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. 2022 Dec 19;13(1):16. doi: 10.1007/s13205-022-03435-5

Reducing the biosynthesis of condensed tannin in winged bean (Psophocarpus tetragonolobus (L.) DC.) by virus-induced gene silencing of anthocyanidin synthase (ANS) gene

Vinayak Singh 1,2, Rayees Ahmad Lone 1,3, Verandra Kumar 1, Chandra Sekhar Mohanty 1,3,✉
PMCID: PMC9763518  PMID: 36561838

Abstract

The Underutilized legume-winged bean (Psophocarpus tetragonolobus (L.) DC.) and its various parts are infested with condensed tannin (CT) or proanthocyanidin (PA). CT has anti-nutritional effect as it adversely affects the digestion of proteins, minerals and vitamin among ruminants and humans. It is also responsible for low protein digestibility and decreased amino acid availability. One of the probable reasons of underutilization of P. tetragonolobus is due to its infestation with CT. Histochemical staining of various tissues of P. tetragonolobus with dimethylcinnmaldehyde (DMACA) developed a deep-blue colour indicating the presence of polyphenolic condensed tannin. Structural monomeric unit catechin and epi-catechin were reported to be responsible for biosynthesis of CT in P. tetragonolobus. The enzyme anthocyanidin synthase (ANS) and its corresponding transcripts were identified and phylogenetically mapped. The transcript was subjected to virus-induced gene silencing (VIGS) through agro-infiltration in P. tetragonolobus for reducing the CT-content. The WbANS-VIGS induced P. tetragonolobus resulted in four-fold decrease of CT as compared to the control P. tetragonolobus. A decrease of 73% of CT level was reported in VIGS silenced Wb-ANS line of P. tetragonolobus. This study resulted and confirmed that, the silencing of (ANS) gene in P. tetragonolobus has a regulatory effect on the condensed tannin biosynthesis. This study will pave way for further manipulation of ANS enzyme for reducing the biosynthesis of the anti-nutrient CT. Reducing the CT content will make this underutilized legume more acceptable.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-022-03435-5.

Keywords: Agro-infiltration, Anthocyanidin synthase, Condensed tannin, Virus-induced gene silencing, Winged bean

Introduction

The legume winged bean (Psophocarpus tetragonolobus (L.) DC.) belongs to family Fabaceae and sub-family Papilionoideae. It is a tropical vegetable with a high nutritional value (Sasiprapa et al. 2015). The immature pods of P. tetragonolobus contain 28 to 45% of protein (Mohanty et al. 2020), 14 to 19% oil and 34 to 40% carbohydrate (Adegboyega et al. 2019). Winged bean has suitable agronomic features which make it ideal for growing in tropics with a high average yield. The meals made from winged bean are effective to fulfil the consumer protein needs. It has long been used as a crop with potential to address issues on nutritional security (Tanzi et al. 2019). But, infestation of this plant with anti-nutrient condensed tannin or proanthocyanidin limits its use and makes it less-acceptable (Mohanty et al. 2021). Exploring and improving the potential of underutilized legume winged bean (Psophocarpus tetragonolobus (L.) DC.) for food and nutrition security is of importance. Efforts are needed to popularize this high-quality nutritional crop and enhance their consumption and commercialization by reducing the anti-nutritional factors is the prime objective of the study. This will provide a sustainable synergy between food and nutrition among the communities.

