RESUMEN
BACKGROUND: The periderm is a protective barrier crucial for land plant survival, but little is known about genetic factors involved in its development and regulation. Using a transcriptomic approach in the cork oak (Q. suber) periderm, we previously identified an RS2-INTERACTING KH PROTEIN (RIK) homologue of unknown function containing a K homology (KH)-domain RNA-binding protein, as a regulatory candidate gene in the periderm. RESULTS: To gain insight into the function of RIK in the periderm, potato (S. tuberosum) tuber periderm was used as a model: the full-length coding sequence of RIK, hereafter referred to as StRIK, was isolated, the transcript profile analyzed and gene silencing in potato performed to analyze the silencing effects on periderm anatomy and transcriptome. The StRIK transcript accumulated in all vegetative tissues studied, including periderm and other suberized tissues such as root and also in wounded tissues. Downregulation of StRIK in potato by RNA interference (StRIK-RNAi) did not show any obvious effects on tuber periderm anatomy but, unlike Wild type, transgenic plants flowered. Global transcript profiling of the StRIK-RNAi periderm did show altered expression of genes associated with RNA metabolism, stress and signaling, mirroring the biological processes found enriched within the in silico co-expression network of the Arabidopsis orthologue. CONCLUSIONS: The ubiquitous expression of StRIK transcript, the flower associated phenotype and the differential expression of StRIK-RNAi periderm point out to a general regulatory role of StRIK in diverse plant developmental processes. The transcriptome analysis suggests that StRIK might play roles in RNA maturation and stress response in the periderm.
Asunto(s)
Proteínas de Plantas/genética , Tubérculos de la Planta/genética , ARN de Planta/metabolismo , Solanum tuberosum/genética , Estrés Fisiológico/genética , Arabidopsis/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Elementos Transponibles de ADN , Flores/genética , Flores/fisiología , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Silenciador del Gen , Proteínas de Plantas/metabolismo , Tubérculos de la Planta/anatomía & histología , Tubérculos de la Planta/citología , Plantas Modificadas Genéticamente , Solanum tuberosum/citologíaRESUMEN
We present a new large-scale three-fold annotated microscopy image dataset, aiming to advance the plant cell biology research by exploring different cell microstructures including cell size and shape, cell wall thickness, intercellular space, etc. in deep learning (DL) framework. This dataset includes 9,811 unstained and 6,127 stained (safranin-o, toluidine blue-o, and lugol's-iodine) images with three-fold annotation including physical, morphological, and tissue grading based on weight, different section area, and tissue zone respectively. In addition, we prepared ground truth segmentation labels for three different tuber weights. We have validated the pertinence of annotations by performing multi-label cell classification, employing convolutional neural network (CNN), VGG16, for unstained and stained images. The accuracy has been achieved up to 0.94, while, F2-score reaches to 0.92. Furthermore, the ground truth labels have been verified by semantic segmentation algorithm using UNet architecture which presents the mean intersection of union up to 0.70. Hence, the overall results show that the data are very much efficient and could enrich the domain of microscopy plant cell analysis for DL-framework.
Asunto(s)
Aprendizaje Profundo , Microscopía , Células Vegetales , Tubérculos de la Planta/citología , Solanum tuberosum/citología , Procesamiento de Imagen Asistido por Computador , Redes Neurales de la ComputaciónRESUMEN
Anthocyanins are antioxidant pigments widely used in drugs and food preparations. Flesh-coloured tubers of the cultivated potato Solanum tuberosum are important sources of different anthocyanins. Due to the high degree of decoration achieved by acylation, anthocyanins from potato are very stable and suitable for the food processing industry. The use of cell culture allows to extract anthocyanins on-demand, avoiding seasonality and consequences associated with land-based-tuber production. However, a well-known limit of cell culture is the metabolic instability and loss of anthocyanin production during successive subcultures. To get a general picture of mechanisms responsible for this instability, we explored both genetic and epigenetic regulation that may affect anthocyanin production in cell culture. We selected two clonally related populations of anthocyanin-producing (purple) and non-producing (white) potato cells. Through targeted molecular investigations, we identified and functionally characterized an R3-MYB, here named StMYBATV. This transcription factor can interact with bHLHs belonging to the MBW (R2R3-MYB, bHLH and WD40) anthocyanin activator complex and, potentially, may interfere with its formation. Genome methylation analysis revealed that, for several genomic loci, anthocyanin-producing cells were more methylated than clonally related white cells. In particular, we localized some methylation events in ribosomal protein-coding genes. Overall, our study explores novel molecular aspects associated with loss of anthocyanins in cell culture systems.
