Figure 3 - uploaded by Pierre Hilson
Content may be subject to copyright.
Detection of nuclear GFP in cells of tomato fruit pericarp. Sections were prepared from 8-daa fruits. A and B, 35Spro:NLS:GUS- GFP tissues showing GFP signal in all fruit tissues. B, Magnified image showing localization of GFP in the nucleus. C and D, Fruit pericarp cross section from 8-daa tomato fruits transformed with PPC2pro:NLS: GUS-GFP showing preferential GFP localization in expanding cells from fruit mesocarp. D, Magnified image of mesocarp showing localization of GFP in polyploid nucleus from large cell. Bars = 400 m m in A and C and 100 m m in B and D.
Source publication
As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collections and ease of cultivation and transformation that enable the analysis of biological processes impossible to investigate in other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collectio...
Contexts in source publication
Context 1
... NLS fused to GFP proved very effective because the fluorescence signal was detected in the nucleus of all cell types in the pericarp and gel of 35Spro:NLS- GFP-GUS fruits (Fig. 3, A and B). In the pericarp of PPC2pro:NLS-GFP-GUS fruits, GFP was absent from the fruit epidermis and from most underlying divid- ing cells and was essentially limited to the large expanding mesocarpic cells (Fig. 3, C and D). This observation is consistent with the hypothesis that malate synthesized via PECase serves as a counterion for ...
Context 2
... because the fluorescence signal was detected in the nucleus of all cell types in the pericarp and gel of 35Spro:NLS- GFP-GUS fruits (Fig. 3, A and B). In the pericarp of PPC2pro:NLS-GFP-GUS fruits, GFP was absent from the fruit epidermis and from most underlying divid- ing cells and was essentially limited to the large expanding mesocarpic cells (Fig. 3, C and D). This observation is consistent with the hypothesis that malate synthesized via PECase serves as a counterion for potassium that accumulates in the vacuole, thus providing the driving force for fruit cell enlargement (Guillet et al., 2002). To conclude, in addition to the information pro- vided by promoter-GUS transgenic lines about ...
Context 3
... produced fruits that remained light orange during ripening and only turned light red when left on the plant (Fig. 4, D and E). Our observa- tions are consistent with the PPC2 profile documented through reporter expression and indicating that the promoter activity is the highest at the early stages of fruit development (Fig. 2) and in the mesocarp (Fig. 3C), i.e. at developmental stages and in fruit tissues less sensitive to light than the exocarp where the 35S- driven expression is very high (Fig. 3A) and the photobleaching more likely to occur. Our results fur- ther illustrate how fruit promoters and constructs developed herein can be used to target specific fruit tissues and alter ...
Context 4
... consistent with the PPC2 profile documented through reporter expression and indicating that the promoter activity is the highest at the early stages of fruit development (Fig. 2) and in the mesocarp (Fig. 3C), i.e. at developmental stages and in fruit tissues less sensitive to light than the exocarp where the 35S- driven expression is very high (Fig. 3A) and the photobleaching more likely to occur. Our results fur- ther illustrate how fruit promoters and constructs developed herein can be used to target specific fruit tissues and alter expression of candidate genes in ...
Citations
... Researchers have successfully developed a strong defense against Tomato mosaic virus (ToMV) infection by expressing solely the coat protein. This approach has also shown positive results in transgenic tomato plants, providing protection against several plant viruses such as Cucumber mosaic virus (CMV) and alfalfa mosaic virus [43,44]. Progress in engineering insect resistance in transgenic tomato has been achieved [45] and [46] (Table 4). ...
