RESUMEN
Fruit softening, an irreversible process that occurs during fruit ripening, can lead to losses and waste during postharvest transportation and storage. Cell wall disassembly is the main factor leading to loss of fruit firmness, and several ripening-associated cell wall genes have been targeted for genetic modification, particularly pectin modifiers. However, individual knockdown of most cell wall-related genes has had minimal influence on cell wall integrity and fruit firmness, with the notable exception of pectate lyase. Compared to pectin disassembly, studies of the cell wall matrix, the xyloglucan-cellulose framework, and underlying mechanisms during fruit softening are limited. Here, a tomato (Solanum lycopersicum) fruit ripening-associated α-expansin (SlExpansin1/SlExp1) and an endoglucanase (SlCellulase2/SlCel2), which function in the cell wall matrix, were knocked out individually and together using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9-mediated genome editing. Simultaneous knockout of SlExp1 and SlCel2 enhanced fruit firmness, reduced depolymerization of homogalacturonan-type pectin and xyloglucan, and increased cell adhesion. In contrast, single knockouts of either SlExp1 or SlCel2 did not substantially change fruit firmness, while simultaneous overexpression of SlExp1 and SlCel2 promoted early fruit softening. Collectively, our results demonstrate that SlExp1 and SlCel2 synergistically regulate cell wall disassembly and fruit softening in tomato.
Asunto(s)
Celulasa , Solanum lycopersicum , Frutas/metabolismo , Solanum lycopersicum/genética , Celulasa/genética , Celulasa/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pectinas/metabolismo , Pared Celular/metabolismoRESUMEN
The Cuscuta genus comprises obligate parasitic plants that have an unusually wide host range. Whether Cuscuta uses different infection strategies for different hosts or whether the infection strategy is mechanistically and enzymatically conserved remains unknown. To address this, we investigated molecular events during the interaction between field dodder (Cuscuta campestris) and two host species of the Solanum genus that are known to react differently to parasitic infection. We found that host gene induction, particularly of cell wall fortifying genes, coincided with a differential induction of genes for cell wall degradation in the parasite in the cultivated tomato (Solanum lycopersicum) but not in a wild relative (Solanum pennellii). This indicates that the parasite can adjust its gene expression in response to its host. This idea was supported by the increased expression of C. campestris genes encoding an endo-ß-1,4-mannanase in response to exposure of the parasite to purified mono- and polysaccharides in a host-independent infection system. Our results suggest multiple key roles of the host cell wall in determining the outcome of an infection attempt.
Asunto(s)
Cuscuta , Parásitos , Solanum lycopersicum , Solanum , Animales , Cuscuta/genética , Interacciones Huésped-Parásitos/genética , Solanum lycopersicum/genética , Solanum/genética , Expresión GénicaRESUMEN
Cell fate maintenance is an integral part of plant cell differentiation and the production of functional cells, tissues, and organs. Fleshy fruit development is characterized by the accumulation of water and solutes in the enlarging cells of parenchymatous tissues. In tomato (Solanum lycopersicum), this process is associated with endoreduplication in mesocarp cells. The mechanisms that preserve this developmental program, once initiated, remain unknown. We show here that analysis of a previously identified tomato ethyl methanesulfonate-induced mutant that exhibits abnormal mesocarp cell differentiation could help elucidate determinants of fruit cell fate maintenance. We identified and validated the causal locus through mapping-by-sequencing and gene editing, respectively, and performed metabolic, cellular, and transcriptomic analyses of the mutant phenotype. The data indicate that disruption of the SlGBP1 gene, encoding GUANYLATE BINDING PROTEIN1, induces early termination of endoreduplication followed by late divisions of polyploid mesocarp cells, which consequently acquire the characteristics of young proliferative cells. This study reveals a crucial role of plant GBPs in the control of cell cycle genes, and thus, in cell fate maintenance. We propose that SlGBP1 acts as an inhibitor of cell division, a function conserved with the human hGBP-1 protein.
