ABSTRACT
Cyclotides are multifunctional plant cyclic peptides containing 28-37 amino acid residues and a pattern of three disulfide bridges, forming a motif known as the cyclic cystine knot. Due to their high biotechnological potential, the sequencing and characterization of cyclotide genes are crucial not only for cloning and establishing heterologous expression strategies, but also to understand local plant evolution in the context of host-pathogen relationships. Here, two species from the Brazilian Cerrado, Palicourea rigida (Rubiaceae) and Pombalia lanata (A.St.-Hil.) Paula-Souza (Violaceae), were used for cloning and characterizing novel cyclotide genes. Using 3' and 5' RACE PCR and sequencing, two full cDNAs, named parigidin-br2 (P. rigida) and hyla-br1 (P. lanata), were isolated and shown to have similar genetic structures to other cyclotides. Both contained the conserved ER-signal domain, N-terminal prodomain, mature cyclotide domain and a C-terminal region. Genomic sequencing of parigidin-br2 revealed two different gene copies: one intronless allele and one presenting a rare 131-bp intron. In contrast, genomic sequencing of hyla-br1 revealed an intronless gene-a common characteristic of members of the Violaceae family. Parigidin-br2 5' and 3' UTRs showed the presence of 12 putative candidate sites for binding of regulatory proteins, suggesting that the flanking and intronic regions of the parigidin-br2 gene must play important roles in transcriptional rates and in the regulation of temporal and spatial gene expression. The high degree of genetic similarity and structural organization among the cyclotide genes isolated in the present study from the Brazilian Cerrado and other well-characterized plant cyclotides may contribute to a better understanding of cyclotide evolution.
Subject(s)
Evolution, Molecular , Gene Expression Regulation, Plant/physiology , Peptides, Cyclic , Plant Proteins , Rubiaceae , Cloning, Molecular , DNA, Complementary , Peptides, Cyclic/biosynthesis , Peptides, Cyclic/genetics , Plant Proteins/biosynthesis , Plant Proteins/genetics , Rubiaceae/genetics , Rubiaceae/metabolism , South America , Species SpecificityABSTRACT
Zantedeschia aethiopica is an evergreen perennial plant cultivated worldwide and commonly used for ornamental and medicinal purposes including the treatment of bacterial infections. However, the current understanding of molecular and physiological mechanisms in this plant is limited, in comparison to other non-model plants. In order to improve understanding of the biology of this botanical species, RNA-Seq technology was used for transcriptome assembly and characterization. Following Z. aethiopica spathe tissue RNA extraction, high-throughput RNA sequencing was performed with the aim of obtaining both abundant and rare transcript data. Functional profiling based on KEGG Orthology (KO) analysis highlighted contigs that were involved predominantly in genetic information (37%) and metabolism (34%) processes. Predicted proteins involved in the plant circadian system, hormone signal transduction, secondary metabolism and basal immunity are described here. In silico screening of the transcriptome data set for antimicrobial peptide (AMP) -encoding sequences was also carried out and three lipid transfer proteins (LTP) were identified as potential AMPs involved in plant defense. Spathe predicted protein maps were drawn, and suggested that major plant efforts are expended in guaranteeing the maintenance of cell homeostasis, characterized by high investment in carbohydrate, amino acid and energy metabolism as well as in genetic information.
Subject(s)
Flowers/genetics , Flowers/metabolism , Transcriptome/genetics , Zantedeschia/genetics , Amino Acid Sequence , Anti-Infective Agents/pharmacology , Carrier Proteins/chemistry , Circadian Rhythm/genetics , Environment , Escherichia coli/drug effects , Flowers/drug effects , Gene Expression Regulation, Plant/drug effects , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/genetics , Ligands , Microbial Sensitivity Tests , Molecular Dynamics Simulation , Molecular Sequence Data , Plant Growth Regulators/metabolism , Plant Immunity/drug effects , Plant Immunity/genetics , Secondary Metabolism/drug effects , Secondary Metabolism/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Staphylococcus aureus/drug effects , Tissue Extracts , Transcription, Genetic/drug effects , Transcriptome/drug effects , Zantedeschia/drug effects , Zantedeschia/immunologyABSTRACT
Meloidogyne spp., plant-parasitic nematodes present worldwide, are intensively studied because of the damage caused to a large variety of agronomically important crops. Several reports indicate that proteins from the Meloidogyne spp. dorsal gland might play an important role to allow proper establishment of a functional nematode feeding site. The precise role of these proteins in the process of feeding cell development is unknown. To gain insights into the function of these secreted M. incognita proteins, we constitutively (ectopically) expressed the nematodes dorsal gland protein 7E12 in tobacco plants. It was found that the number of galls at 8 and 16 days after nematode infection was significantly higher in transgenic plants compared to control plants. Eggs from nematodes in transgenic plants hatched faster than those in control plants. Histological analysis of nematode induced galls in transgenic plants clearly shows a different morphology. Giant feeding cells harbor more vacuoles and an increased amount of cell wall invaginations, while neighboring cells surrounding feeding cells are more numerous. These results suggest that the presence of the 7E12 protein in tobacco accelerates gall formation. This assumption is supported by our data illustrating faster gall formation and egg eclosion in transgenic plants.
