RESUMEN
Divergent architecture of shoot models in flowering plants reflects the pattern of production of vegetative and reproductive organs from the apical meristem. The SELF-PRUNING (SP) gene of tomato is a member of a novel CETS family of regulatory genes (CEN, TFL1, and FT) that controls this process. We have identified and describe here several proteins that interact with SP (SIPs) and with its homologs from other species: a NIMA-like kinase (SPAK), a bZIP factor, a novel 10-kD protein, and 14-3-3 isoforms. SPAK, by analogy with Raf1, has two potential binding sites for 14-3-3 proteins, one of which is shared with SP. Surprisingly, overexpression of 14-3-3 proteins partially ameliorates the effect of the sp mutation. Analysis of the binding potential of chosen mutant SP variants, in relation to conformational features known to be conserved in this new family of regulatory proteins, suggests that associations with other proteins are required for the biological function of SP and that ligand binding and protein-protein association domains of SP may be separated. We suggest that CETS genes encode a family of modulator proteins with the potential to interact with a variety of signaling proteins in a manner analogous to that of 14-3-3 proteins.
Asunto(s)
Proteínas de Plantas/metabolismo , Proteínas de Saccharomyces cerevisiae , Solanum lycopersicum/metabolismo , Proteínas 14-3-3 , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Sitios de Unión/genética , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/genética , Datos de Secuencia Molecular , Mutagénesis , Mutación , Fenotipo , Proteínas de Plantas/genética , Tallos de la Planta/genética , Tallos de la Planta/metabolismo , Plantas Modificadas Genéticamente , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Scrophulariaceae/genética , Scrophulariaceae/metabolismo , Transducción de Señal , Especificidad de la Especie , Transactivadores/genética , Transactivadores/metabolismo , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Vegetative and reproductive phases alternate regularly during sympodial growth in tomato. In wild-type 'indeterminate' plants, inflorescences are separated by three vegetative nodes. In 'determinate' plants homozygous for the recessive allele of the SELF-PRUNING (SP) gene, sympodial segments develop progressively fewer nodes until the shoot is terminated by two consecutive inflorescences. We show here that the SP gene is the tomato ortholog of CENTRORADIALIS and TERMINAL FLOWER1, genes which maintain the indeterminate state of inflorescence meristems in Antirrhinum and Arabidopsis respectively. The sp mutation results in a single amino acid change (P76L), and the mutant phenotype is mimicked by overexpressing the SP antisense RNA. Ectopic and overexpression of the SP and CEN transgenes in tomato rescues the 'indeterminate' phenotype, conditions the replacement of flowers by leaves in the inflorescence and suppresses the transition of the vegetative apex to a reproductive shoot. The SELF-PRUNING gene is expressed in shoot apices and leaves from very early stages, and later in inflorescence and floral primordia as well. This expression pattern is similar to that displayed by the tomato ortholog LEAFY and FLORICAULA. Comparison of the sympodial, day-neutral shoot system of tomato and the monopodial, photoperiod-sensitive systems of Arabidopsis and Antirrhinum suggests that flowering genes that are required for the processing of floral induction signals in Arabidopsis and Antirrhinum are required in tomato to regulate the alternation between vegetative and reproductive cycles in sympodial meristems.