Asunto(s)
Frutas/citología , Frutas/crecimiento & desarrollo , Proteínas de Plantas/genética , Solanum lycopersicum/citología , Sistemas CRISPR-Cas , Ciclo Celular/genética , Diferenciación Celular , Tamaño de la Célula , Pared Celular/genética , Pared Celular/metabolismo , Endorreduplicación , Frutas/genética , Frutas/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Edición Génica , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Mutación , Pectinas/genética , Pectinas/metabolismo , Fenotipo , Células Vegetales , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , PloidiasRESUMEN
Fruit softening, which is a major determinant of shelf life and commercial value, is the consequence of multiple cellular processes, including extensive remodeling of cell wall structure. Recently, it has been shown that pectate lyase (PL), an enzyme that degrades de-esterified pectin in the primary wall, is a major contributing factor to tomato fruit softening. Studies of pectin structure, distribution, and dynamics have indicated that pectins are more tightly integrated with cellulose microfibrils than previously thought and have novel structural features, including branches of the main polymer backbone. Moreover, recent studies of the significance of pectinases, such as PL and polygalacturonase, are consistent with a causal relationship between pectin degradation and a major effect on fruit softening.
Asunto(s)
Frutas/crecimiento & desarrollo , Pectinas/metabolismo , Pared Celular/metabolismo , Almacenamiento de Alimentos , Frutas/metabolismo , Solanum lycopersicum/crecimiento & desarrollo , Solanum lycopersicum/metabolismoRESUMEN
The parasitic flowering plant genus Cuscuta (dodder) is a parasitic weed that infects many important crops. Once it winds around the shoots of potential host plants and initiates the development of penetration organs, called haustoria, only a few plant species have been shown to deploy effective defense mechanisms to ward off Cuscuta parasitization. However, a notable exception is Solanum lycopersicum (tomato), which exhibits a local hypersensitive reaction when attacked by giant dodder (Cuscuta reflexa). Interestingly, the closely related wild desert tomato, Solanum pennellii, is unable to stop the penetration of its tissue by the C. reflexa haustoria. In this study, we observed that grafting a S. pennellii scion onto the rootstock of the resistant S. lycopersicum did not change the susceptibility phenotype of S. pennellii. This suggests that hormones, or other mobile substances, produced by S. lycopersicum do not induce a defense reaction in the susceptible tissue. Screening of a population of introgression lines harboring chromosome fragments from S. pennellii in the genome of the recurrent parent S. lycopersicum, revealed that most lines exhibit the same defense reaction as shown by the S. lycopersicum parental line. However, several lines showed different responses and exhibited either susceptibility, or cell death that extended considerably beyond the infection site. These lines will be valuable for the future identification of key loci involved in the perception of, and resistance to, C. reflexa and for developing strategies to enhance resistance to infection in crop species.
Asunto(s)
Cuscuta/fisiología , Malezas/fisiología , Solanum lycopersicum/fisiología , Solanum/fisiología , Cromosomas de las Plantas/genética , Genoma de Planta/genética , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Fenotipo , Brotes de la Planta/genética , Brotes de la Planta/metabolismo , Brotes de la Planta/fisiología , Solanum/genética , Solanum/metabolismo , Especificidad de la EspecieRESUMEN
The thick cuticle covering and embedding the epidermal cells of tomato (Solanum lycopersicum) fruit acts not only as a protective barrier against pathogens and water loss but also influences quality traits such as brightness and postharvest shelf-life. In a recent study, we screened a mutant collection of the miniature tomato cultivar Micro-Tom and isolated several glossy fruit mutants in which the abundance of cutin, the polyester component of the cuticle, was strongly reduced. We employed a newly developed mapping-by-sequencing strategy to identify the causal mutation underlying the cutin deficiency in a mutant thereafter named gpat6-a (for glycerol-3-phosphate acyltransferase6). To this end, a backcross population (BC1F2) segregating for the glossy trait was phenotyped. Individuals displaying either a wild-type or a glossy fruit trait were then pooled into bulked populations and submitted to whole-genome sequencing prior to mutation frequency analysis. This revealed that the causal point mutation in the gpat6-a mutant introduces a charged amino acid adjacent to the active site of a GPAT6 enzyme. We further showed that this mutation completely abolished the GPAT activity of the recombinant protein. The gpat6-a mutant showed perturbed pollen formation but, unlike a gpat6 mutant of Arabidopsis (Arabidopsis thaliana), was not male sterile. The most striking phenotype was observed in the mutant fruit, where cuticle thickness, composition, and properties were altered. RNA sequencing analysis highlighted the main processes and pathways that were affected by the mutation at the transcriptional level, which included those associated with lipid, secondary metabolite, and cell wall biosynthesis.
