WO2024134685A1

WO2024134685A1 - A recombinant vector comprising 4cl11 orf from ocimum kilimandscharicum, and implementations thereof

Info

Publication number: WO2024134685A1
Application number: PCT/IN2023/051211
Authority: WO
Inventors: Ashok Prabhakar GIRI; Santosh Govind LAVHALE; Kirtikumar Ramesh KONDHARE; Rakesh Shamsunder JOSHI
Original assignee: Council Of Scientific And Industrial Research
Priority date: 2022-12-21
Filing date: 2023-12-21
Publication date: 2024-06-27

Abstract

The present disclosure relates to a recombinant vector. Particularly, the present disclosure relates to a recombinant vector to overexpress 4-coumarate Co-enzyme A ligase 11 gene (4CL11) in plant cells. Further, the present disclosure relates to a binary expression vector comprising a pRI101AN vector with the 4CL11 ORF sequence from Ocimum kilimandscharicum and regulatory elements for overexpressing host 4CL11 gene in plant cells, which results in the accumulation of flavonoids (kaempferol and quercetin as well as their glycoside derivatives), altered lignin content, increased natural product biosynthesis that eventually leads to reduced auxin levels and decreased expression of auxin transport and signaling genes, causing reduced root growth.

Description

A RECOMBINANT VECTOR COMPRISING 4CL11 ORF FROM OCIMUM KILIMANDSCHARICUM, AND IMPLEMENTATIONS THEREOF

TECHNICAL FIELD OF THE INVENTION

The present disclosure relates to a recombinant vector. Particularly, the present disclosure relates to a recombinant vector to overexpress 4-coumarate Co-enzyme A ligase 11 gene (4CL11) in plant cells. Further, the present disclosure relates to a binary expression vector comprising a pRUOlAN vector with the 4CL11 ORF sequence from Ocimum kilimandscharicum and regulatory elements for overexpressing host 4CL11 gene in plant cells, which results in reduced root growth, altered lignin content, and increased natural product biosynthesis.

BACKGROUND AND PRIOR ART OF THE INVENTION

Secondary metabolites are extensively distributed in the plant kingdom. They play an essential role in the growth, development, and reproduction of plants. Moreover, they are known to impart protection to plants against their competitors via allelopathy.

Phenylpropanoids are a large class of plant secondary metabolites derived from aromatic amino acid phenylalanine in most plants or tyrosine in partial monocots, which are produced in response to various biotic or abiotic stress conditions. These stress conditions are comprised of pathogen infections, wounding, UV irradiation, exposure to ozone/pollutants, and other hostile environmental conditions. Several genes and their gene products are enzymes involved in the phenylpropanoid biosynthesis pathway and they show differential molecular responses against numerous stress conditions. Many of them are also known to have or are being explored for medicinal properties.

4-coumarate CoA ligase (EC 6.2.1.12) (4CE) is a crucial enzyme involved in the phenylpropanoid pathway as well as lignin biosynthesis. It serves as the main branch point in the pathway. They are encoded by a multigene family of adenylate-forming enzymes. 4CL convert hydroxy or methoxy cinnamic acid derivatives to the corresponding activated thioesters. Products of 4CL are utilized by various oxygenase, reductases, and transferases for the biosynthesis of lignin, flavonoids, anthocyanins, tannins, aurones, stilbenes, coumarins, suberin, cutin, sporopollenin, etc. It has been reported that 4CLs might play a crucial role in lignin biosynthesis. Lignin is the polymer of H-lignin, G-lignin, and S-lignin monomers. Also, several nonstructural phenylpropanoids synthesized via 4CLs exhibit diverse functions in plant physiology. 4CL enzymes contain two conserved peptide motifs: box I (SSGTTGLPKGV) and box II (GEICIRG). Members of the adenylate-forming enzyme family have a conserved adenosine monophosphate binding domain (box I). Crystal structure analysis suggested that during the catalysis process, enzymes undergo two conformations: adenylate forming and thioester forming. 4CL isoforms with differential substrate affinities can be used to manipulate metabolite flux. The expression of 4CL isoforms can be modulated in response to a specific trigger.

Biocatalytic properties of 4-coumarate-CoA ligase (4CLs) have been explored in the past for commercial applications in fuel, flavor, and natural product synthesis.

One of the reports is PCT Publication No WO1999024561, which describes a method to genetically alter plants through downregulation of 4-coumarate Coenzyme A ligase (4CL Another report Yohei Katsuyama et al. in a research study published in Journal of general microbiology, 2008 titled “Production of curcuminoids by Escherichia coli carrying an artificial biosynthesis pathway” disclosed that the curcuminoids biosynthesis in Escherichia coli can be achieved by expressing 4CL from Lithospermum erythrorhizon and curcuminoid synthase from rice.

Another report, US Patent No. 9187757 covers a method for reducing lignin biosynthesis in a plant by suppressing the expression of 4-coumarate-CoA ligase (4CL) genes or 4CL enzymes.

Another report by Y. Wang et al. in a research study published in Journal of the American Chemical Society, 2011 titled “Structural and Kinetic Analysis of the Unnatural Fusion Protein 4-Coumaroyl-CoA Ligase: Stilbene Synthase” discloses that the resveratrol biosynthesis can be improved in yeast by fusion of A CLl from A. thaliana and stilbene synthase from Vitis vinifera (VvSTS).

Another report by J.H. Jung et al. in a research study published in Plant Molecular Biology, 2016 titled “Precision breeding for RNAi suppression of a major 4-coumarate: coenzyme A ligase gene improves cell wall saccharification from field-grown sugarcane” discloses that the silencing of lignin biosynthesis-specific 4CL isoform can cause a reduction in lignin content.

Based on the literature pertaining to P-glucosidase, it appears there is a need in the art to find an alternative process to overexpress the 4-coumarate-CoA ligase for commercial applications.

OBJECTS OF THE INVENTION

Accordingly, the main objective of the present disclosure is to provide a recombinant vector for overexpressing 4-coumarate Co-enzyme A ligase gene (4CL) in host cells.

An objective of the present disclosure is to provide a recombinant vector comprising 4CL11 ORF sequence from Ocimum kilimandscharicum for overexpressing the host 4CL11 gene in plant cells.

Another objective of the present disclosure is to provide a recombinant binary expression vector comprising a pRUOlAN vector comprising Ok4CLll ORF sequence and regulatory elements, which on integration with the plant cell genome is responsible for overexpressing the host 4CL11 gene in plant cells.

Yet another objective of the present disclosure is to provide a method for transforming the pRIlOlAN vector comprising Ok4CLll ORF sequence and regulatory elements into plant cells. Yet another objective of the present disclosure is to provide a method for overexpressing host 4CL11 gene in plant cells by transforming the pRIlOlAN vector comprising Ok4CLll ORF sequence and regulatory elements into plant cells.

