Introduction
Shelf life of food is the maximum time that the food produce can be stored, during which duration the quality remains acceptable for storage.
Food industries face major challenges in storage and transportation after harvesting of fruits due their highly perishable nature. Ripening is followed by decaying due to accumulation of toxic components which Is the result of increased microbial growth resulting from increased water content due to ripening. The fact that the fruits start ripening at a particular time makes it very difficult to transport fruits since they would ripen and get decayed even before reaching the customer. Prevention of postharvest loss during transportation, storage and selling is critical. It is estimated that more than 25-30% of fruits are lost in the post harvesting stage. Post-harvest deterioration can be decreased by altering the storage conditions.
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Various methods like freezing, heating, drying, modification in your storage conditions and chemical methods are used to prevent spoilage. But these methods cannot assure to prevent the ripening process and also would lead to change in the texture and taste. Hence various recombinant DNA techniques like RNAi and VIGS are being explored to increase the shelf-life of fruits.
Recombinant DNA methods to increase the shelf life of fruits
1. RNAi
RNA interference is a conserved mechanism that involves triggering the silencing of a gene by degradation of Double stranded RNA. This involves mechanism of generation of small interfering RNA through RNAase III, DICER which are small fragments of 20-23 nucleotides. These short fragments mediate the degradation of complementary homologous RNA. The challenge is higher plants with RNAi is that the RNA fragments larger than 30 nucleotides are considered to trigger an anti-viral response leading the host cells shutting down the whole translation response and resulting in the cells leading to non-specific degradation of RNA transcripts.
2. VIGS
Virus infected plants were found to be resistant to infection by the same or closely related strains of same virus. The phenomenon found in plants was coined as cross protection. The molecular basis of this phenomenon was PTGS (Post- transcriptional gene silencing) which is a scenario that results in sequence specific degradation of endogenous mRNAs.
The term Virus Induced gene Silencing was first used by A. van Kammen, to explain the natural process of recovery of plants from Viral infections.
The potential of VIGS is recognized as a tool for downregulation of genes, through degradation of the transcripts. A DNA fragment with minimum of 23 nucleotides, with 100% identity to the targeted gene is a necessity for the silencing to occur.
VIGS involves cloning of the targeted gene sequence into a viral vector which is transferred into the plants or detached fruits. Various vectors have been used as vehicles in VIGS. Vectors of Tobacco Mosaic Virus (TMV), Potato Virus X(PVX) and Tobacco Rattle virus are a few examples which can be used for both protein expression and gene silencing. But some vectors have potent anti-silencing proteins that interfere with the host silencing machinery.
Ethylene
Fruits produce a component, ethylene that is responsible for the ripening of fruits which results is changes in texture, taste and color of fruits. The process of ethylene production is called the ethylene biosynthesis pathway.
A highly conserved regulated pathway, which is carried out by enzyme ACS synthase (1-aminocyclopropane-1-carboxylase synthase). ACS synthase is encoded by a multigene family that show different patterns of growth in response to external factors like weather and temperature. Ethylene is derived from Methionine which is converted into S-adenosylmethionine by S-adenosylmethionine synthetase which is then converted into 1-aminocyclopropane-1-carboxylic acid (ACC) and 5¢-deoxy-5¢methylthioadenosine (MTA) by the enzyme 1-aminocyclopropane-1-carboxylase synthase (ACS) which is the rate limiting step in the ethylene biosynthesis pathway. The final step of conversion is converting the ACC into ethylene, CO2 amd cyanide and this step is catalyzed by ACC oxidase enzyme. The cyanide produced by this reaction is detoxified into b-cyanoalanine by the enzyme b-cyanoalanine synthase, which prevents the accumulation of high concentrations of cyanide.
Ethylene production increases dramatically at number of stages such as abscission, germination and fruit ripening. Also certain hormone expression such as auxin, brassinosteroids, and cytokinin also play an important role in the production of ethylene. Ethylene production is also auto regulated where the production can be auto-stimulated or auto-inhibited. Light also can regulate production of ethylene.
Increase in The Shelf life of tomatoes
Tomatoes are one of the most abundantly consumed fruits, the world consumption of tomatoes is extremely high. It is also one of those fruits that ripens soon, hence decaying soon. It is estimated that more than 30% of the whole produce is wasted due to ripening and decaying also making the transportation very difficult. Hence increasing the shelf life of tomatoes will improve the texture, taste and quality for consumers at the same time it will also ease the transportation for suppliers.
