Puducherry: A team of researchers led by the head of the food science and technology department of said they have developed a simple, rapid and cost-effective method to detect of fruits. Artificial ripening of fruits using calcium commonly called as ‘powder’ or ‘masala’ has been banned under Prevention of Food Adulteration Act and Food Safety and Standards Act. Consumption of fruits ripened artificially using calcium carbide may cause serious health hazards. The team, led by Prathap Kumar Shetty, developed a sensor solution (bio-functionalized gold nanoparticle).
When the solution is mixed with water with which the fruits have been washed, the solution changes colour, if the fruits had been ripened using calcuim carbide. This can be used to prove whether the fruits were ripened using calcium carbide. The team has filed a patent for their invention at regional patent office, Chennai.
The other members of the team were PhD student Ankita J Lakade and postdoctoral scientist K Sundar. “The sensor solution can be used by anyone without any technical expertise. The procedure is very simple and cost effective. Wash the test fruit with 10ml of water and take 1ml of the wash and mix with equal volume of sensor solution in a glass test tube and mix. The change in colour of solution from red to purple indicates that calcium carbide is used for ripening the fruit.
No change in colour of the solution indicates that calcium carbide was not used for ripening the fruit,” said Shetty. He said reputed laboratories have begun validating the sensor solution. “The sensor solution will be released soon after validation. It is a timely invention as there have been a lot of concerns among the consumers on the artificial ripening of fruits such as mangoes. The authorities could not enforce the regulation due to lack of simple analytical methods to ascertain artificial ripening of fruits,” said Shetty. He added that he was motivated to evolve a simple technique to detect artificial ripening of fruits when he served as member of a scientific panel on analysis at Food safety and standards authority of India (FSSAI). He is currently a member of a panel on contaminants in food chain at FSSAI.
The cost of an analysis is less than 20 paise, Shetty said. “The cost will be much lower if the sensor solution is synthesized in commercial scale,” he added. Explaining the concept of the sensor solution, Shetty said calcium carbide contains high levels of arsenic.
“Fruits ripened using calcium carbide always have higher amounts of arsenic than the naturally-ripened fruits. We developed a cheap sensor solution, which exhibits visible changes in colour when it comes in contact with arsenic hydride.
The solution is red in colour and it charges to purple when arsenic hydride comes in contact with it,” he said. The same team had earlier developed a new technique to detect early stages of spoilage of milk under practical condition so that milk can be used before it is spoiled completely.
IntroductionFruit ripening is a complex and irreversible developmental process that involves numerous metabolic, biochemical, physiological and organoleptic alterations. Among these changes, ripening leads to fruit softening, accumulation of sugars, volatile compounds and pigments, reduction of organic acids, etc., making the fruit more attractive for animal consumption, and therefore, facilitating seed dispersal.Fleshy fruits are classified as climacteric or non-climacteric, depending on whether or not they produce autocatalytic ethylene, respectively. Thus, climacteric fruits such as tomato, apple, avocado, and banana are characterized by an increase in the respiration rate and a burst of ethylene at the onset of ripening. In contrast, in non-climacteric fruits, which include strawberry, grape, citrus, and pepper among others, ethylene production remains at low levels and there is no dramatic change in respiration.The role of phytohormones and the transcriptional regulation of climacteric and non-climacteric fruit ripening have been extensively reviewed in the last few years (;;; ). In particular, ethylene perception and signaling have been very well characterized, especially in tomato ( Solanum lycopersicum), which is the most studied model system for fruit ripening (; ). In contrast, the regulatory network involved in non-climacteric fruit ripening has been much less studied. Nevertheless, it is known that abscisic acid rather than ethylene is essential in the control of ripening in strawberry (; ), the established model for non-climacteric fruits.Fruit ripening is of major economic importance for agriculture.