Asunto(s)
Antocianinas/biosíntesis , Técnicas de Cultivo de Célula , Epigénesis Genética , Células Vegetales/metabolismo , Tubérculos de la Planta/metabolismo , Solanum tuberosum/metabolismo , Antocianinas/genética , Epigénesis Genética/fisiología , Tubérculos de la Planta/citología , Solanum tuberosum/citología , Solanum tuberosum/genéticaRESUMEN
KEY MESSAGE: This study established a rapid method for the gene expression analysis in potato tubers. The use of microtubers would be useful for primary evaluation of tuber-expressed genes. In the development of transgenic potato or of potato with other genome modifications (e.g., genome editing or RNA-directed DNA methylation (RdDM) and so on) to improve tuber traits, analysis of the target gene is often difficult because of the long cultivation cycle (3-4 months), large areas required, numerous materials for plant cultivation, and considerable efforts needed to obtain transgenic tubers. We demonstrate here rapid and convenient analysis of gene expression in potato microtubers. Enough microtubers for expression analysis can be induced over about 4 weeks in a simple liquid medium in an Erlenmeyer flask. High-quality RNA and protein can be easily prepared from microtubers and used for northern blot, qRT-PCR, and western blot analyses without further purification. We investigated the expression of two tuber-expressed genes (GBSS1 and Vinv) in microtubers derived from the wild-type and from lines derived from RdDM-mediated transcriptional gene silencing. As expected, the expression of both genes was similar between microtubers and normal tubers. Furthermore, we demonstrated that microtubers can be used in western blot and confocal immunofluorescent microscopy analyses. These results suggest that expression analysis using microtubers is a convenient tool for the analysis of tuber-expressed genes such as GBSS1 and Vinv in potato.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Proteínas de Plantas/genética , Tubérculos de la Planta/genética , Solanum tuberosum/genética , Técnicas de Cultivo de Tejidos/métodos , Western Blotting , Medios de Cultivo , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Microscopía Fluorescente , Proteínas de Plantas/metabolismo , Brotes de la Planta/citología , Tubérculos de la Planta/citología , Tubérculos de la Planta/crecimiento & desarrollo , Plantas Modificadas Genéticamente , ARN de PlantaRESUMEN
Macronutrients in whole plant foods are enclosed inside cells. The metabolic response from these entrapped nutrients may depend on cell-wall porosity, by controlling the passage of digestive enzymes. As non-interacting size mimics of digestive enzymes, we investigated the diffusion of fluorescently-labelled probes across the walls of isolated plant cells from potato tuber, red kidney bean and banana. Diffusion properties of permeable probes, dextran (20-kDa and 70-kDa) and albumin, were quantified, using fluorescence recovery after photobleaching. The consistent reduction of diffusion rate in the presence of cell walls (around 40%) compared to free-diffusion rate was attributed to the limiting porosity of the wall matrix. A combination of the physical barrier effects demonstrated here and non-catalytic binding of enzymes to cell walls limits the hydrolysis of intracellular macronutrients. This and further understanding of the structural basis for the physical barrier properties would help to design foods from plant materials with enhanced nutrition.