SUMMARY OF THE INVENTION

Accordingly, aspects of the present disclosure relate to a recombinant vector. Particularly, the present disclosure relates to a recombinant vector to overexpress 4-coumarate Co-enzyme A ligase 11 gene (4CL1 ) in plant cells. Further, the present disclosure relates to a binary expression vector comprising apRIlOlAN vector with \hc 4CLl I ORF sequence from Ocimum kilimandscharicum and regulatory elements for overexpressing host 4CL11 gene in plant cells results in reduced root growth, altered lignin content and structure, and increased natural product biosynthesis.

In an aspect of the present disclosure, the host 4CL11 in plant cells is overexpressed by transforming a binary expression vector comprising an open reading frame (1731 bp) of 4- coumarate- Co-enzyme A Ligase 11 gene isolated from Ocimum kilimandscharicum (Ok4CLi / ) and one or more transcriptional regulatory sequence into the plant cell, and allowing the vector to integrate into the genome by non-homologous recombination, thus allowing overexpression of the host 4CL11.

In another aspect of the present disclosure, the host 4CL11 gene is overexpressed by transforming a binary expression vector pRIlOlAN comprising Ok4CLll ORF and one or more transcriptional regulatory sequence into the plant cell, and allowing the vector to integrate into the genome by non-homologous recombination, thus allowing overexpression of the host 4CL11. In another aspect of the present disclosure, the Ok4CLll ORF sequence is cloned into the pRIlOlAN vector using the restriction enzymes Ndel and EcoRI to form the pRUOlAN- Ok4CLl /-OE construct.

In yet another aspect of the present disclosure, the pRUOl M -Ok4CLl /-OE construct is flanked by a 35S CaMV promoter for driving the expression of Ok4CLll ORF sequence, followed by the expression of host 4CL11 gene, 5' untranslated region (UTR) of alcohol dehydrogenase gene from Arabidopsis thaliana, and Nopaline synthase terminator. In still another aspect of the present disclosure, the overexpression of host 4CL11 further helps to increase the level of flavonoids (kaempferol and quercetin-derivatives), lignin, and natural product biosynthesis, which may further lead to a reduced root growth phenotype.

In yet another aspect of the present disclosure, the pRI101AN-C>£4CL77-OE construct effects overexpression of host 4CL11 in plant cells selected from but not limited to Nicotiana benthamiana, Arabidopsis thaliana or potato plants resulting in rootless or reduced root growth phenotype of said plants that present disclosure. Plants can be multiplied at a large scale in plant tissue culture using stem nodal cuttings and used in hydroponic, aeroponic, and vertical farming for commercial applications.

DETAILED DESCRIPTION OF THE DRAWINGS

Figure 1: depict (A) schematics of Ok4CL7, -11 and -15 overexpression constructs in pRIlOlAN vector under 35S promoter; (B) Gel electrophoresis showing the confirmation of Ok4CL7, -11 and -15 genes cloning in pRIlOlAN vector by EcoRI and Ndel restriction enzymes digestion.

Figure 2: depict the procedure for the generation of transgenic N. benthamiana plants using leaf as explant for Agrobacterzum-mediated transformation protocol.

Figure 3: depict the screening of putative transgenic lines using RT-PCR: (A) Ok4CLll and (B) Ok4CL7 and Ok4CL15. Gel A (Well No. 1-9: 0k4CLll-0E-Nb (cDNA as template); 10: WT-Nb (cDNA as template); 11: positive control for Ok4CLll PCR (Plasmid); 12: negative control for Ok4CLll PCR; 13: 100 bp ladder); Gel B (Well No. 1 and 8: 100 bp DNA ladder; 2-4: Ok4CLll-Nb (cDNA as template); 5 and 15: (cDNA as template); 6: positive control for Ok4CL7 PCR (Plasmid); 7: negative control for Ok4CL7 PCR; 9-14: Ok4CL15-Nb plants; 16: positive control for Ok4CL15 PCR (Plasmid); 17: negative control for Ok4CL15 PCR) Figure 4: depict the gene expression of Ok4CLs in the individual overexpression lines of N. benthamiana using semi-quantitative PCR: (A) Ok4CL7. (B) Ok4CLll, and (C) Ok4CL15. N. benthamiana L23P was used as a reference gene. The PCR cycle number used for gel imaging is 25. Complementary DNA (cDNA) prepared from wild-type (WT) N. benthamiana plant was used as negative control for PCR.

Figure 5: depict (A) analysis of root phenotypes of N. benthamiana plants overexpressing Ok4CL7, -11 or -15 gene compared to wild-type plants (WT); (B) the number of primary roots per plant; (C) the lateral roots on the primary root; (D) the primary root length (cm). Ok4CLll- OE1 and -2 are the two independent overexpression lines of Ok4CLll, whereas wild-type (WT) is a non-transformed plant of N. benthamiana. Ok4CL7-OE\ and Ok4CL/5-OE\ are the individual overexpression lines of Ok4CL7 and -15 constructs, respectively. Figure 6: depict (A) Relative abundance of auxins (indole-3-acetic acid and indole- 3 -propionic acid), flavonoids and their glycosides (kaempferol, kaempferol-3 -rhamnoside [K3R], kaempferol-3, 7-O-bis-alpha-L-rhamnoside [K3,7R], quercetin 3-[2-caffeoylglucuronide]) in Ok4CLll-OE lines (OE1 and OE2) and wild-type N. benthamiana plants; (B-C) Lignin accumulation in Ok4CL7, -11 and -15 OE lines and wild-type N. benthamiana plants. (D) Expression analysis of phenylpropanoid pathway genes and flavonoid- specific glycosyltransferases. (E) Expression analysis of auxin transport and signaling genes.

Figure 7 (A-E): Depicts the overexpression line confirmation of Q 4CL-1 and -11 in potato (Solanum tuberosum cv. Desiree), and the resultant reduced root growth phenotypes by Ok4CLll overexpression in the potato plant. No such phenotype was observed for Ok4CL7- OE lines. Figure 7B Well Nos: 1-4 Ok4CL7-OE line confirmation; 8-13: Ok4CLll-Q line confirmation; 5: wild type potato plant; 6: Negative control for PCR; 7: positive control Ok4CL7 (166 bp); 14: positive control Ok4CLll (136 bp).

Figure 8: Depicts the schematic diagram of the method of producing rootless phenotype in Nicontiana benthamiana plant.

Figure 9 (A-F): Depicts the supplementation of flavonoids (kaempferol and quercetin) and their glycosides (R, K3,7R and Q3G) delays AR emergence in wild-type Nicotiana benthamiana.

