolution and genome-wide duplications represent the major mechanism for this family expansion. Subsequently, we analyzed their expression under biotic (bacteria and fungus infection), abiotic (cold, drought and salinity) and the phytohormone ABA treatment. These data showed that some members of this family were expressed in various tissues with differentiated expression abundances whereas several members showed no expression under normal growth conditions or various environmental stresses. On the other hand, the expression of many RIP members was regulated by various abiotic and biotic stresses. All these data suggested that specific members of the RIP family in rice might play important roles in biotic and abiotic stress-related biological processes and function as a regulator of various environmental cues and hormone signaling. They may be potentially useful in improving plant tolerance to various abiotic and biotic stresses by over-expressing or suppressing these genes.