Asunto(s)
Pared Celular/química , Musa/citología , Nutrientes/metabolismo , Phaseolus/citología , Solanum tuberosum/citología , Pared Celular/metabolismo , Dextranos/química , Dextranos/metabolismo , Difusión , Enzimas/química , Enzimas/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Hidrólisis , Musa/química , Nutrientes/química , Phaseolus/química , Células Vegetales/química , Tubérculos de la Planta/citología , Porosidad , Solanum tuberosum/químicaRESUMEN
Freeze-drying (FD) has utility for phytonutrient screening but its reliability for starch measurements is unclear. The impact of FD was tested on total (TS), digestible (DS) and resistant starch (RS) for four potato varieties (PC Red, GG Red, GG Yellow, and Dolbec Yellow). The treatments included: (a) tubers boiled and then cooled for 1h at room temperature (RT) (control; Treatment 1) and 24h at 4°C; (b) tubers boiled and then cooled for 1h at RT with subsequent FD (Treatment 2); and (c) raw tubers that underwent FD, then were rehydrated, boiled, and cooled for 1h at RT (Treatment 3). TS and DS content did not differ between the control samples cooled for 1h or 24h with Treatment 1 but RS content at 24h was higher, which indicated starch retrogradation. Cultivar variations were observed in the percent increase in RS between 24h vs. 1h with the greatest increase in Dolbec Yellow (114.5±7.6%). Relative to controls, FD treatments modified measured TS content in three of four varieties including overestimation by 94.2±6.5% and 156.0±5.2% for GG Yellow with Treatments 2 and 3, respectively. FD caused overestimation of DS and underestimation of RS in the same three varieties relative to controls including overestimation of DS in GG Yellow by 122.9±4.7% (Treatment 2) and 205.7±13.8% (Treatment 3). PC Red showed the greatest underestimation in RS content compared to controls of 42.5±9.6% and 61.7±5.4% in Treatment 2 and 3, respectively. Modifications to cooking and rehydration procedures following FD of raw tuber samples did not improve reliability of TS, DS, and RS measurements. Microscopy showed that cells remained intact following cooking whereas cell wall integrity was reduced when FD followed cooking and that cooking followed by FD led to destruction of cellular structure. We conclude that FD leads to unreliable starch measurements, which was supported by morphological microscopic evidence. For accuracy of starch profile measurements, the use of freshly cooked potato samples is essential.
Asunto(s)
Culinaria , Digestión , Manipulación de Alimentos/métodos , Liofilización , Tubérculos de la Planta/química , Solanum tuberosum/química , Almidón/análisis , Pared Celular/química , Análisis de los Alimentos/métodos , Ensayos Analíticos de Alto Rendimiento , Calor , Microscopía , Tubérculos de la Planta/citología , Reproducibilidad de los Resultados , Solanum tuberosum/citología , Factores de TiempoRESUMEN
Potato tuber is a high yielding food crop known for its high levels of starch accumulation but only negligible levels of triacylglycerol (TAG). In this study, we evaluated the potential for lipid production in potato tubers by simultaneously introducing three transgenes, including WRINKLED 1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) and OLEOSIN under the transcriptional control of tuber-specific (patatin) and constitutive (CaMV-35S) promoters. This coordinated metabolic engineering approach resulted in over a 100-fold increase in TAG accumulation to levels up to 3.3% of tuber dry weight (DW). Phospholipids and galactolipids were also found to be significantly increased in the potato tuber. The increase of lipids in these transgenic tubers was accompanied by a significant reduction in starch content and an increase in soluble sugars. Microscopic examination revealed that starch granules in the transgenic tubers had more irregular shapes and surface indentations when compared with the relatively smooth surfaces of wild-type starch granules. Ultrastructural examination of lipid droplets showed their close proximity to endoplasmic reticulum and mitochondria, which may indicate a dynamic interaction with these organelles during the processes of lipid biosynthesis and turnover. Increases in lipid levels were also observed in the transgenic potato leaves, likely due to the constitutive expression of DGAT1 and incomplete tuber specificity of the patatin promoter. This study represents an important proof-of-concept demonstration of oil increase in tubers and provides a model system to further study carbon reallocation during development of nonphotosynthetic underground storage organs.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/genética , Mejoramiento Genético/métodos , Ingeniería Metabólica/métodos , Aceites de Plantas/metabolismo , Tubérculos de la Planta/genética , Tubérculos de la Planta/metabolismo , Solanum tuberosum/genética , Carbohidratos/análisis , Ácidos Grasos/análisis , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Galactolípidos/metabolismo , Genes de Plantas , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Fosfolípidos/metabolismo , Aceites de Plantas/análisis , Aceites de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tubérculos de la Planta/citología , Plantas Modificadas Genéticamente , Solanum tuberosum/citología , Almidón/análisis , Almidón/metabolismo , Transformación Genética , Triglicéridos/metabolismoRESUMEN