Asunto(s)
Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Lípidos de la Membrana/metabolismo , Solanum lycopersicum/enzimología , Secuencia de Aminoácidos , Mapeo Cromosómico , Frutas/anatomía & histología , Frutas/enzimología , Frutas/genética , Frutas/crecimiento & desarrollo , Glicerol-3-Fosfato O-Aciltransferasa/genética , Solanum lycopersicum/anatomía & histología , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , Modelos Moleculares , Mutación , Fenotipo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polen/anatomía & histología , Polen/enzimología , Polen/genética , Polen/crecimiento & desarrollo , Proteínas Recombinantes , Alineación de Secuencia , Análisis de Secuencia de ARNRESUMEN
Host plant penetration is the gateway to survival for holoparasitic Cuscuta and requires host cell wall degradation. Compositional differences of cell walls may explain why some hosts are amenable to such degradation while others can resist infection. Antibody-based techniques for comprehensive profiling of cell wall epitopes and cell wall-modifying enzymes were applied to several susceptible hosts and a resistant host of Cuscuta reflexa and to the parasite itself. Infected tissue of Pelargonium zonale contained high concentrations of de-esterified homogalacturonans in the cell walls, particularly adjacent to the parasite's haustoria. High pectinolytic activity in haustorial extracts and high expression levels of pectate lyase genes suggest that the parasite contributes directly to wall remodeling. Mannan and xylan concentrations were low in P. zonale and in five susceptible tomato introgression lines, but high in the resistant Solanum lycopersicum cv M82, and in C. reflexa itself. Knowledge of the composition of resistant host cell walls and the parasite's own cell walls is useful in developing strategies to prevent infection by parasitic plants.
Asunto(s)
Pared Celular/metabolismo , Cuscuta/metabolismo , Interacciones Huésped-Parásitos , Metabolómica , Parásitos/fisiología , Pelargonium/parasitología , Solanum lycopersicum/parasitología , Animales , Cuscuta/citología , Resistencia a la Enfermedad , Epítopos/metabolismo , Glucanos/metabolismo , Solanum lycopersicum/citología , Análisis por Micromatrices , Pectinas/metabolismo , Pelargonium/citología , Enfermedades de las Plantas/parasitología , Tallos de la Planta/fisiología , Plantas Modificadas Genéticamente , Polisacárido Liasas/metabolismo , Polisacáridos/metabolismo , Xilanos/metabolismoRESUMEN
Solanum pennellii is a wild tomato species endemic to Andean regions in South America, where it has evolved to thrive in arid habitats. Because of its extreme stress tolerance and unusual morphology, it is an important donor of germplasm for the cultivated tomato Solanum lycopersicum. Introgression lines (ILs) in which large genomic regions of S. lycopersicum are replaced with the corresponding segments from S. pennellii can show remarkably superior agronomic performance. Here we describe a high-quality genome assembly of the parents of the IL population. By anchoring the S. pennellii genome to the genetic map, we define candidate genes for stress tolerance and provide evidence that transposable elements had a role in the evolution of these traits. Our work paves a path toward further tomato improvement and for deciphering the mechanisms underlying the myriad other agronomic traits that can be improved with S. pennellii germplasm.