Figure 10 (A-C): Depicts the reduced root growth phenotype in Ok4CLll -OE lines of Arabidopsis thaliana, ecotype Col-0.

SOURCE OF BIOLOGICAL MATERIAL:

Nicotiana benthamiana was taken from CSIR-NCL, Pune, Maharashtra, India. Ocimum kilimandscharicum used to isolate the 4-coumarate Co-enzyme A ligase 11 (Ok4CLl 1) polynucleotide was obtained from CSIR-NCL, Pune, Maharashtra, India. Binary expression vector pRIlOl AN was obtained from DSS Takara Bio India Pvt. Ltd., New Delhi, India. Potato (Solanum tuberosum cv. Desiree) were obtained from the Indian Institute of Science Education and Research (IISER) Pune, and grown in plant tissue culture facility at the CSIR-NCL, Pune (Maharashtra, India). Arabidopsis thaliana, ecotype Col-0 seeds were germinated in vitro on MS medium and plantlets obtained were grown in the plant growth room at the CSIR-NCL, Pune (Maharashtra, India).

DETAILED DESCRIPTION OF THE INVENTION:

As used herein, the terms “Ok4CLs”, when used in the context of the present disclosure, refer to enzyme 4-coumarate-Co-enzyme A ligases (4CLs) from Ocimum kilimandscharicum. Accordingly, embodiments of the present disclosure relates to a recombinant vector. Particularly, the present disclosure relates to a recombinant vector to overexpress 4-coumarate Co-enzyme A ligase 11 gene (4CL11) in host cells. Further, the present disclosure relates to a binary expression vector comprising a pRUOlAN vector with the 4CL11 ORF sequence from Ocimum kilimandscharicum and regulatory elements for host 4CL11 overexpression in plant cells resulting in a reduction in root growth, altered lignin content and structure, and increase in natural product biosynthesis.

Accordingly, the present disclosure provides a binary vector to upregulate or overexpress the host 4CL11 to achieve a reduction in root growth, altered lignin content, and increase in natural product biosynthesis.

In an embodiment of the present disclosure, the host 4CL11 overexpression is effected by introducing a binary expression vector comprising an open reading frame (173 Ibp) of 4- coumarate- Co-enzyme A Ligase 11 gene isolated from Ocimum kilimandscharicum (Ok4CLi 1) and one or more transcriptional regulatory sequence into the plant cell, and allowing the vector to integrate into the genome by non-homologous recombination, thus allowing overexpression of the host 4CL11.

In still another embodiment of the present disclosure, the open reading frame of Ok4CLll is represented by SEQ ID NO: 1.

In yet another embodiment of the present disclosure, the Ok4CLll coding SEQ ID NO: 1 is >Ok4CLll (1731 bp) ATGGCTGCATTAATAAAAGCCCACGTCGAGTTGCAGACTGTGAGAGAAGAAAGTG GAGGGATTGAAGAAATGTCGAAAACTAGAAAGTTGACATGCTCAAACCCTTCTTG GTATTGCCCTGAAACAGGGATATACTCAAGCAAATACCCTTCCATTAGCCTCCCTT CTGACCCTTTTCTTGATGTTGTTTCGTTCATTTTCTCACATGAACATGATGGGTTAC ATGCACTCGTTGATTCAGCATCTGGGCTTTCAGTTCCCTACTCCAAGCTGTTACCTT TGGTGAAATCCATGGCGGCTGGTCTTCATCATTTGGGTGTGAAGCAAGGTGATGTG ATCTTGATTCTGCTGCCCAATTCTGTGTGTTTCCCTTTGATTCTTTTAGGTGCTTTGA GTGTTGGTGCTGTTGTCACCACCCTGAATCCTTTGAGTAGTTTGTTAGAGATAAGA AAACAGGTTCTTGATAGTAATGCAAGTCTTGTATTCTCTGGTATTGGTAGGGTTGA TGAATTAGGTAATGCTTTGGAGGGTTGTCTTGTTGTAGGGGTGCCTGAAGTTTTGG ATTTGAGCTGTGAGAATGTTAATAGTTCTGTTTTTTGTAAGCTCATTTCTAGTGATC CTGAAATGGCACCTAGGCCTAGAATTAAGCAACAGGACACCGCCGCGATTTTGTA CTCGTCTGGCACTACTGGGAAGTCTAAAGGAGTCATGTTGACTCATGGGAACTTCA TAGCCATGATTGAGCTCTTTGTGAGGTTTGAAGCTTCTCTGTATGATTACCCTAGTA CAGCTAATGTCTACTTGGCTGTTGTGCCTATGTTCCATGTTTACGGGCTGTCTCTTT TCGTGTTGGGATTGTTGTCGTTGGGGAGTACTGTTGTCACCATGAGAAAGTTTGAT GGTGATGAAATGGTGAGAGCTATTGATAGATACGGCGTGACTCACCTCCACGCTG TCCCTCCTATACTGGTTGCGTTGACGAAGAGAGCCAAGAACGGTGATGGTAGTGG TTTTCGGAGCTTGAGGCAGGTTTCGTGTGGGGCTGCTCCGTTGAGTGAGAAAAGCA TTGTTGAATTGGTTGAGGCACTCCCCCATGTTGATTTCATTCAGGGTTATGGCATG ACAGAGTCGACCGCGTTGGCAACTCGCGGCTATAATAATGGTGGAGTCAAGAAAT ATTCGTCTGTGGGACTTCTTTCTCCGAATATTGAAGCTAAAGTGGTGGACTGGGTT ACAGGAGCTCTCGTTCCTCCAGGTTCGATCGGTGAGCTTTGGTTGCGCACGCCGGG GAATATGAAAGGGTACTTGAATAATACTGAAGCAACTGTGGGTGCACTTGACAAA GAAGGTTGGCTACACACCGGTGACATAGTTTATTTCGACCAAGAAGGATACTTGT ATGTAATCGATCGCCTGAAAGAAGTAGTCAAGTACAAAGGATTCCAGATTGCTCC AGCGGATTTGGAGGCGGTATTGATGTCACATCCAGAAGTAGCTGACGCGGCTGTT ACAGGTGTGAGAGACGAAGAAGCCGGAGAGATCCCTGTAGCATTCGTCGTTCCAA AAGAAGGTACATCACTCTCCGCGGCCACCCTCATGGACCACGTCGCGAAGCAGGT TGCACCTTATAAGAAGGTGAGAAAGGTGTATTTTCGGTCGTCGATACCGAGATCG

CCGGCCGGAAAAATACTTCGCAGGGAGCTCAAGAACTTGTTCGTTTCAAGGCTTTA G

In still another embodiment of the present disclosure, the Ok4CLll ORF encodes a polypeptide having the amino acid sequence represented by SEQ ID NO: 2, or a fragment or variant thereof. In a preferred embodiment of the present disclosure, the Ok4CLll ORF protein SEQ ID NO: 2 is