Among many factors that regulate potato tuberization, calcium and calcium-dependent protein kinases (CDPKs) play an important role. CDPK activity increases at the onset of tuber formation with StCDPK1 expression being strongly induced in swollen stolons. However, not much is known about the transcriptional and posttranscriptional regulation of StCDPK1 or its downstream targets in potato development. To elucidate further, we analyzed its expression in different tissues and stages of the life cycle. Histochemical analysis of StCDPK1::GUS (ß-glucuronidase) plants demonstrated that StCDPK1 is strongly associated with the vascular system in stems, roots, during stolon to tuber transition, and in tuber sprouts. In agreement with the observed GUS profile, we found specific cis-acting elements in StCDPK1 promoter. In silico analysis predicted miR390 to be a putative posttranscriptional regulator of StCDPK1. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis showed ubiquitous expression of StCDPK1 in different tissues which correlated well with Western blot data except in leaves. On the contrary, miR390 expression exhibited an inverse pattern in leaves and tuber eyes suggesting a possible regulation of StCDPK1 by miR390. This was further confirmed by Agrobacterium co-infiltration assays. In addition, in vitro assays showed that recombinant StCDPK1-6xHis was able to phosphorylate the hydrophilic loop of the auxin efflux carrier StPIN4. Altogether, these results indicate that StCDPK1 expression is varied in a tissue-specific manner having significant expression in vasculature and in tuber eyes; is regulated by miR390 at posttranscriptional level and suggest that StPIN4 could be one of its downstream targets revealing the overall role of this kinase in potato development.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , MicroARNs/genética , Proteínas Quinasas/metabolismo , Solanum tuberosum/enzimología , Regulación Enzimológica de la Expresión Génica , Genes Reporteros , Ácidos Indolacéticos/metabolismo , Proteínas de Transporte de Membrana , Especificidad de Órganos , Fosforilación , Reguladores del Crecimiento de las Plantas/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Tubérculos de la Planta/citología , Tubérculos de la Planta/enzimología , Tubérculos de la Planta/genética , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Proteínas Quinasas/genética , Procesamiento Postranscripcional del ARN , ARN de Planta/genética , Solanum tuberosum/citología , Solanum tuberosum/genética , Solanum tuberosum/crecimiento & desarrolloRESUMEN
Fungi can modify the pH in or around the infected site via alkalization or acidification, and pH monitoring may provide valuable information on host-fungus interactions. The objective of the present study was to examine the ability of two fungi, Colletotrichum coccodes and Helminthosporium solani, to modify the pH of potato tubers during artificial inoculation in situ. Both fungi cause blemishes on potato tubers, which downgrades tuber quality and yield. Direct visualization and estimation of pH changes near the inoculation area were achieved using pH indicators and image analysis. The results showed that the pH of the area infected by either fungus increased from potato native pH of approximately 6.0 to 7.4 to 8.0. By performing simple analysis of the images, it was also possible to derive the growth curve of each fungus and estimate the lag phase of the radial growth: 10 days for C. coccodes and 17 days H. solani. In addition, a distinctive halo (an edge area with increased pH) was observed only during the lag phase of H. solani infection. pH modulation is a major factor in pathogen-host interaction and the proposed method offers a simple and rapid way to monitor these changes.
Asunto(s)
Colletotrichum/fisiología , Helminthosporium/fisiología , Interacciones Huésped-Patógeno , Enfermedades de las Plantas/microbiología , Solanum tuberosum/microbiología , Concentración de Iones de Hidrógeno , Tubérculos de la Planta/química , Tubérculos de la Planta/citología , Tubérculos de la Planta/microbiología , Solanum tuberosum/química , Solanum tuberosum/citologíaRESUMEN
In this study, we analyze 31 transgenic lines and their respective untransformed background lines to determine the transgene effects on targeted structures including the pectin components rhamnogalacturonan I (RG-I) and homogalacturonan (HG), neutral side chains (galactan/arabinangalactan), acetylation of pectin, and cellulose level. Modification arising from the pectin backbone- or pectin side chain transgenic lines either increased or decreased the HG:RG-I ratio, side chain length, and methyl esterification of pectin in the tuber cell wall. The pectin esterification transgenic line exhibited only limited side effects. The cellulose level-targeting transgenic lines yielded an unexpectedly high HG:RG-I ratio and longer pectic side chains. These results clearly demonstrate that in effects of a transgene are not restricted to the direct activity of the targeted enzyme but have consequences for the structure of the cell wall matrix. Analysis of whole cell wall structure is therefore necessary to assess the complete effect, direct and indirect, of a transgene.