Diseases and pests have a substantial effect on tomato production, greatly affecting both the quantity and quality of this crucial vegetable crop. Although fungicides and insecticides have been important in controlling plant diseases and pests, their excessive usage raises significant environmental issues. Vegetable breeders are increasingly concentrating on developing cultivars with natural tolerance to biotic stresses to promote sustainability and environmental friendliness. The change in focus is intended to cultivate tomato cultivars with inherent resistance to diseases and pests, hence decreasing the need for chemical treatments. Advancements in creating high-yielding genetically resistant tomato cultivars are a result of detailed study on the genetic basis of pest and disease resistance in tomato crops, as well as the complex interactions between the host plant and pathogens. For effective breeding programs and pre-breeding activities, scientists and breeders must have access to sources of resistance and a thorough grasp of the genetic complexities involved. This requires examining the genetic composition of both the tomato plants and the different infections that are impacting them. Breeders may generate tomato cultivars with strong resistance to common diseases and pests by using the inherent defensive mechanisms found in certain tomato types via selective crossing. Continuing to study how hosts and pathogens interact and the molecular processes involved in resistance is crucial. This information offers vital insights on how to improve and expand resistance, leading to the creation of cultivars with long-lasting and wide-ranging resistance. Currently, the emphasis on breeding is a proactive and sustainable strategy for transfer of resistances in high yielding tomato cultivars. Researchers aim to develop tomato cultivars that provide high yield and demonstrate tolerance to changing disease and pest stresses by integrating genetic knowledge with sophisticated breeding methods. This comprehensive method protects tomato crops and encourages environmental sustainability by decreasing the need on chemical inputs in agriculture.
... Researchers have successfully developed a strong defense against Tomato mosaic virus (ToMV) infection by expressing solely the coat protein. This approach has also shown positive results in transgenic tomato plants, providing protection against several plant viruses such as Cucumber mosaic virus (CMV) and alfalfa mosaic virus [43,44]. Progress in engineering insect resistance in transgenic tomato has been achieved [45] and [46] (Table 4). ...
Diseases and pests have a substantial effect on tomato production, greatly affecting both the quantity and quality of this crucial vegetable crop. Although fungicides and insecticides have been important in controlling plant diseases and pests, their excessive usage raises significant environmental issues. Vegetable breeders are increasingly concentrating on developing cultivars with natural tolerance to biotic stresses to promote sustainability and environmental friendliness. The change in focus is intended to cultivate tomato cultivars with inherent resistance to diseases and pests, hence decreasing the need for chemical treatments. Advancements in creating high-yielding genetically resistant tomato cultivars are a result of detailed study on the genetic basis of pest and disease resistance in tomato crops, as well as the complex interactions between the host plant and pathogens. For effective breeding programs and pre-breeding activities, scientists and breeders must have access to sources of resistance and a thorough grasp of the genetic complexities involved. This requires examining the genetic composition of both the tomato plants and the different infections that are impacting them. Breeders may generate tomato cultivars with strong resistance to common diseases and pests by using the inherent defensive mechanisms found in certain tomato types via selective crossing. Continuing to study how hosts and pathogens interact and the molecular processes involved in resistance is crucial. This information offers vital insights on how to improve and expand resistance, leading to the creation of cultivars with long-lasting and wide-ranging resistance. Currently, the emphasis on breeding is a proactive and sustainable strategy for transfer of resistances in high yielding tomato cultivars. Researchers aim to develop tomato cultivars that provide high yield and demonstrate tolerance to changing disease and pest stresses by integrating genetic knowledge with sophisticated breeding methods. This comprehensive method protects tomato crops and encourages environmental sustainability by decreasing the need on chemical inputs in agriculture.
... These processes, especially the transfer process, challenge the genetic stability of foreign genes. At the molecular level, the integration site, gene fragment size, copy number, methylation, repeat sequences, trans-inactivation, and co-inhibition of foreign genes can all have an effect on their stable inheritance during asexual and sexual reproduction (Fernandez et al. 2009;Marjanac et al. 2009;Mette et al. 2000;Que et al. 1997). Polymerase chain reaction (PCR) screening of marker genes or reporter genes is one of the main methods used to detect foreign genes. ...