Asunto(s)
Proteínas de Arabidopsis , Genes de Plantas , Meristema/crecimiento & desarrollo , Proteínas de Plantas/genética , Brotes de la Planta/crecimiento & desarrollo , Solanum lycopersicum/genética , Factores de Transcripción , Secuencia de Aminoácidos , Secuencia de Bases , Expresión Génica , Solanum lycopersicum/crecimiento & desarrollo , Meristema/anatomía & histología , Modelos Biológicos , Datos de Secuencia Molecular , Morfogénesis/genética , Mutación , Fenotipo , Brotes de la Planta/anatomía & histología , Plantas Modificadas Genéticamente , Homología de Secuencia de AminoácidoRESUMEN
The Curl (Cu) and Mouse-ear (Me) mutations of tomato cause two seemingly unrelated developmental syndromes with a wide range of pleiotropic phenotypes. Yet, the distinct morphogenic alterations in shoots, leaves, and inflorescences conferred by the two mutations appear to be caused by unchecked meristematic activity that characterizes dominant mutations in Knotted1 (Kn1)-like genes of monocot plants. We have been unable to separate the two closely linked Cu and Me mutations, and they may lie in the same gene. A homeobox-containing class I Kn1-like gene, TKn2, also maps to the same location. Significantly, the dominant mutations are associated with two aberrant modes of TKn2 transcription. Overexpression of the two in-frame wild-type transcripts of TKn2 is associated with the Cu mutation, whereas misexpression of an abundant and oversized fusion mRNA is associated with the Me mutation. Available molecular evidence strongly suggests that the defective Me-TKn2 transcript is generated via a novel splicing event that merges transcripts of two closely linked genes. The translated fusion product is comprised of most of the 5' end of the adjacent PPi-dependent fructose 6-phosphate phosphotransferase (PFP) transcript spliced in-frame to coding position 64 of the TKn2 transcript, leaving the TKn2 homeobox intact. We suggest that class I Kn1-like genes were selected early during evolution to regulate basic programs of aerial meristems and that subtle alterations in their function may be the basis for the wide diversity in growth parameters of shoot systems, leaves, and inflorescences among plant species.
Asunto(s)
Genes de Plantas , Mutación , Solanum lycopersicum/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN/genética , ADN de Plantas/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes Homeobox , Proteínas de Homeodominio/genética , Solanum lycopersicum/crecimiento & desarrollo , Meristema/genética , Meristema/crecimiento & desarrollo , Datos de Secuencia Molecular , Fenotipo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Plantas Tóxicas , Reacción en Cadena de la Polimerasa , Nicotiana/genética , Nicotiana/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
The most distinctive morphogenetic feature of leaves is their being either simple or compound. To study the basis for this dichotomy, we have exploited the maize homeobox-containing Knotted-1 (Kn1) gene in conjunction with mutations that alter the tomato compound leaf. We show that misexpression of Kn1 confers different phenotypes on simple and compound leaves. Up to 2000 leaflets, organized in compound reiterated units, are formed in tomato leaves expressing Kn1. In contrast, Kn1 induces leaf malformations but fails to elicit leaf ramification in plants with inherent simple leaves such as Arabidopsis or in tomato mutant plants with simple leaves. Moreover, the tomato Kn1 ortholog, unlike that of Arabidopsis, is expressed in the leaf primordia. Presumably, the two alternative leaf forms are conditioned by different developmental programs in the primary appendage that is common to all types of leaves.
Asunto(s)
Genes de Plantas , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Solanum lycopersicum/crecimiento & desarrollo , Solanum lycopersicum/genética , Secuencia de Aminoácidos , Clonación Molecular , ADN Complementario , Expresión Génica , Genes Homeobox , Proteínas de Homeodominio/química , Hibridación in Situ , Datos de Secuencia Molecular , Morfogénesis , Fenotipo , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/química , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Zea mays/genéticaRESUMEN
The gene coding for threonine deaminase (TD), the enzyme which catalyzes the first committed step in the biosynthesis of isoleucine, was isolated from tomato as a consequence of its unusual 500-fold upregulation in floral organs. It was subsequently shown that TD is induced in potato leaves in response to wounding, abscisic acid and methyl jasmonate (MeJa). Detailed analysis presented here, reveals an intricate developmental regulation pattern of gene expression in flowers that is operating solely in parenchyma territories. Yet, despite its high pre-existing expression level, TD in flowers can be further induced by MeJa. Induction of TD in flowers as well as in leaves is effective only in the parenchyma domains, irrespective of the prior expression levels. TD is neither expressed nor induced in epidermal, vascular or sporogenous tissues. Promoter analysis in transgenic tomato plants indicates that induction of TD follows identical kinetics in flowers and leaves. Furthermore, the 'conditioning' of developmental upregulation in flowers, the response to MeJa in flowers and leaves, and the parenchyma-specific expression are all mediated by the cis-elements within the proximal 192 bp of the promoter. Promoter elements regulating the correct organ-specific expression are located, however, further upstream. The promoter constructs used in this study can serve as useful tools for expressing extremely high levels of transgenes in specific cells. A scheme explaining tissue-specific response to MeJa, in conjunction with developmental control, is discussed.
