RESUMO
In plants, the amino acids tyrosine and phenylalanine are synthesized from arogenate by arogenate dehydrogenase and arogenate dehydratase, respectively, with the relative flux to each being tightly controlled. Here the characterization of a maize opaque endosperm mutant (mto140), which also shows retarded vegetative growth, is described The opaque phenotype co-segregates with a Mutator transposon insertion in an arogenate dehydrogenase gene (zmAroDH-1) and this led to the characterization of the four-member family of maize arogenate dehydrogenase genes (zmAroDH-1-zmAroDH-4) which share highly similar sequences. A Mutator insertion at an equivalent position in AroDH-3, the most closely related family member to AroDH-1, is also associated with opaque endosperm and stunted vegetative growth phenotypes. Overlapping but differential expression patterns as well as subtle mutant effects on the accumulation of tyrosine and phenylalanine in endosperm, embryo, and leaf tissues suggest that the functional redundancy of this gene family provides metabolic plasticity for the synthesis of these important amino acids. mto140/arodh-1 seeds shows a general reduction in zein storage protein accumulation and an elevated lysine phenotype typical of other opaque endosperm mutants, but it is distinct because it does not result from quantitative or qualitative defects in the accumulation of specific zeins but rather from a disruption in amino acid biosynthesis.
Assuntos
Regulação da Expressão Gênica de Plantas , Prefenato Desidrogenase/genética , Prefenato Desidrogenase/metabolismo , Sementes , Zea mays/enzimologia , Zea mays/genética , Sequência de Aminoácidos , Aminoácidos/metabolismo , Endosperma/enzimologia , Endosperma/genética , Lisina/metabolismo , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Fenilalanina/biossíntese , Fenilalanina/genética , Fenilalanina/metabolismo , Prefenato Desidrogenase/química , Sementes/enzimologia , Sementes/genética , Sementes/metabolismo , Alinhamento de Sequência , Tirosina/metabolismo , Zea mays/classificação , Zea mays/metabolismo , Zeína/metabolismoRESUMO
Plants possess multiple genes encoding calcium sensor proteins that are members of the penta-EF-hand (PEF) family. Characterized PEF proteins such as ALG-2 (apoptosis-linked gene 2 product) and the calpain small subunit function in diverse cellular processes in a calcium-dependent manner by interacting with their target proteins at either their N-terminal extension or Ca2+ binding domains. We have identified a previously unreported class of PEF proteins in plants that are notable because they do not possess the hydrophobic amino acid rich N-terminal extension that is typical of these PEF proteins. We demonstrate that the maize PEF protein without the N-terminal extension has the characteristics of known PEF proteins; the protein binds calcium in the 100 nM range and, as a result of calcium binding, displays an increase in hydrophobicity. Characterization of the truncated maize PEF protein provides insights into the role of the N-terminal extension in PEF protein signaling. In the context of the current model of how PEF proteins are activated by calcium binding, these results demonstrate that this distinctive class of PEF proteins could function as calcium sensor proteins in plants even in the absence of the N-terminal extension.
Assuntos
Proteínas de Ligação ao Cálcio/química , Cálcio/química , Proteínas de Plantas/química , Zea mays/metabolismo , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/classificação , Proteínas de Ligação ao Cálcio/genética , Cátions Bivalentes/química , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Conformação Proteica , Análise de Sequência de Proteína , Deleção de Sequência , Triptofano/químicaRESUMO
Sequence similarity and profile searching tools were used to analyze the genome sequences of Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster for genes encoding three families of histone deacetylase (HDAC) proteins and three families of histone acetyltransferase (HAT) proteins. Plants, animals and fungi were found to have a single member of each of three subfamilies of the GNAT family of HATs, suggesting conservation of these functions. However, major differences were found with respect to sizes of gene families and multi-domain protein structures within other families of HATs and HDACs, indicating substantial evolutionary diversification. Phylogenetic analysis identified a new class of HDACs within the RPD3/HDA1 family that is represented only in plants and animals. A similar analysis of the plant-specific HD2 family of HDACs suggests a duplication event early in dicot evolution, followed by further diversification in the lineage leading to Arabidopsis. Of three major classes of SIR2-type HDACs that are found in animals, fungi have representatives only in one class, whereas plants have representatives only in the other two. Plants possess five CREB-binding protein (CBP)-type HATs compared with one to two in animals and none in fungi. Domain and phylogenetic analyses of the CBP family proteins showed that this family has evolved three distinct types of CBPs in plants. The domain architecture of CBP and TAF(II)250 families of HATs show significant differences between plants and animals, most notably with respect to bromodomain occurrence and their number. Bromodomain-containing proteins in Arabidopsis differ strikingly from animal bromodomain proteins with respect to the numbers of bromodomains and the other types of domains that are present. The substantial diversification of HATs and HDACs that has occurred since the divergence of plants, animals and fungi suggests a surprising degree of evolutionary plasticity and functional diversification in these core chromatin components.