A lack of stability in the expression of Bacillus thuringiensis genes (CRY) and the dialaninophosphate resistance gene (BAR) in transgenic rice plants can lead to the loss of important characters. The genetic stability of transgenic expression in high-generation lines is thus critically important for ensuring the success of molecular breeding efforts. Here, we studied the genetic stability of resistance to insect pests and herbicides in transgenic rice lines at the molecular and phenotypic levels in a pesticide-free environment. Southern blot analysis, real-time polymerase chain reaction, and enzyme-linked immunosorbent assays revealed high stability in the copy numbers and expression levels of CRY1C, CRY2A, and BAR in transgenic lines across different generations, and gene expression levels were highly correlated with protein expression levels. The insecticide resistance of the transgenic rice lines was high. The larval mortality of Chilo suppressalis was 50.25% to 68.36% higher in transgenic lines than in non-transgenic control lines. Percent dead hearts and percent white spikelets were 16.66% to 22.15% and 27.07% to 33.47% lower in transgenic lines than in non-transgenic control lines, respectively. The herbicide resistance of the transgenic rice lines was also high. The bud length and root length ranged were 2.53 cm to 4.20 cm and 0.28 cm to 0.73 cm higher in transgenic lines than in non-transgenic control lines in the budding stage, respectively. Following application of the herbicide Basta, the chlorophyll content of the transgenic lines began to recover 2 d later in the seedling and tillering stages and 3 d later in the booting and heading stages, by contrast, the chlorophyll content of the non-transgenic lines did not recover and continued to decrease. These findings revealed high genetic stability of the resistance to insect pests and herbicides across several generations of transgenic rice regardless of the genetic background.
... Micro-Tom. This causes photobleaching mainly on fruit and leaves expressing that PDS gene has been knocked out [20]. Similarly, a clear manifestation of color change can be observed in Vb-15 to validate lycopene cyclase gene knockout using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated protein-9 (CRISPR/Cas9) vector system delivered by Agrobacterium tumefaciens. ...
... From the entry vector, the M13 primer pair was used to amplify the coding sequence flanked by the att sites using the same PCR conditions described above. The purified product was used for the recombination with pK7m24GW,3 and pEN-L4-PPC-R1 plasmids (Fernandez et al. 2009) via LR clonase II Plus (Thermo Scientific). Recombination was confirmed by amplifying the fragment flanked by the PHOSPHOE-NOLPYRUVATE CARBOXYLASE 2 (PPC2) promoter and AtVTE5 or AtVTE6 using specific primer pairs with the same PCR conditions described above. ...
... In order to address whether phytol recycling is a rate-limiting step for tocopherol accumulation in tomato fruits, transgenic lines expressing AtVTE5 (AT5G04490) or AtVTE6 (AT1G78620) under the control of the fruit-specific PHOS-PHOENOLPYRUVATE CARBOXYLASE 2 (PPC2) promoter were generated (Fig. 1A). This promoter is highly active during the phase of rapid fruit growth, corresponding to cell expansion, and its activity gradually decreases during ripening (Fernandez et al. 2009). The background genotype used for the transformation harbors the wild-type SlGLK2 allele; thus, the pedicellar portion of fruits properly develops chloroplasts, while they are poorly differentiated in the stylar portion (Powell et al. 2012). ...
Tocopherols are potent membrane-bound antioxidant molecules that are paramount for plant physiology and also important for human health. In the past years, chlorophyll catabolism was identified as the primary source of phytyl diphosphate for tocopherol synthesis by the action of two enzymes, PHYTOL KINASE (VTE5) and PHYTHYL PHOSPHATE KINASE (VTE6) that are able to recycle the chlorophyll-derived phytol. While VTE5 and VTE6 were proven essential for tocopherol metabolism in tomato fruits, it remains unknown whether they are rate-limiting steps in this pathway. To address this question, transgenic tomato plants expressing AtVTE5 and AtVTE6 in a fruit-specific manner were generated. Although ripe transgenic fruits exhibited higher amounts of tocopherol, phytol recycling revealed a more intimate association with chlorophyll than with tocopherol content. Interestingly, protein–protein interactions assays showed that VTE5 and VTE6 are complexed, channeling free phytol and phytyl-P, thus mitigating their cytotoxic nature. Moreover, the analysis of tocopherol accumulation dynamics in roots, a chlorophyll-devoid organ, revealed VTE5-dependent tocopherol accumulation, hinting at the occurrence of shoot-to-root phytol trafficking. Collectively, these results demonstrate that phytol recycling is essential for tocopherol biosynthesis, even in chlorophyll-devoid organs, yet it is not the rate-limiting step for this pathway under normal growth conditions.