One of the main challenges for producers is to offer a product at the ripening stage with a good flavor and nutritional value, while also having sufficient shelf life to maintain its quality until it is consumed. This is especially relevant for climacteric fruits highly sensitive to ethylene, and for non-climacteric fruits such as strawberry, which become quickly inedible. Thus, improved ripening and shelf life has been a focus of interest for many scientists in recent decades, using conventional breeding and genetic modification. However, the latter relies on the generation of transgenic plants, which have a very limited commercial use due to the current skepticism of consumers and restrictive government policies. Moreover, transgenic strategies have been based on the modulation of gene expression, which may lead to temporary and/or incomplete knockdown effects, unpredictable off-target effects, and too much background noise.
However, the availability of genome editing tools offers new opportunities to overcome these drawbacks. The Emergence of Genome-Editing TechnologyIn the past decade, new and powerful approaches have emerged enabling the precise editing of a gene of interest.
These approaches are based on the use of nucleases that are targeted to a specific sequence to generate a double-strand break (DSB). DSBs trigger two different repair mechanisms: (i) error-prone non-homologous-end-joining (NHEJ) and (ii) homology-directed repair (HDR). While NHEJ repair results in InDel mutations of variable lengths, HDR can be used to introduce specific point mutations or a sequence of interest, through recombination supplying an exogenous donor template.
To obtain DSBs for genome editing, four major classes of customizable DNA-binding proteins have been engineered so far: meganucleases , zinc-finger nucleases (ZFNs) , transcription activator-like effector nucleases (TALENs) , and RNA-guided DNA endonuclease Cas9. Meganucleases, ZFN, and TALEN rely on the binding and recognition of the nucleases to specific sequences of DNA. Therefore, these approaches require complex engineering processes to produce custom nucleases that target the sequence of interest.
First Genome Editing Approaches in TomatoIn the case of dicot crops, tomato became the ideal candidate for gene editing because of its several advantages, i.e., (i) diploid and high-quality sequenced genome, (ii) ease of transformation, , and (iii) economic importance, being the fourth most important commercial crop in the world. The first reports on genome editing in tomato appeared in 2014 when CRISPR/Cas9 was applied to effectively perform gene functional analysis by stable root transformation, using Agrobacterium rhizogenes. This study was followed by two others, where CRISPR/Cas9 and TALENs were applied to generate mutations in complete plants for the first time, in particular for the ARGONAUTE7 ( SlAGO7) and PROCERA ( PRO) genes, respectively (; ). As in most of the pioneer studies of genome editing in any species, both genes had been functionally characterized already, allowing the functional validation of these new approaches. Particularly, PRO encodes for a DELLA protein that acts as a negative regulator of gibberellin (GA) signaling (; ).
Characterized the vegetative stage of the TALEN-induced pro mutants, which showed a phenotype consistent with an increased GA response, such as tall and slender plants. Besides their role in plant growth and development, the role of GAs in fruit set and fruit ripening have been widely studied.
In fact, a previous report where the spontaneous pro mutant was phenotypically characterized, showed that fruit ripening was significantly delayed and that the Brix index value was higher in the mutant , consistent with a higher GA response in these plants. Therefore, it would be expected that fruit ripening was also altered in the TALEN-induced pro mutants, though did not characterize the fruit phenotype.Two years later, the ZFNs gene editing tool was applied for the first time in tomato to mutagenize the LEAFY COTYLENDON1-LIKE4 ( LIL4) gene, which encodes for a subunit of a heterotrimeric transcription factor. Mutation in LIL4 resulted in a pleiotropic phenotype, including fruits with different sizes and shapes, a reduced number of locules, and absence of placenta. Furthermore, fruits with a paler color and slower ripening were obtained , although how LIL4 regulates this processes is still unknown.
