Functional validation of Winged bean (Psophocarpus tetragonolobus (L.) DC.) anthocyanidin synthase (Wb ANS) gene through virus-induced gene silencing

Abstract Background Histochemical and microscopic observations of various tissues of the underutilized legume winged bean (Psophocarpus tetragonolobus (L.) DC.) indicated that the plant and its various parts are highly infested with condensed tannin (CT). Characterization of CT was carried out through the quantification of its structural-monomeric units catechin and epicatechin. The responsible candidate gene for anthocyanidin synthase (ANS) biosynthesis was identified, phylogenetically mapped and manipulated for lowering the CT-content. Results Virus-induced gene silencing (VIGS) was employed for silencing of WbANS transcript. WbANS-VIGS induction resulted in four-fold decrease in condensed tannin biosynthesis in P. tetragonolobus. Conclusion As condensed tannin adversely affects digestion and considered as an anti-nutrient, so this study might be helpful in future for altering the biosynthesis of condensed tannin by manipulating the ANS-encoding molecular factors.

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Unraveling the regulatory role of miRNAs responsible for proanthocyanidin biosynthesis in the underutilized legume Psophocarpus tetragonolobus (L.) DC.

10.1101/2021.07.24.453638 ◽

2021 ◽

Author(s):

Sagar Prasad Nayak ◽

Priti Prasad ◽

Vinayak Singh ◽

Abhinandan Mani Tripathi ◽

Sumit K Bag ◽

...

Keyword(s):

Condensed Tannin ◽

Epigallocatechin Gallate ◽

Target Prediction ◽

Mature Mirnas ◽

Winged Bean ◽

Pcr Analysis ◽

Psophocarpus Tetragonolobus ◽

Leaf Tissues ◽

Underutilized Legume

The underutilized legume winged bean (Psophocarpus tetragonolobus (L.) DC.) is deposited with various degrees of proanathocyanidin (PA) or condensed tannin (CT) on its seed-coat. PA content of two different lines of P. tetragonolobus was estimated and accordingly they were denoted as high-proanthocyanidin containing winged bean (HPW) and low-proanthocyanidin containing winged bean (LPW). The level of PA-content varied as 59.23 mg/g in HPW and 8.68 mg/g in LPW when estimated through vanillin-HCl assay. The identification and quantification of catechin and epigallocatechin gallate were estimated in a range of 63.8 mg/g and 2.3 mg/g respectively in HPW whereas only epigallocatechin gallate was reported in LPW line with a value of 3 mg/g. A comparative miRNA profiling of the leaf-tissues of these contrasting lines of P. tetragonolobus revealed a total of 139 mature miRNAs. Isoforms of known novel miRNAs were also identified in this study. Differentially expressed miRNAs e.g., miR156, miR396, miR4414b, miR4416c, miR894, miR2111 and miR5139 were validated through qRT-PCR analysis. Target prediction of the identified miRNAs especially miR156, miR396, miR4416b shows that they have a potential role in the proanthocyanidin biosynthesis of P. tetragonolobus. The study will provide the basis for understanding the role of miRNAs in regulating the biosynthesis of proanthocyanidin.

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Proteases of germinating winged-bean (Psophocarpus tetragonolobus) seeds: purification and characterization of an acidic protease

Biochemical Journal ◽

10.1042/bj3130423 ◽

1996 ◽

Vol 313 (2) ◽

pp. 423-429 ◽

Cited By ~ 9

Author(s):

Rajamma USHA ◽

Manoranjan SINGH

Keyword(s):

Physiological Role ◽

Immunoblot Analysis ◽

Protein Band ◽

Winged Bean ◽

Psophocarpus Tetragonolobus ◽

Ph Optimum ◽

Apparent Homogeneity ◽

Sds Page ◽

Storage Protein Mobilization

Two major classes of protease are shown to occur in germinating winged-bean (Psophocarpus tetragonolobus) seeds, by assaying extracts at pH 8.0 and pH 5.1 with [14C]gelatin as substrate. At pH 8.0, the activity profile of the enzyme shows a steady rise throughout the period of germination, whereas the activity at the acidic pH is very low up to day 5 and then increases sharply reaching a peak on day 11, followed by an equally sharp decline. The winged-bean acidic protease (WbAP) has been purified to apparent homogeneity, as attested by a single protein band on both PAGE and SDS/PAGE. WbAP is a monomeric enzyme with a molecular mass of 35 kDa and a pH optimum of 6.0. It is a thiol protease that does not belong to the papain family and it has tightly bound Ca2+ as shown by 45Ca2+-exchange studies. Besides gelatin and casein, it hydrolyses a 29 kDa winged-bean protein, indicating a prospective physiological role for it in storage-protein mobilization. Immunoblot analysis shows that it occurs only in the seeds and sprouting tubers of this plant and also that it is synthesized in developing seeds just before desiccation. It appears that the newly synthesized enzyme is inactive, and activation takes place around day 6 of germination. However, neither the mechanism of activation nor the signal that triggers it is clearly understood.

