Transcriptional alterations associated with overexpression of a chlorogenic acid pathway gene in eggplant fruit

1 Introduction

Eggplants are the same plant family members as tomatoes and peppers, i.e., Solanaceae and the cultural practices and production techniques are almost like those plants (Welbaum, 2015; Knapp and Peralta, 2016). Eggplants originated in India and continued to grow as a wild type, but thanks to breeding techniques that improved these wild varieties' yield, appearance, taste, and color to be consumed and cooked easily (Page et al., 2019). Eggplants have a high amount, i.e., 95 % of chlorogenic acid, a phenolic compound that has proven beneficial for humans and the plant itself (Docimo et al., 2016). Phenolic acids benefit human health significantly because they protect against diseases like diabetes, cancer, and rheumatoid arthritis. Similarly, these phenolic acids are valuable for plants because they help protect the plant against insects and pathogen infestations (Kaushik et al., 2015). Moreover, a high percentage of chlorogenic acid is present in Eggplant compared to other vegetables of Solanaceae.

As a result, increasing the amount of chlorogenic acid in eggplant is a significant breeding objective currently being pursued (Caruso et al., 2017). Hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (SmHQT) plays a critical role in catalyzing chlorogenic acid synthesis (Kaushik and Saini, 2019). While cultivated eggplants contain fewer phenolic acids than wild ones, using wild species in the eggplant breeding program requires additional time to eliminate undesirable genes resulting from the severed connection. Also, planting wild species is difficult because of the duration of the plants and their adaptability (Kaushik et al., 2017). Agrofiltration is the most common technique that allows the expression of transient genes in plant networks (Ahmad et al., 2012). The agroinfiltration approach for gene expression is regularly performed in fleshy fruits such as tomatoes, strawberries, melons, and cucumber and succeed (Guidarelli and Baraldi, 2015). Therefore, changes in the expression of SmHQT gene via agroinfiltration in the eggplant fruits will open new insights.

Several breeding techniques have been adopted for enhancing the effect of chlorogenic acid, but they gave fewer results (Garg et al., 2018; Lu et al., 2020). Certain unwanted genes also interfere with chlorogenic acid production in wild relatives of eggplants, and certain techniques were unable to eradicate these undesirable genes. Recently, certain molecular approaches have enhanced certain genes' qualitative effects by genome editing and transgenic technology (Docimo et al., 2016). With transgenic technology, it is now possible to give sequence-specific traits. Thus, the aim of this study is to enhance SmHQT gene expression in eggplant fruit flesh (Sinha et al., 2019). This P19 gene is well-known for its ability to prevent post-transcriptional gene silence from occurring. As a result, it was also included in the vector (Chu et al., 2000).

The transcripts in the transgenic plant can be contrasted by RNA-seq, as this approach may provide valuable insight into the expression of a transgene (Mahdavi Mashaki et al., 2018; Kaundal et al., 2021). Therefore, here RNA Sequencing (RNA Seq) dependent gene expression of agroinfiltrated fruits were compared. This is because the agroinfiltration method is straightforward, cheap and has maximum transformation efficiency than any other method. With this technique's help, we can efficiently study the function of genes involved in different pathways. Thus, with the help of a low-cost system, we can overexpress the gene of interest, i.e., the one related to chlorogenic acid for its production.

2 Materials and methods

3 Results

3.1

3.1 Agroinfiltration assay and HPLC

Similarly, temporary expressions are also carried out with original promoters. GMO plants are obtained by transforming fruits mediated from Agrobacterium tumefaciens to the greenhouse, 3 DAI. Among fruits that expressed SMHQT too much, some fruits appeared phenotypic variations compared to normal plants. Next, the chlorogenic acid content in agroinfiltrated fruit is more than double the normal fruit (Fig. 1).

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Fig. 1

Comparison of chlorogenic acid concentration (g/mg of FW) of agroinfiltrated plant SmHQT and control fruit samples.A.

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3.3

3.3 Degs analysis and enrichment of gene ontology (GO)

As a result, the difference in DEGs between the OE and control genotypes is determined (Fig. 2). Both conditions generated 3166 and 134 DEGs; however, the DEGs expressed in only one condition were 3 1238 and 1794, respectively, and were significantly expressed in agroinfiltrated and control plants (Fig. 2). The differential expression of each transcript in control vs agroinfiltrated fruits is detailed in Table S1, as are the transcripts themselves. This resulted in discovering about 694 DEGs that were downregulated in response to OE; however, it is unknown if any genes in the control group were silenced. Additionally, it was determined that OE inhibits the synthesis of 169 DEGs, indicating that OE serves as a negative regulator of the chlorogenic biosynthetic pathway. Furthermore, the GO analysis identified a large number of functional categories (Fig. 3), including catalytic activity, transporter activity, and transcription regulatory activity (Fig. 3). As with the cellular component category, when the OE genotype is compared to the control fruit sample, a significant number of DEGs are selected in the OE genotype (Fig. 3).

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Fig. 2

Venn diagrams illustrating the expression of DEG genes in and among control and agroinfiltrated fruits. DEG values were calculated using NGS sequencing data (A). Transcriptome heat map of control vs agroinfiltrated eggplant fruit (B).