... The obtained binary plasmids were transferred to A. tumefaciens strain GV3101::pMP90 and the resulting strains were used to transform tomato (cv. Micro-Tom) cotyledons (Fernández et al., 2009). The presence of the transgenes in kanamycin-resistant tomato plants (T0) was checked by PCR amplification of a 1137-bp fragment extending from the 3 ′ region of the NPTII gene to the 5 ′ -end of the amiSGT foldback coding sequence, and a 686-bp fragment extending from the 5 ′ -end of the amiSGT foldback coding sequence to the 3 ′ end of the tNOS, using primers shown in Supplementary Table S1 and leaf genomic DNA obtained using the cetyltrimethyl ammonium bromide (CTAB) method (Richards et al., 1994). ...
Free and glycosylated sterols localize in the plant cell plasma membrane where in combination with other lipids regulate its structure and function. The role of glycosylated sterols in regulating membrane-associated biological processes is more relevant in plants like tomato (Solanum lycopersicum) where glycosylated sterols are the predominant sterols. A proper ratio of free sterols versus glycosylated sterols has proven to be essential for proper plant performance in several species, but almost nothing is known in tomato. To assess the role of glycosylated sterols in tomato plant and fruit development, we generated transgenic lines of tomato cultivar Micro-Tom expressing two different amiRNAs devised to silence STEROL GLYCOSYLTRANSFERASE 1, the most actively expressed of the four genes encoding sterol glycosyltransferases in this plant. STEROL GLYCOSYLTRANSFERASE 1 gene silencing caused moderate plant dwarfism and reduced fruit size. Analysis of the profile of glycosylated sterols throughout fruit development demonstrated that the maintenance of proper levels of these compounds during the early stages of fruit development is essential for normal fruit growth, since reduced levels of glycosylated sterols trigger a transcriptional downregulatory response that affects genes involved in processes that are critical for proper fruit development, such as seed filling, cell wall extension and auxin signaling.
... The aim of this work was to evaluate the effect of overexpression of GGP and GPP in tomatoes during plant growth and fruit ripening. We employed one constitutive, 35S-CaMV and two different fruit-specific promoters, PPC for green fruit and PG for pink and red fruit (Fernandez et al., 2009). We also shed more light on whether GGP and GPP are involved (or not) in the regulation (positive or otherwise) of the biosynthesis and/or accumulation of AsA during tomato fruit ripening and development. ...
L-Ascorbic acid (AsA), a strong antioxidant, serves as an enzyme cofactor and redox status marker, modulating a plethora of biological processes. As tomato commercial varieties and hybrids possess relatively low amounts of AsA, the improvement of fruit AsA represents a strategic goal for enhanced human health. Previously, we have suggested that GDP-L-Galactose phosphorylase (GGP) and L-galactose-1-phosphate phosphatase (GPP) can serve as possible targets for AsA manipulation in tomato (Solanum lycopersicon L.) fruit. To this end, we produced and evaluated T3 transgenic tomato plants carrying these two genes under the control of CaMV-35S and two fruit specific promoters, PPC2 and PG-GGPI. The transgenic lines had elevated levels of AsA, with the PG-GGP1 line containing 3-fold more AsA than WT, without affecting fruit characteristics. Following RNA-Seq analysis, 164 and 13 DEGs were up- or down-regulated, respectively, between PG-GGP1 and WT pink fruits. PG-GGP1 fruit had a distinct number of up-regulated transcripts associated with cell wall modification, ethylene biosynthesis and signaling, pollen fertility and carotenoid metabolism. The elevated AsA accumulation resulted in the up regulation of AsA associated transcripts and alternative biosynthetic pathways suggesting that the entire metabolic pathway was influenced, probably via master regulation. We show here that AsA-fortification of tomato ripe fruit via GGP1 overexpression under the action of a fruit specific promoter PG affects fruit development and ripening, reduces ethylene production, and increased the levels of sugars, and carotenoids, supporting a robust database to further explore the role of AsA induced genes for agronomically important traits, breeding programs and precision gene editing approaches.