Asunto(s)
Acetatos/metabolismo , Aminoácidos/biosíntesis , Ciclopentanos/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Regiones Promotoras Genéticas , Solanum lycopersicum/fisiología , Treonina Deshidratasa/biosíntesis , Treonina Deshidratasa/genética , Secuencia de Bases , Glucuronidasa/biosíntesis , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Datos de Secuencia Molecular , Oxilipinas , Hojas de la Planta , Mapeo Restrictivo , Solanum tuberosum/metabolismo , TATA BoxRESUMEN
To understand the details of the homeotic systems that govern flower development in tomato and to establish the ground rules for the judicious manipulation of this floral system, we have isolated the tomato AGAMOUS gene, designated TAG1, and examined its developmental role in antisense and sense transgenic plants. The AGAMOUS gene of Arabidopsis is necessary for the proper development of stamens and carpels and the prevention of indeterminate growth of the floral meristem. Early in flower development, TAG1 RNA accumulates uniformly in the cells fated to differentiate into stamens and carpels and later becomes restricted to specific cell types within these organs. Transgenic plants that express TAG1 antisense RNA display homeotic conversion of third whorl stamens into petaloid organs and the replacement of fourth whorl carpels with pseudocarpels bearing indeterminate floral meristems with nested perianth flowers. A complementary phenotype was observed in transgenic plants expressing the TAG1 sense RNA in that first whorl sepals were converted into mature pericarpic leaves and sterile stamens replaced the second whorl petals.
Asunto(s)
Proteínas de Unión al ADN/genética , Genes Homeobox , Genes de Plantas , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Verduras/genética , Proteína AGAMOUS de Arabidopsis , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Proteínas de Unión al ADN/biosíntesis , Expresión Génica , Datos de Secuencia Molecular , Proteínas de Plantas/biosíntesis , ARN sin Sentido/biosíntesis , Homología de Secuencia de AminoácidoRESUMEN
The tomato MADS box gene no. 5 (TM5) is shown here to be expressed in meristematic domains fated to form the three inner whorls-petals, stamens, and gynoecia-of the tomato flower. TM5 is also expressed during organogenesis and in the respective mature organs of these three whorls. This is unlike the major organ identity genes of the MADS box family from Antirrhinum and Arabidopsis, which function in overlapping primordial territories consisting of only two floral whorls each. The developmental relevance of the unique expression pattern of this putative homeotic gene was examined in transgenic plants. In agreement with the expression patterns, antisense RNA of the TM5 gene conferred both early and late alterations of morphogenetic markers. Early defects consist of additional whorls or of a wrong number of organs per whorl. Late, organ-specific changes include evergreen, cauline, and unabscised petals; green, dialytic, and sterile anthers; and sterile carpels and defective styles on which glandular trichomes characteristic of sepals and petals are ectopically formed. However, a complete homeotic transformation of either organ was not observed. The early and late floral phenotypes of TM5 antisense plants suggest that TM5 mediates two unrelated secondary regulatory systems. One system is the early function of the floral meristem identity genes, and the other system is the function of the major floral organ identity genes.
RESUMEN
A gene coding for a polypeptide abundant in tomato floral meristems was isolated and shown to represent a tomato 66.3-kD polyphenoloxidase. Analysis of cDNA clones and a corresponding intronless genomic clone indicated that the plastid-bound 587-residue-long polypeptide, designated P2, contains two conserved copper-binding domains, similar to those found in fungal and mammalian tyrosinases. P2 transcripts and polypeptides are accumulated in the arrested floral primordia of the anantha mutant inflorescences and are equally abundant in primordia of wild-type flowers; the gene continues to be expressed at high levels in developing floral organs. In young expanding leaves, P2 protein is concentrated in palisade cells and in epidermal trichomes. Expression patterns of P2 in plant meristems permit molecular distinction between floral and vegetative primordia, and, in a companion study, comparison with dUTPase suggests that the two genes mark two alternative complementary developmental programs in the floral and vegetative meristems of the tomato plants.