RIN – How CRISPR/Cas9 Converted a Loss-of-Function into a Gain-of-Function MutationA large number of studies have been focussed in the role of different transcription factors (TFs) involved in the ripening process. One of the most investigated TFs for ripening is RIPENING-INHIBITOR gene ( RIN), a member of the SEPALATA ( SEP) class of the MADS-box gene family, first discovered half a century ago when a mutation in this locus ( rin) was found to cause a failure to ripen in tomato (; ). RIN is induced early during ripening, and regulates ethylene-dependent and ethylene-independent pathways that promote ripening. The effect on ripening of the rin mutation has been commercially exploited as hybrid cultivars ( RIN/rin) with an extended shelf life. Due to the importance and clear phenotype of the rin mutation, the RIN locus has been recently targeted using CRISPR/Cas9 to validate the functionality and inheritance of mutations mediated by this approach in tomato. Designed three CRISPR/Cas9 constructs to mutagenize three different regions within the RIN locus. As expected, CRISPR/Cas9-mediated novel mutations at the RIN locus resulted in an inhibition of fruit ripening at the T0 generation.
However, and contrary to the rin mutant, these CRISPR mutants partially initiated the ripening process, and this was interpreted as the result of the presence of wild-type RIN in the T0 generation. Previously, generated knockdown RIN plants using RNA interference, resulting in a fruit ripening that was only partially suppressed, in contrast to the green rin mutant phenotype, and interpreted as due to residual expression of RIN.
Thus, RIN has been considered so far to function as an essential regulator of ripening, and the models have always been based on the idea that rin was a loss-of-function mutation. However, a recent paper has overturned this model.
Firstly, unlike the rin mutant, homozygous CRISPR/Cas9-mediated knockout rin mutants ( RIN-KO) did not fail to ripen, reaching a pale red color. Moreover, a molecular and physiological characterization of these lines showed that most ripening-related parameters were less affected than in the rin mutant. These results suggested that, contrary to what has been considered so far, ripening can be initiated independently of RIN.
Furthermore, they also suggested that the rin mutant protein may have gained a new function, as a partial dominant negative mutation that blocks the initiation of ripening. This hypothesis was supported by the fact that rin mutant allele encodes for a in-frame fusion of RIN and Macrocalyx coding sequences , the latter containing a putative repression motif that might convert rin into a transcriptional repressor. This repressor activity was actually confirmed in vitro. Consistent with this hypothesis, use of CRISPR/Cas9 to generate additional mutations in the rin mutant allele ( rinΔN) resulted in fruits that recovered the initiation of ripening, showing a similar phenotype to that of RIN-KO lines. Furthermore, a molecular and physiological characterization of rinΔN lines showed a partial recovery of most of the ripening markers.
Thus, this study proposes that the rin mutant protein would impair the DNA-binding and activation of ripening-related genes by other master regulators such as NONRIPENING (NOR), COLORLESS NONRIPENING (CNR) (; ), or other SEP homologs.In conclusion, the use of a gene editing approach such as CRISPR/Cas9 has allowed generating alternative knockout alleles, which have changed our current model of fruit ripening, with RIN being necessary to initiate this process, and rin being a loss-of-function mutation. This implies that many results obtained in the past should be reconsidered, and that further experiments should be carried out now that we are closer to defining the actual mechanism. Targeting Fruit TextureWhile RIN/rin hybrid plants are widely used by tomato breeders, the incomplete ripening of these hybrid fruits leads frequently to a poor flavor and a reduced nutritional value. Hence, to modify texture characteristics for an improved shelf life, without reducing tomato organoleptic and nutritional quality, has been a challenge for researchers and breeders for many years.
Fruit softening depends on cell-wall modifying proteins such as polygalacturonase, pectin methylesterase, endo-β-(1,4)-glucanase, β-galactosidase, and expansin. A number of studies have characterized the effect of silencing the expression of genes encoding these proteins in strawberry , and tomato (;,;;;;; ). Silencing of the polygalacturonase gene had no apparent effect on tomato fruit softening (;, ), but it did affect the firmness of strawberry fruits, which even showed a slightly higher °Brix. For the rest of the genes, silencing of their expression has had only very small or no detectable effects on both tomato or strawberry fruit ripening (;;;; ).