... Plasmids pEn-Chimera/ASAT1 and pEn-Chimera/PSAT1 were recombined with pDE-Cas9-Kan R destination vector yielding plasmids pDE-Cas9 ASAT1 and pDE-Cas9 PSAT1 (Figure 1), which were transferred to the A. tumefaciens strain GV3101:: pMP90 prior to transform tomato (cv. Micro-Tom) cotyledons (Fernandez et al., 2009). The presence of the transgenes in kanamycin-resistant tomato plants (T0) was checked by PCR amplification of a 442-bp fragment of the AtU6-26 promoter using primer pairs shown in Supplementary Table 1 and leaf genomic DNA isolated with the cetyltrimethyl ammonium bromide (CTAB) method (Richards et al., 1994). ...
Steryl esters (SE) are stored in cytoplasmic lipid droplets and serve as a reservoir of sterols that helps to maintain free sterols (FS) homeostasis in cell membranes throughout plant growth and development, and provides the FS needed to meet the high demand of these key plasma membrane components during rapid plant organ growth and expansion. SE are also involved in the recycling of sterols and fatty acids released from membranes during plant tissues senescence. SE are synthesized by sterol acyltransferases, which catalyze the transfer of long-chain fatty acid groups to the hydroxyl group at C3 position of FS. Depending on the donor substrate, these enzymes are called acyl-CoA:sterol acyltransferases (ASAT), when the substrate is a long-chain acyl-CoA, and phospholipid:sterol acyltransferases (PSAT), which use a phospholipid as a donor substrate. We have recently identified and preliminary characterized the tomato (Solanum lycopersicum cv. Micro-Tom) SlASAT1 and SlPSAT1 enzymes. To gain further insight into the biological role of these enzymes and SE biosynthesis in tomato, we generated and characterized CRISPR/Cas9 single knock-out mutants lacking SlPSAT1 (slpsat1) and SlASAT1 (slasat1), as well as the double mutant slpsat1 x slasat1. Analysis of FS and SE profiles in seeds and leaves of the single and double mutants revealed a strong depletion of SE in slpsat1, that was even more pronounced in the slpsat1 x slasat1 mutant, while an increase of SE levels was observed in slasat1. Moreover, SlPSAT1 and SlASAT1 inactivation affected in different ways several important cellular and physiological processes, like leaf lipid bo1dies formation, seed germination speed, leaf senescence, and the plant size. Altogether, our results indicate that SlPSAT1 has a predominant role in tomato SE biosynthesis while SlASAT1 would mainly regulate the flux of the sterol pathway. It is also worth to mention that some of the metabolic and physiological responses in the tomato mutants lacking functional SlPSAT1 or SlASAT1 are different from those previously reported in Arabidopsis, being remarkable the synergistic effect of SlASAT1 inactivation in the absence of a functional SlPSAT1 on the early germination and premature senescence phenotypes.
... These processes, especially the transfer process, challenge the genetic stability of foreign genes. At the molecular level, the integration site, gene fragment size, copy number, methylation, repeat sequences, trans-inactivation, and co-inhibition of foreign genes can all have an effect on their stable inheritance during asexual and sexual reproduction (Fernandez et al. 2009;Marjanac et al. 2009; Mette et al. 2000; Que et al. 1997). Polymerase chain reaction (PCR) screening of marker genes or reporter genes is one of the main methods used to detect foreign genes. ...
A lack of stability in the expression of Bacillus thuringiensis genes ( CRY ) and the dialaninophosphate resistance gene ( BAR ) in transgenic rice plants can lead to the loss of important characters. The genetic stability of transgenic expression in high-generation lines is thus critically important for ensuring the success of molecular breeding efforts. Here, we studied the genetic stability of resistance to insect pests and herbicides in transgenic rice lines at the molecular and phenotypic levels in a pesticide-free environment. Southern blot analysis, real-time polymerase chain reaction, and enzyme-linked immunosorbent assays revealed high stability in the copy numbers and expression levels of CRY1C , CRY2A , and BAR in transgenic lines across different generations, and gene expression levels were highly correlated with protein expression levels. The insecticide resistance of the transgenic rice lines was high. The larval mortality of Chilo suppressalis was 50.25% to 68.36% higher in transgenic lines than in non-transgenic control lines. Percent dead hearts and percent white spikelets were 16.66% to 22.15% and 27.07% to 33.47% lower in transgenic lines than in non-transgenic control lines, respectively. The herbicide resistance of the transgenic rice lines was also high. The bud length and root length ranged were 2.53 cm to 4.20 cm and 0.28 cm to 0.73 cm higher in transgenic lines than in non-transgenic control lines in the budding stage, respectively. Following application of the herbicide Basta, the chlorophyll content of the transgenic lines began to recover 2 d later in the seedling and tillering stages and 3 d later in the booting and heading stages, by contrast, the chlorophyll content of the non-transgenic lines did not recover and continued to decrease. These findings revealed high genetic stability of the resistance to insect pests and herbicides across several generations of transgenic rice regardless of the genetic background.
