RESUMO
Agaves resist extreme heat and drought. In A. tequilana var. azul, the central spike of the rosette -containing the shoot apical meristem and folded leaves in early stages of development- is remarkably heat tolerant. We found that the most abundant protein in this organ is a 27 kDa protein. This protein was named mayahuelin to honor Mayáhuel, the agave goddess in the Aztec pantheon. LC-MS/MS analyses identified mayahuelin as a type I RIP (Ribosome Inactivating Protein). In addition to the spike, mayahuelin was expressed in the peduncle and in seeds, whereas in mature leaves, anthers, filaments, pistils, and tepals was absent. Anti-mayahuelin antibody raised against the A. tequilana var. azul protein revealed strong signals in spike leaves of A. angustifolia, A. bracteosa, A. rhodacantha, and A. vilmoriniana, and moderate signals in A. isthmensis, A. kerchovei, A. striata ssp. falcata, and A. titanota, indicating conservation at the protein level throughout the Agave genus. As in charybdin, a type I RIP characterized in Drimia maritima, mayahuelin from A. tequilana var. azul contains a natural aa substitution (Y76D) in one out of four aa comprising the active site. The RIP gene family in A. tequilana var. azul consists of at least 12 genes and Mayahuelin is the only member encoding active site substitutions. Unlike canonical plant RIPs, expression of Mayahuelin gene in S. cerevisiae did not compromise growth. The inhibitory activity of the purified protein on a wheat germ in vitro translation system was moderate. Mayahuelin orthologs from other Agave species displayed one of six alleles at Y76: (Y/Y, D/D, S/S, Y/D, Y/S, D/S) and proved to be useful markers for phylogenetic analysis. Homozygous alleles were more frequent in wild accessions whereas heterozygous alleles were more frequent in cultivars. Mayahuelin sequences from different wild populations of A. angustifolia and A. rhodacantha allowed the identification of accessions closely related to azul, manso, sigüín, mano larga, and bermejo varieties of A. tequilana and var. espadín of A. angustifolia. Four A. rhodacantha accessions and A. angustifolia var. espadín were closer relatives of A. tequilana var. azul than A. angustifolia wild accessions or other A. tequilana varieties.
RESUMO
Fructans are the main storage polysaccharides found in Agave species. The synthesis of these complex carbohydrates relies on the activities of specific fructosyltransferase enzymes closely related to the hydrolytic invertases. Analysis of Agave tequilana transcriptome data led to the identification of ESTs encoding putative fructosyltransferases and invertases. Based on sequence alignments and structure/function relationships, two different genes were predicted to encode 1-SST and 6G-FFT type fructosyltransferases, in addition, 4 genes encoding putative cell wall invertases and 4 genes encoding putative vacuolar invertases were also identified. Probable functions for each gene, were assigned based on conserved amino acid sequences and confirmed for 2 fructosyltransferases and one invertase by analyzing the enzymatic activity of recombinant Agave protein s expressed and purified from Pichia pastoris. The genome organization of the fructosyltransferase/invertase genes, for which the corresponding cDNA contained the complete open reading frame, was found to be well conserved since all genes were shown to carry a 9 bp mini-exon and all showed a similar structure of 8 exons/7 introns with the exception of a cell wall invertase gene which has 7 exons and 6 introns. Fructosyltransferase genes were strongly expressed in the storage organs of the plants, especially in vegetative stages of development and to lower levels in photosynthetic tissues, in contrast to the invertase genes where higher levels of expression were observed in leaf tissues and in mature plants.
Assuntos
Agave/enzimologia , Hexosiltransferases/metabolismo , Componentes Aéreos da Planta/enzimologia , Proteínas de Plantas/metabolismo , beta-Frutofuranosidase/metabolismo , Agave/genética , Sequência de Aminoácidos , Clonagem Molecular , Sequência Conservada , DNA Complementar/análise , DNA Complementar/biossíntese , Éxons , Frutanos/biossíntese , Hexosiltransferases/genética , Íntrons , Dados de Sequência Molecular , Filogenia , Pichia , Componentes Aéreos da Planta/genética , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Transcriptoma , beta-Frutofuranosidase/genéticaRESUMO
Bulbil formation in Agave tequilana was analysed with the objective of understanding this phenomenon at the molecular and cellular levels. Bulbils formed 14-45 d after induction and were associated with rearrangements in tissue structure and accelerated cell multiplication. Changes at the cellular level during bulbil development were documented by histological analysis. In addition, several cDNA libraries produced from different stages of bulbil development were generated and partially sequenced. Sequence analysis led to the identification of candidate genes potentially involved in the initiation and development of bulbils in Agave, including two putative class I KNOX genes. Real-time reverse transcription-PCR and in situ hybridization revealed that expression of the putative Agave KNOXI genes occurs at bulbil initiation and specifically in tissue where meristems will develop. Functional analysis of Agave KNOXI genes in Arabidopsis thaliana showed the characteristic lobed phenotype of KNOXI ectopic expression in leaves, although a slightly different phenotype was observed for each of the two Agave genes. An Arabidopsis KNOXI (knat1) mutant line (CS30) was successfully complemented with one of the Agave KNOX genes and partially complemented by the other. Analysis of the expression of the endogenous Arabidopsis genes KNAT1, KNAT6, and AS1 in the transformed lines ectopically expressing or complemented by the Agave KNOX genes again showed different regulatory patterns for each Agave gene. These results show that Agave KNOX genes are functionally similar to class I KNOX genes and suggest that spatial and temporal control of their expression is essential during bulbil formation in A. tequilana.
Assuntos
Agave/crescimento & desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Plantas/genética , Agave/anatomia & histologia , Agave/genética , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Diferenciação Celular/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/metabolismo , Meristema/genética , Meristema/metabolismo , Dados de Sequência Molecular , Fenótipo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismoRESUMO
Light-regulation of photosynthesis-associated nuclear genes is mediated by multipartite cis-regulatory units located in their promoter regions. The combination, spacing, and relative orientation of transcription factor binding sites in these units influences the assembly of specific multiprotein complexes that control gene expression. In this work, the functional architecture of the conserved modular array 5 (CMA5), a previously characterized minimal light-regulatory unit of rbcS gene promoters, has been analysed. With the aim of defining the sequences of CMA5 that, besides the I- and G-box elements, are essential for CMA5 responsiveness to light and chloroplast-derived signals, a series of mutations affecting the sequences flanking these elements was performed. It was found that an I-box associated module, named IbAM5, is essential for CMA5 functionality and is able to bind nuclear proteins in vitro. The spacing requirements of the I- and G-box elements in achieving adequate combinatorial interaction were also studied as well as the effect of interchanging the relative position of these elements in the native rbcS promoter arrangement. The results show that helical phasing and distance between the I- and G-box motifs are critical to determine the functionality and transcriptional strength of CMA5. Furthermore, it is shown that the relative position of the IbAM5/I-box composite element and the G-box element is not critical for the enhancer activity of CMA5, as long as the proper distance between them is maintained. Taken together, these results suggest that the light-responsive, plastid-dependent activity of CMA5 requires the synergistic interaction of several DNA-binding transcription factors assembled in a higher-order nucleoprotein complex.