Ethylene Biosynthesis in Tomatoes
The ACS synthase is coded by 8 genes is tomatoes, regulated by various abiotic and biotic factors. In most of the climatic fruits the ethylene biosynthesis has two systems. Syste 1 operated during vegetative growth, during which ethylene inhibits its own biosynthesis
And system 2 occurs during the ripening of climatic fruit and during in some during senescence of petals. This feedback mechanism is proposed to integrate ripening of the entire fruit once it has commenced. The mature green fruit only has LE-ACS6 gene expressed but it doesn’t express after the breaker stage. LE-ACS2 and LE-ACS4 are two genes that are expressed after the breaker stage. The literature survey states that LE-ACS2 gene has the highest steady state level of RNA expression and that expression of only LE-ACS2 gene is dependent on ethylene. It has been proposed that the genes responsible for the system 1 are LE-ACS1A and LE-ACS6 in the green fruit. And when ripening is induced LE-ACS1A expression increases and LE-ACS4 is induced. Expression of LE-ACS2 is solely responsible for initiation and maintenance of ethylene biosynthesis. ACS genes are mostly auxin and cyclohexamide regulated. The stability of ACS activity in tomato varied during fruit ripening. In particular, the half-life of ACS activity in green tomato pericarp tissue was shorter than that in ripening pericarp tissue.
Methods used for Extension of shelf-life
Whole genome sequencing: WGS is a technique used for analysis of the complete genetic makeup of a particular organism. It involves the sequencing of both mitochondrial and nuclear genomes and chloroplasts for plants. While capillary approach was one initially used for sequencing of the whole genome, since 2005, the capillary approach has been replaced by high throughput sequencing techniques such as illumina dye sequencing, pyrosequencing and SMRT sequencing. Shotgun strategy, parallelization and template generation via genome fragmentation is the basis that these techniques adapt. Whole genome sequence can generate and provide raw data of an organism’s complete genetic makeup. However, further analysis is required in order to provide biological or functional meaning of this data, such as how this knowledge can be used to prevent diseases. This approach can be used to understand the genetic makeup and function of different genes in tomato and to recognize one gene regulating which can delay the ripening process.
VIGS
The gene that needs to be regulated is recognized and selected using Whole genome sequencing. It has been proposed that LeACS2 gene encodes a key speed restricting enzyme in the ethylene biosynthesis pathway in tomato fruit and suppression of LeACS2 will result in strong inhibition of ethylene production. Thus this gene is the most potential candidate for downregulation in order to delay the ripening process.
The vector of choice is TRV (tobacco rattle virus), this is the vector that is abundantly used for the purpose of gene transfer. VIGS vectors are constructed by cloning a fragment of gene of interest with efficient siRNA generation and no off-target genes into the modified viral genome. And the recombinant virus is then introduced into the plant or fruit using Agarobacterium tumefacians mediated transient expression, in vitro transcribed RNA inoculation or direct RNA inoculation. The transgene gets amplified along with viral RNA either by an endogenous or a viral RNA-dependent RNA polymerase (RdRp) enzyme generating dsRNA molecules after the recombinant virus is inserted into the plant cells. DICER- like enzymes recognizes these molecules which cleave the dsRNA into fragments of 21-25 nucleotides called as small interfering RNAs (siRNA). These siRNA are then recognized by RSIC (RNA induced silencing complex). The RSIC uses the single stranded siRNA and identifies complementary RNA sequences in the cells and then degrades them.
Vector pTRV2 was inserted with 707bp LeACS2 gene using restriction enzymes and cloning methodologies. The resultant vector pTRV2-LeACS2, along with vector pTRV1 in the ratio 1:1 were inserted into Agarobacterium tumefacians through which they were transferred into the plant or separated fruit. The in-filtered fruit is expected to remain yellow at least for the duration of a month. The content of 1-aminocyclopropane-1-carboxylic acid can be measured to confirm the extent. To confirm the Lescs2 suppression at molecular level, semi quantitative RT PCR can be used.
RT-PCR
Real-time also called qPCR (quantitative PCR) that combines amplification of target DNA sequence with the quantification of the concentration of the DNA in the reaction. The primers that anneal outside the region of the LeACS2 gene targeted should be used. Housekeeping gene was used as a comparison for gene regulation quantification. The RNA of the tomato is extracted and cDNA is synthesized using Reverse Transcriptase PCR and then this cDNA is used as a template for real time PCR. The results of Real Time PCR is represented in graphical format of the representation of accumulation of PCR products over time. The data obtained in the form of Ct values has to be used to determine the levels of expression.
The same approach can be used for other fruits for their respective genes. VIGS has been shown to occur for a shorter period of approximately 3 weeks and the efficiency decreases after a month resulting in partial or complete recovery of plants from the silencing. Advantages of VIGS:
VIGS is one of the most widely used plant functional genomics tools. Key metabolic and regulatory genes that are required for plant survival that cannot be studied by mutant analyses can be readily analyzed by VIGS. VIGS that occurs for a short duration of the life cycle of a plant (few weeks) as short-duration conventional VIGS. first advantage is easy and rapid gene silencing. Second, no need for stable plant transformation. Third, partial sequence information is sufficient to silence a gene. Fourth, VIGS can be used for both forward and reverse genetics. Fifth, VIGS can be used to silence genes with multiple copies [e.g. genes from polyploid plants such as wheat (Triticum aestivum)] or multiple family members. Sixth, VIGS can be used to assess the function of genes whose mutation (or antisense-mediated knockdown) is lethal in sexually propagated plants. VIGS-mediated transmission of the gene silencing phenomenon to progeny seedlings can be referred to as ‘non-integration-based transmissible PTGS’. This method has several advantages over conventional short-duration VIGS. One advantage is fewer viral symptoms in the progeny. This has been shown for at least two VIGS vectors, namely TRV and BSMV (H.S. Bennypaul, PhD thesis, Washington State University, 2008). However, the mechanism behind mild viral symptoms in the progeny plants is not yet known. A second advantage is the ability to silence genes that express during seed dormancy, seed germination and seedling emergence. Third, complete systemic silencing can be achieved in root, stem and the cotyledons of a seedling. Finally, seedling vigor, a yield-attributing parameter, can be studied under biotic and abiotic stress.