Condensed tannins (CTs) or proanthocyanidins (PAs) are the group of polyphenolic secondary metabolites synthesized in plants via flavonoid biosynthetic pathway. The oligomers or polymers of flavan-3-ol monomeric units are known as condensed tannins. CTs are the second most common group of natural phenolics after lignins and are commonly found in the plant-kingdom (Tan et al. 1983). The structural complexity of CT endows them with a variety of biochemical properties like: protein interaction, metal chelation and antioxidant activities (Santos et al. 2000). Catechins and epi-catechins are the simplified monomeric flavanol units for CT formation. They are formed by three enzymes namely: leucoanthocyanidin reductase (LAR), anthocyanidin reducatse (ANR) and anthocyanidin synthase (ANS) in anthocyanin biosynthesis pathway. Anthocyanidin synthase (EC 1.14.11.19) is also known as leucoanthocyanidin dioxygenase (LDOX). It is a 2-oxoglutarate ascorbate dependent oxygenase (Karamac et al. 2009). ANS is the first and most active enzyme in anthocyanin formation (Nakajima et al. 2001). ANS oxidizes the leucoanthocyanidin molecules to colored anthocyanidins (pelargonidin, cyanidin and delphinidin) (Pelletier et al. 1997). Colorless anthocyanidins are formed from the unstable anthocyanidins. Anthocyanidin reductase transforms flavan-3-ols to [(–) epiafzelechin, (–) epicatechin and (–) epigallocatechin] (Saito et al.1999). Arabidopsis thaliana ANS was shown to transform the natural substrate leucoanthocyanidin to cis- and trans-dihydroquercetin and quercetin (Xie et al. 2003).The biosynthesis of CT oligomers commences by adding an extension unit to a starter unit (catechin or epicatechin) and then goes on adding extension units in a sequential order. The most common monomeric units in CTs are 2, 3-trans ( +) catechin and 2, 3-cis (–) epicatechin, which have chiral C3 carbon on the C-rings with the opposite stereochemistry. Condensed tannin is formed after polymerization of these monomers (Welford et al. 2001). Tea LAR gene was expressed ectopically in tobacco, resulting in a higher accumulation of epicatechin than catechin, suggesting that LAR is active in epicatechin biosynthesis (Harding et al. 2014). Grape and tea ANRs have shown to have epimerase activity allowing them to transform anthocyanidin to epi-catechin as well in catechin (Harding et al. 2014; Pang et al. 2013). CTs are formed in the cytoplasm of cells and get collected in the vacuoles (Gargouri et al. 2010). CT biosynthesis and aggregation was recorded in a number of species in the plant kingdom Brillouet et al. 2013; Matsui et al. 2004). Concerns have been raised about the transfer of CT from the cytosol to vacuoles and flavan-3-ol monomer polymerization (Liu et al. 2014). Increased CT-content beyond a certain level reduces the seed-protein quality significantly (Hagerman et al. 1998) and disturbs the digestive system of monogastrics. The CT in plants binds to proteins and affects the enzymatic activity and protein solubility thereby affecting the forage quality of the plants. The insurgence of bloating in ruminants is indicative of the forage value of CT in plants. The CT-specific pathway and the involved structural genes encoding LAR and ANR are yet to be fully elucidated in P. tetragonolobus. However, LAR was the first enzyme to be identified in Vitis vinifera (Zhao et al. 2010; Bogs et al. 2005; Pfeiffer et al. 2006) and strawberry (Maugé et al. 2010). The exact details of the CT polymerization and the functional role of various enzymes involved in this pathway need to be elucidated completely (Dikson et al. 2005; Lepiniec et al.2006). Identification of ANS transcript family of P. tetragonolobus opened new frontier to study the biosynthetic mechanism of CT. This family of transcripts might have impacted on transcriptional regulation in CT biosynthesis in plant-tissues. The initial step for discovery of genes, transcripts and transcription factors responsible for the biosynthesis of CT had been determined with leaf transcriptomics of two contrasting CT-lines of P. tetragonolobus (Singh et al. 2017). The present activity elucidates the functional role of ANS-encoding gene and its role in modulating the biosynthesis of catechin and epicatechin which is ultimately responsible for CT biosynthesis in the underutilized legume P. tetragonolobus. Functional validation of gene-encoding ANS is carried out in the present investigation.

The proposed study will shed light on the biological role of detected ANS transcripts and their probable role in modulating the biosynthesis of condensed tannin in P. tetragonolobus. To analyze and functionally characterize the responsible transcripts (Hagerman et al. 1998) virus induced gene silencing (VIGS) was employed through agro-infiltration (Burch-Smith et al. 2004; Robertson et al. 2004) in P. tetragonolobus. A comparative gene-expression profile and their analysis had been carried out. Cotyledonary, hypocotyl and epicotyl explants along with young leaf discs and cotyledonary leaf discs when subjected to infect with A. tumifaciens through agro-infiltration technique, ANS-VIGS construct was made. Phytoenedesaturase (PDS) levels were quantified by qRT-PCR. Lowering of PDS-RNA expression in PDS-VIGS lines in control plants were estimated. Using ANS gene specific primers, qRT-PCR was performed. It was reported that the transcript level of the VIGS plants were reduced. Catechin and epicatechin shown to have in greater quantity in control plant as compared to VIGS silenced expression line of induced P. tetragonolobus.