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Characterization of winged bean (<i>Psophocarpus tetragonolobus</i> (L.) DC.) based on molecular, chemical and physiological parameters

American Journal of Molecular Biology ◽

10.4236/ajmb.2013.34025 ◽

2013 ◽

Vol 03 (04) ◽

pp. 187-197 ◽

Cited By ~ 18

Author(s):

Chandra Sekhar Mohanty ◽

Sushma Verma ◽

Vinayak Singh ◽

Shahina Khan ◽

Priyanka Gaur ◽

...

Keyword(s):

Physiological Parameters ◽

Winged Bean ◽

Psophocarpus Tetragonolobus

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Virus-Induced Gene Silencing-Based Functional Characterization of Genes Associated with Powdery Mildew Resistance in Barley

PLANT PHYSIOLOGY ◽

10.1104/pp.105.062810 ◽

2005 ◽

Vol 138 (4) ◽

pp. 2155-2164 ◽

Cited By ~ 159

Author(s):

Ingo Hein ◽

Maria Barciszewska-Pacak ◽

Katarina Hrubikova ◽

Sandie Williamson ◽

Malene Dinesen ◽

...

Keyword(s):

Powdery Mildew ◽

Gene Silencing ◽

Powdery Mildew Resistance ◽

Functional Characterization ◽

Virus Induced Gene Silencing ◽

Mildew Resistance

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Application of Virus-induced Gene Silencing Technology in Gene Functional Validation of Orchids

Orchid Biotechnology ◽

10.1142/9789812775900_0012 ◽

2007 ◽

pp. 211-223 ◽

Cited By ~ 2

Author(s):

Hsiang-Chia Lu ◽

Hong-Hwa Chen ◽

Hsin-Hung Yeh

Keyword(s):

Gene Silencing ◽

Virus Induced Gene Silencing ◽

Functional Validation

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Simultaneous silencing of two target genes using virus-induced gene silencing technology in Nicotiana benthamiana

Zeitschrift für Naturforschung C ◽

10.1515/znc-2018-0071 ◽

2019 ◽

Vol 74 (5-6) ◽

pp. 151-159

Author(s):

Feng Zhu ◽

Yanping Che ◽

Fei Xu ◽

Yangkai Zhou ◽

Kun Qian ◽

...

Keyword(s):

Gene Silencing ◽

Nicotiana Benthamiana ◽

Target Genes ◽

Function Analysis ◽

Tobacco Rattle Virus ◽

Pcr Analysis ◽

Virus Induced Gene Silencing ◽

Biotic Stresses ◽

Overlap Extension

Abstract Virus-induced gene silencing (VIGS) is an effective strategy for rapid gene function analysis. It is well established that the NAC transcription factor and salicylic acid (SA) signal pathway play essential roles in response to biotic stresses. However, simultaneous silencing of two target genes using VIGS in plants has been rarely reported. Therefore, in this report, we performed VIGS to silence simultaneously the SA-binding protein 2 (NbSABP2) and NbNAC1 in Nicotiana benthamiana to investigate the gene silencing efficiency of simultaneous silencing of two genes. We first cloned the full-length NbNAC1 gene, and the characterization of NbNAC1 was also analysed. Overlap extension polymerase chain reaction (PCR) analysis showed that the combination of NbSABP2 and NbNAC1 was successfully amplified. Bacteria liquid PCR confirmed that the combination of NbSABP2 and NbNAC1 was successfully inserted into the tobacco rattle virus vector. The results showed that the leaves from the NbSABP2 and NbNAC1 gene-silenced plants collapsed slightly, with browning at the base of petiole or veina. Quantitative real-time PCR results showed that the expression of NbSABP2 and NbNAC1 were significantly reduced in 12 days post silenced plants after tobacco rattle virus infiltration compared with the control plants. Overall, our results suggest that VIGS can be used to silence simultaneously two target genes.

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