>Ok4CLll (576 aa)

MAALIKAHVELQTVREESGGIEEMSKTRKLTCSNPSWYCPETGIYSSKYPSISLPSDPFL DVVSFIFSHEHDGLHALVDSASGLSVPYSKLLPLVKSMAAGLHHLGVKQGDVILILLP NSVCFPLILLGALSVGAVVTTLNPLSSLLEIRKQVLDSNASLVFSGIGRVDELGNALEG CLVVGVPEVLDLSCENVNSSVFCKLISSDPEMAPRPRIKQQDTAAILYSSGTTGKSKGV

MLTHGNFIAMIELFVRFEASLYDYPSTANVYLAVVPMFHVYGLSLFVLGLLSLGSTVV TMRKFDGDEMVRAIDRYGVTHLHAVPPILVALTKRAKNGDGSGFRSLRQVSCGAAPL SEKSIVELVEALPHVDFIQGYGMTESTALATRGYNNGGVKKYSSVGLLSPNIEAKVVD WVTGALVPPGSIGELWLRTPGNMKGYLNNTEATVGALDKEGWLHTGDIVYFDQEGY LYVIDRLKEVVKYKGFQIAPADLEAVLMSHPEVADAAVTGVRDEEAGEIPVAFVVPK EGTSLSAATLMDHVAKQVAPYKKVRKVYFRSSIPRSPAGKILRRELKNLFVSRL In still another embodiment of the present disclosure, the Ok4CL7 gene encodes a polypeptide having the amino acid sequence represented by SEQ ID NO: 3, or a fragment or variant thereof. In yet another embodiment of the present disclosure, the Ok4CL7 coding SEQ ID NO: 3 is >Ok4CL7 (1731 bp)

ATGGCTGCATTAATAAAAGCCCACGTCGAATTGCAGACTGTGAAAGAAGACCGTC GTGTGATGGAAGAAATGTCGAAAACTAGAAAGTTGACACACTCCAATCCTTCTTG GTATTGCCCTGAAACAGGAATATACTCCAGCAAATACCCTTCCATCAACCTCCCTT CTGACCCTTTTCTTGATGTTGTTTCGTTCATTTTCTCACATAAACATGATGGGACAC ATGCACTCGTTGATTCAGCATCAGGGCTATCACTTCTCTACTCCAAGCTCTTACCTT TGGTGAAATCCATGGCTGCTGGTCTTCACCATTTGGGTGTCAAGCAAGGTGATGTC ATCTTGATTCTGCTGCCCAATTCTGTGTGTTTCCCTTTAATTCTCTTGGGTGCTTTGA GCGTTGGTGCTGTTGTCACCACCCTGAATCCTTTGAGTAGTTTGTTAGAGATCAAA AAACAGGTTCGTGATAGTAATGCAACTCTAGCATTTTCTGCTATTGGTAGGGTTGA TGAATTAGGTAATGCATTGGAGGGTTGCCTTATAGTAGGTGTGCCTGAAGTTTTAG ATTTGAGCTCTCAGCCTATTAGTGGTTCTGTTTTTCATAAGCTCATTTCTAGTGATC CTGAAATGGCCCCTAGGCCTAGAATTAAGCAGCAGGACACCGCCGCGATTTTATA CTCATCGGGCACTACTGGTAAGTCTAAAGGAGTCATGTTGACTCATGGGAACTTCA TAGCCATGATTGAGCTCTTTGTGAGGTTTGAAGCTTCTCTGTATGATTATCCTAGTA CAGCCAATGTCTACTTGGCTGTTGTGCCTATGTTCCATGTGTATGGGCTGTCTCTTT TCGTTTTGGGATTGTTGTCGTTGGGGAGTACTGTAGTCACCATGAGAAAGTTTGAT GGTGATGAAATGGTGAGAGCTATTGATAGATATGGTATAACTCACCTTCATGCTGT CCCGCCTATACTGATTGCGTTGACGAAGAGAGCCAAGAACGCTGATGGTAATGGT TTTCAGAGCTTGAAACAGGTTTCGTGTGGGGCTGCTCCGTTGAGTGAGAAAAGCAT AGTTGAATTGATTGAGGCACTCCCCCATGTTGACTTCATTCAGGGTTATGGCATGA CAGAATCGACCGCATTGGCAACTCGCGGCTACAATAATGGTGGAGTCAAGAAATA TTCCTCTGTGGGACTTCTTTCTCCAAATATTGAAGCCAAAGTGGTGGACTGGGTTA CAGGAGCTCTCGTTCCTCCAGGATCGATCGGTGAGCTTTGGTTGCGCACACCAGGA AACATGAAAGGGTACTTGAATAATACTGAAGCAACTGTGGGTGCACTTGACAAAG AAGGCTGGCTACACACTGGTGACATAGTTTATTTTGACCAAGAAGGATACTTGTAT GTAATCGATCGCCTGAAAGAAGTCATCAAGTACAAAGGATTCCAGATTGCTCCAG CGGATTTGGAGGCAGTATTGATGTCACATCCAGAAGTAGCTGATGCAGCTGTTAC AGGTGTGAGGGACGAAGAAGCCGGAGAGATCCCTGTGGCATTCGTCGTTCCAAAA GAAGGTACATCACTCTCCGCGGCCACCCTCATGGACCACGTAGCGAAGCAGGTTG CACCTTATAAGAAGGTGAGAAAGGTGTATTTTCGGACATCAATACCGAGATCGCC

TGCCGGAAAAATACTTCGCAGGGAGCTCAAGAACTTGTTTGTTTCCAGGCTTTAG

In a preferred embodiment of the present disclosure, the Ok4CL7 protein SEQ ID NO: 4 is >Ok4CL7 (576 aa)

MAALIKAHVELQTVKEDRRVMEEMSKTRKLTHSNPSWYCPETGIYSSKYPSINLPSDP

FLDVVSFIFSHKHDGTHALVDSASGLSLLYSKLLPLVKSMAAGLHHLGVKQGDVILIL

LPNSVCFPLILLGALSVGAVVTTLNPLSSLLEIKKQVRDSNATLAFSAIGRVDELGNALE

GCLIVGVPEVLDLSSQPISGSVFHKLISSDPEMAPRPRIKQQDTAAILYSSGTTGKSKGV

MLTHGNFIAMIELFVRFEASLYDYPSTANVYLAVVPMFHVYGLSLFVLGLLSLGSTVV

TMRKFDGDEMVRAIDRYGITHLHAVPPILIALTKRAKNADGNGFQSLKQVSCGAAPLS

EKSIVELIEALPHVDFIQGYGMTESTALATRGYNNGGVKKYSSVGLLSPNIEAKVVDW

VTGALVPPGSIGELWLRTPGNMKGYLNNTEATVGALDKEGWLHTGDIVYFDQEGYL

YVIDRLKEVIKYKGFQIAPADLEAVLMSHPEVADAAVTGVRDEEAGEIPVAFVVPKEG

TSLSAATLMDHVAKQVAPYKKVRKVYFRTSIPRSPAGKILRRELKNLFVSRL

In still another embodiment of the present disclosure, the open reading frame of Ok4CL15 is represented by SEQ ID NO: 5.