Materials and methods

Plant material

The ICAR-National Bureau of Plant Genetic Resources (NBPGR) India supplied the winged bean seeds (Psophocarpus tetragonolobus) and these seeds were cultivated in botanic garden of CSIR- National Botanical Research Institute in Lucknow, India for further use. These plants were grown in an augmented design with 3.0 m row length with spacing of 90 cm between rows and 30 cm between the plants. Standard method of cultivation and agronomic practices were followed as per the guidelines of International Board for Plant Genetic Resources (IBPGR) (Mohanty et al. 2013).

Histochemical staining of different plant tissues

The staining for condensed tannin of root, pod, flower and seed tissues of P. tetragonolobus was carried out through ethanol: HCl 6M (1:1) ratio (Hagerman et al. 1998), and 0.1% dimethyl amino cinnamaldehyde (DMACA) (Sigma-Aldrich) (w/v) for about 3 to 6 min and washed thrice with water. All other plant organs were stained for 2 h and flowers were stained for only 20 min. After staining, the tissues were visualized through microscope and photographed at an appropriate magnification (Abeynayake et al. 2011).

Histochemical analysis for GUS expression

The co-cultivated explants were rinsed with sterile water containing cefotaxime (0.25 mgl−1) and blotted on filter-paper. The GUS-histochemical staining was carried out according to the earlier report with minor modifications (Jefferson et al. 1987). These explants were incubated in GUS staining solution containing 1.0 mM of bromo-chloro-indolyl solution (X-Gluc), 0.5 mM ferrocyanide in 50 mM phosphate buffer (pH 7.0) at 37 °C overnight. The glucuronidase(GUS) stained explants were rinsed with sterile distilled water and treated with 70% Ethanol for de-coloration of chlorophyll.

Transcript-VIGS construct preparation

WbANS and WbPDS transcript sequences were extracted from de novo leaf-transcriptome data of comparative condensed-tannin lines of P.tetragonolobus (Singh et al.2017). Effective region of the transcript was selected by SGN-VIGS online tool (Fernandez-Pozo et al. 2015). These transcripts were amplified using gene specific primers (Supplementary Table S1). The purified fragment was cloned into PTRV1 and PTRV2 vector (procured from TAIR). It was digested with XbaI and SacI restriction enzymes (NEB). The digested product of both the insert and vector were ligated with 1 μl of T4 DNA ligase (NEB) (Singh et al. 2016; Senthil-Kumar et al. 2014). The ligated constructs of both the transcripts were transformed into the DH5α strain of E. coli. Amplification of replicase (562 bp), coat protein (351 bp), WbPDS (282 bp) and WbANS (162 bp) were carried out through the basic primers. They were tested in the colonies and were confirmed for positive construct through gene specific PCR amplification.

Plant growth and agro-infiltration

The freshly germinated P.tetragonolobus (4 leaf stage) was subjected to agro-infiltration with TRV construct. WbANS::TRV2 and WbPDS::TRV2 constructs were then transformed into GV3101 Agrobacterium tumefaciens strain. Positive transformants were chosen using colony PCR in Luria–Bertani agar medium (25 mg rifampicin, 50 mg gentamycin, 50 mg kanamycin per litre) (Kumar et al. 2003). The Agrobacterium strains were cultured in 5 ml primary cultures and were injected from individual colonies on plates two days before infiltration and grown for 16 h at 28 °C. The primary culture was injected with 20 ml of secondary culture, which was cultivated for (5–6) hrs. Centrifugation at 3000×g for 5 min at room temperature was used to extract the culture. The pellet was regenerated with the same quantity of induction buffer, 10 mM MES (pH 5.5) and 200 M acetosyringone and was incubated at 28 °C at 80 rpm for (3–4) hour. The culture was then pelleted at 3000×g and resuspended in 5 mM MES (pH 5.5) penetration buffer with OD600 set to (0.8–0.9). The cultures were combined at a ratio of 1:1 (v/v). The abaxial side of the lower leaves was inoculated with TRV1 + ANS::TRV2 Agrobacterium culture using a needleless syringe. TRV1:: TRV2 infiltration in the plants acted as control (empty vector).The inoculated plants in the greenhouse were analysed during the observations(Wang et al. 2018a, b).