Asunto(s)
Genoma de Planta , Solanum/genética , Estrés Fisiológico/genética , Mapeo Cromosómico/métodos , Cromosomas de las Plantas , Elementos Transponibles de ADN , Sitios de Carácter CuantitativoRESUMEN
The pectin polymer homogalacturonan (HG) is a major component of land plant cell walls and is especially abundant in the middle lamella. Current models suggest that HG is deposited into the wall as a highly methylesterified polymer, demethylesterified by pectin methylesterase enzymes and cross-linked by calcium ions to form a gel. However, this idea is based largely on indirect evidence and in vitro studies. We took advantage of the wall architecture of the unicellular alga Penium margaritaceum, which forms an elaborate calcium cross-linked HG-rich lattice on its cell surface, to test this model and other aspects of pectin dynamics. Studies of live cells and microscopic imaging of wall domains confirmed that the degree of methylesterification and sufficient levels of calcium are critical for lattice formation in vivo. Pectinase treatments of live cells and immunological studies suggested the presence of another class of pectin polymer, rhamnogalacturonan I, and indicated its colocalization and structural association with HG. Carbohydrate microarray analysis of the walls of P. margaritaceum, Physcomitrella patens, and Arabidopsis (Arabidopsis thaliana) further suggested the conservation of pectin organization and interpolymer associations in the walls of green plants. The individual constituent HG polymers also have a similar size and branched structure to those of embryophytes. The HG-rich lattice of P. margaritaceum, a member of the charophyte green algae, the immediate ancestors of land plants, was shown to be important for cell adhesion. Therefore, the calcium-HG gel at the cell surface may represent an early evolutionary innovation that paved the way for an adhesive middle lamella in multicellular land plants.
Asunto(s)
Pared Celular/metabolismo , Carofíceas/citología , Carofíceas/metabolismo , Pectinas/metabolismo , Calcio/metabolismo , Adhesión Celular/efectos de los fármacos , Pared Celular/ultraestructura , Celulosa/metabolismo , Carofíceas/efectos de los fármacos , Carofíceas/ultraestructura , Ácido Edético/análogos & derivados , Ácido Edético/farmacología , Epítopos/metabolismo , Análisis por Micromatrices , Modelos Biológicos , Pectinas/química , Pectinas/inmunología , Poligalacturonasa/metabolismo , Polisacárido Liasas/metabolismoRESUMEN
Application of the dintroaniline compound, oryzalin, which inhibits microtubule formation, to the unicellular green alga Penium margaritaceum caused major perturbations to its cell morphology, such as swelling at the wall expansion zone in the central isthmus region. Cell wall structure was also notably altered, including a thinning of the inner cellulosic wall layer and a major disruption of the homogalacturonan (HG)-rich outer wall layer lattice. Polysaccharide microarray analysis indicated that the oryzalin treatment resulted in an increase in HG abundance in treated cells but a decrease in other cell wall components, specifically the pectin rhamnogalacturonan I (RG-I) and arabinogalactan proteins (AGPs). The ring of microtubules that characterizes the cortical area of the cell isthmus zone was significantly disrupted by oryzalin, as was the extensive peripheral network of actin microfilaments. It is proposed that the disruption of the microtubule network altered cellulose production, the main load-bearing component of the cell wall, which in turn affected the incorporation of HG in the two outer wall layers, suggesting coordinated mechanisms of wall polymer deposition.