>Ok4CL15 (1608 bp)

ATGGAGCTGAAAGAAGAGAAGGAATACATTTTCCGATCGAAGCTTCCAGATATCT

ACATTCCCCTCCATCTCCCTCTCCACACATACTGCTTCGAAAACCTCTCACTCCACC

GCGCTCGGCCGTACTTAATCAACGCCGTCACCGGCGAAACCTTCACCCATGCCGA

ATTCGAGCTCACCGCGCGCAGAGTCGGCGCCGGTCTCCACAACCTCGGAATCCGC

AAATCCGACGTCGTCATGCTCCTCCTCCACAACTCGCCTGAATTCGCGTTCGCTTTC

CTAGGAGCCTCCTTCGTCGGCGCGGTCGTCACCACCGCGAATCCTCTCTACACCGC

CTCGGAAATCGCGCTGCAGGTTGAGATCTCGAGGCCGAGGCTTATCATCACACAC

GCCTGTCACGTGGAGAAGGTGAGGCAGCACGCCTCCGCGGATGGCGCCAAAATCG

TGACGATCGACGCTCCGCCGTCGCCGGAGATCGTTCATTTCTCGGATTTGACGAGA

TCTGACGAGAATCGACTTCCTGCAGTCGAAATCCGAGGAGAGGACACGGCGGCGC

TGCCGTTCTCCTCCGGAACCACCGGTCTCCCCAAAGGAGTAATGCTGAGTCACAGA

AACATAATCACGTGCATCTCGCAGCAGGTGGACGGTGAAAATCCGGCGAATCACA

TCGACTGCGAGGATCGGCTTCTCTGCGTGTTGCCGCTGTTTCACGTTTACTCTATGG

TGTCGGTGATGCTCTGCAGTCTCCGCGCCGGAGCGGCGATTGTGATTATGCCGAGA

TTTGAACTGAATGAGTTGATGGAGGTGATACAGAAATATAAGGTGACGATCGCGC

CGTTTGTGCCGCCGATATTGTTAGGCATCGCGAAGAGCCAGACGGCGGCGAAGTT

CGATCTGTCGTCGGTGAGGAGAGTCGTCTGCGGCGCGGCGCCGATGGATCGGAAA CTTGAGCTGTCACTCAAATCAAAACTTCCGAATGCTATTATTGGGCAGGGTTATGG TATGACAGAAGCCTTAGTCCTATCAATGTGTTTAGGTTTCGCCAAGTTTCCGATGA AATTCAAGGCTGGCTCGTGTGGAAATGTGATTAAAAACGCCCGTATGAAGATCAT

CGACCCGGCCACCGGTGCCTCCCTCGACCGGAATCAGAGGGGGGAGATTTGCCTC AAAGGAAGCTCCGTGATGAAAGGTTATTATAAGGATCCGGAGGCGACGAAGAGG ACTATCGACGAGGAGGGGTGGCTGCACACCGGCGATATAGGGTACATTGACGATG

ACGGTGAAGTGTTCATTGTGGACAGGTTGAAGGAATTGATCAAATACAAAGGATT TCATGTTGCTCCTGCTGAACTTGAAGCTCTTCTCATAGCTCATCCCTCCATATCTGA TGCTGCTGTTGTGCCTATGGCCGATGAGGCTGCTGGAGAAGTTCCTGTTGCATTTG

TAGTCAGGGAAAATGGTTCGAAGATTACGGAACTAGAAATCAAGAAATACATCGC GAGCCAGGTGGTGTCGTACAAGCGTATCAAACATGTATTCTTCATTGACAATATTC CAAAAGCCCCCTCTGGTAAAATTCTGAGGAAGAATCTGAGAGCTAGAATGTAA

In a preferred embodiment of the present disclosure, the Ok4CL15 protein SEQ ID NO: 6 is >Ok4CL15 (535 aa)

MELKEEKEYIFRSKLPDIYIPLHLPLHTYCFENLSLHRARPYLINAVTGETFTHAEFELT

ARRVGAGLHNLGIRKSDVVMLLLHNSPEFAFAFLGASFVGAVVTTANPLYTASEIALQ VEISRPRLIITHACHVEKVRQHASADGAKIVTIDAPPSPEIVHFSDLTRSDENRLPAVEIR GEDTAALPFSSGTTGLPKGVMLSHRNIITCISQQVDGENPANHIDCEDRLLCVLPLFHV

YSMVSVMLCSLRAGAAIVIMPRFELNELMEVIQKYKVTIAPFVPPILLGIAKSQTAAKF DLSSVRRVVCGAAPMDRKLELSLKSKLPNAIIGQGYGMTEALVLSMCLGFAKFPMKF KAGSCGNVIKNARMKIIDPATGASLDRNQRGEICLKGSSVMKGYYKDPEATKRTIDEE

GWLHTGDIGYIDDDGEVFIVDRLKELIKYKGFHVAPAELEALLIAHPSISDAAVVPMA

DEAAGEVPVAFVVRENGSKITELEIKKYIASQVVSYKRIKHVFFIDNIPKAPSGKILRKN LRARM

In an embodiment, the present disclosure provides a recombinant binary expression vector for overexpressing 4-coumarate Co-enzyme A ligase 11 gene (4CL11) in host plant cells, the recombinant binary expression vector comprising: a pRUOlAN vector comprising an open reading frame (ORF) of a 4-coumarate- Coenzyme A ligase gene isolated from Ocimum kilimandscharicum (pRI 101 AN-OZACL/ /- overexpression construct); and at least one transcriptional regulatory element, wherein the pRI 101 AN-O CL/ /-ovcrcxprcssion construct comprises the ORF of the Ok4CLll gene ligated into a multiple cloning site (MCS) of the pRUOlAN vector after restriction digestion of the pRIlOlAN vector with Ndel and EcoRI restriction enzyme sites.

In an embodiment of the present disclosure provides, wherein the ORF of the Ok4CLll gene comprises a nucleotide sequence with at least 70% similarity to SEQ ID NO: 1.