RNA isolation and Real-Time PCR analysis

RNA isolation kit (Sigma-Aldrich) was used to extract total RNA from the upper leaves of 3 week-infiltered plants. RNase-free DNaseI treatment (Ambion) was provided to the extracted RNA. Approximately, 5 µg of total RNA and oligo (dT) primers were used for first-strand complementary DNA synthesis. Step One Plus qRT-PCR System was used to perform the PCR (Applied Biosystems 7500). Supplementary Table S1 lists the primers used in qRT-PCR. Three separate biological triplicates and two duplicates for each biological replicates were used for validation.

Comparative quantification of CT

The overall CT-value was measured using the colorimetric method of the modified vanillin-HCl assay in both VIGS and control P. tetragonolobus developed by Hagerman (Hagerman et al. 1981). Dry-leaf powder (~ 50 mg each) was suspended in 1 ml methanol and centrifuged. Approximately, 5 ml of active Vanillin-HCl reagent was added to the supernatant (one part vanillin solution and one part 8% HCl solution in methanol) was incubated for 20 min at 30 °C.The absorbance of the solution was recorded at 500 nm by spectrophotometer (Shimadzu, UV-1601).

Analysis of monomeric components of condensed tannin in P. tetragonolobus

HPLC analysis for catechin (Sigma-Aldrich, Cas No. 154-23-4), epicatechin (Sigma-Aldrich, Cas No.490-46-0) and VIGS line of P. tetragonolobus leaf was carried out on phenomenex Luna RP-C 18 column using high pressure liquid chromatography. The ultra violet (Shimadzu LC-10A, Japan) fitted two fold pump LC-10AT with binary system with UV detector SPD-10A at 254 nm, rheodyne injection valve with a 20 l loop on phenomenex Luna RP-C 18 (4.6 X 250 mm with 5 m pore-size) preceded with column guard of same chemistry. Shimadzu’s class VP series programme was used to incorporate the data. HPLC analysis of the dried powdered leaf-tissues of P.tetragonolobus was performed to produce metabolic profiles.

The HPLC system (Shimadzu LC-10A, Japan) consisted of quaternary pump with vacuum degasser, thermostat column compartment, auto sampler, and UV detector. A reverse-phase column (TARGA, C18, 5μ , 250 × 4.6 mm) was used and the column temperature was maintained at 30 °C. HPLC mobile phase was prepared with solution A: 0.1 mL of orthophosphoric acid dissolved in 900 mL of HPLC grade water and the volume was made up to 1000 mL with water and the solution was filtered through 0.45 μm membrane filter and degassed in a sonicator for 3 min, solution B: acetonitrile. Mobile phase was run using gradient elution: at the time 0.01 min 11% B; at the time 30 min 25% B; at the time 35 to 39 min 100% B; and at the time 40 to 50 min 11% B. The mobile phase flow rate was 1.0 mL/min and the injection volume was 10 μL. The eluents were detected and analyzed at 280 nm.

Confocal analysis

For condensed tannin deposition assay through Oregon green 488 staining was performed. Then 1 mg Oregon green was diluted with 5 ml PBS buffer. The infected leaves were incubated in 1 ml solution at room temperature for (15–30) minutes and washed with PBS solution for 15 min. The leaves which were stained were imaged using a Carl Zeiss LSM 510 META confocal microscope with a 20 × Plan-apochromate lens. Excitation was sustained with a 519 nm diode laser and emission was reported at in 499 nm band pass filter.