Asunto(s)
Pared Celular/química , Pectinas/química , Pectinas/genética , Tubérculos de la Planta/química , Solanum tuberosum/química , Solanum tuberosum/genética , Animales , Tubérculos de la Planta/citología , Solanum tuberosum/citología , Transgenes/genéticaRESUMEN
The digestibility of starchy foods, such as potatoes, can be characterized by the proportion of starch that is rapidly digestible by in vitro hydrolysis (rapidly digestible starch, RDS). This study evaluated the RDS content in a potato germplasm collection consisting of 98 genotypes and identified three advanced lines, Crop39, Crop71 and Crop85, where cooked potato RDS content was significantly lower than that of their respective isolated starches (P < 0.05). In Crop39, Crop71 and Crop85, the properties of their isolated starch did not differ significantly from that of five control lines with higher RDS contents. Cell wall analyses revealed that, compared with other lines tested, Crop39, Crop71 and Crop85 had at least four times the amount of rhamnogalacturonan-I (RG-I) galactan side-chains that were very firmly attached to the wall and requiring 4 M KOH for extraction. Pectin solubilization during cooking was also remarkably low (2-4%) in these three lines compared with other lines tested (7-19%). The findings suggest that possession of higher amounts of RG-I galactan that interact strongly with cellulose may provide a sturdier wall that better resists solubilization during cooking, and effectively impedes access of digestive enzymes for starch hydrolysis in an in vitro model.
Asunto(s)
Pared Celular/química , Pared Celular/fisiología , Células Vegetales/fisiología , Tubérculos de la Planta/citología , Solanum tuberosum/citología , Almidón/químicaRESUMEN
Uridine diphosphate (UDP)-glucose 4-epimerase (UGE) catalyzes the conversion of UDP-glucose to UDP-galactose. Cell wall materials from the cv. Kardal (wild-type, background) and two UGE transgenic lines (UGE 45-1 and UGE 51-16) were isolated and fractionated. The galactose (Gal) content (mg/100g tuber) from UGE 45-1 transgenic line was 38% higher than that of wild-type, and resulted in longer pectin side chains. The Gal content present in UGE 51-16 was 17% lower than that of wild-type, although most pectin populations maintained the same level of Gal. Both UGE transgenic lines showed unexpectedly a decrease in acetylation and an increase in methyl-esterification of pectin. Both UGE transgenic lines showed similar proportions of homogalacturonan and rhamnogalacturonan I within pectin backbone as the wild-type, except for the calcium-bound pectin fraction exhibiting relatively less rhamnogalacturonan I. Next to pectin modification, xyloglucan populations from both transgenic lines were altered resulting in different XSGG and XXGG proportion in comparison to wild-type.
Asunto(s)
Pared Celular/química , Tubérculos de la Planta/química , Polisacáridos/química , Polisacáridos/genética , Solanum tuberosum/química , Solanum tuberosum/genética , Animales , Pared Celular/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Tubérculos de la Planta/citología , Tubérculos de la Planta/genética , Tubérculos de la Planta/fisiología , Plantas Modificadas Genéticamente/genética , Solanum tuberosum/citología , Solanum tuberosum/enzimología , UDPglucosa 4-Epimerasa/genética , UDPglucosa 4-Epimerasa/metabolismoRESUMEN
Tuber wounding induces a cascade of biological responses that are involved in processes required to heal and protect surviving plant tissues. Little is known about the coordination of these processes, including essential wound-induced DNA synthesis, yet they play critical roles in maintaining marketability of the harvested crop and tubers cut for seed. A sensitive "Click-iT EdU Assay" employing incorporation of the thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU), in conjunction with 4',6-diamindino-2-phenylindole (DAPI) counter labeling, was employed to objectively identify and determine the time course and spatial distribution of tuber nuclei that were wound-induced to enter S-phase of the cell cycle. Both labeling procedures are rapid and sensitive in situ. Following wounding, EdU incorporation (indicating DNA synthesis) was not detectable until after 12h, rapidly reached a maximum at about 18h and then declined to near zero at 48h. About 28% of the nuclei were EdU labeled at 18h reflecting the proportion of cells in S-phase of the cell cycle. During the â¼30h in which induced cells were progressing through S-phase, de novo DNA synthesis extended 7-8 cell layers below the wound surface. Cessation of nuclear DNA synthesis occurred about 4 d prior to completion of wound closing layer formation. Initiation of wound periderm development followed at 7 d, i.e. about 5 d after cessation of nuclear DNA biosynthesis; at this time the phellogen developed and meristematic activity was detected via the production of new phellem cells. Collectively, these results provide new insight into the coordination of wound-induced nucleic acid synthesis with associated tuber wound-healing processes.