Assuntos
Acetiltransferases/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Histona Desacetilases/genética , Filogenia , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição , Acetiltransferases/classificação , Acetiltransferases/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Proteína de Ligação a CREB , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Cromatina/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Proteínas Fúngicas/genética , Genoma de Planta , Histona Acetiltransferases , Histona Desacetilase 1 , Histona Desacetilases/classificação , Histona Desacetilases/fisiologia , Dados de Sequência Molecular , Proteínas Nucleares/genética , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/classificação , Proteínas de Saccharomyces cerevisiae/fisiologia , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Homologia de Sequência de Aminoácidos , Sirtuínas/genética , Transativadores/genética , Fatores de Transcrição TFII/genéticaRESUMO
The switch from vegetative to reproductive growth is marked by the termination of vegetative development and the adoption of floral identity by the shoot apical meristem (SAM). This process is called the floral transition. To elucidate the molecular determinants involved in this process, we performed genome-wide RNA expression profiling on maize (Zea mays) shoot apices at vegetative and early reproductive stages using massively parallel signature sequencing technology. Profiling revealed significant up-regulation of two maize MADS-box (ZMM) genes, ZMM4 and ZMM15, after the floral transition. ZMM4 and ZMM15 map to duplicated regions on chromosomes 1 and 5 and are linked to neighboring MADS-box genes ZMM24 and ZMM31, respectively. This gene order is syntenic with the vernalization1 locus responsible for floral induction in winter wheat (Triticum monococcum) and similar loci in other cereals. Analyses of temporal and spatial expression patterns indicated that the duplicated pairs ZMM4-ZMM24 and ZMM15-ZMM31 are coordinately activated after the floral transition in early developing inflorescences. More detailed analyses revealed ZMM4 expression initiates in leaf primordia of vegetative shoot apices and later increases within elongating meristems acquiring inflorescence identity. Expression analysis in late flowering mutants positioned all four genes downstream of the floral activators indeterminate1 (id1) and delayed flowering1 (dlf1). Overexpression of ZMM4 leads to early flowering in transgenic maize and suppresses the late flowering phenotype of both the id1 and dlf1 mutations. Our results suggest ZMM4 may play roles in both floral induction and inflorescence development.
Assuntos
Proteínas de Domínio MADS/fisiologia , Proteínas de Plantas/fisiologia , Zea mays/crescimento & desenvolvimento , Mapeamento Cromossômico , Cromossomos de Plantas , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Duplicação Gênica , Perfilação da Expressão Gênica , Glucuronidase/análise , Hibridização In Situ , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/análise , Reprodução/genética , Sintenia , Triticum/genética , Zea mays/genética , Zea mays/metabolismoRESUMO
Despite a good understanding of genes involved in oil biosynthesis in seed, the mechanism(s) that controls oil accumulation is still not known. To identify genes that control oil accumulation in seed, we have developed a simple screening method to isolate Arabidopsis seed oil mutants. The method includes an initial screen for seed density followed by a seed oil screen using an automated Nuclear Magnetic Resonance (NMR). Using this method, we isolated ten low oil mutants and one high oil mutant. The high oil mutant, p777, accumulated 8% more oil in seed than did wild type, but it showed no differences in seed size, plant growth or development. The high-oil phenotype is caused by the disruption of the GLABRA2 gene, a previously identified gene that encodes a homeobox protein required for normal trichome and root hair development. Knockout of GLABRA2 did not affect LEAFY COTYLEDON 1 and PICKLE expression in developing embryo. The result indicates that in addition to its known function in trichome and root hair development, GLABRA2 is involved in the control of seed oil accumulation.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/fisiologia , Óleos de Plantas/química , Adesivos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Northern Blotting , Southern Blotting , Escuridão , Genes Homeobox , Genes de Plantas , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Luz , Espectroscopia de Ressonância Magnética , Modelos Genéticos , Mutação , Fenótipo , Reguladores de Crescimento de Plantas , Proteínas de Plantas/química , Raízes de Plantas , Estruturas Vegetais , Plantas Geneticamente Modificadas , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , Sementes , Fatores de TempoRESUMO
Maize (Zea mays) endosperm consists of an epidermal-like surface layer of aleurone cells, an underlying body of starchy endosperm cells, and a basal layer of transfer cells. To determine whether surrounding maternal tissues perform a role in specifying endosperm cell fates, a maize endosperm organ culture technique was established whereby the developing endosperm is completely removed from surrounding maternal tissues. Using cell type-specific fluorescence markers, we show that aleurone cell fate specification occurs exclusively in response to surface position and does not require specific, continued maternal signal input. The starchy endosperm and aleurone cell fates are freely interchangeable throughout the lifespan of the endosperm, with internalized aleurone cells converting to starchy endosperm cells and with starchy endosperm cells that become positioned at the surface converting to aleurone cells. In contrast to aleurone and starchy endosperm cells, transfer cells fail to develop in in vitro-grown endosperm, supporting earlier indications that maternal tissue interaction is required to fully differentiate this cell type. Several parameters confirm that the maize endosperm organ cultures described herein retain the main developmental features of in planta endosperm, including fidelity of aleurone mutant phenotypes, temporal and spatial control of cell type-specific fluorescent markers, specificity of cell type transcripts, and control of mitotic cell divisions.