Multiple-sequence alignment and phylogenetic analysis of anthocyanidin synthase gene

MUSCLE software was employed for the sequence alignment of protein sequences of the ANS gene from transcript data of P. tetragonolobus. All protein databases of ANS was created and saved in the ClustalW format. The phylogenetic analysis was carried out at 1000 bootstrap replications using the MEGA 6.0 software (Wang et al. 2018a, b) The Poisson model approach was used to build an unrooted neighbor-joining tree as well as a minimum evolution tree using the same alignment file.

Statistical analysis

Statistical analyses giving the means and standard errors of means (SEM) of Relative gene expression, condensed tannin estimation and degree of fluorescence were performed by one-way ANOVA. For individual comparisons within a group, mean values were separated using Student’s t test at P values. P value < 0.05 to 0.004 in case of relative gene expression which showed significant difference between Control plant (EV) and VIGS-silenced plants. P value < 0.01 to 0.001, showed significant difference between Control plant (EV) and VIGS-silenced plants in case of condensed tannin estimation. When compare the mean value of degree of fluorescence in Control plant (EV) and VIGS-silenced plants, the P value is less than 0.05, showed significant difference.

Result

Localization of condensed tannin in different tissues

Pod, seeds, root and flower tissues of P. tetragonolobus at different stages of their maturity were decolorized in absolute ethanol. Staining of these samples were carried out with 0.1% (w/v) 4-dimethylaminocinnamaldehyde (DMACA) in absolute ethanol and 6 N hydrochloric acid in 1:1 ratio. It resulted in the development of a deep-blue color in seedling plant, leaf, flower, fleshy parts of the pod and seed-coat (Fig. 1a, b, c, d). The deep-blue colour indicates the presence of condensed tannin in all these tissues.

Fig. 1.

Fig. 1

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Localization of condensed tannin in tissues of P.tetragonolobus through DMACA staining a mature seed coat, b Tannin accumulation in different stages of seed development (0–7 days of duration) of P.tetragonolobus seeds, c T.S. of fresh seed containing seed coat, d flower

Establishment of Agrobacterium infection in various explants of P. tetragonolobus

Infection with Agrobacterium tumefaciens was established for transformation in P. tetragonolobus (Singh et al. 2014). A. tumefaciens was used for infection through the binary vector pCAMBIA1311 which consisted of β-glucuronidase (GUS) reporter gene (Supplementary Fig. S1). Cotyledonary, hypocotyl and epicotyl explants along with young leaf discs and cotyledonery leaf discs were infected with A. tumifaciens. It showed patches of blue colour when subjected to GUS histochemical staining (Supplementary Fig. S2). These results confirmed A. tumifaciens infection and transient expression of GUS gene in the transformed explants.

Development of constructs

The P. tetragonolobus ANS-VIGS construct was created by targeting the PDS gene and constructing primers from the conserved region of P.tetragonolobus transcript. The PDS construct was prepared by PCR based amplification of WB-PDS primer and cloned into VIGS vector (Fig. 2a). Due to the PDS gene silencing, 15 to 20 days after inoculation, infiltration of this PDS-VIGS construct resulted in systemic photo-bleaching in the leaves of P. tetragonolobus (Fig. 2b). The bleached areas were initially limited to the veins of the leaves, but the symptoms eventually spread to the majority of the leaf tissues. The expression level of PDS transcript was quantified by qRT-PCR (Fig. 2c). It resulted in 80 to 90% lowering of PDS-RNA expression in PDS-VIGS lines systemic tissue than in control plants (empty vector).PDS-VIGS line P1 and P2 were confirmed by PCR with PDS gene specific primer (282 bp) (Fig. 2d).

Fig. 2.