Asunto(s)
ADN de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Tubérculos de la Planta/fisiología , Solanum tuberosum/fisiología , Núcleo Celular/genética , Núcleo Celular/metabolismo , ADN de Plantas/genética , Indoles , Cinética , Modelos Biológicos , Especificidad de Órganos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tubérculos de la Planta/citología , Tubérculos de la Planta/genética , Fase S , Solanum tuberosum/citología , Solanum tuberosum/genética , Coloración y Etiquetado , Factores de Tiempo , Heridas y LesionesRESUMEN
Pectin is a complex polysaccharide and an integral part of the primary plant cell wall and middle lamella, contributing to cell wall mechanical strength and cell adhesion. To understand the structure-function relationships of pectin in the cell wall, a set of transgenic potato lines with altered pectin composition was analysed. The expression of genes encoding enzymes involved in pectin acetylation, degradation of the rhamnogalacturonan backbone and type and length of neutral side chains, arabinan and galactan in particular, has been altered. Upon crossing of different transgenic lines, some transgenes were not transmitted to the next generation when these lines were used as a pollen donor, suggesting male sterility. Viability of mature pollen was severely decreased in potato lines with reduced pectic arabinan, but not in lines with altered galactan side chains. Anthers and pollen of different developmental stages were microscopically examined to study the phenotype in more detail. Scanning electron microscopy of flowers showed collapsed pollen grains in mature anthers and in earlier stages cytoplasmic protrusions at the site of the of kin pore, eventually leading to bursting of the pollen grain and leaking of the cytoplasm. This phenomenon is only observed after the microspores are released and the tapetum starts to degenerate. Timing of the phenotype indicates a role for pectic arabinan side chains during remodelling of the cell wall when the pollen grain is maturing and dehydrating.
Asunto(s)
Pared Celular/metabolismo , Pectinas/metabolismo , Polen/citología , Polen/crecimiento & desarrollo , Polisacáridos/metabolismo , Solanum tuberosum/citología , Segregación Cromosómica , Cruzamientos Genéticos , Dosificación de Gen , Monosacáridos/metabolismo , Fenotipo , Infertilidad Vegetal/genética , Tubérculos de la Planta/citología , Tubérculos de la Planta/metabolismo , Plantas Modificadas Genéticamente , Polen/anatomía & histología , Polen/ultraestructura , Solanum tuberosum/genética , Solanum tuberosum/ultraestructura , Transformación Genética , Transgenes/genéticaRESUMEN
Mitochondria contain hundreds of proteins but only a few are encoded by the mitochondrial genome. The other proteins are nuclear-encoded and imported into mitochondria. These proteins can be translated on free cytosolic polysomes, then targeted and imported into mitochondria. Nonetheless, numerous cytosolic mRNAs encoding mitochondrial proteins are detected at the surface of mitochondria in yeast, plants and animals. The localization of mRNAs to the vicinity of mitochondria would be a way for mitochondrial protein sorting. The mechanisms responsible for mRNA targeting to mitochondria are not clearly identified. Sequences within the mRNA molecules (cis-elements), as well as a few trans-acting factors, have been shown to be essential for targeting of some mRNAs. In order to identify receptors involved in mRNA docking to the mitochondrial surface, we have developed an in vitro mRNA binding assay with isolated plant mitochondria. We show that naked mRNAs are able to bind to isolated mitochondria, and our results strongly suggest that mRNA docking to the plant mitochondrial outer membrane requires at least one component of TOM complex.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , ARN Mensajero/metabolismo , ARN/metabolismo , Solanum tuberosum/metabolismo , Sitios de Unión , Transporte Biológico , Citosol/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Células Vegetales/metabolismo , Tubérculos de la Planta/citología , Tubérculos de la Planta/genética , Tubérculos de la Planta/metabolismo , Unión Proteica , ARN/química , ARN/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN Mitocondrial , Solanum tuberosum/citología , Solanum tuberosum/genética , Transcripción Genética , Canales Aniónicos Dependientes del Voltaje/genética , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