In an embodiment of the present disclosure provides, wherein the ORF of the Ok4CLll gene encodes a polypeptide having an amino acid sequence with at least 70% similarity to SEQ ID NO: 2.

In a preferred embodiment, the present disclosure provides an Ok4CLl 7-ORF nucleotide sequence comprising at least 70% nucleotide sequence identity with the native sequence. For example, at least 70% identity, 75% identity, 80% identity, 85% identity, 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity, or 100% identity.

In a preferred embodiment, the present disclosure provides an Ok4CLll -ORF polypeptide sequence comprising at least 70% polypeptide sequence identity with the native sequence. For example, at least 70% identity, 75% identity, 80% identity, 85% identity, 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity, or 100% identity.

In another preferred embodiment, the present disclosure provides a binary expression vector comprising nucleotide sequences encoding regulatory elements operably linked to a nucleotide sequence encoding the Ok4CLl 1 -ORF.

In another embodiment of the present disclosure, the host 4CL11 gene is overexpressed by transforming a binary expression vector pRIlOlAN comprising Ok4CLll ORF and one or more transcriptional regulatory sequence into the plant cell, and allowing the vector to integrate into the genome by non-homologous recombination, thus allowing overexpression of the host 4CL11.

In still another embodiment of the present disclosure, the binary expression vector pRIlOlAN may additionally comprises one or more transcriptional regulatory elements selected from but not limited to a promoter, a transcriptional enhancer, a reporter, and a terminator.

In still another exemplary embodiment of the present disclosure, the binary expression vector pRIlOlAN additionally comprises one or more transcriptional regulatory elements selected from but not limited to 3 S CaMV promoter, 5' untranslated region (UTR) of alcohol dehydrogenase gene from Arabidops is thaliana, and Nopaline synthase terminator. In an embodiment of the present disclosure, the binary expression vector pRIlOlAN encoding the Ok4CLll-O operably linked to 35S CaMV promoter, 5' untranslated region (UTR) of alcohol dehydrogenase gene from Arabidopsis thaliana, and Nopaline synthase terminator.

In an embodiment of the present disclosure, the nucleotide construct of at least 70% sequence identity (e.g., 75% identity, 80% identity, 85% identity, 90% identity, 95% identity, 98% identity, 99% identity, or 100% identity) with the nucleotide construct of this invention.

In another embodiment of the present disclosure, the Ok4CLll ORF sequence is cloned into the pRIlOlAN vector using the restriction enzymes Ndel and EcoRI to form the pRUOlAN- Ok4CLl 7-OE construct.

In an embodiment, the present disclosure provides a method for expressing host 4CL11 in a suitable plant cell is effected by stably transforming the suitable plant cell with said pRUOl AN- Ok4CLl 1 -OE construct. In another embodiment, the transforming is effected by Agrobacterium-mediated transformation. It is to be noted that the transformation can also be effected by the methods known in the prior art. Any person having ordinary skill in the art would be able to perform the same with the other known transformation methods.

In an embodiment, the present disclosure provides a method for expressing host 4CL11 in a plant cell, said method comprising the steps of a. providing the pRUOl AN- Ok4CLl 1 -OE construct as disclosed herein; b. providing a plant cell; c. stably transforming the plant cell with said pRI 101 MA-Ok4CLl 7-OE construct to obtain transformed plant cell; d. isolating and identifying the transformed plant cells overexpressing 4CL11 gene; e. regenerating the transformed plant cell overexpressing 4CL11 gene into a plant using plant growth media.

In an embodiment, the present disclosure provides a method for expressing a host 4CL11 gene in a plant, the method comprising: a. providing the pRUOl -Ok4CLl -overexpression construct as claimed in claim 1; b. providing a plant cell, a calli tissue, or a leaf explant; c. stably transforming the plant cell, the calli tissue, or the leaf explant with the pRUOlAN- Ok4CLl 1 -overexpression construct to obtain a transformed plant cell, a transformed calli tissue, or a transformed leaf explant; d. isolating and identifying the transformed plant cell, the transformed calli tissue, or the transformed leaf explant overexpressing the host 4CL11 gene; and e. regenerating the transformed plant cell, the transformed calli tissue, or the transformed leaf explant overexpressing the host 4CL11 gene into the plant using a plant growth media.

In an embodiment of the present disclosure, wherein the plant growth media is defined in Figure 8.

In an embodiment of the present disclosure, wherein the plant cell derived from a leaf or stem tissue obtained from Nicotiana benthamiana, Arabidopsis thaliana, or Solanum tuberosum were used as explants for AgrofeacterzMm-mediated stable transformation.

In an embodiment of the present disclosure, wherein the stable transformation is effected by an Agrobacterium-mediated leaf transformation method.

In an embodiment of the present disclosure, wherein the transformation is effected by an Agrobacterium tumefaciens strain (GV3101 strain) using a freeze and thaw transformation method.

In an embodiment of the present disclosure, wherein the pRI 101 AN-Ok4CLl 7-overexpression construct integrates into the genome of cells of Nicotiana benthamiana, Arabidopsis thaliana, or Solanum tuberosum by non-homologous recombination.

In an embodiment, the present disclosure provides a transgenic plant is produced by the method as claimed in claim 7, wherein the transgenic plant overexpresses a host 4CL11 gene.

In still another embodiment of the present disclosure, the 4CL11 overexpression lines of N. benthamiana, Arabidopsis thaliana, and potato are generated through the Agrobacterium- mediated leaf transformation method as disclosed in Horsch et al. (1985), Clough and Bent (1998) and Banerjee et al. (2006), respectively.

In still another embodiment of the present disclosure, five transgenic lines of Ok4CLll overexpression in N. benthamiana (referred as Ok4CLl /-OE Nb lines) and 4 lines of potato are developed.

In yet another embodiment of the present disclosure, the overexpression lines of 4CL11 in plants or plant cells help to increase the level of flavonoids (such as kaempferol- and quercetinderivatives) in the transgenic cells or plant lines of N. benthamiana as compared to its wildtype. The increased accumulation of flavonoids and their derivatives) and the reduced auxin content, decreased expression of auxin transport and signaling genes further leads to a reduced root growth phenotype.

In still another embodiment of the present disclosure, the 4CL11 overexpression in N. benthamiana plants leads to a reduction in root growth, altered lignin content, and increase in natural product biosynthesis. Reduced root growth is also observed in case of Ok4CLll overexpression in potato or Arabidopsis thaliana plants.

In a preferred embodiment of the present disclosure, the 4CL11 overexpressed N. benthamiana plants can be further used as feedstock for biofuel production.

In an embodiment of the present disclosure, a pRUOlAN vector with the Ok4CLll ORF sequence used for the preparation of rootless plants including but not limited to N. benthamiana and potato is also disclosed. This is the unique function exhibited by Ok4CLll, and not by other two 4CL isoforms like Ok4CL7 and -15.