Fig. 2

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a systemic construct of TRV2:PDS. b VIGS-EV (control plant) and VIGS PDS plant (P1 and P2) positive phenotypic change in leave of silenced plant after 20 day post infiltration, c Real time expression of VIGS-PDS: data represent the mean ± SD. The Real-Time PCR (qRT-PCR) analysis was conducted in three independent experimental runs. Statistical analysis was performed using unpaired two-tailed t test (**p < 0.01) d Gel image showing positive screening of PDS-VIGS plants

Silencing of WbANS

The ANS construct was designed from ANS transcripts. The ANS-VIGS construct was prepared whenWbANS transcript was cloned into VIGS vector. Screening of positive construct was carried out through PCR with coat-protein primers (CP 351 bp) of virus vector and ANS (anthocyanidin 162 bp) gene specific primers of P. tetragonolobus (Fig. 3a). Six positive ANS-VIGS lines were picked up after 4 weeks of transformation using PCR with CP unique primers (Fig. 3b). Using ANS gene specific primers, qRT-PCR was performed with all six silenced plant and control plant, it was reported that the transcript level of the VIGS plants were reduced. When compared to the control plant, the ANS-VIGS lines showed a significant reduction of (up to 80%) of the constituents of CT (Fig. 4a).

Fig. 3.

Fig. 3

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a Systemic construct of TRV2: ANS, b Gel image showing the ANS (162 bp) and CP (351 bp) gene specific primer for screening of positive construct of ANS (Anthocyanidin synthase), c Positive selection of VIGS-ANS plant: positive plant selected through coat protein primer of virus (351 bp)

Fig. 4.

Fig. 4

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a Expression levels in leaves of Control plant (EV) and VIGS-silenced plants. Six silencing events were analyzed (1–6).Error bars represent mean ± SD for three technical replicates for each. The RT-qPCR analysis was conducted in three independent experimental runs. Statistical analysis was performed using unpaired two-tailed t-test varies between p < 0.05–0.004 b Condensed tannin estimation through vanillin assay in control plant (EV) and six VIGS-ANS lines of P.tetragonolobus. Error bars represent mean ± SD for three technical replicates for each. Vanillin-HCl assay was conducted in three independent experimental runs. Statistical analysis was performed using unpaired two-tailed t test varies between p < 0.001–0.01. Levels of the asterisk on the basis of P values calculate through Student’s t test (*P > 0.05, **p > 0.01 and ***P > 0.001)

Condensed tannin estimation through Vanillin-HCl assay

Quantitative estimation of condensed tannin was determined in both silenced and control plant of P. tetragonolobus. A calibration curve of catechin-equivalent standard curve was obtained and expressed in mg/g dry weight. All assays were carried out in triplicates. The total condensed tannin content was estimated by Vanillin-HCl assay. In control plant, the value was 2.6 mg/g of dry weight of leaf sample and in case of all six VIGS-silenced line of P. tetragonolobus the value reduced to range between 1.2 and 0.7 mg/g of dry weight of leaf tissues (Fig. 4b) in all the six VIGS-silenced lines of P. tetragonolobus. So, a significant decrease of 70–73% in condensed tannin content was reported in VIGS-induced P. tetragonolobus lines.

Silencing of ANS transcripts decreased the accumulation of catechin and epicatechin

Catechin and epicatechin showed higher quantity in control as well as VIGS silenced P. tetragonolobus. The quantity was estimated through standard HPLC (Supplementary Fig. S.3). The control plant contained 3.8 mg of catechin per g of dry weight of leaf sample. But, in VIGS-silenced line the concentration of catechin decreased upto 0.28 mg/g. This showed that, catechin concentration decreased up to 92% after silencing of ANS transcript. Same is in the case of epicatechin concentration of VIGS line which decreased up to 71% compared to the control plant (Table 1).

Table 1.

Quantification of Monomeric unit of condensed tannin

Monomeric unit of condensed tannin Control plant (EV Empty vector) P. tetragonolobus VIGS-ANS P. tetragonolobus
Catechin (mg/g) 4.8 ± 0.10 0.28 ± 0.03
Epicatechin (mg/g) 1.8 ± 0.06 0.52 ± 0.08
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Confocal microscopic analysis of the VIGS-induced P. tetragonolobus

The concentration of condensed tannin in both VIGS-silenced as well as control P. tetragonolobus was visualized by confocal microscopy after Oregon green staining. Transverse sections of the leaves of both the lines were observed under microscope. Integrated hexagonal network of cells without any deformities or abnormalities were observed in control plant but in case of VIGS-line, there was breakage in hexagonal networking of cells (Fig. 5a). Oregon green dye formed complex with condensed tannin and the confocal microscopic images showed green fluorescence on interaction. Image J software was used to measure the fluorescence levels of the control and VIGS P. tetragonolobus (http://rsbweb.nih.gov/ij/) and it was reported that in VIGS-line of P. tetragonolobus, the florescence level decreased significantly compared to the control plant (Fig. 5b).This result confirmed that the silencing of anthocyanidin synthase gene in P. tetragonolobus have a regulatory effect on condensed tannin biosynthesis in P. tetragonolobus.