... To address this goal, we used the OP/LhG4 trans-activation system (Moore et al., 1998) to silence TKN-II genes in different stages of fruit development. First, three fruit-specific promoter:LhG4 lines: pCRC:LhG4 (Fernandez et al., 2009), p2A11:LhG4 (Gupta et al., 2021), and pE8:LhG4 (see methods) were crossed to an OP:NLS-mRFP reporter line, to characterize their spatial and temporal expression patterns. The F1 ovaries and fruits were collected at five-day intervals and imaged under a fluorescent stereoscope starting at anthesis. ...
... A, Digitally extracted images displaying the expression patterns of fruit-specific promoters used for developmental stage-specific knockdown of CLASS-II KNOX genes visualized via the NLS-mRFP signal (red). Promoter CRC (Fernandez et al., 2009) top row, promoter 2A11(Gupta et al., 2021 middle row, and promoter E8 bottom row. The first column shows equatorial anthesis-stage carpel sections (bar = 100 mm), the second column shows equatorial 10 DAP fruit sections (bar = 500 mm), the third column shows sectors of equatorial "BR" fruit sections (bar = 500 mm), the fourth column shows sectors of equatorial "RR" fruit sections (bar = 500 mm). ...
... Tomato (S. lycopersicum) M82 cultivar was used as a control and transformation background for the transgenic 35S:amiR-TKN-II, promoter E8:LhG4, OP:amiR-TKN-II lines used in this study. The artificial micro-RNA constructs were generated as described in Supplemental Figure S2 and by Alvarez et al. (2006) using the 35S promoter (Fernandez et al., 2009) or OP/LhG4 system (Moore et al., 1998) in BJ36 plasmid (Eshed et al., 2001) same system was used to subclone 1,095 bp sequence upstream of the Solyc09g089580 (E8) to generate the promoter E8:LhG4 driver. The transgenic tomato plants were generated using previously described protocols (McCormick, 1997). ...
Fruits can be divided into dry and fleshy types. Dry fruits mature through senescence and fleshy fruits through ripening. Previous studies have indicated that partially common molecular networks could govern fruit maturation in these different fruit types. However, the nature of such networks remains obscure. CLASS-II KNOX genes were shown to regulate the senescence of the Arabidopsis (Arabidopsis thaliana) dry fruits, the siliques, but their roles in fleshy-fruit development are unknown. Here, we investigated the roles of the tomato (Solanum lycopersicum) CLASS-II KNOX (TKN-II) genes in fleshy fruit ripening using knockout alleles of individual genes and an artificial microRNA line (35S:amiR-TKN-II) simultaneously targeting all genes. 35S:amiR-TKN-II plants, as well as a subset of tkn-II single and double mutants, have smaller fruits. Strikingly, the 35S:amiR-TKN-II and tknII3 tknII7/+ fruits showed early ripening of the locular domain while their pericarp ripening was stalled. Further examination of the ripening marker-gene RIPENING INHIBITOR (RIN) expression and 35S:amiR-TKN-II rin-1 mutant fruits suggested that TKN-II genes arrest RIN activity at the locular domain and promote it in the pericarp. These findings imply that CLASS-II KNOX genes redundantly coordinate maturation in both dry and fleshy fruits. In tomato, these genes also control spatial patterns of fruit ripening, utilizing differential regulation of RIN activity at different fruit domains.