Mature native periderm that exhibits resistance to excoriation (RE) is the primary defense for potato tubers against abiotic and biotic challenges. However, little is known about the physiology of periderm maturation and associated gene expressions. In this study, periderm maturation events and associated gene expressions were determined in tubers of two diverse potato genotypes (NDTX4271-5R (ND) and Russet Burbank (RB); 2008 and 2009 crops) at four harvest maturities ranging from immature (non-senesced vines and low RE) to mature (senesced vines and high RE). Approximately 104 d after planting, the fine balance of accumulation and loss of periderm phellem cell layers showed signs of subsiding, indicating cessation of cell division by the phellogen. Phellogen radial cell walls thickened as periderm matured throughout the harvests, increasing RE/skin-set. In both genotypes, the cell cycle gene cyclin-dependent kinase B (StCDKB) rapidly down-regulated after the second harvest coinciding with apparent cessation of cell division. Expression patterns of genes encoding epidermal growth factor binding protein (StEBP) and cyclin-dependent kinase regulatory subunit (StCKS1At) were less indicative of phellogen inactivation and periderm maturation. Genes encoding the structural cell wall proteins extensin (StExt1) for ND and extensin-like (StExtlk) for ND and RB remained up-regulated respectively by the second harvest, suggesting involvement with completion of phellem cell accumulation and on-set of periderm maturation. The expression of genes encoding pectin methyl esterase (StPME), StExt1 and a cell wall strengthening "tyrosine-and lysine-rich protein" (StTLRP) increased in phellogen cells from later harvests of ND tubers, but were down regulated in RB tubers; this suggests roles in phellem cell generation and completion of delayed cell wall development in non-meristematic phellogen cells of ND, a red skinned phenotype. StCDKB and StPrePME genes were rapidly down-regulated by the third harvest for both genotypes. Collectively, these results suggest that down-regulation of these genes coordinates with on-set of periderm maturation and skin-set progression.
Asunto(s)
Desarrollo de la Planta/genética , Epidermis de la Planta/citología , Epidermis de la Planta/crecimiento & desarrollo , Tubérculos de la Planta/citología , Tubérculos de la Planta/crecimiento & desarrollo , Solanum tuberosum/crecimiento & desarrollo , Solanum tuberosum/genética , Diferenciación Celular/genética , División Celular/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Variación Genética , GenotipoRESUMEN
Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-¹4C]glucose 1-phosphate, [U-¹4C]sucrose, [U-¹4C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-¹4C]sucrose plus unlabelled equimolar glucose 1-phosphate. C¹4-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced ¹4C incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-¹4C]glucose 1-phosphate or adenosine-[U-¹4C]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro C¹4C-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells.
Asunto(s)
Ciclo del Carbono , Solanum tuberosum/citología , Solanum tuberosum/metabolismo , Almidón/metabolismo , Ciclo del Carbono/efectos de los fármacos , Isótopos de Carbono , Mezclas Complejas , Glucanos/metabolismo , Glucofosfatos/farmacología , Isoenzimas/metabolismo , Tubérculos de la Planta/citología , Tubérculos de la Planta/efectos de los fármacos , Tubérculos de la Planta/fisiología , Tubérculos de la Planta/ultraestructura , Plantas Modificadas Genéticamente , Plastidios/efectos de los fármacos , Plastidios/enzimología , Polisacáridos/metabolismo , Solanum tuberosum/genética , Solanum tuberosum/fisiología , Solubilidad/efectos de los fármacos , Almidón/ultraestructura , Almidón Fosforilasa/metabolismo , Almidón Sintasa/metabolismo , Sacarosa/farmacología , TemperaturaRESUMEN
Potato (Solanum tuberosum) tuber, a swollen underground stem, is used as a model system for the study of dormancy release and sprouting. Natural dormancy release, at room temperature, is initiated by tuber apical bud meristem (TAB-meristem) sprouting characterized by apical dominance (AD). Dormancy is shortened by treatments such as bromoethane (BE), which mimics the phenotype of dormancy release in cold storage by inducing early sprouting of several buds simultaneously. We studied the mechanisms governing TAB-meristem dominance release. TAB-meristem decapitation resulted in the development of increasing numbers of axillary buds with time in storage, suggesting the need for autonomous dormancy release of each bud prior to control by the apical bud. Hallmarks of programmed cell death (PCD) were identified in the TAB-meristems during normal growth, and these were more extensive when AD was lost following either extended cold storage or BE treatment. Hallmarks included DNA fragmentation, induced gene expression of vacuolar processing enzyme1 (VPE1), and elevated VPE activity. VPE1 protein was semipurified from BE-treated apical buds, and its endogenous activity was fully inhibited by a cysteinyl aspartate-specific protease-1-specific inhibitor N-Acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO). Transmission electron microscopy further revealed PCD-related structural alterations in the TAB-meristem of BE-treated tubers: a knob-like body in the vacuole, development of cytoplasmic vesicles, and budding-like nuclear segmentations. Treatment of tubers with BE and then VPE inhibitor induced faster growth and recovered AD in detached and nondetached apical buds, respectively. We hypothesize that PCD occurrence is associated with the weakening of tuber AD, allowing early sprouting of mature lateral buds.