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Fig. 3

Gene Ontology (GO) terms as classified based on molecular function, cellular component and biological process that were discovered in the eggplant fruits overexpressing the SmHQT gene.

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4 Discussion

Eggplant is an essential vegetable in India it is preferred because of its added nutrition advantages and ease of cooking (Taher et al., 2017). Chlorogenic acid is the most common phenolic acid discovered in this eggplant. It is also the most critical and valuable phenolic acid for both the plant and people since it nourishes humans while also protecting the plant from certain pests and viruses (Manach et al., 2004). So far, the trials conducted regarding the enhancement of chlorogenic acid were not very successful and up to the mark. Our study has chosen the agroinfiltration technique to improve the chlorogenic acid content in eggplant fruits (Šilarová et al., 2019). The standardized protocol for the agroinfiltration method was selected. Based on this standardized protocol, we have incorporated the gene for chlorogenic acid synthesis and its enhancement in the fruits of eggplant. By using the agroinfiltration technique, which is very cheap, easy and less laborious to induce the gene of interest into the plant parts and with the help of this, the expression of the gene SmHQT is enhanced because it is the key factor for the synthesis of chlorogenic acid (Yang et al., 2000; Lee and Yang, 2006).

Agroinfiltration is a compatible genetic expression or gene silencing process. It could be used as an alternative to Agrobacterium-mediated traditional approach to producing enzymes used for industrial use and very beneficial in detecting fruit tissue gene functions and protein development (Gleba et al., 2013; Krenek et al., 2015). This approach quickly offers detailed knowledge about gene function. In recent agro-infiltration research, valuable insights into several essential processes, such as promoting activity, gene expression, subcellular protein location, and metabolism, have been given. Agroinfiltration is a flexible process related to RNA-seq that can be used for plant crops, which already have standardized agroinfiltrated protocols (strawberries, tomatoes and grapes) (Rigano et al., 2013; Chialva et al., 2016). Many plants, including the transgenic development of eggplant with healthy gene expression, can take time and can be difficult. For the first time, Eggplant was transformed by Guri and Sink (1988) with a transformation of genes mediated by agrobacterium. Numerous fruitful attempts have been made to refine this technique and produce those transgenic eggplants with different important characteristics such as tolerance to Colorado potato beetle (Arpaia et al., 1997), root-knot nematode (Papolu et al., 2016), and shot and fruit borer (Rai et al., 2013). Although the technology for integrating foreign genes into eggplant has advanced significantly, studies have been conducted to optimize the transformation protocol to produce transgenic plants resistant to abiotic stress like tolerance to salt, drought, and chilling, as well as osmotic stress (Saini and Kaushik, 2019; Ahmad et al., 2019; Kohli et al., 2019; Mansoor et al., 2022). Additionally, several beneficial agronomic traits have been transferred to eggplant through transgenic technologies (Singh et al., 2010).

Processed functional food additives have gained appeal in part due to their incorporation into well-known food products. The protocol for agroinfiltration of SmHQT genes to increase the chlorogenic acid content of eggplant is described here. Eggplant output has risen, as has their appeal. Additionally, bioactive chemicals may be added “naturally” to food products. This effective functionalization results in extra health advantages, which may be achieved via the intentional addition of bioactive substances to dietary items (Sonker et al., 2017). Biotechnology is a parallel advancement that does not confine farmers and clinicians to a small, precarious natural development (Winickoff et al., 2005; Bertheau, 2015). Vegetable genetic modifications to produce a higher concentration of bioactive compounds and incorporate functional foods have unmatched reliability and characteristics. It remains to be seen how this innovation can respond to its participants' varied needs, including the realistic food industry (Pinela et al., 2016; Kamiloglu et al., 2021). Nonetheless, the opportunities for a thriving functional food market and a balanced community are undoubtedly worth pursuing.

5 Conclusion

Eggplants are immense vegetables that are from the Solanaceae family. They are high in valuable phenolic compounds, including chlorogenic acids. In this study, high-quality verbalization research was used to extend the spectrum of eggplant's chlorogenic dangerous component. This agroinfiltration method has gained popularity because of its superiority in fast quality exchange by skipping the typical replication cycle. In a utilitarian assessment of SmHQT, it was concluded that overexpression of SmHQT increased chlorogenic acid content, making it ineffective as a phenolic acid controller. Other interaction partner features discovered using our RNAseq data may restrict the eggplant during overexpression, but this is not a guarantee of success. Using agroinfiltration, we were able to increase the content of chlorogenic acid in eggplants. To evaluate and analyse its efficacy, the SmHQT gene may be overexpressed in the flesh of an agroinfiltrated eggplant fruit. Our findings suggest that agroinfiltration increases the quantity of a key phenolic acid, chlorogenic acid, leading to the overexpression of the SmHQT gene and therefore enhancing the nutritional quality of eggplant fruits.

6 Consent to participate

All authors consent to participate in this manuscript.

7 Consent for publication

All authors consent to publish this manuscript in Journal of King Saud University - Science.

8 Availability of data and material

Data will be available on request to corresponding or first author.

Author contributions

AS drafted the experimental design, performed the experiments. PK, MNA, SA worked in data collection, data analysis and initial draft of manuscript text. PYY revised the manuscript. All authors read the manuscript before communication.