However, silencing another cell-wall related protein, the pectate lyase (PL), has been successfully applied for the modulation of fruit firmness in both species. PL silencing increased fruit firmness without changes in color, size, total soluble solids, or metabolites influencing taste and aroma in both strawberry , and tomato. Particularly in tomato, preliminary analysis of CRISPR/Cas9-induced pl mutants has shown an effect on fruit firmness without altering color and soluble solids content. A further characterization would be necessary to confirm that these CRISPR/Cas9 mutant lines maintain other important agronomical characteristics such as aroma, flavor, yield, color, and resistance to pathogens, all required traits for a successful introduction to the market. Targeting Photoperiodic ResponseAn appropriate timing of flowering is not only essential for plant reproductive success but also to optimize yield in agriculture.
In a search for the genetic component controlling the different day-length sensitivities regulating flowering in tomato, Soyk and colleagues identified SELF PRUNING 5G ( SP5G) as the responsible gene. SP5G is a FLOWERING LOCUS T-like gene that works as a floral repressor controlling flowering under long-day conditions. In this study, the authors generated CRISPR/Cas9-mediated mutations for this gene, obtaining plants that flowered earlier under long-day conditions. Another gene, SELF-PRUNING ( SP), is an ortholog of Arabidopsis TERMINAL FLOWER 1 ( TFL1) and encodes for another flowering repressor in tomato. The sp mutation revolutionized tomato cultivation since it leads to determinate plants with a synchronized burst of flowering and fruit ripening. In order to obtain faster-flowering and determinate growth plants, Soyk and collaborators used CRISPR/Cas9 to generate double sp5g sp mutants, which showed an earlier flowering burst and an earlier fruit ripening than that of the sp single mutant and the wild-type. However, the earlier tomato ripening in the double sp5g sp mutant might be caused simply by the earlier flowering time phenotype of these plants.
Therefore, further studies on fruit ripening dynamics need to be performed to clarify whether SP5G actually modulates actively this process. Targeting Post-Transcriptional RegulationThere are several previous studies demonstrating the importance of post-transcriptional regulation by non-coding RNAs in the control of fruit ripening (;; ). To investigate further, CRISPR/Cas9 has been employed in two studies to identify and characterize post-transcriptional regulators of tomato fruit ripening.In plants, long non-coding RNAs (lncRNAs) are important regulators of gene expression, as they interact with DNA, RNA and proteins.
Interestingly, two lncRNAs, lncRNA1459 and lncRNA1840, have been recently associated with tomato fruit ripening. To investigate further the role of lncRNA1459 in fruit ripening, this gene was stably knocked-out by using CRISPR/Cas9, obtaining CR- lncRNA1459 mutant lines with a delayed fruit ripening. A molecular characterization of this mutant showed that key ripening-related genes involved in lycopene and ethylene biosynthesis, and in signal transduction were down-regulated. Consistently, CR- lncRNA1459 mutant fruits showed a reduction in lycopene accumulation and an inhibition of ethylene production.
Artificial Ripening Of Fruits
However, the mechanism and target genes of lncRNA1459 in its regulation of fruit ripening still need clarification.Another post-transcriptional regulation involved in ripening that has been recently explored is RNA editing. In flowering plants, RNA editing by cytidine-to-uridine (C-to-U) conversion is a widespread process that occurs only in plastids and mitochondrial transcripts and plays an important role in developmental processes such as organelle biogenesis, adaptation to environmental changes and signal transduction. In a recent report, aimed to identify RNA editing factors that might play an essential role in the regulation of tomato fruit ripening. A virus-induced gene silencing (VIGS) assay was performed, targeting 11 RNA editing factor genes putatively related to ripening, positively identifying SlORRM4, which is located in mitochondria.