Asunto(s)
Pared Celular/metabolismo , Celulosa/metabolismo , Chlorophyta/citología , Chlorophyta/metabolismo , Microtúbulos/metabolismo , Pectinas/metabolismo , Anticuerpos Monoclonales/metabolismo , Forma de la Célula/efectos de los fármacos , Pared Celular/efectos de los fármacos , Pared Celular/ultraestructura , Chlorophyta/crecimiento & desarrollo , Chlorophyta/ultraestructura , Dinitrobencenos/farmacología , Glicósido Hidrolasas/farmacología , Inmunohistoquímica , Análisis por Micromatrices , Microtúbulos/efectos de los fármacos , Modelos Biológicos , Polisacáridos/metabolismo , Sulfanilamidas/farmacologíaRESUMEN
Although self-incompatibility (SI) in plants has been studied extensively, far less is known about interspecific reproductive barriers. One interspecific barrier, known as unilateral incongruity or incompatibility (UI), occurs when species display unidirectional compatibility in interspecific crosses. In the wild tomato species Solanum pennellii, both SI and self-compatible (SC) populations express UI when crossed with domesticated tomato, offering a useful model system to dissect the molecular mechanisms involved in reproductive barriers. In this study, the timing of reproductive barrier establishment during pistil development was determined in SI and SC accessions of S. pennellii using a semi-in vivo system to track pollen-tube growth in developing styles. Both SI and UI barriers were absent in styles 5 days prior to flower opening, but were established by 2 days before flower opening, with partial barriers detected during a transition period 3-4 days before flower opening. The developmental expression dynamics of known SI factors, S-RNases and HT proteins, was also examined. The accumulation of HT-A protein coincided temporally and spatially with UI barriers in developing pistils. Proteomic analysis of stigma/styles from key developmental stages showed a switch in protein profiles from cell-division-associated proteins in immature stigma/styles to a set of proteins in mature stigma/styles that included S-RNases, HT-A protein and proteins associated with cell-wall loosening and defense responses, which could be involved in pollen-pistil interactions. Other prominent proteins in mature stigma/styles were those involved in lipid metabolism, consistent with the accumulation of lipid-rich material during pistil maturation.
Asunto(s)
Flores/crecimiento & desarrollo , Flores/metabolismo , Proteoma/metabolismo , Solanum/crecimiento & desarrollo , Solanum/metabolismo , Análisis de Varianza , Proteínas de Plantas/metabolismo , Tubo Polínico/crecimiento & desarrollo , Polinización/fisiología , Proteómica , Reproducción , Ribonucleasas/metabolismo , Autoincompatibilidad en las Plantas con Flores , Factores de TiempoRESUMEN
Cell adhesion in plants is mediated predominantly by pectins, a group of complex cell wall associated polysaccharides. An Arabidopsis mutant, friable1 (frb1), was identified through a screen of T-DNA insertion lines that exhibited defective cell adhesion. Interestingly, the frb1 plants displayed both cell and organ dissociations and also ectopic defects in organ separation. The FRB1 gene encodes a Golgi-localized, plant specific protein with only weak sequence similarities to known proteins (DUF246). Unlike other cell adhesion deficient mutants, frb1 mutants do not have reduced levels of adhesion related cell wall polymers, such as pectins. Instead, FRB1 affects the abundance of galactose- and arabinose-containing oligosaccharides in the Golgi. Furthermore, frb1 mutants displayed alteration in pectin methylesterification, cell wall associated extensins and xyloglucan microstructure. We propose that abnormal FRB1 action has pleiotropic consequences on wall architecture, affecting both the extensin and pectin matrices, with consequent changes to the biomechanical properties of the wall and middle lamella, thereby influencing cell-cell adhesion.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/citología , Adhesión Celular/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabinosa/metabolismo , Adhesión Celular/genética , Clonación Molecular , Galactosa/metabolismo , Aparato de Golgi/metabolismo , Pectinas/metabolismoRESUMEN
Effective proteome profiling is generally considered to depend heavily on the availability of a high-quality DNA reference database. As such, proteomics has long been taxonomically restricted, with limited inroads being made into the proteomes of "non-model" organisms. However, next generation sequencing (NGS), and particularly RNA-Seq, now allows deep coverage detection of expressed genes at low cost, which in turn potentially facilitates the matching of peptide mass spectra with cognate gene sequence. To test this, we performed a quantitative analysis of the proteomes of pollen from domesticated tomato (Solanum lycopersicum) and two wild relatives that exhibit differences in mating systems and in interspecific reproductive barriers. Using a custom tomato RNA-Seq database created through 454 pyrosequencing, more than 1200 proteins were identified, with subsets showing expression differences between genotypes or in the accumulation of the corresponding transcripts. Importantly, no major qualitative or quantitative differences were observed in the characterized proteomes when mass spectra were used to interrogate either a highly curated community database of tomato sequences generated through traditional sequencing technologies, or the RNA-Seq database. We conclude that RNA-Seq provides a cost-effective and robust platform for protein identification and will be increasingly valuable to the field of proteomics.