Fig. 5.

Fig. 5

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Confocal microscopy of Control vs VIGS plant: a Oregon green staining in the leaf of P. tetragonolobus in VIGS silenced plant and control plant (20X magnifications on confocal microscope. Bars = 20 μm). Upper lane of image is Control plant and lower one is VIGSsilenced plant (1). OG oregon green stained (2) DIC and (3) merge of Oregon green and DIC b the degree of fluorescence calculated by IMAGE J software which showed significant decrease in VIGS-ANS silenced plant. Error bars represent the standard deviation of three biological replicates. Asterisks represent Student’s t test: *P < 0.05

Phylogenetic analysis of ANS

Phylogenetic analysis of the conserved ANS protein sequences were carried out among 114 plant species. It grouped them into four well defined clades based upon their proximity with each other. The phylogenetic tree was generated on the basis of their likelihood and similarity upon analysis on MEGA-X software. Four clades were formed indicating the similarity among the plant-taxa. In the first-clade, P. tetragonolobus showed closeness with Spinach oleraceae and out group in the clade was Fragaria esculentum. In second clade, P. tetragonolobus was in close proximity to Carthamus tintctorius and Oryza officinalis (Fig. 6).

Fig. 6.

Fig. 6

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Phylogenetic analysis of Anthocyanidin synthase gene of two P.tetragonolobus sequence with National Center for Biotechnology Information(NCBI) data of anthocyanidin synthase through Mega 6 software

Discussion

The nutritive benefit of the browse shrub mulga is substantially decreased when CT reaches (8–10)% of dietary dry content (Pritchard et al. 1988). P. tetragonolobus is one of the underutilized legume of agricultural importance and contain high protein. Almost all parts of the plant are edible. It has high nutritive value in terms of protein and fatty oil content (Mohanty et al. 2014). But, presence of anti-nutrients is one of the reasons of its lesser and limited use. Condensed tannin is one among the anti-nutrients in P. tetragonolobus. Localization, qualitative and quantitative estimation of condensed tannin through DMACA histochemical staining and Vanillin-HCl assay respectively was carried out in different tissues of the P. tetragonolobus. DMACA staining exhibited a high level of condensed tannin in almost all tissues at different developmental stages of the seed. It has susceptibility to flavonols, since epicatechin is 1000 times more resilient than the structurally similar p-dimethylamino benzaldehyde (Robertson et al. 2004; Treutter et al. 1989). To classify spatial patterns of condensed tannin aggregation in different plant tissues, histochemical staining of plant tissues with DMACA is commonly used. The formation of CTs in tracheophyta inside the thylakoid-derived organelles, the tannosomes was proposed by (Brillouet et al. 2004). Multiple membrane-bound shuttles transport tannosomes from the plastid to the vacuole. HPLC analysis reported flavan-3-ol (catechin) as a general secondary metabolite in seeds and various tissues of this plant. Condensed tannin gets highly accumulated in the ovary, pod, root and fleshy part of the outer seed coat of the plant (Fig. 1). Microscopic observations and histochemical staining indicated that, the seeds are the major sites of accumulation of CTs (Fig. 1e). HPLC analysis of P. tetragonolobus seeds revealed the existence of flavan-3-ols (catechins) as a general secondary metabolite, as well as catechin, epicatechin and various dimeric flavan-3-ols as predominant polyphenol (Table 1). Anthocyanins are synthesized on the endoplasmic reticulum (ER) cytoplasmic surface but accumulate primarily in the vacuole, with glutathione S-transferases (GSTs) thought to be primarily responsible for the transport process As anthocyanidin synthase is the enzyme for synthesis of condensed tannin in P. tetragonolobus. Evolutionary analysis revealed that the WbANS proteins were closely related to Spinacia. P. tetragonolobus protein had been shown similarity with Glycine max. It has been revealed that ANS gene was evolved early in evolution, which might be playing a significant role in the biosynthesis of condensed tannin among the different members of the plant-kingdom.