Asunto(s)
Catecol Oxidasa/genética , Plantas/enzimología , Plantas/genética , Secuencia de Bases , Catecol Oxidasa/metabolismo , Mapeo Cromosómico , ADN/genética , Expresión Génica , Inmunohistoquímica , Datos de Secuencia Molecular , Monofenol Monooxigenasa/genética , Desarrollo de la Planta , Homología de Secuencia de Ácido NucleicoRESUMEN
A meristem-specific gene coding for deoxyuridine triphosphatase (EC 3.6.1.23) (dUTPase) in tomato was isolated, and its developmental expression in vegetative and floral apices was monitored. An 18-kD polypeptide, P18, was isolated as a consequence of its accumulation in arrested floral meristems of anantha mutant plants. The corresponding cDNA isolated from an expression library exhibited a 40 to 60% similarity with the pseudoprotease sequences of poxviruses, genes that have been suggested to encode dUTPases. Enzymatic tests and conservation of peptide motifs common to bacterial and viral genes verified that the P18 cDNA clone indeed represents a eukaryotic dUTPase. Immunogold localization and in situ hybridization experiments showed that polypeptides and transcripts of dUTPase are maintained at high levels in apical meristematic cells of vegetative and floral meristems. dUTPase gene activity is also high in the potentially meristematic cells of the provascular and vascular system. Its expression is lower in the immediate parenchymal derivatives of the apical meristematic cells, and this downregulation marks, perhaps, the transition from totipotency to the first differentiated state.
Asunto(s)
Plantas/enzimología , Plantas/genética , Pirofosfatasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN/genética , Expresión Génica , Inmunohistoquímica , Datos de Secuencia Molecular , Desarrollo de la Planta , Pirofosfatasas/metabolismoRESUMEN
The gene encoding the plant biosynthetic threonine deaminase (Td; EC 4.2.1.16) has been cloned as a result of its unusual upregulation in tomato flowers. The Td gene of tomato encodes a polypeptide of 595 residues, the first 80 of which comprise a putative two-domain transit peptide cleaved at position 51. Comparison of the amino acid sequence with the corresponding enzymes from yeast and bacteria reveals a near identity of the important catalytic regions and greater than 40% overall similarity. The Td gene is unique in the tomato genome and its coding region is interrupted by eight introns. Its expression is greater than 50-fold higher in sepals and greater than 500-fold higher in the rest of the flower than in leaves or roots. Its overexpression, however, is strictly confined to the parenchymal cells of the floral organs. In young tomato leaves, the chloroplast-bound enzyme is found almost exclusively in the subepidermal spongy mesophyll cells.
Asunto(s)
Genes de Plantas , Genes Sintéticos , Fenómenos Fisiológicos de las Plantas , Treonina Deshidratasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Biblioteca de Genes , Datos de Secuencia Molecular , Plantas/enzimología , Plantas/genética , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , TATA Box , Transcripción GenéticaRESUMEN
The molecular evolution and patterns of conservation of clones from four Y chromosome lampbrush loops of Drosophila hydei were investigated. Each loop contains a discrete family of transcribed repeats that are only slightly conserved even in the hydei subgroup species. Sequencing of clones from the four D. hydei loops indicates that all transcribed repeats evolved from A+T-rich elements of the genome. Evidence is presented that suggests a Y-specific family evolved as a result of the transposition of repeated sequences from an autosomal site to the Y chromosome with the concomitant acquisition of transcriptional activity and loss of non-Y sequences. The results support a structural role for the loops in shaping a spermatocyte-specific nuclear organization. Transcribed heterochomatic sequences could play a similar role in nuclear organization in many cell types.
RESUMEN
Using loop-specific DNA clones, we established that the T(X:Y)56 (Hackstein and Hennig 1982) chromosome, formerly thought to be deleted for the Yshort arm and the associated 'nooses' loops is actually an XYS X YL combination. It carries, adjacent to the translocation junction, the complete and uninterrupted set of the two dysfunctional 'nooses' domains. The morphologically altered and functionally defective loops are transcribed at about 50% of the normal rate. Transcripts in one of the two 'nooses' domains are preferentially underrepresented and their distribution in the spermatocyte nucleus is distorted, presumably as a consequence of a spreading effect. No alteration in transcript size or in the correct strand selection, and no variegation of transcription on the single spermatocyte level, were observed. In another translocation T(X:Y)97, in which 'tubular ribbons' were reported to be inactivated (Hess 1970), complete elimination of DNA sequences is observed. A possible mechanism for the position effect inactivation of Y loops in X:Y translocations is discussed briefly.