Challenges and Remedies
Lack of appropriate VIGS vectors
VIGS cannot be used in some plant species because of the lack of appropriate VIGS vectors. This can be overcome in two ways. One way is to use heterologous gene sequences, wherever possible, from plant species that are recalcitrant to VIGS to silence genes in a closely related VIGS-amenable species
Lack of an efficient method for virus vector delivery
Agrobacterium-mediated delivery of binary VIGS vectors is efficiently used in many dicot plants. However, for plants that are recalcitrant to Agrobacterium-mediated transformation, VIGS can be induced by the virus sap inoculation method or RNA transcript inoculation or DNA bombardment. Sap inoculation method involves multiplication of the virus in virus-friendly N. benthamiana or an appropriate host plant and then inoculation of the target species with the sap.
Lack of silencing in certain tissues
Uneven or localized VIGS resulting in a lack of silencing in certain tissues is mainly the result of ineffective virus movement. This can be addressed by maintaining environmental conditions favoring systemic virus movement
Interference from viral symptoms with interpretation of data
Symptoms caused by a higher virus titer can interfere with the interpretation of data. VIGS vectors that produce severe symptoms in host plants should be avoided. Selection of an appropriate virus–host system where viral symptoms are less obvious is important. In forward genetics screens, silencing of certain plant genes can allow more viral replication resulting in severe developmental phenotypes. In such large-scale studies, the viral titer can be quickly estimated by using a Chenopodium (or any local lesion host plant) leaf lesion assay to determine if the phenotype is caused by the silencing of a plant gene or if it is the result of more virus accumulation. Apart from local lesion assay, techniques such as northern blot, ELISA and immunocapture RT-PCR can be used for more specific quantification of excessive virus levels in gene-silenced plants.
Off-target silencing
Off-target silencing can be minimized by careful selection of an insert gene sequence by using publicly available software. The software ‘siRNA-scan’ has a search environment with several integrated components, including a sequence similarity search to identify potential off-targets and efficiency estimation of siRNAs. This software can find a potential region that generates efficient siRNAs for the target gene with no sequence similarity to any other off-target genes in the searched database. Therefore, this can be used to design VIGS constructs or siRNAs with minimal off-targets. If off-targets are predicted using this or a similar computational approach, the trigger sequence which can avoid a perfect siRNA match of less than 11 bp for a predicted off-target gene should be selected for VIGS. A nucleotide identity of less than 11 bp between siRNA and target mRNA reduces chances of silencing. This should prevent off-target effects while maintaining adequate complementarity for target gene silencing.
Low silencing efficiency
Gene silencing in many plants is affected by gene target position, insert length and orientation. Recent studies conclude that for optimum VIGS, insert lengths should be in the range of ∼200 bp to ∼350 bp. Furthermore, siRNA-scan software can be used to assess the potency of 21 nt siRNA generation of a gene fragment before cloning into a VIGS vector. This assessment helps to select highly efficient gene fragments for silencing. A recent study showed that localized expression of a viral silencing suppressor improves virus multiplication and this can increase virus threshold to initiate silencing in distal plant parts. Use of short-inverted repeats of inserts has been shown to enhance silencing in Turnip yellow mosaic virus (TYMV) and Tobacco mosaic virus (TMV)-based VIGS systems.
Limitations inherent to VIGS
Although several of the above-mentioned shortcomings of conventional VIGS have been overcome, a few of them are inherent to VIGS and remain a challenge. Some examples are described below. First, the presence of a virus vector can interfere with the metabolism of the plant and affect results from some plant–microbe interaction studies. Hence, apart from using appropriate controls, it is sometimes necessary to confirm VIGS results using other gene disruption approaches. Second, gene insertion in the VIGS vector can hinder virus multiplication and many viruses are known to delete the gene insert during multiplication and spread. Third, most viral vectors are excluded from meristematic tissue and, therefore, gene silencing in the meristem is not possible in most instances. Fourth, VIGS often results in incomplete silencing of a target gene. Although this is indeed a limitation, a potential trade-off between existences of such a limitation and need for rectifying it should be considered. Because of this characteristic, VIGS is more amenable to study gene functions associated with plant development that would be otherwise lethal and intractable in mutant plants. Finally, genotype of a plant species can affect the performance of VIGS construct. Hence, specific standardization of a VIGS protocol is required for each genotype in some plant species.
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