Asunto(s)
Proteínas de Plantas/genética , Polen/genética , Proteómica/métodos , ARN de Planta/genética , Análisis de Secuencia de ARN/métodos , Solanum lycopersicum/genética , Bases de Datos Genéticas , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/química , Proteínas de Plantas/análisis , Polen/químicaRESUMEN
Numerous evolutionary innovations were required to enable freshwater green algae to colonize terrestrial habitats and thereby initiate the evolution of land plants (embryophytes). These adaptations probably included changes in cell-wall composition and architecture that were to become essential for embryophyte development and radiation. However, it is not known to what extent the polymers that are characteristic of embryophyte cell walls, including pectins, hemicelluloses, glycoproteins and lignin, evolved in response to the demands of the terrestrial environment or whether they pre-existed in their algal ancestors. Here we show that members of the advanced charophycean green algae (CGA), including the Charales, Coleochaetales and Zygnematales, but not basal CGA (Klebsormidiales and Chlorokybales), have cell walls that are comparable in several respects to the primary walls of embryophytes. Moreover, we provide both chemical and immunocytochemical evidence that selected Coleochaete species have cell walls that contain small amounts of lignin or lignin-like polymers derived from radical coupling of hydroxycinnamyl alcohols. Thus, the ability to synthesize many of the components that characterize extant embryophyte walls evolved during divergence within CGA. Our study provides new insight into the evolutionary window during which the structurally complex walls of embryophytes originated, and the significance of the advanced CGA during these events.
Asunto(s)
Evolución Biológica , Pared Celular/química , Carofíceas/química , Lignina/análisis , Polisacáridos/análisis , Anticuerpos Monoclonales , Pared Celular/genética , Pared Celular/ultraestructura , Celulosa/análisis , Carofíceas/genética , Carofíceas/ultraestructura , Ácido Edético/análogos & derivados , Ácido Edético/química , Embryophyta/química , Embryophyta/genética , Embryophyta/ultraestructura , Epítopos , Técnica del Anticuerpo Fluorescente , Genes de Plantas/genética , Glicoproteínas/análisis , Análisis por Micromatrices , Pectinas/análisis , Filogenia , Plantas , Hidróxido de Sodio/químicaRESUMEN
Cell wall disassembly in softening fruits is a complex process involving the cumulative action of many families of wall-modifying proteins on interconnected polysaccharide matrices. One strategy to elucidate the in vivo substrates of specific enzymes and their relative importance and contribution to wall modification is to suppress their expression in transgenic fruit. It has been reported previously that inhibiting the expression of pectate lyase genes by antisense technology in strawberry (Fragaria x ananassa Duch.) fruit resulted in prolonged fruit firmness. This suggested that pectin depolymerization might make a more important contribution to strawberry fruit softening than is often stated. In this present study, three independent transgenic lines were identified exhibiting a greater than 90% reduction in pectate lyase transcript abundance. Analyses of sequential cell wall extracts from the transgenic and control fruit collectively showed clear quantitative and qualitative differences in the extractability and molecular masses of populations of pectin polymers. Wall extracts from transgenic fruits showed a reduction in pectin solubility and decreased depolymerization of more tightly bound polyuronides. Additional patterns of differential extraction of other wall-associated pectin subclasses were apparent, particularly in the sodium carbonate- and chelator-soluble polymers. In addition, microscopic studies revealed that the typical ripening-associated loss of cell-cell adhesion was substantially reduced in the transgenic fruits. These results indicate that pectate lyase plays an important degradative role in the primary wall and middle lamella in ripening strawberry fruit, and should be included in synergistic models of cell wall disassembly.