Assuntos
Sementes/crescimento & desenvolvimento , Zea mays/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Índice Mitótico , Dados de Sequência Molecular , Mutação , Fenótipo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/citologia , Sementes/citologia , Amido/metabolismo , Técnicas de Cultura de Tecidos , Zea mays/genéticaRESUMO
The pale aleurone color1 (pac1) locus, required for anthocyanin pigment in the aleurone and scutellum of the Zea mays (maize) seed, was cloned using Mutator transposon tagging. pac1 encodes a WD40 repeat protein closely related to anthocyanin regulatory proteins ANTHOCYANIN11 (AN11) (Petunia hybrida [petunia]) and TRANSPARENT TESTA GLABRA1 (TTG1) (Arabidopsis thaliana). Introduction of a 35S-Pac1 transgene into A. thaliana complemented multiple ttg1 mutant phenotypes, including ones nonexistent in Z. mays. Hybridization of Z. mays genomic BAC clones with the pac1 sequence identified an additional related gene, mp1. PAC1 and MP1 deduced protein sequences were used as queries to build a phylogenetic tree of homologous WD40 repeat proteins, revealing an ancestral gene duplication leading to two clades in plants, the PAC1 clade and the MP1 clade. Subsequent duplications within each clade have led to additional WD40 repeat proteins in particular species, with all mutants defective in anthocyanin expression contained in the PAC1 clade. Substantial differences in pac1, an11, and ttg1 mutant phenotypes suggest the evolutionary divergence of regulatory mechanisms for several traits that cannot be ascribed solely to divergence of the dicot and monocot protein sequences.
Assuntos
Antocianinas/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Plantas/genética , Sementes/genética , Zea mays/genética , Alelos , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Extensões da Superfície Celular/genética , Extensões da Superfície Celular/fisiologia , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Fenótipo , Filogenia , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetitivas de Aminoácidos/genética , Sequências Repetitivas de Aminoácidos/fisiologia , Sementes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Zea mays/metabolismoRESUMO
Seed-type vacuolar processing enzyme (VPE) activity is predicted to be essential for post-translational proteolysis of seed storage proteins in the protein storage vacuole of developing seeds. To test this hypothesis, we examined the protein profiles of developing and germinating seeds from Arabidopsis plants containing transposon-insertional knockout mutations in the genes that encode the two seed-type VPEs in Arabidopsis, betaVPE, which was identified previously, and deltaVPE, which is described here. The effects of these mutations were studied individually in single mutants and together in a double mutant. Surprisingly, we found that most of the seed protein still was processed proteolytically in seed-type VPE mutants. The minor differences observed in polypeptide accumulation between wild-type and betaVPE mutant seeds were characterized using a two-dimensional gel/mass spectrometric analysis approach. The results showed increased amounts of propolypeptide forms of legumin-type globulins accumulating in mutant seeds. However, the majority of protein (>80%) still was processed to mature alpha- and beta-chains, as observed in wild-type seeds. Furthermore, we identified several legumin-type globulin polypeptides, not corresponding to pro or mature forms, that increased in accumulation in betaVPE mutant seeds compared with wild-type seeds. Together, these results indicate the existence of both redundant and alternative processing activities in seeds. The latter was substantiated by N-terminal sequencing of a napin-type albumin protein, indicating cleavage consistent with previous in vitro studies using purified aspartic protease. Analysis of genome-wide transcript profiling data sets identified six protease genes (including an aspartic protease gene and betaVPE) that shared spatial and temporal expression patterns with seed storage proteins. From these results, we conclude that seed-type VPEs constitute merely one pathway for processing seed storage protein and that other proteolytic enzymes also can process storage proteins into chains capable of stable accumulation in mature seeds.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Sementes/enzimologia , Vacúolos/enzimologia , Sequência de Aminoácidos , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Ensaio Cometa , Eletroforese em Gel Bidimensional , Germinação/genética , Espectrometria de Massas , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes/crescimento & desenvolvimentoRESUMO
Histone proteins play a central role in chromatin packaging, and modification of histones is associated with chromatin accessibility. SET domain [Su(var)3-9, Enhancer-of-zeste, Trithorax] proteins are one class of proteins that have been implicated in regulating gene expression through histone methylation. The relationships of 22 SET domain proteins from maize (Zea mays) and 32 SET domain proteins from Arabidopsis were evaluated by phylogenetic analysis and domain organization. Our analysis reveals five classes of SET domain proteins in plants that can be further divided into 19 orthology groups. In some cases, such as the Enhancer of zeste-like and trithorax-like proteins, plants and animals contain homologous proteins with a similar organization of domains outside of the SET domain. However, a majority of plant SET domain proteins do not have an animal homolog with similar domain organization, suggesting that plants have unique mechanisms to establish and maintain chromatin states. Although the domains present in plant and animal SET domain proteins often differ, the domains found in the plant proteins have been generally implicated in protein-protein interactions, indicating that most SET domain proteins operate in complexes. Combined analysis of the maize and Arabidopsis SET domain proteins reveals that duplication of SET domain proteins in plants is extensive and has occurred via multiple mechanisms that preceded the divergence of monocots and dicots.