Asunto(s)
Apoptosis , Flores/citología , Meristema/citología , Tubérculos de la Planta/citología , Tubérculos de la Planta/crecimiento & desarrollo , Solanum tuberosum/citología , Solanum tuberosum/crecimiento & desarrollo , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Forma del Núcleo Celular/efectos de los fármacos , Frío , Fragmentación del ADN/efectos de los fármacos , Flores/efectos de los fármacos , Flores/ultraestructura , Hidrocarburos Bromados/farmacología , Meristema/efectos de los fármacos , Meristema/metabolismo , Meristema/ultraestructura , Datos de Secuencia Molecular , Oligopéptidos/farmacología , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Tubérculos de la Planta/efectos de los fármacos , Tubérculos de la Planta/ultraestructura , Preservación Biológica , Solanum tuberosum/efectos de los fármacos , Solanum tuberosum/ultraestructuraRESUMEN
Little is known about the coordinate induction of genes that may be involved in agriculturally important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using two diverse potato genotypes and two harvests (NDTX4271-5R and Russet Burbank tubers; 2008 and 2009 harvests). By 5 d after wounding, the closing layer and a nascent phellogen had formed. Phellogen cell divisions generated phellem layers until cessation of cell division at 28 d after wounding for both genotypes and harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB) and cyclin-dependent kinase regulatory subunit (StCKS1At) were induced by 1 d after wounding; these expressions coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) were dramatically up-regulated by 1-5 d after wounding, suggesting involvement with closing layer and later phellem cell layer formation. Wounding up-regulated pectin methyl esterase genes (StPME and StPrePME); StPME expression increased during closing layer and phellem cell formation, whereas maximum expression of StPrePME occurred at 5-14 d after wounding, implicating involvement in later modifications for closing layer and phellem cell formation. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening "tyrosine-and lysine-rich protein," suggested a role in the formation of the closing layer followed by phellem cell generation and maturation. Collectively, the genes monitored were wound-inducible and their expression profiles markedly coordinated with closing layer formation and the index for phellogen layer meristematic activity during wound periderm development; results were more influenced by harvest than genotype. Importantly, StTLRP was the only gene examined that may be involved in phellogen cell wall thickening after cessation of phellogen cell division.
Asunto(s)
Hidrolasas de Éster Carboxílico/genética , Ciclo Celular/genética , Pared Celular/enzimología , Proteínas de Plantas/genética , Tubérculos de la Planta/enzimología , Solanum tuberosum/citología , Solanum tuberosum/enzimología , Hidrolasas de Éster Carboxílico/metabolismo , Pared Celular/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Genotipo , Lípidos , Proteínas de Plantas/metabolismo , Tubérculos de la Planta/citología , Tubérculos de la Planta/genética , Polifenoles/metabolismo , Solanum tuberosum/genéticaRESUMEN
An increasing interest in carotenoids as nutritional sources of provitamin A and health-promoting compounds has prompted a significant effort in metabolic engineering of carotenoid content and composition in food crops. The strategy commonly used in plants is to increase the biosynthetic capacity by altering the carotenogenic enzyme activities. The recent isolation of the Or gene from a cauliflower orange mutant has brought a new endeavor for carotenoid enhancement by increasing the sink strength to sequester and store the synthesized carotenoids. Potato as one of the major staple crops usually accumulates low levels of carotenoids. In this chapter, we describe a detailed protocol for metabolic engineering of carotenoids in potato plants with the Or gene and the analysis of the Or transformants.