In a preferred embodiment of the present disclosure, the composition can also include variations of Ok4CL7, -11 and -15 genes in the pRIlOlAN vector. The variants are generated by methods selected from but not limited to site-directed mutagenesis, intron removal and the like, wherein the variants of said genes comprise at least 70% sequence identity with the native sequence. For example, at least 70% identity, 75% identity, 80% identity, 85% identity, 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity, or 100% identity.

EXAMPLES

Example 1:- Isolation and cloning of Ok4CL7, Ok4CLll and Ok4CL 15 genes: cDNA encoding 4-coumarate Co-enzyme A ligase 11 (Ok4CLl / ) were isolated from Ocimum kilimandscharicum. The cDNA sequence of Ok4CLll has an open reading frame of 1731 bp. A binary expression vector, pRUOlAN, was taken as a basic backbone vector to develop the transgenic plants. This vector consists of a 35S CaMV promoter, 5' untranslated region (UTR) of the alcohol dehydrogenase gene from Arabidopsis thaliana, and the nopaline synthase terminator (depicted in Figures 1 A and B). The purified Ok4CLll was ligated into the multiple cloning site (MCS) of pRUOlAN after restriction digestion of the vector with Ndel and EcoRI restriction enzyme sites in forward and reverse primers, respectively. Cloning of Ok4CLll gene in pRUOlAN vector was confirmed using restriction enzyme digestion (depicted in Figure 1 C), followed by sequencing. Similarly, Ok4CL7 and Ok4CIA5 are also cloned individually in pRUOlAN vector to generate their respective overexpression constructs.

Example 2:- Generation of gene constructs

For the generation of Ok4CL7, Ok4CLll and Ok4CL15 overexpression gene constructs, the longest open reading frame (ORF) of Ok4CL7 (1731 bp), Ok4CU 1 (1731 bp) and Ok4CL15 (1530 bp) were amplified by Polymerase Chain Reaction (PCR) with high-fidelity Phusion DNA Polymerase (Thermo Fisher Scientific, Waltham, United States). Complementary DNA (cDNA) synthesized from leaf of O. kilimandscharicum was used as a template for PCR. Amplified sequences were mobilized individually in a binary vector pRUOlAN between the Cauliflower Mosaic Virus (CaMV) 35S promoter and the nopaline synthase terminator (NOST). For cloning purposes, Ndel restriction enzyme site was added in the forward primer, whereas EcoRI or Sad was added in the reverse primer. Finally, the individual overexpression constructs of these Ok4CLs (35S::Ok4CL7, 35S::Ok4CLll , and 35S::Ok4CL15) were prepared. Similarly, 35S::GUS construct was used to generate a vector control (VC) line. To develop the RNAi construct of Ok4CLll (35S: :Ok4CLl 1 -RNAi), 350 bp unique sequence of sense and antisense strands was cloned on either side of 500 bp intron in the pRUOl binary vector.

Example 3:- Expression of Ok4CL7, Ok4CLll, and Ok4CL15 genes:

Transformation of cells: pRI 101 AN-6RAC7.7, Ok4CLll, and Ok4CL15 constructs were mobilized individually into Agrobacterium tumefaciens (GV3101 strain) using the freeze and thaw transformation method. The resulting binary vector containing the Agrobacterium strain was used to develop the transgenic lines of N. benthamiana. Overexpression lines of three Ok4CL isoforms (Ok4CL7. Ok4CLll and Ok4CL15) in N. benthamiana were generated through the Agrobacterium- mediated leaf transformation method as described by Horsch et al. (1985) (depicted in Figure 2). A total of five independent lines of Ok4CLll overexpression (referred to as Ok4CLll-OEl to Ok4CLll-Q 5) were developed using this method (Fig. 3). Similarly, overexpression lines of Ok4CL7 and Ok4CL15 in N. benthamiana are generated (Fig. 3).

Transformation of Explants:

In another experiment (Figure 9 A-E), three- week old wild-type N. benthamiana seedlings were used to prepare explants (shoot-apex with 2-nodes) for supplementation, whereas in the experiment shown in Figure 9 (F), a single-node explants from the middle region of stem (3-5¹¹¹ nodes from the shoot-apex) from two-month old wild-type N. benthamiana were used as an explant.

Example 4:- Screening of transgenic lines:

Respective transgenic lines were confirmed by reverse transcriptase-PCR (RT-PCR) amplification of target gene from cDNA (synthesized from mRNA) and through PCR from genomic DNA isolated from leaves of putative transformants of N. benthamiana and wild-type plants (as negative control).

Results:

Transformation of cells: Out of 9 putative transformants, 5 lines were found to be true transformants (depicted in Figure 3). For Ok4CL7 overexpression construct, 1 line out of 3 putative transformants is positive, whereas for Ok4CL15 overexpression construct, 2 lines out of 7 putative transformants are confirmed (depicted in Figure 3). The gene expression of Ok4CL7, Ok4CLll and Ok4CL15 in the individual overexpression lines of N. benthamiana-, wherein two lines of Ok4CL7, five lines of Ok4CLll and two lines of Ok4CL15 are shown (Figure 4). Root growth was significantly affected in all transgenic N. benthamiana plants compared to wild-type plant (depicted in Figure 5A). Further, transgenic lines, such as Ok4CL 11 -OE\ and Ok4CLl 7-OE2 showed rootless phenotype. The number of primary and lateral roots was significantly less in Ok4CLll-OE lines compared to wild-type plant (Figures 5B-C). The length of the primary root was also significantly affected in Ok4CLll -OE lines (OE1 and OE2) compared to wild-type N. benthamiana plants (depicted in Figure 5D). Root growth parameters, such as root number, lateral root length and the main root length remained similar to wild-type in Ok4CL15-OEl line, whereas in Ok4CL15-OE line, only root number was increased with the main root length and lateral root numbers being comparable to wild-type N. benthamiana plants (Figures 5B-D).

Transformation of Explants:

• The adventitious root (AR) emergence was initiated on day 3 for control plants, whereas the individual flavonoids (K and Q) or their glycosides (R, K3R, K3,7R, Q3G) showed delayed AR initiation (2-3 days). Moreover, the mixture of these aglycon flavonoids and their glycosides (K + Q + R + K3R + K3,7R + Q3G) was the most effective to delay the AR emergence by 4 days Figure 9(A).

• On day 9, the average number of ARs per plant was significantly higher (p>0.000078) for control (2.35) compared to 1.36 ARs in the mix treatment. Same for day 12, p>0.000001 for control (3.1) compared to 1.84 ARs in the mix treatment. For day 15, p>0.0001 for control (3.38) compared to 2.24 ARs in the mix treatment.