Consistently, CRISPR/Cas9-mediated stable slorrm4 mutants resulted in a delay of ripening, and in a diminution of the respiratory rates and ethylene production compared with the wild-type. Further molecular characterization showed that slorrm4 mutation results in a down-regulation of genes associated with the Krebs cycle and mitochondrial function, and a decrease in the level of proteins essential for the mitochondrial respiratory chain, supporting the essential role of mitochondria in the regulation of ripening. However, the specific mechanisms linking RNA-editing in mitochondria with ripening requires further investigation. Future PerspectivesBesides ripening, other interesting agronomic traits have been modulated recently in tomato using CRISPR/Cas9, such as parthenocarpy (; ), lycopene content , and fruit size, inflorescence branching and plant architecture. Especially relevant is the work of, in which, instead of editing CDS loci, they targeted cis-regulatory elements (CREs) in promoters, obtaining quantitatively different phenotypes.
CRE mutations are widespread in nature and have notably contributed to crop domestication through the alteration of gene expression levels. Thus, targeting CREs with genome-editing technologies offers the possibility to fine-tune gene expression without the common pleiotropic effects observed in complete loss-of-function mutants, opening the door to enhance variability for important agronomic and quality traits. However, a lack of precise knowledge about functional motifs in CREs hampers the current application of this approach.In addition to tomato, the CRISPR/Cas9 gene-editing strategy has been successfully applied in several fruit crop species to date, including examples of climacteric ripening species, such as apple (; ), banana and kiwifruit , and non-climacteric ripening species, such as sweet orange (; ), Duncan grapefruit (, ), grapevine (;; ), watermelon , cucumber , and the woodland and cultivated strawberries (; Table ). Most of these studies have targeted either the Phytoene Desaturase gene ( PDS), or genes to improve pathogen resistance. However, they have opened up the possibility of using CRISPR/Cas9 technology to study or improve fruit ripening in these crops. Among them, strawberry is a species of particular interest because the fast softening of the berries is the main cause of its short shelf life and the source of commercial losses.
Hence, the successful application of gene editing using the CRISPR/Cas9 approach may provide effective solutions for these postharvest issues.It is important to highlight that all these studies are based on the generation of new random mutations mediated by the NHEJ mechanism. However, homologous recombination-based gene targeting (GT) allows a more precise genome editing. GT has already been successfully achieved in several crops, including tomato (; ). In a recent study, GT has been used to extend tomato shelf life by the incorporation of alcobaca ( alc) , an allelic mutation of NOR with a lower impact on fruit quality than nor and rin mutations , into a wild-type genotype. Despite these successful studies, GT is still a very challenging approach due to its low efficiency.
Thus, the optimization of GT in higher plants in general, and in crop species in particular, would provide of a much wider spectrum of possibilities for breeding, allowing the introgression of genes of interest into elite breeding germplasm.In conclusion, genome editing strategies, especially CRISPR/Cas9 are becoming rapidly more efficient and precise. Their application to the coding sequences of TFs, hormones or metabolites biosynthetic enzymes, and hormone receptors, or, alternatively to CREs of these genes may allow a more precise fruit ripening modulation (Figure ).
Importantly, genome editing tools have the possibility of removing transgenes through self- or backcrossing, an important advantage compared to traditional approaches for genetic modification. Moreover, preassembled Cas9 protein-gRNA ribonucleoproteins (RNPs) remove the likelihood of inserting recombinant DNA into the host genome. This particular approach has been demonstrated in the protoplasts of several plant species (;;; ) as a strategy that could be potentially operated outside existing GMO regulatory criteria and gain acceptance from consumers.
However, the recent decision of the Court of Justice of the European Union is a major setback to innovation in EU agriculture, considering the “process” instead of the “product” and putting crops created using gene-editing techniques under GMO regulations (Directive 2001/18/EC). Hopefully, these regulatory decisions will be reconsidered in the future, as there are unquestionable advantages of gene editing to address the challenge of ensuring sufficient food supply for an increasing global population in the current changing climatic conditions.