VIGStechnology has been regarded as one of the quickest methods for determining gene function. At present, more than 40 viruses have been transformed into VIGS vectors and approximately 37 of which can be used for gene silencing in dicotyledons till date (Wang et al. 2021). Establishing a link between viral loading and VIGS response will aid in clarifying mechanisms and optimizing VIGS efficiency in various crop plants (Orzaez et al. 2009). Despite the benefits and utility of VIGS in plant functional genomics research, an important limitation is the uneven distribution of silencing in target tissues. The use of visible reporter genes would allow for the efficient separation of silenced tissues from non-silenced tissues, increasing the sensitivity of subsequent analysis. In VIGS, a reporter gene is required to visualize the silenced region and track silencing efficiency. Phytoene desaturase (PDS) silencing reduced chlorophyll and carotenoid biosynthesis, resulting in decreased photosynthetic activity, and the PDS-silenced plant displayed growth defects (Kim et al. 2017).

Virus-induced gene silencing (VIGS) is a simple method of inhibiting gene expression to determine the function of a specific gene (Brigneti et al. 2004). The preparation of an efficient VIGS construct for ANS transcript along with the silencing by ANS within the plant is influenced by environmental conditions as well as the process of inoculation (Broderick et al. 2013; Singh et al. 2016). The expressed genes were used for optimization of gene silencing (Ruiz et al. 1998: Sha et al. 2014). The PDS-VIGS gene silencing of the PDS gene in chilli pepper leaves resulted in the repression of flavonoid biosynthesis. So, photo-bleaching became a problem for the virus-infected silenced plants (Zhang et al. 2015).

The WB-PDS was effectively silenced using PDS from transcript data of P.tetragonolobus as similar efficiency was found among the members of Solanaceaeous family with TRV and PDS constructs (Senthil-Kumar et al. 2011). The ANS-VIGS mechanism had been used to down-regulate anthocyanidin synthase to gain better understanding of their function in plants. The better understanding of the result of the ANS transcript-silencing on condensed tannin biosynthetic pathway was confirmed through qPCR analysis of different silenced plants which encode ANS. The expression level of ANS gene was decreased in silenced plants. The down-regulated silenced lines accumulated low amount of catechin and epicatechin in the leaves. From these results, it was evident that, the accumulation of condensed tannin increased due to the increased expression of ANS encoding gene in the biosynthetic pathway of condensed tannin. The HPLC analysis of silenced lines of P. tetragonolobus indicated that, catechin and epicatechin concentration was lowered. Previous studies about the condensed tannin showed that, synthesis of condensed tannin started in chloroplast and got accumulated in the vacuole of cell (Gargouri et al. 2010). Accumulation of condensed tannin was confirmed through Oregon green dye and it showed that the accumulation to decrease with the silencing of ANS-encoding genes of P. tetragonolobus (Fig. 5).

Conclusion

An efficient protocol for virus induced gene silencing was developed to down-regulate ANS transcript in P. tetragonolobus. Decreased level of condensed tannin was reported in the WB-ANS-VIGS silenced plant. This silenced line of P. tetragonolobus provided transient expression of ANS-encoding gene. This study will open up new avenues for administering current editing technologies for production of stable line with lowered condensed tannin-level in P. tetragonolobus. This will help in creating protein-rich P. tetragonolobus line with reduced condensed tannin content which can be further utilized for addressing the food and nutrition security.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 2119 KB) (2.1MB, docx)

Acknowledgements

We acknowledge Director, CSIR-NBRI for providing the required infrastrutural facilities for carrying out this activity. The manuscript number provided by the institute is CSIR-NBRI_MS/2021/04/05.

Declarations

Conflict of interest

Conflict of interest the authors declare no conflict of interest.

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