• The mixture of the aglycon flavonoids and their glycosides (K + Q + R + K3R + K3,7R + Q3G) appears to be the most effective to affect root number, length, AR biomass as well as shoot biomass Figure 9(B-E). These effects on the shoot biomass are similar to TIBA and NPA treatment (our previous experiment); however, TIBA and NPA results in complete inhibition of root emergence, leading to rootless explants.

• Figure 9(F) shows images of the single-node stem explants of wild-type N. benthamiana grown on flavonoids and their glycosides to evaluate their effect on root growth inhibition. All the flavonoids and their glycosides are inhibiting the root growth. Abbreviations used for flavonoids and their glycosides: Kaempferol (K), Quercetin (Q), Rutin (R), Kaempferol 3-rhamnoside (K3R), Kaempferol-3,7-O-bis-alpha-L-rhamnoside (K3,7R), Quercetin 3-O-p-D-glucuronide (Q3G), Mix (K+Q+R+K3R+K3,7R+Q3G).

Example 5:- Ok4CLll overexpression study:

The effect of Ok4CLll overexpression on the metabolite contents was studied with the leaf extracts (in 80% methanol) of both transgenic and wild type N. benthamiana using an untargeted LC-MS assay.

Results:

LC-MS assay shows that the level of flavonoids (kaempferol- and quercetin-derivatives) increased in Ok4CLll -OE lines of N. benthamiana compared to wild-type plants (depicted in Figure 6A). The preliminary analysis further shows that the Ok4CLll overexpression in N. benthamiana alters the lignin content compared to wild-type plants (depicted in Figures 6B-C), further adding its potential application in biofuel production.

Example 6:- Ok4CLll overexpression in potato:

AgroZzacterzw -mediated potato leaf transformation is performed and the overexpression line of potato for Ok4CLll gene is generated. The overexpression line is confirmed by reverse transcriptase PCR and root growth phenotype is recorded.

Results:

Of the two potato lines that are tested for Ok4CLl 1 overexpression, one line is confirmed (Figure 7). This line showed a reduced root growth phenotype similar to N. benthamiana overexpression plants.

ADVANTAGES OF THE INVENTION

• The present technique can be used in hydroponic, aeroponic, and vertical agriculture in order to reduce root growth.

• The present technique can be used in biofuel industries to alter the lignin content by overexpressing the 4CL11 isoform in targeted crop plants.

• The present technique can be applied further in natural product biosynthesis.

• Provides overexpression of a specific Ocimum 4CL isoform - Ok4CLll in N. benthamiana leads to a rootless or reduced root growth phenotype, owing to accumulation of specific flavonoids (kaempferol and quercetin) and their glycosides. Further, this phenotype was also shown in potato and Arabidopsis thaliana plants. • Specifically, the invention provides stable overexpression of Ok4CLll in N. benthamiana causes accumulation of specific flavonoids (kaempferol and quercetin) and their glycosides, leading to the rootless or reduced root growth phenotype.

• More specifically, the invention provides generation of overexpression gene construct of the specific 4CL isoform from Ocimum - Ok4CLll in a binary vector pRIlOl and its effect on plant root growth.

• Also, it provides overexpression of Ok4CLll in A. benthamiana causes accumulation of specific flavonoids (kaempferol and quercetin) and their glycosides that could inhibit auxin transport, leading to the rootless phenotype.

• The effect of overexpressing Ok4CLll on root growth was also found in potato and Arabidopsis thaliana plants.

Claims

We Claim:

1. A recombinant binary expression vector for overexpressing 4-coumarate Co-enzyme A ligase 11 gene (4CL11) in host plant cells, the recombinant binary expression vector comprising: a pRUOlAN vector comprising an open reading frame (ORF) of a 4-coumarate- Co-enzyme A ligase gene isolated from Ocimum kilimandscharicum (pRIlOlAN- Ok4CLl 1 -overexpression construct); and at least one transcriptional regulatory element, wherein the pRI 101 AN-OAACL/ /-overcxpression construct comprises the ORF of the Ok4CLll gene ligated into a multiple cloning site (MCS) of the pRUOlAN vector after restriction digestion of the pRUOlAN vector with Ndel and EcoRI restriction enzyme sites.

2. The recombinant binary expression vector as claimed in claim 1, wherein the ORF of the Ok4CLll gene comprises a nucleotide sequence with at least 70% similarity to SEQ ID NO: 1.

3. The recombinant binary expression vector as claimed in claim 1, wherein the ORF of the Ok4CLll gene encodes a polypeptide having an amino acid sequence with at least 70% similarity to SEQ ID NO: 2.

4. The recombinant binary expression vector as claimed in claim 1, wherein the transcriptional regulatory element is a 35S CaMV promoter.

5. The recombinant binary expression vector as claimed in claim 1, wherein the transcriptional regulatory element is a 5' untranslated region (UTR) of an alcohol dehydrogenase gene from Arabidopsis thaliana.

6. The recombinant binary expression vector as claimed in claim 1, wherein the transcriptional regulatory element is a nopaline synthase terminator.

7. A method for expressing a host 4CL11 gene in a plant, the method comprising: a. providing the pRI 101 M\-Ok4CL! 7 -overexpression construct as claimed in claim 1; b. providing a plant cell, a calli tissue, or a leaf explant; c. stably transforming the plant cell, the calli tissue, or the leaf explant with the pRI 101 AN-OHCL/ /-ovcrcxprcssion construct to obtain a transformed plant cell, a transformed calli tissue, or a transformed leaf explant; d. isolating and identifying the transformed plant cell, the transformed calli tissue, or the transformed leaf explant overexpressing the host 4CL11 gene; and e. regenerating the transformed plant cell, the transformed calli tissue, or the transformed leaf explant overexpressing the host 4CL11 gene into the plant using a plant growth media.

8. The method as claimed in claim 7, wherein the plant cell derived from a leaf or stem tissue obtained from Nicotiana benthamiana, Arabidopsis thaliana, or Solanum tuberosum were used as explants for Agrobacterium-mediated stable transformation.

9. The method as claimed in claim 7, wherein the stable transformation is effected by an Agrobacterium-mediated leaf transformation method.

10. The method as claimed in claim 9, wherein the stable transformation is effected by an Agrobacterium tumefaciens strain (GV3101 strain) using a freeze and thaw transformation method.

11. The method as claimed in claim 7, wherein the pRI 101 AN- Ok4CLll-o verexpression construct integrates into the genome of cells of Nicotiana benthamiana, Arabidopsis thaliana, or Solanum tuberosum by non-homologous recombination.

12. A transgenic plant is produced by the method as claimed in claim 7, wherein the transgenic plant overexpresses a host 4CL11 gene.