Asunto(s)
Drosophila/genética , Aberraciones Cromosómicas Sexuales/veterinaria , Translocación Genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Femenino , Regulación de la Expresión Génica , Masculino , Hibridación de Ácido Nucleico , Aberraciones Cromosómicas Sexuales/genética , Espermatocitos/ultraestructura , Espermatogénesis , Transcripción Genética , Cromosoma X/ultraestructura , Cromosoma Y/ultraestructuraRESUMEN
Drosophila hydei clones representing transcribed middle-repetitive sequences from four of six major lampbrush loops of the Y chromosome were isolated. Sequences homologous to each clone are clustered in a particular locus on the Y chromosome, but additional euchromatic sites were found for one of the transcribed clones. In situ hybridization to lampbrush-loops RNA permitted the identification of clones homologous with the two "nooses" loops on YS and with the "clubs" and "tubular ribbons" on the YL arm. Loop-specific nuclear RNA molecules range in size from 10S to 60S. Loop RNA is accumulated in the nucleus and remains attached to the loops during the course of primary spermatocyte growth. It disappears, however, along with the loop structures, during the first meiotic prophase. The structure and function of the Y chromosome and its lampbrush loops are briefly considered in the light of these findings.
Asunto(s)
Drosophila/genética , Cromosomas Sexuales/inmunología , Cromosoma Y/inmunología , Animales , Mapeo Cromosómico , Clonación Molecular , Masculino , Metafase , ARN/genética , Transcripción Genética , Cromosoma Y/ultraestructuraRESUMEN
We sought for cloned sequences of middle repetitive (MR) complexity that mark obligatory heterochromatic regions. Total genome probes were employed in a differential screening procedure to recover X-specific, Y-specific and autosomal specific heterochromatic sequences. X-and Y-linked sequences were recovered in the same experiment. (Y-linked clones will be described elsewhere). All nine independent, non-identical X-specific clones were found to be partially homologous to one another and to type I rDNA insertion. No other X-specific Bam HI or HindIII clones were found. In situ hybridization to normal and inverted chromosomes revealed extensive homology in the heterochromatin spanning the nucleolus organizer (NOR) and the eu-heterochromatin junction. Eleven clones which are underrepresented in polytene chromosomes were selected in another differential screening. None was autosome-specific. Five were of nucleolar origin. Among them a presumptive type II 28SrDNA insertion sequence was clearly localized within the X-chromosome proximal heterochromatin in addition to the known localization of the X and Y nucleolar organizers. We mapped three clones to major sites on the Y chromosome and to secondary autosomal sites. The results are discussed with regard to the complexity of heterochromatin organization.
Asunto(s)
Drosophila melanogaster/genética , Heterocromatina/fisiología , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Clonación Molecular , ADN/genética , Femenino , Masculino , Cromosoma X , Cromosoma YRESUMEN
Localization, as detected by in situ hybridization, of major heterochromatic blocks in interphase nuclei of larval brain and imaginal discs is reported. We conclude that the position of heterochromatic regions in interphase nuclei is correlated with their respective position in metaphase chromosomes and hence, independent of sequence recognition. Furthermore, chromocentral associations of X-, Y- or autosomal-based heterochromatin are not formed in these cells. Homologues do align in close proximity, but heterochromatin plays no role in this arrangement. Heterochromatin, and probably nucleoli, establish their membrane links in situ, and have no prefixed recognition sites. The most intimate association between homologous repetitive sequences was found in the histone locus, but no tendency for clustering was found among loci of multisite euchromatic gene families.
Asunto(s)
Drosophila melanogaster/genética , Heterocromatina/fisiología , Histonas/genética , Animales , Nucléolo Celular/fisiología , Clonación Molecular , Genes , Interfase , Hibridación de Ácido Nucleico , ARN Ribosómico/genéticaRESUMEN
Cytological studies of the developing mycelium of Claviceps suggest that genetic uniformity may be obtained in strains derived from single spores.