Asunto(s)
Fragaria/fisiología , Frutas/fisiología , Pectinas/metabolismo , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Pared Celular/química , Pared Celular/enzimología , Pared Celular/genética , Pared Celular/fisiología , Cromatografía en Gel , Fragaria/química , Fragaria/enzimología , Fragaria/genética , Frutas/química , Frutas/enzimología , Frutas/genética , Pectinas/química , Pectinas/genética , Fenotipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/fisiologíaRESUMEN
Cell wall disassembly in ripening fruit is highly complex, involving the dismantling of multiple polysaccharide networks by diverse families of wall-modifying proteins. While it has been reported in several species that multiple members of each such family are expressed in the same fruit tissue, it is not clear whether this reflects functional redundancy, with protein isozymes from a single enzyme class performing similar roles and contributing equally to wall degradation, or whether they have discrete functions, with some isoforms playing a predominant role. Experiments reported here sought to distinguish between cell wall-related processes in ripening melon that were softening-associated and softening-independent. Cell wall polysaccharide depolymerization and the expression of wall metabolism-related genes were examined in transgenic melon (Cucumis melo var. cantalupensis Naud.) fruit with suppressed expression of the 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene and fruits treated with ethylene and 1-methylcyclopropene (1-MCP). Softening was completely inhibited in the transgenic fruit but was restored by treatment with exogenous ethylene. Moreover, post-harvest application of 1-MCP after the onset of ripening completely halted subsequent softening, suggesting that melon fruit softening is ethylene-dependent. Size exclusion chromatography of cell wall polysaccharides, from the transgenic fruits, with or without exogenous ethylene, indicated that the depolymerization of both pectins and xyloglucans was also ethylene dependent. However, northern analyses of a diverse range of cell wall-related genes, including those for polygalacturonases, xyloglucan endotransglucosylase/hydrolases, expansin, and beta-galactosidases, identified specific genes within single families that could be categorized as ethylene-dependent, ethylene-independent, or partially ethylene-dependent. These results support the hypothesis that while individual cell wall-modifying proteins from each family contribute to cell wall disassembly that accompanies fruit softening, other closely related family members are regulated in an ethylene-independent manner and apparently do not directly participate in fruit softening.
Asunto(s)
Pared Celular/fisiología , Cucumis melo/fisiología , Etilenos/metabolismo , Frutas/fisiología , Cucumis melo/genética , Cartilla de ADN , Etilenos/biosíntesis , Frutas/genética , Plantas Modificadas Genéticamente/fisiología , Polen/fisiología , Reacción en Cadena de la PolimerasaRESUMEN
Most published proteomics studies of bulk plant tissues use a procedure in which proteins are precipitated with trichloroacetic acid (TCA) and acetone (TCA-A), but few attempts have been made to contrast this approach in a systematic way with alternative methods against a spectrum of tissues. To address this, TCA-A was compared with another acetone-based protocol (TCA-B) or a phenol (Phe)-based method, targeting a range of tomato tissues and three species of fruits that contain high levels of contaminating compounds: banana, avocado and orange. The Phe method gave a higher protein yield and typically greater resolution and spot intensity, particularly with extracts from tissues containing high levels of soluble polysaccharides. The methods also generated remarkably different two-dimensional gel electrophoresis (2-DE) protein spot patterns. Peptide mass fingerprinting was used to identify polypeptides that were common to multiple extracts or uniquely present in one extract type. While no clear pattern emerged to explain the basis for the differential protein extraction, it was noted that the Phe method showed enhanced extraction of glycoproteins. These results suggest that the Phe protocol is highly effective with more recalcitrant tissues and that a combination of TCA-A and Phe methods provides enhanced 2-DE based proteomic analyses of most plant tissues.